急性淋巴细胞白血病患儿化疗结束后T淋巴细胞克隆谱系分析

目的了解急性淋巴细胞白血病(ALL)患儿化疗结束后T淋巴细胞免疫重建情况。方法逆转录-聚合酶链反应(RT-PCR)高压变性聚丙烯酰胺凝胶电泳法检测8例正常对照及44例化疗结束后的ALL患儿外周血T细胞受体(TCR)β链可变区(BV)第三互补决定区(CDR3)的克隆谱系。结果白血病化疗结束至48个月TCRBV家族仍可见表达增高或降低(P均0.05)。化疗结束后TCRBVCDR3谱型多态性基本恢复,但寡克隆增生情况仍明显高于正常对照组(P均0.05),小于6个月组差异最明显(P均0.05)。16例普通B细胞急淋(c-ALL)BV8、BV5.1、BV5.2和BV12发生克隆性增生的频率较高,9例前B细胞急淋(pre-B)BV6家族发生克隆性增生较多。结论白血病患儿化疗结束至48个月T细胞免疫尚未完全恢复;T细胞克隆谱系异常以寡克隆增生为主。     

急性白血病患儿外周血T淋巴细胞亚群和NK细胞水平测定及临床意义

为探讨急性白血病患儿外周血T淋巴细胞亚群和NK细胞水平及意义 ,采用流式细胞技术测定 90例急性白血病患儿外周血T淋巴细胞亚群和NK细胞水平。结果 ,急性白血病患儿外周血T淋巴细胞亚群中CD3+ 、CD4+ 、CD4+ CD8+ 均明显低于对照组水平 (P <0 0 1) ,CD8+ 高于对照组水平 (P <0 0 1) ,NK细胞 (CD1 6+ 56+ )明显低于对照组水平 (P <0 0 1) ,经治疗缓解者T淋巴细胞亚群和NK细胞达正常水平。结果表明 ,T淋巴细胞亚群和NK细胞可作为急性白血病辅助诊断指标 ,为临床治疗提供实验依据     

单纯性肾病综合征患儿T淋巴细胞亚群及体液免疫变化

目的　探讨单纯性肾病综合征 (SNS)患儿T淋巴细胞及体液免疫变化。方法　应用流式细胞仪和特定蛋白分析仪对SNS患儿 1 4例活动期和缓解期 (1 2例 )及 1 5例正常儿童外周血T淋巴细胞亚群及血清免疫球蛋白 (Ig)进行检测。结果　SNS患儿活动期组外周血总CD3 、CD4、CD8细胞低于对照组 (t=2 .1 4 ,2 .86　P均 <0 .0 5)。活动期组CD4 、CD8 细胞明显低于缓解期组 (t=2 .49,2 .2 9　P均 <0 .0 5)。活动期组CD4 /CD8 明显低于缓解期组和对照组 (t =2 .75 ,4.1 0　P均<0 .0 5)活动期组和缓解期组血清IgG与对照组比较均有明显降低 (t=2 .75 ,7.0 1　P <0 .0 5 ,0 .0 1 )。结论　SNS患儿在活动期周围血T淋巴细胞数量异常 ,且T淋巴细胞亚群间比例失调 ,且伴体液免疫异常。     

手足口病患儿外周血淋巴细胞亚群变化的临床研究

目的 探讨手足口病(HFMD)患儿外周血T淋巴细胞亚群、B淋巴细胞、NK细胞的变化特点.方法 将41例HFMD患儿分为一般病例组23例,重症病例组18例,选择20例健康儿童作为对照组,采用流式细胞仪检测外周血T淋巴细胞亚群、B淋巴细胞、NK细胞.结果 CD3~+与CD4~+细胞表达:HFMD重症组、一般病例组、对照组依次显著降低(P＜0.01);CD8~+细胞表达:HFMD重症组、一般病例组、对照组依次显著降低(P＜0.05);NK细胞表达:HFMD重症组与一般病例组相似(P＞0.05),皆高于对照组(P＜0.05);B淋巴细胞表达:HFMD重症组、一般病例组、对照组依次升高(P＜0.01).结论 HFMD患儿免疫系统功能紊乱,其中细胞免疫有不同程度的抑制,体液免疫反应亢进.     

