内分泌与代谢病的免疫学发病机制研究
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摘要
第一部分CD8+T细胞在1型糖尿病发病中的作用
目的1型糖尿病是T细胞介导的自身免疫性疾病,其自身免疫过程包括临床糖尿病前期(即胰岛炎期)和显性糖尿病期,最近的遗传学资料及研究结果表明,T细胞可作为预防1型糖尿病的靶细胞。因此,T细胞在1型糖尿病发病中作用的研究是目前1型糖尿病研究的热点。大量组织学分析和T细胞转移实验研究表明,T细胞亚群CD4+T细胞和CD8+T细胞,在1型糖尿病发病中都是必不可少的,但它们在糖尿病进程中各自的作用仍不明确。本研究,旨在探讨CD8+T细胞是否启动了胰岛炎,如果是,CDS+T细胞是怎样启动糖尿病发生的。
方法用NOD鼠和基因工程构建的NOD背景的转基因鼠作为本研究的动物模型,用PCR和流式细胞技术鉴定实验动物的基因型(图2-8)。
过继转移前,先从供鼠分离新鲜脾细胞,通过特异性抗体染色、流式细胞分析,确证所要转的细胞种类,计算所要转移的细胞数量,然后将一定数量的供鼠新鲜脾细胞腹腔注射给受鼠,采尾静脉血监测受鼠血糖,并将未发生糖尿病的受鼠胰腺用免疫组化技术,进行胰岛炎的评估。
过继转移时,首先检验幼年(2周龄)的NOD鼠是否带有致糖尿病的T细胞、NOD鼠T细胞的致糖尿病能力是否随着供鼠年龄的增长而增加,以及是否幼年NOD鼠体内缺少β细胞自身抗原的表达。然后研究β细胞上共刺激分子CD80的表达对CD4+T细胞和CD8+T细胞致糖尿病能力的影响,评估β细胞上表达共刺激分子CD80对未被免疫的静止CD8+T细胞的作用。
用CFSE分子探针标记NOD.scid.CL4 TCR转基因供鼠的脾细胞,将标记的脾细胞过继转移给NOD.scid.InsHA鼠或NOD.scid鼠,用流式细胞技术评价转移后不同时间受鼠的脾、胰腺淋巴结和胰岛中,HA特异性的CL4 CD8+T细胞的增殖和活化通情况。
采用SPSS11.0软件包进行统计分析,两样本率的比较采用四格表资料的x~2检验,多个样本率的比较采用行×列表资料的x~2检验。
结果
1.14天龄NOD鼠的脾细胞被转移给6～8周龄的NOD.scid鼠(n=36)和年龄匹配的NOD.scid.Rip.B7鼠(n=35),在转移脾细胞后9周,当68.6%NOD.scid.Rip.B7受鼠发生糖尿病时,NOD.scid受鼠组尚没有检测到胰岛淋巴细胞浸润(图9)。转移脾细胞后11周,80%的NOD.scid.Rip.B7鼠发生糖尿病(图13 B所示)。但在NOD.scid受鼠组,即使转移后20周,尚没有1例受鼠发生糖尿病。
2.我们将相同数量(2×10~7个脾细胞/受鼠)的、不同周龄的NOD鼠的脾细胞分别转移给相同周龄的NOD.scid受鼠,观察T细胞的致糖尿病能力在不同周龄NOD供鼠的变化。正如图14～17所示,2周龄NOD鼠的脾细胞过继转移给6～8周龄的NOD.scid鼠(受鼠n=36)时,转移后20周仍然没有受鼠发生糖尿病;转移3周龄NOD鼠的脾细胞时(NOD.scid受鼠n=35),转移后13周时开始有受鼠发生糖尿病(5%),到转移后25周时有约30%的受鼠发生糖尿病;当转移4周龄NOD鼠的脾细胞时(NOD.scid受鼠n=36),转移后12周时开始有受鼠发生糖尿病(10%),到转移后20周时有约50%的受鼠发生糖尿病;而转移6周龄NOD鼠的脾细胞时(NOD.scid受鼠n=37),转移5周后(36天)开始有NOD.scid受鼠发生糖尿病,到转移后9周时有约65%的受鼠发生糖尿病,转移后13周时,100%的受鼠发生糖尿病。经多个样本率之间两两比较,在转移后同一时间,不同周龄的NOD供鼠在相同受鼠中诱导的糖尿病发病率有显著性差异(P＜0.001)。
3.幼年(2周龄)NOD鼠的脾细胞不能诱导NOD.scid受鼠发生糖尿病,是否幼年NOD鼠体内缺少β细胞自身抗原的表达?我们进一步用过继转移试验回答上述问题。从已发生糖尿病的NOD鼠分离脾细胞并注射给新生的(n=30)以及1周龄(n=32),2周龄(n=34),3周龄(n=32),6周龄(n=36)的NOD.scid鼠(结果见表2)。所有年龄组的受鼠均在接受转移细胞后5周左右开始发病,转移后9周时所有年龄组受鼠100%发病。尽管受鼠的年龄不同,但它们发生糖尿病的时间却相似(图13A)。经行×列表资料的x~2检验,P＞0.05,说明在转移后同一时间,不同周龄的受鼠糖尿病发病率的差异无统计学意义。
4.从9～10天龄的BDC2.5/NOD.scid鼠的脾脏分离出BDC2.5 CD4+T细胞,将5×10~5个CD4+T细胞转移给6～8周龄的NOD.scid鼠(n=40)和NOD.scid.Rip.B7鼠(n=40)。两组受鼠都是从转移后12天开始发病,到转移后15天时,两组受鼠糖尿病发病率均为100%(图18A),经x~2检验,两组受鼠糖尿病发病率的差异无统计学意义(P＞0.05)。
然后我们将不同数量的BDC2.5 CD4+T细胞转移到年龄匹配NOD.scid和NOD.scid.Rip.B7受鼠,各组受鼠数量均为40只。结果不论我们转移了多少BDC2.5CD4+T细胞,在NOD.scid.Rip.B7受体组和NOD.scid对照组所诱导的糖尿病发生率及发病特点都是相似的(图18A～D),该结果证明了β细胞上共刺激分子CD80的表达对CD4+T细胞的致糖尿病性是没有影响的。
5.从4周龄的NOD.Rag~(null).AI4 TCR转基因鼠分离脾细胞作为活化的CD8 T细胞,将相当于7×10~5个CD8+T细胞的脾细胞转移至NOD.scid(n=35)或NOD.scid.Rip.B7(n=40)鼠(表3)。虽然两组受鼠均发生糖尿病,但发病速度明显不同,转移后24天,当NOD.scid.Rip.B7组受鼠全部发生糖尿病时(发病率100%),NOD.scid对照组糖尿病发病率只有14.3%,经x~2检验,两组发病率有显著性差异(P＜0.001)。转移相当于7×10~4个CD8+T细胞的脾细胞时,转移后45天,NOD.scid.Rip.B7组受鼠全部(n=40)发生糖尿病时(表4),NOD.scid对照组(n=35)糖尿病发病率只有17.1%,经x~2检验,两组发病率的差异有统计学意义(P＜0.001)。
两组试验尽管供鼠与受鼠相同,由于转移的细胞数不同,相同受鼠发生糖尿病的速度却是明显不同的。
从9～10天龄NOD.Rag~(null).AI4 TCR转基因鼠分离脾细胞,将相当于7×10~5个CD8+T细胞的脾细胞转移至NOD.scid.Rip.B7鼠(n=32)和NOD.scid对照鼠(n=30)。31天后NOD.scid.Rip.B7组开始有受鼠发生糖尿病,在转移脾细胞后55天NOD.scid.Rip.B7受鼠100%发生糖尿病(图20A)。静止的CD8+T细胞所诱导的糖尿病的发生及其发病率与已发生糖尿病的AI4 TCR转基因鼠及糖尿病前期的AI4TCR转基因鼠的脾细胞所诱导的糖尿病的发生及其发病率相似。NOD.scid组在转移60天后尚未发现有受鼠发生胰岛炎,但在120天时NOD.scid组的部分受试对象发生外周胰岛炎和胰岛炎(图20B)。
6.将9～10天龄NOD.Rag~(null).AI4 TCR转基因鼠的静止的AI4 CD8+T细胞转移到NOD.scid.β2m~(null).Rip.B7鼠和NOD.scid.Rip.B7鼠各40只。NOD.scid.Rip.B7组被迅速诱发糖尿病(图21所示),在转移后23天开始有受鼠发生糖尿病,转移后35天100%受鼠发生糖尿病。但作为对照,NOD.scid.β2m~(null).Rip.B7组即使在转移后80天也无1例受鼠发生糖尿病。
当我们转移4周龄的AI4 CD8+T细胞给NOD.scid.β2m~(null).Rip.B7鼠和NOD.scid.Rip.B7鼠时,NOD.scid.Rip.B7组迅速发生糖尿病,NOD.scid.β2m~(null).Rip.B7组虽然也有受鼠发病,但发病时间晚,发病率低。
7.将CFSE标记的对流感病毒血球凝集素(HA)特异性的CL4 CD8+ T细胞转移到12只选择性地在β细胞上表达HA基因的NOD.scid.Ins.HA鼠内,1×10~7CL4 CD8+T细胞/受鼠,12只NOD.scid.鼠作为对照。在NOD.scid.Ins.HA受鼠组,转移后第4天,从受鼠胰岛分离到的CL4 CD8+T细胞有高达50%的细胞已经经历了多个循环周期的分化,而从胰腺淋巴结和脾脏分离的CL4 CD8+T细胞却只有少数增殖(图22,23)。转移CL4 CD8+T细胞5天后,由于淋巴细胞严重浸润的胰岛脆性太大,易裂解的缘故,已经无法回收NOD.scid.InsHA受鼠胰岛内的CL4CD8+T细胞。但在NOD.scid对照组,直到转移7天后才在脾脏和PLN首先检测到T细胞增殖(图22,23),而在胰岛中只发现了少量CL4 CD8+T细胞,细胞数并不随时间的延长而增加。
结论
1.幼年NOD鼠的脾细胞含有β细胞特异性T细胞,数量有限,但其致糖尿病能力随着NOD鼠年龄的增长而增加。2周龄NOD小鼠的脾细胞可将糖尿病转移到NOD.scid.Rip.B7小鼠中,说明14天龄的NOD鼠的脾细胞含有β细胞特异性T细胞。在NOD.scid受鼠中没有胰岛炎和糖尿病,也说明了在幼年NOD鼠中的T细胞库和/或致糖尿病的T细胞的数量是有限的。转移不同周龄NOD鼠的脾细胞后,4组受鼠糖尿病的发生时间及发病速度明显不同,供鼠周龄越大,受鼠发病越早,发病速度越快,发病率越高。说明T细胞的致糖尿病能力随着NOD供体鼠年龄的增长而增加。NOD.scid.Rip.B7实验组与NOD.scid对照组糖尿病发生率的明显差异表明,共刺激分子CD80在β细胞上的表达促进了静止T细胞对糖尿病的转移诱导,这给我们提供了T细胞与胰岛β细胞直接作用的证据。
已发生糖尿病的NOD鼠的脾细胞转移给新生的以及1,2,3,6周龄的NOD.scid鼠,尽管受鼠的年龄不同,但它们发生糖尿病的时间却相似,该结果表明,在胰岛炎启动前,新生的和幼年的NOD.scid鼠就已经表达与疾病有关的β细胞自身抗原。
2.β细胞上共刺激分子CD80的表达不影响CD4+T细胞的致糖尿病作用9～10天龄的BDC2.5/NOD.scid鼠的脾细胞转移给6～8周龄NOD.scid.Rip.B7小鼠和NOD.scid小鼠时,在实验组和对照组中观察到的糖尿病的发生率和特点是相同的,并且不论我们转移了多大数量的BDC2.5 CD4+T细胞,在NOD.scid.Rip.B7实验组和NOD.scid对照组所诱导的糖尿病发生率及发病特点都是相似的,该结果证明了β细胞上表达共刺激分子CD80对BDC2.5 CD4+T细胞的致糖尿病能力无影响。
3.β细胞上共刺激分子CD80的表达影响了CD8+T细胞的致糖尿病性,并且在淋巴细胞浸润胰岛过程中对静止的CD8+ T细胞发挥了共刺激作用4周龄的NOD.Rag~(null).AI4αβTCR转基因鼠的脾细胞(CD8+ T细胞已经活化)诱导NOD.scid.Rip.B7组受鼠糖尿病的快速发生表明,β细胞上共刺激分子CD80的表达增强了β细胞特异性CD8+T细胞的致糖尿病能力。9～10天龄NOD.Rag~(null).AI4αβTCR转基因鼠的脾细胞(CD8+ T细胞未被活化)不能将糖尿病转移到NOD.scid受鼠,但对NOD.scid.Rip.B7受鼠诱导的糖尿病的发病率,与4周龄的AI4αβTCR转基因鼠的脾细胞所诱导的糖尿病的发病率相似。这说明,CD8+T细胞与β细胞是直接作用的,而NOD.scid受鼠胰岛炎的低发生率提示共刺激分子CD80在胰岛淋巴细胞浸润过程中对静止的CD8+T细胞发挥了协同刺激作用。
