抗原特异性细胞毒性T细胞体外扩增方法的研究近展

抗原特异性细胞毒性T细胞(cytoxicTlymphocytes,CTLs)在细胞免疫应答中起着重要的效应功能,从而在抗肿瘤和抗感染免疫中发挥着重要作用。由于受到各种因素的限制和影响,抗原特异性CTLs无法在体内有效地诱导和扩增,因此,通过体外诱导和扩增CTLs后再将此CTLs回输到体内是一个比较理想的方法,且具有重要的应用价值,如何高效激活和扩增抗原特异性CTLs是此法能否成功应用的关键。     

检测白色念珠菌p47抗原ELISA法的建立及临床应用价值

目的 :建立检测血清中白色念珠菌相对分子质量 470 0 0 (p47)抗原的实验室方法 ,并探讨其临床应用价值。　方法 :亲和层析纯化白色念珠菌 p47抗原 ,制备抗 p47单抗和多抗 ,建立 EL ISA法检测血清中 p47抗原 ,并以美国 Biom erica公司试剂盒对照 ,同时检测血液病患者和念珠菌培养阳性的患者。　结果 :血液病组 6 9例患者中 ,本法检测 10例阳性 ,Biomerica法 9例阳性 ;培养阳性组 12 6例患者 ,本法检测 8例阳性 ,Biom erica法 6例阳性。两种方法的阳性符合率为 40 %。　结论 :对怀疑侵袭性念珠菌感染的患者 ,检测血清中 p47抗原是一种有应用前景的诊断方法。     

Contemplating immunopeptidomes to better predict them

The identification of T-cell epitopes is key for a complete molecular understanding of immune recognition mechanisms in infectious diseases, autoimmunity and cancer. T-cell epitopes further provide targets for personalized vaccines and T-cell therapy, with several therapeutic applications in cancer immunotherapy and elsewhere. T-cell epitopes consist of short peptides displayed on Major Histocompatibility Complex (MHC) molecules. The recent advances in mass spectrometry (MS) based technologies to profile the ensemble of peptides displayed on MHC molecules – the so-called immunopeptidome – had a major impact on our understanding of antigen presentation and MHC ligands. On the one hand, these techniques enabled researchers to directly identify hundreds of thousands of peptides presented on MHC molecules, including some that elicited T-cell recognition. On the other hand, the data collected in these experiments revealed fundamental properties of antigen presentation pathways and significantly improved our ability to predict naturally presented MHC ligands and T-cell epitopes across the wide spectrum of MHC alleles found in human and other organisms. Here we review recent computational developments to analyze experimentally determined immunopeptidomes and harness these data to improve our understanding of antigen presentation and MHC binding specificities, as well as our ability to predict MHC ligands. We further discuss the strengths and limitations of the latest approaches to move beyond predictions of antigen presentation and tackle the challenges of predicting TCR recognition and immunogenicity.     

Different cytokines regulate antigen uptake and presentation of a precursor dendritic cell line

Langerhans cells (LC) and dendritic cells (DC) need to be activated in order to perform their antigen-presenting function. In this study, we explored the influence of cytokines on the uptake and presentation of protein antigens by the retrovirally immortalized myeloid cell line FSDC. This cell line was generated from mouse fetal skin and was previously shown to have the characteristics of early DC precursors. Both FSDC and bone marrow-derived DC (BM-DC) were more effective in the pinocytosis of FITC-conjugated ovalbumin (FITC-OVA) and dextran (FITC-DX) than B cells or macrophages. Pretreatment of FSDC with granulocyte/macrophage colony-stimulating factor (GM-CSF) ± interleukin (IL)-4 enhanced the pinocytic uptake of FITC-OVA and FITC-DX, but did not induce antigen-presenting capacity. In contrast, untreated FSDC or FSDC pre-incubated with GM-CSF ± IL-4 suppressed T cell responses. Treatment of FSDC with IFN-γ reduced pinocytosis but increased the expression of MHC and co-stimulatory/adhesion molecules and promoted efficient presentation of OVA protein or peptide to the specific DO11.10 T cell hybridoma or to naive CD4⁺ T cells from DO11.10 TCR-transgenic mice. The results suggest that antigen uptake and antigen presentation in DC are regulated by different cytokine signals provided by the surrounding tissue.     

SARS荧光免疫诊断技术研究

目的应用基因工程技术,在昆虫细胞中表达SARS核蛋白基因,产生无感染性的核蛋白抗原,利用此蛋白制备抗原片,用于检测血清中SARS特异性抗体.方法从非典型性肺炎病人血清中提取病毒RNA,通过RT-PCR方法扩增出SARS核蛋白基因片段,将核蛋白基因插入杆状病毒,构建重组昆虫杆状病毒,转染昆虫细胞,收获细胞,经丙酮固定,制成SARS抗原片,建立了SARS荧光免疫方法(IFA).结果用此抗原片检测多份血清,仅与SARS病人血清起反应,而与正常人及其它发热病人血清不起反应.结论利用该方法制备抗原片,不需要P3实验室,操作简便,为诊断与研究SARS提供了方便而安全的方法.     

