Fig. 4
CCR8 inhibition prolongs graft survival and attenuates CD8⁺ TRM activation. (A) Mice were treated with R243 (1 mg/kg) on days 0, 2, 4, and 6 post-transplantation. Grafts and lymph nodes, spleen, and blood were harvested at days 7, 9, 11, and 13 post-transplant. (B) Photos of skin grafts from control and R243-treated mice on days 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25 post-transplant. (C) Graft survival rates in control and R243-treated mice. (D) Histological examination of grafts using H&E staining, showing reduced immune infiltration in R243-treated mice. (E) Representative H&E-stained sections of skin grafts from control and R243-treated mice, illustrating immune cell infiltration and tissue damage. (F) Quantification of CD4⁺ and CD8⁺ T cell infiltration in grafts using immunohistochemistry for CD4 and CD8 markers. (G) Immunohistochemistry of CD69 and CD103 in syngeneic and allogeneic grafts. (H) Flow cytometry analysis showing the percentage of immune cell subsets (T cells, B cells, NK cells, DCs, and macrophages) in skin grafts from control and R243-treated mice. (I) Flow cytometry plots depicting CD69 and CD103 expression on CD4⁺ and CD8⁺ T cells in skin grafts from control and R243-treated mice. (J) Immunofluorescence staining of skin grafts for DAPI (blue), CD4 (cyan), CD8 (white), CD69 (orange), CCR8 (red), FOXP3 (green), and CD103 (yellow) in control and R243-treated mice. (K) Flow cytometry analysis of immune checkpoint markers (PD-1, TIGIT, and CTLA-4) on CD8⁺TRM cells in skin grafts from control and R243-treated mice. (L) Flow cytometry analysis of cytokine production (GZMB, IFN-γ, TNF-α, and IL-2) by CD8⁺TRM cells in skin grafts from control and R243-treated mice. Statistical analysis was performed using the t-test to compare the differences between groups. Bars represent mean ± SD from 10 independent experiments. Statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; “ns” denotes no significant difference.
