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.
Clin Immunol
; 157(2): 216-25, 2015 Apr.
Article
em En
| MEDLINE
| ID: mdl-25728493
ABSTRACT
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.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Antígeno HLA-A2
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Linfócitos T CD8-Positivos
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Diabetes Mellitus Tipo 1
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ELISPOT
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Células Apresentadoras de Antígenos
Tipo de estudo:
Diagnostic_studies
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Observational_studies
Limite:
Adolescent
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Adult
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Animals
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Child
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Female
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Humans
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Male
Idioma:
En
Ano de publicação:
2015
Tipo de documento:
Article