Search

Purpose Vogt-Koyanagi-Harada (VKH) syndrome is an autoimmune disease characterized by inaugural uveomeningitidis and hearing loss and at late stages a depigmentation in eyes and skin. Melanocytes are the cells common to the four affected tissues, namely eye, brain, inner ear, and skin. Melanocytes are therefore considered as the source of self-antigens. The melanocytic proteins tyrosinase-related protein-1 (TRP1), TRP2, tyrosinase, and gp100 have been proposed as the proteins targeted by autoreactive T cells from VKH patients bearing human leukocyte antigen (HLA)-DRB1*04:05, the HLA allele classically associated with VKH disease. The objective of this work was to determine the antigens recognized by a large number of potentially autoreactive CD4 T lymphocytes obtained from the cerebrospinal fluid of one VKH patient who did not express HLA-DRB1*04:05. Methods T cells were isolated from the cerebrospinal fluid of a newly diagnosed HLA-DRB1*14:01,*15:03;-DPB1*01:01,*04:02 patient in the acute phase of the VKH disease and cloned by limiting dilution. Each of the 107 T cell clones, of which 90% were CD4+, was tested for its ability to secrete cytokines upon contact with autologous antigen-presenting cells loaded with either of the melanocytic proteins TRP1, TRP2, tyrosinase, gp100, Melan-A and KU-MEL-1. The sensitivity of our recombinant bacteria-based approach was validated with a CD4 T cell clone with known antigen specificity. The ability of each of the 107 clones to secrete cytokines upon nonspecific stimulation was verified. Results None of the 107 T cell clones was able to secrete tumor necrosis factor-α, interferon-γ, interleukin (IL)-5, or IL-17 upon contact with autologous B cells loaded with any of the six common melanocytic proteins. Nine clones secreted high-level IL-17 upon stimulation with beads coated with antibodies. Conclusions The self-antigens that triggered the VKH disease in this patient probably derive from proteins other than the six melanocytic proteins mentioned above. Further study of antigens that are recognized by potential autoreactive T cells from VKH patients is likely to benefit from testing a broader set of melanocytic proteins.

Antigens encoded by cancer-germline genes are attractive targets for cancer immunotherapy. In this study, we aimed to evaluate the mRNA expression of cancer-germline genes, expression of the encoded proteins in patients with esophageal squamous cell carcinoma (ESCC) and their correlations with clinical characteristics. In addition, the effects of downregulation cancer-germline genes on ESCC cells were assessed in vitro. Our results showed that cancer-germline genes were frequently expressed in ESCC samples. The positive rates of in ESCC samples were: 87% of MAGEA3, 60% of MAGE-A4, 65% of MAGE-C2, and 20% of NY-ESO-1 at mRNA level. MAGE-A3 expression was associated with age, lymph node metastasis and tumor stage (all P

Immunity against Theileria parva is associated with CD8 T-cell responses that exhibit immunodominance, the focussing of the response against limited numbers of epitopes. As candidates for inclusion in vaccines, characterisation of responses against immunodominant epitopes is a key component in novel vaccine development. We have previously demonstrated that the Tp249-59 and Tp1214-224 epitopes dominate CD8 T-cell responses in BoLA-A10 and BoLA-18 MHCI homozygous animals respectively. In this study, peptide-MHCI tetramers for these epitopes, and a subdominant BoLA-A10-restricted epitope (Tp298-106 ), were generated to facilitate accurate and rapid enumeration of epitope-specific CD8 T-cells. During validation of these tetramers a substantial proportion of Tp249-59 -reactive T-cells failed to bind the tetramer, suggesting this population was heterogeneous with respect to the recognised epitope. We demonstrate that Tp250-59 represents a distinct epitope and that tetramers produced with Tp50-59 and Tp49-59 show no cross reactivity. The Tp249-59 and Tp250-59 epitopes utilise different serine residues as the N-terminal anchor for binding to the presenting MHCI molecule. Molecular dynamic modelling predicts that the 2 peptide-MHCI complexes adopt structurally different conformations and TRB sequence analysis show that Tp249-59 and Tp250-59 are recognised by non-overlapping TCR repertoires. Together these data demonstrate that although differing by only a single residue, Tp249-59 and Tp250-59 epitopes form distinct ligands for TCR recognition. Tetramer analysis of T. parva-specific CD8 T-cell lines confirmed the immunodominance of Tp1214-224 in BoLA-A18 animals and showed in BoLA-A10 animals that the Tp249-59 epitope response was generally more dominant than the Tp250-59 response and confirmed that the Tp298-106 response was subdominant. This article is protected by copyright. All rights reserved.

Long-term tumor regressions have been observed in patients following the adoptive transfer of autologous tumor-infiltrating lymphocytes or genetically modified T cells expressing MHC class I-restricted T-cell receptors (TCRs), but clinical trials have not evaluated responses to genetically modified T cells expressing antitumor MHC class II-restricted TCRs. As studies carried out in a murine tumor model system have demonstrated that the adoptive transfer of CD4 T cells could lead to the regression of established tumors, we plan to test the hypothesis that CD4 T cells can also induce tumor regressions in cancer patients. In this study, 2 MAGE-A3-specific TCRs were isolated from a regulatory T-cell clone (6F9) and an effector clone (R12C9), generated from the peripheral blood of 2 melanoma patients after MAGE-A3 vaccination. The results indicated that T cells transduced with 6F9 TCR mediated stronger effector functions than R12C9 TCR. The 6F9 TCR specifically recognized MAGE-A3 and the closely related MAGE-A6 gene product, but not other members of the MAGE-A family in the context of HLA-DPB1*04:01. To test the feasibility of a potential clinical trial using this TCR, a clinical-scale procedure was developed to obtain a large number of purified CD4 T cells transduced with 6F9 TCR. Because HLA-DPB1*04:01 is present in ∼60% of the Caucasian population and MAGE-A3 is frequently expressed in a variety of cancer types, this TCR immunotherapy could potentially be applicable for a significant portion of cancer patients.

The plethora of tumor antigens that have been--and are still being--defined required systematization to provide a comprehensive overview of those tumor antigens that are the most relevant targets for cancer immunotherapy approaches. Here, we provide a new update of a peptide database resource that we initiated many years ago. This database compiles all human antigenic peptides described in the literature that fulfill a set of strict criteria needed to ascertain their actual "tumor antigen" nature, as we aim at guiding scientists and clinicians searching for appropriate cancer vaccine candidates (www.cancerimmunity.org/peptide). In this review, we revisit those criteria in light of recent findings related to antigen processing. We also introduce the 29 new tumor antigens that were selected for this 2013 update. Two of the new peptides show unusual features, which will be briefly discussed. The database now comprises a total of 403 tumor antigenic peptides.

Antitumor cytolytic T lymphocytes (CTLs) recognize peptides derived from cellular proteins and presented on MHC class I. One category of peptides recognized by these CTLs is derived from proteins encoded by "cancer-germline" genes, which are specifically expressed in tumors, and therefore represent optimal targets for cancer immunotherapy. Here, we identify an antigenic peptide, which is derived from the MAGE-A1-encoded protein (160-169) and presented to CTLs by HLA-B*44:02. Although this peptide is encoded by MAGE-A1, processed endogenously and presented by tumor cells, the corresponding synthetic peptide is hardly able to sensitize target cells to CTL recognition when pulsed exogenously. Endogenous processing and presentation of this peptide is strictly dependent on the presence of tapasin, which is believed to help peptide loading by stabilizing a peptide-receptive form of HLA-B*44:02. Exogenous loading of the peptide can be dramatically improved by paraformaldehyde fixation of surface molecules or by peptide loading at acidic pH. Either strategy allows efficient exogenous loading of the peptide, presumably by generating or stabilizing a peptide-receptive, empty conformation of the HLA. Altogether, our results indicate a potential drawback of short peptide-based vaccination strategies and offer possible solutions regarding the use of problematic epitopes such as the one described here.

