Identification of gingipain‐specific I ‐ A b ‐restricted CD 4 + T cells following mucosal colonization with P orphyromonas gingivalis in C 57 BL /6 mice

Summary Chronic periodontitis is associated with P orphyromonas gingivalis infection. Although virulence factors of P . gingivalis are hypothesized to contribute to the pathogenesis of periodontitis, it is unclear whether the local CD 4 + 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 CD 4 + T cells during P. gingivalis oral colonization. The protein sequences of gingipains R gp A and Kgp, and OMP 40 and OMP 41 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 ELIS pot‐based screen revealed those peptide‐primed effector/memory CD 4 + T cells that could be re‐stimulated in vitro with P . gingivalis or the peptide itself to produce interleukin‐17 A or interferon‐γ. Two immunodominant peptides, Kgp 467–477 (p K gp) and RgpA 1054–1064 /Kgp 1074–1084 (p R /Kgp) were identified and engineered to be displayed on I‐A b molecular tetramers. Peptide p R /Kgp is conserved across all sequenced P. gingivalis strains. C 57 BL /6 mice were orally inoculated with P. gingivalis strain 53977 and cervical lymph node cells were stained with phycoerythrin‐conjugated p K gp::I‐A b and p R / K gp::I‐ A b tetramers. We found that only p R /Kgp::I‐A b bound with the desired specificity to gingipain‐specific CD 4 + T cells. The p R /Kgp::I‐A b tetramer complex will allow the identification of effector/memory CD 4 + T cells specific for two virulence factors of P . gingivalis strains associated with periodontal disease.