 |
INTRODUCTION |
Stimulating ligands for 
T lymphocytes are usually composed
of single peptides complexed at the surface of major histocompatibility complex molecules. Some small non-peptidic structures, however, may also constitute specific agonist ligands for T cells, particularly 
T lymphocytes. In human blood, about 3% of T cells initiate their physiological function upon recognition of small phosphorylated non-peptide antigens (phosphoantigens). This cognate interaction involves on the one hand phosphoantigens in the absence of major histocompatibility complex-presenting molecules, and on the other hand,
highly selective receptors
(TCR)1 of 
subtype. In nature, phosphoantigens that can activate human 
T
cells at nanomolar concentrations are produced by Gram-positive and
Gram-negative bacteria and also by some eukaryotic parasites and
plants. Synthetic analogues of natural phosphoantigens are also known,
but their stimulating concentrations for the reactive cells never go
below the micromolar range. Mycobacterium tuberculosis, the
agent of human tuberculosis, produces four distinct phosphoantigens. These molecules share a moiety that is responsible for the potent stimulation of 
cells seen in tuberculosis patients (1). The
structure of this common core is 3-formyl-1-butyl-pyrophosphate, a
recently described phosphoester (2). Its metabolic production might be
related to the non-mevalonate (or so-called Rohmer's) pathway for
isoprenoid precursor biosynthesis (3). 3-formyl-1-butyl-pyrophosphate is produced in very small amounts in slow-growing mycobacteria such as
Mycobacterium tuberculosis and only accumulates to
submicromolar concentrations in culture media from fast-growing
mycobacterial species (4). Getting large amounts of highly bioactive
phosphoantigens by purification routes from such natural sources is
therefore hard to conceive.
Such molecules could prove therapeutically useful for immunotherapeutic
approaches involving 
T cell-mediated immunity, such as
elicitation of anti-infectious protection or antitumor immunity (5, 6).
To address the need for readily available highly bioactive
phosphoantigens, we have developed a synthetic reagent called
bromohydrin pyrophosphate (BrHPP), whose biological properties on human
T cells are optimized compared with those of
3-formyl-1-butyl-pyrophosphate.
 |
MATERIALS AND METHODS |
Chemical Synthesis--
All glassware and equipment were dried
for several hours prior to use. Unless otherwise stated, the reagents
and starting material were from Fluka. Trisodium
(R,S)-3-(bromomethyl)-3-butanol-1-yl-diphosphate (BrHPP) was produced as white amorphous powder by the following procedure. Tosyl chloride (4.8 g, 25 mmol) and
4-(N,N-dimethylamino-) pyridine (3.4 g, 27.5 mmol; Aldrich) were mixed under magnetic stirring with 90 ml of
anhydrous dichloromethane in a 250-ml three-necked flask cooled in an
ice bath. A solution of 3-methyl-3-butene-1-ol (2.2 g, 25 mmol) in
about 10 ml of anhydrous dichloromethane was then slowly introduced
with a syringe through a septum in the flask, and the ice bath was then
removed. The reaction was monitored by silica gel TLC (pentane/ethyl
acetate, 85:15 (v/v)). After 2 h with constant stirring, the
mixture was precipitated by dilution into 1 liter of hexane and
filtered, and the filtrate was concentrated under reduced pressure.
This filtration/suspension step was repeated using diethyl ether, and
the resulting oil was purified by liquid chromatography on silica
gel (pentane/ethyl acetate, 85:15 (v/v)), yielding a yellow oil
of 3-methyl-3-butene-1-yl-tosylate (5.6 g, 23.5 mmol, 94% yield) kept
under dry N2 at 4 °C (positive mode ESI-MS:
m/z 241 [M + H]+;
m/z 258 [M + NH4]+;
m/z 263 [M + Na]+;
MS2 of m/z 258:
m/z 190 (C5H8 loss)).
