Diminished Peripheral CD29hi Cytotoxic CD4+ T Cells Are Associated With Deleterious Effects During SIV Infection

Abstract

Cytotoxic CD4+ T cells (CD4+ CTLs) limit HIV pathogenesis, as evidenced in elite controllers (a subset of individuals who suppress the virus without the need for therapy). CD4+ CTLs have also been shown to kill HIV-infected macrophages. However, little is known about their contribution towards HIV persistence, how they are affected following exposure to immune modulators like morphine, and what factors maintain their frequencies and function. Further, the lack of robust markers to identify CD4+ CTLs in various animal models limits understanding of their role in HIV pathogenesis. We utilized various PBMC samples obtained from SIV infected and cART treated rhesus macaques exposed to morphine or saline and subjected to flow cytometry evaluations. Thereafter, we compared and correlated the expression of CD4+ CTL-specific markers to viral load and viral reservoir estimations in total CD4+ T cells. We found that CD29 could be reliably used as a marker to identify CD4+ CTLs in rhesus macaques since CD29hi CD4+ T cells secrete higher cytotoxic and proinflammatory cytokines following PMA/ionomycin or gag stimulation. In addition, this immune cell subset was depleted during untreated SIV infection. Strikingly, we also observed that early initiation of cART reconstitutes depleted CD29hi CD4+ T cells and restores their function. Furthermore, we noted that morphine exposure reduced the secretion of proinflammatory cytokines/cytotoxic molecules in CD29hi CD4+ T cells. Lastly, increased functionality of CD29hi CD4+ T cells as depicted by elevated levels of either IL-21 or granzyme B hi T Bet+ gag specific responses were linked to limiting the size of the replication-competent reservoir during cART treatment. Collectively, our data suggest that CD4+ CTLs are crucial in limiting SIV pathogenesis and persistence.

Keywords: CD29+ CD4+ T cells; CD4+ CTLs; SIV; biomarker; morphine; reservoirs.

Figure 1

Loss of CD29 hi CD4+ T cells are associated with increased viral loads, declining immune status, and dysregulated NK cell subsets in SIV infected rhesus macaques exposed to either morphine or saline. Viral loads at baseline (day 0), acute (day 14), and chronic (day 245) during untreated SIV infection (A). Changes in percent CD4+ T cells (B), percent CD8+ T cells (C), CD4/CD8 ratio (D), percent CD29+ CD4+ T cells (E), percent CD29+ CD8+ T cells (F), percent CD29+/CD4+/HLA-DR+ (G), percent CD29+/CD8+/HLA-DR+ (H) and CD8+/CD16+ T cells (I) at the three studied time points (day 0, day 14 and day 245) post infection. Evaluating NK cells and comprising subsets that include changes in total NK cells (J), CD16+CD56- NK cells (K), CD16-CD56+ NK cells (L), and CD16-CD56- NK cells (M) at day 0, day 14, and day 245 post infection. (N) Correlogram showing correlations between various immune cell subsets and percent CD29+ CD4+ T cells.. Relationship between either (O) percent CD29+ CD4+ T cells or (P) absolute CD29+ CD4+ T cells and viral load. * shows p < 0.05, ** indicates p < 0.01 while *** denotes p < 0.001 resulting from Wilcoxon matched pairs signed rank tests between paired groups of the different comparisons. ns represents non significant. Per visit comparisons between morphine and saline were also performed using the Mann Whitney U test (non- significant).

Figure 2

Early cART initiation (4 weeks) restores the loss of CD29hi CD4+ T cells and restores polyfunctionality. (A) Representative gating used to obtain CD4+ CD29 hi T cells used for changes in cytokine secretion at different stages of SIV progression. Briefly, CD4+ T cells were obtained from gated CD3+ T cells/lymphocytes/live cells/singlets/Time vs FSC-A populations. Thereafter, CD29 hi populations were then gated out from CD4+ T cells and the extent of CD95 expression and IFN-γ/TNF-α secretion. Following this, the changes of (B) %CD29hi CD4+ T cells (C) %CD95+ CD29hi CD4+ T cells (D) IFN-γ+ CD29 hi CD4+ T cells (E) TNF-α+ CD29hi CD4+ T cells evaluated at baseline, week 2, week 7 and week 22 post SIV infection. (F) Polyfunctionality profiles of CD29hi CD4+ T cells stimulated with PMA/ionomycin. Included pie charts/bar graphs represent time course changes in CD29hi poly functionality based on IFN-γ, TNF-α and CD95 expression (n = 8). Bar graphs show the median percent cytokine expressing cells. All paired comparisons across the different timepoints were performed using the non-parametric Wilcoxon test. * shows p < 0.05 while ** indicates p < 0.01.

