Fig 1: The effect of LEF treatment on CMR- and AVR- mediated immune responses. (A) MLR responses. Recipient splenocytes were isolated at POD 6 (responders) and irradiated naïve SD or BALB/c splenocytes (stimulators) were co-cultured for three days. Data are representative of three independent experiments. (B) Absolute numbers of splenocytes in LEF -treated and normal-saline treated mice recipients. Naïve C57BL/6 mice are shown for comparison (n = 3 mice/group). (C) Representative proportion of CD4+ and CD8+ T cells in recipient splenocytes. A total of 1 × 106 splenocytes were isolated at POD 6, and the percentage of CD4+ and CD8+ T cells was determined by flow cytometry (n = 3 mice/group). (D) Representative proportion of CD19+ B cells in recipient splenocytes. A total of 1 × 106 splenocytes were isolated at POD 6, and the percentage of CD19+ B cells was determined by flow cytometry (n = 3 mice/group). (E) The number of CD3+ T cells in recipient spleen were determined by flow cytometry (n = 3 mice/group). (F) The number of CD19+ B cells in recipient spleen were determined by flow cytometry (n = 3 mice/group). (G) Representative proportion of donor-specific antibodies in recipient serum. Serum was collected from xenograft and allograft recipients at POD 6, and the percent of IgG1, IgG2a, and IgM was determined by flow cytometry (n = 3 mice/group). (H) Serum levels of proinflammatory cytokines. Peripheral blood was collected at POD 6 and IFN-γ serum levels were measured by ELISA (n = 3 mice/group). (I) CD3+ T cells were isolated from naïve C57BL/6 mice, and co-cultured with anti-CD3 and anti-CD28 monoclonal antibody in the absence and presence of LEF for 3 days, the supernatant was collected and measured by ELISA (n = 3 separate experiments). Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the normal saline-treated group.