Induction of Human T‐Cell Tolerance to Pig Xenoantigens Via Thymus

American Journal of Transplantation 2009; 9: 1324–1329 C 2009 The Authors Wiley Periodicals Inc. Journal compilation C 2009 The American Society of Transplantation and the American Society of Transplant Surgeons Brief Communication doi: 10.1111/j.1600-6143.2009.02646.x

Induction of Human T-Cell Tolerance to Pig Xenoantigens via Thymus Transplantation in Mice with an Established Human Immune System

∗ K. Habiro, M. Sykes and Y-G. Yang tation, the best available therapy for end-stage organ fail- ure (1). The successful production of viable pigs with ho- Transplantation Biology Research Center, Massachusetts mozygous deletion of a1,3Gal transferase (2–4) made it General Hospital, Harvard Medical School, Boston, MA possible to avoid both hyperacute rejection (HAR) and *Corresponding author: Yong-Guang Yang, acute humoral xenograft rejection (AHXR) (5,6). However, [email protected] a1,3Gal-deficient porcine xenografts can still be vigorously rejected by T cells, and the use of nonspecific immuno- Thymus xenotransplantation has been shown to in- suppressive drugs has not been successful in preventing duce tolerance to porcine xenografts in mice and to per- a1,3Gal-deficient porcine xenograft rejection without se- mit survival of a 1,3Gal-transferase knockout porcine vere toxicity in primate recipients (5–7). Thus, tolerance kidney xenografts for months in nonhuman primates. induction is likely to be essential for clinical success of We evaluated the ability of porcine thymus xenotrans- xenotransplantation. plantation to induce human T-cell tolerance using a humanized mouse (hu-mouse) model, where a hu- We have recently shown that cotransplantation of human immune system is preestablished by implantation man fetal thymus and CD34+ cells achieves long-term re- of fetal human thymus tissue under the kidney cap- + population with multilineage human lymphohematopoietic sule and intravenous injection of CD34 hematopoietic stem/progenitor cells. Human T-cell depletion with cells and formation of secondary lymphoid organs in im- an anti-CD2 mAb following surgical removal of hu- munodeficient mice (8–10). Furthermore, these humanized man thymic grafts prevented the initial rejection of mice (hu-mice) mediate robust antigen-specific immune re- porcine thymic xenografts in hu-mice. In these hu- sponses and rejection of porcine skin and islet xenografts. mice, porcine thymic grafts were capable of support- We have previously shown that porcine thymus can gen- ing human thymopoiesis and T-cell development, and erate human T cells that are tolerant of the porcine thymic inducing human T-cell tolerance to porcine xenoanti- donor (11). In this study, we used this hu-mouse model gens. Human T cells from these mice responded to investigate the possibility of porcine thymus transplan- strongly to third-party pig, but not to the thymic donor tation to induce human T-cell tolerance in hu-mice with swine leukocyte antigen (SLA)-matched pig stimula- a preestablished human immune system. We observed tors in a mixed lymphocyte reaction (MLR) assay. Anti- pig xenoreactive antibodies declined in these hu-mice, that brief conditioning with depleting anti-human CD2 mAb whereas antibody levels increased in nontolerant ani- results in acceptance of porcine thymic grafts and donor- mals that rejected porcine thymus grafts. These data specific tolerance in human thymic graftectomized hu-mice show that porcine thymic xenotransplantation can in- with a preestablished human immune system. duce donor-specific tolerance in immunocompetent hu-mice, supporting this approach for tolerance induction in clinical xenotransplantation. Materials and Methods Key words: Humanized mouse, pig, thymus transplantation, tolerance, xenotransplantation Animals and human fetal tissues Immunodeficient nonobese diabetic/severe combined immunodeficient Received 09 January 2009, revised 06 March 2009 and (NOD/SCID) mice were purchased from National Cancer Institute accepted for publication 10 March 2009 (Bethesda, MD) or The Jackson Laboratory (Bar Harbor, ME), and housed in a specific pathogen-free microisolator environment. Human fetal thymus and liver tissues of gestational age 17 to 20 weeks were obtained from Advanced Bioscience Resource (Alameda, CA). Porcine fetal thymi were harvested from fetuses (60–70 gestational days) of Massachusetts Gen- Introduction eral Hospital inbred miniature swine (kindly provided by Dr. David H. Sachs) (12). Protocols using human tissues and animals in this study were approved Xenotransplantation from pigs provides a possible solu- by the Massachusetts General Hospital Human Research Committee and tion to the overwhelming scarcity of human organ donors Subcommittee of Research Animal Care, and all of the experiments were that presents a major limiting factor in clinical transplan- performed in accordance with the protocols.

