Bacille Calmette-Guérin Vaccination Enhances Human γδ T Cell Responsiveness to Mycobacteria Suggestive of a Memory-Like Phenotype

Daniel F. Hoft, Robin M. Brown and Stanford T. Roodman
J Immunol July 15, 1998, 161 (2) 1045-1054;

Abstract

Bacille Calmette-Guérin (BCG) immunity can be studied as one experimental model for mycobacterial protective immunity. We have used flow cytometry to investigate human T cell subsets induced by BCG vaccination. PBMC harvested from BCG-vaccinated individuals and controls were stimulated with mycobacterial Ags, and the T cell subsets present after 7 days of in vitro expansion were characterized. The most dramatic expansions induced by mycobacterial Ags were detected in γδ T cells. The γδ T cell expansions measured after in vitro stimulation with mycobacterial Ags were significantly greater in BCG responders compared with nonsensitized controls, indicating that BCG vaccination induced γδ T cell activation associated with enhanced secondary responses. The majority of γδ T cells induced by BCG vaccination were γ9+δ2+ T cells reactive with isoprenyl pyrophosphates. Coculture with CD4+ T cells induced optimal γδ T cell expansion, although IL-2 alone could provide this helper function in the absence of CD4+ T cells. γδ T cells were found to provide helper functions for mycobacterial specific CD4+ and CD8+ T cells as well, demonstrating reciprocal stimulatory interactions between γδ T cells and other T cell subsets. Finally, prominent mycobacterial specific γδ T cell expansions were detected in a subset of unvaccinated controls with evidence for prior sensitization to mycobacterial lysates (elevated mycobacterial specific lymphoproliferative responses). These latter findings are consistent with the hypothesis that exposure to atypical mycobacteria or related environmental Ags may induce γδ T cells cross-reactive with Ags present in the Mycobacterium tuberculosis complex. Our results suggest that γδ T cells may be capable of developing a memory immune-like phenotype, and therefore might be important targets for new vaccines.

Bacille Calmette-Guérin (BCG),3 an attenuated strain of Mycobacterium bovis, is the only vaccine available for prevention of human disease caused by mycobacteria. The protective efficacy of BCG has been controversial, but recent metaanalyses suggest that BCG can reduce the risk of pulmonary TB by 50% and decrease TB-related deaths by 71% (1, 2). The resurgence of TB in many regions of the world, and the increasing occurrence of multiple drug-resistant TB, have renewed interest in the expanded use of BCG and the development of vaccines more effective than BCG. To develop better vaccines, detailed studies characterizing the partially protective immune responses induced by BCG are important. In addition, the molecular techniques required to insert foreign genes into BCG have been developed (3, 4, 5, 6, 7), and detailed knowledge of human immunity induced by BCG will be important for its appropriate use as a live vector.

Recent evidence has shown that T lymphocytes can produce distinct cytokine profiles with opposing effects on resistance and susceptibility (8, 9, 10, 11). Type 1 responses involve the induction of IFN-γ, IL-2, and cytolytic activity, and are associated with resistance to intracellular infections. Type 2 responses are associated with IL-4, IL-5, IL-6, and IL-10 production, cytokines involved in the induction of humoral immunity as well as the down-regulation of type 1 immune responses. Extensive animal data indicate that type 1 CD4+ T cell responses are important in mycobacterial immunity (12, 13, 14, 15, 16, 17, 18, 19, 20, 21). In addition, increased IFN-γ responses have been documented in three human models of protective mycobacterial immunity: in pleural lymphocytes from patients with tuberculous pleuritis (22, 23), in PBMC from persons with asymptomatic Mycobacterium tuberculosis infections (23, 24), and in PBMC from BCG-vaccinated individuals (25, 26). Furthermore, recent human genetic studies have documented that mutations resulting in nonfunctional IFN-γ receptor genes can result in high susceptibility to mycobacteria (27, 28). These findings indicate that type 1 immunity is important in humans for resistance against mycobacterial infections, but the specific subsets of human T cells involved in these type 1 responses have not been well characterized. Human CD4+ T cells, CD8+ T cells, γδ T cells, and CD4,CD8 double-negative αβ T cells all have been shown to be induced by mycobacterial Ags (15, 24, 26, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39), but the specific functions of these different subsets and their relative importance for protective immunity are unknown.

Recent investigations have demonstrated that normal human volunteers not previously vaccinated with BCG and presumably never infected with M. tuberculosis can be divided into two groups defined by their baseline mycobacterial specific lymphoproliferative reactivity: 1) individuals with evidence for previous sensitization to mycobacterial Ags and 2) nonsensitized individuals (26). Comparisons of the immune responses induced by BCG vaccination in these two groups suggested that BCG boosted memory CD4+ T cell responses in previously sensitized individuals, and induced primary CD4+ T cell immunity in previously nonsensitized individuals. We have used a similar strategy of identifying subsets of presensitized and nonsensitized volunteers, and have focused on the identification of the major subsets of T cells induced by BCG vaccination during primary immune responses in nonsensitized individuals. Our results provide evidence for the relative importance of γδ T cells in BCG-induced immunity, and support the hypothesis that γδ T cells can develop a form of memory immune phenotype.

