This study provides evidence that HIV controllers harbor a pool of high avidity memory CD4+ T cell precursors directed against an immunodominant Gag peptide. Memory CD4+ T cells specific for the Gag293 peptide were endowed with rapid growth potential and, importantly, with IFN-γ secretion capacity, suggesting that they would rapidly generate a pool of CD4+ T cells with effector function upon antigenic stimulation in controller patients. The high functional avidity of Gag293 specific cells points to their capacity of initiating recall responses in the presence of minimal amounts of HIV antigen. The high functional avidity could be explained, at least in part, by a high avidity interaction between the TCR and the cognate Gag293 peptide/MHC complex. Thus, the high sensitivity of controller CD4+ T cells to antigen appeared intrinsic, rather than dependent on the antigen presentation context or the cytokine milieu. The Vβ repertoire of tetramer-positive Gag293-specific cells proved diverse, suggesting that multiple clones contributed to the high avidity CD4 response in HIV controllers. This property may favor the long-term persistence of a high avidity response, since the presence of multiple clones reduces the probability of viral escape or of immune senescence. Taken together, these findings suggest that CD4 recall responses to Gag293 are rapid and efficient in the group of controller patients. We propose that the rapid triggering of recall responses may contribute to viral control. A rapid CD4+ T cell response upon occurrence of “viral blips” will keep the immune system in alert, provide immediate help for CD8+ T cells to exert efficient cytotoxic function, and possibly provide direct antiviral effector function 
. This rapid recall response may help keep HIV-1 replication under a low threshold, and avoid the progressive undermining of the immune system associated with repeated viral replication episodes 
The presence of high avidity CD4+ T cells helps explain how HIV controllers maintain an active T cell response in the face of very low viremia. We and others have previously shown that the level of the specific CD4+ T cell responses in HIV controllers exceeds that seen in efficiently treated patients, even though both groups have very low antigenemia 
. The triggering of recall responses at very low antigenic load in the controller group may account for this difference. Emerging evidence suggests that the CD8+ T cell response may also be of high avidity in HIV controllers. In particular, individuals harboring the protective HLA-B27 allele frequently develop a high avidity response against the immunodominant KK10 CD8 epitope, the avidity of the response correlating inversely with viral load 
. The fact that HIV controllers maintain antiviral CD8+ T cells with high cytotoxic potential in spite of their low viral load is also suggestive of a high avidity response 
. Thus, both the CD4+ and the CD8+ T cell compartments may contribute to the high sensitivity to antigen characterizing antiviral responses in the controller group. Studies in mouse models of chronic viral infections have shown that efficient CD8 responses do not persist in the long term without CD4 help 
. Therefore, a high avidity CD4 response may be essential in maintaining the quality of the CD8 response in low viremia conditions.
A correlate of a heightened sensitivity to HIV antigens may be a chronic level of immune activation, due to the recall of cellular responses upon each viral replication episode, however limited. Indeed, we have previously reported on signs of ongoing immune activation in the effector memory CD4+ T cell compartment of HIV controllers, as measured by the expression of HLA-DR, the downregulation of the IL-7 receptor, and the secretion of MIP-1β 
. Other signs of activation include raised levels of LPS in plasma 
and increased expression of HLA-DR within the HIV-specific CD8+ T cells 
as compared to efficiently treated patients. These observations confirm the notion that viral control is achieved through an active immunological process. One should note that excessive chronic activation may be deleterious in the long term, as suggested by a trend toward CD4+ T cell decrease in controller patients with the highest degree of immune activation, even in the persistence of undetectable viral load 
. It will be important in future studies to determine if such individuals show a decrease in T cell functional avidity, which may lead to more prolonged induction of recall responses to achieve viral control, and consequently to prolonged episodes of immune activation. On the other end of the activation spectrum, a few controllers appear to have low HIV-specific CD8 responses and a generally quiescent immune system 
. This phenotype may result from a particularly successful viral control, with an antigenemia so low that it would not activate the high avidity memory T cell population for long periods of time. It also remains possible that non-T cell based, alternate mechanisms of viral control predominate in these rare individuals.
It was intriguing that HIV controllers responded less frequently to the Gag161 peptide than efficiently treated patients, while the quality of the CD4+ T cell response appeared generally better in the former group. This observation pointed to possible changes in the immunodominance pattern associated with the controller status. Responses to Gag161 may have become subdominant in the controller group due to competition by high avidity CD4+ T cells responding to other epitopes, including that present in the Gag293 peptide. Indeed, high avidity has been shown to sharpen immunodominance in mouse models 
. The key mechanism appears to be the increased proliferative capacity of high avidity T cells, which progressively fill the memory T cell niche, a phenomenon accounting for the apparent avidity maturation of T cell responses over time 
. Importantly, in the present study, the duration of HIV-1 infection in the group of efficiently treated patients did not differ significantly from that in the controller group, with median of 12 (7–20) vs. 15 (10–21) years, respectively. Thus, a longer infection time was unlikely to account for the presence of high avidity CD4+ T cells in the controller group.
