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T Cells by Aminobisphosphonate Antigen1
Department of Immunology and Cell Biology, Graduate School of Biostudies and Graduate School of Medicine, Kyoto University, Kyoto, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells respond to nonpeptide Ags such as
pyrophosphomonoesters and alkylamines in a TCR-dependent manner
in the absence of other APCs. Recently, aminobisphosphonates such as
pamidronate have also been shown to activate human T cells. In
the present study, we indicate that activation of primary T
cells by pamidronate strictly depends on the presence of
monocyte-lineage cells, unlike that by pyrophosphomonoesters. Thus,
although pamidronate induced cell clustering, proliferation, and
IFN- production of T cells in the culture of PBMC, it failed
to induce any of these activities in the culture of purified primary
T cells. By adding back the purified monocytes, however, both
cell clustering and IFN- production of T cells by pamidronate
could be restored. The pamidronate-pulsed, but not untreated,
myelomonocytic line, THP-1, was capable of activating the purified
T cells to produce IFN-, which was associated with the
down-regulation of TCR. Furthermore, pamidronate-pulsed THP-1
cells were significantly more susceptible to T cell-mediated
cytotoxicity than untreated THP-1. Also, TCR-defective Jurkat T cells
transfected with TCR genes produced a significant level of IL-2
in response to the pamidronate-pulsed THP-1 cells. These results have
suggested strongly that human T cells are functionally activated
via TCR by aminobisphosphonate Ag presented on the surface of
monocyte lineage cells rather than directly by its free form
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells recognize peptide Ags
presented by MHC class II and class I molecules, respectively
(1). A portion of CD4/CD8 double-negative T cells
has been shown to recognize lipophilic nonpeptide Ags presented by CD1
molecules (2). On the other hand, human T cells
have been indicated to respond to a series of small nonpeptide
compounds, such as pyrophosphomonoesters (3, 4, 5, 6, 7, 8, 9, 10, 11) and
alkylamines (12), derived from various
micro-organisms as well as unprocessed proteins such as staphylococcal
enterotoxin A (13, 14). It was indicated that the
essential component of antigenic structures of the former includes a
short hydrocarbon chain attached to a pyrophosphate group or an amino
group (3). Although the involvement of V2V2-TCRs in
the recognition of these nonpeptide Ags has been demonstrated, the
modes of recognition have remained to be elucidated
(15).
It was reported previously that pyrophosphomonoester Ags induced
proliferation and cytokine production of human T cells in the
absence of other specialized accessory cells, although cell-cell
contact among T cells was necessary for the activation
(16). Based on these findings, several distinct mechanisms
have been proposed for the recognition of such nonpeptide Ags by human
T cells (5). T cells may directly recognize
the free Ags via TCR-like Igs, but they require additional
costimulatory signals for the activation to occur through unidentified
accessory molecules via intimate cell-cell contact. Alternatively, the
Ags may transiently associate with the surface of T cells either
specifically or nonspecifically, and the T cells are activated
only by such presented Ags, for instance via efficient TCR
cross-linking. However, since T cells per se could not be pulsed
effectively with these Ags to activate them, the presentation model has
remained to be proved (17).
