Identification of gingipain-specific I-A(b) -restricted CD4+ T cells following mucosal colonization with Porphyromonas gingivalis in C57BL/6 mice.

Chronic periodontitis is associated with Porphyromonas gingivalis infection. Although virulence factors of P. gingivalis are hypothesized to contribute to the pathogenesis of periodontitis, it is unclear whether the local CD4(+) T-cell-mediated response they elicit prevents or contributes to periodontal bone destruction. We hypothesize that major histocompatibility complex class II I-A(b) -binding peptides existing in Kgp and RgpA are presented to CD4(+) T cells during P. gingivalis oral colonization. The protein sequences of gingipains RgpA and Kgp, and OMP40 and OMP41 of P. gingivalis were scanned using an I-A(b) -binding matrix. From this analysis we identified 53 candidate peptides that had the potential to engage the peptide-binding groove of the I-A(b) molecule of C57BL/6 mice. An ELISpot-based screen revealed those peptide-primed effector/memory CD4(+) T cells that could be re-stimulated in vitro with P. gingivalis or the peptide itself to produce interleukin-17A or interferon-γ. Two immunodominant peptides, Kgp467-477 (pKgp) and RgpA1054-1064 /Kgp1074-1084 (pR/Kgp) were identified and engineered to be displayed on I-A(b) molecular tetramers. Peptide pR/Kgp is conserved across all sequenced P. gingivalis strains. C57BL/6 mice were orally inoculated with P. gingivalis strain 53977 and cervical lymph node cells were stained with phycoerythrin-conjugated pKgp::I-A(b) and pR/Kgp::I-A(b) tetramers. We found that only pR/Kgp::I-A(b) bound with the desired specificity to gingipain-specific CD4(+) T cells. The pR/Kgp::I-A(b) tetramer complex will allow the identification of effector/memory CD4(+) T cells specific for two virulence factors of P. gingivalis strains associated with periodontal disease.