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

  • Chudley, Lindsey1
  • McCann, Katy J.1
  • Coleman, Adam1
  • Cazaly, Angelica M.1
  • Bidmon, Nicole2
  • Britten, Cedrik M.2
  • van der Burg, Sjoerd H.3
  • Gouttefangeas, Cecile4
  • Jandus, Camilla5
  • Laske, Karoline4
  • Maurer, Dominik6
  • Romero, Pedro5
  • Schröder, Helene2
  • Stynenbosch, Linda F. M.3
  • Walter, Steffen6
  • Welters, Marij J. P.3
  • Ottensmeier, Christian H.1, 7
  • 1 University of Southampton, Cancer Sciences Unit, Faculty of Medicine, Experimental Cancer Medicine Centre, Southampton General Hospital, Tremona Road, Southampton, SO16 6YD, UK , Southampton (United Kingdom)
  • 2 Johannes-Gutenberg University GmbH, Translational Oncology, University Medical Center, Mainz, Germany , Mainz (Germany)
  • 3 Leiden University Medical Centre, Department of Clinical Oncology, Leiden, The Netherlands , Leiden (Netherlands)
  • 4 Eberhard-Karls University, Department of Immunology, Institute for Cell Biology, Tübingen, Germany , Tübingen (Germany)
  • 5 Ludwig Institute for Cancer Research, Translational Tumour Immunology, Lausanne, Switzerland , Lausanne (Switzerland)
  • 6 Immatics Biotechnologies GmbH, Tübingen, Germany , Tübingen (Germany)
  • 7 University of Southampton, Somers Cancer Research Building (Mailpoint 824), Cancer Sciences Unit, Faculty of Medicine, Southampton General Hospital, Tremona Road, Southampton, SO16 6YD, UK , Southampton (United Kingdom)
Published Article
Cancer Immunology Immunotherapy
Publication Date
Aug 19, 2014
DOI: 10.1007/s00262-014-1593-0
Springer Nature


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 R2 = 0.5113), but not before IVS. In summary, IVS was shown to be a reproducible method that benefitted from method harmonisation.

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