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Transplant Research Division, Toronto Hospital, Toronto, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and TGFß). We found that fresh C57BL/6 splenic
DC induced IL-2, not IL-4, production. Cells from the sources indicated
inhibited IL-2 and IFN- production and promoted IL-4 and TGFß
production. Inhibition was associated with increased expression of OX-2
on these cells, as defined by semiquantitative PCR and FACS analysis.
By size fractionation, cells expressing OX-2 were a subpopulation of
NLDC145+ cells. Our data imply a role for cells expressing
OX-2 in the regulation of induction of cytokine production by
conventional allostimulatory DC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) with selective sparing
of type 2 cytokine-producing (IL-4, IL-10, and TGFß) cells. A number
of laboratories, including our own, have suggested that Ag-specific
preimmunization via the portal vein
(pv)3 is one protocol that
produces increased graft survival and preferential type 2 cytokine
production in animals (1, 2, 3, 4). We have hypothesized that the
mechanism(s) operating to induce tolerance after pv immunization
reflect similar events to those occurring after oral immunization
(5, 6, 7). How such polarization in CD4+ cytokine-producing cells develops in vivo (8, 9) remains an enigma. Stimulation of T cells depends not only on the cytokine milieu in which Ag recognition occurs, but also on the nature of the APC and the level of expression of various costimulator molecules on those APC. Using a model in which we infused long-term culture-derived donor dendritic cells (DC) as tolerogen, we have also reported that persistent graft survival was dependent upon the existence of functional donor Ag in association with host DC (10). Furthermore, we found that stimulation of spleen responder cells with allogeneic DC in the presence of syngeneic (with the responder cell source) hepatic nonparenchymal mononuclear cell (NPC) preparation led to inhibition of type 1 cytokine production, development of a cell population able to inhibit further stimulation (to type 1 cytokine production) in vitro by fresh DC, and prolonged graft survival in vivo (11). These data were consistent with the notion that induction of suppression of type 1 cytokine production in vitro, and increased graft survival in vivo, is associated with the existence of a "facilitator" cell in the NPC preparation that somehow regulates the functional outcome of allostimulation.
More recent studies have attempted to explore whether expression of novel molecules on APC following pv immunization might be associated with the increased renal graft survival seen in this model. We reported that expression of OX-2, a previously described DC surface Ag (12, 13, 14), was increased in NLDC145+ cells taken from pv-immunized mice and that anti-OX-2 Ab abolished the increased graft survival following pv immunization (15). Interestingly, in a separate study, Borriello et al. also reported that OX-2 provided a costimulatory signal for activated murine T cells, but was unable to increase IL-2 production (16). Our data suggest that OX-2 in fact provides a costimulator signal for preferential IL-4 (TGFß) production (15). Taken with the observations using syngeneic NPC, we have thus asked whether the "facilitator" cell that alters the outcome of stimulation of spleen responder cells with allogeneic DC is itself an OX-2-expressing cell population. The experiments described below are derived from a series of studies designed to test this hypothesis. In addition, we have made use of a recently described growth factor, Flt3 ligand (Flt3L) (17, 18, 19), which is known to increase the proliferation of hematopoietic progenitor cells, to show that mice infused with human Flt3L contain increased numbers of OX-2+ cells in the liver NPC pool and that these latter cells alone, infused i.v. into naive recipients, can induce prolonged survival of renal allografts.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male and female C3H/HEJ and B10.BR (H-2k/k), B10.D2 (H-2d/d), and C57BL/6 (H-2b/b) mice were purchased from the 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.
Monoclonal Abs
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, Manassas, VA; biotinylated, BVD6-24G2); anti-IFN-
(R4–6A2, American Type Culture Collection; biotinylated XMG1.2);
anti-IL-10 (JES5-2A5; biotinylated SXC-1); and phycoerythrin
anti-B7-1/B7-2 (Cedarlane Labs, Hornby, Ontario, Canada).
