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Updated: May 12, 2026
Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells
Published on: February 8, 2018
Head-to-head comparison of nuclear imaging approaches to quantify tumor CD8+ T-cell infiltration
Gerwin G W Sandker1, René Raavé1, Inês F Antunes2
1Department of Medical Imaging, Radboud University Medical Center, Nijmegen, the Netherlands.
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 ([89Zr]Zr-anti-CD8-IgG2asilent), ex vivo 89Zr-labeled ovalbumin-specific CD8+ T cells ([89Zr]Zr-OT-I), and 18F-labeled IL2 ([18F]AlF-RESCA-IL2).
Methods:
B16F10/OVA tumor-bearing C57BL/6 mice (n = 10/group) underwent PET/CT imaging at 72 ([89Zr]Zr-anti-CD8-IgG2asilent), 24 and 48 h ([89Zr]Zr-OT-I), and 10 min ([18F]AlF-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 [89Zr]Zr-anti-CD8-IgG2asilent and [89Zr]Zr-OT-I. Furthermore, their tumor uptake correlated to intratumoral CD8+ T cells presence even though intratumoral distribution patterns differed significantly.
Conclusion:
[89Zr]Zr-anti-CD8-IgG2asilent and [89Zr]Zr-OT-I PET/CT imaging can evaluate intratumoral CD8+ T cell infiltration. [89Zr]Zr-anti-CD8-IgG2asilent might be suited for TME immunophenotyping, while ex vivo labeling visualizes tumor migration and invasion dynamics of tumor-specific T cells. [18F]AlF-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 the correct interpretation of clinical CD8 imaging.
