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Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 1992-2002
CD40-Activated B-Cell Chronic Lymphocytic Leukemia Cells for Tumor
Immunotherapy: Stimulation of Allogeneic Versus Autologous T Cells
Generates Different Types of Effector Cells
By
Raymund Buhmann,
Annette Nolte,
Doreen Westhaus,
Bertold Emmerich, and
Michael Hallek
From the Laboratorium für Molekulare Biologie, Genzentrum,
Medizinische Klinik, Klinikum Innenstadt, and Medizinische Klinik III,
Klinikum Gro hadern, Ludwig-Maximilians-Universität,
München, Germany.
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ABSTRACT |
Although spontaneous remissions may rarely occur in B-cell chronic
lymphocytic leukemia (B-CLL), T cells do generally not develop a
clinically significant response against B-CLL cells. Because this
T-cell anergy against B-CLL cells may be caused by the inability of
B-CLL cells to present tumor-antigens efficiently, we examined the
possibility of upregulating critical costimulatory (B7-1 and B7-2) and
adhesion molecules (ICAM-1 and LFA-3) on B-CLL cells to improve antigen
presentation. The stimulation of B-CLL cells via CD40 by culture on
CD40L expressing feeder cells induced a strong upregulation of
costimulatory and adhesion molecules and turned the B-CLL cells into
efficient antigen-presenting cells (APCs). CD40-activated B-CLL
(CD40-CLL) cells stimulated the proliferation of both
CD4+ and CD8+ T cells. Interestingly,
stimulation of allogeneic versus autologous T cells resulted in the
expansion of different effector populations. Allogeneic CD40-CLL cells
allowed for the expansion of specific CD8+
cytolytic T cells (CTL). In marked contrast, autologous CD40-CLL cells
did not induce a relevant CTL response, but rather stimulated a
CD4+, Th1-like T-cell population that expressed high
levels of CD40L and released interferon- in response to stimulation
by CD40-CLL cells. Together, these results support the view that CD40
activation of B-CLL cells might reverse T-cell anergy against the
neoplastic cell clone, although the character of the immune response
depends on the major histocompatibility complex (MHC)
background on which the CLL or tumor antigens are presented. These
findings may have important implications for the design of cellular
immunotherapies for B-CLL.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
CHRONIC LYMPHOCYTIC leukemia of B-cell
origin (B-CLL) is the most common type of leukemia in the western
hemisphere. Despite a continued effort to improve the outcome of B-CLL
by new chemotherapeutic agents, the disease remains uncurable.
Therefore, it seems rewarding to evaluate alternative treatment options
such as immunotherapy.
Although some cases of spontaneous remission in CLL have been
reported,1 B-CLL cells generally fail to induce a
clinically relevant immune response. Whereas the clinical appearance of
B-CLL often remains stable for years, the total tumor cell burden tends to expand at variable speed without any apparent reaction of the immune
system against the tumor. This may be caused by an impaired T-cell-mediated immune response,2 including a depressed
function of natural killer (NK) cells and antibody-mediated cellular
cytoxicity (ADCC)3,4; a reduced susceptibility of the
leukemic cells towards the effector cells5; or an inability
of the neoplastic cells to function efficiently as antigen-presenting
cells (APCs), similar to other lymphoid malignancies.6,7
In the specific case of B-CLL, the malignant cells are the neoplastic
counterpart of a subpopulation of CD5+ B
cells8,9 that might function as professional APCs and effectively present endogenous tumor antigens to T cells. Moreover, the
B-CLL specific idiotype provides a unique tumor antigen that might be
recognized by the immune system, similar to other lymphoid malignancies
in which this strategy has been tested successfully in clinical
trials.10-12 However, despite their strong expression of
major histocompatibility complex class I (MHC I) and class II (MHC II)
molecules, B-CLL cells are generally ineffective stimulator cells in
mixed lymphocyte reactions.13
It has been demonstrated that expression of MHC molecules is not
sufficient for the induction of a productive immune response and that
adequate expression of adhesion and costimulatory molecules is critical
to stimulate a potent T-cell response. Failure to receive these signals
renders potential effector T cells anerg or
tolerant.14 Among the costimulatory molecules, the B7
family appears to be unique, because it has been demonstrated that B7-1 (CD80) and B7-2 (CD86) are both necessary and sufficient to prevent the
induction of anergy.15-17 In normal and malignant B cells, activation of CD40 seems to be a major stimulus for the induction of
B7-1 and B7-2.7,18
In this study we sought to determine whether B-CLL cells could be
turned into efficient APCs and whether this could reverse T-cell anergy
against the neoplastic cell clone. Stimulation of CD40 on B-CLL cells
in vitro induced a strong upregulation of adhesion and costimulatory
molecules. Repeated stimulation of allogeneic versus autologous T cells
with CD40-CLL cells resulted in different effector populations.