Materials and Methods

Subjects and PBMC

The study protocols were approved by St. Louis University Institutional Review Board (St. Louis, MO). Volunteers were healthy individuals aged 18 to 45 with negative reactions to a 5 T.U. PPD skin test and negative HIV serology at the time of enrollment. Fifty-four volunteers were randomized in a double-blinded fashion into three groups of 18 individuals to receive one of two BCG strains or placebo. A quantity amounting to 3 × 106 CFU of Connaught (Connaught Labs, Swiftwater, PA) or Tice (Organon Teknika, Rockville, MD) BCG was administered intradermally in the left deltoid in 100 μl of saline. The placebo group received 100 μl of saline alone. A total of 53 of the 54 volunteers enrolled in the randomized trial completed the 3-mo follow-up, including evaluations of reactogenicity and serial measurements of immune responses. Of 36 volunteers given BCG in the randomized trial, 86% had ≥5 mm of induration in response to a 5 or 10 T.U. PPD skin test 2 mo after vaccination, and similar PPD responses were measured in recipients of both BCG strains. None of the placebo recipients had detectable induration in response to 10 T.U. PPD skin tests 2 mo postvaccination. Heparinized blood was obtained prevaccination and on days 28 and 56 postvaccination, and PBMC were purified over Histopaque (Sigma, St. Louis, MO). PBMC were either stimulated on the day of harvest in T cell proliferation assays, as described below, or diluted with 7.5% DMSO before CryoMed computer-controlled freezing (Forma Scientific, Marietta, OH) and liquid N2 storage until use in T cell expansion studies.

Ag preparations for in vitro stimulation of PBMC

Whole cell lysates of the Erdman strain of M. tuberculosis were prepared from mid-logarithmic phase cultures grown in glycerol-alanine salts broth. After washing with PBS, mycobacteria were heat killed (80°C for 1 h), then disrupted by sonication or bead vortex, before passage through 0.2-μm filters. Live BCG was prepared from lyophilized vials of commercial BCG vaccine provided by Connaught Labs. A new vial of BCG vaccine was freshly reconstituted in RPMI with glutamine, penicillin/streptomycin, and 10% human AB pooled serum on each day that cultures of PBMC were stimulated with live BCG. Representative vials of the BCG vaccine lot used to stimulate PBMC were cultured on 7H10 agar to confirm the concentration of viable CFU stated by the manufacturer. Isopentenyl pyrophosphate (IPP) (#I0503) and PHA were obtained from Sigma. Preservative- and detergent-free tetanus toxoid was supplied by Connaught Labs. Murine rIL-2 was obtained from Boehringer Mannheim (Indianapolis, IN).

T cell proliferation assays

The kinetics and dose response of proliferative responses in PBMC to the different antigenic preparations were optimized in preliminary experiments. The following protocol was found to result in measurements of optimal responses to mycobacterial and control Ags. PBMC freshly harvested from BCG and placebo recipients on days 0, 28, and 56 postvaccination were diluted to 5 × 105 cells/ml in RPMI medium supplemented with 10% pooled human AB serum, penicillin, streptomycin, glutamine, and β-mercaptoethanol, and stimulated for 6 days with optimal doses of M. tuberculosis whole lysate (2 μg/ml), tetanus toxoid (10 Lf/ml), PHA (1 μg/ml), or medium alone at 37°C with 5% CO2. On the sixth day, cultures were pulsed with 0.5 μCi of tritiated thymidine and further incubated overnight before measuring cell-associated radioactivity. Proliferative responses in the BCG and placebo groups were compared in Mann-Whitney U tests using the Statistica software package (StatSoft, Tulsa, OK).

Analysis of in vitro T cell subset expansion

The timing for T cell subset analyses and the doses of Ags used for in vitro stimulation were optimized in preliminary experiments. Maximal absolute numbers of total viable T cells were present after 7 days of in vitro expansion, when maximal incorporation of tritiated thymidine was measured. PBMC frozen 2 mo after vaccination with BCG or placebo were thawed, washed, and diluted to 1 × 106 cells/ml in RPMI medium supplemented with 10% pooled human AB serum, penicillin, streptomycin, and glutamine (without β-mercaptoethanol). These PBMC were stimulated in vitro for 7 days with 1) medium alone, 2) tetanus toxoid (10 Lf/ml), 3) M. tuberculosis whole lysate (10 μg/ml), 4) live BCG (4000 CFU/2 × 105 PBMC), or 5) 10 μM of IPP plus murine rIL-2 (10 U/ml). Viable cells were recovered, total numbers counted by hemacytometer, and aliquots stained with fluorescent Abs specific for T cell surface markers. The percentages of CD3+CD4+CD8−, CD3+CD4−CD8+, and CD3+αβ TCR−γδ TCR+ T cells were analyzed by three-color flow analysis with a Becton Dickinson FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA). Eight population analyses were performed with the CellQuest software (Becton Dickinson), assigning cutoffs for positive staining in the three individual fluorescent intensity histograms, and gating on lymphocytes based on forward and side scatter. This allowed all eight possible three-color staining combinations to be calculated. The following Ab combinations were used: 1) anti-CD3 (Leu4) FITC, anti-CD8 (Leu2a) PE, and anti-CD4 (Leu3a) PerCP; 2) anti-αβ TCR (WT31) FITC, anti-γδ TCR(γ/δ-1) PE, and anti-CD3 (Leu4) PerCP; and 3) anti-Vγ9 FITC, anti-Vδ2 PE, and anti-CD3 (Leu4) PerCP. All Abs were obtained from Becton Dickinson, except the anti-Vγ9 and anti-Vδ2 Abs obtained from PharMingen (San Diego, CA). The absolute numbers (AN) of different T cell subsets were calculated by multiplying the percentage measured by flow cytometry by the total number of viable cells recovered from the same culture (AN = % × total cells recovered). Expansion indices (EI) were calculated by dividing the AN of a specific T cell subset present after Ag stimulation by the AN of these T cells present after incubation with medium alone (EI = AN with Ag/AN with media). Mann-Whitney U test comparisons of responses in different groups were performed with the Statistica software (StatSoft).

