同种异体移植免疫耐受的进展

探索组织器官移植后的排斥反应,提高移植物的存活率是移植免疫研究者致力攻克的堡垒。20世纪90年代以来,随着器官移植术的广泛开展,人们对移植排斥反应的认识不断加深,对移植免疫耐受提出了新的研究思路和方法。目前普遍认为,解决异体移植排斥反应的关键在于诱导受体对供体的细胞、组织或器官产生免疫耐受,即受体免疫系统对移植物抗原产生特异性免疫无反应性。本文将对近年来阻断特异性免疫应答、诱导免疫偏离、主动免疫诱导同种移植耐受及建立受者体内嵌合体诱导移植耐受等主要方面的进展综述。     

吻合血管同种异体骨移植动物模型的建立

目的:为吻合血管同种异体骨移植提供理想的动物模型。方法:日本大耳白兔雌21只为供体,雄42只为受体,对股骨滋养血管进行显微解剖观测,在此基础上设计吻合血管同种异体股骨干移植术,并设立应用环孢素A的实验组和未予任何免疫抑制剂的对照组。结果:股骨干的滋养孔位于小粗隆前下方3.0mm,滋养动脉起始于旋股外侧动脉,管径0.3mm,斜向外下入滋养孔,干长1.7cm,伴行静脉1条。实验组术后一般情况正常,血管吻合口通畅,骨愈合良好;对照组术后出现脱毛,腹泻,死亡等GVHD现象,术肢出现血管早期栓塞,非感染性坏死组织,骨愈合不良等HVGR征象。结论:本动物模型滋养血管解剖恒定,可操作性强,且能较好地复制免疫排斥反应及体现免疫抑制剂对吻合血管同种异体骨移植的调控作用。     

同种异体移植子宫内膜异位症大鼠模型的建立

目的建立Wistar大鼠同种异体子宫内膜异位症模型。方法采用外科诱导法对24只Wistar雌性大鼠行异体子宫组织移植手术,30只行自体子宫组织移植手术。结果 Wistar大鼠造模12周后,异体移植成模率83.3%,与自体移植成模率76.7%比较差异有统计学意义（P〈0.05）;异体、自体模型异位内膜上皮细胞功能活跃,有类似正位子宫内膜的周期变化和生理特征;且造模后大鼠体液免疫反应敏感性与假手术对照组比较,C3、C4、IgG升高,IgM降低,差异有统计学意义,IgA没有明显变化;异体移植模型IgG比自体移植模型高,差异有统计学意义。结论应用Wistar大鼠进行同种异体移植手术可以成功的建立子宫内膜异位症模型。     

同种异体大鼠耳廓移植模型的建立及意义

目的建立同种异体大鼠外耳廓移植模型,进行异体复合组织移植免疫的实验研究.方法以Lewis大鼠为供体、BN大鼠为受体,应用显微外科技术,进行吻合血管的全耳廓异体移植,观察移植耳排斥反应的大体表现及组织学特征.结果20只受体动物移植术后全部存活,吻合血管即刻通畅率为100%,移植耳廓呈现典型的免疫排斥反应过程,平均存活时间为(7.8±1.7)d.结论大鼠外耳廓移植是进行异体复合组织移植免疫研究良好的动物模型.     

同种异基因骨髓细胞移植诱导嵌合体小鼠生成

采用C57BL/J和BALB/c两种小鼠进行骨髓细胞移植。移植前受体小鼠经 6 0Gy6 0 Coγ射线照射。用细胞染色法和流式细胞术分析了射线对小鼠淋巴细胞的损伤强度和照射后移植了骨髓细胞的小鼠在不同时间内的淋巴细胞亚群变化。结果表明 ,(1 ) 6 0Gyγ射线照射受体鼠 ,能杀伤 95 5 %的外周淋巴细胞 ,但是不影响自身H 2Kb 分子在淋巴细胞表面表达的百分数。残留细胞中CD3+ T细胞、NK1 1 + 淋巴细胞、CD3+ /NK1 1 + 淋巴细胞的阳性率分别由照射前的 40 44%增加到45 30 %、 1 1 3 %增加到 37 41 %和 1 0 7%增加到 5 86 % (P≤ 0 0 1 ) ;(2 )小鼠的淋巴细胞数量从照射后 30d开始恢复 ,到1 90d达到正常值 ;(3 )照射后进行同种异基因骨髓细胞移植后的第 60天 ,受体淋巴细胞表面自身H 2Kb 抗原表达下降 ,CD3+ /NK1 1 + 淋巴细胞百分数增加。随H 2Kb 的抗原表达的恢复 ,CD3+ /NK1 1 + 淋巴细胞百分数下降 ;(4)在细胞移植的第 1 90天 ,受体小鼠 (黑色 )表型呈现出供体鼠 (白色 )颜色特征。在嵌合体小鼠外周淋巴细胞中 ,有 2 5 %为供体H 2Kd 阳性细胞 ,CD3+ /NK1 1 + 淋巴细胞百分数为 1 88% ,明显高于正常值 (P≤ 0 0 1 )。以上结果表明 ,6 0Gy6 0 Coγ射线照射能诱导免疫耐受 ,其耐受性的形成可能与最初保     