Allogeneic CD40-CLL cells activated CD8+, cytolytic T cells
with activity against both native B-CLL and CD40-CLL cells. In marked
contrast, autologous CD40-CLL activated predominantly CD4+
T cells that had no major cytolytic activity. The results suggest that
cellular vaccination studies in B-CLL may use at least two different
strategies that depend on the source of T cells: when using allogeneic,
peripheral blood T cells from healthy donors, CD40-CLL cells are very
potent in expanding CD8+, cytolytic T cells in vitro; this
potential might be used for adoptive immune transfer studies. In
contrast, autologous T cells respond to CD40-CLL cells primarily by an
expansion of CD4+ Th1-like T cells; this might be
exploited and further studied in a trial in which CD40-CLL cells are
directly applied to the patient.
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MATERIALS AND METHODS |
Patients.
After informed consent, peripheral blood samples were obtained from
patients with B-CLL. The diagnosis of B-CLL was based on standard
clinical and laboratory criteria.19 The study included 12 patients (4 women and 8 men; 49 to 82 years of age). Staging was
performed according to the Binet classification.20
Characteristics of the patients studied are summarized in
Table 1.
HeLa/SF cells transfected with CD40L cDNA.
The CD40L coding region was amplified as previously
described.21 Briefly, RNA was isolated from activated human
T cells. Reverse transcription was followed by a two-step polymerase
chain reaction (PCR). The first-step PCR was performed using sense
primers coding for the first 20 nucleotides of the CD40L coding
sequence and antisense primers coding for the last 23 nucleotides of
the CD40L coding sequence. In a second step, the amplified PCR products reamplified using extended primers. The sense primer (5'-GTA GGA ATT CGT CGA CGC CGC CAC CAT GAT CGA AAC ATA CAA CC-3') contains EcoRI and Sal I sites, a strong translational start
site, and the first 20 nucleotides of the CD40L coding sequence. The
antisense primer (5'-GAC TAG TGT CGA CGA ATT CAG AGT TTG AGT AAG
CCA AAG-3') contains the last 23 nucleotides of the CD40L coding
sequence, including the stop codon and EcoRI, Sal I,
and Spe I sites. For each step, 20 cycles were performed
(95°C for 1 minute, 48°C for 30 seconds, and 72°C for 1 minute), followed by one cycle at 72°C for 10 minutes. The 0.8-kb
PCR product was digested with EcoRI, gel-purified, and ligated
into EcoRI-digested pcDNA3.1 vector (purchased from Invitrogen,
NV Leek, The Netherlands). Plasmids containing the CD40L insert were
transfected via electroporation in HeLa/SF cells. Transfectants were
selected by growth in 250 µg/mL G418 and further subcloned. Biologic
activity was determined by costimulation of B-cell proliferation and differentiation.
Isolation of B-CLL cells.
Mononuclear cells (MNCs) from peripheral blood samples were isolated by
centrifugation on a Ficoll/Hypaque (Seromed, Berlin, Germany)
density gradient and depleted from monocytes by overnight adherence to
plastic tissue culture flasks. Subsequently, the nonadherent
lymphocytes were cryopreserved in liquid nitrogen in the presence of
10% dimethyl sulfoxide (DMSO; Sigma, München, Germany). As assessed by flow cytometry, greater than 95%
of these cells typically coexpressed CD19 and CD5 surface molecules.
B-CLL cell culture.
For CD40L-induced activation, freshly isolated B-CLL cells were
cultured as previously described.14 Briefly, CD40L or
mock-transfected NIH3T3 fibroblasts or HeLa/SF cells were
-irradiated at 200 Gy, plated at 5 × 105
cells/well in 6-well plates in media without G418, and incubated overnight at 37°C in a 5% CO2 humidified atmosphere.
Before addition of the B-CLL cells, the feeder layers were washed
twice with phosphate-buffered saline, and tumor cells were cultured at
2 × 106 cells/mL in Iscove's medium (Seromed)
supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin. For
further studies, culture was performed in presence or absence of
interleukin-2 (IL-2; 20 IU/mL), IL-4 (1 IU/mL), and interferon (IFN; 20 IU/mL). For performing functional studies, tumor cells were
harvested after 24, 72, and 120 hours of culture; purified by ficoll
density gradient centrifugation; washed; and analyzed by flow
cytometry. For T-cell restimulation, the activated B-CLL cells
(CD40-CLL) were aliquoted and stored in liquid nitrogen. The
CD40L-transfected NIH3T3 cell line was a generous gift from Dr J. Schultze (Dana-Farber Cancer Insitute, Boston, MA). With respect to
their stimulatory capacity, no differences between CD40L-transfected
NIH3T3 fibroblasts and CD40L-transfected HeLa/SF cells were detected;
therefore, both feeder cell lines are referred to as t-CD40L throughout
the manuscript.
Cytokines and cytokine measurements.
Recombinant human IL-2 (rhIL-2), rhIL-4, and rhIFN were obtained
from Boehringer Mannheim (Mannheim, Germany) and used as indicated.
Cytokine measurements were performed using commercial IL-4 and IFN
enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems,
Wiesbaden-Nordenstadt, Germany) according to the manufacturer's instructions. The detection limits of the assays were 5 to 2,000 pg/mL.
Immunophenotyping.
Immunophenotyping was performed with the following monoclonal
antibodies (MoAbs) conjugated with fluorescein isothiocyanate (FITC),
phycoerythrin (PE), or phycoerythrin cyanine 5 (PE-Cy5): CD3, CD4, CD5,
CD8, CD19, CD20, CD23, CD25, CD28, CD54, CD56, CD58, CD69, CD95,
HLA-ABC, HLA-DR, anti- , anti- (Coulter/Immunotech, Hamburg,
Germany), CD40, CD40L, CD80, CD86, and CD95L (PharMingen, Hamburg,
Germany). Fluorescence was measured with a Coulter Epics XL-MCL
(Coulter Electronics, Miami, FL).
Purification of T cells.
Purification of T cells was performed by negative selection. Briefly,
the monocyte-deprived MNCs from healthy donors or B-CLL patients were
stained with a cocktail of antibodies (CD11b, CD16, CD19, CD36, and
CD56), labeled with goat anti-rat IgG microbeads (Miltenyi Biotec,
Bergisch Gladbach, Germany), and selected using magnetic separation
columns (Miltenyi Biotec). Isolated cells were greater than 95% pure
as determined by immunofluorescent flow cytometry analysis (FACS) and
appeared viable by exclusion of trypan blue and forward/side scatter analysis.
Generation of effector T cells.
Purified T cells from healthy donors or B-CLL patients were stimulated
with -irradiated (75 Gy) CD40-CLL cells or with native B-CLL cells
at different effector to target (E:T) ratios ranging from 10:1 to 5:1.
Stimulation was performed on days 0, 7, 14, and 21. Briefly, T cells
were harvested weekly, washed, and recultured at a concentration of 1 × 106 cell/mL with -irradiated, autologous, or
allogeneic stimulator cells in Iscove's medium (Seromed) supplemented
with 5% human heat-inactivated AB-serum (Serva, Heidelberg, Germany),
2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL
streptomycin. The addition of IL-2 (20 IU/mL) was perfomed 48 hours
after (re)stimulation.
Mixed lymphocyte reaction (MLR).
Irradiated (75 Gy) B-CLL cells and CD40-CLL cells were used as
stimulators, cocultured at 1 × 104 cells/well in a
final volume of 200 µL with allogeneic T cells at 1 × 105 cells/well in 96-well round-bottom plates, and
incubated for 3 days at 37°C in a 5% CO2 humidified
atmosphere. The culture medium used was Iscove (Seromed) supplemented
with 5% heat-inactivated human AB-serum (Serva), 2 mmol/L L-glutamine,
100 U/mL penicillin, and 100 µg/mL streptomycin. All microcultures
were performed in triplicate. During the last 12 hours of the 72-hour
culture period, cells were pulsed with 0.5 µCi [3H]
thymidine (Amersham, Braunschweig, Germany). Cells were harvested onto
glass fiber filters and dried, and the [3H] thymidine
incorporation was measured by scintillation spectrophotometry in a
Wallac Microbeta Plus 1450 scintillation counter (Turku, Finland). The
stimulation indexes (SI) were calculated for each individual experiment
as follows: SI = cpm(T cells + B-CLL cells)/cpm(T
cells).
Cytotoxicity assays.
T-cell-mediated cytotoxicity was determined using a standard 4-hour
[51Cr] release assay.22 Unstimulated and
CD40-stimulated B-CLL cells as well as NK-sensitive K562 cells were
used as targets. Target cells were labeled with 100 µCi of
[51Cr] (Na51CrO2; Dupont, Bad
Homburg, Germany) per 106 cells for 2 hours at 37°C in
a water bath. Thereafter, the cells were washed three times in complete
medium and seeded in v-bottomed microtiter plates at a concentration of
2.5 × 103 cells/well. Indicated numbers of effector
cells were added in triplicate in 200 µL of complete medium.
