Supplementary Figure Legends s6

Supplementary Figure Legends

Inflammation-dependent IL-18 signaling restricts hepatocellular carcinoma growth by enhancing the accumulation and activity of tumor-infiltrating lymphocytes

Geoffrey J. Markowitz, Pengyuan Yang, Jing Fu, Gregory A. Michelotti, Rui Chen, Jianhua Sui,

Bin Yang, Wen-Hao Qin, Zheng Zhang, Fu-sheng Wang, Anna Mae Diehl, Qi-Jing Li,

Hongyang Wang, and Xiao-Fan Wang*

Contains Legends for Supplementary Figures S1-S19.

1 SUPPLEMENTARY FIGURE LEGENDS

Supplementary Fig. S1. Stratification of patients based on IL-18 staining density in intratumoral and peritumoral tissue. Intratumoral (A) and peritumoral (B) tissue samples were immunohistochemically stained for IL-18, and staining density quantified and resultant values plotted on dot plots. Stratification thresholds for highest third, middle third, and lowest third of values are indicated by dotted lines on each plot. (C, D) Representative stains for highest, middle, and lowest density stains for intratumoral (C) and peritumoral (D) tissue at 400X magnification. Statistics: one-way ANOVA with Tukey’s multiple comparisons test, n=46 matched intratumoral and peritumoral samples per group. ****P<0.0001.

Supplementary Fig. S2. Efficient fibrosis induction by carbon tetrachloride and slightly modulated fibrosis burden between WT and IL18R1-/- mice. (A, B) Circulating alanine aminotransferase (A) and circulating aspartate aminotransferase (B) were measured by a colorimetric activity assay in blood harvested at sacrifice from mice receiving either a sham matrigel plug or the Hepa1-6 cell line expressing a GFP-luciferase vector implanted into wild- type or IL18R1-deficient mice on the C57Bl/6 background treated with olive oil or carbon tetrachloride. Gray regions indicate normal ranges for circulating ALT and AST in C57Bl/6 mice. (C, D) Representative alpha-smooth muscle actin staining for activated hepatic stellate cells (C) and Sirius red staining for collage deposition (D) in wild-type or IL18R1-deficient mice on the C57Bl/6 background treated with olive oil or carbon tetrachloride. Statistics: (A, B) one- way ANOVA with Tukey’s multiple comparisons test, n=6-14 per cell-implanted group pooled from 2 experiments. NS non-significant; *P<0.05; **P<0.01; ****P<0.0001.

Supplementary Fig. S3. Multiple cell types produce IL-18 and IL18R1 in HCC. (A-D) Immunohistochemical staining for IL-18 was performed on tumor samples generated in mice via

CCl4 treatment and orthotopic implantation (A), diethylnitrosamine treatment (B), bile duct ligation and orthotopic implantation (C), and diethylnitrosamine and CCl4 treatment (D). Images from two representative fields are shown at 160X magnification. (E) Mice were treated with + + + + + CCl4 and orthotopic tumor implantation, and TCR CD4 T-cells, TCR CD8 T-cells, NK1.1 NK cells, CD31+ endothelial cells (ECs), GFP+ Hepa1-6 tumor cells, CD11b+ myeloid cells, CD11c+ myeloid cells, and CD11b+ CD11c+ myeloid cells were sorted from harvested tumors, mRNA extracted and reverse transcribed, and probed for expression of IL-18 and IL18R1. Relative expression was calculated by normalization to the average expression level of these transcripts in tumor-associated endothelial cells, which express a moderate level of both of these transcripts. Statistics: (B) n=2-5 per cell type; for NK cells, due to limited numbers of cells, sorted cells from 2 or 3 mice were pooled to make a sample.

Supplementary Fig. S4. Enhanced neutrophil-to-lymphocyte ratios and decreased circulating lymphocytes in tumor-bearing IL18R1-/- mice. Complete blood counts were performed on blood harvested at sacrifice from naïve wild-type or IL18R1-deficient mice on the

2 C57Bl/6 background or from mice of these genotypes treated with carbon tetrachloride and receiving orthotopic implantation of the Hepa1-6 cell line expressing a GFP-luciferase vector. Total blood counts as thousands of cells per microliter (A) and the percentages of total counts made up by each population (B) were plotted. Statistics: multiple t-tests, n=8-11 per group for carbon tetrachloride-treated mice pooled over 3 experiments, n=4-6 per group for naïve mice pooled over 2 experiments. *P<0.05; **P<0.01; ***P<0.001.

