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[EN]It is well illustrated that the generation of Tregs is the main mechanism responsible for maintaining immune tolerance during developmental exposure to non-inherited maternal antigen (NIMA). Moreover, the presence of NIMA-specific Tregs in the uterus of pregnant mice promote reproductive fitness by enforcing maternal tolerance to overlapping paternal antigens expressed by the fetus during next-generation pregnancies. However, the reason why perinatal T cell lineage is biased towards immune tolerance is poorly understood. Due to the fact that terminal deoxynucleotidyl transferase (TdT) is not expressed in neonatal T cells in the mouse, neonatal T cells have a less diverse TCR repertoire. This is known to limit their specificity and to increase their affinity for MHC/peptide complexes. At the start of the present work, we postulated that expressing high affinity TCR might be the reason that forces the development of antigen-specific Tregs in neonates. We undertook our study with the aim to investigate the mechanisms underlying mouse NIMA-specific Treg development in the perinatal period. Using 2W1S-OVA+ heterozygous mouse model in which 2W1S antigen was transformed into surrogate NIMA for half of the offspring, we observed an increased frequency of 2W1S-specific Tregs in NIMA-2W1S-exposed animals. Moreover, we also observed that periphery-derived NIMA-2W1S Tregs had a less diverse TCR repertoire and were phenotypically distinct from thymus-derived SELF-2W1S-specific Tregs. In order to investigate whether the lack of diversity was responsible for the development of neonatal NIMA-specific Tregs, we generated transgenic mice where TdT expression was enforced in T cells before birth. We found that transgenic TdT added clonal TCR diversity but did not prevent the development of T cell clones with neonatal type TCR repertoire and did not modify the frequency of neonatal NIMA-specific Tregs. On the contrary, TdT expression increased significantly generation of SELF-speci, [FR]Il est bien illustré que la génération périnatal de Treg est le principal mécanisme responsable du maintien de la tolérance immunitaire fœtale qui se développe suite à l'exposition aux antigènes maternels non-hérités (NIMA). De plus, la présence de Tregs spécifiques des NIMA dans l'utérus des femmes enceintes favorise la capacité de reproduction en renforçant la tolérance maternelle aux mêmes antigènes paternels exprimés par le fœtus pendant les grossesses de prochaine génération. Cependant, la raison pour laquelle la lignée des cellules T fœtales est biaisée en faveur de la tolérance immunitaire est mal comprise. Chez la souris, en raison du manque d'expression de la désoxynucléotidyl transférase terminale (TdT), les cellules T néonatales ont un répertoire de TCR moins diversifié. Ceci est connu pour limiter leur spécificité et augmenter leur affinité pour les complexes CMH / peptide. Au début du présent travail, nous avons émis l'hypothèse que l'expression de TCRs de haute affinité pourrait être la raison qui force le développement de Treg spécifiques chez les nouveau-nés. Nous avons plus particulièrement entrepris notre étude dans le but d'étudier les mécanismes sous-jacents au développement de Tregs spécifiques des NIMA chez la souris pendant la période périnatale. En utilisant le modèle de souris hétérozygotes pour 2W1S-OVA+ dans lequel l'antigène 2W1S a été transformé en NIMA pour la moitié de la progéniture, nous avons observé une fréquence accrue de Tregs spécifiques de 2W1S chez les animaux exposés au NIMA. De plus, nous avons également observé que les Treg NIMA-2W1S dérivés de la périphérie avaient un répertoire de TCRs moins diversifié et étaient phénotypiquement distincts des Tregs spécifiques de SELF-2W1S dérivés du thymus. Afin de déterminer si le manque de diversité était responsable du développement de Tregs néonataux spécifiques de NIMA, nous avons généré des souris transgéniques où l'expression de TdT était appliquée dans les cellules T avant l, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

The current SARS-CoV-2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID-19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS-CoV-2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS-CoV-2 would offer significant relief, although vaccine developers have warned that design, testing and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (i), but the earliest vaccines were based on the live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS-CoV-2 will mutate and may naturally attenuate over time (ii). What makes the current pandemic unique is that, thanks to state-of-the-art nucleic acid sequencing technologies, we can follow in detail how SARS-CoV-2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS-CoV-2 variants across the globe should be of key interest in our fight against the pandemic.

