Transfer of tumor specific immunity with “immune” RNA: prospects for the treatment of human cancer

Summary Ribonucleic acids extracted from specifically sensitized lymphoid cells (I-RNA) have been shown to transfer specific immunoreactivity to normal non-immune lymphoid cells. Evidence for the transfer by I-RNA, of immune responses to tumor-associated antigens of animal and human neoplasms, in vivo and in vitro, is reviewed. Results obtained in our laboratory and in other laboratories indicate that xenogeneic, allogeneic and syngeneic I-RNA extracts mediate specific cytotoxicity to tumor cells, in vitro, and mediate transplantation resistance and tumor rejection responses in vivo. Our results suggest that I-RNA preparations fail to elicit immune responses directed against self antigens. By contrast, I-RNA's directed against non-self tumor-associated antigens appear to induce lymphocytes to effect specific anti-tumor immune responses. The mechanisms responsible for the failure of I-RNA to initiate immune responses against self antigens are not known at present and demand investigation.Preliminary results of a clinical Phase I trial of immunotherapy with xenogeneic I-RNA in selected cancer patients are reviewed. I-RNA might offer promise as a new modality for the immunotherapy of human cancer.Recipient of a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft     

Enhancement of anti-tumor immunity through local modulation of CTLA-4 and GITR by dendritic cells

Cancer vaccines have now demonstrated clinical efficacy, but immune modulatory mechanisms that prevent autoimmunity limit their effectiveness. Systemic administration of mAbs targeting the immune modulatory receptors CTLA-4 and glucocorticoid-induced TNFR-related protein (GITR) on Treg and effector T cells augments anti-tumor immunity both experimentally and clinically, but can induce life-threatening autoimmunity. We hypothesized that local delivery of anti-CTLA-4 and anti-GITR mAbs to the sites where T cells and tumor antigen-loaded DC vaccines interact would enhance the induction of anti-tumor immunity while avoiding autoimmunity. To achieve this goal, DCs transfected with mRNA encoding the H and L chains of anti-mouse CTLA-4 and GITR mAbs were co-administered with tumor antigen mRNA-transfected DCs. We observed enhanced induction of anti-tumor immunity and significantly improved survival in melanoma-bearing mice, without signs of autoimmunity. Using in vitro assays with human DCs, we demonstrated that DCs transfected with mRNA encoding a humanized anti-CTLA-4 mAb and mRNA encoding a soluble human GITR-L fusion protein enhance the induction of anti-tumor CTLs in response to DCs transfected with mRNAs encoding either melanoma or breast cancer antigens. Based on these results, this approach of using local delivery of immune modulators to enhance vaccine-induced immunity is currently being evaluated in a phase I clinical cancer immunotherapy trial.     

DC/C6融合瘤苗防治C6胶质瘤的实验研究

目的：探讨DC/C6融合瘤苗防治C6胶质瘤的疗效及作用机制。方法：采用PEG化学融合方法制备融合瘤苗，应用GFAP-FITC免疫荧光检查进行瘤苗的鉴定；立体定向制备大鼠颅内C6肿瘤模型，于种瘤后5 d经尾静脉注射107融合瘤细胞、107DC以及100 μL PBS，分设为A、B、C 3组，采用Log-rank对数秩检验进行生存分析，并行肿瘤标本HE染色及抗CD8Mcab免疫组化染色。结果： 融合瘤苗GFAP-FITC免疫荧光检查阳性；Log-rank生存分析对数据进行对数秩检验，结果表明A组与B、C组进行比较均有统计学意义（P<0.01）；A组晚期死亡大鼠（>31 d）HE染色见较多的炎性细胞浸润，CD8Mcab免疫组化染色阳性。结论：DC/C6融合瘤苗能够有效的发挥抗原提呈、活化T淋巴细胞的功能，CD8+T细胞参与抗胶质瘤免疫反应。     

Generation of an effective anti-tumor immunity after immunization with xenogeneic antigens

