Increased frequencies and pro-inflammatory cytokine responses of CD4⁺CCR2⁺CCR5⁺ T cells in children with multiple sclerosis. PBMC were stained with appropriate antibodies and isotype controls and initially gated on singlet, live, total CD4⁺CD3⁺ T cells (Supplementary Fig. 1). (A) Dot-plots depicting approach to gating on CD4⁺CCR2⁺CCR5⁺ T cells in representative healthy control (HC), monoADS (Mono) or multiple sclerosis (MS) children. (B) Percentages of circulating CD4⁺CCR2⁺CCR5⁺ T cells in healthy control (n = 17), monoADS (n = 18) and multiple sclerosis (n = 15). 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; U = 71, P = 0.021. (C) Following short-term activation with PMA/ionomycin and Golgi stop, PBMC were stained for both surface markers and intracellular-cytokines. Dot plots are shown for healthy control, monoADS or multiple sclerosis children depicting IFNγ and IL-17 expression by activated CD4⁺CCR2⁺CCR5⁺ T cells. (D and E) Percentages of IFNγ and IL-17 expressing CD4⁺CCR2⁺CCR5⁺ T cells in healthy control (n = 13), monoADS (n = 13) and multiple sclerosis (n = 14). Mann-Whitney U = 19, P = 0.034 for IFNγ and U = 44, P = 0.023 for IL-17.