Limited diversity of peptides related to an alloreactive T cell epitope inthe HLA-B27-bound peptide repertoire results from restrictions at multiplesteps along the processing-loading pathway

The influence of various factors along the processing-loading pathway in li miting the diversity of HLA-B27-bound peptides around a core protein sequen ce was analyzed. The C5 proteasome subunit-derived RRFFPYYV and RRFFPYYVY p eptides are natural B*2705 ligands, The octamer is an allospecific CTL epit ope, Digestion of a 27-mer fragment of C5 revealed that both ligands are ge nerated from this precursor substrate with the 20S proteasome in vitro in a ratio comparable to that in the B*2705-bound peptide pool. The C5 sequence allowed to derive a nested set of six additional peptides with 8-11 residu es containing the core octamer sequence and the Arg2 motif of HLA-B27, none of which was found in the B27-bound pool. Together, low proteasomal yield, disfavored TAP-binding motifs, and low affinity for B*2705 accounted for t he absence of four of the six: peptides, The two remaining differed from th e natural octamer or nonamer ligands only by an additional N-terminal Ser r esidue. Their stability in complex with B*2705 was lower than the respectiv e natural ligands, raising the possibility that N-terminal trimming might h ave favored a shift toward the more stable peptides, The results suggest th at the B*2705-bound peptide repertoire has a highly restricted diversity ar ound a core alloantigenic sequence. This is not explained by a single bottl eneck feature, but by multiple factors, including proteasomal generation, T AP-binding motifs, MHC-binding efficiency, and perhaps optimized stability through N-terminal trimming. Tapasin-dependent restrictions, although not e xcluded, were not required to explain the absence in vivo of the particular peptide set in this study.