Photoaffinity antigens for human γδ T cells

J Immunol. Author manuscript; available in PMC 2009 December 1.

Published in final edited form as:

J Immunol. 2008 December 1; 181(11): 7738–7750.

PMCID: PMC2696061

NIHMSID: NIHMS115632

Photoaffinity antigens for human γδ T cells¹

Ghanashyam Sarikonda,^*^† Hong Wang,^† Kia-Joo Puan,²^† Xiao-hui Liu,³^‡ Hoi K. Lee,^† Yongcheng Song,^§ Mark D. Distefano,^¶ Eric Oldfield,^§ Glenn D. Prestwich,^‡ and Craig T. Morita⁴^*^†

^† Division of Rheumatology, Department of Internal Medicine, University of Iowa College of Medicine, EMRB 400F, Iowa City, IA 52242 USA

^* Interdisciplinary Graduate Program in Immunology, University of Iowa College of Medicine, EMRB 400F, Iowa City, IA 52242 USA

^‡ Department of Medicinal Chemistry, University of Utah School of Pharmacy, Salt Lake City, UT 84112 USA

^§ Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801 and the Center for Biophysics and Computational Biology, 607 South Mathews Avenue, Urbana, Illinois 61801 USA

^¶ Department of Chemistry, University of Minnesota, Minneapolis, MN 55455 USA

⁴ Address correspondence and reprint requests to Craig T. Morita, M.D., Ph.D., Division of, Rheumatology, Department of Internal Medicine and the Interdisciplinary Graduate Program in, Immunology, University of Iowa Carver College of Medicine, EMRB 400F, Iowa City, IA, 52242 USA. E-mail address: Craig-Morita/at/uiowa.edu, Phone (319) 335-9981; FAX (319) 335-7607

²Current address: Division of Cellular and Molecular Research, Humphrey Oei Institute of Cancer Research, National Cancer Centre, 11 Hospital Drive Singapore 169610, Singapore

³Current address: Supergen, Inc., 2401 South Foothill Drive, Salt Lake City, UT 84109

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Abstract

Vγ2Vδ2 T cells comprise the major subset of peripheral blood γ δ T cells in humans and expand during infections by recognizing small, nonpeptide prenyl pyrophosphates. These molecules include (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP), a microbial isoprenoid intermediate, and isopentenyl pyrophosphate (IPP), an endogenous isoprenoid intermediate. Recognition of these nonpeptide antigens is mediated by the Vγ2Vδ2 T cell antigen receptor (TCR). Several findings suggest that prenyl pyrophosphates are presented by an antigen presenting molecule: contact between T cells and APCs is required; the antigens do not bind the Vγ2Vδ2 TCR directly; and antigen recognition is abrogated by TCR mutations in CDRs distant from the putative antigen recognition site. Identification of the putative antigen presenting molecule, however, has been hindered by the inability to achieve stable association of nonpeptide prenyl pyrophosphate antigens with the presenting molecule. In this study, we show that photoaffinity analogs of HMBPP, meta/para-benzophenone-(methylene)-prenyl pyrophosphates (m/p-BZ-(C)-C₅-OPP), can cross-link to the surface of tumor cell lines and be presented as antigens to γ δ T cells. Mutant tumor cell lines lacking MHC class I, MHC class II, β₂-microglobulin, and CD1, as well as tumor cell lines from a variety of tissues and individuals, will all crosslink to and present m-BZ-C₅-OPP. Finally, pulsing of BZ-(C)-C₅-OPP is inhibited by IPP and an inactive analog, suggesting that they bind to the same molecule. Taken together, these results suggest that nonpeptide antigens are presented by a novel antigen presenting molecule that is widely distributed, non-polymorphic, but not classical MHC class I, MHC class II, or CD1.

This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third party. The final, citable version of record can be found at www.jimmunol.org.

Keywords: T cells, T-cell antigen receptors gamma-delta, antigen presentation, photoaffinity, isopentenyl pyrophosphate, antigen recognition, isoprenoid metabolism

Introduction

The γ δ T cell subset, expressing T cell antigen receptors (TCR)⁵ using γ and δ rearranging genes (1), has functional roles in immunity distinct from the αβ T cell subset (2). In humans, the majority of circulating γδ T cells express Vγ2Vδ2 (also termed Vγ9Vδ2) TCRs. Vγ2Vδ2 T cells recognize nonpeptide prenyl pyrophosphate intermediates in isoprenoid biosynthesis such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) (3, 4) and isopentenyl pyrophosphate (IPP) (5). Vγ2Vδ2 T cells can expand during infections to very high numbers accounting for half of the circulating T cells in some patients (reviewed in 2). HMBPP is the most potent antigen described (3) and is produced in the methyl-erythritol phosphate pathway for isoprenoid synthesis used by many Eubacteria, some protozoa, and plant chloroplasts. By recognizing HMBPP produced by many pathogenic bacteria (such as those that cause tuberculosis and gastroenteritis) as well as Apicomplexan parasites (such as those that cause malaria and toxoplasmosis), Vγ2Vδ2 T cells likely play important roles in human immunity to both bacteria and parasites (2).

Vγ2Vδ2 T cells also kill many types of tumor cells in vitro including malignant B cells, melanomas, prostate carcinomas, renal cell carcinoma, epithelial carcinomas, and others. (6–10). This appears due to both TCR mediated and NK receptor mediated tumor cell recognition (11–13). Zoledronate and other bisphosphonates greatly enhance tumor recognition by inhibiting the intracellular farnesyl pyrophosphate synthase enzyme resulting in increases in endogenous IPP (Wang and Morita, unpublished observations and ref. 14, 15, 16). Importantly, treatment of patients with B cell malignancies (17) and metastatic prostate carcinomas (18) with a bisphosphonate and IL-2 to activate and maintain Vγ2Vδ2 T cells led to partial remissions and stable disease in several individuals. Given their broad tumor reactivity, immunotherapy with Vγ2Vδ2 T cells appear to have promise for the treatment of a variety of cancers.

Despite the importance of Vγ2Vδ2 T cells in human immunity to pathogens and their potential for tumor immunotherapy, little is known about the molecular mechanisms for the presentation of prenyl pyrophosphate antigens to these T cells. Although gene transfer studies show that the Vγ2Vδ2 TCR mediates antigen recognition by Vγ2Vδ2 T cells (11), there is no evidence for direct binding of prenyl pyrophosphates to the Vγ2Vδ2 TCR. Attempts to soak prenyl pyrophosphates into crystals of the Vγ2Vδ2 TCR (19) or to demonstrate prenyl pyrophosphate binding to soluble γδ TCR by equilibrium dialysis or microcalorimetry failed (C. T. Morita, unpublished observations). Moreover, although the chemical structural requirements for antigenic activity of prenyl pyrophosphates and other phosphoantigens has been extensively studied (20–23), this knowledge has provided little insight into how the antigens are presented.

Unlike protein antigens, prenyl pyrophosphate antigens do not require antigen uptake, processing, or intracellular loading for presentation (24). Moreover, activation of Vγ2Vδ2 T cells is extremely rapid with calcium flux observed within 90 seconds upon exposure to IPP (24) and metabolic acidification within 10 seconds upon exposure to bromohydrin pyrophosphate (BrHPP) (25). Although rapid, activation of Vγ2Vδ2 T cells by prenyl pyrophosphates still requires cell-cell contact (24, 26) similar to the contact required by αβ T cells during the recognition of peptide antigens presented by MHC class I and II molecules (27). Most human cells are capable of presenting prenyl pyrophosphates (as assessed by indirect stimulation by a bisphosphonate) except those deficient in accessory molecule ligands (6, 28, 29). In contrast, murine, rat, and hamster cell do not present prenyl pyrophosphates or bisphosphonates (26, 28, 30).

