IV型II类反式激活因子基因新变异体的克隆、鉴定和功能测定

为获取有功能的IV型II类反式激活因子基因 (CIITA IV ) ,诱导肿瘤细胞表达MHCII类分子 ,从IFN γ刺激的THP 1细胞中以RT PCR获得CIITA IV ,将其连接到pGEMT easy载体。对所构建的pcDNA3 1 CIITA IV型表达载体进行反复测序后发现 ,所获得的CIITA IV基因存在结构变异 ,在 2 87位插入了 3个核苷酸TAG ,使 2 86 2 88位的AAG改变成为ATAGAG(2 86 2 90 ) ,并引起其他 8个座位核苷酸 (及推导的氨基酸残基 )发生改变。将表达载体转入原先不表达MHCII类分子的HeLa细胞中 ,检测到所获得的IV型CIITA变异体具有诱导人II类分子HLA DR表达的能力。空载体和CIITA IV基因导入的HeLa细胞中 ,DR阳性细胞百分率分别为 0 0 1 %和 37 6 4 %。该基因已从GenBank得到登录号 ,表明这是一个具有诱导HLA DR分子表达功能的IV型CIITA新基因。     

两种HLA-B分子对外周血单个核细胞分泌细胞因子的影响

为了解HLA B2 7和B39分子对外周血单个核细胞 (PBMC )分泌IFN γ和TNF α的影响。我们将外源HLA B 2 70 4和B 390 5 2基因分别表达在HLAI类分子缺陷的K5 6 2细胞表面 ,与PBMC作用 12h后 ,用ELISA法检测IFN γ和TNF α的含量。结果显示 :HLA B2 7分子能显著抑制PBMC分泌IFN γ ,而对TNF α分泌的影响不显著 ;而HLA B39表达于K5 6 2细胞后 ,均不能影响IFN γ、TNF α分泌。提示HLA B2 7分子与HLA B39分子影响PBMC分泌细胞因子的能力不同     

转染MHCII类基因对卵巢癌细胞株免疫特性的影响

建立稳定表达CIITA基因的人卵巢癌细胞株HO CIITA ,分析转染CIITA基因对T细胞体外抗肿瘤免疫应答的影响。以CIITA基因逆转录病毒 (pLXSN/CIITA )转染并筛选获得HO CIITA。用FACS和RT PCR分析HLA以及抗原加工递呈基因表达水平。以磁珠法分离得到正常人外周血CD4 +/CD8+T细胞 ,分别进行混合淋巴细胞反应及细胞因子测定 (ELISA和RT PCR )。结果显示 ,转染CIITA基因后 ,使HO细胞HLAII类分子和LMP7基因表达增高 ,而Ii基因表达由阳性转为阴性 ,未检测到TAP1表达 ;HO和HO CIITA细胞刺激CD4 +T细胞分泌IL 4含量两者有显著差异。刺激 4 8h后达到顶峰 ,前者分泌量约为后者的 1/2 ,但分泌IFN γ无差异 ,RT PCR与ELISA两种测定结果一致。表明转染CIITA基因可增加肿瘤细胞表面MHCII类分子的表达 ,该作用与IFN γ具有协同效应 ;并能诱导CD4 +T细胞表达IL 4。     

肿瘤细胞HLA分子的表达及IFN—γ的调控作用

目的 研究肿瘤细胞表达HLA分子及IFN－γ的调控作用。检测TIL细胞中T细胞亚群的变化,并与PBMC比较。方法 采用FACS检测细胞表达的CD抗原,采用ABC免疫组化染色检测肿瘤细胞HLA－Ⅰ,Ⅱ类分子的表达。以IFN－γ诱导建系的卵巢癌细胞表达HLA分子。结果 明细胞癌患者TIL细胞中的T细胞明显多于大肠癌患者。肿瘤患者PBMC中的T细胞数少于正常对照;肿瘤组织HLA－Ⅰ类抗原的表达明显低于正常组织,HLA－Ⅱ类抗原泊表达高于正常组织。与正常组织相比较,大肠癌患者比肾细胞癌患者HLAⅠ类抗原的表达更低。IFN－γ可诱导建系卵巢癌细胞表达HLA－Ⅰ类抗原,但不能诱导HLA－Ⅱ类抗原的表达。结论 肿瘤组织HLA表达的变化与肿瘤组织中TIL的数有关;IFN－γ能选择性地诱导卵巢癌细胞表达HLA－Ⅰ类分子,在抗肿瘤免疫中具有重要的意义。     

