Extended Data Fig. 2: T cells progressed through cell cycle and proliferated upon activation.

(a) Experimental design and timeline. CD3 T cells were isolated from 3 healthy donors and activated with StemCell αCD2/αCD3/αCD28 in ImmunoCult XF media (Imm) or TransAct αCD3/αCD28 in TexMACS media (Tex). Cells were divided into 7 groups with duration of activation ranging from 0 hours (quiescent T cells) to 72 hours prior to electroporation to introduce the anti-GD2 CAR transgene. When activated T cells were collected for electroporation, their metabolic features were characterized using OMI. Meanwhile, their cell cycle stage and proliferation capacity were assessed using flow cytometry of Hoechst 33342 and Ki-67 staining. These activated T cells were then electroporated to incorporate the anti-GD2 CAR transgene and expanded in corresponding culture media. Genome editing efficiency was determined after 7 days of expansion with GD2 CAR antibody using flow cytometry. (b, c) T cells progressed through cell cycle upon activation. Density plots of Hoechst MFI in T cells activated with (b) Imm and (c) Tex methods. For each donor, Hoechst MFI was normalized to the average of 12-hour-activated group. n = 393,999 cells and 370,802 cells for Imm and Tex activation methods, respectively. (d) Correlation between NAD(P)H α₁ and cell cycle stage (% cells in S/G2/M phase) at electroporation. Each dot represents one sample average, color coded based on the method of activation (Imm or Tex) and duration of activation (0–72 hours). n = 42 samples, Pearson R analysis. (e, f) T cell proliferation (% Ki-67⁺) following activation with (e) Imm method or (f) Tex method. n = 3 donors, Brown-Forsythe and Welch ANOVA test with Dunnett’s post hoc test for multiple comparisons. Bars are mean ± SD. Color lines connected donor-match averages.