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Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 994-1002
T-Cell Receptor-Independent Activation of Clonal Th2 Cells
Associated With Chronic Hypereosinophilia
By
Florence Roufosse,
Liliane Schandené,
Catherine Sibille,
Bernard Kennes,
André Efira,
Elie Cogan, and
Michel Goldman
From the Departments of Immunology and Internal Medicine,
Hôpital Erasme, Université Libre de Bruxelles, Brussels;
the Institute of Pathology and Genetics, Loverval, Gerpinnes; the
Department of Internal Medicine, Centre Hospitalo-Universitaire
Vésale, Montigny le Tilleul; and the Department of Internal
Medicine, Hôpital St. Pierre, Université Libre de
Bruxelles, Brussels, Belgium.
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ABSTRACT |
We recently observed a clonal expansion of
CD3 CD4+ T cells secreting Th2-type
cytokines in patients presenting chronic hypereosinophilia. As clonal T
cells isolated from such patients did not spontaneously secrete
cytokines in vitro, we reasoned that costimulatory signals delivered by
antigen-presenting cells might be required to induce their full
activation. To address this question, we investigated in two such
patients the responses of CD3CD4+ T cells
to dendritic cells (DC). DC elicited proliferation and production of
interleukin-5 (IL-5) and IL-13 by clonal cells from patient 1 and
upregulated their expression of CD25 (IL-2R- ). These effects were
abolished when blocking monoclonal antibodies (MoAbs) against IL-2R-
and IL-2 were added to cocultures, indicating critical involvement of
an autocrine IL-2/IL-2R pathway. Cells from patient 2 were stimulated
by DC to produce Th2 cytokines only when rIL-2 or rIL-15 was added to
cocultures. In both patients, addition of inhibitory MoAbs against
B7-1/B7-2 or CD2 to cocultures resulted in dramatic reduction of
cytokine production and inhibited CD25 upregulation. Thus,
TCR/CD3-independent activation of clonal Th2 cells by DC is an
IL-2-dependent process, which requires signaling through CD2 and CD28.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE IDIOPATHIC hypereosinophilic syndrome
(HES) is defined as blood eosinophilia of unknown origin exceeding
1,500 cells/µL and persisting for over 6 consecutive months. Organ
damage and dysfunction in HES is due to infiltration of tissues by
eosinophils followed by local release of their toxic
content.1-3 The different modes of presentation and disease
courses already observed in initial reports suggest that HES represents
a heterogenous group of disorders. Furthermore, it is well established
that a proportion of patients with HES eventually develop a malignant
hematologic disorder after the initial diagnosis of HES, including
chronic or acute myeloid leukemia, granulocytic sarcoma, acute
lymphoblastic leukemia, and T-cell lymphoma.2 Recent
studies have shed some light on the nature of the primary disorders
leading to expansion of the eosinophilic lineage observed in HES
patients, explaining the clinical heterogeneity of this disease. On one
hand, demonstration of the clonal nature of eosinophils in some HES
patients has provided clear evidence that a primitive disorder of the
eosinophilic lineage, reminiscent of other myeloproliferative
syndromes, can account for hypereosinophilia.4-7 On the
other hand, a subgroup of patients with chronic hypereosinophilia
presenting a monoclonal expansion of T cells has been
identified.8-12 The profile of cytokines produced by the
clonal T cells suggests their direct involvement in the pathogenesis of
hypereosinophilia. Indeed, we and others have demonstrated that these
cells consistently produce high levels of interleukin-5 (IL-5), a
potent eosinophilopoietic cytokine that also contributes to peripheral
activation of eosinophils and promotes their survival through
inhibition of apoptosis.13-16 In some cases, the clonal T
cells also secrete IL-4,8-10 which may favor tissue homing
of eosinophils through upregulation of vascular cell adhesion
molecule-1 (VCAM-1) expression on endothelial cells.17
In our own series of hypereosinophilic patients with an underlying
T-cell disorder, the clonal CD4+ T cells were first
detected because of absent expression of the T-cell receptor (TCR)/CD3
complex on their membrane.8,9 On in vitro activation by
phorbol 12-myristate 13-acetate (PMA) and A23187 calcium ionophore, the
CD3CD4+ cells displayed a Th2
phenotype,18 as indicated by their inability to produce
interferon- (IFN-) and their synthesis of high levels of IL-4,
IL-5, and IL-13, together with variable amounts of IL-2.19 Although the chronic hypereosinophilia observed in these patients and
the clonality of the T cells suggested their persistent activation and
expansion in vivo, these cells were unable to produce cytokines or
proliferate spontaneously in vitro, indicating that they remain dependent on exogenous activating factors. Because of their unique surface phenotype, these clonal T cells represent an ideal tool to
investigate alternative activation pathways of Th2-type cells. In this
study, we sought to determine whether costimulatory signals provided by
antigen-presenting cells (APC) could lead to activation of the clonal
Th2 cells in the absence of signaling through the TCR/CD3 complex. To
this end, we cocultured highly purified
CD3CD4+ T cells from two
hypereosinophilic patients with autologous or allogeneic dendritic
cells (DC) in a series of mixed leukocyte cultures (MLC). We observed
that DC did indeed stimulate CD3CD4+
cells to proliferate, to upregulate their expression of the IL-2R- chain (CD25), and to produce Th2 cytokines. We then investigated the
nature of the stimulatory signals delivered by DC, paying particular
attention to B7/CD28 and lymphocyte function-associated antigen
(LFA)-3/CD2 interactions. Finally, we also specified the role of an autocrine IL-2/IL-2R pathway in this model.