Disodium dihydrogen pyrophosphate (51.5 mmol, 11.1 g) dissolved in
100 ml of deionized water (adjusted to pH 9 with NH4OH) was
passed over a cation exchange DOWEX 50WX8 (42 g, 200 meq of form
H+) column and eluted with 150 ml of deionized water (pH
9). The collected solution was neutralized to pH 7.3 using
tetra-n-butyl ammonium hydroxide and lyophilized. The
resulting hygroscopic powder was solubilized with anhydrous
acetonitrile and further dried by repeated evaporation under reduced
pressure. The resulting Tris (tetra-n-butyl ammonium)
hydrogenopyrophosphate (97.5% purity by HPAEC; see below) was stored
(concentration, ~0.5 M) at
20 °C in anhydrous
conditions under molecular sieves. 100 ml of a solution containing 50 mmol of Tris (tetra-n-butyl ammonium) hydrogenopyrophosphate (0.5 M, 2.5 eq) in anhydrous acetonitrile under magnetic
stirring in a 250-ml three-necked flask cooled in an ice bath were
slowly mixed with 20 mmol (4.8 g) of
3-methyl-3-butene-1-yl-tosylate introduced via a septum with a syringe.
After 20 min, the ice bath was withdrawn, and the reaction was left
under agitation at room temperature for 24 h. The reaction was
analyzed by HPAEC (see below), evaporated, and diluted into 50 ml of a
mixture composed of a solution (98 % volume) of ammonium
hydrogenocarbonate (25 mM) and 2-propanol (2 volume %).
The resulting mixture was passed over a cation exchange DOWEX 50WX8
(NH
, 750 meq) column formerly equilibrated
with 200 ml of the solution (98 % volume) of ammonium
hydrogenocarbonate (25 mM) and 2-propanol (2 volume %).
The column was eluted with 250 ml of the same solution at a slow flow
and collected in a flask kept in an ice bath. The collected liquid was
lyophilized, and the resulting white powder was solubilized in 130 ml
of ammonium hydrogenocarbonate (0.1 M) and completed by 320 ml of acetonitrile/2-propanol (v/v). After agitation, the white
precipitate of inorganic pyro- and mono-phosphates was eliminated by
centrifugation (2100 × g, 10 °C, 8 min). This procedure was repeated three times, the supernatant was collected and
dried, and the resulting oil was diluted in 120 ml of water. Remainders
of unreacted tosylates were extracted three times by chloroform/methanol (7:3 (v/v)) in a separatory funnel, and the water
phase was finally lyophilized. The resulting white powder was again
washed twice by acetonitrile/chloroform/methanol (50:35:15 (v/v)) and
dried under gentle N2 flow. 11.25 mmol of pure
3-methyl-3-butene-1-yl-pyrophosphate triammonium salt were obtained by
this procedure (75% yield) and were then dissolved in 200 ml of water
for oxidation. For 6 mmol of 3-methyl-3-butene-1-yl-pyrophosphate, an
aqueous solution of Br2 (0.1 M) kept at 4 °C
was added dropwise until appearance of a persistent yellowish color,
yielding after evaporation 5.8 mmol (2.3 g) of an acidic solution (pH
2.1) of BrHPP, which was immediately neutralized by passing over
DOWEX 50WX8-200 (NH
, 48 meq). The ammonium
salt of BrHPP obtained after lyophilization was dissolved in water and
separated from bromides by passing through Dionex OnGuard-Ag (2 meq/unit) cartridges and an on-line column of (100 meq, 21 g)
DOWEX 50WX8-200 (Na+) eluted by milli-Q water. Colorless
stock solutions of BrHPP (Na+) were filtered over Acrodisc
25 membranes of 0.2 µM and kept as aliquots at
20 °C.
HPLC--
Final purification of BrHPP was achieved by HPLC
(Spectra system P1000 XR device) on an analytic Symmetry 5 µ C18 column (Waters) eluted at 1 ml/min and 20 °C with the ternary
gradient indicated below. Upstream of detectors, a split of eluent
distributes 190 µl/min in the online MS detector (see below), and the
remaining 810 µl/min was sent to the Waters 996 photodiode array
detector. Single wavelength detection at
= 226 nm was of 7 milliabsorbance units for 6 µg of BrHPP injected in 25 µl
(Rheodyne injector). The gradient program was as follows: solvent A,
acetonitrile; solvent B, 50 mM ammonium acetate; solvent C,
water; 0-7 min, 5% B in C; 7.1-11 min, 100% C; 12-15 min, 100% A;
15-17 min, 100% C.
