非清髓性异基因骨髓移植治疗白血病的动物实验研究

[目的]探讨非清髓性异基因骨髓移植及加供者淋巴细胞输注治疗小鼠白血病的疗效.[方法]荷L7212白血病的615(H-2K)小鼠,于接种白血病细胞后第2天接受60Co-γ射线全身照射(TBI 8.5Gy或5Gy)分为若干组,照射当天移植供鼠BALB/C(H-2d)小鼠的骨髓细胞(5×106)和脾细胞(1.5×107),移植后第2天腹腔注射环磷酰胺(200mg/kg);供者淋巴细胞输注组分别于移植后第7天、14天、21天再次输注供鼠脾细胞5×106、1×107、2×107,观察受鼠的移植物植入、移植物抗宿主病(GVHD)、受鼠生存时间及移植相关并发症等.[结果]非清髓性预处理能保证移植物的稳定植入,非清髓性异基因骨髓移植组小鼠生存时间为22.3±4.8天,与非清髓空白组14.7±3.4天和传统移植组18.3±3.2天比较均有显著性差异(P＜0.05),供者淋巴细胞输注(DLI)组小鼠平均生存时间明显延长,为34.3±2.5天,与非清髓移植组比较均有显著性差异(P＜0.05),且无明显GVHD表现和病理学改变,移植相关并发症减少.[结论]非清髓性异基因骨髓移植能在减轻GVHD的同时保留一定的移植物抗白血病(GVL)效应,移植后行DLI可在减轻移植相关并发症的基础上进一步增强GVL效应.     

髓腔内造血干细胞移植联合供者淋巴细胞输注对GVHD和GVL的影响

 目的　观察髓腔内供者淋巴细胞输注（IBM-DLI）对急性淋巴细胞白血病小鼠异基因外周造血干细胞移植（allo-PBSCT）后移植物抗宿主病（GVHD）和移植物抗白血病效应（GVL）的影响。方法　雌性C57BL／6小鼠为受鼠建立白血病模型,接受全身照射(TBI)预处理后,输注雄性BABL/C小鼠来源的经rhG-CSF动员后的外周造血干细胞,分别经尾静脉（IV）和髓腔内进行DLI,2天后腹腔注射环磷酰胺（CTX）,观察移植后小鼠的生存状态和GVHD发生情况,应用流式细胞仪检测受鼠体内嵌合体形成和CD+4 CD+25调节性T细胞（Treg）比例。结果　IBM-DLI组的受鼠GVHD发生比例和死于白血病的小鼠比例均较IV-DLI组明显减低（P＜0.01）;移植后第7天各组受鼠骨髓中供鼠来源的细胞比例均在95 ％以上;与IV-DLI组比较,脾细胞中Tregs比例在IBM-DLI组明显升高（P＜0.01）。结论　与IV-DLI相比,IBM-DLI有利于减轻GVHD的发生,并保留GVL效应,其机制可能与受鼠体内Tregs细胞比例增高有关。     

非清髓性异基因造血干细胞移植治疗恶性肿瘤

非清髓性异基因造血干细胞移植,主要是通过移植物抗肿瘤效应来根除恶性肿瘤细胞,与经典的清髓性异基因造血干细胞移植相比,其移植相关并发症和病死率降低,对年龄较大、体质差的患者是一种较为安全可靠的治疗手段.     

非清髓性异基因造血干细胞移植治疗恶性肿瘤

非清髓性异基因造血干细胞移植 ,主要是通过移植物抗肿瘤效应来根除恶性肿瘤细胞 ,与经典的清髓性异基因造血干细胞移植相比 ,其移植相关并发症和病死率降低 ,对年龄较大、体质差的患者是一种较为安全可靠的治疗手段     

减轻异基因造血干细胞移植GVHD增强GVT的研究

异基因造血干细胞移植(allogeneic hematopoietic stem cell transplantation,allo-HSCT)是一种有效治疗恶性肿瘤及非恶性疾病的方法,近年来成为治愈某些恶性血液性肿瘤疾病的主要手段之一,明显提高了患者的长期生存率[1];但移植物抗宿主病(graft-versus-host disease,GVHD)仍然是allo-HSCT的主要并发症,是移植相关死亡及影响患者生存质量的重要原因。GVHD是由移植物中所含供者T细胞识别宿主抗原而攻击宿主组织器官所产生的病理损伤,并导致宿主多脏器损伤的一个疾病过程。以T细胞为靶点的免疫抑制剂及T细胞去除术的应用是目前GVHD…     

