THE FUNCTIONAL CHARACTERISATION OF HUMAN INNATE LYMPHOCYTES IN
RENAL FIBROSIS AND CHRONIC KIDNEY DISEASE
Becker M.P. Law BSc (Hons)
Submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
Institute of Health and Biomedical Innovation
School of Biomedical Sciences
Faculty of Health
Queensland University of Technology
2019
To my family, friends,
past and present mentors, and
Ms. Stephanie Tan.
QUT Verified Signature
ii The Functional Characterisation of Human Innate Lymphocytes in Renal Fibrosis and Chronic Kidney Disease
Keywords
Chronic Kidney Disease
Gamma-Delta T Cells
Mucosal Associated Invariant T Cells
Natural Killer Cells
Proximal Tubular Epithelial Cells
The Functional Characterisation of Human Innate Lymphocytes in Renal Fibrosis and Chronic Kidney Disease iii
Abstract
Chronic kidney disease (CKD) is a complex syndrome characterised by gradual loss
of kidney function over time. Irrespective of the initial kidney insult, CKD involves
unsuccessful repair of existing injury and exhibits pathological features including
tubulointerstitial fibrosis, lymphocyte infiltration, proximal tubular atrophy and
hypoxia. For more than a decade there have been no advances in CKD therapeutic
options despite our accumulating understanding of lymphocyte-mediated pathogenesis
from animal models of kidney disease. This research shifts our focus back on to human
lymphocytes, in particular, innate lymphocytes such as natural killer (NK), gamma-
delta (γδ) T and mucosal associated invariant T (MAIT) cells. The objectives are to
decipher the functional roles of innate lymphocytes using ex vivo flow cytometric and
in situ immunofluorescence confocal microscopy analysis of fresh and frozen renal
specimens, as well as in vitro co-culture with kidney parenchymal cells under pro-
fibrotic/hypoxic conditions to examine mechanistic interactions. From the first study,
CD56bright NK cells were found to be a significant producer of pro-inflammatory
cytokine IFN-γ in fibrotic renal biopsies. Following this work, γδ T cells were
examined and found to contribute to CKD via the secretion of pro-inflammatory
cytokine IL-17A. In the final part of this thesis, I describe MAIT cell cytotoxicity of
proximal tubular epithelial cells (PTEC) in human CKD. This research uncovers
several novel immune signature molecules and cellular targets for future development
as potential therapeutics and diagnostics in human CKD.
iv The Functional Characterisation of Human Innate Lymphocytes in Renal Fibrosis and Chronic Kidney Disease
List of Publications
Manuscripts presented in this Thesis
Law BMP, Wilkinson R, Wang X, Kildey K, Lindner M, Rist MJ, Beagley K, Healy H, Kassianos AJ. Interferon-γ production by tubulointerstitial human CD56bright natural killer cells contributes to renal fibrosis and chronic kidney disease progression. Kidney Int. 2017; 92(1). Law BMP, Wilkinson R, Wang X, Kildey K, Lindner M, Beagley K, Healy H, Kassianos AJ. Effector γδ T cells in human renal fibrosis and chronic kidney disease. Nephrol Dial Transplant. 2018. doi: 10.1093/ndt/gfy098. Law BMP, Wilkinson R, Wang X, Kildey K, Giuliani K, Beagley K, Ungerer J, Healy H, Kassianos AJ. Human tissue-resident mucosal-associated invariant T (MAIT) cells contribute to renal fibrosis and chronic kidney disease (CKD). J Am Soc Nephrol. [Manuscript Accepted]
Manuscripts relevant to this Thesis but not incorporated
Wilson GJ, Gois PHF, Zhang A, Wang X, Law BMP, Kassianos AJ, Healy H. The Role of Oxidative Stress and Inflammation in Acute Oxalate Nephropathy Associated With Ethylene Glycol Intoxication. Kidney Int Reports. 2018; 3(5). Kildey K, Law BMP, Muczynski K, Wilkinson R, Healy H, Kassianos A. Identification and Quantitation of Leukocyte Populations in Human Kidney Tissue by Multi-parameter Flow Cytometry. Bio-Protocol . 2018; 8(16). Kildey K, Francis R, Hultin S, Hartfield M, Giuliani K, Law BMP, Wang X, See E, John G, Ungerer J, Wilkinson R, Kassianos A, Healy H. Specialised roles of human natural killer cell subsets in kidney transplant rejection. Frontiers of Physiology. 2019. [Manuscript Submitted]
The Functional Characterisation of Human Innate Lymphocytes in Renal Fibrosis and Chronic Kidney Disease v
Acknowledgements
This work would not be possible without the support and mentorship from my
supervisors, the conjoint renal medical laboratory members, QIMR Berghofer
scientific support and research fundings from RBWH foundation, Pathology
Queensland, QUT Institute of Health and Biomedical Innovation, and the National
Health and Medical Research Council.
vi The Functional Characterisation of Human Innate Lymphocytes in Renal Fibrosis and Chronic Kidney Disease
Thesis Format
This is a thesis by publication and consists of 5 chapters. Chapter 1 presents the
research background, a literature review, the aims and objectives to address the
research problem. The findings are presented in chapters 2, 3 and 4 and will be
presented by publications comprised of three published peer-reviewed manuscripts
accepted in Kidney International, Nephrology Dialysis Transplantation and Journal
of the American Society of Nephrology. The significance and future direction of this
research will be discussed in Chapter 5.
The Functional Characterisation of Human Innate Lymphocytes in Renal Fibrosis and Chronic Kidney Disease vii
Table of Contents
Statement of Original Authorship ............................................................................................. i
Keywords ................................................................................................................................. ii
Abstract ................................................................................................................................... iii
List of Publications ................................................................................................................. iv
Acknowledgements ...................................................................................................................v
Thesis Format.......................................................................................................................... vi
Table of Contents ................................................................................................................... vii
Chapter 1: Literature Review and Research Objectives .................................. 1
Chronic kidney disease .............................................................................................................2 Definition, treatments and outcomes ..............................................................................2 Etiologies and mechanisms of injury ..............................................................................4 Lymphocytes and CKD ..................................................................................................5
Innate Lymphocytes and CKD ..................................................................................................6 Activation ......................................................................................................................7 Recruitment and retention ...............................................................................................7 Tissue residency .............................................................................................................7 Function of Innate Lymphocytes in CKD.......................................................................9
Proximal Tubular Epithelial Cells and CKD ..........................................................................16 PTEC-immune cell cross-talk during renal immune homeostasis ................................16 PTEC-immune cell cross-talk in CKD .........................................................................17 Renal hypoxia: key driver of PTEC injury ...................................................................17
Summary and Research Hypothesis ........................................................................................19
Research Objective and Aims .................................................................................................21
References ...............................................................................................................................22
Chapter 2: Natural Killer Cells in CKD .......................................................... 33
Chapter 3: Gamma-Delta T cells in CKD ........................................................ 65
Chapter 4: Mucosal Associated Invariant T cells in CKD ............................. 95
Chapter 5: General Discussion ....................................................................... 135
Summary of findings .............................................................................................................136
Research in progress and outstanding research questions.....................................................139 Recruitment and retention pathways...........................................................................139 Activation pathways ...................................................................................................142 Other innate lymphocytes in CKD..............................................................................144 Function of IFN-γ and IL-17A in human CKD ..........................................................144
Limitations of the current study ............................................................................................145
Clinical and diagnostic translation ........................................................................................146 Therapeutic translation ...............................................................................................146 Diagnostic translation .................................................................................................146
Concluding remarks ..............................................................................................................148
References .............................................................................................................................149
Chapter 1: Literature Review and Research Objectives 1
Chapter 1: Literature Review and Research Objectives
Chronic Kidney Disease (CKD) is a complicated disorder with
limited information on the role of innate lymphocytes in the pathogenesis
of human kidney disease. The research objective is to examine innate
lymphocyte function in human CKD and the effect of pro-fibrotic/hypoxic
conditions on innate lymphocyte and proximal tubule epithelial cell
interactions.
In this Chapter, I will provide an introduction to this thesis with the
background and literature necessary to understand the objective of this
project, the specific aims of the study, and the findings of this PhD in the
subsequent chapters.
2 Chapter 1: Literature Review and Research Objectives
CHRONIC KIDNEY DISEASE
Chronic kidney disease (CKD) is the most prevalent chronic disease in
Australia.1 One in nine Australian adults exhibit at least one marker of CKD, and the
cost of its management accounts for over a billion dollars per year. CKD challenges
the financial viability of the health care system and its burden on patients and societies
continues to rise.2 In the past decade, there have been no definitive cures or
improvements in the treatments available for CKD, urging the critical development of
new therapies.3
Definition, treatments and outcomes
CKD is a complicated syndrome involving persistent changes to kidney structure
and function (Figure 1.1). The latest classification of CKD, defined by the Kidney
Disease Improving Global Outcomes (KDIGO) initiative, is based on abnormalities of
kidney structure or function that persist for >3 months.4
Treatment of CKD is challenging because patients with progressive chronic
renal failure are mostly asymptomatic and therefore, do not seek medical treatment
until it is too late to be offered preventive therapies. Current medical treatments lack
specificity and offer no treatment to already damaged renal structure. The therapeutic
goal for treating CKD is to preserve existing renal function and to delay end-stage
kidney disease (ESKD) progression.5
Therapy of CKD can be divided into three phases: early, late and kidney
replacement therapy (KRT). Early therapy is targeted against the initiating or
underlying renal disorder since CKD is not a single process and renal insults can come
from a variety of sources. In contrast, treatment of late-stage CKD as a single entity
has been more common because irrespective of initiating insult, eventual development
to ESRD is a shared physiological pathway that underlies the progression of CKD. The
only therapeutic option when CKD has progressed to ESKD is KRT (dialysis or kidney
transplantation). KRT is not able to replace all the functions of our kidneys. It prolongs
life expectancy but comes with reduced quality of life and increased health
complications.6
Chapter 1: Literature Review and Research Objectives 3
Figure 1.1 Kidney anatomy and physiology under homeostatic conditions. (adapted from [7])
The kidneys are a pair of bean-shaped organs that specialise in metabolic waste excretion, nutrient retention, hormone secretion, and fluid and biochemical homeostasis. They are located in the abdominal cavity on either side of the vertebral column and are enclosed by a fibrous capsule. Beneath this enclosure lies the kidney parenchyma which is divided into an outer cortex and inner medulla. The adult kidney is composed of about 1.5 million nephrons – the functional units of the kidney. Each nephron comprises two compartments: the glomerulus and the renal tubules. The glomerular compartment is made up of a bed of capillary loops surrounded by a saclike structure called the Bowman’s capsule. The tubular compartment is an extension of the Bowman’s capsule and includes the proximal tubule, loop of Henle, distal tubule and the collecting duct.
The process of blood filtration by the nephron begins within the glomerulus located at the cortex. The glomerular filtrate flows into the proximal tubule and moves toward the medulla. The majority of nutrients, such as glucose, amino acids, minerals and vitamins, are actively reabsorbed by the highly metabolic proximal tubules during this stage. The ultrafiltrate then drains into the loop of Henle, which is responsible for creating concentration gradients that favour the reabsorption of water. The remaining ultrafiltrate then travels towards the cortex and passes through the distal tubules where pH and water regulation occur. The distal tubule extends to a point in the cortex where multiple nephrons join to form the collecting duct. Finally, further water reabsorption occurs at the collecting duct system to form concentrated urine.7,8
4 Chapter 1: Literature Review and Research Objectives
Etiologies and mechanisms of injury
Injury to the healthy kidney drives cellular responses that lead to inflammation
and tissue repair and the restoration of normal physiology. However, if the response
to injury is inappropriate or not resolved, the dysfunctional nephron can interfere with
overall kidney function by overstressing the remaining nephrons, leading to a cycle of
injury, reduced oxygen diffusion within the tubulointerstitium (renal hypoxia) and
unsuccessful repair. The result of kidney injury and unwanted cellular responses can
lead to a chronic decline in renal function, progressive nephron loss and thus CKD.9
CKD progression is categorised into multiple stages and begins with minimal
kidney injury (CKD stage 1) to ESKD (CKD stage 5). The classification of CKD
stages is determined by the extent of renal barrier dysfunction and performance of
renal excretory function.6 These are measured by albuminuria levels and estimated
glomerular filtration rates (eGFR) respectively and are reliable predictors of CKD
outcomes.6
CKD is an heterogeneous group of disease and has no singular cause.10 Kidney
injury, and the subsequent development of CKD, can be initiated by various conditions
and factors (Table 1.1). However, there are common pathophysiological processes
downstream of the initiating disease/injury that describe the maladaptive response
common to all CKD. These include drop out of the specialised kidney tubules (renal
tubular atrophy), accumulation of mononuclear cells and the development of fibrosis
in the tubulointerstitium.10,11 The pathogenesis of renal fibrosis is a multidimensional
process. In this PhD thesis I will primarily focus on the crucial role of innate
lymphocytes and their impact on proximal tubules in CKD progression.
Table 1.1 Common diseases and conditions that can cause kidney injury and CKD (adapted from
[10])
Diabetes mellitus Hypertension Genetic disorders Infectious diseases
Malignancies Autoimmune disease Urinary tract obstruction Renal toxins
Chapter 1: Literature Review and Research Objectives 5
Lymphocytes and CKD
In CKD, the renal immune system is severely compromised leading to loss of
renal homeostasis as well as loss of kidney function. Immune cells such as
lymphocytes, in particular, are implicated in the progression of CKD as they are
strongly associated with lesions in the tubulointerstitium.10 Clinical studies of human
kidney biopsies have reported strong correlations between lymphocyte numbers,
cytokines and chemokines specific for lymphocyte chemotaxis with progressive loss
of renal function and tubulointerstitial injury.12–16 As a result, many animal models
have been developed for investigating the mechanisms of tubulointerstitial damage by
lymphocytes.
Experimental approaches to induce inflammation and tubulointerstitial injury
include non-immune mediated and immune-mediated murine models. Non-immune
mediated examples can be: drug induced (cadmium chloride, mercury chloride and
adriamycin); extreme diet containing high folic acid, adenine, salt and lipid contents;
ischemia reperfusion injury (IRI) by renal blood vessel clamping; and unilateral ureter
obstruction (UUO) by ureter ligation.17 On the other hand, anti-neutrophil cytoplasmic
antibodies (ANCA), anti-glomerular basement membrane (GBM) antibodies and
genetic modulation causing autoimmune diseases have been utilized to generate
immune-mediated injury in murine kidneys.17
Although attempts in these models to understand lymphocyte function, by
deletion of specific lymphocyte subsets or through inhibition of chemokines and
cytokines, have demonstrated attenuation of renal disease progression, the functional
roles of the different lymphocyte subsets in the progression of human kidney disease
is still not well characterised and require further investigation. In this PhD project, I
will focus on the function of specific innate lymphocyte subsets in human CKD,
which, until now, have received limited attention. The characteristics and roles of these
cells in murine and human kidneys will be discussed in the next section.
6 Chapter 1: Literature Review and Research Objectives
INNATE LYMPHOCYTES AND CKD
Immune cells in humans can be separated in two arms of the immune system:
the innate and adaptive immune system.18 The cells of the innate arm of the immune
system provide critical cellular responses for the rapid sensing and elimination of
foreign substances without self:non-self discrimination. In contrast, cells of the
adaptive immune system are more specifically tuned and involve the recognition of
specific self and non-self antigens. Different lymphocyte subsets participate in both
innate and adaptive immune responses and can be broadly classified into innate and
adaptive lymphocytes.19
Adaptive lymphocytes of the adaptive immune system are immature
lymphocytes that require antigen-specific stimulation to differentiate into mature cells
capable of mounting an immune response. T and B lymphocytes are adaptive
lymphocytes and are characterised by antigen-specific surface receptors -
immunoglobulin on B cells and T cell receptors (TCR) on T cells.
In contrast, innate lymphocytes of the innate immune system comprise of
mature/differentiated cells that can readily respond to stimuli, typically with
production of pro-inflammatory cytokines and cytolysis of target cells. The immune
response driven by innate lymphocytes is an efficient mechanism to limit the spread
of disease, as they do not require priming, maturation, and clonal expansion in the
periphery and are considerably more rapid (hours) compared to the adaptive immune
system (days).19
Innate lymphocytes can be classified into two broad groups: 1) Cells from the
lymphoid lineage but do not express a TCR, and 2) Cells that express an
unconventional TCR but have innate characteristics. Natural killer (NK) cells and the
newly identified innate lymphoid cells (ILCs) are examples of lymphocytes without a
TCR. On the other hand, gamma-delta (γδ) T cells, mucosal associated invariant T
(MAIT) cells, and natural killer T (NKT) cells are examples of innate-like
unconventional T cells that recognise antigen presented via non-traditional antigen-
presenting molecules.19
Chapter 1: Literature Review and Research Objectives 7
Activation
The activity of innate lymphocytes can be controlled via triggering of inhibitory
or activatory receptors on their cell surface or in response to soluble factors
(inflammatory cytokines) via their high expression of cytokine receptors.20 Ligation of
inhibitory receptors will down-regulate the functional activity of innate lymphocytes.
In contrast, the engagement of activatory receptors with cognate ligands or pro-
inflammatory cytokines will drive innate cell proliferation, instantaneous immune
responses and the expression of activation markers (Table 1.2).
Activation of innate lymphocytes also occur by triggering of their TCR with non-
peptide antigen presented via unique antigen-presenting molecules (e.g. MR1 or
CD1d). However, unlike conventional T cells that are dependent on classical antigen
(peptide)-presentation by major histocompatibility complex (MHC) class I and II
molecules, unconventional T cells can also function in a TCR independent manner.21
This dual activation response (i.e. TCR dependent or independent triggering) is
another discriminatory feature of TCR-expressing innate lymphocytes.21
Recruitment and retention
Compared to most adaptive lymphocytes, innate lymphocyte are well known for
their ability to enter and be preferentially accumulate within peripheral tissues.21,22.
Chemokine receptors can drive the chemotaxis of innate lymphocytes into various
organs, including the kidney. In addition, innate lymphocytes typically express various
adhesion molecules to assist in their retention after entry (Table 1.2).
Tissue residency
Contemporary studies support the localisation of distinct tissue-resident innate
(and adaptive) lymphocyte subsets in non-lymphoid tissues.23–25 The concept of tissue-
resident lymphocytes has been best demonstrated by parabiosis experiments, a surgical
technique that joins the circulatory systems of two animals. Lymphocytes in the joint-
circulatory systems are free to travel between the two animals, eventually reaching an
equal distribution of donor and parabiont-derived lymphocytes. In contrast, non-
circulating tissue compartments remain to be populated by endogenous tissue-resident
lymphocytes. Minimal migration is a defining feature of tissue-resident lymphocytes.
Most tissue-resident lymphocytes from the innate or adaptive immune system
are known to express integrins (either or both CD103 and CD49a) that re-enforce
8 Chapter 1: Literature Review and Research Objectives
retention via their engagement with tissue-expressed ligands (E-cadherin and
collagen).24 In addition, common to all tissue-resident cells is the expression of CD69,
a molecule that inhibits the re-entering of lymphocytes into the circulation via
disruption of the S1PR1/S1P chemotaxis pathway. Although not all tissue-resident
lymphocytes express CD69, CD49a or CD103, the co-expression of CD69 with either
CD49a or CD103 is currently considered to be the gold standard for defining tissue
residency and can be used to identify tissue-resident innate lymphocytes (Table
1.2).23–25
Table 1.2. Selected surface molecules implicated in the inhibition, activation, recruitment and retention
of innate lymphocytes in humans
Function Surface molecules Reference Inhibitory receptors PD-1, CTLA-4 [26]
Activatory receptors NKp46, NKp44, NKG2D, DNAM-1, IL-12r, IL-15r, IL-18r
[27–30]
Activation markers CD161, CD69* [27,31]
Chemokine receptors CCR2, CCR5, CXCR3, CX3CR1 [32]
Adhesion molecules LFA-1, VLA-1 CD44, CD49a*, CD103* [25]
*Markers of tissue residency
Chapter 1: Literature Review and Research Objectives 9
Function of Innate Lymphocytes in CKD
The kidneys are a major blood filtering unit and maintain immune system
homeostasis by removing a rich supply of circulating cytokines and bacterial toxins
that can activate innate lymphocytes.11 In CKD, much of the kidney architecture and
immune system is severely compromised, leaving the kidney in a vulnerable state to
be targeted by pathogenic, activated innate lymphocytes. Thus, innate lymphocytes
have garnered tremendous interest in nephrology (predominantly in animal models of
kidney disease) as they are can potentially influence disease progression as first and
immediate responders in the kidney.23
In the following sections, the functions of innate lymphocytes in kidney disease
will be reviewed. However, the discussion of the roles of renal innate lymphocytes in
cancer and transplantation will be excluded. Chronic injury in cancer is not primary
due to dysregulation of the immune response elicited by effector lymphocytes, but
rather the uncontrolled growth of malignant cells. Similarly, the effector function of
lymphocytes in kidney transplantation is an allogeneic immune response between
recipient immune cells and donor tissue, and thus, distinct from the autologous
environment of native CKD. Thus, in these sections, I will focus on the role of innate
lymphocytes in native forms of kidney disease that may play a role in the progression
of CKD.
Natural killer cells
NK cells are unlike adaptive lymphocytes in that they do not express antigen-
specific receptors. They are early responding lymphocytes that survey host tissues to
target injured and dysfunctional cells. They were first discovered 40 years ago for
killing tumour cells without needing prior sensitization. NK cells can be identified by
the expression of cell surface CD56 and CD16 in humans and can be subcategorised
based on CD56 expression levels into high density (CD56bright) and low-density
(CD56dim) populations. Other markers such as NKp46 and CD117 expression on
CD56bright NK cells are useful to differentiate these cells from CD56dim NK cells.33
CD56bright NK cells are known to mediate immune responses via prolific cytokine
production, whilst CD56dim NK cells behave as potent cytotoxic effector cells.34
10 Chapter 1: Literature Review and Research Objectives
NK cells in animal models of kidney disease
Animal experimental models suggest that NK cells contribute to acute forms of
kidney injury.35,36 In mouse models of ischemia reperfusion injury (IRI), NK cells
infiltrate the kidney almost immediately after injury.35 Depletion of NK cells has been
shown to improve renal function in mice with IRI, while adoptive transfer of NK cells
from WT mice to recombination activating gene (RAG)-2-deficient mice that lack
mature lymphocytes, severely reduces kidney function.37 Recent studies suggest the
following sequence of events involving NK cell-mediated injury are likely to take
place during IRI: Firstly, IRI-damaged kidneys drive NK cell migration through
expression of osteopontin (OPN) and C-C chemokine receptor type 5 (CCR5)
ligands;38,39 secondly, NK cells recruited to the kidney drive tubular epithelial cell
apoptosis via a perforin-dependent pathway;37 thirdly, NK cells stimulate tubular
epithelial cells to secrete chemokine (C-X-C motif) receptor ligands 1 and 2 (CXCL1,
CXCL2) to attract neutrophils for further renal destruction.39,40
The role of mouse NK cells in chronic inflammation has been examined to a
lesser extent. In both mouse models of lupus nephritis (CKD caused by systemic lupus
erythematosus) and chronic proteinuric injuries, an increased accumulation of NK
cells has been detected in the diseased kidney.41,42 However, Zheng et al showed that
NK cell depletion by anti asialo-GM1, a glycolipid highly expressed on NK cells, did
not improve adriamycin-induced nephropathy in mice, a model of chronic progressive
glomerular disease.41 It is possible that the pathogenic effect of NK cells in this model
was not demonstrated due to the ineffectiveness of the NK cell depletion. A recent
publication by Victorino et al demonstrated that depletion of NK cells by anti asialo-
GM1 is not an effective way of depleting NK cells in mice kidneys.43 We must
therefore take great care when interpreting and translating findings from mice to
humans.
NK cells in human kidney disease
Extending data of the functional roles of specific NK cell subsets from animals
to humans has been challenging. Animal models are informative, but the difference in
NK cell phenotype between mice and humans do not allow direct translation of
findings.18 Initial studies have identified human NK cells in patient kidneys with
autoimmune disorders (e.g. immunoglobulin (Ig) A nephropathy, pauci-immune
necrotising glomerulonephritis) and infectious diseases (e.g. dengue induced
Chapter 1: Literature Review and Research Objectives 11
glomerulonephritis) by single immunohistochemical staining of CD56 or CD57
antigens.14,44,45 This is an inadequate method for detecting NK cells as various subsets
of T cells are also able to express CD56 or CD57 and furthermore, this technique will
not allow the identification and characterisation of human NK cell (CD56bright vs
CD56dim) subsets.46,47 Human kidney NK cell studies are therefore critical for better
understanding this innate cell population and potentially for the discovery of useful
targeted therapeutics for CKD.
Innate lymphoid cells
ILCs represent a newly recognised family of lymphocytes that are identified as
constituents of the innate immune system.22 ILCs are defined by their lymphoid
morphology and lack rearranged antigen receptors. A commonly accepted
nomenclature of ILCs is based on transcriptomic and functional characteristics. The
ILC family are categorised into three groups (ILC1, ILC2 and ILC3). Group 1 ILCs
include classical NK cells and ILC1. Both express the transcription factor T-bet and
can produce IFN-γ and TNF-α. However, only NK cells can also produce perforin and
granzymes. Group 2 ILCs or ILC2s are known for their expression of GATA3
transcription factor and by their production of IL-4. Lastly, ILC3s express the
transcription factor RORγt and secrete IL-17A. The marked interest in ILC biology in
recent years has uncovered the important roles of ILCs in tissue homeostasis and
defence against pathogens within the lung and gut. Currently, only ILC2s have been
studied in the kidney.
ILCs in kidney disease
Huang et al provided the first evidence that ILC2s are protective in mouse
kidneys with acute injuries.48 The group showed that adoptive transfer of ILC2s is
associated with anti-inflammatory cytokine IL-4 production and increased numbers of
anti-inflammatory macrophages in IRI. Following this study, several groups have
shown that the activation of ILC2s, either by IL-33 or IL233 (an IL-2 and IL-33 fusion
molecule), can improve renal function and reduce inflammatory pathology caused by
lupus, ischemia and glomerular injuries. 49–52. In humans, only one study has shown
that ILC2s can be localised in healthy kidneys.52 Thus, the function of these ILCs and
other subsets of the ILC family in human kidney diseases are so far unclear.
12 Chapter 1: Literature Review and Research Objectives
Gamma-delta T cells
γδ T cells express a unique TCR made up of one γ chain and one δ chain that is
distinct from αβ TCR heterodimers of conventional T cells.21 In humans, two major
subsets of γδ T cells have been identified: tissue surveying Vδ1 and circulatory Vδ2 T
cells.53 γδ T can recognise a wide range of antigens in an MHC-independent manner.
For example, Vδ1 T cells can recognise stress-induced proteins such as MHC class I
polypeptide-related sequence A (MICA) and UL16 binding proteins (ULBPs), while
Vδ2 T cells can recognise bacterial phosphoantigens.54 The specific recognition of
different cellular ligands suggests that Vδ1 T cells play a role in eliminating infected
or stressed/damaged cells, whereas Vδ2 T cells are important players in anti-bacterial
immunity.55
γδ T cells in animal models of kidney disease
Most of our current understanding of the functional roles of γδ T cells in the
kidney comes from experimental animal models. Transgenic mice lacking γδ TCR and
antibody depletion studies have suggested that γδ T cells drive renal pathogenesis. In
these studies, mice lacking γδ T cells have improved renal function, reduced
inflammation and reduced structural lesions.56–58 Supporting these studies, Turner et
al showed in a murine model of crescentic GN that γδ T cells drive inflammation and
injury in the kidney by IL-17A-dependent recruitment of macrophages and
neutrophils.59 In a later study, IL-17A derived from γδ T cells has also been shown to
mediate chemotaxis of T cells and activation of fibroblasts in mice with ureteral
obstruction.60
Interestingly, several experimental models suggest that γδ T cells may have a
regulatory role in chronic kidney disease. In mice and rats with adenine induced renal
injury, γδ T cells were found to express TGF-β, with antibody mediated depletion of
γδ T cells resulting in marked interstitial inflammation and worsening of renal
function. 61,62 In a more recent study, adoptive transfer of a γδ T cell subset, Vδ2 T
cells, was found to prevent Mycobacterium tuberculosis from establishing renal
lesions and confined tuberculosis pathology mostly to the infection site.63 These
contradicting functions of γδ T cells in animal models highlight the importance of
translating this collective work to a human clinical model.
Chapter 1: Literature Review and Research Objectives 13
γδ T cells in human kidney disease
To the best of our knowledge, only two papers have documented γδ T cells in
human kidney disease. These studies showed that γδ T cell numbers are associated
with the severity of renal histopathological injury in renal biopsies of patients with
immunoglobulin A (IgA) nephropathy.64,65 However, the pathogenicity of γδ T cell
subsets in humans still remains unclear. Confirmatory studies in humans are necessary
to uncover the function of these unique T cells in renal diseases.
Natural killer T cells
Natural killer T (NKT) cells are unconventional T lymphocytes that react to non-
peptide antigens presented by MHC-like molecule CD1d.66 NKT cell subsets are
divided into type I and type II NKT cells according to their TCR repertoire. Type I
NKT cells, also known as invariant NKT (iNKT) cells, express a semi-invariant TCR
α chain (Vα24-Jα18 in humans, Vα14-Jα18 in mice) and heterogeneous TCR β chains
(Vβ11 in humans, Vβ2, 7 or 8.2 in mice).66 The unusual and biased TCR αβ-chain
combination permits type I NKT to be activated by glycolipids such as α- and β-
galactosylceramide (α/β-GalCer).67 Type II NKT cells also recognise lipids presented
by CD1d, but with a more diverse TCR repertoire. They are often referred to as diverse
or variant NKT cells and do not respond to α-GalCer stimulation. Instead, type II NKT
cells recognise glycolipid sulfatide or phospholipid phosphatidylglycerol.68,69 NKT
cells have innate-like characteristics as they can be activated either by TCR recognition
of lipid antigens or by inflammatory cytokines alone, allowing them to quickly respond
to their microenvironment and for rapid release of copious amounts of chemokines and
cytokines.70
NKT cells in animal models of kidney disease
Experimental animal models provide strong evidence that NKT cells play a
regulatory role in CKD. CD1d knock out mice that lack both type I and II NKT cells
showed a reduction in renal function, increased tubular death and inflammation in
lupus,71–73 ischemia,74 glomerular injury,75 and tubular injury models.76,77 From
migration studies involving chemokine receptor knockout mice, it is suggested that C-
X-C motif receptor (CXCR) 3, CXCR6 and CCR5 are required for NKT cell
chemotaxis to the tubulointerstitium in crescentic GN and IRI mouse models.74,78,79
14 Chapter 1: Literature Review and Research Objectives
Type I NKT cell-specific depletion examined in Jα18 knockout mice with
glomerular basement membrane injury demonstrated worsening of renal function.80
Similarly, the activation of type I NKT via α-GalCer and its agonist,
glycosphingolipid-1 (GSL-1), has been shown to attenuate glomerular injury.79,81
Similar observations in other mouse models with lupus nephritis provide strong
evidence of the regulatory function of activated type I NKT cells in the kidney.82–85
Due to the absence of specific markers on type II NKT cells, annotating the
functions of type II NKT cells is a challenge. Yang et al have provided the only
evidence that type II NKT cells have a protective role in mice with ischemia-
reperfusion injuries.74 The group demonstrated that mice deficient in type I and II NKT
cells (CD1d knockout mice) have accentuated renal pathology and T cell infiltration
compared to mice deficient in type I NKT alone (Jα18 knockout mice), suggesting a
regulatory function of type II NKT. Yang et al further showed that activation of type
II NKT in Jα18 knockout mice with in vivo sulfatide administration significantly
alleviated ischemia-induced pathophysiology.
