L'IL-33 en immunothérapie anticancéreuse par les lymphocytes T Gamma Delta

Les lymphocytes T Vgamma9Vdelta2 humains constituent la sous-population majoritaire de lymphocytes Tgammadelta dans le sang chez l'adulte sain et représentent 1% à 4% des cellules mononucléées du sang périphérique (PBMC). Ce sont des lymphocytes T non conventionnels activés par des antigènes non peptidiques, les phosphoantigènes (PAgs) sans nécessité de restriction par les molécules du CMH. Ils jouent un rôle essentiel dans l'immunité anti-infectieuse et antitumorale, notamment en sécrétant des cytokines pro-inflammatoires et des molécules lytiques au contact de leurs cellules cibles. L'efficacité des lymphocytes T Vgamma9Vdelta2 en immunothérapie anticancéreuse est aujourd'hui démontrée mais reste fortement limitée en raison de la grande toxicité de l'IL-2 requise pour leur expansion. Toutes les cytokines de la famille de l'IL-2 possèdent la même toxicité intrinsèque, en raison de la chaîne gamma commune à tous les récepteurs de cette famille. Il est donc nécessaire de trouver une molécule alternative à l'IL 2, moins toxique mais tout aussi efficace, dont la transduction du signal ne dépend pas de la chaîne gamma. L'IL-33 est une cytokine de la famille de l'IL-1 appartenant au groupe des alarmines, dont le récepteur ST2/IL-1 RAcP est gamma chain-indépendant. Elle est naturellement présente dans le microenvironnement tumoral et son récepteur ST2 est exprimé sur de nombreuses cellules de l'immunité innée et adaptative. L'IL-33 est une cytokine pluripotente, pouvant induire à la fois des réponses immunitaires de type Th2 ou Th1. Néanmoins, aucune donnée n'était disponible quant à la bioactivité de l'IL-33 sur les lymphocytes T Vgamma9Vdelta2. Mes travaux de thèse ont donc consisté à déterminer si l'IL-33 pouvait potentialiser les fonctions anticancéreuses des lymphocytes T Vgamma9Vdelta2. Au cours de cette étude, nous avons montré que l'IL-33 associée à un PAg induit la prolifération et l'amplification des lymphocytes T Vgamma9Vdelta2 au sein des PBMC. Après amplification, les lymphocytes T Vgamma9Vdelta2 induits avec de l'IL-33 sécrètent des cytokines de type Th1 INF-gamma et TNF-alpha et ont une activité cytotoxique semblable à ceux induits avec de l'IL-2. De plus, nous avons mis en évidence que la prolifération des lymphocytes T Vgamma9Vdelta2 induite par l'IL-33 est issue d'un mécanisme complexe dépendant d'une interaction avec les lymphocytes T CD4. L'ensemble de ces résultats suggère que l'IL-33 pourrait représenter une bonne alternative à l'IL-2 dans les protocoles d'immunothérapie anticancéreuse impliquant des lymphocytes T Vgamma9Vdelta2.Human Vgamma9Vdelta2 T cells represent the most prominent subset of gammadelta T cells in the blood of healthy adults, representing 1 to 4% of peripheral blood mononuclear cells (PBMC). These cells are non-conventional lymphocytes activated by non peptidic antigens, the phosphoantigens (PAgs), without restriction by MHC molecules. They are very important actors of anti-infectious and antitumor immune responses. Indeed, their activation upon a contact with their target cells lead them to secrete pro-inflammatory cytokines and lytic granules mediating cell cytotoxicity. Their efficacy in cancer immunotherapy is now demonstrated but appears strongly limited by the toxicity of IL-2 which is essential for their expansion. All the cytokines of the IL-2 family have the same toxicity owing to the gamma chain shared by all the receptors of the family. Therefore, it appears crucial to find as effective but safer alternative to IL-2 which signaling does not depend on the gamma chain. IL-33 is a member of IL-1 family belonging to the alarmins which its receptor ST2/IL-1RAcP does not require the gamma chain. It is naturally present in the tumor microenvironment and ST2 is expressed on numerous innate and adaptive immune cells. IL 33 is a multipotent cytokine able to sustain both Th2 and Th1 immune responses. However, its bioactivity on Vgamma9Vdelta2 T cells has never been studied. The aim of my PhD thesis consisted in determining if IL-33 could promote Vgamma9Vdelta2 T cell anticancer functions. We found that IL-33 enhances the proliferation and the amplification of PAg-activated Vgamma9Vdelta2 T cells. Moreover, we found that IL-33-induced Vgamma9Vdelta2 T cells display the same anticancer functions than those induced by IL-2, through their secretion of Th1 type cytokines and to their cytotoxic activity towards cancer cells. Interestingly, we found that Vgamma9Vdelta2 T cell proliferation induced by IL-33 is due to a complex mechanism requiring interaction with CD4 T cells. Altogether, these results suggest that IL-33 represents a potential alternative to IL-2 in Vgamma9Vdelta2 T cell-based immunotherapies