CD4 purification/depletion

Thawed PBMC were labeled with magnetic anti-CD4 microbeads (Miltenyi Biotec, Auburn, CA). A proportion of 20 μl of anti-CD4 microbeads for 107 total PBMC resulted in optimal depletions of CD4+ T cells. CD4+ and CD4− T cells were separated in MiniMACS separation columns (Miltenyi Biotec), and aliquots of the separated populations were stained with anti-CD3 (Leu4) FITC, anti-CD8 (Leu2a) PE, and anti-CD4 (Leu3a) PerCP for flow cytometry. Purified CD4+ T cells were found to be greater than 99% CD3+CD4+CD8−. CD4-depleted cells had less than 1% residual CD3+CD4+CD8− T cells. For T cell expansion studies, 1 × 105 purified CD4+ T cells were incubated with 2 × 105 irradiated total autologous PBMC as APCs. A quantity amounting to 2 × 105 total and CD4-depleted cell populations was studied concurrently.

γδ T cell depletion

Thawed PBMC were stained with an unlabeled human γδ T cell pan-specific γ/δ-1 mAb obtained from Becton Dickinson (1 μg for 2 × 106 PBMC). The PBMC were washed, and magnetic goat anti-mouse Ig microbeads (Miltenyi Biotec) were added (20 μl of goat anti-mouse Ig microbeads for 107 total PBMC). γδ T cells were removed in MiniMACS separation columns (Miltenyi Biotec), and aliquots of the final populations were stained with anti-αβ TCR (WT31) FITC, anti-γδ TCR (γ/δ-1) PE, and anti-CD3 (Leu4) PerCP for flow cytometry. In all γδ depletion experiments, less than 0.25% of the residual cells after depletion were γδ TCR+ T cells. In addition, the lack of γδ T cell expansion in depleted cultures after stimulation with M. tuberculosis whole lysate or live BCG further demonstrated the efficacy of the depletion protocol (see Table III⇓).

IFN-γ assay

PBMC were thawed and incubated in 96-well tissue culture plates (2 × 105 cells in 0.2 ml/well) with medium alone, or optimal concentrations of M. tuberculosis whole lysate (2 μg/ml) or live BCG (4000 CFU of Connaught BCG/well) for 6 days at 37°C with 5% CO2. Supernatants were collected and stored at −70°C. IFN-γ levels in these supernatants were measured by ELISA using the cytokine-specific Ab pair 400-45/6 mouse IgG1 anti-human IFN-γ (Chemicon, Temecula, CA) and P-700 rabbit anti-human IFN-γ (Endogen, Boston, MA), followed by ABTS substrate (Kirkegaard & Perry, Gaithersburg, MD). Human rIFN-γ (PharMingen) was used as a standard to determine cytokine concentration. The sensitivity of the IFN-γ assay was 1 ng/ml.

Results

Mycobacterial specific T cell proliferative responses identify important groups for comparison in BCG and placebo recipients

BCG induced significant increases in proliferative responses to M. tuberculosis whole lysates postvaccination compared with placebo. The overall mean responses (±SE) in the placebo (n = 18) and BCG (n = 35) groups were 24,884 (±7,600) and 54,167 (±6,200), respectively (p < 0.004 by Mann-Whitney U test). However, despite the fact that all volunteers included in this vaccine trial were required to have negative PPD skin tests and the absence of any known direct exposure to TB before enrollment, PBMC harvested from some of the volunteers in the placebo and BCG groups prevaccination were highly reactive with the mycobacterial lysates used in our T cell proliferation assays. Other investigators have described similar levels of immunoreactivity with mycobacterial lysates in PBMC from unvaccinated and presumably uninfected individuals, and this baseline immunoreactivity has been attributed to lymphocytes previously sensitized by cross-reactive environmental Ags (26).

The main goal of our present work was to identify the T cell subsets induced by BCG vaccination with enhanced responsiveness to secondary in vitro stimulation with mycobacterial Ags. The inclusion of volunteers with prior sensitization to mycobacteria in comparisons of BCG and placebo groups can complicate efforts to distinguish the treatment effects of BCG vaccination from preexisting immunoreactivity. To facilitate the identification of memory T cell responses induced by BCG in our initial investigations, we excluded volunteers from both BCG and placebo groups with evidence for sensitization to mycobacterial Ags prevaccination. Previously sensitized individuals were defined as individuals with lymphoproliferative responses to M. tuberculosis whole lysate prevaccination greater than 18,392 dpm (mean of prevaccination responses in all 54 volunteers plus 1 SD). To further enhance the sensitivity of our analyses for the detection of T cell responses induced by BCG, we excluded nonresponders from the treatment group defined as BCG recipients with lymphoproliferative responses to M. tuberculosis whole lysate 2 mo postvaccination less than 31,000 dpm (baseline mean for all 54 volunteers plus 2 SDs).

