Memory T-Cell Responses to Vibrio cholerae O1 Infection

Study subjects and overview.The International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B) Dhaka hospital cares for over 20,000 cholera patients annually, most of whom are residents of Dhaka city. Patients older than 6 months who had a positive stool culture for V. cholerae and were without significant comorbid conditions were eligible for inclusion in the present study. All of the participants included presented with severe, acute watery diarrhea and required treatment with intravenous fluids. The study participants were also treated with antibiotics. Individuals with similar socioeconomic backgrounds as patients and who experienced no diarrheal illness in the previous 3 months were selected as healthy controls. For patients, blood samples were obtained during acute infection (the second day of hospitalization) and on days 7 and 30 during the convalescent period. At each time point, T-cell lymphoblast proliferation was measured in response to ex vivo antigenic stimulation with a membrane preparation from V. cholerae O1 (MP), TcpA, VCC, or V. cholerae LPS. These antigens are described in detail below. In addition, vibriocidal titers and serologic responses to antigens TcpA, VCC, and the homologous LPS (V. cholerae-specific serotypes Inaba or Ogawa) were performed. Study participants were enrolled after providing written informed consent. The Research and Ethical Review Committee of the ICDDR,B and the Institutional Review Board of Massachusetts General Hospital approved the present study. The human experimentation guidelines of the U.S. Department of Health and Human Services were followed during the conduct of this research.

Bacteriological examination of patient stools.Cases were confirmed by culturing stool onto taurocholate-tellurite-gelatin and MacConkey agar (28). After overnight incubation of plates, suspected V. cholerae colonies were serologically confirmed by slide agglutination with specific monoclonal antibody for Ogawa or Inaba serotypes (34, 41).

V. cholerae-specific stimulating antigens and controls.A V. cholerae MP was made from the sequenced O1 El Tor strain N16961 grown in vitro in AKI medium (21). Specifically, a culture of N16961 was inoculated into 1 liter of AKI medium (21). The culture was grown in nonaerating conditions to an optical density at 600 nm of 0.3 and then transferred to shaking conditions for growth to stationary phase. Bacteria were pelleted by centrifuging and then sonicated. The sonicated mixture was centrifuged at 1,400 × g for 10 min, and the remaining supernatant was then centrifuged at 14,900 × g for 30 min. The pellet containing the membrane fraction was then suspended in MgCl₂-Tris buffer (5 mM MgCl₂, 10 mM Tris [pH 8.0]) for subsequent experiments. Mass spectrometry analysis indicated that the MP contains a mixture of bacterial proteins, and the most abundant of these proteins are listed in Table 1.

The VCC monomer was used for V. cholerae-specific antigenic stimulation at a concentration of 2.5 ng/ml. We used VCC purified from a non-O1 clinical strain of V. cholerae; this protein is known to be immunologically and biochemically identical to V. cholerae O1 VCC (50). Isolation and purification was conducted as previously described (10). Recombinant TcpA, prepared as described previously, was used at a concentration of 2.5 μg/ml (3). V. cholerae O1 Inaba or Ogawa LPS, matched to the case serotype, was used at a concentration of 2.5 μg/ml. Preparation of V. cholerae O1 LPS was conducted as previously described (33). Positive controls for the assay included purified protein derivative (Statens Serum Institut, Copenhagen, Denmark) and phytohemagglutinin (Murex, Remel, Sweden) at concentrations of 5 and 1 μg/ml, respectively. Samples containing unstimulated cells were also included.

Measurement of V. cholerae antigen-specific antibodies in serum and lymphocyte supernatants.Vibriocidal antibody assays were performed as previously described using guinea pig complement and the homologous matched serotype of V. cholerae El Tor Ogawa (X-25049) or El Tor Inaba (T-19479) as the target organism (38). The vibriocidal titer was defined as the reciprocal of the highest serum dilution resulting in >50% reduction of the optical density compared to the optical density of control wells without serum. Seroconversion was defined as a ≥4-fold increase in vibriocidal titer after acute dehydrating diarrhea.

