title: Cryopreservation of MHC multimers: Recommendations for quality assurance in detection of antigen specific T cells.
abstract: Fluorescence-labeled peptide-MHC class I multimers serve as ideal tools for the detection of antigen-specific T cells by flow cytometry, enabling functional and phenotypical characterization of specific T cells at the single cell level. While this technique offers a number of unique advantages, MHC multimer reagents can be difficult to handle in terms of stability and quality assurance. The stability of a given fluorescence-labeled MHC multimer complex depends on both the stability of the peptide-MHC complex itself and the stability of the fluorochrome. Consequently, stability is difficult to predict and long-term storage is generally not recommended. We investigated here the possibility of cryopreserving MHC multimers, both in-house produced and commercially available, using a wide range of peptide-MHC class I multimers comprising virus and cancer-associated epitopes of different affinities presented by various HLA-class I molecules. Cryopreservation of MHC multimers was feasible for at least 6 months, when they were dissolved in buffer containing 5-16% glycerol (v/v) and 0.5% serum albumin (w/v). The addition of cryoprotectants was tolerated across three different T-cell staining protocols for all fluorescence labels tested (PE, APC, PE-Cy7 and Quantum dots). We propose cryopreservation as an easily implementable method for stable storage of MHC multimers and recommend the use of cryopreservation in long-term immunomonitoring projects, thereby eliminating the variability introduced by different batches and inconsistent stability.
authors.affiliation: Department of Hematology, Center for Cancer Immune Therapy (CCIT), University Hospital Herlev, Herlev, Denmark.
authors.forename: Sine Reker
authors.initials: SR
authors.lastname: Hadrup
authors.forename: Dominik
authors.initials: D
authors.lastname: Maurer
authors.forename: Karoline
authors.initials: K
authors.lastname: Laske
authors.forename: Thomas Mørch
authors.initials: TM
authors.lastname: Frøsig
authors.forename: Sofie Ramskov
authors.initials: SR
authors.lastname: Andersen
authors.forename: Cedrik M
authors.initials: CM
authors.lastname: Britten
authors.forename: Sjoerd H
authors.initials: SH
authors.lastname: van der Burg
authors.forename: Steffen
authors.initials: S
authors.lastname: Walter
authors.forename: Cécile
authors.initials: C
authors.lastname: Gouttefangeas
mesh_terms: D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000737:chemistry; D005434:Flow Cytometry / Q000592:standards*; D005456:Fluorescent Dyes / Q000737:chemistry; D015395:Histocompatibility Antigens Class I / Q000737:chemistry*; D006801:Humans; D007202:Indicators and Reagents / Q000592:standards*; D010455:Peptides / Q000737:chemistry*; D055503:Protein Multimerization; D011786:Quality Control; D045663:Quantum Dots / Q000737:chemistry; D015203:Reproducibility of Results; D013194:Staining and Labeling / Q000592:standards*; D013601:T-Lymphocytes / Q000276:immunology / Q000473:pathology
chemical_list: D003451:Cryoprotective Agents; D005456:Fluorescent Dyes; D015395:Histocompatibility Antigens Class I; D007202:Indicators and Reagents; D010455:Peptides
keywords: MHC multimer; cryopreservation; cryoprotectant; glycerol in T cell staining; quality assurance; recommendations for MHC multimer storage