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Background:  Activated CD4⁺ T cells express CD154 (CD40L), providing co-stimulatory signals to activate B cells and antigen-presenting cells. Previous assays for cell surface or intracellular CD154 were limited by the transient nature of CD154 expression. We describe a new assay for CD154, and use it to study antigen-specific T cells in vaccinated or infected individuals.

Methods:  To enhance CD154 detection, we add fluorochrome-conjugated anti-CD154 during the cell stimulation so that transiently expressed CD154 binds and internalizes fluorescent antibody. We compare this method with cell surface and intracellular methods in fresh PBMC, stimulated with SEB. In addition, antigen-specific cytokine responses are correlated to CD154 expression in CMV-responsive people after stimulation with CMV peptides. Finally, CD4⁺ T-cell responses to HIV-env A peptides are analyzed in healthy people receiving a DNA vaccine. Flow cytometry is used to measure CD3, CD4, CD8, IFN-γ, IL-2, TNF-α, and CD154 simultaneously.

Results: U sing monensin and fluorochrome-conjugated CD154 during stimulation increased the proportion of CD154⁺ cells 2- to 4-fold compared with previous methods. Maximal levels occurred with 6 hours’ stimulation, remaining at these levels for 24 hours. This indicates that we identified cells for which CD154 expression was transient. We confirmed specificity by characterizing SEB-responding T cells: CD154 was confined to those TCR Vb families known to respond to SEB (Vb 12, 17, and 20). Using CMV- or HIV-antigen stimulation, we evaluated antigen-specific responses using CD154. For either CMV- or HIV-stimulated CD4⁺ cells, cells expressing IFN-γ alone, and those expressing multiple cytokines were more likely to express CD154 than cells expressing only IL-2 or TNF-α alone. CMV-specific CD4⁺ T-cells were mostly CD154⁺ IFN-γ⁺ IL-2^­ TNF-α⁺ or CD154⁺ IFN-γ⁺ IL-2^­ TNF-α^­ (35% and 30% of the response, respectively). In contrast, cells unlikely to provide CD154-costimulation (CD154^­ IFN-γ^­ IL-2⁺ TNF-α^­) dominated the HIV-specific CD4⁺ T-cell response (69% of responding cells) to the vaccine.

Conclusions:  This assay improves upon previous methods, overcoming problems of transient expression. It provides a non-lethal alternative to intracellular cytokine measurements for detection of antigen-specific CD4⁺ T-cells; in particular it marks those cells expressing multiple cytokines. Finally, it describes a biologic aspect of antigen-specific responses: ability to provide CD154-mediated costimulatory signals.

Keywords: CD154; antigen-specific; cytokines