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Autoimmunity and Transplantation Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) infiltration. We developed an
i.p. transplant model to define the roles of M and T cells in
xenograft rejection. Nonobese diabetic or BALB/c mice were injected
i.p. with xenogeneic, allogeneic, or syngeneic cells, and the
responding cells in subsequent lavages were assessed by flow cytometry
and adoptive transfer. Neutrophils and monocytes/elicited M were
rapidly recruited in response to xenogeneic pig (PK15 or spleen) cells
and, to a significantly lesser extent, allogeneic cells. These innate
responses preceded T cell infiltration and occurred in their absence in
SCID mice. Syngeneic cells induced negligible neutrophil or M
responses. Neutrophils and M induced by xenogeneic cells in SCID
mice stimulated T cell recruitment after transfer to immunocompetent
mice. T cells in turn were required for M activation and xenogeneic
cell rejection. Thus, M harvested from immunocompetent but not SCID
mice injected with xenogeneic cells expressed activation markers and
rejected xenogeneic cells when transferred into SCID mice. These
findings demonstrate the interdependent roles of M and T cells in
xenograft rejection. The requirement for M reflects their ability to
mount a rapid, local innate response that stimulates T cell recruitment
and, having received T cell help, to act as direct effectors of
rejection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(10), Fc receptors
(6), NK cells (11) and perforin
(12) are each alone not essential for xenograft
rejection.
It is increasingly recognized that innate immune responses mediated by
monocytes/macrophage (M), neutrophils, mast cells, and NK cells
drive and shape adaptive immunity (13, 14). Cells of the
innate immune system evolved to express receptors that recognize
pathogen-associated molecular patterns (PAMPs), in particular disparate
oligosaccharide structures exquisitely different from self (14, 15). Generic expression of these germline-encoded, invariant
receptors allows a rapid response to pathogens. In contrast, T cells of
the adaptive immune system use randomly rearranged receptors that do
not discriminate whether an Ag is or is not pathogen associated.
However, innate immunity provides signals for T cell priming
(15) and thereby focuses adaptive immunity on
pathogen-associated Ags.
M are the dominant infiltrating cell in pancreatic islet or FPP
xenografts undergoing rejection, and some express an unusual
CD4+ phenotype (11). Previously, we
showed that M are required for T cell infiltration and rejection of
xenografts (16). In the present study, our aims were as
follow: first, to investigate whether xenografts elicit an innate
immune response; second, to define the interactions between M and T
cells in xenograft rejection; and finally, to determine whether M
are direct effectors of xenograft rejection. An i.p. transplant (IPT)
model was developed in which xenogeneic cells are injected i.p., and
responding cells are harvested for phenotypic and functional analysis
by flow cytometry and adoptive transfer.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nonobese diabetic (NOD), NOD-SCID, CB17-SCID, and BALB/c mice, aged 6–10 wk, were bred at the Walter and Eliza Hall Institute (Parkville, Australia). Outbred pregnant Landrace sows at 60–90 days of gestation were bred at the Victorian Institute of Animal Science (Werribee, Australia).
Preparation and injection of cells
The adherent epithelial-like pig kidney cell line, PK15, was obtained from American Type Culture Collection (Manassas, VA) and grown in Eagle’s MEM with nonessential amino acids and 5% FCS. Similarly, the adherent fibroblast-like BALB/c-3T3 cell line was obtained from American Type Culture Collection and grown in DMEM containing 5% FCS. Cell lines were harvested by incubation with trypsin and washed three times with mouse tonicity PBS. Spleens, harvested from mice and adult or fetal pigs, were disrupted by gentle extrusion through a fine wire mesh. The resultant single-cell suspensions were treated with 0.16 M NH4Cl to lyse RBC, then washed three times with PBS. All cells were resuspended at 2 x 107/ml in PBS, and 1 x 107 cells were injected i.p. Syngeneic splenocytes were labeled before injection with carboxyfluorescein diacetate succinimidyl ester (Molecular Probes, Eugene, OR) by incubation in a 0.01 mM solution in PBS containing 0.1% BSA for 10 min at 37°C.
