functional properties of human invariant · lúcia de fátima moreira teixeira. functional...

Lúcia de Fátima Moreira Teixeira

FUNCTIONAL PROPERTIES OF HUMAN INVARIANT NATURAL KILLER T CELLS: FROM INFLAMMATION TO TOLERANCE

PROPRIEDADES FUNCIONAIS DAS CÉLULAS HUMANAS

T INVARIANTES “NATURAL KILLER”:

DA INFLAMAÇÃO À TOLERÂNCIA

Tese do 3º Ciclo de Estudos Conducente ao Grau de Doutoramento em CiênciasFarmacêuticas -

- Especialidade de Biologia Celular e Molecular

Trabalho realizado sob a orientação de:

Professora Doutora Anabela Cordeiro-da-Silva (Professora Associada com Agregação

da Faculdade de Farmácia da Universidade do Porto, Porto, Portugal)

Doutora Maria do Carmo Leite-de-Moraes (Directeur de Recherche au CNRS UMR

8147, Hôpital Necker, Paris, France)

Junho, 2011

ii

DE ACORDO COM A LEGISLAÇÃO EM VIGOR, NÃO É PERMITIDA A REPRODUÇÃO

DE QUALQUER PARTE DESTA TESE.

iii

Faculdade de Farmácia da Universidade do Porto

FUNCTIONAL PROPERTIES OF HUMAN INVARIANT NATURAL KILLER T CELLS:

FROM INFLAMMATION TO TOLERANCE

Lúcia de Fátima Moreira Teixeira

iv

The candidate performed the experimental work with a doctoral fellowship

(SFRH/BD/37178/2007) supported by the “Fundação para a Ciência e a Tecnologia”

(FCT; Portugal), which also participated with grants to attend international meetings and

for the graphical execution of this thesis. The Faculty of Pharmacy of the University of

Porto (FFUP; Portugal), the Institute for Molecular and Cell Biology (IBMC; Portugal) and

the National Center for Scientific Research (CNRS; France) provided the facilities and

logistical supports.

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AUTHOR’S DECLARATION

Under the terms of the “Decreto-lei nº 216/92, de 13 de Outubro”, is hereby declared that

the author afforded a major contribution to the conceptual design and technical execution

of the work, interpretation of the results and manuscript preparation of the published

articles included in this dissertation.

Under the terms of the “Decreto-lei nº 216/92, de 13 de Outubro”, is hereby declared that

the following original articles/communications were prepared in the scope of this

dissertation.

SCIENTIFIC PUBLICATIONS

ARTICLES IN INTERNATIONAL PEER-REVIEWED JOURNALS

In the scope of this dissertation

Article 1. Lúcia Moreira-Teixeira, Mariana Resende, Maryaline Coffre, Odile Devergne, Jean-

Philippe Herbeuval, Olivier Hermine, Elke Schneider, Lars Rogge, Frank M. Ruemmele,

Michel Dy, Anabela Cordeiro-da-Silva, and Maria C. Leite-de-Moraes (2011).

Proinflammatory Environment Dictates the IL-17−Producing Capacity of Human Invariant

NKT Cells. J Immunol 186: 5758-5765

Article 2. Lúcia Moreira-Teixeira, Mariana Resende, Odile Devergne, Jean-Philippe Herbeuval,

Olivier Hermine, Elke Schneider, Michel Dy, Anabela Cordeiro-da-Silva, and Maria C.

Leite-de-Moraes. Rapamycin and TGF-β convert invariant Natural Killer T cells into

suppressive Foxp3+ regulatory cells. (To be submitted)

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Participation in other publications in related fields

Article 3. Marie-Thérèse Rubio, Lúcia Moreira-Teixeira, Pierre Milpied, Emmanuel Bachy, Felipe

Suarez, Jean-Antoine Ribeil, David Ghez, Ambroise Marcais, Richard Delarue, Sylviane

Bouguennec, Marie bouillie, Agnès Buzyn, Sophie Caillat-Zucman, Marina Cavazzana-

Calvo, Bruno Varet, Michel Dy, Olivier Hermine* and Maria Leite-de-Moraes*. Invariant

Natural Killer T cells as a predictive factor of acute graft-versus-host disease with a

preserved graft versus leukemia effect in allogeneic haematopoietic stem cell

transplantation. (Submitted for publication)

* The authors have equally contributed to the work

Communications

Oral Communications

1. Lúcia Moreira-Teixeira#, Mariana Resende, Maryaline Coffre, Odile Devergne, Jean-


Michel Dy, Anabela Cordeiro-da-Silva and Maria C. Leite-de-Moraes. Proinflammatory

Environment Dictates the IL-17−Producing Capacity of Human Invariant NKT Cells. World

Immune Regulation Meeting – V, Davos, Switzerland, 24-27 March 2011

2. Lúcia Moreira-Teixeira#, Mariana Resende, Odile Devergne, Jean-Philippe Herbeuval,

Olivier Hermine, Elke Schneider, Michel Dy, Anabela Cordeiro-da-Silva and Maria C.

Leite-de-Moraes. Foxp3-expressing human invariant NKT cells. 13e Colloque Cytokines

du Croisic, Presqu’île du Croisic, France, 10-12 May 2010

2. Marie T. Rubio#, Lucia Teixeira, Pierre Milpied, Emmanuel Bachy, Felipe Suarez,

Richard Delarue, S. Bouguenec, Agnèes Buzyn, Sophie Caillat-Zucman, Bruno Varet,

Olivier Hermine and Maria C. Leite-de-Moraes. Valuer prognostique de la reconstitution

en lymphocytes NKT invariants après greffe de cellules souches hématopoïétiques (CSH)

allogéniques. Congrès de la Société Française d’Hématologie. Paris, France, 18-20 Mars

2010.

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3. Marie T. Rubio#, Lucia Teixeira, Pierre Milpied, Emmanuel Bachy, Felipe Suarez,

David Ghez, Richard Delarue, Agnèes Buzyn, Sophie Caillat-Zucman, Bruno Varet,

Olivier Hermine and Maria C. Leite-de-Moraes. Impact of donor-derived invariant Natural

Killer T (iNKT) cell reconstitution after allogeneic haematopoietic stem cell transplantation.

51st ASH Annual Meeting and Exposition. New Orleans, USA, 5-8 December 2009.

# Presenting author.

Poster Communications

1. Lúcia Moreira-Teixeira#, Mariana Resende, Maryaline Coffre, Odile Devergne, Jean-


Michel Dy, Anabela Cordeiro-da-Silva and Maria C. Leite-de-Moraes. Proinflammatory

Environment Dictates the IL-17−Producing Capacity of Human Invariant NKT Cells.

Second I3S Scientific Retreat, Póvoa de Varzim, Portugal, 5-6 May 2011



Leite-de-Moraes. Foxp3-expressing human invariant NKT cells. XXXVI Annual Meeting of

the Portuguese Society for Immunology, Braga, Portugal, 20-22 September 2010



Leite-de-Moraes. Foxp3-expressing human invariant NKT cells. Second I3S Scientific

Retreat, Póvoa de Varzim, Portugal, 6-7 May 2010

4. Lúcia Teixeira#, Odile Devergne, Jean-Philippe Herbeuval, Olivier Hermine, Michel Dy,

Anabela Cordeiro-da-Silva and Maria C. Leite-de-Moraes. Human invariant Natural Killer

T cells: a new source of IL-17. XXXV Annual Meeting of the Portuguese Society for

Immunology, Lisbon, Portugal, 28-30 September 2009



T cells: a new source of IL-17. 2nd European Congress of Immunology, Berlin, Germany,

13-16 September 2009

viii



T cells: a new source of IL-17. 12e Colloque Cytokines du Croisic, Presqu’île du Croisic,

France, 18-20 May 2009

# Presenting author.

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ACKNOWLEDGMENTS

There are not enough words to express my gratitude to all those who contributed to made

this thesis possible…

First, I would like to acknowledge my supervisors, Professora Doutora Anabela Cordeiro-

da-Silva and Doutora Maria Leite-de-Moraes, for all. To Professora Doutora Anabela, for

the opportunity to give my first steps in the research, even when I was still an

undergraduate student. Those were determinant. Professora Doutora Anabela, I thank

your for your advices, guidance and support. To Doutora Maria, for her support and

guidance throughout theses years in which I have had the privilege of working under her

supervision. Her knowledge, enthusiasm, advices and encouragement were essentials to

the success of this thesis. Maria, I will always be in debt to you.

I would like to thank Doctor Michel Dy, Director of the CNRS UMR 8147, for the

opportunity of developing my research project at his Unit, in which all the necessary

conditions for the success of my work were available.

To all the co-authors of the work performed during this thesis, for all their collaborations

that allowed me to go further in my research. I am likewise grateful to Jérôme and Corine

for their invaluable help. I thank you all for your work and effort you have made to help me

in this quest.

I am very grateful to my friends of the “MLM team”. Bérangère, Séverine, Cristiana and

Marie-Laure, thank you so much for your friendship, support and motivation. Also, I would

like to thank all the trainees that have passed in the laboratory during these years, teach

them was an enriching experience. I would like to extend my gratitude to all the people of

the Unit CNRS UMR 8147 that helped and supported me during these past years: Sarah,

Marie, Aurélie, Amédée, Pierre, Michaël, Rachel, Mélanie, François, Elisa, Pascal,

Pascaline, Emilie, Lucie, Christophe, Ruddy, Esther, Julie, Séverine, Anne-Sophie,

Sophie, Jean-Benoît, Julien, Maud, Geneviève, Zakia, Raouf, Linh, Olivier, Francine,

Odile, Jean-Philippe, Flora, Mireille, Catherine, Fabienne, Camara, Olinda and others.

Thank you all for your welcome and for contributing to a pleasant environment during

these long years. (Bérangère, Sarah, Marie, Amédée and Maxime, tkank you so much for

your friendship and care. I already miss our long and pleasant talks!)

A special word of acknowledgment to all members of the Parasite Disease group for all

the help, support and friendship. A special thanks to Joana Tavares (thank you for your

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guidance in the beginning of my thesis), Sofia (thank you for your support and

encouragement throughout these years), Mariana, Joana Cunha, Ricardo, Nuno, Vasco,

Marta and Inês.

I thank also all the members of the Departamento de Bioquímica of the Faculdade de

Farmácia da Universidade do Porto. A special thanks to D. Casimira for all the support

and friendship.

I am indebted to the Fundação para a Ciência e a Tecnologia for my PhD fellowship

(SFRH/BD/37178/2007 – funded by Programa Operacional Potencial Humano) and for

financial support to attend international meetings; and also to Société Française

d’Immunologie for their training award to attend a meeting.

To all my friends, especially those I met at the Maison du Portugal and who were as a

second family for me in the last three years I spent in Paris. Tiago (the Physicist!), João

(our Pianist!), Sara (the “crazy” Scientist!), Susana, João, Zé, Sandro, Morad, Sophie,

Cécile, André, Tiago, Davide, Wassim, Lilia, Achintya, Cleopatra, Paulo, Caroline,

Marina… Thank you so much!

Finally, (but not less important) I would like to express my sincere gratefulness to my

family and to Vítor Carneiro, for all the love and the ability to make the distance so small…

They encouraged me everyday. Thank you for everything!

O meu sincero Obrigada a todos…

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ABSTRACT

Invariant natural killer T (iNKT) cells are innate immune cells that co-express NK cell

markers with a restricted T cell receptor (TCR) repertoire consisting of Vα14Jα18 and

Vα24Jα18 in mice and humans, respectively. Their unusual TCR enables them to

recognize endogenous and exogenous glycolipid antigens presented by CD1d molecules

expressing by antigen presenting cells (APC). Upon activation, these cells rapidly produce

several cytokines with potent immunomodulatory activities. To date iNKT cells have been

reported to be critical in the regulation of many different types of immune responses,

ranging from self-tolerance and development of autoimmunity or allergies to response to

pathogens and tumours. However, it remains unclear how iNKT cells conceivably play

such apparently diverse roles from one type of immune response to another. Considering

the implication of iNKT cells in several pathologies and the recent clinical trials using

these cells as target, the better understanding of the functional properties of human iNKT

cells becomes critical.

This thesis is focused on the biology of human iNKT cells, namely their effector and

regulatory properties such as IL-17 production and suppressor activity, respectively. Here,

we revealed that human iNKT cell subsets are highly sensitive to environmental cues,

acquiring or losing their functions depending on their maturation stage and the cytokines

encountered during antigenic stimulation. CD161+ iNKT cells, which are intrinsically

endowed with the capacity to produce IL-17, require TGF-β plus IL-1β and IL-23 signalling

during activation to carry out their functional potential. IL-17-producing iNKT cells in adults

belong to both CD4+ and CD4- subsets, co-produce IFN-γ but have restricted ability to co-

produce IL-22. IL-17-producing iNKT cell precursors are already present in cord blood but,

at this stage, they belong predominantly to CD4- subset and are not able to co-produce

IFN-γ. The presence of TGF-β decreases IL-4 and IFN-γ production while increases the

production of IL-10 by human iNKT cells. We also established that the environment plays

a critical role on the suppressive capacity of human iNKT cells. For the first time, we

demonstrated that iNKT cells expressing Foxp3 suppress the proliferation of human CD4

conventional T cells. High levels of Foxp3 are induced in the presence of TGF-β alone but

Foxp3+ iNKT cells require mTOR inhibition to acquire suppressive activity.

The results presented here provide original data on specialized functions of distinct

human iNKT cell subsets and reveal important insight into the environmental cues that

control effector cell polarization of human iNKT cells. These findings may provide

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additional means to manipulate iNKT cell function and improve their use in adoptive

immunotherapy.

Keywords: iNKT, human, IL-17, Foxp3, TGF-β

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RESUMO

As células T invariantes “Natural Killer” (iNKT) são células do sistema imunológico inato

que co-expressam marcadores de células NK com um repertório restrito de receptor de

células T (TCR), composto por Vα14Jα18 e Vα24Jα18 em ratinhos e humanos,

respectivamente. O seu TCR incomum permite-lhes reconhecer antigénios glicolipídicos,

endógenos e exógenos, apresentados por moléculas CD1d expressas à superfície das

células apresentadoras de antigénios (APC). Após ativação, estas células rapidamente

produzem várias citoquinas com forte atividade imunorreguladora. Até à data, as células

iNKT foram descritas como agentes críticos na regulação de diferentes tipos de resposta

imunológica, que vão desde a auto-tolerância e desenvolvimento de doenças autoimunes

ou alérgicas à resposta contra microrganismos patogénicos e tumores. No entanto, ainda

não está claro como de um tipo de resposta imunológica para outro as células iNKT têm

funções aparentemente tão diferentes. Considerando a implicação das células iNKT em

diversas patologias e em ensaios clínicos recentes usando estas células como alvo,

torna-se necessária uma melhor compreensão das propriedades funcionais das células

iNKT humanas.

Esta tese é centrada na biologia das células iNKT humanas, incluindo as suas funções

efetoras e reguladoras, tais como a produção de IL-17 e atividade supressora,

respectivamente. Nós demonstramos que as células iNKT humanas são extremamente

sensíveis aos estímulos ambientais, adquirindo ou perdendo funções dependendo do seu

estado de maturação e das citoquinas encontradas ao longo da estimulação antigénica.

As células iNKT CD161+ estão intrinsecamente dotadas com a capacidade de produzir IL-

17 mas requerem a ação combinada do TGF-β, IL-1β e IL-23 durante a sua ativação para

realizarem essa função. Na idade adulta, ambas as subpopulações, CD4+ e CD4-, das

células iNKT são produtoras de IL-17, que co-produzem com IFN-γ, mas têm uma

capacidade limitada para co-produzir IL-22. Os precursores das células iNKT produtoras

de IL-17 estão presentes no sangue do cordão umbilical, mas, nesta fase, estas células

pertencem predominantemente à subpopulação CD4- e não são capazes de co-produzir

IFN-γ. A presença de TGF-β, durante a ativação antigénica, diminui a produção de IL-4 e

IFN-γ enquanto aumenta a produção de IL-10 pelas células iNKT humanas. Nós

demonstramos também que os factores ambientais desempenham um papel fundamental

na capacidade supressiva destas células. Pela primeira vez, nós demonstramos que as

células iNKT humanas que expressam Foxp3 suprimem a proliferação de células CD4+ T

convencionais. Elevados níveis de Foxp3 são induzidas na presença de TGF-β sozinho,

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mas as células iNKT Foxp3+ requerem a inibição da via de sinalização de mTOR para

adquirir atividade supressora.

Os resultados aqui apresentados fornecem dados originais sobre as funções de

diferentes subpopulações das células iNKT humanas e evidenciam a importância dos

estímulos ambientais na indução de novas funções, efetoras ou reguladoras, pelas

células iNKT humanas. Estas descobertas podem fornecer meios adicionais para a

manipulação da função das células iNKT visando melhorar a sua utilização em

imunoterapia celular adotiva.

Palavras-chave: iNKT, humanos, IL-17, Foxp3, TGF-β

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TABLE OF CONTENTS

Author’s Declaration ..........................................................................................................V

Acknowledgments ............................................................................................................ IX

Abstract ............................................................................................................................ XI

Resumo.......................................................................................................................... XIII

Table of contents ............................................................................................................ XV

Index of figures ............................................................................................................. XVII

Index of tables ................................................................................................................ XX

Abbreviation list ............................................................................................................. XXI

CHAPTER I

INVARIANT NKT CELLS ................................................................................................ 23 A brief history of NKT cells .......................................................................................... 24

iNKT cell development and distribution ........................................................................ 26

iNKT cell agonists ........................................................................................................ 31

iNKT cell activation ...................................................................................................... 34

iNKT cell functions ....................................................................................................... 35

iNKT cell interaction with other cells ............................................................................ 37

iNKT cell heterogeneity ............................................................................................... 39

iNKT cells in human diseases ...................................................................................... 41

CHAPTER II

IL-17 & TH17 CELLS ...................................................................................................... 49 Th1/Th2 paradigm ....................................................................................................... 50

Th17 cell differentiation ............................................................................................... 52

Features of human Th17 cells ..................................................................................... 54

Human Th22 cells ....................................................................................................... 56

Innate IL-17-producing cells ........................................................................................ 56

IL-17 in human health and disease .............................................................................. 58

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CHAPTER III

FOXP3 & TREG CELLS ................................................................................................. 61 Immune tolerance ........................................................................................................ 62

Phenotype of Treg cells ............................................................................................... 62

Foxp3 .......................................................................................................................... 63

Heterogeneity of human Foxp3+ T cells ....................................................................... 65

Mechanisms of suppression ........................................................................................ 66

Induced Treg cells ....................................................................................................... 70

CHAPTER IV

OBJECTIVES AND RESULTS ....................................................................................... 74 I. AIMS OF THE THESIS ............................................................................................. 75

II. RESULTS ................................................................................................................ 76

Article 1. Proinflammatory Environment Dictates the IL-17−Producing Capacity of

Human Invariant NKT Cells. .................................................................................... 76

Article 2. Rapamycin and TGF-β convert invariant Natural Killer T cells into

suppressive Foxp3+ regulatory cells. ....................................................................... 90

CHAPTER V

DISCUSSION AND PERSPERCTIVES ......................................................................... 121 IL-17-producing human iNKT cells: major conditions required ................................... 122

Foxp3+ iNKT cells: suppressors or not suppressors? ................................................. 127

CHAPTER VI

REFERENCES .............................................................................................................. 133

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INDEX OF FIGURES

FIGURE 1. Mouse iNKT cell development and maturation. Mouse iNKT cells arise in

the thymus from a common precursor pool of double-positive (DP) thymocytes.

Those expressing a TCR that binds to CD1d plus self-antigen, expressed by other

DP thymocytes, enter the iNKT cell lineage (blue). Once selected iNKT cell precursor

undergo a series of differentiation steps that ultimately results in iNKT cell pool.

(Adapted from Godfrey et al. 2010). .........................................................................................................28

FIGURE 2. Structure of some glycolipid antigens recognized by iNKT cells. a.

Structure of α-galactosylceramide (α-GalCer), the first known antigen for iNKT cells,

originally extracted from a marine sponge. b. Structure of synthetic analogues of α-

GalCer: OCH and α-C-GalCer. c. Structure of microbial glycolipids recognized by

iNKT cells: GalA-GSL (glycosphingolipid containing galacturonic acid) originally

extracted from Sphingomonas spp. and BbGL-IIc (monogalactosyl diacylglycerol

lipid) originally extracted from Borrelia burgdorferi. (Adapted from Tupin et al. 2007).

.........................................................................................................................................................................................33

FIGURE 3. Models of iNKT cell activation during microbial infection. a. Direct

activation. iNKT cells are activated by recognition of microbial antigens presented by

CD1d molecules on DC surface. b. Indirect activation. iNKT cells are activated by the

combination of IL-12 and IL-18 produced by TLR-stimulated DC and recognition of

endogenous glycolipid antigens. (Adapted from Tupin et al. 2007). .....................................35

FIGURE 4. iNKT cells interacts and modulate the function of many different cell types. iNKT cells directly or indirectly modulate the function of many other cell types,

such as NK cells and T cells. iNKT cell-DC interactions are bidirectional, as iNKT

cells receive signals from DC and vice-versa. Signals can be received through cell-

surface receptors, such as TCR recognizing glycolipid-CD1d complexes, co-

stimulatory receptors, as well as through soluble mediators, such as cytokines.

(Adapted from Cerundolo et al. 2009). ....................................................................................................39

FIGURE 5. iNKT cells and human disease. A causative association between iNKT cells

and disease is poorly defined, but probably involves one of two mechanisms. a. In

the first mechanism, decreased frequency and/or function of iNKT cells negatively

affect their immunoregulatory role and thus diseases associated with failure of

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immune regulation become more common. b. The second mechanism involves a

direct or indirect pathogenic role of iNKT cells, in which iNKT cells respond

inappropriately to self (or non-self) antigens or cytokines, contributing to allergy and

inflammatory diseases. (Adapted from Berzins et al. 2011). .....................................................42

FIGURE 6. Th1/Th2 cross-regulation. Naïve CD4+ T cells differentiate towards Th1 or

Th2 cell subset, depending on the cytokines present during antigenic stimulation.

Each subset secretes cytokines that act in an autocrine manner to give feedback on

the development of their own subset, while inhibiting the other subset. Similarly, the

lineage-specific transcription factors mutually inhibit their expression or function.

(Adapted from Amsen et al., 2009). ..........................................................................................................51

FIGURE 7. Subsets of effector T helper cells. Depending on the cytokine milieu present

at the time of antigenic stimulation, naïve CD4+ T cells can differentiate into various

subsets of T helper cells (Th1, Th2, Th17, Th9 and Th22). For most of T helper cell

differentiation programme, specific transcription factors have been identified as

master regulators. Terminally differentiated T helper cells are characterized by a

specific combination of effector cytokines that orchestrate specific effector functions

of the adaptive immune system. (Adapted from Akdis et al., 2011). ....................................52

FIGURE 8. Heterogeneity of human Th17 cells. Human IL-17-producing T cells could be

subdivided into two distinct subsets: a CCR4+ subset that expresses RORC and

produces IL-17 (Th17) and a CXCR3+ subset that co-expresses RORC and T-bet

and co-produces IL-17 and IFN-γ (Th17/Th1). Whether Th17/Th1 cells are an

intermediate state of Th17 or Th1 cells, or whether they are a distinct and stable

lineage of effector Th cells is still unknown. (Adapted from Annunziato and

Romagnani, 2009). ..............................................................................................................................................55

FIGURE 9. Control of Treg cell function by Foxp3. The transcriptional complexe

involving NFAT and Runx1 activates or represses the genes encoding cytokines

(such as IL-2 and IFN-γ) and Treg cell-associated molecules (such as CD25, CTLA-4

and GITR) in Treg cells and non-Treg cells, depending on the presence of Foxp3.

(Adapted from Sakaguchi et al. 2008). ....................................................................................................65

FIGURE 10. Mechanisms of suppression used by Treg cells. Treg cells can suppress

the proliferation and/or function of non-Treg cells using several mechanisms, which

involve the release of inhibitory cytokines, the induction of cytolysis or metabolic

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disruption of the target cell, and/or modulation of DC maturation and function.

(Adapted from Vignali et al. 2008). ............................................................................................................67

FIGURE 11. Factors regulating the iTreg-Th17 cell balance. TGF-β induces both Foxp3

and ROR-γt expression by antigen-primed naïve T cells. Under non-inflammatory

conditions, mediators like IL-2, IL-27 or retinoic acid (RA) enhance TGF-β-induced

Foxp3 expression, which inhibits ROR-γt, promoting iTreg cell development. During

inflammation, Th17-polarizing cytokines (such as IL-1β, IL-6 and IL-21) enhance

ROR-γt, which in turn inhibits Foxp3 expression, leading to Th17 cell development.

