Transplantation of the bone marrow microenvironment leads to hematopoietic chimerism without cytoreductive conditioning

BACKGROUND: It has been hypothesized that regimens to induce transplantation tolerance and long-term hematopoietic chimerism require recipient conditioning with whole body irradiation or a cytoablative regimen to create space within the marrow microenvironment to permit pluripotent stem cell engraftment. The purpose of this study was to determine if transplantation of an intact bone marrow microenvironment in the form of a bone graft would permit stable hematopoietic stem cell engraftment, shape the repertoire of developing T cells, and induce donor-specific unresponsiveness in the absence of a conditioning regimen. METHODS: Fragments of femur were transplanted under the kidney capsule of recipient mice. At defined time points after bone graft transplantation recipients were assayed for chimerism, bone graft viability, and responses to donor and third party alloantigens in vitro and in vivo. RESULTS: In the absence of an immunological barrier, bone graft transplantation resulted in long-term multi-lineage hematopoietic chimerism in the peripheral blood. Nude bone graft transplantation into SCID recipients resulted in development of donor- derived T cells that underwent negative selection on bone graft derived I-E+ cells within the thymus. Across a fully allogeneic barrier in immunocompetent recipients treated with combined blockade of the CD40 and CD28 pathways bone graft transplantation resulted in long-term donor-specific hyporesponsiveness in vitro and acceptance of donor specific skin grafts. CONCLUSIONS: Transplantation of bone marrow in the form of a bone graft may facilitate the production of hematopoietic chimerism and lead to long-term donor-specific hyporesponsiveness in the absence of a cytoreductive conditioning regimen.     

Infusion of Mesenchymal Stem Cells and Rapamycin Synergize to Attenuate Alloimmune Responses and Promote Cardiac Allograft Tolerance

The inherent immunosuppressive properties and low immunogenicity of mesenchymal stems cells (MSCs) suggested their therapeutic potential in transplantation. We investigated whether MSCs could prolong allograft survival. Treatment involving infusion of MSCs into BALB/c recipients 24 hours after receiving a heart allograft from a C57BL/6 donor significantly abated rejection and doubled graft mean survival time compared to untreated recipients. Furthermore, combination therapy of MSCs and low-dose Rapamycin (Rapa) achieved long-term heart graft survival (>100 days) with normal histology. The treated recipients readily accepted donor skin grafts but rejected third-party skin grafts, indicating the establishment of tolerance. Tolerant recipients exhibited neither intragraft nor circulating antidonor antibodies, but demonstrated significantly high frequencies of both tolerogenic dendritic cells (Tol-DCs) and CD4⁺CD25⁺Foxp3⁺T cells in the spleens. Infusion of GFP⁺C57BL/6-MSCs in combination with Rapa revealed that the GFP-MSCs accumulated in the lymphoid organs and grafts of tolerant recipients. Thus, engraftment of infused MSCs within the recipient's lymphoid organs and allograft appeared to be instrumental in the induction of allograft-specific tolerance when administered in combination with a subtherapeutic dose of Rapamycin. This study supports the clinical applicability of MSCs in transplantation.     

Vascularized thymic lobe transplantation in a pig-to-baboon model: a novel strategy for xenogeneic tolerance induction and T-cell reconstitution

