Figure 1

(A) Gating Strategy for analysis of CD4⁺ and Treg subpopulations: PBMC isolated from a MS patient were stained for multicolour flow cytometry. Cells were first gated on SSC-A vs. SSC-H to exclude doublets, then lymphocytes were gated based on forward scatter (FSC-A) and side-scatter (SSC-A). CD4⁺ cells within the lymphocyte population were gated. Staining of CD25 vs. CD127 identified CD4⁺CD25⁺CD127^loTreg within CD4 gate. Subpopulations were then identified based on Foxp3 vs. CD45RA expression either from CD4⁺ gate or from CD4⁺CD25⁺CD127^loTreg gate. Within CD4⁺ cells five different populations were studied as defined by Miyara et al.⁴³. Treg are thought to be located within populations I, II and III, with activated Th-like Treg thought to lie within population II. Populations IV and V are Foxp3⁻ and CD25⁻ and are respectively defined as activated/memory (CD45RA⁻) and effector CD4⁺ cells (CD45RA⁺). Populations I, II and III were then analysed for chemokine receptor expression (CXCR3, CCR6 or CCR7) to examine frequencies of specific Th-like Treg subsets, showing CXCR3 Th1-like Treg as an example. (B): Comparison of percentage of lymphocyte subsets in HD and MS patients. PBMC from MS patients (n = 36) were compared to PBMC from HD (n = 20) for lymphocyte count (a), CD4⁺cell count (b) and their proportion within lymphocytes (d), and CD4⁺CD25⁺CD127^loFoxp3⁺ Treg count (c) and their proportion within CD4⁺cells (e). Regression analysis for Treg proportion within CD4⁺ cells vs. CD4⁺ cell numbers did not change with change in CD4⁺ cell numbers (f).