Profiles of immune cell infiltration and their clinical significance in head and neck squamous cell carcinoma

It is well established that there exist comprehensive interactions between tumor immunology and tumor biology. Tumor-infiltrating immune cells (TIICs) have been appealing therapeutic targets due to their significance in regulating cancer progression. The purpose of this study was to illustrate the comprehensive landscape of TIICs composition in head and neck squamous cell carcinoma (HNSCC) and their clinical significance. CIBERSORT was applied to calculate the 22 immune cell types proportion in HNSCC and further analysis suggested that six kinds of immune cells (resting memory CD4 T cells, M1 macrophages, resting dendritic cells, resting mast cells, monocytes, and eosinophils) were closely correlated with HNSCC progression. Moreover, memory CD4 T cells may serve as prognosis indicator for HNSCC patients. Collectively, this study uncovered the immune cells infiltration landscape in HNSCC and illustrated their potential relationships with clinical parameters, thereby contributing to the development of customized treatment strategy.     

Exploration of gene expression profiles and immune microenvironment between high and low tumor mutation burden groups in prostate cancer

BackgroundTumor mutation burden (TMB) has been established as a biomarker for response to immune therapy and prognosis in various cancers. However, the association between TMB and prognosis of prostate cancer (PCa) remains unclear. This study aimed to investigate the impact of TMB in biochemical recurrence (BCR) and the immune microenvironment in high and low TMB groups.MethodsMutation data, gene expression, clinicopathological information were downloaded from The Cancer Genome Atlas (TCGA). Mutation types and TMB values were identified. All samples were divided into high and low TMB groups with median TMB value as the cutoff point. The BCR-free survival rates, Differentially expressed genes (DEGs) and immune cells infiltrations in different TMB groups were identified.ResultsThe most common variant type and SNV were single nucleotide polymorphism and C > T. respectively. High TMB level was significantly associated with older age, positive lymph node, higher International Society of Urological Pathology (ISUP) grade, advanced stage and poor BCR-free survival. 132 DEGs were identified and involved in receptor ligand activity and hormone activity. High expression of six core genes UBE2C, PLK1, CDC20, BUB1, CDK1 and HJURP were associated with worse BCR-free survival. The analysis of immune cells infiltration revealed that the amount of activated CD4⁺ memory T cells was significantly different in high and low TMB groups.ConclusionsThe current study comprehensively described the summary of mutation and TMB related DEGs in PCa. TMB was associated with BCR-free survival and the infiltration of activated CD4⁺ memory T cells in the immune microenvironment.     

Peripheral blood neutrophil granulocytes from patients with head and neck squamous cell carcinoma functionally differ from their counterparts in healthy donors

Solid tumors such as head and neck squamous cell carcinoma (HNSCC) display an intense interaction between tumoral factors and the immune system. Functional modulation of tumor-infiltrating and peripheral blood immune cells plays an important role during tumor progression. In this pilot study we compared biological functions of polymorphonuclear granulocytes (PMN) from the peripheral blood of HNSCC patients and healthy subjects. PMN were simultaneously isolated from the peripheral blood of HNSCC patients and healthy donors for functional analysis (apoptosis, production of reactive oxygen species (ROS), cytokine release and immunophenotyping). PMN from HNSCC patients showed a significantly lower inducible production of ROS (P = 0.02) and reduced spontaneous apoptosis (P= 0.008) compared with PMN from healthy donors. Under standard culture conditions, there was no significant difference regarding the release of inflammatory cytokines between PMN from HNSCC patients and PMN from healthy donors. Confirming previous observations, serum concentrations of PMN-related cytokines were significantly higher in the peripheral blood of HNSCC patients than in that of controls. Importantly, immunophenotyping revealed an increased number of immature PMN in PMN fractions isolated from HNSCC patients. Peripheral blood PMN from HNSCC patients and healthy donors show distinct functional differences. The presence of increased numbers of immature stages of PMN in HNSCC patients may partly contribute to the changes observed. After recruitment to and infiltration of the tumor, PMN may be further modulated in the local tumor microenvironment. This pilot study justifies functional analyses of myeloid cells in larger cohorts of patients with HNSCC.     

