Elimination of donor CD47 protects against vascularized allograft rejection in mice

Abstract

CD47 is a ubiquitously-expressed transmembrane glycoprotein that plays a complex role in regulation of cell survival and function. We have previously shown that the interspecies incompatibility of CD47 plays an important role in triggering rejection of cellular xenografts by macrophages. However, the role of CD47 in solid organ transplantation remains undetermined. Here, we explored this question in mouse models of heart allotransplantation. We observed that the lack of CD47 in donor hearts had no deleterious effect on graft survival in syngeneic or single MHC class I-mismatched recipients, in which both wild-type (WT) and CD47 knockout (CD47 KO) mouse hearts survived long-term with no sign of rejection. Paradoxically, elimination of donor CD47 was beneficial for graft survival in signal MHC class II- and class I plus class II-mismatched combinations, in which CD47 KO donor hearts showed significantly improved survival compared to WT donor hearts. Similarly, CD47 KO donor hearts were more resistant than WT hearts to humoral rejection in α1,3-galactosyltransferase-deficient mice. Moreover, a significant prolongation of WT allografts was observed in recipient mice treated with antibodies against a CD47 ligand thrombospondin-1 (TSP1) or with TSP1 deficiency, indicating that TSP1-CD47 signaling may stimulate vascularized allograft rejection. Thus, unlike cellular transplantation, donor CD47 expression may accelerate the rejection of vascularized allografts.

Introduction

CD47 is ubiquitously expressed and acts as a marker of “self” that prevents phagocytosis of hematopoietic cells by signaling through signaling regulatory protein (SIRP)α, a critical inhibitory receptor on macrophages and dendritic cells (DCs) (1–3). Donor CD47 expression was found to be protective after cell transplantation. We have shown that the inability of donor CD47 to interact with recipient SIRPα induces macrophage-mediated rejection of xenogeneic hematopoietic cells (4, 5), and that lack of CD47 on donor cells promotes recipient innate immune cell activation and graft loss after hepatocyte transplantation (6). Furthermore, CD47-SIRPα signaling plays an essential role in repressing recipient CD11c^hiSIRPα⁺CD8α^- DC activation and suppressing allograft rejection after donor specific transfusion (7, 8). Recently, human CD47-transgenic pigs showed great promise in facilitating the induction of mixed chimerism and xenograft tolerance in a pig-to-baboon transplantation model (9).

However, the role of CD47 in vascularized organ transplantation has been relatively poorly explored. The present study was designed to determine the role of intragraft CD47 expression in cardiac rejection. Our results indicate that, in contrast to cell transplantation, donor CD47 is deleterious and elimination of CD47 from donor hearts significantly delays graft rejection by both cellular and humoral mechanisms.

Materials and Methods

Mice

C57BL/6 (B6, H-2^b), B6.C-H2^bm1/ByJ (bm1, H-2^bm1) and B6.C-H-2^bm12KhEg (bm12, H-2^bm12) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). CD47 gene knock-out (CD47 KO) mice on the bm1 background were generated by crossing CD47 KO B6 mice (1) with bm1 mice. Thrombospondin-1 (TSP1) KO mice on the bm12 background were generated by crossing TSP1 KO B6 mice (B6.129S2-Thbs1^tm1Hyn/J; Jackson Laboratory) (1)with bm12 mice. α1,3-galactosyltransferase gene-knockout (GalT-KO) B6 mice were bred in our colony. Female mice (6–8 weeks of age) were used in all experiments. Protocols involving animals were approved by the Institutional Animal Care and Use Committee of Columbia University Medical Center and Jilin University First Bethune Hospital. All animal experiments were performed in accordance with the protocols.

Heterotopic vascularized cardiac transplantation

Heterotopic heart transplantation was performed according to the technique previously described (10). Briefly, donor hearts were transplanted into recipient mice by suturing donor aorta and donor pulmonary artery end-to-side to recipient lower abdominal aorta and inferior vena cava, respectively. GalT-KO recipient mice were immunized with rabbit red blood cells (RBCs; 1×10⁹/mouse) 8 days prior to heart transplantation. Ischemic time averaged 35 minutes in length. Grafts were monitored every other day by abdominal palpation, and rejection was defined as complete cessation of a palpable beat and confirmed by direct visualization at laparotomy. Cardiac grafts were harvested from recipient mice on the day of complete cessation of palpable beat or at the indicated times (for surviving grafts) for histological analysis by hematoxylin and eosin (H&E) staining as previously described (8).

