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,
*
University of Melbourne, Department of Surgery, Royal Melbourne Hospital, Victoria, Australia;
Cardiovascular Biology Laboratory, Harvard School of Public Health, and
Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; and
§
Monash Medical School, Alfred Hospital, Prahran, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and
allograft inflammatory factor-1) were up-regulated in grafts from
IL-4-/- recipients. Computer-assisted analysis of
elastin-stained vessels revealed that the severity of vascular
thickening (percentage of luminal occlusion, mean ± SD,
n = 329) was similar in grafts from
IL-4-/- (63.7 ± 16.9%) and wild-type (69.5 ±
17.6%) recipients. Thus, IL-4 deficiency did not alter primary or
secondary graft survival, infiltration, or vascular thickening. The
selective alterations in monokine expression suggests that alternative
pathways are activated and may compensate in IL-4-/-
mice. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-12 and are down-regulated by IL-4, IL-10, and TGF-ß
(3). In the transplant field, descriptive studies have shown a
predominance of Th1 cytokines in grafts undergoing acute rejection. In
grafts surviving long term, decreases in the Th1 cytokines (IL-2 and
IFN-) with concomitant increases in the Th2 products, IL-4, and
IL-10 have been reported (4, 5, 6). These findings have led to the
hypothesis that Th1-type responses are required for acute rejection,
while Th2 type responses maintain graft acceptance and are possibly
involved in the progression of chronic rejection and
transplant arteriosclerosis (7).
However, the hypothesis that IL-4 may mediate peripheral
tolerance in recipients of long surviving allografts following
transient T cell depletion has been controversial (6, 8, 9, 10). Studies
have shown IL-4 expression in rejecting grafts (11, 12) and mixed
Th1/Th2-type responses, including IFN-, IL-2, and IL-4 expression,
in the allografts that survived long term (13). Functional studies
using mAb or recombinant protein treatments have further contributed to
the perplexity. Anti-IL-4 mAb administration blocked neonatal tolerance
induction, supporting the hypothesis (14). In contrast, administration
of soluble IL-4R, to neutralize IL-4 function in vivo, unexpectedly
prolonged graft survival in mice instead of decreasing it (15). Similar
prolonged allograft survival was seen in IL-4R transgenic mice (15).
Administration of rIL-4 did not significantly alter heart or skin graft
survival (16), and transfusion of IL-4-producing Th2 cells into SCID
recipients caused rapid graft rejection (17). Hence, further
investigations are required to clarify the role of IL-4 in peripheral
tolerance induction and maintenance.
IL-4 has also been implicated as a mediator of chronic rejection. This form of rejection produces a more insidious form of graft decline that occurs later in the transplant course. The histologic hallmarks are progressive parenchyma fibrosis and transplant arteriosclerosis (18). Some studies in rodent models have shown that IL-4 expression is higher in allografts that develop prominent vascular thickening (7, 13, 19). To date there are no functional studies that establish a causative role for IL-4 in the process of myofibrotic expansion of the neointima, the hallmark of chronic rejection.
Recently, the availability of mice with targeted gene deletion of IL-4 (20, 21) has allowed direct study of the effects of this cytokine in vivo. The mice are phenotypically normal, without aberration in B or T cell development. However, after challenge, lymphocytes from IL-4-/- mice produce aberrant Th2 responses to Nipponstrongylus brasiliensis infection or anti-CD3 cross-linking. IL-4-/- mice are particularly useful in studies of tolerance and transplant arteriosclerosis, since both these conditions require long term survival. The first reports using IL-4-/- mice as recipients in transplantation did not reveal differences in the rate of islet or cardiac rejection (22). Lakkis et al. (23) extended this observation by showing that IL-4 was not required for long term survival of murine cardiac allografts that received CTLA4 Ig induction therapy. We have recently confirmed that long term survival can also be induced without IL-4 after a 30-day course of anti-CD4/8 (24). However, no studies to date have examined the functional role of IL-4 in peripheral tolerance or in the development of transplant arteriosclerosis (chronic rejection).