The evidence for a protective role for NKT cells in renal injury is not without
controversy. Studies have also shown that NKT cells can exacerbate autoimmune,
ischemia and infection-related disorders by promoting inflammation.84,86–91 The
differences in experimental observations may be attributed to the age of the mice, and
the quantity and potency of the activation stimulus. For instance, Uchida et al showed
that type I NKT can become anergic from repeated α-GalCer injections.85 In addition,
this group also demonstrated that α-GalCer administration to mice with ischemia leads
to worsened injury in middle-aged mice compared to young mice.88
NKT cells in human kidney disease
Despite this, little is known about NKT cell subsets in humans. To date, only
type I NKT have been identified in the human kidney. The numbers of type I NKT
cells were found to negatively correlate with severity of tubular necrosis, suggesting a
protective function.74 The cellular mechanisms involved in NKT cell chemotaxis and
their immuno-regulatory role in protecting kidneys from injury remain to be fully
elucidated.
Chapter 1: Literature Review and Research Objectives 15
Mucosal-associated invariant T cells
Mucosal-associated invariant T (MAIT) cells are non-classical T cells with
characteristics of both innate and adaptive immune cells. MAIT cells express an αβ
TCR that recognises vitamin B metabolites presented on the MHC class I-related
protein (MR1).21 Human MAIT cells typically express a TCR α chain consisting of
Vα7.2 and Jα18 gene segments paired to a broad range of TCR β chains, including
Vβ13 family genes.92 Another defining feature of human MAIT cells is the high
surface expression of CD161, C-type lectin-like receptor, identified through MR1
tetramer studies.93 Similar to NKT cells, activation of MAIT cells can be TCR-
independent. The innate-like properties of MAIT cells has been demonstrated through
their response to pro-inflammatory cytokines such as IL-12, IL-15 and IL-18 in a TCR-
independent manner during infectious and non-infectious diseases.94 Multiple lines of
evidence have illustrated the defensive role of MAIT cells during bacterial and viral
infections in peripheral organs.95,96 However, emerging insights into their pathogenic
role in inflammatory diseases, including acute and chronic inflammatory injuries, have
recently been discovered.95,96
MAIT cells in kidney disease
The function of MAIT cells in the kidney has not been studied. This is mainly
due to the lack of mouse models available for characterisation and functional studies.
In conventional naïve mouse strains, MAIT cells are relatively rare and it remains
unclear whether they can be located in the kidney.93 There is currently the development
of transgenic mouse strains to circumvent the problem of low MAIT cell numbers in
animal models.97 However, it is uncertain whether these models will recapitulate
human MAIT cell function, highlighting the need to formally examine MAIT cells in
human CKD. To date, MAIT cells have only been identified in human kidney tissue
by the expression of MAIT cell-specific TCR transcripts (Vα7.2-Jα33 and Vα7.2-
Jα12).98,99 The localisation, function and phenotype of human MAIT cells in kidney
disease awaits further investigation.
16 Chapter 1: Literature Review and Research Objectives
PROXIMAL TUBULAR EPITHELIAL CELLS AND CKD
PTEC are important components of the kidney. They form the proximal tubules
of the nephron and are responsible for nutrient reabsorption in the kidney. Together
with the tubulointerstitium, proximal tubules make up over 50% of the kidney and are
major sites of renal injury.100 Metabolically active PTEC are particularly vulnerable to
chronic injury (e.g. hypoxia).101 Cell death of PTEC resulting from chronic injury
ultimately leads to uncontrolled inflammation and formation of non-functional
nephrons. Thus, without the re-establishment of renal immune homeostasis and
resolution of PTEC-mediated inflammation, the development of fibrosis and an
advancement toward CKD ensue.102
Critically, chronic PTEC damage and the subsequent establishment of tubular
atrophy/interstitial fibrosis is a shared process in all forms of CKD and is closely
associated with the presence of inflammatory immune cells within the
tubulointerstitial compartment. The localisation of immune cells within the
tubulointerstitium suggests that they are indeed well-positioned to sense danger signals
and be activated by damaged PTEC.10,11
However, the functional role of human PTEC in modulating local
tubulointerstitial immune responses during the early inflammatory stages of kidney
injury through to the later stages of established CKD are only now being elucidated.
PTEC-immune cell cross-talk during renal immune homeostasis
In the normal kidney, renal immune homeostasis ensures resolution of
inflammation and proper injury repair. Recent studies have shown that PTEC
participate in immune homeostasis and have intrinsic immunoregulatory properties
(expression of surface molecules, soluble factors) designed to dampen inflammation
during inflammatory renal injury (Table 1.3). In fact, our group have illustrated a
number of mechanisms by which human PTEC down-regulate immune responses
under inflammatory conditions. For example, in the presence of inflammatory
cytokines (IFN-γ and TNF-α), PTEC were shown to down-regulate autologous T cell
proliferation and cytokine production via a PD-L1-mediated mechanism.103 Moreover,
Sampangi et al recently demonstrated that inflammatory PTEC could also limit local
inflammation via the production of soluble factor HLA-G.104 Others groups have also
Chapter 1: Literature Review and Research Objectives 17
shown that MHC class II, classically known to activate T cells, is constitutively
expressed on PTEC and can trigger anergy or hyporesponsiveness in T cells. 105,106
PTEC-immune cell cross-talk in CKD
In contrast, within the chronic inflammatory environment of CKD, PTEC are
considered to play a more pathogenic role as key inflammatory and fibrogenic cells
that can accelerate disease progression. In response to chronic injury, PTEC are known
to mediate tubulointerstitial damage through the production of chemokines (CCL2,
CCL5 etc) and pro-inflammatory cytokines (e.g. IL-12), as well as the expression of
immuno-stimulatory molecules (CD112, MICA etc) known to activate distinct
lymphocyte subsets (Table 1.3). Injured PTEC also contribute to the formation of
interstitial fibrosis by a process called epithelial to mesenchymal transition.107 In this
process, myofibroblasts developed from PTEC became capable of synthesizing excess
collagen and pro-fibrotic growth factors such TGF-β and PDGF.101
Renal hypoxia: key driver of PTEC injury
Renal hypoxia is one of the key pathobiological drivers of tubulointerstitial
injury and the common pathway by which chronic kidney disease (CKD) progresses
to ESRD.108 Compromised oxygen delivery in CKD can result from glomerular injury,
rarefaction of peritubular capillaries and renal fibrosis. PTEC, fuelled by mitochondria
and dependent on oxidative phosphorylation, are particularly sensitive to this hypoxic
environment, resulting in cell apoptosis, promotion of interstitial fibrosis and
triggering of inflammatory processes.101 Taken together, the effect of hypoxic injury
on PTEC is an important contributor to CKD progression.
In this PhD project, I will recapitulate the pro-fibrotic microenvironment in
human kidneys by evaluating the interactions of human PTEC with innate
lymphocytes under hypoxic conditions.
18 Chapter 1: Literature Review and Research Objectives
Table 1.3. Selective immuno-modulatory molecules expressed by human PTEC
Molecules Receptors Reference(s) Inhibitory ligands
ICOS-L CD278 (ICOS) [103,109] PD-L1 PD-1 [103] sHLA-G CD85j (ILT2), CD855d
(ILT4), CD158d (KIR2DL4), CD8, CD160
[104,110]
Inhibitory complexes MHC-II TCR [103,105,106]
Activating ligands CD112 CD226 (DNAM-1) [111] MICA, MICB CD314 (NKG2D) [112–114] LLT1 CD161 [115]
Activating cytokines
IL-12 IL-12R [116] IL-15 IL-15R [117] IL-18 IL-18R [118]
Chemoattractants
CCL2 (MCP1) CCR2 [119] CCL5 (RANTES) CCR5 [119,120] ICAM-1 LFA-1 [121,122] VCAM-1 VLA-1 [123] CX3CL1 CX3CR1 [124,125] Osteopontin CD44 [126] CXCL9, CXCL10, CXCL11
CXCR3 [120,127]
Chapter 1: Literature Review and Research Objectives 19
SUMMARY AND RESEARCH HYPOTHESIS
Accumulation of inflammatory lymphocytes and PTEC damage are common
pathological features of CKD. However, to date, little is known about the specific role
of innate lymphocytes in the human kidney and CKD progression. In addition, while
it is known that PTEC are capable of immuno-modulatory functions during early
inflammation, there is currently a knowledge gap regarding the role of PTEC and their
interactions with innate lymphocyte subsets during the more established
hypoxic/inflammatory processes of CKD (Figure 1.2).
I hypothesise that human innate lymphocytes have a pathogenic role in driving
CKD. The pathogenesis of innate lymphocytes is mediated by hypoxic PTEC that are
absent of immuno-regulatory functionality observed under immune homeostasis.
Dysregulated innate lymphocyte and hypoxic PTEC cross-talk during CKD
inadvertently leads to greater numbers of infiltrating inflammatory cells within the
tubulointerstitial compartment. Examination of these innate immune cells and their
interaction/s with PTEC will give us a better understanding of the cellular/molecular
pathways that drive CKD, uncovering novel therapeutic targets for clinical translation
in renal medicine.
20 Chapter 1: Literature Review and Research Objectives
Figure 1.2 PTEC-immune cell interactions in the human kidney.
(a) Cytokine-activated PTEC down-regulate immune responses of adaptive lymphocytes in normal kidney via: (1) immuno-regulatory molecules (MHC-II, PD-L1) and soluble HLA-G; and (2) DC-mediated anti-inflammatory cytokines IL-4 and IL-10. (b) The presence of innate lymphocyte subpopulations in healthy and diseased human kidneys requires further investigation. My PhD will be focussing on innate lymphocytes, NK, γδ T and MAIT cells. (c) The mechanisms of established and persisting inflammation in CKD are not well understood. I hypothesise hypoxic PTEC are pro-inflammatory and play an important role in mediating chronic inflammation through stimulation of pro-fibrotic innate lymphocyte subsets.
Chapter 1: Literature Review and Research Objectives 21
RESEARCH OBJECTIVE AND AIMS
The objective of this PhD project is to identify human innate lymphocyte subsets
and to examine the cross-talk between these lymphocyte subsets and PTEC during
hypoxia. In order to achieve this, I will first identify which innate lymphocyte subsets
are present in human kidneys with tubulointerstitial fibrosis and their localisation
relative to PTEC. Following this, I will examine innate lymphocyte functions when
interacting with PTEC under normal and diseased (hypoxic) conditions. This project
will be divided into three separate aims for the above purpose.
1) To identify, enumerate and phenotype innate lymphocyte subsets from
healthy and diseased human kidney tissue.
2) To analyse the preferential spatial distribution and localisation of identified
innate lymphocyte subsets within diseased human kidneys.
3) To define the immunological drivers of CKD by in vitro co-culture of
primary human PTEC with innate lymphocyte subsets under hypoxic (pro-
fibrotic) conditions.
22 Chapter 1: Literature Review and Research Objectives
REFERENCES
1. Chadban, S. J. et al. Prevalence of kidney damage in Australian adults: The AusDiab kidney study. J. Am. Soc. Nephrol. 14, S131-8 (2003).
2. Wyld, M. L. R. et al. Cost to government and society of chronic kidney disease stage 1-5: a national cohort study. Intern. Med. J. 45, 741–747 (2015).
3. Stefoni, S., Iorio, M., Cianciolo, G., Baraldi, O. & Angelini, M. L. Emerging drugs for chronic kidney disease. Expert Opin. Emerg. Drugs 19, 183–199 (2014).
4. Kidney Disease: Improving Global Outcomes (KDIGO) CKD Work Group. KDIGO 2012 clinical practice guideline for the evaluation and management of chronic kidney disease. Kidney International Supplements 3, (Elsevier, 2013).
5. Little, M. H. Regrow or Repair: Potential Regenerative Therapies for the Kidney. J. Am. Soc. Nephrol. 17, 2390–2401 (2006).
6. Romagnani, P. et al. Chronic kidney disease. Nat. Rev. Dis. Prim. 3, (2017).
7. Kardasz, S. The function of the nephron and the formation of urine. Anaesth. Intensive Care Med. 13, 309–314 (2012).
8. Cullen-McEwen, L., Sutherland, M. R. & Black, M. J. The Human Kidney. Kidney Development, Disease, Repair and Regeneration 1, (Elsevier Inc., 2016).
9. Kriz, W. & LeHir, M. Pathways to nephron loss starting from glomerular diseases - Insights from animal models. Kidney International 67, 404–419 (2005).
10. Kurts, C., Panzer, U., Anders, H.-J. & Rees, A. J. The immune system and kidney disease: basic concepts and clinical implications. Nat. Rev. Immunol. 13, 738–753 (2013).
11. Tecklenborg, J., Clayton, D., Siebert, S. & Coley, S. M. The role of the immune system in kidney disease. Clin. Exp. Immunol. 192, 142–150 (2018).
12. Iványi, B., Hamilton-Dutoit, S. J., Hansen, H. E. & Olsen, S. Acute tubulointerstitial nephritis: phenotype of infiltrating cells and prognostic impact of tubulitis. Virchows Arch. 428, 5–12 (1996).
13. Olsen, S., Hansen, E. S. & Jepsen, F. L. The prevalence of focal tubulo-interstitial lesions in various renal diseases. Acta Pathol. Microbiol. Scand. A. 89, 137–45 (1981).
14. Alexopoulos, E., Seron, D., Hartley, R. B., Nolasco, F. & Cameron, J. S. The Role of Interstitial Infiltrates in IgA Nephropathy: A Study with Monoclonal Antibodies. Nephrol. Dial. Transplant. 4, 187–195 (1989).
15. Lin, Q. et al. Kidney injury molecule-1 expression in IgA nephropathy and its correlation with hypoxia and tubulointerstitial inflammation. Am. J. Physiol. Renal Physiol. 306, F885-95 (2014).
Chapter 1: Literature Review and Research Objectives 23
16. Segerer, S. et al. CXCR3 Is Involved in Tubulointerstitial Injury in Human Glomerulonephritis. Am. J. Pathol. 164, 635–649 (2004).
17. Becker, G. J. & Hewitson, T. D. Animal models of chronic kidney disease: useful but not perfect. Nephrol. Dial. Transplant. 28, 2432–2438 (2013).
18. Colucci, F., Di Santo, J. P. & Leibson, P. J. Natural killer cell activation in mice and men: different triggers for similar weapons? Nat. Immunol. 3, 807–13 (2002).
19. Vermijlen, D. & Prinz, I. Ontogeny of innate T lymphocytes - some innate lymphocytes are more innate than others. Frontiers in Immunology 5, 486 (2014).
20. Seyda, M., Elkhal, A., Quante, M., Falk, C. S. & Tullius, S. G. T Cells Going Innate. Trends Immunol. 37, 546–556 (2016).
21. Godfrey, D. I., Uldrich, A. P., Mccluskey, J., Rossjohn, J. & Moody, D. B. The burgeoning family of unconventional T cells. Nat. Immunol. 16, 1114–1123 (2015).
22. Ebbo, M., Crinier, A., Vély, F. & Vivier, E. Innate lymphoid cells: Major players in inflammatory diseases. Nat. Rev. Immunol. 17, 665–678 (2017).
23. Turner, J.-E., Becker, M., Mittrücker, H.-W. & Panzer, U. Tissue-Resident Lymphocytes in the Kidney. J. Am. Soc. Nephrol. 29, 389–399 (2018).
24. Topham, D. J. & Reilly, E. C. Tissue-Resident Memory CD8+ T Cells: From Phenotype to Function. Front. Immunol. 9, 515 (2018).
25. Gebhardt, T., Palendira, U., Tscharke, D. C. & Bedoui, S. Tissue-resident memory T cells in tissue homeostasis, persistent infection, and cancer surveillance. Immunol. Rev. 283, 54–76 (2018).
26. Parry, R. V et al. CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms. Mol. Cell. Biol. 25, 9543–53 (2005).
27. Hudspeth, K., Silva-Santos, B. & Mavilio, D. Natural Cytotoxicity Receptors: Broader Expression Patterns and Functions in Innate and Adaptive Immune Cells. Front. Immunol. 4, 69 (2013).
28. Teng, M. W. L. et al. IL-12 and IL-23 cytokines: from discovery to targeted therapies for immune-mediated inflammatory diseases. Nat. Med. 21, 719–729 (2015).
29. Nakanishi, K. Unique Action of Interleukin-18 on T Cells and Other Immune Cells. Front. Immunol. 9, 763 (2018).
30. Fehniger, T. A. & Caligiuri, M. A. Interleukin 15: biology and relevance to human disease. Blood 97, 14–32 (2001).
31. Fergusson, J. R., Fleming, V. M. & Klenerman, P. CD161-expressing human T cells. Front. Immunol. 2, 36 (2011).
32. Sokol, C. L. & Luster, A. D. The chemokine system in innate immunity. Cold Spring Harb. Perspect. Biol. 7, (2015).
24 Chapter 1: Literature Review and Research Objectives
33. Allan, D. S. J. et al. TGF-β affects development and differentiation of human natural killer cell subsets. Eur. J. Immunol. 40, 2289–95 (2010).
34. Freud, A. G., Mundy-Bosse, B. L., Yu, J. & Caligiuri, M. A. The Broad Spectrum of Human Natural Killer Cell Diversity. Immunity 47, 820–833 (2017).
35. Ascon, D. B. et al. Phenotypic and Functional Characterization of Kidney-Infiltrating Lymphocytes in Renal Ischemia Reperfusion Injury. J. Immunol. 177, 3380–3387 (2006).
36. Chan, A. J. et al. Innate IL-17A-producing leukocytes promote acute kidney injury via inflammasome and toll-like receptor activation. Am. J. Pathol. 184, 1411–1418 (2014).
37. Zhang, Z.-X. et al. NK Cells Induce Apoptosis in Tubular Epithelial Cells and Contribute to Renal Ischemia-Reperfusion Injury. J. Immunol. 181, 7489–7498 (2008).
38. Zhang, Z.-X. Z.-X. X. et al. Osteopontin expressed in tubular epithelial cells regulates NK cell-mediated kidney ischemia reperfusion injury. J. Immunol. 185, 967–973 (2010).
39. Kim, H. J. et al. TLR2 Signaling in Tubular Epithelial Cells Regulates NK Cell Recruitment in Kidney Ischemia-Reperfusion Injury. J. Immunol. 191, 2657–2664 (2013).
40. Kim, H. J. et al. Reverse signaling through the costimulatory ligand CD137L in epithelial cells is essential for natural killer cell-mediated acute tissue inflammation. Proc. Natl. Acad. Sci. 109, E13–E22 (2012).
41. Zheng, G. et al. NK cells do not mediate renal injury in murine adriamycin nephropathy. Kidney Int. 69, 1159–1165 (2006).
42. Spada, R. et al. NKG2D ligand overexpression in lupus nephritis correlates with increased NK cell activity and differentiation in kidneys but not in the periphery. J. Leukoc. Biol. 97, 583–598 (2015).
43. Victorino, F. et al. Tissue-Resident NK Cells Mediate Ischemic Kidney Injury and Are Not Depleted by Anti–Asialo-GM1 Antibody. J. Immunol. 195, 4973–4985 (2015).
44. Zhao, L., David, M. Z., Hyjek, E., Chang, A. & Meehan, S. M. M2 macrophage infiltrates in the early stages of ANCA-associated pauci-immune necrotizing GN. Clin. J. Am. Soc. Nephrol. 10, 54–63 (2015).
45. Pagliari, C. et al. Human kidney damage in fatal dengue hemorrhagic fever results of glomeruli injury mainly induced by IL17. J. Clin. Virol. 75, 16–20 (2016).
46. Kared, H., Martelli, S., Ng, T. P., Pender, S. L. F. & Larbi, A. CD57 in human natural killer cells and T-lymphocytes. Cancer Immunol. Immunother. 65, 441–452 (2016).
47. Van Acker, H. H., Capsomidis, A., Smits, E. L. & Van Tendeloo, V. F. CD56
Chapter 1: Literature Review and Research Objectives 25
in the Immune System: More Than a Marker for Cytotoxicity? Front. Immunol. 8, 892 (2017).
48. Huang, Q. et al. IL-25 Elicits Innate Lymphoid Cells and Multipotent Progenitor Type 2 Cells That Reduce Renal Ischemic/Reperfusion Injury. J. Am. Soc. Nephrol. 26, 2199–2211 (2015).
49. Düster, M. et al. T cell-derived IFN-γ downregulates protective group 2 innate lymphoid cells in murine lupus erythematosus. Eur. J. Immunol. 48, 1364–1375 (2018).
50. Cao, Q. et al. Potentiating Tissue-Resident Type 2 Innate Lymphoid Cells by IL-33 to Prevent Renal Ischemia-Reperfusion Injury. J. Am. Soc. Nephrol. ASN.2017070774 (2018). doi:10.1681/ASN.2017070774
51. Stremska, M. E. et al. IL233, A Novel IL-2 and IL-33 Hybrid Cytokine, Ameliorates Renal Injury. J. Am. Soc. Nephrol. 28, 2681–2693 (2017).
52. Riedel, J.-H. et al. IL-33-Mediated Expansion of Type 2 Innate Lymphoid Cells Protects from Progressive Glomerulosclerosis. J. Am. Soc. Nephrol. 28, 2068–2080 (2017).
53. Lawand, M., Déchanet-Merville, J. & Dieu-Nosjean, M.-C. Key Features of Gamma-Delta T-Cell Subsets in Human Diseases and Their Immunotherapeutic Implications. Front. Immunol. 8, 761 (2017).
54. Chien, Y. H. & Konigshofer, Y. Antigen recognition by γδ T cells. Immunol. Rev. 215, 46–58 (2007).
55. Vantourout, P. & Hayday, A. Six-of-the-best: unique contributions of γδ T cells to immunology. Nat. Rev. Immunol. 13, 88–100 (2013).
56. Rosenkranz, A. R. et al. Regulatory interactions of αβ and γδ T cells in glomerulonephritis. Kidney Int. 58, 1055–1066 (2000).
57. Savransky, V. et al. Role of the T-cell receptor in kidney ischemia-reperfusion injury. Kidney Int. 69, 233–238 (2006).
58. Hochegger, K. et al. Role of α/β and γ/δ T cells in renal ischemia-reperfusion injury. Am. J. Physiol. Physiol. 293, F741–F747 (2007).
59. Turner, J.-E. et al. IL-17A Production by Renal γδ T Cells Promotes Kidney Injury in Crescentic GN. J. Am. Soc. Nephrol. 23, 1486–1495 (2012).
60. Peng, X. et al. IL-17A produced by both γδ T and Th17 cells promotes renal fibrosis via RANTES-mediated leukocyte infiltration after renal obstruction. J. Pathol. 235, 79–89 (2015).
61. Ando, T. et al. Infiltration of canonical Vgamma4/Vdelta1 gammadelta T cells in an adriamycin-induced progressive renal failure model. J Immunol 167, 3740–3745 (2001).
62. Wu, H. et al. Depletion of T Cells Exacerbates Murine Adriamycin Nephropathy. J. Am. Soc. Nephrol. 18, 1180–1189 (2007).
63. Qaqish, A. et al. Adoptive Transfer of Phosphoantigen-Specific γδ T Cell
26 Chapter 1: Literature Review and Research Objectives
Subset Attenuates Mycobacterium tuberculosis Infection in Nonhuman Primates. J. Immunol. 198, 4753–4763 (2017).
64. Wu, H., Clarkson, A. R. & Knight, J. F. Restricted γδ T-cell receptor repertoire in IgA nephropathy renal biopsies. Kidney Int. 60, 1324–1331 (2001).
65. Falk, M. C. et al. Infiltration of the kidney by αβ and γδ T cells: Effect on progression in IgA nephropathy. Kidney International 47, (1995).
66. Godfrey, D. I., Stankovic, S. & Baxter, A. G. Raising the NKT cell family. Nat. Immunol. 11, 197–206 (2010).
67. Godfrey, D. I. & Rossjohn, J. New ways to turn on NKT cells: Figure 1. J. Exp. Med. 208, 1121–1125 (2011).
68. Tatituri, R. V. V. et al. Recognition of microbial and mammalian phospholipid antigens by NKT cells with diverse TCRs. Proc. Natl. Acad. Sci. 110, 1827–1832 (2013).
69. Blomqvist, M. et al. Multiple tissue-specific isoforms of sulfatide activate CD1d-restricted type II NKT cells. Eur. J. Immunol. 39, 1726–1735 (2009).
70. Kohlgruber, A. C., Donado, C. A., LaMarche, N. M., Brenner, M. B. & Brennan, P. J. Activation strategies for invariant natural killer T cells. Immunogenetics 68, 649–63 (2016).
71. Yang, J.-Q. et al. Immunoregulatory role of CD1d in the hydrocarbon oil-induced model of lupus nephritis. J. Immunol. 171, 2142–2153 (2003).
72. Yang, J. Q. et al. Examining the role of CD1d and natural killer T cells in the development of nephritis in a genetically susceptible lupus model. Arthritis Rheum. 56, 1219–1233 (2007).
73. Baglaenko, Y. et al. Suppression of autoimmunity by CD5 + IL-10-producing B cells in lupus-prone mice. Genes Immun. 16, 311–320 (2015).
74. Yang, S. H. et al. Sulfatide-Reactive Natural Killer T Cells Abrogate Ischemia-Reperfusion Injury. J. Am. Soc. Nephrol. 22, 1305–1314 (2011).
75. Yang, S. H. et al. NKT cells inhibit the development of experimental crescentic glomerulonephritis. J. Am. Soc. Nephrol. 19, 1663–71 (2008).
76. Alhasson, F. et al. NKT cell modulates NAFLD potentiation of metabolic oxidative stress-induced mesangial cell activation and proximal tubular toxicity. Am. J. Physiol. - Ren. Physiol. ajprenal.00243.2015 (2015). doi:10.1152/ajprenal.00243.2015
77. Aguiar, C. & Naffah-de-Souza, C. Administration of α-galactosylceramide improves adenine-induced renal injury. Mol. Med. 21, 1 (2015).
78. Tsutahara, K. et al. The blocking of CXCR3 and CCR5 suppresses the infiltration of T lymphocytes in rat renal ischemia reperfusion. Nephrol. Dial. Transplant. 27, 3799–3806 (2012).
79. Riedel, J.-H. et al. Immature renal dendritic cells recruit regulatory CXCR6(+)
Chapter 1: Literature Review and Research Objectives 27
invariant natural killer T cells to attenuate crescentic GN. J. Am. Soc. Nephrol. 23, 1987–2000 (2012).
80. Mesnard, L. et al. Invariant natural killer T cells and TGF-beta attenuate anti-GBM glomerulonephritis. J. Am. Soc. Nephrol. 20, 1282–92 (2009).
81. Pereira, R. L. et al. Invariant natural killer T cell agonist modulates experimental focal and segmental glomerulosclerosis. PLoS One 7, 1–11 (2012).
82. Singh, A. K. et al. The natural killer T cell ligand α-galactosylceramide prevents or promotes pristane-induced lupus in mice. Eur. J. Immunol. 35, 1143–1154 (2005).
83. Yang, J. Q., Kim, P. J. & Singh, R. R. Brief treatment with iNKT cell ligand α-galactosylceramide confers a long-term protection against lupus. J. Clin. Immunol. 32, 106–113 (2012).
84. Morshed, S. R., Takahashi, T., Savage, P. B., Kambham, N. & Strober, S. Beta-galactosylceramide alters invariant natural killer T cell function and is effective treatment for lupus. Clin. Immunol. 132, 321–33 (2009).
85. Uchida, T. et al. Repeated administration of alpha-galactosylceramide ameliorates experimental lupus nephritis in mice. Sci. Rep. 8, 8225 (2018).
86. Bajwa, A. et al. Dendritic Cell Sphingosine 1-Phosphate Receptor-3 Regulates Th1-Th2 Polarity in Kidney Ischemia-Reperfusion Injury. J. Immunol. 189, 2584–2596 (2012).
87. Li, L. et al. NKT Cell activation Mediates neutrophil IFN- γ production and renal ischemia-reperfusion injury. J. Immunol. 178, 5899–5911 (2007).
88. Uchida, T. et al. Activated natural killer T cells in mice induce acute kidney injury with hematuria through possibly common mechanisms shared by human CD56+ T cells. Am. J. Physiol. Renal Physiol. (2018). doi:10.1152/ajprenal.00160.2018
89. Obata, F. et al. Natural killer T (NKT) cells accelerate Shiga toxin type 2 (Stx2) pathology in mice. Front. Microbiol. 6, 1–10 (2015).
90. Yang, J.-Q. et al. Repeated alpha-galactosylceramide administration results in expansion of NK T cells and alleviates inflammatory dermatitis in MRL-lpr/lpr mice. J. Immunol. 171, 4439–46 (2003).
91. Zeng, D., Liu, Y., Sidobre, S., Kronenberg, M. & Strober, S. Activation of natural killer T cells in NZB / W mice induces Th1-type immune responses exacerbating lupus. J. Clin. Invest. 112, 1211–1222 (2003).
92. Tilloy, F. et al. An invariant T cell receptor alpha chain defines a novel TAP-independent major histocompatibility complex class Ib-restricted alpha/beta T cell subpopulation in mammals. J. Exp. Med. 189, 1907–21 (1999).
93. Reantragoon, R. et al. Antigen-loaded MR1 tetramers define T cell receptor heterogeneity in mucosal-associated invariant T cells. J. Exp. Med. 210, 2305–2320 (2013).
28 Chapter 1: Literature Review and Research Objectives
94. Xiao, X. & Cai, J. Mucosal-Associated Invariant T Cells: New Insights into Antigen Recognition and Activation. Front. Immunol. 8, 1540 (2017).
95. Le Bourhis, L. et al. Antimicrobial activity of mucosal-associated invariant T cells. Nat. Immunol. 11, 701–708 (2010).
96. Kurioka, A., Walker, L. J., Klenerman, P. & Willberg, C. B. MAIT cells: new guardians of the liver. Clin. Transl. Immunol. 5, e98 (2016).
97. Gapin, L. Check MAIT. J. Immunol. 192, 4475–4480 (2014).
98. Lepore, M. et al. Parallel T-cell cloning and deep sequencing of human MAIT cells reveal stable oligoclonal TCRβ repertoire. Nat. Commun. 5, 3866 (2014).
99. Peterfalvi, A. et al. Invariant V 7.2-J 33 TCR is expressed in human kidney and brain tumors indicating infiltration by mucosal-associated invariant T (MAIT) cells. Int. Immunol. 20, 1517–1525 (2008).
100. Chevalier, R. L. The proximal tubule is the primary target of injury and progression of kidney disease: role of the glomerulotubular junction. Am. J. Physiol. - Ren. Physiol. 311, F145–F161 (2016).
101. Liu, B.-C., Tang, T.-T., Lv, L.-L. & Lan, H.-Y. Renal tubule injury: a driving force toward chronic kidney disease. Kidney Int. 93, 568–579 (2018).
102. Gandhi, M., Olson, J. L. & Meyer, T. W. Contribution of tubular injury to loss of remnant kidney function. Kidney Int. 54, 1157–1165 (1998).
103. Wilkinson, R., Wang, X., Roper, K. E. & Healy, H. Activated human renal tubular cells inhibit autologous immune responses. Nephrol. Dial. Transplant. 26, 1483–1492 (2011).