(Adapted from Burgler et al. 2010). ...........................................................................................................72

FIGURE 12. Factors regulating suppressive or effector IL-17-producing capacities of

human iNKT cells. TGF-β has a crucial role in the induction of Foxp3 expression

and acquisition of suppressive activity by human iNKT cells, when combined with

rapamycin. On the other hand, TGF-β combined with IL-1-β and IL-23 dictate IL-17

production by pre-committed CD161+ iNKT cells. Whether these dual functional

properties can be attributed to a single population (a) or to functionally distinct iNKT

cell subpopulations (b) remains to be determined. .......................................................................132

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INDEX OF TABLES

TABLE 1. Classification of NKT cells ............................................................................... 25

TABLE 2. Comparison of human and mouse iNKT cells .................................................. 30

TABLE 3. Comparison of human CD4+ and CD4- iNKT cell subsets ................................ 40

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ABBREVIATION LIST

Ab antibody

α-GalCer alpha-galactosylceramide

AGLs alyered glycolipid ligands

AHR aryl-hydrocarbon receptor

APC antigen presenting cells

cAMP cyclic adenosine monophosphate

BALF bronchoalveolar lavage fluid

CBMC cord blood mononuclear cells

CD cluster of differentiation

CTLA-4 cytotoxic T-lymphocyte antigen 4

DC dendritic cells

DN double-negative

DP double-positive

EAE experimental autoimmune encephalomyelitis

EBV Epstein-Barr virus

ELISA enzyme linked immunosorbent assay

Foxp3 forkhead box P3

GALT gut-associated lymphoid tissue

GITR glucocorticoid-induced TNFR-related protein

GM-CSF granulocyte macrophage colony-stimulating factor

GVH graft-versus-host

HIV human immunodeficiency virus

HSCT hematopoietic stem cell transplantation

IBD inflammatory bowel disease

IDO Indoleamine 2,3-dioxygenase

IFN interferon

iGb3 isoglobotrihexosylceramide

IL interleukin

ILC innate lymphoid cells

iNKT cells invariant natural killer T cells

IRF4 interferon related factor 4

LAG3 lymphocyte-activation gene 3

LLT1 lectin-like transcript-1

LPG lypophosphoglycan

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LPS lipopolysaccharide

LTi cells lymphoid-tissue inducer

MIP macrophage inflammatory protein

mTOR mammalian target of rapamycin

NK cells natural killer cells

NOD mice non-obese diabetic mice

PBMC peripheral blood mononuclear cells

PIM4 phosphatidylinositol tetramannoside

PLZF promyelocytic leukemia zinc finger

RA retinoic acid

RORγt retinoic acid receptor-related orphan receptor γt

RT-PCR reverse transcription-polymerase chain reaction

Runx1 runt-related transcription factor 1

SAP SLAM-associated protein

SLAM signalling lymphocytic activation molecule

SMAD3 mothers against decapentaplegic homolog 3

STAT signal transducer and activator of transcription

TCR T cell receptor

TGF-β transforming growth factor-β

Th cells T helper cells

TLI total lymphoid irradiation

TNF tumour necrosis factor

Treg cells regulatory T cells

XLP X-linked lymphoproliferative syndrome

CHAPTER I

INVARIANT NKT CELLS

CHAPTER I –iNKT Cells

24

A brief history of NKT cells

Natural killer T (NKT) cells are a distinct population of immune cells that are found in mice

and humans and express both a T cell receptor (TCR) as well as classical natural killer

(NK) cell markers, particularly the NK1.1 (CD161) molecule. Nearly two decades ago,

three independent lines of study contributed to the identification of NKT cells. First, M.

Taniguchi and collaborators identified a canonical Vα14-Jα18 TCR (Jα18 was previously

known as Jα218 or Jα15) rearrangement in a set of suppressor hybridomas (Imai et al.,

1986; Koseki et al., 1989). Second, parallel studies from B.J. Fowlkes and R. Budd lead to

the identification of a small population of mouse double-negative (DN) T cells, i.e. CD4-

CD8- T cells, with a repertoire skewed toward TCR Vβ8 usage, suggesting a unique

population distinct from conventional T cells by its DN phenotype and a restricted TCR Vβ

repertoire (Budd et al., 1987; Fowlkes et al., 1987). Third, S. Porcelli and P. Dellabona, in

two independent studies, reported a recurrent Vα24-Jα18 rearrangement in human DN

peripheral blood lymphocytes (Dellabona et al., 1994; Porcelli et al., 1993). These

observations were linked together when a population of interleukin (IL)-4-producing CD4

and DN thymocytes co-expressing NK markers and a biased set of TCR β was identified

(Arase et al., 1992; Bendelac and Schwartz, 1991; Bendelac et al., 1992; Hayakawa et al.,

1992), which combined with a canonical Vα14-Jα18 in mouse and the homologous Vα24-

Jα18 in humans (Lantz and Bendelac, 1994). General interest in these cells increased

with the discovery that, in addition to IL-4 production, they were a potent source of other

immunoregulatory cytokines including interferon (IFN)-γ and tumour necrosis factor (TNF)

(Arase et al., 1992; Yoshimoto and Paul, 1994; Zlotnik et al., 1992). The finding that

mouse and human NKT cells were reactive to cells expressing the MHC class-I-like

molecule CD1d (Bendelac et al., 1994; Bendelac, 1995; Bendelac et al., 1995; Exley et

al., 1997), completed the initial characterization of this population.

NKT cells have long been defined as NK1.1+ T cells, however, it is now known that some

NKT cells do not express NK1.1 and some T cells expressing NK1.1 are not NKT cells,

making the definition based on NK1.1 and TCR too inaccurate. Nowadays, two categories

of T cell are referred to as NKT cells and their properties are summarized in the TABLE 1.

Type I NKT cells (also known as invariant NKT (iNKT) cells) express the invariant Vα24-

Jα18 TCR α-chain associated with the Vβ11 TCR β-chain in human and the homologous

Vα14-Jα18 associated with the Vβ8.2, Vβ7 or Vβ2 in mouse (Lantz and Bendelac, 1994).

Their invariant TCR αβ-chain specifically recognizes the glycosphingolipid antigen α-


25

galactosylceramide (α-GalCer; see below) presented by CD1d (Kawano et al., 1997), and

therefore, human and mouse type I NKT cells are specifically detected by flow cytometry

using CD1d tetramers loaded with α-GalCer antigen (α-GalCer-CD1d tetramers)

(Matsuda et al., 2000). The successful identification of α-GalCer and the development of

gene-manipulated mice that lack NKT cells (Jα18-deficient and CD1d-deficient mice) have

helped to elucidate the remarkable functional diversity of NKT cells from host defense to

immunoregulation (Bendelac et al., 2007; Kronenberg, 2005; Taniguchi et al., 2003). Type

II NKT cells are also CD1d reactive, but they have a more diverse TCR repertoire (Park et

al., 2001) that recognize a range of hydrophobic antigens, including sulfatide (Jahng et al.,

2004), lysophosphatidylcholine (Chang et al., 2008) and even small aromatic (non-lipid)

molecules (Van Rhijn et al., 2004). Much less is known about type II NKT cells because

we lack specific reagents to directly identify them. At present, the best way to study the

function of these cells in vivo is by comparing CD1d-deficient mice (which lack both type I

and type II NKT cells) with Jα18-deficient mice (which lack only type I NKT cells). In this

work, I focused my attention in type I NKT cells, particularly human type I NKT cells,

referred to hereafter as iNKT cells.

TABLE 1. Classification of NKT cells

Type I NKT cells Type II NKT cells

(iNKT cells)

CD1d dependent Yes Yes

α-GalCer reactive Yes No

TCR α-chain Vα24Jα18 (human)

Diverse Vα14Jα18 (mouse)

TCR β-chain Vβ11 (human)

Diverse Vβ8.2, Vβ7 and Vβ2 (mouse)

NK1.1 (CD161) +/- +/-

Adapted from Godfrey et al. 2000


26

iNKT cell development and distribution

iNKT cells are a thymus-dependent T-cell population, but they are developmentally and

functionally distinct from conventional CD4+ and CD8+ T cells. For instance, their

development is absolutely dependent on Runt-related transcription factor (Runx) 1 and

retinoic acid receptor-related orphan receptor (ROR)-γt, which influence but are not

required for the development of conventional T cells (Egawa et al., 2005). There is

convincing evidence that iNKT cells segregate from conventional T-cell development at

the double-positive (DP), i.e. CD4+CD8+, thymocyte stage in the thymic cortex

(Bezbradica et al., 2005; Egawa et al., 2005; Gapin et al., 2001). DP cortical thymocytes

that randomly produce the semi-invariant TCR, that characterizes iNKT cells, are

positively selected by interaction with CD1d molecules expressed by neighboring

thymocytes (Bendelac, 1995; Coles and Raulet, 1994, 2000; Wei et al., 2005). This

positive selection of iNKT cells requires the presentation of an undefined self antigen in

the context of CD1d. Isoglobotrihexosylceramide (iGb3) has been proposed as the

strongest candidate for iNKT selection (Schumann et al., 2006; Wei et al., 2006; Zhou et

al., 2004), however, recent studies showing that mice deficient for the enzyme iGb3

synthase have no apparent defect in iNKT cell development (Porubsky et al., 2007), and

that, in humans, iGb3 synthase does not seem to be expressed (Christiansen et al.,

2008), have challenged that idea. The cortical thymocyte-mediated selection is associated

with a different array of costimulatory signals provided by thymocytes, compared with that

of thymic epithelial cells, which may promote some of the unique characteristics

associated with iNKT cells. Positive selection by cortical thymocytes facilitates, for

instance, co-signalling via signalling lymphocyte activation molecule (SLAM) family

members, and mice deficient to SLAM adaptor protein (SAP), which is required for SLAM

signalling, lack iNKT cells, although normal numbers of conventional T cells are present

(Chung et al., 2005; Nichols et al., 2005; Pasquier et al., 2005). Similarly, in patients with

X-linked lymphoproliferative syndrome (XLP), caused by mutations in SAP, iNKT cell

development is severely impaired (Chung et al., 2005; Ma et al., 2007; Nichols et al.,

2005; Pasquier et al., 2005).

Given that iNKT cells develop following random TCR generation, with diverse TCR β-

chains, it is likely that iNKT cells are also susceptible to negative selection during their

development to eliminate potentially high self-reactive cells. In support of this idea, in the

presence of the potent ligand α-GalCer (Pellicci et al., 2003) or in the presence of CD1d-

transgenic dendritic cells (DC) (Chun et al., 2003) iNKT cell development is abrogated.


27

Once selected, iNKT cells precursors undergo a series of differentiation/maturation steps

that ultimately results in the iNKT cell pool. Based on studies in mice, at least four distinct

iNKT cell development stages have been defined through differences in expression of

CD24, CD44 and NK1.1 (FIGURE 1). The earliest precursor to emerge is defined as

CD24+CD44lowNK1.1low (stage 0). These cells are very rare and apparently non-dividing

(Benlagha et al., 2005). Then, they down regulate CD24 and they enter into a highly

proliferative phase (stage 1: CD24lowCD44lowNK1.1low). CD4- iNKT cells seem to branch

from CD4+ iNKT cells at this stage of development. While still proliferating, maturing iNKT

cells up regulate CD44 (stage 2: CD24lowCD44highNK1.1low) (Benlagha et al., 2002;

Benlagha et al., 2005). The up regulation of NK cell receptors such as NK1.1 define the

next maturation step (stage 3: CD24lowCD44highNK1.1high), and it is accompanied by much

less proliferation (Benlagha et al., 2002; Gadue and Stein, 2002; Pellicci et al., 2002).

These maturation steps are controlled by a variety of signal transducers molecules (e.g.

Fyn, SAP), transcription factors (e.g. NFκB, T-bet, Ets1, Runx1, ROR-γt, Itk, Rlk, AP-1),

and co-stimulatory molecules (e.g. CD28 and ICOS) (D'Cruz et al., 2010; Godfrey and

Berzins, 2007) and are accompanied of several important changes in iNKT cell function.

Importantly, recent studies have shown a critical role of the transcription factor

promyelocytic leukemia zinc finger (PLZF) in directing the effector differentiation of iNKT

cells during thymic development (Kovalovsky et al., 2008; Savage et al., 2008). Mouse

iNKT cells from stage 1 and stage 2 produce high levels of IL-4 and IL-10, but little IFN-γ,

whereas, stage 3 iNKT cells produce abundant IFN-γ but less IL-4 and little if any IL-10

(Benlagha et al., 2002; Gadue and Stein, 2002; Pellicci et al., 2002). Mouse IL-17-

producing iNKT cells (discussed below) originate from a separate pathway of iNKT cell

development that seems to be regulated by RORγt (Michel et al., 2008). At our laboratory,

it was found that IL-17-producing iNKT cells are already present in the thymus, belonging

restrictedly to CD44highNK1.1-CD4- iNKT cells that express ROR-γt. These iNKT cells,

regarded so far as an immature stage of thymic iNKT cell development, fail to generate

other differentiation stages indicating that they are already mature cells. In contrast ROR-

γtneg iNKT cell precursors mature to other stages, but acquire neither ROR-γt expression

nor the ability to secrete IL-17 (Michel et al., 2008). The significance of cytokine

production by thymic iNKT cells is currently unclear, because there is no evidence that

these cells normally become activated, and it remains to be determined whether they

have a distinct physiological function. Recently, it was reported that IL-4 secreted by

thymic iNKT cells is required for the generation of memory-like CD8+ T cells in the thymus

(Lai et al., 2011), suggesting that cytokine production by thymic iNKT cells may play a role

in the development of other T cell subpopulations.


28

FIGURE 1. Mouse iNKT cell development and maturation. Mouse iNKT cells arise in the thymus

from a common precursor pool of double-positive (DP) thymocytes. Those expressing a TCR that

binds to CD1d plus self-antigen, expressed by other DP thymocytes, enter the iNKT cell lineage

(blue). Once selected iNKT cell precursor undergo a series of differentiation steps that ultimately

results in iNKT cell pool. (Adapted from Godfrey et al. 2010).

Human iNKT cells seem to follow a similar process of differentiation during foetal life,

giving rise at birth to an activate/memory phenotype (CD45RO+CD62L-) (D'Andrea et al.,

2000). The earliest detectable iNKT cell precursors are CD4+ and CD161low and CD4-

CD161high iNKT cells arise at later development stages (Baev et al., 2004; Berzins et al.,

2005) but the precise stage at which the CD4- iNKT cell lineage emerges in humans is

unclear. Mouse iNKT cells that emigrate from the thymus mostly do so at stage 2 and

progress to stage 3 in the periphery (FIGURE 1). This seems to be similar in humans, in

which most iNKT cells leave the thymus at the CD4+CD161low stage. But whereas, in

mice, maturation to stage 3 can occur in parallel in the thymus and in the periphery, in

humans, the equivalent maturation step does not seem to occur in the thymus, or at least,

CD161high iNKT cells are extremely rare in human thymus, in contrast to their frequency in

human blood (Baev et al., 2004; Berzins et al., 2005; Sandberg et al., 2004). Moreover,


29

iNKT cells present in human cord blood require an additional stimulus in the periphery to

be completely able to produce cytokines upon antigen activation (Baev et al., 2004;

D'Andrea et al., 2000), contrasting with mouse iNKT cells that become functionally mature

in the thymus (Benlagha et al., 2002; Pellicci et al., 2002).

Another important difference between human and mouse iNKT cells relates to their

frequency (TABLE 2). In mice, iNKT cells represent 0.2-0.5% of lymphocytes in the

thymus, spleen, blood and bone marrow, and 15-35% of liver lymphocytes. In humans,

the frequency of iNKT cells is lower and with a high degree of variability between

individuals. iNKT cells typically represent 0.01-0.1% (ranging from 0.001% to 3%) of

human peripheral blood mononuclear cells (PBMC) (Chan et al., 2009; Gumperz et al.,

2002; Kim et al., 2002; Lee et al., 2002a) and there are similar frequencies of iNKT cells in

human bone marrow and spleen. The frequency of human iNKT cells is lower in the

thymus (~0.001-0.01% of lymphocytes) (Baev et al., 2004; Berzins et al., 2005) and

higher in the liver (~1%) (Lynch et al., 2009) and omentum (10%) (Lynch et al., 2009). In

humans, the frequency of iNKT cells in blood is not directly related to iNKT cell frequency

in the thymus (Berzins et al., 2005). This lack of correlation may reflect the existence of

parallel pathways of iNKT cell maturation in the thymus and periphery mentioned above.

The factors that regulate peripheral iNKT cell development and homeostasis are not

completely understood. Mouse iNKT cells exhibit a basal level of slow proliferation at the

periphery (0-2 divisions per week) that is dependent on IL-15 and to a lesser extent on IL-

7, with little or no requirement for TCR signals (Matsuda et al., 2002). Human iNKT cells

also turn over slowly in the periphery and respond to IL-15 and IL-7 (Baev et al., 2004;

Sandberg et al., 2004). However, distinct peripheral homeostatic requirements of human

CD4+ and CD4- iNKT cells were described. IL-15 receptor is preferentially expressed in

CD4- iNKT cells, which predominantly respond to IL-15. In contrast, CD4+ iNKT cells

mainly express IL-7Rα and are more sensitive to IL-7 (Baev et al., 2004). This may

contribute to the differences observed in human CD4+/CD4- iNKT cell ratio between birth

and adult life. CD4+ iNKT cells are mainly supported by thymic output and survive in

periphery with limited cell division. In contrast, the number of CD4- iNKT cells mostly

depends on peripheral expansion; CD4- iNKT cells are relatively infrequent in the human

thymus, cord blood and neonatal peripheral blood, yet they accumulate in the blood with

age (Baev et al., 2004; Berzins et al., 2005). It is still unclear whether CD4- iNKT cells

derive directly from the CD4+ iNKT cells emigrated from the thymus, or whether there is a

disproportionate peripheral expansion of CD4- iNKT cells in periphery that results in them

becoming a major subset in adults.


30

TABLE 2. Comparison of human and mouse iNKT cells

Human Mouse

Semi-invariant T cell receptor Vα24; Vβ11 Vα14; Vβ8.2, Vβ7 or Vβ2

Development in the thymus

Yes, although functional maturity is reached in the periphery.

Yes, all mature subsets develop in the thymus.

Functionally distinct mature iNKT cell subsets

CD4+CD8-, CD4-CD8-, and CD4-CD8+

CD4+CD8-, CD4-CD8-, NK1.1+ and NK1.1-

Potent cytokine production*

Yes (TNF, IFN-γ, IL-4, IL-10, IL-13 and GM-CSF; IL-17⌘, IL-22⌘ and IL-21⌘)

Yes (TNF, IFN-γ, IL-2, IL-4, IL-10, IL-13, IL-17, IL-22, IL-21 and GM-CSF)

Present at birth Yes, mostly CD4+ iNKT cells at birth. The CD4- iNKT cell subset emerges with age.

No, first detected at ~5 days after birth

Frequency in the blood 0.01-0.1% (highly variable; reported frequencies range from undetectable to > 3%).

0.2-0.5%

Relative frequency in tissues

Highest in liver (~1%) and omentum (~10%) Highest in liver (~30%)

Similar in spleen, blood, bone marrow and lymph nodes (0.01-0.5%)

Similar in thymus, spleen, blood and bone marrow (0.2-0.5%)

Lowest in the thymus (<0.001-0.01%)

Lowest in lymph nodes (0.1-0.2%)

Adapted from Berzins et al. 2011. *The cytokine profile varies between iNKT cell subsets. ⌘Results obtained during my PhD study (presented and discussed below).


31

iNKT cell agonists

Unlike classical MHC-restricted T cells, which are selected for recognition of non-self

compounds, iNKT cells have been found to recognize both self and foreign molecules.

Their semi-invariant TCR, selected during thymic development, enables all iNKT cells to

recognize a specific molecular pattern in which a carbohydrate is attached in an α-

anomeric conformation to the polar head group of a lipid (Kjer-Nielsen et al., 2006; Scott-

Browne et al., 2007). The prototypical lipid of this category, the glycosphingolipid α-

GalCer (FIGURE 2a), was the first agonist described for iNKT cells (Kawano et al., 1997).

α-GalCer, originally extracted from the marine sponge Agelas mauritianus during a screen

for reagents that prevent tumour metastases in mice (Morita et al., 1995), is a very potent

iNKT cell agonist and has been extensively studied with regard to its interaction with

CD1d and the invariant TCR of iNKT cells, its immunomodulatory activities and its

therapeutic properties (Van Kaer, 2005). Since its discovery, many α-GalCer analogues

have been synthesized (FIGURE 2b), including OCH (Miyamoto et al., 2001), β-GalCer, α-

GlcCer, α-C-GalCer (Schmieg et al., 2003) and PBS-57 (Liu et al., 2006b), with higher or

lower affinity for the iNKT cell TCR. Importantly, mammalian cells do not seem to produce

glycolipids in which the carbohydrate is attached to the lipid via an α-linkage, and thus self

antigen (or self antigens) recognized by iNKT cells apparently do not contains this

molecular pattern. The glycolipid iGb3 has been identified as the self-antigen recognized

by iNKT cells (Zhou et al., 2004). However, this glycolipid is not essential for the

development of mouse and human iNKT cells (discussed above), suggesting that other

not yet identified compound (or compounds) also function as iNKT cell self antigen (or self

antigens).

One characteristic and intriguing aspect of iNKT cells is their intrinsic autoreactivity. The

mechanisms implicated need to be determined since we it is still speculate whether iNKT

cells have an intrinsic affinity for CD1d or whether they recognize a particular self-lipid-

CD1d complex. Recently, two papers shed light on these key points. Wun et al. (2011)

found that modifications to the galactosyl head group altered the affinity of iNKT-TCR

binding, while modifications to the lipid tails did not generally affect iNKT-TCR binding.

The authors showed the crystal structures of complexes between the iNKT-TCR and

CD1d using five of the investigated AGLs (altered glycolipid ligands) and reported that the

iNKT cell footprint on each CD1d-AGL was essentially identical, with almost all contacts

between the iNKT-TCR and CD1d conserved. This “lack” of substantial structural

differences suggest that binding of the iNKT-TCR requires enforcement of an “induced fit”

that could alter the kinetics of signalling, consequently the multiple Vβ domain usage in


32

mouse iNKT cells endows these cells with an increased flexibility for recognition of CD1d-

presented lipid antigens (Wun et al., 2011). It remains to be determined whether human

iNKT cells, which do not vary in their Vβ domain usage, have similar flexibility.

The second paper by Mallevaey et al. (2011) investigated iNKT cell ability to recognize

CD1d without exogenous antigen. The authors engineered a set of “sticky” iNKT-TCRs,

randomized at the CDR3β and selected to bind “empty” CD1d and they reported that this

recognition was not necessarily antigen specific. They reported that iNKT cell

autoreactivity can be the result of a hydrophobic motif within the CDR3β loop of the iNKT

TCR that mediates direct recognition with CD1d in an antigen-independent manner

(Mallevaey et al., 2011).

Many different self lipids have been shown to bind CD1d molecules and recent findings

showed that human iNKT cells could be stimulated by lysophosphatidylcholine and

lysosphyngomyelin loaded onto CD1d at the surface of antigen presenting cells (APC)

(Fox et al., 2009). Such reactivity has not yet been extended to mouse iNKT cells, and it is

not clear what role these lysophospholipids might have in iNKT cell positive selection in

the thymus and/or in iNKT cell autoreactivity in the periphery.

Lipids with structural similarity to α-GalCer have also been identified from several

microbial sources (FIGURE 2c). Recent works have demonstrated that iNKT cells

recognize α-glycosphingolipids from Sphingomonas (S. capsulate, S. paucimobilis and S.

wittichii) and Ehrlichia muris (Kinjo et al., 2005; Mattner et al., 2005; Sriram et al., 2005),

in a CD1d-dependent manner. Moreover, it was reported that mouse and human iNKT

cells also recognize α-galactosyl diacylglycerols from Borrelia burgdorferi, the causative

agent of Lyme disease (Kinjo et al., 2006). Other microbial lipids that have been reported

to activate iNKT cells are the Leishmania donovani lypophosphoglycan (LPG) (Amprey et

al., 2004) and the phosphatidylinositol tetramannoside (PIM4) purified from

Mycobacterium leprae (Fischer et al., 2004), although synthetic PIM4 does not stimulate

iNKT cells (Kinjo et al., 2006).


33

FIGURE 2. Structure of some glycolipid antigens recognized by iNKT cells. a. Structure of α-

galactosylceramide (α-GalCer), the first known antigen for iNKT cells, originally extracted from a

marine sponge. b. Structure of synthetic analogues of α-GalCer: OCH and α-C-GalCer. c.

Structure of microbial glycolipids recognized by iNKT cells: GalA-GSL (glycosphingolipid containing

galacturonic acid) originally extracted from Sphingomonas spp. and BbGL-IIc (monogalactosyl

diacylglycerol lipid) originally extracted from Borrelia burgdorferi. (Adapted from Tupin et al. 2007).


34

iNKT cell activation

The identification of microbial iNKT cell antigens provides an explanation for the activation

of iNKT cells by some microorganisms, but does not explain the capacity of iNKT cells to

become activated in response to microorganisms that lack those cognate antigens or

during inflammatory or autoimmune responses. Two major models of iNKT cell activation

during microbial infection have been proposed, a direct and an indirect pathway (FIGURE

3). The direct pathway of activation, involves iNKT cell recognition of specific microbial

lipids as foreign antigens. In contrast, in the indirect pathway, iNKT cells are activated by

recognition of self-antigen in the presence (or not) of co-stimulation by cytokines as IL-12

and IL-18 that are produced by APC upon Toll-like receptor (TLR) signalling (Tupin et al.,

2007). Support for the direct pathway, comes from the identification of bacterial glycolipids

antigens that have a wider distribution, that bind to CD1d and activate iNKT cells (Kinjo et

al., 2005; Kinjo et al., 2006; Mattner et al., 2005; Sriram et al., 2005). However, when APC

are exposed to microbial pathogens, they can also stimulate iNKT cells in a CD1d-

dependent manner that does not require microbial antigens (Brigl et al., 2003; Mattner et

al., 2005), supporting the indirect pathway. This might be mediated through the

recognition of specific self-antigen that is otherwise not present by CD1d molecules in the

steady state and/or iNKT cell sensitivity to self-antigen is increased by the presence of

microbial pathogens. For instance, it was reported that human iNKT cell response to self

antigen-CD1d complexes is amplified by IL-12 produced by DC in response to Salmonella

typhimurium (Brigl et al., 2003). Another study reported that, in response to Schistosoma

mansoni egg antigens, iNKT cells can be activated by self antigens presented by CD1d,

even in the absence of TLR signalling and IL-12 (Mallevaey et al., 2006). Several years

ago, it was demonstrated in our laboratory that proinflammatory cytokine IL-18 associated

with IL-12 activate mouse iNKT cells even in the absence of TCR engagement, reporting

a new type of iNKT cell activation (Leite-De-Moraes et al., 1999). These findings were

recently confirmed by the demonstration that iNKT cells can be stimulated in response to

IL-12 and IL-18 produced by DC activated by Escherichia coli lipopolysaccharide (LPS), in

the absence of TCR stimulation (Nagarajan and Kronenberg, 2007), indicating that

inflammatory cytokines as IL-12 and IL-18, which are produced by TLR-stimulated APC,

are sufficient to induce iNKT cell activation. Others studies have also reported that mouse

iNKT are activated in vivo by different TLR ligands, such as LPS and R848 (Askenase et

al., 2005; Grela et al., 2011). Recently, it was suggested that innate and cytokine-driven

signals, rather than microbial antigens, dominate in iNKT cell activation during microbial

infection (Brigl et al., 2011). Similar mechanisms might be involved in iNKT cell activation

during inflammatory and autoimmune responses, which can implicate production of TLR


35

ligands, release of pro-inflammatory cytokines and/or alterations in glycolipid

homeostasis.

