Optimization of intracellular cytokine staining for the quantitation of antigen-specific CD4+ T cell responses in rhesus macaques.
Standard proliferation assays used for analysis of CD4+ T cell function have significant shortcomings, including limited sensitivity, lack of truly quantitative readouts and significant variability. We have optimized an intracellular cytokine staining (ICS) assay in rhesus macaques which allows us to identify virus-specific CD4+ T cells at the single-cell level with high sensitivity while reducing background staining to a minimum. A variety of parameters were tested to determine the optimal experimental conditions necessary for the detection of antigen-specific CD4+ T cells in macaques. Central to our optimized protocol was the addition of cross-linked costimulatory anti-CD28 and anti-CD49d Mabs, a modification which resulted in up to threefold enhancement of the frequency of TNF-alpha-secreting CD4+ T cells following superantigen- or antigen-specific stimulation. The ICS protocol was also optimized with respect to antigen concentration and duration of antigenic stimulation. These modifications resulted in a convenient and highly reproducible assay with intra- and inter-assay variability of less than 10%. Although cryopreservation of PBMC generally led to a 40% to 80% decrease in the frequency of antigen-specific CD4+ T cells detected by ICS using stimulation with viral proteins, the use of overlapping peptide pools minimized the effects of cryopreservation on ICS responses. The use of more sensitive techniques such as ICS permits delineation of antigen-specific cells at the single cell level and should provide new insights into pathogen-specific immune responses in the rhesus macaque model.
Gauduin
Marie-Claire
MC
New England Primate Research Center, Harvard Medical School, Division of Immunology, One Pine Hill Drive, P.O. Box 9102, Southborough, MA 01772-9102, USA. marie-claire_gauduin@hms.harvard.edu
Kaur
Amitinder
A
Ahmad
Shabbir
S
Yilma
Tilahun
T
Lifson
Jeffrey D
JD
Johnson
R Paul
RP
D000818:Animals; D000941:Antigens / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D016207:Cytokines / Q000032:analysis*; D004306:Dose-Response Relationship, Immunologic; D007371:Interferon-gamma / Q000276:immunology; D008253:Macaca mulatta / Q000276:immunology / Q000378:metabolism* / Q000821:virology; D015302:Simian Immunodeficiency Virus / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology
D000941:Antigens; D016207:Cytokines; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma