In the present study, we characterized in healthy adults the cellular immune responses to a variety of HHV-6 preparations and defined 11 immunodominant CD4+ T cell epitopes. These represent the first HHV-6-specific immunodominant epitopes defined for this virus. Using samples of peripheral blood, we found that the CD4+ T cell response to HHV-6 is characterized by a low frequency of cytokine-producing cells (<0.12%). We expanded the HHV-6-specific population using purified disrupted HHV-6A and HHV-6B preparations to reveal HHV-6-specific responses in T cell lines many orders of magnitude over the background response. Using these in vitro-expanded TCLs, we confirmed the polyfunctional cytokine and potential cytolytic nature of the anti-HHV-6 CD4+ response, and we evaluated the specificity and variant cross-reactivity in the CD4+ T cell response to the 11 HLA-DR-restricted epitopes. MHC tetramers carrying these epitopes were able to detect HHV-6-specific CD4+ T cells in samples of peripheral blood.
Characterization of CD4 T cell epitopes in HHV-6 is complicated by poor knowledge of the immune response to this virus, the low frequency of responding cells, the large number of proteins encoded by the HHV-6 genome (20
), and the lack of a small animal model (30
). We approached the characterization of the T cell response to HHV-6 using a candidate epitope approach. Initial screening of synthetic peptides was performed with fresh PBMCs rather than expanded cell populations, to assess the full diversity of the HHV-6 T cell response and avoid potential skewing toward cells capable of preferential expansion in vitro
. In spite of using fresh PBMCs and a relatively low number of peptides in the pools (4 to 6 peptides), we observed that for some pools eliciting positive responses we were not able to identify individual peptides that met our criteria for a positive response (, pool 53). This potential limitation of a bottom-up, peptide-based approach has already been highlighted in studies of the T cell response to HIV (57
) and hepatitis C virus (HCV) (78
). PBMC responses to candidate epitopes identified in the peptide screening were validated using TCLs raised against viral preparations and individual peptides. In order to obtain a representative sample of T cell responses in healthy adults, TCLs were generated from 6 donors, including the two used in the PBMC studies. The responses of virus-expanded TCLs were found to be variable between individuals. Each candidate epitope was recognized by at least one TCL, but a TCL from any single donor did not recognize all 11 peptides identified. In spite of differences in haplotype, 5 peptides were recognized by at least 4 of the 6 donors (1106, 1162, 1211s, 020, and 083s). Peptide-expanded TCLs generated from PBMCs of two donors recognized each one of the 11 candidate HHV-6 CD4+
T cell epitope peptides. These antipeptide TCLs showed consistent IFN-γ CD4+
T cell responses to both purified viral antigen and heat-inactivated lysate of HHV-6-infected cells, demonstrating that the corresponding epitopes are generated upon processing of the source protein and so represent immunodominant and not cryptic epitopes.
MHC tetramers are very useful reagents in following antigen-specific T cell responses ex vivo
, and we were able to identify epitopes that could be used for tetramer staining in PBMCs. However, the low frequency of responding cells resulted in low signals relative to background staining unless tetramer-positive cells were enriched using magnetic beads prior to flow cytometry. A previous study has indicated that one of the benefits of enrichment prior to staining is the reduction of background staining (62
). We observed this reduction in background staining as well. Tetramer staining signals were significantly above background for the HA and all the 11 HHV-6 tetramers postenrichment compared to only two tetramers before enrichment. However, comparison of the frequencies of tetramer-positive cells before and after enrichment indicates that the enrichment procedure introduces some skewing of the tetramer-positive population. Considering the frequency of tetramer-positive cells after enrichment, HLA-DR1-peptide complexes could be sorted into two well-delineated groups. The first group includes HLA-DR1-peptide complexes with 1096, 083s, and 020 with MHC tetramer staining frequencies of over 8% after enrichment. The relative affinities of peptides in this group for HLA-DR1 are high, with IC50
values below 1,500 nM (range, 80 to 1,500 nM). The second group, with lower staining frequency, includes the remaining eight HHV-6 complexes. Relative affinities in this second group are more variable, with an IC50
range from 220 nM in peptide 1105 to 30 μM in peptide 1106. These findings suggest that the tetramer-positive frequencies observed postenrichment could be a function of not only the preenrichment frequency but also the relative affinity of a peptide for HLA-DR1. The tetramer enrichment procedure potentially could be improved by using covalently attached peptides (16
), particularly for weaker-affinity binders. Also, it is possible that the tetramer enrichment technique might not be appropriate for T cell populations with relatively low functional avidities (62
). Despite these caveats, MHC tetramer staining experiments using peptide-expanded TCLs demonstrated significant correlation between tetramer staining and IFN-γ ICS responses. Thus, HLA-DR1 tetramers carrying the HHV-6 epitopes identified here should be useful in following HHV-6-specific CD4+
T cell responses.
