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Small phosphorylated metabolites from mycobacteria stimulate human γδ T lymphocytes. Although such phosphoantigens could prove useful in the composition of vaccines involving γδ T cell-mediated immunity, their very low abundance in natural sources limits such applications. Here, we describe the chemical production, purification, and bioactivity of a phosphorylated bromohydrin (BrHPP) analogue that mimics the biological properties of natural phosphoantigens. This compound can be obtained in gram amounts, is easy to detect, and is of high stability in aqueous solutions. Whereas unspecific binding of BrHPP to a wide panel of cell surface receptors is not detected even at micromolar concentrations, nanomolar concentrations specifically trigger effector responses of human γδ T lymphocytes. Thus, BrHPP is a novel molecule enabling potent immunostimulation of human γδ T lymphocytes. Small phosphorylated metabolites from mycobacteria stimulate human γδ T lymphocytes. Although such phosphoantigens could prove useful in the composition of vaccines involving γδ T cell-mediated immunity, their very low abundance in natural sources limits such applications. Here, we describe the chemical production, purification, and bioactivity of a phosphorylated bromohydrin (BrHPP) analogue that mimics the biological properties of natural phosphoantigens. This compound can be obtained in gram amounts, is easy to detect, and is of high stability in aqueous solutions. Whereas unspecific binding of BrHPP to a wide panel of cell surface receptors is not detected even at micromolar concentrations, nanomolar concentrations specifically trigger effector responses of human γδ T lymphocytes. Thus, BrHPP is a novel molecule enabling potent immunostimulation of human γδ T lymphocytes. T cell receptor bromohydrin pyrophosphate electrospray ionization mass spectrometry high pressure anion exchange chromatography peripheral blood lymphocytes tumor necrosis factor-α 3-formyl-1-butyl-pyrophosphate interleukin Stimulating ligands for αβ T lymphocytes are usually composed of single peptides complexed at the surface of major histocompatibility complex molecules. Some small non-peptidic structures, however, may also constitute specific agonist ligands for T cells, particularly γδ T lymphocytes. In human blood, about 3% of T cells initiate their physiological function upon recognition of small phosphorylated non-peptide antigens (phosphoantigens). This cognate interaction involves on the one hand phosphoantigens in the absence of major histocompatibility complex-presenting molecules, and on the other hand, highly selective receptors (TCR)1 of γδ subtype. In nature, phosphoantigens that can activate human γδ T cells at nanomolar concentrations are produced by Gram-positive and Gram-negative bacteria and also by some eukaryotic parasites and plants. Synthetic analogues of natural phosphoantigens are also known, but their stimulating concentrations for the reactive cells never go below the micromolar range. Mycobacterium tuberculosis, the agent of human tuberculosis, produces four distinct phosphoantigens. These molecules share a moiety that is responsible for the potent stimulation of γδ cells seen in tuberculosis patients (1Constant P. Davodeau F. Peyrat M.A. Poquet Y. Puzo G. Bonneville M. Fournie J.J. Science. 1994; 264: 267-270Crossref PubMed Scopus (629) Google Scholar). The structure of this common core is 3-formyl-1-butyl-pyrophosphate, a recently described phosphoester (2Belmant C. Espinosa E. Poupot R. Peyrat M.-A. Guiraud M. Poquet Y. Bonneville M. Fournié J.-J. J. Biol. Chem. 1999; 274: 32079-32084Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Its metabolic production might be related to the non-mevalonate (or so-called Rohmer's) pathway for isoprenoid precursor biosynthesis (3Sicard H. Fournié J.J. Infect. Immun. 2000; 68: 4375-4377Crossref PubMed Scopus (34) Google Scholar). 3-formyl-1-butyl-pyrophosphate is produced in very small amounts in slow-growing mycobacteria such asMycobacterium tuberculosis and only accumulates to submicromolar concentrations in culture media from fast-growing mycobacterial species (4Poquet Y. Constant P. Halary F. Peyrat M.A. Gilleron M. Davodeau F. Bonneville M. Fournie J.J. Eur. J. Immunol. 1996; 26: 2344-2349Crossref PubMed Scopus (43) Google Scholar). Getting large amounts of highly bioactive phosphoantigens by purification routes from such natural sources is therefore hard to conceive.Such molecules could prove therapeutically useful for immunotherapeutic approaches involving γδ T cell-mediated immunity, such as elicitation of anti-infectious protection or antitumor immunity (5Poccia F. Gougeon M.L. Bonneville M. Lopez-Botet M. Moretta A. Battistini L. Wallace M. Colizzi V. Malkovsky M. Immunol. Today. 1998; 19: 253-256Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 6Fisch P. Moris A. Rammensee H.G. Handgretinger R. Immunol. Today. 