负载不同形式肝癌抗原的树突状细胞抑瘤功能的比较

目的：探讨不同形式的肝癌抗原修饰的树突状细胞(DC)的抑瘤功能。方法：分别用肝癌H22冻融抗原、H22小分子抗原肽和Hsp70-H22抗原肽复合物修饰DC；用MTT比色法分析DC激活的CTL对H22细胞的杀伤能力，并用RT-PCR法测定脾脏T细胞中IFN-γ mRNA的表达水平；用不同修饰的DC免疫小鼠，观察其对H22肝癌的生长抑制作用。结果：单独的H22肝癌抗原肽修饰的DC不能激活CTL。Hsp70-H22肽复合物修饰的DC激活CTL的能力强于H22肝癌冻融抗原修饰的DC，对H22细胞的杀伤率分别为47．3％和18．3％。各组T细胞中IFN-γ表达水平的变化与杀伤率的变化相一致。用H22肝癌冻融抗原和Hsp70-H22肽复合物修饰的DC免疫小鼠后，均可抑制H22细胞生长，但后者的抑制作用更强，成瘤率仅40％。其他各组的成瘤率均为100％。结论：Hsp70-H22肽复合物是一种DC的强致敏物．可通过激活CTL、诱导CD4^ T细胞分化成Th1型细胞而参与肝癌的免疫排斥。     

Hsp70-肿瘤抗原肽复合物修饰的DC疫苗体内外特异性抗瘤作用

目的 :探讨树突状细胞 (DC)及Hsp70 肿瘤抗原肽复合物修饰后的体内外特异性抗瘤作用。方法 :采用一定的生化技术 ,从H2 2肝癌细胞中提取肿瘤抗原肽 ,并在体外与Hsp70进行结合 ;采用细胞培养技术 ,培养rmGM CSF、rmIL 4诱导的小鼠骨髓细胞 ,体外获取大量的DC ,后者经Hsp70 肿瘤抗原肽复合物修饰后刺激小鼠脾淋巴细胞 ,通过MTT法进行检测淋巴细胞的激活 ;收集上述刺激传代培养的淋巴细胞 ,检测其对H2 2瘤细胞和艾氏腹水癌细胞的杀伤功能 ;采用H2 2瘤细胞肌肉接种和H2 2瘤细胞、艾氏腹水癌细胞腹腔接种 ,对接种小鼠给予经体外修饰的DC回输 ,观察其抑制肿瘤的效果。结果 :Hsp70 肿瘤抗原肽复合物可使DC成熟 ,大量分泌IL 12、TNF α、IL 1β等细胞因子 ,并能够使DC激活小鼠脾淋巴细胞 ;激活后传代培养的淋巴细胞对H2 2瘤细胞能够特异性地杀伤 ,而对艾氏腹水癌细胞无效 ;经Hsp 70 肿瘤抗原肽复合物修饰后的DC可作为一种有效的瘤苗 ,体内能特异性地抑制小鼠H2 2肿瘤生长。结论 :Hsp70 肿瘤抗原肽复合物能够很好地修饰体外诱导获取的DC ,使后者成为一种有效的瘤苗 ,体外能够特异性地激活淋巴细胞 ,体内有效地抑制肿瘤生长     

B7-1/B7-H1相对比例变化对Hsp70-肽复合物抗肿瘤免疫效应的影响

目的：探讨Hsp7肽复合物对B7-1／B7-H1相对比例的影响及真核表达可溶性PD-1(sPD-1)对Hsp70-肽复合物抗肿瘤作用的影响。方法：通过RT-PCR和半定量PCR技术检测Hsp70-肽复合物体外刺激和体内免疫对小鼠脾细胞正调控共刺激分子B7-1和抑制性共刺激分子B7-H1及其受体PD-1表达的影响；体内转染表达sPD-1后，观察Hsp70-肽复合物免疫小鼠的肿瘤生长以及脾淋巴细胞毒性的变化。结果：基因表达检测表明．Hsp70-肽复合物体外刺激的小鼠脾细胞B7-1 mRNA和B7-H1 mRNA的水平随时问而变化，B7-1／B7-H1比值随刺激时间而增高；Hsp70-肽复合物体内免疫小鼠后期脾细胞B7-1表达下降，B7-H1及其受体PD-1表达上调，B7-1／B7-H1比值逆转；体内表达sPD-1可显著增强和延长Hsp70-肽复合物的抑瘤效果；体内表达sPD-1可提高Hsp70-肽复合物免疫的荷瘤小鼠脾细胞的杀伤率。结论：Hsp70-肽复合物的刺激引起共刺激分子B7-1和B7-H1表达的变化，B7-1／B7-H1的比例与激活效应相关，sPD-1通过阻抑B7-H1／PD-1途径、上调B7-1／B7-H1比例，可增强免疫应答，提高Hsp70-肽复合物特异性免疫治疗肿瘤的效应。     