4.胰岛炎的启动需要CD8+ T细胞与胰岛β细胞的直接作用。将静止的AI4CD8+T细胞转移到NOD.scid.β2m~(null).Rip.B7鼠和NOD.scid.Rip.B7鼠时,NOD.scid.Rip.B7组受鼠被迅速诱发糖尿病,但作为对照,NOD.scid.β2m~(null).Rip.B7组受鼠不能被诱导发生糖尿病或胰岛炎。这说明,当NOD.scid.Rip.B7的β细胞失去MHCⅠ类分子的表达,其活化静止CD8+T细胞的能力也随之丧失。也就是说,静止的CD8+ T细胞的活化是因为共刺激分子CD80与表达MHCⅠ类分子的β细胞直接作用的结果。这些结果证明,在胰岛炎的启动中需要β细胞和CD8+T细胞的直接作用。
5.静止的CD8+ T细胞在胰岛内与β细胞抗原相遇。将CFSE标记的CL4CD8+ T细胞转移到NOD.scid.Ins.HA和NOD.scid.受鼠内,第4天,从NOD.scid.InsHA受鼠胰岛分离的CL4 CD8+T细胞,有高达50%已经经历了多个循环周期的分化,而从其胰腺淋巴结和脾脏分离的CL4 CD8+T细胞却只有少数增殖;但是在对照组,转移后7天才在受鼠内检测到CL4 CD8+T细胞明显的生理性增殖。这些结果证明,未被免疫的静止CD8+T细胞是直接进入胰岛,在胰岛内与内源性自身抗原相遇并启动胰岛炎,随后CD8+T细胞在胰岛内增殖和扩增。
本研究的创新点
1.关于CD8+ T细胞在1型糖尿病中的作用及其作用方式的研究,国内至今尚未见有报道;国外虽然有这方面的研究,但尚无定论。本研究利用过继转移的方法,研究发现CD8 T细胞通过与胰岛β细胞直接作用,启动了糖尿病的发生,目前国内外未见报道。
2.利用过继转移实验,发现在糖尿病发病过程中,共刺激分子对静止CD8 T细胞的活化依赖于β细胞上表达MHCⅠ类分子。对此国内尚未见报道。
3.通过CFSE标记静止的CD8 T细胞,首次发现CD8 T细胞是直接进入胰岛,在胰岛内被活化,而不是在胰腺淋巴结等胰腺外的淋巴器官被活化。
第二部分散发性甲旁减自身免疫性发病机制的研究
目的特发性甲旁减(idiopathic hypoparathyroidism,IH)分为散发性甲旁减(sporadic idiopathic hypoparathyroidism,SIH)、Di Geoge综合症、家族性1型、家族性2型(autoimmune polyglandular syndrome type 1,APS-1)。
关于特发性甲旁减自身抗体方面的研究,主要是针对APS-1中的IH患者。APS-1指Addison病、甲状旁腺机能减退症(甲旁减)和慢性粘膜皮肤念珠菌病三者中至少存在两个,还可伴有其他相关的免疫性疾病。由于组成APS-1的其它内分泌病都是典型的自身免疫病,人们很自然地认为IH也是自身免疫性疾病。而散发性甲旁减患者无家族史,也不伴有APS-1包括的自身免疫性疾病,所以,对APS—1的研究不能代替对散发性甲旁减(SIH)的研究。
由于散发性特发甲旁减患者极其罕见,国内外研究甚少,至今病因不明,尚无充分证据说明该病有自身免疫性。本研究,采用间接免疫荧光技术,检测了26例SIH患者血清中的抗甲状旁腺自身抗体,从体液免疫方面探讨SIH的自身免疫性。运用流式细胞技术,分析了10例SIH患者外周血淋巴细胞亚群及调节性T细胞的表达,从细胞免疫方面探讨SIH的自身免疫性。
方法
1、研究对象:散发性特发甲旁减患者26例及其直系亲属112例和健康对照60例。散发性特发甲旁减患者均无甲旁减、肾上腺皮质功能减退或黏膜皮肤念珠菌感染的家族史,患者本人也不伴有肾上腺皮质功能减退或黏膜皮肤念珠菌感染等自身免疫性疾病,排除APS-1型。所有空腹血清标本采集于1995～2006年间,分装后保存于-80℃待检。部分用于检测钙、磷、镁、全段甲状旁腺激素(iPTH)、FT3、FT4、TSH、血清皮质醇(Cortisol)、TPOAb,其余用于检测抗甲状旁腺自身抗体。
另外,采集上述研究对象中10例SIH患者及其38例直系亲属、12例健康对照的新鲜全血,ACD抗凝,分离单个核细胞,用于流式细胞分析。
2、抗甲状旁腺抗体的检测:采用间接免疫荧光技术,选用猴子甲状旁腺作底物抗原,用DAPI染核定位甲状旁腺细胞。结果先在荧光显微镜下观察,抗体滴度1:10阳性者,再按1:100稀释重复上述步骤。然后用共聚焦激光扫描显微镜(CLSM)进行扫描并采集图像。488nm波长的光激发绿色荧光图像(FITC标记的抗体)、356nm波长的光(紫外光,UV)激发蓝色荧光图像(DAPI标记的细胞核)。利用Leica Microsystems Heidelberg GmbH(Leica Confocal Software)分析软件进行图像处理。
3、血清钙、血清磷和血清镁含量检测:分别用偶氮砷Ⅲ法、紫外直接沉淀法和二甲苯胺蓝比色法。
4、血清FT3、FT4、TSH、Cortisol、TPOAb检测:采用电化学发光免疫分析法,iPTH用双抗体夹心法。
5、外周血淋巴细胞亚群的分析:从ACD抗凝的新鲜外周血分离单个核细胞,用荧光标记的抗体染色,洗涤去除未结合的抗体后,应用流式细胞仪进行检测。
6、流式细胞分析:用Becton Dickinson FACScalibur流式细胞仪检测,激发光为氩离子激光488nm谱线,使用荧光校准微球校准仪器,调整各荧光补偿。首先检测对照管,建立二维散点图,在散点图上调整参数前向散射光(FSC)及侧向散射光(SSC),调整阈值,圈定淋巴细胞射门以获取细胞。运行CELLQuest软件(Becton Dickinson,San Jose,CA)采集数据,WinMDI2.8软件分析数据,结果以各细胞亚群占PBMC细胞的百分率表示。
6、统计学分析:正态分布或近似正态分布的计数资料均以均数±标准差表示,非正态分布的计数资料以中位数及四分位间距表示。应用SPSS11.0软件进行数据处理,检验水平α=0.05。
结果
1、一般指标治疗前26例患者血清钙与iPTH水平均明显降低,与直系亲属组及正常对照组比较差异具有统计学意义(P均小于0.01),血清磷水平明显升高,与直系亲属组及正常对照组比较差异具有统计学意义(P均小于0.05),以上各指标在直系亲属组与正常对照组之间无显著性差异(P均大于0.05)。3组血清镁比较虽然无显著性差异,但其中4例SIH患者血清镁略低于正常范围;3组FT3,FT4,TSH,Cortisol及TPOAb等均在正常范围,统计学分析无显著性差异(P均大于0.05)。
2、抗甲状旁腺抗体26例SIH患者中有10例(8例为女性)患者的血清1:10稀释时抗甲状旁腺抗体阳性,阳性率38.46%,其中3例在1:100稀释时仍呈阳性反应。112例患者直系亲属中有11例血清1:10稀释时抗甲状旁腺抗体阳性,阳性率9.82%,60例正常对照组中有4例血清1:10稀释时抗甲状旁腺抗体呈阳性反应,阳性率6.67%,SIH患者组阳性率明显高于直系亲属组与正常对照组(P均小于0.01),直系亲属组与正常对照组抗体阳性率无显著性差异(P＞0.05)。抗体阳性的患者与抗体阴性的患者血清钙、磷、镁水平无显著性差异(P均大于0.05)。两组性别、年龄构成无显著性差异,病程及血清iPTH水平比较差异虽无统计学意义,但抗体阴性的患者病程较抗体阳性的患者有延长的趋势,并且其血清iPTH水平较阳性组患者有降低的趋势。
3、淋巴细胞表型的检测所测的淋巴细胞亚群中,CD4+,CD8+,CD4+比CD8+比值,CD5+,CD19+,CD5+CD19+等在三组研究对象间结果相似,三组之间比较差异无统计学意义。SIH患者外周血中CD4+CD25+调节性T细胞的百分率较健康对照组及患者直系亲属组低,二者比较有显著性差异(P＜0.01),患者直系亲属组CD4+CD25+调节性T细胞的百分率与健康对照组比较,差异无统计学意义(P＞0.05)。
结论
1、散发性特发甲旁减患者体内存在体液免疫功能的改变。本研究中散发性特发甲旁减患者抗体阳性率(38.46%)明显高于亲属组(9.82%)(P＜0.01)及健康对照组(6.67%)(P＜0.01),提示患者体内体液免疫活化。一般认为,自身抗体在发病中起始动作用的疾病如Grave病和重症肌无力,其临床症状和生化异常的严重程度与血清中抗体阳性程度有关,但我们的研究未发现SIH患者血清中的抗甲状旁腺抗体与该病的临床症状和生化异常的严重程度有关。
2、散发性特发甲旁减患者体内存在细胞免疫功能的改变。CD4+CD25+调节性T细胞(Treg)是参与对自身抗原外周耐受的主要T细胞群,也是对外来抗原应答的主要调节性T细胞,对于维持外周免疫耐受有重要意义。有研究表明CD4+CD25+Treg数量减少或功能异常均有可能导致自身免疫病的发生。本研究发现SIH患者外周血中CD4+CD25+T细胞百分比较患者亲属及健康对照组低,提示SIH患者体内免疫调节细胞减少,不能有效的维持自身耐受。患者体内自身免疫稳态破坏,机体更易发生病理性免疫应答,从而激活免疫反应,促使自身免疫性疾病的发生。
总之,散发性特发甲旁减患者体内自身反应性抗体的存在以及CD4+CD25+Treg细胞的改变,表明其自身免疫功能的紊乱。
本研究的创新点:
1、首次对散发性特发甲旁减患者外周血中CD4+CD25+Treg细胞的表达进行了研究,从细胞免疫学角度探讨疾病的自身免疫发病机制,检索近20年的文献,国内外未见任何报道。
2、首次利用流式细胞技术,探讨了散发性特发甲旁减患者外周血中单个核细胞淋巴细胞亚群的表达,检索近20年的文献,国外只有1篇关于特发性甲旁减淋巴细胞亚群的研究报道。
3、利用间接免疫荧光技术,对散发性特发甲旁减患者体内抗甲状旁腺自身抗体的存在进行了研究,从体液免疫角度探讨疾病的自身免疫发病机制,至今国内未见任何报道,国外的研究也很少。
Objective Type 1 diabetes is an chronic autoimmune disease mediated by T cells. The autoimmune process of the disease include a long pre-clinical period of diabetes (i.e.,insulitis)and overt diabetes period.Recent genetic data and preliminary trial results suggest T cells as a target for preventive strategies.The role of T cells in the pathogenesis of type 1 diabetes has become hot points in the study of type 1 diabetes. Sufficient histological analysis and transfer study of T cells suggests that both CD4+ T cells and CDS+ T cells are essential in the development of type 1 diabetes,the relative contribution of these two T cell subsets in triggering insulitis remains poorly defined.Therefore,we put that if CD8 + T lymphocytes launched insulitis? If so, how CD8 + T cells trigerred the diseases?
