Dariusz Izycki

Donor-specific tolerance in fully major histocompatibility complex-mismatched limb allograft transplants under an anti-αβ T-cell receptor monoclonal antibody and cyclosporine A protocol

Background. Recent studies have demonstrated that treatment with &agr;&bgr;–T-cell receptor (TCR) monoclonal antibody and cyclosporine A (CsA) can extend survival in composite tissue allografts (CTA). The purpose of this study was to induce tolerance in fully major histocompatibility complex (MHC)-mismatched rat limb allografts under 7 days of a combined &agr;&bgr;-TCR–CsA protocol. Methods. The authors performed 30 hind-limb allotransplantations across the MHC barrier between Brown Norway donors (BN; RT1n) and Lewis recipients (LEW; RT1l). Isograft and allograft controls received no treatment. The experimental groups received monotherapy of &agr;&bgr;-TCR and CsA or a combination of &agr;&bgr;-TCR and CsA for 7 days only. Donor-specific tolerance and immunocompetence were determined by standard skin grafting in vivo and mixed lymphocyte reaction (MLR) in vitro. The efficacy of immunosuppressive therapy and the level of donor-specific chimerism were determined by flow cytometry. Results. Long-term survival (>350 days) was achieved in allograft recipients (n=6) under the 7-day protocol of combined &agr;&bgr;-TCR–CsA. Donor-specific tolerance and immunocompetence of long-term chimeras were confirmed by acceptance of skin grafts from the donors and rejection of the third-party alloantigens (A×C Irish). At day 120, MLR demonstrated unresponsiveness to the host and donor antigens but strong reactivity against third-party alloantigens. Flow cytometry confirmed the high efficacy of immunosuppressive treatment and the development of donor-specific chimerism (7.6% of CD4+-RT1n+ cells, 1.3% of CD8+-RT1n+ cells, and 16.5% of CD45RA+-RT1n+ cells) in the periphery of tolerated recipients. Conclusions. Combined therapy of &agr;&bgr;-TCR–CsA for 7 days resulted in tolerance induction in fully MHC-mismatched rat hind-limb allografts. Tolerance was directly associated with stable, donor-specific chimerism.

Induction of tolerance in composite-tissue allografts

Background. Transplantation of composite-tissue allograft (CTA) such as the human hand recently became a clinical reality. The high risks associated with the use of lifelong immunosuppression have been the prohibiting factor in the routine use of the CTA transplants. In this article, we present a new approach of inducing long-term, donor-specific tolerance to CTAs without recipient preconditioning and need for chronic immunosuppression. Methods. We have developed a clinically applicable 35-day protocol that induces donor-specific tolerance in a rat hindlimb-transplantation model across major histocompatibility complex (MHC) barrier [Lewis-Brown-Norway (LBN, RT1l/n→F1) to Lewis (LEW, RT1l) by using cyclosporine A (CsA) and a mouse monoclonal antibody against rat &agr;&bgr;-T-cell receptor (TCR) to systemically eliminate alloresponsive cells. Standard skin grafting, flow cytometry (FC), and mixed lymphocyte reaction (MLR) were used to assess efficacy of immunodepletion and confirm donor-specific tolerance and chimerism. Results. Under this protocol long-term tolerance (>720 days) was induced in all (n=5) CTA recipients across the MHC barrier without further need for immunosuppression. Tolerance was confirmed in all limb-allograft recipients by skin grafting in vivo and by MLR in vitro. The animals rejected third-party grafts, indicating immunocompetence. Conclusions. In this CTA model, combined protocol of &agr;&bgr;-TCR monoclonal antibody and CsA resulted in induction of donor-specific tolerance across the MHC barrier without recipient conditioning. We believe that our findings will foster development of new therapeutic strategies and expand clinical applications for composite-tissue transplantation.

Induction of tolerance to hind limb allografts in rats receiving cyclosporine A and antilymphocyte serum: effect of duration of the treatment.