Central and peripheral tolerance mechanisms are expected to hamper the generation of effective immunity against tumors. To break self tolerance against malignant gliomas, we assessed the therapeutic potential of self/foreign antigen cross-reactivity in an immunocompetent rat glioma model. Immunotherapy of tumors using xenogeneic human glioma membrane proteins (HGP) as a vaccine inhibited tumor growth, whereas no significant effect was obtained with rat glioma membrane proteins (RGP). In contrast to RGP, HGP elicited a specific IgG immune response that cross-reacted with RGP. This immune response was found to be mainly a Th1 type response. On tumor sections stained with hematoxylin and eosin, glioma cells are sparse and apoptotic in HGP-immunized rats, whereas control tumors showed condensed and viable cells. Tumor-specific CTL were induced in HGP-immunized rats. Immunohistochemical analysis revealed that a significant number of CD8(+) and CD4(+) cells infiltrated into tumors from HGP-vaccinated rats, whereas RGP vaccination led to only few tumor-infiltrating T cells. Taken together, the data establish the in vivo applicability of the cross-stimulation between self and foreign antigens as an alternative way to break tolerance against the poorly immunogenic gliomas.     

The role of dendritic cells in cancer and anti-tumor immunity

Dendritic cells (DC) are key sentinels of the host immune response with an important role in linking innate and adaptive immunity and maintaining tolerance. There is increasing recognition that DC are critical determinants of initiating and sustaining effective T-cell-mediated anti-tumor immune responses. Recent progress in immuno-oncology has led to the evolving insight that the presence and function of DC within the tumor microenvironment (TME) may dictate efficacy of cancer immunotherapies as well as conventional cancer therapies, including immune checkpoint blockade, radiotherapy and chemotherapy. As such, improved understanding of dendritic cell immunobiology specifically focusing on their role in T-cell priming, migration into tissues and TME, and the coordinated in vivo responses of functionally specialized DC subsets will facilitate a better mechanistic understanding of how tumor-immune surveillance can be leveraged to improve patient outcomes and to develop novel DC-targeted therapeutic approaches.     

蛋白表达的亚细胞定位影响基因免疫诱导的体液免疫应答水平的研究

目的研究表达产物的不同亚细胞定位对基因免疫诱导的体液免疫应答水平的影响,为基因疫苗的设计提供参考。方法利用分子克隆技术,分别构建能表达不同细胞定位的EGFP-HPVl6L1融合蛋白和截短型EGFP-HPV16L1△NLS融合蛋白的重组pcDNA-EGFP-HPV16L1和pcDNA-EGFPHPV16L1△NLS真核表达载体;通过转染CHO细胞,并在倒置荧光显微镜下观察表达产物的细胞定位;用重组pcDNA载体免疫BALB/c小鼠;EGFP作为抗原,ELISA法检测血清抗体吸光度。结果①重组pcDNA-EGFP-HPV16L1转染的CHO细胞核内可见到绿色荧光,pcDNA-EGFP-HPV16L1△NLS真核表达载体转染的CHO细胞质内可见到绿色荧光:②两种不同的重组pcDNA载体均可诱导BALB/c小鼠体液免疫反应,但重组pcDNA-EGFP-HPV16L1△NLS真核表达载体免疫组小鼠IgG抗体A_(450)值显著高于重组pcDNAEGFP-HPV16L1真核表达载体免疫组小鼠IgG抗体A_(450)值(P0.001)。结论表达产物的不同细胞定位可影响基因免疫诱发的体液免疫应答水平,定位于细胞质内的蛋白分子较定位于细胞核的蛋白分子能诱导机体更强的体液免疫反应,这为以增强体液免疫反应为目的的基因疫苗的设计提供了参考。     