Enzyme-linked immunosorbent assay (ELISA)

Serum levels of IgM and IgG antibodies against Galα1–3Galβ1–4GlcNAc (Gal) were quantified by ELISA according to procedures described previously (11). Briefly, serially diluted serum samples were added into the 96-well ELISA plates coated with Gal-BSA (Alberta Research Council, Alberta, Canada), and the bound antibodies were detected using horseradish peroxidase-conjugated polyclonal donkey anti-mouse IgM and IgG antibodies (Accurate Chemical and Scientific Corp., Westbury, NY).

Mixed lymphocyte reaction (MLR)

MLR assay was performed as previously described (7, 8). Briefly, splenocytes prepared from the cardiac allograft recipients and donor mice were used as the responders and allogeneic stimulators, respectively, and responders cultured without allogeneic stimulators were used as unstimulated controls. MLR (i.e., proliferative response) was determined by [³H]-thymidine incorporation assay and data are expressed as cpm values or stimulation index (cpm of stimulated culture/cpm of unstimulated culture).

Skin transplantation

Full thickness tail skins from donor mice were cut into small flaps (approximately 1×1 cm²) and grafted on the dorsum of recipient mice, sutured with 5–0 silk, and secured with Vaseline gauze and bandage for 5 days. Skin graft survival was followed by daily visual inspection and grafts were defined as rejected when <10% of the graft remained viable.

Statistical analysis

Graft survival data were presented as Kaplan-Meier survival curves and differences between groups were analyzed by logrank test using GraphPad Prism (San Diego, CA). Differences between group means were evaluated by one-way ANOVA followed by Bonferroni correction for post-hoc t-test. A p value of <0.05 was considered to be significant.

Results

Lack of CD47 does not induce cardiac graft rejection in syngeneic or single MHC-I-mismatched allogeneic wild-type recipients

We first compared CD47 KO vs. WT B6 mouse heart survival in syngeneic WT B6 mice to determine whether the lack of interaction between donor CD47 and recipient SIRPα can trigger macrophage activation, leading to rejection of solid organ grafts. As expected, WT B6 hearts showed no rejection throughout the observation period of 150 days (Figure 1). Similar to WT grafts, CD47 KO B6 hearts also survived indefinitely (Figure 1A) with no sign of rejection at histology (Figure 1B) in WT B6 mice. Previous studies have shown that cardiac allografts could survive long term when transplanted between B6 and bm1 mice (a single class I-mismatched combination) (12), reflecting an important role for CD4 T cells in cardiac allograft rejection and cardiac allograft vasculopathy (13, 14). Thus, we next assessed whether lack of CD47-SIRPα signaling can stimulate rejection of B6 heart allografts in bm1 recipient mice. Again, both WT and CD47KO B6 hearts showed permanent survival in bm1 mice (Figure 1), despite that skin allografts from the same donors were uniformly rejected within 3 weeks (Figure 1C). The data indicate that, unlike CD47-deficient cellular grafts (e.g., hematopoietic cells and hepatocytes) that induce rapid innate immune cell activation and the associated graft loss after transplantation into CD47^+/+ mice (1–3), CD47 deficiency has no detectable deleterious effects on heart graft survival in syngeneic or single MHC-I-mismatched WT mouse recipients.

Figure 1. WT and CD47 KO cardiac and skin transplantation in syngeneic and single MHC I-mismatched combinations.

(A) Survival times of WT or CD47 KO B6 cardiac grafts in WT syngeneic B6 or allogeneic bm1 mouse recipients (n=5 per group). (B) Cardiac recipient mice were sacrificed at day 150 post-transplantation, and heart grafts were harvested for histological analysis. Shown are H&E staining of representative WT (left) and CD47 KO (right) heart grafts from B6 and bm1 recipients. (C) WT and CD47 KO B6 skin graft survival in syngeneic B6 or a single MHC class I-mismatched bm1 recipients.

Lack of CD47 expression is protective against cardiac allograft rejection

We then investigated whether the lack of CD47 mediates deleterious or protective effects on cardiac allografts in allogeneic combinations, in which the recipients are capable of rejecting WT cardiac allografts. We first compared the survival of CD47 KO vs. WT heart allografts in a MHC-II-mismatched bm12 mice. As shown in Figure 2A, all WT B6 hearts were rejected by 33 days with a median survival time (MST) of 28 days, while most of the CD47 KO B6 hearts survived long-term (p<0.005) in bm12 mice. Similar observation was made in a MHC-I/II-mismatched bm1-to-bm12 combination, in which CD47 KO bm1 hearts had significantly prolonged survival (with a MST of 81 days and approximately 30% of the grafts survived long-term) compared to WT grafts (which were all rejected with a MST of 17.5 days) in bm12 mouse recipients (p<0.05; Figure 2B). Histology revealed that the rejected allografts in both MHC-II- and MHC-I/II-mismatched models had significant mononuclear cell infiltration and myocardial lesions, a typical feature of cell-mediated rejection, which was markedly more severe in WT grafts compared to CD47 KO grafts (Figure 2; right panels). The prolonged survival of CD47 KO cardiac allografts was associated with an inhibition of anti-donor T cell responses. Spleen cells from bm12 mouse recipients of WT B6 hearts, but not from bm12 mice receiving CD47 KO B6 hearts showed significantly enhanced anti-donor MLR compared to T cells from naïve bm12 mice (Figure 3). These results indicate that donor CD47 expression is deleterious to cardiac allograft survival and that elimination of CD47 from the grafts inhibits T cell-mediated rejection.

