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.

Conflict of interest statement

Conflict of interest: S. Koh is the Director of Research at Lion TCR Pte. Ltd., a biotech company developing T cell receptors for treatment of virus-related cancers and chronic viral diseases. A. Bertoletti is a cofounder of Lion TCR Pte. Ltd.

Figures

Figure 1. Lytic and antiviral function of mRNA HBV–specific TCR–electroporated T cells in vitro.

(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).

Figure 2. mRNA HBV–specific TCR–electroporated T cells show antiviral efficacy in vivo.

(A) Schematic representation of the experiment performed to assess the effect of 1 single injection of electroporated effector T cells in high viremic mice reconstituted with haplotype-matched hepatocytes. (B) Viremia changes relative to baseline levels determined after 4 and 6 days in individual mice upon 1 injection of mRNA HBV s183–TCR T cells (n = 5) and in untreated controls (n = 2). (C) ALT levels determined in HBV-infected and uninfected mice receiving a single injection of effector T cells. (D) Schematic representation of the experiment performed to assess the antiviral effect of multiple injections of electroporated effector T cells in high viremic mice reconstituted either with haplotype-matched (n = 3) or -mismatched (n = 3) human hepatocytes and in comparison with mice that were left untreated (n = 3). (E) Median viremia changes determined within each group depicted in D and relative to baseline levels determined in individual mice upon 3 injections of mRNA HBV s183–TCR T cells. Blood was taken 4, 8, and 12 days after the first T cell injection. (F) Median changes in levels of circulating HBeAg were determined by ELISA in all animal groups. BL, baseline.

Figure 3. mRNA HBV–specific TCR–electroporated T cells are specifically recruited and activated in livers of haplotype-matched HBV-infected mice.

(A) Liver tissues of humanized HBV-infected mice and uninfected mice that underwent 3 injections of HBV s183–TCR T cells and were sacrificed 4 days after the third T cell transfer were used for immunofluorescence. Human hepatocytes were identified using human-specific CK18 Abs (red). Transferred human immune cells were visualized using human-specific CD45 Abs (green). (B) Liver tissues of HBV-infected mice adoptively transferred with either HLA-A2– or HLA-A1–presenting human hepatocytes were costained with HBV core–specific Ab (green) and human CD45-specific Ab (red). (C) Transcript levels of human T cell response–related genes (GZMB and IFNG) and human ISGs (MxA, STAT1, TAP1, and HLA-A) were measured by quantitative reverse-transcriptase PCR (qRT-PCR) and normalized against human housekeeping transcripts. Statistical analysis was performed with GraphPad Prism 6 software.

Figure 4. Antiviral and inflammatory events after in vivo multiple injections of different mRNA HBV–specific TCR–electroporated T cells.

(A) Median viremia changes relative to baseline levels were determined as indicated after 4, 8, and 12 days upon multiple injections of mRNA HBV s183–TCR T cells (n = 3), mRNA c18-TCR T cells (n = 3), and mRNA mock TCR T cells (n = 3) as well as in untreated controls (n = 6). (B) Individual reduction (shown as percentages) of viremia relative to baseline levels determined on days 4, 8, and 12 upon transfer of mRNA s183–191 T cells and mRNA c18 T cells. Transcriptional changes of human T cell response–related genes (C, GZMB; E, IFNG) were measured by qRT-PCR and normalized against human housekeeping transcripts. (D) ALT levels were determined in uninfected (n = 3) and HBV-infected mice receiving multiple injections of effector T cells presenting s183 (n = 3), c18 (n = 3), or HCV NS3 as mock control (n = 3) in comparison with HBV-infected control mice (n = 6). (F) Median changes in human IFN-γ serum protein levels were determined by multiplex measurement in HBV-infected (s183 median = 1280 ng/ml or c18 median = 160 ng/ml) as well as uninfected (s183 median = 3.6 ng/ml) mice that received 3 injections of HBV-specific effector T cells relative to HBV-infected control mice (n = 4; median = 0.53 ng/ml).

Figure 5. Adoptive transfer of mRNA HBV–specific TCR–electroporated T cells leads to temporary limited liver inflammation and cell damage.

(A) Schematic representation of the experiment performed to assess the effect of multiple injections of electroporated effector T cells after treatment cessation both on inflammatory and virological parameters. (B) ALT levels were determined in 1 HBV-infected animal shortly before T cell injection (baseline) after receiving 3 injections of HBV-specific c18-TCR T cells (day 12) and 9 days after the last T cell injection (day 21). (C) Longitudinal changes in viremia relative to baseline were determined at 4, 8, 12, and 21 days after the first T cell transfer as depicted in A. (D) Serum protein levels of CCL2 were determined in both 1 HBV-infected control mouse (gray) and 1 mouse receiving multiple T cell injections (c18-TCR), and that was monitored for 21 days. (E) Longitudinal changes in levels of circulating HBeAg were determined by ELISA in the same mice described in D.