趋化性细胞因子对人Tc1和Tc2细胞内Ca~(2+)浓度变化的影响

为了探讨趋化性细胞因子在体外对人Tc1和Tc2亚群细胞内Ca2 + 浓度变化的影响 ,从PBMC中分离纯化CD8+ T细胞 ,在特定细胞因子及细胞因子抗体作用下 ,体外定向诱导出能长期培养的Tc1和Tc2细胞系 ,用免疫荧光染色结合流式细胞术分析对其进行鉴定后 ,通过流式细胞术检测在趋化性细胞因子刺激前后 ,细胞内Ca2 + 浓度的变化。发现受SDF 1作用后 ,Tc1及Tc2细胞内Ca2 + 浓度变化均不明显 ,而IP 10刺激后 ,Tc1及Tc2细胞内Ca2 + 水平在短时间内明显上调 ,且在Tc1胞内的上升幅度远高于Tc2细胞 ,在MIP 1β刺激后 ,也观察到类似趋势 ;受Eotaxin刺激后 ,Tc1及Tc2细胞内Ca2 + 水平均有微小上升 ,在Tc2细胞内的上升幅度略高于Tc1细胞。说明Tc1和Tc2细胞受趋化性细胞因子作用后 ,细胞内Ca2 + 浓度有不同程度的变化 ,且与趋化性细胞因子受体的表达呈现一定的相关性。     

超抗原SEA对Tc1和Tc2细胞分化和增殖的影响

目的　探讨超抗原SEA对Tc1和Tc2亚群细胞增殖的不同影响。方法　以SEA刺激PBMC ,以免疫荧光染色结合流式细胞术比较刺激与未刺激的PBMC中T细胞各亚群比例的变化 ;从PBMC中分离CD8 T细胞 ,定向诱导为Tc1和Tc2细胞系 ,3 H TdR掺入法检测Tc1和Tc2细胞对SEA刺激的反应 ,用免疫荧光染色和流式细胞术检测CD2 5的表达。结果　经SEA刺激后 ,PBMC中的Tc0 /TH0增殖并向Ⅰ型或Ⅱ型分化 ,但SEA所诱导PBMC中的Tc2 /TH2的比例远高于Tc1/TH1;SEA在体外能更有效地促进Tc2亚群细胞的增殖 ;经SEA刺激的Tc2细胞系CD2 5的表达也高于Tc1。结论　SEA优先诱导PBMC中的Tc2 /TH2的分化 ,在体外更有效地促进Tc2细胞增殖     

复发性生殖器疱疹患者外周血趋化性细胞因子及其受体的研究

目的 :在单细胞水平上研究复发性生殖器疱疹 (RGH)外周血T淋巴细胞上CCR2、CCR5、CXCR1、CXCR3的表达 ,并通过观察血清趋化性细胞因子的改变 ,探讨趋化性细胞因子及其受体在RGH发病机理中的作用。方法 :采用ELISA法检测 2 0例RGH患者及 30例正常人血清中白细胞介素 8(IL 8)、γ干扰素诱导的蛋白 10 (IP 10 )、单核细胞趋化蛋白 1(MCP 1)、巨噬细胞炎性蛋白 12 (MIP 12 )活化后可调节的正常T细胞表达和分泌的因子 (RANTES) ,巨噬细胞来源的趋化性细胞因子(MDC)等水平变化 ;同时用双色荧光标记的流式细胞术分析 2 0例RGH患者外周血淋巴细胞表面趋化因子受体CCR2、CCR5、CXCR1、CXCR3的表达情况。结果 :RGH患者血清RANTES水平低于正常对照组 (P <0 0 5 ) ;外周血CXCR1+ T淋巴细胞百分率高于正常对照组 (P <0 0 5 )。结论 :病毒反复感染机体后 ,抑制机体的免疫细胞产生RANTES并通过T淋巴细胞CXCR1+ 的高表达可能在RGH抗病毒感染的免疫机理中起重要作用。     

趋化性细胞因子和T细胞游走的研究进展

主要总结趋化性细胞因子及其受体的新发现 ,趋化性细胞因子受体CXCR4、CCR5、CCR3等与HIV的关系以及趋化性细胞因子及其受体与T细胞游走等方面的研究进展。     