Mass Spectrometry and NMR--
Mass spectrometry was performed
with an LCQ ion trap mass spectrometer (Finnigan MAT, San Jose,
CA). LC-ESI-ion trap MS was performed with a standard Finnigan
ESI source in negative ion mode at a voltage of 4.3 kV. The heated
transfer capillary was kept at a temperature of 200 °C, the sheath
gas (N2) flow was 80 units, and the auxiliary gas
(N2) flow was 15 units. The maximum ion collection time was
set at 800 ms, and two microscans were summed per scan. The scan MS
range was m/z 75-500. MSn was
performed with a commercial nanospray ESI source (The Protein Analysis
Co., Odense, Denmark) using glass capillaries (The Protein Analysis
Co.), which were positioned directly at a distance of about 1 mm from
the entrance hole of the heated transfer capillary with the help of a
stereomicroscope. The capillaries were palladium and gold-coated for
electrical contact. The glass capillaries were filled with 5 µl of
analyte solution. Voltage of the nanospray needle was set to 700 V; no
nebulizer gas was used in this spray mode. The heated transfer
capillary was kept at a temperature of 150 °C. Samples were analyzed
in the negative ion polarity mode. Collision energy was adjusted
manually (range, 15-25%). NMR was done as previously published
(2).
Cell Culture--
Isolation of peripheral blood
lymphocytes (PBL):heparinized peripheral blood was taken from
healthy donors, and PBL were separated on Ficoll-PaqueTM PLUS
(Amersham Pharmacia Biotech), washed three times, and then cultured at
106 cells/ml in culture medium RPMI 1640 with
Glutamax-I (Life Technologies, Inc.), supplemented with 10% AB human
serum, 25 mM Hepes, 100 units/ml penicillin G, 100 µg/ml
streptomycin, and 1 mM sodium pyruvate.
Polyclonal
9
2 T cell lines were specifically raised by incubating
PBL (106/ml) in culture medium with
3-formyl-1-butyl-pyrophosphate (10 nM) plus 100 units/ml
IL2 (Sanofi, France) during 15 days. The expansion of
9
2 T cells
was followed by cytometric analysis, and only cultures showing more
than 95% TCR V
2 positive cells were used for subsequent experiments.
Measurement of Cytokines--
TNF-
release was measured by a
bioassay using TNF-
-sensitive cells (WEHI-13VAR ATCC CRL-2148).
Briefly, 104 
T cells/well were incubated with
stimulus plus 25 units of IL2/well in 100 µl of culture medium during
24 h at 37 °C. Then, 50 µl of supernatant were added to 50 µl of WEHI cells plated at 3 × 104 cells/well in
culture medium plus actinomycin D (2 µg/ml) and LiCl (40 mM) and incubated for 20 h at 37 °C. Viability of
WEHI cells was then measured with a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
assay. 50 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma; 2.5 mg/ml in phosphate-buffered saline) per well were added, and after 4 h of incubation at 37 °C, 50 µl of
solubilization buffer (20% SDS, 66% dimethyl formamide, pH
4.7) were added, and absorbance (570 nm) was measured. Levels of
TNF-
release were then calculated from a standard curve obtained
using purified human rTNF-
(PeproTech, Inc., Rocky Hill, NJ).
Interferon-
released by activated T cells was measured by a sandwich
enzyme-linked immunosorbent assay. 5 × 104 
T
cells/well were incubated with stimulus plus 25 units of IL2/well in
100 µl of culture medium during 24 h at 37 °C. Then, 50 µl
of supernatant were harvested for enzyme-linked immunosorbent assay
using mouse monoclonal antibodies (BIOSOURCE,
Camarillo, CA).
