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International Immunology Advance Access originally published online on April 19, 2007
International Immunology 2007 19(5):657-673; doi:10.1093/intimm/dxm031
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Preferential recognition of a microbial metabolite by human V{gamma}2V{delta}2 T cells

Kia-Joo Puan1, Chenggang Jin1, Hong Wang1, Ghanashyam Sarikonda1, Amy M. Raker1, Hoi K. Lee1, Megan I. Samuelson1, Elisabeth Märker-Hermann2, Ljiljana Pasa-Tolic3, Edward Nieves4, José-Luis Giner5, Tomohisa Kuzuyama6 and Craig T. Morita1

1 Division of Rheumatology, Department of Internal Medicine and the Interdisciplinary Graduate Program in Immunology, University of Iowa College of Medicine, EMRB 400F, Iowa City, IA 52242, USA
2 Division of Rheumatology, Immunology and Nephrology, Department of Internal Medicine IV, Dr. Horst Schmidt Kliniken GmbH, 65191 Wiesbaden, Germany
3 Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA
4 Laboratory for Molecular Analysis and Proteomics, Albert Einstein College of Medicine, Bronx, NY 10461, USA
5 Department of Chemistry, State University of New York-ESF, Syracuse, NY 13210, USA
6 Laboratory of Cell Biotechnology, Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Correspondence to: C. T. Morita; E-mail: craig-morita{at}uiowa.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Human V{gamma}2V{delta}2 T cells are stimulated by prenyl pyrophosphates, such as isopentenyl pyrophosphate (IPP), and play important roles in mediating immunity against microbial pathogens and have potent anti-tumor activity. (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) has been identified as a metabolite in the 2-C-methyl-D-erythritol-4 phosphate (MEP) pathway for isoprenoid biosynthesis that is used by many bacteria and protozoan parasites. We find that HMBPP is the major V{gamma}2V{delta}2 T-cell antigen for many bacteria, including Mycobacterium tuberculosis, Yersinia enterocolitica and Escherichia coli. HMBPP was a 30 000-fold more potent antigen than IPP. Using mutant bacteria, we show that bacterial antigen levels for V{gamma}2V{delta}2 T cells are controlled by MEP pathway enzymes and find no evidence for the production of 3-formyl-1-butyl pyrophosphate. Moreover, HMBPP reactivity required only germ line-encoded V{gamma}2V{delta}2 TCR elements and is present at birth. Importantly, we show that bacterial HMBPP levels correlated with their ability to expand V{gamma}2V{delta}2 T cells in vivo upon engraftment into severe combined immunodeficiency–beige mice. Thus, the production of HMBPP by a microbial-specific isoprenoid pathway plays a major role in determining whether bacteria will stimulate V{gamma}2V{delta}2 T cells in vivo. This preferential stimulation by a common microbial isoprenoid metabolite allows V{gamma}2V{delta}2 T cells to respond to a broad array of pathogens using this pathway.

Keywords: 2-C-methyl-D-erythritol-4 phosphate pathway, microbial immunity, prenyl pyrophosphate antigens, {gamma}{delta} T cells


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
V{gamma}2V{delta}2 T cells are a unique subset of human T lymphocytes comprising 1–4% of total adult peripheral blood T cells (1, 2). They expand during a variety of prokaryotic and eukaryotic protozoan infections such as tuberculosis (3–5), leprosy (6), typhoid fever (7), brucellosis (8), tularemia (9–11), ehrlichiosis (12), malaria (13, 14) and toxoplasmosis (15). Studies in a human peripheral blood lymphocyte-SCID mouse model (hu-PBL-SCID) demonstrated that V{gamma}2V{delta}2 T cells help provide immunity against Escherichia coli, Morganella morganii and Staphylococcus aureus infections (16). Moreover, using rhesus monkeys, we showed that V{gamma}2V{delta}2 T cells expand during resolution of Mycobacterium tuberculosis and Mycobacterium bovis Bacille Calmette-Guérin (BCG) infections, suggesting that {gamma}{delta} T cells also play a role in immunity against mycobacteria (17).

The first natural antigen structurally identified for V{gamma}2V{delta}2 T cells was isopentenyl pyrophosphate (IPP), a metabolite all organisms use to synthesize isoprenoid compounds. Despite the presence of endogenous IPP in humans, there is no evidence that V{gamma}2V{delta}2 T cells mediate autoimmunity, suggesting that they can distinguish between pathogen and host prenyl pyrophosphates under normal conditions (18). Two distinct pathways for IPP synthesis have been delineated that appear to contribute to this specificity (19, 20). The mevalonate pathway is found in most eukaryotes, archaebacteria, some eubacteria and the cytosol of plants. The second pathway, the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (also termed the deoxyxylulose phosphate pathway), is found in most eubacteria, apicomplexan protozoa, cyanobacteria and plant chloroplasts. In the MEP pathway, seven enzymes have been identified: Dxs, Dxr, YgbP, YchB, YgbB, GcpE and LytB (Fig. 1). A MEP pathway metabolite, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) (21, 22) (also termed hydroxy-dimethylallyl pyrophosphate (HDMAPP), has been shown to have potent stimulatory activity for V{gamma}2V{delta}2 T cells (23). Also, the in vitro stimulatory activity of E. coli could be diminished by deletion of the Dxs and GcpE enzymes in the MEP pathway (24) and increased by deletion of the LytB enzyme which is downstream from HMBPP (25). Mycoplasma species that retain MEP pathway enzymes are also able to expand V{gamma}2V{delta}2 T cells in vitro (26). Finally, Listeria monocytogenes, that uses both pathways to make isoprenoid intermediates, loses in vitro bioactivity for V{gamma}2V{delta}2 T cells when GcpE is deleted whereas deletion of mevalonate kinase or HMG-CoA reductase did not affect bioactivity (27). Deletion of LytB in Listeria monocytogenes increases bioactivity 7-fold, a much smaller increase than is noted in E. coli (27). These findings suggest that HMBPP may act as an antigen that allows V{gamma}2V{delta}2 T cells to distinguish exogenous from endogenous prenyl pyrophosphate antigens.


Figure 1
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  		Fig. 1 2-C-methyl-D-erythritol 4-phosphate (MEP) and mevalonate pathways for isoprenoid biosynthesis. The MEP pathway is found in most eubacteria, apicomplexan protozoa and chloroplasts whereas the mevalonate pathway is found in archaebacteria. eukaryotes and the cytoplasm of plants. Genes for MEP enzymes are also termed ispC (dxr), ispD (ygbP), ispE (ychB), ispF (ygbB), ispG (gcpE) and ispH (lytB).