RNA干扰对胶质瘤细胞垂体瘤转化基因影响的实验研究

目的构建表达靶向抑制PTTG基因的表达短发夹结构(shRNA)的RNA干扰载体。方法根据PTTG基因cDNA序列,设计针对PTTG基因的3组干扰序列,并设计1组阴性对照序列,克隆到真核表达载体pGenesil-1中,进行酶切鉴定和测序鉴定。将构建好的4组重组质粒分别命名为PTTG-1组,PTTG-2组,PTTG-3组、阴性对照NC(negativecontrol)组。利用RNA干扰技术,将以上shRNA分别转染细胞中,逆转录聚合酶链式反应(RT-PCR)检测PTTGmRNA表达水平、Western blot检测PTTG蛋白表达水平。结果构建的PTTG RNA干扰序列和阴性对照序列经基因测序证明序列正确。细胞在转染PTTG干扰载体后,提取细胞的总RNA,经RT-PCR电泳发现,在转染24 h后,PTTG-2、PTTG-3即显示出抑制作用,PTTGmRNA表达水平降低,PTTG-1组对目的基因的抑制作用不明显;在转染48 h后,各组均显示出抑制作用,但PTTG-3的抑制作用更明显;转染72 h后,PTTG-1组有微弱的抑制作用,PTTG-2及PTTG-3组仍有显示明显的抑制作用,但PTTG-3抑制作用较PTTG-2更强烈。Western blot检测不同时间PTTG蛋白的变化显示,在转染24 h后,PTTG-1、PTTG-2、PTTG-3组对目的基因的抑制作用均不明显;在转染48 h后,PTTG-1、PTTG-2、PTTG-3组均显示出抑制作用,但PTTG-3的抑制作用更明显;转染h小时后,PTTG-1组没有抑制作用,PTTG-2及PTTG-3组仍有显示明显的抑制作用,但PTTG-3抑制作用较PTTG-2更强烈。结论成功构建了靶向抑制PTTG基因的表达短发夹结构(shRNA)的RNA干扰载体,并筛选了干扰效果最强的序列,为进一步实验打下了基础。     

RNA干扰对老年性痴呆症的治疗机制研究

阿尔茨海默病 (Alzheimer’sdisease ,AD)即老年性痴呆症 ,是一种病因不明的原发性脑退行性疾病 ,是老年人的多发病和常见病 ,占全部痴呆症的 2 /3,已经成为人类的第四号杀手。临床表现为进行性脑功能减退 ,包括近、远期记忆障碍、分析判断力下降、情绪改变、行为异常、晚期意识模糊和智力逐渐丧失 ,最终生活不能自理。脑内病理特征为神经元缺失、细胞外老年斑 (senileplaque ,SP)和细胞内神经原纤维缠结 (neurofibrillarytangles ,NFT)。这些病理改变主要发生于基底前脑、海马和大脑皮质。Aβ沉积还可导致胆碱能神经细胞损害 ,胆碱乙酰…     

Characterization of a novel single-stranded RNA mycovirus in Pleurotus ostreatus

A mycovirus, named oyster mushroom spherical virus (OMSV), was isolated from cultivated oyster mushrooms with a severe epidemic of oyster mushroom Die-back disease. OMSV was a 27-nm spherical virus encapsidating a single-stranded RNA (ssRNA) of 5.784 kb with a coat protein of approximately 28.5 kDa. The nucleotide sequence of the virus revealed that its genomic RNA was positive strand, containing 5784 bases with seven open reading frames (ORF). ORF1 had the motifs of RNA-dependent RNA polymerases (RdRp) and helicase. ORF2 encoded a coat protein. ORF3 to 7 could encode putative polypeptides of approximately 12, 12.5, 21, 14.5, and 23 kDa, respectively, but none of them showed significant similarity to any other known polypeptides. The 5' end of the viral RNA was uncapped and the 3' end was polyadenylated with 74 bases. Genomic structure and organization and the derived amino acid sequence of RdRp and helicase domain were similar to those of tymoviruses, a plant virus group.     

