Unit: Cellular Immunology and Immunogenetics
Director: THEZE Jacques
Infection by HIV leads progressively to marked immune deficiency. The Unit analyses the molecular basis of this dysfunction. The treatment of HIV patients by complementary immunotherapy using IL-2 or IL-7 could supplement our therapeutic arsenal. In addition, we are studying the mechanism of action of IL-2 and some of its mimetics on different forms of receptor expressed differentially by lymphocyte subpopulations.
Molecular basis of T lymphocyte dysfunction in HIV infection (Virginie Mazard-Pasquier and Marko Kryworuchko)
A gradual reduction in the number CD4 T lymphocytes dominates the pathophysiology of HIV infection. Moreover, the study of T lymphocyte reactivity in HIV-positive patients has highlighted major functional abnormalities. In this context, we conducted an analysis of the interleukin-2 system (IL-2) which is the principal interleukin that activates the immune system. The expression of the three chains that make up the IL-2 receptor, and lymphocyte reactivity to IL-2, were studied in several groups of HIV-positive patients. These analyses led us to identify functional abnormalities in the IL-2 / IL-2 receptor system. This defect is reversible: the IL-2 / IL-2 receptor system can be brought back into function in most patients after starting effective anti-retroviral (ARV) treatment.
Detection of protein gp120 in the serum and lymph nodes of HIV-positive patients suggests that this protein plays a role in altering immune responses. By binding to the CD4 molecule, the HIV envelope glycoprotein (gp120) exerts a negative effect on the proliferation of CD4 lymphocytes taken from healthy subjects. Also, we have shown that pre-incubation of CD4 lymphocytes with gp120 reduces expression of the IL-2 receptor αchain. More recently, we observed that gp120 is able to reduce the transduction of IL-2 signals in CD4 lymphocytes. This dysfunction is due to a phosphorylation defect in Jak1 and Jak3 and in STAT5 a and b transcription factors.
In parallel, we have investigated the IL-2 receptor signalling pathway in the CD8 lymphocytes of HIV-positive patients. In this study the CD8 lymphocytes were collected from the patients before ARV then six months after its start. Prior to the treatment a large proportion of the patients showed a defect in the activation of the Jak/STAT pathway after stimulation of the purified CD8 lymphocytes by IL-2. Interestingly, this defect was reversed after six months of treatment.
Our results indicate that the proximal part of the IL-2/IL-2 receptor system's transduction system is directly or indirectly a major target of HIV. Deregulation of the Jak/STAT transduction pathway suggests that more general damage occurs in the function of several cytokine systems and therefore appears to be one of the means used by HIV to induce immune deficiency.
As part of the Grand Programme Horizontal Grand Programme Horizontal HIV/AIDS, we intend to develop a new project to deepen our knowledge of the signalling abnormalities that alter the reactivity of CD4 lymphocytes taken from HIV-positive patients (see below).
Immunotherapy in HIV+ patients: therapeutic potential of IL-2 and IL-7 (Jean-Louis Moreau, Stéphanie Beq and Jean-Hervé Colle in collaboration with J.-F. Delfraissy, Centre Bicêtre, Assistance Publique-Hôpitaux de Paris)
The treatment of HIV-positive patients, from a certain stage in the course of their disease, requires the administration of ARV. In a large fraction of these patients, this type of treatment has proven to be effective both in controlling viral load and in restoring the CD4 count. Given the limited effects of this therapy in numerous patients, the discovery of new therapeutic strategies remains a priority. Cytokines with either immune stimulating properties, such as IL-2, or those such as IL-7 that are likely to affect directly the restoration of the CD4 pool, open up the perspective of new treatments.
