Background: The conditioning regimen to induce chimerism for immune tolerance is usually accompanied by high toxicity and graft-versus-host disease (GVHD). Our aim was to explore a nontoxic strategy for the induction of mixed chimerism by pretreatment with anti-CD25 monoclonal antibody (mAb), cytotoxic T-lymphocyte-associated antigen 4 immunoglobulin (CTLA4Ig) and anti-CD154 mAb. Methods: A total of 30 Lewis recipient rats (RT1l) were randomly divided into control (A and B) and treated (C, D and E) groups. Recipients serving as controls were without any pretreatment (group A) or pretreated with anti-CD25 mAb, CTLA4Ig and anti-CD154 mAb on days 0, 2, 4, 6 and 8 without bone marrow transplantation (BMT; group B). In the treated groups, the recipients were pretreated with anti-CD25 mAb and CTLA4Ig (group C), with anti-CD25 mAb and anti-CD154 mAb (group D) or with anti-CD25 mAb, CTLA4Ig and anti-CD154 mAb (group E) on days 0, 2, 4, 6 and 8 plus BMT [2 × 108 unmodified whole bone marrow cells from Brown Norway donor rats (RT1n)] on day 0. Full-thickness skin grafts from donor-specific Brown Norway rats were grafted to the dorsal thoracic wall of Lewis recipients on day 8. GVHD was assessed after BMT, and chimerism and T cell apoptosis on days 7, 21, 35 and 49 were detected by flow cytometry. Results: GVHD was not observed in any groups. On days 7, 21 and 35, hematopoietic chimerism was present and maintained in the recipients of the 3 treated groups (groups C, D and E), and thereafter disappeared on day 49. The rate of chimerism in group E was significantly higher compared to that in group C on day 7 and that in group C or D on day 21, but there was no significant difference on day 35 among the 3 groups. The rate of T cell apoptosis in group C, D or E was significantly higher than in group A or B on days 14, 21 and 35. The grafted skin survival in group C, D or E was longer than in group A or B, and survival was significantly longer in group E than in group C or D. Conclusion: Preconditioning with anti-CD25 mAb, CTLA4Ig and anti-CD154 mAb could effectively induce chimerism and immune tolerance without GVHD in a major histocompatibility complex-disparate rat model. This strategy may be attractive for induction of transplantation tolerance. T cell apoptosis is one of the important considerations in tolerance induction.

Keywords:
Graft-versus-host disease, Costimulatory signals, Tolerance, Apoptosis, Chimerism
1.
Muramatsu K, Kuriyama R, You-Xin S, Hashimoto T, Matsunaga T, Taguchi T: Chimerism studies as an approach for the induction of tolerance to extremity allografts. J Plast Reconstr Aesthet Surg 2008;61:1009–1015.
2.
Owen RD: Immunogenetic consequences of vascular anastomoses between bovine twins. Science 1945;102:400–401.
3.
Li S, Salgar SK, Kurimoto Y, Yousem S, Pham SM: Mixed chimerism achieved by a nonlethal conditioning regimen induces donor-specific tolerance to lung allografts. J Surg Res 2008;146:289–297.
4.
Sykes M, Szot GL, Swenson KA, Pearson DA: Induction of high levels of allogeneic hematopoietic reconstitution and donor-specific tolerance without myelosuppressive conditioning. Nat Med 1997;3:783–787.
5.
Wang GM, Ma JB, Jin YZ, Feng YG, Hao J, Gao X, Xie SS: Long-term survival of cardiac allografts induced by cyclophosphamide combined with CTLA4Ig-gene transfer mediated by adenoviral vector. Transplant Proc 2006;38:3043–3045.
6.
Thiel MA, Steiger JU, O’Connell PJ, Lehnert AM, Coster DJ, Williams KA: Local or short-term systemic costimulatory molecule blockade prolongs rat corneal allograft survival. Clin Experiment Ophthalmol 2005;33:176–180.
7.
Watanabe T, Miyatake T, Kumamoto H, Mafune N, Kubota S, Okamoto H, Murashita T, Uede T, Yasuda K: Adenovirus-mediated CTLA4 immunoglobulin G gene therapy in cardiac xenotransplantation. Transplant Proc 2004;36:2478–2479.
8.
Yuan X, Dong VM, Coito AJ, Waaga AM, Salama AD, Benjamin CD, Sayegh MH, Chandraker A: A novel CD154 monoclonal antibody in acute and chronic rat vascularized cardiac allograft rejection. Transplantation 2002;73:1736–1742.
9.
Im SH, Barchan D, Maiti PK, Fuchs S, Souroujon MC: Blockade of CD40 ligand suppresses chronic experimental myasthenia gravis by down-regulation of Th1 differentiation and up-regulation of CTLA-4. J Immunol 2001;166:6893–6898.
