TY - JOUR

T1 - More tricks with tetramers

T2 - a practical guide to staining T cells with peptide-MHC multimers

AU - Dolton, Garry

AU - Tungatt, Katie

AU - Lloyd, Angharad

AU - Bianchi, Valentina

AU - Theaker, Sarah M

AU - Trimby, Andrew

AU - Holland, Christopher J

AU - Donia, Marco

AU - Godkin, Andrew J

AU - Cole, David K

AU - Straten, Per Thor

AU - Peakman, Mark

AU - Svane, Inge Marie

AU - Sewell, Andrew K

N1 - © 2015 John Wiley & Sons Ltd.

PY - 2015/9

Y1 - 2015/9

N2 - Analysis of antigen-specific T-cell populations by flow cytometry with peptide-MHC (pMHC) multimers is now commonplace. These reagents allow the tracking and phenotyping of T cells during infection, autoimmunity and cancer, and can be particularly revealing when used for monitoring therapeutic interventions. In 2009, we reviewed a number of 'tricks' that could be used to improve this powerful technology. More recent advances have demonstrated the potential benefits of using higher order multimers and of 'boosting' staining by inclusion of an antibody against the pMHC multimer. These developments now allow staining of T cells where the interaction between the pMHC and the T-cell receptor is over 20-fold weaker (K(D) > 1 mm) than could previously be achieved. Such improvements are particularly relevant when using pMHC multimers to stain anti-cancer or autoimmune T-cell populations, which tend to bear lower affinity T-cell receptors. Here, we update our previous work to include discussion of newer tricks that can produce substantially brighter staining even when using log-fold lower concentrations of pMHC multimer. We further provide a practical guide to using pMHC multimers that includes a description of several common pitfalls and how to circumvent them.

AB - Analysis of antigen-specific T-cell populations by flow cytometry with peptide-MHC (pMHC) multimers is now commonplace. These reagents allow the tracking and phenotyping of T cells during infection, autoimmunity and cancer, and can be particularly revealing when used for monitoring therapeutic interventions. In 2009, we reviewed a number of 'tricks' that could be used to improve this powerful technology. More recent advances have demonstrated the potential benefits of using higher order multimers and of 'boosting' staining by inclusion of an antibody against the pMHC multimer. These developments now allow staining of T cells where the interaction between the pMHC and the T-cell receptor is over 20-fold weaker (K(D) > 1 mm) than could previously be achieved. Such improvements are particularly relevant when using pMHC multimers to stain anti-cancer or autoimmune T-cell populations, which tend to bear lower affinity T-cell receptors. Here, we update our previous work to include discussion of newer tricks that can produce substantially brighter staining even when using log-fold lower concentrations of pMHC multimer. We further provide a practical guide to using pMHC multimers that includes a description of several common pitfalls and how to circumvent them.

KW - Antibodies

KW - CD8-Positive T-Lymphocytes

KW - Flow Cytometry

KW - Fluorescent Dyes

KW - Histocompatibility Antigens Class I

KW - Histocompatibility Antigens Class II

KW - Humans

KW - Major Histocompatibility Complex

KW - Peptides

KW - Protein Multimerization

KW - Receptors, Antigen, T-Cell, alpha-beta

KW - Staining and Labeling

U2 - 10.1111/imm.12499

DO - 10.1111/imm.12499

M3 - Review

C2 - 26076649

VL - 146

SP - 11

EP - 22

JO - Immunology

JF - Immunology

SN - 0019-2805

IS - 1

ER -