LPS-, TcpA-, and VCC-specific IgG and IgA in serum samples were quantified by using standardized enzyme-linked immunosorbent assay (ELISA) procedures as previously described (36, 40). For anti-TcpA detection, ELISA plates were coated with TcpA (1 μg/ml) in carbonate buffer (50 mM, pH 9.6). For LPS- and VCC-specific responses, ELISA plates were coated with 2.5 μg of antigen/ml in phosphate buffered saline (PBS; 10 mM, pH 7.2). Sera were diluted 1:100 for TcpA and VCC antibody testing and 1:50 for LPS, with 0.1% bovine serum albumin in PBS-Tween (10 mM PBS [pH 7.2] containing 0.05% Tween 20). Then, 100 μl of diluted serum/well was added for each antigen. Horseradish peroxidase-conjugated secondary antibodies to human IgG or IgA were applied in separate wells. Plates were washed and developed with ortho-phenylene diamine substrate (Sigma, St. Louis, MO) and 0.012% hydrogen peroxide in 0.1 M sodium citrate buffer. The optical density was measured kinetically at 450 nm for 5 min. The maximal rate of optical density change was expressed as milli-optical density absorbance units per minute. ELISA units were normalized by calculating the ratio of the test sample to a standard of pooled convalescent-phase serum from recovered cholera patients added to each plate (33).

For the collection of lymphocyte supernatants, heparinized blood was diluted in PBS (1:1). Peripheral blood mononuclear cells (PBMC) and serum were isolated by differential centrifugation with Ficoll-Isopaque (Pharmacia, Piscataway, NJ). PBMC were resuspended at a concentration of 10⁷ cells/ml in RPMI medium (Gibco, Carlsbad, CA) and supplemented with 1% penicillin, 1% streptomycin, 1% l-glutamine, 1% sodium-pyruvate, and 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT). PBMC were incubated for 48 h at 37°C in 5% CO₂ in 24-well tissue culture plates without mitogen. Plates were centrifuged at 1,200 × g for 10 min, and the supernatants were collected. A protease inhibitor cocktail containing aprotinin (0.15 μM), leupeptin (10 μM), sodium azide (15 μM), and 4-(amino-ethyl) benzene sulfonyl fluoride (0.2 μM) was added at a concentration of 10 μl/ml of supernatant, and sample aliquots were preserved at −70°C. Supernatants were assayed at a 1:2 dilution in 0.1% bovine serum albumin in PBS-Tween in an ELISA as described above, and responses were detected with rabbit anti-human IgA- and IgG-horseradish peroxidase conjugate. Responses were measured kinetically as previously described (33).

FASCIA.The FASCIA (flow cytometric assay of specific cell-mediated immune response in activated whole blood) method was used as previously described to determine lymphoblast formation in response to antigenic stimulation (14, 48). Briefly, whole blood was collected in a lithium heparin-coated tube and diluted 1:8 in Dulbecco modified Eagle medium supplemented with 1% gentamicin, 1% mercaptoethanol, and 10% heat-inactivated fetal calf serum. Then, 100 μl of stimulating antigen, control antigen, or additional medium was added to 400 μl of diluted blood. After 6 days of in vitro culture at 37°C in 5% CO₂, supernatant was preserved for cytokine analysis, and cells were stained with anti-CD3-FITC, anti-CD4-PerCP, anti-CD8-APC, and anti-CD45R0-PE monoclonal antibodies (Becton Dickinson Immunocytometry Systems [BD], Stockholm, Sweden). An erythrocyte lysing solution of 1.0 μl of ammonium chloride (Orthumune lysing reagents; Ortho Diagnostics, Stockholm, Sweden) was added for 6 min, followed by incubation at room temperature, followed in turn by centrifugation, the removal of supernatants, and washing. Cells were suspended in 2% paraformaldehyde and stored in the dark; within 12 h, acquisition was conducted by fluorescence-activated cell sorting (FACSCalibur [BD]) for standardized 2-min intervals using CellQuest Pro software (Becton Dickinson, San Jose, CA). Analysis was performed with FlowJo software (TreeStar, Inc.). The results are presented as the ratio of lymphoblast count with antigenic stimulation to the count without stimulation (stimulation index). A value of “1” indicates that stimulation is equal in samples with or without V. cholerae antigenic stimulation.

Additional investigation of memory T-cell phenotype.To further define the character of lymphoblasts proliferating in response to V. cholerae-specific antigens, we examined the peripheral blood of five cholera patients for additional T-cell phenotypic markers. On day 2 and day 7 after infection, we used the FASCIA method as described above and identified cell surface markers with anti-CD4-PerCP, anti-CD45R0-PE, anti-CD45RA-FITC, and anti-CCR7-APC monoclonal antibodies (BD).

Statistical analyses.The Wilcoxon signed-rank test was used to compare immunologic responses of cholera patients on different study days, and the Mann-Whitney U test was used to compare immune responses between patients and healthy controls. All reported P values are two tailed. A cutoff of P ≤ 0.05 was the predetermined threshold for statistical significance. Analyses and figure preparation were performed on GraphPad Prism 4.0 and Stata version 9.0 (Stata Corp., Inc., College Station, TX). Geometric means with 95% confidence intervals are shown in figures unless otherwise stated.

