Basic science foundation for clinical composite tissue transplantation

Basic Science Foundation for Clinical Composite Tissue Transplantation K.M. Schuster and C.W. Hewitt

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OMPOSITE tissue allotransplantation (CTA) is a field that has been evolving since the legend of Cosmos and Damian.1 A composite tissue allograft is an anatomic composite of tissues made up of ectodermal and mesodermal elements. Examples of composite tissue allografts include limbs, larynx, mandible, abdominal wall, and vascularized bone marrow.2 Animal models of composite tissue allotransplantation have been developed in a number of species, including rodent, canine, porcine, and primates. The development and progression of these models has paralleled advances in immunomodulation/suppression and strategies for tolerance induction. Central to these strategies is the idea of vascularized bone marrow transplantation (VBMT) with the development of mixed chimerism and tolerance. Until recently, CTA research was confined to the laboratory setting. This changed in September 1998 when a team in Lyon, France transplanted the right distal forearm and hand of a brain-dead donor to a man who had suffered a traumatic amputation of the distal third of his own right forearm 14 years earlier.3 Surgeons at the University of Louisville performed a second human hand allograft in January 1999. Again, this was done for a previous traumatic amputation of the patient’s native hand.4 The team in Lyon repeated the hand transplantation operation in January 2000 as they performed bilateral allotransplantations for a man who had suffered bilateral traumatic amputations 4 years earlier.5 In light of the broad scope of CTA we limit our discussion to two important concepts in CTA, VBMT and sitespecific immunosuppression, as they apply to our primary efforts in the field of CTA. Site-specific immunosuppression has been part of the successful regimen used by both the French and American groups conducting human trials.6,7 IMMUNOMODULATION AND COMPOSITE TISSUE ALLOGRAFTS

The concepts of host tolerance for transplanted allografts and mixed chimerism are not new and many strategies have been investigated to induce these conditions. The effect of pretransplant nonspecific blood transfusion on the improvement of allograft survival was first noted in kidney transplants.8 This observation has been confirmed in other studies.9,10 When applied to CTAs, a very questionable and © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

limited effect of immunosuppression due to nonspecific blood transfusions was demonstrated.11 VBMT is the result of inclusion of perfused bone containing marrow and hematopoetic stem cells in CTA.12 VBMT has been shown to induce stable mixed chimerism and tolerance under certain conditions and immunosuppressive regimens.12–14 Although mixed chimerism has been induced without VBMT experimentally in multiple models,15 these situations are not yet applicable to the clinical setting due to the pretransplant timing of many of the therapies. The one important caveat with VBMT is the observation that, like cellular bone marrow transplantation, it has the capacity to induce graft-versus-host disease (GVHD). We first proposed that CTAs could possibly function to induce chimerism in 1985,16 and confirmed the development of stable mixed chimeras in CTA recipients in 1986.13 Essentially, this was the first evidence that the limb–CTA functioned as a vascularized bone marrow allograft. The impetus for this investigation was the observation of longterm limb allograft survival in rats across a haplotypemismatched histocompatibility barrier (Lewis–Brown Norway donors and Lewis recipients) maintained with relatively low doses of immunosuppression. All animals with longterm allograft survival demonstrated chimerism in peripheral blood and/or spleen with an average level of 19.7%. These animals also demonstrated histologically normal bone marrow in their allografts. Subsequently, rat hindlimb transplantation was performed in a fully allogeneic model.14 Being fully allogeneic allowed for the study of possible development of GVHD. Again, CsA was used in variable dosing regimens for immunosuppression. It was found that animals maintaining their grafts past 100 days posttransplant developed significant lymphoid chimerism, had donor marrow in the transplanted limbs, and eventually developed a wasting syn-

From the Division of Surgical Research, Department of Surgery, University of Medicine and Dentistry of New Jersey–Robert Wood Johnson Medical School, Cooper Health System/University Medical Center, Camden, New Jersey, USA. Address reprint requests to Dr Charles W. Hewitt, Surgical Research, Department of Surgery, 3 Cooper Plaza, Suite 411 Camden, NJ 08103. 0041-1345/01/$–see front matter PII S0041-1345(00)02655-5 1717

