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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1677-1684
CD80-Transfected Acute Myeloid Leukemia Cells Induce Primary
Allogeneic T-Cell Responses Directed at Patient Specific Minor
Histocompatibility Antigens and Leukemia-Associated Antigens
By
Tuna Mutis,
Ellen Schrama,
Cornelis J.M. Melief, and
Els Goulmy
From the Department of Immunohematology and Blood Bank, Leiden
University Medical Center, Leiden, The Netherlands.
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ABSTRACT |
Despite sufficient levels of HLA class I and class II expression,
acute myeloid leukemia (AML) cells usually fail to induce a significant
T-cell response in vitro. Therefore, we investigated whether in vitro
modifications could enhance the T-cell stimulatory properties of AML
cells. AML cells were either cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and tumor
necrosis factor- (TNF-), or transfected with the CD80 (B7.1) gene
and used as stimulator cells for primed and unprimed allogeneic T
cells. Cytokine treatment increased HLA class I and II expression, but
did not induce CD80 on AML cells. Cytokine-treated AML cells
efficiently presented nominal and allo-antigens to primed T-cell
clones, induced strong T-cell proliferation in HLA mismatched mixed
lymphocyte reactions (MLR), but failed to induce primary T-cell
responses from an HLA identical bone marrow donor in MLR. In contrast,
CD80-transfected AML cells induced T-cell proliferation of
HLA-identical bone marrow donor peripheral blood mononuclear cell
(PBMC) in primary MLR, allowing the generation of leukemia reactive
CD4+ T-cell lines and clones. The majority of the
generated oligoclonal (25 of 35) T-cell cultures showed patient
specific reactivity that did not discriminate between patient's
leukemic cells and Epstein-Barr virus (EBV)-transformed B cells
(EBV-LCL). The remaining 10 oligoclonal T-cell cultures recognized only
leukemic cells. One of these latter leukemia reactive oligoclonal T
cells was cloned. The majority of the clones (25 of 29) reacted against both leukemic cells and patient's EBV-LCL. A minority of the T-cell clones with the CD4 phenotype (four of 29) showed strong
HLA-DP restricted reactivity against leukemic cells, but
not against patient's EBV-LCL or against HLA-matched nonleukemic
cells, indicating that their target antigens are preferentially
expressed by leukemic cells. In conclusion, our study shows that the in
vitro allogeneic T-cell response induced by CD80-transfected AML cells
is mainly directed against patient's specific minor histocompatibility
antigens, while antigens preferentially expressed by leukemic cells can also trigger T-cell responses.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
ALLOGENEIC BONE MARROW transplantation
(BMT) is an effective treatment for acute and chronic forms of
leukemia.1-4 Clinical and experimental data indicate that
donor-derived T cells play an important role in eliminating the
residual leukemic cells after BMT.5-8 In the HLA identical
situation, much can be said in favor of donor T cells reactive against
the minor histocompatibility antigens (mHag) expressed on patient's
leukemic cells (reviewed in Goulmy9). Donor-derived
mHag-specific cytotoxic T cells (CTL) isolated from graft-versus-host
disease (GVHD) patients have been shown to effectively
lyse leukemic cells and to inhibit the outgrowth of leukemic cell
precursors.10,11 However, it is unclear whether leukemic
cells express unique antigens that can induce a leukemia cell-specific
T-cell response. Addressing this issue has been difficult because
leukemic cells rarely induce a significant T-cell response in vitro,
which may be caused by the absence of costimulatory molecules such as
CD80. Recent studies suggest that the T-cell stimulatory properties of
leukemic cells can be upregulated. For instance, upon activation via
CD40, B-cell lymphoma or leukemia cells express high levels of HLA and
CD80/CD86 molecules and become efficient antigen-presenting cells (APC) for alloreactive T cells.12,13 Moreover, CD40 stimulated
pre-B leukemia cells have been succesfully used for the in vitro
induction of leukemia reactive autologous CTL from several pre-B
leukemia patients.14 Potent APC with dendritic cell
phenotype can be also generated from malignant CD34+
precursors of chronic myeloid leukemia (CML) cells by culturing with
granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and tumor necrosis factor-
(TNF-).15 In addition, in different mice models,
including a leukemia model, low immunogenic tumor cells that were
genetically engineered to express CD80 became more immunogenic and were
rejected by syngeneic hosts.16-24 In light of these recent
data, we searched for in vitro conditions that could potentiate antigen
presentation by acute myeloid leukemia (AML) cells. In a previous
study, we have shown that AML cells cultured with GM-CSF, IL-4, and
TNF- induced strong T-cell responses in an HLA mismatched
combination. This led to the identification of an allo-major
histocompatibility complex (MHC)-restricted T-cell clone
recognizing leukemic cells and CD34+ early progenitor cells
only.25
Here we have compared the T-cell stimulatory capacities of
cytokine-treated AML cells and AML cells transfected with CD80 cDNA in
HLA identical bone marrow (BM) donor/patient combinations. Cytokine-treated AML cells efficiently presented nominal and
allo-antigens to T-cell clones, induced strong T-cell proliferation in
HLA mismatched, but not in HLA identical combinations. In contrast,
CD80-transfected AML cells were able to induce T-cell proliferation
from HLA identical BM donor. This allowed us to generate leukemia
reactive CD4+ T-cell lines and clones and to analyze the
target cell specificity of these leukemia reactive T cells.
