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Vδ2 T lymphocytes
Immunology Letters 95 (2004) 129–138
Review
Non-peptide antigens activating human V␥9/V␦2 T lymphocytes Mary Poupot, Jean-Jacques Fourni´e∗ d´epartement Oncog´en`ese and Signalisation dans les Cellules H´ematopoi´etiques, Unit´e 563 de l’Institut National de la Sant´e Et de la Recherche M´edicale, Centre de Physiopathologie de Toulouse Purpan, BP3028, 31024 Toulouse, France Received 10 May 2004; received in revised form 25 June 2004; accepted 29 June 2004 Available online 21 July 2004
Abstract Various non-peptidic ligands which specifically activate most of circulating human V␥9/V␦2 T lymphocytes are now known. Most of these are so-called phosphoantigens and directly trigger the V␥9/V␦2 TCR expressing cells, without need for MHC-restricted presentation molecules. Although some potent phosphoantigens currently involved in clinical trials are chemically-synthesized molecules, most of the natural antigens were isolated from microbial cultures. The structures and biosynthesis of phosphoantigens are reviewed here and the possible physiological significance of their recognition by ␥␦ T lymphocytes is discussed. © 2004 Elsevier B.V. All rights reserved. Keywords: Non-peptide antigens; V␥9/V␦2 T lymphocytes; ␥␦T cell
1. Retrospective The biochemical structures of non-peptide antigens which specifically activate circulating human V␥9/V␦2 T lymphocytes provide clues for the possible driving force and significance of this specific subset. Since their first description in the early 90’s, the main human ␥␦ T cell subset in circulating blood has been associated with reactivity to tuberculosis and to B cell tumors [1]. Although initially attributed to recognition of hsp65-derived peptides, it soon became clear that this protein did not account for most of the V␥9/V␦2 T cell specificity [2]. Instead pioneering studies clearly indicated that this T cell subpopulation was reactive to small non-peptide antigens [3,4]. These were characterized in Mycobacterium tuberculosis and related species as a group of four structurally related phosphoesters (so-called TUBag [1–4]), comprising both thymidine and uridine nucleotide-conjugates as well as their non-nucleotide distal pyrophosphoester moieties [5–7]. Parallel studies in Mycobacterium smegmatis identified phosphate-containing antigens as isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophos∗ Corresponding author. Tel.: +335 6274 8364; fax: +335 6274 8386. E-mail address: [email protected] (J.-J. Fourni´e).
0165-2478/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2004.06.013
phate (DMAPP) [8,9]. Although it is now clear that all these findings were correct, at that time, though these apparently conflicting results did not account for the ␥␦ cell reactivity to human tumor cells, which encompassed B cell lymphoma, multiple myeloma, cutaneous, breast and renal carcinoma. Soon, it became obvious though puzzling that actually, a broad set of natural and synthetic phosphorylated compounds is able to selectively trigger the V␥9/V␦2 T cell population, and the phosphate group is their only shared determinant of bioactivity [8–10]; this criterion thus defining phosphoantigens [11]. Novel natural phosphoantigens have been detected in a wide spectrum of microorganisms and in plant extracts [12]. Thus far, the most potent natural phosphoantigen (E)4-hydroxy-3-methylbut-2-enyl-pyrophosphate (HDMAPP) was characterized in Escherichia coli (E. coli) [13,14] but also in plants [15]. This compound is a precursor of a microbial non-mevalonate pathway to IPP and further to isoprenoids [16]. In parallel, non-peptide and non-phosphoantigens molecules that also stimulate in a cross-reactive fashion the same V␥9/V␦2 T lymphocytes were reported. They comprise synthetic and natural alkylamines on the one hand [17] and therapeutic aminobisphosphonates like pamidronate [18,19] on the other. More recently, the V␥9/V␦2-selective antigens from human tumor cells were characterized as endogenous
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metabolites of the mevalonate pathway and most likely as IPP [20]. Since therapeutic aminobisphosphonates inhibit the mevalonate pathway in eukaryote cells [21,22], it appears that their ability to activate human ␥␦ T cells reflects their pharmacological action on the tumoral cell target [20,23]. Altogether, these features now converge into a scenario where the human V␥9/V␦2 T lymphocytes actually scan their targets for the biosynthesis of isoprenoids. This is reviewed below together with its possible physiological significance.
2. The isoprenoid metabolisms All living organisms produce isoprenoids, essential metabolites of their cellular and intercellular biology. Among these, sterols are found from the oldest sediments (hopanoids) to the entire current living world, encompassing prokaryotes to both plant and animal reigns. Rich of about 30,000 different molecules today, isoprenoids constitute the most extraordinarily diverse structural family, comprising, for example, ubiquinones, sterols, terpenes, carotenoids, gibberellins and taxoids. Strikingly enough, the biosynthesis responsible for such a huge structural diversity implies in all living cells the same bottleneck: the repeated condensation of two branched C5 precursors; the isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP). Despite this apparent unity however, an elder divergence has occurred in the biogenesis of these two precursors. Archaebacteria, few bacteria and most eukaryotes, synthesize IPP from acetyl-CoA through the mevalonate pathway (MVA). The MVA pathway involves the initial oxidation of pyruvate into acetyl-CoA by a multimolecular complex initially discovered in mitochondria, the pyruvate deshydrogenase (for review, see [24]). Three acetyl-CoA molecules are condensed into 3-hydroxy-3-methyl-glutarylCoA, which is reduced to mevalonate by the key enzyme HMG-CoA reductase. Then are ATP-dependent steps catalyzed by structurally related enzymes [25], wherein three successive phosphorylations of mevalonate are followed by a decarboxylation step producing IPP. Finally, IPP isomerase produces its DMAPP isomer, and these are C5 precursosrs for downstream metabolites such as sterols. Important pharmacological tools to dissect the MVA pathway are statins and related hypocholesterolemic agents inhibiting HMG-CoA reductase [26], isoprenols such as farnesol and 7-dehydrocholesterol which facilitate its degradation [27] and aminobisphosphonates which inhibit both IPP isomerase and downstream farnesyl PP synthase [21] (Fig. 1). On the other hand, in cyanobacteria, algae, chloroplasts and most eubacteria, IPP is produced by another route involving the 1-deoxy-d-xylulose-5-phosphate (DOXP) [28,29]. The DOXP pathway starts from pyruvate and d-glyceraldehyde-3-phosphate substrates [30] by 1-deoxy-d-xylulose-5-phosphate synthase making 1-deoxyd-xylulose-5-phosphate (DOXP). DOXP is reduced into 2-C-methyl-d-erythritol-4-phosphate [31], transferred into
a nucleotidyl conjugate CDP-methyl-erythritol [32] and phosphorylated by CME-kinase [33] to give CDP-methylerythritol-2-phosphate [34]. This activated precursor is then enzymatically cleaved [35] and releases 2-C-methylerythritol-2,4-cyclodiphosphate [36]. This cyclic intermediate is then successively reduced and submitted to elimination, by at least two metallo-enzymes [37,38] requiring oxygen-free conditions: HDMAPP synthase which produces 4-hydroxy-dimethylallyl diphosphate (HDMAPP) [39] and HDMAPP reductase which provides a 5:1 mixture of IPP and DMAPP [40,41]. Interesting pharmacological tools to investigate the DOXP pathway are fosmidomycin and its related FR900098 prodrug [42,43], benzyl viologen and other oxidants [44]. Downstream to these two ubiquitous C5 precursors in all living cells is a common step of elongation by sequential additions that provides C10, C15 and C20 linear allylic alcohols, respectively geraniol, farnesol and geranylgeraniol. Next to these, are substrates for hundreds of cyclases in numerous paths leading to the generation of a huge diversity of natural isoprenoids [45]. Interestingly enough, most known isoprenoid cyclases and farnesyl PP synthase have co-evolved a similar so-called “isoprenoid synthase fold” structural motif, suggesting strong co-evolutionary relationships [46] (Fig. 1).
