细胞因子在急性移植物抗宿主病中的临床研究

目的:探讨细胞因子IL-21、SIL-2R在异基因造血干细胞移植(allo-HSCT)后急性移植物抗宿主病(aGVHD)发病机理中的作用。方法:观察20例Allo-HSCT患者aGVHD的发病情况,移植前后定期采集20例患者的外周血,采用双夹心酶联免疫吸附法(ELISA)检测其细胞因子IL-21、SIL-2R的浓度。结果:1.异基因造血干细胞移植后20例患者全部获得造血功能重建,中性粒细胞恢复到0.5×109/L及血小板恢复到20×109/L的中位时间分别为移植后13.5天及18天。2.发生aGVHD的患者,IL-21、SIL-2R浓度较移植前明显升高,IL-21、SIL-2R浓度在aGVHD阳性组明显高于aGVHD阴性组(P0.01)。结论:1.细胞因子IL-21、SIL-2R在aGVHD的发病中起重要的正向调节作用,检测异基因造血干细胞移植后患者血清的IL-21、SIL-2R水平有助于预测aGVHD的发生。2.IL-21、SIL-2R与感染无相关性。     

调节性T细胞及间充质干细胞在急性移植物抗宿主病中的研究进展

急性移植物抗宿主病(acute graft versus host disease,aGVHD)的一线治疗方案为糖皮质激素,但药物治疗特异性差,会增加异基因造血干细胞移植(allogeneic hematopoietic stem cell transplantation,allo-HSCT)后感染和复发的风险,而且激素耐药性移植物抗宿主病的治疗效果欠佳,目前尚缺乏标准的二线治疗方案。近年来,随着对具有免疫调节活性细胞的认识,人们发现此类细胞在aGVHD的防治中可能具有独特的作用,因而应用免疫调节性细胞治疗aGVHD引起了广泛关注,其中以调节性T细胞(regulatory T lymphocyte,Treg)和间充质干细胞(mesenchymal stem cells,MSCs)的研究最为广泛。为更好地了解免疫调节性细胞调控aGVHD的机制以及其临床应用的可行性和有效性,该文主要就Treg和MSCs生物学特性以及Treg和MSCs在预防与治疗aGVHD中的作用作一综述。     

异基因造血干细胞移植后激素耐药胃肠道急性移植物抗宿主病的临床分析

目的探讨异基因造血干细胞移植后激素耐药胃肠道急性移植物抗宿主病(aGVHD)的影响因素。方法回顾性分析西安交通大学第一附属医院2012年1月至2019年12月期间行异基因造血干细胞移植,术后发生胃肠道aGVHD 20例患者的临床资料。按照一线糖皮质激素治疗后的反应分为激素敏感组(13例)和激素耐药组(7例)。单因素Logistics回归分析患者性别、年龄、诊断、移植前微小残留病灶、移植类型、供者年龄、供受者关系、供受者ABO血型、输注单个核细胞数、CD34+细胞数、CD3+细胞数、中性粒细胞及血小板植入时间、CMV及EB病毒感染、胃肠道aGVHD发生的时间等与激素耐药胃肠道aGVHD的关系。观察激素耐药患者治疗后的转归,采用Kaplan-Meier生存分析激素耐药及敏感患者的预后差异。结果20例胃肠道aGVHD患者中7例存在激素耐药。胃肠道GVHD发生时间<1个月,激素耐药的风险增加(OR=13.500,95﹪CI=1.197~152.211,P=0.035),患者性别、年龄、诊断、移植前MRD、移植类型、供者年龄、供受者关系、供受者ABO血型、输注的单个核细胞、CD34+细胞、CD3+细胞、中性粒细胞和血小板植入时间、CMV和EB病毒感染均不影响激素耐药(P>0.05)。7例激素耐药胃肠道aGVHD患者均接受二线CD25单克隆抗体治疗,治疗后5例有效,2例无效死亡。与激素敏感组比较,激素耐药组患者1年总生存率(64﹪比52﹪)降低及无进展生存率(28﹪比32﹪)升高,差异无统计学意义(P>0.05)。结论异基因造血干细胞移植后胃肠道aGVHD激素耐药可能与其发生时间相关,发生时间越早,越容易出现激素耐药。     

