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*
Laboratory for Experimental Transplantation and
Rega Institute, University of Leuven, Leuven, Belgium
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Xenotransplant accommodation refers to a phenomenon in which an Xg acquires resistance to Ab and complement-mediated humoral immunity (7). The existence of accommodation appeared from observations that in some cases, when anti-donor XAbs and/or complement had been depleted from a Xg recipient for a period of time, the graft continued to function even after the Ab titer and complement function were restored (7, 8, 9, 10, 11). In a recent study involving CsA-treated rats in which the complement system was temporarily blocked by cobra venom factor, accommodation of hamster heart Xgs was demonstrated. This accommodation involved Th2 lymphocytes and cytokines, and seemed to be maintained by the induction of anti-apoptotic genes (12). The development of T cell xenotransplantation tolerance was also recently demonstrated by grafting thymectomized, T cell-depleted mice with xenogeneic fetal pig thymus and liver tissue. Subsequently, these mice developed specific T cell tolerance for pig Ags and permanently accepted pig skin grafts (13). However, for vascularized Xgs, many non-T cell-mediated immune responses, involving T-independent B cells, NK cells, and macrophages, may lead to rejection of Xgs before T lymphocytes are even activated by xenoantigens (14, 15).
Previous studies from our and other laboratories have shown that a combination therapy of CsA with leflunomide (LF), a novel immunosuppressant, allowed for long term survival of hamster heart Xgs in rats (16, 17). This synergistic effect appeared to result from the different actions of LF and CsA to block T-independent and T-dependent XAb formation, respectively (16). In addition, it was shown that after a short course (2–4 wk), LF could be withdrawn, and xenografts survived indefinitely with CsA treatment alone (16). The mechanisms allowing for this progressive withdrawal of LF from the therapy were investigated here by experiments involving second xenotransplantation, adoptive transfer of hyperimmune serum, and retransplantation of the long term surviving Xgs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inbred 2- to 4-mo-old male PVG rats, weighing ±200 g, were used as recipients. Golden Syrian hamsters, weighing ±100 g, were used as donors. Animals were kept in conventional housekeeping facilities.
Heterotopic heart transplantation
First, hamster hearts were transplanted in the neck of recipient rats. The recipients were anesthetized by Enflurane (Abbott S.A., Ottignies, Belgium) supplemented with oxygen through a semiclosed circuit inhalation system. The aorta and pulmonary artery of the donor were anastomosed to the carotid artery and jugular vein of the recipient by either microsuture or cuff techniques. A second hamster heart was transplanted to the contralateral side of the recipient neck or in the abdominal cavity. Retransplantation was performed by implanting long term surviving hamster heart Xgs to naive recipient rats. The function of the grafts was monitored by daily inspection and palpation. Rejection was determined by the cessation of beating of the graft and was confirmed by histology.
Immunosuppressive therapy
LF (HWA 486, Hoechst, Germany) was provided by Dr. Bartlett (Hoechst, Wiesbaden, Germany) and was suspended in 1% carboxymethylcellulose just before gavage. CsA (Sandimmun, Sandoz, Belgium) was diluted in olive oil and given by gavage.
Serum samples
Whole blood (0.5–2 ml) was taken by heart puncture at various times after transplantation. After 40 min at room temperature, the serum was separated from the blood by centrifugation, and aliquots were stored at -70°C until use.
Isotype analysis of XAbs
The IgM and IgG isotypes of anti-hamster XAbs were determined by flow cytometry using hamster PBMC as targets. Aliquots of 0.5 x 106 PBMC were cultured for 30 min at 4°C with 100 µl of 1/10 diluted serum taken from recipient rats at various times after transplantation. After a secondary staining with FITC-conjugated anti-rat IgM (The Binding Site Ltd., Birmingham, U.K.) or anti-rat whole IgG (ST-AR 17, Serotec, Oxford, U.K.) Abs, the cells were examined by flow cytometry. Results were expressed as relative mean channel fluorescence, which was calculated as the mean channel fluorescence of stained cells divided by the mean channel fluorescence of cells incubated with control serum and counterstained with FITC-conjugated anti-rat IgM or IgG Abs.