Antitumor cytolytic T lymphocytes (CTLs) recognize peptides derived from cellular proteins and presented on MHC class I. One category of peptides recognized by these CTLs is derived from proteins encoded by "cancer-germline" genes, which are specifically expressed in tumors, and therefore represent optimal targets for cancer immunotherapy. Here, we identify an antigenic peptide, which is derived from the MAGE-A1-encoded protein (160-169) and presented to CTLs by HLA-B*44:02. Although this peptide is encoded by MAGE-A1, processed endogenously and presented by tumor cells, the corresponding synthetic peptide is hardly able to sensitize target cells to CTL recognition when pulsed exogenously. Endogenous processing and presentation of this peptide is strictly dependent on the presence of tapasin, which is believed to help peptide loading by stabilizing a peptide-receptive form of HLA-B*44:02. Exogenous loading of the peptide can be dramatically improved by paraformaldehyde fixation of surface molecules or by peptide loading at acidic pH. Either strategy allows efficient exogenous loading of the peptide, presumably by generating or stabilizing a peptide-receptive, empty conformation of the HLA. Altogether, our results indicate a potential drawback of short peptide-based vaccination strategies and offer possible solutions regarding the use of problematic epitopes such as the one described here.

Cette brève revue décrit les principes généraux de l’antigénicité des tumeurs humaines vis-à-vis des lymphocytes T, et en particulier des lymphocytes T cytolytiques. L’existence de ces antigènes spécifiques de tumeurs est à la base de stratégies vaccinales thérapeutiques, c’est-à-dire qui visent à faire régresser des tumeurs existantes ou à prévenir l’apparition de métastases. Nous passons brièvement en revue une série de petits essais cliniques effectués dans le mélanome, et qui ont montré des résultats cliniques encourageants mais limités. L’analyse immunologique de certains de ces patients vaccinés a montré l’importance des réponses lymphocytaires T antitumorales, mais aussi le fait que l’absence d’efficacité clinique est très probablement le résultat d’une résistance tumorale à ces réponses immunitaires. Les recherches actuelles s’orientent donc vers l’étude des principaux mécanismes de cette résistance et des moyens de la contrer

"Cancergermline" genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor specific Ags, which have been used in therapeutic vaccination trials of cancer patients. MAGE3 is expressed in 74% of metastatic melanoma and in 50% of carcinomas of esophagus, head and neck, bladder, and lung. We report here the identification of a new MAGE3 peptide, which is recognized by three different CD4+ T cell clones isolated from a melanoma patient vaccinated with a MAGE3 protein. These clones, which express different TCRs, recognize on HLADR1 peptide ACYEFLWGPRALVETS, which corresponds to the MAGE 3267 282 and the MAGE12267 282 protein sequences. One of the T cell clones, which expresses LFA-1 at a high level, lysed tumor cells expressing DR1 and MAGE3. Another of these DR1-restricted CD4+ clones recognized not only the MAGE-3/12 peptide but also homologous peptides encoded by genes MAGE-1, 2, 4, 6, 10, and 11.

Antigens encoded by MAGE genes are of particular interest for cancer immunotherapy because of their tumoral specificity and because they are shared by many tumors. Antigenic peptide MEVDPIGHLY, which is encoded by MAGE-3 and is known to be presented by human leukocyte antigen (HLA)-B44, is currently being used in therapeutic vaccination trials. We report here that a cytolytic T lymphocyte (CTL) clone, which is restricted by HLA-B*1801, recognizes the same peptide and, importantly, lyzes HLA-B18 tumor cells expressing MAGE-3. These results imply that the use of peptide MEVDPIGHLY can now be extended to HLA-B18 patients. We also provide evidence that, under limiting amounts of protein MAGE-3, HLA B*1801 and B*4403 compete for binding to the peptide.

Amino acids blood levels are remarkably constant, in spite of fasting periods and large variations in diet supply. Systemic homeostasis is particularly important for tryptophan, given its key role in many cellular processes, including immunity and neuronal activity. In humans, tryptophanemia is maintained around 60µM. Here, we showed that tryptophan homeostasis is largely controlled by the post-translational stability of tryptophan 2,3-dioxygenase (TDO), the hepatic enzyme catalyzing the first step of tryptophan catabolism along the kynurenine pathway. TDO, located at the strategic crossroad of the portal and systemic circulation, degrades more than 90% of food-derived tryptophan. High tryptophan levels stabilize the active tetrameric conformation of TDO through binding non-catalytic exosites, promoting rapid blood tryptophan catabolism. In low tryptophan, the lack of tryptophan binding in the exosites destabilizes the tetramer into inactive monomers and dimers, and unmasks a degron. This triggers the polyubiquitination of TDO by SKP1-CUL1-F-box complexes, resulting in proteasome-mediated degradation of TDO and rapid interruption of tryptophan catabolism. Artificial stabilization of tetrameric TDO with the non-metabolizable analog alpha-methyl-tryptophan in vivo stably reduces blood tryptophan levels. Our results uncover a mechanism allowing a rapid adaptation of tryptophan catabolism to ensure quick degradation of excess tryptophan while preventing further catabolism below physiological levels. This ensures a tight control of tryptophanemia, as required for both neurological and immune homeostasis. La concentration plasmatique des acides aminés est remarquablement constante en dépit des variations de nos apports alimentaires. L’homéostasie systémique des acides aminés est particulièrement importante pour le tryptophane, en raison de son rôle clef dans de nombreux processus biologiques, incluant l’immunité et l’activité neuronale. Chez l’homme, la tryptophanémie est en permanence maintenue à 60µM. Nos découvertes révèlent que l’homéostasie du tryptophane est majoritairement contrôlée par la stabilité post-traductionnelle de la tryptophane 2,3-dioxygenase (TDO), l’enzyme hépatique catalysant la première réaction du catabolisme du tryptophane dans la voie métabolique de la kynurénine. Localisée au carrefour de l’artère hépatique et de la veine porte, la protéine TDO occupe une position stratégique pour filtrer le sang et dégrade plus de 90% du tryptophane d’origine alimentaire. Lorsque la tryptophanémie augmente, le tryptophane active la protéine TDO en stabilisant sa conformation tétramérique au moyen de quatre exosites non catalytiques, promouvant ainsi le catabolisme du tryptophane plasmatique. Une fois la tryptophanémie faible, le tryptophane libère les exosites et provoque la déstabilisation du tétramère en dimères et en monomères inactifs, libérant ainsi un motif de dégradation ou dégron reconnu par des ubiquitines ligases SKP1-CUL1-F-box. Elles déclenchent alors la polyubiquitination de TDO, laquelle conduit à la dégradation de l’enzyme par le protéasome et à l’arrêt du catabolisme du tryptophane. In vivo, la stabilisation de la protéine TDO hépatique avec un analogue non métabolisable, l’alpha-méthyl-tryptophane, réduit de manière permanente la concentration plasmatique du tryptophane. Nos résultats décrivent un mécanisme d’adaptation rapide du catabolisme du tryptophane permettant une dégradation de l’acide aminé en excès et prévenant son catabolisme en dessous de sa teneur physiologique. Cette boucle de rétrocontrôle assure une régulation fine de la tryptophanémie dont le maintien est nécessaire à l’homéostasie neurologique et immunitaire. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2021