Recently, another group of compounds, aminobisphosphonates such as
pamidronate, was demonstrated to be capable of activating the primary
human T cells both in vitro and in vivo (18, 19). Aminobisphosphonates contain a short hydrocarbon chain
attached to a bisphosphonate residue, mimicking antigenic
pyrophosphomonoester compounds. In addition, they have an alkyl
amine moiety. Thus, aminobisphosphonates are unique in having the
chemical features of both pyrophosphomonoester and alkylamine
Ags to stimulate T cells. In the present study, we
demonstrate that activation of the primary human V2V2-TCR-bearing
T cells in the peripheral blood by pamidronate is strictly dependent on
the presence of adherent monocyte lineage cells unlike that by
pyrophosphomonoesters. We also show that the primary T cells as
well as the TCR-defective Jurkat cells transfected with V2V2-TCR
genes are activated by a monocyte lineage tumor cell line pulsed with
pamidronate. These results have suggested strongly that human T
cells are activated via V2V2-TCR by pamidronate bound on the
surface of monocyte lineage cells in the blood, but very little by its
free form. Potential clinical application of the present findings will
be also discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monoethyl pyrophosphate (MEP)3 was prepared by allowing ditriethylammonium phosphate in acetonitrile to react with ethyl alcohol in the presence of trichloroacetonitrile and was purified by Q-Sepharose HP anion exchange column chromatography (2.5 cm diameter x 10 cm long) with a linear gradient from 0 through 500 mM triethylammonium bicarbonate buffer, pH 7.5 (3). The product was eluted at 180 mM, lyophilized, resuspended in water, and converted into its sodium salt form by the use of Dowex 50W cation exchange column chromatography (2.5 cm diameter x 10 cm long) as previously described. Pamidronate was purchased from Novartis (Nuernberg, Germany). Isobutyl amine and 3-aminopropyl phosphonic acid were obtained from Aldrich (Chicago, IL), and the latter was converted into the sodium salt form as described above.
Cell lines
A V2V2-TCR-bearing clone, 12G12, was established
previously from the PBMC of a patient with tuberculoid leprosy
stimulated with Mycobacterium tuberculosis H37Ra
(4). A T cell line, MS, was derived from PBMC of a
healthy donor stimulated with 200 µM MEP. They were maintained in the
modified Yssel’s medium supplemented with 10% pooled human AB serum
or human serum albumin by repeated stimulation with 1 µg/ml PHA-L or
200 µM MEP. A human leukemia cell line of myelomonocytic lineage,
THP-1; a TCR-deficient mutant line of Jurkat, J.RT3-T3.5; and a
Burkitt’s lymphoma, Daudi, obtained from American Type Culture
Collection (Manassas, VA) were all maintained in the RPMI 1640
supplemented with 10% FCS.
Cell separations
Primary T cells were enriched as follows.
Ficoll-Paque-purified PBMC (2 x 107) were
suspended in the modified Yssel’s medium and incubated at 37°C in a
25-cm2 flask for 1 h. Nonadherent cells
recovered by gentle pipetting were incubated with Dynabeads (Dynal,
Chantilly, VA) coated with anti-CD4, anti-CD8, and
anti-CD19 mAbs (2 x 108, 2 x
108, and 4 x 108,
respectively) on ice for 1 h, followed by the depletion of
bead-conjugated cells with a magnet. The purity of T cells was
about 95% in the CD3+ cell population as judged
by flow cytometry. For the preparation of accessory cells of monocyte
lineage, Ficoll-Paque-purified PBMC (5 x
107) were suspended in FCS and mixed with 5
x 109 2-aminoethylisothioururonium
bromide-treated sheep RBC. After incubation on ice for 1 h,
rosette-forming T cells were removed by Ficoll-Paque, and the resultant
cells (1 x 107) were treated with Dynabeads
coated with anti-CD19 mAb (4 x 108).
After incubation on ice for 1 h, B cells were removed using a
magnet. Over 95% of the recovered cells were OKM1 positive.
Ag pulsing of THP-1 cells
THP-1 cells (1 x 107) were incubated with 0.1 or 1 mM pamidronate for 1.5 h at 37°C and washed five times with the modified Yssel’s medium before use.
Cell cultures
For the long-term cell expansion assay, PBMC from healthy donors
were cultured at 1.7 x 106/ml in modified
Yssel’s medium along with the indicated concentrations of stimulants
in the presence of 20 U/ml IL-2 (provided by Shionogi, Osaka, Japan).
The cells were passaged every 2 days. On day 14, viable cells were
separated by Ficoll-Paque for the flow cytometric analysis. For the
analysis of IFN--producing cells and detection of IFN- secretion,
the enriched T cells were cultured at 1.7 x
106/ml as described above for 48 and 24 h,
respectively. For the reconstitution experiments, 2 x
105 normal monocyte-enriched cells prepared as
described above or 1.25 x 105 Ag-pulsed
THP-1 cells were added to the culture of enriched T cells.
