The observation that disparate outcomes were not dependent on simple quantitative measures of response magnitude alone prompted us to seek qualitative determinants of CD8⁺ T cell–mediated immune protection in this cohort. Given that downstream cellular effector functions originate from the initial interaction between TCR and cognate peptide–MHC class I antigen, we first examined the clonotypic architecture of immunodominant SIV-specific CD8⁺ T cell populations. In each macaque, the dominant response 2 wk after boosting with Ad5 vectors (the postvaccination [PV] time point) was directed against the CM9 epitope (Fig. 1 C); this is consistent with antigen targeting patterns observed in Mamu-A*01⁺ macaques during primary SIV infection but with substantial skewing away from the typically codominant SL8 epitope (19). After infection, the vaccinated macaques mounted massive anamnestic CD8⁺ T cell responses to the SL8 and CM9 epitopes that together comprised >50% of the total acute phase SIV-specific CD8⁺ T cell population (18). Cognate SL8- and CM9-specific CD8⁺ T cells were visualized directly ex vivo with fluorochrome-conjugated pMamu-A*01 tetrameric complexes and sorted viably by flow cytometry at both the PV time point and 4 wk after infection (the postinfection [PI] time point); an anchored template-switch RT-PCR that reproducibly generates an accurate hierarchical representation of TCRB gene expression was then used to ascertain the clonotypic composition of each sorted population (17). The amino acid sequences of all expressed TCRB CDR3s at both time points are shown for the SL8-specific clonotypes in Fig. S1 and for the CM9-specific clonotypes in Fig. 2. Strikingly, both vaccine-induced antigen-specific CD8⁺ T cell populations in all eight macaques exhibited clonotypic profiles similar to those observed in a previous study of acute SIV infection (17). In particular, the SL8-specific clonotypes expressed predominantly TCRBV14 and TCRBJ1.5 in association with a restricted CDR3 motif characterized by the presence of an arginine residue at position 6 (Fig. S1), whereas the CM9-specific clonotypes exhibited greater CDR3 diversity and a preference for TCRBV13 (Fig. 2). Furthermore, CD8⁺ T cell populations of both specificities elicited by vaccination exhibited the phenomenon of TCRB “sharing” between different macaques (Fig. S1 and Fig. 2); all common TCRB CDR3 sequences were encoded with distinct interindividual nucleotide profiles and restricted to the relevant antigen specificity across the entire dataset (Table S1 and Table I). Such public clonotypes (20) are defined in this study on the basis of CDR3 and TCRBJ identity because of minor 5′ regional variations between detected TCRBV13 sequences (Table S2) and were more prevalent in the motif-dominated SL8-specific CD8⁺ T cell populations, which were generally more polyclonal (median = 16.5 clonotypes; range = 10–38 clonotypes) than the corresponding CM9-specific populations (median = 8 clonotypes; range = 2–16 clonotypes). Overall, these characteristics are remarkably analogous to those observed in primary SIV infection (17), thereby indicating that the intrinsic nature of cognate antigen rather than the context of presentation is a primary determinant of clonotype recruitment in the periphery.

The CD8⁺ T cell responses mounted after SIVmac239 infection were substantially greater in magnitude and mobilized with faster kinetics compared with the corresponding epitope-specific responses in nonvaccinated controls (18). Within these anamnestic responses, which were generally expanded compared with those induced by vaccination (Fig. 1 C), a notable degree of clonotypic preservation was apparent for both SL8-specific (Fig. S1) and CM9-specific (Fig. 2) CD8⁺ T cell populations. Indeed, conservation of clonotype usage was apparent in longitudinal follow-up studies of macaques r96133 and r97113 beyond week 4 after infection (Fig. 3 and not depicted). This is consistent with a previous study in humans, in which vaccine-induced clonotypes specific for the HLA B*2705–restricted HIV-1 p24 Gag KK10 epitope (KRWIILGLNK; residues 263–272) were well represented after infection (21). However, there was also a high degree of clonotype turnover between the PV and PI time points, with evident recruitment of new clonotypes and loss of vaccine-primed clonotypes in both the SL8- and CM9-specific CD8⁺ T cell populations (Fig. S1 and Fig. 2); similar shifts in clonotypic composition were observed in a recent study of vaccinated pigtail macaques (22) and tend to characterize the natural evolution of immunodominant CD8⁺ T cell responses in both HIV and SIV infection (12, 17). Overall, the immunodominant primary SIV-specific CD8⁺ T cell populations characterized in the present cohort exhibited significantly less diversity at the clonotypic level than the corresponding repertoires in nonvaccinated macaques at a similar time point after infection (P = 0.03 for SL8/TL8, and P = 0.008 for CM9 using the Mann-Whitney U test comparing Simpson's diversity indices with standardization for clone sample size) (23). Although we cannot unequivocally attribute repertoire narrowing to antigen preexposure during immunization, it is salutary to anticipate that such reductions in clonotypic diversity might adversely complicate vaccination by facilitating viral escape (17, 24).

