In this study, we found that the activation signals to the target T cells and Tregs can modulate the suppressive function of the Tregs. Increasing the strength of signal or presence of IL-2 can override the Treg-mediated suppression, which we show in the context of three different in vitro models of antigen-specific T cell activation. In contrast, higher frequency of Tregs and stronger stimuli through their TCR results in better suppression of effector T cell activation, The data presented here provides us with better insight into the delicate balance between activation signals and suppressing an antigen specific response.
We showed that the strength of TCR and IL-2 signals affects the magnitude of suppression that can be achieved by the Tregs. However, it remains to be determined whether direct suppression of initial activation of antigen-specific T cells by Tregs extinguishes the immune response at a population level of T cells, without directly inhibiting every responder T cell. This could potentially happen in a scenario where Tregs stop few potent responder T cells from secretion of cytokines, thus limiting the availability of growth factors such as IL-2 to neighboring cells that may require IL-2 for efficient activation. Indeed, addition of IL-2 to in vitro activation cultures overrides Treg-mediated suppression of target T cells during both SEB and allogeneic stimulation. It is also possible that IL-2 changes the activation threshold needed for Treg suppressive capability at the cell-to-cell interaction level. While not mutually exclusive, these data highlight the important double-edged role of IL-2 both in enabling Tregs to suppress and target cells to escape Treg-suppression.
When we stimulated Tregs only in the presence of autologous APCs the suppression observed was less potent compared to suppression by Tregs stimulated with SEB, allogeneic DCs or HIV-peptide. These findings would predict that during the course of an immune response, Tregs that recognize antigens from pathogens develop in parallel to conventional effector/memory T cells. In support of this prediction, we recently provided evidence that Tregs can develop from a naïve T cell precursor when activated in vitro through their TCR 
. Our findings do not rule out the possibility that autologous or bystander Treg activation play a role in the suppression of HIV specific immune responses. However, we speculate that suppression of HIV-specific activation by Tregs recognizing self-antigens would not be efficient unless HIV-specific CD8 cells are at very low frequencies.
During HIV infection, global control of T cell activation by Tregs could conceivably limit cellular targets of HIV, as T cell activation is required for a successful infection 
. In contrast, it has been suggested that HIV-immune dysfunction is associated with suppression of HIV-specific effectors by Tregs, leading to an inefficient immune response against HIV 
. Several reports have shown that depletion of Tregs can amplify CD8+
T cell responses 
. One plausible reason why in our experimental system we did not observe similar results could be that the patient population we used has very potent and high frequencies of CD8+
T cell responses, thus as we showed, rendering them more difficult to suppress. It is also possible that the affinity of CD8+ T cells for this HIV peptide is too strong for autologous Tregs to overcome. Indeed, increasing the dose of peptide concentration by 10-fold was able to overcome the suppression mediated even by the allogeneic Tregs. Further, in our system, it is also highly unlikely that Tregs are activated by CD8+ specific HIV-peptide, therefore they would have to rely on self-antigens in order to be activated and display their suppressive function. In other experimental systems, where T cells are activated by complete HIV antigens or viral particles, Tregs could also potentially recognize HIV antigens and thus conceivably more potent in suppressing HIV-specific activation.
Defining the determinants of Treg-mediated regulation of allogeneic immune responses is essential for therapeutic application of Tregs in transplantation tolerance and control of GVHD 
. We have confirmed that allogeneic stimulation of Tregs renders them capable of suppressing the activation and proliferation of conventional T cells. In order to successfully prevent GVHD after an allograft-transplant, donor Tregs that are transferred or develop in the host should be able to suppress most of allo-specific CD4+ and CD8+ T cells 
. However, our data suggest that Tregs do not necessarily have to recognize the same allo-antigens as the effector T cells. Successful inhibition of allo-specific T cell activation would only require that some Tregs are also activated at the same time, even if through different antigens. Based on these postulates, a potential treatment approach would be to isolate Tregs from donor blood prior to the transplantation, which would then be activated and expanded with allogeneic DCs from the host recipient. These Tregs can then be infused to patients with the transplant. Because Tregs have limited in vitro expansion potential 
, recently identified naïve Tregs, which have greater proliferative capacity could be useful for this approach 
In summary, our results indicate that the strength of TCR signals and IL-2 can override Treg-mediated suppression when Treg numbers and activation are limiting. In the context of HIV, both controlling immune activation and generation of effective HIV-specific immune responses may need to be balanced. Thus, Treg responses during HIV infection may require fine-tuning depending on the stage of the disease. Approaches to modulate this delicate balance will be important when designing vaccines for HIV or other infectious diseases and controlling the immune response during transplantation or in chronic immune activation.