Donor-specific Chimerism Research Articles

Correlation of Chimerism With Graft Size and Revascularization in Vascularized and Nonvascularized Skin Allografts

The high antigenicity of skin components of composite tissue allografts is the most challenging factor in achieving long-term viability after composite tissue allografts transplantation. The vascularization pattern of vascularized skin allografts (VSA) and nonvascularized skin allografts (NVSA) differs significantly and these differences may alter host immune responses. We hypothesized that vascularized grafts contribute to better engraftment and long-term survival. We have tested our hypothesis by transplantation of different sizes (2 x 2 cm, 4 x 4 cm, 6 x 6 cm) of VSA and NVSA across major histocompatibility complex barrier between LBN (RT1(1+n)) and Lewis (RT1(1)) rats. Correlation of revascularization process with development of donor-specific chimerism was tested. We found that the highest chimerism levels (8%-12.2% in VSA groups; 2.53%-3.92% in NVSA groups) were reached as early as 7 days in both VSA and NVSA. Chimerism decreased in both groups during 100-day follow-up and higher chimerism was found only in VSA in late posttransplant period. Revascularization, assessed by the presence of CD31 positive vessels, was significantly higher in VSA compared with NVSA and to controls (P < 0.05). A direct correlation was seen between increased skin diameter and donor chimerism in VSA, whereas inverse correlation was tested in NVSA. We have confirmed that allograft size and vascularization pattern contribute to donor chimerism development and maintenance.

Maintenance of Donor-Specific Chimerism Despite Osteopontin-Associated Bone Fibrosis in a Vascularized Bone Marrow Transplantation Model

The role of vascularized bone allografting is not established in plastic and reconstructive surgery. The authors evaluated the contribution by osteopontin to fibrosis of allografted bone in a vascularized bone marrow transplantation model across a major histocompatibility complex barrier. Thirty-six transplantations were performed between Brown Norway (RT1 n) donors and Lewis (RT1 l) recipients divided into three groups: group 1, isografts between Lewis rats (n = 12); group 2, allografts without treatment (n = 8); and group 3, allografts under a 7-day alphabeta-T-cell receptor/cyclosporine protocol (n = 16). Flow cytometry assessed the presence of chimerism for donor major histocompatibility complex class I (RT1 n) antigens. Immunostaining was used to determine osteopontin expression in grafted and recipient bone, and histologic examination was used to assess bone architecture. Early engraftment of donor bone marrow cells (RT1 n) into the recipient bone marrow compartment was achieved at posttransplantation day 7. This corresponded with osteopontin expression restricted to the endosteum of trabecular bone and was associated with the preservation of hematopoietic cells within donor bone. Cell migration between donor and recipient bone marrow compartments was confirmed by the presence of recipient cells (RT1 l) within the allografted bone and donor-origin cells (RT1 n) within the recipient bone. At posttransplantation day 63, osteopontin expression within allografted bone was associated with allograft bone fibrosis and lack of hematopoietic properties. In contrast, the recipient's contralateral bone demonstrated a highly localized osteopontin expression pattern within the endosteum and active hematopoiesis with the presence of donor-specific (RT1 n) cells and correlated with chimerism maintenance. These results confirm that despite up-regulation of osteopontin expression and fibrosis of allografted bone, vascularized bone marrow transplantation resulted in efficient engraftment of donor cells into the recipient's bone marrow compartment, leading to chimerism maintenance.

Chimerism May Prolong Islet Allograft Acceptance through the Preservation of CD4+CD25+FoxP3+ Treg in Diabetic NOD Mice.

