Non-linear in cis-spliced bacterial MHC class II epitopes are formed in human monocyte-derived dendritic cells and are recognized by human CD4+ T cells

Abstract

Pathogen-derived spliced MHC class II epitopes have not been reported so far. Earlier we studied HLA-DRB*0101-presented peptides derived from P1.7-2,4, a major outer membrane porin (PorA) serosubtype from Neisseria meningitidis, using stable isotope-tagging of the antigens. We found evidence for +114 Da and +270 Da in cis-spliced peptide variants, but could not unequivocally identify their sequence at the time. Here we aimed to confirm this biological phenomenon and determine the identity and immunogenicity of the in-cis-spliced pathogen-derived epitopes. Metabolically 14N and 15N-labeled P1.7-2,4 preps were isolated and mixed 1:1 (protein mass) before pulsing monocyte-derived HLA-DRB*0101 homozygous dendritic cells for 48 hr. MHC peptides were acid-eluted after HLA-DR pull-down, fractionated and analysed using nanoLC-EThcD- MS-MS. Mass spectra of P1.7-2,4-derived peptides were algorithm-searched based on their 14N/15N-ion doublets. Candidate ions were sequenced by using EThcD. Linear and spliced peptides were tested ASVG+114 Da and IGNYTQINAASVG+270 Da were confirmed, present in full 14N-and 15N-labeled form, indicating in-cis transpeptidation. The improved sequence coverage enabled by using EThcD identified the in cis -spliced peptides as IGNYTQINAASVG, C-terminally extended with Gly-Gly or Gly- Gly-Arg. Both linear and in cis -spliced region 8 peptides stimulated clone JS-63 in an HLA-DR-restricted manner. This is the first evidence for the formation of pathogen-derived in cis -spliced epitopes that are recognized by human CD4+ T cells. The mechanism behind the in cis transpeptidation remains to be elucidated, although the overall P1.7-2,4 sequence may inform a protease specificity. Further studies are ongoing to evaluate the biological consequences of these findings.