Nicole Israël

IL-7 Induces Immunological Improvement in SIV-Infected Rhesus Macaques under Antiviral Therapy

Despite efficient antiretroviral therapy (ART), CD4+ T cell counts often remain low in HIV-1-infected patients. This has led to IL-7, a crucial cytokine involved in both thymopoiesis and peripheral T cell homeostasis, being suggested as an additional therapeutic strategy. We investigated whether recombinant simian IL-7-treatment enhanced the T cell renewal initiated by ART in rhesus macaques chronically infected with SIVmac251. Six macaques in the early chronic phase of SIV infection received antiretroviral treatment. Four macaques also received a 3-wk course of IL-7 injections. Viral load was unaffected by IL-7 treatment. IL-7 treatment increased the number of circulating CD4+ and CD8+ memory T cells expressing activation (HLA-DR+, CD25+) and proliferation (Ki-67+) markers. It also increased naive (CD45RAbrightCD62L+) T cell counts by peripheral proliferation and enhanced de novo thymic production. The studied parameters returned to pretreatment values by day 29 after the initiation of treatment, concomitantly to the appearance of anti-IL-7 neutralizing Abs, supporting the need for a nonimmunogenic molecule for human treatment. Thus, IL-7, which increases T cell memory and de novo renewal of naive T cells may have additional benefits in HIV-infected patients receiving ART.

Population-Based Sequencing of the V3 Region of env for Predicting the Coreceptor Usage of Human Immunodeficiency Virus Type 1 Quasispecies

ABSTRACT Genotypic population-based methods could be faster and less expensive than phenotypic recombinant assays for determining human immunodeficiency virus type 1 (HIV-1) coreceptor usage in patient samples, but their clinical use requires good genotype-phenotype correlation and concordance with clonal analyses. We have assessed these requirements by clonal analysis of the V1 to V3 env PCR products of 26 patients infected with subtype B HIV-1. We used the resulting set of molecular clones, all sequenced and characterized using a single-cycle recombinant virus phenotypic entry assay, to reevaluate genotype-phenotype correlations. Combining the previously described 11/25 and net charge rules for the V3 genotype improved the prediction of HIV-1 coreceptor usage. We also evaluated the concordance of population-based and clonal analyses for predicting the coreceptor usage of HIV-1 quasispecies. Our population-based recombinant phenotypic assay and direct sequencing of V3 were similarly sensitive for detecting the presence of minor species in the virus population, and both correlated well with clonal analysis. The improved genotype-phenotype correlation obtained by combining two simple genotypic rules and the good concordance with clonal analyses suggest that direct sequencing of V3 is a valuable alternative to population-based recombinant phenotypic assays.

IL-7 stimulates T cell renewal without increasing viral replication in simian immunodeficiency virus-infected macaques

The main failure of antiretroviral therapy is the lack of restoration of HIV-specific CD4+ T cells. IL-7, which has been shown to be a crucial cytokine for thymopoiesis, has been envisaged as an additive therapeutic strategy. However, in vitro studies suggest that IL-7 might sustain HIV replication in thymocytes and T lymphocytes. Therefore, in the present study, we evaluated the effect of IL-7 on both T cell renewal and viral load in SIVmac-infected young macaques in the absence of antiretroviral therapy. This evaluation was conducted during the asymptomatic phase in view of a potential treatment of HIV patients. We show that IL-7 induces both a central renewal and a peripheral expansion of T lymphocytes associated with cell activation. No alarming modulation of the other hemopoietic cells was observed. No increase in the viral load was shown in blood or lymph nodes. These data strengthen the rationale for the use of IL-7 as an efficient immunotherapy in AIDS.

Positive Regulation of CXCR4 Expression and Signaling by Interleukin-7 in CD4+ Mature Thymocytes Correlates with Their Capacity To Favor Human Immunodeficiency X4 Virus Replication

