Predicting the Prognosis and Immunotherapeutic Response of Triple-Negative Breast Cancer by Constructing a Prognostic Model Based on CD8+T Cell-Related Immune Genes

Objective Triple-negative breast cancer(TNBC)poses a significant challenge for treatment efficacy.CD8+T cells,which are pivotal immune cells,can be effectively analyzed for differential gene expression across diverse cell populations owing to rapid advancements in sequencing technology.By leveraging these genes,our objective was to develop a prognostic model that accurately predicts the prognosis of patients with TNBC and their responsiveness to immunotherapy.Methods Sample information and clinical data of TNBC were sourced from The Cancer Genome Atlas and METABRIC databases.In the initial stage,we identified 67 differentially expressed genes associated with immune response in CD8+T cells.Subsequently,we narrowed our focus to three key genes,namely CXCL13,GBP2,and GZMB,which were used to construct a prognostic model.The accuracy of the model was assessed using the validation set data and receiver operating characteristic(ROC)curves.Furthermore,we employed various methods,including Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway,immune infiltration,and correlation analyses with CD274(PD-L1)to explore the model's predictive efficacy in immunotherapeutic responses.Additionally,we investigated the potential underlying biological pathways that contribute to divergent treatment responses.Results We successfully developed a model capable of predicting the prognosis of patients with TNBC.The areas under the curve(AUC)values for the 1-,3-,and 5-year survival predictions were 0.618,0.652,and 0.826,respectively.Employing this risk model,we stratified the samples into high-and low-risk groups.Through KEGG enrichment analysis,we observed that the high-risk group predominantly exhibited enrichment in metabolism-related pathways such as drug and chlorophyll metabolism,whereas the low-risk group demonstrated significant enrichment in cytokine pathways.Furthermore,immune landscape analysis revealed noteworthy variations between(PD-L1)expression and risk scores,indicating that our model effectively predicted the response of patients to immune-based treatments.Conclusion Our study demonstrates the potential of CXCL13,GBP2,and GZMB as prognostic indicators of clinical outcomes and immunotherapy responses in patients with TNBC.These findings provide valuable insights and novel avenues for developing immunotherapeutic approaches targeting TNBC.

Identification of prognostic molecular subtypes and model based on CD8+ T cells for lung adenocarcinoma

Background:Cytotoxic T lymphocytes(CD8+T)cells function critically in mediating anti-tumor immune response in cancer patients.Characterizing the specific functions of CD8+T cells in lung adenocarcinoma(LUAD)could help better understand local anti-tumor immune responses and estimate the effect of immunotherapy.Methods:Gens related to CD8+T cells were identified by cluster analysis based on the single-cell sequencing data of three LUAD tissues and their paired normal tissues.Weighted gene co-expression network analysis(WGCNA),consensus clustering,differential expression analysis,least absolute shrinkage and selection operator(LASSO)and Cox regression analysis were conducted to classify molecular subtypes for LUAD and to develop a risk model using prognostic genes related to CD8+T cells.Expression of the genes in the prognostic model,their effects on tumor cell invasion,and interactions with CD8+T cells were verified by cell experiments.Results:This study defined two LUAD clusters(CD8+0 and CD8+1)based on CD8+T cells,with cluster CD8+0 being significantly associated with the prognosis of LUAD.Three heterogeneous subtypes(clusters 1,2,and 3)differing in prognosis,genome mutation events,and immune status were categorized using 42 prognostic genes.A prognostic model created based on 11 significant genes(including CD200R1,CLEC17A,ZC3H12D,GNG7,SNX30,CDCP1,NEIL3,IGF2BP1,RHOV,ABCC2,and KRT81)was able to independently estimate the death risk for patients in different LUAD cohorts.Moreover,the model also showed general applicability in external validation cohorts.Low-risk patients could benefit more from taking immunotherapy and were significantly related to the resistance to anticancer drugs.The results from cell experiments demonstrated that the expression of CD200R1,CLEC17A,ZC3H12D,GNG7,and SNX30 was significantly downregulated,while that of CDCP1,NEIL3,IGF2BP1,RHOV,ABCC2 and KRT81 was upregulated in LUAD cells.Inhibition of CD200R1 greatly increased the invasiveness of the LUAD cells,but inhibiting CDCP1 expression weakened the invasion ability of LUAD cells.Conclusion:This study defined two prognostic CD8+T cell clusters and classified three heterogeneous molecular subtypes for LUAD.A prognostic model predictive of the potential effects of immunotherapy on LUAD patients was developed.

