人类白细胞抗原单倍体不合造血干细胞移植后自然杀伤性细胞和T淋巴细胞杀伤抑制性受体表达的研究

目的研究人类白细胞抗原(HLA)单倍体不合的造血干细胞移植后自然杀伤性细胞(NK细胞)和T淋巴细胞杀伤抑制性受体重建的规律。方法2004-04～2004-12对北京大学血液病研究所借助三色和四色荧光标记技术,用流式细胞仪对应用该所GIAC方案(即G:粒细胞集落刺激因子 I:强免疫抑制 A:抗胸腺细胞球蛋白 C:外周血与骨髓联合)进行HLA单倍体不合造血干细胞移植的24例患者(移植前、移植后 30、 60、 90、 120、 180d)及其供者外周血NK细胞及T细胞上杀伤免疫球蛋白类受体(KIR),包括CD158a(KIR2DL1)、CD158b(KIR2DL2)、CD158e(KIR3DL1)和Lectin样受体(CD94/NKG2A)的表达进行了测定。结果根据移植后NK细胞上受体重建规律不同可以分为两组:第1组在移植后 30d,与供者表达水平相比,病人受体表达明显升高(CD94,P=0.013;CD94∶NKG2A,P<0.0001;CD158e,P=0.063),随后逐渐下降,至 180d时重建为供者水平(CD158e、CD94)或仍明显高于供者水平(CD94∶NKG2A、P=0.001);第2组在移植后 30d,与供者表达水平相比,病人受体表达明显降低(CD158a,P=0.016;CD158b,P<0.003),随后逐渐升高,至正常供者水平。移植后患者外周血T淋巴细胞上杀伤抑制性受体的表达规律与NK细胞第1组相近,在 30和(或)60d时,所有KIR及CD94∶NKG2A的表达均明显高于正常供者水平,其后逐渐下降, 180d时降至正常供者水平(CD158a、CD158a/CD158b及CD158e)或仍明显高于供者水平(CD94/NKG2A,CD158b)。结论移植后NK细胞上KIR受体的低表达及CD94∶NKG2A的高表达可能是移植后早期NK细胞杀伤功能低下的主要影响因素;而移植后T细胞上KIR及CD94∶NKG2A的高表达可能有利于移植后的移植物抗宿主疾病(GVHD)及移植物抗白血病(GVL)效应分离。     

人类白细胞抗原不合或单倍体亲属供者造血干细胞移植

异基因造血干细胞移植(allo-HSCT)是重症血液病的主要根治性治疗方法。除人类白细胞抗原(HLA)相合的健康同胞为首选供者外,非血缘、脐带血和亲属HLA不合或单倍体造血干细胞都作为重要的干细胞来源进入了临床。美国的统计资料显示只有不超过40%的患者可以找到HLA相合的同胞供者。白人中找到非血缘HLA相合供者的机会约为75%,但在少数人种中这一几率低于50%。尽管如此,非血缘供者的查询过程耗时长,使得许多急性患者无法在最适宜的时机接受移植。脐带血来源的造血干细胞虽然可以立即得到,对供者不构成任何负担而且运输过程污染机会小,但…     

同胞人类白细胞抗原相合骨髓和外周血造血干细胞混合移植后造血重建分析

,P=0.065).以移植物中CD34+细胞的中位数为界,输入CD34+数量＞2.45×106/ks的78例患者中性粒细胞、血小板植入的时间分别为14.5 d和11 d显著快于CD34+细胞数量≤2.45×106/kg的79例患者(16 d和14 d)(P值分别为0.021、0.010).结论 移植物中CD34+细胞是同胞HLA相合骨髓和allo-HSCT后中性粒细胞和血小板植入的影响因素,病程也是血小板植入的一个影响因素.     

