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  Director : THEZE Jacques (jtheze@pasteur.fr)


  abstract

 

In our study of the immune deficiency induced by HIV infection, we are developing three main avenues of investigation: 1) Deregulation of the IL-2/IL-2 receptor system and of IL-2 dependent signaling routes in the course of HIV infection, 2) Interleukins 2 and 7 as potential immune-therapy in HIV+ patients, 3) Mechanism of action of a human IL-2 mimetic peptide and analysis of its therapeutic potential.



  report

cale

Deregulation of the IL-2/IL-2 receptor system and of IL-2 dependent signaling routes in the course of HIV infection. (L. Bani, D. David, H. Keller, M. Kryworuchko, V. Pasquier.)

The lymphocytes in the bloodstream are devoted to meeting antigenic challenges, regardless of whether these are new or recurrent. The number of sub-populations within this cell contingent, and their functional capacities, are maintained by numerous interactions, including notably those between the cytokines, IL-2 and IL-7, and their receptors. Any long-term disturbance of these interactions alters resistance to infectious agents.

We selected several different groups of HIV+ patients either receiving or not receiving various antiretroviral treatments to analyze the IL-2/IL-2 receptor (IL-2R) system by determining 1) the different lymphocyte sub-populations in the blood and 2) the expression of the three IL-2R chains (abg) and their response to IL-2. Two expression profiles were observed in normal individuals, corresponding to the separation of cells into those involved in non-specific and specific responses. The NK cells and monocytes showed abundant expression of the b and g chains respectively. Most T lymphocytes (CD4, CD8) and B lymphocytes did not show any surface expression of the three IL-2R chains. The g chain was nevertheless present in an intracellular compartment of CD4 lymphocytes.

HIV+ patient in the chronic phase of the infection, accompanied by an elevated viral load, were observed to express the three IL-2R chains on a larger proportion of their CD8 and B lymphocytes, and to a lesser extent on their CD4 cells. Despite this over-expression of the IL-2R chains, the CD8 and NK cells in these patients showed a deficient response to IL-2. In a group of patients receiving HAART — a combination of antiretroviral drugs used for the long-term control of viral load and resulting in a significantly increased CD4 count — we observed that the expression of IL-2 receptor chains had to some extent returned to normal and that CD8 and CD4 responses to this cytokine were restored. The IL-2R functional abnormality that we demonstrated in some patients corresponds to an intrinsic defect of the T lymphocytes and this adds to the other functional alterations already described in the course of HIV infection (diminished capacity to produce IL-2, reduced expression of certain TCR components and TCR signaling defects). Collectively, these anomalies support the idea that chronic HIV infection leads progressively to a specific alteration of several T lymphocyte signaling routes. To describe more precisely both the nature and the site of this defect in the response to IL-2, an ex vivo study in CD8 cells from patients in the chronic phase of the infection, before and after their inclusion in a HAART regiment, is ongoing. Here we shall investigate the activation of IL-2-induced transduction signals, notably by Western blot analysis and EMSA, and the expression and activation of the different factors involved in IL-2 transduction.

Interleukins 2 and 7 as potential immune-therapy in HIV+ patients (S. Beq, D. David, H. Keller, J-L Moreau, J-H. Colle)

The possible use of adjunctive immune-therapy based on stimulation of the immune system by certain Interleukins is today fueling the hope of overcoming the limits of HAART which, when withdrawn, is rapidly followed by the re-initiation of viral replication and a consequent fall in the CD4 count. Additionally, the major side effects associated with HAART, and the emergence of viral resistance to the drugs used, continue to be matters of concern. The administration of IL-2 in HIV infection has already been the subject of numerous clinical trials and in most cases resulted in an increased CD4 count. Recently, we conducted a clinical trial that included the use of IL-2 in a group of patients who, despite continuous HAART and sustained control of the viral load, showed little or no improvement in the CD4 count. The administration in these patients of a few IL-2 cycles, consisting of the subcutaneous injection of IL-2 for 5 days followed by 5 weeks in which HAART was the only treatment given, resulted in a specific increase in CD4 counts to beyond the critical threshold of 200 CD4/mm3 considered as necessary for protection against infectious agents. This increase was maintained for several months after IL-2 was withdrawn. A molecular and functional analysis of the CD4 cells showed that these lymphocytes had clearly improved their capacity for spontaneous survival, associated with enhanced expression of the anti-apoptotic molecule Bcl-2, and had regained their capacity to respond to IL-2. Given the central role played by IL-2 in the immune response, the growth of CD4 cells and the activation of cytotoxic CD8 and NK cells, the specific effects of IL-2 on CD4 cells were difficult to predict. Collectively, the data generated by the different trials, and in particular that we conducted, suggest that IL-2 has a beneficial impact on the homeostasis of the CD4 pool. We shall continue to investigate the mechanism involved in the CD4 restoration that follows IL-2 treatment by characterizing the transcription program expressed by CD4 sub-populations (Affymetrix micro arrays technique). These CD4 sub-populations will be obtained by a cell enrichment procedure based on the expression of phenotype-specific membrane markers.