Method Non-obese diabetic(NOD)mice and NOD background transgenic mice using genetic engineering were used as animal models in our study.PCR and flow cytometry were used to type genotype of experimental animals.
Splenocytes were isolated from donor mice before adoptive transfer experiments. Stained with specific monoclonal antibody and determined the type and number of cells to be transferred by flow cytometry,a certain amount of cells were intraperitoneally to recipient mice.Diabetes was monitored by measuring glucose using tail vein blood of recipient mice.Insulitis was asssessed for those recipient mice which did not develop diabetes by immunohistochemistry.
Adoptive transferred experiments were carried out first to test whether the diabetogenic T cells existed in young(2 weeks old)NOD mice,and the diabetogenic capacity of T cells increased with the age of donor NOD mice,as well as whether cognate autoantigens were absent in young NOD mice,then to determine whether the expression of costimulatory molecules CD80 on beta cells influence the diabetogenic capability of CD4+ and CD8+ T cells,and to value the effects of costimulatory molecules CD80 on beta cells on naive CD8+ T cells.
Spleen cells from NOD.scid.CL4 TCR transgenic mice were labeled with molecular probe of CFSE and transferred to NOD.scid.InsHA mice and NOD.scid mice. Different time post-transfer,HA specific CL4 CD8+ T cells were isolated from the spleen,draining PLN and islets.T cells were stained with mAbs specific for the transgenic TCR,CD69 and CD62L.Proliferation and activation of HA-specific CL4 CD8+ T cells were assessed by flow cytometric analysis.
SPSS 11.0 software was used to process the data,test levelα=0.05.Pearson Chi-Square Tests were used for statistical analysis of diabetes incidence induced in recipient mice.
Results
1 Spleen cells of 14 day-old NOD mice were transferred into adult NOD.scid mice (n=36)and age-matched NOD.scid.Rip.B7 mice(n=35).Diabetes was induced in 68.6%of NOD.scid.Rip.B7 mice by 9 weeks post-transfer while in contrast,no infiltration of lymphocytes in the islets of NOD.scid recipients(as shown in Fig.9). Diabetes was induced in 80%of NOD.scid.Rip.B7 mice by 11 weeks post-transfer (as shown in Fig.13B).In contrast,none of the NOD.scid recipients developed diabetes even by 20 weeks post-transfer.
2 Then,spleen cells from different week-old NOD mice were transferred into adult NOD.scid mice in the same number of cells(2×10~7 cells / recipient).The diabetogenic capacity of T cells in different age of donor NOD mice were observed. As shown in Fig.14～17,none of the NOD.scid recipients(n=36)developed diabetes even by 20 weeks post-transfer when transferred spleen cells from 2 week-old NOD mice.However,when spleen cells from 3 week-old NOD donor mice were transferred, the recipient mice(n=35)began to develope diabetes from 13 weeks post-transfer (5%).There were about 30%of recipient mice developed diabetes by 25 weeks post-transfer.When spleen cells from 4 week-old NOD donor mice were transferred, the recipient mice(n=36)began to develope diabetes from 12 weeks post-transfer (10%)and there were about 50%of recipient mice developed diabetes by 20 weeks post-transfer.When spleen cells from 6 week-old NOD donor mice were transferred, the NOD.scid recipient mice(n=37)began to develope diabetes in earlier time of 5 weeks post-transfer,and there were about 65%of recipient mice developed diabetes by 9 weeks post-transfer,100%by 13 weeks post-transfer.There were significant difference among the diabetes incidence induced by differen age of NOD donor mice by the same time post-transfer(P＜0.001).
3 Spleen cells of young NOD mice(14 day-old NOD mice)could not transfer diabetes to NOD.scid recipient mice.Is it duo to the lack of expression of cognate autoantigens of beta cells? We answered the question by adoptive transferred experiments.Spleen cells were isolated from diabetic NOD mice and injected into neonatal(n=30),1 week-old(n=32),2 week-old(n=34),3 week-old(n=32), and 6 week-old(n=36)NOD.scid mice and the development of diabetes monitored (table 2).All recipient groups began to develop diabetes from 5 weeks post transfer and 100%recipient mice developed diabetes by 9 weeks post-transfer.Irrespective of age,diabetes was induced in all recipients with similar time of onset(Fig.13A).This result demonstrated that disease relevant beta cell autoantigens are expressed in neonatal and young NOD.scid mice,at times prior to the initiation of insulitis.There was no significant difference among different age of recipient mice of the diabetes incidence induced by diabetic NOD donor mice by the same time post-transfer(P＞0.05).
4 Naive splenic BDC2.5 CD4+ T cells were isolated from 9～10 day-old BDC2.5/NOD.scid mice and adoptively transferred into NOD.scid(n=40)and NOD.scid.Rip.B7 mice(n=40),(5×10~5 cells / recipient).Both recipient groups began to develop diabetes at 12 days post transfer,and by 15 days post transfer,100% recipient mice in two groups developed diabetes.There was no significant difference between the two recipient groups of the diabetes incidence(P＞0.05).
To exclude the possibility that the number of BDC2.5 CD4+ T cells transferred masked the effect of CD80,various numbers of BDC2.5 CD4+ T cells were transferred into NOD.scid and NOD.scid.Rip.B7 recipients.Regardless of the numbers of BDC2.5 CD4+ T cells transferred,diabetes was induced in NOD.scid.Rip.B7 and NOD.scid recipients with a similar frequency and kinetics(Fig. 18A-D).These results demonstrated that expression of CD80 by beta cells had no significant effect on the diabetogenic capacity of CD4+ T cells.
5 Activated CD8+ T cells prepared from the spleens of diabetic or 4 week-old NOD.Rag~(null).AI4 TCR transgenic mice,7×10~5 CD8+ T cells were transferred into NOD.scid(n=35)and NOD.scid.Rip.B7 mice(n=40).As demonstrated in table 3, diabetes developed in both NOD.scid and NOD.scid.Rip.B7 recipients,but with markedly different kinetics.All NOD.scid.RipB7 recipients developed overt diabetes by 24 days post-transfer,in contrast,diabetes incidence was only 14.3%in NOD.scid recipients.There were significant difference between the two groups of diabetes incidence(P＜0.001).
When 7×10~4 CD8+ T cells were transferred,all NOD.scid.RipB7 recipients(n=40) developed overt diabetes by 45 days post-transfer,in contrast,diabetes incidence was only 17.1%in NOD.scid recipients(n=35).There were significant difference between the two groups of diabetes incidence(P＜0.001).
CD8+ T cells prepared from the spleens of 9～10 day-old NOD.Rag~(null).AI4 TCR transgenic mice,7×10~5 CD8+ T cells were transferred into NOD.scid(n=30)and NOD.scid.Rip.B7 mice(n=32).As demonstrated in Fig.20A,NOD.scid.Rip.B7 recipient mice began to develop diabetes at 31 days post transfer,100%recipient mice developed diabetes by 55 days post transfer.The onset and incidence of diabetes induced by naive CD8+ T cells was comparable to that of CD8+ T cells prepared from diabetic or pre-diabetic AI4 TCR transgenic mice.Insulitis in the NOD.scid recipients was absent at 60 days post-transfer,but at 120 days post-transfer peri-insulitis and insulitis was detected in some recipients(Fig.20B).
6 Naive AI4 CD8+ T cells were adoptively transferred into NOD.scid.β2m~(null).B7 and NOD.scid.Rip.B7 mice.Diabetes was readily induced in NOD.scid.Rip.B7 mice (Fig.21).It began to develop diabetes at 23 days post transfer,and by 35 days post transfer,all animals in NOD.scid.Rip.B7 recipient mice developed diabetes.In contrast,none of the NOD.scid.β2m~(null).Rip.B7 recipient mice developed diabetes even by 20 days post-transfer.
7 Splenocytes from NOD.scid.CL4 transgenic mice which express an H2Kd-resticted TCR specific for influenza HA were labeled with CFSE.12 mice of NOD.scid.InsHA, which selectively express HA on bete cells,were injected i.p.with 1×10~7 CFSE-labeled spleen cells,12 mice of NOD.scid were used as control.By day 4 post-transfer,～50%of CL4 CD8+ T cells isolated from the islets of NOD.scid.InsHA had undergone multiple rounds of division whereas only a few CL4 CD8+ T cells from PLN and spleen showed evidence of proliferation(Fig.22,23). The lack of recovery of CL4 CD8+ T cells from the islets of NOD.scid.InsHA recipients at 5 days post-transfer or later because of the fragility of the islets with heavy infiltration.However,in NOD.scid recipients group,significant T cell proliferation was first detected in the spleen and PLN 7 days post-transfer(Fig.22,23). The small numbers of CL4 CD8+ T cells were found in the islets of NOD.scid control recipients,but did not increase with time.
Conclusion
1 Spleen cells of young NOD mice contain beta cell-specific T cells,however,the T cell repertoire and/or frequency of diabetogenic T cells are limited.The diabetogenic capacity of T cells increased with the age of NOD mice.
The spleen cells from 2-week-old NOD mice succeeded in transferring diabetes to NOD.scid.RipB7 recipients demonstrated that spleen cells of 14 day-old NOD mice contain beta cell-specific T cells.The lack of insulitis and diabetes in NOD.scid recipients,however,also suggested that the T cell repertoire and/or frequency of diabetogenic T cells are limited in young NOD mice.There were recipients in both groups developed diabetes when transferred splenocyte from different-week-old donors,but the incidence in control recipients was far lower from that of in NOD.scid.Rip.B7 mice(P＜0.001).It suggested that the diabetogenic capacity of T cells increased with the age of donor NOD mice.The significant difference between the two groups demonstrated that the expression of costimulatory molecule CD80 on βcells promopt the naive T cells in transferring induction of diabetes.It provided us an evidence of direct interaction between CD8+ T cells and beta cells.
2 Eexpression of CD80 on beta cell had no significant effect on the diabetogenic capacity of CD4+ T cells
Naive splenic BDC2.5 CD4+ T cells were isolated from 9-10 day-old BDC2.5/NOD.scid mice and adoptively transferred into 6～8-week-old NOD.scid and NOD.scid.Rip.B7 mice.Diabetes,with identical incidence and kinetics,was observed in both NOD.scid.Rip.B7 and control NOD.scid recipients.Regardless of the numbers of BDC2.5 CD4+ T cells transferred,diabetes was induced in NOD.scid.Rip.B7 and NOD.seid recipients with a similar frequency and kinetics.The result demonstrated that beta cell expression of CD80 had no effect on the diabetogenicity of BDC2.5 CD4+ T cells.
3 Beta cell expression of costimulatory molecules CD80 influences the diabetogenic capacity of CD8+ T cells,and CD80 exerts its co-stimulatory function on naive CD8+ T cells during the initiation of islet infiltration.
The accelerated onset of diabetes in NOD.scid.Rip.B7 recipients induced by the splenocytes from 4 week-old NOD.Rag~(null).AI4 TCR transgenic mice indicates that beta cell expression of costimulatory molecules CD80 enhances the diabetogenic capacity of beta cell-specific CD8+ T cells.However,these naive AI4 CD8+ T cells failed to transfer diabetes in NOD.scid recipients.The incidence of diabetes in NOD.scid.Rip.B7 recipients induced by naive AI4 CDS+ T cells was similar to that of induced by AI4 CD8+ T cells from diabetic or pre-diabetic donors.It suggested that CD8+ T cells directly interaced with beta cells,and CD80 played a costimulatory role on naive CD8+ T cells during the lymphocyte infiltrating islets.
