Etude comparative de différents vaccins codant pour la protéine non structurale 3 (ns3) du virus de l'hépatite C

L'infection par le virus de l'Hépatite C conduit fréquemment à une hépatite chronique pouvant évoluer vers un carcinome hépatocellulaire et n'est soignable que dans 40-50% des cas. Le développement d'un vaccin s'avère donc nécessaire mais se heurte à la méconnaissance des corrélats immunitaires de protection et à l'impossibilité d'utiliser des vaccins conventionnels du fait de l'absence de systèmes de réplication disponibles in vitro et in vivo. L'objectif de cette thèse a été d'évaluer à partir de vaccins génétiques et de vecteurs viraux, l'immunogénicité de la protéine non structurale 3 (NS3), impliquée dans la réplication mais également dans l' élimination virale au cours de l'infection naturelle. Pour cela, l'induction d'une réponse cellulaire médiée par les lymphocytes T CD8+ dirigée contre des épitopes décrits dans l'infection naturelle a été testée dans un modèle de souris transgéniques pour la molécule HLA-A2.1. Les vaccins génétiques testés avaient pour objectifs : 1) d'exprimer NS3 sous le contrôle du promoteur du cytomégalovirus (pCI.NS3) ou du réplicon du virus de la forêt de Semliki (SFV) (pSFV.NS3) ou 2) de la cibler au niveau des compartiments riches en molécules de classe II (pLAMP.NS3). Nos résultats ont montré que ces vecteurs, quelle que soit leur nature, ont conduit à l'induction de réponses spécifiques de longue durée mais restreintes à un épitope (sur 4) (aa 1073-1081), la réponse la plus vigoureuse étant obtenue avec le vecteur pCI.NS3. L'expression de la protéine NS3 sous forme de particules virales dérivées du SFV (rSFV.NS3) injectées seules ou en combinaison avec pCI.NS3 n'ont ni permis d'élargir, ni d'accroître la vigueur de cette réponse. La présence du plasmide pCI.NS3 dans le contexte d'une vaccination combinée avec des protéines recombinantes réalisée chez le chimpanzé semble, néanmoins, confirmer un rôle protecteur de la réponse anti-NS3. Globalement, l'ensemble de nos travaux indiquent que de nouvelles stratégies vaccinales doivent être développées afin de potentialiser la réponse anti-NS3.LYON1-BU Santé (693882101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Acute Myeloid Leukemia: Is It T Time?

Acute myeloid leukemia (AML) is a heterogeneous disease driven by impaired differentiation of hematopoietic primitive cells toward myeloid lineages (monocytes, granulocytes, red blood cells, platelets), leading to expansion and accumulation of “stem” and/or “progenitor”-like or differentiated leukemic cells in the bone marrow and blood. AML progression alters the bone marrow microenvironment and inhibits hematopoiesis’ proper functioning, causing sustained cytopenia and immunodeficiency. This review describes how the AML microenvironment influences lymphoid lineages, particularly T lymphocytes that originate from the thymus and orchestrate adaptive immune response. We focus on the elderly population, which is mainly affected by this pathology. We discuss how a permissive AML microenvironment can alter and even worsen the thymic function, T cells’ peripheral homeostasis, phenotype, and functions. Based on the recent findings on the mechanisms supporting that AML induces quantitative and qualitative changes in T cells, we suggest and summarize current immunotherapeutic strategies and challenges to overcome these anomalies to improve the anti-leukemic immune response and the clinical outcome of patients

Costimulatory effects of IL-1 on the expansion/differentiation of CD4+CD25+Foxp3+ and CD4+CD25+Foxp3– T cells

CD4+CD25+forkhead box p3 (Foxp3)+ regulatory T cells (Treg) control peripheral tolerance. Although Treg are anergic when stimulated through the TCR, mature bone marrow-derived, but not splenic, dendritic cells (DC) can induce their proliferation after TCR stimulation in the absence of IL-2. One possibility is that the DC produce proinflammatory cytokines such as IL-1 or IL-6 that function as growth factors for Treg. We have analyzed the costimulatory effects of IL-1 on the expansion of Foxp3+ Treg in vitro. When CD4+CD25+ T cells were cultured in the presence of splenic DC and IL-1, marked expansion of the Foxp3+ T cells was observed. The effects of IL-1 were mediated on CD4+CD25+Foxp3– T cells present in the starting population rather than on the DC or on the CD4+CD25+Foxp3+ T cells. In contrast, stimulation of CD4+CD25+ T cells with plate-bound anti-CD3 and IL-1 in the absence of DC resulted in the outgrowth of a CD4+CD25+Foxp3– T cell population composed of NKT cells and non-NKT, IL-17-producing cells. Foxp3+ Treg purified from mice expressing the reporter gene enhanced GFP in the Foxp3 locus failed to proliferate when costimulated with IL-1. These findings have important implications for the design of protocols for the expansion of CD4+CD25+ T cells for cellular biotherapy

