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

  1. Getahun Abate1,
  2. Joy Eslick2,
  3. Frances K. Newman1,
  4. Sharon E. Frey1,
  5. Robert B. Belshe1,
  6. Thomas P. Monath3 and
  7. Daniel F. Hoft1,2
  1. Departments of
  2. 1Internal Medicine and
  3. 2Molecular Microbiology and Immunology, Saint Louis University, St. Louis, Missouri;
  4. 3Acambis Inc., Cambridge, Massachusetts
  1. Reprints or correspondence: Dr. Daniel Hoft, Div. of Infectious Diseases and Immunology, Dept. of Internal Medicine and Molecular Microbiology, Saint Louis University Health Sciences Center, 3635 Vista Ave., FDT-8N, St. Louis, MO 63110 (hoftdf{at}slu.edu)
 
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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

Smallpox is a severe human disease with mortality >30% [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 >2 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 [4–6]. 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+/αβTCR+, CD8+/αβTCR+, and γδTCR+ 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

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Subjects, Materials, and Methods

SamplesPeripheral 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 vaccinia-specific 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 reagentsVaccinia 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 PerCP–Cy 5.5, anti-CD8 PerCP–Cy 5.5, anti-γδTCR phycoerythrin [PE], anti–granzyme A PE, anti-CD107a PE, and anti–interferon [IFN]–γ allophycocyanin [APC]; BD Biosciences), Cytofix/Cytoperm kits (BD), CFSE (Molecular Probes), phorbol myristate acetate (PMA), ionomycin, and phytohemagglutinin (PHA) (Sigma)

CFSE labeling and expansionPBMCs were labeled for 15 min with 1 μmol/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 stainingOn 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 μL/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 not induce effector molecule expression in these medium-rested T cells. Anti-CD107a PE was added during the PMA/ionomycin stimulation, to stain viable cells with transient surface expression of lysosomal-associated membrane protein–1 (LAMP-1), because of cytolytic degranulation [8]. Restimulated cells were washed with staining buffer containing sodium azide and were stained for detection of CD4, CD8, and γδTCR surface expression. These cells were subsequently permeabilized with Cytofix/Cytoperm solutions, as described by the manufacturer (BD), and then were stained for detection of intracellular IFN-γ and/or granzyme A. The following combinations of stains were used:

  • CFSE/anti–granzyme A PE/anti-CD4 PerCP–Cy 5.5/anti–IFN-γ APC;

  • CFSE/anti–granzyme A PE/anti-CD8 PerCP–Cy 5.5/anti–IFN-γ APC;

  • CFSE/anti-CD107a PE/anti-CD8 PerCP–Cy 5.5/anti–IFN-γ APC; and

  • CFSE/anti-γδTCR PE/anti-CD3 PerCP/anti–IFN-γ APC

Fluorescence-activated cell sorter (FACS) analysisFlow-cytometric acquisition was performed by use of a 4-color FACSCalibur (BD) instrument, and analyses were done using CellQuest (version 3.3; BD) and FlowJo (version 6.2.1; Tree Star) software. A minimum of 10,000 CD4+, CD8+, or γδTCR+ events were acquired. CD4+ and CD8+ T cells were regated from lymphocyte population gates on the basis of forward and side scatter. The proliferating cells were identified as populations with decreased mean FL1 fluorescence intensity and are labeled as CFSElo. The absolute numbers of effector CD4+, CD8+, and γδTCR+ T cells were calculated by multiplying the total number of viable cells recovered times the percentage of the specific T cell subset detected by flow-cytometric analysis

Cytotoxicity assayA standard 51Cr-release cytotoxicity assay was performed with all 9 samples collected in the first trial. Autologous lymphocyte blasts generated by stimulation of PBMCs with PHA (8 μg/mL) for 3 days were used as targets [9]. A portion of blast cells was infected with vaccinia (MOI ⩾1) and labeled with 100 μCi of 51Cr (PerkinElmer) overnight at 37°C. Equal numbers of uninfected blasts were labeled with 100 μCi of 51Cr, for use as control targets. Labeled cells were washed 3 times in RPMI, and 1×104 blasts were transferred to appropriate wells of 96-well plates