4 The initiation of insulitis results from direct interaction between CD8+ T cells and beta cells.
Naive AI4 CD8+ T cells were adoptively transferred into NOD.scid.Rip.B7 and NOD.scid.β2m~(null).B7 mice that are deficient of MHC class I expression,but still with the expression of CD80 ectopically on beta cells.Diabetes was readily induced in NOD.scid.Rip.B7 mice.In contrast,naive AI4 CD8+ T cells failed to induce diabetes or insulitis in NOD.scid.β2m~(null).B7 recipient mice(Fig.21).These results indicated that the loss of MHC classⅠexpression on beta cells in NOD.scid.β2m~(null).B7 mice rendered incapable of priming naive CD8+ T cells.That is to say,activation of naive CD8+ T cells was in fact the result of a direct interaction with host beta cells expressing MHC classⅠand costimulatory molecule CD80.In summary,a direct interaction between beta cells and CD8+ T cells is required for the initiation of insulitis.
5 Naive CD8+ T cells encounter beta cell antigens within the islets.
CFSE-labeled CL4 CD8+ T cells were adoptive transferred to NOD.scid.InsHA and NOD.scid mice.By day 4 post-transfer,～50%of CL4 CD8+ T cells isolated from the islets of NOD.scid.InsHA had undergone multiple rounds of division whereas only a few CL4 CD8+ T cells from PLN and spleen showed evidence of proliferation.But in NOD.scid contrast group,significant T cell proliferation was first detected in the spleen and PLN 7 days post-transfer.These data demonstrated that naive CD8+ T cells traffic directly into the islets,encounter cognate autoantigens and initiate insulitis following proliferation and expansion.
The innovation
1 There has been no report on the role of CD8 + T cells in type 1 diabetes and the mode of its role.Some studies about CD8 T cells and T1D are ongoing abroad, however,there has been no consistent result by now.We explored the autoimmune pathogenesis of type 1 diabetes by studing the role of CD8 + T cells in the triggering of diabetes with adoptive transfer experiments,and found for the first time that the initiation of insulitis results from direct interaction between CD8+ T cells and beta cells.
2 We found for the first time that activation of naive CD8+ T cells was in fact the result of a direct interaction between costimulatory molecule CD80 and host beta cells expressing MHC classⅠ.
3 We found for the first time by CFSE-labelled naive CD8+ T cells that naive CD8+ T cells trafficed directly into the islets and was activated in islets,not in any other lymph organ or tissue like PLN.
PART 2 STUDY OF AUTOIMMUNE MECHANISM OF SPORADIC IDIOPATHIC HYPOPARATHYROIDISM
Object Idiopathic hypoparathyroidism(IH)includes sporadic IH(SIH),Di Geoge syndrome,familial type 1,familial type 2.Familial type 2 is also called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy(APECED)or autoimmune polyglandular syndrome type 1(APS-1).
The study on autoantibodies of idiopathic hypoparathyroidism(IH)is mainly targeted at the IH patients associated with APS-1.Less study is conducted on autoantibodies of SIH.At least two in three disorders of Addison disease, hypoparathyroidism and chronic skin and mucosal candidiasis disease are referred to as APS 1,that may be companied with other concerned immune diseases.Because the other endocrine components of APS 1(i.e.,APECED)are typical autoimmune diseases,it is natural to assume that this is also true of the HP.While sporadic IH without a family history,nor the autoimmune components of APS-1,the study of APS-1 is no substitute for SIH's.
The study on sporadic idiopathic hypoparathyroidism(SIH)is little at home and abroad because this disease is extremely rare.There is no sufficient proof for its autoimmune nature.To explore its autoimmune nature from humoral immunity,we searched for parathyroid autoantibodies in 26 patients with SIH by indirect immunofluorescence techniqes.We also analyzed the expression of lymphocyte subsets in peripheral blood of 10 patients with SIH by flow cytometry and explore its autoimmune nature from cellular immunity.
Method
1.Patients and control subjects After obtaining informed consent,we enrolled 26 patients with SIH and 60 age-and sex- matched healthy individuals as well as 112 family members of the patients with SIH.None of the patients had a family history suggestive of hypoparathyroidism or adrenal insufficiency or clinical features of mucocutaneous candidiasis infection.The type 1 autoimmune polyglandular syndrome was excluded by demonstrating a normal adrenocorticotrophin(ACTH) value and without clinical features of mucocutaneous candidiasis infection.All sera samples were obtained from subjects after 8-10 hours' fasting during1995 to 2006, kept in -80℃to be detected.Some sera were used to detect calcium,phosphorus, magnesium,intact parathyroid hormone(iPTH),FT3,FT4,TSH,Cortisol,TPOAb, and the rest for detection of anti-parathyroid autoantibodies.Fresh whole blood with ACD anticoagulant were collected from 10 of the 26 patients with SIH,38 of the 112 family members and 12 of the 60 healthy controls,and mononuclear cells were separated for flow cytometry analysis.
2.Detection of antibodies against parathyroid Indirect immunofluorescence techniques were used in our study.It was suggested that the antibodies against parathyroid in patients with APECED was a false positive IF results caused by human-specific antimitochondrial antibodies and organ-specific antibodies against parathyroid were extremely rare.To avoid this false positive IF results,we used parathyroid glands from monkey other than from human as substrates.To position the cells of parathyroid glands,we stained the nucleolus with DAPI.The results were firstly observed under fluorescence microscope,if the antibody was positive ina sera titer of 1:10,then repeated the above steps in 1:100 dilution.Then confocal laser scanning microscopy(CLSM)was used to scan and obtain the image.Green fluorescent images(FITC-conjugated antibody)was excitated by the light of 488 nm wavelength,blue fluorescence images(DAPI labeled nuclei)was excitated by the light of 356 nm wavelength(UV).Processing the image with Leica Microsystems Heidelberg GmbH(Leica Confocal Software)analyzing software.
3.Detection of Serum calcium,serum phosphorus and serum magnesium
Serum calcium,phosphorus and magnesium were examined respectively with AzoⅢarsenium method,ultraviolet direct precipitation method and xylidine chromatometry.
4.Detection of Serum FT3,FT4,TSH,Cortisol,TPOAb FT3/FT4 were examined by electrochemiluminescenc immuno-analysis method,iPTH was detected by double antibody sandwich method.
5.Analysis of lymphocyte subsets in peripheral blood PBMCs were isolated from the blood with ACD-anticoagulant of patients and healthy donors as well as family members of patients by means of Ficoll density gradient centrifugation.
6.Flow cytometry
Mononuclear cells were isolated from the fresh blood with ACD anticoagulant, stained with antibody fluorescent-labeled.Dual-color flow cytometry was performed using a CellQuest software and Becton Dickinson FACSCalibur instrument. Argon ion laser of 488 nm spectrum as excitation,equipment was calibrated by the use of fluorescent microspheres calibration,adjusted the fluorescence compensation. Isotype-matched controls were used to determine nonspecific background.Cells labeled with one fluorescent antibody and the other isotype control were used to determine the separation threshold of the cytometer channels.Lymphocytes were electronically gated based on their forward scatter(FSC)and side scatter(SSC) characteristics,and then double-positive cells were detected by analyzing at least 10,000 lymphocytes.In these assays,careful color compensation was performed, and their intra-assay reproducibility was corroborated.Obtained cells with CellQuest software.WinMDI2.8 software was used to analyze the data.
Results
1.General indicator The level of sera iPTH and calcium significantly decreased and sera phosphate significantly increased in 26 patients before treatment compared with the family members and the normal controls.There was no significant difference(P＞0.05)of the indicators above between family members and normal controls.Although there was no significant difference of serum magnesium among the three groups,but the sera magnesium of four patients with SIH were slightly lower than the normal range;The levels of sera FT3,FT4,TSH,Cortisol and TPOAb were all in the normal range of three groups.
2.Detection of anti-parathyroid antibody There were 10(8 cases are female)in 26 patients with positive antibodies against parathyroid when sera were diluted in 1:10,the positive rate was 38.46 percent,three were still positive when sera were diluted in 1:100.There were 11 in 112 family members with positive antibodies when sera were diluted in 1:10,the positive rate was 9.82 percent.There were 4 in 60 normal controls with positive antibodies when sera were diluted in 1:10,the positive rate was 6.67 percent.The positive rate in SIH patients were significantly higher than the family members and normal controls(P＜0.01).There was no significant difference between family members and normal controls(P＞0.05). There was no significant difference between patients with positive antibodies and without antibodies in sera calcium,phosphorus,magnesium levels(P＞0.05).There was no significant difference between patients with positive antibodies and without antibodies in gender and age.Although no significantly difference in duration of illness and level of sera iPTH,the patients without antibodies had a longer duration of illness[(4.4±2.07)years]and a lower serum iPTH[(6.0±3.95)pg/ml]than those of in patients with antibodies[(2.6±1.34)years and(9.6±3.77)pg/ml,respectively].
3.Detection of lymphocytes phenotype The results of the lymphocyte subsets detected in CD4+,CD8+,ratio of CD4+:CD8+,CD5+,CD19+,ratio of CD5+:CD19+ were similar among the three groups of subjects.There were no significantly difference of the lymphocyte subsets above when compared with each other among three groups.The frequencies of the phenotype of CD4+CD25+ were lower in patients than those of in family members and healthy controls.There was no significant difference between family members and healthy controls(P＞0.05).
Conclusion
1.Changes of humoral immune function present in patients with sporadic idiopathic hypoparathyroidism.
The frequencies of antibody against hypoparathyroid in patients(38.46%)was significantly higher than that of in family members(9.82%)(P＜0.01)and in healthy controls(6.67%)(P＜0.01).It was suggested that humoral immune be activated in patients.In organ-specific autoimmune disease,the patients typically has circulating autoantibodies specific for the affacted organ,often these precede the clinical disease by years and persist for yaers after its onset.This is also true for most of autoimmune components of APS 1,but we did not found in the serum of patients with SIH that autoantibodies against parathyroid had something with the clinical symptoms and the severity of the biochemical abnormalities.
2.Changes of cellular immune function present in patients with sporadic idiopathic hypoparathyroidism.CD4+CD25+regulatory T cells(Treg)are the main subset in the peripheral tolerance to self-antigen and the major regulatory T cells responding to foreign antigen.It is important for the maintenance of peripheral tolerance.Some studies showed that both the decrease of number and the dysfunction of CD4 + CD25 + regulatory T cells could lead to autoimmune diseases.We found that the frequencies of CD4 + CD25 + T cells in peripheral blood of SIH patients were lower than the frequencies of family members and healthy controls.It suggested that immunoregulatory cells in patients could not effectively maintain their own tolerance because of the decreased number.The destruction of autoimmune balance in patients made the body easy in pathological immune responses,thus activating immune response to autoimmune diseases.
In short,the presence of self-reactive antibodies and the decrease of CD4 + CD25+ regulatory T cells in patients with SIH showed that there were immune function disorders in these patients.
In conclusion,serum anti-parathyroid antibody exists in SIH patients demonstrated that humoral immune disorder was present in the disease.Decreased frequency of CD4+CD25+ T regulatory cells support the presence of a cellular immune disturbance in patients with SIH.
The innovation
1 There has been no report in the world on the expression of CD4+CD25+ T regulatory cells in patients with SIH by now.We explored the pathogenesis of sporadic idiopathic hypoparathyroidism from cellular immunity for the first time.
2 We studied the changes of lymphocytes in peripheral blood mononuclear cells of patients with SIH by flow cytometry.There was only one paper about it in the past 20 years in the world.
3 We detected anti-parathyroid antibody in the sera of SIH patients by indirect immunofluorescence techniques.No report about this has been seen in China and only little investigation has been conducted in the world.
