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The Journal of Immunology, 2007, 179, 3057 -3064
Copyright © 2007 by The American Association of Immunologists, Inc.
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Central Memory V{gamma}9V{delta}2 T Lymphocytes Primed and Expanded by Bacillus Calmette-Guérin-Infected Dendritic Cells Kill Mycobacterial-Infected Monocytes1

Angelo Martino2, Rita Casetti, Alessandra Sacchi and Fabrizio Poccia3

Unit of Cellular Immunology, "Fabrizio Poccia," National Institute for Infectious Diseases "Lazzaro Spallanzani," Instituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In humans, innate immune recognition of mycobacteria, including Mycobacterium tuberculosis and bacillus Calmette-Guérin (BCG), is a feature of cells as dendritic cells (DC) and {gamma}{delta} T cells. In this study, we show that BCG infection of human monocyte-derived DC induces a rapid activation of V{gamma}9V{delta}2 T cells (the major subset of {gamma}{delta} T cell pool in human peripheral blood). Indeed, in the presence of BCG-infected DC, V{gamma}9V{delta}2 T cells increase both their expression of CD69 and CD25 and the production of TNF-{alpha} and IFN-{gamma}, in contrast to DC treated with V{gamma}9V{delta}2 T cell-specific Ags. Without further exogenous stimuli, BCG-infected DC expand a functionally cytotoxic central memory V{gamma}9V{delta}2 T cell population. This subset does not display lymph node homing receptors, but express a high amount of perforin. They are highly efficient in the killing of mycobacterial-infected primary monocytes or human monocytic THP-1 cells preserving the viability of cocultured, infected DC. This study provides further evidences about the complex relationship between important players of innate immunity and suggests an immunoregulatory role of V{gamma}9V{delta}2 T cells in the control of mycobacterial infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human T cells expressing {gamma}{delta} TCR represent an unique lymphocyte population with unusual tissue distribution and Ag recognition pathway (1). Although generally portrayed as a minor subset, peripheral blood {gamma}{delta} T cells rapidly proliferate following infection with certain pathogens, expanding from 1 to >50% of circulating T cells within a week (2, 3). Conditions leading to responses of {gamma}{delta} T cells are not fully understood, and current concepts of {gamma}{delta} T cells as the "first line of defense," "regulatory T cells," or the "bridge between innate and adaptive immunity" only address facets of their complex behavior. Thus, {gamma}{delta} T cells form an entire lymphocyte system, working through cross-talks with other leukocytes (4, 5).

Found only in primates, V{gamma}9V{delta}2 T cells, the major subset of the circulating {gamma}{delta} T cell pool, are involved in immunity against microbial pathogens and hematological malignancies (6, 7). This subset is activated by two kinds of Ags: 1) nonpeptidic small phosphorylated compounds produced by mammalian cells, such as isopentenyl-pyrosphosphate (IPP),4 or by nonmammalian cells, such as 4-hydroxy-3-dimethylallyl pyrophosphate (8, 9, 10); and 2) a group of nonpeptidic compounds, the aminobisphosphonates, as Zoledronic acid (Zol). These last have been demonstrated to activate V{gamma}9V{delta}2 T cells through the accumulation of mevalonate pathway metabolites, as IPP, subsequently recognized by TCR (11).

Similar to CD4 and CD8 {alpha}β T cells, V{gamma}9V{delta}2 T cells are heterogeneous and comprise distinct populations defined by surface marker expression and effector functions. Naive (CD45RA+CD27+) and central memory (CD45RA–CD27+) V{gamma}9V{delta}2 T cells abound in lymph nodes and lack immediate effector functions. Conversely, effector memory (CD45RA–CD27–) and terminally differentiated (CD45RA+CD27–) V{gamma}9V{delta}2 T cells are poorly represented in the lymph nodes although abounding at the sites of inflammation, and display immediate effector functions (12). The differentiation pathway for the generation of these subsets is uncertain, but circulating effector {gamma}{delta} T cells have been found significantly reduced in several diseases as pulmonary tuberculosis (13). Furthermore, cellular requirements for activation, proliferation, and differentiation of V{gamma}9V{delta}2 T cells remain unclear. There is now ample evidence that {gamma}{delta} T cells and other innate cells exert regulatory influences upon each other (14, 15, 16). Our previous data evidenced the ability of dendritic cells (DC) to potentiate V{gamma}9V{delta}2 T cell activation and cytokine production with a reciprocal effect on their own maturation (17). Although such reports showed the existence of a striking link between both of these cell subsets in vivo (18, 19), the exact role of DC in the activation of {gamma}{delta} T cells, particularly during infections, has not been clarified.

