The dynamics of CD62L shedding is inversely correlated with the expression of CD107a.

<p><b>A.</b> The dynamics of CD62L shedding in CD8+/CD62L+ population was followed over 6hr. CD8+ MART-1 TCR transduced Tcm cells were sorted by FACS as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022560#pone-0022560-g003" target="_blank">figure 3</a>. These sorted cells (CD45RO+/CD62L+) were co-cultured with melanoma lines 526 (HLA-A0201+) and 938 (HLA-A0201−) for the times indicated and analyzed for CD45RO and CD62L. Column on left (co-cultured with 938) and on right (co-cultured with 526). The numbers in each quadrant represent the percent of cells in that quadrant. <b>B.</b> The expression of CD107a correlates with the shedding of CD62L in CD8+/CD62L+ population. CD8+ MART-1 TCR transduced T cells from a different donor were co-cultures as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022560#s2" target="_blank">methods</a>. The cells were gated on CD3/CD8/MART-1+ and then plotted for CD107a and CD62L expression. The samples were collected and stained at the times indicated on right. The fluorophores conjugated antibodies for this analysis were CD107a FITC, MART-1 PE, CD62L APC, CD45RO PE-Cy7, CD8 APC-Cy7 and propidium iodide staining.</p

The loss of CD62L is tumor antigen specific and the expression of CD107a occurs on CD62L negative cells.

<p><b>A.</b> TCR transduced cells were cultured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022560#s2" target="_blank">methods</a>. Shown is the FACS analysis, where cells were gated on CD3/CD8/MART-1+ or CD3/CD8/MART-1−, and then plotted using markers CD45RO and CD62L (left two column plots). The expression of CD107a on each group was then determined and separately plotted in the histograms on right corresponding to column data on left. In each histogram the expression of CD107a on each subset was overlaid reflecting Tem, Tcm and naïve populations. Red (CD45RO+CD62L−) indicates Tem, blue (CD45RO+CD62L+) indicates Tcm, and green (CD45RO−CD62L+) indicates Tn population. <b>B.</b> The cells were processed as described above. By FACS analysis, the cells were gated on CD3/CD8/MART-1+ or CD3/CD8/MART-1−, then plotted using markers CD107a and CD62L in dot plots. The fluorophore conjugated antibodies used in this analysis were CD107a FITC, CD62L PE, CD45RO APC, CD3 APC-Cy7 with propidium iodide staining.</p

Introduction of shedding resistant CD62L into JKF6 hinders CD107a expression following non-specific activation.

<p><b>A.</b> JKF6 lines expressing CD62L and mutant dK-S were activated using PMA/Ionomycin (PMA/Ion) for 4 h, and the surface expression for CD107a and IFN-γ was evaluated by FACS. One representative result from three independent experiments is shown (Mock, mock transduced cells; WT, CD62L transduced JKF6; dK-S, JKF6 transduced with shedding resistant mutant). For CD107a live cell staining, the fluorophores conjugated antibodies were CD107a FITC, CD62L APC, CD3 PE and propidium iodide staining; For intracellular staining of IFN-γ, the fluorophore conjugated antibodies were IFN-γ FITC, CD62L APC, CD3 PE, without propidium iodide staining. <b>B.</b> Plot of the percent positive cells for CD107a and IFN-γ on the surface of JKF6 lines expressing wild type and shedding resistant CD62L mutant. The mean ± STDEV from three independent experiments is displayed, and t-test was used for statistical analysis.</p

The loss of CD62L is specific and correlates with CD107a surface expression.

<p><b>A.</b> MART-1 TCR vector-transduced T cells were co-cultured with melanoma lines 526, 624 (both HLA-A*0201 positive) and 938 (HLA-A*0201 negative) for four hours and then analyzed by FACS. The first column on left, the cells were plotted using differentiation markers CD45RO and CD62L into 4 subsets, the expression of CD107a on each subset was denoted in the histograms to the right. <b>B.</b> Based on data from three independent experiments, the expression of CD107a after co-culture with melanoma lines was determined and mean% ± stdev plotted. Student t-test was used for statistical analysis. The fluorophore conjugated antibodies used in this analysis were CD107a FITC, CD62L PE, CD45RO APC, CD3 APC-Cy7 with propidium iodide staining.</p

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

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

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

Generation of retroviral vectors for the expression of SSX2-specific TCRs.

<p>The coding region of each TCR alpha-chain was amplified by PCR using primers flanked by an NcoI restriction site in the 5′ end and an overhanging sequencing containing the element in the 3′ end. In parallel, each TCR beta-chain was amplified by PCR using a forward primer containing a 5′ overhang that overlaps with the 3′ overhang present in the primer used for the amplification of the alpha-chain. The reverse primer used for amplification of the beta-chain contained a stop codon and an <i>Eco</i>RI restriction site. In a second round of PCR, the products of the alpha- and beta-chain amplifications were pooled, and ligation of both cDNA fragments through the overlapping overhangs was achieved by PCR using external primers. The resulting PCR products were cloned in pMSGV1 vector for retrovirus production. LTR: long terminal repeat, sd: splice donor, sa: splice acceptor, ψ: retrovirus encapsidation signal.</p

TCRs isolated from SSX2-reactive lymphocyte clones.

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