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 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-infected patients, while having no significant effects on cells from HIV-seronegative subjects. To further investigate the effects of IL-7 in the course of HIV-1 infection, we have performed 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 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. Phase 1 clinical trials in chronically HIV-infected patients concomitantly receiving antiretroviral treatment have demonstrated that short-term IL-7 treatment is safe and has transient beneficial effects on peripheral CD4+ T-cell counts without inducing sustained increases in viral replication levels. The LIR-9 protocol was designed to evaluate the immunological, virological and clinical effects of administering 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 (Macacamulatta). 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 administered once a week, s.c., at a dose of 50 micrograms/kg. No antiretroviral treatment was administered. During the acute phase, treatment with IL-7 had distinct effects on the immunological profile of SIV-infected macaques. Specifically, the animals showed a significant increase in the mean absolute numbers of total circulating lymphocytes, affecting both CD4+ T cells and CD8+ T cells, which persisted throughout the treatment period. The effect gradually disappeared within 2-3 weeks of treatment interruption. Importantly, treatment with IL-7 prevented the early depletion of peripheral blood central memory (CM) CD4+ T cells during the acute phase of SIV infection without significantly increasing SIV replication levels. This notwithstanding, CM CD4+ T cells started to decline during the course of IL-7 treatment and became significantly depleted within a few weeks of treatment interruption. Treatment with IL-7 also prevented the depletion of peripheral blood nave (N) CD4+ T cells, and induced significant increases in all subsets of CD8+ T cells (N, CM, EM). In lymphoid tissues (gut-associated lymphoid tissue GALT and lymph nodes), IL-7 did not significantly affect the level of spontaneous apoptosis nor the phenotype of CD4+ T cells, while it increased the proportion of EM CD8+ T cells in lymph nodes. During the post-acute phase, the animals were monitored for several months without further administration of IL-7. Unfortunately, two animals in each group showed early signs of disease progression with unusual manifestations such as neurologic and GI disease in the absence of significant depletion of circulating CD4+ T cells. This picture is in line with previous descriptions of rapid SIV-disease progression (RP) in macaques, reportedly associated with a unique pathogenesis mediated by massive SIV replication in cells of the mononuclear phagocytic lineage rather than CD4+ T cells (Brown et al., J. Virol. 2007). Our preliminary microarray data show distinctive gene expression profiles at baseline in PBMC from RP macaques vs. conventional progressors (CP), with upregulation of B-cell-associated genes (immunoglobulins, MHC class II, B-cell activation markers) and downmodulation of T-cell genes, platelet/megakaryocyte genes and the IL-7 receptor gene. During the chronic phase, low-dose IL-7 treatment (10 micrograms/kg) was restarted in the 4 remaining animals in group 2 at month 8 of SIV inoculation. In all the animals, retreatment induced transient increases in both CD4+ and CD8+ T cells, although higher IL-7 doses were required in some. However, these changes were only transient and peripheral T-cell counts returned to baseline levels during the treatment period. Severe anemia developed in two animals during prolonged IL-7 treatment, although it is unclear whether this effect was related to IL-7 treatment because the other two animals did not show reductions in circulating red blood cells or hemoglobin levels. In parallel with the LIR-9 study, we have started to investigate, both ex vivo and in vivo, the molecular mechanisms of spontaneous T-cell apoptosis in HIV-1-infected subjects, as well as the mechanisms 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. We have demonstrated that the two major pathways of apoptosis (endogenous and exogenous) are both activated in PBMC from HIV-infected subjects during ex vivo culture in the absence of activating 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 associated with a marked 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 insights into the mechanisms of spontaneous apoptosis in HIV-infected subjects and its reduction by IL-7, we also evaluated the levels of expression of several death receptors and their ligands. We found that IL-7 treatment ex vivo results in a significant increase in the expression of two apoptosis-associated receptors, CD95/Fas and lymphotoxin receptor/TNF-receptor III, as well as of the pro-apoptotic cytokine TNF-alpha, despite the reduction of apoptosis levels. CD120b/TNF-receptor II, which is not typically linked to induction of apoptosis, was also upregulated. Altogether, these data suggest a complex immunomodulatory effect of IL-7 with a dominant effect of anti-apoptotic molecules such as Bcl-2, which counteract the action of pro-apoptotic receptors and cytokines. IL-7 is an essential cytokine in the development and homeostasis of the GALT. Thus, we have also investigated the effects of IL-7 on the expression of gut-homing receptors, in particular the chemokine receptor CCR9 and the integrin alpha4-beta7 (a4b7). Preliminary results indicate that IL-7 is a potent inducer of a4b7 expression in both CD4+ and CD8+ T cells, even in the absence of additional activating stimuli. This effect occurs in both phenotypic memory (CD45RO+) and nave (CD45RO-) T cells, and is associated with the induction of the fully activated state of a4b7, as indicated by an increased binding activity of the natural integrin ligands, MAdCAM and HIV-1 gp120. Altogether, these data suggest a dual role for IL-7 in HIV-1 infection: on one side, IL-7 may reduce the levels of apoptosis and increase the pool of CD4+ and CD8+ T cells, thereby favoring the immune reconstitution of subjects concomitantly receiving antiretroviral therapy;on the other side,IL-7 may favor the homing of T cells to the gut, which is a primary site of HIV-1 replication, and, consequently, susceptibility to HIV infection.
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