10.
di Filippo S: Anti-IL-2 receptor antibody vs polyclonal anti-lymphocyte antibody as induction therapy in pediatric transplantation. Pediatr Transplant 2005;9:373–380.
11.
Kaplan B, West P, Neeley H, Martellotto J, Iqbal R, Gangemi A, Hatipoglu B, Benedetti E, Oberholzer J: Use of low-dose tacrolimus, mycophenolate mofetil and maintenance IL-2 receptor blockade in an islet transplant recipient. Clin Transplant 2008;22:250–253.
12.
Pinana JL, Valcarcel D, Martino R, Moreno ME, Sureda A, Briones J, Brunet S, Sierra J: Encouraging results with inolimomab (anti-IL-2 receptor) as treatment for refractory acute graft-versus-host disease. Biol Blood Marrow Transplant 2006;12:1135–1141.
13.
Vallera DA, Blazar BR: T cell depletion for graft-versus-host-disease prophylaxis: a perspective on engraftment in mice and humans. Transplantation 1989;47:751–760.
14.
Abou el-Ezz AY, Boggs SS, Johnson PC, Li H, Patrene KD, Itskowitz MS, Kaufman CL, Ildstad ST: A minimal conditioning approach to achieve stable multilineage mouse plus rat chimerism. Transpl Immunol 1995;3:98–106.
15.
Taylor PA, Lees CJ, Waldmann H, Noelle RJ, Blazar BR: Requirements for the promotion of allogeneic engraftment by anti-CD154 (anti-CD40L) monoclonal antibody under nonmyeloablative conditions. Blood 2001;98:467–474.
16.
Colson YL, Zadach K, Nalesnik M, Ildstad ST: Mixed allogeneic chimerism in the rat: donor-specific transplantation tolerance without chronic rejection for primarily vascularized cardiac allografts. Transplantation 1995;60:971–980.
17.
Engh E, Strøm-Gundersen I, Benestad HB, Rolstad B: Long-term donor chimerism after MHC (RT1) mismatched bone marrow transplantation in the rat: the role of host alloreactive NK cells. Scand J Immunol 2001;54:198–203.
18.
Okayama J, Ko S, Kanehiro H, Kanokogi H, Hisanaga M, Ohashi K, Sho M, Nagao M, Ikeda N, Kanamura T, Akashi S, Nakajima Y: Bone marrow chimerism and tolerance induced by single-dose cyclophosphamide. J Surg Res 2004;120:102–110.
19.
Poynton CH: T cell depletion in bone marrow transplantation. Bone Marrow Transplant 1988;3:265–279.
20.
Hows JM, Chapple M, Marsh JC, Durrant S, Yin JL, Swirsky D, Gordon-Smith EC: Bone marrow transplantation for Fanconi’s anaemia: the Hammersmith experience 1977–89. Bone Marrow Transplant 1989;4:629–634.
21.
Jankowski RA, Ildstad ST: Chimerism and tolerance: from freemartin cattle and neonatal mice to humans. Hum Immunol 1997;52:155–161.
22.
Liang Y, Huang T, Zhang C, Todorov I, Atkinson M, Kandeel F, Forman S, Zeng D: Donor CD8+ T cells facilitate induction of chimerism and tolerance without GVHD in autoimmune NOD mice conditioned with anti-CD3 mAb. Blood 2005;105:2180–2188.
23.
Foster RD, Pham S, Li S, Aitouche A: Long-term acceptance of composite tissue allografts through mixed chimerism and CD28 blockade. Transplantation 2003;76:988–994.
24.
Blaha P, Bigenzahn S, Koporc Z, Sykes M, Muehlbacher F, Wekerle T: Short-term immunosuppression facilitates induction of mixed chimerism and tolerance after bone marrow transplantation without cytoreductive conditioning. Transplantation 2005;80:237–243.
25.
Guo L, Fujino M, Kimura H, Funeshima N, Kitazawa Y, Harihara Y, Tezuka K, Makuuchi M, Suzuki S, Li XK: AdCTLA-4Ig combined with donor splenocytes, bone marrow cells and anti-ICOS antibody treatment induce tolerance in a rat heart transplantation model. Transpl Int 2004;17:15–21.
26.
Blaha P, Bigenzahn S, Koporc Z, Schmid M, Langer F, Selzer E, Bergmeister H, Wrba F, Kurtz J, Kiss C, Roth E, Muehlbacher F, Sykes M, Wekerle T: The influence of immunosuppressive drugs on tolerance induction through bone marrow transplantation with costimulation blockade. Blood 2003;101:2886–2893.
27.
Malek TR, Yu A, Vincek V, Scibelli P, Kong L: CD4 regulatory T cells prevent lethal autoimmunity in IL-2Rβ-deficient mice: implications for the nonredundant function of IL-2. Immunity 2002;17:167–178.
28.
Sadlack B, Merz H, Schorle H, Schimpl A, Feller AC, Horak I: Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 1993;75:253–261.
29.
