CD14+HLA-DRlow/-髓系来源抑制性细胞在Graves病中的检测及临床意义

目的:检测Graves病(Graves' disease,GD)患者外周血中髓系来源抑制性细胞(myeloid-derived suppressor cells,MDSCs)表达情况,探讨MDSCs在GD发生、发展中的作用.方法:收集52例初诊GD患者和30例健康志愿者外周血,以CD14+HLA-DR1ow/-作为MDSCs的免疫标记,分别应用流式细胞术及ELISA方法检测外周血中MDSCs的比例及血浆细胞因子Arg-1,IL-6,G-CSF浓度;并分析MDSCs与GD患者甲状腺功能相关性.结果:GD患者外周血MDSCs比例明显升高,相比对照组差异有统计学意义(P＜0.05),且GD患者病情平稳后,MDSCs水平较治疗前明显下降(P＜0.05);MDSCs水平与GD患者甲状腺功能未见明显相关性;GD患者外周血浆Arg-1水平未见明显升高,IL-6及G-CSF浓度显著升高(P＜0.05).结论:GD患者外周血MDSCs升高,可能是GD发生和发展的重要因素.     

DC-CIK细胞联合替吉奥治疗非小细胞肺癌的疗效观察

目的 观察树突状细胞-细胞因子诱导的杀伤细胞(DC-CIK)联合替吉奥治疗非小细胞肺癌的临床疗效.方法 76例非小细胞肺癌(NSCLC)患者随机分为两组(n=38),对照组单用替吉奥治疗,观察组在对照组治疗的基础上加以DC-CIK细胞治疗.采集观察组患者的外周静脉血进行DC细胞培养和CIK细胞培养.于治疗前与治疗7d后用流式细胞仪检测两组患者外周血中CD4+/CD8+、CD44+NK细胞的表达情况, 以及检测两组患者的白细胞数、血小板数,并观察两组患者的非小细胞肺癌症状.结果 观察组治疗后的外周血中CD4+/CD8+、CD4圾NK细胞百分比分别为(1.65±1.03)、(34.56±8.90)和(18.68±7.98),均显著高于对照组的(1.32±0.70)、(29.07±7.15)和(15.28±8.23),差异均具有统计学意义(均P＜0.05);观察组治疗后CD8+细胞百分比(25.56±8.90)与对照组(26.64±6.77)差异无统计学意义(P＞0.05),呕吐、白细胞降低、血小板降低的患者少于对照组,差异均具有统计学意义(均P＜0.05);观察组患者1年生存率为52.63％明显高于对照组患者的42.11％,差异具有统计学意义(P＜0.05).结论 DC-CIK细胞联合替吉奥治疗非小细胞肺癌能有效提高临床疗效,延长患者的生存时间,是一种安全、有效的治疗方案.     

自体细胞因子诱导的杀伤细胞联合常规化疗治疗中晚期宫颈癌的临床研究

目的 探讨回输自体细胞因子诱导的杀伤细胞(CIK)联合顺铂治疗中晚期宫颈癌的临床疗效,并观察此治疗对患者外周血中Th1/Th2细胞因子的漂移的影响.方法 将我院在2013年2月至2014年2月接收的宫颈癌患者44例,随机分为单纯化疗组(n=22)和联合治疗组(n=22),并选取健康的女性个体20例作为对照组,采用酶联免疫吸附(ELISA)法检测检测所有样本外周血中Th1型细胞因子白细胞介素(IL)-2、干扰素-γ(IFN-γ)的表达水平,Th2型细胞因子IL-4、IL-6、IL-10的表达水平,流式细胞仪检测Th1/Th2细胞的比率变化.同时评价两组患者疗效及生活质量.结果 与健康对照组比较,治疗后两组患者血清中Th1型细胞因子IL-2、IFN-γ的浓度显著升高,且联合治疗组升高程度高于单纯化疗组,而Th2型细胞因子IL-4、IL-6、IL-10的浓度明显降低,且联合治疗组降低程度高于单纯化疗组(P＜0.05).治疗后单纯化疗组和联合治疗组Th1/Th2比率小于对照组,但联合治疗组高于单纯化疗组(P＜0.05).联合治疗组完全缓解率高于单纯化疗组(36.4％比22.7％,P＜0.05).联合治疗组有效率高于单纯治疗组(90.9％比72.7％,P＜0.05).联合治疗组的生活质量总提高率高于单纯化疗组(86.4％比72.7％,P＜0.05).结论 自体CIK细胞联合常规化疗能够改善中晚期宫颈癌的Th1向Th2漂移,是安全并且有效的.     

