HIV-1 infection is characterized by a chronic activation of the immune system and suppressed function of T lymphocytes. Regulatory CD4+ CD25high FoxP3+CD127low T cells (Treg) play a key role in both conditions. Here, we show that HIV-1 positive patients have a significant increase of Treg-associated expression of CD39/ENTPD1, an ectoenzyme which in concert with CD73 generates adenosine. We show in vitro that the CD39/adenosine axis is involved in Treg suppression in HIV infection. Treg inhibitory effects are relieved by CD39 down modulation and are reproduced by an adenosine-agonist in accordance with a higher expression of the adenosine A2A receptor on patients' T cells. Notably, the expansion of the Treg CD39+ correlates with the level of immune activation and lower CD4+ counts in HIV-1 infected patients. Finally, in a genetic association study performed in three different cohorts, we identified a CD39 gene polymorphism that was associated with down-modulated CD39 expression and a slower progression to AIDS.

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The mechanisms by which Regulatory T cells suppress IL-2 production of effector CD4+ T cells in pathological conditions are unclear. A subpopulation of human Treg expresses the ectoenzyme CD39, which in association with CD73 converts ATP/ADP/AMP to adenosine. We show here that Treg/CD39+ suppress IL-2 expression of activated CD4+ T-cells more efficiently than Treg/CD39−. This inhibition is due to the demethylation of an essential CpG site of the il-2 gene promoter, which was reversed by an anti-CD39 mAb. By recapitulating the events downstream CD39/adenosine receptor (A2AR) axis, we show that A2AR agonist and soluble cAMP inhibit CpG site demethylation of the il-2 gene promoter. A high frequency of Treg/CD39+ is associated with a low clinical outcome in HIV infection. We show here that CD4+ T-cells from HIV-1 infected individuals express high levels of A2AR and intracellular cAMP. Following in vitro stimulation, these cells exhibit a lower degree of demethylation of il-2 gene promoter associated with a lower expression of IL-2, compared to healthy individuals. These results extend previous data on the role of Treg in HIV infection by filling the gap between expansion of Treg/CD39+ in HIV infection and the suppression of CD4+ T-cell function through inhibition of IL-2 production.

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HIV-1 infection is characterized by a progressive decline in CD4 + T cells leading to a state of profound immunodeficiency. IL-2 therapy has been shown to improve CD4 + counts beyond that observed with antiretroviral therapy. Recent phase III trials revealed that despite a sustained increase in CD4 + counts, IL-2-treated patients did not experience a better clinical outcome [Abrams D, et al. (2009) N Engl J Med 361(16):1548-1559. To explain these disappointing results, we have studied phenotypic, functional, and molecular characteristics of CD4 + T cell populations in IL-2-treated patients. We found that the principal effect of long-term IL-2 therapy was the expansion of two distinct CD4 + CD25 + T cell populations (CD4 + CD25 lo CD127 lo FOXP3 + and CD4 + CD25 hi CD127 lo FOXP3 hi ) that shared phenotypic markers of Treg but could be distinguished by the levels of CD25 and FOXP3 expression. IL-2-expanded CD4 + CD25 + T cells suppressed proliferation of effector cells in vitro and had gene expression profiles similar to those of natural regulatory CD4 + CD25 hi FOXP3 + T cells (Treg) from healthy donors, an immunosuppressive T cell subset critically important for the maintenance of self-tolerance. We propose that the sustained increase of the peripheral Treg pool in IL-2-treated HIV patients may account for the unexpected clinical observation that patients with the greatest expansion of CD4 + T cells had a higher relative risk of clinical progression to AIDS. molecular profiling | immune-based therapy H IV infection is mainly associated with a progressive decrease in the number of CD4 + T lymphocytes and defective CD4-and CD8-specific T cell responses against HIV and other pathogens. Quantitative and qualitative CD4 T cell reconstitution following introduction of antiretroviral therapy (ART) in HIVinfected patients is often incomplete, leading one to evaluate the impact of immune-based therapy in combination with ART. In the past 20 years, a large set of clinical trials demonstrated that intermittent interleukin (IL)-2 therapy increased the CD4 T cell pool in HIV-infected patients and induced, in a large majority of patients, significant and sustained increases in CD4 T cell count beyond that seen in patients treated with antiviral drugs alone. In particular, recombinant IL-2 therapy raised naive and central memory CD4 + cells expressing CD25, the α-chain of the IL-2 receptor, more than antiretroviral drugs alone (1-6). This population accounted for up to 70% of the total CD4 + T cell pool of IL-2-treated patients and persisted for a long time.The clinical impact of IL-2 was evaluated in two long-run large phase III trials (SILCAAT and ESPRIT) involving almost 6,000 chronically HIV-1-infected patients. Results recently reported showed that, despite a sustained increase in CD4 + T cell counts, over a median follow-up of 7-8 years, IL-2-treated patients did not experience a better clinical outcome (7). These disappointing results have remained unexplained so far.In mice, IL-2 has been shown to be essential for the...

