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Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3479-3490
Effect of Costimulation and the Microenvironment on Antigen
Presentation by Leukemic Cells
By
A.G.S. Buggins,
N. Lea,
J. Gäken,
D. Darling,
F. Farzaneh,
G.J. Mufti, and
W.J.R. Hirst
From the Departments of Haematological Medicine and Molecular
Medicine, King's College School of Medicine and Dentistry, London, UK.
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ABSTRACT |
Costimulatory signals supplied by genetically modified tumor cells
can enable T-cell recognition of tumor-associated antigens that were
previously silent when presented by unmodified tumor cells. Although
the mechanism of the CD80/CD28 costimulation has been studied
extensively in the normal T-cell/antigen-presenting cell
(APC) interactions, it is unclear how expression of CD80 by tumor cells mediates its effect. We demonstrate here that optimal CD80 expression on a leukemic cell enhances T-cell recognition of
alloantigen primarily by lowering the level of T-cell receptor (TCR)
stimulation required for activation. CD80 expression by leukemic cells
leads to increased survival of activated T cells by inducing
upregulation of the antiapoptotic protein BCL-2, but not
BCL-XL. The cytokine microenvironment in which T cells are activated is crucial in determining their differentiation and consequently the nature of the immune response generated. Many tumor
cells produce immunosuppressive cytokines that may not favor the
induction of cell-mediated immunity. In this study, the presence of
CD80 on leukemic cells increased T-cell activation in vitro, but this
did not result in the production of Th1 cytokines. We show that this is
due to a leukemia-derived soluble factor that inhibits the production
of Th1 cytokines. Optimal expression of a costimulatory molecule,
therefore, enhances the ability of leukemic cells to present antigen by
amplifying TCR signals, but the microenvironment generated by leukemic
cells may suppress the immune response required for their eradication.
Thus, strategies aimed at inducing antileukemic immunity by providing
leukemic cells with costimulatory functions must ensure the presence of
an appropriate microenvironment.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ANTIGEN-SPECIFIC T-CELL
activation is a multistep process requiring at least 3 steps for
optimal response: (1) contact with antigen-presenting cells (APCs)
through cell adhesion molecules (CAM); (2) antigen recognition by the
T-cell receptor (TCR); and (3) costimulation (eg, CD80:CD28). In the
early stages of disease, most malignant cells express major
histocompatibility complex (MHC) and CAMs; thus, the
absence of costimulation may be one mechanism by which malignant cells
bearing tumor-associated antigens (TAA) can escape immune surveillance.
Consequently, provision of costimulatory molecules on malignant cells
by a variety of techniques1-3 has been explored as a means
of inducing antitumor immunity. Several studies, both in vivo and in
vitro, have demonstrated that expression of members of the B7 family of
costimulatory molecules (CD80 [B7.1] and CD86 [B7.2]) can induce
immune responses to both the modified cells and, subsequently on
rechallenge, the parental tumor cell.4,5 Gene transfer of
CD80 into human and murine tumor cells,6,7 including
leukemias,1,8,9 has been shown in vitro to increase T-cell
activation, enable generation of tumor-specific cytotoxic T lymphocytes
(CTLs),10 and increase the repertoire of
tumor-specific CTLs.11 Although the role of CD80 in the
context of normal APC:T-cell interaction has been extensively studied,
the mechanism for its activity in the tumor setting has not been
investigated in detail.
The precise role of CD80:CD28 costimulation in T-cell activation has
not been fully established, although it is recognized as playing an
important role in the outcome of T-cell activation. CD28 stimulation
augments production of interleukin-2 (IL-2),12 enabling
activated T cells to progress through the cell cycle,13 and
also upregulates expression of anti-apoptotic proteins14-16 protecting T cells from activation-induced cell death. It is unclear from these studies if CD28 produces its anti-apoptotic effect via a
direct mechanism or via IL-2. Activity of CD28 is balanced by the other
CD80/CD86 receptor, CTLA4, as evidenced by the lymphoproliferative disorder that develops in CTLA4-deficient mice.17 CTLA4 has a higher affinity for both CD80 and CD8618,19 and induces
an opposing signal.20 However, unlike CD28, CTLA4 is only
expressed on the surface by activated T cells. Therefore, the
consequence of costimulation through CD80 is dependent on its level of
expression and also on the relative expression of CD28 and CTLA4 on the
T cell, ie, the activation status.
