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The Journal of Immunology, 1998, 161: 2044-2051.
Copyright © 1998 by The American Association of Immunologists

Induction of Species-Specific Host Accommodation in the Hamster-to-Rat Xenotransplantation Model1

Dengping Yin, Lian Li Ma, Leonard Blinder, JiKun Shen, Howard Sankary, James W. Williams and Anita S.-F. Chong2

Section of Transplantation, Department of General Surgery, Rush Presbyterian St. Luke’s Medical Center, Chicago, IL 60612


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The combination of two immunosuppressants, leflunomide and cyclosporin A (CsA), completely inhibits immune xenoreactions in the hamster-to-Lewis rat xenotransplantation model. In addition, the control of acute xenograft rejection with this combination of immunosuppressants subdues early T-independent xenoreactivity and uncovers a late immune response that can be controlled by CsA alone. We attribute this acquired responsiveness to CsA to a modification in the recipient’s humoral response to the xenograft, and refer to this change as host accommodation. Host accommodation can be induced in Lewis rats receiving hamster hearts by the combination of leflunomide and CsA. A 7-day treatment with leflunomide and CsA was able to convert xenoreactivity from one that was resistant to CsA treatment into one that was controlled by CsA. The presence of the hamster xenograft was critical for the induction of host accommodation since the immunosuppressive regimen, either alone or in combination with a transfusion with donor-specific spleen cells, was unable to modify the anti-hamster reactivity in Lewis rats. When accommodation was induced in the presence of hamster hearts, these accommodated rats were able to acutely reject third-party mouse hearts while under CsA therapy, thus indicating that the host accommodation is species specific. Finally, we demonstrate that host accommodation is associated with a loss in the ability to produce species-specific, T-independent xenoantibodies. These novel observations suggest that xenoreactive T-independent humoral responses can be deleted selectively without significant loss of other innate, Ag-specific T-independent humoral responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Recent success in controlling complement-mediated hyperacute rejection has shifted the focus of xenotransplantation from the control of hyperacute rejection to the control of acute vascular rejection (AVR)3 or delayed xenograft rejection (DXR) (1, 2, 3). There is increasing experimental evidence that xenoantibodies (XAbs) are important mediators of AVR/DXR and that the production of XAbs during AVR/DXR in the pig-to-primate model is remarkably resistant to conventional immunosuppression (4). XAb production can be controlled significantly by triple immunosuppression with cyclophosphamide, cyclosporine, and steroids, leading to extended xenograft survival (1, 5). However, severe bone marrow toxicity limits the extended use of cyclophosphamide (6, 7). Thus, there is an urgent need for alternative approaches to controlling the elicited XAb responses seen in transplantations of pig-to-baboon and, presumably pig-to-human.

The humoral response to xenografts in the concordant model of hamster-to-Lewis rat xenograft transplantation is T cell independent and resistant to conventional T cell-specific immunosuppressive agents. Effective control of xenograft rejection has been achieved with combinations of antiproliferative and immunosuppressive agents (8, 9, 10). These observations were obtained initially by Hasan and his colleagues using the combination of cyclophosphamide and cyclosporine (11, 12, 13, 14). In an extensive study by Murase et al. (15), stable long-term survivals of hamster heart and liver xenografts were achieved successfully by combining six different antiproliferative drugs, including cyclophosphamide, with the immunosuppressant FK506. Most impressive were their observations that a transient treatment with the antiproliferative agents resulted in long-term xenograft survival as long as FK506 treatment was maintained.

We and others have reported that the novel immunosuppressant, leflunomide, in combination with CsA or FK506, completely prevents the rejection of hamster xenografts in rats (16, 17, 18). Cessation of all immunosuppression invariably results in xenograft loss. We referred to this rejection initially as DXR (19) but, more recently, as late xenograft rejection (LXR) to avoid confusion with the rejection of discordant xenografts after complement is inactivated, which is also labeled DXR (19). Histologic examination of the grafts undergoing LXR indicates the presence of large numbers of macrophages, deposited IgM, edema, hemorrhage, and myocyte necrosis. Thus, while LXR is observed in a concordant transplantation model, it has many histologic features of AVR/DXR, as described in discordant systems.

