Figure 2.

Generation of CD22 CART from positively and negatively selected T cells

(A) Schema of the manufacturing protocol used to generate CD22 CART. TR, transduction. (B) Viable total nucleated cells (TNC) are shown between days 2 and 9 of culture. The B-ALL patient sample is indicated with an x. (C) Fold expansion was calculated as TNC on day 9 normalized to TNC on day 2. Lines are used to connect data points from the same donor. (D) CD3⁺ T cell purity was assessed on day 9 after gating on viable cells. (E) (i) The ratio of CD4:CD8 cells was assessed after gating on viable cells. (ii) Transduction was measured as a function of protein L staining on viable CD3⁺ cells, plotted as the mean ± SD of six donors. (iii) %CD25⁺ T cells was assessed on viable CD3⁺ cells, plotted as the mean ± SD of six donors. (iv) PD1 expression was assessed on viable CD3⁺ cells and means ± SDs of PD1⁺ T cells from the six donors at days 2–9 are presented. (F) Naive/stem/central memory-like (T_n/scm-like), central memory (T_cm), effector memory (T_em), and terminal effector (T_eff) T cells were monitored by flow cytometry as CCR7⁺CD45RA⁺, CCR7⁺CD45RA⁻, CCR7⁻CD45RA⁻, and CCR7⁻CD45RA⁺, respectively and gated from the CD3⁺CAR⁺ population. Day 9 means ± SEM (n = 6) are shown. (G) PCA and (H) pathway analysis of gene expression data assessed at days 2, 4, 7, and 9 of culture. Means ± SEM for the six donors are shown. Statistical significance was calculated using a multiple paired t test; ns > 0.05.