Loss of GVL effects is associated with increased PD-1 expression and reduced oligoclonal expansion in allogeneic transplant recipients and can be effectively restored by PD-L1 blockade. (A-F) Established allogeneic (B10.A → B6) or syngeneic transplant recipients (B6 → B6) received 40 × 10⁶ allogeneic (B10.A) or syngeneic (B6) TCR-transduced T cells on day 56 after HCT. Three days after AT, mice were challenged with 1.2 × 10⁶ C1498-OVA cells intravenously. (A) PD-1 expression on transferred cells was analyzed in peripheral blood 7 days after leukemia challenge on syngeneic (white bar) vs allogeneic (black bars) and GFP⁺CD8⁺ T cells. Carboxyfluorescein succinimidyl ester-labeled naive DLIs were used as controls. *P < .05. n = 4 or 5 per group. In consecutive experiments, transplanted mice received either α-PD-L1 blocking antibodies or an isotype control intraperitoneally starting one day before delayed (day 56) AT. A loading dose of 300 μg was used followed by 200 μg on days 57, 59, 62, 65, and 68 after HCT. A lethal dose of leukemia was given on day 59. (B) Survival graphs of cohorts are shown that did not receive AT on day 56 but were injected with PBS instead. **P < .01 between treatment with α-PD-L1 antibody (×) and isotype control (*). n = 10 per group. (C) Syngeneic transplant recipients (B6 → B6) received gene-modified T cells (B6) on day 56 after HCT. No significant survival differences were seen between the treatment groups (α-PD-L1; □ on dashed line) or isotype control (□ on solid line) antibody (n = 8 per group). (D) PD-L1 blockade resulted in significantly increased survival rates in allogeneic transplant recipients. **P < .005 between cohorts that received α-PD-L1 (■ on dashed line) and isotype control (■ on solid line; n = 8 per group). (E) Allogeneic transplant recipients were treated with either α-PD-L1 antibody or isotype control and monitored for GVHD as previously described. (F) Established allogeneic recipients (B10.A → B6) received 2.5 × 10⁶ TCR-transduced B10.A T cells on day 56 after HCT and were treated with α-PD-L1 antibody or isotype control on days 55, 57, and 59. In addition, mice were injected with 1 mg BrdU intraperitoneally daily from day 57 to day 59. On day 60, splenocytes were sorted for GFP⁺ cells, and BrdU expression of gated CD8⁺ T cells was determined. *P < .05 between groups. n = 3 per group. (G) Established allogeneic transplant recipients (B10A → B6) or B6 mice were challenged with 1.2 × 10⁶ C1498-OVA cells, and T cells from the liver were analyzed on day 25 after tumor injection by flow cytometry. Cohorts were treated either with α-PD-L1 or isotype control antibody (200-μg/dose) from day 10 to day 20 every other day. Cells were either gated on CD4 and analyzed for Foxp3 expression (left bar graph). *P < .05. n = 6 per group, gated on CD8 and analyzed for PD-1 expression (middle bar graph). ***P < .001. n = 6 per group, or stimulated with α-CD3/α-CD28, gated on CD8, and analyzed for IFN-γ expression (right bar graph). *P < .05. n = 6 per group. Black bars represent allogeneic recipients; and white bars, syngeneic recipients.