Hematopoietic chimerism and central tolerance created by peripheral-tolerance induction without myeloablative conditioning

Allogeneic hematopoietic chimerism leading to central tolerance has significant therapeutic potential. Realization of that potential has been impeded by the need for myeloablative conditioning of the host and development of graft-versus-host disease (GVHD). To surmount these impediments, we have adapted a costimulation blockade-based protocol developed for solid organ transplantation for use in stem cell transplantation. The protocol combines donor-specific transfusion (DST) with anti-CD154 mAb. When applied to stem cell transplantation, administration of DST, anti-CD154 mAb, and allogeneic bone marrow leads to hematopoietic chimerism and central tolerance with no myeloablation and no GVHD. Tolerance in this system results from deletion of both peripheral host alloreactive CD8+ T cells and nascent intrathymic alloreactive CD8+ T cells. In the absence of large numbers of host alloreactive CD8+ T cells, the transfusion that precedes transplantation need not be of donor origin, suggesting that both allospecific and non-allospecific mechanisms regulate engraftment. Agents that interfere with peripheral transplantation tolerance impair establishment of chimerism. We conclude that robust allogeneic hematopoietic chimerism and central tolerance can be established in the absence of host myeloablative conditioning using a peripheral transplantation tolerance protocol.     

Mixed chimerism and permanent specific transplantation tolerance induced by a nonlethal preparative regimen

The use of allogeneic bone marrow transplantation as a means of inducing donor-specific tolerance across MHC barriers could provide an immunologically specific conditioning regimen for organ transplantation. However, a major limitation to this approach is the toxicity of whole body irradiation as currently used to abrogate host resistance and permit marrow engraftment. The present study describes methodology for abrogating host resistance and permitting marrow engraftment without lethal irradiation. Our preparative protocol involves administration of anti-CD4 and anti-CD8 mAbs in vivo, 300-rad WBI, 700-rad thymic irradiation, and unmanipulated fully MHC-disparate bone marrow. B10 mice prepared by this regimen developed stable mixed lymphohematopoetic chimerism without any clinical evidence of graft-vs.-host disease. Engraftment was accompanied by induction of specific tolerance to donor skin grafts (B10.D2), while third-party skin grafts (B10.BR) were promptly rejected. Mice treated with the complete regimen without bone marrow transplantation appeared healthy and enjoyed long-term survival. This study therefore demonstrates that stable mixed chimerism with donor-specific tolerance can be induced across an MHC barrier after a nonlethal preparative regimen, without clinical GVHD and without the risk of aplasia.     

In vivo selection and chemoprotection after drug resistance gene therapy in a nonmyeloablative allogeneic transplantation setting in dogs

We have previously demonstrated successful in vivo selection, chemoprotection, and modulation of donor chimerism in dogs that received myeloablative allogeneic stem cell transplantation with cells expressing the P140K mutant of the DNA repair enzyme methylguanine methyltransferase (MGMTP140K). Here, we wished to investigate whether in vivo selection, chemoprotection, and modulation of donor chimerism could also be achieved after nonmyeloablative transplantation, which could allow for less toxic transplantation regimens for patients with malignant and genetic diseases. Three dogs received a nonmyeloablative conditioning regimen and infusion of allogeneic stem cells transduced with MGMTP140K. All three dogs had stable gene marking and donor chimerism before receiving a course of O(6) -benzylguanine (O(6) BG)/1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) between days 210 and 589 after transplantation. One to four doses led to a marked increase in gene marking in all dogs. Furthermore, the transduced cells conferred chemoprotection and prevented severe neutropenia. Our results suggest that drug resistance gene therapy is feasible and safe in the nonmyeloablative transplantation setting.     

