Unit: Cellular Immunology and Immunogenetics

Director: THEZE Jacques

Infection by HIV leads progressively to marked immune deficiency. A gradual reduction in the number and function of CD4 T lymphocytes dominates the pathophysiology of HIV infection. This deficiency has a number of causes: depletion of CD4 lymphocytes by viral infection, abnormal activation of the immune system and diminished specific responses against the virus. The Unit study the cellular and molecular mechanisms that cause these dysfunctions, with efforts focused on the investigation of two cytokines (IL-2 and IL-7). These play central roles in the function of the immune system and are implicated in the pathogenesis of HIV infection. Our studies also aim to evaluate the therapeutic potential of these two cytokines. In this context we are studying the mechanism of action of IL-2 on different lymphocyte sub-populations taken from healthy volunteers and on precursors of regulatory lymphocytes isolated from patients receiving this cytokine as treatment.

1) Molecular basis of T-lymphocyte dysfunction in HIV infection (Florence Bugault, Olivier Juffroy, Jean-Louis Moreau and Jean-Hervé Colle in collaboration with J.-F. Delfraissy, Centre Bicêtre, Assistance Publique-Hôpitaux de Paris)

A study evaluating the responsiveness of CD4 and CD8 lymphocytes from HIV-infected patients showed that marked functional abnormalities appear soon after initial infection. To characterize these abnormalities we are exploring the expression and the function of IL-2 and IL-7 receptors in different groups of HIV-infected patients.

IL-2 is a major cytokine of the immune system and is under expressed in HIV-infected patients. By analyzing the expression of the three chains that make up its receptor (IL-2R) and lymphocyte subpopulation responses to IL-2, we were able to identify several dysfunctions of this system in infected patients. We found that although CD4 lymphocytes express the IL-2R, they no longer respond to IL-2. More recently, during the longitudinal follow-up of a group of patients, we used CD8 T lymphocytes to analyze the transduction signals induced by IL-2, and found that purified CD8 lymphocytes from these viremic patients were unable to activate the Jak/STAT signaling pathway. Our study therefore provided a new description of the molecular abnormalities that characterize the lymphocytes of HIV-infected patients.

IL-7 is a crucial cytokine which controls both the thymopoiesis and the homeostasis of peripheral T lymphocytes. We undertook a study of the IL-7 system and its receptor in HIV+ patients and noted that serum IL-7 increased in viremic patients in relation to the magnitude of CD4 lymphopenia, indicative of an IL-7/ CD4 regulatory loop. CD4 lymphocyte expression of the chain specific to the IL-7 receptor (IL-7R alpha or CD127) was close to normal, but its function was altered. This resulted in significantly less IL-7-induced proliferation and reduced induction of the anti-apoptotic molecule Bcl-2. Studies in CD8 lymphocytes showed a very substantial decrease in the expression of IL-7R, more particularly in memory CD8 lymphocytes that are responsible for long-term maintenance of cytotoxic function.

Our results have demonstrated that HIV infection causes the dysfunction of several cytokine systems and their signaling pathways. On a molecular level the mechanisms that could possibly explain these effects are currently the subject of many studies. The HIV glycoprotein envelope (gp120) found in the serum and lymph nodes of HIV-positive patients may play a role in these alterations. Abnormal and prolonged activation of the immune system in these patients could also disorganize their lymphocyte signaling pathways. Our current studies should contribute to a more precise definition of these abnormalities and a deeper understanding of their origins.

2) Effects of anti-retroviral (ARV) therapy and therapeutic potential of IL-2 and IL-7 in HIV+ patients (Florence Bugault, Jean-Louis Moreau and Jean-Hervé Colle. In collaboration with J.-F. Delfraissy, Centre Bicêtre, Assistance Publique-Hôpitaux de Paris and M. Morre, Cytheris S.A. Vanves).

From a certain stage in the course of their disease, the treatment of HIV-infected patients requires the administration of ARV drugs. In a large proportion of these patients, this type of treatment has proven to be effective both in controlling viral load and in restoring the CD4 count. However, the treatment fails to eradicate the virus and does not restore effective antiviral responses. Also, ARV must be taken life-long and unwanted effects can be severe. The discovery of new therapeutic strategies therefore remains a priority and the complementary use of cytokines such as IL-2 or IL-7 paves the way toward new treatments.

We confirmed that the function of the IL-2R and IL-7R is partly restored after prolonged ARV treatment. Interestingly, we observed that signaling defects in the Jak/STAT pathway - which is shared by the IL-2 and IL-7 receptors - were reversed after six months of ARV treatment. This provided a molecular basis justifying the therapeutic combination of ARV with cytokines such as IL-2 or IL-7.

Several clinical trials have already been conducted with IL-2 in patients receiving ARV. These trials demonstrated that IL-2 is able to induce a sustainable increase in the CD4 count, and particularly the naive CD4 cell count. A clinical trial of this type was conducted with a group of patients who, when receiving ARV alone, showed adequate control of their viral load but a low CD4 count (CD4 < 200 cells/mm3) recovery. Here we observed that IL-2 rapidly induced the appearance of a new CD4+/CD25+ lymphocyte subpopulation. The appearance of this new lymphocyte population would appear to be a major effect of treatment with IL-2, as shown by C. Lane (NIH, USA). The mechanisms by which IL-2 induces the emergence of this subpopulation remain to be established. We shall continue with the characterization of this CD4+/CD25+ lymphocyte subpopulation in connection with GPH HIV/AIDS (see below) Also, we have shown that IL-2 rapidly increases the plasma levels of two thymopoietic cytokines, IL-7 and Flt-3L. This suggests that IL-2 immune therapy also induces the production of lymphocytes at a central level.