目的1型糖尿病是T细胞介导的自身免疫性疾病,其自身免疫过程包括临床糖尿病前期(即胰岛炎期)和显性糖尿病期,最近的遗传学资料及研究结果表明,T细胞可作为预防1型糖尿病的靶细胞。因此,T细胞在1型糖尿病发病中作用的研究是目前1型糖尿病研究的热点。大量组织学分析和T细胞转移实验研究表明,T细胞亚群CD4+T细胞和CD8+T细胞,在1型糖尿病发病中都是必不可少的,但它们在糖尿病进程中各自的作用仍不明确。本研究,旨在探讨CD8+T细胞是否启动了胰岛炎,如果是,CDS+T细胞是怎样启动糖尿病发生的。
方法用NOD鼠和基因工程构建的NOD背景的转基因鼠作为本研究的动物模型,用PCR和流式细胞技术鉴定实验动物的基因型(图2-8)。
过继转移前,先从供鼠分离新鲜脾细胞,通过特异性抗体染色、流式细胞分析,确证所要转的细胞种类,计算所要转移的细胞数量,然后将一定数量的供鼠新鲜脾细胞腹腔注射给受鼠,采尾静脉血监测受鼠血糖,并将未发生糖尿病的受鼠胰腺用免疫组化技术,进行胰岛炎的评估。
过继转移时,首先检验幼年(2周龄)的NOD鼠是否带有致糖尿病的T细胞、NOD鼠T细胞的致糖尿病能力是否随着供鼠年龄的增长而增加,以及是否幼年NOD鼠体内缺少β细胞自身抗原的表达。然后研究β细胞上共刺激分子CD80的表达对CD4+T细胞和CD8+T细胞致糖尿病能力的影响,评估β细胞上表达共刺激分子CD80对未被免疫的静止CD8+T细胞的作用。
用CFSE分子探针标记NOD.scid.CL4 TCR转基因供鼠的脾细胞,将标记的脾细胞过继转移给NOD.scid.InsHA鼠或NOD.scid鼠,用流式细胞技术评价转移后不同时间受鼠的脾、胰腺淋巴结和胰岛中,HA特异性的CL4 CD8+T细胞的增殖和活化通情况。
采用SPSS11.0软件包进行统计分析,两样本率的比较采用四格表资料的x~2检验,多个样本率的比较采用行×列表资料的x~2检验。
结果
1.14天龄NOD鼠的脾细胞被转移给6～8周龄的NOD.scid鼠(n=36)和年龄匹配的NOD.scid.Rip.B7鼠(n=35),在转移脾细胞后9周,当68.6%NOD.scid.Rip.B7受鼠发生糖尿病时,NOD.scid受鼠组尚没有检测到胰岛淋巴细胞浸润(图9)。转移脾细胞后11周,80%的NOD.scid.Rip.B7鼠发生糖尿病(图13 B所示)。但在NOD.scid受鼠组,即使转移后20周,尚没有1例受鼠发生糖尿病。
2.我们将相同数量(2×10~7个脾细胞/受鼠)的、不同周龄的NOD鼠的脾细胞分别转移给相同周龄的NOD.scid受鼠,观察T细胞的致糖尿病能力在不同周龄NOD供鼠的变化。正如图14～17所示,2周龄NOD鼠的脾细胞过继转移给6～8周龄的NOD.scid鼠(受鼠n=36)时,转移后20周仍然没有受鼠发生糖尿病;转移3周龄NOD鼠的脾细胞时(NOD.scid受鼠n=35),转移后13周时开始有受鼠发生糖尿病(5%),到转移后25周时有约30%的受鼠发生糖尿病;当转移4周龄NOD鼠的脾细胞时(NOD.scid受鼠n=36),转移后12周时开始有受鼠发生糖尿病(10%),到转移后20周时有约50%的受鼠发生糖尿病;而转移6周龄NOD鼠的脾细胞时(NOD.scid受鼠n=37),转移5周后(36天)开始有NOD.scid受鼠发生糖尿病,到转移后9周时有约65%的受鼠发生糖尿病,转移后13周时,100%的受鼠发生糖尿病。经多个样本率之间两两比较,在转移后同一时间,不同周龄的NOD供鼠在相同受鼠中诱导的糖尿病发病率有显著性差异(P＜0.001)。
3.幼年(2周龄)NOD鼠的脾细胞不能诱导NOD.scid受鼠发生糖尿病,是否幼年NOD鼠体内缺少β细胞自身抗原的表达?我们进一步用过继转移试验回答上述问题。从已发生糖尿病的NOD鼠分离脾细胞并注射给新生的(n=30)以及1周龄(n=32),2周龄(n=34),3周龄(n=32),6周龄(n=36)的NOD.scid鼠(结果见表2)。所有年龄组的受鼠均在接受转移细胞后5周左右开始发病,转移后9周时所有年龄组受鼠100%发病。尽管受鼠的年龄不同,但它们发生糖尿病的时间却相似(图13A)。经行×列表资料的x~2检验,P＞0.05,说明在转移后同一时间,不同周龄的受鼠糖尿病发病率的差异无统计学意义。
4.从9～10天龄的BDC2.5/NOD.scid鼠的脾脏分离出BDC2.5 CD4+T细胞,将5×10~5个CD4+T细胞转移给6～8周龄的NOD.scid鼠(n=40)和NOD.scid.Rip.B7鼠(n=40)。两组受鼠都是从转移后12天开始发病,到转移后15天时,两组受鼠糖尿病发病率均为100%(图18A),经x~2检验,两组受鼠糖尿病发病率的差异无统计学意义(P＞0.05)。
然后我们将不同数量的BDC2.5 CD4+T细胞转移到年龄匹配NOD.scid和NOD.scid.Rip.B7受鼠,各组受鼠数量均为40只。结果不论我们转移了多少BDC2.5CD4+T细胞,在NOD.scid.Rip.B7受体组和NOD.scid对照组所诱导的糖尿病发生率及发病特点都是相似的(图18A～D),该结果证明了β细胞上共刺激分子CD80的表达对CD4+T细胞的致糖尿病性是没有影响的。
5.从4周龄的NOD.Rag~(null).AI4 TCR转基因鼠分离脾细胞作为活化的CD8 T细胞,将相当于7×10~5个CD8+T细胞的脾细胞转移至NOD.scid(n=35)或NOD.scid.Rip.B7(n=40)鼠(表3)。虽然两组受鼠均发生糖尿病,但发病速度明显不同,转移后24天,当NOD.scid.Rip.B7组受鼠全部发生糖尿病时(发病率100%),NOD.scid对照组糖尿病发病率只有14.3%,经x~2检验,两组发病率有显著性差异(P＜0.001)。转移相当于7×10~4个CD8+T细胞的脾细胞时,转移后45天,NOD.scid.Rip.B7组受鼠全部(n=40)发生糖尿病时(表4),NOD.scid对照组(n=35)糖尿病发病率只有17.1%,经x~2检验,两组发病率的差异有统计学意义(P＜0.001)。
两组试验尽管供鼠与受鼠相同,由于转移的细胞数不同,相同受鼠发生糖尿病的速度却是明显不同的。
从9～10天龄NOD.Rag~(null).AI4 TCR转基因鼠分离脾细胞,将相当于7×10~5个CD8+T细胞的脾细胞转移至NOD.scid.Rip.B7鼠(n=32)和NOD.scid对照鼠(n=30)。31天后NOD.scid.Rip.B7组开始有受鼠发生糖尿病,在转移脾细胞后55天NOD.scid.Rip.B7受鼠100%发生糖尿病(图20A)。静止的CD8+T细胞所诱导的糖尿病的发生及其发病率与已发生糖尿病的AI4 TCR转基因鼠及糖尿病前期的AI4TCR转基因鼠的脾细胞所诱导的糖尿病的发生及其发病率相似。NOD.scid组在转移60天后尚未发现有受鼠发生胰岛炎,但在120天时NOD.scid组的部分受试对象发生外周胰岛炎和胰岛炎(图20B)。
6.将9～10天龄NOD.Rag~(null).AI4 TCR转基因鼠的静止的AI4 CD8+T细胞转移到NOD.scid.β2m~(null).Rip.B7鼠和NOD.scid.Rip.B7鼠各40只。NOD.scid.Rip.B7组被迅速诱发糖尿病(图21所示),在转移后23天开始有受鼠发生糖尿病,转移后35天100%受鼠发生糖尿病。但作为对照,NOD.scid.β2m~(null).Rip.B7组即使在转移后80天也无1例受鼠发生糖尿病。
当我们转移4周龄的AI4 CD8+T细胞给NOD.scid.β2m~(null).Rip.B7鼠和NOD.scid.Rip.B7鼠时,NOD.scid.Rip.B7组迅速发生糖尿病,NOD.scid.β2m~(null).Rip.B7组虽然也有受鼠发病,但发病时间晚,发病率低。
7.将CFSE标记的对流感病毒血球凝集素(HA)特异性的CL4 CD8+ T细胞转移到12只选择性地在β细胞上表达HA基因的NOD.scid.Ins.HA鼠内,1×10~7CL4 CD8+T细胞/受鼠,12只NOD.scid.鼠作为对照。在NOD.scid.Ins.HA受鼠组,转移后第4天,从受鼠胰岛分离到的CL4 CD8+T细胞有高达50%的细胞已经经历了多个循环周期的分化,而从胰腺淋巴结和脾脏分离的CL4 CD8+T细胞却只有少数增殖(图22,23)。转移CL4 CD8+T细胞5天后,由于淋巴细胞严重浸润的胰岛脆性太大,易裂解的缘故,已经无法回收NOD.scid.InsHA受鼠胰岛内的CL4CD8+T细胞。但在NOD.scid对照组,直到转移7天后才在脾脏和PLN首先检测到T细胞增殖(图22,23),而在胰岛中只发现了少量CL4 CD8+T细胞,细胞数并不随时间的延长而增加。
结论
1.幼年NOD鼠的脾细胞含有β细胞特异性T细胞,数量有限,但其致糖尿病能力随着NOD鼠年龄的增长而增加。2周龄NOD小鼠的脾细胞可将糖尿病转移到NOD.scid.Rip.B7小鼠中,说明14天龄的NOD鼠的脾细胞含有β细胞特异性T细胞。在NOD.scid受鼠中没有胰岛炎和糖尿病,也说明了在幼年NOD鼠中的T细胞库和/或致糖尿病的T细胞的数量是有限的。转移不同周龄NOD鼠的脾细胞后,4组受鼠糖尿病的发生时间及发病速度明显不同,供鼠周龄越大,受鼠发病越早,发病速度越快,发病率越高。说明T细胞的致糖尿病能力随着NOD供体鼠年龄的增长而增加。NOD.scid.Rip.B7实验组与NOD.scid对照组糖尿病发生率的明显差异表明,共刺激分子CD80在β细胞上的表达促进了静止T细胞对糖尿病的转移诱导,这给我们提供了T细胞与胰岛β细胞直接作用的证据。
已发生糖尿病的NOD鼠的脾细胞转移给新生的以及1,2,3,6周龄的NOD.scid鼠,尽管受鼠的年龄不同,但它们发生糖尿病的时间却相似,该结果表明,在胰岛炎启动前,新生的和幼年的NOD.scid鼠就已经表达与疾病有关的β细胞自身抗原。
2.β细胞上共刺激分子CD80的表达不影响CD4+T细胞的致糖尿病作用9～10天龄的BDC2.5/NOD.scid鼠的脾细胞转移给6～8周龄NOD.scid.Rip.B7小鼠和NOD.scid小鼠时,在实验组和对照组中观察到的糖尿病的发生率和特点是相同的,并且不论我们转移了多大数量的BDC2.5 CD4+T细胞,在NOD.scid.Rip.B7实验组和NOD.scid对照组所诱导的糖尿病发生率及发病特点都是相似的,该结果证明了β细胞上表达共刺激分子CD80对BDC2.5 CD4+T细胞的致糖尿病能力无影响。
3.β细胞上共刺激分子CD80的表达影响了CD8+T细胞的致糖尿病性,并且在淋巴细胞浸润胰岛过程中对静止的CD8+ T细胞发挥了共刺激作用4周龄的NOD.Rag~(null).AI4αβTCR转基因鼠的脾细胞(CD8+ T细胞已经活化)诱导NOD.scid.Rip.B7组受鼠糖尿病的快速发生表明,β细胞上共刺激分子CD80的表达增强了β细胞特异性CD8+T细胞的致糖尿病能力。9～10天龄NOD.Rag~(null).AI4αβTCR转基因鼠的脾细胞(CD8+ T细胞未被活化)不能将糖尿病转移到NOD.scid受鼠,但对NOD.scid.Rip.B7受鼠诱导的糖尿病的发病率,与4周龄的AI4αβTCR转基因鼠的脾细胞所诱导的糖尿病的发病率相似。这说明,CD8+T细胞与β细胞是直接作用的,而NOD.scid受鼠胰岛炎的低发生率提示共刺激分子CD80在胰岛淋巴细胞浸润过程中对静止的CD8+T细胞发挥了协同刺激作用。
