158 Chapter 5 cells (CD3+CD4¯ γδTCR+ lymphocytes) was found only at PBW 1 (Figure 4C), indicating a fast response of γδ T cells after burn injury. The absolute number of γδ T cells steadily increased over time after burn injury (Supplementary Figure 2F). The shift towards a higher abundance of γδ T cells at PBW 1 was confirmed by mapping flow cytometry data of T cells using FlowSOM (clusters 3 and 4; Figure 4D,E) and shows that the majority of the γδ T cells was CD25+, which is a prominent marker for cellular activation [18]. At PBW 1 there was a relative decrease of T cells with a regulatory phenotype (CD25+CD127¯; cluster 1). We did not observe considerable alterations in the cluster containing CD3+CD4¯ T cells (cluster 5) Figure 4. Local T cell response to burn injury. Flow cytometry-based quantification of: (A) Absolute number of T cells (CD3+ lymphocytes) per mg tissue; (B) CD4+/CD4¯ T cell (CD3+ lymphocytes) ratio in tissue; (C) Percentage of T cells (CD3+ lymphocytes) that are γδ T cells (γδTCR+CD4¯ T cells). (D) Unsupervised clustering of T cells (CD3+ lymphocytes) in healthy skin and burn tissue, 5 clusters are highlighted. Node size represents relative size of population and node diagram shows expression level of markers. (E) Percentage of T cells within each cluster. Error bars in E show boxplot, p values were calculated using Mann-Whitney U statistical test, significant differences are indicated by black asterisks: *p < 0.05; **p < 0.01; ***p < 0.001.
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