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IMMUNOBIOLOGY
From the Institute of Haematology, Royal Prince Alfred
Hospital, Sydney, New South Wales, and the Mater Medical Research
Institute, Brisbane, Queensland, Australia.
Limited response to idiotype vaccination in patients with myeloma
suggests that there is a need to develop better immunotherapy strategies. It has been determined that the number of high-potency CMRF44+CD14 Dendritic cells (DCs) are immune surveillance cells
that have a specialized ability to process antigen and to present
relevant peptides to T lymphocytes in association with major
histocompatibility complex class I and II molecules. Engagement of the
T-cell receptor defines specificity, but a productive immune response
also requires the costimulatory molecules CD80 (B7-1) and CD86 (B7-2)
on antigen-presenting cells to bind to their counterreceptors CD28 and
CD152 (CTLA-4) on T lymphocytes. Expression of these costimulatory
molecules plays a major role in determining whether T-cell survival,
apoptosis, anergy, or productive immunity occurs.1-3 There
is now considerable interest in the ability of DCs to generate CD4
helper, CD8 cytotoxic, and humoral responses and to act as cellular
vectors in immunotherapy protocols. Identification and enumeration of
DCs from peripheral blood and optimal conditions for their collection
for immunotherapy trials have been difficult to standardize because of
the lack of specific markers, the heterogeneous nature of the DC
population, and the immature phenotype of DCs in peripheral blood.
Early quantitation methods using complex purification techniques
estimated the number of DCs to be up to 4% of the circulating
mononuclear cell population.4 More recently, the
expression of CMRF44 on CD19 It is paradoxical that idiotype vaccination is considered a
treatment option for patients with myeloma because they respond poorly
to immunization by viral and bacterial antigens, and it has been
recognized for many years that they have a primary immune defect.7 Murine studies have suggested that this defect is associated with antigen presenting cells (APCs).8 There
have been reports that the DCs of patients with myeloma are
functionally normal9,10; however, the criteria used to
define DCs in these latter studies included monocytes-macrophages and
even B cells. Preliminary reports of immunotherapy trials in patients
with myeloma have demonstrated only minor responses.11-14
If immunotherapy using autologous DCs is to be a serious treatment
option for patients with myeloma, it is important that we have a
greater understanding of the number and function of high-potency blood
DCs and the optimal conditions for their collection and administration.
One important factor to consider is that without adequate expression of
costimulating molecules on the APCs, immunotherapy involving idiotype
presentation may induce further T-cell tolerance.
Expression of the B7 family of costimulatory molecules can be modulated
by a range of cytokines. This includes down-regulation by transforming
growth factor (TGF)- Patient selection
Blood dendritic cell assay
Up-regulation of CD80 and CD86 by huCD40LT Human trimeric CD40 ligand was kindly provided by Immunex (Seattle, WA). Ficoll-Hypaque-separated peripheral blood mononuclear cells were cultured with or without huCD40LT (5 µg/mL) and rIL-2 (0.1 µg/mL; R&D Systems, Minneapolis, MN) in 1 mL cultures of RPMI containing 10% fetal calf serum (CSL) at 37°C in 10% CO2. After 24 and 72 hours, cells were harvested and assayed for CMRF44 antigen expression on CD19 CD14PI cells using a
Coulter Epics XL flow cytometer. Four-color staining with anti-CD80 PE
(Becton Dickinson) or anti-CD86 PE (Becton Dickinson), anti-CD19
PE-Cy5 plus anti-CD14 PE-Cy5, CMRF44 (indirect FITC) on viable cells
(propidium iodide) was performed to demonstrate the expression of
costimulatory molecules before and after culture. Similar cultures were
established from mononuclear cells of bone marrow biopsy samples from
patients with myeloma and controls. Plasma cells were detected using
CD38hi expression (Becton Dickinson), and plasma cell
subpopulations were distinguished using anti-CD45 (Becton Dickinson) as
previously described.22 These were either primitive
CD38++CD45++, immature
CD38++CD45+, or mature
CD38++CD45.
Cytokine neutralization and inhibition studies Cultures of peripheral blood cells were established (as per DC assay) with and without anti-TGF- 1, anti-IL-10, or
anti-IFN- (R&D Systems) at concentrations of 0.12 to12 µg/mL.
CD80 expression on DCs was assayed after 24 hours. Normal peripheral
blood cells were similarly cultured in the presence of
rTGF-1 (2-200 pg/mL), and CD80 expression on DCs was
assayed after 24 hours.
