体外大量制备新型gp96肿瘤疫苗及其诱导的特异性抗肿瘤免疫应答分析

旨在体外组装酵母菌表达的gp96 (Recombinant gp96,rgp96) 蛋白与B16.F10黑色素瘤抗原,大量制备新型gp96肿瘤疫苗,并研究其诱导的特异性抗肿瘤免疫应答。利用体外组装的rgp96-肿瘤抗原复合物免疫C57BL/6小鼠,并通过酶联免疫斑点实验、细胞因子染色、杀伤实验技术进行分析,结果显示与单纯rgp96或肿瘤抗原免疫组相比,体外组装的rgp96-肿瘤抗原复合物免疫能够显著抑制B16肿瘤的生长,而且能够明显提高肿瘤特异性T细胞活性。rgp96-肿瘤抗原复合物的抗肿瘤免疫活性与从肿瘤组织中提取的gp96接近。研究结果为大量制备新型gp96肿瘤疫苗提供了依据。     

病毒特异性CD8+T细胞抗病毒免疫的研究进展

病毒利用宿主细胞核酸和蛋白质装置进行增殖,并与宿主细胞表面的受体结合,感染众多靶细胞c一旦建立感染,抗原呈递细胞通过内源性抗原呈递途径加工、呈递病毒抗原,激活机体免疫应答。病毒特异性免疫主要机制是细胞毒性T淋巴细胞作用,清除病原体和感染的靶细胞．同时CD8^ T细胞分化为记忆T细胞,介导再次免疫应答。     

热休克蛋白-多肽复合物在肿瘤和传染性疾病免疫中的作用

热休克蛋白家族中的许多成员如gp96\,HSP90\,HSP70等具有排斥和治疗肿瘤及传染性疾病的免疫原性,进一步研究发现热休克蛋白作为分子伴侣可结合细胞中的肽库,它本身没有抗原性,抗原性由结合的短肽所决定。热休克蛋白将结合的短肽呈递给I类MHC分子,进而激活特异性CTL和记忆性T细胞,引发机体细胞免疫反应。据最新发现gp96还可能有与MHC一样的功能,可直接将结合的多肽抗原呈递给T细胞。近年来对哺乳动物的二种主要热休克蛋白gp96和HSP70的免疫机制和作为治疗性疫苗的优越性进行了详细研究,这为乙型肝炎和乙肝继发性肝癌的免疫治疗提供了新思路。     

N-糖基化修饰对热休克蛋白gp96免疫学功能的影响

文中旨在以N-糖基化位点突变的重组热休克蛋白gp96为对象,研究N-糖基化修饰对其免疫功能的影响。首先利用昆虫表达系统表达野生型和突变型gp96蛋白,并检测其糖基化水平。进一步通过体外和体内实验,利用流式细胞术和酶联免疫吸附试验 (Enzyme linked immunosorbent assay,ELISA) 检测小鼠CD8+IFN-γ+ T细胞亚群和IFN-γ的分泌,查明糖基化对gp96抗原呈递功能的影响,进一步用ATPase试剂盒检测gp96的ATPase活性。最后通过小鼠免疫实验探究糖基化对gp96疫苗佐剂功能和活化流感疫苗特异性T细胞的影响。结果显示,N-糖基化修饰位点突变后,重组gp96蛋白总含糖量下降了27.8%。与野生型重组蛋白相比,突变gp96的抗原呈递能力减弱,同时ATPase活性明显降低。同时与野生型重组gp96相比,突变gp96佐剂活化流感疫苗特异性T细胞水平也明显减少。这些结果表明,N-糖基化修饰参与调节gp96的ATPase活性和抗原呈递功能,进而影响其疫苗佐剂功能,为开发基于gp96的佐剂疫苗提供了依据。     

Gp96蛋白的免疫学及其临床应用研究进展

热休克蛋白Gp96属于HSP90家族,是内质网中最丰富的蛋白质之一,在细胞内发挥着分子伴侣的作用。在天然免疫中,Gp96则通过与Toll样受体等相互作用刺激抗原呈递细胞 (如DC等) 产生各种细胞因子激活免疫系统;而在获得性免疫中,Gp96抗原胶通过抗原交叉呈递给MHC-I类分子,诱发机体抗原特异性细胞毒T细胞免疫应答,清除病原物感染和肿瘤;近年来的研究还发现Gp96具有免疫佐剂的功能。以下从Gp96的生物学特性、免疫学机制以及其在抗病原感染和抗肿瘤免疫中的应用等方面做一小结,为设计以Gp96-抗原肽为新一代疫苗的临床研究提供理论基础。     

