Dendritic cell-based vaccines suppress metastatic liver tumor via activation of local innate and acquired immunity

Background  Dendritic cell (DC)-based vaccines have been applied clinically in the setting of cancer, but tumor-associated antigens (TAAs) have not yet been enough identified in various cancers. In this study, we investigated whether preventive vaccination with unpulsed DCs or peptide-pulsed DCs could offer anti-tumor effects against MC38 or BL6 liver tumors. Methods  Mice were subcutaneously (s.c.) immunized with unpulsed DCs or the recently defined TAA EphA2 derived peptide-pulsed dendritic cells (Eph-DCs) to treat EphA2-positive MC38 and EphA2-negative BL6 liver tumors. Liver mononuclear cells (LMNCs) from treated mice were subjected to ⁵¹Cr release assays against YAC-1 target cells. In some experiments, mice were injected with anti-CD8, anti-CD4 or anti-asialo GM1 antibody to deplete each lymphocyte subsets. Results  Immunization with unpulsed DCs displayed comparable efficacy against both MC38 and BL6 liver tumors when compared with Eph-DCs. Both DC-based vaccines significantly augmented the cytotoxicity of LMNCs against YAC-1 cells. In vivo antibody depletion studies revealed that NK cells, as well as, CD4+ and CD8+ T cells play critical roles in the anti-tumor efficacy associated with either DC-based modality. Tumor-specific cytotoxic T lymphocyte (CTL) activity was generally higher if mice had received Eph-DCs versus unpulsed DCs. Importantly, the mice that had been protected from MC38 liver tumor by either unpulsed DCs or Eph-DCs became resistant to s.c. MC38 rechallenge, but not to BL6 rechallenge. Conclusions  These results demonstrate that unpulsed DC vaccines might serve as an effective therapy for treating metastatic liver tumor, for which TAA has not yet been identified. Shinjiro Yamaguchi and Tomohide Tatsumi contributed equally to this work.     

Contribution of calumin to embryogenesis through participation in the endoplasmic reticulum-associated degradation activity

Calumin is an endoplasmic reticulum (ER)-transmembrane protein, and little is known about its physiological roles. Here we showed that calumin homozygous mutant embryos die at embryonic days (E) 10.5−11.5. At mid-gestation, calumin was expressed predominantly in the yolk sac. Apoptosis was enhanced in calumin homozygous mutant yolk sacs at E9.5, pointing to a possible link to the embryonic lethality. Calumin co-immunoprecipitated with ERAD components such as p97, BIP, derlin-1, derlin-2 and VIMP, suggesting its involvement in ERAD. Indeed, calumin knockdown in HEK 293 cells resulted in ERAD being less efficient, as demonstrated by attenuation in both degradations of a misfolded α1-antitrypsin variant and the ER-to-cytosol dislocation of cholera toxin A1 subunit. In calumin homozygous mutant yolk sac endoderm cells, ER stress-associated alterations were observed, including lipid droplet accumulation, fragmentation of the ER and dissociation of ribosomes from the ER. In this context, the ER-overload response, assumed to be cytoprotective, was also triggered in the mutant endoderm cells, but seemed to fully counteract the excessive ER stress generated due to defective ERAD. Taken together, our findings suggested that calumin serves to maintain the yolk sac integrity through participation in the ERAD activity, contributing to embryonic development.     

钙网蛋白与肿瘤免疫

钙网蛋白是内质网分子伴侣,负责糖蛋白的折叠及维持细胞内Ca2＋平衡。新近研究发现,某些凋亡刺激能诱导肿瘤细胞内的钙网蛋白快速转位到肿瘤细胞膜上,这种表面包被有大量钙网蛋白的肿瘤细胞,能被抗原呈递细胞有效识别和吞噬,由此激发抗同种肿瘤的特异性免疫杀伤效应,提示钙网蛋白在肿瘤免疫治疗中的潜在应用价值。对钙网蛋白在肿瘤免疫中的研究进展作一综述。     