FIGURE 3. Models of iNKT cell activation during microbial infection. a. Direct activation. iNKT

cells are activated by recognition of microbial antigens presented by CD1d molecules on DC

surface. b. Indirect activation. iNKT cells are activated by the combination of IL-12 and IL-18

produced by TLR-stimulated DC and recognition of endogenous glycolipid antigens. (Adapted from

Tupin et al. 2007).

iNKT cell functions

Activation of iNKT cells results in TCR down regulation, proliferation and prolonged

cytokine secretion (Crowe et al., 2003; Harada et al., 2004; Wilson et al., 2003). Secretion

of the prototypical Th1 and Th2 cytokines, IFN-γ and IL-4, respectively, by human iNKT

cells has been thoroughly documented, but they produce many others cytokines, including

IL-2, TNF, IL-5, IL-13, IL-10, (Gumperz et al., 2002; Kim et al., 2002; Lee et al., 2002a),

IL-21, IL-22 and IL-17 (results obtained during my PhD thesis; presented and discussed

below) (Moreira-Teixeira et al., 2011). Human iNKT cells also produce growth factors for

hematopoietic cells such as granulocyte macrophage colony-stimulating factor (GM-CSF)

and chemokines such as macrophage inflammatory protein (MIP)-1α and MIP-1β


36

(Gumperz et al., 2002; Snyder-Cappione et al., 2010). In addition, these cells have

cytolytic activity owing to perforin and Fas ligand (FasL or CD95L) expression (Gumperz

et al., 2002).

The T helper (Th) 1 versus Th2 outcome of iNKT cell activation is not yet completely

understood. It was recently shown that human iNKT cells produce distinct cytokines in

response to increasing TCR signal strength: GM-CSF and IL-13 are activated by exposure

to low doses of α-GalCer, higher levels of α-GalCer increase secretion of these cytokines

and also induce IFN-γ and IL-4, and production of IL-2 requires the highest amount of

antigen (Wang et al., 2008). Self-antigenic stimulation of iNKT cells appears to provide

relatively weak TCR signalling and led mainly to secretion of GM-CSF and IL-13, with little

IFN-γ or IL-4, and generally undetectable IL-2 (Wang et al., 2008). However, in the

presence of cytokines such as IL-12, IL-15 and IL-18 human iNKT cells are able to

produce IFN-γ, but not IL-4, in response to suboptimal TCR stimulation (Salio et al., 2007).

In mouse, IL-12 alone or associated to TCR stimulation favours the production of IFN-γ by

iNKT cells, whereas IL-18 or IL-7 increases the production of IL-4 (Leite-De-Moraes et al.,

1997; Leite-De-Moraes et al., 1998; Leite-De-Moraes et al., 2001; Vicari et al., 1996).

Mouse iNKT cells also produce IFN-γ, but not IL-4, in response to IL-12 plus IL-18 or IL-33

(a Th2 cytokine), even in the absence of TCR stimulation (Bourgeois et al., 2009; Leite-

De-Moraes et al., 1999; Nagarajan and Kronenberg, 2007). Another study, also from our

laboratory, showed that histamine play a major role in the functional properties of mouse

iNKT cells, since a strong decrease in IL-4 and IFN-γ production by activated iNKT cells

was observed in histamine-deficient mice and cytokine production was restored when

exogenous histamine was added to these mice before iNKT cell activation (Leite-de-

Moraes et al., 2009). Moreover, recent studies have shown that different iNKT cell

agonists can elicit distinct iNKT cell response. OCH preferentially induce IL-4 production

(Miyamoto et al., 2001), whereas, others, such as the α-C-GalCer, promote a Th1 bias in

the cytokine production profile of mouse iNKT cells (Schmieg et al., 2003). Together these

observations suggest that the effector functions displayed by iNKT cells are strongly

influenced by the nature, strength and context of the stimulus.


37

iNKT cell interaction with other cells

Not only do iNKT cells have the capacity to rapidly and robustly produce cytokines and

chemokines, they also have the ability to influence the behaviour of many other immune

cells (FIGURE 4). Upon stimulation, activated iNKT cells can alter the strength and

character of immune responses through crosstalk with DC, NK cells, B cells and T cells,

and by shifting cytokine responses to (or from) a Th1, Th2 or Th17 cell-type profile

(Bendelac et al., 2007; De Santo et al., 2008; De Santo et al., 2010; Matsuda et al., 2008). DC maturation is a crucial event of the induction of most adaptive immune responses.

iNKT cell-induced DC maturation has been extensively documented in vitro and in vivo

after mouse iNKT cell stimulation with α-GalCer presented by CD1d (Fujii et al., 2002;

Fujii et al., 2003; Fujii et al., 2004; Kitamura et al., 1999). Upon activation, iNKT cells up

regulate CD40 ligand (CD40L), which interacts with CD40 on DC to induce DC maturation

as evidenced by increased expression of CD86, IL-12 production, and priming of T cell

responses (Fujii et al., 2004; Kitamura et al., 1999). In turn, IL-12 production and up

regulation of CD70 and OX40 ligand expression, by maturing DC, enhance iNKT cell

activation and cytokine secretion (Taraban et al., 2008; Zaini et al., 2007). IL-12 derived

from DC and IFN-γ production resulting from iNKT cell activation promotes a prompt NK

cell activation, including proliferation, up regulation of CD69 expression, additional IFN-γ

secretion and increase in cytotoxic activity (Carnaud et al., 1999; Eberl and MacDonald,

2000; Lisbonne et al., 2004). Likewise, activated human iNKT cells have also been shown

to promote NK cytotoxic function, which is promoted by IL-2 (and enhanced by IFN-γ)

production (Metelitsa et al., 2001).

Beyond the ability to facilitate DC maturation and NK cell activation, iNKT cell derived

cytokines can modulate the recruitment of myeloid progenitors and granulocytes to the

periphery. Upon activation, mouse iNKT cells contribute to the mobilization of myeloid

progenitors and neutrophils from bone marrow to the periphery, through IL-3 and GM-CSF

production (Leite-de-Moraes et al., 2002). More recently, it was also reported that IL-17

production by mouse iNKT cells contribute to neutrophils recruitment to inflammatory sites

(Michel et al., 2007).

The cross-talk between iNKT cells and DC also results in the induction of antigen specific

response by CD4+ and CD8+ T cells (Fujii et al., 2003; Stober et al., 2003), in a CD40-

dependent but IFN-γ-independent manner (Hermans et al., 2003). Thus, iNKT cells

augment both innate and adaptive immune responses as a consequence of DC

maturation. Mouse iNKT cells can support and sustain Th1 responses by facilitating DC


38

maturation and IL-12 production and by activating NK cells, which secrete IFN-γ (Fujii et

al., 2003; Fujii et al., 2004). Under different inflammatory conditions, iNKT cell activation

favours Th2 differentiation by producing IL-4 (Singh et al., 1999). More recently, it has

become clear that mouse iNKT cells inhibit Th17 differentiation, either by a cell contact

dependent (Oh et al., 2011) or independent mechanism (Mars et al., 2009), which

requires IL-4, IL-10 and IFN-γ. In human, a recent study showed that human iNKT cells

down regulate IL-23 production by DC supressing IL-17 production by memory CD4+ T

cells (Uemura et al., 2009). Mouse iNKT cells can also provide effective help for CD8+ T

cells. Activated iNKT cells enhance CD8+ T cell activation, IFN-γ production and cytotoxic

function (Fujii et al., 2003; Silk et al., 2004; Stober et al., 2003). In contrast to the studies

in mice, human iNKT cells demonstrate inhibition rather than enhancement of antigen

specific cytotoxic T cell responses in vitro by the production of Th2 type cytokines (IL-4,

IL-5 and IL-10) (Osada et al., 2005).

In addition to promoting the generation of potent antigen-specific CD4+ and CD8+ T cell

responses, activation of iNKT cells can also provide help to B cells. Human iNKT induce

proliferation of naïve and memory B cells and higher antibody production (Galli et al.,

2003). The interaction between human iNKT cells and B cells requires CD1d molecules

on B cell surface but seems independent of exogenous antigens. In mice, activation of

iNKT cells enhances antibody responses to protein antigens in vivo, through CD40-CD40L

interaction and cytokine release (Galli et al., 2007). More recently, it was described that B

cell receptor (BCR) recognizes specific lipid antigens that are internalized and presented

by CD1d molecules to iNKT cells. As a result, activated iNKT cells provide help for B cell

proliferation and enhance specific antibody response (Barral et al., 2008; Leadbetter et

al., 2008)

As massive iNKT cell-derived cytokine release may strongly influence the subsequent

adaptive immune response, iNKT cell function has to be tightly regulated. Activated

human iNKT cells promote CD4+CD25+Foxp3+ regulatory T (Treg) cell proliferation by a

IL-2 dependent mechanism (Jiang et al., 2005), which in turn can suppress the

proliferation, cytokine production, and cytolytic activity of human iNKT cells by a cell

contact dependent mechanism (Azuma et al., 2003). Although, recent reports have

provided evidence for the reciprocal cross-talk between Treg cells and iNKT cells (La

Cava et al., 2006), the precise mechanism implications of this interaction remains unclear.


39

FIGURE 4. iNKT cells interact and modulate the function of many different cell types. iNKT

cells directly or indirectly modulate the function of many other cell types, such as NK cells and T

cells. iNKT cell-DC interactions are bidirectional, as iNKT cells receive signals from DC and vice-

versa. Signals can be received through cell-surface receptors, such as TCR recognizing glycolipid-

CD1d complexes, co-stimulatory receptors, as well as through soluble mediators, such as

cytokines. (Adapted from Cerundolo et al. 2009).

iNKT cell heterogeneity

It is becoming increasingly clear that iNKT cells can and do respond differently under

different circumstances. Indeed, the functional versatility of iNKT cells is increasingly

being attributed to iNKT cell subsets with distinct cytokine profiles (Godfrey et al., 2010).

Although iNKT cells are defined by their invariant TCR, the population itself is clearly

heterogeneous in its expression of other cell surface markers and differential expression

of the CD4 co-receptor and NK markers have been shown to discriminate between

functionally distinct iNKT cell subsets.


40

Mature iNKT cells from humans and mice can be divided into functionally distinct

CD4+CD8− and CD4−CD8− (DN) subsets (Crowe et al., 2005; Lee et al., 2002a), and

humans also have a CD4−CD8+ iNKT cell subset that is not found in mice (Gumperz et al.,

2002; Kim et al., 2002). Human DN and CD4-CD8+ iNKT cells are highly cytolytic and

produce Th1 type cytokines, whereas CD4+CD8− iNKT cells are broadly associated with

Th0 type immune responses (Gumperz et al., 2002; Lee et al., 2002a), and these subsets

differently regulate DC activity (Liu et al., 2008). In fact, the DC stimulated by the CD4+

iNKT cell subset preferentially induce Th1 responses, whereas the DC stimulated by the

DN iNKT cell subset induce a shift toward Th2 responses (Liu et al., 2008). There is also

marked heterogeneity in the expression of functionally important cell surface markers,

such as adhesion molecules and chemokines receptors, by CD4+ and CD4− iNKT cells

(TABLE 3) (Gumperz et al., 2002; Kim et al., 2002; Lee et al., 2002a; Montoya et al.,

2007), which indicates that additional subsets might also exist and suggesting that they

might be targeted to different tissues and perform different immune functions.

TABLE 3. Comparison of human CD4+ and CD4- iNKT cell subsets

CD4+ CD4-

Effector function Th1 and Th2 cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-13 and IL-10); GM-CSF; FasL

Th1cytokines mostly (IFN-γ and TNF-α); perforin

Chemokine receptors CCR1low, CCR2, CCR4, CCR5, CCR7low, CXCR3, CXCR4

CCR1, CCR2, CCR5, CCR6, CCR7low, CXCR3, CXCR4, CXCR6

Adhesion molecules CD49a, CD62L, CLA, α4β7 CLA, α4β7, CD11ahigh

NK receptors CD161 CD161, 2B4, CD94, NKG2A, NKG2D

Adapted from Kim et al. 2002

Although, in mice, functional distinctions are less obvious for CD4+ and CD4- iNKT cell

subsets, the division of iNKT cells based on NK1.1 expression has revealed striking

differences between mouse NK1.1- and NK1.1+ iNKT cell subsets in the thymus. Thymic

NK1.1- iNKT cells produce large amounts of IL-4 and little IFN-γ, whereas NK1.1+ iNKT


41

cells produce less IL-4 and more IFN-γ (Benlagha et al., 2002; Pellicci et al., 2002). More

recently, several studies have converged on the description of a mouse CD4- NK1.1- iNKT

cell subset that produces large amounts of IL-17 but little IL-4 or IFN-γ and constitutively

expres the receptor for IL-23 and the transcription factor ROR-γt (two features of Th17

cells, discussed below) (Coquet et al., 2008; Lee et al., 2008; Michel et al., 2007; Michel

et al., 2008; Rachitskaya et al., 2008; Yoshiga et al., 2008). These cells, named to as

“iNKT17 cells”, can be further distinguished from other iNKT cells by expression of CCR6,

CD103 and CD121 (Doisne et al., 2009) and are present in thymus, spleen, liver and lung

as a small subset of total iNKT cells (Coquet et al., 2008; Michel et al., 2007; Michel et al.,

2008; Rachitskaya et al., 2008), but are highly represented in peripheral lymph nodes

(Coquet et al., 2008; Doisne et al., 2009). Mouse iNKT cells also produce other Th17

associated cytokines, including IL-22 and IL-21 (Coquet et al., 2007; Coquet et al., 2008;

Goto et al., 2009). Although it remains to be demonstrated how IL-17, IL-22 and IL-21 are

all produced by the same or by distinct iNKT cell subsets, a recent study of our laboratory

clear demonstrated that IL-17 and IL-22 are co-produced by ROR-γt+ iNKT cells (Massot

et al., submitted for publication). The secretion of IL-17 by mouse iNKT17 cells may be

triggered after TCR engagement by glycolipid-CD1d complexes (Doisne et al., 2009;

Michel et al., 2007; Rachitskaya et al., 2008) or after stimulation with IL-1β and/or IL-23

(Massot et al., submitted for publication) and is strongly enhanced in vivo by the presence

of LPS or LPS-activated DC (Doisne et al., 2009). These iNKT17 cells seem to have an

important role in IL-17-associated diseases, including airway induced neutrophilia (Lee et

al., 2008; Michel et al., 2007), ozone-induced asthma (Pichavant et al., 2008) and

collagen-induced arthritis (Yoshiga et al., 2008).

The discovery of this unique iNKT cell subset in mouse, led us to ask if an iNKT cell

subset with similar featues exists in humans.

iNKT cells in human diseases

As more is learned about iNKT cell heterogeneity, it is increasingly apparent that care

must be taken to study each subset separately to better understand the role of these

subsets in human diseases and optimize iNKT cell based therapies.

Many clinical studies have reported a strong association between iNKT cell defects and

increased susceptibility to many autoimmune diseases, cancer and infections (Berzins et

al., 2011). However, for most of these conditions, the iNKT cell defect has been only


42

partially characterized and in some cases has been disputed by contradictory studies.

One problem is that many of the human studies used imprecise methods for the

identification of iNKT cells and carried out only limited analysis of the distribution and/or

function of iNKT cell subsets. Therefore, there is no real consensus in the field about the

implication of iNKT cell deficiencies and functional defects in human diseases, or the

potential for using iNKT cells as a biomarker or therapeutic agent (Rubio et al., submitted

for publication).

FIGURE 5. iNKT cells and human disease. A causative association between iNKT cells and

disease is poorly defined, but probably involves one of two mechanisms. a. In the first mechanism,

decreased frequency and/or function of iNKT cells negatively affect their immunoregulatory role

and thus diseases associated with failure of immune regulation become more common. b. The

second mechanism involves a direct or indirect pathogenic role of iNKT cells, in which iNKT cells

respond inappropriately to self (or non-self) antigens or cytokines, contributing to allergy and

inflammatory diseases. (Adapted from Berzins et al. 2011).

The reported associations between iNKT cells and human disease are grouped into three

categories (Berzins et al., 2011): (i) iNKT cell defects (e.g. decreased frequency and/or

impaired cytokine production) are thought to compromise immune regulation and increase

predisposition to autoimmune diseases, cancer and some infections (FIGURE 5a)

(Bendelac et al., 2007; Terabe and Berzofsky, 2008; Wu and Van Kaer, 2009); (ii) iNKT

cells can be normal in number and functionally competent, but they respond


43

inappropriately to self (or non self) glycolipid antigens or cytokines deploying a pathogenic

immune responses that could contribute to diseases such as atherosclerosis, asthma,

allergy and some skin disorders (FIGURE 5b) (Balato et al., 2009; Bendelac et al., 2007;

Pham-Thi et al., 2006b; Umetsu and Dekruyff, 2010); (iii) iNKT cells do not necessarily

contribute to disease pathology, but the stimulation of iNKT cell function (for example, by

administering glycolipids) might be beneficial for treating the disease (Wu et al., 2009).

iNKT cells in autoimmunity. Evidences for the potential influence of iNKT cells in

autoimmune diseases comes from the finding that various mouse strains that are

genetically susceptible to autoimmunity have defective iNKT cell number and/or function

(Wu and Van Kaer, 2009). For instance, non-obese diabetic (NOD) mice have defects in

iNKT cell frequency and cytokine production (Fletcher and Baxter, 2009; Novak et al.,

2007) and the development of diabetes in these mice can be prevented by adoptive

transfer of iNKT cells or by overexpression of the iNKT cell TCR (Lehuen et al., 1998).

Overexpression of CD1d (Wang et al., 2001) or stimulation of iNKT cells with α-GalCer

also prevents the development of diabetes in NOD mice (Sharif et al., 2001), and in iNKT

cell-deficient (CD1d- or Jα18-deficient) NOD mice diabetes is exacerbated (Fletcher and

Baxter, 2009; Wang et al., 2001). In human, contradictory studies reported either defective

iNKT cell numbers or function in individuals with, or at high risk of, type 1 diabetes (Kent

et al., 2005; Kis et al., 2007; Kukreja et al., 2002; Wilson et al., 1998), no change in iNKT

cell numbers or function (Lee et al., 2002b; Tsutsumi et al., 2006) or increased iNKT cell

numbers in patients with a recent-onset of the disease (Oikawa et al., 2002). Explanations

for the conflicting results include genetic and environmental factors, patient age, stage of

disease and the methods used to characterize iNKT cells. One additional concern is that

the iNKT cell numbers in the blood of NOD mice is normal, despite there being significant

deficiencies of iNKT cells in other tissues (Berzins et al., 2004). It is not known whether

iNKT cells from the peripheral blood provide a better image of the systemic iNKT cell pool

in humans than in mice, but more extensive characterization of human iNKT cells

(particularly from the tissues) is needed before iNKT cell defects can be excluded as a

contributing factor in human type 1 diabetes. Despite conflicting observations in human

type 1 diabetes, altered functions or decreased numbers of iNKT cells have been

observed in patients with rheumatoid arthritis (Linsen et al., 2005; Segawa et al., 2009)

and multiple sclerosis (Araki et al., 2003; Gausling et al., 2001; Illes et al., 2000),

supporting an association between iNKT cell defects and human autoimmune disease.

iNKT cells in tumour immunity. The potential importance of iNKT cells in tumour

surveillance has been established in mice, in which defective iNKT cells predispose to and


44

the adoptive transfer or stimulation of iNKT cells can provide protection against cancer

(Smyth et al., 2000; Swann et al., 2007; Terabe and Berzofsky, 2008). The CD4− iNKT cell

subset seems to have the main role in surveillance of some tumours in mice (Crowe et al.,

2005), and the cytokine profile of CD4− iNKT cells in humans indicates that they might

have a similar role (Gumperz et al., 2002; Lee et al., 2002a). A decrease in the number

and function of iNKT cells has been reported in the peripheral blood of patients with

cancer (Konishi et al., 2004; Molling et al., 2005) as well as in the tissues surrounding

tumours (Lynch et al., 2009; Song et al., 2007) and in the tumours themselves (Balato et

al., 2009). Some studies have been shown that iNKT cells from patients with cancer

produce less IFN-γ than iNKT cells from healthy individuals (Molling et al., 2005; Tahir et

al., 2001), which could potentially decrease the IFN-γ-dependent antitumour activities of

NK cells and CD8+ T cells. Other studies have reported an increased frequency of

intratumour iNKT cells in some patients (Bricard et al., 2009; Tachibana et al., 2005),

suggesting an antitumour role for iNKT cells. Frequency of iNKT cells is reported to

correlate inversely with tumour burden and positively with prognosis (Molling et al., 2007),

although it is not clear whether iNKT cell deficiencies could be the cause or effect of the

cancer.

iNKT cells in host defense. As noted above, the specificity of iNKT cells for certain

bacterial and parasitic lipids suggests that they may assist or modulate immunity to

infection, but few studies showed a role of human iNKT cells in infectious disease. The

identification of Borrelia burgdorferi (Kinjo et al., 2006) and Sphingomonas spp. (Kinjo et

al., 2005; Mattner et al., 2005) antigens that can stimulate iNKT cells suggests that

defects in iNKT cells could predispose to Lyme disease and to certain nosocomial

infections, although this has not been formally tested and the natural role of iNKT cells in

immunity to these microorganisms remains uncertain. However, there is emerging

evidence that iNKT cell deficiency might predispose patients who are infected with

Mycobacterium tuberculosis to acute tuberculosis. A recent study found that iNKT cell

deficiency perfectly predict the presence of active disease in infected individuals, and

treatment for active tuberculosis restore the normal iNKT cell numbers in these patients

(Sutherland et al., 2009). LPG from Leishmania donovani was also identified as an

agonist of mouse iNKT cells (Amprey et al., 2004), however very little is known concerning

the ability of human iNKT cells to respond to parasite antigens. A recent study

demonstrated that human iNKT cells recognize DC infected with Leishmania infantum in

vitro, producing IFN-γ and a potent cytolytic response against infected cells (Campos-

Martin et al., 2006), suggesting that iNKT cells can have an important role in facilitating

the development of a protective Th1 response against Leishmania.


45

iNKT cells may also play a role in viral immunity. As noted earlier, SAP deficiency leads to

a selective loss of iNKT cells in mice and humans (Nichols et al., 2005; Pasquier et al.,

2005). The indirect suggestion that iNKT cells may be involved in the control of Epstein-

Barr virus (EBV) infection comes from the observation that patients with XLP syndrome

(caused by SAP deficiency) are much more sensitive to lethal infection with EBV (Gaspar

et al., 2002). There is also a report of a disseminated varicella infection, after vaccination,

in a patient with deficiency in iNKT cells but no other immune defect (Levy et al., 2003).

Furthermore, iNKT cells are considerably affected by human immunodeficiency virus

(HIV)-1, both in number and function (Li and Xu, 2008; Unutmaz, 2003), suggesting that

iNKT cells may play a role in HIV pathogenesis. CD4+ iNKT cells express the major

receptor used by HIV to enter the cell and, moreover, the co-receptor CCR5 is highly

expressed on most iNKT cells (Kim et al., 2002). Interestingly, CD4+ iNKT cell number

inversely correlates with viral load (Motsinger et al., 2002; van der Vliet et al., 2002) and it

was reported that CD4+ iNKT cells were selectively depleted when exposed to HIV-1 in

vitro (Sandberg et al., 2002), suggesting that CD4+ iNKT cells are targets for HIV infection.

iNKT cells in allergy and inflammation. Several studies have implicated iNKT cells in

allergic and inflammatory responses. Our laboratory is particularly interested in lung

inflammation, more precisely in allergic asthma, and recently demonstrated that Jα18-

deficient mice, which lack iNKT cells, fail to develop allergen-induced airway

hyperreactivity and have reduced airway inflammation after airway challenge with

ovalbumin (Lisbonne et al., 2003). Adoptive transfer of wild-type but not IL-4/IL13-deficient

iNKT cells into Jα18-deficient mice fully restored airway hyperreactivity and airway

inflammation, showing that iNKT cells producing IL-4 and IL-13 are required for the

development of allergen-induced airway hyperreactivity (Akbari et al., 2003). More

recently, a critical role of iNKT cells in asthmatic patients was also reported (Akbari et al.,

2006; Pham-Thi et al., 2006a; Pham-Thi et al., 2006b). Independent studies looking for

the presence of iNKT cells in bronchoalveolar lavage fluid (BALF), lung biopsy or both

from patients with asthma have provided divergent results (Matangkasombut et al.,

2009b). Most of the studies found increased iNKT cell numbers in the BALF and lung of

asthmatic patients (Akbari, 2006; Matangkasombut et al., 2009a; Pham-Thi et al., 2006a),

but some other studies did not (Thomas et al., 2006; Vijayanand et al., 2007). It is

possible that differences in the groups of patients studied, particularly in terms of disease

severity, might have contributed to the divergences observed because a recent study of

patients with a very broad range of asthma severity showed that the frequency of iNKT

cells in the lungs of these patients varied widely with severity of the disease

(Matangkasombut et al., 2009a). Compared with non-asthmatic controls individuals,


46

patients with severe, poorly controlled, asthma had a consistent and very significant

increase in the frequency of BALF iNKT cells, whereas only about half of patients with

well-controlled asthma had detectable increase. Therefore, the absence of detectable

increase of pulmonary iNKT cell numbers in some asthmatic patients (especially patients

with well-controlled asthma) cannot be interpreted as an indication that iNKT cells are not

important in asthma but instead suggests that functional studies must be done to better

understand the role of human iNKT cells in asthma.

iNKT cells as biomarkers. Although defects in iNKT cell number have been widely

reported in patients with autoimmune, infectious or allergic diseases to patients with

cancer, it is clear that a more detailed analysis of iNKT cell subset distribution and

cytokine production is necessary to better define the role of human iNKT cell defects in

the specific context of these diseases and might allow the identification of patterns of iNKT

cell characteristics (e.g. frequency, subset proportions, cell surface antigen expression

and cytokine production) as useful biomarkers for disease diagnosis, prognosis and

selection of treatments.

