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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4347-4357
Downregulation of Antigen-Presenting Cell Functions After
Administration of Mitogenic Anti-CD3 Monoclonal Antibodies in Mice
By
Eric Muraille,
Fabienne Andris,
Bernard Pajak,
K. Martin Wissing,
Thibaut De Smedt,
Fabrice Desalle,
Michel Goldman,
Maria-Luisa Alegre,
Jacques Urbain,
Muriel Moser, and
Oberdan Leo
From the Laboratoire de Physiologie Animale, Département de
Biologie Moléculaire, Université Libre de Bruxelles,
Gosselies, Belgium; the Laboratoire d'Immunologie Expérimentale,
Hôpital Erasme, Brussels, Belgium; and the Department of
Medicine, University of Chicago, Chicago, IL.
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ABSTRACT |
Antibodies against CD3 are widely used as immunosuppressive
agents. Although it is generally assumed that these reagents exert
their immunomodulatory properties by inducing T-cell deletion and/or
inactivation, their precise mechanism of action remains to be
elucidated. Using a murine model, we demonstrate in this report that
administration of anti-CD3 antibodies causes the migration and
maturation of dendritic cells (DC) in vivo, as determined by
immunohistochemical analysis. This maturation/migration process was
followed by selective loss of splenic DC, which resulted in a selective
inhibition of antigen-presenting cell (APC) functions in
vitro. Spleen cells from anti-CD3-treated animals were unable to
productively stimulate naive alloreactive T cells and Th1-like clones
in response to antigen, while retaining the ability to present antigen
to a T-cell hybridoma and Th2 clones. Anti-CD3 treatment was found
to induce a selective deficiency in the ability of spleen cells to
produce bioactive interleukin-12 in response to CD40 stimulation. APC
dysfunction was not observed when nonmitogenic forms of anti-CD3
antibodies were used, suggesting that splenic DC loss was a consequence
of in vivo T-cell activation. Nonmitogenic anti-CD3 monoclonal
antibodies were found to be less immunosuppressive in vivo, raising the
possibility that APC dysfunction contributes to anti-CD3-induced
immunomodulation. Collectively, these data suggest a novel mechanism by
which mitogenic anti-CD3 antibodies downregulate immune responses.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE INITIATION OF an adaptive immune
response to foreign antigen (Ag) is dependent on the interaction
between Ag-specific T lymphocytes and appropriate Ag-presenting cells
(APC). Dendritic cells (DC) have been recognized as the
major APC population able to optimally activate naive T lymphocytes
both in vitro and in vivo. This priming property is related to their
(1) ability to take up, process, and present Ags; (2) high expression
of adhesive and costimulatory molecules able to interact with T-cell
borne counterreceptors; and (3) ability to migrate from peripheral
tissues to T-cell areas in lymphoid organs (for review, see
Hart1 and Bancherau and Steinman2). Recent
studies have demonstrated that these functional features were
differentially expressed according to tissue location and maturation
state. Immature DC are mostly found in peripheral tissues, where they
capture and process Ags while displaying poor T-cell stimulatory
properties. Conversely, mature DC in lymphoid organs have reduced
Ag-presenting capacities, but are potent stimulators of naive T cells,
a property related to their higher expression of costimulatory and
adhesive molecules. This developmental sequence, originally
demonstrated to occur spontaneously in vitro,3,4 has been
recently studied in vivo.5 Collectively, these studies have
led to the concept that immature DC in peripheral organs capture Ag
locally and then migrate via afferent lymphatics to draining lymph
nodes, where they acquire the capacity to activate Ag-specific naive T cells.
Studies related to transplantation have provided many insights into DC
biology and function in vivo. Major histocompatibility complex
(MHC) class II+ cells with DC morphology can
be found in virtually all organs.6 Tissue DC migrate out of
transplanted tissues into the circulation and reach lymphoid organs
such as the spleen.7,8 It is noteworthy that depletion of
donor DC has been shown to correlate with increased kidney and heart
allograft survival.9,10 Taken together, these observations
suggest that migration and maturation of DC represent critical steps
for the initiation of an immune response to a transplanted organ.
