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Increasing evidence emphasizes the pivotal role of CD4 + T cells in orchestrating cancer immunity. Noninvasive in vivo imaging of the temporal dynamics of CD4 + T cells and their distribution patterns might provide novel insights into their effector and regulator cell functions during cancer immunotherapy (CIT). Methods: We conducted a comparative analysis of 89 Zr-labeled anti-mouse (m) and anti-human (h) CD4-targeting minibodies (Mbs) for in vivo positron emission tomography (PET)/magnetic resonance imaging (MRI) of CD4 + T cells in human xenografts, syngeneic tumor-bearing wild-type (WT), and human CD4 + knock-in (hCD4-KI) mouse models. Results: Both 89 Zr-CD4-Mbs yielded high radiolabeling efficiencies of >90%, immunoreactivities of >70%, and specific in vitro binding to their target antigens. The specificity of in vivo targeting of 89 Zr-hCD4-Mb was confirmed by PET/MRI, revealing ~4-fold greater 89 Zr-hCD4-Mb uptake in subcutaneous hCD4 + hematopoietic peripheral blood acute lymphoblastic leukemia tumors (HPB-ALL) than in solid hCD4 - diffuse histiocytic lymphomas (DHL) and 89 Zr-mCD4-Mb uptake in hCD4 + HPB-ALL tumors. In a comparative cross-validation study in anti-programmed death ligand (αPD-L1)/anti-4-1BB-treated orthotopic PyMT mammary carcinoma-bearing hCD4-KI and WT mice, we detected 2- to 3-fold enhanced species-specific 89 Zr-hCD4-Mb or 89 Zr-mCD4-Mb uptake within CD4 + cell-enriched secondary lymphatic organs (lymph nodes and spleens). The 89 Zr-hCD4-Mb uptake in the PyMT tumors was more pronounced in hCD4-KI mice compared to the WT control littermates. Most importantly, MC38 adenocarcinoma-bearing mice treated with a combination of αPD-L1 and anti-lymphocyte-activation gene 3 (αLag-3) antibodies exhibited ~1.4-fold higher 89 Zr-mCD4-Mb uptake than mice that were not responsive to therapy or sham-treated mice. Conclusion: CD4 PET/MRI enabled monitoring of the CD4 + cell distribution in secondary lymphatic organs and the tumor microenvironment, capable of predicting sensitivity to CIT. Our imaging approach will provide deeper insights into the underlying molecular mechanisms of CD4-directed cancer immunotherapies in preclinical mouse models and is applicable for clinical translation., Competing Interests: Competing Interests: ImaginAb holds a patent on the CD4 minibodies (W O 2019/236684 Al), in which AMas is listed as inventor., (© The author(s).)