Rat anti-mouse OX-2 mAbs were prepared by Immuno-Precise Antibodies (Victoria, British Columbia, Canada) following immunization of rats with a crude membrane extract of LPS-stimulated murine DC, followed by fusion with a nonsecreting rat myeloma parent cell line (YB2/3H]l.P2.G11.16Ag.20). Hybridoma supernatants were screened in ELISA using plates precoated with a 40- to 45-kDa preparation of DC extracts run on Western gels (12, 13). Positive clones were rescreened using FACS analysis of Chinese hamster ovary (CHO) cells transduced with a cDNA clone encoding full-length murine OX-2 (14). FITC-conjugated F(ab')2 rabbit anti-rat IgG (noncross-reactive with mouse IgG) from Serotec (Mississauga, Canada) was used as secondary Ab. The mAb selected for further analysis (M3B5) was grown in bulk in a CellMax system (Cellco, Germantown, MD). A crude preparation of rat Ig (30% saturated ammonium sulfate preparation) was used as a control Ig.
In tissue culture assays, where anti-cytokine mAbs were used to confirm the specificity of the assay used, 10 µg/ml of the relevant mAbs was found to neutralize up to 5.0 ng/ml of the cytokine tested.
NLDC145 (anti-mouse DC) was also obtained from Serotec. Recombinant mouse IL-4 was a kind gift from Dr. L. Yang (The Toronto Hospital); mouse recombinant granulocyte-macrophage-CSF was purchased from PharMingen. Recombinant human Flt3L (derived from CHO cells) was a kind gift from Dr. A. B. Troutt (Immunex, Seattle, WA).
Renal transplantation
Renal transplantation was performed essentially as described elsewhere (20). Animals were anesthetized with a combination of halothane and nitrous oxide inhalation using novogesic for postoperative analgesia. Orthotopic renal transplantation was performed using routine procedures. In brief, Donor animals received 200 U heparin, and kidneys were flushed with 2 ml of ice-cold heparinized physiological saline solution before removal and transplantation into recipient animals with left nephrectomy. The graft renal artery was anastomosed to the recipient’s abdominal aorta, and the renal artery was anastomosed to the recipient’s inferior vena cava. The ureter was sewn into the recipient bladder using a small donor bladder patch. All recipients received i.m. injection with cefotetan (30 mg/Kg) on the day of transplantation and for 2 successive days. The remaining host kidney was removed 2 days after transplantation, unless otherwise indicated. Treatment of recipients with pv immunization, by mAbs, or by oral immunization was as described in individual studies.
Portal vein and oral immunization
Portal vein and oral immunization was performed as described earlier (9, 10). All animals were anesthetized with nembutal. A midline abdominal incision was made and the viscera exposed. 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 secured without hematoma formation by gentle pressure using a 2 mm3 gel-foam.
Bone marrow-derived DC for pv immunization were obtained by culture of T-depleted bone marrow cells in vitro with rIL-4 and recombinant granulocyte-macrophage-CSF (10). Staining with NLDC145 and FITC anti-rat IgG, or with FITC anti-CD3, confirmed >95% NLDC145+ and <5% CD3+ cells at day 10 of culture (10). These cells were washed and injected into mice or used for mixed leukocyte cultures.
Preparation of cells
Spleen and bone marrow (10) cell suspensions were prepared
aseptically from individual mice in each experiment. NPC were isolated
essentially as described elsewhere (21). Tissue was first digested at
37°C for 45 min with a mixture of collagenase/dispase, before
separation (15 min at 17,000 rpm at room temperature) over mouse
lymphopaque (Cedarlane Labs). Mononuclear cells were resuspended in
-minimal essential medium supplemented with 2-ME and 10% FCS
(F10). Where cells were obtained from Flt3L injected
mice, animals were treated by i.v. injection of 10 µg/mouse
Flt3L daily for 10 days. After enzyme digestion, recovery of
liver/spleen cells from these mice was markedly increased compared with
saline-injected controls (120 x 106 and 390 x
106 vs 7 x 106 and 120 x
106, respectively).
