C-terminal localization of the HGIRNASFI peptide allows its presentation on the surface of infected cells.
(A) Cell surface expression of MHC class I molecule (Db). LSECs (C57BL/6) were infected with the indicated viruses at MOI of 5 with centrifugal enhancement as described. Db expression was measured by flow cytometry at 16h p.i‥ Fluorescence histograms for a representative experiment are shown. (B) LSECs were infected with the indicated viruses or incubated with a corresponding dose of UV inactivated virus (UV dose– 150J) at an MOI of 0.2 with centrifugal enhancement and co-cultured with HGIRNASFI-specific CTLs at an E:T ratio 3:1. Co-culture was performed for 15h, upon which the T cells were collected and stained for intracellular IFNγ. Where indicated, virus was inactivated by UV light. Two independent experiments were performed, with 4 or 5 wells per experimental condition. Representative dot plots are shown. (C) Relative intensity of signals measured by targeted nanoLC-MS3 from Db-immunoprecipitates of cells infected for 24 hours with indicated viruses at an MOI of 2 with centrifugal enhancement. Two high abundant endogenous control peptides (KALINADEL and AALENTHLL) and a low abundant (FGPVNHEEL) endogenous control peptide were present in all IP samples, indicating valid sample processing in all cases. The MCMV target peptide HGIRNASFI was detected only in the cells infected with the MCMVM45Cterm recombinant. (D) Grouped means +/- SEM of blood CD8 T-cells stained by HGIRNASFI-Db tetramers in bone-marrow chimeric mice, where C57BL/6 mice received TAP-/- or C57BL/6 bone marrow at 3 months before infection with 106 PFU/mouse of MCMVWT (top panel) or MCMVM45Cterm (bottom panel). The experiment was performed three times at 5 mice per group and pooled results are shown. Significance on day 7 p.i. was assessed by a Mann—Whitney U test. ****p < 0.0001, ns—not significant.