In the past several years, novel insights into the role of costimulatory molecules in generating adaptive immune responses have led to the development of new approaches for tolerance induction. We previously demonstrated that preconditioning of diabetes-prone NOD mice with anti-CD8 and combined with blockade of CD40L (anti-CD154) allowed chimerism to be established with 550 cGy TBI. Here, we investigated whether combined costimulatory blockade of CD40L, inducible co-stimulator (ICOS) and OX40L (CD134L) would enhance bone marrow chimerism and promote islet allograft survival in diabetic NOD mice. We also determined whether CD4+/CD25+/FoxP3+ regulatory T-cells (Treg) play an important role in maintenance of tolerance. Spontaneously diabetic NOD mice (blood glucose levels >350 mg/dL) were treated with anti-CD8 antibody (day -3). After 500 cGy TBI (day 0), NOD mice were transplanted with 30 × 106 B10.BR bone marrow cells (day 0) followed by transplantation of 300–400 B10.BR islets under the renal capsule and administered anti-CD154, anti-OX40L, and anti-ICOS mAbs IP (day 0, 1, 2, 3). Chimerism and peripheral blood CD4+/CD25+/FoxP3+ Treg cell expression were quantitated by flow cytometry. Urine and blood glucose levels were monitored. Nonmyeloablatively conditioned mice transplanted with islet allografts and infused with bone marrow (BM) demonstrated acceptance and continued function of islet allografts, with hyperglycemic reversal ≥ 45 (A) and 95 (B) days (Figure 1). Recipients of islet allografts and BM (∼5–12 days), islet allografts and antibody treatment (∼20 days) or islet allografts alone (∼5 days) rejected their grafts. Blood glucose returned to pretreatment hyperglycemic levels in the rejected islet allograft recipient (C) treated in the same manner as the acceptors (A and B) as well as in the control recipients. Donor chimerism at one month post-BMT was 58.5% (A), 89.7% (B) and 0% (C), respectively. Although CD4+/CD25+/FoxP3+ Treg expression was similar between the islet allograft acceptor and rejector recipients, the ratio of CD4+/CD25+/FoxP3+ Treg to CD4+/CD25+/FoxP3− T-effector cells was 2-fold greater in the islet acceptors (3.6 vs. 1.8) (Figure 2). Taken together, our data suggest that prolongation of donor-specific chimerism is associated with tolerance to islet allografts and that CD4+/CD25+/FoxP3+ Treg may be important in maintaining peripheral tolerance. Studies are underway to determine if CD4+/CD25+/FoxP3+ Treg may prevent intra-islet infiltration by autoimmune effector cells, thereby maintaining tolerance to islet allografts. [Display omitted] [Display omitted]

A New Rat Model of Maxilla Allotransplantation

We developed a rat model to test the effects of vascularized maxilla allotransplantation on composite maxillary substructures. Allograft maxilla transplantations were performed across the major histocompatibility barrier between 10 Lewis-Brown-Norway (RT1(n+l)) and 10 Lewis (RT1(l)) recipient rats under cyclosporin A monotherapy. Grafts were dissected along Le-Fort II osteotomy lines based on the common carotid artery and external jugular vein and transplanted to the anterior abdominal wall via microvascular anastomosis. Allografts were examined by tomography, flow cytometry, angiography, and histology. Three of the allografts survived up to 105 days without any signs of rejection. High level of donor-specific chimerism for T-cell and B-cell lineages was maintained in the peripheral blood. The incisors continued to grow; teeth buds, bone, cartilage, and mucosa remained intact. Moderate inflammation of the nasal, oral mucosa, and keratinous metaplasia was noted histologically. We created a maxilla allotransplantation model that allows the study of immunologic responses and demonstrates potential clinical applications based on the growth properties of the allograft.

Chimerism induction in vascularized bone marrow transplants augmented with bone marrow cells

Composite tissue allografts (CTAs) are currently accepted in the clinic; however, long-term immunosuppression is still needed for allograft survival. The presence of donor-specific chimerism may induce tolerance. Thirty-six vascularized bone marrow transplantation (VBMT) allotransplantation were performed across MHC barrier under short-term protocol of 7-day alphabeta-TCRmAb and Cyclosporin A therapy to determine the efficacy of VBMT alone and VBMT augmented with donor bone marrow transplantation (BMT) in chimerism induction. Flow cytometry analysis revealed that VBMT supported with donor BMT directly into the bone resulted in chimerism augmentation and maintenance compared to VBMT. In vivo and in vitro tolerance testing showed prolonged survival of donor skin graft up to 35 days and moderate reactivity in MLR assay that suggests only tolerance induction. Transplantation of vascularized bone without chronic immunosuppression provides a substantial source of bone marrow cells, leading to the development of stable donor-specific chimerism.