 
Although HMBPP appears to be a major microbial antigen, its relationship to other described phosphoantigens, termed TUBag1, TUBag2, TUBag3 and TUBag4 (28), is unclear. The structure of TUBag1 isolated from Mycobacterium fortuitum has been reported as 3-formyl-1-butyl pyrophosphate (3-FBPP) (29). However, this compound has an identical molecular weight and chemical composition to HMBPP. The TUBag3 and TUBag4 antigens are reported to be 3-formyl-butyl conjugates to TTP and UTP (28, 30) but little is known about the presence and relative amounts of unconjugated to nucleotide-conjugated phosphoantigens in bacteria. A closely related structure, 3-formyl-1-pentyl pyrophosphate (3-FPPP) has been proposed for the second phosphoantigen, TUBag2, isolated from E. coli (31) and mycobacteria (2, 32). Also, lysates of gram-positive cocci that use the mevalonate pathway, such as Staphylococcus aureus and group A, B and C Streptococcus, stimulate V{gamma}2V{delta}2 T cells (33, 34, data not shown) suggesting that an additional phosphoantigen (perhaps IPP) exists besides HMBPP and 3-FBPP as the major antigen for bacteria using the mevalonate pathway.

We have recently reported the synthesis of 3-FBPP (proposed as TUBag1) and find that synthetic 3-FBPP has only moderate stimulatory activity (EC50% = ~3 µM) rather than the high stimulatory activity reported (EC50% = ~5–50 nM) and that its NMR spectra does not match that reported for the natural antigen (35). Moreover, we found that the 275 Da compound in mycobacteria that was proposed to be 3-formyl-pentyl pyrophosphate (TUBag2) is actually 6-phosphogluconate, a compound without biological activity for V{gamma}2V{delta}2 T cells (35). Thus, none of the TUBag antigens are 3-formyl-alkyl pyrophosphates leading to uncertainty about their structures and the relative importance of the different phosphoantigens.

To further clarify the structure and relative importance of natural phosphoantigens in different bacteria and to confirm the importance of MEP pathway enzymes in determining in vitro and in vivo stimulation of V{gamma}2V{delta}2 T cells, we isolated bacterial antigens and identified mutations that affect bacterial antigen levels. We find that unconjugated HMBPP is the major bacterial antigen in multiple species using the MEP pathway. Consistent with this, mutations that affect bacterial antigen levels were primarily in enzymes of the MEP pathway or genes regulating this pathway. No evidence for additional enzymes that could produce 3-FBPP was found. The HMBPP metabolite was highly preferentially recognized over IPP by V{gamma}2V{delta}2 T cells including neonatal {gamma}{delta} T cells. Moreover, in the human-PBL-SCID–beige mouse model, only a bacterial mutant with high levels of HMBPP expanded V{gamma}2V{delta}2 T cells. These findings demonstrate a major role for HMBPP in determining activation of V{gamma}2V{delta}2 T cells for bacteria using the MEP pathway.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Antigens
Ethyl pyrophosphate (EPP) was synthesized as described (1). Bromohydrin pyrophosphate was provided by Eric Oldfield (University of Illinois, Urbana-Champaign). HMBPP was synthesized as described (36).

Derivation and maintenance of {gamma}{delta} T-cell clones
T-cell clones were propagated by periodic re-stimulation as described (37). The 12G12, DG.SF68 and CP.1.15 V{gamma}2V{delta}2 T-cell clones have been described (1). AC.2 and AC.8 are fetal liver clones (37) whereas CB.32.26 is a cord blood clone (38).

Purification and characterization of the major antigen from Mycobacterium smegmatis and M. fortuitum
Antigen was purified from 34 l of M. smegmatis and 4 l of M. fortuitum culture grown in Middlebrook 7H9 broth. The culture supernatants were passed through a carbon–Celite column (2) followed by tangential ultrafiltration (1000 MW cutoff, Pall-Filtron, Northborough, MA, USA). Minimal bioactivity (5–10%) was lost during these steps. Compounds in the ultrafiltrates were separated on a Q-Sepharose-Fast flow column (5 x 30 cm) by FPLC using an ammonium acetate gradient. Bioactive fractions were identified by their ability to stimulate the proliferation of a V{gamma}2V{delta}2 T-cell clone and then pooled. The single peak of bioactivity was further purified by HPLC using a DEAE-5PW column (150 x 21.5 mm, Bio-Rad, Hercules, CA, USA) followed by a Mono Q column (Amersham Pharmacia Biotech, Piscataway, NJ, USA) eluted with a triethylammonium bicarbonate gradient. The antigen was further purified using a Luna C18 column (250 x 4.6 mm, Phenomenex, Torrance, CA, USA) under ion pairing conditions with a tertiary solvent system. Solvent A: 100 mM triethylammonium bicarbonate (TEAB), pH 8.0 (prepared by bubbling CO2 through 100 mM TEA until pH 8.0); solvent B: 100 mM TEAB in 10% (v/v) methanol and solvent C: 100 mM TEAB in 50%(v/v) methanol. The column was eluted as follows: 0–10 min isocratic in solvent A at 1 ml/min; 10–70 min linear gradient 0–10% B in A at 1 ml/min; 70–75 min linear gradient 10–50% C in A at 0.5 ml/min; 75–90 min isocratic 50% C in A at 0.5 ml/min. One minute fractions were collected from which 1 µl of each fraction was tested for stimulation of a V{gamma}2V{delta}2 T-cell clone. Note that since this is a volatile buffer system, there is some variation in retention times for identical compounds. Each 1 min fraction was assayed for bioactivity with a {gamma}{delta} T-cell clone and analyzed by electrospray ionization tandem mass spectrometry in the negative mode (precursor ion and product-ion analyses) to identify and quantitate phosphate-containing compounds as previously reported (2). Electrospray ionization tandem mass spectrometry (ES MS/MS) spectra were obtained in negative ion mode using API-III, API 300 and API Qstar Pulsar I mass spectrometers (Applied Biosystems/MDS Sciex, Ontario, Canada), as described (2). The measured accurate mass of the compounds was determined by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry using electrospray ionization in the negative ionization mode on the 7 Tesla spectrometer at the Environmental Molecular Sciences Laboratory. Internal calibration employing IPP and geranyl pyrophosphate was used for accurate mass measurements.

Mutation of E. coli W3110 bacteria
E. coli mutants that carry point mutations in ygbP, ychB, ygbB and gcpE were derived from E. coli W3110 following treatment with N-methyl-N'-nitro-N-nitrosoguanidine as described (39–42). The mutant bacteria were engineered for isoprenoid metabolism through a partial mevalonate pathway by transformation of the parent bacteria with the pTMV20KM plasmid [which includes the Streptomyces sp. strain CL190 mevalonate pathway genes, mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase and isopentenyl disphosphate isomerase plus a kanamycin resistance gene (43)]. Since genes upstream of mevalonate are not included, addition of mevalonate (0.1 mg ml–1) into the media was required for growth (Fig. 1). The DK310 LytBG120D (pTMV20KM) strain was derived from the isopentenyl diphosphate isomerase disruptant strain, DK310, by treatment with N-methyl-N'-nitro-N-nitrosoguanidine and transformation with pTMV20KM (44). The dxr mutant was derived by the insertion of a kanamycin resistance gene into the coding sequence of dxr as described (45) except that the parent bacteria were transformed with the pTMV19 plasmid which includes the Streptomyces sp. strain CL190 mevalonate pathway genes found in pTMV20 plus the HMG-CoA reductase and HMG-CoA synthase genes (43) and addition of mevalonate (0.1 mg ml–1) into the media. The mutation for each strain is detailed in Table 1. ‘Leaky’ mutants were identified by plating bacteria on LB plates lacking mevalonate and culturing overnight at 37°C followed by 10 days at room temperature.