Immunotherapy has revolutionized the treatment of certain cancers by facilitating the antitumor immune response and represents today one of the mainstays of cancer therapy. However, only a subset of patients responds to immunotherapy, which can also lead to serious complications. The tumor microenvironment is composed of multiple and complex cellular and molecular interactions providing to cancer cells not only a supportive framework but promoting also many steps of immunosuppression and tumor progression. To date, the mechanisms that drive the acquisition of these immunosuppressive features are still poorly defined. Tumor-associated macrophages can be highly represented in the tumor microenvironment where they are shaped and become key players in the innate and adaptive immune escape of the tumor cells.Heme oxygenase-1 is the rate-limiting enzyme that catabolizes heme into three major biologically active byproducts which display cytoprotective, antioxidant and immunomodulatory effects. We hypothesized that tumor-associated macrophages might suppress anti-tumor T-cell response through heme oxygenase-1 induction in the tumor microenvironment and macrophage polarization. We showed that heme oxygenase-1 is highly expressed in tumor-associated macrophages. By using a subcutaneous EG7-OVA lymphoma model on genetically engineered mice with a conditional deletion of heme oxygenase-1 in macrophages, our data show that myeloid-restricted heme oxygenase-1 deficiency improves the effect of a therapeutic antitumor immunization by enhancing tumor-infiltrating antitumor CD8+ T-cell proliferation and cytotoxicity and represses tumor growth. Our data suggest a major role of myeloid heme oxygenase-1 in the differentiation and the phenotypic, functional, transcriptional and epigenetic reprograming of tumor-associated macrophages. Myeloid HO-1 inhibition might be considered as a new myeloid HO-1-mediated immune checkpoint blockade. Targeting myeloid compartment could reprogram the tumor microenvironment and synergize with other cancer therapies., Doctorat en Sciences médicales (Médecine), info:eu-repo/semantics/nonPublished

Cancer immunotherapies aim at inducing or stimulating anti-tumor immune responses to eliminate tumor cells and trigger tumor regressions in patients with cancer. Approaches under clinical development comprise cancer vaccines, adoptive transfers of immune cells and immunostimulatory antibodies. Several immunostimulatory antibodies were approved by regulatory agencies for the treatment of various types of cancers. Despite impressive clinical successes, response rates to cancer immunotherapies remain very diverse, and only a minority of patients benefit from the currently available approaches. Novel approaches that target other immunosuppressive pathways are needed to increase the clinical efficacy of cancer immunotherapies. Regulatory T cells or Tregs are immunosuppressive cells that are indispensable to prevent auto-immunity, but in cancer patients they are detrimental because they suppress anti-tumor immunity. Our laboratory previously identified a mechanism of immunosuppression by Tregs which implies production and activation of the immunosuppressive TGF-β1 cytokine. Unlike other cells, activation of TGF-β1 by Tregs requires intervention of two transmembrane proteins named GARP and integrin αvβ8. We developed antibodies against GARP:TGF-β1 complexes that allow to selectively block TGF-β1 activation by Tregs. These could provide significant advantages over global TGF-β1 inhibition, which bears the risk of undesired tumor-promoting effects. Indeed, TGF-β1 exerts vastly diverse effects in cancer, supporting either tumor progression by favoring metastases and inhibiting anti-tumor immunity, or tumor suppression by inhibiting malignant cell proliferation. Here, we show that blocking TGF-β1 activation by Tregs with anti-GARP:TGF-β1 antibodies induces regressions of mouse tumors that are otherwise resistant to anti-PD-1 immunotherapy. We found GARP-expressing Tregs and evidence that they produce TGF-β1 in human melanoma metastases. Finally, we provide evidence that upon administration to tumor-bearing mice, anti-GARP:TGF-β1 antibodies directly bind Tregs and reduce autocrine TGF-β1 signals in these cells. Altogether, our work suggests that blocking production of a single TGF-β isoform emanating from a restricted cellular source is sufficient to potentiate anti-tumor immunity. Anti-GARP:TGF-β1 mAb may serve as a new immunostimulatory drug to improve patient responses to cancer immunotherapies. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2020

Little is known about anti-tumor T cell activity in low antigenic but frequent tumor such as breast carcinoma. We first explored the immunogenicity towards CD8 T cells for 6 primary human breast carcinomas. We screened tumor derived T cell clones for the recognition of tumor specific antigens. We found only 7 T cell clones that were able to recognize mutations. They originate from the same tumor. This demonstrates that anti-tumor T cell activity is possible in primary human breast carcinoma but seems to be rare. Aware of potential selection bias linked to the ability of T cells to grow in vitro and eager to characterize the ex vivo phenotype of the identified anti-tumor T cells, we developed a workflow involving single cell RNAseq analysis of freshly extracted tissue T cells followed by TCR reconstruction in a reporter cell line to evaluate their specificity. This workflow has also been applied to analyze kidney infiltrating T cells from lupus nephritis suffering patients. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

Calreticulin is an ER resident chaperone protein that also plays an important role in buffering the Ca2+ ions in the ER, regulating the calcium homeostasis and calcium-dependent signaling in the cell. In 2013, mutations of the calreticulin encoding gene CALR were shown to be driver mutations of essential thrombocythemia (ET) and myelofibrosis, two diseases that are part of the myeloproliferative neoplasm family. Previous studies in our team have shown that the thrombopoieitin receptor is activated by calreticulin mutants, thereby activating the JAK-STAT pathway of CALR mutated cells. This work focused on establishing a precise comprehension of the thrombopoietin receptor activation mechanisms by calreticulin mutants by studying their interaction in vitro and by establishing a relevant mouse model of disease. In vitro studies have elucidated that calreticulin mutants act as ‘rogue chaperones’ that are capable of escaping the ER and accompanying the thrombopoietin receptor through the secretory pathway. This is shown to be possible through a specific interaction of the lectin domain of calreticulin mutants with the first N-glycan of the thrombopoietin receptor that remains in an immature form even as both proteins progress through the Golgi compartment to the plasma membrane. We also provide evidence of a direct physical interaction between the calreticulin mutant and the thrombopoietin receptor by BRET. We also show that the human and mouse mutant calreticulin proteins are highly similar in their amino-acid sequences, biochemical properties and oncogenic capabilities. This allowed us to create two mutated Calr knock-in mouse models by CRISPR/Cas9 genome editing that reproduce an ET phenotype but with subtle differences that are attributable to the level of STAT5 activation both mutant calreticulin proteins are able to induce through their activation of the thrombopoietin receptor. We also confirm the observation that the calcium buffering role of calreticulin is essential for the myocardium development during embryogenesis and that lack of the C-terminus of the protein is embryonically lethal just like the full knock-out of the protein. This work provides substantial answers to the field of CALR mutated myeloproliferative neoplasms regarding the oncogenic mechanism at the cellular level but also at the level of a whole organism. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

Production of active TGF-β is regulated at a post-translational level and implies the release of the mature cytokine from the inactive latent TGF-β precursor. Several cell-type specific mechanisms of TGF-β activation have been described. In this thesis, we identified that TGF-β1 activation by regulatory T cells (Tregs) implies the transmembrane protein GARP, which binds and presents latent TGF-β1 at the Tregs cell surface. We derived two monoclonal antibodies directed against the GARP/latent TGF-β1 complex and blocking TGF-β1 activation and immunosuppression by human Tregs. The crystal structure of the GARP/latent TGF-β1 complex bound by one blocking antibody reveals how GARP presents latent TGF-β1 for activation at the Treg cell surface and how blocking anti-GARP/latent TGF-β1 antibodies inhibit TGF-β1 activation by human Tregs. These antibodies may serve as therapeutic tools to improve efficiency of cancer immunotherapy. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2018

