TM-mediated cross-linkage with tumor cells results in UniCAR T cell activation and cytokine release. (A) In the UniCAR T cell approach, T cells are engineered to express a universal chimeric antigen receptor (UniCAR) that recognizes the E5B9 peptide of the nuclear antigen La/SS-B. Thus, under physiological conditions UniCAR T cells are switched “OFF”. To engage the killing capabilities of UniCAR T cells against tumor cells (“ON”), an E5B9-tagged target module (TM) recognizing a tumor-associated antigen is required. (B,C) UniCAR T cells were incubated alone or with PC3-PSCA or PC3 wt cells at an E:T ratio of 5:1 in the presence or absence of 5 nM anti-PSCA IgG4-TM. After 24 h, (B) CD69 expression on UniCAR T cells and (C) secretion of TNF, IFN-γ and IL-2 were examined via flow cytometry or ELISA (x: not detectable), respectively. (B) Data of one representative experiment with one T cell donor are shown. (C) Summarized data of three different T cell donors are shown as mean ± SEM. (* p < 0.05, ** p < 0.01, *** p < 0.001 compared to samples w/o the anti-PSCA IgG4-TM; two-way ANOVA with post-hoc Šídák’s multiple comparisons test).