Using the criteria outlined above, 8 of 18 placebo recipients were defined as nonsensitized negative controls, and 23 of 36 BCG recipients were defined as BCG responders. The proportions of volunteers excluded from placebo and BCG groups were not significantly different (p = 0.444 by two-tailed Fischer’s exact t test). There were no differences comparing the nonsensitized control, presensitized control, and BCG responder groups for overall immunoreactivity prevaccination; the mean proliferative responses stimulated by tetanus and PHA were similar in all three groups (data not shown). The results in Figure 1⇓ demonstrate that significant increases in lymphoproliferative responses to M. tuberculosis lysate were detected in the BCG responder group postvaccination (p < 0.01 by Mann-Whitney U test comparing the BCG responder group with each of the three other groups). The increased responses to M. tuberculosis lysate detected in the BCG responder group postvaccination were Ag specific; lymphoproliferation in cultures incubated with medium alone or stimulated with the tetanus control Ag was not increased in the BCG responder group postvaccination (Fig. 1⇓).

FIGURE 1.
FIGURE 1.

M. tuberculosis-specific T cell proliferative responses in BCG recipients and controls 2 mo postvaccination. The pre- to postvaccination increases in proliferative responses (day 56 dpm divided by day 0 dpm) detected in cultures of PBMC incubated with medium alone, tetanus toxoid Ag, or M. tuberculosis whole lysate are shown. The placebo group has been divided into nonsensitized and presensitized individuals with baseline proliferative responses to M. tuberculosis lysate < or > 18,321 dpm, respectively (18,321 dpm = mean response plus 1 SD of all volunteers prevaccination). The BCG vaccine group was divided into responders and nonresponders. BCG responders were defined as volunteers who met the nonsensitized definition prevaccination, and then developed T cell proliferative responses to M. tuberculosis whole lysate 2 mo postvaccination >31,000 dpm (the baseline mean response plus 2 SDs). The fold increase in Mtb lysate-stimulated lymphoproliferation in the BCG responder group was significantly greater than the fold increases in each of the three other groups by Mann-Whitney U test comparisons (*p < 0.01). Points indicate medians; boxes, 25%/75% of cases; whiskers, nonoutlier ranges.

BCG vaccination induces enhanced responsiveness of γδ T cells to secondary in vitro stimulation with mycobacterial lysates and live BCG

To study the specific subsets of T cells induced by BCG vaccination, we analyzed the relative proportions of CD4+, CD8+, and γδ T cells present after secondary in vitro stimulation with mycobacterial Ags. Representative two-parameter dot plots obtained by FACS analysis of PBMC from one BCG responder are shown in Figure 2⇓. Presented are the results of dual labeling for the pan T cell marker, CD3, combined with reagents specific for CD4, CD8, or γδ TCR, after in vitro expansion with tetanus toxoid Ag or M. tuberculosis whole lysate. The percentages of CD4+ and CD8+ T cells were decreased after M. tuberculosis lysate stimulation compared with tetanus toxoid stimulation (Fig. 2⇓, A and B). However, the percentage of γδ T cells was more than 10-fold higher after in vitro expansion with M. tuberculosis whole lysate compared with tetanus toxoid (Fig. 2⇓C). It is also apparent that the populations of CD3+CD4− and CD3+CD8− cells were increased after M. tuberculosis whole lysate stimulation (Fig. 2⇓, A and B). By eight population analyses, the increased percentage of CD3+CD4−CD8− cells was equivalent to the increased percentage of CD3+αβ TCR−γδ TCR+ cells present after M. tuberculosis whole lysate stimulation (data not shown). Therefore, no increase in the CD3+αβ TCR+CD4−CD8− T cell subset previously found to be reactive with mycobacterial lipid (39) was detected.

FIGURE 2.
FIGURE 2.

In vitro stimulation with mycobacterial lysate results in marked expansion in γδ T cells. Shown are two-parameter dot plots of flow analyses indicating the subsets of T cells present in cultures of PBMC from one representative BCG responder after in vitro expansion with tetanus toxoid and M. tuberculosis whole lysate. A, Presents the results of dual staining for CD3 and CD4 surface markers. B, Presents the results of dual staining for CD3 and CD8 surface markers. C, Presents the results of dual staining for CD3 and γδ TCR surface markers.

Table I⇓ presents the overall results for T cell expansion studies with PBMC from nonsensitized controls and BCG responders after in vitro stimulation with media, tetanus, M. tuberculosis whole lysate, or live BCG. The percentages and absolute numbers of CD4+, CD8+, and γδ T cells present after incubation with medium alone or tetanus toxoid were not different comparing PBMC from nonsensitized controls and BCG responders. In addition, CD4+ and CD8+ T cell percentages and absolute numbers were similar in nonsensitized controls and BCG responders after stimulation with M. tuberculosis whole lysate. However, the absolute number of γδ T cells present after in vitro stimulation with M. tuberculosis whole lysate was fourfold higher in BCG responders compared with nonsensitized controls (p < 0.02). All three populations of T cells were increased significantly in BCG responders compared with nonsensitized controls after stimulation with live BCG (p < 0.01). The absolute number of γδ T cells present after in vitro stimulation with live BCG was 142-fold higher in BCG responders compared with nonsensitized controls (p < 0.002). At the dose of live BCG used to stimulate PBMC (4000 CFU/2 × 105 PBMC), the induction of γδ T cell expansion required viable mycobacteria. Similar doses of heat-killed BCG did not induce responses above background levels detected after incubation with medium alone (data not shown).

