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Cancer Immunology, Immunotherapy
Published in: Cancer Immunology, Immunotherapy 10/2009

Open Access 01-10-2009 | Original Article

Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium

Authors: Cedrik Michael Britten, Sylvia Janetzki, Leah Ben-Porat, Timothy M. Clay, Michael Kalos, Holden Maecker, Kunle Odunsi, Michael Pride, Lloyd Old, Axel Hoos, Pedro Romero, for the HLA-peptide Multimer Proficiency Panel of the CVC-CRI Immune Assay Working Group

Published in: Cancer Immunology, Immunotherapy | Issue 10/2009

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Abstract

Purpose

The Cancer Vaccine Consortium of the Cancer Research Institute (CVC-CRI) conducted a multicenter HLA-peptide multimer proficiency panel (MPP) with a group of 27 laboratories to assess the performance of the assay.

Experimental design

Participants used commercially available HLA-peptide multimers and a well characterized common source of peripheral blood mononuclear cells (PBMC). The frequency of CD8+ T cells specific for two HLA-A2-restricted model antigens was measured by flow cytometry.
The panel design allowed for participants to use their preferred staining reagents and locally established protocols for both cell labeling, data acquisition and analysis.

Results

We observed significant differences in both the performance characteristics of the assay and the reported frequencies of specific T cells across laboratories. These results emphasize the need to identify the critical variables important for the observed variability to allow for harmonization of the technique across institutions.

