Fig. 6: Characterization of CUL5 KO primary human CD8⁺ T cells.

a Western blot analysis of CUL5 expression in primary human CD8⁺ T cells with NC (Black) or CUL5 KO (Red). Quantification of CUL5 protein levels normalized against GAPDH is shown (Bottom; n = 3; Two-sided unpaired t-test). b Flow cytometry analysis of GZMB expression in NC or CUL5 KO primary human CD8⁺ T cells before (Left) or after (Right) 6-hour anti-CD3 plus anti-CD28 stimulation. Data are shown in bar chart (Top) as mean ± SEM (Two-sided unpaired t-test, n = 3). c Flow cytometry analysis of IFNG⁺ cells and expression in NC (Black) or CUL5 KO (Red) primary human CD8⁺ T cells after 6-hour anti-CD3 plus anti-CD28 stimulation. Data are shown in bar chart (Top) as mean ± SEM (Two-sided unpaired t-test, n = 3). d, e Flow cytometry analysis of d, GZMB expression as MFI and e, IFNG expression as positive % in total (Left) and MFI in positive population (Right) in NC (Black) or CUL5 KO (Red) primary human CD8⁺ T cells transduced with CAR-CD19 after 6-hour co-culture with NALM6 B cell line. Data are shown in bar chart (Top) as mean ± SEM (Two-sided unpaired t-test, n = 3) and as representative histogram (d, Bottom) or scatter plot (e, Bottom).. f In vitro killing assay of NC (Black) or CUL5 KO (Red) CAR-CD19 primary human CD8⁺ T cells co-cultured with NALM6 B cell line overnight. E:T, T cell to NALM6 B cell ratios. The data presented as % of killing (mean ± SEM; Two-way Anova with Sidak correction, n = 3). g Flow cytometry analysis of CTLA4 expression in NC or CUL5 KO primary human CD8⁺ T cells 2-day post anti-CD3 plus anti-CD28 stimulation. Data are shown as mean ± SEM (Two-sided unpaired t-test, n = 3). h–j TIDE analyses of CUL5 expression correlation with T cell dysfunction signatures and survival benefits in patients with h, ovarian cancer, i, leukemia and j, melanoma. (Left panels: patients with high CUL5 expression; Right panels: patients with low CUL5 expression). Datum points represent biological replicates.