Lls were not functionally impaired, but were instead highly cytotoxic.Decreased CD96 MFI and Absolute Numbers is Associated with Disease ProgressionOne of the hallmarks of HIV-1 disease progression is CD4+ T cell depletion. We have shown that during HIV-1 infection the CD8+ T cell population express lower levels of CD96. These cells produce perforin following stimulation in vitro. Presence of abnormally high fractions of cytotoxic T cells could potentially contribute to immunopathogenesis and increased destruction of CD4+ T cells. Thus, to determine a potential clinical relevance ofCD96, we assessed CD96 expression JSH-23 site relative to CD4+ T cell counts. Due to the relatively small sample size of each group, correlations were assessed on the total number of HIV-1 infected individuals. We found that both the CD96 MFI on CD8+ T cells (r = 0.53 and p , 0.0004, n = 37) and the absolute number of CD96+CD8+ T cells (r = 0.35 and p = 0.03, n = 36) were positively correlated with CD4+ T cell order Ivosidenib counts (Fig. 4A and B). Commonly, high viral loads can be associated with lower CD4+ T cell counts. However, in the viremic individuals included in this study cohort no association was observed (data not shown). Instead, we established that the absolute numbers of TEM cells was positively correlated with viral load in viremic patients regardless of CDCD96 Expression during HIV-1 InfectionFigure 2. CD96 expression is down-modulated by LPS stimulation and up-regulated by TCR engagement. A) Percentage of CD38+ HLADR+ CD8+ T cells as a measure of immune activation. B) Association of CD96 MFI on CD8+ T cells and percentage of CD38+ HLA-DR+ CD8+ T cells (n = 40). C) Percentage of CD96 expression and D) CD96 MFI on CD8+ T cell following stimulation with either LPS, PHA, IL-12/18 and anti-CD3/CD28 for 24 hrs compared to unstimulated cells. Statistical analysis was performed using Student’s T test *p , 0.05, **p , 0.01, ***p , 0.001. Correlations were determined by two-tailed non-parametric Spearman correlations. doi:10.1371/journal.pone.0051696.gexpression (data not shown). Collectively this suggested that disease progression, as determined by CD4+ T cell counts, was correlated with CD96 expression, but was unaffected by viral loads. Instead the presence of virus promoted the expansion of the CD8+ TEM cell pool, but was not directly associated with CD96 expression.DiscussionCD96 is normally expressed by most T cells. However, the function of CD96 expression on T cells still remains elusive. Furthermore, modulations in CD96 expression during disease such as HIV-1 infection and relationship to pathogenesis has not previously been reported. In this study, we have shown that elite controllers significantly differ in their expression of CD96 as compared to noncontrollers. The reduced frequency of CD96 expressing CD8+ T cells were observed in all T cell subsets, although decreased density of CD96 expression was predominantly observed in the TEM population. The absolute numbers of CD96+ CD8+ T cells and the MFI were significantly associated with the peripheral CD4+ T cell counts. Collectively these data suggest that CD96 is potentially causally related to prevention of HIV-1-associated disease progression, although the cross-sectional nature of the study precludes definitive conclusions. Furthermore, we found that presence of LPS (which is thought to drive pathogenesis in HIV-1 disease) promoted CD96 down-regulation in vitro whereas direct TCR stimulation with anti-CD3 and anti-C.Lls were not functionally impaired, but were instead highly cytotoxic.Decreased CD96 MFI and Absolute Numbers is Associated with Disease ProgressionOne of the hallmarks of HIV-1 disease progression is CD4+ T cell depletion. We have shown that during HIV-1 infection the CD8+ T cell population express lower levels of CD96. These cells produce perforin following stimulation in vitro. Presence of abnormally high fractions of cytotoxic T cells could potentially contribute to immunopathogenesis and increased destruction of CD4+ T cells. Thus, to determine a potential clinical relevance ofCD96, we assessed CD96 expression relative to CD4+ T cell counts. Due to the relatively small sample size of each group, correlations were assessed on the total number of HIV-1 infected individuals. We found that both the CD96 MFI on CD8+ T cells (r = 0.53 and p , 0.0004, n = 37) and the absolute number of CD96+CD8+ T cells (r = 0.35 and p = 0.03, n = 36) were positively correlated with CD4+ T cell counts (Fig. 4A and B). Commonly, high viral loads can be associated with lower CD4+ T cell counts. However, in the viremic individuals included in this study cohort no association was observed (data not shown). Instead, we established that the absolute numbers of TEM cells was positively correlated with viral load in viremic patients regardless of CDCD96 Expression during HIV-1 InfectionFigure 2. CD96 expression is down-modulated by LPS stimulation and up-regulated by TCR engagement. A) Percentage of CD38+ HLADR+ CD8+ T cells as a measure of immune activation. B) Association of CD96 MFI on CD8+ T cells and percentage of CD38+ HLA-DR+ CD8+ T cells (n = 40). C) Percentage of CD96 expression and D) CD96 MFI on CD8+ T cell following stimulation with either LPS, PHA, IL-12/18 and anti-CD3/CD28 for 24 hrs compared to unstimulated cells. Statistical analysis was performed using Student’s T test *p , 0.05, **p , 0.01, ***p , 0.001. Correlations were determined by two-tailed non-parametric Spearman correlations. doi:10.1371/journal.pone.0051696.gexpression (data not shown). Collectively this suggested that disease progression, as determined by CD4+ T cell counts, was correlated with CD96 expression, but was unaffected by viral loads. Instead the presence of virus promoted the expansion of the CD8+ TEM cell pool, but was not directly associated with CD96 expression.DiscussionCD96 is normally expressed by most T cells. However, the function of CD96 expression on T cells still remains elusive. Furthermore, modulations in CD96 expression during disease such as HIV-1 infection and relationship to pathogenesis has not previously been reported. In this study, we have shown that elite controllers significantly differ in their expression of CD96 as compared to noncontrollers. The reduced frequency of CD96 expressing CD8+ T cells were observed in all T cell subsets, although decreased density of CD96 expression was predominantly observed in the TEM population. The absolute numbers of CD96+ CD8+ T cells and the MFI were significantly associated with the peripheral CD4+ T cell counts. Collectively these data suggest that CD96 is potentially causally related to prevention of HIV-1-associated disease progression, although the cross-sectional nature of the study precludes definitive conclusions. Furthermore, we found that presence of LPS (which is thought to drive pathogenesis in HIV-1 disease) promoted CD96 down-regulation in vitro whereas direct TCR stimulation with anti-CD3 and anti-C.