Innate and adaptive immune responses both contribute to pathological CD4 T cell activation in HIV-1 infected Ugandans

Abstract

HIV-1 disease progression is associated with persistent immune activation. However, the nature of this association is incompletely understood. Here, we investigated immune activation in the CD4 T cell compartment of chronically HIV-1 infected individuals from Rakai, Uganda. Levels of CD4 T cell activation, assessed as co-expression of PD-1, CD38 and HLA-DR, correlated directly to viral load and inversely to CD4 count. Deeper characterization of these cells indicated an effector memory phenotype with relatively frequent expression of Ki67 despite their PD-1 expression, and levels of these cells were inversely associated with FoxP3+ regulatory T cells. We therefore use the term deregulated effector memory (DEM) cells to describe them. CD4 T cells with a DEM phenotype could be generated by antigen stimulation of recall responses in vitro. Responses against HIV-1 and CMV antigens were enriched among the DEM CD4 T cells in patients, and the diverse Vβ repertoire of DEM CD4 T cells suggested they include diverse antigen-specificities. Furthermore, the levels of DEM CD4 T cells correlated directly to soluble CD14 (sCD14) and IL-6, markers of innate immune activation, in plasma. The size of the activated DEM CD4 T cell subset was predictive of the rate of disease progression, whereas IL-6 was only weakly predictive and sCD14 was not predictive. Taken together, these results are consistent with a model where systemic innate immune activation and chronic antigen stimulation of adaptive T cell responses both play important roles in driving pathological CD4 T cell immune activation in HIV-1 disease.

Figure 1. PD-1, HLA-DR, and CD38 together identify an effector memory CD4 T cell subset elevated in HIV-1 infection.

(A) Identification of CD4 T cells co-expressing PD-1, HLA-DR and CD38 in HIV-1 infected and uninfected subjects. (B) Box and whisker plots showing the median and 10–90 percentile percentage of CD38, HLA-DR and PD-1 triple-expression in CD4 T cells in HIV negative (n = 40) and HIV positive (n = 103) subjects. (C) In order to assess the phenotype of the activated CD4 T cells, PBMC were analyzed by flow cytometry for the expression of CD45RO, CCR7, CD28, and Ki67. Filled line represents CD4 cells triple-positive for CD38, HLA-DR, and PD-1, while dashed line represents data from the overall CD4 compartment and empty filled line is the fluorescence minus one (FMO) control for that channel.

Figure 2. Inverse correlation between FoxP3+ regulatory T cells and PD-1+CD38+HLA-DR+ CD4 T cells.

(A) Box and whisker plots showing the median and 10–90 percentile percentage of FoxP3+CD25+CD127- regulatory CD4 T cells in HIV negative and HIV positive subjects. (B) Spearman rank correlation between regulatory CD4 T cells and CD4 absolute T cell counts. (C) Spearman rank correlation between regulatory CD4 T cells and viral load. (D) Spearman rank correlation between regulatory CD4 T cells and CD38, HLA-DR and PD-1 triple-positive CD4 T cells in blood.

Figure 3. Levels of deregulated effector memory CD4 T cells correlate to and predict HIV-1 disease progression.

(A) Spearman rank correlation between percentages of CD38, HLA-DR and PD-1 triple-expressing CD4 T cells and HIV-1 plasma viral load. (B) Spearman rank correlations between percentages of CD38, HLA-DR and PD-1 triple-expressing CD4 T cells and CD4 counts. (C) Kaplan-Meier analysis comparing the time to AIDS, as defined by CD4 T cell counts less than 250 cells/µl of whole blood. HIV-1 positive subjects with CD38, HLA-DR and PD-1 triple-expressing CD4 T cells above the median (6%) were more likely to advance to AIDS (Log-rank  = 0.001, Hazard Ratio (HR) 0.336, 95% CI = 0.173 to 0.653).

Figure 4. Associations between sCD14 and IL-6 levels in plasma and measures of HIV-1 disease progression.

(A) Box and whisker plot showing sCD14 levels in plasma from HIV-1 infected (n = 103) and HIV-1 uninfected (n = 40) subjects. Spearman rank correlations between sCD14 in plasma and (B) CD4 absolute T cells counts, and (C) viral load. (D) Kaplan-Meier analysis comparing the time to AIDS. HIV-1 positive subjects with sCD14 levels above and below the median (1939 ng/ml) were not statistically different (Log-rank P = 0.185, Hazard Ratio (HR) 0.631, 95% CI = 0.319 to 1.247). (E) Box and whisker plot showing IL-6 levels in plasma from HIV-1 infected (n = 103) and HIV-1 uninfected (n = 40) subjects. Spearman rank correlations between IL-6 in plasma and (F) CD4 absolute T cells counts and (G) viral load. (H) Kaplan-Meier analysis comparing the time to AIDS. HIV-1 positive subjects with IL-6 levels above the median (3.2 pg/ml) more likely to advance to AIDS (Log-rank P = 0.023, HR 0.462, 95% CI = 0.238–0.900).

Figure 5. sCD14 and IL-6 correlate with levels of deregulated effector memory CD4 T cells, but not with Tregs.

(A) Spearman rank correlation between sCD14 in plasma and CD38, HLA-DR and PD-1 triple-positive CD4 T cells in blood. (B) Spearman rank correlation between IL-6 in plasma and CD38, HLA-DR and PD-1 triple-positive CD4 T cells. (C) Spearman rank correlation between IL-6 and sCD14 in plasma. (D) Spearman rank correlation between Tregs and IL-6. (E) Spearman rank correlation between Tregs and sCD14 levels.

Figure 6. CD38+HLA-DR+PD-1+ cells can be driven by antigen in vitro, but display a non-biased Vβ distribution in vivo.

(A) Experiments were conducted to assess the ability to generate the combined expression of CD38, HLA-DR, and PD-1 on CD4 T cells after three days of mitogen stimulation, or six days of antigen or superantigen stimulation of PBMC from four healthy donors. Bars represent mean and standard error of the mean. (B) Example PD-1 expression flow cytometry histogram and contour plot for CD38 and HLA-DR expression on CD4 T cells from one CMV non-responder (top panels), and one CMV responder after six days of stimulation with CMV lysate. (C) In order to characterize the prevalence of antigen specific cells in the CD38, HLA-DR and PD-1 triple-positive CD4 T cell subset, thawed PBMC were stimulated with HIV Gag peptide pool, whole inactivated HIV-1 (HIV_WIV), CMV pp65 peptide pool and lysate, and the superantigen SEB. Grouped columnar scatter plots for responses to Gag peptides or whole inactivated virus (n = 10), CMV lysate responders (n = 18) and SEB responders (n = 18). Cells co-expressing IFNγ and TNFα were considered positive in this assay. Data were analyzed using the paired t test. (D) In order to assess the TCR Vβ repertoire of CD38+HLA-DR+PD-1+ CD4 T cells, the representation of 17 different Vβ specificities were analyzed by flow cytometry. Grouped vertical bar graph showing the mean and standard deviation for 10 HIV-1 infected patients. White bars represent the overall CD4 T cell compartment, while grey bars represent the CD38, HLA-DR, and PD-1 triple-expressing CD4 T cells. There is no statistically significant difference in representation of any Vβ between the two CD4 T cell subsets.