Although the decision to initiate an immune response is dependent on
suitably primed APCs, the cytokine microenvironment plays an important
role in determining the nature of any response generated. Cytokines
mediate their effect by influencing the Th1/Th2 decision during T-cell
differentiation. In addition, the pattern of cytokines secreted by
primed T cells determines which effector arms are recruited in response
to a given antigen. An immune response is only beneficial if the
appropriate effectors are induced, eg, antitumor immunity is generally
considered to require a cell-mediated Th1 response. Many tumor cells
produce cytokines (eg, IL-10,21-23 transforming growth
factor- [TGF-],24-30 and vascular endothelial growth factor [VEGF]31) and other soluble
factors (soluble IL-2 receptor [sIL-2R],32 soluble tumor
necrosis factor receptor [sTNF-R],33 and soluble Fas
ligand [sFas-L]34,35) that may influence the
nature of immune responses. It is possible, therefore, that the
presence of costimulatory molecules may not have the desired effect on
antitumor immunity by initiating T-cell activation in an
immunosuppressive environment, which may inadvertently induce the
generation of tolerance.31,36-38
The aim of this study has been to address the mechanisms underlying the
enhanced T-cell recognition and proliferation when encountering
alloantigen presented by CD80-expressing leukemic cells. We have used
here an acute leukemia model to demonstrate that one of the major roles
of the CD80:CD28 interaction is through the amplification of TCR
signals. In addition, we have assessed the effect of CD80 expression on
enabling clonal expansion by prevention of activation-induced cell
death. To establish the nature of the immune response in this model, we
have analyzed the cytokine profile produced in response to tumor cells
expressing CD80 and compared it with that induced in response to
professional APCs, ie, dendritic cells (DCs). The results of this study
show a clear effect of CD80 on enhancing the initiation of T-cell
activation and survival, but in a tumor microenvironment, the secretion
of soluble factors may inhibit the production of the desired
cell-mediated immune response.
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MATERIALS AND METHODS |
Cells.
U937 cells (a human monocytic leukemia cell line)39 were
obtained from the American Type Culture Collection (ATCC; Rockville, MD) and grown in RPMI/10% fetal calf serum (FCS; Sigma, Dorset, UK).
Peripheral blood mononuclear cells (PBMNCs) and T cells
were obtained from normal donors. T cells were purified by positive selection of CD4 and CD8 cells using immunomagnetic beads (Dynal, Oslo,
Norway) and beads were detached using the detachment reagent (Dynal)
according to the manufacturer's instructions. CTLL-2 cells were
maintained in RPMI/10% FCS and 10 U/mL recombinant human IL-2 (rhIL-2;
Chiron, Harefield, UK). For the generation of peripheral blood-derived DCs, PBMNCs were incubated in RPMI/10% FCS at 37°C and 5% CO2 for 2 to 4 hours, and nonadherent cells were
removed. Adherent cells were then cultured for 7 to 10 days in the
presence of 50 ng/mL granulocyte-macrophage colony-stimulating factor
(GM-CSF; Schering Plough, Suffolk, UK) and 1,000 U/mL IL-4 (Schering
Plough). DC phenotype was confirmed by flow cytometry (see below), with purities ranging from 70% to 85%.
Flow cytometric analysis.
U937 cells were stained using the following antibodies; anti-CD80,
anti-HLA-DR (Becton Dickinson, Oxford, UK), anti-CD86 (Serotec, Oxford, UK), and anti-MHC class I (Cymbus Biosciences, Southampton, UK). Biotinylated-CTLA4.Ig (Ancell Corp, Bayport, MN) was
used to confirm the ability of CD80 to bind to one of its receptors and
was also used in functional blocking studies. Confirmation of DC
phenotype was by dual-labeling using anti-CD1a and anti-CD14 (Serotec;
CD1a+, CD14 ). Flow cytometric analysis
was performed using a FACS Vantage (Becton Dickinson) and Cellquest
software (Becton Dickinson).
Generation of CD80+ U937 cells.
Initially, CD86 U937 cells were obtained by
fluorescence-activated cell sorting (FACS) cells
previously cultured for 48 hours in 20 µg/mL phorbol myristate
acetate (PMA; Sigma), which increases CD86 expression
(data not shown). The sorted CD86 negative cells
failed to express CD86 on further exposure to PMA. U937 cells were
transduced using a myeloproliferative sarcoma virus
(MPSV)-based retroviral vector encoding human CD80 cDNA with Hygromycin-resistance gene as a selectable marker.1,40 Hygromycin-resistant cells were then further sorted by FACS for high
CD80 expression (U937-CD80 cells). Control empty vector infected cells
were selected for Hygromycin resistance and subsequently sorted for
absence of CTLA4.Ig binding (U937-M3P).
Proliferation assays.
PBMNCs or T cells (>98% purity) were cocultured for 5 days at
105/well in triplicate with irradiated control U937-M3Ps,
U937-CD80 cells, or third-party DCs at the stated concentrations. U937
cells were irradiated with 100 Gy and third-party DCs with 35 Gy. On day 5,3H-thymidine (0.5 µCi/well; Amersham International,
Little Chalfont, UK) was added for the final 20 hours. The cells were
then harvested and 3H-thymidine incorporation was measured
by liquid scintillation. Where indicated, CTLA4.Ig (10 ng/mL) was added
to block CD80 interaction with its cognate receptors.
Helper T lymphocyte precursor cells (HTLp) frequency
analysis.