During mycobacterial infections, responses of {gamma}{delta} T cells were described as early as 1989 (20). Dramatic expansion of {gamma}{delta} T cells has been found during bacillus Calmette-Guérin (BCG) vaccination in newborn and adult subjects, and several phosphorylated Ags from mycobacteria have been defined (21, 22). Purified preparations of these molecules can induce an expansion of V{gamma}9V{delta}2 T cells comparable to that induced by BCG stimulation in vitro (23, 24). Nevertheless, triggering of an effective specific antimycobacterial immunity is one of the functions of DC, being an early target of mycobacteria, and central players for the success of TB immunization. Thus, functional and phenotypical features of V{gamma}9V{delta}2 T cells after the engagement of DC infected by BCG or pulsed with phosphoantigens needed further investigations. In this study, we show that human DC harboring live BCG lead to the generation of a population of V{gamma}9V{delta}2 T cells displaying a central memory phenotype (CD45RA–CD27+) but high perforin contents. This subset shows specific cytotoxicity against BCG-infected monocytes, but not against BCG-infected DC. These data show for the first time an unsuspected dissociation of phenotypic immaturity with the functional maturity of human {gamma}{delta} T cells together with their role in discriminating target cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mycobacteria and reagents

In all experiments, BCG from Aventis Pasteur has been used. Lyophilized BCG was resuspended in physiologic solution at ~1 x 106 CFU per 100 µl. BCG viability was verified by CFU assay. In brief, bacteria were plated in Middlebrook 7H10 agar (BD Biosciences) and the BCG colonies were enumerated after incubation at 37°C in humidified air for 15–21 days (25). In some experiment IPP (Sigma-Aldrich) and Zol (Novartis Pharmaceuticals) were used.

Monocyte purification, DC generation, and mycobacterial infection

PBMCs were isolated from buffy coats of healthy donors by density gradient centrifugation using Lympholyte-H (Cederlane Laboratories). Monocytes were positively separated by anti-CD14 magnetic beads (MACS; Miltenyi Biotec) according to the manufacturer’s instructions. The cells were then resuspended in RPMI 1640 (Euroclone) supplemented with 10% FCS (HyClone, Invitrogen Life Technologies), L-glutamine (2 mM), HEPES buffer (10 mM), and gentamicin (10 µg/ml) (Sigma-Aldrich), and cultured for 5 days in the presence of GM-CSF (200 U/ml) and IL-4 (10 ng/ml; Euroclone) to generate immature DC (imDC). Then, imDC were infected for 3 h with single cell suspensions of BCG at a multiplicity of infection of 1 or treated with IPP (20 µg/ml; data not shown) or Zol (50 µM). The infection was conducted in the absence of antibiotics and, after the treatment, cells were washed with PBS to eliminate extracellular bacteria or phosphometabolites. In some experiments, DC were preincubated with mevastatin (2.5 µM; Sigma-Aldrich) before the infection with BCG, or with anti-human MHC class I-related chain A (MICA)-neutralizing mAb (10 µg/ml; R&D Systems) before the coculture with {gamma}{delta} T cells. Viability of infected cells was determined by trypan blue exclusion.

{gamma}{delta} T cell purification and DC coculture

{gamma}{delta} T cells were separated from autologous PBMCs by positive selection using anti-{gamma}{delta}-magnetic beads (MACS; Miltenyi Biotec) according to the manufacturer’s instructions. Purified cell populations contained >98% of viable {gamma}{delta} T cells as assessed by flow cytometry. After an overnight culture in complete medium, purified {gamma}{delta} T cells were added to imDC, BCG-infected DC, or DC treated with IPP or Zol at a 1:1 ratio. In some experiments, BCG was heat killed by incubating it at 80°C for 60 min. The viability of bacteria was assessed by CFU analysis. In other experiments, the {gamma}{delta} T lymphocytes were physically separated from DC by a semipermeable membrane (6.5-mm of diameter, 0.4-µm pore size in 24-well plates; Costar). In some experiments, {gamma}{delta} T cells were preincubated with anti-human NKG2D (10 µg/ml; R&D Systems) neutralizing Ab 1 h before the coculture with DC. To evaluate the role of IL-15 in V{gamma}9V{delta}2 T cell differentiation, IL-15 (Euroclone) was added to DC(BCG)/{gamma}{delta} coculture at three concentrations (5, 10, and 15 nM).

