KDM6A depletion decreases MHC I expression in MEFs and decreases tumor immunogenicity in vivo. (A) Schematic of the protocol used to develop isogenic Kdm6a KO MEF from a C57BL/6J mouse homozygous for an allele of Kdm6a in which exon 3 was flanked with LoxP sites. (B) Heat map of z score for all expressed MHC I genes in Kdm6a WT and KO MEF. (C) Nlrc5 mRNA analysis by qPCR normalized to GAPDH (5 biological replicates; ± SD. Mann-Whitney t test). (D) Flow cytometry analysis of H-2kb in Kdm6a WT or KO MEFs (upper panel). MFI quantification of H-2kb surface expression from upper panel (5 biological replicates; ± SD, Mann-Whitney t test) (lower panel). (E) K-Ras-transformed WT and Kdm6a KO MEFs were injected into left and right flanks respectively of C57BL/6J and NOD-SCID mice and tumors excised and weighed after 3 weeks. The ratio of tumor weights from animal injected with Kdm6a-replete or Kdm6a-deficient K-Ras-transformed fibroblasts was calculated in immunocompetent C57BL/6J and immunodeficient NOD-SCID mice (8 biological replicates; ± SD, Mann-Whitney t test). (F) Flow cytometry quantifications of T cell, NK cells, and macrophage populations in the tumors isolated in panel E. (G) Spearman correlation between KDM6A and NLRC5 or CIITA gene expression and CD8⁺ T-cell infiltration in tumors isolated from various human cancers and correlation between KDM6A expression and T cell infiltrates as determined using Cistrome TIMER 2.0.³⁶