Mice heterozygous for a deletion of the tumor necrosis factor-α and lymphotoxin-α genes: biological importance of a nonlinear response of tumor necrosis factor-α to gene dosage

The tumor necrosis factors (TNF-α and lymphotoxin, or LT-α) are important mediators of the immune and inflammatory responses, and it has been proposed that a positive feedback loop could boost the expression of the TNF to sufficiently high levels to fend off infections. To investigate this phenomenon and its biological consequences, we have generated LT-α/TNF-α knockout mice and compared mice having one or two functional LT-α/TNF-α alleles. In response to lipopolysaccharide (LPS) stimulation, TNF-α levels in the circulation or in the supernatant of macrophage cultures were 20- to 100-fold lower in heterozygous samples than in their wild-type counterparts. This differential increased with the intensity of stimulation and throughout the response, supporting the involvement of a positive feedback loop. Moreover, the heterozygous mice had an increased bacterial load following Listeria monocytogenes infection and exhibited a bimodal response to the association of D -galactosamine and LPS which was similar to that of wild-type mice at low doses of LPS and more like that of homozygous mutants at high doses. These results therefore establish the biological importance of the nonlinear response of TNF-α levels to gene dosage, and these mice provide a unique tool to study how the propensity to produce TNF can determine the immunological fitness of individuals.     

T细胞亚群在肿瘤微环境中的作用

根据T细胞亚群的不同,T细胞在肿瘤免疫中起着不同的重要作用.其能够对肿瘤组织产生不同的影响,进而引起肿瘤免疫的复杂性、双向性.因而深入了解肿瘤微环境中各T细胞亚群的功能及相互作用,能够对肿瘤的治疗提供重要依据.因此,对T细胞不同亚群在肿瘤中的作用进行研究具有重要意义.     

鼻咽癌肿瘤微环境中的免疫逃逸机制

鼻咽癌(NPC)是我国东南部地区常见的头颈部肿瘤,传统放化疗手段对晚期患者的疗效有限,特异性免疫治疗是目前临床探索的新方向.肿瘤细胞通过多因素多机制逃避机体免疫系统的监控打击在肿瘤的发生发展中发挥了重要作用,肿瘤微环境中的免疫逃逸机制为恶性肿瘤侵袭、转移、治疗抵抗及复发提供了有利条件.了解NPC肿瘤微环境中的免疫逃逸机制,有助于寻找肿瘤免疫逃逸机制中的有效治疗靶点,开发相关免疫治疗药物.     

Wnt16b基因扩增影响细胞增殖及肿瘤微环境的研究进展

Wnt蛋白家族是一类在进化上高度保守的家族,与胚胎发育、器官分化相关,在细胞的生长、增殖、分化及凋亡等生物学行为方面发挥重要的调控作用.研究显示,Wnt信号通路紊乱与许多疾病尤其是肿瘤密切相关.Wnt16b作为Wnt蛋白家族的一员,在骨骼的生长分化过程中起关键作用.近年研究发现,Wnt16b基因在多种肿瘤组织中高扩增,提示Wnt16b基因可能在肿瘤的发展、迁移、化疗耐药等方面起重要作用.本文重点就Wnt16b基因扩增与细胞增殖及肿瘤微环境的关联研究进行综述.     

Interleukin (IL)-24 transforms the tumor microenvironment and induces anticancer immunity in a murine model of colon cancer

Interleukin-24 (IL-24) is a novel tumor suppressor and can mediate the induction of Th1-type cytokines from peripheral blood mononuclear cells. The individual properties of IL-24 have been previously examined; however, its in vivo immunological consequences and antitumor properties have not been previously evaluated with respect to colon cancer, the most commonly diagnosed cancer in China. Thus, we evaluated whether IL-24 could inhibit the progression of colon cancer in murine models with intact immune competence and explored the mechanisms underlying the immunological effects of IL-24 on colon cancer progression in vivo. In these murine models, we found that IL-24 promoted CD4⁺ T cells and CD8⁺ T cells to secrete interferon gamma and enhanced the cytotoxicity of CD8⁺ T cells in vivo. More importantly, we demonstrated that IL-24 transformed the tumor microenvironment and enhanced antitumor effects in favor of tumor eradication. Additionally, IL-24 expression correlated inversely with the clinical stage of human colorectal cancer. Thus, our study establishes a role of IL-24 in promoting antitumor immune responses and supports the development of a novel cytokine immunotherapy against colon cancer.     

