CN101252941A - 治疗炎性肠病 - Google Patents
治疗炎性肠病 Download PDFInfo
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- CN101252941A CN101252941A CNA2006800313067A CN200680031306A CN101252941A CN 101252941 A CN101252941 A CN 101252941A CN A2006800313067 A CNA2006800313067 A CN A2006800313067A CN 200680031306 A CN200680031306 A CN 200680031306A CN 101252941 A CN101252941 A CN 101252941A
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Abstract
一种治疗炎性肠病(IBD)的方法，包括从引流患或未患IBD的肠节段的患者前哨淋巴结收集活化或非活化状态的调节性T细胞，通过将这些细胞与细胞因子和从发炎肠节段获得的抗原提取物接触从而任选活化这些细胞，体外扩增这些T细胞，和将扩增的T细胞重新输注回该患者。还披露了获得前哨淋巴结、扩增T细胞、在克罗恩病患者中重建TH1/TH2平衡的方法，和扩增的T细胞、细胞因子和抗原提取物以及由此活化和扩增的T细胞的相应应用。
Description
发明领域
本发明涉及治疗炎性肠病的方法和实施该方法的手段。
发明背景
缺少对炎性肠病(IBD)，例如克罗恩病(CD)和溃疡性结肠炎(UC)的永久性治愈方法。在瑞典，每年有1400位新患者诊断患有IBD，其发病率峰值出现在20岁，从而导致终身要用副作用严重的肾上腺皮质素和/或免疫抑制药物治疗。此外，IBD常导致外科肠切除术和永久的小孔(stoma)，这是主要的不利之处。溃疡性结肠炎还增加了大肠癌症的风险。
据认为，IBD是针对通常无害的粘膜抗原的免疫应答异常所致的自身免疫疾病。根据其中涉及的细胞因子，CD炎症被鉴定为TH1细胞介导的免疫应答。有人提出UC具有TH2细胞介导的应答，虽然这尚未清楚地证实(Fuss I J等，1996.J.Immunol.157：1261-70；Mullin G E等，1996.Inflamm.Bowel Dis.2：16-26)。
细胞因子用作免疫应答的调节剂，因为它们刺激所涉及的细胞元件分化和增殖。CD4+辅助T细胞可分化成两种主要的效应细胞亚群，用IL-12刺激时的TH1和用IL-4刺激时的TH2。TH1细胞分泌IFN-γ从而刺激了抵御胞内微生物，例如病毒或胞内细菌的吞噬细胞和溶细胞性T细胞依赖性反应。TH2细胞分泌IL-4和IL-5从而刺激了抵御寄生虫的嗜曙红细胞/肥大细胞介导的防御机制和IgE产生。TH2还起到下调TH1应答的功能。
发明目的
本发明的一个目的是提供治愈炎性肠病或至少长期控制该病的方法。
本发明的另一目的是提供该方法中所用的手段。
通过以下发明概述、说明本发明的许多附图、其优选实施方式描述以及随附的权利要求书可了解其它目的。
发明概述
本发明基于这样一种发现，即，给予从患者的前哨淋巴结(sentinel lymph node)收集并体外扩增的调节性T细胞(Treg)能永久性治愈(permanently cure)炎性肠病，特别是克罗恩病和/或溃疡性结肠炎，或至少将其维持在不活动或不活跃的状态。从而能减轻，甚至完全抑制肠炎症，而不用求助例如皮质类固醇等药物。
将免疫系统对口服给予的抗原无反应定义为口服耐受。很明显，该机制防止了针对肠道内含物的不适当免疫反应，据信该机制由至少两种途径介导：第一，作为T辅助细胞家族(CD4+)成员的调节性T细胞(Treg，CD4+CD25+)的活化；第二，诱导T细胞的克隆排除或无反应性。推测负责低剂量抗原接触耐受的第一种途径发生在局部肠道淋巴系统，其依赖于细胞因子TGF-β的产生。推测第二种(排除)途径发生在系统淋巴组织中，由滤过性抗原(filtered antigen)激发，并与对没有免疫佐剂存在下静脉内给予的抗原的诱导耐受的机制相似。后一种耐受机制看来在接触高剂量抗原后更突出。
T细胞活化受到严格调节，从而通过避免如自身免疫中所见的过分和错误导向的免疫应答而能在免疫系统中维持平衡。在克罗恩病患者中，肠系膜淋巴结极度增大是T细胞活化不受控的标志。