The purpose of this study was to test the interrelated hypotheses that IL-4 is required for peripheral tolerance induction and development of vascular thickening associated with chronic rejection. To study these questions, we compared vascularized cardiac allografts placed in IL-4-/- recipients with controls placed in wild-type recipients focusing on the subgroups in which long term graft survival was achieved after anti-CD4 and anti-CD8 mAb induction therapy (24). Parameters of peripheral tolerance induction, vascular thickening, and leukocyte infiltration and activation were compared in allografts from tolerant IL-4-/- and wild-type recipients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CBA/CaJ (H-2k) and BALB/c (H-2d) were used as donors of heart allografts. Recipients were C57BL/6J (H-2b; The Jackson Laboratory, Bar Harbor, ME) with and without IL-4 gene deletion (21). Isografts were performed from IL-4-/- donor to IL-4-/- recipient. The targeted gene deletion was backcrossed 12 times onto C57BL/6J, and its presence was confirmed in our laboratory using multiplex PCR assays that amplified a portion of the neomycin cassette and a portion of the targeted exon (www.jax.org).
Murine cardiac transplantation
Vascularized abdominal heterotopic heart grafts from 8- to 12-wk-old male mice were transplanted into age-matched male recipients (n = 33) as previously described (25, 26). Twenty-four of these transplants had been completed as part of the larger body of work comparing graft survival and inflammatory activation among three cytokine knockout recipients (24). In the earlier study we found heterogeneity in onset of late rejection in the IL-4-/- group. The purpose of this study was to characterize further a subset of long term surviving grafts. Transplant function was evaluated by regular palpation graded on a scale from 4 (functioning well) to 0 (not beating) (25).
mAb treatment and assessment of peripheral tolerance
C57BL/6J recipients were either left untreated or were given mAb against CD4 (clone GK1.5, rat IgG2b, American Type Culture Collection, Rockville, MD) and anti-CD8 (clone 2.43, rat IgG2b, American Type Culture Collection) for 1 to 4, 7, 21, and 28 days after transplantation to attenuate acute rejection. Treated recipients received 500 µg of each mAb/mouse i.p. on the indicated days (24). Mice with transplants surviving >80 days were tested for donor Ag-specific tolerance with a second heart attached to the jugular and carotid vessels in the neck of the recipient either from donor-strain (BALB/c) or third party (CBA/CaJ (H-2k)) mice (27). CD4 cells typically recover to within normal range by 3 to 4 wk after cessation of mAb treatment (6, 28). We performed second transplants >80 days after transplantation (>50 days after last mAb injection) when mice are capable of rejecting a third party heart transplant that is used to challenge the recipient. Although placement of second cardiac grafts is technically more difficult, this form of challenge has proved more reliable than skin grafts in demonstrating tolerance (29). Primary and secondary allografts from tested recipients were harvested, and histologic samples were taken when second donor hearts had survived >30 days or for third party hearts when they rejected (palpation score, <1).
Immunohistochemistry
To detect cell surface Ags and cytokines, transverse cryostat
(4-µm) sections from the basal portion of the heart were embedded in
OCT medium (Tissue Tek, Miles, Elkhart, IN) and quickly frozen in
isopentane cooled below -90° C. Sections were fixed with 4%
paraformaldehyde and treated with saponin to increase cell membrane
permeability before addition of primary Ab (30). The Abs used for cell
typing were KT3 (CD3), affinity purified from ascites from nude rats at
the Department of Surgery, Royal Melbourne Hospital (Melbourne,
Australia). The Abs used for cytokine analysis included XMG1-2
(IFN-) obtained from Dr. Charmaine Simeonovic (Australian National
University, Canberra, Australian Capitol Territory); and 125.A8
(IL-2R), F4/80 (macrophages), and B220 (B cells) obtained from Dr.
Thomas Mandel (The Walter and Eliza Hall Institute, Australia). These
were used as supernatant from cell cultures. S4B6 (IL-2) was purchased
from PharMingen (San Diego, CA), and ammonium sulfate-precipitated
11B11 (IL-4) was obtained from Dr. Christina Cheers (University of
Melbourne, Melbourne, Australia). Binding of mAbs was detected by the
four-layer peroxidase-antiperoxidase method described previously (31).
Labeled cells within 10 high power fields/section/mouse were counted
and expressed as mean cells per high powered (x400) field of view
± SD. Control sections, which showed some background staining, were
treated with primary Ab omitted.