传染性单核细胞增多症外周血B淋巴细胞CD21的表达

目的　探讨外周血B淋巴细胞CD2 1 的表达在传染性单核细胞增多症 (IM)发病机制中的作用。方法　用流式细胞术测定IM患儿 38例、新生儿 1 4例、对照组 1 4例的外周血B淋巴细胞CD2 1 表达水平。结果　IM组外周血中B淋巴细胞比例、表达CD2 1 B淋巴细胞数及B淋巴细胞表达CD2 1 位点均高于 2个对照组 (P <0 .0 5 ,P <0 .0 1 )。结论　EB病毒 (EBV)感染可诱导B淋巴细胞持续高表达CD2 1 ,以利于病毒进入细胞并导致潜伏型感染和增殖性感染。     

CD58在儿童急性B淋巴细胞白血病中的表达及其用于微小残留病检测的可行性研究

目的 检测淋巴细胞功能相关抗原-3(CD58)在儿童急性B淋巴细胞白血病(B-ALL)中的表达情况,并探讨其用于儿童B-ALL微小残留病(MRD)检测的可行性。方法 选取2014年1~9月确诊为B-ALL的87例患儿为研究对象,同期选取20例非肿瘤、非血液病且骨髓涂片细胞形态正常的住院患儿作为对照组。应用四色流式细胞术(FCM)检测对照组及B-ALL患儿初诊时、诱导化疗15 d时骨髓标本的CD58表达水平。在诱导化疗33 d时,采用实时荧光定量聚合酶链反应(RT-PCR)和FCM对B-ALL患儿进行MRD检测。结果 87例B-ALL患儿骨髓CD58平均荧光强度(MFI)(91±33)高于对照组(14±6)(P< 0.01);其中44例B-ALL患儿CD58强表达。在诱导化疗过程中,诱导化疗15 d时B-ALL患儿骨髓CD58的表达量(105±22)与初诊时(107±26)比较差异无统计学意义(P> 0.05)。对44例CD58强表达B-ALL患儿进行MRD检测,FCM检测MRD阳性患儿9例,阴性患儿35例;RT-PCR检测MRD阳性患儿11例,阴性患儿33例,42例(95%)检测结果一致,两种检测方法检测MRD差异无统计学意义(P> 0.05)。结论 CD58在B-ALL患儿中高表达且表达稳定,可以作为B-ALL患儿MRD检测的指标。     

儿童急性白血病淋巴细胞变化与化疗和感染相关性探讨

目的分析儿童急性白血病化疗后外周血中淋巴细胞的变化，探讨淋巴细胞值异常与患儿继发免疫紊乱，导致感染及重症感染发生之间的关系。方法急性淋巴细胞白血病(ALL)27例，急性髓性白血病(AML)9例，经化疗骨髓持续缓解(CCR)9—12个月和21—24个月，外周血白细胞≥4．0×10^9／L时，用流式细胞仪、单克隆抗体(美国BD公司)分别检测患儿外周血中CD3+、CD4+、CD8+、CD19+、CD16／56+细胞的百分数，进行统计分析。结果1．随着化疗次数的增加，机体的免疫活性细胞会受到不同程度损害，T细胞亚群的比例明显失调，CD4+细胞百分数下降(P<0．01)，CD8+细胞百分数上升(P<0．05)，CD4+／CD8+细胞比值严重倒置(P<0．01)，CD19+细胞百分数下降(P<0．05)，临床上常见白血病患儿易感染和多发严重感染亦与此有关。2．CD16／56+标记的自然杀伤细胞并不随化疗次数增加而有变化(P>0．05)，在白血病的治疗中有重要意义。3．AML外周血中CD3+、CD4+、CD8+、CD4+／CD8+、CD19+、CD16+／56+细胞百分数无改变(P>0．05)。结论ALL患儿免疫活性细胞随着化疗次数的增加受到明显影响，有必要尽早进行免疫干预治疗，以提高患儿机体的免疫功能，以增强抗感染能力，恢复机体的免疫监督能力。对CD19+细胞减少，在感染发生时，给与丙种球蛋白，可减轻感染程度。     

