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Basic and Experimental Research

The Innate Immune Response and Activation of Coagulation in α1,3-Galactosyltransferase Gene-Knockout Xenograft Recipients

Ezzelarab, Mohamed1; Garcia, Bertha2; Azimzadeh, Agnes3; Sun, Hongtao2; Lin, Chih Che1; Hara, Hidetaka1; Kelishadi, Sean3; Zhang, Tianshu3; Lin, Yih Jyh1; Tai, Hao-Chi1; Wagner, Robert4; Thacker, Jnanesh5; Murase, Noriko1; McCurry, Kenneth1,5; Barth, Rolf N.3; Ayares, David6; Pierson, Richard N. III3; Cooper, David K.C.1,7

Author Information
doi: 10.1097/TP.0b013e318199c34f
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Abstract

The availability of α1,3-galactosyltransferase gene-knockout (GTKO) pigs has enabled the transplantation (Tx) of organs into nonhuman primates (1, 2). Initial studies by Kuwaki et al. (3, 4) indicated that with an immunosuppressive regimen that prevented the T–cell-dependent adaptive immune response, heart grafts survived for 2 to 6 months; graft failure was from a thrombotic microangiopathy (TM) (5).

Subsequently, however, Chen et al. (6) reported both hyperacute rejection and acute humoral xenograft rejection (AHXR) in renal grafts from GTKO pigs in baboons that received an immunosuppressive regimen that did not prevent an elicited antibody response. These in vivo observations correlated with in vitro assays that indicated a significant cytotoxicity associated with the binding of antibodies to GTKO pig cells (7–10).

We here report 12 organs (nine hearts, three kidneys) from GTKO pigs transplanted into baboons that received no therapy, partial regimens, or full therapeutic regimen based on costimulation blockade. With or without an adaptive immune response, the histopathologic features seen were associated with an innate immune response, whereas TM was a constant feature.

METHODS

Animals

Baboons (Papio species, 6–20 kg, of known ABO blood type, n=12: Division of Animal Resources, Oklahoma University Health Sciences Center, Oklahoma City, OK, and the Southwest Foundation for Biomedical Research, San Antonio, TX) were recipients. Homozygous GTKO pigs (n=9, of blood group O (non-A), 10–20 kg, Revivicor, Blacksburg, VA), served as sources of hearts (n=9) and kidneys (n=3) (1,3Gal (Gal) expression (1).

All animal care was in accordance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication No. 86-23, revised 1985). Protocols were approved by the University of Pittsburgh or the University of Maryland Institutional Animal Care and Use Committee.

Surgical Procedures

Anesthesia, intravascular catheter placements in pigs and baboons, heart and kidney excision in pigs, renal Tx, and heterotopic heart Tx (in the abdomen) in baboons have been described previously (12–14). Two independent observers regularly monitored cardiac graft function by palpation. Kidney graft status was monitored by serum creatinine (13–16).

In some cases, after excision of a rejected heart graft, the baboon was followed up for several weeks; remnants of pig aorta and pulmonary artery remained in the baboon. After rejection of a life-supporting kidney graft, the baboon was euthanized.

Immunosuppressive and Supportive Therapy

Baboons received no therapy (n=2), partial immunosuppression (n=7), or full immunosuppression (n=3) (Table 1). Baboons with partial immunosuppression electively received cobra venom factor only (n=2), cobra venom factor+thymoglobulin+leflunomide (n=1), or low-dose anti-CD154 monoclonal antibody [mAb] (ABI193; NovartisPharma, Basel, Switzerland)+low-dose cytotoxic T-lymphocyte antigen 4 -Ig+mycophenolate mofetil (n=2). In two others, technical problems prevented the full therapy from being administered. The full therapeutic regimen consisted of induction with thymoglobulin (Genzyme, Cambridge, MA) and maintenance with anti-CD154 mAb, mycophenolate mofetil (Roche, Basel, Switzerland), and methylprednisolone (17, 18).

TABLE 1
TABLE 1:
Immunosuppressive regimen, graft survival, and complications after GTKO organ transplantation in baboons

Because consumptive coagulopathy (CC) can develop in xenograft recipients (19, 20), and in view of potential anti-CD154 mAb-associated thromboembolic complications (21, 22), heparin was begun on day 0 and gradually increased to maintain the activated partial thromboplastin time (aPTT) at 150 sec (previous experience demonstrated that maintenance of the aPTT more than 150 sec was associated with an increased risk of bleeding). However, this goal was not always achieved.

Monitoring of Recipient Baboons

Blood cell counts, chemistry, and coagulation parameters were measured by standard methods. The serum trough level of the anti-CD154 mAb was monitored by ELISA (maintained at >400 μg/mL in the full regimen) (23), and T/B cell numbers by flow cytometry. Anti-non-Gal antibody levels were determined by flow cytometry, and serum cytotoxicity was determined using GTKO peripheral blood mononuclear cells (7).

