HIV-induced changes in T cell signaling pathways

Abstract

Infection with HIV usually results in chronic activation of the immune system, with profound quantitative and qualitative changes in the T cell compartment. To better understand the mechanistic basis for T cell dysfunction and to discern whether such mechanisms are reversed after effective antiviral treatment, we analyzed changes in signaling pathways of human CD4(+) and CD8(+) T cells from 57 HIV-infected subjects in varying stages of disease progression and treatment, including long-term nonprogressors, progressors, and chronically infected subjects provided effective antiretroviral therapy (responders). A previously described PhosFlow method was adapted and optimized so that protein phosphorylation could be visualized in phenotypically defined subpopulations of CD4(+) and CD8(+) T cells (naive, memory, and effector) by flow cytometry. T cell signaling induced by TCR cross-linking, IL-2, or PMA/ionomycin was found to be blunted within all T cell subpopulations in those with progressive HIV disease compared with long-term nonprogressors and responders. Although alterations in cellular signaling correlated with levels of basal phosphorylation, viral load, and/or expression of programmed death-1, it was the level of basal phosphorylation that appeared to be the factor most dominantly associated with impaired signaling. Notably, provision of effective antiretroviral therapy was associated with a normalization of both basal phosphorylation levels and T cell signaling. These data, in aggregate, suggest that generalized dysfunction of the T cell compartment during progressive HIV disease may be in part dependent upon an increased basal level of phosphorylation, which itself may be due to the heightened state of immune activation found in advanced disease.

FIGURE 1

A, Representative flow cytometric plots of staining and gating strategy of fixed/permeabilized cells used in signaling analyses by PhosFlow. Lymphocytes and singlets were identified in forward and sideward scatter dot plots. Dead cells were excluded by amine stain, and live cells were gated for expression of CD3. The CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells were further separated by expression of CD45RA and CD27 to identify naive (CD45RA⁺CD27⁺), memory (CD45RA⁻CD27⁺), memory-effector (CD45RA⁻CD27⁻), and effector (CD45RA⁺CD27⁻) T cells as indicated. B, Mean frequencies of CD4⁺ and CD8⁺ T cells within the CD3⁺ compartment (left) and of naive, memory, memory-effector, and effector T cells within CD4⁺ (middle) and CD8⁺ T cells (right) in the three HIV-infected groups of LTNP, PROGs, and RESPs. Error bars indicate SD. C, Cell surface expression levels of CD3 on CD3⁺ T cells (left) and CD4 or CD8 on naive, memory, memory-effector, and effector CD4⁺ or CD8⁺ subpopulations (middle and left, respectively) from three HIV-infected groups as indicated. Expression levels of cell surface markers were measured as MFI and normalized (ΔMFI) to an HIV-uninfected standard control, which was included in all experiments. For the box-and-whisker graph, the lines in the boxes represent median values, the boxes range from the 25^th to 75^th percentiles, and the error bars indicate the lowest and highest values. Groups in B and C were compared using the nonparametric two-tailed Mann-Whitney U test; statistically significant differences are indicated by the lines below the plots. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 2

T cell-specific stimulation by TCR cross-linking induces specific signaling analysis, as analyzed by PhosFlow, and Ca²⁺-influx. A, Histograms of MFI of p-Zap70 in CD4⁺ (left) and CD8⁺ T cells (right). PMBCs were stained for expression of CD3, CD4, and CD8 and subsequently incubated with biotinylated Abs binding either CD3 + 28 + 4 or anti-CD3 + 28 + 8 or without Abs. TCR-mediated signaling was activated by cross-linking biotinylated Abs with streptavidin for 15 min. After fixation and permeabilization, cells were stained with an Ab detecting p-Zap70. Levels of p-Zap70 within CD4⁺ and CD8⁺ T cells were analyzed by flow cytometry. B, Bar graphs represent levels of p-Zap70 as quantified by MFI and fold-changes (stimulated over unstimulated) of MFI after specific TCR cross-linking, as indicated below, in CD4⁺ (left) and CD8⁺ T cells (right). C, TCR cross-linking induces cellular Ca²⁺-influx. PBMCs were incubated with biotinylated Abs binding CD3 + 8 before staining with a combination of anti-CD3, anti-CD8, and anti-CD4 Abs. The TCR signaling was activated by adding streptavidin, and Ca²⁺ influx was measured with the fluorescent calcium indicator, Indo-1. Calcium release was measured by flow cytometry over time by the change in emission spectrum from blue to violet (Indo-1 AM). Stimulation with the calcium ionophore, ionomycin, served as a positive control.

