Original Article

Cancer Immunology, Immunotherapy

, Volume 57, Issue 3, pp 289-302

Open Access This content is freely available online to anyone, anywhere at any time.

The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays

  • C. M. BrittenAffiliated withDepartment of Immunohaematology and Blood Transfusion, Leiden University Medical Center Email author 
  • , C. GouttefangeasAffiliated withDepartment of Immunology, University of Tuebingen Email author 
  • , M. J. P. WeltersAffiliated withDepartment of Immunohaematology and Blood Transfusion, Leiden University Medical Center
  • , G. PawelecAffiliated withCentre for Medical Research, University of Tuebingen
  • , S. KochAffiliated withCentre for Medical Research, University of Tuebingen
  • , C. OttensmeierAffiliated withCancer Sciences Division, Southampton University Hospitals
  • , A. ManderAffiliated withCancer Sciences Division, Southampton University Hospitals
  • , S. WalterAffiliated withImmatics Biotechnologies
  • , A. PaschenAffiliated withSkin Cancer Unit of the German Cancer Research Centre, University Clinics of Mannheim
    • , J. Müller-BerghausAffiliated withPaul-Ehrlich-Institute
    • , I. HaasAffiliated withDepartment of Haematology and Oncology, University of Regensburg
    • , A. MackensenAffiliated withDepartment of Haematology and Oncology, University of Regensburg
    • , T. KøllgaardAffiliated withDepartment of Haematology, Centre for Cancer Immune Therapy
    • , P. thor StratenAffiliated withDepartment of Haematology, Centre for Cancer Immune Therapy
    • , M. SchmittAffiliated withDepartment of Immunohaematology and Blood Transfusion, Leiden University Medical CenterThird Department of Internal Medicine, University of Ulm
    • , K. GiannopoulosAffiliated withDepartment of Immunohaematology and Blood Transfusion, Leiden University Medical CenterClinical Immunology Department, Medical University of Lublin
    • , R. MaierAffiliated withDepartment of Immunohaematology and Blood Transfusion, Leiden University Medical CenterResearch Department, Kantonal Hospital St Gallen
    • , H. VeelkenAffiliated withDepartment of Immunohaematology and Blood Transfusion, Leiden University Medical CenterDepartment of Haematology and Oncology, Freiburg University Medical Centre
    • , C. BertinettiAffiliated withDepartment of Immunohaematology and Blood Transfusion, Leiden University Medical CenterDepartment of Haematology and Oncology, Freiburg University Medical Centre
    • , A. KonurAffiliated withDepartment of Immunohaematology and Blood Transfusion, Leiden University Medical CenterThird Medical Department, University Mainz
    • , C. HuberAffiliated withDepartment of Immunohaematology and Blood Transfusion, Leiden University Medical CenterThird Medical Department, University Mainz
    • , S. StevanovićAffiliated withDepartment of Immunology, University of Tuebingen
    • , T. WölfelAffiliated withDepartment of Immunohaematology and Blood Transfusion, Leiden University Medical CenterThird Medical Department, University Mainz
    • , S. H. van der BurgAffiliated withDepartment of Immunohaematology and Blood Transfusion, Leiden University Medical CenterDepartment of Clinical Oncology, Leiden University Medical Center Email author 

Abstract

The interpretation of the results obtained from immunomonitoring of clinical trials is a difficult task due to the variety of methods and protocols available to detect vaccine-specific T-cell responses. This heterogeneity as well as the lack of standards has led to significant scepticism towards published results. In February 2005, a working group was therefore founded under the aegis of the Association for Immunotherapy of Cancer (“CIMT”) in order to compare techniques and protocols applied for the enumeration of antigen-specific T-cell responses. Here we present the results from two consecutive phases of an international inter-laboratory testing project referred to as the “CIMT monitoring panel”. A total of 13 centers from six European countries participated in the study in which pre-tested PBMC samples, synthetic peptides and PE-conjugated HLA-tetramers were prepared centrally and distributed to participants. All were asked to determine the number of antigen-specific T-cells in each sample using tetramer staining and one functional assay. The results of the first testing round revealed that the total number of cells analyzed was the most important determinant for the sensitive detection of antigen-specific CD8+ T-cells by tetramer staining. Analysis by ELISPOT was influenced by a combination of cell number and a resting phase after thawing of peripheral blood mononuclear cells. Therefore, the experiments were repeated in a second phase but now the participants were asked to change their protocols according to the new guidelines distilled from the results of the first phase. The recommendations improved the number of antigen-specific T-cell responses that were detected and decreased the variability between the laboratories. We conclude that a two-step approach in inter-laboratory testing allows the identification of distinct variables that influence the sensitivity of different T-cell assays and to formally show that a defined correction to the protocols successfully increases the sensitivity and reduces the inter-center variability. Such “two-step” inter-laboratory projects could define rational bases for accepted international guidelines and thereby lead to the harmonization of the techniques used for immune monitoring.

Keywords

ELISPOT Tetramer Standardization Interlaboratory testing