The frequency of responding HTLp cells was assessed by limiting
dilution analysis as previously described.41 Briefly,
PBMNCs were added to irradiated stimulator cells (control U937-M3P or U937-CD80) at serial dilutions. Each dilution was performed in replicates of 24, as were wells containing stimulators or responders alone. After 4 days, 100 µL of supernatant was transferred to a new
plate. CTLL-2 cells, previously starved of IL-2 for 24 hours, were then
added at 104/well. 3H-thymidine (0.5 µCi/well) was added after 8 to 10 hours and incorporation was
measured after a further 20 hours. Wells were scored as positive if the
reading exceeded 3 standard deviations above the mean for the
stimulator alone wells. Frequency analysis was calculated by maximum
likelihood statistical analysis, using software kindly provided by Dr
Peter Brookes (Hammersmith Hospital, London, UK).41
Calcium mobilization.
PBMNCs were resuspended to 107/mL in RPMI and labeled with
3 µmol/L Indo-1 (Molecular Probes, Eugene, OR) for 30 minutes at 37°C, washed, and resuspended in RPMI at
106/mL. Cells were then mixed with either U937-M3P or
U937-CD80 cells at a 1:1 ratio. Kinetic studies of intracellular
calcium concentration were performed on a FACS Vantage equipped with
488 nm and UV lines (60 mW). The calcium concentration was determined
by the bound:free Indo-1 ratio (ie, fluorescence at 480 nm:530 nm). By
gating on lymphocytes, leukemic cells were excluded, on the grounds of
both scatter properties and Indo-1 labeling. A concentration of
anti-CD3 (clone OKT-3; Janssen-Cilag, Bucks, UK) was established (ie,
0.1 ng/mL) that, with cross-linking using a final dilution of 1/175 RAM
(Rabbit antimouse Ig; Dako A/S, Glostrup, Denmark), gave
suboptimal activation of lymphocytes in the presence of U937-M3Ps. This
experiment was then repeated either with the addition of 1 µg/mL of
anti-CD28 (clone CD28.2; PharMingen, San Diego, CA) or in
the presence of U937-CD80 cells. Control experiments were performed
using anti-CD28 or U937-CD80 without anti-CD3. Adequate Indo-1 labeling
was confirmed by stimulation with ionomycin (2 µg/mL).
Viability of activated T cells.
Mixed lymphocyte:leukemic cell reactions (MLLRs) were set up using
PBMNCs (2 × 106/mL in RPMI with 10% autologous
serum) mixed at a responder:stimulator ratio of 5:1 with either
irradiated U937-M3Ps or irradiated U937-CD80s and then incubated at
37°C and 5% CO2 for 4 days. Cells were then labeled
using fluorescein isothiocyanate (FITC)-anti-CD69, FITC-anti-CD25 (Bioproducts, Heidelberg, Germany), phycoerythrin (PE)-anti-CD4, and
PE-anti-CD8 (Sigma). Dual-labeled cells were sorted by FACS and then
plated at a concentration of 1 × 105/mL in complete
medium containing 30 U/mL rhIL-2. Sorted T cells from the U937-M3P
MLLRs were also plated in conditioned media from the original U937-CD80
MLLR containing 30 U/mL IL-2. Viabilities were established using trypan
blue exclusion.
Measurement of BCL-2 and BCL-XL.
MLLRs were set up as described above, and a positive control was
incorporated using PBMNCs stimulated with anti-CD3 and anti-CD28. After
4 days, PBMNCs (106) were dual-labeled for surface CD25
(Bioproducts) and intracellular BCL-2 (Dako) or BCL-XL
(Zymed Laboratories Inc, San Francisco, CA) using a commercially
available fix and permeabilization kit (Fix and Perm; Caltag
Laboratories, Burlingame, CA). Cells were initially fixed and labeled
with the unconjugated intracellular antibodies according to the
manufacturer's instructions. Cells were washed in ice-cold
phosphate-buffered saline containing 2.5% FCS (PBSF), and PE-labeled
secondary antibody (rabbit antimouse 1:30 dilution; Dako) was added for
20 minutes at room temperature. After a further wash in PBSF, cells
were incubated for 10 minutes at room temperature with pure mouse Ig
(Sigma) followed by FITC-anti-CD25 for 20 minutes at 4°C. Cells
were then washed in PBSF and resuspended in PBS. Flow cytometric
analysis was performed using a FACS Vantage. Gates were set on the
lymphocyte region. For each experiment, an appropriate isotype-matched
control antibody (IMC) was used to determine the level of nonspecific
binding. The level of protein expression in CD25+ cells was
calculated by subtracting the mean fluorescence intensity (MFI) for the
IMC from that with the specific antibodies. A control assay was set up
to confirm the specificity of the BCL-XL antibody in a flow
cytometric-based assay. Normal PBMNCs were either left unstimulated or
activated for 48 hours with anti-CD3 alone or anti-CD3 and anti-CD28.
Intracellular staining and flow cytometric analysis was as described above.
Cytokine analysis.
Cytokine analysis was performed on 5-day MLLRs using enzyme-linked
immunosorbent assay (ELISA) kits, IL-2, IL-4, IL-10, and  -interferon
(-IFN; Genzyme, Cambridge, MA), as per the manufacturer's instructions. The detection limits were 10, 6, 5, and 6 pg/mL for IL-4,
IL-2, IL-10, and -IFN, respectively.