3. Distribution of the MVA and DOXP pathways in nature Although biochemical data have documented the presence of DOXP pathways in a limited number of microorganisms (see below), the survey of genomic databases has been of great interest to delineate the presence of either MVA or DOXP pathways in nature. Both biosynthesis routes are highly intricated nevertheless, clearly distinct pictures may be drawn for prokaryotes, eukaryotes and in plants. All non-photosynthetic eukaryotes including protozoa (Leishmania and Trypanosoma spp.), most fungi, yeast and animals use only the MVA pathway to make IPP (reviewed in [47]). This pathway has a complex origin, as shown by the phylogeny of its key enzyme HMG-CoA reductase (HMGR). HMGR is split into class 1 isoforms acquired by archaeal and eukaryotic cells and class 2 bacterial HMGR which evolved separately. Since mammals make their sterols only through the MVA pathway, it is the rationale target for the lipidlowering statin class of drugs [48]. Of note, Class 1 HMGR are preferentially inhibited by natural statins. Occasionally, they are found in few eubacteria like Vibrio cholerae, but these species most presumably acquired its gene from archaea by some later lateral gene transfer [49]. In plants and yeast, regulation of the MVA pathway at the HMGR level is well documented. In response to attack by pathogens or wounds, reduction of HMGR involves both sterol-sensor-regulated MAPK cascades which drive selected gene transcriptions and its controlled ubiquitination/degradation [50,51]. High levels
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Fig. 1. DOXP and MVA pathways to isoprenoid biosynthesis. Three examples of final products from isoprenoid biosynthesis are shown. For clarity, names of enzymes have been omitted. Inhibitors are indicated in blue boxes.
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of FPP, of sterols or of phenylalanine inhibit their HMGR activity. In mammalin cells, the HMGR activity is inhibited by statins, by phenylalanine [52] or by a feedback inhibition with aminobisphosphonate-induced FPP accumulation [53]. Thus, statins may reverse by upstream blockade the aminobisphosphonate-induced ␥␦ T cell activation by endogenous phosphoantigens, but not by the synthetic exogenous BrHPP phosphoantigen agonist. Inherited defects in the MVA pathway such as mevalonate kinase deficiency do exist in humans were they are associated with mevalonic-aciduria, hyper-IgD and periodic fever syndromes (HIDS) [54]. The HMGR activity, and thus, the whole MVA pathway, were found increased in cancer cell lines such as leukaemia, nonHodgkin lymphoma [55], mammary and lung adenocarcinoma [56,57], in which HMGR activity is controlled by EGFPI3 mediated signaling [58]. Algae and higher plants cumulate the MVA pathway in the cytosol and in mitochondria, together with a functional DOXP pathway in their chloroplasts but its genes remain encoded in the cell nucleus (reviewed in [28,59,60]). Although these two routes usually lead to different isoprenoid end-products in higher plants: e.g. sterols in the cytoplasm and carotenoids in chloroplasts, under stress conditions, plastidial isoprenoids formed by the DOXP route may cross-talk with the cytosolic MVA pathway for functional complementation [61,62]. At the extreme end of this spectrum, some unicellular green algae and few parasitic eukaryotes like Plasmodium falciparum are exceptions which have not the MVA pathway and solely rely upon their DOXP counterpart. Of note, the plasmodial DOXP pathway is actually located in an intracellular plastid-like compartment called apicoplast [43]. Which of the DOXP or MVA pathway has arisen first along evolution remains unknown, since DOXP only exists in bacteria and plastids where it provides most primary isoprenoids instead of the MVA used by archae [49] (Fig. 2). Nevertheless, the MVA pathway remains more sparsely distributed among bacteria than the prevalent DOXP, since the HMGR and downstream MVA genes are patchily distributed in this group. These comprise Aquificales, Thermotogales, Chlamidiae, Cyanobacteria, Porphyrobacteria, Deinococci, Gram + such as Actinomycetes Streptomyces, Clostridiae, Mycobacteria, Corynebacteria, Nocardiae, Actinoplanae, Bacilli, Spirochaetes and most Proteobacteria of the subgroups ␣ (Rhodopseudomonas, Zymomonas, . . .),  (Bordetella, Neisseiria, Ralstonia, . . .), ␥ (V. cholerae, E. coli, Pseudomonas, Yiersinia, Salmonella, . . .) and (Helicobacter, Campylobacter, . . .) [49,63]. Whereas Streptomyces frequently use the DOXP pathway not only for producing menaquinones but also antibiotics, Actinoplanes switch from using DOXP in exponential growth phase to MVA for making antibiotics in later phases [64]. Therefore, some bacterial species have kept both pathways but for different roles: DOXP for primary metabolism and MVA for secondary metabolites [49]. Of note, Mycobacterium tuberculosis and
E. coli – where only the DOXP pathway is present – are the two species where the complete structures of natural phosphoantigens have been fully established [13,65].