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  		Table 1 Mutations in enzymes in the MEP pathway of Escherichia coli K12 W33110

 
Transposon mutagenesis of the DK310 LytBG120D mutant
Mutant strains were generated by transposon-mediated mutagenesis of the DK310 LytBG120D (pTMV20KM) bacteria using the EZ:TNTM <DHFR-1> Tnp TransposomeTM kit (Epicentre, Madison, WI, USA) (44). Bacterial mutants thus generated were arrayed in a 96-well format and ~15 000 were screened for the loss of bioactivity with the 12G12 V{gamma}2V{delta}2 T-cell clone and for their ability to grow in the absence of mevalonate. To ensure the complete loss of activity, bacteria were further grown at room temperature for 4–7 days. Genomic DNA was isolated from mutants using a MasterPureTM DNA purification kit (Epicentre) and directly sequenced with a pair of primers specific to each end of the transposon at the University of Iowa DNA sequencing facility. The genomic transposition sites were located using BLAST programs maintained at the NCBI web site of the National Library of Medicine (http://www.ncbi.nlm.nih.gov/BLAST/).

Preparing bacterial supernatants and sonicates
To test bacterial supernatants and sonicates for their ability to stimulate V{gamma}2V{delta}2 T cells, E. coli bacteria were grown to late stationary phase in 1 l of LB media in 2.6 l fluted Fernback flasks by incubating for ~24 h at 37°C in an Innova 4400 shaker oscillating at 225 rev/min, this maximizes bioactivity. The bacteria were harvested by centrifugation at 380 x g for 15 min at 4°C. The culture supernatant was removed and the bacteria washed twice with PBS. Bacteria from 1l of culture were suspended in 10 ml of PBS and continuously probe sonicated for 10 min on ice at a 4.5 setting (Sonic Dismembrator Model 550, Fisher Scientific). The sonicated bacteria were centrifuged at 300 x g for 15 min at 4°C. The supernatants from the sonicated bacteria and the culture supernatants were heated in a boiling water bath for 5 min, cooled on ice for 5 min, centrifuged at 16 000 x g for 30 min at 4°C, filter sterilized with a 0.22-µm filter and frozen at –80°C. Note that the heating caused precipitation of protein and other bacterial components that inhibit T-cell proliferation and that give falsely low estimates of bioactivity but did not affect the overall bioactivity for V{gamma}2V{delta}2 T cells (44). Heating also dissociates prenyl pyrophosphates from proteins and other bacterial components that prevent the passage of prenyl pyrophosphates through membrane ultrafiltration units with molecular cutoffs greater than 1000–3000 Da.

V{gamma}2V{delta}2 T-cell proliferation assay and the quantitation of bacterial bioactivity for V{gamma}2V{delta}2 T cells
T-cell proliferation assays were performed as previously described (1). Mean proliferation and standard error of mean of triplicate cultures are shown. To quantitate bioactivity, the reciprocal dilution of the bacterial supernatant or sonicate that gave half-maximal proliferation was determined relative to a standard EPP antigen preparation (44). One unit of bioactivity was the amount of antigen in 1 ml that gave half-maximal antigen-induced proliferation of a V{gamma}2V{delta}2 T-cell clone (usually DG.SF68 or CP.1.15) and corresponds to an HMBPP concentration of 31.6 pM or 31.6 femtomoles ml–1 and an IPP concentration of 3 µM or 3 nmoles ml–1.

Expansion of V{gamma}2V{delta}2 T cells by non-peptide antigens
PBMC were isolated either from leukopacs or buffy coats by density centrifugation over Ficoll–Hypaque (Amersham Pharmacia Biotech). PBMCs (1 x 105) were cultured in 96-well round bottom plates in complete RPMI 1640 (37) alone or in complete RPMI 1640 with 50 µM IPP or 0.316 µM HMBPP. On day 3, 100 µl of supernatant were replaced with complete medium supplemented with 1.7% human serum and 1 nM recombinant human IL-2 (Chiron Corporation, Emeryville, CA, USA). On day 7, the PBMC were harvested, counted, stained with anti-V{delta}2 (BB3, gift from A. Moretta) and anti-CD3 (HIT3a, BD Pharmingen, San Diego, CA, USA) monoclonal antibodies (mAbs) and analyzed by two-color flow cytometry. The Institutional Review Board at the University of Iowa approved these studies.

Expansion of V{gamma}2V{delta}2 T cells by live bacteria
E. coli wild type, LytBG120D and LytBG120D yhjK– bacteria were grown to mid-log phase and stored in LB broth containing 10% glycerol at –80° C until use. To determine colony-forming units, bacteria were washed once with PBS and grown on LB plates. For the transwell assay, 1–3 x 106 bacteria were added in 0.1 ml RPMI 1640 medium to the inner wells (Corning Costar, Kennebunk, ME, USA). The inner well was separated from the outer well by a 0.4-µm membrane. PBMCs (2 x 106) were added to the outer well in 0.9 ml of complete medium. After 4 h, the inner wells were removed leaving the PBMC in culture. On day 3, half of medium was replaced with complete medium supplemented with human serum and recombinant IL-2. On day 6, the PBMC were harvested, counted and V{gamma}2V{delta}2 T cells determined by flow cytometry using anti-V{delta}2 and anti-CD3 mAbs.

V{gamma}2V{delta}2 T-cell proliferation in human-PBL-SCID–beige mice
Homozygous C.B-Igh-1b/GbmsTac-PrkdcSCID-LystbgN7 (C.B-17 SCID–beige) male mice (age 5–6 weeks old) were purchased from Taconic (Germantown, NY, USA) and maintained in microisolator cages. Animals were fed autoclaved food and water and all manipulations were performed in laminar flow cabinets. In vivo expansion of {gamma}{delta} T cells was performed in SCID–beige mice using either HMBPP-activated or unactivated PBMC. To assess the effector capability of V{gamma}2V{delta}2 T cells, PBMC were activated for 24 h in vitro with 0.316 µM HMBPP, washed and 2.5–3 x 107 cells injected i.p. into each mouse in 0.5 ml of RPMI. To assess the in vivo stimulatory capability of mutant bacteria, unactivated PBMC were used. Two hours later, each SCID–beige mouse was injected i.p. with either wild type or LytBG120D (termed lytB– in the figures) E. coli at 1 x 106–1 x 107 bacteria in 0.5 ml of RPMI medium. Alternatively, varying amounts of HMBPP were given in 0.25 ml of PBS. Recombinant human IL-2 (5000 IU) (Chiron, Emeryville, CA, USA) was given i.p. every other day starting on day 0. On day 9, the mice were sacrificed and peritoneal cells were harvested by washing the peritoneum with 4 ml of PBS. The peritoneal cells were counted and analyzed by flow cytometry using anti-V{delta}2 and anti-CD3 monoclonal antibodies to determine the percentage of V{gamma}2V{delta}2 T cells among human CD3+ T cells. The Institutional Animal Care and Use Committee of the University of Iowa approved all animal protocols. Data were tested for statistically significant differences using the non-parametric Mann–Whitney U-test.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Purification of the major antigen for V{gamma}2V{delta}2 T cells from mycobacteria
Although previous studies showed that mycobacterial lysates contain non-peptide antigens that stimulate {gamma}{delta} T cells, there are questions about the relative importance of HMBPP, 3-FBPP and IPP as well as the relative abundance of unconjugated and nucleotide-conjugated compounds (1, 2, 28, 46–48). Therefore, we prepared lysates from various bacteria, including mycobacteria [the BCG vaccine strain of M. bovis, opportunistic (M. avium and M. fortuitum) and environmental (M. smegmatis) species], gram-positive and -negative rods and gram-positive cocci and evaluated them for their ability to stimulate V{gamma}2V{delta}2 T cells. Despite their divergent origins, all bacterial lysates stimulated V{gamma}2V{delta}2 T cells including the lysate from S. aureus, a bacterium that uses the mevalonate pathway for IPP synthesis (Fig. 2A).


