Figure 4.

Effect of CTX Treatment on M11 CAR T Cells, Endogenous T Cells, and Regulatory T Cells (Tregs)

TDLNs, spleens, and tumors were harvested from mice treated as described in Figures 3A and 3B. After digestion, they were subjected to flow cytometry. (A) M11 CAR T cells were tracked by flow cytometry through their GFP label. CTX treatment increased the persistence of M11 CAR T cells in TDLNs (left graph) and spleens (right graph) (numbers are in percent total live cells). Samples: n = 3 per group. Statistics by 2-way ANOVA: ∗∗p < 0.01. (B) The frequency of T cells in AE17om tumors post-treatment was assessed. When used alone, CTX decreased the percentage of CD3+ and CD4+ T cells of live tumor cells. Tumors treated with the combination of CTX plus M11 CAR T cells showed significantly increased infiltration of CD8+ T cells compared to the untreated group, the group treated with M11 CAR T cells only, or the CTX-only group. Samples: n = 3 per group. Statistics by 2-way ANOVA: ∗p < 0.05; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. (C) Treg (CD25+CD3+CD4+FoxP3+) frequency was seen to be significantly decreased in TDLNs of mice treated with CTX alone. This effect was not replicated in the spleens of these mice, where a slight decrease in Tregs was seen in CTX-treated samples, but it was not significant. Tumors treated with CTX and with CTX plus M11 CAR T cells had a significantly smaller percentage of Tregs compared to the Tregs in tumors of untreated mice or mice that were treated with M11 CAR T cells. Samples: n = 3 per group. Statistics by 2-way ANOVA: ∗p < 0.05; ∗∗∗∗p < 0.0001.