For intracellular cytokine analysis, PBMNCs were cultured at
106/mL in the presence of a transwell insert (pore size,
0.4 µm; Falcon, Becton Dickinson) containing U937 cells at
105/mL for 24 hours. Cells were then activated with PMA (10 ng/mL) and Ionomycin (1 µg/mL) overnight in the presence of monensin (1.4 µg/mL; all from Sigma). For neutralizing assays, agents were used at a concentration of 5 µg/mL anti-TGF-, 30 ng/mL
anti-IL-10, 10 µmol/L Indomethacin, 100 U/mL IL-2, and 50 pmol
IL-12. Labeling with conjugated antibodies to cytokines (IL-2-FITC,
IL-4-PE, IL-10-PE, and -IFN-FITC; all from Serotec) was performed
using a commercially available permeabilization kit (Fix and Perm;
Caltag Laboratories). Both irrelevant conjugated antibodies and
unstimulated cells were used as controls to determine the level of positivity.
| |
RESULTS |
Phenotype of U937 cells: Expression of MHC class I and II and absence
of CD80/CD86 on control cells.
In this study, we have used the human acute monocytic leukemia cell
line U937 that has a phenotype similar to many primary acute myeloid
leukemias (AMLs) and, importantly, expresses high levels
of MHC class I and class II (Fig 1A).
Wild-type U937 cells express low levels of CD86, which is upregulated
by PMA. Both CD80 and CD86 have a higher affinity for the negative
regulator CTLA4 than they do for CD28.18 Therefore,
low-level expression of these costimulators will preferentially
activate CTLA4. To eliminate any effect of CD86, U937 cells were
stimulated with PMA for 48 hours, and then CD86
cells were sorted by FACS. These cells remained CD86
and failed to bind CTLA4.Ig (Fig 1B) even with further stimulation with
PMA (data not shown). Using retroviral vectors, we generated 2 cell
lines, 1 expressing CD80 (U937-CD80) and control cells infected with
the control vector (U937-M3P). Costimulation through CD28 is optimal
when CD80 molecules are expressed at high levels.42,43 Therefore, U937-CD80 cells were further sorted for high CD80 expression determined by both anti-CD80 antibody and CTLA4.Ig binding. In addition
to MHC, both U937 cell lines express cell adhesion molecules: ICAM-1
(CD54), LFA-1 (CD11a), and Mac-1 (CD11b) (data not shown).
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| Fig 1.
MHC class I and II and CD80 expression by U937 cells. (A)
Single-color labeling demonstrated that U937 cells have similar levels
of MHC class I and II as DCs. Control U937-M3P cells were
CD80, and U937-CD80 cells had a high level of CD80
expression that was greater than that of DC. (B) Dual-labeling using
anti-CD80 antibody and CTLA4.Ig demonstrated no CD80/CD86 expression on
the control U937-M3P population and high anti-CD80 as well as CTLA4.Ig
binding on the U937-CD80 population.
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CD80 expression increases proliferative responses of allogeneic T
cells to U937 cells.
To compare the responses to CD80+ and control U937 cells,
purified allogeneic T cells (Fig 2A) were
used in 5-day MLLRs. T cells from 5 normal donors demonstrated up to a
16-fold increase (range, 1.7- to 16-fold; mean, 7-fold; median,
3.6-fold) in their response to CD80+ U937 cells compared
with the control (Fig 2A). Despite high levels of CD80 and MHC
expression, responses to U937-CD80 cells were still less than
third-party DCs at a lower stimulator:responder ratio. The specificity
of the enhanced response to CD80 was demonstrated by the ability of its
solubilized high-affinity receptor, CTLA4.Ig, to inhibit T-cell
proliferation (Fig 2B).
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| Fig 2.
CD80 expression increases proliferative responses of
allogeneic T cells to U937 cells. (A) T cells were cocultured with
irradiated U937-M3P, U937-CD80, or third-party DCs. Cumulative data of
proliferative responses from 8 experiments with 5 different donors with
mean ±SE are shown. Stimulator:responder ratios were 1:1 and 1:10 for
the leukemic and DCs, respectively. T cells demonstrated up to a
16-fold increase in their proliferative response to U937-CD80 cells
compared with the control cells. (B) The increased proliferative
response of T cells to CD80+ cells seen in (A) could be
inhibited by soluble CTLA4.Ig (10 ng/mL), demonstrating this is a
CD80-mediated effect. These data are representative of 3 independent
experiments.
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CD80 expression increases the frequency of precursor T cells
activated.