Human immunodeficiency virus type 1 (HIV-1) infection is characterized by chronic immune activation and suppressed T-lymphocyte functions. Here we report that CD73, both a coactivator molecule of T cells and an immunosuppressive ecto-enzyme through adenosine production, is only weakly expressed by CD8+ T cells of HIV-infected patients and only partially restored after successful antiviral treatment. CD73 expression on CD8+ T cells correlates inversely with cell activation both ex vivo and in vitro. However, CD8+ T cells from HIV controllers (HICs), which spontaneously control HIV replication, express CD73 strongly, despite residual immune activation. Finally, we demonstrate that CD73 is involved in the HIV-specific CD8+ T-cell expansion. Thus, we show that CD73 is central to the functionality of HIV-specific CD8+ T cells and that the preservation of HIV-specific CD73+ CD8+ T cells is a characteristic of HICs. These observations reveal a novel mechanism involved in the control of viral replication.

The detection of negative-strand hepatitis C virus (HCV) RNA is a hallmark of replication. A highly sensitive and specific method is required to quantify the very low level of replication inherent to in vitro infection systems. Based on reverse transcription with a tagged primer in the 5' non-coding region of the HCV genome, followed by a nested PCR with a second round of real-time PCR, a novel method is described with improved sensitivity for negative-strand HCV RNA quantification. The lower detection level was 25 copies per reaction of negative-strand HCV RNA, even in the presence of 1 x 10(5) copies of positive-strand HCV RNA. This protocol was applied to the detection of negative HCV strand RNA in the liver of HCV-infected patients as well as in primary human hepatocytes infected in vitro. In both models, and particularly in each of three, independent in vitro infection experiments, this assay permitted the quantitation of HCV replication.

Alternative, non-invasive techniques are necessary to monitor the progression of liver disease during chronic hepatitis C. Firstly, because serum is the most accessible material for studies using qPCR in microplates, gene transcription was compared in 219 selected genes involved in the pathogenesis of hepatitis C virus (HCV) infection between sera, PBMCs and liver samples collected simultaneously from five patients infected chronically. Secondly, using sera, gene profiles were compared between HCV-infected patients (n = 10) and healthy controls (n = 10). In addition, the influence of alcohol intake was examined in patients infected with HCV genotype-1. Firstly, amplifiable mRNAs were obtained in all samples. After amplification, significant correlations were observed between: liver versus serum; liver versus PBMCs; and serum versus PBMCs (r(2) = 0.37, r(2) = 0.54, r(2) = 0.49, respectively). A comparison of gene transcription by gene involved in T- and B-cell markers, adhesion molecules, apoptosis, liver matrix turnover and inflammation, revealed comparable, significant correlations between serum and liver, (r(2) = 0.30, r(2) = 0.60, r(2) = 0.51, r(2) = 0.51, r(2) = 0.26, and r(2) = 0.61 respectively). Secondly, a quantitative analysis of gene expression in sera between genotype-1b-infected patients and healthy controls revealed that 41 genes involved closely in T-cell activation and apoptosis were over-expressed significantly in patients infected with HCV. In these patients, alcohol consumption was associated with an increased expression of six genes involved in the inflammatory response, together with a decrease of genes associated with dendritic cell function. It is concluded that in patients infected with HCV, serum can be used to evaluate expression of liver genes. Further prospective studies are clearly needed to validate the initial results and to define the relevant genes.