title: The development of standard samples with a defined number of antigen-specific T cells to harmonize T cell assays: a proof-of-principle study.
abstract: The validation of assays that quantify antigen-specific T cell responses is critically dependent on cell samples that contain clearly defined measurable numbers of antigen-specific T cells. An important requirement is that such cell samples are handled and analyzed in a comparable fashion to peripheral blood mononuclear cells (PBMC). We performed a proof-of-principle study to show that retrovirally TCR-transduced T cells spiked at defined numbers in autologous PBMC can be used as standard samples for HLA/peptide multimer staining. NY-ESO-1157-165-specific, TCR-transduced CD8+ T cell batches were successfully generated from PBMC of several HLA-A*0201 healthy donors, purified by magnetic cell sorting on the basis of HLA tetramer (TM) staining and expanded with specific antigen in vitro. When subsequently spiked into autologous PBMC, the detection of these CD3+CD8+TM+ T cells was highly accurate with a mean accuracy of 91.6 %. The standard cells can be preserved for a substantial period of time in liquid nitrogen. Furthermore, TM staining of fresh and cryopreserved standard samples diluted at decreasing concentrations into autologous cryopreserved unspiked PBMC revealed that the spiked CD3+CD8+TM+ T cells could be accurately detected at all dilutions in a linear fashion with a goodness-of-fit of over 0.99 at a frequency of at least 0.02 % among the CD3+CD8+ T cell population. Notably, the CD3+CD8+TM+ cells of the standard samples were located exactly within the gates used to analyze patient samples and displayed a similar scatter pattern. The performance of the cryopreserved standard samples in the hands of 5 external investigators was good with an inter-laboratory variation of 32.9 % and the doubtless identification of one outlier.
authors.affiliation: Department of Clinical Oncology, Leiden University Medical Center, Building 1, K1-P, P.O. Box 9600, 2300 RC, Leiden, The Netherlands. sk_singh79@hotmail.com
authors.forename: Satwinder Kaur
authors.initials: SK
authors.lastname: Singh
authors.forename: Bart
authors.initials: B
authors.lastname: Tummers
authors.forename: Ton N
authors.initials: TN
authors.lastname: Schumacher
authors.forename: Raquel
authors.initials: R
authors.lastname: Gomez
authors.forename: Kees L M C
authors.initials: KL
authors.lastname: Franken
authors.forename: Els M
authors.initials: EM
authors.lastname: Verdegaal
authors.forename: Karoline
authors.initials: K
authors.lastname: Laske
authors.forename: Cécile
authors.initials: C
authors.lastname: Gouttefangeas
authors.forename: Christian
authors.initials: C
authors.lastname: Ottensmeier
authors.forename: Marij J P
authors.initials: MJ
authors.lastname: Welters
authors.forename: Cedrik M
authors.initials: CM
authors.lastname: Britten
authors.forename: Sjoerd H
authors.initials: SH
authors.lastname: van der Burg
mesh_terms: D000951:Antigens, Neoplasm / Q000276:immunology*; D001681:Biological Assay / Q000379:methods / Q000592:standards*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D006680:HLA Antigens / Q000276:immunology; D015789:HLA-A2 Antigen / Q000276:immunology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D015166:Monitoring, Immunologic / Q000592:standards*; D015588:Observer Variation; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D012016:Reference Values; D013194:Staining and Labeling; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: D000951:Antigens, Neoplasm; D006680:HLA Antigens; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D011948:Receptors, Antigen, T-Cell