Reflux of Endoplasmic Reticulum proteins to the cytosol inactivates tumor suppressors

In the past decades, many studies reported the presence of endoplasmic reticulum (ER)‐resident proteins in the cytosol. However, the mechanisms by which these proteins relocate and whether they exert cytosolic functions remain unknown. We find that a subset of ER luminal proteins accumulates in the cytosol of glioblastoma cells isolated from mouse and human tumors. In cultured cells, ER protein reflux to the cytosol occurs upon ER proteostasis perturbation. Using the ER luminal protein anterior gradient 2 (AGR2) as a proof of concept, we tested whether the refluxed proteins gain new functions in the cytosol. We find that refluxed, cytosolic AGR2 binds and inhibits the tumor suppressor p53. These data suggest that ER reflux constitutes an ER surveillance mechanism to relieve the ER from its contents upon stress, providing a selective advantage to tumor cells through gain‐of‐cytosolic functions—a phenomenon we name ER to Cytosol Signaling (ERCYS).     

microRNAs在肿瘤免疫中的调控作用

microRNAs（miRNAs）是一类转录后调控基因表达的内源性非编码微小RNA。愈来愈多的研究显示,miRNAs在肿瘤免疫应答中发挥重要调控作用。一方面,miRNAs通过转录后调控ICAM（intercellular adhesion molecule）、B7（CD80／86）和HLA—G（human leucocyte antigen—G）等肿瘤表面分子的表达,影响肿瘤的免疫原性;另一方面,miRNAs通过平衡肿瘤局部的细胞因子微环境或调控肿瘤免疫相关细胞的分化、发育及功能发挥,调节机体抗肿瘤免疫应答。为后续深入研究肿瘤与宿主的相互作用机制,以及发展更有效的肿瘤生物治疗手段,就目前miRNAs在肿瘤免疫中的调控作用的研究进展做一综述。     

Novel functions of herbal medicines in dendritic cells: role of Amomi Semen in tumor immunity

Dendritic cells (DCs) have a major role in regulating immune responses, including tumor immunity and peripheral tolerance. In the present study, we identified novel functions of herbal medicines in DCs by screening 99 herbal medicines, most of which are among the 210 Chinese medicines approved by the Ministry of Health, Labour, and Welfare, Japan. Ethanol extracts were prepared, and a murine epidermal-derived Langerhans cell line, XS106, was used to screen the 99 extracts by analyzing major histocompatibility complex (MHC) class II expression. Amomi Semen (amomum seed), Polyporus (polyporus sclerotium), and Plantaginis Semen (plantago seed) potently activated XS106 and were selected for further analysis. The effects of these extracts on bone marrow-derived DCs (BM-DCs) generated in vitro were then analyzed using surface phenotype (MHC class II, CD80, and CD86) and interleukin (IL)-12p70 production as indicators. BM-DCs treated with Amomi Semen extract exhibited activated phenotypes and secreted IL-12p70. The activation level was similar to that induced by lipopolysaccharides. Finally, an E.G7-OVA tumor model (E.L4-OVA transfectant) was used to examine the anti-tumor effects of Amomi Semen extract. Vaccination of mice with a subcutaneous injection of BM-DCs treated with Amomi Semen extract and OVA peptide significantly inhibited the growth of tumor cells and prolonged survival time compared to controls. Furthermore, therapeutic effects were observed on established tumors. The inhibition rates for both the prophylactic and therapeutic protocols were comparable to those of lipopolysaccharides. These results indicate that Amomi Semen extract potently activate DCs and is potentially useful for DC vaccination.     