联合使用低剂量环磷酰胺有效增强热休克蛋白gp96免疫佐剂功能

前期研究发现热休克蛋白gp96作为分子伴侣,能特异结合乙肝病毒(HBV)表位肽,并将结合的多肽交叉呈递给MHCⅠ类分子,从而激活病毒特异性CD8+ T细胞(CTL).在此基础上,研究BALB/c小鼠模型联合低剂量环磷酰胺(cyclophosphamide,CTX)对热休克蛋白gp96免疫佐剂功能的影响.以gp96或其N端片段(N355)与H-2Kd限制的乙肝病毒核心蛋白表位HBcAg87-95作为佐剂联合低剂量CTX免疫BALB/c小鼠.联合使用低剂量CTX的试验组CD8+T细胞、IFN-γ+CD8+T细胞、乙肝抗原特异T细胞比例显著高于未使用CTX的对照组(P＜0.05),且联合使用CTX的试验组CD4+CD25+调节性T细胞(T Regulatory Cells ,Treg)比例显著低于单独使用gp96和N355试验组(P＜0.05),说明低剂量的CTX能特异性抑制Treg从而导致T细胞的增加.以上研究表明联合使用低剂量的环磷酰胺能有效增强gp96免疫佐剂功能,这为进一步优化gp96佐剂疫苗的使用提供了依据.     

IL-27:保持免疫平衡的重要因子

由EBI-3和p28组成的IL-27是IL-12家族的新成员,主要来源于树突状细胞(dendritic cells,DCs)的分泌。当DCs表面的Toll样受体(Toll like receptor,TLR)受到外界信号刺激的时候,通过激活下游因子MyD88、IRF3、c-Rel以及JNK等通路介导IL-27表达。IL-27受体由WSX-1和gp130两个亚基组成,表达于多种免疫细胞和非免疫细胞表面。IL-27对这些细胞的发育、分化和功能都发挥着不可或缺的作用。IL-27与其受体结合后,通过激活下游的STAT1/STAT3途径诱导na?veT细胞向Th1方向分化,同时抑制其向Th2、Th17和Foxp3+Treg方向分化。B细胞抗体类型的转换、DCs表面共刺激分子的表达和促进Th1反应的能力也受到IL-27的调节。另外,IL-27还可以诱导一些非免疫细胞的表面表达MHC分子,使其具有抗原呈递的功能。更具有临床意义的是,IL-27在许多感染性疾病、自身免疫病和肿瘤中都发挥了重要作用,是相关疾病潜在的作用靶点。     

小鼠热休克蛋白gp96羧基端片段的克隆、表达及纯化

热休克蛋白(Heat shock protein)gp96(Grp94)是近年来新发现的一类糖蛋白,除了分子伴侣的功能外,现有越来越多的献报道了它在先天性免疫和获得性免疫中的重要作用。gp96可以促进抗原呈递细胞的成熟以及细胞因子的分泌。热休克蛋白抗原肽复合体可以引起特异性的细胞毒T淋巴细胞效应,应用这个特点可以设计抗病毒及抗肿瘤药物。但是gp96全长分子量大,蛋白在大肠杆菌中表达量低,不稳定,难纯化。组织提取的gp96又受组织来源和样品量的限制。对gp96的结构和功能的研究带来困难。克隆并表达了小鼠热休克蛋白gp96的羧基端560．751aa约四分之一长的功能片段,该段包含gp96的一个肽结合区和二聚化位点。将该功能片段在大肠杆菌中进行融合表达,纯化后将融合的片段切掉,并对目的片段进行了分析,结果表明该段可能是形成二聚体密切相关的片段,为进一步研究其结构和功能打下基础。     

