Summary This application is concerned with the study of epigenetics events affecting multiple sclerosis (MS) risk and progression. We present preliminary results consistent with widespread differential hypomethylation in peripheral CD19+ B cells at the time of diagnosis, posing a mechanistic link to the clinical efficiency of anti-CD20 antibody treatment for this disease. We build on these results and access to informative and diverse data- and sample-sets to propose in Specific Aim 1 the simultaneous assessment of single-cell gene expression (scRNA-seq), chromatin accessibility (scATAC-seq), and cell surface markers in paired cerebrospinal fluid (CSF) and peripheral blood mononuclear cells from treatment naïve MS patients at the time of clinical onset and well matched controls, and link in Aim 2 to the individuals’ DNA variance to develop global and cell-specific genetic burdens associated with disease expression. In Aim 3 we connect the emerging epigenetic and transcriptome signatures with cell function using the Beacon® system optofluidic platform to visualize and assess cellular phenotypes at the single-cell level. In Aim 4 we implement a targeted trimodal single-cell assay to describe the epigenetic landscape of the principal MS susceptibility locus, the Major Histocompatibility Complex in chromosome 6p21. By systematically integrating single cell chromatin states, DNA variance, and gene expression data, we expect to gain important novel information about pathogenesis. Moreover, we will identify cell-specific epigenetic signatures associated with MS clinical onset, potentially serving as biomarkers of affectation status. The meticulous clinical follow up of study participants offers an opportunity to assess their prognostic potential.