Peripheral CD4^(+)CD8^(+) double positive T cells:A potential marker to evaluate renal impairment susceptibility during systemic lupus erythematosus

Lupus nephritis(LN) has a high incidence in systemic lupus erythematosus(SLE) patients, but there is a lack of sensitive predictive markers. The purpose of the study was to investigate the association between the CD4^(+)CD8^(+)double positive T(DPT) lymphocytes and LN. The study included patients with SLE without renal impairment(SLE-NRI), LN, nephritic syndrome(NS), or nephritis. Peripheral blood lymphocyte subsets were analyzed by flow cytometry. Biochemical measurements were performed with peripheral blood in accordance with the recommendations proposed by the National Center for Clinical Laboratories. The proportions of DPT cells in the LN group were significantly higher than that in the SLE-NRI group(t=4.012, P<0.001), NS group(t=3.240,P=0.001), and nephritis group(t=2.57, P=0.011). In the LN group, the risk of renal impairment increased significantly in a DPT cells proportion-dependent manner. The risk of LN was 5.136 times(95% confidence interval, 2.115–12.473) higher in cases with a high proportion of DPT cells than those whose proportion of DPT cells within the normal range. These findings indicated that the proportion of DPT cells could be a potential marker to evaluate LN susceptibility, and the interference of NS and nephritis could be effectively excluded when assessing the risk of renal impairment during SLE with DPT cell proportion.

Combined TIM-3 and PD-1 blockade restrains hepatocellular carcinoma development by facilitating CD4+ and CD8+T cellmediated antitumor immune responses

BACKGROUND Immune checkpoint inhibitors(ICIs)targeting programmed cell death protein 1(PD-1)and T cell immunoglobulin and mucin domain-containing protein 3(TIM-3)are beneficial to the resumption of anti-tumor immunity response and hold extreme potential as efficient therapies for certain malignancies.However,ICIs with a single target exhibit poor overall response rate in hepatocellular carcinoma(HCC)patients due to the complex pathological mechanisms of HCC.AIM To investigate the effects of combined TIM-3 and PD-1 blockade on tumor development in an HCC mouse model,aiming to identify more effective immunotherapies and provide more treatment options for HCC patients.METHODS The levels of PD-1 and TIM-3 on CD4+and CD8+T cells from tumor tissues,ascites,and matched adjacent tissues from HCC patients were determined with flow cytometry.An HCC xenograft mouse model was established and treated with anti-TIM-3 monoclonal antibody(mAb)and/or anti-PD-1 mAb.Tumor growth in each group was measured.Hematoxylin and eosin staining and immunohistochemical staining were used to evaluate T cell infiltration in tumors.The percentage of CD4+and CD8+T cells in tissue samples from mice was tested with flow cytometry.The percentages of PD-1+CD8+,TIM-3+CD8+,and PD-1+TIM-3+CD8+T cells was accessed by flow cytometry.The levels of the cytokines including tumor necrosis factor alpha(TNF-α),interferon-γ(IFN-γ),interleukin(IL)-6,and IL-10 in tumor tissues were gauged with enzyme-linked immunosorbent assay kits.RESULTS We confirmed that PD-1 and TIM-3 expression was substantially upregulated in CD4+and CD8+T cells isolated from tumor tissues and ascites of HCC patients.TIM-3 mAb and PD-1 mAb treatment both reduced tumor volume and weight,while combined blockade had more substantial anti-tumor effects than individual treatment.Then we showed that combined therapy increased T cell infiltration into tumor tissues,and downregulated PD-1 and TIM-3 expression on CD8+T cells in tumor tissues.Moreover,combined treatment facilitated the production of T cell effector cytokines TNF-α and IFN-γ,and reduced the production of immunosuppressive cytokines IL-10 and IL-6 in tumor tissues.Thus,we implicated that combined blockade could ameliorate T cell exhaustion in HCC mouse model.CONCLUSION Combined TIM-3 and PD-1 blockade restrains HCC development by facilitating CD4+ and CD8+T cell-mediated antitumor immune responses.

