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Transplant Research Division, The Toronto Hospital, Toronto, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) production and increased IL-4 (IL-10)
production. Similarly, in vivo infusion of OX-2:Fc promotes increased
allo- and xenograft (both skin and renal grafts) survival and decreases
the Ab response to sheep erythrocytes. Our data suggest this molecule
might have clinical importance in allo- and
xenotransplantation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We hypothesized that OX-2 might be involved in the delivery of negative "tolerizing" signals to T cells simultaneously encountering Ag. This is in contrast to conventional costimulator molecules (such as CD40, CD80, and CD86) that deliver "costimulatory" signals. One prediction of such a model is that the soluble form of OX-2 (an immunoadhesin) might itself then act as a general immunosuppressant when cells are triggered with Ag. This approach has been used successfully to show that an immunoadhesin (CTLA4Ig), blocking binding of CD80,CD86 with their respective ligands, can prolong survival of vascularized grafts in a number of different models (7). We report data below on the preparation of an immunoadhesin in which the extracellular domains of OX-2 were linked to the Fc region of murine IgG2a (OX-2:Fc) and expressed in a eukaryotic expression system (baculovirus). The OX-2:Fc-expressed molecule was indeed able to promote survival of vascularized and nonvascularized allografts and xenografts in mice and to alter cytokine production in treated recipients. Inhibition of sheep erythrocyte Ab responses was also seen in OX-2:Fc-treated mice. Our data suggest this reagent may have potential in clinical situations where controlled immune suppression is required.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male C3H/HEJ and C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed five per cage and allowed food and water ad libitum. All mice were used at 8–12 wk of age. Male Lewis (LEW) rats (35–40 g) were purchased from Sprague Dawley for use as xenogeneic skin or kidney donors.
mAbs
The following mAbs, all obtained from PharMingen (San Diego, CA)
unless stated otherwise, were used: anti-IL-2 (JES6-1A12;
biotinylated, JES6-5H4); anti-IL-4 (11B11, American Type Culture
Collection (ATCC, Manassas, VA); biotinylated, BVD6-24G2);
anti-IFN- (R4-6A2, ATCC; biotinylated XMG1.2); and
anti-IL-10 (JES5-2A5; biotinylated, SXC-1). Anti-mouse and
anti-rat OX-2 (3B6 and 6A3, respectively) were prepared as
described elsewhere (8). Anti-B7-1 and anti-B7-2 were
obtained from Dr. G. Powers (Roche Pharmaceuticals, Nutley, NJ). All
noncommercial mAbs were grown as ascites in BALB/C nu/nu mice and
purified by ammonium sulfate precipitation and resuspension in PBS
before use. In control groups, a crude preparation of normal rat or
mouse Ig (30% saturated ammonium sulfate preparation) was used. When
injected i.v., 100 µg of Ig was used for each recipient, as described
in individual studies; in vitro assays used 5 µg/ml of the Ig
preparations. These concentrations of mAbs tested in the lymphokine
assays described in vitro (see below) produced relatively equivalent
amounts of inhibition (>10 units of lymphokine neutralized).
Anti-thy 1.2, L3T4, anti-Ly2.2, rabbit complement, and streptavidin HRP were obtained from Cedarlane Labs, Hornby, Ontario.
Preparation of cells
Single cell spleen suspensions were prepared aseptically from
individual mice in each experiment. After centrifugation, cells were
resuspended in 
-MEM supplemented with 2-ME and 10% FCS
(F10).
Portal vein immunization
Portal vein immunization was performed essentially as described earlier (9). All animals were anesthetized with nembutal. Donor-specific (LEW rat or C57BL/6 mouse) bone marrow cells were injected in 0.1 ml through a superior mesenteric vein using a 30-gauge needle. After injection, the needle was rapidly withdrawn and hemostasis was secured without hematoma formation by gentle pressure using a 2-mm3 gel-foam. Complications were seen in less than 10% of mice, and these were excluded from analysis because of hemorrhage postinjection.
Renal transplantation
This procedure was performed essentially as described elsewhere (10). All recipients received i.m. injection with cefotetan (30 mg/kg) on the day of transplantation and for 2 succeeding days. All animals received i.m. cyclosporin A (15 mg/kg) daily for the first 3 days posttransplantation. The remaining host kidney was removed 2 days after transplantation, unless otherwise indicated. Pretreatment of recipients with donor-specific pv immunization with bone marrow cells, or with immunoadhesin, was as described in individual studies.
