Lúcia de Fátima Moreira Teixeira
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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)
D
E ACORDO COM A LEGISLAÇÃO EM VIGOR, NÃO É PERMITIDA A REPRODUÇÃO DE QUALQUER PARTE DESTA TESE.iii
Faculdade de Farmácia da Universidade do Porto
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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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UTHOR’S
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ECLARATION
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.
S
CIENTIFIC
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UBLICATIONS
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)
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-Philippe Herbeuval, Olivier Hermine, Elke Schneider, Lars Rogge, Frank M. Ruemmele, 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
vii
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-Philippe Herbeuval, Olivier Hermine, Elke Schneider, Lars Rogge, Frank M. Ruemmele, 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
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. XXXVI Annual Meeting of the Portuguese Society for Immunology, Braga, Portugal, 20-22 September 2010
3. 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. 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
5. 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. 2nd European Congress of Immunology, Berlin, Germany,
6. 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. 12e Colloque Cytokines du Croisic, Presqu’île du Croisic, France, 18-20 May 2009
ix
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CKNOWLEDGMENTS
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
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!
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A
BSTRACT
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, 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
additional means to manipulate iNKT cell function and improve their use in adoptive immunotherapy.
xiii
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ESUMO
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
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.
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T
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ONTENTS
Author’s Declaration ...V Acknowledgments ... IX Abstract ... XI Resumo... XIII Table of contents ... XV Index of figures ... XVII Index of tables ... XX Abbreviation list ... XXICHAPTER 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
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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I
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IGURES
FIGURE1. 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
FIGURE5. 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). ...42
FIGURE6. 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
FIGURE7.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
FIGURE8.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
FIGURE10. 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
xix
disruption of the target cell, and/or modulation of DC maturation and function. (Adapted from Vignali et al. 2008). ...67FIGURE11. 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
FIGURE12. 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
I
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ABLES
TABLE1.Classification of NKT cells ... 25
TABLE2.Comparison of human and mouse iNKT cells ... 30
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BBREVIATION
L
IST
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
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
C
HAPTER I
CHAPTER I –iNKT Cells
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).
CHAPTER I –iNKT Cells
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) +/- +/-
CHAPTER I –iNKT Cells
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.
CHAPTER I –iNKT Cells
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.
CHAPTER I –iNKT Cells
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
CHAPTER I –iNKT Cells
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 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
CHAPTER I –iNKT Cells
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.
CHAPTER I –iNKT Cells
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
CHAPTER I –iNKT Cells
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
CHAPTER I –iNKT Cells
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).
CHAPTER I –iNKT Cells
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
CHAPTER I –iNKT Cells
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
CHAPTER I –iNKT Cells
(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.
CHAPTER I –iNKT Cells
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-inCD40-dependent 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
CHAPTER I –iNKT Cells
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.
CHAPTER I –iNKT Cells
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.
CHAPTER I –iNKT Cells
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
CCR1 low, CCR2, CCR4, CCR5, CCR7low, CXCR3, CXCR4 CCR1, CCR2, CCR5, CCR6, CCR7low, CXCR3, CXCR4, CXCR6Adhesion molecules
CD49a, CD62L, CLA, α4β7 CLA, α4β7, CD11ahighNK 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
CHAPTER I –iNKT Cells
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
