Inbreeding depression and lack of genetic diversity in inbred mice could mask unappreciated causes of graft failure or remove barriers to tolerance induction. To test these possibilities, we performed heart transplantation between outbred or inbred mice. Unlike untreated inbred mice in which all allografts were rejected acutely (6–16 days posttransplantation), untreated outbred mice had heterogeneous outcomes, with grafts failing early (<4 days posttransplantation), acutely (6–24 days) or undergoing chronic rejection (>75 days). Blocking T cell costimulation induced long-term graft acceptance in both inbred and outbred mice, but did not prevent the early graft failure observed in the latter. Further investigation of this early phenotype established that it is dependent on the donor, and not the recipient, being outbred and that it is characterized by hemorrhagic necrosis and neutrophilic vasculitis in the graft without preformed, high titer antidonor antibodies in the recipient. Complement or neutrophil depletion prevented early failure of outbred grafts, whereas transplanting CD73-deficient inbred hearts, which are highly susceptible to ischemia-reperfusion injury, recapitulated the early phenotype. Therefore, outbred mice could provide broader insight into donor and recipient determinants of allograft outcomes but their hybrid vigor and genetic diversity do not constitute a uniform barrier to tolerance induction.
Inbred mouse strains have been instrumental in uncovering fundamental immunological mechanisms that underlie transplant rejection. The utility of the inbred mouse as a preclinical model for testing transplantation tolerance strategies, however, has been more limited [1, 2]. Administration of biological agents that readily induce tolerance in inbred mice, for example, often fail in large animals and humans. Failure has been attributed to the evolutionary separation (∼65 million years) between mice and humans, reduced exposure of laboratory mice housed in barrier facilities to microbial pathogens, and/or the biological consequences of inbreeding [1, 2]. The latter include inbreeding depression and restricted genetic diversity within and between inbred mouse strains [3-5].
Known discrepancies between mice and humans that can be attributed to evolutionary separation span both innate and adaptive immunity . Humanized mice in which the human immune system has replaced that of the mouse are beginning to elucidate the importance of these differences , but it remains unclear whether they are superior to inbred mice as preclinical models of organ transplantation. The impact of specific-pathogen free (SPF) housing on the ease by which transplantation tolerance can be induced in laboratory mice, on the other hand, is supported by heterologous immunity studies. Mice previously exposed to microbial pathogens generate memory T cells that crossreact with allogeneic MHC molecules and become resistant to tolerance-inducing agents [8-10]. In contrast, naïve laboratory mice harbor less memory T cells and can be readily made to accept MHC-mismatched allografts. As crossreactive memory constitutes a substantial component of the human alloreactive T cell repertoire [11, 12], heterologous immunity could provide one explanation why transplantation tolerance strategies that succeed in mice often fail in humans.
In addition to their rich repertoire of alloreactive memory T cells, humans are an outbred species characterized by considerable heterozygosity and genetic diversity. Both traits contribute to the vigorous yet diverse immune responses of humans; raising the possibility that outbreeding among humans is an important reason why transplantation tolerance or allograft acceptance strategies do not translate uniformly from inbred mice to patients. Except for one study in which outbred mice were found to require more stringent immunosuppression than their inbred counterparts to achieve islet allograft survival , the effect of outbreeding on allograft outcomes, particularly clinically relevant vascularized grafts, has not been tested yet. Here we investigated the effects of outbreeding in the donor, recipient or both on the survival of vascularized heart allografts in mice.
B6 male mice (6–8 weeks old; Jackson Laboratory, Bar Harbor, ME, USA), nonsibling, CD-1 and CF-1 outbred male and female mice (4 weeks old; Charles River Laboratory [CRL], Wilmington, MA, USA) and BALB/c, FVB and SJL inbred male mice (6–8 weeks old) (also from CRL) were housed under SPF conditions. B6 CD73−/− mice were a generous gift from Linda F. Thompson (Oklahoma Medical Research Foundation). Serum and tail snips were obtained prior to transplantation. PCR analysis to type MHC II loci was performed at CRL . All procedures were IACUC approved.