Figure 2
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  		Fig. 2 Characterization of the major bacterial non-peptide antigen for V{gamma}2V{delta}2 T cells. (A) V{gamma}2V{delta}2 T-cell clone, CP.1.15, stimulation with various bacterial sonicates. (B) The major mycobacterial phosphoantigen has a molecular weight of 262 Da. The major phosphoantigen was purified from Mycobacterium smegmatis and Mycobacterium fortuitum by several chromatographic steps followed by ion-pairing reverse-phase chromotography on a Luna C18 column. Bioactive fractions were identified by their stimulation of a V{gamma}2V{delta}2 T-cell clone and analyzed by ES MS/MS for ions containing phosphate residues. Note the strict correlation between bioactivity and the presence of an [M–H]– ion of m/z 261 (corresponding to a 262 Da molecular weight). (C) The m/z 261 ion contains a pyrophosphate moiety and has the chemical composition of C5H11O8P2. The m/z 261 ion was analyzed by tandem mass spectrometry revealing a m/z 159 product ion corresponding to a pyrophosphate moiety. The chemical composition was determined from the accurate molecular mass measured by FT-ICR MS. A C5H11O8P2 composition for the m/z 261 ion was consistent with the calculated mass to within +0.72 parts per million. (D) The m/z 261 ion is common to Mycobacteria and the gram-negative rod bacteria, Yersinia enterocolitica. Antigens were purified from lysates of M. tuberculosis and Y. enterocolitica and from culture supernatants of M. smegmatis and M. fortuitum. The biologically active fractions were resolved by ion-pairing chromatography on a Luna C18 column. Note that since this is a volatile buffer system, there is some variation in retention times for identical compounds. (E) The m/z 261 ion is common to M. smegmatis and Escherichia coli. The biologically active fractions were resolved by ion-pairing chromatography on a Luna C18 column. (F) Phosphoantigen levels during bacterial growth. (Top) Bioactivity levels during M. smegmatis growth. Stationary phase was achieved at day 2–3. Intracellular bioactivity was not determined but in stationary phase cultures constituted <5% of total bioactivity. (Middle) Bioactivity levels during M. fortuitum growth. This mycobacterium grows slower than M. smegmatis. Data is from (49) and is included for comparison. (Bottom) Bioactivity levels during E. coli growth. Note that phosphoantigen levels are maximal during the late stationary phase of growth when the majority of the bioactivity is in the culture supernatant.

 
To identify the compounds responsible for bioactivity in bacteria, the major peak of bioactivity was purified from M. fortuitum and M. smegmatis. Ninety to 95% of the antigenic activity in the supernatant from M. smegmatis passed through an activated charcoal–Celite column that retains nucleotide, nucleotide-conjugated and hydrophobic compounds (1). Thus, nucleotide-conjugated antigens, such as the 5'-UTP-conjugated antigen reported for M. fortuitum (30) and the 5'-dTTP-conjugated antigen reported for M. tuberculosis (28), accounted for, at most, 5–10% of bioactivity in M. smegmatis. Unlike antigenic activity from lysates of heat-killed M. tuberculosis (Fig. 2D), subsequent anion exchange and ion-pairing reverse phase chromatography revealed only one peak of bioactivity from both M. fortuitum and M. smegmatis. This peak of bioactivity for V{gamma}2V{delta}2 T cells on ion-pairing reverse-phase chromatography (Fig. 2B, middle panels) correlated with the presence of an ion with a mass to charge ratio (m/z) = 261 ([M–H]–) (Fig. 2B, upper panels). Further characterization of the m/z 261 ion using product-ion analysis by electrospray ionization tandem mass spectrometry (ES MS/MS) revealed that the m/z 261 ion is pyrophosphorylated, as evidenced by the presence of products ions at m/z 159 (corresponding to HP2O6–), m/z 97 (corresponding to H2PO4–) and m/z 79 (corresponding to PO3–) (Fig. 2C). The measured accurate mass of the m/z 261 ion was 260.993655 as determined by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Based on this weight, the m/z 261 ion matched most closely a chemical composition of C5H11O8P2 (+0.72 ppm error) (Fig. 2C). This is identical to the negative ion [M–H]– of HMBPP and of 3-FBPP. Synthetic HMBPP and 3-FBPP had nearly identical major ions on collision-induced dissociation using an ion trap mass spectrometer (data not shown and 32) precluding the use of this technique to distinguish between the two compounds. Therefore, the major antigen could be either HMBPP or 3-FBPP.

The m/z 261 ion of M. smegmatis and M. fortuitum had similar retention times to TUBag1 in M. tuberculosis and the major antigen in Y. enterocolitica (Fig. 2D) and E. coli (Fig. 2E). Note, that there were minor variations in retention times for identical compounds due to the use of a volatile buffer. Nucleotide-conjugated compounds were not produced by either E. coli or Y. enterocolitica since only one peak of bioactivity was isolated (Fig. 2D and E). To determine if the level of bioactivity was related to the bacterial growth phase, cultures of M. smegmatis, M. fortuitum (data from 49) and E. coli were grown and bioactivity for V{gamma}2V{delta}2 T cells quantitated for different growth phases. In all three bacteria, antigen levels were highest in the late stationary phase with most of the bioactivity present in the culture supernatants (Fig. 2F). The presence of bioactivity in the culture supernatants of actively growing bacteria confirms earlier studies (30, 49) although it is not clear whether the major antigen is actively secreted or just released by dying bacteria. In some other bacterial species, antigenic activity is retained in the cytoplasm (data not shown). Since the highest levels of bioactivity are found in late stationary phase cultures, bioactivity levels for bacteria were determined at this time point.