TGF- 1 or IL-10 (R&D Systems) diluted 1:250 in 0.2 bicarbonate buffer, pH 9.2, was added to the wells of a flat-bottomed
microtiter plate, incubated overnight at 4°C, and washed 3 times with
phosphate-buffered saline (PBS), pH 7.4, containing 0.05% Tween 20 (wash buffer). To assay IL-10, serum was diluted 1:10 with reagent
diluent (1.4% bovine serum albumin, 0.01% Tween 20 in PBS at pH 7.3).
To assay TGF-1, serum was first treated with 2.5 N
acetic acid for 10 minutes, followed by neutralization with 2.7 N NaOH.
Acid-treated serum was diluted 1:30 with reagent diluent.
rTGF-1 and rIL-10 (R&D Systems) were diluted to 50, 25, 12.5, 6.25, 3.12, and 1.56 ng/mL in reagent diluent. Two hundred
microliters each dilution was added to triplicate wells, incubated for
3 hours, and washed 3 times with PBS. Then 200 µL biotinylated
anti-TGF-1 (PharMingen, San Diego, CA) was added to
each well of the TGF-1 plate, and 200 µL polyclonal
anti-IL-10 (R&D Systems) was added to the IL-10 plate. Both plates
were incubated for an hour at room temperature and were washed 3 times
in wash buffer. One hundred microliters StreptAB complex (DAKO,
Carpinteria, CA) was added to the TGF-1 plate and
incubated for 30 minutes. One hundred microliters biotinylated F(ab)2
fragment was added to the IL-10 plate for 1 hour and was washed before
the addition of 100 µL StreptAB complex for 30 minutes. Both plates
were washed 3 times, and then 100 µL o-phenylenediamine solution (20 mg in 20 mL phosphate citrate buffer containing 10 µL
H2O2) was added. Color development stopped with
50 µL of 1.5 M H2SO4. Absorbance was read at
492 nm on a Multiskan RC microplate reader with Genesis software
(Labsystems, Basingstoke, United Kingdom).
Cytoplasmic cytokine expression Expression of the cytokines IL-10, TGF-1, and the
latency-associated peptide (LAP) in the cytoplasm of plasma cells was
determined by flow cytometry. Mononuclear cells separated from bone
marrow on Ficoll-Hypaque were cultured for 1 hour at 37°C in 5%
CO2 in RPMI 10 and then for 5 hours with brefeldin A (10 µg/mL). Bone marrow samples were permeabilized (Harlan Sera-Lab,
Loughborough, United Kingdom) and stained with anti-IL-10 PE (IQ
Products, Groningen, The Netherlands), anti-TGF-1 (R&D
Systems), or anti-LAP (R&D Systems). IL-10-stained cells were washed
and stained with anti-CD38-FITC (Becton Dickinson), whereas the
TGF-1 and LAP-stained cells were washed with PBS
containing 0.5% saponin and 0.5% fetal calf serum and then were
stained with sheep anti-mouse IgG-FITC. Normal mouse serum was used for
blocking, and then these cells were stained with anti-CD38 PE (Becton
Dickinson). Appropriate direct and indirect controls were used, and all
tubes were read within 24 hours.
Number of DCs in peripheral blood of patients with myeloma Flow cytometric DC assay involved an analysis of mononuclear cells after culture at 37°C for 24 hours. A gating strategy that incorporated all possible DCs (Figure 1A) that were PI (Figure 1B) was used. High-potency DC
populations were identified as the
CMRF44+CD14CD19
population (Figure 1D) with CMRF75-FITC-stained cells (Figure 1C) used as the isotype control. Our normal range (0.05%-0.8% of
MNCs; n = 13) was not significantly different from either that reported in the original publication5 or the normal range
established in 2 other independent laboratories (unpublished
observations, D. H., September 2000). The range of values for
DC numbers in myeloma peripheral blood (0.03%-0.8% of MNCs; n = 26)
was not significantly different from the normal range (Figure
2). There was a trend for the mean number
of DCs in the blood of patients with progressive disease (n = 12;
mean, 0.12%) to be lower than in patients with stable disease
(n = 14; mean, 0.18%); however, this difference was not
statistically significant.
CD80 and CD86 expression on dendritic cells The percentage of blood DCs (CMRF44+CD19CD14PI)
expressing CD80 and CD86 are shown for patients with myeloma (n = 26)
and for a control group (n = 12) (Figure
3). Percentage CD80-expressing DCs was
always lower than percentage CD86-expressing DCs, and the
relatively low number of DCs expressing CD80 confirmed the immature DC
phenotype on circulating DCs from patients and controls. There was no
significant difference between the mean percentage CD80+
DCs in the group of patients with myeloma (29%±17% MNCs) compared with a control group (29%±17% MNCs), nor was the percentage CD86 on
DCs of patients with myeloma (85%±10% MNCs) significantly different from that of controls (86%±16% MNCs).