Peritoneal lavage
Mice were killed by CO2 inhalation and lavages were performed immediately by injecting 5.5 ml of PBS containing 5% FCS and 5 U/ml heparin into the peritoneal cavity. After massaging the abdomen, peritoneal fluid was recovered with a needle and syringe. Recovered volumes ranged from 4.8 to 5.5 ml. Cell counts were performed with a hemocytometer. Cells were then centrifuged, and the pellet was resuspended in PBS containing 2% BSA, 0.1% sodium azide, and 0.1% EDTA that had been filtered through a 0.45-µm pore size sterile filter (wash buffer).
Flow cytometry
The mAbs used for flow cytometry, their specificities, and their
sources are listed in Table I
. mAbs were
either directly fluorochrome conjugated or biotinylated and detected
with streptavidin-PE (Caltag, Burlingame, CA). Cells were kept at 4°C
throughout the staining procedure. Cells (at least 1 x
106/well) were incubated with FcIII/II
receptor block (Table I) for 10 min, then incubated with primary mAbs
for 30 min. When using biotinylated primary mAbs, cells were washed
twice with wash buffer, then incubated with streptavidin-PE for 15 min.
After staining, cells were washed twice with wash buffer, then fixed
with 3% (v/v) formaldehyde in PBS. When staining for FcIII/II
receptors, blocking was omitted and the primary mAb was detected with
FITC-conjugated anti-rat Ig (Vector Laboratories, Burlingame, CA).
The number of cells expressing a particular marker was calculated by
multiplying percentages obtained from flow cytometry by the
concentration of cells in lavage fluid. In this way, variability due to
lavage volumes recovered was avoided. To discriminate between
responding cells and injected splenocytes, two-color analysis was
performed with an mAb specific for H-2Dd (Table I) to detect allogeneic BALB/c splenocytes and by carboxyfluorescein
diacetate succinimidyl ester labeling to detect syngeneic NOD
splenocytes.
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Mice were given either a single i.p. injection of cells or two
injections (1 mo apart) and i.p. cells were harvested 1–5 days later
for intracellular cytokine analysis. Lavages were performed as
previously described, with addition of 10 µg/ml brefeldin A (Sigma,
St. Louis, MO) to the lavage buffer maintained at 37°C. Cells at
2–10 x 106/ml were incubated in this
buffer for an additional hour at 37°C to facilitate intracellular
accumulation of cytokines. After staining for surface markers, as
described above, cells were fixed, permeabilized, and stained with
cytokine-specific mAbs (Table I
) according to the protocols of
PharMingen (San Diego, CA).
Induction and recovery of elicited M and neutrophils for
adoptive transfer
CB17-SCID mice were injected i.p. with xenogeneic PK15 cells.
Mice were killed 1 day later, and peritoneal lavages were performed.
Lavaged cells were pelleted by centrifugation and resuspended in 30%
Percoll (Amersham Pharmacia Biotech, Uppsala, Sweden), then loaded onto
a discontinuous 45 and 70% Percoll gradient. The gradient was
centrifuged at 400 x gav for 20
min at room temperature in a swing-out rotor. PK15 cells were mainly
retained in the 30% layer that was discarded. Cells at the 45–70%
Percoll interface, enriched for elicited M and neutrophils, were
collected, washed twice with PBS, and resuspended in PBS for i.p.
injection. The compositions of lavaged cells and subsequent Percoll
fractions were determined by flow cytometry. In several experiments
lavage cells were stained with M142, the mAb specific for mouse MHC
class I (Table I), to enable neutrophils and elicited M to be
differentiated from other cells, including PK15 cells (see Fig. 4A), and then sorted to >99% purity using the MoFlo
(Cytomation, Fort Collins, CO).