The requirement for cell-cell contact coupled with the small size of prenyl pyrophosphates (they are likely monovalent) and their lack of direct binding to the Vγ2Vδ2 TCR suggests that prenyl pyrophosphates are presented by a presenting molecule, similar to peptides presented by MHC class I and class II or lipids presented by CD1. However, unlike peptide antigens, prenyl pyrophosphates do not stably associate with their presenting molecule with high affinity, precluding pulsing of these antigens on APC (24, 26). This type of presentation is similar to that of nonpeptide drugs, such as sulfamethoxazole and lidocaine, that load into MHC class I and II molecules on the cell surface for recognition by CD8 and CD4 αβ T cells (31, 32). Although this recognition is MHC restricted, the drugs do not stably associate with MHC molecules or require internalization (31, 33). Recognition of lipid antigens that load into CD1a or CD1b molecules at the cell surface also show similarities to prenyl pyrophosphate recognition since lipids bind extremely rapidly (as short as 2 minutes) and, again, do not require processing or internalization (34, 35). However, the putative presenting molecule for prenyl pyrophosphates has eluded identification.

Previously, we found that prenyl pyrophosphate antigens did not stably associate with APC with high affinity since EBV-transformed B cells and PBMC pulsed with IPP or mono-ethyl-phosphate followed by washing did not activate Vγ2Vδ2 T cells (24). This direct presentation of prenyl pyrophosphates (24) differs from the indirect stimulation by bisphosphonates which enter cells via fluid phase endocytosis (36) to inhibit FPPS and, thus, “pulse” into APC (37). Our attempts to measure binding of IPP to the APC cell surface suggest that the binding affinity between prenyl pyrophosphates and any putative antigen presenting molecule is very low (data not shown). This property has made efforts to characterize the antigen presenting molecule using natural antigens, such as IPP and HMBPP, difficult.

In this study, we sought a prenyl pyrophosphate antigen that would have both antigenic activity for Vγ2Vδ2 T cells and stable association with the APC cell surface. Here we show that meta/para-benzophenone-(methylene)-prenyl pyrophosphates (m/p-BZ-(C)-C₅-OPP), photoaffinity FPP analogs, are recognized by Vγ2Vδ2 T cells. m/p-BZ-(C)-C₅-OPP stimulate Vγ2Vδ2 T cells even after ultraviolet (UV) crosslinking to the APC surface and extensive washing. This covalent surface crosslinking is inhibited by IPP suggesting that the molecules bind to the same protein on the APC cell surface. We also find that natural prenyl pyrophosphate can rapidly “pulse” onto the surface of APC under optimal conditions. m-BZ-C₅-OPP was able to stably associate with the cell surface of most hematopoietic and non-hematopoietic cell lines including mutant APCs lacking classical MHC class I, β₂m, MHC class II, and CD1 molecules. Thus, m/p-BZ-(C)-C₅-OPP antigens may enable identification of this putative presenting molecule which is predicted to be broadly distributed, functionally non-polymorphic, and not a known presenting molecule.

Materials and Methods

Antigens

HMBPP was synthesized as described (38). Mono-ethyl phosphate (MEP) and mono-ethyl pyrophosphate (EPP) were prepared and purified by anion exchange as described (23, 39). Mono-methyl phosphate, farnesyl pyrophosphate (FPP), and isopentenyl pyrophosphate (IPP) were obtained from Sigma (St. Louis, MO). Phytohemagglutinin-P (PHA) was obtained from Difco (Detroit, MI).

Synthesis of photoaffinity compounds and bromohydrin pyrophosphonate

Syntheses of m/p-BZ-C₅-OPP were performed by minor modifications of the previous method (40). Briefly, dimethylallyl alcohol was first protected as the chloroacetate and then oxidized with t-butyl hydroperoxide and catalytic H₂SeO₃. The resulting aldehyde was reduced with sodium borohydride, and the corresponding allylic alcohols were coupled under Mitsunobu conditions with either 4-benzoylphenol or 3-benzoylphenol to give the protected prenyl benzophenone ethers. The chloroacetate was removed by hydrolysis with methanolic aqueous ammonia, and the allylic alcohols converted to the corresponding allylic chlorides using N-chlorosuccinimide and dimethyl sulfide in dichloromethane. Displacement of the allylic chlorides with tris(tetra-n-butylammonium) hydrogen diphosphate afforded the desired allylic diphosphates, which were then purified by reversed-phase chromatography and characterized by NMR. Syntheses of m/p-BZ-C-C₅-OPP (ether) were performed as described (41). Syntheses of m/p-BZ-C-C₅-OPP (esters) were performed as described (42). Syntheses of m/p-BZ-C-GPP (ethers) were performed as described (43). Syntheses of DATFP-dihydroester-(alkyl)-FPP, DATFP-dh-GPP, and FPP-p-BZ were performed as described (Hovlid, M. L., R. L. Edelstein, F. Lopez-Gallego, S. A. Agger, C. Schmidt-Dannert, S. Sen, D. Shintani, K. Cornish, and M. D. Distefano, manuscript in preparation).

Synthesis of bromohydrin pyrophosphonate (BrHPCP) [[(4-bromo-3-hydroxy-3-methylbutoxy)hydroxyphosphinyl]methyl]-phosphonic acid (triammonium salt) was performed as follows: To a solution of O-isopentenyl methylene-1,1-bisphosphonate triammonium salt (IPCP) (5 mg) in water (1 ml) was added freshly prepared bromine water, dropwise, until the solution was persistently yellow. The yellow color (due to a trace amount of Br₂) was removed by gently blowing N₂ into the solution, which was then used without further purification.

Maintenance of cell lines

Va2 cells are derived from the SV-40-transformed human fibroblast cell line, W1–18 (44–46). Other cell lines used were described previously (24) and include the Burkitt’s lymphoma, Raji, and its MHC class II negative mutant, RJ-2.2.5 (47); the CD3⁻ Jurkat T cell line, JRT3–T3.5 (3); the erythroleukemia cell line, K562 (4); the parent EBV line 721 and the 721.221 mutant that lacks surface expression of HLA-A, -B, and -C (48); and the mutant melanoma cell line, FO-1, that is β₂M deficient (49) and lacks detectable assembled class I molecules (50). Va2 cells were cultured in Dulbecco’s Modified Eagles Medium (DMEM) (Invitrogen, Carlsbad, CA) with 10% FCS (Gemini Bio-Products, West Sacramento, CA) at 37°C in a 10% CO₂ incubator while the other cells were cultured at 37°C in a 5% CO₂ incubator in P-media. P-media is RPMI 1640 supplemented with 20 mM HEPES, 2 mM glutamine, 1 mM pyruvate, 1x MEM non-essential amino acids, 0.5x MEM essential amino acids, 5.5 × 10⁻² mM 2-ME (all from Invitrogen), and 10% FCS (Gemini Bio-Products), and adjusted to pH 7.25 with 2 N NaOH.