Cytotoxicity and cytokine assays
In cultures used to assess induction of cytotoxicity or cytokine
production, responder cells were stimulated with irradiated (2000 rad)
stimulator cells in triplicate in F10. Supernatants were pooled from
replicate wells at 40 h for cytokine assays (see below). No
reproducible differences in cytokine levels have been detected from
cultures assayed between 36 and 54 h of stimulation. In some
experiments, the cultures received 1 µCi/well (at 72 h) of
[3H]TdR, and proliferation was assessed by harvesting
cells 14 h later and counting in a well-type beta-counter.
Where cytotoxicity was measured, cells were harvested and pooled from equivalent cultures at 5 days, counted, and recultured at different E:T rations with 51Cr EL4 (H-2b/b) or P815 (H2d/d) tumor target cells. Supernatants were sampled at 4 h for assessment of specific cytotoxicity.
IL-2 and IL-4 activity were assayed by bioassay using the IL-2/IL-4-dependent cell lines, CTLL-2 and CT4.S, respectively. Recombinant cytokines for standardization of assays was purchased from Genzyme (Cambridge, MA). IL-2 assays were set up in the presence of 11B11 to block potential stimulation of CTLL-2 with IL-4; IL-4 assays were set up in the presence of S4B6 to block IL-2-mediated stimulation. Both the IL-2 and IL-4 assays reproducibly detected 50 pg of recombinant lymphokine added to cultures.
In addition, IL-2, IL-4, IFN-, and IL-10 were assayed using ELISA
assays. For IFN-, the assay used flat-bottom Nunc plates (Life
Technologies, Grand Island, NY) coated with 100 ng/ml R4-6A2.
Varying dilutions of supernatant were bound in triplicate at 4°C,
washed three times, and biotinylated anti-IFN- (XMG1.2) added.
After washing, plates were incubated with streptavidin-horseradish
peroxidase (Cedarlane Labs), developed with appropriate substrate, and
OD405 determined using an ELISA plate reader. rIFN- for
standardization was from PharMingen. IL-10 was similarly assayed
by ELISA, using JES5–2A5 as a capture Ab and biotinylated SXC-1
as developing Ab. rIL-10 for standardization was from PeproTech (Rocky
Hill, NJ). Each assay detected 0.1 ng/ml cytokine. ELISA assays for
IL-2 and IL-4 used JES6-1A12 and 11B11 as capture Abs, with JAS6-5H4 or
BVD6-24G2 as developing Abs. Sensitivity of detection was 20 pg/ml for
each cytokine. Where checked, the correlation between bioassay and
ELISA for IL-2 or IL-4 was excellent (r > 0.90). In
all studies reported below, data are shown from ELISA assays only.
Where cytokine data are pooled from several studies (e.g., Figs. 6
, 8, 9), absolute values of cytokine production were obtained as above using
commercial recombinant cytokines to standardize the assays. In our
hands, supernatants from C3H anti-C57BL/6 cultures, under the
conditions described, reproducibly contain 950 ± 200 and 80
± 25 pg/ml IL-2 and IL-4, respectively.
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Different sources of tissue from renal-grafted female mice receiving DC and kidney allografts from male mice were harvested for RNA extraction as described elsewhere (9). The OD280/260 of each sample was measured, and reverse transcription was performed using oligo (dT) primers (27-7858; Pharmacia, Piscataway, NJ). The cDNA was diluted to a total volume of 100 µl with water and frozen at -70°C until use in PCR reactions with primers for murine GAPDH, B7-1, B7-2, or OX-2. The sense and anti-sense primers were synthesized by the Biotechnology Service Centre (Hospital for Sick Children, Toronto, Canada) using published sequences. 5' primers were 32P end-labeled for PCR and had comparable levels of sp. act. after purification by ethanol precipitation. cDNA (5 µl) was amplified for 35 cycles by PCR, and samples were analyzed in 12.5% polyacrylamide gels followed by overnight (18 h) exposure for autoradiography. In control studies, using H-Y primer sets, this technique reliably detects H-Y mRNA from extracts of female spleen cells to which male cells are added at a concentration of 1:105 (10). Quantitative comparison of expression of different PCR products used densitometric scanning of the autoradiograms: GAPDH sense, 5'-TGATGACATCAAGAAGGTGGTGAAG-3'; GAPDH anti-sense, 5'-TCCTTGGAGGCCATGTAGGCCAT-3'; B7-1 sense, 5'-CCTTGCCGTTACAACTCTCC-3'; B7-1 anti-sense, 5'-CGGAAGCAAAGCAGGTAATC-3'; B7-2 sense, 5'-TCTCAGATGCTGTTTCCGTG-3'; B7-2 anti-sense, 5'-GGTTCACTGAAGTTGGCGAT-3'; OX-2 sense, 5'-GTGGAAGTGGTGACCCAGGA-3'; OX-2 anti-sense, 5'-ATAGAGAGTAAGGCAAGCTG-3'.
