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The Journal of Immunology, 2004, 173: 5827-5834.
Copyright © 2004 by The American Association of Immunologists

Mixed Xenogeneic Chimerism Induces Donor-Specific Humoral and Cellular Immune Tolerance for Cardiac Xenografts1

Yolonda L. Colson2,*, Hong Xu{dagger}, Yiming Huang{dagger} and Suzanne T. Ildstad{dagger}

* Department of Surgery, Division of Thoracic Surgery, Brigham and Women’s Hospital, Boston, MA 02115; and {dagger} Institute for Cellular Therapeutics, Baxter Biomedical Research Building, University of Louisville, Louisville, KY 40202


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Xenotransplantation has been suggested as a potential solution to the critical shortage of donor organs. However, success has been limited by the vigorous rejection response elicited against solid organs transplanted across species barriers. Mixed xenogeneic bone marrow chimeras resulting from the transplantation of a mixture of host and donor marrow (B10 mouse + F344 rat -> B10 mouse) results in donor-specific cross-species transplantation tolerance for subsequent nonvascularized skin and islet grafts. Furthermore, compared with fully xenogeneic chimeras (rat -> mouse), mixed xenogeneic chimeras exhibit superior immunocompetence for infectious agents in vivo and in vitro, suggesting that the immune system is intact. The ability to establish long-term humoral and cellular tolerance for primarily vascularized xenografts in vivo, in the setting of both recipient and donor Ig and effector cell production, has not previously been characterized. Mixed xenogeneic chimeras exhibit donor-specific humoral tolerance as evident by the absence of anti-donor Ab and Ab-dependent donor-specific cytotoxicity in vitro and intravascular IgM deposition within donor-strain (F344) cardiac xenografts in vivo. F344 cardiac xenografts are accepted (median ≥180 days) without clinical or histologic evidence of rejection, suggesting cellular tolerance. In contrast, MHC-disparate third-party mouse (B10.BR) and rat (ACI or WF) grafts are rejected (median of 23 and 41 days, respectively) in association with extensive mononuclear cell infiltration and vascular deposits of mouse IgM. These results demonstrate that mixed xenogeneic chimerism establishes donor-specific humoral and cellular tolerance and permits the successful transplantation of even primarily vascularized xenografts in the setting of intact Ab production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
As cardiac transplantation has become the therapy of choice for a number of cardiac maladies, the number of transplant candidates has grown exponentially. Furthermore, <50% of cardiac allografts survive for >10 years, further compounding the donor organ shortage by adding retransplant for chronic rejection to the list of indications (1). This increasing demand on the limited donor pool has resulted in longer waiting lists, increased numbers of candidate deaths before transplantation, and a patient population with significant morbidity at the time of transplantation.

The use of alternate species as donors has been suggested as a potential means to alleviate the shortage of donor organs. Unfortunately, clinical xenotransplantation has been thwarted by the vigorous rejection response elicited against solid organs transplanted across a species barrier (2, 3, 4). Transplantation of xenografts between widely disparate species has been limited by rejection due to the presence of natural Abs (5, 6, 7, 8, 9). Even when hyperacute rejection is prevented with plasmapheresis, complement depletion, immunoaffinity columns, or the use of more closely related species, long-term xenotransplantation is still limited by robust acute humoral and cellular immune rejection (10, 11, 12, 13, 14, 15, 16). Susceptibility of the xenograft to Ab-mediated rejection varies among tissues, but primarily vascularized xenografts are the most immune accessible, the most rapidly rejected, and, unlike skin grafts, remain sensitive to Ab-mediated destruction long after transplantation (17). Therefore, there is a significant challenge to achieve both humoral and cellular immune tolerance for solid organ transplants by a means that is also applicable to primarily vascularized xenografts.

The induction of donor-specific tolerance through bone marrow (BM)3 chimerism has been suggested as a potential approach to overcome the rejection response against xenografts. Mixed xenogeneic chimerism (mouse + rat -> mouse) resulting from the transplantation of a mixture of host and donor BM into ablated mouse recipients has been shown to confer permanent donor-specific transplantation tolerance for nonvascularized skin and islet xenografts (18, 19, 20). The restriction specificity of both host and donor lymphocytes in mixed chimeras (mouse + rat -> mouse) is to mouse APCs which are present in these mixed chimeras (21, 22). Furthermore, we have demonstrated that this immune restriction results in a relative resistance to graft-vs-host disease, functional cross-species T lymphocytes, and immunocompetence as evidenced by the generation of viral-specific CTLs in vitro and in vivo (19, 22, 23). It would be of obvious clinical benefit if both cellular and humoral tolerance could be induced for primarily vascularized xenografts in an immunocompetent recipient.