4. Structures of phosphoantigens and of related V␥9/V␦2 T cell-specific stimuli Early studies had shown at a clonal level that all V␥9/V␦2 TCR-expressing ␥␦ T cells reactive to mycobacteria also recognized the Daudi lymphoma [66,67], which indicated that these cell types had similar antigens. Further work delineated the receptor for phosphoantigens as the V␥9/V␦2 TCR, and more specifically to its CDR3 regions [68–70]. The crystal structure of this TCR also supports its adaptation to interact with phosphoantigens [71] by its surface exposed lysine residues [70]. The earliest identication of mycobacterial antigens for ␥␦ T cells was incomplete, in describing four distinct but structurally related molecules [TUBag1-4) where TUBag3 and TUBag4 are respectively, UTP and TTP conjugates of a terminal unidentified alkyl moiety found in the two pyrophosphorylated TUBag 1 and TUBag2 [5,7,72,73]. Although parallel reports suggested that, this moiety was isopentenol since Mycobacterium smegmatis-derived IPP or its DMAPP isomer were identified as natural ligands for V␥9/V␦2 T lymphocytes [9], these ubiquitous metabolites hardly accounted for their selective activation in some infectious diseases only (such as tuberculosis and malaria for instance), and were produced in insufficient amounts to reach the IPP bioactive threshold (1 M) on ␥␦ T cells [74,75]. A link between production of phosphoantigens and cell activation/proliferation [11] including IPP biosynthesis [76,77] could resolve this controversial issue. This link was definitively established by the good correlate between DOXP pathway and presence of phosphoantigens among several bacterial species [75] and in P. falciparum [43,78]. Several reports identified a 262 Da phosphoantigen (TUBag1) in distinct highly immunogenic extracts from mycobacteria and from E. coli [79,80]. Using ∼30 g of the highly purified terminal X-PP moiety from the mycobacterial phosphoantigen TUBag3 (UTP-X structure), the TUBag1 antigen (X-PP MW 262) was characterized biochemically as FBPP. More recently though, an Escherichia coli (E.coli) mutated in the last step of the DOXP pathway (ispH syn.to lytB [81]) was found to accumulate 150-times more phosphoantigen than wild-type strains [14]. Based on mg amounts of purified molecule, the structural elucidation of this phosphoantigen unambiguiously defined the XPP structure of MW 262 as 4-hydroxy-dimethylallylPP (HDMAPP) rather than FBPP [13]. HDMAPP was synthesized chemically [82–84] and also produced by bioconversion from MEcPP [38]. Its bioactivity, the specific induction of V␥9/V␦2 T cells at sub-nanomolar HDMAPP concentrations [13,14] confirmed the structural assignment of the natural phosphoantigen HDMAPP. The definitive structure for TUBag2 (MW 276) now remains to be established, to-
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Fig. 2. Distribution of DOXP and MVA pathways in nature. Living organisms using the DOXP and/or MVA pathways for their isoprenoid biosynthesis are shown by the specified boxes on a simplified tree of life. Dotted lines: presence of an incomplete pathway (few genes only are present); smaller inside boxes: some species only; no box: information lacking. Adapted from [45,47,49,113,114].
gether with its link to the DOXP pathway. It also remains to be precisely determined whether 4-hydroxy-dimethylallyl or rather 3-formyl-butyl is actually involved the terminal ester moiety of both mycobacterial nucleotidyl conjugates of X-UTP (TUBag3) and X-TTP (TUBag4). Indeed, both structures may coexist as free esters in most prokaryotes, but the two nucleotide phosphoantigens are not produced by Gram-negative bacteria [77]. Nevertheless, phosphoantigens are also produced by plants such as the Viscum album parasite [12,85].
The V␥9/V␦2 T cell reactivity to IPP has thus, provided an extremely useful tool for investigating these T cell responses using a well-defined and readily available ligand. Although its spontaneous production may be limited in most prokaryote and normal eukaryote cells, it is detected in significant amounts in various types of human cancer cells. Both metabolic blockade and accumulation of biosynthesized IPP determine the phosphoantigen content of cancer cell lines such as Daudi [20]. Therefore rather than a microbial phosphoantigen, IPP now appears as the major eu-
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karyote phosphoantigens. Two tumor cell-derived phosphoantigen are present in lysates of cultured human cancer cell lines (our unpublished observations). Presumably after IPP, DMAPP could represent this second antigen since this IPP isomer has also M bioactivity on human ␥␦ T cells [7,9]. In this regard, IPP and DMAPP clearly confirmed the previously proposed ␥␦ T cell involvement in scanning of eukaryotic farnesylation [9], known to be of the highest importance in Ras-mediated oncogenesis [9,86]. Several synthetic phosphoantigens have been produced in order to enable the rationale development of chemically defined phosphoantigens in large scale amounts compatible with their use in human clinical trials. Initially, these comprised various linear alkyl phosphates most notably like ethyl-pyrophosphate [8], and were followed by more potent agonists like EpoxPP or phosphohalohydrins such as BrHPP [87]. QSAR studies of phosphoantigens have demonstrated the prominent role of their pyrophosphate moieties [8,88], which must enable the hydrolytic cleavage of their pyrophosphate bond [89]. Antagonists of phosphoantigens have thus been defined on the basis of phosphoantigen structures with replacement of the pyrophosphate bond by stable analogues such as methylene bisphosphonates, iminobisphosphonates and mono- or di-fluoromethylene bisphosphonate groups [89]. Common pharmacophores of phosphoantigens have been defined by two computational approaches, which lead to the ranking of calculation-predicted bioactivities in impressively good concordance with the experimental results [90], therefore providing an extremely useful tool for the drug design of next generation of phosphoantigens.
