T cell exhaustion markers and populations
As there is no clear consensus, and just little information about the specific marker combination that describes specific subpopulations of exhausted T cells in patients with SLE, we initially used an unsupervised approach to explore the behavior and clustering of surface markers and transcription factors that have been reported as relevant in exhausted T cells (see methods for the complete list of markers included). As a first step, we generated tSNE maps using concatenated files with an equal number of CD3+ cells from all the patients included in each group. Figures 1A and 1B show the tSNE plots for each group, and the distribution of the included markers across the plots. In order to identify the clustering of these markers in subpopulations, we used a self-organizing maps algorithm [11]. We could identify five subpopulations of CD8+, seven of CD4+ cells and three CD3+CD4-CD8-. The heatmaps and tables showing the relative expression of markers in each cluster across all the identified subpopulations are shown in figure 1C. Overlapping of these populations on the tSNE plots are shown in 1D. Histograms represent the relative expression of a CD3+ CD8+ cell population that seems to be exclusive to the PR-SLE group, in both in panel II and III (2.86% and 3.75% respectively) and which expresses significantly transcription factors and surface markers associated with senescence and depletion.
Populations differentially represented in the PR when compared with the Act-SLE group .
Within the CD4+ compartment, a population of Tbet+CD45RO+ cells identified in the unsupervised analysis, which represents 10.20% (1.74–30.50) of the CD4+ cells in the PR group, was significantly increased compared with the Act-SLE (1.68, IQR: 0.42–2.83) and HS (4.08, IQR: 1.64–5.48). The CD4+EOMES+ cells were also increased in the PR group compared with Act-SLE and HS. The other population that was significantly different between PR and Act-SLE was the CD4+Tbet+ cells, which were increased in the PR group, and similar to the HS.
Within the CD8+ T cells, the proportion of PD1+ cells was apparently increased only in the PR group, even though the difference was only significant when compared with the HS. The CD8+2B4+ cells, identified through the unsupervised clustering, were increased in the PR vs Act-SLE group. The most striking difference was observed in the EOMES+ cells, with a median of 37.6 (24.9–53.2) in the PR group, vs 12.9 (6.78–24.8) in the HS and 8.13 (2.33–20.5) in the Act-SLE groups.