125 Systemic Immune Response in Burn Patients 7, respectively), which were both increased in week 2 and 3. In week 4, CCR4+CCR6¯ Treg numbers were comparable to healthy controls, while the numbers of CCR4+CCR6+ Tregs were still increased. In the CD3¯ lymphocyte cluster only small changes were observed. Figure 4. Unsupervised FlowSOM analysis of lymphocyte subtypes after severe burn injury. FlowSOM plots present proportions of populations and the expression of markers that were used in the lymphocyte flow cytometry panel (CD3, CD4, CD25, CD127, CCR4 and CCR6). (A) Cluster structure based on flow cytometry data of 10 healthy controls and 12 burn wound patients that were observed for 4 weeks. The most pronounced subtypes are encircled by dashed lines: CD4+ T cells (nodes 1-7), Tregs (nodes 6, 7), CD4¯ T cells (nodes 8-12), CD3¯ lymphocytes (nodes 13-16). FlowSOM plots of: (B) Week 1; (C) Week 2; (D) Week 3; (E) Week 4 after burn; (F) Healthy controls. Similar to the analysis of the innate cells, we took a supervised approach on the lymphocyte flow cytometry data of all patients to verify the unsupervised findings (Figure 5). The increase in CD4+ T cells in the second week after burn injury was confirmed, while the number of CD4¯ T cells did not change (Figure 5A,B). A more detailed analysis showed that Treg numbers were increased from PBD 7 until 39 (Figure 5C). Also, we confirmed the increase in chemokine receptors (CCR4 and CCR6) expressing CD4+ T cells and Tregs (Figures 5F,I). Furthermore, we could confirm the increase in CCR4+CCR6¯ Tregs (Figure 5H) after PBD 7, and observed a constant level of CCR4¯CCR6+ CD4+ T cells (Figure 5D). We found more CCR4+CCR6+ CD4+ (non Treg) T cells than Tregs, suggesting that the balance might be tipped, enhancing the inflammation rather than resolving 4
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