In this study, we used a combination of point-mutated pMHCI antigens in both soluble and cell-associated forms, a modified template-switch anchored RT-PCR that detects all expressed TCRBV genes without bias, and polychromatic flow cytometric analysis of cellular function and phenotype to dissect directly ex vivo factors that contribute to the clonal structure of antigen-specific CD8+ T cell populations during persistent infection with CMV and EBV. A number of consistent features emerged from this analysis.
T populations specific for CMV NV9 and EBV GL9 are oligoclonal and generally dominated by one or two prevalent clonotypes (). Furthermore, no consistent TCRBV CDR3 motifs were apparent in either case. Thus, in contrast with some immunodominant CD8+
T cell responses, the peripheral TCR repertoire that can serve as a reservoir for recruitment of antigen-specific clonotypes is diverse and does not appear to be limited by the exacting structural constraints that can be imperative for functional pMHCI engagement (20
). The potential for a given pMHCI complex to be recognized by TCRs with diverse structural features is likely to be a prerequisite for the occurrence of interclonal competition within an antigen-specific CD8+
T cell response.
Second, common antigen-specific clonotypes were observed in different individuals for each of the CMV- and EBV-specific CD8+ T cell populations (). Importantly, these public clonotypes were encoded distinctly at the nucleotide level in different donors and were confined by antigen specificity ( C). In addition, several recurrent patterns were observed at the level of primary TCRB CDR3 structure in the private repertoire specific for each antigen. These data provide evidence that cognate TCR selection in the periphery operates at the protein level as previously described for antibody responses, and implies that ongoing selection for optimal fit guides convergent clonal evolution in vivo.
T cells specific for CMV NV9 were characterized by high levels of avidity for cognate antigen in all donors tested ( A and 4; not depicted). This property is consistent with ongoing competition for antigen in vivo. Curiously, the same did not appear to hold for CD8+
T cells specific for EBV GL9, which exhibited comparatively greater dependency on the pMHCI–CD8 interaction for stable binding and uptake of soluble tetrameric antigen complexes ( B and 3; not depicted) and for activation (). Although the relationship between antigen avidity and affinity is complex, it seems feasible that these EBV-specific CD8+
T cells exhibit weaker TCR–pMHCI interaction affinities. There are several potential explanations for this observation. For example, it is known that high avidity CD8+
T cells are susceptible to apoptosis and clonal deletion under conditions of excess antigen load (37
). This process contributes to the pattern and quality of the CD8+
T cell response to persistent viral infections that evolves over time (40
). Thus, it is reasonable to postulate that high levels of viremia during the acute phase of EBV infection might lead to a predominantly low avidity CD8+
T cell response during the chronic phase, especially given that GL9 is a lytic epitope preferentially expressed in the early stages. In contrast, the early period of viral dissemination during CMV infection might be associated with fewer negative effects on the reactive immune repertoire, thereby allowing the continuous selection and accrual of high avidity clonotypes during the chronic phase of infection (41
). Further studies will be needed to distinguish whether the observed differences in CD8+
T cell avidity for CMV- and EBV-derived epitopes reflect a function of virus biology or an intrinsic property of the epitopes themselves.