系统性红斑狼疮患者外周血CD4+和CD8+T细胞PD-1的表达和意义

目的 探讨程序性死亡分子1 (PD-1)在系统性红斑狼疮(SLE)患者外周血CD4+和CD8+T细胞上的表达及临床意义.方法 应用流式细胞仪检测51例SLE患者和38例健康对照者外周血T细胞亚群表面PD-1表达水平,比较SLE稳定组、活动组和健康对照组以及狼疮肾炎组和无狼疮肾炎组之间CD4+和CD8+T细胞表面PD-1表达的百分比,并分析其与临床表现及实验室检查数据的相关性.结果 SLE活动组CD4+T细胞PD-1表达水平高于健康对照组和不活动组,差异均有统计学意义(P＜0.05).SLE活动组、稳定组CD8+T细胞PD-1表达水平均高于健康对照组,差异有统计学意义(P＜0.05).狼疮肾炎患者CD4+PD-1+和CD8+PD-1+T细胞分别高于无狼疮肾炎患者(P＜0.01).SLE患者中抗dsDNA抗体、抗Sm抗体、抗核小体抗体阳性组外周血CD4+和CD8+T细胞PD-1表达水平均高于对应阴性组.SLE患者CD4+和CD8+T细胞PD-1表达百分率与SLE疾病活动度指数(SLEDAI)、尿蛋白定量呈正相关,与补体C3呈负相关.结论 SLE患者外周血CD4+和CD8+T细胞PD-1表达异常,与SLEDA1和自身抗体产生有明确的相关性.     

血细胞多参数联合分析在大细胞性贫血疾病中的诊断价值

目的 探讨血细胞多参数联合分析在大细胞性贫血疾病中的临床诊断及鉴别诊断价值.方法 用Sysmex XE-5000型全自动血细胞分析仪对急性白血病(AL)组42例、溶血性贫血(HA)组30例、骨髓增生异常综合征(MDS)组27例、巨幼细胞性贫血(MA)组24例、再生障碍性贫血(AA)组16例,多发性骨髓瘤(MM)组15例及健康组50例的抗凝静脉血标本进行红细胞参数(HGB、MCV、MCH、MCHC、RDW-CV)、网织红细胞参数(Het％、LFR、MFR、HFR、Ret-He、IRF)及血小板相关参数(PLT、MPV、PDW、IPF)检测,并作统计分析大细胞性贫血患者各参数的分布规律及临床鉴别诊断.结果 ①所有疾病贫血患者组的RDW-CV值均高于正常对照组,HA组RDW-CV与其他疾病组间差异有统计学意义(P＜0.05).②所有疾病贫血患者组的MFR、HFR、IRF明显高于正常对照组,LFR低于正常对照组,HA组的MFR、HFR、IRF显著高于其他疾病组,差异有统计学意义(P＜0.05).AL、HA、MA、MM组的Ret-He均升高,MDS组与其他疾病组比较,Ret-He差异有统计学意义(P ＜0.05);MDS组与健康对照组比较,Ret-He差异无统计学意义(P＞0.05).③MDS组患者PDW值均高于其余各疾病组,差异有统计学意义(P＜0.05),与正常对照组比较,差异无统计学意义(P＞0.05).结论 血细胞相关参数是诊断大细胞性贫血疾病的重要依据,对于不同大细胞性贫血疾病的诊断及鉴别诊断具有十分重要的价值,为临床医生提供有方向地选择特异性诊断检查.     