4.胰岛炎的启动需要CD8+ T细胞与胰岛β细胞的直接作用。将静止的AI4CD8+T细胞转移到NOD.scid.β2m~(null).Rip.B7鼠和NOD.scid.Rip.B7鼠时,NOD.scid.Rip.B7组受鼠被迅速诱发糖尿病,但作为对照,NOD.scid.β2m~(null).Rip.B7组受鼠不能被诱导发生糖尿病或胰岛炎。这说明,当NOD.scid.Rip.B7的β细胞失去MHCⅠ类分子的表达,其活化静止CD8+T细胞的能力也随之丧失。也就是说,静止的CD8+ T细胞的活化是因为共刺激分子CD80与表达MHCⅠ类分子的β细胞直接作用的结果。这些结果证明,在胰岛炎的启动中需要β细胞和CD8+T细胞的直接作用。
5.静止的CD8+ T细胞在胰岛内与β细胞抗原相遇。将CFSE标记的CL4CD8+ T细胞转移到NOD.scid.Ins.HA和NOD.scid.受鼠内,第4天,从NOD.scid.InsHA受鼠胰岛分离的CL4 CD8+T细胞,有高达50%已经经历了多个循环周期的分化,而从其胰腺淋巴结和脾脏分离的CL4 CD8+T细胞却只有少数增殖;但是在对照组,转移后7天才在受鼠内检测到CL4 CD8+T细胞明显的生理性增殖。这些结果证明,未被免疫的静止CD8+T细胞是直接进入胰岛,在胰岛内与内源性自身抗原相遇并启动胰岛炎,随后CD8+T细胞在胰岛内增殖和扩增。
本研究的创新点
1.关于CD8+ T细胞在1型糖尿病中的作用及其作用方式的研究,国内至今尚未见有报道;国外虽然有这方面的研究,但尚无定论。本研究利用过继转移的方法,研究发现CD8 T细胞通过与胰岛β细胞直接作用,启动了糖尿病的发生,目前国内外未见报道。
2.利用过继转移实验,发现在糖尿病发病过程中,共刺激分子对静止CD8 T细胞的活化依赖于β细胞上表达MHCⅠ类分子。对此国内尚未见报道。
3.通过CFSE标记静止的CD8 T细胞,首次发现CD8 T细胞是直接进入胰岛,在胰岛内被活化,而不是在胰腺淋巴结等胰腺外的淋巴器官被活化。
第二部分散发性甲旁减自身免疫性发病机制的研究
目的特发性甲旁减(idiopathic hypoparathyroidism,IH)分为散发性甲旁减(sporadic idiopathic hypoparathyroidism,SIH)、Di Geoge综合症、家族性1型、家族性2型(autoimmune polyglandular syndrome type 1,APS-1)。
关于特发性甲旁减自身抗体方面的研究,主要是针对APS-1中的IH患者。APS-1指Addison病、甲状旁腺机能减退症(甲旁减)和慢性粘膜皮肤念珠菌病三者中至少存在两个,还可伴有其他相关的免疫性疾病。由于组成APS-1的其它内分泌病都是典型的自身免疫病,人们很自然地认为IH也是自身免疫性疾病。而散发性甲旁减患者无家族史,也不伴有APS-1包括的自身免疫性疾病,所以,对APS—1的研究不能代替对散发性甲旁减(SIH)的研究。
由于散发性特发甲旁减患者极其罕见,国内外研究甚少,至今病因不明,尚无充分证据说明该病有自身免疫性。本研究,采用间接免疫荧光技术,检测了26例SIH患者血清中的抗甲状旁腺自身抗体,从体液免疫方面探讨SIH的自身免疫性。运用流式细胞技术,分析了10例SIH患者外周血淋巴细胞亚群及调节性T细胞的表达,从细胞免疫方面探讨SIH的自身免疫性。
方法
1、研究对象:散发性特发甲旁减患者26例及其直系亲属112例和健康对照60例。散发性特发甲旁减患者均无甲旁减、肾上腺皮质功能减退或黏膜皮肤念珠菌感染的家族史,患者本人也不伴有肾上腺皮质功能减退或黏膜皮肤念珠菌感染等自身免疫性疾病,排除APS-1型。所有空腹血清标本采集于1995～2006年间,分装后保存于-80℃待检。部分用于检测钙、磷、镁、全段甲状旁腺激素(iPTH)、FT3、FT4、TSH、血清皮质醇(Cortisol)、TPOAb,其余用于检测抗甲状旁腺自身抗体。
另外,采集上述研究对象中10例SIH患者及其38例直系亲属、12例健康对照的新鲜全血,ACD抗凝,分离单个核细胞,用于流式细胞分析。
2、抗甲状旁腺抗体的检测:采用间接免疫荧光技术,选用猴子甲状旁腺作底物抗原,用DAPI染核定位甲状旁腺细胞。结果先在荧光显微镜下观察,抗体滴度1:10阳性者,再按1:100稀释重复上述步骤。然后用共聚焦激光扫描显微镜(CLSM)进行扫描并采集图像。488nm波长的光激发绿色荧光图像(FITC标记的抗体)、356nm波长的光(紫外光,UV)激发蓝色荧光图像(DAPI标记的细胞核)。利用Leica Microsystems Heidelberg GmbH(Leica Confocal Software)分析软件进行图像处理。
3、血清钙、血清磷和血清镁含量检测:分别用偶氮砷Ⅲ法、紫外直接沉淀法和二甲苯胺蓝比色法。
4、血清FT3、FT4、TSH、Cortisol、TPOAb检测:采用电化学发光免疫分析法,iPTH用双抗体夹心法。
5、外周血淋巴细胞亚群的分析:从ACD抗凝的新鲜外周血分离单个核细胞,用荧光标记的抗体染色,洗涤去除未结合的抗体后,应用流式细胞仪进行检测。
6、流式细胞分析:用Becton Dickinson FACScalibur流式细胞仪检测,激发光为氩离子激光488nm谱线,使用荧光校准微球校准仪器,调整各荧光补偿。首先检测对照管,建立二维散点图,在散点图上调整参数前向散射光(FSC)及侧向散射光(SSC),调整阈值,圈定淋巴细胞射门以获取细胞。运行CELLQuest软件(Becton Dickinson,San Jose,CA)采集数据,WinMDI2.8软件分析数据,结果以各细胞亚群占PBMC细胞的百分率表示。
6、统计学分析:正态分布或近似正态分布的计数资料均以均数±标准差表示,非正态分布的计数资料以中位数及四分位间距表示。应用SPSS11.0软件进行数据处理,检验水平α=0.05。
结果
1、一般指标治疗前26例患者血清钙与iPTH水平均明显降低,与直系亲属组及正常对照组比较差异具有统计学意义(P均小于0.01),血清磷水平明显升高,与直系亲属组及正常对照组比较差异具有统计学意义(P均小于0.05),以上各指标在直系亲属组与正常对照组之间无显著性差异(P均大于0.05)。3组血清镁比较虽然无显著性差异,但其中4例SIH患者血清镁略低于正常范围;3组FT3,FT4,TSH,Cortisol及TPOAb等均在正常范围,统计学分析无显著性差异(P均大于0.05)。
2、抗甲状旁腺抗体26例SIH患者中有10例(8例为女性)患者的血清1:10稀释时抗甲状旁腺抗体阳性,阳性率38.46%,其中3例在1:100稀释时仍呈阳性反应。112例患者直系亲属中有11例血清1:10稀释时抗甲状旁腺抗体阳性,阳性率9.82%,60例正常对照组中有4例血清1:10稀释时抗甲状旁腺抗体呈阳性反应,阳性率6.67%,SIH患者组阳性率明显高于直系亲属组与正常对照组(P均小于0.01),直系亲属组与正常对照组抗体阳性率无显著性差异(P＞0.05)。抗体阳性的患者与抗体阴性的患者血清钙、磷、镁水平无显著性差异(P均大于0.05)。两组性别、年龄构成无显著性差异,病程及血清iPTH水平比较差异虽无统计学意义,但抗体阴性的患者病程较抗体阳性的患者有延长的趋势,并且其血清iPTH水平较阳性组患者有降低的趋势。
3、淋巴细胞表型的检测所测的淋巴细胞亚群中,CD4+,CD8+,CD4+比CD8+比值,CD5+,CD19+,CD5+CD19+等在三组研究对象间结果相似,三组之间比较差异无统计学意义。SIH患者外周血中CD4+CD25+调节性T细胞的百分率较健康对照组及患者直系亲属组低,二者比较有显著性差异(P＜0.01),患者直系亲属组CD4+CD25+调节性T细胞的百分率与健康对照组比较,差异无统计学意义(P＞0.05)。
结论
1、散发性特发甲旁减患者体内存在体液免疫功能的改变。本研究中散发性特发甲旁减患者抗体阳性率(38.46%)明显高于亲属组(9.82%)(P＜0.01)及健康对照组(6.67%)(P＜0.01),提示患者体内体液免疫活化。一般认为,自身抗体在发病中起始动作用的疾病如Grave病和重症肌无力,其临床症状和生化异常的严重程度与血清中抗体阳性程度有关,但我们的研究未发现SIH患者血清中的抗甲状旁腺抗体与该病的临床症状和生化异常的严重程度有关。
2、散发性特发甲旁减患者体内存在细胞免疫功能的改变。CD4+CD25+调节性T细胞(Treg)是参与对自身抗原外周耐受的主要T细胞群,也是对外来抗原应答的主要调节性T细胞,对于维持外周免疫耐受有重要意义。有研究表明CD4+CD25+Treg数量减少或功能异常均有可能导致自身免疫病的发生。本研究发现SIH患者外周血中CD4+CD25+T细胞百分比较患者亲属及健康对照组低,提示SIH患者体内免疫调节细胞减少,不能有效的维持自身耐受。患者体内自身免疫稳态破坏,机体更易发生病理性免疫应答,从而激活免疫反应,促使自身免疫性疾病的发生。
总之,散发性特发甲旁减患者体内自身反应性抗体的存在以及CD4+CD25+Treg细胞的改变,表明其自身免疫功能的紊乱。
本研究的创新点:
1、首次对散发性特发甲旁减患者外周血中CD4+CD25+Treg细胞的表达进行了研究,从细胞免疫学角度探讨疾病的自身免疫发病机制,检索近20年的文献,国内外未见任何报道。
2、首次利用流式细胞技术,探讨了散发性特发甲旁减患者外周血中单个核细胞淋巴细胞亚群的表达,检索近20年的文献,国外只有1篇关于特发性甲旁减淋巴细胞亚群的研究报道。
3、利用间接免疫荧光技术,对散发性特发甲旁减患者体内抗甲状旁腺自身抗体的存在进行了研究,从体液免疫角度探讨疾病的自身免疫发病机制,至今国内未见任何报道,国外的研究也很少。
Objective Type 1 diabetes is an chronic autoimmune disease mediated by T cells. The autoimmune process of the disease include a long pre-clinical period of diabetes (i.e.,insulitis)and overt diabetes period.Recent genetic data and preliminary trial results suggest T cells as a target for preventive strategies.The role of T cells in the pathogenesis of type 1 diabetes has become hot points in the study of type 1 diabetes. Sufficient histological analysis and transfer study of T cells suggests that both CD4+ T cells and CDS+ T cells are essential in the development of type 1 diabetes,the relative contribution of these two T cell subsets in triggering insulitis remains poorly defined.Therefore,we put that if CD8 + T lymphocytes launched insulitis? If so, how CD8 + T cells trigerred the diseases?