Overall, peptides derived from HHV-6 virion proteins appear to be recognized by CD4+
T cell epitopes more frequently than are peptides from virally encoded proteins in general. In our studies, 10 out of 220 (~4.5%) high-scoring virion peptides were identified as containing CD4+
T cell epitopes, compared to two out of 113 (~1.8%) high-scoring peptides from the translated genome as a whole. In a previous study of CD4+
T cell responses to vaccinia virus, we had observed that T cell epitopes were not found frequently among peptides with the very highest MHC binding prediction scores (nor among the peptides experimentally shown to have the very highest affinity) (11
). One possible factor contributing to the epitope skewing that we observed might have been higher overall MHC binding predictions for the whole-genome set; however, the skewing is observed even when sets in the same prediction score range were compared (normalized combined P9-SYFPEITHI score, 0.54 to 0.67): with 2 out of 9 virion peptides recognized but only 1 out of 84 whole-genome peptides (B). Lower expression levels for proteins selected from the whole translated genome than for virion components do not appear to explain the observed skewing, since the whole-genome set included 19 peptides from six proteins abundantly expressed in persistent infection (U18, U20, U27, U85, U90, and U94) (53
), none of which were identified as T cell epitopes. Finally, the observed epitopes derive from proteins exhibiting early, intermediate-early, and late expression patterns (71
), suggesting that temporal regulation of gene expression is not a critical factor for CD4 T cell immunogenicity in HHV-6. Thus, several factors suggested previously to regulate CD4+
T cell immunogenicity in other scenarios do not appear to explain the skewing toward virion components that we observe for long-term responses to HHV-6. It is possible that HHV-6 may employ immunoevasion strategies to limit generation of CD4+
T cell epitopes in infected cells, as it does for CD8+
T cell epitopes (29
), perhaps employing strategies related to those reported for other herpesviruses (9
One of the major problems in assessing the involvement of HHV-6 in human diseases is the lack of reagents that can be used to track immune responses to this virus, especially reagents that could differentiate between HHV-6 and HHV-7. A previous study that analyzed a large number of CD4+
T cell clones demonstrated that 70% of the clones respond to both HHV-6 and HHV-7 (87
). Sequence analysis of the 11 epitopes reported here reveals that seven of these peptide sequences do not have significant homologies with other human pathogens, including HHV-7 (see Table S2 in the supplemental material). Thus, these epitopes should be useful in tracking immune responses specific for HHV-6, although it should be noted that it remains to be directly experimentally verified that reactivity to these epitopes is elicited upon infection/reactivation of HHV-6. The four remaining peptides, 083s, 1017, 1106, and 1211s, have significant homology with HHV-7 (see Table S2). HHV-6A but not HHV-6B is characterized by neurotropism (2
), and so it would be advantageous to have reagents to corroborate if relapses in diseases like MS are associated with immune responses to HHV-6A rather than HHV-6B, as suggested by Fogdell-Hahn et al. (25
). Sequence analysis of the 11 epitope peptides and cross-reactivity studies () suggest that they might not fulfill this requirement. An epitope screening strategy focused on variable regions together with a fine mapping study could in principle provide immunological reagents to differentiate between responses to HHV-6 variants, although it would be preferable to have sequencing of a larger number of variant strains to select variant-specific sequences.
Overall, we found that IFN-γ-secreting CD4+
T cells responding to HHV-6 represented less than 0.1% of the total CD4+
T cell population. This can be contrasted to the related betaherpesvirus HCMV, for which much higher responding-cell frequencies, typically 2 to 6% of total T cells, were observed (55
). Like HHV-6, HCMV infection usually is asymptomatic with establishment of a lifelong chronic latency, and the continued presence of virus has been suggested to be important in maintaining high responding-cell frequencies. In our experiments, we observed relatively low levels of IL-2 compared to other cytokines in supernatants of PBMCs or TCLs challenged with inactivated HHV-6 viral preparations. Moreover, IL-2 levels did not increase substantially after expansion of HHV-6-specific subpopulations using heat-inactivated or detergent-disrupted (not shown) virus preparations, although levels of other cytokines were significantly increased ( and ). Previous publications have demonstrated impaired IL-2 production and cell proliferation of PBMCs and the induction of IL-10 in cells infected with HHV-6 (24
). Consequently, it is reasonable to suggest that the combined effects of impaired IL-2 production and the induction of IL-10 by HHV-6 could limit the expansion of HHV-6 T cell responses. Despite the differences in responding-cell frequencies, the targets of the response are similar, with most HCMV-specific CD4+
T cells targeting abundant virion proteins (68
), as we observed also for CD4+
T cells recognizing HHV-6.
In summary, we report that IFN-γ can be used as a surrogate marker of the cellular response to HHV-6. Using HHV-6 viral preparations, lysates of infected cells, and peptides, we observed very low frequencies of responding CD4+ T cells. The response mapped preferentially to peptides derived from abundant virion proteins. Although the T cell response to HHV-6 was much lower than that to the related betaherpesvirus HCMV, homologous proteins were targeted. Finally, we present seven virus-specific peptide sequences that could be used to track immune responses to HHV-6.