应用基因重组病毒表达产物筛选FBL3小鼠白血病肿瘤特异性抗原…

肿瘤特异性抗原是肿瘤免疫研究中心课题，众多的研究表明Ｔ细胞在肿瘤免疫中起重要作用，因而寻长Ｔ细胞所识别的肿瘤特异性表位并以为瘤苗的靶抗原有重要的研究价值，以一系列重组Ｆ－ＭｕＬＶｇａｇ病毒载体，用已建立的ＦＢＬ３－ＣＴＬ克隆筛选，并通过Ｈ－２Ｉ类分子结合实验，精确地测得Ｆ－ＭｕＬＶｇａｇ中一个能被为ＦＢＬ３－ＣＴＬ克隆所识别的表位（ＣＣＬＣＬＴＶＦＬ，ｐ８５～９３），并以此ｐ８５～９３合成肽免疫动     

检测Epstein-Barr病毒特异性细胞毒性T淋巴细胞方法的建立及其初步研究

目的建立一种非放射性、简便易行的可检测特异性细胞毒性Ｔ淋巴细胞的方法，并且初步应用于Ｅｐｓｔｅｉｎ－Ｂａｒ病毒的细胞免疫应答。方法用重组的ＥＢＶ－ＬＭＰ１痘苗病毒、ＴＫ＋痘苗病毒和杆状病毒系统表达的ＥＢＶ－ＬＭＰ１蛋白分别免疫Ｂａｌｂ／Ｃ小鼠，用Ｐ８１５细胞和乳酸脱氢酶法检测ＥＢ病毒特异性细胞毒性Ｔ细胞的杀伤效应。结果重组ＥＢＶ－ＬＭＰＩ痘苗病毒免疫组原发ＣＴＬ水平和体外诱生的二次ＣＴＬ水平均高于ＴＫ＋痘苗病毒免疫组和正常组；杆状病毒系统表达的ＥＢＶ－ＬＭＰ１蛋白免疫组的ＣＴＬ水平也明显高于正常鼠。结论本法可以较好的反映ＥＢ病毒特异性细胞毒性Ｔ细胞的水平，而且再一次说明ＬＭＰ１基因能够诱发特异性的细胞免疫。     

GM-CSF重组腺病毒扩增的骨髓树突状细胞体外经肿瘤抗原刺激后诱导抗肿瘤免疫应答

用ＧＭ－ＣＳＦ重组腺病毒感染小鼠骨髓细胞，观察扩增到的骨髓树突状细胞的生物学功能。结果表明，ＧＭ－ＣＳＦ重组腺病毒一次性感染骨髓细胞，１ｗ后能从一只小鼠股骨中扩增到２．４×１０６～３．３×１０６树突状细胞，扩增到的骨髓树突状细胞表达ＭＨＣ－Ⅱ、ＭＨＣ－Ⅰ、Ｂ７－１、Ｂ７－２和Ｉ－ＣＡＭ－１等免疫分子，分泌一定水平的ＧＭ－ＣＳＦ，对同种异体Ｔ细胞具有很强的刺激活性，体外经Ｂ１６肿瘤瘤苗刺激后免疫同系小鼠，能诱导出抗原特异性的ＣＴＬ活性，使免疫动物产生更强的保护性免疫反应，抵抗Ｂ１６细胞的攻击。提示可用ＧＭ－ＣＳＦ重组腺病毒从骨髓中扩增足够数量的树突状细胞，用于肿瘤的免疫治疗和基因治疗。     

检测Epstein—Barr病毒特异性细胞毒性T淋巴细胞方？…

目的 建立一种非放射性、简便易行的可检测特异性细胞毒性Ｔ淋巴细胞的方法，并且初步应用于Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒的细胞免疫应答。方法 用重组的ＥＢＶ－ＬＭＰ１痘苗病毒、ＴＫ＾＋痘苗病毒和杆状病毒系统表达的ＥＢＶ－ＬＭＰ！蛋白分别免疫Ｂａｌｂ／Ｃ小鼠，用Ｐ８１５细胞和乳酸脱氢酶法检测ＥＢ病毒特异性细胞毒性Ｔ细胞的杀伤效应。结果 重组ＥＢＶ－ＬＭＰ１痘苗病毒免疫组原发ＣＴＬ水平和体外诱生的二次ＣＴＬ     

