Universidade de Lisboa
Faculdade de Ciências
Departamento de Química e Bioquímica
Impact of
Foxp3+ regulatory invariant NKT cells
in the allergic airways disease
Marta Isabel de Carvalho Ferreira Gomes
Dissertação
Mestrado em Bioquímica
Bioquímica Médica
2013
Universidade de Lisboa
Faculdade de Ciências
Departamento de Química e Bioquímica
Impact of
Foxp3+ regulatory invariant NKT cells
in the allergic airways disease
Marta Isabel de Carvalho Ferreira Gomes
Dissertação orientada pelo Profº DoutorLuís Graça
e pela Profª Doutrora Margarida Telhada
Mestrado em Bioquímica
Bioquímica Médica
2013
Este trabalho é dedicado à minha mãe,
cujas escolhas fizeram com que me tornasse
na pessoa que sou hoje.
Agradecimentos
Porque esta tese não foi só um conjunto de folhas impressas, há pessoas a quem não posso deixar
de agradecer,
Um gigante OBRIGADA à Sílvia Almeida, pessoa que me orientou, que me ajudou e que me
ensinou quase tudo o que aprendi naquele laboratório. Sem ela esta tese não teria sem dúvida
sido a mesma. Obrigada por não desesperar quando eu ao princípio não acertava uma, obrigada
pelas horas passadas no laboratório por minha causa e obrigada por todas as boas conversas que
tivemos;
Obrigada à Raquel, não só pelos debates de trabalho, nem pelas boleias para o IGC, mas
principalmente pelo companheirismo e pelos bons fins de tarde no lab;
Obrigada à Ana Água-Doce, pela ajuda com as minhas experiências, pela boa disposição e
pelos dias de hambúrguer;
Obrigada ao Luís Graça, que me acolheu no laboratório mesmo depois da que espero ter
sido a pior entrevista da minha vida;
Obrigada também aos restantes membros da UNICEL, Jorge “Jaquinzinho”, Marta
Monteiro, Vanessa Oliveira e Alexandre Costa. Cada um contribuiu para os bons momentos que
passei naquele laboratório.
E porque este ano não foi só de trabalho,
Obrigada ao Nuno, pela presença mesmo à distância, pela paciência, pelo carinho e por
acreditar mais nas minhas capacidades do que eu própria. Obrigada também pelo exemplo de
esforço e por me mostrar que os sonhos são possíveis de realizar;
Obrigada à Sara, a minha companheira de sempre. Não há palavras para agradecer a
verdadeira amizade;
Obrigada também à Joaninha, à Lenita e à Fá, amigas sem as quais o caminho até aqui não
teria sido tão bom como foi;
Por fim, um obrigada do tamanho do mundo à minha mãe e à minha avó, as pessoas que sempre
se esforçaram para que eu chegasse até aqui.
VII
Resumo
As células NKT invariantes Foxp3+ demonstram propriedades imunossupressoras após
indução da expressão de Foxp3, tendo por isso sido designadas “células NKT reguladoras Foxp3+”.
Estas células partilham vários marcadores fenotípicos com as células T reguladoras, tais como a
expressão de CD25, GITR e CTLA-4, não perdendo, contudo, as suas características de células iNKT,
nomeadamente a expressão de PLZF. Muito embora estas células tenham sido identificadas in vivo
em nódulos linfáticos cervicais de ratinhos protegidos de encefalomielite autoimune
experimentalmente induzida (EAE), após administração de α-galactosilceramida, o seu estudo tem
sido feito recorrendo à conversão in vitro de células iNKT. Assim sendo, para que se verifique a
conversão para células que expressem Foxp3, as células iNKT são isoladas por citometria de fluxo e
colocadas em cultura com TGF-β, IL-2 e anti-CD28, na presença de anti-CD3 imobilizado em placa
de cultura.
Apesar do facto das células iNKT Foxp3+ terem já sido caracterizadas, várias características
fenotípicas permanecem ainda por estudar. Além disso, a possibilidade de estas células exercerem
um impacto semelhante ao apresentado pelas células T reguladoras em algumas patologias nunca
foi avaliada. Um bom modelo para testar esse possível impacto é o modelo murino de indução da
doença alérgica das vias respiratórias através da administração de ovalbumina (OVA). Neste
modelo está descrito que a transferência adoptiva de células T Foxp3+CD25+CD4+ por via sistémica
tem a capacidade para suprimir as principais características da doença. Assim sendo, este trabalho
tem como objectivos: (1) caracterizar a população de células iNKT Foxp3+ e (2) avaliar se estas
exercem um impacto protector in vivo num modelo de ratinho da doença alérgica das vias
respiratórias induzida por OVA.
Para optimizar o processo de obtenção de células iNKT Foxp3+, células iNKT isoladas de
fígado e de baço de ratinhos C57BL/6J foram purificadas por citometria de fluxo e colocadas em
cultura com TGF-β, IL-2 e anti-CD28 na presença de anti-CD3 imobilizado em placa durante 4 dias.
Após conversão, foi possível demonstrar que não existem diferenças fenotípicas significativas
entre células iNKT Foxp3+ de fígado e de baço, dado que ambas as populações expressam de igual
forma os marcadores de superfície Nrp-1, CTLA-4, GITR, CD103, PD-1 e NKG2D, não expressando
NK1.1 nem CD62L. Foi também demonstrado que células iNKT recentemente saídas do timo
apresentam uma maior propensão para conversão em células que expressem Foxp3. Os ensaios
efectuados basearam-se num estudo recente que demonstrou que células NKT recentemente
VIII
saídas do timo apresentam uma expressão característica de neuropilina-1 (Nrp-1), um receptor
transmembranar comummente expresso pelas células T reguladoras. Embora tanto as células iNKT
Nrp-1+ do fígado como as do baço apresentem maior propensão para conversão que as células
Nrp-1-, esta conversão é superior nas células do baço.
No que diz respeito aos receptores de quimiocinas, este estudo demonstrou que as células
iNKT Foxp3+ expressam CXCR3 e CXCR6, não expressam CCR7 e expressam pouco CXCR5. Este
padrão de expressão é semelhante ao já descrito na literatura para as células iNKT, podendo
indicar que estas células migram para locais de infecção e inflamação.
Vários modelos murinos têm sido descritos como uma excelente ferramenta para estudar
a doença alérgica das vias respiratórias, já que estes apresentam um conjunto de características
típicas da asma alérgica em seres humanos. Foi demonstrado que estes animais desenvolvem
infiltrados inflamatórios nas vias respiratórias, onde se observa em secções de pulmão e em
lavados broncoalveolares, eosinófilia e hiperplasia de células calciformes. Estes animais também
apresentam um aumento na concentração sérica de IgE e IgG1 específica para alergénio, bem
como citocinas Th2 e hiperplasia das células calciformes. Os modelos de imunização activa têm por
base a administração de um antigénio, tanto numa pré-imunização, como numa re-exposição por
via intra-nasal, de maneira a mimetizar a resposta alérgica a estímulos exógenos. Na fase de re-
exposição, o contacto com o alergénio desencadeia uma resposta inflamatória nas viaas
respiratórias. O uso de antigénios proteicos como alergénios permite um melhor controlo e
reprodutibilidade do modelo, dado que é possível controlar a administração de uma determinada
concentração numa determinada localização. No caso particular destes modelos de doença
alérgica das vias respiratória, o antigénio mais comummente utilizado é a ovalbumina de ovo de
galinha. No entanto, o facto de que mesmo pequenas variações no protocolo, como por exemplo a
via de imunização, o adjuvante utilizado, a dose de antigénio administrada, e o background
genético dos animais, podem influenciar os resultados do estudo, fazem com que seja crucial uma
caracterização prévia do modelo a utilizar.
Para analisar o desenrolar das respostas imunes características do protocolo escolhido de
indução da doença alérgicas das vias respiratórias através de OVA, ratinhos Thy1.1 foram
sensibilizados por via intraperitoneal com 10µg de OVA-Alum, aos dias 0, 7 e 14, e re-estimulados
por via intranasal, aos dias 21, 22 e 23 com 50µg de OVA em solução salina. Como grupo de
controlo, foram também incluídos no estudo ratinhos Thy1.1 não sensibilizados. Ao dia 24 todos
IX
os animais foram sacrificados por injecção letal, tendo sido recolhido sangue, pulmões, nódulos
linfáticos e lavado broncoalveolar.
Desta análise foi possível concluir que neste modelo de doença alérgica das vias
respiratórias induzida por OVA se verifica um aumento na percentagem de eosinófilos presentes
no lavado broncoalveolar, e infiltrado inflamatório junto das vias respiratórias. É igualmente visível
um aumento das concentrações séricas de ambas as imunoglubulinas testadas. No entanto, este
modelo não demonstra qualquer alteração nas concentrações de citocinas Th2 no pulmão. A
possibilidade da ocorrência de uma resposta Th1 foi excluída pela ausência de IgG2a específica
para OVA no soro dos animais sensitizados.
Finalmente, para testar o possível impacto de células iNKT reguladoras Foxp3+, procedeu-
se à transferência de células iNKT CD25+Foxp3+, células T CD4+CD25+Foxp3+ ou células iNKT Foxp3-
por via intratraqueal para ratinhos Thy1.1 onde a doença alérgica das vias respiratórias foi
induzida pelo protocolo descrito. A partir destes procedimentos foi possível verificar que, como
esperado, ratinhos que receberam células T reguladoras apresentam um decréscimo na
percentagem de eosinófilos presentes no BAL e menores infiltrados inflamatórios no pulmão,
sendo também visível uma diminuição na concentração sérica de IgE. No caso de ratinhos para os
quais foram transferidas células iNKT Foxp3+, estes demonstraram um ligeiro decréscimo quer na
percentagem de eosinófilos presentes no BAL, quer no infiltrado inflamatório junto das vias
respiratórias. Pelo contrário, a transferência de células iNKT Foxp3- não exerceu qualquer efeito
sobre o número de eosinófilos, aumentando significativamente a concentração sérica de IgE. A
presença das células transferidas foi confirmada pela sua presença nos nódulos linfáticos
drenantes (nódulos linfáticos do mediastino).
Em conclusão este estudo mostra que as células NKT invariantes Foxp3+ do fígado e do
baço apresentam capacidade de conversão e fenótipo semelhante, partilhando muitas das suas
características fenotípicas com as células T reguladoras. Estas células expressam também CXCR3 e
CXCR6, mas não expressam CCR7 e poucas expressam CXCR5. Ao analisar o impacto das células
NKT invariantes Foxp3+ na doença alérgica das vias respiratórias através de transferências
adoptivas, foi possível concluir que estas parecem possuir a capacidade de diminuir ligeiramente a
eosinofilia característica da doença. No entanto, esta é a única característica da doença onde estas
células parecem ter algum impacto.
X
O facto do modelo de doença alérgica das vias respiratórias utilizado não permitir o
correcto estudo de respostas Th2, provavelmente devido ao facto da estirpe utilizada apresentar
uma tendência para respostas Th1, sugere que mais experiências são necessárias, baseadas numa
diferente estirpe de ratinho, para esclarecer de modo claro esta questão.
Palavras-chave: células NKT invariantes Foxp3+, doença alérgica das vias respiratórias, células T
reguladoras, regulação imunitária, modelo de ratinho
XI
Abstract
Foxp3+ invariant NKT cells are a subset of iNKT cells that display immunosuppressive
properties. These cells share many phenotypic hallmarks with regulatory T cells, while retaining its
NKT cell characteristics. Despite the fact that Foxp3+ iNKT cells have been already characterized,
many phenotypical characteristics remained unstudied. Moreover, a possible impact of those cells
in the prevention of inflammatory pathologies has never been addressed. A good model to test
this issue is OVA-induced allergic airways disease in mice, where Foxp3+CD25+CD4+ T cells have
been described to suppress some manifestations of the disease. Therefore, this work aimed to
further characterize the Foxp3+ invariant NKT cell population and evaluate its impact in vivo on a
mouse model of OVA-induced allergic airways disease.