The increased T-cell proliferation induced by CD80 may be explained by
either an increased frequency of precursor T cells activated or
activation of the same number of precursors receiving a greater
proliferative stimulus (ie, effects on the initiation of T-cell
activation or later events). To test this, HTLp frequencies (measured
using the IL-2-dependent CTLL-2 cell line) to CD80+ and
CD80 U937 cells were measured by limiting dilution
analysis. CD80 expression consistently increased the precursor
frequency (mean, 4.1-fold; range, 2.8- to 5.5-fold;
Fig 3A). Calculated HTLp frequencies correlated with the proliferation observed in bulk cultures. The possibility that CD28 stimulation by CD80 induced a nonspecific proliferation was excluded by analysis of the mean values of the negative wells. This showed similar background levels for
CD80+ and CD80 stimulators (Fig 3B). At
the lowest dilution (2.5 × 103 cells/well), the
majority of wells were negative, so any positive wells were likely to
have contained single precursors. Because the readout for the HTLp
frequency assay is a sensitive IL-2 bioassay, a comparison of the
values of the positive wells at the lowest dilution also indicates the
amount of IL-2 secreted after activation of a single precursor. This
analysis showed only a 78% increase in IL-2 production in the presence
of CD80+ stimulators (Fig 3B). We can conclude therefore
that CD28 stimulation may increase IL-2 secretion, but that, in this
model of antigen presentation by leukemic cells, this is of less
significance than the effect on precursor activation.
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| Fig 3.
CD80 expression by tumor cells increases the frequency of
precursor T cells activated. (A) The data show calculated HTLp
frequencies (log10 scale) to U937-M3P and U937-CD80 cells
for 5 individuals as described in Materials and Methods. Precursor
frequencies were measured by a limiting dilution assay. The frequencies
were increased up to 5-fold by CD80 expression and correlated with the
proliferation observed in bulk cultures. (B) This bar chart represents
the mean ± SE 3H-thymidine incorporation by the
IL-2-dependent cell line, CTLL-2, for the wells of the bottom dilution
from the 5 experiments shown in (A). The values from wells scored
negative or positive are shown, when either U937-M3P ( ) or U937-CD80
( ) cells were used. 3H-thymidine incorporation of the
negative wells was similar in both U937-M3P and U937-CD80 assays,
demonstrating a comparable baseline level. Mean CTLL-2 proliferation in
the positive wells was 78% greater in the presence of U937-CD80 cells.
This suggests that, after activation, precursors stimulated by
U937-CD80 cells produce more IL-2.
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CD28 stimulation lowers the threshold for TCR triggering.
The observed increase in precursor activation suggests that CD80
stimulation of CD28 acts in part by regulating TCR signaling. To test
this, the effect of CD28 stimulation on calcium mobilization after
suboptimal TCR stimulation was studied. Using the defined suboptimal
concentration of cross-linked anti-CD3, TCR stimulation alone in the
presence of U937-M3P cells induces a minimal increase in intracellular
calcium (Fig 4A). However, simultaneous TCR
and CD28 stimulation either by anti-CD28 (Fig 4B) or by the presence of
CD80-expressing tumor cells (Fig 4C) enhanced calcium mobilization. Neither CD28 cross-linking (Fig 4D) nor U937-CD80 cells (data not
shown) induced calcium mobilization in the absence of TCR stimulation.
Therefore, CD28 stimulation through CD80 expression by tumor cells
lowers the threshold of TCR triggering, ie, enables the initiation of
T-cell activation at a lower level of TCR stimulation.
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| Fig 4.
CD28 stimulation lowers the threshold for TCR triggering.
Calcium mobilization in normal Indo-1-loaded T cells, before and after
TCR stimulation with anti-CD3 followed by cross-linking with polyclonal
rabbit antimouse Ig antibody (RAM). The plots show intracellular
calcium concentrations (ratio of bound:free Indo-1) over a 240-second
time period. The white line represents the median calcium concentration
at each 10-second time slice. In (A), a suboptimal level of anti-CD3
was determined that, with cross-linking in the presence of U937-M3P
cells, gave poor median calcium mobilization. Costimulation through
CD28 by addition of a specific activating antibody at the same time as
anti-CD3 markedly increased the number of activated T cells (B) not
seen using anti-CD28 alone (D). Enhanced T-cell activation was also
seen when CD28 stimulation was provided by CD80+
U937-CD80 cells instead of U937-M3Ps (C). U937-CD80 cells, in the
absence of anti-CD3, also failed to trigger calcium mobilization (data
not shown).
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CD80 expression enhances survival of activated T cells.
After a 4-day MLLR, activated T cells stimulated in the presence of
U937-M3P cells were found to express similar levels of the
high-affinity IL-2 receptor (CD25) as those stimulated in the presence
of U937-CD80 cells. The percentages of CD25+ T cells were
12.5% and 12.6%, and the levels of CD25 expression given as the MFI
were 105 au and 109 au for U937-M3P and U937-CD80, respectively. These
activated cells were sorted and returned to culture with exogenous IL-2
(30 U/mL). Analysis of the viability and numbers of sorted
CD25+ T cells demonstrated that, after 7 days in culture,
viability of cells stimulated by U937-CD80 cells decreased to almost
one third (Fig 5A). The cell count was only
marginally reduced (Fig 5B), indicating a combination of continued
proliferation and death. By day 7, T cells that had been activated in
the presence of U937-M3P cells had undergone activation-induced cell
death and there were no viable cells present. When T cells from the
U937-M3P MLLR were cultured in the presence of conditioned medium from
the U937-CD80 MLLR as well as rhIL-2, there was an improvement in both
the cell count and the viability, suggesting that CD28 stimulation
induces the secretion of another T-cell growth factor.
