Vagaries of the ELISpot assay: specific detection of antigen responsive cells requires purified CD8(+) T cells and MHC class I expressing antigen presenting cell lines.
Quantification of antigen-specific CD8(+) T cells is important for monitoring infection, vaccination, and response to therapy in cancer and immune-mediated diseases. Cytokine enzyme-linked-immunospot (ELISpot) assays are often used for this purpose. We found that substantial spot formation in IFNγ ELISpot assays occurred independently of CD8(+) T cells even when classical MHC class I restricted peptides are used for stimulation. Using fractionated cells and intracellular cytokine staining, the non-CD8(+) T cell IFNγ production was attributed to the CD4(+) T cell fraction. We therefore refined a cell line-based ELISpot assay combining HLA-A*0201 expressing K562 cells for antigen presentation with purified CD8(+) T cells and demonstrated that it specifically detected CD8(+) T cell responses with detection limits comparable to traditional ELISpot assays and dextramer-based quantification. The assay was further adapted to whole antigen responses with antigen (pre-proinsulin)-expressing HLA-A*0201K562 cells. Thus, we revealed and corrected a weak spot of the CD8(+) ELISpot assay.
Technische Universität Dresden, DFG-Center for Regenerative Therapies Dresden, Fetscherstr. 105, 01307 Dresden, Germany; Paul Langerhans Institute Dresden, Germany; Forschergruppe Diabetes e.V., Ingolstädter Landstraße 1, 85764 Neuherberg, Germany.
Yannick F
YF
Fuchs
Technische Universität Dresden, DFG-Center for Regenerative Therapies Dresden, Fetscherstr. 105, 01307 Dresden, Germany.
Gregor W
GW
Jainta
Technische Universität Dresden, DFG-Center for Regenerative Therapies Dresden, Fetscherstr. 105, 01307 Dresden, Germany.
Denise
D
Kühn
Technische Universität Dresden, DFG-Center for Regenerative Therapies Dresden, Fetscherstr. 105, 01307 Dresden, Germany.
Carmen
C
Wilhelm
Technische Universität Dresden, DFG-Center for Regenerative Therapies Dresden, Fetscherstr. 105, 01307 Dresden, Germany.
Marc
M
Weigelt
Technische Universität Dresden, DFG-Center for Regenerative Therapies Dresden, Fetscherstr. 105, 01307 Dresden, Germany.
Anne
A
Karasinsky
Technische Universität Dresden, DFG-Center for Regenerative Therapies Dresden, Fetscherstr. 105, 01307 Dresden, Germany.
Bhaskar
B
Upadhyaya
Forschergruppe Diabetes e.V., Ingolstädter Landstraße 1, 85764 Neuherberg, Germany; Institute of Diabetes Research, Helmholtz Zentrum München, and Forschergruppe Diabetes, Klinikum rechts der Isar, Technische Universität München, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany.
Anette-G
AG
Ziegler
Technische Universität Dresden, DFG-Center for Regenerative Therapies Dresden, Fetscherstr. 105, 01307 Dresden, Germany; Paul Langerhans Institute Dresden, Germany; Institute of Diabetes Research, Helmholtz Zentrum München, and Forschergruppe Diabetes, Klinikum rechts der Isar, Technische Universität München, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany. Electronic address: ezio.bonifacio@crt-dresden.de.
Ezio
E
Bonifacio
D000293:Adolescent; D000818:Animals; D000938:Antigen-Presenting Cells / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D016022:Case-Control Studies; D002648:Child; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology*; D058501:Enzyme-Linked Immunospot Assay / Q000379:methods*; D005260:Female; D015789:HLA-A2 Antigen / Q000276:immunology*; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology; D020014:K562 Cells; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D055815:Young Adult
C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D015395:Histocompatibility Antigens Class I; D007371:Interferon-gamma
Autoantigen; CD8(+) T cells; Diabetes; ELISpot; MHC class I