Fig. 5: 4-1BB versus CD28 CARs: proliferation and survival.

a T cells expressing FHVH33-CD828Z (28) or FHVH33-CD8BBZ (BB) were labeled with CFSE and cultured with irradiated BCMA-K562 cells or NGFR-K562 cells. Changes in CAR⁺ T-cell numbers during the 4-day culture are shown (n = 6). All bar graphs in this figure show mean ± s.e.m.; all statistics are paired two-tailed t-tests, P < 0.05 was considered statistically significant. b BCMA-specific proliferation was represented by dividing the CFSE MFI of T cells stimulated with BCMA-K562 by the CFSE MFI of T cells stimulated with NGFR-K562. BCMA-specific CFSE dilution and proliferation were greater with FHVH33-CD828Z T cells (28) than FHVH33-CD8BBZ (BB) T cells (n = 6). c T cells expressing FHVH33-CD828Z (28) or FHVH33-CD8BBZ (BB) were cultured overnight with either BCMA-K562 cells or NGFR-K562 cells and stained with annexin V to detect apoptosis. As a measure of BCMA-specific apoptosis, the %annexin V⁺ CAR⁺ T cells was calculated as the percentage annexin V⁺ CAR⁺ T cells after BCMA-K562 stimulation minus the percentage annexin V⁺ CAR⁺ T cells after NGFR-K562 stimulation. The percentages of annexin V⁺ CAR⁺ cells were higher for FHVH33-CD828Z versus FHVH33-CD8BBZ for CD4⁺ (P = 0.017) and CD8⁺ T cells ⁽P = 0.007); n = 5. d T cells expressing FHVH33-CD828Z (28) or FHVH33-CD8BBZ (BB) were stimulated with BCMA-K562 cells. CAR⁺ T cells were quantified at the beginning of culture and 7 days later. Changes in CAR⁺ T-cell numbers between initiation and day 7 of culture are shown (n = 4). e After the culture described in d, T cells were stained for annexin V. Mean ± s.e.m. %annexin V⁺ CAR⁺ T cells is shown (n = 4). f Mean ± s.e.m. of CD4:CD8 ratios of CFSE-labeled 28 and BB CAR⁺ T cells after the 4-day culture from a are shown (n = 6).