Regulatory T cells (Treg) are a suppressive subset of helper T cells that controls immune responses using a variety of mechanisms. Despite initially being thought of as a homogenous population, Treg cells were recently found to adapt their function to their environment and acquire specialized phenotypes depending on their tissue of residence. The molecular mechanisms underlying this functional and tissular adaptation remain to be fully elucidated.In the course of this work, we studied the role of transcription factor c-Maf in the differentiation and the function of Treg cells. For this, we used a murine model invalidated for c-Maf specifically in Treg cells. Transcription factor c-Maf is preferentially expressed by intestinal Treg cells, particularly among the RORγt+ Treg subset, which controls immune responses directed towards the gut microbiota. We have shown that the differentiation of RORγt+ Treg cells results from the integration of multiple environmental signals, highlighting the plasticity of Treg specialization. Furthermore, we have shown that c-Maf is required for the differentiation of RORγt+ Treg cells as well as the expression of the anti-inflammatory cytokine IL-10 by intestinal Treg cells, thus endowing Treg cells with the ability to control homeostatic Th17 responses in the intestine. Mice deficient for c-Maf in Treg cells exhibit an exacerbated Th17 response and spontaneously develop colitis. However, c-Maf-deficient mice develop fewer polyps in a model of colitis-associated colon cancer. This suggests that c-Maf expression in Treg cells plays a beneficial role on intestinal homeostasis at steady state, but is detrimental in a tumoral context.Our results allow a better understanding of the mechanisms involved in the specialization of intestinal Treg cells. The knowledge of these mechanisms is crucial for the identification of new therapeutic targets in the context of inflammatory bowel disease and colon cancer., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Targeting GARP on human Tregs: a novel approach for the immunotherapy of cancer?

Until now, DNA vaccination against cancer failed to demonstrate a relevant activity in clinic or in a therapeutic setting in preclinical models. This thesis shows two strategies to improve DNA vaccine activity. The first is the optimization of the plasmid itself, by codon optimization and a rational antigen choice. The second is the combination of the vaccines with other complementary therapies: 1) Immune checkpoint blockade in P815 mastocytoma. 2) Oncolytic virus in B16F1 melanoma. 3) Tumor resection in orthotopic GL261 glioblastoma. The common outcome was the dramatic increase in survival in the combination group, explained by the presence of active and antigen-specific T cells in the tumor. Hence, vaccine optimization and rational combined therapies overcome the current limitations of DNA vaccines. This work opens the way to new therapies for the treatment of cancer patients., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

One of the immunosuppressive pathways acting in the tumor microenvironment relies on the degradation of tryptophan (Trp) into kynurenine (Kyn), orchestrated by two distinct enzymes: indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3 dioxygenase (TDO). Local tryptophan depletion combined with kynurenine production strongly impair cellular immunity and make IDO1 and TDO endowing immunosuppressive properties. Like IDO1, TDO is considered as a relevant drug target to improve the efficacy of cancer immunotherapy. However, its role in various immunotherapy settings has not been fully characterized. Here we report that the small molecule inhibitor of TDO (PF06845102/EOS200809) modulates kynurenine and tryptophan concentration in the plasma, liver and tumor and increases the ability of checkpoint inhibitor anti-CTLA4 to induce rejection of CT26 tumors expressing TDO. To better characterize TDO as a therapeutic target, we also used TDO-KO mice. These mice have drastically increased levels of tryptophan in the blood, because they cannot degrade dietary tryptophan. In the MC38 colon carcinoma mouse model, we found that anti-tumor activity of immune checkpoint inhibitors anti-CTLA4 or anti-PD1 was higher in TDO-KO compared to the wild-type mice. Because these MC38 tumors did not express TDO, we related this improved immune response to the high systemic levels of tryptophan in TDO-KO mice. In agreement with this hypothesis, the effect of anti-PD1 was abolished in TDO-KO mice fed with a tryptophan-low diet that normalized their blood tryptophan level. Moreover, treatment of mice with an IDO1 inhibitor improved the efficacy of anti-PD1 in wild-type but not in TDO-KO mice. Since MC38 tumors express IDO1, its immunosuppressive activity limits the efficacy of anti-PD1 treatment in wild-type mice but appears to be overcome in TDO-KO mice due to the high levels of tryptophan. In addition, TDO-KO mice were more resistant to liver metastases than their wild-type counterpart. These res, (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