Histopathology and Immunohistopathology of Porcine Heart and Kidney Grafts

Biopsies of xenografts were obtained 30 min after reperfusion, at the time of graft excision, or at the time of euthanasia. Tissues were fixed in 10% formalin and embedded in paraffin. Sections of 4 μm were stained with hematoxylin-eosin and with martius-scarlet-blue (MSB) for light microscopy. Immunohistochemical (IHC) staining for IgM, IgG, C3, C4d, fibrin and platelets, neutrophils (myeloperoxidase), macrophages (CD68), T lymphocytes (CD3), and B lymphocytes (CD20) was performed (6). Primate tissue factor (TF) was stained using an anti-human TF antibody (American Diagnostica, Stamford, CT).

Quantitative Real-Time Polymerase Chain Reaction Measurement of Primate TF mRNA Levels in Heart Xenografts

Total RNA was extracted from the graft or native heart using Trizol (Life Technologies, Grand Island, NY). RNA content was measured using 260/280 UV spectrophotometry. Briefly, total RNA pellets were suspended in RNase-free water, followed by treatment with DNase I (Life Technologies, Rockville, MD). RNA (3 μg) from each sample was used for reverse transcription with an oligo dT (Life Technologies) and Superscript II (Life Technologies). Polymerase chain reaction mixture was prepared using SYBR Green PCR Master Mix (PE Applied Biosystems, Foster City, CA). Primers for baboon-TF were 5′-TGCTTTTACACAGCAGACACAGAGT-3′ (forward) and 5′-AAGACCCGTGCCAAGTACGT-3′ (reverse); baboon-β-actin were 5′-TGGAAGAATGCGGCTCATATT-3′ (forward) and 5′ TACTATCCAATCCTAGAAAGAACATG-3′ (reverse). Thermal cycling conditions were 10 min at 95°C, followed by 40 cycles of 95°C for 15 sec, and 60°C for 1 min on an ABI PRISM 7000 Sequence Detection System (PE Applied Biosystems).

RESULTS

Pig Heart and Kidney Graft Survival and Complications

With no immunosuppression (n=2), graft survival was less than 24 hr, though in one case this was because the graft had to be excised because it was too large to close the abdomen (Table 1). Both hearts were excised, and both baboons were followed for monitoring of their immune response. Graft survival was prolonged to 2 to 12 days in baboons receiving partial immunosuppression (heart Tx, n=5; kidney Tx, n=2), with graft failure from AHXR. In baboons receiving the full regimen (n=3), with one exception, graft survival was extended to 5 and 8 weeks (Table 1). In 7 of the 12 baboons, features of CC were detected (Table 1). CC was identified clinically, that is, bleeding, and through laboratory parameters (rapidly falling platelet count, falling hematocrit, and prolongation of PT and aPTT even after the discontinuation of heparin infusion) (24), which were seen at the time of cessation of graft function or euthanasia.

Immunologic Monitoring

In those that received the full regimen, T and B cell counts remained low, as reported previously (3, 4). There was a trend for an increase in neutrophils and mononuclear as rejection developed. The platelet count was variable, depending on immunosuppressive therapy and the adequacy of heparinization, but generally fell as TM developed.

Anti-non-Gal IgM was present in all baboons before Tx, but only one had anti-non-Gal IgG. In those baboons that received no or partial immunosuppressive therapy and were followed up for more than 8 days (in the presence of a functioning graft or after graft excision), there was an increase in both anti-non-Gal IgM and IgG binding on flow cytometry, and increased lysis of GTKO peripheral blood mononuclear cells in the serum cytotoxicity assay, indicating sensitization to non-Gal antigens.

In baboons that received full therapy, there was no increase in antibody binding or serum cytotoxicity to GTKO peripheral blood mononuclear cells, indicating that this regimen had prevented sensitization, as reported previously (3, 4, 17, 18, 25).

Histopathology of GTKO Pig Hearts and Kidneys

The histopathological findings at the time of graft excision or recipient euthanasia are summarized in Table 2. Of importance, as early as 1 hr after reperfusion, most grafts showed features of a humoral response (IgM, IgG, and complement deposition on the vascular endothelium). Irrespective of the period of graft survival, features of a humoral response were present in 10 of 12 at the time of graft excision (Figs. 1 and 2). Furthermore, all excised grafts showed features of TM (widespread platelet and fibrin deposition) in the interstitial capillaries and large vessels (Fig. 2), except the heart graft electively excised after 2.5 hr.