Numerous 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 with a group of patients who, when receiving ARV alone, showed adequate control of viral load but a low CD4 count (CD4 < 200 cells/mm3). We observed a rapid increase in blood CD4 cells in the course of this trial. We showed that after a few cycles of IL-2 treatment, CD4 lymphocyte expression of the anti-apoptotic molecule Bcl-2 was enhanced at the same time that spontaneous survival was lengthened and IL-2 reactivity was restored. Also, in parallel with the laboratory headed by C. Lane (NIH, USA) we observed that treatment with IL-2 induced the appearance of a new subpopulation, CD4+/CD25+. This population persists for at least several months after the IL-2 administration and may account for up to 70 % of the CD4 cells. Emergence of the CD4+/CD25+ population would appear to be a major effect of IL-2 treatment. We are continuing to characterise this lymphocyte subpopulation as part of the GPH HIV/AIDS (see above)
Interleukin-7 is a major cytokine that controls thymopoiesis and the number of lymphocytes residing in the peripheral compartment and is also likely to increase the T lymphocyte repertoire and their specific responses. We conducted a longitudinal study in a group of patients and observed a positive correlation between plasma IL-7 levels prior to ARV and restoration of the CD4 count subsequent to ARV treatment. This suggests that IL-7 plays a direct role in the mechanism controlling the CD4 pool in HIV-positive patients. Alternatively, IL-7 levels may reflect the capacity of patient lymphoid organs to restore their CD4 count. This study showed that IL-7 treatment in patients receiving ARV should help restore the CD4 count. A pre-clinical trial of IL-7 is now ongoing in the treatment of HIV-positive patients. Since T lymphocytes in the blood may be a major target of IL-7 treatment, we undertook to study the expression and the functionality of the IL-7 receptor expressed by T lymphocytes in different groups of HIV-positive patients. This study is conducted in a complementary manner with that of N. Israêl and F. Barré Sinoussi (Unité de Biologie des Rétrovirus, Institut Pasteur).
IL-2/IL-2 receptor mechanism of action, description of new mimetics (Virginie Mazard-Pasquier, Valérie Guez and Thierry Rose)
An in-depth investigation into the mechanism by which the receptor recognises IL-2 and its mimetics is ongoing. Three chains, known as a, b, andγ, may enter into the composition of the IL-2 receptor. Differences in the level of expression of these chains in different lymphocyte sub-types result in the production of several forms of this receptor which possibly involve different signalling cascades. A very high affinity receptor (Kd = 10pM) is produced by assembly of the three chains (αβγ). The βγ combination produces a lower affinity receptor (Kd = 1nM). Our results suggest the existence of a new form of IL-2 receptor composed solely of β chains. The biological pertinence of β chain dimers is currently in the process of validation. The transmission of signals dependent upon this receptor is currently under study.
Peptide P1-30, an IL-2 receptor agonist, corresponds to the first 30 residues of IL-2. NK cells and CD8low lymphocytes, which constitutively express the β chain, are stimulated by P1-30. Ultracentrifugation experiments have shown the formation in solution of P1-30 tetramers that have an affinity for the preformed dimers of the extracellular portion of the βchain (Kd ? 150μM). In a similar manner to the crystalline structure of erythropoietin that was resolved in the absence and in the presence of its agonist, we produced a model of the manner in which a β2-type receptor would function. This model enabled us to put forward a map of the putative contacts between the peptide or IL-2 and the receptor. To identify the molecular basis of the affinity and the specificity of future agonists, we undertook to perform a virtual screening process of peptide libraries (using adjustment by molecular dynamics). The best candidates from this set of molecules will be selected in solution then in several cell types corresponding to the targets likely to be of therapeutic benefit.
Grand Programme Horizontal HIV/AIDS: "Gene expression and signalling defects in the pathogenesis of HIV infection"
Seven Institut Pasteur units from three different departments are involved in this project which is split into three sub-programmes. This GPH aims to analyse in CD4 lymphocytes the genes and the transduction signals involved in patient resistance to AIDS or susceptibility to AIDS development. It also aims to characterise IL-2 mechanism of 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 programme. The Unité d'ImmunoGénétique Cellulaire is directly implicated in two sub-programmes. In collaboration with the Unité d'Immunologie Moléculaire (O. Acuto) a focus will be made on abnormalities in the signalling pathways induced by TCR in order to understand the very early abnormalities that cause a reduction in the reactivity of CD4 T lymphocytes. In collaboration with the Immunoregulation laboratory (L. Rogge) we have undertaken to investigate CD4 restoration during IL-2 therapy by combining characterisation of a purified CD4+/CD25+ population with a study of functional genetics. The patients will be drawn from study ANRS 118 (Prof. Y. Levy and J.-F. Delfraissy).
Keywords: HIV, CD4 lymphocytes, IL-2, IL-2 receptor, IL-2 mimetics, IL-7, IL-7-receptor, transduction signal, transcription, immunotherapy