Flow cytometric analysis
For the detection of T cells, the cultured cells were
two-color stained using PE-conjugated anti-CD3 (Becton Dickinson,
Mountain View, CA) and FITC-conjugated anti-V2-TCR. For the
cytoplasmic staining of IFN-, the cells were first stained with
FITC-conjugated anti-V2-TCR, fixed with 1% paraformaldehyde,
permealized with 0.5% saponin, and then stained with PE-conjugated
anti-IFN- (PharMingen, San Diego, CA). The stained cells were
analyzed with a FACScan analyzer (Becton Dickinson). Controls with
isotype-matched Abs established the quadrants in such a way that >99%
of the cells were in the double-negative region.
Proliferation assay
T cell lines were cultured at 1.5 x
105/100 µl in modified Yssel’s medium in
round-bottom 96-well plates with varying concentrations of Ags in
triplicate. After 36 h the cells were pulsed with
[3H]thymidine (2 µCi/well; 1 Ci = 37
GBq) for 12 h, harvested, and assayed by liquid scintillation
(3).
ELISA
Culture supernatants were harvested and examined for IFN-
using a high sensitivity human IFN- ELISA system (Biotrak; Shinjuku,
Tokyo, Japan).
Cell aggregation assay
Purified T cells were cultured at 1.2 x
106/ml in modified Yssel’s medium in 48-well
plates either alone or with 200 µM MEP or 10 µM pamidronate. Cell
aggregation was observed under the microscope.
Cytotoxicity assay
THP-1 cells preincubated with medium or 1 mM pamidronate at
37°C for 1 h were washed five times and labeled with 100 µCi
of Na51Cr for 1 h. A T cell clone,
12G12, was added to the labeled THP-1 cells (1 x
104) at E:T cell ratios of 5:1, 10:1, and 20:1
and incubated for 5 h. Chromium release was measured on a gamma
counter, and the specific release (percentage) was calculated as
follows: (experimental release - spontaneous release)/(maximum
release - spontaneous release) x 100.
TCR gene transfection
cDNA was synthesized from 5 µg of total RNA extracted from a
T cell clone, 12G12, using a SuperScript II preamplification
system (Life Technologies, Gaithersburg, MD), and the full-length TCR
- and -chain cDNAs were obtained by PCR using primers
within the 5'-untranslated regions of the V2 and V2 genes and
3'-untranslated regions of the C and C genes, respectively. The
primers used were ggg-ctc-gag-gac-acc-gct-tta-caa-cga and
ggg-tct-aga-gtg-agg-ttc-tct-gtg-t for TCR -chain and
ggg-ctc-gag-aac-act-tgt-gtg-ttg-gtt-ca and
ggg-gga-tcc-agt-gta-tca-ctt-gta-gga-g for TCR -chain, respectively.
They were linked to BstXI adaptors and subcloned into the
expression plasmid pEF-BOS provided by Dr. Shigekazu Nagata (Osaka
University, Suita, Osaka, Japan) to yield pEF-BOS- and pEF-BOS-,
respectively. A TCR--Jurkat mutant, J.RT3-T3.5
obtained from American Type Culture Collection, was transfected with
the digested pEF-BOS- and pEF-BOS- (20 µg each) together with
0.7 µg of pST-NeoB digested with XhoI by electroporation.
The cells were plated into twenty 96-well round-bottom plates and
selected with 1 mg/ml Geneticin (Life Technologies) beginning on day 2.
Wells containing TCR-expressing transfectants were identified by
flow cytometry. The cloned CD3+
TCR+ transfectants were tested for IL-2
production by stimulation with anti-CD3 and PMA, and the positive
clones were selected.
