转染II类分子反式激活因子基因突变体抑制猪SLA的表达

为观察CIITA基因突变体对猪SLA表达的影响 ,用脂质体转染法将pcDNA3及CIITA突变体pcDNA3mCIITA2、pcDNA3mCIITA3和pcDNA3mCIITA4转染入猪血管内皮细胞系PIEC和猪B淋巴细胞系L2 3。利用流式细胞术 /RT PCR检测各种突变体对猪SLA DR、 DQ分子 /mRNA组成性和诱导性表达的影响。发现两种突变体pcDNA3mCIITA3和pcDNA3mCIITA4对PIEC/L2 3细胞SLAII类分子的表达起明显的抑制作用 ,而空载体pcDNA3和不具有起始密码子的突变体pcDNA3mCIITA2无此作用。提示pcDNA3mCIITA3和pcDNA3mCIITA4能抑制猪SLAII类分子的表达。     

构建可抑制HLA-DR/DQ表达的MHC-Ⅱ类分子反式激活因子基因的突变体及其机制

目的：构建能抑制MHC—Ⅱ类分子表达的MHC—Ⅱ类分子反式激活因子(MHC class Ⅱ transactivator，CⅡTA)基因的突变体．并探讨其抑制MHC—Ⅱ类分子表达的机制。方法：用PCR、酶切及连接技术，构建不含起始密码子的pcDNA3mCⅡTA2，含起始密码子的pcDNA3mCⅡTA3以及含起始密码子及NLS(nuclear localization signal)的pcDNA3mCⅡTA4突变体。用脂质体转染法，将上述3种突变体及空载体pcDNA3转入Hela细胞和Raji细胞中。用流式细胞术和RT-PCR法，观察他们对Hela／Raji细胞HLA—DR／DQ分子的诱导性和组成性表达的影响。将mCⅡTA4转移到对四环素浓度依赖的质粒pU-HD10-3上，通过改变培养环境中四环素的浓度，调节外源CⅡTA突变体的表达量，观察突变体的表达量与MHC-Ⅱ类分子受抑率的关系。结果：细胞和基因水平证实，pcDNA3mCⅡTA3和pcDNA3mCⅡTA4对Hela／Raji细胞HLA-DR／DQ的表达均有明显的抑制作用；而pcDNA3mCⅡTA2和空载体pcD-NA3无此作用。MHC-Ⅱ类分子被抑制的程度与外源转入CⅡTA突变体(pUHD10-3mCⅡTA4)的量明显相关。结论：成功地构建pcDNA3mCⅡTA3和pcDNA3mCⅡTA4，并能抑制HLA-Ⅱ类分子的表达。初步证实CⅡTA突变体是通过与胞内的野生型CⅡTA竞争性结合反式激活蛋白，来抑制MHC-Ⅱ类分子的转录和表达。     

一种新的CIITA显性负向突变体的构建和功能研究

II类反式激活因子(CIITA)参与MHC II类基因转录表达的调控,本研究采用RT-PCR等分子克隆技术构建含起始密码子和NLS序列,但是删除N和P/S/T结构域的CIITA突变体质粒pCDNA3.1(+)mCIITA,用脂质体转染法将上述突变体及pCDNA3.1(+)空载体转入来源于BALB/c小鼠的1B4.B6细胞株,用流式细胞术和RT-PCR观察I-A分子表达的影响,并通过混合淋巴细胞反应观察C3H小鼠CD4+T细胞对转入突变体及空载体的1B4.B6细胞的免疫应答强度。结果在细胞和分子水平证实pCDNA3.1(+)mCIITA对1B4.B6细胞I-A的表达起明显抑制作用,强阳性克隆抑制率为90%以上。细胞已基本丧失了对受者CD4+T细胞的刺激能力。以上结果为减低同种异体/异种器官细胞性排斥提供了新的思路和手段。     

CIITA反义RNA抑制MHCII类分子表达

为探索利用CIITA (MHCclassIItransactivator)基因反义RNA抑制MHCII类分子表达的可能性 ,为CIITA的应用研究奠定基础。利用RT PCR扩增能与CIITAcDNA第 95至 5 0 0bp互补的片段 ,并构建成pcDNA3/CIITAa 重组质粒 ,脂质体转染法将其转入人HeLa/Raji细胞和猪PIEC/L2 3细胞 ,流式细胞术动态检测反义RNA对人 /猪MHCII类分子的抑制率和抑制程度。结果显示HeLa、Raji、PIEC和L2 3四种细胞MHCII类分子受抑制率分别为 4 6 7% ( 7/ 15 ) ,4 0 % ( 6 / 15 ) ,4 6 7% ( 7/ 15 )和33 3% ( 5 / 15 )。典型受抑克隆细胞MHCII类分子受抑程度高达 85 % ,反义RNA对MHCII类分子的抑制时间持续 35～ 5 0d。CIITA反义RNA能有效抑制人 /猪MHCII类分子的表达 ,它在自身免疫和移植免疫中有一定的应用前景     

