In this study, we characterized the TCR repertoire of CD8 T-cell populations specific for the immunodominant Mane-A*10-restricted SIV Gag164-172 KP9 epitope in pigtail macaques. TCR repertoires have not previously been investigated in pigtail macaques, and it was necessary to sequence the TRB constant region to ensure unbiased amplification (29
). Several single nucleotide substitutions were detected within the sequenced constant region, although only two of these substitutions were present exclusively in pigtail macaque sequences. Our pigtail macaque TRB amplifications therefore included novel primers based on these sequences.
Several intriguing insights into the molecular characteristics of KP9-specific CD8 T-cell populations emerged. Remarkably, only 1 out of 161 different KP9-specific clonotypes was found in more than one animal, suggesting that the KP9-specific response is mediated by a diverse repertoire of clonotypes that are highly “private” to individual animals in this outbred populations. This contrasts with TCR repertoires dominated by “public” or shared repertoires between individuals which occur either when a particular TCR has a selection advantage over others (2
) or when unusual structural features limit the number of reactive clonotypes (24
). The selection of highly “private” TCR repertoires suggests that the bound KP9 epitope presents sufficient physical features to elicit recognition by multiple clonotypes but does not have any unusual features that might restrict recognition or give some clonotypes a selection advantage over others (36
). Our findings that there was significant recognition of mutated KP9 variants and a diverse phenotype of KP9-specific CD8 T cells are also consistent with the broad TCR repertoire we identified. Future analyses of the TCR repertoires of subpopulations of KP9-specific CD8 T cells with different avidities (as identified by using lower concentrations of the tetramer) are also suggested by these studies (30
Despite the overall diversity of the KP9-specific repertoire, some animals did demonstrate a bias toward the use of a particular Vβ gene within this response (for example, Vβ 6.8 in animal 5821). These patterns were not consistent across all of the animals studied, however. Notably, the Vβ bias was not reflected in the CDR3 sequences, with multiple CDR3 sequences occurring in conjunction with individual Vβ segments. The CDR3 loops are likely to be more important in the specificity of antigen recognition than TRBV-encoded CDR1 and CDR2 (17
). In this study, the CDR3 sequences did not show any common features or motifs. CDR3 lengths were highly variable, with an average of 14 amino acids, but the dominant clonotypes, even within an individual macaque, contained CDR3 loops of various lengths. Long TRB CDR3 sequences have been associated with improved recognition of emerging escape mutants and long-term viral control in HLA-B8+
HIV-infected humans (10
). In the latter study, however, the CDR3 sequences were highly conserved between individuals and were paired with common Vβ and Jβ segments; this is in marked contrast to the KP9-specific repertoire.
In mice, study of a single TCR binding to the same MHC molecule presenting two different peptides has shown that the CDR3 loop is crucial for TCR cross-reactivity (31
). This is consistent with studies with SIV-infected Mamu-A*01+
rhesus macaques, where a restricted repertoire directed toward the Tat TL8 epitope was associated with consistent patterns of viral escape (29
). It is therefore conceivable that the wide repertoire of KP9-specific CD8 T cells in general, and the diverse CDR3 lengths and sequences in particular, contributes to the recognition of potential escape variants, therefore limiting successful viral escape to mutations such as the dominant K165R mutation (14
). Such a mechanism appears to account for the development of escape at the Gag CM9 epitope in rhesus macaques (7
), although escape at CM9 generally occurs much later than escape at the KP9 epitope (3
). Additionally, repertoire breadth resulting from multiple reactive clonotypes within the naïve T-cell repertoire appears to contribute significantly to the immunodominance of the Gag CM9 epitope (18
) and may also account, at least in part, for the immunodominance of the KP9 epitope.
The mechanistic basis for the availability of a large repertoire of potentially reactive clonotypes specific for the KP9 epitope may lie in the structural nature of the antigen. Manipulations of a murine influenza virus epitope have elegantly demonstrated the impact of epitope structural features on TCR repertoire diversity (37
). A diverse TCR repertoire directed toward the wild-type H-2Db
-restricted PA224 epitope with a prominent central arginine residue was transformed into a more uniform and less diverse repertoire when the arginine residue was mutated to alanine (37
). Thus, the highly diverse KP9-specific repertoire suggests that the KP9 epitope complexed to Mane-A*10 may have prominent structural features that enable the selection of broad TCR specificities. Structural studies of the KP9 epitope bound to Mane-A*10 would help to profile the features that contribute to TCR repertoire diversity in this case.