5. Aminobisphosphonates are metabolically related to phosphoantigens The potent and selective bioactivity of therapeutic aminobisphosphonates such as pamidronic acid for V␥9/V␦2 TCRexpressing ␥␦ T cells has been a discovery rich in surprises. It was initially based on the finding that several patients with multiple myeloma (MM) treated with the well-established osteoporosis inhibitor pamidronic acid presented significantly high numbers of blood-borne ␥␦ T cells [19]. Pamidronateactivated ␥␦ T cells in vitro secrete cytokines (IFN␥), proliferate and exhibit strong cytotoxicity against various cancer cell lines [18]. Nevertheless, the bioactivity of aminobisphosphonates required that accessory “APC” cells be treated with this drug prior to the assay on ␥␦ T cells [91], suggesting that these compounds were either pulsed on the APC, where they may be recognized further by the ␥␦ TCR [92]. At that time, it could not be excluded that aminobisphosphonates exerted their known pharmacological activity on these “APC”, which as secondary event might become more antigenic for the ␥␦ T cells, since most eukaryotic cell lines are targeted by these drugs [93], and then accumulate IPP [21,22,53,94]. Recently, it was definitively established that for bioactivity on ␥␦ T lymphocytes, the aminobisphosphonates require to be inter-
nalised and exert a statin-sensitive effect, namely inhibiting the endogenous MVA pathway [20]. Thus, the aminobisphosphonates cause a pharmacological inhibition of the mevalonate pathway into treated cells, leading to IPP bioaccumulation and create a highly phosphoantigenic phenotype. This accounts for the potent and selective bioactivity of aminobisphosphonates on human ␥␦ T cells [20] and reconciled earlier conflicting data showing that despite the proven bioactivity of M pamidronate concentrations on human ␥␦ T in fresh PBL cultures, the same concentrations were barely active in direct V␥9/V␦2 TCR+ cell activation measured by real-time microphysiometry (unpublished results). In agreement with the implication of the pharmacological action of amino-bisphosphonates as the rationale for their action on ␥␦ T cell activation [95], non-amino bisphosphonates inhibitors for osteoporosis such as etidronate or clodronate neither inhibit the MVA pathway nor enable ␥␦ T cell activation by etidronate- or clodronate-treated cells, respectively. On the contrary, more potent aminobisphosphonates analogues for both MVA inhibition and ␥␦ T cell activation are now available, such as zoledronate or ibandronate [92]. The established use of aminobisphosphonates in various clinical approaches against skeletal morbidity [96–101] now enables to re-evaluate their interest for human ␥␦ T cell stimulation in cancer immunotherapy. A recent clinical study involving a cohort of MM patients demonstrated that in vivo treatment with pamidronate and IL2 of patients selected for ␥␦ T lymphocytes responding to pamidronate in vitro, an objective regression of the autologous tumor mass was recorded [102]. In other cancer patients treated with the more potent aminobisphosphonate zoledronate, the effector functions of ␥␦ T lymphocytes were induced in vivo, whereas proliferation of na¨ıve and central memory V␥9/V␦2 T cells progressively reduced. Over a 3-month-follow-up, a concomitant increase in phosphoantigen-induc1ed IFN␥−secretion characteristic of the most mature compartment of effector memory cells was detected [103]. Thus, the finding of ␥␦ T cell activation by a therapeutic agent targeting some cancer morbidity finally permitted to identify the structure of phosphoantigens naturally produced by cancer cell and activating these same ␥␦ T lymphocytes. Current clinical studies are now under way to evaluate the potency of in vivo ␥␦ T lymphocytes activation by potent therapeutic aminobisphosphonates combinations.
6. Alkylamines: V␥9/V␦2 T cell-specific antigens different from phosphoantigens V␥9/V␦2 T lymphocytes respond in vitro to a second class of small, hydrophilic non-peptide antigens, structurally composed of non-phosphate short alkyl chains bearing a terminal amine group. These alkylamines comprise one to five carbons but the organic moiety does not present the same structural requirements than the alkyl moiety from phosphoantigens for bioactivity; prototypic bioactive alkylamines are ethylamine and sec-butylamine. Second, this class of non-
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peptide antigens does not require MHC class I, or class II or the related CD1 molecules for presentation, although response to these ligands needs cell-to-cell contact with the ␥␦ T lymphocyte. Response to alkylamines requires cell surface expression of the V␥9/V␦2 TCR by the responsive ␥␦ T cell, but in contrast with aminobisphosphonates, there is not yet evidence for internalization or processing of alkylamines for their activity. The bioactivity of alkylamines is usually measured within micromolar-to-millimolar concentration ranges. These molecules are not produced from either MVA or DOXP pathways, but are largely spread in nature as components from edible plants such as wine or green tea, or secreted in culture medium from several bacteria. Listeria monocytogenes, Bacteroides fragilis, Proteus morganii, Clostridium perfringens, and Salmonella typhimurium produce alkylamines in concentrations able to activate the ␥␦ T cell responses. Altogether, the alkylamines are a separate class of antigens which presumably upon ingestion of food or beverages, may stimulate in vivo the innate physiological response of ␥␦ T cells from persons following adequate regimens. Since these response comprise mainly Th1 cytokines, it is thought that alkylamine-rich diets may contribute to counter food allergies [104].
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So one may envision the appearance of phosphoantigenreactive T cells in primates as the reflect of an evolutive emergence of immunological effectors serving the surveillance of infectious or transformed cells. Although, such type of pressure has also led to earlier appearance of phagocytic cells, these latter react to conserved PAMP ligands with a much broader distribution than phosphoantigens. It is tempting to speculate that phosphoantigens may represent the link between widely spread PAMP and highly focussed peptide antigens which define the exquisite specificity of MHCrestricted ␣ T lymphocytes. To challenge this view, it may be of interest to evaluate whether the phylogenetic emergence of phosphoantigen-specific CDR3 sequences from TCRV␥9 and TCRV␦2 chains in apes may be dated to the same time as exposure to some phosphoantigen-producing pathogens that exerted strong selective pressure for these primate hosts.
Acknowledgements: The author’s laboratory is supported by INSERM, l’ARC, and the European Union TBVAC Program.
References 7. Significance of the ␥␦ T cell response to non-peptide antigens A broad but selective ability of ␥␦ T lymphocytes to rapidly detect a large set of non-peptide ligands is typical of pathogen-associated molecular recognition patterns, which innate immunity involves to trigger adaptative immunity [105]. In this case though, the germline–encoded tolllike receptors (TLR) are scanning for conserved microbial metabolites to detect both infectious non-self and abnormal self [106]. By contrast, V␥9/V␦2 TCR are produced through somatic recombination thereby reflecting a process of adaptive immunity, even if these receptors appear to play a similar role as scanners for conserved metabolic signatures of both infectious non-self (e.g. DOXP pathway) and abnormal self (e.g. increased MVA pathway). Interestingly, while ligandinduced activation through TLR enable a potent priming of adaptative immunity via maturation of monomyeloid cells [107–109], activated V␥9/V␦2 TCR+ cells also promote DC maturation [110], in much the same way as proinflammatory microbial lipids [111]. Phosphoantigen-reactive ␥␦ T or ␣Gal ceramide responsive NKT cells [112] behave similarly in terms of cell mediating adjuvant effects, with a role parallel to authentic TLR ligands known as immunological adjuvants. In addition to this role nevertheless, both cell subsets may also deliver direct Th or cytolytic effector functions, in addition to their marked propensity to increase their frequency in blood of previously sensitized persons. Altogether, such features depict non-peptide antigens-responsive ␥␦ T cells as a self-renewed pool of killers and adaptative immunity tuners.
