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Blood, Vol. 110, Issue 8, 2931-2939, October 15, 2007
Allorestricted T cells with specificity for the FMNL1-derived peptide PP2 have potent antitumor activity against hematologic and other malignancies
Blood Schuster et al.
110: 2931
Supplemental materials for: Schuster et al, Vol. 110, Issue 8, 2931-2939.
Files in this Data Supplement:
- Table S1. Prediction scores of selected peptides derived from FMNL1 (JPG, 52.9 KB)
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- Table S2. HLA-Typing of HLA-A2− LCL (JPG, 57.6 KB)
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- Figure S1. Generation of the FMNL1-specific antibody (JPG, 85.8 KB)
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(A) Human FMNL1 sequence (Swiss-Prot database, Accession No. O95466). Matched peptides detected by mass spectrometry analysis are shown (bold and framed) covering 24% of the protein. (B) Four different monoclonal antibody supernatants (5C9, 5B12, 6F2, 5A1) were tested in a Western blot using 50 µg of cell lysates from 293 HEK cells transfected with pCMV-FMNL1 (F) and pCMV-Vector alone (M).
- Figure S2. Peptide competition assay (JPG, 27.4 KB)
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Peptide candidates derived from FMNL1 were investigated for their potential binding ability to HLA-A2. Therefore, different FMNL1-derived peptides were loaded on T2 cells and afterwards pulsed with the tyrosinase-derived peptide (Tyr). Flu was used as positive control. IPS as well as peptide pulsed T2 cells, which were afterwards not pulsed with tyrosinase, were used as negative controls. Tyrosinase-specific recognition was investigated by the tyrosinase-specific T-cell clone IVSB in a (51Cr)-release assay at an effector:target ratio of 2.5:1.Error bars indicate the standard deviation of tested duplicates.
- Figure S3. T-cell receptor (TCR) analysis of the FMNL1-PP2-specific T-cell clone SK22 (JPG, 85.2 KB).
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(A) Analysis of the TCR  -chain repertoire of clone SK22 was performed using 34 single V alpha segment-specific primers. The primers V14 (band at 401bp) and V14.1 (band at 459bp) amplified identical in-frame CDR3-sequences. Control primers were used to amplify the constant chain (547 bp). Additional fuzzy bands did not result in any readable sequence. (B) TCR  -chain analysis was performed using 37 V beta segment-specific primers. The primers V13 (bands at 278 and 632bp) and V14 (bands at 187 and 541bp) resulted in identical in-frame CDR3-sequences. The primers for V6.2 and V6.3 (bands at 171 and 439bp) resulted in amplified sequences with out-off-frame gene rearrangements. Additional fuzzy bands did not result in any readable sequence. Control primers were used to amplify the constant chain (351bp). (C) The FMNL1-specific T-cell clone was stained with a FITC-conjugated antihuman V14-specific antibody and a PE-conjugated antihuman CD8 antibody (right plot). The isotype control is shown in the left plot.
- Figure S4. Crossreactivity against HLA-A*3303 (JPG, 19.5 KB)
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IFN -release was investigated by ELISA to test the FMNL1-PP2-specific T-cell clone against C1R cells transfected with HLA-A*3303 at an effector:target ratio of 1:2. C1R cells transfected with HLA-A*0201 were used as positive control and untransfected C1R cells as well as C1R cells transfected with HLA*6601 as negative controls. Error bars indicate the standard deviation of tested duplicates. Results shown are representative for two experiments.
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