157 Local Immune Response in Burn Patients Figure 3. Local macrophage response to burn injury. (A) Flow cytometry gating strategy for detection of differentiation stages of monocytic cells (classical, intermediate or non-classical, as based on CD14 and CD16). (B) Flow cytometry-based quantification of percentage of monocytic cells within classical, intermediate, non-classical gates. (C) CD68 immunohistochemical DAB staining of a representative section of healthy skin and burn tissue (from 15 days post burn) (black scale bar = 100 μm). (D) CD68+ area of tissue sections. (E) Flow cytometry-based quantification of absolute number of macrophages (CD68+ monocytic cells) per mg tissue. (F) Unsupervised clustering of macrophages (CD68+ monocytic cells) in healthy skin and burn tissue, 3 clusters are highlighted. Node size represents relative size of population and node diagram shows expression level of markers. (G) Percentage of macrophages (CD68+ monocytic cells) within each cluster. Error bars in G 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. Burn injury causes shifts in the lymphocyte composition and increases total T cells at PBW 2-3 T cell (CD3+ lymphocyte) numbers rose significantly at PBW 2-3 (Figure 4A), in line with the total lymphocyte increase (Figure 1D). A shift towards more CD4+ T cells was detected in burn tissue compared to healthy skin and were highest in burn tissue from PBW 3 as the CD4/CD8 T cell ratio (CD3+CD4+/CD3+CD4¯ ratio) was higher in burn tissue than in healthy skin (Figure 4B and Supplementary Figure 2E). An increase in the proportion of γδ T 5
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