Microphysiometry--
The cell acidification rate was monitored
using a cytosensor microphysiometer (Molecular Devices, Crawley, UK),
which measures pH of extracellular fluid using a silicon-based method
(7). The raw data from sensor output give mV = f(t), which may be converted to pH = f(t). The system allows cells (8 × 105) disposed in sensor chambers to be irrigated (flow on
period, 90 s) by low buffered RPMI medium (Molecular Devices)
containing phosphoantigen or not; then, during a flow off period (30 s), the sensor data are used to calculate a slope, giving
pH/
t and referred as to the acidification rate. In
each experiment, 32 × 105 
T cells were
resuspended in 30 µl of low buffered medium plus 10 µl of melted
low temperature-melting agarose at 37 °C. 10 µl of the mixture
were rapidly spotted on a cytosensor cell capsule. After 10 min the
cell capsule was assembled and loaded in the sensor chamber of a
microphysiometer. The experiments were run at 37 °C, and the
low buffered medium (pH 7.4) was perfused at 100 µl/min.
Phenotype Analysis by Flow Cytometry--
5 × 105 cells were washed in phosphate-buffered saline
containing 5% fetal calf serum and incubated for 30 min at
4 °C with anti-CD3-PE and anti-
2-fluorescein isothiocyanate
monoclonal antibodies (Beckman Coulter) or isotypic controls.
Samples were then washed in phosphate-buffered saline, 5% fetal calf
serum and immediately acquired by an EPICS XL flow cytometer (Beckman Coulter).
 |
RESULTS |
Molecular Overlay of 3-Formyl-1-butyl-pyrophosphate and
BrHPP--
Former structure-activity relationship studies of natural
and synthetic phosphoantigens have shown that among monoesters of pyrophosphate (8, 9), several organic esters with chemical reactivity
(e.g. of Sn-2 type) (10) presented 
cell-stimulating bioactivities higher than that of single chain alkyl, such as ethyl
pyrophosphate. In addition to these parameters, a topological fit of
the alkyl chain clearly contributes to optimize recognition by the

TCR. To select a synthetic phosphoantigen matching
3-formyl-1-butyl-pyrophosphate (3fbPP) as much as possible, we overlaid
this latter compound and several synthetic compounds. The
bromohydrin phosphate BrHPP was selected for its good superimposition
to 3fbPP (arbitrarily shown as the same enantiomers in Fig.
1) and two other 
cell-stimulating phosphoantigens, isopentenyl pyrophosphate and ethyl pyrophosphate (8, 9). Both 3fbPP and BrHPP molecules distribute hydrogen bonds from the carbonyl and hydroxyl groups similarly in their surrounding volume while aligning for the remaining part of the molecule. Shorter homologues (C4) of BrHPP have been produced and were
described elsewhere, but they do not present bioactivity for human T
lymphocytes (10). In addition, this compound may be conveniently
derived from oxidation of isopentenyl pyrophosphate (see below).

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 1.
Molecular envelopes of synthetic
BrHPP and of some natural phosphoantigens:
3-formyl-1-butyl-pyrophosphate and isopentenyl pyrophosphate.
Molecular envelopes of mycobacterial 3-formyl-1-butyl-PP,
isopentenyl-PP, and BrHPP were computed for molecules in triacidic form
under stretched conformation. Molecular volumes were as follows:
3-formyl-1-butyl-PP, 198 Å3; isopentenyl-PP, 191 Å3; BrHPP, 199 Å3. Molecular surfaces were as
follows: 3-formyl-1-butyl-PP, 170 Å2; isopentenyl-PP, 163 Å2; BrHPP, 176 Å2. Computing and images were
generated with Alchemy 2000 (Tripos) and Swiss PDBViewer 6.3.
|
|
Chemical Synthesis of BrHPP--
The procedure for BrHPP synthesis
was modified from Ref. 11 and is summarized in Fig.