Statistical analysis
In studies with multiple groups, ANOVA was performed to compare significance. In some cases (as defined in individual circumstances), pairwise comparison between groups was also subsequently performed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a previous manuscript (11), we reported that C3H spleen cells, stimulated in the presence of syngeneic NPC and allogeneic (C57BL/6) DC, produced a cell population able to inhibit generation of IL-2 from fresh spleen cells stimulated with C57BL/6 DC and capable of inhibiting C57BL/6 renal allograft rejection in vivo. To ask whether this function of NPC was MHC restricted or not, we performed the following study.
C57BL/6 (H2b/b) spleen cells were stimulated in vitro with
B10.BR (H2k/k) bone marrow-derived DC in the
presence/absence of the following NPC: C57BL/6, B10.BR, and B10.D2
(H2d/d). In addition, control cultures were incubated with
the NPC only. Proliferation and IL-2/IL-4 production was measured in
one aliquot of these primary cultures. In addition, at 5 days cells
were harvested from another set of the primary cultures, washed, and
2 x 105 cells added to cultures containing 5 x
106 fresh C57BL/6 spleen cells and B10.BR DC. Proliferation
and cytokine production was measured in these latter cultures in
standard fashion. Data pooled from three equivalent studies are shown
in Fig. 1, A and B.
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A). In contrast, IL-4 production was enhanced. NPC alone,
whether syngeneic or allogeneic to the responder cells, produced no
obvious effect (groups 3–5, Fig. 1A). Furthermore, cells
from primary cultures receiving the DC + NPC mixture were able to
inhibit proliferation and IL-2 production (while promoting IL-4
production) from fresh spleen cells stimulated in secondary cultures
with the same (B10.BR) DC (see Fig. 1B). However, the data
in Fig. 1 make another important point. The same inhibition of
proliferation/IL-2 production in primary cultures was seen using either
B10.BR NPC (MHC matched with the DC stimulus; group 8, Fig. 1A) or with third-party B10.D2 NPC (MHC mismatched with both
spleen responder cells and allogeneic stimulator DC; group 7, Fig. 1A). Again no obvious effect was seen in cultures stimulated
with B10.BR or B10.D2 NPC alone (groups 4 and 5, Fig. 1A).
Finally, cells taken from primary cultures stimulated with DC and NPC
from either B10.BR or B10.D2 could also inhibit proliferation/IL-2
production from secondary C57BL/6 spleen cell cultures stimulated with
B10.BR DC; again, cells taken from primary cultures with NPC alone
produced no such inhibition (Fig. 1B). Thus, the inhibition
of proliferation/IL-2 production and enhancement of IL-4 production
seen in primary cultures, as well as the induction of suppression
measured in secondary cultures, all induced by NPC, are not MHC
restricted. Specificity of inhibition/suppression induced by hepatic NPC
One interpretation of the data shown in Fig. 1 and elsewhere is
that NPC deliver a signal to DC-stimulated cells that is distinct from
the Ag-specific signal provided by the DC themselves (and is MHC
nonrestricted). This signal modulates the Ag-specific signal
provided by the DC. To assess the Ag-specificity of the
immunoregulation described in Fig. 1, the following experiment was
performed.