Humoral chimerism and humoral xenotolerance have not been investigated in the setting of increased immunocompetence associated with mixed chimerism. In this study, we demonstrate that mixed xenogeneic chimeras (B10 mouse + F344 -> B10 mouse) exhibit intact Ab production of both mouse and rat Ig isotypes without the generation of anti-donor Ab or Ab-mediated cytotoxicity against donor Ags, even though donor Ags are present within the host. In contrast, unconditioned controls produced anti-donor Ab after BM infusion. The induction of cellular and humoral tolerance in mixed xenogeneic chimeras is confirmed in vivo as donor-specific cardiac xenografts are permanently accepted with a median survival time (MST) of >180 days without evidence of IgM deposition or cellular infiltrates. In contrast, third-party mouse and rat xenografts were readily rejected as characterized by diffuse intravascular deposition of host IgM, extensive myocyte necrosis, and mononuclear cell infiltration. These findings indicate that mixed xenogeneic chimeras exhibit humoral as well as cellular chimerism, with the resultant induction of donor-specific humoral and cellular tolerance for even primarily vascularized xenografts. With the recent demonstration that mixed chimerism can be achieved in presensitized allogeneic recipients (24, 25), mixed xenogeneic chimerism may offer a clinically viable approach to expand the pool of donor organs and induce drug-free, long-term tolerance for solid organ xenografts in the future.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Male C57BL/10SnJ (B10) and B10.BRSgSn (B10.BR) mice 6–8 wk of age were purchased from The Jackson Laboratory (Bar Harbor, ME). Male Fisher 344 (F344) rats 4–8 wk old were purchased from Harlan Sprague Dawley (Indianapolis, IN). Pregnant female rats were also purchased to provide newborn F344, Wistar Furth (WF), and ACI rats (25–30 g) necessary for cardiac xenograft donors. Animals were housed and cared for in a specific pathogen-free facility at the Pittsburgh Cancer Institute and subsequently at the Institute for Cellular Therapeutics at the University of Louisville, under the guidelines instituted by the respective animal care committees.

Mixed xenogeneic reconstitution (B10 + F344 -> B10)

Xenogeneic reconstitution was performed using the method modified in 1992 to enhance mixed xenogeneic engraftment and survival and has been our standard approach since that time (19). Briefly, B10 male recipients were lethally irradiated with a single dose of 950 cGy from a 137Cs source (Nordion, Ottawa, Ontario, Canada). Using sterile technique, BM was flushed from the femurs and tibias of donors with medium 199 (Invitrogen Life Technologies, Grand Island NY) containing 50 µl/ml gentamicin using a 22-gauge needle. BM was mechanically resuspended and filtered through sterile nylon mesh. The cells were centrifuged at 300 x g for 10 min and resuspended in medium 199. Syngeneic B10 marrow was T cell-depleted (TCD) by treatment with rabbit anti-mouse brain sera and guinea pig complement (Life Technologies) (18, 26). Dilutions of rabbit anti-mouse brain and complement were previously determined to maximize T cell lysis with a 2–3 log reduction confirmed by flow cytometry while minimizing nonspecific killing. Cells were washed twice and resuspended for injection. Rat BM was washed and administered untreated (non-TCD) as this was previously shown to result in stable rat lymphoid chimerism, whereas TCD rat BM does not (18, 19, 23, 27). Recipients received 5 x 106 TCD B10 and 40 x 106 non-TCD F344 cells (B10 + F344 -> B10) within 4–6 h of radiation via BM injection into the tail vein using a 27-gauge needle. Increased evidence of graft-vs-host disease has not been noted in this model despite the use of untreated rat BM. Radiation controls were performed to confirm adequacy of the lethal radiation dose.

Characterization of chimeras by flow cytometry

Recipients were characterized for engraftment using flow cytometry (FACS II; BD Biosciences, Mountain View, CA) to determine the percentage of PBLs bearing H-2b (B10) and F344 (Rt1a1) surface markers (19, 23, 27). Briefly, peripheral blood was collected into heparinized plastic serum vials and 200 µl of medium 199 was added to each vial. After thorough mixing, the suspension was layered over 2 ml of room temperature Lymphocyte Separation Medium (Organon Teknik, Durham, NC) and centrifuged at 37°C (400 x g for 25 min). The buffy coat layer was aspirated from the saline interface and washed. Lymphocytes were stained with mouse or rat anti-class I mAb for 45 min at 4°C. Anti-H-2b-FITC (BD Pharmingen, San Diego, CA) was used for anti-class I staining of B10 cells. Biotinylated anti-rat class I mAb were generously supplied by Dr. H. Kunz and Dr. T. J. Gill and counterstained with streptavidin -FITC (BD Pharmingen).