Applications of Bilateral Vascularized Femoral Bone Marrow Transplantation for Chimerism Induction Across the Major Histocompatibility (MHC) Barrier

Bilateral vascularized bone marrow transplant (VBMT) model was designed to induce chimerism across the major histocompatibility (MHC) barrier under combined alphabeta T-cell receptor monoclonal antibody and cyclosporine A (alphabeta-TCRmAb/CsA) protocol. Seventeen transplants were performed between BN(RT1) donors and Lewis(RTI) recipients. Group I, isograft controls; Group II, allografts rejection controls; Group III, allografts under 7-day protocol of alphabeta-TCRmAb/CsA. Donor bilateral femoral bones were bilaterally anastomosed to the abdominal aorta and inferior vena cava of recipient. At day 7 posttransplantation, all bone flaps were viable. Groups I and III survived without signs of rejection. In Group III, peak level of chimerism in peripheral blood was evaluated at day 21 (24.2%), at day 63 declined to 1.5%, and was maintained at this level thereafter. Donor-derived cells were present in the bone marrow of recipients at 28.2% at day 21 posttransplant. Histology confirmed viability of bone marrow cells in isograft during the entire follow-up and up to 35 days in treatment Group III. Bilateral VBMT induced donor-specific chimerism across the MHC barrier under the immunomodulatory protocol of alphabeta-TCRmAb/CsA.

39: Long Term Survival of Composite Hemiface/Mandible/Tongue Tissue Allograft Permitted by Donor Specific Chimerism

39: Long Term Survival of Composite Hemiface/Mandible/Tongue Tissue Allograft Permitted by Donor Specific Chimerism

Role of Thymus in Operational Tolerance Induction in Limb Allograft Transplant Model

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 alphabeta- T-cell receptor (TCR)/ cyclosporine A (CsA) protocol. A total of 62 limb allograft transplants were studied. Euthymic (group A) and thymectomized (group B) Lewis recipients (LEW, RT1(1)) received vascularized hind-limb allografts from hybrid Lewis x Brown-Norway (F1), (LBN, RT1(1+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. Isograft transplants survived indefinitely. For thymectomized rats, the median survival time (MST) of limb allograft in non-treated recipients was 7 days; monotherapy with alphabeta-TCR extended MST to 16 days, and CsA therapy extended it to 30 days. Using the alphabeta-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 alphabeta-TCR or CsA extended MST to 13 or 22 days, respectively. Treatment with alphabeta-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. 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 alphabeta-TCR/CsA protocol.

Trafficking of Donor-Derived Bone Marrow Correlates With Chimerism and Extension of Composite Allograft Survival Across MHC Barrier

We proposed to evaluate differences between recipient’s immune response to vascularized skin and combined vascularized skin/bone allografts, under a 7-day αβ-TCR plus cyclosporine (CsA) treatment protocol. Thirty-six transplantations were performed in six groups: group I (isograft control-vascularized skin graft; n = 6); group II (isograft control-combined vascularized skin/bone graft; n = 6); group III (allograft rejection control group-vascularized skin graft; n = 6); group IV (allograft rejection control-combined vascularized skin/bone graft; n = 6); group V (allograft treatment-vascularized skin graft; n = 6); and group VI (allograft treatment-combined vascularized skin/bone graft; n = 6). Isograft transplantations were performed between Lewis rats and allografts were transplanted across the MHC barrier from Brown Norway to Lewis rats. In the allograft treatment group, a combined αβ-TCR+CsA protocol was applied for 7 days. All groups were compared clinically, immunologically and histologically. Statistical significance was determined with two-tailed Student’s t test. Indefinite graft survival was achieved in the isograft control group (>300 days). Allograft rejection controls rejected within 5 to 9 days posttransplant; chimerism levels were undetectable (<.5%). Allografts under the αβ-TCR+CsA protocol had significantly extended survival when skin was combined with bone (61–125 days) compared to vascularized skin allografts (43–61 days). Lymphoid macrochimerism was significantly higher in group VI than group V. Histology confirmed skin and bone viability. Combined vascularized skin/bone allografts had higher and sustained levels of donor-specific chimerism and extended allograft survival.

