Cytokine dysregulation is a central driver of chronic inflammatory diseases such as multiple sclerosis (MS). Here, we sought to determine the characteristic cellular and cytokine polarization profile in patients with relapsing–remitting multiple sclerosis (RRMS) by high-dimensional single-cell mass cytometry (CyTOF). Using a combination of neural network-based representation learning algorithms, we identified an expanded T helper cell subset in patients with MS, characterized by the expression of granulocyte–macrophage colony-stimulating factor and the C-X-C chemokine receptor type 4. This cellular signature, which includes expression of very late antigen 4 in peripheral blood, was also enriched in the central nervous system of patients with relapsing–remitting multiple sclerosis. In independent validation cohorts, we confirmed that this cell population is increased in patients with MS compared with other inflammatory and non-inflammatory conditions. Lastly, we also found the population to be reduced under effective disease-modifying therapy, suggesting that the identified T cell profile represents a specific therapeutic target in MS.