Method: Previous work identified peptides predicted to fit the binding groove of the MHC class II I-Ab heterodimer. ELISPOT identified two immunodominant epitopes: pR/Kgp, shared by two Pg virulence factors, Arg- (R) and Lys-gingipain (Kgp) (RgpA*1054-64, Kgp*1074-1084), and pKgp (Kgp*467-477). These peptides were each engineered in-frame with His-tagged I-Ab β-chain and were assembled with biotinylated I-Ab α-chain into pMHCII monomers in S2 insect cells. Monomers were purified using nickel-affinity and size-exclusion chromatography, and were tetramerized with PE-conjugated streptavidin. pMHCII tetramers were used to detect antigen-experienced (CD44Hi), Pg antigen-specific memory CD4+ T-cells with flow cytometry. Non-specific staining was evaluated post hoc by tetramer-bound CD8+ T-cells.
Result: pR/Kgp::I-Ab tetramer identified Pg antigen-specific CD44Hi CD4+ T-cells (0.13%) in mice subcutaneously-inoculated with Pg with very low background staining on (0.001% of CD8+ T-cells) and in sham-inoculated mice (0.0004% of CD44Hi CD4+ T-cells). However, pKgp::I-Ab tetramer yielded an unsuccessful reagent due to low specificity and non-specificity in experimental conditions (0.053% CD44Hi CD4+ T-cells; 0.018% CD8+ T-cells) and sham-inoculated control mice (0.067% CD44Hi CD4+ T-cells).
Conclusion: We have selected, engineered and produced a pMHCII tetramer that identifies a subset of CD4+ T-cells specific for an immunodominant epitope conserved among Pg virulence factors RgpA and Kgp. This tetramer will allow us determine the phenotype of Pg -specific CD4+ T-cells in vivo. We are currently exploring differences in efficacy between pR/Kgp and pKgp tetramers due to potential differences in purification.
Keywords: Animal, Immunology, Pathogen recognition and Periodontal disease