Preparative Hepatic Irradiation (HIR) for Transplantation of Human Induced Pluripotent Stem Cell (iPSC)-Derived Hepatocyte-Like Cells (iHep) in Mouse Livers

Abstract

anecdotal data suggest an interaction between checkpoint blockade and radiation therapy (RT). Using a murine model of melanoma, we tested the hypothesis that RT alters immune checkpoint ligand expression on tumor cells, and modulates immune checkpoint expression on tumor infiltrating lymphocytes (TIL). Materials/Methods: The B16 melanoma cell line was maintained in DMEM + 10% FBS. Two times 10 B16 cells were injected SQ into C57BL/6 mice. Tumors were treated to 10 Gy x 1 using a small animal irradiator (SARRP). In some studies mice were treated with PD-1 antibody blockade (10 mg/kg). Tumors were harvested following treatment and TIL were isolated for flow cytometry. Results: Splenic and tumor-draining lymph node lymphocytes did not express PD-1, TIM-3, or LAG-3; however, TIL upregulated PD-1, TIM-3, and LAG-3 (p< 0.05). RT resulted in an obvious increase in CD8 TIL (p< 0.05); in addition, RT increased the proportion of CD8 TIL expressing TNF-a and IFN-g (p < 0.05), consistent with activation. However, a high proportion (50-80%) of TIL continued to express the negative regulatory checkpoint proteins PD-1 and TIM-3. Interestingly, LAG-3 expression was significantly decreased post-RT (p Z 0.05). B16 melanoma expressed the checkpoint ligand PD-L1, and RT (in vitro) further increased expression 24-48 hr post-RT (p < 0.05). When mice received PD-1 blockade, RT, or combined therapy, significant tumor growth inhibition occurred: PD-1 blockade 45 16%; RT 33 6%; combined therapy 22 5% (p < 0.05). PD-1 blockade as single-therapy failed to significantly increase tumor infiltration of lymphocytes, whereas combined PD-1 blockade with RT resulted in increased TIL compared to PD-1 blockade or no treatment (p < 0.05). However, with combined therapy, TIL expressed TIM-3 and PD-1 in high frequency (30-60%), and LAG-3 to a lesser extent (10%). Conclusions: RT-mediated lymphocyte migration to the tumor microenvironment resulted in upregulation of the negative regulatory checkpoint molecules PD-1, TIM-3, and LAG-3. RT + PD-1 blockade increased TIL, but with continued expression of negative checkpoints. Additionally, RT upregulated PD-L1 on tumor cells. These data provide a rationale for combining RT with the blockade of multiple checkpoints simultaneously, especially PD-1/PD-L1. Author Disclosure: T.J. Harris: None. C. Jackson: None. N. Gandhi: None. P. Tran: None. C.G. Drake: G. Consultant; Bristol-Myers Squibb in the past.