IL-2 releasing assay
Jurkat transfectants were cultured in 96-well round-bottom plates at 2 x 105 cells/well in the presence or the absence of varying concentrations of MEP or pamidronate together with 10 ng/ml PMA. When indicated, THP-1 cells pulsed with pamidronate (1.25 x 105) or Daudi cells (2 x 105) were added to the culture. After 24 h, supernatant was harvested, and IL-2 activity was assessed by the CTLL-2 assay. Namely, IL-2-dependent CTLL-2 cells were cultured at 3 x 103/well with or without the culture supernatants at a final dilution of 1/5 for 24 h in triplicate, pulsed with [3H]thymidine (0.5 µCi/well) for an additional 7 h, and harvested for the counting of radioactivity by a liquid scintillation beta counter.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells
PBMCs derived from a healthy donor were incubated with 10 µM
pamidronate in the presence of 20 U/ml IL-2 for 2 wk and examined for
the proportion of V2-TCR+ T cells in
CD3+ cell population. Although 20 U/ml of IL-2
alone failed to induce the expansion of
V2-TCR+ T cells (2.1 to 1.2%; Fig. 1
A), the percentage of
V2-bearing T cells markedly increased after culture with pamidronate
at a level comparable to those with representative stimulants, MEP and
isobutyl amine (IBA; Fig. 1, B, C, and
E). Since the total cell number increased from 3 x
106 to 5 x 107, it
was calculated that the absolute number of
V2-TCR+ cells expanded roughly 1000-fold
(3.6 x 104 to 3.5 x
107) during the 2 wk of culture with pamidronate.
Dose-response analysis revealed that pamidronate exhibits comparable
efficiency as MEP to stimulate V2-TCR+ cells,
which is nearly 100 times more efficient than IBA (Fig. 1F).
Pamidronate at 100 µM or more, however, resulted in the significant
reduction of the activity most likely due to toxicity (Fig. 1F). We previously reported that a pyrophosphate (P-O-P)
backbone in pyrophosphomonoester Ags was critical for eliciting higher
activity (3). For instance, the elimination of one
phosphate residue from isopentenyl pyrophosphate significantly reduced
the stimulatory activity. We then removed a phosphonate residue along
with a hydroxyl group from pamidronate and examined its stimulatory
activity for T cells. As shown in Fig. 1, D and
F, 3-aminopropyl phosphonate totally failed to expand
T cells at any concentration up to 1 mM, although the compound retained
the amino group. These results suggested a critical requirement for
bisphosphonate moiety (P-C-P) with a hydroxyl group of pamidronate for
the stimulating activity of T cells. Identical results were
obtained in PBMCs from three independent healthy donors (data not
shown).
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T cells by pamidronate
We then examined the stimulatory activity of pamidronate for the
homogenous human T cell lines, clone 12G12 and polyclonal line
MS, compared with that of MEP. MEP stimulated the proliferation of both
T cell lines in a dose-dependent manner (Fig. 2A) as reported previously
(3). Unexpectedly, however, pamidronate exhibited no
detectable stimulatory activity for either T cell line
throughout the entire dose range up to 1 mM (Fig. 2A). This unresponsiveness was unlikely to be due to the
increased susceptibility to the toxicity by pamidronate, since the
lines precultured with 10 µM pamidronate normally proliferated in
response to MEP (data not shown). We, therefore, re-evaluated the
stimulatory activity of pamidronate for the primary T cells
using IFN- production as an indicator of activation. As
shown in Fig. 2B, pamidronate (10 µM) induced abundant
IFN- secretion in the culture of whole PBMC comparable to the effect
of MEP (data not shown). When PBMC had been depleted of dish-adherent
cells, however, pamidronate failed to induce IFN- secretion over the
background level (Fig. 2B). To identify the
IFN--producing cells, intracellular staining of IFN- was
performed. The primary T cells were enriched up to 95% in the
CD3+ population by extensive depletion of
adherent cells followed by the additional elimination of
CD4+, CD8+, and
CD19+ cells. As shown in Fig. 2C, MEP
induced IFN- production selectively in the
V2-TCR+ cell population (Fig. 2C,
b), while pamidronate again failed to do so (Fig. 2C, c). Then, we added back the
monocyte/macrophage-enriched fraction of PBMC to the T
cell-enriched population and incubated them with MEP or pamidronate. As
also shown in Fig. 2C, pamidronate induced IFN-
production in the V2-TCR+ cells (Fig. 2C, f) at a level comparable to that of MEP (Fig. 2C, e). As expected from Fig. 2B,
addition of either normal T cells or B cells failed to restore
the IFN- production of T cells produced by pamidronate (data
not shown). These results indicated that, unlike pyrophosphomonoesters
and alkylamines, pamidronate effectively activates primary T
cells in peripheral blood only in the presence of accessory cells of
monocyte lineage.