幽门螺杆菌感染患者血清尿素酶B亚单位测定及意义

目的探讨幽门螺杆菌(Helicobacter pylori,H.pylori)尿素酶B亚单位(Urease sulbunit B,UreB)核心多肽作为疫苗抗原的可能性.方法应用酶免疫法测定H.pylori感染患者血清游离Ure B及各型免疫复合物.结果61例H.pylori感染患者血清中,游离Ure B及Ure B-IgG、Ure B-IgA、Ure B-IgM任一项阳性者60例,占98.4%,其中游离Ure B阳性率63.9%,UreB-IgG为62.3%,Ure B-IgA为75.4%,UreR-IgM为78.7%.结论H.pylori感染患者血清中存在Ure B,Ure B核心多肽能引起特异性免疫应答,Ure B核心多肽可望成为疫苗抗原.     

Cyfra21-1、CA125 测定、B超检查对卵巢癌诊断及监测价值比较

目的：评价细胞角蛋白19片段抗原21-1(Cyfra21-1)、CA125测定和B超检查在卵巢癌诊断和监测中的价值。方法：对23例卵巢良性病变及25例卵巢癌患者进行血清Cyfra21-1、CA125免疫放射测定和B超检查，并进行比较。结果：Cyfra21-1、CA125测定和B超检查鉴别卵巢良性病变和卵巢癌的敏感性分别为84．0％，80．0％，84．0％。特异性分别为100．0％，78．5％，82．6％。3种方法联合应用，对卵巢癌总的阳性检出率为100．0％，对工期卵巢癌检出率为80．0％(4／5)。对18例卵巢癌患者动态观察结果显示，Cyfra21-1和CA125水平与病情进展一致。结论：联合应用Cyfra21-1、CA125测定和B超检查对诊断卵巢癌具有重要价值，Cyfra21-1和CA125可作为诊断早期复发卵巢癌的监测指标。     

供者抗原门静脉输注对大鼠皮肤移植存活的影响

目的：探讨特异性抗原诱导免疫耐受的作用。方法：通过门静脉途径输注脾细胞、骨髓细胞,观察皮肤移植模型。结果：受体接受供体脾细胞（DSC）、骨髓细胞（DBMC）及同时接受（DSC）和（DBMC）后能诱导受体产生免疫耐受。结论：肝脏特殊的免疫功能在免疫耐受产生中起重要作用。     

Differential processing of influenza nucleoprotein in human and mouse cells

To investigate how early events in antigen processing affect the repertoire of peptides presented by MHC class I molecules, we compared the presentation of the influenza A nucleoprotein epitope 265 – 273 by HLA-A3 class I molecules in human and mouse cells. Mouse cells that express HLA-A3 failed to present the NP265 – 273 peptide when contained within the full-length nucleoprotein, to HLA-A3-restricted human cytotoxic T lymphocytes. However, when the epitope was generated directly in the cytosol using a recombinant vaccinia virus that expressed the nonamer peptide, mouse cells were recognized by HLA-A3-restricted CTL. Poor transport of the peptide by mouse TAP was not responsible for the defect as co-infection of mouse cells with recombinant vaccinia viruses encoding the full-length nucleoprotein and the human TAP1 and TAP2 peptide transporter complex failed to restore presentation. These results therefore demonstrate a differential processing of the influenza nucleoprotein in mouse and human cells. This polymorphism influences the repertoire of peptides presented by MHC class I molecules at the cell surface.     

IL-10 prevents the differentiation of monocytes to dendritic cells but promotes their maturation to macrophages

Human monocytes cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-13 for 7 days differentiate into cells with the morphology and function of dendritic cells (DC). We have investigated the effect of IL-10 on this differentiation pathway. In the presence of IL-10 cells did not develop DC morphology, did not express CD1a and had lower levels of MHC class II. IL-10 promoted the differentiation of large cells with the morphology, cytochemistry and membrane phenotype of macrophages, including staining for nonspecific esterase and high levels of CD14, CD16 and CD68. The effect of IL-10 was dose dependent and was best appreciated when the cytokine was added at the initiation of the culture, as addition on day 3 was less inhibitory. When added to already differentiated DC on day 6, IL-10 caused only a modest reduction of MHC class II and CD1a expression, and no acquisition of the macrophage markers CD14, CD16 and CD68. Prolonged incubation up to 5 days with IL-10 did not induce a shift of differentiated DC to macrophages. On the other hand, the macrophages obtained by culturing for 7 days with GM-CSF+IL-13+IL-10 did not shift to DC upon removal of IL-10 for up to 3 days. Thus, the effect of IL-10 on monocyte differentiation, occurs only at the precursor level and confers an irreversible phenotype. From a functional point of view, cells cultured in the presence of IL-10 were poor stimulators of allogeneic cord blood T cells in mixed lymphocyte reaction (MLR) and presented tetanus toxin (TT) to specific T cell lines with much less efficiency than control DC. In contrast, IL-10-cultured DC showed 7 times greater endocytosis of FITC-dextran. This increased endocytosis was mostly mediated via the mannose receptor, as demonstrated by blocking with unlabeled mannose. In conclusion, IL-10 inhibits DC differentiation from monocytes and, in a substantial proportion of the cells, promotes the differentiation to mature macrophages. Intriguingly, IL-10 inhibits antigen presentation while it stimulates endocytic activity.