Fig. 4: CUL5 KO causes proteomic alterations in primary CD8⁺ T cells.

a DIA-MS signals of the CUL5 protein in NC (Black) or CUL5 (Red) KO primary CD8⁺ T cells at the T0, T8 and T16 conditions determined by mass spectrometry (MS). Data are shown as mean ± SEM (Two-sided multiple t-test, n = 3). b Flow cytometry analysis of CUL5 expression in primary CD8⁺ T cells before (Black) or after (Blue) 2-day activation by anti-CD3/CD28 beads, with 2-day activated CUL5 KO cells as a control (Red). Data are shown as mean ± SEM (Two-sided unpaired t-test, n = 3). c Western blot analysis of CUL5 expression in primary CD8⁺ T cells treated as in b. Neddylated CUL5 (Bottom) was quantified, with Histone H3 as internal control. d Volcano plot of differentially expressed proteins between CUL5 KO and non-targeting (NC) primary CD8⁺ T cells at T0 (Left) and T16 (Right) conditions as quantified by DIA-MS (n = 3). e–g Flow cytometry validation of immunological markers (CD25, CD5, CD137, ICOS, PD1, CTLA4 and CD62L) altered in CUL5 KO primary CD8⁺ T cells at T0 (e) and/or T16 (e–g) conditions. Data are shown as mean ± SEM (Two-sided unpaired t-test, n = 3). h Live cell numbers of NC (Black) and CUL5 (Red) KO primary CD8⁺ T cells before and after 16-hour anti-CD3 plus anti-CD28 stimulation. Data are shown as mean ± SEM (Two-sided unpaired t-test, n = 3). i Growth curve of tumors from E.G7-OVA cells inoculated s.c. into C57BL/6 mice. Data are shown as mean ± SEM. The back arrow indicates the time of sub-lethal irradiation followed by immediate adoptive transfer of Cas9/OT-I cells with NC (Black), CTLA4 (Blue), CUL5 (Pink) and CTLA4/CUL5 double (Red) KO. (Two-way ANOVA with Tukey correction; n = 4–5). Datum points represent biological replicates.