第二军医大学学报  2018, Vol. 39 Issue (11): 1202-1208   PDF    
CCR7loPD-1hi滤泡辅助性T细胞在系统性红斑狼疮患者外周血中的比例及临床意义
康子健, 刘亚群, 张志国, 刘耀阳, 徐沪济     
海军军医大学(第二军医大学)长征医院风湿免疫科, 上海 200003
摘要: 目的 检测系统性红斑狼疮(SLE)患者外周血中CCR7loPD-1hi滤泡辅助性T细胞(Tfh)的比例,并探讨其临床意义。方法 收集31例SLE患者、29例类风湿关节炎(RA)患者、12例干燥综合征(SS)患者和37名健康对照的外周血样本。采用流式细胞术检测Tfh表面C-X-C趋化因子受体3(CXCR3)、诱导型共刺激分子(ICOS)和信号淋巴细胞激活分子家族成员5(SLAMF5)的表达水平,以及外周血中CCR7loPD-1hi Tfh和CCR7hiPD-1lo Tfh的比例。分析SLE患者外周血中CCR7loPD-1hi Tfh的比例与临床指标、成浆细胞比例的相关性。结果 相比CCR7hiPD-1lo Tfh,CCR7loPD-1hi表面CXCR3、ICOS和SLAMF5的表达水平较高(t=3.73、5.06、8.27,P均 < 0.01)。SLE和RA患者外周血中CCR7loPD-1hi Tfh的比例分别为5.79%(2.97%,8.95%)和4.57%(2.51%,7.60%),均高于健康对照组[2.93%(1.63%,5.21%),U=314.5、332.5,P均 < 0.01],而SS患者外周血中CCR7loPD-1hi Tfh的比例与健康对照相比差异无统计学意义(U=183.0,P>0.05)。SLE患者外周血中CCR7loPD-1hi Tfh的比例与系统性红斑狼疮疾病活动指数(SLEDAI)、抗双链DNA抗体滴度、成浆细胞比例均呈正相关(r=0.447 1、0.517 4、0.466 9,P均 < 0.05)。结论 SLE患者外周血中CCR7loPD-1hi Tfh比例增加与SLEDAI升高和成浆细胞比例的增加相关,检测患者外周血Tfh亚群可间接反映生发中心及B淋巴细胞的功能状态,对SLE的诊断、监测和预后有重要意义。
关键词: 系统性红斑狼疮     滤泡辅助性T细胞     CCR7受体     程序性死亡蛋白1     类风湿关节炎     干燥综合征    
Proportion of CCR7loPD-1hi follicular helper T cell in peripheral blood of systemic lupus erythematosus patients and its clinical role
KANG Zi-jian, LIU Ya-qun, ZHANG Zhi-guo, LIU Yao-yang, XU Hu-ji     
Department of Rheumatology and Immunology, Changzheng Hospital, Navy Medical University(Second Military Medical University), Shanghai 200003, China
Supported by Young Science Fund of National Natural Science Foundation of China (81302578).
Abstract: Objective To explore the proportion of CCR7loPD-1hi follicular helper T cell (Tfh) in peripheral blood of the patients with systemic lupus erythematosus (SLE) and its clinical role. Methods Peripheral blood samples were collected from 31 SLE patients, 29 rheumatoid arthritis (RA) patients, 12 Sjögren's syndrome (SS) patients and 37 healthy controls. Flow cytometry was used to detect the expression levels of C-X-C chemokine receptor 3 (CXCR3), inducible costimulator (ICOS) and signaling lymphocytic activation molecule family member 5 (SLAMF5) on surface of Tfh, and the frequencies of CCR7loPD-1hi Tfh and CCR7hiPD-1lo Tfh in peripheral blood. The correlation between the proportion of CCR7loPD-1hi Tfh in peripheral blood of SLE patients and clinical indicators and the proportion of plasmablasts was analyzed. Results The expression levels of CXCR3, ICOS and SLAMF5 were significantly higher on the surface of the CCR7loPD-1hi Tfh compared with those of the CCR7hiPD-1lo Tfh (t=3.73, 5.06 and 8.27; all P < 0.01). The frequencies of CCR7loPD-1hi Tfh in the peripheral blood of the SLE and RA patients were 5.79% (2.97%, 8.95%) and 4.57% (2.51%, 7.60%), respectively, which were both significantly higher than that of the healthy controls (2.93%[1.63%, 5.21%], U=314.5 and 332.5, both P < 0.01). There was no difference in the frequency of CCR7loPD-1hi Tfh in peripheral blood between SS patients and healthy controls (U=183.0, P>0.05). The proportion of CCR7loPD-1hi Tfh in peripheral blood of the SLE patients was positively correlated with systemic lupus erythematosus disease activity index (SLEDAI), serum anti-double-stranded DNA (dsDNA) titers and the proportion of plasmablasts (r=0.447 1, 0.517 4 and 0.466 9; all P < 0.05). Conclusion Increased proportion of CCR7loPD-1hi Tfh in peripheral blood of the SLE patients is associated with increased SLEDAI and increased proportion of plasmablasts; and detecting the Tfh subsets can indirectly reflect the functional status of germinal center and B lymphocytes, which is of great significance for diagnosis, monitoring and prognosis of SLE.
Key words: systemic lupus erythematosus     follicular helper T cells     CCR7 receptors     programmed death 1     rheumatoid arthritis     Sjögren's syndrome    