104. Sampangi, S. et al. The Mechanisms of Human Renal Epithelial Cell Modulation of Autologous Dendritic Cell Phenotype and Function. PLoS One 10, e0134688 (2015).
105. Singer, G. G. et al. Stimulated renal tubular epithelial cells induce anergy in CD4+T cells. Kidney Int. 44, 1030–1035 (1993).
106. Frasca, L. et al. Interferon-γ-treated renal tubular epithelial cells induce allospecific tolerance. Kidney Int. 53, 679–689 (1998).
107. Fragiadaki, M. & Mason, R. M. Epithelial-mesenchymal transition in renal fibrosis - evidence for and against. International Journal of Experimental Pathology 92, 143–150 (2011).
108. Nangaku, M. Chronic Hypoxia and Tubulointerstitial Injury: A Final Common Pathway to End-Stage Renal Failure. J. Am. Soc. Nephrol. 17, 17–25 (2005).
109. De Haij, S. et al. Renal tubular epithelial cells modulate T-cell responses via ICOS-L and B7-H1. in Kidney International 68, 2091–2102 (2005).
110. Sampangi, S. et al. Human proximal tubule epithelial cells modulate autologous B-cell function. Nephrol. Dial. Transplant. 30, 1674–1683 (2015).
111. Kraus, A. K. et al. The role of T cell costimulation via DNAM-1 in kidney
Chapter 1: Literature Review and Research Objectives 29
transplantation. PLoS One 11, e0147951 (2016).
112. Luo, L. et al. The role of HIF-1 in up-regulating MICA expression on human renal proximal tubular epithelial cells during hypoxia/reoxygenation. BMC Cell Biol. 11, 91 (2010).
113. Peraldi, M. N. et al. Oxidative stress mediates a reduced expression of the activating receptor NKG2D in NK cells from end-stage renal disease patients. J Immunol 182, 1696–1705 (2009).
114. Song, H. et al. Transforming growth factor-beta1 regulates human renal proximal tubular epithelial cell susceptibility to natural killer cells via modulation of the NKG2D ligands. Int. J. Mol. Med. 36, 1180–1188 (2015).
115. Llibre, A. et al. Expression of lectin-like transcript-1 in human tissues. F1000Research 5, 2929 (2016).
116. Timoshanko, J. R., Kitching, A. R., Holdsworth, S. R. & Tipping, P. G. Interleukin-12 from intrinsic cells is an effector of renal injury in crescentic glomerulonephritis. J. Am. Soc. Nephrol. 12, 464–71 (2001).
117. Weiler, M., Kachko, L., Chaimovitz, C., Van Kooten, C. & Douvdevani, A. CD40 ligation enhances IL-15 production by tubular epithelial cells. J. Am. Soc. Nephrol. 12, 80–7 (2001).
118. Yang, Y. et al. IL-37 inhibits IL-18-induced tubular epithelial cell expression of pro-inflammatory cytokines and renal ischemia-reperfusion injury. Kidney Int. 87, 396–408 (2015).
119. Lai, K. N., Leung, J. C. K., Chan, L. Y. Y., Guo, H. & Tang, S. C. W. Interaction between proximal tubular epithelial cells and infiltrating monocytes/T cells in the proteinuric state. Kidney Int. 71, 526–538 (2007).
120. Cockwell, P., Calderwood, J. W., Brooks, C. J., Chakravorty, S. J. & Savage, C. O. S. Chemoattraction of T cells expressing CCR5, CXCR3 and CX3CR1 by proximal tubular epithelial cell chemokines. Nephrol. Dial. Transplant 17, 734–744 (2002).
121. Frishberg, Y., Meyers, C. M. & Kelly, C. J. Cyclosporine A regulates T cell-epithelial cell adhesion by altering LFA-1 and ICAM-1 expression. Kidney Int. 50, 45–53 (1996).
122. Bishop, G. A. & Hall, B. M. Expression of leucocyte and lymphocyte adhesion molecules in the human kidney. Kidney Int 36, 1078–85. (1989).
123. Seron, D., Cameron, J. S. & Haskard, D. O. Expression of VCAM-1 in the normal and diseased kidney. Nephrol. Dial. Transplant. 6, 917–922 (1991).
124. Kassianos, A. J. et al. Fractalkine-CX3CR1-dependent recruitment and retention of human CD1c + myeloid dendritic cells by in vitro-activated proximal tubular epithelial cells. Kidney Int. 87, 1153–1163 (2015).
125. Chakravorty, S. J., Cockwell, P., Girdlestone, J., Brooks, C. J. & Savage, C. O. S. Fractalkine expression on human renal tubular epithelial cells: Potential role in mononuclear cell adhesion. Clin. Exp. Immunol. 129, 150–159 (2002).
30 Chapter 1: Literature Review and Research Objectives
126. Zhang, Z.-X. X. et al. Osteopontin expressed in tubular epithelial cells regulates NK cell-mediated kidney ischemia reperfusion injury. J Immunol 185, 967–973 (2010).
127. Arai, Y. et al. Salt suppresses IFNγ inducible chemokines through the IFNγ-JAK1-STAT1 signaling pathway in proximal tubular cells. Sci. Rep. 7, 46580 (2017).
Chapter 1: Literature Review and Research Objectives 31
32 Chapter 1: Literature Review and Research Objectives
Chapter 2: Natural Killer Cells in CKD 33
Chapter 2: Natural Killer Cells in CKD
My PhD journey began with the study of the innate lymphocyte
population, natural killer (NK) cells, in human CKD. NK cells have been
reported to contribute to the pathogenesis of animal models of kidney
disease. However, the utility of mouse models to recapitulate the human
immune response remains uncertain due to the differences in mouse and
human NK cell biology. In this chapter, I present my findings regarding
human NK cell subsets in CKD.
QUT Verified Signature
Chapter 2: Natural Killer Cells in CKD 35
ABSTRACT
Natural killer (NK) cells are a population of lymphoid cells that play a significant role
in mediating innate immune responses. Mouse studies suggest a pathological role for
NK cells in models of kidney disease. In this study, we characterised, for the first time,
the NK cell subsets present in human native kidneys with tubulointerstitial fibrosis,
the pathological hallmark of chronic kidney disease.
Using multi-colour flow cytometry, we detected significantly elevated numbers of
total NK cells (CD3-CD56+) in diseased biopsies with tubulointerstitial fibrosis
compared with diseased biopsies without fibrosis and healthy kidney tissue. At a
subset level, numbers of both the CD56dim NK cell subset and, in particular, the
CD56bright NK cell subset, were significantly elevated in fibrotic kidney tissue.
However, only numbers of CD56bright NK cells correlated significantly with loss of
kidney function. Expression of the tissue-retention and activation molecule CD69 on
CD56bright NK cells was significantly increased in fibrotic biopsies compared with non-
fibrotic kidney tissue, indicative of a pathogenic phenotype. Further flow cytometric
phenotyping revealed selective co-expression of activating receptor CD335 (NKp46)
and differentiation marker CD117 (c-kit) on CD56bright NK cells. Multi-colour
immunofluorescent staining of fibrotic kidney tissue localised the accumulation of NK
cells within the tubulointerstitium, with CD56bright NK cells (NKp46+ CD117+)
identified as the source of pro-inflammatory cytokine interferon (IFN)-γ within the NK
cell compartment.
Collectively, our data indicate that activated, IFN-γ-producing CD56bright NK cells are
positioned to play a key role in the fibrotic process and, thus, progression to chronic
kidney disease.
36 Chapter 2: Natural Killer Cells in CKD
INTRODUCTION
Natural killer (NK) cells are a specialised subpopulation of innate lymphocytes that
play a significant role in immune surveillance of stressed autologous cells. NK cells
receive activation signals when damaged cells display reduced or aberrant major
histocompatibility complex (MHC) class I and/or express cellular stress ligands that
engage with activating receptors on NK cells. This triggering can lead to NK cell
proliferation, production of inflammatory cytokines and cytotoxic activity.1
Human NK cells are defined as CD3-/CD56+ cells that can be subcategorised based on
expression levels of CD56 (neural cell adhesion molecule, NCAM) into low density
(CD56dim) and high density (CD56bright) subsets.2 These NK cell subsets differ in
distribution, phenotype and function. CD56dim NK cells represent the majority of
peripheral blood NK cells.3 They express high levels of CD16 (FcγRIII, the low
affinity receptor for the Fc portion of immunoglobulin G), can express CD57 (a marker
of terminal differentiation) and behave as potent cytotoxic effector cells.4-6 In contrast,
CD56bright NK cells are preferentially enriched in human secondary lymphoid and
peripheral tissues.7 CD56bright NK cells are CD16-/low and mediate immune responses
by secreting large amounts of pro-inflammatory cytokines (e.g. interferon (IFN)-γ).4,8
Compared to CD56dim NK cells, CD56bright NK cells express higher levels of activating
receptor NKp46 and can express CD117 (c-kit, the receptor for stem cell factor).4,6,9
Mouse studies have highlighted the importance of NK cells in mediating ischaemic
acute kidney injury (AKI)10-12 and, to a lesser extent, in the progression of chronic
kidney disease (CKD).13 In this lupus nephritis model, Spada et al reported an
accumulation of NK cells (NKp46+ cells) with activated phenotype and increased
functional capacity (elevated IFN-γ production) in diseased kidneys compared with
kidneys from pre-diseased mice, concluding that they play a pathogenic role in the
disease process.13 In contrast, a study by Zheng et al has suggested that NK cells do
not play a role in adriamycin-induced nephropathy in mice.14 These conflicting
findings may result from the differing mouse models analysed and highlight potential
problems with extrapolating murine findings to human native kidney disease.
Initial immunohistochemical (IHC)-based evaluations of NK cells in diseased human
kidney biopsies have been made, although this methodology is not amenable to the
Chapter 2: Natural Killer Cells in CKD 37
multi-parameter labelling required to unequivocally define NK cells and, in particular,
NK cell subsets. Interstitial NK cells (based on CD57 expression) have been reported
in native kidney biopsies from patients with IgA nephropathy,15 whilst the presence of
NK cells (CD16+ cells) have also been described in interstitial lesions of human
crescentic glomerulonephritis (GN).16 Intragraft NK cells (CD56+ cells) in kidney
transplant biopsies have also been reported to associate with interstitial fibrosis and
poor clinical outcomes.17,18 However, single staining for these antigens is not
sufficiently specific to directly identify NK cells given the broader expression of these
markers on T cell subpopulations.19,20 Furthermore, expression of both CD57 and
CD16 antigens within the NK cell compartment is primarily restricted to CD56dim NK
cells, highlighting an under-representation of markers identifying CD56bright NK cells
in these previous studies. These short comings can be addressed by multi-parameter
staining methodologies that accurately detect, quantify and phenotype NK cell subsets
in human kidney disease.
We previously demonstrated that human native kidneys with tubulointerstitial fibrosis,
the pathological hallmark of CKD, have significantly elevated numbers of total
lymphocytes compared to non-fibrotic renal tissue.21 In this present study, we extend
this multi-colour flow cytometric-based approach to demonstrate that diseased native
biopsies with interstitial fibrosis have significantly increased numbers of
tubulointerstitial NK cells compared to non-fibrotic biopsies, with activated CD56bright
NK cells identified as a key producer of IFN-γ.
38 Chapter 2: Natural Killer Cells in CKD
RESULTS
Identification of NK cell subsets in human kidney tissue.
Healthy and diseased kidney tissue was enzymatically digested to obtain single cells
for flow cytometric analysis. Using a gating strategy outlined in Figure 1, we were
able to separate CD45+ leukocytes into granulocytes with higher side scatter (SSC)
and mononuclear cells (MNC). These MNC were further divided into CD14+
monocyte and CD14- lymphocyte populations. Lymphocytes were then delineated into
CD3+ T cells, CD19+ B cells and CD3- CD19- double negative (DN) cells. Within this
DN population, total NK cells were identified as CD56+ cells, with CD56dim CD16+
and CD56bright CD16-/low NK cell subsets defined for the first time in diseased human
kidney tissue.
Significantly elevated numbers of CD56bright NK cells in diseased biopsies with
interstitial fibrosis.
We enumerated NK cells in healthy and diseased kidney tissue, with diseased biopsies
stratified based on the absence or presence of interstitial fibrosis. Quantification with
Flow-Count fluorospheres revealed a significant increase in total NK cells in diseased
biopsies with interstitial fibrosis compared with diseased biopsies without fibrosis and
healthy kidney tissue (Figure 2a). At a subset level, both CD56dim and, in particular,
CD56bright NK cell subsets were significantly elevated in fibrotic biopsies compared
with non-fibrotic biopsies and healthy tissue (Figure 2b-c). Notably, scatter plot
correlations between NK cell numbers and the degree of fibrosis showed significant
correlations for total NK cells (r = 0.6038, P<0.0001) and CD56bright NK cells (r =
0.6970, P<0.0001) (Figure 2d-f). Collectively, these results associate CD56bright NK
cells with renal interstitial fibrosis.
Absolute numbers of CD56bright NK cells correlate significantly with loss of kidney
function.
In addition to interstitial fibrosis, NK cell counts were correlated to kidney function
(estimated glomerular filtration rate; eGFR). Total NK cell numbers were significantly
higher in patients with moderate-severe kidney dysfunction (eGFR<60
ml/min/1.73m2) (Figure 3a). However, of the two NK cell subsets, numbers of only
CD56bright NK cells correlated significantly with loss of kidney function (Figure 3b-c).
We also correlated NK cell numbers to primary diagnoses of patients, with diseased
Chapter 2: Natural Killer Cells in CKD 39
biopsies stratified into glomerular immune-mediated, glomerular non-immune-
mediated and non-glomerular diseases. Interestingly, this revealed that CD56bright NK
cells were significantly elevated within glomerular non-immune-mediated and non-
glomerular diseases compared with healthy controls (Figure 3d-f). Notably, no
significant associations between NK cell counts and histological levels of interstitial
inflammation in the kidney biopsies were observed (Supplementary Figure 1).
The proportional representation of CD56bright NK cells in healthy human tissue,
including kidneys, has recently been reported by Carrega et al.7 In our present study,
we extended this work by assessing the percentage of CD56bright NK cells among total
NK cells isolated from healthy and diseased kidney tissue. The proportion of CD56bright
NK cells (mean: 55.3%) was higher in fibrotic kidney tissue compared to both non-
fibrotic (mean: 38.6%) and healthy kidney tissue (mean: 36.5%) (Figure 4a).
Stratification of diseased biopsies based on kidney function revealed a similar pattern
with significantly higher proportion of CD56bright NK cells in patients with an
eGFR<60 (mean: 53.0%) compared to patients with an eGFR≥60 (mean: 32.0%)
(Figure 4b-c). Collectively, these data show that CD56bright NK cell numbers and
distribution are related to kidney pathogenesis and functional outcome.
Significantly elevated CD69 expression on CD56bright (NKp46+ CD117+) NK cells
in diseased biopsies with interstitial fibrosis.
We next examined the phenotypes of kidney NK cells in healthy and diseased kidney
tissue. Whilst expression levels of tissue-retention and activation marker CD69 on
CD56dim NK cells (Figure 5a) were comparable between healthy and diseased kidney
tissue, the expression of CD69 on CD56bright NK cells was significantly elevated in
diseased biopsies with interstitial fibrosis compared with non-fibrotic biopsies (Figure
5b). These results suggest that the local environment within fibrotic kidneys retains
and directs CD56bright NK cells toward an activated, pathogenic phenotype.
We further characterised the two NK cell subsets in fibrotic biopsies using a
combination of surface antigens expressed on human blood and tissue NK cells (Figure
5c). Activating receptor NKp46 represents a reliable marker for human NK cell
identification.22 Analysis of fibrotic kidney tissue showed NKp46 expression restricted
to the NK cell compartment, with higher expression on CD56bright NK cells. Previous
40 Chapter 2: Natural Killer Cells in CKD
human studies have used CD117 (c-kit) to discriminate between CD56dim and
CD56bright NK cell subsets.7,9 Notably, in fibrotic kidney tissue, CD117 was expressed
at low levels on CD56bright NK cells, with minimal to no expression detected on
CD56dim NK cells. Differences between human CD56dim and CD56bright NK cells also
include the expression of chemokine receptors.23,24 Chemokine receptor profiling
showed CXCR3 expressed on T cells and, within the NK cell compartment, on
CD56bright NK cells, whilst CX3CR1 was expressed exclusively on CD56dim NK cells.
Collectively, these data provide a panel of surface antigens for more precisely
identifying and distinguishing human NK cell subsets in fibrotic kidney tissue.
Tubulointerstitial localization of human NK cells in fibrotic kidney tissue.
Based on our phenotyping data, we used NKp46 immunofluorescent (IF) staining to
examine the localisation of NK cells in human kidney tissue. Consistent with our flow
cytometric analyses, a prominent accumulation of NKp46+ cells was detected in
diseased biopsies with interstitial fibrosis compared with non-fibrotic biopsies (Figure
6). In fibrotic biopsies, we identified NKp46+ cells within the tubulointerstitial
compartment, adjacent to proximal tubular epithelial cells (PTEC), defined as tubular
cells expressing aquaporin-1.25 Notably, NKp46+ cells localised to sites of
tubulointerstitial injury (inflammation, tubular atrophy), where the NK cells are well
positioned to play a significant role during disease progression.
Tubulointerstitial CD56bright NK cells (NKp46+ CD117+) are a key source of IFN-
γ in fibrotic kidney tissue.
Among the most prominent cytokines produced by NK cells is IFN-γ, a pro-
inflammatory cytokine implicated in driving renal injury in both human and
experimental kidney diseases.26 Thus, we examined the source of IFN-γ in fibrotic
kidney tissue by IF staining, demonstrating, for the first time, the co-localisation of
IFN-γ with tubulointerstitial NK (NKp46+) cells (Figure 7). Quantitative analysis from
three fibrotic donor biopsies demonstrated that 44.68%±6.09% of IFN-γ-expressing
cells were NKp46 positive, whilst 32.33%±8.23% of cells that expressed NKp46 also
expressed IFN-γ.
We used the selective co-expression of NKp46 and CD117 by CD56bright NK cells
(identified earlier in Figure 5c) to discriminate them from CD56dim NK cells and define
Chapter 2: Natural Killer Cells in CKD 41
the NK subset-specific source of IFN-γ. Subsequent IF analysis of fibrotic kidney
tissue showed strong IFN-γ expression in tubulointerstitial CD56bright (NKp46+
CD117+) NK cells alone (Figure 8). Taken together, these findings support, for the
first time to our knowledge, a specialised role for kidney CD56bright NK cells in the
fibrotic process through the production of IFN-γ.
42 Chapter 2: Natural Killer Cells in CKD
DISCUSSION
NK cells are important components of innate immunity and have been implicated in
the progression of kidney disease. In murine studies, NK cells are commonly
associated with AKI.10-12,27,28 However, the role for NK cells in CKD remains unclear
and is complex. Furthermore, our understanding of human NK cells in CKD has been
limited by methodological shortcomings, with previous IHC-based studies using
markers not adequately specific for NK cells or expressed only by one subset of NK
cells.15,16 In this present study, we have used a multi-colour based approach to
investigate the absolute numbers, distribution, phenotype and function of NK cells in
fibrotic human kidneys. Our results find an association between absolute numbers of
tubulointerstitial NK cells, in particular CD56bright NK cells, with both the tissue
pathology of CKD (presence of interstitial fibrosis) and loss of kidney function
(decreased eGFR). Notably, we identified activated CD56bright NK cells as an important
source of IFN-γ in fibrotic biopsies. Collectively, these results provide, for the first
time, a comprehensive mapping of NK cell subsets in fibrotic kidney disease, ascribing
a role in CKD to CD56bright NK cells.
NK cells have been identified in most compartments of the human body. Although
CD56dim NK cells are the predominant subset in human peripheral blood, CD56bright
NK cells are more abundantly represented in most peripheral tissues, with the relative
distribution of the two NK cell subsets dependent on the tissue/organ analysed.29
Carrega et al recently identified human CD56bright NK cells in healthy kidney tissue.7
Interestingly, the percentage of CD56bright NK cells among total NK cells in healthy
kidney tissue in our study (Figure 4; mean: 36.5%) mirrored the findings of this
previous study (mean: 37%). We have extended this work to pathological conditions
to demonstrate elevated proportions and absolute numbers of CD56bright NK cells in
diseased native biopsies with interstitial fibrosis and loss of kidney function. This is
consistent with previous findings in other organs that human CD56bright NK cells are
preferentially populated at inflammatory sites and comprise the major NK cell
population in inflamed tissues.30
Recent data indicate that there is further heterogeneity within the human CD56bright
NK cell compartment, with evidence of: (1) circulating CD56bright NK cells that traffic
through human tissue and (2) tissue-resident CD56bright NK cells (recently described
Chapter 2: Natural Killer Cells in CKD 43
in the uterus, liver and lymphoid tissues) that are permanently retained in situ.31-33 In
contrast, no tissue-resident human CD56dim NK cells have been described to date.
Victorino et al recently examined the relative contributions of circulating and tissue-
resident NK cells in a mouse model of ischaemic AKI, concluding that kidney tissue-
resident NK cells (and not circulating NK cells) were potent mediators of tissue
injury.12 However, direct translation of mouse kidney NK cell phenotype and function
to humans is not possible with important differences like the absence of CD56
expression on murine NK cells. Therefore, whether the human CD56bright NK cells
identified in our study are recruited during inflammatory responses from the
circulation or are permanently tissue resident is an important point of discussion.
Tissue-resident CD56bright NK cells require a mechanism to prevent egress from tissues
into the blood. Although originally identified as an activation marker, CD69 is now
also considered an important molecule in retaining immune cells in tissues34 and thus,
has been used to identify tissue-resident CD56bright NK cells in humans.29 Based on this
classification, the elevated CD69 MFI levels on CD56bright NK cells (Figure 5b)
compared to CD56dim NK cells (Figure 5a) in our study suggest the presence of tissue-
resident CD56bright NK cells within the total kidney NK cell population. However, the
expression of CD117 on our CD56bright NK cells, a marker that is absent on tissue-
resident CD56bright NK cells from human bone marrow, spleen, lymph nodes and
uterine tissue,31,35 would indicate that the CD56bright NK cells likely represent a
circulating population recruited into the kidney. Future studies will be required to
clarify this apparent dichotomy.
Another possible mechanism for tissue-specific recruitment and/or retention of NK
cells is via the engagement of chemokine receptors. We highlighted NK subset-
specific differences in levels of chemokine receptors (CXCR3 on CD56bright NK cells
and CX3CR1 on CD56dim NK cells), consistent with previous studies of human blood
NK cells23. Both CXCR3 and CX3CR1 have been implicated in human
tubulointerstitial injury and loss of kidney function.36,37 It is thus tempting to speculate
that these chemokine receptors are pivotal in NK cell recruitment and retention in
human CKD.
Increased numbers of IFN-γ-producing interstitial mononuclear cells have been
previously reported in kidney biopsies of patients with diffuse proliferative lupus
44 Chapter 2: Natural Killer Cells in CKD
nephritis.38 However, there has been minimal research into the subset-specific source
of this cytokine in human CKD. In this present study, we identified CD56bright NK cells
(NKp46+ CD117+) as a key source of IFN-γ within the tubulointerstitial compartment
of fibrotic kidneys.
IFN-γ is a pleiotropic cytokine produced by activated immune cells, including NK
cells.26 The pro-inflammatory roles of IFN-γ in kidney disease include: (1) M1
macrophage activation,39 (2) modulation of effector T cell responses,40 (3) induction
of MHC class I and II molecules for antigen presentation41 and (4) upregulation of
chemokines that augment immune cell infiltration.37 In addition to its pro-
inflammatory effects, IFN-γ has also been reported to limit kidney disease progression
and preserve renal function.42-44 These studies highlight the complex nature of IFN-γ
dependent on the context of the kidney disease process. Our findings of a significant
association of IFN-γ-producing CD56bright NK cells with loss of kidney function
support a more pro-inflammatory, pathogenic role for this human NK cell subset. This
concept is reinforced by the detection of CD56bright NK cells in areas of interstitial
inflammation/tubular atrophy, often in direct contact with aquaporin-1-expressing
PTEC. Notably, PTEC have been previously reported to induce IFN-γ secretion by NK
cells under in vitro diseased conditions.45 Here we demonstrate, for the first time, the
in situ representation of this PTEC-NK cell interaction.
On the basis of our results, we propose that tubulointerstitial CD56bright NK cells
receive activation signals within the diseased microenvironment to acquire a pro-
inflammatory (IFN-γ-producing) role in CKD. Importantly, our current study provides
the first human evidence of functional correlations to IFN-γ-producing mouse NK cells
described previously in nephritic kidneys.13 Further dissection of CD56bright NK cells
and their interactions with PTEC is now required for the development of therapeutics
capable of blocking the activation of this immune cell population in fibrotic kidney
disease.
Chapter 2: Natural Killer Cells in CKD 45
MATERIALS AND METHODS
Kidney tissue specimens
Renal cortical tissue was obtained with informed patient consent from the
macroscopically/microscopically healthy portion of tumour nephrectomies or native
diseased biopsies, following approval by the Royal Brisbane and Women’s Hospital
Human Research Ethics Committee (2002/011 and 2006/072). Healthy cortical tissue
was obtained from 11 donors (5 females/6 males) of mean age 58±6, whilst diseased
clinical biopsies were obtained from 37 donors (21 females/16 males) of mean age
57±18. A range of primary diagnoses was sampled, including 18 glomerular immune-
mediated (lupus nephritis, crescentic GN, membranoproliferative GN, necrotizing GN,
pauci-immune GN, IgA nephropathy, membranous nephropathy and minimal change
disease), 7 glomerular non-immune-mediated (amyloidosis, focal segmental
glomerulosclerosis and diabetic nephropathy) and 12 non-glomerular (interstitial
nephritis, light chain-related proximal tubulopathy, cast nephropathy,
arterionephrosclerosis and hypertensive nephropathy) etiologies (Supplementary
Table 1).
Fresh biopsies were taken with a 16-gauge biopsy needle (Biopsybell, Mirandola,
Italy) and immediately divided for: 1) tissue dissociation (1-5mm of a core biopsy); 2)
freezing in Tissue-Tek OCT compound (Sakura, Torrance, CA, USA) for IF analysis;
and 3) fixation in formalin for assessing levels of interstitial fibrosis/tubular atrophy
by renal histopathologists blinded to experimental results. For assessment of renal
interstitial fibrosis, formalin-fixed 4μm sections were stained with Masson's trichrome,
and the proportion of fibrotic area in the cortex was quantified over 20 high-power
fields. Biopsies displaying ≥5% interstitial fibrosis were deemed fibrotic, based on the
Banff 97 working classification of renal pathology.46 According to this criterion,
diseased specimens were then grouped into biopsies without (n=21; 13 females/8
males; mean age 55±21; mean eGFR 65±29 ml/min/1.73m2; mean urine protein to
creatinine ratio (uPCR) 371±253 mg/mmol) or with interstitial fibrosis (n=16; 8
females/8 males; mean age 60±9; mean eGFR 34±21 ml/min/1.73m2; mean uPCR
318±269 mg/mmol). Kidney function (eGFR) was calculated using the MDRD method
by AUSLAB (Queensland Health, Brisbane, Australia).
Tissue dissociation for flow cytometric analysis
46 Chapter 2: Natural Killer Cells in CKD
Healthy kidney tissue and diseased biopsies were digested with 1mg/ml collagenase P
(Roche, Mannheim, Germany) in the presence of 20μg/ml DNase I (Roche) (250μl
volume) for 15 min and then further digested with 10μg/ml trypsin/4μg/ml EDTA
(Life Technologies, Grand Island, NY, USA) (500μl volume) for 10 min.
Flow cytometry
Single cell suspensions were initially stained with LIVE/DEAD® Fixable Near-IR
Dead Cell Stain Kit (Life Technologies) to exclude non-viable cells. Cells were then
incubated with Human TruStain FcX™ Blocking Solution (Biolegend, San Diego,
CA, USA) at room temperature for 5-10 min and then stained on ice for 30 min with
combinations of test (0.25μg per antibody) (Table 1) or isotype-matched control
antibodies in cold FACS buffer [0.5% BSA (Sigma, St Louis, MO, USA) and 0.02%
sodium azide (Sigma) in PBS]. Flow-Count Fluorospheres™ (Beckman Coulter, Brea,
CA, USA) were used for direct determination of absolute counts following the
manufacturer’s recommendations. Briefly, target cell concentrations (cells/μl) were
calculated as: total number of target cells counted/total number of Fluorospheres
counted x Flow-Count Fluorospheres™ concentration. This value was then multiplied
by the total sample volume to obtain absolute counts for each target cell population.
Total cell counts were then normalized to cell numbers per cm3 of tissue, in which the
volume of renal tissue was calculated as: πr2 x length of biopsy tissue, where the radius
(r) of a 16 gauge biopsy is 0.8mm. Cell acquisition was performed on an LSR Fortessa
(BD) and data analyzed with FlowJo software (TreeStar, Ashland, OR, USA).
Immunofluorescence staining
Frozen 7μm tissue sections from three fibrotic renal biopsies were fixed with 25%
ethanol:75% acetone at room temperature for 5 min, followed by a protein block with
Background Sniper Blocking Reagent for 30 min (Biocare Medical, Concord, CA,
USA). Sections were subsequently probed with combinations of anti-IFN-γ (Goat
polyclonal IgG; R&D Systems, Minneapolis, MN, USA), anti-NKp46 (Monoclonal
Mouse IgG2b; Clone 195314; R&D Systems), anti-Aquaporin-1 (Rabbit polyclonal
IgG; Santa Cruz, Dallas, TX, USA) and anti-CD117 (Rabbit polyclonal IgG; Agilent
Technologies, Santa Clara, CA, USA) or isotype-matched control antibodies at room
temperature for 1 hour. Fluorescent detection was obtained by secondary incubation
with combinations of AlexaFluor-488 anti-goat IgG, AlexaFluor-555 anti-mouse IgG
Chapter 2: Natural Killer Cells in CKD 47
and AlexaFluor-647 anti-rabbit IgG (all from Life Technologies) at room temperature
for 30 min. Nuclei were stained with DAPI (Sigma). Slides were coverslipped in
fluorescence mounting medium (Agilent). A Zeiss 780 NLO confocal microscope
(Carl Zeiss, Hamburg, Germany) was used for fluorescence microscopy. Image
acquisition and analysis were performed using ZEN software (Carl Zeiss).
Quantitative expression was undertaken from 3 fibrotic donor samples, counting a
randomly selected 1mm2 area from 4 separate slides for each donor.
Statistics
All statistical tests were performed using Prism 7.0 analysis software (GraphPad
Software, La Jolla, CA, USA). Multiple comparisons were performed using a Kruskal-
Wallis test with Dunn’s post-test. P values ≤0.05 were considered statistically
significant. Absolute NK cell numbers were correlated with levels of interstitial
fibrosis in diseased biopsies by Spearman correlation analysis.
48 Chapter 2: Natural Killer Cells in CKD
DISCLOSURE
All the authors declared no competing interests.
ACKNOWLEDGEMENTS
The work was funded by Pathology Queensland, a Royal Brisbane and Women’s
Hospital (RBWH) Research Grant, the Kidney Research Foundation and a National
Health and Medical Research Council (NHMRC) Project Grant GNT1099222. BL was
supported by a Pathology Queensland PhD Scholarship. The authors would like to
thank the tissue donors and clinicians, particularly renal histopathologist, Dr Leo
Francis (Queensland Health), for assessment of interstitial fibrosis levels in kidney
biopsies.
