Intrahepatic CD206+ macrophages contribute to inflammation in advanced viral-related liver disease.

BACKGROUND & AIMS: Liver inflammation is key in the progression of chronic viral hepatitis to cirrhosis and hepatocellular carcinoma. The magnitude of viral replication and the specific anti-viral immune responses should govern the degree of inflammation, but a direct correlation is not consistently found in chronic viral hepatitis patients. We aim to better define the mechanisms that contribute to chronic liver inflammation. METHODS: Intrahepatic CD14+ myeloid cells from healthy donors (n=19) and patients with viral-related liver cirrhosis (HBV, HBV/HDV or HCV; n=15) were subjected to detailed phenotypic, molecular and functional characterisation. RESULTS: Unsupervised analysis of multi-parametric data showed that liver disease was associated with the intrahepatic expansion of activated myeloid cells mainly composed of pro-inflammatory CD14+HLA-DRhiCD206+ cells, which spontaneously produced TNFα and GM-CSF. These cells only showed heightened pro-inflammatory responses to bacterial TLR agonists and were more refractory to endotoxin-induced tolerance. A liver-specific enrichment of CD14+HLA-DRhiCD206+ cells was also detected in a humanised mouse model of liver inflammation. This accumulation was abrogated following oral antibiotic treatment, suggesting a direct involvement of translocated gut-derived microbial products in liver injury. CONCLUSIONS: Viral-related chronic liver inflammation is driven by the interplay between non-endotoxin-tolerant pro-inflammatory CD14+HLA-DRhiCD206+ myeloid cells and translocated bacterial products. Deciphering this mechanism paves the way for the development of therapeutic strategies specifically targeting CD206+ myeloid cells in viral-related liver disease patients. Lay summary: Viral-related chronic liver disease is driven by intrahepatic pro-inflammatory myeloid cells accumulating in a gut-derived bacterial product-dependent manner. Our findings support the use of oral antibiotics to ameliorate liver inflammation in these patients.

Immunoprevalence and immunodominance of HLA-Cw*0801-restricted T cell response targeting the hepatitis B virus envelope transmembrane region.

HLA-C-restricted T cells have been shown to play an important role in HIV control, but their impact on protection or pathogenesis in other viral infections remains elusive. Here, we characterized the hierarchy of HLA class I-restricted hepatitis B virus (HBV) epitopes targeted by CD8 T cells in HBV-infected subjects. The frequency of CD8 T cells specific for a panel of 18 HBV epitopes (restricted by HLA-A∗0201/03/07 [hereinafter HLA-A0201/03/07], -A1101, -A2402/07, -B5801, -B4001, -B1301, and -Cw0801) was quantified in a total of 59 subjects who resolved HBV infection. We found that the HLA-Cw0801-restricted epitope comprised of Env residues 171 to 180 (Env171-180) is immunoprevalent in the Southeast Asian subjects (10/17 HLA-Cw0801-positive subjects) and immunodominant in the majority of HLA-Cw0801-positive subjects able to control HBV infection. HLA-Cw0801-restricted Env171-180-specific CD8 T cells recognized endogenously produced HBV surface antigen (HBsAg) and tolerated amino acid variations within the epitope detected in HBV genotypes B and C. In conclusion, we demonstrate that the HLA-Cw0801-restricted Env171-180 T cell response is an important component of the HBV-specific adaptive T cell immunity in Asians infected with HBV. Thus, HLA-C restricted T cells might play an important role in various viral infections.

Building and optimizing a virus-specific T cell receptor library for targeted immunotherapy in viral infections.

Restoration of antigen-specific T cell immunity has the potential to clear persistent viral infection. T cell receptor (TCR) gene therapy can reconstitute CD8 T cell immunity in chronic patients. We cloned 10 virus-specific TCRs targeting 5 different viruses, causing chronic and acute infection. All 10 TCR genetic constructs were optimized for expression using a P2A sequence, codon optimization and the addition of a non-native disulfide bond. However, maximum TCR expression was only achieved after establishing the optimal orientation of the alpha and beta chains in the expression cassette; 9/10 TCRs favored the beta-P2A-alpha orientation over alpha-P2A-beta. Optimal TCR expression was associated with a significant increase in the frequency of IFN-gamma+ T cells. In addition, activating cells for transduction in the presence of Toll-like receptor ligands further enhanced IFN-gamma production. Thus, we have built a virus-specific TCR library that has potential for therapeutic intervention in chronic viral infection or virus-related cancers.

Mobilizing monocytes to cross-present circulating viral antigen in chronic infection.

Selection of antigens for therapeutic vaccination against chronic viral infections is complicated by pathogen genetic variations. We tested whether antigens present during persistent viral infections could provide a personalized antigenic reservoir for therapeutic T cell expansion in humans. We focused our study on the HBV surface antigen (HBsAg), which is present in microgram quantities in the serum of chronic HBV patients. We demonstrated by quantitative fluorescent microscopy that, out of 6 professional APC populations in the circulation, only CD14 monocytes (MNs) retained an HBsAg depot. Using TCR-redirected CD8+ T cells specific for MHC-I-restricted HBV epitopes, we showed that, despite being constantly exposed to antigen, ex vivo-isolated APCs did not constitutively activate HBV-specific CD8+ T cells. However, differentiation of HBsAg+ CD14 MNs from chronic patients to MN-derived DCs (moDCs) induced cross-presentation of the intracellular reservoir of viral antigen. We exploited this mechanism to cross-present circulating viral antigen and showed that moDCs from chronically infected patients stimulated expansion of autologous HBV-specific T cells. Thus, these data demonstrate that circulating viral antigen produced during chronic infection can serve as a personalized antigenic reservoir to activate virus-specific T cells.

A practical approach to immunotherapy of hepatocellular carcinoma using T cells redirected against hepatitis B virus.

Hepatocellular carcinoma (HCC) cells often have hepatitis B virus (HBV)-DNA integration and can be targeted by HBV-specific T cells. The use of viral vectors to introduce exogenous HBV-specific T-cell receptors (TCR) on T cells to redirect their specificity is complex and expensive to implement in clinical trials. Moreover, it raises safety concerns related to insertional mutagenesis and potential toxicity of long-lived HBV-specific T cells in patients with persistent infection. To develop a more practical and safer approach to cell therapy of HCC, we used electroporation of mRNA encoding anti-HBV TCR. Approximately 80% of CD8(+) T cells expressed functional HBV TCR 24 hours postelectroporation, an expression efficiency much higher than that obtained by retroviral transduction (~18%). Antigen-specific cytokine production of electroporated T cells was efficient within 72-hour period, after which the redirected T cells lost their HBV-specific function. Despite this transient functionality, the TCR-electroporated T cells efficiently prevented tumor seeding and suppressed the growth of established tumors in a xenograft model of HCC. Finally, we established a method for large-scale TCR mRNA electroporation that yielded large numbers of highly functional clinical-grade anti-HBV T cells. This method represents a practical approach to cell therapy of HCC and its inherently self-limiting toxicity suggests potential for application in other HBV-related pathologies.Molecular Therapy-Nucleic Acids (2013) 2, e114; doi:10.1038/mtna.2013.43; published online 13 August 2013.