To obtain as many Foxp3+ invariant NKT cells as possible converted in vitro, sorted iNKT
cells were cultured for 4 days with TGF-β, IL-2 and anti-CD28 in the presence of plate-bound anti-
CD3. Upon conversion it was possible to show that there are no significant phenotypical
differences between liver and splenic Foxp3+ iNKT cells, as both populations have similar
expression of CD25, Nrp-1, CTLA-4, GITR, CD103, PD-1 and NKG2D, although differ in CD62L and
NK1.1 expression. It was also shown that recent thymic emigrant iNKT cells are more prone to
conversion into Foxp3-expressing iNKT cells. Regarding chemokine receptors, as iNKT cells, Foxp3+
iNKT cells express CXCR3 and CXCR6, do not express CCR7 and express almost no CXCR5, in the
same way as Foxp3- NKT cells. Upon adoptive cell transfers of Foxp3+ iNKT cells, OVA-sensitized
mice showed a small decrease airway eosinophila.
Keywords: Foxp3+ iNKT cells, allergic airways disease, regulatory T cells, immune regulation,
mouse model
Table of Contents
XIII
Table of contents
Agradecimentos ....................................................................................................................... V
Resumo .................................................................................................................................. VII
Abstract .................................................................................................................................. XI
Table of contents ................................................................................................................... XIII
List of Abreviations ................................................................................................................. XV
Introduction ............................................................................................................................. 1
The biology of NKT cells .................................................................................................................. 4
Tissue distribution ....................................................................................................................... 5
Development and Maturation .................................................................................................... 6
Activation .................................................................................................................................... 8
Functions ..................................................................................................................................... 9
Foxp3+ NKT regulatory cells....................................................................................................... 11
Immune responses in the lung ...................................................................................................... 12
Maintenance of Tolerance ........................................................................................................ 13
Allergic airways disease ............................................................................................................. 14
NKT cells in the allergic airways disease ................................................................................... 16
Aims of the work ........................................................................................................................... 17
Material and Methods ............................................................................................................ 19
Experimental mice ......................................................................................................................... 21
Ethics statement............................................................................................................................ 21
Isolation, culture and conversion of iNKT cells into Foxp3+ NKTcells ........................................... 21
Phenotypic characterization ......................................................................................................... 22
Induction of Allergic airways disease ............................................................................................ 23
Intratracheal adoptive cell transfers ............................................................................................. 23
Bronchoalveolar Lavage (BAL) analysis ......................................................................................... 23
Table of Contents
XIV
Lymph nodes analysis .................................................................................................................... 24
Histology ........................................................................................................................................ 24
Immunoglobulin and cytokine quantification ............................................................................... 25
Statistical Analysis ......................................................................................................................... 25
Results ................................................................................................................................... 27
Splenic iNKT cells cultured for 4 days present the best Foxp3+ iNKT cells recovery efficiency .... 29
Splenic and liver Foxp3+ regulatory iNKT cells express phenotypic characteristics of regulatory T
cells but lower levels of NKT cells phenotypic markers ................................................................ 31
Recent thymic emigrant iNKT cells are more prone to conversion into Foxp3+ iNKT cells ........... 33
Foxp3+ iNKT cells express the chemokines receptors CXCR3 and CXCR6 ..................................... 34
Allergic airways disease can be induced in mice upon OVA sensitization .................................... 36
Impact of Foxp3+ iNKT cells in the allergic airways disease .......................................................... 39
Discussion .............................................................................................................................. 45
Concluding remarks ....................................................................................................................... 50
References ............................................................................................................................. 51
List of Abbreviations
XV
List of Abbreviations
α-GalCer α-galactosylceramide
AAD Allergic airways disease
AHR Airway hyperresponsivness
APC Antigen-presenting cell
BAL Bronchoalveolar lavage
BCR B-cell receptor
CCR CC chemokine receptor
CD Cluster of differentiation
CTLA-4 Cytotoxic T-lymphocyte antigen 4
CXCR CXC chemokine receptor
DC Dendritic cell
DN Double negative
DP Double positive
EAE Experimental autoimmune encephalomyelitis
ELISA Enzyme-linked immunosorbent assay
GITR Glucocorticoid-induced TNFR-related protein
H&E Haematoxylin/eosin
IFN Interferon
Ig Immunoglobulin
IL Interleukin
iNKT Invariant natural killer T
i.p. Intraperitoneal injection
i.t. Intratracheal injection
MHC Major histocompatibility complex
NK Natural killer
NKT Natural killer T
Nrp-1 Neuropilin-1
OVA Ovalbumin
PAMP Pathogen-associated molecular pattern
List of Abbreviations
XVI
PAS Periodic acid-Schiff
PBS Phosphate buffered saline
PD-1 Programmed cell death 1
PRR Pathogen recognition receptor
RAG Recombination activating gene
s.c. Subcutaneous
TCR T-cell receptor
TGF Transforming growth factor
Th T helper
Treg Regulatory T
Introduction
Introduction
3
The immune system consists of a well-organized network of cells and tissues that defends
an organism against foreign substances. There are two components in this system, the innate
and the adaptive immunity. The important difference between them is that while innate
immunity recognizes broad, conserved large molecular patterns of pathogens, adaptive
immune response is highly specific and recognizes small sequences in a given molecule
Innate immunity is composed by physical and chemical barriers, proteins and especially by
cells that provide the first and quick response against foreign “invaders”. The most common
cells of the innate system are phagocytes (macrophages, dendritic cells, and neutrophils), mast
cells, eosinophils, basophils and natural killer (NK) cells. These cells express on their surface
specialized receptors, pathogen recognition receptors (PRRs), which recognize conserved
pathogen-associated molecular patterns (PAMPs). Because these PAMPs are not produced by
the host organism, their signal can trigger innate immune responses such as phagocytosis,
induction of synthesis of anti-microbial peptides or release of nitric oxide. This signal can also
recruit lymphocytes (adaptive cells) to the site of infection and regulate some of their effector
mechanisms, induce the expression of inflammatory cytokines and chemokines and, most
importantly, lead to the expression of co-stimulatory molecules on antigen-presenting cells
(APCs) that are critical for T cell activation.
On the other hand, the adaptive immune system is mostly composed by two types of cells,
B and T lymphocytes, also called B and T cells, respectively. This component of the immune
system is characterized by two hallmarks: specificity and memory. B cells can recognize soluble
antigens via the B-cell receptor (BCR). After antigen recognition, B cells proliferate and
differentiate into IgM-producing cells, the plasma cells, being also able to differentiate to
produce other antibody isotypes (isotype switch) through a T cell dependent process. T cell
activation is achieved through the recognition of antigens presented by MHC molecules,
expressed by professional APCs by a T cell receptor (TCR). Activation of naïve T cells leads to
clonal expansion and to the generation of effector T cells. Some activated B and T cells mature
to long-living memory B and T cells, providing the basis for a fast and highly specific response
to re-infection. Unlike the receptors of innate immunity, BCRs and TCRs are specific for their
cognate antigen being generated in immature B and T cells by recombination of different gene
segments. This process undergoes several control mechanisms such as positive and negative
selection, avoiding/minimizing the generation of unresponsive TCRs or BCRs as well as the
Introduction
4
recognition of host derived structures. However, the generation of a subset of auto-reactive T
cells, regulatory T cells characterized as CD25+Foxp3+CD4+ T cells, is also crucial to control
immune responses and prevent autoimmunity.
Despite the fact that the immune system can be artificially divided into two components,
some cells show simultaneous features of both innate and adaptive immunity. NKT cells fall
into this category, expressing a TCR and producing T-like cytokines while also exhibiting
properties and functions of NK cells.
When the immune system control mechanisms fail, and self molecules or innocuous
antigens are mistakenly recognized as threats triggering an immune response, this may give
rise to the development of autoimmune diseases or allergies.
The biology of NKT cells
The name “NK T cells” was first published in 1995, to describe mouse NK1.1+ TCR αβ+ T
cells that express uniquely a Vα14 TCR [1]. However, the studies leading to the discovery of NKT
cells started long before. In 1987 three independent studies described the existence of mouse T
cells that expressed intermediate, rather than high, levels of the αβ-TCR, with a two- to three-fold
higher frequency of Vβ8 than conventional T cells and lacked expression of CD4 and CD8 [2-4]. Just
a few years later, other groups reported the existence of a subset of T cells of the αβ lineage that
expressed NK1.1, a molecule that was thought to be exclusively expressed by NK cells [5]. During
the subsequent years numerous studies were conducted, and by the mid nineties, it became
widely accepted that mouse NKT cells are a distinct T cell lineage [6]. Since then, NKT cells were
also identified in humans [7, 8], non-human primates [9, 10] and rats [11]. Despite the fact that
the term “NKT cell” was first used to refer to the expression of NK1.1 by these T cells, it is now
used as an abbreviation of “Natural Killer T cells” [6].
Nowadays NKT cells are narrowly defined as a T cell lineage expressing NK lineage
receptors, in addition to CD1d-restricted αβ-TCRs. Because of their CD1d-restricted TCRs, these
cells present specificity for glycolipid antigens presented by CD1d molecules [12-14]. NKT cells can
be broadly divided into two different types, type I or invariant NKT (iNKT) cells and type II NKT
cells. Type I NKT cells are characterized by expressing an invariant TCR α-chain Vα14-Jα18 in mice
Introduction
5
and Vα24-Jα18 in humans and a limited, but not invariant, TCR β-chain, usually Vβ8.2, Vβ7 or Vβ2
in mice and Vβ11 in humans [2, 3, 15]. Type II NKT cells do not express the invariant TCR α-chain
characteristic of type I NKT cells, but among their heterogeneous collection of TCRs they also
express biased TCR repertoires, being Vα3.2-Jα9/Vβ8 and Vα8/Vβ8 the most over-represented
ones [16]. Another important difference between these two types of NKT cells is related to their
antigen specificity, which does not overlap. While type I NKT cells recognize a marine sponge-
derived α-galactosilceramide (α-GalCer) [14], closely related microbial α-glycuronylceramides [17]
and the mammalian glycolipids isoglobotrihexosylceramide (iGb3) [18] and disialoganglioside GD3
[19], irrespective of their β chain, type II NKT cells recognize a range of hydrophobic antigens such
as sulfatide [20], lysophospatidilcholine [21] and small aromatic (non-lipid) molecules [19]. Besides
being less abundant, type II NKT cells are also less understood since they are more difficult to
identify. While Type I NKT cells are easily isolated with α-GalCer-loaded CD1d tetramers, this is not
an easy task when regarding type II NKT cells due to their broad repertoire. However, efforts have
been made through the usage of antigens to which these cells selectively respond, such as
sulfatide or sulfatide-loaded CD1d tetramers [20, 22]. While the majority of NKT cells in mice are
type I, in human the majority of NKT cells are type II [23].
Since the work described in this dissertation focuses only on mice invariant NKT cells,
hereafter the term “NKT cells” will refer only to invariant or Type I NKT cells.
Tissue distribution
The distribution of NKT cells in mice has been well characterized, being known that they
are present wherever conventional T cells can be detected but with a pattern that is very tissue-
specific as they represent up to 30% of liver T cells, but only around 2.5% of T cells in the spleen,
mesenteric and pancreatic lymph nodes and 0.5% of the T cell population in the blood and
peripheral lymph nodes. In the thymus, NKT cells make around 0.5% of the total cells, but up to 5%
of recent thymic emigrants found in the spleen. These cells can also be detected in bone marrow,
fat tissue, lung and gastrointestinal mucosa. Concerning their intra-tissue localization, it is known
that they reside in liver sinusoids and within the marginal zone and red pulp in the spleen [24-27].
In humans, even though tissue distribution is not so-well characterized, it is known that type I NKT
cells appear to be ten times less frequent in all locations when compared to the mouse [24].
Introduction
6
Development and Maturation
To explain the development of the of the NKT cell lineage more than one model was
proposed. However, the most widely accepted model proposes that the lineage is instructed only
after TCR expression and interaction with NKT ligands (figure 1) [24]. Numerous studies indicate
that the generation of an appropriate semi-invariant CD1d-restricted TCR is the most important
step in the development of NKT cells, since mice that lacked RAG-1 and RAG-2 molecules or the
Jα18 TCR segment do not have NKT cells [28]. Thus, the first detectable stage of NKT cell
development is identified after TCR rearrangement. At this stage, thymocytes have a CD24high
CD4int CD8int phenotype (double positive (DP) stage) during which these precursors may contact
with CD1d-expressing cortical thymocytes and undergo positive selection proceeding to a CD4high
CD69high CD8neg stage (stage 0) [29, 30]. It is after this stage that cells start downregulating CD24,
entering an important expansion phase where they acquire a memory phenotype. There are still
few studies about negative selection in NKT cells, however there are some evidences that the NKT
cell repertoire also undergoes a negative selection process, most likely in the early development
stages [31].