Supernatants were collected, and the released [51Cr] was
measured in a -counter (LKB-Wallac 1282, Uppsala, Sweden). Spontaneous release was determined by incubation of target cells in
medium alone, and maximum release was determined by resuspending the
wells with 10% Triton X-100. Specific lysis was determined for each
individual experiment as follows: specific lysis (%) = [(experimental
[51Cr] release  spontaneous [51Cr]
release)/(maximum [51Cr] release spontaneous
[51Cr] release)] × 100.
Statistical analysis.
Differences between experimental groups were analyzed using the
 2 test and the Student's t-test.
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RESULTS |
B-CLL cells lack costimulatory molecules.
B7 costimulatory molecules play a crucial role in the induction of a
T-cell-mediated immune response. Lack to receive costimulatory signals
after antigen presentation renders T cells anergic or tolerant.23,24 This may be a mechanism by which CLL cells
escape the immune response. Therefore, we determined the cell surface expression of MHC, adhesion, and costimulatory molecules by
immunophenotyping of freshly isolated B-CLL cells obtained from 12 patients. The results are summarized in
Table 2. The expression level of the surface molecules on B-CLL cells was classified according to their mean
fluorescence intensity (MFI). All patients tested expressed high (MFI
1.5 to 2.5 logarithm) or even very high (MFI >2.5 logarithm) levels
of MHC class I and II molecules. The adhesion molecules ICAM-1 (CD54)
were undetectable in 3 of 12 patients, were expressed at intermediate
(MFI 0.5 to 1.5 logarithm) in 8 of 12 patients, and were expressed at
high levels in 1 of 12 patients. LFA-3 (CD58) was undetectable in 3 of
12 patients and was expressed at low to intermediate levels in 9 of 12 patients. The costimulatory molecule B7-1 (CD80) was not detectable in
8 of 12 patients or showed only low expression (MFI >0.2 to 0.5 logarithm) in 4 of 12 patients. B7-2 (CD86) was not expressed in 2 of
12 patients, whereas 10 of 12 patients expressed it at low to
intermediate levels. CD40 was detectable in all B-CLLs (12/12) at low
or intermediate levels. Together, these experiments indicated that
B-CLL cells showed a markedly reduced expression of costimulatory
molecules, especially of B7-1 (CD80).
Stimulation of B-CLL cells by t-CD40L in the presence of IL-4
efficiently upregulates costimulatory molecules.
In the next step, we stimulated freshly isolated B-CLL cells by t-CD40L
and mock-transfected feeder cells in the presence of IL-4 (1 IU/mL; see
Materials and Methods). Stimulation by t-CD40L induced a cluster
formation of B-CLL cells caused by adhesion to the stimulator cells and
a hairy-cell like morphology in some of the B-CLL cells
(Fig 1A and B). No aggregation or
morphologic changes were observed by stimulation with mock controls.
May-Grünwald-Giemsa staining of cytospin smears showed that the
size of CD40-CLL cells increased due to an expansion of both nuclei and
cytoplasm. Moreover, CD40-CLL showed a relatively high degree of
vacuolization, corresponding to an activated state of B-lymphoid cells
(Fig 1C). No plasmacytoid differentiation was seen.
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| Fig 1.
(A through C) Morphology of t-CD40L-stimulated CLL
(CD40-CLL) cells after 3 days of culture. (A and B) Typical cluster
formation of CD40-CLL cells that surround the CD40L-transfected NIH 3T3
fibroblasts (original magnifications: [A] × 100; [B] × 650).
(C) CD40-CLL cells stained by May-Grünwald-Giemsa (original
magnification × 650).
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To optimize the cytokine cocktail used for stimulation of B-CLL cells,
B-CLL cells were stimulated by t-CD40L or mock-transfected feeder cells
for 3 days in the presence of various cytokines such as IL-2 (20 IU/mL), IL-4 (1 IU/mL), and IFN (20 IU/mL).
Figure 2A and B shows that maximal
expression of B7-1 and B7-2 was induced by t-CD40L when combined with
IL-4. IL-4 alone or in combination with mock stimulation only mediated
a slight increase of B7-2 expression on the B-CLL cell surface (data
not shown). Stimulation by t-CD40L in combination with IL-2 and IFN
even reduced the expression of B7-1 when compared with stimulation
without cytokines. The addition of IFN induced only an upregulation
of Fas (CD95) but had no other effects (data not shown). Taken
together, the combination of t-CD40L and IL-4 seemed optimal for
enhancing the expression of important costimulatory molecules on B-CLL
cells and was therefore used in all subsequent experiments.
