Study of the unconventional cytomegalovirus-specific HLA-E-restricted T cells in kidney transplanted patients

L'infection à Cytomégalovirus Humain (HCMV) demeure une cause majeure de morbidité chez les individus immunodéficients. L'immunité antivirale étant primordiale dans la gestion du virus chez l'hôte, la compréhension du rôle de chacun des effecteurs immuns dans la lutte contre le HCMV est cruciale pour améliorer à l'avenir la prise en charge de ces patients. Dans cette étude, nous avons caractérisé ex vivo par une approche qualitative et quantitative les lymphocytes T CDS non conventionnels restreints par HLA-E et spécifiques du peptide signal d'UL40 (LT HLAEuL4o) chez des patients transplantés rénaux (n=119) et des individus sains (n=25). Nous montrons que leur développement est lié à l'infection à HCMV du receveur et est spécifique de la souche virale infectante. Présents chez un individu HCMV• sur trois, les LT HLA-EuL4o s'amplifient de manière quasi-clonale pouvant représenter jusqu'à 40% des LT CDS circulants. De plus, il apparaît que l'allèle HLA-A *02 et le génotype HLA-E*01 :01101 :03 sont des facteurs associés à la génération de ces populations. De phénotype effecteur-mémoire, les LT HLAEuL4o sont doués de capacités cytotoxique et cytokinique (TNF-a, IFN-y, IL-2). Ils reconnaissent, outre le peptide nominal, des nonamères relativement proches, dont certains correspondent à des peptides signaux issus des protéines HLA-I présentés physiologiquement par HLA-E, posant ainsi la question d'une réactivité autologue et/ou allogénique potentielle. En conclusion, nos résultats soulignent l'importance d'étudier le rôle des LT HLA-EuL4o dans la protection contre le HCMV et leur possible impact dans un contexte de transplantation.Human Cytomegalovirus (HCMV) infection remains a major cause of morbidity in immunodeficient individuals. Antiviral immunity being essential in the management of the virus in the host, understanding the role of each of the immune effectors in the anti-HCMV immune response is crucial to improve the upcoming management of these patients. ln this study, we characterized ex vivo, by a qualitative and quantitative approach, the unconventional UL40-specific HLA-E-restricted CDS T lymphocytes (LT HLA-EuL4o) · in kidney transplant patients (n = 119) and healthy volonteers (n = 25). We show that their development is related to HCMV infection of the recipient and is specific for the infectious viral strain. LT HLA-EuL40 cells are quasiclonal populations present in approximately 30% of the HCMV• individuals and can account for up to 40% of circulating CDS T cells. ln addition, it appears that the HLA-A*02 allele and the HLA-E*01 :01/01 :03 genotype are factors associated with the generation of these populations. LT HLA-EuL4o are effector-memory CDS T cells capable of cytotoxicity and cytokine production (TNF-a, IFN-y, IL-2). ln addition to the nominal peptide, these cells recognize a set of relatively close nonamers, including signal peptides derived from HLA-I proteins presented physiologically by HLA-E, thus raising the question of potential autologous and/or allogeneic reactivity. ln conclusion, our results highlight the importance of studying the role of l T HLA-EuL4o in the protection against HCMV and their possible impact in a transplant context

INTERNALISATION DES EXOSOMES DE MELANOME PAR LES LYMPHOCYTES T CD8+ ET IMPACT SUR LEURS FONCTIONS : APPORT DU CYTOMETRE IMAGEUR

International audienc

Human gut microbiota-reactive DP8α regulatory T cells, signature and related emerging functions

International audienceIn mice, microbiota-induced Tregs both maintain intestinal homeostasis and provide resistance to immuno-pathologies in the adult. Identifying their human functional counterpart therefore represents an important goal. We discovered, in the human colonic lamina propria and blood, a FoxP3-negative IL-10-secreting Treg subset, which co-expresses CD4 and CD8α (hence named DP8α) and displays a TCR-reactivity against Faecalibacterium prausnitzii , indicating a role for this symbiotic bacterium in their induction. Moreover, supporting their role in intestinal homeostasis, we previously reported both their drastic decrease in IBD patients and their protective role in vivo against intestinal inflammation, in mice. Here, we aimed at identifying the genomic, phenotypic and functional signatures of these microbiota-induced Tregs, towards delineating their physiological role(s) and clinical potential. Human F. prausnitzii -reactive DP8α Treg clones were derived from both the colonic lamina propria and blood. RNA-sequencing, flow cytometry and functional assays were performed to characterize their response upon activation and compare them to donor- and tissue-matched FoxP3 + Treg clones. DP8α Tregs exhibited a unique mixed Tr1-like/cytotoxic CD4 + T cell-profile and shared the RORγt and MAF master genes with mouse gut microbiota-induced FoxP3 + Tregs. We revealed their potent cytotoxic, chemotactic and IgA-promoting abilities, which were confirmed using in vitro assays. Therefore, besides their induction by a Clostridium bacterium, DP8α Tregs also partake master genes with mouse microbiota-induced Tregs. The present identification of their complete signature and novel functional properties, should be key in delineating the in vivo roles and therapeutic applications of these unique human microbiota-induced Tregs through their study in pathological contexts, particularly in inflammatory bowel diseases

