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. 2013 Apr 26;8(4):e61384. doi: 10.1371/journal.pone.0061384

Figure 4. Characterization of functionality of a TCR isolated from rare antigen specific T cells.

Figure 4

(A) Cells double-positive for HLA-A2/Her2neu369–377 PE- and BV421-conjugated multimers were FACS sorted. The left FACS plot shows CD8 and MHC multimer staining of the cell fraction enriched with Her2/neu specific MHC-multimer. The right FACS plot serves as a negative control that was stained in the same way, but for MACS enrichment only Strep-Tactin backbone was used. Ten single-cell samples were prepared and processed for TCR sequencing. Full-length variable domain sequences of α- and β-chain full-length variable domain sequences from one specific TCR was identified and is shown in the table. (B) Sequence from TCR 4A was expressed in Jurkat76 T cells by retroviral gene transfer. Left FACS plot shows A2/Her2/neu369–377 staining and right FACS plot shows staining with an irrelevant MHC multimer. (C) Sequence from TCR 4A was expressed in human PBMCs by retroviral gene transfer. Left FACS plot shows A2/Her2/neu369–377 staining and right FACS plot shows staining with an irrelevant MHC multimer. (D) For functional testing 2×103 peptide-loaded T2 cells (1 µM) labeled with 51Cr were incubated for 4 h with TCR-4A modified PBLs at different effector/target ratios (E/T ratios). After 4 h free 51chrome was quantified in the supernatant. TCR-specificity was controlled by co-incubation with GFP-transduced and non transduced (n.t.) PBMCs. All conditions were tested in duplicates. (E) For detection of peptide specific release 1×105 T2-cells were pulsed with an equal number of Her2/neu peptide titrated from 10−5–10−9 M concentration. Equal numbers of transduced (black bar) and non-transduced (grey bar) T cells were co-incubated for 4 hours and IFNγ concentration in the supernatant was measured by ELISA. As positive control T cells were stimulated with CD3/CD28 and as control for peptide specificity T2-cells were pulsed with 1 µM of an irrelevant peptide. IFNγ concentration in the supernatant was measured by ELISA. (F) For detection of tumor specific activity105 tumor cells and equal numbers of transduced (black bar) and non transduced (grey bar) human PBMCs were co-incubated for 4 hours and IFNγ concentration in the supernatant was measured by ELISA. As positive control T cells were stimulated with CD3/CD28. All conditions were tested in triplicates.