At our laboratory, we report donor-derived invariant NKT cell recovery as a novel post-

transplant predictive factor of the absence of acute graft-versus-host (GVH) disease after

human allogeneic hematopoietic stem cell transplantation (HSCT) with the particularity of

being associated with preserved antitumour responses (Rubio et al., submitted for

publication). HSCT is a curative therapy for hematologic malignancies but GVH disease is

one of the most serious complications. It was reported that the number of iNKT cells was

lower in patients with, than in those without, GVH disease after HSCT (Haraguchi et al.,

2004). Furthermore, a conditioning regimen involving fractionated total lymphoid

irradiation (TLI) plus anti-thymocyte globulin, which is a strategy to increase the number of

iNKT cells and protect against GVH disease in a mouse model (Lan et al., 2001; Lan et

al., 2003), decreased the incidence of acute GVH disease after HSCT in humans (Lowsky

et al., 2005). We have performed a longitudinal analysis of the immune reconstitution of

peripheral blood iNKT cells, as well as conventional T cells and NK cells, in patients

having received an allogeneic HSCT for diverse malignant hematological diseases and we

correlated the results with the development of acute GVH disease, underlying

hematological disease relapse and mortality. In both univariate and multivariate time

dependent Cox analyses, the recovery of a high iNKT/T cell ratio appears as an

independent predictive factor of the absence of acute GVH disease. Importantly, despite

developing less acute GVH disease, patients within the high iNKT/T cell ratio group did

not experience a higher risk of relapse and had a significantly improved overall survival


47

than those with a poor recovery. Moreover, we have analyzed the factors influencing iNKT

cell recovery after transplantation and we show that iNKT cells emerging in this setting are

functional and from the donor (Rubio et al., submitted for publication). Our results might

serve as the basis of future trials aiming at modulating the number and function of iNKT

cells in order to improve the prognosis of allogeneic HSCT, which remains a procedure

associated with a high mortality and morbidity.

iNKT cells as therapeutic targets. Several approaches to iNKT cell based immunothery

have entered clinical trials. The aim of such therapies is to increase the number of iNKT

cells and/or stimulate them to produce cytokines that achieve a desired immunoregulatory

effect. A phase I study involving the intravenous injection of α-GalCer in twenty-four

patients with advanced solid tumours was the first to be reported (Giaccone et al., 2002).

In that study, no dose-limiting toxicity was observed, and the therapy was well tolerated.

One day after injection, iNKT cell numbers decreased in the periphery, possibly due to

TCR down regulation induced by activation (Crowe et al., 2003) and/or migration to

tissues, and increased serum levels of IFN-γ, IL-12 and GM-CSF were detected in several

patients. However, no antitumour effect was observed clinically. In other clinical trials, α-

GalCer-loaded DC or in vitro-expanded iNKT cells were administrated (Chang et al., 2005;

Ishikawa et al., 2005; Motohashi et al., 2006; Nieda et al., 2004; Uchida et al., 2008). All

therapies were well tolerated and some immunological responses, including increased

iNKT cell numbers, increased levels of IFN-γ and IL-12 and activation of NK cells and T

cells, were reported. However, no tumour regression was reported although disease

progression stabilised in some patients in all trials.

iNKT cell-based immunotherapy, although well tolerated, has been disappointing

indicating that these approaches might need to be refined because the high variability of

iNKT cell frequency and subset distribution between individuals makes it difficult to predict

the size and cytokine profile of the iNKT cell response to each individual. It might become

useful to pre-assay the iNKT cell frequency and subset distribution in patients to facilitate

the design of customized therapies. It is also difficult to predict whether iNKT cells

enhance or attenuate antitumour effect, because they produce both Th1- and Th2-type

cytokine. Thus, the use of in vitro expanded CD4- iNKT cell subset may elicit a favourable

anti-tumour response. The use of iNKT cell agonists that preferentially induce a Th1-

deviated response such as α-C-GalCer in cancer therapy also may be effective.


48

Although iNKT cells exhibit potent therapeutic properties in a number of diseases, iNKT

cell activation can also exacerbate disease such as allergic and inflammatory reactions.

Thus, a deeper knowledge on the functional proprieties of human iNKT cells is crucial for

a better understanding of their potential contribution to a variety of pathogenic processes

and for a rational design of iNKT cell-based immunotherapeutic strategies.

CHAPTER II

IL-17 & TH17 CELLS

CHAPTER II – IL17 & Th17 Cells

50

Th1/Th2 paradigm

In 1986 Mosmann and Coffman discovered that effector Th cells are not a uniform

population, but can be subdivided into two major groups, one making IFN-γ and the other

producing IL-4 (Mosmann et al., 1986). The IFN-γ-secreting subset has been termed Th1

cells, while IL-4-producing cells were named Th2 cells. Since then, it has been clearly

demonstrated that the differentiation towards one or other subset is largely determined by

the set of transcription factors they express, whose induction depends on the cytokines

present in the microenvironment during stimulation of naïve T cell by APC (FIGURE 6).

Once differentiated, each Th cell subset secretes a confined set of cytokines and has

distinct functions in the immune system. Th1 cells are essential for clearing intracellular

bacteria and viruses but, when not regulated, Th1 cells have a pathogenic role in several

autoimmune disorders (Szabo et al., 2003; Trinchieri, 2003). Th1 differentiation is induced

by IL-12 (Hsieh et al., 1993; Manetti et al., 1993), which is mainly produced by monocytes

and DC, and IFN-γ (Lighvani et al., 2001), which is secreted by already differentiated Th1

cells, NK cells and iNKT cells. IFN-γ activates signal transducer and activator of

transcription 1 (STAT1) an, in turn, STAT1 signalling induces the T-box transcription factor

TBX21 (also known as T-bet) (Afkarian et al., 2002), which is a master regulator of Th1

differentiation (Mullen et al., 2001; Szabo et al., 2000). T-bet potentiates expression of

Ifng gene and up regulates the inducible chain of IL-12 receptor (IL-12Rβ2) (Mullen et al.,

2001; Mullen et al., 2002), expression of which enables IL-12 signalling through STAT4

and further potentiates IFN-γ production (Kaplan et al., 1996b; Thierfelder et al., 1996).

The transcription factor regulating Th2 differentiation, trans-acting T-cell-specific

transcription factor GATA-3 (Zhang et al., 1997; Zheng and Flavell, 1997), is instead

activated by IL-4 receptor signalling via STAT6 (Kaplan et al., 1996a; Shimoda et al.,

1996; Takeda et al., 1996). IL-4 can be provided by mast cells, basophils, iNKT cells,

eosinophils or previously differentiated Th2 cells. GATA-3 autoreactivates its own

expression and drives epigenetics changes that enable expression of Th2 cytokine cluster

(Pai et al., 2004; Yamashita et al., 2004; Zhu et al., 2004). Secreting the cytokines IL-4,

IL-5, IL-9, IL-13 and IL-25 (also known as IL-17E), Th2 cells have a major role in defense

against helminthes and other extracellular pathogens (Anthony et al., 2007; Paul and Zhu,

2010). However, when not regulated, Th2 cell response can lead to allergic disorders,

such as allergy and asthma (Barrett and Austen, 2009; Paul and Zhu, 2010). Overall,

cytokines secreted by Th1 or Th2 cell subsets not only determine their effector function

but they also act in an autocrine manner to give a positive feedback and promote their


51

own development and expansion (Amsen et al., 2009). Moreover, they can reciprocally

inhibit the function of other sets of Th cells, as cytokines produced by Th1 cells could

negatively regulate the function of Th2 cells and vice-versa (Coffman and Carty, 1986;

Mosmann et al., 1986). Mutual inhibition of Th1 and Th2 cells is also mediated by the

lineages specific transcription factors. The Th1 cell transcription factor T-bet inhibits Th2

development by binding to their master regulator GATA-3, preventing it from activation of

Th2-specific genes (Hwang et al., 2005). Likewise, GATA-3 can down regulate STAT4, a

transcription factor important for Th1 development (Usui et al., 2003) (FIGURE 6).

FIGURE 6. Th1/Th2 cross-regulation. Naïve CD4+ T cells differentiate towards Th1 or Th2 cell

subset, depending on the cytokines present during antigenic stimulation. Each subset secretes

cytokines that act in an autocrine manner to give feedback on the development of their own subset,

while inhibiting the other subset. Similarly, the lineage-specific transcription factors mutually inhibit

their expression or function. (Adapted from Amsen et al., 2009).

This Th1/Th2 paradigm has recently been complemented with a third effector Th cell

subset, the Th17 cells, named based on their hallmark cytokine IL-17A (simply referred as

IL-17 in this manuscript) (Harrington et al., 2005; Park et al., 2005). Furthermore, it is

likely that much more than three effector Th cell subsets exist (FIGURE 7). Accordingly, it

has been proposed that a population of IL-9-producing T cells constitute a separate

subset, called Th9 cells (Dardalhon et al., 2008; Goswami and Kaplan, 2011; Veldhoen et

al., 2008), and recent reports suggest the existence of Th22 cells, a subset secreting IL-

22, which has previously been associated with Th17 cells (Duhen et al., 2009; Nograles et

al., 2009; Trifari et al., 2009).


52

FIGURE 7. Subsets of effector T helper cells. Depending on the cytokine milieu present at the

time of antigenic stimulation, naïve CD4+ T cells can differentiate into various subsets of T helper

cells (Th1, Th2, Th17, Th9 and Th22). For most of T helper cell differentiation programme, specific

transcription factors have been identified as master regulators. Terminally differentiated T helper

cells are characterized by a specific combination of effector cytokines that orchestrate specific

effector functions of the adaptive immune system. (Adapted from Akdis et al., 2011).

Th17 cell differentiation

Th17 cells were recognized as an independent subset of effector Th cells, distinct from

Th1 and Th2 cell subsets, by the identification of differentiation factors and transcription

factors that are unique to Th17 cells. Three independent groups simultaneously reported

that a combination of IL-6 plus transforming growth factor β (TGF-β) induced the

differentiation of mouse naïve T cells into Th17 cells (Bettelli et al., 2006; Mangan et al.,

2006; Veldhoen et al., 2006). This finding was surprising, since TGF-β has been

associated mainly with anti-inflammatory effects such as the development of Treg cells

(discussed below) and inhibition of Th1 and Th2 cell differentiation (Li et al., 2006; Li et

al., 2007). Recent studies have been shown that TGF-β promotes Th17 cell differentiation

by inhibiting the differentiation of Th1 and Th2 cells (Das et al., 2009) and differentiation of


53

Th17 cells can occurs in the absence of TGF-β signalling but leads to the generation of

pathogenic Th17 cells that co-express ROR-γt and T-bet (Ghoreschi et al., 2010).

IL-6 seems to be the decisive factor that favours Th17 cells. IL-6 receptor signalling

activates STAT3, which subsequently induces the master transcription factors of Th17

differentiation, ROR-γt and ROR-α (Ivanov et al., 2006; Yang et al., 2008c). However, full

induction of ROR-γt is only reached in the presence of TGF-β (Zhou et al., 2008).

Activation of ROR-γt induces transcription of the Il17 gene and causes expression of the

receptor for IL-23, a characteristic feature of Th17 cells (Ivanov et al., 2006; Zhou et al.,

2007). The cytokine IL-23 is a heterodimeric molecule, sharing the p40 subunit with the

Th1 cytokine IL-12 but differing from IL-12 because of its unique p19 subunit (Oppmann et

al., 2000). Unlike IL-12, IL-23 does not induce Th1 cells but instead appears to be

important for amplifying and/or stabilizing the Th17 cell phenotype, characterized by

sustained IL-17 production (Aggarwal et al., 2003; McGeachy et al., 2007).

T cells generally require cytokines of the common gamma chain family such as IL-2 for

their survival. Surprisingly, Th17 cells are not dependent on IL-2, but might rather be

inhibited by IL-2 (Laurence et al., 2007). In turn, IL-21, another member of IL-2 cytokine

family, promotes and/or sustains Th17 cell differentiation in an autocrine manner (Korn et

al., 2007; Nurieva et al., 2007; Zhou et al., 2007). Moreover, IFN-regulatory factor 4 (IRF4)

has been shown to be essential for Th17 cell development by acting via the induction of

IL-21 (Brustle et al., 2007; Huber et al., 2008). The proinflammatory cytokines IL-1β and

TNF-α have also been shown to amplify the development of Th17 cells (Sutton et al.,

2006; Veldhoen et al., 2006). However, none of these cytokines can substitute for TGF-β

or IL-6 to promote mouse Th17 cell differentiation. Aryl-hydrocarbon receptor (AHR) has

also been associated with the development of mouse Th17 cells and the presence of AHR

agonists seems to be crucial for optimal differentiation of Th17 cells (Veldhoen et al.,

2009). Taken together, IL-6 and TGF-β are the key mediators to induce differentiation of

naïve T cells towards Th17 cells in mice, a process that is enhanced by IL-1β and TNF-α.

In addition, IL-21 and IL-23 seem to be important for the expansion and stabilization of

Th17 cells. As expected, Th1 and Th2-promoting cytokines, such as IL-12, IFN-γ and IL-4,

inhibit the induction of IL-17 (Harrington et al., 2005; Park et al., 2005).

While development of mouse Th17 cells has been intensively studied, the optimal

conditions for the differentiation of human Th17 cells remain uncertain. Initially, TGF-β

was not considered to be a differentiation factor for human Th17 cells. On the contrary,

TGF-β was found to inhibit the differentiation of human precursors into Th17 cells, which


54

was promoted by IL-6 plus IL-1β (Acosta-Rodriguez et al., 2007a; Wilson et al., 2007).

These studies, however, did not control for endogenous sources of TGF-β such as serum

and platelets and did not use truly naïve T cell precursors. When naïve T cells from cord

blood were cultured in serum-free medium, the essential role of TGF-β in human Th17

differentiation was confirmed and appeared to be dose-dependent (Manel et al., 2008;

Volpe et al., 2008; Yang et al., 2008a). In serum-free conditions, it was found that TGF-β

induces the expression of RORC, the human homologous of mouse ROR-γt, but

paradoxically inhibits its transcriptional activity, thereby preventing IL-17 expression. The

association of inflammatory mediators counteracts that inhibition, promoting IL-17

production (Manel et al., 2008). The inflammatory mediators include IL-1, IL-21, IL-6 and

IL-23, however, the relative role of each one are still debatable (Manel et al., 2008; Volpe

et al., 2008; Yang et al., 2008a). The role of TGF-β on IL-17 production by human memory

T cells, however, seems to be different from its effects on naïve T cells. At doses shown to

be necessary for IL-17 production by naïve T cells, IL-17 production from memory T cells

is inhibited (Evans et al., 2007; Liu and Rohowsky-Kochan, 2008). A recent paper claimed

that TGF-β did not have a direct critical role on the differentiation of naïve T cells into

Th17 cells, but rather indirectly favours IL-17 expansion by suppressing T-bet expression

and Th1 development (Santarlasci et al., 2009). Overall, the data suggest that similar

cytokines are involved in mouse and human Th17 cell development, however, differences

exist and further investigations need to be conducted to elucidate the factors regulating

human Th17 cell development.

Features of human Th17 cells

Studies in humans also showed that Th17 cells are different than in mice because all

human Th17 cells and their precursors express CD161 whereas NK1.1 expression has

never been reported in mouse Th17 cells. In human, IL-17-producing T cells originate

from CD161+CD4+ T cell precursors, detectable in both human cord blood and newborn

thymus (Cosmi et al., 2008). Moreover, virtually all IL-17-producing, RORC- and IL-23R-

expressing, CD4+ T cells were found within the CD161+ cell fraction of circulating T cells

from healthy adults, and also within the CD161+ cell fraction infiltrating the inflamed skin of

patients with psoriasis or the inflamed gut of patients with Crohn’s disease (Cosmi et al.,

2008; Kleinschek et al., 2009). CD161+ Th17 cells expressed a broad TCR repertoire and

were not restricted to CD1d (Cosmi et al., 2008), excluding the possibility that CD161+

Th17 cells belonged to the CD1d-restricted NKT (iNKT cells or type II NKT) cell


55

population. Although CD161 has been identified as a novel surface marker for human

Th17 cells, its functional significance is still unclear.

Human Th17 cells are currently defined as cells that produce IL-17 and IL-17F but they

are also able to produce TNF-α, IL-6, IL-22, IL-21 and IL-26 (Acosta-Rodriguez et al.,

2007b; Annunziato et al., 2007; Caprioli et al., 2008; Liu and Rohowsky-Kochan, 2008;

Manel et al., 2008; Pene et al., 2008; Volpe et al., 2008; Wilson et al., 2007). Interestingly,

a large fraction of human peripheral blood T cells co-expresses IL-17 and IFN-γ, as well

as RORC and T-bet (Acosta-Rodriguez et al., 2007b; Annunziato et al., 2007).

Additionally, human Th17 cells were found to express IL-12Rβ apart from IL-23R and

stimulation of these cells in the presence of IL-12 down regulated RORC and up regulated

T-bet, enabling them to produce IFN-γ in addition to IL-17 (Annunziato et al., 2007;

Annunziato and Romagnani, 2009). Human Th17 cells are characterized by the

expression of the chemokine receptor CCR6, and two distinct subsets of IL-17-producing

T cells were identified based on the expression of additional chemokine receptors: CCR4-

expressing subset that produce IL-17 and IL-22, and CXCR3-expressing subset that

produce IL-17 together with IFN-γ (Acosta-Rodriguez et al., 2007b; Annunziato et al.,

2007; Singh et al., 2008). The dual appearance of these antagonistic cytokines in the

same cell is intriguing and it remains undetermined if these so-called Th17/Th1 cells are a

distinct and stable lineage of effector Th cells, or whether they are an intermediate state of

Th17 or Th1 cells (FIGURE 8).

FIGURE 8. Heterogeneity of human Th17 cells. Human IL-17-producing T cells could be

subdivided into two distinct subsets: a CCR4+ subset that expresses RORC and produces IL-17

(Th17) and a CXCR3+ subset that co-expresses RORC and T-bet and co-produces IL-17 and IFN-γ

(Th17/Th1). Whether Th17/Th1 cells are an intermediate state of Th17 or Th1 cells, or whether

they are a distinct and stable lineage of effector Th cells is still unknown. (Adapted from Annunziato

and Romagnani, 2009).


56

In contrast to what was found in the mouse (Quintana et al., 2008; Veldhoen et al., 2009),

AHR agonists did not enhance but rather inhibited the generation of Th17 cells from

human naïve T cells (Ramirez et al., 2010; Trifari et al., 2009). AHR agonists have been

shown to reduce the expression of IL-23R and RORC in human T cells, without affecting

T-bet or GATA-3 (Ramirez et al., 2010), which may explain the negative effect of AHR

agonists on human IL-17 production. Importantly, in humans, AHR activation did not only

decrease the number of Th17 cells but also primed naïve CD4+ T cells to produce IL-22

without IL-17 and IFN-γ (Ramirez et al., 2010). Moreover, knocking down RORC inhibited

the production of IL-17 and IL-22 by human T cells, whereas targeting AHR specifically

inhibited the production of IL-22 (Trifari et al., 2009). Overall, the data suggest that IL-17

and IL-22 are regulated differently in human T cells.

Human Th22 cells

IL-22 was originally described in mice and humans as a Th17 associated cytokine.

However, a distinct subset of human memory T cells has recently been shown to produce

IL-22, but neither IL-17 nor IFN-γ (Duhen et al., 2009; Trifari et al., 2009). Differentiation of

IL-22 producing T cells, now named Th22 cells, could be promoted by stimulation of naive

T cells in the presence of IL-6 and TNF-α or by interaction with plasmacytoid DC or

Langerhans cells, and appears to be independent of RORC but highly dependent of AHR

activation (Duhen et al., 2009; Fujita et al., 2009; Trifari et al., 2009). Although the findings

so far indicate an important role of AHR in regulating IL-22 production, it remains to be

determined whether AHR is a master transcription factor of Th22 cell differentiation.

Human Th22 cells express the chemokine receptor CCR6 and the skin-homing receptors

CCR4 and CCR10, which raise the possibility that these cells may be important in skin

homeostasis and pathology (Duhen et al., 2009; Trifari et al., 2009). In this line of

evidence, a subset of T cells secreting IL-22 alone was found in the blood and skin of

patients with atopic eczema (Eyerich et al., 2009b). At this time, a specific Th22 cell

lineage has yet to be described in mice.

Innate IL-17-producing cells

In addition to Th17 cells, innate immune cells, including γδ T cells, iNKT cells, innate

lymphoid cells (ILC) and myeloid cells, also produce IL-17 during inflammatory responses


57

(Li et al., 2010; Lockhart et al., 2006; Michel et al., 2007; Passos et al., 2010; Takatori et

al., 2009). A recent report demonstrated that during thymic selection, CD27+ γδ T cells

exhibit high levels of T-bet and become IFN-γ producers, whereas CD27- γδ T cells

constitutively express ROR-γt and become producers of IL-17 (Ribot et al., 2009). IL-17-

producing γδ T cells express hallmarks of Th17, such as CCR6, AHR, IL-1R and IL-23R

(Haas et al., 2009; Martin et al., 2009; Riol-Blanco et al., 2010; Sutton et al., 2009).

Stimulation of this γδ T cell subset with a combination of IL-1 and IL-23 readily induces IL-

17 in a TCR-independent manner (Sutton et al., 2009), and AHR activation promotes co-

production of IL-22 by activated IL-17-producing γδ T cells (Martin et al., 2009). IL-17-

producing γδ T cells have been also reported in humans (Fenoglio et al., 2009; Peng et

al., 2008), however few co-produce IL-22 (Ness-Schwickerath et al., 2010). A very recent

study has demonstrated that, like human Th17 cells, human circulating γδ T cells that

produce IL-17 express the distinctive marker CD161 (Maggi et al., 2010). Moreover,

CD161 expression identifies cord blood γδ T cells that express RORC and IL-23R mRNA

and can be induced to differentiate into IL-17-producing cells by IL-1β and IL-23 (Maggi et

al., 2010). Two other studies have also demonstrated that human naïve γδ T cells are

efficiently polarized towards IL-17 production by TCR activation in association with a

combination of polarizing cytokines known to be involved in Th17 cell differentiation

(Caccamo et al., 2011; Ness-Schwickerath et al., 2010).

ILC family includes lymphoid tissue-inducer (LTi) cells and NK cells, which have different

functions but have been shown to be development related (Crellin et al., 2010; Cupedo et

al., 2009). These IL-17-producing ILC are present mainly in the intestinal tract, express

and require RORC (or ROR-γt, in mouse) for development and function and their IL-17

production is regulated by IL-23 (Buonocore et al., 2010; Geremia et al., 2011). It has

been also reported that mucosal NK cells expressing NKp44 (in humans) or NKp46 (in

mouse) markers secrete large amounts of Th17-associated cytokine IL-22 with little if any

IFN-γ production or cytotoxic activity, belong to a different lineage from conventional NK

cells (Cella et al., 2009; Luci et al., 2009; Sanos et al., 2009; Satoh-Takayama et al.,

2008). Like innate IL-17-producing cells, these NK-22 cells are dependent of RORC but

following stimulation with IL-23 they express IL-22 (not IL-17) (Cella et al., 2009; Sanos et

al., 2009; Satoh-Takayama et al., 2008).


58

IL-17 in human health and disease

The receptor for IL-17 can be found in a wade range of tissues such as lung, kidney, liver,

intestine and skin; and is expressed on many non-immune cells like fibroblasts, epithelial

cells, endothelial cells, keratinocytes or marrow stromal cells but also in myeloid cells

such as DC, macrophages and neutrophils (Akdis et al., 2011; Ishigame et al., 2009; Lin

et al., 2009; Moseley et al., 2003; Yao et al., 1995). Consistent with the broad expression

pattern of its receptor, IL-17 acts on a variety of cells, which respond by up regulating

expression of proinflammatory cytokines, chemokines, and metalloproteases (Kolls and

Linden, 2004; McKenzie et al., 2006). The induction of these proinflammatory mediators

leads to inflammation and tissue destruction, thereby implicating IL-17 in several

autoimmune disorders. In contrast to this pathogenic role, IL-17 promotes the recruitment

of neutrophils (Laan et al., 1999; Ye et al., 2001) and induces expression of anti-microbial

peptides by immune and non-immune cells (Liang et al., 2006), protecting the host from

potential pathogenic organisms.