Targeting GARP on human Tregs: a novel approach for the immunotherapy of cancer?

Les bases scientifiques de l’immunothérapie du cancer.

Les cellules T régulatrices dérivées du thymus (nTreg) sont essentielles pour le maintien de la tolérance périphérique et la prévention des maladies auto-immunes. La caractérisation de ces cellules ainsi que la compréhension de leurs modes d’action sont deux étapes importantes afin de pouvoir les utiliser en thérapie. Nous avons montré que les nTreg sont une population de cellules très hétérogènes. Grâce à deux marqueurs associés au métabolisme de l’ATP, le CD26 et le CD39, et au marqueur de maturation CD45RA, nous avons séparé les nTreg en 5 populations distinctes, chacune représentant un stade de maturation différent. Les facteurs micro-environnementaux dictent cette maturation ainsi que les différentes fonctions des nTreg. En effet, chaque population a une production cytokinique et une activité suppressive qui lui est propre et qui dépend du micro-environnement. De plus, la distribution de ces différentes populations reste stable chez un individu mais elle est variable d’un individu à l’autre. D’un point de vue clinique, nous avons observé une accumulation des cellules mémoires matures et des nTreg « anormaux » CD25- ou CD127+ dans les maladies inflammatoires chroniques. Le profil CD39/CD26 des nTreg pourrait donc représenter un biomarqueur sanguin pour le suivi de ces maladies. La fonction et le nombre des nTreg sont diminués dans de nombreuses maladies auto-immunes et allo-immunes. Le transfert adoptif de ces cellules semble donc être une stratégie thérapeutique prometteuse. Cependant, l’expansion des nTreg est compliquée en raison de leur faible proportion dans le sang et de leur instabilité fonctionnelle dans certaines conditions. La génération d’iTreg stables et suppressifs est donc essentielle. Nous avons décrit un nouveau protocole permettant de générer des iTreg à partir de cellules T naïves, avec une forte activité suppressive peu importe le contexte. L’utilisation du PGE2 et de la rapamycine, en plus du TGFb et de l’IL-2 utilisé classiquement pour génér, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