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MATERIALS AND METHODS |
Patients.
We recently evaluated seven patients fulfilling the diagnostic criteria
of HES with blood eosinophil levels above 1,500/µL. In three of these
patients, flow cytometric immunophenotyping of circulating leukocytes
showed the existence of an abnormal CD3CD4+ lymphocyte subset. Two of them
were available for the present study. Patient 1 (P1) was a 20-year-old
woman presenting with severe pruritis, eczema, and tenosynovitis of the
right ankle. At presentation, circulating leukocyte count was
16,900/µL, including 8,923 eosinophils and 4,630 lymphocytes.
CD4+ T cells represented 87% of total lymphocytes and were
composed of 88% CD3CD4+ cells (3,545 CD3CD4+ cells per µL) and 12%
CD3+CD4+ cells. Serum IgE and IgM levels were
340 U/mL (normal, <20) and 310 mg/dL (normal, 40 to 250),
respectively, soluble CD25 level was 180 U/mL, and serum IL-5 was 10 pg/mL by enzyme-linked immunosorbent assay (ELISA)
(Endogen, Woburn, MA). Five-year follow-up has been characterized by progressive increase in absolute count of the aberrant
circulating population of helper T cells (reaching 4,800/µL) and
persistence of marked hypereosinophilia (reaching 17,082/µL). Patient
2 (P2) was a 21-year-old woman also presenting with severe pruritis and
eczema, as well as cyclic angioedema. Circulating leukocyte count was
14,800/µL, including 9,102 eosinophils and 3,419 lymphocytes.
CD4+ T cells represented 87% of total lymphocytes and were
composed of 83% CD3CD4+ cells (2,469 CD3CD4+ cells/µL) and 17%
CD3+CD4+ cells. Serum IgE and IgM levels
reached 15,640 U/mL and 1,253 mg/dL, respectively, soluble CD25 level
was 175 U/mL and serum IL-5 was below detection threshold (2 pg/mL).
The clinical and biological findings were consistent with the diagnosis
of Gleich's syndrome.20 Two-year follow-up has been
characterized by good response to glucocorticoid treatment, as
evidenced by normalization of eosinophil levels. Although the
CD3CD4+ cell population had decreased
significantly (662/µL) over this period, tapering of steroid dosage
was quickly followed by recurrence of hypereosinophilia and clinical
manifestations. Neither patient presented clinical evidence of
lymphoma, such as enlarged lymph nodes or hepatosplenomegaly. Bone
marrow aspiration showed abundant eosinophil precursors and absence of
blastic cells. P1 had received glucocorticoids and IFN-, but was out
of treatment for 7 months at the time of the study, while cells from P2
were collected before initiation of glucocorticoid therapy.
Cell purification.
Circulating leukocytes were obtained from both patients by
cytapheresis, after informed consent, and from buffy coats of healthy blood donors. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway) according to the manufacturer's instructions. After washing with Hanks' balanced salt solution (HBSS) (GIBCO, Life Technologies, Paisley, UK), PBMC were resuspended in cold-conservation medium composed of 80% RPMI 1640 (Bio Whittaker/Boehringer Ingelheim, Verviers, Belgium), 10% dimethyl sulfoxide (Sigma Chemical Co, St
Louis, MO) and 10% heat-inactivated fetal calf serum (FCS) (Bio
Whittaker/Boehringer Ingelheim). Cells were stored in liquid nitrogen
until tested.
Purified CD3CD4+ cells from patients and
CD3+CD4+ cells from healthy subjects were
obtained by negative selection. PBMC were thawed and treated with
Lymphokwik T (One Lambda, Los Angeles, CA) according to the
manufacturer's instructions. The remaining cells were resuspended in
culture medium (RPMI 1640 supplemented with 10% FCS and 40 µg/mL
gentamicin (Schering-Plough, Kennelworth, NJ) at 10 × 106/mL, and incubated with mouse monoclonal antibodies
(MoAb) against CD8, CD14, CD19, and CD56 (Becton Dickinson, Mountain
View, CA), as well as against CD3 for patients, for 30 minutes at
4°C. After washing with HBSS, cells were resuspended in culture
medium and incubated with sheep anti-mouse IgG-coated magnetic
Dynabeads (Dynal, Oslo, Norway) for 45 minutes at 4°C. Coated cells
were removed with a magnet, leaving purified
CD3CD4+ (patients) or
CD3+CD4+ (healthy subjects) cells in
suspension. No contaminating B cells, monocytes, or natural killer
(NK) cells were detected. The
CD3CD4+ cell preparations contained less
than 0.5% CD3-positive cells and more than 98.5% CD4-positive cells,
as assessed by flow cytometry. The monoclonality of the purified
CD3CD4+ cells was established by
Southern blotting and polymerase chain reaction (PCR) analysis for TCR
genes (not shown).