因为IBD是一种炎症，毗邻炎性部位的肠系膜淋巴结主要负责该部位的引流。处于沿炎性区域的直接途径上的第一批淋巴结称为前哨淋巴结。
按照本发明的第一优选方面，从引流IBD肠节段的患者淋巴结(前哨淋巴结)收集活化状态的调节性T细胞，体外扩增并重新输注入该患者中。
按照本发明的第二优选方面，从引流健康肠节段，即未受IBD影响的肠节段的患者淋巴结收集非活化状态的调节性T细胞，体外扩增并重新输注入该患者中。如此收集的活化以及非活化的调节性T细胞可以被合适的物质，例如细胞因子在体外(进一步)活化。
按照本发明的第三优选方面，分别从引流患或未患IBD的肠节段的淋巴结收集活化状态或非活化状态的调节性T细胞并体外扩增。扩增可以是连续或有间隔的，例如间插有休息期的阶段。联用一种或多种细胞因子和炎性肠节段的抗原提取物来活化(刺激)扩增的调节性T细胞。或者，在扩增前或各扩增阶段之间以这种方式活化(刺激)从引流患或未患IBD的肠节段的淋巴结收集调节性T细胞。选自IL-2、IL-7、IL-10、TGF-β、TSLP的一种细胞因子，优选两种或多种细胞因子，例如含有IL-2、IL-7、IL-10、TGF-β，特别是TGF-β1、TSLP的细胞因子混合物可与炎性肠节段的抗原提取物联用以获得Treg活化。还优选利用抗-CD3、抗-CD28和雷帕霉素提供补充Treg活化(additional Treg activation)。扩增后，将扩增和活化的调节性T细胞重新输注入该患者中。
用自体调节性T细胞的优点在于副作用低，甚至没有副作用而且疗效高。
按照第三优选方面的第一种改变形式，从引流患或未患IBD肠节段的克罗恩病患者淋巴结收集活化状态的调节性T细胞，联用炎性部位的抗原提取物和用于扩增的低剂量细胞因子IL-2来活化，优选再联用抗IFN-γ和/或TNF-α的抗体，从而能抑制TH1偏移(skewing)和TH2扩增。还优选以一定间隔，例如每天到每5天，特别是在1周或更长时期内，特别是在3-4周时期内每天条件化(condition)培养基。由此获得了TH2细胞群，该细胞群可用于自身输血从而在患者中建立TH1/TH2平衡。用CD3抗体补充活化可支持扩增。为改善TH2偏移，扩增中还可联用抗CD28抗体和抗IL-12抗体。
按照第三优选方面的第二种改变形式，从引流患或未患IBD肠节段的溃疡性结肠炎患者淋巴结收集活化状态的调节性T细胞，联用炎性部位的抗原提取物和用于扩增的细胞因子IL-2、IL-12及IFN-α中的一种或多种(优选再联用抗IL-4和/或抗IL-10抗体)来活化。还优选以一定间隔，例如每天到每5天，特别是在1周或更长时期内，特别是在3-4周时期内每天条件化培养基。可以提供额外的刺激，例如CD3和CD28，特别是接近扩增期结束时。
本发明的第四优选方面提供了用药学上可接受的载体配制的一种或多种细胞因子，来体外活化从IBD患者，特别是受IBD影响的前哨淋巴结收集的调节性T细胞。
本发明的第五优选方面提供一种获得引流IBD肠节段的前哨淋巴结的方法，该方法包括将淋巴染色试剂，例如专利蓝(patent blue)注射入IBD患者肠道的病损区域，鉴定该试剂染色的淋巴组织，任选用缝线标记该染色组织并切除该染色组织。仔细研究切除的组织(例如用显微镜检查)，继而将引流IBD肠节段的前哨淋巴结从组织上切下。
本发明的第六优选方面提供一种从引流IBD肠节段的前哨淋巴结分离调节性T细胞的方法，该方法包括对该淋巴结施加压力，收集由此被挤出该淋巴结的细胞混悬液，并分离该混悬液的调节性T细胞。
本发明的第七优选方面提供从引流IBD肠节段的前哨淋巴结分离的调节性T细胞。
随附的权利要求书包括了本发明的其它优选方面。
下文将参考由许多附图说明的本发明优选但非限制性的实施方式来更详细地解释本发明。
附图简述
图1显示通过两种表面标记的表达鉴定了Treg样品中活化的Treg；
图2显示了功能性Treg试验；
图3显示了提取自扩增的Treg的mRNA的RT-PCR试验；
图4显示了扩增前后Treg群的调节能力。
图5显示了发炎的肠切片的内源性提取物对人免疫细胞的活化作用。
图6显示源自前哨淋巴结的类似活化的Treg的剂量响应。
图7显示了作为TH2标记的CCR5的表达。
图8显示了UC患者的Treg中IL-4的分泌。
图9显示了CD患者的Treg中IFN-γ的分泌。
优选实施方式描述
实施例1.鉴定引流炎性肠节段的前哨淋巴结
