非清除性异基因骨髓移植后输注供者淋巴细胞治疗白血病的实验研究

目的探讨非清除性异基因骨髓移植(allo-BMT)后供者淋巴细胞输注(DLI)是否能减少移植相关并发症、相关死亡和减轻移植物抗宿主病(GVHD),是否能增强移植物抗白血病(GVL)效应.方法荷L615白血病的615(H-2k)小鼠,于接种白血病细胞后第3天接受60Coγ射线全身照射(TBI,5Gy),照射当天移植供鼠BALB/c(H-2d)小鼠的骨髓细胞(3×107)和脾细胞(1×107),移植后第2天腹腔注射环磷酰胺(200mg/kg),分别于移植后第14天和第21天输注供鼠脾细胞(2×107)或移植后第14天输注经氢化可的松(HC)和环孢菌素A(CsA)处理后的供鼠脾细胞(5×107),观察其抗白血病作用.结果移植后第21天输注供鼠淋巴细胞组和移植后14天输注经HC、CsA处理后的淋巴细胞组小鼠生存期明显延长,分别为(45.1±12.8)d和＞50d,而非清除性allo-BMT组为(26.2±3.6)d,第14天输注供鼠淋巴细胞组为(29.3±3.7)d,差异有显著性(P＜0.01),且无明显GVHD发生.结论非清除性allo-BMT后早期输注经HC和CsA处理的供者淋巴细胞或延迟加用DLI可在减轻移植相关并发症的基础上,增强GVL效应,提高长期无病生存率.     

小鼠非清髓性半相合骨髓移植造血的重建

目的：探讨在非清髓性预处理条件下,纯化的供鼠NK细胞对MHC半相合小鼠骨髓移植（BMT）造血重建的影响。 方法：以CB6F₁^H-2d/b（BALB/c^H-2d×C57BL／6^H-2b）小鼠为受者,C57BL／6^H-2b小鼠为供者。60只受者小鼠根据不同照射剂量及不同NK细胞输注量随机分为10组,每组6只,经9.0、7.0、6.0 Gy 3个剂量⁶⁰Co照射。经9.0 Gy照射后分为单纯照射组（Ⅰ）、半相合BMT组（Ⅱ）。经7.0 Gy照射后分为单纯照射组（Ⅲ）、半相合BMT组（Ⅳ）、NK细胞移植1组（Ⅴ）和NK细胞移植2组（Ⅵ）。经6.0 Gy照射后分为单纯照射组（Ⅶ）、半相合BMT组（Ⅷ）、NK细胞移植1组（Ⅸ）和NK细胞移植2组（X）。在照射后4 h移植供鼠骨髓细胞,其中NK细胞移植1组（Ⅴ、Ⅸ）每只受鼠移植前输注纯化供鼠NK细胞1×106,NK细胞移植2组（Ⅵ、X）每只受鼠移植前输注供鼠NK细胞5×10⁵。以血常规、体重、生存期等变化为指标,观察60 d。 结果：①生存期：Ⅰ组为（5.83±0.98）d,其他组小鼠生存期均大于60 d。②移植后第10天时白细胞及血小板计数：Ⅴ、Ⅵ组WBC和PLT分别为（1.05±0.30）×10⁹•L^-1、（53.50±12.11）×10⁹•L^-1及（0.95±0.26）×10⁹•L^-1、（47.17±11.82）×10⁹•L^-1,明显高于Ⅲ、Ⅳ组（P<0.01）;Ⅸ、X组WBC和PLT分别为（0.98±0.34）×10⁹•L^-1、（50.67±12.58）×10⁹•L^-1及（0.88±0.29）×10⁹•L^-1、（45.33±14.36）×10⁹•L^-1,明显高于Ⅶ、Ⅷ组（P<0.05）。 结论：MHC半相合BMT小鼠移植前输注纯化的供鼠NK细胞促进其造血重建。     

