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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.
Introduction
Adoptive T cell therapy with receptor-modified T cells is emerging as a promising strategy to treat cancers (1). T cells are, however, not only capable of killing cancer cells, but are also essential for the control of viral infection. T cells can be engineered to express virus-specific (human CMV [HCMV], EBV, HIV, hepatitis B virus [HBV], hepatitis C virus [HCV], SARS) T cell receptors (TCRs) (2–6). Virus-specific TCR-redirected T cells have shown protective capacity in animal models (7). In humans, the antiviral efficacy of virus-specific TCR-redirected T cells has not yet been demonstrated, even though adoptive transfer of in vitro–expanded autologous virus-specific T cells has shown remarkable efficacy in EBV and HCMV reactivation occurring in immunocompromised patients (8, 9).
Virus-specific T cells are known to also be protective in the setting of HBV infection. Deletion of HBV-specific CD8+ T cells in HBV-infected chimpanzees blocks HBV clearance (10), and in patients who resolve the infection (11), HBV control is associated with the presence of polyfunctional CD8+ and CD4+ T cell responses targeting multiple HBV antigens (11). Thus, HBV-specific T cells are determinant to clear the virus. On the other hand, patients with chronic HBV infection have severe quantitative and functional defects in HBV-specific T cell response (12, 13), and current treatments are unable to resolve chronic HBV infection. This is mostly due to the inability of treatments based on polymerase inhibitors to target the HBV persistent form, the so-called covalently closed circular DNA (cccDNA) in infected hepatocytes. Thus, despite efficient suppression of viral replication, cccDNA persists within the hepatocyte nucleus, causing viral relapse after treatment discontinuation. Complete elimination of the cccDNA remains very challenging, and it may even be an unrealistic goal, since low cccDNA amounts were shown to persist even after resolution of acute infection (14). However, substantial immune-mediated destruction of infected cells or the induction of substantial cccDNA destabilization and silencing appears mandatory for achieving immune control and hence functional cure (15).
The reconstitution of HBV-specific CD8+ T cell response through HBV-specific TCR transfer can constitute an effective therapy for functional HBV cure. Experimental evidence showing that an adoptive transfer of HBV immunity can lead to an HBV curative effect has already been reported in humans. Adoptive transfer of bone marrow, previously primed with HBV vaccine (16) or HBV infection (17), spawned HBV control in chronic HBV patients. Similarly, liver transplantation of an HBV-infected liver in an HBV-immune patient was followed by viral clearance (18).
Genetic modification of T cells to express HBV-specific receptors, either a chimeric antigen receptor (CAR) (19, 20) or TCR (6), may supplement the deleted or functionally exhausted HBV-specific T cells in chronic HBV patients and, after adoptive transfer, achieve HBV control. T cells obtained from lymphocytes derived from HBV chronically infected patients and engineered to express HBV-specific receptors (6) were shown to produce antiviral cytokines and selectively eliminate HBV-expressing cells in vitro and in animal models (6, 19, 20).
HBV-specific TCR redirected T cells have already been used in a patient with HBV-related hepatocellular carcinoma (HCC) (21), where they caused a substantial drop of HBsAg produced by DNA integrated in HCC cells (22). However, despite this use, toxicity concerns have hampered their implementation in the therapy of patients chronically infected with HBV. Of note, HBV-specific T cells cannot only selectively lyse HBV-infected hepatocytes, but can also trigger inflammatory events within the liver (23). Since T cells transduced by viral vectors stably express TCR, their unchecked expansion might lead to progressive liver toxicities difficult to clinically manage.
To bypass this problem, we engineered T cells that transiently express HBV-specific TCRs for 3 to 5 days through messenger RNA electroporation (24). These cells are not genetically modified, and because of their limited life span, they can be adoptively transferred in escalating doses and their potential toxicities more easily managed. We have demonstrated that such mRNA TCR–redirected T cells are still able, despite their transient expression of TCR, to control the expansion of HBV-expressing hepatoma cells in mice (24), though their potential antiviral activity was not tested.
In the present study, we aimed to test the antiviral and pathogenetic activity of human T cells that transiently express HBV-specific TCR in vitro and in HBV-infected human liver chimeric uPA/SCID/ILγR2 (USG) mice (25). We demonstrate that engineered T cells transiently expressing HBV-TCR are specifically recruited to the liver of HBV-infected mice, harboring human hepatocytes expressing HLA-A2–matched haplotype, and that repeated administrations of these T cells induce a progressive but timely controlled virus-specific immune-mediated reduction of serological and intrahepatic HBV viral loads.