Evidence from vaccination studies indicates a protective role for the Mamu-A*01–restricted CM9-specific CD8⁺ T cell response (9); this is consistent with experiments demonstrating that strict biological constraints operate in this region of the virus and limit the potential for escape through antigenic mutation (25, 26). We therefore hypothesized that the clonotypic constitution of CM9-specific CD8⁺ T cell populations could determine virologic outcome in macaques infected with SIV. Initially, we explored this possibility retrospectively using data reported in a previous study of 12 rhesus macaques acutely infected with SIVmac251 (17). Among several candidate measures of clonal composition at week 5 after infection, the best predictor of postprimary set point pVL proved to be the number of public clonotypes within the CM9-specific CD8⁺ T cell population (P = 0.0006 and r² = 0.71; Fig. 4 A and not depicted); this parameter is solely a measure of interindividual clonotype sharing within the antigen-specific repertoire and is distinct from both clonal diversity and TCR sequence. Multivariate linear regressions were also considered using stepwise model building to test for the influence of other potentially predictive parameters; such variables included absolute CD4⁺ and CD8⁺ T cell counts, mean log₁₀ pVL at time points before week 6, and additional measures of clonality such as the total number of clonotypes and the proportion of the response that comprised public clonotypes. None of these covariates lead to a model that was significantly more predictive of postprimary set point pVL (i.e., mean log₁₀ pVL from week 6 to 16) than the simple model based on the number of public clonotypes (unpublished data). Similarly, the inclusion of these other variables did not substantially change estimates of the relationship between the number of public clonotypes and postprimary set point pVL. Furthermore, the results were unaffected by small changes in parameterization of the response variable, such as modeling the mean log₁₀ pVL or using the area under the curve from week 10 to 16, or by altering the definition of public clonotype at the amino acid level as follows: (a) matching TCRBV, TCRB CDR3, and TCRBJ; (b) matching TCRB CDR3 and TCRBJ; and (c) matching TCRB CDR3 alone. Subsequently, we tested the hypothesis generated from this analysis using the dataset described in this study; indeed, the rationale for the current study was based on the initial finding, and this second stage was conducted prospectively with investigator blinding. There was a significant inverse relationship between the number of public clonotypes at week 2 after vaccination and the mean log₁₀ pVL from week 6 to 16 (Pearson: r = −0.81 and P = 0.014; Spearman: r = −0.82 and P = 0.015). The linear regression parameter (slope = −0.55) indicates that the estimated effect of each public clonotype was a mean reduction in postprimary pVL of 0.55 log₁₀ RNA copies/ml during this period (Fig. 4 B). Notably, no such correlation with outcome was observed for clonal diversity per se (Pearson: P = 0.11) despite a strong association between this parameter and the number of public clonotypes (Pearson: r = 0.85 and P = 0.007; diversity of clonotypes with standardization for clone sample size) (23). There was no significant relationship between the number of public clonotypes at week 4 after infection and mean log₁₀ pVL from week 6 to 16 (Pearson: P = 0.92). As described, no other measures of clonality were significant predictors of postprimary set point pVL, either alone or as covariates (unpublished data); similarly, we could find no clonotypic correlates of either central memory CD4⁺ T cell preservation (18, 27, 28) or the number of challenges required for infection to occur. Finally, to validate the association further, the original cohort was revisited with public clonotypes assigned according to the same definition but compiled using the entire dataset from all 20 macaques. This expanded range for the number of public clonotypes exerted only a minimal influence on a simple linear regression performed against mean log₁₀ pVL from week 6 to 16 (P = 0.007 and r² = 0.54). In all analyses, results using the computed area under the pVL trajectory