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

Background: 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. Aim: 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. Methods: Sublethally irradiated athymic Balb/c mice were injected with 10⁷ human hepatoma cells followed 10 days later by transplantation of 2 × 10⁶ 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 α-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. Results: 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 mm³ in controls. Serum αFP 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. IFNγ production increased two-fold. Conclusion: Oral immune regulation towards HCC antigens effectively enhanced the antitumor 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 IFNγ production.