Recent observations have demonstrated that, in addition to Ag-specific
stimuli such as allogeneic grafts or contact allergens,11 a
variety of factors, including microbial products (lipopolysaccharide [LPS]) or inflammatory mediators (tumor necrosis factor-
[TNF-] and, to a lesser extent, interleukin-1 [IL-1]) can induce
DC migration in vivo. Injection of LPS causes a massive migration of DC
from skin, heart, kidney, and intestine, as well as maturation and movement of spleen DC from the marginal zone into the T-cell
area.5,12
The observation that inflammatory stimuli mobilize DC from tissues led
us to investigate the effect of in vivo administration of anti-CD3
monoclonal antibodies (MoAbs) on APC distribution and function
.13,14 Anti-CD3 antibodies have been successfully used
to prevent or treat allograft rejections. However, before exerting
their immunosuppressive properties, mitogenic anti-CD3 antibodies
trigger the in vivo release of multiple cytokines, including IL-2,
IL-4, interferon- (IFN-), and TNF-.15-17 In this
study, we demonstrate that administration of anti-CD3 MoAbs induce
profound changes in the APC population in vivo, suggesting a novel
mechanism by which these antibodies may downregulate immune responses.
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MATERIALS AND METHODS |
Animals, Cell Lines, and Reagents
Six- to 8-week-old female BALB/c (H-2d), CBA
(H-2k) CB17, and CB17 SCID mice were purchased from Charles
River Wiga (Sulzfeld, Germany). Six- to 8-week-old A/J and B10.A mice
were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were
maintained in pathogen-free conditions in our animal facility.
The OVA-specific T-cell hybridoma DO.11.1018 was kindly
provided by P. Marrack (Howard Hughes Medical Institute, Denver, CO).
The A.E7 Th1 clone, specific for pigeon cytochrome C (PCC)3 + Iek 19 was a kind gift of R.H. Schwartz
(National Institutes of Health, Bethesda, MD). The HG-4 Th2 clone was
generated from the draining nodes of A/J (H-2a) mice primed
with 300 µg human gamma globulins (HGG) in complete Freund adjuvant (CFA). The ovalbumine peptide
(323-339)-specific Th clones were derived as described.20
Briefly, spleen cells (5 × 106) from naive DO11.10
mice (kindly provided by Anne O'Garra, DNAX, Palo Alto, CA) were
cultured in 24-well plates with 0.5 µg/mL of OVA(323-339) in the
presence of recombinant IL-12 (rIL-12) and 10 µg/mL anti-IL-4 (clone
11B11; ATCC, Rockville, MD) to promote the development of
Th1-like cells or 200 U/mL IL-4 and 100 ng/mL anti-IL-12 (clone C17.8;
kindly provided by G. Trinchieri, Wistar Institute, PA21)
to favor the differentiation toward a Th2 phenotype. T-cell clones
(106/well) were restimulated biweekly in a 24-well plate
with 107 irradiated syngeneic splenocytes/well and the
relevant Ag. At 48 hours, the cells were expanded 5-fold into medium
containing 10 U/mL rIL-2 and rested for at least 7 days. The clones
used in this study were referred to as Th1 or Th2 based on the cytokine profile (IFN- or IL-4/IL-5/IL-10) secreted in response to Ag stimulation.
The antibodies 145-2C11 (hamster IgG, anti-CD3; ATCC) and PARSI-19
(hamster IgG, anti-p-azophenylarsonate; generated in our laboratory) were purified from ascitic fluids. The m3-2C11
MoAb22 is a chimeric anti-CD3 antibody comprising the
variable region of the 145-2C11 clone and the heavy and light constant
regions of murine 3 and k origin.
In Vivo Treatment
BALB/c mice were injected intravenously (IV) into the lateral tail vein
with anti-CD3 antibodies solubilized in pyrogen-free NaCl (0.9%).
Control animals were injected with the same volume of diluent or with
hamster control IgG.
In Vitro Responses
The complete medium used in all experiments was RPMI 1640 (Seromed
Biochem KG, Berlin, Germany) supplemented with 2% HY ULTROSER (a
serum-free media purchased from GIBCO BRL, Merelbeke, Belgium), penicillin, streptomycin, nonessential aminoacids, sodium pyruvate, 2-mercaptoethanol (2-ME), and L-glutamine (Flow ICN
Biomedicals, Bucks, UK). Sephadex G10-depleted responder cells and
T-cell hybridomas were stimulated in vitro as described in the figure
legends. Supernatants were collected after 24 or 48 hours of culture,
frozen, and assayed for IL-2 content by a bioassay using a subclone of
the CTL.L cell line insensitive to murine IL-4. T-cell
clones (0.3 to 1.0 × 105/well) were incubated in the
presence of irradiated spleen cells (3.0 to 6.0 × 105/well) and nominal Ag [0.3 µmol/L of PCC, 1 mg of
HGG, or 0.2 µg/mL of OVA (323-339)] in 96-well plates, and cell
proliferation was determined by 3H-thymidine incorporation.