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T cells only in the
presence of adherent accessory cells
It was reported previously that the activation of T cells
by pyrophosphomonoesters in the culture was consistently associated
with their clustering (16). To determine whether this was
also the case for pamidronate, whole PBMC were cultured with MEP or
pamidronate for 48 h. Both MEP and pamidronate induced visible
cell clustering in the culture of whole PBMC (data not shown). When
enriched T cells depleted of adherent cells were used, however,
no significant cell clustering was observed in the presence of
pamidronate (Fig. 3A), while
MEP still induced the cell clustering (Fig. 3B). By adding
back the purified monocytes in the PBMC, cell clustering was restored
in the culture with pamidronate (Fig. 3C) to a level
comparable to that produced by MEP (Fig. 3D). The results
indicated that the intimate cell-cell contact considered to be required
for the activation of T cells was also dependent on the presence
of adherent accessory cells for pamidronate but not for MEP.
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T cells and becomes more
susceptible for their cytotoxic activity
We next addressed the possibility that the adherent accessory
cells were required to "present" pamidronate for T cells.
For the effective Ag preloading, the presenting cells need to be
treated with much higher doses of Ags than under regular conditions.
However, pamidronate at 100 µM and more was progressively toxic for
normal monocytes in blood as reported above (see also Fig. 1
F), and the attempts to effectively preload them with
pamidronate were severely hampered. We, therefore, attempted the
pulsing experiments using a monocyte lineage leukemic cell line, THP-1,
which were resistant to pamidronate treatment even up to 1 mM. THP-1
cells were incubated with medium or pamidronate (0.1 or 1 mM) for
1.5 h, extensively washed, and then added to the enriched T
cell population depleted of adherent cells. The primary T cells
produced little IFN- in response to untreated THP-1 (Fig. 4A, a). However,
THP-1 pretreated with pamidronate induced the production of IFN- in
the primary V2V2-TCR+ cells in a
dose-dependent manner (Figs. 4A, b and
c). Although not shown, incubation of THP-1 with pamidronate
for as briefly as 10 min could activate T cells to produce
IFN- (data not shown). It was noted that the level of V2-TCR
expression on T cells was significantly down-modulated in the
cultures stimulated with pamidronate-pulsed THP-1 cells in a
dose-dependent manner (Figs. 4A, b and
c), suggesting the active engagement of TCR. We then
examined whether pulsing of THP-1 cells with pamidronate affected
their susceptibility to the cytolytic activity of T cells as
well. As shown in Fig. 4B, THP-1 cells pretreated with
pamidronate were lysed by 12G12 T cell clone more efficiently
than untreated THP-1 cells in the short-term (5-h) assay. These results
suggested strongly that the primary T cells are activated in
response to pamidronate bound on the surface of accessory cells, but
not to the free pamidronate.
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T cells by pamidronate presented by THP-1 is
mediated by V2V2-TCR
Finally, to confirm the direct involvement of TCR in the
recognition of pamidronate, we transfected a TCR-defective
Jurkat cell line, J.RT3-T3.5, with V2 TCR- and V2 TCR
-chain cDNAs originated from the 12G12 clone. A stable transfectant
expressing the TCR along with CD3 was obtained (Fig. 5A), which responded to the
anti-CD3 stimulation as revealed by the production of IL-2 (Fig. 5B). Like the original 12G12 clone, the transfectant did not
produce IL-2 in response to free pamidronate, while it did so in
response to free MEP (Fig. 5B). When the Jurkat transfectant
was cultured with THP-1 pretreated with 1 mM pamidronate, however,
significant IL-2 production was observed, which was comparable to that
in Daudi cells, the well-known target tumor of T cells.