Chapter 2: Natural Killer Cells in CKD 49
REFERENCES
1. Spits H, Di Santo JP. The expanding family of innate lymphoid cells: regulators
and effectors of immunity and tissue remodeling. Nat Immunol 2011; 12: 21-
27.
2. Caligiuri MA. Human natural killer cells. Blood 2008; 112: 461-469.
3. Angelo LS, Banerjee PP, Monaco-Shawver L et al. Practical NK cell
phenotyping and variability in healthy adults. Immunol Res 2015; 62: 341-356.
4. Cooper MA, Fehniger TA, Caligiuri MA. The biology of human natural killer-
cell subsets. Trends Immunol 2001; 22: 633-640.
5. Nagler A, Lanier LL, Cwirla S et al. Comparative studies of human FcRIII-
positive and negative natural killer cells. J Immunol 1989; 143: 3183-3191.
6. Michel T, Poli A, Cuapio A et al. Human CD56bright NK Cells: An Update. J
Immunol 2016; 196: 2923-2931.
7. Carrega P, Bonaccorsi I, Di Carlo E et al. CD56(bright)perforin(low)
noncytotoxic human NK cells are abundant in both healthy and neoplastic solid
tissues and recirculate to secondary lymphoid organs via afferent lymph. J
Immunol 2014; 192: 3805-3815.
8. Fehniger TA, Shah MH, Turner MJ et al. Differential cytokine and chemokine
gene expression by human NK cells following activation with IL-18 or IL-15
in combination with IL-12: implications for the innate immune response. J
Immunol 1999; 162: 4511-4520.
9. Matos ME, Schnier GS, Beecher MS et al. Expression of a functional c-kit
receptor on a subset of natural killer cells. J Exp Med 1993; 178: 1079-1084.
10. Kim HJ, Lee JS, Kim JD et al. Reverse signaling through the costimulatory
ligand CD137L in epithelial cells is essential for natural killer cell-mediated
acute tissue inflammation. Proc Natl Acad Sci U S A 2012; 109: E13-22.
11. Zhang ZX, Wang S, Huang X et al. NK cells induce apoptosis in tubular
epithelial cells and contribute to renal ischemia-reperfusion injury. J Immunol
2008; 181: 7489-7498.
12. Victorino F, Sojka DK, Brodsky KS et al. Tissue-Resident NK Cells Mediate
Ischemic Kidney Injury and Are Not Depleted by Anti-Asialo-GM1 Antibody.
J Immunol 2015.
50 Chapter 2: Natural Killer Cells in CKD
13. Spada R, Rojas JM, Perez-Yague S et al. NKG2D ligand overexpression in
lupus nephritis correlates with increased NK cell activity and differentiation in
kidneys but not in the periphery. J Leukoc Biol 2015; 97: 583-598.
14. Zheng G, Zheng L, Wang Y et al. NK cells do not mediate renal injury in
murine adriamycin nephropathy. Kidney Int 2006; 69: 1159-1165.
15. Alexopoulos E, Seron D, Hartley RB et al. The role of interstitial infiltrates in
IgA nephropathy: a study with monoclonal antibodies. Nephrol Dial
Transplant 1989; 4: 187-195.
16. Furuichi K, Wada T, Iwata Y et al. Upregulation of fractalkine in human
crescentic glomerulonephritis. Nephron 2001; 87: 314-320.
17. Hidalgo LG, Sis B, Sellares J et al. NK cell transcripts and NK cells in kidney
biopsies from patients with donor-specific antibodies: evidence for NK cell
involvement in antibody-mediated rejection. Am J Transplant 2010; 10: 1812-
1822.
18. Shin S, Kim YH, Cho YM et al. Interpreting CD56+ and CD163+ Infiltrates
in Early versus Late Renal Transplant Biopsies. Am J Nephrol 2015; 41: 362-
369.
19. Markey AC, MacDonald DM. HNK-1 antigen is not specific for natural killer
cells. J Invest Dermatol 1989; 92: 774-775.
20. Trinchieri G. Biology of natural killer cells. Adv Immunol 1989; 47: 187-376.
21. Kassianos AJ, Wang X, Sampangi S et al. Increased tubulointerstitial
recruitment of human CD141hi CLEC9A+ and CD1c+ myeloid dendritic cell
subsets in renal fibrosis and chronic kidney disease. Am J Physiol Renal
Physiol 2013; 305: F1391-1401.
22. Sivori S, Vitale M, Morelli L et al. p46, a novel natural killer cell-specific
surface molecule that mediates cell activation. J Exp Med 1997; 186: 1129-
1136.
23. Campbell JJ, Qin S, Unutmaz D et al. Unique subpopulations of CD56+ NK
and NK-T peripheral blood lymphocytes identified by chemokine receptor
expression repertoire. J Immunol 2001; 166: 6477-6482.
24. Berahovich RD, Lai NL, Wei Z et al. Evidence for NK cell subsets based on
chemokine receptor expression. J Immunol 2006; 177: 7833-7840.
25. Bedford JJ, Leader JP, Walker RJ. Aquaporin expression in normal human
kidney and in renal disease. J Am Soc Nephrol 2003; 14: 2581-2587.
Chapter 2: Natural Killer Cells in CKD 51
26. Imig JD, Ryan MJ. Immune and inflammatory role in renal disease. Compr
Physiol 2013; 3: 957-976.
27. Kim HJ, Lee JS, Kim A et al. TLR2 signaling in tubular epithelial cells
regulates NK cell recruitment in kidney ischemia-reperfusion injury. J
Immunol 2013; 191: 2657-2664.
28. Zhang ZX, Shek K, Wang S et al. Osteopontin expressed in tubular epithelial
cells regulates NK cell-mediated kidney ischemia reperfusion injury. J
Immunol 2010; 185: 967-973.
29. Bjorkstrom NK, Ljunggren HG, Michaelsson J. Emerging insights into natural
killer cells in human peripheral tissues. Nat Rev Immunol 2016; 16: 310-320.
30. Dalbeth N, Gundle R, Davies RJ et al. CD56bright NK cells are enriched at
inflammatory sites and can engage with monocytes in a reciprocal program of
activation. J Immunol 2004; 173: 6418-6426.
31. Lugthart G, Melsen JE, Vervat C et al. Human Lymphoid Tissues Harbor a
Distinct CD69+CXCR6+ NK Cell Population. J Immunol 2016; 197: 78-84.
32. Hudspeth K, Donadon M, Cimino M et al. Human liver-resident
CD56(bright)/CD16(neg) NK cells are retained within hepatic sinusoids via
the engagement of CCR5 and CXCR6 pathways. J Autoimmun 2016; 66: 40-
50.
33. Manaster I, Mandelboim O. The unique properties of uterine NK cells. Am J
Reprod Immunol 2010; 63: 434-444.
34. Shiow LR, Rosen DB, Brdickova N et al. CD69 acts downstream of interferon-
alpha/beta to inhibit S1P1 and lymphocyte egress from lymphoid organs.
Nature 2006; 440: 540-544.
35. Montaldo E, Vacca P, Chiossone L et al. Unique Eomes(+) NK Cell Subsets
Are Present in Uterus and Decidua During Early Pregnancy. Front Immunol
2015; 6: 646.
36. Segerer S, Banas B, Wornle M et al. CXCR3 is involved in tubulointerstitial
injury in human glomerulonephritis. Am J Pathol 2004; 164: 635-649.
37. Kassianos AJ, Wang X, Sampangi S et al. Fractalkine-CX3CR1-dependent
recruitment and retention of human CD1c myeloid dendritic cells by in vitro-
activated proximal tubular epithelial cells. Kidney Int 2015; 87: 1153-1163.
52 Chapter 2: Natural Killer Cells in CKD
38. Masutani K, Akahoshi M, Tsuruya K et al. Predominance of Th1 immune
response in diffuse proliferative lupus nephritis. Arthritis Rheum 2001; 44:
2097-2106.
39. Ricardo SD, van Goor H, Eddy AA. Macrophage diversity in renal injury and
repair. J Clin Invest 2008; 118: 3522-3530.
40. Kitching AR, Holdsworth SR, Tipping PG. IFN-gamma mediates crescent
formation and cell-mediated immune injury in murine glomerulonephritis. J
Am Soc Nephrol 1999; 10: 752-759.
41. Wilkinson R, Wang X, Roper KE et al. Activated human renal tubular cells
inhibit autologous immune responses. Nephrol Dial Transplant 2011; 26:
1483-1492.
42. Oldroyd SD, Thomas GL, Gabbiani G et al. Interferon-gamma inhibits
experimental renal fibrosis. Kidney Int 1999; 56: 2116-2127.
43. Poosti F, Bansal R, Yazdani S et al. Selective delivery of IFN-gamma to renal
interstitial myofibroblasts: a novel strategy for the treatment of renal fibrosis.
FASEB J 2015; 29: 1029-1042.
44. Kitching AR, Turner AL, Semple T et al. Experimental autoimmune anti-
glomerular basement membrane glomerulonephritis: a protective role for IFN-
gamma. J Am Soc Nephrol 2004; 15: 1764-1774.
45. Luo L, Lu J, Wei L et al. The role of HIF-1 in up-regulating MICA expression
on human renal proximal tubular epithelial cells during
hypoxia/reoxygenation. BMC Cell Biol 2010; 11: 91.
46. Racusen LC, Solez K, Colvin RB et al. The Banff 97 working classification of
renal allograft pathology. Kidney Int 1999; 55: 713-723.
Chapter 2: Natural Killer Cells in CKD 53
TITLE AND LEGENDS
Table 1. Antibodies used for flow cytometric staining.
Antigen Clone Fluorochrome Source
CD45 HI30 Brilliant Violet 510 Biolegend
CD14 M5E2 Alexa Fluor 700 Biolegend
CD3 HIT3a APC BD Biosciences
CD19 HIB19 PE BD Biosciences
CD56 HCD56 PerCP/Cy5.5 Biolegend
CD16 3G8 PE-CF594 BD Biosciences
CD69 FN50 Brilliant Violet 785 Biolegend
CD335 (NKp46) 9E2 Brilliant Violet 421 Biolegend
CD117 104D2 PE-Cy7 Biolegend
CXCR3 G025H7 Brilliant Violet 711 Biolegend
CX3CR1 2A9-1 FITC Biolegend
54 Chapter 2: Natural Killer Cells in CKD
Figure 1. Identification of natural killer (NK) cell subsets in human kidney tissue.
Gating strategy used to identify total NK cells (CD3- CD19- CD56+ lymphocytes) and
NK cell subpopulations (CD56dim and CD56bright NK cells) in human kidney tissue.
Representative flow cytometric data from one of sixteen individual fibrotic renal
biopsies are shown. An identical gating strategy was used for healthy kidney tissue
and non-fibrotic renal biopsies.
Chapter 2: Natural Killer Cells in CKD 55
Figure 2. Significantly elevated NK cell numbers in diseased biopsies with
interstitial fibrosis. (a-c) Absolute numbers of total NK cells (a), CD56dim NK cells
(b) and CD56bright NK cells (c) in healthy kidney tissue and diseased biopsies without
and with fibrosis. Values for individual donors are presented; bars represent means.
*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, Kruskal-Wallis test. (d-f) Spearman
correlation analyses of absolute numbers of total NK cells (d), CD56dim NK cells (e)
and CD56bright NK cells (f) versus percentages of interstitial fibrosis in diseased
biopsies.
56 Chapter 2: Natural Killer Cells in CKD
Figure 3. CD56bright NK cell numbers correlate significantly with loss of kidney
function. (a-c) Absolute numbers of total NK cells (a), CD56dim NK cells (b) and
CD56bright NK cells (c) in healthy kidney tissue and diseased biopsies with eGFR≥60
(CKD I-II) and <60 (CKD III-V). Values for individual donors are presented; bars
represent means. *p<0.05, **p<0.01, ***p<0.001, Kruskal-Wallis test. (d-f) Absolute
numbers of total NK cells (d), CD56dim NK cells (e) and CD56bright NK cells (f) in
healthy kidney tissue and diseased biopsies with glomerular immune-mediated,
glomerular non-immune-mediated and non-glomerular primary diagnoses. Values for
individual donors are presented; bars represent means. *p<0.05, **p<0.01, Kruskal-
Wallis test.
Chapter 2: Natural Killer Cells in CKD 57
Figure 4. Significantly increased proportion of CD56bright NK cells with loss of
kidney function. Frequency of CD56bright NK cells among total NK cells in healthy
kidney tissue and (a) diseased biopsies without and with fibrosis and (b) diseased
biopsies with eGFR≥60 (CKD I-II) and <60 (CKD III-V). Values for individual donors
are presented; bars represent means, with mean values presented in parentheses.
*p<0.05, Kruskal-Wallis test. (c) Contour plots (gated on CD3- CD19- double negative
lymphocytes) highlighting the relative proportions of CD56dim and CD56bright NK cell
subsets from representative diseased biopsies with eGFR≥60 (CKD I-II) (top panel)
and eGFR<60 (CKD III-V) (bottom panel). Percentage values of CD56bright NK cells
among total NK cells are presented.
58 Chapter 2: Natural Killer Cells in CKD
Figure 5. Human CD56bright NK cells in fibrotic kidney tissue display an activated
phenotype. (a-b) Surface expression of CD69 on CD56dim NK cells (a) and CD56bright
NK cells (b) in healthy kidney tissue and diseased biopsies without and with fibrosis.
Values of median fluorescence intensity (MFI) for individual donors are shown; bars
represent means, with mean values presented in parentheses. *p<0.05, Kruskal-Wallis
test. (c) Relative expression of CD69, NKp46, CD117, CXCR3 and CX3CR1 by
CD56bright NK cells compared to CD56dim NK cells, T cells and isotype control in
fibrotic kidney tissue. Representative flow cytometric data from eight individual
experiments are shown.
Chapter 2: Natural Killer Cells in CKD 59
Figure 6. Co-localization of human NK cells with proximal tubular epithelial cells
(PTEC). Immunofluorescent staining of frozen kidney sections from diseased biopsies
without (left panel) and with interstitial fibrosis (middle/right panels) stained for
NKp46 (red), PTEC marker Aquaporin-1 (white) and DAPI (blue). NKp46+ cells are
circled (right panel). Scale bars represent 100μm (left/middle panels) and 10μm (right
panel). Representative results for three individual donor experiments are shown.
60 Chapter 2: Natural Killer Cells in CKD
Figure 7. Human NK cells produce pro-inflammatory cytokine IFN-γ in fibrotic
kidney tissue. Immunofluorescent labelling of frozen fibrotic kidney tissue stained for
NKp46 (red; left panel) and IFN-γ (green; middle panel). Co-localisation is visualised
by the yellow merge of red and green (right panel). NKp46+ IFN-γ+ cells are circled.
Scale bars represent 100μm for large frames and 10μm for insets. Representative
results for three individual donor experiments are shown.
Chapter 2: Natural Killer Cells in CKD 61
Figure 8. Human CD56bright NK cells are a key source of IFN-γ in fibrotic kidney
tissue. Immunofluorescent labelling of frozen fibrotic kidney tissue stained for NKp46
(red; first panel), CD117 (orange; second panel) and IFN-γ (green; third panel). Co-
localisation is visualised by merging the three colours (fourth panel). NKp46+ CD117+
IFN-γ+ cells are circled. Scale bars represent 100μm for large frames and 10μm for
insets.
62 Chapter 2: Natural Killer Cells in CKD
Supplementary Table 1. Clinical and histological features of patients at the time of
kidney biopsy.
*Interstitial Inflammation; Nil = not present, Minimal = up to 10%, Mild = 10-25%,
Moderate = 26–50% and Severe
Chapter 2: Natural Killer Cells in CKD 63
Supplementary Figure 1. NK cell numbers do not significantly correlate with
levels of inflammatory activity in diseased biopsies. Absolute numbers of total NK
cells (a), CD56dim NK cells (b) and CD56bright NK cells (c) in healthy kidney tissue
and diseased biopsies grouped based on histological scoring of interstitial
inflammation (Nil = not present, Minimal = up to 10%, Mild = 10-25%, Moderate =
26–50% and Severe = greater than 50%). Values for individual donors are presented;
bars represent means.
64 Chapter 2: Natural Killer Cells in CKD
Chapter 3: Gamma-Delta T cells in CKD 65
Chapter 3: Gamma-Delta T cells in CKD
In this chapter, I moved on to study the role of gamma-delta (γδ) T
cells in human CKD. γδ T cells are a unique type of innate lymphocyte as
they express a T cell receptor consisting of gamma and delta chains. From
animal studies of CKD, γδ T cells contribute to kidney pathogenesis via
the production of pro-inflammatory cytokine IL-17A. However, there is
currently only limited information about the pathogenesis of γδ T cells in
human kidneys. To confirm the findings regarding γδ T cells in animal
models of kidney disease, I present here my ex vivo and in situ analysis of
human γδ T cells in CKD.
QUT Verified Signature
Chapter 3: Gamma-Delta T cells in CKD 67
ABSTRACT
Background: γδ T cells are effector lymphocytes recognised as key players during
chronic inflammatory processes. Mouse studies suggest a pathological role for γδ T
cells in models of kidney disease. Here, we evaluated γδ T cells in human native
kidneys with tubulointerstitial fibrosis, the pathological hallmark of chronic kidney
disease.
Methods: γδ T cells were extracted from human kidney tissue and enumerated and
phenotyped by multi-colour flow cytometry. Localisation and cytokine production by
γδ T cells was examined by immunofluorescent microscopy.
Results: We detected significantly elevated numbers of γδ T cells in diseased biopsies
with tubulointerstitial fibrosis compared with diseased biopsies without fibrosis and
healthy kidney tissue. At a subset level, only numbers of Vδ1+ γδ T cells were
significantly elevated in fibrotic kidney tissue. Expression levels of CD161, a marker
of human memory T cells with potential for innate-like function and IL-17A
production, were significantly elevated on γδ T cells from fibrotic biopsies compared
with non-fibrotic kidney tissue. Flow cytometric characterisation of CD161+ γδ T cells
in fibrotic biopsies revealed significantly elevated expression of natural killer cell-
associated markers CD56, CD16 and CD336 (NKp44) compared with CD161- γδ T
cells, indicative of a cytotoxic phenotype. Immunofluorescent analysis of fibrotic
kidney tissue localised the accumulation of γδ T cells within the tubulointerstitium,
with γδ T cells identified, for the first time, as a source of pro-inflammatory cytokine
IL-17A.
Conclusions: Collectively, our data suggest that human effector γδ T cells contribute
to the fibrotic process and thus, progression to chronic kidney disease.
68 Chapter 3: Gamma-Delta T cells in CKD
INTRODUCTION
T lymphocytes are broadly subcategorised based on their T cell receptor (TCR) type
into classical alpha/beta (αβ) T cells and the unconventional gamma/delta (γδ) T cells.
Despite representing the more minor population of human T lymphocytes, γδ T cells
are now established as specialised cells that play a major role in bridging local innate
and adaptive immune responses.
Like classical adaptive αβ T cells, γδ T cells undergo clonal expansion and exhibit
antigen-specific memory in response to TCR-dependent triggering.1 However, while
αβ TCR are reactive to peptides in the context of major histocompatibility complex
(MHC) class I or II molecules, γδ TCR recognise a broad range of antigens (soluble or
membrane proteins, phospholipids) in an MHC-independent fashion.2 Like natural
killer (NK) cells, γδ T cells also respond to stress-induced ligands expressed by
aberrant cells that engage with activating receptors on their cell surface. This triggering
process in the absence of antigen processing and presentation allows γδ T cells to act
in the innate phase of the immune response.3 In response to these collective activatory
signals, γδ T cells perform diverse effector functions including potent cytotoxic
activity and pro-inflammatory cytokine production (e.g. interleukin (IL)-17A).4
γδ T cells comprise only a small fraction of total peripheral blood lymphocytes, but
are preferentially enriched in peripheral tissues, especially at epithelial surfaces of the
skin, lungs, reproductive tract and intestines.5,6 Notably, the pathogenesis of many
inflammatory diseases in these tissues involves the accumulation of γδ T cells.4,6
However, their functional role in human native kidney disease remains poorly defined.
The function of γδ T cells has been examined in murine models of acute and chronic
kidney disease (CKD). Mouse studies of ischaemic acute kidney injury ascribe a
pathogenic role for γδ T cells as mediators between innate and adaptive immunity,7,8
whilst in acute crescentic glomerulonephritis (GN)9 and more chronic models of anti-
glomerular basement membrane (GBM) GN10 and interstitial fibrosis,11 there is
evidence that γδ T cells promote immune-mediated kidney injury. However, in a
chronic model of focal segmental glomerulosclerosis (FSGS), depletion of γδ T cells
was found to exacerbate the disease process, suggestive of a more regulatory role.12
These conflicting findings may be attributed to differing mouse strains, disease models
Chapter 3: Gamma-Delta T cells in CKD 69
and γδ T cell depletion methods and highlight the importance of caution in translating
murine findings to human native kidney disease.
An initial immunohistochemical (IHC)-based study of human kidney biopsies from
patients with IgA nephropathy reported associations between interstitial γδ T cell
numbers and disease progression.13 Despite the potential pathological significance of
γδ T cells highlighted by this study, larger confirmatory studies in a broader range of
human kidney diseases are yet to be performed.
We previously showed that human native kidneys with tubulointerstitial fibrosis, the
pathological hallmark of CKD, have significantly elevated numbers of total
lymphocytes compared to non-fibrotic renal tissue.14 In this present study, we focus
this multi-parameter flow cytometric-based approach to accurately detect, quantify
and phenotype γδ T cells in human kidney disease. We demonstrate that diseased
native biopsies with interstitial fibrosis have significantly increased numbers of
tubulointerstitial γδ T cells compared to non-fibrotic biopsies, with γδ T cells
exhibiting an innate-like cytotoxic phenotype and identified as a producer of IL-17A.
70 Chapter 3: Gamma-Delta T cells in CKD
MATERIALS AND METHODS
Kidney tissue specimens
Kidney cortical tissue was obtained with informed patient consent from the
macroscopically/microscopically healthy portion of tumour nephrectomies or native
diseased biopsies, following approval by the Royal Brisbane and Women’s Hospital
Human Research Ethics Committee (2002/011 and 2006/072). Healthy cortical tissue
was obtained from 18 donors (8 females/10 males) of mean age 58±7, whilst diseased
clinical biopsies were obtained from 69 donors (34 females/35 males) of mean age
56±16. A range of primary diagnoses was sampled, including 28 glomerular immune-
mediated (lupus nephritis, crescentic GN, membranoproliferative GN, necrotizing GN,
pauci-immune GN, IgA nephropathy, membranous nephropathy and minimal change
disease), 24 glomerular non-immune-mediated (fibrillary GN, focal segmental
glomerulosclerosis, renal amyloidosis and diabetic nephropathy) and 17 non-
glomerular (interstitial nephritis, light chain-related proximal tubulopathy,
arterionephrosclerosis and hypertensive nephropathy) etiologies.
Fresh biopsies were taken with a 16-gauge biopsy needle (Biopsybell, Mirandola,
Italy) and immediately divided for: 1) tissue dissociation (1-5mm of a core biopsy); 2)
freezing in Tissue-Tek OCT compound (Sakura, Torrance, CA, USA) for
immunofluorescence (IF) analysis; and 3) fixation in formalin for assessing levels of
interstitial fibrosis/tubular atrophy by renal histopathologists blinded to experimental
results. Biopsies displaying ≥5% interstitial fibrosis were deemed fibrotic, based on
the Banff 97 working classification of renal pathology.15 According to this criterion,
diseased specimens were then grouped into biopsies without (n=31; 16 females/15
males; mean age 57±20; mean eGFR 61±31ml/min/1.73m2) or with interstitial fibrosis
(n=38; 18 females/20 males; mean age 56±14; mean eGFR 38±23ml/min/1.73m2).
Kidney function (eGFR) was calculated using the CKD-EPI method by AUSLAB
(Queensland Health, Brisbane, Australia).
Tissue dissociation for flow cytometric analysis
Healthy kidney tissue and diseased biopsies were digested with 1mg/ml collagenase P
(Roche, Mannheim, Germany) in the presence of 20μg/ml DNase I (Roche) (250μl
volume) for 15 min and then further digested with 10μg/ml trypsin/4μg/ml EDTA
(Life Technologies, Grand Island, NY, USA) (500μl volume) for 10 min.
Chapter 3: Gamma-Delta T cells in CKD 71
Flow cytometry
Single cell suspensions were initially stained with LIVE/DEAD® Fixable Near-IR
Dead Cell Stain Kit (Life Technologies) to exclude non-viable cells. Cells were then
incubated with Human TruStain FcX™ Blocking Solution (Biolegend, San Diego,
CA, USA) at room temperature for 5-10 min and then stained on ice for 30 min with
combinations of test (Table 1) or isotype-matched control antibodies in cold FACS
buffer [0.5% BSA (Sigma, St Louis, MO, USA) and 0.02% sodium azide (Sigma) in
PBS]. Flow-Count Fluorospheres™ (Beckman Coulter, Brea, CA, USA) were used for
direct determination of absolute counts following the manufacturer’s
recommendations and as described previously.14,16 Cell acquisition was performed on
an LSR Fortessa (BD Biosciences, San Jose, CA, USA) and data analyzed with FlowJo
software (TreeStar, Ashland, OR, USA).
IF staining
Frozen 7μm tissue sections from three fibrotic renal biopsies were fixed with 25%
ethanol:75% acetone at room temperature for 5 min, followed by a protein block with
Background Sniper Blocking Reagent (Biocare Medical, Concord, CA, USA) for 30
min. Sections were subsequently probed with combinations of anti-IL-17/IL-17A
(Goat polyclonal IgG; R&D Systems, Minneapolis, MN, USA), anti-TCR γδ
(Monoclonal mouse IgG1; Clone B1; Biolegend) and anti-Aquaporin-1 (Rabbit
polyclonal IgG; Santa Cruz, Dallas, TX, USA) or isotype-matched control antibodies
at room temperature for 1 hour. Fluorescent detection was obtained by secondary
incubation with combinations of AlexaFluor-488 anti-goat IgG, AlexaFluor-555 anti-
mouse IgG and AlexaFluor-647 anti-rabbit IgG (all from Life Technologies) at room
temperature for 30 min. Nuclei were stained with DAPI (Sigma). Slides were
coverslipped in fluorescence mounting medium (Agilent Technologies, Santa Clara,
CA, USA). A Zeiss 780 NLO confocal microscope (Carl Zeiss, Hamburg, Germany)
was used for fluorescence microscopy. Image acquisition and analysis were performed
using ZEN software (Carl Zeiss).
Statistics
All statistical tests were performed using Prism 7.0 analysis software (GraphPad
Software, La Jolla, CA, USA). Comparisons between paired groups were performed
using a Wilcoxon matched-pairs signed rank test and multiple comparisons were
72 Chapter 3: Gamma-Delta T cells in CKD
performed using a Kruskal-Wallis test with Dunn’s post-test. Absolute cell numbers
were correlated with patient eGFR and levels of interstitial fibrosis in diseased biopsies
by Spearman correlation analysis. P values ≤0.05 were considered statistically
significant.
Chapter 3: Gamma-Delta T cells in CKD 73
RESULTS
Identification of γδ T cells in human kidney tissue.
Healthy and diseased kidney tissue was enzymatically digested to obtain single cells
for multi-colour flow cytometric analysis. Using a gating strategy outlined in
Supplementary Figure 1, we separated CD45+ leukocytes into granulocytes with
higher side scatter (SSC) and mononuclear cells (MNC). These MNC were further
divided into CD14+ monocyte and CD14- lymphocyte populations. Total T cells were
then defined as CD3+ lymphocytes, with γδ T cells (CD3+ TCR γδ+) identified within
this total T cell population.
Absolute numbers of human γδ T cells correlate significantly with loss of kidney
function.
We enumerated total T cells in healthy and diseased kidney tissue, with diseased
biopsies stratified based on patient kidney function (eGFR). Quantification with Flow-
Count fluorospheres revealed a significant increase in total T cell numbers in diseased
biopsies from patients with reduced kidney function (eGFR<60ml/min/1.73m2)
compared with diseased biopsies from patients with normal kidney function
(eGFR≥60ml/min/1.73m2) and healthy kidney tissue (Figure 1A). Within the total T
cell population, numbers of γδ T cells were also significantly elevated in diseased
biopsies from patients with reduced kidney function (Figure 1B). Further analysis
showed weak but significant negative correlations between patient eGFR and both
total T cell numbers (r = -0.3622, P=0.0052) and γδ T cell numbers (r = -0.3660,
P=0.0047) (Figure 1C-D). These data demonstrate that kidney γδ T cell numbers are
related to functional outcome.
Significantly elevated numbers of human γδ T cells in diseased biopsies with
interstitial fibrosis.
In addition to kidney function, diseased biopsies were grouped based on the
histological absence or presence of interstitial fibrosis, the characteristic feature of all
forms of chronic kidney disease. This approach revealed significantly elevated
numbers of total T cells (Figure 2A) and γδ T cells (Figure 2B) in diseased biopsies
with interstitial fibrosis compared with diseased biopsies without fibrosis and healthy
kidney tissue. Notably, scatter plot correlations between cell numbers and the degree
of fibrosis showed significant correlations for total T cells (r = 0.5218, P<0.0001) and
74 Chapter 3: Gamma-Delta T cells in CKD
γδ T cells (r = 0.5974, P<0.0001) (Figure 2C-D). Collectively, these data associate γδ
T cells with renal interstitial fibrosis.
Diseased biopsies with interstitial fibrosis contain significantly elevated numbers
of Vδ1+ γδ T cells.
For identification purposes, human γδ T cells are commonly subdivided based on their
expression of one of two variable regions of TCR-δ, Vδ1 or Vδ2.17 Using our flow
cytometric approach, we were able to identify both Vδ1+ and Vδ2+ γδ T cells in human
kidney tissue (Figure 3A). Notably, Vδ1+ γδ T cell numbers were significantly higher
in fibrotic biopsies compared with healthy tissue (Figure 3B), whilst Vδ2+ γδ T cell
numbers were elevated, but not significantly, in diseased biopsies with interstitial
fibrosis (Figure 3C). A significant correlation between cell numbers and the degree of
interstitial fibrosis was only observed for the Vδ1+ γδ T cell subset (r = 0.7986,
P=0.0048) (Figure 3D-E).
Human CD161+ γδ T cells in fibrotic kidneys display an innate-like cytotoxic
phenotype.
C-type lectin receptor, CD161, has been identified as a marker of human T cells with
a memory phenotype,18,19 capacity for innate-like function20 and with the potential to
produce IL-17A.18 Thus, we next examined CD161 expression levels on T cells in our
healthy and diseased kidney tissue. Although expression levels of CD161 on total T
cells were similar between healthy and diseased tissue (Figure 4A), the expression of
CD161 on γδ T cells was significantly elevated (P<0.05) in diseased biopsies with
interstitial fibrosis compared with non-fibrotic biopsies (Figure 4B-C). We did not
observe significant differences in CD161 expression levels between Vδ1+ and Vδ2+ γδ
T cells from fibrotic biopsies (Figure 4D).
Further characterisation of CD161+ γδ T cells in fibrotic biopsies revealed significantly
elevated expression levels of NK cell-associated markers CD56 (neural cell adhesion
molecule, NCAM), CD16 (FcγRIII, the low affinity receptor for the Fc portion of
immunoglobulin G) and natural cytotoxicity receptor NKp44 (CD336) compared with
CD161- γδ T cells (Figure 5A-C). These results suggest that the local environment
within fibrotic kidneys directs γδ T cells toward an innate-like cytotoxic function.