BACKGROUND: This laboratory has previously demonstrated the induction of allogeneic tolerance by vascularized thymic lobe (VTL) transplantation in miniature swine. We report here our initial attempt to induce tolerance by VTL transplantation in the clinically relevant, discordant, pig-to-baboon model of xenotransplantation. METHODS: Six baboons received xenografts of hDAF VTLs. Four of these baboons also received omental thymic tissue implants. All recipients were treated with an immunosuppressive conditioning regimen that included thymectomy, splenectomy, extracorporeal immunoadsorption of anti-alpha Gal antibodies, and T-cell depletion. Two control baboons received sham operations, of which one also received 5x10 hDAF porcine thymocytes/kg intravenously. RESULTS: Transplanted VTL grafts supported early thymopoiesis of recipient-type immature thymocytes, and facilitated engraftment of nonvascularized thymic omental implants. Recipients of the VTL grafts demonstrated donor-specific unresponsiveness in MLR assays, development of peripheral CD45RAhigh/CD4 double positive (DP) cells, and positive cytokeratin staining of thymic stroma in the grafts for 2 months following xenotransplantation. The control baboons did not show these markers of thymic reconstitution. The eventual return of Gal natural antibodies led to the destruction of graft epithelial cells and the rejection of all VTL grafts by 3 months posttransplantation. CONCLUSIONS: VTL transplantation from hDAF swine to baboons induced early thymopoiesis in the recipients and donor-specific cellular unresponsiveness in vitro. When coupled with additional strategies aimed at silencing humoral rejection, VTL transplantation may significantly prolong xenograft survival and result in long-term tolerance.     

Intragraft delivery of 16, 16-dimethyl PGE2 induces donor-specific tolerance in rat cardiac allograft recipients

The stable prostaglandin E2 analogue, 16,16-dimethyl PGE2 (di-M-PGE2) was continuously infused by osmotic pump directly into rat heterotopic cardiac allografts. Intragraft delivery of 20 micrograms/kg/day di-M-PGE2 for 2 weeks completely prevented graft rejection for more than 150 days (n = 10), while untreated Buffalo recipients rejected Lewis cardiac allografts within 8 days after transplantation (mean survival time = 7.4 +/- 0.5 days, n = 5). When given for only 1 week, 20 micrograms/kg/day had a partial effect, since 60% of recipients accepted grafts long-term and 40% experienced rejection by day 14 (n = 5). In contrast, systemic intravenous administration of 20 micrograms/kg/day di-M-PGE2 for 2 weeks could not prolong graft survival (MST = 7.0 +/- 0.0 days, n = 3), and the higher dose of 200 micrograms/kg/day resulted in death by day 2 (n = 5). Long-term BUF recipients of LEW cardiac allografts accepted LEW donor strain skin grafts for more than 35 days while rejecting third-party Wistar Furth skin grafts in a normal fashion (MST = 7.3 +/- 0.5 days, n = 3), indicating the induction of donor-specific tolerance. Long-surviving LEW cardiac allografts retransplanted into naive BUF recipients were rejected within 7 days (MST = 6.7 +/- 0.5 days, n = 3), indicating no change in graft immunogenicity. Therefore, a 14-day infusion of di-M-PGE2 directly into a strongly MHC-mismatched cardiac allograft uniformly has resulted in long-term engraftment and the development of recipient donor-specific tolerance.     

Mesenchymal Stem Cells Facilitate the Induction of Mixed Hematopoietic Chimerism and Islet Allograft Tolerance without GVHD in the Rat

Induction of hematopoietic chimerism and subsequent donor-specific immune tolerance via bone marrow transplantation is an ideal approach for islet transplantation to treat type-1 diabetes. We examined the potential of mesenchymal stem cells (MSCs) in the induction of chimerism and islet allograft tolerance without the incidence of graft-versus-host disease (GVHD). Streptozotocin-diabetic rats received a conditioning regimen consisting of antilymphocyte serum and 5 Gy total body irradiation, followed by an intraportal co-infusion of allogeneic MSCs, bone marrow cells (BMCs) and islets. Although all the recipients rejected the islets initially, half of them developed stable mixed chimerism and donor-specific immune tolerance, shown by the engraftment of donor skin and second-set islet transplants and acute rejection of a third-party skin. The engraftment of the primary islet allografts with stable chimerism was achieved by the addition of a 2-week peritransplant administration of 15-deoxyspergualin (DSG). Without MSCs, none of the recipients treated with DSG developed chimerism or reversal of diabetes. GVHD was not observed in any of the recipients infused with MSCs (0/15), whereas it occurred in 4/11 recipients without MSCs. These results indicate a potential use of MSCs for induction of hematopoietic chimerism and subsequent immune tolerance in clinical islet transplantation.     