趋化性细胞因子及其受体和对疾病的免疫干预

趋化性细胞因子是一类重要的免疫调节因子 ,为介绍有关趋化性细胞因子 /趋化性细胞因子受体在抗肿瘤免疫反应和自身免疫性疾病中所起的重要作用 ,以及特异性趋化性细胞因子受体阻断剂的应用研究新进展。趋化性细胞因子与趋化性细胞因子受体的相互作用是IL 12诱导的抗肿瘤T细胞向肿瘤局部浸润的必备因素之一 ,当运用CCR5的特异性阻断剂TAK 779时 ,几乎完全阻断了IL 12的抗肿瘤作用 ;在II型胶原蛋白诱导的小鼠关节炎模型上 ,体内给予CCR5的阻断剂TAK 779后 ,关节炎的发病率明显降低 ,临床症状得到显著改善 ;在动物实验中 ,运用抗CCR5的抗体或CXCR3配体Mig的中和抗体可缓解移植物抗宿主反应 (GVHD) ;此外 ,CCR5和CXCR4在HIV感染宿主细胞时是必需的。所以针对趋化性细胞因子及受体的应用研究将为许多疾病的药物治疗提供新的手段     

IL-2和IL-4联合诱导B细胞死亡受体CCR3的表达及其功能研究

目的　研究在IL 2和IL 4作用下 ,趋化性细胞因子受体CCR3在人生发中心 (germinalcenter,GC)B细胞上的表达及其功能特性。方法　采用流式细胞术检测人GCB细胞上CCR3表达和在CCR3配体eotaxin作用下B细胞的凋亡 ,实时定量RT PCR和Northernblot法检测GCB细胞内CCR3mRNA的表达 ,淋巴细胞趋化和黏附试验检测B细胞的趋化和黏附能力。结果　人GCB细胞极低表达趋化性细胞因子受体CCR3,经IL 2和IL 4作用后 ,GCB细胞高表达CCR3,但此时CCR3不能在其配体作用下诱导GCB细胞的趋化和黏附功能 ,而是诱导GCB细胞凋亡。结论　IL 2和IL 4联合诱导人GCB细胞CCR3表达 ,CCR3可能具有死亡受体的功能。     

人肠道正常粘膜组织与外周血中记忆性IL-22~+T淋巴细胞频率及表型的比较

目的比较人肠道正常粘膜组织与外周血中IL-22+T淋巴细胞的频率及其表型特征。方法分离人肠道正常粘膜与外周血中单个核细胞,anti-CD3+anti-CD28刺激后,采用流式细胞术(FACS)检测IL-22的产生及其与IFN-γ、IL-17的关系,分析IL-22+T淋巴细胞CD45RO,CD62L,CCR7,CCR6,CCR10,CCR4等表面分子的表达。结果与anti-CD3+anti-CD28刺激外周血中CD4+和CD8+T淋巴细胞产生少量的IL-22(0.6%;0.57%)相比,肠道粘膜CD4+T细胞产生大约3.15%的IL-22,CD8+T淋巴细胞产生4%左右的IL-22。此外,肠道粘膜CD4+和CD8+T细胞中存在一群产生IL-22并独立于Th1、Th17,Tc1、Tc17的细胞亚群。肠道粘膜IL-22+T细胞表达较高比例的CD45RO,其中部分细胞表达CCR7,而较少表达CD62L。进一步研究表明,肠道粘膜CD4+IL-22+和CD8+IL-22+T细胞表达较高水平的CCR10(55.3%;73.9%),部分细胞表达CCR6或CCR4。结论人肠道正常粘膜组织中IL-22主要由效应型或中央型记忆T细胞产生,部分IL-22+T细胞独立于Th1、Th17,Tc1、Tc17细胞亚群。     

系统性红斑狼疮患者的Th细胞亚群平衡失调的初步研究

为分析Th亚群平衡失调在系统性红斑狼疮 (SLE )发生发展中的变化特点 ,以ELISA法检测血清IL 10、IL 12水平 ,以细胞内细胞因子的流式细胞术检测SLE患者PBMC的不同Th细胞亚群分泌细胞因子的变化特点 ,应用三色荧光标记技术分析Th1/Th2细胞表型。结果显示 :SLE患者血清IL 10水平显著高于正常人 ,而IL 12呈低水平表达。SLE患者CD4 + IFN γ IL 10 + 细胞亚群百分率显著高于正常人 ,IFN γ+ IL 10 与IFN γ IL 10 + 细胞亚群的比值明显降低 ,IFN γ+ IL 10 + 双阳性的CD4 + T细胞亦显著增多。SLE患者的CD4 + CCR5 CCR3+ 细胞亚群百分率与正常人比较显著升高 ,CD4 + CCR5 + CCR3 细胞亚群与正常人比较 ,示发现有显著差异 ,CD4 + CCR5 + CCR3 /CD4 + CCR3+ CCR5 比值显著低于正常对照组。这些结果提示 :SLE高水平IL 10与IL 12的低水平表达呈负相关 ;患者体内分泌IL 10的Th细胞数增多 ;Th细胞表面趋化因子受体的表达提示SLE存在CCR5 CCR3+ 细胞亚群的优势活化、数量增多 ,CCR5 + CCR3 /CCR3+ CCR5 细胞比例失调 ,从而导致免疫网络平衡被破坏。     

