Generation of T-cell receptors targeting a genetically stable and immunodominant cytotoxic T-lymphocyte epitope within hepatitis C virus non-structural protein 3

Abstract

Hepatitis C virus (HCV) is a major cause of severe liver disease, and one major contributing factor is thought to involve a dysfunction of virus-specific T-cells. T-cell receptor (TCR) gene therapy with HCV-specific TCRs would increase the number of effector T-cells to promote virus clearance. We therefore took advantage of HLA-A2 transgenic mice to generate multiple TCR candidates against HCV using DNA vaccination followed by generation of stable T-cell-BW (T-BW) tumour hybrid cells. Using this approach, large numbers of non-structural protein 3 (NS3)-specific functional T-BW hybrids can be generated efficiently. These predominantly target the genetically stable HCV genotype 1 NS3(1073-1081) CTL epitope, frequently associated with clearance of HCV in humans. These T-BW hybrid clones recognized the NS3(1073) peptide with a high avidity. The hybridoma effectively recognized virus variants and targeted cells with low HLA-A2 expression, which has not been reported previously. Importantly, high-avidity murine TCRs effectively redirected human non-HCV-specific T-lymphocytes to recognize human hepatoma cells with HCV RNA replication driven by a subgenomic HCV replicon. Taken together, TCR candidates with a range of functional avidities, which can be used to study immune recognition of HCV-positive targets, have been generated. This has implications for TCR-related immunotherapy against HCV.

Fig. 1.

(a) Screening summary for the BW-fused HHD T-BW hybrid clones. Cytokine production against HHD or HLA-A2 target cells loaded with NS3₁₀₇₃. (b) IL-2 secretion upon stimulation with HHD splenocytes loaded with NS3₁₀₇₃. (c) IL-2 and (d) IFN-γ secretion upon stimulation with T2 cells loaded with NS3₁₀₇₃ (▪) or NS3₁₄₀₆ (□). Depicted fold induction is given as the ratio of cytokine concentration in co-cultures containing peptide-loaded target (10 µg indicated peptide ml⁻¹) over the unloaded control (0 µg indicated peptide ml⁻¹). Mean values of duplicate co-cultures from one experiment are shown. Amount of cytokine released (pg ml⁻¹) is shown on the top of each bar. *Not detected.

Fig. 2.

(a) Summary of TCR gene usage and CDR3 coding sequences of the nine IL-2⁺IFN-γ⁺ T-BW hybrid clones. (b) Affinity to the NS3₁₀₇₃/HLA-A2 pentamer. Indicated T-cell clones were stained with R-PE-labelled NS3₁₀₇₃/HLA-A2 pentamer (black line unfilled) or control R-PE-labelled HBV core_18–27 pentamer (grey filled) and labelled with FITC-labelled anti-mouse CD3 antibody and analysed by FACS. Histograms of fluorescence intensity of pentamer staining were gated on the live CD3⁺ population.

Fig. 3.

Functional avidity of T-BW hybrid clones (I8H4, •; I8A4, ○; I4G7, ▪; I4F8, □) tested against T2 cells loaded with descending concentration of NS3₁₀₇₃. Comparative results were obtained in three separate experiments.

Fig. 4.

Cross-reactivity against other viral peptides that share similarity with the NS3₁₀₇₃ peptide sequence encoded by the DNA vaccine (genotype 1a). Overnight IL-2 production in T-BW hybrid clones against T2 cells loaded with the indicated viral peptide (10 µg peptide ml⁻¹) was measured for each T-cell clone and is shown as a percentage of IL-2 production by genotype 1a of the NS3₁₀₇₃ peptide. Amino acids that differ from the genotype 1a are indicated in bold. Mean values and sd of triplicate co-cultures are shown. Flu, Flu-NA₂₃₁; gt, genotype.

Fig. 5.

Responses to the alanine-substituted peptide analogues of NS3₁₀₇₃. Overnight IL-2 production in T-BW hybrid clones (I8H4, •; I8A4, ○; I4G7, ▪; I4F8, □) co-cultured with T2 cells loaded with descending concentrations of the alanine-substituted peptides of NS3₁₀₇₃ (substituted from positions 1 to 9). Mean values of duplicate co-cultures are shown.

Fig. 6.

HLA-A2 expression in the B-lymphocyte C1R-A2 cell line (a, filled grey) and the hepatoblastoma Huh-6 cell line (b, filled grey) loaded with NS3₁₀₇₃ peptide compared with that of the C1R-null cells (solid line no fill) and T2 cells (dotted line). (c, d) IL-2 secretion in T-BW hybrid clones following co-culture with (c) C1R-A2 cells (▪) or C1R-null (□) cells or (d) Huh-6 cells loaded with 10 µg NS3₁₀₇₃ peptide ml⁻¹. Mean values and sd of duplicate co-cultures are shown. Similar results were obtained in two separate experiments.

Fig. 7.

Cytokine release against the Huh-7/Lunet-derived HCV replicon cells. (a) IL-2 concentration in T-BW hybrid clone I8H4, I8A4, I4F8 or I4G7 co-cultured with the Lunet-HlaA2-neoET (R-neo/A2) replicon cells that harbour both the HCV Con1-ET subgenomic replicon and HLA-A2, or co-cultured with control cell lines Lunet-blr/neo ET (R-neo) or Lunet-HlaA2 neo (A2) that harbour only the HCV replicon or HLA-A2, respectively. (b) IL-2 and IFN-γ concentration in the T-BW hybrid clones co-cultured with Lunet-HlaA2-neoET (R-neo/A2) HCV replicon cells, or the control Lunet-HlaA2 neo (A2) cells that express HLA-A2 but no HCV replicon, that were loaded with NS3₁₀₇₃ peptide (genotype 1a, 10 µg ml⁻¹). Co-cultures consisted of 1×10⁵ of indicated Lunet cells and T-BW cells in the ratio 1 : 1 or 1 : 5. Mean values and sd of duplicate co-cultures are shown.

Fig. 8.

Multiple effector functions in the TCR-expressing naïve HCV-non-specific CD8 T-cells co-cultured with peptide-loaded T2 cells or the endogenous viral peptide in HCV Con1 genotype 1b replicon cells. (a) Intracellular IFN-γ staining on mouse (mu)Vβ⁺humanCD8⁺ lymphocytes in transduced T-cells co-cultured overnight with peptide (NS3₁₀₇₃ 1a or 1b)-loaded T2 cells or controls, and (b) intracellular TNF-α and IL-2 staining in the IFN-γ⁺ population in (a). (c) Bioluminescence of HCV replicon-encoded luciferase activity in Lunet-HlaA2-Luc-ubi-neo Con1 replicon cells that have been co-incubated with human T-cells transduced with I4F8 or I8H4 TCR, as assessment of antiviral inhibition specific to the endogenous NS3₁₀₇₃ viral peptide (1b). (d) Anti-HCV effect on Lunet-HlaA2-Luc-ubi-neo Con1 replicon cells, and (e) hepatocellular injury caused by TCR-transduced T-cells in quadruple co-cultures. Mean values and sd are given and expressed as percentage relative light units for luciferase (where mock corresponds to 100 %), and international units (I.U.) per litre for aspartate transaminase. * Indicates P value <0.01 (Student’s t-test) compared with mock. Dotted line indicates the cut-off value of background bioluminescence in empty wells.