PBMCs expanded with an optimal concentration of vaccinia (MOI 0.02) for 10 days were used as effectors. The effectors were washed in RPMI and incubated at 90:1, 30:1, and 10:1 ratios with targets for 5 h at 37°C. Culture supernatants were harvested and counted by use of a MicroBeta counter (PerkinElmer). Vaccinia-specific cytotoxicity was calculated as follows: Formula

[3H]-thymidine incorporation assayPBMCs (2×105 cells/well) were stimulated in round-bottom 96-well plates with antigens or were allowed to rest in medium for 7 days. On day 6, [3H]-thymidine (1 μCi/well) was added, and cells were further incubated for 18 h. Cultures were harvested on glass fiber filters and read by use of a MicroBeta counter. Stimulation indices were calculated by dividing counts per minute for antigen-stimulated cells by counts per minute for medium-rested controls

Statistical analysesMann-Whitney U tests were used to analyze differences in the expansions of effector T cells in the first trial, which compared vaccinated and unvaccinated volunteers. Wilcoxon matched-pairs tests were used in the second trial, to compare responses detected in pre- and postvaccination samples. Correlations between the results of flow-cytometric assays and 51Cr-release assays were studied by use of Spearman’s&amp;rank analysis

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Results

CFSE-based assay optimization to measure vaccinia-specific immunityFigure 1 shows a schematic of the CFSE-based assay. We found that a 1 μmol/L concentration of CFSE enabled us to easily identify proliferating cells in the FL1 channel without significant lateral absorption in the FL2, -3, and -4 channels. In addition, we found that limiting the time of PMA/ionomycin stimulation of T cells to 2 h prevented down-modulation of CD4 expression. Restimulation with live vaccinia, instead of PMA/ionomycin, after the 10-day in vitro expansion period gave similar results but was associated with decreased detection of antigen-induced effector molecule expression. In addition, the PMA/ionomycin stimulation performed after 10 days of rest in medium in vitro did not activate T cells to express effector molecules such as IFN-γ

Figure 1
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Figure 1

Schematic of vaccinia-specific assay linking proliferation and effector function. Carboxyfluorescein succinimidyl ester (CFSE)–labeled peripheral blood mononuclear cells (PBMCs) (1×106) were incubated with live vaccinia (MOI 0.02) or medium for 10 days. These in vitro–expanded PBMCs were then stimulated with phorbol myristate acetate/ionomycin in the presence of GolgiStop (BD) and stained for surface and intracellular molecules. Flow-cytometric analysis was performed by use of a 4-color FACSCalibur instrument (BD). FACS, fluorescence-activated cell sorter; PMA, phorbol myristate acetate

Detection of vaccinia-specific memory CD4+ T cells capable of both proliferation and effector functionsPBMCs from vaccinated individuals had a significantly higher proliferation of IFN-γ–producing CD4+ T cells after 10 days of in vitro stimulation with live vaccinia (figure 2). Figure 2A shows typical FACS plots for PBMCs harvested from vaccinia-naive and vaccinia-immune individuals. Essentially no CD4+ T cells were found to proliferate in medium-rested PBMCs from vaccinia-naive and vaccinated individuals or in cultures of PBMCs harvested from vaccinia-naive individuals after in vitro stimulation with live vaccinia (MOI 0.02). In contrast, PBMCs from vaccinia-immune individuals were found to markedly proliferate after in vitro stimulation with live vaccinia. Figure 2B shows that the absolute numbers of antigen-specific CFSElo/IFN-γ+ (proliferating and IFN-γ–expressing) CD4+ T cells in PBMCs from vaccinated individuals (n=7) were significantly higher than the absolute numbers in PBMCs from vaccinia-naive individuals (P<.05, Mann-Whitney U test). The median absolute numbers of CD4+/CFSElo/IFN-γ+ T cells were 11 and 20,748 for vaccinia-naive and vaccinia-immune individuals, respectively. Similarly, figure 2C shows the results from a second trial, in which PBMCs were collected from 11 other volunteers before and 45 days after vaccination with the Dryvax strain of vaccinia. There was a significant increase in CD4+/CFSElo/IFN-γ+ T cells after vaccination with vaccinia (P<.01, Wilcoxon matched-pairs test). The median absolute numbers of CD4+/CFSElo/IFN-γ+ T cells were 97 and 30,316 for pre- and postvaccination responses, respectively. Of note, the vaccine-induced fold increases in vaccinia-specific CD4+/CFSElo/IFN-γ+ T cell responses (>300-fold) were, in general, >100-fold higher than the stimulation indices detected by standard [3H]-thymidine incorporation lymphoproliferation assays (data not shown)