During the progress to a CD24low mature stage there are three distinct stages. First, a
CD44low NK1.1neg stage (stage 1) where cells only produce IL-4 and no IFN-γ and after which cells
undergo a massive cellular expansion. Second, a CD44high NK1.1neg stage (stage 2) where cells
produce not only IL-4, but also IFN-γ. After this phase, the majority of cells migrate to peripheral
tissues where they stop proliferating and express NK lineage markers like NK1.1, NKG2D, NKG2A,
Ly49, C/I and G2. Thus, the third stage (stage 3) occurs simultaneously in the periphery and in the
thymus with cells having a CD44high NK1.1pos phenotype and producing more IFN-γ than IL-4. The
small subset of NKT cells that stay in the thymus become long-lived resident cells [24, 32, 33].
Moreover, during these three stages a downregulation of 30%-50% in the expression of CD4
occurs, originating double negative (DN) NKT cells [24, 29].
Introduction
7
Figure 1. iNKT cell development. Schematic representation of the invariant NKT cell developmental process in the thymus.
A recent study has proposed that recent thymic emigrant NKT cells can be recognized
through the expression of Nrp-1, a transmembrane receptor for class 3 semaphorins and vascular
endothelium growth factor isoforms [34]. This protein is expressed in a wide range of tissues
including immature thymocytes, and mediates diverse cellular functions. Nrp-1 is also known as a
marker of Foxp3+ regulatory T cells [35] being involved in their suppressive activity through long-
lasting interactions with dendritic cells (DCs) [36].
CD4
Thymus
Periphery
CD24high
CD69highCD4
CD4
CD4
CD4
CD4
CD4
DNDN
DN
CD4
CD4
DN
DN
DN
DP
CD24low
CD44low
NK1.1-
Vα14-Jα18
CD24low
CD44high
NK1.1-
CD24low
CD44high
NK1.1+
CD24low
CD44high
NK1.1+
DPTCR CD1d
IL-4 + IFN-γ
IL-4
IFN-γ
IFN-γ
CD4
CD4
CD4
Stage 0
Stage 1
Stage 2
Stage 3
Introduction
8
The development of NKT cells is regulated by the transcription factor PLZF, with this factor
being induced both in very early stages right after positive selection (CD4high CD69high thymocytes)
and in mature NKT cells. However, its expression downregulates as the cells mature. Despite the
fact that PLZF is necessary for full functionality (e.g. PLZF expression, expression of high levels of
some surface markers and cytokine production upon activation), additional factors are necessary
for complete differentiation [37, 38].
Activation
CD1d is a protein that presents lipid antigens through TCR interactions. Thus, it activates
mostly NKT cells, but also some other lipid-specific T cells. CD1d is constitutively expressed by
APCs such as DCs, macrophages and B cells, especially marginal zone B cells [39]. Besides, it is also
expressed in cortical thymocytes, Kupffer cells, hepatocytes and endothelial cells lining liver
sinusoids, where a great number of NKT cells are found in mice [40]. Moreover, similar to what
happens with MHC class II molecules, the majority of solid tissues and non-antigen presenting
hematopoietic cells express low or undetectable levels of CD1d [24].
To become activated, a NKT cell needs to recognize an antigen presented by an APC
through CD1d molecules. The antigenic presentation can be either direct, for exogenous antigens,
or indirect, for endogenous antigens (figure 2).
Figure 2. iNKT cell activation. (a) Schematic representation of the direct iNKT cell activation. (b) Schematic representation of the indirect iNKT cell activaton. (Adapted from Van Kaer et al. 2013 [27])
Introduction
9
Several studies have characterized the cascade of activation events that NKT cells undergo
after direct exogenous activation, unveiling that this process is based on reciprocal activation
between NKT cells and DCs. The process starts with the presentation of endocitized glycolipid
antigens by resting DCs to NKT cells, via CD1d-antigen complexes. This first contact will permit NKT
cells to upregulate CD40L and rapidly express Th1 and Th2 cytokines and chemokines. On the
other hand, CD40L-CD40 cross-linking will induce an upregulation of CD40, B7.1, B7.2 and IL-12 by
DCs which will enhance NKT cell activation and cytokine production, especially a higher IFN-γ
production [41]. The propagation of this reaction will lead to activation of NK cell cytolysis and to
upregulation of DC costimulatory properties and MHC-mediated antigen presentation [42, 43].
Thus, this response will lead to priming of adaptive immune responses. Furthermore, NKT cells can
also provide direct and indirect help to B cells for antibody production [44, 45]. These data shows
that the major immune role of NKT cells is the ability to activate innate and adaptive immunity,
rather than to act directly as effector cells.
An indirect NKT-cell activation occurs when APCs are firstly activated through Toll-like
receptors, triggering the loading of CD1d with endogenous glycolipid antigens. This process will
lead to NKT activation not only by antigen presentation but also by proinflammatory cytokine
production such as IL-12 by APCs [27, 46]. It is also important to refer that the quality of NKT cell
activation and the type of produced cytokines is influenced by the binding affinity, concentration
and stability of antigen-CD1d complexes [47-49]. An example is the fact that localization of CD1d
molecules in lipid rafts helps to stabilize the CD1d-antigen complex [49].
There are many exogenous and self CD1d-restricted glycolipid antigens that stimulate NKT
cells, being α-GalCer the first to be discovered and the most studied. How these antigens are
loaded onto CD1d molecules can fine-tune NKT responses. For example, if CD1d loads less
hydrophobic antigens on the cell surface, it can result in a biased Th2 cytokine response [50]. By
contrast, if CD1d intracellularly loads hydrophobic antigens it leads to the transport of the CD1d-
antigen into lipid rafts regions, leading NKT cells to produce IFN-γ [51].
Functions
NKT cells have been described as cells implicated in immune responses against infectious
agents, tumors and tissue grafts, and in regulating a variety of autoimmune and inflammatory
Introduction
10
diseases. Other studies have further revealed a role of iNKT cells in regulating hematopoiesis [52].
However, the contribution of NKT cells to these immune responses is described to be either
protective or exacerbative, depending on the particular disease investigated and the experimental
model or mouse strain used [27].
Infection
Several studies have reported a protective role of NKT cells against bacterial, parasitic and
viral infections [24]. Interestingly, this protective role has been also described for infections
mediated by microorganisms that lack obvious NKT cell antigens. The way NKT cells are activated
can vary depending on the type of infection, but the fact that some microorganisms upregulate
CD1d expression might contribute to this. By contrast, several viruses downregulate CD1d
expression, presumably as an immune evasion mechanism. NKT cell protective effects often
involve their capacity to produce large amounts of IFN-γ and activate other cell types such as DCs,
NK cells, and cytotoxic CD8+ T cells. However, these effects are usually only effective within a
narrow time window around the time of infection, thus limiting its therapeutic applications [53].
Tumor immunity
Although the paradox between the ability of NKT cells to both promote and suppress
immune responses, a great number of studies in mice have provided evidence that NKT cells
contribute to natural tumor immunity against a variety of tumors. Moreover, it was published that
activation of NKT cells with potent agonists leads to strong antimetastatic responses in mice. A
variety of mechanisms involved in the anti-tumor activities of NKT cells have been described, being
the most common the activation of other cell types such as NK cells, cytotoxic T cells, Th1 cells,
and γδ T cells, with consequent production of perforin, IFN-γ and other pro-inflammatory
cytokines, and direct lysis of suppressive myeloid lineage cells [51, 53].
Tissue graft rejection
NKT cells have been shown to play a critical role in allograft models where tolerance is
induced by blocking co-stimulatory receptors and were also described as essential for long-term
survival of corneal allografts [27, 54]. However, these cells can also contribute to allograft
rejection, as observed in a model of pancreatic islet transplantation into the liver [55]. NKT cells
Introduction
11
were further related to graft versus host disease, as several studies provided evidence that these
cells can prevent the disease in an IL-4-dependent manner [56].
Autoimmunity
As seen in experimental models of type 1 diabetes, multiple sclerosis, lupus and arthritis,
NKT cells usually play a protective role in autoimmunity. However, in some models, and
depending on the mouse stain or treatment protocol used, NKT cell activation exacerbates rather
than protects against autoimmune responses [53]. A good example is the role of NKT cells in type I
diabetes, where some studies revealed a protective role during the development of the disease
[51, 57] and others highlighted its deleterious role [51, 58]. A possible explanation for these
contradictory results is proposed by a study that suggested an influence of the genetic background
of the mice in the role of NKT cells in the development of the disease [59]. Thus, the functional
effects of NKT cells on a given autoimmune disease probably depends on the disease itself, on the
NKT cell subset being studied, on the stage of the disease, and on the genetic background of the
host or animal model being used [51]. Despite this paradox, protection against disease is usually
associated with enhanced Th2 and/or reduced Th1 responses against the target antigens involved.
Furthermore, additional studies have described that NKT cells can promote anergy in pathogenic T
cells, and induce the generation of regulatory Foxp3+ T cells and suppressive myeloid-lineage cells
[53].
Allergic disease and asthma
The contribution of NKT cells to the immune response to allergens will be more carefully
discussed after the next section.
NKT cells have been also related to a number of other pathologies such as atherosclerosis,
cirrhosis, colitis, metabolic diseases, ischemia-reperfusion injury and inflammation-induced
preterm delivery [24, 27].
Foxp3+ NKT regulatory cells
As increasing evidences support the existence of functional subpopulations of NKT cells, a
new subset of invariant NKT cells was identified in cervical lymph nodes of mice protected from
Introduction
12
experimental autoimmune encephalomyelitis (EAE) following α-GalCer administration [60]. Since
this population displays strong immunosuppressive properties upon Foxp3 upregulation, it was
termed Foxp3+ NKT regulatory cells. Interestingly, this subset presents regulatory T cell phenotypic
hallmarks, including CD25, GITR and CTLA-4, while retaining its NKT cell characteristics, namely,
PLZF expression. Like regulatory T cells, that can develop in particular contexts at the periphery
when activated in the presence of TGF-β, regulatory NKT cells can also occur in vivo, in a TGF-β rich
mucosal environment. However, there is no evidence that these Foxp3+ NKT cells develop
naturally in the thymus. Nevertheless, the Foxp3+ NKT regulatory cells have been studied in vitro
upon isolation with CD1d-loaded tetramers and culture with TGF-β, IL-2 and anti-CD28 in presence
of plate-bound anti-CD3, conditions known to convert conventional T cells into Foxp3-expressing T
cells [60].
Immune responses in the lung
The lung is in permanent contact with the external environment due to its huge surface area.
Therefore, the physical barrier between external inhaled material and the internal tissues formed
by the pulmonary epithelium has a pivotal role in the maintenance of pulmonary homeostasis,
protecting from microbes and noxious stimuli that overcome the mucociliary barrier. Within the
epithelium, goblet and Clara cells (surfactant-secreting cells) adhere together forming an
impermeable barrier. Adding to its function as a physical barrier, the pulmonary epithelium is also
immunologically active. Epithelial cells can regulate immune reactions and recruit cells of the
innate and adaptive system by being able to secrete a wide range of cytokines and chemokines in
response to danger. These responses can be triggered through the recognition of PAMPs, present
in viruses, bacteria, fungi, protozoa and multicellular parasites, by PRRs [61]. Apart from epithelial
cells, DCs and macrophages also play a pivotal role in pulmonary immune responses. DCs provide
a mechanism for continuous immune surveillance of the airway luminal surface, as they have the
ability to directly patrol the airway lumen by extruding through the epithelial barrier directly into
the lumen. Their simultaneous capacity to function as APCs and to produce cytokine makes them
pivotal in bridging innate and adaptive immunity through T cell activation. In addition to these
classical DCs, plasmacytoid, inflammatory and interferon-producing killer DCs are involved in lung
Introduction
13
immunity. However, while plasmacytoid DCs are also resident within the lung, inflammatory and
interferon-producing killer DCs migrate to the lung only during inflammatory processes [62].