Modified CD4(+) T-cell response in recipients of old cardiac allografts

With an increasing demand, organs from elderly donors are more frequently utilized for transplantation. Herein, we analyzed the impact of donor age on CD4(+) T-cell responses with regard to regulatory and effector mechanisms. Young (3months) BM12 recipients were engrafted with young or old (18months) B6 cardiac allografts. Systemic CD4(+) T-cell responses and intragraft changes were monitored and compared to age-matched syngenic transplant controls. While elderly, nonmanipulated hearts contained significantly elevated frequencies of donor-derived leukocytes prior to transplantation, allograft survival was age-independent. T-cell activation, however, was delayed and associated with a compromised immune response in mixed lymphocyte cultures (MLR; P=0.0002) early after transplantation (day 14). During the time course after transplantation, recipients of old grafts demonstrated an augmented immune response as shown by significantly higher frequencies of activated CD4(+) T-cells and a stronger in vitro alloreactivity (MLR; ELISPOT; P<0.01). In parallel, frequencies of regulatory T-cells had increased systemically and overall fewer CD4(+) T-cells were detected intragraft. Interestingly, changes in the CD4(+) T-cell response were not reflected by graft morphology. Of note, transplantation of young and old syngenic hearts did not show age-related differences of the CD4(+) T-cells response suggesting that old grafts can recover from a period of short cold ischemia time. Our data suggest that donor age is associated with an augmented CD4(+) T-cells response which did not affect graft survival in our model. These findings contribute to a better understanding of the immune response following the engraftment of older donor organs.     

Simultaneous blockade of co-stimulatory signals, CD28 and ICOS, induced a stable tolerance in rat heart transplantation

An inducible co-stimulator (ICOS), a recently identified co-stimulatory receptor with a close structural homology of CD28 and CTLA4, is expressed on activated T cells. Anti-ICOS antibody was demonstrated to be effective on prolongation of graft survival after liver transplantation in rats. In this study, we investigated the potency of tolerance induction using the antibody combined with a recombinant adenovirus vector containing CTLA-4Ig cDNA (AdCTLA-4Ig) in rat heart transplantation model. Using a DA-to-Lewis rat heart transplantation model, an anti-rat ICOS antibody and AdCTLA-4Ig were simultaneously administered i.v. into recipients. The tissue specimens from the grafts were removed on various days after transplantation for histological evaluation. Donor-strain skin and heart grafts, and third-party heart allografts were challenged in the recipients with a long-term surviving graft. Splenocytes from the tolerance-induced recipients were used for adoptive transfer study. Anti-ICOS antibody alone did not prolong the survival of heart allograft. AdCTLA-4Ig monotherapy significantly prolonged the survival of heart allograft (Group 4). With a combination of Anti-ICOS antibody and AdCTLA-4Ig, all recipients were resulted in a long-term allograft acceptance for more than 200 days (Group 8). When challenged donor-strain skin grafts in the tolerant rats of Group 4, the skin was rejected, which also lead to a rejection of primary heart allografts. The recipients in Group 8 also rejected donor-strain skin grafts with no rejection of the primary heart grafts. These recipients accepted secondary heart grafts from donor-strain but not third-party. In Group 8 long-term survival recipients showed a high population of CD4+CD25+ regulatory T cell in peripheral blood, and in adoptive transfer study subtraction of these CD4+CD25+ T cells accelerate the rejection of heart graft in secondary irradiated recipients. The present results demonstrated that anti-ICOS antibody combined with AdCTLA-4Ig potently induces a stable immune tolerance after heart allografting in rat, which is mediated by the induction of CD4+CD25+ regulatory T cells. This strategy may be attractive for clinical employment to induce transplantation tolerance.     