Intraosseus Transplantation of Donor-Derived Hematopoietic Stem and Progenitor Cells Induces Donor-Specific Chimerism and Extends Composite Tissue Allograft Survival

We investigated the effect of the intraosseous allotransplantation of the donor-derived hematopoietic stem cells (HSC) CD90 + on chimerism induction and survival of rat hind limb transplants. Eighteen rat hind limb transplantations were performed between Lewis-Brown-Norway and Lewis rats in three groups. Isograft and allograft rejection controls received no treatment. In the experimental group, 0.8 to 1.2 × 10 6 of separated and purified CD90 + HSC cells were transplanted intramedullary into the bone marrow cavity of the recipient’s tibia during opposite hind limb transplantation, without immunosuppressive therapy. Transplants from isograft group survived indefinitely. Allograft controls rejected transplants on day 7 posttransplant. The injection of separated and purified CD90 + cells of the donor origin extended survival of the transplanted limbs up to 15 days in group III. We introduced a novel method of transplantation of the CD90 + cells of the donor origin into the recipient’s bone marrow cavity. This technique resulted in extended allograft survival, without immunosuppressive therapy.

Development and Maintenance of Donor-Specific Chimerism in Semi-Allogenic and Fully Major Histocompatibility Complex Mismatched Facial Allograft Transplants

The clinical application of composite tissue allograft transplants opened the discussion on the restoration of facial deformities by allotransplantation. We introduce a hemifacial allograft transplant model to investigate the rationale for the development of operational tolerance across a major histocompatibility complex (MHC) barrier. Thirty rats were studied in five groups of six animals each. The composite hemiface isograft transplantations were performed in group 1. Allograft rejection controls included semi-allogenic transplantations from LBN (RT1(1+n) donors (group 2) and fully allogenic transplantations from ACI (RT1a) donors (group 3) to LEW (RT1(1)) recipients. In the allograft treatment groups, recipients of LBN (group 4) and ACI donors (group 5) were treated with cyclosporine A monotherapy (16 mg/kg/day, tapered to 2 mg/kg/day). Face allografts were evaluated clinically and histologically. Donor-specific chimerism for MHC class I RT1n and RT1a antigens was assessed by flow cytometry. Mixed lymphocyte reaction for donor-specific tolerance in vitro was tested at day 160 posttransplant. Isograft controls survived indefinitely. All nontreated allografts rejected within 5 to 8 days posttransplant. Long-term survival was achieved in 100% of LBN (up to 400 days) and ACI (up to 330 days) recipients. At day 160, posttransplant donor-specific chimerism was present in recipients of LBN (10.14% CD4/RT1n, 6.38% CD8/RT1n, 10.02% CD45RA/RT1n) and ACI (17.54% CD4/RT1a, 9.28% CD8/RT1a) transplants, and mixed lymphocyte reaction confirmed tolerance in recipients of LBN transplants and moderate reactivity in recipients of ACI allografts. Operational tolerance was induced in hemiface allograft transplants across an MHC barrier under cyclosporine A monotherapy protocol. It was associated directly with the presence of multilineage donor-specific chimerism.

Different routes of donor-derived hematopoietic stem cell transplantation for donor-specific chimerism induction across MHC barrier

Transplantation of donor-derived stem cells can improve organ allograft survival in animal models. This study was designed to investigate the effect of different routes of bone marrow cell (BMC) transplantation on donor-specific tolerance induction across MHC barrier under short-term CsA monotherapy and αβTCR/CsA treatment protocols. Forty-eight BMC transplantations were performed between BN(RT1 n) donors and LEW(RT1 1) recipients. Intraosseous and intravenous BMC transplantation was studied in six groups of eight animals each receiving 35 × 10 6 ( n = 4) and 70 × 10 6 ( n = 4) bone marrow cells. Groups I and II (controls) received BMC transplantation but no treatment, groups III and IV CsA monotherapy, and groups V, VI αβTCR/CsA protocol for 7 days. Flow cytometry monitored immunodepletion and donor-specific chimerism for MHC class I RT1 n/CD4, RT1 n/CD8 and RT1 n/CD45RA antigens. All animals survived without graft-versus-host disease. At day 63 under CsA monotherapy a low level of chimerism for RT1 n/CD4 was induced after intraosseous (1.9%) and intravenous (0.8%) transplantation of (70 × 10 6) BMC. Under αβTCR/CsA protocol chimerism for RT1 n/CD4 revealed 6.5% and 0.9% in intraosseous and intravenous (70 × 10 6) BMC transplantation, respectively. The total number of chimerism in intraosseous and intravenous (70 × 10 6) BMC transplantation groups was 9.9% and 3.4%, respectively. Following intraosseous BMC transplantation under αβTCR/CsA protocol chimerism was 50% higher in a group receiving 70 × 10 6 (9.9%) vs 35 × 10 6 (4.9%) BMC. Intraosseous transplantation of donor BMC under αβTCR/CsA protocol was 75% more efficient in induction of donor-specific chimerism compared to intravenous transplantation.