2000; 21: 187-191Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). To address the need for readily available highly bioactive phosphoantigens, we have developed a synthetic reagent called bromohydrin pyrophosphate (BrHPP), whose biological properties on human T cells are optimized compared with those of 3-formyl-1-butyl-pyrophosphate.DISCUSSIONThis paper details the structure, chemical synthesis, and biological property of bromohydrin pyrophosphate, a novel molecule activating human γδ T lymphocytes. Based on the molecular overlay of this compound with the natural γδ T cell ligand found in mycobacteria, it was hoped that this synthetic compound could mimic the biological properties of the naturally occurring 3fbPP,i.e. activation of a specific γδ T cell subset in human blood (see Ref. 19Hayday A.C. Annu. Rev. Immunol. 2000; 18: 975-1026Crossref PubMed Scopus (950) Google Scholar for a review). We describe a convenient and straightforward mode of synthesis for producing BrHPP. This simple method is based on pyrophosphorylation of the tosylated C5 precursor, followed by stoichiometric oxidation of the pyrophosphoester product in aqueous bromine. Because the compound carries a chiral C3, the resulting product is a racemic mixture used without further resolution of enantiomers. This straightforward and inexpensive synthesis is followed by a purification scheme involving solvent precipitation, LC, and HPLC to eliminate residual inorganic phosphate and bromide. The final stock solutions of BrHPP (Na+) salts are very stable and can be stored for several months without degradation. Little information about structural changes of the organic moiety of phosphorylated metabolites is usually drawn from HPLC-MS in negative mode, thereby limiting its use as an analytical tool. The mass spectral data of BrHPP presented here demonstrate a highly sensitive detection of BrHPP in aqueous phases, and its bromine content enables unambiguous detection for pharmacological follow-up studies. For the reasons listed above, BrHPP appears to be a promising lead candidate for therapeutic explorations among synthetic phosphoantigens.In vitro cultures of bulk human lymphocytes carried out in the presence of 100 nm BrHPP and IL2 lead to the systematic expansion of T lymphocytes, which express the phosphoantigen-reactive γ9δ2 TCR, and no other cell subset. This has been described previously for total lymphocyte populations stimulated by crude extracts from M. tuberculosis and is known to rely upon the presence of several stimulating phosphoantigens. Here, BrHPP also acts as a phosphoantigen agonist, because BrHPP dephosphorylation abolishes this bioactivity. Exposure to BrHPP also elicits TNF-α and interferon-γ release, indicating that the full range of γδ T cell effector response is activated by this ligand. Microphysiometric analysis showed that this activation results from exposure to a typical activating agonist (20Rabinowitz J.D. Beeson C. Wulfing C. Tate K. Allen P.M. Davis M.M. McConnell H.M. Immunity. 1996; 5: 125-135Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Its early signal transduction involves a BrHPP dose-dependent extracellular acid release, as was shown for αβ T cells stimulated with peptide-major histocompatibility complex tetramers (21Boniface J.J. Rabinowitz J.D. Wulfing C. Hampl J. Reich Z. Altman J.D. Kantor R.M. Beeson C. McConnell H.M. Davis M.M. Immunity. 1998; 9: 459-466Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Whereas high bioactive doses of BrHPP triggered a strong acidification burst followed by a sustained intracellular signaling, suboptimal BrHPP concentrations (5 nm) led to barely detectable signaling. When rapidly exposed to saturating BrHPP concentrations, γδ cells respond by extracellular acidification within about 10 s, indicating that little (if any) intermediate processing of the stimulating BrHPP occurs prior to triggering the T cell reaction.In summary, although synthetic BrHPP presents the same biological properties as natural phosphoantigens, the possibility of synthesizing gram amounts from simple procedures bypasses the production drawbacks of the natural counterparts. This makes BrHPP an attractive candidate for investigations of selective γδ T cell-based immunomodulation approaches. Future studies will evaluate the potential of this novel immunostimulating molecule in subunit vaccines where γδ T cell contribution in vivo is expected to be beneficial, such as antituberculous immunity and protection against acute leukemia.APPENDIXReceptor/ligand bindings that were tested and found to be unaffected by 10 μm BrHPP are listed in Table I. The binding assays were performed as follows. For each receptor binding assay, the respective reference ligand was tested in duplicate at a minimum of eight concentrations to obtain a competition curve to validate the