Figure 2
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Figure 2

Induction by vaccinia vaccination of significant increases in CD4+ T cells capable of antigen-specific proliferation and interferon (IFN)–γ expression. A Typical flow-cytometric 2-parameter dot plot results obtained by use of the carboxyfluorescein succinimidyl ester (CFSE)–based assay, for representative vaccinia-naive and vaccinia-immune individuals. Lymphocytes gated on the basis of forward and side scatter and then regated on CD4+ cells were analyzed for CFSE and IFN-γ expression by use of FlowJo software. The no. in the upper left quadrant of each dot plot refers to the percentage of CD4+/CFSElo/IFN-γ+ T cells detected after in vitro stimulation. APC, allophycocyanin. B CD4+/CFSElo/IFN-γ+ responses from 7 vaccinia-immune and 2 vaccinia-naive individuals. After 10 days of in vitro stimulation with live vaccinia, the absolute nos. of CD4+/CFSElo/IFN-γ+ T cells in PBMCs from vaccinia-immune individuals were significantly higher than in PBMCs from vaccinia-naive individuals. *P<.05, Mann-Whitney U test. C CD4+/CFSElo/IFN-γ+ responses from 11 volunteers studied before and 45 days after vaccination. PBMCs were allowed to rest in medium or were stimulated with live vaccinia (MOI 0.02) for 10 days. The absolute nos. of CD4+/CFSElo/IFN-γ+ T cells were significantly higher in postvaccination PBMCs. **P<.01, Wilcoxon matched-pairs test. Medians (points) interquartile ranges (boxes) and extreme quartile ranges (whiskers) are shown in panels B and C

Detection of vaccinia-specific memory CD8+ T cells capable of both proliferation and effector functionFigure 3 shows vaccinia-specific CD8+ T cell responses. Figure 3A shows FACS plots from a representative vaccinia-naive and a representative vaccinia-immune individual. PBMCs from the vaccinated individual showed a marked increase in CD8+/CFSElo/IFN-γ+ T cells (14%) after 10 days of in vitro stimulation with live vaccinia, compared with PBMCs from the vaccinia-naive individual. Figure 3B and 3C shows CD8+ T cell responses detected in PBMCs from all volunteers recruited into the first trial (7 vaccinated and 2 vaccinia-naive individuals) and the second trial (11 subjects studied before vaccination and 45 days after vaccination), respectively. The absolute numbers of CD8+/CFSElo/IFN-γ+ T cells were significantly higher in samples from vaccinated individuals in the first trial (P<.05, Mann-Whitney U test) and also in samples collected 45 days after vaccination in the second trial (P<.01, Wilcoxon matched-pairs test). The median absolute numbers of CD8+/CFSElo/IFN-γ+ T cells were 20 and 3400 for vaccinia-naive and vaccinia-immune individuals and 18 and 3500 for pre -and postvaccination responses, respectively