Flow cytometry.
Flow cytometric analysis of surface phenotype was performed by two- and
three-color immunofluorescence using fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, and peridinin-chlorophyll-a-protein (PerCP)-conjugated MoAbs. Surface antigens of T cells were stained with
MoAbs against TCR-/ , CD3, CD4, CD8, CD7, CD27, CD25, CD2, CD28,
CD80, CD40L, and CD45RO from Becton Dickinson and CD86 from Pharmingen
(San Diego, CA). Surface antigens of DC were stained with MoAbs
against HLA-DR, CD14, CD80 (Becton Dickinson), CD86 (Pharmingen), CD83
(Immunotech, Marseille, France), CD40 (Biosource, Camarillo, CA), CD1a (Dako, Glostrup,
Denmark) and the corresponding isotype-matched irrelevant MoAbs. Data
were collected on 10,000 viable cells using a FACScan flow cytometer
(Becton Dickinson).
The percentage of apoptotic lymphocytes was determined by two methods.
At the end of cell cultures, the CD3CD4+
cells were directly analyzed by flow cytometry. Apoptotic cells could
be distinguished from surviving lymphocytes by their decreased forward
scatter and increased side scatter. Secondly, cells from each culture
condition were stained with FITC-conjugated Annexin-V (Pharmingen) and
propidium iodide (PI) (Pharmingen) according to the manufacturer's
instructions, before fluorescence-activated cell sorting (FACS)
analysis. Comparison of the two methods showed that all surviving cells
according to forward and side scatter parameters were
Annexin-V-negative and excluded PI, whereas the apoptotic cells were
mostly positive for both markers with a minute proportion of
Annexin-V-positive PI-negative cells.
Flow cytometry was also used for the detection of intracytoplasmic
cytokine expression in lymphocyte subsets. To this end, total T cells
(CD4+ and CD8+) were isolated from PBMC of both
patients and healthy control subjects using the same procedure as
described above, except that only MoAbs against CD14, CD19, and CD56
were used. These cells were incubated at 106/mL with
Brefeldine A (Sigma Chemical Co) at 10 µg/mL, alone or combined with
50 ng/mL PMA (Sigma Chemical Co) and 0.1 µg/mL calcium ionophore
A23187 (Calbiochem-Behring, San Diego, CA) for 6 hours in culture
medium (37°C, 5% CO2). Surface antigens were stained on aliquots of 2 × 105 cells with FITC- or
PE-conjugated anti-CD8 MoAb, and PerCP-conjugated anti-CD3 MoAb.
Staining of CD8 was preferred to CD4 because of important
downregulation of surface CD4 expression on T cells after in vitro
stimulation. Cells were fixed with FACS Lysing Solution (Becton
Dickinson) for 10 minutes at room temperature in the dark, washed, and
then permeabilized with 0.5 mL FACS Permeabilizing Solution (Becton
Dickinson) in the same conditions. Intracellular cytokines were stained
with PE- or FITC-conjugated MoAbs against IL-2, IFN-, IL-4 (Becton
Dickinson), and IL-5 (Pharmingen). Negative controls for cytokine
expression were provided by unstimulated cells treated only with
Brefeldine A and by intracellular staining with isotype-matched
irrelevant PE- or FITC-conjugated MoAbs. Triple-stain flow cytometry
permitted distinct analysis of cytokine expression in gated
CD3+CD8 (equivalent to
CD3+CD4+) and
CD3CD8 (equivalent to
CD3CD4+) cells.
Stimulation of CD4+ cells with mitogenic agents in
vitro.
Purified CD3CD4+ cells from patients or
CD3+CD4+ cells from healthy subjects (5 × 105/mL) were stimulated using 1 ng/mL PMA alone or combined
with either 0.1 µg/mL A23187 or 1 µg/mL anti-CD28 MoAb (clone
CD28.2) (Immunotech). In addition, cells were also incubated in wells coated with the anti-CD3 MoAb OKT3 (Orthoclone OKT3, Cilag,
Switzerland) in the presence of soluble anti-CD28 MoAb (1 µg/mL). All
culture supernatants were harvested after 48 hours for measurement of cytokine concentrations.
Determination of cytokine levels in culture supernatants.
Commercial ELISA kits were used to determine concentrations of IL-12
and IL-13 (Biosource). Other cytokine concentrations were measured by
two-site sandwich ELISA using antibodies from Genzyme for IL-2,
Chromogenix (Mölndal, Sweden) for IFN-, Mabtech (Stockholm,
Sweden) for IL-4, and Pharmingen for IL-5 and IL-10.
Generation of DC from PBMC.