To establish that rat BM has engrafted, the existence of multiple lineages of donor and host origin was assessed in peripheral blood by flow cytometry. This analysis was performed using species-specific lineage mAbs against rat vs mouse: {alpha}{beta}TCR+ T cells, anti-rat {alpha}{beta}TCR (R73) vs anti-mouse {alpha}{beta}TCR (H57-597); CD8+ T cells, anti-rat CD8 (OX8) vs anti-mouse CD8 (53-6.7); CD4+ T cells, anti-rat CD4 (OX-35) vs anti-mouse CD4 (RM4-5); B cells, anti-rat CD45RA (OX-33) vs anti-mouse B220 (RA3-6B2); and NK cells, anti-rat NKR-P1A (10/78) vs anti-mouse NK1.1 (PK136); macrophages, anti-rat CD11b (WT.5) vs anti-mouse CD11b (M1/70). All mAbs were from BD Pharmingen.

ELISA characterization of mouse and rat Ig subsets

Serum samples were assayed for the presence of mouse IgG1, IgG2a, IgG2b, IgM, and rat IgM and IgG production using a sandwich ELISA for isotype detection (28). Briefly, flat-bottom Immulon microtiter plates were coated with 50 µl of respective capture Abs (goat anti-mouse: {gamma}1, {gamma}2a, {gamma}2b, µ, or goat anti-rat µ or {gamma}) from BD Pharmingen. Plates were prepared and incubated overnight at room temperature before rinsing off excess mAb with distilled water. Residual binding capacity of the plate was blocked with 0.25% BSA and Tween 20. Standard B10 and F344 sera were used as respective positive and negative controls for the various capture mAb. Controls and experimental sera (50 µl) were plated against each of the various capture mAb. After 1-h incubation at room temperature, plates were washed five times and agitated for several minutes with each wash. The presence of mouse or rat Ig "captured" by the various isotype-specific mAb was detected by the addition of HRP-labeled goat anti-mouse Ig or goat anti-rat Ig, respectively. Plates were again washed five times following a 1-h incubation at room temperature and the color reaction was developed with 100 µl of ABTS (Sigma-Aldrich, St. Louis, MO) substrate buffer containing a 1/1000 dilution of 30% H2O2 for 20 min at room temperature. Absorbencies were read on a microtiter plate reader at 405 mm. Serum levels of each Ab isotype from chimeras were normalized to syngeneic B10 recipients to account for reconstitution in irradiated recipients in the following manner. The full scale of absorbencies from positive controls (syngeneic reconstituted B10 recipients for mouse Ig and normal F344 for rat Ig) was calculated and established as the 100% serum Ab control. Absorbencies for mouse Ig in F344 rats and rat Ig in B10 mice were used as 0% Ab controls. A semiquantitative assessment of serum levels for a given isotype in a chimera was then calculated by normalizing the absorbency of a given chimera sample to the range of absorbencies detected in the 0 and 100% Ab controls.

Ab-dependent cytotoxicity assay

Serum was collected from unmanipulated B10 mice or F344 rats, nonirradiated B10 mice sensitized to F344 Ags by prior injection of F344 BM (sensitized), irradiated B10 recipients that failed to engraft with F344 BM (nonchimeras), and B10 + F344 -> B10 chimeras. Collected sera was stored at –20°C until needed for analysis, at which time it was decomplemented at 50°C for 30 min, serially diluted from 1/2 to 1/1024, and placed in 96-well round-bottom plates. A total of 2.5 x 104 donor 51Cr-labeled target splenocytes was added to each well, incubated at 37°C for 30 min, and washed at 300 x g. Following a second 30-min incubation at 37°C in the presence of rabbit complement titered for minimal nonspecific killing (1/8), supernatants were collected and counted on a gamma counter (Titertek System; Skatron Instruments, Lier, Norway; DP5000, Beckman Coulter, Fullerton, CA). Cytotoxicity was determined by the formula: percent cytotoxicity = 100% x ((experimental – spontaneous)51Cr/(maximum – spontaneous)51Cr). Spontaneous 51Cr release for controls with medium and complement, or Ab alone, was <20% of total release. An H-2-specific antiserum (IgM) was used as a positive control, with donor-specific lysis resulting in 65–100% maximum cytotoxicity of splenocytes in this assay. Statistical analysis of cytotoxic activity between groups of animals was performed using unpaired two-tailed t test analysis.