系统性红斑狼疮(systemic lupus erythematosus,SLE)是一种累及多器官、多系统的慢性自身免疫性疾病,其病理特点为自身抗体大量产生,并与体内相应的自身抗原形成免疫复合物,沉积于组织和器官,从而导致损伤[1]。SLE的发病机制尚不清楚,但目前认为其主要与T淋巴细胞、B淋巴细胞功能紊乱导致的免疫稳态失衡有关[1]

滤泡辅助性T细胞(follicular helper T cell,Tfh)是CD4+ T淋巴细胞的一个亚群,在二级淋巴器官生发中心为B淋巴细胞提供活化信号,促进体细胞高频突变及抗体类型转换[2]。Tfh通过表达转录因子B细胞淋巴瘤6(B cell lymphoma 6,BCL-6)、C-X-C趋化因子受体(C-X-C chemokine receptor,CXCR)5、诱导型共刺激分子(inducible costimulator,ICOS)及程序性死亡蛋白1(programmed death 1,PD-1)等分子调节生发中心的B淋巴细胞的发育、增殖、分化,促进抗体分泌细胞形成[3-4]。Tfh还可以通过分泌白细胞介素(interleukin,IL)21(IL-21)促进生发中心形成、B淋巴细胞增殖和分化以及大量抗体的产生[5]

Tfh不仅存在于生发中心,亦存在于外周血中。外周血Tfh亚群紊乱与多种自身免疫性疾病有关,但其表型特征、临床意义以及在自身免疫性疾病发病中的作用仍然不清楚。研究发现,在小鼠和人体内可根据C-C趋化因子受体7(C-C chemokine receptor 7,CCR7)和PD-1的表达量,将Tfh分为2种功能状态:CCR7hiPD-1lo(CCR7高表达、PD-1低表达)静息型Tfh和CCR7loPD-1hi(CCR7低表达、PD-1高表达)效应型Tfh,其中CCR7loPD-1hi Tfh的比例增加提示Tfh活化[6]。本研究通过检测SLE患者外周血单个核细胞(peripheral blood mononuclear cell,PBMC)中CCR7loPD-1hi Tfh的比例,分析其与临床指标和成浆细胞的相关性,从而揭示其在SLE发病机制中的作用。

1 资料和方法 1.1 研究对象

纳入2018年2月至2018年7月在我院风湿免疫科就诊的31例SLE患者,均符合1997年美国风湿病协会(American College of Rheumatology,ACR)的SLE诊断标准[7],且均行系统性红斑狼疮疾病活动指数(systemic lupus erythematosus disease activity index,SLEDAI)测评。纳入同期在我院风湿免疫科就诊的类风湿关节炎(rheumatoid arthritis,RA)患者29例(均符合1987年ACR的RA诊断标准[8])、干燥综合征(Sj?gren’s syndrome,SS)患者12例(均符合2002年ACR的SS诊断标准[9]),以及在我院体检中心行健康体检的健康对照37名。本研究经我院医学伦理委员会审批,所有受试者均签署知情同意书。

1.2 主要试剂与仪器

活细胞染料、别藻蓝蛋白-花青素7(allophycocyanin-cyanine 7,APC-cy7)标记的抗CD4、异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记的抗CD45RA、AlexFlour 647标记的抗CXCR5、Brilliant VioletTM 421标记的抗CCR7、多甲藻黄素叶绿素蛋白-花青素5.5(peridinin chlorophyll protein-cyanine 5.5,PerCP-cy5.5)标记的抗PD-1、藻红蛋白(phycoerythrin,PE)标记的抗信号淋巴细胞激活分子家族成员5(signaling lymphocytic activation molecule family member 5,SLAMF5)、PE-CFTM594标记的抗CXCR3、藻红蛋白-花青素7(phycoerythrin-cyanine 7,PE-cy7)标记的抗ICOS、别藻蓝蛋白(allophycocyanin,APC)标记的抗CD19、PerCP-cy5.5标记的抗CD27、PE-cy7标记的抗CD38和PE标记的抗免疫球蛋白(immunoglobulin,Ig)D(IgD)抗体均购自美国Biolegend公司。人淋巴细胞分离液购自美国Sigma公司。使用美国BD公司LSRFortessa型流式细胞仪进行检测。

