Immunohistochemistry using the antibody reacting with the C terminus of Foxp3 detected only mononuclear cells (Treg cells), but the antibody reacting with the N terminus highlighted cholangiocarcinoma cells as well as Treg cells (Fig. 4A). The cytoplasm

as well as nucleus of tumor cells was positive in several cases. However, because R428 mw Foxp3 is a transcription factor, the nuclear pattern was evaluated as functional expression. Consequently, 21 of 54 (39%) cholangiocarcinomas tested positive for Foxp3 by the antibody reacting with the N terminus. The relation between the IgG4 reaction and Foxp3 expression in cholangiocarcinoma cells is shown in Fig. 5. In cases of positivity for Fopx3, the number of IgG4-positive cells was significantly higher than in cases of negativity for Foxp3. RT-PCR analysis demonstrated that a cholangiocarcinoma cell line, HuCCT1, expressed the mRNA of Foxp3, but close examination using four sets of primers corresponding to exons 1, 3, 10-12, and 12 revealed a lack of exon 3 (Fig. 6), suggesting the presence of a splicing variant of Foxp3 in cholangiocarcinoma cells. Moreover, RT-PCR and ELISA revealed that HuCCT1 cells expressed IL-10 mRNA (Fig. 6) and protein in the culture medium at 7.8-15.6 pg/mL. IgG4 is important to the pathogenesis of IgG4-related diseases. SP600125 nmr However, patients with pancreatic

adenocarcinomas accompanying IgG4 reactions and/or elevated serum IgG4 levels4, 18-20 and with pancreatic and biliary cancers arising from IgG4-related diseases20-22 have been reported, though a cause-and-effect

relationship between IgG4 reactions MCE and cancers has yet to be demonstrated. Moreover, in IgG4-nonrelated diseases, including primary sclerosing cholangitis, IgG4 reactions were found to various degrees.23, 24 Therefore, the presence of IgG4-positive cells is not a histological hallmark of IgG4-related diseases, and IgG4 reactions are speculated to be nonspecific in several pathological conditions, including cholangiocarcinomas. The present study also demonstrated the presence of extrahepatic cholangiocarcinoma cases with abundant IgG4 reaction, though there was no significant difference in IgG4-positive cell counts among anatomical locations of extrahepatic cholangiocarcinomas (common bile ducts, gallbladder, and the Papilla of Vater). The significance and mechanisms of IgG4 reactions in cancers as well as IgG4-related diseases are still unknown, but we speculated that cancer cells directly participate in the histogenesis of IgG4 reactions. Because the regulatory cytokine IL-10 is known to induce the differentiation of IgG4-positive plasma cells or favor B cell switching to IgG4 in the presence of IL-4,5, 6 we noted the IL-10–related regulatory cytokine network around cholangiocarcinoma tissue in this study.