COSTIMULATION BLOCKADE IN PIG ARTERY PATCH XENOTRANSPLANTATION – A SIMPLE MODEL TO MONITOR THE ADAPTIVE IMMUNE RESPONSE IN NONHUMAN PRIMATES

Animals

Baboons (Papio species, n=14; Division of Animal Resources Oklahoma University Health Sciences Center, Oklahoma City, OK) weighing 6-10kg and of known ABO blood type, were recipients of GTKO pig artery grafts. GTKO pigs (n=7) of blood group O (nonA) weighing 7-20kg (Revivicor, Inc., Blacksburg, VA), generated by nuclear transfer/embryo transfer from modified fibroblasts from Large White/Landrace/Duroc cross-breed pigs (1, 11), served as sources of carotid artery patches.

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 in pigs and baboons, and intravascular catheter placements in baboons have been described previously (12-14). In the source GTKO pig, a length of carotid artery was excised, and stored in University of Wisconsin solution in ice (at approximately 4°C) until transplanted into the baboons. The ischemic period was approximately 6h (n=10) or 20h (n=4). (Some grafts were transported to us by colleagues at the University of Maryland. The ischemic period did not correlate with transplant outcome). The artery was divided longitudinally to form a patch, which was cut into two for insertion into two baboons. Each patch was approximately 2×1cm. In the recipient baboon, the abdomen was opened through a midline incision, and the abdominal aorta was exposed. After partial heparinization (100U/kg i.v.), the aorta was clamped immediately distal to the renal vessels and at the bifurcation, and opened longitudinally. The artery patch was sutured in place as an onlay graft using 5.0 polypropylene sutures (Figure 1). The clamps were then released, and, after determining there was good flow to both legs, the abdomen was closed.

Immunosuppressive regimens

Recipients received either no immunosuppressive therapy (Group 1), anti-CD154mAb-based (Group 2), or CTLA4-Ig-based (Group 3) immunosuppression (Tables 1 and 2). However, baboons in all three groups received induction cobra venom factor (CVF; Complement Technology, Inc., Tyler, Texas) and methylprednisolone (on days −1, 0, and 1), which may prevent the occasional incidence of hyperacute rejection that has been reported after GTKO organ xenotransplantation; it has been our policy to administer CVF to all baboon recipients of GTKO pig heart grafts. The decision not to include corticosteroids for maintenance in the regimen was based on the observations of Yamada and his colleagues mentioned above (8,9). Anti-CD154mAb (ABI193) was generously provided by Novartis Pharma, Basel, Switzerland). Numerous previous measurements in other baboons have demonstrated that, at the dose given, the serum level is maintained at >200μg/mL throughout the course of the experiment. ATG was obtained from Genzyme, Cambridge, MA, and mycophenolate mofetil (MMF) was obtained from Roche, Nutley, NJ. CTLA4-Ig (Abatacept) was obtained, from BMS, Princeton, NJ. (Belatacept [LEA 29Y] was not available to us). CTLA4-Ig was also not measured during the course of the experiments, but was measured in retrospect. At the dose given, the serum level was >150μg/mL. In contrast to most of our studies involving pig organ Tx in baboons, maintenance corticosteroid therapy was not included in the regimen (based on the observations by Yamada and colleagues mentioned above [8,9])

Anticoagulation

Despite the importance of anticoagulation when using anti-CD154mAb in the pig-to-baboon xenoTx model (15, 16), it was initially felt that this would be unnecessary in the artery patch model (Group 2). However, when anti-CD154mAb was administered alone (without anticoagulation), the abdominal aorta thrombosed at the site of the graft within 48h (Table 2), suggesting that, in the presence of anti-CD154mAb, anticoagulation was required. In all subsequent experiments in Group 2, either continuous heparin (n=3) or intermittent ketorolac (n=3) (17) was administered (Table 1). Heparin and ketorolac were equally successful in preventing artery thrombosis. The single baboon in Group 3 that received 3 doses of anti-CD154mAb received heparin throughout the course of study.

