Fig. 2 | Nature Communications

Fig. 2

From: Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response

Fig. 2

Incorporation of TRuCs in TCR complexes and functional analysis of endogenous TCR in Jurkat cells stably transduced with empty control vector or vectors encoding the indicated CAR or TRuC transgenes. The transgene expression was linked to GFP via a T2A sequence. a Cells were lysed and TCR complexes separated by Blue Native PAGE. After Western blot TCR complexes were stained using an anti-CD3ζ antibody. “0” denotes the natural TCR complex, “1” and “2” denote TCR complexes with one or two TRuCs, respectively. b Complex formation of TRuC variants with TCR subunits. Upon cell lysis, TRuCs and CARs were immunopurified using the anti-F(ab’)2 antibody, and then separated by reducing SDS-PAGE. As a control, the procedure was also applied to the lysis buffer alone. (Co)-purified proteins were detected using the described antibodies by western blot. Data in a and b show representative results of three experiments. c Transduced cells were stained with an anti-CD3ε antibody and analyzed by flow cytometry. d The effect of ε-TRuC on activation of an influenza HA-specific TCR. Jurkat T cells expressing the HA1.7 TCR (Jurkat cell line CH7C17) were transduced with a control vector (black) or vectors encoding for the ε-TRuC (light blue), the 28ζ (red) or BBζ CAR (purple). Cells were co-cultured with DapDR1-ICAM1 antigen-presenting cells loaded with different amounts of the HA306–318 peptide. After 6 h, upregulation of the activation marker CD69 was quantified by flow cytometry. The graphs in c and d show representative data of two independent experiments. Data were analyzed using a parametric two-way ANOVA test, followed by Dunnett’s multiple comparison test to compare means of the indicated sample to the control sample (Vector Control) at each HA concentration. ****Adjusted-P ≤ 0.0001. Error bars depict standard deviation. Samples were measured in triplicates

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