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. 2017 Sep 6;7:10713. doi: 10.1038/s41598-017-11126-y

Figure 3.

Figure 3

Functional activity of TCR-CAR. (a) Primary peripheral T cells isolated from a healthy donor were mock electroporated (tinted) or electroporated with mRNA encoding Radium-1 (blue) or DMF5 (red) constructs. After 12 hours, cells were stained with the indicated antibodies or multimer and analysed by flow cytometry. (b) The same cells were co-incubated 18 hours later with APCs loaded with the indicated peptides for 5 hours (grey = no peptide, white = TGFbR2 peptide and black = MART-1 peptide). The presence of the degranulation marker CD107a was performed to monitor T-cell stimulation. Data are representative of two separate experiments. Values were normalized to that of Radium-1 TCR CAR. Mean ± Standard error of the mean (SEM). N = 2. **P < 0.0001, *P < 0.001, 2-way ANOVA followed by Tukey’s multiple comparison test. (c) same as in (b) but here Radium-1 TCR-CAR was compared to the full-length Radium-1 TCR. DMF5 was included as a control for M1 loading. Mean ± SEM, N = 2. **P < 0.0001, 2-way ANOVA followed by Tukey’s multiple comparison test. (d) DMF5 (black) or Radium-1 (grey) TCR-CAR and full-length constructs expressing T cells were analyzed for the indicated cytokine response in the CD8 population. Target cells were loaded (+) or not (−) with the antigenic peptide and incubated with the T cells at an E:T ratio of 1:2. As a control mock transfected T cells (white) were tested in the same conditions or alone. Intracellular flow cytometry readings were collected after 6 hours of co-culture and % of positive cells were plotted. Mean ± SEM, N = 3. Unpaired t-test was used as statistical analysis. (e) same as in (d) where specific lysis of target cells loaded with the indicated peptide was analyzed. Transduced Tc with the indicated constructs were incubated at different E:T ratios with target and lysis was monitored by BLI cytotoxic assay. Luminescence readings were collected after 10 hours of co-culture. Mean ± SEM, N = 3. Unpaired t-test performed between indicated group and corresponding Mock group. Ranges for unpaired t-test were as follows *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.00001.