System analysis based on the T cell exhaustion‑related genes identifies CD38 as a novel therapy target for ovarian cancer

Ovarian cancer(OV)is highly heterogeneous tumor with a very poor prognosis.Studies increasingly show that T cell exhaustion is prognostically relevant in OV.The aim of this study was to dissect the heterogeneity of T cell subclusters in OV through single cell transcriptomic analysis.The single RNA-sequencing(scRNA-seq)data of five OV patients were analyzed,and six major cell clusters were identified after threshold screening.Further clustering of T cell-associated clusters revealed four subtypes.Pathways related to oxidative phosphorylation,G2M checkpoint,JAK-STAT and MAPK signaling were significantly activated,while the p53 pathway was inhibited in the CD8+exhausted T cells.The standard marker genes of CD8+T cell exhaustion were screened to develop a T-cell related gene score(TRS)based on random forest plots in TCGA cohort.The patients with low TRS have better prognosis compared to the patients with high TRS in both TCGA and GEO.In addition,most genes included in the TRS showed significant differences in expression levels between the high-and low-risk groups.Immune cell infiltration was analyzed using the MCPcounter and xCell algorithms,which revealed significant differences between the two risk groups,indicating that the different prognoses may stem from the respective immune landscapes.In addition,CD38 knockdown in OV cell lines increased apoptosis and inhibited invasion in vitro.Finally,we performed a drug sensitivity analysis and identified six potential drug candidates for OV.To summarize,we identified the heterogeneity and clinical significance of T cell exhaustion in OV and built a superior prognostic model based on T cell exhaustion genes,which can contribute to the development of more precise and effective therapies.

The Mechanisms of CD8+ T Cells Exhaustion in the Tumor Microenvironment and Immune Therapy

In the tumor immune microenvironment, CD8+ T cells differentiate towards functional failure. The exhaustion of CD8+ T cells (Tex) showed varying degrees of effect dysfunction, loss of proliferation ability, and sustained high expression of a variety of inhibitory receptors, with metabolic and epigenetic changes. Tex cells are heterogeneous, including several subsets with different characteristics at different stages of differentiation. Immune checkpoint inhibitors (ICIs) can restore the effect or function of Tex cells, indicating that this T cell subset plays a key role in tumor immunotherapy. The understanding of the mechanism of CD8+ T cell exhaustion will be helpful to the implementation of tumor immunotherapy. This article reviews the production, differentiation and functional characteristics of Tex cells and their relationship with tumor immunotherapy.

电针调节CD8^(+)T细胞对帕金森病模型小鼠黑质区神经元损伤的保护机制研究

目的:观察早期电针干预对CD8^(+)T细胞浸润帕金森病(Parkinson’s disease,PD)模型小鼠黑质区酪氨酸羟化酶(tyrosine hydroxylase,TH)及CD8^(+)T细胞相关细胞因子表达的影响,探讨早期电针对PD的神经元保护效应及机制。方法:将雄性C57BL/6小鼠随机分为空白组、模型组、电针组、左旋多巴组,每组15只;除空白组外,其余各组均腹腔注射1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)构建PD小鼠模型。造模成功电针组选取“足三里”“悬钟”进行干预14天,治疗结束后进行转棒、旷场测试,并观察黑质区TH表达及CD8^(+)T细胞表型标记物CD103^(+)T细胞、干扰素γ(IFNγ)蛋白及颗粒酶B(GzmB)蛋白的表达。结果:造模结束后,模型组小鼠毛色暗淡无光,精神萧瑟,活动减少,肌肉颤动,而电针组及左旋多巴组小鼠精神状态良好。与模型组相比,电针组及左旋多巴组小鼠掉棒潜伏时间延长(P<0.01),运动总距离延长且经过区域中心的速度改善(P<0.01),黑质区TH阳性表达增加(P<0.01),CD8^(+)CD103^(+)T细胞共定位表达率降低(P<0.01),IFNγ含量下调(P<0.05,P<0.01),GzmB表达下降(P<0.05,P<0.01)。结论:早期电针干预可以提高PD模型小鼠的运动能力,增加黑质区TH表达并改善神经元损伤,其机制与电针减少黑质区CD8^(+)CD103^(+)T细胞浸润,进一步抑制IFNγ、GzmB分泌,最终降低细胞毒性并提高机体免疫功能有关。