1.3 实验方法 1.3.1 PBMC的制备

用乙二胺四乙酸(ethylenediaminetetraacetic acid,EDTA)抗凝管采集所有研究对象外周静脉血各5 mL,加入5 mL磷酸盐缓冲液(phosphate buffer saline,PBS)稀释、混匀,然后加至含5 mL人淋巴细胞分离液的离心管中,1 100×g离心23 min。吸取中间白膜层,PBS洗涤2次,加入含0.5%胎牛血清的1 mL流式染色缓冲液重悬细胞,即得PBMC悬液。

1.3.2 细胞表面染色

调整PBMC悬液细胞密度为1×107/mL,取100 μL(1×106个细胞)细胞悬液于流式管中,分别加入活细胞染料和抗CD4、抗CD45RA、抗CXCR5、抗CXCR3、抗CCR7、抗ICOS、抗PD-1、抗SLAMF5抗体。另取1×106个细胞,分别加入活细胞染料和抗CD19、抗CD27、抗IgD、抗CD38抗体。4 ℃孵育30 min,PBS洗涤2次,加入300 μL流式染色缓冲液重悬后使用流式细胞仪进行检测,并采用FlowJo软件进行数据分析。

1.4 统计学处理

应用GraphPad Prism 7.0软件进行统计学分析。呈正态分布且方差齐性的计量资料以x±s表示,组间比较采用t检验或方差分析;偏态分布的计量资料以中位数(下四分位数,上四分位数)表示,两组间比较采用Mann-Whitney U检验;计数资料以例数和百分数表示,组间比较采用χ2检验;相关性分析采用Spearman相关分析。检验水准(α)为0.05。

2 结果 2.1 各组的基线资料

表 1。健康对照37名,男4名、女33名,年龄(42.49±11.29)岁;SLE患者31例,男3例、女28例,年龄为(40.29±13.06)岁;RA患者29例,男3例、女26例,年龄(63.10±10.95)岁;SS患者12例,男1例、女11例,年龄(50.75±11.80)岁。各组间的性别构成差异无统计学意义(χ2=0.07,P>0.05);SLE、SS患者年龄与健康对照差异均无统计学意义(t=-0.77、2.12,P均>0.05),而RA患者年龄大于健康对照,差异有统计学意义(t=7.09,P<0.01)。RA患者C3、C4、IgM和IgE水平均高于SLE患者(t=5.12、4.66,U=207.00、198.50;P均<0.01)。SS患者红细胞沉降率(erythrocyte sedimentation rate,ESR)、C反应蛋白(C-reactive protein,CRP)、C4、IgA水平低于RA患者(U=78.50、73.50,t=-2.53,U=68.50;P<0.05,P<0.01)。各组间病程、白细胞计数、淋巴细胞计数、IgG水平差异均无统计学意义(H=4.04、F=0.04、F=0.50、H=3.07,P均>0.05)。

表 1 1各组临床资料比较 Tab 1 Comparison of clinical characteristics among different groups

2.2 CCR7loPD-1hi Tfh与CCR7hiPD-1lo Tfh表面分子的表达情况

根据CCR7和PD-1的表达情况,可将健康对照外周血CD4+CD45RA-CXCR5+Tfh分为CCR7loPD-1hi Tfh和CCR7hiPD-1lo Tfh(图 1A)。相比CCR7hiPD-1lo Tfh,CCR7loPD-1hi Tfh表面CXCR3、ICOS和SLAMF5表达水平较高(t=3.73、5.06、8.27,P均<0.01;图 1B)。

图 1 CCR7hiPD-1lo Tfh与CCR7loPD-1hi Tfh设门方法及表面分子表达差异 Fig 1 Gate strategy and distinct phenotypes of CCR7hiPD-1lo Tfh and CCR7loPD-1hi Tfh subsets A: Gate strategy to identify CCR7hiPD-1lo Tfh and CCR7loPD-1hi Tfh subsets in Tfh; B: MFI of CXCR3, ICOS and SLAMF5 expression levels on CCR7hiPD-1lo Tfh and CCR7loPD-1hi Tfh subsets. Tfh: Follicular helper T cell; CCR7: C-C chemokine receptor 7; PD-1: Programmed death 1; hi: High expression; lo: Low expression; HC: Healthy control; SLE: Systemic lupus erythematosus; CXCR: C-X-C chemokine receptor; ICOS: Inducible costimulatory; SLAMF5: Signaling lymphocytic activation molecule family member 5; MFI: Mean fluorescence intensity. **P < 0.01. n=12, x±s