移植物抗宿主病和移植物抗白血病相关细胞因子的研究进展

异基因造血干细胞移植（allogeneic hemotoic stem cell transplantation,allo-HSCT）是治疗血液系统恶性肿瘤的有效方法,治疗效果主要依赖于移植物抗白血病效应（graft-versus-leukemia,GVL）,可以清除残存肿瘤细胞,达到完全治愈恶性血液病的目的。然而,GVL与allo-HSCT的并发症移植物抗宿主病（graft-versus-host disease,GVHD）紧密相连。通过合理调节一些细胞因子的作用可以达到降低GVHD、保留GVL的目的。该文就GVHD与GVL发生机制中相关细胞因子作用的研究进展做一综述。     

IL-10在输血相关性移植物抗宿主病小鼠模型中的免疫调节作用

目的:IL-10在输血相关性移植物抗宿主病小鼠模型中的免疫调节作用。方法:取BALB/c实验小鼠免疫活性淋巴细胞,分别输注于BALB/c小鼠(设为A组)及BALB/c裸鼠(设为B组),建立TA-GVHD模型,观察小鼠症状,HE染色判断小鼠肝、肺、小肠、皮肤病理变化情况;采用双夹心酶联免疫吸附法(ELISA)检测两组小鼠血清IL-10浓度;用逆转录聚合酶链反应法RT-PCR检测移植后外周血单个核细胞中IL-10的表达。结果:A组中2只死亡(12.5%),B组中3只死亡(18.75%),共5只死亡,29只存活,两组死亡率比较无明显差异(P>0.05)。B组小鼠累及肝、肺、小肠和皮肤病理损伤程度较A组严重;存活小鼠IL-10浓度较死亡小鼠明显升高(P2<0.05);存活小鼠IL-10 mRNA表达阳性率96.55%明显高于死亡小鼠(20.00%)。结论:IL-10在输血相关的移植物抗宿主病小鼠模型中发挥负向免疫调节--免疫抑制作用。     

脐带间充质干细胞辅助治疗糖皮质激素耐药的慢性移植物抗宿主病5例并文献复习

目的:探讨脐带间充质干细胞输注治疗糖皮质激素耐药的慢性移植物抗宿主病的疗效和安全性。方法:5例糖皮质激素耐药的慢性移植物抗宿主病患者在原有免疫抑制剂治疗基础上联合脐带间充质干细胞治疗,2~4次为1个疗程,每次间隔1周。对患者进行定期随访观察其治疗效果、移植相关死亡、输注相关不良事件和复发率。结果:5例患者接受脐带间充质干细胞输注后2例获得完全缓解(CR)、2例获得部分缓解(PR),1例患者死亡。2例CR患者分别在脐带间充质干细胞治疗368、452d后停用免疫抑制剂,随访1~1.5年慢性移植物抗宿主病无复发;2例PR患者在脐带间充质干细胞治疗84、96d后开始进入免疫抑制剂减量阶段,目前病情仍稳定并存活。1例患者死于原发病无复发性肺部严重感染。治疗过程中及治疗后未观察到与治疗有关的副作用。新鲜制备脐带间充质干细胞的细胞活力(92%~95%)高于液氮冻存37℃水浴复苏细胞活力(72%~76%)。结论:脐带间充质干细胞辅助治疗可以改善糖皮质激素耐药的慢性移植物抗宿主病的临床症状且不增加恶性血液病复发率,新鲜制备间充质干细胞活性高于液氮冻存复苏细胞。     

