Isolation and functional characterization of hepatitis B virus-specific T-cell receptors as new tools for experimental and clinical use.

T-cell therapy of chronic hepatitis B is a novel approach to restore antiviral T-cell immunity and cure the infection. We aimed at identifying T-cell receptors (TCR) with high functional avidity that have the potential to be used for adoptive T-cell therapy. To this end, we cloned HLA-A*02-restricted, hepatitis B virus (HBV)-specific T cells from patients with acute or resolved HBV infection. We isolated 11 envelope- or core-specific TCRs and evaluated them in comprehensive functional analyses. T cells were genetically modified by retroviral transduction to express HBV-specific TCRs. CD8+ as well as CD4+ T cells became effector T cells recognizing even picomolar concentrations of cognate peptide. TCR-transduced T cells were polyfunctional, secreting the cytokines interferon gamma, tumor necrosis factor alpha and interleukin-2, and effectively killed hepatoma cells replicating HBV. Notably, our collection of HBV-specific TCRs recognized peptides derived from HBV genotypes A, B, C and D presented on different HLA-A*02 subtypes common in areas with high HBV prevalence. When co-cultured with HBV-infected cells, TCR-transduced T cells rapidly reduced viral markers within two days. Our unique set of HBV-specific TCRs with different affinities represents an interesting tool for elucidating mechanisms of TCR-MHC interaction and dissecting specific anti-HBV mechanisms exerted by T cells. TCRs with high functional avidity might be suited to redirect T cells for adoptive T-cell therapy of chronic hepatitis B and HBV-induced hepatocellular carcinoma.

Characterization of T Antigens, Including Middle T and Alternative T, Expressed by the Human Polyomavirus Associated with Trichodysplasia Spinulosa.

UNLABELLED: The polyomavirus tumor (T) antigens play crucial roles in viral replication, transcription, and cellular transformation. They are encoded by partially overlapping open reading frames (ORFs) located in the early region through alternative mRNA splicing. The T expression pattern of the trichodysplasia spinulosa-associated polyomavirus (TSPyV) has not been established yet, hampering further study of its pathogenic mechanisms and taxonomic relationship. Here, we characterized TSPyV T antigen expression in human cell lines transfected with the TSPyV early region. Sequencing of T antigen-encoded reverse transcription-PCR (RT-PCR) products revealed three splice donor and acceptor sites creating six mRNA splice products that potentially encode the antigens small T (ST), middle T (MT), large T (LT), tiny T, 21kT, and alternative T (ALTO). Except for 21kT, these splice products were also detected in skin of TSPyV-infected patients. At least three splice products were confirmed by Northern blotting, likely encoding LT, MT, ST, 21kT, and ALTO. Protein expression was demonstrated for LT, ALTO, and possibly MT, with LT detected in the nucleus and ALTO in the cytoplasm of transfected cells. Splice site and start codon mutations indicated that ALTO is encoded by the same splice product that encodes LT and uses internal start codons for initiation. The genuineness of ALTO was indicated by the identification of acetylated N-terminal ALTO peptides by mass spectrometry. Summarizing, TSPyV exhibits an expression pattern characterized by both MT and ALTO expression, combining features of rodent and human polyomaviruses. This unique expression pattern provides important leads for further study of polyomavirus-related disease and for an understanding of polyomavirus evolution. IMPORTANCE: The human trichodysplasia spinulosa-associated polyomavirus (TSPyV) is distinguished among polyomaviruses for combining productive infection with cell-transforming properties. In the research presented here, we further substantiate this unique position by indicating expression of both middle T antigen (MT) and alternative T antigen (ALTO) in TSPyV. So far, none of the human polyomaviruses was shown to express MT, which is considered the most important viral oncoprotein of rodent polyomaviruses. Coexpression of ALTO and MT, which involves a conserved, recently recognized overlapping ORF subject to positive selection, has not been observed before for any polyomavirus. As a result of our findings, this study provides valuable new insights into polyomavirus T gene use and expression. Obviously, these insights will be instrumental in further study and gaining an understanding of TSPyV pathogenicity. More importantly, however, they provide important leads with regard to the interrelationship, functionality, and evolution of polyomaviruses as a whole, indicating that TSPyV is a suitable model virus to study these entities further.

T Cells Engineered to Express a T-Cell Receptor Specific for Glypican-3 to Recognize and Kill Hepatoma Cells In Vitro and in Mice.

BACKGROUND & AIMS: Cancer therapies are being developed based on our ability to direct T cells against tumor antigens. Glypican-3 (GPC3) is expressed by 75% of all hepatocellular carcinomas (HCC), but not in healthy liver tissue or other organs. We aimed to generate T cells with GPC3-specific receptors that recognize HCC and used them to eliminate GPC3-expressing xenograft tumors grown from human HCC cells in mice. METHODS: We used mass spectrometry to obtain a comprehensive peptidome from GPC3-expressing hepatoma cells after immune-affinity purification of human leukocyte antigen (HLA)-A2 and bioinformatics to identify immunodominant peptides. To circumvent GPC3 tolerance resulting from fetal expression, dendritic cells from HLA-A2-negative donors were cotransfected with GPC3 and HLA-A2 RNA to stimulate and expand antigen-specific T cells. RESULTS: Peptide GPC3367 was identified as a predominant peptide on HLA-A2. We used A2-GPC3367 multimers to detect, select for, and clone GPC3-specific T cells. These clones bound the A2-GPC3367 multimer and secreted interferon-γ when cultured with GPC3367, but not with control peptide-loaded cells. By genomic sequencing of these T-cell clones, we identified a gene encoding a dominant T-cell receptor. The gene was cloned and the sequence was codon optimized and expressed from a retroviral vector. Primary CD8(+) T cells that expressed the transgenic T-cell receptor specifically bound GPC3367 on HLA-A2. These T cells killed GPC3-expressing hepatoma cells in culture and slowed growth of HCC xenograft tumors in mice. CONCLUSIONS: We identified a GPC3367-specific T-cell receptor. Expression of this receptor by T cells allows them to recognize and kill GPC3-positive hepatoma cells. This finding could be used to advance development of adoptive T-cell therapy for HCC.