Identification d'un nouveau mécanisme de contrôle de l'homéostasie des lymphocytes T iNKT et MAIT

Les cellules Invariant Natural Killer T (iNKT) représentent une sous-population particulière de cellules T qui se distingue par son développement, ses fonctions et les ligands qu elle reconnaît. Chez l homme, les cellules iNKT expriment le réarrangement Va24-Ja18/Vb11 et reconnaissent des glycosphingolipides présentés par la molécule monomorphe du CMH de classe I CD1d. De plus, elles produisent rapidement de grandes quantités de cytokines, et sont ainsi considérées comme des cellules T ayant des caractéristiques innées. Les mécanismes moléculaires qui régulent l homéostasie descellules iNKT ne sont pas complètement compris. La protéine XIAP (X-linked Inhibitor of Apoptosis) est un inhibiteur physiologique des caspases 3, 7 et 9. Des mutations du gène XIAP sont à l origine du syndrome lymphoprolifératif lié à l X de type 2 (XLP-2), un déficit immunitaire primitif (DIP) caractérisé par une susceptibilité accrue à l infection par le virus Epstein Barr (EBV). Les patients souffrant du XLP-2 présentent une forte réduction de leur nombre de cellules iNKT dans le sang. Au cours de mon travail de thèse, j ai montré que XIAP est requis pour la survie des cellules iNKT humaines. Cette fonction de XIAP corrèle avec un phénotype pro-apoptotique des cellules iNKT qui n est pas retrouvé dans les cellules T conventionnelles. La susceptibilité accrue à l apoptose des cellules iNKT est observée en utilisant des stimuli de la voie intrinsèque ou extrinsèque de l apoptose. Les cellules iNKT, contrairement aux cellules T conventionnelles expriment des quantités élevées de protéines pro-apoptotiques comme les caspases 3 ou 7 ou Bid. Ce phénotype proapoptotique est acquis de manière précoce, puisqu il est déjà présent dans des cellules iNKT de thymus ou de sang de cordon. L extinction de XIAP dans des cellules iNKT et l analyse de patients déficients en XIAP indiquent que XIAP est un inhibiteur efficace de l apoptose dans les cellules iNKT alors qu il n a qu un effet modéré dans les cellules T conventionnelles. J ai ensuite montré que le phénotype pro-apoptotique des cellules iNKT est dépendant de l expression du facteur de transcription PLZF. Celui-ci est déjà connu comme étant nécessaire à l acquisition des fonctions effectrices de cescellules. De manière concordante, la surexpression de PLZF dans des cellules T conventionnelles conduit à un phénotype pro-apoptotique et à une augmentation de l expression de la caspase 3. Récemment, une deuxième population de cellules T invariantes, les cellules MAIT (Mucosal Associated Invariant T) a été décrite. Ces cellules expriment un TCR semi-invariant Va7.2-Ja33 et partagent avec les cellules iNKT certaines caractéristiques qui en font des cellules T innées comme les cellules iNKT. De la même manière que les cellules iNKT, les cellules MAIT ont un phénotype proapoptotiqueet sont diminuées dans le sang des patients déficients en XIAP. Ce phénotype proapoptotique est aussi dépendant de PLZF. De manière intéressante, un patient déficient en XIAP et ayant un nombre normal de cellules iNKT a été identifié. Ce patient n a pas encore rencontré l EBV, suggérant que la diminution des cellules iNKT chez les patients déficients en XIAP est due à une apoptose augmentée dans un contexte d infection par l EBV. Enfin, j ai obtenu des données préliminaires suggérant que l EBV utilise un mécanisme d échappement aux cellules iNKT en diminuant l expression de CD1d à la surface des cellules B. Mon travail de thèse a donc permis d identifier une voie de régulation inconnue des lymphocytes T innés qui dépend de XIAP et de PLZF. PLZF est donc un facteur clé pour la différentiation et l homéostasie des cellules T innées en régulant l acquisition de leurs fonctions effectrices et en limitant leur survie. Ces observations ont aussi permis d identifier le premier DIP associé à un déficit en cellules MAIT. Enfin, ces résultats suggèrent un rôle des cellules iNKT dans le contrôle de l infection par l EBV.Invariant natural killer T (iNKT) lymphocytes represent a peculiar T cell-lineage that differs from conventional T cells by its development, function, and ligands it recognizes. In humans, iNKT cells express an invariant TCR made of the V?24-J?18/V?11 rearrangement, which recognizes glycosphingolipids presented by the MHC class I monomorphic molecule CD1d. Moreover, they rapidly produce high amounts of cytokines when stimulated and are thus considered as innate-like T cells. The molecular mechanisms that control the homeostasis of iNKT are poorly understood. XIAP (X-linked Inhibitor of Apoptosis) is a physiological inhibitor of caspases 3, 7 and 9 and is mutated in the X-linked lymphoproliferation syndrome 2 (XLP-2), a rare primary immunodeficiency (PID) characterized by a peculiar susceptibility to Epstein-Barr virus (EBV) infection. Patients with a XIAP deficiency exhibit a strong reduction of their iNKT cells in blood. Here, I report that XIAP is required for the survival of iNKT cells in humans. The requirement of XIAP correlates with a pro-apoptotic phenotype of iNKT cells that is not observed in conventional T cells. The increased susceptibility to apoptosis of iNKT cells was observed upon stimuli that trigger either extrinsic or intrinsic apoptosis pathways. iNKT cells by contrast to conventional T cells express elevated amounts of pro-apoptotic molecules including caspases 3 or 7 and Bid. The pro-apoptotic phenotype of iNKT cells is early acquired since iNKT cells from cord blood and thymus display a similar pro-apoptotic phenotype. Knock-down of XIAP in iNKT cells and analysis of XIAP-deficient humans indicate that XIAP is a potent inhibitor of apoptosis in iNKT cells while it has only a moderate effect in conventional T cells. I also show that this pro-apoptotic phenotype of iNKT cells is dependent of the expression of the transcription factor PLZF. This factor is already known to be necessary for the acquisition of the effector functions of these cells. Conversely, over expression of PLZF in conventional T cells leads to a pro-apoptotic phenotype and to an increased expression of caspase 3. Recently, a second invariant T cell subpopulation, the mucosal associated invariant T (MAIT) cells was identified both in humans and mice. These cells express a semi-invariant TCR made of V?7.2-J?33 rearrangements and share with iNKT cells a number of developmental, functional and phenotypical features that lead to consider MAIT cells as innate-like T cells like iNKT cells. Similarly, MAIT cells also exhibit a pro-apoptotic phenotype and are decreased in XIAP-deficient humans. The pro-apoptotic phenotype of MAIT cells is also dependent on PLZF. Interestingly, one XIAP-deficient patient with normal iNKT cell number was identified. This patient has not yet encountered EBV, suggesting that reduction of iNKT cells in XIAP-deficient patients is likely due to increased apoptosis in the context of EBV infection. I also show that EBV might have an escape mechanism from iNKT cells by down-regulating the expression of CD1d on the surface of B cells. My thesis works identify a previously unknown pathway controlling innate T cell homeostasis depending on XIAP and PLZF. PLZF is thus a key factor involved in the differentiation and the homeostasis of innate T cells by regulating the acquisition of their effector functions and their survival. I also identified the first PID associated with a defect in MAIT cells. Finally, these results provide evidences that iNKT cells might play a role against EBV infection.PARIS5-Bibliotheque electronique (751069902) / SudocSudocFranceF

