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Blood, Vol. 112, Issue 5, 1876-1885, September 1, 2008
Cytotoxic T lymphocytes directed to the preferentially expressed antigen of melanoma (PRAME) target chronic myeloid leukemia
Blood Quintarelli et al.
112: 1876
Supplemental materials for: Quintarelli et al
Files in this Data Supplement:
- Figure S1. Peptide titration experiments for PRAME-specific CTLs generation (JPG, 30 KB)
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The optimal peptide concentration for the generation of PRAME-specific T cells was determined in preliminary titration experiments loading pAPCs and aAPCs with decreasing concentration of PRAME-peptides (from 50 to 0.05 µM). The figure shows the titration of ALY-peptide for the generation of ALY-specific CTLs in one representative HLA-A*02 donor, using a specific IFN-γ ELIspot assay. CTLs generated using 5µM of ALY-peptide produced significant higher numbers of IFNγ+ SFC when stimulated with the specific peptide (black bars) as compared to CTLs generated using 0.5 or 0.05 µM of the same peptide (85 ± 6 IFNγ+ SFC/1 × 105 cells vs. 12 ± 2 IFNγ+ SFC/1 × 105 or 25 ± 9 IFNγ+ SFC/1 × 105, respectively). The number of IFNγ+ SFC was negligible when T cells were tested in ELIspot assay using media alone (white bars) or an irrelevant peptide (gray bars).
- Figure S2. Generation of CTLs specific for other tumor associated antigens (JPG, 59.5 KB)
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Using the same culture conditions optimized for the generation of PRAME-CTLs we evaluated whether this approach could be extended to generate CTL targeting other TAA. (A) shows the frequency of IFNγ+ T cells, using the Elispot assay, in CTL lines generated from HLA-A*02+ healthy donors against peptides derived from the following TAA: WT1 (RMFPNAPYL), MART-1 (ELAGIGILTV), Tyr (RLVDDFLLV), PR3 (VLQELNVTV), MAGE-A3 (KVAELVHFL) and hTERT (ILAKFLMWL and RLVDDFLLV). Numbers on the bars indicate the number of donors for whom CTL lines were successfully generated. (B–C) show the specificity and functionality of ELA-specific CTLs generated from a representative healthy donor. Tetramer staining and cytotoxic activity are shown in panel B and C, respectively.
- Figure S3. Sensitivity of PRAME-CTLs healthy donor-derived by peptide titration (JPG, 40.6 KB)
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(A) Since only two out of nine PRAME-CTL lines showed specific binding with the ALY-tetramer at FACS analysis, we incubated ALY-tetramer+ CTLs (black bars) and ALY-tetramer negative CTLs (gray bars) with decreasing concentration of ALY-peptide (from 200 to 0.02 nM) to characterize their αβTCR avidivty. The figure shows that ALY-tetramer+ CTLs produced significant higher numbers of IFNγ+ SFC at the 0.2 nM peptide concentration as compared to ALY-tetramer negative CTLs (600 IFNγ+ SFC/1 × 105 cells vs. 35 ± 8 IFNγ+ SFC/1 × 105). In addition, (B) shows that PRAME-specific T cell lines generated from two CML patients (#3 and #1) produced IFNγ+ SFC only if exposed to more than 2 nM peptide concentration.
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