IL-2 mechanism of action: control of the equilibrium between activation/differentiation and maintenance of homeostasis (P. Chastagner, J. Reddy)
We have sought to identify new genes whose expression is under the control of IL-2. A subtractive CDNA approach was employed for a cell line able to grow in IL-2 or IL-4. From this we were able to select 66 different transcripts. The expression kinetics of 8 of these transcripts suggested that they are induced during activation/differentiation phenomena triggered by IL-2 in T lymphocytes. The first group of these genes code for factors involved in transcription control e.g. CCCTC-binding factor, Jun inhibitor factor-1 and transcription factors E2F-4 and AMP-responsive element-binding protein, zhx-1. IL-2 also increases the expression of a-tubulin, a cytoskeletal protein, and multifunctional proteins such as b-catenin and nucleolin. In addition, we demonstrated that TNF-b is one of the first genes to be induced by IL-2 through a mechanism identified previously in the induction of the IL-2Ra chain (activation of the Jak/STAT pathway and presence of a binding site for STAT5 upstream of the TNF-b gene). The study of the expression of these genes was continued in vivo using mice where the IL-2 gene is either disrupted or not (IL-2-/-, IL-2-/+). IL-2-/- mice are characterized in terms of secondary lymph organs by increased proliferation of T lymphocytes but a thymus that is normal both in structure and function. Our studies in IL-2 deficient mice showed that some of the genes coding for secondary lymph organs were not expressed. As far as these organs are concerned, other genes of the TNF group (TNF-a, LT-b, TNFR1 and TNFR2) have been shown in the same manner as TNF-b - to be under the control of IL-2. The critical role played by IL-2 in the expression of these genes suggests that they may be involved in controlling the activation and maintenance of homeostasis in peripheral T cells. Recently, we showed that the proliferation and lesser susceptibility to opoptois of T cells taken from IL-2-/- mice is associated with enhanced expression of cFLIP, a protein that inhibits the activation of TNF-R- or FAS-dependent apoptosis.
Characterization of a new human RIL-2b agonist (R. Eckenberg,, J-L. Moreau, F. Gesbert.)
In the framework of our structure-function studies of human IL-2, we characterized a new agonist of the RIL-2 b chain known as peptide p1-30. This peptide consists of the first 30 amino acids of IL-2. In order to elucidate its mechanism of action, we undertook to study the physicochemical and biological properties of this new molecule. A determination of its secondary and quaternary structure in solution showed that it folded into an a helix and associated to form a tetramer. Studies conducted using cell lines expressing one or several chains of human IL-2R, or with specific antibodies directed against these chains showed that only the b chain is essential for peptide's activity. Synergy was observed when this peptide was used in combination 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. In view of the elective association of this peptide with the b chain, we undertook to characterize the signaling cascade triggered by p1-30. The transduction signals induced by p1-30 were studied. In a similar manner to IL-2, p1-30 induces phosphorylation of the SHC adaptor protein and activation of protein kinase p56lck. In the IL-2/RIL-2 system, these two proteins are specifically recruited by RIL-2b.
Unlike IL-2, p1-30 induces phosphorylation of the kinase Tyk2 but does not lead to activation of the Jak1 or Jak3 proteins, nor STAT5s. At a transcriptional level, p1-30 induces Bcl-2 expression and synergetic induction with IL-2 of the proto-oncogenes c-myc, c-jun and c-fos. This could explain the synergetic effect of these two molecules on cell proliferation.
Work has also been performed to identify potential cell targets for p1-30 in PBMCs. Here, p1-30 induces LAK activity and production of IFN-g. CD69, an early marker of cell activation, is detected exclusively in NK and CD8low sub-populations, both being involved in cytotoxic phenomena and presenting constitutive expression of the IL-2R b chain.
Our studies of p1-30 also aim to determine its potential use in therapy and to identify derivatives with a higher therapeutic index.
Involvement of IL-2 and its receptor in the immunopathology associated with HIV infection (D. David, H. Keller, L. Bani, S. Beq, V. Pasquier, M. Kryworuchko, J-H. Colle.)
We analyzed the expression of the three IL-2R chains on the surface of lymphocytes in blood taken from normal individuals and from patients suffering from chronic infection with HIV. Two profiles were observed in the normal individuals, representing the separation of cells into those involved in non-specific versus innate responses. NK cells and monocytes show abundant expression of the b and g chains respectively. When lymphocytes were examined (T CD4, T CD8 and L B) none of the three chains was detected on the surface. The g chain is nevertheless expressed by T, CD4, CD8, NK and L B cells but remains in the intracellular compartment. HIV+ patients show strong expression of the three RIL-2 chains on the surface of CD8 and L B. In these patients, the L B react well to this cytokine but the CD8 cells are insensitive and the NK cells show reduced reactivity.
These biological parameters were studied in patients receiving HAART and in whom the viral load had fallen below the limit of detection. Under these conditions we noted that as the CD4 count increased, the CD8 cells acquired reactivity to IL-2 and the reactivity of the NK cells was restored. It has been known for some time that the CD4 cells of HIV+ patients have a diminished capacity to produce IL-2 and this is considered to play an important role in the immune deficiency of these patients. The abnormalities we have demonstrated in the expression and function of the IL-2R chains would also appear to contribute to this defect. More recently, IL-2R expression and function were analyzed in patients enrolled for discontinuous IL-2 treatment because their CD4 count remained below the critical threshold despite the fact that they were receiving HAART and their viral load was effectively reduced. Once the treatment was started, we noted a rapid rise in the CD4 count associated with the following changes in the CD4 cells: a decrease in susceptibility to cell death by apoptosis, an increase in Bcl-2 expression and the acquisition of reactivity to IL-2.
In most patients receiving HAART, a decrease in viral load is generally accompanied by restored CD4 count and reactivity to IL-2. Studies conducted in the last group of patients showed a new facet of immune deficiency not dependent on a persistent active viral process. These studies will be continued to further elucidate the IL-2 mechanism of action in vivo and will be broadened to the therapeutic evaluation of IL-2.