FACS assessment of anti-donor Ab

Serum was harvested from control syngeneic radiation chimeras (5 x 106 B10 -> irradiated B10 recipient), unconditioned B10 recipients injected with the mixed B10 + F344 BM inocula (sensitized), and mixed xenogeneic chimeras. Splenocytes isolated from F344 rats were incubated with 5 µl of the designated serum at 4°C for 25 min. Samples were washed twice and incubated with goat anti-mouse Ig FITC (BD Pharmingen) to detect anti-donor Abs within the respective sera samples that remained adherent to the F344 splenocytes. To avoid false positive results due to rat B cell nonspecific staining with goat anti-mouse Ig FITC, samples were counterstained with anti-rat CD4 and CD8 PE, such that flow cytometric analysis of anti-donor Ab (FITC+) was assessed on rat CD4+ and CD8+ T cells. Results were displayed as average fluorescence intensity ± SD.

Heterotopic cardiac transplantation

One to 2 mo after reconstitution, cardiac xenografts from newborn rat F344, WF, and ACI donors (30–35 g) or adult B10 and B10.BR mice were transplanted into xenogeneic chimeras as primarily vascularized grafts using the method previously reported by Corry et al. (29). Briefly, following harvest of the donor heart, the recipient intra-abdominal aorta and inferior vena cava (IVC) were exposed. The arterial anastomosis was performed between the recipient abdominal aorta and the donor graft aorta using 10–0 Novafil (Davis & Geck, Danbury, CT). Similarly, the venous anastomosis was completed between the recipient IVC and the donor pulmonary artery. Spontaneous cardiac contractility returned with perfusion of the xenograft. Xenograft survival was assessed daily based on the presence and quality of the graft heartbeat graded from 0 (no palpable beat) to 4 (visual pulsation). Acute rejection of cardiac xenografts was defined as the cessation of graft pulsations and confirmed by histologic analysis.

Histologic assessment of cardiac graft cellular rejection

Tissue samples for analysis were obtained at the time of rejection or at a predetermined date in long-term survivors. Heart grafts were snap frozen with Freon and liquid nitrogen and stored at –70°C until the time of analysis. Frozen sections (4 µm) were processed with methanol before treatment with H&E stains. Coverslips were placed over tissue samples after washing samples with PBS and sealed with glycerin sealant. Slides from the various cardiac donors were stained and read in a double-blind fashion. As defined by Billingham (30) for human cardiac rejection, histologic evidence of cardiac rejection was obtained by evaluation of perivascular and interstitial spaces for evidence of a mononuclear diffuse or, later in rejection, a diffuse mixed neutrophil, macrophage, and lymphoid cellular infiltrate. In addition, myocytolysis, myocyte necrosis, and interstitial hemorrhage marked the progression from moderate to severe rejection.

Immunohistochemical staining of intravascular Ig deposition

Sections for immunohistochemical staining of mouse IgM were pretreated with 0.3% H2O2/PBS and blocked for nonspecific protein interactions with 5% goat serum before incubation with biotin-labeled goat anti-mouse IgM mAb (Vector Laboratories, Burlingame, CA) for 45 min at room temperature. Slides were washed in PBS before incubation with the avidin-biotinylated enzyme complex within the Vectastain ABC peroxidase kit (Vector Laboratories) and detection was performed with 3,3-diaminobenzidine. 3,3-Diaminobenzidine solution was prepared immediately before use and tissue sections were incubated at room temperature until color was visible (~5 min). The reaction was stopped by a 5-min wash in distilled water. Slides were permanently mounted using VectaMount (Vector Laboratories). All slides were read in a double-blind fashion, examined for evidence of intravascular Ig deposition, and scored for the degree of deposition using a modification of the previously defined scoring system by Hammond et al. (31). Briefly, intravascular mouse IgM deposition in vivo was graded as: 0, no to weak diffuse staining; 1, <10% of endothelial cells positive; 2, moderate (10–50%) positive staining; 3, heavy (>50%) positive; and 4, massive Ig deposition.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mixed xenogeneic chimeras exhibit cellular and humoral donor chimerism

Mixed xenogeneic chimeras (B10 + F344 -> B10) were prepared by transplantation of 5 x 106 B10 + 40 x 106 F344 BM cells into conditioned (950 cGy) B10 recipients. All recipients were assessed for evidence of PBL chimerism at 28 days following reconstitution via flow cytometric analysis. Chimerism ranged from 1.5 to 99.0%. Analysis of multilineage chimerism including T, B, NK, and macrophages in a representative experiment is presented in Table I. In all chimeras tested (n = 11), donor rat-derived T cells ({alpha}{beta}TCR, CD4 and CD8), B cells, NK cells, and macrophages were present. Results from a representative chimera are depicted in Fig. 1. These findings suggest that engraftment of the rat BM stem cell has occurred rather than selected lineage progenitors and that individual lineage chimerism is regulated independently. In contrast to mixed chimeras, donor rat lineages were not detectable in unconditioned B10 mice given the same B10 + F344 mixed BM inoculum, indicating that engraftment of donor BM did not occur in these unconditioned recipients.


