It was noted that liver transplants in mice are spontaneously accepted, but hepatocyte transplants acutely rejected, suggesting the immunoregulatory activities of liver non-parenchymal cells. We have identified that the hepatic stellate cells (HSC), the stromal cells in the liver, are immensely immunosuppressive and can effectively protect islet transplants via induction of myeloid derived suppressor cells (MDSC). Addition of HSC into dendritic cell (DC) culture promoted development of MDSC, instead of DC, which was mediated by soluble factors. Upon activation, HSC produced VEGF, GM-CSF, and G-CSF. These factors have been shown to promote MDSC in cancer environment. However, we could not find these factors were critical in the induction of MDSC by HSC. The 100-250KD portion of HSC culture supernatant had the greatest influence on MDSC induction. Electrophoresis analysis showed unique bands. Peptide sequence analysis of the bands identified two groups of molecules: 1) extracellular matrices, which were expected, and 2) complement, including complement component 3 (C3), which was beyond expectation since C3 is mainly produced by hepatocytes and generally exists in serum. Interestingly, HSC from C3^-/- mice lost ability to protect islet allografts and induce MDSC, indicating absolute requirement of HSC-produced C3 which did not found to undergo alternative slicing and post-translational modifications. We demonstrated that HSC produced complement activation factor B and factor D (tested by Zymosan alternative pathway compensation assay using serum from factor B^-/- or D^-/- mice) which then enhanced C3 cleavage to activation products iC3b and C3d. Addition of exogenous iC3b, but not C3d, into the DC culture led to the differentiation of MDSC with potent immune inhibitory function. These findings provided novel mechanistic insights into the differentiation of myeloid cells mediated by local tissue cells, and may assist in the development of MDSC based therapy in clinical settings.

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