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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.Cite this article
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 