小鼠H-2半相合非清髓同种异基因造血干细胞移植模型的建立及其相关研究

为了探讨小鼠主要组织相容性抗原 (H 2 )半相合非清髓移植中预处理方案的可行性、移植后的造血重建、嵌合体水平及GVHD的发生情况 ,以CB6F1小鼠为受鼠 ,分为 3组 ,移植前 5天开始给予预处理 ,A组给予清髓(10 .5Gy)预处理方案 ,B组给予全身照射 (2Gy) Ara C Cy ,C组为全身照射 (2Gy) Ara C Cy Flu ,对所有受鼠均未进行GVHD防治。所有受鼠在第 0天经尾静脉注射C5 7BL 6小鼠混合细胞悬液 (2× 10 7骨髓细胞 1×10 7脾细胞 ) ,然后观察其造血恢复、植入及移植物抗宿主病 (GVHD)的情况。结果表明 :A组植入结果始终为完全供者型嵌合体 ,B和C组则为混合型或完全供者型嵌合体 ,其中B组混合嵌合体保持在 80 %以下 ,且在移植 5 0天以后有下降趋势 ,而C组嵌合体水平保持在 80 %以上 ,其嵌合接近或为完全供者型 ,移植 5 0天以后仍保持稳定。由于没有给予GVHD防治措施 ,各组均发生不同程度的GVHD ,其中A组受鼠GVHD的发生率和死亡率明显高于B和C两组 (P≤ 0 .0 1) ,但B和C两组之间无显著差异。结论 :以氟达拉宾为主的非清髓预处理方案 ,能够在受者体内形成稳定且持久的植入 ,并通过在受者体内形成混合性嵌合体 ,诱导供体对受体的特异免疫耐受 ,减少或避免GVHD的发生。     

Mixed chimerism induced without lethal conditioning prevents T cell– and anti-Galα1,3Gal–mediated graft rejection

Gal alpha 1,3Gal-reactive (Gal-reactive) antibodies are a major impediment to pig-to-human xenotransplantation. We investigated the potential to induce tolerance of anti-Gal-producing cells and prevent rejection of vascularized grafts in the combination of alpha 1,3-galactosyltransferase wild-type (GalT(+/+)) and deficient (GalT(-/-)) mice. Allogeneic (H-2 mismatched) GalT(+/+) bone marrow transplantation (BMT) to GalT(-/-) mice conditioned with a nonmyeloablative regimen, consisting of depleting CD4 and CD8 mAb's and 3 Gy whole-body irradiation and 7 Gy thymic irradiation, led to lasting multilineage H-2(bxd) GalT(+/+) + H-2(d) GalT(-/-) mixed chimerism. Induction of mixed chimerism was associated with a rapid reduction of serum anti-Gal naturally occurring antibody levels. Anti-Gal-producing cells were undetectable by 2 weeks after BMT, suggesting that anti-Gal-producing cells preexisting at the time of BMT are rapidly tolerized. Even after immunization with Gal-bearing xenogeneic cells, mixed chimeras were devoid of anti-Gal-producing cells and permanently accepted donor-type GalT(+/+) heart grafts (>150 days), whereas non-BMT control animals rejected these hearts within 1-7 days. B cells bearing receptors for Gal were completely absent from the spleens of mixed chimeras, suggesting that clonal deletion and/or receptor editing may maintain B-cell tolerance to Gal. These findings demonstrate the principle that induction of mixed hematopoietic chimerism with a potentially relevant nonmyeloablative regimen can simultaneously lead to tolerance among both T cells and Gal-reactive B cells, thus preventing vascularized xenograft rejection.     

阻断共刺激信号途径对造血干细胞移植后致敏受者免疫耐受排斥的影响简

本研究旨在应用CTLA4Ig及anti-CD154分别阻断致敏小鼠的B7/CD28及CD40/CD154共刺激信号途径,探讨其对异基因造血干细胞（HSC）植入的影响,为临床应用异基因造血干细胞移植（HSCT）治疗致敏受者免疫排斥提供实验基础.实验将BALB/c小鼠分为4组：①移植前7d致敏组;②移植前7d致敏同时应用CTLA4Ig＋anti-CD154组;③正常小鼠应用CTLA4Ig及anti-CD154的鼠源性对照抗体;④正常小鼠空白对照组.每组15只,于移植当天给小鼠8 Gy照射,然后将荧光标记的C57BL/6小鼠骨髓干细胞经尾静脉注射小鼠体内进行移植,于不同时间点取血或器官组织用流式细胞术检测荧光细胞归巢,同时记录生存状况,监测其造血重建水平.结果表明,与致敏鼠相比,CTLA4Ig＋ anti-CD154能促进异基因HSC在致敏受者体内植入,诱导免疫耐受,延长其生存期并在28 d内完成造血重建.结论：应用CTLA4Ig及anti-CDl5能诱导致敏受者对异基因HSC的免疫耐受,促进其植入,并完成造血重建.     

