Extended Data Figure 9: The response to PD-L1 blockade is dependent on CD8⁺ T cells and clonal expansion of HCC-directed CD8⁺ T cells.

MUP-uPA and MUP-uPA/Iga^−/− mice were placed on HFD and treated with anti-PD-L1, as described in Extended Data Fig. 2a. a, At the end of the treatments, mouse weights were measured (n = 3, 2, 4, 3, 3, 7, 3, 7). b, MUP-uPA mice treated with anti-PD-L1 were analysed for serum IgA by ELISA (n = 10, 6). c, Liver/body weight ratio of indicated strains kept on HFD that received the indicated treatments and were of the indicated ages (6 months, 11 months) (n = 4, 6, 9, 19, 6, 2, 4, 15, 11, 17, 3, 14, 7, 20). d, e, Paraffin-embedded and frozen liver sections from HCC-bearing MUP-uPA mice were stained with Oil Red O, haematoxylin and eosin, or Sirius Red and analysed (n = 4 or 5). The experiments were repeated at least two times. Low and high magnifications are shown in e to demonstrate the absence of tumour-invading immune cells in a mouse that failed to respond to anti-PD-L1 and their presence within a tumour of a treatment responsive mouse. Non-responsiveness to anti-PD-L1 treatment correlates with a fibrotic tumour stroma. Scale bars: haematoxylin and eosin, 250 μm; Sirius Red, 250 μm; Oil Red O, 50 μm. f, Response to PD-L1 blockade is dependent on CD8⁺ T cells. HCC-bearing MUP-uPA/Cd8a^−/− mice (n = 3) were treated with anti-PD-L1 for 8 weeks and tumour multiplicity was determined. g, Two-dimensional plot showing the frequency of the top ten TCRα CDR3 sequences expressed by CD8⁺ T cells from spleens and livers of HCC-bearing MUP-uPA mice. h, CD8⁺ T cells were sorted from spleens and livers of normal-chow- and HFD-fed WT mice (n = 5 mice), their RNA was extracted, and TCR α-chain (top) and β-chain (bottom) CDR3 sequences were amplified and analysed by deep sequencing (ten samples). The panels show the clonality, the frequency of the top 50 TCR α- and β-chain sequences, and the percentage of productive unique TCR α- and β-chain sequences. i–k, CD8⁺ T cells were sorted from spleens and livers of HCC-bearing mice of the indicated strains and treatments (n = 13 mice, 26 samples as indicated) and their TCR α- and β-chain CDR3 sequence diversity was analysed. Shown are the clonality (i), the diversity (inverse Simpson’s index) (j), and percentage of productive unique TCR β-chain sequences in the indicated strains (k). Note that TCR sequencing data of WT, MUP-uPA, and MUP-uPA/Iga^−/− mice are also shown in Fig. 4g, h and Extended Data Fig. 9h. l–n, Splenocytes from the indicated mice treated with or without anti-PD-L1 were stimulated overnight with alpha-fetoprotein, stained as indicated, and analysed by flow cytometry. Shown are the percentage of IFNγ⁺CD107a⁺ cells (n = 3, 3, 2, 5, 3, 2, 3, 4, 4) (l) and IFNγ⁺TNF⁺ cells (n = 4, 3, 5, 4) (m) gated on CD8⁺ T cells. n, The percentage of IFNγ⁺TNF⁺ cells gated on CD4⁺ T cells (n = 3, 3, 2, 4, 3, 2, 3, 6, 4). Two-sided t-test (a–d, f, h–n) and Mann–Whitney test (l) were used to determine significance. *P < 0.05; **P < 0.01; ***P < 0.001. N values for each group in each panel are provided from left to right accordingly.