NUF2基因表达与肺腺癌免疫浸润及临床预后的相关性分析

目的： 研究NUF2与肺腺癌（lung adenocarcinoma，LUAD）免疫细胞浸润及预后的关系。方法： 采用Oncomine数据库分析NUF2在LUAD和肺鳞癌（lung squamous cell carcinoma，LUSC）组织中的表达水平；用GEPIA和PrognoScan数据库评估NUF2对LUAD和LUSC患者预后的影响；采用TIMER数据库分析LUAD中NUF2表达和免疫细胞浸润水平的相关性；采用LinkedOmics数据库进行NUF2相关差异表达基因的GO功能注释和KEGG通路分析。RT-qPCR和Western Blot检测NUF2在肺腺癌细胞中的表达，并采用集落形成实验研究NUF2对肺腺癌细胞增殖的影响。结果： NUF2在LUAD和LUSC组织中的表达水平显著高于正常组织（P<0.01）。NUF2高表达与LUAD患者的总生存率和无疾病生存率负相关（P<0.05），但与LUSC患者的总生存率无相关性。NUF2表达与LUAD组织中B细胞、CD4⁺ T细胞、巨噬细胞和树突状细胞的浸润水平显著负相关（P<0.01）。NUF2相关基因富集在氧化磷酸化等通路，参与的生物学过程有细胞周期检查点和DNA重组等。NUF2在人肺腺癌细胞中高表达，且敲减NUF2可抑制肺腺癌细胞集落形成（P<0.01）。结论： NUF2高表达与LUAD免疫细胞浸润水平降低和不良预后相关，有望成为LUAD潜在的预后标志物和免疫相关治疗靶点。     

The regulation of CD4+ T cell immune responses toward Th2 cell development by prostaglandin E2

As an important immune mediator, PGE2 plays an important role in the immune tolerance, autoimmune diseases, immune regulation and tumor immunotolerance. PGE2 is considered to be a promising candidate for the control of the immune diseases. To further understand the immuno-modulating effects of PGE2 on CD4+ T cells, in vitro investigation was conducted in the present study. The results showed that PGE2 inhibited the proliferation of T cells in vitro in a dose-dependent manner. Gene expression profiling showed that 1716 genes were down regulated and 73 genes were up regulated with a change of 1.5 fold. Several signal transduction pathways were involved, such as TNF-α and NF-kB signaling pathway, T cell receptor signaling pathway, IL-2 signaling pathway, and MAPK pathway. The results showed that PGE2 inhibited IFN-γ, TNF-α and IL-4 production by CD4+ T cells 24 h after cell culture. A comparison between IFN-γ and IL-4 production showed that PGE2 enhanced the relative ratio of IL-4 to IFN-γ in CD4+ T cells culture, and regulated CD4+ T cells toward Th2 cell development. The results of the present study indicated that PGE2 has the potential to treat Th1-mediated inflammatory diseases by regulating CD4+ T cells toward Th2 cell immune response.     

免疫细胞治疗临床研究及相关产业现状与未来发展趋势

随着医学技术的不断进步和科学研究的不断深入,肿瘤免疫细胞治疗作为一种新的治疗方式受到社会各界的广泛关注,成为辅助肿瘤治疗的一种新手段。过继免疫细胞治疗相继出现了不同类型的细胞——T淋巴细胞、细胞因子诱导的杀伤（CIK）细胞、树突状细胞（DC）-CIK细胞、自然杀伤（NK）细胞和肿瘤浸润性淋巴细胞（TIL）细胞等。日前开展的多种类型的免疫细胞临床研究在肿瘤治疗中已经体现出显著的疗效,肿瘤免疫治疗成为快速发展的研究领域。如何选择肿瘤免疫细胞治疗方法和时机,治疗过程中的质量控制和质量管理应当怎么做,如何得到更有说服力的循证医学证据,是值得深入探索的重要问题。对目前的肿瘤免疫细胞治疗现状和未来5年发展做一概述,初步探讨临床研究中的质量控制和质量管理方向。     

A novel ceRNA axis involves in regulating immune infiltrates and macrophage polarization in gastric cancer

