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32 VOLUME 11 | NUMBER 1 | JANUARY 2005 NATURE MEDICINE

Marked prolongation of porcine renal xenograft survival in baboons through the use of α1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissueKazuhiko Yamada1, Koji Yazawa1, Akira Shimizu1, Takehiro Iwanaga1, Yosuke Hisashi1, Matthew Nuhn1, Patricia O’Malley1, Shuji Nobori1, Parsia A Vagefi1, Clive Patience2, Jay Fishman3, David K C Cooper1, Robert J Hawley2, Julia Greenstein2, Henk-Jan Schuurman2, Michel Awwad2, Megan Sykes1 & David H Sachs1

The use of animal organs could potentially alleviate the critical worldwide shortage of donor organs for clinical transplantation. Because of the strong immune response to xenografts, success will probably depend upon new strategies of immune suppression and induction of tolerance. Here we report our initial results using α-1,3-galactosyltransferase knockout (GalT-KO) donors and a tolerance induction approach. We have achieved life-supporting pig-to-baboon renal xenograft survivals of up to 83 d with normal creatinine levels.

Overcoming antibody-mediated immunity, consisting of both natural and induced antibody responses, is of particular importance to the success of xenotransplantation. Natural antibodies are present in humans and Old World primates without known immunization and are directed almost exclusively toward the single epitope, galactosyl-α1,3,-galactose (Gal), ubiquitously present on the surface of pig cells1,2. In previous studies, hyperacute rejection of porcine organ xenografts has been avoided by the extracorporeal immunoadsorption of natural antibodies3,4 and through complement inhibition5. But the inexorable return of antibodies against the Gal epitope has thwarted most efforts to date by triggering acute humoral xenograft rejection6. To avoid this problem, α1,3-galactosyltransferase gene knockout pigs (GalT-KO) have recently been produced which do not express the Gal epitope7–9. These animals have now made it possible to study pig-to-baboon xeno-grafts in the absence of effects of natural antibodies against Gal. Here we report our initial trial of these animals as donors of life- sustaining, orthotopic, renal xenografts, using a protocol directed toward the

induction of T-cell tolerance through concomitant transplantation of vascularized donor thymus tissue, which has previously been shown to induce allogeneic transplantation tolerance10–12.

We have performed 11 experimental transplants of kidney plus vascularized thymus from homozygous GalT-KO donors, 5 as thymokidneys13,14 and 6 as vascularized thymic lobes10,15 (VTL). Recipients were treated with a protocol directed at tolerance induc-tion, including thymectomy, splenectomy, T-cell depletion, anti-CD154 monoclonal antibody and mycophenolate mofetil, with or without low-dose steroids (see Supplementary Methods online). Immunosuppression was tapered slowly from day 30, with rate of taper dictated by following the serum creatinine. The taper of immunosup-pression was not complete in any animal.

Data for all GalT-KO transplants performed are provided in Table 1. Five of the experimental transplants were considered evalu-able with respect to the effects of the tolerance-induction regimen (in the others the regimen could not be completed for unrelated causes, as indicated in Table 1). Serum creatinine levels and graft survivals for these 5 transplants are shown in Fig. 1a and Fig. 1c, respectively. Values for three control GalT-KO kidneys transplanted without thymic tissue are shown in Fig. 1b and Fig. 1c.

The three evaluable experimental VTL recipients (B127, B135 and B113) survived with functioning and grossly normal kidneys until days 31, 56 and 68 after operation (Fig. 1a), when they died of infection, myocardial infarction and during anesthesia to replace an infected intravenous line, respectively. The two evaluable recipients of thy-mokidneys (B118 and B134) both survived longer than 80 d (Fig. 1a). In B134, no steroids were utilized. This animal maintained normal renal function until day 83, when it suddenly died; the only apparent cause was a small myocardial infarct seen on histology (probably related to the flushing of a misplaced carotid arterial catheter). Grossly and histologi-cally, the renal graft showed no evidence of rejection (Fig. 1d).

As indicated in Table 1, immunosuppression had to be discontin-ued early in two experimental animals (B115 and B129), causing rapid rejection in both. The other four experimental animals died either of causes unrelated to the transplant (B125 and B117) or from apparent serum sickness (B132 and B133), following prophylactic treatment with human intravenous immunoglobulin in an attempt to prevent throm-botic microangiopathy, which has been seen as a prominent feature of rejection in heart xenografts in our laboratory. All four of these animals died with normal creatinine levels and normal kidney histology. The thymic graft in the thymokidney of B117 (day 16) showed viable thymic tissue with a presence of Hassal’s corpuscles (Fig. 1f). In contrast to the recipients of kidneys plus vascularized thymus grafts, the three control recipients of renal xenografts alone rejected their grafts on days 20, 33, and 34 (Fig. 1b,c). The histological findings were consistent with cel-lular and antibody-mediated rejection.

