SARS患者康复期淋巴细胞功能状态及T细胞受体Vβ24个亚家族表达的格局(英文)

Lymphocyte immunological function and 24 T cell receptor V beta subfamilies expression in convalescent patients with severe acute respiratory syndrome

背景:严重急性呼吸道综合征(severe acuterespi ratory syndrome,SARS)是由SARS相关冠状病毒感染引起的急性传染性疾病。但目前其康复期患者细胞免疫状态仍有待进一步研究。目的:观察SARS患者康复期淋巴细胞功能状态和T细胞受体(TCR)Vβ24个亚家族表达的格局。设计:前后对照观察。单位:广东省中医院中心实验室。对象:选择2003-01/04在广州中医药大学第二附属医院收治的76例治愈的SARS患者,全部病例均符合"非典型肺炎的临床诊断标准"、"非典型肺炎重症病例诊断标准、出院标准"及《中医临床诊疗术语证候部分》的诊断标准,男30例,女46例,平均(32±11)岁。选择10名同期健康到院体检者,女5名,男5名,平均(32±7)岁,所有受试对象对检测项目知情同意。方法:①TCRVβ24个亚家族的表达检测:取受试对象全血2mL,用EDTA-K2抗凝管抗凝。在流式细胞术检测专用管中,分别加入各种荧光素标记的mAb:抗CD3、抗CD4、抗CD8、抗CD25、抗CD28、抗HLA-DR、PC5-抗CD3mAb、TCRVβ1(PE+FITC)、Vβ2(PE+FITC)、Vβ3(FITC)、Vβ4(PE+FITC)、Vβ5.1(PE+FITC)、Vβ5.2(PE)、Vβ5.3(PE)、Vβ7.1(PE+FITC)、Vβ7.2(FITC)、Vβ8(FITC)、Vβ9(PE)、Vβ11(PE)、Vβ12(FITC)、Vβ13.1(PE)、Vβ13.2(PE)、Vβ13.6(PE+FITC)、Vβ14(FITC)、Vβ16(FITC)、Vβ17(PE+FITC)、Vβ18(PE)、Vβ20(FITC)、Vβ21.3(FITC)、Vβ22(PE+FITC)和Vβ23(PE)10μL,然后加入抗凝血50μL。混匀后,于室温避光孵育15min,再加入溶血素溶血、洗涤。最后将沉淀溶在300μLPBS中,用CoulterESP流式细胞仪进行检测。②T细胞亚群,T、B细胞的活化状态及Ts细胞和Tc细胞百分率的检测:每次收集5000个细胞,用仪器所带软件分别计算T细胞亚群(CD3、CD4和CD8)、活化的T、B细胞(CD3+/CD25+、CD3+/HLA-DR+、和CD3-/HLA-DR+)、Ts细胞和Tc细胞(CD8+/CD28+、CD8+/CD28-)的百分率。主要观察指标:①T细胞亚群(CD3、CD4和CD8)的表达。②活化的T、B细胞(CD3+/CD25+、CD3+/HLA-DR+、和CD3-/HLA-DR+)的表达。③Ts细胞和Tc细胞(CD8+/CD28+、CD8+/CD28-)的百分率。④TCRVβ24个亚家族的表达。结果:所有受试对象均进入TCRVβ24个亚家族检测结果分析,74例SARS患者及健康对照者10名均进入其余检测结果分析。①T细胞亚群的检测结果:CD4+细胞的百分率均数低于参考值[(33.33±6.64)%,(43±9)%,P<0.01],CD8+细胞的百分率高于参考值[(34.07±6.40)%,(30±9)%,P<0.01]。②活化的T、B细胞表达检测:表达HLA-DR的T、B细胞增加,而表达CD25的T细胞减少。有53例CD25+细胞的百分率低于正常参考值,有64例CD3+/CD25+T细胞的百分率低于正常参考值。只有CD3+/HLA-DR+和CD3-/HLA-DR+细胞的百分率高于正常参考值,其中表达CD3+/HLA-DR+的T细胞增加36例,表达CD3-/HLA-DR+的T细胞增加30例。③Ts细胞和Tc细胞的百分率:Ts细胞(CD8+/CD28-)的百分率较参考值增高[(28.75±7.31)%,(15.99±5.1)%,P<0.01];Tc细胞(CD8+/CD28+)的百分率则低于参考值[(5.99±3.60)%,(13.2±4.1)%,P<0.01]。其中有39例Tc细胞的百分率降低,有48例Ts细胞的百分率升高,CD4+与CD8+T细胞的比值均在正常参考值范围内。④TCRVβ24个亚家族表达:康复期SARS患者TCRVβ24个亚家族表达中,TCRVβ14的表达最高,TCRVβ5.3、和23表达次之,出现TCRVβ24个亚家族表达的不同。正常对照组中只有TCRVβ14表达最高,与SARS患者的结果相一致,但TCRVβ24个亚家族的分布格局不同。两组进行比较,Vβ1、Vβ5.2、Vβ5.3、Vβ7.2、Vβ9、Vβ11、Vβ13.1、Vβ13.2、Vβ17、Vβ18、Vβ22和Vβ23的表达差异显著,康复期SARS患者组均高于正常对照组(P<0.05-0.01)。结论:康复期SARS患者CD8+CD28-T细胞数的升高可导致CD8+T细胞数升高;而Ts细胞数的增加,因其分泌的抑制因子增多,从而造成CD3+和CD4+T细胞数降低。SARS患者康复期TCRVβ24个亚家族在T细胞上的表达不同。CD3+/HLA-DR+和CD3-/HLA-DR+T细胞数的升高,可能与T、B细胞的活化较晚有关。

BACKGROUND： Severe acute respiratory syndrome （SARS） is caused by a genetically novel coronavirus that is caused by acute infectious disease. It is not yet clear for the immunology function of SARS patients in their convalescent stage. OBJECTIVE： To study the effects on T lymphocyte, and the titer profiling of 24 T cell receptor （TCR） V β subfamilies expressions in SARS convalescent patients. DESIGN ： A self-control observation SETTING ： Central