RT-PCR
To measure relative differences in transcript levels between long term surviving cardiac transplants from IL-4-/- and wild-type recipients, we used a semiquantitative 32P RT-PCR technique described in detail previously (13, 24, 32). PCR amplification for the glyceraldehyde-3-phosphate dehydrogenase (G3PDH)3 was performed to assess potential variations in cDNA or total RNA loading between samples. The mean G3PDH obtained from at least three analyses was assessed to normalize other transcript level measurements obtained from the same cDNA sample. Corrected values were derived by dividing the measured amount of incorporated 32P for the transcript of interest by the mean G3PDH value for the same cDNA sample.
Vascular analysis
Paraffin sections were treated with Verhoeff elastin stain to visualize the internal elastic lamina (33). The severity of vascular thickening (percentage of luminal occlusion) as well as the frequency (percentage of diseased vessels) were analyzed in two sections from each graft that met the criteria for experimental tolerance. Microscopic images of each elastin-stained vessel (total of 329, mean of 12.7 vessels/graft) were captured, and the percentage of luminal occlusion was tabulated by tracing the internal elastic lamina and the lumen with the ScionImage 1.59 software (National Institutes of Health, Baltimore, MD). The frequency of the disease in the graft was defined as the percentage of diseased (>0% luminal occlusion) vessels. The mean ± SD for all grafts in each transplant subgroup are reported.
Statistical analysis of RT-PCR and morphometric data
Results are given as the mean per subgroup ± SD, which was derived from the mean per graft. The data were subjected to multiple analysis of variance without replication (StatView 4.1, Abacus Concepts, Berkeley, CA). If the multiple analysis of variance was significant, individual comparisons were made by Student’s t test, and the level of significance was corrected by Bonferroni’s method. To evaluate significance in tolerance induction, Fisher’s exact test was used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peripheral tolerance assessments comparing
IL-4-/- and wild-type mice on the inbred C57BL/6
background are shown in Table I
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Untreated IL-4-/- and wild-type recipients rejected
BALB/c allografts in 8 to 9 days, confirming that IL-4 is not required
for acute rejection. In cardiac allografts from wild-type recipients
treated with anti-CD4/CD8, five of eight (63%) hearts survived
long term with a mean survival time of 99 ± 26 days as reported
previously (24). Three mice accepted donor (BALB/c) second hearts for
>30 days, while two mice challenged with third party hearts (CBA)
rejected them in 11 and 16 days. Thus, treatment with anti-CD4/CD8
produced acceptance of a second donor-strain allograft, but rejection
of third party graft consistent with donor-specific peripheral
tolerance defined by conventional criteria in the tolerance field
(34, 35).
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Evaluation of inflammatory activation in graft parenchymal tissue
Immunoperoxidase staining was compared in primary cardiac
allografts with survival >110 days from the IL-4-/-
and wild-type recipients. In both groups, allograft tissue showed areas
of mononuclear cell infiltration involving the parenchymal and
perivascular regions. The infiltrate in these hearts consisted of both
T cells (CD3 positive, Fig. 1a) and macrophages
(F4/80 positive, Fig. 1b), with a few scattered B
cells (B220) and IL-2R-positive cells (not shown). As summarized in
Table II, counting the number of cells
per field of view confirmed that the levels of CD3-, F4/80-, B220-, and
IL-2R-positive cells were equivalent in hearts from both
IL-4-/- and wild-type groups. Rejecting grafts, which
failed at <80 days with palpation scores of 0 at harvest, showed the
expected higher expression of CD3 and a trend toward higher macrophage
(F4/80) expression compared with long term surviving grafts. Again,
similar immunoperoxidase staining was seen in allografts from
IL-4-/- and wild-type recipients. As expected, isografts,
had significantly lower numbers of infiltrating cells. In addition,
primary and secondary allografts in these two groups of mice showed
similar patterns of mononuclear cell infiltration. Interestingly, in
both primary and secondary allografts from IL-4-/- and
wild-type recipients, the inflammatory cell infiltration was not
associated with impaired ventricular function. The mean palpation score
(on the scale 0–4) for primary BALB/c hearts in the
IL-4-/- group was 1.8 ± 0.8, which was similar to
that in the wild-type group (2.0 ± 0.9). Secondary BALB/c hearts
were also beating well (2.3 ± 1.2 in IL-4-/- and
2.3 ± 0.9 in wild-type group). These are crude demonstrations of
preserved heart beat.