Specific tolerance induction across a xenogeneic barrier: production of mixed rat/mouse lymphohematopoietic chimeras using a nonlethal preparative regimen

The development of safe methods for inducing donor-specific tolerance across xenogeneic barriers could potentially relieve the critical shortage of allograft donors that currently limits the applicability of organ transplantation. We report here that such tolerance can be induced in a xenogeneic combination (rat----mouse) using a nonmyeloablative and nonlethal preparative regimen. Successful induction of chimerism and donor-specific transplantation tolerance required pretreatment of recipients with monoclonal antibodies (mAbs) against NK1.1, Thy-1.2, CD4 and CD8, followed by administration of 3 Gy whole body radiation (WBI), 7 Gy thymic irradiation, and infusion of T cell-depleted rat bone marrow cells (BMC). Rat cells appeared among peripheral blood lymphocytes (PBL) of such recipients by 2-3 wk, and rat T cells by 2-5 wk following bone marrow transplantation (BMT). Donor-type rat skin grafts placed 4 mo after BMT were accepted, while simultaneously placed non-donor-type rat skin grafts were promptly rejected. In addition to its clinical potential, the ability to induce donor-specific tolerance across xenogeneic barriers using such a nonlethal preparative regimen provides a valuable model for the study of mechanisms of xenogeneic transplantation tolerance.     

Extrathymic T Cell Deletion and Allogeneic Stem Cell Engraftment Induced with Costimulatory Blockade Is Followed by Central T Cell Tolerance

A reliable, nontoxic method of inducing transplantation tolerance is needed to overcome the problems of chronic organ graft rejection and immunosuppression-related toxicity. Treatment of mice with single injections of an anti-CD40 ligand antibody and CTLA4Ig, a low dose (3 Gy) of whole body irradiation, plus fully major histocompatibility complex–mismatched allogeneic bone marrow transplantation (BMT) reliably induced high levels (>40%) of stable (>8 mo) multilineage donor hematopoiesis. Chimeric mice permanently accepted donor skin grafts (>100 d), and rapidly rejected third party grafts. Progressive deletion of donor-reactive host T cells occurred among peripheral CD4⁺ lymphocytes, beginning as early as 1 wk after bone marrow transplantation. Early deletion of peripheral donor-reactive host CD4 cells also occurred in thymectomized, similarly treated marrow recipients, demonstrating a role for peripheral clonal deletion of donor-reactive T cells after allogeneic BMT in the presence of costimulatory blockade. Central intrathymic deletion of newly developing T cells ensued after donor stem cell engraftment had occurred. Thus, we have shown that high levels of chimerism and systemic T cell tolerance can be reliably achieved without myeloablation or T cell depletion of the host. Chronic immunosuppression and rejection are avoided with this powerful, nontoxic approach to inducing tolerance.     

Stable mixed chimerism and tolerance using a nonmyeloablative preparative regimen in a large-animal model

Bone marrow transplantation (BMT) has considerable potential for the treatment of malignancies, hemoglobinopathies, and autoimmune diseases, as well as the induction of transplantation allograft tolerance. Toxicities associated with standard preparative regimens for bone marrow transplantation, however, make this approach unacceptable for all but the most severe of these clinical situations. Here, we demonstrate that stable mixed hematopoietic cell chimerism and donor-specific tolerance can be established in miniature swine, using a relatively mild, non-myeloablative preparative regimen. We conditioned recipient swine with whole-body and thymic irradiation, and we depleted their T-cells by CD3 immunotoxin-treatment. Infusion of either bone marrow cells or cytokine-mobilized peripheral blood stem cells from leukocyte antigen-matched animals resulted in stable mixed chimerism, as detected by flow cytometry in the peripheral blood, thymus, and bone marrow, without any clinical evidence of graft-versus-host disease (GvHD). Long-term acceptance of donor skin and consistent rejection of third-party skin indicated that the recipients had developed donor-specific tolerance.     