View this table:
Table I.

T cell subset expansion in PBMC from nonsensitized controls and BCG responders after in vitro stimulation with control and mycobacterial antigensa

The occurrence of both maximal total absolute numbers of T cells and maximal incorporation of tritiated thymidine at the day 7 time point used in our analyses (data not shown) indicates that our results included measurements of T cell expansion, not just increased viability. Comparisons of the absolute numbers of γδ T cells present before and after stimulation with mycobacteria provide further direct evidence for T cell expansion during our experimental protocol. The absolute numbers of γδ T cells present in cultures of PBMC before in vitro stimulation were always less than 10,000 (1–5% of 2 × 105 mononuclear cells/well). The mean absolute numbers of γδ T cells detected after stimulating cultures of PBMC from BCG responders with M. tuberculosis lysate or live BCG (Table I⇑) were 6.4- to 8.5-fold higher than the maximal starting number of γδ T cells.

Figure 3⇓ presents the same data shown in Table I⇑ for M. tuberculosis whole lysate and live BCG stimulation, but expressed as the EI for each T cell subset (calculated by dividing the absolute number of a T cell subset present after stimulation with M. tuberculosis whole lysate or live BCG by the absolute number of the same T cell subset present after incubation with medium alone). When the data were analyzed in this manner, the only significant differences between BCG responders and nonsensitized controls were detected in comparisons of the expansion of γδ T cells.

FIGURE 3.
FIGURE 3.

CD4+, CD8+, and γδ T cell expansions after stimulation with M. tuberculosis whole lysate and live BCG. Shown are the same data presented in Table I⇑, but analyzed as the EI for each T cell subset induced by M. tuberculosis whole lysate and live BCG. The EI is defined as the absolute number of a specific T cell subset present after Ag stimulation, divided by the absolute number of the same T cell subset present in the same PBMC incubated with medium alone. The legends for each bar type identify the T cell subset being analyzed and the Ag used to induce T cell expansion. CD4, indicates CD3+CD4+CD8− T cells; CD8, indicates CD3+CD4−CD8+ T cells; and γδ, indicates CD3+αβ TCR−γδ TCR+ T cells. Statistically significant differences in Mann-Whitney U test comparisons between BCG responder and nonsensitized controls are indicated. Points indicate medians; boxes, 25%/75% of cases; whiskers, nonoutlier ranges.

The majority of γδ T cells induced by BCG vaccination are γ9+δ2+ T cells

Increased γ9+δ2+ T cells have been detected in healthy PPD+ persons asymptomatically infected with M. tuberculosis compared with individuals presenting with active TB (36). These results suggested that γ9+δ2+ T cell responses may be important for protective mycobacterial immunity. We next studied the specific subsets of γ and δ TCR protein subunits expressed in the γδ T cells induced by BCG vaccination. We used mAbs specific for the γ9 and δ2 TCR polypeptide subunits to study the proportion of γδ T cells expressing these genotypes in PBMC from three different BCG responders after in vitro expansion with M. tuberculosis whole lysate (Table II⇓). In all three cases, γ9 and δ2 were detected on the majority of γδ T cells expanding after M. tuberculosis whole lysate stimulation. The percentages of CD3+γ9+ and CD3+δ2+ present were virtually identical, strongly suggesting that most of the expanding γδ T cells were CD3+γ9+δ2+, although the γ9- and δ2-specific reagents available could not be used to simultaneously label individual cells to confirm this conclusion. In any case, these results indicate that BCG vaccination induces subsets of γδ T cells with TCR expression similar to the γδ T cells previously found to be increased in individuals with asymptomatic M. tuberculosis infections and decreased in people with active TB (36).

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Table II.

γ 9+δ 2+ cells are the predominant γδ T cells induced by BCGa

γδ T cells induced by BCG are responsive to IPP plus IL-2

γδ T cells from PPD+ persons have been shown previously to be reactive with protease-resistant components present in mycobacterial lysates (40, 41, 42), and recent investigations have demonstrated that these γδ T cells proliferate in response to prenyl pyrophosphate derivatives (43, 44, 45). To determine whether BCG vaccination induced γδ T cells reactive with phosphorylated isoprenoidal compounds, we compared cultures of PBMC from BCG responders and nonsensitized controls after stimulation with purified IPP. In preliminary experiments, we found that IPP alone did not stimulate γδ T cell expansions. However, high levels of γδ T cell expansion were induced in PBMC from BCG responders with a combination of IPP plus IL-2. Incubation with IL-2 alone had no effect on CD4+, CD8+, or γδ T cell subsets in PBMC from nonsensitized controls and BCG responders (data not shown). The absolute numbers of CD4+, CD8+, and γδ T cells present after stimulation of PBMC from six nonsensitized controls and eight BCG responders with IPP plus IL-2 are shown in Figure 4⇓. A significant increase in γδ T cell expansion induced by IPP plus IL-2 was detected in BCG responders compared with nonsensitized controls (p < 0.029 by Mann-Whitney U test). Because IPP is thought to be a specific stimulus for γδ T cells (46), these results suggest that the important helper function provided by CD4+ T cells for γδ T cell expansion is the secretion of IL-2. Unexpectedly, significant increases in the expansion of both CD4+ and CD8+ T cells were induced by IPP plus IL-2 in PBMC from BCG responders (p < 0.007 and p < 0.015, respectively). These latter results suggested the possibility that γδ T cells may provide important helper functions for the induction of CD4+ and CD8+ T cells during Ag-specific immune responses.

