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Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 384-392
FOCUS ON HEMATOLOGY
From the Medizinische Poliklinik Wuerzburg, Julius-Maximilians
Universität Wuerzburg, Wuerzburg, Germany.
Bisphosphonates are well-known inhibitors of osteoclastic bone
resorption, but recent clinical reports support the possibility of
direct or indirect antitumor effects by these compounds. Because bisphosphonates share structural homologies with recently
identified
Bisphosphonates are the treatment of choice for
diseases that involve excessive bone resorption and have been shown to
be effective in preventing osteolytic bone disease in several different malignancies, including multiple myeloma (MM).1,2
Chemically, bisphosphonates are synthetic analogues of endogenous
pyrophosphate. Variation of their side chains contributes to the
different relative potency of bisphosphonates. However, the precise
mechanisms whereby bisphosphonates inhibit bone resorption are still
not completely understood.3
Recent data raise the possibility that certain bisphosphonates also
exert antitumor effects. In this context, results of a large,
randomized, double-blind, placebo-controlled study4 showed
significant improvement in the survival rates of a subgroup of patients
with MM who entered the trial receiving intravenous pamidronate
treatment in addition to salvage chemotherapy. In addition, objective
remission or inhibition of disease progression has been reported in
patients with MM who underwent pamidronate treatment
alone.5 Furthermore, in experimental models of human breast
cancer, bisphosphonates were found to reduce tumor burden in
skeleton.6
Several studies demonstrate the presence of a T-cell-mediated immune
response against MM.7,8 Most experiments have focused on
The structural relationship between bisphosphonates and defined Patients
Reagents
Cytofluorometric analysis For evaluation of cell expansion, PBMC were harvested after a 7-day culture period and were analyzed using 2-color flow cytometry (FACScan; Becton Dickinson, Heidelberg, Germany). In some experiments, cell number per well was counted to calculate the expansion of absolute cell numbers. Monoclonal antibodies (mAb) used included fluorescein isothiocyanate (FITC)-conjugated antipan  TCR (TCR1), anti-V9 variable light chain (TiA), anti-V2 variable light chain (BB3) (all from Coulter-Immunotech, Hamburg, Germany), and anti-  TCR (WT3; Becton Dickinson, Mountain View, CA) or
phycoerythrin (PE)-conjugated anti-CD3, anti-CD19, anti-CD14, and
anti-CD16/CD56 (all from Coulter-Immunotech). To identify activated
peripheral blood (PB) or bone marrow (BM) T cells,
FITC-conjugated antipan TCR (TCR1) and PE-conjugated
anti-CD25 ( chain IL-2 receptor; Coulter-Immunotech) or
PE-conjugated anti-CD69 (Coulter-Immunotech) mAb were used for 2-color
flow cytometry analysis. For identification of malignant BM plasma
cells, the following panel of mAbs was used: FITC-conjugated anti-CD45
(Coulter-Immunotech), PE-conjugated anti-CD38 (Becton Dickinson,
Heidelberg, Germany), FITC-conjugated anti-CD19 (Coulter-Immunotech),
and PE-conjugated anti-CD138 (B-B4/Syndecan-1; Biermann, Bad Nauheim,
Germany). Myeloma plasma cells were defined as
CD45low,(+)/CD38bright,++ and
CD19 / CD138++ cells. In all experiments,
10 000 viable cells were analyzed using forward/side-scatter gating.
Isotype-matched mAbs were used as controls.