Mutation of genes in the MEP pathway identifies HMBPP as the primary bacterial antigen for V{gamma}2V{delta}2 T cells
Given its product-ion spectra, chemical composition and the complete delineation of the MEP pathway, we hypothesized that the m/z 261 ion phosphoantigen was HMBPP rather than 3-FBPP. To test this hypothesis using a genetic approach, we made E. coli strains with mutations in enzymes of the MEP pathway, the pathway that produces HMBPP and IPP in E. coli (Table 1). Since this pathway is essential for viability, these mutants were derived from an E. coli strain that was first modified to contain a partial mevalonate pathway. The mevalonate pathway synthesizes IPP in mammals but does not make HMBPP or any other MEP pathway intermediate (Fig. 1). Mutations in MEP pathway enzymes upstream from HMBPP (YgbP, YgbB and GcpE), that completely abrogated growth in the absence of mevalonate, also markedly reduced bioactivity of V{gamma}2V{delta}2 T cells (Fig. 3A and Table 1). Conversely, when the downstream enzyme LytB was mutated, the bacteria showed a 300- to 1500-fold increase in V{gamma}2V{delta}2 T-cell bioactivity (Fig. 3A). As expected, this elevated level of bioactivity found in LytBG120D mutant bacteria could be reduced to wild-type levels by adding fosmidomycin (FMM), a specific inhibitor of the upstream enzyme, deoxyxylulose-5-phosphate reductoisomerase (dxr). The level of HMBPP appears to be tightly regulated since bacteria with point mutations that greatly slowed but did not completely eliminate growth had similar bioactivity levels as wild-type bacteria in late stationary phase cultures (Table 1). The requirement for GcpE for biological activity confirms previous results (24, 25) and we now show that other enzymes in the pathway are similarly required.


Figure 3
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  		Fig. 3 Bioactivity of E. coli with mutations in the MEP pathway. (A) Loss of bioactivity of E. coli with mutations in the MEP pathway correlates with mevalonate-dependent growth. Sonicates and culture supernatant were prepared from 1 l of wild-type Escherichia coli and MEP pathway-defective E. coli strains complemented with the mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase and isopentenyl disphosphate isomerase genes from the mevalonate pathway. FMM, (20 µg ml–1) was included to partially block the dxr enzyme and mevalonate (1 mg ml–1) was added to support isoprenoid biosynthesis through the mevalonate pathway by enzymes introduced into the bacteria. One unit of bioactivity corresponds to 31.6 femtomoles of HMBPP per milliliter. The order of the mutants (left to right) is the same as Table 1. Note that only those bacteria with complete lack of mevalonate-independent growth had low levels of bioactivity despite the presence of fosmidomycin in all mutant cultures. (B) Bioactivity of LytBG120D mutant E. coli mutated by transposons. Mutants were screened for bioactivity for V{gamma}2V{delta}2 T cells and for mevalonate-dependent growth. Mutants are as in Table 2.

 
As it is possible that 3-FBPP is produced as a side metabolite from HMBPP by a novel enzyme, we performed transposon mutagenesis of the LytBG120D strain that accumulates high levels of bioactivity to identify genes required for bioactivity. Since transposons can insert throughout the bacterial genome, this technique should identify genes required for bioactivity for V{gamma}2V{delta}2 T cells potentially including genes not in the MEP pathway. Approximately 15 000 mutants were screened for their bioactivity for {gamma}{delta} T cells and for their ability to grow independently of mevalonate (Table 2). Twenty-seven clones had lower bioactivity compared with the LytBG120D bacteria (Fig. 3B). Direct genomic sequencing of these mutants revealed that 23 out of 27 of mutants had a transposon inserted into a known gene in the MEP pathway identifying five out of six upstream enzymes from HMBPP. These mutants did not grow in the absence of mevalonate, since they lacked the ability to synthesize IPP through the MEP pathway (Table 2 and Fig. 3B).


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  		Table 2 Transposon mutants of DK310 LytBG120D Escherichia coli bacteria

 
Two additional genes, fldA and sppA, were found to be important in the synthesis of HMBPP. fldA encodes flavodoxin I that functions as an electron donor for GcpE in the synthesis of HMBPP (21). sppA (not previously reported) encodes a signal peptide peptidase that cleaves signal peptides and may be required for enzyme activity. Mutation of triose phosphate isomerase (tpi) also reduced bioactivity since it is required to convert dihydroxyacetone phosphate to glyceraldehyde-3-phosphate, a precursor for the MEP pathway. Mutation of yhjK reduced bioactivity of the LytBG120D mutant but when deleted in wild-type E. coli {gamma}{delta} bioactivity and bacterial growth were normal (K. -J. Puan and C. T. Morita, unpublished data). yhjK encodes a transmembrane signaling protein that likely regulates cyclic diguanylate monophosphate levels and that may, in turn, regulate HMBPP pool size only in the LytBG120D strain. Two mutants, cysB and yaeD, required mevalonate for growth but their HMBPP levels remained unaltered. yaeD encodes a phosphatase involved in LPS synthesis; cysB encodes a protein involved in the regulation of cysteine synthesis and may be required for activity of the mutant LytBG120D enzyme. Importantly, no enzyme that could convert HMBPP to 3-FBPP was identified. These genetic studies complement our structural analysis (Fig. 1) and functional tests on synthetic 3-FBPP (35) and suggest that HMBPP, rather than 3-FBPP, is the major antigen for bacteria using the MEP pathway.

HMBPP is a highly potent phosphoantigen and its recognition can be mediated by V{gamma}2V{delta}2 T-cell TCRs that are present at birth
To verify that HMBPP stimulates V{gamma}2V{delta}2 T cells, synthetic HMBPP was tested for its ability to stimulate several V{gamma}2V{delta}2 T-cell clones including fetal liver clones (AC.2 and AC.8) that use the invariant V{gamma}2 (V{gamma}9) chain (50); a cord blood clone, CB32.26 (38); and adult clones, 12G12, DG.SF68 and CP.1.15. For all clones, HMBPP was 30 000-fold more antigenic than IPP (Fig. 4A, half-maximal proliferation for HMBPP and IPP for the AC.2 and DG.SF68 clones was 36 pM and 1 µM, respectively, and unpublished data) and 100- to 300-fold more antigenic than bromohydrin pyrophosphate, a synthetic phosphoantigen. To confirm that the V{gamma}2V{delta}2 TCR mediated HMBPP recognition, a V{gamma}2V{delta}2 TCR transfectant, DBS43, was tested and found to release IL-2 in response to HMBPP (unpublished data). HMBPP also stimulated the expansion of V{gamma}2V{delta}2 T cells from normal donors (Fig. 4B). The fetal liver clones AC.2 and AC.8 use the invariant V{gamma}2 chain that is found in 10–30% of adult V{gamma}2V{delta}2 TCR (50). Reactivity to HMBPP by fetal and cord blood clones confirms ours and other's earlier studies showing that cord blood V{gamma}2V{delta}2 T cells respond to HMBPP in mycobacterial lysates and to IPP, and that these responses are present at birth (38, 51–53). These results suggest that reactivity to HMBPP is a property of most V{gamma}2V{delta}2 T cells, including those expressing invariant V{gamma}2 chains.


Figure 4
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  		Fig. 4 HMBPP is the most immunogenic non-peptide antigen for V{gamma}2V{delta}2 T cells. (A) HMBPP is the most immunogenic natural phosphoantigen. V{gamma}2V{delta}2 T-cell clones from fetal liver (AC.2), cord blood (CB.32.26) and adult synovial fluid (DG.SF68) were cultured for 48 h with different concentrations of the indicated antigens in the presence of mitomycin C-treated Va-2 cells. Proliferation was measured by 3H-thymidine incorporation. Error bars are ± 1 standard error of the mean. (B) HMBPP expands V{gamma}2V{delta}2 T cells in PBMC. PBMC were cultured in either medium alone, or medium supplemented with 50 µM IPP or 0.316 µM HMBPP. On day 7, PBMC were harvested and analyzed by two-color flow cytometry using anti-V{delta}2 and anti-CD3 specific mAbs.