CD103^+CD8^+T细胞在结直肠癌组织中的浸润分布及其临床意义

目的:研究组织驻留CD8^+T细胞(CD103^+CD8^+T细胞)在结直肠癌(colorectal cancer,CRC)组织中浸润程度及分布特征,分析其浸润程度与患者临床病理特征及预后的关系。方法:选用上海芯超生物科技有限公司的88例结肠癌HColA180Su14和77例直肠癌HRec-Ade180Sur-03组织芯片,应用免疫荧光染色法分别检测CRC组织及相应癌旁组织中CD103^+CD8^+T细胞的浸润分布特征及程度,Wilcoxon秩和检验比较CRC及癌旁组织中CD103^+CD8^+T细胞浸润程度,χ2检验分析CRC中CD103^+CD8^+T细胞浸润程度与患者临床病理特征的关系;Kaplan-Meier生存分析CD103^+CD8^+T细胞浸润程度与患者预后的关系,拟合Cox模型评价不同指标与患者预后的关系。结果:CRC组织中CD103^+CD8^+T细胞浸润程度与癌旁组织比较差异无统计学意义(P>0.05),有远处转移患者中CD103^+CD8^+T细胞高度浸润的比率显著低于无远处转移患者(P<0.01),CD103^+CD8^+T细胞浸润程度与患者其他临床病理特征无明显相关(P>0.05)。Kaplan-Meier生存分析显示,CD103^+CD8^+T细胞高度浸润患者的OS较低度浸润患者显著延长(54.42%vs 25.00%,P<0.05),多因素Cox显示,病理分级(P<0.01)和CD103^+CD8^+T细胞高度浸润(P<0.05)均可作为CRC患者预后的独立影响因素。结论:CRC组织中CD103^+CD8^+T细胞浸润与预后相关,提示其在CRC发生发展过程中发挥重要作用。

Prognostic value of programmed death.1, programmed death-ligand 1, programmed death-ligand 2 expression, and CD8(+) T cell density in primary tumors and metastatic lymph nodes from patients with stage T1.4N+M0 gastric adenocarcinoma

Background: Anti-programmed death-1/programmed death-ligand 1(PD-1/PD-L1) immunotherapy has been proved to be effective on gastric cancer in ongoing clinical trials. However, the value of PD-L1 in predicting responses of patients with gastric cancer to anti-PD-1/PD-L1 immunotherapy is controversial. Some studies suggested that intra-and inter-tumoral heterogeneity of PD-L1 expression might explain the controversy.This study aimed to analyze the expression of PD-L1, PD-L2, and PD-1 as well as CD8(+) T-cell density in primary tumors and lymph nodes from patients with stage T1-4 N+M0 gastric adenocarcinoma to explore the heterogeneity of PD-1 signaling pathway molecules.Methods: In primary tumors and metastatic as well as non-metastatic lymph nodes from patients with stage T1-4 N+M0 gastric adenocarcinoma, we detected PD-L1 and PD-L2 expression with immunohistochemistry. CD8(+)T-cell density in primary tumors and PD-1 expression on CD8(+)T cells were detected with immunofluorescence. Univariate analysis was used to determine the prognostic values of them. Cox proportional hazard regression model was used to identify independent risk factors that affect patients' overall survival and disease-free survival.Results: Among 119 eligible patients who had undergone surgical resection, the positive rate of PD-L1 was higher in metastatic lymph nodes than in primary tumors(45.4% vs. 38.7%, P = 0.005); the positive rate of PD-1 on CD8(+)T cells was significantly higher in primary tumors and metastatic lymph nodes than in tumor-free lymph nodes(both P < 0.001). The intensity of PD-1 expression on CD8(+) T cells in primary tumors and in metastatic lymph nodes were stronger than that in tumor-free lymph nodes from the same patient. Beside, the positive rate of PD-L2 did not show any differences between primary tumors and metastatic lymph nodes. In multivariate analysis, PD-L1 expression,PD-L2 expression, a low density of CD8(+) T cells in primary tumors, and PD-1 expression on CD8(+) T cells in primary tumors were associated with poor prognosis.Conclusion: The expression of PD-L1 is heterogeneous in primary tumors and in metastatic lymph nodes from patients with stageT1-4 N+M0 gastric adenocarcinoma, which might explain the inconsistent results in assessing the prognostic value of PD-L1 expression in previous studies.