非清髓性骨髓移植的动物实验研究

目的　建立非清髓性骨髓移植的小鼠动物模型 ,探讨非清髓预处理条件下能否实现异基因造血细胞的植活 ,探讨输入的异基因造血细胞数量和移植植活的关系。方法　以雌性C5 7BL/ 6小鼠为异基因骨髓移植的受者 ,雄性BALB/c小鼠为异基因骨髓移植的供者 ,采用非致死剂量的环磷酰胺腹腔注射 (2 0 0mg/kg)加γ射线全身照射(5Gy)为非清髓性预处理方案 ,将移植受鼠分为A、B、C 3组 ,每组 10只 ,分别给予 2× 10 7、8× 10 6和 2× 10 6个供鼠骨髓细胞 ,移植后第 36天用聚合酶链式反应检测受鼠骨髓细胞内Y染色体特异性基因检测移植物植活。结果　在移植A、B和C组中分别有 8、3和 0只小鼠检测到了异基因造血细胞的植活 ,经过统计学检验发现A组小鼠的植活比例高于其余两组。结论　非清髓的预处理条件下可以实现异基因骨髓移植的成功植入 ;加大移植物中造血前体细胞的数量有助于克服移植供、受者间主要组织相容性 (MHC)间的差异和促进移植物植活     

Nonmyeloablative Conditioning Generates Autoantigen‐Encoding Bone Marrow That Prevents and Cures an Experimental Autoimmune Disease

Autoimmune diseases result from chronic targeted immune responses that lead to tissue pathology and disease. The potential of autologous hematopoietic stem cells transplantation as a treatment for autoimmunity is currently being trialled but disease relapse is an issue. We have previously shown in a mouse model of experimental autoimmune encephalomyelitis (EAE) that the transplantation of bone marrow (BM) transduced to encode the autoantigen myelin oligodendrocyte glycoprotein (MOG) can prevent disease induction. However these studies were performed using lethal irradiation to generate BM chimeras and a critical factor for translation to humans would be the ability to utilize low toxic preconditioning regimes. In this study, treosulfan was used as a nonmyeloablative agent to generate BM chimeras encoding MOG and assessed in models of EAE induction and reversal. We find that treosulfan conditioning can promote a low degree of chimerism that is sufficient to promote antigen specific tolerance and protect mice from EAE. When incorporated into a curative protocol for treating mice with established EAE, nonmyeloablative conditioning and low chimerism was equally efficient in maintaining disease resistance. These studies further underpin the potential and feasibility of utilizing a gene therapy approach to treat autoimmune disease.     

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.     

Incidence of humoral sensitization in HLA partially mismatched hematopoietic stem cell transplantation

As part of the 15th International Histocompatibility and Immunogenetics Workshop (IHIWS), seven centers participated in a collaborative project to determine whether any significant humoral sensitization occurred post-transplant among recipients of HLA partially mismatched hematopoietic cell transplants (HCTs). A total of 140 donor/recipient pairs were enrolled with a total of 367 pre-and post-transplant sera analyzed. The majority of the samples (69.1%) were obtained within 30–90 days post-HCT. HLA-specific antibodies were defined using single antigen bead assays on a Luminex™ platform with a positive cutoff value of 1000 normalized median fluorescence intensity (MFI). There was an overall incidence of post-HCT sensitization toward donor HLA mismatches of 5.7%; however, all cases were among recipients of one HLA haplotype-mismatched grafts under nonmyeloablative, pre-transplant conditioning. Among the one haplotype-mismatched recipients, 15.7% (8/51) developed donor HLA-specific antibodies and 29.4% also had antibodies directed toward third party HLA antigens. Among the donor-specific antibodies, 9.8% were directed toward HLA class I antigens; 7.8% were against class II antigens; and 2.0% had both class I and II specificity. The relative strength of post-transplant antibodies was low with no significant difference in the mean maximum MFI values between third party and donor-specific antibodies. Because only a small number (10.2%) of the post-transplant samples were obtained 180 days or more post-HCT, longer term study is needed to evaluate any clinical relevance of these low-to-moderate levels of donor-specific antibody in one haplotype-mismatched recipients, as well as to determine whether any other antibodies occur at later times.     

Revisiting the role of hematopoietic stem cell transplantation in chronic lymphocytic leukemia

Since the advent of hematopoietic stem cell transplantation more than 40 years ago, numerous methods of transplantation have been developed, modified and improved upon. Although hematopoietic stem cell transplantation has been used in a variety of malignant diseases since then, its use in the treatment of chronic lymphocytic leukemia has recently started to gain interest. Patients with chronic lymphocytic leukemia are generally elderly, and because of its relatively benign course, they were not considered suitable candidates for hematopoietic stem cell transplantation. Nonetheless, there have been marked improvements in transplantation techniques, including better conditioning regimens that have decreased treatment-related morbidity and mortality. In this article, the authors review the most recent data on hematopoietic stem cell transplantation in chronic lymphocytic leukemia as well as the change in risk stratification based on newer prognostic factors and its impact on treatment decisions in chronic lymphocytic leukemia.     

