Fig. 6: Combined targeting of T and COTL1^high NK cells enhances immunotherapy responses in tsMHC-I^impaired HCC.

a, A schematic illustration showing Hep-53.4-bearing mice treated with DTA-1, αPD-L1, combination or isotype control. b, The representative images and statistical results of whole-liver morphology from Hep-53.4-bearing orthotopic HCC model (each n = 5; isotype versus αPD-L1, P = 0.0079, DTA-1, P = 0.0079, DTA-1 + αPD-L1, P = 0.0079; αPD-L1 versus DTA-1, P = 0.4365, DTA-1 + αPD-L1, P = 0.0238). The dashed white lines mark the tumour boundary. c, The flow cytometry analysis of IFN-γ production in CD49a⁺CD49b⁻ NK cells (each n = 5; isotype versus αPD-L1, P = 0.0079, DTA-1, P = 0.0079, DTA-1 + αPD-L1, P = 0.0079; αPD-L1 versus DTA-1, P = 0.0556, DTA-1 + αPD-L1, P = 0.0079). d, The flow cytometry analysis of IFN-γ production in CD8⁺ T cells (each n = 5; isotype versus αPD-L1, P = 0.0079, DTA-1, P = 0.0317, DTA-1 + αPD-L1, P = 0.0079; αPD-L1 versus DTA-1, P = 0.0952, DTA-1 + αPD-L1, P = 0.1508). e, The flow cytometry analysis of T_reg frequencies (each n = 5; isotype versus αPD-L1, P = 0.0556, DTA-1, P = 0.0079, DTA-1 + αPD-L1, P = 0.0079; DTA-1 + αPD-L1 versus αPD-L1, P = 0.1508, DTA-1, P = 0.0079). f, The mean fluorescence intensity of MHC-I in Hep-53.4-implanted tumours from WT mice determined by mean values of five random FOVs (each n = 4, isotype versus αPD-L1, P = 0.0286, DTA-1, P = 0.0286, DTA-1 + αPD-L1, P = 0.0286; DTA-1 + αPD-L1 versus αPD-L1, P = 0.0286, DTA-1, P = 0.0286; αPD-L1 versus DTA-1, P = 0.6857). g, A schematic illustration showing Hep-53.4-bearing Zfp683^+/+ or Zfp683^−/− mice treated with αPD-L1 or combination with DTA-1. h, Representative images of whole-liver morphology from WT or Zfp683^−/− mice treated with αPD-L1 or combination with DTA-1. The dashed white lines mark the tumour boundary. i, The statistical results of maximum tumour diameter from WT or Zfp683^−/− mice treated with αPD-L1 or combination with DTA-1 (each n = 5, Zfp683^+/+–αPD-L1 versus Zfp683^+/+–αPD-L1 + DTA-1, P = 0.0476, Zfp683^−/−–αPD-L1, P = 0.0635; Zfp683^−/−–αPD-L1 + DTA-1 versus Zfp683^−/−–αPD-L1, P = 0.8492, Zfp683^+/+–αPD-L1 + DTA-1, P = 0.0079). j, The representative images and statistical results showing the average number of CD8⁺Granzyme B⁺ cells of five FOVs (500 μm × 500 μm) in Hep-53.4 hepatoma (each n = 4, each mouse five FOVs; Zfp683^+/+–αPD-L1 versus Zfp683^+/+–αPD-L1 + DTA-1, P = 0.0286, Zfp683^−/−–αPD-L1, P = 0.0286; Zfp683^−/−–αPD-L1 + DTA-1 versus Zfp683^−/−–αPD-L1, P = 0.4857, Zfp683^+/+–αPD-L1 + DTA-1, P = 0.0286). k, A schematic illustration showing NK-deficient mice receiving intratumoural injection of sgCtrl or sgTnfrsf18 NK cells. l, Representative images and statistical results of whole-liver morphology from NK-deficient mice receiving an intratumoural injection of sgCtrl or sgTnfrsf18 NK cells (each n = 6; P = 0.0043). Scale bar, 1 cm. The dashed white lines mark the tumour boundary. m, The tumour tissues from five patients with MHC-I^impaired HCC were collected and rapidly dissected into clusters that were treated with or without autologous PBMC-induced COTL1^high NK cells plus atezolizumab or ragifilimab for 3 days (Methods). n, The representative immunostaining of EVOCs using cleaved caspase 3 and Ki-67 staining was performed. Scale bars, 100 μm. o, The quantitation results are shown (n = 4 FOVs for each five patients; Ki-67, NC versus αPDL1 + COTL1^high NK, P = 0.4291, GITR agonist + COTL1^high NK, P = 0.0024, αPDL1 + GITR agonist + COTL1^high NK, P < 0.0001; cleaved caspase 3, NC versus αPDL1 + COTL1^high NK, P = 0.4020, GITR agonist + COTL1^high NK, P = 0.4612, αPDL1 + GITR agonist + COTL1^high NK, P = 0.0026). p, The representative immunostaining of EVOCs using Pan-CK and HLA-ABC staining was performed. Scale bars, 150 μm. The quantitation results are shown (each n = 5, NC versus αPDL1 + GITR agonist + COTL1^high NK, P = 0.0079). P values were determined by unpaired two-sided Mann–Whitney test in c–f, i, j, l, o and p. Data are shown as the mean ± s.e.m. In the box plots, the centre line is the median, box limits are the first and third quartiles and whiskers are 1.5× interquartile range. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant. Ab, antibody. NC, negative control. Icons in m created in BioRender; You, W. https://biorender.com/7uda7v9 (2026).

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