同种异体骨骨折临床及预防的进展

大段骨缺损是矫形外科较普遍又棘手的问题，由于自体骨来源有限，人工假体无生物学活性，易发生松动和疲劳断裂等并发症，而异体骨来源丰富，形状大小不受限制，并具有生物学活性，有较好的应用前景。临床长期随访结果常发生异体骨骨折并发症〔１，２〕，影响手术效果。为...     

同种异体MHC抗原识别和免疫耐受

间接识别在同种异体移植排斥反应中起重要作用。免疫反应早期局限于供体ＭＨＣ抗原的少数显性决定簇，然后可转移到供体ＭＨＣ抗原的隐性决定簇和受体的交叉反应性抗原。利用供体基序肽等方法特异性干扰免疫反应过程诱导免疫耐受是移植免疫领域的新进展。     

同种异体松质骨移植的生物力学研究

目的：探讨同种异体松质骨移植后的力学性能变化以及不同力学环境对其的影响。方法：在40只家兔前肢尺骨中段进行同种异体松质骨移植，并使左右侧植骨块分别承受正常生理载荷与低载荷，动物分批处死后取出植骨区标本进行骨密度值、三点弯曲、平均骨小梁厚度等测试。结果：同种异体松质骨愈合时的骨密度值、最大弯矩、平均骨小梁厚度逐渐上升。在移植后第16周时，和低载荷侧比较，正常载荷侧的上述指标都明显优于低载荷侧（P值分别＜0.01，0.05，0.05）。结论：同种异体松质骨移植后的力学性能变化与它所承受的载荷大小有较强的相关性。     

超抗原SEB在同种异体细胞移植小鼠中诱导的免疫耐受及其特征

目的 探讨超抗原葡萄球菌肠毒素B（SEB）体内诱导的免疫耐受性及其特征。方法 采用MHC不同的两种小鼠进行淋巴细胞移植。用流式细胞术和混合淋巴细胞培养技术，检测受体鼠T细胞亚群的变化，供体鼠H－2K^d分子在受体鼠体内表达的阳性率与免疫应答性。结果 （1）注射SEB可选择性地降低CD4^ T细胞和CD4^ T/H-2K^b 细胞的百分率，而不影响CD8^ T细胞的数量。在SEB注射的21d，抑制作用最强，其对CD4^ T细胞和CD4^ T/H-2K^b 细胞的抑制率分别是6.24%和23.38%。（2）在进行异源性MHC淋巴细胞移植时注射SEB，于移植细胞后21d，受体小鼠肝脏淋巴细胞上开始表达供体小鼠H－2K^d分子，至移植后40d，表达率达到高峰7.90%。与此同时，受体鼠外周淋巴细胞对供体鼠淋巴细胞的免疫应答能力也明显降低。结论 SEB能诱导小鼠产生免疫耐受，免疫耐受的形成与受体鼠内CD4^ T细胞克隆的明显减少有关。     

同种异体半月板移植的研究进展

膝关节半月板具有重要的生物力学功能，它是维持膝关节正常生理功能的重要组成部分，它可以增加关节软骨面之间的接触面积，减小关节面单位面积上的压力，其楔形结构可增加膝关节的稳定性，缓冲纵向压力。半月板切除后必然导致韧带松驰，关节不稳，软骨面磨损加快，加速膝关节退行性改变，为了避免这些并发症的发生，     