All xenogeneic transplants were performed in mice that were pretreated one day earlier by i.v. infusion of 100 µl anti-asialo-GM1 (to deplete NK cells; Ref. 11) and 250 µl of rabbit anti-mouse lymphocyte serum. In addition, these mice received 30 x 106 LEW rat bone marrow cells (treated with anti-rat lymphocyte serum and complement) via the pv on the day preceding transplantation. All other treatment was as described for renal allografts.
Ab-forming assays
Mice were immunized i.v. in 0.5 ml PBS with 4 x 108 washed sheep red blood cells (SRBC), (Cedarlane). Spleen cell suspensions were prepared 7 days following immunization, and Ab-forming-cells (AFC) to SRBC were enumerated using the Cunningham modification of the Jerne plaque assay, as described elsewhere (12). In some cases, mice also received i.v. infusions of OX-2:Fc (or control mouse Ig). See individual experiments for details.
Cytotoxicity and cytokine assays
In cultures used to assess induction of cytotoxicity or cytokine
production, C3H responder cells were stimulated with equal numbers of
mitomycin C-treated (45 min at 37°C) spleen stimulator cells in
triplicate in F10. Supernatants were pooled at 48 h from
replicate wells and assayed in triplicate in ELISA assays for
lymphokine production as described below. In our hands, no reproducible
differences in cytokine levels have been detected from cultures between
36 and 50 h of culture. In some experiments, the culture wells
later (at 72 h) received 1 µCi/well of
[3H]TdR, and proliferation was measured by
harvesting the contents of the well 14 h later and counting in a
well-type beta counter. When cytotoxicity was measured, cells were
harvested at 5 days and pooled from replicate wells, counted, and
cultured at various E:T ratios with 51Cr 72
h spleen Con A blasts as target cells. Supernatants were sampled at
4 h for assessment of specific cytotoxicity.
IL-2, IL-4, IFN-, and IL-10 were assayed using ELISA assays. For
IFN-, the assay used flat-bottom 96-well Nunc plates (Life
Technologies, Grand Island, NY) coated with 100 ng/ml R4-6A2. Varying
volumes of supernatant were bound in triplicate at 4°C and washed
3x, and biotinylated anti-IFN- (XMG1.2) was added. After
washing, plates were incubated with streptavidin-HRP (Cedarlane) and
developed with appropriate substrate, and OD405
was determined using an ELISA plate reader. Recombinant IFN- for
standardization was obtained from PharMingen. IL-10 was assayed using a
similar ELISA system, with JES5-2A5 as the capture Ab, and biotinylated
SXC-1 as developing Ab. Recombinant IL-10 for standardization of this
assay was obtained from Pepro Tech (Rocky Hill, NJ). Each assay
reliably detected cytokine levels in the range 0.01 to 0.1 ng/ml. ELISA
assays for IL-2 and IL-4 used JES6-1A12 and 11B11 as capture Abs, with
biotinylated JES6-5H4 or BVD6-24G2 as developing Abs. Sensitivity of
detection was 20 pg/ml for each cytokine.
Production and large scale expression of an OX-2:Fc fusion protein in a baculovirus expression system
The Baculovirus Expression Vector System (BEVS), one of the most powerful and versatile eukaryotic expression systems available, was chosen for expression of a construct of murine OX-2 linked to a murine IgG2a-Fc region (7).
The V-C domains of OX-2 were first amplified from a mouse cDNA preparation encoding full-length OX-2 (see Ref. 1) using the following primer pairs: sense, 5'-CCGTCGACCAAGTGGAAGTG-3'; antisense, 5'-ACGGATCCTTTGTCCAGACTCTGCTT-3'.
A vector encoding the cDNA for a previously described murine IgG2a Fc was obtained from Dr. Terry Strom (Harvard University, Boston, MA) (7) and amplified using the following primer pairs: sense, 5'-ACGGATCCTGCCCAGGGATTGTGGTT-3'; antisense, 5'-AATCTAGACCCAAGGCAGTG-3'.