| |
MATERIALS AND METHODS |
Cells.
Peripheral blood mononuclear cells (PBMC) or BM cells were obtained by
Ficoll-opaque density centrifugation. Patient PBMC that contained
morphologically more than 95% malignant cells were assigned as AML
cells. PBMC, BM, and AML cells were cryopreserved until usage.
Epstein-Barr virus transformed B cells (EBV-LCL).
PBMC were incubated with EBV for 1.5 hours at 37°C. EBV transformed
B cells (EBV-LCL) were maintained in RPMI-1640 plus 10% fetal calf
serum (FCS).
T-cell clones.
The HLA-DR15 restricted, hsp65 reactive T-cell clone R2F10 was obtained
from Dr T. Ottenhoff (Leiden University Medical Center [LUMC],
Leiden, The Netherlands), The HLA-DPB1* 1301 and HLA-DRB1* 1302 specific alloreactive T-cell clones were obtained from Dr S. de Koster
(LUMC, Leiden, The Netherlands). The antigen specificities of these
clones were described previously.26,27
Cytokine treatment of AML cells.
AML cells were thawed and cultured in the presence of 800 U/mL GM-CSF
(kindly provided by Dr S. Osanto, LUMC, Leiden, The Netherlands), 500 U/mL IL-4 (Genzyme, Leuven, Belgium), 50 U/mL TNF- (Genzyme) in RPMI
1640 (GIBCO, Grand Island, NY) supplemented with 10% FCS,
100 U/mL penicillin, 100 µg/mL streptomycin (GIBCO) during different
periods (72 hours to 14 days).
Transfection of AML cells with plasmid vectors.
AML cells were thawed and cultured 16 hours in RPMI supplemented with
10% FCS and antibiotics (penicillin 100 U/mL, streptomycin 100 µg/mL). A total of 6 to 8 × 106 viable AML cells
were electroporated at 960 F and 240 V in the presence of 25 mg of
plasmid DNA. pCDNA-1 plasmid containing the full-length CD80 (B7.1)
cDNA was a gift of Dr S. Schoenberger (LUMC, Leiden, The Netherlands).
Phenotypic analysis.
AML cells were labeled with fluorescein isothiocyanate (FITC) or
phycoerythrin (PE)-conjugated monoclonal antibodies (MoAbs) against
HLA-DR, CD34 (Becton Dickinson, Mountain View, NY) CD80, CD86 (Ancell, Lâufelfingen, Switzerland), or HLA
class I (w6/32, Dr A. Mulder, LUMC, Leiden, The Netherlands) and
analyzed by flow cytometry (fluorescence-activated cell sorting
[FACS]). All AML cells used in the experiments were
>95% CD34+, negative for CD80, and expressed low levels
of CD86 before culturing with cytokines or CD80 transfection.
Leukemia reactive T-cell lines and T-cell clones were labeled with FITC
or PE-conjugated MoAbs against CD4 and CD8 (both from Beckton
Dickinson) and analyzed by flow cytometry (FACS). The T-cell
clones, 2.4.1 and 2.4.8, were labeled >95% with anti-CD4 antibodies.
Generation of Leukemia Reactive T-Cell Lines and Clones
Stimulator cells.