Les lymphocytes T (LT) CD8+ sont les médiateurs clés de la réponse immunitaire adaptative dirigée contre les pathogènes intracellulaires et les cellules tumorales. Lors d’une infection ou d’une immunisation, ils sont activés en réponse à leur antigène spécifique, prolifèrent et se différencient en cellules effectrices. Les LT CD8+ effecteurs participent à l’élimination du pathogène grâce à leurs fonctions cytotoxiques et à la production de cytokines. Les cellules activées subissent ensuite une phase de contraction à l’issue de laquelle ne subsistera qu’une petite population hétérogène de cellules mémoires qui contribuera à la protection à long terme de l’organisme lors d’infections ultérieures par le même pathogène. Les LT CD8+ peuvent également acquérir un phénotype de cellule mémoire dans des conditions physiologiques en l’absence de reconnaissance antigénique. Ces LT CD8+ mémoires non conventionnels peuvent apparaître dans le thymus (“LT CD8+ mémoires innés thymiques” (TIM) ou en périphérie (“LT CD8+ mémoires virtuels” (VM)). Ces sous-populations de LT CD8+ mémoires conventionnels et non conventionnels peuvent être activés sans reconnaissance antigénique par des cytokines inflammatoires, acquérir des fonctions cytotoxiques et produire des quantités importantes d’IFNγ. Elles contribuent ainsi à la première ligne de défense de l’organisme au même titre que d’autres cellules de l’immunité innée comme les cellules NK, NKT et γδ. Le programme transcriptionnel impliqué dans ces processus de différenciation alternative est mal connu. Eomesodermin (Eomes), un facteur de transcription apparenté à T-bet, joue un rôle central dans l’acquisition du phénotype mémoire et dans la fonction de ces cellules. Cependant, malgré le rôle crucial d’Eomes dans ce contexte, les signaux responsables de son induction et du maintien de son expression ne sont pas clairement établis. Dans ce travail, nous avons montré que les voies de signalisation induites par les interférons (IFN) de type I, Doctorat en Sciences médicales (Médecine), info:eu-repo/semantics/nonPublished

DNA vaccination against cancer has become a promising strategy for inducing a specific and long-lasting antitumor immunity. However, DNA vaccines fail to generate potent immune responses when used as a single therapy. To enhance their activity into the tumor, a DNA vaccine against murine P815 mastocytoma was combined with antibodies directed against the immune checkpoints CTLA4 and PD1. The combination of these two strategies delayed tumor growth and enhanced specific antitumor immune cell infiltration in comparison to the corresponding single therapies. The combination also promoted IFNg, IL12 and granzyme B production in the tumor microenvironment and decreased the formation of liver metastasis in a very early phase of tumor development, enabling 90% survival. These results underline the complementarity of DNA vaccination and immune checkpoint blockers in inducing a potent immune response, by exploiting the generation of antigen-specific T cells by the vaccine and the ability of immune checkpoint blockers to enhance T cell activity and infiltration in the tumor. These findings suggest how and why a rational combination therapy can overcome the limits of DNA vaccination but could also allow responses to immune checkpoint blockers in a larger proportion of subjects.

The DA strain of Theiler’s virus has a remarkable ability to evade the immune response of the host and to cause persistent infections of the central nervous system. During the chronic phase of infection, the virus is primarily detected in microglial cells, infiltrating macrophages and oligodendrocytes in the white matter of the spinal cord. The non-structural L* protein encoded by Theiler's virus was shown to enhance viral replication in macrophages in vitro and to be required for the establishment of persistent infections. Previous work in our lab revealed that L* directly interacts with RNase L and thereby inhibits the activity of this antiviral enzyme. Interestingly, RNase L antagonism by L* is a highly species-specific process; indeed, L* inhibits mouse RNase L but not its orthologues from other tested species. On the one hand, we characterized further the molecular mechanisms of RNase L antagonism by the Theiler’s virus L* protein. We identified a novel strategy of RNase L inhibition in which, upon binding to ankyrin repeats 1 and 2 of the mouse RNase L, L* competitively inhibits 2-5A binding to RNase L. Using chimeric MHV viruses expressing L*, we demonstrated that L* is able to counteract RNase L activity in vivo, in infected mice. On the other hand, we took advantage of the species-specificity of L* activity to identify the natural host of Vilyuisk human encephalitis virus (VHEV). VHEV was isolated from mice that were inoculated with CSF from a patient suffering from chronic encephalitis. It is therefore unclear whether the virus derives from the human sample or from the mice used to isolate the virus. We observed that L* of VHEV specifically binds to and inhibits mouse but not human RNase L, thereby showing that VHEV is of mouse origin and suggesting that it is a contaminant that originated from the mice that were used to inoculate the human sample., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2017