经导管动脉栓塞化疗联合射频消融和细胞因子诱导的杀伤细胞输注治疗小肝癌

目的 探讨经导管动脉栓塞化疗(TACE)联合射频消融(RFA)后行自体细胞因子诱导的杀伤细胞(CIK)输注治疗对小肝癌的疗效.评价CIK细胞过继免疫治疗后患者免疫指标的变化.方法 2004年2月至2006年2月本院收治的小肝癌患者43例,分为两组,所有患者均行TACE联合RFA治疗.其中研究组患者21例,均完成4次以上自体CIK细胞输注治疗,每次回输CIK细胞的数量为(1.1～1.5)×1010m,检测研究组患者在CIK治疗前后外周血T淋巴细胞亚群及NK细胞水平的变化;对照组患者22例,未行CIK细胞过继免疫治疗.两组患者均每1～2个月评价肿瘤情况.结果 (1)对照组治疗后第1、2和3年的肝内累积复发率分别为9.1%、22.3%和27.3%;术后1、2和3年的生存率分别为95.5%、81.8%和68.2%.研究组第1、2和3年的肝内累积复发率分别是9.5%、14.3%和19.0%;术后1、2和3年的生存率分别为95.2%、85.7%和76.2%.两组生存率差异无统计学意义(P=0.558).(2)研究组CIK细胞治疗前后外周血T淋巴细胞亚群检测显示CD3+、CD4+、CD56+(NK)效应细胞的比例和CD4+/CD8+比值显著上升(P<0.05),CD8+和CD3+CD56+效应细胞比例下降(P<0.05).结论 TACE序贯联合RFA和CIK细胞过继免疫细胞治疗小肝癌可以提高患者的机体免疫水平,可能对降低肿瘤复发,延长小肝癌患者的生存期有一定作用.     

慢性阻塞性肺疾病急性加重期患者外周血Th17细胞的表达及临床意义

目的 探讨外周血Th17细胞在慢性阻塞性肺疾病急性加重期(AECOPD)患者中的表达及临床意义.方法 以260例慢性阻塞性肺疾病(COPD)患者为研究对象,按临床表现分为COPD稳定期组(130例)和AECOPD组(130例),同时选取50例健康体检者为对照组.采用流式细胞术检测外周血Th17细胞水平,酶联免疫吸附法检测外周血细胞因子IL-17水平,同时检测C-反应蛋白(CRP)及白细胞计数(WBC)水平,记录COPD评估测试(CAT)评分.结果 AECOPD组患者外周血Th17细胞、IL-17、CRP及WBC水平显著高于COPD稳定期组和正常对照组(P＜0.05);经过相应治疗后,AECOPD组患者外周血Th17细胞、IL-17、CRP、WBC水平及CAT评分均较治疗前明显降低,差异具有统计学意义(P＜0.05).相关性分析显示,AECOPD组患者外周血Th17细胞水平与CAT评分呈显著正相关(r=0.91,P＜0.01).外周血Th17细胞水平与炎性因子IL-17也呈正相关(r=0.89,P＜0.01),而AECOPD组患者外周血Th17细胞水平与CRP(r =0.084,P＞0.05)、WBC(r=0.063,P＞0.05)水平无显著相关性.ROC曲线分析显示,外周血Th17细胞水平的曲线下面积(AUC)为0.868(95％ CI0.774～0.918),以外周血Th17细胞水平3.72作为临界值时,其诊断AECOPD的敏感性为88.6％,特异性为86.4％,其显著优于CRP和WBC指标.结论 外周血Th17细胞在AECOPD患者中高表达,其可作为预测COPD急性加重期的有效指标,具有一定的临床运用价值.     