Serum adoptive transfer experiment
Anti-hamster IgM XAb-containing serum (0.5 ml), taken from CsA-treated rats that rejected hamster heart Xgs with the production of IgM, but not IgG, XAbs, were injected i.v. into rats carrying long term (6 wk) surviving hamster Xgs or into naive rats carrying a newly transplanted Xg (1 h after transplantation).
Histopathology and immunohistochemistry
Graft samples for histology were fixed in 10% formalin solution, paraffin embedded, sectioned, and stained with hematoxylin and eosin. Graft samples for immunopathology were snap-frozen in prechilled isopentane and stored at -70°C. Specimens were cut into 4-µm sections in a cryostat at -25°C, air-dried, fixed in ice-cold acetone for 10 min, and washed with PBS. For immunofluorescence staining, each section was incubated with FITC-conjugated anti-rat IgM (The Binding Site), IgG (ST-AR 17, Serotec), C3 (Cappel, Organon Teknika, Turnhout, Belgium), and anti-human C6, shown to be cross-reactive with rat C6 (18). Before use, the Abs were absorbed with hamster tissues. Sections were washed with PBS and mounted in 50% glycerol in PBS. Sections of naive hamster hearts were used as negative controls. For immunoperoxidase staining, sections were incubated overnight with a rabbit Ab for P-selectin (PharMingen, San Diego, CA) and rat mAb for E-selectin (PharMingen). The sections were then stained with labeled streptavidin-biotin reagents (Dako LSAB kit, peroxidase, Dako, Carpenteria, CA).
Statistics
Results were statistically analyzed by two-sample Student’s t test or by the log rank or Wilcoxon’s test when appropriate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Table I
shows Xg survival after
withdrawal of LF from the CsA/LF combination immunosuppression. As
demonstrated previously (16), when CsA was combined with LF and
administrated continuously, all hamster heart Xgs survived (group 1).
When LF was withdrawn after 1 wk (group 2), Xgs were rejected after a
mean survival time (MST) of 10 ± 1 days. In contrast, when LF was
withdrawn after 2 (group 3) or 4 (group 4) wk, respectively, 7 of 9 and
12 of 12 grafts survived under CsA monotherapy. However, if both drugs
were withdrawn on day 30, Xgs were invariably rejected (5 of 5) after
an MST of 39 ± 2 days (group 5).
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While a combination of LF/CsA resulted in a continuous absence of
XAb formation (Fig. 1A), when LF was
withdrawn after 1 wk, a rapid increase of mainly IgM anti-donor
XAbs was noticed (Fig. 1B). In contrast, when LF was
given for 2 (Fig. 1C) or 4 (data not shown) wk, no
XAbs occurred after withdrawal of LF. However, when both LF and CsA
were withdrawn after 4 wk, a rapid rise of predominantly IgG
anti-donor XAbs was detected (Fig. 1D).
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As a 2- or 4-wk course of LF was sufficient to block IgM XAb
formation and to induce long term Xg survival in CsA-treated rats, it
was hypothesized that LF had induced tolerance in T-independent B
lymphocytes. To verify this hypothesis, second xenotransplantations
were performed 6 wk after the first xenografting. As shown in Figure 2A, when the second Xgs were
transplanted in the rats that received only 2-wk LF treatment, the
second Xgs invariably underwent an acute rejection within 4 days after
transplantation (MST = 3 ± 1 days; n = 5).
Interestingly, this process of second Xg rejection did not influence
the survival of the first Xgs. In contrast, in the 4-wk LF-treated
group, four of five second Xgs were accepted indefinitely. Again,
second xenografting did not influence the survival of the first Xgs. In
some cases (Fig. 2C), the surviving first Xg was
removed 3 days before second xenotransplantation in the 4-wk LF group.
This did not significantly affect the acceptance of the second
Xg.
