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, Kgp_467-477 (pKgp) and RgpA_1054-1064/Kgp_1074-1084 (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.