Ivo, no matter if HCV+ or HCV-negative (Table 1; Figures 1,2). All round, 32 (9/28) of liver samples tested ex vivo demonstrated CD1d-reactivity. 5/14 HBV/HCV-negative and 0/3 HBV+ nNOS Gene ID subjects developed significant levels of CD1d-specific IFN. 1/5 IHL from HCV+ subjects with documented history of alcohol abuse and 3/5 other HCV+ IHL produced 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; variety: undetectable- comparable to mitogen). Ultimately, substantial IL-13 could possibly 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 had been constant with our prior benefits of a substantial population of largely non-invariant Th1-biased human hepatic CD1d-reactive T cells with or TXB2 custom synthesis without having HCV infection, most readily detectable in CHC (19,21,22). Apparently, human hepatic iNKT activity was reasonably rare. Non-invariant CD1d responses have been somewhat significantly less readily detectable straight ex vivo than in vitro from both HCV+ and HCV-negative subjects. CD1d-specific IFN was most regularly detected in comparison to 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 in the presence or absence of various stimuli and activation determined by FACS measurement of up-regulation of CD69 and IFN production (Figure 3). A substantial fraction of handle highly-enriched iNKT line cells responded to CD1d (Figure 3A,B). As expected given their low frequency in human IHL, iNKT-specific ligand GalCer did not stimulate several IHL ex vivo (not shown), while iNKT stimulation is well-known to quickly result in activation of first iNKT and then NK cells (each CD69 up-regulation and IFN production), followed by other immune cells downstream (9;292). Even so, 2 co-stimuli known to become active with CD1d for no less than murine iNKT (IL-12) (50) and for all sorts of CD1d-reactive T cells (19,21,22,33,48) (`Total’=PMA), IL-12 and PMA, each developed 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 delivers an alternative to PMA. Importantly, CD1d mAb particularly lowered 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). Hence, a substantial fraction ofJ Viral Hepat. Author manuscript; available in PMC 2014 August 01.Yanagisawa et al.Pagehuman IHL, bigger than the common proportion of antigen-specific T cells (e.g. 1;17), is straight 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 without having history of alcohol To date, only limited CD1d expression has been shown in human liver. These are at trace levels inside standard hepatocytes (26,27), elevated expression by biliary epithelia in PBC (27) and in HCV infection (21), by unidentified cells adjacent to hepatic stellate cells in HCV cirrhosis (20), and on hepatic mononuclear cell surface in normal liver (22). Figure four shows hepatocyte CD1d surface expression.