CD80 and CD86 expression on peripheral blood cells after huCD40LT stimulation The effect of huCD40LT with or without IL-2 on the expression of CD80 and CD86 on autologous B cells (CD19+) and monocytes (CD14+) was studied over 72 hours as an internal control for the effect on DCs. B cells from patients with myeloma (n = 12) and from controls (n = 6) had low percentages of CD80+ and CD86+ cells on day 0. Up-regulation of CD80 and CD86 on B cells of the control group and the patients with myeloma was attributed almost entirely to huCD40LT rather than to IL-2 (Figure 4). CD80 and CD86 up-regulation on B cells, however, was greater in the controls than in the patients with myeloma. Few monocytes expressed CD80 on day 0, but most expressed CD86. In contrast to B cells, up-regulation of CD80 expression on the monocytes of patients with myeloma (n = 12) was the same as in the control group (n = 6) and was caused by IL-2, not huCD40LT. Neither huCD40LT nor IL-2 significantly altered the expression of CD86 on monocytes.
CD80 and CD86 expression on DCs after huCD40LT stimulation There was a significant up-regulation of the percentage of CD80+ DCs in the control group (n = 10) after stimulation with huCD40LT plus IL-2 for 24 hours. Up-regulation of CD80 on DCs from patients with myeloma occurred but was significantly less than normal in patients with stable disease (n = 9), whereas DCs from patients with progressive disease (n = 7) completely failed to up-regulate CD80 expression in response to stimulation by huCD40LT and IL-2 (Figure 5). These latter patients had clinical or laboratory features of progressive disease, which included active lytic lesions, low hemoglobin level, and increasing M-protein or serum thymidine kinase levels. Percentage of CD86+ DCs in the control group was high without (mean, 86%) and with (mean, 89%) stimulation in vitro with huCD40LT and IL-2. There was no apparent defect in CD86 expression on the DCs of patients with myeloma.
Effect of anti-TGF 1, anti-IL-10, or anti-IFN- to determine
whether these cytokines were responsible for the failure of huCD40LT
and IL-2 to up-regulate CD80 expression. Figure
6A illustrates that either
anti-TGF1 or anti-IL-10 (1.25 µg/mL) could
independently neutralize the failure to up-regulate CD80 expression on
the DCs of a patient with progressive-stage myeloma. Three other
patients in progressive-stage disease demonstrated the same phenomenon.
However, for 6 patients who had stable disease, the addition of either
anti-TGF-1 or anti-IL-10 did not significantly enhance
the up-regulation of CD80 expression on DCs (Figure 6B). The addition
of anti-IFN- had no significant effect on the expression of CD80 on
the DCs of the patients studied (Figure 6A,B). Neutralization of any
inhibitory effect on CD80 up-regulation was dose dependent and was
achieved at antibody concentrations of more than 1 µg/mL (Figure
6C).
In the presence of huCD40LT and IL-2, there was a normal up-regulation
of CD80 expression on the monocytes of all patients with myeloma in
stable and in progressive disease. Unlike DCs, CD80 up-regulation on
monocytes was attributed almost exclusively to IL-2 rather than to
huCD40LT (Figure 4). Also in contrast to DCs, there was no further
up-regulation of CD80 on the monocytes of patients with myeloma after
the addition of either anti-IL-10 or anti-TGF- Effect of rTGF- 1 to cultures of normal
mononuclear cells (n = 5) stimulated by huCD40LT and IL-2 caused a
dose-dependent inhibition of the expected CD80 up-regulation (Figure
6D,E). A significant inhibition of CD80 up-regulation was demonstrated with concentrations of rTGF-1 as low as 2 pg/mL.
Inhibition of CD80 up-regulation by rTGF-1 was more
marked on monocytes than on DCs.
Serum TGF- 1 and IL-10 were measured in the serum of
the same patients tested for the CD80 response to huCD40LT + IL-2
and a group of controls (n = 12). Serum IL-10 levels were within the
normal range (0-30 µg/L) for all 10 patients with myeloma. One
patient had a modestly elevated TGF-1 level at 29 µg/L (normal range = 0-20 µg/L).