癌胚抗原基因重组疫苗的构建及免疫效果观察

目的：探讨含癌胚抗原（CEA）部分编码基因的真核重组质粒pIRES-CEAⅢ用于CEA阳性肿瘤免疫治疗可能性。方法：应用基因重组技术构建了含CEA信号肽和Ⅲ区编码序列的真核表达质粒pIRES，CEAⅢ，肌注免疫BALB／c小鼠，再分别应用野生型小鼠肝癌细胞H22及CEAcDNA全序列转染的Hn细胞进行小鼠皮下接种，观察基因重组疫苗对CEA阳性肿瘤的抑制作用及其诱导小鼠细胞毒性T淋巴细胞（CTL）特异性杀伤CEA阳性肿瘤细胞的能力。结果：pIRES-CEAⅢ真核重组质粒免疫组小鼠CEA阳性肿瘤的生长速度趋缓，瘤块较小，与对照组小鼠（免疫空载体质粒和接种野生型H控细胞）相比有明显差异（P〈0．05）；经pIRES-CEAⅢ重组质粒免疫的小鼠脾细胞对H22．CEA（＋）细胞的杀伤率明显升高，差异显著（P〈0．01）；但对H22细胞则没有明显的杀伤作用。结论：pIRES-CEAⅢ作为基因疫苗可以抑制CEA阳性肿瘤在小鼠体内的生长，诱导小鼠CTL对CEA阳性肿瘤细胞的特异性杀伤。     

TIM2转基因细胞瘤苗对小鼠作用的初步研究

目的 观察小鼠TIM2转基因H22肝癌细胞瘤苗在小鼠体内的成瘤作用 ,初步研究其免疫原性.方法 构建TIM2基因真核表达载体pIRES2-EGFP - TIM2,脂质体法转染H22细胞,制备得到TIM2基因修饰的H22细胞瘤苗,建立小鼠肝癌移植瘤模型,观察其在小鼠体内的成瘤作用.结果 成功得到TIM2基因修饰的H22 细胞瘤苗,免疫接种小鼠后,可明显抑制小鼠体内肿瘤的发生和发展;给予H22-TIM2接种的小鼠CD4亚群、CD4/CD8比值显著高于H22-EGFP 细胞接种组、荷瘤对照组和正常对照组.结论 TIM2基因修饰H22细胞后,可显著降低H22细胞的体内成瘤性,抑制小鼠肿瘤生长,同时.在体内具有一定的免疫原性,为进一步探讨TIM2 在肿瘤生物治疗中的作用提供了初步的实验依据.     

体外构建的HSP70—肝癌抗原肽诱导抗原肽特异性免疫反应

目的：研究体外构建的HSP70-肝癌抗原肽复合物诱导针对肝癌的特异性免疫反应能力,为该复合物的临床应用奠定基础。方法：在体外构建HSP70-肝癌抗原肽复合物,联合应用粒/巨细胞集落刺激因子（GM-CSF）及白介素-4-（IL-4）直接从志愿者外周血中培养出DC;以HSP70、HSP70-肝癌抗原肽、抗原肽分别刺激DC,DC激活同源的T淋巴细胞产生细胞毒性T淋巴细胞（CTL）,检测其杀伤72细胞和肝癌细胞系的能力。结果：HSP70-肝癌抗、抗原肽均可诱导CD8^ 的抗原肽特异性CTL,而前者的诱导效果更强。结论：体外构建的HSP70-抗原肽复合物具有免疫原性,HSP70可以增强抗原肽诱导特异性免疫反应的能力,HSP70-抗原肽复合物有可能作为肽疫苗用于临床肿瘤免疫治疗。     

