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. 2021 Dec 31;24:385–399. doi: 10.1016/j.omto.2021.12.019

Figure 3.

Figure 3

Flow cytometry analysis of peripheral circulating and tumor-infiltrating CAR-T cells

(A) T cell count in peripheral blood. Peripheral blood was analyzed weekly via flow cytometry. T cell count per 100 μL was quantified in BBζ CAR8 (pink), 28ζ CAR8 (green), 28BB CAR8 (orange), and BBζ CAR4/8 (blue) CAR-T cells and CD8 (black) and CD4/8 (gray) UNT cells. (B) Exhaustion of peripheral circulating T cells. The percentage of PD-1 high-expressing T cells was quantified in BBζ CAR8 (pink), 28ζ CAR8 (green), 28BB CAR8 (orange), and CD8 (black) UNT cells on day 7. P values are defined by unpaired two-tailed t-tests ( ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001). (C) Tumor-infiltrating T cell percentage. At the endpoint of the animal study, tumor-infiltrating human CD45+ lymphocytes were isolated. T cell infiltration of BBζ (pink), 28ζ (green), 28BBζ (orange), and BBζ CD4/8 (blue) CAR-T cells and CD8 (black) and CD4/8 (gray) UNT cells in tumors was quantified. The percentage was defined by human CD45 population divided by total viable cells. (D) Percent CD4 and CD8 TILs. (E) CAR+ percentage. (F and G) Exhaustion marker expression (PD-1 high; F) and the memory T cell subset (CD45RO+CD45RA−; G) of CAR4/8 BBζ CAR-T cells on day 72 were analyzed. ZsGreen signal was used to define CAR+ population. P values are defined by unpaired two-tailed t-tests ( ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001).