自杀基因在异基因骨髓移植中的应用

异基因骨髓移植(allo-BMT)是治愈恶性血液系统疾病的重要方法之一，但其并发症移植物抗宿主病(GVHD)限制了其在临床上的发展应用。国内外不少研究表明自杀基因转染T淋巴细胞可控制移植物抗宿主病。     

NK细胞与异基因骨髓移植

近年来研究发现，自然杀伤细胞在异基因骨髓移植中也起飞丰独特的作用，宿主的静息状态自然杀伤细胞能排斥异基因骨髓细胞；而供者的活化自然杀菌细胞输注宿主体内能促进骨髓植入和造血重建，还能够预防异基因骨髓移植的严重并发症－移植物抗宿主病，并增强抗肿瘤效应。本文综述了这些方面的研究近况和新进展。     

骨髓间充质干细胞在异基因骨髓移植中的研究进展

骨髓间充质干细胞(MSCs)是骨髓基质细胞的前体细胞,分泌多种造血相关因子,表达多种黏附因子,同时具有免疫调节功能,在造血微环境发挥重要作用.因此,利用MSCs做滋养层,体外扩增造血干细胞(HSC),临床联合移植MSCs和HSC,提高造血重建的能力,减少或减轻移植物抗宿主病(GVHD)的发生,具有很广阔的研究前景.     

骨髓间充质干细胞在异基因骨髓移植中的研究进展

骨髓间充质干细胞（MSCs）是骨髓基质细胞的前体细胞,分泌多种造血相关因子,表达多种黏附因子,同时具有免疫调节功能,在造血微环境发挥重要作用。因此,利用MSCs做滋养层,体外扩增造血干细胞（HSC）,临床联合移植MSCs和HSC,提高造血重建的能力,减少或减轻移植物抗宿主病（GVHD）的发生,具有很广阔的研究前景。     

TRAIL-induced cell death cooperates with IFN-gamma activation in the graft-versus-tumor effect against colon tumors

The graft-versus-tumor (GVT) effect that occurs following allogeneic bone marrow transplantation (BMT) and donor lymphocyte infusion (DLI) is currently being subjected to intensive investigation because of clinical evidence for GVT efficacy against leukemia. In this report, we investigate the efficacy and molecular mechanisms of GVT against solid tumors, using a modification of the mouse parent-to-F1 BMT model. Mouse Colon26 cells in which tumor necrosis factor related apoptosis-inducing ligand (TRAIL) receptor expression was stably knocked down were transplanted to investigate the role of the TRAIL-TRAIL receptor system in the GVT effect. In addition, Fas ligand-(FasL) deficient mice on a C57BL6 (B6) background were used as donors, to determine the significance of the Fas-FasL system for the antitumor effect. The group that received B6 DLI followed by preconditioning with 950 rad irradiation underwent tumor reduction associated with the induction of IFN-gamma, TRAIL and tumor-cell apoptosis. In vitro cultured Colon26 cells were resistant to TRAIL but susceptible to the combination of IFN-gamma and TRAIL in a TRAIL-dose-dependent manner. The infusion of lymphocytes from FasL-defective donors reduced the tumor progression, although efficacy was decreased in the TRAIL receptor knockdown tumors but not in wild-type ones, compared with infusion of B6-derived lymphocytes. The findings indicate that GVT activity against subcutaneous colon tumors is efficiently induced by preconditioning with irradiation and allogeneic DLI, and that TRAIL and IFN-gamma act cooperatively in the antitumor effect.     

Immunomodulation following chemotherapy

Summary In the last decade, immunomodulation has emerged as a mode of therapy capable of mediating the regression of cancer in some patients. This article reviews our experience with immunomodulation following transplant and non-transplant chemotherapy. We used interferon and cyclosporine A following conventional chemotherapy in a non-transplant setting for a B 16 melanoma in a murine model. This combination generated cells with MHC-unrestricted cytotoxicity. We have also used immunotherapy in the transplant setting with IL-2 activated PBSC in patients with breast cancer. Of the 28 patients treated, 20 developed GVHD and the average time to reconstitution was 12 days (comparable to a control group). This article also raises the possibility of extending immunomodulation to breast cancer patients in the nontransplant setting to induce an antitumor immune response following cytoreductive chemotherapy.Presented at the symposium "New Approaches in the Therapy of Breast Cancer", Georgetown University Medical Center, Washington DC, October 1994, generously supported by an education grant from Bristol-Myers Squibb.     

