Fig. 4: Tumor antigens drive clonal expansion of ChAT-expressing CD4⁺ T cells in HCC-bearing livers.

a,b, Circos plots showing the distribution of TCR types among GFP⁺ and GFP^– T cells from control (a) and HCC-bearing (b) mice. T cells of the same TCR type share TCRα and TCRβ chains of the same amino acid sequences. The top 30 TCRs are numbered and highlighted with different colors. c, CDR3 sequences of clonotypes encoding TCR 1. V, D and J segments and N nucleotides and P nucleotides at V(D)J junctions are denoted with different colors. Nucleotides that are mismatched between clonotypes are shaded. d, Composition of ChAT–GFP⁺ and ChAT–GFP^– cells among T cells bearing the indicated TCR 1 clonotypes color-coded by cell clusters. Each bar represents an individual clonotype and is labeled with the clone ID as shown in c. Horizontal axis labels indicate cell numbers. e, Percentage of ChAT–GFP⁺ cells among CD4⁺ T cells from normal or HCC-bearing livers of Chat-GFP or Chat-GFP; OT-II mice (n = 6 in the control, HCC and OT-II control groups; n = 5 in the OT-II HCC group). Statistical significance was assessed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test, and data are representative of three independent experiments. f,g, Representative flow cytometry plots (f) and quantification (g) of percentages of ChAT–GFP⁺ cells among CD4⁺ T cells expressing TCR Vβ5⁺ (transgenic TCR) or TCR Vβ5^– (natural TCRs) in livers of Chat-GFP; OT-II mice (n = 7 in control; n = 6 in HCC). h, Plasmids for simultaneous CRISPR–Cas9-mediated deletion of Trp53/Pten plus overexpression of Myc and tetracycline-on (Tet-On) inducible OVA. i, Experimental protocol for inducing OVA expression. Dox was added to drinking water following palpable HCC onset; FACS, fluorescence-activated cell sorting. j,k, Representative flow cytometry plots (j) and quantification (k) of percentages of ChAT–GFP⁺ cells among CD4⁺ T cells expressing TCR Vβ5⁺ or TCR Vβ5^– in livers of mice that were left untreated or treated with Dox-containing drinking water following HCC onset. Data are the summary of two independent experiments (n = 3 mice per group). In e, g and k, each dot represents an individual mouse. Data are shown as mean ± s.e.m. In g and k, P values were determined by two-way ANOVA with Sidak’s multiple comparisons test.

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