Harnessing IL-33 for gamma delta T cell-based immunotherapies

Les lymphocytes T Vgamma9Vdelta2 humains constituent la sous-population majoritaire de lymphocytes Tgammadelta dans le sang chez l'adulte sain et représentent 1% à 4% des cellules mononucléées du sang périphérique (PBMC). Ce sont des lymphocytes T non conventionnels activés par des antigènes non peptidiques, les phosphoantigènes (PAgs) sans nécessité de restriction par les molécules du CMH. Ils jouent un rôle essentiel dans l'immunité anti-infectieuse et antitumorale, notamment en sécrétant des cytokines pro-inflammatoires et des molécules lytiques au contact de leurs cellules cibles. L'efficacité des lymphocytes T Vgamma9Vdelta2 en immunothérapie anticancéreuse est aujourd'hui démontrée mais reste fortement limitée en raison de la grande toxicité de l'IL-2 requise pour leur expansion. Toutes les cytokines de la famille de l'IL-2 possèdent la même toxicité intrinsèque, en raison de la chaîne gamma commune à tous les récepteurs de cette famille. Il est donc nécessaire de trouver une molécule alternative à l'IL 2, moins toxique mais tout aussi efficace, dont la transduction du signal ne dépend pas de la chaîne gamma. L'IL-33 est une cytokine de la famille de l'IL-1 appartenant au groupe des alarmines, dont le récepteur ST2/IL-1 RAcP est gamma chain-indépendant. Elle est naturellement présente dans le microenvironnement tumoral et son récepteur ST2 est exprimé sur de nombreuses cellules de l'immunité innée et adaptative. L'IL-33 est une cytokine pluripotente, pouvant induire à la fois des réponses immunitaires de type Th2 ou Th1. Néanmoins, aucune donnée n'était disponible quant à la bioactivité de l'IL-33 sur les lymphocytes T Vgamma9Vdelta2. Mes travaux de thèse ont donc consisté à déterminer si l'IL-33 pouvait potentialiser les fonctions anticancéreuses des lymphocytes T Vgamma9Vdelta2. Au cours de cette étude, nous avons montré que l'IL-33 associée à un PAg induit la prolifération et l'amplification des lymphocytes T Vgamma9Vdelta2 au sein des PBMC. Après amplification, les lymphocytes T Vgamma9Vdelta2 induits avec de l'IL-33 sécrètent des cytokines de type Th1 INF-gamma et TNF-alpha et ont une activité cytotoxique semblable à ceux induits avec de l'IL-2. De plus, nous avons mis en évidence que la prolifération des lymphocytes T Vgamma9Vdelta2 induite par l'IL-33 est issue d'un mécanisme complexe dépendant d'une interaction avec les lymphocytes T CD4. L'ensemble de ces résultats suggère que l'IL-33 pourrait représenter une bonne alternative à l'IL-2 dans les protocoles d'immunothérapie anticancéreuse impliquant des lymphocytes T Vgamma9Vdelta2.Human Vgamma9Vdelta2 T cells represent the most prominent subset of gammadelta T cells in the blood of healthy adults, representing 1 to 4% of peripheral blood mononuclear cells (PBMC). These cells are non-conventional lymphocytes activated by non peptidic antigens, the phosphoantigens (PAgs), without restriction by MHC molecules. They are very important actors of anti-infectious and antitumor immune responses. Indeed, their activation upon a contact with their target cells lead them to secrete pro-inflammatory cytokines and lytic granules mediating cell cytotoxicity. Their efficacy in cancer immunotherapy is now demonstrated but appears strongly limited by the toxicity of IL-2 which is essential for their expansion. All the cytokines of the IL-2 family have the same toxicity owing to the gamma chain shared by all the receptors of the family. Therefore, it appears crucial to find as effective but safer alternative to IL-2 which signaling does not depend on the gamma chain. IL-33 is a member of IL-1 family belonging to the alarmins which its receptor ST2/IL-1RAcP does not require the gamma chain. It is naturally present in the tumor microenvironment and ST2 is expressed on numerous innate and adaptive immune cells. IL 33 is a multipotent cytokine able to sustain both Th2 and Th1 immune responses. However, its bioactivity on Vgamma9Vdelta2 T cells has never been studied. The aim of my PhD thesis consisted in determining if IL-33 could promote Vgamma9Vdelta2 T cell anticancer functions. We found that IL-33 enhances the proliferation and the amplification of PAg-activated Vgamma9Vdelta2 T cells. Moreover, we found that IL-33-induced Vgamma9Vdelta2 T cells display the same anticancer functions than those induced by IL-2, through their secretion of Th1 type cytokines and to their cytotoxic activity towards cancer cells. Interestingly, we found that Vgamma9Vdelta2 T cell proliferation induced by IL-33 is due to a complex mechanism requiring interaction with CD4 T cells. Altogether, these results suggest that IL-33 represents a potential alternative to IL-2 in Vgamma9Vdelta2 T cell-based immunotherapies