Transplantation and tissue processing
Vascularized, heterotopic cardiac transplantation was performed as described . Vascular anatomy was similar between inbred and outbred mice and surgical mortality was equivalent in the two groups (3.8% and 4.3%, respectively). Grafts were monitored daily and harvested upon cessation of palpable heartbeat or after 75 days. Serum was collected at harvest and graft tissue was snap frozen and embedded in OCT (Sakura Finetek, Torrance, CA, USA), or paraffin and stained with hematoxylin and eosin (H&E). Histological analysis was performed by AJD in a blinded fashion. Donor splenocytes and thymocytes were harvested at time of surgery.
15 mg/kg MR1 (anti-CD40L mAb) and 15 mg/kg CTLA-4-Ig (BioExpess and BioXCell, West Labanon, NH, USA) were given i.p. on days 0, 2, 4 and 6 to induce long-term graft survival. C3 was depleted by injecting 10 units cobra venom factor (CVF; Quidel Corporation, San Diego, CA, USA) in 3 divided doses i.p. 1 day before transplantation. Neutrophils were depleted by injecting 250 μg anti-Ly6G (1A8) antibody (BioXCell) i.v. 1 day prior to transplantation . Adenosine analog 5′-N-ethylcarboxamidoadenosine (NECA) (Sigma Aldrich, St. Louis, MO, USA) was administered to CD-1 donors (0.1 mg/kg) and CD-1 recipient mice (0.01 mg/kg) i.p. 4 h prior to transplantation.
A panel of RBCs from 10 outbred mice were incubated with pretransplant serum (diluted 1:2) from 7 outbred mice that had accelerated graft loss for 30 min at 37°C, centrifuged at 400g, resuspended and examined for agglutination. If agglutination was absent, the plate was washed and 10-μg anti-mouse-Ig (Chemicon, Temecula, CA, USA) added to each well before reassessing for agglutination.
To measure preformed antibodies to donor MHC, serum was diluted 1:2 and incubated with thymocytes from their respective donors for 20 min at 4°C, stained with anti-IgM and anti-IgG antibodies (BD Biosciences, San Jose, CA, USA) and analyzed on an LSRII cytometer.
Antibody deposits were examined in allografts transplanted between outbred mice. As a positive control, we transplanted BALB/c hearts into C57Bl/6 mice presensitized by transplanting BALB/c skin grafts10 days earlier. Allografts were harvested at 6 or 12 h posttransplantation and frozen in OCT. Six-micrometer sections were fixed, blocked (Vectabond ABC Blocking Kit, Vector Laboratories, Burlingame, CA, USA) and stained with FITC-conjugated anti-IgG or anti-IgM (Zymed Laboratories, San Francisco, CA, USA) at room temperature for 1 h. To identify neutrophils, sections were incubated with unconjugated anti-Ly6G and biotin conjugated CD31 (eBioscience, San Diego, CA, USA) overnight at 4°C. After incubating with secondary antibodies, the tissue was counterstained with DAPI (Molecular Probes, Eugene, OR, USA).
Enzyme-linked immunosorbent assay (ELISA)
C3 serum levels were measured pre-transplantation and on days 1, 5, 7 and 11 after transplantation using mouse C3 ELISA kit (Kamiya Biomedical, Seattle, WA, USA) per manufacturer's instructions.
Flow cytometry data is shown as mean fluorescent intensity (MFI) and standard error of mean (SEM) and analyzed by unpaired t-test. Presence or absence of early graft failure was analyzed using a two-sided Fischer's exact test. Median survival time (MST) comparisons were performed by Gehan–Breslow–Wilcoxon test.
Genetic diversity of outbred mouse stocks
CD-1 and CF-1 outbred mouse stocks, defined as closed populations of genetically variable animals that are bred to maintain maximum heterozygosity , were used as either donors or recipients of heart grafts. Mice from either stock displayed outbred vigor judged by greater body weight and larger litter size than inbred strains (http://www.criver.com). To determine their diversity, we genotyped class II H-2 loci by PCR. Of 12 alleles that were genotyped, four (p, b, q and u) were represented in the CD-1 (n = 364) and five (p, b, q, u and k) in the CF-1 stock (n = 79), resembling the diversity of an isolated "island" population . The extent of heterozygosity at these loci was 0.60 and 0.74 in the CD-1 and CF-1 stocks, respectively, which is similar to the average heterozygosity of feral mice and humans . Therefore, the outbred mouse stocks used in this study are somewhat limited in their diversity but maintain a significant degree of heterozygosity typical of outbred populations.
