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. 2021 May 18;11:10476. doi: 10.1038/s41598-021-88448-5

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Comparison of Treg subpopulations in HD and MS patients. Subpopulations were gated based on CD45RA and Foxp3 either within CD4⁺ cells (Population I-V) or CD4⁺CD25⁺CD127^loTreg (population I–III). Values were expressed as the percentage of cells within each population. (A) Representative FACS profile of PBMC from HD (Left Column) and an MS patients (Right Column) gated on CD4⁺ (Top Row) and CD4⁺CD25⁺CD127^loTreg (Bottom Row) showing increased frequency of Foxp3^hiCD45RA⁻ cells in Population II in MS patients compared to HD; 2.45% vs. 1.28% when gated on CD4⁺, and 32.6% vs. 20.3% when gated on CD4⁺CD25⁺CD127^lo Treg. (B–D) shows comparison of all MS patients (n = 36) with HD (n = 20). (B) CD4⁺ cells had lower proportion of Population I (p = 0.001) in MS (n = 36) than HD (n = 20). (C) As proportion of CD4⁺CD25⁺CD127^loTreg, significant difference was noted both in population I and II, Population I was significantly lower (p = 0.004) and Population II was significantly greater (p = 0.0017) in MS (n = 36) than HD (n = 20). (D) Proportion of Population IV (activated/memory effectors) in CD4⁺ was greater in MS (n = 36) compared to HD (n = 20) (p < 0.000001). Proportion of Population V (Foxp3⁻CD45RA⁺) within CD4⁺ cells was significantly lesser in MS than in HD (p < 0.000001). (E–G) shows comparison of subpopulations in treatment naïve MS patients (n = 12) to HD (n = 20) using similar analysis as in B–D. (E) Treatment naïve MS patients had significantly lower proportion of cells in Population I in CD4⁺ gated cells than HD (p = 0.043). There was no significant difference between Population II or Population III. (F) In CD4⁺CD25⁺CD127^loTreg gate Population II was significantly greater in untreated MS than HD (p = 0.018) but there was no difference in population I or III although Population I was low. (G) In CD4⁺ gated cells, MS patients had more Population IV (p = 0.0018) and less Population V (p = 0.0016) than HD.