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Review
Non-peptide antigens activating human V␥9/V␦2 T lymphocytes Mary Poupot, Jean-Jacques Fourni´e∗ d´epartement Oncog´en`ese and Signalisation dans les Cellules H´ematopoi´etiques, Unit´e 563 de l’Institut National de la Sant´e Et de la Recherche M´edicale, Centre de Physiopathologie de Toulouse Purpan, BP3028, 31024 Toulouse, France Received 10 May 2004; received in revised form 25 June 2004; accepted 29 June 2004 Available online 21 July 2004
Abstract Various non-peptidic ligands which specifically activate most of circulating human V␥9/V␦2 T lymphocytes are now known. Most of these are so-called phosphoantigens and directly trigger the V␥9/V␦2 TCR expressing cells, without need for MHC-restricted presentation molecules. Although some potent phosphoantigens currently involved in clinical trials are chemically-synthesized molecules, most of the natural antigens were isolated from microbial cultures. The structures and biosynthesis of phosphoantigens are reviewed here and the possible physiological significance of their recognition by ␥␦ T lymphocytes is discussed. © 2004 Elsevier B.V. All rights reserved. Keywords: Non-peptide antigens; V␥9/V␦2 T lymphocytes; ␥␦T cell
1. Retrospective The biochemical structures of non-peptide antigens which specifically activate circulating human V␥9/V␦2 T lymphocytes provide clues for the possible driving force and significance of this specific subset. Since their first description in the early 90’s, the main human ␥␦ T cell subset in circulating blood has been associated with reactivity to tuberculosis and to B cell tumors [1]. Although initially attributed to recognition of hsp65-derived peptides, it soon became clear that this protein did not account for most of the V␥9/V␦2 T cell specificity [2]. Instead pioneering studies clearly indicated that this T cell subpopulation was reactive to small non-peptide antigens [3,4]. These were characterized in Mycobacterium tuberculosis and related species as a group of four structurally related phosphoesters (so-called TUBag [1–4]), comprising both thymidine and uridine nucleotide-conjugates as well as their non-nucleotide distal pyrophosphoester moieties [5–7]. Parallel studies in Mycobacterium smegmatis identified phosphate-containing antigens as isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophos∗ Corresponding author. Tel.: +335 6274 8364; fax: +335 6274 8386. E-mail address: [email protected] (J.-J. Fourni´e).
0165-2478/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2004.06.013
phate (DMAPP) [8,9]. Although it is now clear that all these findings were correct, at that time, though these apparently conflicting results did not account for the ␥␦ cell reactivity to human tumor cells, which encompassed B cell lymphoma, multiple myeloma, cutaneous, breast and renal carcinoma. Soon, it became obvious though puzzling that actually, a broad set of natural and synthetic phosphorylated compounds is able to selectively trigger the V␥9/V␦2 T cell population, and the phosphate group is their only shared determinant of bioactivity [8–10]; this criterion thus defining phosphoantigens [11]. Novel natural phosphoantigens have been detected in a wide spectrum of microorganisms and in plant extracts [12]. Thus far, the most potent natural phosphoantigen (E)4-hydroxy-3-methylbut-2-enyl-pyrophosphate (HDMAPP) was characterized in Escherichia coli (E. coli) [13,14] but also in plants [15]. This compound is a precursor of a microbial non-mevalonate pathway to IPP and further to isoprenoids [16]. In parallel, non-peptide and non-phosphoantigens molecules that also stimulate in a cross-reactive fashion the same V␥9/V␦2 T lymphocytes were reported. They comprise synthetic and natural alkylamines on the one hand [17] and therapeutic aminobisphosphonates like pamidronate [18,19] on the other. More recently, the V␥9/V␦2-selective antigens from human tumor cells were characterized as endogenous
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metabolites of the mevalonate pathway and most likely as IPP [20]. Since therapeutic aminobisphosphonates inhibit the mevalonate pathway in eukaryote cells [21,22], it appears that their ability to activate human ␥␦ T cells reflects their pharmacological action on the tumoral cell target [20,23]. Altogether, these features now converge into a scenario where the human V␥9/V␦2 T lymphocytes actually scan their targets for the biosynthesis of isoprenoids. This is reviewed below together with its possible physiological significance.
2. The isoprenoid metabolisms All living organisms produce isoprenoids, essential metabolites of their cellular and intercellular biology. Among these, sterols are found from the oldest sediments (hopanoids) to the entire current living world, encompassing prokaryotes to both plant and animal reigns. Rich of about 30,000 different molecules today, isoprenoids constitute the most extraordinarily diverse structural family, comprising, for example, ubiquinones, sterols, terpenes, carotenoids, gibberellins and taxoids. Strikingly enough, the biosynthesis responsible for such a huge structural diversity implies in all living cells the same bottleneck: the repeated condensation of two branched C5 precursors; the isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP). Despite this apparent unity however, an elder divergence has occurred in the biogenesis of these two precursors. Archaebacteria, few bacteria and most eukaryotes, synthesize IPP from acetyl-CoA through the mevalonate pathway (MVA). The MVA pathway involves the initial oxidation of pyruvate into acetyl-CoA by a multimolecular complex initially discovered in mitochondria, the pyruvate deshydrogenase (for review, see [24]). Three acetyl-CoA molecules are condensed into 3-hydroxy-3-methyl-glutarylCoA, which is reduced to mevalonate by the key enzyme HMG-CoA reductase. Then are ATP-dependent steps catalyzed by structurally related enzymes [25], wherein three successive phosphorylations of mevalonate are followed by a decarboxylation step producing IPP. Finally, IPP isomerase produces its DMAPP isomer, and these are C5 precursosrs for downstream metabolites such as sterols. Important pharmacological tools to dissect the MVA pathway are statins and related hypocholesterolemic agents inhibiting HMG-CoA reductase [26], isoprenols such as farnesol and 7-dehydrocholesterol which facilitate its degradation [27] and aminobisphosphonates which inhibit both IPP isomerase and downstream farnesyl PP synthase [21] (Fig. 1). On the other hand, in cyanobacteria, algae, chloroplasts and most eubacteria, IPP is produced by another route involving the 1-deoxy-d-xylulose-5-phosphate (DOXP) [28,29]. The DOXP pathway starts from pyruvate and d-glyceraldehyde-3-phosphate substrates [30] by 1-deoxy-d-xylulose-5-phosphate synthase making 1-deoxyd-xylulose-5-phosphate (DOXP). DOXP is reduced into 2-C-methyl-d-erythritol-4-phosphate [31], transferred into
a nucleotidyl conjugate CDP-methyl-erythritol [32] and phosphorylated by CME-kinase [33] to give CDP-methylerythritol-2-phosphate [34]. This activated precursor is then enzymatically cleaved [35] and releases 2-C-methylerythritol-2,4-cyclodiphosphate [36]. This cyclic intermediate is then successively reduced and submitted to elimination, by at least two metallo-enzymes [37,38] requiring oxygen-free conditions: HDMAPP synthase which produces 4-hydroxy-dimethylallyl diphosphate (HDMAPP) [39] and HDMAPP reductase which provides a 5:1 mixture of IPP and DMAPP [40,41]. Interesting pharmacological tools to investigate the DOXP pathway are fosmidomycin and its related FR900098 prodrug [42,43], benzyl viologen and other oxidants [44]. Downstream to these two ubiquitous C5 precursors in all living cells is a common step of elongation by sequential additions that provides C10, C15 and C20 linear allylic alcohols, respectively geraniol, farnesol and geranylgeraniol. Next to these, are substrates for hundreds of cyclases in numerous paths leading to the generation of a huge diversity of natural isoprenoids [45]. Interestingly enough, most known isoprenoid cyclases and farnesyl PP synthase have co-evolved a similar so-called “isoprenoid synthase fold” structural motif, suggesting strong co-evolutionary relationships [46] (Fig. 1).
