Materials and MethodsĪ C1R–HLA-B*18:01 cell pellet (5 × 10 8 cells) was ground in a Retsch mixer mill MM 400 under cryogenic conditions, resuspended in 0.5% IGEPAL, 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and protease inhibitors (cOmplete protease inhibitor cocktail tablet Roche Molecular Biochemicals) at a density of 5 × 10 7 cells/ml and incubated with rotation for 1 h at 4☌ ( 15). These results underscore the exquisite specificity of the immune system but highlight the potential danger of self-reactivity by T cells expanded in response to common viral infection. A significant proportion of T cells raised against the EBV epitope cross-reacted with this HLA-B*18:01–binding self-peptide, and structural studies revealed the molecular basis of this cross-reactivity. A peptide corresponding to this sequence from the cleavage and polyadenylation specific factor 3–like protein (CPSF3L) was shown by peptide elution/mass spectrometry to be presented by the HLA-B*18:01 molecule on the surface of human cells. In the present study, we have identified a human protein sequence (DELEIKAY, termed DEL) with sequence homology to the octamer EBV epitope. Recently, we investigated the CD8 + T cell response to the immediate early, BZLF1 Ag of EBV and described an octamer peptide 173SELEIKRY 180 (termed SEL) that is recognized by HLA-B*18:01 + individuals ( 14). EBV infection is associated with several autoimmune conditions, including MS, for which a molecular mimicry mechanism has been proposed ( 13). The lymphotropic γ-1 herpesvirus, EBV, is widespread in all human populations, infecting ∼95% of the adult population worldwide ( 11, 12). These results illustrate how aberrant immune responses and immunopathological diseases could be generated by EBV infection. To our knowledge, this is the first report confirming the natural presentation of a self-peptide cross-recognized in the context of self-HLA by EBV-reactive CD8 + T cells. Structural studies revealed that the self-peptide–HLA-B*18:01 complex is a structural mimic of the EBV peptide–HLA-B*18:01 complex, and that the strong antiviral T cell response is primarily dependent on the alanine/arginine mismatch at position 7. A diverse array of TCRs was expressed by the cross-reactive T cells, with variable functional avidity for the self-peptide, including some T cells that appeared to avoid autoreactivity by a narrow margin, with only 10-fold more of the self-peptide required for equivalent activation as compared with the EBV peptide. A significant proportion of CD8 + T cells raised from some healthy individuals against this EBV epitope cross-reacted with the self-peptide. This self-peptide was shown to bind stably to HLA-B*18:01, and peptide elution/mass spectrometric studies showed it is naturally presented by this HLA molecule on the surface of human cells. In this study, we describe a human self-peptide (DELEIKAY) that is a homolog of a highly immunogenic EBV T cell epitope (SELEIKRY) presented by HLA-B*18:01. EBV represents a potentially important factor in the pathogenesis of several T cell–mediated autoimmune disorders, with molecular mimicry a likely mechanism. These data also provide a powerful approach to identify and monitor B cells in the PB that correspond to clonally amplified populations in the CNS in MS and other inflammatory states.T cell cross-reactivity underpins the molecular mimicry hypothesis in which microbial peptides sharing structural features with host peptides stimulate T cells that cross-react with self-peptides, thereby initiating and/or perpetuating autoimmune disease. B cells are strong candidates for autoimmune effector cells in MS, and these findings suggest that CNS-directed autoimmunity may be triggered and supported on both sides of the BBB. Some clusters of related IgG-VH appeared to have undergone active diversification primarily in the CNS, while others have undergone active diversification in the periphery or in both compartments in parallel. For the first time to our knowledge, we found that a restricted pool of clonally related B cells participated in robust bidirectional exchange across the BBB. We applied deep repertoire sequencing of IgG heavy chain variable region genes (IgG-VH) in paired cerebrospinal fluid and PB samples from patients with MS and other neurological diseases to identify related B cells that are common to both compartments. However, it is unclear whether antigen-experienced B cells are shared between the CNS and the peripheral blood (PB) compartments. In multiple sclerosis (MS) pathogenic B cells likely act on both sides of the blood-brain barrier (BBB).
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