Combination of anti‐CD4 antibody treatment and donor lymphocyte infusion ameliorates graft‐versus‐host disease while preserving graft‐versus‐tumor effects in murine allogeneic hematopoietic stem cell transplantation

Allogeneic hematopoietic stem cell transplantation (allo‐HSCT) is not only a well‐established immunotherapy for hematologic malignancies, but is potentially useful for treating solid tumors refractory to available therapies. However, application of allo‐HSCT to solid tumors is limited, despite the beneficial antitumor effects, by the risk of graft‐versus‐host disease (GVHD). CD4⁺ T cells have been implicated in several aspects of GVHD, and suppress antitumor CD8⁺ T‐cell responses. In the present study, we investigated clinically applicable allo‐HSCT protocols designed to maximize antitumor effects while reducing the risk of GVHD. We used a mouse model of allo‐HSCT with s.c. tumors. We found that myeloablative conditioning was associated with better inhibition of tumor growth but with severe acute GVHD. Early treatment with anti‐CD4 mAb substantially ameliorated GVHD while preserving antitumor effects, leading to improved survival in myeloablative allo‐HSCT. Late treatment with anti‐CD4 mAb also ameliorated GVHD to some extent. Donor lymphocyte infusion in GVHD mice treated with anti‐CD4 mAb further suppressed tumor growth without exacerbating GVHD. Collectively, our results suggest that myeloablative allo‐HSCT followed by anti‐CD4 mAb treatment and donor lymphocyte infusion could be a potent and safe immunotherapy for patients with cancers refractory to available therapies.     

MHC半相合脾加骨髓细胞诱发H22荷瘤鼠的抗肿瘤效应

目的：观察MHC半相合脾加骨髓细胞移植抗小鼠H22实体瘤的效果。方法：以皮下接种H22肝癌细胞的BALB/c×C57BL/6杂交F1代雌性小鼠为受鼠,以健康雌性F1、雄性C57BL/6、雄性C3H小鼠为MHC全相合、半相合、不相合供鼠,观察移植后的抑瘤情况；观察供鼠细胞经~(60)Co照射的MHC半相合移植对WBC、生化和嵌合体的影响；比较供鼠细胞经与不经~(60)Co照射的MHC半相合移植的GVHD情况。结果：供鼠细胞经/不经~(60)Co照射的MHC半相合移植小鼠的肿瘤明显较小,与单纯化疗未进行移植者比较,差异具有统计学意义(P＜0．05)；但受鼠未经化疗预处理的MHC半相合细胞输注没有出现抗肿瘤效应；供鼠细胞经7．5 Gy ~(60)Co照射的MHC半相合移植能明显降低GVHD反应,且对外周血白细胞、生化无不良影响。结论：经7．5 Gy~(60)Co照射的MHC半相合脾加骨髓细胞移植能对H22肝癌细胞产生移植物抗肿瘤效应并降低GVHD反应。     

Analysis of the Clonal Expansion of TCR VβT Cells in Patients

Objective To investigate the clonal expansion of T cell receptor(TCR)Vβ subfamily T cells which were considered as GVL effective cells after donor lymphocytes infusion(DLI)in patients with relapse chronic myelogenous leukemia(CML)after allogeneic bone marrow transplantation(allo-BMT).Methods The CDR3 of TCR Vβ24 subfamily genes were amplified in samples of peripheral blood mononuclear cells at different time points before and after DLI,which were drawn from 2 cases of relapse CML treated by allo-BMT,to observe the usage of TCR Vβrepertoire.The PCR products were further labeled with fluorescent and analyzed by genescan technique for identification of the CDR3 size,to evaluate the clonality of the detectable TCR VβT cells.Results Only 4-11 VβT subfamily T cells could be identified in CML cases before DLI,and 12-21 Vβ subfamily T cells could be deected in samples from CML which display remission after DLI.Genescan analysis showed that new clonal expansion TCR Vβ subfamily T cells could be found in samples after DLI.Conclusion The skew distribution of TCR Vβ subfamily T cells could be found on patients with relapse CML after allo-BMT,and this skewing pattern may stage to stage to normal pattern during the complete remission.The GVL effect may exert through some clonal expansion TCR Vβ subfamily T cells during the treatment of DLI in relapse CML.     

rhGM-CSF After Allogeneic Bone Marrow Transplantation From Unrelated Donors: A Pilot Study of Cyclosporine and Prednisone as Graft-Versus-Host Disease Prophylaxis