1324 Thymus Xenotransplantation in Humanized Mice

Ta b l e 1 : Hu-mice used in this study % human Human chimerism1 % human chimerism4 Hu-mouse Time of Pig thymus thymopoiesis Recipients’ I.D. CD45+ CD3+ sacrufuce2 Graft in pig thymus3 CD45+ CD3+ BTI-322-conditioned 2084 51.8 4.2 20 Survived Yes 20.6 16.0 2090 53.8 4.3 28 Survived Yes 72.9 51.2 3607 78.6 16.2 20 Survived Yes 88.4 53.3 3809 62.7 22.9 20 Survived Yes 45.5 10.7 3859 19.2 12.5 20 Survived Yes 4.0 2.75 3877 26.5 22.5 20 Survived Yes 59.3 34.4 Nonconditioned 2151 75.3 56.1 20 Rejected N/D 69.5 19.7 217 41.4 13.6 12 Rejected N/D 87.0 73.5 673 46.3 10.9 20 Rejected N/D 23.6 2.8 3875 59.3 45.2 20 Rejected N/D 45.1 25.4 1Shown are human chimerism in PBMCs 1 week prior to porcine thymus transplantation, and numbers represent percentages of human CD45+ and CD3+ cells in a live lymphocyte gate. 2Weeks after porcine thymus transplantation. 3The establishment of human thymopoiesis is determined as the presence of >5 × 106 human thymocytes with normal phenotypic distribution of double negative, double positive, CD4 single positive and CD8 single positive cell populations. 4Shown are human chimerism in PBMCs at the day of sacriﬁce, and numbers represent percentages of human CD45+ and CD3+ cells in a live lymphocyte gate.

Hu-mouse preparation Mixed lymphocyte reaction (MLR) assay Humanized NOD/SCID mice were created as previously described (8–10). Hu-mouse splenocytes (4 × 105/well) were cultured with an equal num- Briefly, female NOD/SCID mice (7–10 weeks old) were conditioned with ber of irradiated (30 Gy) stimulator cells (PBMCs from thymic donor swine sublethal (2–3 Gy) whole body irradiation. Eight to 20 h later, mice were leukocyte antigen [SLA]-matched pigs or third-party pigs) or without stimula- implanted with human fetal thymus (Thy) and liver (Liv) tissue fragments tor cells (background) in AIM-V medium containing 10% human AB serum 3 ◦ 3 measuring about 1 mm under the kidney capsule, and injected (i.v.) with (Sigma, St Louis, MO) at 37 Cin5%CO2. Cells were pulsed with [ H]- 1–5 × 105 CD34+ human fetal liver cells (FLCs). CD34+ FLCs were purified TdR on day 5 and incorporation of [3H]-TdR was measured 18 h later. Cell from same donor by the magnetic-activated cell sorter (MACS) separa- proliferation is presented as net counts per minute (cpm) (i.e. background tion system using anti-human CD34 microbeads (Miltenyi Biotec, Auburn, cpm subtracted; the background counts were less than 10% of third-party- CA). Levels of human hematopoietic cells in peripheral blood of the re- stimulated cultures for all samples). constituted mice were determined by flow cytometric (FCM) analysis using the following mAbs: anti-human CD3, CD4, CD8, CD45 and isotype Complement-dependent killing assay control mAbs (all purchased from BD Bioscience, San Jose, CA). FACS Anti-pig antibodies in hu-mouse sera were measured as previously de- analysis was performed on a FACScalibur (BD Bioscience, San Jose, CA). scribed (13). Briefly, target cells (i.e. porcine PBMCs) were suspended at The investigators performing this study have been producing consistent 5 × 106/mL in Medium 199 (Cellgro, Herdon, VA) with 2% FCS (Sigma, St. results using this protocol for several years, with approximately 80% suc- Louis, MO), and added (25 lL/well) to the 96 well U-bottom plates (Corning, cess rates in most experiments. The surgical procedure is well tolerated NY) containing serially diluted heat-inactivated mouse serum (25 lL/well). in mice and no graft-versus-host disease (GVHD) has been seen in the Cells were cultured for 15 min at 37◦C, washed with 2% FCS medium reconstituted hu-mice. Hu-mice used in this study were from different co- and incubated at 37◦ C for 30 min with rabbit complement (1:8 diluted horts, but all had >4% of human CD3+ cells in peripheral blood mononu- in 2% FCS medium). Cells were then stained with 10 lLof10lg/mL clear cells (PBMCs) 1 week prior to BTI-322 treatment and porcine thymus 7-Aminoactinomycin (7 AAD; Sigma, St. Louis, MO), and the frequen- transplantation. cies of dead cells were determined by FCM analysis. The percentage of killing is presented as the percentage of 7 AAD-stained cells (background subtracted). Porcine thymus transplantation Hu-mice underwent surgical removal of human thymic grafts by nephrectomy 9–15 weeks after human Thy/Liv/CD34+ FLC transplantation, which Results was followed immediately by transplantation of a fetal porcine thymus frag- ment measuring about 1 mm3 into the capsular space of the contralateral Long-term acceptance of porcine thymus grafts kidney. Hu-mouse recipients were treated with two injections (i.p.) of anti- and human T-cell reconstitution in human thymic human CD2 mAb (BTI-322; 100 lg/mouse) or phosphate buffered saline graftectomized hu-mice after brief conditioning (PBS) 7 days and 1 day before porcine thymus transplantation. Hu-mice + + with anti-CD2 mAb were bled every 3 weeks and the levels of human CD45 and CD3 cells in PBMCs were determined by FCM analysis as described above. The sur- Porcine fetal thymi were transplanted in hu-mice after vival or rejection of porcine thymic grafts was confirmed at the time when FCM analysis confirmed human lymphohematopoietic cell these hu-mice were sacrificed for analyses between 12 and 28 weeks after reconstitution, approximately 10–14 weeks after human + porcine thymic transplantation. Thy/Liv/CD34 FLC transplantation (Table 1). To prevent