ABSTRACT The emergence of X4 human immunodeficiency virus type 1 (HIV-1) variants in infected individuals is associated with poor prognosis. One of the possible causes of this emergence might be the selection of X4 variants in some specific tissue compartment. We demonstrate that the thymic microenvironment favors the replication of X4 variants by positively modulating the expression and signaling of CXCR4 in mature CD4+ CD8− CD3+ thymocytes. Here, we show that the interaction of thymic epithelial cells (TEC) with these thymocytes in culture induces an upregulation of CXCR4 expression. The cytokine secreted by TEC, interleukin-7 (IL-7), increases cell surface expression of CXCR4 and efficiently overcomes the downregulation induced by SDF-1α, also produced by TEC. IL-7 also potentiates CXCR4 signaling, leading to actin polymerization, a process necessary for virus entry. In contrast, in intermediate CD4+ CD8− CD3− thymocytes, the other subpopulation known to allow virus replication, TEC or IL-7 has little or no effect on CXCR4 expression and signaling. CCR5 is expressed at similarly low levels in the two thymocyte subpopulations, and neither its expression nor its signaling was modified by the cytokines tested. This positive regulation of CXCR4 by IL-7 in mature CD4+ thymocytes correlates with their high capacity to favor X4 virus replication compared with intermediate thymocytes or peripheral blood mononuclear cells. Indeed, we observed an enrichment of X4 viruses after replication in thymocytes initially infected with a mixture of X4 (NL4-3) and R5 (NLAD8) HIV strains and after the emergence of X4 variants from an R5 primary isolate during culture in mature thymocytes.

Naïve T-Cell Depletion Related to Infection by X4 Human Immunodeficiency Virus Type 1 in Poor Immunological Responders to Highly Active Antiretroviral Therapy

ABSTRACT The reasons for poor CD4+ T-cell recovery in some human immunodeficiency virus (HIV)-infected subjects despite effective highly active antiretroviral therapy (HAART) remain unclear. We recently reported that CXCR4-using (X4) HIV-1 could be gradually selected in cellular reservoirs during sustained HAART. Because of the differential expression of HIV-1 coreceptors CCR5 and CXCR4 on distinct T-cell subsets, the residual replication of R5 and X4 viruses could have different impacts on T-cell homeostasis during immune reconstitution on HAART. We examined this hypothesis and the mechanisms of CD4+ T-cell restoration by comparing the virological and immunological features of 15 poor and 15 good immunological responders to HAART. We found a high frequency of X4 viruses in the poor immunological responders. But the levels of intrathymic proliferation of the two groups were similar regardless of whether they were infected by R5 or X4 virus. The frequency of recent thymic emigrants in the poor immunological responders was also similar to that found in the good immunological responders, despite their reduced numbers of naïve CD4+ T cells. Our data, rather, suggest that the naïve T-cell compartment is drained by a high rate of mature naïve cell loss in the periphery due to bystander apoptosis or activation-induced differentiation. X4 viruses could play a role in the depletion of naïve T cells in poor immunological responders to HAART by triggering persistent T-cell activation and bystander apoptosis via gp120-CXCR4 interactions.

An intronic polymorphic repeat sequence modulates interleukin-1 alpha gene regulation.

Recently, we characterized a polymorphism within IL-1 alpha intron 6 as a variable number of a 46 bp tandem repeat (ranging from 5 to 18 repeats). We now analyse whether this polymorphism could play a role in IL-1 alpha gene regulation. We have found that reporter gene expression driven by the IL-1 alpha promoter or a heterologous promoter was decreased by increasing numbers of the repeat sequence corresponding to the most frequent alleles seen in the human population. Furthermore, we showed that the transcription factor Sp1 can bind to the 46 bp sequence. Finally, we were unable to show a statistically-significant relation between in vitro IL-1 alpha production and the number of repeats although there was a clear trend towards an inverse relation. Taken together, these results are consistent with a negative regulatory role for IL-1 alpha intron 6 repeat sequence on IL-1 alpha basal gene transcription.

Over-expression of Bcl-2 does not protect cells from hypericin photo-induced mitochondrial membrane depolarization, but delays subsequent events in the apoptotic pathway.

Hypericin (HY) is a powerful photo‐inducer of apoptosis in Jurkat cells as measured by caspase‐3 activation, cell shrinkage, phosphatidylserine (PS) exposure and the appearance of hypoploid DNA. These processes are preceded by rapid Bcl‐2‐independent mitochondrial transmembrane depolarization and a drop in cytoplasmic pH. Pre‐incubation of cells with inhibitors of the mitochondrial permeability transition pore, such as cyclosporin A or bongkrekic acid, does not protect cells from mitochondrial membrane potential (Δψ m) decrease. However, monitoring of mitochondrial entrapped calcein by confocal fluorescence imaging gives clear evidence of HY photo‐induced mitochondrial permeability. This should be considered as the result of a non‐specific alteration of mitochondrial membrane integrity brought about by lipid peroxidation. Nevertheless, synthesis of the anti‐apoptotic protein Bcl‐2 appears to delay the subsequent time course of PS exposure and to reduce caspase‐3 activation and the fraction of cells which become hypoploid. We interpret this partially protective effect as the consequence of a direct interaction of Bcl‐2 with cytosolic cytochrome c previously released from mitochondria upon Δψ m decrease and/or of Bcl‐2 inhibition of the deleterious retro‐effect of caspase‐3 on the mitochondrial permeability transition pore and/or the mitochondrial membrane components.