猕猴单倍体相合非清髓造血干细胞联合间充质干细胞移植的实验研究

目的 用非清髓预处理建立猕猴单倍体相合造血干细胞移植(HSCT)模型，研究间充质干细胞(MSC)在单倍体相合移植中的作用。方法采用健康、单倍体相合的猕猴亲子配对，受非清髓性预处理用氟达拉滨 环磷酰胺 ^60Co(200cGy)全身照射 兔抗人胸腺细胞球蛋白，移植物抗宿主病(GVHD)预防用环孢菌素A、霉酚酸酯、抗CD25单抗。实验分为单纯造血干细胞(HSC)移植组和HSC联合MSC移植组；检测供受体嵌合水平，观察造血恢复、GVHD等情况。结果单倍体相合的子代猕猴采用非清髓预处理方案，可获稳定植入?比较了单纯非清髓HSC移植组和HSC联合MSC移植组的造血恢复，发现造血恢复时间主要与嵌合状态有关；MSC可促进植入；相同条件下，HSC联合MSC移植组更容易形成供受混合嵌合；GVHD的发生率低。结论成功建立了猕猴非清髓性单倍体相合HSCT的模型，非清髓HSCT联合MSC可能在单倍体相合移植中有较好效果。     

Inhibition of CD26 peptidase activity significantly improves engraftment of retrovirally transduced hematopoietic progenitors

It has previously been shown that inhibition of CD26 (DPPIV/dipeptidylpeptidase IV) peptidase activity improves homing of hematopoietic stem cells (HSCs) to the bone marrow and increases engraftment efficiency. Here, we demonstrate that treatment of retrovirally transduced mouse bone marrow cells with the tri-peptide Diprotin A (Ile-Pro-Ile), a specific inhibitor of CD26, significantly enhances engraftment of retrovirally transduced HSCs. Treatment of transduced bone marrow cells with Diprotin A permitted long-term expression of a retrovirally encoded MHC class I gene on multiple hematopoietic cell lineages after transplantation of a suboptimal number of transduced cells. Secondary transfer experiments revealed that expression of the transduced MHC class I gene resulted from engraftment of transduced HSCs. Expression of the allogeneic MHC class I antigen on bone marrow-derived cells following transplantation of Diprotin A-treated cells was sufficient to induce transplantation tolerance. Therefore, inhibition of CD26 activity significantly enhances engraftment of limited numbers of genetically modified HSCs, resulting in physiologically relevant levels of gene transfer.     

Cellular basis of graft versus host tolerance in chimeras prepared with total lymphoid irradiation

BALB/c mice given allogeneic (C57BL/Ka) bone marrow cells after toal lymphoid irradiation become stable chimeras approximately 80% donor- type and 20% host-type cells in the spleen. The chimeras doe not develop graft vs. host disease (GVHD). Purified cells of C57BL/Ka origin from the chimeras mediated GVHD in lightly irradiated C3H (third party), but not in BALB/c (host-strain) mice. Thus graft vs. host tolerance in the chimeras could not be explained by complete immunodeficiency of donor-type cells, serum blocking factors, or suppressor cells of host (BALB/c) origin. Clonal deletion or suppression of lymphocytes reactive with host tissues remain possible explanations. The transfer of donor-type chimeric spleen cells to BALB/c recipients given 500-550 rad whole-body irradiation WBI led to stable mixed chimerism in approximately 50% of recipients. The cells were presumably acting as tolerogens because similarly irradiated BALB/c mice given (BALB/c X C57BL/Ka)F1 spleen or bone marrow cells also became stable mixed chimeras.     