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  		Table I. Multilineage chimerism in mixed xenogeneic chimerasa

 


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  		FIGURE 1. Detection of donor- and host-derived cells of lymphoid and myeloid lineages in mixed xenogeneic chimeras using two-color flow cytometry. Multilineage typing was performed on chimeras (n = 11) that exhibited levels of donor chimerism ranging from 3 to 99%. The x-axis shows staining with FITC-conjugated mAbs against mouse lineages. On the y-axis, the mAbs stain for the different cell lineage markers against rat. The cell subpopulations tested included T cells ({alpha}{beta}TCR, CD8, CD4), NK cells (rat NKR-P1A vs mouse NK1.1), B cells (rat CD45RA vs mouse B220), and CD11b (macrophages). The results of a representative chimera are presented.

 
Humoral chimerism was assessed by ELISA immunodetection of Ig in serum samples collected from mixed xenogeneic chimeras, transplanted irradiated B10 recipients which failed to engraft (nonchimeras), and nonirradiated B10 recipients which were transplanted and thereby "sensitized" to F344 Ags. Mixed xenogeneic chimeras (n = 9) demonstrate mouse IgM, IgG1, IgG2a, and IgG2b production when compared semiquantitatively to normal B10 mice (Fig. 2). In addition, chimeras demonstrated significant levels of rat IgM and IgG production at ~90 and 60–70% of normal rat controls, even 3 mo after reconstitution. This is in contrast to the absent or minimal levels of rat IgG detected in conditioned recipients that failed to engraft (nonchimeras) or in unconditioned recipients given the same mixed B10 + F344 BM inoculum (i.e., sensitized; Fig. 3). These findings suggest that both cellular and humoral chimerism are present in mixed xenogeneic chimeras as a result of stable engraftment of donor rat marrow and not merely due to the persistence of lymphocytes and/or Abs in the BM inoculum following transplantation.



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  		FIGURE 2. Humoral chimerism in mixed xenogeneic chimeras as evident by the production of mouse Ig isotypes. Murine Ig isotypes were semiquantitatively assessed by sandwich ELISA detection of isotypes within recipient sera using isotype-specific mAbs. Results from mixed xenogeneic chimeras (n = 9) were normalized to absorbencies present in syngenically reconstituted recipients.

 


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  		FIGURE 3. Humoral chimerism is present in mixed xenogeneic chimeras as assessed by the presence of rat IgG in recipient sera as detected by ELISA. Sera were collected from B10 mice, F344 rats, mixed xenogeneic chimeras (n = 8), nonchimeras (n = 3), and nonirradiated transplant recipients (i.e., sensitized n = 4). Levels are semiquantitative as absorbencies are normalized to levels present in control F344 rats.

 
In vitro evidence of donor-specific humoral tolerance in mixed xenogeneic chimeras

Donor-specific tolerance for cellular Ags has previously been demonstrated in mixed xenogeneic chimeras using the proliferative MLR and cytotoxic cell-mediated lympholysis in vitro assays (18, 22). However, humoral tolerance, as defined by the absence of anti-donor Abs in the recipient, has not been investigated. Ab-dependent donor-specific cytotoxicity was measured in recipient sera following transplantation. Sera was collected from mixed xenogeneic chimeras and four control groups which did not exhibit humoral or cellular chimerism: normal B10 mice, F344 rats, B10 nonchimeras, and B10 sensitized to F344 Ags. Sera from all animals were incubated in vitro with 51Cr-labeled F344 rat splenocytes and complement. The percentage of 51Cr release and cell lysis are measures of the cytotoxic activity secondary to anti-donor Abs present within the serum sample. As shown in Fig. 4A, mixed xenogeneic chimeras do not exhibit evidence of donor-specific cytotoxicity over that seen in control B10 mouse and donor F344 rat sera.



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  		FIGURE 4. Ab-dependent donor-specific cytotoxicity is not detectable in mixed xenogeneic chimeras. A, Sera from B10 mice, F344 rats, mixed xenogeneic chimeras (n = 10), nonchimeras (n = 3), and sensitized nonirradiated recipients (n = 6) were assayed for anti-donor cytotoxic Abs using a 51Cr cytotoxic assay. B, Sera from control syngeneic radiation chimeras (n = 4), sensitized, nonirradiated recipients (n = 4), and mixed xenogeneic chimeras (n = 13) were assayed using flow cytometric analysis for detection of anti-donor F344 Ab. For all groups, statistical significance was p < 0.05 compared with mixed xenogeneic chimeras by Student’s t test analysis.

