The impact of Treg on HIV progression has been the subject of some debate (24
). Adding to the confusion is the variety of approaches used to identify Treg. The combination of markers most commonly used includes CD4+
, and CD4+
. Additionally, the majority of studies have focused on peripheral Treg, while comparatively few have sampled mucosal tissues despite the severe impact HIV infection is known to have on the gut (12
). For these reasons, we used a multiparameter flow cytometry approach to determine the phenotype of Treg present in the blood and rectal mucosae and established a suppression assay to evaluate Treg function in subjects from across the clinical spectrum.
Regardless of the method we used to identify Treg, a significantly increased frequency was evident in the rectal mucosae of noncontrollers compared to controllers or seronegative subjects. This is in agreement with previous studies examining Treg in lymphoid and mucosal tissues of HIV+
). We did not observe a corresponding increase in peripheral blood of noncontrollers, in contrast to what has been reported previously (71
). This discrepancy may be due to differences in patient characteristics, specifically a lower median CD4 count in the noncontroller cohort, and/or due to differences in Treg gating strategies.
This increase in frequency was observed as a percentage of CD4+
T cells; however, the number of Treg normalized per 10,000 CD3+
T cells was not increased in noncontrollers relative to the other groups. This finding suggests that HIV may preferentially deplete conventional CD4+
T cells while sparing the regulatory population; alternatively, both populations may be infected and depleted to a similar extent, but Treg may be more quickly replaced. Indeed, multiple mechanisms may influence Treg numbers in the mucosae, including recruitment and trafficking (3
), increased survival (50
), active proliferation (3
), and peripheral conversion of non-Treg into Treg (56
). A recent study of SIV-infected rhesus macaques found high levels of HIV DNA present in Treg but low levels of HIV RNA, indicating Treg were permissive to viral entry and integration, but not productively infected. Additionally, Treg proliferated along with the total CD4+
T cell pool but were less susceptible to SIV-related cell death, resulting in an increased percentage and absolute number of Treg (2
). A similar situation may be at play in chronic HIV infection. Treg themselves are a target of viral infection (67
), and the balance between proliferation, immune activation, and death may be what keeps Treg numbers stable as non-Treg decline.
Importantly, controllers maintain low mucosal Treg frequencies similar to seronegative subjects. Although this might simply be a reflection of the low level of plasma viremia and relative CD4+
T cell preservation, controllers do experience a significant loss of gut CD4+
T cells compared to uninfected persons without a corresponding increase in Treg frequency. Our group has previously reported that HIV-specific CD4+
T cell responses in the rectal mucosae of controllers were of greater magnitude and more polyfunctional than responses in noncontrollers (33
). Additionally, controllers had a significantly lower number of Treg and higher ratio of conventional T cells to Treg in the periphery compared to HIV-negative controls, a situation that could potentially allow for more robust anti-HIV responses.
The level of T cell activation was significantly higher in noncontrollers compared to controllers or seronegative patients in both the blood and mucosal compartments, as expected. CD38–PD-1 coexpression was higher in the rectal compartment compared to the blood for all patient groups, and the difference in activation was comparable for CD4+ T cells and CD8+ T cells. Mucosal Treg likewise expressed increased levels of CD38 and PD-1, measured by MFI, compared to blood Treg. Noncontroller Treg had a significantly higher CD38 MFI in the mucosae compared to controllers or seronegative subjects as well as increased CD38 MFI in the periphery compared to controllers only. Thus, it appears that mucosal Treg of noncontrollers are highly activated and may be responding to increased levels of immune activation in the gut. In support of this, the frequency of rectal Treg exhibited a positive correlation with both plasma viral load and CD4 and CD8 mucosal T cell activation. Importantly, significant correlations between Treg frequency, viral load, and immune activation were seen only in the gut, suggesting that in addition to the early assault endured during primary infection, this compartment continues to be a dynamic site of HIV replication throughout the course of disease.
The gastrointestinal mucosa must maintain a delicate balance between tolerance to dietary and commensal organisms and responsiveness to pathogenic intruders. A number of studies have underscored the importance of Treg in maintaining a healthy gut environment (48
). Treg suppressive function in the blood and lymph node of HIV+
subjects has been examined in numerous studies (1
), but to our knowledge, this is the first to perform functional experiments on mucosal Treg in HIV infection. Studies of peripheral Treg function found a decreased suppressive capacity in Treg from patients with high viral loads and low CD4 counts (54
). In contrast, Kinter et al. described an increased capacity for suppression in Treg isolated from lymph nodes of HIV+
). Despite difficulties purifying mucosal Treg, we did observe Treg-mediated suppression of non-Treg proliferation as well as a decrease in coexpression of CD25 and OX40. The degrees of suppression of proliferation were similar in all patient groups for both blood and mucosal cultures.
Interestingly, noncontrollers did have significantly greater suppression of CD25-OX40 coexpression in mucosa compared to controllers. Griseri et al. recently described an important role for OX40 expression on Treg in a mouse model of colitis (42
). OX40 provided survival signals necessary for the accumulation of Treg in the colon, and under inflammatory conditions, OX40+
Treg were able to suppress colitogenic T cell responses, presumably by competing with effector T cells for access to OX40L, thereby limiting their ability to sustain an effective response. Further studies will be necessary to determine whether expression of OX40 on mucosal Treg of humans has a role in the suppression of effector responses in HIV.
As the body of work examining Treg in HIV infection continues to grow, a model of how Treg are impacted by infection and how they in turn impact disease progression is beginning to emerge. Although Treg can be infected and depleted by HIV, their frequency relative to conventional CD4+ T cells increases in progressive infection. This increase is most evident in the lymphoid tissues and gastrointestinal tract, where the majority of early HIV replication occurs. The relationship between mucosal Treg frequency, viral load, and immune activation in our study suggests that the frequency of Treg increases in the gastrointestinal mucosa in response to high levels of immune activation in HIV noncontrollers. They may then suppress antiviral immune responses, contributing to an environment permissive to ongoing viral replication, which in turn further exacerbates immune activation. Additional studies will be required to address the mechanisms driving the accumulation of Treg in mucosal tissues and to test whether strategies designed to modulate mucosal Treg function could boost adaptive responses and/or limit immune activation in HIV noncontrollers.