CN114786688A - 治疗多发性硬化症中鞘内施用调节性t细胞 - Google Patents
治疗多发性硬化症中鞘内施用调节性t细胞 Download PDFInfo
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Abstract
本发明涉及在治疗多发性硬化症中鞘内施用的包含CD3+CD4+CD25+CD127‑调节性T细胞的医药产品。
Description
本发明涉及在治疗中临床使用的包含CD3+CD4+CD25+CD127-调节性T细胞的医药产品。该产品在多发性硬化症的治疗中鞘内施用。
多发性硬化症(MS)是一种免疫介导的疾病，其中对髓鞘(myelin sheath)敏感的自身免疫常规T细胞(Tconv)会破坏血脑屏障并破坏中枢神经系统(CNS)的神经元。据推测，CD4+CD25高CD127-FoxP3+调节性T细胞(Treg)可能通过对Tconvs施加的抑制活性来抑制这种破坏。
Treg淋巴细胞占所有外周血淋巴细胞的约1％，但对于维持其自身组织的耐受性是重要的。缺乏调节性T细胞会导致许多自身免疫性疾病和超敏反应，如X连锁免疫缺陷综合征、多内分泌腺病和肠病(IPEX)患者中所见。这种自身免疫综合征之一也是多发性硬化症。
Treg淋巴细胞可以被称为“智能类固醇”，因为作为类固醇，它们抑制炎症反应并发挥免疫抑制作用，但相比之下，Treg细胞的生理抑制作用仅涉及病理反应(例如针对其自身组织)。临床试验的结果，包括本发明人的观察结果表明，用Treg淋巴细胞进行治疗是安全的，并且不会削弱针对外来危险抗原(病毒、细菌、癌细胞)的免疫反应。
本发明提供了包含CD3+CD4+CD25+CD127-调节性T细胞的医药产品经由鞘内注射的施用方式。
本发明的主题是包含CD3+CD4+CD25+CD127-调节性T细胞的医药产品。
该产品鞘内施用，用于治疗被诊断患有多发性硬化症的患者。
该产品鞘内施用。
该产品施用于多发性硬化症。
附图说明
图1-显示研究中的临床结果
在整个试验过程中，患者接受了方案计划的神经学检查。使用EQ-5D问卷(EQ-5D)评估生活质量，并使用MSFC量表的部分和EDSS量表来监测身体/神经状态，例如定时25英尺步行(FWT)、显性(9-HPT P)和非显性(9-HPT L)九孔插板测试(9-HPT)和节奏听觉串行加法测试(PASAT)。在整个随访过程中，静脉内和鞘内施用的患者的分数分别呈现为中位数(最小-最大)，点代表原始数据。
图1S-显示CONSORT流程图
图2-使用MRI显示CNS疾病的进展
在整个试验过程中，患者接受了方案计划的MRI检查。最重要的变化表现为以下变化的指数：FLAIR序列上斑块总体积、FLAIR序列上五个最大斑块的体积和斑块数量。“y”轴上变化的指数是根据从第“0”天(紧邻施用Tregs之前；被视为“100”)开始的变量的单个值计算的，并按比例计算以下检查中的变化。对比增强病变值和微出血数的变化以绝对数表示。在整个随访过程中，静脉内和鞘内施用的患者的指数和绝对值分别呈现为中位数(最小-最大)，点代表原始数据。组间差异用线连接并用星号(*)标记，特定组中随时间的变化用线和井号(#)标记。
图2S-使用MRI显示CNS疾病进展方面的附加数据
在整个试验过程中，患者接受了计划的MRI检查。这些图表显示了整个试验过程中CNS特定结构(灰质、白质、脑、脑皮层、脑实质、脑脊液)的体积变化以及灰质和白质的T1低信号。数据表示为“y”轴上的变化的指数，其中第“0”天的变量的单个值被视为“100”，并按比例计算以下检查中的变化。在整个随访过程中，静脉内和鞘内施用的患者的指数分别呈现为中位数(最小-最大)，点代表原始数据。
图3-显示整个研究中Tregs和Tconvs的水平