Strategies to develop chimerism in vascularized skin allografts across MHC barrier

In this study, we investigated the effects of 7-day-protocols of alphabeta-T-cell receptor monoclonal antibody (alphabeta-TCRmAb), cyclosporine A (CsA), and tacrolimus (FK-506) immunosuppressive monotherapies, and their combinations on the survival of vascularized skin allografts (VSA). Forty-two transplantations of VSA across a strong MHC barrier were performed between ACI (RT1a) donors and Lewis (RT1(l)) recipients in seven groups. Isograft and allograft rejection controls received no treatment. Treatment groups received a 7-day protocol of alphabeta-TCRmAb, CsA, or FK-506 monotherapy, or a combination of alphabeta-TCRmAb/CsA and alphabeta-TCRmAb/FK-506. VSA transplants were evaluated on a daily basis. Donor-specific chimerism was determined by flow cytometry (FC). The combined protocols of alphabeta-TCRmAb/FK-506 and alphabeta-TCRmAb/CsA significantly prolonged VSA survivals compared to monotherapy groups ( P < 0.005). FC analysis revealed 15.82% of donor-specific chimerism on day 7 under the alphabeta-TCRmAb/CsA protocol and a gradual chimerism decline on day 63 posttransplant. The significant extension of VSA survival achieved under 7-day protocols of combined therapies was directly associated with the presence of donor-specific chimerism.

Facial tissue allograft transplantation

A hemifacial allograft transplant model was used to investigate the rationale for development of functional tolerance across an MHC barrier. Thirty hemiface transplantations were performed in five groups of six Lewis (RT1 1) rat recipients each. Isografts were performed in group 1. Transplants were obtained from semiallogenic LBN(RT1 1+n) in group 2 and from fully allogenic ACI(RT1 a) in group 3 donors, which served as allograft rejection controls. Group 4 grafts using LBN donors and group 5 using ACI donors in addition received CsA monotherapy (16 mg/kg/d for 1 week) and maintained at 2 mg/kg/d. Signs of graft rejection were sought daily. Isograft controls survived indefinitely. All nontreated allografts were rejected within 5 to 8 days posttransplant. Eighty-three percent of face-transplant recipients from LBN donors and 67% from ACI donors did not show any signs of rejection up to 270 days and 200 days, respectively. Flow cytometry at day 63 in LBN recipients showed the presence of donor-specific chimerism for MHC class I RT1 n antigens, namely 3.39% CD4/RT1 n; 1.01% CD8/RT1 n T-lymphocytes; and 3.54% CD45RA/RT1 n B-lymphocytes. In ACI recipients the chimerism test revealed 10.55% CD4/RT1 a and 4.59% of CD8/RT1 a T-lymphocytes. MLR assay at day 160 posttransplant revealed suppressed responses against LBN donor antigens in group 4, but moderate reactivity to ACI donor antigens in group 5. Functional tolerance toward hemifacial allograft transplants induced across MHC barrier using a CsA monotherapy protocol was associated with the presence of donor-specific chimerism in T- and B-cell subpopulations.

Tolerance Induction in Composite Facial Allograft Transplantation in the Rat Model