3. Distribution of the MVA and DOXP pathways in nature Although biochemical data have documented the presence of DOXP pathways in a limited number of microorganisms (see below), the survey of genomic databases has been of great interest to delineate the presence of either MVA or DOXP pathways in nature. Both biosynthesis routes are highly intricated nevertheless, clearly distinct pictures may be drawn for prokaryotes, eukaryotes and in plants. All non-photosynthetic eukaryotes including protozoa (Leishmania and Trypanosoma spp.), most fungi, yeast and animals use only the MVA pathway to make IPP (reviewed in [47]). This pathway has a complex origin, as shown by the phylogeny of its key enzyme HMG-CoA reductase (HMGR). HMGR is split into class 1 isoforms acquired by archaeal and eukaryotic cells and class 2 bacterial HMGR which evolved separately. Since mammals make their sterols only through the MVA pathway, it is the rationale target for the lipidlowering statin class of drugs [48]. Of note, Class 1 HMGR are preferentially inhibited by natural statins. Occasionally, they are found in few eubacteria like Vibrio cholerae, but these species most presumably acquired its gene from archaea by some later lateral gene transfer [49]. In plants and yeast, regulation of the MVA pathway at the HMGR level is well documented. In response to attack by pathogens or wounds, reduction of HMGR involves both sterol-sensor-regulated MAPK cascades which drive selected gene transcriptions and its controlled ubiquitination/degradation [50,51]. High levels
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Fig. 1. DOXP and MVA pathways to isoprenoid biosynthesis. Three examples of final products from isoprenoid biosynthesis are shown. For clarity, names of enzymes have been omitted. Inhibitors are indicated in blue boxes.
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of FPP, of sterols or of phenylalanine inhibit their HMGR activity. In mammalin cells, the HMGR activity is inhibited by statins, by phenylalanine [52] or by a feedback inhibition with aminobisphosphonate-induced FPP accumulation [53]. Thus, statins may reverse by upstream blockade the aminobisphosphonate-induced ␥␦ T cell activation by endogenous phosphoantigens, but not by the synthetic exogenous BrHPP phosphoantigen agonist. Inherited defects in the MVA pathway such as mevalonate kinase deficiency do exist in humans were they are associated with mevalonic-aciduria, hyper-IgD and periodic fever syndromes (HIDS) [54]. The HMGR activity, and thus, the whole MVA pathway, were found increased in cancer cell lines such as leukaemia, nonHodgkin lymphoma [55], mammary and lung adenocarcinoma [56,57], in which HMGR activity is controlled by EGFPI3 mediated signaling [58]. Algae and higher plants cumulate the MVA pathway in the cytosol and in mitochondria, together with a functional DOXP pathway in their chloroplasts but its genes remain encoded in the cell nucleus (reviewed in [28,59,60]). Although these two routes usually lead to different isoprenoid end-products in higher plants: e.g. sterols in the cytoplasm and carotenoids in chloroplasts, under stress conditions, plastidial isoprenoids formed by the DOXP route may cross-talk with the cytosolic MVA pathway for functional complementation [61,62]. At the extreme end of this spectrum, some unicellular green algae and few parasitic eukaryotes like Plasmodium falciparum are exceptions which have not the MVA pathway and solely rely upon their DOXP counterpart. Of note, the plasmodial DOXP pathway is actually located in an intracellular plastid-like compartment called apicoplast [43]. Which of the DOXP or MVA pathway has arisen first along evolution remains unknown, since DOXP only exists in bacteria and plastids where it provides most primary isoprenoids instead of the MVA used by archae [49] (Fig. 2). Nevertheless, the MVA pathway remains more sparsely distributed among bacteria than the prevalent DOXP, since the HMGR and downstream MVA genes are patchily distributed in this group. These comprise Aquificales, Thermotogales, Chlamidiae, Cyanobacteria, Porphyrobacteria, Deinococci, Gram + such as Actinomycetes Streptomyces, Clostridiae, Mycobacteria, Corynebacteria, Nocardiae, Actinoplanae, Bacilli, Spirochaetes and most Proteobacteria of the subgroups ␣ (Rhodopseudomonas, Zymomonas, . . .),  (Bordetella, Neisseiria, Ralstonia, . . .), ␥ (V. cholerae, E. coli, Pseudomonas, Yiersinia, Salmonella, . . .) and (Helicobacter, Campylobacter, . . .) [49,63]. Whereas Streptomyces frequently use the DOXP pathway not only for producing menaquinones but also antibiotics, Actinoplanes switch from using DOXP in exponential growth phase to MVA for making antibiotics in later phases [64]. Therefore, some bacterial species have kept both pathways but for different roles: DOXP for primary metabolism and MVA for secondary metabolites [49]. Of note, Mycobacterium tuberculosis and
E. coli – where only the DOXP pathway is present – are the two species where the complete structures of natural phosphoantigens have been fully established [13,65].