C57BL/6 spleen responder cells were stimulated with B10.D2 or B10.BR
bone marrow-derived DC in the presence/absence of NPC from B10.BR or
B10.D2 mice. Proliferation and cytokine production was measured in
aliquots of these cultures as before. In addition, further aliquots of
cells harvested from these primary cultures were added to cultures of
fresh C57BL/6 spleen cells stimulated with B10.BR (Fig. 2B) or B10.D2 (Fig. 2C) DC. Again, proliferation and cytokine production was
measured. Data pooled from three such studies are shown in Fig. 2.
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A) recapitulates the
observations made in Fig. 1 and show that NPC inhibit proliferation and
IL-2 production from DC-stimulated responder cells in an Ag- and
MHC-unrestricted fashion. However, the data in Fig. 2, B and
C clearly show that adoptive transfer of inhibition using
cells from these primary cultures occurs in an Ag-restricted fashion,
dictated by the Ag-specificity of the DC used in the primary cultures,
not of the NPC used for induction of suppression. Thus, these auxiliary
cells in the NPC population have a functional property of being
"facilitator cells for induction of suppression." Note that in
other studies (data not shown) where the final assay system involved
measuring cytotoxicity to allogeneic target cells, a similar inhibition
of lysis (rather than cytokine production) was seen using cells
harvested from primary cultures stimulated with DC and hepatic NPC (see
11 . Hepatic cell preparations from Flt3L-treated mice are a potent source of DC and "facilitator" cells
We have reported at length that pv infusion of alloantigen, or
i.v. infusion of liver-derived allogeneic mononuclear cells induces
operational unresponsiveness in recipient animals (10, 20, 22, 23). The
total hepatic mononuclear cell yield from normal mice is of the order
of 5 x 106 cells/mouse. To increase the yield, and
explore the possibility that the liver itself might be a source both of
allostimulatory DC and "facilitator" cells, we exposed two C57BL/6
mice for 10 days to daily i.v. infusions of 10 µg/mouse human
CHO-derived Flt3L, a known growth factor for DC (18). Liver
tissue was harvested and pooled from these donors, and mononuclear
cells were prepared as described in Materials and Methods
(mean 130 x 106 cells/donor). These cells were
further subjected to subfractionation by size using unit gravity
sedimentation techniques (24). A typical size profile for recovered
cells is shown in Fig. 3 (one of three
studies).
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were tested as follows. Firstly, cells were stained with
FITC-labeled mAbs to B7-1, B7-2, NLDC145, and rat anti-mouse OX-2
(M3B5) with FITC anti-rat IgG as secondary Ab. In addition, mRNA
extracted from the different cell samples were assayed by PCR for
expression of GAPDH, B7-1, B7-2, and OX-2. Data are shown in Figs. 3
(pooled from three separate studies) and
4 (representative PCR data from one
experiment). Further aliquots of the cells were used to stimulate fresh
C3H spleen responder cells in culture. Proliferation and cytokine
assays were performed as before (Fig. 1), and, in addition, cells were
taken from these primary cultures and added to fresh secondary cultures
of C3H spleen responder cells and C57BL/6 bone marrow-derived DC.
Again, proliferation and cytokine production was assayed from these
secondary cultures. Data pooled from three studies of this type are
shown in Fig. 5, A and
B. Finally, cells from the various fractions were infused
i.v. into two C3H mice per group, which also received C57BL/6
renal allografts as Ag challenge. Spleen cells were harvested from
these individual mice 10 days after transplantation and restimulated in
culture with C57BL/6 or B10.D2 DC, again with cytokines measured at
40 h (see Fig. 6).
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show that distinct subpopulations of slow-sedimenting
cells express OX-2 in the cells harvested from Flt3L-treated
mice when compared with cells expressing B7-1 and/or B7-2. In general,
expression of OX-2 and B7-2 occurred in equivalent subpopulations.
Faster-sedimenting cells (Fx3 and Fx4 in Fig. 3), while staining for
NLDC145, were positive by fluorescence mainly for B7-1, not B7-2 or
OX-2. Similar conclusions were reached both by FACS analysis of cell
populations (Fig. 3) and by PCR analysis of mRNA (Fig. 4).