Cell preparations and culture PBMC and BMMC were obtained by centrifugation of heparinized peripheral blood or bone marrow aspirates over Ficoll-Hypaque gradients (Pharmacia, Uppsala, Sweden); 1×105 cells were cultured in 96-well round-bottom microtiter wells (Nunc, Wiesbaden, Germany) for indicated time intervals (3 to 7 days) at 37°C in humidified atmosphere (5% CO2). Medium consisted of RPMI 1640 (Gibco, Life Technologies, Karsruhe, Germany) supplemented with 10% pooled human AB serum, L-glutamine (Gibco; 2 mmol/L), 1% penicillin-streptomycin (Seromed, Berlin, Germany), and, when indicated, 10 U/mL IL-2 (generously provided by W. Sebald; Theodor-Boveri Institute, University of Wuerzburg, Germany). For T-cell depletion
experiments, BMMC were depleted of T cells by negative selection
procedures using magnetic-activated cell sorting (MACS). In brief, BMMC
were incubated with FITC-labeled anti-TCR (TCR1) mAb or
IgG-isotype control mAb followed by anti-FITC magnetic microparticles
(MACS system; Miltenyi Biotec, Bergisch Gladbach, Germany). After 2 washing steps, the cells were passed through a strong magnetic field, and effluent cells were evaluated for residual T cells by
staining with PE-labeled anti-TCR (TCR1). The negative
selected BMMC consisted of less than 0.5% TCR positive cells,
whereas cell viability (by trypan blue exclusion test) and number of BM
plasma cells (as identified by
CD45low,(+)/CD38bright,++ and
CD19/CD138++ expression) remained
unchanged after this procedure. Purification and enrichment of T
cells for proliferation assays was performed by similar
negative-selection procedures. To deplete PBMC from T cells and
natural killer (NK) cells, PBMC were preincubated with FITC-labeled
anti- TCR (T Cell Diagnostics) and anti-CD 16 (Coulter-Immunotech) mAb before they were labeled with anti-FITC magnetic microparticles and MACS separation. Isolated cells consisted of 25% to 30% T cells and less than 0.5% T cells or
NK cells, as determined by FACS analysis. Viability was confirmed by
trypan blue exclusion test and forward/side-scatter gating.
Proliferation assay In round-bottom microtiter wells, 4×104 purified ( T cell and NK cell) PBMC
were cultured with pamidronate (4 µmol/L), IPP (4 µmol/L), phytohemagglutinin (PHA; 1 µg/mL), or medium alone for 96 hours in a
5% CO2 humidified atmosphere. Absolute cell number of
T cells in these PBMC cultures was 1.0 to 1.2 × 104 T cells per well. Medium consisted of RPMI 1640 supplemented with 10% pooled human AB serum, L-glutamine
(2 mmol/L), and 1% penicillin-streptomycin. After a culture period of
48 hours, IL-2 (10 U/mL) was added. During the last 12 hours of the
96-hour culture period, cells were pulsed with 1 µCi
[3H] thymidine (Amersham, Braunschweig, Germany). Cells
were harvested with a semiautomated sample harvester, and
[3H] thymidine incorporation was measured in a liquid
scintillation counter (Beckman, München, Germany). Results are
shown as cpm (geometric mean + SD) of triplicate cultures.
Cytoine analysis Quantification of cytokines (IFN-, granulocyte
macrophage-colony-stimulating factor [GM-CSF], tumor necrosis factor
[TNF]-) in PBMC supernatants was performed by enzyme-linked
immunosorbent assay (Endogen, Woburn, MA). Supernatants were collected
after 4, 12, 24, 48, and 72 hours and stored at  80°C after
centrifugation (5000g for 10 minutes) until analysis was
performed according to the manufacturer's instructions. Samples were
analyzed in triplicate. The sensitivity of the assays used was less
than 2 pg/mL for IFN-, less than 2 pg/mL for GM-CSF, and less than 5 pg/mL for TNF-, respectively. Intracellular cytokine staining was
performed to determine IFN- production of T cells at the
single-cell level. Monensin (2 µmol/L; Sigma) was added for 2 hours
to the cells in culture to cause intracellular accumulation of newly
synthesized proteins. Cells were harvested and stained for surface
expression of TCR- by PE-conjugated anti- TCR (TCR1).
After they were washed with PBS/2% fetal calf serum (FCS), cells were
fixed with 4% paraformaldehyde in PBS for 30 minutes at room
temperature. Cells were washed with PBS/2% FCS and permeabilized with
0.5% saponin (Sigma) in PBS for 30 minutes at room temperature.