titration. In parallel, 10 μm BrHPP was tested in duplicate in each assay. Following incubation, the membranes or cells in suspension were rapidly harvested onto glass fiber filters with an ice-cold buffer using a cell harvester. Bound radioactivity was then measured with a scintillation counter (Betaplate, Wallac). The specific radioligand binding to the receptors is defined as the difference between total binding and nonspecific binding determined in the presence of an excess of unlabelled ligand. Results are expressed as percent inhibition of control specific binding obtained in the presence of BrHPP. Conc. represents the concentration causing a half-maximal inhibition of control specific binding.Table IReceptor-ligands unaffected by BrHPP Stimulating ligands for αβ T lymphocytes are usually composed of single peptides complexed at the surface of major histocompatibility complex molecules. Some small non-peptidic structures, however, may also constitute specific agonist ligands for T cells, particularly γδ T lymphocytes. In human blood, about 3% of T cells initiate their physiological function upon recognition of small phosphorylated non-peptide antigens (phosphoantigens). This cognate interaction involves on the one hand phosphoantigens in the absence of major histocompatibility complex-presenting molecules, and on the other hand, highly selective receptors (TCR)1 of γδ subtype. In nature, phosphoantigens that can activate human γδ T cells at nanomolar concentrations are produced by Gram-positive and Gram-negative bacteria and also by some eukaryotic parasites and plants. Synthetic analogues of natural phosphoantigens are also known, but their stimulating concentrations for the reactive cells never go below the micromolar range. Mycobacterium tuberculosis, the agent of human tuberculosis, produces four distinct phosphoantigens. These molecules share a moiety that is responsible for the potent stimulation of γδ cells seen in tuberculosis patients (1Constant P. Davodeau F. Peyrat M.A. Poquet Y. Puzo G. Bonneville M. Fournie J.J. Science. 1994; 264: 267-270Crossref PubMed Scopus (629) Google Scholar). The structure of this common core is 3-formyl-1-butyl-pyrophosphate, a recently described phosphoester (2Belmant C. Espinosa E. Poupot R. Peyrat M.-A. Guiraud M. Poquet Y. Bonneville M. Fournié J.-J. J. Biol. Chem. 1999; 274: 32079-32084Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Its metabolic production might be related to the non-mevalonate (or so-called Rohmer's) pathway for isoprenoid precursor biosynthesis (3Sicard H. Fournié J.J. Infect. Immun. 2000; 68: 4375-4377Crossref PubMed Scopus (34) Google Scholar). 3-formyl-1-butyl-pyrophosphate is produced in very small amounts in slow-growing mycobacteria such asMycobacterium tuberculosis and only accumulates to submicromolar concentrations in culture media from fast-growing mycobacterial species (4Poquet Y. Constant P. Halary F. Peyrat M.A. Gilleron M. Davodeau F. Bonneville M. Fournie J.J. Eur. J. Immunol. 1996; 26: 2344-2349Crossref PubMed Scopus (43) Google Scholar). Getting large amounts of highly bioactive phosphoantigens by purification routes from such natural sources is therefore hard to conceive. Such molecules could prove therapeutically useful for immunotherapeutic approaches involving γδ T cell-mediated immunity, such as elicitation of anti-infectious protection or antitumor immunity (5Poccia F. Gougeon M.L. Bonneville M. Lopez-Botet M. Moretta A. Battistini L. Wallace M. Colizzi V. Malkovsky M. Immunol. Today. 1998; 19: 253-256Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 6Fisch P. Moris A. Rammensee H.G. Handgretinger R. Immunol. Today. 2000; 21: 187-191Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). To address the need for readily available highly bioactive phosphoantigens, we have developed a synthetic reagent called bromohydrin pyrophosphate (BrHPP), whose biological properties on human T cells are optimized compared with those of 3-formyl-1-butyl-pyrophosphate. DISCUSSIONThis paper details the structure, chemical synthesis, and biological property of bromohydrin pyrophosphate, a novel molecule activating human γδ T lymphocytes. Based on the molecular overlay of this compound with the natural γδ T cell ligand found in mycobacteria, it was hoped that this synthetic compound could mimic the biological properties of the naturally occurring 3fbPP,i.e. activation of a specific γδ T cell subset in human blood (see Ref. 19Hayday A.C. Annu. Rev. Immunol. 