Phosphoantigens and butyrophilin 3A1 induce similar intracellular activation signaling in human TCRVγ9+ γδ T lymphocytes

International audienceHuman γδ cells expressing TCRVγ9 are T lymphocytes with great potential for cancer immunotherapy and unconventional pattern of antigen specificity. These HLA-unrestricted lymphocytes are specifically reactive to non-peptide metabolites (phosphoantigens) and to the butyrophilin 3A (BTN3A/CD277) protein. Whether recognition of such highly different structures trigger the same activation signaling pathway remains unclear, however. Here we combined fluorescent cell barcoding and phosphoflow analysis of TCRVγ9(+) T lymphocytes to compare simultaneously the level of several signaling phosphoproteins after activation by phosphoantigen (BrHPP) or by anti-BTN3A (monoclonal antibody 20.1). This approach shows that the same pathways involving ZAP70, PLCγ2, Akt, NFκB p65, MAPK p38 and Erk1, were induced by either of these stimuli. These data strongly suggest the TCRVγ9(+) T lymphocytes detect phosphoantigens and butyrophilin A3 by the same recognition process

IL-33-expanded human Vγ9Vδ2 T cells have anti-lymphoma effect in a mouse tumor model

International audienceFrom several years, the anticancer effects of Vγ9 T lymphocytes make these cells good candidates for cancer immunotherapies. However, the proved efficacy of γδ Τ cell-based cancer immunotherapies in some clinical trials was minimized due to the inherent toxicity of IL-2, which is essential for the combination therapy with Phosphoantigen (PAg). Recently, we showed that IL-33, a γ chain receptor-independent cytokine, was able to induce the in vitro proliferation of PAg-activated Vγ9 T cells, which were fully functional expressing IFN-γ and TNF-α and showing in vitro anti-tumor cytotoxicity. We proposed IL-33 as an alternative to IL-2 for Vγ9 T cell-based cancer immunotherapies, and have therefore evaluated the efficacy of this cytokine in preclinical investigations. This study shows that human Vγ9 T cells are able to proliferate in a mouse model with the combination of PAg and rhIL-33, and that IL-33-expanded Vγ9 T cells can prevent tumor growth in a mouse lymphoma model

TCRVγ9 γδ T Cell Response to IL-33: A CD4 T Cell–Dependent Mechanism

International audienceThe availability of specific stimuli to induce the anticancer cytotoxicity of human TCRVγ9-expressing T lymphocytes has allowed the development of γδ T cell-based cancer immunotherapies. However, the stringent dependence of such strategies on the inherently toxic IL-2 has raised safety concerns for patients, justifying a search for alternative methods for inducing γδ T cell stimulation. IL-33 is a γ-chain receptor-independent cytokine of the IL-1 superfamily that is expressed by endothelial cells from a tumor microenvironment and can sustain Th1 and Th2 immune responses. Therefore, we investigated its ability to support the stimulation of human TCRVγ9(+) γδ T cells. In this study, we report that IL-33 efficiently sustained the in vitro activation of Vγ9 T lymphocytes by synthetic phosphoantigens, zoledronate, and a BTN3A1 Ab in the absence of an exogenous supply of IL-2. IL-33 was as potent as IL-2 in allowing the proliferative amplification of Vγ9 T cells isolated from PBMC following activation by the synthetic phosphoantigen bromohydrin pyrophosphate. IL-33 also induced an identical maturation into TNF-α- and IFN-γ-producing Th1 effector memory cells, and IL-33-stimulated cells showed an equivalent cytotoxicity for various tumor cells in vitro. Finally, we found that the bioactivity of IL-33 on the Vγ9 T cell was indirectly mediated through contact with CD4 T cells and IL-2 production by CD4 T cells and Vγ9 T cells themselves. These data posit IL-33 as an alternative to IL-2 for Vγ9 T cell-based cancer immunotherapies

IL-7 expands lymphocyte populations and enhances immune responses to sipuleucel-T in patients with metastatic castration-resistant prostate cancer (mCRPC).