Interleukin-7 is another cytokine that possesses the potential to improve certain aspects of HIV-related immune deficiency. IL-7 is an essential factor in the production of T lymphocytes by the thymus. It is involved in controlling the number of peripheral lymphocytes and has the capacity to increase the intensity of specific antigen responses. Recent studies have shown that IL-7 blood levels are often high in HIV patients. A positive correlation was observed in one study between plasma IL-7 levels and viral load. Other studies have shown an inverse correlation where an increase in IL-7 is accompanied by a decrease in the CD4 count. In order to clearly establish the exact relation between plasma IL-7 and HIV infection, we are currently conducting a longitudinal study in patients where IL-7 levels and CD4 counts are recorded before and after their inclusion in a HAART regiment. In the perspective of conducting a clinical trial with IL-7, we have begun to study T lymphocyte response to IL-7 in different groups of HIV patients since these cells are considered as the major target of IL-7 treatment.

Mechanism of action of a human IL-2 mimetic peptide and analysis of its therapeutic potential. (E.Astier, P. Chastagner, J-L Moreau, F. Gesbert).

In the framework of a structure-function study of human IL-2, we characterized a new agonist of the IL-2R b chain known as peptide p1-30. This peptide consists of the first 30 amino acids of IL-2. A study of its physicochemical properties in solution showed that the molecules fold to from helixes and become organized into tetramers. Physicochemical studies showed that these tetramers associate with dimers of human IL-2R b chain. In vitro, P1-30 induces similar biological activities to those of a growth or survival factor in cell lines expressing certain combinations of the three human IL-2R chains. We plan to study the signaling routes activated by IL-2Rb2 dimers and investigate more deeply the mechanisms involved in IL-2 transduction signals via the bg, abg and b2 heterodimers. Synergy was observed with IL-2, IL-4, IL-9 and IL-15, i.e. all cytokines which include the gc chain in the composition of their receptor. We undertook to determine the potential cell targets of p1-30 among the lymphocytes in the bloodstream. The induction of LAK activity and the secretion of IFN-g have been observed. At the same time, we noted that cells of the NK and CD8low sub-populations, which express constitutively the IL-2R b chain, showed an activated cell phenotype after stimulation by p1-30. The studies conducted on p1-30 are of fundamental value since they provide a more precise description of the function and organization of the IL-2 receptor. These studies also open the way toward future therapeutic applications. The results generated by our modeling studies show the formation of a (p1-30)4/(IL-2Rb)2 complexe and are currently in the process of validation in cell systems (COS cells transfected with two copies of the human IL-2R b gene carrying either HA or Myc tags). The stœchiometry of the IL-2Rb complexes will be verified by cross-linking and FRET experiments. Results obtained in our laboratory and by others suggest that p1-30 can activate cytotoxic cells with a higher therapeutic index than IL-2. In our continuation along this avenue of research, we have conducted studies in a transgenic mouse model that expresses the b chain of human IL-2R. This model will also serve to investigate whether changing some of the residues that make up the hydrophobic region of the p1-30 molecule allow us to augment its biological activity.

Keywords: VIH, lymphocytes, IL-2, IL-2R, IL-7, signal transduction, transcription, immunotherapy, lL-2 mimetic



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  BREGEAT Annick. E mail : abregeat@pasteur.fr COLLE Jean-Hervé. Pasteur. E mail : jhcolle@pasteur.fr

GESBERT Franck. Pasteur. E mail : fgesbert@pasteur.fr

BEQ Stéphanie. PhD student. E mail : sbeq@pasteur.fr

KRYWORUCHKO Marko. Postdoc E mail : mkrywor@pasteur.fr

PASQUIER Virginie PhD student. E mail : vmazard@pasteur.fr

Ingeneer : MOREAU Jean-Louis. Pasteur. émail : jlmoreau@pasteur.fr

Technician : CHASTAGNER Patricia. Pasteur émail : pchasta@pasteur.fr


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