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| Fig 5.
CD28 stimulation reduces death and promotes expansion of
T cells. PBMNCs were activated in the presence of U937-M3Ps or
U937-CD80s in a 4-day MLLR. Activated T cells were sorted and cultured
in exogenous IL-2 (30 U/mL). Activated T cells from the U937-M3P MLLR
were also cultured in conditioned medium from the U937-CD80 MLLR. In
this experiment, T cells activated in the presence of U937-CD80 cells
() demonstrated 38% viability by day 7 (A), but viable cell numbers
were still 80% of the original count (B), suggesting that there was an
expansion of a specific population. T cells cultured in the presence of
U937-M3P cells ( ) demonstrated zero viability by day 7. The addition
of conditioned medium from the MLLR that used U937-CD80 cells as
stimulators to these cells improved their viability ( ). These data
are the mean of 3 independent experiments ± SE.
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U937-CD80 cells increase BCL-2 but not BCL-XL.
Clonal expansion of T cells is partly dependent on the upregulation of
antiapoptotic proteins such as BCL-2 and BCL-XL, leading to
the prevention of activation-induced cell death. To determine whether
observed differences in T-cell survival were due to altered expression
of antiapoptotic proteins, levels of BCL-2 and BCL-XL were
measured in responding T cells after 4 days of stimulation with either
U937-M3P or U937-CD80 cells. Dual-color flow cytometry was used to
detect expression of these antiapoptotic proteins in activated T cells
(ie, CD25+). Cells stimulated by U937-CD80 cells
demonstrated a 40% increase in expression of BCL-2 (measured as MFI)
compared with those stimulated by U937-M3P cells (P = .023 by
paired t-test; Fig 6A). Although reports suggest that the major antiapoptotic protein upregulated by
CD28 ligation is BCL-XL, we were not able to detect any
expression in activated T cells even when the stimulator cells
expressed CD80 (Fig 6B). Activation with anti-CD3 and anti-CD28 induced BCL-XL expression, which was not seen with anti-CD3 alone.
CD28 stimulation mediated by CD80 expression on tumor cells is
therefore less effective in inducing antiapoptotic proteins.
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| Fig 6.
U937-CD80 cells cause upregulation of BCL-2 but not
BCL-XL. PBMNCs were activated in the presence of irradiated
U937-M3Ps or U937-CD80s in a 4-day MLLR. BCL-2 ( ) and
BCL-XL () levels were measured in the activated
(CD25+) population by dual surface and intracellular flow
cytometry as described in Materials and Methods. U937-CD80 cells
stimulated a 40% greater expression of BCL-2 in CD25+
cells measured by MFI compared with stimulation with U937-M3Ps
(P = .023, paired t-test). There was no significant
increase in BCL-XL with either stimulator. These data are
the mean ± SE of 4 experiments. Expression of BCL-XL in
CD25+ cells stimulated by either (1) anti-CD3 anti-CD28
or (2) U937-M3P or U937-CD80 cells. Cells were dual-labeled for
anti-CD25 and anti-BCL-XL after 48 or 96 hours of
stimulation, respectively. The histograms show expression of
BCL-XL in the CD25+ population. Labeling with
control antibodies gave the same level of fluorescence as that with
anti-BCL-XL after stimulation with anti-CD3 alone.
Therefore, T-cell receptor ligation alone is insufficient to induce
BCL-XL expression. The addition of CD28 cross-linking
induced expression of BCL-XL, which was not observed when
CD28 stimulation was through CD80 expression on U937 stimulator cells.
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U937-CD80 cells fail to induce a Th1 cytokine profile.
The pattern of cytokines produced by T cells plays a pivotal role in
the nature of an immune response to a given antigen. Secretion of Th1
and Th2 cytokines produced in response to U937-M3P, U937-CD80, or
third-party DCs were compared. As anticipated, third-party DCs produced
a Th1 cytokine pattern (-IFN, IL-2, and no Th2 cytokines). In 4 of 5 donors, there was no detectable cytokine response induced by either
CD80+ or CD80 U937 cells despite
moderate proliferation. With 1 donor that had identical proliferative
responses to U937-CD80 cells and third-party DCs, there was
significantly less Th1 cytokine production, but also detectable
secretion of Th2 cytokines (Fig 7). In this
experiment, the production of IL-4 was particularly enhanced by the
expression of CD80 on U937 cells. These data indicate that, although
CD80 expression on U937 cells increases T-cell activation and
proliferation, there is a failure to induce the secretion of
appropriate Th1 cytokines in response to alloantigen.
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[in a new window]
| Fig 7.
U937-CD80 cells may induce a Th2 cytokine profile. T
cells were cocultured with irradiated U937-M3P, U937-CD80, or
third-party DCs in a 5-day MLLR and cytokine ELISAs were performed on
the resultant supernatant. DCs produced a Th1 cytokine pattern as
anticipated (high levels of -IFN and IL-2). Of 5 experiments using
different responders, only 1 demonstrated detectable cytokine
production. This was more of a Th2 cytokine pattern (IL-4 and IL-10
were detected and IL-2 and -IFN were much lower than induced by the
DCs). There was no detectable cytokine production (IL-2, IL-4, IL-10,
or -IFN) from 5-day irradiated U937 cells alone.
|
|
Soluble factors secreted by U937 cells inhibit Th1 cytokine
production.