前哨淋巴结技术已应用了十年，其对恶性肿瘤，主要是恶性黑色素瘤和乳腺癌进行疾病分期(staging)。利用注射入肠道中病损区域的淋巴染色试剂，例如专利蓝(CAS 129-17-9)和专利蓝V(CAS 3536-49-0；常以钙螯合的二聚体提供)描绘癌症的淋巴引流液，从而在外科手术期间鉴定前哨淋巴结。利用缝线或其它手段标记如此鉴定的前哨淋巴结。然后以常规方式切除肠道病损以及未受影响的边缘区和肠系膜，包括血管和局部淋巴结。仔细研究切除的组织，鉴定并除去前哨淋巴结。
该方法应用于溃疡性结肠炎或克罗恩病患者中引流发炎和未受影响肠道的前哨淋巴结。此外，收集患者的静脉血样品。然后在实验室中处理外周血样品、淋巴结和炎性病损的样本以及未受疾病影响的肠道节段的样本以进行免疫学分析和T细胞扩增。利用菲科尔-泛影钠(Ficoll-Hypaque)(法玛西亚安玛山姆公司(Pharmacia，Amersham))纯化外周血白细胞(PBL)。利用松散配合的玻璃匀浆器(loose fit glass homogenizer)轻柔施压以获得淋巴结细胞的单细胞悬液。用杜恩斯(Dounce)匀浆器匀浆组织片段，97℃变性5分钟来制备肠道样品的抗原提取物。然后对这些细胞进行功能分析。
实施例2.分离T细胞群
利用瑞典乌普萨拉市安玛山姆生物科技公司(Amersham Biosciences，Uppsala，Sweden)的菲科尔-泛影钠正(Ficoll-Hypaque Plus)纯化血液样品或暗黄层的淋巴细胞和单核细胞。用PBS稀释所研究的暗黄层或血液样品，将其小心地层叠在菲科尔-泛影钠溶液上，然后以400g离心该两相系统30分钟。在菲科尔溶液和血浆之间的中间相收集淋巴细胞和单核细胞，而红细胞和粒细胞在试管的底部获得。利用巴斯德移液管(Pasteur pipette)取出淋巴细胞层，用HBSS洗涤这些细胞以除去过量的菲科尔-泛影钠正、血浆和血小板。不立即使用这些细胞时，将它们37℃保存在含有10％HuS、1％PeSt和1％谷氨酰胺的RPMI培养基中。利用德国伯格施格拉德巴赫市米尔泰尼生物科技公司(MiltenyiBiotec，Bergisch Gladbach，Germany)的自动MACS分离器(autoMACS Separator)纯化常规的CD4+Th细胞和CD4+CD25+细胞。计数细胞，300g离心10分钟，完全吸出上清液，按照每107个细胞用90μL MACS缓冲液(PBS配制的0.5％BSA、2mM EDTA、0.01％叠氮钠)和10μL生物素-抗体混合物来溶解细胞。这样所有非CD40+细胞群均被标记。4℃培育10分钟后，每107个细胞加入20μL抗-生物素微珠，4℃再培育15分钟。用约20体积的MACS缓冲液洗涤细胞，300g离心10分钟，完全除去上清液，按照每108个细胞用500μL MACS缓冲液来重悬细胞。用自动MACS分离器进行磁性分离以消除样品中的非CD4+细胞。为分离CD4+/CD25+细胞与CD4+细胞群，计数CD4+细胞，300g离心10分钟，完全除去上清液，按照107个细胞用90μL MACS缓冲液和10μL CD25微珠来溶解沉淀物。4℃培育15分钟后，如上所述洗涤细胞，用自动MACS分离器进行正向选择来分离CD25+细胞。通过流式细胞术分析所有细胞群的等份试样。
实施例3.通过流式细胞术表征细胞
采用流式细胞术(FACS)研究分离的细胞群上不同表面标记的表达。将细胞分配入4mL FACS试管，用2mL FACS缓冲液(用PBS配制的2％FCS、0.05％叠氮钠)洗涤，300·g离心10分钟，倒去上清液，将沉淀物重悬在100μL FACS缓冲液中。加入7μL待用的各抗体，4℃培育至少30分钟，进而再用FACS缓冲液洗涤细胞一次，如上所述离心并重悬在1mL用于细胞固定和红细胞裂解的FACS裂解溶液中，混合，4℃培育10分钟，然后再次如上所述洗涤和离心一次。倒去上清液，按每试管用500μL FACS缓冲液重悬细胞，然后利用美国新泽西州富兰克林湖市(Franklin Lakes，NJ，USA)的贝克坦迪金森公司FACS校验仪(Becton Dickinson FAC SCalibur instrument)进行分析。
实施例4.增殖试验
研究了纯化的CD4+CD25+细胞的抑制能力。37℃用25μL PBS配制的5μg/mL抗-CD3 IgG温育圆底96-孔板90分钟。用200μL PBS洗涤各孔3次，然后加入3种不同的细胞群。每孔加入用25Gy照射的100μL 500,000细胞/mLCD4-细胞连同30 000 CD4+CD25-应答细胞和1μg/mL可溶性CD28(终浓度)。将不同数量的CD4+CD25+细胞加入各孔，从而产生许多不同的CD4+CD25-/CD4+CD25+细胞比例。将相同数量的CD4+CD25-细胞加入一组额外孔中的应答细胞作为对照。