Blocking T cell costimulation induces long-term allograft acceptance in outbred mice
We hypothesized that a standard tolerogenic regimen that induces allograft acceptance in inbred mice would be less effective in outbred mice. This was tested by comparing the survival of heart allografts transplanted between disparate inbred mouse strains (BALB/c to B6, n = 41) to that of allografts transplanted between nonsibling outbred mice (CD-1 to CD-1, n = 33, or CF-1 to CD-1, n = 28). Recipients were either left untreated or received a combination of CTLA4-Ig and anti-CD154 (MR1) to block T cell costimulation at the time of transplantation. As expected, T cell costimulation blockade induced 100% allograft acceptance (graft survival >75 days) in the inbred group while all untreated recipients rejected their allografts acutely (MST = 8 days; Figure 1A). In contrast, graft outcomes were not uniform in the outbred group (Figure 1). In untreated mice, grafts failed either very early (1–4 days after transplantation, 10/35 or 29%), acutely (6–24 days, 19/35 or 54%) or underwent chronic rejection (>75 days, 6/35 or 17%) with graft MST remaining similar to that of the inbred group (10 days vs. 8 days, p = 0.52). In treated, outbred mice, the response was dichotomous with 7/26 (27%) losing their grafts very early and 19/26 (73%) achieving long-term graft survival. The rate of graft acceptance in the treated, outbred group was significantly greater than that in the untreated outbred group (73% vs. 17%, p < 0.0001). Treated, outbred recipients that did not manifest the unusual early graft failure went on to have 100% (19/19) long-term allograft acceptance similar to the treated inbred group. Allograft acceptance after costimulation blockade occurred independent of outbred stock combination used (69% in CD-1 to CD-1 and 80% in CF-1 to CD-1, n = 16 and 10, respectively, p = 0.67). Histopathological analysis revealed mild cellular infiltration, minimal fibrosis and focal chronic vasculopathy (intimal thickening) in long-term accepted allografts in the treated inbred and outbred groups, while long-term surviving allografts in the untreated outbred group exhibited chronic rejection, ranging from severe to focal (Figure 1C). These data indicate that genetic diversity and heterozygosity (hybrid vigor) lead to more heterogeneous graft outcomes in untreated mice but do not constitute a uniform barrier to graft acceptance after induction therapy.
Increased early graft failure in outbred mice
An unexpected finding that emerged from the above experiments is that all treated, outbred mice that lost their allografts (27%) did so between 1 and 4 days after transplantation (Figure 1B). This accelerated graft loss was also observed in a similar proportion (29%) of untreated mice in the outbred group (Figure 1B), but in none (0/41) of the untreated or treated mice in the inbred group (p < 0.0001; Figure 1A). Early graft failure was not influenced by the particular outbred stock combination used (33% in CD-1 to CD-1 and 21% in CF-1 to CD-1, p = 0.40). The gross morphology and histopathology of early graft failure were distinct from classical acute cellular rejection (Figure 2). Allografts that failed very early were significantly enlarged and were dark in color. Histopathology revealed extensive hemorrhagic necrosis with neutrophilic margination and focal neutrophilic arteritis without mononuclear cell infiltration (Figure 2, left panels). In contrast, acutely rejected hearts in the untreated outbred group exhibited typical severe acute lymphocytic infiltration with arteritis (Figure 2, right panels). These findings suggest that the unusually high early graft failure observed exclusively in the outbred group is caused by an inflammatory or innate immune process.
Early graft failure is dependent on donor's outbred status
Data from mouse and human studies suggest that allograft outcomes are not only determined by the recipient's genetic background but also by that of the donor. To investigate whether recipient or donor outbred status is responsible for early graft failure, we transplanted inbred (BALB/c) hearts into outbred CD-1 mice and vice versa. Donors and recipients were weight matched to avoid unanticipated surgical complications that could arise from transplanting grafts into size-mismatched recipients. In untreated recipients, early graft failure was observed in 7/26 (27%) of inbred recipients of outbred hearts (outbred to inbred [O to I] group) but in none of the outbred recipients of inbred grafts (inbred to outbred [I to O] group; Figure 3A). The donor outbred status effect was also observed in immunosuppressed recipients. Costimulation blockade induced 100% allograft acceptance in the I to O group, while 5/10 (50%) of grafts failed in the first 4 days in a similarly treated O to I cohort (Figure 3B). Analysis of all outbred to outbred (O to O) or O to I transplantation experiments showed that graft outcome (early failure, acute rejection and chronic rejection) did not correlate with the degree of MHC class II mismatch between donor and recipient (Figure 3C).