Engineering of bone marrow cells with fas-ligand protein-enhances donor-specific tolerance to solid organs

Effective immunomodulation to induce tolerance to tissue/organ allografts is attained by infusion of donor lymphocytes endowed with killing capacity through ectopic expression of a short-lived Fas-ligand (FasL) protein. The same approach has proven effective in improving hematopoietic stem and progenitor cell engraftment. This study evaluates the possibility of substitution of immune cells for bone marrow cells (BMC) to induce FasL-mediated tolerance to solid organ grafts. Expression of FasL protein on BMC increased the survival of simultaneously grafted vascularized heterotopic cardiac grafts to 90%, as compared to 30% in recipients of naïve BMC. Similar results were obtained for skin allografts implanted into radiation chimeras at 1 week after bone marrow transplantation. Further reduction of preparative conditioning to busulfan resulted in acceptance of donor skin implanted at 2 weeks after transplantation of naïve and FasL-coated BMC, whereas third-party grafts were acutely rejected. The levels of donor chimerism were in the range of 0.7% to 12% at the time of skin grafting, with higher levels in recipients of FasL-coated BMC. It is concluded that FasL-mediated abrogation of alloimmune responses can be effectively attained with BMC. There is no threshold of donor chimerism, but tolerance to solid organs evolves during the process of donor-host mutual acceptance.     

Mixed chimerism achieved by a nonlethal conditioning regimen induces donor-specific tolerance to lung allografts

BACKGROUND: Graft rejection and toxicity associated with chronic immunosuppressive therapy remain a major problem in lung transplantation (Tx). Mixed hematopoietic chimerism has been shown to produce long-lasting donor-specific transplant tolerance without immunosuppressive drugs in animal models; however, most conditioning regimens required to achieve mixed chimerism are too toxic for clinical use. The aim of this study was to develop a nonlethal conditioning regimen to induce tolerance to lung allografts. METHODS: Four to 6-wk old ACI (RT1.A(a)) and Wistar Furth (RT1.A(u)) rats were used as organ donors and recipients, respectively. The recipient conditioning regimen included: 10 mg/animal antilymphocyte globulin (on day-5), 1 mg/kg/d tacrolimus (days 1 to 10), total body irradiation (500 cGy; day 0), and donor bone marrow (DBM) Tx (100 x 10(6) T-cell depleted cells on day 0 following irradiation). Six weeks after DBM Tx, chimeric animals received orthotopic left lung Tx. Graft survival was monitored by chest X-ray and histology. RESULTS: Long-term DBM engraftment was observed: hematopoietic chimerism in the peripheral blood was 12.4 +/- 3.4%, 36.7 +/- 14.1%, and 31.9 +/- 14.1% at 30 d, 6 mo, and 16 mo following DBM Tx, respectively. There was no graft versus host disease. Chimeric recipients (RT1.A(u)) permanently accepted (>400 d) donor-specific lungs (RT1.A(a); n = 8), yet rapidly rejected (<8 d) third party hearts (RT1.A(l); n = 5). Graft (lung) tolerant (>150 d) chimeric recipients accepted secondary donor-specific heart grafts (>150 d; n = 4) but rejected third party heart grafts (<7 d; n = 3). Graft tolerant recipients demonstrated reduced (P < 0.05) in vitro donor-specific lymphoproliferative response and cytotoxicity, and no evidence of acute or chronic graft rejection. CONCLUSION: Mixed chimerism achieved by a nonlethal conditioning regimen induced long-term donor-specific tolerance to lung allografts.     