肿瘤抗原p185蛋白免疫鼠脾细胞的抗瘤效应研究

目的 研究肿瘤抗原ｐ１８５蛋白作为肿瘤疫苗所诱导的抗肿瘤效应。方法 亲和层析纯化的ｐ１８５蛋白皮下注射正常ＢＡＬＢ／ｃ小鼠,取脾细胞分别与ｐ１８５蛋白、３Ｔ３－ｍｅｕ细胞体外共培养,以单独佐剂注射组的小鼠脾细胞为对照,观察其培养上清中ＨＦＮ－γ和ＴＮＦ－α活性。将免疫鼠脾淋巴细胞经ｐ１８５抗原和低剂量的ＩＬ－２体外培养１周后,再用ＣＤ３抗体刺激扩增,用流式细胞仪分析其细胞表型。３Ｔｅ－ｎｅｕ和３Ｔ     

巨噬细胞型骨髓基质细胞对肿瘤抗原提呈的体外实验研究

目的　探讨骨髓基质细胞对肿瘤抗原的提呈功能。方法　小鼠骨髓贴壁细胞经 Ｇ Ｍ Ｃ Ｓ Ｆ 诱导,形成以成熟巨噬细胞为主的基质细胞,用小鼠红白血病细胞 Ｆ Ｂ Ｌ３ 肿瘤抗原刺激,然后再与 Ｆ Ｂ Ｌ３ 肿瘤抗原致敏的 Ｔ 淋巴细胞混合培养。结果　骨髓基质细胞经 Ｆ Ｂ Ｌ３ 肿瘤抗原刺激后, Ｔ Ｎ Ｆα和 Ｉ Ｌ１β的分泌水平明显升高,经抗原预激的骨髓基质细胞能特异性地刺激同种抗原致敏的 Ｔ 淋巴细胞增殖和分泌高水平的 Ｉ Ｌ２ 。单抗阻断试验发现, Ｍ Ｈ ＣⅡ类分子和 Ｂ７２ 分子的联合阻断能有效地抑制致敏 Ｔ 淋巴细胞分泌 Ｉ Ｌ２ 。结论　本实验证实骨髓基质细胞具有抗原提呈功能, Ｍ Ｈ ＣⅡ类分子和 Ｂ７２ 分子在其抗原提呈中发挥了重要作用。     

弱酸洗脱提取的肿瘤抗原肽致敏的树突状细胞对CTL的体内外激活效应

弱酸洗脱提取小鼠红白血病细胞膜表面肿瘤抗原肽,用于致敏树突状细胞（ｄｅｎｄｒｉｔｉ ｃｅｌｌｓ,ＤＣ）观察其对细胞毒性Ｔ淋巴细胞（ＣＴＬ）的体内外激活效应。方法以ＰＨ３．３的枸橼酸－磷酸盐缓冲液室温下处理ＦＢＬ－３小鼠红白血病细胞５ｍｉｎ,流式细胞术检测洗脱效率,弱酸洗脱的抗原肽经ＳｅｐＰａｋＣ１８柱提取纯化,用于致敏ＤＣ,检测其体外刺激ＣＴＬ细胞株增殖的能力及体内激发特异性抗肿瘤免疫应答的能力。     

CA-Hb3抗原触发的成纤维细胞诱导大肠癌患者的特异性CTL反应

大肠癌患者的成纤维细胞经阳离子脂质体导入ＣＡ Ｈｂ３抗原,作为抗原刺激细胞诱导自身外周血淋巴细胞的细胞毒Ｔ淋巴细胞（ＣＴＬ）反应。实验成功地激活了抗原特异性ＣＴＬ,获得的ＣＴＬ在体外对亲本肿瘤细胞有显著的细胞毒活性,其培养上清中ＩＦＮ γ和ＴＮＦ α分泌水平增高。同时制备自身肿瘤的荷瘤鼠模型并进行过继治疗,ＣＴＬ一次过继能够延缓肿瘤的形成和生长,降低荷瘤鼠的死亡率,多次过继能够完全治愈肿瘤。以流式细胞仪检测细胞的免疫表型,显示ＣＴＬ主要为ＣＤ３＋ＣＤ８＋ＣＤ２８＋Ｔ细胞亚群。研究结果表明以ＣＡ Ｈｂ３抗原触发的成纤维细胞可以诱导大肠癌患者自身的抗原特异性ＣＴＬ反应。     