Cytoplasmic expression of TGF- 1
(range, 0%-38%) and IL-10 (range, 0%-6%) in the cytoplasm of plasma
cells (CD38++) of 13 different bone marrow samples from
patients with myeloma. The percentage of plasma cells expressing
TGF-1 was significantly higher (P < .01)
in the 7 patients with progressive disease (mean, 16.4%) than in the 6 patients with stable disease (mean, 2.9%). Cytoplasmic IL-10
expression was only detected in the plasma cells of 3 patients, all of
whom also had small but detectable levels of TGF-1
expression. In contrast, LAP expression was high (more than 80%
positive plasma cells) in samples from 8 patients but lower (10%-80%
positive plasma cells) in samples from 5 patients. The 5 patients with
the highest TGF-1 expression had LAP expression of more
than 90%. No obvious correlation existed, however, between LAP
expression and either disease stage or TGF-1 expression. Figure 7 demonstrates low
TGF-1 (Figure 7B), high LAP (Figure 7C), and absent
IL-10 expression (Figure 7E) in plasma cells (CD38++) of a
patient with myeloma with appropriate isotype controls (Figure 7A,D).
The failure of intensive chemotherapy with autologous stem cell
support to provide a cure for patients with myeloma has led to
considerable interest in immunotherapy protocols for patients with this
disease, and several clinical trials involving immunotherapy have
already begun.11-14 The most commonly used protocols
involve priming autologous monocyte-derived DCs with idiotype to
stimulate a T-cell response The availability of a specific marker (CMRF44) on high-potency human
blood DCs has allowed the introduction of a simple and reproducible
assay for DCs in blood and has provided a new tool to study DC
biology.5,6 The studies in this report have demonstrated that (1) the number of DCs in the peripheral blood of patients with
multiple myeloma is relatively normal throughout the course of the
disease; (2) peripheral blood DCs have an immature phenotype; (3)
huCD40LT + IL-2 up-regulates the expression of the costimulatory molecule CD80 on normal DCs during 24-hour culture; (4) up-regulation of CD80 after huCD40LT and IL-2 stimulation on blood DCs (but not
monocytes) from patients with myeloma is reduced in stable disease and
absent in progressive disease; (5) this apparent functional defect in
the DCs of patients with myeloma is caused by TGF- If DCs from patients with myeloma are to be used as cellular vectors in immunotherapy protocols, it is important to determine whether these cells have a functional defect. There have been some controversial findings concerning DC biology in myeloma. It was originally reported that these cells are infected with the Kaposi sarcoma herpes virus (KSHV or HHV-8)23; however, many others have failed to reproduce these observations.24-26 In contrast to the current study, 2 studies on the phenotype and function of DCs from patients with myeloma did not report any abnormality.9,10 The reason for these differences may relate to the difficulties in defining DC populations and comparing analyses of DCs between laboratories. The other studies do not specifically analyze the high-potency DCs marked by the antigen CMRF44. Our studies have used the new monoclonal antibody (CMRF44) to isolate high-potency DCs and, by ensuring that these cells did not express either CD14 or CD19, have focused on a standardized DC population in blood. Previous studies included cytokine-generated DC populations involving prolonged periods of in vitro culture, which may have corrected any in vivo microenvironment defects. Our methodology detected a defect in the early up-regulation of an essential costimulatory molecule (CD80) on the high-potency blood DCs of patients with myeloma. This may be a possible cause of the primary immune defect seen in these patients. Although the expression of CD86 did not appear to be impaired, good evidence suggests that CD80 expression is essential for the induction of anti-tumor immunity3,27 and cannot be replaced by CD86. TGF- Other studies have demonstrated that cross-linking of CTLA-4 induces
the production of TGF- Oreffo et al35 reported that osteoclast-derived cells in
culture can activate TGF- If immunotherapy is to succeed in patients with myeloma, it is
important to recognize that the dysfunctional immune state that exists
in these patients will result in a poor response to vaccination
procedures. The real challenge for immunotherapy may well be to learn
from the deficiencies in current protocols and to recognize that
multiple defects in the immune response must be considered and
overcome. In this study we have identified one of these defects, and we
suggest that the up-regulation of CD80 expression on DC ex vivo may be
an important new strategy for immunotherapy and that it may be
important to determine which patients will benefit from ex vivo
stimulation. A proposed mechanism for the inhibition of the activation
of high-potency DCs ex vivo is represented in Figure
8. Clearly, the DCs of some patients fail
to respond to huCD40LT stimulation and remain dysfunctional. Reasonable
evidence now suggests that failure to up-regulate the expression of
costimulatory molecules on DCs will at best produce immunologic
ignorance and at worst induce tolerance.2,3
Submitted November 21, 2000; accepted July 5, 2001.
Supported by the Anthony Rothe Memorial Trust and Foundation IV.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Ross D. Brown, Institute of Haematology, Royal Prince Alfred Hospital, Missenden Rd, Camperdown, NSW 2120, Australia; e-mail: rbrown{at}haem.rpa.cs.nsw.gov.au.
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1 and
interleukin-10
Top
Abstract
Introduction
hence, an idiotype vaccination. However,
patients with myeloma respond poorly to immunization by viral and
bacterial antigens,