To test the hypothesis that soluble factors derived from U937 cells
directly influence cytokine secretion, cytokine production by PBMNCs
stimulated by PMA/ionomycin in the absence or presence of U937 cells
was compared by intracellular flow cytometric analysis. U937 cells were
separated by a transwell insert enabling soluble factors produced by
the leukemic cells to interact with the activated T cells, but avoiding
the influence of cell:cell contact. Dual labeling of Th1 (-IFN and
IL-2) and Th2 (IL-4 and IL-10) was performed as described in Materials
and Methods. Resting PBMNCs had very low levels of cytokine production,
whereas stimulated ones demonstrated increased levels of IL-2 and
-IFN. Both of these cytokines were inhibited when cells were
stimulated in the presence of U937s
(Fig 8A). No significant
levels of IL-4 or IL-10 were detected in any of the assays, but they
were only short-term assays, ie, before peak secretion occurs. Attempts
to overcome the observed inhibition by either blocking known Th1
inhibitors (TGF-, IL-10, and prostaglandin E2
[PGE2]; not shown) or overcoming it with
concurrent addition of exogenous IL-2 or IL-12 were unsuccessful (Fig
8B). However, preincubation of PBMNCs with exogenous IL-2 for 4 hours
before exposure to U937 cells overcame the inhibitory effect (Fig 8C).
This preventative action of IL-2 on cytokine inhibition was not seen
with either IL-12 or GM-CSF even with preincubation times of up to 24 hours (data not shown).
View larger version (85K):
[in this window]
[in a new window]
| Fig 8.
Soluble factors secreted by U937 cells inhibit Th1
cytokine production. (A) FACS analysis of intracellular cytokines
(-IFN and IL-4, IL-2, and IL-10) was performed on PBMNCs that had
been stimulated with PMA/ionomycin in the absence or presence of U937
cells separated by a 0.4-µm pore membrane. PBMNCs stimulated in the
absence of U937 cells were almost exclusively producing -IFN and
IL-2. Those activated in the presence of U937 cells showed a
much-reduced number producing -IFN and IL-2. (B) The same experiment
in the presence of reagents to either block potential Th1 inhibitors
(neutralizing antibodies to TGF-, IL-10, or [not shown here]
indomethacin to block PGE2) or overcome them (IL-2 and
IL-12) showed all had no effect when added concurrently with U937
cells. (C) PBMNCs were incubated with IL-2 for 4 hours
before the addition of U937 cells and were then analyzed for -IFN
production by intracellular staining as described above. Preincubation
with IL-2 overcame the effect of the leukemia-derived inhibitory factor
on -IFN production.
|
|
| |
DISCUSSION |
In this study, we have demonstrated that CD80 expression on a leukemic
cell increases T-cell recognition and proliferation primarily by
lowering the threshold for TCR signal transduction and also by
enhancing survival signals. However, in this model, soluble factors
secreted by the leukemic cells inhibit cytokine production, thus
preventing the development of the desired cell-mediated immunity.
Several studies have shown enhanced T-cell proliferative responses to
CD80-expressing tumor cells by generating a costimulatory signal
through ligation of CD28 on T cells.4,5 The role of CD28
ligation during T-cell activation by professional APCs has generally
been considered to be primarily by promoting IL-2 secretion and, hence,
growth and survival. However, we show here that, when CD80+
tumor cells are used as nonprofessional APCs, the primary mechanism for
enhancing T-cell recognition is through amplification of TCR signals,
ie, during the initial activation phase. These results are consistent
with the finding of reduced TCR occupancy required for T-cell
activation in the presence of CD28 stimulation.44 A
consequence of this augmentation of TCR signaling is the potential recognition of previously silent antigens, a hypothesis supported by
the finding of an expanded T-cell repertoire generated against CD80+ tumor cells.11
Although initiation of T-cell activation is not dependent on CD28, CD28
plays an important role in the survival of activated T cells. It is now
becoming clear that, in addition to effects on IL-2 secretion, CD28
plays a role in T-cell survival through upregulation of the
antiapoptotic proteins, BCL-XL, and, to a lesser extent,
BCL-2.14-16 We have been able to show here in vitro that
expression of CD80 by tumor cells induces the upregulation of BCL-2,
delaying the death observed in T cells activated in the absence of
CD80-mediated costimulation. Although additional cross-linking of CD28
with TCR stimulation induced BCL-XL expression, no
BCL-XL expression could be detected when CD28 was
stimulated by CD80+ U937 cells. Several studies suggest
that signaling through CD28 has a more profound effect on
BCL-XL than BCL-2,45 which may be due in part
to CD28-mediated IL-2 secretion, because signaling through the IL-2 receptor (high and intermediate affinity) itself may
induce BCL-XL. One mechanism to explain the failure of
CD80-expressing U937 cells to induce BCL-XL may therefore
be a consequence of the inhibition of IL-2 secretion by a U937-derived
soluble factor(s). Equally, the inhibitory factors may act on
downstream components of the CD28 signaling pathway blocking both
cytokine and BCL-XL synthesis.