In contrast to acute xenograft rejection of hamster hearts by Lewis rats, LXR can be controlled completely by CsA monotherapy (19, 20). We interpret these observations as indicating that the mechanism of LXR is different from that of acute xenograft rejection. Recent studies suggest that a modification of the immune response in the Lewis rat to the hamster xenografts was responsible for this acquired sensitivity to CsA. First, the rejection of a second hamster heart transplanted into an accommodated, but not in a normal, Lewis rat could be controlled by CsA alone (21). Second, the production of XAbs was inhibited by CsA in accommodated Lewis rats, but not in normal freshly transplanted Lewis rats (22, 23, 24). Third, the XAbs produced during acute xenograft rejection, but not during LXR, were able to induce the hyperacute rejection of freshly transplanted hamster hearts in naive Lewis rats (21). We have referred to this modification of the host immune reaction as host accommodation, and hypothesize that this modification reflects an inactivation or tolerance of the T-independent xenoantibody response. In this work, we describe a series of experiments that focus on further defining the conditions required for the induction of host accommodation in this rodent model of xenotransplantation, and the mechanism of host accommodation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Transplantation model

Lewis rats, BALB/c mice, and Golden Syrian hamsters were purchased from Harlan Labs (Indianapolis, IN). In some transplants, (C57BL6/SJL)F1 mice that were used were a gift from Dr. Arjay Sharma (Nextran, Princeton, NJ). Hamster or mouse hearts were heterotopically transplanted into the abdomen or the right groin of the recipients following a modified protocol described by Ono et al. (25). In some experiments, a third transplant, of mouse or hamster heart, was transplanted into the left groin of Lewis rat recipients.

Immunosuppression

Cyclosporine (20 mg/kg/day) in oral form (Sandimmune, Sandoz, East Hanover, NJ) was suspended by sonication in water, while leflunomide (5–20 mg/kg/day; custom synthesized for research purposes) was suspended in 1% carboxymethyl cellulose. Both drugs were administered by gavage.

Histology and immunohistochemistry

All heart grafts were harvested, embedded in OCT, and immediately snap frozen in liquid nitrogen. The hearts were sectioned (5 µm) and stained with hematoxylin and eosin (H&E). Other sections for immunohistochemical staining were subject to the standard avidin-biotin conjugate (ABC) method. Briefly, microsections were fixed with cold acetone, then incubated serially with 0.015% H2O2, 5% goat serum, anti-rat IgM or anti-rat IgG (MARM-4 or MARG-2; Serotec USA, Washington DC), biotinylated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA), and then with horseradish peroxidase-conjugated streptavidin (Zymed Labs, South San Francisco, CA). Chromogen, 3,3'-diaminobenxidine solution was added, and the slides were counterstained with Mayer’s hematoxylin. For all histologic sections, isotype-matched controls of purified rabbit or goat Ig were performed in parallel.

Quantification of hamster-specific IgM and IgG Abs

Quantification of hamster-specific Abs was performed as previously described (16, 19). A total of 5 x 105 golden Syrian hamster or BALB/c mouse erythrocytes was incubated with diluted heat-inactivated test serum or control naive Lewis rat serum (1/20 dilution) for 30 min at 4°C. Erythrocytes were washed in 4% (w/v) sodium citrate/PBS, then stained with phycoerythrin-conjugated F(ab')2 anti-rat IgM or FITC-conjugated F(ab')2 anti-rat IgG (Jackson ImmunoResearch). After staining, the erythrocytes were washed, fixed in 1% Formalin, and analyzed using a flow cytometer (Ortho Cytoron Absolut, Ortho Diagnostic Systems, Raritan, NJ).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Leflunomide and CsA are both required for the induction of host accommodation

It has been established that a transient treatment with leflunomide and CsA induces host accommodation in the hamster-to-rat xenotransplantation model (17, 21, 26) (Table IGo). In contrast, hamster hearts are rejected in 4 to 15 days when treated with CsA (20 mg/kg/day) alone (21) (Table IGo), and in 7.7, 52, 77, and 59 days when treated with leflunomide alone at doses of 5, 10, 15, and 20 mg/kg/day, respectively (16). We first asked whether host accommodation can be induced with leflunomide monotherapy, or whether it required the combination of leflunomide and CsA. Lewis rats receiving a hamster heart were treated with leflunomide, at doses of 10, 15, and 20 mg/kg/day, for 14 days, then maintained on CsA (20 mg/kg/day). While immunosuppression with leflunomide monotherapy (10–20 mg/kg/day) resulted in the complete inhibition of xenoreactive IgM production, host accommodation was not induced and the hamster hearts were rejected in 5 to 6 days after immunosuppression was switched to CsA monotherapy (Table IGo). Xenograft rejection was associated with a 5.6-fold increase in the levels of hamster-reactive IgM (Fig. 1Gob). These elevated levels of xenoreactive IgM were comparable with those observed during acute xenograft rejection in the presence of CsA (Fig. 1Goa). Collectively, these observations indicate that effective immunosuppression with leflunomide monotherapy is not sufficient for inducing host accommodation, and suggest the induction of the accommodated state requires additional inhibition of T cell function by CsA.