Figure 3
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Figure 3

Induction by vaccinia vaccination of significant increases in CD8+ T cells capable of both antigen-specific proliferation and interferon (IFN)–γ expression. A Typical flow-cytometric 2-parameter dot plot results obtained with peripheral blood mononuclear cells (PBMCs) from representative vaccinia-naive and vaccinia-immune individuals. Lymphocytes gated on the basis of forward and side scatter and then regated on CD8+ cells were analyzed for carboxyfluorescein succinimidyl ester (CFSE) and IFN-γ expression by use of FlowJo software. The no. in the upper left quadrant of each dot plot refers to the percentage of CD8+/CFSElo/IFN-γ+ T cells detected after in vitro stimulation. APC, allophycocyanin. B CD8+/CFSElo/IFN-γ+ responses from 7 vaccinia-immune and 2 vaccinia-naive individuals. After 10 days of in vitro stimulation with live vaccinia, the absolute nos. of CD8+/CFSElo/IFN-γ+ T cells in PBMCs from vaccinia-immune individuals were significantly higher than in PBMCs from vaccinia-naive individuals. *P<.05, Mann-Whitney U test. C CD8+/CFSElo/IFN-γ+ responses from 11 volunteers studied before and 45 days after vaccination. PBMCs were allowed to rest in medium or were stimulated with live vaccinia (MOI 0.02) for 10 days. The absolute nos. of CD8+/CFSElo/IFN-γ+ T cells were significantly higher in postvaccination PBMCs. *P<.01, Wilcoxon matched-pairs test. Medians (points) interquartile ranges (boxes) and extreme quartile ranges (whiskers) are shown in panels B and C

Direct correlation of vaccinia-specific CD8+ T cell responses detected by the CFSE-based flow-cytometric assay with cytotoxic T lymphocyte (CTL) activityPBMCs from the first trial were further studied for expression of granzyme A, as well as for cytolytic activity, by use of a standard 51Cr-release assay after 10 days of in vitro expansion with live vaccinia (MOI 0.2). In addition, PBMCs harvested before and after vaccination in the second trial were studied for absolute numbers of CD8+CFSElo/CD107a+ T cells. Granzyme A was used as a marker for cytolytic granules, and CD107a (LAMP-1) transient expression on the cell surface was used as a marker for degranulation. Figure 4A and 4B shows markedly higher absolute numbers of CD8+/CFSElo/granzyme A+ and CD8+/CFSElo/CD107a+ T cells in PBMCs from vaccinated individuals, respectively. Median absolute numbers of CD8+/CFSElo/granzyme A+ T cells (figure 4A) were 86-fold higher in vaccinated individuals than in vaccinia-naive individuals (P<.05, Mann-Whitney U test). In the second trial, median absolute numbers of CD8+/CFSElo/CD107a+ T cells (figure 4B) were 85-fold higher after vaccination than before vaccination (P<.01, Wilcoxon matched-pairs test). PBMCs from vaccinated individuals showed an increase in cytolytic activity against vaccinia-infected autologous targets that was dependent on the number of effector cells added (figure 4C). The percentage of cytolytic activity measured in these 51Cr-release assays directly correlated with the proportions of CD8+/CFSElo/IFN-γ+ (r=0.7; P<.05) and CD8+/CFSElo/granzyme A+ (r=0.8; P<.05) T cells (figure 4D). The results of analysis of CD107a expression in CD8+/CFSElo T cells in samples from the first trial, although not performed with all samples (n=5), showed a trend toward correlation with CTL activity (r=0.5). Anti-CD107a staining was performed with all samples from the second trial (n=11), but 51Cr-release assays were not performed with these samples. Similar to the results described for CD4+ T cell responses, the vaccinia-specific fold increases in CD8+ T cell responses detectable by the flow-cytometric assay were >100-fold higher than the increases detected by 51Cr-release assays