FIGURE 4.
FIGURE 4.

IPP plus IL-2 induce significant increases in γδ T cells, as well as in CD4+ and CD8+ T cells, in BCG responders. PBMC harvested from BCG responders and nonsensitized controls were incubated with 10 μM IPP plus 10 U/ml rIL-2, and the T subsets present after 7 days were analyzed by flow cytometry, as described in Figures 2⇑ and 3⇑. Shown are the absolute numbers of each T cell subset in nonsensitized control and BCG responder groups. IL-2 alone had no effects on T cell subsets. Statistically significant differences in Mann-Whitney U test comparisons between BCG responder and nonsensitized controls are indicated. Points indicate medians; boxes, 25%/75% of cases; whiskers, nonoutlier ranges.

Effects of CD4 and γδ T cell depletions on the reactivity with IPP

Our next experiments were designed to determine whether the enhanced γδ T cell responsiveness detected after BCG vaccination was due to an intrinsic difference in γδ T cells or simply a manifestation of an increased helper function provided by mycobacterial specific memory CD4+ T cells. In addition, we wanted to rule out the possibility that the CD4+ and CD8+ T cell expansions shown in Figure 4⇑ were due to a direct activation of these cells by IPP + IL-2. To accomplish these goals, we compared the expansions of T cells induced by IPP + IL-2 in total PBMC, CD4-depleted PBMC, and γδ-depleted PBMC harvested from four BCG responders 2 mo postvaccination (Fig. 5⇓). The protocols used to deplete CD4+ and γδ T cells were highly efficient, reducing the residual percentages of CD4+ and γδ T cells to less than 1% of the original populations (data not shown). In addition, it can be seen in Figure 5⇓A that CD4+ and γδ T cells were nearly undetectable in CD4- and γδ-depleted cultures, respectively, after the in vitro expansions with IPP + IL-2. The absolute numbers (Fig. 5⇓A) and expansion indices (Fig. 5⇓B) of γδ T cells induced by IPP + IL-2 were unaffected by the depletion of CD4+ T cells. In addition, it is clear from these experiments that CD4+ and CD8+ T cells are not directly induced to expand by IPP + IL-2 because these T cell subsets actually decreased after stimulation in γδ-depleted cultures. These results indicate that the enhanced γδ T cell responsiveness detected after BCG vaccination was not due simply to an increased helper function provided by mycobacterial specific memory CD4+ T cells, but was related to an intrinsic difference in γδ T cells postvaccination.

FIGURE 5.
FIGURE 5.

Interactions between CD4+ and γδ T cells in BCG-induced immunity. Total, CD4-depleted, and γδ-depleted PBMC from BCG responders were stimulated with 10 μM IPP and IL-2. Seven days later, the absolute numbers of CD4+, CD8+, and γδ T cells present were analyzed by flow cytometry. CD4+ and γδ T cells were depleted with immunomagnetic beads, as described in Materials and Methods. Shown are the mean (± SE) results from four separate experiments with PBMC from individual BCG responders. A, Presents absolute numbers of T cell subsets. B, Presents expansion indices, as calculated in Figure 3⇑. CD4 depletion had no effect on the expansion of γδ T cells induced with IPP. CD4+ and CD8+ T cells were not reactive with IPP in the absence of γδ T cells.

γδ T cell depletion inhibits CD4+ and CD8+ T cell expansion, but does not prevent IFN-γ production

To follow up on the interesting possibility that γδ T cells may provide important helper functions for the induction of CD4+ and CD8+ T cells, we compared T cell expansions induced by M. tuberculosis whole lysate and live BCG in total and γδ-depleted PBMC from four separate BCG responders (Table III⇓). The depletion of γδ T cells resulted in significant decreases in the expansions of both CD4+ and CD8+ T cells in cultures stimulated with either M. tuberculosis whole lysate or live BCG. For example, the mean number of CD4+ T cells present after in vitro stimulation with live BCG was 10-fold lower in cultures of γδ T cell-depleted PBMC compared with total PBMC (p < 0.021). These results provide further evidence for the hypothesis that γδ T cells provide important helper functions for the optimal expansion of CD4+ and CD8+ T cells. However, IFN-γ responses induced by in vitro stimulation with M. tuberculosis whole lysate and live BCG were similar in cultures of γδ T cell-depleted and total PBMC. Therefore, these results indicate that γδ T cells are not necessary for the induction of mycobacterial specific IFN-γ responses in PBMC, at least not during secondary in vitro stimulation.