FACS analysis

The following FITC-, PE-, PerCP-, or APC- conjugated Abs: CD25, CD69, CD27, CD45RA, CD62L, CCR7, NKG2D, and V{delta}2 (BD Biosciences) were used for direct immunofluorescence staining to characterize the phenotype of {gamma}{delta} T cells. Isotype-matched mAbs (BD Biosciences) were used in all experiments as controls. In brief, the cells were washed twice in PBS, 1% BSA, and 0.1% sodium azide, and were stained with the mAbs for 15 min at 4°C. The cells were then washed and analyzed using a FACSCalibur instrument with CellQuest software (BD Biosciences). The following PE- and allophycocyanin-conjugated anti-TNF-{alpha} and IFN-{gamma} mAbs (BD Pharmingen) were used for intracellular immunostaining to characterize V{delta}2 T cells producing cytokines. Moreover, perforin and granzyme PE were assessed by intracytoplasmic staining using mAbs (BD Biosciences).

Proliferation assay

In some experiments, {gamma}{delta} T cells were labeled with CFSE (2.5 {propto}M; BD Pharmingen). In brief, purified {gamma}{delta} T cells were incubated with CFSE at room temperature for 5 min. The cells were washed three times using PBS supplemented with 5% FCS and resuspended in complete medium. Labeled {gamma}{delta} T cells were cultured with imDC, BCG-infected DC, or DC treated with IPP or Zol. After 6 days of culture, {gamma}{delta} T cell proliferation was assessed by flow cytometry.

Cytolytic assay

Human monocytic THP-1 cells and autologous monocytes were labeled with CFSE (2.5 {propto}M), according to the manufacture’s instructions. After CFSE labeling, THP-1 and monocytes were infected with BCG. Cytotoxic function of V{gamma}9V{delta}2 T cells derived from the coculture with imDC or BCG infected DC was evaluated by adding CFSE-labeled THP-1 or autologous monocytes infected or not with BCG (ratio of 10:1 and 1:1, respectively). After 8 h of coculture, CFSE signaling was assessed by flow cytometry. Cell-mediated cytotoxicity was assessed also by measuring lactate dehydrogenase (LDH) release in the supernatants of the cultures by using Cytotox 96 nonradioactive cytotoxicity assay (Promega) according to the manufacture’s instructions and expressed as a percentage of cytotoxicity.

Statistical analysis

Statistical analysis was determined using a Mann-Whitney U test. Values of p < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Human DC infected with live BCG-activated V{gamma}9V{delta}2 T cells inducing a rapid production of TNF-{alpha} and IFN-{gamma}

To ascertain the influence of DC upon resting autologous V{gamma}9V{delta}2 T cells during BCG infection or specific Ag pulsing, we compared the ability of BCG-infected or Zol-pulsed DC to induce phenotypic and functional activation of V{gamma}9V{delta}2 T cells. DC were generated from purified monocytes of healthy donors by culturing with GM-CSF and IL-4 for 5 days. ImDC were infected with BCG at a multiplicity of infection of 1 or with pulsed Zol (50 µM) for 3 h. Then, DC were extensively washed and cocultured with autologous purified {gamma}{delta} T cells for 24 h. The percentage of CD25+ and CD69+ V{gamma}9V{delta}2-activated T cells was analyzed by flow cytometry. In the presence of BCG-infected DC, V{gamma}9V{delta}2 T cells comprised >30% of CD25+ and CD69+ cells without any other exogenous stimulation, whereas any effect was observed in the presence of untreated DC. As expected, Zol-pulsed DC led to the 20% CD25+ and 22% CD69+ of V{gamma}9V{delta}2 T cells (Fig. 1A). As previously observed, DC pulsed with IPP are not efficient in the activation of V{gamma}9V{delta}2 T cells (data not shown) because their immunogenicity is immediately lost upon washing.