以T细胞受体为基础的免疫疗法研究进展

T细胞是机体抗肿瘤免疫的核心,以T细胞功能调控为基础的免疫检查点疗法已经在多种肿瘤的临床治疗中取得了重大突破,以基因工程化T细胞为基础的过继性免疫细胞疗法在血液瘤治疗中取得了重要进展,免疫治疗已经对肿瘤的临床治疗产生了深刻变革,成为肿瘤临床治疗策略的重要组成部分。T细胞受体（T cell receptor,TCR）赋予了T细胞识别肿瘤抗原的特异性,能够识别由主要组织相容性复合体（major histocompatibility complex,MHC）呈递的包括胞内抗原在内的广泛肿瘤抗原,具有高度的抗原敏感性,因而具有广泛的抗肿瘤应用前景。2022年第一款TCR药物的上市开启了TCR药物开发的新纪元,多项TCR药物临床研究表现出潜在的肿瘤治疗价值。本文综述了以TCR为基础的免疫治疗策略研究进展,包括T细胞受体工程化T细胞（T cell receptor-engineered T cell,TCR-T）和TCR蛋白药物,以及基于TCR信号的其他免疫细胞疗法,以期为以TCR为基础的免疫治疗策略开发提供参考。     

Toll样受体通路调节Tregs功能的研究进展

模式识别受体Toll样受体(Toll like receptors,TLRs)是固有免疫中免疫受体的代表,进化上十分保守,对生物体的生存极为重要。TLRs通过内源或外源的配体启动信号转导,激活下游一系列重要的基因表达与活化。研究表明调节性T细胞(Regulatory T cell,Treg)在维持机体外周免疫耐受和阻止移植排斥反应等方面发挥核心作用。Treg细胞表达某些TLRs,包括TLR2、TLR4、TLR5、TLR7、TLR8、TLR9等。TLRs的活化可能直接或间接地影响(主要是活化) Treg的增殖和免疫抑制功能,这种调节与感染、自身免疫病和癌症的发生密切相关。其中热休克蛋白作为TLRs配体分子对于Treg的调节发挥了重要的作用。因此,了解TLRs通路对研究Treg免疫调控机制、新药物研发和靶向治疗有重大意义。文中简要介绍了TLRs通路调节Treg免疫功能的相关研究进展。     

Dendritic cells pulsed with gp96-peptide complexes derived from human hepatocellular carcinoma (HCC) induce specific cytotoxic T lymphocytes

Dendritic cells (DCs) are one of the most potent antigen-presenting cells (APCs) capable of activating immune responses. Different forms of tumor antigens have been used to load DCs to initiate tumor-specific immune responses. Heat shock proteins (HSPs) are considered natural adjuvants which have the ability to chaperone peptides associated with them presented efficiently by interaction with professional APCs through specific receptors. In the present study, we used HSP, gp96-peptide complexes, derived from human hepatocellular carcinoma (HCC) cells as antigens for pulsing DCs. We found that gp96-peptide complexes derived from HCC cells induced the maturation of DCs by enhancing expression of human leukocyte antigen class II, CD80, CD86, CD40, and CD83. The matured DCs stimulated a high level of autologous T cell proliferation and induced HCC specific cytotoxic T lymphocytes, which specifically killed HCC cells by a major histocompatability complex (MHC) class I restricted mechanism. These findings demonstrate that DCs pulsed with gp96-peptide complexes derived from HCC cells are effective in activating specific T cell responses against HCC cells.     

Evolution of the immunomodulatory role of the heat shock protein gp96.

In mammals, certain heat shock proteins (hsps) participate in specialized responses to stressors associated with pathogens or tumors, and as such, act as agents of immune surveillance interacting with both innate and adaptive immunity. We are investigating the conservation of this role throughout the class of vertebrates. We have shown that in Xenopus as in mammals, gp96, the major resident of the endoplasmic reticulum, generates MHC-restricted thymus-dependent immunity in vivo and CR in vitro against minor histocompatibility (H) antigens. By as yet unclear mechanisms that may involve classical MHC-unrestricted cytotoxic CD8+ T cells, gp96 also elicits peptide-specific responses against MHC-class I-negative tumors in adult frogs that may involve cytotoxic NK, MHC-unrestricted CD8+ T and NK/T cells. In naturally MHC class I-deficient but immunocompetent Xenopus larvae, gp96 also generates an innate type of anti-tumor response that is independent of chaperoned peptides. Finally, in a subset of Xenopus sIgM+ B cells, a substantial fraction of gp96 is directed to the cell surface by an active process that is upregulated by bacterial stimulation. This may allow gp96 to access the extracellular compartment without necrosis. Given the dual abilities of gp96 to chaperone antigenic peptides and to modulate innate immune responses, we propose that stimulated B cells that are up-regulating surface gp96 can directly interact with antigen presenting cells (APC) and/or T helper (Th) cells to trigger or amplify immune responses.     