Method Non-obese diabetic(NOD)mice and NOD background transgenic mice using genetic engineering were used as animal models in our study.PCR and flow cytometry were used to type genotype of experimental animals.
Splenocytes were isolated from donor mice before adoptive transfer experiments. Stained with specific monoclonal antibody and determined the type and number of cells to be transferred by flow cytometry,a certain amount of cells were intraperitoneally to recipient mice.Diabetes was monitored by measuring glucose using tail vein blood of recipient mice.Insulitis was asssessed for those recipient mice which did not develop diabetes by immunohistochemistry.
Adoptive transferred experiments were carried out first to test whether the diabetogenic T cells existed in young(2 weeks old)NOD mice,and the diabetogenic capacity of T cells increased with the age of donor NOD mice,as well as whether cognate autoantigens were absent in young NOD mice,then to determine whether the expression of costimulatory molecules CD80 on beta cells influence the diabetogenic capability of CD4+ and CD8+ T cells,and to value the effects of costimulatory molecules CD80 on beta cells on naive CD8+ T cells.
Spleen cells from NOD.scid.CL4 TCR transgenic mice were labeled with molecular probe of CFSE and transferred to NOD.scid.InsHA mice and NOD.scid mice. Different time post-transfer,HA specific CL4 CD8+ T cells were isolated from the spleen,draining PLN and islets.T cells were stained with mAbs specific for the transgenic TCR,CD69 and CD62L.Proliferation and activation of HA-specific CL4 CD8+ T cells were assessed by flow cytometric analysis.
SPSS 11.0 software was used to process the data,test levelα=0.05.Pearson Chi-Square Tests were used for statistical analysis of diabetes incidence induced in recipient mice.
Results
1 Spleen cells of 14 day-old NOD mice were transferred into adult NOD.scid mice (n=36)and age-matched NOD.scid.Rip.B7 mice(n=35).Diabetes was induced in 68.6%of NOD.scid.Rip.B7 mice by 9 weeks post-transfer while in contrast,no infiltration of lymphocytes in the islets of NOD.scid recipients(as shown in Fig.9). Diabetes was induced in 80%of NOD.scid.Rip.B7 mice by 11 weeks post-transfer (as shown in Fig.13B).In contrast,none of the NOD.scid recipients developed diabetes even by 20 weeks post-transfer.
2 Then,spleen cells from different week-old NOD mice were transferred into adult NOD.scid mice in the same number of cells(2×10~7 cells / recipient).The diabetogenic capacity of T cells in different age of donor NOD mice were observed. As shown in Fig.14～17,none of the NOD.scid recipients(n=36)developed diabetes even by 20 weeks post-transfer when transferred spleen cells from 2 week-old NOD mice.However,when spleen cells from 3 week-old NOD donor mice were transferred, the recipient mice(n=35)began to develope diabetes from 13 weeks post-transfer (5%).There were about 30%of recipient mice developed diabetes by 25 weeks post-transfer.When spleen cells from 4 week-old NOD donor mice were transferred, the recipient mice(n=36)began to develope diabetes from 12 weeks post-transfer (10%)and there were about 50%of recipient mice developed diabetes by 20 weeks post-transfer.When spleen cells from 6 week-old NOD donor mice were transferred, the NOD.scid recipient mice(n=37)began to develope diabetes in earlier time of 5 weeks post-transfer,and there were about 65%of recipient mice developed diabetes by 9 weeks post-transfer,100%by 13 weeks post-transfer.There were significant difference among the diabetes incidence induced by differen age of NOD donor mice by the same time post-transfer(P＜0.001).
3 Spleen cells of young NOD mice(14 day-old NOD mice)could not transfer diabetes to NOD.scid recipient mice.Is it duo to the lack of expression of cognate autoantigens of beta cells? We answered the question by adoptive transferred experiments.Spleen cells were isolated from diabetic NOD mice and injected into neonatal(n=30),1 week-old(n=32),2 week-old(n=34),3 week-old(n=32), and 6 week-old(n=36)NOD.scid mice and the development of diabetes monitored (table 2).All recipient groups began to develop diabetes from 5 weeks post transfer and 100%recipient mice developed diabetes by 9 weeks post-transfer.Irrespective of age,diabetes was induced in all recipients with similar time of onset(Fig.13A).This result demonstrated that disease relevant beta cell autoantigens are expressed in neonatal and young NOD.scid mice,at times prior to the initiation of insulitis.There was no significant difference among different age of recipient mice of the diabetes incidence induced by diabetic NOD donor mice by the same time post-transfer(P＞0.05).
4 Naive splenic BDC2.5 CD4+ T cells were isolated from 9～10 day-old BDC2.5/NOD.scid mice and adoptively transferred into NOD.scid(n=40)and NOD.scid.Rip.B7 mice(n=40),(5×10~5 cells / recipient).Both recipient groups began to develop diabetes at 12 days post transfer,and by 15 days post transfer,100% recipient mice in two groups developed diabetes.There was no significant difference between the two recipient groups of the diabetes incidence(P＞0.05).
To exclude the possibility that the number of BDC2.5 CD4+ T cells transferred masked the effect of CD80,various numbers of BDC2.5 CD4+ T cells were transferred into NOD.scid and NOD.scid.Rip.B7 recipients.Regardless of the numbers of BDC2.5 CD4+ T cells transferred,diabetes was induced in NOD.scid.Rip.B7 and NOD.scid recipients with a similar frequency and kinetics(Fig. 18A-D).These results demonstrated that expression of CD80 by beta cells had no significant effect on the diabetogenic capacity of CD4+ T cells.
5 Activated CD8+ T cells prepared from the spleens of diabetic or 4 week-old NOD.Rag~(null).AI4 TCR transgenic mice,7×10~5 CD8+ T cells were transferred into NOD.scid(n=35)and NOD.scid.Rip.B7 mice(n=40).As demonstrated in table 3, diabetes developed in both NOD.scid and NOD.scid.Rip.B7 recipients,but with markedly different kinetics.All NOD.scid.RipB7 recipients developed overt diabetes by 24 days post-transfer,in contrast,diabetes incidence was only 14.3%in NOD.scid recipients.There were significant difference between the two groups of diabetes incidence(P＜0.001).
When 7×10~4 CD8+ T cells were transferred,all NOD.scid.RipB7 recipients(n=40) developed overt diabetes by 45 days post-transfer,in contrast,diabetes incidence was only 17.1%in NOD.scid recipients(n=35).There were significant difference between the two groups of diabetes incidence(P＜0.001).
CD8+ T cells prepared from the spleens of 9～10 day-old NOD.Rag~(null).AI4 TCR transgenic mice,7×10~5 CD8+ T cells were transferred into NOD.scid(n=30)and NOD.scid.Rip.B7 mice(n=32).As demonstrated in Fig.20A,NOD.scid.Rip.B7 recipient mice began to develop diabetes at 31 days post transfer,100%recipient mice developed diabetes by 55 days post transfer.The onset and incidence of diabetes induced by naive CD8+ T cells was comparable to that of CD8+ T cells prepared from diabetic or pre-diabetic AI4 TCR transgenic mice.Insulitis in the NOD.scid recipients was absent at 60 days post-transfer,but at 120 days post-transfer peri-insulitis and insulitis was detected in some recipients(Fig.20B).
6 Naive AI4 CD8+ T cells were adoptively transferred into NOD.scid.β2m~(null).B7 and NOD.scid.Rip.B7 mice.Diabetes was readily induced in NOD.scid.Rip.B7 mice (Fig.21).It began to develop diabetes at 23 days post transfer,and by 35 days post transfer,all animals in NOD.scid.Rip.B7 recipient mice developed diabetes.In contrast,none of the NOD.scid.β2m~(null).Rip.B7 recipient mice developed diabetes even by 20 days post-transfer.
7 Splenocytes from NOD.scid.CL4 transgenic mice which express an H2Kd-resticted TCR specific for influenza HA were labeled with CFSE.12 mice of NOD.scid.InsHA, which selectively express HA on bete cells,were injected i.p.with 1×10~7 CFSE-labeled spleen cells,12 mice of NOD.scid were used as control.By day 4 post-transfer,～50%of CL4 CD8+ T cells isolated from the islets of NOD.scid.InsHA had undergone multiple rounds of division whereas only a few CL4 CD8+ T cells from PLN and spleen showed evidence of proliferation(Fig.22,23). The lack of recovery of CL4 CD8+ T cells from the islets of NOD.scid.InsHA recipients at 5 days post-transfer or later because of the fragility of the islets with heavy infiltration.However,in NOD.scid recipients group,significant T cell proliferation was first detected in the spleen and PLN 7 days post-transfer(Fig.22,23). The small numbers of CL4 CD8+ T cells were found in the islets of NOD.scid control recipients,but did not increase with time.
Conclusion
1 Spleen cells of young NOD mice contain beta cell-specific T cells,however,the T cell repertoire and/or frequency of diabetogenic T cells are limited.The diabetogenic capacity of T cells increased with the age of NOD mice.
The spleen cells from 2-week-old NOD mice succeeded in transferring diabetes to NOD.scid.RipB7 recipients demonstrated that spleen cells of 14 day-old NOD mice contain beta cell-specific T cells.The lack of insulitis and diabetes in NOD.scid recipients,however,also suggested that the T cell repertoire and/or frequency of diabetogenic T cells are limited in young NOD mice.There were recipients in both groups developed diabetes when transferred splenocyte from different-week-old donors,but the incidence in control recipients was far lower from that of in NOD.scid.Rip.B7 mice(P＜0.001).It suggested that the diabetogenic capacity of T cells increased with the age of donor NOD mice.The significant difference between the two groups demonstrated that the expression of costimulatory molecule CD80 on βcells promopt the naive T cells in transferring induction of diabetes.It provided us an evidence of direct interaction between CD8+ T cells and beta cells.
2 Eexpression of CD80 on beta cell had no significant effect on the diabetogenic capacity of CD4+ T cells
Naive splenic BDC2.5 CD4+ T cells were isolated from 9-10 day-old BDC2.5/NOD.scid mice and adoptively transferred into 6～8-week-old NOD.scid and NOD.scid.Rip.B7 mice.Diabetes,with identical incidence and kinetics,was observed in both NOD.scid.Rip.B7 and control NOD.scid recipients.Regardless of the numbers of BDC2.5 CD4+ T cells transferred,diabetes was induced in NOD.scid.Rip.B7 and NOD.seid recipients with a similar frequency and kinetics.The result demonstrated that beta cell expression of CD80 had no effect on the diabetogenicity of BDC2.5 CD4+ T cells.
3 Beta cell expression of costimulatory molecules CD80 influences the diabetogenic capacity of CD8+ T cells,and CD80 exerts its co-stimulatory function on naive CD8+ T cells during the initiation of islet infiltration.
The accelerated onset of diabetes in NOD.scid.Rip.B7 recipients induced by the splenocytes from 4 week-old NOD.Rag~(null).AI4 TCR transgenic mice indicates that beta cell expression of costimulatory molecules CD80 enhances the diabetogenic capacity of beta cell-specific CD8+ T cells.However,these naive AI4 CD8+ T cells failed to transfer diabetes in NOD.scid recipients.The incidence of diabetes in NOD.scid.Rip.B7 recipients induced by naive AI4 CDS+ T cells was similar to that of induced by AI4 CD8+ T cells from diabetic or pre-diabetic donors.It suggested that CD8+ T cells directly interaced with beta cells,and CD80 played a costimulatory role on naive CD8+ T cells during the lymphocyte infiltrating islets.