流式细胞术分析、代表性流式直方图和门控策略[A]。流式数据的分析从前向对比侧向散射点阵图和淋巴门(P1)的生成开始。这用于创建CD3对比CD4点阵图和CD3+CD4+T细胞门(P2)。该门用于分析Tregs细胞(左列中的点阵图)和CD4+Tconv细胞(右列中的点阵图)。建立了涵盖CD127-CD25高细胞的Tregs门(P3-左列)，并在FoxP3对比Helios点阵图中验证了在该门中FoxP3的表达，然后用其生成三个门：所有Tregs FoxP3+(P4左列)、胸腺tTregs FoxP3+Helios+(P5)和外周pTregs FoxP3+Helios-(P6)。所有Tregs(CD3+CD4+CD25高CD127-FoxP3+)、胸腺tTregs(CD3+CD4+CD25高CD127-FoxP3+Helios+)和外周pTregs(CD3+CD4+CD25高CD127-FoxP3+Helios-的水平)显示在图表[B]中。此外，从TregsFoxP3+门和TconvsFoxP3-门生成CD62L对比CD45RA点阵图，以评估幼稚/记忆细胞的百分比。Tregs(CD3+CD4+CD25高CD127-FoxP3+)和Tconvs(CD3+CD4+CD25低/-CD127+FoxP3-)内的Tn(CD62L+CD45RA+-Q2)、Tcm(CD62L+CD45RA—Q1)和Tem(CD62L-CD45RA--Q3)的水平显示在图表[C]中。由所有三个Treg门(P4到P6-[A]中的左列直方图)分析表达以下标记的Tregs FoxP3+的百分比：CCR10、CXCR4、CCR4、CD103、CCR8、CD18、CD39、CD73、CTLA-4、PD-1、4-1BB和OX40。示例直方图显示了CD39表达的分析([A]中左列直方图中的Q2-1象限)，也对其他标记执行了相同的操作。为进行Tregs分析而获取的文件也用于评估相同标记在Tconv细胞上的表达。Tconvs通过使最初用于Treg分析的P3门和P4门的位置反转而发现。建立了涵盖CD127+CD25低/-细胞的Tconv门([A]中的P3右列直方图)，并在FoxP3对比Helios的点阵图中验证了该门中缺乏FoxP3，然后将其用于生成Tconvs FoxP3-门([A]中的P4右列直方图)。然后由右列直方图P4门分析表达以下标记的Tconvs的百分比：CCR10、CXCR4、CCR4、CD103、CCR8、CD18、CD39、CD73、CTLA-4、PD-1、4-1BB和OX40。该示例显示了CD39表达的分析([A]中右列直方图中的Q2-1象限)。表达标记的Tregs和Tconvs(来自P4门)的水平显示为热图。对聚类树进行排序以找到在Tregs和Tconvs之间具有表达差异大的标记[热图D，详细水平也示于图3S中]。进行了类似的聚类分析以比较和对比胸腺tTregs(CD3+CD4+CD25高CD127-FoxP3+Helios+，来自[A]左列直方图中的P5)和外周pTregs(CD3+CD4+CD25高CD127-FoxP3+Helios-，来自[A]中的左列直方图中的P6)[热图E]。
[A]中所示的流式分析中阳性信号的截止是基于同种型对照和荧光减一(FMO)门控建立的。箭头显示门控的层级。减少了特定点阵图中的点数以清楚地显示种群。流式细胞仪由独立的服务运营商进行操作认证(OQ)，并使用CS&T珠粒(BDBioscience，USA)进行定期质量控制。在图表[B]和[C]中，分别显示iv.组和tc.组中Tregs制剂施用时(第“0”天)、施用后+3个月、+6个月和+12个月的细胞百分比。结果显示为中位数(最小-最大)，点代表原始数据。整个图中的星号[*]表示显著差异。
图3S-显示整个研究中Tregs和Tconvs亚群的水平(与图3中的热图对应的百分比值)
A.分别显示每个试验组中Tregs制剂施用时(第“0”天)、施用后+3个月、+6个月和+12个月的表达趋化因子受体或整联蛋白的Tregs和Tconvs的亚群的百分比。显示了Tregs(CD3+CD4+CD25高CD127-FoxP3+)和Tconvs(CD3+CD4+CD25低/-CD127+FoxP3-)内CCR10+、CXCR4+、CCR4+、CD103+、CCR8+和CD18+细胞的百分比。结果显示为中位数(最小-最大)，点代表原始数据。
B.分别显示每个试验组中Tregs制剂施用时(第“0”天)、施用后+3个月、+6个月和+12个月的表达受体和其他对Tregs功能重要的分子的Tregs和Tconvs的亚群的百分比。显示了Tregs(CD3+CD4+CD25高CD127-FoxP3+)和Tconvs(CD3+CD4+CD25低/-CD127+FoxP3-)内CD39+、CD73+、CTLA-4+、PD-1+、4-1BB+和OX40+细胞的百分比。结果显示为中位数(最小-最大)，点代表原始数据。