American Journal of Transplantation 2009; 9: 1324–1329 1325 Habiro et al.

Figure 1: Depletion of human T cells in peripheral lymphoid tissues, but not in the thymus of hu-mice treated with BTI-322. Hu-mice with >5% of human CD3+ T cells in PBMCs were treated (i.p.) with BTI-322 (100 lg; N = 8) or PBS (N = 3) at days –7 and –1, and splenocytes and thymocytes were analyzed by flow cytometry at day 0. Flow cytometric profiles of representative hu-mice are shown. initial rejection of porcine thymic grafts, hu-mouse recipi- porcine thymus implantation. Long-term survival (20–28 ents were treated with two injections (i.p.) of anti-human weeks) of porcine thymic grafts was seen in all BTI-322- CD2 mAb (BTI-322) to deplete human T cells 7 days and treated hu-mice, but not in any of the control hu-mice that 1 day prior to porcine thymic transplantation. Because received human thymic graftectomy alone (Table 1). Fur- BTI-322 treatment mediates efficient human T-cell deple- thermore, human T-cell reconstitution was detected in BTI- tion in peripheral lymphoid tissues (10), but not in the thy- 322-treated, human thymic graftectomized hu-mice be- mus (Figure 1), all hu-mouse recipients had the human tween 6 and 9 weeks after porcine thymus transplantation thymic graft removed (by nephrectomy) at the time of (Figure 2A). FCM analysis of porcine thymic grafts showed

Figure 2: Human T-cell reconstitution in BTI-322-treated, human thymic graftectomized hu-mice after porcine thymus transplantation. Human T-cell recovery and thymopoiesis was assessed by ﬂow cytometry in BTI-322-treated, human thymic graftectomized hu-mice after porcine thymus transplantation (n = 6; these mice are the same BTI-322-treated hu-mice presented in Table 1). (A) Levels of human CD45+ (closed symbols) and CD3+ T (open symbols) cells in PBMCs at the indicated times (mean ± SD); (B) Staining of single cell suspension of a representative porcine thymic graft with anti-human CD4 and CD8 mAbs.