 
To confirm that the lack of cytotoxicity was attributable to the absence of anti-donor Ab in the sera of mixed xenogeneic chimeras and not attributable to insensitivity of the assay, sera from control syngeneic B10 chimeras (B10 -> B10, n = 4); sensitized, nonirradiated B10 recipients (B10 + F344 -> B10, n = 4); and mixed xenogeneic chimeras (n = 13) were analyzed by flow cytometry for the presence of anti-donor (F344) Ab. F344 splenocytes were incubated with the designated sera followed by an anti-mouse Ig FITC secondary Ab. No anti-donor Ab was detected in mixed xenogeneic chimeras (Fig. 4B). In contrast, both assays demonstrated significant anti-F344 donor cytotoxicity and anti-donor Ab in recipients of F344 BM that failed to exhibit donor PBL chimerism, regardless of whether recipients received irradiation (nonchimeras, Fig. 4A) or not (sensitized, Fig. 4B). These findings confirm that mixed xenogeneic chimeras that exhibit both cellular and humoral chimerism also exhibit humoral tolerance, having prevented the development of donor-specific cytotoxic Abs in vivo despite the in situ production of mouse and rat serum Igs and the presence of persistent donor Ag in spleen and peripheral blood.

Mixed xenogeneic chimeras permanently accept donor-specific cardiac xenografts

To assess whether these findings of chimerism and tolerance have in vivo clinical relevance, mixed xenogeneic chimeras received one of three MHC-disparate, primarily vascularized cardiac grafts: donor F344 rat (Rt1Al), third-party rat (WF; Rt1Au; Rt1Aa), or third-party mouse (B10.BR; H-2k). Each graft was placed heterotopically within the abdomen with an anastomosis between the donor aorta and intra-abdominal aorta of the recipient, and between the donor pulmonary artery and recipient IVC. Graft survival was assessed by daily palpation of the spontaneously beating xenograft and confirmed by subsequent histologic analysis. These primarily vascularized cardiac xenografts, unlike skin and islet grafts, are directly exposed to the host circulation and immune system and thus are directly accessible targets for cellular and humoral rejection.

Donor-specific F344 cardiac xenografts were permanently accepted (n = 9; MST >180 days) by B10 + F344 -> B10 xenochimeras (Fig. 5). In contrast, third-party ACI and WF rat or B10.BR mouse cardiac grafts were acutely rejected in a time course similar to that of unmanipulated B10 controls (B10.BR; n = 5, MST = 23 and ACI or WF; n = 9, MST = 41). Furthermore, the prolonged survival of F344 cardiac xenografts was specific for B10 + F344 -> B10 xenochimeras, as F344 xenografts transplanted into unmanipulated B10 mice were rejected within 12 days of transplantation. These studies evaluated donor solid organ tolerance with levels of chimerism ranging from 1.5 to 99%. Since tolerance would be most likely to fail in recipients with low levels (i.e., <10%) of donor chimerism, donor tolerance and third-party rejection were of particular interest in this chimera subset. Analysis of this subset revealed that there was no difference in the level of donor chimerism among chimeras that accepted F344 donor xenografts and those that rejected third-party cardiac grafts (3.0 ± 0.6% vs 4.6 ± 0.6%, p = 0.1).



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  		FIGURE 5. Survival of donor-specific or MHC-disparate third-party mouse and rat cardiac grafts in mixed xenogeneic chimeras reconstituted with a mixture of B10 mouse and F344 rat BM (B10 + F344 -> B10). Unmanipulated B10 mice were used as normal controls. Intra-abdominal heterotopic cardiac transplantation was performed 4–6 wk after BM reconstitution. Transplanted grafts were followed for >100 days. Rejection was assessed by palpation and confirmed by histologic assessment.

 
Evidence for cellular and humoral tolerance toward donor-specific cardiac xenografts in mixed xenogeneic chimeras

It has been shown previously that even with control of hyperacute rejection, cardiac xenograft rejection is associated with intravascular IgM deposition and extensive cellular infiltration (11). To investigate whether subclinical damage to the transplanted xenografts was present, donor-specific xenografts were electively removed at fixed time points and assessed for histologic evidence of cellular or humoral rejection between 16 and 200 days following transplantation into mixed xenogeneic chimeras. Syngeneic B10 cardiac grafts were harvested from mixed xenogeneic chimeras at similar time points to serve as histologic negative controls. No histologic evidence of cellular rejection was present in any of the donor F344 cardiac xenografts (Fig. 6A). Furthermore, donor-specific cardiac xenografts transplanted into mixed xenogeneic chimeras had no evidence of significant intravascular IgM Ab deposition of host origin (Table II and Fig. 6B), even though we have demonstrated that mouse IgM is present within the serum and has direct access to the xenograft. This is in contrast to the extensive mononuclear cell infiltration, myocyte necrosis, and marked intravascular deposits of mouse IgM present in third-party ACI, WF, or B10.BR grafts which appear as early as 16 days after cardiac transplantation (Fig. 6, C and D). These findings are consistent with cellular and humoral rejection, respectively.