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XAb formation after the second xenotransplantation is shown in
Figure 3. After a second Xg, rats that
received only a 2-wk course of LF developed significant levels of
anti-donor IgM XAbs (Fig. 3A), which correlated
with the rejection of these second Xgs. In contrast, in the rats that
initially received a 4-wk course of LF, no IgM XAbs were detectable
(Fig. 3B). In both cases, IgG XAbs were not detected
(Fig. 3, C and D), indicating that the CsA
treatment efficiently blocked T-dependent IgG XAb formation.
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The observation that in 2-wk LF-treated rats, IgM XAbs provoked
after the second xenotransplantation destroyed only the second, but not
the first, Xgs suggested that the first Xgs had become resistant to
this IgM XAb-mediated rejection, and, hence, that some form of
accommodation was achieved. This was further investigated by an
experiment involving adoptive transfer of anti-donor IgM XAbs taken
from CsA-treated rats rejecting a hamster heart Xg. We previously
showed that these rats produced only IgM XAbs (16). Transfer of 0.5 ml
of this serum provoked hyperacute Xg rejection in naive rats treated
with CsA only (MST = 30 ± 20 min; n = 5;
Table II). In contrast, the same
procedure did not cause rejection of primary Xgs in the 2-wk LF-treated
group when injected 6 wk after transplantation (Table II). Hence, the
latter Xgs had become resistant to IgM XAbs. Together with the presence
of the normal level of lytic complement in these rats (data not shown),
this observation fits with the definition of accommodation.
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Interestingly, after retransplantation in recipients treated with
CsA only, the accommodated Xgs were rejected within 2 to 5 days
(MST = 3.5 ± 1 days; n = 5) and provoked an
induction of significant levels of anti-hamster IgM XAbs (Fig. 4). Hence, it appeared that the
xenoantigens that were resistant to IgM XAbs in the accommodated hearts
were able to provoke an IgM XAb-mediated rejection. This suggested that
the process underlying accommodation of Xgs was reversible after
retransplantation.
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The histology of all rejected hamster hearts, including first,
second, and retransplanted Xgs, showed similar signs of acute vascular
rejection. The histology of a hamster heart rejected by an untreated
rat is shown in Figure 5A;
intravascular thrombosis, interstitial hemorrhage, edema, myocardial
necrosis, and polymorphonuclear cell infiltration were noticed. In
contrast, long term surviving Xgs (Fig. 5B) showed
normal vessels without intima proliferation, a healthy myocardium, and
the absence of immune cell infiltration.
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A) and complement C6
(Fig. 6B). Long term surviving first Xgs showed an
absence of this deposition, even after rejection of second Xgs in the
2-wk LF group (Fig. 6, C and D). In the
2-wk LF group, deposition of IgM (Fig. 6E) and C6
(Fig. 6F) was clearly found in rejected second
hamster hearts. In contrast, in the 4-wk LF group, both the long term
surviving first (Fig. 6, G and H) and the
accepted second Xgs (Fig. 5
, I and J)
showed an absence of deposition of IgM and complement. After
retransplantation in CsA-treated recipients, accommodated hamster
hearts were rejected and exhibited vessel wall deposition of both IgM
(Fig. 5K) and C6 (Fig. 5L).
Immunoperoxidase staining showed that after retransplantation, there
was a progressive induction of the expression by the adhesion molecules
of P-selectin (Fig. 7,
A–D) and E-selectin (Fig. 7, E–H)
on vessel endothelial cells of accommodated hamster hearts.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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First, the capacity of rats carrying long surviving Xgs to reject a second hamster heart graft was investigated. A 2-wk course of LF failed to prevent IgM XAb formation after second xenografting, and these XAbs bound to and provoked rejection of these second Xgs. In contrast, the primary grafts survived in the presence of these XAbs and functional complement. This fits with the definition of Xg accommodation. Accommodation was further confirmed by the experiment involving transfer of IgM XAbs. IgM XAb (0.5 ml) containing serum provoked hyperacute Xg rejection in naive rats, but did not affect the survival of the accommodated grafts. The phenomenon of accommodation was first described in recipients of Red Blood cell group ABO-incompatible kidney allografts by Alexandre and co-workers (8, 10, 11). It was also observed in some cases following transplantation of organs into patients with circulating anti-donor HLA Abs (19). Graft accommodation was demonstrated after xenotransplantation (10, 20, 21). In experiments using plasmapheresis and immunosuppression to prolong the survival of swine-to-baboon renal Xgs, one recipient had a graft function for >20 days despite the presence of circulating anti-graft Abs (10). Accommodation may have an important implication for the eventual clinical application of xenotransplantation, since it would allow progressive withdrawal of immunosuppressive regimens aimed at depleting and/or suppressing XAbs and/or complement from Xg recipients.
