Abstract

We developed a carboxyfluorescein succinimidyl ester (CFSE)–based flow-cytometric assay that can detect different subsets of vaccinia-specific T cells capable of both antigen-specific expansion and protective effector functions. Proliferation and effector functions were detected by CFSE dilution and intracellular staining, respectively. Absolute numbers of CD4+/CFSElo/interferon (IFN)–γ+, CD8+/CFSElo/IFN-γ+, CD8+/CFSElo/granzyme A+, and CD8+/CFSElo/CD107a+ T cells present after in vitro stimulation with live vaccinia were significantly higher in immunized individuals (P<.05). These CD4+ and CD8+ T cell increases were >2 log higher than increases detectable by standard lymphoproliferation and cytotoxicity assays. Vaccinia-specific CD8+/CFSElo/IFN-γ+ and granzyme A+ T cell responses were significantly correlated with the results of standard 51Cr-release cytolytic assays (P<.05). Furthermore, vaccinia induced antigen-specific memory γδ T cells. We demonstrate that vaccinia induces robust memory effector CD4+, CD8+, and γδ T cells, all of which are relevant for protection against smallpox. CFSE-based flow-cytometric assays will be useful in evaluating cell-mediated immune responses induced by new smallpox vaccines

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