Differential susceptibility of human thymic dendritic cell subsets to X4 and R5 HIV-1 infection

Objectives:Human thymus can be infected by HIV-1 with potential consequences on immune regeneration and homeostasis. We previously showed that CD4 thymocytes preferentially replicate CXCR4 tropic (X4) HIV-1 dependently on interleukin (IL)-7. Here we addressed the susceptibility of thymic dendritic cells (DC) to HIV-1 infection. Methods:We investigated the replication ability of CXCR4 or CCR5 (R5) tropic HIV-1 in thymic micro-explants as well as in isolated thymic CD11clowCD14− DC, CD11chighCD14+ DC and plasmacytoid DC subsets. Results:Thymic tissue was productively infected by both X4 and R5 viruses. However, X4 but not R5 HIV-1 replication was enhanced by IL-7 in thymic micro-explants, suggesting that R5 virus replication occurred in cells other than thymocytes. Indeed, we found that R5 HIV-1 replicated efficiently in DC isolated from thymic tissue. The replicative capacity of X4 and R5 viruses differed according to the different DC subsets. R5 but not X4 HIV-1 efficiently replicated in CD11chighCD14+ DC. In contrast, no HIV-1 replication was detected in CD11clowCD14− DC. Both X4 and R5 viruses efficiently replicated in plasmacytoid DC, which secreted interferon-α upon HIV-1 exposure. Productive HIV-1 infection also caused DC loss, consistent with different permissivity of each DC subset. Conclusions:Thymic DC sustain high levels of HIV-1 replication. DC might thus be the first target for R5 HIV-1 infection of thymus, acting as a Trojan horse for HIV-1 spread to thymocytes. Furthermore, DC death induced by HIV-1 infection may affect thymopoiesis.

High level of Bcl-2 counteracts apoptosis mediated by a live rabies virus vaccine strain and induces long-term infection

We report here that rabies virus strains, currently used to immunize wildlife against rabies, induce not only caspase-dependent apoptosis in the human lymphoblastoid Jurkat T cell line (Jurkat-vect), but also a caspase-independent pathway involving the apoptosis-inducing factor (AIF). In contrast, a strain of neurotropic RV that does not induce apoptosis did not activate caspases or induce AIF translocation. Bcl-2 overproduction in Jurkat T cells (Jurkat-Bcl-2) abolished both pathways. ERA infection and production were similar in Jurkat-vect and Jurkat-Bcl-2 cells, indicating Bcl-2 has no direct antiviral effects. Bcl-2 production is naturally upregulated by day 3 in ERA-infected Jurkat-vect cultures. The increase in Bcl-2 levels seems to be controlled by the virus infection itself and results in the establishment of long-term, persistently infected cultures that continue to produce virus. Thus, in infections with live RV vaccine strains, infected cells may be productive reservoirs of virus in the long term. This may account for the high efficacy of live rabies vaccines.

Disease progression in macaques with low SIV replication levels: on the relevance of TREC counts.

Background:An attenuated immunodeficiency virus has been long considered innocuous. Nevertheless, converging data suggest that low levels of viral replication can still provoke AIDS. Pathogenesis of these attenuated infections is not understood. Objectives:To determine the pathogenicity of a long-term attenuated infection and to delineate T-cell dynamics during such an infection. Methods:This is a cross-sectional study of 12 rhesus macaques infected with SIVΔnef for 8 years. We evaluated apoptosis (annexin V), activation (HLA-DR, Ki67), and newly generated T cells (TCR excision circle: TREC). Results:Infection with SIVΔnef induced pathological CD4 T-cell depletion after 8 years of infection. Virus replication and CD8 T-cell activation positively correlated with the rate of disease progression. The frequency of TREC within CD8+CD45RA+ cells increased in SIVΔnef-infected animals compared to age-matched non-infected controls. Moreover, in the cohort of infected animals, TREC+CD45RA+CD4+ T-cell counts correlated strongly with non-progression to AIDS. The animal with the lowest rate of disease progression exhibited a 115-fold increase in TREC+CD45RA+CD4+ T-cell counts compared to age-matched non-infected controls. In contrast, the animal showing the fastest rate of progression to AIDS displayed 600-fold lower TREC+CD45RA+CD4+ T-cell counts compared to age-matched non-infected controls. Conclusions:Our results suggest that the thymus plays a major role in the pathogenesis of an attenuated SIV infection and that a sustained thymic output could maintain CD4 T-cell homeostasis in the context of low viral loads.