Fig. 4: WNT11 knockdown in tumor cells stimulates CD8⁺ T-cell proliferation and CD8⁺ T-cell-mediated tumor cell killing via AFF3.

a–d Flow cytometry analysis of the proliferation and apoptosis of CD8⁺ T-cells in MC38 (a, b) and Panc02 (c, d) liver metastases between scramble and shWnt11 group. e, f FACS analysis of CD8⁺ T-cell proliferation (CFSE low) in cocultures with MC38 (e) or Panc02 (f) scramble and shWnt11 cancer cells. g. Schematic representation of an in vitro T-cell killing assay. OVA-expressing tumor cells were cocultured in different ratios with CD8⁺ T-cells isolated from OVA-specific T-cell receptor transgenic (OT1) mice that had been preactivated using an OVA peptide and IL-2 plus IL-7 treatment. h Titration assay involving coculture at different effector-to-target ratios assessing the sensitivity to cytotoxic T-cell killing of MC38-OVA scramble, shWnt11, and shWnt11-shAff3 cells. i Alterations in AFF3 and β-catenin protein levels in MC38 cells following treatment with WNT11 and inhibitors of CAMKII, JNK, and PKC as indicated. j Knockdown of β-catenin abrogated the AFF3-induced increase in expression following Wnt11 knockdown. k, l Knockdown of WNT11 increased the nuclear translocation of β-catenin. m FACS analysis of CD8⁺ T-cell proliferation (CFSE low) in cultured with conditioned media derived from MC38 cells treated with WNT11 and CAMKII, JNK, and PKC inhibitors as indicated. n qPCR analysis of the expression of Camk2b in MC38 cells following CAMKII knockdown. o Aff3 mRNA levels were detected using qPCR in MC38 cells following CAMKII knockdown and WNT11 treatment as indicated. p FACS analysis of CD8⁺ T-cell proliferation (CFSE low) in cultured with conditioned media derived from MC38 cells with CAMKII knockdown and WNT11 treatment as indicated. q Titration assay involving coculture at different effector-to-target ratios assessing the sensitivity to cytotoxic T-cell killing of MC38-OVA scramble and shCamk2b cells with or without WNT11 treatment. b, d P-values were calculated using a Student’s t test (two-tailed), n = 5 biological replicates. e, f, m–p P-values were calculated using a one-way ANOVA followed by a Tukey’s multiple comparison test, n = 3 biological replicates. h, q P-values were calculated using a two-way ANOVA, n = 3 biological replicates. Data are presented as the mean ± SD. Source data are provided as a Source Data file.