 cross-restriction, high antigen sensitivity, and polyfunctionality in both the CD4+ and CD8+ T cell subsets.
Exploration of the TCR repertoire specific for the most immunoprevalent CD4 epitope in HIV-1 Gag revealed a repertoire markedly biased toward the TRAV24 and TRBV2 gene families. Highly prevalent public motifs and public clonotypes were preferentially shared by HIV controllers, suggesting that particular TCR determinants contributed to the efficiency of the antiviral CD4 response in these patients. This notion was reinforced by functional analysis of the most prevalent public clonotypes, as these were able to confer HLA-cross-restricted, highly sensitive, polyfunctional responses against Gag antigens, indicative of superior function. These findings, which to our knowledge provide the first assessment of the HIV-specific CD4+ T cell repertoire at the clonotypic level, emphasize that intrinsic TCR determinants, rather than low antigenemia, specify the remarkable properties of the cellular response in controlled HIV infection.
It was noteworthy that both the TRAV24 and TRBV2 repertoires specific for Gag293 showed a high degree of motif and clonotype sharing. So far, most studies of the human TCR repertoire have focused on TRB genes, but recent analyses suggest that the human TRA repertoire is even more diverse (38). Our findings provide evidence for a dual bias in controlled HIV infection, with both TCR chains showing a significant degree of motif conservation. The most prevalent TRAV24 clonotype (AFKAAGNKLT) was present in 6 of 8 controllers (75%) studied. In comparison, CD8 TRB public clonotypes reported in previous studies of the HIV-specific repertoire were shared by 2–4 patients (13, 14, 39, 40). When the analysis was extended to less-stringent definitions of TCR biases, close to half of TRAV24 and TRBV2 clonotypes shared defined public motifs, while the corresponding frequencies were of 29% and 10%, respectively, for clonotypes of treated patients. Such strong bias in the Gag293-specific TCR repertoire of controllers could not be attributed to amplification of a few clonotypes during culture, as the repertoire of controllers remained highly diverse, with a median of 36 distinct TRBV2 clonotypes per 100 CDR3 sequences. In addition, similar frequencies of public motifs were detected in vitro and ex vivo when the TCR repertoire of controllers could be analyzed in both conditions. Rather, these findings suggest that strong selective pressures (further discussed in the supplemental material) shaped the Gag293-specific repertoire during the establishment of viral control.
The coexpression of TRAV24 and TRBV2 public clonotypes generated functional TCRs that, for some combinations, displayed a remarkably high affinity for Gag293–MHC II complexes. Human TCRs generally show affinities in the 1 to 100 μM range by SPR analysis (36). MHC II–restricted TCRs show a trend for a lower affinity as compared with MHC I–restricted TCRs, with average KD values of 70 versus 35 μM, respectively. With a KD of 0.86 μM for the Gag293-DR11 complex, F24 demonstrated one of the highest affinities reported so far for an MHC I– restricted TCR, ranking second in a list of 22 human and mouse TCRs for which structural data are available (36). The molecular determinants of a high-avidity interaction for the Gag293-MHC complex appear to depend both on the TRAV24 and TRBV2 chains and on the restricting HLA allele. The most conserved CDR3 residues in public motifs represent likely candidates for contact residues with Gag293. However, conserved CDR1/CDR2 residues may also contact the Gag293 peptide, considering the predominance of the TRAV24 and TRBV2 families. Future structural studies should help determine the respective roles of the CDR3 versus the CDR1/2 regions in shaping high-avidity interactions for Gag293-MHC complexes. The expression of high-affinity TCRs for Gag293 in controllers resulted in high antigen sensitivity as measured by CD69 induction, with a good correlation between the two parameters (r = 0.85). Thus, TCR biophysical properties largely accounted for TCR function, consistent with multiple studies in animal models (41, 42). It should be noted, however, that in some studies TCRs with very high or supraphysiological affinities were found to have reduced functions (43). A proposed explanation is that a peptide-MHC (pMHC) that interacts too tightly with a very high-affinity TCR cannot disengage to contact additional TCRs and thus does not achieve serial TCR triggering and T cell response amplification (44). We did not observe decreased functions for the TCR/pMHC II combinations showing the highest affinities by SPR, suggesting that TCR affinities in the order of 1 μM are still in the physiological range for optimal TCR function in human CD4+ T cells.