Promoting angiogenesis within the tumor microenvironment: The secret life of murine lymphoid IL‐17‐producing γδ T cells

After two decades in the shadow of their αβ counterparts, γδ T cells have recently gathered significant attention following the discovery that they produce IL‐17 in various mouse models of infection and autoimmune disease. In contrast, the secretion of large amounts of IFN‐γ by γδ T cells has long been known, and has been tightly linked to their anti‐tumor function. In this issue of the European Journal of Immunology, a study unexpectedly reports that the lymphoid γδ T cells that infiltrate tumor foci induced in the mouse skin produce very little IFN‐γ, but abundant IL‐17. In fact, these γδ T cells are the major source of IL‐17 within the tumor microenvironment, where they appear to promote angiogenesis, and thus tumor growth. This Commentary discusses the relevance of these interesting findings in the context of the currently paradoxical pro‐ versus anti‐tumor roles of IL‐17 in cancer immunology.     

人类肿瘤表达细胞因子基因的临床意义

目的:探讨IL-2,IL-4,IL-6,IL-10,IL-13和IFNγ在肿瘤发生发展及治疗诊断中作用。方法:用RTPCR技术或RNA点杂交技术检测183份肿瘤细胞或肿瘤组织中上述6种细胞因子的基因表达状况。结果:在183份不同的肿瘤细胞或组织中Th2类细胞因子的表达显著增加,其中IL-4,IL-6,IL-10和IL-13的表达率达65％,70.5％,83.6％和61.7％,而Th1类细胞因子IFNγ和IL-2的表达率仅达12.6％和22.9％。结论:Th1/Th2漂移与肿瘤的发生发展密切相关,有可能造成机体的免疫抑制,有利于肿瘤细胞的免疫逃逸。     

Lactic acid in alternative polarization and function of macrophages in tumor microenvironment

In developing tumor, macrophages are one major immune infiltrate that not only contributes in shaping up of tumor microenvironment (TME) but also have the potential of determining the fate of tumor in terms of its progression. Phenotypic plasticity of macrophages primarily channelizes them to alternative (M2) form of tumor associated macrophages (TAM) in the TME. One of the key tumor derived components that plays a crucial role in TAM polarization from M1 to M2 form is lactic acid and has prominent role in progression of malignancy. The role of lactic acid as signalling molecule as well as an immunomodulator has recently been recognized. This review focuses on the mechanism and signalling that are involved in lactic acid induced M2 polarization and possible therapeutic strategies for regulating lactic acidosis in TME.     

肿瘤炎性微环境与树突状细胞研究进展

肿瘤免疫是近年来的研究热点,已知肿瘤微环境尤其是炎性微环境在促进肿瘤发生、发展过程中起重要作用。肿瘤浸润的树突状细胞（DC）多存在表型未成熟化、分布异常及功能障碍等现象,这可能是肿瘤诱导机体免疫耐受的重要机制之一。肿瘤炎性微环境可通过多种途径调节DC的分化和成熟,相关信号通路和分子机制正逐步得到阐明,此将为DC疫苗的研制和肿瘤免疫治疗提供新的希望。     

T cells which do not express membrane tumor necrosis factor-α activate macrophage effector function by cell contact-dependent signaling of macrophage tumor necrosis factor-α production