 
Bacterial HMBPP levels determine the magnitude of in vitro and in vivo expansion of V{gamma}2V{delta}2 T cells
If HMBPP is a major determinant of V{gamma}2V{delta}2 T-cell reactivity to bacteria, we reasoned that increasing HMBPP levels would results in stronger V{gamma}2V{delta}2 T-cell responses. To determine if the levels of HMBPP in bacteria influence V{gamma}2V{delta}2 T-cell expansion in vitro, PBMC were co-cultured with live LytBG120D that overproduce HMBPP, LytBG120D yhjK– mutant bacteria that had extremely low bioactivity levels, and wild-type bacteria with moderate bioactivity levels in a transwell system. V{gamma}2V{delta}2 T cells expanded slightly more with LytBG120D bacteria than with wild type, but much less with LytBG120D yhjK– bacteria that had extremely low levels of bioactivity (Fig. 5).


Figure 5
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  		Fig. 5 Bacterial HMBPP levels determine in vitro expansion of V{gamma}2V{delta}2 T cells. Live wild-type or mutant bacteria (1 or 3 x 106) were added to the inner well of a transwell where they were separated from PBMC in the outer well by a 0.4-µm membrane. After 4 h, the inner wells were removed. On day 6, PBMC were harvested, counted and V{gamma}2V{delta}2 T cells determined by flow cytometry using anti-V{delta}2 and anti-CD3 mAbs. Data shown are from one donor and are representative of results with seven donors.

 
Since V{gamma}2V{delta}2 T cells and prenyl pyrophosphate recognition is restricted to primates, direct testing in vivo is difficult. The hu-PBL-SCID–beige model provides a small animal model where human PBMC are transplanted into immunodeficient SCID–beige mice. Transplanted V{gamma}2V{delta}2 T cells have been shown to proliferate when preactivated with antigen prior to transplantation where they help provide immunity to infection with bacteria through their production of IFN-{gamma} (16). Therefore, we used this model system to determine the effects of differing levels of bacterial HMBPP on the expansion of V{gamma}2V{delta}2 T cells in vivo.

SCID–beige mice were engrafted with HMBPP-activated PBMC (containing 1–5% V{gamma}2V{delta}2+ T cells) and subsequently infected with bacteria. Peritoneal cells were harvested 9 days later and analyzed by flow cytometry. Mice that received HMBPP-activated PBMC, followed by either LytBG120D E. coli (that have very high bioactivity) or Morganella morganii, (with more modest levels of bioactivity) showed expansion of V{delta}2+ T cells that was dose dependent but not significantly different between the two bacteria (Fig. 6A and B). Similarly, wild-type and LytBG120D bacteria elicited roughly similar levels of V{delta}2+ T-cell expansion. This result is consistent with previous in vitro studies showing that LPS alone could stimulate antigen-activated V{gamma}2V{delta}2 T cells to expand and secrete IFN-{gamma} (54). These findings suggest that after non-peptide antigen stimulation in vitro, subsequent in vivo V{gamma}2V{delta}2 T-cell expansion is less dependent on antigen levels.


Figure 6
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  		Fig. 6 Bacterial HMBPP levels determine in vivo expansion of unactivated V{gamma}2V{delta}2 T cells in the hu-PBL-SCID–biege mouse model. (A, B) Escherichia coli LytBG120D or Morganella morganii expand HMBPP-activated V{gamma}2V{delta}2 T cells in a dose-dependent manner. SCID–beige mice were reconstituted with 3 x 107 HMBPP-activated PBMC i.p. and subsequently challenged with increasing numbers of E. coli LytBG120D or M. morganii bacteria. After 9 days, cells were harvested and V{gamma}2V{delta}2 T cells determined by two-color flow cytometry. (A) Two-color flow cytometric analysis of representative mice with or without bacteria. (B) Bacterial dose response of HMBPP-activated V{gamma}2V{delta}2 T cells. Total V{gamma}2V{delta}2 T cells were up to 3-fold greater in mice receiving bacteria. (C) Expansion of HMBPP-activated V{gamma}2V{delta}2 T cells by both wild-type and LytBG120D E. coli. SCID–beige mice were reconstituted with HMBPP-activated PBMC as in (A). Note the similar increases in V{delta}2+ T cells in mice receiving wild-type bacteria and LytBG120D bacteria that have elevated levels of HMBPP. (D) Expansion of unactivated V{gamma}2V{delta}2 T cells is dependent on HMBPP levels. SCID–beige mice were reconstituted with unactivated PBMC followed by infection with either wild-type or LytBG120D bacteria. Left and right panels represent two independent experiments with three mice per group and 10 mice per group, respectively. *P < 0.01. Note that only mice receiving the LytBG120D bacteria showed expansion of V{gamma}2V{delta}2 T cells.

 
In contrast, when unactivated PBMC were used for engraftment, significantly higher levels of V{delta}2+ T-cell expansion were found only with infection with LytBG120D E. coli as compared with wild-type E. coli or non-infected controls (Fig. 6D, right). V{delta}2+ T-cell expansion was also dependent on bacterial numbers, as shown in an independent experiment with a different donor (Fig. 6D, left). These expansions occurred in the absence of exogenously added IL-2. Thus, bacterial HMBPP levels likely play an important role in determining in vivo responses by V{gamma}2V{delta}2 T cells.

To determine if HMBPP could directly stimulate V{gamma}2V{delta}2 T cells in vivo, unactivated PBMC were transplanted into SCID–beige mice and then stimulated with synthetic HMBPP. HMBPP stimulated the expansion of resting V{gamma}2V{delta}2 T cells such that their absolute numbers and percentage of CD3 T cells were significantly increased (Fig. 7). Unlike expansions with bacterial infection, this expansion was dependent on exogenous IL-2 (data not shown).


Figure 7
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  		Fig. 7 Synthetic HMBPP stimulates in vivo expansion of V{gamma}2V{delta}2 T cells. SCID–beige mice were reconstituted with 3 x 107 unactivated PBMC i.p. and subsequently challenged with varying amounts of HMBPP. Human IL-2 (5000 IU) was given i.p. every other day starting on day 0. After 9 days, cells were harvested by peritoneal lavage and the expansion of V{gamma}2V{delta}2 T cells assessed by two-color flow cytometry: (top) V{gamma}2V{delta}2 T cells as % of CD3+ T cells; (bottom) total V{gamma}2V{delta}2 T-cell numbers. Similar increases in V{gamma}2V{delta}2 T cells (as a % of CD3+ T cells) were noted in two additional experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In this study, we show that the level of HMBPP in bacteria is a major factor in determining in vivo responses in the hu-PBL-SCID–beige mouse model by V{gamma}2V{delta}2 T cells. We find that HMBPP is the primary antigen for V{gamma}2V{delta}2 T cells in mycobacteria and in the gram-negative rods, Escherichia coli and Yersinia enterocolitica. We confirm and extend previous studies by showing that mutations in all six enzymes upstream of HMBPP in the MEP pathway abolished or greatly diminished bioactivity, whereas mutation of the downstream LytB enzyme greatly increased bioactivity. Infection with the LytBG120D mutant also expanded V{gamma}2V{delta}2 T cells in the hu-PBL-SCID–beige mouse model. The magnitude of the V{gamma}2V{delta}2 T-cell expansion was related to the HMBPP levels in the bacteria, and synthetic HMBPP was highly active on a molar basis in stimulating V{gamma}2V{delta}2 T cells both in vitro and in vivo. Since the MEP pathway is widely distributed in many important human pathogens including Mycobacteria, gram-negative bacteria and apicomplexan protozoa, recognition by V{gamma}2V{delta}2 T cells of a metabolite in this pathway allows V{gamma}2V{delta}2 T cells to combat infection by a broad range of microbial pathogens.