Laboratory, Guangdong Provincial Hospital of Traditional Chinese Medicine PARTICIPANTS： Seventy-six cured SARS patients who received treatment in the Second Hospital Affiliated to Guangzhou University of Traditional Chinese Medicine between January and April 2003. All the patients corresponded to ＂clinical diagnostic criteria of atypical pneumonia＂, ＂ diagnostic criteria of severe atypical pneumonia and discharge criteria＂ and ＂clinical diagnostic criteria and discharge criteria of severe acute respiratory syndrome＂. The involved patients, 30 male and 46 female, averaged （32±11）years old. Another 10 subjects who simultaneously received health examination in the same hospital, 5 male and 5 female, aged （32±7）years, were involved in the study. Informed consents of detected items were obtained from all the subjects. METHODS; （1） Detecting the expression of 24 T cell receptor （TCR） V β subfamilies in SARS convalescent patients： Peripheral blood （2 mL） was collected from the healthy convalescent subjects, and EDTA-K2 was used as anticoagulant. In the flow cytometry detection tubes, 10 μL various fluorescein-labeled mAb, such as anti-CD3, anti-CD4, anti-CD8, anti-CD25, anti-CD28, anti-HLA-DR, anti-CD3mAb conjugated with PC5, TCR Vβ1 （PE±FITC）, Vβ2（PE±FITC）, Vβ3 （FITC）, Vβ4（PE±FITC）, Vβ5.1 （PE±FITC）, Vβ5,2（PE）, Vβ5.3（PE）, Vβ7.1 （PE±FITC）, Vβ7.2（FITC）, Vβ8（FITC）, Vβ9 （PE）, Vβ11 （PE）, Vβ12（FITC）, Vβ13.1 （PE）, Vβ13.2（PE）, Vβ13.6（PE±FITC）, Vβ14（FITC）, Vβ16（FITC）, Vβ17 （ PE±FITC ）, Vβ18 （ PE ）, Vβ20 （ FITC ）, Vβ 21.3 （FITC）, Vβ22 （ PE±FITC ）and Vβ23 （ PE ）, was added in special flow tubes, and then 50 μL whole blood was added. The mixed solution was incubated away from light for 15 minutes. After erythrocytolysin being added, mixed solution was washed. Finally, cell deposit was dissolved in 300 μL phosphate buffer solution （PBS）.Coulter ESP flow cytometer was used for detection. For the analysis of TCR expression, an electronic gate was set on these cells and at least 5000 events per sample were collected. Three-color cytofluorimetric analysis was performed using a Coulter ESP flow cytometer. （2）Detecting the T cell subset, activated T and B cells, and the percentage of Ts and Tc cells： 5000 cells were collected and used to calculate the expression of T cells （CD3, CD4 and CD8）, the activated T and B cells （CD3^＋/CD25^＋, CD3^＋/HLA-DR^± and CD3-/HLA-DR^＋）, as well as the percentage of Ts and Tc cells by Coulter ESP flow cytometer and its software. MAIN OUTCOME MEASURES ： （1）The change of T cell subset（CD3,CD4, and CD8） from SARS convalescent patients. The change of activated T and B celIs（CD3^＋/CD25^＋, CD3^＋/HLA-DR^＋ and CD3VHLA-DR^±）. （3）The percentage of Ts and Tc celIs（CD8^＋/CD28^＋, CD8^＋/CD28^＋） in convalescent patients.