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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-012-1351-0) contains supplementary material, which is available to authorized users.
Highlights
To evaluate immunotherapies for cancer, it is needed to show the specific ability or capacity of the product to achieve a defined biological effect based on its expected mechanism of action
Information on the existing frequency of antigenspecific T cells in cell samples is derived from pre-testing in central laboratories or by consensus, and this can only serve as an approximation of the real number of antigenspecific cells present in a given sample [1,2,3,4]; especially, in the field of cancer immunotherapy, an estimation of frequency may fail to reveal subtle aspects of assay protocols that allow the accurate detection of tumor-specific T cells at the generally low frequencies reported in patients
We aimed at generating standard samples with defined number of antigen-specific CD8-T cells spiked into autologous peripheral blood mononuclear cells (PBMC) for use in HLA/peptide multimer experiments following a similar gating strategy as applied to patient PBMC samples
Summary
To evaluate immunotherapies for cancer, it is needed to show the specific ability or capacity of the product to achieve a defined biological effect based on its expected mechanism of action. In order to generate reference material, it has been proposed to spike PBMC with previously isolated T cell clones (TCC) with known reactivity [6, 7] This is not a robust option because TCC have a limited life span and the production of quality standard samples requires huge expansions from a single TCC, meaning that new TCC (of constant quality) should be generated constantly. TCR-transduced T cells spiked into autologous PBMC may form a valuable tool to increase the accuracy of the measurements of immune responses and for performing periodic quality controls of assays They can be used to compare the measurements of different laboratories within proficiency panels, and as such, they can be used to optimize and standardize immunomonitoring assays. They offer the possibility to fully compare the results of one laboratory to another
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