IL-12 determinations were performed using a bioassay based on the
ability of IL-12 to induce IFN- secretion. Briefly, spleen cells
isolated from control or anti-CD3-treated animals were incubated with
graded doses of an activating anti-CD40 MoAb (clone 3/23; kindly
provided by G. Klaus, National Institute for Medical Research, London,
UK23). Supernatants were collected after 48 hours, and the
amount of IFN- was determined by enzyme-linked immunosorbent assay
(ELISA) as previously described. Standard curves were generated by
incubating both control and anti-CD3-treated cell populations with
recombinant murine IL-12 (kindly provided by S. Wolf, Genetics Institute, Cambridge, MA). The addition of neutralizing anti-IL-12 MoAbs (clone C17.8) was found to completely inhibit anti-CD40-induced IFN- secretion, thus establishing the specificity of the assay for
IL-12. Control rat IgG2a antibodies (clone IR418) were kindly provided
by H. Bazin (Université Catholique de Louvain, Brussels, Belgium).
Purification of Low-Density Spleen Cells
Spleens were digested with collagenase (CLSIII; Worthington Biochemical
Corp, Freehold, NJ) and separated into low- and high-density fractions
on a BSA gradient (Bovuminar Cohn fraction V powder; Armour
Pharmaceutical Co, Tarrytown, NY), as previously
described.24
Cytofluorometric Analysis
Cells were analyzed by flow cytometry with a FACScan cytometer (Becton
Dickinson, Mountain View, CA). The cells were preincubated with 2.4G2
(a rat antimouse Fc receptor MoAb; ATCC) for 10 minutes before staining
to prevent antibody binding to FcR and further labeled with
phycoerythrin (PE)-coupled B220 (Pharmingen, San Diego, CA). The
antibodies 145-2C11, 14.4.4-S (anti-I-Ek,d; ATCC), and N418
(anti-CD11c; ATCC) were purified from ascitic fluids and coupled to
fluorescein isothiocyanate (FITC) or biotin in our laboratory. Cells
were gated according to size and scatter to eliminate dead cells and
debris from analysis.
Immunohistochemistry
Cryosections.
Tissue samples were frozen in isopentane ( 80°C) and 8-µm
cryostat sections were prepared. Samples were fixed in acetone for 10 minutes, air-dried, treated with 3% H2O2 in
phosphate-buffered saline (PBS) for 30 minutes to block endogenous
peroxydase, and incubated in PBS containing 0.5% of blocking reagent
(Roche Diagnostics, Belgium, Brussels) for 30 minutes.
Immunohistowax process.
Spleens were fixed for 3 days in Immunohistofix (Sanver TECH, Boechout,
Belgium) followed by dehydratation in acetone for 6 hours. Tissues were
embedded in Immunohistowax (Sanver TECH), sectioned at 3 to 6 µm,
deembedded by washing in acetone for 10 minutes, and transferred to PBS.
Immunostaining.
Sections were incubated for 20 minutes with the monoclonal rat
antimouse antibody 2.4G2 to Fc receptors to prevent nonspecific staining. Slides were washed in PBS, incubated for 1 hour at room temperature with biotinylated MoAbs (10 µg/mL) in 0.5% PBS-BR, and
washed in PBS: M1/70 (anti-CD11b/Mac-1; ATCC), N418 (anti-CD11c), RM4-5
(anti-mouse-CD4; Pharmingen), NLDC-145 (anti-DEC-205; ATCC), GL1
(anti-CD86; ATCC), and LO-MD6 (a kind gift of H. Bazin). Slides were
then incubated in avidin-biotin-peroxidase complex (Vectastain ABC kit;
Vector Laboratories, Burlingame, CA) and stained with a solution of
diaminobenzidine tetrahydrochloride with metal enhancer (DAB tablets,
SigmaFAST; Sigma, St Louis, MO) or incubated in avidin-biotin-alkalin
phosphatase complex (Vectastain ABC kit, AK-5000; Vector Laboratories)
and stained with alkaline phosphatase substrate kit I (SK-5100, red;
Vector Laboratories) or kit III (SK-5300, blue; Vector Laboratories).
The sections were counterstained with methyl green, dehydrated, and
mounted in Poly-Mount (Polysciences, Warrington, PA).
Digitized images were captured using a Ikegami CCD color camera
(Ikegami Tsushinki, Tokyo, Japan) and analyzed using
CorelDraw 7 software (Corel, Ottawa, Ontario, Canada).