恶性疟CTL表位重组疫苗的构建及在人类HLA—A2和HLA—B51细胞中的 …

ＣＴＬ是红前期恶性疟免疫防护的关键环节。ＭＨＣ分子多态性和严格的种属特异性是限制ＣＴＬ恶性疟重组疫苗研制的重要因素。本文选用中国人群常见的ＨＬＡＩ类分子Ａ－２，１和Ｂ５１限制的恶履疟ＣＴＬ抗原表位ＴＲ２６和ＳＨ６，进行表位ＤＮＡ序列合成并将其克隆地真核表达载体，构建单表位ＤＮＡ重组疫苗，同时应用同尾酶克隆技术，将上述表位ＤＮＡ序列串联构建成双表位ＤＮＡ重组疫苗ｐｃＤＮＡ３．１ＴＳ。将以上克隆在含相     

抗人黑素瘤双特异性抗体增强LAK细胞毒效应的实验研究

抗肿瘤单克隆抗体的高度特异性与杀伤细胞对肿瘤的细胞毒效应相互结合起来，是增强ＬＡＫ细胞抗肿瘤作用的重要途径。本研究用化学连接法制备了既可识别淋巴细胞表面ＣＤ３抗原又可识别ＬｉＢｒ黑素瘤细胞表面肿瘤相关抗原的双特异性抗体ＣＤ３－ＨＢ８７５９。ＦＡＣＳ分析证实，ＣＤ３－ＨＢ８７５９具有结合两种抗原的活性，４ｈ＾５１Ｃｒ释放细胞毒实验结果表明，ＣＤ３－ＨＢ８７５９在诱导相可显著增强ＬＡＫ对ＬｉＢｒ细胞的     

A single amino acid variation within an immunodominant AKR/Gross MuLV cytotoxic T-lymphocyte epitope leads to a loss in immunogenicity

C57BL/6 mice characteristically generate vigorous H-2K(b)-restricted cytotoxic T lymphocytes (CTL) directed against an immunodominant CTL epitope (KSPWFTTL) expressed by endogenous AKR/Gross murine leukemia viruses (MuLV). These AKR/Gross MuLV-specific CTL do not efficiently recognize tumor cells induced by Friend/Moloney/Rauscher (FMR) MuLV, which express the highly homologous peptide RSPWFTTL. In this report, we not only confirm the inefficient recognition of FMR tumors by AKR/Gross MuLV-specific CTL, but also demonstrate that RSPWFTTL is poorly immunogenic in C57BL/6 mice. To gain insight into the mechanism(s) contributing to the inefficient recognition of FMR MuLV-induced tumors, we examined the RSPWFTTL dissociation rate from H-2K(b) as well as the ability for RSPWFTTL to diminish CTL effector functions by T-cell antagonism. In contrast to immunogenic peptides, which form stable MHC class I-peptide complexes having slow dissociation rates, poorly immunogenic peptides characteristically have faster dissociation rates. On the basis of a cell-surface MHC class I peptide stabilization assay, the dissociation rate of RSP-WFTTL from H-2K(b) is characterized by a half-life that is nearly identical to the half-life of KSPWFTTL. In addition, we could find no evidence for antagonistic inhibition of AKR/Gross MuLV-specific CTL over a wide concentration range of RSPWFTTL. Analysis of the role of the transporter associated with antigen processing (TAP), by use of recombinant vaccinia and Sindbis viruses expressing a hydrophobic amino-terminal endoplasmic reticulum (ER) targeting sequence coupled to RSPWFTTL, indicated that RSPWFTTL cell-surface presentation can be dramatically enhanced when directly targeted into the ER.     

Genetic analysis of the endogenous C3H murine leukemia virus genome: evidence for one locus unlinked to the endogenous murine leukemia virus genome of C57BL/6 mice.

The genetics of expression of the endogenous C3H AKR-type murine leukemia virus (MuLV) was followed serologically in crosses with C57BL/6 and NIH Swiss mice. The serologically defined phenotypes in backcrosses with NIH Swiss mice were shown to correlate with the segregation of proviral DNA. The segregation patterns in backcrosses of NIH Swiss and C57BL/6 mice are most consistent with a single AKR-type MuLV genome in C3H mice. Linkage analysis of this locus shows no linkage with the genes for esterases 1 or 3, glucose-6-phosphate dehydrogenase, the β-hemoglobin chain, H-2, or the agouti color locus. In NIH × (C57BL/6 × C3H) mice the linkage of the C3H-MuLV locus with the single C57BL/6-MuLV locus was evaluated. The results demonstrate that the MuLV gene of C3H is not linked to the C57BL/6-MuLV gene.     