Figure 2. Donor CD47 exacerbates vascularized allograft rejection.

(A) WT or CD47 KO B6 heart transplantation in MHC class II-mismatched bm12 recipients. Left, graft survival; Right, representative H&E sections of WT (top; rejected at day 20) and CD47 KO (bottom; a surviving graft harvested at day 20) B6 heart allografts. (B) WT or CD47 KO bm1 heart transplantation in MHC class I-plus class II-mismatched bm12 recipients. Left, graft survival; Right, representative H&E sections of WT (top; rejected at day 18) and CD47 KO (bottom; rejected at day 52) bm1 heart allografts.

Figure 3. Reduced anti-donor MLR in the recipients of CD47 KO cardiac allografts.

Spleen cells were prepared from bm12 mice receiving WT or CD47KO B6 hearts, and non-transplanted naïve bm12 mice (Ctrl) at day 20 post-transplantation, cultured with or without B6 stimulators (i.e., irradiated B6 splenocytes), and proliferative responses were measured 48 (left) or 72 (right) hours later. Shown are cpm values (mean±SDs).

Lack of CD47 inhibits antibody-mediated graft loss in a syngeneic mouse model of cardiac transplantation

We next compared the survival of Gal^+/+ WT vs. CD47 KO hearts in syngeneic GalT-KO recipients to determine the role of CD47 in antibody-mediated humoral rejection. Because naïve GalT-KO mice have low levels of natural antibodies against Gal and the majority of these antibodies are IgM, GalT-KO recipient mice were immunized with rabbit RBCs to elevate anti-Gal antibody levels 8 days prior to heart transplantation, as previously described (15). ELISA confirmed the production of anti-Gal IgG in the immunized GalT-KO recipient mice (Figure 4A). Most rabbit RBC-immunized GalT-KO B6 mice rejected WT B6 heart grafts between 2 and 3 weeks following transplantation, and the rejected grafts showed typical features of humoral rejection, such as massive interstitial hemorrhage and edema (Figure 4B). In contrast, all CD47 KO B6 heart grafts survived long-term (Figure 4B). These results indicate that intragraft CD47 expression also exacerbates antibody-mediated (i.e., humoral), T cell-independent cardiac rejection.

Figure 4. Improved survival of CD47 KO compared to WT B6 heart grafts in rabbit RBC-immunized GalT-KO B6 mouse recipients.

GalT-KO B6 mice were immunized with rabbit RBCs, followed 8 days later by heterotopic heart transplantation from WT or CD47 KO B6 donors (n=4 per group). (A) Serum levels of anti-Gal IgG measured immediately prior to and after 22 days following the injection of rabbit RBCs. (B) Donor heart graft survival (Left) and H&E staining of representative WT and CD47 KO heart grafts (Right).

TSP1 accelerates allograft rejection

To understand the mechanisms of how intragraft CD47 expression exacerbates vascularized allograft rejection, we investigated the effect of blockade of CD47 interaction with its ligand, TSP1, on WT allograft survival. Treatment with anti-TSP1 antibodies significantly improved the survival of bm1 heart allografts in bm12 mice (Figure 5A). We also compared the survival of WT and CD47 KO B6 skin allografts in bm12 mice with or without TSP1 deficiency. Similar to heart allografts, CD47 KO skin allografts survived significantly longer than WT B6 skin allografts in bm12 mice (Figure 5B). The survival of WT B6 skin allografts in TSP1 KO bm12 mice was also significantly prolonged compared to those in WT bm12 mice (Figure 5B). Taken together, our results implicate that TSP1 accelerates vascularized allograft rejection, and this deleterious effect is likely largely mediated by TSP1 signaling via its receptor CD47 in the donor graft.

Figure 5. TSP1 promotes allograft rejection in mice.