Patlolla V, Zhong X, Reed GW, Mandelbrot DA: Efficacy of anti-IL-2 receptor antibodies compared to no induction and to antilymphocyte antibodies in renal transplantation. Am J Transplant 2007;7:1832–1841.
30.
Villegas EN, Lieberman LA, Mason N, Blass SL, Zediak VP, Peach R, Horan T, Yoshinaga S, Hunter CA: A role for inducible costimulator protein in the CD28-independent mechanism of resistance to Toxoplasma gondii. J Immunol 2002;169:937–943.
31.
Shiraishi T, Yasunami Y, Takehara M, Uede T, Kawahara K, Shirakusa T: Prevention of acute lung allograft rejection in rat by CTLA4Ig. Am J Transplant 2002;2:223–228.
32.
Nabeyama K, Yasunami Y, Toyofuku A, Nakano M, Satoh M, Matsuoka N, Ono J, Kamada M, Uede T, Todo S, Ikeda S: Beneficial effects of costimulatory blockade with anti-inducible costimulator antibody in conjunction with CTLA4Ig on prevention of islet xenograft rejection from rat to mouse. Transplantation 2004;78:1590–1596.
33.
Cheung ST, Tsui TY, Wang WL, Yang ZF, Wong SY, Ip YC, Luk J, Fan ST: Liver as an ideal target for gene therapy: expression of CTLA4Ig by retroviral gene transfer. J Gastroenterol Hepatol 2002;17:1008–1014.
34.
Pan XC, Guo L, Deng YB, Naruse K, Kimura H, Sugawara Y, Makuuchi M: Further study of anti-ICOS immunotherapy for rat cardiac allograft rejection. Surg Today 2008;38:815–825.
35.
Pan H, Lu HM, Hu WM, Tian BL, Liu XB, Zhang ZD, Mai G: Anti-CD25 mAb, anti-IL2 mAb, and IL2 block tolerance induction through anti-CD154 mAb and rapamycin in xenogeneic islet transplantation. Transplant Proc 2007;39:3452–3454.
36.
Camirand G, Rousseau J, Ducharme ME, Rothstein DM, Tremblay JP: Novel Duchenne muscular dystrophy treatment through myoblast transplantation tolerance with anti-CD45RB, anti-CD154 and mixed chimerism. Am J Transplant 2004;4:1255–1265.
37.
Kanaya K, Tsuchida Y, Inobe M, Murakami M, Hirose T, Kon S, Kawaguchi S, Wada T, Yamashita T, Ishii S, Uede T: Combined gene therapy with adenovirus vectors containing CTLA4Ig and CD40Ig prolongs survival of composite tissue allografts in rat model. Transplantation 2003;75:275–281.
38.
Wekerle T, Sayegh MH, Hill J, Zhao Y, Chandraker A, Swenson KG, Zhao G, Sykes M: Extrathymic T cell deletion and allogeneic stem cell engraftment induced with costimulatory blockade is followed by central T cell tolerance. J Exp Med 1998;187:2037–2044.
39.
Lu L, Li W, Zhong C, Qian S, Fung JJ, Thomson AW, Starzl TE: Increased apoptosis of immunoreactive host cells and augmented donor leukocyte chimerism, not sustained inhibition of B7 molecule expression are associated with prolonged cardiac allograft survival in mice preconditioned with immature donor dendritic cells plus anti-CD40L mAb. Transplantation 1999;68:747–757.
40.
Honey K, Bemelman F, Cobbold SP, Waldmann H: High dose bone marrow transplantation induces deletion of antigen-specific T cells in a Fas-independent manner. Transplantation 2000;69:1676–1682.
41.
Li JM, Gorechlad J, Larsen CP, Waller EK: Apoptotic donor leukocytes limit mixed-chimerism induced by CD40-CD154 blockade in allogeneic bone marrow transplantation. Biol Blood Marrow Transplant 2006;12:1239–1249.
42.
Hoerbelt R, Johnston DR, Shoji T, Houser SL, Hasse RS, Ledgerwood LG, Iribarne A, Allan JS, Sayegh MH, Sachs DH, Madsen JC: Combination treatment with donor-specific transfusions and cyclosporine a induces long-term survival of cardiac allografts in miniature swine. Transplantation 2005;80:1275–1282.
43.
Yang L, du Temple B, Khan Q, Zhang L: Mechanisms of long-term donor-specific allograft survival induced by pretransplant infusion of lymphocytes. Blood 1998;91:324–330.
44.
Wells AD, Li XC, Li Y, Walsh MC, Zheng XX, Wu Z, Nuñez G, Tang A, Sayegh M, Hancock WW, Strom TB, Turka LA: Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance. Nat Med 1999;5:1303–1307.
45.
van Parijs L, Abbas AK: Homeostasis and self-tolerance in the immune system: turning lymphocytes off. Science 1998;280:243–248.
© 2011 S. Karger AG, Basel
2011
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.