In this study, we have evaluated the peptide-specific CD4+ T cell response to 126 overlapping A10L peptides and identified 7 peptides with positive responses. We also measured the IC50, intrinsic dissociation half-life, DM-mediated half-life, and DM-susceptibility of the corresponding HLA-DR1-A10L peptide complexes. Within this data set, DM-mediated half-life is the primary contributory factor of peptide immunogenicity and can predict CD4 T cell epitopes with high accuracy. In another set of peptides from the entire vaccinia virus genome, epitopes again were distinguished by their longer DM-mediated half-lives.
Although T cell response to each individual A10L peptide was only evaluated in donor SL131, we were able to verify some of the peptides in other vaccinia virus-vaccinated or nonvaccinated HLA-DR1 donors (Fig. S2
). Moreover, we did not observe any positive responses against A10L peptides in PBMC or CD4 TCL from non-vaccinia-immune HLA-DR1 donors (Fig. S1A, Fig. S2E
and data not shown). Whether these positive A10L peptides identified here are in general immunodominant vaccinia epitopes would need to be evaluated in additional donors.
As noted above, other factors beside MHC-peptide interaction may be involved in epitope selection. Endo-peptidases have been demonstrated to either enhance or destroy epitopes available to MHC II molecules (12
). A peptide cannot induce immune response if it is degraded by proteases due to the prominent protease cleavage site within the peptide. Protein expression level also must play a role. Besides the interaction between MHC II molecules and peptides, the binding between T cell receptor (TCR) and MHC-peptide complex has also been shown to be important for peptide immunogenicity, both in CD4 (44
) and CD8 contexts (59
). It is possible that a strong interaction between TCR and MHC II-peptide complex can compensate for the low stability of MHC II-peptide complexes. Finally, the asymmetrical T cell repertoire and TCR frequency would in some case influence peptide immunogenicity and epitope selection (13
). Despite these caveats, our data still indicate that DM-mediated half-life is a primary factor that governs peptide-specific CD4 T cell responses (–). Notably, Isamu Hartman and colleagues recently using a cell-free antigen processing system composed of HLA-DR1, DM and cathepsins successfully identified known and novel immunodominant epitopes from various antigens (21
), which further demonstrates a role for DM in epitope selection.
Significant progress has been made on understanding the role of MHC II-peptide complex stability in peptide immunogenicity (17
), which can be utilized to develop more effective epitope prediction algorithms. Most current CD4 T cell epitope prediction algorithms are based on either binding affinity to MHC II molecules, including P9 and IEDB evaluated in this study, or characteristics of endogenously processed peptides associated with MHC II proteins, including Syfpeithi in this study (30
). Consistently, we found that the predictive abilities of the three evaluated computational algorithms were similar with those of IC50
and intrinsic half-life. Here, we have shown that DM-mediated half-life predicted CD4 T cell epitopes better than the tested prediction algorithms. The effect of DM on antigen presentation and epitope selection was also observed in previous reports (10
). Thus, a method to take DM-mediated peptide dissociation into account in epitope prediction algorithms would be likely to increase their accuracy.
DM catalyzes peptide exchange in a specialized compartment for loading peptides onto MHC II molecules (5
). The reported half time of MHC II residence in the peptide loading compartment, corresponding to the time for which they exposed to the action of DM, has been measured at ~4 to 6 hrs (61
). Notably, in our ROC analysis, when the threshold of DM-mediated half-life was set to 6 hrs, the prediction of epitopes was highly efficient with 100% sensitivity and 96 % specificity (). Considering this, our observation may indicate that the half-lives of MHC II-peptide complexes in the presence of DM should be longer than the MHC II transit time in order for them to get presented and selected as epitopes.
DM has been shown to be associated with several autoimmune diseases including rheumatoid arthritis (RA), but the mechanism by which DM mediates susceptibility to RA is still unknown (22
). The expression of DM, as well as the DM:DR ratio, were found to be decreased significantly in RA patients (64
). It was also found that the autoantigen type II collagen in RA and autoantigen glutamate decarboxylase in type I diabetes both show DM-dependent presentation in those autoimmune disease models (9
). Therefore, it is possible that due to the down regulation or deficiency of DM in antigen presenting cells, self peptides bound to MHC II alleles that are susceptible to DM-mediated dissociation and normally have short DM-mediated half-lives can get presented and cause autoimmune disease. This idea is supported by the observation that the repertoire of autoimmune-encephalitogenic T cells consists primarily of T cells specific for short-lived MHC complexes due to tolerance deletion of T cells that recognize long-lived complexes in an experimental allergic encephalomyelitis study (14
). This negative correlation between peptide immunogenicity and strength of HLA-DR binding were also seen in other autoimmune animal studies (66
), and multiple sclerosis patients study (26
), which further support the hypothesis that autoreactive T cells escape negative selection due to the low stability of corresponding MHC-peptide complexes. Thus, the importance of central and peripheral tolerance can likely explain the apparent paradox that foreign pathogenic epitopes form stable and DM-resistant complexes, as seen in this study and previous reports (10
), while autoimmune self-epitopes form complexes with low affinity and kinetic stability (14
In conclusion, we have shown that long DM-mediated half-life is a distinguishing feature of CD4 T cell epitopes, and that DM-mediated kinetic stability can effectively predict CD4 T cell epitopes in an example of a human infectious disease.