Natural course of chronic hepatitis B is characterized by changing patterns of programmed death type-1 of CD8-positive T cells

AIM:To investigate if and how programmed death type-1(PD-1)expression affects the natural course of hepatitis B virus(HBV)infection. METHODS:Sixty-four patients in different natural stages of chronic HBV infection were enrolled in this study.PD-1 expression in total T cells was detected by flow cytometry.Levels of total CD8+T cell responses and proliferation in relation to PD-1 expression levels were analyzed with intracellular staining and PD-1/ PD-L1 blockage. RESULTS:The PD-1 expression in T cells was dynamically changed during the natural course of chronic HBV infection,did not significantly increase in the immune tolerance phase,and returned to normal in the inactive virus carrier stage.Blockage of the PD-1/PD-L1 pathway could not affect the T-cell response in the immune tolerance and inactive virus carrier stages of chronic HBV infection.However,it could significantly restore the T-cell response in the immune clearance stage of chronic HBV infection.Furthermore,the PD-1 expression level in T cells was associated with the alanine aminotransferase level during the immune clearance stage of chronic HBV infection. CONCLUSION:The PD-l/PD-L1 pathway plays a different role in T-cell response during the natural course of chronic HBV infection.

Specific CD8^+ T cell response immunotherapy for hepatocellular carcinoma and viral hepatitis

Hepatocellular carcinoma (HCC), chronic hepatitis B (CHB) and chronic hepatitis C (CHC) are characterized by exhaustion of the specific CD8+ T cell response. This process involves enhancement of negative co-stimulatory molecules, such as programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte antigen-4 (CTLA-4), 2B4, Tim-3, CD160 and LAG-3, which is linked to intrahepatic overexpression of some of the cognate ligands, such as PD-L1, on antigen presenting cells and thereby favouring a tolerogenic environment. Therapies that disrupt these negative signalling mechanisms represent promising therapeutic tools with the potential to restore reactivity of the specific CD8+ T cell response. In this review we discuss the impressive in vitro and in vivo results that have been recently achieved in HCC, CHB and CHC by blocking these negative receptors with monoclonal antibodies against these immune checkpoint modulators. The article mainly focuses on the role of CTLA-4 and PD-1 blocking monoclonal antibodies, the first ones to have reached clinical practice. The humanized monoclonal antibodies against CTLA-4 (tremelimumab and ipilimumab) and PD-1 (nivolumab and pembrolizumab) have yielded good results in testing of HCC and chronic viral hepatitis patients. Trelimumab, in particular, has shown a significant increase in the time to progression in HCC, while nivolumab has shown a remarkable effect on hepatitis C viral load reduction. The research on the role of ipilimumab, nivolumab and pembrolizumab on HCC is currently underway.

Human immune suppression is inducible by trichosanthin via CD8 cell-mediated pathway

Thichosanthin(Tk), a polypeptide with 249 amino acid residues isolated and purified from a Chinese medicinal herb, showed the capability of inducing abortion and was able to inhibit tumor growth and HIV replication. Owing to sequence homology of the peptide with a ribosomeinactivating protein, the downward activity of Tk was suggested to be related to its cytotoxic property. We report here, however, that Tk could exert potent inhibitory effects on human lymphoproliferative responses in vitro to allogeneic, mitogenic and soluble antigens with 50% inhibition doses ranged between 0.05 and 0.5 μg/ml. The lowresponsiveness caused by Tk was not due to toxic cytolysis. Rather, evidences suggested that, in the dose range adopted, the Tk-induced inhibition was attributable, at least in part, to immune suppression, in view of (1) Tk was more effective in the early stage of alloreactivity; (2)Suppression also occurred if responder cells were pulsetreated with Tk rather than cocultured; (3) Irradiated Tk-pulsed cells were capable of inducing suppression in a Tk-free culture; (4) Suppression could also be transferred by the supernatants of Tk-pulsed cultured cells; (5) Tkinduced immune suppression was diminished by depletion of CD8+ cells from the culture, and, finally; (6) Adding CD8+ cells back to the culture could restore the suppres Trichosanthin-induced humall immune suppression sion. Thus the possibility that Tk might function as a down-regulator by immunological mechanisms in human immune responses is discussed.

