混合骨髓移植及移植后供者淋巴细胞输注导致急性白血病复发1例

我科收治1例急性髓细胞性白血病(AML-M2a)患者,持续完全缓解(CR)11月,外周血干细胞动员失败后行HLA半相合混合骨髓移植(MBMT),移植后短期复发,经化疗后获得完全缓解(CR2),后行供者淋巴细胞输注(DLI),DLI后20天白血病复发,现报道如下.     

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.     

供体淋巴细胞输注联合伊马替尼治疗异基因造血干细胞移植后Ph+白血病复发的初步临床观察

 目的 观察供体淋巴细胞输注（DLI）联合伊马替尼治疗异基因移植后复发的Ph+白血病的疗效。方法 以实时定量聚合酶链反应（RQ-PCR）及荧光原位杂交（FISH）方法检测5例Ph+白血病患者移植后微小残留病灶（MRD）,在复发早期进行DLI联合伊马替尼治疗。伊马替尼初始剂量300～400 mg／d,DLI采用递增剂量方案,首次剂量为0.5×106／kg ~ 5×106／kg,间隔时间为1 ~ 4周。观察治疗反应及相关并发症。结果 5例患者中有2例慢性髓细胞白血病（CML）患者发生分子学反应,其中1例达分子学缓解;而2例高危Ph+急性淋巴细胞白血病（Ph+ ALL）患者及1例早期血液学复发的慢性髓细胞白血病第二次慢性期（CML-CP2）患者治疗无反应。复发后随访40 d～14个月,5例应用伊马替尼后均未发生严重并发症。DLI后1例发生Ⅳ度急性移植物抗宿主病（aGVHD）,2例发生Ⅰ～Ⅱ度aGVHD,1例发生肺部感染。结论 伊马替尼联合DLI可有效治疗移植后复发的CML慢性期患者,副作用可耐受,但对复发的Ph+ ALL及CML急变期患者疗效有限。     

供体细胞嵌合率的动态定量检测在供体淋巴细胞回输中的应用

近年来，大量的临床实践证实，供体淋巴细胞回输(donor lymphocyte infirsion，DLI)是治疗异基因造血干细胞移植(allo-HSCT)后白血病复发最有效的手段之一。我们对6例移植后早期复发或移植物被排斥的患者DLI前后供体细胞嵌合率(DC)进行了动态定量检测，并尝试探究DC的定量检测在DLI中的意义。     

供者淋巴细胞输注联合化疗治疗异基因造血干细胞移植后白血病复发的疗效观察

目的 观察供者淋巴细胞输注(DLI)联合化疗治疗异基因造血干细胞移植(Allo-HSCT)后白血病复发的疗效。方法 对Allo-HSCT后白血病复发的4例患者予分次逐渐提高单次剂量方案(EDR．)的DLI联合化疗治疗，观察白血病血液学完全缓解(CR)率、无病生存(DFS)率、移植物抗宿主病(GVFtD)和骨髓抑制发生率。结果 4例均获得血液学CR，CR率100％，STR-PCR测定证实恢复为供者基因型；随访70d～14个月，1例DLI后50天发生慢性GVHD，发生率25％,无1例发生急性GVHD、全血细胞减少和骨髓抑制，3例无病生存(DFS)，1例于DLI后70天死于CMV性间质性肺炎。随访至今，3例DFS患者血象和骨髓象一直正常。结论 EDR DLI联合化疗治疗Allo-HSCT后白血病复发，疗效显著,副作用小,应用安全。     

急性髓性白血病混合造血干细胞移植后DLI+IL-2治疗的临床研究

目的:探讨急性髓性白血病(acute myeloid leukemia,AML)患者在接受自体外周血干细胞混合人类白细胞抗原(human leukocyte antigen,HLA)半相合异体骨髓移植(Mixed-HSCT)后,继予供体淋巴细胞输注+白介素2(DLI+IL-2)治疗的疗效。方法:对23例AML患者在完全缓解期采用TBI+VEMAC预处理方案,实施Mixed-HSCT。造血恢复后给予DLI+IL-2治疗1-8次。结果:所有患者均获得造血重建,中性粒细胞(ANC)≧0.5×109/L的中位时间为14(12-17)天,白细胞(WBC)≧4.0×108/L的中位时间为17(16-21)天。血小板(PLT)≧20×108/L的中位时间为21(19-23)天,PLT≧50×108/L的中位时间为25(24-27)天。+16至+21天时骨髓检查示恢复期骨髓象,无移植物抗宿主病(graft versus host disease,GVHD)发生,有6例形成混合嵌合体(46XX/46XY)。经过3年以上随访,存活15例,长期无病存活率(DFS)为65.2%。结论:Mixed-HSCT后应用DLI+IL-2治疗对急性髓性白血病患者的长期无病生存有积极意义。     