FIGURE 3

Signaling is comparable between T cells from HIV-uninfected individuals and HIV-infected LTNP, but blunted in T cells from HIV PROGs. Comparing stimulated over unstimulated cells, fold-changes in phosphorylation were analyzed in naive, memory, memory-effector, and effector subpopulations of CD4⁺ (no effector T cells analyzed) and CD8⁺ T cells. Protein phosphorylation after specific stimulation was analyzed between (A–C) HIV-infected individuals and (D–F) LTNP and HIV-uninfected individuals for (A and D) Lck and Zap70 after TCR stimulation, (B and E) Stat5 after stimulation with IL-2, and (C and F) ERK1/2 and p38 after stimulation with PMA/ionomycin. The groups were compared using the nonparametric two-tailed Mann-Whitney U test; statistically significant differences are indicated by the lines below the plots. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 4

Higher levels of HIV VL are associated with blunted cellular signaling. Fold-changes in phosphorylation (x-axis) after stimulation (as indicated on top) were correlated with levels of HIV VL (y-axis) in CD4⁺ and CD8⁺ T cell subpopulations (as indicated on left). These analyses were performed for a subset of LTNPs and PROGs, those that had detectable VL. Two-tailed Spearman's rank correlation was used to analyze the relationship between signaling and levels of VL; statistically significant correlations are indicated. *, p < 0.05; **, p < 0.01; ***, p < 0.001; R, correlation coefficient.

FIGURE 5

Levels of PD-1 expression are correlated with cellular signaling in HIV infection. A, Mean cell surface expression levels of PD-1 on naive, memory, memory-effector, and effector subpopulations of CD4⁺ and CD8⁺ T cells (left and right, respectively) from three HIV-infected groups. Expression levels of PD-1 were measured as MFI and normalized (ΔMFI) to an HIV-uninfected standard control, which was included in all experiments. Error bars indicate SE. HIV-infected groups were compared using the nonparametric two-tailed Mann-Whitney U test, statistically significant differences are indicated by the lines below the plots. B, Fold-changes in phosphorylation (x-axis) after stimulation (as indicated on top) were correlated with expression levels of PD-1 (y-axis) in CD4⁺ and CD8⁺ T cell subpopulations (as indicated on left). Two-tailed Spearman's rank correlation was used to analyze the relationship between signaling and levels of VL; statistically significant correlations are indicated. *, p < 0.05; **, p < 0.01; ***, p < 0.001; R, correlation coefficient.

FIGURE 6

Basal phosphorylation levels are elevated in T cells from HIV PROGs. Levels of basal phosphorylation were analyzed in naive, memory, memory-effector, and effector subpopulations of CD4⁺ (left; no effector T cells analyzed) and CD8⁺ T cells (right). Protein phosphorylations were analyzed for (A) Lck and Zap70, (B) Stat5, and (C) ERK1/2 and p38. The HIV-infected groups were compared using the nonparametric two-tailed Mann-Whitney U test; statistically significant differences are indicated by the lines below the plots. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 7

High levels of basal phosphorylation are associated with blunted signaling in HIV infection. Fold-changes in phosphorylation (x-axis) after stimulation (as indicated on top) were correlated with basal levels of phosphorylation (y-axis) in CD4⁺ and CD8⁺ T cell subpopulations (as indicated on left). Two-tailed Spearman's rank correlation was used to analyze the relationship between signaling and levels of VL; statistically significant correlations are indicated. *, p < 0.05; **, p < 0.01; ***, p < 0.001; R, correlation coefficient.

FIGURE 8

Overview of significances of correlations analyzed using a heat map representation. Values of p from previous correlation analyses are here color-coded (*, p < 0.05 in dark gray; **, p < 0.01 in gray; ***, p < 0.001 in light gray; not significant (ns) in black). Columns show p values for correlation of signaling (Lck and Zap70 after TCR stimulation; ERK1/2 and p38 after stimulation with PMA/ionomycin; and Stat5 after IL-2 stimulation) with (i) basal phosphorylation (left; compare Fig. 8), (ii) VL (middle; compare Fig. 4), and (iii) cell surface expression of PD-1 (right; compare Fig. 5). Corresponding CD4⁺ and CD8⁺ T cell subpopulations analyzed are ordered in rows as indicated on the right.