BackgroundIncreasing evidence suggests that the lncRNA-miRNA-mRNA regulatory network is highly correlated with gastric cancer (GC) development. However, a prognosis-associated lncRNA-miRNA-mRNA network remains to be identified in GC.MethodsDifferentially expressed genes (DEGs) were screened by integrating 6 microarray datasets using the RRA method. Hub genes were identified by analysing their degrees in a PPI (protein–protein interaction) network. Upstream miRNAs and lncRNAs of hub genes were predicted by miRTarBase and miRNet, respectively. Key genes, miRNAs and lncRNAs were identified by evaluating their expression and prognosis in GEPIA and Kaplan-Meier plotter, respectively. A key lncRNA-miRNA-mRNA network was constructed in Cytoscape, and the correlations were analysed in the ENCORI database. We also evaluated the mRNA expression of ceRNA axes in the TIMER and Oncomine databases and their correlation with prognosis in GC patients with different clinical features using Kaplan-Meier plotter. In addition, correlations between mRNA and immune infiltrating cells in GC were investigated by the TIMER database. Finally, several experiments were conducted to verify our analyses.ResultsForty-two upregulated and 86 downregulated DEGs were obtained from the “RRA” integrated analysis. Eight of the 20 hub genes were identified as key genes by analysing their expression and prognosis. Seventeen miRNAs were predicted to target key genes, and low expression of 4 miRNAs suggested poor outcome in GC. Furthermore, 155 lncRNAs were predicted to target 4 key miRNAs, and only 5 lncRNAs were highly expressed, suggesting poor outcomes in patients with GC. Then, the H19-miR-29a-3p-COL1A2 axis was constructed by correlation analysis. In addition, COL1A2 was positively correlated with lymphatic metastasis, immune infiltrating cell levels, markers of monocytes, tumour-associated macrophages (TAMs), and M2 macrophages but not M1 macrophages in GC. The experimental results revealed that the H19-miR-29a-3p-COL1A2 axis may promote macrophage polarization from M1 to M2 in GC.ConclusionsA novel lncRNA-miRNA-mRNA axis was identified and may be involved in regulating immune cell infiltration and macrophage polarization, which may provide new treatment strategies for GC.     

胰岛素受体底物1低表达促进头颈部鳞状细胞癌的侵袭转移

目的:研究胰岛素受体底物1(insulin receptor substrate 1,IRS-1)在头颈部鳞状细胞癌( head and neck squamous cell carcinoma,HNSCC)侵袭转移中的作用及其可能的机制.方法:应用定量RT-PCR法检测HNSCC患者淋巴结转移灶和原发灶癌组织中IRS-1 mRNA的表达,以及不同转移能力头颈部肿瘤细胞株上IRS-1 mRNA的表达.用Taqman探针法检测miR-9及U6的相对表达量.利用小干扰RNA技术阻抑IRS-1表达,肿瘤细胞侵袭实验检测IRS-1干扰前后对细胞侵袭能力的影响.用定量RT-PCR和Western blot检测上皮－间叶转化(epithelial-mesenchymal transition,EMT)的标志物E-cadherin及vimentin mRNA和蛋白的表达;用SRB法检测细胞存活率.结果:(1) HNSCC患者淋巴结转移灶中IRS-1 mRNA的表达低于原发灶(P＜0.05);高转移性HNSCC细胞株中IRS-1mRNA的表达低于低转移性细胞株(P＜0.05).(2)下调IRS-1表达,肿瘤细胞的侵袭能力增强,上皮间叶转化标志物E-cadherin表达下降,同时伴有miR-9表达的升高.结论:IRS-1低表达可能通过上皮间叶转化和上调miR-9而增强细胞的侵袭能力,促进HNSCC的转移.     