FITC-conjugated anti-IFN- (Coulter-Immunotech) was added to
permeabilized cells and incubated for 30 minutes. Afterward cells were
washed with PBS/0.5% saponin and finally with PBS/2% FCS. Samples
were analyzed on a FACScan flow cytometer. For control, samples were
incubated with an irrelevant isoptype-matched mAb. Specificity of
anti-IFN- mAb was demonstrated by preincubation of a 100- to
1000-fold molar excess of recombinant IFN-, together with the
anti-IFN- mAb for 1 hour before it was added to the
sample. This procedure resulted in greater than 90% inhibition of
IFN- detection.
Cell lines T-cell lines were established by culturing
1 × 105 freshly isolated PBMC in standard medium
(RPMI 1640 media supplemented with 10% pooled AB human serum, 2 mmol/L
L-glutamine, and 1% antibiotics) with a single dose of
aminobisphosphonate (40 µmol/L pamidronate). After 48 hours,
IL-2 (50 U/mL) was added to the cultures, and cells were
periodically restimulated with IL-2 (50 U/mL) every 4 to 6 days. After
2 to 3 weeks, more than 90% of the cells expressed the V9V2 TCR,
as determined by flow cytometry. Daudi, U266, and RPMI 8226 cell lines
were obtained from the ATCC (Rockville, MD). All cell lines were grown
in RPMI 1640 media supplemented with 10% FCS and 2 mmol/L
L-glutamine.
Quantification of plasma cell number Number of plasma cells in BMMC of patients with MM was calculated by counting the number of viable cells per well and by cytofluorometric identification of plasma cells using FACS analysis (CD45low,(+)/CD38bright,++, CD19/CD138++). On day 5 of culture,
plasma cell numbers in treated and control (medium alone) cultures were
counted, and results were shown as percentage of control culture
according to the following calculation: [plasma cell number in treated
(IPP or pamidronate) culture]/[plasma cell number in control culture
(medium alone)] × 100.
Cytotoxicity assay A standard 4-hour chromium Cr 51 release assay was performed. In brief, target cell lines (Daudi, RPMI 8226, U266, and allogeneic PHA blasts) were labeled with 100 µCi 51Cr, and 5000 cells/well were incubated in round-bottom triplicate wells with the pamidronate-reactive V9V2 T-cell line at the indicated
effector/target (E/T) ratios. After 4 hours, the amount of
51Cr released into supernatant was measured as cpm and
expressed as specific lysis according to the following formula: % specific lysis = % specific 51Cr release = (effector
induced cpm spontaneous cpm/maximum cpm spontaneous
cpm) × 100. Spontaneous cpm represents the amount of
51Cr released by target cells incubated without effector
cells, and maximum cpm was obtained by lysis with 1 mol/L HCl.
Statistical analysis Results are expressed as mean ± SD. The Student t test was used to determine statistical significance of detected differences. P < .05 was considered significant.
Comparison of different bisphosphonates to stimulate T cells. In
contrast, the non-aminobisphosphonates (clodronate and etidronate) were
inactive even at very high concentrations (greater than 1000 µmol/L).
All aminobisphosphonates exhibited a lower stimulating activity than
IPP, which is known as a potent natural T-cell ligand
(half-maximal activities: IPP, 0.2 µmol/L; alendronate, 0.9 µmol/L;
ibandronate, 1.0 µmol/L; pamidronate, 4 µmol/L). Flow cytometric
analysis revealed no significant expansion of other PBMC subpopulations
(monocytes, B cells, NK cells, and T cells) after a 7-day
culture in the presence of the bisphosphonates under study (data not
shown). The relative increase of CD3+ T lymphocytes
in response to IPP or aminobisphosphonates (as shown in Figure 1A) also
reflects an increase in absolute cell numbers, as determined by
counting the number of T cells per well on day 7. Figure 1B
shows a dose-dependent absolute increase of T cells in the
presence of pamidronate in 1 representative experiment, whereas the
nonaminobisphosphonate clodronate failed to expand T cells. This
selective T-cell outgrowth required the presence of low doses of
IL-2 (10 U/mL).
