Fig 2: NF‐kB‐IL‐16 axis was involved in the chemotaxis and proliferation of T cells as well as Th1/Th2 differentiation induced by RBPJL overexpression. (a) Representative scatter plots compare the differences between EV and RBPJL and those between RBPJL and RBPJL (p.P476S) (red represents upregulation, blue represents downregulation, and grey represents no difference). (b) IL‐16 expression levels in CMs from EV, RBPJL and RBPJL (p.P476S) overexpression were measured and identified by quantitative measurement of 40 human cytokine arrays. (c) mRNA expression of IL‐16 was determined by reverse transcription‐polymerase chain reaction. (d) Chemotaxis of CD4+ and CD8+ T cells as analysed by transwell assay. IgG was used as a control for anti‐IL‐16. (e, f) The effect of CMs from KYSE150 cells on the proliferation of T cells was assessed by the loss of CFSE fluorescence. (g, h) Sorted naïve CD4+ T cells were stimulated with anti‐CD3/CD28 and human IL‐2 and incubated in the presence of CMs for 5 days, followed by staining with CD4, T‐bet and GATA3 antibodies for flow cytometry analysis. Percentages of CD4+ T‐bet+ and CD4+ GATA3+ T cells are presented in h. (i) Immunoblot analysis of phospho‐IκBα, total IκBα, phospho‐NF‐κB/p65 and total NF‐κB/p65 in KYSE150 and KYSE510 treated as indicated, and β‐actin was used as a loading control. The ratio of p‐IκBα to IκBα and that of p‐NF‐κB to NF‐κB were quantitated. Data are presented as mean ± standard deviation (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001. The data represent three independent technical replicates.

Fig 3: RBPJL (p.P476S) inhibited Th2/Th1 differentiation induced by RBPJL overexpression-derived CM. (a) Sorted naïve CD4+ T cells were stimulated by anti-CD3/CD28 and human IL-2, and subsequently incubated in the presence of CMs from cells overexpressing the four full-length and mutant genes for 5 days, followed by staining with CD4, T-bet and GATA3 antibodies for flow cytometry analysis. (b) Percentages of CD4+ T-bet+ and CD4+ GATA3+ T cells. (c) The levels of mRNA expression of IL-10, TGF-B1 and TNF-B mRNA in CD4+ T cells were determined by RT-PCR analysis. (d) IFN-? concentration in CD4+ T cells was determined by ELISA. Data in b–d are presented as mean ± standard deviation (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001. The data represent three independent technical replicates.

Fig 4: RBPJL (p.P476S) inhibited the therapeutic effects of RBPJL in a PBMCs‐CDX oesophagus cancer model in the presence/absence of toripalimab. (a) Representative flow cytometric plots of human CD45+CD3+CD8+, CD45+CD3+CD4+ T cells and mice CD45+CD3+ T cells in the blood of PBMC‐CDX NSG mice. Visual examination (b), weight (c) and volume (d) of isolated tumors in mice subcutaneously injected with KYSE150 cells and treated as indicated. (e) Representative immunohistochemistry images of CD4, CD8 and Ki67 are shown in randomly selected tumors. Scale bar, 200 μm. The number of positive cells from 3–5 fields was counted. (f) The average positive ratio from each section was identified. Data in c‐f are presented as mean ± standard deviation (n = 6), *P < 0.05, **P < 0.01. The data represent three independent technical replicates.

Fig 5: CM of RBPJL knockdown cells inhibited the chemotaxis and proliferation of T cells and Th1/Th2 differentiation induced by CM from RBPJL‐overexpressing cells. (a) Representative images of RBPJL and IL‐16 staining in responsive oesophagus and lung lesions as well as liver lesions. Scale bars represent 200 μm. The number of positive cells from 3 to 5 fields was counted. The average positive ratio from each section was identified. (b) The chemotaxis of CD4+ and CD8+ T cells as analysed by transwell assay. (c) Effects of CMs from KYSE150 cells on the proliferation of T cells were assessed by loss of CFSE fluorescence. (d) Sorted naïve CD4+ T cells were stimulated by anti‐CD3/CD28 and human IL‐2, and subsequently incubated in the presence of CMs for 5 days, followed by staining with CD4, T‐bet and GATA3 antibodies for flow cytometry analysis. Percentages of CD4+ T‐bet+ and CD4+ GATA3+ T cells are shown. Data are presented as the mean ± standard deviation (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001. The data represent three independent technical replicates.