重组FN多肽CH50抑制黑色素瘤B16细胞体内转移的研究

目的研究重组纤黏连蛋白(FN)多肽CH50对小鼠黑色素瘤B16细胞体内转移的影响,以探讨CH50多肽抑制肿瘤转移的可能分子机制。方法体外培养黑色素瘤B16细胞,用荧光染料CFSE标记,接种脾脏后24h取脾、肝、肺做冰冻切片,观察肿瘤细胞在3种组织中的侵袭情况。从脾脏接种B16细胞,建立体内肿瘤转移动物模型,采用基于流体动力学的体内基因转染方法于小鼠体内表达CH50多肽,RT-PCR检测CH50 mRNA在肝组织的表达,Western印迹检测CH50多肽的表达。通过比较原位肿瘤结节及转移结节在数量、大小、分布上的差异及检测原位肿瘤组织中MMP-2、MMP-9表达差异,观察CH50多肽的治疗效果。结果注射24h后即可在脾脏形成荧光结节。pCH510质粒通过尾静脉注射后,可在肝组织中检测到CH50 mRNA及CH50多肽的表达。从脾脏接种B16细胞后第14天可在脾脏形成原发肿瘤,至第35天肝脏表面已形成转移瘤结节,成功建立了体内器官间(脾转肝)肿瘤转移动物模型。体内转染表达CH50多肽能抑制肿瘤生长、侵袭和转移,抑制原位肿瘤结节中MMP-2、MMP-9的表达。结论CH50多肽可以通过对MMP-2、MMP-9蛋白表达的抑制作用来抑制黑色素瘤B16细胞的成瘤能力和体内侵袭、转移能力。     

B7-1基因转染小鼠肝癌细胞诱导的抗瘤效应研究

目的：研究共刺激分子B7-1在抗肿瘤免疫中的作用.方法：体外观察转染B7-1基因及空载体的小鼠肝癌细胞株H22与同源小鼠脾细胞混合培养后,测定淋巴细胞增殖指数、CTL,于小鼠皮下接种不同H22细胞后观察肿瘤生长情况.结果：转染B7-1基因的H22细胞体外可刺激淋巴细胞增殖,增强CTL的杀伤活性,接种小鼠后成瘤潜伏期和荷瘤鼠存活期明显延长（P＜0.05）.结论：转入B7-1基因的小鼠肝癌细胞H22能增强免疫原性,能有效诱导CTLs介导的抗肿瘤免疫反应.     

小鼠肝癌细胞H22在肿瘤淋巴道转移研究中的应用

目的：探讨小鼠肝癌细胞H22在肿瘤淋巴道转移研究中的应用。方法：将小鼠肝癌细胞株H22分别接种于Km小鼠左腹股沟部（A组）和左后肢爪垫皮下（B组）,于接种后分批处死小鼠。H-E染色观察A组移植瘤及B组胴淋巴结、腹股沟淋巴结;透射电镜下观察A组移植瘤内毛细淋巴管超微结构。结果：A组小鼠移植瘤周边区可见毛细淋巴管,其内皮细胞亚细胞结构发生改变。B组淋巴结发生癌转移的概率和程度与接种细胞数相关。结论：小鼠肝癌（H22）模型可作为一种重要的肿瘤移植性模型而在肿瘤的淋巴道转移研究中发挥作用。     

HSP70多肽复合物修饰树突细胞抗胰腺癌研究

目的：研究肿瘤热休克蛋白70（HSP70）多肽复合物修饰树突状细胞激活淋巴细胞治疗胰腺癌的策略和方法。方法：采用低渗裂解，ConA—Sepharose亲和层析柱及ADP—Agarose亲和层析柱，从小鼠胰腺癌（MPC83）瘤块中纯化HSP70多肽复合物；纯化出的70KD蛋白修饰小鼠骨髓来源诱导树突细胞（DC）并制备树突细胞HSP70多肽肿瘤疫苗，MTT法检测修饰后DC增殖活性；用修饰后DC激活小鼠脾淋巴细胞，MTT法检测激活淋巴细胞在不同效靶比下对MPC83的体外杀伤活性。结果：获得较高纯度分子量为70kD左右的蛋白质；50～100ng HSP70多肽复合物可修饰10^4树突细胞，每克瘤块能获取HSP70多肽复合物约100μg；来自MPC83细胞瘤块HSP70多肽复合物激活的淋巴细胞能特异性杀伤MPC83细胞。结论：采用低压亲和层析柱可从胰腺癌瘤块中获得较高纯度HSP70多肽复合物，HSP多肽复合物DC疫苗用于胰腺癌细胞免疫治疗能获得体外杀伤效果，为临床胰腺癌生物免疫治疗奠定基础。     

Involvement of CD91 and scavenger receptors in Hsp70‐facilitated activation of human antigen‐specific CD4+ memory T cells