Given its lymphopoietic properties, IL-7 has a promising therapeutic potential and in immune deficient patients should broaden the T-lymphocyte repertoire and increase their specific responses. We conducted a longitudinal study of plasma IL-7 levels in a group of HIV+ patients before and during treatment with ARV. A positive correlation was demonstrated between IL-7 levels prior to treatment and the magnitude of CD4 restoration in the course of ARV treatment. This suggests that IL-7 is involved in controlling the CD4 lymphocyte compartment in HIV-positive patients. This study also indicated that IL-7 treatment in patients receiving ARV should promote CD4 restoration. Since the response to IL-7 is in part regulated by negative feedback from its own receptor, this type of regulation and its reversibility are currently under study to predict the effects of its in vivo administration. Also, we studied the effect of ARV on the normal function of the IL-7R.

3) IL-2 Receptor: demonstration of a new form, signal transduction mechanisms and differential responses of lymphocyte subpopulations (Virginie Mazard-Pasquier, Jean-Louis Moreau, Anne-Hélène Pillet and Thierry Rose)

Three chains, alpha, beta and gamma, enter into the composition of the IL-2 receptor (IL-2R). The level at which each chain is expressed depends on the lymphocyte subtype. Two functional forms of IL-2R have already been described: assembly of all three chains produces a high affinity receptor (Kd = 10pM) whereas combination of the beta and gamma chains forms a receptor of far lower affinity (Kd = 1nM). Our recent results suggest that in the absence of the gamma chain a third form of IL-2R can be formed, made up of two beta chains. In confirmation of this, the construction then the expression of hybrid beta chains carrying HA or Myc epitopes enabled us to demonstrate the existence of beta/beta dimers by co-immunoprecipitation. Likewise, the expression of hybrid beta chains fused with fluorescent proteins derived from GFP (beta-CFP or beta-YFP) allowed us to study IL-2Rbeta/IL-2Rbeta interactions on the surface of transfected COS and HEK cells. We used confocal microscopy to analyze the fluorescence generated by the energy transfer (FRET) from one subunit to the other after irradiation. At the same time we produced models of beta/beta associations and propose that this complex is as stable as the IL-2Rbeta/IL-2Rgamma heterodimer previously described.

NK lymphocytes respond to IL-2 both spontaneously and very intensely. IL-2R beta chains play a critical role in the early activation of NK lymphocytes. Given that these cells express a large number of beta chains on their surface, but few gamma chains, we assume that the beta dimers identified play a physiological role here at the NK cell surface. We have explored the signaling pathways activated by IL-2R in NK lymphocytes and have isolated the signaling complex used by these lymphocytes after stimulation by IL-2. This complex, which is made up of IL-2Rbeta chains and JAK3 and STAT5 kinases, does not include an IL-2R gamma chain, demonstrating that IL-2Rbeta dimers are functional in NK lymphocytes. (in collaboration with S. Pellegrini).

In order to respond to IL-2, T lymphocytes - unlike NK cells - need to be co-activated by a signal from their specific receptor (TCR). The role played by this co-activation has not yet been understood but it may control the expression of high-affinity IL-2R or affect the steps which control the cell activation and division that characterize the response to IL-2. To decide between these two hypotheses, we undertook a study of transgenic mice that constitutively express the human IL-2R beta chain. Their CD4 lymphocytes respond to IL-2 by phosphorylation of STATs and MAPKs but they fail to proliferate because the cell division inhibitor p27kipl continues to be expressed at a high level after exposure to IL-2. Lymphocyte proliferation in response to IL-2 is acquired only after repression of p27kipl, obtained after TCR stimulation. By contrast, direct inhibition by IL-2 of p27kipl expression in the CD8 lymphocytes of transgenic mice triggers proliferation of cytotoxic lymphocytes in the absence of co-stimulation by TCR. The expression of IL-2R is therefore sufficient to determine the responsiveness of cytotoxic T lymphocytes to IL-2. By contrast, as far as CD4 lymphocytes are concerned, this competence is acquired only after engagement of the antigen's specific receptor (in collaboration with F. Gesbert).

4) Grand Programme Horizontal HIV/AIDS: "Gene expression and signaling defects in the pathogenesis of HIV infection"

Six units and three platforms in different departments of the Pasteur Institute, along with two clinical departments of the Assistance Publique - Hôpitaux de Paris, are involved in this project which is organized as three sub-programs. This GPH aims to analyze - in CD4 lymphocytes - the genes and the transduction signals involved in patient resistance to AIDS or susceptibility to the development of full-blown AIDS. It also aims to characterize the mechanism of action of treatment with IL-2 in HIV+ patients receiving ARV. It is steered by a committee made up of M. Muller-Trutwin, L. Chakrabarti, O. Acuto. L. Rogge and F. Arenzana-Seisdedos. Prof. Jacques Thèze is coordinating the work. Our unit is directly implicated in two sub-programs. We are studying the central memory CD4 lymphocytes of infected patients who are able to control their viral load spontaneously ("HIV Controllers", recently characterized by O. Lambotte and J.-F. Delfraissy). These studies are being conducted by S. Potter (grant from the Australian government) and by L. Chakrabarti. Also, work has been undertaken with the Immune Regulation Laboratory (L. Rogge) to investigate the mechanism of CD4 restoration after combined treatment with ARV and IL-2 by associating characterization of a purified CD4+/CD25+ population with a study of functional genetics. The patients are drawn from study ANRS 118 (Prof.  Y. Levy)

Keywords: HIV, CD4 lymphocytes, IL-2, IL-2R, IL-7, IL-7R, receptors and signal transduction, immunotherapy

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