C.分别显示每个试验组中Tregs制剂施用时(第“0”天)、施用后+3个月、+6个月和+12个月的tTregs(CD3+CD4+CD25高CD127-FoxP3+Helios+)和pTregs(CD3+CD4+CD25高CD127-FoxP3+Helios-)的亚群。仅显示显著不同的亚群：CCR10+、CD103+、CD39+、CD73+和CTLA-4+。结果显示为中位数(最小-最大)，点代表原始数据。
图4-显示整个研究中血清细胞因子水平
用静脉内或鞘内注射Tregs治疗的患者血清中细胞因子的水平以热图呈现。对聚类树进行排序以找到这两组患者之间水平形成对比的细胞因子簇(详细水平也在图4S中)。分别显示每个试验组中Tregs制剂施用时(第“0”天)、施用后+14天、+3个月、+6个月、+9个月和+12个月的细胞因子水平。星号[*]标记了这两组患者之间水平显著不同的细胞因子。
图4S-显示整个研究中血清细胞因子水平
(与图4中的热图对应的百分比值)
分别显示每个试验组中Tregs制剂施用时(第“0”天)、施用后+14天、+3个月、+6个月、+9个月和+12个月的患者血清中的细胞因子水平。组间水平显著不同的细胞因子以粗体标记。这些包括TGFα和与炎症相关的MCP3、CXCL8和IL1RA。结果显示为中位数(最小-最大)，点代表原始数据。
本发明通过以下实施例进行说明，但不限于此。
研究方案
该研究是根据赫尔辛基宣言原则进行的。该方案已在EudraCT数据库中以编号2014-004320-22注册，并获得了格但斯克医学院机构审查委员会(Institutional ReviewBoard of the Medical University of Gdańsk)的批准(编号NKBBN/414/2012和NKBBN/414-163/2017)。在开始任何医疗程序之前，在招募时收到了所有参与者的书面知情同意书。
14名MS患者(18-55岁)被招募到用静脉内(iv.n＝11)或鞘内(tc.n＝3)Tregs治疗的两组中(表1和图1S)。来自iv.组的一名患者在随访期间因怀孕退出试验。纳入标准如下：复发-缓解型MS(根据McDonald标准或修订版McDonald标准来诊断)，在过去一年至少复发1次或在前2年至少复发2次，伤残状况扩展量表(EDSS)最高4分，提供书面知情同意书的能力以及用于抽血的合适的静脉通路。最重要的排除标准是任何免疫抑制，包括在施用Tregs制剂前最多6个月施用的干扰素β。唯一的例外是糖皮质激素，它可以只作为复发的治疗方法而施用。其他排除标准包括：其他自身免疫性疾病；诊断为免疫缺陷；活动性感染的存在或病史，包括乙型肝炎、丙型肝炎、HIV、结核病(TB)、全身性真菌感染；任何恶性肿瘤史；诊断为血细胞减少症；血栓形成活动升高或既往血栓形成史；纳入前最近2年因心血管事件住院；定义为视乳头水肿的颅内压升高；任何视网膜病变；动脉高血压；大量白蛋白尿的存在或病史；患者与手术相关的过度焦虑；研究者认为可能影响安全参与试验的任何医疗状况；已知活跃的酒精或药物滥用；妊娠试验阳性(对于女性对象)，在研究期间和停药后4个月内(在适当时)不愿意使用有效的避孕措施：在研究期间或停药后4个月内(在适当时)(对于男性对象)有生育意愿。
随访开始于施用Tregs(第“0”天)时并持续12个月，访问时间为：施用后+14天、+3个月、+6个月、+9个月和+12个月。测量的终点包括治疗副作用的数量和强度、年度复发次数、EDSS量表恶化至少1分、多发性硬化症功能复合(MSFC)量表的变化、根据MAGNIMS 2015共识的MRI变化、生活质量问卷(QOL)变化、外周血淋巴细胞免疫表型变化和血清细胞因子水平变化。
Tregs的制造和施用
Tregs的制备是在与我们之前的试验[10-13]类似的良好生产规范(GMP)条件下制造的。