Measurement of anti-pig antibodies

Anti-pig (nonGal) antibody levels were measured by flow cytometry using GTKO porcine aortic endothelial cells (PAEC) as target cells. Baboon sera (20μL) were heat-inactivated for 30min at 56°C, and then incubated with 0.1-0.2×10⁶ PAEC for 30min at 4°C. PAEC were washed and incubated for 30min at 4°C with secondary antibodies (1:20) - anti-human FITC-IgM (μ-chain-specific) and FITC-IgG (γ-chain-specific) (Invitrogen, Carlsbad, CA). Negative controls were obtained by incubating the target cells with secondary anti-human antibodies only (and no serum). Relative mean fluorescence intensity (MFI) was calculated by dividing the MFI value for each sample by the negative control. Samples were analysed using LSR II flow. Data were analyzed using Flowjo or WinMDI software.

Mixed leukocyte reaction (MLR)

MLR were carried out before Tx and between days 21-28 after Tx. Baboon peripheral blood mononuclear cells (PBMC) and irradiated GTKO pig PBMC were isolated from heparinized blood by centrifugation at 700g for 30min over Ficoll-Paque PLUS (GE Healthcare, Piscataway, NJ). The total mononuclear cell fraction was washed twice with PBS (Invitrogen, Carlsbad, CA) and contaminating red cells were lysed with ammonium chloride/potassium solution, when necessary. PBMC were then resuspended in AIM V medium (Invitrogen). Baboon PBMC were used as responders (0.4×10⁶ cells/well) and irradiated GTKO pig PBMC as stimulators (0.4×10⁶ cells/well), with a responder:stimulator ratio of 1:1. ³H-thymidine (1μCi/well) was added during the last 16h of incubation. Cells were harvested and analyzed by beta-scintillation counter (PerkinElmer, Waltham, MA). Samples were tested in quadruplicate and the mean of ³H-thymidine uptake was calculated. Stimulation index (SI) was calculated by dividing the value of proliferation in response to GTKO PBMC by the value of proliferation in response to autologous PBMC.

Monitoring of CD4⁺ and CD8⁺ T cells

Whole blood samples were collected before any therapy, and 2-3 weeks after Tx. FITC-conjugated mouse anti-human CD3ε (Cat# 556611), PEcy7-conjugated anti-CD4 (Cat# 557852), and PE-conjugated anti-CD8 (Cat# 555367) antibodies were obtained from BD Pharmingen (San Diego, CA). Percentages of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells were determined by flow cytometry. Whole blood samples were analysed using LSR II flow. All data were analyzed using Flowjo or WinMDI software.

Histopathology and immunohistopathology of porcine artery grafts

Pig artery patch xenografts were isolated at euthanasia. For conventional histology, tissues were fixed in 10% formalin and embedded in paraffin. Sections (4μm) were stained with hematoxylin and eosin (H&E) for light microscopy. Staining for IgM, IgG, complement (C3), CD3⁺, CD4⁺, and CD8⁺ T cells, CD20⁺ B cells, neutrophils (myeloperoxidase), macrophages (CD68⁺), and platelets (CD42⁺) was performed on paraffin sections, as previously described (6, 7)

Recipient baboon survival and clinical complications

In Group 1, one graft ruptured into the lumen of adherent small intestine on day 14 (presenting as acute anemia); the baboon was euthanized (Table 2). One Group 2 baboon was euthanized following thrombosis of the abdominal aorta within 48h at the site of the graft; this complication presented with paresis of the lower limbs. Postmortem examination of the abdominal aorta demonstrated no technical reason for the development of thrombosis. One Group 3 baboon was euthanized on day 14 following a catheter complication. All other grafts in all groups remained in situ without complication for 28-33 days, at which time the baboons were electively euthanized.

Antibody responses to GTKO pig artery grafts

Baboon IgM antibodies directed to pig nonGal antigens were present in all baboons before artery Tx (Figure 2). Low titers of anti-nonGal IgG were also detectable in all recipients. In Group 1, there was an increase in both anti-nonGal IgM and IgG titers within 14 days, indicating sensitization to nonGal antigens (Figure 2A). While IgM was declining by day 28, IgG continued to rise (probably due to antibody Isotype class switch). When anti-CD154mAb was administered alone (Group 2A), a very slight increase in IgM was observed in all four recipients on day 14, which decreased to pre-transplant levels by day 28. There was no detectable increase in IgG on days 14 or 28, except in one recipient, where a possible very slight increase was measured (Figure 2B). In the one recipient that received anti-CD154mAb combined with induction ATG and MMF maintenance (Group 2B), there was no increase in IgM or IgG (Figure 2B).