白血病小鼠经不同预处理方案的骨髓移植后移植细胞归巢特点

本研究构建白血病小鼠模型,通过这个模型探讨经不同预处理方案骨髓移植后骨髓细胞的归巢特点。采用非清髓性和放、化疗两种清髓性预处理方案。非清髓性预处理方案为5 Gy 60Coγ射线全身照射(A组),放疗清髓性方案为9 Gy 60Coγ射线全身照射(B组),化疗清髓性预处理方案为大剂量化疗(C组)。经上述预处理方案处理的白血病小鼠分别在骨髓移植后24、48、72、96 h检测受鼠外周血、骨髓和脾中有核细胞数和供鼠阳性细胞数,计算供鼠骨髓细胞在受鼠骨髓和脾的归巢率。结果显示:供鼠骨髓细胞在受鼠体内的表现分别为:A组是先归巢再出巢;B组是归巢,出巢,再归巢;C组同B组,但是在脾中的归巢比在骨髓中延迟。比较3组的归巢率显示,供鼠骨髓细胞在骨髓的归巢率在A组早期比B组和C组高,随着时间的推移,部分细胞出巢,归巢率迅速下降至各组中最低,为(0.90±0.09)%(P<0.05);B、C两组早期归巢率较低,随着时间推移,细胞出巢后再次归巢,第4天的归巢率迅速上升,B组升至最高,为(2.17±0.26)%(P<0.05);在脾中的归巢率与骨髓中相似,C组仍有延迟。结论:经非清髓性预处理的白血病小鼠骨髓移植术后,早期归巢率较高,72 h后明显减低;经放疗清髓性预处理小鼠,其早期归巢率较低,72 h后明显增高;经化疗清髓性预处理小鼠,早期和72 h后归巢率无明显变化,归巢率介于其他两组之间。     

Alloresistance to engraftment of allogeneic donor bone marrow is mediated by an Lyt-2+ T cell in mixed allogeneic reconstitution (C57BL/10Sn + B10.D2/nSn----C57BL/10Sn)

In the mixed allogeneic reconstitution (B10 + B10.D2----B10) model, alloresistance to engraftment of allogeneic donor results if the syngeneic component of the mixed bone marrow inoculum is not depleted of Lyt-2+ cells before transplantation. Resultant experimental animals repopulate as fully syngeneic, reject B10.D2 skin allografts, and are reactive to B10.D2 lymphoid cells in vitro, as assessed by mixed lymphocyte culture proliferative and cellular cytotoxicity assays. In contrast, depletion of Lyt-2-reactive cells from the syngeneic component of the mixed bone marrow inoculum results in mixed lymphopoietic chimerism and specific in vivo transplantation tolerance to B10.D2 allogeneic donor skin grafts and in vitro unreactivity to B10.D2 lymphoid elements. Full reactivity to third party is evident both in vitro and in vivo in these animals. This model may be helpful in further study of the syngeneic host-type cell phenotypes responsible for alloresistance to bone marrow engraftment.     

异基因骨髓源间充质干细胞移植形成稳定的嵌合体并诱导免疫耐受的研究

目的　探讨器官移植中间充质干细胞诱导免疫耐受的可能性。方法　雌性C57BL/6小鼠致死量照射后 ,移植同基因骨髓细胞和异基因骨髓源间充质干细胞 ,150d后以流式细胞仪检测其骨髓及脾脏的T细胞嵌合状态 ;以H3 TdR混合淋巴细胞反应 (MLR)和刀豆蛋白A(ConA)诱导增殖实验检测细胞移植组小鼠对供者来源细胞和有丝分裂原的反应性 ;以皮肤移植实验观测细胞移植组小鼠对供者来源组织器官移植物的反应性。结果　在细胞移植组小鼠脾脏中检测到供者来源T细胞占受者脾细胞 5 97% ;MLR反应中 ,细胞移植组小鼠的平均刺激指数 (SI)为 1 79,未处理组小鼠的平均SI为 7 2 8;在ConA诱导增殖实验中 ,细胞移植组小鼠的平均SI为 3 1 92 ,未处理组小鼠的平均SI为 3 4 99。细胞移植组小鼠供者来源皮肤移植物存活 >90d ,未处理组平均为 8d。结论　异基因骨髓源间充质干细胞移植后可形成稳定嵌合体 ,并诱导特异性免疫耐受的产生 ,使供者来源的皮肤移植物长期存活     