Since CD-1 animals were originally derived from the Swiss mouse stock , we then asked whether early graft failure is determined by genetic determinants specific to the Swiss background. To answer this question, we performed heart transplants using inbred Swiss strains as either donors (SJL to C57Bl/6, n = 23) or as both donors and recipients (SJL to FVB, n = 9). Of these, only 1/23 (3%) (p = 1.0 compared to inbred to inbred [I to I] group) in the former and none in the latter group exhibited the early graft failure phenotype, indicating that the Swiss background of CD-1 mice does not account for the high incidence of early graft failure observed in the O to I (Figures 2A and B) or in the O to O (Figure 1B) transplantation experiments.
Role of antibodies in pathogenesis of early graft failure
The histopathology of early graft failure described above bears striking resemblance to that of hyperacute rejection observed in sensitized transplant recipients who harbor preformed antibodies against donor ABO or HLA antigens [19, 20]. We, therefore, tested for such antibodies in the pretransplantation serum of mice that went on to develop early graft failure. Neither pretransplantation serum from seven such recipients (all outbred) nor serum from additional 10 untransplanted outbred mice caused agglutination of blood from a panel of 10 unrelated outbred mice (data not shown), indicating absence of significant preformed hemagglutinins. These results are consistent with reported lack of demonstrable expression or function of blood group antigens in mice [21, 22]. We then tested whether recipients that went on to develop early graft failure had preformed IgG or IgM antibodies against donor MHC by incubating pretransplantation serum with donor thymocytes. As shown in Figure 4A, no antidonor IgG antibodies were detected while a small increase in IgM antibodies over that found in pretransplantation serum of one cohort of mice (O to I) that developed acute rejection could be identified, with the caveat that all groups had very low IgM levels to start with. These data indicate the absence of significant preformed IgG or IgM anti-MHC antibodies in recipients that develop early graft failure but do not rule out the presence of antibodies directed at other tissue antigens. To explore the latter possibility, we assessed antibody binding to graft tissue 6 and 12 h after transplanting outbred hearts into inbred recipients. IgM and IgG could not be detected at 6 and 12 h in grafts that already displayed early histopathologic evidence of hemorrhagic necrosis (Figure 4B). Grafts harvested from prior experiments that had already developed conspicuous hemorrhagic necrosis at 24 h after transplantation stained strongly with fluorescein-conjugated anti-IgM or anti-IgG antibodies (micrographs not shown). The presence of significant tissue damage and hemorrhage at the 24 h time point, however, makes it likely that fluorescein conjugated antibodies bound to complement and Ig in the extravasated blood rather than in the heart tissue. These data, therefore, rule out the presence of significant, preformed, antidonor antibodies in recipients that develop early graft failure but do not exclude a contribution of low titer antibodies to the observed pathology.
Early graft failure is dependent on complement activation and neutrophils
Because of the characteristics of the early graft failure phenotype observed so far, we sought to investigate innate mechanisms that could contribute to this process. We first depleted circulating C3 in outbred donor and recipient mice by administering cobra venom factor (CVF) around the time of transplantation. We found that C3 depletion completely abrogated early graft failure (n = 0/20, p = 0.02 compared to untreated [O to O] group; Figure 5A), but as C3 levels returned to baseline, five heart grafts failed between days 8 and 18 after transplantation with histopathologic manifestations of hemorrhagic necrosis (Figure 5A). These results establish a cause–effect relationship between the complement cascade and early graft failure. They also confirm that the early hemorrhagic necrosis phenotype is not a reflection of an unanticipated high surgical failure rate but is a consequence of a biological process dependent on complement activation.
