Cd3 percp cy5

Naïve CD4⁺ T cells were isolated from spleens of wild-type C57BL/6 J mice (8–14 weeks; both sexes). In brief, dissected mouse spleens were gently ground through 70 µm cell strainer mesh (Falcon), and naïve CD4⁺/CD25⁺ (regulatory; T_reg) or CD4⁺/CD25^- (effector; T_eff) T cells were isolated and collected using a CD4⁺CD25⁺ T cell isolation kit (Miltenyi Biotec; #130-091-041) and magnetic separation columns (Miltenyi Biotec, #130-042-201 and #130-042-401) according to manufacturer’s protocol. Purity of T cell populations were verified by using flow cytometry (Supplementary Fig. 6a; CD3-PerCP-Cy5.5 1:100, eBioscience, #45-0031-80; CD4-Pacific Blue 1:100, eBioscience, #57-0042-82; and Foxp3-APC 1:100, eBioscience, #17-5773-80; FlowJo v.10.6.2). Isolated CD4⁺ T cells were resuspended in RPMI 1640 containing 10% FBS, 1% PSQ, 1% sodium pyruvate, 1% HEPES, and 1% nonessential amino acids, and plated on poly-L-lysine coated coverslips >1 h before recording. Whole-cell recording of CD4⁺ T cells was carried out within 48 h after isolation.

PBMC sample processing was performed at the same time for each triplet (PwMS at VIS1&VIS2 and HC), to minimize differences within processing. The MojoSort™ Human CD4 T Cell Isolation Kit (#480010, BioLegend, San Diego, CA, USA) was used for CD4⁺ T cell enrichment, in accordance with the manufacturer’s instructions.
To assess whether CD4⁺ T cell enrichment was achieved, flow cytometry was performed in a BD LSRFortessa™ X-20 Cell Analyzer (BD Biosciences, Franklin Lakes, NJ, USA) on a subset of samples (before and after magnetic enrichment). The following antibodies were used: CD14-eFluor450 (#48014941, clone 61D3, eBioscience™, Thermo Fisher Scientific, Waltham, MA, USA); CD19-Alexa Fluor 647 (#302222, clone HIB19, BioLegend, San Diego, CA, USA); CD3-PerCP-Cy5.5 (#45003741, clone OKT3, eBioscience™, Thermo Fisher Scientific, Waltham, MA, USA); CD4-BV711 (#317439, clone OKT4, BioLegend, San Diego, CA, USA); and CD56-PE-Cy7 (#25056741, clone CMSSB/NCAM, eBioscience™, Thermo Fisher Scientific, Waltham, MA, USA).
Regarding the gating strategy, CD4⁺ T cells were defined as CD3⁺CD4⁺CD19^-CD56^- single cells (see Supplementary Figs. S6, 7 online). A detailed protocol and additional details regarding the methodology used for CD4⁺ T cell enrichment and flow cytometry may be found in Sect. 2 of the Supplementary Information.