Untreated THP-1 cells induced no detectable IL-2 activity. Although not
shown, parental Jurkat cells did not produce IL-2 in response to any of
these stimulants. The results strongly suggested that V2V2-TCR
was indeed involved directly in the recognition of pamidronate
presented on the surface of THP-1 cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells play significant roles
during intracellular infections, such as tuberculosis
(20), malaria (21), ehlrichosis
(22), and many others (23, 24, 25, 26, 27, 28, 29, 30, 31). Evidence
indicates that human T cells bearing V2V2-TCR, which constitute
the vast majority of T cells in healthy adults, respond to
unique Ags in the extracts and/or supernatants of these bacteria and
protozoa and are activated with respect to proliferation, cytokine
production, and cytotoxic activity (17). These Ags include
nonpeptide molecules such as a wide variety of small organic
pyrophosphomonoesters (3, 4, 5, 7) and alkylamines
(12) as well as unprocessed protein Ags
(32, 33, 34). Although it has been indicated that the response
takes place in a V2V2-TCR-dependent manner based on substantial
evidence including the TCR gene transfer studies (15),
knowledge of how human T cells recognize such molecules has
remained elusive.
It was indicated previously that activation of T cells by
pyrophosphomonoester and alkylamine Ags required no other specialized
APCs (3, 12). On the other hand, it was also reported that
intimate cell-cell contact among T cells themselves or with
other cell types, such as B cell lines, was necessary for activation of
T cells (16). Thus, for instance, the solitary
T cells hardly showed calcium influx in response to such Ags,
while those lightly centrifuged to effect cell-cell contact exhibited
significant calcium influx (12). These results could be
interpreted to indicate that these Ags had to associate with the cell
surface, including the T cell themselves, to activate them via
TCR-cross linking for instance. However, previous attempts to elicit
the activation of T cells by pulsing T or B cells with
these Ags were unsuccessful (16).
Recently, a new class of nonpeptide Ags, aminobisphosphonates, was
demonstrated to be capable of activating human T cells both in
vivo and in vitro, including proliferation and IFN- production
(18, 19). In the present study the features of human
T cell activation by pamidronate were analyzed compared with
those by pyrophosphomonoesters. We found that the bisphosphonate moiety
(P-C-P) of pamidronate was essential for the activation of T
cells as in the case of MEP, in which the pyrophosphate moiety (P-O-P)
was critical (3). Since an amino group of pamidronate was
also reported to be necessary for T cell activation (18, 19), it appears that pamidronate has the structural features of
both pyrophosphomonoesters and alkylamines to stimulate T cells.
In contrast to the latter Ags, however, it was indicated that the
activation of primary T cells by pamidronate was strictly
dependent on the presence of adherent cells in peripheral blood. Thus,
the production of IFN- by primary V2-TCR+
cells was completely abrogated by the depletion of adherent cells and
could be fully restored by adding back the monocyte-enriched fraction.
Conforming to this, pamidronate totally failed to induce the
proliferation of homogenous T cell lines, while MEP could
successfully do so. Unlike MEP, the induction of T cell
clustering by pamidronate was also dependent on the presence of
monocytes. These results suggested strongly that monocyte lineage cells
play a crucial role in activation of primary human T cells by
pamidronate.
A monocyte lineage leukemic line, THP-1, preincubated with pamidronate
followed by extensive washing could efficiently induce IFN-
production by purified T cells, while either untreated THP-1 or
free pamidronate was totally without effect under the same condition.
This is the first direct indication to our knowledge that the primary
T cells are functionally activated by nonpeptide Ag presented on
the cell surface. Pamidronate could be functionally loaded almost
instantly (within 10 min) on the surface of THP-1 cells. Therefore, it
appears that pamidronate directly attaches to the surface of monocyte
lineage cells without requiring intracellular processing, unlike the
peptide loading to MHC molecules for protein Ags (35, 36, 37, 38, 39).