2. A fully detailed description of this
synthesis, which yields the racemate of BrHPP, due to asymmetry of the C3 position, is given under "Materials and Methods." Thus, in the absence of further enantiomer resolution of the synthetic mixture, the produced BrHPP compound corresponds to the racemic structure represented in Fig. 2. The produced BrHPP was essentially devoid of unreacted reagents when using the separation scheme based on
differential solvent reprecipitation, as described under "Materials
and Methods." 4-20% of the recovered material still corresponded to
other products (e.g. phosphate), warranting a final step of
HPLC separation (see below). At this step, BrHPP was obtained as a
triammonium salt, which was found to interfere with several cell
culture assays (data not shown). It was then converted to BrHPP
(Na+ form) by cation exchange. This latter form is
stable in aqueous solutions and can be stored at
20 °C for 4 months without detectable structural degradation.
Chromatographic and Structural Assignment of BrHPP--
Because of
the halohydrin structure of BrHPP, its chromatographic analysis
could not be undertaken by HPAEC as described for natural
phosphoantigens (12), because hydroxide eluents of HPAEC rapidly
catalyzed an epoxide rearrangement by HBr elimination (data not shown).
Therefore, a chromatographic procedure for BrHPP analysis was based
upon use of near-neutral pH eluents. The stock solutions of BrHPP were
analyzed by ion pair reverse-phase C18 HPLC and monitored by UV-visible
diode array and MS detection following a procedure described
previously, with minor modifications (see "Materials and Methods"
and Ref. 1). As shown in Fig. 3A, several UV-absorbing and
phosphorylated contaminants eluted close to the major peak of BrHPP
(sample injected: 0.75 mM, Rt = 5.3 min.). Ion-trap MS in negative mode of the collected fractions gave a
unique set of signals at m/z 341 and 343, as
expected for the pseudomolecular anion of BrHPP. Its composition,
C5H12P2O5Br, was
supported by the relative abundance of natural bromine isotopes evidenced by zoom scan (Fig. 3B). Negative mode
MS2 of m/z 341 showed a unique
fragment of m/z 261 corresponding to an epoxide
rearrangement after HBr loss. Subsequent MS3 of
m/z 341, corresponding to MS2 of
m/z 261, showed its [(M
H)
H20] fragment (m/z 243) as well as
diagnostic ions for pyrophosphate and phosphate
(m/z 159, P2O6H;
m/z 97, PO4H2) moieties,
respectively. 1H NMR (D2O) confirmed the BrHPP
structure:
1.41 (s, 3H), Me; 2.06 (t, J = 6 Hz, 2H), CH2; 3.56 and 3.60 (AB, J = 11 Hz, 2H), CH2Br; 4.10 (m, 2H), CH2O.
31P NMR (D2O) was as follows:
10.38 (d, J = 19.5 Hz);
11.04 (d,
J = 19.5 Hz).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 3.
Chromatographic isolation and mass
spectrometry of BrHPP. A, BrHPP was analyzed using
reverse-phase HPLC and diode array detection in UV over the range
of 200-400 nm. The major fraction absorbing at low wavelength (226 nm)
and eluting around 5 min corresponds to BrHPP, as identified by MS
analysis. The dotted box shows the BrHPP fraction collected
for MS and biological analysis. B, ion trap MS analysis of
the BrHPP fraction in negative ionization mode. Full scan analysis of
the HPLC eluate was obtained using the standard LC-MS coupling and ESI
source, whereas MSn and zoom scan were done by
nanospray of the purified BrHPP. The main BrHPP spectrum shows two
(M H) pseudomolecular ions with equal relative
abundance (zoom scan) due to natural bromine isotopes. Their respective
MS2s show only HBr loss, both leading to the same epoxide
rearrangement (m/z 261). The latter ion
fragments further in MS3 by water loss and generation of
both P and PP daughter ions.
|
|
Biological Properties of BrHPP: Receptor Profiling of
BrHPP--
To search for further putative receptors for BrHPP, a high
concentration (10 µM) of this molecule was assayed for
inhibition of the selective binding of various reference radioligands
on their nominal receptors (13). BrHPP was not found to interfere significantly with any of the 70 binding assays tested (see
"Appendix"). This suggested that mammalian receptors for BrHPP
might be restricted to the formerly characterized V
9/V
2-encoded
antigen receptors borne by phosphoantigen-reactive human 
T
lymphocytes (1, 8, 9, 14-16).