These two fragments were digested with BamHI and fused using T4 ligase at the corresponding BamHI sites. The product was purified by gel electrophoresis, digested with SalI and XbaI, and cloned into a pBK vector after digestion of the vector with the same enzymes. After sequencing to ensure in-frame ligation, the OX-2:Fc fragment in the pBK vector was amplified using the following primer pairs to introduce SmaI and XbaI sites for cloning into a pAcGP67A transfer vector for use in a baculovirus system: sense, 5'-GGGCCCGGGGTCGACCAAGTGGTG-3'; antisense, 5'TCTAGATCATTTACCCGGAGT-3'.
Spodoptera frugiperda (Sf9) cells for transfection were grown in IPL-41 medium (Life Technologies) as a monolayer culture at 27°C without CO2 supplement. BaculoGold linearized wild-type virus DNA (0.5 µg) was mixed with 5 µg recombinant transfer vector containing the OX-2:Fc fragment and used for transfection of Sf9 cells. After 4–5 days of infection, the supernatant was collected from this cotransfection plate, virus was grown to high titer in fresh cultures, and Sf9 was infected at a high multiplicity of infection. Supernatant containing OX-2:Fc fusion protein was eluted from a protein G Sepharose 4 Fast Flow gel column (Pharmacia Biotech, Toronto, Canada) with 0.1 M glycine buffer (pH 3.0) and fractionated on SDS-PAGE, and samples containing the OX-2:Fc fusion protein were combined and dialyzed against 1x PBS without Ca2+. A typical yield of OX-2:Fc fusion protein (nonoptimized) from supernatant collected after 4 days of infection was of the order of 1 µg/ml. Characterization of OX-2:Fc in the respective samples prepared relied on an ELISA in which rat anti-murine OX-2 (3B6; Ref. 8) was bound to the wells of microtiter plate (incubation overnight at a concentration of 100 ng/well), followed by blocking with Tween buffer, incubation with test reagent, and development using a secondary alkaline-phosphatase-coupled goat anti-mouse Fc (Cedarlane) and substrate.
Statistical analysis
Comparison of skin graft survival in different groups relied on a nonparametric, Mann-Whitney U test. For comparison of cytokine production in different groups assayed in vitro, initial ANOVAs were performed, followed by pair-wise comparison of relevant groups using a Student t test (see legends to Figures and Tables).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mixed lymphocyte cultures were set up in triplicate using equal numbers of responder C3H mouse or LEW rat spleen cells and mitomycin C-treated allogeneic stimulator cells (see footnotes to Tables I and II). Some cultures received titrated amounts of OX-2:Fc or normal mouse Ig. Supernatants were harvested and tested at 48 h for cytokine production. Cultures received 1 µCi/well of [3H]TdR at 72 h and were harvested 14 h later to assess proliferation. In other replicate cultures, cells were pooled at day 5 and assayed for CTL using 51Cr-labeled, 72 h Con A spleen cell targets. Data pooled from three studies of this type are shown in Tables I and II.
These data demonstrate that OX-2:Fc at a concentration of 50 ng/ml
inhibits induction of CTL, proliferation, and IL-2 (or IFN-)
production in responder allostimulated cells of either mouse or rat
origin. These results are in keeping with our earlier data using mAb to
rat OX-2 to detect the murine molecule, which had suggested a
cross-reactivity between murine and rat OX-2 (4).
Role of OX-2:Fc in promotion of graft survival in vivo
Spleen adherent cells taken from C3H mice receiving donor-specific pv immunization with bone marrow cells showed increased numbers of OX-2+ cells (4, 8). Under these conditions, allogeneic graft survival was increased (13), while administration of anti-OX-2 mAbs could reverse this increased graft survival (4). To assess whether passive administration of soluble OX-2:Fc would also produce increased graft survival, we performed the following study.
Groups of six per group C3H/HEJ mice received C57BL/6 skin allografts
or renal transplants as described in Materials and Methods.
Control groups received i.v. infusions of saline alone at 2-day
intervals, or crude mouse Ig (10 µg/mouse). An experimental group
received 10 µg/mouse OX-2:Fc i.v. at daily intervals. All groups
received injections for 10 days. Graft survival was followed daily with
data pooled from two such studies shown in Figs. 1
and
2.
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), mice received daily i.v.
infusion of OX-2:Fc, followed by anti-OX-2 mAb (3B6; see Ref.