1Transplantation Biology Research Center, Massachusetts General Hospital, Boston, Massachusetts 02129, USA. 2Immerge BioTherapeutics, Cambridge, Massachusetts 02139, USA. 3Infectious Disease Unit, Massachusetts General Hospital, Boston, Massachusetts, 02129 USA. Correspondence should be addressed to D.H.S. (sachs@helix.mgh.harvard.edu).

Published online 26 December 2004; doi:10.1038/nm1172

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NATURE MEDICINE VOLUME 11 | NUMBER 1 | JANUARY 2005 33

All kidney plus thymus xenograft recipients showed proteinuria at levels of 1–4+ by dipstick (Multistix 10SG, Bayer), but serum protein levels were readily managed by administration of human serum albu-min to keep total protein in the normal range. Control animals (kid-ney alone) showed dipstick levels of 3–4+ and unlike the experimental animals, they required more human serum albumin, their total protein was not readily controlled to normal levels, and they developed edema despite this treatment (Table 1).

Renal xenografts from the evaluable experimental animals showed no evidence of rejection histologically, and immunohistochemistry showed no evidence of IgG deposition (Fig. 1e). Control kidneys showed severe cellular and humoral rejection, and bright staining for IgG in glomeruli and peritubular capillaries (Fig. 1g).

Cytokeratin staining of the vascularized thymic lobe grafts in experimental animals with functioning kidney xenografts revealed well-preserved thymic architecture and plentiful cytokeratin-positive thymic epithelial cells (Supplementary Fig. 1 online). Immunohistochemistry utilizing a primate-specific anti-CD4 monoclonal antibody with no cross-reaction with porcine CD4 showed that the CD4+CD3– baboon mononuclear cells in the graft were clustered around cytokera-tin- positive thymic epithelial cells (Supplementary Fig. 1 online), suggesting the presence of thymopoiesis in the transplanted thymic graft. Small arteries in the grafts were intact without signs of arteritis.

T-cell depletion and immunosuppression early after transplanta-tion led to general mixed lymphocyte reaction hyporesponsiveness during the first post-operative month. However, long-term survivors

B113 and B134 both developed pig-specific unresponsiveness in mixed lymphocyte reaction, when tested on days 56 and 71 respectively, by which times they were receiving much reduced immunosuppression. In the longest- surviving baboon (B134), which had a functioning thymokidney graft for 83 d, we observed the return of immunocom-petence in the cell-mediated lympholysis assay by day 78 (Fig. 1h).

The presence of cytotoxic antibodies to nonGal antigens was assessed by antibody-mediated cytotoxic assays on periperal blood lympho-cytes (PSL). Low levels of such antibodies were present in most of the baboons in this study (Supplementary Fig. 2 online), but they only increased in titer after transplant in control recipients, that received kidney grafts without vascularized thymic tissue (with the caveat that testing has been performed on PBL and not on endothelial cells).

We consider these results very encouraging. To our knowledge, an 83-d graft survival with normal function and histology is the longest life-supporting xenograft yet reported. Using a regimen directed toward tolerance induction and changing only the source of the donor organ from standard to GalT-KO miniature swine, we have observed an increase in renal xenograft survival time from a maximum of 30 d (n = 11, median 16 days)14 to over 80 d (Fig. 1a). Furthermore, unlike control animals prolonged with immunosuppression alone, the pig kidneys in the evaluable recipients of kidney plus thymus continued to function until the time of death. At autopsy, the grafts appeared normal on gross inspection and showed only mild, focal thrombotic microangi-opathy microscopically, suggesting that additional small changes to the treatment regimen may permit the kind of prolonged survival required for eventual clinical applications.

Note: Supplementary information is available on the Nature Medicine website.

ACKNOWLEDGMENTSThe authors wish to thank I. McMorrow for her review of the manuscript, S. Arn for herd management and quality control and M. Doherty, E. Samelson-Jones and B. Wong for help in manuscript preparation. D.H. Sachs, A. Shimizu, J. Fishman, D.K. Cooper and K. Yamada were consultants to Immerge Biotherapeutics, Inc. This work was supported in part by the US National Institutes of Health Program Project 1P01A45897 and by a Sponsored Research Agreement between the Massachusetts General hospital and Immerge Biotherapeutics, Inc.

COMPETING INTERESTS STATEMENTThe authors declare competing financial interests (see the Nature Medicine website for details).

Figure 1 Results of GalT-KO renal transplantation. Serum creatinine levels following renal xenotransplantation: (a) in 2 long-term GalT KO thymokidney survivors (red lines) and 3 long-term GalT KO VTL plus kidney graft survivors (blue lines); (b) in three control animals that received only a renal graft. (c) Renal graft survival of GalT-KO kidney alone (white bars) and GalT-KO thymokidney or VTL plus kidney (black bars). Black arrows on the ends of the black bars indicate that the animal died with normal renal function. Histological and immunological examinations of the longest survivor of GalT-KO thymokidney xenotrans-plan tation (B134), included: (d) microscopic appearance at day 60 biopsy (H&E, magnification ×100); (e) absence of anti-porcine IgG deposition by immunofluorescence on day 83, compared to (g) diffuse anti-porcine IgG deposition in the glomeruli and peritubular capillaries in graft of control animal B126 on day 34; (h) absence of a cytotoxic T lymphocyte response of B134 (day 78) to porcine stimulators and targets compared to normal response of a naive baboon (solid lines), and normal cytotoxic T lymphocyte responses of both B134 and a naive baboon to allogeneic baboon stimulators and targets (dashed lines). (f) Also shown is histology of vascularized thymic grafts in B117 on day 16 (H&E staining at low power, magnification ×100).