Macrophages are long-lived residents of the airways that can be classified into two main
groups after activation. While classically activated macrophages are activated by IFN-γ and
lipopolysaccharide and have potent microbicidal properties and promote strong IL-12-mediated
Th1 responses, alternatively activated macrophages are activated upon exposure to a variety of
molecules such as interleukins, TGF-β, immune complexes of lipopolysaccharide or glucocorticoids
and support Th2-associated Th1 responses. Furthermore, alveolar macrophages are the
predominant immune effector cells resident in both alveolar spaces and conducting airways [61].
When pathogens escape lung innate defenses, T cell responses are required for ultimate
control of the infection. CD4+ T helper cells are especially important as they provide a great variety
of functions such as helping B cells in mounting antibody responses, providing feedback to DCs,
enhancing and maintaining CD8+ T cells, mediating macrophage activation, performing cytotoxic
functions and inducing mucosal tissue-genes that contribute to host defense [63]. The phenotype
and functions of these cells differ depending on the stimulating conditions to which they are
exposed. Thus, after antigen presentation by DCs, and depending on the cytokine milieu, naïve
CD4+ T cells can differentiate into distinct subsets, namely Th1, Th2 and Th17 that can be
distinguished by specific transcription-factors and cytokines they produce. While Th1 cells express
T-bet and produce IFN-γ, Th2 cells express GATA-3 and produce IL-4, IL-5, and IL-13 [64]. Th17 cells
are characterized by expression of RORγt and IL-17 production [65]. In addition to their capacity to
differentiate into effector cells, CD4+ T cells can also acquire regulatory properties, mainly
acquiring Foxp3 expression, becoming specialized in suppressing effector T cell responses.
Maintenance of Tolerance
Despite permanent contact with large amounts of potentially immunogenic antigenic
material, the lung presents a number of control mechanisms to avoid constant induction of
immune responses and memory effector cells development. These tightly regulated mechanisms
promote what is called immune tolerance. The fact that even small inflammation can compromise
the respiratory function, makes this process vital. Among a variety of tolerance processes, the
presence of regulatory T cells has been described as a key mechanism of tolerance to inhaled
Introduction
14
allergens. These cells have been found within the lung parenchyma and airway lumen, as well as in
the draining lymph nodes after inhaled allergen challenge [66].
The crucial role of CD4+ regulatory T cells in maintaining immunological tolerance results
from the capacity of controlling excessive inflammation by suppression of effector T cell activity.
This process also leads to restoration and/or maintenance of the lung homeostatic environment
[63]. There are several mechanisms by which regulatory T cells can exert their suppressive activity.
Due to the high expression of adhesion molecules, regulatory T cells are recruited by APCs,
especially DCs, out-competing antigen-specific naïve T cells in aggregating around the APCs.
Moreover, these cells can modulate dendritic cell function by hindering the activation of other T
cells, in manners such as the downregulation of CD80 and CD86 molecules expressed by DCs, in a
CTLA-4-dependent mechanism. Some regulatory T cells may also kill or inactivate effector T cells
by secreting granzime/perforin or immunosuppressive cytokines such as IL-10. These mechanisms
can occur in a synergistic and sequential manner, depending on the particular immune response
[67]. Two main subsets of CD4+ Foxp3+ regulatory T cells have been described, namely, thymus-
derived natural regulatory T (nTreg) cells and peripherally induced regulatory (iTreg) cells.
However, generation of iTreg cells can only occur in a TGF-β and IL-2-rich environment. Besides
CD4+ regulatory T cells, other cell types show regulatory capacities, generally through the
expression of anti-inflammatory receptors such as CTLA-4, or PD-1, or the secretion of suppressive
cytokines. These cells include several APC populations, mast cells, CD8+ T cells, γδT cells, NK cells
and NKT cells [61]. Although tolerance mechanisms are tightly controlled, inflammatory responses
against innocuous antigens such as the ones present in pollens or dust can occur, leading to the
development of an allergic disease.
Allergic airways disease
Allergic airways disease (AAD) is a pathology caused by a Th2-driven inflammatory
response to innocuous antigens, leading to increased eosinophilia and IgE production and airway
hyperresponsiveness (AHR) (figure 3). Moreover, evidences show that during this process, the
pulmonary epithelium barrier function is impaired with increased epithelium permeability [68]. In
addition to airway inflammation, airway remodeling (the phenomenon of structural changes in the
airways, namely airway wall thickness), and excessive mucus secretion from goblet cells are also
Introduction
15
evident. The combination of these processes leads to airway narrowing and therefore, reduced
lung function [69, 70].
Figure 3. Allergic airway inflammation. Schematic representation of the Th2-driven inflammatory response to allergens. (adapted from Brusselle et al. 2013 [71])
After antigen presentation by APCs, type 2 helper CD4+ T cells produce an array of
cytokines that will, directly or indirectly, influence the development of the allergic response
through different pathways. While IL-4 and IL-13 are involved in inducing B cell responses, driving
the production of allergen-specific IgE, IL-5 is necessary for the development and survival of
eosinophils and IL-9 activates mast cells. Furthermore, once IL-13 is produced, it can promote the
Introduction
16
survival and migration of eosinophils and the activation of macrophages to create an allergic cell
phenotype[72]. IgE-mediated activation of mast cells, basophils and macrophages leads to rapid
release of proinflammtry mediators that induce mucus secretion, muscle cell contraction and
microvascular leakage. This leakage results in edema within the airway wall, damaging the
epithelium and preventing mucus clearance, thus leading to narrowing of the airway lumen.
Chemoatractors produced by these innate cells will recruit more leukocytes to the inflammation
site, including eosinophils and CD4 T cells [73]. Eosinophils are granulocytes with the capacity to
produce a great variety of secretory products and express a wide range of receptors, which
indicates that these cells can exert functions beyond the role of basic granulocytes. In fact,
eosinophils show an important modulatory role in AAD, contributing to inflammation, AHR and
airway remodeling. AHR is the increased capacity of airways to narrow after exposure to non-
specific stimuli [73], and in addition to eosinophils, it can also be caused by mast cells and
basophils, via prostaglandins, leukotrienes and histamine secretion [74]. Airway remodeling is
thought to occur as a result of an imbalance in regeneration and repair mechanisms, resulting in
abnormal regulation of extra-cellular matrix components [69]. Distinct features of airway
remodeling are subepithelial fibrosis and an increase in myocyte muscle mass [75]. Eosinophils can
also produce stem cell factor and nerve growth factor to support the growth and survival of mast
cells, and cytokines such as IL-4 and IL-13 to stimulate the release of eotaxin. Eotaxin can directly
contribute to tissue damage and recruitment of more eosinophils and T cells [73].
As the late response of the allergic process develops, the overall inflammatory infiltrate
may include, in addition to eosinophils, significant numbers of monocytes, neutrophils and
platelets together with representatives from a variety of subpopulations of T cells, other than Th2
cells [76].
NKT cells in the allergic airways disease
Although NKT cells are not the predominant T cell in the allergic lung, they are known to
play an important role in AAD induced in mice by allergens such as ovalbumina (OVA) or ragweed,
respiratory viruses, environmental pollutants such as ozone, house dust mite extracts, IL-25, iNKT
cell antigens, and apoptotic respiratory epithelial cells [77]. Moreover, NKT cells can also play a
protective role in lung inflammation, as shown in some experimental models [78, 79]. The number
Introduction
17
of NKT cells in the lung varies quite significantly, as they can account for from less than 1% of
lymphocytes of naïve mice, to more than 50% after NKT cell stimulation [80]. One study suggested
that in the lungs, NKT cells are activated by recognition of self antigens that are exposed after
noxious antigens alter the mucosal environment of the respiratory tract [81]. The variety of
mechanisms to which NKT cells have been associated occur both in the early and late phases of
the AAD. In the early phase, NKT cells could facilitate sensitization by using endogenous or
exogenous lipid antigens as adjuvants for the initial maturation and polarization of airways DCs
that induce Th2 responses. During the late allergic response, NKT cells may contribute to the
exacerbation of allergic features by rapidly secreting cytokines and chemokines, which may serve
to modulate T cell polarization and/or the recruitment of T cells, eosinophils, basophils, and
neutrophils. In addition, the cytokines produced by NKT cells could also contribute to the
hypersecretion of mucus, AHR and airway remodeling, characteristic features of the late phase of
the AAD that are influenced by Th2 cell-derived cytokines [82]. One study has even showed that, in
mice, IL-4 and IL-13-producing NKT cells are essential for the development of allergen induced
AHR [81]. Despite all these evidences, there are also numerous contradictory results regarding the
pro- or anti-inflammatory role of NKT cells in the AAD. Further research on this subject would be
necessary to clarify these apparently conflicting observations.
Aims of the work
In 2010 the hosting laboratory published a study describing the existence of Foxp3+
invariant NKT cells [60]. The fact that these cells share many common features with regulatory
CD4+ T cells, such as upregulation of Foxp3, expression of CD25, CTLA-4 and GITR and suppressive
function in vitro, resulted in the term “Foxp3+ NKTreg cells”. However, contrary to regulatory T
cells, the possible regulatory properties of these cells in a physiological context have not yet been
well established. An example where the suppressive role of CD4+ regulatory T cells has been
extensively revised is in the context of the induced allergic airways disease, in which mouse
models share many pathology features with human asthma. In this model, regulatory T cells were
described as essential for the suppression of inappropriate immune responses [83]. Moreover,
another study showed that adoptive transfers of regulatory T cells were able to suppress the
allergic inflammation and AHR [66]. Whether Foxp3+ iNKT cells would have an impact on this
inflammatory response was never addressed.
Introduction
18
Therefore, this work aimed to further characterize the regulatory capacity of Foxp3+ iNKT cell
population and evaluate its impact, in vivo, upon adoptive cell transfers on a mouse model of OVA-
induced allergic airways disease. To achieve this goal, the work has been organized in four tasks:
i. Optimization of the conversion process into Foxp3+ iNKT cells;
ii. Phenotypical characterization of Foxp3+ iNKT cells;
iii. Characterization of the OVA-induced allergic airways disease;
iv. Study of the impact of Foxp3+ iNKT cells in the allergic airways disease.
Material and Methods
Material and Methods
21
Experimental mice
C57BL/6J (H-2b, B6), B6.Cg-IgHaThy1aGp1a/J (H-2b, Thy1.1) obtained from The Jackson
Laboratory (Bar Harbor, ME) and Foxp3gfp knockin mice (H-2b) obtained from the University of
Washington (Seattle, WA) were bred and maintained in specific pathogen-free conditions at the
Instituto Gulbenkian de Ciência (Oeiras,Portugal) and the Instituto de Medicina Molecular (Lisbon,
Portugal).
Ethics statement
All animal work was conducted in compliance with the Portuguese and European laws
(Portaria 1005/92 and Directive 86/609/EEC, respectively), and following the FELASA
recommendations.
Isolation, culture and conversion of iNKT cells into Foxp3+ NKTcells
Spleens and livers of C57BL/6J or Foxp3gfp mice were harvested and smashed through a
70µm cell strainer in complete RPMI (RPMI 16401 1X with GlutaMAX, supplemented with 10%
(v/v) heat inactivated Foetal Bovine Serum (FBS), 10mM HEPES, 100U/mL penicillin / 100µg/mL
streptomycin, 100mM sodium pyruvate, 0,05mM 2-mercaptoethanol and 0,025mg/mL gentamicin
from Gibco) to obtain a homogeneous cell suspensions. Liver cell suspensions were fractionated
through a 33% (v/v) normo-osmotic Percoll solution (Sigma) by centrifugation at 720g without
brake, for 30min at room temperature. Both cell suspensions were depleted of erythrocytes by
resuspending in ammonium chloride 0.155M, potassium hydrogenocarbonate 0.01M and EDTA
0.00014M. Liver cells were then incubated with PE-labeled PBS57-loaded mCD1d tetramer
(provided by NIH Tetramer Core Facility), anti-mouse-CD19-APC, anti-mouse-CD25-PE-Cy7 and
anti-mouse-TCR-β-APC-eFluor780 (eBioscience). Splenic cell suspensions were pre-incubated with
purified anti-CD16/32 (eBioscience), followed by incubation with the PE-labelled PBS57-loaded
mCD1d tetramer (given by NIH Tetramer Core Facility) for iNKT cell enrichment. After
centrifugation at 300g, for 3min, at 4ªC, anti-PE magnetic microbeads (Miltenyi Biotec) were
added to the cell suspensions. The magnetically labeled fraction was isolated by loading the
suspension through MACS LS Columns (Miltenyi Biotec). The eluted iNKT enriched fraction was
Material and Methods
22
incubated with anti-CD19-APC, anti-CD25-PE-Cy7 and anti-TCR-β-APC-eFluor780. iNKT cells were
finally sorted on a FACS Aria (BD Biosciences) based on CD19-CD25-TCR-βintTet+ expression. For
conversion controls, the negative fraction recovered from MACS LS Columns was stained with anti-
mouse-CD25-PE-Cy7, anti-mouse-TCR-β-APC-eFluor780 and anti-mouse-CD4-PerCP-Cy5.5
(eBioscience) and CD4+ T cells sorted based on CD25-TCR-bhighCD4+ expression. In some
experiments cell suspensions were also stained with purified anti-recombinant Neuropilin-1 goat
IgG (R&D Systems) followed by donkey anti-goat IgG-FITC (Santa Cruz biotechnology) and iNKT
cells sorted based on Nrp-1 positive or negative expression. Sorted iNKT and CD4+ T cells were
cultured for 4 days (3, 4 and 5 days in one experiment) in complete RPMI with 5ng/mL of TGF-β
(R&D Systems), 5ng/mL of IL-2 (eBioscience) and 2µg/mL of anti-CD28 (eBioscience) in round-
bottom-96-well plates with 3µg/mL of plate-bound anti-CD3. In all experiments cultures were
performed with 30000 cells per well, except when cells were sorted based on their Nrp-1
expression, where 22000 cells per well were cultured.
