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A live-cell assay to detect antigen-specific CD4+ T cells with diverse cytokine profiles

Nature Medicine volume 11, pages 1113–1117 (2005)Cite this article

Abstract

Recently activated, but not resting, CD4+ T cells express CD154, providing costimulatory signals to B cells and antigen-presenting cells (APCs). Therefore, de novo CD154 expression after stimulation identifies antigen-specific CD4+ T cells. Previous assays were limited by the transient nature of surface CD154 expression; we overcame this by including fluorescently conjugated CD154-specific antibody during stimulation. Our assay is fully compatible with intracellular cytokine staining, and can be used for stimulations as long as 24 h. Notably, it is nonlethal, providing a means to purify viable antigen-specific CD4+ T cells for further analysis. Using this assay, we found that stimulated cells expressing tumor necrosis factor (TNF)-α, interleukin (IL)-2 or interferon (IFN)-γ were predominantly CD154+. Furthermore, some cells expressing none of these cytokines also expressed CD154, suggesting that CD154 marks cells with other effector functions. For vaccine- or pathogen-specific responses, we found substantial heterogeneity in expression of CD154 and cytokines, suggesting previously unrecognized diversity in abilities of responding cells to stimulate APCs through CD40.

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Figure 1: Optimization of CD154 detection.
Figure 2: Relationship between CD154 and cytokine expression.
Figure 3: Characterization of antigen-specific T cells.
Figure 4: Viable isolation of CD4+CD154+ T cells.

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Acknowledgements

This work is supported by the Intramural Research Program of the US National Institutes of Health, Vaccine Research Center, National Institute of Allergy and Infectious Disease.

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  1. ImmunoTechnology Section, Vaccine Research Center, National Institute of Allergy and Infectious Disease, National Institutes of Health, 40 Convent Drive, Room 5509, Bethesda, 20892, Maryland, USA

    Pratip K Chattopadhyay, Joanne Yu & Mario Roederer

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  1. Pratip K Chattopadhyay
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Correspondence to Mario Roederer.

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Chattopadhyay, P., Yu, J. & Roederer, M. A live-cell assay to detect antigen-specific CD4+ T cells with diverse cytokine profiles. Nat Med 11, 1113–1117 (2005). https://doi.org/10.1038/nm1293

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