PBMC were isolated from buffy coats obtained from healthy blood donors
or from cytapheresis of P1. DC were generated by culturing plastic-adherent PBMC with 800 IU/mL recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) (Leucomax) and 500 IU/mL rIL-4 (both kindly provided by Schering-Plough) in enriched
culture medium (RPMI 1640 supplemented with 10% FCS, 2 mmol/L
L-glutamine [GIBCO], 1% nonessential amino acids [GIBCO], 50 µmol/L 2-mercapto-ethanol [GIBCO], and 40 µg/mL gentamycin), as
previously described by Romani et al.21 After 6 days, DC
were harvested, washed, and incubated at 4 × 105/mL
in the presence of 1 µg/mL LPS from Escherichia coli (strain 0128:B12) (Sigma, Bornem, Belgium) in 24-well flat-bottom culture plates for a further 24 hours. The resulting DC displayed a mature phenotype, as assessed by high levels of CD40, CD80, and CD86 expression, and positivity for surface CD83 by flow cytometry (not
shown). For all MLCs, DC were irradiated using 3,000 rads.
MLCs.
All MLCs were performed in enriched culture medium. Purified
CD3CD4+ cells (2 × 105
cells/well) obtained from both patients were cocultured with allogeneic
(P1 and P2) or autologous (P1 only) DC (6.7 × 103
cells/well) in 96-well sterile round-bottom microtiter plates, for 5 days, at 37°C and 5% CO2. In some experiments with P2
cells, rIL-2 (150 U/mL) or rIL-15 (16.7 ng/mL) (Genzyme) was added to the MLC. To determine the nature of the costimulatory molecules involved in activation of the CD3CD4+
cells during cocultures, blocking MoAbs against CD2 (IgG1 isotype), CD80 (IgM), CD86 (IgG2b) (10 µg/mL) (Pharmingen), or CD40 (IgG1) (100 ng/mL) (Genzyme) were added to successive MLCs. Isotype-matched irrelevant MoAbs were used as controls at the same concentrations. To
further assess the role of B7/CD28 interactions, soluble CTLA4-Ig (Ancell, Bayport, MN) or control mouse IgG2a (10 µg/mL) was also added to MLCs. Finally, cocultures were performed in the presence of
inhibitory concentrations (10 µg/mL) of an anti-IL-2R- MoAb (Genzyme) alone or combined with an anti-IL-2 MoAb (Genzyme) or with
corresponding isotype-matched irrelevant MoAbs (IgG2a and IgG1,
respectively). After 5 days of MLC, proliferation of
CD3CD4+ cells was assessed by
3H-thymidine uptake during the following 16 hours, culture
supernatants were harvested for measurement of cytokine concentrations,
and the remaining cells were resuspended in fresh medium to determine surface expression of CD3, CD4, and CD25, as well as the proportion of
apoptotic cells by flow cytometry.
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RESULTS |
Phenotype and cytokine profile of clonal T cells from two patients with
chronic hypereosinophilia.
Lymphocyte phenotyping showed a population of
CD3CD4+ lymphocytes in two patients (P1
and P2) with chronic hypereosinophilia. These aberrant cells, which did
not express / or / TCR (not shown), represented 88% and
83% of total CD4+ lymphocytes in P1 and P2, respectively.
As shown in Fig 1, the CD3-negative helper
T cells from both patients expressed CD2 and CD28, but neither CD80 nor
CD86 (not shown). Furthermore, they expressed the CD45RO isoform
characteristic of memory T cells, while lacking CD40L, CD7, and CD27.
Finally, the chain of the IL-2 receptor (CD25) was absent on cells
from P1, while it was weakly expressed on cells from P2 (Fig 1).
Distribution of surface antigens on the CD3-positive helper T-cell
population from P1 and P2 was similar to that of CD4+ T
cells obtained from normal subjects.
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| Fig 1.
Surface phenotype of clonal T cells from two
hypereosinophilic patients. PBMC from two hypereosinophilic patients
and from a healthy blood donor were stained with FITC-, PE-, or
PerCP-conjugated MoAbs against CD4, CD3, CD2, CD28, CD45RO, CD25, CD7,
and CD27 antigens. Flow cytometric determination of surface phenotype
is shown after gating on CD4+ (healthy subject) or
CD3CD4+ (patients) lymphocytes. Data were
obtained on more than 10,000 viable cells.
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The clonal nature of the CD3CD4+ cell
population from both patients was established by Southern blot and PCR
analysis of the TCR -chain and -chain genes, respectively (not
shown). Cytogenetic analysis of initial blood samples from P1 at time
of presentation showed a normal karyotype, but clonal T cells obtained
for the present study displayed chromosomal abnormalities characterized by partial deletions of chromosomes 6 and 10. In contrast, circulating leukocytes obtained from P2 displayed a normal karyotype at the time of investigation.
