CD8⁺ T cells of Listeria monocytogenes-infected mice recognize both linear and spliced proteasome products

CD8⁺ T cells responding to infection recognize pathogen-derived epitopes presented by MHC class-I molecules. While most of such epitopes are generated by proteasome-mediated antigen cleavage, analysis of tumor antigen processing has revealed that epitopes may also derive from proteasome-catalyzed peptide splicing (PCPS). To determine whether PCPS contributes to epitope processing during infection, we analyzed the fragments produced by purified proteasomes from a Listeria monocytogenes polypeptide. Mass spectrometry identified a known H-2K^b-presented linear epitope (LLO_296-304) in the digests, as well as four spliced peptides that were trimmed by ERAP into peptides with in silico predicted H-2K^b binding affinity. These spliced peptides, which displayed sequence similarity with LLO_296-304, bound to H-2K^b molecules in cellular assays and one of the peptides was recognized by CD8⁺ T cells of infected mice. This spliced epitope differed by one amino acid from LLO_296-304 and double staining with LLO_296-304- and spliced peptide-folded MHC multimers showed that LLO_296-304 and its spliced variant were recognized by the same CD8⁺ T cells. Thus, PCPS multiplies the variety of peptides that is processed from an antigen and leads to the production of epitope variants that can be recognized by cross-reacting pathogen-specific CD8⁺ T cells. Such mechanism may reduce the chances for pathogen immune evasion.