细胞外ATP与肿瘤免疫

ATP是最重要的胞内代谢产物之一,也是一种重要的信号分子。研究发现,某些凋亡刺激能诱导肿瘤细胞内ATP释放到细胞外,这种释放到细胞外的ATP能促进吞噬细胞对凋亡细胞的吞噬,由此激发特异性抗肿瘤免疫杀伤效应,提示细胞外ATP在肿瘤免疫治疗中的潜在应用价值。本文就细胞外ATP在肿瘤免疫中的研究进展作一综述。     

Exosome DNA: Critical regulator of tumor immunity and a diagnostic biomarker

Nanosized cellular vesicles “exosome” contains a variety of biological cargo including DNA fragments from cell-of-origin. Despite its biological stability and clinical utility in tumor diagnosis, exosome DNA (ExoDNA) is very little studied as compare with exosome RNA. Cytoplasmic accumulation of damaged DNA from nucleus and mitochondria often leads to its packaging in exosomes by yet unknown pathways. ExoDNA modulates tumor immunity via paracrine interactions and activation of cytosolic DNA sensor pathways, for example, STING, cGAS, and so forth in specific immune cell subsets. In addition to priming tumor immunity, ExoDNA is also emerging as a critical regulator of check-point immunotherapy. As a useful diagnostic biomaterial, ExoDNA contains a variety of clinically relevant tumor-specific mutations representing multiple genes (e.g., EGFR, BRAF, RAS, IDH, and HER2), thus making it a promising “liquid biopsy” material for therapy recommendations. Hence, ExoDNA in addition to tumor immunity modulation, is also emerging as a suitable diagnostic material for personalized therapy in cancer. Here, we review the current status of ExoDNA research and its potential uses in tumor biology.     

Organ-specific pancreatic tumor growth properties and tumor immunity

We established a model of orthotopic injection of a syngeneic pancreatic tumor cell line in C57BL/6 mice and evaluated the effects of organ site on induction of immunity to a tumor-specific antigen, MUC1. Mice were challenged with a syngeneic pancreatic adenocarcinoma cell line that expressed MUC1 (Panc02-MUC1) by orthotopic injection into the pancreas, or by subcutaneous injection. Tumor cells injected into the pancreas grew much faster than those injected subcutaneously. Mice challenged subcutaneously with Panc02-MUC1 rejected tumors or developed slowly growing tumors that were negative for MUC1 expression. In contrast, mice challenged orthotopically into the pancreas developed progressive tumors that were positive for MUC1 expression. Sera from mice that rejected Panc02-MUC1 (tumor-immune mice) showed no detectable IgG1 and IgM titers against the MUC1 tandem-repeat peptide, whereas mice with progressive tumor growth had significant titers of IgG1 and IgM specific for MUC1. This suggests that the humoral immune response was ineffective in mediating tumor rejection. The results show that the growth properties and immunological rejection of pancreatic tumors is affected by the organ site at which the tumor grows. Received: 25 April 1998 / Accepted: 7 October 1998     

The RB tumor suppressor at the intersection of proliferation and immunity: relevance to disease immune evasion and immunotherapy

The retinoblastoma tumor suppressor (RB) was the first identified tumor suppressor based on germline predisposition to the pediatric eye tumor. Since these early studies, it has become apparent that the functional inactivation of RB is a common event in nearly all human malignancy. A great deal of research has gone into understanding how the loss of RB promotes tumor etiology and progression. Since malignant tumors are characterized by aberrant cell division, much of this research has focused upon the ability of RB to regulate the cell cycle by repression of proliferation-related genes. However, it is progressively understood that RB is an important mediator of multiple functions. One area that is gaining progressive interest is the emerging role for RB in regulating diverse features of immune function. These findings suggest that RB is more than simply a regulator of cellular proliferation; it is at the crossroads of proliferation and the immune response. Here we review the data related to the functional roles of RB on the immune system, relevance to immune evasion, and potential significance to the response to immune-therapy.     

