Proteasome-Generated cis-Spliced Peptides and Their Potential Role in CD8⁺ T Cell Tolerance

The human immune system relies on the capability of CD8⁺ T cells to patrol body cells, spot infected cells and eliminate them. This cytotoxic response is supposed to be limited to infected cells to avoid killing of healthy cells. To enable this, CD8⁺ T cells have T Cell Receptors (TCRs) which should discriminate between self and non-self through the recognition of antigenic peptides bound to Human Leukocyte Antigen class I (HLA-I) complexes-i.e., HLA-I immunopeptidomes-of patrolled cells. The majority of these antigenic peptides are produced by proteasomes through either peptide hydrolysis or peptide splicing. Proteasome-generated cis-spliced peptides derive from a given antigen, are immunogenic and frequently presented by HLA-I complexes. Theoretically, they also have a very large sequence variability, which might impinge upon our model of self/non-self discrimination and central and peripheral CD8⁺ T cell tolerance. Indeed, a large variety of cis-spliced epitopes might enlarge the pool of viral-human zwitter epitopes, i.e., peptides that may be generated with the exact same sequence from both self (human) and non-self (viral) antigens. Antigenic viral-human zwitter peptides may be recognized by CD8⁺ thymocytes and T cells, induce clonal deletion or other tolerance processes, thereby restraining CD8⁺ T cell response against viruses. To test this hypothesis, we computed in silico the theoretical frequency of zwitter non-spliced and cis-spliced epitope candidates derived from human proteome (self) and from the proteomes of a large pool of viruses (non-self). We considered their binding affinity to the representative HLA-A^*02:01 complex, self-antigen expression in Medullary Thymic Epithelial cells (mTECs) and the relative frequency of non-spliced and cis-spliced peptides in HLA-I immunopeptidomes. Based on the present knowledge of proteasome-catalyzed peptide splicing and neglecting CD8⁺ TCR degeneracy, our study suggests that, despite their frequency, the portion of the cis-spliced peptides we investigated could only marginally impinge upon the variety of functional CD8⁺ cytotoxic T cells (CTLs) involved in anti-viral response.