The CFSE analysis identified a population of Gag293-specific cells with high proliferative capacity in HIV Controller cell lines, which was consistent with the presence of a pool of high avidity CD4+ T cells. The number of divisions undergone by Tet+ cells was heterogeneous, with only a fraction reaching 5 generations and above. This may reflect a range of avidities for the Gag293 antigen, with only a fraction of Tet+ cells being endowed with high avidity and thus high proliferative capacity. This notion is also supported by the shape of the functional avidity curves, which suggests the presence of both high and low avidity populations within the pool of Gag293-specific cells from HIV Controllers. However, the high avidity component was absent in the Gag293-specific CD4+ T cell population from treated patients, independent of the method of analysis (functional avidity, tetramer avidity, or proliferation of Tet+ cells). One should note that the growth ratio of CD4+ T cell lines depended on the intrinsic proliferative capacity of specific cells but also on the frequency of these specific cells at the initiation of culture. We have previously shown that the frequency of p24 Gag-specific cells measured ex vivo by intracellular cytokine staining was approximately 3 fold higher in HIV controllers than in efficiently treated patients 
. The analysis of ex vivo ELISPOT responses to the Gag293 peptide also showed a trend for higher values in the controller group. Thus, it is likely that both an increased precursor frequency and a higher proliferative capacity contributed to the efficient growth of CD4+ T cell lines from HIV controllers. Both properties may also contribute to the long term persistence of CD4 responses in these patients.
The genetic background may play a role in conferring a better ability to mount high avidity CD4+ T cell responses. The increased frequency of HLA DRB1*0701 in the controller group could suggest a beneficial effect of this allele on the development of anti-HIV CD4 responses. However, since we did not detect an association between the presence of HLA DRB1*0701 and the level or avidity of the CD4 response within the controller group, we speculate that this allele may play a role in initially facilitating viral control, rather than in maintaining high avidity CD4+ T cells. Alternatively, HLA DRB1*0701 may be in linkage disequilibrium with a protective MHC class I allele associated with viral control. A beneficial effect of the HLA DRB1*13 alleles on CD4 responses has also been suggested 
, though we did not detect a significant effect in our study. It will be important to confirm these findings in cohorts of patients powered for large scale genetic studies. An intrinsic advantage in CD4+ T cell growth capacity may also promote efficient CD4 responses in controllers. Van Grevenynghe et al. 
have reported an increased growth capacity of controller CD4+ T cell lines in response to polyclonal stimulation, as compared to cell lines derived from efficiently treated patients or even from healthy donors. These authors demonstrated a role for the activation of the PI-3 kinase pathway, and the resultant inactivation of the downstream apoptosis inductor FOXO3a, in this particular growth phenotype. We did note a trend for higher growth ratios in controller CD4+ T cell cultures in the presence of CMV peptides, even in the absence of a positive IFN-γ ELISPOT response (not shown). However, an increase in growth propensity only partially accounts for the CD4 response characteristics observed in HIV controllers. High avidity CD4+ T cells were directed against HIV but not CMV, pointing towards a selective advantage in the induction of anti-HIV responses.
The selection of high avidity gag-specific CD4+ T cells may result from a lower exposure to HIV antigens during the acute infection stage, when the repertoire of responding T cells is initially shaped. The few reported cases of acute HIV-1 infection followed by spontaneous viral control support the notion of a lower viral peak in patients who acquire a controller status 
. Mouse models indicate that low antigen exposure is associated to the development of a high avidity response, since only the high avidity T cells receive sufficient signals through the TCR to proliferate in the long term 
. Such a scenario may predominate in patients who spontaneously control HIV replication. The presence of high avidity T cells may in turn stabilize the controller status by limiting viral replication episodes.
On the other hand, we cannot rule out that high avidity Gag-specific CD4+ T cells are selected but subsequently lost in progressor patients. Since high avidity CD4+ T cells are the first to respond in the presence of low HIV antigen amounts, they may be the first to get activated in the presence of replicating HIV, and may represent the initial wave of target cells available to the virus. HIV is known to preferentially infect HIV-specific cells 
, and among those it may well preferentially infect the most readily activated population. A recent report suggests that responses to several CD4 epitopes can be detected during the acute infection stage but are subsequently lost in progressor patients, which supports the idea of a rapid culling of the CD4 repertoire 
. Another reason for the loss of high avidity CD4+ T cells may be senescence due to overstimulation by high antigenic loads in progressor patients. The observation that high avidity CD8 responses can be lost after acute HIV infection supports such a model 
. An important area of future research will be to elucidate mechanisms that protect high avidity CD4+ T cells from depletion in HIV controllers.
In conclusion, this study provides evidence for the presence of high avidity CD4+ T cells directed against Gag in HIV controllers. It is remarkable that the distinctive properties of HIV-specific T cells in Controllers, including high proliferative potential 
, polyfunctionality 
and high cytotoxic capacity per cell 
, are all known attributes of high avidity T cells 
. Thus, high avidity may underlie many of the characteristics of an efficient adaptive immune response against HIV. The presence of high avidity T cells has been associated with control of chronic viral infections in mice 
, monkeys 
, and humans 
. Since high avidity also confers long-term memory and rapid reactivation in presence of antigen 
, it represents a desirable property to be induced by candidate T cell vaccines against HIV.