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     

重组人Hsp70提呈的H22肝癌细胞混合抗原肽治疗小鼠肿瘤的研究

目的：探讨B22肝癌细胞混合抗原肽体内抑制小鼠肿瘤的最佳方式。方法：采用冻融，低渗振荡，加热沉淀及酸处理等方法从H22肝癌细胞中制备抗原肽；将接种过H22瘤细胞的小鼠分别给予Hsp70-H22肽复合物注射，复合物＋pCH510质粒注射或化疗后给予复合物＋质粒注射，观察肿瘤抑制情况。结果：上述方法制备的抗原肽为混合肽，其与Hsp70形成的复合物体内能够抑制小鼠肿瘤生长，并且这种复合物可与pCH510质粒协同完全抑制10^5接种的瘤细胞形成肿 瘤，但不能完全抑制10^6接种的瘤细胞形成肿瘤，然而这种双因素与化疗进行衔接则可完全抑制10^6接种的瘤细胞形成肿瘤。结论：H22肝癌细胞肿瘤混合抗原肽经rhHsp70提呈可诱导H22荷瘤小鼠产生特异性免疫保护作用，与其它因素联合应用可产生更好的治疗效果。     

体外构建的HSP70-肝癌抗原肽诱导抗原肽特异性免疫反应1

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T2细胞和肝癌细胞系的能力.结果HSP70-抗原肽、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.     

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。     

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。     

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。     

自身骨髓瘤抗原致敏树突细胞介导特异性CTL反应的研究

目的：研究多发性骨髓瘤(MM)患者肿瘤冻融物致敏的树突细胞(DC)能否诱导特异性细胞毒T淋巴细胞(CTL)反应。方法：用MM患者骨髓CD34^ 细胞诱生DC。将MM患者骨髓瘤冻融物冲击致敏DC。MTT法检测骨髓瘤抗原致敏及非致敏DC诱导的自身T细胞对不同靶细胞(患者骨髓瘤细胞、K562细胞)的杀伤率。结果：骨髓瘤冻融物致敏DC诱导的自身T细胞对患者骨髓瘤细胞的杀伤远大于对K562细胞的杀伤。结论：患者骨髓瘤冻融物致敏的DC能有效诱导自身T细胞特异性抗瘤免疫。     

不同免疫途径对抗原肽修饰DC疫苗免疫效应的影响

目的探讨不同的免疫途径对抗原肽修饰树突状细胞(DC)疫苗免疫效果的影响。方法用鸡卵清蛋白(OVA)MHCⅠ限制性多肽SIINFEKL(OVAp257-264)包被小鼠骨髓来源的DC,通过皮下、腹腔、静脉或肌肉注射免疫小鼠,7天后行体内细胞毒性T淋巴细胞杀伤实验(InvivoCTL)分析CTL杀伤活性和细胞内IFN-γ染色(ICS)分析免疫小鼠脾脏CD8+细胞产生IFN-γ的情况。结果免疫7天后,InvivoCTL结果显示SIINFEKL修饰的DC皮下、腹腔、静脉或肌肉注射免疫小鼠其特异性CTL杀伤效应分别是37.3%±7.3%、61.0%±4.2%、56.9%±3.6%和10.8%±2.3%;ICS结果示四组小鼠产生IFN-γ的CD8+细胞占总CD8+细胞的比例分别是0.31%±0.07%、0.85%±0.12%、0.76%±0.14%和0.15%±0.04%。结论不同免疫途径可明显影响抗原肽修饰DC疫苗的免疫效应,其中腹腔注射诱发的抗原特异性CTL反应最强,静脉注射者次之,而皮下注射特别是肌肉注射较弱,提示通过腹腔注射DC疫苗可能是安全、高效的免疫途径。     

CTL识别的HLA-A2限制性人卵巢癌相关抗原OVA66表位的鉴定

目的：鉴定CTL识别的HLA—A2限制性人卵巢癌相关抗原OVA66表位。方法：以细胞因子从外周血单个核细胞(PBMC)中诱导树突状细胞(DC)，通过形态学观察和流式细胞术进行鉴定。用表位预测法选取并合成两种肽分子，分别脉冲成熟的DC，并刺激HLA—A2^ 健康人自体CD8^ T细胞，1wk后，用脉冲肽的自体PBMC以每7d的间隔刺激该CD8^ T细胞3次。以共接受4次抗原肽刺激的T细胞作为CTL，用乳酸脱氢酶(LDH)释放试验，检测CTL对靶细胞的杀伤效应。用酶联免疫斑点法(ELISPOT)．检测CTL中抗原特异性分泌IFN-γ的T细胞数。结果：形态学和流式细胞术的结果显示．PBMC可诱生成熟的DC。肽1235(FLPDHINIV)诱导的CTL．可特异性杀伤1235脉冲的T2细胞和OVA66^ 、HLA—A2^ 的SW480细胞，且L235诱导的特异性分泌IFN-γ的T细胞数增加。结论：卵巢癌相关抗原OVA66的HLA—A2限制性CTL表位1235．能激发对肿瘤抗原的特异性免疫应答，为制备肿瘤特异性肽疫苗奠定了实验基础。     

