T-B interactions yield CD3⁺ B cells and CD20⁺ T cells with pathogenic features in multiple sclerosis

Classically, B and T cells are differentiated by their mutually exclusive expression of surface markers such as CD3 and CD20, but it is now established that a few percent of T cells in human blood display also CD20. We analyzed CD3⁺CD20⁺ cells in detail by combining single-cell proteogenomics, FACS, and ImageStream. Thereby, we identified CD3⁺ B cells and found that about 40% of plasmablasts displayed CD3 in both blood and tonsils. In tonsils, where T-B interactions occur, about 80% of follicular helper T (T_fh) cells displayed CD20. In vitro, coactivation of T and B cells induced marker exchange via trogocytosis. We refer to the resulting cells as "masquerading" lymphocytes and analyzed them in multiple sclerosis (MS). CD20⁺ T cells were markedly enriched in cerebrospinal fluid, exhibited cytotoxic gene signatures, accumulated in perivascular inflammatory cuffs, and showed increased myelin antigen reactivity. Anti-CD20 therapy (ofatumumab) not only depleted masquerading lymphocytes but also prevented their reinduction. Together, we identify CD3⁺ B cells as footprints of T-B interactions thereby shifting the paradigm of lymphocyte-specific surface markers and implicate masquerading lymphocytes as active contributors to MS immunopathology.