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). Cytokines
were generally expressed in areas of focal infiltrate in perivascular
or parenchymal regions. Figure 1, d and f, shows
cytokine expression in serial sections of a representative long term
surviving cardiac allograft in a wild-type recipient. IL-2 (Fig. 1d) was strongly expressed in isolated clusters of
small mononuclear cells. IL-4 staining was present in areas of focal
infiltrate with diffuse positivity. Clear staining of IL-4 could be
seen around individual cells in parenchymal regions in hearts from
wild-type recipients (Fig. 1e). No IL-4 staining
above background (Fig. 1c) levels was seen in hearts
from the IL-4-/- recipients. IFN- was also seen
associated with clusters of small and large mononuclear cells in
perivascular areas, with some mononuclear cells being strongly positive
in hearts from both IL-4-/- and wild-type recipients
(Fig. 1f). Hence, clear positive cytokine expression
was seen in allografts from both IL-4-/- and wild-type
recipients.
To identify and quantitate potential alterations in the alloimmune
response associated with IL-4 deficiency, we used semiquantitative
32P RT-PCR assays to measure relative differences in
intragraft gene transcripts levels (n = 5 in
each group; Fig. 2). Selected monokine
expression patterns were studied, as IL-4 typically suppresses
macrophage responses (36). In the transplanted hearts from
IL-4-/- recipients IL-12, allograft inflammatory factor-1
(AIF-1) and TNF- transcript levels were significantly higher
compared with those in wild-type controls. However, monocyte
chemoattractive protein-1 and inducible nitric oxide synthase levels
were not altered by the absence of recipient sources of IL-4 (Fig. 2),
indicating that IL-4 may selectively regulate monokine expression.
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). T cell activation, as
measured by Th1-type cytokine transcripts and IFN- and IL-2
transcripts were comparable in both groups. Transcripts for IL-4 were
not above background levels in IL-4-/- recipients, as
would be expected in mice with a IL-4 gene deletion. The Th2-type
cytokine, IL-10, produced by both T cells and monocytes, was not
significantly altered in IL-4-/- recipients compared with
wild-type recipients. Detection of cytokines by immunoperoxidase
staining confirmed the presence or the absence of the protein implied
by detection of mRNA in the RT-PCR results. Taken together, these
results imply that allografts from IL-4-/- recipients
show decreased macrophage suppression, but have T cell activation
levels comparable to those in wild-type recipients. Vascular characteristics of cardiac allografts
Elastin staining of long term surviving cardiac allografts
from IL-4-/- and wild-type recipients showed diffuse,
prominent, obliterative vascular lesions. There was concentric vascular
thickening diffusely involving both small and large vessels. The
expanded neointima involved interposition of mononuclear cells and
occasional myointimal cells (Fig. 3A). Quantitative
comparisons were made by tabulating the severity of vascular thickening
(Fig. 3B). Computer-assisted morphometric analysis of
329 elastin-positive vessels showed that the mean percent luminal
occlusion was 63.7 ± 16.9% from grafts in IL-4-/-
recipients (n = 5) compared with 69.5 ±
17.6% in wild-type recipients (n = 5,
p = NS). As expected, IL-4-/- isografts
had significantly less luminal occlusion (8.7 ± 5.7%,
n = 6, p < 0.0001 compared with both
allograft groups). The frequency of the disease measured as the
percentage of diseased vessels (luminal occlusion, >0%) in the grafts
from IL-4-/- recipients was 92.3 ± 12.5%
(n = 56 vessel cross-sections) and thus
comparable to 99.2 ± 1.9% (n = 63) in
wild-type recipients (p = NS). In isografts,
only 24.5 ± 7.5% of the vessels (n = 68)
were affected by minor amounts of thickening (p
< 0.0001 compared with both allograft groups).