子痫前期患者外周血树突状细胞与T细胞亚群的变化

目的 观察子痫前期患者外周血中的树突状细胞亚群与T细胞亚群相关细胞因子的变化.方法 实验组为子痫前期患者32例,对照组为未孕妇女20例,正常妊娠妇女20例.采集研究对象外周血细胞,流式细胞术检测全血细胞中髓系树突状细胞(mDC)和浆细胞样树突状细胞(pDC);分离外周血单个核细胞,经胞内细胞染色检测Th1、Th2、Th17细胞数量及Th1/Th2比值.结果 子痫前期组mDC百分比(0.33±0.12)％和mDC/pDC比值(2.96±1.65)均高于正常妊娠组,二组数据有明显差异(P＜0.05);pDC百分比(0.16±0.13)％较正常妊娠组(0.21 ±0.12)％有所下降,二组差异有统计学意义(P＜0.05).子痫前期组IFN-γ、IL-4和IL-17的百分比分别为(18.67 ±1.96)％、(1.88±0.51)％和(1.36±0.59)％,与正常妊娠组相比均有显著性差异(P＜0.01).子痫前期组mDC/pDC比率和Th1/Th2之间呈显著正相关(r=0.637,P＜0.01);Th17表达率与pDC表达率之间呈负相关(r=-0.670,P＜0.05),与mDC/pDC比率之间呈显著正相关(r=0.772,P＜0.01).结论 子痫前期患者外周血中树突状细胞亚群和Th1、Th2、Th17型细胞因子异常表达,可能是患者发生免疫失衡的重要原因.     

干扰素β1b对Th17细胞介导的复发-缓解型多发性硬化无效

目的 探讨干扰素β1b(IFN-β1b)在复发-缓解型多发性硬化(RRMS)中的疗效与外周血中Th17细胞的关系.方法 收集11例RRMS患者,给予6个月IFN-β-1b治疗.评估治疗前后扩展残疾状态评分(EDSS)及核磁共振(MRI)T2病灶数的变化.并通过流式细胞术检测治疗前患者外周血单个核细胞(PBMC)中Th17细胞的数量.结果 11例RRMS患者根据EDSS评分的变化分为治疗有效组和无效组.两组之间EDSS评分及MRI T2病灶数在治疗前无统计学差异(P＞0.05),治疗后有效组EDSS评分和MRI T2病灶数较无效组明显减少(P＜0.05).治疗前PBMC中Th17细胞数量在无效组较有效组明显增加(P＜0.05).结论 IFN-β-1b治疗对Th17细胞介导的RRMS无明显疗效.     

类风湿关节炎患者Th17和CD161+Th17细胞水平的变化

目的 观察类风湿关节炎(RA)患者和健康对照组外周血中CD4+ IL-17+T细胞(Th17)和CD4+ CD161+ IL-17+T细胞(CD161+Th17)的水平,探讨其在RA发病过程中的意义.方法 采用流式细胞术测定36例RA患者和11例健康对照组外周血中Th17细胞及CD161+ Th17的细胞百分率.结果 RA患者外周血Th17及CD161+ Th17细胞水平明显高于健康对照组(P＜0.01)且与疾病活动指数(DAS28)、血沉和C-反应蛋白水平呈显著正相关(P ＜0.05);RA患者和健康对照组外周血中CD161+ Th17细胞百分率明显高于Th17细胞百分率(P＜0.01);Th17细胞百分率与CD161+ Th17细胞百分率显著正相关(P＜0.01).结论 Th17及CD161+ Th17细胞在RA患者外周血中均增高且与疾病活动度正相关.     

慢性乙型肝炎患者外周血细胞因子诱导的杀伤细胞的动态观察

目的:动态观察慢性乙型肝炎患者细胞因子诱导的杀伤(CIK)细胞的增殖及杀伤活性。方法:抽取10例健康人及20例慢性乙型肝炎患者的外周血,常规分离单个核细胞(PB-MC),加IFN-γ、IL-2及抗人CD3单克隆抗体(mAb)后培养,诱导CIK细胞形成。于培养后3、6、12、24及30 d,取培养的 细胞,用流式细胞仪检测CIK细胞表面CD3、CD4和CD8以及CD4、CD25、CD3、CD56和CD95、CD28分子的表达水平、增殖及杀伤活性。结果:慢性乙型肝炎患者CIK细胞的增殖及杀伤活性均低于正常对照组。培养不同时间乙肝患者CIK细胞的增殖倍数、杀伤活性和上述表面标志的表达水平,均较培养前明显增高,于培养后12 d达高峰。结论:慢性乙型肝炎患者CIK细胞的增殖倍数、杀伤活性均低于正常人,但明显高于培养前;这可能是导致HBV感染持续发展的原因之一。     