人卵巢癌细胞HLA分子与其相关基因表达的研究

目的：探讨人卵巢癌细胞中HLA分子及其相关基因的表达与IFN-γ诱导HLA分子表达的相互关系。方法：采用Western blot，免疫组化和流式细胞术，检测卵巢癌细胞中HLA分子表达，以RT-PCR技术，分子卵巢癌细胞TAP，LMP和MHCⅡ类分子反式激活蛋白（CⅡTA）基因表达。结果：被检测的11株卵巢癌细胞中，HLA-I类分子异常的表达率达为45%，而HLA-I类分子表达的异常与TAP1，TAP2，LMP2和LMP74种基因表达的异常有关，卵巢癌细胞或其他肿瘤细胞HLA-Ⅱ类分子的表达与CⅡTA基因的表达一致。组成性或诱导性表达CⅡTA基因的肿瘤细胞，经IFNγ作用后，其HLA-I，-Ⅱ类分子的表达增强；而诱导后仍不表达CⅡTA基因的肿瘤细胞，其HLA分子的表达无增强作用。结论：卵巢癌细胞中TAP和LMP基因表达的缺陷，是引起HLA-I类分子表达异常的重要因素。提示CⅡTA基因参与了调控肿瘤细胞HLA-Ⅰ，-Ⅱ类分子的表达。     

转染MHCⅡ类基因对卵巢癌细胞株免疫特性的影响

建立稳定表达CIITA基因的人卵巢癌细胞株HO-CIITA，分析转染CIITA基因对T细胞体外抗肿瘤免疫应答的影响。以CIITA基因逆转录病毒(pLXSN/CIITA)转染并筛选获得HO-CIITA。用FACS和RT-PCR分析HLA以及抗原加工递呈基因表达水平。以磁珠法分离得到正常人外周血CD4 ／CD8 T细胞，分别进行混合淋巴细胞反应及细胞因子测定(ELISA和RT-PCR)。结果显示，转染CIITA基因后，使HO细胞HLA Ⅱ类分子和LMP7基因表达增高，而Ⅱ基因表达由阳性转为阴性，未检测到TAP1表达；HO和HO-CIITA细胞刺激CD4 T细胞分泌IL-4含量两者有显著差异。刺激48h后达到顶峰，前者分泌量约为后者的1／2，但分泌IFN-γ无差异，RT-PCR与ELISA两种测定结果一致。表明转染CIITA基因可增加肿瘤细胞表面MHC Ⅱ类分子的表达，该作用与IFN-γ具有协同效应；并能诱导CD4 T细胞表达IL-4。     

IFN-γ对人胃癌细胞侵袭力及转移力的调节作用

用IFN γ处理胃癌细胞 ,采用流式细胞仪分析胃癌细胞表面抗原分子的表达水平 ,用附壁试验检测胃癌细胞对细胞外基质的亲和力 ,用细胞聚集试验检测胃癌细胞的聚集能力以研究IFN γ对胃癌细胞侵袭力及转移力相关因素的调节作用。结果表明 ,经高浓度和低浓度IFN γ处理后 ,胃癌细胞表面ICAM 1及HLAI表达均显著增加 ,CD44表达显著减少 ,胃癌细胞对细胞外基质的亲和力及胃癌细胞聚集程度均降低。提示IFN γ可增加胃癌细胞ICAM 1及HLAI的表达 ,抑制CD44表达 ,抑制胃癌细胞对细胞外基质的亲和力及胃癌细胞的聚集作用 ,因此可抑制胃癌细胞的侵袭力和转移力。     

肝癌细胞中HLA I类重链基因表达与启动子区域甲基化的相关性

目的:研究肝癌细胞株中DNA甲基化与HLAI类分子异常表达的相关性。方法:应用MSP技术对相关细胞系的HLA I类分子重链A、B、C位点启动子区域CpG岛的甲基化状态进行分析,Real-time PCR检测HLAI类分子重链mRNA水平的表达情况,Western blot检测RNA干扰后HLAI类分子重链表达情况。结果:在8株肝癌细胞系中HLA I类分子重链A、C位点启动子区域CpG岛存在甲基化;将启动子区域的DNA甲基化与相关基因表达数据相比较显示二者没有关联性;在RNA干扰DNA甲基化转移酶3a或3b的肝癌细胞系SMMC7721中,比较基因干扰前后HLA I类分子重链蛋白表达无显著变化。结论:在研究的肝癌细胞系中DNA甲基化没有参与调控肝癌中HLA I类分子的异常表达。     