人类白细胞抗原不合/单倍体造血干细胞移植的临床和试验研究——访北京大学人民医院北京大学血液病研究所所长黄晓军教授

黄晓军,男。主任医师、教授、博士生导师。现任北京大学人民医院血液病研究所所长。兼任中华医学会血液学分会第七届委员会常务委员、中国医师协会血液科医师分会第一届委员会会长、中华医学会血液学分会造血干细胞移植学组组长、中国抗癌协会血液肿瘤专业委员会委员、北京市抗     

单倍体造血干细胞移植治疗难治复发NK／T细胞淋巴瘤1例

患者,男,16岁,2007年5月下旬出现鼻咽部疼痛,有异物感,随后出现持续发热,体温最高40℃。鼻咽部内镜示：鼻咽部顶后壁弥漫性隆起。病理检查示：非霍奇金淋巴瘤,T—NK型。免疫染色瘤细胞CD45RO（＋）,CD56（＋）,GrB（＋）,CD20（-）。体检：一般情况可,全身浅表淋巴结未触及肿大;咽部充血,软腭饱满,左侧弥漫隆起,无溃疡,扁桃体不大;心肺未见明显异常,肝脾不大。诊断非霍奇金淋巴瘤NK／T细胞型Ⅱ期B。给予BCHOP化疗1次,部分缓解。后给予鼻咽部放疗,     

母供子非去T细胞性单倍体异基因造血干细胞移植治疗恶性血液病

异基因造血干细胞移植（allo-HSCT）是治愈恶性血液病的有效手段，但HLA相合（血缘相关或无关）供者来源的不足极大限制了allo—HSCT的应用。随着研究的进展，单倍体半相合allo-HSCT逐渐应用于临床，它有来源广、易获得、移植快等优点，使其成为allo-BMT研究的热点。我们对18例恶性血液病开展了母供子非去T细胞性单倍体allo-HSCT，探讨其有效性及毒副反应。     

杀伤细胞免疫球蛋白样受体基因多态性对造血干细胞移植后巨细胞病毒感染的影响

  目的 探讨杀伤细胞免疫球蛋白样受体（KIR）基因的多态性对造血干细胞移植后巨细胞病毒（CMV）感染的影响。方法 选择2005年10月至2011年5月行造血干细胞移植供受体138对,移植前采用序列特异性引物聚合酶链反应（PCR-SSP）对供受体KIR基因分型,移植后2周起,每周采用荧光免疫组化法检测移植后患者外周血CMVpp65抗原,分析在CMV阳性组和CMV阴性组,供受体抑制性和激活性KIR基因以及供受体KIR单体型频率的差异。结果 供受体KIR基因频率分布和供受体AA,AB,BB单体型频率分布差异无统计学意义。供体2DS2、2DS4*003-007基因在CMV阳性组频率明显低于CMV阴性组（8%比16%,P=0.0420;3%比13%,P=0.0050）,受体KIR基因在CMV阳性组和阴性组频率的差异无统计学意义。供体BB单体型CMV感染率明显低于AA单体型（36.84%比64.38%,P=0.0299）,受体各单体型间CMV感染率差异无统计学意义。多因素分析发现2DS4*003-007,BB单体型与CMV感染有关。结论 供体KIR 基因多态性与造血干细胞移植后CMV感染相关。        

HLA同胞全相合异基因造血干细胞移植后T细胞重建的规律

目的通过胸腺近输出功能和T细胞受体克隆谱型来研究同胞全相合骨髓移植后T细胞免疫重建。方法应用实时定量PCR的方法定量检测23例同胞全相合骨髓移植后不同时间及70例正常供者T细胞受体重排删除环（TRECs）水平;应用RT-PCR扩增其中10例患者骨髓移植后及5例正常供者外周血的T细胞受体β链可变区（TCRBV）24个家族的基因序列,通过基因扫描的方法判断TCRBV家族的克隆表达、互补决定区3（CDR3）克隆性质及计算TCRBV家族的利用率。结果70例正常人外周血单个核细胞中TRECs为（3351．1±3711．1）拷贝／10^5细胞,年龄与TRECs含量呈负相关。移植前患者TRECs定量检测结果为（307．9±433．3）拷贝／10^5细胞,明显低于正常人。移植后1～5个月患者TRECs水平均明显下降,部分患者未能检测到TRECs。移植后6个月TRECs明显升高并持续1年;移植后24个月TRECs水平有较大提高并接近移植前状态。移植后2～15个月,10例患者在7～16个BV家族有表达,且多克隆表达率为48％,单克隆及寡克隆分布在24个BV家族。结论在移植后早期TRECs为低水平并持续一段时间。移植后6个月,胸腺产生一定数量的初始T细胞并且TCRBV CDR3谱型显示BV家族的利用及多克隆的表达均在增加。     

