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Recently activated, but not resting, CD4[sup.+] 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[sup.+] 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[sup.+] T cells for further analysis. Using this assay, we found that stimulated cells expressing tumor necrosis factor (TNF)-[alpha], interleukin (IL)-2 or interferon (IFN)-[gamma] were predominantly CD154[sup.+]. 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., Author(s): Pratip K Chattopadhyay [1]; Joanne Yu [1]; Mario Roederer (corresponding author) [1] CD4[sup.+] T-cell responses are commonly recognized by the ex vivo expression of various cytokines, such as IFN-[gamma] [...]

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. [ABSTRACT FROM AUTHOR]