TABLE 2
TABLE 2:
Graft histopathology at the time of graft failure
FIGURE 1.
FIGURE 1.:
Histopathologic features of failed heart grafts. Graft failure occurred after 24 hr in Experiment 2, showing (A) hemorrhage, thrombosis, and infarction (hematoxylin-eosin, ×400), (B) fibrin deposition (martius-scarlet-blue [MSB], ×400), and (C) platelet accumulation (immunohistochemical [IHC], ×400). IgM (D), IgG (E), and complement C3 (F) deposition (all indicated by brown staining-arrows) were present in the heart of Experiment 3 that underwent failure at 7 days from acute humoral xenograft rejection (×400). Graft failure occurred on day 6 in Experiment 6, showing significant neutrophil (hematoxylin-eosin, ×200) (G) and macrophage (H) infiltration, with relatively less T (I) and B (J) cell infiltration (all IHC, ×200, stained brown).
FIGURE 2.
FIGURE 2.:
Histopathologic features of the heart graft that failed after 8 weeks (Experiment 12), showing (A) thrombosis and infarction (hematoxylin-eosin, ×200), (B) fibrin deposition (martius-scarlet-blue [MSB] ×400), and (C) massive platelet accumulation (immunohistochemical [IHC], ×400). A neutrophil infiltrate can be seen in the interstitium (IHC, ×400) (D); these cells are also present in the vessels of the graft (E), but there are relatively few infiltrating macrophages (IHC,200) (F), and few T (G) and B (H) lymphocytes (stained brown) (magnification ×200).

With no immunosuppression, the main features in the failed grafts were of the humoral response and TM together with cellular infiltration in the form of neutrophils and macrophages only (Fig. 1A–C). With partial immunosuppression, the grafts showed features of AHXR and TM (Fig. 1D–J), but some grafts showed no or minimal antibody and complement deposition. T and B cell infiltrates were not a constant feature in these grafts. With the full immunosuppressive regimen, myocardial ischemia and TM were the prominent features (Fig. 2) with features of a humoral response at the time of excision; T and B cell infiltrates were minimal.

Primate Tissue Factor Expression in Failed Xenografts

Five of the heart grafts were examined for primate TF expression. These grafts showed strong expression of TF in the thrombosed vessels and less expression in the interstitium (Fig. 3A,B). To further elucidate possible sources of the primate TF in the failed pig hearts, double staining for macrophages and primate TF was performed; this showed colocalization of TF with CD68+ cells (macrophages) in both the thrombosed vessels and in the interstitium of the graft (Fig. 3C,D), suggesting a role for these cells in the initiation of TM through the expression of TF. Primate TF was detected in high levels compared with lower levels in a normal baboon heart and none in a normal pig heart (Fig. 3E).

FIGURE 3.
FIGURE 3.:
Staining for primate tissue factor (TF) in heart grafts that rejected at day 12 (A; Experiment 5) and at 8 weeks (B; Experiment 12), showing strong staining in the thrombosed vessels and less staining in the interstitium (arrows) (×600). Colocalization of primate TF (red stain) and macrophages (stained for CD68, brown) in heart grafts excised on day 12 (C, Experiment 5) and at 8 weeks (D, Experiment 12) is indicated by arrows (×600). Biopsies from three other rejected xenografts showed similar results. Primate TF mRNA levels in a rejected porcine heart xenograft by quantitative real-time polymerase chain reaction (E); the heart failed from thrombotic microangiopathy at 8 weeks (Experiment 12) and showed high levels of primate TF. Tissue from a normal baboon heart was used as a positive control, while a porcine heart was used as a negative control to ensure the specificity of the primate TF primer. Real-time polymerase chain reaction data were plotted as the ΔRn fluorescence signal versus the cycle number. The expression of each gene was normalized to actin mRNA content and calculated relative to control using the comparative CT method.

DISCUSSION

Hyperacute rejection follows the Tx of wild-type pig organs in nonhuman primates (14, 26), and has been reported after GTKO kidney (6) and heart (27) Tx in baboons, although this complication was not seen in the initial series of studies (3, 4, 16).

In the present series, the hearts transplanted in the absence of any immunosuppressive therapy (Experiments 1 and 2) showed some features of vascular injury as early as 2.5 hr after Tx. The heart graft excised at 24 hr showed typical features of antibody-mediated rejection. There was a neutrophil infiltrate in both grafts, and macrophages were present in the thrombi in the vessels. In both cases, as it was too early for an elicited antibody response to have developed, the histopathologic features must have been related to the innate immune system. This experience also indicated that CC can occur as early as 24 hr after Tx.

When only partial immunosuppressive therapy was administered (Experiments 3–7), or when therapy was unable to be administered adequately (Experiments 8 and 9), graft failure was associated with AHXR which occurred before or after an elicited anti-non-Gal antibody response, with a cellular infiltrate consisting mainly of neutrophils and macrophages, T and B lymphocytes being less obvious. Features of TM were widespread, and CC occurred in five of the seven baboons. The predominant histopathologic features were, therefore, associated with an innate immune response. The results in these experiments correlate well with those of Chen et al. (6), and demonstrate that similar histopathology is seen in hearts and kidneys.

