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. 2019 Oct 30;9:15650. doi: 10.1038/s41598-019-51961-9

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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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T-stochastic neighbour embedding (t-SNE) analysis identifies three CD4⁺ T cell populations expressing CXCR5 pre and post QIV. (a) Gating strategy and t-SNE analysis of live CD3⁺CD4⁺ lymphocytes in all participants at all time-points. Heatmaps depict the relative expression of each antigen, the scale from high (red) to low (blue) expression. (b) Representative t-SNE analysis of live CD3⁺CD4⁺ lymphocytes of one C-HIV participant showing the relative expression of T cell markers at Day 0 and Day 7 post QIV. Three populations of interest defined by CD32 and CXCR5 expression were gated on all participants and labelled Populations 1, 2 and 3 (bottom right panel). (c) Characterisation of P1–3 using histograms derived from t-SNE analysis. Data from concatenated file of all individuals’ data. Left panels show P1 (red), P2 (blue) and P3 (purple). Histograms, right panel, show the relative expression of T cell markers measured. Panels show the frequency of P1 (d), P2 (e) and P3 (f) from t-SNE analysis, at each time point. (g) Representative gating and t-SNE analysis of one C-HIV participant showing live CD19⁺ lymphocytes of all participants at Day 0 and Day 7 showing appearance of CD38^hiCD27^hiCXCR3^hi antibody secreting cells at Day 7. (h) Frequency of CXCR3^hi ASCs from t-SNE analysis at each time point. Statistical analysis was performed using a Friedman test (p value below graph) and Dunn’s multiple comparisons test (p value above graph). Individual data points are shown: HC, filled green circles; E-HIV, filled orange circles; C-HIV, open orange circles.