EAE was induced in Foxp3-Cre-YFP mice brain (from which meninges were removed) and spinal cord were collected 14 days and 50 days after EAE immunization and enzymatically digested. Cells were then incubated with fluorochrome-labeled antibodies and antibodies for CITE-seq analysis of CD69, P2RX7 and CXCR6 protein expression all from BioLegend. Antigen-experienced (CD44+, Foxp3-YFP-) TCRβ+ CD11b- conventional CD4+ T cells were then sorted by flow cytometry from both spinal cord and brain from day 14 and day 50 EAE mice. Between 1000 and 16000  viable cells per sample were encapsulated into droplets using Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 with Feature Barcoding technology for Cell Surface Protein, according to manufacturer's protocol (10X Genomics CG000206 Rev D user guide), with adjustments to fit TotalSeq-A antibodies.