Lymphocytes transiently expressing virus-specific T cell receptors reduce hepatitis B virus infection
Janine Kah, Sarene Koh, Tassilo Volz, Erica Ceccarello, Lena Allweiss, Marc Lütgehetmann, Antonio Bertoletti, Maura Dandri
Janine Kah, Sarene Koh, Tassilo Volz, Erica Ceccarello, Lena Allweiss, Marc Lütgehetmann, Antonio Bertoletti, Maura Dandri
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Research Article Immunology Infectious disease

Lymphocytes transiently expressing virus-specific T cell receptors reduce hepatitis B virus infection

Abstract

Adoptive transfer of T cells engineered to express a hepatitis B virus–specific (HBV-specific) T cell receptor (TCR) may supplement HBV-specific immune responses in chronic HBV patients and facilitate HBV control. However, the risk of triggering unrestrained proliferation of permanently engineered T cells raises safety concerns that have hampered testing of this approach in patients. The aim of the present study was to generate T cells that transiently express HBV-specific TCRs using mRNA electroporation and to assess their antiviral and pathogenetic activity in vitro and in HBV-infected human liver chimeric mice. We assessed virological and gene-expression changes using quantitative reverse-transcriptase PCR (qRT-PCR), immunofluorescence, and Luminex technology. HBV-specific T cells lysed HBV-producing hepatoma cells in vitro. In vivo, 3 injections of HBV-specific T cells caused progressive viremia reduction within 12 days of treatment in animals reconstituted with haplotype-matched hepatocytes, whereas viremia remained stable in mice receiving irrelevant T cells redirected toward hepatitis C virus–specific TCRs. Notably, increases in alanine aminotransferase levels, apoptotic markers, and human inflammatory cytokines returned to pretreatment levels within 9 days after the last injection. T cell transfer did not trigger inflammation in uninfected mice. These data support the feasibility of using mRNA electroporation to engineer HBV TCR–redirected T cells in patients with chronic HBV infection.

Authors

Janine Kah, Sarene Koh, Tassilo Volz, Erica Ceccarello, Lena Allweiss, Marc Lütgehetmann, Antonio Bertoletti, Maura Dandri

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Figure 1

Lytic and antiviral function of mRNA HBV–specific TCR–electroporated T cells in vitro.

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Lytic and antiviral function of mRNA HBV–specific TCR–electroporated T c...
(A) Activated T cells were electroporated with HBV s183–TCR or c18-TCR mRNA, and TCR expression was determined 24 hours after electroporation. Mock electroporated T cells served as negative control. Shown are representative plots. The percentages of HLA-A2/pentamer+ cells out of CD8+ or CD8− T cells are indicated. (B) TCR expression on electroporated cells was measured longitudinally from 24 hours to 96 hours. Electroporated T cells were cocultured with their respective peptide-pulsed T2 cells for 18 hours, and the frequencies of IFN-γ–producing CD8+ T cells out of total lymphocytes were quantified. (C) The ability of mRNA TCR–electroporated T cells to lyse HepG2.2.15 HBV–producing cells at 1:3, 1:5, and 1:100 E:T ratios within 24 hours after T cell addition was compared with that of retroviral transduced (TCR RV) T cells. (D) Sensitivity of T cell activation, displayed as percentage of maximum IFN-γ response using mRNA TCR–electroporated T cells compared with retroviral-transduced T cells (upper panel). MRNA HBV s183–TCR–electroporated T cells were cocultured with HBV s183–191 genotype B (FLLTKILTI) or genotype A/C/D (FLLTRILTI) peptide-loaded T2 cells. The percentages of CD8+ or CD8− T cells producing IFN-γ are indicated (lower panel). (E) Mock, mRNA HBV s183–TCR, or c18-TCR–electroporated T cells were cocultured with either HepG2 or HepG2.2.15 cells for 24 hours. The percentages of CD8+ or CD8− T cells producing IFN-γ are indicated. (F) mRNA HBV s183–TCR or c18-TCR–electroporated T cells were cocultured with mock or HBV-infected HepG2-NTCP for 24 hours, and IFNG gene expression was determined using NanoString analysis. (G) Mock or mRNA HBV s183–TCR–electroporated T cells were cocultured with HepG2.2.15 cells at a 1:3 E:T ratio for 24 hours, and intracellular HBV DNA was quantified by real-time quantitative PCR (qPCR). AST levels were determined in coculture media. Shown are means of percentage reduction in intracellular HBV DNA ± SD (black bars) and means of AST ± SD (gray bars) from 3 independent experiments (right panel).