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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


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


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


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.


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


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


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


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


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


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.


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.


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


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.


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.


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


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.


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]


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.


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.


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.


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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).

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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


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).

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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).


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).

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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.



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


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.


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


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


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


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.


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


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


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.


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


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


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.


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.


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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


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.


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.


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.


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.


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.


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.


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.


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.


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


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.


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.



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


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.


MATERIALS AND METHODS


Kidney cortical tissue was obtained with informed patient consent from the






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.



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,


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).


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





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


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.


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


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


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.


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.


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


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.


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.


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


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.


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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


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.


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.


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.


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.


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.


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.


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.


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.


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.



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


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.


METHODS


Kidney cortical tissue was obtained with informed patient consent from the






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).



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,


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).


Healthy kidney tissue and diseased biopsies were dissociated and processed for flow

cytometric analysis according to our published methodology.25 Briefly, kidney tissue


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





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-


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


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.


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


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.


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).


Significantly elevated numbers of human MAIT cells in diseased biopsies with


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


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.


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


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


that hypoxic PTEC activate cytotoxic MAIT cells within the inflammatory/fibrotic

tubulointerstitium, which, in turn, leads to PTEC damage/necrosis.


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


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


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-


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.


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


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.


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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.


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.


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).


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.


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.


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.


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.


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.


Supplementary Table 1. Clinical and histological features of patients at the time of

kidney biopsy.


Supplementary Table 2. Antibodies used for flow cytometric staining.


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 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


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).


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).


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


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.


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.


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.


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.


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.


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.


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.


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'


IL-15 Anti-IL-15 monoclonal antibody; Mikβ1


IL-18 Recombinant IL-18 human binding protein, tadekinig alfa



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.


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