大鼠异基因骨髓移植急性移植物抗宿主病模型的建立

目的建立较稳定的异基因骨髓移植急性移植物抗宿主病动物模型,为异基因骨髓移植后的急性移植物抗宿主病（aGVHD）的相关研究提供实验参照。方法以雄性SD大鼠为供鼠,雌性Wistar大鼠为受鼠,受体大鼠随机分成A、B、C、D、E 5组,移植当天所有受鼠均接受8.5 GY的全身照射（TBI）,于照射后4～6 h内,A组回输等量培养液,B组经尾静脉输注供鼠骨髓细胞（2×10^8个/kg）,C、D、E组分别回输供鼠骨髓细胞（2×10^8个/kg）＋不同比例的脾细胞。观察各组大鼠生存期、外周白细胞计数、及有无aGVHD的临床及病理表现。结果A组大鼠于15d内全部死亡,外周血白细胞计数明显减低,骨髓病理示造血组织减少,提示死于造血衰竭。B、C、D、E组大鼠外周血白细胞计数均有明显恢复,B组大鼠8只存活超过50 d,C、D、E组大鼠均于50 d观察期内死亡,并有aGVHD的临床表现及病理表现,但C组大鼠aGVHD的程度较轻且时间不集中,其中D、E组大鼠可于相对集中的时间内观察到典型aGVHD临床及病理。结论TBI预处理的方式是可行的,单纯输入异基因骨髓细胞不能引起明显的aGVHD,骨髓细胞与脾细胞1∶1及1∶1.5混合组均可作为异基因骨髓移植后理想的aGVHD动物模型。     

aGVHD在allo-HSCT后小鼠早期弥漫性肺损伤的作用及机理研究

为了探讨异基因造血干细胞移植（allo-HSCT）急性移植物抗宿主病（aGVHD）在早期肺损伤中的作用及机理,本研究构建C57BL/6→BALA/C小鼠allo-HSCT aGVHD模型,动态对移植后小鼠的肺脏进行CT、肺组织病理和肺组织内TNFα和IFNγ检测.单纯照射（A组）、同基因移植（B组）和异基因移植（C组）小鼠aGVHD发生率分别为0%,0%和100%.移植后＋3,＋7天3组小鼠肺部未见影像学异常,A组＋12天（濒死前）10只小鼠有两只出现双肺弥漫性毛玻璃状渗出影,B组＋12,＋14天小鼠未出现影像学异常,C组＋14天（aGVHD后3天）10只小鼠有6只出现双肺弥漫性毛玻璃状渗出影.3组小鼠移植后＋3天肺组织病理病变类似：轻度间质性肺炎.A组：＋7天肺上皮细胞水肿、增生;＋12天肺泡上皮细胞坏死,淋巴细胞浸润,肺泡腔出血、蛋白物渗出.B组：＋7天病变较＋3天减轻,＋14天恢复正常.C组：＋7天肺毛细血管高度扩张、充血,淋巴细胞浸润;＋14天肺组织水肿,淋巴细胞浸润与巨噬细胞浸润,蛋白物渗出,血管周围性炎症.肺组织内TNFα组内比较：A组＋7天低于＋3天;B组＋3天达高峰,＋7和＋14天呈下降趋势;C组＋7天达高峰,＋7与＋14天比较无差异（P=0.816）.IFNγ组内比较：A组＋7天高于＋3天;B组随时间延长呈上升趋势,但不同时间比较没有差异（P值分别为0.521,0.118,0.340）;C组＋7天达高峰,＋14天呈下降趋势.研究表明,aGVHD是allo-HSCT后造成早期非感染性肺损伤的原因之一,淋巴细胞和TNFα参与aGVHD肺损伤的发病机理,IFNγ在aGVHD发生后水平下降可能与晚期非感染性弥漫性肺损伤中肺纤维化有关。     

未成熟树突状细胞防治移植物抗宿主病的实验研究

目的：观察供者未成熟树突状细胞（imDC）刺激自体T细胞增殖的能力,探讨利用imDC防治移植物抗宿主病（GVHD）临床应用的可行性。方法：Ak健康供者外周血分离单核细胞,采用重组人粒细胞巨噬细胞集落刺激因子（rhGM-CSF）和白细胞介素（IL）4联合培养4d,诱导其分化成imDC;培养7d,分化成mDC。并通过倒置显微镜和HE染色观察细胞形态、流式细胞仪检测细胞表型。采用MLR方法,构建GVHD发生机制的模型,比较供者imDC和mDC刺激自体T细胞增殖的能力。结果：（1）培养4天后细胞具有典型的imDC特征,CDla、CD83和双抗分别表达为55．79％、64、67％和46、67％,成熟标志CD83表达较低;培养7天后具有典型mDC特征,CDla、CD83和双抗表达分别为61．56％、82．40％和64．12％,成熟标志CD83表达较高。（2）MLR法共孵育72小时后,加入CCK-8检测OD值,imDC组与对照组比较无统计学意义（P〉0．05）,不能刺激自体T细胞增殖（SI〈1．00）;mDC组与对照组、imDC组比较均有显著统计学意义（P〈0．01）,能够刺激自体T细胞增殖（SI〉2．00）。结论：供者imDC能够诱导自体T细胞低反应,有望用来防治GVHD。     