4 The initiation of insulitis results from direct interaction between CD8+ T cells and beta cells.
Naive AI4 CD8+ T cells were adoptively transferred into NOD.scid.Rip.B7 and NOD.scid.β2m~(null).B7 mice that are deficient of MHC class I expression,but still with the expression of CD80 ectopically on beta cells.Diabetes was readily induced in NOD.scid.Rip.B7 mice.In contrast,naive AI4 CD8+ T cells failed to induce diabetes or insulitis in NOD.scid.β2m~(null).B7 recipient mice(Fig.21).These results indicated that the loss of MHC classⅠexpression on beta cells in NOD.scid.β2m~(null).B7 mice rendered incapable of priming naive CD8+ T cells.That is to say,activation of naive CD8+ T cells was in fact the result of a direct interaction with host beta cells expressing MHC classⅠand costimulatory molecule CD80.In summary,a direct interaction between beta cells and CD8+ T cells is required for the initiation of insulitis.
5 Naive CD8+ T cells encounter beta cell antigens within the islets.
CFSE-labeled CL4 CD8+ T cells were adoptive transferred to NOD.scid.InsHA and NOD.scid mice.By day 4 post-transfer,～50%of CL4 CD8+ T cells isolated from the islets of NOD.scid.InsHA had undergone multiple rounds of division whereas only a few CL4 CD8+ T cells from PLN and spleen showed evidence of proliferation.But in NOD.scid contrast group,significant T cell proliferation was first detected in the spleen and PLN 7 days post-transfer.These data demonstrated that naive CD8+ T cells traffic directly into the islets,encounter cognate autoantigens and initiate insulitis following proliferation and expansion.
The innovation
1 There has been no report on the role of CD8 + T cells in type 1 diabetes and the mode of its role.Some studies about CD8 T cells and T1D are ongoing abroad, however,there has been no consistent result by now.We explored the autoimmune pathogenesis of type 1 diabetes by studing the role of CD8 + T cells in the triggering of diabetes with adoptive transfer experiments,and found for the first time that the initiation of insulitis results from direct interaction between CD8+ T cells and beta cells.
2 We found for the first time that activation of naive CD8+ T cells was in fact the result of a direct interaction between costimulatory molecule CD80 and host beta cells expressing MHC classⅠ.
3 We found for the first time by CFSE-labelled naive CD8+ T cells that naive CD8+ T cells trafficed directly into the islets and was activated in islets,not in any other lymph organ or tissue like PLN.
PART 2 STUDY OF AUTOIMMUNE MECHANISM OF SPORADIC IDIOPATHIC HYPOPARATHYROIDISM
Object Idiopathic hypoparathyroidism(IH)includes sporadic IH(SIH),Di Geoge syndrome,familial type 1,familial type 2.Familial type 2 is also called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy(APECED)or autoimmune polyglandular syndrome type 1(APS-1).
The study on autoantibodies of idiopathic hypoparathyroidism(IH)is mainly targeted at the IH patients associated with APS-1.Less study is conducted on autoantibodies of SIH.At least two in three disorders of Addison disease, hypoparathyroidism and chronic skin and mucosal candidiasis disease are referred to as APS 1,that may be companied with other concerned immune diseases.Because the other endocrine components of APS 1(i.e.,APECED)are typical autoimmune diseases,it is natural to assume that this is also true of the HP.While sporadic IH without a family history,nor the autoimmune components of APS-1,the study of APS-1 is no substitute for SIH's.
The study on sporadic idiopathic hypoparathyroidism(SIH)is little at home and abroad because this disease is extremely rare.There is no sufficient proof for its autoimmune nature.To explore its autoimmune nature from humoral immunity,we searched for parathyroid autoantibodies in 26 patients with SIH by indirect immunofluorescence techniqes.We also analyzed the expression of lymphocyte subsets in peripheral blood of 10 patients with SIH by flow cytometry and explore its autoimmune nature from cellular immunity.
Method
1.Patients and control subjects After obtaining informed consent,we enrolled 26 patients with SIH and 60 age-and sex- matched healthy individuals as well as 112 family members of the patients with SIH.None of the patients had a family history suggestive of hypoparathyroidism or adrenal insufficiency or clinical features of mucocutaneous candidiasis infection.The type 1 autoimmune polyglandular syndrome was excluded by demonstrating a normal adrenocorticotrophin(ACTH) value and without clinical features of mucocutaneous candidiasis infection.All sera samples were obtained from subjects after 8-10 hours' fasting during1995 to 2006, kept in -80℃to be detected.Some sera were used to detect calcium,phosphorus, magnesium,intact parathyroid hormone(iPTH),FT3,FT4,TSH,Cortisol,TPOAb, and the rest for detection of anti-parathyroid autoantibodies.Fresh whole blood with ACD anticoagulant were collected from 10 of the 26 patients with SIH,38 of the 112 family members and 12 of the 60 healthy controls,and mononuclear cells were separated for flow cytometry analysis.
2.Detection of antibodies against parathyroid Indirect immunofluorescence techniques were used in our study.It was suggested that the antibodies against parathyroid in patients with APECED was a false positive IF results caused by human-specific antimitochondrial antibodies and organ-specific antibodies against parathyroid were extremely rare.To avoid this false positive IF results,we used parathyroid glands from monkey other than from human as substrates.To position the cells of parathyroid glands,we stained the nucleolus with DAPI.The results were firstly observed under fluorescence microscope,if the antibody was positive ina sera titer of 1:10,then repeated the above steps in 1:100 dilution.Then confocal laser scanning microscopy(CLSM)was used to scan and obtain the image.Green fluorescent images(FITC-conjugated antibody)was excitated by the light of 488 nm wavelength,blue fluorescence images(DAPI labeled nuclei)was excitated by the light of 356 nm wavelength(UV).Processing the image with Leica Microsystems Heidelberg GmbH(Leica Confocal Software)analyzing software.
3.Detection of Serum calcium,serum phosphorus and serum magnesium
Serum calcium,phosphorus and magnesium were examined respectively with AzoⅢarsenium method,ultraviolet direct precipitation method and xylidine chromatometry.
4.Detection of Serum FT3,FT4,TSH,Cortisol,TPOAb FT3/FT4 were examined by electrochemiluminescenc immuno-analysis method,iPTH was detected by double antibody sandwich method.
5.Analysis of lymphocyte subsets in peripheral blood PBMCs were isolated from the blood with ACD-anticoagulant of patients and healthy donors as well as family members of patients by means of Ficoll density gradient centrifugation.
6.Flow cytometry
Mononuclear cells were isolated from the fresh blood with ACD anticoagulant, stained with antibody fluorescent-labeled.Dual-color flow cytometry was performed using a CellQuest software and Becton Dickinson FACSCalibur instrument. Argon ion laser of 488 nm spectrum as excitation,equipment was calibrated by the use of fluorescent microspheres calibration,adjusted the fluorescence compensation. Isotype-matched controls were used to determine nonspecific background.Cells labeled with one fluorescent antibody and the other isotype control were used to determine the separation threshold of the cytometer channels.Lymphocytes were electronically gated based on their forward scatter(FSC)and side scatter(SSC) characteristics,and then double-positive cells were detected by analyzing at least 10,000 lymphocytes.In these assays,careful color compensation was performed, and their intra-assay reproducibility was corroborated.Obtained cells with CellQuest software.WinMDI2.8 software was used to analyze the data.
Results
1.General indicator The level of sera iPTH and calcium significantly decreased and sera phosphate significantly increased in 26 patients before treatment compared with the family members and the normal controls.There was no significant difference(P＞0.05)of the indicators above between family members and normal controls.Although there was no significant difference of serum magnesium among the three groups,but the sera magnesium of four patients with SIH were slightly lower than the normal range;The levels of sera FT3,FT4,TSH,Cortisol and TPOAb were all in the normal range of three groups.
2.Detection of anti-parathyroid antibody There were 10(8 cases are female)in 26 patients with positive antibodies against parathyroid when sera were diluted in 1:10,the positive rate was 38.46 percent,three were still positive when sera were diluted in 1:100.There were 11 in 112 family members with positive antibodies when sera were diluted in 1:10,the positive rate was 9.82 percent.There were 4 in 60 normal controls with positive antibodies when sera were diluted in 1:10,the positive rate was 6.67 percent.The positive rate in SIH patients were significantly higher than the family members and normal controls(P＜0.01).There was no significant difference between family members and normal controls(P＞0.05). There was no significant difference between patients with positive antibodies and without antibodies in sera calcium,phosphorus,magnesium levels(P＞0.05).There was no significant difference between patients with positive antibodies and without antibodies in gender and age.Although no significantly difference in duration of illness and level of sera iPTH,the patients without antibodies had a longer duration of illness[(4.4±2.07)years]and a lower serum iPTH[(6.0±3.95)pg/ml]than those of in patients with antibodies[(2.6±1.34)years and(9.6±3.77)pg/ml,respectively].
3.Detection of lymphocytes phenotype The results of the lymphocyte subsets detected in CD4+,CD8+,ratio of CD4+:CD8+,CD5+,CD19+,ratio of CD5+:CD19+ were similar among the three groups of subjects.There were no significantly difference of the lymphocyte subsets above when compared with each other among three groups.The frequencies of the phenotype of CD4+CD25+ were lower in patients than those of in family members and healthy controls.There was no significant difference between family members and healthy controls(P＞0.05).
Conclusion
1.Changes of humoral immune function present in patients with sporadic idiopathic hypoparathyroidism.
The frequencies of antibody against hypoparathyroid in patients(38.46%)was significantly higher than that of in family members(9.82%)(P＜0.01)and in healthy controls(6.67%)(P＜0.01).It was suggested that humoral immune be activated in patients.In organ-specific autoimmune disease,the patients typically has circulating autoantibodies specific for the affacted organ,often these precede the clinical disease by years and persist for yaers after its onset.This is also true for most of autoimmune components of APS 1,but we did not found in the serum of patients with SIH that autoantibodies against parathyroid had something with the clinical symptoms and the severity of the biochemical abnormalities.
2.Changes of cellular immune function present in patients with sporadic idiopathic hypoparathyroidism.CD4+CD25+regulatory T cells(Treg)are the main subset in the peripheral tolerance to self-antigen and the major regulatory T cells responding to foreign antigen.It is important for the maintenance of peripheral tolerance.Some studies showed that both the decrease of number and the dysfunction of CD4 + CD25 + regulatory T cells could lead to autoimmune diseases.We found that the frequencies of CD4 + CD25 + T cells in peripheral blood of SIH patients were lower than the frequencies of family members and healthy controls.It suggested that immunoregulatory cells in patients could not effectively maintain their own tolerance because of the decreased number.The destruction of autoimmune balance in patients made the body easy in pathological immune responses,thus activating immune response to autoimmune diseases.
In short,the presence of self-reactive antibodies and the decrease of CD4 + CD25+ regulatory T cells in patients with SIH showed that there were immune function disorders in these patients.
In conclusion,serum anti-parathyroid antibody exists in SIH patients demonstrated that humoral immune disorder was present in the disease.Decreased frequency of CD4+CD25+ T regulatory cells support the presence of a cellular immune disturbance in patients with SIH.
The innovation
1 There has been no report in the world on the expression of CD4+CD25+ T regulatory cells in patients with SIH by now.We explored the pathogenesis of sporadic idiopathic hypoparathyroidism from cellular immunity for the first time.
2 We studied the changes of lymphocytes in peripheral blood mononuclear cells of patients with SIH by flow cytometry.There was only one paper about it in the past 20 years in the world.
3 We detected anti-parathyroid antibody in the sera of SIH patients by indirect immunofluorescence techniques.No report about this has been seen in China and only little investigation has been conducted in the world.
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