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  		Table I. Both leflunomide and CsA are required for the induction of host accommodation

 


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  		FIGURE 1. a, Quantitation of the levels of circulating anti-hamster IgM and IgG during AXR, AXR with CsA (20 mg/kg/day) treatment (AXR + CsA), and LXR. b, Quantitation of the levels of circulating anti-hamster IgM and IgG in Lewis recipients of hamster hearts treated with leflunomide (Lef) at 10 or 20 mg/kg/day for 14 days (d14), then switched to CsA (20 mg/kg/day) until rejection (Rej). c, Quantitation of the levels of circulating anti-hamster IgM and IgG in Lewis recipients of hamster hearts treated with leflunomide (Lef) for 7 or 14 days and CsA (20 mg/kg/day) for the duration of the experiment. Data are presented as mean channel fluorescence (MCF) and represent the means of three to six individual animals per group (±SE).

 
Time course for treatment with leflunomide and CsA to induce host accommodation

The next series of experiments were designed to determine the minimum time of immunosuppression required for the induction of host accommodation. It has been reported previously that a 21-day treatment with leflunomide and CsA allowed subsequent hamster heart survival to be maintained on CsA alone (17, 21, 26). We demonstrate in this study that the minimum time of combination treatment with leflunomide and CsA that allowed hamster hearts to be maintained on CsA alone was 7 days (Table IIGo). Extended survival of the hamster hearts corresponded with effective inhibition of the XAb production by CsA after either a 7- or 14-day treatment with leflunomide and CsA (Fig. 1Goc). In contrast, Lewis recipients of hamster hearts that were treated with 20 mg/kg/day of CsA alone produced hamster-reactive IgM by 4 to 16 days posttransplantation (Fig. 1Goa). These results confirm and extend previous observations that a transient treatment with the combination of leflunomide and CsA effectively subdues the T-independent XAb response and uncovers a T-dependent response that is controllable by CsA alone (21, 22, 23, 24).


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  		Table II. Time course of combination treatment

 
In contrast to CsA, leflunomide (15 mg/kg/day) was unable to maintain long-term survival of the hamster grafts in accommodated Lewis rats (Table IGo). Following 21 days of combined treatment with leflunomide (5 mg/kg/day) and CsA (20 mg/kg/day), an immunosuppressive dose of 15 mg/kg/day of leflunomide resulted in severe anemia by 24 or 56 days posttransplantation, and the Lewis recipients were sacrificed. At this time, the xenograft hearts were beating weakly. Histologic examination revealed signs of intimal thickening and mononuclear infiltration, similar to hamster hearts maintained on leflunomide monotherapy for the duration of the experiment (data not shown) (16). These observations are consistent with those previously reported by Lin et al. (27) in a hamster into PVG rat transplantation model, and indicate that CsA and control of T cell xenoreactivity are critical for the maintenance of long-term graft survival.

Induction of host accommodation requires the presence of the hamster heart grafts

It is possible that potent immunosuppression with leflunomide and CsA resulted in a nonspecific loss in the immune reactivity, and an apparent induction of host accommodation. We reasoned that if this were the case, then host accommodation should be induced in the absence of the xenograft. We therefore investigated whether treatment with leflunomide and CsA in the absence of a xenograft would result in a subsequent inability of Lewis rats to reject hamster hearts while under CsA alone. As presented in Table IIIGo, Lewis rats pretreated with leflunomide and CsA for 14 days, before transplantation with a hamster heart, were able to reject hamster hearts in 4 to 7 days while on CsA monotherapy.