Lymphocyte expression of EZH2 is associated with mortality and secondary infectious complications in sepsis

Recent studies have shown that epigenetic factors may affect immune responses. We previously reported that histone methyltransferase enhancer of zeste homolog 2 (EZH2) was involved in the innate inflammatory responses both in animal model of sepsis and in septic patients. In this study, we prospectively evaluated EZH2 expression kinetics in peripheral CD4+ and CD8+ T cells and HLA-DR expression in CD14+ cells from 48 patients with sepsis and 48 healthy controls. Results showed higher level of EZH2 in CD4+ T cells and CD8+ T cells in sepsis patients than in controls. Meanwhile, EZH2 expression was correlated with CD27 status on T cells. Mean fluorescence intensity (MFI) of EZH2 in CD8+ T cells on day 1 independently predicted death in septic patients. Also, the combination of CD8+ T cell EZH2 expression with APACHEII and SOFA score could enhance the prognostic predictive ability. Moreover, multivariate logistic regression analysis showed that increased expression (proportion and MFI) of EZH2 in CD4+ and CD8+ lymphocytes on day 3 were independently associated with nosocomial infection in septic patients. Additionally, spearman correlation analysis indicated that the levels of EZH2 in CD4+ T cells and CD8+ T cells correlated to CD14+ cells-expressing HLA-DR in patients with sepsis at each time point. Overall, these findings suggest that EZH2 in CD4+ T cells or/and CD8+ T cells may be a novel biomarker for predicting adverse outcomes (mortality and secondary infectious complications) in patients with sepsis.     

基于GEO和TCGA数据库筛选恶性胸膜间皮瘤免疫治疗疗效预测关键基因

目的 探索免疫治疗在恶性胸膜间皮瘤（malignant pleural mesothelioma,MPM）中免疫疗效相关关键分子及其可能的机制。方法 2018年7月至2020年7月,纳入GEO数据库中GSE117358的表达谱进行分析;首先,根据是否对免疫治疗是否有效分为两组：免疫治疗反应组和免疫治疗无反应组,同时分析两组之间差异有统计学意义基因;其次,分析两组差异有统计学意义基因在基因本体（gene ontology,GO）生物学行为及京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes,KEGG）富集信号通路等方面的差异;最后,对差异有统计学意义基因构建蛋白-蛋白互作网络,筛选在生物学行为中的重要模块及关键基因,并对关键基因在TCGA数据库中进行整合分析验证。结果 在免疫治疗反应组（12个样本）及免疫治疗无反应组（12个样本）中共筛选出1 025个差异基因,相对于免疫治疗无反应组,在免疫治疗反应组中有782个上调和243个下调基因。GO和KEGG分析显示,这些差异基因的功能主要集中在细胞因子受体通路、细胞黏附通路、T细胞受体通路及...     

A robust signature associated with patient prognosis and tumor immune microenvironment based on immune-related genes in lung squamous cell carcinoma

BackgroundLung squamous cell carcinoma (LUSC) is one common type of lung cancer. Immune-related genes (IRGs) are closely associated with cancer prognosis. This study aims to screen the key genes associated with LUSC and establish an immune-related prognostic model.MethodsBased on the Cancer Genome Atlas (TCGA) database, we screened the differentially expressed genes (DEGs) between LUSC and normal samples. Intersecting the DEGs with the immune-related genes (IRGs), we obtained the differentially expressed IRGs (DEIRGs). Univariate as well as multivariate Cox regression analyses were performed to identify the survival-associated IRGs and establish an immune-related prognostic model. The relationship between the prognostic model and tumor-infiltrating immune cells was analyzed by TIMER and CIBERSORT.ResultsA total of 229 DEIRGs were screened, and 14 IRGs associated with survival were identified using univariate Cox analysis. Among the 14 IRGs, six genes were selected out using Lasso and multivariate Cox analyses, and they were used to build the prognostic model. Further analysis indicated that overall survival (OS) of high-risk groups was lower than that of low-risk groups. High risk score was independently related to worse OS. Moreover, the risk score was positively correlated with several immune infiltration cells. Finally, the efficacy of the prognostic model was validated by another independent cohort GSE73403.ConclusionThe DEIRGs described in the study may have the potential to be the prognostic molecular markers for LUSC. In addition, the risk score model could predict the OS and provides more information for the immunotherapy of patients with LUSC.     

