Extended Data Fig. 8: Delineating the effects of Chat on Tregs in HCC.
From: Tumor-specific cholinergic CD4+ T lymphocytes guide immunosurveillance of hepatocellular carcinoma
a Percentage of Foxp3+ Tregs among CD4+ T cells in HCC-bearing livers of Chatfl/fl and CD4-Cre;Chatfl/fl mice, representative of three independent experiments. Each dot represents an individual animal (n=11 mice in Chatfl/fl and n=12 mice in Chatfl/fl;CD4-Cre). b-d Representative flow cytometry plots (b), and quantification of the percentages of CTLA-4+ cells among Tregs (c) and Tconvs (d), in HCC-bearing livers from Chatfl/fl and Chatfl/fl;CD4-Cre mice on day 16 of HCC induction. In (c) and (d), each dot represents an individual mouse (n=6 mice per group). e Quantification of the percentages of induced CD25+ cells among Foxp3+CD4+ Tregs and Foxp3-CD4+ Tconvs isolated from purified splenic CD4+CD25- T cells of Chatfl/fl and Chatfl/fl;CD4-Cre mice. CD4+CD25- T cells were enriched with negative selection microbeads and stimulated with the indicated ratios of anti-CD3/28 microbeads. CD25 expression was analyzed by flow cytometry at the indicated time points. P values were determined by unpaired, two-tailed t-test, three replicates for each condition, representative of two independent experiments. f Representative flow cytometric plots of CD25+ Foxp3+ CD4+ Tregs in blood of Foxp3-YFP mice that were treated with either anti-CD25 antibody (α-CD25) or control IgG. Data are representative of 2 mice/group analyzed on day 18 of antibody treatment. g,h Percentage of CD4+ T cells (g) and NK cells (h) among MNCs from HCC-bearing livers of mice treated with anti-CD4 antibodies (α-CD4), anti-NK1.1 antibodies (α-NK1.1) or IgG/PBS control. Each dot represents an individual mouse (n=8 mice in the IgG/PBS group; n=9 mice in α-CD4 and α-NK1.1 groups). i,j Quantification of numbers of tumor nodules/mm2 in H&E-stained sections (i), and percentages of CD25+ Tregs in HCC-bearing livers (j), of Foxp3Cre and Chatfl/fl;Foxp3Cre mice on day 22 of HCC induction. Each dot represents an individual mouse. In (i), n=8 mice in the Foxp3Cre group and n=7 mice in the Chatfl/fl;Foxp3Cre group. In (j), n=8 mice in Foxp3Cre and n=6 mice in Chatfl/fl;Foxp3Cre. In (a), (c), (d), and (g-j), data are the mean ± SEM. P values were determined by unpaired, two-tailed t-test. Related to Figs. 6 and 7.