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  		Table III. Role of the xenograft in the induction of host accommodation1

 
Further evidence of the importance of Ag in the induction of host accommodation came from the next series of experiments. It has been reported that donor-specific transfusion with spleen cells synergizes with a variety of immunosuppressive protocols to induce tolerance to allografts in mice, rats, and humans (28, 29, 30, 31). Lin et al. reported that immunization of rats with hamster spleen cells (20 x 106/mouse) under leflunomide monotherapy (20 mg/kg/day) could induce T-independent accommodation to a second challenge of hamster splenocytes (32). We therefore tested whether hamster spleen cells could induce host accommodation and allow late xenoreactivity to hamster hearts to be subdued by CsA alone. Hamster spleen cells (50 x 106/Lewis rat) were administered i.v. on day 0 into Lewis rats treated with leflunomide and CsA for 14 days. On day 14 posttransplantation, immunosuppression was switched to CsA monotherapy and the Lewis rats were transplanted with a hamster heart (Table IIIGo). All hamster hearts were rejected in 4 to 10 days, indicating that donor-specific transfusion of hamster spleen cells could not substitute for a hamster heart to induce host accommodation to hamster hearts, and that host accommodation is at least partially tissue specific.

Specific induction of host accommodation to hamster hearts

The importance of xenograft, and the inability of hamster spleen cells to induce host accommodation to hamster hearts led us to hypothesize that this process could also be species specific. Hamster hearts were transplanted into Lewis rats treated for 14 days with leflunomide and CsA to induce host accommodation. These accommodated Lewis rats were then maintained on CsA for the duration of the experiment. A second hamster heart was transplanted into these accommodated rats after 7 to 14 days of CsA monotherapy. Both first and second hamster hearts were maintained on CsA monotherapy for >=77 and >=58 days, respectively (Table IVGo). When a third-party mouse (BALB/c) heart was transplanted into these accommodated Lewis rats, the mouse hearts were rejected in 3 to 4 days, despite continuous CsA therapy. This rate of rejection of the mouse heart is comparable with the rejection by naive Lewis rats on CsA therapy.


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  		Table IV. Host accommodation is species-specific

 
Histologic examination revealed that the mouse heart was acutely rejected with severe arterial necrosis and thrombosis, edema, myocardial necrosis, and modest mononuclear infiltration (Fig. 2GoG). Immunohistologic analysis indicated extensive rat IgM deposition (brown stain) and a dense infiltrate of ED1-positive cells (brown cells) in the myocardium of the rejected mouse hearts (Fig. 2Go, H and I). In contrast, both hamster hearts appeared normal with minimal signs of rejection when examined at the time of mouse heart rejection (Fig. 2Go, A and D). There were minimal IgM deposition (Fig. 2Go, B and E) and only scattered infiltrating ED1-positive cells (Fig. 2Go, C and F) in the myocardium of both first and second hamster hearts.



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  		FIGURE 2. Lewis rats accommodated to hamster hearts are able to reject a third-party mouse heart. Lewis rats were transplanted with hamster hearts on days 0 and 14 to 27, and mouse hearts were transplanted on days 74 to 82. These Lewis rats were treated with leflunomide (Lef) for 14 days, and CsA (20 mg/kg/day) for the duration of the experiment. Hearts were harvested on the day of rejection of the mouse heart. A–C and D–F are sections from the first and second hamster hearts, respectively, and G–I are sections from the third-party mouse hearts. A, D, and G are hematoxylin and eosin (H&E)-stained sections; B, E, and H are immunohistochemical demonstrations of deposited IgM; and C, F, and I are ED-1 mAb stainings of tissue macrophages.

 
These immunohistochemical observations of IgM deposition in only the mouse xenografts were consistent with the levels of circulating XAbs. Accommodated Lewis rats did not make hamster-reactive Abs while under CsA monotherapy. The rejection of a third-party mouse heart was accompanied by a 5.6-fold increase in the levels of anti-mouse IgM; however, no detectable increase in the levels of anti-hamster IgG was observed (Fig. 3Gob). These data are in contrast to the levels of cross-reactive anti-hamster Abs observed during the acute rejection of mouse hearts by Lewis rats (Fig. 3Goc). At the time of acute rejection of mouse hearts in untreated or CsA-treated Lewis rats, the levels of anti-mouse IgM were elevated 9- and 7.6-fold, respectively (Fig. 3Goc), while the levels of cross-reactive anti-hamster IgM in untreated or CsA-treated Lewis rats were elevated 3.4- and 3.1-fold, respectively (Fig. 3Goc). These observations collectively suggest that host accommodation involves a species-specific loss in T-independent, hamster-specific Ab responses in Lewis rats.



