A small proportion of multiple sclerosis (MS) patients develop new disease activity soon after starting anti-CD20 therapy. This activity does not recur with further dosing, possibly reflecting deeper depletion of CD20-expressing cells with repeat infusions. We assessed cellular immune profiles and their association with transient disease activity following anti-CD20 initiation as a window into relapsing disease biology. Peripheral blood mononuclear cells from independent discovery and validation cohorts of MS patients initiating ocrelizumab were assessed for phenotypic and functional profiles using multiparametric flow cytometry. Pretreatment CD20-expressing T cells, especially CD20^dimCD8⁺ T cells with a highly inflammatory and central nervous system (CNS)-homing phenotype, were significantly inversely correlated with pretreatment MRI gadolinium-lesion counts, and also predictive of early disease activity observed after anti-CD20 initiation. Direct removal of pretreatment proinflammatory CD20^dimCD8⁺ T cells had a greater contribution to treatment-associated changes in the CD8⁺ T cell pool than was the case for CD4⁺ T cells. Early disease activity following anti-CD20 initiation was not associated with reconstituting CD20^dimCD8⁺ T cells, which were less proinflammatory compared with pretreatment. Similarly, this disease activity did not correlate with early reconstituting B cells, which were predominantly transitional CD19⁺CD24^highCD38^high with a more anti-inflammatory profile. We provide insights into the mode-of-action of anti-CD20 and highlight a potential role for CD20^dimCD8⁺ T cells in MS relapse biology; their strong inverse correlation with both pretreatment and early posttreatment disease activity suggests that CD20-expressing CD8⁺ T cells leaving the circulation (possibly to the CNS) play a particularly early role in the immune cascades involved in relapse development.