Effects of T cell depletion in radiation bone marrow chimeras. II. Requirement for allogeneic T cells in the reconstituting bone marrow inoculum for subsequent resistance to breaking of tolerance

The ability of normal recipient-type lymphocytes to break tolerance in long-term allogenic radiation chimeras has been investigated. Reconstitution of lethally irradiated mice with a mixture of syngeneic and allogeneic T cell-depleted (TCD) bone marrow (BM) has previously been shown to lead to mixed chimerism and permanent, specific tolerance to donor and host alloantigen (3-5). If allogeneic T cells are not depleted from the reconstituting inoculum, complete allogeneic chimerism results; however, no clinical evidence for GVHD is observed, presumably due to the protective effect provided by syngeneic TCD BM. This model has now been used to study the effects of allogenic T cells administered in reconstituting BM inocula on stability of long-term tolerance. We have attempted to break tolerance in long-term chimeras originally reconstituted with TCD or non-TCD BM by challenging them with inocula containing normal, nontolerant recipient strain lymphocytes. tolerance was broken with remarkable ease in recipients of mixed marrow inocula in which both original BM components were TCD. In contrast, tolerance in chimeras originally reconstituted with non-TCD allogeneic BM was not affected by such inocula. Susceptibility to loss of chimerism and tolerance was not related to initial levels of chimerism per se, but rather to T cell depletion of allogeneic BM, since chimeras reconstituted with TCD allogeneic BM alone (mean level of allogeneic chimerism 98%) were as susceptible as mixed chimeras to the tolerance-breaking effects of such inocula. The possible contribution of GVH reactivity to this resistance was investigated using an F1 into parent strain combination. In these animals, the use of non-TCD F1 BM inocula for reconstitution did not lead to resistance to the tolerance-breaking effects of recipient strain splenocytes. These results suggest that the ability of T cells in allogeneic BM inocula to confer resistance to late graft failure may be related to their graft-vs.-host reactivity, even in situations in which they do not cause clinical GVHD. These findings may have relevance to the mechanism whereby T cell depletion of allogeneic BM leads to an increased incidence of late graft failure in clinical BM transplantation situations.     

Bone marrow transplantation combined with gene therapy to induce antigen-specific tolerance and ameliorate EAE.

Hematopoietic stem cell (HSC) transplantation is a potential therapy that can offer multiple sclerosis patients a radical, potentially curative treatment. Using experimental autoimmune encephalomyelitis (EAE) as a model, we previously reported that retrovirally transduced B cells expressing myelin basic protein (MBP), MBP Ac1-11, or myelin oligodendrocyte glycoprotein p35-55 induced tolerance and reduced symptoms. Here, we extend our tolerance approach using bone marrow (BM) cells expressing full-length phospholipid protein (PLP) in a model for relapsing, remitting EAE. Using GFP expression as a marker, we found that up to 50% of cells were positive for transgene expression in peripheral blood after 900 rad irradiation and transduced BM transplantation, and expression was stable in hematopoietic lineages for over 10 weeks. Upon challenge, T cell proliferation in response to PLP p139-151 was reduced and EAE was completely abolished in a pretreatment protocol. In addition, protection from EAE could be achieved with PLP-transduced BM cells given on day 12 after immunization, a potential therapeutic protocol. Finally, the protective effect of PLP-expressing BM could also be observed using a nonmyeloablative protocol, albeit with lower efficacy. Our results suggest that HSC may be useful to achieve long-lasting tolerance to protect mice from EAE and possibly to promote CNS repair in ongoing EAE.     

