T was not directly associated with CD96 expression.Epigenetic Reader Domain 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-CD28, but not PHA, resulted in increased per cell CD96 density. We also observed that cells lacking CD96 on CD8+ Tcells represented a population that produced both IFNc and perforin following stimulation. All together, these data suggest that changes in CD96 expression may be a useful additional marker to measure 25837696 overall effector function and disease progression during HIV-1 infection. In HIV-1 infected individuals chronic immune activation is a common feature where increased CD8+ T cell activation is associated with CD4+ T cell depletion [23,24]. During HIV-1 infection there is also evidence that bacterial translocation occurs as LPS and bacterial DNA have been detected in the blood of HIV-1 infected individuals [20,25]. CD96 is abundantly expressed on resting T cells, but interestingly we found that in vitro LPS stimulation of PBMCs from healthy donors decreased CD96 expression. It has already been reported that CD96 can be shed during chronic disease such as Hepatitis B infection [18]. Autophagy Correspondingly, we observed that CD96 expression was decreased during chronic HIV-1 infection in our cohort. The total CD8+ T cell population of elite controllers was also found to have decreased frequencies of CD96+ expressing cells compared to healthy controls, although density per cell was maintained. These data indicate that inflammatory responses to LPS is a contributing mechanism by which downregulation of CD96 expression is induced. LPS translocation may therefore to some extent explain the down-regulation of CD96 observed in the subjects of this cohort. In contrast to LPS, direct TCR stimulation in vitro 
instead increased the density of CD96 expression. This is in accordance with previous studies that report upregulation of CD96 on T cells uponCD96 Expression during HIV-1 InfectionFigure 3. CD8+ T cells lacking CD96 produce perforin and IFNc following stimulation with PMA/ionomycin. FACS sorted CD96+ and CD96neg were stimulated with PMA/ionomycin and assessed for A) IFNc and B) perforin production in an ELISPOT assay. Bars represent the mean value 6 SD of three independent experiments and show spot forming units (SFU) per 5000 cells. Statistical analysis was performed using Student’s T test *p , 0.05. doi:10.1371/journal.pone.0051696.gactivation [9]. However, TCR triggering by PHA resul.T 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-CD28, but not PHA, resulted in increased per cell CD96 density. We also observed that cells lacking CD96 on CD8+ Tcells represented a population that produced both IFNc and perforin following stimulation. All together, these data suggest that changes in CD96 expression may be a useful additional marker to measure 25837696 overall effector function and disease progression during HIV-1 infection. In HIV-1 infected individuals chronic immune activation is a common feature where increased CD8+ T cell activation is associated with CD4+ T cell depletion [23,24]. During HIV-1 infection there is also evidence that bacterial translocation occurs as LPS and bacterial DNA have been detected in the blood of HIV-1 infected individuals [20,25]. CD96 is abundantly expressed on resting T cells, but interestingly we found that in vitro LPS stimulation of PBMCs from healthy donors decreased CD96 expression. It has already been reported that CD96 can be shed during chronic disease such as Hepatitis B infection [18]. Correspondingly, we observed that CD96 expression was decreased during chronic HIV-1 infection in our cohort. The total CD8+ T cell population of elite controllers was also found to have decreased frequencies of CD96+ expressing cells compared to healthy controls, although density per cell was maintained. These data indicate that inflammatory responses to LPS is 
a contributing mechanism by which downregulation of CD96 expression is induced. LPS translocation may therefore to some extent explain the down-regulation of CD96 observed in the subjects of this cohort. In contrast to LPS, direct TCR stimulation in vitro instead increased the density of CD96 expression. This is in accordance with previous studies that report upregulation of CD96 on T cells uponCD96 Expression during HIV-1 InfectionFigure 3. CD8+ T cells lacking CD96 produce perforin and IFNc following stimulation with PMA/ionomycin. FACS sorted CD96+ and CD96neg were stimulated with PMA/ionomycin and assessed for A) IFNc and B) perforin production in an ELISPOT assay. Bars represent the mean value 6 SD of three independent experiments and show spot forming units (SFU) per 5000 cells. Statistical analysis was performed using Student’s T test *p , 0.05. doi:10.1371/journal.pone.0051696.gactivation [9]. However, TCR triggering by PHA resul.