NIRF/PET imaging of ?d T cells via metabolic glycoengineering and bioorthogonal labeling

Non-invasive tracking of ?d T cells may help to predict patient responsiveness and therapeutic efficacy. In this study, we developed an efficient ?d T cell NIRF/PET imaging strategy by combining metabolic glycoengineering and bioorthogonal click chemistry. Initially, ?d T cells were incubated with N-azidoacetyl-D-mannosamine-tetraacylated, and azide (N-3) groups were then incorporated onto the surface of ?d T cells via metabolic glycoengineering to obtain N-3-?d T cells. Next, Cy5.5 dibenzylcyclooctyne (DBCO-Cy5.5) and the short half-life (68)Galabeled chelator Ga-68-NETA-DBCO were conjugated with N-3-?d T cells via in vitro or in vivo bioorthogonal metalfree click chemistry for NIRF and PET imaging, respectively. In Daudi tumor-bearing mice, Cy5.5-labeled ?d T cells migrated specifically to tumor sites, and adoptive cells were observed clearly via non-invasive NIRF imaging over 10 days. Furthermore, in the pre-targeted PET images, high tumor uptake of Ga-68-NETA-DBCO was observed in mice three days post-injection of N-3-?d T cells, which was 1.5-fold higher than that of control mice. Interestingly, dead-Cy5.5-?d T cells also showed high tumor targeting and good tumor retention, peaking at day 1 and retaining NIRF signal up to 10 days, indicating a high potential of ?d T cells as a drug delivery system. These findings demonstrated that a bioorthogonal labeling method followed by NIRF/PET imaging maps the biodistribution, migration, and tumor-homing efficiency of adoptive ?d T cells in vivo, representing a new potential ?d T cell imaging technology.