将各板维持于37℃4或5天，然后向各孔中加入1μCi[3H]胸腺嘧啶，各板温育18小时，然后在-20℃冷冻。融化细胞，用美国康涅狄格州哈姆登市汤姆技术公司(TOMTEC，Hamden，CT，USA)的细胞收集器将孔内含物转移至芬兰土尔库市华莱士公司(Wallac，Turku，Finland)的玻璃纤维滤膜。将芬兰土尔库市华莱士公司的MeltiLex A-融化闪烁器片(Melt-on scintillator sheet)置于该滤膜的顶端，85℃融化。利用芬兰土尔库市华莱士公司的1205贝塔普雷特液体闪烁计数器(1205 Betaplate Liquid Scintillation Counter)检测放射性。
实施例5.FoxP3的TGF-β1诱导
如上所述从暗黄层制备CD4+CD25-细胞。制备不同供体的PBMC，并照射(25Gy)。然后在含或不含1ng/mL TGF-β1的6-孔板中，按照2mL/孔的体积用1.25×106个PBMC同种异型活化(allo-activate)3×106个CD4+细胞。各板维持于37℃。取细胞样品进行流式细胞术分析，在第2、5和7天提取RNA。
实施例6.RNA提取和cDNA合成
如下所述从不同细胞群提取RNA用于随后的PCR试验。按照每5-6×106个细胞用1mL美国加利福尼亚州卡尔斯巴德市英杰公司(Invitrogen，Carlsbad，CA，USA)的TRIzol来裂解细胞，然后立即于-70℃冷冻保存样品，或在室温培育5分钟，再按照每mL TRIzol试剂0.2mL的用量加入氯仿，剧烈振荡试管。2-3分钟后，在4℃以12,000g离心15分钟。将透明的水相转移至新试管，第一步骤中每用1mL TRIzol，则用0.5mL异丙醇沉淀RNA，室温下培育RNA 10分钟，然后在4℃以12,000g离心10分钟。除去上清液，第一步骤中每用1mLTRIzol，则用1mL 75％的乙醇洗涤沉淀物。旋振各试管，4℃以7,500g离心5分钟，随后短时风干沉淀物(5-10分钟)，溶解于不含RNA酶的水中，55℃培育10分钟。按照生产商的方案利用美国加利福尼亚州赫拉克勒斯市拜尔雷得公司(Bio-Rad，Hercules，CA，USA)的艾斯克利普特cDNA合成试剂盒(iScript cDNASynthesis Kit)合成cDNA。
实施例7.聚合酶链式反应(PCR)和实时定量PCR
PCR利用不同细胞群的合成的cDNA。PCR的目的是检测FoxP3、GAPDH和RNA聚合酶II(RPII)的表达。引物购自瑞典胡丁厄市赛博基因AB公司(Cybergene AB，Huddinge，Sweden)。其序列为：FoxP3，正向-CAGCACATTCCCAGAGTTCCTC，反向-GCGTGTGAACCAGTGGTAGATC；GAPDH，正向-GAAGGTGAAGGTCGGAGTC，反向-GAAGATGGTGATGGGATTTC；RPII，正向-GCACCACGTCCAATGACAT，反向-GTGCGGCTGCTTCCATAA。常规PCR使用德国法兰克福市新英格兰生物实验室公司(New England Biolabs，Frankfurt amMain，Germany)的ThermoPol反应缓冲液和Tag DNA聚合酶，而定量实时PCR使用美国加利福尼亚州赫拉克勒斯市拜尔雷得公司的iQSYBR绿超级混合物(iQSYBR Green Supermix)。按照以下方案分别用美国加利福尼亚州赫拉克勒斯市拜尔雷得公司的麦萨克勒尔热循环仪(MyCycler Thermal Cycler)和艾萨克勒尔iQ实时PCR检测系统(iCycler iQ Real-Time PCR Detection System)进行反应：95℃、30秒，48-65℃、30秒和72℃、30秒，至少重复循环40次。对于定量实时PCR反应，采用如Ginzinger D G，Exp.Hematol.2002 30：503-12所述的相对定量方法。用2％琼脂糖凝胶分离PCR产物，利用紫外光激发掺入的溴化乙锭来检测各条带。
实施例8.研究从IBD患者的前哨淋巴结获得的Treg