The clinical efficacy of immunostimulatory antibodies targeting the PD-1 pathway in cancer is likely mediated by CD8 T cells recognizing tumor-specific antigens. Whether breast carcinomas (BCs) are infiltrated by such cells is not known. We wished to obtain evidence for the presence of tumor-specific CD8 T cells in primary BCs. Random sets of CD8 T cell clones were derived from 6 BCs that we screened for recognition of candidate tumor-specific antigenic peptides, which were encoded either by mutated genes or by cancer-germline genes. One of these sets contained 6 different clones that recognized 4 mutant peptides, thus demonstrating that some primary BCs are immunogenic. The corresponding tumor displayed a DNA mismatch repair deficiency associated with a high number of mutations compared to the other 5 tumors. In these 5 tumors, the estimated number of presented mutant antigenic peptides was very low or zero, likely explaining the absence of detectable tumor-specific T cells. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2016

Peptides recognized by cytolytic T lymphocytes (CTL) on tumors usually originate from the degradation of cellular proteins by the proteasome. Understanding the parameters involved in the production of a given antigenic peptide is important to define the most appropriate vaccination modality for this peptide. In this work, we studied the unconventional processing of two tumor antigens deriving from the differentiation protein gp100PMEL17 and the cancer specific protein MAGE-A4. Peptide splicing is a novel mechanism of production of peptides relying on the proteasome and involving the linkage of fragments originally distant in the parental protein. In the first study of this work, we describe a new antigenic peptide, which is presented by HLA-A3 and comprises two non-contiguous fragments of the melanoma differentiation antigen gp100PMEL17 spliced together in the reverse order to that in which they appear in the parental protein. Contrary to the previously described spliced peptides, which are produced by the association of fragments of 3 to 6 amino acids, the peptide described here results from the ultimate association of an 8-amino acid fragment with a single arginine residue. As described before, peptide splicing takes place in the proteasome by transpeptidation involving an acyl-enzyme intermediate linking one of the peptide fragment to a catalytic subunit of the proteasome. Interestingly, we observe that the peptide causing the nucleophilic attack on the acyl-enzyme intermediate must be at least three-amino acid long in order to give rise to a spliced peptide. The spliced peptide produced from this reaction therefore bears an extended C-terminus that needs to be further trimmed in order to produce the final antigenic peptide. We show that the proteasome is able to perform the final trimming step required to produce the antigenic peptide described here. In the second part of this work, studying the processing of the MAGE-A4 peptide NYKRCFPVI143-151, we observed that proteasome inhibition improved its presentation, suggesting that the proteasome does not produce the peptide but rather degrades it. Interestingly, the degradation efficiency of the antigenic peptide depends on the proteasome subtype. Indeed, our results based on cellular and in vitro experiments showed that the standard proteasome is more efficient at degrading the peptide than the 11 immunoproteasome. Because the peptide processing was independent from the proteasome, we investigated the role of alternatives proteases. Our results showed that two different proteases processed the peptide: a serine protease and a metalloprotease. In conclusion, we show that the peptide NYKRCFPVI143-151 is (1) degraded by the standard proteasome and (2) produced by two other proteases, which are still under investigation. Les peptides antigéniques reconnus par les lymphocytes T cytolytiques (CTL) à la surface des cellules proviennent majoritairement de la dégradation des protéines par le protéasome. La compréhension des paramètres qui influencent la production d’un peptide antigénique donné est primordiale pour définir la meilleure modalité de vaccination contre ce peptide. Dans ce travail, nous avons étudié l’apprêtement non conventionnel de deux antigènes tumoraux. Dans une première étude, nous avons décrit un nouveau peptide antigénique présenté par HLA-A3 à la surface de cellules de mélanomes. Cet antigène est formé par l’association de deux fragments non-contigus de la protéine de différentiation gp100PMEL17. Ce phénomène, appelé épissage peptidique se produit au sein du protéasome et implique la formation d’un nouveau lien peptidique entre des fragments à l’origine distants dans la protéine dont ils proviennent. Le mécanisme d’épissage peptidique se produit suite à l’attaque nucléophile d’un intermédiaire acyl-enzyme par le groupement N-terminal d’un fragment peptidique présent dans la chambre catalytique du protéasome. Contrairement aux peptides épissés précédemment étudiés et qui résultent de l’association de fragments de 3 à 6 acides aminés, le peptide décrit dans ce travail provient de l’association d’un fragment de 8 acides aminés avec un seul résidu arginine. Notre travail a montré que le peptide responsable de l’attaque nucléophile devait contenir au minimum 3 acides aminés. Le fragment peptidique résultant de cet épissage est ensuite raboté pour produire le peptide antigénique final. La deuxième étude présentée a quant à elle étudié l’apprêtement de l’antigène tumoral NYKRCFPVI143-151 dérivé de MAGE-A4. De manière intéressante, nous avons observé que le peptide était dégradé de manière différentielle par les différents sous-types de protéasome puisque le protéasome standard dégrade le peptide de manière plus importante que l’immunoprotéasome. Le fait que ce peptide n’est pas produit par le protéasome nous a amené à étudier l’implication de protéases alternatives. Nous avons pu démontrer que deux protéases différentes sont impliquées dans l’apprêtement de l’antigène : une sérine protéase et une métalloprotéase. Ces deux protéases sont actuellement en cours d’identification. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2015

In our laboratory, we generated a transgenic mouse strain, named Tirp-10B, which develops inducible melanomas upon 4-OH Tamoxifen treatment. These melanomas express the P1A tumor antigen. Despite the induction of anti-P1A specific CD8 T cells after immunization, we did not observe any effect on the incidence of tumor development. These results indicate the presence of an immunosuppressive mechanism directed against P1A-specific CTLs. The adoptive transfer of P1A TCR transgenic CD8 T cells did not induce tumor rejection although these T cells infiltrated the tumors, were activated and produced IFNγ and TNFα. However, we showed that they disappeared very quickly by apoptosis. These results contribute to the description of an immunosuppressive mechanism present in the melanoma environment involved in tumor escape. Au sein du laboratoire, un modèle transgénique murin de mélanomes inductibles a été développé. Chez ces souris, nommées TiRP-10B, l’administration de 4-OH Tamoxifène induit le développement de mélanomes qui expriment l’antigène tumoral P1A. Malgré l’induction d’une réponse lymphocytaire T CD8 anti-P1A à la suite d’une immunisation, nous n’avons pas observé d’effet sur l’incidence des tumeurs. Ces résultats indiquent l’existence d’un mécanisme de résistance immunitaire spécifique de l’antigène tumoral P1A. Le transfert adoptif de lymphocytes T CD8 TCR transgéniques anti-P1A ne permet pas le rejet des tumeurs induites alors que ces lymphocytes sont capables d’infiltrer les tumeurs, de s’activer et de produire de l’IFNγ et du TNFα. Cependant, ils disparaissent très rapidement par apoptose. L’ensemble des travaux réalisés contribue à la description d’un mécanisme d’immunosuppression impliqué dans l’échappement tumoral. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2015

The prevalence of alcohol use disorders in Europe is 7.5%. Alcohol dependence is traditionally considered a brain disorder where ethanol interacts with several receptors for neurotransmitters. Pharmacological treatments of addiction currently target these receptors but demonstrated only limited therapeutic effect, and the relapse rate is still very high. This PhD work was dedicated to the study of alterations of the gut in alcohol-dependence. We observed important alterations of the gut microbiota in a large fraction of alcohol-dependent subjects, that was associated with a leaky gut barrier, which enabled the translocation of bacterial products from the gut lumen to the systemic circulation. These bacterial products induced an inflammatory response that correlated with psychological symptoms of alcohol dependence including depression, anxiety and craving. The results of this thesis open a new field of research for the treatment of alcohol dependence, targeting the gut microbiota. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2014