Skin Grafting
BALB/c mice were grafted with tail skin from A/J (class I and class II
MHC mismatch) as follows. Graft beds were created by excising
approximately 0.5 cm2 of skin on the right thoracic wall
and skin grafts were covered by a double layer of Vaseline gauze and
plaster bandage, which were removed on day 8 after skin grafting.
Grafts were considered rejected when no more viable tissue was visible.
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RESULTS |
Anti-CD3 Administration Induces Dendritic Cell
Movement and Maturation
Anti-CD3 MoAbs (50 µg) were injected IV into naive adult mice and
APC populations visualized in serial sections using specific antibodies
(Fig 1A). As expected,5
CD11c+ DC and macrophage-like, CD11b+ cells
were found in the marginal zone, between the red and white pulp in
naive or saline-treated animals (Fig 1A, left panels). Injection of
anti-CD3 MoAbs led to the rapid (6 hours posttreatment) redistribution of some CD11b- and most CD11c-expressing cells to the
white pulp in the T-cell area surrounding the central arteriole (Fig
1A, central panels). Forty-eight hours after anti-CD3
administration, the overall number of splenic DC was strongly reduced
both in the marginal zone and in the T-cell area, whereas the number
and location of CD11b-expressing cells was found unaltered compared with control spleens (Fig 1A, right panels). Few cells in the marginal
zone were labeled with antibodies to the costimulatory molecule CD86
before treatment, whereas the expression of the DEC-205 marker was
barely detectable (Fig 1B, left panels). Anti-CD3 administration led
to a redistribution of DC markers in the white pulp in the area
surrounding the central arteriole (where most CD4+ T cells
are located, data not shown). However, note that cells expressing CD11c
and CD86 could still be found in the marginal zone 6 hours
posttreatment (Fig 1B, central panels). Concomitantly, anti-CD3
injection led to the increased expression of CD86 and DEC-205, 2 markers known to be upregulated during DC maturation2 (Fig
1B, central panels). These observations strongly suggest that
anti-CD3 MoAbs induced the maturation and migration of DC from the
marginal zone to the T-cell area. As previously noted, a strong
reduction in the expression of all DC markers was observed in the
spleen 48 hours after anti-CD3 administration (Fig 1, right panels).
The distribution of IgD+ cells did not change in a
significant manner during the first 24 hours after treatment (Fig 1 and
our own unpublished observations). However, note that the
distribution of B cells was affected at 48 hours because of the
increased size of the T-cell area after anti-CD3 administration (Fig
1).
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| Fig 1.
Anti-CD3 administration induces DC movement and
maturation. (A) Immunoperoxidase staining of cryostat sections or (B)
alkaline phosphatase staining of embedded serial sections of spleens
from control (hamster Ig) or anti-CD3 injected mice (50 µg IV), 6 and 48 hours posttreatment. Sections were stained with antibodies to
CD11b, CD11c, IgD, Dec-205, and CD86 as indicated in the figure.
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The selective loss of splenic DC induced by anti-CD3 at 48 hours was
further confirmed by flow cytometry (Fig
2). DC-enriched, low-density spleen cells isolated from control and
treated animals were double-stained with antibodies to a B-cell marker
(CD45R), a DC marker (CD11c), and MHC class II molecules
(I-Ed). The data confirmed that administration of
anti-CD3 antibodies led to a selective depletion of DC from the
low-density splenic population (see the lower right panel in each
contour plot) identified as I-Ed+, CD45R
(Fig 2A through C) or CD11c+, CD45R (Fig
2D through F). The effect of anti-CD3 administration was reversible,
because control levels of DC were found in mice tested 15 days after
treatment.
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| Fig 2.
Selective reduction of DC number after anti-CD3
administration. BALB/c mice were treated on day 0 with control antibody
or anti-CD3 (50 µg IV) and analyzed on days 2 and 15 for
I-Ed, CD45R, and CD11c expression. Pooled low-density
spleen cells from 3 individuals in each group were analyzed by 2-color
immunofluorescence after staining with PE-labeled anti-CD45R and
FITC-labeled anti-I-Ed (A through C) or PE-labeled
anti-CD45R and FITC-labeled anti-CD11c (D through F).
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Because DC in nonlymphoid organs often fail to express the CD11c
marker, we monitored the presence of MHC class II+ cells in
the hearts of control and anti-CD3-treated mice
(Fig 3A). Cells expressing MHC class II
molecules in control hearts displayed a DC-like morphology, in
agreement with a previous finding suggesting that DC represent the
major MHC class II+ APC population in this
organ.25 Anti-CD3 administration led to a marked
reduction in DC-like numbers in the heart 48 hours posttreatment (Fig
3A). When sections of lymph nodes were analyzed, a marked increase in
CD11c+ cells was observed in sections from mice injected
with anti-CD3 MoAbs 6 hours earlier (Fig 3B). DC numbers returned to
near control levels 48 hours after treatment (Fig 3B). Collectively,
these data suggest that anti-CD3 MoAb-treatment profoundly affects the tissue distribution and phenotype of DC, with different effects depending on timing and tissue location.
