Figure 1. Dimensional Reduction Analysis and Unsupervised clustering of subpopulations of CD3+ T cells using markers associated with cell exhaustion in Panels 2 and 3 (described previously in methods).
A) t-Distributed Stochastic Neighbor Embedding (tSNE) plots for each study group (HS, Act-SLE and PR-SLE). Individual cell subsets of CD3+ cells from each subject in each group were randomly down-sampled to an equal number of events and concatenated in a single file. B) Relative expression of selected markers shown in tSNE plots. C) Heat maps of selected markers using the Self Organizing Maps (FlowSOM) algorithm according to the fluorescence intensity of the markers by flow cytometry. Eight clusters were identified and named Pop 0 to Pop 7). The heatmap shows the relative fluorescence intensity and hierarchical clustering of each of the markers included in the subpopulations generated. Percentage relative to CD3+ cells is shown in tables below. D) Distribution of clusters identified in C overlaid on the t-SNE plots in each study group. In both panels, a CD8+ subpopulation that expresses exhaustion and senescence markers was identified exclusively in the PR-SLE group. Overlaid histograms (HS: green; Act-SLE: red; PR-SLE: purple) show relative frequencies of most abundant markers.
Figure 2. Bar graphs showing the frequencies (mean ±SEM) of lymphocytes populations derived from traditional manual gating and FlowSOM clusters identified in the heatmap in panel a. Mann Whitney U test was performed to compare between two groups and differences are represented by the following symbols: * p < 0.05. ** p < 0.01. *** p < 0.001. **** p < 0.0001. HS (healthy subjects); Act-SLE (active SLE); PR-SLE (prolonged remission SLE).