慢性乙型肝炎患者外周血淋巴细胞上CXCR3表达的初步观察

目的：探讨趋化性细胞因子受体CXCR3在慢性乙型肝炎症发生机制中的作用。方法：采用流式细胞术，检测炎症活动程度不同患者外周血淋巴细胞表面CXCR3的表达，及其在CD4^ 和CD8^ T细胞上的分布。结果：慢性乙肝患者外周血CXCR3^ 淋巴细胞和单核细胞明显增多，以CXCR3^ CD8^ T细胞的增加更明显。结论：趋化因子受体CXCR3与其配体的相互作用，可能在募集淋巴细胞及炎症形成过程中有重要作用。     

体外扩增γδ T细胞的肿瘤组织趋向性

 摘要 目的 旨在考察体外扩增的γδ T细胞向结直肠癌细胞迁移的能力。方法 采用密度梯度离心法分离外周血单个核细胞（Peripheral blood mononuclear cells,PBMCs）,并利用固相化抗T细胞受体（T cell receptor,TCR）γδ抗体进行两周的体外扩增。对扩增的γδ T进行免疫荧光染色和流式细胞仪分析或流式细胞仪分选。采用Bioplex 200流体芯片系统分析细胞因子和趋化因子的表达情况。采用transwell小室进行趋化试验。结果 体外扩增的γδ T细胞主要表达CCR5和CXCR3两种趋化因子受体。四种结直肠癌细胞系和十种结直肠癌肿瘤组织中存在CCR5和CXCR3配体的表达。体外扩增的γδ T细胞在TCR激活的情况下可以大量产生Th1和Th2型细胞因子,进一步募集γδ T细胞。特别是其产生的干扰素γ（Interferon γ,IFN-γ）能够提高结直肠癌细胞CXCR3配体的表达量,从而进一步促进γδ T细胞的趋近。结论 我们的研究结果为γδ T细胞过继免疫治疗结直肠癌提供了重要指征。     

Induced regulatory T cells suppress Tc1 cells through TGF-β signaling to ameliorate STZ-induced type 1 diabetes mellitus

Type 1 diabetes mellitus (T1D) is a chronic autoimmune condition in which the immune system destroys insulin-producing pancreatic β cells. In addition to well-established pathogenic effector T cells, regulatory T cells (Tregs) have also been shown to be defective in T1D. Thus, an increasing number of therapeutic approaches are being developed to target Tregs. However, the role and mechanisms of TGF-β-induced Tregs (iTregs) in T1D remain poorly understood. Here, using a streptozotocin (STZ)-induced preclinical T1D mouse model, we found that iTregs could ameliorate the development of T1D and preserve β cell function. The preventive effect was associated with the inhibition of type 1 cytotoxic T (Tc1) cell function and rebalancing the Treg/Tc1 cell ratio in recipients. Furthermore, we showed that the underlying mechanisms were due to the TGF-β-mediated combinatorial actions of mTOR and TCF1. In addition to the preventive role, the therapeutic effects of iTregs on the established STZ-T1D and nonobese diabetic (NOD) mouse models were tested, which revealed improved β cell function. Our findings therefore provide key new insights into the basic mechanisms involved in the therapeutic role of iTregs in T1D.     

