Cervical lymph node B cell subset resists depletion and sustains lytic Epstein Barr-virus signature in patients with multiple sclerosis

B cells have arisen as key in multiple sclerosis (MS) pathogenesis by the high efficacy of B cell depleting CD20 monoclonal antibodies and by the identification of Epstein-Barr virus (EBV) as a prerequisite factor. We previously demonstrated an expansion of memory and double negative (DN) B cells in the deep cervical lymph nodes (dcLNs) of newly diagnosed patients with MS (pwMS), with DN B cells showing a molecular signature consistent with lytic EBV infection. Effect of anti-CD20 therapies for pwMS require sustained administration, which led us to hypothesize that incomplete effects on the dcLNs, a natural site for lymphocyte priming towards the central nervous system (CNS), might be sustaining the pathology. Using single-cell transcriptomics and paired sampling, we characterized the effects of anti-CD20 therapies on dcLNs compared to circulation and to other therapies. We observed switched (SM) and unswitched (USM) memory B cells to be depleted uniquely by anti-CD20 in both dcLNs and circulation, whereas DN B cells of the dcLNs were not. Moreover, EBV lytic signature was retained in DN B cells during anti-CD20, and using clonal trajectory we saw that the DN B cells continued to contribute to the plasmablast differentiation within dcLNs. EBV targeting of expanded memory CD8 T cells, as well as CD8 T cell exhaustion, were retained during anti-CD20 therapy within the dcLNs. Our findings suggest a sustained, EBV-driven process in pwMS during anti-CD20 in the CNS-targeting priming dcLNs, with therapeutic implications to be investigated further.