Conclusions

Three key recommendations emerged that would likely reduce assay variability and thus move toward harmonizing of this assay. (1) Use of more than two colors for the staining (2) collect at least 100,000 CD8 T cells, and (3) use of a background control sample to appropriately set the analytical gates. We also provide more insight into the limitations of the assay and identified additional protocol steps that potentially impact the quality of data generated and therefore should serve as primary targets for systematic analysis in future panels. Finally, we propose initial guidelines for harmonizing assay performance which include the introduction of standard operating protocols to allow for adequate training of technical staff and auditing of test analysis procedures.
Appendix
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Literature
1.
Mocellin S, Mandruzzato S, Bronte V, Lise M, Nitti D (2004) Part I: vaccines for solid tumours. Lancet Oncol 5:681–689PubMedCrossRef
2.
Finke LH, Wentworth K, Blumenstein B, Rudoplph NS, Hoos A (2007) Lessons from randomized phase III studies with active cancer immunotherapies—outcomes from the 2006 meeting of the Cancer Vaccine Consortium (CVC). Vaccine 25:B97–B109PubMedCrossRef
3.
4.
Hoos A, Parmiani G, Hege K, Sznol M, Loibner H, Eggermont A, Urba W, Blumenstein B, Sacks N, Keilholz U, Nichol G, the Cancer Vaccine Clinical Trial Working Group (2007) A clinical development paradigm for cancer vaccines and related biologics. J Immunother 30:1–15PubMedCrossRef
5.
Yee C, Greenberg P (2002) Modulating T cell immunity to tumours: new strategies for monitoring T cell responses. Nat Rev Cancer 2:409–419PubMedCrossRef
6.
Appay V, Douek DC, Price DA (2008) CD8+ T cell efficacy in vaccination and disease. Nat Med 14:623–628PubMedCrossRef
7.
Connerotte T, Van PA, Godelaine D, Tartour E, Schuler-Thurner B, Lucas S, Thielemans K, Schuler G, Coulie PG (2008) Functions of Anti-MAGE T cells induced in melanoma patients under different vaccination modalities. Cancer Res 68:3931–3940PubMedCrossRef
8.
Schmitt M, Schmitt A, Rojewski MT, Chen J, Giannopoulos K, Fei F, Yu Y, Gotz M, Heyduk M, Ritter G, Speiser DE, Gnjatic S, Guillaume P, Ringhoffer M, Schlenk RF, Liebisch P, Bunjes D, Shiku H, Dohner H, Greiner J (2008) RHAMM-R3 peptide vaccination in patients with acute myeloid leukemia, myelodysplastic syndrome, and multiple myeloma elicits immunologic and clinical responses. Blood 111:1357–1365PubMedCrossRef
9.
Adams S, O’Neill DW, Nonaka D, Hardin E, Chiriboga L, Siu K, Cruz CM, Angiulli A, Angiulli F, Ritter E, Holman RM, Shapiro RL, Berman RS, Berner N, Shao Y, Manches O, Pan L, Venhaus RR, Hoffman EW, Jungbluth A, Gnjatic S, Old L, Pavlick AC, Bhardwaj N (2008) Immunization of malignant melanoma patients with full-length NY-ESO-1 protein using TLR7 agonist imiquimod as vaccine adjuvant. J Immunol 181:776–784PubMed
10.
Speiser DE, Baumgaertner P, Voelter V, Devevre E, Barbey C, Rufer N, Romero P (2008) Unmodified self antigen triggers human CD8 T cells with stronger tumor reactivity than altered antigen. Proc Natl Acad Sci USA 105:3849–3854PubMedCrossRef
11.
Rosenberg SA, Yang JC, Restifo NP (2004) Cancer immunotherapy: moving beyond current vaccines. Nat Med 10:909–915PubMedCrossRef
12.
Curigliano G, Spitaleri G, Pietri E, Rescigno M, de Braud F, Cardillo A, Munzone E, Rocca A, Bonizzi G, Brichard V, Orlando L, Goldhirsch A (2006) Breast cancer vaccines: a clinical reality or fairy tale? Ann Oncol 17:750–762PubMedCrossRef
13.
Diefenbach CS, Gnjatic S, Sabbatini P, Aghajanian C, Hensley ML, Spriggs DR, Iasonos A, Lee H, Dupont B, Pezzulli S, Jungbluth AA, Old LJ, Dupont J (2008) Safety and immunogenicity study of NY-ESO-1b peptide and montanide ISA-51 vaccination of patients with epithelial ovarian cancer in high-risk first remission. Clin Cancer Res 14:2740–2748PubMedCrossRef
14.
Darrah PA, Patel DT, De Luca PM, Lindsay RW, Davey DF, Flynn BJ, Hoff ST, Andersen P, Reed SG, Morris SL, Roederer M, Seder RA (2007) Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major. Nat Med 13:843–850PubMedCrossRef
15.
Duvall MG, Precopio ML, Ambrozak DA, Jaye A, McMichael AJ, Whittle HC, Roederer M, Rowland-Jones SL, Koup RA (2008) Polyfunctional T cell responses are a hallmark of HIV-2 infection. Eur J Immunol 38:350–363PubMedCrossRef
16.
Precopio ML, Betts MR, Parrino J, Price DA, Gostick E, Ambrozak DR, Asher TE, Douek DC, Harari A, Pantaleo G, Bailer R, Graham BS, Roederer M, Koup RA (2007) Immunization with vaccinia virus induces polyfunctional and phenotypically distinctive CD8(+) T cell responses. J Exp Med 204:1405–1416PubMedCrossRef
17.
de Vries I, Bernsen MR, Lesterhuis WJ, Scharenborg NM, Strijk SP, Gerritsen MJ, Ruiter DJ, Figdor CG, Punt CJ, Adema GJ (2005) Immunomonitoring tumor-specific T cells in delayed-type hypersensitivity skin biopsies after dendritic cell vaccination correlates with clinical outcome. J Clin Oncol 23:5779–5787PubMedCrossRef
18.
Janossy G, Barry SM, Breen RA, Hardy GA, Lipman M, Kern F (2008) The role of flow cytometry in the interferon-gamma-based diagnosis of active tuberculosis and its coinfection with HIV-1—a technically oriented review. Cytometry B Clin Cytom 74(Suppl 1):S141–S151PubMed
19.