Although only in one baboon, when CTLA4-Ig was given alone (Group 3A), there were increases in both IgM and IgG on days 14 and 28 (Figure 2C). When CTLA4-Ig was administered with ATG+MMF (Group 3B), there was a slight increase in IgM on days 14 and 28 in three recipients (Figure 2C), and a moderate increase in IgG by day 14 in at least two recipients (one of which had received three doses of anti-CD154mAb in the induction phase [Table 2]), maintained to day 28. In the remaining two, there was a slight rise in IgG.

Antibody and complement deposition in GTKO pig artery grafts

With no immunosuppression (Group 1), antibody (IgM and IgG) and complement (C3) deposition were detected in all three aortic patches (Figure 3). When either anti-CD154mAb or CTLA4-Ig was given alone (Groups 2A and 3A), or when CTLA4-Ig was administered with ATG+MMF (Group 3B), antibody and complement deposition were still detected in the grafts (Figure 3). When anti-CD154mAb was administered with ATG+MMF (Group 2B), IgG deposition was detected, with minimal IgM and no complement deposition.

Baboon PBMC proliferation in response to pig nonGal antigens

PBMC were not obtained in every baboon, either due to complications (Table 2) or impaired cell viability (probably related to high levels of anti-CD154mAb). On MLR, with no immunosuppression (Group 1), two recipients developed increased PBMC proliferation in response to GTKO PBMC after Tx, indicating sensitization (Figure 4). Recipients that received anti-CD154mAb alone (Group 2A) showed reduced PBMC proliferation, while in the one recipient that received CTLA4-Ig alone (Group 3A), PBMC proliferation was not reduced. In recipients that received either the full anti-CD154 mAb (Group 2B) or CTLA4-Ig (Group 3B)-based regimen, PBMC proliferation was inhibited.

CD4⁺ and CD8⁺ T cell kinetics in artery patch recipients

The numbers of CD4⁺ (Figure 5A) and CD8⁺ (Figure 5B) T cells were monitored before and 21-28 days after Tx. With no immunosuppression (Group 1) or when CTLA4-Ig alone was administered, both CD4⁺ and CD8⁺ T cells increased after Tx. When anti-CD154mAb was given alone, low levels of CD4⁺ T cells were maintained after Tx, while low levels of CD8⁺ T cells were maintained in 2 of 4 baboons. When anti-CD154mAb or CTLA4-Ig was combined with ATG+MMF, low numbers of CD4⁺ and CD8⁺ T cells were maintained. There was a tendency towards low numbers of B cells after Tx when either of the full regimens was administered (not shown).

Cellular infiltration in GTKO pig artery grafts

With no immunosuppression (Group 1), there was intense cellular infiltration in all layers of the artery patch (intima, media, and adventitia), consisting mainly of T cells (CD4⁺ and CD8⁺), some B cells, with abundant macrophages and neutrophils (Figure 6A). When anti-CD154mAb (Group 2A) was given alone, moderate T cell (with fewer CD4⁺T than CD8⁺T cells) and B cell infiltration was seen, with stronger infiltration of neutrophils and macrophages (Figure 6B). Although only in one recipient, when the anti-CD154mAb-based full regimen was used (Group 2B), there was no cellular infiltration, except for minimal macrophage infiltration (Figure 6C). When CTLA4-Ig was used alone (Group 3A) there was intense T cell and moderate B cell infiltration, with moderate neutrophil and macrophage infiltration (Figure 6D). When the full CTLA4-Ig-based regimen was administered (Group 3B), there were fewer CD4⁺ and CD8⁺ T cell and no B cell infiltrates, but extensive neutrophil and macrophage infiltrates were present (Figure 6E).

Platelet deposition in GTKO pig artery patch grafts

Platelet deposition was detected in all artery patch grafts in Groups 1 and 3 (except the single baboon in Group 3 that received a heparin infusion) (Figure 7). Platelet deposition was prevented only when heparin was administered Group 2); when ketorolac replaced heparin, platelet deposition could still be detected.