Derivation of and culture conditions for Vγ2Vδ2 T cell clones

T cell lines and clones were maintained by periodic stimulation with PHA-P. 1–2×10⁵ T cells per well were cultured in 1 ml RPMI 1640 supplemented as for P-media but with the addition of rIL-2 (1–4 nM, Proleukin, Novartis) and 2% human AB serum (Atlanta Biologicals, Lawrenceville, GA) with irradiated (4000 rad) allogeneic PBMC (2×10⁵) and an equal mix of irradiated (5000 rad) EBV-transformed B cells (DG. EBV and CP. EBV) (5×10⁵ total) as feeder cells and PHA-P (1/4000 final dilution) in 24 well plates (Linbro, MP Biomedicals, Irvine, CA). The derivation of the CD8αα⁺ 12G12 and DG.SF68 and the CD4⁺ HF.2 Vγ2Vδ2 T cell clones has been described (39, 51, 52).

Treatment of APCs

APCs were treated with either mitomycin C (unfixed) or with glutaraldehyde (fixed). For mitomycin C treatment, APCs (1–3×10⁷ cells/ml) in Dulbecco’s phosphate buffered saline (PBS) without calcium or magnesium were incubated with fresh mitomycin C (Sigma, MO) (100 μg/ml) for 1 h at 37°C in a 5% CO₂ incubator, then washed three times in PBS, and resuspended in either PBS or P-medium for further use. For glutaraldehyde fixation, APCs were adjusted to 1–3×10⁷ cells/ml in PBS and reacted with 0.05% glutaraldehyde (EM grade, Sigma, MO) for 15 seconds at room temperature while vortexing. The reaction was stopped by adding an equal volume of 0.2 M L-lysine (in H₂O at pH 7.4) followed by incubation for two minutes. The fixed cells were then washed three times in PBS and resuspended in either PBS or P-medium for further use.

Pulsing and UV crosslinking of m/p-BZ-(C)-C₅-OPP on APC

Following mitomycin C-treatment, APC were resuspended in ice cold PBS to a concentration of 1×10⁷ cells per ml. The cell suspension (200 μl) was added to wells of a 24 well plate. 200 μl of m/p-BZ-C₅-OPP antigen was then added to each well and the cells and antigen incubated with or without 350 nm UV light treatment for 90 minutes on ice. The cells were transferred to 15 ml conical tubes (BD Falcon, BD Biosciences, USA), washed 3 times with 10 ml ice cold PBS at 4°C, and resuspended in P-media for use. In some experiments, the cells were washed first in ice cold PBS and then exposed to UV light while in other experiments they were first exposed to UV light and then washed 3 times with ice cold PBS. Long wavelength UV light (350 nm) was used in order to avoid protein damage. For inhibition of photoaffinity antigen binding by IPP or BrHPCP, APC were incubated with IPP or BrHPCP (an inactive analog of bromohydrin pyrophosphate) in P-media with serum for 30 minutes on ice followed by the addition of a suboptimal dose of either m-BZ-C₅-OPP or m-BZ-C-C₅-OPP ether and exposed to UV light for 90 minutes. The APC were then washed 3 times with 4°C PBS and used as APC with Vγ2Vδ2 T cell clones. Alternatively, BrHPCP was incubated with mitomycin C treated Va2 for 30 minutes followed by the addition of m-BZ-C-C₅-OPP ether and 12G12 T cells.

Pulsing of prenyl pyrophosphate antigens on antigen presenting cells

Mitomycin C-treated or glutaraldehyde-fixed APCs were added at 1×10⁴ – 1×10⁵ cells per 100 μl PBS into wells of 96-well round bottom plates (Corning Inc., Corning, NY) and incubated with antigens at 37°C in a 5% CO₂ incubator for between 5 and 120 minutes. APCs were washed in the plate five to seven times with PBS either at room temperature or at 4°C and resuspended in 100 μl P-media for further assays. A Vγ2Vδ2 T cell clone was then added to the antigen-pulsed APCs and proliferation assessed by adding 1 μCi ³H-thymidine at 24 hours followed by harvesting 16–24 hours later. Each pulsed or unpulsed APC group was also cultured with the same T cell clone in the continuous presence of antigens such as mono-ethyl pyrophosphate, IPP, HMBPP, or m-BZ-C₅-OPP, or with the mitogen, PHA-P, as positive controls for each APC group. No proliferation was noted in the absence of T cells. Also, there was no stimulation of T cells in wells pulsed with antigen in the absence of APC.

T cell proliferation and cytokine release assays

T cell proliferation assays were performed as described (53). Briefly, T cells were plated in duplicate or triplicate in round bottom 96 well plates at 5–10×10⁴ T cells per well with 1×10⁵ irradiated (7,000 rads) allogeneic PBMC or mitomycin C-treated allogeneic tumor cells as APC. Because the Vγ2Vδ2 T cell response to prenyl pyrophosphate antigens is not MHC restricted (54), allogeneic cells are suitable APCs. The cultures were pulsed with 1 μCi of ³H-thymidine (2 Ci/mmol) on day 1 and harvested 16–24 hours later using a Tomtec 96 well harvester. The samples were then counted using a Wallac Betaplate scintillation counter. The mean proliferation and standard error of the mean (SEM) of triplicate (or occasionally duplicate) cultures are shown. For cytokine release, culture supernatants were removed after 24 h and TNF-α or IFN-γ levels determined by sandwich ELISA (R&D Systems, Minneapolis, MN) on single or duplicate cultures. For statin inhibition experiments, APC were preincubated with mevastatin (Sigma, MO) for 30 minutes followed by the addition of set amounts of the stimulatory compounds in the continued presence of mevastatin. T cells were then added after 60 minutes either directly to the APC (for HMBPP and m-BZ-C₅-OPP) or after washing the APC and resuspending in mevastatin containing media (for risedronate). Similar results are obtained if the prenyl pyrophosphate are pulsed on the APC.

Results

Benzophenone reaction

Because prenyl pyrophosphate antigens do not stably associate with the putative antigen presenting molecule it has been difficult to determine its identity (24). A similar lack of stable association is found for nonpeptide drugs presented as antigens by MHC class I or class II molecules to CD4 and CD8 αβ T cells (31). To overcome this problem, we have studied bioactive photoactivatable analogs of prenyl pyrophosphates to covalently link the prenyl pyrophosphate antigen to the APC surface. The farnesyl pyrophosphate analog, m-BZ-C₅-OPP, is comprised of a benzophenone (BZ) photophore linked to an HMBPP molecule via the hydroxyl group (40). m-BZ-C₅-OPP is a chemically stable compound that can be reversibly activated using long wavelength (which avoids protein damage) UV light. When activated, m-BZ-C₅-OPP reacts with C-H bonds in close proximity (Fig. 1).

Figure 1

Mechanism of cross-linking photoaffinity FPP/HMBPP analogs

m/p-BZ-(C)-C₅-OPP compounds stimulate Vγ2Vδ2 T cells

In in vitro experiments, m/p-BZ-C₅-OPP function as analogs of isoprenoid pyrophosphates since they can crosslink to farnesyl pyrophosphate synthase (FPPS) and other prenyl synthases (40). Structurally, m/p -BZ-C₅-OPP resemble both FPP (through their spacing of C-C double bonds) and HMBPP (where the hydroxyl attached to C4 is now an ether or ester bond) (see Fig. 2 and and33 for structures). Therefore, to determine whether m/p-BZ-(C)-C₅-OPP compounds are recognized by Vγ2Vδ2 T cells, m-BZ-C₅-OPP and p-BZ-C₅-OPP were tested for their ability to induce proliferation of Vγ2Vδ2 T cells. Similar to IPP and EPP, both compounds stimulated DG. SF68 Vγ2Vδ2 T cells in a dose dependent manner (Fig. 2A). The concentrations that induced half maximum proliferation were 0.4 μM, intermediate between IPP (1–3 μM) and HMBPP (0.0000316 μM). Thus, as predicted based on their structure, both m-BZ-C₅-OPP and p-BZ-C₅-OPP are recognized as antigens by Vγ2Vδ2 T cells.

