Blocking PD-1 restores the TNF-α production by CD8
T-cells from chronic spinal cord injury (SCI) mice. Splenocytes (1 × 106) isolated from uninjured mice were stimulated ex vivo with PMA/ionomycin in the presence of brefeldin A for four hours (group: CT). Splenocytes (1 × 106) isolated from chronic SCI mice were also stimulated ex vivo with the same condition as the CT group, except that 10 μg/mL anti-PD-1 blocking antibody (group: SCI + αPD-1) or 10 μg/mL rat IgG2a, κ isotype (group: SCI + Isotype) were added to the culture. (A) Representative dot plots show the percentage of IFN-γ+ cells in gated CD4+ T-cells. (B) Bar graph represents the mean ± SEM percentage of IFN-γ+ cells in CD4+ T-cells. (C) Representative dot plots show the percentage of TNF-α+ cells in gated CD8+ T-cells. (D) Bar graph represents the mean ± SEM percentage of TNF-α+ cells in CD8+ T-cells. Ten thousand events gated on live singlets were collected. n = 9 for CT, n = 11 for SCI + Isotype, n = 11 for SCI + αPD-1. Data are pooled across three independent experiments. *P < 0.05, **P < 0.01, n.s. no significant difference was detected, P > 0.05, one-tailed Student’s t-test.