Figure 4
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Figure 4

Expression of effector molecules important for cytolysis of vaccinia-infected targets by memory CD8+ T cells induced by vaccinia vaccination. A Carboxyfluorescein succinimidyl ester (CFSE)–labeled peripheral blood mononuclear cells (PBMCs) from 7 vaccinia-immune and 2 vaccinia-naive individuals allowed to rest in medium or stimulated with live vaccinia for 10 days. After in vitro stimulation with live vaccinia, the absolute nos. of CD8+/CFSElo/granzyme A+ T cells in PBMCs from vaccinia-immune individuals were significantly higher than in PBMCs from vaccinia-naive individuals. *P<.05, Mann-Whitney U test. B CFSE-labeled PBMCs from pre- and postvaccination paired samples tested for expression of CD107a. The absolute nos. of CD8+/CFSElo/CD107a+ T cells were significantly higher in postvaccination PBMCs than in prevaccination PBMCs. *P<.01, Wilcoxon matched-pairs test. Medians (points) interquartile ranges (boxes) and extreme quartile ranges (whiskers) are shown in panels A and B. C Induction of robust cytotoxic T lymphocyte (CTL) responses by vaccinia vaccination. PBMCs expanded with vaccinia (MOI 0.02) for 10 days were used as effectors. Autologous phytohemagglutinin (PHA) blasts (infected or uninfected with vaccinia) were labeled with 51Cr and used as targets. Three effector:target ratios (90:1, 30:1, and 10:1) were used, and 51Cr release in supernatants was measured by use of a MicroBeta counter. PBMCs from the first trial (n=9) were used, and only PBMCs from vaccinated individuals (n=7) displayed cytolytic activity against vaccinia-infected target cells. D Correlation of flow-cytometric detection of antigen-specific memory effector T cells with results of a standard 51Cr-release CTL assay. The percentage of CD8+/CFSElo/granzyme A+ T cells and the percentage of CD8+/CFSElo/IFN-γ+ T cells expanded after 10 days of stimulation with vaccinia were studied for correlation with the percentage of lysis (effector:target ratio, 90:1) obtained by use of the 51Cr-release assays in Spearman’s&amp;rank tests

Detection of vaccinia-specific memory γδ T cellsWe have previously seen that bacille Calmette-Guérin and canarypox vaccination can induce memory γδTCR+ T cells [10,11]. We next asked whether vaccinia vaccination can also induce antigen-specific memory responses in γδTCR+ T cells. As with CD4+ and CD8+ T cells, marked increases of γδTCR+/CFSElo/IFN-γ+ T cells were detected in postvaccination PBMCs after in vitro stimulation with live vaccinia. Figure 5A shows representative FACS dot plots indicating an obvious expansion of γδTCR+/CFSElo/IFN-γ+ T cells after stimulation with live vaccinia in vitro only in PBMCs collected after vaccination. Figure 5B shows that, compared with PBMCs collected before vaccination with vaccinia, PBMCs collected 45 days after vaccination with vaccinia had significantly larger numbers of γδTCR+/CFSElo/IFN-γ+ T cells after in vitro stimulation. The median absolute number of vaccinia-expanded γδTCR+/CFSElo/IFN-γ+ T cells was 237-fold higher with postvaccination responses than with prevaccination responses (P<.01, Wilcoxon matched-pairs test)

Figure 5
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Figure 5

Induction of significant increases in memory γδTCR+ T cells capable of both antigen-specific proliferation and interferon (IFN)–γ expression by vaccinia vaccination. A Typical flow-cytometric 2-parameter dot plot results obtained for a representative vaccinia-immune and a representative vaccinia-naive individual. Lymphocytes were gated on the basis of forward and side scatter and then were regated on γδTCR+ cells and analyzed for carboxyfluorescein succinimidyl ester (CFSE) and IFN-γ expression by use of FlowJo software. The no. in the upper left quadrant of each dot plot refers to the percentage of CD3+/γδTCR+/CFSElo/IFN-γ+ T cells detected after in vitro stimulation. APC, allophycocyanin. B CFSE-based flow-cytometric assay, which was used to study γδTCR+ T cell responses induced by vaccinia vaccination with peripheral blood mononuclear cells (PBMCs) collected from 11 volunteers before and 45 days after vaccination. PBMCs were allowed to rest in medium or were stimulated with live vaccinia (MOI 0.02) for 10 days. The absolute nos. of CD3+/γδTCR+/CFSElo/IFN-γ+ T cells were significantly higher in postvaccination PBMCs. **P<.01, Wilcoxon matched-pairs test. Medians (points) interquartile ranges (boxes) and extreme quartile ranges (whiskers) are shown