IL-17 in host defense. The role of IL-17 in host defense against pathogens has been

characterized extensively in mouse models, with the general consensus that IL-17 is

necessary for protective immunity against bacteria, fungus and parasite (Higgins et al.,

2006; Huang et al., 2004; Ishigame et al., 2009; Kelly et al., 2005; Meeks et al., 2009;

Shibata et al., 2007). In human, however, little direct information exists regarding the

importance of IL-17 in resistance to infection. Consistent with the finding that, in mice, IL-

17 is required to protect the host against Candida albicans infection (Huang et al., 2004),

a recent study reported a decreased number of CCR6+ IL-17-producing T cells in patients

with chronic mucocutaneous candidiasis (Eyerich et al., 2008), a disease recently

associated with genetic defects related to IL-17R (Puel et al., 2011). Moreover, two other

studies also reported that patients with hyper-IgE syndrome (HIES), in which a mutation in

STAT3 impairs an efficient Th17 response, have recurrent oro-pharyngeal candidiasis (Ma

et al., 2008; Milner et al., 2008). Indeed, treatment of skin keratinocytes with Th17

cytokines markedly increases anti-candicidal activity in vitro and activated T cells from

HIES patients are unable to induce this anti-candicidal activity. An important role for IL-22

against C. albicans infection has also been suggested. Memory IL-22-producing CD4+ T

cells specific to C. albicans were detected in humans (Liu et al., 2009) and, when

associated with TNF-α, IL-22 seems to inhibit the growth of C. albicans and to conserve

the integrity of the epidermal barrier in an in vitro skin infection model (Eyerich et al.,


59

2011). Moreover, neutralizing antibodies to IL-22 and IL-17 were found at high levels in

human with chronic mucocutaneous candidiasis (Puel et al., 2010), which may explain the

susceptibility of these patients to mucosa Candida infection.

IL-17 in chronic inflammation and autoimmunity. Recent data have been shown that

tissue-infiltrating Th17 cells are present in diverse chronic inflammatory disease, including

psoriasis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease (IBD),

atopic eczema and allergic asthma (Annunziato et al., 2007; Eyerich et al., 2009a;

Kleinschek et al., 2009; Pene et al., 2008). Peripheral blood from allergic asthmatic

patients also harbor elevated numbers of Th17 cells compared to healthy controls and the

plasma concentrations of IL-17 and IL-22 tend to increase with the severity of the disease

(Zhao et al., 2010). Moreover, IL-17 is increased in lung tissue, BALF and sputum from

asthmatic patients (Ivanov and Linden, 2007), which correlates with the presence of

neutrophils in the sputum from asthmatic patients with increasing disease severity

(Bullens et al., 2006), suggesting that IL-17 contributes to local accumulation of

neutrophils in asthma and to disease severity. These findings are in agreement with the

data from studies in mouse models that reveal the importance of IL-17 in the induction

and maintenance of allergic airway disease (Finkelman et al., 2010). Although a positive

correlation between IL-22 mRNA and serum total IgE levels was found in asthmatic

children (Farfariello et al., 2011), the role of this cytokine in allergic asthma is still elusive,

even in the mouse model, in which IL-22 seems to be required for the onset of allergic

asthma, but functions as a negative regulator of established allergic inflammation

(Besnard et al., 2011).

Th17 cells and downstream cytokine, IL-17, are also elevated in the injured psoriatic skin

(Lowes et al., 2008; Pene et al., 2008). IL-17 induces antimicrobial peptides and

metalloproteases that are commonly found in psoriatic skin (Liang et al., 2006; Wolk et al.,

2006). It has been reported that IL-22 synergizes with IL-17 to enhance the induction of

antimicrobial peptides from keratinocytes (Liang et al., 2006). Recently, IL-22 but not IL-

17-producing T cells have also been identified in psoriatic skin samples (Duhen et al.,

2009; Eyerich et al., 2009b). Increased levels of IL-22 in the serum and elevated

expression of IL-22 mRNA in injured skin from psoriatic patients have also been reported

(Lo et al., 2010; Wolk et al., 2004). IL-22 induces hyperplasia, abnormal differentiation,

and the expression of many psoriatic marker genes such as psoriasin from keratinocytes

(Boniface et al., 2005; Sa et al., 2007), which may indicate a pathogenic role for IL-22 in

psoriasis.

Biopsies from inflamed colonic tissue of patients with Crohn’s disease and ulcerative


60

colitis showed increased levels of IL-17 (Nielsen et al., 2003), suggesting a pivotal role for

IL-17 in IBD. Additionally, CD4+ T cells from peripheral blood and tissues of Crohn’s

disease patients express high levels of IL-17 (Annunziato et al., 2007; Kleinschek et al.,

2009). IL-17 stimulates production of metalloproteases and the release of pro-

inflammatory cytokines in colonic subepithelial myofibroblasts (Yagi et al., 2007), which

may contribute to the chronic inflammation characteristic of the disease. In addition, IL-17

inhibits the proliferation of intestinal epithelial cells (Schwartz et al., 2005), suggesting

that, besides contributing to inflammation and tissue damage in IBD, IL-17 also interferes

with the repair mechanism important for the maintenance of the tissue integrity. In addition

to IL-17, IL-22 expression is also augmented in patients with IBD (Andoh et al., 2005;

Brand et al., 2006), and IL-22 levels seem to correlate with disease severity (Schmechel

et al., 2008). Despite the fact that IL-22 is correlated with disease activities in IBD

patients, data from several mouse models suggest that IL-22 exert protective functions in

IBD by enhancing epithelial innate defense mechanisms and barrier integrity (Pickert et

al., 2009; Sugimoto et al., 2008; Zenewicz et al., 2008).

IL-17 as therapeutic target. As pathogenic roles of IL-17 were suggested in most human

autoimmune diseases, clinical trials to inhibit Th17 development or neutralize IL-17 have

been carried out. Targeting the IL-6R with a monoclonal antibody (Ab) or blocking IL-1R

with an antagonist were both found to be effective approaches to the treatment of

rheumatoid arthritis and Crohn’s disease (Geyer and Muller-Ladner, 2010; Patel and

Moreland, 2010). Although the precise mechanism of action of these molecules is

unknown, it is possible that the observed therapeutic effects are due, at least in part, to

inhibition of Th17 differentiation. Treatment with a monoclonal Ab specific to p40 subunit

of IL-12 and IL-23 has been successfully used in psoriasis and Crohn’s disease (Griffiths

et al., 2010; Kimball et al., 2011; Leonardi et al., 2008; Sandborn et al., 2008). In Crohn’s

disease, the Ab caused a local reduction of the levels of IL-12 and IL-23, so the effects

cannot be attributed specifically to the IL-23-IL-17 axis. Unlike the results obtained for

psoriasis and Crohn’s disease, monoclonal Ab specific to p40 subunit of IL-12 and IL-23

produced no substantial therapeutic effects in a trial to treat patients with relapsing-

remitting multiple sclerosis (Segal et al., 2008). Monoclonal antibodies against IL-17 or IL-

17R and a soluble IL-17R have been developed for clinical application. Humanized IL-17

antibodies have produced favourable results in the treatment of rheumatoid arthritis,

psoriasis and uveitis (Genovese et al., 2010; Hueber et al., 2010). Overall, neutralization

of IL-17 or inhibition of IL-12 and IL-23 (p40), IL-1 or IL-6 activity, which are important for

the production of IL-17 seems beneficial for the treatment of autoimmune diseases.

CHAPTER III

FOXP3 & TREG CELLS

CHAPTER III – Foxp3 & Treg Cells

62

Immune tolerance

Immunological tolerance, which is defined as the lack of immune responsiveness towards

an antigen, is based on the ability of the immune system to discriminate between self and

non-self and can be acquired by central or peripheral mechanisms. Central tolerance

occurs during early ontogeny and leads to elimination of self-reactive lymphocytes by

clonal deletion in the thymus. However, a large number of self-reactive lymphocytes

escapes this central process of negative selection and is exported to the periphery, where

it forms a pool of potentially dangerous lymphocytes. In the periphery, tolerance is

maintained through a variety of mechanisms that control the quality and quantity of

immune responses. One important component of peripheral tolerance is represented by

lymphocytes with regulatory functions, including iNKT cells (discussed earlier) and Treg

cells (Sakaguchi, 2004; Taniguchi et al., 2003).

Treg cells develop in the thymus as a distinct cell lineage predestined to suppress

immune responses and they have a vital role in preventing autoimmune disease, such as

type 1 diabetes (Sakaguchi et al., 2001; Shevach et al., 2006), and limiting chronic

inflammatory diseases, such as asthma and IBD (Coombes et al., 2005; Xystrakis et al.,

2006), but they also block beneficial responses by preventing sterilizing immunity to some

pathogens (Belkaid, 2007; Rouse et al., 2006) and reducing antitumour immunity

(Kretschmer et al., 2006).

Phenotype of Treg cells

The key marker of Treg cell lineage is the forkhead box p3 (Foxp3) (Fontenot et al., 2003;

Hori et al., 2003)(discussed below), although as an intracellular transcription factor its

usage in Treg cell identification is restricted. Human and mouse Treg cells have been

traditionally characterized as CD4+ T cells that constitutively express the IL-2 receptor α-

chain (CD25) (Baecher-Allan et al., 2001; Sakaguchi et al., 1995). However, human

activated T cells up regulate CD25 expression and, only CD4+ T cells with the highest

levels of CD25 expression are suppressive in humans (Baecher-Allan et al., 2001). Treg

cells typically express cytotoxic T lymphocyte antigen 4 (CTLA-4, also known as CD152)

and glucocorticoid-induced TNF-receptor-related protein (GITR, also known as

TNFRSF18), but these markers are also expressed by non-regulatory T cells after


63

activation (McHugh et al., 2002; Shimizu et al., 2002; Takahashi et al., 2000). Recently,

several groups showed that human CD4+ T cells that express low levels of IL-7 receptor

α-chain (CD127) manifest suppressive activity (Liu et al., 2006a; Seddiki et al., 2006a),

suggesting the CD127 marker as an alternative to CD25 for the delineation of human Treg

cells. However, CD4+ T cells tend to down regulate CD127 expression upon activation

(Mazzucchelli and Durum, 2007). Therefore, even CD127 expression cannot accurately

discriminate Treg cells from activated non-regulatory T cells. Several other molecules

have also been identified on the cell surface of Treg cells, such as L-selectin (CD62L),

CCR4, ICOS, HLA-DR, CD95, neuropilin-1, CD103, OX40 and others (Sakaguchi et al.,

2010). However the expression of these markers is not restricted to Treg cells.

Foxp3

An important progress in the understand of Treg cells came with the identification of the

key transcription factor Foxp3 that is required for their development, maintenance and

function (Fontenot et al., 2003; Hori et al., 2003). Mice and humans that lack Foxp3

develop profound autoimmune-like lymphoproliferative disease that clearly emphasizes

the importance of Treg cells in the maintenance of peripheral tolerance (Bennett et al.,

2001; Brunkow et al., 2001; Wildin et al., 2001). In fact, Foxp3 gene was originally

identified as the defective gene in the mouse strain Scurfy, which succumb to X-linked

recessive autoimmune and inflammatory disorders as a result of uncontrolled activation of

CD4+ T cells (Brunkow et al., 2001). Additionally, humans with mutation in the Foxp3 gene

develop the disease called immunodysregulation, polyendocrinopathy and enteropathy, X-

linked syndrome (IPEX), which is characterized by severe multi-organ autoimmune

disease, allergy and IBD, that develops early in infancy (Bennett et al., 2001; Wildin et al.,

2001). The clinical and immunological similarities between IPEX in humans and the

autoimmune and inflammatory disorders observed in mice following depletion of Treg cells

triggered a number of studies that provided clues on the crucial role of Foxp3 for the

development and function of Treg cells. In mice, Foxp3 is mainly expressed in Treg cells

and ectopic Foxp3 expression can phenotypically convert effector T cells to regulatory T

cells (Hori et al., 2003). Moreover, transfer of CD4+CD25+ T cells but not CD4+CD25- T

cells into Scurfy mice or Foxp3-deficient mice prevents disease development (Fontenot et

al., 2003). Other mouse cells, such as CD8+ T cells, iNKT cells and macrophages can

also express Foxp3 (Mayer et al., 2011; Monteiro et al., 2010; Zorro Manrique et al.,

2011), however in CD8+ T cells, this expression is not necessarily correlated to


64

suppressive properties (Zorro Manrique et al., 2011).

Although Foxp3 has proved to be a unique marker of mouse Treg cells, its role in human

Treg cells is ambiguous. Human adult Treg cells also express high levels of Foxp3

whereas CD25- T cells do not (Roncador et al., 2005; Yagi et al., 2004) and Foxp3

appears to be required for human Treg cell development and function (Bennett et al.,

2001; Wildin et al., 2001). However, expression of Foxp3 alone is clearly not sufficient for

regulatory function as a significant percentage of human activated T cells express Foxp3

but do not possess regulatory activity (Allan et al., 2007; Gavin et al., 2006; Morgan et al.,

2005; Tran et al., 2007; Wang et al., 2007). It appears that only those cells that express

stable and high levels of Foxp3 during activation have suppressive function, while those

with transient or low levels of Foxp3 expression do not seem to be suppressive (Gavin et

al., 2006; Miyara et al., 2009; Wang et al., 2007).

It has been shown that Foxp3 can interact with a number of transcription factors, such as

NFAT (Torgerson et al., 2009; Wu et al., 2006), Runx1 (Ono et al., 2007), and NF-κB

(Bettelli et al., 2005), which have important roles in regulating T cell activation and

differentiation of effector T cells (Rao et al., 1997; Taniuchi et al., 2002). A model have

been proposed in which these transcription factors promote or inhibit the transcription of

genes encoding for cytokines and surface molecules in Treg cells and non-Treg cells,

depending on the presence of Foxp3 (Sakaguchi et al., 2008) (FIGURE 9). According to

the model, binding of Foxp3 to the transcription factors NFAT and Runx1 blocks their

ability to transcribe cytokines such as IL-2 and IFN-γ, while at the same time increases the

transcription of Treg cell-associated molecules, such as CD25, CTLA-4 and GITR (Chen

et al., 2006; Ono et al., 2007; Wu et al., 2006). This process mediates suppression of

effector T cell function and renders Treg cells highly dependent of exogenous IL-2, which

is mainly produced by activated effector T cells. In contrast, in the absence of Foxp3 the

transcriptional complex transcribes IL-2 and IFN-γ and represses Treg cell-associated

molecules facilitating the activation of effector T cells. So, the up regulation of Foxp3 in

activated T cells could be one component of the homeostatic programme initiated by

these cells to exert negative feedback during the immune response.


65

FIGURE 9. Control of Treg cell function by Foxp3. The transcriptional complex involving NFAT

and Runx1 activates or represses the genes encoding cytokines (such as IL-2 and IFN-γ) and Treg

cell-associated molecules (such as CD25, CTLA-4 and GITR) in Treg cells and non-Treg cells,

depending on the presence of Foxp3. (Adapted from Sakaguchi et al. 2008).

Heterogeneity of human Foxp3+ T cells

As suggested above, human Foxp3+ T cells are functionally heterogeneous, including

suppressive and non-suppressive T cells. Three distinct human Foxp3+ T cell

subpopulations were recently identified, based on CD45RA, CD25 and Foxp3 expression:

(i) CD45RA+CD25+Foxp3low naïve Treg cells (Fritzsching et al., 2006; Miyara et al., 2009;

Seddiki et al., 2006b; Valmori et al., 2005), (ii) CD45RA-CD25highFoxp3high effector Treg

cells, which represent different stages of Treg cell differentiation and are both potently

suppressive in vitro; and (iii) CD45RA-CD25+Foxp3low non-Treg cells, which do not

suppress effector cells in vitro and produces proinflammatory cytokines, including IL-2,

IFN-γ and IL-17 (Miyara et al., 2009). Naïve Treg cells are the main subset of Treg cells in

utero (Darrasse-Jeze et al., 2005) and are enriched in cord blood (Takahata et al., 2004),

whereas effector Treg cells are more prevalent in adults and in elderly people (Miyara et

al., 2009). Effector Treg cells are similar to mouse Treg cells in terms of CD25 and other

Treg cell markers, potent suppressive activity and apparent anergic state (Baecher-Allan

et al., 2001; Jonuleit et al., 2001; Levings et al., 2001) and they are apt to die by apoptosis

upon antigenic stimulation (Fritzsching et al., 2005). In contrast, naïve Treg cells are

highly resistant to apoptosis (Fritzsching et al., 2005) and upon antigen stimulation they

proliferate, differentiate and acquire an effector Treg cell phenotype as they up regulate

Foxp3, CD25 and CTLA-4 (Miyara et al., 2009). Despite these differences, the ability of


66

naïve and effector Treg cells to suppress conventional T cells is comparable (Miyara et

al., 2009).

Mechanisms of suppression

Treg cells suppress the proliferation of naïve T cells and their differentiation to effector T

cells. They can also suppress cytokine production of differentiated CD4+ T cells and CD8+

T cells and the function of other cell types, such as NK cells, NKT cells, B cells,

macrophages and DC (Miyara and Sakaguchi, 2007; Shevach, 2009; Tang and

Bluestone, 2008). Human Treg cells must be activated through their TCR to be

functionally suppressive (Baecher-Allan et al., 2001; Dieckmann et al., 2001; Jonuleit et

al., 2001; Taams et al., 2002). The success of suppression depends on the strength of T

cell stimulation. Activation of responder T cells in the presence of strong TCR stimulation

or strong co-stimulatory signals impairs suppression by Treg cells, as does the presence

of growth-promoting cytokines (Baecher-Allan et al., 2001; Baecher-Allan et al., 2002).

These data suggests that human Treg cells are not able to suppress cytokine production

and/or proliferation in conditions in which responder T cells are strongly activated. In

addition, in proinflammatory conditions, human Treg cells can be induced to produce IL-17

(Beriou et al., 2009). IL-17+Foxp3+ Treg cells have been found in human peripheral blood

(Ayyoub et al., 2009; Beriou et al., 2009; Miyara et al., 2009; Voo et al., 2009) and,

although, they are suppressive in the presence of low TCR stimulation, they lose the

ability to suppress and acquire the capacity to secrete IL-17 when they are strongly

activated in the presence of proinflammatory cytokines (Beriou et al., 2009). Overall, it

seems that an increase in effector T cell resistance and a decrease in Treg cell function

underlie the loss of suppression in the presence of strongly activation factors.

The precise molecular mechanisms of suppression by human Treg cells remain to be

determined. However, in vitro and in vivo mouse studies have implicated several

molecules and mechanisms in Treg cell-mediated suppression (Shevach, 2009; Vignali et

al., 2008). From a functional perspective, the mechanisms of Treg cell-mediated

suppression can be divided into four basic groups: (i) suppression by inhibitory cytokines,

(ii) suppression by cytolysis, (iii) suppression by metabolic disruption, and (iv) suppression

by modulation of DC maturation and function (FIGURE 10).


67

FIGURE 10. Mechanisms of suppression used by Treg cells. Treg cells can suppress the

proliferation and/or function of non-Treg cells using several mechanisms, which involve the release

of inhibitory cytokines, the induction of cytolysis or metabolic disruption of the target cell, and/or

modulation of DC maturation and function. (Adapted from Vignali et al. 2008).

Inhibitory cytokines. Although the role of IL-10 and TGF-β as suppressive mediators is

undisputed, whether they contribute to the function of Treg cells is still controversial

(Shevach, 2006). This is in part due to the general perception that suppressive function of

Treg cells is dependent on cell contact with the target cells. Indeed, in vitro studies using

neutralizing antibodies or T cells that are unable to produce or respond to IL-10 and TGF-

β suggested that these cytokines might not be essential for Treg cell function (Jonuleit et

al., 2001; Thornton and Shevach, 1998). However, this is in contrast with data from in vivo

studies showing that IL-10 and TGF-β are required for the Treg cell mediated control of

disease in IBD and allergic asthma models (Annacker et al., 2003; Hawrylowicz and

O'Garra, 2005; Huber et al., 2011; Joetham et al., 2007; Li et al., 2007). Moreover, cell-

surface-bound TGF-β can also mediate suppression in a cell contact dependent

mechanism (Nakamura et al., 2001). IL-35 is another, recently identified, inhibitory


68

cytokine that is preferentially produced by Treg cells in mice and required to their maximal

suppressive activity (Collison et al., 2007; Collison et al., 2009). However, two

contradictory studies exist in humans (Bardel et al., 2008; Chaturvedi et al., 2011). The

first one showed that, in humans, IL-35 is produced by activated T effector cells but not by

Treg cells (Bardel et al., 2008), while a more recent one showed that human Treg cells

express and require IL-35 for maximal suppressive activity (Chaturvedi et al., 2011). So,

the contribution of IL-35 to the suppressive function of human Treg cells is still unclear.

Cytolysis. Granzymes and galectins are secreted molecules that potentially play a role in

Treg cell-mediated suppression. Activated human Treg cells have been shown to express

granzyme A and to kill autologous CD4+ and CD8+ T cells and other cell types in a

perforin-dependent, Fas-FasL-independent manner (Grossman et al., 2004). In mice,

granzyme B is up regulated upon Treg cell activation and it has been suggested that Treg

cells kill responder cells by a granzyme B-dependent mechanisms and that granzyme B-

deficient Treg cells show reduced capacity to suppress proliferation in vitro (Gondek et al.,

2005). In contrast to these reports where granzymes seem to act as effector molecules

produced by Treg cells, a recent study reported that granzyme B produced by responder

T cells inhibits the effector functions of Treg cells (Ashley and Baecher-Allan, 2009). In

this study, activated human CD4+ T cells express granzyme B and actively kill a special

fraction of Treg cells, called DR+ Treg cells, in response to strong TCR stimulation (Ashley

and Baecher-Allan, 2009). Galectin-1 (also known as LGALS1), is preferentially

expressed by Treg cells and has been shown to be up regulated after Treg cell activation

(Garin et al., 2007). Moreover, blocking of galectin-1 markedly reduced the inhibitory

effects of human and mouse Treg cells, and Treg cells from galectin-1-deficient mice had

reduced activity (Garin et al., 2007). Human Treg cells also constitutively express

intracellular galectin-10 and specific inhibition of galectin-10 seems to abrogate their

suppressive function (Kubach et al., 2007).

Metabolic disruption. Suppressive mechanisms that interfere with the metabolism of

responder T cells have also been described. For instance, Treg cells were shown to

contain high concentrations of cyclic adenosine monophosphate (cAMP) and to suppress

effector T cell function through direct transfer of cAMP to activated T cells via membrane

gap junctions (Bopp et al., 2007). This second messenger is known to be a potent inhibitor

of proliferation, differentiation and IL-2 synthesis in T cells (Bodor et al., 2001). Three

other studies described the importance of CD39 and CD73, both expressed on activated

Treg cells, for the generation of adenosine, which suppressed effector T cell function

through activation of the adenosine receptor A2 (A2AR) (Borsellino et al., 2007; Deaglio et


69

al., 2007; Kobie et al., 2006). Furthermore, several studies have demonstrated that Treg

cells mediate suppression by inhibiting the induction of IL-2 mRNA (and mRNA for other

effector cytokines) in the responder T cells (Takahashi et al., 1998; Thornton and

Shevach, 1998). Treg cells do not produce but require IL-2 for their maintenance and

function (Fontenot et al., 2005), and they are characterized by high expression of CD25,

the high-affinity receptor for IL-2, therefore they could absorb the IL-2 produced by

effector T cells and mediate suppression as a result of direct cytokine consumption. A

recent study suggested that Treg cells inhibit T cell effector function, in vitro and in vivo,

by inducing cytokine (specially IL-2) deprivation-mediated apoptosis (Pandiyan et al.,

2007)

Modulation of DC function. In addition to the direct effect on effector T cells, Treg cells

might also modulate the maturation and/or function of DC. Treg cells either abrogate the

antigen-presenting activity of the DC or promote the secretion of suppressive factors by

the target DC. Treg cells constitutively express CTLA-4 and down regulation of the co-

stimulatory molecules CD80 and CD86 on DC cell surface, in a CTLA-4 dependent

manner, has been reported (Oderup et al., 2006). This was recently confirmed in vivo

(Wing et al., 2008) and suggests that inhibition of CD80 and CD86 expression by Treg

cells impairs the capacity of the DC to stimulate naive T cells trough CD28 co-signalling.

Additionally, CTLA-4 ligation of CD80 and CD86 can induce DC to express indoleamine

2,3-dioxygenase (IDO), a potent regulatory molecule which is known to induce the

production of pro-apoptotic metabolites from the catabolism of tryptophan, resulting in the

suppression of effector T cells (Fallarino et al., 2003; Grohmann et al., 2002). Another cell

surface molecule that has been suggested to play a role in DC-mediated Treg cell

suppression is lymphocyte activation gene 3 (LAG3), a CD4 homolog that binds MHC

class II molecules on DC, resulting in suppressed DC maturation and activation (Liang et

al., 2008). Two additional molecules that have been implicated in Treg cell suppression

are fibrinogen-like protein 2 (FLG2) and neuropilin 1 (Nrp-1), both interacting with DC

(Sarris et al., 2008; Shalev et al., 2008). Furthermore, it is still not clear what the primary

target is for many of the mechanisms described above since suppression by cytolysis,

adenosine or cAMP could be directed against DC and/or effector T cells, and inhibitory

cytokines could also influence both populations.