Chapter 3: Gamma-Delta T cells in CKD 75
Tubulointerstitial localisation of human γδ T cells in fibrotic kidney tissue.
We next examined the localisation of γδ T cells in human kidney tissue using IF
microscopy. In line with our flow cytometric data, an increased presence of γδ T cells
was detected in diseased biopsies with interstitial fibrosis compared with non-fibrotic
kidney tissue (Figure 6). Moreover, γδ T cells were identified within the
tubulointerstitial compartment, in apposition to proximal tubular epithelial cells
(PTEC), defined as tubular cells expressing aquaporin-1.21 Notably, γδ T cells
localised to sites of tubulointerstitial injury (inflammation, tubular atrophy),
suggesting these cells participate in the progression of kidney fibrosis.
Tubulointerstitial γδ T cells are a source of IL-17A in fibrotic kidney tissue.
Previous studies have reported tubulointerstitial IL-17A-expressing T cells in the
kidneys of patients with acute GN22 and lupus nephritis.23,24 We extended this work to
examine the localisation of IL-17A in our fibrotic biopsies by IF staining,
demonstrating, for the first time, the co-expression of IL-17A with tubulointerstitial γδ
T cells (Figure 7). Although only a small proportion of IL-17A-expressing cells were
shown to be γδ T cells, those IL-17A+ γδ T cells were often evident adjacent to PTEC
(Figure 7), further supporting a specialised functional role for kidney γδ T cells in the
fibrotic process.
76 Chapter 3: Gamma-Delta T cells in CKD
DISCUSSION
γδ T cells represent a functionally specialised lymphocyte population that act as a
bridge between the innate and adaptive immune responses. Murine models support a
functional role for γδ T cells in the pathogenesis of inflammatory kidney diseases.9-11
However, our understanding of human γδ T cells in CKD has, until now, been
constrained by methodological limits. In this present study, we have used a multi-
colour based approach to study the absolute numbers, phenotype and function of γδ T
cells in fibrotic human kidneys. We demonstrate, for the first time, an association
between absolute numbers of γδ T cells, in particular Vδ1+ cells, with the tissue
pathology of CKD (presence of interstitial fibrosis). We have also identified γδ T cells
in fibrotic kidney tissue with both innate-like cytotoxic potential (expression of CD56,
CD16 and NKp44) and as a source of proinflammatory IL-17A, consistent with
elevated CD161 expression levels on these cells. These findings suggest that human
γδ T cells are of profound importance in interstitial fibrosis and thus, contribute to the
immunopathogenesis of CKD.
The accumulation of human γδ T cells has been shown to positively correlate with the
severity of inflammatory diseases in other target organs (skin, brain).25-27 With respect
to human kidney disease, the abundance of γδ T cells, as assessed by
immunohistochemistry, has been associated with disease progression (eGFR decline)
in a restricted cohort of patients with IgA nephropathy.13 Here, we demonstrate, for
the first time, irrespective of primary diagnosis, significant correlations between
absolute γδ T cell numbers and lower eGFR.
We further extend these observations to demonstrate significant associations between
γδ T cell counts and histological severity of renal interstitial fibrosis. Previous
evidence linking human γδ T cells to fibrosis largely comes from studies of the
prototypic human fibrotic disease, systemic sclerosis.28,29 In particular, Vδ1+ γδ T cells
have been shown to accumulate in the skin of systemic sclerosis patients.28 In line with
these findings, we demonstrated significantly elevated numbers of only Vδ1+ γδ T cells
– not Vδ2+ γδ T cells – in diseased biopsies with interstitial fibrosis. Wu et al have
previously reported that Vδ1+ γδ T cells from IgA nephropathy kidney biopsies have
a restricted TCR repertoire, suggesting the clonal expansion of individual Vδ1+ γδ T
cells in the kidney.30 It is tempting to speculate that, indeed, the increased numbers of
Chapter 3: Gamma-Delta T cells in CKD 77
Vδ1+ γδ T cells in our fibrotic biopsies may be similarly driven by a set of conserved
antigens in the diseased kidney.
Although originally identified as an NK cell marker, CD161 expression is consistently
associated with a memory phenotype in human T cells19, including γδ T cells.18 The
functional role of this molecule in human T cells has not yet been fully defined, with
reports of both costimulatory20,31 and inhibitory effects32,33 following CD161 ligation.
However, previous studies have implicated CD161+ γδ T cells in inflammatory
diseases such as multiple sclerosis.34,35 Here, we report elevated CD161 MFI levels on
γδ T cells in diseased biopsies with interstitial fibrosis compared to non-fibrotic
biopsies, suggesting that the local inflammatory milieu within fibrotic kidneys skews
γδ T cells toward a unique memory phenotype.
CD161 has also been recently identified as a phenotypic marker of human T cells with
a functional potential for innate-like activity, including upregulated expression of
cytotoxic proteins.20 Notably, the cytotoxic potency of human γδ T cells has been
shown to correlate with expression levels of NK-associated markers CD16,36,37
CD5638,39 and NKp44.40 In line with these previous studies, we show increased
expression of these three molecules on CD161+ γδ T cells within our fibrotic biopsies.
Thus, we provide the first evidence of a cytotoxic effector γδ T cell population within
human native kidney disease, marked by the expression of CD161.
In addition to this innate-like response, CD161 has been applied as a phenotypic
marker of IL-17A-expressing human T cells, including γδ T cells.18 In our present
study, we identify γδ T cells as a source, although minor, of IL-17A within the
tubulointerstitial compartment of fibrotic kidneys. Experimental models of kidney
disease have illustrated the pivotal role of IL-17A in promoting tissue injury,41 with
recent mouse studies highlighting that IL-17A production by renal γδ T cells, in
particular, contributes significantly to the immunopathogenesis of acute crescentic
GN9 and renal fibrosis.11 However, the translation of this work to the clinical setting
has been more difficult to demonstrate. Although human studies outside the kidney
have reported IL-17A+ γδ T cells under chronic inflammatory conditions,25,27,42 until
now, there have been no reports of equivalent IL-17A-expressing γδ T cells in human
CKD.
78 Chapter 3: Gamma-Delta T cells in CKD
IL-17A is a pleiotropic cytokine that directly promotes renal inflammation by: (1)
stimulating neutrophil recruitment,9 (2) driving macrophage differentiation43 and (3)
augmenting cytokine/chemokine production by non-haematopoietic cells, including
PTEC.44,45 An in vitro study by van Kooten et al showed IL-17A enhances the
production of IL-6, IL-8, MCP-1 and complement C3 by human PTEC,44 whilst we
have reported increased C3 expression by in vivo human PTEC from diseased biopsies
with overall cortical inflammation.46 We propose that the in vivo inflammatory signal
for this C3 production may, in fact, be provided by IL-17A+ γδ T cells. This concept
is reinforced by our in situ detection of IL-17A-expressing γδ T cells in areas of
interstitial inflammation/tubular atrophy, often in direct contact with PTEC. Our
observed co-localisation of PTEC with γδ T cells may also represent a cytotoxic
interaction potentiating the disease process, supporting a recent in vitro observation by
Chen et al of PTEC cell line HK-2 killing by γδ T cells.47 We postulate that the potency
of the low-frequency γδ T cell population in the process of tubulointerstitial fibrosis
lies in their specialised dual capacity for cytokine production (IL-17A secretion that
drives renal inflammation) and cytotoxic activity (PTEC killing). Future studies will
be required to dissect the relative contribution of these cytokine-producing versus
cytotoxic γδ T cell functions in the immunopathogenesis of human CKD.
Collectively, these results provide a comprehensive characterisation of human γδ T
cells in fibrotic kidney disease. Based on our results, we propose that tubulointerstitial
γδ T cells receive stimulatory signals within the fibrotic microenvironment to acquire
a pathogenic, effector functionality pivotal in the progressive loss of kidney function.
The tubulointerstitial localisation of γδ T cells, often adjacent to damaged PTEC,
would suggest an important functional role for these lymphocytes in cortical interstitial
fibrosis. It should be noted that a recent study has established tight relationships
between cortical and medullary fibrosis.48 The role of leukocytes, including γδ cells,
in this medullary scarring will be an area for future investigations. Importantly, our
current study also provides human functional correlations to pathogenic mouse γδ T
cells previously reported in experimental studies of kidney disease.9,11 A deeper
understanding of kidney γδ T cell biology, in particular, the functional role of different
γδ T cell subsets in fibrotic kidney disease, is now required for the development of
therapeutics capable of blocking the recruitment or activation of this previously
untargeted immune cell population.
Chapter 3: Gamma-Delta T cells in CKD 79
ACKNOWLEDGEMENTS
The authors would like to thank the tissue donors and clinicians, particularly renal
histopathologist, Dr Leo Francis (Queensland Health), for assessment of interstitial
fibrosis levels in kidney biopsies.
CONFLICT OF INTEREST STATEMENT
All the authors declared no competing interests. The results presented in this paper
have not been published previously in whole or part, except in abstract form.
AUTHORS’ CONTRIBUTIONS
B.L., R.W., K.B., H.H. and A.J.K. conceived and designed the study; B.L., X.W.,
K.K., M.L. and A.J.K. carried out experiments and analysed the data; B.L., R.W., H.H.
and A.J.K. drafted the paper; all authors revised and approved the final version of the
manuscript.
FUNDING
The work was funded by Pathology Queensland, a Royal Brisbane and Women’s
Hospital (RBWH) Research Grant, the Kidney Research Foundation and a National
Health and Medical Research Council (NHMRC) Project Grant GNT1099222. BL was
supported by a Pathology Queensland – Study, Education and Research Committee
(SERC) PhD Scholarship.
80 Chapter 3: Gamma-Delta T cells in CKD
REFERENCES
1. Morita CT, Jin C, Sarikonda G, Wang H. Nonpeptide antigens, presentation
mechanisms, and immunological memory of human Vγ2Vδ2 T cells:
discriminating friend from foe through the recognition of prenyl pyrophosphate
antigens. Immunol Rev 2007; 215: 59-76.
2. Lafont V, Sanchez F, Laprevotte E et al. Plasticity of γδ T cells: Impact on the
anti-tumor response. Front Immunol 2014; 5: 622.
3. Patil RS, Bhat SA, Dar AA, Chiplunkar SV. The Jekyll and Hyde story of IL17-
producing γδ T cells. Front Immunol 2015; 6: 37.
4. Papotto PH, Ribot JC, Silva-Santos B. IL-17+ γδ T cells as kick-starters of
inflammation. Nat Immunol 2017; 18: 604-611.
5. Hayday AC. γδ T cells and the lymphoid stress-surveillance response.
Immunity 2009; 31: 184-196.
6. Fay NS, Larson EC, Jameson JM. Chronic inflammation and γδ T cells. Front
Immunol 2016; 7: 210.
7. Hochegger K, Schatz T, Eller P et al. Role of αβ and γδ T cells in renal
ischemia-reperfusion injury. Am J Physiol Renal Physiol 2007; 293: F741-747.
8. Savransky V, Molls RR, Burne-Taney M, Chien CC, Racusen L, Rabb H. Role
of the T-cell receptor in kidney ischemia-reperfusion injury. Kidney Int 2006;
69: 233-238.
9. Turner JE, Krebs C, Tittel AP et al. IL-17A production by renal γδ T cells
promotes kidney injury in crescentic GN. J Am Soc Nephrol 2012; 23: 1486-
1495.
10. Rosenkranz AR, Knight S, Sethi S, Alexander SI, Cotran RS, Mayadas TN.
Regulatory interactions of αβ and γδ T cells in glomerulonephritis. Kidney Int
2000; 58: 1055-1066.
11. Peng X, Xiao Z, Zhang J, Li Y, Dong Y, Du J. IL-17A produced by both γδ T
and Th17 cells promotes renal fibrosis via RANTES-mediated leukocyte
infiltration after renal obstruction. J Pathol 2015; 235: 79-89.
12. Wu H, Wang YM, Wang Y et al. Depletion of γδ T cells exacerbates murine
adriamycin nephropathy. J Am Soc Nephrol 2007; 18: 1180-1189.
13. Falk MC, Ng G, Zhang GY et al. Infiltration of the kidney by αβ and γδ T cells:
effect on progression in IgA nephropathy. Kidney Int 1995; 47: 177-185.
Chapter 3: Gamma-Delta T cells in CKD 81
14. Kassianos AJ, Wang X, Sampangi S, Muczynski K, Healy H, Wilkinson R.
Increased tubulointerstitial recruitment of human CD141hi CLEC9A+ and
CD1c+ myeloid dendritic cell subsets in renal fibrosis and chronic kidney
disease. Am J Physiol Renal Physiol 2013; 305: F1391-1401.
15. Racusen LC, Solez K, Colvin RB et al. The Banff 97 working classification of
renal allograft pathology. Kidney Int 1999; 55: 713-723.
16. Law BMP, Wilkinson R, Wang X et al. Interferon-g production by
tubulointerstitial human CD56bright natural killer cells contributes to renal
fibrosis and chronic kidney disease progression. Kidney Int 2017; 92: 79-88.
17. Kalyan S, Kabelitz D. Defining the nature of human γδ T cells: a biographical
sketch of the highly empathetic. Cell Mol Immunol 2013; 10: 21-29.
18. Maggi L, Santarlasci V, Capone M et al. CD161 is a marker of all human IL-
17-producing T-cell subsets and is induced by RORC. Eur J Immunol 2010;
40: 2174-2181.
19. Takahashi T, Dejbakhsh-Jones S, Strober S. Expression of CD161 (NKR-P1A)
defines subsets of human CD4 and CD8 T cells with different functional
activities. J Immunol 2006; 176: 211-216.
20. Fergusson JR, Smith KE, Fleming VM et al. CD161 defines a transcriptional
and functional phenotype across distinct human T cell lineages. Cell Rep 2014;
9: 1075-1088.
21. Bedford JJ, Leader JP, Walker RJ. Aquaporin expression in normal human
kidney and in renal disease. J Am Soc Nephrol 2003; 14: 2581-2587.
22. Velden J, Paust HJ, Hoxha E et al. Renal IL-17 expression in human ANCA-
associated glomerulonephritis. Am J Physiol Renal Physiol 2012; 302: F1663-
1673.
23. Crispin JC, Oukka M, Bayliss G et al. Expanded double negative T cells in
patients with systemic lupus erythematosus produce IL-17 and infiltrate the
kidneys. J Immunol 2008; 181: 8761-8766.
24. Zickert A, Amoudruz P, Sundstrom Y, Ronnelid J, Malmstrom V, Gunnarsson
I. IL-17 and IL-23 in lupus nephritis - association to histopathology and
response to treatment. BMC Immunol 2015; 16: 7.
25. Cai Y, Shen X, Ding C et al. Pivotal role of dermal IL-17-producing γδ T cells
in skin inflammation. Immunity 2011; 35: 596-610.
82 Chapter 3: Gamma-Delta T cells in CKD
26. Hvas J, Oksenberg JR, Fernando R, Steinman L, Bernard CC. γδ T cell receptor
repertoire in brain lesions of patients with multiple sclerosis. J Neuroimmunol
1993; 46: 225-234.
27. Laggner U, Di Meglio P, Perera GK et al. Identification of a novel
proinflammatory human skin-homing Vγ9Vδ2 T cell subset with a potential
role in psoriasis. J Immunol 2011; 187: 2783-2793.
28. Giacomelli R, Matucci-Cerinic M, Cipriani P et al. Circulating Vδ1+ T cells
are activated and accumulate in the skin of systemic sclerosis patients. Arthritis
Rheum 1998; 41: 327-334.
29. White B, Yurovsky VV. Oligoclonal expansion of Vδ1+ γδ T-cells in systemic
sclerosis patients. Ann N Y Acad Sci 1995; 756: 382-391.
30. Wu H, Clarkson AR, Knight JF. Restricted γδ T-cell receptor repertoire in IgA
nephropathy renal biopsies. Kidney Int 2001; 60: 1324-1331.
31. Aldemir H, Prod'homme V, Dumaurier MJ et al. Cutting edge: lectin-like
transcript 1 is a ligand for the CD161 receptor. J Immunol 2005; 175: 7791-
7795.
32. Rosen DB, Cao W, Avery DT et al. Functional consequences of interactions
between human NKR-P1A and its ligand LLT1 expressed on activated
dendritic cells and B cells. J Immunol 2008; 180: 6508-6517.
33. Le Bourhis L, Dusseaux M, Bohineust A et al. MAIT cells detect and
efficiently lyse bacterially-infected epithelial cells. PLoS Pathog 2013; 9:
e1003681.
34. Schirmer L, Rothhammer V, Hemmer B, Korn T. Enriched CD161high CCR6+
γδ T cells in the cerebrospinal fluid of patients with multiple sclerosis. JAMA
Neurol 2013; 70: 345-351.
35. Poggi A, Zocchi MR, Costa P et al. IL-12-mediated NKRP1A up-regulation
and consequent enhancement of endothelial transmigration of Vδ2+ TCR γδ+
T lymphocytes from healthy donors and multiple sclerosis patients. J Immunol
1999; 162: 4349-4354.
36. Couzi L, Pitard V, Sicard X et al. Antibody-dependent anti-cytomegalovirus
activity of human γδ T cells expressing CD16 (FcγRIIIa). Blood 2012; 119:
1418-1427.
Chapter 3: Gamma-Delta T cells in CKD 83
37. Chen Z, Freedman MS. CD16+ γδ T cells mediate antibody dependent cellular
cytotoxicity: potential mechanism in the pathogenesis of multiple sclerosis.
Clin Immunol 2008; 128: 219-227.
38. Thedrez A, Harly C, Morice A, Salot S, Bonneville M, Scotet E. IL-21-
mediated potentiation of antitumor cytolytic and proinflammatory responses of
human Vγ9Vδ2 T cells for adoptive immunotherapy. J Immunol 2009; 182:
3423-3431.
39. Alexander AA, Maniar A, Cummings JS et al. Isopentenyl pyrophosphate-
activated CD56+ γδ T lymphocytes display potent antitumor activity toward
human squamous cell carcinoma. Clin Cancer Res 2008; 14: 4232-4240.
40. Correia DV, Fogli M, Hudspeth K, da Silva MG, Mavilio D, Silva-Santos B.
Differentiation of human peripheral blood Vδ1+ T cells expressing the natural
cytotoxicity receptor NKp30 for recognition of lymphoid leukemia cells. Blood
2011; 118: 992-1001.
41. Cortvrindt C, Speeckaert R, Moerman A, Delanghe JR, Speeckaert MM. The
role of interleukin-17A in the pathogenesis of kidney diseases. Pathology
2017; 49: 247-258.
42. Hu C, Qian L, Miao Y et al. Antigen-presenting effects of effector memory
Vγ9Vδ2 T cells in rheumatoid arthritis. Cell Mol Immunol 2012; 9: 245-254.
43. Ge S, Hertel B, Susnik N et al. Interleukin 17 receptor A modulates monocyte
subsets and macrophage generation in vivo. PLoS One 2014; 9: e85461.
44. Van Kooten C, Boonstra JG, Paape ME et al. Interleukin-17 activates human
renal epithelial cells in vitro and is expressed during renal allograft rejection. J
Am Soc Nephrol 1998; 9: 1526-1534.
45. Woltman AM, de Haij S, Boonstra JG, Gobin SJ, Daha MR, van Kooten C.
Interleukin-17 and CD40-ligand synergistically enhance cytokine and
chemokine production by renal epithelial cells. J Am Soc Nephrol 2000; 11:
2044-2055.
46. Wilkinson R, Wang X, Kassianos AJ et al. Laser capture microdissection and
multiplex-tandem PCR analysis of proximal tubular epithelial cell signaling in
human kidney disease. PLoS One 2014; 9: e87345.
47. Chen H, You H, Wang L, Zhang X, Zhang J, He W. Chaperonin-containing T-
complex Protein 1 Subunit zeta Serves as an Autoantigen Recognized by
84 Chapter 3: Gamma-Delta T cells in CKD
Human Vδ2 gammadelta T Cells in Autoimmune Diseases. J Biol Chem 2016;
291: 19985-19993.
48. Farris AB, Ellis CL, Rogers TE, Lawson D, Cohen C, Rosen S. Renal
Medullary and Cortical Correlates in Fibrosis, Epithelial Mass,
Microvascularity, and Microanatomy Using Whole Slide Image Analysis
Morphometry. PLoS One 2016; 11: e0161019.
Chapter 3: Gamma-Delta T cells in CKD 85
TITLE AND LEGENDS
Table 1. Antibodies used for flow cytometric staining.
Antigen Clone Fluorochrome Source
CD45 HI30 Brilliant Violet 510 Biolegend
CD14 M5E2 Alexa Fluor 700 Biolegend
CD3 HIT3a APC BD Biosciences
TCR γδ 11F2 PE-Cy7 BD Biosciences
TCR Vδ1 TS8.2 FITC Thermo Scientific
TCR Vδ2 B6 Brilliant Violet 421 Biolegend
CD161 HP-3G10 Brilliant Violet 785 Biolegend
CD56 HCD56 PerCP/Cy5.5 Biolegend
CD16 3G8 PE-CF594 BD Biosciences
CD336 (NKp44) p44-8 PE BD Biosciences
86 Chapter 3: Gamma-Delta T cells in CKD
Figure 1. Human γδ T cell numbers correlate significantly with loss of kidney
function. (A-B) Absolute numbers of total T cells (A) and γδ T cells (B) in healthy
kidney tissue (n=17) and diseased biopsies with eGFR≥60 (n=20) and eGFR<60
(n=38). Values for individual donors are presented; horizontal bars represent means.
**p<0.01, ***p<0.001, ****p<0.0001, Kruskal-Wallis test with Dunn’s post-test. (C-
D) Spearman correlation analyses of absolute numbers of total T cells (C) and γδ T
cells (D) versus patient eGFR.
Chapter 3: Gamma-Delta T cells in CKD 87
Figure 2. Significantly elevated human γδ T cell numbers in diseased biopsies
with interstitial fibrosis. (A-B) Absolute numbers of total T cells (A) and γδ T cells
(B) in healthy kidney tissue (n=17) and diseased biopsies without (n=27) and with
fibrosis (n=31). Values for individual donors are presented; horizontal bars represent
means. ***p<0.001, ****p<0.0001, Kruskal-Wallis test with Dunn’s post-test. (C-D)
Spearman correlation analyses of absolute numbers of total T cells (C) and γδ T cells
(D) versus percentages of interstitial fibrosis in diseased biopsies.
88 Chapter 3: Gamma-Delta T cells in CKD
Figure 3. Diseased biopsies with interstitial fibrosis have significantly elevated
numbers of Vδ1+ γδ T cells. (A) Dot plot (gated on CD3+ lymphocytes) identifying
Vδ1+ and Vδ2+ γδ T cells in human kidney tissue. Representative dot plot from one of
seven individual fibrotic renal biopsies is presented. An identical gating strategy was
used for healthy kidney tissue and non-fibrotic renal biopsies. (B-C) Absolute numbers
of Vδ1+ γδ T cells (B) and Vδ2+ γδ T cells (C) in healthy kidney tissue and diseased
biopsies without and with fibrosis. Values for individual donors are presented;
horizontal bars represent means. **p<0.01, Kruskal-Wallis test with Dunn’s post-test.
(D-E) Spearman correlation analyses of absolute numbers of Vδ1+ γδ T cells (D) and
Vδ2+ γδ T cells (E) versus percentages of interstitial fibrosis in diseased biopsies.
Chapter 3: Gamma-Delta T cells in CKD 89
Figure 4. Significantly elevated CD161 expression on γδ T cells in diseased
biopsies with interstitial fibrosis. (A-B) Surface expression of CD161 on total T cells
(A) and γδ T cells (B) in healthy kidney tissue (n=5) and diseased biopsies without
(n=6) and with fibrosis (n=16). Values of median fluorescence intensity (MFI) for
individual donors are shown; horizontal bars represent means. *p<0.05, Kruskal-
Wallis test with Dunn’s post-test. (C) Representative histogram of CD161 expression
on γδ T cells from diseased biopsies without and with interstitial fibrosis compared
with isotype control. (D) Surface expression of CD161 on Vδ1+ and Vδ2+ γδ T cells
in fibrotic biopsies. Values of median fluorescence intensity (MFI) for individual
donors are shown, with lines connecting paired samples for each individual donor.
90 Chapter 3: Gamma-Delta T cells in CKD
Figure 5. Human CD161+ γδ T cells in fibrotic kidneys display an innate-like
cytotoxic phenotype. (A-C) Surface expression of cytotoxic markers (CD56, CD16,
NKp44) on CD161+ versus CD161- γδ T cells from fibrotic biopsies. Values of median
fluorescence intensity (MFI) for individual donors are shown, with lines connecting
paired samples for each individual donor. *p<0.05, **p<0.01, Wilcoxon matched-
pairs signed rank test.
Chapter 3: Gamma-Delta T cells in CKD 91
Figure 6. Co-localisation of human γδ T cells with proximal tubular epithelial
cells (PTEC). Immunofluorescent staining of frozen kidney sections from diseased
biopsies without (left panel) and with interstitial fibrosis (middle/right panels) stained
for TCR γδ (red), PTEC marker Aquaporin-1 (white) and DAPI (blue). γδ T cells are
circled (right panel). Scale bars represent 100μm (left/middle panels) and 10μm (right
panel). Representative results for three individual donor experiments are shown.
92 Chapter 3: Gamma-Delta T cells in CKD
Figure 7. Human γδ T cells produce proinflammatory cytokine IL-17A in fibrotic
kidney tissue. Immunofluorescent labelling of frozen fibrotic kidney tissue stained for
Aquaporin-1 (white; first panel), TCR γδ (red; second panel) and IL-17A (green; third
panel). Co-expression of TCR γδ and IL-17A is visualised by the yellow merge of red
and green (fourth panel). IL-17A+ γδ T cells are circled. Scale bars represent 100μm
for large frames and 10μm for insets. Representative results for three individual donor
experiments are shown.
Chapter 3: Gamma-Delta T cells in CKD 93
Supplementary Figure 1. Identification of γδ T cells in human kidney tissue.
Gating strategy used to identify total T cells (CD3+ lymphocytes) and γδ T cells (CD3+
TCR γδ+ lymphocytes) in human kidney tissue. Representative flow cytometric data
from one of 38 individual fibrotic renal biopsies are shown. An identical gating
strategy was used for healthy kidney tissue and non-fibrotic renal biopsies.
94 Chapter 3: Gamma-Delta T cells in CKD
Chapter 4: Mucosal Associated Invariant T cells in CKD 95
Chapter 4: Mucosal Associated Invariant T cells in CKD
Following these NK and γδ T cell studies, I examined the role of
mucosal associated invariant T (MAIT) cells in human CKD. MAIT cells
are also an innate unconventional T lymphocyte population that express a
biased αβ TCR. Due to the inherent rarity of MAIT cells in mice, to date,
there are no adequate animal models available for MAIT cell
characterisation and functional assays. My work on MAIT cells in human
CKD therefore presents the first functional characterisation of MAIT cells
in human kidneys. In addition, unique to my NK and γδ T cell work, in
this MAIT cell study, I examined the interactions of this innate lymphocyte
population with proximal tubule epithelial cells (PTEC) under hypoxia, a
major driver of CKD in humans.
QUT Verified Signature
Chapter 4: Mucosal Associated Invariant T cells in CKD 97
ABSTRACT
Background: Mucosal-associated invariant T (MAIT) cells represent a specialised
lymphocyte population associated with chronic inflammatory disorders. Despite this,
little is known about MAIT cells in diseases of the kidney. Here, we evaluated MAIT
cells in human native kidneys with tubulointerstitial fibrosis, the pathological hallmark
of chronic kidney disease (CKD).
Methods: MAIT cells were identified, enumerated and phenotyped from human
kidney tissue by multi-colour flow cytometry. Localisation of MAIT cells was
performed by immunofluorescence microscopy. MAIT cells and human primary
proximal tubular epithelial cells (PTEC) were co-cultured under hypoxic (1% O2)
conditions to examine mechanistic tubulointerstitial interactions.
Results: MAIT cells (CD3+ TCR Vα7.2+ CD161hi) were identified in healthy and
diseased kidney tissue, with expression of tissue-resident markers (CD103/CD69)
detected on MAIT cells in both states. Enumeration of MAIT cells showed
significantly elevated numbers in diseased biopsies with tubulointerstitial fibrosis
compared with diseased biopsies without fibrosis and healthy tissue. Furthermore,
expression levels of CD69, also an established marker of lymphocyte activation, were
significantly increased on MAIT cells from fibrotic biopsies. Immunofluorescent
analyses of fibrotic kidney tissue identified MAIT cells accumulating adjacent to
PTEC. Notably, MAIT cells activated in the presence of human PTEC under hypoxic
conditions, modelling the fibrotic micro-environment, displayed significantly up-
regulated expression of CD69 and cytotoxic molecules (perforin/granzyme B), with a
corresponding significant increase in PTEC necrosis also observed in these co-
cultures.
Conclusions: Collectively, our data indicate that human tissue-resident MAIT cells in
the kidney may contribute to the fibrotic process of CKD via complex interactions with
PTEC.
98 Chapter 4: Mucosal Associated Invariant T cells in CKD
INTRODUCTION
The global burden of chronic kidney disease (CKD) has risen dramatically in recent
years, largely driven by demographic expansion (population growth and ageing) and
the increased prevalence of diabetes and hypertension worldwide.1 Regardless of its
origins, CKD is characterised pathobiologically by fibrosis within the tubulointerstitial
compartment, the interstitial tissue adjoining the renal tubules. The pathology of
tubulointerstitial fibrosis is underpinned by the sustained presence of inflammatory
immune cells within the local micro-environment.2 Most studies of CKD
immunobiology have focused on conventional T lymphocytes reactive to classical
major histocompatibility complex (MHC)-peptide antigen complexes. However, less
is known about the contribution and function of unconventional T cell subsets in
driving this pathogenic fibrotic process in CKD.
Mucosal-associated invariant T (MAIT) cells are a specialised subset of
unconventional (non-MHC-restricted) T cells that have emerged as key players in
immunity and pathological inflammation. MAIT cells are characterised by a highly
restricted αβ T cell receptor (TCR) that recognises small molecule antigens in the
context of the non-classical MHC-related molecule 1 (MR1).3-5 Human MAIT cells
express a semi-invariant TCRα chain (Vα7.2 coupled with restricted Jα segments)
associated with a limited repertoire of TCRβ chains (including Vβ13 and Vβ2).6 They
are classically defined in humans by their co-expression of TCR Vα7.2 and high levels
of the C-type lectin receptor CD161.7 Human MAIT cells also express several cytokine
receptors under steady state conditions, including IL-7Rα, IL-12R and IL-18Rα,
allowing them to respond to innate interleukins in a TCR-independent manner.8,9 Once
activated in a TCR-dependent and/or –independent manner, MAIT cells display
immediate effector function through the production of cytotoxic effector molecules
(perforin and granzyme B). The majority of MAIT cells in humans also express CD8,10
a signature consistent with their potent cytotoxic activity against target cells.11
MAIT cells are abundant within human peripheral tissues, particularly in the liver and
mucosal tissues, such as lung and gut.12 Indeed, MAIT cells have a distinct chemokine
receptor profile, including CCR5 and CXCR3 expression, consistent with their
capacity to home to peripheral tissues.13 Moreover, MAIT cells expressing markers
compatible with tissue-resident lymphocytes (CD103 and CD69) have been identified
Chapter 4: Mucosal Associated Invariant T cells in CKD 99
in human peripheral tissue,14 highlighting their immunological importance in the local
micro-environment under healthy and diseased conditions. In healthy individuals,
MAIT cells contribute to local protective immunity by maintaining epithelial and
mucosal layer integrity and eliciting anti-microbial responses.8,15,16 However, recent
studies have suggested a pathogenic function for these cells in chronic inflammatory
diseases, with increased numbers of MAIT cells identified in tissue lesions of patients
with inflammatory bowel disease,17 obesity,18 psoriasis,19 multiple sclerosis20 and
rheumatoid arthritis.21 In contrast, their functional contributions to the pathogenesis of
the tubulointerstitial fibrosis of human CKD have not been studied.