Organ transplant specificity of tolerance to skin grafts with heart or kidney grafts plus nondepleting anti-CD4 monoclonal antibody (RIB 5/2) and intravenous donor alloantigen administration

BACKGROUND: CD4+ T cells play an essential role in allograft rejection. Monoclonal anti-rat CD4 antibody, RIB 5/2, has been shown to modulate the CD4 glycoprotein without eliminating recipient T cells. A single dose of monoclonal anti-rat CD4 antibody RIB 5/2 plus donor splenocytes results in donor-specific unresponsiveness to heart and kidney allografts, but not skin allografts. This study examined whether tolerance to the more resistant skin graft could also be achieved with RIB 5/2. METHODS: Buffalo (RT1(b)) recipients were given a single dose (20 mg/kg) of monoclonal antibody RIB 5/2 IP plus IV Lewis (RT1(l)) splenocytes (25 x 10(6)) 21 days before Lewis heart, kidney, or skin grafts. In addition, Lewis skin was grafted either simultaneously with or after long- term Lewis heart or kidney allograft acceptance (>50 days). RESULTS: While IV alloantigen plus RIB 5/2 results in long-term acceptance of both heart and kidney, skin allografts are rejected when transplanted alone. Simultaneous transplantation with a Lewis kidney, but not with a Lewis heart, resulted in long-term Lewis skin graft acceptance. However, recipients tolerant to Lewis kidney or heart alone will not accept subsequent Lewis skin grafts, while recipients of simultaneous Lewis skin and kidney grafts subsequently accept a second Lewis, but not third-party Brown Norway (RT1(n)), skin graft. CONCLUSION: RIB 5/2 plus Lewis donor splenocytes tolerize for donor-specific heart and kidney but not skin grafts. However, Lewis skin grafted simultaneously with a Lewis kidney, but not Lewis heart, is accepted and protects a subsequent donor-specific Lewis skin graft.     

Alpha beta TCR+ T cells play a nonredundant role in the rejection of heart allografts in mice.

BACKGROUND: Although the transplantation of solid organs and cellular grafts is a clinical routine, the morbidity and mortality associated with immunosuppression is significant. This could be avoided by the induction of donor-specific tolerance. To develop targeted antirejection strategies and regimens to induce donor-specific tolerance, cell populations in the recipient-mediating rejection of solid organ and cellular grafts must be defined. In this study we examined the role of alpha beta-TCR+ cells in the rejection of allogeneic heart grafts, by use of knockout (KO) mice deficient in the production of alpha beta-TCR+ T cells. METHODS: C57BL/6-TcrbtmlMom (alpha beta-KO) and C57BL6/J (B6) recipient mice were transplanted with B10.BR/SgSnJ (B10.BR) or BALB/c heart allografts. Animals also received bone marrow from normal B10.BR donors, followed by donor-specific or third-party heart transplants. RESULTS: Naive B6 control mice rejected B10.BR and BALB/c grafts within 16 days. In striking contrast, B10.BR and BALB/c heart allografts were indefinitely accepted in unmanipulated alpha beta-KO mice. The immune responsiveness was restored after bone marrow transplantation from normal donors. After bone marrow transplantation major histocompatibility-disparate BALB/c third-party heart grafts were rejected, whereas donor-specific grafts were still accepted. CONCLUSIONS: alpha beta-TCR+ T cells play a nonredundant role in the rejection of heart allografts in mice. Bone marrow chimerism is associated with donor-specific transplantation tolerance.     

Evidence of donor-specific cellular suppressor activity in donor-specific cell-mediated lympholysis unresponsiveness in renal transplant patients

Twenty patients with well-functioning kidney grafts from one-haplotype-mismatched related donors, were studied 1-10 years after transplantation (A). Another group of six patients were studied at various times after transplantation (B). The presence of donor-specific transplantation tolerance, using mixed lymphocyte culture (MLC) and cell-mediated lympholysis (CML) tests was investigated, as well as the possible existence of cells with suppressive activity. All recipients were transfused prior to transplantation and treated with conventional immunosuppression. The patients in group A showed MLC reactivity against donor and third-party cells, indicating a allogeneic response capacity. The CML activity against the donor was low, however, and remained low also following removal of adherent cells. The CML activity toward third-party cells was within the normal range of unmatched individuals. In group B, two of six recipients and high postoperative CML activity against the donor. Both recipients showed clinical signs of rejection. In the remaining four recipients, the antidonor CML reactivity one week after transplantation was lower than the preoperative level. The decrease was even more pronounced at 12 months, although the reactivity against third-party cells was unaltered. The CML reactivity from unrelated fourth-party individuals toward donors was suppressed when cells from recipients with long-term functioning kidneys were added to the cell cultures. The results suggest the presence of a donor-specific cellular suppressor mechanism underlying the donor-specific CML unresponsiveness in recipients with long-term-functioning kidney allografts.     