Over the past decade, studies using murine models have led to the demonstration that CD4+ T helper (Th) cells play a critical role in the control of cancer progression. Additional support for their importance comes from the growing body of recent clinical/translational research data demonstrating the importance of tumor-infiltrating T and B lymphocytes in long-term patient survival for various types of cancer, including breast cancer (BC). As the key population coordinating adaptive immune responses, the role(s) played by individual Th subsets in cancer immunity remains largely controversial. The Th1 subset has uniquely been shown to have a clear anti-tumor effect, guiding CD8+ cytotoxic T cells-mediated direct tumor cell lysis through IFN-γ secretion. Although the negative regulatory role played by Treg cells has been extensively studied in cancer, its prognostic value along with that of Th2 and Th17 cells have not been clearly demonstrated in patients. T follicular helper (Tfh) cells, a recently characterized Th subset that plays a primary role in the generation of B cell memory in secondary lymphoid organs, have not been previously described infiltrating solid tumors. The principal objective of this thesis was to perform an in-depth characterization of tumor-infiltrating CD4+ T cells (TIL) and Th subsets in human BC, where very little is currently known.Using whole genome microarrays, we analyzed the gene expression profiles of TIL relative to their counterparts from the axillary lymph nodes and peripheral blood. Applying a novel approach, we compared TIL profiles with public microarray data for Th subsets, demonstrating: 1) the presence of all major Th subsets (Th1, Th2, Th17, Treg as well as Tfh) in the TIL, 2) the TIL are effector memory rather than central memory cells, 3) the TIL are concomitantly activated and suppressed and 4) TIL from tumors with extensive lymphoid infiltrates are more activated/less suppressed in the TCR/CD3 signaling pathway, producing higher levels and a wider panel of Th cytokines than TIL from minimally-infiltrated tumors.We also performed in vitro experiments to study tumor microenvironment effects on TIL by treating normal CD4+ T cells from healthy donor blood with primary tumor supernatants (SN). Tumor SN largely reproduces the TIL profile in normal Th cells, totally suppressing their activation and inhibiting their cytokine production. Intriguingly, the highly restricted number of cytokines induced by tumor SN included several tumor-promoting factors, such as IL-8 and TNF. SN from an extensively-infiltrated tumor was found to be less immune-suppressive than SN from minimally-infiltrated tumors. In line with this, TIL from minimally-infiltrated tumors are closer to SN-treated (suppressed) activated donor cells whereas TIL from extensively-infiltrated tumors are more similar to activated cells without SN treatment.These results led us to further investigate the observed differences between TIL from extensive and minimally-infiltrated tumors. Genes characterizing Th1 and Tfh cells were enriched in the extensively-infiltrated tumors. PD-1hiCD200hi Tfh cells were specifically detected in extensively-infiltrated tumors by flow cytometry and these cells were determined to be the major source of the chemokine CXCL13. Immunohistochemical analysis demonstrated highly-organized tertiary lymphoid structures (TLS) within the tumor, containing a CD4+/CD8+ T cell zone and a B cell zone with reactive germinal centers where Tfh cells and follicular dendritic cells (FDC) are resident. Their presence suggests the origin of an effective memory anti-tumor immune response.Finally, we generated Tfh- and Th1-specific gene signatures reflecting differences between extensive and minimal TIL and tested their prognostic value in large-patient-scale public data sets. Our Tfh signature predicts better 10-year disease-free survival for all BC subtypes, outperforming the Th1 signature, suggesting that Tfh cells play a more central role than Th1 cells in anti-tumor immunity. CXCL13 is the determinant gene of our Tfh signature, showing particularly strong prognostic power for the HER2+ subtype. Additionally, these signatures also predict a better response to neoadjuvant chemotherapy.This thesis research has demonstrated that a previously undetected Th subset, Tfh cells, infiltrates solid tumors and shown that their presence signals enhanced anti-tumor immunity. Durant cette dernière décennie, des travaux menés dans des modèles murins ont permis de mettre en évidence le rôle crucial joué par les lymphocytes T auxiliaires CD4+ (Th) dans le contrôle de la progression des cancers. De plus, de nombreuses études cliniques et/ou translationnelles récentes corroborent ces observations en montrant une corrélation entre l’importance de l’infiltration intra-tumorale par les lymphocytes T et B et la survie à long terme des patients atteints de différents types de cancer, dont le cancer du sein. En tant que chefs d’orchestre de la réponse immune adaptative, les rôles spécifiques des sous-populations des cellules Th restent controversés. Les Th1 sont la seule population exerçant une claire réponse anti-tumorale, qui est liée à la sécrétion d’IFN-γ, une cytokine primordiale à l’action des lymphocytes T cytotoxiques CD8+. Bien que le rôle néfaste des T régulateurs (Treg) a été largement étudié dans le cancer, leur implication pronostique ainsi que celle des Th2 et Th17 n’ont pas encore été clairement démontrées. La présence d’une sous-population de CD4, les T auxiliaires folliculaires (Tfh), cellules clés dans la différenciation des lymphocytes B mémoires au sein des organes lymphoïdes secondaires, n’a jamais été décrite dans les cancers solides. Le but principal de ce travail est de caractériser les sous-populations des lymphocytes T CD4+ infiltrant la tumeur (TIL) en prenant comme modèle le cancer du sein humain. A l’heure actuelle, il existe très peu de données sur les TIL CD4 dans ce type de cancer.Nous avons d’abord établi le profil génique des TIL en les comparant avec ceux provenant des ganglions axillaires ou du sang périphérique. En appliquant une nouvelle approche, nous avons comparé les profils des TIL avec les données publiques de sous-populations de Th et démontré que :1) toutes les sous-populations de cellules Th (Th1, Th2, Th17, Treg et Tfh) infiltrent la tumeur, 2) les TIL ont un phénotype plus proche de celui des cellules mémoires effectrices que des cellules mémoires centrales, 3) les TIL sont simultanément activés et supprimés et 4) les TIL provenant des tumeurs massivement infiltrées («extensives») par des lymphocytes sont mieux activés et moins supprimés que les TIL des tumeurs peu infiltrées («minimales») dans la voie de signalisation TCR et produisent des cytokines d’une quantité plus élevée et d’une répertoire plus large.Nous avons également effectué des expériences in vitro pour étudier l’effet de l’environnement tumoral sur les TIL en traitant des CD4 normaux (provenant des donneuses saines) par le surnageant (SN) extrait des tumeurs fraiches. Le SN tumoral induit un profil génique proche de celui des TIL en inhibant l’activation et la production de cytokines de ces cellules stimulées. Curieusement, parmi le peu de cytokines induites par le SN tumoral, des facteurs pro-tumoraux comme IL-8 et TNF sont détectés. Le surnageant provenant d’une tumeur «extensive» est moins immunosuppresseur que ceux des tumeurs «minimales». Conformément, les TIL provenant des tumeurs «minimales» ont un profil génique proche des cellules normales activées et traitées (supprimées) par le SN tumoral tandis que les TIL des tumeurs «extensives» ressemblent aux cellules activées non traitées.Ces résultats nous avaient guidés à investiguer plus profondément les différences observées entre les TIL des tumeurs «extensives» et «minimales». Les gènes caractéristiques des Th1 et Tfh sont enrichis dans les tumeurs «extensives». Les cellules Tfh PD1hiCD200hi sont spécifiquement détectées par cytométrie de flux dans les tumeurs «extensives» et sont identifiées comme les producteurs principaux de la chimiokine CXCL13. L’examen par immunohistochimie a permis de détecter des structures lymphoïdes tertiaires (TLS) dans la tumeur, composées d’une zone T (CD4 et CD8) et d’une zone B au sein de laquelle se trouve parfois un centre germinatif actif contenant des Tfh et des cellules dendritiques folliculaires (FDC). La présence de ces structures suggère l’origine d’une réponse immune mémoire anti-tumorale.Finalement, nous avons établi des signatures géniques spécifiques aux Tfh et Th1 et recherché leur impact pronostique dans deux bases de données publiques à grande échelle. Notre signature Tfh est positivement corrélée avec la survie à 10 ans des patientes de tous les sous-types de cancer du sein, et est plus performante que la signature Th1. Ceci suggère que les Tfh pourraient jouer un rôle plus crucial que les Th1 dans la réponse immune anti-tumorale. CXCL13 est le gène déterminant de notre signature Tfh et son expression est fortement associée à une meilleure survie chez les patientes du sous-type HER2+. De plus, ces signatures prévoient également une meilleure réponse à la chimiothérapie néoadjuvante (préopératoire).Cette étude a démontré qu’une nouvelle sous-population de CD4, les Tfh, infiltre la tumeur solide et leur présence indique l’existence d’une immunité anti-tumorale renforcée., Doctorat en Sciences biomédicales et pharmaceutiques, info:eu-repo/semantics/nonPublished