The reasons for the preferential amplification of high-affinity Gag293-specific TCRs in controllers compared with treated patients remain to be elucidated. A particular genetic background or a limited duration of acute infection may have promoted the amplification of such TCRs in controllers (45). Conversely, patients who initially progressed to disease may have lost the high-avidity CD4+ T cell population due to preferential infection and depletion, a notion supported by the restricted CD4 TCR repertoire observed in treated patients, and by rapid loss of certain CD4 specificities after the acute infection stage (6). It will be informative to determine whether other immunoprevalent HIV epitopes elicit a response pattern similar to that observed for Gag293, with the induction of a highly biased, high-affinity TCR repertoire in controlled infection. The preferential detection of Gag-specific CD4 responses in HIV controllers (20) suggests that several regions of Gag may be targeted by high-affinity TCRs, though this remains to be investigated. High-affinity CD4 TCRs are endowed with strong proliferative capacity (41), which helps explain how they could reach such a dominance in controlled HIV infection. The high affinity of controller TCRs also translated into efficient cytokine secretion upon Gag293 peptide stimulation. Primary CD4+ T cells transduced with the F24 TCR showed EC50 values ranging between 10–8 M and 10–9 M peptide for the different cytokines studied, indicative of a remarkably high antigen sensitivity for the CD4+ T cell subset. Polyfunctionality, a property that has been associated with HIV control (37), showed a clear dependence on TCR affinity. Comparison of the F24, F25, and F5 TCRs showed that a difference of 1.1 log in affinity resulted in a 2-log difference in the peptide concentration at which half the specific cells retained 3 functions. Thus, TCR affinity appears critical in determining the range of cytokines produced, particularly at low antigen dose. Of interest, not all cytokines were equally dependent on the strength of the TCR signal, with TNF-α showing the lowest requirements and IFN-γ the most stringent. The notion of a hierarchy in cytokine production is consistent with a report showing that the affinity of Gag-specific T cell clones dictated their cytokine expression profile (10). It is relevant that for CD4+ T cells, in contrast to CD8+ T cells, IFN-γ is the most “demanding” cytokine, as it can be produced only by high-avidity cells upon limiting antigenic stimulation. IFN-γ plays a key role in CD4+ T cell helper function, through its capacity to upregulate MHC II in APCs and prime them for efficient antigen presentation. In addition, IFN-γ production by tissue CD4+ T cells plays an underappreciated role in the recruitment of immune effectors, through the triggering of chemokine cascades (46). In controlled HIV infection, high-avidity Gag-specific CD4+ T cells may play a similar role, by keeping the immune system in constant alert and rapidly recruiting CD8+ T cells and NK cells to sites of HIV replication upon the occurrence of viral replication blips. In addition, IFN-γ secretion may play a direct antiviral role, through the induction of IFN-stimulated genes (ISGs) that inhibit HIV replication (47). For instance, Th1 cells are thought to be less infectable than Th2 cells due to their higher expression of the HIV restriction factor APOBEC3G (48). Highly differentiated Th1 effectors that reexpress CD45RA appear particularly resistant to HIV infection (49). Gag-specific CD4+ T cells in controllers maintain a Th1 differentiation status with persistent IFN-γ production (22), raising the possibility that such cells possess a degree of resistance to HIV infection. This would be consistent with studies that documented a lower susceptibility of controller CD4+ T cells to HIV replication in vitro (50). Thus, high TCR affinity, with the associated capacity for IFN-γ expression at low antigen dose, may contribute to HIV control at several levels, through sensitive immune surveillance, rapid triggering of helper functions, and direct antiviral effector mechanisms.
Of interest, the F24 TCR was able to confer polyfunctional cytokine responses when transferred into CD8+ T cells. The responses were MHC II restricted, indicating that the F24 TCR could interact with the Gag293–MHC II complex in the absence of the CD4 coreceptor. The CD4 molecule is not thought to contribute significantly to the affinity of the pMHC/TCR/CD4 complex but plays an important role in relocating the kinase Lck close to the TCR/CD3 signaling complex (51). The lower cytokine responses in CD8+ than in CD4+ T cells may thus result from a lower number of Lck molecules available for triggering intracellular signals. The transfer of the F24 TCR to CD8+ T cells still conferred the 5 functions tested at the 10–5 M to 10–7 M antigen dose, suggesting that transduced CD8+ T cells could become efficient effectors in foci of productive HIV replication.