PMC

IFN-γ (A), IL-4 (B) and IL-6 (C) production by the splenocytes from B6 recipients of bm12 heart grafts assessed at 10 days post-Tx by ELISPOT. Data represent mean ± SEM of triplicate wells from three mice per group.

bm12 cardiac grafts transplanted into B6 B7.1- or B7.2-deficient recipients had a median survival time of greater than 56 days. When treated with MIH6 anti-PDL1 mAb, B7.2-deficient recipients rejected all allografts within 10 days (B), while allografts survived long-term in B7.1- deficient recipients (A). N= 6 for each group.

(A) bm12 hearts were transplanted into PD1KO recipients and the recipients were treated with 2H11 anti-PDL1 mAb. The survival time of cardiac allografts was significantly shortened as compared to controls. (B) bm12 hearts were transplanted into B7.2-deficient B6 recipients and the recipients treated with 2H11. Bm12 cardiac grafts transplanted into B6 B7.2-deficient recipients had a median survival time of greater than 56 days, while 2H11 precipitated allograft loss. N= 6 for each group.

(A) bm12 cardiac grafts transplanted into B6 WT recipients had a median survival time of greater than 56 days. Anti-PD1 mAbs treatment had similar allograft survival times to untreated group; however, allograft rejection was remarkably accelerated when recipients were treated with dual-blockade MIH6 anti-PDL1 mAb. (B) Bm12 cardiac grafts transplanted into a PD1-deficient recipients had a median survival time of greater than 56 days. When PD1KO recipients were treated with anti-PDL1 mAb, (MIH6) allograft rejection was remarkably accelerated despite PD1 deficiency. N= 6 for each group.

The hearts from bm12 mice were transplanted into B6 WT recipients and treated with 2H11 anti-PDL1 mAb, which exclusively blocks PDL1: B7.1 pathway. (A) 2H11 treatment significantly increased vasculopathy score by 56 days after transplantation as compared with no treatment. ISHLT grade was also increased, although differences did not reach statistical significance. Values shown represent mean ± SEM histological score obtained from 3 individual mice. (B) Representative photomicrographs of elastin staining show advanced vasculopathy in 2H11-treated allografts. Bars, 200 μm.

FACS analysis of splenocytes from B6 recipients of bm12 hearts with or without 2H11 treatment at 10 -14 days post-Tx showed that 2H11 treatment resulted in a significantly lower percentage of CD4⁺CD25⁺Foxp3⁺ regulatory T cells than controls (A). The percentage of CD4⁺ and CD8⁺ effector/memory phenotype (CD44^highCD62L^low) was similar between the 2H11-treated and control recipients (B and C). Bar graphs show means ±SEM. All results are representative of at least three different sets of experiments.

Fourteen days after B6 WT skin was transplanted into Balb/c WT or PDL1KO recipients, spleens and lymph nodes from recipients were harvested and CD4+ T cells were isolated. CD4+ T cells were then cultured with irradiated CD11c+ cells from Flt-3L-injected B6 WT or PDL1KO mice. Cell-free supernatants of individual wells were removed after 48 h of incubation with 2H11 or control IgG and analyzed by a Luminex for IFN-γ production. 2H11 mildly increased IFN-γ-production by CD4+ T cells when PDL1 was present on both APCs and T cells (A), however no effect was seen when PDL1 was absent on both cells (B). In addition, 2H11 did not enhance IFN-γ production when PDL1 was absent on CD11c+ cells alone (D). 2H11 increased IFN-γ production when PDL1 was present on the APC side but absent in T cells (C). Bar graphs show means ±SEM. All results are representative of at least three different sets of experiments with 3 mice.