1326 American Journal of Transplantation 2009; 9: 1324–1329 Thymus Xenotransplantation in Humanized Mice

Ta b l e 2 : Antithymic donor and third-party MLR MLR (cpm; mean ± SD of triplicate) Ratio (antidonor/ Hu-mice BTI-322 Pig thymus graft Thymic donor Third party anti-third party) 2084 Yes Survived 13,231 ± 1,989 131,794 ± 2,883 0.10 2090 Yes Survived 121 ± 25 7,046 ± 512 0.01 3607 Yes Survived 725 ± 225 2,476 ± 652 0.29 2151 No Rejected 155,581 ± 10,262 112,080 ± 4,528 1.39 673 No Rejected 2,561 ± 11 2,327 ± 247 1.10 normal phenotypic distribution of double negative, dou- cytolytic antibodies remained undetectable, or in animals ble positive, CD4 single positive and CD8 single positive with pretransplant anti-pig antibodies, were markedly re- human thymocytes in all these mice (Figure 2B). Porcine duced in posttransplant sera from BTI-322-treated hu-mice thymocytes remained detectable in some porcine thymic with long-term acceptance of porcine thymic xenografts grafts, but no porcine cells were detected in peripheral (Figure 3B). Despite the strong T-cell responses against lymphoid organs in these hu-mice (data not shown). These third-party stimulators (Table 2), the absence of antibod- data showed that transient human T-cell depletion permit- ies to the thymic donor was associated with an absence of ted porcine thymic engraftment in human thymic graftec- anti-third-party pig antibodies in the sera from these mice. tomized hu-mice, and that de novo human thymopoiesis and T-cell development from human lymphoid progenitors occurred in the porcine thymic grafts. Discussion

Previous studies have shown that thymic transplantation Analysis of porcine donor antigen-specific T-cell is a particularly promising approach toward inducing T- responses and antibody production in porcine cell tolerance across highly disparate xenogeneic barriers. thymus-transplanted hu-mice Porcine thymus transplantation induces robust xenograft Spleen cells were prepared from hu-mouse recipients at tolerance in T-cell-depleted, thymectomized mice, in which various times (12–28 weeks) after porcine thymus trans- the mouse recipients of porcine thymic grafts demonstrate plantation, and analyzed for MLR responses against the donor-specific tolerance in an MLR assay and accept skin thymic donor and third-party pig stimulators. As shown in grafts from the porcine thymic donors (17,18). Porcine thy- Table 2, human T cells from hu-mice with long-term porcine mus transplantation has also been shown to prolong sur- thymic graft survival (i.e. those treated with BTI-322) ex- vival of porcine aGal-deficient kidney xenografts in non- hibited donor-specific nonresponsiveness to the porcine human primates (5). Furthermore, we have previously thymic donors. Human T cells from these hu-mice prolifer- shown that human T cells developing in porcine thymic ated significantly in response to third-party pig stimulators, grafts in immunodeficient mice exhibit specific unrespon- but not to the porcine thymic donor stimulators. However, siveness to the major histocompatibility complex (MHC) human T cells from hu-mice that rejected porcine thymic of the porcine thymic donor (11). In this study, we ex- grafts (i.e. those that received human thymectomy alone) plore the possibility of inducing human T-cell tolerance to exhibited strong responses to both thymic donor and third- porcine xenoantigens by thymic xenotransplantation in hu- party stimulators. mice with a preestablished human immune system. We show that long-term acceptance of porcine thymic grafts We also assessed the levels of anti-pig xenoreactive an- and donor-specific tolerance can be achieved in hu-mice tibodies in the sera of hu-mouse recipients prior to and whose human thymus grafts are removed and that receive after porcine thymus transplantation by a complement- injection of depleting anti-CD2 mAb. dependent cytotoxicity assay (13). Because human B cells developing in a1,3Gal (Gal)-expressing mice are expected Human thymopoiesis in porcine thymic grafts and human T- to be tolerant of the Gal epitope (14,15), anti-pig antibodies cell reconstitution have been reported in immunodeficient produced by hu-mice should predominantly react to non- mice after implantation of porcine thymus and human fe- Gal xenoantigens. With the exception of one mouse, all hu- tal liver (as a source of human lymphoid progenitors) tis- mice had low or almost undetectable levels of anti-non-Gal sues side by side under the recipient renal capsule (11). xenoreactive antibodies prior to porcine thymic transplan- This observation indicates that porcine thymus can support tation (Figure 3), which is similar to previous observations human thymopoiesis and T-cell development. However, it in non-human primates (16). However, high levels of in- was unknown whether circulating human lymphoid produced anti-pig antibodies were detected in posttransplant genitors can migrate into a porcine thymus as efficiently sera from all hu-mice that rejected porcine thymic grafts as they do in a human thymus. In this study, we trans- (Figure 3A). Interestingly, sera from these hu-mice exhib- planted porcine thymus (without human fetal liver) into ited comparable killing activity against the porcine thymic hu-mice with a preestablished human lymphohematopoi- donor and third-party porcine cells. In contrast, anti-pig etic system. Thus, the observed human thymopoiesis in

American Journal of Transplantation 2009; 9: 1324–1329 1327 Habiro et al.

