Fig. 7: NOTCH1 activation is associated with T-cell proliferation in vivo. | Nature Communications

Fig. 7: NOTCH1 activation is associated with T-cell proliferation in vivo.

From: Viral transduction of primary human lymphoma B cells reveals mechanisms of NOTCH-mediated immune escape

Fig. 7

a Representative IHC staining of NOTCH1 in CLL lymph node biopsies. b Graphical scheme of the mass cytometry analysis from nuclear NOTCH1 negative (n = 3) or positive (n = 4) specimens. Created with BioRender.com. c Multiplexed IMC image example of a NOTCH1 positive specimen with an enlarged area of the proliferative center (PC). Representative t-SNE plots from an individual patient specimens are presented for CD19, CD4, and CD8 cell populations (n = 4). d Intensity of cellular signal per given cell was calculated using the HistoCat software. PD-L1 signal in CD19-gated cells in PC and non-PC areas (p < 0.0001), PD-L1 (p = 0.0079) and KI67 (p = 0.011) in CD19-gated PC-cells from samples with a positive or negative NOTCH1 nuclear staining. e Total CD4 signal, KI67, and PD-1 in CD4-gated cells in PCs of NOTCH1 positive or negative samples. f Total CD8 signal and PD-1 in CD8-gated cells in PCs of NOTCH1 positive or negative samples (**p = 0.0044). g Graphical scheme of the in vivo experiment. CLL cells were transduced with an empty vector or NOTCH1ΔPEST and intraperitoneally injected into male 8–10 week-old NSG mice. Autologous T cells were cultured with IL-2, α-CD3, and α-CD28 for 7 days prior to injection. Created with BioRender.com. h Engraftment of human CD19+ cells in the peritoneal cavity (left) and spleen (right) of mice injected with NOTCH1ΔPEST (n = 8) or an empty vector (n = 8) transduced CLL cells. In total 16 mice were analyzed using cells from 3 independent donors. Each symbol refers to an individual patient sample. i Quantification of human autologous CD4+/CD8+ T cells. Mean value was obtained from 3 independent experiment using different donor cells. Each symbol refers to an individual patient sample (p = 0.007). j Ratio of human CD4+/CD8+ T cells in the peripheral blood of a cohort of treatment-naive CLL patients with mutated (n = 13) or wild-type NOTCH1 (n = 34). Cohorts are shown as median (d–f) or mean ± SEM (h–j). Statistical analysis was done by paired (h, i) or unpaired t-tests (d–f, j) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and not significant (ns) P > 0.05.

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