Characterization of a library of 20 HBV-specific MHC class II-restricted T cell receptors

Abstract

CD4⁺ T cells play an important role in the immune response against cancer and infectious diseases. However, mechanistic details of their helper function in hepatitis B virus (HBV) infection in particular, or their advantage for adoptive T cell therapy remain poorly understood as experimental and therapeutic tools are missing. Therefore, we identified, cloned, and characterized a comprehensive library of 20 MHC class II-restricted HBV-specific T cell receptors (TCRs) from donors with acute or resolved HBV infection. The TCRs were restricted by nine different MHC II molecules and specific for eight different epitopes derived from intracellularly processed HBV envelope, core, and polymerase proteins. Retroviral transduction resulted in a robust expression of all TCRs on primary T cells. A high functional avidity was measured for all TCRs specific for epitopes S17, S21, S36, and P774 (half-maximal effective concentration [EC₅₀] <10 nM), or C61 and preS9 (EC₅₀ <100 nM). Eight TCRs recognized peptide variants of HBV genotypes A to D. Both CD4⁺ and CD8⁺ T cells transduced with the MHC II-restricted TCRs were polyfunctional, producing interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and granzyme B (GrzB), and killed peptide-loaded target cells. Our set of MHC class II-restricted TCRs represents an important tool for elucidating CD4⁺ T cell help in viral infection with potential benefit for T cell therapy.

Keywords: CD4+ T cells; HBV clearance; MHC class II-restricted T cell receptors; T cell help; TCR expression; adoptive T cell therapy; chronic hepatitis B; hepatitis B virus infection; hepatocellular carcinoma; retroviral transduction.

Graphical abstract

Figure 1

Identification and cloning of TCRs from HBV-specific CD4⁺ T cell clones (A) MHC II alleles of donors 1–5 with acute or resolved HBV infection. For donor 3, high-resolution MHC II typing was not available (n.a.) due to limited material resources. n.d., none detected. (B) Overview of the procedure: PBMCs from donors with acute or resolved HBV infection were stimulated with 1 μM of selected HBV peptides derived from core, envelope, or polymerase proteins. After two weeks, HBV-specific TNF-α or TNF-α/IFN-γ-secreting CD4⁺ T cells were sorted by fluorescence-activated cell sorting and expanded from a single-cell level by limiting dilution cloning. After two further weeks of expansion with the addition of feeder cells and IL-2, clones were screened for HBV specificity and TCR α and β chains were identified by Sanger sequencing. Codon-optimized variable α and β domains (TRAV and TRBV) were cloned into the retroviral vector MP71 in combination with murine constant domains (mTRAC and mTRBC), including an additional cysteine (Cys) residue to increase pairing. TCR transduction rates were determined via flow cytometry by staining the mTRBC, here plotted against the cell volume (forward scatter, FSC). TCRs were phenotypically and functionally characterized through co-cultures of TCR-transduced T cells with peptide-pulsed target cells; i.e. HLA-matched B-LCLs or fibroblasts. (C) Final panel of identified TCRs. Clone and TCR names are indicated in square boxes. Peptide specificities from HBV core (C), envelope (preS/S), or polymerase (P) proteins are written below with the number indicating the peptide starting residue within the respective antigen. (D) Transduction rates (•, left y-axis) and average number of integrates per cell (x, right y-axis) of a representative cell batch. The average vector copy number per bulk cell population, i.e., including both transduced and non-transduced cells, was measured in a multiplex qPCR of the viral woodchuck hepatitis virus postregulatory element relative to the genomic single-copy gene PTBP2. (E) MFI of TCR⁺ populations in flow cytometry from four independent transductions normalized to mean of each experiment. Square boxes below TCRs indicate peptide specificities.

Figure 2

Verification of MHC II restrictions of HBV-specific TCRs TCR-transduced CD4⁺ T cells were co-cultured at an effector to target cell ratio of 2:1 with MHC II knockout (KO) fibroblasts or Raji-derived B-LCLs, stably transfected to express a single MHC II molecule and pulsed with 1 μM of target peptide. Each TCR was matched with target cells (limited to availability) co-expressing the corresponding MHC α and β chains of the respective donor; single MHC II transfectant cells are named after their respective MHC II β chain allele. TNF-α secretion was determined via ELISA and is shown relative to values from co-culture with the original B-LCLs of the respective donor. Data points represent mean values ±SD from triplicates. Controls without peptide were consistently below 5%, with the exception of TCR 2F2_C91, which showed similarly high TNF-α secretion during co-culture with HLA-DRB1∗13:01 target cells with or without peptide (data not shown). Square boxes at the top left of each graph indicate peptide specificities.