A PBS 80 80 2151 3875 60 60

40 40

% Killing 20 20

0 0 6 18 54 162 486 6 18 54 162 486 Serum Dilution

B BTI-322 80 80 2084 2090 60 60

40 40 Figure 3: Lack of anti-pig cytolytic antibodies in sera of BTI-322-treated 20 20 hu-mouse recipients of porcine thymic grafts. Sera were collected = 0 0 from PBS-injected (A) (N 2) and 6 18 54 162 486 6 18 54 162 486 BTI-322-treated (B) (N = 4) hu-mice 2 80 80 weeks before (pretransplant; /)and % Killing 3859 3877 20 or 28 weeks after porcine thymus 60 60 transplantation (at the time of sacrifice; posttransplant, •/◦), and the levels of 40 40 cytolytic antibodies against the thymic donor (closed symbols) and third-party 20 20 (open symbols) pig targets were measured by a complement-dependent killing assay. Each figure represents 0 0 6 18 54 162 486 6 18 54 162 486 one hu-mouse recipient. Numbers in upper right of each figure represent Serum Dilution animal ID. porcine thymic grafts in these hu-mice indicates that hu- producing B cells, but does prevent the sensitization of man lymphoid progenitors can migrate from the bone this response, thereby avoiding an increase in anti-Gal marrow into the porcine thymus grafts to initiate de antibodies after thymic xenotransplantation (19). The re- novo human thymopoiesis. The ability of porcine thymi to sults presented here raise the interesting possibility that efficiently recruit human lymphoid progenitors and support human natural antibody-producing B cells may be toler- human T-cell development further supports the possibility ized by porcine thymic transplantation. However, unlike T that porcine thymic transplantation could be a clinically rel- cells in the tolerized hu-mice that exhibited donor SLA- evant approach to inducing xenograft tolerance. specific unresponsiveness in MLR (Table 2), neither antidonor nor anti-SLA-mismatched third-party pig antibodies Porcine thymic transplantation also prevented the were detected in the sera of these mice. Similarly, sim- production of anti-non-Gal xenoreactive antibodies in BTI- ilar levels of antidonor and anti-third-party pig antibodies 322-conditioned hu-mice (Figure 3). Most remarkably, anti- were detected in sera from hu-mice that rejected porcine non-Gal antibodies declined following porcine thymic trans- thymic grafts. These data suggest that human anti-pig plantation in tolerant animals that had these antibodies antibodies mainly recognize highly conserved non-MHC prior to porcine thymus transplantation. These results sug- antigens and/or shared epitopes of the SLA proteins, as gest that either the T-celltolerance induced by thymic xeno- previously reported in a pig-to-baboon xenotransplantation transplantation leads to loss of T-cell-dependent xenoanti- model (16). body production, or the human natural antibody-producing B cells themselves are tolerized by porcine thymic trans- In summary, our data demonstrate that the hu-mouse plantation. Previous studies in Gal-deficient mice have model used in this study provides a useful in vivo shown that T-cell tolerance induced by porcine thymic model system for characterizing human anti-pig xenoim- xenotransplantation does not tolerize anti-Gal antibody- mune responses and testing immunosuppressive or