EB病毒相关T/NK细胞淋巴增殖性疾病的造血干细胞移植现状

正华中科技大学同济医学院附属同济医院余秋霞曹阳*李登举,武汉430030     

HLA配型不合/单倍型造血干细胞移植供者淋巴细胞输注量与急性移植物抗宿主病的关系

急性移植物抗宿主病（aGVHD）是单倍型造血干细胞移植（HSCT）最常见的并发症之一。其发生率和病死率都较高，而且发病机制非常复杂。T细胞起着核心作用，移植物内T细胞过多可导致GVHD，而去除T细胞或T细胞亚群可以预防GVHD，但移植物被排斥和白血病／淋巴瘤的复发率增多。本研究检测59例HLA配型不合／单倍型HSCT供者淋巴细胞及T细胞亚群数，并分析其与受者aGVHD的关系。     

The expression of NK cell inhibitory receptors on cytotoxic T cells in B-cell chronic lymphocytic leukaemia (B-CLL)

Immune surveillance of tumours is mediated by cytotoxic T cells (CTL) that recognise tumour antigen. Reduced reactivity of CTL towards tumour cells could thus lead to disease progression and loss of tumour control. In B-cell chronic lymphocytic leukaemia (B-CLL), the function of tumour-reactive CTL seems to correlate inversely to disease stage. Inhibitory NK cell receptors are known to suppress the CTL response upon interaction with major histocompatibility complex (MHC) class I and increased expression of such receptors on CTL may inhibit the anti-tumour response. So, the aim of this study was to investigate the expression of NK cell inhibitory receptors on CTL in B-CLL patients and if such expression correlated to disease stage. CD8+ T cells from B-CLL patients in Binet stage A (n = 26) and stage C (n = 14) and healthy controls (n = 14) were analysed for the expression of killer immunoglobulin-like receptors (KIR) CD158a (KIR2DL1), CD158b (KIR2DL2), CD158e (KIR3DL1) and the C-type lectin receptor CD94, by flow cytometry analysis. Patients with advanced disease (Binet stage C) had a significantly greater percentage of CTL expressing CD158b, CD158e and CD94 than patients with non-progressive disease (Binet stage A) and healthy controls. Stage C patients also had a significantly higher percentage of CTL expressing CD158a than stage A patients. No statistically significant differences were found between Binet A patients and healthy controls. Our results suggest that increased expression of KIR and CD94 on CTL in advanced stage B-CLL may potentially contribute to the impaired anti-tumour immune response in these patients.     

Functional inhibitory human leucocyte antigen class I receptors on natural killer (NK) cells in patients with chronic NK lymphocytosis

Chronic natural killer (NK) lymphocytosis is a rare disorder characterized by an indolent clinical course. Despite high NK cell numbers, many patients present with only mild clinical symptoms, and are often asymptomatic. NK cells are equipped with a range of receptors that bind human leucocyte antigen (HLA)-class I molecules. The killer immunoglobulin-like receptors (KIR, CD158) bind groups of HLA alleles, the CD94/NKG2 receptors bind HLA-E, and the CD85j (ILT2, LIR-1) receptor binds to the relatively non-polymorphic alpha3 domain of HLA molecules. Inhibitory HLA class I receptors silence NK cells against cells expressing normal levels of HLA class I. Analysis of NK cells in six patients with chronic NK lymphocytosis revealed a high level of the inhibitory CD94/NKG2A receptor on all NK cells. In four patients, KIR were absent, in one patient a single KIR was expressed in the absence of self-ligand, and in one patient CD85j and multiple KIR were expressed. Cytotoxicity assays demonstrated that all HLA class I receptors were functional. The ability of monoclonal antibodies to block the receptors and allow killing of autologous target cells established that both receptor and ligand expression were adequate for inhibitory function. We propose that the silent behaviour of NK cells in patients with chronic NK lymphocytosis is due to effective inhibitory HLA class I receptors.     

