Fig. 2: CUL5 knockout (KO) promotes sustained proliferation and enhances effector functions later after stimulation. | Nature Communications

Fig. 2: CUL5 knockout (KO) promotes sustained proliferation and enhances effector functions later after stimulation.

From: Cullin-5 deficiency promotes chimeric antigen receptor T cell effector functions potentially via the modulation of JAK/STAT signaling pathway

Fig. 2

a–d Changes in the percentage of CUL5 KO-CD19 CAR T cells generated by sgRNA#4690 from Days 10–20 (a) and 20–30 (b) or by sgRNA#8377 from Days 10–20 (c) and 20–30 (d). (n = 4 in a and b; n = 3 in c and d) (e) Representative flow plots of CellTrace Violet (CTV)-labeled control (Ctrl) or CUL5 KO-CD19 CAR T-cell division assays. GFP− fractions are unmodified CAR T cells, whereas GFP+ fractions are CUL5 KO-CAR T cells. f, g CTV mean fluorescence intensity (MFI) after stimulation at either earlier (f, Days 0–3, n = 3) or later time points (g, Days 4–7, n = 5) in Ctrl or CUL5 KO-CD19 CAR T cells. h, i Ki-67+ cell percentage after stimulation during earlier (h, Days 0–3, n = 4) or later (i, Days 4–7, n = 5) culture periods in Ctrl or CUL5 KO-CD19 CAR T cells. j Intracellular cytokine staining of Ctrl or CUL5 KO-CD19 CAR T cells stimulated with K562 or Raji cells. Representative flow plots of interferon-gamma (IFN-γ) and interleukin (IL)-2 are shown. k, l Intracellular cytokine staining for IFN-γ (k) and IL-2 (l). (n = 5) (m) CD25 MFI after Raji stimulation in Ctrl or CUL5 KO-CD19CAR T cells. Data were expressed as mean ± SEM. (n = 3) (n) Phenotype analysis of Ctrl and CUL5 KO-CD19 CAR T cells for CD45RA and C-C chemokine receptor 7 (CCR7) on Day 30. o, p Percentages of CD45RA( − )-CCR7(−) effector memory T cells and CD45RA( − )-CCR7(+) central memory T cells after CD3/CD28 beads stimulation. q, r Percentages of CD45RA( − )-CCR(−) effector memory T cells and CD45RA( − )-CCR7(+) central memory T cells after Raji cell stimulation. s CCR7 MFI after Raji stimulation. (n = 5 in (o–s) Data from three to five different donors are acquired and each dot represents data from each donor. The paired two-sided Student’s t-test was used for (a–d, f–i, and o–s). The two-way ANOVA with the Šidák multiple comparisons test was used for (k and l). The multiple unpaired t-test was used for (m). Source data are provided as a Source Data file.

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