非去髓性造血干细胞移植治疗髓性白血病的成就与展望

近年来提出了非去髓性造血干细胞移植( NST)的策略 ,认为骨髓移植 ( BMT)的作用是通过移植供者淋巴细胞产生过继免疫或通过移植后供者淋巴细胞输注 ( DLI)清除受者来源的残余肿瘤细胞 ,而非移植前超大剂量的化疗与全身照射 ( TBI)。在恶性血液病中采用较易耐受的预处理方案还可以避免和减少移植相关毒性与死亡率以及晚期并发症 ,有助于提高长期存活率。有关 NST用于治疗急性髓性白血病 ( AML)和骨髓增生异常综合征( MDS)的原理与理论基础的依据为 :( 1 )超大剂量放疗、化疗无法完全清除 AML/MDS患者体内的异常细胞 ;( 2 )通过免…     

异基因造血干细胞移植治疗母细胞性浆细胞样树突细胞肿瘤

 【摘要】　目的　探索采用增强预处理强度的异基因造血干细胞移植（allo-HSCT）联合快速递减免疫抑制剂和供者淋巴细胞输注（DLI）策略治疗母细胞性浆细胞样树突状细胞肿瘤（BPDCN）的效果。方法　2009年7月至2011年5月,南方医院血液科确诊2例BPDCN,例1患者以皮肤浸润起病,经皮肤病理活组织检查确诊CD+4 CD+56 LCA+ TdT+ CD+43 BPDCN侵犯皮下及真皮层,移植前经联合化疗处于完全缓解;例2患者以骨髓浸润起病,先后误诊为急性淋巴细胞白血病和急性非淋巴细胞白血病,后经流式细胞术免疫分型诊断CD+4 CD+56 CD+123 BDCA-1+ BPDCN,移植前未缓解。接受以全身放疗联合环磷酰胺为基础的增强预处理allo-HSCT,供者均来源于同胞;采用环孢素A（CsA）联合短疗程甲氨蝶呤（MTX）预防移植物抗宿主病（GVHD）,单倍体相合移植加用兔抗人类胸腺细胞免疫球蛋白（ATG）;移植后2个月开始快速减停免疫抑制剂,采用流式细胞术监测微小残留病（MRD）指导或采用预防性DLI防治复发。结果　移植后2例患者均获完全供者植入及完全缓解,其中例1处于持续缓解,+6个月行DLI诱发IV度皮肤及肠道急性GVHD,经联合免疫抑制治疗后控制,+243天死于血栓性微血管病、弥漫性肺泡出血;例2患者+60天复发,经化疗联合DLI、白细胞介素-2治疗后未缓解,+101天死于败血症、弥漫性血管内凝血。结论　BPDCN以CD+4 CD+56 CD+123 CD+43 TdT+ 树突状细胞来源的肿瘤细胞浸润皮肤和（或）骨髓、临床进程呈高度侵袭为典型特征,增强预处理的allo-HSCT联合快速递减免疫抑制剂和MRD监测指导DLI对早期BPDCN可有效控制疾病发展,但对于难治复发患者仍需更多研究。     

环孢素诱导白血病细胞系Nalm—6细胞凋亡

目的：研究环孢素（环孢霉素A，CsA）在体外对B－细胞性白血病细胞株Nalm-6细胞增殖的影响，并探讨其可能的作用机制。方法：应用细胞形态学、DNA凝胶电泳等方法检测在体外CsA对B－细胞性白血病细胞株Nalm-6细胞增殖的作用。结果：临床耐受剂量CsA能明显抑制Nalm-6细胞增殖，且证实诱导细胞凋亡是CsA抑制增殖的主要机理之一。结论：CsA能有效地通过诱导人类B－细胞性白血病细胞株凋亡而起到抑制增殖的作用。     

环孢素A联合化疗方案治疗复发、难治性白血病

目的：探讨用环孢素A联合化疗方案治疗复发、难治性白血病的疗效。方法：18例急性非淋巴细胞白血病患者，采用CsA+DA方案治疗6例，CsA+MAE方案治疗12例。结果：CsA+DA组完全缓解率为33％。CsA+MAE组完全缓解率为50％。两组病例的有效率及完全缓解率经统计学分析没有显著差异，P值均大于0.5。结论：环孢素能够逆转难治性白血病的耐药性，在难治性白血病的治疗中有一定作用，但缓解后的治疗需要引起重视。     

Infusion of CD4+ donor lymphocytes induces the expansion of CD8+ donor T cells with cytolytic activity directed against recipient hematopoietic cells.

