IFNg secretion (pg/mL) by TCR-5 transduced lymphocytes upon coculture with indicated targets.

<p>IFNg secretion (pg/mL) by TCR-5 transduced lymphocytes upon coculture with indicated targets.</p

Analysis of expression and biological function of codon-optimized TCR-5 in human lymphocytes transduced with clinical grade retroviral vector supernatants.

<p>A) Flow cytometry analysis of tetramer staining of T cells transduced with vector supernatants produced by stable packaging line clones A8, A10, C3, D8, F2 and H2. Clones were previously selected as those displaying the highest expression of viral RNA using dot plot analysis. Results of tetramer and CD8 staining of T cells are shown for three patients. B) IFNg ELISA of supernatants from cocultures of T cells transduced with vectors derived from clones A8, A10, C3, D8, F2 and H2. HLA-A*0201+ SSX2+ melanoma cells 624, HLA-A*0201- SSX2+ melanoma cells 938 and HLA-A*0201+ SSX2- cells CosA2 were used as targets.</p

Scatter plot for <i>q</i>² against <i>r</i>² (calculated on the training set) for the three real 3D-QSAR models (red crosses) and for those obtained with randomized cross-reactivities (black circles).

<p>The real 3D-QSAR models are well-separated from the random cases, implying robust real models.</p

Analysis of recognition of other genes by TCR-5.

<p>Peripheral blood T cells expressing TCR-5 were cocultured overnight with T2 cells previously pulsed with the serial dilutions of the indicated peptides. Results of IFNg concentration in culture supernatants are expressed as average of duplicates in a representative experiment. Sequence alignment of the tested peptides is shown in the figure legend for A) SSX-family genes and B) non-SSX genes with overlapping sequences. IGSF22: immunoglobulin superfamily member 22, ARHGAP1: Rho GTPase-activating protein 1, GPR82: Probable G-protein coupled receptor 82, PHF8: histone lysine demethylase PHF8, LIPM: lipase member M, SYT14: synaptotagmin-14, TCOF1: treacle protein, RBL2: retinoblastoma-like protein 2, FRAS1: extracellular matrix protein FRAS1. Prediction of binding affinity to HLA-A2*0201 is shown for each peptide, expressed as dissociation constant (K_D, nM).</p

Selected peptides.

<p>Selected peptides.</p

Codon optimization and replacement of TCR constant regions with murine sequences.

<p>A) Schematic representation of the three constructs generated for the expression of TCR-5 and derivatives. LTR: long terminal repeat, sd: splice donor, sa: splice acceptor, ψ: retrovirus encapsidation signal, MC: mouse TCR constant region, 2A: linker peptide. B) Analysis of expression of TCR-5 variants by tetramer staining. OKT3-stimulated lymphocytes were transduced twice with the corresponding TCR-expressing vector and stained with anti-CD3, anti-CD8 and SSX2_41-49 tetramers one week after transduction. Representative results from three independent experiments. Values in parentheses represent the mean fluorescence intensity of tetramer staining within the CD8 T cell population. C) ⁵¹Cr-release assay for the evaluation of antigen-specific cytolysis induced by TCR-5-transduced lymphocytes after four-hour coculture with the indicated target cells. Percentage of lysis depicted for each target cell line at different effector:target ratios is the average of duplicates in a representative experiment of three independent experiments. UT: untransduced T cells used as negative control of unspecific lysis, WT: Wild-type TCR, Co Op: copon-optimized TCR, MCR: codon-optimized TCR with mouse constant region.</p

The heavy atom RMSD to the X-ray structure of the parental peptide Melan-A_26–35A27L (ELAGIGILTV) versus the predicted cross-reactivity for the ELX set: a correlation can be observed (correlation coefficient:−0.84).

<p>In fact, an increased RMSD to the parental peptide corresponds to a lower predicted cross-reactivity.</p

TCRs isolated from SSX2-reactive lymphocyte clones.

<p>TRAV: TCR alpha-chain variable region.</p><p>TRBV: TCR beta-chain variable region.</p

Correlation between the predicted and experimental cross-reactivities for the external test set (6 complexes).

<p>The correlation coefficient is 0.92; the slope is 0.97 and the intercept is −2.1.</p

The backbone RMSD to the X-ray structure of the parental peptide Melan-A_26–35A27L (ELAGIGILTV) or to the predicted structure of the parental peptide Melan-A_26–35 (peptide 22; EAAGIGILTV) versus the experimental cross-reactivity: no correlation can be observed.

<p>The backbone RMSD to the X-ray structure of the parental peptide Melan-A_26–35A27L (ELAGIGILTV) or to the predicted structure of the parental peptide Melan-A_26–35 (peptide 22; EAAGIGILTV) versus the experimental cross-reactivity: no correlation can be observed.</p