抗CD28单抗和IL-15对CIK增殖和杀肿瘤作用的研究

CIK是肿瘤过继性细胞免疫治疗中的免疫效应细胞。为使CIK在实验室里能被更有效地诱导增殖并赋予其更强的杀肿瘤效应,我们在CIK常规培养环境中加入抗CD28单抗和IL-15,探讨抗CD28单抗和IL-15对CIK增殖和杀肿瘤效应。取人外周血单个核细胞(PBMC),预先以常规方法诱导CIK,然后加入抗CD28单抗和IL-15与CIK共培养。用全自动五分类血液分析仪计数CIK增殖率;用流式细胞术测定CIK中粒酶B、穿孔素和CD107a等分子的表达率;用ELISA方法检测CIK分泌IL-10、IL-12、INF-γ和TNF-α水平;用乳酸脱氢酶释放法测定CIK对人肺癌细胞株(A549)、乳腺腺癌细胞株(MFC-7)和人黑素瘤细胞株(HME1)的杀伤活性。PBMC经常规CIK诱导培养以后再加入抗CD28单抗和IL-15与对照组比较,前者细胞增殖率明显增强(P<0.05);在CIK培养体系中加入抗CD28单抗和IL-15可促进颗粒酶B、穿孔素和CD107a等分子的表达率进一步增强(P<0.05);加入抗CD28单抗和IL-15,培养8d后CIK对A549、MFC-7和HME1细胞杀伤活性分别为82.2%、59.3%和70.6%,与对照组(分别为60.9%、49.6%和48.4%)相比差异有统计学意义(P<0.05);在培养体系中加入抗CD28单抗和IL-15,培养8d后其细胞因子IFN-γ、TNF-α分泌水平显著高于对照组(P<0.05),组间IL-10和IL-12的分泌量未见显著差异(P>0.05)。实验说明在CIK培养体系中加入抗CD28单抗和IL-15可增加CIK增殖率并提高其抗肿瘤效应。     

Transfusion of multi-factors activated immune cells as a novel treatment for patients with chronic hepatitis B.

BACKGROUND AND OBJECTIVES: Host-virus interactions play a central role in determining the prognosis of hepatitis B virus infection. Multi-factors activated immune cells (MAICs) are autologous peripheral blood mononuclear cells activated with anti-CD3 monoclonal antibody, interleukin-2 and interferon-gamma. The present pilot clinical trial was designed to determine whether adoptively transferred MAICs inhibit the replication of hepatitis B virus and is safe for patients. STUDY DESIGN: Fourteen patients with chronic hepatitis B were enrolled in the study. A total of (1.5-4.0)x10(9) peripheral blood mononuclear cells were isolated from each patient and activated by anti-CD3 monoclonal antibody, interleukin-2 and interferon-gamma in vitro for 10 days to produce MAICs. Cell phenotypes and levels of cytokine secretion were determined during cell culture. Patients were followed up for 1 year after the transfusion of MAICs. RESULTS: After 10 days of culture, peripheral blood mononuclear cells were expanded to a mean fold of 4.68+/-1.78 and activated effectively, as demonstrated by CD25 expression and cytokine secretion. Cell activation peaked on day 4. All patients accepted the MAICs transfusion with some slight adverse events, and fulminant hepatitis and bilirubinemia were not observed. Significant HBV inhibition was observed in 8 out of 14 patients, in which 5 patients achieved complete response (defined as that serum HBV DNA levels below the detection limit, occurrence of HBeAg seroconversion and normalization of ALT were observed together after MAICs transfusion and kept for at least 6 months) and 3 patients achieved partial response (defined as that serum HBV DNA levels below the detection limit, occurrence of HBeAg disappearance and normalization of ALT were observed together in 6 months after MAICs transfusion, but kept for less than 6 months). CONCLUSION: These findings strongly suggest that MAICs transfusion, which effectively inhibits the replication of hepatitis B virus, is safe for patients.     

