Harmonisation of short-term in vitro culture for the expansion of antigen-specific CD8^sup +^ T cells with detection by ELISPOT and HLA-multimer staining

• Published in: Cancer immunology, immunotherapy Vol. 63; no. 11; p. 1199
• Main Authors: Chudley, Lindsey, Mccann, Katy J, Coleman, Adam, Cazaly, Angelica M, Bidmon, Nicole, Britten, Cedrik M, van der Burg, Sjoerd H, Gouttefangeas, Cecile, Jandus, Camilla, Laske, Karoline, Maurer, Dominik, Romero, Pedro, Schröder, Helene, Stynenbosch, Linda F; M, Walter, Steffen, Welters, Marij J; P, Ottensmeier, Christian H
• Format: Journal Article
• Language: English
• Published: Heidelberg Springer Nature B.V 01.11.2014
• Subjects: Antigens; Cancer research; Consortia; Immunology; Immunotherapy; Laboratories; Lymphocytes; Medical research; Methods; Oncology; Peptides; Vaccines; United Kingdom > UK; Europe; Germany
• ISSN: 0340-7004; 1432-0851
• DOI: 10.1007/s00262-014-1593-0

Ex vivo ELISPOT and multimer staining are well-established tests for the assessment of antigen-specific T cells. Many laboratories are now using a period of in vitro stimulation (IVS) to enhance detection. Here, we report the findings of a multi-centre panel organised by the Association for Cancer Immunotherapy Immunoguiding Program to investigate the impact of IVS protocols on the detection of antigen-specific T cells of varying ex vivo frequency. Five centres performed ELISPOT and multimer staining on centrally prepared PBMCs from 3 donors, both ex vivo and following IVS. A harmonised IVS protocol was designed based on the best-performing protocol(s), which was then evaluated in a second phase on 2 donors by 6 centres. All centres were able to reliably detect antigen-specific T cells of high/intermediate frequency both ex vivo (Phase I) and post-IVS (Phase I and II). The highest frequencies of antigen-specific T cells ex vivo were mirrored in the frequencies following IVS and in the detection rates. However, antigen-specific T cells of a low/undetectable frequency ex vivo were not reproducibly detected post-IVS. Harmonisation of the IVS protocol reduced the inter-laboratory variation observed for ELISPOT and multimer analyses by approximately 20 %. We further demonstrate that results from ELISPOT and multimer staining correlated after (P < 0.0001 and R ^sup 2^ = 0.5113), but not before IVS. In summary, IVS was shown to be a reproducible method that benefitted from method harmonisation.[PUBLICATION ABSTRACT]