慢性乙型肝炎外周血中T细胞亚群和NK细胞的变化及临床意义

目的观察慢性乙型肝炎患者外周血中T细胞亚群及NK细胞含量的变化,进一步了解慢性乙型肝炎患者的免疫功能状况。方法收集48例慢性乙型肝炎患者作为实验组,其中HBe Ag阳性20例,阴性28例,选择26名健康人群作为正常对照组,采用流式细胞仪检测两组血清中CD3+T细胞、CD4+T细胞、CD8+T细胞和NK细胞的含量。结果与正常对照组比较,慢性乙型肝炎患者外周血中CD3+T细胞、CD4+T细胞和NK细胞的含量明显降低,而CD8+T细胞含量升高,差异具有统计学意义(P0.05),而HBe Ag阳性和阴性的慢性乙型肝炎患者之间的差异无统计学意义(P0.05)。结论慢性乙型肝炎患者免疫功能低下,这对患者免疫功能的判断、疾病进展、治疗及预后有一定的指导意义。     

乙型肝炎肝硬化患者外周血T淋巴细胞亚群和NK细胞的变化及临床意义

目的：探讨乙肝肝硬化患者外周血中T淋巴细胞亚群及NK细胞含量的变化,了解乙肝肝硬化患者的免疫功能状况。方法乙肝肝硬化患者81例,并按Child-Pugh分为A、B、C三级,26例健康者作为健康对照,采用流式细胞仪检测血清中CD3＋ T淋巴细胞、CD4^＋ T淋巴细胞、CD8^＋ T淋巴细胞和NK细胞的含量。结果 Child-Pugh B级和C级的乙肝肝硬化患者外周血中CD3^＋ T淋巴细胞、CD4^＋ T淋巴细胞和NK 细胞的含量明显降低,而CD8^＋ T淋巴细胞含量升高,与健康对照组比较,差异有统计学意义（P＜0．05）,而Child-Pugh A级的乙肝肝硬化患者T淋巴细胞亚群变化不明显（P＞0．05）。结论乙肝肝硬化患者的机体免疫功能低下,并且随着其肝功能下降及Child-Pugh 分级的增加而显著,对判断乙肝肝硬化患者的病情及预后具有重要的临床价值。     

Primary CD56+ T/NK cell lymphoma of the colon

Primary T/natural killer (NK) cell lymphoma of the colon is extremely rare. Despite the advances in histological and immunophenotypic studies, the diagnosis of primary T/NK cell lymphoma of the colon can be delayed because the early symptoms and colonoscopic findings may be very similar to those of inflammatory bowel diseases such an Crohn's colitis, and most physicians have little available information on this group of neoplasms. Moreover, florid nonspecific inflammatory infiltrates would not allow characterization of the tumor cells in such an inflammatory background. Herein, we describe a patient who initially presented with features that were clinically and colonoscopically similar to Crohn's colitis. Three months later, he had cecal bleeding and perforation, and primary T/NK cell lymphoma of the colon was diagnosed through immunophenotypic and genotypic studies of surgical specimens. Received: March 5, 2001 / Accepted: August 24, 2001 Reprint requests to: D.K. Lee     

CD16+ CD56- NK cells in the peripheral blood of cord blood transplant recipients: a unique subset of NK cells possibly associated with graft-versus-leukemia effect

A marked increase in CD16+ CD56- NK cells in the peripheral blood (PB) was observed in a cord blood transplant (CBT) recipient with refractory acute myeloid leukaemia (AML) in association with attaining molecular remission. CD16+ CD56- NK cells isolated from the patient became CD16+CD56+NKG2D+ when they were cultured in the presence of IL-2. Although cultured CD16+CD56- NK cells retained the killer-cell immunoglobulin receptor (KIR)-ligand (KIR-L) specificity and the patient's leukemic cells expressed corresponding KIR ligands, they killed patient's leukemic cells expressing ULBP2. The cytotoxicity by cultured CD16+CD56- NK cells was abrogated by anti-ULBP2 antibodies. When leukemic cells obtained at relapse after CBT were examined, both the ULBP2 expression and susceptibility to the cultured NK cells decreased in comparison to leukemic cells obtained before CBT. An increase in the CD16+CD56- NK cell count (0.5 x 10(9)/L or more) in PB was observed in seven of 11 (64%) CBT recipients but in none of 13 bone marrow (BM) and eight peripheral blood stem cell (PBSC) transplant recipients examined during the similar period after transplantation. These findings suggest an increase in CD16+CD56- NK cells to be a phenomenon unique to CBT recipients and that mature NK cells derived from this NK cell subset may contribute to the killing of leukemic cells expressing NKG2D ligands in vivo.     

