Vascularized Thymic Lobe Transplantation in Miniature Swine: Thymopoiesis and Tolerance Induction Across Fully MHC-Mismatched Barriers
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Vascularized thymic lobe transplantation in miniature swine: Thymopoiesis and tolerance induction across fully MHC-mismatched barriers Chisako Kamano*, Parsia A. Vagefi*, Naoki Kumagai, Shin Yamamoto, Rolf N. Barth, John C. LaMattina, Shannon G. Moran, David H. Sachs, and Kazuhiko Yamada† Transplantation Biology Research Center, Massachusetts General Hospital͞Harvard Medical School, Massachusetts General Hospital-East, Building 149-9019, 13th Street, Boston, MA 02129 Edited by Laurie H. Glimcher, Harvard School of Public Health, Boston, MA, and approved January 5, 2004 (received for review October 21, 2003) As the major site of self-nonself discrimination in the immune by prevascularization of autologous thymic tissue grafts within a system, the thymus, if successfully transplanted, could potentially kidney’s subcapsular space (prepared as single-donor composite carry with it the induction of central tolerance to any other organ grafts: thymokidneys). We have recently reported the ability of or tissue from the same donor. We have recently developed a these composite grafts to induce allogeneic tolerance (1, 5, 6), technique for transplantation of an intact, vascularized thymic lobe suggesting that prevascularization of the thymic tissue before (VTL) in miniature swine. In the present study, we have examined transplantation was crucial for success in large animals (1, 6). the ability of such VTL allografts to support thymopoiesis and Although thymokidneys were able to induce tolerance (1, 6), induce transplantation tolerance across fully MHC-mismatched the full potential of vascularized thymus transplantation will only barriers. Six miniature swine recipients received fully MHC-mis- be realized if the scope of its applicability can be broadened to matched VTL grafts with a 12-day course of tacrolimus. Three of additional organs, less amenable to the preparation of composite these recipients were thymectomized before transplantation and grafts. In this regard, the ability to perform thymus transplan- accepted their VTL allografts long-term, with evidence of normal tation as an isolated vascularized thymic lobe (VTL) graft could thymopoiesis. In contrast, three euthymic recipients rejected their permit thymic-facilitated tolerance induction with any solid VTL allografts. Donor renal allografts, matched to the donor VTL organ or tissue transplanted simultaneously. We recently re- grafts, were transplanted without immunosuppression into two of ported our first attempts to perform VTL transplantation across the three thymectomized recipients, and one of the three euthymic minor mismatched barriers in Massachusetts General Hospital recipients. These renal allografts were accepted by thymectomized miniature swine, and have demonstrated that (i) the technique recipients, but rejected by the euthymic recipient in an accelerated of VTL transplantation in swine can be performed successfully, fashion. This study thus demonstrates that successful transplan- and (ii) minor antigen-mismatched VTL allografts have the tation of a vascularized thymus across a fully MHC-mismatched ability to support thymopoiesis (7). barrier induces tolerance in this preclinical, large-animal model. Given our initial success across minor antigen-mismatched This procedure should enable studies on the role of the thymus in barriers, we have now extended our work to a greater antigenic transplantation immunology as well as offer a potential strategy disparity. We report here the successful application of VTL graft for tolerance induction in clinical transplantation. transplantation to the induction of tolerance and thymopoiesis across two-haplotype, fully MHC-mismatched barriers in thymus transplantation ͉ vascularization thymectomized miniature swine recipients. Our data indicate that VTL grafts induce donor-specific systemic tolerance while he clinical application of organ transplantation is currently maintaining immunocompetence to third-party antigens. Our Tlimited by a critical shortage of donor organs, as well as by results also demonstrate that the host thymus interferes with the the requirement for chronic, nonspecific immunosuppressive induction of tolerance in this model. We believe that this therapy for the remainder of a patient’s life. Therefore, new technique offers a promising strategy to support long-term strategies directed toward both the supply of organs and the thymopoiesis and to induce transplantation tolerance across induction of tolerance, have become major goals in the field of allogeneic or even xenogeneic barriers. transplantation immunology. The induction of tolerance to transplanted organs or tissues could