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. 2021 Mar 23;12:639818. doi: 10.3389/fimmu.2021.639818

Figure 3.

Figure 3

The dimerization of the CD28-TMD depends on a core of four amino acids. (A) Diagram representing the amino acid sequence of the wild type and four mutants of the CD28-TMD. A representative example of MYC and the mCherry expression for each mutant is shown. (B) A representative example of CFSE dilution of a mixed population of CD3+/−CAR+/− T cells restimulated with anti-CD3/CD28 beads and unstimulated conditions. Representative CFSE MFI ratios (/CAR negative CD4 T cells) for low, intermediate, and high CAR expression with or without stimulation are shown (C) The normalized CFSE MFI ratio for CD3-CARlow/int/high was calculated by dividing the MFI of each of these population with the MFI of CD3−mCherry− cells within the same culture. A summary of results using T cells from four unrelated donors in two independent experiments is shown. (D) Proliferation of purified CD3−CAR+ T cells in response to plate-bound anti-CD28 stimulation. Results represent the mean of three independent experiments. (E) CD28 or the Myc-tag of CD3−CAR+ T cells were immunoprecipitated. Western blot analysis of the input (5% of the whole cell lysate) as well as of the precipitated was performed using anti-CD28 (clone D2Z4E) and anti-Myc (clone 9B11). Results are representative of two independent experiments for each condition. The two-way ANOVA was used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001.