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  		FIGURE 6. Histologic assessment of cardiac grafts using H&E staining (original magnification, x40) and immunohistochemical analysis of intravascular mouse IgM deposition. A and B,: F344 cardiac xenografts examined at 200 days after transplantation in B10 + F344 -> B10 chimeras. Note that tissue architecture has been maintained and there is no evidence of mononuclear infiltration, myocyte necrosis (A) or intravascular mouse IgM deposition (B) within the specimens. C and D,: ACI cardiac xenografts examined 16 days after transplantation in B10 + F344 -> B10 chimeras exhibit evidence of extensive tissue destruction and mononuclear cell infiltration (C) and significant intravascular mouse IgM deposition (arrows, D).

 

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  		Table II. Intravascular deposition of mouse IgM in primarily vascularized cardiac grafts

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Xenotransplantation is currently limited by hyperacute rejection due to the presence of preformed natural Abs in the host (5, 6, 7, 8). Furthermore, the presence of anti-species Ab may be tissue specific, obscuring the general distinction of discordant vs concordant status. For example, the rat-mouse combination is considered concordant for solid organs but has been demonstrated to be discordant for BM (32). More importantly, even when hyperacute rejection due to these preformed anti-species Abs is avoided through the use of concordant species, {alpha}-Gal{alpha}1,3Gal knockouts, pretransplant plasmapheresis, or complement depletion, acute rejection of xenografts still occurs secondary to vigorous humoral and cellular effector mechanisms (10, 11, 12, 14, 15, 16). Xenotransplantation cannot become a clinical reality until these acute events of xenoreactivity can be overcome.

After Billingham et al. (33) demonstrated that neonatal allogeneic BM chimerism was associated with donor-specific transplantation tolerance and the subsequent success achieved in adult mouse recipients with the use of lethal irradiation, there has been considerable interest in the use of BM chimerism to induce permanent donor-specific transplantation tolerance for solid organ and cellular grafts within and across species barriers (21, 27, 33, 34, 35). This interest was further fueled by the demonstration that the transplantation of rat BM cells could rescue ablated mice recipients and that these fully xenogeneic (rat -> mouse) chimeras exhibited donor-specific tolerance to rat skin (36, 37, 38). However, poor survival of fully xenogeneic animals and transient engraftment limited studies of tolerance (3, 36, 37, 38, 39). Improved survival of conditioned rat recipients has been achieved with transplantation of massive inocula of 300 x 106 mouse BM cells and recipients exhibited donor-specific tolerance to vascularized xenografts. However, the significance and clinical relevance of this observation are compromised since these recipients are relatively immunoincompetent with B and T cells of only mouse origin (40). This immune incompetence is functionally reflected by the delayed rejection of even third-party xenografts in these animals.

Mixed xenogeneic chimerism (mouse + rat -> mouse), from the transplantation of a mixture of syngeneic T cell-depleted mouse and untreated donor rat BM results in excellent recipient survival and stable hemopoietic chimerism (19, 20). Furthermore, all recipients with ≥0.5% rat PBL chimerism exhibit donor-specific tolerance, both in vitro as assessed by MLR and cell-mediated lympholysis, and in vivo for nonvascularized skin and islet xenografts (18, 19, 20).

Both rat- and mouse-derived T cells develop normally in the xenogeneic thymus, with tolerance toward both mouse and rat Ags. Interestingly, positive selection of developing lymphocytes occurs efficiently, with both rat and mouse T cells selected for immune restriction to host (mouse) MHC Ags (22, 23). The restriction of Ag presentation to host elements present in mixed xenogeneic chimeras, prepared as outlined in this manuscript, has been demonstrated to result in a relative resistance to graft-vs-host disease and to the superior immunocompetence present following mixed reconstitution (19, 22, 23, 41). These mixed xenogeneic chimeras demonstrate that both mouse and rat T cells that develop in a mixed xenogeneic environment can recognize virally infected cells of the host by the generation of viral-specific CTL activity in vivo (22). In contrast, fully xenogeneic chimeras fail to mount an effective cytotoxic cellular response and all animals succumb to influenza A viral infection. Although the cellular immune response of mixed xenogeneic chimeras (mouse to rat) has been studied, the regulation of Ab production and humoral tolerance, particularly in the setting of primarily vascularized xenografts, had not been investigated before the current study.