Hsp70 plays several roles in the adaptive immune response. Based on the ability to interact with diverse peptides, extracellular Hsp70:peptide complexes exert profound effects both in autoimmunity and in tumor rejection by evoking potent T cell responses to the chaperoned peptide. The interaction with receptors on APC represents the basis for the immunological functions of Hsp70 and a critical point where the immune response can be regulated. Various surface proteins (e.g. CD91, scavenger receptors (SR)) have been implicated in binding of Hsp70. In this study, antigenic peptides from tetanus toxin and influenza hemagglutinin complexed to human stress‐inducible Hsp70 were found to enhance the proliferation and cytokine production of human antigen‐specific CD4⁺ T cells. This was demonstrated in proliferation experiments using human monocytes as APC. Proliferated antigen‐specific cells were detected combining HLA‐DRB1^*0401 or HLA‐DRB1^*1101 tetramer and CFSE staining. Treating monocytes with CD91 siRNA diminished these effects. Additional blocking of SR by the SR ligand fucoidan completely abolished enhanced proliferation and production of Th1 and Th2 cytokines. Taken together, our data indicate that in the human system, CD91 and members of the SR family efficiently direct Hsp70:peptide complexes into the MHC class II presentation pathway and thus enhance antigen‐specific CD4⁺ T cell responses.     

Mutations in the substrate binding site of human heat‐shock protein 70 indicate specific interaction with HLA‐DR outside the peptide binding groove

Heat‐shock protein 70 (Hsp70)–peptide complexes are involved in MHC class I‐ and II‐restricted antigen presentation, enabling enhanced activation of T cells. As shown previously, mammalian cytosolic Hsp70 (Hsc70) molecules interact specifically with HLA‐DR molecules. This interaction might be of significance as Hsp70 molecules could transfer bound antigenic peptides in a ternary complex into the binding groove of HLA‐DR molecules. The present study provides new insights into the distinct interaction of Hsp70 with HLA‐DR molecules. Using a quantitative binding assay, it could be demonstrated that a point mutation of amino acids alanine 406 and valine 438 in the substrate binding pocket led to reduced peptide binding compared with the wild‐type Hsp70 whereas HLA‐DR binding remains unaffected. The removal of the C‐terminal lid neither altered the substrate binding capacity nor the Hsp70 binding characteristics to HLA‐DR. A truncated variant lacking the nucleotide binding domain showed no binding interactions with HLA‐DR. Furthermore, the truncated ATPase subunit of constitutively expressed Hsc70 revealed similar binding affinities to HLA‐DR compared with the complete Hsc70. Hence, it can be assumed that the Hsp70–HLA‐DR interaction takes place outside the peptide binding groove and is attributed to the ATPase domain of HSP70 molecules. The Hsp70‐chaperoned peptides might thereby be directly transferred into the binding groove of HLA‐DR, so enabling enhanced presentation of the peptide on antigen‐presenting cells and leading to an improved proliferation of responding T cells as shown previously.     

Involvement of cell-cell adhesion molecules in liver colonization by metastatic murine lymphoma/lymphosarcoma variants

Metastatic variant sublines of the murine RAW117 large cell lymphoma or lymphosarcoma have been established in vitro by sequential cycles of harvesting of liver tumor nodules after intravenous inoculation of tumor cell suspensions into syngeneic BALB/c mice. After five and tenin vivo selections for liver colonization, variant sublines RAW117-H5 and -H10, respectively, were established, and these formed significantly more surface liver tumors than the parental RAW117-P line. RAW117 sublines were tested for their abilities to adhere to embryonic mouse liver or brain cells in anin vitro cell-cell adhesion assay. Liver colonizing RAW117-H10 cells adhered with greater selectivity to liver cells than to brain cells. Parental RAW 117-P cells were more homotypically adhesive, but they were nonselective in their organ cell adhesion properties. We examined RAW117 cells for the presence of liver cross-reactive antigens using polyclonal xenoantibody preparations directed against embryonic murine liver cells. These antibody preparations block organ-specific homotypic adhesion of embryonic murine liver cellsin vitro. The amount of fetal liver antigen(s) expressed on RAW117 sublines correlated with liver colonization potentials (H10 > H5 > P) in quantitative absorption assays. Treatment of the highly metastatic RAW117-H10 subline with polyclonal anti-embryonic murine liver F(ab)₂ or Fab antibody fragments had no effect on RAW117-H10 cell viability or growthin vitro orin vivo, but inhibited liver colonization (median liver tumor colonies reduced from > 200 to 0) and prolonged life expectancy. In contrast, pretreatment of RAW 117-H 10 cells with polyclonal anti-H-2 did not modify thein vivo biologic properties of these metastatic cells.Address correspondence to this author in Houston.     

