title: Harmonisation of short-term in vitro culture for the expansion of antigen-specific CD8(+) T cells with detection by ELISPOT and HLA-multimer staining.
abstract: 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.
authors.affiliation: Cancer Sciences Unit, Faculty of Medicine, Experimental Cancer Medicine Centre, Southampton General Hospital, University of Southampton, Tremona Road, Southampton, SO16 6YD, UK, ll4@soton.ac.uk.
authors.forename: Lindsey
authors.initials: L
authors.lastname: Chudley
authors.forename: Katy J
authors.initials: KJ
authors.lastname: McCann
authors.forename: Adam
authors.initials: A
authors.lastname: Coleman
authors.forename: Angelica M
authors.initials: AM
authors.lastname: Cazaly
authors.forename: Nicole
authors.initials: N
authors.lastname: Bidmon
authors.forename: Cedrik M
authors.initials: CM
authors.lastname: Britten
authors.forename: Sjoerd H
authors.initials: SH
authors.lastname: van der Burg
authors.forename: Cecile
authors.initials: C
authors.lastname: Gouttefangeas
authors.forename: Camilla
authors.initials: C
authors.lastname: Jandus
authors.forename: Karoline
authors.initials: K
authors.lastname: Laske
authors.forename: Dominik
authors.initials: D
authors.lastname: Maurer
authors.forename: Pedro
authors.initials: P
authors.lastname: Romero
authors.forename: Helene
authors.initials: H
authors.lastname: Schröder
authors.forename: Linda F M
authors.initials: LF
authors.lastname: Stynenbosch
authors.forename: Steffen
authors.initials: S
authors.lastname: Walter
authors.forename: Marij J P
authors.initials: MJ
authors.lastname: Welters
authors.forename: Christian H
authors.initials: CH
authors.lastname: Ottensmeier
mesh_terms: D018414:CD8-Positive T-Lymphocytes / Q000166:cytology*; D019411:Clinical Laboratory Techniques; D058501:Enzyme-Linked Immunospot Assay / Q000379:methods*; D005858:Germany; D006680:HLA Antigens / Q000737:chemistry*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000276:immunology; D009426:Netherlands; D015203:Reproducibility of Results; D013194:Staining and Labeling; D013557:Switzerland; D006113:United Kingdom
chemical_list: D006680:HLA Antigens