Jean-Louis Moreau

CD127 expression and regulation are altered in the memory CD8 T cells of HIV-infected patients--reversal by highly active anti-retroviral therapy (HAART).

HIV infection activates abnormally the immune system and the chronic phase is accompanied by marked alterations in the CD8 compartment. The expression of CD127 (IL‐7R alpha chain) by memory CD8 T lymphocytes in HIV‐infected patients is analysed and reported. The memory CD8 T cell subset was characterized by expression of CD45RA and CD27 markers, and CD127 cell surface expression was measured ex vivo by four‐colour flow cytometry. HIV infection was associated with a fall in the proportion of CD127+ cells among memory CD8 lymphocytes that resulted in a higher CD127– CD45RA–CD27+ CD8 T cell count in HIV‐infected patients. Diminished CD127 cell surface expression [mean fluorescence intensity (MFI)] by positive cells was also observed in this subset. The data suggest that these defects were reversed by highly active anti‐retroviral therapy (HAART). The regulation of CD127 expression was also studied in vitro. Down‐regulation of CD127 by interkeukin (IL)‐7 was observed in memory CD8 lymphocytes from healthy donors and HAART patients. Expression of CD127 by memory CD8 lymphocytes cultured in the absence of IL‐7 confirmed that IL‐7R regulation is altered in viraemic patients. Under the same experimental conditions, memory CD8 lymphocytes from HAART patients were shown to express CD127 at levels comparable to cells from healthy individuals. Altered CD127 cell surface expression and defective CD127 regulation in the memory CD8 T lymphocytes of HIV‐infected patients are potential mechanisms by which these cells may be impeded in their physiological response to endogenous IL‐7 stimulatory signals. Our data suggest that these defects are reversed during the immune reconstitution that follows HAART.

Regulatory dysfunction of the interleukin-7 receptor in CD4 and CD8 lymphocytes from HIV-infected patients--effects of antiretroviral therapy.

Summary: Despite an increase in plasma IL-7 levels, the CD4 T-cell pool decrease progressively in HIV-infected patients. Here we report on our tests to check the hypothesis that defects in the IL-7 receptor system might be involved in this phenomenon. The cell surface expression of CD127 was measured ex vivo in CD4 and CD8 T lymphocytes drawn from 3 groups of HIV patients. IL-7 function was also followed in vitro by measuring IL-7-driven T-cell proliferation, the induction of the CD25 activation marker, and overexpression of the antiapoptotic molecule Bcl-2. Untreated viremic patients showed a slight but significant decrease in CD127 expression on the surface of their CD4 lymphocytes. By contrast, CD127 expression was substantially altered on the surface of CD8 T lymphocytes taken from untreated viremic patients. IL-7-induced overexpression of the antiapoptotic molecule Bcl-2 was dramatically altered in viremic patients, whereas IL-7-dependent CD25 induction and T-cell proliferation were reduced. Highly active antiretroviral therapy partially corrected these defects in patients with an undetectable viral load and CD4 counts of more than 400 cells/&mgr;L. The effects of HAART were less pronounced in patients with undetectable VL but low CD4 counts (<250 cells/&mgr;L). The IL-7 receptor is dysfunctional in the CD4 and CD8 lymphocytes of HIV-infected patients. This may be due to abnormal activation of the immune system in HIV-infected patients and may contribute to the reduced CD4 count and the altered function of the CD8 compartment.

NK Cells and Polymorphonuclear Neutrophils Are Both Critical for IL-2-Induced Pulmonary Vascular Leak Syndrome

The mechanism of IL-2-induced vascular leak syndrome (VLS) is still poorly understood. Cells of both innate and adaptive immune systems have been implicated, but no definitive conclusions have been reached concerning their respective roles. In this study we report a new mouse model of IL-2-induced pulmonary VLS used to obtain a detailed analysis of the early events (sequestration of polymorphonuclear neutrophils and bronchoconstriction) and late events (modifications in the cell and protein content of bronchoalveolar lavages, followed by edema) that characterize this lung injury. This model and knockout animals are used to reconsider the importance of the different leukocyte lineages in early and late events. Recombinase-activating gene 2−/− mice are used to demonstrate that adaptive lymphocytes, including NK T cells, are not required for pulmonary VLS induction. By contrast, results obtained with newly described recombinase-activating gene 2−/−/IL-15−/− mice indicate that NK cells play a key role in both early and late events. In parallel, polymorphonuclear neutrophil depletion is used to evaluate the contributions made by these cells to the late alterations occurring in the lung. Furthermore, when used in combination with inhibition of NO synthase, granulocyte depletion was completely effective in protecting mice from the late events of IL-2-induced pulmonary VLS. Together our results indicate that both NK and PMN cells play a central role in the late events of IL-2-induced VLS.

