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. 2019 Dec 19;9:19471. doi: 10.1038/s41598-019-55852-x

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© The Author(s) 2019

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Phenotypic analysis of the lymphoid lineage. (a) Statistical t-SNE map obtained from bin-wise testing for differential abundance between the two studied groups (CON, n = 11; early MS, n = 11) using edgeR statistical framework (with negative binomial GLM) and 10% FDR adjustment. The colour spectrum corresponds to FDR-adjusted P values, ranging from 1 (blue) to ≤ 0.05 (red), detecting significant differences between the group in the CD4⁺ T cell subset. (b) Representative single-cell t-SNE map indicates the expression of CCR7, IL-7R and CD62L. The colour spectrum represents an expression level (red, high expression; dark blue, no expression). (c) t-SNE map highlights CD4⁺ T cell subset (green dots). Heat map and cluster analysis within the CD4⁺ T cell cluster from all samples on the basis of the mean expression of analysed markers. Identified clusters are indicated by dendrograms. Heat colours show overall marker expression levels. The bar graph shows mean cluster frequencies as % of parent (black number) and % of total cells (grey number). Cluster 1 and 13 (* in red) are quantified as differentially abundant subsets between the groups. (d) The graph shows differences in frequency (%) of cluster 1 and 13 between the two studied groups. (e) Median expression levels as shown in dot plot representation for randomly selected cells (n = 256/cluster) in the cluster 1 (orange), 13 (green) and all other clusters (blue). The selected markers are identified as defining markers for cluster 1 and 13. (f) The t-SNE map, heat map and cluster analysis of CD8⁺ T cells from all samples on the basis of the mean expression of analysed markers. Cluster 5 (* in red) is quantified as differentially abundant subsets between the groups. (g) The graph shows differences in frequency (%) of cluster 5 between the two studied groups. (h) Median expression levels as shown in dot plot representation for randomly selected cells (n = 256/cluster) in the cluster 5 (orange) and all other clusters (blue). A P value < 0.05 at 10% FDR was considered statistically significant, determined using GLMM (*P < 0.05; **P < 0.01, unadjusted).