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and 6), it was found that optimal direct
stimulation (or proliferation and IL-2 production) was seen from
B7-1-expressing cells (Fx3 and Fx4 in Fig. 5A), while only
OX-2 expressing cells (Fx1 and Fx2 in Figs. 3 and 4) were capable of
producing the inhibitory effects defined earlier (Figs. 1 and 2) in the
two-stage culture system (Fig. 5B). These same cells (Fx1
and Fx2) were in turn able, after i.v. infusion, to polarize cells from
mice given renal allografts to produce predominantly IL-4, IL-10, and
TGFß production on restimulation in vitro (Fig. 6). These data are
consistent with the notion that after Flt3L treatment of
mice expansion of a population of immunostimulatory DC occurs within
the liver, which also contains another distinct population of
(facilitator) cells that promote immunoregulation. Evidence that cell populations with "facilitator" activity from the liver of Flt3L-treated mice prolong graft survival in vivo
Because we have reported elsewhere that there is a good
correlation between treatments (such as pv immunization) that decrease
IL-2 production and increase IL-4 production from restimulated cells
and prolongation of graft survival (4, 10), and that increased
expression of OX-2 is also independently associated with increased
graft survival after pv immunization (15), we next asked whether cells
isolated from Flt3L-treated mice, which induced inhibitory
function in vitro (see Figs. 1, 2 and 5) and expressed increased
amounts of OX-2 (Figs. 3 and 4), were themselves capable of promoting
increased graft survival in vivo.
Groups of two C57BL/6 mice received i.v. infusions of 10 µg/mouse
Flt3L for 10 days as before. Cells were isolated from the
liver by enzyme digestion and fractionated by unit gravity
sedimentation. Four pools of cells were recovered, and an aliquot
stained as before in FACS with anti-OX-2. Groups of two C3H mice
received 10 x 106 cells i.v. from the four separate
pools. A control group received saline injections only. Over the next
48 h, all mice received C57BL/6 renal transplants. All mice
received cyclosporin (CsA) (10 mg/Kg) on the day of renal
transplantation. The data in Fig. 7 are
pooled from three studies of this type (representing six mice/group)
and show the animal survival in these five different groups.
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that only hepatic cells expressing OX-2
(Fx1 and Fx2; see Figs. 3 and 4) were capable of promoting increased
graft survival after i.v. infusion. Comparison of these data with those
in Fig. 5 confirm that these cell populations were also those
identified, using a two-stage culture assay system, as cells with
functional "facilitator" activity (see also Figs. 1 and 2). Note
that though our conclusions are restricted by the use of only six
mice/group in this study, there was no significant difference in
survival between groups receiving NPC-Fx1 or NPC-Fx2 in this
experiment, in keeping with relatively equivalent levels of OX-2
expression in these fractions (Fig. 3). Anti-OX-2 mAb in vitro reverses regulation induced by hepatic NPC
In a final study, we asked whether anti-OX-2 mAb M3B5, added
to cultures of C3H spleen responder cells, allogeneic (C57BL/6) DC and
NPC from C57BL/6 mice, could prevent the inhibition of IL-2 production
in primary cultures, and the development of cells able to inhibit such
cytokine responses from freshly stimulated responder cells in secondary
cultures (see Figs. 1, 2, and 5). Data in Figs. 8 and 9 are
pooled from three studies of this type. Primary cultures were of two
types, containing C3H responder spleen cells and C57BL/6 DC alone (Fig. 8), or the same mixture with added C57BL/6 NPC (Fig. 9). Subsets of
these cultures contained in addition either 5 µg/ml of anti-B7-1,
anti-B7-2, or anti-OX-2. Supernatants from responder cells
stimulated in the presence of DC only were assayed after 60 h for
cytokine production (Fig. 8). For the primary cultures incubated with
both DC and NPC, supernatants were harvested at 60 h and tested
for cytokine production (Fig. 9A). In addition, cells were
harvested after 5 days, washed, and added to secondary cultures of
fresh C3H responder cells with fresh C57BL/6 DC. No mAbs were added at
this second culture stage. Data for cytokine production of these
secondary cultures are shown in Fig. 9B.