Cytokine-treated or CD80-transfected leukemic cells of an AML patient
with AML-M1 subclassification were used as stimulator cells. The HLA typing of the stimulator leukemic cells
was: HLA-A2, -A24, -B60, -DR13 (DRB1*1302), -DR7 (DRB1*07), -DR52
(Dw26) (-DRB3*0301), -DPB1*0401,*1301.
Responder cells.
PBMC from the HLA identical sibling BM donor of the
patient were used as responder cells. Irradiated
stimulator cells (3 × 106 cells) were
cocultured with an equal amount of responder cells in 5 mL of T-cell
culture medium at 37°C and 5% CO2. On day 6, 20 U/mL
of r-IL-2 was added. On day 8, the T-cell cultures were tested and
restimulated with irradiated untransfected AML cells, and on day 15, leukemia reactive T-cell lines were semicloned by limiting dilution at
5,000 cells/well in 96-well round bottom microtiter plates in the
presence of a feeder cell mixture consisting of 30 Gy irradiated PBMC
from six random blood donors (1 × 106 c/mL), 30 Gy
irradiated leukemic cells (2.5 × 105 cells/mL), 20 U/mL r-IL-2 (Cetus, Emeryville, CA) and 1%
Leucoagglutinin-A (Pharmacia, Uppsala, Sweden). IL-2 (20 U/mL)
containing medium was added into the cultures every 72 hours. Cloning
was performed by limiting dilution at 0.3 cells/well in 96-well round
bottom microtiter plates in the presence of the feeder-cell mixture
described above. The T-cell clones were expanded in the presence of
r-IL-2 and restimulated each week with the feeder-cell mixture.
T-cell proliferation assays.
Responder T cells (104 cells/well) and irradiated (30 Gy)
stimulator cells (5 × 104 cells/well) were cocultured
in 96-well flat bottom microtiter plates for 88 hours.
Antigens were added in the assay. Sixteen hours before harvesting the
cells were labeled with 0.5 Ci of 3H-thymidine. The
3H-thymidine incorporation was determined by liquid
scintillation counting. The results are expressed as the mean of
triplicate cultures.
Mixed lymphocyte reactions (MLR).
Irradiated stimulator cells and responder cells (both 105
cells/well) were cocultured in 96-well round bottom microtiter plates for 5 days. Sixteen hours before harvesting the cells were labeled with
0.5 Ci of 3H-thymidine. The 3H-thymidine
incorporation was determined by liquid scintillation counting. The
results are expressed as the mean of triplicate cultures. The standard
error of the mean (SEM) of the results never exceeded 15%.
Cytokine measurements.
T cells (4 × 104 cells/well) were stimulated with
irradiated stimulator cells (1.5 × 105 cells/well) or
with phorbol myristate acetate (PMA) (1 ng/mL) plus
iIonomycine (1 pg/mL) in 96-well round bottom microtiter plates
containing 200 µL culture medium. After 72 hours, cell-free supernatants were harvested. The interferon- (IFN-) and IL-4 release in the supernatants was measured by cytokine-specific sandwich
enzyme-linked immunosorbent assay (ELISA) assays following the
instructions of the manufacturer (CLB, Amsterdam, The Netherlands).
| |
RESULTS |
GM-CSF, IL-4, and TNF--treated AML cells efficiently present
nominal and allo-antigens to established T-cell clones.
To assess the effect of cytokines on surface molecules involved in
antigen presentation, AML cells were cultured with GM-CSF, IL-4, and
TNF-, and the expression levels of HLA and costimulatory molecules
were measured by FACS. Cytokine treatment enhanced the expression of
HLA molecules, but did not induce CD80 on AML cells, not even after 14 days of culture. Expression levels of CD86 and CD34 were not affected
by cytokines (Fig 1A). Subsequently,
untreated and cytokine-treated AML cells were used as APC for
antigen-specific and alloreactive T-cell clones. As shown in Fig 1B,
untreated, HLA-DR15, DRB1*1302 positive AML cells presented the
mycobacterial recombinant 65-kD protein (hsp65) and its peptide to an
HLA-DR15 restricted, hsp65 specific T-cell clone. Untreated AML cells
also stimulated the DR13-specific alloreactive T-cell clone. Cytokine treatment significantly enhanced antigen presentation by AML cells. Both antigen-specific and alloreactive T-cell clones showed
substantially higher levels of proliferative responses. When the
cytokines were used separately, antigen presentation by AML cells
cultured with IL-4 was also significantly increased (Fig 1B). A
synergistic effect of TNF- with IL-4 was observed, whereas the
addition of GM-CSF did not augment antigen presentation by AML cells
(Fig 1B). Similar phenotypical and functional results were obtained using three other AML cells derived from patients with AML-M1 or AML-M5
classifications (data not shown).
