<p>Efficient application of immunotherapy necessitates advanced whole-body imaging techniques to monitor sites of immune cell activation. Deoxycytidine kinase (dCK), a key enzyme in the deoxynucleotide salvage pathway, is upregulated in proliferating immune cells and can be targeted by the radiotracers [¹⁸F]FAC (preclinical) and [¹⁸F]CFA (clinical), allowing for noninvasive monitoring of immune activation in lymphatic organs via positron emission tomography (PET). In this study, we aimed to assess the efficacy of [¹⁸F]FAC in detecting immune activation upon immune checkpoint inhibitor therapy (CIT). In vitro, activated T cells and macrophages exhibited significantly higher [¹⁸F]FAC uptake compared to their naïve counterparts. In vivo, preclinical [¹⁸F]FAC-PET/MRI revealed a CIT-induced significant increase in [¹⁸F]FAC uptake in tumor-draining lymph nodes (TDLNs) compared to contralateral lymph nodes, independent of tumor responsiveness. This phenomenon was absent in TDLNs of sham-treated experimental mice. Ex vivo cell sorting further confirmed elevated [¹⁸F]FAC uptake in T cells from TDLNs following CIT. Consistently, [¹⁸F]CFA-PET/CT imaging in metastatic melanoma patients demonstrated CIT-induced enhanced regional LN uptake. Together, these findings establish a strong correlation between CIT-induced immune activation and [¹⁸F]FAC/[¹⁸F]CFA uptake, underscoring the critical role of TDLNs in cancer immuotherapy. The radiotracers [¹⁸F]FAC and [¹⁸F]CFA provide valuable tools for noninvasive monitoring of immune cell activation, potentially unveiling tumor-microenvironment-related resistance mechanisms and advancing the utility of PET imaging in immunotherapy monitoring and patient stratification.</p>