Previous studies have suggested that T cell contact-dependent signaling of macrophages (MΦ) is mediated by membrane tumor necrosis factor-α (memTNF-α), based on the observation that anti-TNF-α could inhibit T cell-mediated MΦ activation. The current report confirms that anti-TNF-α does inhibit activation of interferon-γ (IFN-γ)-primed MΦ by paraformaldehyde-fixed activated T cells. However, the involvement of membrane molecules other than memTNF-α in the contact-dependent signaling is suggested by two lines of evidence. First, the T_H2 clone, AK8, displayed neither secreted TNF-α/β nor memTNF-α/β detectable by bioassay or immunofluorescence. Nonetheless, AK8 cells were equally effective, on a per cell basis, in contact-dependent signaling of MΦ activation as T_H2 and T_H1 cells which do express memTNF-α. Second, the expression of memTNF-α by the T_H clone, D10.G4, is maximal 24 h after activation, whereas the ability of this clone to activate MΦ is maximal at 6–8 h of activation and declines thereafter. Since TNF-α is known to play a critical role in activation of MΦ effector function, it was hypothesized that T cell membrane components other than memTNF-α might signal MΦ production of TNF-α, thus allowing autocrine TNF-α stimulation of MΦ effector function. In support of this, it is demonstrated that paraformaldehyde-fixed activated T_H2 cells can induce de novo production and release of TNF-α by MΦ. This effect was not an artifactual result of paraformaldehyde fixation since paraformaldehyde-fixed resting T cells did not induce TNF-α gene expression. Previous studies have demonstrated a role for autocrine TNF-α stimulation in LPS induction of effector function in recombinant IFN-γ-primed MΦ. The current study confirms that TNF-α plays a critical role in T cell contact-dependent signaling of MΦ but indicates that memTNF on the T cells may not be a sine qua non factor for contact-dependent signaling. The data suggest that other T cell membrane molecules contribute to activation of MΦ effector function by stimulation of MΦ TNF-α production.     

结肠癌组织中STC1的表达及其与肿瘤微环境的关系

目的:探讨斯钙素1(STC1)在结肠癌组织中的表达,分析STC1与结肠癌预后的相关性,并探讨其与结肠癌肿瘤微环境的联系。方法:用UALCAN数据库(http://ualcan.path.uab.edu/index.html)和Oncomine数据库(https://www. oncomine.org)分析STC1在正常结肠组织及结肠癌组织中的表达水平;RT-qPCR及Western blot检测结肠癌及其癌旁组织中STC1的表达;使用OncoLnc(http://www.oncolnc.org/)分析工具评估STC1表达水平与结肠癌临床预后的相关性。氯化钴缺氧和脂多糖诱导处理结肠癌HT-29细胞,RT-qPCR和Western blot检测细胞STC1的表达水平变化;运用TIMER分析工具(https://cistrome.shinyapps.io/timer/),分析STC1与缺氧诱导因子1α(HIF1α)及免疫调节的相关性。结果:STC1在结肠癌组织中的表达水平显著高于正常结肠组织,并且STC1的高表达与结肠癌的分期和不良预后呈正相关关系(P0.01)。在结肠癌HT-29细胞中,缺氧能够显著诱导STC1表达,STC1的表达水平与HIF1α的水平存在正相关关系;炎症模拟条件下,STC1的表达水平显著升高,STC1与炎症分子的表达和肿瘤组织中免疫细胞的浸润水平存在密切关系。结论:STC1在结肠癌组织中高表达,是结肠癌不良预后的一个重要的分子标志物,并且STC1与结肠癌肿瘤微环境,特别是与结肠癌的缺氧及免疫调节存在密切关系。     

Cytokine stimulation of T lymphocytes regulates their capacity to induce monocyte production of tumor necrosis factor-α, but not interleukin-10: Possible relevance to pathophysiology of rheumatoid arthritis

Previous studies in the laboratory have shown that the pro-inflammatory cytokine tumor necrosis factor (TNF)-α plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA). The mechanisms involved in regulating monocyte/macrophage cytokine production are not yet fully understood, but are thought to involve both soluble factors and cell/cell contact with other cell types. We and others have previously demonstrated that T cells activated through the T cell receptor/CD3 complex induce monocyte TNF-α production by contact-mediated signals. In this report, we investigated further whether T cells activated by cytokines in the absence of T cell receptor stimulation also regulate monocyte cytokine production. T cells were activated in an antigen-independent manner using the cytokines interleukin (IL)-15 or IL-2 alone, or in combination with IL-6 and TNF-α. Subsequently, T cells were fixed and incubated with monocytes. Fixed, cytokine-stimulated T cells induced monocytes to secrete TNF-α in a dose-dependent manner, but did not induce secretion of IL-10, a potent endogenous down-regulator of TNF-α and other pro-inflammatory cytokines. Stimulation of monocyte TNF-α was markedly inhibited when T cells were physically separated from monocytes within the tissue culture well, confirming that T cell contact is necessary. T cell acquisition of monocyte-activating capacity was shown to be dependent on the period of cytokine stimulation, with T cells activated for 8 days more effective than T cells activated for shorter periods. Addition of interferon-γ or granulocyte/macrophage colony-stimulating factor to the T cell/monocyte cultures enhanced T cell induction of monocyte TNF-α by threefold and ninefold, respectively. The results from this model of cognate interaction suggest that cytokine-stimulated T cells, interacting with macrophages in the rheumatoid synovial membrane, may contribute to the continuous excessive production of TNF-α observed in the RA joint, and to the imbalance of pro-inflammatory cytokines over anti-inflammatory cytokines.     

