However, CD25+FoxP3+cells in the culture with -HpSC increased to 14.8%(Fig. generate CD25+Foxp3effector cells, HpSC selectively expanded CD4+CD25+Foxp3+cells in an IL-2 dependent manner. These expanded CD4+CD25+Foxp3+cells showed regulatory T (Treg) cell activity in effectively inhibiting T cell proliferation in responses to anti-CD3 mAb or alloantigens in a MHC nonspecific fashion. The Treg cells were expanded from the CD4+CD25+population with the help of IL-2, impartial of B7-H1 and TGF-. Administration of HpSC into allogeneic recipients resulted in expansion of CD4+CD25+FoxP3+cells in vivo. == Conclusion == Liver stromal HpSC acted as non-professional APC, and preferentially expanded CD25+FoxP3+Treg cells, which may contribute to immune regulation in the liver. Keywords:Hepatic stellate cell, Regulatory T cell, Dendritic cell, Antigen Presentation, IL-2, B7-H1, TGF-, Immune regulation, T cell response == Introduction == Organ transplantation has been successfully practice for decades, but the outcome of cell transplantation remains disappointing. This is the case in animal models; liver allografts in many species are spontaneous accepted without requirement of immunosuppression (13). Portal venous infusion or oral administration of an antigen (4,5) leads to antigen-specific tolerance (4,5). However, hepatocyte transplants are acutely rejected, apparently resulting from immune attacks because syngeneic hepatocyte transplants survive indefinitely (6). This suggests Demethoxycurcumin that liver non-parenchymal cells play an important role in protecting parenchymal cell from rejection. We have recently tested this hypothesis in mouse liver stromal cells, called hepatic stellate cells (HpSC) that are well known to participate in the repairative processes following liver injury. HpSC, upon activation, possess potent immunomodulatory activity. They markedly suppressed allogeneic T cell immune responses via induction of T cell apoptosis, which was partially mediated by B7-H1 expressed on HpSC (7). Co-transplantation of allogeneic islets with HpSC effectively guarded islet allografts from rejection (8). In this study, we demonstrate that HpSC, upon activation by interferon (IFN)-, a key cytokine released by activated T cells during inflammation, acquire the ability to present antigens and selectively expanded CD25+forkhead box P3 (FoxP3)+T regulatory cells (Treg) in an IL-2 dependent manner. == Materials and Methods == == Animals == Male C57BL/6J (B6; H-2b) and BALB/c (H-2d) mice were purchased Demethoxycurcumin from Jackson Laboratory (Bar Harbor, ME). Thy1.1+(BALB/c background) and B7-H de1ficient (B6 background) mice were kindly provided by Drs. Hyam I. Levitsky and Lieping Chen of Johns Hopkins University Medical School (Baltimore, MD), respectively. All animals were maintained in the specific pathogen-free facility at the Lerner Research Institute, Cleveland Clinic (Cleveland, OH). All animals received care according to the criteria of NIH. == Preparation of HpSC == HpSC were isolated from mouse liver NPC as previously described (7), and cultured (105/ml) in cell culture flask (25 cm2surface area) (Nunclon, Roskilde, Denmark) with RPMI-1640 (Mediatech Inc., Herndon, VA) supplemented with 20% fetal calf serum (FCS) in a 5% CO2in air at 37C for 710 days. Cell Demethoxycurcumin viability was greater than 90% as determined by trypan blue exclusion. The purity of HpSC was determined by desmin immunostaining and the typical light microscopic appearance of the lipid droplets. Activation of HpSC was accomplished by incubation with 200 U/ml IFN- (R & D Systems, Minneapolis, MN) for the last 72 hours of culture (subsequently referred to as -HpSC). == Culture of DC == B6 bone marrow cells were cultured in complete RPMI-1640 medium made up of 10% FCS in the presence of recombinant mouse GM-CSF (4 ng/ml) and IL-4 (1000 U/ml) (both from Schering-Plough, Kenilworth, NJ) as previously described (9). DC were isolated from non-adherent cells released from the proliferating cell clusters and purified by positive sorting with CD11c mAb coated magnetic beads (Milteny Biotec, Auburn, CA). == mAb and Flow cytometry == To examine expression of surface molecules, cells were stained with mAbs against CD3, CD4, CD25, CD40, CD54, CD80, CD86, MHC class I (H-2b), II (I-Ab), Thy1.1 (BD. PharMingen, San Diego, CA) or B7-H1 antigen (eBioscience, San Diego, CA). Staining for FoxP3 was performed using an intra-cellular staining kit (eBioscience). Appropriate isotype control antibodies were used in all experiments. For CFSE dilution assay, T cells (107/ml) were Rabbit Polyclonal to FZD10 incubated with 0.5 M CFSE (Molecular Probes, Eugene, OR) for 10 min at 37C. Cells were analyzed using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). == T cell purification == CD4+T cells were purified using magnetic beads via positive or unfavorable (bead-coated cocktail antibodies) selection with >99% purity as determined by flow cytometry. CD4+CD25+and CD4+CD25cells were purified using magnetic bead-coated anti-CD25 mAb with purity >99%. == Proliferation and.