Techniques to improve the direct ex vivo detection of low frequency antigen-specific CD8+ T cells with peptide-major histocompatibility complex class I tetramers.
The ability to quantify and characterize antigen-specific CD8+ T cells irrespective of functional readouts using fluorochrome-conjugated peptide-major histocompatibility complex class I (pMHCI) tetramers in conjunction with flow cytometry has transformed our understanding of cellular immune responses over the past decade. In the case of prevalent CD8+ T cell populations that engage cognate pMHCI tetramers with high avidities, direct ex vivo identification and subsequent data interpretation is relatively straightforward. However, the accurate identification of low frequency antigen-specific CD8+ T cell populations can be complicated, especially in situations where T cell receptor-mediated tetramer binding occurs at low avidities. Here, we highlight a few simple techniques that can be employed to improve the visual resolution, and hence the accurate quantification, of tetramer binding CD8+ T cell populations by flow cytometry. These methodological modifications enhance signal intensity, especially in the case of specific CD8+ T cell populations that bind cognate antigen with low avidities, minimize background noise, and enable improved discrimination of true pMHCI tetramer binding events from nonspecific uptake.
Chattopadhyay
Pratip K
PK
Immunotechnology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Melenhorst
J Joseph
JJ
Ladell
Kristin
K
Gostick
Emma
E
Scheinberg
Phillip
P
Barrett
A John
AJ
Wooldridge
Linda
L
Roederer
Mario
M
Sewell
Andrew K
AK
Price
David A
DA
D000941:Antigens / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods*; D015395:Histocompatibility Antigens Class I / Q000737:chemistry* / Q000276:immunology*; D006801:Humans; D010455:Peptides / Q000737:chemistry* / Q000276:immunology*; D055503:Protein Multimerization
D000941:Antigens; D015395:Histocompatibility Antigens Class I; D010455:Peptides