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| Fig 2.
Phenotypic characterization of B-CLL cells after 3 days
of stimulation by CD40L or mock-transfected NIH3T3 fibroblasts in the
presence of IL-2 (20 IU/mL), IL-4 (1 IU/mL), and IFN (20 IU/mL).
Blank areas represent the isotype-matched control antibodies and the
solid areas represent the fluorescence distribution of the MoAbs tested
as assessed by flow cytometric analysis. The results shown are from one
experiment and are representative of three independent experiments.
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To determine the optimal length of t-CD40L stimulation for full
activation of B-CLL cells, time course experiments were performed. B-CLL cells were stimulated with t-CD40L for 24, 72, and 120 hours. As
soon as 24 hours after stimulation by t-CD40L, a significant upregulation of ICAM-1 (CD54), LFA-3 (CD58), and Fas (CD95) was detectable (Fig 3B). Expression of
costimulatory molecules B7-1 and B7-2 reached its maximum on day 3 of
t-CD40L stimulation. Mock stimulation (Fig 3C) in the presence of IL-4
(1 IU/mL) caused a slight increase of the adhesion molecules ICAM-1 and
LFA-3, as well as of the costimulatory molecule B7-2. No upregulation was detected for the costimulatory molecules B7-1 and Fas (CD95). Table 3 summarizes the data obtained with
leukemic cells from 12 patients after a 3-day t-CD40L stimulation in
the presence of IL-4 (1 IU/mL). In all cases, CD40-CLL cells showed an
increased expression of B7-1 and B7-2 (Table 3). CD40 expression was
further increased in 8 cases. ICAM-1 and LFA-3 were also upregulated in most cases, except if they were already expressed at intermediate levels before t-CD40L stimulation (ICAM-1: patients no. 6, 7, and 10;
LFA-3: patients no. 7 through 10). Expression of both MHC class I and
II molecules was further increased to high or very high levels in those
cases in which the expression was lower before CD40 stimulation.
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| Fig 3.
Phenotypic characterization of unstimulated B-CLL cells
and B-CLL cells stimulated in the presence of IL-4 (1 ng/mL) for 24, 72, and 120 hours by either CD40L-transfected or mock-transfected
NIH3T3 fibroblasts. The results shown are from one experiment and are
representative of three independent experiments.
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CD40L-activated B-CLL cells retain the immunophenotypic
characteristics of neoplastic cells.
To exclude that treatment with t-CD40L induced the expansion of normal
rather than neoplastic B cells, immunophenotypic analyses of light
chain restriction and CD5 expression were performed on CD40-CLL cells.
As shown in one representative experiment, CD40-CLL cells showed the
phenotype of B-CLL cells as demonstrated by coexpression of CD5 and
CD19 and by light chain restriction (Fig
4).
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| Fig 4.
Representative immunophenotypic characterization of B-CLL
cells before and after 3 days of CD40-stimulation in the presence of
IL-4 (1 IU/mL) as determined by CD5/CD19 positivity and light chain
restriction (patient no. 3). The results shown are from one experiment
and are representative of three independent experiments.
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CD40-CLL cells induce a proliferative T-cell response.
We then investigated whether CD40-CLL cells provided a proliferative
stimulus to T cells. For this purpose, we used highly (>95%)
purified allogeneic T cells and incubated them in the presence or
absence of IL-2 (20 IU/mL) with -irradiated (75 Gy) native CLL and
CD40-CLL cells for 72 hours. CD40-CLL cells were prepared by
prestimulation with t-CD40L for 24, 72, and 120 hours. T-cell proliferation was assessed by incorporation of
[3H]thymidine added during the last 12 hours of the
experiment. The highest stimulatory capacity of the CD40-CLL cells was
achieved on day 3 of t-CD40L prestimulation
(Fig 5). The experiments shown are
representative for 3 different allogeneic T-cell donors and different
cases of B-CLL examined.
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| Fig 5.
Proliferative responses of purified allogeneic
CD3+ T cells in the absence of IL-2 to -irradiated
B-CLL cells (patient no. 12) either unstimulated or stimulated for 24, 72, and 120 hours by CD40L-transfected NIH3T3 fibroblasts.
[3H] Thymidine incorporation was assessed for the last 12 hours of a 3-day culture. Appropriate controls (CD3+ T
cells and B-CLL cells) were always less than 1,500 cpm. The stimulation
index was calculated as cpm(T cells + B-CLL
cells)/cpm(T cells). Results are representative for
three independent experiments and are expressed as the mean ± SD of
the stimulation index.
