We have used an influenza virus infection model to address the relationship between the naturally occurring age-associated decline in repertoire diversity and the response of aged animals to a newly encountered pathogen. We showed using in vivo limiting dilution analysis that the precursor frequency of T cells specific for NP in naive, young C57BL/6 mice is ~10-fold lower than the precursor frequency of T cells specific for PA. The CD8 T cell responses to NP and PA in young mice are equidominant (31
) and are mediated predominantly by a restricted repertoire of Vβ8.3+
T cells and a diverse repertoire of Vβ7+
T cells, respectively (36
). However, analysis of the response of aged mice to de novo influenza virus infection exhibited a markedly altered immunodominance, characterized by a preferential loss of NP reactivity. In addition, TCR repertoire analysis with both TCR Vβ antibodies and CDR3 spectratyping showed age-associated perturbations in the repertoire of NP- and PA-specific CD8 T cells that were unique in individual mice. Importantly, the impact of age-related contraction of the T cell repertoire on the CD8 T cell response to influenza virus infection was mimicked by thymectomy, which accelerated the age-associated loss of NP responsiveness. Finally, the age-dependent failure to develop an NP-specific response after primary infection generally correlated with poor heterosubtypic protection. Together, these data provide experimental evidence that the age-associated decline in CD8 T cell repertoire diversity can greatly impact the response to new infections, and the development of heterosubtypic immunity. Importantly, perturbations in the repertoire of T cells specific for influenza virus epitopes for which there is a low precursor frequency and limited TCR diversity, lead to the selective development of holes in the repertoire for a typically immunodominant viral epitope.
T cell repertoire is frequently characterized by either exhaustive sequence analysis of individual cells or spectratype analysis of bulk populations of T cells, and different information is obtained by the two techniques. The repertoires of Vβ8.3+
NP-specific T cells and Vβ7+
PA-specific T cells in C57BL/6 mice have been extensively characterized by clonal sequencing. 45 β-chain sequences have been reported for Vβ8.3+
NP-specific T cells, 3 of which were public (shared between individuals), and 241 different β-chain sequences have been reported for Vβ7+
PA-specific T cells, none of which were public. Individual mice expressed an average of 8 different NP Vβ8.3 sequences and 21 different PA Vβ7 sequences (36
). More recently, sequence analysis of NP-specific T cells was extended to include those expressing Vβ4+
TCRs, and only four different clonotypes were identified, leading to the conclusion that the Vβ4 component of the primary NP response in young mice was restricted and public, analogous to the Vβ8.3 component (37
). A broader picture of repertoire diversity can be assessed by spectratype analysis, as CDR3 diversity reflects the overall complexity of T cell populations. Whereas naive repertoires show a characteristic Gaussian distribution of CDR3 length diversity, oligoclonal responses are reflected by the emergence of distinct peaks (46
). These peaks sometimes reflect expansion of a single clone, but this is not the case for the responses to NP and PA. For example, for the dominant Vβ8.3 and Vβ7 responses to NP and PA in C57BL/6 mice, there have been shown to be several different sequences with the same CDR3 length represented within the dominant 9- and 6-aa spectratype peaks, respectively (36
). Thus, our spectratype analysis was comprehensive in that it spanned all TCR Vβ families. The response in individual young mice to both NP and PA was diverse. This heterogeneity in the fine specificity of epitope-specific T cells is predicted by the previous demonstration that the naive repertoire to specific peptides in individual mice is unique (48
). Because of stochastic age-associated declines in the naive repertoire, the extreme heterogeneity in individual aged mice is not unexpected. Importantly, because of the low precursor frequency and the comparatively restricted diversity in the TCR repertoire of NP-specific T cells in young mice, approximately half of aged mice developed a hole in the repertoire and were unable to generate an effective response to NP.
T cell responses to de novo antigens are not dependent solely on naive precursors. Because T cell recognition is highly degenerate (49
), and there is a surprising degree of cross reactivity in the T cell responses to seemingly unrelated viral antigens (51
), it has been suggested that fortuitously cross-reactive memory cells may make an important contribution to the response to newly encountered antigens in mice as the naive repertoire becomes increasingly constrained with age (53
). Indeed, aged mice show a high proportion of memory–phenotype T cells in the periphery. This complicates the simple interpretation of our data, in that our analyses did not distinguish the relative contributions of naive and cross reactive memory cells to the antiviral CD8 T cell response in individual mice. This does not, however, lessen the significance of our data which demonstrate that age-associated loss of repertoire diversity dramatically impacts the response to de novo antigens, and can, in some cases, lead to loss of responsiveness to a particular epitope. Moreover, it is likely that, because of structural constraints, the NP epitope elicits only a restricted repertoire of naive T cells (39
), and is less likely to induce cross reactivity in heterologous memory cells. Thus, cross reactive memory cells may only make a minor contribution to the NP response in naive aged mice. Defining the exact role of these cells in the response of aged mice to primary influenza virus infection will require further study.