Unfortunately, however, the attempt to perform similar Ag-pulsing
experiments, which requires the transient exposure of cells with much
higher doses of Ags than during regular stimulation, using the primary
monocytes was hampered by the toxicity of pamidronate for them. The
pulsed exposure of normal monocytes with pamidronate at 0.1 mM, which
was a minimal dose to detect the stimulatory effect for T cells
in THP-1 cells, resulted in considerable cell death in normal
monocytes. The selective toxicity of pamidronate against monocyte
lineage cells including osteoclasts may well reflect the selective
binding of pamidronate to these cells.
The present results indicate that pamidronate exhibits nearly 100 times
more efficient stimulation of T cells than IBA, while
3-aminopropyl phosphonate was totally ineffective, although all these
compounds share the essential amino group. It is therefore tempting to
speculate that an additional bisphosphonate moiety markedly enhances
the stimulatory activity of alkylamine by causing efficient binding of
the molecules to the selected cell surface, whereas a monophosphonate
not only is incapable of inducing cellular binding, but interferes with
the activity of alkylamine. Since it is shown that a hydroxyl group
attached to a bisphosphonate enhances the selective binding of
pamidronate to the bone matrix (40), it may also
contribute to the molecular binding to the cell surface. In any case,
the exact molecular basis for the binding of pamidronate to the THP-1
and monocytes remains to be further verified.
The direct engagement of TCR in the recognition of
cell-associated pamidronate was indicated by several independent
observations. First, the expression level of V2-TCR on the primary
T cells was specifically down-regulated by stimulation with
THP-1 pulsed with pamidronate. Second, the Ag-pulsed THP-1 cells were
significantly more susceptible than untreated ones to cytotoxic
activity by T cells. Finally, TCR-defective Jurkat cells
transfected with TCR genes could specifically secrete a
significant level of IL-2 in response to the pamidronate-pulsed THP-1
at a level comparable to that by Daudi cells. These results suggested
strongly that functional TCR engagement, TCR cross-linking for
instance, occurs efficiently only when pamidronate is presented on the
cell surface of accessory cells. At present, how TCR interacts
with the ligand on the cell surface remains unknown, but our
unpublished results indicate that the J1.2 gene-encoded sequence of
the TCR -chain plays a critical
role.4
Aminobisphosphonate compounds were originally developed as therapeutic
drugs for osteoporosis based on structural similarities with
pyrophosphoric acid (40). It was reported that
aminobisphosphonates caused apoptosis of osteoclasts by inhibiting a
rate-limiting step in the cholesterol biosynthesis pathway (18, 19). Since osteoclasts belong to the monocyte lineage, it is
tempting to speculate that the activation of T cells and
targeted cytotoxicity by them might also contribute to the toxic effect
of aminobisphosphonates on osteoclasts in vivo. The peak serum
concentrations achieved in the therapeutic pamidronate in humans are
estimated to be 0.5–4 µM (18, 19). Considering the
preferential targeting to selective tissues such as bones, potent
activation of T cells with 1–10 µM pamidronate in the present
experiments may well be significant in vivo as well. More recently,
pamidronate is also used for patients with multiple myeloma and other
tumors accompanied by hypercalcemia, with significant beneficial
effects in a proportion of patients. T cells stimulated with
these nonpeptide Ags, on the other hand, have potential cytotoxic
activity toward a wide spectrum of tumors. Indeed, our unpublished
results indicate that primary human T cells can kill various
types of tumor cells pulsed with pamidronate far more efficiently than
untreated tumor cells (Y. Tanaka, S. Yamashita, F. Miyagawa, Y. Kato,
and N. Minato, manuscript in preparation). Thus, the present
results may provide a new strategy for immunotherapy in human
malignancy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nagahiro Minato, Department of Immunology and Cell Biology, Graduate School of Medicine, Kyoto University, Yoshidakonoe-Cho, Sakyo-Ku, Kyoto 606-8501, Japan.
3 Abbreviations used in this paper: MEP, monoethyl pyrophosphate; IBA, isobutyl amine.
4 F. Miyagawa, Y. Tanaka, S. Yamashita, K. Danno, M. Uehara, B. Mikami, and N. Minato. Submitted for publication.
Received for publication December 1, 2000. Accepted for publication February 26, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), pamidronate (
),
3-aminopropyl phosphonate (•), or IBA (
), and the percentage of