小鼠→大鼠异种移植耐受模型的建立

目的：用非清髓方法建立小鼠→大鼠混合嵌合体模型，探讨免疫耐受机制。方法：给SD大鼠亚致死全身照射(TBI)后，4h内输入Balb／c小鼠骨髓细胞(BMC)，2d后腹腔注射环磷酰胺(CTX)，分别于BMT后30、60和90d，检测小鼠源性BMC在大鼠体内植活情况。通过皮肤移植、迟发超敏反应(DTH)和混合淋巴细胞反应(MLR)检查，探讨其耐受机制。结果：经处理大鼠外周血可测出小鼠源性嵌合体，皮肤移植、DTH和MLR检查显示对Balb／c小鼠产生特异性耐受，且较持久。结论：应用7．5Gy TBI 腹腔注射50mg／kg CTX 供体BMT、可成功建立小鼠→大鼠混合嵌合体模型诱导特异性耐受，嵌合体与耐受有关系。     

Neonatally tolerant rats actively eliminate donor-specific lymphocytes despite persistent chimerism

Rats from the allotype-marked PVG-RT7^b and PVG-RT1^u-RT7^b strains were injected at birth with semi-allogeneic F₁ bone marrow (BM) cells from athymic nude rats (PVG-rnu/rnu x PVG-RT1^u-rnu/rnu) to induce neonatal tolerance. As adults, 97% of the animals accepted donor-specific allogeneic skin grafts and a majority (65%) of rats were chimeric, expressing the major histocompatibility complex class I and allotype marker of the donor strain. Similar results were obtained when PVG-RT1^u-RT7^b rats were injected at birth with fully allogeneic PVG-rnu/rnu nude BM cells: as adults, 94% accepted donor-specific skin allografts and 76% of recipients were chimeric. Donor-derived CD4T cells, CD8T cells and B cells were found in low numbers (< 2%) in peripheral blood of rats made tolerant by F₁ BM cells. A large proportion of T cells bore the phenotype of recent thymic emigrants, suggesting that they were newly produced. All the evidence was consistent with clonal deletion tolerance, induced centrally within the thymus. The thymus was chimeric and thymocytes failed to respond in vitro to alloantigens of the donor-specific haplotype; donor-specific skin allografts survived indefinitely on athymic nude recipients reconstituted with CD4⁺CD8^? thymocytes or peripheral CD4T cells from tolerant animals. The chimeric state was interesting, since the PVG and PVG-RT1^u rat strains contain a natural killer (NK) cell system that rapidly eliminates (within 24 h) intravenously injected allogeneic or semi-allogeneic lymphocytes – a phenomenon known as allogeneic lymphocyte cytotoxicity or ALC. When neonatal tolerant rats were tested, the ALC index (a measure of cell killing) was unchanged in nonchimeric tolerant rats and significantly altered (reduced killing), but not abolished in chimeric animals. Hence, the injection of allogeneic BM cells which induced specific tolerance in the T cell population failed to tolerize the NK cell system, allowing the constant killing of newly produced donor-derived lymphocytes and putting at risk the very survival of the allogeneic BM cells. This has interesting implications for clinical transplantation.     

Mixed hematopoietic chimerism and transplantation tolerance

Durable transplantation tolerance can be reliably achieved by inducing engraftment of hematopoietic cells in recipients initially depleted of T-lymphocytes. Engraftment of donor pluripotent hematopoietic stem cells (PPHSC) produces mixed hematopoietic chimeras in which both host and donor cells coexist and are tolerant of each other. The major mechanism of tolerance in these chimeras is central, intrathymic clonal deletion, which is induced and maintained by immigration of both host and donor marrow-derived cells to the host thymus, ensuring the ongoing central deletion of donorand host-reactive cells. In this article, approaches developed in our laboratory to induce stable mixed hematopoietic chimerism and specific central deletional allogeneic and xenogeneic tolerance without toxic or myeloablative host conditioning are reviewed.     

A semiquantitative PCR technique for detecting chimerism in hamster-to-rat bone marrow xenotransplantation

Although bone marrow transplantation has been used to induce donor-specific tolerance in many allogeneic models, similar effort in xenogeneic transplantation is met with obstacles like more severe graft versus host disease (GVHD). We are currently engaged in developing a GVHD-free hamster-to-rat xenotransplantation model using splenectomy, total body irradiation, and donor bone marrow transplantation. To test donor cell chimerism, particularly in the solid tissues, we developed a semiquantitative polymerase chain reaction (PCR) method using primers specific for hamster beta-actin and mitochondrial cytochrome C oxidase I and II (MCO I and II) genes and rat sex determination region on the Y chromosome (SRY) gene. Using this method, we estimated the level of hamster cells chimerism in rats subjected to splenectomy, total body irradiation (10 Gy), and hamster bone marrow transplantation (3 x 10(8) cell/recipient) and observed high levels of donor cells in all recipient tissues tested.     