8) at 2-day intervals from the time of transplantation for
12 days. A further control group in this study received donor-specific
pv immunization followed by renal allotransplantation. In this study
(Fig. 3), treatment with anti-OX-2 Ab abolished both the increased
survival following pv immunization, as described elsewhere
(4), and the increased survival following infusion of
OX-2:Fc.
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and 3
. Data pooled from six
mice/group are shown in Fig. 4. Once
again, infusion of OX-2:Fc resulted in significant prolongation of
xenografts in this combination, suggesting that OX-2:Fc delivers an
immunoregulatory signal(s) effective in preventing both xeno- and
allograft rejection.
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Increased survival following pv immunization is associated not
only with elevated expression of OX-2 but also with altered cytokine
production (increased IL-4 and IL-10, and decreased IL-2 and IFN-)
in stimulated cells from grafted mice (4). To assess the
potential changes in cytokine production in mice receiving renal grafts
as well as OX-2:Fc, we performed the following study.
Groups of three per group C3H mice received C57BL/6 renal allografts as
above, along with mouse Ig or OX-2:Fc daily (for 10 days). Thereafter,
all mice/group were sacrificed (day 11), spleen cell suspensions were
prepared, and cells were stimulated in vitro with mitomycin C-treated
C57BL/6 spleen cells. Cytokines were measured in the supernatant of
these cultures as before. Data pooled from two such studies (six
individual spleen cell preparations tested/group) are shown in Table III. It is clear that infusion of OX-2:Fc
not only increased graft survival (Figs. 1 and 2) but also altered
polarization in cytokine production away from type 1 cytokines (IL-2,
IFN-) toward type 2 cytokines (IL-4 and IL-10), in a similar fashion
to data we have reported earlier following pv immunization
(4).
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We postulated that engagement of OX-2 with its ligand (OX-2L)
leads to a physiological immunosuppression of immune responses
(6). Accordingly, we next asked whether mice immunized
with SRBC to produce anti-SRBC AFC would also be inhibited (for AFC
production) by in vivo infusion of OX-2:Fc. As before, mice received
i.v. infusion of 10 µg/mouse OX-2:Fc or control mouse Ig beginning on
the day of SRBC immunization (with 4 x 108
SRBC i.v.). Spleen cells were harvested at 7 days, and IgM and IgG AFC
were assayed using plaque assays described in Materials and
Methods. Data in Table IV show
pooled results from two studies (a total of 12 mice/group). Infusion of
OX-2:Fc led to a 2.5- to 3-fold inhibition of the anti-SRBC AFC
response in this study, consistent with a generalized immunosuppressive
effect induced by this molecule.
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In the next series of studies, we asked whether the decreased
response to allogeneic stimulation, as assessed by inhibition of CTL or
IL-2 cytokine production, required the continual presence of OX-2:Fc or
only transient incubation in the presence of OX-2:Fc. In this study,
bulk cultures of responder C3H and C57BL/6 stimulator cells were
incubated for 4, 24, 48, or 96 h with OX-2:Fc (50 ng/ml), before
washing and replating in large 24-well culture plates. In this study,
supernatants were harvested at 60 h from all cultures for assay of
IL-2 production, while CTL activity was measured at 6 days of culture.
In other experiments, OX-2:Fc was added only after 24, 48, or 96 h
of culture, and cytokine production/CTL activity measured. Data pooled
from three studies of each type are shown in Table V.
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). However, delaying addition until beyond 48 h of culture
resulted in a failure to inhibit the induction of CTL/production of
IL-2 (upper half of Table V). These data suggest that OX-2
delivers its immunoregulatory signal(s) during the first phase of T
cell activation and that it is required throughout this phase for
optimal inhibition. Inhibition of allostimulation in vitro by combined treatment with OX-2:Fc and anti-B7 Ab
A large body of data suggests that T cell activation itself
requires costimulation following engagement of, among others, the
costimulator molecules B7-1 and B7-2 expressed on APC (14, 15). We thus investigated whether simultaneous blockade of
costimulation (by anti-B7 Ab) along with provision of a tolerizing
signal (using OX-2:Fc) would lead to more profound allosuppression. In
the first of such studies in vitro, responder spleen cells were
incubated with spleen stimulator cells in triplicate in the presence of
OX-2:Fc, F(ab')2 fragments of anti-B7-1 or
anti-B7-2, or a combination of these reagents. Supernatants were
harvested for assay of cytokine production, and CTL were measured at
day 5. Data pooled from three such studies are shown in Table VI.
