CCR2⁺CCR5⁺ Teff cells are enriched within CD4⁺CD161⁺ T cells, particularly in children with multiple sclerosis, who exhibit an abnormal balance between disease-implicated CD4⁺CD161⁺CCR2⁺CCR5⁺ Teffs and Tregs. PBMC were stained with appropriate antibodies and isotype controls and initially gated on singlet, live, total CD4⁺CD3⁺ T cells (Supplementary Fig. 1). (A) Flow cytometry dot-plots depicting approach to gating on CD4⁺CD161⁺ T cells in representative healthy control (HC), monoADS (Mono) or multiple sclerosis (MS) children. (B) Percentages of circulating CD4⁺CD161⁺ T cells in healthy control (n = 10), monoADS (n = 13) and multiple sclerosis (n = 12). Two-tailed non-parametric Mann-Whitney U-test for independent groups of monoADS and multiple sclerosis z-scores using the mean and SD of the healthy control group; U = 10, P < 0.001. (C) Flow cytometry dot plots depicting co-expression of CCR2 and CCR5 within CD4⁺CD161⁺ T cells, in representative healthy control, monoADS, or multiple sclerosis children. (D) Percentages of circulating CCR2⁺CCR5⁺ T cells among total CD4⁺ T cells and among CD4⁺CD161⁺ T cells in healthy control (n = 7), monoADS (n = 10) and multiple sclerosis (n = 8); Mann-Whitney U = 13, P = 0.016. (E) The ratio between the frequency of CCR2⁺CCR5⁺ T cells (within CD4⁺CD161⁺ T cells) and the frequency of Tregs in individual children with multiple sclerosis (n = 8) compared to either monoADS (n = 9) or healthy control (n = 8); Mann-Whitney U = 10, P < 0.001.