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  		FIGURE 3. Lewis rats were transplanted with hamster hearts (a) or mouse hearts (c), and the levels of anti-hamster or anti-mouse IgM were quantified by flow cytometry using Lewis rat or BALB/c erythrocytes. Sera were harvested during AXR, AXR with CsA (20 mg/kg/day) treatment (AXR + CsA), and LXR. In a second series of experiments, Lewis rats were transplanted with hamster hearts on days 0 and 14 to 27, and mouse hearts were transplanted on days 74 to 82 (c). In a third series of experiments, Lewis rats were transplanted with mouse hearts on days 0 and 14 to 27, and hamster hearts were transplanted on days 74 to 82 (d). These Lewis rats were treated with leflunomide (Lef) for 14 days and CsA (20 mg/kg/day) for the duration of the experiment, and transplants were performed on the days indicated by the arrows. Sera were harvested on the indicated days posttransplantation. All data are presented as mean channel fluorescence (MCF) and represent the means of three to six individual animals per group (±SE).

 
Specific induction of host accommodation to mouse hearts

The observation that Lewis rats can be specifically accommodated to hamster hearts, yet retain the ability to reject mouse hearts while under CsA therapy, is a novel and potentially significant one. To extend this observation and to further characterize the specificity of the accommodation process, we performed the converse experiment. We tested whether Lewis rats could be specifically accommodated to mouse hearts, yet retain the ability to reject hamster hearts while under CsA. Presented in Table IVGo are data indicating that specific accommodation could also be induced to mouse hearts. In addition, the data suggest that accommodation in this combination is also species specific, as five of six Lewis rats accommodated to mouse heart were able to reject the hamster heart while retaining the accommodated mouse heart (Table IVGo).

We also quantified the levels of anti-hamster and anti-mouse IgM in these accommodated rats. Five of the six Lewis rats accommodated to mouse hearts were unable to produce mouse-reactive Abs while under CsA monotherapy, but were able to reject a third-party hamster heart. The rejection of the hamster heart was accompanied by a 6.3-fold increase in the levels of anti-hamster IgM, while no detectable increase observed in the levels of cross-reactive anti-mouse IgM was observed (Fig. 3God). These data are in contrast to the increase in levels of cross-reactive anti-mouse IgM during the acute rejection of hamster hearts by Lewis rats (Fig. 3Goa). At the time of acute rejection of hamster hearts in untreated or CsA-treated Lewis rats, the levels of anti-mouse IgM were elevated 11.3- and 8.6-fold, respectively (Fig. 3Goc). The levels of cross-reactive anti-hamster IgM in untreated or CsA-treated Lewis rats were elevated 4.8- and 4.7-fold, respectively (Fig. 3Goc). These observations collectively support the notion that host accommodation involves a species-specific inactivation of T-independent, xenograft-specific B cell responses in Lewis rats.

Specificity of xenoreactive Abs during the acute rejection of hamster hearts: challenge with mouse heart transplants

It has been reported previously that the XAbs produced by Lewis rats acutely rejecting hamster hearts are cross-reactive to mouse Ags (33, 34). We have extended those findings and have characterized the cross-reactivity of the Abs produced during acute rejection. Pooled serum from rats with acutely rejected hamster xenografts was adsorbed on hamster or mouse erythrocytes, and the amount of hamster-reactive or mouse-reactive Abs was quantified. When serum was adsorbed with hamster erythrocytes, a significant decrease in hamster-reactive IgM was observed. But when sera were adsorbed with mouse cells, no significant reduction in the amount of IgM capable of binding hamster cells was observed (Fig. 4Goa). In contrast, when we quantified the IgM, produced during the acute rejection of hamster hearts, that cross-reacted with mouse cells, we observed that mouse cells were most effective at adsorbing the cross-reactive IgM (Fig. 4Gob). These results suggest that only a small percentage of XAbs mediating the rejection of hamster hearts reacted with mouse cells. Similar results were obtained with serum from Lewis rats with acutely rejected mouse heart. Mouse cells were most effective at adsorbing the mouse-reactive IgM, while hamster and mouse cells were equally effective at absorbing out the hamster cross-reactive IgM Abs (Fig. 4Go, c and d).