Phenotypic characterization
Cultured cells were incubated with purified anti-mouse-CD16/32 (eBioscience) to block
unspecific binding of antibodies to Fc receptors. All the labelling reaction were performed in FACS
Buffer (PBS1X, 2% (v/v) FBS, 0.02% (w/v) NaN3) using titrated concentrations of the antibodies
indicated. For staining of surface markers, cells were incubated with anti-mouse-CD25-PE-Cy7,
anti-mouse-CD25-AlexaFluor488, anti-mouse-CD4-PerCPCy5.5, anti-mouse-CD3e-APC, anti-mouse-
TCR-β-APC-eFluor780, anti-mouse-NK1.1-PE-Cy7, anti-mouse-CD69-FITC, anti-mouse-CD279(PD-
1)-PE-Cy7, anti-mouse-CD62L-APC, anti-mouse-NKG2D-Biotin, anti-mouse-CD103-Biotin, anti-
mouse-CD152(CTLA-4)-Biotin, streptavidin-PerCP-Cy5.5 and streptavidin-APC from eBioscience,
anti-mouse-GITR-PE from BD Pharmingen, purified anti-rNeuropilin-1 goat IgG from R&D Systems
and donkey anti-goat IgG-FITC from Santa Cruz biotechnology. For staining of chemokine
receptors, cultured cells were incubated with anti-mouse-CD25-APC-eFluor780, anti-mouse-
CD197(CCR7)-PE-Cy7 and anti-mouse-CD183(CXCR3)-PE from eBioscience, mouse CXCR6 rat IgG-
APC from R&D Systems, anti-mouse-CXCR5-Biotin from BD-Pharmingen and streptavidin-PerCp-
Cy5.5 from eBioscience. Intracellular stainings with anti-mouse/rat-Foxp3-eFluor450 (eBioscience)
were performed using the Foxp3 Staining buffer set from eBioscience for cell permeabilization and
fixation, according to the manufacturer´s instructions. Stained cells were analysed by flow
Material and Methods
23
cytometry using LSR FortessaII (BD Biosciences) and the results analysed by FlowJo software (Tree
Star).
Induction of Allergic airways disease
8 to 12-week old Thy1.1 female mice were sensitized by receiving 100µL intraperitoneal
(i.p.) injections of 10µg of OVA-Alum (Grade V, Sigma) at day 0, 7 and 14 of experiment. The OVA
was coupled to endotoxin-free aluminum hydroxide (Alugel-S, Serva) by gently shaking in an
orbital incubator at 4ªC for a minimum of 1 hour. At days 21, 22 and 23 the mice were challenged
through the intranasal route with 50µg OVA in 50µL of saline (B Braun) while anaesthetized with
isoflurane (IsoFlo, Abbot).
Intratracheal adoptive cell transfers
Splenic iNKT and CD4+ T cells of Foxp3gfp mice, sorted and cultured as described, were
incubated with anti-CD25-PE-Cy7 (eBiosciences) for sorting based on Foxp3 and CD25 expression.
Three different subpopulations were isolated: CD25+ Foxp3+ iNKT cells, Foxp3- iNKT cells and CD25+
Foxp3+ CD4+ T cells. Before injecting, cells were carefully washed by centrifugation to remove any
contaminating FBS and resuspended in saline. Intratracheal (i.t.) injections were performed on the
exposed trachea of anaesthetized mice using and a 27g needle. After injection, the incision was
sutured with catgut sutures (SMI). Mice were anaesthetized with a subcutaneous (s.c.) injection of
a mixture of ketamine (75mg/KgBW; Imalgene 1000, Merial) and medetomidine (1mg/KgBW;
Domitor, Pfizer) in saline, and the anaesthesia reverted with a s.c. injection of atipamezole
(1mg/KgBW; Antisedan, Pfizer). 4 previously sensitized Thy1.1 mice were injected with each
isolated subpopulation. As a control, 4 naïve mice were i.t. injected with saline.
Bronchoalveolar Lavage (BAL) analysis
Mice were sacrificed with an i.p. injection of T-61 (Embutramide 200mg,Intervet). BAL was
performed by inserting a tube in the exposed trachea to slowly flush in and out 3x1mL of FACS
Buffer (PBS 1x, 2% NBS and 0,02% azide). Samples of BAL fluid were kept for cell counting with
Material and Methods
24
trypan blue using a haemocytometer. For cell recovery, the BAL fluid was centrifuged at 260g for
3min at 4°C. For flow cytometry analysis, cells were incubated with purified anti-mouse-CD16/32
(eBioscience), anti-mouse-I-A/I-E-PerCP-Cy5.5, anti-mouse-CD193(CCR3)-AlexaFluor647
(BioLegend), anti-mouse-Gr1-eFluor450, anti-mouse-CD90.1(Thy1.1)-APC-eFluor780 and anti-
mouse-CD90.2(Thy1.2)-PE-Cy7(eBioscience). Flow cytometry analysis was performed on LSR
FortessaI (BD Biosciences) and results analysed by FlowJo software (Tree Star). To confirm results,
differential cell counts were performed on cytospin samples stained with Diff-Quick Stain Kit (IMEB
Inc.). Two hundred cells were counted in random regions using a x40 magnification.
Lymph nodes analysis
Mediastinal and inguinal lymph nodes were harvested and processed smashed through a
70µm cell strainerin FACS buffer. Recovered cells were counted with trypan blue using a
haemocytometer. For flow cytometry analysis, mediastinal lymph nodes cells were incubated with
purified anti-mouse-CD16/32 (eBioscience), anti-mouse-CD90.1(Thy1.1)-PerCP-Cy5.5, anti-mouse-
CD90.2(Thy1.2)-APC-eFluor780, anti-mouse-CD25-PE-Cy7, anti-mouse-CD4-AlexaFluor405
(Invitrogen) and PE-labeled PBS57-loaded mCD1d tetramer (given by NIH Tetramer Core Facility).
Flow cytometry analysis was performed on LSR FortessaI (BD Biosciences) and results analysed by
FlowJo software (Tree Star).
Histology
Isolated lung lobes were fixed in 10% (v/v) formalin (3.7% v/v formaldehyde) for at least
24 hours. Staining with haematoxylin/eosin (H&E) and the periodic acid-Schiff solution (PAS) was
performed by the Histology service of the Instituto de Medicina Molecular. Slides were
photographed using a Leica DM2500 microscope and a Leica DFC420 camera.
Material and Methods
25
Immunoglobulin and cytokine quantification
Immunoglobulin and cytokine concentrations were determined by ELISA in serum and lung
homogenates, respectively. For serum isolation, blood was collected by cardiac puncture and the
serum separated by centrifugation at 5000rpm for 10min. Lung homogenates were prepared in
1mL of ELISA buffer and the concentrations of IL-4, IL-5, IL-13 and IFN-γ were measured using the
respective murine ELISA kit from Peprotech. With the exception of the coating buffer (0.1M
sodium carbonate, pH 9.5) and the stop solution ( 1M H2SO4), ELISA reagents, ELISA buffer (PBS 1x,
1% (w/v) Bovine Serum Albumin and 0,1% (v/v) Tween-20), ELISA wash (PBS 1x and 0,1% Tween)
and substrate solution (TMB solution, equal parts of substrate reagent A and B, BD OptEIA) were
freshly prepared. Serum concentrations of OVA-specific IgG1 and IgG2a and total IgE were
measured using grade V OVA from Sigma as capture for OVA-specific IgG1 and IgG2a, OVA-specific
IgG1 standard from Abcam, IgG1 and IgG2a detection antibodies from Southern Biotech and IgE
ELISA Set (capture, standard and detection antibody) from BD OptEIA.
Statistical Analysis
Statistical significance was determined using a two-tailed unpaired t test. Statistical
analysis was performed using the GraphPad Prism Software. p values under 0.05 were considered
significant (*p<0.05, **p<0.01, ***p<0.001)
Results
Results
29
Splenic iNKT cells cultured for 4 days present the best Foxp3+ iNKT cells recovery
efficiency
The experimental design of this project required a relatively high number of Foxp3-
expressing iNKT cells, raising the need to recover as many cells as possible from each mouse. The
fact that less cells can be obtained from Foxp3gfp mice when comparing to C57BL/6J mice, made
mice availability a limiting factor in some experiments. Thus, optimizing the conversion process of
iNKT cells into Foxp3+ iNKT cells was a crucial step in this work.
As previously established in the hosting laboratory, upon culture with TGF-β, IL-2 and
anti-CD28 in the presence of plate-bound anti-CD3, isolated iNKT cells can be converted into
Foxp3+ NKT cells [60]. However, the number of converted cells is related to proliferation,
conversion and death rates, which can vary depending on factors such as days of culture or the
organ from which the cells are isolated. Spleen and liver are the organs from where more NKT cells
can be recovered. Thus, the same number of sorted iNKT cells from spleen and liver of 10
C57BL/6J mice were cultured for 3, 4 or 5 days in order to evaluate their conversion efficiency and
the number of recovered cells. As conversion controls, sorted splenic CD4+CD25- T cells were
similarly cultured and analyzed. All cells were stained to exclude dead cells prior to Foxp3
expression analysis.
Even though the number of total cells increased during the 5 days of culture in all three
populations (splenic NKT cells, liver NKT cells and CD4+ T cells), their conversion rates into Foxp3+
cells varied. iNKT cells isolated from the spleen showed conversions of 28% ± 1,60 at day 3, 23,5%
± 1,72 at day 4 and 16,9 ± 0,79 at day 5. The day when more Foxp3+ cells (≈27500 cells) were
present was day 4. iNKT cells from the liver showed conversions of 23,8% ± 0,81 at day 3, 18,3% ±
0,80 at day 4 and 12,2 ± 1,53 at day 5, also presenting more Foxp3+ cells (≈16500 cells) at day 4.
On the contrary, CD4+ T cells showed conversions of 78,9% ± 1,29 at day 3, 92,1% ± 0,87 at day 4
and 89,8 ± 1,19 at day 5, presenting more Foxp3+ cells (≈111000 cells) at day 5 (figure 4). Taken
together, these data show that it is possible to obtain more Foxp3+ iNKT cells when iNKT cells are
isolated from the spleen and cultured for 4 days.
Results
30
Figure 4. iNKT cells convert more efficiently into Foxp3+ iNKT cells upon 4 days of culture. Splenic and liver iNKT cells and splenic CD4
+ T cells were FACS sorted and cultured (30000 cells/well) for 3, 4 or 5 days with IL-
2 and anti-CD28 and with or without TGF-β in the presence of plate-bound anti-CD3. Data show Foxp3 expression upon 3, 4 and 5 days of culture and the number of Foxp3
+ cells obtained per well. In flow
cytometry dotplots cells were gated on live cells prior to Foxp3/CD25 analysis. Data are expressed as mean±SD. All results are representative of triplicates. Data are representative of four independent experiments.