2.3 SLE、RA和SS患者外周血中CCR7loPD-1hi Tfh和CCR7hiPD-1lo Tfh的比例

SLE、RA和SS患者外周血中CD4+CD45RA-CXCR5+ Tfh的比例与健康对照相比差异均无统计学意义(P均>0.05,图 2A)。与健康对照相比,SLE患者外周血中CCR7loPD-1hi Tfh的比例增多[5.79%(2.97%,8.95%)vs 2.93%(1.63%,5.21%),U=314.5,P<0.01;图 2B],CCR7hiPD-1lo Tfh的比例降低[17.00%(10.50%,27.60%)vs 25.20%(16.55%,44.90%),U=341.5,P<0.01;图 2C]。RA患者CCR7loPD-1hi Tfh的比例与健康对照相比亦增加[4.57%(2.51%,7.60%)vs 2.93%(1.63%,5.21%),U=332.5,P<0.01;图 2B],而CCR7hiPD-1lo Tfh的比例与健康对照相比差异无统计学意义(U=413.5,P>0.05;图 2C)。SS患者CCR7loPD-1hi Tfh和CCR7hiPD-1lo Tfh的比例与健康对照相比差异均无统计学意义(U=183.0、208.0,P均>0.05;图 2B2C)。

图 2 Tfh(A)、CCR7loPD-1hi Tfh(B)和CCR7hiPD-1lo Tfh(C)在HCs和SLE、RA、SS患者外周血中的比例 Fig 2 Proportions of Tfh (A), CCR7loPD-1hi Tfh (B) and CCR7hiPD-1lo Tfh (C) in peripheral blood of SLE, RA and SS patients and HCs Tfh: Follicular helper T cell; CCR7: C-C chemokine receptor 7; PD-1: Programmed death 1; lo: Low expression; hi: High expression; HC: Healthy control (n=37); SLE: Systemic lupus erythematosus (n=31); RA: Rheumatoid arthritis (n=29); SS: Sj?gren's syndrome (n=12). **P < 0.01

2.4 SLE患者外周血中CCR7loPD-1hi Tfh比例与临床指标及成浆细胞比例的相关性

SLE患者外周血中CCR7loPD-1hi Tfh的比例与患者年龄、病程无明显相关性(r=-0.041 2、0.123 3,P均>0.05),而与SLEDAI、抗双链DNA(double-stranded DNA,dsDNA)抗体滴度以及成浆细胞(CD19+IgD-CD38++)比例呈正相关(r=0.447 1、0.517 4、0.466 9,P均<0.05)。见图 3

图 3 SLE患者外周血中CCR7loPD-1hi Tfh比例与临床指标及成浆细胞比例的相关性 Fig 3 Correlation analysis between clinical data, frequency of plasmablasts and percentage of CCR7loPD-1hi Tfh subset in peripheral blood of SLE patients SLE: Systemic lupus erythematosus; CCR7: C-C chemokine receptor 7; PD-1: Programmed death 1; lo: Low expression; hi: High expression; Tfh: Follicular helper T cell; SLEDAI: Systemic lupus erythematosus activity index; dsDNA: Double-stranded DNA

3 讨论

Tfh的主要功能是辅助B淋巴细胞增殖、分化并促进抗体生成,调节体液免疫。因此,Tfh比例及功能失调与诸多自身免疫性疾病的发生、发展有关。SLE是一种以大量自身抗体产生为特征的自身免疫性疾病,患者机体内生发中心反应亢进,自身反应性B淋巴细胞显著增加,这种病理改变与Tfh异常密切相关[10]。Odegard等[11]利用狼疮小鼠模型进行研究,发现Tfh增加可以促进小鼠生发中心B淋巴细胞IgG分泌增多,导致狼疮病情进展,而清除体内Tfh病情则改善。Tfh不仅可在生发中心与B淋巴细胞相互作用调节体液免疫,亦可在外周血及SLE患者的肾组织中发挥作用[12-13]。由于人淋巴组织样本采集受限,分析患者外周血Tfh成为一种具有重要临床意义的替代策略。外周血Tfh与生发中心Tfh具有相似的表型和B淋巴细胞辅助功能[14]。利用不同的表面标志,Tfh可被细分为多种亚群,不同亚群的功能存在差异。Choi等[15]发现CXCR5hiICOShiPD-1hi(CXCR5、ICOS、PD-1均高表达)Tfh在SLE患者体内增加,与SLEDAI和抗dsDNA抗体滴度相关。Le Coz等[16]根据CXCR3和CCR6将Tfh分成Tfh1、Tfh2和Tfh3,其中Tfh2扩增与SLE病情相关。但是,既往研究均缺乏对不同自身免疫性疾病中Tfh的对比以及Tfh与B淋巴细胞亚群的相关性分析。