Modification of anti-tumor immunity by tolerogenic dendritic cells

Immunosuppressive functions of glucocorticoids (GC) can be mediated via various mechanisms, including the modulation of dendritic cells (DC). Our study investigates the effects of tolerogenic GC-treated DCs on NK and T cell anti-tumor responses in OT-1/Rag^?/? mice, expressing a transgenic TCR in CD8⁺ T cells. The effects caused by GC-treated DCs were compared to the responses to immunogenic, CpG-activated DCs. The effects of DCs on anti-tumor immune responses were analyzed using the EG7 tumor model, where the tumor cells express the peptide epitope recognized by OT-1 T cells. We observed that immunization with CpG and peptide-treated DCs protected against tumor growth by activation of NK cell response. Also, immunogenic DCs induced the expansion of cytotoxic CD8⁺OT-1 cells, expressing activation markers CD44 and CD69 and producing IFNγ. In contrast, the peptide and GC-treated DCs in OT-1 mice increased the numbers of immature Mac-1⁺CD27^? NK cells as well as Foxp3⁺ and IL-10 secreting CD8⁺OT-1 cells with suppressive properties. We conclude that the generation of tolerogenic DCs is one of many immunosuppressive mechanisms that can be induced by GC. Our study demonstrated that tolerogenic DCs modify anti-tumor immune response by suppressing NK cell activity and stimulating the formation of IL-10-secreting CD8⁺ Tregs.     

PD-1 silencing improves anti-tumor activities of human mesothelin-targeted CAR T cells

Chimeric antigen receptor T (CAR T) cell therapy is a new pillar in cancer therapeutics, and has been successfully used for the treatment of cancers, including acute lymphoblastic leukemia and solid cancers. Following immune attack, many tumors upregulate inhibitory ligands which bind to inhibitory receptors on T cells. For example, the interaction between programmed cell death protein 1 (PD-1) on activated T cells and its ligands (widely known as PD-L1) on a target tumor limits the efficacy of CAR T cells therapy against poorly responding tumors. Here, we use mesothelin (MSLN)-expressing human ovarian cancer cells (SKOV3) and human colon cancer cells (HCT116) to investigate whether PD-1–mediated T cell exhaustion affects the anti-tumor activity of MSLN-targeted CAR T cells. We utilized cell-intrinsic PD-1-targeting shRNA overexpression strategy, resulting in a significant PD-1 silencing in CAR T cells. The reduction of PD-1 expression on T cell surface strongly augmented CAR T cell cytokine production and cytotoxicity towards PD-L1-expressing cancer cells in vitro. This study indicates the enhanced anti-tumor efficacy of PD-1-silencing MSLN-targeted CAR T cells against several cancers and suggests the potential of other specific gene silencing on the immune checkpoints to enhance the CAR T cell therapies against human tumors.     

Combination of lentivector immunization and low-dose chemotherapy or PD-1/PD-L1 blocking primes self-reactive T cells and induces anti-tumor immunity

In the last two decades, anti-cancer vaccines have yielded disappointing clinical results despite the fact that high numbers of self/tumor-specific T cells can be elicited in immunized patients. Understanding the reasons behind this lack of efficacy is critical in order to design better treatment regimes. Recombinant lentivectors (rLVs) have been successfully used to induce antigen-specific T cells to foreign or mutated tumor antigens. Here, we show that rLV expressing a murine nonmutated self/tumor antigen efficiently primes large numbers of self/tumor-specific CD8(+) T cells. In spite of the large number of tumor-specific T cells, however, no anti-tumor activity could be measured in a therapeutic setting, in mice vaccinated with rLV. Accumulating evidence shows that, in the presence of malignancies, inhibition of T-cell activity may predominate overstimulation. Analysis of tumor-infiltrating lymphocytes revealed that specific anti-tumor CD8(+) T cells fail to produce cytokines and express high levels of inhibitory receptors such as programmed death (PD)-1. Association of active immunization with chemotherapy or antibodies that block inhibitory pathways often leads to better anti-tumor effects. We show here that combining rLV vaccination with either cyclophosphamide or PD-1 and PD-L1 blocking antibodies enhances rLV vaccination efficacy and improves anti-tumor immunity.     