Day 3
Day 4
Day 5
day 3 day 4 day 50
10
20
30
40
% F
oxp
3+ c
ell
s
day 3 day 4 day 50
1.010 4
2.010 4
3.010 4
# Fo
xp3
+ cel
ls
16.6
77.1
27.8
67.5
Foxp3
CD
25
0.5
98.0
day 3 day 4 day 50
10
20
30
40
% F
oxp
3+ c
ell
s
day 3 day 4 day 50
1.010 4
2.010 4
3.010 4
# Fo
xp3
+ ce
lls
18.3
79.0
23.4
72.4
12.5
85.9
Foxp3
CD
25
1.8
95.4
day 3 day 4 day 50
20
40
60
80
100
% F
oxp
3+ c
ell
s
day 3 day 4 day 50
3.010 4
6.010 4
9.010 4
1.210 5
# Fo
xp3
+ ce
lls
93.1
5.6
79.4
15.7
91.2
6.9
8.2
84.9
Foxp3
CD
25
iNKT cells - Spleen iNKT cells - Liver CD4+ T cells
TGF-β TGF-β TGF-βNo TGF-β No TGF-β No TGF-β
22.9
71.8
Foxp3
CD
25
Foxp3C
D2
5Foxp3
CD
25
Foxp3
CD
25
Foxp3
CD
25
Foxp3
CD
25
1.8
96.9
0.2
99.3
8.3
83.9
1.4
98.0
0.5
99.2
1.5
94.5
Results
31
Splenic and liver Foxp3+ regulatory iNKT cells express phenotypic characteristics
of regulatory T cells but lower levels of NKT cells phenotypic markers
Foxp3+ regulatory iNKT cells have been described as cells that display, upon culture with
TGF-β, many phenotypic markers of regulatory T cells without losing their NKT cell nature.
However, these observations were made regarding only iNKT cells isolated from the spleen [60]. It
has been shown that iNKT cells can display differences in the expression of characteristic
phenotypic markers depending on the organ where they are located [84]. Furthermore, one study
has revealed there are not only phenotypical but, more importantly, functional differences
between splenic and liver NKT [85]. Therefore, it was important to further characterize the
phenotype of Foxp3+ iNKT cells converted from both spleen and liver in order to access possible
differences between them.
Having established an optimal time point for iNKT cells conversion into Foxp3 expressing
cells, equal numbers of sorted iNKT cells isolated from both spleen and liver and splenic CD4+ T
cells of C57BL/6J mice were cultured for 4 days. Upon culture, the expression of Foxp3 and
different surface markers was analyzed by flow cytometry (figure 5). Results showed that Foxp3+
NKT cells presented heterogeneous expression of CD4 and intermediate expression of CD3 and
TCR-β, which are characteristic of iNKT cells. Regarding NK cells markers, converted iNKT cells
were NK1.1 - but the majority expressed NKG2D. Despite the fact that both splenic and liver Foxp3+
iNKT cells were CD25+, Nrp-1+, CTLA-4+, GITR+ and CD103+, typical characteristics of regulatory T
cells, there were also some differences between converted iNKT cells and induced CD4+ T cells.
While CD4+ T cells were positive for CD62L, Foxp3+ iNKT cells showed no expression of this
regulatory T cell marker. Moreover, the expression of programmed cell death protein 1 (PD-1 or
CD279) was slightly higher on Foxp3+ iNKT cells. CD69, an early activation marker, was the only
analyzed protein whose expression was not equal among the three different populations. While
Foxp3+ iNKT cells from the spleen showed a heterogeneous expression of this marker, most Foxp3+
iNKT cells from the liver presented low expression and the majority of Foxp3+ CD4+ T cells did not
express it.
In conclusion, these data suggest that there are no significant phenotypic differences between
Foxp3+ iNKT cells from spleen and liver, and that both populations share phenotypic characteristics
of regulatory T cells.
Results
32
Figure 5. Phenotype of splenic and liver Foxp3+ iNKT cells. iNKT cells isolated from spleen and liver and
splenic CD4+CD25
- T cells were FACS sorted and cultured with TGF-β for 4 days. Histograms represent the
expression of surface phenotypic markers of Foxp3+ (black) and Foxp3
- (pink) cells within iNKT or CD4
+ T cell
populations. All results are representative of triplicates. Data are representative of three independent experiments.
CD4 CD3 TCRβ NK1.1 NKG2D CD25
CTLA-4 GITR CD103 PD-1
Foxp3-Foxp3+
iNKTSpleen
iNKTLiver
CD4+
iNKTSpleen
iNKTLiver
CD4+
CD62L CD69
Nrp-1
Results
33
Recent thymic emigrant iNKT cells are more prone to conversion into Foxp3+ iNKT
cells
Nrp-1 is a transmembrane receptor for class 3 semaphorins and vascular endothelium
growth factor isoforms that is expressed in a wide variety of tissues and cells which mediates
diverse cellular functions. Among these function is its involvement in prolonging the long
interactions between DCs and Foxp3+ CD4+ T cells where Nrp-1 is constitutively express, thus
enhancing regulatory T cell suppressive activity [36]. In iNKT cells, a recent study showed that the
expression of Nrp-1 indicates that these cells are recent thymic emigrants [34].
As previously observed (figure 5), most Foxp3+ iNKT cells express Nrp-1 after conversion
with TGF-β, enlightening its similarities with Foxp3+ regulatory T cells. However, whether there are
differences in the conversion capacities of recent thymic emigrant iNKT cells remains unexplored.
To address this question, iNKT cells isolated from both spleen and liver were sorted based on Nrp-
1 positive (only ≈8% of total iNKT cells) or Nrp-1 negative cells and cultured with TGF-β, IL-2 and
anti-CD28 in the presence of plate-bound anti-CD3 for 4 days. Cells were analyzed by flow
cytometry to access their conversion efficiency. As expected, splenic iNKT cells presented a higher
conversion percentage than liver iNKT cells (figure 6). However, despite the fact that in both
subpopulations Nrp-1+ cells showed better conversion efficiencies than Nrp1- cells, this difference
was much more pronounced in iNKT cells isolated from the spleen. While in Nrp-1+ splenic iNKT
cells 22,7% ± 3,7 converted into Foxp3+ cells, in Nrp-1- cells only 11,9% ± 1,4 acquired this
phenotype. In liver iNKT cells, while in Nrp-1+ cells 8,29% ± 0,4 converted into Foxp3+ cells, only
5,5% ± 1,6 of Nrp-1- cells converted. Thus, these results suggest that recent thymic emigrants are
more prone to conversion into Foxp3+ regulatory iNKT cells, although this tendency is more
pronounced in iNKT cells that migrate to the spleen when compared with liver recent thymic
emigrant iNKT cells.
Results
34
Figure 6. Nrp-1+ iNKT cells are more prone to conversion than Nrp-1
- iNKT cells. iNKT cells isolated from
spleen and liver were FACS sorted based on positive or negative expression of Nrp-1 and cultured with TGF-β for 4 days. Data show the percentage of conversion of cultured cells into Foxp3
+ iNKT cells and their
expression of Nrp-1 upon conversion. Results are representative of duplicates for Nrp-1+ iNKT cell cultures
and triplicates for Nrp-1+ iNKT cell cultures. Data are expressed as mean±SD. *p<0.05. Data are
representative of two independent experiments.
Foxp3+ iNKT cells express the chemokines receptors CXCR3 and CXCR6
Cellular trafficking and activity of immune cells is largely coordinated by chemokines, it is
thus important to study the expression of chemokine receptors in each cell subpopulation. Since
iNKT cells are involved in a wide range of functions, the expression of chemokine receptors on
these cells has already been addressed [86]. While the majority of iNKT cells express receptors for
extra-lymphoid tissue or inflammation-related cytokines, only a few subsets express secondary
lymphoid tissue-homing cytokine receptors. However, as the chemokine receptors expressed by
Foxp3+ iNKT cells remained largely unstudied, it was important to characterize the surface
expression of some chemokine receptors known to be expressed by iNKT cells. The chosen
Foxp3- Foxp3+
Spleen Liver
Foxp3 Foxp3
Foxp3 Foxp3
CD
25
CD
25C
D2
5
CD
25
Nrp-1+ Nrp-1-0
10
20
30
% F
oxp
3+
cell
s
Nrp-1+ Nrp-1-0
10
20
30
% F
oxp
3+
cell
s
25.3
59.9
8.3
85.4
4.6
86.8
12.1
78.1
Nrp-1+
Nrp-1-
Nrp-1 Nrp-1
Nrp-1Nrp-1
Foxp3- Foxp3+
Foxp3- Foxp3+ Foxp3- Foxp3+
*
Results
35
chemokine receptors were CCR7 and CXCR5, which were identified as central conductors of
migration to the secondary lymphoid organs [87], CXCR3, which is abundantly expressed by iNKT
cells [86] as well as by the regulatory T cells that co-localize with Th1 cells, and CXCR6, which is
also expressed on iNKT cells, controlling the selective accumulation of these cells in the liver,
playing a role in basal accumulation in the lungs and being involved in several inflammatory
diseases[88].
In accordance with the previous experiments, equal numbers of sorted iNKT cells isolated from
both spleen and liver and splenic CD4+ T cells of C57BL/6J mice were cultured for 4 days, at which
time the expression of Foxp3 and the indicated chemokine receptors was analyzed by flow
cytometry (figure 7). It was observed, that like the majority of iNKT cells, most Foxp3+ iNKT cells
from both spleen and liver expressed CXCR3 and CXCR6, not expressing CCR7 and almost no
CXCR5. Results also showed that the Foxp3+ iNKT cells expression of chemokine receptors was
similar to that of CD4+ regulatory T cells, although Foxp3+ iNKT cells showed higher mean
florescent intensities. In conclusion, even after culture, Foxp3+ iNKT cells retain the characteristic
chemokine receptor-pattern of iNKT cells.
Figure 7. Expression of chemokine receptors in Foxp3+ iNKT cells. iNKT cells isolated from spleen and liver
and splenic CD4+CD25
- T cells were FACS sorted and cultured with TGF-β for 4 days. Histograms represent
the expression of chemokine receptors of Foxp3+ (black) and Foxp3
- (pink) cells within iNKT or CD4
+ T cell
populations. All results are representative of triplicates.
CCR7 CXCR3 CXCR6
Foxp3-Foxp3+
iNKTSpleen
iNKTLiver
CD4+
CXCR5
Results
36
Allergic airways disease can be induced in mice upon OVA sensitization
Mouse models have been reported to be an excellent tool to study the features of AAD, as
it displays a range of hallmarks of the human disease. These animals have been shown to develop
inflammatory infiltrates in the lungs (as shown in lung sections and in brochoalveolar lavages),
airways eosinophilia, mucus hyper-secretion and hyper-production of allergen-specific IgE and Th2
cytokines [89]. Active immunization models rely on the delivery of an antigen both in a
preimmunization and in a challenge phase, in order to mimic the allergic response to exogenous
stimuli. In the challenge phase, an allergen is introduced in the airways triggering the
inflammatory response. The use of defined protein antigens (such as OVA) as allergens makes the
model more controlled and reproducible, given that a determined amount of antigen can be
delivery at a particular site. In these models, the most commonly used antigen is chicken egg
ovalbumin (OVA), since it leads to a significant increase in antigen-specific IgE responses, numbers
of eosinophils and lymphocytes in peribronchiolar tissue and BAL, Th2-cytokine production and
serum IgE levels [89]. However, the fact that differences in protocol (e.g. the route of
immunization, adjuvant, antigen dose and genetic background) can importantly influence the
outcome of the disease, makes the characterization of a determined model prior to its study of
pivotal importance.
