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. 2015 Jun;96(Pt 6):1463-1477.
doi: 10.1099/vir.0.000065. Epub 2015 Jan 27.

Nef Promotes Evasion of Human Immunodeficiency Virus Type 1-infected Cells From the CTLA-4-mediated Inhibition of T-cell Activation

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Free PMC article

Nef Promotes Evasion of Human Immunodeficiency Virus Type 1-infected Cells From the CTLA-4-mediated Inhibition of T-cell Activation

Mohamed El-Far et al. J Gen Virol. .
Free PMC article

Abstract

CTLA-4 is a negative regulator of T-cell receptor-mediated CD4(+) T-cell activation and function. Upregulation of CTLA-4 during human immunodeficiency virus type 1 (HIV-1) infection on activated T cells, particularly on HIV-specific CD4(+) T cells, correlates with immune dysfunction and disease progression. As HIV-1 infects and replicates in activated CD4(+) T cells, we investigated mechanisms by which HIV-1 modulates CTLA-4 expression to establish productive viral infection in these cells. Here, we demonstrate that HIV-1 infection in activated CD4(+) T cells was followed by Nef-mediated downregulation of CTLA-4. This was associated with a decreased T-cell activation threshold and significant resistance to CTLA-4 triggering. In line with these in vitro results, quantification of pro-viral HIV DNA from treatment-naive HIV-infected subjects demonstrated a preferential infection of memory CD4(+)CTLA-4(+) T cells, thus identifying CTLA-4 as a biomarker for HIV-infected cells in vivo. As transcriptionally active HIV-1 and Nef expression in vivo were previously shown to take place mainly in the CD3(+)CD4(-)CD8(-) [double-negative (DN)] cells, we further quantified HIV DNA in the CTLA-4(+) and CTLA-4(-) subpopulations of these cells. Our results showed that DN T cells lacking CTLA-4 expression were enriched in HIV DNA compared with DN CTLA-4(+) cells. Together, these results suggested that HIV-1 preferential infection of CD4(+)CTLA-4(+) T cells in vivo was followed by Nef-mediated concomitant downregulation of both CD4 and CTLA-4 upon transition to productive infection. This also highlights the propensity of HIV-1 to evade restriction of the key negative immune regulator CTLA-4 on cell activation and viral replication, and therefore contributes to the overall HIV-1 pathogenesis.