人白细胞介素-10在大肠杆菌中的克隆与表达

目的:构建人IL-10原核表达载体,并对表达产物进行鉴定。方法:应用RT-PCR技术从ConA诱导的单核细胞中扩增出IL-10成熟肽cDNA片段,将其克隆到PGEM-T后载体测序,双酶切回收目的片段构建IL-10原核表达载体PET28a-IL10。PET28a-IL10转化大肠杆菌BL21(DE3),经IPTG诱导表达后对其表达产物进行SDS-Page和Western Blot分析。结果:SDS-Page电泳显示IL-10在大肠杆菌中得到了表达,分子量为21000,Western Blot分析显示表达产物可于IL-10多抗特异反应。结论:IL-10在大肠杆菌中成功表达,具有免疫原性。为其进一步生物学研究奠定了基础。     

Influence of genetic polymorphisms of cytokine genes in the outcome of HLA-matched allogeneic stem cell transplantation in a South East Asian population

Non-HLA gene polymorphisms have been shown to be associated with the risk of graft-versus-host disease (GVHD) and outcome of allogeneic haematopoietic stem cell transplantation (AHSCT). This study aims to investigate the role of IL6, TNFα, IL10, IL2 and IL12 gene polymorphisms in the outcome of AHSCT in a South East Asian population. A total of 67 patients and 59 donors who underwent HLA-identical matched sibling AHSCT were available for analysis. There was no significant association between the different cytokine genotypes of patients with the incidence and severity of acute GVHD. Patients with IL2 1661T allele and patients who received donor stem cells who had IL2 1661G allele appeared to have reduced incidence of cGVHD. Patients who received donor stem cells with IL12 11881C allele are found to be associated with better disease free survival. These results suggest a possible role of IL2 and IL12 gene polymorphisms in the outcome of AHSCT in a South East Asian population.     

Advances in the treatment of acute graft‐versus‐host disease

Allogeneic hematopoietic stem cell transplantation (HSCT) has been widely used for the treatment of hematologic malignant and non‐malignant hematologic diseases and other diseases. However, acute graft‐versus‐host disease (GVHD) is a life‐threatening complication of allogeneic transplantation. Acute GVHD may occur in 30% of transplant recipients, which is a syndrome of erythematous skin eruption, cholestatic liver disease and intestinal dysfunction, resulting from the activation of donor T lymphocytes by host antigen‐presenting cells, resulting in an immune‐mediated inflammatory response. Recent scientific advances in the understanding of the pathogenesis involved in the development of acute GVHD and clinical investigation have provided more effective therapeutic strategies for acute GVHD. This review focuses on major scientific and clinical advances in the treatment of acute GVHD.     

Effect and mechanism of acute graft versus host disease on early diffuse murine lung injury following allogeneic stem cell transplantation