The cytokine profile of the CD3CD4+ cell
population was first determined using flow cytometry after
intracellular staining. In the absence of in vitro stimulation, no
cytokines were detected. After 6 hours of incubation with PMA and
A23187 ionophore, a clearly distinct cytokine profile was observed as
compared with control CD3+CD4+ cells
(Fig 2). Indeed, a significant proportion
of the CD3CD4+ cells expressed IL-4
(77% for P1, 69% for P2) and IL-5 (95% for P1, 69% for P2), whereas
IFN- was virtually absent. Furthermore, most
CD3CD4+ cells produced IL-2 (82% for
P1, 69% for P2). The cytokine profile of
CD3+CD4+ cells from both patients was
comparable to that of control CD4+ cells from normal
subjects (percentage of CD3+CD4+ cells
producing IL-2 was 30% and 31% in P1 and P2, respectively, for
IFN-, 29% and 30%; IL-4, 3% and 6%, and IL-5, 3% and 5%).
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| Fig 2.
Cytokine profile of clonal T cells from two
hypereosinophilic patients. Total T cells (CD4+ and
CD8+) were isolated from PBMC obtained from two
hypereosinophilic patients and a healthy blood donor by negative
selection. After a 6-hour culture (106 cells/mL) with
Brefeldine A (10 µg/mL) in the absence or in the presence of PMA (50 ng/mL) and A23187 (0.1 µg/mL), cell membranes were stained with FITC-
or PE-conjugated anti-CD8 and PerCP-conjugated anti-CD3 MoAbs. The T
cells were fixed and permeabilized before staining cytokines with PE-
or FITC-conjugated MoAbs against IL-2, IFN-, IL-4, or IL-5.
Intracytoplasmic expression of cytokines was analyzed by flow cytometry
after gating on CD3CD8 (equivalent to
CD3CD4+) lymphocytes for patients and
CD3+CD8 (equivalent to
CD3+CD4+) lymphocytes for the control
subject. Filled histograms represent staining for cytokines in cells
that have been stimulated with PMA and A23187. Negative controls (solid
lines) are provided by unstimulated cells that have been incubated in
the presence of Brefeldine A alone.
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Cytokine synthesis by clonal T cells cultured with mitogens.
As shown in Table 1, the Th2 cytokine
profile of the clonal T cells was confirmed by measurement of IL-5,
IL-13, and IFN- concentrations in supernatants of purified
CD3CD4+ lymphocytes incubated for 48 hours with PMA and either A23187 ionophore or anti-CD28 MoAb. IL-4 was
also produced, although at lower levels than IL-13, especially after
stimulation with PMA and anti-CD28 MoAb. In the following experiments,
Th2 activities were therefore assessed by IL-5 and IL-13 measurements.
In addition to IL-4, IL-5, and IL-13,
CD3CD4+ cells from both patients
produced high levels of IL-2 in these conditions. As expected by their
phenotype, these clonal T cells did not respond to immobilized anti-CD3
MoAb alone or combined with soluble anti-CD28 MoAb, neither in terms of
cytokine secretion (levels of IL-5 and IL-13 in 48-hour culture
supernatants remained below detection threshold levels for both
patients) nor in terms of proliferation (3H-thymidine
incorporation after 48 hours of culture: 46 and 128 cpm for patients 1 and 2, respectively, versus 185,348 cpm for normal CD4+ T
cells purified from a healthy donor).
DC induce cytokine synthesis by clonal T cells.
We sought to determine whether signals provided by DC would lead to
activation of clonal T cells despite absence of signaling through the
TCR/CD3 complex. Coculture of purified
CD3CD4+ cells from patient 1 for 5 days
with autologous or allogeneic DC indeed resulted in the induction of
IL-5 and IL-13 synthesis, whereas IFN- remained below detection
levels in corresponding supernatants (Table
2). Only low levels of IL-2 (<700 pg/mL) were measured in these
conditions, presumably because of reuptake by the clonal T cells during
the 5-day culture period (see below). Furthermore, the clonal cells
displayed marked proliferation during T-DC cocultures (Table 2). As
similar results were obtained with autologous versus allogeneic DC in
these experiments (which was consistent with the lack of TCR/CD3
expression by the clonal T cells), allogeneic DC were used in further
experiments for the sake of greater availability.
In contrast to patient 1, clonal T cells from patient 2 were not
stimulated by DC to secrete cytokines
(Table 3). In parallel, we observed a
higher degree of apoptosis among P2 clonal cells: after 5-day coculture
with DC, mean percentage of apoptotic T cells was 59.1% ± 2.63%
in patient 2 (mean ± standard error of mean [SEM], n = 8) versus
36.1% ± 2.79% in patient 1 (n = 9, P = .0003 using
Mann-Whitney's test). We reasoned that addition of a T-cell
survival-promoting factor such as rIL-2 or rIL-15 could influence both
their survival and their responses to DC. As shown in Table 3,
incubation of clonal P2 cells alone with a high concentration of rIL-2
resulted in enhanced survival and proliferation, but induced the
secretion of only low levels of cytokines. Indeed, the presence of both
rIL-2 and DC in cocultures was required to induce secretion of high
amounts of IL-5 and IL-13 by clonal P2 cells.
Activation signals delivered by DC involve B7/CD28 and CD2 pathways.
