Fig. 7. Inactivation of SOX4 gene inhibits emergence of MHC class I deficient TNBC cells during selection by cytotoxic T cells.

(A) Contour plots showing enrichment of HLA-ABC low/negative populations following a 24 h co-culture of SOX4^+/+ or SOX4^−/− BT549 human TNBC cells with human T cells expressing a NY-ESO-1 TCR at the indicated E:T ratios. (B) Quantification of HLA-ABC low/negative cells for indicated gene edited BT549 tumor populations following co-culture with CD8⁺ T cells as described in (A). Data are summarized as mean ± S.E.M. (C-D) BT549 tumor cells were pretreated with indicated concentrations of integrin αvβ6/8 mAb for 72 h and then co-cultured with CD8⁺ T cells. Contour (C) and summary (D) plots of HLA-ABC low/negative BT549 TNBC cells following co-culture with CD8⁺ T cells for 24 h at indicated E:T ratios. Isotype control Ab was used to define MHC-I negative populations. (E) Human BT549 TNBC cells expressing wild-type (WT) or low levels of HLA-ABC (HLA^low) were sorted and then pre-treated with indicated concentrations of integrin αvβ6/8 mAb followed by co-culture with NY-ESO-1 specific CD8⁺ T cells at the indicated E:T ratios. (F) BT549 TNBC cells were transduced with a doxycycline inducible SOX4 cDNA construct followed by FACS-based enrichment of HLA-ABC^high cells; tumor cells were then pre-treated for 48 h with the indicated concentrations (ng/ml) of doxycycline (DOX). Numbers of surviving wild-type (WT) or HLA-ABC^high tumor cells were quantified after co-culture with CD8⁺ T cells for 24 h. (G-J) Characterization of emergence of MHC class I deficient TNBC cells in vivo. (G) Contour plots showing expression of MHC-I (H-2K^b) in Py8119 tumors derived from mice treated with either control IgG Abs or integrin αvβ6/8 mAbs. (H) Quantification of MHC-I^low (H-2K^b) cells shown in (G), represented as a percentage of total cells (left) or as MFI (right). (I) mRNA levels of indicated genes relative to β-actin in sorted MHC-I^high (black) and MHC-I^low (red) murine TNBC cells derived from isotype control IgG treated tumors as shown in (G) or (J) in whole tumors from mice treated as described in (G). Data in [A-D, G-J] are representative of at least two independent experiments. Data in [E, F] are representative of three independent experiments. A two-way ANOVA with Dunnett’s post hoc test [B, D, E, and F] and an unpaired Student t-test [H-J] were used to determine significance, ***P < 0.001; **P < 0.01; *P < 0.05; n.s., not significant. See also Figure S7.