Supplementary Fig. S5. NK and NKT cells in liver and tumor are not significantly different between WT and IL18R1-/- mice. (A) Representative flow cytometry contour dot plots of non- tumor-liver- and tumor-infiltrating NK1.1+ and TCR+ cells in wild-type (left) and IL18R1- deficient (right) mice treated with carbon tetrachloride and tumor implantation. (B) Quantification of NK1.1+, NK1.1+ TCR+, and TCR+ cells in non-tumor-liver tissue from wild- type and IL18R1-deficient mice in (A). (C) Quantification of NK1.1+, NK1.1+ TCR+, and TCR+ cells in tumor tissue from wild-type and IL18R1-deficient mice in (A). Statistics: (B, C) one-way ANOVA with Sidak’s multiple comparisons test, n=9-12 per group for non-tumor liver samples pooled from 3 experiments, n=12-15 per group for tumor samples pooled from 4 experiments. NS non-significant.

Supplementary Fig. S6. Myeloid cell accumulation in liver and tumor are not significantly different between WT and IL18R1-/- mice. (A) Representative flow cytometry contour dot plots of non-tumor-liver and tumor-infiltrating CD11b+ and TCR+ cells in wild-type (left) and IL18R1-deficient (right) mice treated with carbon tetrachloride and tumor implantation. (B) Representative flow cytometry contour dot plots of Gr-1 expression on CD11b+ non-tumor-liver- and tumor-infiltrating cells in wild-type (left) and IL18R1-deficient (right) mice treated with carbon tetrachloride and tumor implantation. (C) Quantification of CD11b+, CD11b+ Gr-1bright, and CD11b+ Gr-1medium cells in non-tumor-liver tissue from wild-type and IL18R1-deficient mice in (A) and (B). (D) Quantification of CD11b+, CD11b+ Gr-1bright, and CD11b+ Gr-1medium cells in tumor tissue from wild-type and IL18R1-deficient mice in (A) and (B). Statistics: (C, D) one- way ANOVA with Sidak’s multiple comparisons test, n=4 per group for non-tumor liver samples, n=6 per group for tumor samples pooled from 2 experiments. NS non-significant.

Supplementary Fig. S7. Quantification of NK and T-cell populations in WT and IL18R1-/- mice. Quantification of NK1.1+, NK1.1+ TCR+, TCR+, TCR+CD4+, TCR+CD8+, and TCR+ CD4+ CD25+ was performed as a percentage of events gated on Forward Scatter (Area) and Side Scatter (Area) for size exclusion for live cells and Forward Scatter (Area) and Forward Scatter (Height) for singlet selection in non-tumor-liver tissue (A) and tumor tissue (B). Statistics: (A, B) multiple t-tests, n=13-16 per group pooled over 4 experiments for non-tumor-liver tissue and n=20-23 per group pooled over 6 experiments for tumor tissue. *P<0.05; **P<0.01; ***P<0.001.

Supplementary Fig. S8. Representative flow cytometry of T-cell populations in non-tumor liver, tumor, and spleen in WT and IL18R1-/- mice. (A-C) Representative flow cytometry

3 contour dot plots of TCR+ CD8+ and TCR+ CD4+ cells in wild-type (left) and IL18R1-deficient (right) mice treated with carbon tetrachloride and tumor implantation in non-tumor-liver tissue (A), tumor (B), and spleen (C).

Supplementary Fig. S9. Alternative tumor models yield similar skewing of T-cell populations in the tumor by loss of IL18R1. (A) Representative flow cytometry contour dot plots of TCR+ tumor-infiltrating CD8+ and CD4+ cells in wild-type (left) and IL18R1-deficient (right) mice treated with bile duct ligation and tumor implantation. (B) Quantification of TCR+, TCR+CD8+, and TCR+ CD4+ cells in tumor tissue from (A). (C) Representative flow cytometry contour dot plots of TCR+ tumor-infiltrating CD8+ and CD4+ cells in wild-type (left) and IL18R1-deficient (right) mice treated with diethylnitrosamine (DEN) and carbon + tetrachloride (CCl4) to induce inflammatory tumorigenesis. (D) Quantification of TCR , TCR+CD8+, and TCR+ CD4+ cells in tumor tissue from (C). Statistics: (B, D) one-way ANOVA with Sidak’s multiple comparisons test, n=7-8 per group for bile duct ligation samples pooled from 2 experiments, n=9 per group for DEN + CCl4 samples pooled from 2-3 experiments per genotype. NS non-significant; *P<0.05; **P<0.01; ***P<0.001.