BrHPP Activates Proliferation and Cytokine Release by TCR

+ T Lymphocytes--
T lymphocytes respond to
antigenic activation by proliferating, secreting cytokines, and/or
mediating toxicity for target cells. Accordingly, when they are
stimulated by the mycobacterial phosphoantigen 3fbPP, 
T cells
expressing the V
9/V
2-encoded TCR expand specifically in culture
and secrete TNF-
(2). Thus we tested whether increasing
concentrations of BrHPP would lead to the selective outgrowth of the
V
9/V
2 T lymphocytes in in vitro cultures of PBL. The
result of a representative experiment (of 10 independent ones) is shown
in Fig. 4A. BrHPP proved to be
as potent a stimulus as whole mycobacterial extract for the in
vitro expansion of the V
9/V
2 T cells. This property was
abrogated by dephosphorylation of BrHPP using alkaline phosphatase in
the culture, demonstrating that BrHPP owes its bioactivity on 
T cells to its pyrophosphate moiety, a characteristic hallmark of all
natural and synthetic phosphoantigens (1, 17). In addition to
proliferative responses, human 
T cells reactive to
phosphoantigens frequently secrete cytokines in response to antigen
stimulation. When 
T cells were exposed to BrHPP, the production
of high levels of interferon-
(Fig. 4C) and TNF-
(Fig.
4D) in the culture medium correlated strongly with the
concentrations of BrHPP used. These observations indicate that BrHPP
presents the same T cell-stimulating property as natural
phosphoantigens.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 4.
BrHPP mimics the natural
 T cell stimulus found in crude
hydrosoluble mycobacterial extract. A, changes of cell
morphology (lymphocytes gated in the ellipsoid window) and of TCR
 + phenotype in cultures of total lymphocytes with 100 nM BrHPP (numbers indicate the ratio of  T
cells over total lymphocytes). B,  T cell outgrowth
ensues after long term culture of peripheral blood lymphocytes
stimulated with mycobacterial extract, BrHPP, but not with
dephosphorylated BrHPP (culture medium with BrHPP and containing 1 unit/ml Escherichia coli alkaline phosphatase), as compared
with unstimulated cultures. M.tb., M. tuberculosis. C and D, secretion of
interferon- (IFN- ) (C) and TNF-
(D) by cultured  T cells in response to BrHPP.
|
|
BrHPP Causes Early Activation of Specific 
T
Cells--
Intracellular transduction of activating
signals delivered by encounter of the natural phosphoantigen 3fbPP
involves extracellular acid release by reactive 
T cells (2). A
similar pathway of signal transduction could be expected for
recognition of a phosphoantigen mimicking 3fbPP, such as BrHPP. We
tested this using CytosensorTM microphysiometry, to detect whether

T cell activation with BrHPP led to early acid release, as
already reported for 
T cells (18). Fig.
5A shows that a transient (10 min) pulse of 
cells with BrHPP does result in acid release (7). As illustrated in Fig. 5B, the dose-response relationship in
this experiment matched the dose responses for cytokine release (Fig. 4, C and D). Whereas this early metabolic
response was not sustained at low BrHPP concentrations (5 and 25 nM), the initial burst was followed by sustained signaling
for at least 1 h when cells were triggered by high BrHPP
concentrations (50-100 nM, Fig. 5A). In this
latter case, the metabolic burst presented a particularly rapid onset,
as evidenced by comparing the raw data from an unstimulated chamber
(Fig. 5C, lower trace) and from a chamber
perfused with BrHPP (Fig. 5C, upper trace). When
taking into account the delay for BrHPP diffusion onto the
cells (7 s in these assays), the mV collapse recorded in the perfused
chamber indicated that extracellular acidification by 
T cells
had occurred about 10 s after exposure to BrHPP. Thus, under
saturating concentrations, phosphoantigen recognition by the 
TCR
leads to very rapid activating events.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 5.