Background. This study assessed the ability of antilymphocyte serum (ALS) and cyclosporine A (CsA) to induce tolerance for hind limb composite tissue allograft in rats without chronic immunosuppression. Methods. Hind limb transplantations were performed in Lewis-Brown-Norway (LBN, RT1l+n) and Lewis (LEW, RT1l) rats. Treatment consisted of ALS only (0.4 mL/kg), CsA only (16 mg/kg), and a combination of CsA and ALS, and it was administered 12 hr before surgery at three different intervals (7, 14, and 21 days). Long-term survivors were tested for tolerance by standard skin grafting from the recipient (LEW), the donor (LBN), and the third party (ACI, RT1a) 60 days after cessation of the treatment and by mixed lymphocyte reaction at 100 days. T-cell lines were analyzed with flow cytometry. Results. Single use of ALS in all treatment intervals did not prolong allograft survival. Single use of CsA extended survival up to 23 days in the 21-day protocol group. CsA and ALS caused indefinite survival in two of six rats in the 14-day protocol and in all six rats in the 21-day protocol (>420 days). The six long-term survivors in the 21-day protocol accepted the skin grafts from the donor (LBN) and the recipient (LEW) and rejected third-party grafts (ACI). Tolerant animals showed a donor-specific hematopoietic chimerism of 35% to 42% in the peripheral blood. Mixed lymphocyte reaction assay demonstrated tolerance to the host and donor alloantigens and increased response to the third party. Conclusions. Administration of CsA and ALS for 21 days induced donor-specific tolerance in the recipients of the rat hind limb composite tissue allografts. The mechanism of tolerance should be investigated further.

Role of thymus in operational tolerance induction in limb allograft transplant model

Background. In this study, we evaluated the role of host thymus in tolerance induction in composite tissue allografts (CTA) across major histocompatibility complex (MHC) barrier during a 7-day αβ- T-cell receptor (TCR)/ cyclosporine A (CsA) protocol. Materials and Methods. A total of 62 limb allograft transplants were studied. Euthymic (group A) and thymectomized (group B) Lewis recipients (LEW, RT1l) received vascularized hind-limb allografts from hybrid Lewis × Brown-Norway (F1), (LBN, RT1l+n) donors. Mixed lymphocyte reaction (MLR) and skin grafting assessed donor-specific tolerance in vitro and in vivo, respectively. Flow cytometry determined the efficacy of immunosuppressive protocols and the presence of donor-specific chimerism. Immunocytochemistry revealed the presence of donor-specific cells in the lymphoid organs of recipients. Results. Isograft transplants survived indefinitely. For thymectomized rats, the median survival time (MST) of limb allograft in non-treated recipients was 7 days; monotherapy with αβ-TCR extended MST to 16 days, and CsA therapy extended it to 30 days. Using the αβ-TCR/CsA protocol, the MST of allografts was 51 days. For euthymic rats, the MST of limb allograft in non-treated recipients was 7 days; monotherapy with αβ-TCR or CsA extended MST to 13 or 22 days, respectively. Treatment with αβ-TCR/CsA resulted in indefinite allografts survival (MST=370 days). MLR and skin grafting confirmed donor-specific tolerance in euthymic recipients. Flow cytometry showed stable chimerism in the euthymic rats and transient chimerism in thymectomized limb recipients. Immunoperoxidase staining revealed the persistence of donor-derived cells in the lymphoid tissues of euthymic recipients. Conclusion. We found that the presence of thymus was imperative for the induction of donor-specific tolerance in rat hind-limb composite tissue allografts using a αβ-TCR/CsA protocol.