肿瘤相关成纤维细胞与成纤维细胞活化蛋白在肿瘤免疫治疗中的研究进展

除了依赖于肿瘤细胞自身的恶性增殖以外,肿瘤的发生和发展还依赖于肿瘤细胞与肿瘤间质微环境的相互作用。肿瘤间质中存在的肿瘤相关成纤维细胞（tumor-associatedfibroblasts,TAF）能够诱导免疫抑制,是肿瘤免疫治疗中的一大障碍。在TAF上存在一种成纤维细胞激活蛋白（fibroblast activationprotein,FAP）,它在细胞表面发挥作用,是一种膜丝氨酸肽酶,是Ⅱ型丝氨酸蛋白酶家族成员之一,具有二肽肽酶及胶原酶活性,在肿瘤微环境中表达FAP的肿瘤相关成纤维细胞是最早被鉴定的一种肿瘤间质细胞类型。它由肿瘤问质中的成纤维细胞与癌细胞相互作用而活化,是肿瘤微环境中最主要的宿主细胞,具有促进肿瘤细胞生长、侵袭及免疫抑制的作用,而且基因组稳定不易耐药,有望成为肿瘤免疫治疗的新靶标。就靶向TAF和FAP在肿瘤免疫治疗中的研究做一综述,为基于肿瘤间质微环境的免疫治疗提供参考。     

Integrated regulation of autophagy and apoptosis by EEF2K controls cellular fate and modulates the efficacy of curcumin and velcade against tumor cells

Endoplasmic reticulum (ER) stress induces both autophagy and apoptosis yet the molecular mechanisms and pathways underlying the regulation of these two cellular processes in cells undergoing ER stress remain less clear. We report here that eukaryotic elongation factor-2 kinase (EEF2K) is a critical controller of the ER stress-induced autophagy and apoptosis in tumor cells. DDIT4, a stress-induced protein, was required for transducing the signal for activation of EEF2K under ER stress. We further showed that phosphorylation of EEF2K at Ser398 was essential for induction of autophagy, while phosphorylation of the kinase at Ser366 and Ser78 exerted an inhibitory effect on autophagy. Suppression of the ER stress-activated autophagy via silencing of EEF2K aggravated ER stress and promoted apoptotic cell death in tumor cells. Moreover, inhibiting EEF2K by either RNAi or NH125, a small molecule inhibitor of the enzyme, rendered tumor cells more sensitive to curcumin and velcade, two anticancer agents that possess ER stress-inducing action. Our study indicated that the DDIT4-EEF2K pathway was essential for inducing autophagy and for determining the fate of tumor cells under ER stress, and suggested that inhibiting the EEF2K-mediated autophagy can deteriorate ER stress and lead to a greater apoptotic response, thereby potentiating the efficacy of the ER stress-inducing agents against cancer.     

Arabidopsis OTU1, a linkage‐specific deubiquitinase,is required for endoplasmic reticulum‐associated protein degradation

Endoplasmic reticulum (ER)‐associated degradation (ERAD) is part of the ER protein quality‐control system (ERQC), which is critical for the conformation fidelity of most secretory and membrane proteins in eukaryotic organisms. ERAD is thought to operate in plants with core machineries highly conserved to those in human and yeast; however, little is known about the plant ERAD system. Here we report the characterization of a close homolog of human OTUB1 in Arabidopsis thaliana, designated as AtOTU1. AtOTU1 selectively hydrolyzes several types of ubiquitin chains and these activities depend on its conserved protease domain and/or the unique N‐terminus. The otu1 null mutant is sensitive to high salinity stress, and particularly agents that cause protein misfolding. It turns out that AtOTU1 is required for the processing of known plant ERAD substrates such as barley powdery mildew O (MLO) alleles by virtue of its association with the CDC48 complex through its N‐terminal region. These observations collectively define AtOTU1 as an OTU domain‐containing deubiquitinase involved in Arabidopsis ERAD.     

