A culture amplified multi-parametric intracellular cytokine assay (CAMP-ICC) for enhanced detection of antigen specific T-cell responses.
Ex-vivo T-cell responses to vaccines and some viral antigens are not always detectable by conventional functional T-cell assays such as lympho-proliferation assays, IFN-gamma ELIspot and intracellular cytokine staining (ICC). In this study we describe the development, optimisation and utilisation of a culture amplified multiparametric intracellular cytokine assay (CAMP-ICC) to detect antigen specific T-cells that may be present at low frequencies and small primed responses typical of those induced by DNA vaccines in humans. CFSE labelled PBMCs are cultured for 10 days with antigens of interest. Low concentrations of exogenous proliferative and anti-apoptotic cytokines are added to assist in amplification of the antigen specific, but not background responses. On day 10 the cultured cells are re-challenged with or without antigen as for the standard ICC and then stained with fluorescent monoclonal antibodies of interest. Various conditions, including concentration, day of administration and length of incubation were tested employing the cytokines, IL-2, IL-7, IL-15 and IL-21 alone or in combination. CMV lysate, CEF peptides and measles viral lysate were used in the optimisation of the CAMP-ICC. We found that addition of 0.5 ng/ml of IL-15 in combination with 0.1 ng/ml of IL-21 at day 5 of culture enhanced proliferation of antigen specific responses whilst maintaining low background. The CAMP-ICC provides much more information than conventional functional T-cell assays allowing simultaneous determination of proliferative capacity and cytokine production by both CD4 and CD8+ T cells.
HIV Immunovirology Laboratory, St. Vincent's Centre for Applied Medical Research, Sydney, NSW, Australia. m.munier@cfi.unsw.edu.au
C Mee Ling
CM
Munier
John J
JJ
Zaunders
Susanna
S
Ip
David A
DA
Cooper
Anthony D
AD
Kelleher
D000328:Adult; D000911:Antibodies, Monoclonal / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D005452:Fluoresceins / Q000737:chemistry; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D019409:Interleukin-15 / Q000276:immunology* / Q000494:pharmacology; D007376:Interleukin-2 / Q000276:immunology; D015851:Interleukin-7 / Q000276:immunology; D007378:Interleukins / Q000276:immunology* / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism; D008213:Lymphocyte Activation; D013194:Staining and Labeling; D013388:Succinimides / Q000737:chemistry; D000098546:Interleukin-21
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D005452:Fluoresceins; D019409:Interleukin-15; D007376:Interleukin-2; D015851:Interleukin-7; D007378:Interleukins; D013388:Succinimides; D007371:Interferon-gamma; D000098546:Interleukin-21