In vivo tracking of bioorthogonally labeled T-cells for predicting therapeutic efficacy of adoptive T-cell therapy.

Non-invasive tracking of T-cells may help to predict the patient responsiveness and therapeutic outcome. Herein, we developed bioorthogonal T-cell labeling and tracking strategy using bioorthogonal click chemistry. First, ovalbumin (OVA) antigen-specific cytotoxic T-cells (CTLs) were incubated with N-azidoacetyl-D-mannosamine-tetraacylated (AcManNAz) for incorporating azide (N) groups on the surface of CTLs via metabolic glycoengineering. Subsequently, azide groups on the CTLs were chemically labeled with near infrared fluorescence (NIRF) dye, Cy5.5, conjugated dibenzylcyclooctyne (DBCO-Cy5.5) via bioorthogonal click chemistry, resulting in Cy5.5-labeled CTLs (Cy5.5-CTLs). The labeling efficiency of Cy5.5-CTLs could be readily controlled by changing concentrations of AcManNAz and DBCO-Cy5.5 in cultured cells. Importantly, Cy5.5-CTLs presented the strong NIRF signals in vitro and they showed no significant changes in the functional properties, such as cell viability, proliferation, and antigen-specific cytolytic activity. In ovalbumin (OVA)-expressing E.G-7 tumor-bearing immune-deficient mice, intravenously injected Cy5.5-CTLs were clearly observed at targeted solid tumors via non-invasive NIRF imaging. Moreover, tumor growth inhibition of E.G-7 tumors was closely correlated with the intensity of NIRF signals from Cy5.5-CTLs at tumors after 2-3 days post-injection. The Cy5.5-CTLs showed different therapeutic responses in E.G-7 tumor-bearing immune-competent mice, in which they were divided by their tumor growth efficacy as 'high therapeutic response (TR (+))' and 'low therapeutic response (TR (-))'. These different therapeutic responses of Cy5.5-CTLs were highly correlated with the NIRF signals of Cy5.5-CTLs at targeted tumor tissues in the early stage. Therefore, non-invasive tracking of T-cells can be able to predict and elicit therapeutic responses in the adoptive T-cell therapy.