Protective Effect of IL-7 against Spontaneous T-Cell Apoptosis

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Interleukin-7 (IL-7) is a non-redundant cytokine that plays a critical role in the regulation of the T-cell compartment of the immune system. Due to its ability to maintain the homeostasis of the T-lymphocyte pool and restore it under conditions of lymphopenia, IL-7 is currently under clinical investigation as a potential immune-reconstitution agent in various forms of immunodeficiency, including HIV-1 infection. We previously demonstrated that IL-7 induces a dramatic reduction in the levels of spontaneous ex vivo apoptosis in both CD4+ and CD8+ T cells derived from HIV-1-infected patients, while having no significant effects on cells from HIV-1-seronegative subjects. A significant inverse correlation was documented between the sensitivity to the antiapoptotic effect of IL-7 and the circulating CD4 counts, suggesting that subjects with a more profound immunologic impairment may be more sensitive to the beneficial effects of IL-7. To further investigate the effects of IL-7 in the course of HIV-1 infection, we have designed a study (LIR-9) of in vivo administration of IL-7 in a well-established nonhuman primate model of AIDS. Previous in vivo studies in macaques chronically infected with SIV had demonstrated that IL-7 administration causes a marked increase in naive T-cell counts, as well as proliferation and activation of both CD4+ and CD8+ memory T cells, without augmenting the viral load. Clinical trials are currently under way with the aim of evaluating the safety and efficacy of IL-7 in HIV-1-infected patients who are concomitantly receiving antiretroviral treatment. The LIR-9 protocol was designed to evaluate the immunological, virological and clinical effects of recombinant macaque IL-7, produced in a fully glycosylated form, during the acute and chronic phases of infection with a pathogenic SIV isolate, SIVmac251, in juvenile Rhesus macaques. The animals (n=12) were divided into 2 groups: group 1 (n=6) received only SIV, while group 2 (n=6) received IL-7 and SIV. In the latter group, IL-7 was inoculated once a week, s.c., at a dose of 50 g/kg. During the acute phase of SIV infection, IL-7 was administered for 7 weeks starting 8 days before SIV inoculation in order to temporally dissociate the early induction of CD4+ T-cell proliferation by IL-7 from the early phase of SIV replication, since a marked cellular proliferation could increase the pool of target cells capable of supporting high-level SIV replication. As expected, we did observe an initial increase in T-cell proliferation documented by expression of the intracellular cell-cycle marker Ki67, but the proliferation levels returned to baseline levels at the end of week 1, when the animals were inoculated with SIV. During the acute phase, we observed distinct effects of IL-7 on the immunological profile of treated macaques. Specifically, the animals showed a significant increase in the mean absolute numbers of circulating total lymphocytes, CD4+ T cells and CD8+ T cells, which persisted throughout the treatment period. The effect gradually disappeared within 2-3 weeks after treatment interruption. Interestingly, the effect was more marked on circulating CD8+ T cells, with persistent increases in all the various CD8+ T-cell subsets throughout the treatment period. Among CD4+ T cells, there was a significant increase of nave cells only at day 14 post-SIV inoculation, which prevented the drop in nave CD4+ T cells that occurred in the control group, as well as a significant increase in central memory CD4+ T cells at days 7 and 14 post-infection. The latter effect, however, failed to prevent a dramatic loss of these cells at day 21. No significant differences were observed in the levels of SIV replication between the two groups of animals, as assessed by the plasma levels of SIV p27 antigen. During the post-acute phase, the animals are being monitored for several parameters without further administration of IL-7, which will be restarted in group 2 at month 9 of SIV inoculation and continued indefinitely until the development of clinical signs of disease progression. Unfortunately, two animals in each group showed an extremely rapid clinical progression, manifested by unusual signs of neurologic and GI disease in the absence of significant depletion of circulating CD4+ T cells, in line with previous descriptions of rapid SIV-disease progression in macaques. The study will now continue with the remaining animals, which seem to be following a standard course of disease progression. However, this reduction in the number of animals represents an important limitation to the significance of the data that will derive from this study. In parallel with the LIR-9 study, we have started to investigate, both in vitro and in vivo, the molecular mechanisms of spontaneous T-cell apoptosis in HIV-1-infected subjects, as well as the mechanism of anti-apoptotic activity of IL-7. In vivo studies, which are currently under way, are conducted by microarray analysis of mRNA transcription profiles in IL-7-treated macaques and untreated controls followed longitudinally during the acute phase of SIV infection. In vitro studies are aimed at evaluating the expression and activation of various apoptosis-related molecules, including caspase 3, 8 and 9, Bcl-2-family anti- and pro-apoptotic proteins, cell surface-expressed death receptors and their respective ligands. We have demonstrated that the two pathways of apoptosis (endogenous and exogenous) are both activated in PBMC from HIV-1-infected subjects during ex vivo culture in the absence of activatory signals. Likewise, the reduction of apoptosis mediated by IL-7 was found to affect both apoptosis pathways. In line with previous data, the effect of IL-7 was shown to be consistently associated with an increase in the intracellular levels of Bcl-2. Moreover, we documented a dramatic decrease in the level of activation of the pro-apoptotic protein Bax in both CD4+ and CD8+ T cells from HIV-1-infected subjects treated with IL-7 ex vivo. To gain further insight into the mechanisms of spontaneous apoptosis in HIV-1-infected subjects and its reduction by IL-7, we also evaluated the levels of several apoptosis-associated death receptors and their ligands. Although elevations of apoptosis-related cytokines and cytokine receptors were observed in most patients, none was consistently detected in all subjects in parallel with the level of T-cell apoptosis, suggesting that a variety of factors are implicated in the induction of apoptosis in PBMC from HIV-1-infected subjects. Likewise, the protective effect of IL-7 was not consistently associated with a reduction of any specific marker.