While patient selection and clinical management have reduced high-dose IL-2 (HDIL2) immunotherapy toxicities, the immune mechanisms that underlie HDIL2-induced morbidity remain unclear. Here we show that dose-dependent morbidity and mortality of IL-2 immunotherapy can be modeled in human immune system (HIS) mice. Depletion of human T cell subsets during the HDIL2 treatment reduces toxicity, pointing to the central function of T cells. Preferential expansion of effector T cells secondary to defective suppressive capacity of regulatory T (Treg) cells after HDIL2 therapy further underscores the importance of Treg in the maintenance of immune tolerance. IL-2 toxicity is induced by selective depletion or inhibition of Treg after LDIL2 therapy, and is ameliorated in HDIL2-treated HIS mice receiving the PIM-1 kinase inhibitor, Kaempferol. Modeling IL-2 pathophysiology in HIS mice offers a means to understand the functions of effector and regulatory T cells in immune-mediated toxicities associated with cancer immunotherapy.

Cytotoxicity is the capacity for immune cells to kill infected or malignant cells in order to eliminate pathogens and tumours through different mechanisms including the exocytosis of perforin-containing cytosolic granules. This crucial property is usually restricted to specialized innate and adaptive lymphocytes such as natural killer (NK) cells and CD8 T cells. T lymphocytes differentiate in the thymus and are delivered to the peripheral blood as naive T cells committed to either the CD8 or the CD4 lineage. CD8 T cells are programmed to acquire cytotoxic effector functions under the control of the transcription factor (TF) Runx3. The fate of CD4 T cells is to acquire multiple helper functions through the action of the TF ThPOK that promotes CD4 helper functions and restricts the CD8 cytotoxic program. However, this restriction is not absolute as cytotoxic CD4 (CD4CTX) T cells differentiate in vivo, indicating that the multipotency of human naive CD4 T cells includes the ability to acquire perforin expression and potent cytotoxicity in vitro and ex vivo. This cytotoxic potential correlates with outcome in human pathology and mediates protection against viral challenge and tumour eradication in murine models. CD4CTX T cells are terminally differentiated effector memory T cells that accumulate during cytomegalovirus chronic infection and ageing. They are phenotypically and functionally related to T helper type 1 (Th1)-effector memory cells. However, whether they belong to the Th1 pathway or constitute a separate specialized helper T cell subset is unknown. In this work, we show that CD4CTX T cell differentiation is an integral part of the Th1 pathway. Indeed, CD4 T cells acquire cytotoxic potential early in the memory differentiation process as central memory Th1 but not Th2 and Th17 cells are epigenetically primed to develop a cytotoxic program. The expression of perforin and other cytotoxic genes present a stepwise increase profile that is specific of the Th1 differ, Doctorat en Sciences médicales (Médecine), info:eu-repo/semantics/nonPublished

Des études récentes menées chez l’homme et la souris ont suggéré que la diversité du répertoire TCR pourrait jouer un rôle dans la protection contre des pathogènes à haut pouvoir mutagène. Afin d’étudier le répertoire des lymphocytes T CD4, nous avons utilisé un modèle de souris TCRβ transgéniques exprimant une chaine β spécifique du peptide env122-141 dans le contexte du MHCII. Suite à l’immunisation des souris TCRβ transgéniques avec des cellules dendritiques pulsées avec le peptide env, une rapide prolifération et une restriction du répertoire des lymphocytes T Vα2 CD4 spécifiques est observée. L’analyse de la diversité du répertoire de ces cellules par séquençage à haut débit, a montré l’émergence d’un répertoire plus divers dans des souris déplétées en lymphocytes T régulateurs. Ces résultats suggèrent qu’en plus du rôle des Tregs dans le contrôle de la magnitude de la réponse immunitaire, ces cellules pourraient également contrôler la diversité du répertoire des lymphocytes T suite à une stimulation antigénique., Recent studies conducted in mice and humans have suggested a role for the TCR repertoire diversity in immune protection against pathogens displaying high antigenic variability. To study the CD4 T cell repertoire, we used a mouse model in which T cells transgenically express the TCRβ chain of a TCR specific to a MHCII-restricted peptide, env122-141. Upon immunization with peptide-pulsed dendritic cells, antigen-specific Vα2+ CD4+ T cells rapidly expand and display a restricted TCRα repertoire. In particular, analysis of receptor diversity by high-throughput TCR sequencing in immunized mice suggests the emergence of a broader CDR3 Vα2 repertoire in Treg-depleted mice. These results suggest that Tregs may play a role in the restriction of the CD4 T cell repertoire during an immune response, raising therefore the possibility that in addition to controlling the magnitude of an immune response, regulatory cells may also control the diversity of TCRs in response to antigen stimulation., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