Successful bone marrow transplantation with split lymphoid chimerism in DiGeorge syndrome

A female infant with DiGeorge syndrome associated with severe T-cell immunodeficiency underwent a successful bone marrow transplantation from her HLA-identical, mixed leukocyte culture-nonreactive brother at 5 months of age. Mature circulating T cells and mitogen-induced proliferative responses were detectable at 10 days posttransplant, and by 8 months posttransplant functional T- and B-cell reconstitution was documented by normal responses to mitogens and normal levels of serum immunoglobulins as well asin vitro andin vivo T-cell reactivity to specific antigens and production of specific antibody to T cell-dependent antigensin vivo. Phytohemagglutinin-induced interleukin-2 production and cell surface interleukin-2 receptor expression improved posttransplant, with normal production values observed by 8 months posttransplant. Histologic examination of appendix and thoracic lymph node obtained 9 and 17 months posttransplant, respectively, revealed near-normal lymphoid architecture, with germinal center formation providing morphologic confirmation of reconstitution. Stable split lymphoid chimerism with T cells of donor origin and B cells remaining recipient in origin was documented by sex chromosome analysis. Two years posttransplant the subject remains free of serious infections. In conclusion, this case indicates that bone marrow transplantation can produce peripheral immunoreconstitution without need for significant thymic influence, most likely by providing a source of postthymic T cells, and that bone marrow transplantation should be considered a therapeutic option in patients with DiGeorge syndrome associated with severe T-cell deficiency.     

The practical application of chimerism analyses in allogeneic stem cell transplant recipients: Blood chimerism is equivalent to marrow chimerism

Background

Chimerism defines the amount of donor versus recipient hematopoiesis following allogeneic stem cell transplant (SCT). PCR-based analyses of short tandem repeats (STRs) are commonly used and are accurate and applicable to allogeneic transplant recipients. These analyses are performed on blood and marrow aspirates, but it is unknown if analyses of both are required. We performed a retrospective analysis of 42 consecutive adult allogeneic SCT recipients at our institution to determine if both sample types are needed.

Methods

Chimerism status was determined by multiplex PCR and capillary electrophoresis of STRs. Analyses were performed at 30, 60, and 90 days after SCT on both unfractionated blood and unfractionated marrow aspirate.

Results

PCR analyses of STRs for chimerism performed on unfractionated blood highly correlated with results obtained using unfractionated marrow aspirates at 30, 60, or 90 days following transplant (p < 0.0001 for each time point). Overall and relapse-free survival of patients experiencing full donor chimerism was not statistically different from patients demonstrating mixed chimerism at days 30, 60, and 90 following SCT.

Conclusions

PCR-based chimerism analyses on blood provide similar information as marrow aspirate analyses. These are unique results suggesting that chimerism analyses may be assessed on peripheral blood alone.     

The cellular composition of human lymph nodes after allogenic bone marrow transplantation: an immunohistological study

Using immunohistological techniques, the cellular composition of lymph nodes was assessed in 18 patients who had died 15 to 326 days after allogeneic bone marrow transplantation for leukaemia. The lymph nodes showed reduced cellularity of the cortex and paracortex, dilated sinuses and no lymphoid follicles. The majority of leucocytes were T lymphocytes with an inversion of the normal T4:T8 ratio. No cells were detected expressing immature cortical thymocyte antigens, using NA1/34 and OKT10, but an excess of T11 (E rosette receptor)+ cells over the sum of T4+, T8+ and HNK1+ cells raised the possibility of the presence of immature cells. B lymphocytes were extremely rare and present as clusters in only two patients. Despite this, plasma cells were prominent in many cases and their number increased with time post transplant. The predominant immunoglobulin heavy chain class was IgA in seven cases, IgG in three cases, IgM in two cases and IgE in one case with no relationship between dominant class and days post transplant. In patients with graft-versus-host disease (GvHD), there was a significantly lower T4:T8 ratio but no increase in expression of lymphocyte activation markers. Pyknotic leucocytes were present in half of the cases with GvHD and none of the other cases. No differences were detected in patients who had received marrow purged with monoclonal antibodies (Campath-I or UCHT1). Chimeric studies on three recipients of one haplotype matched marrow, using a monoclonal antibody specific for HLA-A2 and A28 antigens, showed a significant influx of donor cells by 56 days but this did not appear to be an immediate prelude to full morphological reconstitution.     