Treatment of T-prolymphocytic leukemia with nonmyeloablative allogeneic stem cell transplantation

AIM: T-prolymphocytic leukemia (T-PLL) is a rare disease of the elderly characterized by a high white blood cell count and organomegaly, and is currently incurable. Our aim was to elicit graft-versus-leukemia reactions in a patient with T-PLL. METHODS: A 52-yr-old woman with refractory T-PLL underwent a nonmyeloablative regimen followed by allogeneic peripheral blood stem cell transplantation (a "minitransplant") from her HLA-matched sibling. RESULTS: There was no treatment related toxicity other than neutropenia. Engraftment was successful. The patient experienced no graft-versus-host disease (GVHD) at any time but, on day 84 after transplantation, had a relapse in the central nervous system. Despite infusion of donor lymphocytes and intralumbar chemotherapy, she died on day 157 of systemic disease. CONCLUSION: The reasons why treatment may have failed are discussed (nature of disease, disease progression, treatment schedule).     

HLA‐haploidentical nonmyeloablative stem cell transplantation: induction to tolerance without passing through mixed chimaerism

There are few reports of unmanipulated HLA‐haploidentical nonmyeloablative stem cell transplantation (NST) using only pharmacological acute graft‐vs.‐host disease (GVHD) prophylaxis. We present here a successful case of unmanipulated HLA‐haploidentical NST for mediastinal large B cell lymphoma that was resistant to autologous peripheral blood stem cell transplantation (PBSCT). The conditioning regimen consisted of fludarabine, busulfan and rabbit anti‐T‐lymphocyte globulin (ATG) in addition to rituximab. GVHD prophylaxis was performed using tacrolimus and methylprednisolone 1 mg/kg. The patient had rapid engraftment, with 100% donor chimaerism in the lineages of both T cells and granulocytes on day +12, but developed no GVHD clinically. The patient is still in complete remission past day +1020, with no sign of chronic GVHD without receiving immunosuppressive agents. HLA‐haploidentical NST may be performed without utilizing mixed chimaerism.     

CBA法研究HLA全相合与单倍体相合移植患者血清细胞因子表达的差异性

本研究旨在探讨非清髓异基因造血干细胞HLA全相合移植（HLA-identical nonmyeloablative allogeneichematopoietic stem cell transplantation,iNAHSCT）和单倍体相合移植（HLA-haploidentical nonmyeloablative allogeneichematopoietic stem cell transplantation,hiNAHSCT）患者血清细胞因子表达的差异性,用流式细胞微珠阵列法（flow cy-tometric bead array,CBA）检测20例白血病患者移植前后不同时间点IL-2、IL-4、IL-6、IL-10、TNF-ɑ、γ-IFN、IL-17浓度的变化。结果表明,IL-2分别在iNAHSCT及hiNAHST后1周、2周起表达上调,两类移植各时间点比较无差异（P〉0.05）;γ-IFN都在移植后4周起上调,两类移植后的浓度无差异（P〉0.05）;IL-4在iNAHSCT及hiNAHSCT中分别于移植后2周、移植后1周起增高,iNAHSCT的IL-4浓度高于hiNAHSCT（P〈0.01）;IL-6在iNAHSCT及hi-NAHSCT中分别在移植后1周、移植后2周起上调,移植后4周为峰值,且hiNAHSCT的IL-6浓度高于iNAHSCT（P〈0.02）;IL-10在iNAHSCT、hiNAHSCT中分别于从移植后1、2周起增高,iNAHSCT中IL-10的表达高于hiNAH-SCT（P〈0.05）。TNF-ɑ在hiNAHSCT中从移植后1周、在iNAHSCT中从移植后2周起上调;hiNAHSCT中TNF-ɑ增高的幅度高于iNAHSCT（P〈0.01）。IL-17分别于hiNAHSCT及iNAHSCT后移植后1周、移植后4周时增高,hi-NAHSCT IL-17增高的幅度高于iNAHSCT的（P〈0.05）。结论：两类移植后所检测到的细胞因子水平均明显增高,移植后4周为峰值。IL-6、TNF-ɑ、IL-17在hiNAHSCT中增高明显,而IL-4,IL-10在iNAHSCT中增高显著。