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Table III.

γδ T cell depletion inhibits CD4+ and CD8+ T cell expansion but does not prevent IFN-γ production

Similar profiles of mycobacterial specific T cell expansion are measurable in presensitized controls

After characterizing the mycobacterial specific T cell expansions in BCG responders, it was of interest to compare these results with the T cell expansions inducible with mycobacterial Ags in previously sensitized placebo recipients. Figure 6⇓ presents the expansion indices for CD4+, CD8+, and γδ T cells induced by Mtb whole lysate, live BCG, and IPP plus IL-2 in PBMC harvested from the 10 placebo recipients with evidence for previous mycobacterial sensitization (baseline proliferative responses to Mtb lysate >18,392 dpm) recruited into our randomized vaccine trial. Similar to the results detected in BCG responders, γδ T cells appeared to be the most prominent T cell subset reactive with mycobacterial Ags in PBMC from presensitized controls. These results suggest that γδ T cells induced by cross-reactive environmental Ags are at least partially responsible for the baseline reactivity with mycobacterial lysates detected in a subpopulation of individuals not infected with M. tuberculosis or previously vaccinated with BCG.

FIGURE 6.
FIGURE 6.

T cell expansion studies in presensitized controls. PBMC harvested from the 10 volunteers recruited into the placebo group who met the criteria to be defined as previously sensitized to mycobacterial Ags were stimulated with M. tuberculosis whole lysate, live BCG, or IPP plus IL-2, and T cell expansions were analyzed as in Figure 3⇑. Shown are the expansion indices for CD4+, CD8+, and γδ T cells after each of the three in vitro stimuli. Points indicate medians; boxes, 25%/75% of cases; whiskers, nonoutlier ranges.

Discussion

Many recent studies of mycobacterial immunity have focused on CD4+ T cell responses induced by secreted mycobacterial proteins (13, 15, 19, 47, 48, 49, 50, 51, 52). Extensive animal data indicate that the Th1 type of CD4+ T cells associated with high levels of IFN-γ secretion after antigenic stimulation is important in mycobacterial immunity (12, 13, 14, 15, 16, 17, 18, 19, 20, 21). Major secreted mycobacterial proteins have been shown to induce protective Th1 cell responses in animal models, and are being developed as candidate subunit vaccines (50, 51, 53, 54). Much less is known about the specific subsets of human immune responses important for protection against mycobacteria. In our present work, we have focused on a more general approach with the major goal of identifying human T cell subsets induced by BCG vaccination, rather than a screen for mycobacterial Ags that could induce Th1 responses. The minimal expansions in total CD4+ and CD8+ T cells measured in our experiments may underestimate the importance of these cell types in mycobacterial specific responses induced by BCG vaccination. Specific measurements of the expansion of activated subpopulations of CD4+ and CD8+ T cells may be more sensitive for the detection of contributions by these T cell subsets. Nevertheless, our results indicate that γδ T cells are also important in the mycobacterial specific secondary immune responses induced by BCG vaccination.

In earlier studies, we have found that BCG vaccination consistently induced significant immunologic reactivity with M. tuberculosis whole cell lysates, but only limited reactivity with M. tuberculosis culture filtrates or the purified Ag85 complex (25, 55, and unpublished observations). Relatively poor induction of immunity directed against secreted mycobacterial components may have contributed to the variable efficacy rates reported in previous BCG vaccine trials (56). However, no other vaccines have yet induced higher levels of protection than BCG in animal models, and BCG can induce at least partial protection against TB in humans (1, 2). Therefore, it is important to fully characterize the immune responses induced by BCG to understand the basis of this partial protection. Because we previously found that in vitro stimulation with M. tuberculosis whole lysate provided a sensitive method for distinguishing BCG-induced immunity, we have concentrated on the identification of T cell subsets reactive with this mycobacterial Ag preparation. We now demonstrate that γδ T cell responses are a major component of the M. tuberculosis whole lysate-specific secondary immune responses induced by BCG vaccination. The parallel observation that in vitro stimulation with live BCG also resulted in a relative predominance of γδ T cell expansion in PBMC from BCG-vaccinated individuals further supports the potential relevance of γδ T cells for in vivo responses to natural mycobacterial infections.

The fact that significant increases in mycobacterial specific γδ T cell expansion were detected after BCG vaccination suggests that memory-like immune responses were induced by BCG in γδ T cells. We are unaware of any previous reports of γδ T cell memory. Classically, immune memory involves rapid, potent, and prolonged immune responses after secondary stimulation, and reflects a combination of an increased precursor frequency of specifically reactive lymphocytes and a heightened sensitivity to the Ags concerned (reviewed in Refs. 57–61). However, it is unclear whether memory cells represent a unique subset of lymphocytes or are the result of differential activation. In addition, the requirements for maintenance of memory immune responses are not well understood. The secondary γδ T cell expansions induced in PBMC from BCG recipients were rapid and potent, characteristic of an increased precursor frequency of responding cells and/or an enhanced sensitivity to antigenic stimulation. Future studies will need to examine the duration of enhanced responsiveness, surface phenotype, and TCR sequence diversity of these mycobacterial specific memory-like human γδ T cells induced by BCG vaccination. The identification of increased γδ T cell responsiveness in a subset of PPD-negative, unvaccinated controls with evidence of prior sensitization to mycobacteria (Fig. 6⇑) is consistent with the hypothesis that these persons have long-lasting γδ T cell memory.