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| Fig 3.
Altered DC distribution in peripheral lymphoid and
nonlymphoid organs after anti-CD3 administration. Alkaline
phosphatase staining of cryostat section of (A) hearts and (B)
mesenteric lymph nodes (serial sections) from control and
anti-CD3-injected mice (50 µg IV). Sections were stained with
antibodies to MHC class II, CD4 and CD11c, as indicated in the figure.
A higher magnification of MHC II+ cells in the heart of
control mice is also shown.
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Anti-CD3 Injection Results in Reduced
Ag-Presenting Capacities
Because DC represent the major APC population able to activate naive T
cells and CD4+ Th1 clones,1,2 we evaluated the
Ag-presenting capacity of unselected spleen cell populations from
control (normal hamster Ig-treated) and anti-CD3-treated animals 48 hours postinjection.
As shown in Fig 4, spleen cells from
anti-CD3-treated animals failed to activate naive alloreactive T
cells to proliferation (Fig 4A) and IL-2 secretion (Fig 4B). A mix of
equal numbers of spleen cells from control and antibody-treated mice
stimulated T cells at intermediate levels, suggesting that defective
accessory functions were not maintained through active suppression (Fig 4A and B). By contrast, both APC populations were able to present an
agonist peptide to an Ag-specific T-cell hybridoma (Fig 4C), showing
that anti-CD3-treated spleen cells retained the ability to activate
a costimulatory-independent T-cell line.
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| Fig 4.
Defective APC function in anti-CD3-treated mice.
Spleen cells from control antibody-treated or anti-CD3-treated
BALB/c mice (50 µg IV) were irradiated and used as accessory cells to
stimulate (A and B) G10-nonadherent responding cells from CBA mice (3 × 105/well) or (C) ovalbumin-specific T-cell hybridoma
mice (3 × 105/well) in the presence of 1 µg/mL of
ovalbumin peptide. Proliferation (A) and IL-2 production (B and C) were
assayed as described in Materials and Methods. Results are expressed as
counts per minute (cpm) of [3H] TdR
incorporation by responder cells (A) or IL-2-dependent cell line CTL.L
(B and C). These results are representative of 3 independent
experiments.
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Murine Th1 and Th2 clones display distinct accessory cell requirements.
In particular, Th1 clones have been shown to require an adherent cell
population to optimally proliferate in response to Ag, whereas Th2
clones can be stimulated by Ag presented by purified B
cells.26 We have confirmed these observations in our
laboratory and found a good correlation between the T-helper phenotype
of murine clones and their APC requirement (as determined on 5 Th1 and
5 Th2 clones, data not shown). To further characterize the effect of
anti-CD3 treatment on splenic APC function, 4 representative murine
helper clones (identified as Th1 and Th2 based on their pattern of
cytokine secretion) were stimulated by splenic accessory cells from
anti-CD3-treated and control mice. The Th1 and Th2 helper clones
used in this study displayed distinct APC requirements, as shown by the
selective capacity of G10-passed splenic accessory cells to optimally
activate Th2-type lymphocytes to proliferation. Spleen cells from
animals previously injected with anti-CD3 antibodies failed to
optimally stimulate both murine Th1 clones in the presence of nominal
Ag, while retaining the ability to efficiently present Ag to Th2 clones
(Fig 5).
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| Fig 5.
APC from anti-CD3-treated mice support the growth of
the Th2, but not Th1, Ag-specific clones. Spleen cells from control
antibody-treated or anti-CD3-treated mice (50 µg IV) were
irradiated and used as accessory cells to stimulate the Th1 clones AE.7
and CLOVA 1.1 or the Th2 clones HG.4 and CLOVA 2.9 in the presence of
nominal Ag. Clones were also stimulated by Ag presented by G10-depleted
splenic accessory cells for the sake of comparison. Results are
expressed as cpm of [3H] TdR incorporation by responder
cells. These results are representative of 3 independent experiments.