Potential stem cell marker CD44 is constitutively expressed in permanent cell lines of head and neck cancer

BACKGROUND: Despite significant advances in the use of diagnosis and therapy to treat head and neck squamous cell carcinoma (HNSCC), the prognosis has improved only marginally in the last decades. Thus, there is an enormous need for better understanding of tumor biology and reversely novel immunotherapeutic approaches. It is becoming increasingly obvious that stem cells play an important role in tumor development and progression. The identity of these cells and the underlying cellular and molecular mechanisms are mostly unknown in HNSCC to date. MATERIALS AND METHODS: Solid HNSCC tumors, as well as permanent HNSCC cell lines, were analyzed by flow cytometry concerning the expression of different putative stem cell marker proteins. RESULTS: Distinct populations of CD44 expressing potential stem cells could be identified in solid tumors of HNSCC patients with strong individual deviations. Surprisingly, the potential stem cell marker CD44 was found to be constitutively expressed on the surface of all the permanent HNSCC cell lines analyzed. CONCLUSION: CD44+ 'tumor stem cells' may play a key role in the establishment of permanent HNSCC cell lines, selecting especially robust cell entities that might drive the progression and metastasis of HNSCC. Individual analysis of 'tumor stem cell' markers will be an important tool for innovative therapies and for determining the prognosis of patients with HNSCC.     

Reduced function of CD4+25+ regulatory T cell fraction in silicosis patients

The quality and quantity of CD4+25+ regulatory T cells (Treg) in silicosis patients (SIL) were examined and compared with results from healthy donors (HD) because SIL often develop autoimmune diseases along with pulmonary disorders. Peripheral blood mononuclear cells from 57 SIL and 50 HD were analyzed for Treg. Treg frequency and clinical parameters were subjected to a factor analysis. Treg and CD4+25- T cells (Tneg) from five HD and five SIL, sorted by flow-cytometer, were used for functional assays of Treg, the expression pattern of Treg specific genes (FoxP3, GITR and CTLA-4) and activation-related genes (CD122 and CD123). Although the actual frequency of Treg did not differ between SIL and HD, the age-corrected level was reduced in SIL. The factor analysis showed that Treg frequency was positively associated with the serum level of IL-2. The inhibitory effect of Treg on Tneg activation was decreased when the Treg:Tneg ratio was 1:1/4 to 1/2. In addition, Treg dominancy of FoxP3 and CTLA-4 expression and Tneg dominancy of CD132 expression found in HD were lost in SIL. These results indicated that the Treg fraction in SIL may be substituted with chronically activated T cells due to recurrent exposure to silica, resulting in a reduction in the frequency and function of Treg. Since the reduction of Treg may precede the clinical manifestation, as silicosis may be a pre-clinical status for autoimmune diseases, control of Treg function using cell and/or gene therapy may be a good way to manage autoimmune disease.     

Identification of the collagen family as prognostic biomarkers and immune-associated targets in gastric cancer

BackgroundGastric cancer has extremely high morbidity and mortality. Currently, it is lack of effective biomarkers and therapeutic targets for guiding clinical treatment. In this study, we aimed to identify novel biomarkers and therapeutic targets for gastric cancer.MethodsDifferentially expressed genes (DEGs) between gastric cancer and normal tissues were obtained from Gene Expression Omnibus (GEO). Core genes were identified by constructing protein-protein interaction network of DEGs. The expression of core genes was verified in Gene Expression Profiling Interactive Analysis (GEPIA), UALCAN and clinical samples. Further, the mutation, DNA methylation, prognostic value, and immune infiltration of core genes were validated by cBioPortal, MethSurv, Kaplan-Meier plotter, and Tumor Immune Estimation Resource (TIMER) databases. Additionally, drug response analysis was performed by Cancer Therapy Response Portal (CTRP).ResultsA total of seven collagen family members were identified as core genes among upregulated genes. And copy number amplification may be involved in the upregulation of COL1A1 and COL1A2. Importantly, the collagen family was associated with the poor prognosis of patients with metastasis. Among them, COL1A1 had a higher hazard ratio (HR) for overall survival than other members (HR = 2.33). The correlation between DNA methylation levels at CpG sites of collagen family members and the prognosis was verified in gastric cancer. Besides, collagen family expression was positively correlated with macrophages infiltration and the expression of M2 macrophages markers. Further, collagen expression was related to the sensitivity and resistance of gastric cancer cell lines to certain drugs.ConclusionsThe collagen family, especially COL1A1, COL1A2, and COL12A1, may act as potential prognostic biomarkers and immune-associated therapeutic targets in gastric cancer.     