The correlation between levels of Il-7rα expression and responsiveness to Il-7 is lost in Cd4 lymphocytes from Hiv-infected patients

Measurements of Bcl-2 and CD25 expression suggested that IL-7R function is modified in CD4 lymphocytes of untreated viraemic patients. The extent of IL-7R function restoration post-HAART was analysed. A positive linear relationship was demonstrated between IL-7Rα expression and the magnitude of IL-7-induced responses in healthy individuals, whereas this relationship is lost in HIV-infected patients, suggesting that viraemic patients suffer a receptor signaling transduction defect in IL-7R function. IL-7 responsiveness is only partly restored by HAART.

Contribution of the H- and L-chains and of the binding site to the idiotypic specificities of mouse anti-GAT antibodies

The contribution of the H- and L-chains to the structure of the main idiotype of anti-poly (Glu60-Ala30-Tyr10) (GAT) antibodies has been studied. This idiotype has been previously divided into four types of specificity: (1) the highly conserved idiotypic specificity (h.c. GAT) is expressed by anti-GAT antibodies from the guinea-pig, rat and mice; (2) the public specificity (p. GAT) is expressed in an identical form by all anti-GAT antibodies from all strains of mice tested and by all hybridoma products (HP) with anti-GAT activity; (3) the strain-restricted specificity (s.r. GAT-1) is only expressed by anti-GAT antibodies from strains with Ig-1a, Ig-1c and Ig-1c allotypic markers; and finally (4) the individual specificity i1-GAT defined on HP G5 is also expressed by most of the hybridoma protein with anti-poly (Glu50-Tyr50) (GT) activity. In this paper we show that h.c.GAT, p.GAT and i1-GAT require the interaction of H- and L-chains to be expressed: (1) isolated H- and L-chains from HP G5 did not express these specificities; and (2) recombinant molecules composed of H- and L-chains from HP with anti-GAT activity and an irrelevant myeloma protein (MOPC21) never expressed h.c.GAT, p.GAT and i1-GAT. We next investigated the relationship between the GAT binding site and the p.GAT, h.c.GAT and s.r.GAT-1 idiotypic specificities. GAT and GT were not able to inhibit the binding to s.r.GAT-1 while they inhibit the idiotypic binding to p.GAT and h.c.GAT. A GAT fragment of mol. wt 3000 was also shown to inhibit the binding of p.GAT and h.c.GAT to the appropriate sera. Thus p.GAT and h.c.GAT are very close to the GAT combining site while s.r.GAT-1 represents an idiotypic specificity located outside the GAT binding site.

In vitro induction and expression of interleukin 2 receptor in a clonal T helper cell differentiation model

Induction and expression of interleukin 2 (IL 2) receptor have been studied using a poly( Glu60 Ala30 Tyr10 ) (GAT)-specific T cell clone of mouse origin. This clone (52-3) has been characterized and it exhibits functional properties of T helper (TH) cells: it leads to a specific anti-DNP response in the presence of DNP-GAT and DNP-primed B cells and it secretes biological activities which can induce polyclonal B cell proliferation and IgM secretion. In vitro this clone mimics the activation stages of normal T lymphocytes and can be obtained under two states of differentiation. depending on the antigen-specific signal provided by antigen-presenting cells (APC). The expression of IL 2 receptor by this clone has been studied by (i) its response to IL 2, (ii) its capacity to absorb IL 2 bioactivity, and (iii) its reactivity with monoclonal antibody 7D4 specific for mouse IL 2 receptor. All the results indicate that the unstimulated state does not express the IL 2 receptor while the activated state does. Clone 52-3 has been compared with clone 14-1.6 that derives from a TH cell line and expresses the IL 2 receptor constitutively. 52-3 offers a good experimental model for studying in vitro, in a clonal TH cell population, the detailed mechanism of IL 2 receptor induction.

Human IL-Rβ chains form IL-2 binding homodimers

Two types of functional interleukin-2 receptor (IL-2Ralpha/IL-2Rbeta/gammac and IL-2Rbeta/gammac) have already been characterized in humans. Here we describe a new form consisting of IL-2Rbeta/beta homodimers that assemble spontaneously in the absence of gammac. Co-transfection of COS-7 cells with constructs expressing IL-2Rbeta chains tagged with either HA or MYC sequences results in the formation of IL-2Rbeta:HA/IL-2Rbeta:MYC complexes detectable by coimmunoprecipitation. The formation of these IL-2Rbeta:HA/IL-2Rbeta:MYC dimers is also observed in the absence of IL-2. Moreover, in COS cells expressing chimeras of IL-2Rbeta fused to fluorescence reporters such as IL-2Rbeta:ECFP and IL-2Rbeta:EYFP, we also observed specific FRET at the surface of living cells, as expected for dimer formation. Transiently transfected COS-7 cells expressing IL-2Rbeta bind 125I-labeled IL-2 (homodimers, Kd = 1nM) as cells expressing both IL-2Rbeta and gammac chains (heterodimers, Kd = 1 nM). IL-2Rbeta/IL-2Rbeta could represent either a decoy receptor or a new form of IL-2R involved in signaling when gammac expression is low.

