Extended Data Fig. 2: The variation of tumor MHC class I reshapes immune infiltration of HCC.

a, Flow cytometry histogram showing the protein expression of MHC-I (H-2K^b/H-2D^b) on vector and B2m^OE Hep-53.4 cells. b, Schematic representing the construction of a mouse model bearing vector and B2m^OE Hep-53.4 hepatoma. c, Representative images and statistical results of whole-liver morphology from vector and B2m^OE Hep-53.4-bearing orthotopic HCC models (each n = 5, P = 0.0159). Scale bars, 1 cm. d, Flow cytometry analysis of tumor-infiltrating T and NK cells in vector and B2m^OE Hep-53.4-bearing orthotopic HCC tumors (each n = 5; T cells, P = 0.0079; NK cells, P = 0.0159). e, Representative mIHC staining and statistical results showing the number of NKp46⁺ and CD8⁺ cells per FOV (500 μm * 500 μm) in vector and B2m^OE Hep-53.4 hepatoma (each n = 5, each mouse five FOVs, T cells, P < 0.0001; NK cells, P < 0.0001). Scale bars, 50 μm. f, Flow cytometry histogram showing the protein expression of MHC-I (H-2K^b/H-2D^b) on Hepa1-6 cells. g, Flow cytometry histogram showing the protein expression of MHC-I (H-2K^b/H-2D^b) on sgCtrl and sgB2m Hepa1-6 cells. h, Schematic representing the construction of a mouse model bearing Hepa1-6 hepatoma with variable MHC-I expression backgrounds. i, Representative images and statistical results of whole-liver morphology from Hepa1-6-bearing orthotopic HCC models with variable MHC-I expression (each n = 6; sgCtrl: sgB2m 1:0 versus 1:1, P = 0.6190, 0:1, P = 0.0087; 1:1 versus 0:1, P = 0.0130). Scale bars, 1 cm. j, Representative mIHC staining of NKp46 and CD8 in WT, WT/B2m KO mixed (1:1) and B2m KO Hepa1-6 bearing orthotopic HCC tumors (CD8⁺ T: sgCtrl: sgB2m 1:0 versus 1:1, P = 0.6834, 0:1, P = 0.0728; 1:1 versus 0:1, P = 0.0005; NK cell: sgCtrl: sgB2m 1:0 versus 1:1, P = 0.0016, 0:1, P = 0.2236; 1:1 versus 0:1, P = 0.0051). Scale bars, 50 μm. k, Statistical results showing the number of NKp46⁺ and CD8⁺ cells per FOV (650 μm * 650 μm) in CD8 in WT, WT/B2m KO mixed (1:1) and B2m KO Hepa1-6 bearing orthotopic HCC tumors (each n = 5 and each mouse four FOVs; NK cell: sgCtrl: sgB2m 1:0 versus 1:1, P = 0.0152, 0:1, P = 0.3095; 1:1 versus 0:1, P = 0.0043; T cell: sgCtrl: sgB2m 1:0 versus 1:1, P > 0.9999, 0:1, P = 0.0022; 1:1 versus 0:1, P = 0.0022). l, Representative flow cytometry images of depleting NK, CD8⁺ T and both in orthotopic Hep-53.4 tumors. m, Representative images of whole-liver morphology from Hep-53.4-bearing orthotopic HCC models treated with isotype antibody, anti-asialo GM1, anti-CD8 and combination. Scale bars, 1 cm. n, Statistical results of maximum tumor diameter in Hep-53.4-bearing orthotopic HCC models treated with isotype antibody, anti-asialo GM1, anti-CD8 and combination (each n = 5; Isotype versus anti-asialo GM1, P = 0.0079, anti-CD8, P = 0.0079, anti-asialo GM1+ anti-CD8, P = 0.0079; anti-asialo GM1 versus anti-CD8, P = 0.4365, anti-asialo GM1+ anti-CD8, P = 0.5714). o, Flow cytometry analysis of representative cases of CD49⁺ (COTL1^high) NK cells in human HCC tumors between over 80% tsMHC-I⁺ fraction and less than 80% tsMHC-I⁺ fraction. Frequencies of CD49⁺ (COTL1^high) NK cells in relative to CD45⁺ immune cells (n = 11 vs 13; P = 0.0030). p, Integration analysis of 101 HCC tumors showed the distribution of COTL1^high NK cells between over 80% tsMHC-I⁺ fraction and less than 80% tsMHC-I⁺ fraction (P = 0.0380). q, Label transfer analysis of our data and GSE235863 (ICB-treated patients) by symphony R package showing COTL1^high NK cells have a higher fraction in responders. P values were determined by unpaired two-sided Mann-Whitney tests (c-e, i-k and n-p). Data are shown as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

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