Skip to main content
. Author manuscript; available in PMC: 2023 Feb 19.
Published in final edited form as: Sci Transl Med. 2022 Nov 9;14(670):eabm1463. doi: 10.1126/scitranslmed.abm1463

Fig. 4. Distinct signaling domains differentially affects proliferation, long-term expansion, markers of memory, and exhaustion.

Fig. 4.

(A) The timeline for arrayed proliferation assays in (B and C) is shown. Primary human CD4 and CD8 T cells were separately transduced with the 8 CARs in (B and C) and stimulated 1:1 with irradiated CD19+ or CD19- K562 cells every three days. Proliferation was assessed by CTV dilution every 9 days. (B) Relative proliferation of each CAR was quantified by the relative decrease in mean fluorescence intensity (MFI), representing the dilution of CTV dye, between two donors of CD4 or CD8 T cells. The color legend ranges from less proliferative (dark blue) to more proliferative (yellow). The x-axis indicates the day the cells were stained. White boxes are representative of CAR T cells that dropped out of culture. (C) Histograms of CTV staining of CD4 or CD8 CAR T cells in a representative donor on selected days are shown. Data summarized in (B). Both donors are shown in fig. S4A and B. AU, arbitrary units. (D) Quantification of the cumulative expansion of CD4 T cells engineered with either CD40 (purple), 4-1BB (light blue), or CD28 (dark blue) CARs and stimulated as described in (A). Co-cultures were measured every three days starting on day 3 by flow cytometry and counting beads. The y-axis measures cumulative fold-expansion every three days. (Significance was derived from a linear mixed effects model, for CD40 comparisons, all p < .001). (E) Cells were transduced and stimulated as described in (A). Every 9 days in culture, cells were rested for 6 days without additional stimulation and assessed for surface expression of PD1, LAG3, and TIM3. (F) Percentage of CAR T cells expressing 0 to 3 of the exhaustion markers PD1, TIM3, LAG3 after day 6 and day 15 as described in (E). All CARs, markers, and time points are shown in fig. S4D and E. (G) CD27 surface expression on CD8 CAR T cells was measured over 33 days, as in (E). Percentage of CD27-high cells is shown for each CAR and day on the right. All CARs and time points are shown in fig. S4G. Significance in (F and G) was assessed using a repeated measures (RM) ANOVA model, (FDR < 0.05:*, < 0.01:**, < 0.001:***, < 0.0001:****).