T cell clonal expansions frequently develop in aged individuals, and contribute to the age-associated decline in repertoire diversity (21
) and to compromised antiviral CD8 T cell immunity (50
). Aged mice showing signs of clonal expansions indicated by Vβ perturbations in the peripheral blood CD8 T cells were excluded from our study. However, because the NP-specific precursor repertoire appears sensitive to any perturbation in the naive repertoire, it is likely that the presence of clonal expansions would further constrain the diversity of the antiviral repertoire as described in the current studies (21
How do our data fit in with other studies on the impact of aging on the de novo response to influenza virus infection? Reduced primary CD8 T cell responses to influenza virus in aged mice, including delayed viral clearance, have been reported (12
), consistent with generalized defects in the immune response to influenza virus in elderly people (9
). In a previous study, it was shown that aged mice had a reduced frequency of NP-specific T cells and a corresponding reduction in overall NP-specific effector function, but responses to other epitopes were not examined (12
). In contrast, other reports found no functional defect in the response of aged naive CD8 T cells (14
). Consistent with this, we have shown that despite being found in small numbers, NP-specific CD8 T cells elicited in infected aged mice are as functional as those elicited in younger mice, as determined by IFNg ELISpot analysis (unpublished data). Our data reconcile this apparent inconsistency by showing that the loss of CD8 T cell responses to particular epitopes can be explained by age-associated development of “holes” in the naive T cell repertoire, rather than functional defects, which is consistent with the findings that the CD8 cells present in aged mice are fully functional.
What are the consequences of a loss of reactivity to NP and corresponding shifts in immunodominance for protective immunity? In the current studies, we examined the impact of the loss of NP responsiveness on the immediate recall response to secondary influenza virus infection and showed a general correlation between the ability to clear heterosubtypic virus and the generation of a strong NP response after primary infection. This was surprising to us, as we anticipated that in the absence of the immunodominant NP epitope, compensatory responses to known or hidden epitopes would emerge (42
). However it has been shown that epitopes may vary in protective efficacy (60
In terms of repertoire considerations, in our studies, aged mice generated a comparable CD8 T cell response in terms of numbers of CD8 T cells elicited in the lung and BAL after infection. However, there was a clear shift in immunodominance. Because we only measured responses to three influenza virus epitopes (NP, PA, and PB1), in many cases we failed to identify the viral epitopes to which the aged mice responded. Despite the declining responsiveness to NP, aged mice appeared able to control the infection. However, consistent with other reports (12
), viral clearance was delayed in aged mice (unpublished data), likely a consequence of shifts in immunodominance. Shifts in immunodominance can also have unexpected effects on the response to other epitopes. For example, it has been suggested that in the face of an enhanced PA response, the NP response is suppressed (40
). However, this was not the case in the current studies, as the NP response remained either greatly diminished or absent in a high percentage of aged mice after infection with a virus from which the PA/Db
epitope could not be generated (41
). Whereas it has been shown that the primary responses to NP and PA are relatively equidominant in C57BL/6 mice, the NP response is extremely immunodominant in the recall response (47
). Thus, there may be an important impact of age-associated loss of NP responsiveness on the secondary recall responses of those mice with PA-dominated responses after primary infection. For example, we have previously shown that vaccination of young mice with the PA peptide elicited a strong epitope-specific response, but failed to protect upon challenge with wild-type virus. In addition, it appeared that vaccination with PA actually exerted a detrimental effect on subsequent protection under certain circumstances (40
). Therefore, the dominant primary response to PA in aged mice may have a profound impact on protective immunity.
However, because the impact of aging on the development of long-lasting protective immunity is complex, there are likely other contributing factors in addition to effects on the CD8 T cell repertoire. For example, CD4 signals are essential during the priming phase for the generation of good memory (64
). As CD4 T cell function is impaired in aged mice (5
), it is essential to determine how this contributes to impaired development of protective immunity after de novo infection of aged mice. We are currently examining this question.
Collectively, our findings may have important implications for the future design of cellular vaccines intended for the elderly human population. In addition to declining innate immunity and impaired humoral immune responses, the loss of T cell repertoire diversity needs to be considered. The potential for the development of holes in the T cell repertoire of aged individuals argues against the feasibility of epitope-based vaccination strategies for the elderly. In addition, the emphasis on improving adjuvants to overcome the defective immunity of the aged may be inappropriate, as even the most potent adjuvants will not augment the normal protective CD8 T cell response to infection in the absence of antigen-specific precursors, and may even result in priming of nonprotective or detrimental responses in aged individuals. Rather, newer strategies need to focus on boosting preexisting memory T cell responses present within aged individuals. In addition, it may be desirable to prime cellular immunity before severe loss of thymic output, suggesting that more vaccinations during middle age may be indicated. Therapeutic approaches for improving survival and maintenance of naive T cells, prolonging thymic output, and reconstituting the repertoire of the elderly through hematopoietic stem cell reconstitution should also be considered (6