Figure 1
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  		FIGURE 1. BCG-infected DC induce V{gamma}9V{delta}2 T cell activation and cytokine production. A, CD25 and CD69 activation marker expression was measured on purified V{gamma}9V{delta}2 T cells after 24 h of culture with untreated DC, BCG-infected DC, or Zol-pulsed DC. B, In some experiments, DC were infected with heat-killed BCG or infected with BCG and separated from {gamma}{delta} T cells by a semipermeable membrane. The surface marker expression was analyzed by flow cytometry. Some representative cytofluorimetric panels are shown. Numbers indicate the percentage of CD25+ and CD69+ V{gamma}9V{delta}2 T cells. C, TNF-{alpha} and IFN-{gamma} production by purified V{gamma}9V{delta}2 T cells was assessed by intracellular staining after 16 h of culture with DC, BCG-infected DC, or Zol-pulsed DC and analyzed by flow cytometry. Bars, Percentage of cytokine-producing V{gamma}9V{delta}2 T cells. Results are expressed as means (±SD) of 10 different experiments and statistical analysis was determined by Mann-Whitney U test. *, p < 0.05 was significant.

 
DC infected with heat-killed BCG did not induce the activation of V{gamma}9V{delta}2 T cells, indicating that an active infection is required (Fig. 1B). As known, BCG infection induces a phenotypic maturation of DC comparable to that induced by classical maturation stimuli as LPS. Because DC maturated by LPS did not have effects on V{gamma}9V{delta}2 T cells (data not shown), it is possible to hypothesize that the maturation status of DC is not sufficient for the {gamma}{delta} T cell activation without Ag. The optimal activation achieved by V{gamma}9V{delta}2 following BCG-infected DC coculture was dependent on cell-to-cell contact, as their separation by a permeable membrane (transwell) partially abrogated their activation (Fig. 1B).

Finally, to investigate whether the phenotype activation of V{gamma}9V{delta}2 T cells induced by live BCG-infected DC was associated to their functionality, we analyzed the TNF-{alpha} and IFN-{gamma} production by V{gamma}9V{delta}2 T cells. The percentage of TNF-{alpha}+ and IFN-{gamma}+ V{gamma}9V{delta}2 T cells was evaluated by flow cytometry after overnight DC coculture. In contrast to untreated DC coculture, DC infected with BCG induced a strong frequency of TNF-{alpha}+ and IFN-{gamma}+ V{gamma}9V{delta}2 T cells, which was abrogated when DC were physically separated from {gamma}{delta} T cells (Fig. 1C). As expected, Zol-pulsed DC induced a significantly lower percentage of cytokine-producing V{gamma}9V{delta}2 T cells. Besides confirming the activation of {gamma}{delta} T cells by BCG-infected DC, these data suggest that live BCG-infected DC express more relevant Ags or undefined costimulatory molecules with respect to bisphosphonate-pulsed DC for the activation of V{gamma}9V{delta}2 T cells, with an important role of cell contact.

As previously observed, membrane-bound molecules have been implicated in DC-induced {gamma}{delta} T cell activation (16). However, during BCG infection in vitro, the inhibition of DC costimulatory molecules, such as CD40, CD80, CD86, or Fas, through neutralizing Abs does not affect V{gamma}9V{delta}2 T cells activation (data not shown).

V{gamma}9V{delta}2 T cells cocultured with BCG-infected DC proliferate in the absence of IL-2

As previously observed, {gamma}{delta} T cells are poor IL-2 producers, but upon Ag stimulation, they can proliferate in the presence of Th cells or exogenous IL-2 in vitro and in vivo (26). We investigated whether BCG-infected DC are able to sustain V{gamma}9V{delta}2 T cell proliferation without exogenous IL-2 (Fig. 2). We cocultured BCG-infected DC with purified {gamma}{delta} T cells, previously labeled with CFSE, for 6 days and analyzed the results by flow cytometry. The ability of BCG-infected, untreated, or Zol-pulsed DC to induce {gamma}{delta} T cell proliferation was compared. In addition, we performed a standard {gamma}{delta} T cell proliferation assay using IPP/IL-2 as positive control. We observed that both BCG-infected and Zol-pulsed DC induced {gamma}{delta} T cell proliferation, even if BCG infection induced more cell divisions. Proliferation was not observed in {gamma}{delta} T cells cocultured with untreated DC without exogenous IL-2. These data suggest that BCG-infected DC-induced proliferation does not require any further stimulation.


