Fig 2: Peripheral non-specific and HBV-specific Th9/Tc9 cells in hepatitis B-related hepatocellular carcinoma (HCC). (A) Peripheral blood mononuclear cells (PBMCs) were isolated from all enrolled subjects, including normal controls (NC, n=11), chronic hepatitis B (CHB) patients (n=27), and hepatitis B-related HCC patients (n=22). PBMCs were stimulated with either phorbol myristate acetate (50 ng/ml)+ionomycin (1 µg/ml) (for non-specific stimulation) or HBsAg (for HBV-specific stimulation) in the presence of brefeldin A (10 µg/ml) for 6 hours, and were then stained with anti-CD3, anti-CD4, anti-CD8, and anti-IL-9. CD3+CD4+IL-9+ Th9 cells and CD3+CD8+IL-9+ Tc9 cells were analyzed by flow cytometry. The representative flow dots for non-specific and HBV-specific Th9 and Tc9 cells in NC, CHB, and hepatitis B-related HCC patients were shown. (B) The percentage of peripheral non-specific Th9 cells was compared among NC, CHB, and hepatitis B-related HCC patients. Significance was assessed using one-way ANOVA and SNK-q test. (C) The percentage of peripheral HBV-specific Th9 cells was compared between CHB and hepatitis B-related HCC patients. Significance was assessed using Student t test. (D) The percentage of peripheral non-specific Tc9 cells was compared among NC, CHB, and hepatitis B-related HCC patients. Significance was assessed using one-way ANOVA and SNK-q test. (E) The percentage of peripheral HBV-specific Tc9 cells was compared between CHB and hepatitis B-related HCC patients. Significance was assessed using Student t test.

Fig 3: Liver-infiltrating non-specific and HBV core-specific Th9/Tc9 cells in hepatitis B-related hepatocellular carcinoma (HCC). Intrahepatic lymphocytes (IHLs) were isolated from fresh HCC specimens and non-tumor site liver specimens in thirteen HCC patients (seven in stage A and six in stage B) who underwent hepatic carcinectomy. IHLs were stimulated with either phorbol myristate acetate (50 ng/ml)+ionomycin (1 µg/ml) (for non-specific stimulation) or HBsAg (for HBV-specific stimulation) in the presence of brefeldin A (10 µg/ml) for 6 hours, and were then stained with anti-CD3, anti-CD4, anti-CD8, and anti-IL-9. CD3+CD4+IL-9+ Th9 cells and CD3+CD8+IL-9+ Tc9 cells were analyzed by flow cytometry. (A) The percentage of liver-infiltrating non-specific Th9 cells was compared between non-tumor site and tumor site. Significance was assessed using Student t test. (B) The percentage of liver-infiltrating HBV-specific Th9 cells was compared between non-tumor site and tumor site. Significance was assessed using Student t test. (C) The percentage of liver-infiltrating non-specific Tc9 cells was compared between non-tumor site and tumor site. Significance was assessed using Student t test. (D) The percentage of liver-infiltrating HBV-specific Tc9 cells was compared between non-tumor site and tumor site. Significance was assessed using Student t test.

Fig 4: IL-9 level in hepatitis B-related hepatocellular carcinoma (HCC). (A) Serum IL-9 concentration was measured by ELISA in normal controls (NC, n=11), chronic hepatitis B (CHB) patients (n=27), and hepatitis B-related HCC patients (n=22). Significance was assessed using one-way ANOVA and SNK-q test. (B) Serum IL-9 level was compared among hepatitis B-related HCC patients in BCLC stage A (n=7), stage B (n=7), stage C (n=5), and stage D (n=3). Significance was assessed using one-way ANOVA. (C) Serum IL-9 was also compared between hepatitis B-related HCC patients with cirrhosis (n=14) and without cirrhosis (n=8). Significance was assessed using Student t test. (D) Serum IL-9 was also measured in hepatitis B-related HCC patients who underwent hepatic carcinectomy (n=13) or TACE (n=9), and was compared between baseline and 2 months post therapy. The dotted line presented lower detection limit for IL-9 (3.9 pg/mL). Significance was assessed using paired t test.

Fig 5: Influence of Th9 cells to non-specific and HBV-specific CD8+ T cells in hepatitis B-related hepatocellular carcinoma (HCC). 104 of peripheral CD8+ T cells from normal controls (NC, n=6), chronic hepatitis B (CHB) patients (n=13), and hepatitis B-related HCC patients (n=10) were co-cultured with HepG2.2.15 cells in a direct contact manner in the presence or absence of 104 of autologous Th9 cells or anti-IL-9 neutralization antibody (5 µg/mL). Anti-CD3/CD28 (1 µg/mL) was added for maintenance of non-specific T cell response. Supernatants were harvested 48 hours post co-culture. (A) Percentage of target HepG2.2.15 cell death was measured by LDH release, and was compared among groups in non-specific manners. Significance was assessed using one-way ANOVA and SNK-q test. (B) IFN-? and (C) TNF-a, which secreted by non-specific CD8+ T cells, was measured by ELISA, and was compared among groups. Significance was assessed using one-way ANOVA and SNK-q test. 104 of peripheral CD8+ T cells from CHB patients (n=9), and hepatitis B-related HCC patients (n=11) were co-cultured with HepG2.2.15 cells in a direct contact manner in the presence or absence of 104 of autologous Th9 cells or anti-IL-9 neutralization antibody (5 µg/mL). HBV core 18-27 epitope (sequence: FLPSDFFPSV; 5 µg/mL) was added for maintenance of HBV-specific T cell response. Supernatants were harvested 48 hours post co-culture. (D) Percentage of target HepG2.2.15 cell death was measured by LDH release, and was compared among groups in HBV core-specific manners. Significance was assessed using one-way ANOVA and SNK-q test. (E) IFN-? and (F) TNF-a, which secreted by HBV core-specific CD8+ T cells, was measured by ELISA, and was compared among groups. The dotted line presented lower detection limit for IFN-? (1.56 pg/mL) and TNF-a (1.95 pg/mL), respectively. Significance was assessed using one-way ANOVA and SNK-q test.