Since neutrophil infiltration and neutrophilic vasculitis were prominent features of grafts that failed very early (Figure 2), we then investigated the effect of neutrophil depletion on the incidence of early graft failure. As shown in Figure 5B, only 2/25 (8%) of neutrophil-depleted inbred recipients of outbred allografts developed hemorrhagic necrosis, which is significantly less than the incidence observed in control, neutrophil-replete mice (7/26 or 27%, p = 0.03). Of the two grafts that had hemorrhagic necrosis in the neutrophil-depleted group, one failed on day 2 with significant neutrophil infiltration, suggesting incomplete neutrophil depletion of the recipient. The other failed on day 7, again with conspicuous neutrophil but no mononuclear cell infiltration, suggesting that early graft failure phenotype could have been precipitated by return of neutrophils to the circulation. These data indicate that neutrophils are important contributors to the pathogenesis of early graft failure.
Increasing the transplanted tissue's susceptibility to ischemia-reperfusion injury recapitulates early graft failure
The pathology and mechanisms of early graft failure described so far suggest that donor factors, possibly related to susceptibility of donor tissues to ischemia-reperfusion (I/R) injury, contribute to the early graft failure phenotype. To test this hypothesis, we investigated whether transplanting heart allografts from inbred CD73−/− donors, known to have exaggerated tissue I/R responses , recapitulates early graft loss otherwise typical of outbred grafts. CD73 is an ectonucleotidase expressed on endothelial cells that downregulates I/R injury by catalyzing the hydrolyis of extracellular AMP to the anti-inflammatory metabolite adenosine . We found that CD73−/− B6 hearts transplanted to BALB/c mice exhibit high incidence of early graft failure (4/18 or 22%) similar to that of outbred allografts transplanted to inbred recipients (7/26 or 27%, p = 1.0). Histopathology confirmed that early graft failure was due to hemorrhagic necrosis and neutrophilic vasculitis. Conversely, treating donors and recipients with NECA, a broad adenosine receptor agonist, abrogated early graft loss in the O to O model (n = 0/14, p = 0.02). These results suggest that donor factors that underly susceptibility to I/R injury also play a role in the pathogenesis of early graft failure after transplantation.
It has been postulated that hybrid vigor and genetic diversity of the outbred human population could explain why immune therapies that are successful in inbred mouse models sometimes fail in humans . In this study, we found that costimulation blockade induces long-term allograft acceptance in outbred mouse stocks implying that hybrid vigor cannot account for the discrepancy between humans and inbred mice but does not completely rule out the contribution of genetic diversity. The outbred mouse stocks used in this study have been bred to maintain maximum heterozygosity (thus, hybrid vigor) but are still somewhat limited in their genetic diversity compared to the human population . It is possible then that costimulation blockade may fail to induce allograft acceptance if a more diverse outbred mouse population was studied. Such a population, known as diversity outbred (DO) mice, has been recently generated but remains limited in its availability to investigators (http://jaxmice.jax.org/strain/009376.html; . Investigating allograft outcomes in these mice in the future should resolve whether simultaneous presence of maximal heterozygosity (hybrid vigor) and allelic variation (diversity) impedes allograft acceptance. Moreover, it is possible that the contribution of these genetic variables is only apparent when outbred mice are housed in non-SPF facilities to increase the repertoire of alloreactive memory T cells through heterologous immunity.
A potentially interesting observation in the O to O transplantation group (Figure 1B) is the occurrence of spontaneous long-term allograft survival (>75 days), albeit with significant chronic rejection, in approximately 17% of untreated recipients. The reason for this phenomenon was not addressed in this study but could be related to matching between donors and recipients at non-H-2 loci. By performing a large number of heart transplants between inbred mouse strains matched at either H-2 or non-H-2 loci, Peugh et al. found spontaneous long-term graft survival in 25% of recipients matched at H-2 but mismatched at non-H-2 loci (and vice versa) , suggesting that non-H-2 loci modulate the rejection response. Identifying such loci may be a difficult undertaking at present as it is likely that many loci with additive or opposite effects (quantitative trait loci) exist. Moreover, the spontaneous long-term allograft survival phenotype observed in our study all but disappeared when only donors or recipients were outbred (Figure 3A), implying a complex interplay between donor and recipient determinants. This warrants careful analysis in the future.
