Article Figures & Data
Figures
- FIGURE 1.
Cleavage of SSX-2 oligopeptides by proteasome. A set of 15 overlapping peptides covering the entire SSX-2 protein sequence (in one-letter code) was digested by human proteasome and analyzed by mass spectrometry. The numbers of each peptide correspond to the position of the first and last amino acid of the peptide within the protein. A cleavage is indicated by a solid vertical bar, which intersects with the peptide in which it was detected. Bars with dotted line and asterisks indicate that the same cleavage site was detected in two overlapping peptides.
- FIGURE 2.
Enumeration of SSX-241–49-specific cells in LAU 50 TILN and isolation of specific clonal populations. A, Short term-cultured TILN from patient LAU 50 were stained with PE-labeled A2/SSX-241–49 multimers and anti-CD8FITC mAb and analyzed by flow cytometry. The number in the upper right quadrant represents the percentage of multimer+ cells within CD8+ T lymphocytes. B, Clone LAU 50 E2.4 was stained with PE-labeled A2/SSX-241–49 (left) or A2/CAMEL1–11 (right) multimers and anti-CD8FITC mAb and analyzed by flow cytometry.
- FIGURE 3.
Functional characterization of SSX-241–49 specific CTL clone LAU 50 E2.4. A, Lytic activity of the clone was tested in a 51Cr release assay against T2 cells at an E:T ratio of 10:1 in the absence (∗) or in the presence of exogenously added peptides at the indicated concentrations. Left, Peptide SSX-241–49 (♦), SSX-240–49 (⋄) or MelanA26–35 A27L (○) were tested. Lysis of T2 cells pulsed with proteasome-digested peptide products was also tested (right). Three-fold dilutions, starting from 1 μM, of the precursor peptide SSX-237–58 encompassing the sequence of SSX-241–49 was added directly to T2 cells (•). In parallel, the precursor peptide was incubated with proteasome for 0 (▴) or 30 (▪) min at 37°C; the reaction was stopped by addition of 2% TFA; and samples were lyophilized, resuspended in medium to a concentration of 1 μM of the precursor peptide at time 0, and added in 3-fold dilution steps to T2 cells. B, Recognition of transfected COS-7 cells. Cells transfected with plasmids encoding HLA-A2, SSX-2, or SSX-4 (as indicated) were cocultured with effector cells at an E:T ratio of 2.5:1. The production of IFN-γ was measured by ELISA.
- FIGURE 4.
Lytic activity of SSX-241–49-specific clone LAU 50 E2.4 against melanoma cell lines. A, mRNA expression of SSX-2 in melanoma cell lines. RT-PCR was performed as described in Materials and Methods. SK-MEL-37 cells had already been shown to express SSX-2 and SK-MEL-23 cells had been shown not to express it (12 ). pcDNA plasmid containing SSX-2 cDNA was used as a positive control. B, Chromium-labeled target cells were incubated with effector cells at indicated E:T ratios in the absence (•) or presence (○) of SSX-241–49 synthetic peptide (0.1 μM). HLA-A2 expression was assessed by mAb staining and flow cytometry analysis (data not shown).
Tables
Peptide Sequence Score No. of Spots SSX-25–13 DAFARRPTV 17 5 SSX-27–15 FARRPTVGA 15 5 SSX-215–24 AQIPEKIQKA 16 3 SSX-216–24 QIPEKIQKA 21 4 SSX-240–49 MKASEKIFYV 16 39 SSX-241–49 KASEKIFYV 22 69 SSX-250–59 YMKRKYEAMT 15 3 SSX-253–61 RKYEAMTKL 15 4 SSX-257–65 AMTKLGFKA 16 4 SSX-258–67 MTKLGFKATL 17 7 SSX-259–67 TKLGFKATL 19 12 SSX-2103–111 RLQGISPKI 23 6 -
a HLA-A2-binding peptides (as predicted by motif-based algorithm) for which the C terminus could be generated in vitro by proteasome digestion of 22-aa precursors (Fig. 1⇑). Theoretical binding score given by motif-based prediction algorithm in SYFPEITHI database (http://www.syfpeithi.de; Ref. 20 ). Number of spots obtained by IFN-γ ELISPOT of LAU 50 TILN. Short term-cultured TILN from patient LAU 50 (2 × 104 cells/well) were tested by IFN-γ ELISPOT in the presence of T2 cells (5 × 104 cells/well) alone or in the presence of the indicated peptide in duplicates. The number of spots obtained in the absence of peptide or in the presence of control peptide HIV-RT476–484 was 3 and 13, respectively.
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