Figure 2

Photoaffinity analogs of FPP/HMBPP are antigens for Vγ2Vδ2 T cells

Figure 3

m-BZ-C₅-OPP can be crosslinked onto the surface of antigen presenting cells by UV light for stimulation of Vγ2Vδ2 T cells

To determine the specificity of recognition by Vγ2Vδ2 T cells, other photoaffinity analogs (both meta- and para-substituted) with ether and ester linkages to the benzophenone group were tested (Fig. 2B, C, 3C, D). The linkage and spacing of the C₅-OPP group from the benzophenone group was extremely important in determining bioactivity. Compounds that had ester-linked C₅-OPP groups spaced 1 methylene group away from the benzophenone moiety (m/p-BZ-C-C₅-OPP esters) were extremely active, requiring only slightly higher concentrations for 1/2 maximum stimulation compared with HMBPP (the most potent prenyl pyrophosphate described) with ½ maximum stimulation at 50–60 pM versus 32 pM for HMBPP (Fig. 3C, ,4C,4C, and ref. 3). Changing the ester linkage to an ether linkage (m/p-BZ-C-C₅-OPP ethers) reduced bioactivity by 38–72-fold (Fig. 3C). Removing the methylene spacer (to give m/p-BZ-C₅-OPP ethers) further reduced bioactivity by 140–316-fold (Fig. 2A versus versus3C).3C). Vγ2Vδ2 T cells showed equal or slight preferential recognition of para-BZ-(C)-C₅-OPP compounds compared to their meta- isomers (Fig. 2–4). This was most pronounced for p-BZ-C-GPP and m-BZ-C-GPP compounds which differed by ~10-fold in activity (Fig. 2C). Compounds with DATFP photoaffinity groups attached to longer chain FPP and GPP moieties or with the benzophenone group linked to FPP via the pyrophosphate moiety had little or no activity (Fig. 2C). These results show that Vγ2Vδ2 T cells recognize isoprenoid photoaffinity compounds in a structure specific manner and that the linkage to and spacing from the benzophenone moiety determine bioactivity levels.

Figure 4

Photoaffinity antigens act as direct antigens for Vγ2Vδ2 T cells

Since recognition of prenyl pyrophosphate antigens by Vγ2Vδ2 T cells requires the pyrophosphate moiety, we sought to determine if recognition of m-BZ-C₅-OPP shows a similar requirement. Neither the crosslinked benzophenone photophore without the pyrophosphate moiety nor a 4-maleimido-BZ derivative stimulated Vγ2Vδ2 T cells (Fig 2D) suggesting that Vγ2Vδ2 T cell recognition of m-BZ-C₅-OPP is dependent on the presence of the pyrophosphate moiety and not the benzophenone group.

m-BZ-C₅-OPP can stably associate with APC after photocrosslinking and are presented directly like prenyl pyrophosphates

Previously, we and others have found that prenyl pyrophosphate antigens, including IPP and mono-ethyl phosphate, do not stably associate with APC (24, 26). To determine if the FPP photoaffinity analog, m-BZ-C₅-OPP, can stably associate with APC after UV crosslinking, we incubated APC in media only, with IPP, or with m-BZ-C₅-OPP for 90 minutes on ice. During this incubation, the cells were either exposed to UV light to induce crosslinking of m-BZ-C₅-OPP or they were left unexposed. After extensive washing, the cells were then used as APC to stimulate the 12G12 Vγ2Vδ2 T cell clone. Unlike APC pulsed with m-BZ-C₅-OPP (and not exposed to UV light), APC exposed to UV light during pulsing stimulated Vγ2Vδ2 T cells to proliferate even after extensive washing (Fig. 3A). UV treatment alone did not affect the APC, since APC exposed to UV light in either the absence or presence of IPP did not stimulate Vγ2Vδ2 T cells. The cells were competent for presentation since they were able to present m-BZ-C₅-OPP when the antigen was continuously present (Fig. 3A).

This recognition of m-BZ-C₅-OPP on DG. EBV B cells was dose and UV dependent. Even after extensive washing, the APC with UV crosslinked m-BZ-C₅-OPP retained the ability to stimulate the CD4⁺ Vγ2Vδ2 T cell clone, HF.2, to proliferate in a dose dependent fashion whereas non-UV crosslinked m-BZ-C₅-OPP did not (Fig. 3B). Similarly, UV crosslinking of the m/p-BZ-C-C₅-OPP ether- and ester-linked compounds also resulted in stable association with the APC that was resistant to washing (Fig. 3C). Crosslinked antigens required somewhat higher concentrations for ½ maximal stimulation compared with antigen present continuously (~33-fold and ~13-fold higher for the ester and ether compounds, respectively) (Fig. 3C). Besides stimulating proliferative responses, the photoaffinity antigens also stimulated the release of TNF-α and IFN-γ in a dose dependent manner (Fig. 3D). These findings show that m/p-BZ-(C)-C₅-OPP compounds stimulate Vγ2Vδ2 T cell cytokine and proliferative responses and that these compounds retains their immunogenicity when crosslinked to the APC surface.

To determine the mechanism by which m/p-BZ-(C)-C₅-OPP compounds stimulate Vγ2Vδ2 T cells, we inhibited the response of Vγ2Vδ2 T cells with the statin, mevastatin. We have found that compounds that act as direct antigens for Vγ2Vδ2 T cells (i.e. prenyl pyrophosphates) or as mitogens (e.g. PHA) are much less sensitive to statin inhibition than compounds (e.g. bisphosphonates and alkylamines) that act indirectly by inhibiting FPPS causing IPP accumulation (Wang and Morita, manuscript in preparation). Therefore, we used mevastatin to inhibit Vγ2Vδ2 T cell responses induced by p-BZ-C₅-OPP in comparison with HMBPP and risedronate. Whereas mevastatin inhibited 50% of the response to the FPPS inhibitor, risedronate, at 0.07 μM (Fig. 4A), mevastatin concentrations of 23–42 μM (328–600-fold higher) were required to inhibit p-BZ-C₅-OPP responses (Fig. 4A, B). These levels were similar to the 80 μM mevastatin concentrations that were required to inhibit 50% of the HMBPP responses by the CD4 HF.2 clone (HMBPP presented by CP. EBV) or the CD8αα 12G12 clone (HMBPP presented by Va2) (Fig. 4A, B).

A second characteristic of prenyl pyrophosphate antigens is their recognition by Vγ2Vδ2 T cells in the absence of other cells due to presentation by daughter T cells (24). In contrast, bisphosphonates generally require the presence of APC to stimulate proliferation of Vγ2Vδ2 T cells (37) (although this may be due in part to toxicity associated with the inhibition of FPPS). To determine if the photoaffinity antigens require APC for presentation, the 12G12 and HD.108 Vγ2Vδ2 T cell clones were incubated with m/p-BZ-C-C₅-OPP compounds, IPP, and HMBPP in the presence or absence of Va2 cells as APC. Like IPP and HMBPP, the m/p-BZ-C-C₅-OPP photoaffinity compounds stimulated Vγ2Vδ2 T cell proliferation in the complete absence of APC with similar lower magnitude responses and shifted dose response curves (Fig. 4C). Thus, photoaffinity compounds function as direct antigens for Vγ2Vδ2 T cells rather than as pharmacological inhibitors of FPPS.