Fourth, clonal dominance within the hierarchical structure of CD8+
T cell populations specific for CMV NV9 and EBV GL9 is a function of avidity for antigen. Thus, dominant clonotypes exhibited high levels of avidity directly ex vivo coupled to more sensitive functional response profiles, whereas the inverse applied to subdominant clonotypes (–). These data provide evidence that interclonal competition for antigen is a formative influence during the maintenance of CD8+
T cell populations specific for persistent DNA viruses. In addition, this process can be reflected by interclonal phenotypic dissociations, with high avidity clonotypes preferentially driven toward senescence presumably as a consequence of successful antigen sequestration (). This latter point is reinforced by the finding that CD8+
T cell clonotypes with identical specificities can be equivalent in terms of their potential to respond functionally when presented with sufficient densities of cognate antigen (). Studies in murine models indicate that the sensitivity with which specific T cell populations recognize antigen can evolve during the development of an effector response independent of changes in TCR usage; the mechanisms involved include changes in membrane composition and the basal phosphorylation status of signaling molecules, and altered topographical organization of TCR and coreceptor (43
). There is also some evidence for avidity maturation within antigen-specific T cell populations due to positive selection of cognate TCRs with both optimal affinity for pMHC and slower dissociation kinetics resulting in longer dwell times (8
). In the present study, the observation that intrinsic avidity properties cosegregate with distinct clonotypes suggests that TCR-dependent mechanisms are the principal determinants of response optimization during antigen-driven CD8+
T cell expansion in chronic infection. Although the relationship between antigen avidity and functional sensitivity is nonlinear, such progressive evolution toward high avidity CD8+
T cell usage within the confines of peripheral T cell repertoire is potentially advantageous in terms of improved effector function delivery and control of viral replication (48
Fifth, the data indicate that the differential compensatory role of CD8 enables the recruitment and maintenance of low avidity clonotypes within the antigen-specific CD8+
T cell response, albeit at mostly subdominant frequencies, as suggested previously by studies of clones derived in vitro (27
). Indeed, it is apparent that this process, which affects both overall avidity and functional sensitivity in a manner dependent on CD8 expression levels and other variables (29
), can enable low avidity clonotypes to coexist quite competitively in some instances with their higher avidity associates (, , and ). Such coreceptor-mediated effects could serve to promote clonotypic diversity within antigen-experienced memory T cell pools and confer potential evolutionary advantages in the face of pathogens with variable antigenicity (21
). Interestingly, a recent study concluded that an affinity threshold mechanism operates during the peripheral selection and expansion of antigen-specific CD4+
T cell populations to limit the competitive advantage of clonotypes with the highest avidity and prevent monopolization of the response (12
). It is possible that these distinct mechanisms for engendering clonotypic diversity reflect differences in the biophysical properties of the corresponding T cell coreceptors; thus, whereas CD8 has been shown to stabilize TCR–pMHCI complexes at the cell surface (52
), the affinity of the pMHCII–CD4 interaction is substantially lower. Presumably, there is also a threshold in CD8+
T cell populations beyond which further increases in avidity either fail to confer any additional competitive advantage or actually become deleterious. The present study does not examine these issues, but it seems likely that such a threshold might occur to limit peripheral antigen-driven selection when the optimal activation window is exceeded by clonotypes with excessively high avidity that escape thymic editing; under these circumstances, clonotypes with lower avidities might become dominant. Other scenarios can also be envisaged in which low avidity clonotypes might predominate. For example, a cross-reactive clonotype driven to proliferate by high avidity recognition of the true cognate antigen might be detected with wild-type pMHCI tetrameric complexes bearing the partial agonist peptide due to the compensatory capacity of the CD8 coreceptor. Further work is required to dissect these complex relationships that are created by the existence of antigen-specific CD8+
T cells in the setting of a complex and dynamic system rather than in isolation.
Many different selection forces can potentially interact to define the clonotypic structure of a T cell population activated in response to a specific antigen. These formative processes include antigen-induced apoptosis, avidity-mediated competition, precursor frequency, senescence and exhaustion, and structural constraints (2
). The relative impact of each of these factors under any given circumstance will likely vary according to the nature and dose of antigen, the duration of exposure, and the conditions under which it is encountered. In this study, we provide direct ex vivo evidence that competitive effects operate within CD8+
T cell populations specific for epitopes derived from persistent DNA viruses to generate hierarchies that are dominated by clonotypes with high avidity for antigen during human infection; concurrently, compensatory mechanisms mediated through the CD8 coreceptor act to mitigate this competitive advantage. These coincident processes likely serve to optimize the delivery of antiviral effector function while maintaining clonotypic diversity within the reactive memory T cell pool.