The functions of MAIT cells in established mouse models of kidney disease are
unknown because of their low prevalence in common laboratory mouse strains,6
underlining the importance of investigating these unconventional T cells in clinical
(human) bio-specimens. However, human studies have also been hampered by limited
access to kidney tissue samples. To date, MAIT cells have only been detected in human
kidney tissue by the expression of MAIT cell-specific TCR transcripts (Vα7.2-Jα33
and Vα7.2-Jα12).22,23
In this present study, we show that MAIT cells are present in healthy human kidney
tissue and display a tissue-resident phenotype. We demonstrate significantly increased
numbers of MAIT cells in diseased native biopsies with tubulointerstitial fibrosis
compared to non-fibrotic biopsies and healthy kidney tissue. Moreover, the
accumulation of MAIT cells in fibrotic kidneys is restricted to the tubulointerstitial
compartment, often in direct contact with proximal tubular epithelial cells (PTEC).
Critically, our data point to damaged PTEC as critical drivers of MAIT cell activation
and cytotoxicity within the inflammatory/fibrotic micro-environment and thus,
progression to CKD.
100 Chapter 4: Mucosal Associated Invariant T cells in CKD
METHODS
Kidney tissue specimens
Kidney cortical tissue was obtained with informed patient consent from the
macroscopically/microscopically healthy portion of tumour nephrectomies or native
diseased biopsies, following approval by the Royal Brisbane and Women’s Hospital
Human Research Ethics Committee (2002/011 and 2006/072). Healthy cortical tissue
was obtained from 11 donors (3 females/8 males) of mean age 59±9, whilst diseased
clinical biopsies were obtained from 47 donors (18 females/29 males) of mean age
50±17. A range of primary diagnoses were sampled, including 18 glomerular immune-
mediated (lupus nephritis, crescentic glomerulonephritis (GN), membranoproliferative
GN, pauci-immune GN, IgA nephropathy, membranous nephropathy and minimal
change disease), 20 glomerular non-immune-mediated (fibrillary GN, focal segmental
glomerulosclerosis, renal amyloidosis and diabetic nephropathy) and 9 non-glomerular
(interstitial nephritis, arterionephrosclerosis and hypertensive nephropathy) etiologies
(Supplementary Table 1).
Fresh biopsies were taken with a 16-gauge biopsy needle (Biopsybell, Mirandola,
Italy) and immediately divided for: 1) tissue dissociation (1-5mm of a core biopsy); 2)
freezing in Tissue-Tek OCT compound (Sakura, Torrance, CA, USA) for IF analysis;
and 3) fixation in formalin for assessing levels of interstitial fibrosis/tubular atrophy
by renal histopathologists blinded to experimental results. For assessment of renal
interstitial fibrosis, formalin-fixed 4μm sections were stained with Masson's trichrome,
and the proportion of fibrotic area in the cortex was quantified over 20 high-power
fields. Biopsies displaying ≥5% interstitial fibrosis were deemed fibrotic, based on the
Banff 97 working classification of renal pathology.24 According to this criterion,
diseased specimens were then grouped into biopsies without (n=17; 5 females/12
males; mean age 44±17; mean eGFR 69±27 ml/min/1.73m2) or with interstitial fibrosis
(n=30; 13 females/17 males; mean age 53±16; mean eGFR 38±23 ml/min/1.73m2).
Kidney function (estimated glomerular filtration rate; eGFR) was calculated using the
CKD-EPI method by AUSLAB (Queensland Health, Brisbane, Australia).
Tissue dissociation for flow cytometric analysis
Healthy kidney tissue and diseased biopsies were dissociated and processed for flow
cytometric analysis according to our published methodology.25 Briefly, kidney tissue
Chapter 4: Mucosal Associated Invariant T cells in CKD 101
was digested with 1mg/ml collagenase P (Roche, Mannheim, Germany) in the
presence of 20μg/ml DNase I (Roche) (250μl volume) for 15 min. Supernatant from
this initial dissociation step was collected for cytokine analysis. Dissociated tissue was
then further digested with 10μg/ml trypsin/4μg/ml EDTA (Invitrogen, Grand Island,
NY, USA) (500μl volume) for 10 min.
Flow cytometry
Single cell suspensions were initially stained with LIVE/DEAD® Fixable Near-IR
Dead Cell Stain Kit (Life Technologies) to exclude non-viable cells. Cells were then
incubated with Human TruStain FcX™ Blocking Solution (Biolegend, San Diego,
CA, USA) at room temperature for 5-10 min and then stained on ice for 30 min with
combinations of test (Supplementary Table 2) or isotype-matched control antibodies
in cold FACS buffer [0.5% BSA (Sigma, St Louis, MO, USA) and 0.02% sodium
azide (Sigma) in PBS]. Flow-Count Fluorospheres™ (Beckman Coulter, Brea, CA,
USA) were used for direct determination of absolute counts as outlined in our
published methodology.25 Cell acquisition was performed on an LSR Fortessa (BD
Biosciences, San Jose, CA, USA) and data analyzed with FlowJo software (TreeStar,
Ashland, OR, USA).
Cytokine detection
Dissociation supernatants were harvested and levels of soluble proteins were
determined using the LEGENDplex™ Human Inflammation Panel multiplex bead-
based assay (Biolegend) according to the manufacturer’s instructions. Cytokine values
were normalized to pg per cm3 of tissue.
Immunofluorescence staining with tyramide signal amplification (TSA)
Frozen 7μm tissue sections were fixed with 25% ethanol:75% acetone at room
temperature for 5 min. Endogenous peroxidase activity was quenched with
BLOXALL™ Endogenous Peroxidase and Alkaline Phosphatase Blocking Solution
(Vector Laboratories, Burlingame, CA, USA) at room temperature for 15 min,
followed by a protein block with 10% Donkey Serum (Merck-Millipore, Burlington,
MA, USA) at room temperature for 20 min. Sections were sequentially probed with
anti-TCR Vα7.2 (Monoclonal mouse IgG1; Clone 3C10; Biolegend) and anti-
Aquaporin-1 (Rabbit polyclonal IgG; Santa Cruz, Dallas, TX, USA) or isotype-
102 Chapter 4: Mucosal Associated Invariant T cells in CKD
matched control antibodies at room temperature for 30 min. Fluorescent detection was
obtained by incubation with horseradish peroxidase (HRP)-conjugated F(ab’)2
fragment donkey anti-mouse IgG and donkey anti-rabbit IgG secondary antibodies
(both from Jackson ImmunoResearch, West Grove, PA, USA) at room temperature for
20 min, followed by addition of TSA Plus Cyanine 3 (Cy3) and TSA Plus Cyanine 5
(Cy5) (both from Perkin Elmer, Waltham, MA, USA) at room temperature for 10 min.
Nuclei were stained with DAPI (Sigma). Slides were coverslipped in fluorescence
mounting medium (Agilent Technologies, Santa Clara, CA, USA). A Zeiss 780 NLO
confocal microscope (Carl Zeiss, Hamburg, Germany) was used for fluorescence
microscopy. Image acquisition and analysis were performed using ZEN software (Carl
Zeiss). Quantitative analysis of MAIT cells was undertaken from healthy kidney tissue
(n=4), non-fibrotic kidney biopsies (n=6) and fibrotic kidney biopsies (n=5), counting
TCR Vα7.2+ cells in a randomly selected 1mm2 area from four separate slides for each
donor. The final count presented for each donor is the mean value (mean cells/mm2)
from the four separate slides.
Isolation and culture of human primary PTEC
PTEC were purified from the macroscopically/microscopically healthy portion of
tumor nephrectomies following the method of Glynne and Evans26 and cultured in
Defined Medium (DM) as previously described.27 All PTEC underwent no more than
3 passages prior to use in this study.
Hypoxic treatment of human primary PTEC to mimic the fibrotic micro-
environment
PTEC were cultured in DM in 96-well flat-bottom plates to 70-80% confluence. To
prevent further proliferation, PTEC were irradiated with 3000cGy and then further
cultured for 72 hours in 200μl fresh DM for normoxic PTEC (21% O2) or, for hypoxic
PTEC (1% O2), in 200μl fresh DM for 72 hours in an Invivo2 1000 Hypoxia
Workstation (Ruskinn Laftec, Bayswater North, Victoria, Australia). For hypoxic
conditions, DM was pre-treated at 1% O2 for 24 hours prior to use. Expression of
hypoxia inducible factor 1α (HIF-1α) by hypoxic PTEC alone was confirmed by
Western blotting as previously described.28
Human MAIT cell isolation
Chapter 4: Mucosal Associated Invariant T cells in CKD 103
Leukocyte-rich buffy coats were obtained from healthy blood donors (Australian Red
Cross Blood Service). Mononuclear cells were isolated from buffy coats using
SepMate™ isolation tubes (Stemcell Technologies, Vancouver, Canada) and Ficoll-
Paque™ Plus density gradient centrifugation (Amersham Biosciences, Uppsala,
Sweden). MAIT cells were enriched by positive immuno-magnetic selection using the
EasySep™ Human CD8 Positive Selection Kit II (Stemcell Technologies) and were
further purified by staining and flow cytometry sorting of live CD3-FITC+ (BD
Biosciences), CD8-PerCP-Cy5.5+, TCR Vα7.2-Brilliant Violet 605+ and CD161-
Brilliant Violet 785+ (all from Biolegend) events. These procedures routinely yielded
MAIT cell preparations of >99% purity.
PTEC-MAIT cell co-cultures
Human MAIT cells were resuspended at 1x106 cells/ml in Complete Medium (CM)
consisting of RPMI 1640, supplemented with 30% heat-inactivated fetal bovine serum
(FBS), 100U/ml penicillin, 100μg/ml streptomycin, 2mM L-glutamine, 1mM sodium
pyruvate, 0.1mM non-essential amino acids, 10mM HEPES buffer solution (all from
Invitrogen) and 50μM 2-mercaptoethanol (Sigma). MAIT cells (100,000 cells in CM,
100μl volume) were added to the pre-conditioned PTEC (without removal of PTEC
culture medium) and co-cultured for 24 hours under normoxic or hypoxic conditions
(10% FBS final concentration; 300μl final volume). Where indicated, recombinant
human IL-12p70 (10ng/ml; Biolegend), IL-15 (100ng/ml; Biolegend) and IL-18
(50ng/ml; R&D Systems, Minneapolis, MN, USA) were added to cultures.
Culture supernatants were harvested and levels of soluble proteins (perforin and
granzyme B) were determined using the LEGENDplex™ Human CD8/NK Panel
multiplex bead-based assay (Biolegend). Cells were harvested and stained with
LIVE/DEAD® Fixable Near-IR Dead Cell reagent (to exclude dead cells), CD45-
Brilliant Violet 510 (to exclude PTEC) and CD69-PE antibodies (BD Biosciences) or
appropriate isotype controls for flow cytometric assessment of surface antigen
expression on live MAIT cells. A fold change in protein expression under normoxic
or hypoxic conditions was calculated as the value in the presence of PTEC/the value
in the absence of PTEC.
104 Chapter 4: Mucosal Associated Invariant T cells in CKD
PTEC from in vitro experiments were harvested by trypsin treatment and stained for
Annexin-V and propidium iodide (PI) using the Annexin-V Apoptosis Detection kit I
(BD Biosciences) according to the manufacturer’s instructions. The percentage of
Annexin-V+ PI+ necrotic cells was determined by flow cytometry. A fold change in
PTEC death under normoxic or hypoxic conditions was calculated as the value in the
presence of MAIT cells/the value in the absence of MAIT cells.
Statistics
All statistical tests were performed using Prism 7.0 analysis software (GraphPad
Software, La Jolla, CA, USA). Comparisons between paired groups were performed
using a Wilcoxon matched-pairs signed rank test and multiple comparisons were
performed using a Kruskal-Wallis test with Dunn’s post-test. Absolute cell numbers
were correlated with patient eGFR and levels of interstitial fibrosis in diseased biopsies
by Spearman correlation analysis. P values ≤0.05 were considered statistically
significant.
Chapter 4: Mucosal Associated Invariant T cells in CKD 105
RESULTS
Identification of tissue-resident MAIT cells in healthy human kidney tissue.
Healthy kidney tissue was enzymatically digested to obtain single cells for flow
cytometric analysis. Using the gating strategy outlined in Figure 1A, we were able to
separate CD45+ leukocytes into granulocytes with higher side scatter (SSC) and
mononuclear cells. Total T cells were then defined as CD3+ mononuclear cells. Within
this T cell compartment, MAIT cells were identified as a discrete TCR Vα7.2+ CD161hi
population.
Phenotypic analysis showed MAIT cells in healthy human kidney tissue to be
predominantly CD8+ and express signature cytokine receptors IL-18Rα (CD218a) and
IL-7Rα (CD127) and chemokine receptors CCR5 (CD195) and CXCR3 (CD183)
(Figure 1B). In humans, the expression of CD103 and CD69 has been used to
discriminate tissue-resident from circulating lymphocytes.29,30 Notably, expression of
both markers was identified on kidney MAIT cells (Figure 1B). These results
demonstrate, for the first time, that human MAIT cells are present in healthy kidney
tissue and display a tissue-resident phenotype.
Absolute numbers of human MAIT cells correlate significantly with loss of kidney
function.
An identical gating strategy to that used for healthy kidney tissue was applied to
identify MAIT cells in diseased biopsies. Notably, we observed equivalent flow
cytometric profiles in diseased biopsies to those presented in Figure 1A for healthy
kidney tissue (data not shown).
We enumerated MAIT cells in healthy and diseased kidney tissue, with diseased
biopsies stratified based on patient kidney function (eGFR). Results revealed a
significant increase in MAIT cell numbers in diseased biopsies from patients with
reduced kidney function (eGFR<60ml/min/1.73m2) compared with diseased biopsies
from patients with normal kidney function (eGFR≥60ml/min/1.73m2) and healthy
kidney tissue (Figure 2A). Further analysis showed significant negative correlations
between patient eGFR and MAIT cell numbers (r = -0.4669, P=0.0009) (Figure 2B).
106 Chapter 4: Mucosal Associated Invariant T cells in CKD
Significantly elevated numbers of human MAIT cells in diseased biopsies with
interstitial fibrosis.
In addition to kidney function, diseased biopsies were grouped based on the
histological absence or presence of interstitial fibrosis, the characteristic feature of all
patterns of CKD. Significantly elevated numbers of MAIT cells were identified in
diseased biopsies with interstitial fibrosis compared with diseased biopsies without
fibrosis and healthy kidney tissue (Figure 2C). Notably, a significant correlation
between MAIT cell numbers and the degree of interstitial fibrosis was also observed
(Figure 2D). Collectively, these results associate MAIT cells with CKD pathogenesis
and loss of kidney function.
We also correlated MAIT cell numbers to primary diagnoses of patients, with diseased
biopsies stratified into glomerular immune-mediated, glomerular non-immune-
mediated and non-glomerular diseases. However, no significant differences in MAIT
cell numbers between disease groupings were observed (Supplementary Figure 1A).
Significantly elevated CD69 expression on MAIT cells in diseased biopsies with
interstitial fibrosis.
We next examined the phenotypes of kidney MAIT cells in healthy and diseased
kidney tissue. Expression levels of surface markers TCR Vα7.2, CD161, CD8, IL-
18Rα, IL-7Rα, CCR5 and CXCR3 on MAIT cells were comparable between healthy
and diseased kidney tissue (data not shown). As for healthy kidneys, MAIT cells in
diseased kidney biopsies displayed a tissue-resident phenotype, with expression of
both CD103 and CD69 (Figure 3). Whilst CD103 expression levels were similar
between healthy and diseased kidney tissue (Figure 3A), MAIT cell expression of
CD69, a marker of tissue-residence, but also lymphocyte activation, was significantly
elevated in diseased biopsies with interstitial fibrosis compared with non-fibrotic
biopsies (Figure 3B-C). No significant differences in CD103 or CD69 expression
levels were observed when diseased biopsies were stratified based on primary
diagnoses of patients (Supplementary Figure 1B-C). These results indicate that the
local environment within fibrotic kidneys directs MAIT cells toward a more activated
state.
Significantly elevated IL-18 in diseased biopsies with interstitial fibrosis.
Chapter 4: Mucosal Associated Invariant T cells in CKD 107
The supernatant from dissociated tissue samples was analysed for cytokine levels. Pro-
inflammatory cytokines TNF-α (Figure 4A) and IL-1β (Figure 4B) were significantly
elevated in the supernatant of dissociated fibrotic biopsies compared with healthy
tissue. The critical role of innate cytokines, including IL-18, is established in MAIT
cell activation.31 Notably, IL-18 levels were significantly increased in diseased
biopsies with interstitial fibrosis compared with non-fibrotic biopsies (Figure 4C). Our
data suggest the inflammatory micro-environment within fibrotic kidneys contributes
to MAIT cell activation.
Co-localisation of MAIT cells with PTEC in fibrotic kidney tissue
We next examined the localisation of MAIT cells in human kidney tissue using TCR
Vα7.2 immunofluorescent (IF) staining. TCR Vα7.2+ cells were identified in healthy
kidney tissue (Figure 5A), supporting the concept of tissue-resident MAIT cells.
Consistent with our flow cytometric quantitative data, an increased presence of MAIT
cells was detected in diseased biopsies with interstitial fibrosis (Figure 5C-D)
compared with non-fibrotic diseased biopsies (Figure 5B) and healthy kidney tissue
(Figure 5A). Quantitative analysis was performed, confirming a significantly increased
accumulation of MAIT cells in fibrotic biopsies (Figure 5E). Notably, MAIT cells
were identified within the tubulointerstitial compartment, adjacent to PTEC, defined
as tubular cells expressing aquaporin-1.32 Moreover, in fibrotic kidney tissue, MAIT
cells localised to sites of tubulointerstitial injury (inflammation, tubular atrophy),
suggesting these cells are positioned to play a role during fibrotic disease progression.
MAIT cells activated in the presence of hypoxic PTEC produce significantly
increased levels of perforin and granzyme B
Renal hypoxia is an established driver of tubulointerstitial inflammation/fibrosis and
thus, progression to CKD.33 We postulated that the activated phenotype of MAIT cells
in fibrotic kidney tissue may be driven by adjacent PTEC in the hypoxic micro-
environment. Therefore, we established an in vitro co-culture system to model this
human CKD setting and thus, investigate the functional capacity of hypoxic PTEC to
activate MAIT cells.
We isolated human MAIT cells by immuno-magnetic selection and flow cytometry
sorting and examined CD69 expression levels on MAIT cells co-cultured with pre-
conditioned normoxic (21% O2) or hypoxic (1% O2) PTEC in the absence or presence
108 Chapter 4: Mucosal Associated Invariant T cells in CKD
of interleukin (IL-12p70, IL-15, IL-18) stimulation. There was minimal expression of
CD69 on freshly isolated MAIT cells and MAIT cells cultured in the absence of
interleukin stimulation (Figure 6A). MAIT cell expression of CD69 was upregulated
in response to interleukin stimulation, with highest levels detected in hypoxic PTEC
co-cultures (Figure 6A). Over seven individual PTEC-MAIT cell co-culture
experiments, the fold change in MAIT cell CD69 expression (MFI in the presence of
PTEC/MFI in the absence of PTEC) was significantly increased under hypoxic
conditions, but not normoxic conditions (Figure 6B). Moreover, the fold change in
MAIT cell CD69 expression under hypoxic conditions was significantly elevated
compared with normoxic conditions (Figure 6B).
The production of cytotoxic effector molecules (perforin and granzyme B) by MAIT
cells was also examined. MAIT cells activated in the presence of hypoxic PTEC
secreted the greatest levels of both perforin and granzyme B (Figure 7A and 7C).
Notably, the fold change in MAIT cell production of perforin and granzyme B
(concentration in the presence of PTEC/concentration in the absence of PTEC) was
significantly elevated under hypoxic conditions (Figure 7B and 7D). For both
cytotoxic molecules, the fold change under hypoxic conditions was also significantly
increased compared with the fold change under normoxic conditions (Figure 7B and
7D). No significant differences between hypoxic and normoxic conditions were
observed for other detectable analytes (granulysin, granzyme A, IFN-γ, IL-17A) (data
not shown).
Significantly increased PTEC necrosis in inflammatory/hypoxic co-cultures.
We next examined the possible functional role of these cytotoxic molecules in driving
PTEC damage. PTEC injury was assessed in this in vitro co-culture model by Annexin-
V/PI staining, with highest levels of PTEC necrosis (% Annexin-V+ PI+ cells) detected
in hypoxic co-cultures in the presence of interleukin stimulation (Figure 8A-B). The
fold change in PTEC necrosis (% Annexin-V+ PI+ cells in the presence of MAIT
cells/% Annexin-V+ PI+ cells in the absence of MAIT cells) was significantly elevated
under hypoxic conditions, but not normoxic conditions (Figure 8C). Furthermore, the
fold change in PTEC necrosis under hypoxic conditions was significantly elevated
compared with normoxic conditions (Figure 8C). Taken together, these data suggest
Chapter 4: Mucosal Associated Invariant T cells in CKD 109
that hypoxic PTEC activate cytotoxic MAIT cells within the inflammatory/fibrotic
tubulointerstitium, which, in turn, leads to PTEC damage/necrosis.
110 Chapter 4: Mucosal Associated Invariant T cells in CKD
DISCUSSION
MAIT cells have been previously identified in various human peripheral organs, with
preferential enrichment within the liver and at mucosal barriers, including the
gastrointestinal tract.8,10 Initial reports of MAIT cells in human kidney tissue have been
made, although only by detection of MAIT cell-specific TCR transcripts (Vα7.2-Jα33
and Vα7.2-Jα12).22 Our present study is the first to unequivocally identify MAIT cells,
based on cell surface expression of both TCR Vα7.2 and high levels of CD161, in
healthy and diseased human kidney tissue.
Our phenotypic data report human kidney MAIT cells are a population of tissue-
resident lymphocytes. They co-express tissue-residency markers CD103 (the α chain
of the αEβ7 integrin) and CD69 (a type II C-lectin receptor), both molecules directly
involved in tissue accumulation and retention.34,35 Tissue-resident cells represent a
distinct population of lymphocytes that are non-circulating and establish residency in
peripheral tissues under steady-state conditions. These tissue-resident lymphocytes
self-renew independently of circulating precursors and serve as local sentinels in
response to pathogens and stress ligands.36 Indeed, tissue-resident lymphocytes have
been described in almost every peripheral organ, with evidence that residents greatly
outnumber recirculating cells within mouse non-lymphoid tissues, including the
kidney.37
Kidney-residing lymphocytes have been examined in experimental murine studies,38-
43 yet translation of this work to human clinical samples remains limited. A
longitudinal examination of repeat kidney biopsy samples from lupus nephritis (LN)
patients reported the expansion and persistence of individual T cells clones for up to 6
years, providing evidence of tissue-resident lymphocytes in LN pathogenesis.44 We
previously reported the accumulation of IFN-γ-producing CD69+ natural killer (NK)
cells in kidney biopsies with interstitial fibrosis, proposing a functional role for these
putative tissue-resident lymphocytes in human CKD.45 In this present study, we
provide the first evidence of tissue-resident MAIT cells in human kidney tissue, in line
with published reports of tissue-resident MAIT cells in the human liver,46 gastric
mucosa14 and female genital mucosa.47
Chapter 4: Mucosal Associated Invariant T cells in CKD 111
The formation of tissue-resident lymphocytes is largely dependent on local
environmental cues, including cytokines IL-15 and transforming growth factor (TGF)-
β.48,49 In particular, a recent investigation documented a pivotal role for TGF-β in
driving the development of kidney-resident lymphocytes in mice.40 In this study, Ma
et al showed that TGF-β promotes trans-endothelial migration of effector T cells and
thereby, residency in the kidney, by upregulating chemokine receptor CXCR3 on these
lymphocytes.40 In our present study, we have similarly identified strong expression of
CXCR3 on tissue-resident MAIT cells in human kidney tissue. Given the central role
of TGF-β in human kidney homeostasis and disease,50,51 we speculate that an
analogous TGF-β-mediated pathway may promote the extravasation and subsequent
formation of human kidney-resident MAIT cells.
Human MAIT cells have been linked to the immunopathogenesis of non-renal chronic
inflammatory diseases across multiple peripheral organs (gut, skin, brain).12,17,19,20,52
In particular, Hegde et al recently highlighted a pro-fibrogenic role for human MAIT
cells in hepatic fibrosis, the final common pathway for chronic liver injury.53 Here, for
the first time in a renal model, we demonstrate significant correlations between
absolute MAIT cell numbers and both loss of kidney function (lower eGFR) and
histological severity of CKD (levels of interstitial fibrosis) in humans.
Although CD69 is constitutively expressed on resident lymphocytes to limit egress
from peripheral tissues, the molecule is also established as a marker of activation on
effector cells.54 Notably, we detected significantly elevated levels of CD69 on MAIT
cells in diseased biopsies with interstitial fibrosis compared to non-fibrotic biopsies,
with a concomitant increase in pro-inflammatory cytokines TNF-α, IL-1β and IL-18
in fibrotic biopsies. These data are consistent with the concept that the inflammatory
milieu within fibrotic kidneys skews MAIT cells toward an activated phenotype. A
similar concept has been previously reported in patients with systemic lupus
erythematosus (SLE), with the activation status of MAIT cells, based on CD69
expression levels, shown to correlate with disease activity as well as plasma
concentrations of inflammatory cytokines, including IL-18.55
The contribution of cytokine-mediated inflammation in the pathogenesis of CKD is
well established. In particular, the literature ascribes functional roles to innate
112 Chapter 4: Mucosal Associated Invariant T cells in CKD
cytokines IL-12p70, IL-15 and IL-18 in renal pathobiology.56,57 These molecules are
all potent pro-inflammatory cytokines secreted primarily by monocytes/macrophages
and dendritic cells.58 Due to their constitutive expression of interleukin receptors,
MAIT cells can be activated by combinations of these innate cytokines in the absence
of TCR ligation.55,59 In turn, this leads to cytotoxic licensing of MAIT cells.60,61
However, until now, the role of MAIT cells in sensing these pro-inflammatory signals
in CKD has not been evaluated.
In our fibrotic biopsies, MAIT cells were detected in areas of tubulointerstitial injury
(inflammation/fibrosis), often in direct contact with PTEC. Renal hypoxia is a key
pathobiological driver of this tubulointerstitial injury in CKD, driven by a loss of
peritubular capillaries, reduced efficiency of oxygen diffusion and increased oxygen
consumption in the diseased kidney.2,33 PTEC are particularly sensitive to hypoxia due
to their dependence on aerobic oxidative metabolism,62 in turn leading to cellular stress
and initiation of pro-inflammatory responses in the tubulointerstitial compartment.2,28
Thus, in order to define the functional interactions between co-localised PTEC and
MAIT cells in human CKD, we established an in vitro co-culture model under hypoxic
conditions. Importantly, we showed that in association with pro-inflammatory
interleukins, hypoxic PTEC dramatically enhance MAIT cell activation and cytotoxic
licensing (perforin and granzyme B production). Furthermore, we demonstrated
significantly increased PTEC necrosis in this inflammatory/hypoxic co-culture. This
supports the concept that tubulointerstitial MAIT cells are poised to sense PTEC-
derived danger signals in the CKD micro-environment that, in turn, leads to their
activation and cytotoxic activity (PTEC killing).
The perforin-granzyme B cytotoxic pathway has been implicated in the
pathophysiology of chronic inflammatory diseases,63,64 whilst animal models of
kidney disease have highlighted a role for the perforin-granzyme B pathway in
promoting chronic tissue injury.65 Our data provide the first human evidence
suggesting MAIT cells respond to activatory signals from PTEC within the
inflammatory/fibrotic kidney micro-environment by skewing to a cytotoxic (perforin
and granzyme B-producing) phenotype and function. Future studies of PTEC-MAIT
cell interactions will be required to elucidate the relative contribution of TCR-
Chapter 4: Mucosal Associated Invariant T cells in CKD 113
dependent (triggered by MR1-antigen complexes) versus TCR-independent (cytokine-
induced) signalling pathways in this activation process.
In contrast to these inflammatory/hypoxic conditions, we also showed that MAIT cells
activated in the presence of normoxic (healthy) PTEC produce significantly reduced
levels of granzyme B (Figure 7D). These findings are suggestive of an immuno-
inhibitory role for healthy PTEC under homeostatic conditions and are reminiscent of
previous studies from our group demonstrating PTEC-mediated immuno-regulation of
T cell, B cell and dendritic cell function.27,66-68
Collectively, these results provide the first comprehensive characterisation of MAIT
cells in healthy and diseased human kidneys. Our identification of tissue-resident
MAIT cells in healthy kidneys suggests they play an important role in homeostatic
maintenance and protection against microbial infections (e.g. uropathogenic bacteria).
In contrast, under the inflammatory/fibrotic conditions of CKD, we propose that
kidney MAIT cells are activated by immuno-stimulatory danger signals that skew
them towards a more pathogenic functionality (Figure 9). A deeper understanding of
the mechanisms regulating kidney MAIT cell immunity in health and disease is now
essential to stimulate the development of novel therapeutic strategies for the treatment
of CKD. In particular, the cross-talk between these MAIT cells and other discrete
immune cell populations that we have previously identified in inflammatory/fibrotic
CKD (e.g. TGF-β-producing CD1c+ DC;69 IFN-γ-secreting CD56bright NK cells;45 and
IL-17A-expressing γδ T cells70) will be a significant area for future clinical
investigation. Indeed, it will be critical to extend on the limitations of our studies and
establish how these multiple immune cell populations collectively drive
inflammatory/fibrotic CKD. Further functional examination of these distinct human
immune cell populations (including MAIT cells) in renal fibrosis will be necessary to
unequivocally define the unique, non-redundant role of MAIT cells in CKD
pathogenesis.
114 Chapter 4: Mucosal Associated Invariant T cells in CKD
AUTHOR CONTRIBUTIONS
Each author has participated sufficiently in the work to take public responsibility for
the content. B.L., R.W., K.W.B., J.U., H.H. and A.J.K. conceived and designed the
study; B.L., X.W., K.K., K.G. and A.J.K. carried out experiments and analysed the
data; B.L., R.W., H.H. and A.J.K. drafted the paper; all authors revised and approved
the final version of the manuscript.
DISCLOSURE
All the authors declared no competing interests.
ACKNOWLEDGEMENTS
The work was funded by Pathology Queensland, a Royal Brisbane and Women’s
Hospital (RBWH) Research Grant, the Kidney Research Foundation and National
Health and Medical Research Council (NHMRC) Project Grants (GNT1099222 and
GNT1161319). BL was supported by a Pathology Queensland – Study, Education and
Research Committee (SERC) PhD Scholarship. KG was supported by an Australian
Government Research Training Program (RTP) Scholarship. The authors would like
to thank the tissue donors and clinicians, particularly renal histopathologist, Dr Leo
Francis (Queensland Health), for assessment of interstitial fibrosis levels in kidney
biopsies.