PURPOSE: Autologous monocyte-derived dendritic cells (DCs) electroporated with synthetic mRNA (TriMixDC-MEL) are immunogenic and have antitumor activity as a monotherapy in patients with pretreated advanced melanoma. Ipilimumab, an immunoglobulin G1 monoclonal antibody directed against the cytotoxic T-lymphocyte-associated protein 4 receptor that counteracts physiologic suppression of T-cell function, improves the overall survival of patients with advanced melanoma. This phase II study investigated the combination of TriMixDC-MEL and ipilimumab in patients with pretreated advanced melanoma. PATIENTS AND METHODS: Thirty-nine patients were treated with TriMixDC-MEL (4 × 10(6) cells administered intradermally and 20 × 10(6) cells administered intravenously) plus ipilimumab (10 mg/kg every 3 weeks for a total of four administrations, followed by maintenance therapy every 12 weeks in patients who remained progression free). Six-month disease control rate according to the immune-related response criteria served as the primary end point. RESULTS: The 6-month disease control rate was 51% (95% CI, 36% to 67%), and the overall tumor response rate was 38% (including eight complete and seven partial responses). Seven complete responses and one partial tumor response are ongoing after a median follow-up time of 36 months (range, 22 to 43 months). The most common treatment-related adverse events (all grades) consisted of local DC injection site skin reactions (100%), transient post-DC infusion chills (38%) and flu-like symptoms (84%), dermatitis (64%), hepatitis (13%), hypophysitis (15%), and diarrhea/colitis (15%). Grade 3 or 4 immune-related adverse events occurred in 36% of patients. There was no grade 5 adverse event. CONCLUSION: The combination of TriMixDC-MEL and ipilimumab is tolerable and results in an encouraging rate of highly durable tumor responses in patients with pretreated advanced melanoma.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS), for which current treatments are unable to prevent disease progression. Based on its neuroprotective and neuroregenerating properties, leukemia inhibitory factor (LIF), a member of the interleukin-6 (IL-6) cytokine family, is proposed as a novel candidate for MS therapy. However, its effect on the autoimmune response remains unclear. In this study, we determined how LIF modulates T cell responses that play a crucial role in the pathogenesis of MS. We demonstrate that expression of the LIF receptor was strongly increased on immune cells of MS patients. LIF treatment potently boosted the number of regulatory T cells (Tregs) in CD4+ T cells isolated from healthy controls and MS patients with low serum levels of IL-6. Moreover, IL-6 signaling was reduced in the donors that responded to LIF treatment in vitro. Our data together with previous findings revealing that IL-6 inhibits Treg development, suggest an opposing function of LIF and IL-6. In a preclinical animal model of MS we shifted the LIF/IL-6 balance in favor of LIF by CNS-targeted overexpression. This increased the number of Tregs in the CNS during active autoimmune responses and reduced disease symptoms. In conclusion, our data show that LIF downregulates the autoimmune response by enhancing Treg numbers, providing further impetus for the use of LIF as a novel treatment for MS and other autoimmune diseases.