Study population.Sixteen patients were enrolled in the study, and fifteen completed 30 days of follow-up. All participants had cholera and severe dehydration upon initial clinical evaluation. Ten apparently healthy adults were included as healthy controls. Demographic and clinical features comparing cholera patients and healthy controls are shown in Table 2.

Vibriocidal and antigen-specific antibody responses.VCC, LPS, and TcpA-specific antibody responses and vibriocidal titers were measured in sera on days 2, 7, and 30 and are shown in Table 3. On day 2 of infection, the geometric mean vibriocidal titer was 35 (95% confidence interval, 13 to 97), and this increased to 4,300 (3,000 to 6,300) by day 7 after infection (P < 0.001). All 16 patients demonstrated seroconversion.

We observed significant humoral responses to V. cholerae-specific antigens. VCC-specific IgG antibody titers peaked on day 30 and were significantly higher than titers on day 2 (P = 0.027). TcpA- and V. cholerae O1 LPS-specific IgG levels were highest on day 7 (P < 0.001 and P < 0.001 compared to day 2). Patients demonstrated peak IgA responses on day 7 for all antigens, and these levels were significantly higher than day 2 measurements (P = 0.001 for TcpA- and LPS-specific antibodies, P < 0.001 for VCC-specific antibodies). LPS-, VCC-, and TcpA-specific antibody responses and vibriocidal titers were significantly greater on day 7 compared to responses observed in healthy controls.

Memory T-cell responses.Using the FASCIA method to measure lymphoblast proliferation, we observed significant increases in V. cholerae-specific memory T cells on day 7 after infection; these results are shown in Fig. 1. T-cell memory responses after stimulation by VCC and MP peaked on day 7 and decreased by day 30 (P = 0.013 and P = 0.001 for day 7 compared to day 2). Proliferation in response to TcpA increased by day 7 (P = 0.013) and remained elevated until day 30. Compared to healthy controls, the proliferation of memory T cells in response to VCC was significantly elevated on day 7 and day 30 (P < 0.001 and P < 0.001). Differences in proliferation between HC and patient lymphoblasts were significant on both day 7 (P < 0.001) and day 30 (P = 0.035). Stimulation with V. cholerae O1 LPS, a T-cell independent antigen, did not generate any significant differences in memory T-cell proliferation during the 30-day period after infection.

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FIG. 1.

Memory T-cell responses to V. cholerae antigens. Memory T cells are defined as CD4⁺/CD45R0⁺ cells. *, P ≤ 0.05 for day 2 compared to day 7 or day 30; ∧, P ≤ 0.05 for healthy controls (HC) compared to day 7 or day 30.

Immune responses to VCC.The antigen VCC generated a more robust T-cell memory lymphoblast response than that observed to other V. cholerae antigens assayed (Fig. 1). In addition, high-magnitude humoral responses to VCC were measured (Table 3). We also used the antibody in lymphocyte supernatant assay to assess mucosal immune responses to VCC (9). On day 7 after infection, lymphocytes stimulated in the gut mucosa transiently circulate in the peripheral blood (40). At this time, we observed a significant increase in VCC-specific antibodies secreted from circulating lymphocytes compared to day 2. These results are shown in Fig. 2.

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FIG. 2.

VCC-specific antibody responses in supernatants of lymphocytes circulating on days 2 and 7 after infection.

Memory T-cell function and phenotype.We used known markers of T-cell function to further determine the nature of the memory T-cell populations after V. cholerae infection. The CD45RA marker was used to exclude intermediate memory populations (CD45R0⁺ CD45RA⁺ cells) from the analysis (17). The memory T-cell CD4⁺ CD45R0⁺ CD45RA⁻ population was on average 80% CCR7⁻ after 6 days ex vivo stimulation, and a representative flow cytometry plot is shown in Fig. 3. Lymphoblast populations displaying these markers indicate an effector memory population. The remaining CD4⁺ CD45R0⁺ CD45RA⁻ cells were CCR7⁺, markers consistent with a central memory T-cell phenotype.

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FIG. 3.

Proportion of central memory cells in the proliferating memory T-cell population. On average, 20% of CD4⁺ CD45R0⁺ CD45RA⁻ memory T cells had the CCR7⁺ marker, a finding consistent with the central memory phenotype. Plots above are representative of the five patients sampled. (A) Lymphoblast and lymphocyte population after 6-day ex vivo stimulation with V. cholerae MP. (B) Lymphoblast gated CD4⁺ population. (C) CD4⁺ gated CD45R0⁺ CD45RA⁻ population. (D) CD4⁺ CD45R0⁺ CD45RA⁻ gated CCR7⁺ population. CCR7⁺ gates were placed by comparing a CCR7 unstained control to a CCR7 stained plot.