沙眼衣原体感染对HeLa细胞MHCⅠ、Ⅱ类分子表达的影响

目的　探讨沙眼衣原体 (Chlamydiatrachomatis ,Ct)K型感染对HeLa细胞MHCⅠ、Ⅱ类分子表达的影响。方法　用免疫荧光法和流式细胞术等方法 ,对Ct感染和未感染的HeLa细胞MHCⅠ类分子表达水平和IFN γ诱导的HeLa细胞MHCⅡ类分子表达水平进行检测 ,同时对IL 10抗体的影响也作了研究。结果　Ct感染细胞MHCⅠ类分子表达水平和IFN γ诱导的感染细胞MHCⅡ类分子表达水平 ,随Ct感染剂量增加和感染时间延长而下降 ,与正常未感染细胞比较上述差异均具有统计学意义 (P <0 .0 1)。IL 10抗体能部分抑制感染细胞MHCⅠ类分子表达下调 ,但对IFN γ诱导的感染细胞MHCⅡ类分子表达下调无显著影响。结论　Ct感染可下调感染细胞MHCⅠ类分子和IFN γ诱导的细胞MHCⅡ类分子表达水平 ,这可能是造成衣原体持续感染的重要原因。感染过程中分泌的IL 10在下调感染细胞MHCⅠ类分子表达过程中起一定作用 ,而对IFN γ诱导的感染细胞MHCⅡ类分子表达水平下调无显著性影响     

TPA和IFN-γ活化角朊细胞对T淋巴细胞增殖和细胞因子分泌的作用

角朊细胞激活对T淋巴细胞增殖和细胞因子分泌的作用。TPA和IFN γ联合作用 ,激活角朊细胞表达表面分子 ,FACS检测MHCI、II类分子和CD80、CD86分子水平 ,RT PCR检测B7 H1共刺激分子表达。由激活角朊细胞提供T淋巴细胞激活的第二信号 ;绵羊红细胞花环沉淀分离纯化T细胞 ,抗CD3单抗提供第一信号 ,建立体外混合培养系统 ,3 H TdR渗入法检测T细胞增殖 ,ELISA检测培养上清细胞因子浓度。结果显示 ,TPA和IFN γ联合作用可诱导角朊细胞表达B7 H1共刺激分子 ,并上调其MHCII类分子。以B7 H1为第二信号 ,可以协同抗CD3单抗对T细胞的增殖作用 ,并呈现独特的细胞因子分泌格局。因此角朊细胞激活后表达B7 H1共刺激分子 ,此第二信号可刺激T淋巴细胞增殖 ,对其功能分化具有调节作用     

Reduced human leukocyte antigen expression in advanced‐stage Ewing sarcoma: implications for immune recognition

Ewing sarcoma (EWS) is a tumour most commonly arising in bone, although on occasion in soft tissue, with a poor prognosis in patients with refractory or relapsed disease, despite multimodal therapy. Immunotherapeutic strategies based on tumour‐reactive T and/or natural killer cells may improve the treatment of advanced‐stage EWS. Since cellular immune recognition critically depends on human leukocyte antigen (HLA) expression, knowledge about HLA expression in EWS is crucial in the design of cellular immunotherapeutic strategies. Constitutive and IFNγ‐induced HLA class I expression was analysed in EWS cell lines (n = 6) by flow cytometry, using antibodies against both monomorphic and allele‐specific antigens. Expression of antigen processing pathway components and beta‐2 microglobulin (β2m) was assessed by western blot. Expression of class II transactivator (CIITA), and its contribution to HLA class II expression, was evaluated by qRT‐PCR, transduction assays, and flow cytometry. β2m/HLA class I and class II expression was validated in EWS tumours (n = 67) by immunofluorescence. Complete or partial absence of HLA class I expression was observed in 79% of EWS tumours. Lung metastases consistently lacked HLA class I and sequential tumours demonstrated a tendency towards decreased expression upon disease progression. Together with absent or low constitutive expression levels of specific HLA class I loci and alleles, and differential induction of identical alleles by IFNγ in different cell lines, these results may reflect the existence of an immune escape mechanism. Inducible expression of TAP‐1/‐2, tapasin, LMP‐2/‐7, and the β2m/HLA class I complex by IFNγ suggests that regulatory mechanisms are mainly responsible for heterogeneity in constitutive class I expression. EWSs lack IFNγ‐inducible HLA class II, due to lack of functional CIITA. The majority of EWS tumours, particularly if advanced‐stage, exhibit complete or partial absence of both classes of HLA. This knowledge will be instrumental in the design of cellular immunotherapeutic strategies for advanced‐stage EWS. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

