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

Getahun Abate Joy Eslick 1 Frances K. Newman Sharon E. Frey Robert B. Belshe Thomas P. Monath 0 Daniel F. Hoft () 1 0 Acambis Inc., Cambridge, Massachusetts 1 Molecular Microbiology and Immunology, Saint Louis University , St. Louis, Missouri 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)-g+, CD8+/CFSElo/IFN-g+, 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 12 log higher than increases detectable by standard lymphoproliferation and cytotoxicity assays. Vaccinia-specific CD8+/CFSElo/ IFN-g+ 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 gd T cells. We demonstrate 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. - Smallpox is a severe human disease with mortality 130% [1]. The World Health Organization declared smallpox eradicated in 1980 after relentless campaigns of mass vaccination with live attenuated vaccinia strains [2]. Consequently, vaccinia has not been used in the general population for 12 decades. However, virulent smallpox virus stocks still exist, and the possibility of deliberate release of smallpox as a bioweapon has become a significant concern. It is believed that optimal protection against smallpox requires both boosting of immunity in individuals previously vaccinated [3] and primary immunization of individuals born after the eradication of smallpox. The live attenuated vaccine strains of vaccinia used for eradication of smallpox have been associated with serious adverse effects. Currently, new smallpox vaccines are in different stages of clinical development [46]. These vaccines have been produced with currently acceptable culture methods, and some are more attenuated than vaccinia, which should decrease the risks associated with vaccination [4, 6]. New vaccines must be compared in detail with the reference standard, vaccinia, for their abilities to induce or boost relevant immunity. Cell-mediated immunity is an essential component of vaccinia-specific immunity [3]. Although several assays have been used to measure vaccinia-specific immunity, there is still a need to develop and apply robust assays that can measure memory effector T cells. Our aim was to develop a carboxyfluorescein succinimidyl ester (CFSE) based flow-cytometric assay to reliably measure distinct subsets of vaccinia-specific T cells. The present study demonstrates that vaccination with vaccinia (Dryvax; Wyeth Laboratories) induces memory effector CD4+/abTCR+, CD8+/abTCR+, and gdTCR+ T cells capable of both antigen-specific proliferation and effector functions. These results suggest that all 3 of these T cell responses are important for protective immunity against smallpox. SUBJECTS, MATERIALS, AND METHODS Samples. Peripheral blood mononuclear cells (PBMCs) were purified over histopaque (Sigma) from whole blood collected in 2 trials. The first trial involved a total of 9 individuals (laboratory personnel), 7 of whom had previously been vaccinated against smallpox with vaccinia and 2 of whom were immunologically naive with respect to vaccinia. All 7 previously vaccinated individuals were vaccinated during childhood, and all 7 developed an ulcerative lesion after recent revaccination with commercially available vaccinia. Most assays completed with samples harvested from these 9 individuals were performed with freshly separated PBMCs. Aliquots of these PBMCs were frozen in liquid nitrogen for later comparisons of assays. The second trial (Acambis H400-002) involved 11 other volunteers studied before and 45 days after vaccination with vaccinia. All PBMCs collected in the second trial were stored frozen in liquid nitrogen before use in immunological assays. PBMCs collected in the first trial were mainly used to optimize the CFSE-based flow-cytometric assay for the detection of vacciniaspecific immunity. PBMCs collected in the second trial were used to confirm the applicability of the optimized assay in a clinical trial setting by use of pre- and postvaccination samples. The study protocols were approved by the Saint Louis University Institutional Review Board. Informed consent was obtained from all participants. Virus stock and reagents. Vaccinia virus stock for in vitro stimulation of PBMCs was prepared from vaccinia grown in BSC-40 cells (continuous African green monkey kidney cell line), as described elsewhere [7]. The concentration of the virus stock was determined by use of a plaque assay with the same cell line, and aliquots were frozen at 70 C. A plaque assay performed regularly with frozen aliquots indicated no decline in viability during the study period (data not shown). The following reagents were used: monoclonal antibodies (anti-CD3 peridinin chlorophyll protein [PerCP], anti-CD4 PerCPCy 5.5, anti-CD8 PerCPCy 5.5, anti-gdTCR phycoerythrin [PE], antigranzyme A PE, anti-CD107a PE, and antiinterferon [IFN] g allophycocyanin [APC]; BD Biosciences), Cytofix/Cytoperm kits (BD), CFSE (Molecular Probes), phorbol myristate acetate (PMA), ionomycin, and phytohemagglutinin (PHA) (Sigma). CFSE labeling and expansion. PBMCs were labeled for 15 min with 1 mmol/L CFSE (Molecular Probes) in PBS before being washed with RPMI 1640 medium. CFSE-labeled PBMCs (1 106) were expanded with live vaccinia or were allowed to rest in medium (1 mL of RPMI with 10% heat-inactivated pooled human AB serum, glutamine, and penicillin/streptomycin) in polystyrene round-bottom tubes (17 100 mm; BD) for 10 days at 37 C in 5% CO2. Live vaccinia suspensions were prepared from thawed aliquots that had been sonicated twice for 30 s at room temperature in a water-bath sonicator. An MOI of 0.02 and a 10-day expansion period were selected as optimal conditions for in vitro stimulation of vaccinia-specific T cells in preliminary experiments. Surface and intracellular staining. On day 10 of in vitro stimulation, expanded and medium-rested cells were incubated with 50 ng/mL PMA (Sigma) and 750 ng/mL ionomycin (Sigma), in the presence of 0.7 mL/mL GolgiStop (BD), for 2 h, to maximize intracellular expression of immune effector molecules already up-regulated by antigen-specific stimulation. After 10 days of rest in medium alone, PMA/ionomycin stimulation was shown to no (...truncated)