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DC represent the major cell population able to secrete IL-12, a
cytokine known to favor the differentiation of uncommitted T-cell
precursors into Th1-like cells and to sustain the in vitro proliferation of Ag-stimulated Th1 clones.27,28 We
therefore tested the ability of unselected spleen cells from control
and treated mice to produce IL-12 in response to CD40 signaling, as determined by a sensitive bioassay. As shown in
Fig 6, spleen cells from
anti-CD3-treated mice displayed reduced secretion of bioactive
IL-12 molecules when stimulated by activating anti-CD40 antibodies in
vitro. The specificity of the assay for IL-12 was demonstrated using a
blocking antibody to IL-12. Although we were unable to detect the
production of immunoreactive p70 heterodimeric form of IL-12 under
these experimental conditions, anti-CD3 treatment was found to inhibit
the in vitro production of p40-containing molecules (as assayed by
ELISA) in response to anti-CD40 stimulation (data not shown).
Collectively, these data suggest that spleen cells from
anti-CD3-treated mice retain the ability to capture, process, and
present protein Ags in association with MHC class II molecules but are
unable to provide the accessory signals required for the in vitro
stimulation of naive and Th1-type murine T cells.
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| Fig 6.
Downmodulation of anti-CD40-induced IL-12 secretion
after anti-CD3 treatment. Unselected spleen cells (106
cells/well) from control and anti-CD3-injected mice (48 hours
posttreatment, 50 µg IV) were stimulated in vitro by an activating
rat IgG2a anti-CD40 MoAb (3 µg/mL) or an equivalent amount of an
isotype-matched, control antibody (clone IR418). When indicated,
cultures were supplemented with a neutralizing antibody to the IL-12
subunit p40 (rat IgG2a C17.8, 5 µg/mL) or with an equivalent amount
of an isotype-matched, control antibody (clone IR418). Supernatants
were harvested 48 hours later and assayed as described in Materials and
Methods. The results represent the mean of 3 independent cultures
(assayed in duplicate) and are representative of 3 independent
experiments. The detection limit of the assay was 0.2 pg/mL.
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Only Mitogenic Anti-CD3 MoAbs Affect Splenic Accessory Cell
Functions
To evaluate the role of T-cell activation in the downregulation of
splenic APC functions, mice were injected with a single dose of
mitogenic hamster anti-CD3 antibodies (clone 145-2C11) or chimeric
anti-CD3 antibodies comprising the variable regions of the 145-2C11
clone and the heavy and light constant regions of murine 3 and
k origin. This chimeric molecule displays very low affinity for
murine FCR, resulting in marked reduction of its mitogenic
properties.22 As expected, injection of the m3 isotype
induced a strong downmodulation of TCR cell surface expression as
detected using both anti-CD3 (Fig 7A)
and anti-TCRV reagents (data not shown), but failed to induce the
systemic release of serum IL-2 observed in response to hamster
anti-CD3 antibodies (Fig 7B). To investigate the in vivo
immunosuppressive properties of mitogenic and nonmitogenic anti-CD3
MoAbs under the experimental conditions used throughout this study (a
single injection regimen), we monitored the survival of MHC mismatched
A/J tail skin grafts that were transplanted on the day of antibody
treatment onto BALB/c mice. The administration of mitogenic anti-CD3
MoAbs significantly prolonged the mean graft survival
(Fig 8), whereas animals injected with the
nonmitogenic anti-CD3 MoAb displayed a normal rejection response
when compared with control, untreated mice. We next established the
effect of mitogenic and nonmitogenic anti-CD3 MoAbs on APC functions. As shown in Fig 9, spleen cells
isolated from animals injected with nonmitogenic forms of anti-CD3
antibodies were able to efficiently stimulate alloreactive naive T
cells to proliferate, whereas spleen cells from mice treated with the
mitogenic form of anti-CD3 MoAbs displayed reduced APC functions.
Flow cytometry studies confirmed that nonmitogenic anti-CD3
antibodies failed to downregulate CD11c+ splenic DC numbers
(data not shown). Finally, no effect of mitogenic anti-CD3
antibodies on APC function was observed in SCID mice (data not shown),
in agreement with the idea that APC dysfunction induced by in vivo
administration of anti-CD3 antibodies is a consequence of T-cell
activation.
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| Fig 7.
In vivo administration of m3-anti CD3 and mitogenic
anti-CD3 administration. (A) BALB/c mice were treated on day 0 with
control antibody, m3-anti-CD3, or anti-CD3 (30 µg IV) and
analyzed on day 1 for CD3 expression. Pooled spleen cells from 3 individuals in each group were analyzed by immunofluorescence after
staining with FITC-labeled anti-CD3. (B) BALB/c mice (5 per group)
were treated with control antibody, m3-anti CD3, or anti-CD3.
Mice were bled 2 hours later and the serum IL-2 activity was tested by
ELISA. Results are expressed as the mean absorbance (492 nm) ± SD of
individual determinations.