Induction of tolerance in a rat model of laryngeal transplantation

Background. The major limitation preventing expansion of laryngeal transplantation as a therapeutic modality is the necessity of lifelong immunosuppression. In this report, we describe an immunomodulatory strategy for tolerance induction in laryngeal allotransplantation that permits escape from chronic immunosuppression. Materials and Methods. Larynges were transplanted from Lewis-Brown-Norway (RT1l/n, F1) donors to Lewis (RT1l) recipients. Recipients received 7 days of treatment with tacrolimus and mouse anti-rat &agr;&bgr; T–cell-receptor (TCR) monoclonal antibodies. Histology, mixed lymphocyte reaction (MLR), skin grafting, and flow cytometry assessed functional tolerance, efficacy of immunodepletion, and donor-specific chimerism. Results. All 10 recipients survived until sacrifice at 100 days. Histology suggested functional allograft tolerance. Skin grafting, MLR, and flow cytometry revealed that tolerance is neither donor-specific nor related to systemic immunocompromise. Conclusions. In this rat laryngeal-transplantation model, functional tolerance was induced under combined tacrolimus and &agr;&bgr; TCR protocol. Mechanisms responsible for this tolerance induction require future elucidation.

Composite vascularized skin/bone transplantation models for bone marrow-based tolerance studies

There is an ongoing need to understand the mechanisms of bone marrow-based allograft tolerance. This is important in clarifying the diverse variables influencing the ultimate outcome of the solid organ and composite tissue transplants. To establish bone marrow transplantation as a routine clinical application, further experimental studies should be conducted to overcome the obstacles related to the bone marrow transplantation. These obstacles include graft versus host disease, immunocompetence, and toxicity of the conditioning regimens. For these purposes, novel experimental models are needed. In an attempt to provide a reliable research tool for bone marrow-based tolerance induction studies, we introduced different experimental models of modified vascularized skin/bone marrow (VSBM) transplantation technique for tolerance induction, monitoring, and maintenance studies. In this skin/bone transplantation model, the technical feasibility of concurrent or consecutive transplantation of the combination of bilateral vascularized skin, vascularized bone marrow, or vascularized skin/bone marrow transplants was investigated. Isograft transplantations were performed between genetically identical Lewis (LEW, RT11) rats. Five different experimental designs in 5 groups of 5 animals each were studied. Group I: Bilateral vascularized skin (VS) transplantation; group II: bilateral vascularized skin/bone transplantation; group III: vascularized skin transplantation on one side and vascularized skin/bone transplantation on the contralateral side; group IV: vascularized bone transplantation on one side and vascularized skin/bone transplantation on the contralateral side; group V: vascularized bone transplantation on one side and vascularized skin transplantation on the contralateral side. Successful transplantations were performed in all groups. The survival of the isograft transplants was evaluated clinically and histologically. All skin flaps remained pink and pliable and grew new hair. The viability of the compact bone, bone marrow and skin at 100 days posttransplant was confirmed by histologic evaluation, and bone marrow revealed active hematopoiesis. Bilateral skin/bone transplantation model may serve as an ex-perimental tool to study new strategies in tolerance induction by altering the amount of the immunogenic load in the form of skin transplant and bone marrow delivery in the vascularized form, allowing for expedited engraftment of stem and progenitor cells.

215. Vascularized bone marrow transplantation extends survival of vascularized skin allografts

Purpose Vascularized bone marrow allotransplantation is known to be the most effective way of multilineage chimerism induction in the experimental and clinical models. In this study, we introduce combined vascularized skin/vascularized bone transplantation model (VS/VB) to evaluate the effect of the VB transplantation on tolerance induction across strong MHC barrier, under αβ-TCRmAb+CsA treatment protocol. Methods Thirty-six-transplantations between ACI (RT1a) donors to Lewis (RT1I) recipients were performed in three experimental groups of six animals each. In isograft control group and allograft rejection control group no treatment was applied. The experimental group III of vascularized skin/vascularized bone allograft TCR/CsA for 7 days only. The efficacy of (VS/BM) was treated with ab immunosuppressive treatment and the level of chimerism in the peripheral blood of recipients were determined by flow cytometry (FC). Standard H+E technique was applied for the evaluation of the grade of graft rejection. Results The Isograft Control Group I transplants survived indefinitely. In allograft rejection control transplants were rejected between 5 and 9 days post-transplant. The survival time was significantly (p Conclusion Transplantation of the vascularized skin with vascularized bone resulted in significant prolongation of the allograft survival. Allotransplantation of the vascularized bone with marrow natural micro-anatomic environment increases of the donor-derived stem and progenitor cells engraftment resulted in the establishment of the stable, donor specific chimerism.