To explore the effect and pathogenssis of acute graft-versus-host disease (aGVHD) on early diffuse lung injury in allogeneic hematopoietic stem cell transplantation (allo-HSCT), we established an aGVHD model of C57BL/6→BALB/c mice. Chest computed tomography (CT) scans, histopathology and the levels of cytokines including tumor necrosis factor α (TNFa) and Interferon (IFNg) in lungs were dynamically detected in recipient mice after transplantation. The incidence of aGVHD was respectively 0%, 0% and 100% in simple irradiation group (A), syngeneic transplant group(B) and allogeneic transplant group (C). Chest CT scans of recipient mice were normal in 3 groups on days +3 and +7 after transplantation. CT showed that two of ten mice had bilateral lung diffuse infiltrate on day +12 (on the brink of death) in group A and 6 of 10 mice had bilateral lung diffuse infiltrate on day +14 (3 d after aGVHD occurring) in group C, and were normal on days +12 and +14 in group B after transplantation. Histopathology of lungs in the 3 groups was similar, consisting of minor interstitial pneumonitis on day +3. Group A showed edema, hyperplasia of epithelial cells and widened alveolar interval on day +7, and epithelial cell necrosis, lymphocyte infiltration, hemorrhage, protein leakage, and local consolidation on day +12. The histopathology of group B showed slight edema of epithelial cells on +7 day, which were slighter than that on day +3, and virtually normal on day +14. The histopathology in group C was characterized by the significant expansion and congestion of capillaries, and lymphocyte infiltration on day +7, the acute pneumonitis was present involving tissue edema, lymphocyte and macrophage infiltration, protein leakage and perivascular inflammation on day +14. In group A, the levels of TNFa were lower on day +7 than on day +3. In group B, the levels of TNFa attained a peak on day +3, which decreased on days +7 and +14. In group C, the levels of TNFa were highest on day +7 and there was a significant difference between those on days +7 and +14 (P=0.816). In group A, the levels of IFNg on day +7 were higher than on day +3. In group B, the levels of IFNg increased progressively, but the comparison of IFNg levels in different times had no statistical significance (P=0.521, 0.118, 0.340). In group C, the levels of IFNg attained a peak by day +7 and decreased on day +14. aGVHD is the main cause of early non-infectious lung injury. T lymphocytes and TNFa are possibly implicated in the pathogenesis of acute GVHD-induced lung injury. The decreased levels of IFNg in lung tissues following transplantation might be associated with pulmonary fibrosis in late non-infectious pulmonary complications.     

TNFR2 signaling modulates immunity after allogeneic hematopoietic cell transplantation

Tumor necrosis factor-α (TNF-α) signaling through TNF receptor 2 (TNFR2) plays a complex immune regulatory role in allogeneic hematopoietic cell transplantation (HCT). TNF-α is rapidly released in the circulation after the conditioning regimen with chemotherapy and/or radiotherapy. It activates the function of donor alloreactive T cells and donor Natural Killer cells and promotes graft versus tumor effects. However, donor alloreactive T cells also attack host tissues and cause graft versus host disease (GVHD), a life-threatening complication of HCT. Indeed, anti-TNF-α therapy has been used to treat steroid-refractory GVHD. Recent studies have highlighted another role for TNFR2 signaling, as it enhances the function of immune cells with suppressive properties, in particular CD4⁺Foxp3⁺ regulatory T cells (Tregs). Various clinical trials are employing Treg-based treatments to prevent or treat GVHD. The present review will discuss the effects of TNFR2 signaling in the setting of allogeneic HCT, the implications for the use of anti-TNF-α therapy to treat GVHD and the clinical perspectives of strategies that specifically target this pathway.     

Therapeutic effects of hydrogen on chronic graft‐versus‐host disease

The incidence of chronic graft‐versus‐host disease (cGVHD) is rising recent years, which has been the leading cause of non‐transplantation mortality post allogenetic hematopoietic stem cell transplantation (HSCT). Imbalance of inflammatory cytokines and fibrosis plays critical roles in the pathogenesis of cGVHD. Recent studies showed that molecular hydrogen has anti‐inflammatory, antioxidant, anti‐fibrosis effects. Therefore, we hypothesized that molecular hydrogen may have therapeutic effects on cGVHD. To determine whether hydrogen could protect mice from cGVHD in an MHC‐incompatible murine bone marrow transplantation (BMT) model, survival rates of mice were calculated, and skin lesions were also evaluated after BMT. This article demonstrated that administration of hydrogen‐rich saline increased survival rate of cGVHD mice. Administration of hydrogen‐rich saline after transplantation also reduced skin lesions of cGVHD mice. Previously, we reported the therapeutic effects of hydrogen on acute GVHD. However, there was no report on the therapeutic effects of hydrogen on cGVHD mice. It is suggested that hydrogen has a potential as an effective and safe therapeutic agent on cGVHD. This study will provide new ideas on the treatment of cGVHD and has important theoretical values.     