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| Fig 8.
Effect of m3-anti CD3 and hamster anti-CD3
administration on allogeneic skin graft survival. BALB/c mice were
injected with 30 µg of anti-CD3 MoAbs and grafted with
MHC-mismatched A/J tail skin on the day of antibody treatment. Control
animals were injected with PBS. P < .001 for hamster versus
m3 anti-CD3 or control, PBS-treated animals; P is not
significant for m3 anti-CD3 versus control, PBS-treated
animals.
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| Fig 9.
Effect of m3-anti-CD3 and mitogenic anti-CD3
administration on APC functions. Spleen cells from control
antibody-treated mice, m3-anti-CD3-treated mice, or
anti-CD3-treated mice (50 µg IV) were irradiated and used as
accessory cells to stimulate G10-nonadherent responding cells from CBA
mice (3 × 105/well). Results are expressed as cpm of
[3H] TdR incorporation by responder cells.
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DISCUSSION |
Despite extensive clinical and animal experience, the precise
mechanisms by which anti-CD3 antibodies inhibit in vivo
transplantation responses have not yet been firmly established. Because
CD3, the molecular target of most therapeutic anti-CD3
antibodies, is a 17- to 20-kD transmembrane molecule that is a part of
a multimolecular, Ag-specific receptor complex selectively expressed by
T cells, inhibition of T-cell functions has been proposed as the key
mechanism of action of these antibodies in vivo.
Accordingly, in vivo administration of anti-CD3 MoAbs has been shown
to induce (1) peripheral T-cell depletion, due to receptor-mediated opsonization and killing,29-32 increased adhesion to
vascular endothelium,33 and activation-induced cell
death34; (2) Ag-specific receptor blockade and/or
downmodulation35; and (3) induction of a state of cell
unresponsiveness related to T-cell anergy.36,37
The present study demonstrates that administration of anti-CD3
antibodies caused a marked but selective inhibition of splenic accessory cell functions, suggesting an additional mechanism by which
these antibodies can downmodulate in vivo immune responses.
Injection of anti-CD3 MoAbs appears to induce the same phenomenon of
DC maturation and peripheral cell loss previously observed in response
to LPS.5 Anti-CD3 MoAbs induced the migration of splenic
DC from the marginal zone to the T-cell area and the simultaneous
upregulation of the activation-associated markers DEC-205 and CD86 (Fig
1). By analogy with studies performed in LPS-treated mice, we interpret
the loss of DC from the heart (Fig 3A) and the increase in DC numbers
observed in lymph nodes (Fig 3B) as the consequence of
anti-CD3-induced migration of DC from nonlymphoid organs to lymph
nodes via the lymphatic route.
Although the molecular mechanisms that control migration and
recruitment of DC are not well defined, it has been recently demonstrated that systemic administration of recombinant TNF-, and
to a lesser extent of IL-1, induces movement of DC from nonlymphoid organs into lymph nodes.12 Because anti-CD3 MoAbs cause
the in vivo production of TNF- and other inflammatory
mediators,16 it is reasonable to assume that DC migration
after anti-CD3 administration is secondary to the in vivo release of
inflammatory mediators by polyclonally activated T cells. In support of
this conclusion, no effect on APC function was observed in SCID mice
injected with mitogenic anti-CD3 MoAbs (data not shown) or in
immunocompetent mice treated with nonmitogenic forms of anti-CD3
(Fig 7C).
It has been recently suggested that DC undergo 3 discrete sequential
stages of differentiation, identified as immature, mature, and
apoptotic.38 In particular, in vitro studies have suggested that functional maturation of DC into potent APC is ended by apoptotic cell death and that no reversion to the immature phenotype was observed. We have recently confirmed this hypothesis in vivo and shown
that, after LPS-induced maturation, DC undergo apoptotic cell death in
situ.39 Therefore, loss of splenic DC observed 48 hours
after anti-CD3 administration probably reflects the apoptotic cell
death that follows DC maturation in vivo.