1328 American Journal of Transplantation 2009; 9: 1324–1329 Thymus Xenotransplantation in Humanized Mice tolerance-inducing reagents/protocols. Furthermore, our 8. Lan P, Tonomura N, Shimizu A, Wang S, Yang YG. Reconstitu- data indicate that human T-cell tolerance to porcine tion of a functional human immune system in immunodeficient xenoantigens can be achieved by thymic xenotransplan- mice through combined human fetal thymus/liver and CD34 +cell tation in hu-mice with a preestablished human immune transplantation. Blood 2006; 108: 487–492 [Epub 2006 Jan 12]. system, and suggest a potential approach to inducing tol- 9. Tonomura N, Habiro K, Shimizu A, Sykes M, Yang YG. Antigen- specific human T-cell responses and T cell-dependent production erance in clinical xenotransplantation. of human antibodies in a humanized mouse model. Blood 2008; 111: 4293–4296. 10. Tonomura N, Shimizu A, Wang S et al. Pig islet xenograft rejection Acknowledgments in a mouse model with an established human immune system. Xenotransplantation 2008; 15: 129–135. The authors thank Drs. Joshua Mollov and Takashi Onoe for critical review 11. Nikolic B, Gardner JP, Scadden DT, Arn JS, Sachs DH, Sykes of this manuscript, Mr. Orlando Moreno for outstanding animal husbandry M. Normal development in porcine thymus grafts and specific and Ms. Kelly Walsh for expert assistance with the manuscript. This work tolerance of human T cells to porcine donor MHC. J Immunol was supported by grants from NIH (P01 AI 045897 and R01 AI064569); 1999; 162: 3402–3407. K.H. is supported by a fellowship from JDRF (3-2007-667). 12. Sachs DH, Leight G, Cone J, Schwarz S, Stuart L, Rosenberg S. Transplantation in miniature swine. I. Fixation of the major histocompatibility complex. Transplantation 1976; 22: 559– References 567. 13. Horner BM, Cina RA, Wikiel KJ et al. Predictors of organ allo- 1. Yang YG, Sykes M. Xenotransplantation: Current status and a per- graft tolerance following hematopoietic cell transplantation. Am J spective on the future. Nat Rev Immunol 2007; 7: 519–531. Transplant 2006; 6: 2894–2902. 2. Lai L, Kolber-Simonds D, Park KW et al. Production of alpha-1,3- 14. Yang YG, deGoma E, Ohdan H et al. Tolerization of anti-Gala1–3Gal galactosyltransferase knockout pigs by nuclear transfer cloning. natural antibody-forming B cells by induction of mixed chimerism. Science 2002; 295: 1089–1092. J Exp Med 1998; 187: 1335–1342. 3. Dai Y, Vaught TD, Boone J et al. Targeted disruption of the 15. Ohdan H, Yang YG, Shimizu A, Swenson KG, Sykes M. Mixed alpha1,3-galactosyltransferase gene in cloned pigs. Nat Biotech- chimerism induced without lethal conditioning prevents T cell- and nol 2002; 20: 251–255. anti-Gala1,3Gal-mediated graft rejection. J Clin Invest 1999; 104: 4. Kolber-Simonds D, Lai L, Watt SR et al. Production of {alpha}- 281–290. 1,3-galactosyltransferase null pigs by means of nuclear transfer 16. Buhler L, Xu Y, Li W, Zhu A, Cooper DK. An investigation of the with fibroblasts bearing loss of heterozygosity mutations. Proc specificity of induced anti-pig antibodies in baboons. Xenotrans- Natl Acad Sci U S A 2004; 101: 7335–7340. plantation 2003; 10: 88–93. 5. Yamada K, Yazawa K, Shimizu A et al. Marked prolongation of 17. Lee LA, Gritsch HA, Sergio JJ et al. Specific tolerance across a porcine renal xenograft survival in baboons through the use of discordant xenogeneic transplantation barrier. Proc Natl Acad Sci [alpha]1,3-galactosyltransferase gene-knockout donors and the co- USA 1994; 91: 10864–10867. transplantation of vascularized thymic tissue. Nat Med 2005; 11: 18. Zhao Y, Swenson K, Sergio JJ, Arn JS, Sachs DH, Sykes M. Skin 32–34. graft tolerance across a discordant xenogeneic barrier. Nature Med 6. Kuwaki K, Tseng YL, Dor FJMF et al. Heart transplantation in ba- 1996; 2: 1211–1216. boons using [alpha]1,3-galactosyltransferase gene-knockout pigs 19. Rodriguez-Barbosa JI, Zhao Y, Houser S, Zhao G, Sykes M. Fetal as donors: Initial experience. Nat Med 2005; 11: 29–31. porcine thymus engraftment, survival and CD4 reconstitution in 7. Tseng YL, Moran K, Dor FJ et al. Elicited antibodies in baboons alphaGal-KO mice is impaired in the presence of high levels of exposed to tissues from alpha1,3-galactosyltransferase gene- antibodies against alphaGal. Xenotransplantation 2003; 10: 24– knockout pigs. Transplantation 2006; 81: 1058–1062. 40.

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