A new autophagy-related checkpoint in the degradation of an ERAD-M target

The endoplasmic reticulum (ER) harbors elaborate quality control mechanisms to ensure proper folding and post-translational modifications of polypeptides targeted to this organelle. Once an aberrant protein is detected, it is dislocated from the ER and routed to the proteasome for destruction. Autophagy has been recently implicated in the elevation of the ER stress response; however, the involvement of this pathway in selective removal of ER-associated degradation (ERAD) substrates has not been demonstrated. In the present study, we show that an ER membrane lesion, associated with the accumulation of the yeast ERAD-M substrate 6Myc-Hmg2p elicits the recruitment of Atg8 and elements of the cytosol to vacuole targeting (CVT) to the membrane, leading to attenuation in the degradation process. Deletion of peptide:N-glycanase (PNG1) stabilizes this association, a process accompanied by slowdown of 6Myc-Hmg2p degradation. Truncation of the unstructured C-terminal 23 amino acids of 6Myc-Hmg2p rendered its degradation PNG1-independent and allowed its partial delivery to the vacuole in an autophagy-dependent manner. These findings demonstrate a new conduit for the selective vacuolar/lysosomal removal of ERAD misfolded proteins by an autophagy-related machinery acting concomitantly with the proteasome.     

Role of type 2 innate lymphoid cell and its related cytokines in tumor immunity

Type 2 innate lymphoid cells (ILC2s) have multiple functions that can respond to allergic diseases, parasite infection, metabolic homeostasis, tissue repair, and adipose metabolism homeostasis. In these diseases, ILC2s can be activated by various inflammatory cytokines released by damaged cells. Activated ILC2s produce different type 2 cytokines, including interleukin (IL)-4, IL-5, IL-9, and IL-13, which involved in the pathogenesis of many diseases. In recent years, the relationship between ILC2s and tumor diseases has attracted more and more attention. The role of ILC2s in tumor immunity depends on its surface molecules and cytokine context. This review aims to conclude tumorigenic and antitumorigenic roles of ILC2s, and the characters of ILC2s-related cytokines in tumor diseases to provide a comprehensive overview of the impact of ILC2s in tumor immunity.     

Protein S-palmitoylation in immunity

S-palmitoylation is a reversible posttranslational lipid modification of proteins. It controls protein activity, stability, trafficking and protein–protein interactions. Recent global profiling of immune cells and targeted analysis have identified many S-palmitoylated immunity-associated proteins. Here, we review S-palmitoylated immune receptors and effectors, and their dynamic regulation at cellular membranes to generate specific and balanced immune responses. We also highlight how this understanding can drive therapeutic advances to pharmacologically modulate immune responses.     

The role of dendritic cells in tumor microenvironments and their uses as therapeutic targets

Dendritic cells (DC), which consist of several different subsets, specialize in antigen presentation and are critical for mediating the innate and adaptive immune responses. DC subsets can be classified into conventional, plasmacytoid, and monocyte-derived DC in the tumor microenvironment, and each subset plays a different role. Because of the role of intratumoral DCs in initiating antitumor immune responses with tumor-derived antigen presentation to T cells, DCs have been targeted in the treatment of cancer. By regulating the functionality of DCs, several DC-based immunotherapies have been developed, including administration of tumor-derived antigens and DC vaccines. In addition, DCs participate in the mechanisms of classical cancer therapies, such as radiation therapy and chemotherapy. Thus, regulating DCs is also important in improving current cancer therapies. Here, we will discuss the role of each DC subset in antitumor immune responses, and the current status of DC-related cancer therapies.     