Characterization of a monoclonal antibody directed against the NH2 terminal area of interleukin-2 (IL-2) and inhibiting specifically the binding of IL-2 to IL-2 receptor β chain (IL-2Rβ)

An anti-human IL-2 mAb (19B11β) was found to selectively block the binding of IL-2 to TS1β cells expressing the interleukin-2 receptor β (IL-2Rβ) without affecting binding to TS1α cells expressing the IL-2Rα receptor. It also specifically inhibits the IL-2 driven cell proliferation in TS1β cells. These observations have lead to the hypothesis that its epitope is related to an IL-2 area involved in binding with IL-2Rβ chain. This epitope was identified using various peptides covering the N-terminal half (including α helix A) of the 133 amino acids of IL-2. MAb 19B11gb does not recognize peptides 30–54 and 44–54 but recognizes peptides 1–22 and 1–30 with a good affinity. Furthermore, threonine in position no. 3 was found to be critical for the binding of mAb 19B11β. A relationship between the epitope of mAb 19B11β and the glycosylation of the IL-2 molecule was observed. This further demonstrates that the NH2 terminal area of IL-2 is critical for IL-2IL-2Rβ interactions. Two other mAbs were studied during the course of this work. They served as control for the study of mAb 19B11β and provide some additional insight concerning the question of IL-2IL-2R structure-function. MAb 16F11α selectively blocks the IL-2 binding to TS1α cells. The epitope of mAb 16F11 is conformational and it was not possible to study the corresponding IL-2IL-2Rα region of interaction. Epitope of mAb 3H9 is localized between residues 30 and 54 and does not affect the binding of IL-2 to IL-2Rα.

IL-2Rβ Agonist P1–30 Acts in Synergy with IL-2, IL-4, IL-9, and IL-15: Biological and Molecular Effects

From the sequence of human IL-2 we have recently characterized a peptide (p1–30), which is the first IL-2 mimetic described. P1–30 covers the entire α helix A of IL-2 and spontaneously folds into a α helical homotetramer mimicking the quaternary structure of a hemopoietin. This neocytokine interacts with a previously undescribed dimeric form of the human IL-2 receptor β-chain likely to form the p1–30 receptor (p1–30R). P1–30 acts as a specific IL-2Rβ agonist, selectively inducing activation of CD8 and NK lymphocytes. From human PBMC we have also shown that p1–30 induces the activation of lymphokine-activated killer cells and the production of IFN-γ. Here we demonstrate the ability of p1–30 to act in synergy with IL-2, -4, -9, and -15. These synergistic effects were analyzed at the functional level by using TS1β, a murine T cell line endogenously expressing the common cytokine γ gene and transfected with the human IL-2Rβ gene. At the receptor level, we show that expression of human IL-2Rβ is absolutely required to obtain synergistic effects, whereas IL-2Rα specifically impedes the synergistic effects obtained with IL-2. The results suggest that overexpression of IL-2Rα inhibits p1–30R formation in the presence of IL-2. Finally, concerning the molecular effects, although p1–30 alone induces the antiapoptotic molecule bcl-2, we show that it does not influence mRNA expression of c-myc, c-jun, and c-fos oncogenes. In contrast, p1–30 enhances IL-2-driven expression of these oncogenes. Our data suggest that p1–30R (IL-2Rβ)2 and intermediate affinity IL-2R (IL-2Rβγ), when simultaneously expressed at the cell surface, may induce complementary signal transduction pathways and act in synergy.

Characterization of three monoclonal antibodies specifically directed against the ligand binding site area of the p55 subunit of the mouse interleukin-2 receptor

Three new rat monoclonal antibodies (MAbs) (5A2, 125A8 and 135D5) directed against the mouse interleukin-2 receptor (IL-2R) were isolated. They were obtained after immunization of LOU rats with 14.1.6 T helper cell clones. These three MAbs recognize the p55 subunit of the IL-2R and compete with the binding of previously characterized MAbs AMT13 and 3C7 specific for this p55 subunit [Moreau et al. (1987) Eur. J. Immun. 15, 723-727]. They recognize the same (or closely related epitopes) since they reciprocally compete with each others binding. Scatchard plot analysis of the data from inhibition experiments clearly indicate that they recognize with very high affinity the ligand binding site area of the p55 subunit of the IL-2R. The properties of the Fab fragment prepared from 5A2 and 135D5 indicate that at saturation one intact IgG molecule binds two IL-2R molecules.