Figure 3

CD29hi CD4+ T cells identify cytotoxic CD4+ T cells (CD4+ CTLs) with superior cytotoxicity, expression of inflammatory surface molecules and cytokines following stimulation with PMA/ionomycin in cART treated rhesus macaques exposed to saline or morphine during chronic infection. The expression percent CD11a (A), percent CD11b (B), the chemokine receptor CX3CR1 (C), percent Eomes (D) and percent T bet (E) was evaluated and compared between CD29lo vs. CD29hi T cells. Similarly, cytotoxicity was evaluated based on percent granzyme B (F), percent Granzyme B hi (G), and Granzyme B hi and T bet co-expression (H). In addition, the expression of percent IL-4 (I), percent CD107a (J), percent IL-21 (K), percent IFN-γ (L), percent TNF-α (M), dual co-expression of IFN-γ and TNF-α (N) and IL-21 and IFN-γ co-expression (O) was evaluated. The changes in the secretion of cytokines and cytotoxic molecules IL-21 (P), CD107a (Q), TNF-α (R), IFN-γ (S), IFN-γ and TNF-α (T) and IL-21 and IFN-γ (U) within CD29 hi CD4+ T cells following morphine exposure were evaluated using ICS. Representative gating of CD29 hi versus CD29 lo cells expressing multiple cytokines such as IL-21, CD107a, TNF-α and IFN-γ (V). Polyfunctional profile of CD29hi vs CD29lo CD4+ T cells responding to PMA/ionomycin. (I) Representative manual gating of CD29hi vs CD29lo CD4+ T cells used to analyze polyfunctionality. (II) and (III) Pie charts and the bar plots show the frequency of CD29+ or CD29- CD4+ T cells expressing different combinations of cytokines (TNF-α, IFN-γ, IL-21, IL-4 and CD107a) in n=11 rhesus macaques. Paired comparisons between CD29lo and CD29hi CD4+ T cells were performed using the non-parametric Wilcoxon test. Comparisons between morphine and saline were also performed in CD29+ CD4+ T cells using the Mann Whitney U test. For SPICE analyses, pie charts showing cytokine secretion patterns together with arc plots show the predominant cytokine being secreted. Bar graphs denote median cytokine expressing cells. Simultaneously, the extent of cytokine polyfunctionality was compared within CD29lo vs CD29 hi CD4+ T cell subsets with aid of the Wilcoxon test. For all comparisons, * shows p < 0.05, ** indicates p < 0.01 while *** denotes p < 0.001. ns represents non significant. Red dots denote morphine treated while blue dots represent rhesus macaques exposed to saline.

Figure 4

CD29 hi CD4+ T cells express elevated inflammatory cytokines and cytotoxic molecules following stimulation with gag peptides in cART treated rhesus macaques exposed to saline or morphine during chronic infection. Comparative expression of inflammatory cytokines and cytotoxic molecules between CD29 hi vs CD29 lo CD4+ T cells was performed as depicted by percent IFN-γ (A), percent CD107a (B), percent IL21 (C), percent TNF-α (D), percent IL-4 (E), percent IFN-γ+ TNF-α + and CD107a- (F), percent IFN-γ- TNF-α + and CD107a+ (G), Granzyme B hi (H), MFI of Granzyme B+ hi (I), Granzyme B+ hi T bet+ (J) gag specific CD4+ T cells. Immune modulation by morphine on CD29 hi CD4+ CTLS was analyzed for the expression of various cytokines such as percent IFN-γ (K) percent IL21 (L) percent CD107a (M), percent TNF-α (N), percent Granzyme B+ hi T bet+ cells (O). Scheme for correlation analysis between gag specific IL-21 or Granzyme B hi T bet+ CD29 hi CD4+ T cells and QVOA measures of replication competent reservoirs in total CD4+ T cells (P). Correlation between gag-specific IL-21+ CD29hi CD4+ T cell frequencies and QVOA (Q). Correlation between gag specific Granzyme B+ hi T bet+ CD29hi CD4+ T cell frequencies and QVOA (R). Paired comparisons between CD29 lo and CD29 hi CD4+ T cells were performed using the non-parametric Wilcoxon test. Comparisons between morphine and saline were also performed in CD29+ CD4+ T cells using the Mann Whitney U test. For all comparisons, * shows p < 0.05, ** indicates p < 0.01 while *** denotes p < 0.001. ns signifies non significant. Red dots denote morphine treated while blue dots represent rhesus macaques exposed to saline.