Controversy exists regarding the role of costimulation in the Th1/Th2
decision, but cytokines appear to play a more important role in
determining the nature of an immune response to a given antigen. The
microenvironment generated by a malignant cell is clearly different to
that induced by professional APCs. Tumor cells produce many cytokines
(eg, TGF- and IL-10), soluble cytokine receptors (eg, sIL-2R), and
other biological response modifiers (eg, PGE2) that are
known to inhibit cell-mediated immunity. Despite enhanced proliferative
responses, T cells stimulated by leukemic cells in this model were
generally unable to produce detectable levels of cytokines. In 1 exception, in which there was detectable cytokine production, the
profile was more of a Th2 than a Th1 pattern. In this model, a
leukemia-derived soluble factor(s) inhibits Th1 cytokine synthesis, but
does not prevent proliferation; costimulation is therefore enabling
T-cell activation to occur in a nonpermissive environment for the
generation of cell-mediated immunity. This may explain the synergistic
effect seen in immune gene therapy studies of coexpressing CD80 with
T-cell growth factors (eg, IL-2,46 IL-7,47 and
IL-1248). This concept is supported by the observation in
this study that preincubation of PBMNCs with IL-2 overcomes the
inhibitory effect of U937 cells on T-cell cytokine production. However,
importantly, the inability of IL-2 to prevent this inhibition when
administered concurrently with leukemic cells suggests that, to be
successful in a therapeutic setting, a state of minimal residual
disease would have to be created before the administration of
gene-modified cells.
The findings of this study have several implications for understanding
the mechanisms by which leukemic cells may evade immune rejection, eg,
absence of response to donor leukocyte infusions after leukemic relapse
after allogeneic bone marrow transplantation. Absence of costimulatory
molecules on leukemic cells may result in either ignorance or deletion
of antigen-specific T cells depending on the level of antigen
expressed. If an antigen is expressed at a low level, there is an
inadequate TCR signal for T-cell activation and the antigen will not be
recognized. If an antigen is expressed at a high level, signaling
through the TCR alone is sufficient to induce T-cell activation, but
the lack of survival signals would result in activation-induced cell
death and ultimately clonal deletion. Expression of costimulatory
molecules by a leukemic cell enables T cells to recognize lower levels
of antigen that induce the survival signals required for clonal
expansion of leukemia-reactive T cells, but the microenvironment
generated by the leukemia may create a further defense against immune
rejection. Tumor-derived factors have been shown to have numerous
effects on T cells,25-27 APCs,30,49 and
effector functions.50 Primary T-cell responses in the
presence of immunosuppressive cytokines, eg, IL-1051,52 or
TGF-,53 have been shown to lead to the induction of
tolerance rather than productive immunity. Therefore, activation of
leukemia-reactive T cells by expression of costimulatory molecules on
the leukemic cell may lead to active tolerance if the leukemic cells
generate an immunosuppressive microenvironment. This may partly explain variable results with CD80-modified tumor cells and the observed association between positive effect and inherent
immunogenicity.54 There are known potent Th1 cytokine
inhibitors (IL-10,22 TGF-,22,50 and
PGE251), but from blocking studies (IL-10,
TGF-, and PGE2) and direct assays (IL-10 and TGF-),
we have no evidence to suggest that cytokine inhibition by U937 cells
is caused by any of these. Although cell lines may not be
representative of their primary counterparts, in all 10 primary AML
cases we have studied so far, a similar effect of leukemia-derived
factor(s) on synthesis of IL-2 and -IFN has been observed (data not
shown). Therefore, the data presented here using U937 cells may be
extended to human leukemias.
Several powerful methods are now available to elicit CTL responses to
tumor-associated antigens in vitro and in vivo. It remains to be
addressed how developing tumors bearing such antigens fail to be
rejected; absence of costimulation remains a candidate mechanism. The
provision of costimulation has the potential to expose previously silent antigens to recognition by specific T cells; however,
tumor-elaborated factors may prevent the generation of effective
antitumor immunity.
| |
ACKNOWLEDGMENT |
The authors thank Prof D. Vergani (University College Hospital, London,
UK) and Dr M. Peakman (King's College School of Medicine and
Dentistry, London, UK) for critical reading of the manuscript.
| |
FOOTNOTES |
Submitted December 29, 1998; accepted July 15, 1999.
Supported by the Kay Kendall Leukaemia Fund and the Leukaemia Research
Fund of the United Kingdom.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to W.J.R. Hirst, MD, Department
of Haematological Medicine, King's College School of Medicine and
Dentistry, Leukaemia Sciences Laboratories, The Rayne Institute, 123 Coldharbour Lane, London, UK, SE5 9NU.
| |
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TOP
ABSTRACT
INTRODUCTION