curve from week 6 to 16 were consistent with those derived using the mean pVL for the same period. No significant correlations were observed for parallel analyses of SL8/TL8-specific CD8⁺ T cell clonotypes in either dataset (unpublished data); these negative findings act as an important control and are not unexpected given the propensity with which the infecting virus mutates at this site with minimal biological constraints to evade immune recognition and curtail the efficacy of the cognate response (17, 18, 29–31). Thus, although SL8/TL8-specific CD8⁺ T cell populations are typically polyclonal in acute SIV infection and exhibit a substantial degree of TCR sharing (Fig. S1 and Fig. 3), they are neither protective nor clonotypically diverse (17, 30). Instead, the monomorphic nature of SL8/TL8-specific TCRs, which presumably require a TCRB CDR3 motif to engage a relatively featureless antigen regardless of interindividual sharing (32), predisposes to viral escape at the level of TCR recognition (17); this contrasts with the diverse range of clonotypes that can be mobilized in response to the CM9 epitope, and in the face of such considerations, the clonotypic composition of the SL8/TL8-specific response with respect to the degree of TCR sharing becomes largely irrelevant. Public clonotype usage can therefore be associated with either favorable or unfavorable biological outcomes according to the nature of the targeted epitope and the characteristics of the available cognate repertoire. Consequently, it is important to emphasize that the predictive value of “publicity” within epitope-specific CD8⁺ T cell responses is context dependent.

To investigate the mechanistic basis for the observed clonotypic association with virologic outcome, we attempted to discern the biological properties conferred by public TCR usage that distinguish such T cells from those with identical antigen specificity derived from the private repertoire. Initially, we reasoned that differential TCR/pMamu-A*01 engagement might trigger subtly different cellular outputs in the composite antigen-specific T cell populations. Indeed, recent studies linking the functional attributes of HIV-specific CD8⁺ T cells to pVL in infected individuals provide a potential precedent for such a link (3, 11, 12, 14). We therefore assessed the functional output of CM9-specific CD8⁺ T cell populations in macaques r96133 and r97113 at the prechallenge time point, thereby excluding any confounding impact of replicating SIV on these cells. These macaques were selected on the basis that they epitomize the association expounded in the present study; specifically, despite similar levels of clonal diversity, r97113 had a favorable outcome after infection and a CD8⁺ T cell response to CM9 that was predominantly public, whereas the opposite applied to r96133 (Figs. 2 and ​and3).3). In each case, both IFN-γ and TNF-α production were triggered in response to antigen with almost identical peptide sensitivities for the former effector function (Fig. 5, A–C). Screening for additional soluble factors released on antigen encounter yielded similarly uniform results overall, although PBMCs from r96133 failed to produce significant levels of MIP-1α and were comparatively meager with respect to lymphotoxin A secretion (Fig. 5 B). Furthermore, these vaccine-induced CD8⁺ T cell populations exhibited indistinguishable effector memory (CD28⁻CD45RA⁺CD95⁺) phenotypic profiles (Fig. 5 D); interestingly, this suggests that the impaired maturational profile characteristic of HIV-specific CD8⁺ T cells in infected individuals arises as a consequence of virus exposure (33, 34). Thus, consistent with the findings of a recent mouse study (35), no major disparities were apparent in the interactions of these constitutively distinct CM9-specific CD8⁺ T cell populations with cognate wild-type antigen. Functional and phenotypic homogeneity irrespective of clonotypic composition was also observed in multiparametric analyses of CM9-specific CD8⁺ T cell populations at the PI time point (n = 6; Fig. S2 and not depicted). Collectively, these data suggest that such approaches are insufficient in isolation to monitor the efficacy of T cell responses with respect to biological outcome.