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Graft survival is enhanced by infusion of OX-2:Fc along with anti-B7-1 and anti-B7-2 Abs
In a final study we asked whether the synergism in inhibition of
allostimulation seen in Table VI was mimicked in vivo in a renal
allograft model. Groups of C3H mice received renal allografts (see
Materials and Methods) along with i.v. infusion of OX-2:Fc
alone (10 µg/mouse daily x five doses), a mixture of
F(ab')2 anti-B7-1 and anti-B7-2 (100
µg/mouse daily x five doses), or a combination of OX-2:Fc and
the mAbs. A control group received saline injections only. Graft/animal
survival is shown for the individual groups in Fig. 5.
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. The mixture of
anti-B7-1 and anti-B7-2 also produced some increase in renal
graft survival in this model. However, the most striking effect seen
was in the group receiving combined treatment with mAbs and OX-2:Fc,
where a profound increase in survival beyond that seen with any
treatment alone was observed (p < 0.05,
Mann-Whitney U test). Note that in this experiment the
cumulative dose of OX-2:Fc given was less (50 µg) that that given in
the study shown in Fig. 2, further emphasizing the synergy in
inhibition of graft rejection brought about by combinations of OX-2:Fc
and anti-B7 mAbs. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and conversely IL-4, IL-10, and/or IL-13
regulate in a reciprocal fashion the activation of type 1 and type 2
cytokine-producing cells (17). It has recently been found
that this in turn is reflected in the differential expression on the
surface of those T cells of two distinct molecules, each belonging to
the IL-1R family, namely IL-18R (on Th1 cells) and ST2L (on Th2 cells)
(20).
Using a PCR-based subtraction hybridization method we reported evidence
that implicated a novel molecule, OX-2, in the regulation of graft
rejection following pv immunization (4). Our preliminary
data used an Ab directed to the rat OX-2 molecule and thus depended
upon an unexplained cross-reactivity between mouse and rat OX-2
(4). However, more recently we have confirmed that a mAb
to murine OX-2, 3B6, which detects a molecule on the surface of DCs,
plays a role in regulating cytokine production after allostimulation
(8). Borriello et al. also recently reported that OX-2
expression was not a costimulator for induction of IL-2 and IFN-
synthesis (3). We hypothesized, based on these data, that
engagement of OX-2 on the surface of DCs would be a negative signal for
type 1 cytokine production (1, 4).
The data in this manuscript are from results of experiments designed to
test this hypothesis, using an immunoadhesin in which the extracellular
domains of the OX-2 molecule were linked to the Fc region of murine
IgG2a. The gene encoding the latter had in turn been modified to
eliminate the Fcr binding region and the
complement binding region (7). After expression in a
baculovirus system, we could show that addition of OX-2:Fc to
allostimulated cells in vitro led to significant inhibition of
proliferation, CTL induction, and type 1 cytokine (IL-2, IFN-)
production (see Tables I and II). Furthermore, this inhibition was seen
using both murine and rat responder cells (Table II), in keeping with
our hypothesis that there was cross-reactivity between the murine and
rat molecules (in this case presumably at the level of their natural
binding ligands). Infusion of this reagent into animals receiving renal
or skin allografts produced significantly increased graft survival,
parallel to the levels seen after pv immunization (see Figs. 1 and 2),
an effect that was itself abolished after simultaneous infusion of mAb
to OX-2 (3B6; see Fig. 3). Furthermore, cells taken from grafted mice
receiving OX-2:Fc infusions showed a reversal in polarization of
cytokine production after restimulation in vitro, with an increase in
IL-4 and IL-10 production, and concomitant decreases in IL-2 and
IFN- production (Table III).
