Radiation Therapy Sensitizes a Poorly Immunogenic Breast Cancer to PD-1 Blockade

We have shown that radiation therapy (RT) converts poorly immunogenic tumors resistant to antibodies (mAbs) against CTLA-4 into susceptible ones (Demaria et al). In part, the synergy between RT and anti-CTLA-4 was due to RT-induced upregulation on cancer cells of molecules that facilitate the formation of an immune synapse between target and effector CD8 T cells activated by treatment (Ruocco et al). Programmed death-1 (PD-1) is a checkpoint receptor upregulated on T cells shortly after activation and expressed at high levels on exhausted T cells. Anti-PD-1 mAbs have shown marked clinical activity in some cancer patients but the majority of patients do not respond. Here we tested the hypothesis that RT can sensitize poorly immunogenic tumors to immunotherapy targeting PD-1. BALB/c mice were inoculated s.c. with the syngeneic poorly immunogenic TSA breast cancer cells. When tumors became palpable mice were randomly assigned to one of 4 treatment groups: control, RT, anti-PD-1 mAb and RT + anti-PD-1 mAb. RT was delivered exclusively to the primary tumor in 8 Gy fractions on days 13, 14, and 15 post-tumor inoculation. PD-1 blocking mAb RMP1-14 was given on day 15 and every 4 days thereafter, and mice were followed for tumor growth. In a separate experiment, mice were euthanized on day 20 to characterize tumor-infiltrating lymphocytes and development of CD8+ T cells specific for the tumor epitope AH1 by pentamer analysis. TSA tumors were resistant to PD-1 blockade. RT significantly delayed tumor growth (p < 0.01), but tumor regression was seen only in 1 of 6 mice. In contrast, all mice given RT + RMP1-14 completely rejected tumors by day 25. Splenic AH1-specific CD8+ T-cells were markedly increased (4.6%) with RT + RMP1-14 compared to control (1.7%) or RMP1-14 (1.8%) or RT (2.9%) treated mice (p < 0.05). Compared to controls, RT-treated mice showed a significant increase in CD8+ T cells expressing high levels of PD-1 (CD8+ PD-1hi) (67% vs 36%, p < 0.01). PD-1 ligands PDL-1 and PDL-2 were also upregulated by RT on TSA cells and tumor-infiltrating myeloid cells, suggesting that PD-1 interaction with its ligands may limit activity of anti-tumor T cells. Anti-PD-1 also significantly decreased PD-1hi exhausted CD8+ T cells (14% RMP1-14 versus 36% control, p < 0.01; and 30% RT + RMP1-14 versus 67% RT, p < 0.01). In contrast, CD8+ T cells expressing activation markers CD69 and CD137 and low levels of PD-1 were increased significantly in tumors of mice treated with RT (64%) compared to control (42%) (p < 0.001) irrespective of the treatment with RMP1-14. Results suggest that anti-PD-1 mAb selectively decreases exhausted CD8+PD-1hi TILs, while enhancing the RT-elicited priming and activation of tumor-specific CD8+ T cells and support testing this combination in the clinic.