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We evaluated the histologic status of the second hearts used to
assess donor Ag-specific tolerance. These were transplanted 80 days
after the first transplant without further immunosuppression given to
the recipients. These second donor-strain hearts harvested >30 days
after challenge showed histologic features of parenchymal rejection
similar to those in the primary hearts harvested at 110 days (data not
shown). The extensive T cell and macrophage infiltrate seen in these
well-beating grafts was equivalent to that seen at >110 days in
primary cardiac allografts (Table II). Second donor-strain hearts
showed similar RT-PCR patterns in both IL-4-/- and
wild-type recipients (Table III).
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). The
severity of vascular thickening measured as the mean percent luminal
occlusion was 63.3 ± 17.2% in second grafts
(n = 3, 45 vessels) placed in
IL-4-/- recipients compared with 48.2 ± 18.1% in
second grafts (n = 3, 55 vessels) placed in
wild-type recipients (p = ns). The degree of
vascular occlusion was similar to that in the primary heart grafts that
had been in place for >110 days. This shows an accelerated alloimmune
response to the second donor-specific challenge hearts despite
continued graft function. Hence, in this study the recipient state of
experimental tolerance did not completely protect the second
donor-strain grafts from cardiac rejection given that there were
features of acute and chronic rejection by histologic assessment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our finding that IL-4 does not have an essential role in the
development of vascular thickening in this model adds to a growing body
of evidence from the studies examining the functional role of IL-4 in
transplants. Others have demonstrated, and we have confirmed, that IL-4
is not required for acute cardiac, skin, or pancreatic islet rejection
in nonimmunosuppressed recipients (22, 23, 37). Studies by Lakkis et
al. demonstrated that CTLA4 Ig prolonged cardiac graft survival to a
similar extent in wild-type and IL-4-/- recipients.
Tolerance tests employing challenge with second graft donor tissue were
not performed, but the findings did indicate that IL-4 was not
obligatory for long term graft acceptance (23, 37). This is similar to
the results of our previous study, using a 30-day course of
immunosuppression, showing that allografts placed in
IL-4-/- recipients had graft survival equal to that in
wild-type animals (24). In the same study we demonstrated that gene
deletion of IFN- or IL-10 in the recipient environment was
associated with decreased allograft survival. Hence, in all three
processes (long term graft survival, acute rejection, and chronic
rejection), removal of functional IL-4 did not inhibit the cellular
processes leading to these disorders, suggesting that IL-4 is neither
necessary nor sufficient to sustain these responses.
Our findings support the idea that IL-4-/- mice have a selective defect in Th2 responses as proposed by Kopf and coworkers in their analysis of IL-4 knockout mice (20). They found that Th2 responses in vitro to anti-CD3-mediated activation were impaired, but delayed-type hypersensitivity responses were intact. Interestingly, IL-4-/- mice are resistant to killing or abscess formation from Salmonella typhimurium, showing that IL-4 may actually promote instead of protecting against the pathologic process to this insult (38).
Factors other than IL-4 are likely to play a role in allograft
acceptance and development of vascular thickening. We have examined
only IL-4-mediated responses, but other inflammatory mediators alone or
in combination may contribute to allograft acceptance or chronic
rejection. One major activity associated with the Th2-type response is
deactivation or suppression of macrophage responses. To screen for
possible compensatory factors, we used RT-PCR assays to compare
relative transcript levels in a series of graft cDNAs from each
tolerant recipient group. Our evaluation of selected macrophage factors
showed differences in allografts from IL-4-/- and
wild-type recipients, indicating that macrophage suppression was
altered by IL-4 deficiency. We found an increase in monokines (IL-12,
AIF-1, and TNF-) in IL-4-/- recipients compared with
wild-type animals. However, transcript levels for monocyte
chemoattractive protein-1 and inducible nitric oxide synthase were
similar in IL-4-/- and wild-type recipients. These
findings suggest that the lack of IL-4 interrupted only some of the
macrophage suppression pathways. It is possible that these or other
macrophage factors may have contributed to vascular thickening in the
absence of IL-4.
Interestingly, we did not see an increase in other T cell cytokines,
i.e., IL-2, IFN-, and IL-10, in primary heart allografts from
IL-4-/- recipients compared with that in wild-type
animals. This was concordant with the study by Lakkis et al. (23) in
which long surviving hearts from IL-4-/- and wild-type
mice treated with CTLA4-Ig did not have significant differences in
transcripts for IL-2, IL-10, IL-13, or IFN-. These findings are
inconsistent with the hypothesis that IL-4 inhibits Th1-like responses.
