Visualizing γδ T cells by very late antigen-4-targeted positron emission tomography

Purpose γδ T cell–based immunotherapy has been rolled out as a promising treatment strategy for malignant tumors due to their potent anti-tumor cytotoxicity, ease of expansion, and unrestricted MHC feature. However, the dynamics and outcomes of γδ T cells in tumor sites are poorly understood. Reported strategies rely on ex vivo biolabeling, significantly limiting the application of γδ T cell molecular imaging. Herein, we investigated whether VLA-4 (very late antigen-4), a crucial component in the effective trafficking of lymphocytes, could serve as a biomarker to non-invasively visualize γδ T cells. Methods VLA-4-targeted tracer, 68 Ga-LLP2A, was evaluated in MDA-MB-231- and A549-bearing mice with adoptive transfer of γδ T cells by longitudinal PET/CT imaging. Imaging data were verified by ex vivo biodistribution studies, and the co-localization of CD3 and VLA-4 was validated by immunohistochemistry studies. Results 68 Ga-LLP2A showed high specificity to VLA-4-expressing γδ T cells in both in vitro and tumor-bearing mice with adoptive transfer of γδ T cells. Longitudinal PET imaging of 68 Ga-LLP2A in tumor-bearing mice with adoptive transfer of γδ T cells showed an increasing tumor tracer uptake, revealing the tumor-specific homing of γδ T cells. The presence of VLA-4-expressing γδ T cells in tumors was confirmed via histological analysis. Conclusion To the best of our knowledge, we reported the first molecular probe, 68 Ga-LLP2A, for in vivo imaging of γδ T cells in live tumors, which advances PET imaging of γδ T cells and supports the translation of imaging agents for immunotherapeutic monitoring.