Many previous reports have demonstrated that mycobacterial components can stimulate the expansion of human γδ T cells in vitro (24, 33, 34, 35, 62). Mycobacterial extracts have been shown to preferentially stimulate γ9+δ2+ T cells (40, 41, 42). More recently, isoprenyl pyrophosphates have been identified as minimal molecular components with γδ T cell stimulatory capacity (43, 44, 45). Increased numbers and function of γ9+δ2+ T cells have been identified in PPD+ persons asymptomatically infected with M. tuberculosis compared with individuals with active TB (36). Because healthy PPD+ persons are assumed to have developed protective immune responses that control their M. tuberculosis infections, these latter results suggested that increases in γ9+δ2+ T cells may be important for protection. Our results provide the first evidence for vaccine-induced up-regulation of human γδ T cells. Human immunity induced by BCG vaccination can be used as a second model for protective mycobacterial immunity, and our data support the hypothesis that γ9+δ2+ T cells may be important for protective immunity. Furthermore, the reactivity of these BCG-induced γδ T cells with prenyl pyrophosphates suggests that these nonproteinaceous compounds should be further evaluated as possible vaccine components.

Previous investigators have reported that CD4+ T cells provide important helper functions for the expansion of γδ T cells induced by mycobacterial extracts (63, 64). However, a combination of IPP, a stimulus presumed to be specific for γδ T cells (46), plus IL-2 could induce high levels of γδ T cell expansion in PBMC harvested from BCG-vaccinated individuals (Fig. 4⇑), even in the absence of CD4+ T cells (Fig. 5⇑). These results indicate that the production of IL-2 is the key helper function for γδ T cell expansion provided by CD4+ T lymphocytes, consistent with the findings of previous investigators (46, 64). In addition, we have presented evidence that γδ T cells provide helper functions for CD4+ and CD8+ T lymphocytes (Figs. 4⇑ and 5⇑, and Table III⇑), which may be important for in vivo regulation of protective immunity.

In contrast to the demonstrated importance of γδ T cells for the in vitro expansion of CD4+ and CD8+ T cells in our studies, mycobacterial specific IFN-γ responses were similar in the presence or absence of γδ T cells during in vitro stimulation. A large amount of data has accumulated supporting the importance of IFN-γ responses in protective mycobacterial human immunity (22, 23, 24, 25, 26, 27, 28). The absence of an effect of γδ T cell depletion on IFN-γ responses in our model system appears contradictory to the hypothesis that γδ T cells are important in mycobacterial protective immunity. However, it has been shown recently that γδ T cells can produce high levels of IFN-γ responses (65), and even if αβ T cells or NK cells are responsible for the majority of IFN-γ production measured in our experimental system, γδ T cells may make an important contribution to type 1 cytokine responses in vivo. In addition, our results do not rule out the possibility that γδ T cells may be important during the initial primary immune stimulation in vivo for the induction of IFN-γ responses in αβ T cells. It is also possible that the γδ T cells induced by BCG may be important for other immune effector functions, such as T cell cytotoxicity or the induction of macrophage-mediated mycobactericidal activity.

Finally, we have demonstrated that increased mycobacterial specific γδ T cell expansions are detectable in a subset of unvaccinated and presumably uninfected controls with high baseline reactivity with mycobacterial Ags in lymphoproliferation assays. Previous investigators have assumed that high baseline reactivity with mycobacteria was due to previous exposure to cross-reactive environmental Ags, but the nature of the Ags involved in this cross-reactive priming has been unknown. Our results suggest that environmental priming can induce sensitization of mycobacterial reactive γδ T cells. An association between prior sensitization to mycobacterial Ags and reduced efficacy of BCG has been reported (66, 67, 68, 69). Placebo-controlled trials of BCG in populations with high prevalence of previously activated γδ T cells may result in measurements of lowered vaccine efficacy due to the presence of preexisting protective γδ T cells in placebo recipients.

Acknowledgments

We thank Jan Tennant for her excellent work as nurse coordinator of our Bacille Calmette-Guérin vaccine trial, and all of the volunteers who generously contributed their time and effort for this study. We also thank Drs. Robert Belshe, Lynn Dustin, Ranjit Ray, and Shewangizaw Worku for their review of our manuscript and helpful comments.

Footnotes

  • ↵1 This investigation was supported by National Institutes of Health Vaccine Treatment and Evaluation Unit Contract NO1-AI-45250.

  • ↵2 Address correspondence and reprint requests to Dr. Daniel F. Hoft, Division of Infectious Diseases and Immunology, St. Louis University Health Sciences Center, St. Louis, MO 63110. E-mail address: hoftdf{at}slu.edu

  • ↵3 Abbreviations used in this paper: BCG, Bacille Calmette-Guérin; EI, expansion indices; IPP, isopentenyl pyrophosphate; PE, phycoerythrin; PerCP peridinin chlorophyll protein; PPD, purified protein derivative from Mycobacterium tuberculosis; TB, tuberculosis.

  • Received October 29, 1997.
  • Accepted March 12, 1998.

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The Journal of Immunology
Vol. 161, Issue 2
15 Jul 1998
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