Figure 2
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  		FIGURE 2. BCG-infected DC sustain V{gamma}9V{delta}2 T cell proliferation. Purified {gamma}{delta} T cells were labeled with CFSE before the culture with DC, BCG-infected DC, or Zol-pulsed DC. After 6 days of culture, {gamma}{delta} T cell proliferation was analyzed by flow cytometry. Results of a representative experiment are expressed as cell number counts.

 
V{gamma}9V{delta}2 T cells expanded by BCG-infected DC show central memory phenotype, but high perforin content

To assess the differentiation pattern of V{gamma}9V{delta}2 T cells expanded by BCG-infected DC, staining for CD45RA and CD27 was performed on {gamma}{delta} T cells after 6 days of coculture with BCG-infected DC. As expected, the majority of V{gamma}9V{delta}2 T cells purified from buffy coats showed a central memory (CM) phenotype (CD45RA–CD27+). Surprisingly, V{gamma}9V{delta}2 T cells expanded by BCG-infected DC did not change their differentiation pattern of central memory cells, remaining CD45RA–CD27+ cells (Fig. 3A). However, in contrast to other experimental conditions, the CM V{gamma}9V{delta}2 T cell population derived from BCG-infected DC may be distinguished in two subsets on the basis of perforin expression: perforinlow or perforin high+ V{gamma}9V{delta}2 T cells (Fig. 3B). Finally, the expression of granzyme in all conditions was also compared, observing a slight but significant increase in V{gamma}9V{delta}2 T cells derived from BCG-infected DC coculture (Fig. 3C).


Figure 3
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  		FIGURE 3. BCG-infected DC expand central memory perforinhigh+ V{gamma}9V{delta}2 T cells. A, To assess the differentiation state of V{gamma}9V{delta}2 T cells, CD45RA and CD27 molecule expression was analyzed on a V{gamma}9V{delta}2 T cell surface by flow cytometry. A representative experiment of 15 is shown. B and C, Perforin (B) and granzyme A (C) were measured by intracellular staining on V{gamma}9V{delta}2 T cells after 6 days of culture with different experimental conditions. Numbers indicate the percentage of positive cells.

 
Phenotypic characterization of V{gamma}9V{delta}2 T cells CM perforinhigh

As previously observed, other markers define the central memory population of lymphocytes (27). Then, the total population was stained for CD28, CCR7, and CD62L expression as well as NK receptors. First, we stained perforinhigh/low V{gamma}9V{delta}2 T cells for CD62L and CCR7 lymph node homing receptors and we observed that the perforinhigh V{gamma}9V{delta}2 T cell subset did not express CCR7 or CD62L receptors in contrast to perforinlow V{gamma}9V{delta}2 T cells (Fig. 4A), indicating that perforinhigh V{gamma}9V{delta}2 T cells are not able to reach lymphoid organs. In contrast, the total population of V{gamma}9V{delta}2 T cells was homogeneous for CD28 expression (Fig. 4B), indicating that the majority of V{gamma}9V{delta}2 T cells derived from BCG-infected DC displays a central memory phenotype being CD45RA–CD27+CD28+.


Figure 4
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  		FIGURE 4. BCG-infected DC expand NKG2D– V{gamma}9V{delta}2 T cells whereas only perforinhigh+ V{gamma}9V{delta}2 T cells did not express lymphoid homing receptors. Purified {gamma}{delta} T cells were cultured in the presence of DC, BCG-infected DC, or Zol-pulsed DC. Then, after 6 days, V{gamma}9V{delta}2 T cells were stained for CCR7 and CD62L (A), CD28 (B), and NKG2D (C) analyzed by flow cytometry.

 
Activation of V{gamma}9V{delta}2 T cells is modulated by a range of NK cell receptors, as NKG2D receptors providing a costimulatory signal in the recognition of infected or tumor cells. We analyzed the expression of NKG2D receptors on {gamma}{delta} T cells, observing that V{gamma}9V{delta}2 T cells expanded by BCG-infected DC did not express NKG2D in contrast with other experimental conditions (Fig. 4C). No difference in the expression of other NKG receptors was observed (data not shown). The absence of the NKG2D receptor on the surface of V{gamma}9V{delta}2 T cells suggests a different capacity to recognize and respond to target cells.