RNA is taking its Toll: Impact of RNA‐specific Toll‐like receptors on health and disease

RNA‐sensing Toll‐like receptors (TLRs) are often described as antiviral receptors of the innate immune system. However, the past decade has shown that the function and relevance of these receptors are far more complex. They were found to be essential for the detection of various bacterial, archaeal, and eukaryotic microorganisms and facilitate the discrimination between dead and living microbes. The cytokine and interferon response profile that is triggered has the potential to improve the efficacy of next‐generation vaccines and may prevent the development of asthma and allergy. Nevertheless, the ability to recognize foreign RNA comes with a cost as also damaged host cells can release nucleic acids that might induce an inappropriate immune response. Thus, it is not surprising that RNA‐sensing TLRs play a key role in various autoimmune diseases. However, promising new inhibitors and antagonists are on the horizon to improve their treatment.     

Induction of adaptive immunity by flagellin does not require robust activation of innate immunity

The ability of TLR agonists to promote adaptive immune responses is attributed to their ability to robustly activate innate immunity. However, it has been observed that, for adjuvants in actual use in research and vaccination, TLR signaling is dispensable for generating humoral immunity. Here, we examined the role of TLR5 and MyD88 in promoting innate and humoral immunity to flagellin using a prime/boost immunization regimen. We observed that eliminating TLR5 greatly reduced flagellin‐induced cytokine production, except for IL‐18, and ablated DC maturation but did not significantly impact flagellin's ability to promote humoral immunity. Elimination of MyD88, which will ablate signaling through TLR and IL‐1β/IL‐18 generated by Nod‐like receptors, reduced, but did not eliminate flagellin's promotion of humoral immunity. In contrast, loss of the innate immune receptor for profilin‐like protein (PLP), TLR11, greatly reduced the ability of PLP to elicit humoral immunity. Together, these results indicate that, firstly, the degree of innate immune activation induced by TLR agonists may be in great excess of that needed to promote humoral immunity and, secondly, there is considerable redundancy in mechanisms that promote the humoral immune response upon innate immune recognition of flagellin. Thus, it should be possible to design innate immune activators that are highly effective vaccine adjuvants yet avoid the adverse events associated with systemic TLR activation.     

热休克蛋白gp96-肽复合物诱导的抗肿瘤免疫

目的: 用不同种类肿瘤提取的热休克蛋白 (HSP)gp96 肽复合物纯化后免疫动物,观察其诱导抗肿瘤免疫的效应,并初步探讨其机制。方法: 从肿瘤细胞中制备HSPgp96 肽复合物, 观察其免疫诱导作用。用不同剂量及不同来源的gp96 肽复合物免疫 6组小鼠后, 用H22癌细胞攻击, 观察各组小鼠的肿瘤发生率、瘤重及瘤组织的形态学变化并与对照组相比较。观察gp96 肽复合物对小鼠腹腔巨噬细胞分泌一氧化氮 (NO)和杀瘤活性的影响。结果: 获得的纯化HSPgp96 肽复合物的Mr为 96 000。gp96 肽复合物的免疫效果与其剂量有关, 以 18μg/只的效果最明显。交叉免疫实验证明, 免疫小鼠能抵抗同种肿瘤细胞的攻击, 而对异种肿瘤细胞的则无免疫保护作用。gp96 肽复合物可刺激小鼠腹腔巨噬细胞分泌NO同时杀瘤活性增高。结论: 从肿瘤细胞中分离纯化的HSPgp96 肽复合物免疫小鼠后, 可获得特异性的抗肿瘤作用; 其对肿瘤细胞的杀伤作用与gp96 肽复合物能增强小鼠腹腔巨噬细胞分泌NO增多有关。     

Nucleic acid agonists for Toll-like receptor 7 are defined by the presence of uridine ribonucleotides

Toll-like receptor 7 (TLR7) mediates innate responses by responding to viral RNA in endocytic compartments. However, the molecular pattern recognised by TLR7 and whether it differs between RNA of viral and self origin remains unclear. Here, we identify nucleic acids that act as TLR7 agonists for mouse and human cells. We show that uridine and ribose, the two defining features of RNA, are both necessary and sufficient for TLR7 stimulation, and that short single-stranded RNA (ssRNA) act as TLR7 agonists in a sequence-independent manner as long as they contain several uridines in close proximity. Consistent with the notion that TLR7 lacks specificity for sequence motifs, we show that it is triggered equally efficiently by viral or self RNA delivered to endosomes. Our results support the notion that TLR7 recognises uracil repeats in RNA and that it discriminates between viral and self ligands on the basis of endosomal accessibility rather than sequence.     