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| Fig 1.
(A) Surface expression of HLA class I, HLA class II, and
CD80, CD86, and CD34 molecules on untreated AML cells and AML cells
cultured with GM-CSF, IL-4, and TNF-. I, No MoAb; II, anti-CD80;
III, anti-CD86; IV, anti-DR; V, anticlass I; VI, CD34. Cells were
labeled with fluorescein-conjugated MoAbs and analyzed on a FACS. (B)
Proliferative responses of hsp65 (aa418-427)-specific T-cell clone
R2F10 and HLA-DR13-specific alloreactive clone against untreated or
cytokine treated (72 hours) AML cells. Antigens (hsp65, 5µg/mL; and
hsp65 peptide 418-427, 1 µg/mL) were added in the assay. The results
are expressed as stimulation index (cpm in the presence of stimulator
[APC]-cpm in the absence of stimulator/cpm in the absence of the
stimulator).
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CD80 transfected AML cells, but not cytokine-treated AML cells,
induce T-cell proliferation in HLA identical MLR.
Antigen presentation by AML cells was significantly enhanced by GM-CSF,
IL-4, and TNF-, but this treatment did not induce CD80 expression as
discussed above. Because CD28/CD80 signaling pathway is considered
essential for the induction of primary immune responses, we evaluated
the role of CD80 in an HLA identical setting. Malignant cells from a
patient with AML M-1 classification were transfected with a CD80
containing plasmid vector pCDNA-1. The transfections showed only in
some attempts a transient expression of CD80 on a small fraction of AML
cells (Fig 2A). CD80 transfected or
cytokine-treated AML cells were used as stimulator cells in primary MLR
using PBMC from HLA-identical sibling BM donor as responder cells.
Control responder cells were HLA mismatched PBMC(HLA-mismatched MLR)
(Fig 2B). Cytokine treatment potentiated the stimulatory capacity of
AML cells significantly in an HLA-mismatched MLR (Fig 2B).
Cytokine-treated AML cells were however not able to induce proliferation in HLA-identical MLR. In contrast, CD80-transfected AML
cells stimulated both HLA mismatched and HLA identical PBMC (Fig 2B).
Unmodified AML cells, AML cells transfected with the empty plasmid
(mock transfection), and AML cells that underwent electroporation
without DNA were able to trigger HLA mismatched PBMC, but did not
induce T-cell proliferation from HLA identical PBMC (Fig 2B).
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| Fig 2.
(A) Expression of CD80 on AML cells transfected with
pCDNA-B7 plasmid vector. CD80 expression was determined by FACS 48 hours after transfection of the cells. I, pCDNA-B7- transfected cells + no MoAb; II, untransfected cells + anti-CD80; III,
mock-transfected cells + anti-CD80; IV, pCDNA-B7- transfected cells + anti-CD80. (B) HLA mismatched and HLA identical MLR reactions
induced by cytokine-treated or CD80-transfected AML cells. Control
stimulator cells are AML cells transfected with empty plasmid (mock
transfection), AML cells electroporated without DNA, and untreated AML
cells. Results are expressed as the mean cpm of triplicate cultures.
The SEM did not exceed 15%.
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The specificity of allogeneic T-cell response induced by
CD80-transfected AML cells.
CD80-transfected AML cells were subsequently used as stimulator cells
for unprimed T cells of the HLA identical BM donor to generate leukemia
reactive T-cell lines. Two independent T-cell lines displayed strong
proliferative activity against AML cells and did not recognize
autologous PBMC (Fig 3A). The T-cell line (TCL#2) showed some proliferation against patients' EBV-LCL suggesting the presence of patient specific, rather than leukemia-specific T
cells.
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| Fig 3.