本研究结果示,根据CCR7和PD-1的表达情况,Tfh可被细分为CCR7loPD-1hi Tfh和CCR7hiPD-1loTfh;相比CCR7hiPD-1lo Tfh,CCR7loPD-1hi Tfh表面CXCR3、ICOS和SLAMF5的表达水平较高,说明CCR7loPD-1hi Tfh具有更强的B淋巴细胞辅助能力。CXCR3是一种趋化因子受体,与相应趋化因子结合后可介导Tfh迁移至多种外周炎性组织,特别是肾组织[17]。ICOS和SLAMF5与T淋巴细胞、B淋巴细胞之间相互作用密切相关。ICOS通过与B淋巴细胞表面ICOS配体结合促进B淋巴细胞活化,SLAMF5可增加T淋巴细胞、B淋巴细胞之间的黏附从而产生有效的免疫反应[18]。阻断ICOS和SLAMF5均可抑制B淋巴细胞分化和抗体产生,下调体液免疫的强度[19-20]。Choi等[15]也证实CCR7loPD-1hi Tfh具有更强的分泌IL-21的能力。为了进一步探究CCR7loPD-1hi在自身免疫性疾病中的作用,本研究对不同亚群Tfh进行分析,结果显示CD4+CD45RA-CXCR5+ Tfh的比例在SLE、RA、SS患者和健康对照之间差异均无统计学意义(P均>0.05),但SLE患者外周血中CCR7loPD-1hi Tfh的比例较健康对照增加(P<0.01),CCR7hiPD-1lo Tfh比例降低(P<0.01);RA患者外周血中CCR7loPD-1hi Tfh的比例与健康对照相比亦增加(P<0.01),而CCR7hiPD-1lo Tfh比例差异无统计学意义(P>0.05);SS患者外周血中2个亚群Tfh的比例较健康对照差异均无统计学意义(P>0.05)。研究发现与RA和SS患者相比,SLE患者中自身反应性抗体的种类更加多样[21],Tfh亚群的紊乱可能是SLE自身抗体多样的重要原因。本研究通过相关性分析发现,CCR7loPD-1hi Tfh比例与SLE患者的SLEDAI、抗dsDNA抗体滴度以及CD19+IgD-CD38++成浆细胞比例呈正相关(r=0.447 1、0.517 4、0.466 9,P均<0.05),提示CCR7loPD-1hi Tfh是一种效应型Tfh亚群,可通过辅助B淋巴细胞产生自身抗体促进疾病的发生和发展。

PD-1是含有288个氨基酸的Ⅰ型跨膜蛋白,细胞外含IgV样结构,细胞内含免疫受体酪氨酸依赖抑制基序(immunoreceptor tyrosine-based inhibitory motif,ITIM)和免疫受体酪氨酸依赖转换基序(immunoreceptor tyrosine-based swith motif,ITSM)。PD-1与程序性死亡蛋白配体1(programmed death-ligand 1,PD-L1)结合后,ITIM、ITSM可发生磷酸化,招募蛋白酪氨酸酶,从而使下游分子去磷酸化,转导负向信号,降低T淋巴细胞活化程度及炎症反应[22]。PD-1曾被认为发挥免疫耐受作用,敲除PD-1可以使小鼠产生自身免疫反应,上调PD-1可以改善自身免疫症状[23]。但是多项研究表明,PD-1/PD-L1在自身免疫性疾病患者中表达水平升高,且PD-1hi Tfh具有更强的激活B淋巴细胞能力[15, 24]。这些研究结论并不一致,提示PD-1信号在生发中心对Tfh起着精细的调节作用。上调PD-1虽然可以减少Tfh的数量,但是其具有更强的分泌IL-4和IL-21的能力,从而促进生发中心B淋巴细胞的生长以及高亲和性抗体分泌细胞的形成[4]

综上所述,本研究表明SLE患者外周血中CCR7loPD-1hi Tfh比例增加与SLEDAI升高、成浆细胞比例的增加相关,检测患者外周血Tfh亚群可间接反映生发中心及B淋巴细胞的功能状态,对于疾病诊断、监测和预后有重要意义,针对该群细胞进行干预可能成为治疗SLE的潜在策略。

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