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for adoptive cotransfer
BALB/c mice were injected i.p. with xenogeneic, allogeneic, or
syngeneic cells and peritoneal lavages were performed 5 days later. For
M enrichment, CD4-, CD8-, and B220-positive cells were depleted
using MACS magnetic microbeads conjugated to mAbs specific for these
markers (Miltenyi Biotec, Auburn, CA). The cellular composition of
lavages, before and after MACS separations, was determined by flow
cytometry. Equal numbers of postdepletion, xenogeneic-, allogeneic-, or
syngeneic-stimulated lavage cells were injected i.p. into CB17-SCID
recipients along with 1 x 107 PK15 cells.
Recipient mice were also given 0.1 mg i.p. of the CD4 T cell-depleting
mAb, GK1.5, to deplete any remaining CD4 T cells that may have been
transferred.
Statistics
Results were analyzed by Student’s two-tailed t
test, using Microsoft Excel software. A p 
0.05 was
considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Xenogeneic pig (PK15 or spleen) cells stimulated rapid recruitment
of neutrophils, identified by their GR-1high
(17), intermediate side scatter (SSC) profile when
injected i.p. into NOD (Fig. 1A) or BALB/c mice (data not
shown). Within 1 day, peritoneal neutrophils were markedly and
significantly increased (
10-fold; p < 0.05) in
response to xenogeneic cells compared with syngeneic spleen cells or
PBS (Fig. 1B). Neutrophil numbers increased slightly 1 day
after injecting allogeneic spleen cells, but not significantly compared
with controls (Fig. 1B). Neutrophil responses to xenogeneic
cells were significantly greater than those to allogeneic cells
(xenogeneic vs allogeneic spleen cells, 5.8-fold difference
(p = 0.003; Fig. 1B); PK15 cells vs
3T3 cells, 5.1-fold difference (p = 0.008)).
Xenogeneic cell stimulated-monocytes/elicited M were identifiable by
their F4/80int, GR1int,
intermediate SSC profile (17). This profile was clearly
distinct from the F4/80very high,
GR1low, high SSC profile of resident M
(17) recovered from PBS-injected mice (Fig. 1A). Subsequent differentiation of elicited M was
evidenced by the gradual increase in F4/80 expression and SSC (Fig. 1A). Within 2 days of injecting xenogeneic cells,
monocyte/elicited M numbers were significantly higher than in PBS or
syngeneic cell recipients (Fig. 1B). Allogeneic spleen or
3T3 cells also induced significant increases in monocyte/elicited M
compared with controls (Fig. 1B). However, monocyte/elicited
M responses to xenogeneic cells were significantly greater than to
allogeneic cells (xenogeneic vs allogeneic spleen cells, 2.4-fold
difference (p = 0.005); PK15 cells vs 3T3
cells, 2.1-fold difference (p = 0.007)).
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B). All CD4
cells coexpressed CD3, and an mAb specific for the CD8 
-chain was
used to demonstrate that CD8 T cells expressed the CD8
heterodimer (data not shown). Initially, CD4 T cells exceeded CD8 T
cells in all responses (Fig. 1B), but by day 5 of xenogeneic
responses there were as many CD8 T cells (CD4:CD8 T cell ratio,
1.09 ± 0.18). In contrast, allogeneic 3T3 cells always elicited
more CD4 than CD8 T cells, with a significantly higher CD4:CD8 T cell
ratio (2.14 ± 0.48; p = 0.002) by day 5. B cell
numbers increased 1.6- to 2.5-fold in response to either xenogeneic
cells or allogeneic cells compared with PBS-injected controls (data not
shown). DX5+ NK cells increased in response to
allogeneic (days 1 and 2) and especially xenogeneic splenocytes (days
2–5) in BALB/c mice, but NK cell numbers were very low (Fig. 1B) and were not increased at all in some experiments. NK
cell responses were not assessed in NOD mice, which are NK cell
deficient. There was little or no response of any kind to syngeneic
cells (Fig. 1B), demonstrating that the changes detected
were to foreign cells rather than to trauma or cells per se. Similar
results were obtained in C57BL6 and BALB/c mice (data not shown). Intraperitoneal responses in SCID mice
Xenogeneic or allogeneic cells were injected into SCID mice to
investigate the T cell dependence of responses. As in immunocompetent
mice, neutrophils and elicited M dominated in lavages from NOD-SCID
mice 1 day after injecting xenogeneic PK15 cells (Fig. 2A). In contrast, few or no
neutrophils or elicited M were detected in response to syngeneic
splenocytes; lavages comprised predominantly F4/80very
high (data not shown), high SSC resident M (Fig. 2A). Responses to xenogeneic, allogeneic, or syngeneic
splenocytes or PBS were also compared in CB17-SCID mice (Fig. 2B). As with immunocompetent mice, xenogeneic splenocytes
elicited stronger neutrophil and elicited M responses than
allogeneic splenocytes, and the response to syngeneic splenocytes was
minimal (Fig. 2B).