However, it is possible that other factors have taken the suppressive
role of IL-4 in the knockout animals. Taken together, the search for
compensatory factors will ultimately depend upon establishing causal
linkages demonstrating that putative factors control the processes of
tolerance and/or transplant arteriosclerosis. This will require more
functional studies demonstrating that the absence of a factor
interrupts the process and that its presence promotes it.
Descriptive studies first implicated IL-4 as a key mediator for tolerance based on the presence of IL-4 gene transcripts, protein product, or both in long surviving allografts from tolerant rats and mice (4, 6, 10). IL-4 expression was also increased in cardiac allografts undergoing chronic rejection, and the levels were reduced in concert with arteriosclerosis by various immunosuppressive strategies (13, 19). Our studies using IL-4-/- recipients have brought new important functional data to this area in the transplant field.
In this study, experimental induction of peripheral tolerance, defined as long term function of a primary allograft with acceptance of a secondary donor allograft and rejection of a third party graft (34, 35) could be achieved in >50% of IL-4-/- and wild-type recipients after a 30-day course of immunosuppression. Despite continued graft function, both primary and second hearts showed evidence of ongoing rejection: mononuclear infiltration, inflammatory activation, and vascular thickening. Others have also shown that rat kidney grafts can develop primary graft destruction and chronic rejection and yet fulfill the conventional definition of peripheral tolerance by accepting donor second grafts and rejecting third party grafts (39).
These findings raise an interesting question: have these recipients achieved a clinically applicable level of tolerance? We are of the opinion that the presence of active rejection is incompatible with the tolerant state. In addition to acceptance of a second donor graft and rejection of a third party graft, the parenchymal and vascular tissue should be substantially free of histologic signs of acute and chronic rejection. Hence, the definition of tolerance in experimental model systems should be redefined. As we and others using peripheral tolerance models have not typically achieved this degree of peripheral tolerance, the question of whether true peripheral tolerance can prevent the development of chronic rejection is still unanswered.
Gene manipulation of IL-4 (deletion in our study or addition in others (40)) has failed to modify rejection responses. Along these lines, Davies et al. reported that anti-IL-4 mAb treatment could not break established peripheral tolerance in vivo. In studies of the transfer of infectious tolerance, only partial inhibition of transferable cell activity was seen. They concluded that IL-4 played a role in tolerance, but the tolerant state was not maintained by the continuous systemic presence of IL-4 (41). Thus, it appears that IL-4 is not the sole mediator of peripheral tolerance.
In conclusion, long term graft survival can be induced in MHC class I- and II-mismatched cardiac allograft recipients regardless of recipient IL-4 deficiency. However, histologic analysis showed that true peripheral tolerance was not achieved despite preserved graft function. Although similar patterns were seen in histology and immunohistochemistry among allografts from IL-4-/- and wild-type recipients, the increased sensitivity of the RT-PCR analysis of heart transplants revealed impaired macrophage suppression in IL-4-/- recipients compared with that in wild-type recipients. These or other potential pathways may have contributed to the ongoing alloimmune response observed in these long term surviving heart transplants. In addition, even though the recipient environment was devoid of IL-4, cardiac allografts developed prominent vascular thickening associated with chronic rejection. Further functional studies need to be completed to define the cytokines alone or in combination that mediate tolerance and chronic rejection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mary E. Russell, Cardiovascular Biology Laboratory, Harvard School of Public Health, 677 Huntington Ave., Boston, MA 02115. E-mail address:
3 Abbreviations used in this paper: G3PDH, glyceraldehyde-3-phosphate dehydrogenase; AIF-1, allograft inflammatory factor-1.
Received for publication October 10, 1997. Accepted for publication March 11, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in long-surviving mouse heart allografts after brief CD4-monoclonal antibody therapy. Transplantation 59:559.[Medline]
, interleukin 4 and interleukin-10 knockout mice: recipient environment alters rejection. J. Clin. Invest. 100:2449.[Medline]
knockout recipients. Am. J. Pathol. 152:359.[Abstract]
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