Table 1.
IFNγ secretion (pg/ml) by T cell clones from healthy donor H4a
| Clone ID | T2 + HBV peptideb | T2 + WT1 peptideb | 293-A2-GFPc | 293-A2-WT1c |
|---|---|---|---|---|
| P1A10 | 56 | >2000d | 42 | >2000 |
| P1G11e | 67 | >2000 | 53 | 177 |
| P2H11 | 48 | >2000 | 59 | >2000 |
| P5A7 | 24 | >2000 | 40 | 370 |
| P5D2 | 35 | >2000 | 56 | >2000 |
| P5D4 | 34 | >2000 | 23 | >2000 |
| P6H3 | 40 | >2000 | 0 | >2000 |
| P8A6 | 20 | >2000 | 17 | >2000 |
| P8B5 | 35 | >2000 | 3 | >2000 |
| P9G11 | 29 | >2000 | 29 | >2000 |
CD8+ PBL from healthy donor H4 were stimulated in vitro 2 times with the WT1:126-134 peptide as described in the Material and Methods section. Bulk culture #6 was cloned by limiting dilution, and 130 growth-positive wells were screened for recognition of peptide and transfectants. 108 of those demonstrated specific peptide and transfectant reactivities, and data from 10 of those are presented here. IFNγ in coculture supernatants was measured by ELISA.
T2 cells were pre-loaded with 1 μg/ml of a control peptide from HBV with high binding affinity for HLA-A*0201 or WT1:126-134 ~1 hour prior to the coculture.
293 cells stably expressing high levels of HLA-A*0201 by means of retroviral transduction and antibiotic selection were transiently transfected with GFP cDNA as a negative control or full-length WT1 cDNA ~24 hours prior to the coculture.
underlined values indicate IFNγ secretion >100 pg/ml and >2X background with any negative control target cell.
TCR sequencing of cDNA from all clones except P1G11 indicated the presence of a single α chain (TRAV12-1*01; CDR3: CVVNTPPNTDKLIF) and a single β chain (TRBV7-2*01; CDR3: CASTPFTSGSGWDEQFF). Clone P1G11 contained a distinct TCR α chain (TRAV6*02; CDR3: CAFSGCARQLTF) and β chain (TRBV10-3*01; CDR3: CAISESMASGDNNEQFF).