Until recently, the mechanisms underlying accommodation were unclear, but several possible explanations have been forwarded. One mechanism may involve a decrease in Ab-Ag interactions. This may be due to a change in the affinity or specificity of the anti-graft Abs or to a change of the expression of the target Ags in the donor organ (22). It was recently reported that accommodation of porcine endothelial cells was induced in an in vitro cell culture system by incubation with low concentrations of polyclonal human IgG Abs (23). The induced resistance of the accommodated porcine endothelial cells to complement-mediated lysis was correlated with a down-regulation of expression of an vascular cell adhesion molecule (23). A second mechanism underlying accommodation may be that with continued stimulation of endothelial cells by low levels of Abs and/or complement, the sensitivity of these cells to injury is decreased (22). Such a mechanism has been demonstrated in vitro, as continued stimulation of endothelial cells with endotoxin or IL-1 caused the cells to become resistant to restimulation (24). A third mechanism of accommodation may be one involving certain protective cellular and molecular mechanisms within the grafts, as suggested by the recent work of Bach et al. (12). It was shown that in a model also involving CsA-treated rats but in which the complement system was temporarily blocked by cobra venom factor, surviving hamster heart grafts became resistant to humoral rejection despite deposition of IgM xenoantibodies and complement activation within the graft (12). Interestingly, the endothelial cells of these accommodated Xgs showed an up-regulation of anti-apoptotic genes, and the grafts were infiltrated with Th2-type lymphocytes (12).
The data presented in the present study provide in vivo evidence supporting the regulation of xenoantigen expression as a potential mechanism for accommodation. We clearly showed that the accommodated hamster hearts exhibited a strongly decreased expression or disappearance of several xenoantigens on the endothelial cells. This was supported by two points. First, anti-hamster IgM, whether provoked (by second hamster Xgs) or adoptively transferred, failed to bind to or to cause rejection of the accommodated hamster grafts. Second, by detecting adhesion molecules that are believed to be important expressers of xenogeneic epitopes (22, 25), we found that the endothelial expression of both P-selectin and E-selectin was dramatically down-regulated in the accommodated Xgs. Interestingly, this decrease in xenoantigen expression was reversible when the accommodated grafts were retransplanted to a naive recipient. Indeed, after retransplantation, the graft endothelial cells showed a progressive re-expression of both P- and E-selectins. This process correlated well with IgM XAb formation, IgM deposition, and graft rejection. These findings are consistent with a recent report showing that antisense-mediated down-regulation of GpIIIa, another adhesion molecule of potential relevance in the human-to-pig Xg recognition, results in a significant decrease in the binding of IgM natural Abs (26). As for the mechanisms leading to the reappearance of the xenoantigens, as seen in our study, they may be related to the process of harvesting, cold storage, and reperfussion of the Xgs during retransplantation (27, 28). This increased expression of xenoantigens together with an increased vulnerability to Ab-mediated injury probably explain the rejection after retransplantation of tolerated Xgs.
When comparing the characteristics of accommodation achieved in the present study with those reported by Bach et al., the mechanisms leading to accommodation may be relevant. When accommodation was obtained by blocking complement, while XAb formation was not suppressed, accommodation apparently depended on active mechanisms, such as Th2 lymphocytes and an up-regulation of protective genes in endothelial cells (12). In contrast, when accommodation was achieved by blocking XAb formation, while leaving the complement system intact as in our study, accommodation was based on a passive mechanism involving down-regulation of xenoantigens.
