Azido labeling of T cells allows conjugation of DBCO cytokines to generate potent cytokine-dependent inflammatory phenotypes. A, ELISA quantification of the amount of DBCO-IL-12 conjugated onto one million T cells at various DBCO-IL-12 concentrations (n = 3, one-way ANOVA and Tukey’s test). B, Viability data for T cells receiving no IL-12, conjugated with DBCO IL-12 and presented with soluble IL-12 (no DBCO label) in media in in vitro culture for 7 d (n = 3). C, Representative phenotyping data and (D) heatmap data showing memory phenotype markers and activation and exhaustion markers, for T cells conjugated with DBCO-IL-12 (“NP + conjugated IL-12;” green), treated with continuously soluble IL-12 (“NP + continuous IL-12;” blue), or temporarily exposed to IL-12 for duration of reaction time (“NP + transient IL-12;” purple) at different cytokine concentrations cultured in vitro for 7 d (n = 3, two-way ANOVA). E, Anti-B16-F10 tumor cell cytolytic activities of Pmel-1 T cells conjugated with DBCO-IL-12 (“NP + conjugated IL-12;” green), treated with soluble IL-12 (“NP + continuous IL-12;” blue), or temporarily exposed to IL-12 (“NP + transient IL-12;” purple) (n = 6, one-way ANOVA and Tukey’s test).