通过外科手术收集总共20位IBD患者的前哨淋巴结并研究。前哨淋巴结获得的淋巴细胞(SEAL)的特征在于表达了表面标记CD4、CD8、CD3、T细胞受体(TCR)、CD25、CD69、CD45RA、CD45RO和CD14。检测了CD4+CD25高Treg群的数量(图1)。发现与引流未受影响肠区域的前哨淋巴结中Treg数量相比，引流炎性肠节段的前哨淋巴结中Treg细胞数量正常。
然而，从引流炎性节段的前哨淋巴结纯化的Treg(CD4+CD25高)和从无炎症的肠节段纯化的Treg(CD4+CD25高)在对T细胞的调节性活化中存在实质性不同：引流炎性部位的前哨淋巴结的Treg在调节T细胞活化中表现不理想(图2)。换言之，引流受影响区域的前哨淋巴结的Treg的T细胞应答调节能力明显受损。
实施例9.调节性T细胞的扩增
对于Treg诱导和扩增，用IL-2、CD28、同种异体饲养细胞和TGF-β刺激外周血的CD4+CD25-T细胞。扩增后，如FACS所检测，超过85％的CD4+T细胞表达高水平的CD25，表明了Treg表型(未显示)。RT-PCR证明扩增的(细胞)群含有Treg标记转录物FoxP3，扩增之前的CD4+CD25-细胞群中不含该转录物(图3)。
检验诱导并扩增的Treg细胞群的功能以证明其抑制T细胞活化的能力。1∶10比例便能抑制T辅助细胞增殖(图4)，证明扩增的CD4+CD25+FoxP3+细胞群(图3)显示出Treg调节特性。引流炎性肠节段的IBD患者前哨淋巴结中Treg的数量看来正常；它们显示不足以抑制受刺激T细胞的应答。通过刺激和扩增原初T细胞，可以提供高效的T细胞调节特性。
实施例10.利用从前哨淋巴结获得的调节性T细胞识别从炎性部位获得的抗原
将从前哨淋巴结、负淋巴结(negative node)获得的调节性T细胞、肠道上皮细胞和外周血白细胞在浓度不同的肠道细胞匀浆液中培育5-7天。用炎性区域的抗原提取物刺激前哨淋巴结的Treg观察到增殖(图5)。
然而，当用从非炎性部位获得的抗原提取物刺激从前哨淋巴结获得的调节性T细胞时，未见或仅有极其微弱的应答(未显示)。克罗恩病患者的结果与溃疡性结肠炎患者的相似。
盲肠和乙状结肠中，引流受影响区域的前哨淋巴结均识别从炎性部位获得的同一抗原提取物并增殖(图6)。这些结果表明共同的抗原负责T细胞的克隆扩增。观察到的高度自发增殖是前哨淋巴结中体内克隆性T细胞扩增(clonal Tcell expansion)所致。
从前哨淋巴结获得的调节性T细胞显示细胞因子表达提高。在UC患者中，前哨淋巴结的CD4+T细胞是CD45RO+/CCR5+，这提示针对炎性粘膜的Th2炎性应答状态增大(图7)。
用炎性部位的抗原提取物活化后，UC患者的前哨淋巴结分泌的IL-4(一种Th2细胞因子)数量增多(图8)。
抗原特异性活化后，从克罗恩病患者获得的调节性T细胞的反应是TH1细胞因子IFN-γ的产量增加(图9)。
如CCR5表达增加(图7)和TH2细胞因子，例如IL-4产量增加(图8)所示，这些结果表明，从UC患者前哨淋巴结获得的T细胞针对炎性部位抗原的增殖性应答增大，从前哨淋巴结获得的调节性T细胞的反应是TH2应答。从克罗恩病患者前哨淋巴结获得的T细胞显示相反的活化模式，其主要产生TH1细胞因子，例如IFN-γ。
UC溃疡、或UC或克罗恩病患者的肠道炎性部位的内皮细胞经历凋亡或坏死，然后被专职抗原呈递细胞(APC)内吞。APC内吞分解的内皮细胞，经淋巴管迁移至引流淋巴结(前哨淋巴结)，在该处APC加工源自内皮细胞的蛋白质(抗原)并将其呈递至T细胞。T细胞得到活化，经历克隆扩增并离开淋巴结以找寻炎症区域，在该区域它们变为效应细胞并导致炎症。
实施例11.治疗IBD
可通过以下步骤治疗IBD，特别是克罗恩病和/或溃疡性结肠炎：收集引流炎性区域的患者前哨淋巴结的调节性T细胞，体外扩增它们并活化，通过输注将它们送回该患者体内。具体地说，以抗原特异性方式体外扩增调节性T细胞的优点在于克隆扩增的Treg累积在前哨淋巴结中。在GMP条件下扩增IBD患者前哨淋巴结的Treg，利用含有IL-2、IL-7、IL-10、TGF-β的细胞因子混合物连同炎性肠节段的抗原提取物活化以实现Treg的抗原特异性扩增。采用所述的RT-PCR、FACS和体外抑制试验评估活化效力(图4)。可利用抗-CD3、抗-CD28和雷帕霉素的补充活化以优化IBD Treg的扩增和功能性。或者，如果引流炎性肠节段的前哨淋巴结的Treg的数量和/或功能不令人满意，可以考虑采用相同方案扩增引流非炎性节段的前哨淋巴结的Treg。该备选方法的原理是非炎性节段可以视作经适当调节，因此Treg是有功能的，虽然其数量太少以致于不能在毗邻肠节段中维持调节作用。将任一种类的扩增Treg作为自身输血输回以有序地恢复免疫调节。