IPP and the BrHPCP analog inhibit pulsing and recognition of m-BZ-(C)-C₅-OPP by Vγ2Vδ2 T cells

Since both IPP and m-BZ-C₅-OPP induce the proliferation of Vγ2Vδ2 T cells, we sought to determine if they bind to the same sites on the APC surface. To test this, APC were preincubated with varying concentrations of IPP and then 0.316 μM m-BZ-C₅-OPP was added and the cells exposed to UV light. Following UV treatment, the APC were washed and tested for their ability to stimulate Vγ2Vδ2 T cells. When no competing antigen was present, m-BZ-C₅-OPP crosslinked efficiently to the surface of DG. EBV and induced proliferation of HF.2 Vγ2Vδ2 T cells (Fig. 5A). However, preincubation with IPP inhibited m-BZ-C₅-OPP crosslinking to the APC in a dose dependent fashion, decreasing their ability to stimulate HF.2 T cells. Similarly, the inactive bromohydrin pyrophosphate analog, bromohydrin pyrophosphonate (BrHPCP) (structure shown in Fig. 5A) blocked crosslinking of the m-BZ-C-C₅-OPP ether antigen to the APC surface as evidenced by the inhibition of the proliferation of the HF.2 and 12G12 clones with increasing concentrations of BrHPCP (Fig. 5B). The continuous presence of BrHPCP also inhibited stimulation by the m-BZ-C-C₅-OPP ether antigen even when not crosslinked (Fig. 5C) confirming an earlier study (20). Note that BrHPCP did not stimulate Vγ2Vδ2 T cells to proliferate or secrete TNF-α nor was there any evidence for direct toxicity (Fig. 5C and data not shown). These findings demonstrate that IPP/BrHPCP compete with m-BZ-(C)-C₅-OPP antigens for binding sites on the APC cell surface, preventing stable crosslinking and presentation.

Figure 5

IPP and the inactive BrHPP analog, BrHPCP, inhibit pulsing and recognition of m-BZ-(C)-C₅-OPP by Vγ2Vδ2 T cells

Photoaffinity antigens can be presented by a broad array of hematopoietic and non-hematopoietic tumor cell lines

The ability to covalently attach the prenyl pyrophosphate analog to a molecule on the APC surface allowed us to test tumor cells from a variety of different lineages for expression of the presenting molecule without the complicating possibility of self presentation by Vγ2Vδ2 T cells to each other. We found that virtually any human tumor cell line, irrespective of tissue origin or developmental stage, was able to present the m-BZ-C₅-OPP antigen to Vγ2Vδ2 T cells (Table 1). In contrast, none of the 6 murine hematopoietic cell lines tested were able to present HMBPP or the bisphosphonate, risedronate, to Vγ2Vδ2 T cells (Table 1). This suggests that the putative antigen presenting molecule for prenyl pyrophosphate antigens is broadly distributed like classical MHC class I molecules but is functionally non-polymorphic.

Table I

Human but not murine tumor cells from a variety of cell lineages serve as antigen presenting cells for m-BZ-C₅-OPP/prenyl pyrophosphates

Prenyl pyrophosphate antigens can be pulsed onto APC without crosslinking

During our survey of different tumor cell lines, we noted some, such as Va2, SH-5YSY, and HT-1080, that presented pulsed m-BZ-C₅-OPP without UV crosslinking (Fig. 6A). When pulsed in RPMI with fetal calf serum, the Va2 cell line was able to present m-BZ-C₅-OPP without UV crosslinking, but required a 200-fold higher concentration during pulsing to elicit a similar half maximal response to that observed in the continuous presence of the antigen (0.21 versus 42 μM, Fig. 6B). IPP could also be pulsed onto Va2, but required a 100-fold higher concentration to elicit a similar level of response to the continuous presence of the antigen (Fig. 6B). Thus, prenyl pyrophosphate antigens can be pulsed onto APC without covalent linkage albeit inefficiently under standard conditions.

Figure 6

Prenyl pyrophosphate antigens can be pulsed onto antigen presenting cells without UV crosslinking

In earlier attempts at pulsing prenyl pyrophosphates, we used EBV-transformed B cells or PBMC as APC, with moderate potency antigens such as IPP, mono-methyl-phosphate (MMP), or EPP, or low concentrations of HMBPP in bacterial lysates (24). Since APC may differ in their ability to present prenyl pyrophosphates, we compared the ability of three tumor cell lines (the EBV-transformed B cell line, DG. EBV, Va2, and SH-5YSY) to present m-BZ-C₅-OPP (without UV crosslinking) to the NKG2D⁺, CD8αα⁺ Vγ2Vδ2 T cell clone, DG. SF68 (Fig. 6C).

DG. EBV B cells stimulated Vγ2Vδ2 T cell proliferative responses similar to or higher than the other presenter cell lines. However, compared to Va2, DG. EBV cells required antigen concentrations for half maximal responses (at the optimal APC number) that were 17-fold higher for MEP (exp. 1) and 12-fold higher for IPP (exp. 2) (Fig. 6C). The concentrations required by SH-5YSY were intermediate between the two cell lines. Thus, the Va2 and SH-5YSY cell lines were more effective presenter cells than was DG. EBV.

Va2 are more effective presenter cells, in part, because they express MICA, ULBP2, and ULBP3 ligands that bind to the NKG2D receptors expressed on the surface of the CD8 αα⁺ Vγ2Vδ2 DG.SF68 T cell clone. We have previously shown that NKG2D binding to its ligands enhances Vγ2Vδ2 T cell responses to prenyl pyrophosphates (12) and DG. EBV lacks such NKG2D ligands (data not shown). However, there are likely additional accessory molecule interactions or other factors that enhance recognition since the CD4⁺ Vγ2Vδ2 T cell clone, HF.2 (which lacks NKG2D) also requires 16-fold higher HMBPP concentrations with DG. EBV as compared to Va2 (half maximal proliferation at 0.53 versus 0.033 nM for DG. EBV versus Va2, Fig. 6D). Thus, prenyl pyrophosphate antigens can be pulsed onto antigen presenting cells, although this is difficult to demonstrate using IPP and B cells.

m-BZ-C₅-OPP pulsing is inhibited by serum and media components

Since prenyl pyrophosphates can pulse onto APC, albeit inefficiently, even without UV crosslinking (Fig. 6), we sought to optimize conditions for pulsing of m-BZ-C₅-OPP onto APC. Unlike earlier experiments where media with serum was used (Fig. 6B), we pulsed m-BZ-C₅-OPP onto APC in PBS either with or without serum in the presence or absence of UV light to crosslink the antigen (Fig. 7). Following pulsing, the APC were washed and resuspended in media with serum and cultured with the 12G12 clone. To rule out changes in the APC due to UV exposure or the lack of serum, control APC were treated as above in the absence of m-BZ-C₅-OPP, then assessed for their ability to present m-BZ-C₅-OPP added with the T cells (Fig. 7A, bottom panels). In the absence of serum, pulsing of m-BZ-C₅-OPP was very efficient following crosslinking to the APC (Fig. 7A, top right panel). Importantly, even without UV crosslinking, m-BZ-C₅-OPP could be pulsed onto the APC with similar efficiency (0.33 versus 0.20 μM respectively, Fig. 7A, top left panel versus top right panel). Thus, pulsing was more efficient in the absence of serum, and this effect was more pronounced for m-BZ-C₅-OPP that had not been crosslinked (21-fold for uncrosslinked versus 5-fold for UV-crosslinked). These results suggest that an unknown serum component inhibits the efficient pulsing of m-BZ-C₅-OPP onto APC.