（4）Analysis of the 24 TCR V 13 subfamilies from SARS patients in convalescence. RESULTS ： All data were explored to analyze the expression profiling of 24 TCR Vβ subfamilies, the data from 74 SARS patients and 10 healthy controls were explored to other result analysis. （1）The detecting results of T cell subset： The percentage of CD4^＋T cell mean value was lower than the reference value [（33.33±6.64）% vs.（43±9）%, P 〈 0.01]. The percentage of CD8^＋T cell mean value was higher than the reference value[（34.07±6.40）% vs.（30±9）%, P 〈 0.01]. The expression of activated T and B cells： Percentage of HLA-DR T and B cell was increased while the percentage of CD25^＋ T-cell was decreased compared with referenc9 values. In 53 out of 74 patients, the percentage of CD25^＋ T cells was lower than the reference value, and 64 patients had a lower percentage in CD3^＋/CD25^＋ T cells. The percentages of CD3^＋/HLA-DR^＋ and CD3^＋/HLA-DR^＋ cells were higher than the normal reference value. T cells expressing higher CD3^＋/HLA-DR^＋ were found in 36 patients, and T cells expressing higher CD3VHLA-DR^＋ were found in 30 patients.（3）The ratios of Ts and Tc cells： The percentage of Ts cells which expressed CD8^＋/CD28^＋ was increased compared with reference value [（28.75±7.31）% vs.（15.99±5.1）%, P 〈 0.01], while the percentage of Tc cells which expressed CD8^＋/CD28^＋ was decreased [（5.99±3.60）% vs. （13.2±4.1）%, P 〈 0.01]. Thirty-nine patients were found to possess the lower Tc cells and forty-eight patients were found to possess the higher Ts cells. The ratios of both CD4^＋ and CD8^＋ T cells were in the normal reference value. （4）24 TCR Vβ subfamilies expressions in T cells： It was noteworthy that Vβ14 had a highest percentage in all 24 subfamilies, and followed by Vβ 5.3, and Vβ 23 in the convalescent patients. The percentage of Vβ 14 was the highest in the normal controls, which was consistent with the results of SARS IDatients. But the other subfamilies expression patterns were different. There were significant differences between Vβ1, Vβ5.2, Vβ5.3, Vβ7.2, Vβ9, Vβ11, Vβ13.1, Vβ13.2, Vβ17, Vβ18, VI322 and Vβ23. In the convalescent period, each TCR Vβ expression of SARS patients was higher than that of controls （P 〈 0.05-0.01）.CONCLUSION： In SARS convalescent patients, the increased CD8^＋CD28^＋ T cell may elevate CD8^＋ T cell number; Meanwhile, the reduced CD3^＋ and CD4^＋ T cell number may be corresponding to the increased Ts cell number. For some inhibiting factor secreted by Ts cell was also increased. The usage pattern of 24 TCR Vβ subfamilies in SARS patients is different from that of control group. The increase of percentage of CD3^＋/HLA-DR^＋ and CD3^＋/HLA-DR^＋ T cell may be related to the late response of activated T and B cells.