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  		FIGURE 4. Cross-reactivity of xenoreactive Abs produced during the acute rejection of hamster (a and b) or mouse hearts (c and d). Sera harvested during AXR were pooled (n = 3–5), and diluted 1/5 in PBS. Diluted sera (150 µl) were incubated for 30 min, and at 4°C, with 50 µl of packed Lewis rat, hamster, or mouse erythrocytes (x-axis). This adsorption was repeated three times, and the levels of IgM in the preadsorbed sera that bound to hamster (a and d) or mouse (b and c) erythrocytes were quantified by flow cytometry, as described in Figure 1Go. Data are presented as mean channel fluorescence of triplicates ± SE, and are the representative of two to three experiments.

 
We next tested whether the T-independent XAbs produced by Lewis rats were also cross-reactive with mouse hearts. We observed that Lewis rats that had acutely rejected hamster hearts, while under CsA monotherapy, were able to elicit the hyperacute rejection of mouse hearts (Table VGo). The rejections correlated with elevated levels of IgM that reacted to both hamster and mouse erythrocytes during the acute rejection of a hamster heart, in either untreated or CsA-treated Lewis rats (Fig. 3Goc).


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  		Table V. Specificity of xenoreactive serum during AXR or LXR1

 
Finally, we tested whether T-dependent, anti-hamster Abs produced during LXR by accommodated Lewis rats cross-reacted with mouse Ags. Cessation of CsA therapy in accommodated Lewis rats resulted in the rejection of the hamster heart in 6 to 8 days (Fig. 3Goc). On the day of LXR, mouse hearts transplanted into the Lewis rats were hyperacutely rejected within 10 to 20 min (Table VGo). These rejections correlated with elevated levels of IgM that reacted to both hamster and mouse erythrocytes during LXR of a hamster heart (Fig. 3Goc). These observations indicate that the T-dependent Abs produced by accommodated Lewis rats also cross-react with mouse heart Ags.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous histologic analyses of the spleen of Lewis rats upon transplantation of a hamster heart suggested that both T-independent and T-dependent responses were involved in the process of acute xenograft rejection (35). It is possible to suppress the T-dependent response with agents that block T cell activation, and to demonstrate that the remaining T-independent response is able to acutely reject hamster xenografts. However, it has not been possible to suppress only the T-independent response; thus, T-dependent xenoreactive responses have been poorly characterized to date. The studies presented in this work, as well as recent published studies (20, 21, 23, 26), suggest that a transient treatment with leflunomide and CsA eliminates the T-independent, but not the T-dependent response. Thus, this model system provides a unique opportunity to investigate T-dependent xenograft-specific responses.

It has been reported previously that the XAbs produced in the rat during the acute rejection of the hamster heart xenografts also cross-react with and mediate the hyperacute rejection of mouse cardiac grafts (33, 34). We have extended those findings and report in this work that T-independent XAbs, produced during the acute rejection of hamster grafts in the presence of CsA, can also mediate the hyperacute rejection of mouse hearts. This observation suggests that the repertoire of T-independent XAbs produced during the acute rejection of a hamster heart can be divided into those that are hamster specific and those that cross-react with hamster and mouse Ags.

It has been reported that a transient treatment with leflunomide and CsA, after transplantation of a hamster heart, converts the anti-hamster response from one that is CsA resistant to one that is controlled completely by CsA alone. We and others have concluded that treatment with leflunomide and CsA eliminates the host’s T-independent response, but retains the T-dependent response that is controllable by CsA monotherapy (20, 21, 23, 26). Earlier published studies indicate that this T-dependent anti-hamster response is distinct from the T-independent response, and demonstrate that the T-dependent XAbs have reduced ability to induce C3 deposition and hyperacute rejection of hamster hearts (21). We have referred to this change in xenograft-specific response as host accommodation.

We demonstrate in this study that both CsA and leflunomide are required for the induction of host accommodation, as is the presence of the xenograft. Furthermore, hamster spleen cells were unable to induce host accommodation to hamster hearts. Recent studies by Lin et al. reported that immunizing with hamster spleen cells in the presence of leflunomide induced T-independent B cell tolerance and the inability to respond to a second challenge of hamster spleen cells (32). Their observations along with ours collectively suggest that host accommodation is at least partially tissue specific.