Effect of storage conditions on memory effector T cells in the CFSE-based assayAs shown in figures 2⇑⇑–5, fresh samples (used in studies of cells from the first trial) and frozen samples (used in studies of cells from the second trial) of PBMCs could be used to detect vaccinia-specific T cell responses. In addition, PBMCs from 2 vaccinated individuals were studied fresh, after 1 day of incubation at room temperature, and after freezing in DMSO with liquid nitrogen, all in parallel (data not shown). In the new assay, these parallel studies with matched samples gave similar expansions of effector memory T cells after stimulation with live vaccinia

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Discussion

Vaccines that are protective against intracellular pathogens induce effector memory T cells. The mechanisms involved in memory T cell generation are not fully understood. However, after the initial encounter with an antigen, T cells pass through different phases, including programmed expansion, differentiation into effector cells, and programmed contraction [12–14]. Not all memory cells are able to provide critical protective effector functions. Using Listeria monocytogenes infection in mice as a model, Lauvau et al. showed that both heat-killed and live Listeria species elicit memory CD8+ T cells, but only those induced by live Listeria species proliferate and provide protective effector functions [15]. Similarly, a study of HIV-infected individuals indicated that long-term nonprogressors had markedly higher levels of effector T cells capable of expansion and effector molecule expression, compared with HIV-infected individuals with AIDS [16]. Therefore, reliable measurements of pathogen-specific T cell responses should identify populations that both proliferate and produce effector molecules when stimulated with a relevant antigen

We have optimized a CFSE-based assay for detection of vaccinia-specific cell-mediated immunity. The effector memory T cells identified by our assay proliferate and express the effector molecules IFN-γ, granzyme A, and CD107a after in vitro stimulation with live vaccinia. We used expression of IFN-γ, granzyme A, and CD107a as measures of effector function, because of their direct associations with intracellular microbicidal activity and antigen-specific cytolysis of target cells [17–19]. IFN-γ increases the ability of cells to inhibit poxvirus replication [20]. Granzyme A, a serine protease component of cytolytic granules, induces apoptosis by a caspase-independent pathway [21]. CD107a (LAMP-1) is present in membranes of cytotoxic granules and is transiently expressed on cell surfaces as a result of degranulation [18]. Therefore, the vaccinia-specific memory T cells detectable in our new assay are predicted to be critically important for protective immunity against virulent smallpox. However, it will be important to show that T cells detectable by this CFSE-based assay correlate with protection against poxvirus replication in humans. This question can be addressed by assessing how well these in vitro immune responses predict the ability to limit vaccinia replication in vivo. Clinical protocols are being designed to study this important question

Replication-competent vaccinia strains are known to induce both humoral and cellular immune responses [3, 5]. Specific immunoglobulin has been associated with beneficial effects when given as a postexposure treatment in humans [22]. Antibody responses peak 4–6 weeks after vaccination [23, 24] and wane, to some degree, over time. However, increases in vaccinia-specific antibody levels are maintained for 50 years after primary vaccination [3, 25], with virus-specific memory B cells comprising 0.1% of the total IgG+ B cells [25]