Long-term survival of donor-specific pancreatic islet xenografts in fully xenogeneic chimeras (WF rat----B10 mouse).

We recently reported that reconstitution of lethally irradiated B10 mouse recipients with 40 x 10(6) untreated WF rat bone marrow cells resulted in stable fully xenogeneic chimerism (WF rat----B10 mouse). In these animals, the tolerance induced for skin xenografts was highly MHC specific in that donor-specific WF rat skin grafts were significantly prolonged while MHC-disparate third-party xenografts were rapidly rejected (median survival time [MST] = 9 days). We have now examined whether islet cell xenografts placed under the renal capsule of chimeras rendered diabetic with streptozotocin would be accepted and remain functional to maintain euglycemia. Animals were prepared, typed for chimerism at 6 weeks, and diabetes induced with streptozotocin. Donor-specific WF (Rt1Au) islet cell xenografts were significantly prolonged (MST greater than 180 days) in WF----B10 chimeras, while MHC-disparate third-party F344 rat (Rt1A1) grafts were rejected with a time course similar to unmanipulated B10 mice (MST = 8 days). The transplanted donor-specific islet cells were functional to maintain euglycemia, since removal of the grafts at from 100 to 180 days in selected individual chimeras uniformly resulted in return of the diabetic state. These data suggest that donor-specific islet cell xenografts are accepted and remain functional in mice rendered tolerant to rat xenoantigens following bone marrow transplantation.     

Compromised kidney graft rejection response in Vervet monkeys after withdrawal of immunosuppressants tacrolimus and sirolimus

BACKGROUND: In nonprimates, organ allografts are often not rejected after withdrawal of immunosuppression. In this study, we examined whether such a phenomenon also occurs in primates. METHODS: Vervet monkeys were transplanted with renal allografts and treated for 60 days with tacrolimus, or tacrolimus plus sirolimus. The drugs were totally withdrawn on day 61. The survival of the monkeys was monitored, and their response to donor- or third party-derived alloantigens was examined in vivo and in vitro. RESULTS: The majority (80-100%) of the grafts survived for at least additional 30 days with no signs of acute rejection. The compromised rejection is donor-specific, because recipient monkeys failed to reject a donor-derived skin graft, but a third-party skin graft was rejected. In vitro mixed lymphocyte reaction and interleukin-2 production in the mixed lymphocyte reaction between the recipients and their donors or between the recipients and a third party had no discernable patterns, and thus did not reflect the in vivo status of the immune system. Although the recipients could not reject the graft acutely after drug withdrawal, the kidney grafts and the donor-derived skin grafts had pathological findings of chronic rejection. CONCLUSIONS: The rejection response of the monkeys to an established graft after withdrawal of immunosuppression is compromised. The compromised rejection is specific and is not due to a permanent alteration of the immune system by the initial drug treatment. The allografts are not inert but have low levels of interaction with the recipient immune system.     