The vascular endothelium has been demonstrated to be the target of Ab deposition in humoral xenoreactivity. Since there is no direct vascular access between nonvascularized xenografts and the recipient circulation, it has been suggested that the in vivo acceptance of skin and islet xenografts by BM chimeras is secondary to immunologic seclusion. If mixed xenogeneic chimerism was to result in the induction of donor-specific tolerance only for nonvascularized xenografts, the clinical benefits of this approach to tolerance would be limited. Therefore, in the present study, we evaluated mixed xenogeneic chimeras for the induction of humoral tolerance to primarily vascularized cardiac xenografts. BM cells engrafted in mixed xenogeneic chimeras and produced functional progeny within the recipient mouse hemopoietic microenvironment, as evidenced by the presence of donor and recipient T and B lymphocytes and both mouse and rat Ig isotypes in the peripheral blood.

Notably, the chimeras also exhibited donor-specific humoral and cellular tolerance, with long-term donor cardiac xenograft survival in vivo and the absence of Ab-dependent cytotoxic activity toward donor Ags in vitro. Transplantation of the cardiac xenograft in the heterotopic position results in the rapid detection of graft loss and allows direct contact between the recipient immune system and the donor vasculature. Tolerance for primarily vascularized cardiac xenografts was highly donor strain specific, with permanent acceptance of donor-specific cardiac xenografts for over 6 mo without clinical or histologic evidence of cellular or humoral rejection.

It was previously reported that fully xenogeneic chimeras accept donor cardiac xenografts without the induction of cytotoxic Abs (40). However, these studies were limited by the relative incompetence of the recipient immune system and the relative immunosuppression present following fully xenogeneic reconstitution as reflected by a significant delay, up to 200 days in rejection of third-party rat hearts. Furthermore, <1% of recipient lymphocytes were present in these fully xenogeneic chimeras and normal levels of Ab production were not demonstrated following reconstitution, making the ability of these chimeras to mount an effective host Ab response against the donor xenograft suspect. In the present study, the mixed xenogeneic chimeras mounted both humoral and cellular effector mechanisms of rejection against third-party mouse and rat cardiac grafts in a fashion similar to the response by normal B10 recipients. The current study further supports previous evidence for the relative superior immune competence associated with mixed xenogeneic reconstitution, not only by demonstrating intact Ab levels of both donor and recipient isotypes, but also by the prevention of cellular and humoral graft vs host immune responses.

The current study demonstrates that the strict cotolerance present in mixed xenogeneic BM chimeras results in donor-specific transplantation tolerance for vascularized xenografts even in the setting of intact Ab production. We have not investigated the acceptance of primarily vascularized xenografts in the setting of natural or preformed anti-donor Ab to avoid the additional variable of hyperacute rejection. However, recent studies in this laboratory and others have demonstrated that immunologic memory in recipients presensitized against alloantigens or Gal epitopes can be erased with mixed murine reconstitution (24, 25). This success suggests that future investigation of mixed xenogeneic chimerism involving discordant species may be important to our understanding of cross-species tolerance.

In summary, mixed xenogeneic chimerism from the transplantation of a mixture of mouse and rat BM induces long-term donor-specific humoral and cellular xenotolerance for primarily vascularized cardiac grafts. This tolerance occurs in the setting of intact donor and recipient Ab production and, as previously demonstrated, cellular immune competence, suggesting that mixed xenogeneic chimerism may provide a future strategy for the induction of permanent donor-specific tolerance for vascularized xenografts in an immune competent recipient.


   Acknowledgments
 
We extend our appreciation for the surgical expertise and excellent animal care provided by Katheryn Zadach and for the assistance with electronic images by Dr. Kathleen Haley and Solomon Azouz.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This research has been supported in part by National Institutes of Health R29-AI-49033 and R01-HL-074150 (to Y.L.C.); The Leukemia Society of America (to Y.L.C.); American College of Surgeons Scholarship (to Y.L.C.); American Association for Thoracic Surgery (to Y.L.C.); National Institutes of Health Grant R01 HL63442 (to S.T.I.); The Juvenile Diabetes Foundation (to H.X., Y.H., and S.T.I.); The Commonwealth of Kentucky Research Challenge Trust Fund (to S.T.I.); The Jewish Hospital Foundation (to S.T.I.); and the University of Louisville Hospital (to S.T.I.). Back

2 Address correspondence and reprint requests to Dr. Yolonda L. Colson, Department of Surgery, Division of Thoracic Surgery, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115. E-mail address: ylcolson{at}bics.bwh.harvard.edu Back

3 Abbreviations used in this paper: BM, bone marrow; IVC, inferior vena cava; MST, median survival time; TCD, T cell depleted. Back

Received for publication August 8, 2003. Accepted for publication August 20, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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