Figure 3

Recognition of physiologically processed HBV epitopes TCR-transduced CD4⁺ T cells were co-cultured at an effector to target cell ratio of 2:1 with HLA-matched B-LCLs that had been pre-incubated for 4 h with 10 or 1 μg/mL of core protein (A) or small (S) envelope protein (B). TNF-α secretion was determined after 16 h of co-culture via ELISA. Data points represent mean values ±SD from triplicates. Square boxes at the top left of each graph indicate peptide specificities.

Figure 4

Recognition of peptide variants of major HBV genotypes TCR-transduced CD4⁺ T cells were co-cultured at an effector to target cell ratio of 2:1 with HLA-matched B-LCLs pulsed with 1 μM of peptide from HBV genotypes A, B, C, and D. TNF-α secretion was determined via ELISA after 16 h of co-culture and is shown relative to values from co-culture with genotype A (Gt A), since Gt A peptides had been used for initial T cell stimulation and isolation of HBV-specific CD4⁺ T cell clones. Co-cultures without peptide (w/o pep.) served as negative control. Data points represent mean values ±SD from triplicates. Square boxes at the top left of each graph indicate peptide specificities. TCR 1D4_P774 was not included in this assay, since the P774 peptide is conserved across all four genotypes.

Figure 5

Functional avidity of MHC II-restricted TCRs determined with proliferation assay TCR-transduced CD4⁺ T cells were co-cultured at an effector to target cell ratio of 2:1 with HLA-matched B-LCLs titrating the amount of target peptide from 1 μM to 100 pM. Proliferation was assessed through integration of ³H-thymidine after 72 h of co-culture. Results are expressed as stimulation index, i.e., cpm of a stimulated sample divided by cpm of the unloaded control. Data points represent mean values ±SD from triplicates. All indicated EC₅₀ values were calculated with a non-linear dose-response ordinary fit. EC₅₀ values for C91- and C113-specific TCRs could not be calculated (n/a) because they did not reach a plateau of proliferation at the highest peptide concentration. Square boxes at the top left of each graph indicate peptide specificities.

Figure 6

Cytokine and GrzB secretion of TCR-transduced CD4⁺ or CD8⁺ T cells CD4⁺ and CD8⁺ T cells were co-transduced and then separated by positive selection through magnetic-activated cell sorting prior to the experiment with purities ≥98%. TCR-transduced CD4⁺ (A) or CD8⁺ (B) T cells were co-cultured with HLA-matched B-LCLs, then pulsed with 1 μM of target peptide (w/ peptide). Brefeldin A was added 1 h after co-culture start to retain cytokines in the endoplasmic reticulum (ER). TNF-α (blue bars),IL-2 (red bars), IFN-γ (green bars) and GrzB (gray bars) were measured via intracellular cytokine staining and flow cytometry after 14 h of co-culture in CD4⁺ TCR⁺ or CD8⁺ TCR⁺ subsets, respectively. Co-cultures without peptide (w/o peptide) served as negative control (empty bars in respective colors). Data points represent mean values ±SD from triplicates. Square boxes below TCRs indicate peptide specificities.

Figure 7

Cytotoxic capacity of TCR-transduced CD4⁺ and CD8⁺ T cells CD4⁺ and CD8⁺ T cells were co-transduced and then separated by positive selection through magnetic-activated cell sorting to purities of ≥98%. TCR-transduced CD4⁺ (red) or CD8⁺ (blue) T cells were co-cultured for 24 h with single MHC II transfectant fibroblasts pulsed with 1 μM of peptide (w/ pep) at an effector to target (E:T) cell ratio of 1:1 (dark color), 0.3:1 (medium color), or 0.1:1 (light color) or without peptide (w/o pep) at E:T ratio 1:1 (gray). Cytotoxicity was assessed via the adherence of target cells measured through electrical impedance and is given as a cell index normalized to the starting point of each co-culture. Considering the technical requirements of this assay, only TCRs were included, for which adherent single MHC II transfectant fibroblasts were available. (A) Cytotoxicity kinetics for exemplary TCRs 1C11_C61 and 3H6_C91. Data points were acquired every 30 min and represent mean values from triplicates. (B) Endpoint cytotoxicity after 24 h of co-culture for exemplary TCRs. The normalized cell index is given relative to killing of target cells without peptide at the highest E:T of 1:1, with the exception of TCR 2F2_C91(∗), where samples with peptide at an E:T of 0.1:1 are set to 100%. Data points represent mean values ±SD from triplicates. Square boxes at the top left of each graph indicate peptide specificities.