It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell receptor (TCR) triggering, but how segregation would occur and whether it can initiate signaling is unclear. Using structural and biophysical analysis we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of new structures characterized by spontaneous sub-micron scale CD45 and kinase segregation, called ‘close-contacts’, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the unexpectedly potent signaling effects of local CD45 and kinase segregation. TCR ligands could heighten signaling simply by holding receptors in close-contacts.

Multiple sclerosis (MS) is a heterogenous autoimmune disease in which autoreactive lymphocytes attack the myelin sheath of the central nervous system (CNS). B lymphocytes in the cerebrospinal uid (CSF) of MS patients contribute to in ammation and secrete oligoclonal immunoglobulins. Epstein-Barr virus (EBV) infection has been linked to MS epidemiologically, but its pathological role remains unclear. Here we demonstrate high-a nity molecular mimicry between the EBV transcription factor EBNA1 and the CNS protein GlialCAM, and provide structural and in-vivo functional evidence for its relevance. A cross-reactive CSF-derived antibody was initially identi ed by single-cell sequencing of the paired-chain B cell repertoire of MS blood and CSF, followed by protein microarray-based testing of recombinantly expressed CSFderived antibodies against MS-associated viruses. Sequence analysis, a nity measurements, and the crystal structure of the EBNA1-peptide epitope in complex with the autoreactive Fab fragment allowed for tracking the development of the naïve EBNA1-restricted antibody to a mature EBNA1/GlialCAM crossreactive antibody. Molecular mimicry is facilitated by a post-translational modi cation of GlialCAM. EBNA1 immunization exacerbates the mouse model of MS and anti-EBNA1/GlialCAM antibodies are prevalent in MS patients. Our results provide a mechanistic link for the association between MS and EBV, and could guide the development of novel MS therapies. Main TextThe presence of oligoclonal bands (OCB) in cerebrospinal uid (CSF) and the e cacy of B cell depleting therapies emphasize the importance of B cells in the pathobiology of multiple sclerosis (MS) 2 . Anti-viral antibodies against mumps, measles, varicella-zoster, and Epstein-Barr Virus (EBV) are often present in MS 4,5 , but their relevance is unclear. Anti-EBV antibody titers in over 99% of MS patients provide evidence for an epidemiological link between MS and EBV 6 . Symptomatic infectious mononucleosis during EBV infection increases risk for MS 7 . Molecular mimicry between virus and self-antigens is a potential mechanism that might explain this association 8 . Antibodies against certain EBV nuclear antigen 1 (EBNA1) regions have been found in MS patients, including the region AA365-426 5,9-12 , which we describe here in our identi cation of molecular mimicry between EBNA1 and the glial cellular adhesion molecule GlialCAM. The potential signi cance of this mimicry in the pathophysiology of MS is described in detail.The B cell repertoire in MS CSF plasmablasts is highly clonal CSF and blood samples were obtained from MS patients during the onset of disease (clinically isolated syndrome, n=5) or an acute episode of relapsing-remitting MS (n=4). Patients with a CSF pleocytosis of >10 cells/µl were selected (Extended Data Table 1, Supplementary Discussion). Single B cells were sorted by ow cytometry (Extended Data Fig. 1a,b). Characteristic phenotypic differences of B cells in blood and CSF were observed 13,14 , including (i) high plasmablast (PB) counts in CS...

TCR-signaling strength generally correlates with peptide-MHC binding affinity; however, exceptions exist. We find high-affinity, yet non-stimulatory, interactions occur with high frequency in the human T cell repertoire. Here, we studied human TCRs that are refractory to activation by pMHC ligands despite robust binding. Analysis of 3D affinity, 2D dwell time, and crystal structures of stimulatory versus non-stimulatory TCR-pMHC interactions failed to account for their different signaling outcomes. Using yeast pMHC display, we identified peptide agonists of a formerly non-responsive TCR. Single-molecule force measurements demonstrated the emergence of catch bonds in the activating TCR-pMHC interactions, correlating with exclusion of CD45 from the TCR-APC contact site. Molecular dynamics simulations of TCR-pMHC disengagement distinguished agonist from non-agonist ligands based on the acquisition of catch bonds within the TCR-pMHC interface. The isolation of catch bonds as a parameter mediating the coupling of TCR binding and signaling has important implications for TCR and antigen engineering for immunotherapy.