Cell surface expression of the endoplasmic reticular heat shock protein gp96 is phylogenetically conserved.

In mammals, the heat shock protein gp96 complexed to antigenic peptides elicits T cell adaptive immunity. By itself, however, gp96 can evoke responses that are characteristic of innate immunity. Interestingly, this protein, which resides in the endoplasmic reticulum, is expressed on the surface of certain mouse tumor cells. Given that heat shock proteins are highly conserved, we investigated whether the cell surface expression of gp96 is also evolutionarily conserved. Our data reveal that gp96, most likely containing the endoplasmic reticulum retention motif (KDEL), is expressed on the surface of three different Xenopus lymphoid tumor cell lines, each derived from a different spontaneously arising thymic tumor. Levels of expression differ among the tumor lines tested, with more immunogenic tumors expressing greater amounts of surface gp96. Moreover, a high level of gp96 surface expression is detectable on a subset of Xenopus normal nontransformed splenic lymphocytes (mainly surface IgM+ B cells) but not on other normal cells, including macrophages and nucleated erythrocytes. Surface expression of a gp96 protein homologue occurs also on some, but not all, T and B cell clones derived from peripheral blood cells of the channel catfish, as well as on lymphocyte-like cells, but not on erythrocytes from the hagfish, a primitive agnathan vertebrate lacking markers of an adaptive immune system. gp96 is actively directed to and retained on the plasma membrane of Xenopus lymphocytes and tumor cells and hagfish lymphocyte-like cells by a process that requires vesicular transport. This selective surface expression of gp96 on some immune cells from different vertebrate classes is consistent with an ancestral immunological role of gp96 as danger-signaling molecule.     

热休克蛋白gp96作为抗原载体的研究进展

gp96是存在于真核生物细胞内质网中的分子量约为96kD的热休克蛋白(又称GRP94)。它属于HSP90家族,是胞质HSP90的旁系同源蛋白。研究证实从小鼠肿瘤组织中分离的gp96注射小鼠后,可使小鼠获得针对该肿瘤细胞的特异细胞免疫力。随后发现这种特异性免疫不是由gp96引起,而是由其结合的小肽诱发。gp96受体的发现给这种现象的解释提供了线索。人们提出了多种假说来解释这种现象,其中一些得到了广泛的支持。另外,gp96还参与免疫调节过程。完全了解gp96在免疫系统中的作用机制对开发新型药物如肿瘤和病毒感染治疗性疫苗具有重要意义 。     

Testing the role of gp96 as peptide chaperone in antigen processing

gp96 is a 96-kDa glycoprotein of the endoplasmic reticulum that is believed to be involved in antigen processing as an intermediate carrier of peptides for presentation by major histocompatibility complex (MHC) class I molecules. This function implies that gp96 carries a large array of different peptides that represent the antigenicity of the cell and can serve all MHC class I molecules. So far, the evidence regarding these peptides is largely indirect and based on experiments where mice immunized with gp96 from tumor or virus-infected cells developed T cellular immune responses with the corresponding specificities. We analyzed by mass spectrometry peptides isolated from gp96 and found a number of different peptides derived from the proteins of different cellular compartments but mostly cytoplasm and nucleus. The sequences of these peptides provide information on the specificity of antigen processing and reveal structural requirements for binding to gp96 that only partially correspond to those of peptides presented by MHC class I molecules. The yield of peptides extracted from gp96 was far substoichiometric with an estimated occupancy of this chaperone of between 0.1% and 0.4%. These results strongly argue against a regular role for gp96 as a peptide chaperone in antigen processing.     

Association of peptides with heat shock protein gp96 occurs in vivo and not after cell lysis.

Immunization of mice and rats with gp96 preparations isolated from syngeneic cancers has been shown to elicit protective immunity to a number of cancers. The specific immunogenicity of gp96 preparations derives from the antigenic peptides chaperoned by the gp96 molecule and not from gp96 molecules per se. Studies reported here demonstrate that the association of peptides with gp96 occurs in vivo and is not a procedural artifact which occurs in vitro after cell lysis. This demonstration has a bearing on the proposed functional role of HSP peptide association in antigen processing and presentation by MHC I molecules.     