Block of the differentiation of CM V{gamma}9V{delta}2 T cells by BCG-infected DC is partially dependent on the lack of IL-15

Given the particular phenotype NKG2D– of V{gamma}9V{delta}2 T cells derived from BCG-infected DC coculture, we assessed the role of NKG2D-MICA interaction in this phenomenon. By preculturing DC and {gamma}{delta} T cells with neutralizing Abs against MICA and NKG2D, respectively, we observed that these molecules do not affect the activation and phenotypic differentiation of the resulting V{gamma}9V{delta}2 T cells after 6 days of culture in terms of perforin content and CM phenotype (Fig. 5, A and B). Furthermore, to investigate whether BCG was able to alter the mevalonate pathway of DC inducing the production of cellular phosphoantigens, we preincubated DC with mevastatin before the infection and {gamma}{delta} coculture. We observed that there was no influence in the activation of {gamma}{delta} T cells (Fig. 5, A and B).


Figure 5
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  		FIGURE 5. Block of the differentiation of perforin high V{gamma}9V{delta}2 T cells is partially dependent on IL-15. V{gamma}9V{delta}2 T cells and DC were preincubated with anti-NKG2D and MICA or mevastatin (Mev), respectively, before coculture. A, The expression of CD25 and CD69 was evaluated after 24 h of coculture. B, The presence of perforin was evaluated after 6 days of coculture. Data were analyzed by flow cytometry. A representative experiment is shown. In some experiments, IL-15 was added to the DC(BCG)/{gamma}{delta} coculture for 6 days, and the expression of CD45RA and CD27 was analyzed by flow cytometry (C). One representative experiment of six is shown.

 
The block of differentiation of V{gamma}9V{delta}2 T cells could be explained by the role played by IL-15 in the differentiation pathway of CM V{gamma}9V{delta}2 T cells into effector cells. Because human DC are not able to produce IL-15 during BCG infection in vitro, the lack of this cytokine could be associated to the immature phenotypical differentiation in our model. To asses the role of IL-15, we performed the same experiments, adding three concentrations of IL-15 (5, 10, and 15 nM) in the BCG-infected DC/{gamma}{delta} coculture for 6 days. We observed that, in the presence of 15 nM of IL-15, >40% of V{gamma}9V{delta}2 T cells differentiate into effector cells being CD45RA–CD27– cells (Fig. 5C). This indicates that the lack of IL-15 production is partially involved in the incomplete phenotypical differentiation of effector V{gamma}9V{delta}2 T cells.

CM perforinhigh V{gamma}9V{delta}2 T cells display cytotoxic activity against BCG-infected monocytes and THP-1 target cells, but not against cocultured BCG-infected DC

To investigate the functions of V{gamma}9V{delta}2 T cells expanded from BCG-infected DC, we first examined their killing activity against cocultured DC using CFSE-labeled DC. As previously published (20), the loss of CFSE events is related to the cell-mediated lysis. After 6 days of culture, we did not observe a loss of signal for labeled DC (Fig. 6A). This is also confirmed by the physical parameters and release of LDH in the supernatants (Fig. 6B), indicating that cocultured, infected DC are not affected in their viability. Furthermore, we cultured CFSE-labeled THP-1 cells as a target in DC/{gamma}{delta} cocultures. We used V{gamma}9V{delta}2 T cells from uninfected or BCG-infected DC cocultures as effector cells in a cytotoxic assay against uninfected or BCG-infected THP-1 cells. As shown in Fig. 7A, V{gamma}9V{delta}2 T cells expanded from BCG infected DC strongly killed both uninfected and BCG infected THP-1 cells. As control, V{gamma}9V{delta}2 T cells derived from uninfected DC cocultures lacked of any cytotoxic activity.


Figure 6
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  		FIGURE 6. BCG-infected DC were not killed by cocultured V{gamma}9V{delta}2 T cells. To assess the cytolytic activity of {gamma}{delta} T cells against autologous BCG-infected DC, DC were labeled with CFSE after BCG infection and cultured with autologous {gamma}{delta} T cells. After 1 and 6 days of culture, the CFSE signal was analyzed by flow cytometry (A). Supernatants from DC-, DC/{gamma}{delta}- and BCG-infected DC/{gamma}{delta}-cultures were analyzed for the presence of LDH by a nonradiometric LDH assay kit (B).