Figures

Fig. 1.
Fig. 1.. Phenotypic characterization and permissiveness to HIV-1 infection of CD4+CTLA-4+ T cells. Total CD4+ T cells were isolated by negative selection using magnetic beads, and stimulated with immobilized CD3 (1 µg ml−1) and soluble CD28 antibodies (0.5 µg ml−1) for 72 h. (a) Representative surface staining for surrogate activation markers co-expressed with CTLA-4 (intracellular) on activated CD4+ T cells. Analysis was performed on gated CD3+CD4+ T cells that excluded the viability dye LIVE/DEAD. (b) Enrichment of CTLA-4high T cells by sorting CD25+ T cells using flow cytometry. Left panel: CTLA-4 and CD25 levels on activated T cells prior to sorting. Right panels: purity of the two populations CTLA-4hi and CTLA-4lo after cytometry sorting. (c) Representative HIV p24 intracellular staining in CD25+CTLA-4hi (upper panels) and CD25CTLA-4lo (lower panels) T cells infected with HIV-1Nef+ or HIV-1ΔNef at 48 h post-infection. SSC, side scatter. (d) Mean±sd frequency of HIV-infected cells in CD25+CTLA-4hi and CD25CTLA-4lo T cells when cells were infected with HIV-1Nef+ or HIV-1ΔNef (n = 3). Paired t-test P values are indicated.
Fig. 2.
Fig. 2.. HIV-1 Nef downregulates CTLA-4 expression in HIV-infected primary CD4+ T cells. CD4+ T cells were activated for 48 h as in Fig. 1 and exposed to HIV for an additional 48 h. (a) Representative dot-plots showing the percentage of HIV p24+ cells (left panels) expressing CTLA-4 (intracellular and extracellular), CD4 and CD3 after infection with HIV-1ΔNef (upper panels) or HIV-1Nef+ (lower panels). Numbers in each quadrant refer to the percentage of HIV p24+ T cells expressing CTLA-4, CD4 and CD3. (b) Percentages (upper panels) and MFI (lower panels) of CTLA-4, CD4 and CD3 following infection with HIV-1Nef+ or HIV-1ΔNef (n = 22). Paired t-test P values are indicated. (c) Left panels: representative FACS data for CTLA-4 (upper panels) and CD3 expression (lower panels) on total GFP+ and GFP cells after infection with GFP-HIV-1ΔNef, GFP-HIV-1Nef+, GFP-SIVcpzNef+ and GFP-SIVmac239Nef+ (representative of n = 3). Right panels: histograms showing the frequency of CTLA-4+ and CD3+ cells from the GFP+ subpopulations (infected cells) for the four different viruses at 48 h post-infection (n = 3). Paired t-test P values are indicated; ns, not significant.
Fig. 3.
Fig. 3.. CTLA-4 downregulation by Nef coincides with enhanced IL-2 production in primary CD4+ T cells. CD4+ T cells were activated for 48 h as in Fig. 1, exposed to HIV-1 for an additional 48 h and restimulated via CD3/CD28 antibodies (plate-coated CD3 antibodies, 0.1 µg ml−1; soluble CD28 antibodies, 0.1 µg ml−1). (a) Left panel: frequency of CTLA-4+ cells on the HIV p24+ subpopulations from HIV-1ΔNef- compared with HIV-1Nef+-infected cells at 48 h post-infection (n = 5). Percentages (middle panel) and MFI (right panel) of IL-2-expressing T cells measured by intracellular staining in the total CD4+ T cells (including both HIV p24+ and p24) infected with HIV-1Nef+ or HIV-1ΔNef upon 15 h of TCR restimulation (the same donors as in the left panel). (b) Representative dot-plots showing the frequency of IL-2-expressing cells in the HIV p24+ and p24 populations from cells infected with HIV-1Nef+ and HIV-1ΔNef compared with non-infected control cells. (c) Percentages (left panel) and MFI (right panel) of IL-2+ cells within the p24+ or p24 populations in cells infected with HIV-1Nef+ or HIV-1ΔNef. (d) IL-2 production was measured by ELISA at 24 h following TCR restimulation (plate-coated CD3 antibodies, 0.1 µg ml−1; soluble CD28 antibodies, 0.1 µg ml−1) of cells infected with HIV-1Nef+ or HIV-1ΔNef and uninfected cells (Mock) from three different donors. (e) Levels of IL-2 production measured by ELISA after TCR and CTLA-4 simultaneous cross-linking on CD4+ T cells infected with either HIV-1Nef+ or HIV-1ΔNef in eight donors. Simultaneous TCR and CTLA-4 cross-linking was carried out using beads coated with CD3 and CD28 antibodies (35 and 7 ng/20×106 beads, respectively; four beads per cell) and a fixed concentration (1 µg/20×106 beads) of CTLA-4 antibodies. Relative IL-2 values were calculated by dividing the concentration of IL-2 produced upon CTLA-4 and TCR simultaneous cross-linking by the concentration of IL-2 produced after TCR stimulation alone. Nef-mediated CTLA-4 downregulation in cells infected with HIV-1Nef+ (as shown in Fig. 2) was confirmed in each experiment prior to CTLA-4 triggering. Paired t-test P values are indicated; ns, not significant.