卵巢癌细胞株冻融抗原负载的树突状细胞诱导CTL抗卵巢癌免疫应答的体外研究

目的研究卵巢癌冻融抗原负载的树突状细胞(dendriticcells,DC)诱导细胞毒性T淋巴细胞(CTL)体外杀伤卵巢癌细胞的细胞毒性效应。方法利用免疫磁珠分离法(MACS)分离纯化脐血CD34 细胞并在体外诱导分化为DC,用反复冻融法从卵巢癌细胞系SKOV3中提取的可溶性相关抗原负载DC。流式细胞学检测负载抗原后DC表面各种分化相关抗原的表达,ELISA法检测DC上清中IL12的表达,混合淋巴细胞反应(MLR)测定DC体外刺激T细胞增殖的能力,MTT法检测抗原负载DC激活的抗原特异性CTL对卵巢癌细胞的杀伤作用。结果与未经抗原负载的DC相比,经卵巢癌抗原负载的DC不仅能更高地表达各种DC分化相关抗原CD1α(73.35%±2.94%vs34.1%±2.35%)、CD83(73.9%±8.46%vs54.68%±3.26%)、CD80(91.95%±2.48%vs52.53%±3.18%)、HLADR(70.05%±2.35%vs48.7%±2.07%)以及CD54(88.9%±5.52%vs71.45%±2.29%),同时具有更强的刺激同种异体T淋巴细胞增殖和IL12分泌的能力(P均<0.05)。此外,卵巢癌细胞SKOV3冻融抗原负载DC激活的CTL在体外对SKOV3的杀伤率为77.35%,显著高于未经抗原负载的DC(P=0.0001)。结论经卵巢癌细胞冻融抗原负载DC激活的CTL在体外具有更强的增殖能力和杀伤卵巢癌细胞的作用。     

转染自体胃癌细胞总RNA的树突状细胞诱导个体化免疫治疗的体外实验

目的探讨转染自体胃癌细胞总RNA的树突状细胞(DC)体外介导抗胃癌的免疫效应。方法制备短期培养的原代胃癌细胞。用rhGM-CSF、rhIL-4和TNF-α体外诱导胃癌患者外周血单个核细胞(PBMC)中DC的发育和成熟,并转染自体肿瘤细胞总RNA,激活自体T细胞产生CTL,用CCK-8试剂盒检测CTL的杀伤活性。应用流式细胞术及混合淋巴细胞培养技术检测DC的免疫功能状态。用ELISA法测定IL-12和INF-γ的水平。结果转染自体肿瘤细胞总RNA的成熟DC,不仅可高表达MHC-I、II类分子及CD80、CD83和CD86协同刺激分子,并可获得高效刺激自体或异体T细胞增殖的能力。转染RNA的成熟DC,分泌IL-12的水平及其刺激产生的CTL培养上清液中INF-γ的水平显著高于单纯成熟DC及未成熟DC;且CTL对自体胃癌细胞的杀伤率显著高于异体组。结论转染自体胃癌细胞总RNA的成熟DC能够体外诱导产生对自体肿瘤细胞具有高度抗原特异性杀伤活性的CTL。     

Efficient induction of a Her2-specific anti-tumor response by dendritic cells pulsed with a Hsp70L1-Her2(341-456) fusion protein