With full immunosuppression, there was deposition of IgM, IgG, and complement, and neutrophil and macrophage infiltration, but there was minimal T and B cell infiltration, and no evidence of a T–cell-dependent elicited antibody response (Experimetns 11 and 12). These findings confirm that an immunosuppressive regimen based on costimulation blockade can prevent sensitization to pig antigens, correlating with previous reports (3, 4). The presence of IgM and IgG antibody deposition in grafts that survived for 5 and 8 weeks might be due to minimal but continuous production of natural antibodies throughout this period. Although careful inspection of multiple sections could identify T cells in the graft, there were few, as reported previously (3–5, 25, 28). The absence of an elicited antibody response and the absence of a response in mixed leukocyte reaction (3, 4, 25) are indicators that AHXR and TM may not be associated solely with T-cell activation.

Innate immunity is a prerequisite for effective adaptive immunity (reviewed in Fox and Harrison [29]), but, in contrast, there is evidence that T cells might be a prerequisite for the innate immune cellular response. It may be relevant that the heart graft that functioned for 8 weeks showed minimal T- and B-cell infiltrates and few macrophages.

The potential factors involved in the development of TM have been discussed elsewhere (30, 31). Endothelial cell activation with the expression of porcine TF (32), inducing a change from an anticoagulant to a procoagulant state, which is almost certainly associated with dysregulation of coagulation (33), seems a likely initial mechanism, but it has been suggested that TF expression by recipient cells might play a role in the initiation of coagulation in xenografts (34). A recent report has shown that contact with porcine aortic endothelial cells can induce TF expression on both human platelets and monocytes (35). Our own data would strongly support the conclusion that activation of recipient cells is a significant factor, though the precise role of primate TF in the development of TM in porcine xenografts has not been clarified.

Although neutrophils have been shown to play a role in the rejection of xenografts (36–39), their role in the pig-to-baboon model has not been addressed. Neutrophils can discriminate between allogeneic and xenogeneic cells, resulting in endothelial cell activation in the absence of natural antibody and complement (36). When antibody and complement are present, adhesion of neutrophils to porcine aortic endothelial cells is increased (39). These effects are not related to the expression of Gal on the porcine cells (37). Macrophage infiltration was a prominent finding after the Tx of organs from wild-type pigs into baboons reported by Wu et al. (40), and has also been documented in islet xenografts (41).

Features of CC were seen in 7 of 12 baboons irrespective of the time that had elapsed since the graft was inserted. This observation may be associated with the presence of platelets and macrophages (and possibly neutrophils) in the graft as these cells can, under certain circumstances, express TF (42). There is an evidence to suggest that these recipient cells circulating within the vasculature contribute to TF expression, perhaps more than do vascular endothelial cells (43, 44). It remains possible that coagulation pathway activation can occur without a requirement for significant antibody deposition or complement activation, and is a primary trigger for early graft injury, as suggested previously (45). In several studies in both human and animal models of sepsis (which is a severe form of endothelial cell activation), TF expression on these cells and on platelets coincided with the development of systemic inflammatory and hypercoagulable states (42, 46). Coagulation and inflammation are vital elements of the innate immune system, and there is considerable interaction between the coagulation and inflammation pathways (47, 48). In the present study, we were not able to identify any specific coagulation markers that indicated that CC would or would not develop.

In conclusion, our data suggest that (1) irrespective of the presence or absence of the adaptive immune response, early or late xenograft rejection is associated with activation of the innate immune system; and (2) porcine endothelial cell activation by immunoglobulin/complement and TF expression by innate immune cells may both contribute to the development of TM. The roles of monocytes and platelets in xenograft rejection, and particularly in the development of TM and CC, have perhaps been hitherto underestimated. Therapeutic strategies targeted at innate immunity should reduce both innate and adaptive immunity, facilitating xenograft survival, and might result in a more clinically successful immunosuppressive regimen.

ACKNOWLEDGMENTS

The authors thank Walter Schuler, Ph.D., and his colleagues at the Novartis Institutes for Biomedical Research (Basel, Switzerland) for making ABI793 available to them, Roche Pharmaceuticals (Nutley, NJ) for generously providing mycophenolate mofetil, and Genzyme (Cambridge, MA) for the gift of thymoglobulin. They also thank Weihua Liu from Dr. Garcia' laboratory for expert help and technical support.

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Keywords:

α1,3-Galactosyltransferase gene-knockout; Baboon; Costimulatory blockade; Consumptive coagulopathy; Heart; Kidney; Pig; Xenotransplantation

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