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To monitor rejection, xenogeneic PK15 cells were injected into
immunocompetent or SCID mice, and recoveries were compared over time.
Within the first 2 days, there was considerable variability and no
clear difference between immunocompetent and SCID mice (Fig. 3D). However, by day 3 PK15
cell recovery from immunocompetent mice was markedly reduced, and
thereafter absent, in contrast to the continued recovery of PK15 cells
from SCID mice (Fig. 3, B–D). The rapid loss of PK15 cells
from immunocompetent compared with SCID mice along with the strong T
cell response in the former indicates that xenogeneic cells are subject
to T cell-dependent rejection. Additionally, in three experiments we
found that PK15 cells were rejected within 1 day of injection into
immunocompetent mice (n = 4) that had been primed with
PK15 cells 28 days previously, consistent with a memory T cell
response.
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and neutrophils on T cell
recruitment
M- and neutrophil-enriched PK15 cell-depleted lavage cells from
PK15 cell-injected SCID mice were transferred i.p. to BALB/c mice to
examine their effect on T cell recruitment. PK15 cell-elicited,
Percoll-enriched lavage cells from SCID mice, comprising 78%
neutrophils and elicited M and 3% PK15 cells (Fig. 4A), stimulated significantly
more CD4 and CD8 T cell recruitment within 1 day of transfer to BALB/c
mice than did control cells from PBS-injected SCID mice, comprising
15% (1.1 x 106) neutrophils and M (Fig. 4B), or PK15 cells transferred alone in excess (5 x
106/mouse; Fig. 4B). By day 3, about
4.5 times more CD4 T cells had been recruited after transfer of PK15
cell-elicited neutrophils and M compared with cells from
PBS-injected SCID mice (Fig. 4C). However, by this time PK15
cells alone had induced more T cell recruitment, presumably because of
the greater number transferred (Fig. 4C). When elicited M
and neutrophils from SCID mice injected with PK15 cells were sorted to
>99% purity and transferred i.p. to BALB/c mice, significantly higher
numbers of CD4 and CD8 T cells were recruited within 1 day than after
transfer of control lavage cells from PBS-injected SCID mice (Fig. 4D).
Requirement for T cells in M responses to xenogeneic cells
The requirement for T cells in M responses to xenogeneic cells
was examined by comparing M from PK15 cell-injected immunocompetent
and SCID mice. Within 5 days of PK15 cell injection into
immunocompetent NOD mice, there was a 3.5-fold increase in M that
expressed MHC class II (p = 0.005) compared
with mice injected with syngeneic splenocytes (Fig. 5). This was reflected by an increase in
the total number of MHC class II+ M from
3.5 x 105 ± 1.6 x
105 to 4.9 x 105 ±
2.1 x 105/ml. M harvested from
immunocompetent NOD mice 5 days after xenogeneic vs syngeneic
stimulation also expressed significantly higher levels of CD80
(1.6-fold increase; p = 0.0001; Fig. 5), CD40 (1.4-fold
increase; p = 0.04; data not shown), and FcRIII/II
(1.9-fold increase; p = 0.003; data not shown). In
contrast, M harvested from NOD-SCID mice 5 days after PK15 cell
injection did not express MHC class II or increased levels of CD80
(Fig. 5) or CD40 (data not shown) compared with M from syngeneic
cell-injected controls. FcRIII/II expression was not examined in
SCID mice. Similar results were obtained when responses to xenogeneic
and allogeneic cells were compared in BALB/c and CB17-SCID mice (data
not shown).