A dual regulatory role of Arabidopsis calreticulin-2 in plant innate immunity

Calreticulin (CRT) is an endoplasmic reticulum-resident calcium-binding molecular chaperone that is highly conserved in multi-cellular eukaryotes. Higher plants contain two distinct groups of CRTs: CRT1/CRT2 and CRT3 isoforms. Previous studies have shown that bacterial elongation factor Tu receptor (EFR), a pattern-recognition receptor that is responsible for pathogen-associated molecular pattern-triggered immunity, is a substrate for Arabidopsis CRT3, suggesting a role for CRT3 in regulating plant defense against pathogens. Here we report that Arabidopsis CRT2 is another regulator of plant innate immunity. Despite significantly increased salicylic acid levels and constitutive expression of the systemic acquired resistance-associated marker genes PR1, PR2 and PR5, transgenic plants over-expressing CRT2 displayed reduced resistance to virulent Pseudomonas syringae pv. tomato DC3000 (PstDC3000). A (45)Ca(2+) overlay assay and a domain-swapping experiment further demonstrated that the negatively charged C-terminal tail of CRT2 is responsible for its high calcium-binding capacity and function in regulating the endogenous salicylic acid level. In addition, over-expression of the His173 mutant of CRT2 greatly enhanced plant defense against PstDC3000, supporting the existence of a self-inhibition mechanism that can counteract the effects of salicylic acid-dependent immune responses. These results suggest that CRT2 functions through its N-terminal domain(s) as a self-modulator that can possibly prevent the salicylic acid-mediated runaway defense responses triggered by its C-terminal calcium-buffering activity in response to pathogen invasion.     

Ablation of T cell immunity differentially influences tumor risk in inbred BD rat strains

Inbred rat strains BDIX and BDIV are constitutionally susceptible and resistant, respectively, to the development of malignant peripheral nerve sheath tumors (MPNST) induced by neonatal exposure to N-ethyl-N-nitrosourea (EtNU). They represent a model system for analysis of molecular and cellular processes underlying differential cancer susceptibility. A point mutation in the Neu/ErbB-2 gene is an early marker of Schwann precursor cells at high risk of malignant conversion and is diagnostic of the resulting MPNST predominantly developing in the trigeminal nerves. Initially considerable amounts of Neu/ErbB-2-mutant cells arise in nerve tissue of both rat strains subsequently disappearing in resistant BDIV rats, but persisting and giving rise to MPNST in susceptible BDIX animals. An almost identical cellular immune response—sequentially involving macrophages, T helper- and cytotoxic T lymphocytes—is mounted in the trigeminal nerves of EtNU-treated rats of both strains. In this study, T cell maturation was prevented by neonatal thymectomy following EtNU-exposure. While resistance against MPNST development significantly decreased in BDIV rats MPNST incidence and survival time remained unaltered in thymectomized BDIX rats. Contrary to euthymic animals a number of both thymectomized BDIV and BDIX rats developed MPNST lacking the Neu/ErbB-2-mutation. This suggests that Schwann cells initiated by other genetic alterations can progress to full malignancy in immune-compromised rats only. T cell-dependent resistance against tumorigenesis originating from non-Neu/ErbB-2-mutant Schwann precursors might thus be shared by both strains while BDIV T lymphocytes additionally prevent the development of Neu/ErbB-2-mutant MPNST. Rat strain-specific differences in the interaction of T lymphocytes with (pre)malignant Neu-mutant cells may thus critically contribute to susceptibility and resistance towards EtNU-induced MPNST development.     

Antigen loading of DCs with irradiated apoptotic tumor cells induces improved anti-tumor immunity compared to other approaches

Dendritic cells (DCs) serve as central regulators of adaptive immunity by presenting antigens and providing necessary co-signals. Environmental information received by the DCs determines the co-signals delivered to the responding adaptive cells and, ultimately, the outcome of the interaction. DCs loaded with relevant antigens have been used as therapeutic cellular vaccines, but the optimal antigen loading method has not been determined. We compared different methods to load class I and class II epitopes from the male antigenic complex, HY, onto DCs for the potency of the immune response induced in vivo. Co-incubation of female DCs with HY peptides, RNA or cell lysate from HY expressing tumor induced immune responses equivalent to male DCs. In contrast, female DCs incubated with irradiated, apoptotic HY expressing tumor cells (or male B cells) generated a stronger immune response than male DCs or female DCs loaded using any of the other methods. DC loading with apoptotic tumor resulted in complete protection against high dose HY-expressing tumor challenge whereas 100% lethality was observed in groups receiving DCs that were loaded with peptides, RNA, or lysate. We conclude that signals provided to the DCs by apoptotic cells substantially augment the potency of DC vaccines.