To kill or not to kill – The role of the tumor microenvironment in shaping group 1 ILC functions

Group 1 innate lymphoid cells (ILC) comprise two major IFN-γ producing populations, namely Natural Killer (NK) cells, and ILC1s. Recent studies have revealed a complex and diverse composition of group 1 ILC subsets infiltrating different tumors. In this review, we will outline the commonalities and differences between group 1 ILC subsets in both mice and humans, discuss how the tissue and tumor microenvironment shapes their phenotype and functions, as well as describe their contrasting roles in the response to different cancers.     

NK cells in the tumor microenvironment: Prognostic and theranostic impact. Recent advances and trends

NK cells orchestrate the tumor destruction and control metastasis in a coordinated way with other immune cells of the tumor microenvironment. However, NK cell infiltration in the tumor microenvironment is limited, and tumor cells have developed numerous mechanisms to escape NK cell attack. As a result, NK cells that have been able to infiltrate the tumors are exhausted, and metabolically and functionally impaired. Depending this impairment the prognostic and theranostic values of NK cells differ depending on the studies, the type of cancer, the stage of tumor and the nature of the tumor microenvironment. Extensive studies have been done to investigate different strategies to improve the NK cell function, and nowadays, a battery of therapeutic tools are being tested, with promising results.     

rpoB作为蜡样芽胞杆菌群快速检测的标志基因的实验研究

目的探讨常规PCR和实时荧光定量PCR（Q-PCR）方法检测蜡样芽胞杆菌群rpoB基因的特异性和敏感性。方法提取蜡样芽胞杆菌群和其他各种对照细菌的基因组DNA,合成蜡样芽胞杆菌群rpoB基因扩增引物,采用常规PCR和SYBRgreen实时定量PCR两种方法扩增rpoB基因片段,并将PCR产物克隆到pMD18-T载体后进行DNA测序。结果常规PCR和Q-PCR均能扩增出蜡样芽胞杆菌群rpoB基因的174bpDNA片段,而各种对照菌株均未见扩增。序列比对发现蜡样芽胞杆菌群细菌在该片段中存在5处核苷酸的不同,差异率为2.88%。以炭疽芽胞杆菌基因组DNA系列稀释作为扩增模板显示常规PCR最小检出量为3.42pg,Q-PCR的敏感性达到171fg,3次重复实验显示Q-PCR检测rpoB基因的灵敏度为（3.32×101±7.45×100）拷贝。结论以rpoB基因为检测靶基因的Q-PCR方法具有高度的特异性和良好的敏感性,能实现对蜡样芽胞杆菌群快速而准确的检测。     

Somatic mutational profiles of stage II and III gastric cancer according to tumor microenvironment immune type