附图说明
图1.通过表面标记CD4+的表达和IL-2受体CD25的高水平表达(CD4+CD25高)，鉴定Treg。
图2.功能性Treg试验。用从外周血纯化并用抗CD3和抗CD28活化的CD4+CD25-T辅助细胞培育引流炎性肠节段(空心圆圈)和非炎性节段(实心圆圈)的前哨淋巴结的纯化Treg。在最后16小时期间向培养液中加入3H胸腺嘧啶。
图3.扩增Treg细胞。用IL-2、TGF-β、抗-CD28和饲养细胞活化CD4+CD25-细胞以刺激Treg细胞扩增。采用RT-PCR证明从扩增的CD4+CD25+Treg提取的mRNA和FoxP3转录物，显示扩增的细胞群是Treg。
图4.调节经刺激的应答T辅助细胞。含有CD4+CD25-细胞的预扩增细胞群不能调节(实心圆圈)，而扩增的CD4+CD25+细胞可以获得足够的调节(空心圆圈)。
图5.活化人免疫细胞。利用从溃疡性结肠炎患者的发炎肠切片获得的内源性抗原提取物活化从前哨淋巴结、非前哨淋巴结、肠道获得的细胞以及外周血淋巴细胞。
图6.剂量反应图。显示了从引流盲肠和乙状结肠的前哨淋巴结获得的调节性T细胞被从同一炎性肠切片获得的内源性抗原提取物活化的的剂量反应。
图7.CCR5表达作为TH2的标记。从UC患者前哨淋巴结获得的调节性T细胞中CD4+CD45RO+记忆细胞上CCR5的表达升高是TH2活化的标志。
图8.UC患者的调节性T细胞中TH2细胞因子IL-4的分泌。用炎性抗原提取物刺激从非前哨淋巴结(nSLN)和前哨淋巴结(SLN)获得的细胞48小时。采用夹心ELISA检测培养上清液中分泌的IL-4。
图9.从克罗恩病患者的前哨淋巴结获得的调节性T细胞分泌TH1细胞因子IFN-γ。用炎性抗原提取物刺激从非前哨淋巴结(nSLN)和前哨淋巴结(SLN1和SLN2)获得的细胞48小时。采用夹心ELISA检测培养上清液中分泌的IFN-γ。
Claims (45)
1.一种治疗炎性肠病的方法，其包括(a)从引流患或未患IBD的肠节段的患者前哨淋巴结收集活化或非活化状态的调节性T细胞，(b)体外扩增所收集的T细胞，和(c)将扩增的T细胞重新输注回该患者。
2.如权利要求1所述的方法，其特征在于，所述炎性肠病是克罗恩病。
3.如权利要求2所述的方法，其特征在于，所述炎性肠病是溃疡性结肠炎。
4.如权利要求1-3中任一项所述的方法，其特征在于，收集活化状态的所述调节性T细胞。
5.如权利要求3或4所述的方法，其特征在于，从引流患IBD肠节段的淋巴结收集所述T细胞。
6.如权利要求5所述的方法，其特征在于，联用一种或多种细胞因子与从发炎肠节段获得的抗原提取物来补充活化所收集的T细胞。
7.如权利要求6所述的方法，其特征在于，先活化再扩增。
8.如权利要求6所述的方法，其特征在于，活化与扩增同时进行。
9.如权利要求6所述的方法，其特征在于，先扩增再活化。
10.如权利要求6所述的方法，其特征在于，所述一种或多种细胞因子选自：IL-2、IL-7、IL-10、TGF-β1、TSLP。
11.如权利要求6所述的方法，其特征在于，包括用抗-CD3、抗-CD28和/或雷帕霉素再活化所收集的T细胞。
12.如权利要求1-3中任一项所述的方法，其特征在于，收集非活化状态的所述调节性T细胞。
13.如权利要求12所述的方法，其特征在于，从引流未患IBD肠节段的淋巴结收集所述调节性T细胞。
14.如权利要求12或13所述的方法，其特征在于，联用一种或多种细胞因子与从发炎肠节段获得的抗原提取物来活化所收集的T细胞。
15.如权利要求14所述的方法，其特征在于，先活化再扩增。
16.如权利要求14所述的方法，其特征在于，活化与扩增同时进行。
17.如权利要求14所述的方法，其特征在于，先扩增再活化。
18.如权利要求14所述的方法，其特征在于，所述一种或多种细胞因子选自：IL-2、IL-7、IL-10、TGF-β1、TSLP。
19.如权利要求14-18所述的方法，其特征在于，包括用抗-CD3、抗-CD28和/或雷帕霉素再活化所收集的T细胞。
20.如权利要求2所述的方法，其特征在于，从引流患或未患IBD肠节段的淋巴结收集调节性T细胞，将它们与从炎性部位获得的抗原提取物和低剂量细胞因子IL-2接触使之活化，并扩增。
21.如权利要求20所述的方法，其特征在于，还包括将所述调节性T细胞与抗IFN-γ和/或TNF-α的抗体接触来活化，从而抑制TH1偏移和TH2扩增。
