Combined CD8 and granzyme B PET/CT reveals divergent immune responses to radiotherapy plus PD-1 blockade

Tiivistelmä

Background Combined radiotherapy (RT) and PD-1 blockade can elicit durable antitumor immunity, yet responses remain heterogeneous and difficult to predict with conventional anatomical imaging. Noninvasive visualization of functional immune activity could provide mechanistic insights into treatment response. Here, we evaluated a dual-tracer PET imaging strategy targeting CD8 + T-cell localization and granzyme B (GZMB)-associated cytotoxic activity during radiotherapy plus PD-1 blockade. Methods Female BALB/c mice bearing CT26 colorectal or 4T1 breast tumors were divided into phosphate-buffered saline (PBS), PD-1 blockade, radiotherapy (RT), or combined PD-1 blockade plus RT groups. 89 Zr-DFO-anti-CD8 PET/CT characterized baseline immune phenotype and treatment-associated CD8+ cell localization, whereas serial 68 Ga-NOTA-granzyme B-targeted probe (GZP) PET/CT assessed early granzyme B-associated effector activity. Tumor uptake was quantified as percentage injected dose per cubic centimeter (%ID/cc), and imaging findings were related to tumor growth and immunohistochemical staining. Results In CT26 tumors, the combination group showed significantly increased 89 Zr-DFO-anti-CD8 uptake at 120 hours (10.09 ± 1.05%ID/cc versus 7.01 ± 0.60%ID/cc in PBS; p < 0.001) and increased 68 Ga-NOTA-GZP uptake on day 9 (0.64 ± 0.13%ID/cc; p < 0.05 versus PBS and RT and p < 0.01 versus PD-1 blockade). In 4T1 tumors, combination treatment showed increased 89 Zr-DFO-anti-CD8 uptake at 72 hours (5.88 ± 0.60%ID/cc; p < 0.05 versus PBS group), whereas mean GZP uptake was not significantly different from monotherapy groups. Importantly, animals classified as responders based on a pre-defined day-20 tumor-growth inhibition (TGI) threshold exhibited higher GZP uptake on days 6 and 9 compared with non-responders. Conclusion Dual-tracer PET provides complementary information on CD8 + T-cell localization and GZMB-associated cytotoxic activity. Notably, the model-dependent CD8 and GZP signals in 4T1 tumors suggest that T-cell recruitment may not necessarily reflect effective cytotoxic function, supporting the complementary value of functional imaging alongside localization-based approaches. The findings are exploratory and hypothesis-generating and support further preclinical and prospective clinical investigation.