Supplementary Fig. S10. Slightly increased proportions of CD44+ T-cells in IL18R1-/- mice compared to WT. (A, B) Representative flow cytometry contour dot plots of TCR+ CD8+ (A) and TCR+CD4+ (B) splenic CD44+ cells in wild-type (left) and IL18R1-deficient (right) mice. (C) Quantification of TCR+CD8+ CD44+ and TCR+ CD4+ CD44+ cells in spleens from (A) and (B). (D, E) Representative flow cytometry contour dot plots of TCR+ CD8+ (D) and TCR+CD4+ (E) splenic CD44+ cells in wild-type (left) and IL18R1-deficient (right) mice treated with carbon tetrachloride and tumor implantation. (F) Quantification of TCR+CD8+ CD44+ and TCR+ CD4+ CD44+ cells in spleens from (D) and (E). Statistics: (C, F) one-way ANOVA with Sidak’s multiple comparisons test, n=4-5 per group for naïve samples pooled from 2 experiments, n=6-8 per group for carbon tetrachloride samples pooled from 2 experiments. NS non-significant; *P<0.05; **P<0.01.

Supplementary Fig. S11. No significant changes in CD4+ CD25+ cells, CD4+ FoxP3+ cells, or CD4+ CD25+ FoxP3+ cells. (A) Representative flow cytometry contour dot plots of CD25 and FoxP3 expression in TCR+ CD4+ tumor-infiltrating cells in wild-type (left) and IL18R1- deficient (right) mice treated with carbon tetrachloride and tumor implantation. (B) Quantification of CD25+, FoxP3+, and CD25+ FoxP3+ cells in tumor tissue from (A). Statistics: (B) one-way ANOVA with Sidak’s multiple comparisons test, n=14-16 per group pooled from 4 experiments. NS non-significant.

Supplementary Fig. S12. Modestly decreased PD-1 in CD8+ T cells in IL18R1-/- mice compared with WT mice. (A, B) Representative flow cytometry contour dot plots of PD-1 expression on TCR+ CD4+ (A) and TCR+ CD8+ (B) tumor-infiltrating cells in wild-type (left) and IL18R1-deficient (right) mice treated with carbon tetrachloride and tumor implantation. (C)

4 Quantification of PD-1+ cells in TCR+ CD4+ and TCR+ CD8+ cells in tumor tissue from (A) and (B). (D) Quantification of the mean fluorescence intensity of PD-1 in TCR+ CD4+ and TCR+ CD8+ cells in tumor tissue from (A) and (B). (E, F) Representative flow cytometry contour dot plots of CTLA-4 expression on TCR+ CD4+ (E) and TCR+ CD8+ (F) tumor- infiltrating cells in wild-type (left) and IL18R1-deficient (right) mice treated with carbon tetrachloride and tumor implantation. (G) Quantification of CTLA-4+ cells in TCR+ CD4+ and TCR+ CD8+ cells in tumor tissue from (E, F). (H) Quantification of the mean fluorescence intensity of CTLA-4 in TCR+ CD4+ and TCR+ CD8+ cells in tumor tissue from (E, F). Statistics: (C, D, G, H) unpaired T-tests, n=3-4 per group for PD-1, n=11-13 per group for CTLA-4 pooled from 3 experiments. NS non-significant; *P<0.05; **P<0.01.

Supplementary Fig. S13. Slight differences in cytokine production, no differences in granzyme production in T-cells between WT and IL18R1-/- mice. (A-F) Hepa1-6 cells expressing a GFP-luciferase vector were implanted into wild-type or IL18R1-deficient C57Bl/6 + + + + mice treated with CCl4. Tumor-infiltrating TCR CD4 and TCR CD8 cells in wild-type and IL18R1-deficient mice were stained for: TNF and IL-2 (A-C) or GzmA and GzmB (D-F). Single- and double-stained populations were quantified as percentages of TCR+ CD4+ and TCR+ CD8+ cells. Statistics: (A-F) one-way ANOVA with uncorrected Fisher’s LSD test, n=11- 13 per group pooled over 3 experiments for (A-C), n=8-9 per group pooled over 2 experiments for (D-F). NS non-significant.

