FLASH Radiotherapy Mitigates Radiation-Induced Lymphopenia and Prevents Immunosuppression via Chk1-STAT3 Axis Modulation in a Preclinical Thoracic Irradiation Model

Abstract Background and Aims Radiation-induced lymphopenia (RIL) is a frequent side effect of conventional radiation therapy (CONV RT), due to the high radiosensitivity of circulating lymphocytes. Ultra-high dose rate “FLASH” RT may preferentially spare normal tissue while maintaining tumor control. This study evaluates the impact of single-fraction and multi-fraction thoracic FLASH RT on lymphocyte preservation, apoptosis, and immunosuppressive signaling in mice. Methods We compared the immunological impact of thoracic FLASH RT and CONV RT in C57BL/6 mice using single-fraction (17 Gy) and multi-fraction (2 Gy × 5) regimens using the Mobetron (IntraOp). Longitudinal blood sampling was performed at multiple time-points post-irradiation through facial vein bleed with flow cytometry analysis for CD4+, CD8+, CD19+, and NK cells to assess lymphocyte counts, apoptotic lymphocytes through Annexin V staining, and immune suppression by examining regulatory T cells (Tregs) and PD-1/PD-L1 expression. Mechanistic studies included immunofluorescence and Western blot analyses of splenic tissues to evaluate Chk1 and STAT3 signaling pathways. Results In single-fraction RT, FLASH significantly reduced lung and heart fibrosis (p < 0.0001) at 28 weeks post-RT. The FLASH effect was also seen acutely on circulating immune cells, with significantly reduced lymphocyte apoptosis and accelerated recovery of CD4 ⁺ , CD8 ⁺ , CD3 ⁺ , NK, and B cell populations compared to CONV RT in both single-fraction and multi-fraction regimens. Conversely, CONV RT induced long-lasting increases in Tregs and sustained PD-1 and PD-L1 expression on T- and B-cells at 2- and 5-months post-irradiation in both fractionation regimens. Within the spleen, we also found CONV RT induced sustained activation of the Chk1–STAT3 pathway in CD45+ immune cells, which correlates with increased PD-1/PD-L1 expression. Conclusion FLASH RT mitigates RIL, reduces lymphocyte apoptosis, and prevents long-term immunosuppression by reduced activation of the Chk1–STAT3 pathway. These findings suggest FLASH RT may confer immunological advantages over CONV RT to enhance therapeutic efficacy.