Induction of kidney transplantation tolerance across major histocompatibility complex barriers by bone marrow transplantation in miniature swine

Previous studies from our laboratory have shown that permanent lymphohematopoietic chimerism can be induced in MHC-disparate miniature swine by bone marrow transplantation after lethal total-body irradiation. The purpose of the present study was to determine in this large animal model whether such chimerism would lead to permanent tolerance to a vascularized allograft without a requirement for exogenous immunosuppression. Eight miniature swine that had received MHC-mismatched BMT more than five months earlier underwent kidney transplantation (KTx) from a donor MHC matched (n = 5) or MHC mismatched (n = 3) with the BMT donor. All animals had regained in vitro responsiveness to third-party MHC antigens, as measured by mixed lymphocyte reaction (MLR), before KTx but remained nonresponsive to MHC antigens of the BMT donor and self. All three animals that received KTx mismatched for BMT donor MHC rejected promptly (mean survival time 7.0 days). Of the five animals that received KTx matched for BMT donor MHC, four showed no evidence of rejection and have functioning KTx greater than 200 days after KTx. The fifth animal had excellent renal function for 60 days but then developed a slowly rising BUN and serum creatinine, and died 75 days after KTx. The course of this animal's rejection is consistent with that previously described for rejection due to minor antigen disparities. The difference in survival of KTx matched or mismatched for the MHC of the BMT donor was statistically significant (P = 0.0062). The survival of KTx matched for the MHC of the BMT donor was significantly different from that of control animals without BMT receiving KTx mismatched for MHC (P = 0.0018). We therefore conclude that BMT is an effective means for induction of tolerance to an MHC mismatched KTx in this large animal model.     

New method for thyroid transplantation across major histocompatibility complex barriers using allogeneic bone marrow transplantation

BACKGROUND: It has been shown that allogeneic bone marrow transplantation (BMT) after lethal irradiation elicits donor-specific tolerance for organ or tissue transplantation across major histocompatibility complex (MHC) barriers. Recently, we have demonstrated that the portal venous (p.v.) administration of donor bone marrow cells (BMCs) elicits donor-specific tolerance across MHC barriers by only two administrations of an immunosuppressant (CsA or FK-506). In our study, using the central and intrahepatic tolerance-inducing system, we have established a new method for thyroid transplantation with BMT that would be more applicable to humans. METHODS: In addition to sublethal (6-5 Gy) irradiation, recipient B6 (H-2b) mice received injections i.p. with the myeloablative drug busulfan (BU) on day -2 to provide a sufficient "space" for the donor hematopoietic cells to expand in the recipients. To induce the intrahepatic tolerance, donor BALB/c (H-2d) BMCs were treated with neuraminidase (Neu), which enhances the trapping of i.v. injected BMCs in the liver. After the injection of Neu-treated BMCs, the thyroid organs from the BALB/c mice were engrafted under the renal capsules. RESULTS: A 90% graft survival rate was obtained over 100 days by a combination of BU administration, 6 Gy irradiation, and i.v. injection of Neu-treated BMCs [BU+6 Gy+(Neu) i.v.], and a 70% graft survival rate was obtained by [BU+5 Gy+(Neu) i.v.]. However, the graft survival rate significantly decreased when either the BU or Neu treatment was omitted. T cells collected from the tolerant recipients suppressed the proliferative responses to donor alloantigens. CONCLUSIONS: Using both BU and Neu treatments, we have succeeded in inducing long-term tolerance and preventing the rejection of thyroid allografts by the single-day protocol.     

Posttransplant administration of allochimeric major histocompatibility complex class-I-molecules induces true transplantation tolerance

BACKGROUND: Allochimeric class-I major histocompatibility complex (MHC) molecules that contain donor-type immunogenic epitopes displayed on recipient-type sequences were shown to induce transplantation tolerance when administered at the time of transplantation. Here, we investigated the ability of posttransplant allochimeric administration to induce tolerance and concomitantly inhibit chronic rejection. METHODS: Allochimeric (alpha1h(1/u))-RT1.Aa class-I MHC antigenic extracts were administered by way of the portal vein into ACI recipients of Wistar-Firth (WF) hearts at days +3, +7, and +10 posttransplantation in conjunction with subtherapeutic oral cyclosporine. RESULTS: Delayed posttransplant allochimeric administration induced donor-specific transplantation tolerance to rat cardiac allografts. In contrast, delayed delivery of unaltered donor- or recipient-type MHC extracts failed to prolong allograft survival. In addition, histopathologic examination or estimation of transplant vascular sclerosis by neointimal index assessment, following delayed allochimeric therapy, revealed intact global architecture and minimal intimal thickening, respectively. CONCLUSION: Allochimeric MHC class-I therapy is a unique and novel clinically applicable approach for induction of "true" transplantation tolerance where chronic rejection is concomitantly abrogated.     