Our study also helps to address the question of the structure of the 262 Dalton phosphoantigen (TUBag1). On transposon mutagenesis of the LytBG120D E. coli mutant, no genes were identified that could encode an enzyme that would produce 3-FBPP. Moreover, our studies on synthetic 3-FBPP (35) show that this compound has only low to moderate activity for V{gamma}2V{delta}2 T cells rather than the high activity reported for TUBag1 (29) and has a different NMR spectra than that reported for TUBag1 (29). Similarly, the 275 Dalton compound from mycobacteria that was proposed as 3-formyl-pentyl pyrophosphate is actually 6-phosphogluconate, a biologically inactive compound (35). Nucleotide conjugated forms of TUBag1 do not contribute significant amounts of bioactivity in both gram-negative rods and rapid growing mycobacteria. Also, none of the other metabolites in the MEP pathway have significant bioactivity for V{gamma}2V{delta}2 T cells (31). Taken together and with other reported genetic studies and structural studies on phosphoantigens (23, 24, 26, 27, 55, 56), we conclude that HMBPP is most likely the 262 Dalton antigen isolated from mycobacteria and from gram-negative rods that use the MEP pathway.

Using transposon and chemical mutagenesis, we have identified all of the genes of enzymes in the MEP pathway as affecting bioactivity levels for V{gamma}2V{delta}2 T cells. Mutations in any of these genes blocked bacterial growth on media without mevalonate, and decreased bioactivity of the LytBG120D mutant to wild-type levels. We also found that the fldA gene, encoding flavodoxin I, was essential both for the MEP pathway and for bioactivity. Flavodoxin contains a flavin mononucleotide and donates electrons to a number of iron-containing proteins (57, 58). One protein that likely requires flavodoxin activity is GcpE, a [4Fe-4S] protein that, via two one-electron transfers, catalyses the synthesis of HMBPP (59). After disrupting the fldA gene, HMBPP was not produced and bacteria stopped growing; complementation of mutants with flavodoxin restored growth (44). LytB is also a [4Fe-4S] protein (60) and flavodoxin may be required for its enzymatic activity. Although the mutants had lower bioactivity for V{gamma}2V{delta}2 T cells, the ability of mutant bacteria to stimulate V{gamma}2V{delta}2 T cells was not completely abolished. This bioactivity is probably due to either HMBPP produced by residual MEP enzyme activity or to IPP. Consistent with this latter hypothesis, eubacteria that use the mevalonate pathway, such as Staphylococcus and Streptococcus, also contain a phosphoantigen that is likely to be IPP (unpublished data, 33, 34).

The V{gamma}2V{delta}2 TCR mediates recognition of HMBPP (61) and V{gamma}2V{delta}2 T cells do not require antigenic selection to enrich for rare reactive clones. We previously showed that in cord blood, V{gamma}2V{delta}2 T cells expand to high numbers when cultured with M. tuberculosis lysates (51) and that cord blood V{gamma}2V{delta}2 T-cell clones isolated without antigenic stimulation respond to non-peptide antigens (38). Here, we demonstrate that fetal liver clones and a cord blood clone respond to HMBPP like adult V{gamma}2V{delta}2 T cells. This strong reactivity for HMBPP is found in many cord blood and fetal V{gamma}2V{delta}2 clones (38) including those carrying the germ line-encoded invariant V{gamma}2 gene sequence (such as AC.2 and AC.8) (50). This invariant V{gamma}2 junctional sequence is commonly expressed by V{gamma}2V{delta}2 T cells since it was found in 11–30% of V{gamma}2J{gamma}1.2 rearrangements from nine children (50), in 10–17.6% of V{gamma}2J{gamma}1.2 rearrangements from five adults (62) and in 6.5% of functional V{gamma}2J{gamma}1.2 rearrangements before and 11.9% after IPP stimulation of one donor (63). There is also likely to be selection for more reactive V{gamma}2V{delta}2 T cells during infancy as evidenced by the predominance of V{gamma}2V{delta}2 T cells expressing V{gamma}2 chains using the J{gamma}1.2 region and with a hydrophobic residue in the CDR3{delta} region that are not commonly seen in fetal V{gamma}2V{delta}2 T cells (64).

A recent estimate of precursor frequency of naive CD8 T cells specific for the H-2Db-restricted GP33-41 epitope of lymphocytic choriomeningitis virus was one in 200 000 (65), whereas V{gamma}2V{delta}2 T cells constitute one in 618 T cells in cord blood (38) and one in 25–100 T cells in adults (1, 66, 67). Since most V{gamma}2V{delta}2 T-cell clones isolated from adults by sorting and lectin stimulation respond to mycobacterial lysates [10 reactive/10 clones, C. T. Morita unpublished observation, 11/14 clones (68), and 25/26 clones (67) for 46/50 clones (92%)], it is likely that the majority of adult V{gamma}2V{delta}2 T cells respond to HMBPP. Thus, unlike {alpha}ß T cells specific for peptides, a previous encounter with a specific bacteria is not required to amplify adult HMBPP-specific V{gamma}2V{delta}2 T cells since earlier infections or exposure to endogenous IPP has amplified further the already high percentage of reactive V{gamma}2V{delta}2 T cells (69). This ability of V{gamma}2V{delta}2 T cells to recognize HMBPP may be vital in containing infections prior to the onset of adaptive {alpha}ß T-cell and B-cell responses.

Since murine {gamma}{delta} T cells do not respond to prenyl pyrophosphate antigen, the hu-PBL-SCID–beige mouse model offers a small animal model to study human V{gamma}2V{delta}2 T-cell functions in vivo. Previous studies using the hu-PBL-SCID model frequently relied on the prior activation of PBMC in vitro with an agonistic anti-CD3 antibody (70). In another study, V{gamma}2V{delta}2 T cells were activated in vitro with the alkylamine, isobutylamine, prior to transfer to generate V{gamma}2V{delta}2 T-cell responses in vivo (16). Activating PBMC with HMBPP in vitro increased the responsiveness of the V{gamma}2V{delta}2 T cells to subsequent infection with different E. coli bacteria. This is analogous to CD8+ {alpha}ß T cells where initial priming with antigen ex vivo sensitizes them for greater proliferation and differentiation (71, 72). In contrast, the elevated levels of HMBPP found with mutant LytBG120D E. coli stimulated V{gamma}2V{delta}2 T-cell expansion in SCID–beige mice engrafted without requiring preactivation with antigen or exogenously added IL-2. Similarly, synthetic HMBPP was able to expand transferred V{gamma}2V{delta}2 T cells in hu-PBL-SCID–beige mice but this expansion required exogenously added IL-2 similar to the requirement noted for in vivo stimulation of primates and human V{gamma}2V{delta}2 T cells (73–75).

