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

The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8(+) T lymphocytes by structural and functional assays
The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8(+) T lymphocytes by structural and functional assays
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
ELISPOT, tetramer, standardization, interlaboratory testing
0340-7004
289-302
Britten, C.M.
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Gouttefangeas, C.
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Welters, M.J.P.
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Pawelec, G.
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Koch, S.
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Ottensmeier, C.
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Mander, A.
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Walter, S.
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Paschen, A.
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Muller-Berghaus, J.
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Haas, I.
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Mackensen, A.
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Kollgaard, T.
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Thor Straten, P.
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Schmitt, M.
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Giannopoulos, K.
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Maier, R.
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Veelken, H.
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Bertinetti, C.
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Konur, A.
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Huber, C.
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Stevanovic, S.
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Wolfel, T.
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Van der Burg, S.H.
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Britten, C.M.
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Gouttefangeas, C.
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Welters, M.J.P.
c9970409-fd91-4bc3-bb3b-d7e4a1be4f17
Pawelec, G.
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Koch, S.
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Ottensmeier, C.
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Mander, A.
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Walter, S.
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Paschen, A.
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Muller-Berghaus, J.
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Haas, I.
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Mackensen, A.
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Kollgaard, T.
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Thor Straten, P.
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Schmitt, M.
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Giannopoulos, K.
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Maier, R.
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Veelken, H.
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Bertinetti, C.
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Konur, A.
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Huber, C.
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Stevanovic, S.
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Wolfel, T.
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Van der Burg, S.H.
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Britten, C.M., Gouttefangeas, C., Welters, M.J.P., Pawelec, G., Koch, S., Ottensmeier, C., Mander, A., Walter, S., Paschen, A., Muller-Berghaus, J., Haas, I., Mackensen, A., Kollgaard, T., Thor Straten, P., Schmitt, M., Giannopoulos, K., Maier, R., Veelken, H., Bertinetti, C., Konur, A., Huber, C., Stevanovic, S., Wolfel, T. and Van der Burg, S.H. (2008) The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8(+) T lymphocytes by structural and functional assays. Cancer Immunology Immunotherapy, 57 (3), 289-302. (doi:10.1007/s00262-007-0378-0).

Record type: Article

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

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More information

Published date: March 2008
Keywords: ELISPOT, tetramer, standardization, interlaboratory testing

Identifiers

Local EPrints ID: 62695
URI: https://eprints.soton.ac.uk/id/eprint/62695
ISSN: 0340-7004
PURE UUID: 407791b6-2312-4e17-8913-155d596d4652

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Date deposited: 04 Sep 2008
Last modified: 13 Mar 2019 20:27

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Contributors

Author: C.M. Britten
Author: C. Gouttefangeas
Author: M.J.P. Welters
Author: G. Pawelec
Author: S. Koch
Author: C. Ottensmeier
Author: A. Mander
Author: S. Walter
Author: A. Paschen
Author: J. Muller-Berghaus
Author: I. Haas
Author: A. Mackensen
Author: T. Kollgaard
Author: P. Thor Straten
Author: M. Schmitt
Author: K. Giannopoulos
Author: R. Maier
Author: H. Veelken
Author: C. Bertinetti
Author: A. Konur
Author: C. Huber
Author: S. Stevanovic
Author: T. Wolfel
Author: S.H. Van der Burg

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