 

Figure 7
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  		FIGURE 7. {gamma}{delta} T cells derived from BCG-infected DC culture kill uninfected or BCG-infected THP-1 cells and autologous BCG-infected monocytes. Uninfected or BCG-infected THP-1 (A) and autologous uninfected or BCG-infected monocytes (B) were labeled with CFSE and cultured in the presence of {gamma}{delta} T cells derived from DC or BCG-infected DC cocultures. After 12 h, CFSE-labeled THP-1 and monocytes were analyzed by flow cytometry. C, Cytotoxicity was measured by a nonradioactive assay kit evaluating LDH release in the supernatants of DC/{gamma}{delta}- and DC(BCG)/{gamma}{delta}-cultures in the presence of BCG-infected monocytes as a target. Results are expressed as percentage of cytotoxicity. D, After killing, V{gamma}9V{delta}2 T cell population was analyzed for the expression of CD45RA and CD27 and the presence of perforin. Data were analyzed by flow cytometry. A representative experiment is shown.

 
Finally, using primary healthy or BCG-infected autologous monocytes as a target, we found that only V{gamma}9V{delta}2 T cells expanded by BCG-infected DC show cytotoxic activity against infected, but not uninfected, monocytes (Fig. 7B). These data were confirmed by LDH assay, confirming that {gamma}{delta} T cells derived from BCG-infected DC were able to kill >90% of infected monocytes. (Fig. 7C).

V{gamma}9V{delta}2 T cells CM perforinhigh are not recovered after the killing

To analyze the possibility that the engagement of target cells induces the differentiation of CM cytotoxic V{gamma}9V{delta}2 T cells into cytotoxic effector cells, we analyzed by flow cytometry the expression of CD45RA and CD27 after the killing assay. No differences were observed in the phenotype of V{gamma}9V{delta}2 T cells after killing for CD45RA and CD27 expression (Fig. 7D). However, we observed that the population of perforinhigh was not recovered (Fig. 7D). These data suggest that the infection of DC with BCG expand phenotypically immature, but functionally competent, cytotoxic V{gamma}9V{delta}2 T cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Although it is becoming increasingly clear that the V{gamma}9V{delta}2 T cell subset is important for both innate and adaptive immune response, the cellular requirement for the activation of innate-like T cells, including V{gamma}9V{delta}2 T cells, still remains undefined. A mutually costimulatory relationship was found between {gamma}{delta} T cells and DC, but little is known about the role of DC in the functionality of {gamma}{delta} T cells, especially during infections (14). In this study, we show that BCG infection of human monocyte-derived DC leads to a particularly rapid and strong activation of cocultured V{gamma}9V{delta}2 T cells. This results in a rapid up-regulation of activation markers and production of proinflammatory cytokines. Furthermore, the long-term coculture of infected DC and {gamma}{delta} T cells expands functional competent cytotoxic, but phenotypically immature V{gamma}9V{delta}2 T cell population. This particular population does not display lymph node homing receptors and shows cytotoxic activity against infected target cells.

Because both BCG and phosphoantigens, such as IPP, induce similar expansion of V{gamma}9V{delta}2 T cells, it has been suggested that these Ags may be useful as components of the new tuberculosis vaccine. However, it has been shown that IPP-expanded V{gamma}9V{delta}2 T cells do not arrest the growth of intracellular mycobacteria, whereas those expanded with BCG inhibit intracellular mycobacterial growth (24). Little is known about the role of DC in the differentiation of V{gamma}9V{delta}2 T cells following the phosphoantigen or BCG stimulation. Our data show that BCG-infected DC rapidly activate V{gamma}9V{delta}2 T cells in terms of CD25 and CD69 up-regulation and TNF-{alpha}/IFN-{gamma} production vs untreated DC coculture. Furthermore, DC pulsed with phosphometabolites, such as IPP, did not induce the same level of activation of BCG-infected DC. Conversely, as expected, Zol-pulsed DC induce the activation of V{gamma}9V{delta}2 T cells, but at a lower level than BCG-infected DC. The reactivity of V{gamma}9V{delta}2 T cells to phosphoantigen stimulation alone was not comparable to that induced by DC, suggesting that their presence plays a complementary role in the phenotypical and functional activation. As previously observed, membrane-bound as well as soluble factors have been implicated in DC-induced {gamma}{delta} T cell activation (16). In our model, blocking of several costimulatory molecules, as CD40, CD80, CD86, or Fas, on BCG-infected DC before coculturing with {gamma}{delta} T cells, did not change our observations; nevertheless, the involvement of proteic elements was confirmed by the treatment of BCG-infected DC with trypsin, which abrogated any V{gamma}9V{delta}2 T cell activation (data not shown).

