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

Cancer Immunol Immunother. 2014 Nov;63(11):1199-211. doi: 10.1007/s00262-014-1593-0. Epub 2014 Aug 19.


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 (2) = 0.5113), but not before IVS. In summary, IVS was shown to be a reproducible method that benefitted from method harmonisation.

Publication types

  • Multicenter Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • CD8-Positive T-Lymphocytes / cytology*
  • Clinical Laboratory Techniques
  • Enzyme-Linked Immunospot Assay / methods*
  • Germany
  • HLA Antigens / chemistry*
  • Humans
  • Leukocytes, Mononuclear / immunology
  • Netherlands
  • Reproducibility of Results
  • Staining and Labeling
  • Switzerland
  • United Kingdom


  • HLA Antigens