Defective NKT cell development in mice and humans lacking the adapter SAP, the X-linked lymphoproliferative syndrome gene product

SAP is an adaptor protein expressed in T cells and natural killer cells. It plays a critical role in immunity, as it is mutated in humans with X-linked lymphoproliferative syndrome (XLP), a fatal immunodeficiency characterized by an abnormal response to Epstein-Barr virus (EBV) infection. SAP interacts with the SLAM family receptors and promotes transduction signal events by these receptors through its capacity to recruit and activate the Src kinase FynT. Because it has been previously established that FynT is selectively required for the development of NKT cells, we examined NKT cells in SAP-deficient mice and in humans with XLP. In the absence of SAP, the development of NKT cells is severely impaired both in mice and in humans. These results imply that SAP is a potent regulator of NKT cell development. They also identify for the first time a defect in NKT cells associated with a human primary immunodeficiency, revealing a potential role of NKT cells in the immune response to EBV

CD70 Deficiency Associated With Chronic Epstein-Barr Virus Infection, Recurrent Airway Infections and Severe Gingivitis in a 24-Year-Old Woman

Most of the few patients with homozygous CD70 deficiency described to date suffered from EBV-related malignancies in early childhood. We present a woman with CD70 deficiency diagnosed in adulthood. She presented in childhood with recurrent airway infections due to encapsulated bacteria, herpes zoster and a fulminant EBV infection followed by chronic EBV infection with mild lymphoproliferation and severe gingivitis/periodontal disease with high EBV viral load in saliva and gingival plaques as an adult. Up to the age of 24 years she developed no malignancy despite constant EBV viremia since primary EBV infection 15 years previously. Immunologic evaluation in childhood showed hypogammaglobulinemia with impaired polysaccharide responsiveness. She has been stable on immunoglobulin substitution with no further severe viral infections and no bacterial airway infections in adulthood. Targeted panel sequencing at the age of 20 years revealed a homozygous CD70 missense mutation (ENST00000245903.3:c.2T>C). CD70 deficiency was confirmed by absent CD70 expression of B cells and activated T cell blasts. The patient finished high school, persues an academic career and has rarely sick days at college. The clinical course of our patient may help to counsel parents of CD70-deficient patients with regard to prognosis and therapeutic options including haematopoetic stem cell transplantation