Chapter 4: Mucosal Associated Invariant T cells in CKD 115
REFERENCES
1. Xie Y, Bowe B, Mokdad AH, Xian H, Yan Y, Li T et al.: Analysis of the Global
Burden of Disease study highlights the global, regional, and national trends of
chronic kidney disease epidemiology from 1990 to 2016. Kidney Int 94: 567-
581, 2018
2. Kawakami T, Mimura I, Shoji K, Tanaka T, Nangaku M: Hypoxia and fibrosis
in chronic kidney disease: crossing at pericytes. Kidney Int., Suppl. [Mini
Review] 4: 107-112, 2014
3. Huang S, Gilfillan S, Cella M, Miley MJ, Lantz O, Lybarger L et al.: Evidence
for MR1 antigen presentation to mucosal-associated invariant T cells. J Biol
Chem 280: 21183-21193, 2005
4. Miley MJ, Truscott SM, Yu YY, Gilfillan S, Fremont DH, Hansen TH et al.:
Biochemical features of the MHC-related protein 1 consistent with an
immunological function. J Immunol 170: 6090-6098, 2003
5. Treiner E, Duban L, Bahram S, Radosavljevic M, Wanner V, Tilloy F et al.:
Selection of evolutionarily conserved mucosal-associated invariant T cells by
MR1. Nature 422: 164-169, 2003
6. Tilloy F, Treiner E, Park SH, Garcia C, Lemonnier F, de la Salle H et al.: An
invariant T cell receptor alpha chain defines a novel TAP-independent major
histocompatibility complex class Ib-restricted alpha/beta T cell subpopulation
in mammals. J Exp Med 189: 1907-1921, 1999
7. Martin E, Treiner E, Duban L, Guerri L, Laude H, Toly C et al.: Stepwise
development of MAIT cells in mouse and human. PLoS Biol 7: e54, 2009
8. Dusseaux M, Martin E, Serriari N, Peguillet I, Premel V, Louis D et al.: Human
MAIT cells are xenobiotic-resistant, tissue-targeted, CD161hi IL-17-secreting
T cells. Blood 117: 1250-1259, 2011
9. Turtle CJ, Delrow J, Joslyn RC, Swanson HM, Basom R, Tabellini L et al.:
Innate signals overcome acquired TCR signaling pathway regulation and
govern the fate of human CD161(hi) CD8alpha(+) semi-invariant T cells.
Blood 118: 2752-2762, 2011
10. Reantragoon R, Corbett AJ, Sakala IG, Gherardin NA, Furness JB, Chen Z et
al.: Antigen-loaded MR1 tetramers define T cell receptor heterogeneity in
mucosal-associated invariant T cells. J Exp Med 210: 2305-2320, 2013
116 Chapter 4: Mucosal Associated Invariant T cells in CKD
11. Le Bourhis L, Dusseaux M, Bohineust A, Bessoles S, Martin E, Premel V et
al.: MAIT cells detect and efficiently lyse bacterially-infected epithelial cells.
PLoS Pathog 9: e1003681, 2013
12. Kumar V, Ahmad A: Role of MAIT cells in the immunopathogenesis of
inflammatory diseases: New players in old game. Int Rev Immunol 37: 90-110,
2018
13. Dias J, Leeansyah E, Sandberg JK: Multiple layers of heterogeneity and subset
diversity in human MAIT cell responses to distinct microorganisms and to
innate cytokines. Proc Natl Acad Sci U S A 114: E5434-E5443, 2017
14. Booth JS, Salerno-Goncalves R, Blanchard TG, Patil SA, Kader HA, Safta AM
et al.: Mucosal-Associated Invariant T Cells in the Human Gastric Mucosa and
Blood: Role in Helicobacter pylori Infection. Front Immunol 6: 466, 2015
15. Kurioka A, Walker LJ, Klenerman P, Willberg CB: MAIT cells: new guardians
of the liver. Clin Transl Immunology 5: e98, 2016
16. Le Bourhis L, Martin E, Peguillet I, Guihot A, Froux N, Core M et al.:
Antimicrobial activity of mucosal-associated invariant T cells. Nat Immunol
11: 701-708, 2010
17. Serriari NE, Eoche M, Lamotte L, Lion J, Fumery M, Marcelo P et al.: Innate
mucosal-associated invariant T (MAIT) cells are activated in inflammatory
bowel diseases. Clin Exp Immunol 176: 266-274, 2014
18. Magalhaes I, Pingris K, Poitou C, Bessoles S, Venteclef N, Kiaf B et al.:
Mucosal-associated invariant T cell alterations in obese and type 2 diabetic
patients. J Clin Invest 125: 1752-1762, 2015
19. Teunissen MBM, Yeremenko NG, Baeten DLP, Chielie S, Spuls PI, de Rie
MA et al.: The IL-17A-producing CD8+ T-cell population in psoriatic lesional
skin comprises mucosa-associated invariant T cells and conventional T cells. J
Invest Dermatol 134: 2898-2907, 2014
20. Willing A, Leach OA, Ufer F, Attfield KE, Steinbach K, Kursawe N et al.:
CD8(+) MAIT cells infiltrate into the CNS and alterations in their blood
frequencies correlate with IL-18 serum levels in multiple sclerosis. Eur J
Immunol 44: 3119-3128, 2014
21. Cho YN, Kee SJ, Kim TJ, Jin HM, Kim MJ, Jung HJ et al.: Mucosal-associated
invariant T cell deficiency in systemic lupus erythematosus. J Immunol 193:
3891-3901, 2014
Chapter 4: Mucosal Associated Invariant T cells in CKD 117
22. Lepore M, Kalinichenko A, Colone A, Paleja B, Singhal A, Tschumi A et al.:
Parallel T-cell cloning and deep sequencing of human MAIT cells reveal stable
oligoclonal TCRbeta repertoire. Nat Commun 5: 3866, 2014
23. Peterfalvi A, Gomori E, Magyarlaki T, Pal J, Banati M, Javorhazy A et al.:
Invariant Valpha7.2-Jalpha33 TCR is expressed in human kidney and brain
tumors indicating infiltration by mucosal-associated invariant T (MAIT) cells.
Int Immunol 20: 1517-1525, 2008
24. Racusen LC, Solez K, Colvin RB, Bonsib SM, Castro MC, Cavallo T et al.:
The Banff 97 working classification of renal allograft pathology. Kidney Int
55: 713-723, 1999
25. Kildey K, Law B, Muczynski K, Wilkinson R, Healy H, Kassianos A:
Identification and quantitation of leukocyte populations in human kidney tissue
by multi-parameter flow cytometry. Bio-protocol 8: e2980, 2018
26. Glynne PA, Evans TJ: Inflammatory cytokines induce apoptotic and necrotic
cell shedding from human proximal tubular epithelial cell monolayers. Kidney
Int 55: 2573-2597, 1999
27. Kassianos AJ, Sampangi S, Wang X, Roper KE, Beagley K, Healy H et al.:
Human proximal tubule epithelial cells modulate autologous dendritic cell
function. Nephrol Dial Transplant 28: 303-312, 2013
28. Wang X, Wilkinson R, Kildey K, Potriquet J, Mulvenna J, Lobb RJ et al.:
Unique molecular profile of exosomes derived from primary human proximal
tubular epithelial cells under diseased conditions. J Extracell Vesicles 6:
1314073, 2017
29. Sathaliyawala T, Kubota M, Yudanin N, Turner D, Camp P, Thome JJ et al.:
Distribution and compartmentalization of human circulating and tissue-
resident memory T cell subsets. Immunity 38: 187-197, 2013
30. Thome JJ, Yudanin N, Ohmura Y, Kubota M, Grinshpun B, Sathaliyawala T
et al.: Spatial map of human T cell compartmentalization and maintenance over
decades of life. Cell 159: 814-828, 2014
31. Xiao X, Cai J: Mucosal-Associated Invariant T Cells: New Insights into
Antigen Recognition and Activation. Front Immunol 8: 1540, 2017
32. Bedford JJ, Leader JP, Walker RJ: Aquaporin expression in normal human
kidney and in renal disease. J Am Soc Nephrol 14: 2581-2587, 2003
118 Chapter 4: Mucosal Associated Invariant T cells in CKD
33. Mimura I, Nangaku M: The suffocating kidney: tubulointerstitial hypoxia in
end-stage renal disease. Nat Rev Nephrol 6: 667-678, 2010
34. Mackay LK, Braun A, Macleod BL, Collins N, Tebartz C, Bedoui S et al.:
Cutting edge: CD69 interference with sphingosine-1-phosphate receptor
function regulates peripheral T cell retention. J Immunol 194: 2059-2063, 2015
35. Hadley GA, Bartlett ST, Via CS, Rostapshova EA, Moainie S: The epithelial
cell-specific integrin, CD103 (alpha E integrin), defines a novel subset of
alloreactive CD8+ CTL. J Immunol 159: 3748-3756, 1997
36. Fan X, Rudensky AY: Hallmarks of Tissue-Resident Lymphocytes. Cell 164:
1198-1211, 2016
37. Steinert EM, Schenkel JM, Fraser KA, Beura LK, Manlove LS, Igyarto BZ et
al.: Quantifying Memory CD8 T Cells Reveals Regionalization of
Immunosurveillance. Cell 161: 737-749, 2015
38. Frost EL, Kersh AE, Evavold BD, Lukacher AE: Cutting Edge: Resident
Memory CD8 T Cells Express High-Affinity TCRs. J Immunol 195: 3520-
3524, 2015
39. Casey KA, Fraser KA, Schenkel JM, Moran A, Abt MC, Beura LK et al.:
Antigen-independent differentiation and maintenance of effector-like resident
memory T cells in tissues. J Immunol 188: 4866-4875, 2012
40. Ma C, Mishra S, Demel EL, Liu Y, Zhang N: TGF-beta Controls the Formation
of Kidney-Resident T Cells via Promoting Effector T Cell Extravasation. J
Immunol 198: 749-756, 2017
41. Victorino F, Sojka DK, Brodsky KS, McNamee EN, Masterson JC, Homann
D et al.: Tissue-Resident NK Cells Mediate Ischemic Kidney Injury and Are
Not Depleted by Anti-Asialo-GM1 Antibody. J Immunol 195: 4973-4985,
2015
42. Skon CN, Lee JY, Anderson KG, Masopust D, Hogquist KA, Jameson SC:
Transcriptional downregulation of S1pr1 is required for the establishment of
resident memory CD8+ T cells. Nat Immunol 14: 1285-1293, 2013
43. Mackay LK, Minnich M, Kragten NA, Liao Y, Nota B, Seillet C et al.: Hobit
and Blimp1 instruct a universal transcriptional program of tissue residency in
lymphocytes. Science 352: 459-463, 2016
44. Winchester R, Wiesendanger M, Zhang HZ, Steshenko V, Peterson K,
Geraldino-Pardilla L et al.: Immunologic characteristics of intrarenal T cells:
Chapter 4: Mucosal Associated Invariant T cells in CKD 119
trafficking of expanded CD8+ T cell beta-chain clonotypes in progressive
lupus nephritis. Arthritis Rheum 64: 1589-1600, 2012
45. Law BMP, Wilkinson R, Wang X, Kildey K, Lindner M, Rist MJ et al.:
Interferon-g production by tubulointerstitial human CD56bright natural killer
cells contributes to renal fibrosis and chronic kidney disease progression.
Kidney Int 92: 79-88, 2017
46. Tang XZ, Jo J, Tan AT, Sandalova E, Chia A, Tan KC et al.: IL-7 licenses
activation of human liver intrasinusoidal mucosal-associated invariant T cells.
J Immunol 190: 3142-3152, 2013
47. Gibbs A, Leeansyah E, Introini A, Paquin-Proulx D, Hasselrot K, Andersson
E et al.: MAIT cells reside in the female genital mucosa and are biased towards
IL-17 and IL-22 production in response to bacterial stimulation. Mucosal
Immunol 10: 35-45, 2017
48. Mackay LK, Wynne-Jones E, Freestone D, Pellicci DG, Mielke LA, Newman
DM et al.: T-box Transcription Factors Combine with the Cytokines TGF-beta
and IL-15 to Control Tissue-Resident Memory T Cell Fate. Immunity 43: 1101-
1111, 2015
49. Mackay LK, Rahimpour A, Ma JZ, Collins N, Stock AT, Hafon ML et al.: The
developmental pathway for CD103(+)CD8+ tissue-resident memory T cells of
skin. Nat Immunol 14: 1294-1301, 2013
50. Sureshbabu A, Muhsin SA, Choi ME: TGF-beta signaling in the kidney:
profibrotic and protective effects. Am J Physiol Renal Physiol 310: F596-F606,
2016
51. Kassianos AJ, Wang X, Sampangi S, Muczynski K, Healy H, Wilkinson R:
Increased tubulointerstitial recruitment of human CD141hi CLEC9A+ and
CD1c+ myeloid dendritic cell subsets in renal fibrosis and chronic kidney
disease. Am J Physiol Renal Physiol 305: F1391-1401, 2013
52. Haga K, Chiba A, Shibuya T, Osada T, Ishikawa D, Kodani T et al.: MAIT
cells are activated and accumulated in the inflamed mucosa of ulcerative
colitis. J Gastroenterol Hepatol 31: 965-972, 2016
53. Hegde P, Weiss E, Paradis V, Wan J, Mabire M, Sukriti S et al.: Mucosal-
associated invariant T cells are a profibrogenic immune cell population in the
liver. Nat Commun 9: 2146, 2018
120 Chapter 4: Mucosal Associated Invariant T cells in CKD
54. Simms PE, Ellis TM: Utility of flow cytometric detection of CD69 expression
as a rapid method for determining poly- and oligoclonal lymphocyte activation.
Clin Diagn Lab Immunol 3: 301-304, 1996
55. Chiba A, Tamura N, Yoshikiyo K, Murayama G, Kitagaichi M, Yamaji K et
al.: Activation status of mucosal-associated invariant T cells reflects disease
activity and pathology of systemic lupus erythematosus. Arthritis Res Ther 19:
58, 2017
56. Kitching AR, Turner AL, Wilson GR, Semple T, Odobasic D, Timoshanko JR
et al.: IL-12p40 and IL-18 in crescentic glomerulonephritis: IL-12p40 is the
key Th1-defining cytokine chain, whereas IL-18 promotes local inflammation
and leukocyte recruitment. J Am Soc Nephrol 16: 2023-2033, 2005
57. Azzi S, Gallerne C, Romei C, Le Coz V, Gangemi R, Khawam K et al.: Human
Renal Normal, Tumoral, and Cancer Stem Cells Express Membrane-Bound
Interleukin-15 Isoforms Displaying Different Functions. Neoplasia 17: 509-
517, 2015
58. Carroll HP, Paunovic V, Gadina M: Signalling, inflammation and arthritis:
Crossed signals: the role of interleukin-15 and -18 in autoimmunity.
Rheumatology (Oxford) 47: 1269-1277, 2008
59. Ussher JE, Bilton M, Attwod E, Shadwell J, Richardson R, de Lara C et al.:
CD161++ CD8+ T cells, including the MAIT cell subset, are specifically
activated by IL-12+IL-18 in a TCR-independent manner. Eur J Immunol 44:
195-203, 2014
60. Sattler A, Dang-Heine C, Reinke P, Babel N: IL-15 dependent induction of IL-
18 secretion as a feedback mechanism controlling human MAIT-cell effector
functions. Eur J Immunol 45: 2286-2298, 2015
61. Kurioka A, Ussher JE, Cosgrove C, Clough C, Fergusson JR, Smith K et al.:
MAIT cells are licensed through granzyme exchange to kill bacterially
sensitized targets. Mucosal Immunol 8: 429-440, 2015
62. Epstein FH: Oxygen and renal metabolism. Kidney International 51: 381-385,
1997
63. Hiebert PR, Granville DJ: Granzyme B in injury, inflammation, and repair.
Trends Mol Med 18: 732-741, 2012
Chapter 4: Mucosal Associated Invariant T cells in CKD 121
64. Boivin WA, Cooper DM, Hiebert PR, Granville DJ: Intracellular versus
extracellular granzyme B in immunity and disease: challenging the dogma. Lab
Invest 89: 1195-1220, 2009
65. Fujinaka H, Yamamoto T, Feng L, Nameta M, Garcia G, Chen S et al.: Anti-
perforin antibody treatment ameliorates experimental crescentic
glomerulonephritis in WKY rats. Kidney Int 72: 823-830, 2007
66. Wilkinson R, Wang X, Roper KE, Healy H: Activated human renal tubular
cells inhibit autologous immune responses. Nephrol Dial Transplant 26: 1483-
1492, 2011
67. Sampangi S, Kassianos AJ, Wang X, Beagley KW, Klein T, Afrin S et al.: The
Mechanisms of Human Renal Epithelial Cell Modulation of Autologous
Dendritic Cell Phenotype and Function. PLoS One 10: e0134688, 2015
68. Sampangi S, Wang X, Beagley KW, Klein T, Afrin S, Healy H et al.: Human
proximal tubule epithelial cells modulate autologous B-cell function. Nephrol
Dial Transplant 30: 1674-1683, 2015
69. Kassianos AJ, Wang X, Sampangi S, Afrin S, Wilkinson R, Healy H:
Fractalkine-CX3CR1-dependent recruitment and retention of human CD1c
myeloid dendritic cells by in vitro-activated proximal tubular epithelial cells.
Kidney Int 87: 1153-1163, 2015
70. Law BM, Wilkinson R, Wang X, Kildey K, Lindner M, Beagley K et al.:
Effector gammadelta T cells in human renal fibrosis and chronic kidney
disease. Nephrol Dial Transplant 34: 40-48, 2019
122 Chapter 4: Mucosal Associated Invariant T cells in CKD
TABLE AND FIGURE LEGENDS
Figure 1. Identification and phenotyping of MAIT cells in healthy human kidney
tissue. (A) Gating strategy used to identify MAIT cells (CD3+ TCR Vα7.2+ CD161hi
mononuclear cells) in healthy human kidney tissue. Representative flow cytometric
data from one of 11 individual donors are shown. (B) Relative expression of TCR
Vα7.2, CD161, CD8, IL-18Rα, IL-7Rα, CCR5, CXCR3, CD103 and CD69 by MAIT
cells (black unfilled) and total T cells (grey filled) compared to isotype (dashed).
Representative data from 4-11 individual donors are shown.
Chapter 4: Mucosal Associated Invariant T cells in CKD 123
Figure 2. MAIT cell numbers correlate significantly with loss of kidney function
and degree of interstitial fibrosis. (A) Absolute numbers of MAIT cells in healthy
kidney tissue and diseased biopsies with eGFR≥60 and eGFR<60. Values for
individual donors are presented; horizontal bars represent medians, with interquartile
range also presented. *p<0.05, **p<0.01, Kruskal-Wallis test with Dunn’s post-test.
(B) Spearman correlation analysis of absolute numbers of MAIT cells versus patient
eGFR. (C) Absolute numbers of MAIT cells in healthy kidney tissue and diseased
biopsies without and with fibrosis. Values for individual donors are presented;
horizontal bars represent medians, with interquartile range also presented. *p<0.05,
**p<0.01, Kruskal-Wallis test with Dunn’s post-test. (D) Spearman correlation
analysis of absolute numbers of MAIT cells versus percentages of interstitial fibrosis
in diseased biopsies.
124 Chapter 4: Mucosal Associated Invariant T cells in CKD
Figure 3. Activated MAIT cells in diseased biopsies with interstitial fibrosis. (A-
B) Surface expression of CD103 (A) and CD69 (B) on MAIT cells in healthy kidney
tissue and diseased biopsies without and with fibrosis. Values of median fluorescence
intensity (MFI) for individual donors are shown; horizontal bars represent medians,
with interquartile range also presented. *p<0.05, Kruskal-Wallis test with Dunn’s post-
test. (C) Representative histogram of CD69 expression on MAIT cells from healthy
kidney tissue (black unfilled) and diseased biopsies without (grey filled) and with
(black filled) interstitial fibrosis compared to isotype (dashed).
Chapter 4: Mucosal Associated Invariant T cells in CKD 125
Figure 4. Significantly elevated pro-inflammatory cytokines in diseased biopsies
with interstitial fibrosis. (A-C) Levels of TNF-α (A), IL-1β (B) and IL-18 (C) in the
supernatant of dissociated healthy kidney tissue (n=10) and diseased biopsies stratified
based on the absence (n=11) or presence of interstitial fibrosis (n=19). Values for
individual donors are presented; horizontal bars represent medians, with interquartile
range also presented. *p<0.05, Kruskal-Wallis test with Dunn’s post-test.
126 Chapter 4: Mucosal Associated Invariant T cells in CKD
Figure 5. Co-localisation of human MAIT cells with proximal tubular epithelial
cells (PTEC). Immunofluorescent staining of frozen sections from healthy kidney
tissue (A), non-fibrotic diseased kidney tissue (B) and fibrotic diseased kidney tissue
(C-D) stained for TCR Vα7.2 (red), Aquaporin-1 (white) and DAPI (blue). MAIT cells
are circled. Scale bars represent 100μm (A-C) and 20μm (D). (E) Quantification of
MAIT cells from healthy kidney tissue (n=4), non-fibrotic kidney biopsies (n=6) and
fibrotic kidney biopsies (n=5). Values for individual donors are presented; horizontal
bars represent medians, with interquartile range also presented. **p<0.01, Kruskal-
Wallis test with Dunn’s post-test.
Chapter 4: Mucosal Associated Invariant T cells in CKD 127
Figure 6. MAIT cell activation in the presence of hypoxic PTEC. (A) CD69
expression by MAIT cells freshly isolated (0h) or after 24 hour culture without (-) or
with (+) pre-conditioned PTEC under normoxic or hypoxic conditions in the absence
(24h) or presence (24h Interleukins) of IL-12p70, IL-15, IL-18. Surface expression
was measured by flow cytometry (gated on live, single, CD45+ cells) and expressed as
the median fluorescence intensity (MFI). One representative donor experiment is
shown. (B) Fold changes (MFI in the presence of PTEC/MFI in the absence of PTEC;
+/- PTEC) in CD69 levels on interleukin-stimulated MAIT cells under normoxic and
hypoxic conditions for seven individual donor PTEC experiments. Symbols represent
individual donor PTEC experiments; the representative donor experiment from Figure
6A is identified using open circles. Horizontal bars represent medians, with
interquartile range also presented. *p<0.05, Wilcoxon matched-pairs signed-rank test.
128 Chapter 4: Mucosal Associated Invariant T cells in CKD
Figure 7. MAIT cells activated in the presence of hypoxic PTEC produce
significantly increased levels of cytotoxic molecules. (A, C) Perforin (A) and
granzyme B (C) production by MAIT cells following 24 hour culture without (-) or
with (+) pre-conditioned PTEC under normoxic or hypoxic conditions in the absence
(24h) or presence (24h Interleukins) of IL-12p70, IL-15, IL-18. One representative
donor experiment is shown. (B, D) Fold changes (concentration in the presence of
PTEC/concentration in the absence of PTEC; +/- PTEC) in cytotoxic molecule
production by interleukin-stimulated MAIT cells under normoxic and hypoxic
conditions for seven individual donor PTEC experiments. Symbols represent
individual donor PTEC experiments; the representative donor experiment from Figure
7A and 7C is identified using filled triangles. Horizontal bars represent medians, with
interquartile range also presented. *p<0.05, Wilcoxon matched-pairs signed-rank test.
Chapter 4: Mucosal Associated Invariant T cells in CKD 129
Figure 8. Significantly increased PTEC death in inflammatory/hypoxic co-
cultures. (A-B) PTEC viability after 24 hour culture without (-) or with (+) MAIT
cells under normoxic or hypoxic conditions in the absence (24h) or presence (24h
Interleukins) of IL-12p70, IL-15, IL-18. The percentage of Annexin-V+ propidium
iodide+ necrotic cells was determined by flow cytometry. Results from one
representative donor experiment are presented in the dot plots (A) and by bar graph
(B). (C) Fold changes (% Annexin-V+ PI+ cells in the presence of MAIT cells/%
Annexin-V+ PI+ cells in the absence of MAIT cells) in PTEC necrosis (in the presence
of interleukins) under normoxic and hypoxic conditions for six individual donor PTEC
experiments. Symbols represent individual donor PTEC experiments; the
representative donor experiment from Figure 8A-B is identified using open circles.
Horizontal bars represent medians, with interquartile range also presented. *p<0.05,
Wilcoxon matched-pairs signed-rank test.
130 Chapter 4: Mucosal Associated Invariant T cells in CKD
Supplementary Table 1. Clinical and histological features of patients at the time of
kidney biopsy.
Chapter 4: Mucosal Associated Invariant T cells in CKD 131
Supplementary Table 2. Antibodies used for flow cytometric staining.
132 Chapter 4: Mucosal Associated Invariant T cells in CKD
Supplementary Figure 1. MAIT cell numbers and phenotype do not significantly
correlate with primary diagnoses of patients. Absolute numbers of MAIT cells (A),
and MAIT cell expression of CD103 (B) and CD69 (C) in healthy kidney tissue and
diseased biopsies with glomerular immune-mediated (GI), glomerular non-immune-
mediated (GNI) and non-glomerular (NG) primary diagnoses. Values for individual
donors are presented; horizontal bars represent medians, with interquartile range also
presented.
Chapter 4: Mucosal Associated Invariant T cells in CKD 133
134 Chapter 4: Mucosal Associated Invariant T cells in CKD
Chapter 5: General Discussion 135
Chapter 5: General Discussion
Chronic kidney disease (CKD) continues to be a pathophysiological
conundrum within the field of nephrology. Although there is ongoing
progress in the area of detecting and diagnosing human CKD, the
pathophysiological process/es behind the disease remain elusive,
hampering the clinical development of effective diagnostics and therapies.
As such, research groups have focused on animal models to elucidate the
pathogenesis of CKD, with much attention focused on the roles of
lymphocytes.1
However, although informative, the inherent differences in mouse
and human biology will always remain a barrier to the direct translation of
these findings to humans. In addition, for some innate lymphocytes, such
as MAIT cells, there are limited animal models currently available that are
suitable. Therefore, the overarching aim of my PhD project was to uncover
the functions of human immune cells of the innate lymphocyte lineage
using ex vivo, in situ and in vitro analyses of clinical specimens from a
cohort of CKD patients. The collective findings of this PhD thesis,
including the identification of activated innate lymphocytes and their
mechanistic roles in kidney diseases, offer novel diagnostic and
therapeutic targets for the treatment of CKD.
136 Chapter 5: General Discussion
SUMMARY OF FINDINGS
The series of three papers that comprises my PhD thesis are the first to show the
potential functional roles of kidney innate lymphocytes and their associations with
human CKD. Based on the findings of this thesis, it is tempting to speculate that innate
lymphocyte subsets, NK, γδ T and MAIT cells, play a pathogenic role in CKD. Firstly,
I demonstrated significant associations between absolute numbers of these cells in
kidney biopsy specimens and both aggravated tissue pathology (levels of interstitial
fibrosis) and reduced kidney function (decreased eGFR). I observed that the intrarenal
innate lymphocytes studied in this project were exclusively localised in the
tubulointerstitium, with each possessing dedicated cytokine or cytotoxic molecule
profiles capable of causing direct or indirect damage to the kidney. Finally, this thesis
has presented the first human evidence that: NK cell subset, CD56bright NK cells, are a
significant producer of IFN-γ in fibrotic kidneys (Chapter 2); γδ T cells display a
cytotoxic phenotype and are a source of IL-17A in the tubulointerstitium (Chapter 3);
and MAIT cells have the capacity to develop into cytotoxic effector cells that can
secrete perforin and granzyme B in the CKD microenvironment (Chapter 4).
Human NK cell subsets can be identified based on the density of CD56
expression on their cell surface, as well as the presence or absence of CD16. The work
presented in this thesis is the first to identify human NK cell subsets, cytokine-
producing CD56bright and cytotoxic CD56dim NK cells, in diseased human kidneys.
Results from this PhD suggest that kidney NK cells play a critical role in driving
disease progression via the production of inflammatory cytokines, rather than acting
as cytotoxic effector cells. This is supported by the following findings: 1. Only
CD56bright NK cells greatly associated with worsening of renal pathology and function;
2. CD56bright NK cells were the predominant NK cell subpopulation in fibrotic biopsies
compared to non-fibrotic and healthy kidney tissue; 3. CD56bright NK cells expressed
CD69 in fibrotic kidneys, an effector phenotype observed in activated lymphocytes; 4.
CD56bright NK cells were major producers of IFN-γ in fibrotic kidneys. Taken together,
of the two NK cell subsets, I speculate that CD56bright NK cells are the key human NK
cell subset that contributes to CKD progression and therefore, should be targeted for
the treatment of CKD.
In this PhD, I also explored the functions of innate lymphocytes of the T cell
lineage, including γδ T and MAIT cells. The ex vivo evaluation of γδ T cells in healthy
Chapter 5: General Discussion 137
and diseased kidney tissue showed an increased accumulation of γδ T cells in fibrotic
biopsies. Further examination revealed γδ T cells in fibrotic kidney biopsies exhibit a
cytotoxic phenotype and are a source of IL-17A. Although γδ T cells have been
previously implicated as drivers of pathology in experimental models of renal
disease,2–6 my data is the first to show the pathogenic potential of γδ T cells in human
CKD.
MAIT cell were the final population of unconventional T lymphocytes studied
in this project. MAIT cells have only been previously identified in human kidneys
through the detection of specific TCR transcripts (Vα7.2-Jα33 and Vα7.2-Jα12).7,8
Furthermore, their role in CKD has been elusive, until now, due to a lack of suitable
animal models.9 In this PhD, I have provided the first evidence to unequivocally show
that MAIT cells, defined by their co-expression of TCR Vα7.2 and high levels of
CD161, are located in the tubulointerstitium of healthy and diseased human kidneys.
Furthermore, I showed that MAIT cells have enhanced activation (increased CD69)
and killing capacity (perforin/granzyme B production) when cultured with PTEC
under inflammatory/hypoxic conditions reminiscent of the CKD micro-environment.
Taken together, my results suggest that tubulointerstitial MAIT cells are a significant
driver of tissue injury in CKD by skewing to a cytotoxic phenotype and function
following complex interactions with damaged proximal tubules.
Collectively, the findings presented in this thesis provide strong evidence of the
pathogenic roles of NK, γδ T and MAIT cells in CKD. Previous studies in experimental
models of kidney disease have implicated pro-inflammatory cytokines IFN-γ and IL-
17A in driving inflammatory and fibrotic processes.2,3,10,11 Here, I extend these studies
by providing the first human evidence of the production of IFN-γ and IL-17A by
kidney CD56bright NK cells and γδ T cells respectively. Further to orchestrating the
inflammatory disease process/es of CKD, my results suggest that innate lymphocytes,
γδ T and MAIT cells, also participate in the destruction of renal architecture, as
indicated by their expression of cytolytic markers or the production of cytotoxic
effector molecules, perforin and granzyme B (Figure 5.1).
138 Chapter 5: General Discussion
Figure 5.1. Overview of the findings presented in this PhD.
Human NK cell subsets (CD56bright and CD56dim NK), γδ T and MAIT cells were identified in healthy kidneys, with MAIT cells displaying a tissue-resident phenotype (CD69+CD103+) (a). In fibrotic/CKD kidneys, tubulointerstitial CD56bright NK cells with high expression of CD69 and CXCR3 were shown to secrete pro-inflammatory cytokine IFN-γ (b), whilst CD161+ γδ T cells expressed CD16 and NKp44 and were a source of pro-inflammatory cytokine IL-17A (c). Human MAIT cells cultured with PTEC under in vitro hypoxic/inflammatory conditions, modelling the fibrotic micro-environment, displayed significantly upregulated levels of CD69, perforin and granzyme B, with a corresponding increase in PTEC cell death observed in co-cultures (d).