To analyze the development of of OVA-induced AAD, Thy1.1 mice (n=4) were sensitized at
days 0, 7 and 14 with i.p injections of 10 µg of OVA-alum, and challenged intranasally at days 21,
22 and 23 with 50 µg of OVA in saline (figure 8a). As a control group, untouched Thy1.1 mice were
also included (n=3). All mice were sacrificed by lethal injection of T-61 at day 24. Since eosinophilia
is one of the most prominent features of the AAD, the presence of these cells in the BAL was
analyzed. Cells present in the BAL of each mouse were counted using trypan blue to exclude dead
cells (figure 8b). Although all OVA-sensitized mice presented a marked increase in total cell
numbers when compared with naïve mice, the numbers varied significantly among subjects. BAL
cells were analyzed by flow cytometry to differentiate cell populations based on size, granularity
and the surface expression of MHC class II, Gr1 and CCR3 (figure 8c). Again, it was possible to see
the increase in cell numbers in the BAL of sensitized mice, as well as the higher percentages of
neutrophils and, importantly, of eosinophils (figure 8d). These results were confirmed by
differential cell counts of Diff-Quick-stained cytospin slides (figure 8d). Based on total cell numbers
and flow cytometry results, it was possible to calculate the number of eosinophils present in the
Results
37
BAL (figure 8e). Taken together, these data confirmed that OVA-sensitized mice displayed a greatly
increased airway eosinophilia, although its extent varied among mice. To further confirm the
presence of AAD features in lung tissue, lung sections were stained with H&E (figure 8f) and with
PAS (figure 8f inset), which identifies the mucus-secretory goblet cells, as the mucus is easily
identified by a magenta coloration. These results showed that OVA-sensitized mice presented
significant inflammatory infiltrates and hyperplasia of mucus-producing goblet cells.
In addition, while the levels of OVA-specific IgG1 and total IgE were increased in OVA-
sensitized mice when compared with naïve mice (figure 8g), no significant increase in the levels of
IL-4, IL-5 and IL-13 in lung homogenates of these mice were observed (figure 8h). Again, the
obtained values varied considerably among subjects. Serum levels of IgG2a were undetectable by
ELISA, making a Th1-biased response highly unlikely to be present or significant.
In conclusion, in this model of OVA-induced AAD, sensitized mice showed BAL
eosinophilia, hyperplasia of goblet cells, and increased levels of serum OVA-specific IgG1 and total
IgE, characteristic features of the AAD.
Results
38
Figure 8. Induction of allergic airways disease. (A) Protocol. Female Thy1.1 mice (n=4) were sensitized with 10µg of OVA in 100µL of Alum i.p and intranasally challenged with 50µg OVA in 50µL of saline as indicated. As control, a group of unsensitized Thy1.1 mice (n=3) was also studied. (B) Number of total viable cells in the BAL. Cells were counted based on trypan blue exclusion. (C) Flow cytometry analysis of BAL cells. Cells were gated on viable cells based on size (FSC) and granularity (SSC) and on MHC class II negative expression to exclude APCs. Eosinophils percentage was determined based on CCR3
+Gr1
int expression. Neutrophils
percentage was determined based on CCR3+Gr1
hi expression. Lymphocyte percentage was determined
based on CCR3+Gr1
neg expression. Indicated percentages were calculated based on total viable cells.
Naive OVA-sensitized0
5
10
15
Total IgE
IgE
(
g/m
L)
Naive OVA-sensitized0
20
40
60
80
100
OVA-specific IgG1
IgG
1 (
g/m
L)
Naive OVA-sensitized0
2.010 6
4.010 6
6.010 6
8.010 6
1.010 7
# To
tal c
ell
s
0 7 14 21 22 23 24Days
Sensitization Sensitization Sensitization Challenges
Sacrifice
B C Naive
OVA-sensitized
22.7
FSC
SSC
2.4
0.2
4.1
CCR3
Gr1
12.1
MHC II
FSC
SSC
3.3
73.4
2.5
CCR3
Gr1
MHC II
91.9
D
Naive OVA-sensitized0
20
40
60
80
100
Flow Cytometry
% E
osi
no
ph
ils
Naive OVA-sensitized0
20
40
60
80
100
Cytospin%
Eo
sin
op
hil
s
E F
G
A
Naive OVA-sensitized0
110 0 6
210 0 6
310 0 6
410 0 6
# Eo
sin
op
hil
s
IL-4
Naive OVA-sensitized0
500
1000
1500
pg/
mL
/ 10
0mg
lun
g
IL-5
Naive OVA-sensitized200
250
300
350
400
pg/
mL
/ 10
0mg
lun
g
IL-13
Naive OVA-sensitized80
100
120
140
160
pg/
mL
/ 10
0mg
lun
g
H
Naive OVA-Sensitized
*
*
*
*****
Results
39
(D) Percentage of eosinophils in the BAL. Percentages were calculated based on flow cytometry analysis (as shown in C) and in the number of eosinophils counted on Diff-Quick-stained cytospin slides. Eosinophils were counted out of 200 cells. (E) Number of eosinophils in the BAL. Numbers were calculated based on the total number of eosinophils showed in (B) and on the percentages resultant of flow cytometry analysis. (F) Histological sections of lung tissue using 200x magnification (400x inset). Sections were stained with H&E and PAS (inset) to confirm inflammatory cell infiltration and goblet cell hyperplasia. (G) Concentration of serum OVA-specific IgG1 and total IgE. Values were determined by ELISA. (H) Concentration of the Th2 cytokines IL-4, IL-5 and IL-13 in lung homogenates. Values were determined by ELISA. Data are expressed as mean±SD. *p<0.05, **p<0.01, ***p<0.001. Data are representative of three different experiments.
Impact of Foxp3+ iNKT cells in the allergic airways disease
Studies conducted in mouse models of OVA-induced AAD have shown that adoptive
transfers of CD4+CD25+Foxp3+ regulatory T cells could ameliorate some important features of the
disease [66, 90]. It was therefore decided to use a similar approach to investigate if adoptive
transfer of Foxp3+ iNKT cells could exert a protective impact on AAD.
OVA-sensitized Thy1.1 mice received adoptive transfers of 100000 CD25+Foxp3+CD4+ T
cells, CD25+Foxp3+ NKT cells or Foxp3- NKT cells in the trachea at day 20 (figure 9a). Transferred
cells were previously sorted and cultured for 4 days in the presence of plate-bound anti-CD3, anti-
CD28, IL-2 and TGF-β as described (figure 4). To sort cells based on Foxp3 expression, splenic cells
from Foxp3gfp knock-in mice were used. Mice were then challenged with OVA according to the
protocol. As controls, sensitized mice did not receive adoptive cell transfer and non-sensitized
Thy1.1 mice were injected with saline in the trachea. Each group consisted of 4 mice. All mice
were sacrificed by lethal injection of T-61 at day 24.
Mice that received adoptive cell transfers appeared to present higher number of cells in
the BAL (figure 9b). However, because the number of BAL cells varied substantially among
subjects, it was difficult to observe clear differences between groups. Naïve mice that received i.t.
injections of saline presented very low numbers of cells in the BAL.
Flow cytometry analysis of BAL (figure 9c and 9d) showed that mice that received Foxp3+
CD4+ T cells presented a significant decrease in the percentage of eosinophils when compared with
untreated mice. Although non-statistically significant, mice injected with Foxp3+ iNKT cells also
presented a decrease in the percentage of eosinophils in the BAL. These differences were also
visible in the flow cytometry dotplot profiles, which present changes between the groups, namely
a shift towards Gr1 negative cells.
Results
40
Figure 9. Impact of Foxp3+ iNKT cell in the allergic airways disease. (A) Protocol. Female Thy1.1 mice were
sensitized with 10µg of OVA in 100µL of Alum i.p. To prepare the adoptive transfers, splenic CD4+ T cells and
NKT cells from Foxp3gfp
mice were FACS sorted and cultured for 4 days with IL-2, anti-CD28 and TGF-β in the presence of plate-bound anti-CD3. Cells were then FACS sorted based on their expression of CD25 and Foxp3. Groups of 4 mice received intra-tracheal injections of 100000 CD25
+Foxp3
+CD4
+ T cells, CD25
+Foxp3
+
Salin
e
No cells
T ce
lls
+
CD4
+
Foxp
3
NKT ce
lls
+
Foxp
3 N
KT cells
-
Foxp
3
0
250
500
750
1000
# B
AL
cell
s(x
104)
OVA-sensitized
0 7 14 21 22 23 24Days
Sensitization Sensitization Sensitization Challenges
Sacrifice
16 20
Cell sortingand culture
AdoptivetransfersA
BC
D
Salin
e
No cells
T ce
lls
+
CD4
+
Foxp
3
NKT ce
lls
+
Foxp
3 N
KT cells
-
Foxp
3
0
50
100
150
200
# Eo
sin
op
hil
s(x
104)
Salin
e
No cells
T ce
lls
+
CD4
+
Foxp
3
NKT ce
lls
+
Foxp
3 N
KT cells
-
Foxp
3
0
20
40
60
80
% E
osi
no
ph
ils
OVA-sensitized OVA-sensitized
E
Gr1
53.8
6.1
7.3
CCR3
OVA-No cells
23.2
7.3
22.9
CCR3
Gr1
OVA-Foxp3+ T cells
5.1
0.2
12.3
CCR3
Saline
Gr1
34.6
5.9
15.1
CCR3
Gr1
OVA-Foxp3+ NKT cells
48.1
8.9
5.1
CCR3
Gr1
OVA-Foxp3- NKT cells
Saline OVA-No cells OVA-Foxp3+
T cells
OVA-Foxp3-
NKT cellsOVA-Foxp3+
NKT cells
**
**
*
Results
41
NKT cells or Foxp3- NKT cells. At the indicated time point, mice were intranasally challenged with 50µg OVA
in 50µL of saline. As controls, a group of unsensitized mice that received intra-tracheal injections of saline and a group of sensitized mice that did not received intra-tracheal injections were also studied. (B) Number of total viable cells in the BAL. Cells were counted based on trypan blue exclusion. Data are expressed as mean±SD. (C) Flow cytometry analysis of BAL cells. Cells were gated on viable cells based on size (FSC) and granularity (SSC) and on MHC class II negative to exclude APCs. Eosinophils percentage was determined based on CCR3
+Gr1
int expression. Neutrophils percentage was determined based on CCR3
+Gr1
hi expression.
Lymphocyte percentage was determined based on CCR3+Gr1
neg expression. Indicated percentages were
calculated based on total viable cells. (D) Percentages and total numbers of eosinophils in the BAL. Eosinophil numbers were calculated based on the total cell numbers showed in (B) and on the percentages resultant of flow cytometry analysis. Data are expressed as mean±SD. (E) Histological sections of lung tissue using 200x magnification (400x inset). Sections were stained with H&E and PAS (inset) to confirm inflammatory cell infiltration and goblet cell hyperplasia. *p<0.05, **p<0.01. Data are representative of three different experiments.
Based on BAL total cell numbers and percentages obtained by flow cytometry is it possible
to calculate the number of eosinophils present in the BAL of mice from the different groups.
However, due to the fact that there are important variations in total cell numbers, the number of
eosinophils also varied within the same group, making it difficult to identify a clear tendency.
Naïve mice presented both low eosinophil percentages and numbers.
Differences in the inflammatory infiltrates present in the lungs of mice that received
different treatments were also assessed, as well as the presence of goblet cells hyperplasia. To do
so, lung sections of mice from different groups were stained with H&E (figure 9e) and with PAS
(figure 9e inset). Pictures of the stained lung sections show that OVA-sensitized mice presented a
greater inflammatory than naïve mice. It was also evident that mice that received Foxp3+ cells
showed reduced infiltrates when compared to mice that did not received any cells. All OVA-
sensitized mice showed goblet cells hyperplasia.
A possible impact of Foxp3+ iNKT cells in the levels of serum OVA-specific IgG1 and total
IgE was also addressed (figure 10a). Results showed that there were no significant differences in
the serum concentration of OVA-specific IgG1 between differently-treated groups of sensitized
mice, as values varied considerably among mice of the same group. Mice injected with Foxp3+
CD4+ T cells showed a 2-fold decrease in the concentration of total IgE, in opposition to mice that
received Foxp3- iNKT cells, which showed increased serum IgE concentrations in all but one
mouse. Regarding Foxp3+ iNKT cells, mice that were injected with these cells did not show any
changes in their total IgE levels. As expected, non-sensitized mice did not show appreciable levels
of OVA-specific IgG1 or total IgE. Despite the fact that the AAD is a consequence of a Th2
Results
42
response, to confirm the possible contribution of Th1 immune response, serum levels of OVA-
specific IgG2a were also measured. Results showed that none of the mice presented OVA-specific
IgG2a in its serum, confirming the absence of a Th1-biased response in this model. Furthermore,
the concentration of the Th2 cytokines IL-4, IL-5 and IL-13 in lung homogenates of the differently
treated mice was also measured, but despite some variability among mice, results obtained by
ELISA showed no differences in any cytokine concentration among the various groups (figure 10b).