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). This effect was more pronounced in terms
of IgG AFC than IgM AFC, which may reflect the greater T dependence of
the former and a mechanism of action of OX-2:Fc that encompasses a
functional inhibition of Th cell function. Similarly, infusion of
OX-2:Fc produced significantly increased survival of renal xenografts
in C3H mice receiving LEW grafts along with pv immunization of LEW bone
marrow cells (Fig. 4). As assayed by kinetic studies using an
allogeneic MLR reaction in vitro, delivery of this immunoregulatory
signal occurs within the first 24–48 h following Ag encounter,
although, once cells have been activated, addition of exogenous OX-2:Fc
did not produce as significant a decrease in the resulting immune
response (Table V). We proposed initially that OX-2 may represent a (member of a family of) molecule(s) that, in contrast to the B7 family, provides not costimulation but instead delivers an immunoregulatory signal to activated T cells (4, 6). Based on the lack of immunoreceptor tyrosine-based inhibitory motif (ITIM)/immunoreceptor tyrosine-based activation motif (ITAM) signaling motifs in the cytoplasmic domain of OX-2 (1), we suggested that the natural ligand of OX-2 might deliver this signal, although the cell(s) expressing this putative ligand has not been definitively identified. However, using a similar approach with a solubilized form of OX-2, Preston et al. suggested that macrophages may express a counterreceptor for OX-2 (21). There are no data, to our knowledge, indicating binding of OX-2 directly to T cells themselves. If indeed OX-2 modifies T cell signaling indirectly, by acting on macrophages (or APC in general), one mechanism for immunosuppression might involve OX-2-mediated regulation of expression of the costimulatory molecules B7-1 (B7-2) and/or CD40. We recently found that OX-2+ bone marrow-derived DC could inhibit the allostimulation induced by other B7-1+ (and B7-2+) DC (6). However, even these data do not address the issue of whether the inhibition seen reflects a direct action on T cells or an indirect one mediated by an altered stimulatory function of DC.
Based on a model in which OX-2 delivers an immunoregulatory signal
independent of any effect mediated by B7-1 (or B7-2) expression, we
hypothesized that there would be a synergy for immunosuppression if
blockade of costimulation was coupled with signaling via OX-2. The data
in Table VI and Fig. 5 provide some support for such a model. Profound
immunosuppression both in vitro (as measured by proliferation and CTL
and cytokine production) and in vivo (renal allograft survival) was
seen in a situation where encounter with alloantigen occurred in the
presence of both OX-2:Fc and F(ab')2 anti-B7-1 and anti-B7-2.
We believe these data are best explained by considering that the two
strategies (blockade of costimulation and presentation of OX-2
immunoadhesin) act independently to reduce or prevent allostimulation
in these models.
Taken together our data support a hypothesis that increased expression of OX-2 on the surface of DC following pv immunization provides an important negative signal that results in decreased graft rejection and an alteration in the polarization of cytokine production in such animals. More generally, it seems that engagement of its natural ligand (which we have referred to as OX-2L), results in a profound suppression of responses to nominal Ag, alloantigen, and xenoantigen, which may have relevance in a number of human diseases, including infection, autoimmunity, transplantation, and malignancy.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Reginald Gorczynski, CCRW 2-855, The Toronto Hospital, 200 Elizabeth Street, Toronto, M5G2C4, Ontario, Canada. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cells; OX-2:Fc, immunoadhesin linking extracellular region of OX-2 to murine IgG2a Fc; pv, portal venous immunization; AFC, Ab-forming cell; LEW, Lewis; BN, Brown Norway.
