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by Gerwin G.W. Sandker,  René Raavé,  Inês F. Antunes, Milou Boswinkel,  Lenneke Cornelissen, Gerben M. Franssen,  VJanneke Molkenboer-Kuenen,  Peter J. Wierstra,  Iris M. Hagemans,  Erik F.J. de Vries,  Johan Bussink,  Gosse Adema,  Martijn Verdoes,  Erik H.J.G. Aarntzen,  Sandra Heskamp on behalf of the TRISTAN Consortium

Immunology, 15. Oktober 2024. doi: 10.1101/2024.10.13.618082.

Abstract

Many immunotherapies focus on (re)invigorating CD8+ T cell anti-cancer responses and different nuclear imaging techniques have been developed to measure CD8+ T cell distributions. In vivo labeling approaches using radiotracers primarily show CD8+ T cell distributions, while ex vivo labeled CD8+ T cells can show CD8+ T cell migration patterns, homing, and tumor infiltration. Currently, a comprehensive head-to-head comparison of in vivo and ex-vivo cell labeling with respect to their tumor and normal tissue targeting properties and correlation to the presence of CD8+ T cells is lacking, yet essential for correct interpretation of clinical CD8+ imaging applications. Therefore, we performed a head-to-head comparison of three different CD8+ T cell imaging approaches: 1) 89Zr-labeled DFO-conjugated Fc-silent anti-CD8 antibody ([89Zr]Zr-anti-CD8-IgG2asilent), 2) ex vivo 89Zr-oxine labeled ovalbumin-specific CD8+ T cells ([89Zr]Zr-OT-I cells), and 3) 18F-labeled IL2 ([18F]AlF-RESCA-IL2).

Methods B16F10/OVA tumor-bearing C57BL/6 mice (n=10/group) received intravenously one of the three radiopharmaceuticals. PET/CT images were acquired starting 72 h ([89Zr]Zr-anti-CD8-IgG2asilent), 24 and 48 h ([89Zr]Zr-OT-I cells), and 10 min ([18F]AlF-RESCA-IL2) post injection. Subsequently, ex vivo biodistribution analysis of the radiopharmaceuticals was performed followed by flow cytometric analysis to evaluate the number of intratumoral CD8+ T cells. Additionally, the intratumoral radiolabel distributions was assessed by autoradiography and immunohistochemistry (IHC) on tumor slices.

Results [89Zr]Zr-anti-CD8-IgG2asilent, [89Zr]Zr-OT-I cells, and [18F]AlF-RESCA-IL2 showed uptake in CD8-rich tissues, with preferential targeting to the spleen. Biodistribution analysis showed tumor uptake above blood level for all radiopharmaceuticals, except [18F]AlF-RESCA-IL2. For all three approaches, the uptake in the tumor-draining lymph node was significantly higher compared with the contralateral axial lymph node, suggesting that all approaches allow evaluation of immune responses involving CD8+ T cells. Tumor uptake of [89Zr]Zr-anti-CD8-IgG2asilent (R2=0.65, p<0.01) and [89Zr]Zr-OT-I cells (R2=0.74, p<0.01) correlated to the number of intratumoral CD8+ T cells (flow cytometry). The intratumoral distribution pattern of the radiosignal was different for ex vivo and in vivo radiolabeling techniques. The short half-life of 18F precluded autoradiography assessment of [18F]AlF-RESCA-IL2.

Conclusion We show that [89Zr]Zr-anti-CD8-IgG2asilent and [89Zr]Zr-OT-I cells PET/CT imaging can be used to evaluate intratumoral CD8+ T cells, even though their normal tissues and intratumoral distribution patterns are significantly different. Based on their characteristics, [89Zr]Zr-anti-CD8-IgG2asilent might be most useful to immunophenotyping the TME, while the ex vivo cell labeling approach visualizes CD8+ T cell migrations patterns and the permissiveness of tumors for invasion, whereas [18F]AlF-RESCA-IL2 allows for rapid recurrent imaging and might prove useful for tracking rapid changes in CD8+ T cell distributions. In conclusion, our head-to-head comparison of the three prototype CD8+ T cell labeling approaches provides new insights which can aid in correct interpretation of clinical CD8 imaging and may guide in the selection of the optimal imaging approach for the research question of interest.