Induction of transplantation tolerance by intraportal injection of allogeneic bone marrow cells: Possible implications for intrauterine bone marrow transplantation across major histocompatibility barriers

Abstract. Intraportal inoculation of C57BL/6 marrow cells into sublethally (400 rad) irradiated BALB/c recipients resulted in durable chimerism and the permanent acceptance of C57BL/6 skin allografts. Sublethally irradiated recipients of a similar number of marrow cells inoculated systemically did not develop chimerism or any significant prolongation of the survival of C57BL/6 skin allografts. Consequently, lethal graft-versus-host disease developed only in recipients of intraportal marrow allografts (80%). The intraportal injection of allogeneic C57BL/6 marrow cells into nonirradiated recipients resulted in significant, although not permanent, prolongation of skin allograft survival without durable chimerism, suggesting that the introduction of alloantigens intraportally may favor the induction of nonresponsiveness to alloantigens even across strong major histocompatibility barriers. The relevance of these findings is discussed regarding the intraportal inoculation of allogeneic bone marrow cells for the treatment of genetic disorders in utero through the induction of neonatal tolerance.     

Cardiac allograft survival across major histocompatibility complex barriers in the rhesus monkey following T lymphocyte-depleted autologous marrow transplantation. III. Late allograft rejection

We have studied organ allograft survival in rhesus monkeys conditioned with myeloablative total-body irradiation and T cell-depleted autologous bone marrow transplantation then given a heterotopic MHC-mismatched cardiac allograft in the immediate postmyeloablative period. This model has enabled us to investigate the role of T cells in vascularized organ allograft rejection. We previously reported (1) that recipients of marrow depleted of T cells below a critical threshold (0.16% residual marrow T cells, or 0.14 x 10(5) infused T cells/kg) experienced a period of freedom from acute rejection associated with a profound nonspecific immune deficiency (determined by skin grafting). Resolution of the nonspecific immune deficiency was associated with late graft rejection. In the present report, we correlate the results of peripheral immune reconstitution studies and direct immunohistochemical analysis with allograft status in order to study T cell subsets involved in late rejection. We report that, in contrast with CD8+/CD28- T cells, CD16+ NK cells, and CD20+ B cells, late allograft rejection was associated with the return of peripheral CD4+ T cells and CD8+/CD28+ T cells, suggesting a critical role for one or both of these subsets in late allograft rejection in this model.     

Influence of endotoxin on graft-versus-host disease after bone marrow transplantation across major histocompatibility barriers in mice

Much clinical and experimental data suggest that infection and graft-versus-host disease (GVHD) are intimately associated, and that bacterial endotoxin (ET), a potent immunostimulant, influences the severity of GVHD. We have used a cell-wall-deficient mutant of Escherichia coli (E coli J5) to study the effect of active and passive immunization against ET in a murine model of GVHD induced by major histocompatibility antigens. CBA/Ca (H-2k) mice were irradiated and grafted with 1 X 10(7) bone marrow cells from C57BL/B6 (H-2b) donors. Groups of mice were immunized against J5: either actively immunized with killed J5 cells or pure J5 lipopolysaccharide, or passively immunized with rabbit anti-J5 antiserum (R alpha J5). Controls included irradiation controls, negative controls (syngeneic graft), positive controls (conventional mice receiving allogeneic graft), mice immunized with normal rabbit serum, Freund's adjuvant (FA), or human serum albumin (HSA) in FA. Active immunization with J5 exacerbated the effects of GVHD as indicated by increased weight loss (P = 0.002) and earlier death (P = 0.043). In contrast, immunization with HSA protected against weight loss (P = 0.028), and improved survival (P = 0.008). Passive immunization with J5 had no effect. These observations support the hypothesis that ET influences the pathogenesis of GVHD, and provide a useful model for studying the effects of ET in a well-defined immunological system.     