Figure 7

Stable association of m-BZ-C₅-OPP and other prenyl pyrophosphates with APC is impaired by serum

Since pulsing of m-BZ-C₅-OPP onto APC was most efficient when the pulsing reaction was carried out without serum, we extended these observations to natural prenyl pyrophosphate antigens. We incubated HMBPP in either media with or without serum, or in PBS without serum (Fig. 7B). When HMBPP was continuously present, serum had little effect on Vγ2Vδ2 T cell proliferation (Fig. 7B, left bottom panel). However, pulsing of HMBPP onto APC was better in media without serum and better still in PBS without serum (Fig. 7B, top panels). Thus, pulsing of both natural and synthetic prenyl pyrophosphates onto APC is inhibited by serum, and by other media components.

Prenyl pyrophosphate antigens pulse rapidly onto APC

Since we found that prenyl pyrophosphate antigens could be pulsed efficiently onto APC, we sought to determine the kinetics of nonpeptide antigen pulsing. Mitomycin C-treated or glutaraldehyde-fixed Va2 cells were incubated with HMBPP for varying lengths of time (5 to 120 minutes) in PBS without serum after which the APC were washed extensively. The pulsed APC were then incubated with the CD4⁺ HF.2 clone and γδ T cell proliferation (Fig. 8) and release of TNF-α (data not shown) measured. In agreement with our previous observations (24), fixing APC with glutaraldehyde had no affect on pulsing of HMBPP. In fact, glutaraldehyde-fixed APC were better than mitomycin C-treated APC at presenting nonpeptide antigens to Vγ2Vδ2 T cells (Fig. 8). Within 5 minutes of incubation with the prenyl pyrophosphate antigen, HMBPP, ~75% of the antigenic activity of HMBPP was already associated with the APC. Pulsing of HMBPP onto glutaraldehyde-fixed APC peaked at 45 minutes of incubation, while mitomycin C-treated APC required 60–90 minutes of incubation. These results demonstrate that the putative antigen presenting molecule on the APC associates with prenyl pyrophosphates very rapidly (<5 minutes). These data are consistent with the rapid activation of γδ T cells that we and others have observed (24–26), and with our IPP binding results (data not shown).

Figure 8

Prenyl pyrophosphate antigens pulse rapidly onto the APC cell surface

Recognition of prenyl pyrophosphates does not require APC expression of classical MHC Class I, MHC Class II, β₂M dependent, or CD1 molecules

We earlier demonstrated that Vγ2Vδ2 T cells do not require prenyl pyrophosphates to be internalized or processed for presentation, and do not require professional antigen presenting cells (24). To determine if a known antigen presenting molecule was required for presentation of prenyl pyrophosphates, we tested mutant APC (24) that lacked these molecules and found their absence on the APC had no effect on prenyl pyrophosphate recognition and that monoclonal antibodies to these molecules did not inhibit prenyl pyrophosphate recognition (55). However, since Vγ2Vδ2 T cells are as efficient as dendritic cells at presenting peptide antigens to αβ T cells (56) and can present prenyl pyrophosphates to each other (24), we could not exclude that a known antigen presenting molecule on the Vγ2Vδ2 T cells themselves, was presenting to daughter T cells in these experiments. To exclude presentation by Vγ2Vδ2 T cells to each other, we directly crosslinked m-BZ-C₅-OPP to different APC cell lines that lack expression of known antigen presenting molecules and used the crosslinked APC to stimulate Vγ2Vδ2 T cells. In this way, we could avoid any possible presentation by Vγ2Vδ2 T cells since the antigens are covalently linked to the APC. We found that MHC class II-negative APC (24) including the transcription factor mutant RAJI Burkitt’s lymphoma cell line, RJ-2.2.5; the J.RT3–T3.5 thymoma; and the erythroleukemia, K-562, were able to present crosslinked m-BZ-C₅-OPP to Vγ2Vδ2 T cells (Fig. 9). Similarly, 721.221, that lacks HLA-A, -B, and -C expression; the erythroleukemia cell line, K-562, that lacks MHC class I; as well as the melanoma cell line, FO-1, that lacks β₂M expression, all presented m-BZ-C₅-OPP to Vγ2Vδ2 T cells (Fig. 9). Also, expression of CD1a, CD1b, CD1c, and CD1d molecules was not required since the EBV-transformed B cell line, DG. EBV, and the Burkitt’s cell line, RAJI, both lack these molecules (57) yet still present m-BZ-C₅-OPP. These data clearly demonstrate, therefore, that recognition of prenyl pyrophosphate antigens by Vγ2Vδ2 T cells does not require classical MHC class I, MHC class II, β₂m, or CD1 expression by the APC.

Figure 9

Expression of classical MHC class I molecules, β₂M-dependent molecules, MHC class II molecules, and CD1 molecules is not required for the presentation of prenyl pyrophosphate antigens to Vγ2Vδ2 T cells

Discussion

The molecular basis for prenyl pyrophosphate recognition by human Vγ2Vδ2 T cells is poorly understood due to the inability to identify an antigen presenting molecule or to measure binding of the Vγ2Vδ2 TCR to these compounds. We sought photoaffinity prenyl pyrophosphate antigens that would stably crosslink to the APC surface to aid in these studies. To achieve this goal, we have used m/p-BZ-(C)-C₅-OPP ester- and ether-linked photoaffinity analogs of FPP and HMBPP (40). We had previously found that m-BZ-C₅-OPP and p-BZ-C₅-OPP were substrates for three bacterial prenyl transferases, and underwent efficient chain elongation to polyprenyl diphosphates (40). We report here that these compounds also stimulate Vγ2Vδ2 T cells at lower concentrations than IPP. Recognition of the m/p-BZ-(C)-C₅-OPP ester- and ether-linked photoaffinity compounds was greatly affected by the type of linkage and the spacing from the benzophenone moiety and required the presence of the pyrophosphate moiety. Based on statin sensitivity and APC independence, recognition of m/p-BZ-(C)-C₅-OPP was clearly due to their direct antigenic activity rather than any ability to inhibit FPPS. Importantly, m/p-BZ-(C)-C₅-OPP antigens retained immunogenicity even after UV crosslinking to the APC surface. IPP and a non-stimulatory pyrophosphonate analog of BrHPP (BrHPCP) blocked the covalent crosslinking of m-BZ-(C)-C₅-OPP to the APC cell surface suggesting that they bind to the same sites on the APC as do the m-BZ-(C)-C₅-OPP antigens. m-BZ-C₅-OPP was able to stably associate with the cell surface of human hematopoietic and non-hematopoietic cell lines, including ones lacking known antigen presenting molecules, for stimulation of Vγ2Vδ2 T cells. Thus, the molecule(s) that the photoaffinity antigens bind to are broadly distributed, functionally non-polymorphic, and not a known antigen presenting molecule.