22.如权利要求20或21所述的方法，其特征在于，包括以一定间隔，例如每天到每5天，特别是在1周或更长时期内，特别是在3-4周时期内，每天条件化培养基。
23.如权利要求20-22中任一项所述的方法，其特征在于，包括联用抗CD28抗体和抗IL-12抗体来活化，从而改善TH2偏移。
24.如权利要求3所述的方法，其特征在于，从引流患或未患IBD肠节段的淋巴结收集调节性T细胞，将它们与从炎性部位获得的抗原提取物和细胞因子IL-2、IL-12和IFN-α中的一种或多种接触来活化，并扩增。
25.如权利要求24所述的方法，其特征在于，将调节性T细胞与CD3和/或CD28接触以提供补充活化。
26.药学上可接受的载体配制的一种或多种细胞因子在体外活化从IBD患者收集的调节性T细胞中的应用。
27.如权利要求26所述的应用，其特征在于，所述IBD是克罗恩病。
28.如权利要求26所述的应用，其特征在于，所述IBD是溃疡性结肠炎。
29.一种获得引流受炎性肠病(IBD)影响的肠节段的前哨淋巴结的方法，该方法包括(a)将淋巴染色试剂注射入IBD患者肠道的病损区域，(b)鉴定该试剂染色的淋巴组织，(c)任选用一根或多根缝线标记该染色的组织，(d)切除该染色组织，(e)检查染色的组织以鉴定引流IBD肠节段的前哨淋巴结，将所鉴定的前哨淋巴结从该组织上切下。
30.如权利要求29所述的方法，其特征在于，所述IBD是克罗恩病。
31.如权利要求29所述的方法，其特征在于，所述IBD是溃疡性结肠炎。
32.一种从引流IBD肠节段的切离前哨淋巴结分离调节性T细胞的方法，该方法包括(a)对该前哨淋巴结施加压力，(b)收集被挤出该前哨淋巴结的细胞混悬液，(c)分离该混悬液的调节性T细胞。
33.一种调节性T细胞，其从引流受炎性肠病(IBD)影响的肠节段的前哨淋巴结分离。
34.如权利要求31所述的调节性T细胞，其特征在于，所述IBD是克罗恩病。
35.如权利要求31所述的调节性T细胞，其特征在于，所述IBD是溃疡性结肠炎。
36.一种扩增用于治疗IBD的调节性T细胞的方法，其包括(a)用合适培养基提供从IBD患者的前哨淋巴结收集的调节性T细胞，(b)将它们与细胞因子接触来活化这些细胞，(c)扩增所活化的细胞。
37.如权利要求36所述的方法，其特征在于，扩增期间进行一些或全部的活化。
38.一种在克罗恩病患者中重建TH1/TH2平衡的方法，其包括：(a)从引流患或未患克罗恩病的肠节段的患者前哨淋巴结收集活化状态的调节性T细胞，(b)联用炎性部位的抗原提取物和低剂量细胞因子IL-2来活化如此收集的T细胞，(c)扩增这些细胞，(d)任选在扩增期间将这些细胞与抗IFN-γ和/或TNF-α的抗体接触来抑制TH1偏移和TH2扩增。
39.如权利要求38所述的方法，其特征在于，包括以一定间隔条件化培养基。
40.如权利要求39所述的方法，其特征在于，每天到每5天进行条件化。
41.如权利要求39所述的方法，其特征在于，包括使这些细胞与CD3抗体接触来刺激扩增。
42.选自以下的细胞因子对于活化调节性T细胞的应用，所述调节性T细胞从IBD患者的前哨淋巴结获得：IL-2、IL-7、IL-10、TGF-β1、TSLP。
43.抗原提取物连同细胞因子对于活化调节性T细胞的应用，所述抗原提取物从IBD患者的发炎肠节段获得，所述调节性T细胞从该患者的前哨淋巴结获得。
44.如权利要求43所述的应用，其特征在于，所述细胞因子选自：IL-2、IL-7、IL-10、TGF-β1、TSLP。
45.分别如权利要求42和43-44所述应用的组合。
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| CN102732480A (zh) * | 2012-06-11 | 2012-10-17 | 中山大学 | N-乙酰半胱氨酸对中央记忆性t细胞的绝对数量在体外扩增上的应用 |
| CN103911341A (zh) * | 2014-01-26 | 2014-07-09 | 山东迪博生物技术有限公司 | Th1细胞亚群的制备方法及其在制备抗肿瘤细胞制剂中的应用 |
| CN110051698A (zh) * | 2019-03-18 | 2019-07-26 | 天津大学 | 基于微囊化处理的肠道原生菌基因改造的方法 |
| CN111374989A (zh) * | 2020-03-16 | 2020-07-07 | 中山大学附属第五医院 | 用于治疗炎症性肠病的药物 |