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), M were not able to kill xenogeneic
cells efficiently, if at all, in the absence of T cells. To determine
whether M could kill xenogeneic cells if given T cell help, they
were recovered from BALB/c mice 5 days after PK15 cell injection,
purified, and cotransferred with PK15 cells to CB17-SCID mice. As
before, we found that the number of MHC class II+
M increased after xenogeneic PK15 cell, but not syngeneic 3T3 cell,
injection. Approximately 70% of the transferred cells were M, up to
7% were neutrophils, and <10% were T cells. The SCID recipients were
also injected with a CD4 T cell-depleting mAb (GK1.5), and T cells were
not detected in peritoneal lavages. Recovery of PK15 cells from
recipients of PK15 cell-stimulated M was approximately 2.6-fold
lower than that from recipients of 3T3 cell-stimulated M after only
1 day and was very low or negligible after 2 days, demonstrating that
PK15 cell-stimulated M transferred rapid rejection (Fig. 6). Subsequently, transfer of at least
5 x 106 M was shown to be required for
efficient rejection of 1 x 107 PK15 cells.
PK15 cell rejection was faster after the transfer of M than after
the transfer of primed, unfractionated lavage cells (Fig. 6) or primed
T cells alone (data not shown).
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To further examine the mechanisms of elicited M- and
neutrophil-mediated T cell recruitment and T cell-mediated activation
of M during xenoresponses, cytokine expression by NOD M and CD4 T
cells was examined by two-color flow cytometry. The proportion of cells
expressing IFN- or TNF-, but not IL-4, increased within 1 day of
secondary PK15 cell injection (Fig. 7A), as did cell numbers (data
not shown). In the xenogeneic response, 47 ± 7% of the
TNF-+ cells were
F4/80int-elicited M (Fig. 7A), with
a corresponding increase in F4/80+
TNF-+ cells in xenogeneic-stimulated compared
with control lavages (Fig. 7B). Approximately 15% of
CD4+ cells expressed TNF-, but
GR1+cells did not appear to be
TNF-+ (data not shown). In the xenogeneic
response, 21 ± 6% of IFN-+ cells were
CD4+, with an absolute increase in the numbers of
CD4+ IFN-+ cells (Fig. 7B). Restimulation in vitro with PMA and ionomycin led to a
further increase in the numbers of TNF-+ and
IFN-+ cells, but IL-4+
cells remained undetectable (data not shown). Similar results were
obtained with cells lavaged 2 and 4 days after secondary or 1 and 5
days after primary PK15 cell injection (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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responses
that precede the influx of T cells and occur in the absence of T cells
in SCID mice. Innate immunity is attributed to receptors that evolved
to recognize PAMPs clearly distinguishable from self (14, 15). Our findings imply that grafts express PAMP-like molecular
patterns recognized by innate immune receptors. The PAMPs recognized by
many of the innate receptors identified to date are oligosaccharides
(14). Oligosaccharide differences between species are
exemplified by the strong, preformed Ab response of primates to
Gal(1, 3) Gal expressed on nonprimate, xenogeneic endothelial cells
(18). Primate NK cells directly recognize
oligosaccharides, including Gal(1, 3) Gal moieties, expressed by
porcine endothelial cells in vitro (19, 20), and a range
of oligosaccharides mediate interactions between monocytes and
xenogeneic RBC (21, 22). The stronger stimulation of
innate immunity by xenografts compared with allografts would be
consistent with the idea that they express disparate nonpolymorphic
molecules, whereas allograft disparities are largely restricted to the
polymorphic MHC proteins.