Photoaffinity derivatives of antigens, GTP, and other ligands have been used to dissect various aspects of cellular functions, and to define the binding of antigenic peptides to MHC class I. For instance, photoreactive derivatives of cyclosporins have been used to demonstrate the binding of cyclosporins to cyclophilins, and subsequent complex formation with calcineurin (58). Photoaffinity derivatives of antigenic peptides have been used to demonstrate that cell surface MHC class I glycoproteins do bind peptide antigens, and that this interaction takes place even in the absence of the αβ TCR (59). Further, a photoreactive derivative of the P. berghei antigenic peptide, P.b. CS 249–260, bound to cell associated MHC class I molecules (60), and was used to determine the peptide binding motif for the H-2K^d molecule (61). The same photoreactive peptide has also been used to demonstrate that the avidity of TCR-ligand interactions is strengthened by CD8 on T cells (62), and that CD8β (but not CD8α) was involved in p56 binding in lipid rafts. And recently, it was used to demonstrate that the α chain of the αβ TCR is involved not just in binding to the ligand, but is also involved in enhancing the CD8-TCR interaction (63). Other photoreactive probes have been used to identify the nucleotide binding sites in human IL-2 (64), GTP binding proteins that are biologically active in the T lymphocyte and thymocyte plasma membranes (65), and the active sites of enzymes. These studies demonstrate the usefulness of photoaffinity ligands/antigens to identify and isolate interacting or binding proteins.

For our study, we used benzophenone compounds that were originally developed as analogs of farnesyl pyrophosphate (40). These compounds are photoactivatable substrates for isoprenoid pathway enzymes such as farnesyl pyrophosphate synthase, farnesyl transferase, geranylgeranyl pyrophosphate synthase, and undecaprenyl pyrophosphate synthase, and can label these enzymes. Since we had shown that Vγ2Vδ2 T cells recognize farnesyl pyrophosphate (5, 24), we reasoned that these analogs might also be recognized. Indeed, m-BZ-C₅-OPP stimulated Vγ2Vδ2 T cells to proliferate like other prenyl pyrophosphates, even after photocrosslinking to the cell surface. This stimulation by m-BZ-C₅-OPP (which has a large aromatic benzophenone moiety at the end of the 5 carbon alkenyl chain (Fig. 1)) is consistent with our finding that the carbon chain closest to the pyrophosphate moiety plays the critical role in determining Vγ2Vδ2 T cell stimulation (21). The type of linkage and spacing from the benzophenone group was very important in determining bioactivity with the highest activity noted with ester linkage of the alkenyl pyrophosphate spaced 1 carbon from the benzophenone group. In many cases, Vγ2Vδ2 T cells could also distinguish between the meta and para isomers of BZ-(C)-C₅-OPP compounds similar to their ability to distinguish between the (R)- and (S)-stereoisomers of the chiral phosphoantigens, bromohydrin pyrophosphate and 3,4-epoxy-3-methyl-1-butyl pyrophosphate (66) and the (E)- and (Z)- forms of HMBPP (67). Recognition of m-BZ-C₅-OPP also requires the pyrophosphate moiety, since the benzophenone (BZ) photophore and the 4-maleimide derivative of benzophenone (both lacking the pyrophosphate moiety) failed to stimulate Vγ2Vδ2 T cell proliferation. Thus, like synthetic and natural phosphoantigens (21), recognition of m/p-BZ-(C)-C₅-OPP antigens is critically dependent on the phosphate moiety and adjacent alkenyl chain. Large moieties such as a benzophenone attached to the alkenyl chain or a ribonucleotide phosphate attached to the pyrophosphate group do not interfere with recognition if spaced sufficiently far away from the C₅-OPP structure.

Although, like bisphosphonates, m-BZ-C₅-OPP binds to FPPS, it has only very low activity as an inhibitor requiring 250 μM for 20% inhibition of FPPS activity as compared with 20% stimulation of Vγ2Vδ2 T cells at a 2,500-fold lower concentration of 0.1 μM. Thus, it is likely to function as a direct antigen. Supporting this mechanism of stimulation of Vγ2Vδ2 T cells, the response of Vγ2Vδ2 T cells to m-BZ-C₅-OPP is highly resistant to mevastatin inhibition. This is identical to prenyl pyrophosphate responses (Fig. 4A, B) and unlike bisphosphonate and alkylamine responses which are very sensitive to statin inhibition (Fig. 4A and Wang and Morita, manuscript in preparation). The photoaffinity antigens can also stimulate Vγ2Vδ2 T cells in the absence of additional antigen presenting cells like prenyl pyrophosphates (Fig. 4C and ref. 24). Thus, m/p-BZ-(C)-C₅-OPP antigens function as direct antigens for Vγ2Vδ2 T cells rather than as indirect stimulators through pharmacological inhibition of FPPS.

Although recognition of m-BZ-C₅-OPP by Vγ2Vδ2 T cells was specific and direct, it was not clear if the benzophenone photophore was crosslinking specifically or nonspecifically to the cell surface. To address this question, we used IPP and the biologically inactive pyrophosphonate analog of BrHPP, BrHPCP, to compete with m-BZ-(C)-C₅-OPP antigens for binding to the APC surface. As expected if m-BZ-(C)-C₅-OPP and IPP/BrHPCP were competing for the same specific binding sites, the stimulatory activity of photocrosslinked m-BZ-(C)-C₅-OPP for γδ T cells was diminished in a dose dependent manner by the presence of IPP or BrHPCP during ultraviolet crosslinking. This result strongly suggests that IPP and m-BZ-C₅-OPP compete for the same binding sites on the APC surface. Our results also would suggest an alternative explanation for the specific inhibitory activity of pyrophosphonate (methylene diphosphonate) and difluorodiphosphonate analogs of bromohydrin and iodohydrin pyrophosphate (20). Since BrHPCP prevents the crosslinking of m/p-BZ-(C)-C₅-OPP compounds (Fig. 5), it likely competes for the same binding sites on the cell surface like IPP. We speculate that rather than blocking dephosphorylation of phosphoantigens due to their non-hydrolyzable phosphonate bonds, these phosphonate compounds compete for binding with prenyl pyrophosphate antigens to the proposed presenting molecule. Unlike phosphoantigens, bound pyrophosphonate compounds are not recognized by the Vγ2Vδ2 TCR because of their structural differences from pyrophosphate compounds. Such inhibition of binding would be predicted to result in antigen specific antagonism but not to affect Vγ2Vδ2 T cell mitogen responsiveness identical to what was observed (20).