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34 VOLUME 11 | NUMBER 1 | JANUARY 2005 NATURE MEDICINE

Received 19 October; accepted 24 November 2004Published online at http://www.nature.com/naturemedicine/

1. Galili, U., Wang, L., LaTemple, D. C., & Radic, M. Z. Subcell. Biochem. 32, 79–106 (1999).

2. Good, A. H. et al. Transplant. Proc. 24, 559–562 (1992).3. Xu, Y. et al. Transplantation 65, 172–179 (1998).4. Kozlowski, T. et al. Transplantation 67, 18–30 (1999).5. Kobayashi, T. et al. Transplantation 64, 1255–1261 (1997).

6. Lambrigts, D., Sachs, D. H., & Cooper, D. K. Transplantation 66, 547–561 (1998).7. Lai, L. et al. Science 295, 1089–1092 (2002).8. Kolber-Simonds, D. et al. Proc. Natl. Acad. Sci. USA 101, 7335–7340 (2004).9. Dai, Y. et al. Nat. Biotechnol. 20, 251–255 (2002).10. Kamano, C. et al. Proc. Natl. Acad. Sci. USA 101, 3827–3832 (2004).11. Yamada, K. et al. J. Immunol. 164, 3079–3086 (2000).12. Yamada, K. et al. Transplantation 76, 530–536 (2003).13. Yamada, K. et al. Transplantation 68, 1684–1692 (1999).14. Barth, R. N. et al. Transplantation 75, 1615–1624 (2003).15. LaMattina, J. C. et al. Transplantation 73, 826–831 (2002)

Table 1 Clinical data on recipients of GALT-KO kidneys

Transplant Animal ID CVF Immunosuppression Animal survival Creatinine (mg/dl)a Proteinuria symptomsb Cause of death

VTL+Kidney B113 Yes TCD-1 68 1.4 + / NoneAnesthetic complication during replacement of infected central catheter

B135 Yes TCD-2 56 0.7 + / None Acute myocardial infarction

B127 No TCD-2 31 1.9 + / None Pneumonia

B125 Yes TCD-2 4 1.0 + / NoneCerebral infarction associated with a central catheter

B115 Yes TCD-1 13 3.5 + / NoneHemorrhagic colitis; immunosup-pression stopped day 11; rejection (histologically proven)

B129 Yes TCD-2 33 8.5 + / NoneGastric bleeding; immunosuppres-sion stopped day 22; rejection (histologically proven)

Thymokidney B134 Yes TCD-2* 83 1.0 + / None Acute myocardial infarction

B118 Yes TCD-2 81 4.0 + / NonePneumonia (rejection episode reversing at time of death)

B132 No TCD-2 26 1.1 + / NonePresumed serum sickness (associated with IVIG)

B133 Yes TCD-2 18 1.0 + / NonePresumed serum sickness (associated with IVIG)

B117 Yes TCD-1 16 1.0 + / NoneRespiratory failure following presumed aspiration of peanut

Kidney alone B126 Yes TCD-2 34 2.7 + / SevereSevere uremia and proteinuria Rejection (histologically proven)

B114 Yes PTCD 33 + / SevereSevere uremia and proteinuria Rejection (histologically proven)

B131 Yes PTCD* 20 5.2 + / SevereSevere uremia and proteinuria Rejection (histologically proven)

TCD-1, T-cell depletion regimen-1 (3 doses of antithymocyte globulin (ATG) plus 3–4 doses of rat antibody specific for human CD2; anti-CD154 monoclonal antibody, mycophenolate mofetil (MMF) and steroids); TCD-2, T cell depletion regimen-2 (100 cGy whol body irradiation on day –6, 2 doses of LoCD2, 2 doses of ATG; anti-CD154 monoclonal antibody, MMF and steroids); TCD-2*, TCD-2 regimen without steroids; PTCD, partial T-cell depletion regimen (3 doses of ATG and 3 doses of LoCD2) anti-CD154 monoclonal antibody, MMF and steroids; PTCD*, PTCD regimen but tacrolimus instead of anti-CD154 monoclonal antibody ; CVF, cobra venom factor (14 doses from day 1 to 13); 1V1G, human intravenous immunoglobulin. aSerum creatinine level at time of death. bProteinuria was positive as measured by dipstick in all animals, but could not be quantified because of contamination of urine with other materials; maximal facial and leg edema was graded as none, moderate or severe.

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