Heat shock proteins (HSPs) have been shown to interact with antigen-presenting cells (APCs), especially dendritic cells (DCs). HSPs act as potent adjuvants, inducing a Th1 response, as well as antigen-specific CD8(+) cytotoxic T lymphocytes (CTL) via cross-presentation. Our previous work has demonstrated that Hsp70-like protein 1 (Hsp70L1), a new member of the Hsp70 subfamily, can act as a powerful Th1 adjuvant in a DC-based vaccine. Here we report the efficient induction of tumor antigen-specific T cell immune response by DCs pulsed with recombinant fusion protein of Hsp70L1 and Her2(341-456), the latter of which is a fragment of Her2/neu (Her2) containing E75 (a HLA-A2 restricted CTL epitope). The fusion protein Hsp70L1-Her2(341-456) promotes the maturation of DCs and activates them to produce cytokines, such as IL-12 and TNF-α, and chemokines, such as MIP-1α, MIP-1β and RANTES. Taken together, these results indicate that the adjuvant activity of Hsp70L1 is maintained in the fusion protein. Her2-specific HLA-A2.1-restricted CD8(+) CTLs can be generated efficiently either from the Peripheral blood lymphocytes (PBL) of healthy donors or from the splenocytes of immunized HLA-A2.1/K(b) transgenic mice by in vitro stimulation or immunization with DCs pulsed with the Hsp70L1-Her2(341-456) fusion protein. This results in more potent target cell killing in an antigen-specific and HLA-A2.1-restricted manner. Adoptive transfer of splenocytes from transgenic mice immunized with Hsp70L1-Her2(341-456)-pulsed DCs can markedly inhibit tumor growth and prolong the survival of nude mice with Her2(+)/HLA-A2.1(+) human carcinomas. These results suggest that Hsp70L1-Her2(341-456)-pulsed DCs could be a new therapeutic vaccine for patients with Her2(+) cancer.     

Induction of T cell responses against autologous ovarian tumors with whole tumor cell lysate-pulsed dendritic cells.

The loading of dendritic cells (DCs) with whole tumor cell lysates may circumvent the facts that few tumor-specific antigens have been identified in human solid tumors. The present study was designed to investigate whether ovarian cancer cells lysate-pulsed DCs activate T cell responses against autologous ovarian tumors. Incubation of T cells with autologous tumor cell lysate-pulsed DCs stimulated proliferation of autologous T cells. T cells primed by autologous tumor cell lysate-pulsed DCs showed significant killing activity against autologous tumor cells, which could be blocked by anti-MHC-class-I and anti-CD8 mAb. By contrast, T cells primed by autologous unpulsed DCs alone or tumor lysates alone failed to exhibit significant killing activity. In addition, T cells primed by DCs pulsed with allogeneic tumor cell lysates or with autologous normal cell lysate or by these cell lysates alone did not induce the increase in the autologous tumor killing activity. As additional controls, T cells stimulated with autologous tumor lysate-pulsed DCs express no increase in the lysis of autologous monocytes, allogeneic ovarian tumor cells and other cell lines including K562, Daudi and Molt-4. Furthermore, T cells stimulated with autologous tumor lysate-pulsed DCs could produce the considerable amounts of cytokines such as GM-CSF, TNF-alpha and IFN-gamma. The data in the present study suggest that whole tumor cell lysates-pulsed DCs could activate T cell responses against autologous ovarian tumor cells, and that these pulsed DCs may be used as a new approach for the specific immunotherapy of ovarian cancer patients.     

结核杆菌Hsp65与EGFP融合基因的构建及DC疫苗的制备

目的: 构建结核杆菌H37Rv株Hsp65与增强型绿色荧光蛋白 (EGFP)的融合基因pEGHsp65, 并以其转染小鼠的树突状细胞 (DC), 制备抗结核的DC疫苗。方法: 采用PCR技术, 从培养的结核杆菌H37Rv株中抽提Hsp65基因,克隆到含有EGFP基因的质粒pEGFP- C1中, 构建pEGHsp65融合基因。以其转染Hela细胞, 在共聚焦激光扫描荧光显微镜下观测不同时间荧光表达的强弱, 并用RT- PCR检测Hsp65mRNA的表达。以pEGHsp65融合基因转染小鼠骨髓细胞经GM- CSF和IL -4诱导分化的DC, 用MTT比色法检测DC疫苗刺激未致敏脾细胞的增殖。结果: 用EcoRⅠ和BglⅡ双酶切鉴定证实, H37Rv株Hsp65DNA已插入重组表达载体pEG -FP- C1中。将融合基因转染入Hela细胞, 48h转染率最高;用RT- PCR在mRNA水平上可检测到Hsp65mRNA的表达。用MTT比色法检测表明, 融合基因转染的DC能激活并引起未致敏的脾细胞增殖。结论: 成功地构建pEGHsp65融合基因和以其制备的DC疫苗, 为进一步观察其治疗结核病的效应奠定了基础。     