Figure 10. (A) Concentration of serum OVA-specific IgG1 and total IgE. Values were determined by ELISA. (B) Concentration of the Th2 cytokines IL-4, IL-5 and IL-13 in lung homogenates. Values were determined by ELISA. Data are expressed as mean±SD. *p<0.05. Data are representative of three different experiments.
To investigate the presence of transferred cells, the mediastinal lymph nodes (draining
lymph nodes) were analyzed. As the donor mice were Thy1.2 and the recipients Thy1.1,
transferred cells can be recognized by the expression of CD90.2. Flow cytometry analysis of the
IL-4
Sal
ine
No c
ells
T c
ells
+
CD4
+
Foxp3
NKT c
ells
+
Foxp3
NKT c
ells
-
Foxp3
0
200
400
600
800
1000
pg/
mL
/ 10
0mg
lun
g
OVA-specific IgG1
Sal
ine
No c
ells
T c
ells
+
CD4
+
Foxp3
NKT c
ells
+
Foxp3
NKT c
ells
-
Foxp3
0
100
200
300
400
IgG
1 (
g/m
L)
OVA-sensitized
A
B
OVA-sensitized
Total IgE
Sal
ine
No c
ells
T c
ells
+
CD4
+
Foxp3
NKT c
ells
+
Foxp3
NKT c
ells
-
Foxp3
0
5
10
1530
60
90
IgE
(
g/m
L)
OVA-sensitized
IL-5
Sal
ine
No c
ells
T c
ells
+
CD4
+
Foxp3
NKT c
ells
+
Foxp3
NKT c
ells
-
Foxp3
0
200
400
600
800
pg/
mL
/ 10
0mg
lun
g
IL-13
Sal
ine
No c
ells
T c
ells
+
CD4
+
Foxp3
NKT c
ells
+
Foxp3
NKT c
ells
-
Foxp3
0
100
200
300
pg/
mL
/ 10
0mg
lun
g
OVA-sensitized OVA-sensitized
*
Results
43
mediastinal lymph nodes of OVA-sensitized mice that received or did not receive i.t. injections of
Foxp3+ iNKT cells showed that some transferred cells can be visualised in these lymph nodes
(figure 11). Although the percentage of these cells varied among subjects and was low, this
population was always distinguishable. Further analysis confirmed that these cells were CD1d-
tetramer+, CD25+ and the majority CD4+ and Foxp3+ (data not shown).
Figure 11. Flow cytometry analysis of mediastinal (draining) lymph nodes. Cells were gated on viable lymphocytes based on size (FSC) and granularity (SSC). Transferred cells were identified based on Thy1.2 (CD90.2) expression, as recipient mice were Thy1.1 (n=3). Data are expressed as mean±SD. p<0.05. Data are representative of three different experiments.
Taken together these data indicate that, even though in smaller proportions then
regulatory T cells, the adoptive transfer of Foxp3+ iNKT cells appears to reduce the percentage of
eosinophils present in airways, as well as inflammatory lung infiltrates. However, these cells
present no impact in the production of serum OVA-specific IgG1 and total IgE or Th2 cytokines in
the lung. The presence of the transferred cells can be confirmed by the observation of some of
these cells in the draining lymph nodes.
0.003 0.055
Thy1.1
Th
y1.2
No cells Foxp3+ NKT cells Transferred cells
No cells Foxp3+ iNKT cells0.00
0.02
0.04
0.06
0.08
% T
hy1
.2+ c
ells
*
Discussion
Discussion
47
Regulatory Foxp3+ iNKT cells have been described as iNKT cells that upregulate Foxp3 in a
TGF-β-dependent manner [60]. These cells have been characterized as sharing several phenotypic
markers with regulatory T cells, while maintaining NKT characteristics such as PLZF and NKG2D
expression. Furthermore, and very importantly, these cells also display suppressor activity [60].
Although these cells have been identified in vivo in cervical lymph nodes of mice protected from
EAE following α-GalCer administration, to have substantial numbers of these cells for appropriate
characterization, they must to be converted in vitro from sorted iNKT cells. However, it remained
largely unknown whether iNKT cells from different organs, namely from spleen and liver, where
higher numbers of iNKT cells are present, could convert differently or exhibit different
phenotypes. Therefore, this was one of the goals adressed in this work. Despite the fact that the
efficiency of conversion of iNKT cells into Foxp3+ cells can vary significantly within experiments
(data not shown), the relative conversion percentages, as well as the phenotypic markers
expressed remain constant.
A study conducted in 2011 showed that Nrp-1, a transmembrane receptor
characteristically expressed on regulatory T cells, is also specifically expressed by recent thymic
emigrant iNKT cells, identified as Nrp-1+ cells [34]. In this work, it is shown Nrp-1+ iNKT cells are
more prone to conversion when compared with Nrp1- iNKT cells. Although this difference in the
acquisition of expression of Foxp3 is present in both liver and splenic iNKT cells, it is more
pronounced in the splenic iNKT cell population.
The expression of chemokine receptors on the surface of cells coordinates their migration
patterns. This work demonstrates that both liver and splenic Foxp3+ iNKT cells, like iNKT cells,
express CXCR3 and CXCR6, exhibit no expression of CCR7 and almost no expression of CXCR5.
Since migration is pivotal for the regulation of the immune-cross-talk of leukocytes, the expression
of these receptors may provide some hints on possible localizations and functions exerted by
these Foxp3-expressing cells.
CXCR3 is an inflammatory chemokine receptor that is rapidly induced on naïve T cells
following activation, remaining expressed in Th1 CD4+ T cells, effector CD8+ T cells, NK cells and
NKT cells. In innate lymphocytes, such as NKT cells, its expression is thought to be involved in their
Discussion
48
homing to sites of infection and inflammation [91]. Interestingly, a study performed in humans
suggested a role for CXCR3 in recruiting inflammatory T cells into the BAL during allergic asthmatic
responses [92].
CXCR6 is a chemokine receptor that is highly up-regulated by NKT cells following thymic
positive selection, and it is also involved in the accumulation of these cells in the liver. This
chemokine receptor has also been implicated in lymphocyte accumulation in several inflammatory
diseases. A study on this topic described that apart from controling NKT cell accumulation in the
liver and lungs, CXCR6 also plays a critical role in the activation and production of cytokines [88].
CCR7 and CXCR5 have been identified as central controllers of migration to the secondary
lymphoid organs. The fact that CCR7 is not expressed by Foxp3+ iNKT cells did not come as a
surprise, since it is only expressed by immature NKT cells within the NKT cell population. Another
study has even suggested that CCR7 responsiveness may be down-regulated during NKT cell
maturation in the periphery [86]. However, the presence of CCR7 on Foxp3+ iNKT cells was studied
due to the fact that some publications have described an important role of this receptor in the
migration and function of a subset of regulatory T cells [93, 94]. The existence of a very small
subset of CXCR5-expressing Foxp3+ iNKT might indicate that these cells have the ability to enter
the B-cell zone in germinal centers. In conclusion, it can be assumed that Foxp3+ iNKT cells have
the ability to migrate to sites of infection and inflammation.
Apart from expression of Foxp3, CD25, CTLA-4, GITR and CD103, which has already been
well established for these cells, this work shows that Foxp3+ iNKT cells also share the expression of
Nrp-1 and PD-1 with Foxp3+ regulatory T cells. These results are in conformity with the fact that
Foxp3 is the transcription factor responsible for the activation of genes that encode regulatory T
cell-associated molecules, such as CD25, CTLA-4, and GITR [67]. Due to the described similarities
with Foxp3+ regulatory T cells, one can admit that these cells might exert regulatory functions in
vivo. Supporting this possibility are the functions that these molecules play in regulatory T cells.
CTLA-4 is crucial in promoting the downregulation of CD80 and CD86 by DCs [67], GITR is involved
in enhancement of suppression [95], Nrp-1 prolongs interactions with immature DCs [36], CD69
confers an enhanced ability for cells to accumulate in different tissues [96], and PD-1 limits T-cell
activity in peripheral tissues presenting itself as the last chance to avoid T-cell-mediated
Discussion
49
destruction of self [97]. It is based on this possibility that this work also aimed to address if Foxp3+
iNKT cells exert an impact in a disease were regulatory T cells have been described as playing a
protective role. OVA-induced AAD was the chosen condition, due to the fact that two different
studies have shown that adoptive transfers of regulatory T cells could attenuate airway
inflammation in mice [66, 90]. This attenuation is translated in the reduction of AAD hallmark
manifestations such as, eosinophilia in the BAL, increased serum IgG1 and IgE concentration and
the production of Th2 cytokines, features that I addressed in this study.
Despite the fact that, due to its Th2-prone responses, BALB/c mice constitute a more
suitable strain to use as a model of OVA-induced AAD [98], in this work AAD was induced in
C57BL/6J mice, a strain described as Th1-prone and presenting low susceptibility to AAD [99]. This
was necessary due to the use of Foxp3gfp mice on a C57BL/6J background, from where Foxp3+ cells
for adoptive cell transfers were sorted. Therefore, and because even small variations can influence
the outcome, it was necessary to characterize OVA-induced AAD in this mouse strain. Results of
this characterization showed that, as expected, upon sensitization and challenge with OVA (figure
8a), mice presented BAL eosinophilia and lung inflammatory infiltrates, goblet cells hyperplasia,
and increased levels of serum OVA-specific IgG1 and total IgE. However, OVA-sensitized mice did
not present high levels of Th2 cytokines in the lung. This discrepancy might be justified by its Th1-
biased immune response phenotype, or may be due to the necessity of adjustments in the
sensitization protocol. An interesting review on mouse models of AAD discussed the fact that
different papers on AAD report variations in several parameters of the experimental protocol,
namely the adjuvant, dose and genetic background, which was reflected by distinct types of
allergic responses [89]. Moreover, it stated that conflicting results regarding the role of mediators
such as IL-5 or IgE can be attributed, at least in part, to these intrinsic differences.
Finally, CD25+Foxp3+ iNKT cells were intratracheally injected into OVA-sensitized mice to
study a possible impact in AAD. As a control, CD25+Foxp3+CD4+ T cells and Foxp3- iNKT cells were
injected in independent groups of mice. The fact that in our experiments adoptive cells transfers
were performed locally (intratracheal) and not systemically, as in the majority of the models
published, is imposed by the low numbers of Foxp3-expressing iNKT cells that can be recovered
from spleens of Foxp3gfp mice. The results obtained showed that the adoptive transfer of Foxp3+
iNKT cells appear to reduce the percentage of BAL eosinophils and lung inflammatory infiltrates,
even though this reduction is not as marked with the transfer of regulatory T cells. This conclusion
Discussion
50
does not take into account the number of eosinophils in the BAL, as the total number of BAL cells
showed high variation within the groups. Also, no impact could be observed in the production of
serum OVA-specific IgG1 and total IgE, but, as expected, regulatory T cell exerted a diminishing
effect in serum IgE concentration. Regarding Foxp3- iNKT cells, their adoptive transfer resulted in
no significant differences in eosinophila or OVA-specific IgG1 when compared with OVA-sensitized
mice. However, these cells potentiated a significant increase in total IgE concentration in the
serum. The presence of the transferred cells in the host mice could be confirmed by the
observation of a population of these cells in the draining lymph nodes.
Concluding remarks
This study showed that splenic and liver Foxp3+ iNKT cells present similar conversion
capacities and a similar phenotype. In addition, they express the surface markers CD25, Nrp-1,
CTLA-4, GITR, CD103, PD-1 and CD69, whose expression they share with regulatory T cells, they
also express the chemokine receptors CXCR3 and CXCR6, do not express CCR7 and express almost
no CXCR5. Moreover, it was shown that recent thymic emigrant iNKT cells, identified as Nrp-1+
cells, are more prone to conversion into Foxp3-expressing cells. Due to the fact that Foxp3+iNKT
cells and Foxp3+ regulatory T cells share so many characteristics, the impact of Foxp3+ iNKT cells in
the of AAD was assessed. This showed that these cells can slightly decrease eosinophilia, the only
hallmark of AAD where they appear to have a significant impact. The presence of transferred cells
in the host mice was confirmed by their presence in the draining lymph nodes.
The fact that the studied model of OVA-induced AAD is Th1-biased did not allow a proper
study of Th2 responses, the immune responses more relevant for AAD, suggesting that more
experiments should be performed using a different mouse strain.
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