Suppression of hepatocellular carcinoma growth via oral immune regulation towards tumor-associated antigens is associated with increased NKT and CD8+ lymphocytes.

Oral immune regulation towards viral proteins was previously shown to modulate the anti-HBV immune response. Adoptive transfer of orally immunomodulated lymphocytes suppressed the growth of hepatocellular carcinoma (HCC) expressing HBsAg in athymic mice. NKT lymphocytes play a role in the defense against tumor growth. To evaluate the effect of oral immune regulation towards HCC-associated antigens or HBV proteins on growth of HBsAg-expressing HCC, and to determine the role of NKT lymphocytes in immune modulation. Sublethally irradiated athymic Balb/c mice were injected with 10(7) human hepatoma cells followed 10 days later by transplantation of 2 x 10(6) splenocytes from naive donor mice. Immune modulation was performed via feeding of HCC-extracted proteins or HBV antigens (HBsAg + Pre S1 + Pre S2). The control group was fed with bovine serum albumin (BSA). Mice were followed for survival, tumor volume, and serum alpha-fetoprotein levels. To determine the role of NKT cells in tumor suppression, cytokine expression and FACS analysis for CD4+, CD8+, and NK1.1+ T lymphocyte subsets were performed. Oral immune regulation towards HCC-extracted proteins induced complete tumor suppression in recipient mice. Mortality rates were 0% in HCC-immune-regulated mice, compared with an 80% mortality rate using HBV antigens and a 100% mortality rate in control mice. Oral immune regulation towards HCC prevented weight loss. No visible tumor mass was observed in orally immune-regulated mice as compared with 112 mm(3) in controls. Serum alphaFP levels were 0.9, 378 and 1,358 ng/ml in HCC, HBV immune-regulated and controls, respectively. Immune regulation towards HCC antigens significantly increased the NK1.1+ T lymphocytes/CD4+ and CD8+/CD4+ ratios. IFNgamma production increased two-fold. Oral immune regulation towards HCC antigens effectively enhanced the anti-tumor immune response, thus suppressing the growth of HCC in mice. This effect was associated with an increased NKT,CD8+/CD4+ lymphocyte ratio and may be mediated via enhancement of IFNgamma production.

Shibolet O ，Alper R ，Zlotogarov L ，Thalenfeld B ，Engelhardt D ，Rabbani E ，Ilan Y ... -  《ONCOLOGY》

Inducing oral immune regulation of hepatitis B virus envelope proteins suppresses the growth of hepatocellular carcinoma in mice.

Gotsman I ，Alper R ，Klein A ，Rabbani E ，Engelhardt D ，Ilan Y ... -  《CANCER》

NKT and CD8 lymphocytes mediate suppression of hepatocellular carcinoma growth via tumor antigen-pulsed dendritic cells.

Dendritic cells (DCs) are antigen presenting cells that play a role in T-cell activation. Liver-associated natural killer T lymphocytes (NKTs) are a unique subset of lymphocytes that may be important in antitumor immunity. Hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) expresses hepatitis B virus surface antigen (HBsAg) on its cell surface and may serve as a tumor-associated antigen. The aim of the study was to evaluate the antitumor effect of DC pulsed with tumor or viral-associated antigens in HBV-expressing HCC in mice and to determine the role of NKT lymphocytes in this process. Balb/c mice were sublethally irradiated and transplanted with Hep3b HCC cell line, followed by transplantation of naive splenocytes. DCs were separated using CD11c beads and pulsed with HBV-enveloped proteins (group A), HCC cell lysate (group B), or BSA (control group C). Mice were followed for survival and tumor size. To determine the mechanism of the antitumor effect, intrasplenic and intrahepatic lymphocyte subpopulations were analyzed by FACS for NKT, CD4 and CD8 markers. Tumor-associated antigens-specific IFNgamma ELISPOT, T-cell proliferation assays and serum cytokine analysis were performed. Treatment with tumor-associated antigen-pulsed DC significantly improved survival (40% and 50% as compared with 0% in groups A, B, and control group C, respectively; p < 0.005). Tumor size decreased to 12.8 +/- 0.4 and 0 from 60.4 +/- 0.9 mm(3) in groups A, B, and control group C, respectively (p < 0.005). Adoptive transfer of HBV or Hep3b-associated antigens-pulsed DC induced a 6-fold increase in peripheral CD8(+) lymphocytes (from 1% in control mice to 6% and 5.5% in groups A and B, respectively), along with a decrease in CD4(+) lymphocytes (from 3.5% in controls to 1.4% and 2.3% in A and B, respectively; p < 0.005). The CD8(+)/CD4(+) ratio increased from 0.28 in controls to 4.28 and 2.39 in groups A and B, respectively (p < 0.005). Intrasplenic NKT cells increased from 7% in control mice to 7.98% and 14.6% in groups A and B, respectively. In contrast, an opposite shift was observed inside the liver. Intrahepatic lymphocyte analysis showed a marked increase in CD4(+) and a decrease in CD8(+) lymphocytes in treated groups. The intrahepatic CD4(+) number increased from 0.5% in controls to 2.15% and 25.8% in groups A and B, respectively (p < 0.005). In contrast, a significant decrease in the intrahepatic CD8(+) numbers was observed (from 7% in controls to 1.0% and 2.4% in groups A and B, respectively; p < 0.005). A significant increase was noted in HBV-specific IFNgamma spot-forming T-cell colonies from 0.0 to 8.8 +/- 1.7 and 1.8 +/- 2.9 in groups C, A, and B, respectively (p < 0.005). Similarly, a significant increase in the HBV-specific T-cell stimulation index, from 0.8 +/- 0.2 to 7.2 +/- 0.4, in groups C and B, respectively, was noted (p < 0.002). IFNgamma and IL12 serum levels increased significantly in treated groups. IFNgamma and IL12 serum levels increased to 380 +/- 30 and 400 +/- 20, and 960 +/- 40 and 760 +/- 60 in groups A and B, compared with 150 +/- 16 and 490 +/- 40 pg/ml in control mice (p < 0.005). Tumor antigen-pulsed DCs effectively suppressed the growth of hepatocellular carcinoma in mice. This effect was associated with enhanced NKT and CD8(+) lymphocyte function and augmentation of the antitumor/antiviral-specific IFNgamma production.

Shibolet O ，Alper R ，Zlotogarov L ，Thalenfeld B ，Engelhardt D ，Rabbani E ，Ilan Y ... -  《INTERNATIONAL JOURNAL OF CANCER》

Suppression of hepatocellular carcinoma by transplantation of ex-vivo immune-modulated NKT lymphocytes.

Margalit M ，Shibolet O ，Klein A ，Elinav E ，Alper R ，Thalenfeld B ，Engelhardt D ，Rabbani E ，Ilan Y ... -  《INTERNATIONAL JOURNAL OF CANCER》

The role of intrahepatic CD8+ T cell trapping and NK1.1+ cells in liver-mediated immune regulation.

Shibolet O ，Alper R ，Zolotarov L ，Trop S ，Thalenfeld B ，Engelhardt D ，Rabbani E ，Ilan Y ... -  《CLINICAL IMMUNOLOGY》