Perfusion of
 T cells with BrHPP stimulates a
rapid metabolic activation witnessed by early extracellular
acidification. A, pulsing of  T cells in
cytosensor chambers with increasing concentrations of BrHPP induce a
sustained extracellular acidification. B, microphysiometric
titration of the BrHPP dose effect on  cells. The area below each
acidification curve over 1 h in the presence of BrHPP was
calculated, normalized to 100% using the largest response, and plotted
as a function of BrHPP concentration. C, a physiological
delay of around 10 s separates exposure to BrHPP and the metabolic
response of  cells, evidenced by a relative drop in redox
potential (mV) of the stimulated versus unstimulated trace
(arrows).
|
|
 |
DISCUSSION |
This paper details the structure, chemical synthesis, and
biological property of bromohydrin pyrophosphate, a novel molecule activating human 
T lymphocytes. Based on the molecular overlay of this compound with the natural 
T cell ligand found in
mycobacteria, it was hoped that this synthetic compound could mimic the
biological properties of the naturally occurring 3fbPP,
i.e. activation of a specific 
T cell subset in human
blood (see Ref. 19 for a review). We describe a convenient and
straightforward mode of synthesis for producing BrHPP. This simple
method is based on pyrophosphorylation of the tosylated C5 precursor,
followed by stoichiometric oxidation of the pyrophosphoester product in
aqueous bromine. Because the compound carries a chiral C3, the
resulting product is a racemic mixture used without further resolution
of enantiomers. This straightforward and inexpensive synthesis is followed by a purification scheme involving solvent precipitation, LC, and HPLC to eliminate residual inorganic phosphate and bromide. The
final stock solutions of BrHPP (Na+) salts are very stable
and can be stored for several months without degradation. Little
information about structural changes of the organic moiety of
phosphorylated metabolites is usually drawn from HPLC-MS in negative
mode, thereby limiting its use as an analytical tool. The mass spectral
data of BrHPP presented here demonstrate a highly sensitive detection
of BrHPP in aqueous phases, and its bromine content enables unambiguous
detection for pharmacological follow-up studies. For the reasons listed
above, BrHPP appears to be a promising lead candidate for
therapeutic explorations among synthetic phosphoantigens.
In vitro cultures of bulk human lymphocytes carried out in
the presence of 100 nM BrHPP and IL2 lead to the systematic
expansion of T lymphocytes, which express the phosphoantigen-reactive
9
2 TCR, and no other cell subset. This has been described
previously for total lymphocyte populations stimulated by crude
extracts from M. tuberculosis and is known to rely upon the
presence of several stimulating phosphoantigens. Here, BrHPP also acts
as a phosphoantigen agonist, because BrHPP dephosphorylation abolishes this bioactivity. Exposure to BrHPP also elicits TNF-
and
interferon-
release, indicating that the full range of 
T cell
effector response is activated by this ligand. Microphysiometric
analysis showed that this activation results from exposure to a typical activating agonist (20). Its early signal transduction involves a BrHPP dose-dependent extracellular acid release, as was
shown for 
T cells stimulated with peptide-major
histocompatibility complex tetramers (21). Whereas high bioactive doses
of BrHPP triggered a strong acidification burst followed by a sustained intracellular signaling, suboptimal BrHPP concentrations (5 nM) led to barely detectable signaling. When rapidly
exposed to saturating BrHPP concentrations, 
cells respond by
extracellular acidification within about 10 s, indicating that
little (if any) intermediate processing of the stimulating BrHPP occurs
prior to triggering the T cell reaction.
In summary, although synthetic BrHPP presents the same biological
properties as natural phosphoantigens, the possibility of synthesizing
gram amounts from simple procedures bypasses the production drawbacks
of the natural counterparts. This makes BrHPP an attractive candidate
for investigations of selective 
T cell-based immunomodulation
approaches. Future studies will evaluate the potential of this novel
immunostimulating molecule in subunit vaccines where 
T cell
contribution in vivo is expected to be beneficial, such as
antituberculous immunity and protection against acute leukemia.