To investigate the nature of the activation signals delivered by DC to
clonal T cells, blocking MoAbs to B7-1, B7-2, CD2, or CD40 were added
to cocultures of purified CD3CD4+ T
cells with DC. For the above-mentioned reason, rIL-2 was added to
cultures performed with P2 cells. Addition of anti-CD80 MoAb to T-DC
cocultures had no significant effect on IL-5 and IL-13 production. In
contrast, addition of anti-CD86 MoAb strongly inhibited IL-5
(Fig 3) and IL-13 production by cells of both P1 and P2
(mean percentages of inhibition in two independent experiments: 77.5% and 83.4% for IL-5, and 88.1% and 80% for IL-13, in P1 and P2, respectively). Blocking both CD86 and CD80 almost completely abolished cytokine production by the clonal T cells (mean percentages of inhibition in two independent experiments: 97.1% and 94.25% for IL-5,
and 99.45% and 95.7% for IL-13, in P1 and P2), and this was confirmed
using soluble CTLA4-Ig in parallel experiments. Thus, signaling through
CD28 appears to play a crucial role in activation of the clonal T
cells. Costimulation through CD2 also contributes to this process, as
indicated by the major reduction of IL-5 and IL-13 production on
addition of anti-CD2 MoAb to T-DC cocultures (mean percentages of
inhibition in three independent experiments: 85.6% and 80% for IL-5
and 95.1% and 67% for IL-13 in P1 and P2, respectively). Furthermore,
in cocultures prepared with P1 cells, proliferation of clonal cells was
inhibited by addition of anti-CD86 MoAb alone (mean percentage of
inhibition in two independent experiments: 73.5%) or combined with
anti-CD80 MoAb (mean percentage of inhibition in two independent
experiments: 99.4%) as well as by addition of anti-CD2 MoAb (mean
percentage of inhibition in three independent experiments: 99.5%). In
cocultures prepared with P2 cells in which a high dose of exogenous
IL-2 sufficient to drive T-cell proliferation was added, such
inhibition was not observed. The specificity of these findings was
ascertained by control experiments using irrelevant isotype-matched
MoAbs. Moreover, the addition of a blocking anti-CD40 MoAb had no
significant effect in this system.
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| Fig 3.
Synthesis of Th2 cytokines by clonal T cells requires
signaling through CD28 and CD2 molecules. Purified
CD3CD4+ cells from hypereosinophilic
patients were cocultured with mature allogeneic irradiated DC in the
absence (patient 1) or in the presence (patient 2) of 150 U/mL rIL-2 at
a DC:T-cell ratio of 1:30. In successive experiments, CTLA4-Ig (10 µg/mL) or blocking MoAbs against B7-1 (CD80), B7-2 (CD86), CD2 (all
used at 10 µg/mL), CD40 (100 ng/mL), or isotypic controls at the same
concentrations were added to cocultures. After 5 days, supernatants
were harvested for measurement of IL-5 concentrations by ELISA. Solid
bars represent IL-5 synthesis during cocultures in the presence of the
above-mentioned blocking molecules, and diamonds represent IL-5
production in the presence of corresponding isotypic controls. Results
are expressed as percentage of IL-5 levels reached in the presence of
blocking MoAbs compared with T-DC cocultures prepared in the absence of
these MoAbs. Data from one of two experiments, which yielded similar
results for each coculture condition, are shown.
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Activation of clonal T cells depends on an autocrine
IL-2/IL-2R- pathway.
We finally investigated the role of endogenous IL-2 production in the
responses of T cells from patient 1 during cocultures with DC. First,
we observed that addition of a blocking anti-IL-2R- (CD25) MoAb to
cocultures of P1 cells with DC dramatically decreased their
proliferative responses and enhanced their apoptosis rate. In parallel,
the secretion of both IL-5 and IL-13 was profoundly inhibited in the
presence of the anti-CD25 MoAb. These effects of IL-2R- blockade
were further enhanced by addition of a neutralizing anti-IL-2 MoAb
(Table 4). Consistent with
these findings, we observed that clonal cells from patient 1 upregulated their membrane expression of CD25 during cocultures with
DC, allowing them to respond to their own secretion of IL-2. As shown
in Fig 4, this CD25 upregulation depends
both on CD28 and CD2 signaling, as it is prevented by the addition of
CTLA4-Ig or blocking anti-CD2 MoAb. To investigate the effects of IL-2
on the synthesis of Th2 cytokines independently from its effects on
survival and proliferation, cocultures were prepared using clonal cells
from P2 in the presence of an alternative T-cell survival promoting
factor, rIL-15. In these experiments, anti-IL-2R- MoAb specifically
inhibited cytokine production (99% inhibition for IL-5 and 86%
inhibition for IL-13) without affecting T-cell survival or
proliferation (Table 5). Taken together,
these experiments establish that costimulatory signals delivered by DC
initiate an IL-2/IL-2R autocrine loop, which is critically involved in
the survival and proliferation of the
CD3CD4+ cells, as well as in their
secretion of cytokines.
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| Fig 4.