Received for publication January 19, 1999. Accepted for publication May 11, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 D.-X. Chen, H. He, and R. M. Gorczynski Synthetic peptides from the N-terminal regions of CD200 and CD200R1 modulate immunosuppressive and anti-inflammatory effects of CD200-CD200R1 interaction Int. Immunol., March 1, 2005; 17(3): 289 - 296. [Abstract] [Full Text] [PDF]
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 S. Voigt, G. R. Sandford, G. S. Hayward, and W. H. Burns The English strain of rat cytomegalovirus (CMV) contains a novel captured CD200 (vOX2) gene and a spliced CC chemokine upstream from the major immediate-early region: further evidence for a separate evolutionary lineage from that of rat CMV Maastricht J. Gen. Virol., February 1, 2005; 86(2): 263 - 274. [Abstract] [Full Text] [PDF]
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S. Zhang, H. Cherwinski, J. D. Sedgwick, and J. H. Phillips Molecular Mechanisms of CD200 Inhibition of Mast Cell Activation J. Immunol., December 1, 2004; 173(11): 6786 - 6793. [Abstract] [Full Text] [PDF]
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F. Fallarino, C. Asselin-Paturel, C. Vacca, R. Bianchi, S. Gizzi, M. C. Fioretti, G. Trinchieri, U. Grohmann, and P. Puccetti Murine Plasmacytoid Dendritic Cells Initiate the Immunosuppressive Pathway of Tryptophan Catabolism in Response to CD200 Receptor Engagement J. Immunol., September 15, 2004; 173(6): 3748 - 3754. [Abstract] [Full Text] [PDF]
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M. Foster-Cuevas, G. J. Wright, M. J. Puklavec, M. H. Brown, and A. N. Barclay Human Herpesvirus 8 K14 Protein Mimics CD200 in Down-Regulating Macrophage Activation through CD200 Receptor J. Virol., July 15, 2004; 78(14): 7667 - 7676. [Abstract] [Full Text] [PDF]
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R. Gorczynski, Z. Chen, Y. Kai, L. Lee, S. Wong, and P. A. Marsden CD200 Is a Ligand for All Members of the CD200R Family of Immunoregulatory Molecules J. Immunol., June 15, 2004; 172(12): 7744 - 7749. [Abstract] [Full Text] [PDF]
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 M. D. Rosenblum, E. Olasz, J. E. Woodliff, B. D. Johnson, M. C. Konkol, K. A. Gerber, R. J. Orentas, G. Sandford, and R. L. Truitt CD200 is a novel p53-target gene involved in apoptosis-associated immune tolerance Blood, April 1, 2004; 103(7): 2691 - 2698. [Abstract] [Full Text] [PDF]
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G. J. Wright, H. Cherwinski, M. Foster-Cuevas, G. Brooke, M. J. Puklavec, M. Bigler, Y. Song, M. Jenmalm, D. Gorman, T. McClanahan, et al. Characterization of the CD200 Receptor Family in Mice and Humans and Their Interactions with CD200 J. Immunol., September 15, 2003; 171(6): 3034 - 3046. [Abstract] [Full Text] [PDF]
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Y.-H. Chung, R. E. Means, J.-K. Choi, B.-S. Lee, and J. U. Jung Kaposi's Sarcoma-Associated Herpesvirus OX2 Glycoprotein Activates Myeloid-Lineage Cells To Induce Inflammatory Cytokine Production J. Virol., April 16, 2002; 76(10): 4688 - 4698. [Abstract] [Full Text] [PDF]
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 D. A. Clark, J.-W. Ding, G. Yu, G. A. Levy, and R. M. Gorczynski Fgl2 prothrombinase expression in mouse trophoblast and decidua triggers abortion but may be countered by OX-2 Mol. Hum. Reprod., February 1, 2001; 7(2): 185 - 194. [Abstract] [Full Text] [PDF]
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 A. D. Dick, C. Broderick, J. V. Forrester, and G. J. Wright Distribution of OX2 Antigen and OX2 Receptor within Retina Invest. Ophthalmol. Vis. Sci., January 1, 2001; 42(1): 170 - 176. [Abstract] [Full Text]
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R. M. Gorczynski, K. Yu, and D. Clark Receptor Engagement on Cells Expressing a Ligand for the Tolerance-Inducing Molecule OX2 Induces an Immunoregulatory Population That Inhibits Alloreactivity In Vitro and In Vivo J. Immunol., November 1, 2000; 165(9): 4854 - 4860. [Abstract] [Full Text] [PDF]
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R. M. Gorczynski, Z. Chen, D. A. Clark, J. Hu, G. Yu, X. Li, W. Tsang, and S. Hadidi Regulation of Gene Expression of Murine MD-1 Regulates Subsequent T Cell Activation and Cytokine Production J. Immunol., August 15, 2000; 165(4): 1925 - 1932. [Abstract] [Full Text] [PDF]
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W.-P. Min, R. Gorczynski, X.-Y. Huang, M. Kushida, P. Kim, M. Obataki, J. Lei, R. M. Suri, and M. S. Cattral Dendritic Cells Genetically Engineered to Express Fas Ligand Induce Donor-Specific Hyporesponsiveness and Prolong Allograft Survival J. Immunol., January 1, 2000; 164(1): 161 - 167. [Abstract] [Full Text] [PDF]
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) received no asialo-GM1 or anti-mouse lymphocyte
serum.