Therapeutic potential of low-dose IL-2 in a chronic GVHD patient by in vivo expansion of regulatory T cells

Chronic graft-versus-host disease (cGVHD) is a common complication following allogeneic hematopoietic stem cell transplantation (HSCT), which is characterized by autoimmune like inflammatory responses and reduced levels of regulatory T cells (Tregs). Recently, the use of low-dose IL-2 has been reported to selectively increase Tregs and therefore facilitate immune regulation and promote clinical improvements in cGVHD patients. In this report, we describe the case of a cGVHD patient who was treated with daily low-dose IL-2 therapy. Our observations demonstrate that low-dose IL-2 could induce significant expansion of Tregs in vivo leading to improved Treg/Th17 ratios. The patient showed moderate clinical benefits suggesting that multiple factors may be involved in the immunological responses. Therefore, while the therapeutic potential of low-dose IL-2 is promising, strategic approaches may be needed to induce a clinically significant and sustained Treg effect.     

单倍体造血干细胞移植对儿童重型再生障碍性贫血治疗的并发症分析

目的分析儿童重型再生障碍性贫血(SAA)单倍体造血干细胞移植和同胞全相合移植并发症发生率。方法回顾性分析2010年1月1日至2018年12月31日于苏州大学附属儿童医院进行治疗的SAA患儿(56例),分为治疗组(单倍体造血干细胞移植,35例),对照组(同胞全相合移植,21例)。其中患儿年龄、诊断距离移植时间、总单个核细胞数、总CD34+细胞数、中位随访时间、粒细胞植入时间、血小板植入时间及等级资料[供受体血型相合程度、急性移植物抗宿主病(aGVHD)分级、广泛性慢性移植物抗宿主病(cGVHD)分级、出血性膀胱炎分级、渗漏综合征分级]采用Mann-Whitney U检验。性别、疾病种类、供体与受体性别、预处理方案、CD20单抗的使用、HLA配型、供体来源、移植物来源、植入综合征、巨细胞病毒(CMV)血症、EB病毒(EBV)血症、死亡人数、植入失败人数和骨髓增殖不良人数使用卡方检验进行组间分析。生存曲线以及累积发生率用Kaplan-Meier法绘制,并使用Log-rank检验分析组间差异。结果与对照组比较,治疗组CD19+B细胞的重建延迟,而CD16+CD56+NK细胞数量移植后6个月开始增加,逐渐达到对照组的水平。与对照组比较,治疗组aGVHD、cGVHD、植入综合征和骨髓增殖不良累积发生率(14.29﹪±7.64﹪比57.14﹪±8.36﹪,11.67﹪±7.75﹪比61.59﹪±9.65﹪,9.53﹪±6.41﹪比74.86﹪±7.43﹪,0.0﹪比14.29﹪±5.91﹪)均升高,差异具有统计学意义(P<0.05),两组CMV与EBV感染,5年总体生存率(OS)、无失败生存率(FFS)、无GVHD失败存活率(GFFS)、Ⅲ-Ⅳ度aGVHD及广泛性cGVHD累积发生率比较差异无统计意义(P>0.05)。结论单倍体移植是治疗儿童SAA的有效治疗方案,但与同胞全合造血干细胞移植比较,其GVHD发生率较高,植入综合征和骨髓增殖不良的发生率较高,优化单倍体移植方案有望降低并发症,提高儿童重型再生障碍性贫血的生活质量。     

CD4+ T cells in aged or thymectomized recipients of allogeneic stem cell transplantations

Background

CD4+CD25highFOXP3+ regulatory T (Treg) cells, which include thymus-derived and peripherally induced cells, play a central role in immune regulation, and are therefore crucial to prevent graft-versus-host disease (GVHD). The increasing use of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for elderly patients with thymus regression, and our case of allo-HSCT shortly after total thymectomy, raised questions about the activity of thymus-derived Treg cells and peripherally induced Treg cells, which are otherwise indistinguishable.

Results

We found that despite pre-transplant thymectomy or older age, both naïve and effector Treg cells, as well as naïve and effector conventional T cells, proliferated in allo-HSCT recipients. Higher proportions of total Treg cells 1 month post allo-HSCT, and naïve Treg cells 1 year post allo-HSCT, appeared in patients achieving complete chimera without developing significant chronic GVHD, including our thymectomized patient, compared with patients who developed chronic GVHD.

Conclusions

Treg cells that modulate human allogeneic immunity may arise peripherally as well as in the thymus of allo-HSCT recipients.