In the 4-wk LF group, CsA-treated rats had lost their capacity to produce IgM anti-hamster Abs even after withdrawal of LF and transplantation of a second Xg. This was not due to the fact that in this group LF was withdrawn only 2 wk before performing the second transplantation. Indeed, we have successfully performed second transplantations without rejection in 4-wk LF-treated rats 2 and 3 mo after the first transplantation (data not shown). The absence of circulating anti-donor XAbs after second xenografting was also not due to an adsorption by the tolerated first Xg, since 1) deposition of XAbs was detected in neither the first nor the second Xgs; and 2) removal of the first Xgs 3 days before the second xenotransplantation did not result in an appearance of XAbs in the circulation (data not shown), and the second Xgs were accepted as well as in rats with the first Xgs in place. Hence, B cell unresponsiveness was induced. This unresponsiveness was limited to the T-independent (CsA-insensitive) XAb formation. Indeed, the capacity to make T-dependent XAbs remained, as withdrawal of also CsA resulted in a prompt formation of mainly IgG XAbs. In rodents as well as in other mammalian species, B cells are continuously produced in the bone marrow during postnatal life. Due to their limited lifespan, peripheral immunocompetent B cells are continuously replaced by newly formed B cells emerging from the bone marrow (29, 30). In rats, the majority of the peripheral immunocompetent B cell pool turns over in approximately 4 wk (31). Several studies have demonstrated that Ag recognition by surface IgM in immature B cells induces clonal anergy rather than activation (32, 33). A first element to explain the T-independent B cell tolerance occurring after 4 wk of LF could thus be that this period of time is needed to block the activation of peripheral immunocompetent B cells. As this pool can be expected to be renewed after 4 wk, thereafter the newly formed B cells may be tolerized by the xenoantigens. However, maintenance of the latter tolerance by these xenoantigens remains unclear in the present study. Indeed, we showed that the xenoantigens that were the mediators and also the targets of the IgM XAbs were not detectable in the long surviving Xgs. This paradox may be explained by a difference in the relative concentration needed to tolerize compared with that needed to stimulate immature B cells to form XAbs.
Another explanation for the kinetics of the B cell tolerance may be that in the hamster-to-rat model, IgM XAbs are produced by a specific subpopulation of B lymphocytes with a limited self-renewing capacity. Once tolerized by LF, this subpopulation may be unable to repopulate even after withdrawal of treatment. CD5 B lymphocytes may constitute such a subpopulation. It is generally known that CD5 B cells are a primitive B cell subpopulation with very limited self-renewing capacity (33). It has also been suggested that CD5+ B cells are important for the production of T-independent IgM Abs (34, 35). In addition, we recently found that CD5+ B cells were the major source of T-independent IgM XAbs in rats. This was based on the experiments in which SCID mice were reconstituted with a highly purified CD5+ or CD5- rat B cell population and subsequently challenged with hamster cells; only the CD5+ B cell-reconstituted SCID produced anti-hamster IgM XAbs (Y. Lin and M. Waer, manuscript in preparation).
In conclusion, we suggest that depending on the duration of immunosuppression with LF, the mechanism of long term survival of hamster hearts in rats treated with CsA is based in part on graft accommodation and in part on T-independent B cell tolerance. Transient inhibition of XAb formation in the early period after xenografting allowed for the development of accommodation in Xgs. This accommodation was based on a decreased expression of xenoantigens. In a later phase, a degree of tolerance developed for T-independent B cells. Both, accommodation and T-independent B cell tolerance may allow for the progressive withdrawal of immunosuppressants after xenotransplantation.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mark Waer, Laboratory for Experimental Transplantation, University of Leuven, Onderwÿs en Navorsing, Herestraat 49, B-3000, Leuven, Belgium. E-mail address:
3 Abbreviations used in this paper: Xg, xenograft; XAb, xenoantibody; CsA, cyclosporin; LF, leflunomide; MST, mean survival time.
Received for publication May 7, 1997. Accepted for publication September 22, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and diffuse endothelial activation. Transplant. Proc. 25:293.
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, First
Xgs; 
, second Xgs.