HINT1 peptide/Hsp70 complex induces NK‐cell‐dependent immunoregulation in a model of autoimmune demyelination

Heat shock proteins (Hsps) interact with the immune system and have been shown to contribute to immunoregulation. As efficient chaperones, Hsps bind many peptides and these complexes have many yet‐to‐be‐clarified functions. We have shown that Hsp70 is complexed within the mouse CNS with peptide CLAFHDISPQAPTHFLVIPK derived from histidine triad nucleotide‐binding protein‐1 (HINT1_38–57/Hsp70). Only this complex, in contrast to other peptides complexed with Hsp70, was able to prevent experimental autoimmune encephalomyelitis (EAE) by induction of immunoregulatory mechanisms dependent on NK cells. Pretreatment of proteolipid protein peptide 139–151 (PLP_139–151) sensitized SJL/J mice with HINT1_38–57/Hsp70 prevented the development of EAE, suppressed PLP_139–151‐induced T‐cell proliferation, and blocked secretion of IL‐17. HINT1_38–57/Hsp70 stimulation of NK cells depended on synergistic activation of two NK‐cell receptors, CD94 and NKG2D. NK cells with depleted CD94 or with blocked NKG2D did not inhibit PLP_139–151‐induced spleen cell (SC) proliferation. The HINT1_38–57/Hsp70 complex enhanced surface expression of the NKG2D ligand—H60. Downstream signaling of CD94 and NKG2D converged at the adaptor proteins DAP10 and DAP12, and in response to HINT1_38–57/Hsp70 stimulation, expression of DAP10 and DAP12 was significantly increased in NK cells. Thus, we have shown that the HINT1_38–57/Hsp70 complex affects NK‐cell function by enhancing NK‐cell‐dependent immunoregulation in the EAE model of autoimmune demyelination.     

The heat shock protein Hsp70 enhances antigen-specific proliferation of human CD4+ memory T cells

Heat shock proteins (HSP) can interact with a wide variety of peptides and the resulting HSP:peptide complexes are known to be highly immunogenic. The ability of HSP:peptide complexes to elicit CD8+ T cell responses by cross-presentation of exogenous antigen via MHC class I is well known. In contrast, their role in the activation of CD4+ T cells is less clearly defined, although several recent studies in mice and T cell lines suggest an involvement of HSP in the presentation of antigenic peptides via MHC class II. In this study we have investigated the potential of antigenic peptides from tetanus toxin and influenza hemagglutinin complexed to the human stress-inducible Hsp70 to enhance activation and proliferation of human memory CD4+ T cells. Hsp70:peptide complexes were found to amplify the proliferation of antigen-specific CD4+ T cells as confirmed by HLA-DR tetramer staining. Complex formation of the antigenic peptide with Hsp70 was absolutely required to elicit an antigen-specific amplification. This effect was most pronounced at low doses of antigen and decreasing APC/CD4+ T cell ratios. Taken together, we show the potential of Hsp70 to enhance antigen-specific CD4+ T cell proliferation and to increase the immunogenicity of presented peptides in human CD4+ T cells.     

Members of the heat-shock protein 70 family promote cancer cell growth by distinct mechanisms

Whereas the stress-inducible heat-shock protein 70 (Hsp70) has gained plenty of attention as a putative target for tumor therapy, little is known about the role of other Hsp70 proteins in cancer. Here we present the first thorough analysis of the expression and function of the cytosolic Hsp70 proteins in human cancer cells and identify Hsp70-2, a protein essential for spermatogenesis, as an important regulator of cancer cell growth. Targeted knock-down of the individual family members by RNA interference revealed that both Hsp70 and Hsp70-2 were required for cancer cell growth, whereas the survival of tumorigenic as well as nontumorigenic cells depended on Hsc70. Cancer cells depleted for Hsp70 and Hsp70-2 displayed strikingly different morphologies (detached and round vs. flat senescent-like), cell cycle distributions (G2/M vs. G1 arrest) and gene expression profiles. Only Hsp70-2 depletion induced the expression of macrophage inhibitory cytokine-1 that was identified as a target of P53 tumor-suppressor protein and a mediator of the G1 arrest and the senescent phenotype. Importantly, concomitant depletion of Hsp70 and Hsp70-2 had a synergistic antiproliferative effect on cancer cells. Thus, highly homologous Hsp70 proteins bring about nonoverlapping functions essential for cell growth and survival.