MicroRNAs in Tumor Exosomes Drive Immune Escape in Melanoma

International audienceMicroRNAs (miRNAs), small noncoding RNAs that regulate gene expression, exist not only in cells but also in a variety of body fluids. These circulating miRNAs could enable intercellular communication. miRNAs are packaged in membrane-encapsulated vesicles, such as exosomes, or protected by RNA-binding proteins. Here we report that miRNAs included in human melanoma exosomes regulate the tumor immune response. Using microscopy and flow cytometry, we demonstrate that CD8+ T cells internalize exosomes from different tumor types even if these cells do not internalize vesicles as readily as other immune cells. We explored the function of melanomaderived exosomes in CD8+ T cells and showed that these exosomes downregulate T-cell responses through decreased TCR signaling and diminished cytokine and granzyme B secretions. The result reduces the cells’ cytotoxic activity. Using mimics, we found that miRNAs enriched in exosomes— such as Homo sapiens (hsa)-miR-3187-3p, hsa-miR-498, hsa-miR-122, hsa-miR149, and hsa-miR- 181a/b —regulate TCR signaling and TNFα secretion. Our observations suggest that miRNAs in melanoma-derived exosomes aid tumor immune evasion and could be a therapeutic target

Defining the Immune Checkpoint Landscape in Human Colorectal Cancer Highlights the Relevance of the TIGIT/CD155 Axis for Optimizing Immunotherapy

International audienceWhile immune checkpoint (IC) therapies, particularly those targeting the PD-1/PD-L1 axis, have revolutionized the treatment of melanoma and several other cancers, their effect remains very limited in colorectal cancer (CRC). To define a comprehensive landscape of ICs in the human CRC tumor microenvironment (TME), we evaluated, using multiparametric flow cytometry, their ex vivo expression via tumor-infiltrating lymphocytes (TILs) (n = 40 CRCs) as well as that of their respective ligands on tumor and myeloid cells (n = 29). Supervised flow cytometry analyses showed that (i) most CD3+ TILs expressed PD-1 and TIGIT and, to a lesser extent, Tim-3, Lag3 and NKG2A, and (ii) EpCAM+ tumor cells and CD11b+ myeloid cells differed in their IC ligand expression profile, with a strikingly high expression of CD155 by tumor cells. An in situ analysis of IC and their ligands using immunohistochemistry on paraffin sections of CRC confirmed the overexpression of TIGIT and its ligand, CD155, in the TME. Most interestingly, an unsupervised clustering analysis of IC co-expression on CD4+ and CD8+ TILs identified two tumor subgroups, named IChigh and IClow. Altogether, our findings highlight the TIGIT/CD155 axis as a potential target that could be used in combination IC therapy in CRC

Phase I/II clinical trial of adoptive cell transfer of sorted specific T cells for metastatic melanoma patients

International audienceAdoptive cell transfer (ACT) of tumor-specific T lymphocytes represents a relevant therapeutic strategy to treat metastatic melanoma patients. Ideal T-cells should combine tumor specificity and reactivity with survival in vivo, while avoiding autoimmune side effects. Here we report results from a Phase I/II clinical trial (NCT02424916, performed between 2015 and 2018) in which 6 metastatic HLA-A2 melanoma patients received autologous antigen-specific T-cells produced from PBMC, after peptide stimulation in vitro, followed by sorting with HLA-peptide multimers and amplification. Each patient received a combination of Melan-A and MELOE-1 polyclonal specific T-cells, whose specificity and anti-tumor reactivity were checked prior to injection, with subcutaneous IL-2. Transferred T-cells were also characterized in terms of functional avidity, diversity and phenotype and their blood persistence was evaluated. An increase in specific T-cells was detected in the blood of all patients at day 1 and progressively disappeared from day 7 onwards. No serious adverse events occurred after this ACT. Clinically, five patients progressed and one patient experienced a partial response following therapy. Melan-A and MELOE-1 specific T-cells infused to this patient were diverse, of high avidity, with a high proportion of T lymphocytes coexpressing PD-1 and TIGIT but few other exhaustion markers. In conclusion, we demonstrated the feasibility and safety of ACT with multimer-sorted Melan-A and MELOE-1 specific T cells to metastatic melanoma patients. The clinical efficacy of such therapeutic strategy could be further enhanced by the selection of highly reactive T-cells, based on PD-1 and TIGIT co-expression, and a combination with ICI, such as anti-PD-1