4. Structures of phosphoantigens and of related V␥9/V␦2 T cell-specific stimuli Early studies had shown at a clonal level that all V␥9/V␦2 TCR-expressing ␥␦ T cells reactive to mycobacteria also recognized the Daudi lymphoma [66,67], which indicated that these cell types had similar antigens. Further work delineated the receptor for phosphoantigens as the V␥9/V␦2 TCR, and more specifically to its CDR3 regions [68–70]. The crystal structure of this TCR also supports its adaptation to interact with phosphoantigens [71] by its surface exposed lysine residues [70]. The earliest identication of mycobacterial antigens for ␥␦ T cells was incomplete, in describing four distinct but structurally related molecules [TUBag1-4) where TUBag3 and TUBag4 are respectively, UTP and TTP conjugates of a terminal unidentified alkyl moiety found in the two pyrophosphorylated TUBag 1 and TUBag2 [5,7,72,73]. Although parallel reports suggested that, this moiety was isopentenol since Mycobacterium smegmatis-derived IPP or its DMAPP isomer were identified as natural ligands for V␥9/V␦2 T lymphocytes [9], these ubiquitous metabolites hardly accounted for their selective activation in some infectious diseases only (such as tuberculosis and malaria for instance), and were produced in insufficient amounts to reach the IPP bioactive threshold (1 M) on ␥␦ T cells [74,75]. A link between production of phosphoantigens and cell activation/proliferation [11] including IPP biosynthesis [76,77] could resolve this controversial issue. This link was definitively established by the good correlate between DOXP pathway and presence of phosphoantigens among several bacterial species [75] and in P. falciparum [43,78]. Several reports identified a 262 Da phosphoantigen (TUBag1) in distinct highly immunogenic extracts from mycobacteria and from E. coli [79,80]. Using ∼30 g of the highly purified terminal X-PP moiety from the mycobacterial phosphoantigen TUBag3 (UTP-X structure), the TUBag1 antigen (X-PP MW 262) was characterized biochemically as FBPP. More recently though, an Escherichia coli (E.coli) mutated in the last step of the DOXP pathway (ispH syn.to lytB [81]) was found to accumulate 150-times more phosphoantigen than wild-type strains [14]. Based on mg amounts of purified molecule, the structural elucidation of this phosphoantigen unambiguiously defined the XPP structure of MW 262 as 4-hydroxy-dimethylallylPP (HDMAPP) rather than FBPP [13]. HDMAPP was synthesized chemically [82–84] and also produced by bioconversion from MEcPP [38]. Its bioactivity, the specific induction of V␥9/V␦2 T cells at sub-nanomolar HDMAPP concentrations [13,14] confirmed the structural assignment of the natural phosphoantigen HDMAPP. The definitive structure for TUBag2 (MW 276) now remains to be established, to-
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Fig. 2. Distribution of DOXP and MVA pathways in nature. Living organisms using the DOXP and/or MVA pathways for their isoprenoid biosynthesis are shown by the specified boxes on a simplified tree of life. Dotted lines: presence of an incomplete pathway (few genes only are present); smaller inside boxes: some species only; no box: information lacking. Adapted from [45,47,49,113,114].
gether with its link to the DOXP pathway. It also remains to be precisely determined whether 4-hydroxy-dimethylallyl or rather 3-formyl-butyl is actually involved the terminal ester moiety of both mycobacterial nucleotidyl conjugates of X-UTP (TUBag3) and X-TTP (TUBag4). Indeed, both structures may coexist as free esters in most prokaryotes, but the two nucleotide phosphoantigens are not produced by Gram-negative bacteria [77]. Nevertheless, phosphoantigens are also produced by plants such as the Viscum album parasite [12,85].
The V␥9/V␦2 T cell reactivity to IPP has thus, provided an extremely useful tool for investigating these T cell responses using a well-defined and readily available ligand. Although its spontaneous production may be limited in most prokaryote and normal eukaryote cells, it is detected in significant amounts in various types of human cancer cells. Both metabolic blockade and accumulation of biosynthesized IPP determine the phosphoantigen content of cancer cell lines such as Daudi [20]. Therefore rather than a microbial phosphoantigen, IPP now appears as the major eu-
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karyote phosphoantigens. Two tumor cell-derived phosphoantigen are present in lysates of cultured human cancer cell lines (our unpublished observations). Presumably after IPP, DMAPP could represent this second antigen since this IPP isomer has also M bioactivity on human ␥␦ T cells [7,9]. In this regard, IPP and DMAPP clearly confirmed the previously proposed ␥␦ T cell involvement in scanning of eukaryotic farnesylation [9], known to be of the highest importance in Ras-mediated oncogenesis [9,86]. Several synthetic phosphoantigens have been produced in order to enable the rationale development of chemically defined phosphoantigens in large scale amounts compatible with their use in human clinical trials. Initially, these comprised various linear alkyl phosphates most notably like ethyl-pyrophosphate [8], and were followed by more potent agonists like EpoxPP or phosphohalohydrins such as BrHPP [87]. QSAR studies of phosphoantigens have demonstrated the prominent role of their pyrophosphate moieties [8,88], which must enable the hydrolytic cleavage of their pyrophosphate bond [89]. Antagonists of phosphoantigens have thus been defined on the basis of phosphoantigen structures with replacement of the pyrophosphate bond by stable analogues such as methylene bisphosphonates, iminobisphosphonates and mono- or di-fluoromethylene bisphosphonate groups [89]. Common pharmacophores of phosphoantigens have been defined by two computational approaches, which lead to the ranking of calculation-predicted bioactivities in impressively good concordance with the experimental results [90], therefore providing an extremely useful tool for the drug design of next generation of phosphoantigens.