The initial studies described herein focused on serial samples from DNA/poxvirus-vaccinated macaques subsequently challenge with SIV or SHIV. A common observation, regardless of the vaccination type or challenge virus, was the retention of only a small subset of vaccine-induced clonotypes after challenge (stable TCRs in Fig. ). Many clonotypes induced by vaccination were not detectable after viral challenge, and many new clonotypes, not detected prechallenge, appeared after challenge. These observations could suggest that only a limited number of KP9-specific clonotypes induced by vaccination can expand and respond effectively to a viral challenge.
Our studies, using an unbiased TCR sequencing approach, complement a recently reported study that used TRBV-specific primers to examine the SIV-specific repertoire in vaccinated rhesus macaques (33
). In the latter study, diverse Vβ usage after prime/boost vaccination was also observed, although a transient narrowing of Vβ usage was detected early, but not late, after SHIV89.6
challenge. In contrast to our studies, they found common CDR3 sequences within particular Vβ PCR products from SIV-specific cytotoxic T lymphocytes present in several rhesus macaques. The differences may reflect, in part, the different methodologies used for repertoire analysis but perhaps more likely reflect biological differences in the nature of the antigenic epitopes targeted and the SIV/macaque models used.
Understanding how to induce effective CD8 T cells that can respond to challenge and limit immune escape is a key goal of future T-cell-based vaccination strategies. A recent study has shown that therapeutic vaccination can modulate CD8 T-cell repertoires in HIV-infected individuals (40
), although the long-term impact of such manipulations on viral control has not been evaluated. Investigating the preferred clonotypic characteristics of effective CD8 T cells and how to elicit them through prophylactic or therapeutic vaccination remains an important field of enquiry. Single-cell cloning of CD8 T cells may contribute to our understanding through in vitro studies of clonal T-cell efficacy (25
). However, our data raise theoretical concerns about the representative nature of such approaches because individual antigen-specific clonotypes clearly exhibit differential abilities to expand upon viral exposure and hence presumably to assist in the control of viremia. Furthermore, such procedures can skew the biological properties of individual CD8 T-cell clones, regardless of TCR expression. Thus, detailed characterization of antigen-specific clonotypes directly ex vivo will perhaps prove to be more informative.
The study presented here was limited by the modest number of pigtail macaques studied at serial time points. Additionally, although we attempted to clone nearly 100 separate TCRs at each time point, we cannot be sure that we captured the entire population of CD8 T-cell clonotypes, particularly in cases where low numbers of KP9-specific CD8 T cells were present in the samples (e.g., prechallenge in animal 6267). These sampling issues are especially relevant to highly polyclonal populations, such as those specific for KP9 in animals 5821 and 5827. Further, although we observed a great degree of clonotypic diversity, even within individual pigtail macaques on the same vaccination/challenge protocol, studies of larger numbers of animals receiving identical vaccines might reveal subtle patterns that are not yet apparent. For example, the VV/FPV-vaccinated animals had moderately higher numbers of clonotypes and exhibited greater clonotypic diversity compared to DNA/FPV vaccinees (Fig. ). This observation, which could have several underlying mechanistic explanations, suggests that different vaccine formulations can elicit different repertoires specific for the same antigen. Thus, while the nature of the antigen is likely the primary determinant of which clonotypes can be recruited, the mode of delivery might dictate which clonotypes are actually recruited; importantly, such differences might translate into differential outcomes after challenge. However, first and foremost, confirmation of these findings in larger comparative studies is warranted. Another limitation of this study is that we restricted our analysis to TCR β chains. To confirm the overall diversity of the KP9-specific TCR repertoire, studies should be extended to the TCR α chains that pair with the TCR β chains described here (18
). Such studies could ultimately lead to a structural analysis of TCR engagement with the KP9/Mane-A*10 complex and a more detailed understanding of the generation of TCR repertoire diversity and the emergence of viral escape.
It is clear that the immunodominant Gag KP9-specific CD8 T-cell response is a key component of adaptive immunity during vaccination and SIV or SHIV challenge in pigtail macaques. In this study, we have begun to characterize the diverse nature of this useful CD8 T-cell response at the clonotypic level. These first insights into the substantial TCR repertoire that can be mobilized in response to KP9 reveal the diversity of CD8 T-cell populations that recognize this epitope and suggest that, within this complexity, there might be multiple features that contribute to biological outcome. Further detailed phenotypic and functional studies of antigen-specific clonotypes in different vaccine/challenge models will, we hope, clarify the central determinants of successful CD8 T-cell-mediated immunity and potentially guide vaccine development.