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In another set of experiments, we analyzed the proliferative response
of highly purified allogeneic CD4+ and CD8+ T
cells. For this purpose, we used -irradiated (75 Gy) native CLL and
CD40-CLL cells (day 3) and incubated them for 72 hours with different
ratios of highly (>95%) purified CD4+ and
CD8+ T-cell subpopulations as well as unpurified peripheral
blood mononuclear cells (PBMCs). T-cell proliferation was
assessed by [3H]thymidine incorporation during the last
12 hours of the experiment. A representative experiment is shown in
Fig 6. CD40-CLL cells but not native CLL
cells induced a significant T-cell proliferation, regardless of whether
unpurified PBMCs or CD4+ or CD8+ T cells were
used. Moreover, CD4+ T cells showed a significant stronger
proliferative response than CD8+ T cells. The strongest
response was seen with PBMCs; this result is readily explained by the
presence of some additional immune effector cells (eg, NK cells) or
APCs (eg, dendritic cells) in the unpurified PBMC fraction.
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| Fig 6.
Proliferative response of purified allogeneic
CD4+ and/or CD8+ T cells as well as
PBMCs not further purified to -irradiated native and CD40-stimulated
B-CLL cells. Different CD4/CD8 ratios were tested. [3H]
Thymidine incorporation was assessed for the last 12 hours of a 3-day
culture and determined in cpm ± SD of triplicate determinations. The
results shown are from one experiment and are representative for three
independent experiments.
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Different effector cells are induced by subsequent stimulation of
allogeneic versus autologous T cells with CD40-CLL cells.
In the next step we determined whether CD40-CLL cells could be used to
stimulate T-cell differentiation and expansion. For this purpose, we
used highly purified allogeneic and autologous T cells and stimulated
them weekly (days 0, 7, 14, and 28) with -irradiated (75 Gy) native
and CD40-CLL cells at a ratio of 5:1. In both settings, it was possible
to expand large numbers of T cells in the presence of CD40-activated
B-CLLs and exogenous IL-2 (20 IU/mL). In contrast, the expansion was
not possible with native B-CLL cells, even in the presence of IL-2.
When T cells were monitored by flow cytometric analysis, we found that
only CD40-activated B-CLLs were able to induce activation markers (CD25
and CD95). A marked difference was seen with regard to the CD4/CD8
ratio. In the allogeneic setting, consecutive stimulations with
CD40-CLL cells caused a relative and absolute increase of CD8+ T cells (up to 50% after 3 restimulations) in 4 of 5 cases investigated. In marked contrast, repetitive stimulations in the
autologous setting caused an expansion of CD4+ T cells (up
to 90% after 3 restimulations) in 6 of 6 cases studied. No increase of
CD16+/CD56+ NK cells was observed.
To further characterize the different effector functions of allogeneic
versus autologous T cells, we performed 4-hour standard chromium
release tests (see Materials and Methods). Native B-CLL cells, CD40-CLL
cells, and NK-sensitive K562 cells were used as targets. These
different effector cells were expanded until day 28 and then
restimulated with CD40-CLL cells and native CLL cells in the presence
of IL-2. A significant, cytolytic activity of T cells was only seen if
allogeneic T cells from healthy donors were used
(Fig 7B). At this time point, allogeneic T
cells lysed both native B-CLL cells and CD40-CLL cells. No response
against K562 cells was detected. Furthermore, no T-cell response was
observed with T cells before stimulation with CD40-CLL (Fig 7A). In
marked contrast, autologous T cells expanded by repetitive stimulation with CD40-CLL cells showed only a slight lytic activity against CD40-CLL cells and no lytic activity against native B-CLL cells or the
NK-sensitive K562 cell line (Fig 7D). Taken together, the stimulation
of allogeneic versus autologous T cells by CD40-CLL cells induced a
fundamentally different response in that CD8+ cells with
cytolytic activity could only be expanded in the
allogeneic system.
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| Fig 7.
Cytolytic response of unstimulated and activated
allogeneic and autologous T cells as assessed in a standard 4-hour
chromium release assay. A total of 2.5 × 103 B-CLL cells
( ), CD40-CLL cells ( ), and NK-sensitive K562 cells ( ) were
placed in 96-well v-bottom plates and T cells were added at E:T ratios
of 3:1, 10:1, and 30:1 in a final volume of 200 µL. Cytolytic
response was expressed as the percentage specific lysis. (A) Cytolytic
response of unstimulated T cells and (B) cytolytic response of T cells
restimulated three times with CD40-CLL cells in an allogeneic
setting. (C) Cytolytic response of unstimulated T cells
and (D) cytolytic response of T cells restimulated three times with
CD40-CLL cells in an autologous setting (patient no. 12). The results
are representative for three independent experiments in the allogeneic
system and three independent experiments in the autologous system.
