Role of dentritic epidermal T lymphocytes in immune rejection of skin allograft in mice and its mechanism

Objective To explore the role of dentritic epidermal T lymphocytes (DETCs) in immune rejection of skin allograft in mice and its related mechanism. Methods (1) Full-thickness skin was harvested from back of one male wild type (WT) C57BL/6 mouse. Epithelial cells were isolated for detection of the expression of DETCs and their phenotype with flow cytometer. Another male WT C57BL/6 mouse was used to harvest full-thickness skin from the back. Epidermis was isolated for observation of the morphological characteristics of DETCs with immunofluorescence technology. (2) Four male green fluorescence protein (GFP)-marked C57BL/6 mice, 7 female WT C57BL/6 mice (group WT), and 7 female γδT lymphocytes δ gene knock-out (GK) C57BL/6 mice (group GK) were used. Full-thickness skin in the size of 1.4 cm×1.4 cm on the back of mice in groups WT and GK were excised, and the wounds were transplanted with full-thickness skin in the size of 1.2 cm×1.2 cm obtained from male GFP-marked C57BL/6 mice. The survival time of skin grafts was affirmed with small animal in vivo imager and naked eyes and recorded. (3) Two male WT C57BL/6 mice were used to isolate epithelial cells. Cells were inoculated into 48-well plate and divided into activation group (A) and control group (C) according to the random number table, with 4 wells in each group. Cells in group A were treated with 10 μL concanavalin A in the concentration of 2 μg/mL for 24 hours, while those in group C with PBS in the same volume as that in group A. The expression of interferon γ in DETCs was detected with flow cytometer. (4) Four male GFP-marked C57BL/6 mice were used as donors. Fourteen female WT C57BL/6 mice were used as receptors and divided into interferon γ neutralizing group (IN) and control group (C) according to the random number table, with 7 mice in each group. The skin transplantation model of C57BL/6 male to C57BL/6 female was established as in part (2). Before surgery and 72 hours after, mice in group IN were intraperitoneally injected with 200 μL interferon γ neutralizing antibody in the concentration of 1 mg/mL, and those in group C with normal saline in the same volume as that in group IN. The survival time of skin grafts was observed and recorded using the methods in part (2), and the result of group IN was compared with that of group GK in part (2). The survival curve of skin grafts was processed with Log-rank (Mantel-Cox) test. Results (1) The positive expression rate of DETCs in epithelial cells of skin in mouse was 7.27%, and they were all CD3⁺ cells. DETCs were found to be scattered in the epidermis of skin in mouse with dendritic morphology. (2) The survival time of skin grafts of mice in group GK was 22-35 d, obviously longer than that in group WT (12-16 d, χ²=14.10, P<0.001). (3) Expression of interferon γ was detected in 22.70% DETCs in group A, which was obviously higher than that in group C (0.51%). (4) The survival time of skin grafts of mice in group IN was 19-24 d, which was obviously longer than that in group C (12-16 d, χ²=13.60, P<0.001) but close to that in group GK as in part (2) (χ²=0.06, P=0.810). Conclusions DETCs are involved in promotion of immune rejection of skin allograft probably by secreting interferon γ.