PURPOSE: Donor lymphocyte infusions (DLIs) provide effectivetherapy for patients with relapsed chronic myeloid leukemia after allogeneic bone marrow transplantation. Previous studies have suggested that depletion of CD8+ T cells from the infused donor lymphocytes can reduce the incidence of graft-versus-host disease associated with DLI without reducing antileukemia activity. In this situation however, the immune effector cells responsible for tumor rejection have not been identified. The goal of this study was to characterize these effector populations. EXPERIMENTAL DESIGN: We studied three representative patients with relapsed chronic myeloid leukemia who achieved complete molecular remission after receiving CD8+ T-cell-depleted DLI from HLA-identical sibling donors. Effector T cells were characterized in patient samples after in vitro stimulation and functional assessment. T-cell clones relevant to the immune response were then isolated and further characterized. RESULTS: Analysis of peripheral blood samples collected after DLI indicated the presence of a high frequency of circulating host-reactive cytolytic CD8+ T cells secreting IFN-gamma. These HLA class I-restricted CTLs were specific for recipient minor histocompatibility antigens (mHAs) because they did not recognize target cells of donor origin. One CTL clone was further expanded in vitro and shown to recognize a broadly expressed mHA presented by HLA-B5701. Using a molecular approach, we demonstrated that this clone was expanded in peripheral blood and marrow after DLI. It was not detected before DLI. CONCLUSIONS: These results indicate that CD4+ DLI elicits a potent allogeneic response mediated by mHA-specific CD8+ T cells.     

Role of nonmyeloablative allogeneic stem-cell transplantation after failure of autologous transplantation in patients with lymphoproliferative malignancies.

PURPOSE: Conventional allogeneic stem-cell transplantation (SCT) after a prior failed autograft is associated with a transplant-related mortality rate of 50% to 80%. The aim of the current study was to evaluate the safety and efficacy of sibling, HLA-matched, nonmyeloablative allogeneic SCT with donor lymphocyte infusion (DLI) in patients with lymphoid malignancy after failure of autologous SCT. PATIENTS AND METHODS: A total of 38 patients with refractory, progressive, or relapsed disease after autologous SCT were entered onto this study. The conditioning regimen consisted of the humanized monoclonal antibody CAMPATH-1H, fludarabine, and melphalan. Fifteen of 35 assessable patients received DLI after SCT. RESULTS: Sustained neutrophil engraftment was achieved in 37 recipients, and platelet engraftment was achieved in 35 patients. The estimated transplant-related mortality was 7.9% at day 100 and 20% at 14 months, the median duration of follow-up. Eight patients experienced grade I/II acute graft-versus-host disease (GVHD) after transplantation, but no grade III/IV GVHD was observed in this setting. However, grade III/IV GVHD occurred in seven patients who received DLI. The actuarial overall survival at 14 months was 53%, with a progression-free survival of 50%. DLI produced a further response in three of 15 recipients. CONCLUSION: Nonmyeloablative allogeneic SCT after CAMPATH-1H-containing conditioning is a relatively safe option compared with conventional allogeneic transplantation for patients who have failed previous autologous SCT. The low incidence of early GVHD enabled the subsequent administration of DLI to improve further clinical responses in this poor-risk group of lymphoma and myeloma patients.     

Direct cloning of leukemia-reactive T cells from patients treated with donor lymphocyte infusion shows a relative dominance of hematopoiesis-restricted minor histocompatibility antigen HA-1 and HA-2 specific T cells.

Donor T cells recognizing hematopoiesis-restricted minor histocompatibility antigens (mHags) HA-1 and HA-2 on malignant cells play a role in the antileukemia effect of donor lymphocyte infusion (DLI) in patients with relapsed leukemia after allogeneic stem cell transplantation. We quantified the contribution of HA-1 and HA-2 specific T cells to the total number of leukemia-reactive T cells in three HA-2 and/or HA-1 positive patients responding to DLI from their mHag negative donors. Clinical responses occurring 5-7 weeks after DLI were accompanied by an increase in percentages HLA-DR expressing T cells within the CD8+ T cell population. To clonally analyze the leukemia-reactive immune response, T cells responding to the malignancy by secreting IFNgamma were isolated from peripheral blood, directly cloned, and expanded. Tetramer analysis and specific lysis of peptide-pulsed target cells showed that 3-35% of cytotoxic T lymphocyte (CTL) clones isolated were specific for HA-1 or HA-2. TCR VB analysis showed oligoclonal origin of the HA-1 and HA-2 specific CTL clones. The HA-1 and HA-2 specific CTL clones inhibited leukemic progenitor cell growth in vitro. The relatively high frequency of HA-1 and HA-2 specific T cells within the total number of tumor-reactive T cells illustrates relative immunodominance of mHags HA-1 and HA-2.     