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CD40-CLL activated autologous CD4+ T cells show a Th1-type
cytokine pattern.
In the next step we tried to characterize the autologous,
CD4+ T cells with respect to their cytokine release
pattern. For this purpose, we restimulated 1 × 105
autologous T cells with 2 × 104
paraformaldehyde-fixed (1%) CD40-CLL cells. The supernatant was collected 48 hours later and analyzed for IL-4 and IFN.
Figure 8 summarizes the data of 6 patients.
In 4 of 6 patients tested, we detected predominantly IFN, suggesting
a Th1-like immune response.
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| Fig 8.
Autologous T cells of 6 different patients were
challenged with CD40-CLL cells at an E:T ratio of 5:1. Supernatants
were collected 48 hours later and IL-4 and IFN were measured by
ELISA (detection limit of the IL-4 assay is <5 pg/mL; detection limit
of the IFN assay is <5 pg/mL).
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| |
DISCUSSION |
This report investigates the potential of CD40L-stimulated CLL
(CD40-CLL) cells to activate effector T cells for tumor vaccination. The essential finding of this report is that the quality of the autologous T-cell response against CD40-CLL cells differs dramatically from the allogeneic T-cell response against these cells. So far, it was
known that CD40-ligation could be used in B-CLL cells to upregulate
adhesion and costimulatory molecules.25-27 Our study confirmed these findings by showing that stimulation of B-CLL cells by
t-CD40L resulted in a significant upregulation of both adhesion and
costimulatory molecules in B-CLL cells in a time-dependent manner,
regardless of previous treatment with cytostatic drugs. CD40-CLL cells
were able to stimulate both the allogeneic and the autologous T-cell
proliferation. The addition of IL-4 further enhanced the expression of
both B7-1 and B7-2, whereas IFN or IL-2 reduced it compared with
t-CD40L alone (Fig 2).
A high expression of B7-1 is critical for the induction of a CTL
response.28-30 To induce an effective immune response
against B-CLL cells, consecutive T-cell stimulations were performed
with CD40-CLL cells. Allogeneic CD40-CLL cells strongly stimulated both
CD4+ as well as CD8+ T cells, as previously
described.27 In marked contrast, stimulation of autologous
CD40-CLL cells strongly favored the outgrowth of CD4+, but
not CD8+ T cells. With respect to the effector T-cell
function, there were also marked differences. A cytolytic activity was
only induced with allogeneic T cells stimulated by CD40-CLL cells. This
resulted in a significant alloantigen-specific cytotoxic activity
against both CD40-CLL and naive B-CLL cells, similar to previous
findings.27 In marked contrast, the stimulation of
autologous T cells by CD40-CLL cells induced the expansion of a
predominantly CD4+, Th1-like effector cell population
without cytolytic activity. Moreover and in contrast to findings in
follicular lymphoma and acute lymphoblastic leukemia, CD40-CLL cells
did not allow to expand autologous T cells with cytolytic
activity.31,32
The inability of CD40-CLL cells to stimulate CD8+ T cells
in the autologous system can be explained by several alternative mechanisms. First, CD40 expressed on B-CLL cells may costimulate CD4+ T cells rather than CD8+ T
cells.33 Second, autologous peripheral blood T lymphocytes might be less efficient in mounting a cytolytic response against lymphoma cell antigens than T cells derived from the bone marrow or
tumor-infiltrating lymphocytes.32 Third, most if not all the anti-idiotype immune responses reported to date have been by
CD4+ cells (both Th1- and Th2-type).34-36
Fourth, a distinct pattern of costimulatory molecules expressed on
CD40-CLL cells may induce the preferential stimulation of Th1-like,
CD4+ T cells; for example, it is known that
CD8+ T cells require higher densities of B7-1 to attain an
equivalent level of activation as CD4+ T cells, and
CD40-CLL cells may just not express enough B7-1 and/or B7-2 to
activate CD8+ T cells.28 Sixth, the cytokine
secretion by stimulated T cells themselves may contribute to the
modulation of costimulatory molecules on CD40-CLL cells. These
different mechanisms act probably in concert in regulating the
threshold by which an ongoing T-cell response is maintained.