Chapter 5: General Discussion 139
RESEARCH IN PROGRESS AND OUTSTANDING RESEARCH QUESTIONS
Several questions remain unanswered in this PhD. For example, although I have
provided evidence supporting a pathogenic functional role for kidney NK, γδ T and
MAIT cells, the mechanisms involved in their recruitment and activation have not been
demonstrated. Furthermore, there are still other innate lymphocytes that have yet to be
examined. In the following section, I will provide perspectives on these questions for
future research.
Recruitment and retention pathways
During the progression of CKD, the kidney enters a chronic inflammatory state
mediated by immune and metabolic related disorders. In humans, this condition is
associated with increased accumulation of innate lymphocytes, aggravated pathology,
and worsening kidney function. Indeed, our in situ immunofluorescence images have
revealed that NK, γδ T, and MAIT cells are primarily located in the tubulointerstitial
compartment of diseased kidneys. Therefore, identifying and blocking the mechanistic
pathways involved in the recruitment and retention of these innate lymphocytes offers
novel clinical opportunities for the treatment of CKD.
Chemokine receptors
There are several putative pathways by which innate lymphocytes can be
recruited and retained in the kidney. From animal models of kidney disease, it is clear
that recruitment of leukocytes from the vasculature to sites of inflammation via
chemokine receptors is critical in the development of fibrosis.12,13 Chemokine-
chemokine receptor interactions that have been implicated in human renal diseases
include CXCL10/CXCL11-CXCR3 and fractalkine-CX3CR1.14–18 Supporting these
studies, our ex vivo analysis of kidney biopsies by flow cytometry revealed NK subset-
specific differences in expression of chemokine receptors (CXCR3 on CD56bright NK
cells and CX3CR1 on CD56dim NK cells), consistent with previous studies of human
blood NK cells.19 These findings suggest that these (and potentially other as yet
undefined) chemokine receptors are pivotal in NK subset-specific recruitment in
human CKD. Indeed, I also showed kidney MAIT cells possess a distinct chemokine
receptor profile, including expression of CXCR3, consistent with their capacity to
home to peripheral tissues. These collective data suggest that targeted inhibition of
CXCR3 in human CKD may be of clinical benefit by inhibiting the accumulation of
140 Chapter 5: General Discussion
both pathogenic CD56bright NK cells and MAIT cells within the diseased
tubulointerstitium.
Adhesion molecules
Besides chemokine receptors, innate lymphocytes can also up-regulate various
surface molecules to enhance tissue retention in non-lymphoid organs. For instance,
in studies of mouse kidneys, CD69 and CD103 have been implicated in
tubulointerstitial injury and loss of kidney function.20 Up-regulation of CD69 on
activated lymphocytes has been shown to interfere with lymphocyte chemotaxis
towards S1P (sphingosine-1-phosphate), a chemoattractant highly concentrated in the
lymphatic and blood vasculature.21 Furthermore, the expression of adhesion molecule
CD103 is known to enhance retention of lymphocytes in inflammatory diseases of
various peripheral tissues.22
Using multi-colour flow cytometry, I report, for the first time, the expression of
CD69 and CD103 on human kidney innate lymphocytes. Notably, I detected high
density expression of CD69 on MAIT cells and CD56bright NK cells in fibrotic kidney
biopsies. While unconfirmed, it is also likely that other unconventional T cells (γδ T
and NKT cells) in the kidney also express CD69, since a substantial proportion of total
CD3+ T cells in human kidney biopsies in this PhD study were shown to express this
surface molecule. Expression of CD103 was only evaluated on MAIT cells in this
thesis. Future investigations should evaluate if other innate lymphocytes also share this
phenotype of high CD103 expression.
Tissue-residency
Over the past decade, evidence of tissue-resident lymphocytes that can
permanently reside in various organs have challenged the notion that lymphocytes are
simply circulating cells that have transient access to non-lymphoid tissue.20,22,23
Therefore, it is possible that my observed accumulation of innate lymphocytes during
CKD arises from the expansion of kidney-resident cells rather than circulating
lymphocytes actively recruited into the kidney. The phenotype of MAIT cells in
healthy kidney tissue in this PhD study resembles tissue-resident cells, with co-
expression of CD69 and CD103. Furthermore, I showed MAIT cells in fibrotic
biopsies similarly expressed high levels of CD103 and enhanced CD69 levels. These
results suggest that tissue-resident MAIT cells identified in healthy kidneys may
indeed expand under diseased conditions to drive the progression of CKD.
Chapter 5: General Discussion 141
Based on this initial indication of tissue residency, I speculate that human MAIT
cells in healthy kidneys serve as sentinels to maintain barrier integrity and protect
against microbial pathogens. However, while CD69 and CD103 are the most widely
used markers to infer tissue residency, neither unequivocally denotes permanent tissue
residence.20,22,23 To substantiate my existing data and to determine the tissue residency
status of other innate lymphocytes in the kidney, further ex vivo investigations will be
required. Examples of future experimental approaches may include flow cytometric
detection of other tissue-resident associated markers (e.g. CD49a and CXCR6) and the
RNA sequencing of transcription factors involved in tissue residency (e.g. Hobit and
Blimp1).20,22,23
NK subset differentiation
Within the human kidney NK cell compartment, CD56bright NK cells were, in
particular, identified as a major contributor in the pathogenesis of CKD. I
demonstrated that CD56bright NK cells were the predominant NK cell subset in diseased
native biopsies, with numbers of CD56bright NK cells also significantly associating with
interstitial fibrosis and decline of kidney function. CD56dim to CD56bright NK cell
differentiation is a potential recruitment strategy that could lead to the increased
proportion and elevated cell numbers of CD56bright NK cells in fibrotic kidneys. In
CKD patients, the fibrotic kidney is hypoxic and concentrated with the pro-fibrotic
cytokine, TGF-β.24 Multiple lines of in vitro evidence have illustrated that hypoxia or
TGF-β can indeed convert peripheral blood CD56dim NK cells into CD56bright NK
cells.25–27 More recently, the combination of hypoxia and TGF-β have been shown to
significantly reduce the cytotoxic functionality of CD56dim NK cells and, in addition,
drive an up-regulation in CD56 expression levels.27 It would therefore be interesting
to examine if CD56dim NK cells within human kidneys are capable of differentiating
into CD56bright NK cells in response to the pro-fibrotic (hypoxia/increased TGF-β)
micro-environment of CKD.
142 Chapter 5: General Discussion
Activation pathways
Innate lymphocytes, in contrast to adaptive lymphocytes, can rapidly respond to
the local microenvironment in order to mount immediate immune responses. Strict
regulation of innate lymphocyte activity from activatory signals is therefore important
in maintaining appropriate immune responses during disease. However, proper
regulation of these immune responses is absent in CKD, leading to a perturbed micro-
environment with excess stimulatory signals that can activate local or tissue-surveying
innate lymphocytes. These immuno-stimulatory signals may be provided to
tubulointerstitial innate lymphocytes by local immune cells or by adjacent kidney
parenchymal cells (e.g. PTEC). Innate immune cell signalling pathways may involve:
(1) TCR-independent signalling pathways (cellular stress ligands/cytokines) and/or (2)
TCR-dependent activation.
Activation of innate lymphocytes is a vital trigger required to drive effector
function. Until now, there has been very limited information about the expression and
function of activatory receptors on kidney innate lymphocytes in humans. I present in
this thesis the detection of several activatory receptors on innate lymphocytes with
putative functional roles in human CKD.
TCR independent activation
Innate lymphocytes can be activated independently of TCR stimulation
following engagement of their activatory receptors with soluble factors (cytokines)
and surface molecules (cellular stress ligands). Cytokine receptors that are implicated
in the activation of innate lymphocytes include IL-18Rα and IL-7Rα.28,29 Other
activatory receptors such as CD161, NKG2D, NKp44 and NKp46 have established
functional roles in innate lymphocyte activation via direct contact with their cognate
ligands.30–32
During this PhD, I showed that human kidney MAIT cells constitutively express
receptors such as CD161, IL-18Rα and IL-7Rα under both homeostatic and diseased
conditions. Furthermore, human γδ T cells in fibrotic kidneys also expressed CD161,
in addition to other functional markers such as NKp44. Finally, I demonstrated that
human kidney NK cells express NKp46, a molecule involved in recognition of
stressed/damaged cells. Detection of these receptors that have established roles in
T/NK cell activation provides future targets for blocking studies and paves the way for
the discovery of novel therapeutic targets.
Chapter 5: General Discussion 143
TCR dependent activation
A subpopulation of innate lymphocytes (unconventional T cells) also express a
TCR that can be triggered by interacting with non-peptide antigens presented via non-
classical antigen-presenting molecules, such as MR1 and CD1d. Currently, there are
no reports of MR1 and CD1d expression in human native kidney disease. Future
research should therefore examine the expression of these molecules on antigen-
presenting cells in the kidney to identify novel sources of innate lymphocyte
activation.
Role of PTEC in activating innate lymphocytes during CKD
During CKD, the kidney is characterised by hypoxia, chronic inflammation and
proximal tubule injury.33,34 I hypothesised that proximal tubules transition into
immuno-stimulatory cells that lack the ability to regulate innate lymphocytes during
CKD due to ongoing hypoxic injury. Supporting this concept, in this thesis, I showed
that hypoxic PTEC promote MAIT cell activation and cytolytic activity during
inflammation and may explain a causal role for hypoxia in driving chronic
inflammation during CKD. However, it is unclear whether this immuno-stimulatory
attribute of hypoxic PTEC can be observed in interactions with other innate
lymphocytes; this will require further co-culture assays to examine this hypothesis for
NK cells and γδ T cells.
It is possible that MAIT cells and other innate lymphocytes are activated by
PTEC in a TCR-independent mechanism. Our laboratory is currently examining a
potential activation pathway of γδ T and MAIT cells involving PTEC triggering of
dendritic cells (DC) in the kidney (Giuliani et al, unpublished data). Rather than
directly stimulating innate lymphocytes, we propose that PTEC activate γδ T and
MAIT cells by directing DC to secrete IL-1β, an activator of γδ T cells,35 and IL-18,
an activator of MAIT cells.36 Notably, the DC production of IL-1β and IL-18 in our
model requires PTEC to be injured by hypoxia, reinforcing our hypothesis of a
dysregulated immune response caused by hypoxic PTEC. Thus, blocking either DC or
innate lymphocyte activation may present a potential therapeutic pathway for CKD.
In summary, further studies of cell-cell interactions in human CKD will be
required to elucidate the relative contribution of TCR-dependent versus TCR-
independent (cellular stress ligand-driven or cytokine-induced) signalling pathways in
innate lymphocyte activation.
144 Chapter 5: General Discussion
Other innate lymphocytes in CKD
It is important to note that other innate lymphocytes (NKT cells and ILCs) that
were not the focus of this PhD project remain to be investigated in the context of
human CKD. As previously mentioned in the literature review (Chapter 1), there is
still only limited information about the function of NKT cells and ILCs in the human
kidney and their roles in CKD. To date, type I NKT cells and ILC2s are the only
subsets of NKT cells and ILCs respectively that have been detected in human
kidneys.37,38 In experimental animal models of acute and chronic kidney disease, both
type 1 NKT and ILC2s have been associated with an anti-inflammatory role with the
potential to reduce pathology and improve kidney function.38–49 However, these anti-
inflammatory functions for type 1 NKT and ILC2s have yet to be investigated in
human models of kidney disease.
Function of IFN-γ and IL-17A in human CKD
In this PhD, I was able to present in vitro data suggesting that the functional role
of MAIT cell-derived cytotoxic molecules (granzyme B and perforin) may be to induce
PTEC necrosis under hypoxic/inflammatory conditions. Nevertheless, I was unable to
elucidate the downstream functional role of NK cell-derived IFN-γ and γδ T cell-
derived IL-17A in human CKD.
Mouse models of kidney disease have highlighted the pro-inflammatory roles of
IFN-γ and IL-17A. Both cytokines are capable of driving the pathology and function
of CKD by activating immune cell subsets and upregulating chemokines that augment
immune cell infiltration.50,51 However, there are reports of opposing functional roles
for these cytokines in animal models, suggesting dual functionality in the kidneys. For
example, a blockade/deficiency of IFN-γ or IL-17A in mice has been reported to
enhance tubular injury and interstitial fibrosis compared with control animals.52,53
Furthermore, the administration of IFN-γ or IL-17A has been shown to drive anti-
fibrotic effects and preserve renal function.54–56 These collective studies underscore
the complicated nature of using animal models to recapitulate the functions of IFN-γ
and IL-17A in humans. Further studies are required to clarify the functional roles of
these cytokines in human CKD, possibly with humanised mouse models that are more
compatible with those of humans.
Chapter 5: General Discussion 145
LIMITATIONS OF THE CURRENT STUDY
The investigative pursuit of the functions of innate lymphocytes in human CKD
is not without technical complications. Firstly, our existing multi-parameter flow
cytometric workflow was limited by the number of surface antigens (up to 15) able to
be detected in each biopsy sample.57 It was therefore not possible to detect all innate
lymphocyte subsets and perform comprehensive immunophenotyping in each clinical
specimen. Secondly, this flow cytometric approach relied heavily on enzymatic
digestion of fresh renal tissue to facilitate analysis at a single cell level. However, we
observed that certain surface markers, including CCR6, a chemokine receptor
implicated in T cell chemotaxis and a marker for IL-17A-secreting effector T cells,58
were particularly sensitive to enzymatic cleavage during this digestion process.
Thirdly, we were unable to evaluate the functional phenotype (ie cytokine profile) of
innate lymphocytes by intracellular staining due to the size of the biopsies and thus,
the low numbers of cells available for flow cytometric analysis following
fixation/permeabilisation. Future investigations of human innate lymphocyte subsets
should consider the sensitivity of the technique, size of the sample and antigen stability
prior to processing to ensure optimal and most efficient use of valuable clinical kidney
samples.
146 Chapter 5: General Discussion
CLINICAL AND DIAGNOSTIC TRANSLATION
There have been no major improvements in treating and diagnosing CKD in the
past decade.59 The results from this PhD highlight several immune signature molecules
and cellular populations that may be developed as potential therapeutic and diagnostic
targets.
Therapeutic translation
The potential therapeutic translation of this work may be to target the recruitment
and activation pathways of human innate lymphocytes. I propose that innate
lymphocyte infiltration/retention and activation can be blocked by specific antagonists
or humanised monoclonal antibodies against activatory and chemokine receptors.
Similarly, targeting the ligands for innate lymphocyte recruitment and activation may
also serve as a potential mechanism to inhibit CKD progression. Further investigation
is now required to examine whether existing immunotherapies (from non-renal
models) targeting the novel recruitment and activation pathways uncovered in this PhD
can be repurposed for human CKD (Table 5.1).
Diagnostic translation
Future translation of this work will also include examining the diagnostic utility
of innate immune cell signature molecules as early biomarkers of human CKD
pathology. The ultimate objective of this translational work will be the identification
of suitable biomarkers to predict patient risk of developing CKD, biomarkers of
disease progression or exacerbation, as well as biomarkers of treatment response and
prognosis. For instance, the excretion of kidney parenchymal/immune cells in urine
has been associated with tubulointerstitial damage and loss of renal function.60 Future
studies could examine the diagnostic utility of urinary PTEC and immune cells as
biomarkers of CKD. PTEC (CD45- CD10+ CD13+ cells) and innate lymphocytes could
be isolated from freshly collected urine and examined for cell numbers, viability and
phenotype.60 Bio-samples (urine, serum) from CKD patients and healthy controls
could also be assessed for soluble signalling proteins/complexes, including
cytotoxic/pro-inflammatory molecules IFN-γ, IL-17, perforin and granzyme B.
Chapter 5: General Discussion 147
Table 5.1. Potential therapeutic targets and drugs available for human CKD.
Target Drug name Therapeutic value in CKD Reference
CX3CR1 ligands
Anti-fractalkine monoclonal antibody, KANAb001 (E6011)
Blocking CD56dim NK cell infiltration
[61]
CXCR3 CXCR3 antagonists, VUF10085 and TAK-779
Blocking CD56bright NK cell infiltration
[62]
CXCR3 ligands
Anti-CXCL10 monoclonal antibodies, MDX-1100 and NI-0801
Blocking CD56bright NK cell infiltration
[63]
NKG2D Anti-NKG2D monoclonal antibody, NNC0142-0002
Blocking MAIT cell activation [64]
IL-12
Anti-IL-12 monoclonal antibodies, ustekinumab (CNTO-1275), briakinumab (ABT-874; J695) and the 'SMART anti-IL-12 antibody'
Blocking MAIT cell activation [65]
IL-15 Anti-IL-15 monoclonal antibody; Mikβ1
Blocking MAIT cell activation [66]
IL-18 Recombinant IL-18 human binding protein, tadekinig alfa
Blocking MAIT cell activation [67]
148 Chapter 5: General Discussion
CONCLUDING REMARKS
Collectively, this thesis is the first to identify NK, γδ T and MAIT cells and
examine their potential functional roles in human CKD. Our data provide potential
therapeutic targets to prevent the recruitment, retention and activation of human innate
lymphocytes in the kidney. Further pre-clinical studies blocking these kidney innate
lymphocyte subsets in humanised mouse models and PTEC co-cultures will be
required to determine the therapeutic value of targeting these novel immune cells.
Existing monoclonal and drug therapies targeting these pathways in non-renal diseases
may be repurposed for treatment of CKD in the future.
Chapter 5: General Discussion 149
REFERENCES
1. Becker, G. J. & Hewitson, T. D. Animal models of chronic kidney disease: useful but not perfect. Nephrol. Dial. Transplant. 28, 2432–2438 (2013).
2. Peng, X. et al. IL-17A produced by both γδ T and Th17 cells promotes renal fibrosis via RANTES-mediated leukocyte infiltration after renal obstruction. J. Pathol. 235, 79–89 (2015).
3. Turner, J.-E. et al. IL-17A Production by Renal T Cells Promotes Kidney Injury in Crescentic GN. J. Am. Soc. Nephrol. 23, 1486–1495 (2012).
4. Rosenkranz, A. R. et al. Regulatory interactions of αβ and γδ T cells in glomerulonephritis. Kidney Int. 58, 1055–1066 (2000).
5. Savransky, V. et al. Role of the T-cell receptor in kidney ischemia-reperfusion injury. Kidney Int. 69, 233–238 (2006).
6. Hochegger, K. et al. Role of α/β and γ/δ T cells in renal ischemia-reperfusion injury. Am. J. Physiol. Physiol. 293, F741–F747 (2007).
7. Lepore, M. et al. Parallel T-cell cloning and deep sequencing of human MAIT cells reveal stable oligoclonal TCRβ repertoire. Nat. Commun. 5, 3866 (2014).
8. Peterfalvi, A. et al. Invariant V 7.2-J 33 TCR is expressed in human kidney and brain tumors indicating infiltration by mucosal-associated invariant T (MAIT) cells. Int. Immunol. 20, 1517–1525 (2008).
9. Reantragoon, R. et al. Antigen-loaded MR1 tetramers define T cell receptor heterogeneity in mucosal-associated invariant T cells. J. Exp. Med. 210, 2305–2320 (2013).
10. Kitching, A. R., Holdsworth, S. R. & Tipping, P. G. IFN-gamma mediates crescent formation and cell-mediated immune injury in murine glomerulonephritis. J. Am. Soc. Nephrol. 10, 752–9 (1999).
11. Ikezumi, Y., Atkins, R. C. & Nikolic-Paterson, D. J. Interferon-γ augments acute macrophage-mediated renal injury via a glucocorticoid-sensitive mechanism. J. Am. Soc. Nephrol. 14, 888–898 (2003).
12. Panzer, U. et al. Chemokine Receptor CXCR3 Mediates T Cell Recruitment and Tissue Injury in Nephrotoxic Nephritis in Mice. 18, 2071–2084 (2007).
13. VIELHAUER, V. et al. Journal of the American Society of Nephrology. J. Am. Soc. Nephrol. 12, 919–931 (2001).
14. Romagnani, P. et al. Role for interactions between IP-10/Mig and CXCR3 in proliferative glomerulonephritis. J. Am. Soc. Nephrol. 10, 2518–26 (1999).
15. Enghard, P. et al. CXCR3+CD4+ T cells are enriched in inflamed kidneys and urine and provide a new biomarker for acute nephritis flares in systemic lupus erythematosus patients. Arthritis Rheum. 60, 199–206 (2009).
16. Kassianos, A. J. et al. Fractalkine-CX3CR1-dependent recruitment and retention of human CD1c + myeloid dendritic cells by in vitro-activated proximal tubular epithelial cells. Kidney Int. 87, 1153–1163 (2015).
150 Chapter 5: General Discussion
17. Chakravorty, S. J., Cockwell, P., Girdlestone, J., Brooks, C. J. & Savage, C. O. S. Fractalkine expression on human renal tubular epithelial cells: Potential role in mononuclear cell adhesion. Clin. Exp. Immunol. 129, 150–159 (2002).
18. Cockwell, P., Calderwood, J. W., Brooks, C. J., Chakravorty, S. J. & Savage, C. O. S. Chemoattraction of T cells expressing CCR5, CXCR3 and CX3CR1 by proximal tubular epithelial cell chemokines. Nephrol. Dial. Transplant 17, 734–744 (2002).
19. Campbell, J. J. et al. Unique subpopulations of CD56+ NK and NK-T peripheral blood lymphocytes identified by chemokine receptor expression repertoire. J. Immunol. 166, 6477–82 (2001).
20. Turner, J.-E., Becker, M., Mittrücker, H.-W. & Panzer, U. Tissue-Resident Lymphocytes in the Kidney. J. Am. Soc. Nephrol. 29, 389–399 (2018).
21. Shiow, L. R. et al. CD69 acts downstream of interferon-alpha/beta to inhibit S1P1 and lymphocyte egress from lymphoid organs. Nature 440, 540–4 (2006).
22. Topham, D. J. & Reilly, E. C. Tissue-Resident Memory CD8+ T Cells: From Phenotype to Function. Front. Immunol. 9, 515 (2018).
23. Gebhardt, T., Palendira, U., Tscharke, D. C. & Bedoui, S. Tissue-resident memory T cells in tissue homeostasis, persistent infection, and cancer surveillance. Immunol. Rev. 283, 54–76 (2018).
24. Meng, X. M., Nikolic-Paterson, D. J. & Lan, H. Y. TGF-β: The master regulator of fibrosis. Nature Reviews Nephrology 12, 325–338 (2016).
25. Keskin, D. B. et al. TGFbeta promotes conversion of CD16+ peripheral blood NK cells into CD16- NK cells with similarities to decidual NK cells. Proc. Natl. Acad. Sci. 104, 3378–3383 (2007).
26. Allan, D. S. J. et al. TGF-β affects development and differentiation of human natural killer cell subsets. Eur. J. Immunol. 40, 2289–95 (2010).
27. Cerdeira, A. S. et al. Conversion of peripheral blood NK cells to a decidual NK-like phenotype by a cocktail of defined factors. J. Immunol. 190, 3939–48 (2013).
28. Dinarello, C. A., Novick, D., Kim, S. & Kaplanski, G. Interleukin-18 and IL-18 Binding Protein. Front. Immunol. 4, 289 (2013).
29. Carrette, F. & Surh, C. D. IL-7 signaling and CD127 receptor regulation in the control of T cell homeostasis. Semin. Immunol. 24, 209–17 (2012).
30. Fergusson, J. R., Fleming, V. M. & Klenerman, P. CD161-expressing human T cells. Front. Immunol. 2, 36 (2011).
31. Bauer, S. et al. Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 285, 727–9 (1999).
32. Koch, J., Steinle, A., Watzl, C. & Mandelboim, O. Activating natural cytotoxicity receptors of natural killer cells in cancer and infection. Trends in
Chapter 5: General Discussion 151
Immunology 34, 182–191 (2013).
33. Tecklenborg, J., Clayton, D., Siebert, S. & Coley, S. M. The role of the immune system in kidney disease. Clin. Exp. Immunol. 192, 142–150 (2018).
34. Kurts, C., Panzer, U., Anders, H.-J. & Rees, A. J. The immune system and kidney disease: basic concepts and clinical implications. Nat. Rev. Immunol. 13, 738–753 (2013).
35. Sutton, C. E. et al. Interleukin-1 and IL-23 Induce Innate IL-17 Production from γδ T Cells, Amplifying Th17 Responses and Autoimmunity. Immunity 31, 331–341 (2009).
36. Xiao, X. & Cai, J. Mucosal-Associated Invariant T Cells: New Insights into Antigen Recognition and Activation. Front. Immunol. 8, 1540 (2017).
37. Yang, S. H. et al. Sulfatide-Reactive Natural Killer T Cells Abrogate Ischemia-Reperfusion Injury. J. Am. Soc. Nephrol. 22, 1305–1314 (2011).
38. Riedel, J.-H. et al. IL-33-Mediated Expansion of Type 2 Innate Lymphoid Cells Protects from Progressive Glomerulosclerosis. J. Am. Soc. Nephrol. 28, 2068–2080 (2017).
39. Mesnard, L. et al. Invariant natural killer T cells and TGF-beta attenuate anti-GBM glomerulonephritis. J. Am. Soc. Nephrol. 20, 1282–92 (2009).
40. Cao, Q. et al. Potentiating Tissue-Resident Type 2 Innate Lymphoid Cells by IL-33 to Prevent Renal Ischemia-Reperfusion Injury. J. Am. Soc. Nephrol. ASN.2017070774 (2018). doi:10.1681/ASN.2017070774
41. Stremska, M. E. et al. IL233, A Novel IL-2 and IL-33 Hybrid Cytokine, Ameliorates Renal Injury. J. Am. Soc. Nephrol. 28, 2681–2693 (2017).
42. Pereira, R. L. et al. Invariant natural killer T cell agonist modulates experimental focal and segmental glomerulosclerosis. PLoS One 7, 1–11 (2012).
43. Riedel, J.-H. et al. Immature renal dendritic cells recruit regulatory CXCR6(+) invariant natural killer T cells to attenuate crescentic GN. J. Am. Soc. Nephrol. 23, 1987–2000 (2012).
44. Singh, A. K. et al. The natural killer T cell ligand α-galactosylceramide prevents or promotes pristane-induced lupus in mice. Eur. J. Immunol. 35, 1143–1154 (2005).
45. Yang, J. Q., Kim, P. J. & Singh, R. R. Brief treatment with iNKT cell ligand α-galactosylceramide confers a long-term protection against lupus. J. Clin. Immunol. 32, 106–113 (2012).
46. Morshed, S. R., Takahashi, T., Savage, P. B., Kambham, N. & Strober, S. Beta-galactosylceramide alters invariant natural killer T cell function and is effective treatment for lupus. Clin. Immunol. 132, 321–33 (2009).
47. Uchida, T. et al. Repeated administration of alpha-galactosylceramide ameliorates experimental lupus nephritis in mice. Sci. Rep. 8, 8225 (2018).
152 Chapter 5: General Discussion
48. Huang, Q. et al. IL-25 Elicits Innate Lymphoid Cells and Multipotent Progenitor Type 2 Cells That Reduce Renal Ischemic/Reperfusion Injury. J. Am. Soc. Nephrol. 26, 2199–2211 (2015).
49. Düster, M. et al. T cell-derived IFN-γ downregulates protective group 2 innate lymphoid cells in murine lupus erythematosus. Eur. J. Immunol. 48, 1364–1375 (2018).
50. Law, B. M. P. et al. Effector γδ T cells in human renal fibrosis and chronic kidney disease. Nephrol. Dial. Transplant 34, 40–48 (2019).
51. Law, B. M. P. et al. Interferon-γ production by tubulointerstitial human CD56bright natural killer cells contributes to renal fibrosis and chronic kidney disease progression. Kidney Int. 92, 79–88 (2017).
52. Carvalho-Pinto, C. E. et al. Autocrine production of IFN-gamma by macrophages controls their recruitment to kidney and the development of glomerulonephritis in MRL/lpr mice. J. Immunol. 169, 1058–67 (2002).
53. Thorenz, A. et al. IL-17A blockade or deficiency does not affect progressive renal fibrosis following renal ischaemia reperfusion injury in mice. J. Pharm. Pharmacol. 69, 1125–1135 (2017).
54. Poosti, F. et al. Interferon gamma peptidomimetic targeted to interstitial myofibroblasts attenuates renal fibrosis after unilateral ureteral obstruction in mice. Oncotarget 7, 54240–54252 (2016).
55. Mohamed, R. et al. Low-Dose IL-17 Therapy Prevents and Reverses Diabetic Nephropathy, Metabolic Syndrome, and Associated Organ Fibrosis. J. Am. Soc. Nephrol. 27, 745–65 (2016).
56. Oldroyd, S. D., Thomas, G. L., Gabbiani, G. & El Nahas, A. M. Interferon-gamma inhibits experimental renal fibrosis. Kidney Int. 56, 2116–27 (1999).
57. Kildey, K. et al. Identification and Quantitation of Leukocyte Populations in Human Kidney Tissue by Multi-parameter Flow Cytometry. BIO-PROTOCOL 8, 1–28 (2018).
58. Singh, S. P., Zhang, H. H., Foley, J. F., Hedrick, M. N. & Farber, J. M. Human T cells that are able to produce IL-17 express the chemokine receptor CCR6. J. Immunol. 180, 214–21 (2008).
59. Stefoni, S., Iorio, M., Cianciolo, G., Baraldi, O. & Angelini, M. L. Emerging drugs for chronic kidney disease. Expert Opin. Emerg. Drugs 19, 183–199 (2014).
60. Van der Hauwaert, C. et al. Isolation and Characterization of a Primary Proximal Tubular Epithelial Cell Model from Human Kidney by CD10/CD13 Double Labeling. PLoS One 8, e66750 (2013).
61. Imai, T. & Yasuda, N. Therapeutic intervention of inflammatory/immune diseases by inhibition of the fractalkine (CX3CL1)-CX3CR1 pathway. Inflamm. Regen. 36, 9 (2017).
62. Nedjai, B. et al. CXCR3 antagonist VUF10085 binds to an intrahelical site
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distinct from that of the broad spectrum antagonist TAK-779. Br. J. Pharmacol. 172, 1822–1833 (2015).
63. Van Raemdonck, K., Van den Steen, P. E., Liekens, S., Van Damme, J. & Struyf, S. CXCR3 ligands in disease and therapy. Cytokine and Growth Factor Reviews 26, 311–327 (2015).
64. Vadstrup, K. & Bendtsen, F. Anti-NKG2D mAb: A New Treatment for Crohn’s Disease? Int. J. Mol. Sci. 18, (2017).
65. Teng, M. W. L. et al. IL-12 and IL-23 cytokines: from discovery to targeted therapies for immune-mediated inflammatory diseases. Nat. Med. 21, 719–729 (2015).
66. Morris, J. C. et al. Preclinical and phase I clinical trial of blockade of IL-15 using Mikbeta1 monoclonal antibody in T cell large granular lymphocyte leukemia. Proc. Natl. Acad. Sci. U. S. A. 103, 401–6 (2006).
67. Gabay, C. et al. Open-label, multicentre, dose-escalating phase II clinical trial on the safety and efficacy of tadekinig alfa (IL-18BP) in adult-onset Still’s disease. Ann. Rheum. Dis. 77, annrheumdis-2017-212608 (2018).