Over the past decade, studies using murine models have led to the demonstration that CD4+ T helper (Th) cells play a critical role in the control of cancer progression. Additional support for their importance comes from the growing body of recent clinical/translational research data demonstrating the importance of tumor-infiltrating T and B lymphocytes in long-term patient survival for various types of cancer, including breast cancer (BC). As the key population coordinating adaptive immune responses, the role(s) played by individual Th subsets in cancer immunity remains largely controversial. The Th1 subset has uniquely been shown to have a clear anti-tumor effect, guiding CD8+ cytotoxic T cells-mediated direct tumor cell lysis through IFN-γ secretion. Although the negative regulatory role played by Treg cells has been extensively studied in cancer, its prognostic value along with that of Th2 and Th17 cells have not been clearly demonstrated in patients. T follicular helper (Tfh) cells, a recently characterized Th subset that plays a primary role in the generation of B cell memory in secondary lymphoid organs, have not been previously described infiltrating solid tumors. The principal objective of this thesis was to perform an in-depth characterization of tumor-infiltrating CD4+ T cells (TIL) and Th subsets in human BC, where very little is currently known. Using whole genome microarrays, we analyzed the gene expression profiles of TIL relative to their counterparts from the axillary lymph nodes and peripheral blood. Applying a novel approach, we compared TIL profiles with public microarray data for Th subsets, demonstrating: 1) the presence of all major Th subsets (Th1, Th2, Th17, Treg as well as Tfh) in the TIL, 2) the TIL are effector memory rather than central memory cells, 3) the TIL are concomitantly activated and suppressed and 4) TIL from tumors with extensive lymphoid infiltrates are more activated/less suppressed in the TCR/CD3 signaling pathway, producing higher levels and a wider panel of Th cytokines than TIL from minimally-infiltrated tumors. We also performed in vitro experiments to study tumor microenvironment effects on TIL by treating normal CD4+ T cells from healthy donor blood with primary tumor supernatants (SN). Tumor SN largely reproduces the TIL profile in normal Th cells, totally suppressing their activation and inhibiting their cytokine production. Intriguingly, the highly restricted number of cytokines induced by tumor SN included several tumor-promoting factors, such as IL-8 and TNF. SN from an extensively-infiltrated tumor was found to be less immune-suppressive than SN from minimally-infiltrated tumors. In line with this, TIL from minimally-infiltrated tumors are closer to SN-treated (suppressed) activated donor cells whereas TIL from extensively-infiltrated tumors are more similar to activated cells without SN treatment. These results led us to further investigate the observed differences between TIL from extensive and minimally-infiltrated tumors. Genes characterizing Th1 and Tfh cells were enriched in the extensively-infiltrated tumors. PD-1hiCD200hi Tfh cells were specifically detected in extensively-infiltrated tumors by flow cytometry and these cells were determined to be the major source of the chemokine CXCL13. Immunohistochemical analysis demonstrated highly-organized tertiary lymphoid structures (TLS) within the tumor, containing a CD4+/CD8+ T cell zone and a B cell zone with reactive germinal centers where Tfh cells and follicular dendritic cells (FDC) are resident. Their presence suggests the origin of an effective memory anti-tumor immune response. Finally, we generated Tfh- and Th1-specific gene signatures reflecting differences between extensive and minimal TIL and tested their prognostic value in large-patient-scale public data sets. Our Tfh signature predicts better 10-year disease-free survival for all BC subtypes, outperforming the Th1 signature, suggesting that Tfh cells play a more central role than Th1 cells in anti-tumor immunity. CXCL13 is the determinant gene of our Tfh signature, showing particularly strong prognostic power for the HER2+ subtype. Additionally, these signatures also predict a better response to neoadjuvant chemotherapy. This thesis research has demonstrated that a previously undetected Th subset, Tfh cells, infiltrates solid tumors and shown that their presence signals enhanced anti-tumor immunity. Durant cette dernière décennie, des travaux menés dans des modèles murins ont permis de mettre en évidence le rôle crucial joué par les lymphocytes T auxiliaires CD4+ (Th) dans le contrôle de la progression des cancers. De plus, de nombreuses études cliniques et/ou translationnelles récentes corroborent ces observations en montrant une corrélation entre l’importance de l’infiltration intra-tumorale par les lymphocytes T et B et la survie à long terme des patients atteints de différents types de cancer, dont le cancer du sein. En tant que chefs d’orchestre de la réponse immune adaptative, les rôles spécifiques des sous-populations des cellules Th restent controversés. Les Th1 sont la seule population exerçant une claire réponse anti-tumorale, qui est liée à la sécrétion d’IFN-γ, une cytokine primordiale à l’action des lymphocytes T cytotoxiques CD8+. Bien que le rôle néfaste des T régulateurs (Treg) a été largement étudié dans le cancer, leur implication pronostique ainsi que celle des Th2 et Th17 n’ont pas encore été clairement démontrées. La présence d’une sous-population de CD4, les T auxiliaires folliculaires (Tfh), cellules clés dans la différenciation des lymphocytes B mémoires au sein des organes lymphoïdes secondaires, n’a jamais été décrite dans les cancers solides. Le but principal de ce travail est de caractériser les sous-populations des lymphocytes T CD4+ infiltrant la tumeur (TIL) en prenant comme modèle le cancer du sein humain. A l’heure actuelle, il existe très peu de données sur les TIL CD4 dans ce type de cancer. Nous avons d’abord établi le profil génique des TIL en les comparant avec ceux provenant des ganglions axillaires ou du sang périphérique. En appliquant une nouvelle approche, nous avons comparé les profils des TIL avec les données publiques de sous-populations de Th et démontré que :1) toutes les sous-populations de cellules Th (Th1, Th2, Th17, Treg et Tfh) infiltrent la tumeur, 2) les TIL ont un phénotype plus proche de celui des cellules mémoires effectrices que des cellules mémoires centrales, 3) les TIL sont simultanément activés et supprimés et 4) les TIL provenant des tumeurs massivement infiltrées («extensives») par des lymphocytes sont mieux activés et moins supprimés que les TIL des tumeurs peu infiltrées («minimales») dans la voie de signalisation TCR et produisent des cytokines d’une quantité plus élevée et d’une répertoire plus large. Nous avons également effectué des expériences in vitro pour étudier l’effet de l’environnement tumoral sur les TIL en traitant des CD4 normaux (provenant des donneuses saines) par le surnageant (SN) extrait des tumeurs fraiches. Le SN tumoral induit un profil génique proche de celui des TIL en inhibant l’activation et la production de cytokines de ces cellules stimulées. Curieusement, parmi le peu de cytokines induites par le SN tumoral, des facteurs pro-tumoraux comme IL-8 et TNF sont détectés. Le surnageant provenant d’une tumeur «extensive» est moins immunosuppresseur que ceux des tumeurs «minimales». Conformément, les TIL provenant des tumeurs «minimales» ont un profil génique proche des cellules normales activées et traitées (supprimées) par le SN tumoral tandis que les TIL des tumeurs «extensives» ressemblent aux cellules activées non traitées. Ces résultats nous avaient guidés à investiguer plus profondément les différences observées entre les TIL des tumeurs «extensives» et «minimales». Les gènes caractéristiques des Th1 et Tfh sont enrichis dans les tumeurs «extensives». Les cellules Tfh PD1hiCD200hi sont spécifiquement détectées par cytométrie de flux dans les tumeurs «extensives» et sont identifiées comme les producteurs principaux de la chimiokine CXCL13. L’examen par immunohistochimie a permis de détecter des structures lymphoïdes tertiaires (TLS) dans la tumeur, composées d’une zone T (CD4 et CD8) et d’une zone B au sein de laquelle se trouve parfois un centre germinatif actif contenant des Tfh et des cellules dendritiques folliculaires (FDC). La présence de ces structures suggère l’origine d’une réponse immune mémoire anti-tumorale. Finalement, nous avons établi des signatures géniques spécifiques aux Tfh et Th1 et recherché leur impact pronostique dans deux bases de données publiques à grande échelle. Notre signature Tfh est positivement corrélée avec la survie à 10 ans des patientes de tous les sous-types de cancer du sein, et est plus performante que la signature Th1. Ceci suggère que les Tfh pourraient jouer un rôle plus crucial que les Th1 dans la réponse immune anti-tumorale. CXCL13 est le gène déterminant de notre signature Tfh et son expression est fortement associée à une meilleure survie chez les patientes du sous-type HER2+. De plus, ces signatures prévoient également une meilleure réponse à la chimiothérapie néoadjuvante (préopératoire). Cette étude a démontré qu’une nouvelle sous-population de CD4, les Tfh, infiltre la tumeur solide et leur présence indique l’existence d’une immunité anti-tumorale renforcée., Doctorat en Sciences biomédicales et pharmaceutiques, info:eu-repo/semantics/published