Target cell specificity of leukemia reactive T cells
generated by CD80-transfected AML cells. (A) Leukemic-cell reactivity
of two T-cell lines generated against CD80-transfected AML cells.
T-cell lines were tested against untransfected AML cells and patients'
EBV-LCL after second stimulation. (B) Proliferative activity of
oligoclonal T-cell cultures (semiclones) generated from T-cell line #1.
The results are expressed as the mean cpm of triplicate cultures. The
SEM did not exceed 15%. (C) Preferential recognition of leukemic cells
by four T-cell clones generated from a type II semiclone shown in Fig
3B.
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Because the activity of the T-cell line, TCL#1, was preferentially
directed against AML cells (Fig 3A), this T-cell line was analyzed in
more detail. First, several semiclones were generated by dilution to a
cell concentration of 5,000 cells per well. As shown in Fig 3B, two
types of semiclones could be identified: the majority of the obtained
semiclones (n = 25) showed type I reactivity, which did not
discriminate between leukemic cells and EBV-LCL. Type II semiclones (n = 10) showed strong proliferation towards AML cells with little or no
proliferation against patients' EBV-LCL. One of these type II
semiclones (designated as 2.4), was subjected to limiting dilution at
0.3 cell per well. The limiting dilution showed several T-cell clones
(n = 25), which did not discriminate between EBV-LCL and leukemic cells
(data not shown), four CD4+ T-cell clones (ie, 2.4.1, 2.4.6, 2.4.7, and 2.4.8 ) that strongly proliferated against AML cells
and did not recognize patients' EBV-LCL (Fig 3C). These
clones showed a very low reactivity against autologous PBMC (1,000 to
1,700 cpm) (Fig 3C).
The recognition of AML cells by the four leukemia reactive T-cell
clones was blocked by a MoAb against HLA-DP (B7/21), but not by MoAbs
against HLA-DR (B8.11.2), -DQ (SPV.L3), or against HLA class-I (W6/32)
indicating the HLA-DP restricted recognition of the target antigens
(Fig 4A).
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| Fig 4.
HLA-restriction and the target cell specificity of
leukemia reactive T-cell clones 2.4.1 and 2.4.8. (A) MoAbs against
HLA-DR (B.8.11.2), -DP( B7/21), -DQ(SPV.L3) and HLA class I (W6/32)
were added in the proliferation assays at a final dilution of 1:200.
The results are expressed as % inhibition of the proliferative
response in the absence of antibodies. (B) The proliferative response
of HLA-DP-restricted T-cell clones, 2.4.1 and 2.4.8, against leukemic
cells and nonleukemic cells. The data represent the summary of eight
independent experiments. HLA-DPB1*1301-specific and
HLA-DRB1*1302-specific T-cell clones were used as control. Besides
patients' EBV-LCL, autologous BM, and PBMC, two HLA-DP and DR-matched
PBMC from unrelated donors were tested as stimulator
cells.
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The fine specificity of the AML cell recognition was subsequently
analyzed in more detail. Clones 2.4.1 and 2.4.8 were tested for
proliferation against several stimulator cells including patients' EBV-LCL, autologous PBMC, autologous BM cells, and PBMC derived from
HLA-DP matched healthy individuals. One HLA-DP specific and two HLA-DR
specific alloreactive clones were used as controls. In Fig 4B, the
results of eight independent experiments are summarized. The T-cell
clones, 2.4.1 and 2.4.8, showed strong proliferation against AML cells
in all experiments, but never proliferated against patients' EBV-LCL.
The clones showed weak proliferative activity against autologous PBMC,
autologous BM, and PBMC derived from HLA-DP matched individuals. The
control class II specific alloreactive T cells recognized both leukemic
and nonleukemic cells efficiently (Fig 4B). The low reactivity of
clones 2.4.1 and 2.4.8 against nonleukemic cells could be inhibited by
anti-HLA-DP antibodies and was never observed when HLA mismatched PBMC
or BM cells were used as stimulator cells (data not shown), thereby
ruling out a nonspecific proliferation. This low reactivity against
nonleukemic cells could not be enhanced by culturing stimulator cells
with cytokines like GM-CSF, IL-4, IFN-, or TNF- (data not shown).
Leukemia reactive T-cell clones secrete high levels of IFN-, but
little or no IL-4 in response to leukemic cells.