口腔鳞癌细胞系HLAI类分子异常表达及其机制探讨

为探讨两个口腔鳞癌细胞系 (KB和Tca81 1 3)中HLAI类抗原的表达水平及其异常的分子机制。利用流式细胞术、Westernblot检测细胞系中HLAI类抗原在蛋白水平的表达 ;RT PCR检测细胞系在转录水平的表达。结果两个口腔鳞癌细胞系中HLAI类抗原蛋白水平的表达下调 ;RT PCR结果显示Tca81 1 3细胞系中A、B、C、LMP2、LMP7、LMP1 0基因的mRNA表达下调 ;在KB细胞系中除了A、B、C、LMP7、LMP1 0基因的mRNA表达下调 ,LMP2、PA2 8α基因的mRNA表达显著减少或缺失 ;而TAP1、TAP2、Tapasin基因在mRNA水平的表达无改变。IFN γ诱导后纠正了多个基因在mRNA水平的异常表达。两个口腔鳞癌细胞系中HLAI类抗原表达下调 ,且多基因在转录水平的异常也即转录效率的低下很可能是该下调的重要原因     

Structural and functional characteristics of a dominant‐negative isoform of porcine MHC class II transactivator

The MHC class II transactivator, CIITA, is critical for MHC class II gene expression in all species studied to date. We isolated an interferon (IFN)‐γ‐inducible isoform of porcine CIITA (pCIITA′) encoding a protein of 566 amino acids (aa) with significant homology to human CIITA (hCIITA). Analysis indicated that pCIITA′ lacks the entire GTP‐binding domain that is important for nuclear translocation and activation of target genes by hCIITA. In pCIITA′ this region is replaced by a 14‐aa motif with homology to several signalling peptide sequences. Expression of pCIITA′ in porcine (ST‐IOWA) and human (HeLa) cell lines resulted in suppression of IFN‐γ‐stimulated MHC class II gene expression, at the protein and mRNA levels. We also identified two IFN‐γ‐inducible variants of hCIITA, hCIITAlo and hCIITA′ from Hela cells, both exhibiting dominant‐negative suppression of MHC class II gene expression. Interestingly, hCIITA′ encodes a predicted protein of 546 aa with a strikingly similar organization to pCIITA′ including the 14‐aa GTP‐binding domain‐replacement motif in which 10 out of 14 amino acids are identical to the pig sequence. Expression of hCIITA′ and hCIITAlo sequences in Hela cells suppressed IFN‐γ‐induced MHC class II gene expression. hCIITAlo, a predicted 303‐aa protein with deleted GTP‐binding and carboxy‐terminal domain, displayed a more subtle suppression of IFN‐γ‐induced MHC class II expression. These in vitro data indicate that there may be a role in vivo for isoforms of CIITA that can suppress full‐length CIITA‐mediated MHC class II gene expression. Both humans and now, potentially, pigs are candidate donors for organ and tissue allografts and xenografts, respectively. Regulation of MHC class II gene expression by manipulation of CIITA isoform expression in humans and pigs may provide a useful strategy for attenuation of T‐cell‐mediated cellular rejection.     

Absence of nonhematopoietic MHC class II expression protects mice from experimental autoimmune myocarditis

Experimental autoimmune myocarditis (EAM) is a CD4⁺ T‐cell‐mediated model of human inflammatory dilated cardiomyopathies. Heart‐specific CD4⁺ T‐cell activation is dependent on autoantigens presented by MHC class II (MHCII) molecules expressed on professional APCs. In this study, we addressed the role of inflammation‐induced MHCII expression by cardiac nonhematopoietic cells on EAM development. EAM was induced in susceptible mice lacking inducible expression of MHCII molecules on all nonhematopoietic cells (pIV?/? K14 class II transactivator (CIITA) transgenic (Tg) mice) by immunization with α‐myosin heavy chain peptide in CFA. Lack of inducible nonhematopoietic MHCII expression in pIV?/? K14 CIITA Tg mice conferred EAM resistance. In contrast, cardiac pathology was induced in WT and heterozygous mice, and correlated with elevated cardiac endothelial MHCII expression. Control mice with myocarditis displayed an increase in infiltrating CD4⁺ T cells and in expression of IFN‐γ, which is the major driver of nonhematopoietic MHCII expression. Mechanistically, IFN‐γ neutralization in WT mice shortly before disease onset resulted in reduced cardiac MHCII expression and pathology. These findings reveal a previously overlooked contribution of IFN‐γ to induce endothelial MHCII expression in the heart and to progress cardiac pathology during myocarditis.