Production of therapeutic iduronate-2-sulfatase enzyme with a novel single-stranded RNA virus vector

The Sendai virus vector has received a lot of attention due to its broad tropism for mammalian cells. As a result of efforts for genetic studies based on a mutant virus, we can now express more than 10 genes of up to 13.5 kilo nucleotides in a single vector with high protein expression efficiency. To prove this benefit, we examined the efficacy of the novel ribonucleic acid (RNA) virus vector harboring the human iduronate-2-sulfatase (IDS) gene with 1,653 base pairs, a causative gene for mucopolysaccharidosis type II, also known as a disorder of lysosomal storage disorders. As expected, this novel RNA vector with the human IDS gene exhibited its marked expression as determined by the expression of enhanced green fluorescent protein and IDS enzyme activity. While these cells exhibited a normal growth rate, the BHK-21 transformant cells stably expressing the human IDS gene persistently generated an active human IDS enzyme extracellularly. The human IDS protein produced failed to be incorporated into the lysosome when cells were pretreated with mannose-6-phosphate, demonstrating that this human IDS enzyme has potential for therapeutic use by cross-correction. These results suggest that our novel RNA vector may be applicable for further clinical settings.     

RNA干扰沉默Aurora A基因表达对胶质瘤细胞增殖及凋亡的影响

目的 探讨RNA干扰技术抑制人胶质瘤U251细胞中Aurora A基因的表达,研究其对U251细胞增殖及凋亡的影响。 方法 设计并合成特异性针对Aurora A基因的siRNA,转染至U251细胞中,采用RT-PCR和Western blotting检测Aurora A mRNA和蛋白的表达情况,同时利用四甲基偶氮唑盐(MTT)实验和流式细胞仪观察转染后U251细胞增殖抑制率和细胞凋亡,透射电镜观察细胞凋亡超微结构改变。 结果 转染Aurora A siRNA后,U251细胞Aurora A mRNA表达受到抑制(P<0.01),蛋白表达水平降低;U251细胞增殖的抑制率和细胞凋亡率显著增高 (P<0.01),且U251细胞发生了显著的凋亡形态改变。 结论 体外合成的针对Aurora A基因的特异性siRNA成功地抑制了U251细胞中Aurora A基因的表达,并能抑制胶质瘤细胞增殖、促进凋亡,提示Aurora A可能成为胶质瘤基因治疗的新靶点。     

Inhibition of Viruses by RNA Interference

RNA-mediated interference (RNAi) is a recently discovered process by which dsRNA is able to silence specific gene functions. Although initially described in plants, nematodes and Drosophila, the process is currently considered to be an evolutionarily conserved process that is present in the entire eukaryotic kingdom in which its original function was as a defense mechanism against viruses and foreign nucleic acids. Similarly to the silencing of genes by RNAi, viral functions can be also silenced by the same mechanism, through the introduction of specific dsRNA molecules into cells, where they are targeted to essential genes or directly to the viral genome in case RNA viruses, thus arresting viral replication. Since the pioneering work of Elbashir and coworkers, who identified RNAi activity in mammalian cells, many publications have described the inhibition of viruses belonging to most if not all viral families, by targeting and silencing diverse viral genes as well as cell genes that are essential for virus replication. Moreover, virus expression vectors were developed and used as vehicles with which to deliver siRNAs into cells. This review will describe the use of RNAi to inhibit virus replication directly, as well as through the silencing of the appropriate cell functions.