2000; 18: 975-1026Crossref PubMed Scopus (950) Google Scholar for a review). We describe a convenient and straightforward mode of synthesis for producing BrHPP. This simple method is based on pyrophosphorylation of the tosylated C5 precursor, followed by stoichiometric oxidation of the pyrophosphoester product in aqueous bromine. Because the compound carries a chiral C3, the resulting product is a racemic mixture used without further resolution of enantiomers. This straightforward and inexpensive synthesis is followed by a purification scheme involving solvent precipitation, LC, and HPLC to eliminate residual inorganic phosphate and bromide. The final stock solutions of BrHPP (Na+) salts are very stable and can be stored for several months without degradation. Little information about structural changes of the organic moiety of phosphorylated metabolites is usually drawn from HPLC-MS in negative mode, thereby limiting its use as an analytical tool. The mass spectral data of BrHPP presented here demonstrate a highly sensitive detection of BrHPP in aqueous phases, and its bromine content enables unambiguous detection for pharmacological follow-up studies. For the reasons listed above, BrHPP appears to be a promising lead candidate for therapeutic explorations among synthetic phosphoantigens.In vitro cultures of bulk human lymphocytes carried out in the presence of 100 nm BrHPP and IL2 lead to the systematic expansion of T lymphocytes, which express the phosphoantigen-reactive γ9δ2 TCR, and no other cell subset. This has been described previously for total lymphocyte populations stimulated by crude extracts from M. tuberculosis and is known to rely upon the presence of several stimulating phosphoantigens. Here, BrHPP also acts as a phosphoantigen agonist, because BrHPP dephosphorylation abolishes this bioactivity. Exposure to BrHPP also elicits TNF-α and interferon-γ release, indicating that the full range of γδ T cell effector response is activated by this ligand. Microphysiometric analysis showed that this activation results from exposure to a typical activating agonist (20Rabinowitz J.D. Beeson C. Wulfing C. Tate K. Allen P.M. Davis M.M. McConnell H.M. Immunity. 1996; 5: 125-135Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Its early signal transduction involves a BrHPP dose-dependent extracellular acid release, as was shown for αβ T cells stimulated with peptide-major histocompatibility complex tetramers (21Boniface J.J. Rabinowitz J.D. Wulfing C. Hampl J. Reich Z. Altman J.D. Kantor R.M. Beeson C. McConnell H.M. Davis M.M. Immunity. 1998; 9: 459-466Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Whereas high bioactive doses of BrHPP triggered a strong acidification burst followed by a sustained intracellular signaling, suboptimal BrHPP concentrations (5 nm) led to barely detectable signaling. When rapidly exposed to saturating BrHPP concentrations, γδ cells respond by extracellular acidification within about 10 s, indicating that little (if any) intermediate processing of the stimulating BrHPP occurs prior to triggering the T cell reaction.In summary, although synthetic BrHPP presents the same biological properties as natural phosphoantigens, the possibility of synthesizing gram amounts from simple procedures bypasses the production drawbacks of the natural counterparts. This makes BrHPP an attractive candidate for investigations of selective γδ T cell-based immunomodulation approaches. Future studies will evaluate the potential of this novel immunostimulating molecule in subunit vaccines where γδ T cell contribution in vivo is expected to be beneficial, such as antituberculous immunity and protection against acute leukemia. This paper details the structure, chemical synthesis, and biological property of bromohydrin pyrophosphate, a novel molecule activating human γδ T lymphocytes. Based on the molecular overlay of this compound with the natural γδ T cell ligand found in mycobacteria, it was hoped that this synthetic compound could mimic the biological properties of the naturally occurring 3fbPP,i.e. activation of a specific γδ T cell subset in human blood (see Ref. 19Hayday A.C. Annu. Rev. Immunol. 2000; 18: 975-1026Crossref PubMed Scopus (950) Google Scholar for a review). We describe a convenient and straightforward mode of synthesis for producing BrHPP. This simple method is based on pyrophosphorylation of the tosylated C5 precursor, followed by stoichiometric oxidation of the pyrophosphoester product in aqueous bromine. Because the compound carries a chiral C3, the resulting product is a racemic mixture used without further resolution of enantiomers. This straightforward and inexpensive synthesis is followed by a purification scheme involving solvent precipitation, LC, and HPLC to eliminate residual inorganic phosphate and bromide. The final stock solutions of BrHPP (Na+) salts are very stable and can be stored for several months without degradation. Little information about structural changes of the organic moiety of phosphorylated metabolites is usually drawn from HPLC-MS in negative mode, thereby limiting its use as an analytical tool. The mass spectral data of BrHPP presented here demonstrate a highly sensitive detection of BrHPP in aqueous phases, and its bromine content enables unambiguous detection for pharmacological