Head-to-head comparison of nuclear imaging approaches to quantify tumor CD8+ T-cell infiltration

Abstract Introduction Many immunotherapies focus on (re)invigorating CD8+ T cell anti-cancer responses. Different nuclear imaging techniques have been developed to measure CD8+ T cell distributions. Comprehensive comparisons of in vivo and ex vivo T cell labeling methods with respect to tumor and normal tissue targeting and correlation with CD8⁺ T cell presence are lacking, but essential for accurate clinical interpretation. We performed a head-to-head comparison of three CD8+ T cell imaging approaches: 89Zr-labeled Fc-silent anti-CD8 antibody (89ZrZr-anti-CD8-IgG2asilent), ex vivo 89Zr-labeled ovalbumin-specific CD8+ T cells (89ZrZr-OT-I), and 18F-labeled IL2 (18FAlF-RESCA-IL2). Methods B16F10/OVA tumor-bearing C57BL/6 mice (n=10/group) underwent PET/CT imaging at 72 (89ZrZr-anti-CD8-IgG2asilent), 24 and 48 h (89ZrZr-OT-I), and 10 min (18FAlF-RESCA-IL2) pi. Subsequently, biodistribution analysis was performed followed by flow cytometry to evaluate intratumoral CD8+ T cell numbers. Intratumoral radiolabel distributions were assessed by autoradiography and immunohistochemistry. Results All approaches showed uptake in CD8-rich tissues, with preferential spleen targeting. Biodistribution analyses showed tumor uptake exceeded blood level for 89ZrZr-anti-CD8-IgG2asilent and 89ZrZr-OT-I. Furthermore, their tumor uptake correlated to intratumoral CD8+ T cells presence even though intratumoral distribution patterns differed significantly. Conclusion 89ZrZr-anti-CD8-IgG2asilent and 89ZrZr-OT-I PET/CT imaging can evaluate intratumoral CD8+ T cell infiltration. 89ZrZr-anti-CD8-IgG2asilent might be suited for TME immunophenotyping, while ex vivo labeling visualizes tumor migration and invasion dynamics of tumor-specific T cells. 18FAlF-RESCA-IL2 uptake did not correlate to the intratumoral CD8+ T cell presence. Here, we provide new insights to guide the selection of imaging strategies for assessing relevant immunotherapy-specific aspects of the TME and support correct interpretation of clinical CD8 imaging.