Fig. 3

Relative expression of checkpoint receptors and memory T cell subsets in CAR- and TAC-engineered T cells. T cells were transduced with HER2-TAC, or a second-generation anti-HER2 CAR including the CD28 (28ζ CAR) or 4-1BB (BBζ CAR) costimulatory receptor domains, or a vector control (tNGFR). Engineered T cells are stained for surface marker expression and CD4⁺NGFR⁺ or CD8⁺NGFR⁺ populations are analyzed by flow cytometry for (a) expression of checkpoint receptors PD-1, LAG-3, and TIM-3. All data is normalized to TAC-engineered T cells, and lines represent the mean of four donors. Each donor is represented by a unique symbol to highlight donor-to-donor variations. Multiple t-test is used to determine significance. For the gating strategy see Supplementary Fig. 14. b Memory T cell subsets of TAC-, 28ζ CAR-, and BBζ CAR-T cells, relative to T cells engineered with a vector control (tNGFR). T cell subsets are defined as naïve (CD45RA⁺, CCR7⁺), central memory (CM) (CD45RA⁻, CCR7⁺), effector memory (EM) (CD45RA⁻, CCR7⁻), and terminal effectors (EMRA) (CD45RA⁺, CCR7⁻). Lines represent the mean of four donors. Multiple t-test is used to determine significance. c CD27 and CD28 expression; representative histograms show data from one donor, median fluorescence intensity is indicated. Data are from two independent experiments with four different donors. For both (b) and (c), the gating strategy is shown in Supplementary Fig. 15