Molecular definition of retrovirus-induced antigens recognized by tumour-specific H-2-restricted cytolytic T lymphocytes

The antigenic specificities of H-2-restricted, tumour-specific cytolytic T lymphocytes (CTL) were studied at the molecular level using CTL from BALB.B and C57BL/6 (H-2b) mice sensitized to an H-2b Gross murine leukaemia virus (MuLV)-induced tumour. Target cells were produced by the double transfection of mouse L cells (H-2k) with the cloned H-2Kb or H-2Db gene and retroviral DNA derived from a molecular clone of Akv MuLV (closely related to Gross MuLV). Doubly transfected L cells which express either H-2Kb or H-2Db antigen and retroviral antigens are lysed in a virus-specific manner by Gross MuLV-immune CTL. The existence of two independent Gross MuLV-immune CTL subpopulations, one restricted by H-2Kb and the other by H-2Db, is thus confirmed. Gross MuLV-immune CTL from both BALB.B and C57BL/6 mice killed L cells that express Akv MuLV gag gene products and H-2Kb or H-2Db antigen. In contrast, only CTL from C57BL/6 mice killed L cells that express Akv MuLV env gene products and H-2Kb or H-2Db. This indicates that specific recognition of MuLV-induced antigens by CTL can be selective and varies according to the origin of the CTL.     

Genetic control of natural immunity to ecotropic mouse leukemia viruses: immune response genes.

Humoral immune response to ectropic leukemia viruses in AKR and C57BL/6 mice was controlled by a gene that mapped in linkage group IX. Mice of the AKR strain had an immune nonresponsive allele of this gene, whereas mice of the C57BL/6 strain had an immune responsive allele. Antibody against murine leukemia virus (MuLV) reacted primarily with p15 protein of the viral envelope. It was concluded that the failure to find antibody production in AKR mice was the result of a genetic immunological defect, rather than the result of immunological tolerance that was induced by the persistent viremia of endogenous MuLV.     

Analysis of the helper virus in murine retrovirus-induced immunodeficiency syndrome: evidence for immunoselection of the dominant and subdominant CTL epitopes of the BM5 ecotropic virus

In genetically susceptible strains, such as C57BL/6 (B6) mice, LP-BM5 causes murine AIDS (MAIDS). LP-BM5 is a complex mixture of murine leukemia viruses (MuLV) that includes replication competent ecotropic (BM5eco) and mink cell focus inducing (MCF), and replication defective (BM5d) MuLV. At present, for the BM5eco virus, sequence information on only the gag region is available. In this paper, we describe for the first time the sequencing of the entire BM5eco viral genome as well as analysis of homology with two other previously sequenced and well-characterized MuLVs, Emv-11 and Emv-2, the latter constituting the parental virus for BM5eco. We propose that the detailed sequence comparisons herein provide cogent evidence that BM5eco utilizes variations in cytotoxic T lymphocytes (CTL) epitopes as an immune escape mechanism. This CTL evasion mechanism may contribute substantially to the underlying prototypic susceptibility of B6 mice to LP-BM5-induced MAIDS.     

Polymorphism in the major core protein (p30) of murine leukemia viruses as identified by mouse antisera.

Antisera prepared in C57BL/6 mice against the AKR K36 leukemia cells detect] group-specific and class-specific antigenic determinants on the major core protein (p30) of murine leukemia virus (MuLV). Sera that contain a predominance of antibodies against class-specific antigens react preferentially with the p30 proteins of ecotropic MuLV; these sera do not react with the p30 proteins of xenotropic MuLV. The use of MuLV class-specific antisera in immunological assays enables the assignment of p30 antigens in viruses and tissues to ecotropic or xenotropic origins.     

Identification of murine leukemia viral antigens detected on mouse cells by H-2 alloantisera.

H-2 alloantisera have been previously reported to contain antibodies against murine leukemia viral antigens, but the nature of the viral antigens on mouse cells which interact with these antibodies has not been established. We have found that H-2 alloantisera recognize components of molecular weight 70 000-80 000 mouse lymphocytes and leukemia cells. These components were also detected by a goat antiserum against the murine leukemia virus (MuLV) glycoprotein (gp 70) and are therefore closely related to or identical with that viral protein. Although most H-2 alloantisera detected gp 70-like molecules on lymphocytes and leukemia cells from a great variety of mouse strains, only one H-2 alloantiserum was found to interact with a gp 70 component on cells from C57BL/10 and C57BL/6 mice. Animals such as C57BL/10 mice that lacked the component reacting with most H-2 alloantisera showed increased serum levels of anti-MuLV antibodies after injection of B10.A spleen cells having a gp 70 component detectable by other H-2 alloantisera. In contrast, strains with cells reactive to antiviral antibodies in the H-2 alloantisera had low responses to MuLV antigens after a similar immunization procedure. Serum levels of anti-MuLV antibodies in both groups of mice, however, were increased after injection of Freund's adjuvant. These observations suggest that anti-MuLV antibodies in mouse alloantisera may arise from a response to viral antigens on the immunizing cells and general stimulation of the immune system.     