70

Induced Treg cells

Although Foxp3+ Treg cell subsets have been considered to be a naturally occurring

thymus-derived population, similar Foxp3+ Treg cells can also be induced from naïve

CD4+ T cells in the periphery (so-called induced Treg cells or iTreg cells). Conditions that

favor the peripheral induction of Foxp3 include suboptimal TCR stimulation (during chronic

inflammation and/or during normal homeostasis) and suboptimal co-stimulation, with a

particularly important role for the immunoregulatory cytokine TGF-β (Curotto de Lafaille

and Lafaille, 2009). Additionally, IL-2 and vitamin A metabolite retinoic acid (RA) facilitate

induction of Foxp3 in peripheral Foxp3- CD4+ T cells. For instance, in mice, iTreg cells are

likely prominent in the gut-associated lymphoid tissue (GALT) where chronic exposure to

food, commensal or environmental antigens and the high numbers of CD103+ DC (which

produce RA and TGF-β) probably facilitates their generation (Benson et al., 2007;

Coombes et al., 2007; Kang et al., 2007; Mucida et al., 2007; Schambach et al., 2007;

Sun et al., 2007).

In humans, naïve CD4+ T cells can also be induced to express Foxp3 in the presence of

TGF-β, RA or both (Kang et al., 2007; Tran et al., 2007; Wang et al., 2009). Foxp3+ iTreg

cells converted from cord blood CD4+CD25- T cells in the presence of TGF-β plus RA

have been described as more suppressive than those induced in the presence of either

TGF-β or RA alone (Kang et al., 2007). In adults, however, only Foxp3+ iTreg cells

induced by TGF-β plus RA seems to have a potent and stable suppressive function

(Wang et al., 2009), while TGF-β or RA induced Foxp3+ T cells are neither anergic nor

suppressive (Tran et al., 2007; Wang et al., 2007; Wang et al., 2009), suggesting once

more that human CD4+ T cells require additional factors, besides the functional and

phenotypic changes induced by Foxp3 expression, to acquire full suppressive activity.

Contrary to the promoting factors described below, cytokines and transcription factors that

induce the differentiation of other Th cell types (e.g., Th1, Th2, Th9 and Th17 cells)

antagonize CD4+ T cell differentiation into Foxp3+ iTreg cells (Zhou et al., 2009). For

instance, a suppressive role in Foxp3 regulation has been found for the Th2-specific

transcription factor GATA-3 (Mantel et al., 2007). This study revealed that IL-4-induced

GATA-3 binds to palindromic GATA-site in the Foxp3 promoter, which leads to

transcriptional repression (Mantel et al., 2007). IL-4 also activates STAT6, which binds to

Foxp3 promoter and inhibits gene expression (Takaki et al., 2008). The proinflammatory

cytokines IL-1β, IL-6 and IL-21, which are involved in Th17 cell differentiation, have also

been reported to suppress Foxp3 expression (Fantini et al., 2007; Nurieva et al., 2007;


71

Yang et al., 2008b). As discussed earlier, differentiation of Th17 cells and conversation of

iTreg cells require TGF-β (Bettelli et al., 2006) and, ROR-γt and Foxp3 are both induced

during the early phase of a TGF-β-mediated response, but Foxp3 wins out by shutting

down ROR-γt (Yang et al., 2008b; Zhou et al., 2008). However, in the presence of

proinflammatory cytokines, which induce the expression of ROR-γt, Foxp3 induction is

effectively antagonized (Burgler et al., 2010; Yang et al., 2008b; Zhou et al., 2007). These

data suggest a tight regulation of Foxp3–ROR-γt balance during Th cell differentiation, in

which Foxp3-mediated inhibition of Th17 cell differentiation dominates under non-

inflammatory conditions, while Foxp3 expression (and therefore, iTreg cells development)

is suppressed by ROR-γt during inflammation (FIGURE 11). Others factors regulating the

iTreg-Th17 cell balance seems to exist. For instance, IL-27 inhibits Th17 cell

differentiation (Batten et al., 2006; Stumhofer et al., 2006) whereas enhances TGF-β-

induced Foxp3 expression (Ouaked et al., 2009), in a STAT1-dependent manner. In some

cells, this balance seems to be regulated in a way that allows simultaneous expression of

both Foxp3 and ROR-γt (Ayyoub et al., 2009; Burgler et al., 2009; Lochner et al., 2008;

Voo et al., 2009). Runx1 was recently shown to form a complex with ROR-γt and to

cooperate with it to promote Th17 cell differentiation (Lazarevic et al., 2011; Zhang et al.,

2008). On the other hand, Runx1 induced by TGF-β has been reported to be involved in

the development and suppressive function of Foxp3+ iTreg cells (Klunker et al., 2009).

Thus, functional plasticity in cells that co-express ROR-γt and Foxp3 may be governed by

the ability of Runx to cooperate with either transcription factors.

Recently the mammalian target of rapamycin (mTOR) signalling pathway was proposed to

be an intrinsic signalling pathway that controls the balance between effector Th cells and

iTreg cells (Thomson et al., 2009). It was found that mTOR regulates the differentiation of

Th cells (Delgoffe et al., 2011; Lee et al., 2010) and mTOR-deficient Th cells are unable to

up regulate subset-specific transcription factors, such as GATA-3, T-bet, and ROR-γt,

being therefore unable to differentiate into effector Th cells (Delgoffe et al., 2009). Instead,

mTOR-deficient cells have a tendency to differentiate into iTreg cells (Delgoffe et al.,

2009) and mTOR inhibition by rapamycin enhances Foxp3 transcription inducing iTreg

cells (Sauer et al., 2008). Accordingly, in human, rapamycin was found to increase the

number of Foxp3+ Treg cells by promoting the selective expansion of thymus-derived Treg

cells (Basu et al., 2008; Strauss et al., 2007), and to induce suppressor functions on total

human CD4+ T cells (Valmori et al., 2006). Moreover, some clinical studies have

confirmed that rapamycin modulates Treg cells in vivo (Hendrikx et al., 2009; Lu et al.,

2010)


72

FIGURE 11. Factors regulating the iTreg-Th17 cell balance. TGF-β induces both Foxp3 and

ROR-γt expression by antigen-primed naïve T cells. Under non-inflammatory conditions, mediators

like IL-2, IL-27 or retinoic acid (RA) enhance TGF-β-induced Foxp3 expression, which inhibits

ROR-γt, promoting iTreg cell development. During inflammation, Th17-polarizing cytokines (such

as IL-1β, IL-6 and IL-21) enhance ROR-γt, which in turn inhibits Foxp3 expression, leading to Th17

cell development. (Adapted from Burgler et al. 2010).

Besides Foxp3+ Treg cells, there are other types of regulatory CD4+ T cells that can be

induced from naïve CD4+ T cells in the periphery. For instance, CD4+ T cells secreting IL-

10 and TGF-β, called Tr1 cells, are produced in vitro by antigenic stimulation of naïve T

cells in the presence of IL-10 (Groux et al., 1997). Tr1 cells do not express Foxp3, yet

their properties in vitro are very similar to those of Foxp3+ Treg cells. They exhibit

diminished proliferation in response to antigenic stimulation, have suppressive functions,

produce little to no IL-2, and display activated cell-surface phenotype (Vieira et al., 2004).

Tr1 cells induced by rapamycin plus IL-10 have been shown to mediate stable,

alloantigen-specific tolerance in pancreatic islet cell transplantation (Battaglia et al., 2006),

However, at present, there is little evidence for the contribution of Foxp3-non-expressing

Treg cells to the maintenance of natural self-tolerance in humans.

A variety of other T cell subpopulations including, mouse and human, CD8+, γδ T cells and

macrophages have also been reported to express Foxp3 and to exhibit

immunosuppressive activity (Casetti et al., 2009; Jarvis et al., 2005; Kang et al., 2009;

Kapp et al., 2006; Li et al., 2011; Siegmund et al., 2009; Zorro Manrique et al., 2011).

And, recently, Foxp3+ iNKT cells with regulatory functions were described in mouse

(Monteiro et al., 2010). In vivo, Foxp3+ iNKT cells were found in the cervical lymph node of


73

mice protected from experimental autoimmune encephalomyelitis (EAE) following α-

GalCer treatment. Moreover, Foxp3 expression was induced in activated iNKT cells, in

vivo and in vitro, by a TGF-β-dependent mechanism. These induced Foxp3+ iNKT cells

displayed Treg cell phenotypic hallmarks and were suppressive in vitro (Monteiro et al.,

2010). Of note, TGF-β was found to be critical to the induction of Foxp3 expression in all

these T cell subpopulations (Li et al., 2011; Monteiro et al., 2010; Siegmund et al., 2009).

CHAPTER IV

OBJECTIVES AND RESULTS

CHAPTER IV – Objectives and Results

75

I. AIMS OF THE THESIS

The purpose of this thesis was to better understand the biology of human iNKT cells and

determine the major factors influencing their functional properties, either cytokine

production or suppressor activity. We chose to divide this thesis into two major parts

where in vitro approaches using primary cells were employed to improve the

understanding of human iNKT cell physiology, namely:

1. Determine whether an iNKT cell subset with similar features of mouse iNKT17 cells

exists in humans.

Both mouse and human iNKT cells can produce IL-4 and IFN-γ following TCR

engagement, endowing them with pro-Th1 as well as pro-Th2 functions. They also display

Th17 properties, since our laboratory recently identified an IL-17-producing mouse iNKT

cell subset implicated in airway tissue injury. It mirrors the recently described Th17 subset

that plays a key role in autoimmune diseases and inflammation unique from Th1 and Th2

cells. Concerning human iNKT cells, it remained to be determined whether cells can also

secrete IL-17. Here we solved this question.

2. Establish whether Foxp3+ Treg cells have a human iNKT cell counterpart.

In addition to influence immune responses by their ability to produce cytokines, iNKT cells

can also act as immunosuppressors. Thus, another important point addressed in this

thesis was to evaluate the suppressive activity of human iNKT cells. Here we revealed

that the environment play a major role in the acquisition of Foxp3 expression by human

iNKT cells and demonstrated that these cells are fully capable to inhibit the proliferation of

CD4+ T lymphocytes.


76

II. RESULTS

Article 1. Proinflammatory Environment Dictates the IL-17−Producing Capacity of Human Invariant NKT Cells.

Lúcia Moreira-Teixeira, Mariana Resende, Maryaline Coffre, Odile Devergne, Jean-


Michel Dy, Anabela Cordeiro-da-Silva, and Maria C. Leite-de-Moraes

In J Immunol 186 (2011): 5758-5765

In the present study, we investigated whether an iNKT cell subset with similar features of

mouse iNKT17 cells exists in humans.

Main Results: - Human peripheral blood iNKT cells activated ex vivo did not produce detectable levels of

IL-17 and required TGF-β, IL-1β and IL-23 signalling in association with TCR stimulation

to become able to produce IL-17, which they co-produced with IFN-γ.

- Human iNKT cells were able to produce other Th17-associated cytokines, such as, IL-22

and IL-21, independently of TGF-β, IL-1β and IL-23.

- Both CD4+ and CD4- iNKT cell subsets were equally able to produce IL-17 and IL-22,

whereas IL-21 was mainly produced by CD4+ iNKT cell subset.

- IL-17-producing iNKT cells differ from conventional Th17 cells by their restricted ability to

co-produce IL-22. However, in similarity to Th17 cells, human iNKT cells responded to

AHR activation by decreasing their IL-17 and enhancing their IL-22 secretion.

- Human IL-17-producing iNKT cells originate exclusively from CD161+ precursors. Among

the CD161+ fraction both CCR6+ and CCR6- peripheral blood iNKT cells could produce IL-

17.

- Cord blood iNKT cells behaved differently since IL-17 producers belong predominantly to

the CD4- subset and derived exclusively from the small CD161+CCR6+ iNKT cell subset.


77

Moreover, in contrast to peripheral blood, IL-17-producing iNKT cells from cord blood did

not co-produce IFN-γ.

Conclusions: IL-17 production by human iNKT cells depends on two critical parameters, namely an

intrinsic program and a proinflammatory environment. Indeed, solely the iNKT cells having

acquired CD161 expression were able to differentiate into IL-17 producers, which emerge

when TGF-β, IL-1β and IL-23 were present in the environment.


78


79


80


81


82


83


84


85


86


87


88


89


90

Article 2. Rapamycin and TGF-β convert invariant Natural Killer T cells into

suppressive Foxp3+ regulatory cells.

Lúcia Moreira-Teixeira, Mariana Resende, Odile Devergne, Jean-Philippe Herbeuval,

Olivier Hermine, Elke Schneider, Michel Dy, Anabela Cordeiro-da-Silva, and Maria C.

Leite-de-Moraes

To be submitted

In the present study, we investigated whether Foxp3+ Treg cells have a human iNKT cell

counterpart.

Main Results: - Human iNKT cells did not express detectable levels of Foxp3 ex vivo.

- TGF-β induced de novo Foxp3 expression in human iNKT cells, independently of their

maturation state. TGF-β-induced Foxp3+ iNKT cells were CD25+ but they did not acquire a

complete Treg phenotype, as they were not necessary CTLA-4+ or GITR+.

- Both CD4+ and CD4- iNKT cell subsets were induced to express Foxp3, however, CD4-

iNKT cells displayed limited plasticity for TGF-β polarization.

- TGF−β strongly affected the effector function of human iNKT cells, it impaired the

production of effector cytokines, IL-4 and IFN-γ, whereas increased the production of the

inhibitory cytokine IL-10 by iNKT cells. However, TGF-β alone failed to confer them a

suppressive activity.

- Inhibition of mTOR pathway by rapamycin was critical to sustain TGF-β-induced Foxp3

expression over time and, especially, to confer suppressive activity to Foxp3+ iNKT cells.

Conclusions:

TGF-β induces de novo Foxp3 expression in human iNKT cells and impairs their effector

functions; however, iNKT cells require mTOR inhibition to become fully suppressive

Foxp3+ regulatory cells.


91

Immunobiology

RAPAMYCIN AND TGF-β CONVERT HUMAN INVARIANT NATURAL KILLER T

CELLS INTO SUPPRESSIVE FOXP3+ REGULATORY CELLS

Lúcia Moreira-Teixeira1,2, Mariana Resende1,2, Odile Devergne1, Jean-Philippe

Herbeuval1, Olivier Hermine1, Elke Schneider1, Michel Dy1, Anabela Cordeiro-da-Silva2,

and Maria C. Leite-de-Moraes1

1Unité Mixte de Recherche (UMR) 8147, Centre National de la Recherche Scientifique

(CNRS), Faculté de Médecine René Descartes, Paris V, Hôpital Necker, 161 rue de

Sèvres, 75015, Paris, France. 2IBMC - Instituto de Biologia Molecular e Celular and Faculdade de Farmácia

Universidade do Porto, Rua Campo Alegre 823, 4150-180 Porto, Portugal.

Corresponding author: Maria C. LEITE-DE-MORAES,

CNRS UMR 8147,

161 rue de Sèvres,

75015, Paris, France;

Tel: +33144495394, Fax: +33144490676;

e-mail: [email protected]

Short title: Foxp3+ human iNKT cells

Word counts: text: 4614; abstract: 192; Figures: 7; Supplemental Figures: 3, References:

58.

Keywords: NKT cells, Foxp3, CD1d, α-GalCer, human, TGF-β, rapamycin, cord blood

Abbreviations: α-GalCer (alpha-galactosylceramide), CBMC (cord blood mononuclear

cell), iNKT (invariant Natural Killer T), PBMC (peripheral blood mononuclear cell), TGF-β

(transforming growth factor-beta), Th (T helper).


92

ABSTRACT

Invariant Natural Killer T (iNKT) cells compose a particular T cell population that rapidly

produce diverse cytokines and consequently influence the outcome of several immune

responses. Here we found that TGF-β induces the expression of Foxp3 in both peripheral

and cord blood iNKT cells, respectively considered as more and less experienced cells.

Both CD4+ and CD4- iNKT cells could express this transcription factor. Foxp3+ iNKT cells

were CD25+ but not necessarily CTLA4+ or GITR+, even though the expression of these

markers was up regulated in iNKT lymphocytes exposed to TGF-β. The latter decreased

IL-4 and IFN-γ and increased IL-10 production independently of Foxp3 expression while

IL-17 was undetected. High levels of Foxp3 were induced in the presence of TGF-β but

Foxp3+ iNKT cells required rapamycin to become suppressive. Both peripheral and cord

blood Foxp3+ iNKT cells equivalently suppressed the proliferation of conventional

autologous and heterologous CD4+ T cells in a cell contact dependent manner. Our

findings demonstrate that human iNKT cells become suppressive in the presence of TGF-

β plus rapamycin and provide new insights into the understanding iNKT cell biology and

new tools for their clinical use in future cell therapy based trials.


93

INTRODUCTION

Invariant Natural Killer T (iNKT) cells have elicited a lively interest in the last years

because of their implication in several immune responses and their great potential for

therapeutic modulation1-4. They constitute a distinct subpopulation of T lymphocytes that

are positively selected by CD1d molecules and co-express a highly restricted TCR

repertoire, composed of a single invariant Vα14Jα18 chain in mice and a Vα24Jα18 chain

in humans preferentially paired with limited TCR Vβ chains1,2. Another typical feature of

this population is the expression of receptors like CD161 (homologue of mice NK1.1) and

NKG2D5,6 that they share with NK cells.

In contrast with their conventional T cell counterpart, iNKT cells recognize CD1d-bound

glycolipids rather than peptides7-9. In response to these ligands, iNKT cells promptly

produce large amounts of distinct cytokines7-13, enabling them to regulate autoimmune

diseases, inflammation, antitumour responses, allergic asthma and antimicrobial host

responses14-19. iNKT cells are therefore commonly acknowledged as belonging to the

immunoregulatory T cell population. This immunoregulatory power of iNKT cells was

generally consider as their capacity to shift the balance in favour of a pro-Th1 or pro-Th2

immune responses. For instance, iNKT cells can amplify asthma severity or contribute to

protection against tumour metastasis by supporting a Th2 or Th1 responses,

respectively16,20,21. iNKT cells also act as effector cells, as in the case of IL-17-producing

iNKT cells, by enhancing lung inflammatory responses10,22. In addition to the diverse role

attributed to iNKT cells to amplify the immune responses, these cells can also contribute

to dampen the immune system by their ability to promote regulatory T (Treg) cell

expansion via their ability to secrete IL-223. Here, we demonstrated that in the presence of

TGF-β and rapamycin, human iNKT cells can express Foxp3 and become the equivalent

to conventional Foxp3+ Treg cells24 and suppress the proliferation of conventional CD4+ T

cells.


94

MATERIALS AND METHODS

Cell preparation. Peripheral and cord blood mononuclear cells (PBMC and CBMC) were

collected from healthy donors at the Etablissement Français du Sang or the Necker

Enfants Malades Hospital, Paris. Experiments were performed in accordance with the

Helsinki Declaration, with informed consent from each donor or the donor’s family. PBMC

and CBMC were prepared by Ficoll-Hypaque centrifugation (GE Healthcare) and further

used for in vitro culture or cell sorting.

iNKT cell expansion. PBMC and CBMC were cultured in 24-well plates (Falcon) at a

density of 106 cells per well in RPMI 1640 medium containing antibiotics, 10% FBS, 200

mM glutamine and 10 mM HEPES (all from Invitrogen) with α-GalCer (100 ng/ml; Alexis

Biochemicals) in the presence of absence of recombinant human (rh) TGF-β (5 ng/ml;

R&D Systems). When indicated, rapamycin (Sigma-Aldrich) was added at 20nM at the

start point of culture. rhIL-2 (50 ng/ml, Immunotools) was added to the culture 24 hours

later. After 2 weeks, cells were collected and washed extensively and their viability was

determined by trypan blue exclusion.

Cell purification and sorting. Fresh PBMC or CBMC were labelled with CD1d-tetramer-

PE (provided by National Institutes of Health Tetramer Facility), and iNKT cells were

magnetically enriched with anti-PE magnetic beads (Miltenyi Biotec). CD1d-tetramer+ cells

were further purified by electronic cell sorting on a FACSAria (BD Biosciences). CD1d-

tetramer+ cells were cultured in 96-well plates (Falcon) with anti-CD3 plus anti-CD28 and

anti-CD2 coated beads (Miltenyi Biotec) in a cell: bead ratio of 1:1 in the presence of

rhTGF-β. Cultured cells were analysed for Foxp3 expression. In some experiments,

positive fraction was labelled with anti-CD4 (eBioscience). CD1d-tetramer+CD4+ and

CD1d-tetramer+CD4- cell subsets were then sorted. CD1d-tetramer+ subsets were

cultured with autologous mononuclear cells depleted on iNKT cells (negative fraction from

magnetic enrichment, used as feeder) and expanded with α-GalCer in the presence of

rhTGF-β. After 2 weeks, cells were analysed for Foxp3 expression. For T cell proliferation

assay, CD25- CD4+ T cells were sorted from PBMC or CBMC after staining with anti-CD4

and anti-CD25 (eBioscience). Expanded iNKT cells were stained with CD1d-tetramer and

anti-CD25, and the following populations were sorted: CD1d-tetramer+CD25+ and CD1d-

tetramer+CD25-.


95

Surface and intracellular staining. Fresh or cultured PBMC or CBMC were analysed by

flow cytometry using the CD1d-tetramer and the following Abs: anti-CD3 (Immunotools),

anti-CD4, anti-CD25, anti-GITR (eBioscience). Intracellular analysis of Foxp3 (clone

236A/E7) and CTLA4 (both from eBioscience) was performed after fixation and

permeabilization using Foxp3 staining buffers (eBioscience). Isotype-matched antibodies

were used to define marker settings. For intracellular cytokine staining, expanded iNKT

cells were incubated for 5 h with phorbol myristate acetate (PMA, 25 ng/ml), ionomycin (1

µg/ml) and brefeldin A (10 µg/ml; all from Sigma-Aldrich). Cells were stained with CD1d-

tetramer, and then fixed, washed, and permeabilized using Foxp3 staining buffers and

incubated with anti-Foxp3, anti-CD4, anti-IL-4, anti-IL-10, anti-IL-17 and anti-IFN-γ (all

from eBioscience). Cells were acquired in a FACSCanto II (BD Bioscience) and analysed

using FlowJo software (Tree Star).

Suppression assay. Suppressor activity of expanded iNKT cells was assessed in a

coculture assay set up with autologous or heterologous CD25- CD4+ T cells labelled with 5

µM CFSE (Invitrogen), used as responder T cells. Cells were incubated at a concentration

of 2-5 x 104/well in serum-free X-Vivo 15 medium (Lonza) supplemented with 5 % human

normal serum (Dynal Biotech) and stimulated with anti-CD3 plus anti-CD28 and anti-CD2

coated beads (Miltenyi Biotec) in a cell: bead ratio of 1:1, with or without increasing

numbers of sorted CD1d-tetramer+CD25+ cells. In some experiments, anti-GITR (10

µg/ml; R&D Systems) was added. In some other experiments, supernatants from CD1d-

tetramer+CD25+ cells were used. Supernatants were collected from CD1d-tetramer+CD25+

cells stimulated for 24 or 48 h with anti-CD3 plus anti-CD28 and anti-CD2 coated beads in

the presence or not of responder T cells. Proliferation was assessed after 4 days of

stimulation by cytometric analysis on a FACSCanto II using FlowJo software.

Real-Time RT-PCR. Total RNA was extracted from sorted CD1d-tetramer+CD25+ cells

with the RNeasy Micro kit (Qiagen), and first strand cDNAs were prepared using RT2 First

Strand Kit (SABioscience). Template cDNAs were characterized using the Human T-cell

Anergy and Immune Tolerance PCR Array with the RT2 SYBR Green / ROX qPCR Master

Mix (all from SABioscience) on the StepOnePlusTM Real-Time PCR System (Applied

Biosystems). Fold changes in gene expression between the RNA from iNKT cells cultured

in the presence of TGF-β and rapamycin or in the presence of medium were calculated

using the ΔΔCt method in the PCR Array Data Analysis (SABioscience).

Statistical analysis. Differences between data sets were analysed by Mann-Whitney U

test.


96

RESULTS

TGF-β induces Foxp3 expression in human iNKT cells

We set out to evaluate Foxp3 expression in human iNKT cells (Figure 1A) freshly isolated

from both cord and peripheral blood mononuclear cells (CBMC and PBMC), respectively

considered as functionally immature or mature iNKT cells25,26. As shown in Figure 1B and

1C, both populations were negative for the transcription factor ex vivo, in contrast to

conventional CD4+CD25+ Treg cells, which were used as a positive control.

Given the critical role ascribed to TGF-β in the differentiation of conventional human CD4+

T cells into Treg cells27, we verified whether this cytokine had a similar effect on iNKT

cells. To this end, we cultured PBMC or CBMC with α-GalCer, the iNKT cell-specific

antigen, and IL-2, with or without TGF-β. Two weeks later, the proportion of iNKT cells

among total mononuclear cells increased at least 100 fold (from ~ 0.1% ex vivo to 10 –

40% after culture; data not shown) and Foxp3 expression was consistently induced in

both CBMC and PBMC, when iNKT cells were exposed to TGF-β (Figure 1D and 1E,

right). In the absence of TGF-β no detectable Foxp3 was induced (Figure 1D and 1E, left).

TGF-β-induced Foxp3 expression was not dependent of α-GalCer activation neither from

antigen presenting cells (APC), since ex vivo sorted iNKT cells up regulated Foxp3

expression when activated with anti-CD3 plus anti-CD28 and anti-CD2 beads in the

presence of TGF-β (Figure S1A).

These results show that natural Foxp3+ iNKT cells are undetectable in both cord and

peripheral blood but that Foxp3 expression is easily induced in the presence of TGF-β.