食管癌贲门癌患者T淋巴细胞、NK细胞免疫功能检测及意义

目的本研究检测食管癌、贲门癌患者的免疫功能,寻找肿瘤患者的病情量化指标,用来预测和判定临床治疗效果。方法采集本院胸外科手术前的食管癌贲门癌患者115例及359例正常体检者的血液标本,应用流式细胞术检测外周血T淋巴细胞表面CD3、CD4、CD8及NK细胞(CD56)的表达。结果食管癌、贲门癌组与健康对照组相比较CD3、CD4有明显下降(P0.05),而CD8、CD56有明显的增高(P0.05)。将癌症组分为淋巴结转移组和无淋巴转移组,两组间CD3、CD4、CD8、CD56的差异均无统计学意义(P0.05)。结论食管癌外周血T细胞免疫检测有助于了解疾病的发展进程。     

非小细胞肺癌患者外周血T细胞亚群、NK细胞与临床分期及预后的相关性

目的分析非小细胞肺癌(NSCLC)患者外周血T细胞亚群、NK细胞的表达,及其与临床分期及预后的相关性。方法采用流式细胞术检测96例NSCLC患者、30例肺部良性疾病及健康体检者外周血T细胞亚群、NK细胞的表达,分析其与肺癌临床分期的关系,对患者随访2年,比较化疗后不同表达水平之间的生存率。结果 NSCLC组患者CD+3、CD+4/CD+8及NK细胞比例较对照组和肺良性疾病组明显降低,而CD+8细胞比例则明显升高(P0.05);Ⅲ-Ⅳ期NSCLC患者CD+3及NK细胞比例较Ⅰ-Ⅱ期患者明显降低,而CD+8细胞比例则明显升高(P0.05);CD+3、CD+4/CD+8及NK细胞与临床分期呈负相关(r=-0.715、-0.673、-0.593,P0.05),CD+8与临床分期呈正相关(r=0.672,P0.05);CD+4/CD+8上升组化疗后2年生存率为82.0%,明显高于未上升组57.6%,差异均有统计学意义(P0.05)。结论 NSCLC患者外周血T细胞亚群、NK细胞分布异常,且与临床分期密切相关,联合检测可客观反映免疫功能状态及病情程度,对NSCLC的治疗及预后具有一定的指导作用。     

The imbalance between NKG2A and NKG2D expression is involved in NK cell immunosuppression and tumor progression of patients with hepatitis B virus-related hepatocellular carcinoma

Background

Immunosuppression in a tumor microenvironment is associated with enhanced tumor progression. Natural killer group 2 (NKG2) family proteins, including inhibitory receptors and activators, can be used as attractive targets for immunotherapy of immune checkpoint inhibition. We further explore the expression level prognostic value of NKG2A and NKG2D in hepatitis B virus-related hepatocellular carcinoma (HBV-HCC).

Methods

This study was a prospective study involving 92 patients with HBV-HCC, 16 patients with HBV-related liver cirrhosis, 18 patients with CHB, and 38 healthy donors. We analyzed the expression and related functions of NKG2A, NKG2D, and the NKG2A/NKG2D ratio in the peripheral blood of patients with HBV-HCC and analyzed tumor progression. The tissue samples from patients with HBV-HCC were further used for multiple immunofluorescence and immunohistochemistry.

Results

In patients with HBV-HCC with tumor progression, the ratio of NKG2A/NKG2D is higher in NK cells and T cells. The Kaplan–Meier survival curve showed that the NKG2A/NKG2D ratio on NK cells could predict tumor progression in patients with HBV-HCC, and that an increase in this ratio was associated with inhibition of NK cell function. The Cancer Genome Atlas (TCGA) database was further used to verify that the higher the NKG2A/NKG2D ratio, the shorter the progression-free survival of patients with HCC, and the more likely the immune function was suppressed.

Conclusions

The imbalance between NKG2A and NKG2D of NK cells is involved in NK cell immunosuppression, and the increase of the NKG2A/NKG2D ratio is related to the tumor progression of HBV-HCC.