Refined single-cell profiling captures a CCR5^high CD4⁺ cytotoxic T-cell precursor in multiple sclerosis

Decades of immunological and genetic studies highlight CD4⁺ T cells as initiators of central nervous system (CNS) pathology in individuals who develop multiple sclerosis (MS). A low-frequency CD4⁺ T-cell population termed T helper 17.1 (Th17.1) has been argued to play a central role during MS onset due to differences in abundance, yet little is known about their functional heterogeneity and distinctive mode of action in MS.

Features that distinguished Th17.1 from Th17 and Th1 cells in the blood were explored using spectral flow cytometry. We focused on the brain-homing properties of Th17.1 in MS by analysing paired blood and cerebrospinal fluid (CSF) as well as blood before and after natalizumab treatment. Identified Th17.1 effector signatures were further analysed using purified subsets for single-cell transcriptomics and corresponding in vitro stimulation assays.

Our screens reveal an CCR5^high Th17.1 cluster that is enriched in CSF, but also selectively reduced in post-versus pre-natalizumab blood samples from people with MS. The phenotypical and transcriptional signature of this Th17.1 cluster matched and pointed to a highly receptive and pre-cytotoxic state. Indeed, particularly CCR5^high Th17.1 cells upregulated cytolytic proteins by strongly responding to IL-12, while secretion of these proteins was only found when adding both IL-12 and IL-18 in vitro.

We show that low-frequency, circulating Th17.1 cells harbour a unique MS-associated CCR5^high cluster with enhanced cytotoxic and CNS-infiltrating potential. This CD4⁺ cytotoxic T-cell precursor could be a promising target for future research aimed at monitoring disease-relevant immune perturbations for precision medicine in MS.

ZonMw (09150171910036), Stichting MS Research (19-1075 MS, 23-490 g MS), European Union's Horizon Europe Research and Innovation Actions (101137235; BEHIND-MS) and the Swiss State Secretariat for Education, Research and Innovation (SERI), Erasmus MC Foundation (10502/02).