Keilholz U, Martus P, Scheibenbogen C (2006) Immune monitoring of T cell responses in cancer vaccine development. Clin Cancer Res 12:2346s–2352sPubMedCrossRef
20.
van der Burg SH (2008) Therapeutic vaccines in cancer: moving from immunomonitoring to immunoguiding. Expert Rev Vaccines 7:1–5PubMedCrossRef
21.
Britten CM, Janetzki S, van der Burg SH, Gouttefangeas C, Hoos A (2007) Toward the harmonization of immune monitoring in clinical trials: quo vadis? Cancer Immunol Immunother 57:285–288PubMedCrossRef
22.
Cox JH, Ferrari G, Kalams SA, Lopaczynski W, Oden N, D’souza MP (2005) Results of an ELISPOT proficiency panel conducted in 11 laboratories participating in international human immunodeficiency virus type 1 vaccine trials. AIDS Res Hum Retrovirus 21:68–81CrossRef
23.
Bednarek MA, Sauma SY, Gammon MC, Porter G, Tamhankar S, Williamson AR, Zweerink HJ (1991) The minimum peptide epitope from the influenza virus matrix protein. Extra and intracellular loading of HLA-A2. J Immunol 147:4047–4053PubMed
24.
Valmori D, Fonteneau JF, Lizana CM, Gervois N, Lienard D, Rimoldi D, Jongeneel V, Jotereau F, Cerottini JC, Romero P (1998) Enhanced generation of specific tumor-reactive CTL in vitro by selected Melan-A/MART-1 immunodominant peptide analogues. J Immunol 160:1750–1758PubMed
25.
Walker EB, Disis ML (2003) Monitoring immune responses in cancer patients receiving tumor vaccines. Int Rev Immunol 22:283–319PubMedCrossRef
26.
Heijnen IA, Barnett D, Arroz MJ, Barry SM, Bonneville M, Brando B, D’hautcourt JL, Kern F, Totterman TH, Marijt EW, Bossy D, Preijers FW, Rothe G, Gratama JW (2004) Enumeration of antigen-specific CD8+ T lymphocytes by single-platform, HLA tetramer-based flow cytometry: a European multicenter evaluation. Cytometry B Clin Cytom 62:1–13PubMedCrossRef
27.
Maecker HT, Rinfret A, D’Souza P, Darden J, Roig E, Landry C, Hayes P, Birungi J, Anzala O, Garcia M, Harari A, Frank I, Baydo R, Baker M, Holbrook J, Ottinger J, Lamoreaux L, Epling CL, Sinclair E, Suni MA, Punt K, Calarota S, El-Bahi S, Alter G, Maila H, Kuta E, Cox J, Gray C, Altfeld M, Nougarede N, Boyer J, Tussey L, Tobery T, Bredt B, Roederer M, Koup R, Maino VC, Weinhold K, Pantaleo G, Gilmour J, Horton H, Sekaly RP (2005) Standardization of cytokine flow cytometry assays. BMC Immunol 24:6–13
28.
Horton H, Thomas EP, Stucky JA, Frank I, Moodie Z, Huang Y, Chiu YL, McElrath MJ, De Rosa SC (2007) Optimization and validation of an 8-color intracellular cytokine staining (ICS) assay to quantify antigen-specific T cells induced by vaccination. J Immunol Methods 323:39–54PubMedCrossRef
29.
Asai T, Storkus WJ, Whiteside TL (2000) Evaluation of the modified ELISPOT assay for gamma interferon production in cancer patients receiving antitumor vaccines. Clin Diagn Lab Immunol 7:145–154PubMed
30.
Maecker HT, Hassler J, Payne JK, Summers A, Comatas K, Ghanayem M, Morse MA, Clay TM, Lyerly HK, Bhatia S, Ghanekar SA, Maino VC, Delarosa C, Disis ML (2008) Precision and linearity targets for validation of an IFN gamma ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides. BMC Immunol 9:9PubMedCrossRef
31.
Ogg GS, Jin X, Bonhoeffer S, Dunbar PR, Nowak MA, Monard S, Segal JP, Cao Y, Rowland-Jones SL, Cerundolo V, Hurley A, Markowitz M, Ho DD, Nixon DF, McMichael AJ (1998) Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science 279:2103–2106PubMedCrossRef
32.
Boon T, Coulie PG, Van den Eynde BJ, van der Bruggen P (2006) Human T cell responses against melanoma. Annu Rev Immunol 24:175–208PubMedCrossRef
33.
Coulie PG, Somville M, Lehmann F, Hainaut P, Brasseur F, Devos R, Boon T (1992) Precursor frequency analysis of human cytolytic T lymphocytes directed against autologous melanoma cells. Int J Cancer 50:289–297PubMedCrossRef
34.
Meidenbauer N, Hoffmann TK, Donnenberg AD (2003) Direct visualization of antigen-specific T cells using peptide-MHC-class I tetrameric complexes. Methods 31:160–171PubMedCrossRef
35.
Britten CM, Gouttefangeas C, Welters MJ, Pawelec G, Koch S, Ottensmeier C, Mander A, Walter S, Paschen A, Muller-Berghaus J, Haas I, Mackensen A, Kollgaard T, thor SP, Schmitt M, Giannopoulos K, Maier R, Veelken H, Bertinetti C, Konur A, Huber C, Stevanovic S, Wölfel T, and van der Burg SH (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 Immunol Immunother 57:289–302
36.
Chan C, Feng F, Ottinger J, Foster D, West M, Kepler TB (2008) Statistical mixture modeling for cell subtype identification in flow cytometry. Cytometry A 73:693–701PubMed
37.
Janetzki S, Panageas KS, Ben-Porat L, Boyer J, Britten CM, Clay TM, Kalos M, Maecker HT, Romero P, Yuan J, Kast WM, Hoos A (2008) Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI). Cancer Immunol Immunother 57:303–315PubMedCrossRef
Metadata
Title
Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium
Authors
Cedrik Michael Britten
Sylvia Janetzki
Leah Ben-Porat
Timothy M. Clay
Michael Kalos
Holden Maecker
Kunle Odunsi
Michael Pride
Lloyd Old
Axel Hoos
Pedro Romero
for the HLA-peptide Multimer Proficiency Panel of the CVC-CRI Immune Assay Working Group
Publication date
01-10-2009
Publisher
Springer-Verlag
Published in
Cancer Immunology, Immunotherapy / Issue 10/2009
Print ISSN: 0340-7004
Electronic ISSN: 1432-0851
DOI
https://doi.org/10.1007/s00262-009-0681-z

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