负载K-ras抗原的DCs诱导CTLs对胰腺癌的体外杀伤作用

目的:研究负载K-ras (12-Val)抗原的树突状细胞(DCs)活化的特异性细胞毒性T淋巴细胞(CTLs)对胰腺癌的体外杀伤作用.方法:联合应用粒细胞-巨噬细胞集落刺激因子( rhGM-CSF)和白细胞介素4(IL-4)诱导培养外周血DCs.表达K-ras突变体的胰腺癌细胞株全瘤、单纯K-ras突变体多肽和K-ras突变体表位肽阳离子纳米颗粒分别致敏DCs.流式细胞仪测定DCs表面标志;~3H-TdR掺入法检测细胞增殖,~(125)I-UdR法检测CTL杀伤效应,ELISA试剂盒检测IL-12和IFN-γ.结果:与单纯K-ras(12-Val)突变体多肽相比,K-ras(12-Val)突变体表位肽阳离子纳米颗粒在低浓度时即可被DCs有效提呈(P＜0.05);负载全瘤抗原的DCs诱导产生的CTL与负载单纯K-ras(12-Val)突变体多肽、K-ras(12-Val)突变体表位肽阳离子纳米颗粒诱导组相比对Patu8988(K-ras+)及SW1990(K-ras-)胰腺癌细胞均有明显杀伤活性(P＜0.05),负载单纯K-ras(12-Val)突变体多肽、K-ras(12-Val)突变体表位肽阳离子纳米颗粒的DCs诱导产生的CTL对Patu8988(K-ras+)细胞株有特异性杀伤活性(P＜0.05),而对SW1990(K-ras-)细胞株无杀伤作用(P＞0.05).结论:低浓度K-ras(12-Val)突变体表位肽阳离子纳米颗粒作用后,在短时间即可被DCs有效提呈,且诱导产生的CTL对表达K-ras(12-Val)突变体的胰腺癌细胞株有特异性杀伤活性.     

Heat shock proteins as "danger signals": eukaryotic Hsp60 enhances and accelerates antigen-specific IFN-gamma production in T cells.

The heat shock proteins (HSP) gp96, Hsp70 and Hsp60 activate professional antigen-presenting cells (APC) to secrete proinflammatory cytokines and to express costimulatory molecules. Here, we analyze the impact of Hsp60 as a hypothetical danger signal on the antigen-specific activation of T cells derived from DO11.10 TCR-transgenic mice. The release of IFN-gamma, induced by the antigenic OVA(323-339)-peptide, is increased and accelerated dramatically by the addition of Hsp60 to ex vivo purified populations of T cells and peritoneal macrophages (PEC), while the antigen-specific IL-2 production or proliferation of the T cells remain unchanged. In contrast, "effector" T cells, undergoing secondary stimulation, displayed almost unchanged activation kinetics in the presence of Hsp60. The presence of Hsp60 induces IFN-gamma and up-regulation of CD69 in T cell/PEC cocultures even in the absence of antigenic peptide and this induction of IFN-gamma is strictly dependent on the ability of the macrophages to produce IL-12. Taken together, our data strongly suggest that the presence of eukaryotic mitochondrial Hsp60 allows antigen-specific IFN-gamma secretion under conditions when an antigenic stimulus alone is not sufficient to activate T cells.     

Presentation of lipopeptide by dendritic cells induces anti-tumor responses via an endocytosis-independent pathway in vivo

Cross-presentation by DCs is the major mechanism by which exogenous antigens activate CTLs. However, the mechanisms of entry and presentation of vaccine peptides by DCs remain unclear. In this study, we determined that the mechanisms of antigen presentation differed between nonlipidated and monopalmitoylated peptide antigens. We found that a nonlipidated long peptide could be taken up by DCs and that the peptide could be colocalized with early endosomes. The uptake of nonlipidated peptides by DCs was inhibited at low temperatures or by the depolymerization of actin filaments or microtubules. In contrast, lipidated peptides were internalized by DCs at low temperatures, and internalization was not inhibited when actin filaments or microtubules were depolymerized. Moreover, lipidated peptide, but not nonlipidated peptide, was internalized by nonphagocytic Jurkat cells. The endosomal/lysosomal and proteasomal degradation pathways were necessary for nonlipidated presentation leading to the activation of CD8(+) T cells, but the proteasomal degradation pathway alone was sufficient to process lipidated peptides for MHC class I presentation. We further found that lipidated peptides could enhance peptide-specific T cell responses in vitro and in vivo and induced stronger antitumor responses than nonlipidated peptides. Taken together, our results demonstrate that DCs present lipidated peptides through an endocytosis-independent pathway to promote strong anti-tumor effects in vivo.