Clinical application of composite tissue allograft transplants opened discussion on the restoration of facial deformities by allotransplantation. The authors introduce a hemifacial allograft transplant model to investigate the rationale for the development of functional tolerance across the major histocompatibility complex barrier. Eighteen rats in three groups were studied. The composite hemifacial allotransplantations including the ear and scalp were performed between Lewis-Brown Norway (RT1l+n) and Lewis (RT1l) rats and isotransplantations were performed between Lewis rats. Isograft controls (n = 6) and allograft controls (n = 6) did not receive treatment. Allografts in treatment group (n = 6) were treated with cyclosporine A 16 mg/kg/day during the first week; this dose was tapered to 2 mg/kg/day over 4 weeks and maintained at this level thereafter. Functional tolerance to face allografts was evaluated clinically and histologically. Donor-specific chimerism was assessed at days 21 and 63 by flow cytometry. In vitro evaluation of donor-specific tolerance was performed by mixed lymphocyte reaction at day 160 after transplantation. Isograft controls survived indefinitely. All nontreated allografts were rejected within 5 to 7 days after transplantation, as confirmed by histopathologic analysis. Five of six face allografts under the cyclosporine A protocol showed no signs of rejection for up to 240 days and remained alive and under evaluation, whereas one animal showed signs of rejection at day 140. This was reversed by adjustment of the cyclosporine A dose. At day 21 after transplantation, flow cytometric analysis of the donor-specific chimerism showed 1.11 percent of double-positive CD4FITC/RT1Ac-Cy7 and 1.43 percent of double-positive CD8PE/RT1Ac-Cy7 T-cell populations in the peripheral blood of hemiface allotransplant recipients. The chimerism level of double-positive CD4FITC/RT1Ac-Cy7 T cells increased to 3.39 percent, whereas it remained stable for the double-positive CD8PE/RT1Ac-Cy7 T-cell population at day 63 after transplantation (1.00 percent). The mixed lymphocyte reaction assay at day 160 after transplantation revealed donor-specific tolerance to donor (Lewis-Brown Norway) antigens and strong reactivity to the third-party (ACI) alloantigens. In this study, donor-specific chimerism and functional tolerance were induced in hemifacial allograft transplants across the major histocompatibility complex barrier under cyclosporine A monotherapy protocol. This model will allow further studies on tolerance induction protocols.

FUNCTIONAL TOLERANCE IN FACIAL TISSUE ALLOGRAFT TRANSPLANTS

O133* Aims: We introduce a hemi-facial allograft transplant model to investigate the rationale for development of functional tolerance across MHC barrier. Materials: Thirty composite hemiface transplantations were performed in 5 groups of 6 rats each. Lewis (RT1l) rats served as recipients in all groups. Isograft transplants were performed in Group 1. Transplants from semi-allogenic LBN(RT1l+n) (Group 2) and fully-allogenic ACI(RT1a) (Group 3) donors served as allograft rejection controls. Group 4 (LBN donors) and Group 5 (ACI donors) received CsA monotherapy of 16 mg/kg/day for 1 week, tapered to 2 mg/kg/day over 4 weeks and continued at this dose level thereafter. Signs of graft rejection were evaluated daily. Flow cytometry evaluated donor specific chimerism for MHC class I RT1n and RT1a antigens at days 21 and 63 post-transplant. Mixed lymphocyte reaction (MLR) for donor specific tolerance was tested at day 160 post-transplant. Histological grading of graft rejection was evaluated after H-E staining. Results: Isograft controls survived indefinitely. All non-treated allografts were rejected within 5 to 9 days post-transplant. 83% of face-transplant recipients from LBN donors and 67% from ACI donors did not show any signs of rejection up to 270 days and 200 days respectively. The rejection signs were reversed by CsA dose adjustments in remaining allografts. Donor specific chimerism at day 63 in LBN recipients showed 3.39% CD4/RT1n, 1.01% CD8/RT1n T-lymphocytes and 3.54% CD45RA/RT1n B-lymphocytes; and in ACI recipients 10.55% CD4/RT1a and 4.59% CD8/RT1a T-lymphocytes. MLR assay at day 160 post-transplant revealed suppressed responses against LBN donor antigens in Group 4, but moderate reactivity to the ACI donor antigens in Group 5. Grade II rejection pattern was established in skin biopsies of rejecting LBN and ACI recipients at days 140 and 180 post-transplant. Conclusion: Functional tolerance was induced in hemifacial allograft transplants across MHC barrier under CsA monotherapy protocol. This was associated with the presence of donor specific chimerism in T- and B-cell subpopulations.

