2899 - Remodeling the Tumor-Immune Microenvironment by Radiotherapy and Anti-BTLA Blockade Enhance Anti-PD-L1 Efficacy

B. Wang¹, and L. Zhao²; ¹StateKey Laboratory of Holisticntegrative Managementof Gastrointestinal Cancersand Department of RadiationOncology,Xijing Hospital,Fourth Military MedicalUniversity,Xian, China, Xi’an, China, ²StateKey Laboratory of Holisticntegrative Managementof Gastrointestinal Cancersand Department of RadiationOncology,Xijing Hospital,Fourth Military MedicalUniversity, Xian, China

Purpose/Objective(s): Immune checkpoint inhibitors (ICIs) have transformed cancer immunotherapy (CIT) and improved clinical outcomes in immunogenic tumors with high tumor mutational burden (TMB), but currently only 12.5% of cancer patients respond. Tumors with relatively low TMB, mesenchymal remodeling, and relatively few tumor-infiltrating lymphocytes are ineffective for ICI mono- or dual-agent therapy and require the development of novel combination therapeutic strategies to improve anti-tumor efficacy. Immune priming by local radiotherapy (RT) could promote the conversion of immunocold tumors into inflammatory or hot tumors, thereby re-sensitizing them to CIT, but the immunogenic effect of RT can be counteracted by escape mechanisms in the tumor immune microenvironment(TME), leading to T-cell exhaustion and tumor immune tolerance. Therefore, this study aims to investigate the reported anti-tumor effects and mechanisms of programmed cell death ligand 1 (PD-L1) combined with the B and T lymphocyte attenuator (BTLA) synergistic RT in tumor mouse models. It is expected to provide theoretical support for clinical trials. Materials/

Methods: MC38 and 4T1 cells were injected in the left flanks of C57BL/6 and BAlB/c mice models for efficacy and survival studies. Mice were randomized into four groups, including vehicle, PD-L1, PB(PDL1+BTLA), and PB+RT(8Gy*3F). Mice were euthanized when tumor volumes reached 1500-2000 mm³. A comparison of treatment groups was performed using the log-rank test with p<0.05 considered significant. The cell mixture was harvested from mice tumor tissue and spleen. Immune cell makers were stained by antibodies and then analyzed by flow cytometry. In vitro, Flow cytometry fluorescence quantitative analysis, and immunofluorescence was used to detect the expression of PD-L1 and BTLA. RT-PCR and western blot were performed on the expression of PERK/ATF4/CHOP in B cells.
Results: When the defined tumor volume endpoint was reached, the IPB+RT group delayed tumor volume and improved the survival of the mouse model more than other groups. Furthermore, flow cytometry and immunofluorescence suggested that inoculation upregulated the expression of PD-L1 and BTLA on B cells. This explained the anti-tumor capacity of PB in combination with radiotherapy. In addition, CXCL9 and CXCL13 were upregulated in serum from B cells and 4T1 cells co-incubation. Finally, we examined PERK, ATF4, CHOP PD-L1, and BTLA protein levels by RT-PCR and Western blot after treatment with the PD-L1, PB(PD-L1+BTLA) and PB+RT(8Gy*3F) in B cells. The results indicated that radiation up-regulated PD-L1 and BTLA overexpression, which depended on the PERKP-ATF4-CHOP pathway.
Conclusion: This study proposes that sequential combination therapy can potentially enhance treatment efficacy in PD-L1 tolerance cancer patients and provides insights into how preexisting anti-BTLA blockade and radiotherapy can influence anti-PD-L1 efficacy by remodeling the TME.