The addition of anti-B7-1 or anti-B7-2 to DC-stimulated spleen
cultures led to inhibition of cytokine production (Fig. 8), while in
contrast anti-OX-2 mAb led an increase in IL-2 production in these
primary cultures (Fig. 8). We have reported similar findings elsewhere
(45). Interestingly, anti-OX-2 abolished the inhibition of cytokine
production caused by NPC in these primary cultures (Fig. 9A;
see also Figs. 1, 2, and 5). In addition, anti-OX-2 prevented the
functional development of a cell population capable of transferring
inhibition of cytokine production to freshly stimulated spleen cells
(Fig. 9B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and increased IL-4, IL-10, IL-13, and TGFß (5, 11). Which, if any, of these cytokine changes is directly and causally
implicated nevertheless remains obscure.
Further analysis of the cell population able to induce tolerance after
pv immunization led to the somewhat paradoxical observation that donor
DC represented an excellent tolerizing population (10). Because
Ag-pulsed DC are conventionally thought of as representing an optimal
immunizing regime, we were interested in the mechanism(s) activated
following DC pv immunization that led to tolerance (29). It is already
clear that DC themselves represent an extremely heterogeneous
population, in terms of origin, cell surface phenotype, turnover in
vivo, and possibly function (30, 31). In the mouse lymph node, at least
three discrete populations were identified, one of which comprised
small CD8+NLDC145+ cells, likely of lymphoid
origin, with an immature phenotype, and whose numbers were profoundly
increased (100x) following Flt3L treatment in vivo (30)
(administration of the latter has been reported to lead to
proliferation of DC and other cells of hematopoietic origin (32)).
These cells resembled the interdigitating DC found in the T cell areas
of the splenic white pulp (31) and have been implicated in regulation
of immunity induced by other (myeloid-derived) DC (30, 33, 34).
A variety of other studies have indicated that the induction of immunity (vs tolerance) following Ag presentation was intrinsically dependent upon the coexistence of other signaling ligands at the surface of DC (interacting with appropriate counterligands on the surface of other cells, e.g., stimulated T cells) (35, 36, 37). We speculated that infusion of DC via the pv induced tolerance by coopting another cell population, distinguishable by expression of unique cell surface ligands, whose biological function was to facilitate induction of tolerance, not immunity, when Ag was presented in association with otherwise immunogenic DC; some preliminary evidence supporting this hypothesis was recently reported (11). We have elected to refer to this as a facilitator cell. Moreover, because pv immunization has been shown to be associated with increased expression of a novel molecule, OX-2, previously reported to be expressed on DC (12, 14, 15), we wondered whether this molecule would in fact serve as a "marker" for the hypothetical facilitator cell described. Experiments reported above are consistent with such a hypothesis.
We have shown that within the hepatic NPC population there is a subset
of cells able to inhibit stimulation by allogeneic DC in a non-MHC
restricted fashion (see Figs. 1 and 2) and able to induce the
development of an Ag-specific immunoregulatory cell population in vitro
(see Figs. 1 and 2). The non-MHC-restricted nature of this
"facilitator" cell interaction leads us to believe that it
functions by providing an accessory signal (a regulatory, not a
costimulatory, signal) to the DC that stimulates T cells in the
allogeneic MLR we describe in a fashion analogous to the original
description of costimulatory interactions (38). As a result, the
stimulated lymphocytes alter their cytokine production profile (with
decreased IL-2 production and proliferation), and become able to
regulate the immune response seen from freshly stimulated lymphocytes
(see Figs. 1B and 2B). Most interestingly,
following expansion of DC in vivo by Flt3L treatment, we
have been able to show that in fact the liver itself contains both an
immunostimulating population (large cells by velocity sedimentation
analysis) and this putative "facilitator" cell population (see
Figs. 3–7). Furthmore, the latter biological activity resides within a
slow-sedimenting (small size) NLDC145+ cell population
expressing preferentially both cell-surface B7-2 and OX-2 (see Figs. 3 and 4). When we investigated whether this same population of cells was
active in vivo in regulating graft tolerance, we found again that after
prior Flt3L treatment the liver contained a population of
cells that transferred increased renal graft acceptance (Fig. 7) and in
parallel altered the cytokine production profile of immunized mice
toward increased IL-4 and TGFß and decreased IL-2 and IFN-
production (Fig. 6).
