In an effort to understand the relationship of two key features of the early T cell response to HIV-1 we examined the simultaneous expression of both activation and differentiation markers on total and antigen-specific T cells from treatment naïve adults in early HIV-1 infection before, during and after anti-retroviral therapy. Our data suggests that there may be a link between the activation and maturation stage of CD8+ T cells. The skewed, less differentiated maturation profile of HIV-1-specific T cells was more pronounced at higher CD8+ T cell activation levels. We also found that increased numbers of fully differentiated HIV-1-specific CD8+ T cells associated with higher CD4+ T cell counts suggesting that the presence of mature Gag-specific CD8+ T cells may be protective. However the HIV-1 Gag-specific T cell differentiation profile was not readily altered by suppression of T cell activation by anti-retroviral treatment.
We compared CMV- and HIV-1-specific responses in early infection. Previous studies of differences between CMV- and HIV-specific-responses have suggested that the failure of HIV-specific cells to reach a more differentiated phenotype may explain their inability to fully control HIV replication 
. The magnitude of CD8+ IFN-γ+ and IFN-γ+IL-2+ T cell responses to CMV pp65 were considerably higher than those for HIV-1 Gag. CMV pp65-specific CD8+ T cells had a greater proportion of the more mature LM (CD27−CD28−) cells compared with Gag-specific CD8+ T cells. The level of activation on all subsets of pp65-specific cells was much lower than on Gag-specific cells. These data suggest that a low activation state, a high magnitude of CMV specific IFN-γ and IL-2 responses, and a high proportion of mature CMV specific CD8+ T effector cells may be important features in the control of CMV.
It has been suggested that distinct T cell maturation profiles, associated with responses to different viruses, are established for the purpose of generating memory or maintaining latency 
. A recent report suggested that protective Class I B*27 HIV-1 KK10-specific CD8+ T cells have a intermediate differentiation phenotype (CD27+CD28−CD45RA-CCR7-) that was indistinguishable from CD8+ T cells specific for other less-protective HIV-1 epitopes 
. This may indicate that an intermediate differentiation phenotype can be associated with protection. That said, in agreement with our finding that a more differentiated HIV-1 specific CD8+ T cell response is associated with higher CD4 counts two recent clinical studies have suggested that more differentiated CD8+ T cell responses are associated with lower HIV-1 viral load set point, or long-term non-progressor status in HIV-1 disease 
. Lower CD8+ T cell activation may mark or induce a state of less differentiated total and CD4+ T cell responses. This in turn may amount to a lower fraction of IM CD4+ T cells expressing CCR5 and lower cell turn-over, rendering the CD4+ T cell population less susceptible to infection. The reverse state - of higher CD8+ T cell activation - may associate with increased CD4+ T cell loss due to cytokine forced expansion of EM CD4+ T cells into an IM pool. This stimulation, in the absence of cognate antigen could lead to apoptosis, or direct viral infection and cell death.
We found that patients with the highest numbers of EM Gag-Specific CD4+ T cells had the lowest levels of T cell activation, suggesting that – in contrast to our findings on CD8+ T cell responses – that a less differentiated CD4+ T cell response may be associated with better clinical status. Our data is consistent with a report suggesting a less differentiated CD4+ T cell response marks long-term non-progression in HIV disease, a state of low CD8+ T cell activation 
Repeated stimulation of Gag-specific T cells, by persistent HIV-1 replication, may induce high levels of activation and block maturation into a mature effector phenotype. Based on our model linking activation to maturation impairment, we reasoned that in the absence of repeated antigen stimulation the proportion of Gag-specific T cells with a low activation, LM phenotype may increase. We therefore examined maturation and activation following anti-retroviral therapy and reduction of circulating virus to undetectable levels. While a significant decline in activation levels on all CD8+ T cell subsets was apparent, reduced antigen load had different effects on total, CMV- and HIV-1-specific T cell differentiation profiles. Following therapy, the CMV-specific CD4+ IM T cell pool did expand as the LM pool shrunk. This suggests that HIV-1 viremia, or generalized immune activation may influence the differentiation profile of CMV specific CD4+ T cell responses. Among total CD8+ T cells there was a significant reduction in the IM and a corresponding increase in the LM fraction. As activation declines the total IM CD8+ T cell fraction may mature, or die by apoptosis, shifting the population to an LM profile. In contrast, despite a significant reduction of activation levels on Gag-specific CD8+ T cells, there was no corresponding shift in the maturation profile of Gag specific CD8+ T cells. Taken together, these data suggest that high activation and viral load does not prevent the maturation of Gag-specific cells, although these effects may be apparent on T cells of other specificities. The Gag-specific T cells that we measured may have already become replicatively senescent due to repeated stimulation 
The mechanism by which maturation and activation are associated prior to treatment is not clear from our results. The relative paucity of LM HIV-specific CD8+ T cells may be due to increased susceptibility of these to apoptosis, even after ART suppression of activation. That said, the failure of CD8+ T cells to mature may result from manipulation of signaling pathways in responding HIV-1 specific CD8+ T cells by a viral product, such as secreted Nef, which has been shown to upregulate PD-1 
. Indeed PD-1, which is associated with anergy of antigen-specific cells, is upregulated on HIV- but not CMV-specific CD8+ T cells 
and could prevent maturation to an effector cell type. It has also been suggested that lack of IL-2 production by HIV-1-specific CD8+ T cells may explain their inability to down-regulate CD27 
. Recently it was shown that CD27 down-regulation can be blocked by lack of expression of its ligand, CD70 
which requires both antigen and cytokines, including IL-2 for expression. In our study the proportion of Gag-specific CD8+ IL-2 expressing cells was very low () supporting the idea that the absence of IL-2 producing HIV-1-specific T cells may be unable to prevent CD27 down-regulation 
We observed that HIV-specific T cells are predominantly immature and highly activated at early stages of infection. Although activation is reduced by anti-retroviral therapy immature Gag-specific cells continue to predominate. In a previous report we demonstrated that initiation of anti-retroviral therapy within the first month of HIV-1 infection associated with improved viral control and clinical outcomes once therapy was halted, compared to those who start anti-retroviral therapy one month or later into infection 
. In our current study, persons initiated therapy no earlier than 2 months after acquiring HIV-1, by which time a sub-optimal HIV-1 specific T cell differentiation profile was established. It would be of interest to examine the activation and maturation profile of cells from individuals treated within one month of infection. Earlier intervention with anti-retroviral therapy may lower T cell activation during very early infection, alter the T cell differentiation phenotype of responding CD8+ T cells, or both, in a manner which confers enhanced T cell mediated control of HIV-1.