follow-up studies. For the reasons listed above, BrHPP appears to be a promising lead candidate for therapeutic explorations among synthetic phosphoantigens. In vitro cultures of bulk human lymphocytes carried out in the presence of 100 nm BrHPP and IL2 lead to the systematic expansion of T lymphocytes, which express the phosphoantigen-reactive γ9δ2 TCR, and no other cell subset. This has been described previously for total lymphocyte populations stimulated by crude extracts from M. tuberculosis and is known to rely upon the presence of several stimulating phosphoantigens. Here, BrHPP also acts as a phosphoantigen agonist, because BrHPP dephosphorylation abolishes this bioactivity. Exposure to BrHPP also elicits TNF-α and interferon-γ release, indicating that the full range of γδ T cell effector response is activated by this ligand. Microphysiometric analysis showed that this activation results from exposure to a typical activating agonist (20Rabinowitz J.D. Beeson C. Wulfing C. Tate K. Allen P.M. Davis M.M. McConnell H.M. Immunity. 1996; 5: 125-135Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Its early signal transduction involves a BrHPP dose-dependent extracellular acid release, as was shown for αβ T cells stimulated with peptide-major histocompatibility complex tetramers (21Boniface J.J. Rabinowitz J.D. Wulfing C. Hampl J. Reich Z. Altman J.D. Kantor R.M. Beeson C. McConnell H.M. Davis M.M. Immunity. 1998; 9: 459-466Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Whereas high bioactive doses of BrHPP triggered a strong acidification burst followed by a sustained intracellular signaling, suboptimal BrHPP concentrations (5 nm) led to barely detectable signaling. When rapidly exposed to saturating BrHPP concentrations, γδ cells respond by extracellular acidification within about 10 s, indicating that little (if any) intermediate processing of the stimulating BrHPP occurs prior to triggering the T cell reaction. In summary, although synthetic BrHPP presents the same biological properties as natural phosphoantigens, the possibility of synthesizing gram amounts from simple procedures bypasses the production drawbacks of the natural counterparts. This makes BrHPP an attractive candidate for investigations of selective γδ T cell-based immunomodulation approaches. Future studies will evaluate the potential of this novel immunostimulating molecule in subunit vaccines where γδ T cell contribution in vivo is expected to be beneficial, such as antituberculous immunity and protection against acute leukemia. APPENDIXReceptor/ligand bindings that were tested and found to be unaffected by 10 μm BrHPP are listed in Table I. The binding assays were performed as follows. For each receptor binding assay, the respective reference ligand was tested in duplicate at a minimum of eight concentrations to obtain a competition curve to validate the titration. In parallel, 10 μm BrHPP was tested in duplicate in each assay. Following incubation, the membranes or cells in suspension were rapidly harvested onto glass fiber filters with an ice-cold buffer using a cell harvester. Bound radioactivity was then measured with a scintillation counter (Betaplate, Wallac). The specific radioligand binding to the receptors is defined as the difference between total binding and nonspecific binding determined in the presence of an excess of unlabelled ligand. Results are expressed as percent inhibition of control specific binding obtained in the presence of BrHPP. Conc. represents the concentration causing a half-maximal inhibition of control specific binding.Table IReceptor-ligands unaffected by BrHPP Receptor/ligand bindings that were tested and found to be unaffected by 10 μm BrHPP are listed in Table I. The binding assays were performed as follows. For each receptor binding assay, the respective reference ligand was tested in duplicate at a minimum of eight concentrations to obtain a competition curve to validate the titration. In parallel, 10 μm BrHPP was tested in duplicate in each assay. Following incubation, the membranes or cells in suspension were rapidly harvested onto glass fiber filters with an ice-cold buffer using a cell harvester. Bound radioactivity was then measured with a scintillation counter (Betaplate, Wallac). The specific radioligand binding to the receptors is defined as the difference between total binding and nonspecific binding determined in the presence of an excess of unlabelled ligand. Results are expressed as percent inhibition of control specific binding obtained in the presence of BrHPP. Conc. represents the concentration causing a half-maximal inhibition of control specific binding. We thank Sanofi-Synthélabo (Labège) for kind gifts of rIL2 and G. Cassar (IFR Claude de Préval) for advice with cytometry assays. We acknowledge A. T. N. Joly for critical reading of this manuscript.
Espinosa et al. (Tue,) studied this question.