A principal finding in our study is the identification of a dramatic, early graft failure phenotype characterized by neutrophilic vasculitis and hemorrhagic necrosis in 29 of a total of 97 outbred grafts (30%), but in only 1 of a total of 94 inbred grafts (1%), transplanted to either inbred or outbred mice. All transplants reported in this study were contemporaneous and were performed by the same microsurgeon (Q.L.). Moreover, surgical mortality, which occurred in the first day after transplantation due to failure of the vascular anastomosis, was equivalent in the outbred and inbred graft groups (3.8% and 4.3%, respectively). These facts make it less likely that the early graft failure phenotype was a consequence of high technical failure rate in mice that received outbred grafts. The most compelling evidence that early graft failure is a bona fide biological phenotype is its abrogation when specific biological mediators were eliminated (for example, after complement or neutrophil depletion) and its reappearance at later time points when the mediators had rebounded.
The timing and histopathologic hallmarks of the early graft failure phenotype and its dependence on complement but not T cell costimulation strongly indicate that it is caused by innate or inflammatory mechanisms. Although we did not identify the initial trigger of the inflammatory cascade that led to acute hemorrhagic necrosis, one possibility is the binding of complement fixing, low titer antibodies that evaded detection by either serology or immunofluorescence. Carroll and colleagues have shown that natural IgM antibodies present in low titers initiate I/R injury in the heart, intestine and skeletal muscles . These antibodies bind to self-antigens exposed after tissue ischemia and are potent activators of the complement cascade . Alternatively, IgM antibodies with antidonor allospecificities may have been present in recipients that exhibited the early graft failure phenotype. Another possibility is that hemorrhagic necrosis was triggered by the alternative pathway of complement activation, which is independent of antibody binding to the affected tissue. The role of the alternative pathway in the pathogenesis of ischemia reperfusion injury has been established in a variety of experimental models .
The predominance of the neutrophil among inflammatory cells infiltrating grafts undergoing hemorrhagic necrosis is not surprising. Neutrophils are attracted to sites of I/R injury via many mediators, including products of complement activation. The early graft failure phenotype observed in our experiments bears striking resemblance to accelerated rejection of cardiac allografts by IFNγ-deficient mice  and to accelerated rejection of IFNγ receptor-deficient renal allografts by wildtype recipients . In both cases, microscopic examination of the grafts revealed intense neutrophilic infiltration and parenchymal necrosis. As in our model, neutrophil depletion prevented accelerated rejection, further underscoring the importance of the neutrophil as a mediator of early inflammatory events after organ transplantation.
Another principal finding in our study is that the early graft failure phenotype was dependent on the breeding status of the donor and not the recipient. That donor factors, independent of MHC matching, influence graft outcomes has been documented in both mice and humans . Several studies have pointed to the role of the donor complement system in influencing graft outcomes. For example, donor C3 deficiency leads to prolonged survival of kidney allografts while donor deficiency of decay-accelerating factor (DAF) accelerates cardiac allograft rejection in mice [33, 34]. What donor factors influenced graft outcomes in our study is unclear. We tested whether differences in expression of the complement regulatory molecules DAF and Crry among donors correlate with presence or absence of early graft failure but did not find a significant correlation (Reichenbach, Lakkis & Heeger, unpublished). Our observation that inbred CD73−/− donors recapitulate early graft failure suggests that determinants of tissue susceptibility to I/R in the donor influence graft outcomes after transplantation. This warrants future studies to test whether CD39, CD73 and adenosine receptor expression in outbred mice correlates with graft outcomes.
The model of hemorrhagic necrosis described in this manuscript underscores the importance of the donor in shaping graft outcomes and provides an opportunity to gain better understanding of the inflammatory events that lead to graft injury. Interrupting these events could prevent the adverse consequences of I/R injury on early graft function and possibly temper innate responses that adversely affect long-term allograft outcomes .
We acknowledge Dr. Linda Thompson (Oklahoma Medical Research Foundation) for critical reading of the manuscript and Dr. Peter Heeger (Mount Sinai School of Medicine) for assistance with complement regulatory molecule measurements. This work was funded by NIH grant AI064343 to DMR, WDS and FGL. DKR was funded by NIH/NIAID T32 grant AI 074490.
The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.