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. 2022 May 13;23(10):5448. doi: 10.3390/ijms23105448

Figure 1.

Figure 1

Functional characterization of chimeric antigen receptor (CAR) T cells and CAR T cell exhaustion in vitro. (A) T cells proliferation analysis two and four days after transduction with Anti carbonic anhydrase IX (CAIX) CD8alpha/4-1BB CAR-expressing anti-programmed cell death ligand-1 (PD-L1) IgG4 (anti-CAIX/4-1BB/anti-PD-L1 IgG4), or ZsGreen (anti-CAIX/4-1BB/ZsGreen), anti CAIX CD28 CAR-expressing anti-PD-L1 IgG4 (anti-CAIX/CD28/anti-PD-L1 IgG4) compared to anti-B cell maturation antigen (BCMA) 4-1BB CAR-expressing ZsGreen (anti-BCMA/4-1BB/ZsGreen negative control). (B) Percentage of CAR+ T cells and (C) Proliferation of CAR+ T cells two and four days after transduction. (D) Percentage of CAR T cells fourteen days after initial transduction, representing the long-term stable expression of CAR by integration of lentiviruses into the T cell genome. (E) IgG secretion by anti-CAIX/anti-PD-L1 IgG4 CAR T cells determined by ELISA (Sigma-Aldrich). (F) Cytotoxicity of Skrc59 CAIX+/PD-L1+ renal carcinoma cells promoted by CAR T cells in different proportions of effector cells per tumor cells (E:T). The results presented represent the mean ± SD of CAR T cells from three donors in triplicate. (G) CAR T cell exhaustion in vitro. CAR T cells were activated with anti-CD3/CD28 beads and cultured for five days with 10 ng/mL of IL-7 and IL-15, followed by co-culture with skrc59 CAIX+/PD-L1+ cells for another four days for induction of exhaustion. The expression of the exhaustion markers PD-1, TIM-3, CTLA-4, as well as interleukin 2 (IL-2) were determined by flow cytometry within live CD45 positive-gated cells. Co-expression of exhaustion markers was determined by Boolean analysis in the FlowJo Software (FlowJo LLC, Ashland, OR, USA). * p < 0.05 compared to anti-BCMA/ZsGreen CAR T cells.