The importance of the appropriate Ag in the induction of accommodation to hamster hearts is further illustrated by the ability of these animals to reject a third-party mouse heart. Conversely, Lewis rats accommodated to mouse hearts were able to reject hamster hearts. Collectively, these novel observations suggest that the induction of host accommodation is both species specific and tissue specific. These observations complement and extend observations reported recently by Lin et al. (36) of the induction of specific tolerance across xenogeneic barriers in the hamster heart into nude rat transplantation model. They demonstrated that host accommodation in their model was based on species-specific NK cell tolerance and a non-species-specific tolerance of the T-independent xenoreactive response.

The specificity of the host accommodation in this xenotransplantation model is especially striking because sensitization to hamster hearts also sensitizes the recipient to mouse hearts. In fact, both the T-dependent and the T-independent Abs produced during the rejection of a hamster heart cross-react with mouse Ags. To explain the Ab specificity observed during the different rejection events, we have diagrammed in Figure 5Go the clones of B cells that are stimulated by a hamster xenograft. In naive Lewis rats, the hamster graft stimulates both hamster-specific and hamster-mouse cross-reactive B clones, and both T-independent and T-dependent clones are stimulated. CsA is able to inhibit the activation and expansion of only the T-dependent B cell clones (Fig. 5Goa). Host accommodation in the presence of a hamster graft results in the elimination or inactivation of T-independent B cell clones that are either hamster specific or hamster-mouse cross-reactive (Fig. 5Gob). The T-independent B cell clones that are mouse specific are not affected by the accommodation process, and can subsequently mediate the rejection of a freshly transplanted mouse heart in the presence of CsA therapy (Fig. 5Gob). The T-dependent, anti-hamster or anti-mouse B cells that are not tolerized are immunosuppressed with continuous CsA monotherapy (Fig. 5Gob). Finally, when CsA treatment is stopped, the accommodated Lewis rats reject the hamster heart by a process termed LXR. T-dependent B cells that are either hamster specific or hamster-mouse cross-reactive become activated to produce Abs during LXR.



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  		FIGURE 5. a, Transplantation of hamster hearts results in the clonal expansion of both T-independent (CsA-resistant; circles) and T-dependent (CsA-sensitive; squares) B cells that are either hamster specific or are hamster and mouse reactive. b, Accommodation of Lewis rat to hamster hearts results in the loss in the ability of T-independent, hamster-reactive B cells to respond to a second hamster heart (indicated by "X"). Immunosuppression with CsA alone is required for the continued suppression of the T-dependent anti-hamster response, but is unable to prevent the T-independent, mouse-reactive B cell-mediated rejection of mouse hearts. c, Cessation of all immunosuppression results in LXR that is mediated by T-dependent B cell clones that are either hamster specific or hamster- and mouse-reactive clones.

 
In conclusion, we have made the novel observation that host accommodation can be induced in Lewis rats receiving hamster hearts by the combination of leflunomide and CsA, but not with either of these agents alone. The presence of the hamster xenograft was critical for the induction of host accommodation since the immunosuppressive regimen, alone or in combination with donor-specific transfusion of spleen cells, was unable to modify the anti-hamster reactivity in Lewis rats. We also report that when accommodation was induced in the presence of hamster hearts, these Lewis rats were able to acutely reject third-party mouse hearts while under CsA therapy. Finally, host accommodation in Lewis recipients of hamster hearts is associated with the loss in the ability to produce T-independent, hamster-reactive Abs. This novel observation that host accommodation is Ag specific suggests that xenoreactive T-independent humoral responses can be selectively deleted without significant loss of other innate Ag-specific T-independent humoral responses.


   Acknowledgments
 
We thank Dr. Ian Boussy for his helpful comments and assistance in the preparation of the manuscript. We also thank Drs. Ajay Sharma and John Logan, Nextran (Princeton, NJ), for the gift of (C57B6/SJL)F1 mice.


   Footnotes
 
1 This work is funded in part by a grant from National Institutes of Health (AI34061). Back

2 Address correspondence and reprint requests to Dr. Anita S.-F. Chong, Section of Transplantation, Department of General Surgery, Rush Presbyterian St. Luke’s Medical Center, 1653 West Congress Parkway, Chicago, IL 60612. E-mail address: Back

3 Abbreviations used in this paper: AVR, acute vascular rejection; CsA, cyclosporin A; DXR, delayed xenograft rejection; HRP, horseradish peroxidase; LXR, late xenograft rejection; XAb, xenoantibody. Back

Received for publication February 2, 1998. Accepted for publication April 15, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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