DIFFERENT ROUTES OF DONOR DERIVED HEMATOPOIETIC STEM CELLS TRANSPLANTATION FOR DONOR SPECIFIC CHIMERISM INDUCTION ACROSS MHC BARRIER

P940 Aims: Transplantation of donor–derived stem cells can improve organ allograft survival in animal models. This study was designed to investigate the effect of different routes of stem cell transplantation (SCT) on donor specific tolerance induction across MHC barrier under short-term CsA monotherapy and αβTCR/CsA treatment protocols. Methods: Forty-eight SCT were performed between BN (RT1n) donors and Lewis (RT1l) recipients. Intraosseous and intravenous SCT was studied in 6 groups of 8 animals each receiving 35x106 (n=4) and 70x106 (n=4) SCT. Groups I and II served as controls and received SCT but no treatment. Groups III and IV received CsA monotherapy for 7-days, Groups V and VI were under αβTCR/CsA protocol for 7-day. Flow cytometry analysis was used for evaluation of immunodepletion of T-lymphocytes and multilineage donor specific chimerism for MHC class-I (RT1n) for RT1n/CD4, RT1n/CD8 and RT1n/CD45RA antigens in peripheral blood of recipients at post-transplant days 7, 21, 35 and 63. Results: All animals survived without sign of graft versus host disease. At day 63 non-treatment groups receiving 70x106 SCT developed 4.3% of multilineage donor-specific chimerism following intraosseous transplantation and 1.0% following intravenous transplantation. At day 7 under CsA monotherapy and 70x106 SCT, chimerism level for 1.5% RT1n/CD4 was induced in intraosseous and 10.5% in intravenous transplanted groups. At day 63 following intraosseous transplantation chimerism for RT1n/CD4 was stable, but following intravenous transplantation significantly declined to 0.8%. Under αβTCR/CsA protocol T-lymphocyte subpopulation was significantly depleted at day 7 and repopulated to pre-transplant level at day 63. After intraosseous transplantation of 70x106 SCT at day 7 chimerism was 5.7% for RT1n/CD4, and increased up to 6.5% at day 63. In contrast in intravenously transplanted groups at day 7 chimerism was higher and showed 23.7% for RT1n/CD4 antigens, however declined to 0.9% at day 63. Multilineage chimerism was 75% higher in intraosseous (9.9%) when compared with intravenous (3.4%) transplant groups receiving 70x106 SCT at day 63. Following intraosseous SCT under αβTCR/CsA protocol, multilineage chimerism was 50% higher in group receiving 70x106 stem cells (9.9%) compared to 35x106 stem cells (4.9%). Conclusions: Stem cell transplantation under αβTCR/CsA protocol showed better engraftment of donor cells following intraosseous delivery leading to development of higher level (75%) of multilineage donor-specific chimerism when compared with intravenous transplantation. High level of chimerism in intravenously transplanted groups was transient, whereas intraosseous transplantation induced stable chimerism, which was maintained up to 63 days post-transplantation. This strategy of intraosseous stem cell transplantation under αβTCR/CsA protocol could be applied for chimerism and tolerance induction during transplantation of composite and solid organ allografts.

Development of donor‐specific chimerism and tolerance in composite tissue allografts under αβ‐T‐cell receptor monoclonal antibody and cyclosporine a treatment protocols

Recently, we induced donor-specific tolerance to rat hindlimb allografts under a 35-day course of alphabeta-T-cell receptor monoclonal antibody (alphabeta-TCR mAb) and cyclosporine A (CsA). In this report, we investigated the role of shorter alphabeta-TCR/CsA protocols on tolerance induction. We performed 52 hindlimb transplantations, between Lewis-Brown-Norway (LBN, F1) donors and Lewis recipients to test the impact of 21-, 7-, and 5-day protocols of combined alphabeta-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. All transplants under 5, 7, and 21 days of combined alphabeta-TCR/CsA therapy survived over 350 days. Clinical tolerance and immunocompetence were confirmed by skin grafting in vivo and MLR in vitro. Flow cytometry revealed a high level of donor chimerism in the peripheral blood of long-term survivors. The extension of survival of limb allografts and allo-unresponsiveness were directly associated with the development of stable chimerism in the tolerant recipients.

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

Recent studies have demonstrated that treatment with alphabeta-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 alphabeta-TCR-CsA protocol. 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 alphabeta-TCR and CsA or a combination of alphabeta-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. Long-term survival (>350 days) was achieved in allograft recipients (n=6) under the 7-day protocol of combined alphabeta-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 (AxC 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. Combined therapy of alphabeta-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.

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.