5. Aminobisphosphonates are metabolically related to phosphoantigens The potent and selective bioactivity of therapeutic aminobisphosphonates such as pamidronic acid for V␥9/V␦2 TCRexpressing ␥␦ T cells has been a discovery rich in surprises. It was initially based on the finding that several patients with multiple myeloma (MM) treated with the well-established osteoporosis inhibitor pamidronic acid presented significantly high numbers of blood-borne ␥␦ T cells [19]. Pamidronateactivated ␥␦ T cells in vitro secrete cytokines (IFN␥), proliferate and exhibit strong cytotoxicity against various cancer cell lines [18]. Nevertheless, the bioactivity of aminobisphosphonates required that accessory “APC” cells be treated with this drug prior to the assay on ␥␦ T cells [91], suggesting that these compounds were either pulsed on the APC, where they may be recognized further by the ␥␦ TCR [92]. At that time, it could not be excluded that aminobisphosphonates exerted their known pharmacological activity on these “APC”, which as secondary event might become more antigenic for the ␥␦ T cells, since most eukaryotic cell lines are targeted by these drugs [93], and then accumulate IPP [21,22,53,94]. Recently, it was definitively established that for bioactivity on ␥␦ T lymphocytes, the aminobisphosphonates require to be inter-
nalised and exert a statin-sensitive effect, namely inhibiting the endogenous MVA pathway [20]. Thus, the aminobisphosphonates cause a pharmacological inhibition of the mevalonate pathway into treated cells, leading to IPP bioaccumulation and create a highly phosphoantigenic phenotype. This accounts for the potent and selective bioactivity of aminobisphosphonates on human ␥␦ T cells [20] and reconciled earlier conflicting data showing that despite the proven bioactivity of M pamidronate concentrations on human ␥␦ T in fresh PBL cultures, the same concentrations were barely active in direct V␥9/V␦2 TCR+ cell activation measured by real-time microphysiometry (unpublished results). In agreement with the implication of the pharmacological action of amino-bisphosphonates as the rationale for their action on ␥␦ T cell activation [95], non-amino bisphosphonates inhibitors for osteoporosis such as etidronate or clodronate neither inhibit the MVA pathway nor enable ␥␦ T cell activation by etidronate- or clodronate-treated cells, respectively. On the contrary, more potent aminobisphosphonates analogues for both MVA inhibition and ␥␦ T cell activation are now available, such as zoledronate or ibandronate [92]. The established use of aminobisphosphonates in various clinical approaches against skeletal morbidity [96–101] now enables to re-evaluate their interest for human ␥␦ T cell stimulation in cancer immunotherapy. A recent clinical study involving a cohort of MM patients demonstrated that in vivo treatment with pamidronate and IL2 of patients selected for ␥␦ T lymphocytes responding to pamidronate in vitro, an objective regression of the autologous tumor mass was recorded [102]. In other cancer patients treated with the more potent aminobisphosphonate zoledronate, the effector functions of ␥␦ T lymphocytes were induced in vivo, whereas proliferation of na¨ıve and central memory V␥9/V␦2 T cells progressively reduced. Over a 3-month-follow-up, a concomitant increase in phosphoantigen-induc1ed IFN␥−secretion characteristic of the most mature compartment of effector memory cells was detected [103]. Thus, the finding of ␥␦ T cell activation by a therapeutic agent targeting some cancer morbidity finally permitted to identify the structure of phosphoantigens naturally produced by cancer cell and activating these same ␥␦ T lymphocytes. Current clinical studies are now under way to evaluate the potency of in vivo ␥␦ T lymphocytes activation by potent therapeutic aminobisphosphonates combinations.
6. Alkylamines: V␥9/V␦2 T cell-specific antigens different from phosphoantigens V␥9/V␦2 T lymphocytes respond in vitro to a second class of small, hydrophilic non-peptide antigens, structurally composed of non-phosphate short alkyl chains bearing a terminal amine group. These alkylamines comprise one to five carbons but the organic moiety does not present the same structural requirements than the alkyl moiety from phosphoantigens for bioactivity; prototypic bioactive alkylamines are ethylamine and sec-butylamine. Second, this class of non-
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peptide antigens does not require MHC class I, or class II or the related CD1 molecules for presentation, although response to these ligands needs cell-to-cell contact with the ␥␦ T lymphocyte. Response to alkylamines requires cell surface expression of the V␥9/V␦2 TCR by the responsive ␥␦ T cell, but in contrast with aminobisphosphonates, there is not yet evidence for internalization or processing of alkylamines for their activity. The bioactivity of alkylamines is usually measured within micromolar-to-millimolar concentration ranges. These molecules are not produced from either MVA or DOXP pathways, but are largely spread in nature as components from edible plants such as wine or green tea, or secreted in culture medium from several bacteria. Listeria monocytogenes, Bacteroides fragilis, Proteus morganii, Clostridium perfringens, and Salmonella typhimurium produce alkylamines in concentrations able to activate the ␥␦ T cell responses. Altogether, the alkylamines are a separate class of antigens which presumably upon ingestion of food or beverages, may stimulate in vivo the innate physiological response of ␥␦ T cells from persons following adequate regimens. Since these response comprise mainly Th1 cytokines, it is thought that alkylamine-rich diets may contribute to counter food allergies [104].
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So one may envision the appearance of phosphoantigenreactive T cells in primates as the reflect of an evolutive emergence of immunological effectors serving the surveillance of infectious or transformed cells. Although, such type of pressure has also led to earlier appearance of phagocytic cells, these latter react to conserved PAMP ligands with a much broader distribution than phosphoantigens. It is tempting to speculate that phosphoantigens may represent the link between widely spread PAMP and highly focussed peptide antigens which define the exquisite specificity of MHCrestricted ␣ T lymphocytes. To challenge this view, it may be of interest to evaluate whether the phylogenetic emergence of phosphoantigen-specific CDR3 sequences from TCRV␥9 and TCRV␦2 chains in apes may be dated to the same time as exposure to some phosphoantigen-producing pathogens that exerted strong selective pressure for these primate hosts.
Acknowledgements: The author’s laboratory is supported by INSERM, l’ARC, and the European Union TBVAC Program.
References 7. Significance of the ␥␦ T cell response to non-peptide antigens A broad but selective ability of ␥␦ T lymphocytes to rapidly detect a large set of non-peptide ligands is typical of pathogen-associated molecular recognition patterns, which innate immunity involves to trigger adaptative immunity [105]. In this case though, the germline–encoded tolllike receptors (TLR) are scanning for conserved microbial metabolites to detect both infectious non-self and abnormal self [106]. By contrast, V␥9/V␦2 TCR are produced through somatic recombination thereby reflecting a process of adaptive immunity, even if these receptors appear to play a similar role as scanners for conserved metabolic signatures of both infectious non-self (e.g. DOXP pathway) and abnormal self (e.g. increased MVA pathway). Interestingly, while ligandinduced activation through TLR enable a potent priming of adaptative immunity via maturation of monomyeloid cells [107–109], activated V␥9/V␦2 TCR+ cells also promote DC maturation [110], in much the same way as proinflammatory microbial lipids [111]. Phosphoantigen-reactive ␥␦ T or ␣Gal ceramide responsive NKT cells [112] behave similarly in terms of cell mediating adjuvant effects, with a role parallel to authentic TLR ligands known as immunological adjuvants. In addition to this role nevertheless, both cell subsets may also deliver direct Th or cytolytic effector functions, in addition to their marked propensity to increase their frequency in blood of previously sensitized persons. Altogether, such features depict non-peptide antigens-responsive ␥␦ T cells as a self-renewed pool of killers and adaptative immunity tuners.
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