Occurrence of nodular lymphocyte-predominant hodgkin lymphoma in hermansky-pudlak type 2 syndrome is associated to natural killer and natural killer T cell defects

Hermansky Pudlak type 2 syndrome (HPS2) is a rare autosomal recessive primary immune deficiency caused by mutations on b3A gene (AP3B1 gene). The defect results in the impairment of the adaptor protein 3 (AP-3) complex, responsible for protein sorting to secretory lysosomes leading to oculo-cutaneous albinism, bleeding disorders and immunodeficiency. We have studied peripheral blood and lymph node biopsies from two siblings affected by HPS2. Lymph node histology showed a nodular lymphocyte predominance type Hodgkin lymphoma (NLPHL) in both HPS2 siblings. By immunohistochemistry, CD8 T-cells from HPS2 NLPHL contained an increased amount of perforin (Prf) + suggesting a defect in the release of this granules-associated protein. By analyzing peripheral blood immune cells we found a significant reduction of circulating NKT cells and of CD56brightCD162 Natural Killer (NK) cells subset. Functionally, NK cells were defective in their cytotoxic activity against tumor cell lines including Hodgkin Lymphoma as well as in IFN-c production. This defect was associated with increased baseline level of CD107a and CD63 at the surface level of unstimulated and IL-2-activated NK cells. In summary, these results suggest that a combined and profound defect of innate and adaptive effector cells might explain the susceptibility to infections and lymphoma in these HPS2 patients.peer-reviewe

Germline IKAROS dimerization haploinsufficiency causes hematologic cytopenias and malignancies

IKAROS is a transcription factor forming homo- and heterodimers and regulating lymphocyte development and function. Germline mutations affecting the IKAROS N-terminal DNA binding domain, acting in a haploinsufficient or dominant-negative manner, cause immunodeficiency. Herein, we describe 4 germline heterozygous IKAROS variants affecting its C-terminal dimerization domain, via haploinsufficiency, in 4 unrelated families. Index patients presented with hematologic disease consisting of cytopenias (thrombocytopenia, anemia, neutropenia)/Evans syndrome and malignancies (T-cell acute lymphoblastic leukemia, Burkitt lymphoma). These dimerization defective mutants disrupt homo- and heterodimerization in a complete or partial manner, but they do not affect the wild-type allele function. Moreover, they alter key mechanisms of IKAROS gene regulation, including sumoylation, protein stability, and the recruitment of the nucleosome remodeling and deacetylase complex; none affected in N-terminal DNA binding defects. These C-terminal dimerization mutations are largely associated with hematologic disorders, display dimerization haploinsufficiency and incomplete clinical penetrance, and differ from previously reported allelic variants in their mechanism of action. Dimerization mutants contribute to the growing spectrum of IKAROS-associated diseases displaying a genotype-phenotype correlation

Stepwise Development of MAIT Cells in Mouse and Human

Mucosal-associated invariant T (MAIT) cells display two evolutionarily conserved features: an invariant T cell receptor (TCR)α (iTCRα) chain and restriction by the nonpolymorphic class Ib major histocompatibility complex (MHC) molecule, MHC-related molecule 1 (MR1). MR1 expression on thymus epithelial cells is not necessary for MAIT cell development but their accumulation in the gut requires MR1 expressing B cells and commensal flora. MAIT cell development is poorly known, as these cells have not been found in the thymus so far. Herein, complementary human and mouse experiments using an anti-humanVα7.2 antibody and MAIT cell-specific iTCRα and TCRβ transgenic mice in different genetic backgrounds show that MAIT cell development is a stepwise process, with an intra-thymic selection followed by peripheral expansion. Mouse MAIT cells are selected in an MR1-dependent manner both in fetal thymic organ culture and in double iTCRα and TCRβ transgenic RAG knockout mice. In the latter mice, MAIT cells do not expand in the periphery unless B cells are added back by adoptive transfer, showing that B cells are not required for the initial thymic selection step but for the peripheral accumulation. In humans, contrary to natural killer T (NKT) cells, MAIT cells display a naïve phenotype in the thymus as well as in cord blood where they are in low numbers. After birth, MAIT cells acquire a memory phenotype and expand dramatically, up to 1%–4% of blood T cells. Finally, in contrast with NKT cells, human MAIT cell development is independent of the molecular adaptor SAP. Interestingly, mouse MAIT cells display a naïve phenotype and do not express the ZBTB16 transcription factor, which, in contrast, is expressed by NKT cells and the memory human MAIT cells found in the periphery after birth. In conclusion, MAIT cells are selected by MR1 in the thymus on a non-B non-T hematopoietic cell, and acquire a memory phenotype and expand in the periphery in a process dependent both upon B cells and the bacterial flora. Thus, their development follows a unique pattern at the crossroad of NKT and γδ T cells