In this study, we investigated the impact of declining antigen load on the persistence, phenotypic status andfunctionality of HIV-specific CD8 T cell populations at the clonal level. A marked degree of clonotypic turnover was observed within HIV-specific CD8 T cell populations as a consequence of antigen decay after the initiation of HAART or upon the emergence of viral epitope mutations. Furthermore, new cognate clonotypes emerged under conditions of limited antigen load. In contrast, the contemporaneous CMV-specific CD8 T cell repertoires remained stable. Antigen decay led to changes in the phenotype of HIV-specific CD8 T cells and to the acquisition of novel functions; CMV-specific CD8 T cells remained polyfunctional throughout and maintained their phenotypic profiles. HIV-specific CD8 T cell clonotypes that persisted over time exhibited functional and phenotypic changes that paralleled alterations in antigen load. Moreover, particular clonotypes that became dominant after antigen decay were selected for their higher functional capacities. Overall, these data provide clear evidence of a functional reconstitution within the HIV-specific CD8 T cell compartment upon antigen decay, which can be attributed to two non-exclusive mechanisms: (i) gain of function by persistent clonotypes; and, (ii) selection of clonotypes with high levels of functional sensitivity.
After antigen decay, we observed increases in CD127 and CD45RA expression and decreases in PD-1 expression within HIV-specific CD8 T cell populations, consistent with previous reports (22
).However, these changes were not drastic; indeed, HIV-specific CD8 T cells remained predominantly CD27+
. The functional improvement of HIV-specific CD8 T cell populations observed after antigen decay was primarily due to the acquisition of TNF production. Of note, the loss of TNF production has been described as an early functional marker of exhaustion (10
). Furthermore, CMV-specific CD8 T cell populations in the present study exhibited similar functional and phenotypic properties throughout all time points studied. Thus, the gain of TNF production is limited to the HIV-specific CD8 T cell compartment and can be attributed to antigen decay.It should be noted that we cannot comment directly on changes in cytolytic activity because CD107 mobilization is an indicator of degranulationand may not reflect the expression of key cytolytic molecules, which show substantial heterogeneity within CD8 T cell populations (46
). Nonetheless, it is intriguing to postulate that the improved functionality of wild type epitope-specific CD8 T cells observed after viral escape might not only be the consequence of antigen decay but could also contribute to the generation and maintenance of HIV escape mutants. In addition, althoughthe functional improvementof HIV-specific CD8 T cells was partially sustained after the discontinuation of HAART, this was not associated with improved control of viral replication in the 4 subjects studied in this context.Given that a previous report has suggested such an association in a small number of individuals(48
), further work is warranted to determine the conditions under whichthese parameters can be linked.
HIV-specific CD8 T cell repertoires tend to be oligoclonal and skewed during chronic HIV-1 infection, likely as a consequence of several factors including avidity-based selection(41
), the loss of high avidity clonotypes (49
) and a relative lack of precursor T cell diversity due to decreased thymic output(50
). In the present study, we observed that reduced antigenic stimulation resulted in dynamic HIV-specific CD8 T cell repertoire changes, including the emergence of new clonotypes, modifications of clonal dominance and reductions in the overall numbers of constituent clonotypes. Of note, we observed a degree of cross-reactivity between wild type and variant epitopes in subject 7. In this scenario, TCR repertoire alterations were minimal, consistent with a degree of ongoing antigenic drive mediated by the variant epitope. Thus, cross-reactivity can modify the impact of epitope mutation on the wild type antigen-specific CD8 T cell repertoire.
The emergence of new clonotypes after antigen decay was observed after the institution of HAART and following the emergence of viral epitope mutations. However, the origins of these clonotypes remain to be elucidated. One possibility is that these clonotypes exit secondary lymphoid organs as antigen loaddecreases. De novo
priming of new clonotypes is also known to occur during persistent viral infections(51
), and may even be enhanced by improved CD4 T cell help during HAART and residual low level antigen persistence (52
). With the application of deep sequencing approaches to the evaluation of mutational immune escape, it is becoming clear that wild type antigen frequently persists to some extent within complex mixtures of viral variants and it is probable that such viral quasispecies establish an equilibrium with the residual cognate CD8 T cell population by priming new functional clonotypes(54
). Finally, the apparent emergence of new clonotypes may simply represent a relative increase in the frequency of previously primed memory CD8 T cell clonotypes that were harbored below the level of detection at earlier time points(55
). The application of more sensitive methodologies for TCR repertoire evaluation could help to resolve these issues(56
Our experiments also allowed us to determine whether the overall restoration of functional capacity within HIV-specific CD8 T cell populations was the consequence of a functional improvement at the single cell level or the preferential survival of poly-functional CD8 T cells. Overall, the data indicate that both mechanisms contribute to the observed functional restoration within HIV-specific CD8 T cell populations under conditions of limited antigenic stimulation. Thus, antigen decay, due either to effective HAART or the emergence of epitope mutations, led to a significant change in the TCR repertoiredue to the selection of clonotypes with high levels of functional sensitivity. In addition, individual clonotypes were shown to undergo functional improvement under the same conditions. Consistent with these observations, Reeset al.
showed that antigen load in peptide-immunized mice shaped the CD4 T cell repertoire, and that high antigen load led to the emergence of clonotypes with low levels of functional sensitivity (57
). Moreover, it was reported previously that CD8 T cells with high levels of antigen sensitivity exhibited superior effector functions resulting in more efficient antiviral activity compared to CD8 T cells with poor antigen sensitivity(41
). In addition, Almeida et al.
showed thatepitope-specific CD8 T cell clones endowed with high levels of antigen sensitivity displayed superior functionality, proliferated more efficiently and mediated more potent HIV-1 suppressive activity(6
). Our data suggest that clonotypes with higher levels of antigen sensitivity could also have a superior capacity to persist in vivo
, thereby highlighting the need to define optimal concentrations of antigen for the generation of long-lived memory CD8 T cell clonotypes with maximal antiviral efficacy. Collectively, these findings have implications for vaccination and lend support to immunotherapeutic strategies that aim to induce polyclonal responses under conditions of HAART (65