Th17及Tc17细胞在博来霉素致系统性硬化病小鼠模型外周血、皮肤和肺组织中的表达

目的 研究T辅助细胞17(Th17)及T细胞毒细胞17(Tc17)在博来霉素致系统性硬化病(SSc)小鼠模型外周血、皮肤和肺组织的表达及意义.方法 30只雌性BALB/c小鼠随机分为3组:对照组(A组),博莱霉素注射4周无或轻度肺纤维化(PF)组(B组),明显PF组(C组).观察小鼠皮肤、肺部炎症和纤维化(Ashcroft评分)病变,流式细胞计数检测外周血、皮肤和肺部CD4+、CD8+、CD4+IL-17+Th17、CD8+IL-17+Tc17细胞的比例,荧光定量PCR(RT-PCR)检测小鼠皮肤和肺部维甲酸相关孤独受体(RORγt)、白细胞介素(IL) -17A mRNA的表达,ELISA检测外周血IL-17的含量,并分析这些指标的相关性.结果 皮肤羟脯氨酸含量和PF评分C组[(3.07±1.26) μg,/mg和4.0±1.41]、B组[(2.43±0.61) μg/mg和1.50±0.76]较A组[(1.45±0.40) μg/mg和0.60±0.70]明显增加,C组肺羟脯氨酸的含量较A组和B组明显增多(P均＜0.05).与A组比较,B组和C组外周血、皮肤和肺组织CD4+细胞数明显增多,CD8+细胞数明显减少,Th17细胞比例明显增加,C组外周血、肺组织、B和C组皮肤Tc17细胞明显增加(P均＜0.05);B组和C组外周血、肺组织和皮肤Th17/CD4+CD8+[ (1.41±0.36)％、(1.79±0.77)％],[(2.58±1.07)％、(5.23±2.34)％]和[(3.50±1.20)％、(4.02±1.32)％]较A组(0.71±0.25)％、(1.15±0.59)％、(0.99±0.46)％明显增加,Tc17/CD4+CD8+肺组织C组(1.62±0.53)％较A组(1.00±0.47)％,皮肤B组(1.70±0.70)％和C组(1.63±0.63)％较A组(1.11±0.34)％明显增高(P均＜0.05).与A组比较,B组和C组皮肤、C组肺组织IL-17A、RORγt mRNA的表达量、外周血IL-17含量明显增高(P均＜0.05).外周血Th17细胞、[L-17含量与肺部炎症、PF评分、肺和皮肤羟脯氨酸含量、皮肤炎症密切正相关(P＜0.01);皮肤和肺部Th17和Tc17细胞分别与皮肤和肺部炎症和纤维化评分、皮肤和肺部羟脯氨酸的含量密切正相关(P均＜0.01).结论Th17和Tc17细胞在SSc小鼠模型外周血、皮肤、肺组织的比例增高,且以Th17细胞为主,其表达与皮肤、肺部炎症和纤维化病变密切相关,并可通过分泌IL-17参与SSc的发病.     

Low expression of CD39+/CD45RA+ on regulatory T cells (Treg) cells in type 1 diabetic children in contrast to high expression of CD101+/CD129+ on Treg cells in children with coeliac disease

Type 1 diabetes (T1D) and coeliac disease are both characterized by an autoimmune feature. As T1D and coeliac disease share the same risk genes, patients risk subsequently developing the other disease. This study aimed to investigate the expression of T helper (Th), T cytotoxic (Tc) and regulatory T cells (T_reg) in T1D and/or coeliac disease children in comparison to healthy children. Subgroups of T cells (Th : CD4⁺ or Tc : CD8⁺); naive (CD27⁺CD28⁺CD45RA⁺CCR7⁺), central memory (CD27⁺CD28⁺CD45RA⁻CCR7⁺), effector memory (early differentiated; CD27⁺CD28⁺CD45RA⁻CCR7⁻ and late differentiated; CD27⁻CD28⁻CD45RA⁻CCR7⁻), terminally differentiated effector cells (TEMRA; CD27⁻CD28⁻CD45RA⁺CCR7⁻) and T_reg (CD4⁺CD25⁺FOXP3⁺CD127⁻) cells, and their expression of CD39, CD45RA, CD101 and CD129, were studied by flow cytometry in T1D and/or coeliac disease children or without any of these diseases (reference group). Children diagnosed with both T1D and coeliac disease showed a higher percentage of TEMRA CD4⁺ cells (P < 0·05), but lower percentages of both early and late effector memory CD8⁺ cells (P < 0·05) compared to references. Children with exclusively T1D had lower median fluorescence intensity (MFI) of forkhead box protein 3 (FoxP3) (P < 0·05) and also a lower percentage of CD39⁺ and CD45RA⁺ within the T_reg population (CD4⁺CD25⁺FOXP3⁺CD127⁻) (P < 0·05). Children with exclusively coeliac disease had a higher MFI of CD101 (P < 0·01), as well as a higher percentage of CD129⁺ (P < 0·05), in the CD4⁺CD25^hi lymphocyte population, compared to references. In conclusion, children with combined T1D and coeliac disease have a higher percentage of differentiated CD4⁺ cells compared to CD8⁺ cells. T1D children show signs of low CD39⁺/CD45RA⁺ T_reg cells that may indicate loss of suppressive function. Conversely, children with coeliac disease show signs of CD101⁺/CD129⁺ T_reg cells that may indicate suppressor activity.     