使用可更换的无菌样本系用HEPA封闭式FACS分拣机(Influx，BDBioscience，USA)从患者的静脉外周血(450ml)中将细胞分离至以下表型CD3+CD4+CD25高CD127-lin-doublet-。分拣本身基于使用来自德国Miltenyi Biotec的GMP级单克隆抗体(荧光染料/类/克隆)：抗CD4(Vio-blue，IgG1，M-T466)、抗CD25(PE，IgG1，3G10)和抗CD127(APC，IgG1，MB15-18C9)对细胞进行染色和门控。分拣后Tregs的平均纯度为约98％(范围97％至100％)。使用来自波兰BDBiosciences的单克隆抗体(荧光染料/类/克隆)：抗CD3(PacificBlue，IgG1，UCHT1)、抗CD4(V-500，IgG1，RPA-T4)、抗CD8(PerCP，IgG1，SK1)、抗CD19(PerCP，IgG1，4G7)、CD14(PerCP，IgG2b，
)、抗CD16(PerCP-Cy5.5，IgG1，3G8)、抗CD25(PE，IgG1，M-A251)和抗CD127(APC，IgG1，hIL-7R-M21)从Tregs的分拣后样本中另外确认了表型和杂质。
对于静脉内施用，使用临床级抗CD3/抗CD28珠粒(Miltenyi Biotec)、白细胞介素2(阿地白介素，诺华)和灭活自体血清进行Treg扩增最长达14天[中位数(最小-最大)＝11(10-14)]。在整个扩增过程中，培养基(X-Vivo20，Lonza)补充有10％血清和1000UI/ml的IL2。在扩增开始时将珠粒以1:1的比例添加到细胞中，然后在+7、+8和+9天的传代期间恢复1:1的比例。将培养物从珠粒上洗出，并在培养的最后24-48小时内留在10％血清和低水平的IL2(100UI/ml)中。具有自体CD4+Tconvs的前哨培养在10％血清和低水平IL2(100UI/ml)中进行，作为功能测试的T应答者来源。释放的最终产物：将FoxP3表达保持在90％以上[中位数(最小-最大)＝91％(90-97)]；将CD62L表达保持在80％以上[中位数(最小-最大)＝87％(81-95)]；通过了IFNγ抑制试验并且微生物检测为阴性。培养物的质量控制在+7天和产物释放时进行。如前所述[14]进行IFNγ抑制试验。简而言之，将来自扩增培养物的Tregs样本(从珠粒中洗出并静置至少24小时)与自体Tconv前哨细胞以1:1的比例共培养。对照仅由Tconvs或Tregs的培养物组成，受刺激或未受刺激以产生IFNγ。紧接在试验之前，用细胞示踪剂CFSE(CFDA kit Thermo，USA)对Tconvs进行染色，以将它们与Tregs区分开来，因此可以在试验结束时分别给出IFNγ阳性Tregs和IFNγ阳性Tconvs的比例。根据制造商的描述，用细胞内染色试剂盒(BDBiosciences，波兰)进行培养物的刺激和染色。用50ng/ml佛波醇12-肉豆蔻酸酯13-乙酸酯、500ng/ml离子霉素(Sigma，波兰)和2μl/ml细胞因子释放抑制剂GolgiPlug(BDBiosciences，波兰)刺激培养物5小时。然后，用抗IFNγ抗体对细胞进行染色。试验的阳性读数是与仅使用Tconvs的培养物中产生的IFNγ相比，Tconvs与Tregs共培养产生的IFNγ的抑制为至少25％[中位数(最小-最大)＝69％(52-95)]。Tregs产生的IFNγ从未超过细胞的2％。微生物安全性通过以下来确认：来自扩增培养基的上清液的微生物培养结果阴性(BD Bactec系统，欧洲BDBiosciences)，来自扩增培养基的上清液的内毒素测试阴性(Endosafe-PTS Endotoxin Cartridge/Cartridge reader，Charles River，USA)，来自扩增培养基的上清液的革兰氏染色阴性(Gram Stain Kit，BDBiosciences，欧洲)，以及产物中不存在HBV、HCV、HIV-1和HIV-2遗传物质(Cobas MPX，Roche，欧洲)。对与可能的产品污染相关的患者任何不良症状进行跟踪，直到释放后所有微生物结果被确认为阴性。Tregs的即用型制剂必须从组织机构中释放后2小时内施用。最终剂量为40×106个Tregs/kg b.w.。释放后，将制剂完全洗出，悬浮在250ml注射用0.9％NaCl(Polfa，华沙)中，然后以缓慢静脉输注施用于患者。