Fig. 4.
Fig. 4.. CTLA-4 downregulation is associated with decreased T-cell activation threshold and enhanced HIV-1 replication upon CTLA-4 triggering. CD4+ T cells were activated for 48 h as in Fig. 1, exposed to HIV for an additional 24 h, and restimulated via beads coated with CD3/CD28 and CTLA-4 antibodies or matched isotype control for an additional 24 h (for IL-2 quantification), 4 days (for HIV DNA quantification) or 9 days (for HIV p24 quantification). (a) Levels of IL-2 production measured by ELISA in a representative subject. Simultaneous TCR and CTLA-4 cross-linking was carried out using decreasing concentrations of CD3 and CD28 antibodies (CD3: 280–35 ng/20×106 beads; CD28: 56–7 ng/20×106 beads; four beads per cell) and a fixed concentration (1 µg/20×106 beads) of CTLA-4 antibodies on cells infected with HIV-1Nef+ or HIV-1ΔNef. (b) Fold increase in IL-2 production from HIV-1Nef+ over HIV-1ΔNef under simultaneous TCR and CTLA-4 cross-linking (mean±sd from four subjects). (c) CTLA-4-dependent inhibition of viral replication as monitored by quantification of total HIV DNA at day 4 following TCR (CD3 and CD28 antibodies 35/7 ng/20×106 beads, respectively; four beads per cell) and CTLA-4 (1 µg/20×106 beads) cross-linking (or isotype control: Iso-IgG) on HIV-1Nef+- and HIV-1ΔNef-infected cells (n = 4). (d) HIV p24 production measured by ELISA at day 6 following simultaneous TCR and CTLA-4 cross-linking on cells infected with eGFP-HIV-1 virus expressing Nef compared with the ΔNef virus. Shown are the p24 production levels from both viral infections under simultaneous TCR and CTLA-4 cross-linking relative to TCR+ Isotype activation (n = 3). Prior to TCR/CTLA-4 triggering, CD4+ T cells infected for 24 h with eGFP viruses were treated with and maintained in 5 µM azidothymidine-containing medium to prevent reinfection. Paired t-test P values are indicated.
Fig. 5.
Fig. 5.. Enrichment in pro-viral HIV DNA in memory CD4+CTLA-4+ and memory DN CTLA-4 T cells from treatment-naive subjects. (a) Gating strategy used to sort CTLA-4hi, CTLA-4med and CTLA-4 subsets within memory CD45RACD4+ T cells from PBMCs of treatment-naive HIV-infected subjects. SSC, side-scatter. (b) Frequency of cells harbouring integrated HIV DNA in each sorted cell subset. CD4, CD4 count (mm–3); VL, viral load [log (copies ml−1)]. (c) Frequency of CD3+CD4CD8 DN T cells from treatment-naive HIV-infected subjects compared with HIV-negative and cART-treated subjects (n = 13 in each group). Non-parametric Mann–Whitney two-tailed test P values are indicated. (d) Left panel: CTLA-4 levels on total DN T cells from HIV-negative (n = 3) and HIV-1 viraemic subjects (n = 3). Right panel: CTLA-4 expression on DN T cells expressing (or not) CD45RA in a representative HIV-infected subject. (e) Quantification of integrated HIV DNA in memory (CD45RA) and naive (CD45RA+) subsets of both CD4+ and DN T cells in four HIV-infected viraemic subjects. CD4, CD4 count (mm–3); VL, viral load [log (copies ml−1)].
Fig. 6.
Fig. 6.. Expression of activation markers on memory CD4+CTLA-4+ and memory DN CTLA-4 T cells. PBMCs from HIV-infected and uninfected subjects were analysed for the surface expression of HLA-DR and the intracellular expression of Ki67 on gated CD3+ T-cell subsets ex vivo. (a) Expression of Ki67 (upper panels) and HLA-DR (lower panels) on memory CD4+ T-cells (CTLA-4hi) and memory DN T cells (CTLA-4) from a representative treatment-naive HIV-infected subject and a representative HIV-negative control. (b) HLA-DR and Ki67 expression in these subsets (six subjects per group). (c) Expression of the activation marker Ki67 on the memory fraction of DN T cells (CTLA-4) from viraemic HIV-infected subjects (n = 17) compared with aviraemic cART-treated subjects (n = 15), elite controllers (EC, n = 15) and HIV-negative subjects (n = 15). Non-parametric Mann–Whitney two-tailed test P values are shown in (b) and (c). (d) Correlation between the expression of Ki67 on the memory fraction of DN T-cells (CTLA-4) and plasma viral load in HIV-infected treatment naive subjects (n = 17). Spearman correlation r (rho) and P values are indicated in (d).

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