Pairing cells of different sizes in a microfluidic device for immunological synapse monitoring

International audienceAnalyzing cell–cell interaction is essential to investigate how immune cells function. Elegant designs have been demonstrated to study lymphocytes and their interaction partners. However, these devices have been targeting cells of similar dimensions. T lymphocytes are smaller, more deformable, and more sensitive to pressure than many cells. This work aims to fill the gap of a method for pairing cells with different dimensions. The developed method uses hydrodynamic flow focusing in the z-direction for on-site modulation of effective channel height to capture smaller cells as single cells. Due to immune cells' sensitivity to pressure, the proposed method provides a stable system without any change in flow conditions at the analysis area throughout experiments. Paired live cells have their activities analyzed with calcium imaging at the immunological synapse formed under a controlled environment. The method is demonstrated with primary human T lymphocytes, acute myeloid leukemia (AML) cell lines, and primary AML blasts

Regulatory CD4+CD25hi T cells conserve their function and phenotype after granulocyte colony-stimulating factor treatment in human hematopoietic stem cell transplantation.

International audienceAcute graft-versus-host disease (aGVHD), mediated by CD4(+) and CD8(+) effector T cells, is a life-threatening complication in hematopoietic stem cell transplantation. CD4(+)CD25(hi) regulatory T cells (T(reg)) have been shown to modulate tolerance to aGVHD in murine models. Based on these observations, we examined their role in the prevention of aGVHD in patients who underwent transplantation with peripheral blood-mobilized hematopoietic stem cells after administration of granulocyte colony-stimulating factor. The effects of the G-CSF on the phenotype, frequency, and function of CD4(+)CD25(hi) T cells were analyzed in grafts and after transplantation to determine whether these cells were regulatory T cells. CD4(+)CD25(hi) T cells could be detected at the same frequency before and after granulocyte colony-stimulating factor administration in the donors' peripheral blood. The isolation of these cells from the grafts or from the recipients' peripheral blood after transplantation revealed that they were suppressive to the same extent as T(reg) isolated from healthy volunteers. Their number and frequency were estimated in the grafts and the results indicated that protection against aGVHD was not dependent on the T(reg) amount transferred to the recipients. Similarly there was no correlation between the number of circulating CD4(+)CD25(hi) T cells in the recipients' peripheral blood during the early period after transplantation and the outcome of aGVHD

Patients suffering from acute graft-versus-host disease after bone-marrow transplantation have functional CD4+CD25hiFoxp3+ regulatory T cells.

International audienceAcute Graft-Versus-Host Disease (aGVHD), mediated by CD4(+) and CD8(+) effector T cells, is a life-threatening complication in hematopoietic stem cell (HSC) transplantation. Naturally-occurring CD4(+)CD25(hi)(Foxp3(+)) regulatory T cells (T(reg)) have been shown to modulate tolerance to aGVHD in murine graft models. In this report, we investigated their role in the prevention of aGVHD in patients transplanted with bone-marrow-derived HSC. When CD4(+)CD25(hi)Foxp3(+) T cells were isolated from bone-marrow grafts, they showed no suppressive activity. The analysis of their function in patients suffering from aGVHD after transplantation revealed a gain of suppressive activity indicating their inability to control the aGVHD induction. Thus, our findings clearly demonstrate that CD4(+)CD25(+) and CD4(+)CD25(hi)Foxp3(+) T cells, when administered in steady-state physiological conditions, do not influence the outcome of aGVHD after bone-marrow transplantation

Modulation of the TCR stimulation strength can render human activated CD4+ T cells suppressive.

International audienceIn this study, we explored the potential of human naive CD4(+) T cells to acquire regulatory properties upon stimulation. We demonstrated that, in vitro, pre-activated naive CD4(+)CD25(-)CD45RA(+) T cells could become anergic and suppressive CD4(+)CD25(+) T cells upon lower intensity TCR stimulation. These CD4(+)CD25(+) T cells generated in vitro potently suppress the proliferation of allogenic CD4(+)CD25(-) T cells independently of cytokines and in a contact-dependent manner. Our data indicate that expression of Foxp3 is not necessary to induce the suppressive T cell activity. We demonstrate that these CD4(+)CD25(+) T cells are unresponsive upon re-stimulation and that their suppressive activity is transient. However, we showed that the anergy and the suppressive function could be reversed by increasing the stimulus and their level of activation. We concluded from our data that these anergy and suppressive activities are related to a fine tuning of TCR activation threshold