Cardiac allograft survival across major histocompatibility complex barriers in the rhesus monkey following T lymphocyte-depleted autologous marrow transplantation. IV. Immune reconstitution

Studies of postmyeloablative immune reconstitution have been reported for allogeneic bone marrow transplantation and also for non-T cell-depleted autologous/syngeneic BMT. However, there is a paucity of information regarding immune recovery following T cell-depleted autologous/syngeneic BMT. We have developed a primate transplantation tolerance model in which rhesus monkeys were conditioned with total-body irradiation and extensively T cell-depleted autologous BMT and given a major histocompatibility complex-mismatched heterotopic cardiac allograft. This model provided an opportunity to study peripheral immune recovery following T cell-depleted autologous BMT. Limiting dilution analysis was used to quantify marrow T cells following depletion (2.8% to 25.6% marrow T cells predepletion, 0.00014% to 0.036% residual marrow T cells postdepletion). We found that (1) hematopoietic engraftment was prompt despite extensive marrow T cell depletion, (2) reconstitution of CD4+ helper T cells and CD8+ cytotoxic T cells were substantially delayed (6-12 months) compared with the recovery of CD8+ suppressor T cells, CD16+ NK cells, and CD20+ B cells, (3) distinction between CD8+ cytotoxic T cells and CD8+ suppressor T cells by the CD28 marker was critical in revealing the markedly discrepant recoveries of those subsets, and (4) immune reconstitution resembled that observed in recipients of T cell-depleted allogeneic and non-T cell-depleted autologous/syngeneic BMT, suggesting that the pattern of immune recovery following BMT is not substantially influenced by either allogeneic effects or the number of transferred T cells over a range of values.     

Presentation of donor major histocompatibility complex antigens by bone marrow dendritic cell-derived exosomes modulates allograft rejection

BACKGROUND: Dendritic cells secrete a population of "antigen-presenting vesicles," called exosomes, expressing functional class I and II major histocompatibility complex (MHC) and co-stimulatory molecules. The subcutaneous administration of syngeneic exosomes expressing tumor antigens has been shown to induce specific antitumor immune responses in vivo. The authors hypothesized that antigen presentation by exosomes, depending on the context of their administration, may induce tolerance rather than immunity. METHODS: The authors therefore tested the capacity of exosomes derived from donor bone marrow dendritic cells, given before transplantation, to modulate heart allograft rejection. RESULTS: The authors show here that donor type but not syngeneic exosomes induced a significant prolongation of allograft survival, with a few recipients having long-term graft survival. During the first week after transplantation, allografts from exosome-treated rats displayed a significant decrease in graft-infiltrating leukocytes and in the expression of interferon-gamma mRNA compared with allografts from untreated animals. Moreover, when tested in vitro, spleen CD4+ T cells from exosome-treated recipients displayed a significant decrease in anti-donor responses, suggesting a decrease in anti-donor T-cell responses. However, the authors also found that allogeneic donor-derived exosomes increased anti-donor MHC class II alloantibody production. CONCLUSIONS: The authors demonstrate an effect of allogeneic exosomes on the modulation of immune responses in vivo, suggesting that, like donor cells, exosomes can stimulate or regulate antigen-specific immune responses.     

Cardiac allograft survival across major histocompatibility complex barriers in the rhesus monkey following T lymphocyte-depleted autologous marrow transplantation. II. Prolonged allograft survival with extensive marrow T cell depletion

In the present study, we tested the possibility that a vascularized allograft might induce immunological tolerance in a myeloablated host, similar to the tolerance induced by allogeneic bone marrow grafts. To this end, we developed a rhesus monkey model consisting of myeloablative total-body irradiation and T cell-depleted autologous marrow transplantation followed by MHC-mismatched heterotopic cardiac allograft implantation. Limiting dilution analysis was used to quantify residual marrow T cells following depletion. We found that (1) allograft survival was substantially prolonged in the absence of immunosuppressive drugs (median survival = 160 days) over that seen in controls treated identically but receiving non-T cell-depleted marrow (median survival = 14 days); (2) there was a correlation between allograft survival prolongation and the extent of marrow T cell depletion, with a maximum survival of 329 days associated with a residual marrow T cell content of 0.00014%; (3) nonspecific immune deficiency--and, possibly, specific unresponsiveness of limited duration (determined by cryopreserved donor and third-party skin grafting)--contributed to the rejection-free period seen in recipients of extensively depleted marrow; (4) late allograft rejection occurred in 3 of 3 long-term survivors, thereby demonstrating that permanent tolerance was not induced by the allograft across MHC barriers; and (5) as few as 1.4 x 10(4) infused marrow T cells/kg were sufficient to mediate acute allograft rejection, a threshold approximately 10-fold lower than that reported for the induction of acute graft-versus-host disease following allogeneic bone marrow transplantation.     