Expansion of cytotoxic CD3+ CD56+ cells from peripheral blood progenitor cells of patients undergoing autologous hematopoietic cell transplantation.

Immunotherapy may potentially improve the outcome of autologous hematopoietic cell transplantation (HCT). Poor effector cell proliferation and marginal antitumor activity limit attempts to use immunotherapy. We have characterized the ex vivo expansion, up to 1000-fold, of CD3+ CD56+ lymphocytes from the peripheral blood lymphocytes (PBL) of healthy donors. Expanded cells termed cytokine-induced killer (CIK) cells induce non-major histocompatibility complex-restricted lysis of tumor cells and demonstrate cytolytic activity superior to lymphokine-activated killer cells without the requirement of interleukin (IL)-2 treatment in vivo. To determine whether cytolytic cells could be expanded from patient material, we evaluated samples of peripheral blood progenitor cells (PBPCs) from 25 patients undergoing autologous HCT. The PBPCs were expanded by priming with interferon-gamma followed by anti-CD3 monoclonal antibody and IL-2 the next day. Fluorescence-activated cell sorting analysis was performed on days 0, 15, 21, and 28 of cell culture. The median T-cell content rose from 15.3% (range, 1.1% to 89.7%) on day 0 to 97.2% (range, 83.6% to 99.5%) by day 15. By day 21, T cells expanded 21.8-fold (range, 1.7- to 420.0-fold) and CD3+ CD56+ cells expanded 44.8-fold (range, 5.1- to 747.0-fold). CIK cells were used as effector cells against B-cell lymphoma targets (OCI-Ly8) with a median of 24% (range, 3% to 67%) and 42% (range, 6% to 96%) specific lysis of target cells on days 21 and 28, respectively. CIK cells derived from PBL of 2 additional patients with acute myelogenous leukemia demonstrated 39% and 78% specific lysis of OCI-Ly8 and 26% and 58% specific lysis of autologous leukemic blasts at an effector:target ratio of 40:1. CIK cells may be expanded from granulocyte colony-stimulating factor-mobilized PBPCs of patients undergoing autologous HCT. CIK cells may provide a potent tool for use in posttransplantation adoptive immunotherapy.     

Clinical expansion of cytokine induced killer (CIK) cells

Cytokine induced killer (CIK) cells are polyclonal T cells that can be expanded from marrow or peripheral blood lymphocytes with potent non-MHC-restricted cytotoxicity against a variety of tumor target cells. Earlier work had established the superiority of CIK cells over LAK cells in terms expansion and cytotoxicity. It has been studied extensively both in vitro and in murine experiments with promising killing activity against a wide range of haematological malignancies. Early clinical trials of CIK cells have reported safety and feasibility data as well as possible efficacy and have provided the framework for further clinical studies. As a robust and easily expandable cell population, its role in clinical adoptive immunotherapy should be investigated for translation into a good manufacturing practice (GMP) manufacturing process. Large scale culture to generate adequate number of CIK cells for clinical use involves up-scaling from flasks to culture bags carried out in a GMP compliant cell therapy facility, use of GMP compliant materials and reagents, efficient harvesting procedures to remove foreign elements and strict release criteria with respect to viability, sterility and potency of the product. All the steps should preferably to be done in a closed system, in order to minimize the chance of contamination. A phase I/II clinical study on the use of allogeneic CIK cells has been conducted to define the role of CIK cells in the management of relapse post stem cell transplant with the aim of looking at feasibility of GMP expansion, toxicity and efficacy. A separate trial looking at autologous CIK cells was also conducted. Allogeneic CIK cells were successfully generated for a total of 24 patients including from patients’ own leukepheresed cells in 5 patients who have no access to further donor cells. The median CD3 + T cell expansion was 9·33 (1·3–38·97) fold and CD3 + CD56 + NK-like T cells expansion was 27·77 (2·59–438·93) fold. CIK cells were infused into 16 patients who have either failed or progressed after initial response to various individualized chemotherapy regimen and donor lymphocyte infusion (DLI), for a total of 55 infusions at doses ranging from 10 to 200 million CD3/kg. Evidence of efficacy as defined by a demonstrable response attributable to CIK cell infusion was observed in five patients with minimal toxicity. Other published examples to augment CIK efficacy include co-culture of CIK with myeloma idiotype or CA19-9 peptide-pulsed autologous dendritic cells. Redirecting CIK cells by bispecific antibodies to target has been shown for ovarian carcinoma and B cell lymphoma, as well as B cell ALL by engineering CIK cells to express anti-CD19 receptor. Exploiting the heterogeneous subsets within the bulk CIK cell culture is another avenue for enhancing its potency.     