(A) Survival of WT bm1 hearts in MHC class I plus class II-mismatched bm12 recipients with or without anti-TSP1 antibody treatment (n=5 per group). (B) Survival of WT B6 skin allografts in WT (CD47^+/+ graft in TSP1^+/+ bm12; n=8) or TSP1 KO (CD47^+/+ graft in TSP1^−/− bm12; n-6) bm12 mice, and of CD47 KO B6 skin allografts in WT bm12 mice (CD47^−/− graft in TSP1^+/+ bm12; n=8).

Discussion

CD47 is ubiquitously expressed and plays crucial role in cell adhesion and migration, self-recognition by macrophages, and inflammatory responses (16). In the present study we show that donor CD47 is involved in cardiac graft rejection by both cellular and humoral mechanisms. The lack of CD47 expression in allografts significantly prolonged the survival of MHC class II- or class I plus class II-mismatched cardiac allografts. Elimination of CD47 from donor heart allografts achieved long-term (>100 days) survival in mice without any immunosuppressive treatment, whereas all WT allografts were uniformly rejected in the same strain combinations. Furthermore, elimination of CD47 from donor grafts prevented humoral rejection of Gal-expressing hearts in syngeneic GalT-KO mice. However, the lack of CD47 in donor grafts is insufficient to inhibit heart allograft rejection in a fully MHC-mismatched (B6 into BALB/c) combination without additional treatment (data not shown). Nonetheless, these results demonstrate that the role of donor CD47 in heart transplantation is contradictory to its role in cellular transplantation. In the latter case, the lack of CD47 expression on donor cells induces rapid innate immune cell activation and graft rejection (2, 6–8).

In addition to be a ligand for SIRPα, CD47 also serves as a receptor of TSP1 (16). TSP1 is a matricellular protein produced by a variety of cells including platelets, megakaryocytes, epithelial cells, endothelial cells, cardiac myocytes, neointimal smooth muscle cell, stromal cells, and T cells (17, 18). TSP1 expression is upregulated in tissues following ischemia-reperfusion injury (19, 20) and in allografts undergoing rejection (18, 21). In cardiac allotransplantation, TSP1 elevation in the grafts correlates with the development of cardiac allograft vasculopathy (18). Previous studies have shown that CD47 signaling plays an important role in tissue injury, and that blockade of TSP1-CD47 signaling using antibodies or gene silencing increases ischemic tissue survival and angiogenesis (22, 23). We recently observed that lack of CD47 significantly improves the survival and function of endothelial cells (ECs) both in vitro and in vivo, and that TSP1-CD47 signaling induces EC senescence and cell cycle arrest (24, 25). These studies suggest that TSP1-CD47 signaling may contribute to allograft rejection and that the observed prolongation of CD47 KO allograft survival is likely due to reduced injury by TSP1. Consistent with this possibility, in the present study we observed that allograft survival was significantly prolonged in recipient mice treated with anti-TSP1 antibodies or with genetic deficiency of TSP1.

Although CD47-SIRPα signaling is critical for preventing phagocytosis of hematopoietic cells, CD47-deificient thymic (26), skin (Figure 1C) (27) and heart (Figure 1A-B) grafts could survive long-term in syngeneic WT mice. Furthermore, no detectable difference was found in survival or histology between WT and CD47 KO B6 heart allografts in a single MHC class I-mismatched bm1 mice, in which WT donor heart grafts showed durable survival with no sign of rejection at histology (Figure 1A-B). These results indicate that, unlike cellular transplantation, elimination of donor CD47 does not significantly exacerbate rejection of intact tissue or organ grafts. Our previous studies in CD47 mixed bone marrow chimeras have shown that the lack of CD47 expression on non-hematopoietic cells can induce macrophage tolerance to CD47-deficient cells (3). Thus, it is possible that WT recipient macrophages populated in the CD47 KO organ grafts may become tolerant of CD47-deficient cells and therefore do not cause graft rejection. Further studies are needed to firmly determine the role of macrophages in the rejection of CD47-deficient cardiac allografts.

Previous studies have provided strong evidence demonstrating the critical role for CD47 incompatibility in xenograft rejection by macrophages, supporting the potential of improving xenotransplantation by genetic modification of CD47 (4–9). However, the present study suggests that CD47 expression in donor grafts may be deleterious for vascularized organ transplantation. Further mechanistic studies in this area are expected to provide novel insight into CD47 biology and facilitate the production of genetically modified pigs for clinical xenotransplantation.

Abbreviations:

DC

dendritic cell

Gal

Galα1–3Galβ1–4GlcNAc

GalT

α1,3-galactosyltransferase

MLR

mixed lymphocyte reaction

SIRP

signal regulatory protein

TSP-1

thrombospondin-1

WT

wild-type