Cyclosporine and prednisone were administered as graft-versus-host disease (GVHD) prophylaxis to nine patients undergoing marrow transplant from HLA matched, unrelated donors. RhGM-CSF was administered at a dose of 250 μ;g/m² daily to all patients. The median day of neutrophil recovery to 500/mm³ was Day 16. Four patients developed Grade II acute GVHD and four developed Grade 111 acute GVHD. One patient, who survived only 25 days, did not develop GVHD at all. One patient developed systemic infection within the first 28 days after marrow infusion. Comparison of these data to a prior series of patients undergoing bone marrow transplant (BMT) from unrelated donors who were treated with rhGM-CSF along with methotrexate and cyclosporine for GVHD prophylaxis suggests that rhGM-CSF is well-tolerated, neutrophil recovery may be earlier but the severity of GVHD does not appear reduced. Selection of the GVHD prophylaxis regimen may affect the hematopoietic response to cytokine therapy. Further trials with rhGM-CSF in patients undergoing BMT from unrelated donors are required.     

Isolated extramedullary relapse of acute lymphoblastic leukemia in a submandibular salivary gland

Salivary glands are uncommonly involved in leukemia. Moreover, isolated leukemic relapse in salivary glands is an extremely rare event. We report a young adult male with acute lymphoblastic leukemia who presented with an isolated extramedullary relapse of his leukemia in a left sided submandibular salivary gland just before his planned allogeneic bone marrow transplant. The patient underwent a wide surgical excision of the involved salivary gland then he received a conditioning protocol composed of total body irradiation and cyclophosphamide followed by a successful allograft.     

HLA-CW基因分型在造血干细胞移植中的意义探讨

目的探讨HLA-CW位点基因分型在造血干细胞移植(hematopoicetic stem cell transplantation-HSCT)中的作用,对HLA-CW基因位点相合与不相合造血干细胞移植的效果进行分析.方法采用聚合酶链反应-序列特异性引物扩增(polymerase chain reaction-sequence specific primers,PCR-SSP)方法对HSCT供、受体共42个样本进行HLA-CW位点等位基因分析.结果HLA-CW基因全相合者9例造血重建,存活200 d以上7例,1例存活303d复发,2例存活44 d(死于感染),无病生存率(66.7±3.4)%.1例(11.1%)发生急性移植物抗宿主病(aGVHD),3例(33.3%)发生慢性移植物抗宿主病(cGVHD).HLA-CW基因不相合者12例造血重建,存活200 d以上6例,1例存活105 d复发,6例存活75 d死亡,无病生存率(41.7±5.4)%(P＜0.05).6例(50%)发生aGVHD;5例(41.7%)发生cGVHD(P＞0.05).结论HLA-CW基因不相合导致的aGVHD及相关死亡率明显高于CW基因相合者,显示CW基因不相合也是造成aGVHD发生率增高和影响移植效果的重要因素.     

Immunomodulation with dendritic cells and donor lymphocyte infusion converge to induce graft vs neuroblastoma reactions without GVHD after allogeneic bone marrow transplantation

Background:

Mounting evidence points to the efficacy of donor lymphocyte infusion (DLI) and immunisation with tumour-pulsed dendritic cells (DC) in generating graft vs leukaemia reactions after allogeneic bone marrow transplantation (BMT). We assessed the efficacy of DLI and DC in generating potent graft vs neuroblastoma tumour (GVT) reactions following allogeneic BMT.

Methods:

Mice bearing congenic (H2K^a) Neuro-2a tumours were grafted with allogeneic (H2K^b) T-cell-depleted bone marrow cells. Tumour-pulsed donor DC (DC^Neuro2a) were inoculated (on day +7) in conjunction with donor (H2K^b) and haploidentical (H2K^a/b) lymphocytes.

Results:

Murine Neuro-2a cells elicit immune reactions as efficient as B lymphoma in major histocompatibility complex antigen-disparate mice. Lymphopenia induced by conditioning facilitates GVT, and transition to adaptive immunity is enhanced by simultaneous infusion of and DC^Neuro2a and lymphocytes devoid of graft vs host (GVH) activity (H2K^a/b). In variance, the efficacy of DC-mediated immunomodulation was diminished by severe graft vs host disease (GVHD), showing mechanistic dissociation and antagonising potential to GVT.

Conclsions:

The GVHD is not a prerequisite to induce GVT reactivity after allogeneic BMT, but is rather detrimental to induction of anti-tumour immunity by DC-mediated immunomodulation. Simultaneous inoculation of tumour-pulsed donor DC and DLI synergise in stimulation of potent GVT reactions to the extent of eradication of established NB tumours.