The heat shock protein gp96: a receptor-targeted cross-priming carrier and activator of dendritic cells

Heat shock proteins like gp96 (grp94) are able to induce specific cytotoxic T-cell (CTL) responses against cells from which they originate and are currently studied in clinical trials for use in immunotherapy of tumors. We have recently demonstrated that gp96 binds to at least one yet unidentified receptor restricted to antigen-presenting cells (APCs) like dendritic cells (DCs) but not to T cells. Moreover we have shown, that for CTL activation by gp96-chaperoned peptides receptor-mediated uptake of gp96 by APCs is required. Lately, we have discovered a second function of gp96 when interacting with professional APCs. Gp96 is able to mediate maturation of DCs as determined by upregulation of MHC class II, CD86 and CD83 molecules, secretion of pro-inflammatory cytokines IL-12 and TNF-alpha and enhanced T-cell simulatory capacity. Furthermore, the gp96 receptor(s) are down-regulated on mature DCs, suggesting that the gp96 receptor(s) behave similar to other endocytic receptors like CD36, mannose receptor etc. Our findings now provide additional evidence for the remarkable immunogenicity of gp96: first, the existence of specific gp96 receptors on APCs and second, the capacity to activate dendritic cells which is strictly required to enable these highly sophisticated APCs to prime CTL responses.     

Binding of antigenic peptide to the endoplasmic reticulum-resident protein gp96/GRP94 heat shock chaperone occurs in higher order complexes. Essential role of some aromatic amino acid residues in the peptide-binding site

Vaccination with heat shock protein gp96-antigenic peptide complexes produces a powerful specific immune response against cancers and infectious diseases in some experimental animal models, and gp96-peptide complexes are now being tested in human clinical trials. gp96 appears to serve as a natural adjuvant for chaperoning antigenic peptides into the immune surveillance pathways. A fundamental issue that needs to be addressed is the mechanism of binding of antigenic peptide to gp96. Here, we show using scanning transmission electron microscopy that recombinant gp96 binds peptide in stable multimeric complexes, which may have biological significance. To open the possibility for genetically engineering gp96 for improved immunogenicity and to understand if molecular recognition plays a role in the binding of antigenic peptide, we mutagenized some specific aromatic amino acids in the presumed peptide-binding pocket. Replacement of Tyr-667 or Tyr-678 to Ala reduced affinity for peptide whereas conversion of Trp-654 to Tyr increased peptide binding. Similarly, changing Trp-621 to Phe or Leu or Ala or Ile negatively affected peptide binding whereas changing Trp-621 to Tyr or Val positively affected peptide binding. Probing the peptide microenvironment in gp96-peptide complexes, suggested that hydrophobic interactions (and perhaps hydrogen bonding/stacking interactions) may play a role in peptide loading by gp96.     

Molecular and cellular requirements for enhanced antigen cross-presentation to CD8 cytotoxic T lymphocytes

MHC class I-mediated cross-priming of CD8 T cells by APCs is critical for CTL-based immunity to viral infections and tumors. We have shown previously that tumor-secreted heat shock protein gp96-chaperoned peptides cross prime CD8 CTL that are specific for genuine tumor Ags and for the surrogate Ag OVA. We now show that tumor-secreted heat shock protein gp96-chaperoned peptides enhance the efficiency of Ag cross-priming of CD8 CTL by several million-fold over the cross-priming activity of unchaperoned protein alone. Gp96 also acts as adjuvant for cross-priming by unchaperoned proteins, but in this capacity gp96 is 1000-fold less active than as a peptide chaperone. Mechanistically, the in situ secretion of gp96-Ig by transfected tumor cells recruits and activates dendritic cells and NK cells to the site of gp96 release and promotes CD8 CTL expansion locally. Gp96-mediated cross-priming of CD8 T cells requires B7.1/2 costimulation but proceeds unimpeded in lymph node-deficient mice, in the absence of NKT and CD4 cells and without CD40L. Gp96-driven MHC I cross-priming of CD8 CTL in the absence of lymph nodes provides a novel mechanism for local, tissue-based CTL generation at the site of gp96 release. This pathway may constitute a critically important, early detection, and rapid response mechanism that is operative in parenchymal tissues for effective defense against tissue damaging antigenic agents.