Ruslana Alper

Oral tolerance is the induction of immunological hyporesponsiveness towards orally administered antigens. Tolerance initiation involves induction of anti-inflammatory (Th2) lymphocytes, with downregulation of pro-inflammatory (Th1) lymphocytes. The liver was previously shown to play a critical role in oral tolerance induction. The aim of the present study was to test whether liver-associated-lymphocytes expressing the NK1.1 marker (NK1.1+ LAL) are substantial for induction of oral tolerance in an experimental colitis model. Colitis was induced in C57 mice by intracolonic instillation of trinitrobenzensulfonic acid (TNBS). Mice received five oral doses of colonic proteins extracted from TNBS-colitis colonic wall. Anti-NK1.1 monoclonal antibodies were injected before tolerance induction. Colitis was assessed by standard clinical, macroscopic, and microscopic scores. Serum IFN-gamma, TGF-beta1, and IL4 levels were measured by enzyme-linked immunosorbent assay. To evaluate the role of NK1.1+ LAL in keeping the balance between immunogenic and tolerogenic subsets of cells, we tested whether peripheral lymphocytes harvested from tolerized and NK1.1-depleted nontolerized mice can adoptively transfer the tolerance into naive irradiated rats. Depletion of NK1.1+ LAL prevented immune tolerance induction in the experimental colitis model. NK1.1+ LAL-depleted nontolerized mice, disclosed severe clinical, macroscopic, and microscopic parameters of colitis. These mice had significantly lower TGF-beta1, IL4, and higher IFN-gamma serum levels, and their lymphocytes failed to transfer the tolerance into naive animals. In contrast, the feeding of colitis-extracted proteins, without NK1.1+ LAL depletion, markedly alleviated the disease. Tolerized mice had higher IL4 and TGF-beta1 and lower IFN-gamma serum levels, and adoptive transfer of their suppressor splenocytes markedly alleviated colitis in naive recipients. NK1.1+ LAL plays a critical role in oral tolerance induction. Depletion of this subset of LAL prevents a shift from Th1 to a Th2 type of immune response, hindering the ability to induce immune tolerance.

Concanavalin A (ConA) induces natural killer T (NKT) cell-mediated liver damage. Glucocerebroside (GC) is a naturally occurring glycolipid. Our aims were to determine the effect of GC in a murine model of ConA-induced hepatitis. Mice in groups A and B were treated with GC 2 h before and 2 h following administration of ConA, respectively; group C mice were treated with ConA; group D mice was treated with GC; group E mice did not receive any treatment. Liver damage was evaluated by serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and liver histology. The immune effect of GC was determined by fluorescence-activated cell sorter analysis of intrahepatic and intrasplenic NKT lymphocytes, measurement of cytokine levels, and Western blot analysis for STAT 1, 4, 6, and NF-kappaB expression. The effect of GC on NKT cell proliferation was assessed in vitro. Serum AST and ALT levels were markedly reduced in GC-treated group A mice compared with nontreated group C animals, and histological damage was markedly attenuated in group A. The beneficial effect of GC was associated with a 20% decrease of intrahepatic NKT lymphocytes, significant lowering of serum IFN-gamma levels, and decreased STAT1 and STAT6 expression. In vitro administration of GC led to a 42% decrease of NKT cell proliferation in the presence of dendritic cells but not in their absence. Intraperitoneally administered radioactive GC was detected in the liver and bowel. Administration of GC led to amelioration of ConA hepatitis associated with an inhibitory effect on NKT lymphocytes. GC holds promise as a new immune-modulatory agent.

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.