A number of therapeutic vaccines have been evaluated in patients with metastatic melanoma. Tumor regressions were observed in 20% of the vaccinated patients. Complete and long-term clinical responses have been occasionally observed. Some researchers have used autologous dendritic cells loaded with antigenic peptides to immunize patients, because dendritic cells are considered very effective in antigen presentation to T cells. A first objective of our work was to evaluate the anti-vaccine CTL responses in several patients vaccinated with dendritic cells loaded with antigens derived from the protein MAGE-3. The vaccine was able to induce a response in four of the five analyzed patients. This response was polyclonal in three of the four responding patients. The frequencies of anti-vaccine CTL ranged from 1/1 000 to 1/100 000 of blood CD8 T cells. While these responses were generally low, they seemed to be associated with tumor regressions. In one patient, we were able to extend our analysis beyond the anti-vaccine CTL response and we analyzed the T cell response directed against various antigens expressed by the tumor. We identified a CTL clone directed against one of these antigens. This antigen was previously unknown and derived from protein MAGE-C2. The frequency of anti-MAGE-C2 CTL in the blood increased after vaccination. Unlike the anti-vaccine CTL, this CTL clonotype was found to accumulate in cutaneous metastases. It is tempting to think that it played an important role in the regression of some of these metastases. We also decided to examine the functional profiles of the anti-vaccine CTL that were isolated from the blood of patients. Since many publications associate the expression of several surface markers with a functional differentiation status of T cells, we analyzed the phenotype of the anti-vaccine CTL clones using differentiation markers described in the literature, namely CCR7 and CD45RA. All the analysed clones had lost the expression of CCR7, and some of them strongly expressed CD45RA. In contradiction with published differentiation models, those CCR7-/CD45RA+ clones did not seem to have reached a terminally differentiated status, as they kept important proliferative capacities. Our observations revealing cyclic variations of CD45RA expression depending on the antigenic contacts, we propose that CD45RA re-expression on CCR7- CD8 T cells is a consequence of a prolonged lack of antigenic contact rather than the sign of a terminal differentiation. (SBIM 3) -- UCL, 2012

Most antigenic peptides recognized by CD8 T lymphocytes are produced through degradation of intracellular proteins by the proteasome. However, some antigenic peptides are produced by a proteasome-independent pathway, which is poorly characterized. MAGE-A3168-176 is a tumor antigenic peptide presented by HLA-A1 and widely used for vaccination of melanoma patients. We observed that proteasome and tripeptidyl peptidase inhibitors failed to block presentation of the antigen by tumor cells. However, processing of this peptide occured in the cytosol because TAP-inhibition prevented its presentation. To characterize the cytosolic peptidase producing MAGE-A3168-176 we setup an in vitro digestion assay using a 20-mer precursor peptide encompassing the sequence of the antigenic peptide. We observed that only the cytosolic fraction was able to produce the antigenic peptide from this precursor. This production was abolished by treating the cytosolic fraction with o-phenanthroline, a broad-spectrum inhibitor of metallopeptidases. This inhibitor also blocked the presentation of MAGE-A3168-176 by tumor cells. Processing of the antigenic peptide was observed in HLA-A1 cells electroporated by the 20-mer precursor or by a precursor modified at the C- and N-terminus to exclude exopeptidase activity. With both precusors, the processing was inhibited by treating cells with o-phenanthroline concluding to a major role of a cytosolic metalloendopeptidase. One such enzyme is insulin-degrading enzyme (IDE). We observed that depletion of IDE abolished the capacity of a cytosolic fraction to produce the antigenic peptide. Furthermore, recombinant IDE was able to produce the peptide in vitro from the 20-mer peptide. Using a siRNA approach we observed that a reduction of IDE expression in tumor cells resulted in a reduced capacity of MAGE-A3168-176 presentation. Lastly, half-life studies revealed that in melanoma cells proteasome and IDE act as parallel ways for the degradation of MAGE-A3 contributing together to a different set of peptides. La majorité des peptides antigéniques reconnus par les lymphocytes T cytolytiques sont produits à partir de la dégradation des protéines cellulaires qui implique principalement le protéasome. Certains peptides antigéniques sont cependant produits par une voie qui ne dépend pas du protéasome. Le peptide antigénique dérivé de la protéine tumorale MAGE-A3 (EVDPIGHLY) et présenté par le HLA-A1 a été utilisé dans de nombreux essais de vaccinations chez des patients souffrant de mélanomes métastatiques. Au cours de ce travail, nous avons observé que ni l’inhibition du protéasome ni celle de la tripeptidyl peptidase II ne compromettait la présentation de l’antigène par des cellules tumorales. La présentation de l’antigène étant dépendante du transporteur TAP, cela indiquait que MAGE-A3/HLA-A1 était produit dans le cytosol. Nous avons caractérisé l’activité produisant le peptide à partir d’un précurseur de 20 acides aminés comprenant la séquence du peptide antigénique. Nous avons observé que seule la fraction cytosolique était capable de produire le peptide antigénique. La fraction cytosolique traitée avec un inhibiteur de métallopeptidases, l’orthophénanthroline, ne produisait plus le peptide antigénique. Ce même inhibiteur diminuait la présentation du peptide antigénique par des cellules tumorales. La présentation de l’antigène dans des cellules HLA-A1+/ MAGE-A3- à partir du précurseur de 20 acides aminés ou d’un autre modifié à ses deux extrémités indiquait que la production de l’antigène dépendait d’une endopeptidase. La caractérisation de la présentation de l’antigène indiquait que l’insulin-degrading enzyme (IDE) était vraisemblablement la peptidase qui produisait le peptide. Nous avons ensuite observé qu’une IDE recombinante était capable de produire le peptide antigénique et qu’une déplétion de l’IDE abolissait la capacité de production du peptide antigénique par la fraction cytosolique. Le rôle de l’IDE dans la production du peptide antigénique a finalement été confirmé en observant une inhibition de la présentation de l’antigène dans des cellules transfectées avec des siRNA spécifiques de l’IDE. Enfin, nous avons étudié la dégradation intracellulaire de MAGE-A3 et observé que l’IDE et le protéasome représentaient deux voies de dégradation parallèles qui ensemble contribueraient à la diversité des peptides. (SBIM 3) -- UCL, 2012

