Table 2: Validation runs for the selection and amplification of Melan-A- and MELOE-1- specific T lymphocytes from HLA-A2 melanoma patient-derived PBMC.

Run ID123123
PeptideMelan-AA27LMELOE-136-44

Number of stimulated PBMC

Number of sorted cells and fraction of specific T cells107 6 107
(9.3%)(34%)(38%)(11%)(16%)(9%)
Theoretical number of specific T cells1
Recovered number of sorted specific T cells
Sorting yield242%39%11%22%60%19%
Final number of amplified specific T cells and amplification yield3
( 43500)( 10000)( 62000)( 16700)( 1900)( 25900)
Average number of cell divisions151316141015
Purity of amplified T cells495%99%92%90%91%93%
Activity of amplified T cells on the cognate peptide582%71%72%75%53%50%

The theoretical number of specific cells is estimated from the % of tetramer-positive cells in the sorted population. Sorting yields are calculated by dividing the number of rosetted T cells after sorting by the theoretical number of specific T cells in the sorted populations. Amplification factors are estimated from the final number of amplified T cells divided by the number of rosetted T cells seeded for amplification on feeder cells. Purity of sorted and amplified T cells is assessed by tetramer/CD8 double staining. % of TNF producing T cells among tetramer positive cells : reactivity of amplified T cells is assessed by tetramer/TNF double staining, after peptide activation.