Virus-specific CD4+ T cell responses in chronic HCV infection in blood and liver identified by antigen-specific upregulation of CD154.

BACKGROUND & AIMS: Virus-specific CD4+ T cells play a major role in hepatitis C virus (HCV) infection. Viral clearance is associated with vigorous and multispecific CD4+ T cell responses, while chronic infection has been shown to be associated with weak or absent T cell responses. Most of these studies, however, have used functional assays to analyse virus-specific CD4+ T cell responses. Therefore, the important question, of whether virus-specific CD4+ T cells are completely absent or primarily impaired in specific effector functions during chronic infection, has yet to be analysed in detail. METHODS: To address this issue, a novel assay, where CD4+ T cell frequencies can be determined by de novo CD154 (CD40 ligand) expression in response to HCV antigens, was used in a cohort of chronically infected HCV patients and patients who spontaneously resolved HCV infection. These responses were compared to functional assays, such as the IFN-gamma ELISpot and flow cytometry-based proliferative assays. RESULTS: Our results reveal that using the CD154 assay, virus-specific CD4+ T cells are readily detectable during chronic HCV infection albeit at a lower frequency when compared to patients who spontaneously resolved the infection. Importantly, no CD4+ T cell responses were detectable from these patients when using functional assays. Finally, these cell populations were enriched in the intrahepatic compartment. CONCLUSIONS: Our findings suggest that HCV-specific CD4+ T cell responses are readily detectable in chronic HCV infection and enriched in the infected liver.

Cell activation of human macrophages by lipoteichoic acid is strongly attenuated by lipopolysaccharide-binding protein.

Lipoteichoic acid (LTA) represents immunostimulatory molecules expressed by Gram-positive bacteria. They activate the innate immune system via Toll-like receptors. We have investigated the role of serum proteins in activation of human macrophages by LTA from Staphylococcus aureus and found it to be strongly attenuated by serum. In contrast, the same cells showed a sensitive response to LTA and a significantly enhanced production of tumor necrosis factor alpha under serum-free conditions. We show that LTA interacts with the serum protein lipopolysaccharide-binding protein (LBP) and inhibits the integration of LBP into phospholipid membranes, indicating the formation of complexes of LTA and soluble LBP. The addition of recombinant human LBP to serum-free medium inhibited the production of tumor necrosis factor alpha and interleukins 6 and 8 after stimulation of human macrophages with LTA in a dose-dependent manner. Using anti-LBP antibodies, this inhibitory effect could be attributed to soluble LBP, whereas LBP in its recently described transmembrane configuration did not modulate cell activation. Also, using primary alveolar macrophages from rats, we show a sensitive cytokine response to LTA under serum-free culture conditions that was strongly attenuated in the presence of serum. In summary, our data suggest that innate immune recognition of LTA is organ-specific with negative regulation by LBP in serum-containing compartments and sensitive recognition in serum-free compartments like the lung.

Aggregates are the biologically active units of endotoxin.

For the elucidation of the very early steps of immune cell activation by endotoxins (lipopolysaccharide, LPS) leading to the production and release of proinflammatory cytokines the question concerning the biologically active unit of endotoxins has to be addressed: are monomeric endotoxin molecules able to activate cells or is the active unit represented by larger endotoxin aggregates? This question has been answered controversially in the past. Inspired by the observation that natural isolates of lipid A, the lipid moiety of LPS harboring its endotoxic principle, from Escherichia coli express a higher endotoxic activity than the same amounts of the synthetic E. coli-like hexaacylated lipid A (compound 506), we looked closer at the chemical composition of natural isolates. We found in these isolates that the largest fraction was hexaacylated, but also significant amounts of penta- and tetraacylated molecules were present that, when administered to human mononuclear cells, may antagonize the induction of cytokines by biologically active hexaacylated endotoxins. We prepared separate aggregates of either compound 506 or 406 (tetraacylated precursor IVa), mixed at different molar ratios, and mixed aggregates containing both compounds in the same ratios. Surprisingly, the latter mixtures showed higher endotoxic activity than that of the pure compound 506 up to an admixture of 20% of compound 406. Similar results were obtained when using various phospholipids instead of compound 406. These observations can only be understood by assuming that the active unit of endotoxins is the aggregate. We further confirmed this result by preparing monomeric lipid A and LPS by a dialysis procedure and found that, at the same concentrations, only the aggregates were biologically active, whereas the monomers showed no activity.