β整合素家族在儿童急性T淋巴细胞白血病的表达及其临床意义

目的探讨β整合素家族成员在儿童急性T淋巴细胞白血病(T-ALL)的表达及临床意义。方法收集22例初诊T-ALL患儿和21例对照(非恶性血液病患者和骨髓移植供者)的骨髓标本,采用实时荧光定量PCR方法,检测β整合素家族各成员的m RNA表达;并利用整合素抑制剂精氨酸-甘氨酸-天冬氨酸(RGD)肽作用于Jurkat细胞,采用CCK 8法和流式细胞术检测Jurkat细胞生存率和凋亡情况。结果与对照组相比,T-ALL患儿的整合素β_2、β_3、β_5的m RNA表达水平显著下调(P0.05)。在外周血白细胞计数100×10~9/L、第33天骨髓不缓解或MRD阳性的T-ALL患儿中,整合素β_3表达水平较高(P0.05);复发T-ALL患儿整合素β_5的表达高于无复发T-ALL患儿(P0.05)。整合素β3高表达T-ALL患儿的EFS率、OS率均低于β_3低表达者(P0.05)。与未处理组比较,RGD肽处理的Jurkat细胞生存率较低、凋亡率均高(P0.05)。结论β整合素可能通过影响细胞的增殖和凋亡而影响T-ALL的发生发展,整合素β_5的表达与T-ALL复发风险密切相关,整合素β_3的表达与T-ALL患儿治疗反应及预后密切相关。     

支原体肺炎患儿辅助性T淋巴细胞亚群TH1、TH2细胞状况

目的　探讨肺炎支原体肺炎急性期患儿外周血淋巴细胞亚群以及辅助性T淋巴细胞亚群TH1、TH2细胞的改变 ,为免疫治疗的可能性提供依据。方法　采用流式细胞仪技术 (FACS)检测了3 5例支原体肺炎急性期患儿外周血T淋巴细胞亚群以及NK细胞和B细胞 ,同时通过检测分泌细胞因子γ干扰素 (IFN γ)、白细胞介素 4(IL 4)的CD+ 4细胞方法 ,测出相应TH1、TH2细胞的百分比 ,并与2 8例正常儿童进行比较。同时对 3 5例支原体肺炎患儿中的 3 0例进行了血清免疫球蛋白及精制结核菌素 (PPD)皮肤试验。全部患儿均系在我院住院的患儿 ,男 15例 ,女 2 0例 ,年龄 3～ 13岁 ,平均 9岁。对照组男 14例 ,女 14例 ,年龄 3～ 12岁 ,平均 7岁。结果　支原体肺炎患儿急性期外周血CD+ 3 、CD+ 4T细胞百分率为 68 0 0± 6 66及 3 7 86± 5 84,较对照组 63 71± 7 92及 3 4 54± 6 2 3高 ,(P <0 0 5) ;患儿外周血TH1细胞百分率为 14 13± 8 46,对照组为 2 0 77± 6 89,两者差异有非常显著意义 (P =0 0 0 1)。NK细胞及TH1/TH2比值在患儿组降低 (P分别为 <0 0 1和 <0 0 5)。两组间CD8、TH2、B细胞百分率及CD4/CD8比值差异无显著意义。 3 0例患儿之血清免疫球蛋白与同龄正常儿童比较 ,IgG全部正常 ;IgA升高 7例 ;IgM升高 4例。皮肤P     

急性白血病儿童缓解前后血T淋巴细胞嗜银蛋白检测的意义

目的通过对急性白血病(AL)儿童T淋巴细胞(T细胞)增殖活性的rDNA和rRNA的核仁组成区嗜银蛋白(Ag-NORs)测定,以了解患儿T细胞功能状态。方法分别对42例初发AL儿童化疗前与临床完全缓解后及30例正常儿童(对照组),用图像分析法测定血T细胞Ag-NORs(银染区)/细胞核面积(I.S%)。结果AL化疗前I.S%为5.06%±1.36%,与对照组7.51%±1.06%比较有显著差异(t=8.238 P<0.001),但在完全缓解后I.S%升至7.17%±0.98%,与化疗前比较(t=7.073 P<0.001);与对照组比较无差异(t=1.403 P>0.05)。结论在初发未化疗AL儿童中,Ag-NORs水平降低,提示患儿有T细胞免疫功能下降。     

Recent research on chimeric antigen receptor T cells in children with refractory/relapsed acute lymphoblastic leukemia北大核心CSCD

目前儿童复发难治性急性淋巴细胞白血病的治疗仍处于困境,即使提高化疗强度或联合造血干细胞移植,仍有部分患儿预后差,生存期短。嵌合抗原受体T细胞(chimeric antigen receptor T-cell,CAR-T)免疫疗法通过基因工程修饰T细胞,并利用不依赖于人类白细胞抗原途径识别肿瘤特异性抗原,靶向结合目标抗原细胞,触发免疫反应,从而发挥持续的抗白血病效应。作为发展最为迅速的肿瘤免疫疗法,CAR-T细胞在多种血液肿瘤的治疗中取得了突破性的进展,但目前国内尚未建立全面的CAR-T细胞研发生产体系和规范的临床诊治方案。该文就CAR-T细胞在儿童复发难治性急性淋巴细胞白血病中的研究进展作一综述。     