Transplantation Proceedings, 33, 1717–1719 (2001)

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drome consistent with GVHD. Maintenance of the grafts to 100 days posttransplant required continuous therapeutic dosing of CsA. This study demonstrated, for the first time, that stable mixed lymphoid chimeras could be induced with VBMT across a major histocompatibility barrier. A different semiallogeneic (Lewis to Lewis–Brown Norway) transplant experiment was then performed.12,17 This study was significant because no immune modulation was necessary because the host musculoskeletal tissues could not be rejected due to the immunogenetics of the combination. However, bone marrow derived from the donor would theoretically respond to semiallogeneic host tissue to produce GVHD. Of the transplanted animals, 37.5% developed GVHD and 62.5% showed no signs of rejection (tolerance) or GVHD. The levels of donor chimerism were determined in each group, with the tolerant animals showing low-level chimerism (18.3% of the cells) and the GVHD animals showing much higher levels of donor chimerism (60.2% of the cells). The tolerant animals also displayed immune competence when exposed to third-party antigens, whereas the GVHD animals were nonreactive to thirdparty antigen, possibly due to increased TGF-␤ expression in the transplanted marrow.18 The occurrence of GVHD in the aforementioned study, although important, should be of little concern in the current clinical application of CTA. GVHD would be unlikely in a patient receiving minimally functioning or nonfunctioning hematopoietic marrow as in the case of hand transplantation.19 This was the case in an anecdotal report of the first modern human hand transplant where there was no evidence of peripheral lymphoid chimerism.20 The expansion of CTA to entire limbs may increase the number of stem cells available for transplant, possibly facilitating mixed chimerism, tolerance, and/or GVHD. VBMT may offer the ideal opportunity for the study of the induction of mixed chimerism and tolerance. A VBMT also contains the transplanted microenvironment that supports the marrow. In this case, both the microenvironment and marrow are syngeneic. However, the stromal microenvironment and marrow are allogeneic in conventional cellular marrow transplants. Thus, VBMT may offer an alternative to conventional bone marrow transplantation. Because the rate of GVHD may be lower in VBMT, it may offer a unique advantage. SITE-SPECIFIC IMMUNOSUPPRESSION

Site-specific, or topical, immunosuppression has been utilized as part of the immunosuppressive regimen in clinical hand transplantation. Because skin has been shown to be the most immunogeneic tissue in CTA, it is intuitive that systemic immunosuppressive therapy may be reduced if skin rejection can be treated with topical immunosuppression. The first two hand transplant patients in the modern era had successful treatment of rejection with topical clobetasol, a high-potency glucocorticoid.6,7 The major focus of our research concerning site-specific

SCHUSTER AND HEWITT

immunosuppression has been on CsA. We initially reported improved skin allograft survival in rats with topical CsA, but only when there was initial coverage by systemic CsA. It has also been shown that the immunomodulating effects of topical CsA are synergistic with topical anti-inflammatory agents, particularly topical steroids.21 Vehicles that provided strong hydrophilic properties led to deposition of the lipophilic immunosuppressive within the skin and significant local effects. Vehicles that possessed strong solvent properties and were highly polar conferred properties allowing transdermal penetration of the immunosuppressive. This transdermal penetration provided for local as well as significant systemic effects of the immunosuppressive.22 The mechanisms by which topical CsA induces its local immunomodulating effects have yet to be fully elucidated. We have, however, made several observations during experiments with a site-specific model involving allogeneic skin grafts in rats. These observations included decreased expression of MHC class I and II in the CsA-treated allogeneic skin as compared with control grafts. Infiltration of the transplanted tissue by CD4-positive lymphocytes and mononuclear inflammatory cells was also decreased in the CsA-treated animals versus the control group. These local immunosuppressive effects all occurred without systemic effects.23 CONCLUSIONS