Expression of Foxp3 is not restricted to but belongs predominantly to the CD4+ iNKT-cell subset Knowing that human iNKT cells are heterogeneous as to their expression of CD46,28, we

addressed the question whether the ability to become Foxp3+ was associated with a

distinct cell subset. It turned out that both CD4+ and CD4- iNKT cells from peripheral blood

shared this potential (Figure 2A). However, the frequency of Foxp3+ cells was significantly

higher in gated CD4+ than CD4- iNKT cells (Figure 2B). Similarly, higher levels of Foxp3+

cells were found among CD4+ iNKT cells sorted ex vivo and activated with α-GalCer in the

presence of TGF-β (Figure S1B).

The data suggest that although both CD4+ and CD4- iNKT cell subsets could express

Foxp3, CD4+ iNKT cells are more prone to express this transcription factor.


97

Surface markers of Foxp3+ iNKT cells Since Treg cells are characterized by constitutive surface expression of the IL-2 receptor

α-chain (CD25)24, we verified whether this feature applied also to Foxp3+ iNKT cells. We

found that the addition of TGF-β significantly increased CD25 expression on expanded

iNKT cells and Foxp3+ iNKT cells were mainly CD25+ whatever they origin, cord or

peripheral blood (Figure 3A and 3B). However, Foxp3+ iNKT cells were not necessarily

CTLA4+ or GITR+, even though the expression of these markers was up regulated in TGF-

β-expanded iNKT cells (Figure 3A and 3B). Together, these results indicate that although

TGF-β induces Foxp3 expression by iNKT cells following antigen activation, this effect is

not necessarily accompanied by the acquisition of a typical Treg phenotype.

TGF-β inhibits IL-4 and IFN-γ but increases IL-10 production by iNKT cells

A fundamental prerequisite for the immunoregulatory functions of iNKT cells in

autoimmune diseases, tumour surveillance, allergic asthma and infectious disease is their

ability to produce cytokines that determine the orientation of the immune response, such

as IL-4 and IFN-γ1,2,29. This characteristic together with the implication of TGF-β in the

peripheral differentiation of selective cytokine-producing T cell subsets30,31, led us to

assess its effect on the cytokine profile of expanded iNKT cells. We found that IL-4 and

IFN-γ production was decreased when iNKT cells had been exposed to TGF-β (Figure 4A

and 4B). Our data also revealed that cytokines were predominantly produced by the

Foxp3- iNKT cell fraction (Figure 4A and 4B). Knowing that Foxp3 expression can be

induced by TCR engagement, and that its up regulation in iNKT cells results in

hyporesponsiveness in terms of cytokine production, we postulate that it might represent a

means to switch off prolonged activation. Although, cytokine inhibition was independently

of Foxp3 expression since even Foxp3- iNKT cells failed to produce IL-4 and produce less

IFN-γ when expanded in the presence of TGF-β (Figure 4A and 4B).

Further, we examined whether exposure of iNKT cells to TGF-β affected their production

of IL-10, a cytokine naturally produced by Treg cells24. We found that TGF-β increased IL-

10 production by human cord and peripheral iNKT cells (Figure 4A and 4B, respectively),

but IL-10+ cells were also observed among Foxp3- iNKT cells (Figure 4A and 4B). Foxp3+

iNKT cells did not produce IL-17 (Figure 4A and 4B), conversely to a recent report

showing the production of this cytokine by human Foxp3+ Treg cells32.

TGF-β does not suffice to confer suppressive activity to Foxp3+ iNKT cells

Foxp3 has been identified as a key transcription factor initiating the regulatory program

that confers the Treg phenotype and suppressor functions to conventional CD4+CD25- T


98

cells33-36. To test whether human Foxp3+ iNKT cells also acquire suppressive activity,

iNKT cells from cord or peripheral blood were expanded with α-GalCer plus IL-2 in the

presence or absence of TGF-β and sorted two weeks later according to their CD25

expression. We used this approach to enrich iNKT in Foxp3+ cells (Figure 3) because it

was not possible to obtain long term viable Foxp3+ iNKT cells to perform our suppressive

test if we use a Foxp3 intracellular staining to perform the electronic cell sorting. To

assess the in vitro suppressive potency of CD25+ iNKT cell subset, we tracked the

proliferation of labelled CD4+CD25- T cells (responder T cells) by measuring CFSE

dilutions in cocultures with different ratios of CD25+ iNKT cell subset during 4-day

stimulation with anti-CD3 plus anti-CD28 and anti-CD2 beads. Contrasting with freshly

sorted natural Treg (CD4+CD25+) cells, which drastically hampered responder cell

proliferation (Figure 5A), CD25+ iNKT cells failed to exert a suppressive activity whether

they had been expanded in the presence of TGF-β or not (Figure 5A).

We repeated these experiments with more experienced iNKT cells derived from peripheral

blood, with similar results (Figure 5B). This incapacity of Foxp3+ iNKT cells to suppress

the proliferation of conventional CD4 T cells was confirmed by using CD25+CD4+ iNKT

cells to increase the number of Foxp3-expressing iNKT cells, since we found that Foxp3

expression was higher in CD4+ iNKT cells (Figure 2A and 2B), but we failed to observe

any suppressive effect (data not shown).

Since the suppressive activity of Treg cells can also be indirect through modulation of

APC function, we performed an in vitro suppression assay in the presence of APC but

once more we failed to observe any suppressive effect (data not shown).

Consequently, our results demonstrate that TGF-β-induced Foxp3 expression in human

iNKT cells was not sufficient to confer them a suppressive activity. In accordance with

these findings, it has been reported that mere TCR stimulation can induce Foxp3

expression in apparently naïve human conventional Foxp3-CD4+ T cells without conferring

suppressive activity37. Further stimuli are necessary to transform TGF-β-induced Foxp3+

iNKT cells into suppressor cells.

Rapamycin synergizes with TGF-β to confer suppressive activity to Foxp3+ iNKT

cells

In order to determine whether TGF-β-induced Foxp3+ iNKT cells could suppress the

proliferative response of conventional CD4+ T cells, we added the mTOR inhibitor

rapamycin to the TGF-β cultures because it was already reported that stimulation of

human CD4+ T cells in the presence of rapamycin results in a highly increased suppressor

function as compared with that of CD4+ T cells stimulated in the absence of the drug38.


99

We found that about 70% of iNKT cells from both, cord and peripheral blood, expressed

Foxp3 when expanded in the presence of TGF-β plus rapamycin (Figure 6A) in contrast to

cells cultured with TGF-β alone (Figure 1D and 1E). Further, we analysed the influence of

rapamycin on the maintenance of Foxp3 expression by iNKT cells and we observed that

this mTOR inhibition was important to retain Foxp3 expression over time (Figure 6B).

Foxp3+ iNKT cells obtained with the addition of rapamycin were CD25high but once more

were not necessary CTLA4+ and GITR+ (Figure S2A) and they failed to produce IL-4 and

IFN-γ but they produced IL-10 (Figure S2B).

Regardless the similitude to TGF-β-expanded iNKT cells, the addition of rapamycin

allowed iNKT cells to acquire a suppressive activity, since they suppressed the

proliferation of autologous or heterologous CD25- CD4+ T cells (Figure 6C and 6D). It is

noteworthy that both peripheral and cord blood iNKT cells were capable to display a

suppressive activity in these conditions. Moreover, even whether cord blood is considered

as less experienced iNKT cells and we have recently demonstrated that they have a

distinct cytokine profile13, here they were equally capable to suppress the proliferation of

CD4+ T cells (Figure 6E).

To get inside into the mechanisms implicated in the suppressive activity of Foxp3+ iNKT

cells induced by mTOR inhibition, we sorted CD25+ iNKT cells from rapamycin plus TGF-β

culture, stimulated them and further tested the effect of their cell culture supernatant on

the proliferation of conventional CD25- CD4+ T cells. CD25+ iNKT cell supernatant did not

inhibit T cell proliferation (Figure 7A), indicating that human iNKT cells suppress T cell

proliferation in a cell contact dependent mechanism. Nevertheless, the suppressive effect

of human iNKT cells was not affected by GITR blockage (Figure 7B). We did not observed

any apoptosis of conventional CD25- CD4+ T cells when cocultured with CD25+ iNKT cells

(data not shown). By RT-PCR we analysed some gene candidates to explain the

suppressor effect of iNKT cells. We observed that the expression of Foxp3, IRF-4, IL-2RA,

CCR4 and galectin-3 (LGALS3) mRNAs was enhanced when rapamycin and TGF-β were

added to culture (Figure S3). However, no significant enhancement was found concerning

the expression of FasL or Granzyme B mRNA (data not shown) suggesting that others

molecules are predominantly implicated.


100

DISCUSSION

Our findings demonstrated that TGF-β plays a crucial role in the induction of Foxp3

expression and, more importantly, in the acquisition of suppressive activity by human

iNKT cells, when combined with the mTOR inhibitor rapamycin. Consequently, we

demonstrated that Foxp3+ iNKT cells are equivalent to conventional Foxp3+ induced

regulatory T (iTreg) cells. A recent report39 show that TGF-β induces Foxp3 expression on

mouse and human iNKT cells, in agreement with our results, and that mouse Foxp3+ iNKT

cells are suppressive. In our system, Foxp3 induced by TGF−β failed to confer

suppressive activity to human iNKT cells. The addition of rapamycin was critical to

transform Foxp3+ iNKT cells into suppressor cells. Our results are in agreement with the

literature since several studies reported that a significant number of human activated T

cells express Foxp3 but do not acquire regulatory activity37,40-43. It has been reported that

sustained Foxp3 expression in Treg cells is necessary for maintenance of the Treg cell

phenotype and suppressor function36,44,45. Accordingly, we found that inhibition of mTOR

pathway by rapamycin sustains TGF-β-induced Foxp3 expression over time, conferring

suppressive activity to human Foxp3+ iNKT cells.

Cord and peripheral blood iNKT cells can express distinct surface markers, for instance

the majority (>90%) of cord blood iNKT cells are CD4+CD161low, while in the peripheral

blood up to 95% are CD161+ and the frequency of CD4+ varies among the subjects but

generally is inferior to 50%13,25. The cytokine profile of cord blood is also distinct from

those of peripheral blood iNKT cells, the first been more pro-Th2 than the latter13,25,26.

Globally, cord blood iNKT cells, compared to peripheral blood, produce less cytokines and

require further priming/differentiation event to behave as fully functional cells of immunity.

Considering these findings we could expect some differences in the ability of iNKT cells

from cord and peripheral blood to express Foxp3 and to become suppressor cells. This

was not the case because they similarly suppressed the proliferation of conventional CD4+

T cells. Hence, at birth iNKT cells have already the potential to become suppressors.

Further, we found that Foxp3 expression requires iNKT cell TCR cross-linking but is

independent of antigen presenting cells as ex vivo sorted iNKT cells cultured with anti-

CD3, anti-CD2 plus anti-CD28 coated beads and TGF-β expressed this factor, showing

that iNKT cells are intrinsically endowed with the capacity to become suppressors.

TGF-β has been reported as a major factor for the differentiation of naïve CD4+ T cells

into both Th17 cells and Treg cells46. One of the mechanisms implicated is the ability of


101

TGF-β to inhibit Th1 and Th2 cytokine production47. We found that TGF-β inhibits IL-4 and

IFN-γ production by activated iNKT cells and that Foxp3+ iNKT cells, in contrast to their

Foxp3- counterpart, did not produce these cytokines. Knowing that human iNKT cells can

produce IL-17 in the presence of TGF-β, IL-1β and IL-2313 and that a fraction of

conventional Treg cells can also secrete this cytokine32,48, we explored this possibility but

we detected no IL-17 production by Foxp3+ iNKT cells. Equally, IL-17-producing iNKT

cells did not express Foxp3 (data not shown). Results obtained here and our previous

report13 suggest that human iNKT cells are different from conventional T cells since, in

contrast to the latter49,50, we observed no Foxp3-RORC double-positive iNKT cells that

could represent a common step in the differentiation of these cells into functional IL-17

producers or suppressors .

All cytokines were not dampened in the presence of TGF-β since Foxp3+ iNKT cells

produced IL-10, a cytokine considered as a suppressive mediator for Treg cells51. The

possible mechanisms of suppression by human Treg cells are: (i) suppression by

inhibitory cytokines, (ii) suppression by cytolysis, (iii) suppression by metabolic disruption,

and (iv) suppression by modulation of dendritic cells (DC) maturation and function.

Despite the presence of IL-10, our findings suggest that soluble factors are not required

for iNKT cells to suppress the proliferation of conventional CD4+ T cells since

supernatants from activated iNKT cells were inactive. Suppression by cytolysis is also

unlikely as we detected no sign of apoptosis in the target cells neither any enhancement

in the expression of FasL or Granzyme B mRNAs by suppressor iNKT cells. We can also

discard the fourth possibility, as we used no APCs or DC in our suppressive test. The

mechanism we favour is that Foxp3+ CD25+ iNKT cells absorb the IL-2 produced in the

system and mediate suppression of CD4+ T cell proliferation as a result of direct cytokine

consumption. In agreement with this hypothesis, rapamycin, which confer the suppressive

activity of iNKT cells, strongly up regulated the CD25 marker in iNKT cells.

Several studies have provided evidences for a role of iNKT cells in promoting tolerance in

a variety of experimental models, including models of autoimmune disorders, transplant

tolerance, burn injury-induced immune suppression, and antigen-specific tolerance14,52-55.

These studies clearly establish that iNKT cells play a role in inducing and/or maintaining

peripheral tolerance. The mechanisms by which they mediate their tolerogenic effects are

still unclear but it was reported that they are independent of IL-4 and IL-10 in autoimmune

diabetes56. These iNKT cell cytokine-independent regulatory effects could be explained by

the strong suppressive properties displayed by Foxp3+ iNKT cells, described here. It has

been demonstrated that iNKT cells can prevent type I diabetes through inhibition of


102

autopathogenic T cell expansion and full differentiation, rendering them unable to destroy

pancreatic islets 57. Interestingly, this immunoregulatory function of iNKT cells required cell

contact58, which mirrors Foxp3+ iNKT cell regulatory function and our findings that

cytokines are not always the principal mediator of iNKT cell action on the immune

responses.

In conclusion, our findings clearly demonstrated that human iNKT cells are endowed with

the capacity to become suppressor cells when stimulated in the presence of TGF-β plus

rapamycin. Foxp3+ iNKT cells are likely implicated in tolerogenic immune responses and

could contribute to dampen autoimmune inflammation as those observed in diabetes. We

demonstrated further the human iNKT cell plasticity and the major role of the environment

in controlling their functional properties, then providing additional means for manipulating

iNKT cell function, and thereby improve their use in adoptive immunotherapy.


103

ACKNOWLEDGMENTS

We are especially indebted to the National Institutes of Health Tetramer Facility for

providing CD1d/PBS57 tetramers. We are grateful to Jérôme Mégret and Corinne Garcia-

Cordier (Necker Institute) for cell sorting and we also appreciated the help of Olivier

Hermine’s and Jean-Philippe Herbeuval’s teams in preparing human peripheral and cord

blood cells. We are indebted to the technical assistance of Séverine Diem.

This work was supported by institute funds from de Centre National de la Recherche

Scientifique (CNRS), University René Descartes-Paris V. L.M.T. was supported by

fellowship from Fundação para a Ciência e Tecnologia (FCT) and the Programa

Operacional Potencial Humano (POPH), Portugal (Grant: SFRH/BD/37178/2007 and

PTDC/SAU-FCT/101017/2008). L.M.T. and M.R. were supported by Portugal-France

cooperation: Programa Pessoa (2009-2010) and/or fellowship FCT/CNRS 2009.


104

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FIGURE LEGENDS

Figure 1. Foxp3 expression by human iNKT cells. (A) Representative FACS profile of ex vivo iNKT cells gated based on the expression of

both CD1d-tetramer and CD3 markers. (B, C) Representative FACS profile showing the

expression of intracellular Foxp3 (solid line histograms) in iNKT cells (left) or CD4+

CD25high cells (right) from fresh cord (B) or peripheral blood (C). The shaded histogram

represents the isotype control. Data are representative of 3 to 5 independent donors. (D,

E) Representative FACS profile of Foxp3 expression among gated iNKT cells from cord

(D) or peripheral blood (E) expanded with α-GalCer plus IL-2 in the presence of medium

(left) or TGF-β (right). The shaded histogram represents the isotype control and

percentages are indicated. Data are from 10 CBMC and 20 PBMC donors.

Figure 2. Foxp3 expression by human CD4+ and CD4- iNKT cell subsets. (A) Intracellular staining of Foxp3 in CD4+ or CD4- iNKT-cell subsets (gated as shown in

the left graph) from peripheral blood cultured with α-GalCer plus IL-2 in the presence of

medium (shaded histograms) or TGF-β (solid line histograms). Percentages are indicated.

(B) Frequency of cells expressing Foxp3 among gated CD4+ or CD4- iNKT cell subsets

from peripheral blood cultured as described in (A). Each symbol represents one donor;

horizontal bars indicate median. The p values were determined by a Mann-Whitney U test.

Data are from 20 donors.

Figure 3. Phenotype of Foxp3+ iNKT cells.

(A, B) Expression of Foxp3 and CD25, CTLA4 or GITR by iNKT cells expanded from cord

(A) or peripheral blood (B) with α-GalCer plus IL-2 in the presence of medium (top) or

TGF-β (bottom). Percentages are indicated in each quadrant. Data are representative of

at least 3 donors.

Figure 4. Cytokine production by human Foxp3+ iNKT cells.

(A, B) Intracellular staining of Foxp3 and IL-4, IFN-γ, IL-10 or IL-17 in gated iNKT cells

from cord (A) or peripheral blood (B) cultured with α-GalCer plus IL-2 in the presence of

medium (top) or TGF-β (bottom), and restimulation for 5 h with PMA plus ionomycin and

brefeldin A. Percentages are indicated in each quadrant. Data are representative of at

least 3 donors.


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Figure 5. TGF-β does not confer suppressive activity to human iNKT cells.

(A, B) CD25+ cells were sorted from cord (A) and peripheral blood (B) iNKT cultured with

α-GalCer plus IL-2 in the presence of medium or TGF-β and tested for their suppressive

activity in vitro. Histograms represent CFSE dilution of labelled heterologous CD4+CD25-

responder T cells cultured alone (left) or at a 1:1 ratio with CD25+ iNKT cells or Treg cells.

Data are representative of 8 independent experiments.

Figure 6. Human Foxp3+ iNKT cells induced by TGF-β plus rapamycin are

suppressive (A) Representative FACS profile of Foxp3 expression in gated iNKT cells from cord

(CBMC) or peripheral blood (PBMC) expanded with α-GalCer plus IL-2 in the presence of

TGF-β plus rapamycin. The shaded histogram represents the isotype control.

Percentages are indicated. Data are from 15 CBMC donors and 20 PBMC donors. (B)

Frequency of Foxp3-expressing cells among gated iNKT cells expanded with α-GalCer

plus IL-2 in the presence of TGF-β alone or TGF-β plus rapamycin at different times of

culture. Data are representative of 3 donors. (C, D) CD25+ cells were sorted from iNKT

cells cultured with α-GalCer plus IL-2 in the presence of TGF-β plus rapamycin and tested

for their suppressive activity in vitro. Histograms represent CFSE dilution of labelled

autologous (C) or heterologous (D) CD4+CD25- responder T cells cultured alone (shaded

histograms) or at a 1:1 ratio with CD25+ iNKT cells. Data are representative of 3 to 8

independent experiments. (E) Inhibition of proliferation by CD25+ iNKT cells from 8 CBMC

and 8 PBMC donors normalized to proliferation of responder cells alone, assessed by

CFSE dilution as described in (D). Each symbol represents one donor; horizontal bars

indicate median. The p values were determined by a Mann-Whitney U test. Data are from

8 CBMC and 8 PBMC donors.

Figure 7. Human Foxp3+ iNKT cells suppress T cell proliferation in a cell-contact dependent mechanism.

(A) CD25+ cells were sorted from iNKT cells cultured with α-GalCer plus IL-2 in the

presence of TGF-β plus rapamycin and tested for their suppressive activity in vitro.

Histograms represent CFSE dilution of labelled heterologous responder T cells cultured

alone (shaded histogram) or at a 1:1 ratio with CD25+ iNKT cells (cell-contact) or with the

supernatant obtained from activated CD25+ iNKT cells (supernatant) (solid line

histograms). Data are representative of 2 independent experiments. (B) CFSE dilution of

labelled heterologous responder T cells cultured alone (shaded histogram) or at a 1:1 ratio

with CD25+ iNKT cells in the presence (solid line histograms) or absence (dashed line

histograms) of anti-GITR. Data are representative of 3 independent experiments.


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FIGURES

Figure 1


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Figure 2


113

Figure 3


114

Figure 4


115

Figure 5


116

Figure 6


117

Figure 7


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Figure S1. Foxp3 expression by human iNKT cells is not dependent of α-GalCer

activation neither from APC. (A) Intracellular expression of Foxp3 by iNKT cells sorted ex vivo and cultured with anti-

CD3 plus anti-CD28 and anti-CD2 coated beads plus IL-2 in the presence of medium

(dashed line histogram) or TGF-β (solid line histogram). Data are representative of 3

donors. (B) Intracellular staining of Foxp3 by CD4+ or CD4- iNKT cell subsets sorted ex

vivo and cultured with α-GalCer plus IL-2 in the presence of TGF-β and autologous PBMC

depleted on iNKT cells. Data are representative of 3 donors.


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Figure S2. Phenotype and cytokine production by iNKT cells expanded in the

presence of TGF-β plus rapamycin.

(A) Expression of Foxp3 and CD25, CTLA4 or GITR in gated iNKT cells expanded from

cord (CBMC) or peripheral blood (PBMC) with α-GalCer plus IL-2 in the presence of TGF-

β plus rapamycin. Percentages are indicated in each quadrant. Data are representative of

at least 3 donors. (B) Intracellular staining of Foxp3 and IL-4, IFN-γ or IL-10 in gated iNKT

cells expanded from cord (CBMC) or peripheral blood (PBMC) as described in (A) and

restimulation for 5 h with PMA plus ionomycin and brefeldin A. Percentages are indicated

in each quadrant. Data are representative of 5 donors.


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Figure S3. Fold induction of Foxp3, IRF4, IL-2RA, CCR4 and LGALS3 in human iNKT

cells by TGF-β plus rapamycin.

Real-time RT-PCR analysis of the expression of Foxp3, IRF4, IL-2RA, CCR4 and

LGALS3 in sorted CD25+ iNKT cells obtained after two weeks of culture with α-GalCer

plus IL-2 in the presence of medium or TGF-β plus ramamycin. The results were

normalized to the following housekeeping genes: β2M, HPRT1, RPL13A, GAPDH, ACTB.

Fold induction is represented relative to the expression in CD25+ iNKT cells cultured in the

presence of TGF-β and rapamycin to that culture in the presence of medium. Data are

representative of 2 cord blood and 2 peripheral blood donors.

CHAPTER V

DISCUSSION AND PERSPERCTIVES

CHAPTER V – Discussion and Perspectives

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Since the discovery of iNKT cells over 20 years ago, it has become clear that they

possess distinct functional properties allowing them to influence a large range of immune

responses. Whether their diversified functional capacities can be attributed to a single

population sensitive to environment cues or to functionally distinct iNKT cell

subpopulations remains to be determined. Several teams study the biology of iNKT cells

and the majority of these studies center on mouse iNKT cells. During my thesis, we

focused our attention on human iNKT cells and we demonstrated that CD161+ iNKT cells

are intrinsically endowed to become IL-17 producers although they depend on TGF-β, IL-

1β and IL-23 to carry out this potential. In addition to dictate IL-17 production, when

combined with proinflammatory signalling, TGF-β played a crucial role in the induction of

Foxp3 expression and, more importantly, in the acquisition of suppressive activity by

human iNKT cells, when combined with mTOR inhibition. Our results revealed the

functional plasticity of human iNKT cells and the major influence of cytokines present in

their environment to drive their suppressive or effector IL-17-producing capacities. We will

discuss these points below.

IL-17-producing human iNKT cells: major conditions required

In mice, iNKT17 cells comprise a distinct lineage that retains ROR-γt expression during

their development, does not express the NK1.1 marker and, contrary to conventional T

cells, emigrate from the thymus full capable to secrete IL-17 without polarization (Michel et

al., 2007; Michel et al., 2008). We found that human IL-17-producing iNKT cells behave

differently. Indeed, they originate from a lineage-committed precursor bearing the CD161

marker as human CD161+ iNKT cells, but not their CD161- counterparts, are endowed

with the capacity to secrete IL-17. However, in contrast to mouse iNKT cells, the capacity

of human iNKT cells to secrete IL-17 is only effective when TGF-β, IL-1β and IL-23 are

present during the stimulation. Expression of CD161 by IL-17-producing iNKT cells is in

agreement with the finding that CD161 is a marker for human conventional IL-17-

producing T cells (including CD4+, CD8+ and DN αβ T cells and γδ T cells) (Maggi et al.,

2010), which raises the question of its functional significance. Although CD161 has neither

activating nor inhibitory signalling motifs, CD161 acts as both an activating and inhibitory

receptor, depending on the cell type. Lectin-like transcript 1 (LLT1) has been identified as

a physiological ligand for human CD161 (Kamishikiryo et al., 2011; Rosen et al., 2005).