Innate immune responses to xenografts may impact on the cell-mediated
response and contribute to the strength and complexity of xenograft
compared with allograft rejection. The current study provides evidence
for a role of innate immunity in the recruitment of T cells. M and
neutrophils elicited by xenogeneic cells in SCID mice induced T cell
recruitment upon transfer to immunocompetent mice. Furthermore, we have
previously demonstrated that M are required, at least locally, for T
cell infiltration of FPP xenografts (16). These results
are consistent with the well-documented chemoattractant properties of
neutrophils and monocytes (23, 24), mediated by the
release of chemokines such as IFN-inducible protein-10 and cytokines
such as TNF-. In turn, chemokines, including macrophage inflammatory
protein-1 and -, IL-8, and KC (the murine homologue of
growth-related oncogene-) have been detected in skin allografts
during the innate response before the induction of alloreactive T cells
(25, 26), and blockade of monokine induced by IFN-
prevents circulating alloantigen-specific T cells from entering grafts
(27). As in responses to various pathogens
(24), neutrophils appeared to be the first cells recruited
into xenografts. Neutrophils release both monocyte and T cell
chemoattractants, and it has been suggested that they respond directly
to pathogens and initiate cell-mediated immunity (24, 28).
Cell-specific expression of cytokine protein was quantified during the
xenograft response for the first time in the present study.
TNF-+ cells, approximately 50% of which were
monocytes/elicited M, were induced by xenogeneic cells consistent
with previous semiquantitative PCR studies of cytokine RNA expression
in FPP and pig proislet xenografts (29).
IFN-+ CD4 cells were also induced by
xenogeneic cells, consistent with previous studies (30).
The phenotype of the remaining IFN-+ cells was
not investigated in detail, although IFN-+,
CD8+ cells have been detected (A. Fox,
unpublished observation), and other cells, such as NK cells and M,
may express IFN- (31). IL-4+
cells were not detected, although others have reported that levels of
IL-4 RNA are increased in xenografts (29, 30).
Cell-associated IL-4 protein may be harder to detect than whole
graft-associated IL-4 RNA, or IL-4 expression may differ in the
different xenograft models.
Xenogeneic cells elicited T cell responses that coincided with
rejection. Furthermore, rejection was accelerated in mice that had
previously been primed and did not occur in T cell-deficient SCID mice,
demonstrating its T cell dependence. However, it has never been clear
whether T cells directly reject grafts or are required to activate
other effectors. M are, in fact, the dominant infiltrating cell in
xenografts undergoing rejection and are well-known effectors against
autoimmune or infected target cells (32, 33, 34). We show here
that T cells are required for M activation and for the first time
that activated M can directly reject xenogeneic cells.
Whether rejection by xenoactivated M is specific and, if so, by what
mechanism is currently being investigated. Activation of M that
could kill cells, including self cells, nonspecifically would be
deleterious. That self cells are normally not killed by activated M
indicates that mechanisms operate at the level of either M or the T
cells involved in their activation to direct effector potential toward
foreign targets. Also, the range of targets that activated M can
kill in vitro is limited by the cytotoxic potential that M acquire
during activation and by the sensitivity of different targets to such
toxicity (35). For example, L929 cells are sensitive to
TNF--mediated killing and P815 cells to NO-mediated killing by
LPS/IFN--activated, casein-induced M, whereas allogeneic cells
are not sensitive to either (36). In preliminary
experiments we have found that PK15 cell rejection in SCID mice is
greater after transfer of xenogeneic- than allogeneic-stimulated M.