Comparison of the immunogenicity of the human papillomavirus (HPV)-16/18 vaccine and the HPV-6/11/16/18 vaccine for oncogenic non-vaccine types HPV-31 and HPV-45 in healthy women aged 18-45 years

Protection against oncogenic non-vaccine types (cross-protection) offered by human papillomavirus (HPV) vaccines may provide a significant medical benefit. Available clinical efficacy data suggest the two licensed vaccines (HPV-16/18 vaccine, GlaxoSmithKline Biologicals (GSK), and HPV-6/11/16/18 vaccine, Merck & Co., Inc.) differ in terms of protection against oncogenic non-vaccine HPV types -31/45. The immune responses induced by the two vaccines against these two non-vaccine HPV types (cross-reactivity) was compared in an observer-blind study up to Month 24 (18 mo post-vaccination), in women HPV DNA-negative and seronegative prior to vaccination for the HPV type analyzed (HPV-010 [NCT00423046]). Geometric mean antibody titers (GMTs) measured by pseudovirion-based neutralization assay (PBNA) and enzyme-linked immunosorbent assay (ELISA) were similar between vaccines for HPV-31/45. Seropositivity rates for HPV-31 were also similar between vaccines; however, there was a trend for higher seropositivity with the HPV-16/18 vaccine (13.0-16.7%) versus the HPV-6/11/16/18 vaccine (0.0-5.0%) for HPV-45 with PBNA, but not ELISA. HPV-31/45 cross-reactive memory B-cell responses were comparable between vaccines. Circulating antigen-specific CD4+ T-cell frequencies were higher for the HPV-16/18 vaccine than the HPV-6/11/16/18 vaccine (HPV-31 [geometric mean ratio [GMR] =2.0; p=0.0002] and HPV-45 [GMR=2.6; p=0.0092]), as were the proportion of T-cell responders (HPV-31, p=0.0009; HPV-45, p=0.0793). In conclusion, immune response to oncogenic non-vaccine HPV types -31/45 was generally similar for both vaccines with the exception of T-cell response which was higher with the HPV-16/18 vaccine. Considering the differences in cross-protective efficacy between the two vaccines, the results might provide insights into the underlying mechanism(s) of protection.     

基于转录组测序探究PM2.5损伤肺上皮细胞 BEAS-2B的相关机制

目的 研究 PM2.5 对肺上皮细胞 BEAS-2B 基因表达的影响,并探讨其可能作用的信号通路。方法 BEAS-2B细胞分为对照组（PBS处理）和PM2.5组（200 mg/L PM2.5处理）,干预24 h后提取各组细胞总RNA进行高通 量测序。所得的序列经质控,与参考基因组比对,获得用于后续转录本组装、表达量计算的 mapped data（reads）后,比 对到 6 大数据库［NR、Swiss-Prot、Pfam、COG（Evolutionary genealogy of genes）、GO（Gene Ontology）、KEGG（Kyoto Encyclopedia of Genes and Genomes）］注释,利用差异分析软件DESeq2筛选出差异表达基因（DEGs）,运用生物信息学 分析方法对DEGs进行GO和KEGG生物功能等分析,采用qRT-PCR法检测5个关键基因（FOSB、FOSL1、MUC5AC、 CSF2、IL-6）进行验证。结果 PM2.5组和对照组与转录组的数据比较后共获得961个DEGs,其中PM2.5组上调表达 453个,下调表达508个。通过GO和KEGG功能分析后发现这些DEGs主要参与了细胞蛋白质的磷酸化、免疫调节过 程、信号转导途径的调节以及凋亡途径的调控过程,并显著富集于IL-17信号通路。qRT-PCR结果显示,PM2.5组 FOSB、FOSL1与IL-6的相对表达量显著上调（P＜0.05）,这与RNA-seq测序结果一致。结论 PM2.5可能通过调控 “IL-17信号通路”中关键基因的表达,加重了细胞的炎症反应,促进了细胞凋亡等生命进程。