In keeping with the phenotypical analysis, the functional studies
presented in this study indicate that anti-CD3 treatment induces a
selective APC deficiency characterized by the loss of cells able to
provide costimulation to naive T cells and Th1 clones and to secrete
the proinflammatory cytokine IL-12. The ability of spleen accessory
cells to capture and efficiently present Ag to a Th2 clone is not
affected by anti-CD3 treatment (Fig 5). Accordingly, we have
recently demonstrated that administration of anti-CD3 MoAbs
selectively downregulates the secretion of Th1-type cytokines (IL-2 and
IFN-) upon in vivo restimulation, whereas the production of Th2-type
cytokines was only marginally affected (IL-4) or upregulated
(IL-10).40 Thus, both the relative resistance of Th2-like T
cells to anergy induction in vitro41 and in
vivo40,42 and the selective APC deficiency induced in vivo
by anti-CD3 MoAbs may explain why, despite intense
immunosuppression, mitogenic anti-CD3 antibodies often induce a
humoral response comprising both anti-xenotypic and anti-idiotypic
antibodies that neutralize the drug and abrogate its immunosuppressive
activity.43,44
Recently, nonmitogenic MoAbs have been produced and shown in animal
models to retain immunosuppressive properties while minimizing toxicity.22,45 However, nonmitogenic antibodies to CD3
displayed reduced immunomodulatory potential both in vivo and in vitro
when compared with mitogenic forms.45 Indeed, as
demonstrated in the present study, only mitogenic anti-CD3 MoAbs
were able to achieve immunosuppression after a single, moderate dose
injection regimen.45 Moreover, and of potential relevance
for this study, nonmitogenic anti-CD3 MoAbs forms were found unable
to suppress generation of cytotoxic T cells in
vitro36,45,46 or to cause long-term anergy in the
CD8+ T-cell subset.47 Based on the
well-described capacity of DC2 and IL-1248,49
to potentiate cytotoxic T lymphocytes (CTL) development,
it is tempting to speculate that downregulation of DC numbers
contributes to the long-term efficacy of mitogenic anti-CD3-based
therapy. Finally, although the precise contribution of APC cell
dysfunction to the anti-CD3-induced immunosuppression in vivo is
difficult to establish, numerous studies have suggested that inhibition
of accessory cell functions through disruption of the CD28 and CD40L
signaling has strong therapeutic potential.50,51 The
observation that both cyclosporine52 and
steroids4 affect DC activity and/or survival further
suggests that the clinical efficacy of several immunosuppressive
therapies may in part be due to their ability to affect accessory cell
functions in vivo.
Maturation and migration of DC from the periphery into T-cell areas
have been recognized as important steps leading to T-cell sensitization
and therefore initiation of an Ag-specific immune response.1,2 The observation that these phenotypic and
migratory changes are induced in vivo by anti-CD3 Abs can be viewed
as serious constraint to the use of mitogenic anti-TCR complex MoAbs as
immunosuppressive agents. However, as demonstrated by early studies, a
strong correlation between interstitial DC depletion and delayed graft
rejection has been established,10 suggesting that DC loss
from peripheral organs is beneficial to allograft survival. The
findings reported herein suggest therefore the possibility of
anticipating the first anti-CD3 administration 24 to 48 hours before
organ transplantation to avoid DC maturation in the presence of alloAgs.
Finally, inhibition of APC functions may contribute to the long-term
efficacy of anti-CD3-based therapies or even play a role in the
induction of immune tolerance/suppression after discontinuation of
antibody therapy. Indeed, DC loss/redistribution may favor stimulation
of alloreactive T cells by accessory cells presenting alloAgs in an
inadequate, costimulatory-deficient fashion, leading to T-cell
unresponsiveness. Further studies are thus required to better evaluate
the importance of APC downmodulation in the achievement of in vivo immunosuppression.
| |
ACKNOWLEDGMENT |
The authors thank P. Veirman, G. Dewasme, M. Swanepoel, and F. Tielemans for technical assistance and thank H. Bazin (Université Catholique de Louvain), J.A. Bluestone (University of Chicago, Chicago,
IL), G. Klaus (National Institute for Medical Research, London, UK), P. Marrack (University of Colorado Health Sciences Center, Denver, CO), A. O'Garra (DNAX, Palo Alto, CA), R.H. Schwartz (National Institutes of
Health, Bethesda, MD), and G. Trinchieri (Wistar Institute,
Philadelphia, PA) for providing reagents used in this study. The
scientific responsibility is assumed by the authors.
| |
FOOTNOTES |
Submitted November 30, 1998; accepted July 26, 1999.
This work was founded by the Belgian Program in Interuniversity Poles
of Attraction initiated by the Belgian State, Prime Minister's Office,
Science Policy Programming and by a Research Concerted Action of the
Communauté Française de Belgique. Additional financial
support was given by the Fonds Emile Defay. M.M. is a Research
Associate from the FNRS (Belgium).
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 Oberdan Leo, PhD, Laboratoire
de Physiologie Animale, Université Libre de Bruxelles, Rue Pr
Jeneer et Brachet 12, 6041 Gosselies, Belgium.
| |
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