Super-resolution immunohistochemistry study on CD4 and CD8 cells and the relation to macrophages in human cochlea

Recently, the human cochlea has been shown to contain numerous resident macrophages under steady-state. The macrophages accumulate in the stria vascularis, among the auditory nerves, and are also spotted in the human organ of Corti. These macrophages may process antigens reaching the cochlea by invasion of pathogens and insertion of CI electrode. Thus, macrophages execute an innate, and possibly an adaptive immunity. Here, we describe the molecular markers CD4 and CD8 of T cells, macrophage markers MHCⅡ and CD11b, as well as the microglial markers TEME119 and P2Y12, in the human cochlea. Immunohistochemistry and the advantageous super-resolution structured illumination microscopy(SR-SIM) were used in the study. CD4^+ and CD8^+ cells were found in the human cochleae. They were seen in the modiolus in a substantial number adjacent to the vessels, in the peripheral region of the Rosenthal's canal, and occasionally in the spiral ligament. While there are a surprisingly large number of macrophages in the stria vascularis as well as between the auditory neurons,CD4^+ and CD8^+ cells are hardly seen in these areas, and neither are seen in the organ of Corti. In the modiolus,macrophages, CD4^+ and CD8^+ cells appeared often in clusters. Interaction between these different cells was easily observed with SR-SIM, showing closely placed cell bodies, and the processes from macrophages reaching out and touching the lymphocytes. Otherwise the CD4^+ and CD8^+ cells in human cochlear tissue are discretely scattered. The possible roles of these immune cells are speculated.

Expression of PD1 and BTLA on the CD8^+T Cell and γδT Cell Subsets in Peripheral Blood of Non-Small Cell Lung Cancer Patients

Objective To investigate the expression and regulation of programmed cell death protein 1(PD1),B lymphocyte and T lymphocyte attenuator(BTLA)in peripheral blood of patients with non-small cell lung cancer(NSCLC);to examine the correlation of the mRNA levels between PD and BTLA in NSCLC.Methods Flow cytometry was used to detect the expression of PD1 and BTLA on the surfaces of CD8^+T cells andγδ+T cells in the peripheral blood samples collected from 32 in-patients with stage IV NSCLC and 30 healthy individuals.We compared the expression of PD1 and BTLA on the surfaces ofγδ+T cells in the NSCLC patients with bone metastasis before and after the treatment of zoledronic acid.The correlations of PD1 and BTLA,as well as their ligands were analyzed using Pearson correlation analysis with the cBioPortal data platform.Results The frequency of PD1 on the surfaces of CD8^+T cells was significantly higher than that of theγδT cells in both healthy controls(t=2.324,P=0.024)and NSCLC patients(t=2.498,P=0.015).The frequency of PD1 on CD8^+T cells,rather than onγδ+T cells,was significantly upregulated in advanced NSCLC patients compared with that in healthy controls(t=4.829,P<0.001).The PD1+BTLA+γδT cells of the healthy controls were significantly lower than that of the NSCLC patients(t=2.422,P=0.0185).No differences in percentage of PD1+γδ+and BTLA+γδ+T cells were observed in 7 NSCLC patients with bone metastasis before and after zoledronic acid treatment.PD1 was positively correlated with BTLA in both lung adenocarcinoma(r=0.54;P<0.05)and lung squamous cell carcinoma(r=0.78;P<0.05).Conclusions The upregulation of co-inhibitory molecules occurs on the surfaces of both CD8^+T cells andγδT cells in advanced NSCLC,suggesting that these molecules were involved in regulating the inactivation of CD8^+T cells andγδ+T cells,immune escape and tumor invasion.

Glutamine deprivation impairs function of infiltrating CD8^(+)T cells in hepatocellular carcinoma by inducing mitochondrial damage and apoptosis

BACKGROUND The functions of infiltrating CD8^(+)T cells are often impaired due to tumor cells causing nutrient deprivation in the tumor microenvironment.Thus,the mechanisms of CD8^(+)T cell dysfunction have become a hot research topic,and there is increased interest on how changes in metabolomics correlate with CD8^(+)T cell dysfunction.AIM To investigate whether and how glutamine metabolism affects the function of infiltrating CD8^(+)T cells in hepatocellular carcinoma.METHODS Immunohistochemical staining and immunofluorescence were performed on surgically resected liver tissues from patients.Differentially expressed genes in infiltrating CD8^(+)T cells in hepatocellular carcinoma were detected using RNA sequencing.Activated CD8^(+)T cells were co-cultured with Huh-7 cells for 3 d.The function and mitochondrial status of CD8^(+)T cells were analyzed by flow cytometry,quantitative real-time polymerase chain reaction,and transmission electron microscopy.Next,CD8^(+)T cells were treated with the mitochondrial protective and damaging agents.Functional alterations in CD8^(+)T cells were detected by flow cytometry.Then,complete medium without glutamine was used to culture cells and their functional changes and mitochondrial status were detected.RESULTS There were a large number of infiltrating PD-1+CD8^(+)T cells in liver cancer tissues.Next,we cocultured CD8^(+)T cells and Huh-7 cells to explore the regulatory effect of hepatoma cells on CD8^(+)T cells.Flow cytometry results revealed increased PD-1 expression and decreased secretion of perforin(PRF1)and granzyme B(GZMB)by CD8^(+)T cells in the co-culture group.Meanwhile,JC-1 staining was decreased and the levels of reactive oxygen species and apoptosis were increased in CD8^(+)T cells of the co-culture group;additionally,the mitochondria of these cells were swollen.When CD8^(+)T cells were treated with the mitochondrial protective and damaging agents,their function was restored and inhibited,respectively,through the mitochondrial damage and apoptotic pathways.Subsequently,complete medium without glutamine was used to culture cells.As expected,CD8^(+)T cells showed functional downregulation,mitochondrial damage,and apoptosis.CONCLUSION Glutamine deprivation impairs the function of infiltrating CD8^(+)T cells in hepatocellular carcinoma through the mitochondrial damage and apoptotic pathways.