Prenyl pyrophosphates may bind to a plasma protein prior to their presentation at the APC cell surface. A soluble protein could bind IPP or HMBPP and inhibit presentation, to limit Vγ2Vδ2 T cell responses. Alternatively, a soluble protein could enhance presentation by binding IPP or HMBPP, and then transferring them to cell surface molecules for presentation. For example, apolipoprotein E binds the exogenous α–galactosyl ceramide lipid antigen for uptake and presentation by CD1d to αβ NKT cells (68). In this study, we found that binding of m-BZ-C₅-OPP to the APC, as measured by stimulation of γδ T cell proliferation, was inhibited by serum and by non-protein components of RPMI media. In the absence of serum and media components, natural prenyl pyrophosphate antigens, which were earlier reported not to associate with the APC cell surface (24), could be shown to stably associate with APC. However, this association is not very efficient, since it required 100–1,000 fold more antigen during pulsing to achieve the same stimulation as observed when the antigen was continuously present. These results suggest that unknown components of serum and media can diminish the binding of the negatively charged prenyl pyrophosphate antigens to the APC surface. Apolipoprotein A1 has been proposed to bind to the Vγ2Vδ2 TCR to enhance recognition of the F1 ATPase β subunit (69). It is possible that this lipoprotein interferes with prenyl pyrophosphate antigen binding to the putative presenting molecule. Serum albumin binding of the hydrophobic alkenyl chain of prenyl pyrophosphate could also compete for binding. Alternatively, this inhibition could be due to dephosphorylation of the antigens by alkaline phosphatase that is present in serum, since incubation of BrHPP with cells resulted in hydrolysis of the pyrophosphate moiety presumably through the action of cell surface alkaline phosphatase (20). RPMI media contains divalent cations, amino acids, and other compounds that are absent in PBS and that might interfere with binding of pyrophosphate antigens to the APC surface.

In the absence of serum and media components, we found that the binding of prenyl pyrophosphate antigens with APC was rapid, being detectable within 5 minutes (the least amount of time required for experimental manipulation) (Fig. 8). This binding of pyrophosphate antigens with the APC likely takes seconds since we found that ¹⁴C-IPP binding with APC was extremely rapid, taking only 30 seconds (minimum time required for experimental manipulation) to achieve near maximal binding. Although rapid, IPP binding showed very low affinity and was difficult to accurately measure (data not shown). It is unlikely that the prenyl pyrophosphate antigens require internalization for presentation since they can be pulsed onto APC that have been fixed with glutaraldehyde, supporting our previous observations (24).

Most tumor cells of human origin can present prenyl pyrophosphate antigens to Vγ2Vδ2 T cells (Table 1). These results, taken together with previous studies, might suggest that prenyl pyrophosphates associate nonspecifically with the APC cell surface for recognition. However, we and others have found that only APC of human origin can present nonpeptide prenyl pyrophosphate antigens to γδ T cells, since APC from mice and other species fail to stimulate Vγ2Vδ2 T cells (Table 1 and ref. 28, 30). Moreover, we now demonstrate that IPP and HMBPP can be pulsed onto the APC cell surface. Although the lack of presentation by xenogeneic cells could reflect species differences in accessory and/or costimulatory molecules (28), these results rule out the simple model where prenyl pyrophosphate antigens associate with the APC cell surface nonspecifically to stimulate Vγ2Vδ2 T cells.

Earlier studies could not rule out that Vγ2Vδ2 T cells were presenting nonpeptide antigens to daughter Vγ2Vδ2 T cells since recognition required the continuous presence of antigen. Since we could covalently-link m-BZ-C₅-OPP to the APC surface, human cell lines lacking known antigen presenting molecules could be tested for presentation of m-BZ-C₅-OPP to Vγ2Vδ2 T cells in the absence of soluble antigen, thus ruling out antigen presentation by the Vγ2Vδ2 T cells. Using the m-BZ-C₅-OPP photoaffinity antigen, we find that Vγ2Vδ2 T cells do not require classical MHC class I (HLA-A, HLA-B, and HLA-C), MHC class II, or CD1a, CD1b, CD1c, or CD1d molecules on APC for prenyl pyrophosphate recognition. These findings suggest that a novel cell surface molecule is functioning to present these antigens. However, this putative presenting molecule would be predicted to be widely distributed and non-polymorphic, given that most tumor cells (except for those lacking accessory molecules) can present antigen to Vγ2Vδ2 T cells despite coming from different tissues and different individuals.

Further supporting the existence of a presenting molecule is the restriction of recognition of prenyl pyrophosphate antigens to Vγ2Vδ2 T cells. We have shown that recognition is TCR mediated since transfection of Vγ2Vδ2 TCR cDNAs into the TCR⁻ mutant of the αβ T cell tumor, Jurkat, confers responsiveness to prenyl pyrophosphate antigens (11), and since recognition is blocked by mAbs to the γδ TCR (39, 70). Moreover, only Vγ2Vδ2 γδ T cell clones respond to the prenyl pyrophosphate antigens (39, 71, 72). Mutation of the Vγ2Vδ2 TCR in the Vγ2 and Vδ2 CDR3 regions and other CDRs can abolish prenyl pyrophosphate recognition while preserving anti-TCR mAb responses (Wang and Morita, manuscript in preparation and 73, 74, 75). However, there is no evidence for direct binding to prenyl pyrophosphates to soluble Vγ2Vδ2 TCRs (data not shown and 19). And, unlike murine γδ TCR recognition of T22 MHC class Ib molecules (76), there is no conserved amino acid motif in the Vδ2 CDR3 region that could mediate antigen binding. These results coupled with the small size of phosphoantigens (minimum recognition unit is methyl phosphate (21)), support the existence of an antigen presenting molecule.

Among the various stimulating compounds for γδ T cells, we hypothesize that only prenyl pyrophosphates are directly presented on the APC cell surface to the Vγ2Vδ2 TCR. Supporting this assertion, prenyl pyrophosphate recognition can be extremely rapid (10 seconds) (24, 25) and is not abolished by glutaraldehyde fixation of the APC (24). In contrast, we and others have found that stimulation of human Vγ2Vδ2 T cells by bisphosphonates (14–16), alkylamines (77, 78), and certain tumor cells (16) is indirect and mediated by the intracellular accumulation of isopentenyl pyrophosphate. However, it is unclear how this intracellular isopentenyl pyrophosphate is detected at the cell surface. We speculate that there exists an intracellular pathway where the putative antigen presenting molecule encounters IPP (and perhaps HMBPP from intracellular pathogens) in the cell leading to their transport to the cell surface. Evidence that this pathway utilizes transport by multi-drug related protein 5 transport has recently been reported (79). The ability to covalently attach a prenyl pyrophosphate analog to a molecule on the APC surface using photocrosslinking is, therefore, a significant advance and should assist in identifying this putative antigen presenting molecule for Vγ2Vδ2 T cells.

Acknowledgments

We thank Dr. David Leslie for technical assistance. We thank Chenggang Jin, Grafechew Workelamahu, Diana Colgan, Kristin Ness, Masashi Suzuki, and Amy Raker for critical review of the manuscript.

Footnotes

¹This work was supported by grants from the NIH National Institute of Arthritis and Musculoskeletal and Skin Disease (AR45504), the National Institute of Allergy and Infectious Diseases (Midwest Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research, AI057160), and the National Cancer Institute (CA113874) to C.T.M., the National Institute of Neurological Disorders and Stroke (NS29632) to G.D.P., and the National Institutes of General Medical Sciences (GM073216 to E.O. and GM58442 to M.D).

⁵Abbreviations used in this paper: β₂M, β₂-microglobulin; BZ, benzophenone; BrHPCP, bromohydrin pyrophosphonate; BrHPP, bromohydrin pyrophosphate; DATFP, diazo-3,3,3-trifluoropropionyloxy; EPP, ethyl-pyrophosphate; FPP, farnesyl-pyrophosphate; FPPS, FPP synthase; GPP, geranyl-pyrophosphate; HMBPP, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate; IPP, isopentenyl pyrophosphate; m, meta; MEP, mono-ethyl-phosphate; MMP, mono-methyl-phosphate; OPP, pyrophosphate; p, para.

Disclosures

The authors have no financial conflict of interest.

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