Despite their broad reactivity for prenyl pyrophosphates, our study shows that V{gamma}2V{delta}2 T cells can distinguish between foreign (HMBPP) and self-phosphoantigens (IPP) that are structurally very similar. Although both IPP and HMBPP stimulate similar responses at optimal concentrations, HMBPP is ~30 000-fold more potent on a molar basis. This recognition of HMBPP can be mediated by V{gamma}2V{delta}2 TCRs using a germ line-encoded, invariant V{gamma}2 chain allowing a significant proportion of cord blood V{gamma}2V{delta}2 T cells to recognize foreign pathogens. Furthermore, V{gamma}2V{delta}2 T cells expand early [between the age of 1–3 years (69)] leading to their conversion to a memory phenotype. As a result, by adulthood >98% of circulating V{gamma}2V{delta}2 T cells are memory T cells (data not shown) (76). Thus, in humans >3 years old, V{gamma}2V{delta}2 T cells can mount memory responses to primary infections of bacteria and protozoa for which the rest of the adaptive immune system ({alpha}ß T cells and follicular B cells) is naive. This ability parallels that of marginal zone B cells that are programmed to mount rapid and intense antibody responses to blood-borne pathogens (77, 78). Similar to V{gamma}2V{delta}2 T cells, some marginal zone B cells use their invariant or VH-restricted antibody receptors to recognize non-peptide antigens found in both pathogens and self. But for some marginal zone B cells the targets are phosphorylcholine (phospholipids) or polysaccharide compounds (79).

The ability of V{gamma}2V{delta}2 T cells to preferentially recognize a foreign metabolite is also reminiscent of pattern recognition by Toll-like receptors (TLRs) of the innate immune system. Each TLR recognizes conserved structures produced by or in response to different microbes (80). Moreover, like V{gamma}2V{delta}2 TCR recognition of endogenous IPP, some TLRs, such as TLR9, also recognize endogenous DNA under certain conditions (81). The microbial TLR ligands are abundant, distributed in a wide array of microorganisms, and predominantly non-peptidic. Similarly, HMBPP is present in a wide array of both prokaryotic and eukaryotic microorganisms that use the MEP pathway. V{gamma}2V{delta}2 T cells also express TLR2 and the recognition of non-peptide antigens is enhanced by the presence of TLR ligands either directly (82) or indirectly through their stimulation of IFN-{alpha}/ß from antigen-presenting cells (83, 84).

V{gamma}2V{delta}2 T cells may be particularly important in immunity to infections caused by intracellular bacteria or protozoa that subvert the innate and adaptive immune systems. Many of the infections that expand V{gamma}2V{delta}2 T cells are by intracellular microbes [reviewed in (85, 86)] and the expansion of V{gamma}2V{delta}2 T cells correlated with clearance of mycobacteria in rhesus monkeys (17). In the hu-PBL-SCID mouse model, V{gamma}2V{delta}2 T cells also help to protect mice from infections with E. coli, S. aureus and M. morganii by the production IFN-{gamma} and other cytokines (16). V{gamma}2V{delta}2 T cells can recognize cells infected with M. tuberculosis, M. bovis BCG and Salmonella typhimurium (7, 87–89) and kill the infected cells through perforin- and Fas ligand-dependent pathways (90–93). Released bacteria and malarial parasites can then be killed by granulysin (88, 90, 93–97). Activated V{gamma}2V{delta}2 T cells secrete a variety of cytokines and chemokines [chemokine production is reviewed in (98)]. Most V{gamma}2V{delta}2 T cells secrete TH1 cytokines such as IFN-{gamma}, tumor necrosis factor-{alpha} and other inflammatory cytokines (37, 99). They also secrete inflammatory chemokines such as MIP-1{alpha} (CCL3), MIP-1ß (CCL4), lymphotactin (XCL1) and RANTES (CCL5) (100–102). V{gamma}2V{delta}2 T cells can also kill bacteria by secreting the cathelicidin, LL-37, which has an anti-bacterial effect on Brucella suis (103). Besides their direct role in microbial immunity, V{gamma}2V{delta}2 T cells may also be important for the maintenance of tissue integrity and to speed tissue repair through the production of connective tissue growth factors (104, 105) and metalloproteinases (106). They may also serve to regulate {alpha}ß T cell and innate immune responses as has been shown in mice [reviewed in (107, 108)].

Besides responding to HMBPP, V{gamma}2V{delta}2 T cells also recognize the endogenous IPP metabolite when overproduced by certain tumor cells (109) or by pharmacological inhibition of farnesyl pyrophosphate synthase by bisphosphonates or alkylamines (109–111). This overproduction of IPP appears to determine V{gamma}2V{delta}2 T-cell recognition of some B-cell tumors (109). V{gamma}2V{delta}2 T cells also recognize and kill a wide variety of tumor cells including prostate carcinomas, renal cell carcinomas, nasopharyngeal carcinomas and colon carcinomas probably through non-TCR mediated, NK receptor recognition (112–116). Since immunotherapy with V{gamma}2V{delta}2 T cells can control B cell malignancies (75), V{gamma}2V{delta}2 T cells may naturally perform tumor surveillance and could be used for immunotherapy of a number of different cancers.

In summary, the preferential recognition of the exogenous isoprenoid metabolite, HMBPP, over endogenous isoprenoids is likely to play a central role in the immune function of V{gamma}2V{delta}2 T cells and parallels antigen recognition by adaptive marginal zone B cells and pattern recognition by innate cells. Exploiting this unique property of V{gamma}2V{delta}2 T cells may result in new vaccines for bacterial infections and new immunotherapies for malignancies.


   Acknowledgements
 
This work was supported in part by grants from the National Institute of Arthritis and Musculoskeletal and Skin Disease (RO1 AR45504), the National Institute of Allergy and Infectious Diseases (Midwest Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research, U54 AI057160), the Arthritis Foundation and the Carver Research Foundation. We thank M. Curtiss and D. Colgan for critical review of the manuscript. The accurate mass measurements were performed at the W. R. Wiley Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the U.S. Department of Energy's Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. Pacific Northwest National Laboratory is operated for the Department of Energy by Battelle.


   Abbreviations
 
EPP, ethyl pyrophosphate
ES MS/MS, electrospray ionization tandem mass spectrometry
3-FBPP, 3-formyl-1-butyl pyrophosphate
FT-ICR, Fourier transform ion cyclotron resonance
HDMAPP, hydroxy-dimethylallyl pyrophosphate
HMBPP, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate
hu-PBL-SCID, human peripheral blood lymphocyte-SCID mouse model
IPP, isopentenyl pyrophosphate
MEP, 2-C-methyl-D-erythritol-4 phosphate
TEAB, triethylammonium bicarbonate
TLR, Toll-like receptor

   Notes
 
Transmitting editor: W. Yokoyama

Received 21 December 2006, accepted 23 February 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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