Activation of clonal T cells by DC during cocultures is
associated with upregulation of IL-2R- (CD25) expression. Purified
CD3CD4+ cells from patient 1 were cultured
alone or with mature autologous irradiated DC in the absence or in the
presence of CTLA4-Ig (10 µg/mL), blocking MoAb against CD2 (10 µg/mL) or corresponding isotypic controls at a DC:T cell ratio of
1:30. Cells were harvested after 5 days and stained with
fluoro-conjugated MoAbs against CD3, CD4, and CD25. Flow cytometric
analysis of surface expression of CD25 is shown on at least 10,000 viable cells after gating on CD3CD4+
lymphocytes. Data from one of two experiments with similar results for
each condition are shown.
|
|
| |
DISCUSSION |
Absent or low membrane expression of the TCR/CD3 complex on helper T
cells has been observed in several pathological settings including
(retro-)viral infections,22-24 chronic antigenic
stimulation,25 and cancer.26,27 In our
hypereosinophilic patients, we have failed so far to show any
retroviral sequences and the TCR/CD3-negative phenotype of their clonal
T cells was stable throughout T-DC cocultures, as well as after
prolonged culture in the presence of rIL-2 (data not shown), indicating
that the loss of TCR/CD3 expression did not depend on continuous
exposure to a putative exogenous antigen. Whatever the cause of their
lack of TCR/CD3 expression, these clonal T cells are likely to be
involved in the pathogenesis of hypereosinophilia through secretion of
high levels of IL-5.
In this study, we first observed that freshly explanted and purified
CD3CD4+ cells from our patients did not
proliferate and were unable to produce cytokines spontaneously in
vitro, as established by flow cytometry showing absence of
intracytoplasmic cytokines, and by absence of measurable cytokine
release into culture supernatants. Addition of mitogenic factors, which
bypass physiological activation pathways such as PMA in combination
with either A23187 ionophore or anti-CD28 MoAb, was necessary to elicit
cytokine production in vitro. In these conditions, the clonal cells
secreted high levels of IL-5, IL-4, and IL-13, but were unable to
produce IFN-, indicating their Th2 nature.18,19 Thus,
these cells could be incriminated for both hypereosinophilia, through
IL-5 production,28 and high serum IgE levels, through IL-4
and IL-13 production.29 The absence of spontaneous
activation in vitro contrasted with clinical evidence that the clonal T
cells were in an activated state in vivo. Indeed, the long-standing
hypereosinophilia observed in both patients indicated persistent
secretion of Th2-type cytokines, as eosinophils quickly undergo
apoptosis in the absence of specific survival-promoting cytokines such
as IL-5.15,16 Furthermore, the monoclonality of these cells
suggested constant expansion of the cell population. The divergence
between in vivo and in vitro behavior of the
CD3CD4+ cells argues against
constitutional activation of signaling pathways in the clonal T cells,
as has been described for transformed lymphocytes infected with human
T-cell lymphotropic virus type 1 (HTLV-1) in adult T-cell
leukemia lymphoma.30 They suggest on the contrary that the
clonal CD3CD4+ cell population remains
dependent on exogenous signals for both proliferation and cytokine
production. In this study, we focused on accessory signals delivered by
antigen-presenting cells,31 by performing cocultures
between CD3CD4+ cells and DC previously
incubated with LPS. Indeed, LPS-induced maturation of DC enhances their
T-cell-stimulating properties by upregulating their expression of
LFA-3, B7-1, B7-2, and CD40, as well as their secretion of
IL-12.32 We found that DC were able to induce both
proliferation and cytokine production by clonal cells obtained from
patient 1 in the absence of TCR/CD3 signaling. Despite secretion of
IL-12 by DC, the T cells produced IL-5 and IL-13 and remained unable to
produce IFN-, consistent with recent data demonstrating
unresponsiveness of mature Th2 cells to IL-12.33 Clonal
cells obtained from patient 2 differed in that addition of rIL-2 or
rIL-15 to cocultures was necessary to observe efficient activation by
DC. The different activation requirements of P1 and P2 cells during
cocultures probably reflects different thresholds of
cytokine-deprivation induced apoptosis in these cells. Indeed, complete
blockade of IL-2/IL-2R interactions in P1 cells led to high levels of
apoptosis and thus abrogated their responses to DC alone, leading to a
situation similar to that observed with P2 cells. Such divergence in
survival requirements may be explained by the fact that P1 and P2 cells
were obtained at different time points in disease course. Indeed, cells
were harvested from P1 several years after initial symptoms and
discovery of hypereosinophilia during a treatment-free period
characterized by severe eczematous lesions, whereas cells were obtained
from P2 at time of diagnosis, only 5 months after first appearance of
clinical manifestations. Moreover, P1 cells displayed chromosomal
abnormalities, suggestive of progression toward T-cell malignancy,
whereas P2 had a normal karyotype. Although not yet capable of
autonomous proliferation, clonal cells from P1 may thus be less
dependent on growth factors for survival than those from P2. When
cocultures using P2 cells were prepared in the presence of rIL-15 to
promote T-cell survival instead of rIL-2, addition of anti-IL-2R-
MoAb specifically inhibited Th2 cytokine production without affecting
proliferation or apoptosis. Thus, IL-2 was found critical not only to
protect clonal T cells from apoptosis, but also to induce their
proliferation and synthesis of cytokines.
