Unit: Cellular Immunology and Immunogenetics
Director: THEZE Jacques
Infection by HIV leads progressively to marked immune deficiency, and the Unit has undertaken to analyse the molecular basis of this dysfunction. In the treatment of this infection, it may be possible to add immune therapy to the therapeutic arsenal, bolstering the AntiRetroVirals (ARV) currently employed. With this in view, we are exploring the therapeutic potential of interleukin-2 (IL-2) and interleukin-7 (IL-7) and on a biochemical level are studying the mechanism of action of IL-2 on different forms of receptor expressed by lymphocyte subpopulations.
1) Molecular basis of T lymphocyte dysfunction in HIV infection (Florence Bugault, Stéphanie Beq, Olivier Juffroy, Jean-Louis Moreau and Jean-Hervé Colle in collaboration with J.-F. Delfraissy, Centre Bicêtre, Assistance Publique-Hôpitaux de Paris)
A gradual reduction in the number CD4 T lymphocytes dominates the pathophysiology of HIV infection. Moreover, the study of T lymphocyte reactivity has highlighted major functional abnormalities. To characterize these abnormalities we are exploring the roles played by IL-2 and IL-7 in different groups of HIV-positive patients.
IL-2 is a major activating cytokine of the immune system. Analyzing the expression of the three chains that make up its receptor (IL-2R) and also the IL-2 reactivity of lymphocyte subpopulations, enabled us to identify major dysfunctions of the IL-2/IL-2R system in HIV+ patients. More recently, during the longitudinal follow-up of a group of patients, we used CD8 T lymphocytes to analyze IL-2 transduction signals. At the start of the study, purified CD8 lymphocytes from viremic patients were unable to activate the Jak/STAT signalling pathway. Interestingly, this defect in the Jak/STAT pathway was reversed after six months of ARV treatment. Our study therefore furnished new data concerning the nature of the molecular abnormalities that characterize the lymphocytes of HIV+ patients.
IL-7 is a crucial cytokine that 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. Viremic patients show abnormalities in the expression of the chain specific to the IL-7 receptor (IL-7R alpha), and we also discovered that IL-7 induces significantly less proliferation in these patients. Likewise, induction of the anti-apoptotic molecule bcl-2 is reduced or abolished in these viremic patients. We also noted that these abnormalities were largely corrected in the group of patients receiving ARV.
Our results have demonstrated that HIV infection causes damage to the function of several cytokine systems and to their signalling 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. In support of this we have shown that by binding to its receptor, gp120 exerts a negative effect on the proliferation and signalling of CD4 lymphocytes taken from healthy subjects. Similarly, gp120 in viremic patients may lead to a blockade of the Jak/STAT pathways shared by IL-2 and IL-7.
2) Immune therapy in HIV+ patients: therapeutic potential of IL-2 and IL-7 (Florence Bugault, Stéphanie Beq, 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-positive patients requires the administration of ARV. 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 should not be taken life-long and their unwanted effects can be substantial. 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.
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 phenotype cell count. Our laboratory conducted a clinical trial of this type in a group of patients who, when receiving ARV alone, showed a low CD4 count (< 200 cells/mm3) despite adequate control of the viral load. In the course of this trial we observed that IL-2 induced the appearance of a new lymphocyte subpopulation, CD4+/CD25+. The appearance of this new lymphocyte population would appear to be a major effect of treatment with IL-2, as suggested by C. Lane NIH, USA. The mechanisms by which IL-2 induces the emergence of this subpopulation remain to be established. We showed in a recent study that IL-2 rapidly increases the plasma levels of two thymopoietic cytokines, IL-7 and Flt-3L. This suggests that IL-2 immune therapy induces the production of lymphocytes at a central level. We shall continue with the characterization of this subpopulation of CD4+/CD25+ lymphocytes as part of GPH HIV/AIDS (see below)
Given its lymphopoietic properties, IL-7 therefore has a promising therapeutic potential since IL-7 given to immune deficient patients should be able to 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 the treatment. This suggests that IL-7 is implicated in the control of the CD4 pool in HIV-positive patients. This study also indicated that IL-7 treatment in patients receiving ARV should promote CD4 restoration. A preclinical study of IL-7 is in progress in connection with this. Since the response to IL-7 is in part regulated by negative feedback on its receptor, this type of regulation and its reversibility are currently under study to predict the effects of its in vivo administration. Also, we verified that ARV do not alter the normal function of IL-7R. This study is conducted in a complementary manner with that of N. Israêl and F. Barré Sinoussi (Unité de Biologie des Rétrovirus, Institut Pasteur).
3) IL-2 receptor: demonstration of a new form, signal transduction mechanisms and differential responses of lymphocyte subpopulations (Virginie Mazard-Pasquier, Jean-Louis Moreau and Thierry Rose)
Three chains, alpha, beta and gamma, may enter into the composition of the IL-2 receptor (IL-2R). The level at which each chain is expressed depends on the lymphocyte sub-type. Two functional forms of IL-2R have already been described. Firstly, the assembly of all three chains produces a very 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 the existence of a third form of IL-2R made up of two beta chains. 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 identify beta/beta dimers on the surface of transfected COS and HEK cells. We used confocal microscopy to measure the fluorescence resulting from the energy transfer (FRET) from one subunit to the other after irradiation. At the same time, beta/gamma and beta/beta combinations were modelled with or without IL-2. We use these molecular models to predict key contacts in the recognition and association of the different chains of the IL-2R system.
NK lymphocytes respond to IL-2 both spontaneously and very intensely. Given that these cells show considerable surface expression of beta chains but little gamma chain expression, we assume that beta dimers play a physiological role here. In order to verify whether this beta/beta receptor switches on a specific pathway, we are exploring the various alternatives to the Jak/STAT pathway using purified human NK lymphocytes (in collaboration with S. Pellegrini).
In order to respond to IL-2, T lymphocytes, unlike NK calls, 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. It may control the expression of high-affinity IL-2R or affect the mechanism which control the cell activation or division necessary for 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 do not 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 that follows TCR stimulation. By contrast, direct inhibition by IL-2 of p27kipl expression in the CD8 lymphocytes of transgenic mice allows these cells to respond 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 signalling defects in the pathogenesis of HIV infection"
Seven Institut Pasteur units from four different departments are involved in this project which is split into three sub-programs. This GPH aims to analyse - 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 characterise the mechanism of IL-2 action in vivo. It is steered by a committee made up of Mr. Muller-Trutwin, Mr. O. Acuto, Mr. L. Rogge and Mr. F. Arenzana-Seisdedos. Prof. Jacques Thèze is coordinating the work. Our unit is directly implicated in two sub-programs. In collaboration with the Unité d'Immunologie Moléculaire (O. Acuto) we are studying the CD4 lymphocytes of infected patients who are able to control their viral load spontaneously, i.e. those recently characterized by J.-F. Delfraissy as "HIV Controller" patients. These studies are being conducted by S. Potter (grant from the Australian government). In collaboration with the Immunoregulation laboratory (L. Rogge) we have undertaken 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 will be 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