Neutrophils inhibit CD8^(+)T cells immune response by arginase-1 signaling in patients with sepsis

BACKGROUND:Patients with sepsis often exhibit an acute inflammatory response,followed by an immunosuppressive phase with a poor immune response.However,the underlying mechanisms have not been fully elucidated.METHODS:We sought to comprehensively characterize the transcriptional changes in neutrophils of patients with sepsis by transcriptome sequencing.Additionally,we conducted a series of experiments,including real-time quantitative polymerase chain reaction(RT-qPCR)and flow cytometry to investigate the role of arginase-1 signaling in sepsis.RESULTS:Through the analysis of gene expression profiles,we identified that the negative regulation of T cell activation signaling was enriched,and the expression of arginase-1 was high in neutrophils from patients with sepsis.Furthermore,we conducted flow cytometry and found that the function of CD8^(+)T cells in septic patients was impaired.Moreover,neutrophils from septic patients inhibited the percentage of polyfunctional effector CD8^(+)T cells through arginase-1.Additionally,the proportions of granzyme B^(+)IFN^(-)γ^(+)CD8^(+)T and TNF^(-)α^(+)IFN^(-)γ^(+)CD8^(+)T cells increased after inhibition of arginase-1 signaling.CONCLUSION:The impaired effector function of CD8^(+)T cells could be restored by blocking arginase-1 signaling in patients with sepsis.

Host and viral factors contributing to CD8+ T cell failure in hepatitis C virus infection

Virus-specific CD8+ T cells are thought to be the major anti-viral effector cells in hepatitis C virus (HCV) infection. Indeed, viral clearance is associated with vigorous CD8+ T cell responses targeting multiple epitopes. In the chronic phase of infection, HCV-specific CD8+ T cell responses are usually weak, narrowly focused and display often functional defects regarding cytotoxicity, cytokine production, and proliferative capacity. In the last few years, different mechanisms which might contribute to the failure of HCV-specific CD8+ T cells in chronic infection have been identified, including insufficient CD4+ help, deficient CD8+ T cell differentiation, viral escape mutations, suppression by viral factors, inhibitory cytokines, inhibitory ligands, and regulatory T cells. In addition, host genetic factors such as the host’s human leukocyte antigen (HLA) background may play an important role in the efficiency of the HCV- specific CD8+ T cell response and thus outcome of infection. The growing understanding of the mechanisms contributing to T cell failure and persistence of HCV infection will contribute to the development of successful immunotherapeutical and -prophylactical strategies.

HIV-Specific IL-2^+ and/or IFN-γ^+ CD8^+ T Cell Reponses during Chronic HIV-1 Infection in Former Blood Donors