Our group showed that human tumor-infiltrating CD8 T lymphocytes (CD8 TIL), freshly isolated from solid tumors or ascites, secreted low levels of IFN-γ upon ex vivo stimulation, in contrast to blood CD8 T cells. A few hours of treatment with galectin ligand N-acetyllactosamine resulted in an increased cytokine secretion. Our working hypothesis is that, in the tumor environment, recently activated TIL harbor a set of glycans that are either more numerous or better ligands for galectin-3. Galectin-3 is abundant in many solid tumors and carcinomatous ascites, and can thus bind to surface glycoproteins of TIL and form lattices that would thereby reduce the motility of surface receptors implicated in T cell activation. This results in a loss of functions. Our experiments have confirmed these results on a large number of tumor samples : more than 80% of the CD8 TIL samples responded to a treatment with a galectin ligand. Treating TIL with anti-galectin-3 antibody had a similar effect. We have also shown that the CD4 TIL also secreted low levels of IFN-γ upon ex vivo stimulation and that increased secretion was observed after a short incubation with a galectin ligand. We also showed that the cytotoxic activity of CD8 TIL was increased after a treatment with a galectin ligand. Two polysaccharides of plant origin that are in clinical development were identified as highly effective in increasing the lytic activity of TIL and their secretion of IFN-γ. In tumor-bearing mice that received a vaccine aimed at inducing an anti-tumor CD8 T cell response, one of the two polysaccharides was injected. This led to tumor rejection in half of the mice, whereas all control mice died. In non-vaccinated mice, the polysaccharide had no effect by itself. These data argue for a therapeutic approach combining vaccination and injection of galectin ligands. A clinical trial based on these discoveries is planned. Melanoma-bearing patients will be included. They will be immunized with peptides and injected systemically with DAVANAT, one of the two polysaccharides that we tested in vitro. Notre groupe avait montré que des lymphocytes T infiltrant les tumeurs (TIL) CD8 fraîchement isolés d’infiltrats tumoraux humains sécrétaient peu ou pas d’IFN-γ suite à leur stimulation ex vivo, par opposition aux lymphocytes T CD8 du sang. Une incubation de quelques heures de ces TIL avec un ligand de galectine, du N-acetyllactosamine, induisait une augmentation de leur sécrétion de cytokines. Notre hypothèse de travail est la suivante : dans l’environnement tumoral, les TIL récemment activés présenteraient à leur surface plus de motifs glycosylés qui seraient des ligands de la galectine-3. Cette lectine, retrouvée souvent en grande quantité dans les tumeurs solides et les ascites carcinomateuses, pourrait alors se lier aux glycoprotéines à la surface des TIL et former un réseau réduisant la mobilité des récepteurs de surface impliqués dans l’activation des lymphocytes T. Ceci se traduirait notamment par une perte de fonction. Nos expériences lors de cette thèse ont permis de reproduire ces résultats sur un grand nombre de prélèvement tumoraux et de montrer que plus de 80 % des échantillons de TIL CD8 répondaient au traitement avec un ligand de galectine. Un traitement avec un anticorps anti-galectine-3 avait un effet similaire. Notre travail a permis de montrer que les TIL CD4, aussi, produisaient peu d’IFN-γ suite à leur stimulation ex vivo et que cette fonction était augmentée suite à une courte incubation avec un ligand de galectine. Nous avons également montré que l’activité cytotoxique des TIL CD8 était augmentée par un traitement avec un ligand de galectine. Deux polysaccharides d’origine végétale, en cours de développement clinique, ont été identifiés comme très efficaces pour augmenter la sécrétion d’IFN-γ par des TIL stimulés ex vivo, ainsi que leur capacité lytique. Pour un de ces polysaccharide, nous avons montré que chez des souris porteuses de tumeurs, une vaccination destinée à amplifier une réponse T CD8 anti-tumeur, combinée avec des injections du polysaccharide, menait au rejet de la tumeur chez la moitié des souris. Chez les animaux non vaccinés, le polysaccharide n’avait aucun effet visible. Ces données plaident en faveur d’une approche thérapeutique combinant vaccination et injection de ligands de galectines. Un essai clinique basé sur ces découvertes est envisagé très prochainement pour des patients porteurs de mélanome qui seront vaccinés avec des peptides et injectés de façon systémique avec du DAVANAT, un des deux polysaccharides testés in vitro lors de ce travail. (MED 3) -- UCL, 2011

Antigens presented by MHC class I molecules to CD8+ T lymphocytes consist of peptides of 8-11 amino acids, corresponding to continuous fragments of intracellular proteins. However, some antigenic peptides were found to be created by the splicing of two distinct fragments of the respective parental proteins. Peptide splicing represents a newly described mode of production of antigenic peptides whereby two noncontiguous peptide fragments are joined together after the excision of an intervening segment. Three spliced antigenic peptides have been described. The first example is derived from fibroblast growth factor-5 (FGF-5) and is made up of two fragments of five and four residues. Another is a peptide produced from melanosomal protein gp100 by splicing of two fragments of three and six residues. The third example is a human minor histocompatibility antigenic peptide created by a polymorphism in the SP110 gene. This epitope is composed of two noncontiguous fragments of four and six residues which are spliced together in the reverse order to that in which they appear in the parental protein. In the two latter cases, splicing involves the excision of a short intervening segment of four or six residues and was shown to occur in the proteasome by transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by the N terminus of a peptide fragment present at the active site. In a first part of this work, we studied the mechanism of production of the FGF-5 spliced peptide, for which the length of the intervening segment (40 aa) is much longer than the two other described examples. We confirmed that the proteasome also produces this spliced peptide by transpeptidation and we showed that reducing the length of the intervening segment increased the production of the spliced peptide. We also tested the possibility of trans-splicing (i.e., splicing of fragments from two distinct substrates) and observed that this event can occur in the cell between two peptide precursors with the same efficiency as cis-splicing. However, trans-splicing between two distinct proteins was far less efficient than splicing of peptide fragments from a single protein substrate and unlikely contributes to the production of the spliced peptides derived from FGF-5 and gp100. The production of the three spliced peptides by the standard proteasome and the immunoproteasome was then compared in details, using a cellular approach and an in vitro approach. We showed that both proteasome types can produce spliced peptides although they differ in their efficiency of production of each peptide. The FGF-5 and gp100 peptides are produced more efficiently by the standard proteasome, while the SP110 peptide is better produced by the immunoproteasome. By showing that splicing depends on the efficiency of production of the splicing partners, these results support the transpeptidation model of peptide splicing. In collaboration with Dr. Paul Robbins from the National Cancer Institute in Bethesda, we have identified a new spliced antigenic peptide, which is derived from the melanosomal protein tyrosinase. This original peptide is composed of two spliced and rearranged peptide fragments, each one containing an aspartate residue resulting from asparagine deamidation. The processing of this antigenic peptide involves retrotranslocation of tyrosinase molecules from the endoplasmic reticulum (ER) to the cytoplasm, deamidation of presumably glycosylated asparagines by peptide-N-glycanase, cleavage and splicing by the standard proteasome, and transport of the resulting peptide into the ER via the TAP transporter. The potential role of an additional, yet unidentified, protease during this process is also discussed in this work. (BIOL 3) -- UCL, 2011