avoid the morbidity asso- Materials and Methods ciated with prolonged immunosuppression. The extension of Animals. Animals were selected from our herd of MHC-inbred tolerance-inducing regimens from allogeneic to xenogeneic bar- miniature swine (8, 9) at 2–4 months of age (juvenile animals) riers holds the additional promise of alleviating the worldwide to serve as VTL donors, and swine of 3–6 months of age were shortage of organs. Therefore, tolerance induction strategies used as recipients. have represented a major goal of preclinical transplantation research. Experimental Groups. Six recipient pigs received two-haplotype As the origin of T cell immunity, the thymus has the unique ͞ fully MHC-mismatched VTL grafts. Three of these animals potential to tolerize allogeneic and or xenogeneic responses to (group A) were completely thymectomized 3 weeks before transplanted organs and tissues (1). This laboratory has previ- CELL BIOLOGY ously reported a strategy for the induction of xenogeneic im- munological tolerance in the pig-to-mouse model by directly This paper was submitted directly (Track II) to the PNAS office. transplanting nonvascularized porcine thymic tissue beneath the Abbreviations: CML, cell-mediated lymphocytotoxicity; FACS, fluorescence-activated cell renal capsular space of mice (2, 3). However, attempts to sorter; FK506, tacrolimus; HBSS, Hanks’ balanced salt solution; MLR, mixed lymphocyte perform similar nonvascularized thymic tissue transplants in reaction; PAA, pig allele-specific antigen; PBL, peripheral blood lymphocyte; VTL, vascu- large animals have had limited success (1, 4). Therefore, by using larized thymic lobe. miniature swine as a large-animal model, the potential of *C.K. and P.A.V. contributed equally to this work. vascularized thymic tissue to induce tolerance has been exam- †To whom correspondence should be addressed. E-mail: [email protected]. ined. Vascularized thymic transplants were initially performed © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0306666101 PNAS ͉ March 16, 2004 ͉ vol. 101 ͉ no. 11 ͉ 3827–3832 Downloaded by guest on September 27, 2021 transplantation whereas the other three (group B) remained of cells was performed by three-color staining. The staining euthymic. procedure was performed as follows: 1 ϫ 106 cells were resus- pended in flow cytometry buffer (HBSS containing 0.1% BSA Immunosuppressive Therapy. Tacrolimus (FK506; Fujisawa, Deer- and 0.1% NaN3) and were incubated for 30 min at 4°C with field, IL) was administered for 12 days, starting on the day of saturating concentrations of a FITC-labeled mAb. After two transplantation (day 0). In an attempt to minimize side effects washes, the secondary phycoerythrin-conjugated Ab was added associated with high peak levels, the drug was infused continu- and cells were incubated for 30 min at 4°C. After a further wash, ously through an infusor pump (Baxter Health Care, Deerfield, the final biotinylated Ab was added and incubated for 30 min. IL). Dosing was started at 0.15 mg per kg per day and was ͞ The cells were washed, and cytochrome was added for an 8-min subsequently adjusted to maintain a blood level of 30–40 ng ml. incubation to stain the biotinylated Ab. Cells were then washed Whole-blood levels were determined by microparticle enzyme twice and were analyzed by FACScan. immunoassay (Tacrolimus II, IMx system, Abbott Laboratories). Abs for Detection of Thymopoiesis and Peripheral Chimerism. Thy- Surgery. Complete thymectomy in the recipient. Complete thymec- mocyte development and peripheral chimerism were assessed by tomy was performed before allogeneic transplantation in three immunohistochemistry and FACS analyses using the murine recipients as described (10). Briefly, the pretracheal muscles anti-pig mAbs 74–12-4 (IgG2b, anti-swine CD4), 76–2-11 were retracted, exposing the cervical thymus and trachea from the cervicothoracic junction to the mandibular area. The cervi- (IgG2a, anti-swine CD8), 76–7-4 (IgG2a, anti-swine CD1) (11), cal thymus was excised, after which the mediastinal thymus was MSA4 (IgG2a, anti-swine CD2, BB23–8E6 (IgG2b, anti-swine removed through a sternotomy. CD3), 2–27-3a (IgG2a, anti-swine class I), and 10–9 [anti-swine VTL transplantation. The procedure of VTL graft transplantation pig allele-specific antigen (PAA) (12)]. has been reported (7). Two animals in group A (thymectomized), and two animals in group B (nonthymectomized) had VTL grafts Cell-Mediated Lymphocytotoxicity (CML) Assay. CML assays were transplanted to the s.c. neck space by using the internal carotid performed as described (10, 13–15). Briefly, lymphocyte cultures ϫ 6 ϫ 6 artery and internal jugular vein; in the remaining two recipients containing 4 10 responder and 4 10 stimulator PBLs VTL grafts were transplanted in the retroperitoneum by using (irradiated with 2,500 cGy)