Memory T cells are also protective against poxviruses. Mice with decreased CD4 or major histocompatibility complex (MHC) class II expression and double-knockout mice deficient in MHC class I– and MHC class II–restricted activities have enhanced susceptibility to intranasal challenge with pathogenic poxviruses [26]. Patients with T cell immunodeficiency have an increased risk of complications after smallpox vaccination, and adoptive transfers of immune lymphocytes to humans with severe forms of smallpox disease have been associated with enhanced survival [22]. As with B cell responses, cell-mediated T cell responses peak within a few weeks after administration of vaccinia [27], and increased vaccinia-specific T cell responses can be detected for 70 years after vaccination [3]

The present study has provided further confirmation that vaccinia-specific memory T cells are important components of vaccinia-specific immunity. Vaccinia induced antigen-specific memory CD4+ and CD8+ T cells that proliferate and express immune effector molecules after in vitro stimulation with live vaccinia. PBMCs from vaccinia-immune individuals had markedly higher cytolytic activity against autologous blasts infected with vaccinia. This cytolytic activity was directly correlated with the numbers of CD8+/CFSElo/granzyme A+ (r=0.8; P<.05) and CD8+/CFSElo/IFN-γ+ (r=0.7; P<.05) T cells detectable in our assays. These correlations confirm that the CD8+ T cell responses detected in flow-cytometric analysis represent functional effector T cell responses

In addition to CD4+ and CD8+ conventional αβTCR+ T cells, γδTCR+ T cells are important components of innate and adaptive immunity. γδTCR+ T cells proliferate robustly in response to various pathogens [11, 28, 29], produce cytokines and effector molecules important for limiting the multiplication of intracellular pathogens [11, 28], are capable of developing enhanced memory responses after specific vaccinations [10, 11], and are increasingly recognized in viral immunity [28, 30, 31]. To our knowledge, the present study is the first to show the presence of vaccinia-specific γδTCR+ T cells in humans

IFN-γ is a key cytokine involved in immunity to intracellular pathogens [32–34]. IFN-γ production by vaccinia-specific memory γδTCR+ T cells further suggests that these cells are important for protective immunity. Indeed, in the mycobacterial model, the frequency of γδTCR+ T cells producing IFN-γ was found to represent half of all pathogen-specific IFN-γ–producing T cells [35]. In addition, individual γδ T cells can be more efficient producers of IFN-γ, compared with αβTCR+ T cells [35]. These IFN-γ–producing γδTCR+ T cells can play a crucial role in the control of life-threatening viral infections [36] as well as in antibody isotype switching [37]

γδTCR+ T cells are enriched in epithelial surfaces and mount potent, early responses to invading pathogens. γδTCR+ T cells respond to antigens presented by antigen-presenting cells [38, 39] but are not restricted by conventional MHC class I or class II presentation [39]. They can be activated by nonpeptide phosphorylated microbial metabolites [40], broadening the potential for recognition of invading pathogens. γδTCR+ T cells express several chemokines and chemokine receptors that enable them to migrate to epithelial surfaces and also attract other immune cells relevant for protective immunity [41, 42]. In the present study, γδ T cell responses were detected only in postvaccination PBMCs, indicating that vaccinia-specific γδTCR+ T cells developed immune memory. These features indicate that γδTCR+ T cell responses may be important for vaccine-induced mucosal and systemic protection against smallpox and other pathogens

In conclusion, the CFSE-based flow-cytometric assay reliably measures memory T cells generated after vaccination with vaccinia and may be an ideal tool for testing the immunogenicity of new smallpox vaccines. The present study has shown that, not only CD4+ and CD8+ T cells, but also γδTCR+ T cells play a role in vaccinia-specific immunity

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Footnotes

  • ↵Presented in part: Experimental Biology 2004, Washington, DC, 17–21 April 2004 (abstract 554.13)

    Potential conflicts of interest: none reported

    Financial support: National Institutes of Health (grants RO1-AI-48391 and NO1-AI-25464)

  • Received October 4, 2004.
  • Accepted May 6, 2005.
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  1. J Infect Dis. (2005) 192 (8): 1362-1371. doi: 10.1086/444423
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