123

LLT1 is expressed by TLR-activated plasmacytoid DC, monocyte-derived DC and B cells

and CD161-LLT1 interaction leads to the inhibition of NK cell cytotoxicity and IFN-γ

production (Aldemir et al., 2005; Rosen et al., 2005; Rosen et al., 2008). However, this

interaction in combination with TCR signalling activates T cells to secrete IFN-γ (Aldemir

et al., 2005), which suggest that NK cells and T cells are differentially regulated by CD161

activation. iNKT cells share characteristic from both NK cells and T cells, thereby we can

speculate that, in the absence of TCR signalling, CD161 will act as an inhibitory receptor

(inhibiting iNKT cell cytotoxicity and cytokine production) but, when combined with TCR

signalling, CD161 will act as a co-activating receptor for iNKT cells (promoting their

effector functions). Understand the possible role of CD161 activation on human iNKT cell

function, in particular on IL-17 production, will be a challenge for coming times.

Our findings suggest that CD161+ iNKT cells are committed to become IL-17 producers.

However, a favourable cytokine environment is critical to allow IL-17 production by iNKT

cells. Among these critical cytokines we have TGFβ. TGF-β has been reported as a major

factor for the differentiation of naïve CD4+ T cells into both Th17 cells and Treg cells.

During polarization, TGF-β and proinflammatory cytokines polarize naïve T cells toward a

Th17 phenotype, whereas TGF-β alone induces Treg cells (Bettelli et al., 2006). Similarly,

we reported the dual role of TGF-β in inducing IL-17 production or Foxp3 expression by

human iNKT cells. The requisite for TGF-β in the development of human Th17 cells is

currently under debate. We clearly found that TGF-β plays a crucial role in the induction of

IL-17 production by human iNKT cells since, in the absence of TGF-β, the cytokines IL-1β

and IL-23 were not able to polarize cord blood iNKT cells towards IL-17 production. We

also found that TGF-β strongly inhibits IL-4 and IFN-γ production by activated iNKT cells.

It has been suggested that TGF-β promotes Th17 differentiation by suppressing the

generation of Th1 and Th2 cells (Das et al., 2009; Santarlasci et al., 2009). Human iNKT

cells are able to promptly produce IFN-γ as well as IL-4 following activation. Knowing that

IL-4 inhibits IL-17 production by mouse iNKT cells (Michel et al., 2007) and both Th1 and

Th2 cytokines inhibit Th17 cell differentiation (Park et al., 2005), our findings indicate that

inhibition of IL-4 and IFN-γ by TGF-β may facilitate the induction of IL-17 production by

human iNKT cells without, however, exclude a direct effect of TGF-β on human iNKT17-

cell differentiation.

Previous reports suggested that cord and adult peripheral blood iNKT cells are

functionally distinct (Baev et al., 2004; Eger et al., 2006), and respectively considered as

less and more experienced cells. Here, we demonstrated that both cord and adult


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peripheral blood iNKT cells were able to produce IL-17 under polarizing conditions. IL-17-

producing iNKT cells from cord blood belong predominantly to the minor CD4- cell subset

and derived exclusively from the CD161+CCR6+ cell subset whereas IL-17-producing

iNKT cells from adult peripheral blood derived either from CD161+CCR6+ or

CD161+CCR6- cells, and belong to both CD4+ and CD4- cell subsets. Cord blood and

thymic iNKT cells are comparatively similar since CD161+ iNKT cells are also extremely

rare in human thymus, (Baev et al., 2004; Berzins et al., 2005; Sandberg et al., 2004), but

they already have the potential to secrete IL-17 when stimulated by their TCR in

association with TGF-β, IL-1β plus IL-23 (data not shown). This finding suggests that, like

in mice, human iNKT17 cells develop in the thymus. The frequency of CD161+ iNKT cells

is enhanced in the periphery with age, suggesting a further differentiation or accumulation

since, in humans, thymic cell export is restricted after birth. This is in contrast with their

mouse counterpart, which are recent thymic emigrants (Milpied et al., 2011). Despite their

accumulation in periphery, only a fraction of circulating CD161+ iNKT cells in adults

produce IL-17 following stimulation. Moreover, IL-17 expression is not concomitant with

CCR6 or CD4 expression, as in case of conventional T cells (Cosmi et al., 2008), which

portends further studies to identify the factors defining the developmental program of

human iNKT17 cells, with RORC being a plausible candidate, as demonstrated in mouse

(Michel et al., 2008). The transcription factor RORC has been shown to play a key role on

IL-17-producing cells, being necessary and sufficient for induction of IL17 (Cua and Tato,

2010; Korn et al., 2009). Accordingly, we found that TGF-β, IL-1β and IL-23 increase

RORC mRNA expression by iNKT cells. However, it is still unclear whether the lineage-

committed precursor for IL-17-producing iNKT cells constitutively express RORC or

whether polarizing cytokines induces its expression. It has been reported that cord blood

CD161+ cells express RORC mRNA ex vivo suggesting that at least a fraction of these

cells could be iNKT cells (Cosmi et al., 2008; Maggi et al., 2010). We were not able to

detect RORC, at the protein level, in ex vivo human iNKT cells although RORC was easily

detected in human iNKT cells after activation (data not shown). Our preliminary data

revealed that although all IL-17-producing iNKT cells expressed RORC, not all RORC-

expressing iNKT cells produced IL-17 (data not shown), similarly to results obtained in

mice (Michel et al., 2008). This finding suggests that although necessary for IL-17

induction, RORC expression by activated human iNKT cells does not imply necessary IL-

17 production.

Another difference between cord and peripheral blood iNKT cells is that, in contrast to

peripheral, cord blood IL-17-producing iNKT cells did not co-produce IFN-γ. Co-production

of IL-17 and IFN-γ has been also reported in human Th17 cells and γδ T cells (Annunziato


125

et al., 2007; Boniface et al., 2010; Ness-Schwickerath et al., 2010). TGF-β, IL-1β and IL-6

polarize human γδ T cells into IL-17 single-positive γδ T (γδ T17) cells but the addition of

IL-23 to polarizing cytokines appears to convert γδ T17 cells into IL-17- plus IFN-γ-

producing γδ T (γδ T17/1) cells (Ness-Schwickerath et al., 2010). A similar effect has been

seen for human Th17 clones where IL-23 converted a subset of Th17 cells into Th17/1

cells (Annunziato et al., 2007). This effect of IL-23 does not apply to human IL-17 iNKT

cells since both cord and peripheral blood iNKT cells were polarized in the presence of IL-

23 but only iNKT cells from adult peripheral blood displayed the potential to co-produce

both cytokines. The study with human Th17 clones found an even higher degree of Th17

cell conversion to Th17/1 cell phenotype with IL-12 (Annunziato et al., 2007). Mouse iNKT

cells constitutively express IL-12R, which is up regulated upon activation (Zhu et al.,

2007). Higher levels of IL-12R expression by human peripheral blood IL-17-producing

iNKT cells may explain the co-production of IFN-γ. Several reports have demonstrated

that neonatal innate cells, including professional APCs, produce insufficient levels of IL-12

to program Th1 effector cells (Arulanandam et al., 1999; Garcia et al., 2000; Holt and

Jones, 2000). Because IL-12 production seems to increase with age, we can speculate

that IL-12 secreted by α-GalCer-activated APC in our in vitro culture converts most of the

responding IL-17 single-positive iNKT cells to IL-17- plus IFN-γ-producing iNKT cells in

adult peripheral blood. However, even if confirmed, this hypothesis does not explain the

other differences found between cord and peripheral blood IL-17-producing iNKT cells.

Moreover, cord blood IL-17-producing iNKT cells failed to co-produce IFN-γ even when

cultured with APC from adult peripheral blood (data not shown). Thus other factors

intrinsic to iNKT cell development at the periphery from foetal to adult life may dictate the

functional disparities observed between cord and adult peripheral blood iNKT cells.

IL-22, an important cytokine produced by Th17 cell lineage (Korn et al., 2009), was also

secreted by human iNKT cells. Nevertheless, differences exist since IL-17-producing iNKT

cells have restricted ability to co-produce IL-22 and iNKT cells did not require the

presence of polarizing cytokines to secrete IL-22. These findings suggest that distinct

mechanisms are implicated in the regulation of IL-17 and IL-22 production by human iNKT

cells. The existence of IL-22 single-positive cells has also been described for conventional

CD4+ T cells, γδ T cells and ILC (Cella et al., 2009; Duhen et al., 2009; Ness-Schwickerath

et al., 2010; Trifari et al., 2009). Naïve CD4+ T cells differentiate into Th22 cells in the

presence of IL-6 and TNF-α. The combination of IL-1β, IL-6 and TNF-α favours

development of IL-22- plus IL-17-producing CD4+ T cells, whereas addition of TGF-β

inhibits Th22 cell differentiation (Duhen et al., 2009). Future experiments are needed to


126

characterize the role of these cytokines on IL-22 production by human iNKT cells. AHR is

one of the major factors involved in the regulation of IL-22 production by human T cells

(Trifari et al., 2009). We found that FICZ, an AHR agonist, promoted the production of IL-

22 by human iNKT cells while inhibited IL-17-producing iNKT cell development, thereby

changing the balance of IL-22- versus IL-17-producing iNKT cells. The mechanism of

AHR agonist mediated regulation of IL-22 and IL-17 production remains to be determined.

It has become apparent that IL-17 and IL-22 responses are important for host protection

against potential pathogenic organisms (trough neutrophil recruitment and the induction of

antimicrobial peptides) or, in contrast, have a pathologic role in diverse chronic

inflammatory disorders (by promoting the expression of proinflammatory mediators)

(Iwakura et al., 2011; O'Connor et al., 2010). Given the identification of lysophospholipid

messengers, which are present at elevated levels during inflammatory responses, as new

endogenous ligands of human iNKT cells (Chang et al., 2008; Fox et al., 2009), it can be

expected that endogenous pathways exist to activate iNKT cells during inflammatory

responses. We found high numbers of IL-17- and IL-22-producing cells among circulating

iNKT cells from a patient with Crohn’s disease. Peripheral blood cells are not necessary

representative of the in situ inflammation and further studies of the site of active disease

are required to confirm the presence of these cells, but our result suggests that IL-17-

producing iNKT cells may have an important role in inflammatory response during Crohn’s

disease.

A critical role for iNKT cells has been suggested in asthmatic patients (Akbari et al., 2006;

Pham-Thi et al., 2006a; Pham-Thi et al., 2006b). Compared with non-asthmatic controls

individuals, patients with severe and poorly controlled asthma have increased numbers of

pulmonary iNKT cells. IL-17 is also increased in pulmonary specimens from asthmatic

patients (Ivanov and Linden, 2007), which correlates with the presence of neutrophils in

the sputum from patients with increasing disease severity (Bullens et al., 2006). Recently,

our laboratory reported that mouse IL-17-producing iNKT cells are enriched in the lung,

where they contribute to airway inflammatory responses (Michel et al., 2007). Although

functional studies must be done to better understand the role of human iNKT cells in

asthma, the existing data raises the possibility that human iNKT cells in the lung of

asthmatic patients may contribute to local accumulation of neutrophils and to disease

severity by the production of IL-17.

In this pathological context, recent studies have demonstrated that pathogenicity of IL-17-

secreting conventional T cells is influenced by their ability to co-secrete others cytokines.

For instance, it has been reported that IL-10-producing Th17 cells fail to up regulate the


127

proinflammatory chemokines crucial for central nervous system inflammation, retaining

the pathogenic potential of Th17 cells (McGeachy et al., 2007). However human iNKT

cells have the potential to produce either IL10 or IL-17, we did not find co-production of

both cytokines by iNKT cells. In contrast to IL-10 effect, GM-CSF has been recently

reported as a critical factor for promoting the proinflammatory functions of Th17 cells

(Codarri et al., 2011; El-Behi et al., 2011). Human iNKT cells are an important source of

this cytokine (Gumperz et al., 2002) and it needs to be investigated whether IL-17-

producing iNKT cells co-produce GM-CSF or not. Interestingly, IL-23 signalling has a

pivotal role in the inhibition of IL-10 and the promotion of GM-CSF co-production by IL-17-

producing T cells (Codarri et al., 2011; El-Behi et al., 2011; McGeachy et al., 2007),

determining the pathogenic potential of Th17 cells. We found that IL-23 is implicated on

IL-17 production by human iNKT cells but further studies are required to understand

whether human IL-17-producing iNKT cells can be generated and can have a distinct

cytokine profile in the absence of IL-23 signalling.

Foxp3+ iNKT cells: suppressors or not suppressors?

Similarly to conventional CD4+ T cells, we found that human iNKT cells are induced to

express Foxp3 by TGF-β. In the course of this thesis, Monteiro et al. (2010) reported that

TGF-β induces Foxp3 expression on mouse and human iNKT cells, in agreement with our

results, and that mouse Foxp3+ iNKT cells are suppressive. In our in vitro system, Foxp3

induced by TGF-β failed to confer suppressive activity to human iNKT cells. Emerging

data suggest that human Foxp3+ T cells are not functionally homogenous as a significant

number of human activated T cells express Foxp3 but do not acquire regulatory activity

(Allan et al., 2007; Gavin et al., 2006; Morgan et al., 2005; Tran et al., 2007; Wang et al.,

2007). Multiple factors may control both the cellular susceptibility to induction of Foxp3

and the capacity of cells expressing Foxp3 to manifest suppressive activity. For instance,

it has been reported that transfection of Foxp3 is less efficient in reprogramming human

memory T cells into suppressive cells (Yagi et al., 2004). Human iNKT cells display a

memory phenotype as shown by surface expression of CD45ROhigh and CD62Llow

(D'Andrea et al., 2000; van der Vliet et al., 2000), and could therefore not respond to or

lack components of the Foxp3 driven regulatory pathway. However, we found that human

Foxp3+ iNKT cells are completely able to acquire suppressive activity following activation

in the presence of TGF-β and rapamycin, suggesting that Foxp3 pathway is intact on iNKT

cells. It has been reported that sustained Foxp3 expression in Treg cells is necessary for


128

maintenance of the Treg cell phenotype and suppressor function. Loss of Foxp3 or its

diminished expression in Treg cells lead to acquisition of effector proprieties including

production of cytokines such as IL-4, IL-17 and IFN-γ (Miyara et al., 2009; Wan and

Flavell, 2007; Williams and Rudensky, 2007). Accordingly, we found that inhibition of

mTOR pathway by rapamycin sustains TGF-β-induced Foxp3 expression over time,

conferring suppressive activity to human Foxp3+ iNKT cells. The induction of Foxp3 by

TGF-β depends on the activation of the transcription factor SMAD3 (mothers against

decapentaplegic homologue 3) that cooperate with NFAT to induce Foxp3 gene

expression (Tone et al., 2008). Interestingly, signalling through the mTOR pathway can

inhibit activation of SMAD3 by TGF-β (Song et al., 2006), providing an explanation for the

observation that mTOR inhibition by rapamycin supports Foxp3 up regulation.

Additionally, rapamycin blocks proliferation of conventional effector T cells whereas

promotes selective survival and growth of highly suppressive Treg cells (Battaglia et al.,

2005; Strauss et al., 2007). It has been suggest that this differential response of effector T

cells and Treg cells is due to their divergent activation-cell death response in the presence

of rapamycin. Treg cells up regulate anti-apoptotic and down regulate pro-apoptotic

proteins in the presence of rapamycin being resistant to apoptosis, while effector T cells

do exactly the opposite become highly sensitive to apoptosis (Strauss et al., 2009). We

can speculate that, similarly, TGF-β-induced Foxp3+ iNKT cells will be more resistant to

apoptosis than Foxp3- iNKT cells, being selectively expanded in the presence of

rapamycin. Overall, the effect observed on the expression of Foxp3 by human iNKT cells

in the presence of rapamycin may be explain as the sum of the two effects, the promotion

of Foxp3 induction by TGF-β and the selective proliferation of Foxp3+ iNKT cells, which

become resistant to apoptosis in the presence of rapamycin. Importantly, our results

pointed out another major difference between mouse and human iNKT cells. Whereas

TGF-β signalling is sufficient to induce suppressive properties to mouse iNKT cells

(Monteiro et al., 2010), human iNKT cells required the combined action of TGF-β and

mTOR inhibition to effectively become suppressive Foxp3+ cells.

It has been proposed that the up regulation of Foxp3 by CD4+ T cells after activation is a

mechanism for attenuating the activation of these cells (Ziegler, 2006). Foxp3 binds to the

transcription factors NFAT and Runx1 and blocks their ability to transcribe cytokine,

thereby, suppressing effector T cell function (Ono et al., 2007; Wu et al., 2006). We found

that TGF-β strongly inhibits IL-4 and IFN-γ production by activated iNKT cells. It is

tempting to speculate that Foxp3 induction is the factor responsible for such effect.

However, the inhibition of effector cytokines by TGF-β was independent of Foxp3


129

expression as Foxp3- iNKT cells also failed to produce IL-4. Moreover, we observed that

Foxp3 expression decreased over time but the production of IL-4 was not recovered, even

when iNKT cells were restimulated in the absence of TGF-β (data not shown), suggesting

that the inherent program for IL-4 production was altered. A recent study reported that

inhibition of Th1 and Th2 cell differentiation by TGF-β is specifically due to TGF-β down

regulation of STAT4 and GATA-3 expression (Das et al., 2009). Cross-inhibition of Th cell

subsets is an important mechanism to ensure expansion of the polarized populations and

is carried out by polarizing cytokines but also by transcription factors. For instance, T-bet

directly inhibits Th2 differentiation by binding to its master regulator GATA-3 (Hwang et

al., 2005). Likewise, GATA-3 can down regulate STAT4 (Usui et al., 2003) and Foxp3

(Mantel et al., 2007) and thereby inhibits Th1 and Treg cell differentiation, respectively.

RORC is expressed by both Th17 cells and Treg cells (Burgler et al., 2009; Lochner et al.,

2008; Zhou et al., 2008). In Treg cells, Foxp3 suppresses transcriptional activity of RORC

by direct interaction, thereby inhibiting Th17 cell differentiation (Ichiyama et al., 2008;

Zhou et al., 2008). In turn, it has been recently reported that RORC inhibits Foxp3

expression in human T cells suppressing regulatory T cell-specific programs (Burgler et

al., 2010). We demonstrated that TGF-β is required to obtain both IL-17-producing and

Foxp3+ iNKT cells, but we did not observed the co-expression of these two molecules by

iNKT cells. A possible explanation is that RORC is implicated in the inhibitory effect of IL-

1β and IL-23, which are critical to induce IL-17 production, on Foxp3 expression by iNKT

cells. RORC inhibitors has been recently identified (Huh et al., 2011), opening

perspectives to the development of RORC-targeted therapeutic agents to attenuate

inflammatory responses and autoimmune disease. Understand how TGF-β, Foxp3 and

RORC regulate human iNKT cell function will be of crucial importance to predict the

potential effect of this therapeutic approach on iNKT cells. In addition, our findings show

that human iNKT cells are a useful model to better understand the interactions of all these

factors since they are potentially capable to secrete IL-4, IFN-γ, IL-17, IL-22 and become

Foxp3+ suppressor cells.

Despite inhibition of effector cytokines production, TGF-β promotes IL-10 secretion by

human iNKT cells. IL-10 is an inhibitory cytokine that has been implicated in the

suppressive function of Treg cells in vivo (Hawrylowicz and O'Garra, 2005). However, IL-

10 secretion by human iNKT cells was not involved in their suppressive activity since the

supernatants of activated CD25+ iNKT cells were unable to efficiently suppress responder-

cell proliferation, suggesting that soluble factors are not implicated in the regulatory effects

observed. Most reports have shown that Treg cells suppress immune response through

cell contact-dependent mechanisms having either responder T cells or APC as target


130

(Shevach, 2009). Monteiro et al. (2010) reported that mouse Foxp3+ iNKT cells suppress

responder T cell proliferation through a cell contact-dependent mechanism mediated by

GITR. In contrast, we found that human Foxp3+ iNKT cell-mediated suppression is not

GITR-dependent. Moreover, our data excluded suppression by modulation of APC

activation and/or function, as APC were absence from our in vitro suppressive assay. We

also failed to detect apoptosis of CD4+ cells in the presence of Foxp3+ iNKT cells (data not

shown). Consequently, further investigations are required to depict the precise

mechanism of human iNKT cell-mediated suppression.

Several studies have provided evidences for a role of iNKT cells in promoting tolerance in

a variety of experimental models, including models of autoimmune disorders, transplant

tolerance, burn injury-induced immune suppression, and antigen-specific tolerance

(Godfrey and Kronenberg, 2004; Kim et al., 2006; Palmer et al., 2006; Wu and Van Kaer,

2009; Yang et al., 2007). These studies clearly establish that iNKT cells play a role in

inducing and/or maintaining peripheral tolerance, yet the mechanisms by which they

mediate their tolerogenic effects are still unclear. As iNKT cells are known to produce a

wide variety of cytokines, one possibility is that they provide an essential source of

immunoregulatory cytokines such as IL-10, or that they can shift the balance away from

proinflammatory processes by producing Th2 cytokines such as IL-4. However, studies

using IL-4 and IL-10 deficient mice have demonstrated that the secretion of these

cytokines is dispensable for iNKT cells to mediate regulatory effects in many systems,

such as in autoimmune diabetes (Chen et al., 2005). These iNKT cell cytokine-

independent regulatory effects could be explained by the strong suppressive properties

displayed by Foxp3+ iNKT cells, described here. It has been demonstrated that iNKT cells

can prevent type I diabetes through inhibition of autopathogenic T cell expansion and full

differentiation, rendering them unable to destroy pancreatic islets (Beaudoin et al., 2002).

Interestingly, this immunoregulatory function of iNKT cells required cell contact and was

independent of cytokines, including IL-4 and IL-10 (Novak et al., 2005), which mirrors

Foxp3+ iNKT cell regulatory function. Defects in iNKT cell number have been reported in

patients with GVH disease after HSCT (Haraguchi et al., 2004). Accordingly, a longitudinal

analysis of the immune reconstitution of peripheral blood iNKT cells in patients having

received an allogeneic HSCT for diverse malignant hematological diseases, performed at

our laboratory, show that the recovery of a high iNKT/T cell ratio appears as a predictive

factor of the absence of acute GVH disease (Rubio et al., submitted for publication).

Based on our finding that human Foxp3+ iNKT cells suppressed allogeneic T cell

proliferation, we can speculate that iNKT cells may abrogate GVH disease through


131

suppression of alloreactive T cell activation and proliferation. This hypothesis is being

investigated.

A number of observations suggest that a major role of iNKT cells is to serve as a type of

regulatory T cell that can drive downstream immune responses along either pro-

inflammatory or silencing pathways. Support for this view comes first from findings that

iNKT cells produce a wide variety of cytokines; and that mice genetically deficient in iNKT

cells show defects not only in resistance to microbial infections and in tumour

immunosurveillance but also in establishing peripheral tolerance and preventing

autoimmunity (Bendelac et al., 2007; Kronenberg, 2005). Research interest in human

iNKT cells increased markedly following reports of iNKT cell defects in patients with

autoimmune diseases, cancer and infections (Balato et al., 2009; Berzins et al., 2011;

Swann et al., 2007; Terabe and Berzofsky, 2008; Wu and Van Kaer, 2009). The reported

associations between iNKT cells and human disease have been grouped into distinct

categories. From one side, iNKT cell defects, such as reduced number or impaired

cytokine production, may compromise immune regulation and increase predisposition to

autoimmune diseases, cancer and some infections. We can speculate that defects in

iNKT cell-mediated suppression of autopathogenic T cell clones may contribute to

autoimmune diabetes; and that a compromised production of IL-17 by iNKT cells in

response to pathogens may lead to infection persistence. On the other side, iNKT cells

can be normal in number and functionally competent, but they respond inappropriately to

self-antigens contributing to pathogenic immune responses that could lead to chronic

allergic and inflammatory disorders. The elevated levels of endogenous ligands for human

iNKT cells during inflammatory responses (Chang et al., 2008; Fox et al., 2009) support

this idea. Based in our findings, is it tempting to speculate that chronic activation of iNKT

cells in the inflammatory context of Crohn’s disease contribute to the exacerbation of the

disease. Finally, iNKT cells do not necessary contribute to disease pathology, rather they

may exert beneficial effects in preventing and/or treating the disease. This fits with our

finding that recovered iNKT cell numbers inversely correlates with the development of

GVH disease after HSCT and with our hypothesis that iNKT cells have an essential role in

suppression of alloreactive T cells in these patients. Overall, the evidences suggest that,

despite their small population size, human iNKT cells have potent effects on immune

responses, facilitating different outcomes in different contexts.


132

In conclusion, we have provided evidences of a human iNKT cell lineage that

differentiates into IL-17-producing cells under an inflammatory environment and that could

be implicated in chronic inflammatory disorders. Therefore, we provided further evidences

for a notable heterogeneity in the iNKT cell pool, supporting the idea that care must be

taken to study each subset separately to understand the role of these cells in human

diseases and optimize iNKT cell based therapies. Moreover, our results indicate that the

local microenvironment, through provision of specific factors, directs multiple possible

alternate fates that human iNKT cell subsets can adopt (FIGURE 12). These findings

suggest that human iNKT cells exhibit more functional plasticity than previously

appreciated, providing additional means for manipulating iNKT cell function, and thereby

improve their use in adoptive immunotherapy. Although several questions remains

unresolved, this is an important first step towards the manipulation of iNKT cell function as

a future therapeutic benefit to patients.

FIGURE 12. Factors regulating suppressive or effector IL-17-producing capacities of human

iNKT cells. TGF-β has a crucial role in the induction of Foxp3 expression and acquisition of

suppressive activity by human iNKT cells, when combined with rapamycin. On the other hand,

TGF-β combined with IL-1β and IL-23 dictate IL-17 production by pre-committed CD161+ iNKT

cells. Whether these dual functional properties can be attributed to a single population (a) or to

functionally distinct iNKT cell subpopulations (b) remains to be determined.

CHAPTER VII

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