216. Development of donor specific chimerism and tolerance in composite tissue allografts under ab-t cell receptor monoclonal antibody and cyclosporine a treatment protocols

Purpose Recently, we induced donor specific tolerance to rat hind-limb allografts under 35 days course of ab TCR mAb and Cyclosporine A. In this report, we investigated the role of shorter ab-TCR/CsA protocols on the tolerance induction. Materials and Methods We performed fifty-two hind-limb transplantations, between Lewis-Brown-Norway (LBN, F1) donors and Lewis recipients to test the impact of 21, 7 and 5 day protocols of combined ab-TCR/CsA treatment on tolerance induction. Donor specific tolerance and immunocompetence were tested by mixed lymphocyte reaction (MLR) in vitro and by standard skin grafting in vivo. The efficacy of immunosuppressive protocol and donor specific chimerism was assessed by flow cytometry. Results All transplants under 5,7, and 21 days of combined ab-TCR/CsA therapy survived over 350 days. Clinical tolerance and immunocompetence were confirmed by skin grafting in vivo and MLR in vitro. Flow cytornetry revealed high level of donor chimerism in the peripheral blood of the long term survivors. Conclusion The extention of survival of limb allografts and allounresponsiveness were directly associated with the development of stable chimerism in the tolerated recipients.

214. Induction of the donor specific chimerism and tolerance in fully MHC mismatched limb allograft recipients

Purpose Induction of donor-specific tolerance in composite tissue allografts (CTA) remains one of the most challenging goals in transplant surgery. Recently we have developed a protocol (ab TCR mAb/ CsA) for tolerance induction in the semiallogeneic rat hind-limb transplantations!. In this report we induced tolerance in the fully MHC mismatched allograft recipients under, combined ab-TCR/CSA protocol. Methods Thirty transplantations across strong MHC barrier were performed between Brown-Norway donors (RT1n) and LEW recipients. Isograft and allograft rejection control groups received no treatment. Experimental groups received either rat abTCR or CsA or combination of abTCR and CsA at the day of transplantation and for 7 days thereafter. The efficacy of immunosuppressive treatment and chimerism were monitored by flow cytometry FC. Donor specific tolerance and immunocompetence of the limb recipients were determined in vivo by secondary skin graft from the recipient (LEW), the donor (BN) and the third party (ACI) graft. Mixed lymphocyte reaction (MLR) was performed for the assessment of donor specific tolerance in vitro. Results Only fully mismatched allograft recipients under abTCR/CsA protocol survived indefinite (over 250 days). Three color FC analysis at day 120 post-transplant demonstrated stable, multilineage, donor specific chimerism in the periphery of the tolerated recipients (CD4+PE/RT1n+APC-7.6% and 1.3% of CD8+PE/RT1n+APC positive T cell sub-populations and CD45RA+PE/RT1n+APC-16.5% B cell population). Donor specific tolerance and immunocompetence in vivo was confirmed by acceptance of the secondary skin graft from the donor and rejection of the third-party grafts. MLR revealed no reactivity to host (LEW) and donor (BN) antigens but strong reactivity to the third-party alloantigens. Conclusions Donor specific tolerance was induced under 7 days protocol of abTCR/CsA therapy in the fully mismatched limb allograft transplanrs. Stable mixed chimerism was achieved without the need for the myeloablative bone marrow modification of the recipients and without the need for chronic immunosuppression.