Expansion of V
T-cell-stimulating capacity of aminobisphosphonates, primary cultures of freshly isolated PBMC from 6 different healthy donors were analyzed
for T-cell stimulation by pamidronate. Determination of the
percentage of T cells after 7 days demonstrated that pamidronate
induced a significant expansion of T cells in all donors tested,
though some interindividual differences could be observed (Figure
2A,B). Previous studies have demonstrated
that phosphorylated nonpeptidic ligands for T cells (eg, IPP)
preferentially induce an expansion of the V9V2
subpopulation.19 To determine T-cell subsets that
are stimulated by aminobisphosphonates, primary T cells from
healthy persons were incubated with pamidronate, and V
gene expression was determined by 2-color FACS analysis after 7 days of
culture. Results show that T cells, which were expanded in the
presence of pamidronate, exclusively expressed the V9 and the V2
genes (Figure 2B); no significant proliferation of T cells expressing
other variable genes was detected.
Proliferative response of naive T cells to IPP and pamidronate by [3H]
thymidine incorporation. For this purpose, T-cell- and
NK-cell-depleted PBMC of healthy donors were incubated with IPP or
pamidronate for 96 hours. After 48 hours, IL-2 (10 U/mL) was added, and
cells were exposed to [3H] thymidine during the last 12 hours of the culture period. In line with the results obtained with
unpurified PBMC cultures, IPP and pamidronate induced a significant
proliferation of purified T cells in the presence of IL-2
(Figure 3). In contrast, purified T
cells did not proliferate in response to nonaminobisphosphonates or in
the absence of exogenous IL-2 (data not shown).
Kinetics of IL-2-independent T cells by aminobisphosphonates was
followed by the determination of CD25 (-chain of IL-2R) and CD69 expression on T cells during a 72-hour culture period of PBMC cultures. As shown in Figure 4, both IPP
and pamidronate induced CD25 (Figure 4A) and CD69 (Figure 4B)
expression on a large fraction of T cells in the absence of
exogenous IL-2. Induction of CD25 and CD69 on T cells by
pamidronate was dose dependent, with significant up-regulation at
concentrations as low as 0.4 µmol/L. Similar to T cells, the
induction of CD69 expression on T cells was more rapid starting
at 24 hours, whereas CD25 expression was first seen after 48 hours of
the culture period. These results confirm that T cells can
recognize aminobisphosphonates such as pamidronate in the absence of
exogenous cytokines such as IL-2. In contrast, proliferative responses
of T cells (as shown in Figure 1A,B and Figure 3) was dependent
on exogenous IL-2, indicating that the proliferation of T cells
requires additional signals.
Cytokine production by aminobisphosphonate-activated
T-cell stimulation induces
the release of a variety of cytokines (IFN-, TNF-, IL-2, and GM-CSF), particularly Th1-type cytokines.10 To investigate
the functional consequences of T-cell stimulation by
aminobisphosphonates, cytokine concentrations in supernatants of
pamidronate-treated PBMC were measured. Results show a significant
increase of IFN- concentrations detectable after 24 to 48 hours of
culture (Figure 5A). A similar secretion
pattern for GM-CSF and a slight increase of TNF- (not significant)
concentrations was observed in pamidronate-treated culture
supernatants, whereas cytokine concentrations in control cultures
(medium alone) remained at a low level (data not shown). In addition,
IL-4 concentrations did not change during the culture period (data not
shown). Because cytokine production by other mononuclear cells in
pamidronate-treated PBMC cultures could not be excluded, single-cell
analysis of cytokine production was performed by intracellular staining
of IFN-. As shown for a representative donor, few (12%) T
cells cultured with medium alone expressed significant intracellular
levels of IFN-, whereas 41% and 57% of the T cells,
respectively, were positive for IFN- on stimulation with pamidronate
or IPP (Figure 5B).
Cytotoxicity of pamidronate-activated T
cells is their nonmajor histocompatibility complex (MHC)-restricted cytolytic activity against various tumor targets, particularly of
hematopoietic origin.10 For determination of the lytic
potential of aminobisphosphonate-activated T cells,
pamidronate-induced T-cell lines were generated from PBMC (as
described in "Materials and methods"). Cytotoxicity against 2 previously known T-cell targets (Burkitt lymphoma cell line
Daudi and myeloma cell line RPMI 8226) and another myeloma cell line
(U266) was investigated in a 4-hour standard 51Cr release
assay, in which allogeneic PHA-induced peripheral blood leucocyte blasts served as a control. Results showed
that the pamidronate-stimulated T-cell line exhibited strong
lytic activity against Daudi and RPMI 8226 targets and intermediate
cytotoxicity against U 266 targets. However, no significant killing of
allogeneic PHA blasts was observed (Figure
6).