Correlation between levels of immunoglobulins and immune complexes in plasma of C57BL/6 and C57L/J mice infected with MAIDS retrovirus.

Infection with helper-free, defective MAIDS murine leukemia virus (MuLV) caused a rapid polyclonal activation of B cells in 0.75-, 2-, and 6-month-old C57L/J mice (H-2b, Fv-1n/n), similar to that in C57BL/6 mice (H-2b, Fv-1b/b), which was recognized by elevated plasma immunoglobulin concentrations. However, changes in plasma immunoglobulin levels differed in C57BL/J and C57BL/6 mice. In C57L/J mice, infection resulted in a rapid increase in plasma IgM and IgG2a, and the elevation of IgG2a persisted undiminished for 21 weeks. Levels of IgG2b also became slightly elevated, but those of IgG1 and IgG3 were not significantly affected. Plasma of 6 to 7-month-old C57BL/6 mice contained already high levels of IgM (30-40 mg/ml), which persisted undiminished in uninfected mice but decreased progressively in infected mice to 10% of the original concentration during 25 weeks of observation. In C57BL/6 mice, plasma IgG1 and IgG2b as well as IgG2a became similarly elevated after infection but also only transiently. Their levels began to decrease progressively about 10 weeks after infection and fell to far below the maximum concentration observed. The drastic loss of plasma IgM and IgGs observed in C57BL/6 mice during the later stages of MAIDS MuLV infection did not seem to be a consequence of the polyclonal activation of B cells per se but seemed to reflect additional immunological abnormalities arising in infected C57BL/6 but not C57L/J mice. In both mouse strains these changes in plasma Ig levels correlated with the formation of Ig-containing immune complexes that bound to high-affinity, protein-binding ELISA plates in the absence of antigen coating, which may represent unusual forms of self-antigen-antibody complexes.     

Serological and virological analysis of NIH (NIH X AKR) mice: evidence for three AKR murine leukemia virus loci.

The segregation of murine leukemia virus (MuLV) loci in backcrosses of AKR mice with NIH Swiss, C57BL/6, and BALB/c mice was followed by serological and virological assays as well as by DNA hybridization. In the backcross, NIH × (NIH × AKR), three main phenotypes of virus expression were observed, including viremic mice without any detectable free antibody against MuLV, nonviremic mice with high titers of antibody, and nonviremic mice without any detectable antibody. Of the three phenotypes, only the nonviremic, no-antibody mice were segregants for the lack of MuLV proviral DNA by DNA hybridization, by AKR-MuLV type-specific antigen expression, and by second backcross analysis. These mice represented approximately 13% of the population, suggesting the presence of three AKR-MuLV loci in AKR mice. In backcrosses of AKR mice with C57BL/6 mice only the antibody-positive, nonviremic and no-antibody, nonviremic phenotypes were observed. The segregation of AKR-MuLV loci in these crosses, analyzed serologically, was also consistent with the presence of three AKR-MuLV loci in AKR mice.     

Modification of thymic cell subsets induced by long-term cocaine administration during a murine retroviral infection producing AIDS.

LP-BM5 murine leukemia virus (MuLV) infection and cocaine administration are known to impair the murine immune system. We have developed a murine model to study the effect of daily cocaine administration and retrovirus infection on the lymphoid cell populations of the thymus. C57BL/6 female mice were studied following chronic cocaine administration for 11 weeks with simultaneous LP-BM5 MuLV infection. Cocaine administration reduced body and thymus weight, significantly reduced the number of CD8+ cells in the thymus, and partially prevented thymus enlargement due to lymphoid cell proliferation induced by LP-BM5 MuLV infection. Retrovirus infection was associated with a decrease in the percentage and absolute number of Thy 1.2+, CD4+, and CD8+ cells in the thymus, an effect potentiated by cocaine administration. Therefore cocaine impairs thymic function by altering the number of cells expressing T cell differentiation markers in MAIDS.