Childhood blastic NK cell leukemia successfully treated with L-asparagenase and allogeneic bone marrow transplantation

Blastic NK cell lymphoma/leukemia is a rare and highly malignant neoplasia in both adults and children. It is characterized by lymphoblastoid morphology without cytoplasmic granules and immature NK cell immunophenotypes (CD56+, CD57-, CD16-). It has predilection for extranodal organ involvement, and the prognosis of affected patients is extremely poor under the current chemotherapy. We present a 14-year-old girl who was diagnosed as having blastic NK cell leukemia with mediastinal, pleural, and pericardial involvement. Immunophenotyping of her leukemic cells showed positive for CD2, CD5, CD7, CD34, CD56, HLA-DR, and cytoplasmic CD3. T cell receptor (TCR) and Immunoglobulin heavy chain genes were not rearranged. She received chemotherapy for acute lymphoblastic leukemia incorporating L-asparaginase (L-asp) which successfully induced complete remission. Bone marrow transplantation (BMT) from her HLA-identical sibling was conducted after two courses of consolidation therapy. Expression of aspargine synthetase (AS) protein in the leukemic cells at diagnosis was examined by an immunocytochemical method. She remains in hematological remission for over 36 months after BMT. The expression of AS protein was negative, suggesting that the leukemic cells were sensitive to L-asp. Induction and consolidation therapy incorporating L-asp followed by allo-BMT might be a promising treatment for child hood blastic NK cell leukemia, but more samples of the rare leukemia need to be studied before any definitive conclusions can be drawn.     

免疫组库测序分析新生儿脓毒症患者外周血T细胞受体β链CDR3的多样性

目的 采用免疫组库测序的方法分析新生儿脓毒症患者外周血T细胞受体（T cell receptor，TCR）β链互补决定区3（complementarity determining region 3，CDR3）的多样性，探讨新生儿脓毒症的可能发病机制。 方法 12例新生儿脓毒症确诊患儿为病例组，9例胎龄、出生体重及日龄相匹配的健康足月儿为对照组。采用Omega公司核酸纯化试剂盒（SQ Blood DNA Kit II）从外周血样品中提取DNA，对TCR β链CDR3进行多重PCR扩增，然后对产物进行高通量测序，分析其TCR β链CDR3多样性及表达的差异。 结果 病例组和对照组的TCR β链CDR3长度和类型相似，且均呈高斯分布。采用D50和香农-威纳指数作为多样性的评价指标，显示病例组TCR β链CDR3多样性低于对照组，差异有统计学意义（P<0.05）。针对TCR β链V片段48个基因的使用频率进行比较，其中TRBV10-3、TRBV2和TRBV20-1的使用频率病例组高于对照组，差异有统计学意义（P<0.05）；对TCR β链J片段13个基因的使用频率进行比较，其中TRBJ2-3、TRBJ2-5和TRBJ2-7的使用频率病例组高于对照组，差异有统计学意义（P<0.05）。 结论 新生儿脓毒症患者外周血TCR β链CDR3多样性发生了明显改变，提示这可能与新生儿脓毒症的免疫发病机制有关。 引用格式:     

An aggressive systemic juvenile xanthogranuloma clonally related to a preceding T‐cell acute lymphoblastic leukemia

Juvenile xanthogranuloma (JXG) is a disorder of disputed origin thought to be related to the dermal/interstitial macrophage. A 5‐year‐old female presented with an aggressive systemic JXG that developed 5 months after the diagnosis of T‐cell acute lymphoblastic leukemia (T‐ALL). Examination of the T‐cell receptor gamma (TCR‐γ) rearrangement in T‐ALL blasts, JXG infiltrated lymph node biopsies and micro‐dissected JXG histiocytes revealed an identical bi‐allelic TCR‐γ rearrangement in all samples, thus providing evidence for a clonal relationship between T‐ALL and JXG in this case. Pediatr Blood Cancer 2011;56:859–862. © 2011 Wiley‐Liss, Inc.     

Immunological Reconstitution in Children After Completing Conventional Chemotherapy of Acute Lymphoblastic Leukemia is Marked by Impaired B‐cell Compartment

Humoral and cellular immunity were studied in 28 children completing conventional treatment of standard‐risk (SR) or intermediate‐risk (IR) acute lymphoblastic leukemia (ALL). Both naïve and memory B cells were most severely affected and showed slow recovery during the 2‐year follow‐up, while the T‐cell compartment showed only minor changes. Immunoglobulins and IgG subclasses, components, and antibodies against vaccine‐preventable diseases were not significantly affected. In conclusion, immune recovery after conventional chemotherapy for SR and IR ALL is marked by B‐cell depletion, but otherwise did not show any severe deficiencies in lymphocyte function.     