To gain insight into the cytokines secreted by leukemia reactive T-cell
clones 2.4.1 and 2.4.8, production of IFN- and IL-4 was measured in
response to patients' leukemic cells, donor PBMC, and PMA/ionomycin
(Fig 5). Both T-cell clones secreted high
levels of IFN-, but hardly detectable levels of IL-4 in response to patients' AML cells. A similar cytokine secretion profile was found
after nonspecific stimulation with PMA/ionomycin, suggesting that both
clones displayed a Th1 like cytokine secretion profile. Both T-cell
clones showed significantly lower, but detectable, IFN- responses to
donor PBMC, similar to the low reactivity observed in the T-cell
proliferation studies, which suggested that the target antigens of the
clones may be expressed at low levels on nonleukemic cells (Fig 5).
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| Fig 5.
Cytokine secretion by leukemia reactive T-cell clones.
T-cell clones, 2.4.1 and 2.4.8, were stimulated with the
indicated irradiated stimulator cells or with PMA/ionomycin
for 72 hours. The release of IFN- and IL-4 in the
culture medium was assessed by cytokine-specific sandwich ELISA
and expressed as the mean of the triplicate cultures.
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DISCUSSION |
In this study, we have generated leukemia-cell reactive T-cell lines
and clones from unprimed T cells in an HLA identical BM donor/patient
combination using CD80-transfected AML cells. Our results indicate that
the majority of allogeneic T-cell responses against AML cells is
directed at patients' specific minor histocompatibility antigens. A
minority of the T cells can also be triggered by antigens that are
preferentially expressed by leukemic cells.
Donor-derived T cells play a key role in the the graft-versus-leukemia
(GvL) effect seen after BMT or after donor lymphocyte infusions. It is
therefore important to characterize the leukemia-associated target
antigens recognized by donor derived T cells. These studies have been
hampered because leukemic cells are not potent APC and usually fail to
induce T-cell responses in vitro. We therefore searched for in vitro
conditions that would improve the antigen presenting function of AML
cells and facilitate the generation of leukemia reactive T cells in HLA
identical combinations. First, we explored the effect of GM-CSF,
TNF-, and IL-4 on the antigen presenting function of AML cells.
These cytokines are known to generate dendritic, superior APC from
monocytes, myeloid cell precursors, or malignant precursors of CML
cells.15,28-30 Cytokine treatment significantly enhanced
HLA expression levels of AML cells, but did not induce CD80 expression,
neither increased the low expression levels of CD86 in four different
AML cells classified as AML-M1 or AML M5. AML cells with other
subclassifications were not tested in this study. Although the
possibility remains that other AML types may respond to cytokines
differently, our results indicate that unlike CD34+, CML
precursors,15 CD34+ AML-M1 and AML-M5 cells
seem not to change their phenotype into dendritic-like cells after
culture with GM-CSF, TNF-, and IL-4. After cytokine treatment, AML
cells efficiently presented nominal antigens to T-cell clones, induced
T-cell proliferation in HLA mismatched MLR, but failed to induce
primary T-cell response in HLA identical MLR. Additional attempts to
generate T-cell lines using cytokine-treated AML cells were also
unsuccessful (data not shown). Thus, despite the expression of low
levels of costimulatory CD86 molecule, cytokine-treated AML cells did
not costimulate unprimed T cells sufficiently. Upon the introduction of
CD80 gene, AML cells became adequate stimulator cells and triggered
significant T-cell proliferation from HLA identical PBMC. This finding
underscores the importance of CD80/CD28 interactions during the
induction of primary antitumor T-cell responses and is consistent with
some recent data where costimulation via CD80, but not via CD86, was associated with a clear antitumor effect.17,31 It is not
clear yet why CD80 provides a superior costimulation than CD86. It has been suggested that CD80 and CD86 are involved in the differentiation of reciprocal T-cell subsets. CD80 costimulation may drive naive T
cells to differentiate into Th1-like cells involved in inflammatory and
antitumor responses.31 CD86-costimulated T cells may in contrast differentiate into Th2 type cells, which inhibit
antiinflammatory responses (reviewed in Lu et al32). The
cytokine secretion profile of the leukemia reactive T-cell clones
generated in this study resembles a Th1-like pattern, which again
supports the role of CD80 in the induction of these T cells. However,
it should be noted that the CD80-transfected AML cells that we used
coexpressed low levels of CD86. We, therefore, cannot exclude the
possibility that the proper costimulatory signals are generated by the
synergistic action of both CD80 and CD86, as also suggested by other
investigators.19,31
AML cells transfected with the CD80 gene enabled us to investigate the
target cell specificity of leukemia reactive T cells in an HLA
identical patient/donor combination. The generated T-cell lines and
clones contained mainly CD4+, proliferative T cells without
detectable cytotoxic activity (data not shown). In our experiments, we
were unable to generate CD8+ CTL using CD80-transfected AML cells. It
is possible that CD4+ T cells display an in vitro growth
advantage. Yet, these results imply that CD4+ T cells may
significantly contribute to antileukemia reactivity after BMT. Our
analysis indicates that leukemic cells induce different types of
CD4+ T cells with distinct target cell specificities. The
majority of T cells induced by leukemic cells do not discriminate
leukemic from nonleukemic cells of the patient. Although we have not
performed a thorough analysis of the reaction patterns, these
"patient-specific" T cells are most probably directed to host
specific mHag, which are present on both leukemic and nonleukemic
cells. This also supports the general notion that mHag expressed by
leukemic cells are important targets of post-BMT T-cell responses
(reviewed in Goulmy9).