Supplementary Fig. S14. Representative flow cytometry of differentiation and cytokine production of CD4+ T-cells from WT and IL18R1-/- mice. (A) Representative flow cytometry contour dot plots of IFN production from wild-type and IL18R1-deficient Th1-differentiated CD4+ T-cells stimulated with different doses of IL-18. (B) Representative flow cytometry contour dot plots of IL-17a production from wild-type and IL18R1-deficient Th17-differentiated CD4+ T-cells stimulated with IL-18.

Supplementary Fig. S15. Representative flow cytometry of WT and IL18R1-/- CD8+ T-cell survival and proliferation upon reactivation and stimulation. (A) Representative flow cytometry contour dot plots of 7AAD surface staining and CellTrace Violet dilution from wild- type and IL18R1-deficient CD8+ T-cells upon reactivation and stimulation with different doses of IL-18 with or without tumor conditional media (TCM). Surviving cells are defined as 7AADloCellTraceVioletlo and are the boxed regions on the plot. (B) Representative flow cytometry histograms of CellTrace Violet dilution from wild-type and IL18R1-deficient CD8+ T- cells upon reactivation and stimulation with different doses of IL-18 with or without TCM.

Supplementary Fig. S16. Tumor-infiltrating NK cells have altered cytokine and transcription factor production between WT and IL18R1-/- mice. (A-L) Hepa1-6 cells expressing a GFP-luciferase vector were implanted into wild-type or IL18R1-deficient C57Bl/6 + - mice treated with CCl4. Tumor-infiltrating NK1.1 TCR cells in wild-type and IL18R1-

5 deficient mice were stained for: IFN and IL-17a (A-C); TNF and IL-2 (D-F); T-bet and Eomes (G-I); or GzmB and GzmA (J-L). Single- and double-stained populations were quantified as percentages of NK1.1+ TCR- cells. Statistics: (A-L) unpaired T-tests, n=11-13 per group for (A-F) pooled from 3 experiments, n=8-9 per group for (G-L) pooled from 2 experiments. NS non-significant; *P<0.05; **P<0.01.

Supplementary Fig. S17. Representative immunohistochemistry showing intratumoral, peritumoral, and marginal tissue stained for IL-18 from patients. Representative intratumoral (400X magnification), marginal (100X magnification), and peritumoral staining (400X magnification) for IL-18 from three patients. Sample 4: low intratumoral density, low peritumoral density, middle difference in staining density; Sample 5: low intratumoral density, low peritumoral density, middle difference in staining density; Sample 6: low intratumoral density, middle peritumoral density, middle difference in staining density.

Supplementary Fig. S18. Differences in IL-18 staining density between intratumoral and peritumoral tissue are more closely linked to peritumoral staining density. Staining density quantified from each patient’s tumor tissue was subtracted from staining density in the matched peritumoral tissue, and the resultant values plotted on dot plots (left panel). Stratification thresholds for highest third of differences, middle third of differences, and lowest third of differences are indicated by dotted lines on each plot. Resultant values were alternatively grouped based on groups of thirds of patients stratified based on intratumoral IL-18 staining (top right panel) or peritumoral IL-18 staining (bottom right panel) identified in Supplementary Fig. S1. Statistics: one-way ANOVA with Tukey’s multiple comparisons test, n=46 matched peritumoral and intratumoral samples per group. NS non-significant; ****P<0.0001.

Supplementary Fig. S19. No difference in survival based on expression of IL18R1 in patient samples. (A, B) Kaplan-Meier curves for overall survival (left) and disease-free survival (right) of the cohort of 138 patients stratified into the highest third, middle third, or lowest third of patients based on IL18R1 staining density in tumor tissue (A) or in peritumoral tissue (B). (C) Kaplan-Meier curves for overall survival (left) and disease-free survival (right) of the cohort of 138 patients stratified into the highest third, middle third, or lowest third of patients based on differences in IL18R1 staining density, calculated in a similar fashion as used in Supplementary Fig. S14 and Fig. 7. Statistics: (A-C) Log-rank Mantel-Cox tests, n=46 per group.