CTLA-4 Ig腺病毒基因治疗联合供体细胞输注诱导混合嵌合体和大鼠心脏移植耐受

目的　在心脏移植手术中实施CTLA 4Ig腺病毒基因治疗并联合术后输注供体骨髓细胞 ,诱导异基因大鼠心脏移植耐受 ,并对相关机制进行研究。方法　将异基因DA大鼠的心脏移植给受体LEW大鼠 ,同时经门静脉输注供体DA的脾细胞 (SC ,3× 10 8)、CTLA 4Ig腺病毒 [( 1～ 5 )× 10 9PFU ml],第 4天由舌静脉输注DA的骨髓细胞 (BMC ,3× 10 8)。观察、记录心脏移植物的存活时间。并对皮肤移植的受体作同样处理 ,观察皮肤移植存活情况。通过MLR、IL 2逆转实验及嵌合体的测定 ,探讨耐受机理 ,并检测了CTLA 4Ig的体内表达、TH1 TH2型细胞因子的表达。结果　单用CTLA 4Ig腺病毒基因治疗 ,或CTLA 4Ig腺病毒基因治疗联合单独的供体脾细胞或骨髓细胞能不同程度地延长异基因心脏移植物的存活 ,但不能延长皮肤移植物的存活。脾细胞、CTLA 4Ig腺病毒和骨髓细胞(SC Ad BMC)处理组的心脏移植物存活时间明显超过其它各耐受诱导组 ,并且能够诱导皮肤耐受。RT PCR实验证明 ,在受体内不同的组织CTLA 4Ig基因的表达量有所不同 ,并且随着时间的推移表达下降。TH1和TH2型细胞因子的检测显示 ,耐受大鼠体内未发现这两类细胞因子的偏移现象。MLR证明耐受大鼠的免疫应答表现为供体特异性降低 ,IL 2逆转实验、嵌合体检测表明 ,该耐受可能与     

反向骨髓移植诱导异种造血嵌合体的研究

目的应用大鼠-小鼠骨髓移植模型,探讨反向骨髓移植诱导特异性免疫耐受的作用机制。方法首先诱导形成嵌合体S大鼠,再将此嵌合体大鼠的骨髓反向移植给Balb／c小鼠,监测存活率,GVHD发病率,做混合淋巴细胞培养,检测脾细胞增殖及TNF-α、INF-γ与IL-4产量,监测移植后小鼠嵌合率的变化。结果对照组小鼠观察期间出现严重GVHD表现,反向移植组小鼠仅出现轻度GVHD,生存期明显延长,TNF-α与INF-γ产量明显降低,而IL-4升高,移植后嵌合率较稳定,与对照组相比有显著性差异。结论有受者嵌合体的骨髓移植可以减轻急性GVHD,诱导出较长时间存在的异种造血嵌合体。     

Double negative Treg cells promote nonmyeloablative bone marrow chimerism by inducing T-cell clonal deletion and suppressing NK cell function

The establishment of immune tolerance and prevention of chronic rejection remain major goals in clinical transplantation. In bone marrow (BM) transplantation, T cells and NK cells play important roles for graft rejection. In addition, graft-versus-host-disease (GVHD) remains a major obstacle for BM transplantation. In this study, we aimed to establish mixed chimerism in an irradiation-free condition. Our data indicate that adoptive transfer of donor-derived T-cell receptor (TCR) αβ(+) CD3(+) CD4(-) CD8(-) NK1.1(-) (double negative, DN) Treg cells prior to C57BL/6 to BALB/c BM transplantation, in combination with cyclophosphamide, induced a stable-mixed chimerism and acceptance of C57BL/6 skin allografts but rejection of third-party C3H (H-2k) skin grafts. Adoptive transfer of CD4(+) and CD8(+) T cells, but not DN Treg cells, induced GVHD in this regimen. The recipient T-cell alloreactive responsiveness was reduced in the DN Treg cell-treated group and clonal deletions of TCRVβ2, 7, 8.1/2, and 8.3 were observed in both CD4(+) and CD8(+) T cells. Furthermore, DN Treg-cell treatment suppressed NK cell-mediated BM rejection in a perforin-dependent manner. Taken together, our results suggest that adoptive transfer of DN Treg cells can control both adoptive and innate immunities and promote stable-mixed chimerism and donor-specific tolerance in the irradiation-free regimen.