Multispecific and heterogeneous recognition of the gag protein by cytotoxic T lymphocytes (CTL) from HIV-infected patients: factors other than the MHC control the epitopic specificities.

The HIV gag polyprotein is a major target for recognition by CTL in infected humans. Using recombinant vaccinia viruses (rVV) expressing truncations of the p24gag, and the p18gag, p15gag and HIV-2 p56gag proteins, the characterization of epitope regions recognized by in vitro-stimulated peripheral blood mononuclear cells (PBMC) from 18 infected patients has been studied. The gag-specific response of most individuals is polyclonal and multispecific, and interindividual variations between target epitope regions were frequently observed, despite shared MHC alleles. As CTL may play an important role in the control of HIV replication in infected hosts, these results have important implications for designing vaccine strategies.     

Anti-CD48 (murine CD2 ligand) mAbs suppress cell mediated immunity in vivo

With the identification of murine CD48 as a homolog of the humanCD2 ligand LFA-3 (CD58) and as a ligand itself for murine CD2,the antl-murine CD48 mAb HM48-1 was administered intravenouslyto investigate the role of CD48 In cell mediated immunity invivo. Antl-CD48 mAb diminished the contact sensitivity responseto the hapten trlnltrophenol (TNP). mAb also inhibited in vivopriming for the subsequent generation of secondary, TNP-speclflc,cytotoxic T lymphocytes (CTL) in vitro. The inhibitory effectwas most effective in the afferent or inductive phase of immunityfor CTL, while antl-CD48 mAb was most inhibitory for the efferentor ellcltatlve phase of contact sensitivity. Addition of antl-CD48mAb directly to secondary CTL cultures also completely inhibitedCTL generation, while addition to the lytic assay showed onlyminimal inhibition of CTL activity. Combining cells from mAbtreated and untreated animals showed no evidence for suppressorcells. Further experiments revealed that mAb administered invivo, as well as to culture, Inhibited development of primary,alloantlgen-speclflc CTL in vitro. Mixed lymphocyte reactionand phytohemagglutlnln proliferation were partially suppressedby mAb administered in vivo or in vitro, whereas other mltogenlcresponses remained unaffected. Flow cytometrlc analysis revealeda moderate down modulation of CD48, CD3 and CD8 after treatmentwith anti-CD48. However, this did not represent T cell depletionsince CD2, Thy-1.2 and ig expression did not change. These resultssupport a major unrecognized role for CD48 In diverse aspectsof cell mediated immunity, affecting both CD4⁺ and CD8⁺ effectorT cell function. The anti-CD48 mAb functions not by depletingrelevant T cell populations, but rather by altering the arrayof cell surface receptors, and subsequent responses to primaryand secondary antlgenlc challenge.     

Partial recovery of senescence and differentiation disturbances in CD8+ T cell effector‐memory cells in HIV‐1 infection after initiation of anti‐retroviral treatment

Immune senescence as well as disturbed CD8⁺ T cell differentiation are a hallmark of chronic HIV infection. Here, we investigated to what extent immune senescence is reversible after initiation of anti‐retroviral treatment (ART). Peripheral blood mononuclear cells (PBMCs) from a cohort of HIV patients with different disease courses, including untreated viral controllers (n = 10), viral non‐controllers (n = 16) and patients on ART (n = 20), were analysed and compared to uninfected controls (n = 25) by flow cytometry on bulk and HIV‐specific major histocompatibility complex (MHC) class I tetramer⁺ CD8⁺ T cells for expression of the memory markers CCR7 and CD45RO, as well as the senescence marker CD57 and the differentiation and survival marker CD127. Furthermore, a subset of patients was analysed longitudinally before and after initiation of ART. Frequencies of CD57⁺CD8⁺ T cells decreased after initiation of ART in central memory (Tcm) but not in effector memory T cell populations (TemRO and TemRA). The frequency of CD127⁺CD8⁺ cells increased in Tcm and TemRO. We observed a reduction of CD127^– T cells in Tcm, TemRO and partially in TemRA subsets after initiation of ART. Importantly, HIV‐specific CD8⁺ TemRO cells predominantly displayed a CD127^–CD57⁺ phenotype in untreated HIV‐patients, whereas the CD127⁺CD57^– phenotype was under‐represented in these patients. The frequency of the CD127⁺CD57^–CD8⁺ T cell subpopulation correlated strongly with absolute CD4⁺ counts in HIV‐infected patients before and after initiation of ART. These findings can be interpreted as a phenotypical correlate of CD8⁺ memory T cell differentiation and the premature ‘ageing’ of the immune system, which was even observed in successfully virally suppressed HIV patients.