Interestingly, in our analysis, T-cell responses to AML cells were not
confined to patients' specific antigens. Some T-cell lines and clones
reacted preferentially against AML cells. These cells strongly
proliferated in response to leukemic cells, but did not recognize
patients' nonleukemic EBV-LCL suggesting that the ligands were absent
on B cells. In a detailed analysis, two leukemia reactive, HLA-DP
restricted T-cell clones showed strong proliferative reactivity and
IFN- secretion in response to leukemic cells and never reacted to
patients' EBV-BLCL. The clones however showed a weak, but
reproducible, reactivity against donor PBMC, donor BM, as well as
against two unrelated HLA matched PBMC. Our results thus indicate that
the target antigens of the latter clones are (1) strongly expressed by
leukemic cells, (2) are absent on EBV-LCL, but (3) are expressed at low
levels on nonleukemic myeloid cells. We, therefore, consider that the
target antigens of these HLA-DP restricted clones are
"leukemia-associated" rather than "leukemia-specific." The
preferential reactivity of T-cell clones exclude the possibility that
they may react to antigens that are derived from extracellular sources
such as FCS. It is possible that the target antigens are derived from
membrane-associated or secreted proteins, as it is the case for the
vast majority of HLA class II associated peptides.33
To our knowledge, a genuinely leukemia cell-specific T-cell clone has
not yet been reported. Attempts to generate CTL responses against
CML-specific Bcr/Abl fusion sequence have not been successful. Other
T-cell clones that were initially considered as leukemia-specific appeared to recognize host specific mHag.34-37
Lineage-specific differentiation antigens or developmentally regulated
antigens can be recognized by T cells. Recently, Dolstra et
al38 have described a leukemia-associated mHag antigen
expressed only by EBV-LCL and by leukemic cells of B-cell origin. We
have also identified an allo-HLA restricted T-cell clone that
recognizes an antigen that is shared only by AML cells and
CD34+ early precursor cells, indicating that its expression
is developmentally regulated.25 Also, in other models,
tumor reactive T cells are often directed to differentiation antigens,
developmentally regulated embryonal antigens, or antigens that are
overexpressed by tumor cells, rather than tumor-specific
antigens.39 As in other tumors, also in leukemia tumor-cell
selectivity rather than genuine tumor-specificity may result in
leukemic load reduction. The leukemia reactive T-cell clones isolated
in this study support these findings and will be used for the
biochemical identification of such leukemia-associated antigens.
| |
FOOTNOTES |
Submitted December 29, 1997;
accepted April 22, 1998.
Supported by grants from the Dutch Cancer Foundation and the J.A Cohen
Institute for Radiopathology and Radiation Protection.
Address reprint requests to Tuna Mutis, MD, PhD, The Department of
Immunohematology and Blood Bank, Leiden University Medical Center,
Albinusdreef 2, 2333 ZA, Leiden, The Netherlands.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
We thank Prof Dr F. Claas and Dr M. Oudshoorn for critically reading
the manuscript.
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Abstract
Introduction
Abstract