Article Figures & Data
Figures
- Figure 1.
Amplification of TCR genes and cloning into retroviral plasmid vectors. A, Schematic representation of the TCR-expressing cassette. Abbreviations: LTR, long terminal repeats; ψ, packaging signal; SA, splice acceptor site; WRE, Woodchuck hepatitis virus posttranscriptional regulatory element. B, TCR amplification and cloning procedures. Detailed procedures are described in the Materials and Methods section. Vec' indicates anchor sequence to provide common sequence for amplification by the common primer sets. Anchor sequence is extended by Vec'' during PCR amplification to provide 30 nucleotide overlap (designated as Vec) with the destination plasmid. Steps 1 to 4 were shown only for TCRβ chain for simplicity. For the α chain, Steps 2 to 4 are performed in a separate tube using different primers: HTTCR#F in Step 2 and HTTCR#D and #E in Step 4.
- Figure 2.
Construction of TCR-expressing retroviral vectors from tumor antigen–specific T-cell clones. A, Expression of the TCR transgene after retroviral transduction. Bulk plasmids were used for production of retroviral particles. Polyclonally activated T cells that were transduced by retroviral JD-TCR (Vβ16) or KQ-TCR (Vβ5.3)–expressing vectors were stained by Vβ subtype–specific antibodies. B, Binding of TCR gene–transduced T cells to a specific MHC/peptide tetramer. HLA-B*35–restricted NY-ESO-1(94-102) peptide–specific KQ-TCR and HLA-A*02–restricted NY-ESO-1(157-165) peptide–specific JD-TCR–transduced T cells were stained with the corresponding tetramers and analyzed by flow cytometry. C, Recognition of NY-ESO-1–expressing cancer cell lines. TCR gene–transduced T cells were cocultured with cancer cells for 6 hours in the presence of Monensin. Expression of IFNγ and TNFα was examined by intracellular staining. Each experiment was performed at least twice with similar results.
- Figure 3.
Construction and characterization of TCR gene library of peripheral polyclonal T cells. Vβ subtype frequencies in peripheral T cells and TCR gene library–transduced J.RT3 were compared. Frequencies of Vβ subtype–expressing CD3+ T cells in PBMC and TCR gene library–transduced J.RT3 were determined by flow cytometry. Because of the limited availability of Vβ subtype–specific antibodies, total percentages of peripheral T cells that were stained by any of these antibodies were 59%, 62%, and 57% for 3 donors. Frequency for J.RT3 was normalized to the corresponding percentages in peripheral T cells. A, Frequencies of Vβ subtype–expressing CD3+ PBMC were plotted against the corresponding values in J.RT3. B, Mean frequencies for CD3+ PBMC and J.RT3 were compared. Each bar shows mean frequency and the standard deviation. Statistical significance is shown as * (P < 0.05) and ** (P < 0.01) by the paired t test. Each experiment was performed at least twice with similar results.
- Figure 4.
Construction of tumor-derived TCR gene library. A, Procedures for TCR gene library construction and screening. (1) Tumor specimens that were enriched with tumor-reactive T cells were selected for experiments. (2) TCR genes were amplified from cDNA and (3) randomly assembled into the destination plasmid vector together with the β-chain constant region-P2A fusion gene fragment. (4) Activated peripheral T cells from healthy individuals were transduced with the TCR library and sorted for tumor antigen specificity using the MHC/peptide tetramer reagent. (5) TCR transgenes were amplified from genomic DNA of the sorted tetramer-stained cells and (6) reassembled into the destination vector. (7) Activated T cells were transduced by the secondary library and tested for tumor antigen specificity. B, Randomly paired TCR-expressing libraries were constructed from mixtures of normalized cDNA of HLA-A*02–restricted NY-ESO-1(157-165)–specific AL and irrelevant T-cell clones. Polyclonally activated T cells were transduced by retroviral libraries and stained by A*02/NY-ESO-1(157-165) tetramer. C, Tetramer+ T cells in B were sorted, and integrated TCR-expressing transgenes were amplified and reassembled in TCR-expressing plasmid to create the secondary library. Polyclonally activated T cells were transduced by the secondary libraries and stained by A*02/NY-ESO-1(157-165) tetramer. D, Staining of T cells transduced with the tumor-derived TCR library by HLA-Cw*03/NY-ESO-1(92-100) tetramer. Activated T cells were transduced with the tumor-derived TCR-expression retroviral library. Two days after transduction, cells were stained by the tetramer followed by CD8. Each experiment was performed at least twice with similar results.
- Figure 5.
Characterization of library-derived tumor antigen–specific TCR. A, Tetramer staining of T cells transduced with the secondary TCR gene library. B, Reactivity of the secondary TCR gene library–transduced T cells against the cognate peptide. Transduced T cells were cocultured with NY-ESO-1(92-100) peptide–pulsed or –unpulsed Cw*03+ target cells for 6 hours in the presence of Monensin and intracellularly stained for IFNγ following cell-surface CD8 staining. C, Reactivity to cancer cells. Cw*03+NY-ESO-1+ A2780 were treated with or without IFNγ for 2 days and were used as target cells in intracellular IFNγ staining of tumor #3–derived bulk secondary TCR library–transduced T cells. D, Therapeutic effect of secondary TCR gene library–transduced T cells. Cw*03+NY-ESO-1+ A2780 were treated in vitro with IFNγ for 2 days and subcutaneously inoculated into NSG mice. On day 3, mice were infused with the tumor #3–derived bulk secondary TCR library–transduced or –untransduced T cells. Controls were left untreated. Tumor growth was monitored by measuring tumor diameters. Statistical significance (P < 0.05) is shown as * by t test. Each experiment was performed at least twice with similar results.
Additional Files
Supplementary Data
- Figure S1 - Fig. S1 describes about the destination plasmid.
- Figure S2 - Fig. S2 shows the tetramer-staining of MHC class I-restricted TCR gene-engineered T cells.
- Figure S3 - Fig. S3 shows the antigen-specific reactivity of MHC class II-restricted TCR gene-engineered T cells.
- Figure S4 - Fig. S4 describes about the recovery of antigen-specific TCR from plasmid mixtures.
- Figure S5 - Fig. S5 describes characterization of clones in the library by DNA fingerprinting.
- Figure S6 - Fig. S6 describes tetramer staining of library-derived TCR gene-engineered T cells.
- Figure S7 - Fig. S7 describes recognition of Glycine-substituted peptides by library-derived TCR.
- Tables S1 and S2 - Tables S1 and S2 describe sequences of primers and TCR, respectively.
Volume 6, Issue 5