While this suggests a degree of M specificity, it may be simply that
xenogeneic cells are a stronger stimulus for induction of M with
greater cytotoxicity than allogeneic cells. At least two reports
indicate M specificity for targets. First, allogeneic fetal pancreas
segments were rejected at a normal rate when cotransplanted with a
xenograft, even though the xenograft was rejected more rapidly
(4). Second, M that dominated peritoneal infiltrates
after IPT of allogeneic Meth A tumor cells killed allogeneic, but not
syngeneic, cells in vitro and in vivo, and this killing could only be
inhibited by cells of the same allogeneic haplotype and was cell-cell
contact dependent (36, 37). Mechanisms for specific
recognition of targets by effector M have not been described. As we
have demonstrated that xenogeneic cells are recognized by the innate
immune system, it is tempting to speculate that receptors such as the
PAMP recognition receptors may be involved in directing and limiting
the cytotoxicity of effector M toward foreign targets.
T cells were required for the generation of M that could reject
xenografts, a requirement classically attributed to the provision of
IFN- (33, 38, 39). We demonstrated that xenogeneic
cells induce IFN-+ CD4 T cells. However, as
islet xenograft rejection is only slightly delayed in IFN--deficient
mice (10), other cytokines may be involved in M
activation. Signals from Ag-specific T cells may also influence the
cytotoxic potential of M and hence the targets they can kill. In
preliminary experiments we could recover MHC class
II+ M from PK15 cell-injected SCID mice if
they were also given PK15 cell-stimulated, but not allogeneic
cell-stimulated, T cells. This coincided with PK15 cell rejection
specifically in the former. The sites at which activated M can act
as effectors may also be governed by Ag-specific T cells. This is
indicated by various studies demonstrating a requirement for T cell
infiltration of grafts for M to be present in large numbers
(40, 41). Whether this results from increased M
infiltration or proliferation locally is not known. The requirement for
local T cells is particularly apparent in studies showing that i.v.
injected xenograft-activated M do not infiltrate and reject renal
subcapsular FPP grafts in T cell-deficient SCID mice (A. Fox,
unpublished observation). This need for T cells is bypassed in the IPT
model, by delivering activated M to the graft site.
Concluding remarks
Xenografts in particular are recognized by the innate immune
system, and xenograft rejection requires interactions between innate
and adaptive immunity. Although attention has been focused on the role
of innate immunity in Ag presentation and T cell priming, the present
study highlights the important role of innate immunity in T cell
recruitment. In turn, T cell help is required for M activation and
M-mediated graft rejection. It is likely that general adjuvant
effects of innate immunity mediated by cytokines and chemokines impact
on many aspects of the cell-mediated response and contribute to the
strength and complexity of xenograft compared with allograft rejection.
Innate immunity is a fertile area for the development of novel
therapies to prevent adaptive immunity and graft rejection.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Leonard C. Harrison, Autoimmunity and Transplantation Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia.
3 Abbreviations used in this paper: FPP, fetal pig pancreas; M, macrophage; IPT, i.p. transplant; NOD, nonobese diabetic; PAMP, pathogen-associated molecular pattern; SSC, side scatter.
Received for publication April 20, 2000. Accepted for publication November 6, 2000.
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 R. M. Sutherland, B. S. McKenzie, Y. Zhan, A. J. Corbett, A. Fox-Marsh, H. M. Georgiou, L. C. Harrison, and A. M. Lew Anti-CD45RB antibody deters xenograft rejection by modulating T cell priming and homing Int. Immunol., August 1, 2002; 14(8): 953 - 962. [Abstract] [Full Text] [PDF]
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 A. Fox-Marsh and L. C. Harrison Emerging evidence that molecules expressed by mammalian tissue grafts are recognized by the innate immune system J. Leukoc. Biol., March 1, 2002; 71(3): 401 - 409. [Abstract] [Full Text] [PDF]
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Y. Zhan, J. L. Brady, R. M. Sutherland, and A. M. Lew Without CD4 Help, CD8 Rejection of Pig Xenografts Requires CD28 Costimulation But Not Perforin Killing J. Immunol., December 1, 2001; 167(11): 6279 - 6285. [Abstract] [Full Text] [PDF]
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), allogeneic (
), or syngeneic (
) splenocytes or
PBS (
; mean and SD; n = 4–6/group and time).
Results are for NOD mice, except for NK cells, which are for BALB/c
mice.
