The goal of therapeutic vaccinations in HIV-1 infection is to provide enough antigenic stimulation so that the subject can mount an immune response in order to effectively control viral replication without the presence of an antiretroviral regimen. Recent studies of HIV-1-infected individuals with natural immune control have shown that an effective immune response is characterized by the presence of a polyfunctional CD8+
T cell response to the virus. 
Since the control of HIV-1 has been shown to be more dependent on the quality and specificity of the CD8+
T cell response rather than the quantity of the response alone, 
we evaluated CD8+
T cell polyfunctional response in a previously completed DC-HIV-1 peptide vaccine trial. 
Interestingly, the increased monofunctional response after vaccination correlated with the modest IFNγ response detected using the ELISPOT assay. However, although assessing single cell IFNγ production showed that there were transient and modest responses to the vaccine peptides, permutation comparison analysis showed no significant difference in the CD8+
T cell polyfunctional response post-vaccine. In fact, it appeared that the CD8+
T cell response to Gag peptide was more polyfunctional before the vaccine was given.
The minimal immunologic response to this DC-peptide vaccine could be due to its low immunogenicity. We examined an alternative hypothesis that Treg activity affected immunologic response in the trial. Depletion of Treg in the cord blood of HIV-1-exposed-uninfected neonates resulted in the augmentation of CD4+
T cell HIV-1-specific immune responses. 
Also, Treg isolated from HIV-1-infected patients have been shown to suppress cytokine secretion. 
Because of the major role that Treg, especially the antigen-specific Treg induced in the periphery, play in the immune response to chronic infections, it was important for us to delineate the effects of Treg in this particular study. The objective was to assess whether Treg activity should be a co-factor in the evaluation of immune-based therapeutic trials in HIV-1.
In our study, there was a trend of increased Treg frequency observed in a majority of subjects after DC-HIV peptide vaccination, with a modest median increase of 30% in the 12 subjects. Sixty four percent of vaccine non-responders by ELISPOT assay had increases in Treg frequency compared to 83% of ELISPOT assay responders. As Treg can accumulate in the lymphoid tissues, 
the modest increase in Treg frequency in peripheral blood as well as the lack of correlation between Treg frequency and vaccine response detected by ELISPOT assay could be due to migration of the expanded Treg population to the lymph nodes where T cells are being activated by the vaccine. Interestingly, the CD8+
T cell polyfunctional response appeared greater prior to the vaccine, suggesting that the increase in Treg frequency and Treg activity, could have affected the ability of CD8+
T cells to secrete cytokines. It is also important to note that when we checked the Treg frequencies 3 months after the vaccine was given, the levels decreased to baseline or lower than baseline in 14/14 subjects evaluated (data not shown) suggesting that the increased frequencies were vaccine related.
More important than the Treg frequency is the suppressive function, which we have shown to have an effect on the vaccine response, as evinced by the increased polyfunctionality of the CD8+
T cell response to Gag peptide after Treg depletion. Since the subjects have all been virally suppressed on a stable ART regimen, there is not enough antigenic stimulation, which is believed to be necessary for continuous anti-HIV-1 effector function. 
Upon receipt of the DC vaccine, it is likely that the Treg population was initially activated and expanded, as has been shown in mouse models. 
Indeed, in the study by O'Gorman et al. 
, antigen-specific memory CD4+
T cells only acquire responsiveness after repeated antigenic stimulation. In our study, the high expression levels of the IL-2 receptor α chain, CD25, on Treg could have allowed them to sequester increased levels of IL-2 produced by the T cells upon antigen recognition. Thus, although our DC vaccine was able to elicit an immune response after the second dose, the increased expansion of Treg after the first dose might have significantly limited the total CD8+
T cell immune activity, leading to the modest and transient response.
The specific mechanism by which the Treg exerted suppression of polyfunctional responses of CD8+
T cells remains to be determined. However, it should be noted that there was no change in the expression of CTLA4 and GITR expression in Treg post-vaccine, suggesting that there could have been other mechanisms of suppression that were increased after vaccine administration leading to the increased suppressive function. Antigen-specific induced Treg such as type 1 regulatory T cells (Tr1) mediate suppression by IL10 secretion, which we were not able to test. Indeed, Lopez et al., 
point to evidence indicating that the pre-treatment with immunoinhibitory drugs improves immune responses of cancer patients to DC therapy mainly because these drugs eliminate the Treg population. As the removal of the entire Treg population will also lead to autoimmunity 
, it is important to initially determine which specific Treg mechanism is responsible for immune suppression, and then, define the Treg subsets mediating suppression. In the context of enhancing CD8+
T cell responses against HIV-1, the goal would be inhibiting Treg suppression without causing a significant manifestation of autoimmunity.
Since polyfunctional T cell responses have been associated with better immunologic control of HIV-1, as shown in elite controllers, 
Treg appear to play a significant role in inhibiting effective immune responses to the virus in untreated patients. However, this inhibition may be beneficial for the host since persistent immune response will lead to persistent immune activation, which has been shown as a predictor of HIV-1 disease progression. 
Moreover, activated T cells will also be easily targeted by the virus, thus contributing to progressive infection. Indeed, lower numbers of Treg have been associated with higher levels of immune activation in HIV-1 infection. 
Treg therefore seem to be maintaining a balance between persistent infection and decreased immune activation. As the CD4+
T cell population becomes depleted later in the disease, untreated patients would experience both increased immunosuppression and immune activation due to the eventual Treg depletion. Although Treg may not be exerting a considerable effect in virally suppressed patients on ART, an increase in the immune response to the virus, such as after administration of a therapeutic vaccine, may tilt this delicate balance held in check by Treg towards a proinflammatory T cell response. This will lead the Treg to counteract by increasing their frequency and suppressive effects thus preventing any resulting immune activation.
We believe that this is the first study to assess Treg activity in HIV-1 therapeutic vaccines. Although we were able to evaluate Treg frequencies in the 17 subjects who received the vaccine, we were only able to evaluate polyfunctional response in 7 subjects. It is important to note, however, that in all the 7 subjects, increased polyfunctionality was observed after Treg depletion implying that the DC vaccine did indeed increase polyfunctional CD8+ T cell activity but was masked by Treg expansion and increased suppressive function. Further research should be conducted to investigate the role of Treg in other immunotherapeutic trials for HIV-1. The information from these studies can guide investigators in developing better strategies to enhance HIV-1 immune control.