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J Leukoc Biol. 2013 Mar;93(3):449-55. doi: 10.1189/jlb.1012489. Epub 2012 Dec 21.

Technical advance. Measurement of iNKT cell responses at the single-cell level against rare HIV-1-infected dendritic cells in a mixed culture.

Author information

1
Center for Infectious Medicine, Department of Medicine,Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden.

Abstract

iNKT cells recognize lipid antigens, such as α-GalCer, presented in complex with CD1d expressed by DCs. Exposure of DCs to HIV-1 can lead to productive infection, and it was demonstrated recently that HIV-1 inhibits CD1d surface expression in an apparent mode of immune evasion. However, studies of the interaction between T cells, including iNKT cells and HIV-infected DCs in vitro, are hampered by the low frequency of productive infection in DCs. Here, we demonstrate the utility of full-length HIV-1 modified to express eGFP to address this problem. This virus allowed identification of single, rare productively infected cells in a mixed DC population by fluorescence microscopy and enabled detailed studies of the interaction of such cells with individual iNKT cells. iNKT cell responses to α-GalCer presented by HIV-1-positive and -negative DCs were quantified by intracellular IFN-γ staining in iNKT cells forming conjugates with DCs. Whereas complex formation was observed between iNKT cells and uninfected and infected DCs, only iNKT cells in contact with uninfected DCs produced IFN-γ. This microscopy assay, based on full-length HIV-1 modified to express eGFP, thus allows detailed evaluation of HIV-1 immune-evasion mechanisms in rare virus-infected live DCs.

PMID:
23264676
PMCID:
PMC3579026
DOI:
10.1189/jlb.1012489
[Indexed for MEDLINE]
Free PMC Article

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