Sipuleucel-T (sip-T) is a Food and Drug Administration (FDA)-approved autologous cellular immunotherapy for metastatic castration-resistant prostate cancer (mCRPC). We hypothesized that combining sip-T with interleukin (IL)-7, a homeostatic cytokine that enhances both B and T cell development and proliferation, would augment and prolong antigen-specific immune responses against both PA2024 (the immunogen for sip-T) and prostatic acid phosphatase (PAP). Fifty-four patients with mCRPC treated with sip-T were subsequently enrolled and randomized 1:1 into observation (n=26) or IL-7 (n=28) arms of a phase II clinical trial (NCT01881867). Recombinant human (rh) IL-7 (CYT107) was given weekly×4. Immune responses were evaluated using flow cytometry, mass cytometry (CyTOF), interferon (IFN)-γ ELISpot, 3H-thymidine incorporation, and ELISA. Treatment with rhIL-7 was well tolerated. For the rhIL-7-treated, but not observation group, statistically significant lymphocyte subset expansion was found, with 2.3-2.6-fold increases in CD4+T, CD8+T, and CD56bright NK cells at week 6 compared with baseline. No significant differences in PA2024 or PAP-specific T cell responses measured by IFN-γ ELISpot assay were found between rhIL-7 and observation groups. However, antigen-specific T cell proliferative responses and humoral IgG and IgG/IgM responses significantly increased over time in the rhIL-7-treated group only. CyTOF analyses revealed pleiotropic effects of rhIL-7 on lymphocyte subsets, including increases in CD137 and intracellular IL-2 and IFN-γ expression. While not powered to detect clinical outcomes, we found that 31% of patients in the rhIL-7 group had prostate specific antigen (PSA) doubling times of >6 months, compared with 14% in the observation group. Treatment with rhIL-7 led to a significant expansion of CD4+ and CD8+ T cells, and CD56bright natural killer (NK) cells compared with observation after treatment with sip-T. The rhIL-7 treatment also led to improved antigen-specific humoral and T cell proliferative responses over time as well as to increased expression of activation markers and beneficial cytokines. This is the first study to evaluate the use of rhIL-7 after sip-T in patients with mCRPC and demonstrates encouraging results for combination approaches to augment beneficial immune responses

Comprehensive landscape of neutralizing antibody and cell-mediated response elicited by the 1/5 fractional dose of 17DD-YF primary vaccination in adults

Abstract The present study aimed at evaluating the YF-specific neutralizing antibody profile besides a multiparametric analysis of phenotypic/functional features of cell-mediated response elicited by the 1/5 fractional dose of 17DD-YF vaccine, administered as a single subcutaneous injection. The immunological parameters of each volunteer was monitored at two time points, referred as: before (Day 0) [Non-Vaccinated, NV(D0)] and after vaccination (Day 30–45) [Primary Vaccinees, PV(D30–45)]. Data demonstrated high levels of neutralizing antibodies for PV(D30–45) leading to a seropositivity rate of 93%. A broad increase of systemic soluble mediators with a mixed profile was also observed for PV(D30–45), with IFN-γ and TNF-α presenting the highest baseline fold changes. Integrative network mapping of soluble mediators showed increased correlation numbers in PV(D30–45) as compared to NV(D0) (532vs398). Moreover, PV(D30–45) exhibited increased levels of Terminal Effector (CD45RA+CCR7−) CD4+ and CD8+ T-cells and Non-Classical memory B-cells (IgD+CD27+). Dimensionality reduction of Mass Cytometry data further support these findings. A polyfunctional cytokine profile (TNF-α/IFN-γ/IL-10/IL-17/IL-2) of T and B-cells was observed upon in vitro antigen recall. Mapping and kinetics timeline of soluble mediator signatures for PV(D30–45) further confirmed the polyfunctional profile upon long-term in vitro culture, mediated by increased levels of IFN-γ and TNF-α along with decreased production of IL-10. These findings suggest novel insights of correlates of protection elicited by the 1/5 fractional dose of 17DD-YF vaccine