Activation by aminobisphosphonates of bone marrow T cells from patients with MM, BMMC from 24 patients with MM
were cultured with pamidronate, IPP, or medium alone. After 72 hours,
the percentage of CD25 expressing T cells was evaluated by
FACS analysis. In 14 of 24 (58%) patients, a significant increase of
CD25 expression on BM- T cells was observed in both pamidronate- and IPP-treated BMMC cultures. Results of 3 representative patients are shown in Figure 7. Similar to
the PBMC of healthy donors, CD25 expression on other mononuclear
cell populations (eg, T cells and NK cells) remained
stable during the culture period. Therefore, BM- T-cell
stimulation could be induced by pamidronate in a significant proportion
of patients with MM.
Cytoreductive effects of IPP and pamidronate in multiple myeloma Our previous unpublished experiments have shown that in vitro culture of the bone marrow biopsy specimens from patients with MM, taken 24 hours after pamidronate infusion (90 mg intravenously) revealed a significant outgrowth of T cells in the presence of
low-dose IL-2 (10 U/mL). In addition, the quantification of viable
plasma cells before and after 1 week of culture showed a significant
decrease (30%-40%) of plasma cell number. This cytoreductive effect
could not be observed in bone marrow biopsy specimens cultured without
IL-2 or in specimens from patients with MM who have not received
pamidronate before BM biopsy (data not shown). To confirm these
preliminary observations, the effect of IPP and pamidronate on
autologous plasma cells was determined by counting the total number of
viable plasma cells on day 5 in BMMC cultures of 24 patients with MM.
As illustrated in Table 1, IPP and
pamidronate induced a significant reduction of plasma cells in BMMC
cultures (IPP, P = .0345; pamidronate, P = .0002)
compared with control cultures ( = medium with 10 U/mL IL-2).
Although the range of plasma cell decrease was relatively wide, there
seemed to be a correlation with T-cell activation. Patients with
MM who had significant up-regulation of CD25 expression on T
cells during BMMC culture had more prominent plasma cell decreases (% plasma cells after 5 days compared to control cultures: IPP, 87.0% ± 28.4%; pamidronate, 65.9% ± 38.4%; pamidronate in patients
with CD25+ T cells, 54.8% ± 28.8%). Plasma
cell decrease was independent of the initial BM plasma cell number
because the effect was observed in patients with high and low levels of
BM plasma cell infiltration.
Antiplasma cell activity by pamidronate is mediated by T cells in pamidronate-mediated
antiplasma cell activity, BMMC cultures from 3 patients with MM were
performed under standard conditions and after depletion of T
cells. These BMMC and BMMC () cultures were
challenged with increasing concentrations of pamidronate, and the
percentage of viable plasma cells was compared to that of control
cultures (medium with 10 U/mL IL-2) after 5 days of exposure (Figure
8). Pamidronate induced a dose-dependent
reduction of plasma cells in all BMMC cultures without T-cell
depletion. In contrast, T-cell depletion abrogated the
antiplasma cell effect in 2 patients (patients 1 and 2) but had no
effect on the BMMC cultures of the third patient (patient 3).
Interestingly, the activation of BM- T cells (increase of CD25
expression) was demonstrated only in patients 1 and 2, whereas no
T-cell activation was observed in patient 3 (data not shown).
These data confirm the important role of T cells in
pamidronate-mediated plasma cell cytotoxicity, but they indicate that
in certain patients additional mechanisms may contribute to this
effect.
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T cells by aminobisphosphonates and
induction of antiplasma cell activity in multiple myeloma
Top
Abstract
Introduction
= IPP; 
= pamidronate; 
= etidronate; 
= clodranate; 
= alendronate; and 
= ibandronate. (B) Proliferation of 
= clodranate + IL-2; 
= pamidronate; and 
chain) expression on
BM 