Importantly, {gamma}{delta} T cell activation induced by BCG-infected DC was associated to the expansion of phenotypically immature but cytotoxic {gamma}{delta} T cells. We found that the highest proliferation of {gamma}{delta} T cells is sustained by BCG-infected DC without exogenous cytokine stimulation. However, V{gamma}9V{delta}2 T cells expanded by BCG-infected DC showed a fully competent cytotoxicity, but a phenotypical immaturity being CM but perforinhigh+ cells. Nevertheless, they preserved the viability of cocultured BCG-infected DC in contrast to that of freshly infected monocytes. This event could be partially explained by the absence of the NKG2D receptor on V{gamma}9V{delta}2 T cells. Indeed, NKG2D has been implicated in the killing of mycobacterial-infected cells through its interaction with MICA on their surface (28). Thus, we can hypothesize that the lack of NKG2D on V{gamma}9V{delta}2 T cells may explain the failure in the killing of cocultured BCG-infected DC. As previously observed for NK cells (29), V{gamma}9V{delta}2 T cells could play a role in the homeostasis of the immune response during bacterial infection, preserving the Ag presentation by APCs and controlling the spreading of the infection by killing recruited, infected monocytes. Furthermore, the capacity of this subset to kill uninfected tumor cells, as THP-1 cells, could represent another mechanism in the still-unclear antitumor effect of BCG.

The fact that DC potentiate cytokine but not cytolytic responses of Ag-stimulated {gamma}{delta} T cells has been recently reported (30). However, BCG infection and the complex machinery of DC could represent a source of different stimulations explaining the diversity of V{gamma}9V{delta}2 T cells derived. Possible explanation for the incomplete phenotypic differentiation could be represented by the role played by IL-15 in the differentiation pathway of CM V{gamma}9V{delta}2 T cells into cytotoxic cells (TEMRA) (13). Because human DC are not able to produce IL-15 during BCG infection in vitro (31), the lack of this cytokine could be associated to the immature phenotypical differentiation in our model. Indeed, adding IL-15 in DC/{gamma}{delta} coculture, effector CD45RA–CD27– V{gamma}9V{delta}2 T cells were partially generated. Consistent with these data, after the encounter with target cells, V{gamma}9V{delta}2 T cells derived from BCG-infected DC did not show any change in their phenotypic differentiation, indicating that the functionality of V{gamma}9V{delta}2 T cells is not related to the phenotype.

We therefore conclude that BCG-infected DC stimulate and expand a specific V{gamma}9V{delta}2 T cell population that can functionally recognize mycobacterial-infected target cells. This study provides further evidences of the complex relationship between important players of innate immunity, and suggests the possible mechanisms through which V{gamma}9V{delta}2 T cells and DC could cooperate during human mycobacterial infection and BCG vaccination.


   Acknowledgments
 
We thank Dr. Giovanni Auricchio for revising the entire manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants from the Italian Ministry of Health, Ricerca Corrente and Finalizzata (04.126) and by the TB-VAC FP6 European Project. Back

2 Address correspondence and reprint requests to Dr. Angelo Martino, Unit of Cellular Immunology, National Institute for Infectious Diseases, "Lazzaro Spallanzani," Instituto di Ricovero e Cura a Carattere Scientifico, Via Portuense 292, Rome, Italy. E-mail address: martino{at}inmi.it Back

3 This article is dedicated to Fabrizio Poccia, who died June 12, 2007. The Unit of Cellular Immunology has been renamed in his memory. Back

4 Abbreviations used in this paper: IPP, isopentenyl-pyrosphosphate; Zol, Zoledronic acid; DC, dendritic cell; BCG, bacillus Calmette-Guérin; imDC, immature DC; LDH, lactate dehydrogenase; CM, central memory. Back

Received for publication March 5, 2007. Accepted for publication June 12, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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