T‐cell‐rich HLA‐haploidentical hematopoietic stem cell transplantation for relapsed/refractory pediatric Philadelphia chromosome‐positive acute lymphoblastic leukemia without posttransplant tyrosine kinase inhibitor therapy

Intensive chemotherapy with tyrosine kinase inhibitor (TKI) improves the prognosis of patients with Philadelphia chromosome‐positive acute lymphoblastic leukemia (Ph‐ALL). However, the prognosis of cases of relapsed or refractory Ph‐ALL remains poor. Here, we aimed to assess the efficacy of T‐cell‐rich HLA‐haploidentical hematopoietic stem cell transplantation (TCR‐haplo‐HSCT) in eight patients with relapsed or refractory pediatric Ph‐ALL. Transplant‐related mortality was observed in two patients. All patients discontinued TKI after receiving TCR‐haplo‐HSCT. The 3‐year probability of overall survival and event‐free survival was 75.0 and 62.5%, respectively. These results indicate the efficacy of TCR‐haplo‐HSCT for relapsed/refractory pediatric Ph‐ALL.     

T细胞受体双基因标志检测对ALL患儿化疗效应评价初探

目的     

Prognostic factors for relapsed childhood acute lymphoblastic leukemia: impact of allogeneic stem cell transplantation--a report from the Kyushu-Yamaguchi Children's Cancer Study Group

BACKGROUND: The treatment results of childhood acute lymphoblastic leukemia (ALL) with a first relapse were retrospectively analyzed to determine prognostic factors. In particular, an attempt was made to clarify whether stem cell transplantation (SCT) had any advantages over chemotherapy. PROCEDURES: Of the 407 children with ALL diagnosed between 1984 and 1996, 117 suffered from a relapse before December 1999. The patients were treated differently according to the protocols of each institution. The potential prognostic factors examined were: the time of initial diagnosis, gender, immunophenotype of leukemic blasts and the NCI-risk classification at initial diagnosis, the site of relapse, the time of relapse (early: within 18 months after diagnosis, intermediate: other than either early or late relapse, late: later than 6 months after the discontinuation of front-line chemotherapy), and the treatment after relapse (chemotherapy alone and SCT). RESULTS: A second complete remission (CR2) was achieved in 90 patients (77%) and thirty of them maintained CR2, thus resulting in an event-free survival rate (EFS) of 25.1% and an overall survival rate of 26.1%. The significant prognostic factors identified by a multivariate analysis included the time of relapse (EFS: early 16.2%, intermediate 23.9%, late 35.1%, P = 0.012) and the treatment after relapse (EFS: SCT 30.3%, chemotherapy 22.0%, P = 0.049). When patients with an isolated bone marrow relapse and continuous CR2 for more than 3 months were analyzed, the treatment in CR2 was the only independent prognostic factor (EFS: SCT 60.2%, chemotherapy 25.7%, P = 0.005). CONCLUSIONS: In children with ALL and a first relapse, the time of relapse and the treatment after relapse were found to be independent prognostic factors. Allogeneic SCT in CR2 showed significantly better results than chemotherapy in patients with an isolated bone marrow relapse.     

T cell receptor V gene usage by CD4+ and CD8+ peripheral blood T lymphocytes in immune thrombocytopenic purpura

AIM: To identify T cell expansions, i.e. increased frequencies of T cells using a particular T cell receptor (TCR) V alpha or V beta gene segment, in patients with immune thrombocytopenic purpura (ITP). METHODS: The TCR repertoires of CD4+ and CD8+ peripheral blood lymphocytes of 16 patients with chronic ITP were analysed by staining with a panel of anti-TCR V alpha and V beta antibodies followed by flow cytometry. RESULTS: Four of the 16 patients exhibited a total of 6 expansions of CD8+ T cells using a particular V beta segment, but no expansions were detected in the CD4+ subset. For three of the expansions where a follow-up blood sample after treatment with intravenous immunoglobulin was available, only one expansion remained. CONCLUSION: Overall T cell expansion frequency was the same as in healthy individuals. However, the presence of expansions that normalized with treatment suggests the presence of specific T cells implicated in the pathogenesis of ITP.