Unit: Antiviral immunity, Biotherapy and Vaccines 

Director: GOUGEON Marie-Lise

Our research activity is focused on the study of mechanisms involved in CD4 T cell homeostasis in HIV-infection, and in particular we analyse viral strategies involved in the destruction by apoptosis of both infected and non-infected CD4 T cells. We are also involved in the study of innate effectors, such as γ T lymphocytes and NK cells, and we focus on the expression and the role of NK receptors on T lymphocyte subsets to understand how they modulate antiviral cytotoxic activity. Finally, our team is involved for several years in immunological evaluation of IL-2 immunotherapy trials in order to determine the impact of this cytokine on quantitative and qualitative restoration of the CD4 T cell compartment in HIV-infected persons.

1- Molecular bases of HIV-triggered T lymphocyte apoptosis (Eric Ledru, Luzia de Oliveira-Pinto, Elena Ritsou)

We have discovered ten years ago that HIV induces apoptosis not only in infected CD4 T cells but also in non infected cells, and we were thus interested in determining the molecular bases of this phenomenon and its conseqences on HIV-specific immunity. We have shown that apoptosis of bystander cells is the consequence of the chronic stimulation of the immune system by HIV proteins persistently expressed, which activate apoptotic programmes involving both death receptors and the mitochondria. CD95, TNF-RI and TNF-RII play an important role in the induction of apoptosis in lymphocytes from HIV-infected persons, their expression is upregulated in both CD4 and CD8 T cells and their ligation by agonistic antibodies or their ligands induces ex-vivo a strong apoptotic signal. The contribution of these receptors to the destruction of CD4 T cells is suggested by the correlation between the level of death-receptor-induced apoptosis and the ex-vivo number of CD4 T cells.

The study of the molecular bases of TNF-RI and TNF-RII-induced apoptosis in patients'lymphocytes indicates that it is independent of the in vivo expression of adaptor proteins TRAAD, FLICE and RIP, while it is associated with the negative regulation of Bcl-2 and in vivo activation of Caspases-3 and -8 (de Oliveira Pinto et al. Blood 99 :1666, 2002). We have also recently reported that a variant of IL-4 ((IL-4δ13) is strongly associated with deprivation apoptosis in primary lymphocytes and T cell clones as well, and its expression is increased in HIV-dependent apoptosis (Ledru et al. Blood, 101 :3102, 2003). The functional consequences of premature apoptosis in T lymphocytes from HIV-infected patients are the destruction of CD8 T cells with CTL phenotype (CD45R0+, perforine+, Bcl-2-) and that of CD4 T helper cells, particularly the IL-2-producers, IL-2 being essential for the différentiation of HIV-specific effectors. The proapoptotic mechanisms triggered by HIV and the consequences on T cell homeostasis and antiviral immunity have been discused recently (ML Gougeon, Nature Review Immunology, 3 :392,2003)

2- Role of NKR on the control of antiviral cytotoxic activity and modulation by cytokines (Isabelle Liberman, Delphine Marsac in collaboration with Pr. René Roué and Dr. Thierry Debord, Hôpital des Armées, Bégin).

Cytotoxic activity of Natural Killer cells is controlled by activating receptors (KARs) and inhibiting receptors (KIRs). KIRs ligands are MHC class-I molecules and, in the presence of a normal cell, the positive signal transmitted by the KAR is inhibited by the negative signal delivered by the KIR, preventing the destruction of the normal cell by NK. Tumor cells or viral-infected cells may lack expression of MHC class-I molecule while expressing KAR ligands, leading to the activation of the cytolytic activity of NK cells and further destruction of the target tumor cell.

Some viruses have developped strategies to protect infected cells from cytotoxic lymphocytes, one of them being to modulate KIRs expression. We have shown that KIRs are not only expressed by NK cells but also by γ T lymphocytes and αβ CD4 and CD8 T lymphocytes. In HIV-infected persons, KIRs expression is increased on the surface of NK cells, CD4 and CD8 T cells, and it is positively correlated with the viral load and with disease evolution. Functional regulation of cytotoxic T cells by exogenous cytokines (IL-10, IL15 and TGFß) and by KIRs can be studied following short-term stimulation and multiparametric FACS analysis. We thus showed that IL-15 decreases KIR expression on CD4 and CD8 T lymphocytes from HIV-infected persons, restoring the cytotoxic activity of these cells and the production of TNF-α and IFN-γ. We continue to characterize regulatory mechanisms of antiviral cytotoxic activity.

3- T cell dynamics and antiviral immunity in HIV+ patients receiving IL-2 (Peggy Masdehors, Corinne Guitton, Sylvie Rouyre, Béatrice Poirier-Baudoin in collaboration with Pr. Yves Levy, Hôpital Henri Mondor, Créteil)

Combined antiretroviral therapy inhibiting HIV reverse transcriptase and protease induces a clinical benefit in a large fraction of chronically HIV-infected patients, while reducing the plasmatic viral load to indetectable levels and restoring the level of CD4 T cells. However, because these potent therapies show severe adverse effects, including metabolic complications (lipodystrophy syndrome, hyperlipidaemia, diabetes), new therapeutic strategies are required to spare antiretroviral regimens. For example, antiretroviral therapy has been associated with IL-2 immunotherapy to restore qualitatively and quantitatively the pool of CD4 T lymphocytes.

With the aim to better understand the mechanisms whereby IL-2 induces a significant increase in CD4 T cells in HIV+ patients, we have developped several methodological approaches to detect ex-vivo recent thymic emigrants (TREC), to identify lymphocytes submitted to homeostatic proliferation through the detection of Ki67 molécule, and to identify CD4 and CD8 T cell subsets programmed for apoptosis. In parallel, CD4 helper cells and CD8 cytotoxic cells are detected following a short term stimulation with HIV peptides and multiparametric FACS analysis. We have thus shown that IL-2 immunotherapy allows the long-term preservation of HIV-specific CD4 effectors, their frequency being significantly increased compared to the one found in patients receiving antiretroviral drugs alone. In addition, we have shown that the dynamics of CD4 T cells is modified in the presence of IL-2, this cytokine increasing lymphocyte survival and inducing their peripheral proliferation. The possible influence of IL-2 immunotherapy on thymic regeneration is actually evaluated.

Keywords: Apoptosis, Innate immunity, HIV, IL-2, immunotherapy

Activity Reports 2003 - Institut Pasteur

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