Data obtained with cells of both patients showed that B7/CD28 and
LFA-3/CD2 interactions were critically involved in their TCR-independent activation, as indicated by profound inhibition of
proliferation (P1) and cytokine production (both P1 and P2) when
cocultures were performed in the presence of blocking antibodies that
interfered with these costimulatory pathways. In patient 1, these
effects were in part related to CD28/CD2-dependence of CD25
upregulation on clonal T cells. Several investigators have reported
that simultaneous ligation of CD2 and CD28 molecules with specific
MoAbs in vitro induces prolonged T-cell proliferation compared with
stimulation with anti-CD3 and anti-CD28 MoAb.34-36 Ligation
of CD2 and CD28 on human T cells was previously shown to induce IL-2
production, as well as upregulation of the IL-2R- and - chains,
resulting in autocrine IL-2-dependent stimulation.34,36-39 Our data obtained in patient 1 are consistent with such a role for CD2
and CD28 pathways in TCR-independent activation of T cells.
The abnormal T cells from our patients share some features with Sezary
cells that are found in peripheral blood of patients with advanced
stages of cutaneous T-cell lymphoma (CTCL), including their mature
CD4+CD45RO+CD7CD25
phenotype,40,41 a Th2-type cytokine profile,42
and their clonal nature43 in most cases. The importance of
the CD28-signaling pathway in the activation of Sezary cells has been
demonstrated in a recent report by McCusker et al.44 As
with our CD3CD4+ cells, Sezary cells
have a paradoxical proliferative defect in that, although they are
malignant lymphomatous cells, they do not proliferate spontaneously in
vitro, nor do they respond to the combined action of anti-CD3 and
anti-CD28 MoAbs. However, cocultures of purified
CD4+CD7 cells with allogeneic
growth-inactivated PBMC in the presence of anti-CD3 MoAb induced
proliferation of Sezary cells, and addition of CTLA4-Ig or of combined
anti-B7 MoAbs to these cultures led to substantial inhibition of cell
growth. These results indicate that although signaling through CD28 is
critical for Sezary cell growth following engagement of the TCR/CD3
complex, another as of yet unidentified signal provided by APC is
required for activation of Sezary cells. Similarly, we observed that
stimulation of clonal T cells from patient 1 with a combination of
anti-CD2 and anti-CD28 MoAbs was not sufficient to elicit optimal
cytokine synthesis (data not shown), suggesting that additional
costimulatory signals delivered by DC were indeed operative in our
coculture experiments.
It has been suggested that the nature of signals leading to T-cell
activation can skew the subsequent cytokine profile. Activation of
mature T cells through the alternative CD2-dependent pathway has been
shown to induce IL-4 production more efficiently than engagement of
CD3,45 and signaling through CD28 was involved in the
differentiation of naive CD4+ T cells toward Th2
cytokine-producing cells.46,47 The specific involvement of
the CD28 ligands B7-1 and B7-2 in functional differentiation of target
cells remains a controversial issue.48 Several studies suggest that ligation of CD28 by CD80 preferentially induces Th1 cytokines, whereas CD86 preferentially induces Th2
cytokines.49,50 However, the fact that anti-CD86 MoAb often
inhibited Th2 cytokine production more efficiently than anti-CD80 MoAb
as in our own study could simply reflect different levels and/or
kinetics of expression of these molecules on APC.51,52
Clinical observations indicate that chronic hypereosinophilia is
sometimes associated with a premalignant lymphoproliferative condition
as some HES patients will eventually develop a full-blown T-cell
lymphoma.53 Identification of a monoclonal population of
CD3 helper T cells could be a predictive marker of
malignant transformation.54-56 Understanding the activation
requirements of such cells could lead to more efficient therapeutic
approaches both for the control of eosinophilia and for prevention of
malignant evolution. Our observations that TCR-independent activation
of the CD3CD4+ cells can be provided by
APC through an IL-2-dependent process, which requires signaling
through CD2 and CD28 molecules may be useful for defining new
therapeutic strategies for hypereosinophilic patients with a profile
similar to ours.
| |
ACKNOWLEDGMENT |
We thank Alain Crusiaux for excellent technical assistance.
| |
FOOTNOTES |
Submitted November 4, 1998; accepted March 22, 1999.
Supported by the Fonds de la Recherche Scientifique Médicale and
the Télévie Programme (Belgium). F.R. is a research fellow of the Fonds National de la Recherche Scientifique (Smithkline Beecham
Biologicals fellowship).
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 Michel Goldman, MD, Hôpital Erasme,
Department of Immunology, 808 route de Lennik, B-1070 Brussels,
Belgium; e-mail: mgoldman{at}ulb.ac.be.
| |
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ABSTRACT
INTRODUCTION