对于鞘内治疗的患者，根据上述释放标准检查100万(1×106)个新鲜分离的Tregs(未扩增)，然后悬浮在10ml 0.9％NaCl中。之后，在L4/L5或L5/S1腰椎穿刺期间通过穿刺针以缓慢注射施用。注射后进行6小时的卧床方案。
临床评估
除了在实地探访时进行常规身体/神经系统检查外，还由经过认证的神经科医生根据EDSS和MSFC量表对患者进行评估[15]以监测疾病进展并根据EQ-5D问卷监测生活质量[16]。进行了以下实验室测试(仅显示显著异常值)：全血细胞计数、代谢、肾功能全套和肝功能全套、C反应蛋白水平、尿液分析。
MRI评估
脑部MRI根据MAGNIMS 2015标准方案(3D T1加权、3D T2-FLAIR、3D T2加权和单剂量钆增强后T1加权成像，非间隙切片厚度均≤3mm，DWI序列(≤5-mm切片厚度，1,5TeslaMagnetom Aera，西门子，德国)进行。在施用后+3个月、+6个月和+12个月的访问期间进行MRI。使用BrainMagix软件(比利时，布鲁塞尔)和Philips Intellispace Portal 10对病变及其进展进行评估，由两名观察者计算斑块和对比增强斑块的总数。
免疫应答
使用十色板进行免疫表型分析，以跟踪外周血中的CD3+CD4+CD25高CD127-FoxP3+Tregs和CD3+CD4+CD25低/-CD127+FoxP3-Tconvs。在这两个群体中，跟踪对这些亚群的功能重要的抗原的表达。我们特定地基于以下表型确定了幼稚/记忆亚群的百分比：幼稚/Tn(CD62L+CD45RA+)、中央记忆/Tcm(CD62L+CD45RA-)和效应子记忆/Tem(CD62L-CD45RA-)。基于转录因子Helios在外周[pTreg Helios(-)]和胸腺[tTreg Helios(+)]亚群中的表达，将CD3+CD4+CD25高CD127-FoxP3+Tregs进一步分开[17](图2S)。
在该程序中，使用了购自波兰BDBiosciences的以下抗人单克隆抗体(荧光染料/类/克隆)：抗CD3(PacificBlue/IgG1/UCHT1或V500-C/IgG1/克隆SK7)、抗CD4(PerCP或AlexaFluor700/IgG1/RPA-T4)、抗CD25(PE或BV786/IgG1/M-A251)、抗CD127(FITC或BUV737/IgG1/hIL-7R-M21)、抗CD45RA(PE-Cy7/IgG1/L48)、抗CD73(BUV737/IgG1/AD2)、抗CD279(BV605/IgG1/EH12.1)、抗CD137(BV650/IgG1/4B4-1)、抗CD134(BV711/IgG1/ACT35)、抗CD152(BV786/IgG1/BNI3)抗CD18(FITC/IgG1/L130)、抗CD184(PE-CF594/IgG1/12G-5)、抗CD194(BV605/IgG1/1G1)、抗CD39(BV650/IgG1/TU66或BUV737/IgG1/TU66)、和抗CD103(BUV395/IgG1/Ber-ACT8)。抗CD62L(APC-Cy7/IgG1/3B5)由USA的Invitrogen提供；FoxP3染色试剂盒和抗Helios(eFluor450/IgG1/22F6)由USA的ebioscience/thermoFisher提供；抗CCR8(PerCP/IgG1/91704)和抗CCR10(PE/IgG1/314305)由UK的R&D/biotechne提供。
在luminex分析仪(Merck，USA)上，用基于珠粒的多重分析测量以下38种细胞因子的血清水平：IFNα2、IFNγ、IL10、IL12p40、IL12p70、IL13、IL15、sCD40L、IL17、IL2、IL1RA、IL1α、IL1β、IL3、IL4、IL5、IL6、IL9、TNFα、TNFβ、EGF、FGF-2、TGF-α、G-CSF、GM-CSF、VEGF、FLT-3L、IL7、嗜酸细胞活化趋化因子(Eotaxin)、CX3CL-1、CXCL-1、MCP-3、CCL22、IL8、IP-10、MCP-1、MIP-1α、和MIP-1β。所有测定均根据制造商的说明进行。
