Flow‐Cytometric Detection of Vaccinia‐Induced Memory Effector CD4⁺, CD8⁺, and γδTCR⁺T Cells Capable of Antigen‐Specific Expansion and Effector Functions

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+/CFSE10/interferon (IFN)-gamma(+), CD8(+)/CFSE10/IFN-gamma(+), CD8(+)/CFSE10/granzyme A(+), and CD8(+)/CFSE10/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 12 log higher than P <= .05 increases detectable by standard lymphoproliferation and cytotoxicity assays. Vaccinia-specific CD8(+)/CFSE10/ IFN-gamma(+) and granzyme A(+) T cell responses were significantly correlated with the results of standard Cr-51-release cytolytic assays (P < .05). Furthermore, vaccinia induced antigen-specific memory gamma delta T cells. We demonstrate P < .05 that vaccinia induces robust memory effector CD4(+), CD8(+), and gd 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.