We aimed to determine somatic mutational profiles of stage II/III gastric cancers (GCs) according to their tumor microenvironment immune types (TMITs), which classify cancer based on co‐assessment of PD‐L1 expression and CD8⁺ tumor infiltrating lymphocytes. Eighty patients with stage II/III GC were classified as follows: TMIT I (PD‐L1⁺/CD8^High), TMIT II (PD‐L1^?/CD8^Low), TMIT III (PD‐L1⁺/CD8^Low), and TMIT IV (PD‐L1^?/CD8^High). Deep targeted sequencing using a panel of 170 cancer‐related genes was performed on an Illumina HiSeq‐2500 system. Most frequently mutated genes included GNAQ (41.3%), TP53 (38.8%), CREBBP (35.0%), and MAP3K1 (35.0%). PIK3CA mutations were observed more frequently in TMIT I (45.8%) and III (66.7%), than in II (12.0%) and IV (8.0%). Other genes with enriched mutations within TMIT I included ATM (33.3%), BRCA2 (33.3%), MAP3K4 (29.2%), and FLT4 (25.0%). FGFR3, MAP3K1, and RUNX1 mutations were more frequently found in TMIT II. TMIT III had a unique somatic mutation profile harboring enriched mutations of histone modifiers including CREBBP and KMT2A, and we found FGFR2 amplification exclusively within TMIT IV. Fuzzy clustering analysis based on somatic mutation frequencies identified a hypermutated group (cluster 1) and a hypomutated group (cluster 2). Cluster 1 had significant associations with TMIT I, EBV⁺ GCs, and MSI‐H GCs (P = .023, .014, and .004), and had better overall survival (P = .057) than Cluster 2. TMIT I, EBV⁺, and MSI‐H GCs were estimated to have greater tumor mutational burden (P = .023, .003, and .015). By analyzing somatic mutation profiles according to TMIT classification, we identified TMIT‐specific genetic alterations that provide clues for biological linkage between GC genetics and microenvironment.     

Expression and cleavage of tumor necrosis factor-α and tumor necrosis factor receptors by human monocytic cell lines upon direct contact with stimulated T cells

Tumor necrosis factor-α (TNF-α) is a potent cytokine in inflammatory processes. A variety of mechanisms that modulate its activity have been described, one being its binding to soluble receptors (sTNFR). In this study, we demonstrate that human monocytic cells such as THP-1 respond to direct contact with a membrane preparation of stimulated HUT-78 cells by producing TNF-α and by releasing sTNFR-p75, but not sTNFR-p55, with different kinetics. TNF-α concentration peaked after 12 h of contact and then decreased, whereas sTNFR-p75 production increased progressively upon cell/cell contact. The decrease in TNF-α concentration is not due to trapping of TNF-α by its soluble receptors or other soluble or cell-associated molecules, but rather to a proteolytic activity associated to THP-1 cells. On the other hand, the increase in sTNFR-p75 release does not result from an increase in the cleavage of pre-existing cell-associated sTNFR-p75 but from an increase in TNFR-p75 expression, immediately followed by the cleavage of its extracellular domain. Phenylmethylsulfonylfluoride, a serine protease inhibitor, has a negative effect on both TNF-α degradation and sTNFR-p75 release by THP-1 cells. Thus, there may be an enzymatic activity associated to THP-1 cells that plays an important role in the neutralization of TNF-α activity both by degrading the molecule and by cleaving its receptors at the cell surface.     

Partial purification and characterization of a tumor necrosis factor-α converting activity

Tumor necrosis factor (TNF)-α is initially synthesized as an extracellular membrane-associated 26-kDa protein that is further cleaved at Ala⁷⁶-Val⁷⁷ to yield the soluble 17-kDa form. Recently, peptide-hydroxamate metallopro-teinase inhibitors have been reported to block the proteolytic processing of TNF-α, thus suggesting that the putative TNF-α converting enzyme (TACE) is a zinc-dependent metalloendopeptidase. In this report, we characterize a TNF-α converting activity (TACA) that cleaves in vitro the human 26-kDa TNF-α at the physiological processing site. The chromatography steps followed for purification and the use of a panel of proteinase inhibitors indicate that the enzyme responsible for TACA is a membrane glycosylated metalloendopeptidase which is most likely different from the matrix-degrading metalloproteinases. The failure of TACA to process a Val⁷⁷→Gly⁷⁷ precursor mutant emphasizes the importance of hydrophobic residue at P1' position. In addition, TACA is not able to cleave the mouse pro-TNF-α and does not catalyze in vitro the processing of other transmembrane proteins susceptible to metalloproteinase-mediated shedding, such as interleukin-6 or TNF receptors. These studies suggest the existence of an enzyme specific for TNF-α within the metalloproteinases involved in the processing/shedding of a number of cytokines and cytokine receptors.