Ivo, no matter if HCV+ or HCV-negative (Table 1; Figures 1,2). General, 32 (9/28) of liver samples tested ex vivo demonstrated CD1d-reactivity. 5/14 HBV/HCV-negative and 0/3 HBV+ subjects developed significant levels of CD1d-specific IFN. 1/5 IHL from HCV+ subjects with documented history of FLAP Purity & Documentation alcohol abuse and 3/5 other HCV+ IHL developed readily detectable CD1d IFN responses (Figure 2E,F; Table 1). Measurable CD1d-reactivity of HCV+ IHL was 7, 20, and 59 of mitogen IFN responses (Table 1), comparable to HCV-negative subjects (median=34 of mitogen; range: undetectable- comparable to mitogen). Lastly, substantial IL-13 might be detected in response to CD1d from some subjects ex vivo (Figure 2G), consistent with modest levels detected from in vitro IHL cultures (19). In summary, ex vivo results were consistent with our preceding outcomes of a substantial population of largely non-invariant Th1-biased human hepatic CD1d-reactive T cells with or with out HCV infection, most readily detectable in CHC (19,21,22). Apparently, human hepatic iNKT activity was comparatively rare. Non-invariant CD1d responses had been somewhat less readily detectable straight ex vivo than in vitro from both HCV+ and HCV-negative subjects. CD1d-specific IFN was most regularly detected when compared with other cytokines tested. Proportion of hepatic CD1d-reactive T cells ex vivo Subsequent, we addressed the fraction of IHL capable of responding to CD1d ex vivo. IHL were co-incubated with C1R CD1d or controls inside the presence or absence of diverse stimuli and activation determined by FACS measurement of up-regulation of CD69 and IFN production (Figure 3). A substantial fraction of manage highly-enriched iNKT line cells responded to CD1d (Figure 3A,B). As anticipated offered their low frequency in human IHL, iNKT-specific ligand GalCer did not stimulate numerous IHL ex vivo (not shown), even though iNKT stimulation is well-known to swiftly lead to activation of 1st iNKT and then NK cells (each CD69 up-regulation and IFN production), followed by other immune cells downstream (9;292). However, 2 co-stimuli identified to be active with CD1d for at the very least murine iNKT (IL-12) (50) and for all kinds of CD1d-reactive T cells (19,21,22,33,48) (`Total’=PMA), IL-12 and PMA, each and every made comparable and substantial proportions of CD1d-responsive IHL (Figure 3A,B). IL-12 has not previously been shown to co-stimulate CD1d-specific non-invariant NKT responses, so this provides an option to PMA. Importantly, CD1d mAb especially reduced the proportion of CD69+ and IFN-producing IHL, demonstrating CD1d-dependency of those responses (Figure 3A,B), as previously for IHL and other NKT cell populations (19,21,22,33,48). For that reason, a substantial fraction ofJ Viral Hepat. Author manuscript; offered in PMC 2014 August 01.Yanagisawa et al.Pagehuman IHL, bigger than the standard proportion of antigen-specific T cells (e.g. 1;17), is directly CD1d-reactive ex vivo.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSelective hepatocyte cell surface CD1d up-regulation in active CHC with no history of alcohol To date, only limited CD1d expression has been shown in human liver. They are at trace levels inside normal hepatocytes (26,27), enhanced expression by biliary epithelia in PBC (27) and in HCV infection (21), by unidentified cells adjacent to hepatic CDK7 Storage & Stability stellate cells in HCV cirrhosis (20), and on hepatic mononuclear cell surface in standard liver (22). Figure 4 shows hepatocyte CD1d surface expression.