Enhanced anti-tumor effect of dendritic cells modified by CIITA gene in hepatocellular carcinoma-bearing mice]

AIM: To study the effect of MHC class II transactivator(CIITA ) on the expression of MHC class I/II molecules, CD80 and CD86 molecules on dendritic cells(DCs), and explor the use of DCs modified by CIITA gene in the biotherapy of tumors. METHODS: We transfected the mouse CIITA gene by lipofectamine into DCs drived from mouse bone marrow. The expression of MHC class I/II, CD80, and CD86 molecules was detected by flow cytometry. 40 mice bearing hepatcellular carcinoma were divided into 4 groups: group 1 were treated with a para-tumor injection of PBS, group 2 DCs, group 3 modified DCs, and group 4 modified DCs pulsed with H22 tumor antigen. Then the anti-tumor effect of DCs modified by mCIITA was evaluated by measuring the tumor size. RESULTS: After transfection of mCIITA, the expression rate of MHC class I/II molecules of DCs increased from 74.2%/66.7% to 93.6%/91.4%, and that of CD80/CD86 increased from 52.3%/60.5% to 89.7%/91.5%. After immunization, the growth of H22 tumors in group 4 mice was significantly inhibited compared with that in other three groups (P<0.05 at 3, 4, 5, 6, and 7 weeks versus groups 1, 2; P<0.05 at 5, 6, 7 weeks versus group 3). CONCLUSION: The expression of MHC I/II, CD80 and CD86 on DCs was regulated markedly by transfection of mCIITA. mCIITA transfection also enhanced the anti-tumor effect of DCs. Our results provided a new method for biotherapy of tumors with DCs.     

Different requirements of ICAM-1/LFA-1 adhesion in allorecognition and self-restricted antigen recognition by class II-specific T cell clones

We have analyzed the influence of non-antigen-specific interactions between ICAM-1 and LFA-1 in target recognition by allospecific and antigen-specific Tcells at the clonal level, using human and mouse fibroblasts transfected with HLA-DR1 or DR2 with or without co-expression of ICAM-1, as antigen-presenting cells. The results show a great heterogeneity in the requirements for ICAM-1/LFA-1 interactions for antigen-specific and alloreactive T cell responses and this requirement may depend on the avidity of any particular interaction. The data also show that for most alloreactive clones, ICAM-1/LFA-1 adhesion is not sufficient to facilitate efficient T cell recognition of its target molecule. HLA class II recognition by a large proportion of the DR1- and DR2-specific alloreactive clones studied was different for class II molecules expressed on murine or human fibroblasts compared to human lymphoid cells, and was independent of ICAM-1 expression on the stimulator cells. The inability of some T cell clones to recognize HLA-class II expressed on non-lymphoid cells suggests the absence of specific epitopes and could be due to the lack of the relevant peptides, either because they are derived from species-specific proteins or to differences in processing of endogenous antigen in the transfected stimulator cells.     

In the absence of Ii, MHC class II molecules display a distinct set of self peptides

The influence of the invariant (Ii) chain on antigen presentation by MHC class II molecules is well established. This study addresses whether the absence of Ii leads merely to failure of presentation of certain peptides or to display of novel peptides from endogenous proteins, using transfectants expressing HLA-DR alone, DR + Ii, or DR + Ii + DM. Western blotting revealed that, in the absence of Ii and DM, DR molecules form complexes with multiple intracellular proteins, furthermore, HPLC traces of peptides acid extracted from DR molecules expressed with or without Ii were markedly different. T cells were then used as “probes” of peptide occupancy of DR1. Most anti-DR1 alloreactive T cell clones raised against DR1 PBMC did not recognise DR1 in the absence of Ii and DM. Responses of clones that recognized the DR1⁺Ii⁻DM⁻ transfectants were augmented by co-expression of Ii and DM. In contrast, anti-DR1 clones generated against the DR1⁺Ii⁻DM⁻ transfectants failed to respond to human DR1-B-LCL. Responses to the DR1⁺Ii⁻DM⁻ transfectants were abolished by co-expression of Ii and DM in the transfected cell line, excluding simple lineage-specific allorecognition. These results suggest that, in the absence of Ii, class II molecules display a distinct set of peptides, generated as a result of interactions with proteins early in the biosynthetic pathway. If circumstances arise in vivo when the ratio of Ii to MHC class II is reduced, this may lead to the display of “illegitimate” self peptides, and the consequent interruption of self tolerance.     