With the success of the first hand transplants, we can be certain that the technical expertise for CTA is available. The significant concern in CTA is the need for a toxic immunosuppression regimen to maintain the graft. Both the French and American teams have demonstrated success with current immunosuppressive regimens, without adverse consequences. There has been no mortality due to immunosuppression, and morbidity has been minimal.6,7 There has been no evidence of life-threatening infection secondary to the immunosuppression. The only reported morbidities have been hyperglycemia, transient increases in creatinine, and a herpesvirus infection, which was reversed with acyclovir.6 Immunosuppression-induced malignancies have not been seen, although significant conclusions cannot be drawn at this early stage. Although the complications of immunosuppression have been minor, the episodes of rejection have been equally minor and easily reversed.6,7 Tolerance is the goal in solid-organ transplantation and composite tissue allotransplantation. VBMT offers a unique opportunity to study methods of tolerance induction. Sitespecific immunosuppression without systemic administration would be an acceptable alternative to help induce tolerance. Continued study of the mechanisms underlying this form of therapy may lead to the development of predominantly localized therapies for CTA. There has been some concern regarding the risk of GVHD with regard to VBMT in CTA. Dubernard et al reported a lack of donor cells in the peripheral circulation and replacement of donor Langerhans cells in the epider-

A BASIC FOUNDATION

mis of the graft with cells of recipient origin.20 These findings seem to indicate an insufficient number of stem cells in the allograft to induce chimerism. It is doubtful that transplantation of any CTA containing long bone would, in a clinical sense, carry enough stem cells to induce mixed chimerism or GVHD. With continued clinical success, we do not have to look far into the future for the next milestone in CTA. Whole-limb or near-whole-limb transplants can probably now be considered as the safety of hand transplantation has been established. REFERENCES 1. Black KS, Hewitt CW, Fraser LA, et al: N Engl J Med 3:368, 1982 2. Llull R, Beko KR, Black KS, et al: Transplant Rev 6:175, 1992 3. Dubernard JM, Owen E, Herzberg G, et al: Lancet 353:1515, 1999 4. Jensen JN, Mackinnon SE: J Reconstr Mircrosurg 16:57, 2000 5. Altman LK: New York Times, January 15, 2000 6. Dubernard JM, Owen E, Herzberg G, et al: Lancet 353:1315, 1999 7. Jones JW, Gruber SA, Barker JH, et al: N Engl J Med 343:468, 2000 8. Opelz G, Sengar DP, Mickey MR, et al: Transplant Proc 5:253, 1973

1719 9. Van Es AA, Marquet RL, van Rood JJ, et al: Lancet 1:506, 1977 10. Sanfilippo F, Thacker L, Vaughn WK: Transplantation 49:25, 1990 11. Black KS, Hewitt CW, Woodard TL, et al: J Microsurg 3:162, 1982 12. Hewitt CW, Ramsamooj R, Patel MP, et al: Transplantation 50:766, 1990 13. Hewitt CW, Black KS, Dowdy SF, et al: Transplantation 41:39, 1986 14. Hewitt CW, Black KS, Henson LE, et al: Transplant Proc 20:272, 1988 15. Wekerle T, Sykes M: Transplantation 68:459, 1999 16. Black KS, Hewitt CW, Fraser LA, et al: Transplantation 39:365, 1985 17. Yazdi B, Patel MP, Ramsamooj R, et al: Transplant Proc 23:739, 1991 18. Tatem LD, Hirpara S, Dalsey RM, et al: Transplant Proc 29:2194, 1997 19. Ramsamooj R, Llull R, Black KS, et al: Plast Reconstr Surg 104:1365, 1999 20. Kanitakis J, Jullien D, Claudy A, et al: Lancet 354:1820, 1999 21. Llull R, Lee TP, Vu AN, et al: Transplantation 59:1483, 1995 22. Hewitt CW, Black KS: Transplant Proc 28:922, 1996 23. Black KS, Patel MP, Patel AP, et al: Transplant Proc 23:120, 1991