CTLA4-Ig-based conditioning regimen to induce tolerance to cardiac allografts

BACKGROUND: Transplant rejection and toxicity associated with chronic immunosuppressive therapy remain a major problem. Mixed hematopoietic chimerism has been shown to produce tolerance to solid organ transplants. However, currently available protocols to induce mixed hematopoietic chimerism invariably require toxic pre-conditioning. In this study, we investigated a non-toxic CTLA4-Ig-based protocol to induce donor-specific tolerance to cardiac allografts in rats. METHODS: Fully mismatched, 4 to 6 week old ACI (RT1.A(a)) and Wistar Furth (RT1.A(u)) rats were used as cell/organ donors and recipients, respectively. Recipients were treated with CTLA4-Ig 2 mg/kg/day (on days 0, 2, 4, 6, 8), tacrolimus 1 mg/kg/day (daily, from days 0 to 9), and a single dose of anti-lymphocyte serum (10 mg) on day 10, soon after total body irradiation (300 cGy) and donor bone marrow (100 x 10(6) T-cell depleted cells) transplantation (BMT). Six weeks after BMT, chimeric animals received heterotopic heart transplants. RESULTS: Hematopoietic chimerism was 18.8 +/- 10.6% at day 30, and was stable (24 +/- 10%) at 1 year post-BMT; there was no graft versus host disease. Chimeric recipients (RT1.A(u)) permanently accepted (>360 days) donor-specific (RT1.A(a); n = 6) hearts, yet rapidly rejected (<9 days) third-party hearts (RT1.A(l); n = 5). Graft (heart) tolerant (>100 days) recipients accepted donor-specific secondary skin grafts (>200 days) while rejected the third-party skin grafts (<9 days). Lymphocytes of graft tolerant animals demonstrated hyporesponsiveness in mixed lymphocyte cultures in a donor-specific manner. Tolerant graft histology showed no obliterative arteriopathy or chronic rejection. CONCLUSIONS: The CTLA4-Ig based conditioning regimen with donor BMT produced mixed chimerism and induced donor- specific tolerance to cardiac allografts.     

Induction of carbon monoxide in donor animals prior to organ procurement reduces graft immunogenicity and inhibits chronic allograft dysfunction

BACKGROUND: Nonspecific inflammatory damages occurring prior to organ transplantation reduce long-term graft survival. Here, we tested the beneficial effects of carbon monoxide (CO) induction by methylene chloride (MC). METHODS: Fischer-344 (F-344 Rat) or Dark Agouti (DA Rat) donor animals were either treated with MC four hours prior to organ removal or remained untreated. Kidneys were transplanted into Lewis (LEW) recipients. The low responder strain combination (F-344-->LEW) was studied for long-term graft changes. Dendritic cells (DCs) migration and early changes were followed in additional groups of a high responding donor/recipient strain combination (DA-->LEW). Native kidneys of uninephrectomized, age-matched normal animals served as controls. RESULTS: Following MC application COHb peaked within two hours in donor animals. Renal function and morphology improved significantly in renal allografts of CO induced donor animals and were comparable to native controls long-term (24 wks). Early after transplantation (24 hr) donor-derived DCs, CD4+ T-cells and alloreactive T-cells were significantly reduced following the engraftment of organs from treated donors. In addition, a trend towards a Th1/Th2 shift and a significant intragraft reduction of CD3 mRNA expression was observed. CONCLUSION: Donor treatment for the induction of CO reduced graft immunogenicity and inhibited chronic allograft nephropathy.     

Costimulation blockade of both inducible costimulator and CD40 ligand induces dominant tolerance to islet allografts and prevents spontaneous autoimmune diabetes in the NOD mouse

Costimulation blockade is a promising strategy for preventing allograft rejection and inducing tolerance. Using a fully allogeneic mouse model, we tested the effectiveness of the combined blockade of the CD40 ligand and the inducible costimulator (ICOS) on islet allograft survival and in the prevention of autoimmune diabetes in the NOD mouse. Recipients treated with blocking monoclonal antibodies (mAbs) to ICOS and the CD40 ligand had significant prolongation of graft survival, with 26 of 28 functioning for >200 days. Long-term engrafted mice maintained antidonor proliferative and cytotoxic responses, but donor-specific immunization did not induce graft rejection, and challenge with second, same donor but not third-party grafts resulted in long-term acceptance. The immunohistology of tolerant grafts demonstrated the presence of CD4(+)CD25(+) T-cells expressing Foxp3, and islet/kidney composite grafts from tolerant mice, but not from mice lacking lymphocytes, were accepted indefinitely when transplanted into na?ve B6 mice, suggesting that recipient T-cells were necessary to generate dominant tolerance. Combined anti-ICOS and anti-CD40 ligand mAb therapy also prevented diabetes in NOD mice, with only 11% of treated recipients developing diabetes compared with 75% of controls. These data demonstrate that the blockade of CD40 ligand and ICOS signaling induces islet allograft tolerance involving a dominant mechanism associated with intragraft regulatory cells and prevents autoimmune diabetes in NOD mice.     