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| WO2007027132A1 (en) | 2005-08-31 | 2007-03-08 | Ith Immune Therapy Holdings Ab | Treatment of inflammatory bowel disease |
| EP2062970A1 (en) | 2007-11-26 | 2009-05-27 | Txcell | Compositions for treating an intestinal inflammatory condition |
| WO2014104305A1 (ja) * | 2012-12-28 | 2014-07-03 | 持田製薬株式会社 | プレセプシン測定による炎症性腸疾患の診断 |
| CA2930784C (en) | 2013-11-22 | 2023-01-31 | Cellectis | Method for generating batches of allogeneic t cells with averaged potency |
| JP2018188408A (ja) * | 2017-05-11 | 2018-11-29 | 国立大学法人群馬大学 | 制御性ｔ細胞増強剤並びにそれを含む医薬及び食品組成物 |
| GB201714777D0 (en) * | 2017-09-14 | 2017-11-01 | Univ London Queen Mary | Agent |
| RU2728696C2 (ru) * | 2018-12-28 | 2020-07-30 | Федеральное государственное бюджетное учреждение науки институт биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова Российской академии наук (ИБХ РАН) | Моноклональное антитело к интерферону бета-1а человека |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102732480A (zh) * | 2012-06-11 | 2012-10-17 | 中山大学 | N-乙酰半胱氨酸对中央记忆性t细胞的绝对数量在体外扩增上的应用 |
| CN103911341A (zh) * | 2014-01-26 | 2014-07-09 | 山东迪博生物技术有限公司 | Th1细胞亚群的制备方法及其在制备抗肿瘤细胞制剂中的应用 |
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| CN110051698A (zh) * | 2019-03-18 | 2019-07-26 | 天津大学 | 基于微囊化处理的肠道原生菌基因改造的方法 |
| CN111374989A (zh) * | 2020-03-16 | 2020-07-07 | 中山大学附属第五医院 | 用于治疗炎症性肠病的药物 |
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| NO20081539L (no) | 2008-05-29 |
| EP1933854B1 (en) | 2014-06-25 |
| CA2619735A1 (en) | 2007-03-08 |
| JP2009506109A (ja) | 2009-02-12 |
| US20090311228A1 (en) | 2009-12-17 |
| KR101475800B1 (ko) | 2014-12-23 |
| EA200800662A1 (ru) | 2008-08-29 |
| NO341189B1 (no) | 2017-09-04 |
| AU2006285446A1 (en) | 2007-03-08 |
| EA015989B1 (ru) | 2012-01-30 |
| EP1933854A1 (en) | 2008-06-25 |
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