However, the exact factors preventing the outgrowth of autologous
CD8+ effector T cells with antileukemic activity remain to
be elucidated. It seems highly unlikely that autologous, peripheral
blood T lymphocytes are intrinsically incapable of mounting a CTL
response in B-CLL patients, because the generation of tumor
cell-specific CTLs from the peripheral blood of B-CLL patients has been
recently demonstrated.37
It will be of interest to learn whether the CD4+ Th1 cells
stimulated by CD40-CLL cells are able to induce B-CLL cell death, eg,
by triggering a Fas-dependent apoptotic pathway, as shown in human
tonsillar and Burkitt's lymphoma B cells.38-40 Preliminary experiments in our laboratory showed that CLL cells were indeed rapidly
eliminated in coculture with these CD4+ T cells (R. Buhmann, unpublished data).
The CD40-CD40L interaction is crucial for the immune response. The
ability to trigger and to regulate the induction and expression of
CD40L on CD4+ cells appears to be of paramount importance
to the generation of the T-cell-dependent antigen
response.41,42 An excess of CD40-expressing leukemia cells
might interfere with these cognate T-cell responses and provoke an
aquired CD40L deficiency syndrome in B-CLL.43 Previous
studies and our data suggest that these defects can be restored at
least in part by an effective T-cell activation, with lymphoma or
leukemia cells expressing costimulatory molecules at sufficient
density.6,7,31 Activation of naive T cells can be brought
by any APC, as long as sufficient levels of one or more accessory
molecules are expressed. Later, during the process of T-cell activation
and differentiation, the requirements for full T-cell stimulation
decrease, as T cells become more responsive to a particular
antigen.42,44,45 Thus, an APC that is only weakly
stimulatory for naive T cells can become an efficient stimulator for
preactivated T cells. Accordingly, native B-CLL cells might have
properties of weakly stimulating APCs that need the concomitant presence of strong APCs (such as CD40-CLL cells or dendritic cells) to
fully activate T cells.
Taken together, our results suggest that CD40 activation of B-CLL cells
may reverse T-cell anergy against the neoplastic clone. The results
might provide new perspectives for the immunotherapy of B-CLL, similar
to previous observations in follicular lymphoma7,31 and
pre-B acute lymphoblastic leukemia (ALL).6
Most importantly, the clinical application to produce tumor vaccines or
to generate stimulator cells for adoptive immune transfer strategies in
B-CLL by this approach will meet less practical limitations than in other lymphoid malignancies, because B-CLL cells are readily obtained from peripheral blood. Moreover, the identity of CD40-CLL cells can be
rapidly tested by measuring or light chain restriction and
CD5/CD19 coexpression on the tumor cell surface by flow cytometry. This
will facilitate the practical implementation of these approaches in the
adjuvant therapy of B-CLL.
| |
ACKNOWLEDGMENT |
The authors are indebted to many individuals for their help in the
preparation of this report: Dr Joachim Schultze, Dr Gabriella Pichert,
Doris Schmitter, and Dr John Gribben for their instruction in the
initial stage of the study; Dr Susanne Danhauser-Riedl for her
unconditional assistance in flow cytometry; Dr Heidi Feldmann and
Bettina Meier for their excellent expertise in computer aided design of
the figures; and, finally, Karin Schulz for her support in molecular biology.
| |
FOOTNOTES |
Submitted November 17, 1997; accepted October 31, 1998.
M.H. was supported by the Wilhelm-Sander-Stiftung and the Bayerische
Forschungsstiftung. R.B. was supported by a fellowship of the Fritz
Thyssen-Stiftung. A.N. was supported by a fellowship of the Deutsche Forschungsgemeinschaft.
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 Michael Hallek, MD, Genzentrum,
Feodor-Lynen-Str. 25, D-81377 München, Germany; e-mail:
hallek{at}lmb.uni-muenchen.de.
| |
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E. Gitelson, C. Hammond, J. Mena, M. Lorenzo, R. Buckstein, N. L. Berinstein, K. Imrie, and D. E. Spaner
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Clin. Cancer Res.,
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[Abstract]
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C.-M. Wendtner, D. M. Kofler, H. D. Theiss, C. Kurzeder, R. Buhmann, C. Schweighofer, L. Perabo, S. Danhauser-Riedl, J. Baumert, W. Hiddemann, et al.
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Blood,
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[Abstract]
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A. M. Krackhardt, S. Harig, M. Witzens, R. Broderick, P. Barrett, and J. G. Gribben
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Blood,
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[Abstract]
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P. Chu, W. G. Wierda, and T. J. Kipps
CD40 activation does not protect chronic lymphocytic leukemia B cells from apoptosis induced by cytotoxic T lymphocytes
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TOP
ABSTRACT
INTRODUCTION