FIGURE 1. SSc patients express increased levels of PD-1, TIGIT and TIM-3 in defined peripheral blood lymphocyte subsets.

PBMCs were isolated from SSc patients and healthy controls and the cells stained with a 16-color antibody panel for flow cytometric analysis. (A) Single live CD19⁻CD14⁻ events were gated from flow cytometry data of a representative subject and t-SNE dimension reduction was applied with lineage marker channels used for clustering. Populations were gated manually as in Supplementary Fig. 1 and events overlaid on the t-SNE map to show positioning of 12 separate subsets of immune cells. (B) t-SNE plots showing the expression of PD-1, TIGIT, TIM-3 and LAG-3 on immune cell subsets mapped as in A (color scale depicts fluorescence intensity as a measure of expression level). (C–E) Using manual gating, the percentage of PD-1⁺ (C), TIGIT⁺ (D), TIM-3⁺ (E) and LAG-3⁺ (F) cells within CD4⁺CD25^loCD45RO⁺ Tconv cells, CD4⁺CD25^hiCD127^loCD45RO⁺ Tregs, CD8⁺CD45RO⁺ T cells, γδ TCR⁺ T cells, iNK T cells, CD16⁺CD56^med NK cells and CD16⁻CD56^hi NK cells was determined. Each symbol represents an individual subject. Mean ± SEM is indicated. iNKT cells only shown if >20 events were recorded. Statistics was done with t test with false discovery rate adjustment * p≤ 0.05