统计分析
使用软件Statistica 12.0(Statsoft，波兰)计算数据。使用ClustVis软件(https://biit.cs.ut.ee/clustvis/#mathematics)进行聚类分析。采用非参数检验进行分析。P≤0.05被认为具有统计学意义。
结果
1.1.安全性
在整个试验过程中没有报告严重的不良事件。Tregs静脉内(iv.)治疗的患者出现中度不良反应。最常见的不良反应是CNS病变的复发和进展。有趣的是，Tregs鞘内(tc.)施用的患者中没有发现不良反应(表2)。
生活质量分析显示使用EQ-5D表格进行的自我评估没有恶化。在整个随访过程中，两组的结果相似(所有测试p＞0.05，图1)。
1.2.功效—临床
在整个研究过程中，使用EDSS量表评估的临床状态在各组之间没有差异[Kruskal-Wallis ANOVA：第0天：H＝0.18，p＝0.66；6m：H＝0.36，p＝0.54；12m：H＝0.029，p＝0.86](图1)。然而，在tc.组内和iv.组内EDSS量表中的一年恶化分别为0至0.3和0至1。在iv.组中，10名对象中有3名在EDSS量表上表现出高于1分的恶化。在鞘内治疗的患者中没有观察到这种恶化。在随访期间，静脉内治疗的5名患者出现总计12次复发，频率为每年1次至3次。同时，在tc.组中未观察到复发。
在整个研究中，使用MSFC量表评估的临床状态在任何组中都没有变化，并且在任何量表成分中组间没有差异(所有测试p＞0.05，图1)。
1.3.功效—MRI
与iv.组相比，MRI扫描分析显示该疾病在tc.组中的活动性较低(图2)。
FLAIR序列显示，在整个随访期间，CNS中斑块的总体积在iv.组中增加，而在tc.组中没有变化[弗里德曼方差分析：iv.：χ2＝12.79p＝0.005；tc.：χ2＝4.5p＝0.21]。组间差异在随访6个月和12个月时显著[Kruskal-Wallis ANOVA：3m：H＝1.65，p＝0.19；6m：H＝6.14，p＝0.013；12m：H＝5.33，p＝0.047]。当比较组间五个最大斑块的体积[Kruskal-WallisANOVA:3m：H＝0.01，p＝0.91；6m：H＝7.77，p＝0.005；12m：H＝2.34，p＝0.067]和新斑块的数量[Kruskal-Wallis ANOVA：3m：H＝3.76，p＝0.15；6m：H＝5.10，p＝0.076；12m：H＝4.61，p＝0.091]时，看到差异。有趣的是，随访期间斑块总体积增加的原因是iv.组中的斑块数越来越多[斑块数的弗里德曼方差分析：iv.：χ2＝20.77，p＝0.0001；tc.：χ2＝5.5，p＝0.13]，而不是现有最大斑块的变化[5个最大斑块的平均体积的弗里德曼方差分析：iv.：χ2＝3.66，p＝0.30；tc.：χ2＝3.90，p＝0.27]。此外，在iv.组中对比增强的T1病变在试验结束时显著下降。tc.患者的情况并非如此，因为在整个随访过程中，未在该组中看到这些病变[弗里德曼方差分析：iv.：χ2＝11.41，p＝0.009；tc.：所有数字“0”]。主要CNS结构的体积或T1低强度的体积在各组之间均无差异(图3S)。
1.4.免疫应答
1.4.1.Tregs亚群
在整个随访期间或组间的FoxP3+Tregs和Tconvs的水平无明显变化(所有测试p＞0.05，图3)。然而，无论Tregs的施用途径如何，在所有患者的若干测量亚群中，Tregs都与Tconvs不同。当考虑所有患者，将Tregs与Tconvs进行比较时，Tregs主要包含Tcm表型(50％或更多)，而Tconvs主要包含Tn表型(50％或更多)(图3C和表1S)。我们还发现Tregs表达了若干种受体，例如趋化因子受体CCR10、CXCR4、CCR4、整合素CD103、外核苷酸酶CD39，以及两种共刺激分子CTLA-4和4-1BB，它们在Tconvs上几乎检测不到(图3S-A、B和表1S)。通过聚类分析证实了Tregs和Tonvs在这些受体表达中的差异(图3D)。