Changes in T lymphocyte subsets in mice with CT26 colon tumors after treatment with donor lymphocyte infusion

The objective of this study was to detect changes in T lymphocyte subpopulations in mice with CT26 subcutaneous colon cancer after treatment with donor lymphocyte infusion (DLI) and cyclophosphamide (CP) chemotherapy. A colon cancer model was established by subcutaneous injection of CT26 carcinoma cells into BALB/C mice. The mice were randomized into different treatment groups. We recorded survival times, tumor growth inhibition rates, histopathological changes, and T lymphocyte subsets in peripheral blood of the mice. Mice treated with DLI and CP survived 33.5?±?5.02 days, which was significantly longer than the survival time of untreated control mice (16.7?±?2.98 days, P?<?0.01). In addition, the tumor inhibitory rate was higher in mice treated with DLI and CP (89.3 %) than that in mice treated with CP or DLI alone (67.1 and 34.5 %, respectively). There were higher levels of T lymphocytes that were CD3⁺ and CD4⁺ in mice treated with DLI alone or the combination of CP and DLI (P?<?0.05), and the ratio of CD4⁺/CD8⁺ cells was significantly improved in these mice (P?<?0.05). DLI combined with chemotherapy significantly prolonged survival and inhibited tumor growth in mice with CT26 colon cancer. This treatment might also improve immune function in these mice. Donor spleen cells that include high numbers of allogeneic lymphocytes and a few stem cells could induce a graft-versus-tumor effect, leading to elimination of residual cancer cells. This indicates that it is potentially a feasible adoptive cellular immunotherapy strategy for the management of solid tumors.     

Minor histocompatibility antigen-specific T cells with multiple distinct specificities can be isolated by direct cloning of IFNgamma-secreting T cells from patients with relapsed leukemia responding to donor lymphocyte infusion.

Graft-vs-leukemia reactivity after donor lymphocyte infusion (DLI) can be mediated by donor T cells recognizing minor histocompatibility antigens (mHags) on recipient hematopoietic cells. To study the diversity of cells involved in this immune response, hematopoietic cell reactive T cells were directly clonally isolated from peripheral blood of patients entering complete remission after DLI. T cells were briefly stimulated with bone marrow cells from patients pretransplant, and IFNgamma-secreting T cells were directly clonally isolated, and expanded. Cytotoxic T-lymphocyte (CTL) clones from individual patients used multiple distinct HLA-restricting molecules and varied in reactivity against patient-derived normal and/or malignant hematopoietic cells. For each patient, CTL clones specific for known immunodominant mHags as well as distinct unknown mHags were found. Within individual patients, CTL clones using the same HLA-restricting element could show differential recognition patterns, indicating further diversity in mHag reactivity. CTL clones from individual patients exhibiting identical specificities could show oligoclonal origin. In conclusion, the direct cloning technique shows that the response to hematopoietic cells after DLI is directed against multiple distinct mHags, including but not limited to known immunodominant mHags, implying that immunotherapy with T cells against multiple mHag specificities may be more effective in eradicating malignant cells.     

Donor Lymphocyte Infusion for the Treatment of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation

The results of donor lymphocyte infusion (DLI) for treatment of relapse after bone marrow transplantation (BMT) are reviewed. Durable complete remission can be achieved at the molecular level for a majority (more than 70%) of patients with CML, when treated at early relapse. Results are less favourable for acute leukemias, although useful responses have been reported. Data are scarce though promising for myelodysplastic syndromes and multiple myeloma. Major treatment-associated toxicities are GVHD and bone marrow aplasia. The latter complication can be predicted by evaluating the level of residual donor-derived hematopoiesis. Modification of infused cells (CD8 negative selection or transduction with a suicide gene), addition of peripheral blood stem cells, and early implementation of escalating doses may counteract the complications and increase the response rate. Response rate is variably influenced by the presence of chronic GVHD after initial BMT, T-cell depleted BMT, underlying disease and stage at relapse, and the level of mixed chimerism. DLI is a direct demonstration of the graft-versus-leukemia effect (GVL). Because GVL after BMT is sometimes the predominant cause of cure, it may be advisable in such situations to redirect the conditioning regimens for BMT towards engraftment and less immediate cytotoxicity.     

