P2X7 Receptor Inhibition Improves CD34 T-Cell Differentiation in HIV-Infected Immunological Nonresponders on c-ART

<div><p>Peripheral CD4+ T-cell levels are not fully restored in a significant proportion of HIV+ individuals displaying long-term viral suppression on c-ART. These immunological nonresponders (INRs) have a higher risk of developing AIDS and non-AIDS events and a lower life expectancy than the general population, but the underlying mechanisms are not fully understood. We used an <i>in vitro</i> system to analyze the T- and B-cell potential of CD34+ hematopoietic progenitor cells. Comparisons of INRs with matched HIV+ patients with high CD4+ T-cell counts (immune responders (IRs)) revealed an impairment of the generation of T-cell progenitors, but not of B-cell progenitors, in INRs. This impairment resulted in the presence of smaller numbers of recent thymic emigrants (RTE) in the blood and lower peripheral CD4+ T-cell counts. We investigated the molecular pathways involved in lymphopoiesis, focusing particularly on T-cell fate specification (Notch pathway), survival (IL7R-IL7 axis) and death (<i>Fas</i>, <i>P2X7</i>, <i>CD39/CD73</i>). <i>P2X7</i> expression was abnormally strong and there was no <i>CD73</i> mRNA in the CD34+ cells of INRs, highlighting a role for the ATP pathway. This was confirmed by the demonstration that <i>in vitro</i> inhibition of the P2X7-mediated pathway restored the T-cell potential of CD34+ cells from INRs. Moreover, transcriptomic analysis revealed major differences in cell survival and death pathways between CD34+ cells from INRs and those from IRs. These findings pave the way for the use of complementary immunotherapies, such as P2X7 antagonists, to restore T-cell lymphopoiesis in INRs.</p></div

Limiting dilution assays (LDAs) to determine the T-cell and B-cell differentiation potential of CD34+ cells.

<p>(A) LDA design. Conditions for determining the potential of CD34+ cells to generate T cells ^a and B cells ^b are shown. For further details, see the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005571#sec011" target="_blank">materials and methods</a>. (B, C) Cell cultures on D21 positive for T-cell precursors (B) defined as CD45RA^highCD7+CD5+CD1a+ cells, and B cells (C) defined as CD79a^intra+ cells. (D) Analysis of the T-cell potential of CD34+ cells. Each point on the graph represents the mean value from three independent experiments. (E) The presence of T-cell precursors and B cells was assessed with the ELDA webtool, applying the maximum likelihood method to the Poisson model. Mean values (min-max) for three experiments are indicated for each group and each set of conditions. NS for <i>P</i>>0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Characteristics of the subjects included in the study.

<p>Characteristics of the subjects included in the study.</p

Analysis of lymphopoiesis in peripheral blood.

<p>(A) Gating strategy to identify recent thymic emigrants, RTEs. (B) Percentage of RTEs among CD4+ T lymphocytes in HIV-uninfected individuals (HIV-, <i>n</i> = 5), HIV+ IRs (<i>n</i> = 9) and INRs (<i>n</i> = 9). Mean values ± SEM are presented. The Kruskal-Wallis test was used for comparisons of three groups. NS for <i>P</i>>0.05, *<i>P</i><0.05, **<i>P</i><0.01. (C) Correlation between RTE frequencies and absolute CD4+ cell counts. Spearman’s rank correlation analysis was performed to determine the slope. ****<i>P</i><0.0001. (D) Percentage of circulating CD34+ cells in the peripheral blood of HIV-uninfected subjects (HIV-, <i>n</i> = 18), HIV+ IRs (<i>n</i> = 16) and INRs (<i>n</i> = 16). Median values are shown. The Kruskal-Wallis test was used for comparisons of three groups, NS for <i>P</i>>0.05. (E) Percentage of CD38^low cells among circulating CD34+ cells in some HIV-uninfected subjects (HIV-, <i>n</i> = 7), IRs (<i>n</i> = 11) and INR (<i>n</i> = 6). Median values are shown. The Kruskal-Wallis test was used for comparisons of three groups, NS for <i>P</i>>0.05.</p

Transcriptomic analysis of CD34+ cells in HIV-infected IRs and INRs.

<p>(A) Genes differentially expressed in the CD34+ cells of IRs (<i>n</i> = 7) and INRs (<i>n</i> = 10) are shown. Each column represents an individual sample, and each row, an individual gene, with normalized expression level indicated on a color scale (red = upregulation, green = downregulation). (B) The top 5 biological functions in Ingenuity analysis, based on activation <i>z</i>-score, an algorithm predicting the degree of activation or inactivation of the genes of the group concerned. Rank in the top 5 is indicated by the number after the #. <i>P</i>-values are shown. Biological functions upregulated (positive <i>z</i>-score) in IR patients are shown in red, and biological functions downregulated in IR patients (negative <i>z</i>-score) are shown in green.</p

Immune activation and inflammation in HIV-uninfected individuals and HIV-infected patients.

<p>(A) Percentage of CD38^high cells among CD8+ T lymphocytes (HIV-, <i>n</i> = 18; IRs, <i>n</i> = 16; INRs, <i>n</i> = 16). (B, C, D) Plasma concentrations of IL-6 (B), CRP (C) and sCD14 (D) in HIV-uninfected (HIV-, <i>n</i> = 3) and HIV-positive individuals (IRs, <i>n</i> = 15; INRs, <i>n</i> = 16). Bars indicate the mean and standard error. The Kruskal-Wallis test was used to assess differences between groups. NS for <i>P</i>>0.05, *<i>P</i><0.05, **<i>P</i><0.01.</p

Analysis of IL7R polymorphisms and Notch activation in CD34+ cells from IRs and INRs.

<p>(A) <i>IL7RA</i> polymorphisms in HIV-infected patients. The allelic frequency of each SNP is shown. Fisher’s exact test was used to compare the distribution of SNPs between IRs (<i>n</i> = 9) and INRs (<i>n</i> = 10). NS for <i>P</i> >0.05. (B) Expression of the Notch target gene <i>HES1</i> in purified CD34+ cells from HIV-uninfected subjects (<i>n</i>≥3), IRs (<i>n</i> = 4) and INRs (<i>n</i> = 6) exposed overnight to IgG1-Fc (IgG), Delta-like 4 (DLL-4), and DLL-4 plus hIL-7 (DLL-4+IL7) (5 μg/mL for DLL-4 and 5 ng/mL for IL-7). The mean and standard error are shown. Kruskal-Wallis and Friedman tests were used to compare differences in mRNA levels between groups, for each set of conditions (unpaired data), and between sets of conditions for the same group (paired data), respectively. *<i>P</i><0.05, **<i>P</i><0.01.</p