此外，所有患者中大约20％的Tregs不表达转录因子Helios，表明这些细胞起源于外周(图3B)。考虑到这一点，我们进行了更深入的分析，将Tregs分为胸腺FoxP3+Helios(+)tTregs和外周FoxP3+Helios(-)pTregs。当进行比较时，tTregs含有更高百分比的CCR10+细胞、CD103+细胞、CD73+细胞和CD39+细胞，而pTregs含有更高百分比的CTLA-4+细胞(图3S-C和表1S)。聚类分析证实CCR10、CD103、CD39的高表达和CTLA-4受体的低表达使tTregs与pTregs不同(图3E)。
1.4.2.细胞因子
该研究还包括测量的患者血清中38种不同的细胞因子的阵列。当与静脉内治疗的患者相比时，鞘内治疗的患者显示出更高水平的一些与炎症相关的因子，例如MCP-3、IL1RA和IL8。有趣的是，tc.组中脑营养因子TGFα的水平也比iv.组中的高(表2S，图4S)。tc.组与iv.组中位于同一簇中的MCP-3、IL1RA的水平不同(图4)。在整个随访过程中，试验组之间或每组内的测量的其他细胞因子水平没有差异。
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Claims (4)
1.用于多发性硬化症中临床使用的包含CD3+CD4+CD25+CD127-调节性T细胞的医药产品。
2.根据权利要求1所述的产品，所述产品鞘内施用于被诊断为多发性硬化症的患者。
3.根据权利要求1所述的产品，所述产品是鞘内施用的。
4.根据权利要求1所述的产品，所述产品在多发性硬化症的治疗中施用。
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| AU2005204338A1 (en) * | 2005-03-02 | 2006-09-21 | Sydney West Area Health Service | Treatment for multiple sclerosis |
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| US20170038394A1 (en) * | 2014-04-16 | 2017-02-09 | Genovie Ab | Method for the identification of cd4+ regulatory t-cells for use in the treatment of inflammatory and autoimmune diseases |
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| US20140004133A1 (en) * | 2012-06-29 | 2014-01-02 | Regenex LLC | Treatment method for relapsing-remitting multiple sclerosis |
| US20170038394A1 (en) * | 2014-04-16 | 2017-02-09 | Genovie Ab | Method for the identification of cd4+ regulatory t-cells for use in the treatment of inflammatory and autoimmune diseases |
| US20180362928A1 (en) * | 2015-12-17 | 2018-12-20 | Gdanski Uniwersytet Medyczny | Method for ex vivo expansion of regulatory t cells |
| WO2018024895A1 (en) * | 2016-08-05 | 2018-02-08 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Immunotherapeutic uses of ex vivo generated foxp3+ regulatory t cells |
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