Quantifying MHC dextramer-induced NFAT activation in antigen-specific T cells as a functional response parameter.
MHC-multimers are reagents used for the detection and enumeration of antigen-specific T cells (ASTs). These reagents exploit the mechanism by which T cell receptors (TCR) on cytotoxic CD8 T cells recognize specific antigens in the context of a major histocompatibility complex (MHC) molecule during antigen presentation. MHC-multimers are fluorescently-labeled dextran polymers that carry MHC Class I molecules and peptide sequences that can be modified to represent specific cognate sequences of the antigen of interest with dextramers having a 10-fold multiplicity of the MHC/peptide combination within a single multimer. Since the binding of antigen-specific dextramers mimics antigen presentation to the TCR, the present study sought to determine whether this TCR engagement on the AST was sufficient to elicit a functional T cell response. The effect of binding of CMV specific dextramers on the activation of the NFAT signal transduction cascade was assessed in peripheral blood from bone marrow transplant recipients previously determined to be positive for CMV-ASTs (CASTs). NFAT activation was quantified by measuring nuclear translocation of NFAT1 in CD8+ CASTs and CD8+ non-CASTs by imaging flow cytometry. Our results demonstrate that an increase in the nuclear localization of NFAT1 was detectable in the CASTs following the CMV-dextramer binding and could be observed as early as 10min post-exposure. The NFAT1 activation correlated with a downstream functional response in the form of interferon gamma production. Sample preparation, temperature, and duration of dextramer exposure were important parameters affecting the dextramer-induced NFAT activation with 2h exposure in whole blood at room temperature being the optimal of the conditions tested. Intra- and inter-individual heterogeneity was observed with regards to the NFAT activation in the CASTs. Importantly, no effect of the dextramers was observed in the CD8+ non-CASTs, and therefore dextramer negative cell populations. Exposure to PMA/ionomycin following dextramer exposure resulted in a homogeneous NFAT activation in both the dextramer-positive but NFAT1 nonresponsive CAST and non-CAST cells. Thus, the data demonstrate that binding of antigen-specific dextramers to ASTs specifically results in activation of NFAT, that the NFAT activation correlates with a downstream functional response and that the response can be heterogeneous. This functional parameter may provide insight to the issue whether enumeration alone of ASTs is a sufficient parameter to assess an individual's immune status against a specific antigen.
Department of Flow and Image Cytometry, Roswell Park Cancer Institute, Buffalo, NY 14263, USA. Electronic address: orla.maguire@roswellpark.org.
Orla
O
Maguire
Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
George L
GL
Chen
Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Theresa E
TE
Hahn
Immudex, DK-2100, Copenhagen, Denmark.
Liselotte
L
Brix
Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Philip L
PL
McCarthy
Department of Flow and Image Cytometry, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Paul K
PK
Wallace
Department of Flow and Image Cytometry, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Hans
H
Minderman
D017951:Antigen Presentation; D003587:Cytomegalovirus / Q000276:immunology; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D005786:Gene Expression Regulation; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D019044:Image Cytometry / Q000295:instrumentation / Q000379:methods*; D007371:Interferon-gamma / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007938:Leukemia / Q000276:immunology / Q000473:pathology / Q000628:therapy; D008213:Lymphocyte Activation / Q000187:drug effects; D008285:Major Histocompatibility Complex; D050778:NFATC Transcription Factors / Q000819:agonists / Q000235:genetics / Q000276:immunology*; D010799:Phycoerythrin / Q000737:chemistry; D061251:Primary Cell Culture; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000276:immunology*; D013194:Staining and Labeling / Q000379:methods; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000276:immunology* / Q000473:pathology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D066027:Transplant Recipients
D005456:Fluorescent Dyes; D050778:NFATC Transcription Factors; C494006:NFATC2 protein, human; D011948:Receptors, Antigen, T-Cell; D010799:Phycoerythrin; D015759:Ionomycin; D007371:Interferon-gamma; D013755:Tetradecanoylphorbol Acetate
Antigen specificity; Dextramers; Imaging flow cytometry; NFAT