Differential Expression of Subspecies of Polyomavirus and Murine Leukemia Virus Enhancer Core Binding Protein, PEBP2, in Various Hematopoietic Cells

The core sequence of the enhancer of murine leukemia virus (MuLV) long terminal repeat is highly conserved in a large number of MuLV strains and appears to play an essential role when SL3-3 or Moloney strains induce T cell lymphoma in mice. We found by using the electrophoretic mobility shift assay that a polyomavirus enhancer core-binding protein, PEBP2, bound to this core motif of MuLV. We also noted that PEBP2 in several hematopoietic cell lines derived from B lymphocyte, macrophage and myelocyte lineages migrated significantly faster than the authentic PEBP2 detected in NIH3T3 (ibroblasts. Interestingly, PEBP2 detected in the cell lines of T lymphocyte lineage appeared to contain both types, which were indistinguishable in electrophoretic mobility from those of NIH3T3 and of B lymphocyte, macrophage and myelocyte lineages. The treatment of the nuclear extract containing PEBP2 with phosphatase generated PEBP3, which is a subcomponent of PEBP2 and retained the same DNA-binding specificity as PEBP2. The altered mobility of hematopoietic cell-derived or T lymphocyte-derived PEBP2 was found to be due to the alteration of the mobility of PEBP3. Based on the distinct mobility of PEBP2/3 of T lymphocytes from those of other hematopoietic cells, we discuss the implication of PEBP2 in MuLV-induced T cell leukemia and T cell-specific gene expression.     

Donor lymphocyte infusion leads to diversity of specific T cell responses and reduces regulatory T cell frequency in clinical responders

T cell responses against malignant cells play a major role in maintaining remission and prolonging overall survival in patients after allogeneic stem cell transplantation and donor lymphocyte infusion (DLI) due to graft-versus-leukemia effect. For better characterization of the T cell responses, we assessed frequency and diversity of leukemia-associated antigen (LAA)-specific cytotoxic T cells using ELISpot and pMHC multimer assays and analyzed the frequency of regulatory T cells (Treg) as well as cytokine profiles before/after DLI. The data were correlated to the clinical course of patients. Significantly more LAA-derived T cell epitopes (p = 0.02) were recognized in clinical responders (R) when compared to nonresponders (NR). In addition, pMHC multimer-based flow cytometry showed a significantly higher frequency of LAA-specific T cells in R versus NR. The frequency of Treg in R decreased significantly (p = 0.008) while keeping stable in NR. No differences in T cell subset analysis before/after DLI were revealed. Clinical responders were correlated to specific immune responses and all clinical responders showed an increase of specific immune responses after DLI. Cytokine assays using enzyme-linked immunosorbent assay showed a significant increase of IL-4 after DLI. Taken together, an increase of specific CTL responses against several LAA after DLI was detected. Moreover, this study suggests that enhanced LAA diversity in T cell responses as well as decreasing numbers of Treg contribute to clinical outcome of patients treated with DLI.     

Differential expression of subspecies of polyomavirus and murine leukemia virus enhancer core binding protein, PEBP2, in various hematopoietic cells.

The core sequence of the enhancer of murine leukemia virus (MuLV) long terminal repeat is highly conserved in a large number of MuLV strains and appears to play an essential role when SL3-3 or Moloney strains induce T cell lymphoma in mice. We found by using the electrophoretic mobility shift assay that a polyomavirus enhancer core-binding protein, PEBP2, bound to this core motif of MuLV. We also noted that PEBP2 in several hematopoietic cell lines derived from B lymphocyte, macrophage and myelocyte lineages migrated significantly faster than the authentic PEBP2 detected in NIH3T3 fibroblasts. Interestingly, PEBP2 detected in the cell lines of T lymphocyte lineage appeared to contain both types, which were indistinguishable in electrophoretic mobility from those of NIH3T3 and of B lymphocyte, macrophage and myelocyte lineages. The treatment of the nuclear extract containing PEBP2 with phosphatase generated PEBP3, which is a subcomponent of PEBP2 and retained the same DNA-binding specificity as PEBP2. The altered mobility of hematopoietic cell-derived or T lymphocyte-derived PEBP2 was found to be due to the alteration of the mobility of PEBP3. Based on the distinct mobility of PEBP2/3 of T lymphocytes from those of other hematopoietic cells, we discuss the implication of PEBP2 in MuLV-induced T cell leukemia and T cell-specific gene expression.