Applications of bilateral vascularized femoral bone marrow transplantation for chimerism induction across the major histocompatibility (MHC) barrier: part II

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.     

Experimental cardiac allograft survival across major histocompatibility complex barriers in the rhesus monkey following T lymphocyte-depleted autologous marrow transplantation. I. In vitro T lymphocyte depletion studies

We have developed a rhesus monkey model consisting of myeloablative total-body irradiation and T lymphocyte-depleted autologous bone marrow transplantation followed by MHC-mismatched heterotopic cardiac allograft implantation that has provided an opportunity to study the role of marrow T cells in cardiac allograft rejection. In order to assess quantitatively the effects of low numbers of residual marrow T cells following depletion, methods to deplete rhesus marrow extensively and to detect residual T cells following depletion at levels below the sensitivity of standard assays have been developed. A rhesus marrow limiting dilution assay has been developed that quantifies less than 1 T cell in 10(5) marrow cells and is superior to traditional detection methods by at least 3 logs. In a direct comparison of four T cell depletion methods, effective depletion has been achieved with complement-mediated cytotoxicity (C'MC), erythrocyte rosetting, and counterflow centrifugal elutriation (CCE), the latter with a simplified single-flow rate protocol. Median marrow T cell depletions of 2.1, 1.1, and 3.1 logs, and total nucleated cell losses of 40%, 61%, and 42% respectively, have been observed. A reported use of ricin A-chain-like toxins for the enhancement of C'MC was of low efficacy with rhesus peripheral blood T cell targets. CCE followed by C'MC has resulted in a median 4.8 logs depletion with residual marrow T cell contents less than 0.001%. Thus, C'MC, E-rosetting, and particularly CCE are effective methods of T cell depletion--and, when used in combination, extensively eliminate marrow T cells. A rhesus marrow limiting dilution assay detects residual T cells at these low levels. These techniques provide a basis for the quantitative study of the role of T cells in organ graft rejection following T lymphocyte-depleted autologous marrow transplantation.     

Pituitary transplantation: cyclosporine enables transplantation across a minor histocompatibility barrier

Pituitary glands from neonatal donors were transplanted to the median eminence of hypophysectomized adult rats. Rats with transplants were then treated for 2 weeks with the immunosuppressive drug cyclosporine. For 5 weeks thereafter, blood was drawn at regular intervals for determination of serum thyroxine, prolactin, and luteinizing hormone. Cyclosporine-treated recipients of grafts with minor histocompatibility differences had normal levels of thyroxine and prolactin, whereas untreated animals did not. In addition, the treated animals responded to oophorectomy with a marked elevation in serum luteinizing hormone. This evidence indicates that cyclosporine enables successful transplantation across a minor histocompatibility barrier. It also suggests that these grafts interact with the hypothalamus. Transplantation across a major histocompatibility barrier was unsuccessful even in the presence of cyclosporine.     

Stable mixed hematopoietic chimerism permits tolerance of vascularized composite allografts across a full major histocompatibility mismatch in swine

This study tested the hypothesis that vascularized composite allografts (VCA) could be accepted in a robust model of hematopoietic chimerism by injecting allogeneic bone marrow cells (BMC) into swine fetuses. Outbred Yorkshire sows and boars were screened to ensure the absence of the major histocompatibility (MHC) allele SLA^cc of inbred MGH miniature swine and then mated. Bone marrow harvested from an SLA^cc swine donor was T‐cell depleted and injected intravenously into the fetuses between days 50–55 of gestation. After birth, the piglets were studied with flow cytometry to detect donor cells and mixed lymphocyte reactions (MLR) and cell‐mediated lympholysis (CML) assays to assess their response to donor. Donor‐matched VCAs from SLA^cc donors were performed on four chimeric and two nonchimeric swine. The results showed donor cell engraftment and multilineage macrochimerism after the in utero transplantation of adult BMC, and chimeric animals were unresponsive to donor antigens in vitro. Both control VCAs were rejected by 21 days and were alloreactive. Chimeric animals accepted the VCAs and never developed antidonor antibodies or alloreactivity to donor. These results confirm that the intravascular, in utero transplantation of adult BMC leads to donor cell chimerism and donor‐specific tolerance of VCAs across a full MHC barrier in this animal model.     