Assessment of peripheral tolerance in anti-CD4 treated C57BL/6 mouse heart transplants recipients.

The study was designed to compare second heart and skin grafts and in vitro assays as a means of assessing peripheral tolerance in C57BL/6 mice. Vascularized heterotopic BALB/c hearts were placed in C57BL/6 recipients treated with anti-CD4, GK1.5 (1 mg total per 20 g mouse i.p. on days 0, 1, 2, 3). Those mice in which hearts survived for >60 days were challenged with donor and third-party (CBA) skin grafts or with second heart grafts, of donor or third-party origin, attached to the carotid artery and jugular vein. In vitro alloreactivity was assessed by mixed lymphocyte reactions (MLR) and cell mediated lympholysis (CML) using recipient spleen cells. Parenchymal damage, cellular infiltration and vascular disease were assessed from the histology of long-term allografts and isografts. Allografts in untreated recipients were rapidly rejected while isografts survived > 100 days. Primary allografts in anti-CD4 treated recipients also survived > 100 days, as did donor strain secondary heart transplants given at >60 days after the first graft. Third-party hearts were rapidly rejected, as were donor and third-party skin grafts placed on recipients with long-term allografts. These recipients showed low MLR response to both donor and third-party stimulators and donor-specific suppression of CML at 60 days post graft. Long-surviving heart allografts all showed evidence of parenchymal damage and vascular intimal thickening. Thus in the BALB/c to C57BL/6 donor-recipient strain combination, hearts, but not skin grafts, could be used to demonstrate peripheral tolerance, which seemed to be both organ and major histocompatibility complex (MHC) specific. Despite long survival, BALB/c hearts all showed evidence of parenchymal damage and vascular intimal thickening, a sign of chronic rejection.     

Successful tolerance induction under CD40 ligation in a rodent small bowel transplant model: first report of a study with the novel antibody AH.F5

BACKGROUND: Intestinal transplantation has been hampered by high rates of intestinal allograft rejection. One mechanism of altering rejection in other organ transplant models has been blockade of second set T-cell costimulatory signals. AH.F5, a novel hamster anti-rat monoclonal antibody to CD154, blocks CD40-dependent T-cell costimulation. We hypothesized that blockade of this pathway might abrogate rejection in a rodent orthotopic survival model of intestinal transplantation. METHODS: Eight groups were studied with different dosing schema, including syngeneic transplants (group 1), untreated allogeneic transplants (group 2), allogeneic transplants plus multiple doses of AH.F5 alone given IV or s.c. (groups 3 and 4), allogeneic transplants plus donor splenocyte preconditioning with and without single dose AH.F5 (groups 5 and 6), and donor splenocyte preconditioning followed by multiple doses of AH.F5 with and without thymectomy (groups 7 and 8). RESULTS: Control animals all died within 12 days of transplantation, whereas antibody-alone and splenocytes-alone resulted in modest prolongation of survival to 16 days. Only animals treated with splenocytes before transplantation and AH.F5 survived long-term (>60 days, group 8). These animals tolerated donor-specific skin grafts, rejected third-party grafts, and fed normally. However, their weight gain was subnormal and they demonstrated intestinal muscular thickening, which might represent chronic rejection. Thymectomy prevented the induction of tolerance. CONCLUSIONS: AH.F5 prevents acute intestinal allograft rejection in combination with donor-specific splenocyte preconditioning. We achieved long-term survival and the animals appeared tolerant. Central conditioning is essential for success with this antibody when used alone. Further studies with different dosing regimens or second agents seem warranted.