Adenoviral transfer of a single donor-specific MHC class I gene to recipient bone marrow cells can induce specific immunological unresponsiveness in vivo

We investigated the delivery of a donor-specific MHC class I gene, H-2K(b), using a newly constructed replication-defective recombinant adenovirus (AdSV40K(b)) to recipient tissue before transplantation as a means of inducing donor-specific immunological unresponsiveness. AdSV40K(b) was able to transduce both a fibroblast cell line and freshly isolated bone marrow cells (BMCs) resulting in cell surface expression of H2-K(b) protein. Intravenous infusion of AdSV40K(b)-transduced syngeneic CBA/Ca (H-2(k)) BMCs into CBA recipient mice treated with an anti-CD4 monoclonal antibody 27 days before transplantation of a fully MHC-mismatched, C57BL/10 (H-2K(b+)), cardiac allograft resulted in significant long-term graft survival when compared with mice receiving the same dose of syngeneic BMCs transduced with a control adenovirus, AdRSVbetagal. Despite the induction of H-2K(b)-specific hyporesponsiveness following pretreatment with AdSV40K(b)-transduced CBA BMCs, persistence of H-2K(b) mRNA in central or peripheral tissues could not be demonstrated by RT-PCR. This result was in contrast to the observed persistence of K(b) mRNA both in the periphery and thymus following the infusion of transgenic CBK (H-2(k) + K(b)) BMCs. We conclude that ex vivo adenoviral gene transfer of a single donor MHC class I gene to recipient BMCs in combination with transient depletion of CD4(+) cells is sufficient to induce long-term graft survival of a fully allogeneic cardiac graft. In addition, detectable microchimerism is not a prerequisite for graft survival.     

同基因造血干细胞移植免疫重建诱导小鼠皮肤移植耐受

背景：器官移植耐受的最佳效果是能够诱导对移植抗原的特异性免疫耐受。目的：探讨小鼠异基因皮肤移植后,通过受体同基因造血干细胞移植重建免疫系统诱导移植皮肤免疫耐受的可行性。方法：取BALB/c小鼠骨髓。以C57BL/6小鼠为供体,BALB/c小鼠为受体,进行异基因皮肤移植;32只受体鼠随机均分为4组：移植对照组、环孢素A组、照射组和骨髓移植组。结果与结论：照射组小鼠10d内全部死亡,外周血白细胞数呈持续性降低;而骨髓移植组小鼠长期存活,白细胞数全身照射后6d降到最低,之后持续性增高,照射后21d与环孢素A组比较差异无显著性意义（P〉0.05）,移植皮肤存活时间显著长于其他各组（P〈0.01）,其淋巴细胞浸润及组织结构破坏明显减少,小鼠脾细胞对供体小鼠脾细胞增殖反应显著降低。说明同基因骨髓细胞移植重建免疫系统可显著延长小鼠移植皮肤存活时间,可诱导供者特异性免疫耐受。     

非清髓性骨髓移植诱导异基因受者小鼠免疫耐受的实验研究

本研究通过非清髓性预处理方案联合髓腔内骨髓移植（IBM-BMT）建立异基因小鼠免疫耐受模型,并探讨其诱导耐受的机理.受鼠为雌性C57BL/6（H-2^b,B6）小鼠,于第0天接受^60Co γ线全身照射（TBI）,4小时内输注雄性BALB/c（H-2^d）小鼠来源的骨髓细胞（BMC）,2天后腹腔注射环磷酰胺（CTX）.通过皮肤移植、混合淋巴细胞反应（MLR）检测耐受状态,并通过体外过继转移实验、IL-2逆转实验等探讨免疫耐受的机制.结果显示,经骨髓移植的B6小鼠对BALB/c小鼠的皮肤移植物平均存活时间（MST）＞150天,较对照组明显延长（P＜0.01）;骨髓移植后第90天,受鼠（黑色）表型开始呈现供鼠（白色）颜色特征.MLR结果证明,B6小鼠获得供体特异性耐受,该耐受可以被IL-2逆转且可被过继转移;所有受鼠均未出现GVHD表现.结论：非清髓预处理联合髓腔内骨髓移植可以有效地诱导异基因小鼠免疫耐受,克隆无能、抑制细胞存在及嵌合体产生均参与耐受的形成.