DataSheet_1_Human gut microbiota-reactive DP8α regulatory T cells, signature and related emerging functions.pdf

In mice, microbiota-induced Tregs both maintain intestinal homeostasis and provide resistance to immuno-pathologies in the adult. Identifying their human functional counterpart therefore represents an important goal. We discovered, in the human colonic lamina propria and blood, a FoxP3-negative IL-10-secreting Treg subset, which co-expresses CD4 and CD8α (hence named DP8α) and displays a TCR-reactivity against Faecalibacterium prausnitzii, indicating a role for this symbiotic bacterium in their induction. Moreover, supporting their role in intestinal homeostasis, we previously reported both their drastic decrease in IBD patients and their protective role in vivo against intestinal inflammation, in mice. Here, we aimed at identifying the genomic, phenotypic and functional signatures of these microbiota-induced Tregs, towards delineating their physiological role(s) and clinical potential. Human F. prausnitzii-reactive DP8α Treg clones were derived from both the colonic lamina propria and blood. RNA-sequencing, flow cytometry and functional assays were performed to characterize their response upon activation and compare them to donor- and tissue-matched FoxP3+ Treg clones. DP8α Tregs exhibited a unique mixed Tr1-like/cytotoxic CD4+ T cell-profile and shared the RORγt and MAF master genes with mouse gut microbiota-induced FoxP3+ Tregs. We revealed their potent cytotoxic, chemotactic and IgA-promoting abilities, which were confirmed using in vitro assays. Therefore, besides their induction by a Clostridium bacterium, DP8α Tregs also partake master genes with mouse microbiota-induced Tregs. The present identification of their complete signature and novel functional properties, should be key in delineating the in vivo roles and therapeutic applications of these unique human microbiota-induced Tregs through their study in pathological contexts, particularly in inflammatory bowel diseases.</p

Low levels of natural anti-a-N-acetylgalactosamine (Tn) antibodies are associated with COVID-19

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A novel and efficient approach to high-throughput production of HLA-E/peptide monomer for T-cell epitope screening

International audienceOver the past two decades, there has been a great interest in the study of HLA-E-restricted αβ T cells during bacterial and viral infections, including recently SARS-CoV-2 infection. Phenotyping of these specific HLA-E-restricted T cells requires new tools such as tetramers for rapid cell staining or sorting, as well as for the identification of new peptides capable to bind to the HLA-E pocket. To this aim, we have developed an optimal photosensitive peptide to generate stable HLA-E/pUV complexes allowing high-throughput production of new HLA-E/peptide complexes by peptide exchange. We characterized the UV exchange by ELISA and improved the peptide exchange readout using size exclusion chromatography. This novel approach for complex quantification is indeed very important to perform tetramerization of MHC/peptide complexes with the high quality required for detection of specific T cells. Our approach allows the rapid screening of peptides capable of binding to the non-classical human HLA-E allele, paving the way for the development of new therapeutic approaches based on the detection of HLA-E-restricted T cells

Low Levels of Natural Anti-α-N-Acetylgalactosamine (Tn) Antibodies Are Associated With COVID-19

Human serum contains large amounts of anti-carbohydrate antibodies, some of which may recognize epitopes on viral glycans. Here, we tested the hypothesis that such antibodies may confer protection against COVID-19 so that patients would be preferentially found among people with low amounts of specific anti-carbohydrate antibodies since individual repertoires vary considerably. After selecting glycan epitopes commonly represented in the human anti-carbohydrate antibody repertoire that may also be expressed on viral glycans, plasma levels of the corresponding antibodies were determined by ELISA in 88 SARS-CoV-2 infected individuals, including 13 asymptomatic, and in 82 non-infected controls. We observed that anti-Tn antibodies levels were significantly lower in patients as compared to non-infected individuals. This was not observed for any of the other tested carbohydrate epitopes, including anti-αGal antibodies used as a negative control since the epitope cannot be synthesized by humans. Owing to structural homologies with blood groups A and B antigens, we also observed that anti-Tn and anti-αGal antibodies levels were lower in blood group A and B, respectively. Analyses of correlations between anti-Tn and the other anti-carbohydrates tested revealed divergent patterns of correlations between patients and controls, suggesting qualitative differences in addition to the quantitative difference. Furthermore, anti-Tn levels correlated with anti-S protein levels in the patients’ group, suggesting that anti-Tn might contribute to the development of the specific antiviral response. Overall, this first analysis allows to hypothesize that natural anti-Tn antibodies might be protective against COVID-19.SCOPUS: ar.jinfo:eu-repo/semantics/publishe