骨髓移植诱导特异性免疫耐受同种皮肤移植的动物实验研究

目的　证实通过动物实验模型的骨髓移植可以诱导同种皮肤移植的免疫耐受。方法　将 114只日本白色家兔和Dutch家兔分为对照组和实验组 ,日本白色家兔作为供体 ,Dutch家兔作为受体。对照组 ,在不使用免疫抑制剂的情况下 ,将 12只日本白色家兔与 12只Dutch家兔行相同面积的背部全厚皮肤互换移植 ,观察其成活时间。实验组 ,将 4 5只日本白色家兔和 4 5只Dutch家兔行全厚皮肤移植的同时行骨髓移植 ,然后将作为受体的Dutch家兔分为A ,B ,C ,D四组 ,分别行非致死量的γ射线全身照射的骨髓细胞移植及同种皮肤移植 ,观察移植皮肤的成活时间。结果　对照组 ,供体与受体移植皮肤的平均成活时间分别为 (12 .0± 1.7)天和 (10 .3± 1.3)天。实验组 ,A ,B ,C ,D四组移植皮肤的平均成活时间分别为 (6 1.0± 7.2 )、(80 .7± 10 .4 )、(78.8± 12 .7)、(88.0± 6 .0 )天。结论　通过骨髓移植导特异性免疫耐受同种皮肤移植的动物实验 ,旨在为临床应用提供了理论基础及可靠依据 ,为同种组织重建提供一个新方法     

骨髓移植诱导特异性免疫耐受同种皮肤移植的动物实验研究

目的证实通过动物实验模型的骨髓移植可以诱导同种皮肤移植的免疫耐受.方法将114只日本白色家兔和Dutch家兔分为对照组和实验组,日本白色家兔作为供体,Dutch家兔作为受体.对照组,在不使用免疫抑制剂的情况下,将12只日本白色家兔与12只Dutch家兔行相同面积的背部全厚皮肤互换移植,观察其成活时间.实验组,将45只日本白色家兔和45只Dutch家兔行全厚皮肤移植的同时行骨髓移植,然后将作为受体的Dutch家兔分为A,B,C,D四组,分别行非致死量的γ射线全身照射的骨髓细胞移植及同种皮肤移植,观察移植皮肤的成活时间.结果对照组,供体与受体移植皮肤的平均成活时间分别为(12.0±1.7)天和(10.3±1.3)天.实验组,A,B,C,D四组移植皮肤的平均成活时间分别为(61.0± 7.2)、(80.7± 10.4)、(78.8± 12.7)、(88.0± 6.0)天.结论通过骨髓移植导特异性免疫耐受同种皮肤移植的动物实验,旨在为临床应用提供了理论基础及可靠依据,为同种组织重建提供一个新方法.     

骨髓移植诱导免疫耐受家兔全耳同种移植

目的为了减轻免疫诱导过程中对受体的侵袭,进一步探讨以家兔为动物模型,通过异体骨髓移植诱导同种皮肤移植免疫耐受.方法通过骨髓腔内直接注射异体骨髓细胞,进行骨髓移植.结果皮片的平均存活时间是(88.0±6.0)天(P<0.001).为了探讨其普遍性,我们用此诱导方法,还进行了同种全耳移植.平均存活时间是(146.0±15.1)天,并有1只移植耳存活时间已超过1年,未见任何排斥反应.结论本研究证明了以家兔为动物模型,在不使用免疫抑制剂的情况下,通过骨髓腔内直接注射异体骨髓细胞,进行骨髓移植的方法,不仅在皮肤移植上可以诱导长期稳定的特异性免疫耐受,而且在实质性器官耳的同种移植上取得了良好的效果.     

骨髓移植诱导免疫耐受家兔全耳同种移植

目的　为了减轻免疫诱导过程中对受体的侵袭 ,进一步探讨以家兔为动物模型 ,通过异体骨髓移植诱导同种皮肤移植免疫耐受。方法　通过骨髓腔内直接注射异体骨髓细胞 ,进行骨髓移植。结果　皮片的平均存活时间是 (88.0± 6 .0 )天 (P <0 .0 0 1 )。为了探讨其普遍性 ,我们用此诱导方法 ,还进行了同种全耳移植。平均存活时间是 (1 4 6 .0± 1 5 .1 )天 ,并有 1只移植耳存活时间已超过 1年 ,未见任何排斥反应。结论　本研究证明了以家兔为动物模型 ,在不使用免疫抑制剂的情况下 ,通过骨髓腔内直接注射异体骨髓细胞 ,进行骨髓移植的方法 ,不仅在皮肤移植上可以诱导长期稳定的特异性免疫耐受 ,而且在实质性器官耳的同种移植上取得了良好的效果