人源白细胞介素21对外周血及脐血来源CIK细胞产生和抗肿瘤活性的作用研究

目的：研究新近发现的免疫调节因子——白细胞介素21（IL-21）对外周血及脐血来源的CIK细胞产生及抗肿瘤活性的体外作用。方法：采集分离正常人的外周血及脐血单个核细胞，加用细胞因子诱导培养CIK细胞，在有无人源IL-21（200ng／μl）培养条件下，检测CIK细胞表达及杀伤K562细胞和急性白血病患者肿瘤细胞活性的变化；检测培养上清IFN-γ的浓度及杀伤活性以及RT-PCR法检测培养细胞的IFN-γ RNA表达的不同。结果：在人源IL-21的作用下，培养14天时，①CIK细胞的产生由17．5％升至26．5％（外周血来源）；33．8％升至55．9％（脐血来源）。②CIK细胞对K562细胞的杀伤作用由24．0％升至52．2％（外周血来源）；35．1％升至79．7％（脐血来源）；脐血来源CIK细胞对13例急性白血病患者肿瘤细胞杀伤作用由27．4％升至58．3％。③外周血来源培养上清IFN-γ的浓度上升了近一倍，对K562的杀伤作用增加了近一倍；脐血来源培养上清IFN-γ的浓度上升了三倍多，对K562的杀伤作用增加了近二倍；④外周血和脐血来源培养细胞的IFN-γ RNA表达均明显增高。结论：人源IL-21可增加外周血及脐血来源CIK细胞产生及增强其抗肿瘤活性，通过增加IFN-γ的表达产生为其作用机制之一，提示IL-21在增强肿瘤免疫治疗中具有潜在的临床应用前景。     

Myeloid-derived suppressor cells are increased in frequency but not maximally suppressive in peripheral blood of Type 1 Diabetes Mellitus patients

Type 1 Diabetes Mellitus (T1D) results from the destruction of insulin-producing beta cells in the pancreas by autoreactive T cells. Myeloid derived suppressor cells (MDSCs) are a recently identified immune cell subset that down-regulate T cells. Whether defects in MDSC numbers or function may contribute to T1D pathogenesis is not known. We report here that MDSCs are unexpectedly enriched in peripheral blood of both mice and patients with autoimmune diabetes. Peripheral blood MDSCs from T1D patients suppressed T cell proliferation in a contact-dependent manner; however, suppressive function could be enhanced with in vitro cytokine induction. These findings suggest that native T1D MDSCs are not maximally suppressive and that strategies to promote MDSC suppressive function may be effective in preventing or treating T1D.     