CIITA M1-RNA抑制HeLa细胞表面MHC II类抗原的表达

探讨MHC II类转录激活因子(CIITA)的M1-RNA对细胞表面MHC II类分子表达的抑制。M1-RNA是核糖核酸酶P的催化活性单位,设计并克隆针对CIITA第452、629位点的M1-RNA(分别为M1-452-GS、M1-629-GS)及其相应的CIITA靶基因,分别插入pUC19、pGEM-7zf(+)载体,进行细胞外切割活性筛选。将细胞外切割作用明显的M1-629-GS亚克隆入psNAV载体(psNAV-M1-629-GS,pA629)并稳定转染HeLa细胞株,流式细胞术检测经典的MHC II类抗原(HLA-DR、-DP、-DQ)的表达,RT-PCR检测CIITA的mRNA水平。在重组人γ干扰素诱导下,pA629阳性HeLa细胞株表面HLA-DR、-DP抗原表达分别降低了83.03%及89.91%;同时CIITA的mRNA含量明显减少(P<0.05)。CIITA的M1-RNA抑制了自身mRNA含量,从而阻止其调控的MHC II类分子的表达,为移植物抗宿主病的研究提供了一种新方法。     

MHCII类反式激活蛋白调控肿瘤细胞HLA分子的表达

为研究肿瘤细胞MHCII类分子表达及对IFN γ诱导HLA分子表达的反应性与CIITA表达的关系 ,本文采用Westernblot、免疫组化和流式细胞术检测肿瘤细胞HLA分子表达 ,以RT PCR检测肿瘤细胞CIITA基因表达。肿瘤细胞HLAII类分子表达与CIITA表达一致 ;组成性或诱导性表达CIITA的肿瘤细胞 ,经IFN γ作用后其HLAI、II类分子表达增高 ;IFN γ诱导后仍不表达CIITA的肿瘤细胞 ,其对IFN γ促HLA表达的作用不反应。提示 ,某些肿瘤细胞对IFN γ不能诱导HLA分子表达与CIITA诱导性表达缺陷有关。表面CIITA参与调控肿瘤细胞HLAI、II类分子表达。     

Tumor rejection by gene transfer of the MHC class II transactivator in murine mammary adenocarcinoma cells

The murine mammary adenocarcinoma cell line TS/A is a highly malignant MHC class II-negative tumor. We show that transfection of TS/A cells with the MHC class II transactivator CIITA renders them MHC class II-positive and highly immunogenic in vivo. These cells were fully rejected by 51% of syngeneic recipients and had a significantly lower growth rate in the remaining 49% of animals. This directly correlated to the amount of MHC class II molecules expressed in the transfected tumor. Tumor rejecting animals were protected against rechallenge with the parental TS/A tumor. The rejection required CD4(+) and CD8(+) T cells. CD4(+) T cells were fundamental in the priming phase of the antitumor response. CTL-specific for a peptide of the envelope gp70 of an endogenous ecotropic retrovirus were identified and explained the specificity of the effector mechanism of rejection against the TS/A and the antigenically related C26 carcinoma cells but not against the unrelated gp70-negative syngeneic fibrosarcoma F1F cells. This is the first example of successful tumor vaccination by genetic transfer of CIITA. These results open the way to a possible use of CIITA for increasing both the inducing and the effector phase of the antitumor immune response.     

Single nucleotide polymorphisms in MHC2TA, the gene encoding the MHC class II transactivator (CIITA)

The MHC class II transactivator (CIITA) is the master regulator for HLA-D (DP, DQ, DR) gene expression. In this report the coding and promoter regions of the CIITA gene, MHC2TA, were evaluated for polymorphisms in 50 normal Caucasian individuals. Allele frequencies were obtained for four separate single nucleotide (nt) polymorphisms (SNPs) identified in the MHC2TA coding region: nt 1614 (C-->G), nt 2509 (G-->A), nt 2536 (T-->G), and nt 2791 (G-->A). MHC2TA sequence analysis of 100 chromosomes from these 50 individuals revealed a SNP in MHC2TA promoter (p) III at nt (-)155 (A-->G), but none in CIITA pI or pIV. In addition, we demonstrate the presence of splice variant at a previously undiscovered intron, accounting for a three nt (TAG) insertion at position 474 that was originally described in association with one of the disease-causing CIITA cDNA mutations in bare lymphocyte syndrome.