Results
mRNA TCR–expressing T cells display antiviral efficacy in vitro. HBV-specific T cells transiently expressing HLA-A0201–restricted HBV-specific TCRs (HBV envelope s183-TCR and HBV core c18-TCR) can be engineered in vitro through direct electroporation of mRNA encoding for specific TCR variable α and β chains in activated human T cells (Figure 1, A and B). A detailed report of the method used has been previously published (24, 26). The HBV-specific TCR mRNA–electroporated T cells acquire HBV specificity and are activated only after incubation with target cells presenting the specific HLA-class I/HBV viral peptide complex (ref. 24 and Figure 1B). Since mRNA does not integrate into the host cell genome, the TCR expression and ability to be activated upon antigen recognition declined with time and was lost after 96 hours (Figure 1B). We then tested the cytotoxicity of HBV-specific TCR mRNA–electroporated T cells against target cells that express HBV proteins and compared their killing ability to that of retroviral transduced T cells that permanently expressed HBV-specific TCR. At high effector-to-target (E:T) ratios of 1:5 and 1:3, HBV-specific TCR mRNA–electroporated T cells efficiently lysed 70% and 98% of the target cells, respectively, within 24 hours and their killing ability was similar to that of T cells stably expressing TCR within 24 hours, but less cytotoxic beyond that, as TCR expression declined (Figure 1C). To mimic more closely the situation in vivo where few T cells could encounter their targets, we tested a low E:T ratio of 1:100. Our results showed that HBV-specific TCR mRNA–electroporated T cells, similar to T cells stably expressing TCR, could still recognize and kill few target cells even at a low E:T ratio (Figure 1C). To further characterize the TCR mRNA–electroporated T cells, we tested their sensitivity to being activated at various peptide concentrations compared with that of T cells stably expressing TCR. We found that the sensitivity of HBV s183–TCR mRNA–electroporated T cells was similar to that of stably transduced T cells and that activation could be observed at 1 pg/ml (Figure 1D). The ability of HBV s183–TCR mRNA–electroporated T cells to recognize different viral mutants was also tested by stimulating electroporated T cells with HBV s183–191 genotype B peptide (FLLTKILTI)– or HBV genotype A/C/D peptide (FLLTRILTI)–loaded T2 cells, and similar frequencies of IFN-γ–producing T cells were detected (Figure 1D). The sensitivity of HBV c18-TCR–engineered T cells (6) and the ability of HBV c18 CD8+ T cell lines to recognize variant epitopes (27) have been previously described.
Lytic and antiviral function of mRNA HBV–specific TCR–electroporated T cellFigure 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).
We then investigated whether TCR mRNA–electroporated T cells can recognize not only HBV peptide–pulsed target cells, but hepatocyte-like cells producing HBV virions from stable HBV-DNA integrations (HepG2.2.15) and in HBV-infected cells (HepG2-NTCP), and whether these engineered T cells could suppress HBV replication in vitro. The expression of HBV s183–TCR allows activation of HBV s183–TCR T cells when cocultured with HepG2.2.15, while mock electroporated T cells were not activated and did not produce any IFN-γ (Figure 1E). Coculture of HBV s183–TCR T cells with HepG2 cells (non–HBV producing) did not cause any level of T cell activation (Figure 1E). This was further confirmed in an HBV-infected HepG2-NTCP system in which significant IFNG gene expression was measured in coculture of HBV s183–TCR T cells with HBV-infected HepG2-NTCP, but not with mock electroporated T cells (Figure 1F). Importantly, coculture of HBV s183–TCR T cells with HepG2.2.15 at a 1:3 E:T ratio for 18 hours caused direct lysis of about 69.5% and a 35% inhibition of HBV-DNA production in HepG2.2.15, accompanied by an increase in aspartate aminotransferase (AST) detected in the supernatant (Figure 1G). Similar results were obtained with T cells electroporated with a TCR specific for the HLA-A0201/core 18–27 complex (HBV c18-TCR T cells) (Figure 1, C, E–G). Taken together, these data show that electroporation of HBV TCR mRNA in T cells generates HBV-specific T cells able to recognize, inhibit, and lyse HepG2 cells producing HBV virions.
mRNA HBV–specific TCR–electroporated T cells display antiviral efficacy in vivo. To assess the in vivo antiviral effects of adoptively transferred human T cells transiently expressing an HBV-specific TCR, in a first set of experiments, peripheral blood mononuclear cells (PBMCs) of an HLA-A201+ healthy subject were used. Note that human T cells and human hepatocytes were allogenic, but shared HLA-A0201 expression. After being cultured for 1 week in the presence of IL-2 and anti-CD3 to enrich the fraction of T cells, cells were electroporated with HBV s183–TCR as described in Methods. After 24 hours, HLA-tetramer staining showed that the frequency of pentamer-positive CD8+ T cells ranged between 20% and 25% (data not shown) and that 0.5 million effector HBV s183–TCR T cells were adoptively transferred in each viremic mouse (≥109 HBV-DNA copies/ml) reconstituted with HLA-A2+ hepatocytes (HBV+A2+ mice). As shown in Figure 2, A and B, already 1 single i.p. injection of mRNA HBV s183–TCR T cells caused a drop of viremia in all 5 mice (median Δ0.5 log), which was detected at day 4. However, HBV-DNA values returned to the levels determined in the same animals before treatment at day 6. No decrease of viremia was determined in untreated controls (n = 2). Of note, although relatively high and variable levels of alanine aminotransferase (ALT) are present in this model of liver regeneration, ALT levels appeared exclusively elevated in HBV-infected mice receiving the activated T cells (Figure 2C), since ALT measured in uninfected animals also receiving haplotype-matched T cells remained comparable to levels determined in untreated controls. |
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发表于 2018-4-19 18:49