细胞因子诱导的杀伤细胞的生物学特性研究

目的:探讨细胞因子诱导的杀伤细胞(CIK)的生物学特性。方法:健康人非贴壁单个核细胞用含IFN-γ、IL-1β、IL-2、CD3单抗诱导CIK细胞。以LAK细胞作为对照。流式细胞仪和免疫细胞化学染色检测细胞表型特征;乳酸脱氢酶释放法分析细胞毒活性。结果:诱导2周后,CIK细胞的增殖率达到高峰,CD3⁺细胞占95％以上;第3周细胞生长进入平台期。诱导15d时,CD3⁺CD56⁺NKT亚群占16.5％,比例在2-4周区间内无明显变化。LAK细胞增殖缓慢,显著低于同期CIK细胞增殖率(P＜0.01)。不同效:靶比例CIK细胞对肝癌细胞BeL-7402的特异性溶解率显著高于LAK细胞(P＜0.01)。免疫细胞化学染色结果显示,CIK细胞高度表达HLA-DR和CD54抗原,NKT细胞体积较CD3⁺CD56^-细胞略大,细胞表面有大量伪足。结论:CIK细胞具有高度增殖能力,其体外杀瘤活性明显优于LAK细胞。诱导14-21d,CIK细胞增殖率和CD3⁺CD56⁺阳性细胞率均达到高峰,此时期的CIK细胞适合临床应用。     

Deregulated cytokine network and defective Th1 immune response in multiple myeloma

Intracellular cytokine production by peripheral blood mononuclear cells (PBMC) was analysed in 51 patients with multiple myeloma (MM), 22 with monoclonal gammopathy of undetermined significance (MGUS) and 20 healthy subjects, as a parameter of immunological dysfunction in MM. An increased proportion of T cells and HLA-DR+ cells producing IL-6 was observed in MM patients with active disease (at diagnosis and relapsing) compared with patients in remission and with MGUS, whereas no difference of IFN-gamma+, IL-2+ PBMC between patients and controls was evident. Determination of serum cytokine levels demonstrated that the imbalanced IL-6 production by T cells and the defective anti-tumour Th1 cell activity were related to elevated levels of IL-6 and IL-12. In vitro studies of PHA- and anti-CD3/anti-CD28 MoAbs stimulation of PBMC demonstrated the ability of lymphocytes from MM patients to differentiate towards the Th1 subset in the presence of rIL-12. By contrast, addition of exogenous rIL-6 impaired IFN-gamma production by rIL-12-prompted T cells. Inhibition of Th1 polarization of the immune response by IL-6 was direct on T cells and not mediated by dendritic cells (DC). Evaluation of the ability of MM-derived DC to stimulate cell proliferation of allogenic T lymphocytes and produce IL-12 in vitro, in fact, suggested that MM-derived DC were functionally active. Taken as a whole, these results indicate that a deregulated cytokine network occurs in active MM. They also suggest that increased IL-6 production by peripheral T lymphocytes contributes to the immune dysfunction observed in MM, and enables tumour cells to escape immune surveillance by preventing the anti-tumour Th1 immune response.     

Selective loss of CD4+ CD45R+ T cells in peripheral blood of multiple myeloma patients

The phenotypic distribution of T-lymphocyte subsets in peripheral blood from multiple myeloma (MM) patients shows a reduced proportion of CD4+ cells and a normal proportion of CD8+ cells. The decrease in CD4+ cells could be due to a random process, with all types of CD4+ cells being equally affected, or it could reflect a nonrandom process with selected subsets preferentially reduced. In order to distinguish between these possibilities, double immunofluorescence analysis was performed on blood samples from patients with MM, patients with monoclonal gammopathy of unknown significance (MGUS), and age-matched normal donors, using monoclonal anti-CD4 or anti-CD8 paired with antibodies to the common leukocyte marker Lp220 (CD45R) or 4B4 (CDw29). Normal peripheral blood lymphocytes (PBL) include two phenotypically and functionally distinct CD4+-cell subsets, identified as CD4+ Lp220+ 4B4-and CD4+ Lp220- 4B4+, whereas the majority of CD8+ cells expresses Lp220 (70–85%). MM patients had a highly significant selective reduction of the CD4+ Lp220+ subset compared with normal controls (P<0.001). Although the percentage of CD4+ Lp220- cells was also reduced in some MM patients relative to normal donors, most of MM patients had an elevated Lp220-/Lp220+ ratio of CD4+ cells (P<0.001). The proportion of the two CD8+ subsets was also markedly abnormal. In the set of patients studied the abnormalities within the CD4+ and CD8+ lymphocytes were exclusive to patients with MM since patients with MGUS had normal proportion of CD4+ and CD8+ subsets.