In a final attempt to explore the role for OX-2 expression itself in
this regulatory function, we stimulated fresh spleen cells with DC
alone or in the presence of anti-B7-1, anti-B7-2, or
anti-OX-2. Note that other studies (data not shown) have confirmed
that even the bone marrow-derived DC used contains small numbers of
OX-2+ cells (R.M.G., unpublished observations). Unlike
anti-B7-1 and anti-B7-2, which decreased cytokine production, a
result in keeping with the hypothesized role for these as costimulator
molecules (39, 40, 41), anti-OX-2 produced a small but significant
(1.7- to 2.5-fold in three studies) increase in IL-2 production in this
system (Fig. 8). Most important, however, inclusion of anti-OX-2
mAb in a system where exogenous "facilitator" cells were added
(from NPC) blocked completely the induction of inhibition normally seen
in such cultures (Figs. 1 and 2; compare with Fig. 9B).
These data are consistent with the concept that OX-2 delivers a
regulatory, not a costimulatory, signal in this situation.
How do our data fit within the evolving framework of understanding in
the heterogeneity of DC? As noted above, there has been speculation
that a separate population of CD8+NLDC145+
DC of lymphoid origin, which proliferates in response to
Flt3L, might be responsible for immunoregulation. Other data
have implicated IL-10 as a cytokine that might modify
development/maturation of DC into a population expressing increased
amounts of B7-2 and capable of inducing tolerance (42). The role of
regulation of expression of Fas as a controlling feature in this regard
is unexplored (34). Our data is the first, to our knowledge, that
implicates another molecule, OX-2, or its natural ligand, in the
delivery of a tolerizing signal, perhaps in association with
alterations in expression of B7-2, Fas, etc. It is intriguing that
while there is clearly a key role for intrathymic DC in the regulation
of selftolerance (29), natural expression of OX-2 was initially first
described on thymic DC (as well as within the brain) (12); there is as
yet no evidence to suggest that this represents a functionally relevant
expression for OX-2 in this location. However, other independent data
have also implied an immunoregulatory role for OX-2 expression, again
as assayed by altered cytokine production in vitro from cells
stimulated in the presence/absence of expressed OX-2 (16).
We have also reported that following pv immunization there is a
measurable expansion in numbers of populations of
TCR+ cells capable of adoptive transfer of increased
graft survival to naive recipients (43, 44). Little is known concerning
the nature of the Ag recognized by these cells and why, as a
population, their numbers are preferentially increased following pv
immunization. We speculate that this may be explainable ultimately in
terms of a differential susceptibility of TCR+ vs
ßTCR+ cells to immunoregulatory signals delivered
following OX-2 expression.
In conclusion, we report for the first time that functional heterogeneity in the DC pool may be understandable in terms of differential expression of OX-2 on the cell surface. Expression of this molecule seems to give cells the capability to induce immunoregulation, increased renal graft survival (and altered cytokine production both in vivo and in vitro). We have suggested that such OX-2-expressing cells be referred to as "facilitator" cells (for tolerance induction). The mechanism by which they produce this effect, and the potential involvement of other signaling molecules at the cell surface (including Fas and perhaps the ligand for OX-2), remains to be explored.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. R. M. Gorczynski, CCRW 2-855, Transplant Research Division, Toronto Hospital, 200 Elizabeth Street, Toronto, Ontario, Canada M5G2C4. E-mail address:
3 Abbreviations used in this paper: pv, portal vein; NPC, hepatic mononuclear nonparenchymal cells; DC, dendritic cell(s); Flt3L, Flt3 ligand; CHO, Chinese hamster ovary; CsA, cyclosporin A.
Received for publication July 20, 1998. Accepted for publication October 7, 1998.
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References |
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