Objective Conflicting data have been generated from previous studies to determine which kind of relationship exists between HIV-1 specific CD8 Tcell responses and HIV-1 viral load or CD4 count over the course of infection.In this study,153 HIV-1 infected LTNPs were enrolled to investigate the role of HIV-1 specific CD8 T-cell responses in chronic HIV-1 infection among HIV-1 infected former blood donors.Methods The patients were stratified into three groups according to CD4 count：CD4≥500 cells/μL;350 cells/μL≤CD4〈500 cells/μL;CD4〈350 cells/μL.PBMCs were isolated from the patients＇ anticoagulated blood samples.IL-2 and IFN-γ secretions of CD 8 T cells against 17 HIV-1 consensus B full peptide pools were analyzed by using ICS assay.Results An overall inverse correlation were observed between CD4 count and plasma viral load.Although no significant difference was observed during the comparisons of frequency/breadth of HIV-1 specific CD8 T cell responses,CD4 count stratification analysis showed that different correlation pattern existed in three strata：as for patients whose CD4 counts were less than 350 cells/μL,no significant correlations were identified between frequency/breadth of HIV-1 specific CD8 T cell responses and CD4 count/viral load;as for patients whose CD4 counts ranged from 350 cells /μL to 500 cells/μL,significant correlation was only observed between the response breadth of IL-2＋IFN-γ＋ CD8 T cells and CD4 count;however,as for patients whose CD4 counts were more than 500 cells/μL,direct correlations were identified between IL-2＋IFN-γ＋/IL-2＋/IFN-γ＋ CD8 T cells and viral load or CD4 count.Conclusions Universal consistent inverse correlation was only indentified between CD4 count and viral load.The relationship between HIV-1 specific CD8 T cell responses and CD4 count/viral load varied in different CD4 strata,which showed that better preserved CD4 T cells were correlated with better CD8 T cell functions.

TARGETED ACTIVATION OF CD_(8)^(+) CELLS AND INFECTION OF β_2-MICROGLOBULIN-DEFICIENT MICE FAIL TO CONFIRM A PRIMARY PROTECTIVE ROLE FOR CD_(8)￣(+) CELLS IN EXPERIMENTAL LEISHMANIASIS

CD8+ T cells play an important role in the immunologic control of intracellular pathogens,particularly viruses,Leishmania are obligate intracellular parasites of macrophages in the mammalian host,and previous studies using deletion of CD8+ cells by administration of mAb to infected animals have suggested a protective role for these cells.Two complementary approaches were used to define more carefully the role of CD8+ cells in leishmaniasis.In BALB/c mice susceptible to Leishmania major(L. major)infection, targeted activation of CD8+ cells was attempted by immunization with nonapeptides derived from the conserved major outer surface protein of the organism,8p63, that contained the consensus binding motif for MHC class 1 H-2kd molecules.Two of the nonapeptides induced CTL activity in subsequently infected BALB/c mice that could be elicited against p815 cells pulsed either with peptide or lysates of L.major.purified CD8+ T cells from immunized mice had elevated levels of IFN-γ mRNA transcripts as compared to unimmunized mice.Despite evidence for activation of CD8+ cells,none of the mice immunized with nine different peptides alone or in combination were protected from progressive disease. In a second series of experiments, β2-microglobulin deficient mice that lack CD8+ cells were infected with L. major and the course of infection monitored.These mice cured disease as'rapidly as β2-m +/- and +/+ littermates,and cure was associated with comparable levels of IFN-Y mRNA in the draining lymph node population. Neither of these approaches was able to confirm a substantive role for CD8+ T cells in the primary protective response to L.major.

Effect of Mg^(2+)level on the functions of CD8^(+)T lymphocytes and NK cells in patients with COVID-19

Objective:To investigate the changes of Mg^(2+) levels in serum and peripheral blood mononuclear cells(PBMCs)of patients with COVID-19 and its effects on the functions of CD8^(+)T lymphocytes and NK cells.Methods:A total of 165 COVID-19 patients hospitalized in Ezhou Central Hospital from January 20 to February 20,2020 were divided into mild/common group(98 cases)and severe/critical group(67 cases).At the same time,34 healthy persons were selected as the control group.Peripheral blood was collected and PBMCs were isolated,the level of Mg^(2+) in serum and PBMCs was detected.The subsets of CD8^(+)T lymphocytes and NK cell and the expression levels of their surface inhibitory molecular PD-1 and activator molecular NKG2D were detected by flow cytometry.The correlation between Mg^(2+) concentration and the expression levels of PD-1 and NKG2D was also analyzed.Results:Compared with the control group,the concentration of Mg^(2+) in serum and PBMCs,the counts of CD8^(+)T lymphocytes and NK cell in patients with mild/common and severe/critical groups were significantly reduced(P<0.05),while the expression level of surface inhibitory molecular PD-1 were significantly increased(P<0.05),while the expression level of the activation molecule NKG2D were significantly decreased(P<0.05).However,the changes of the above indicators in patients with severe/critical group were greater than those in the mild/common group(P<0.05).In addition,the Mg^(2+) concentration in COVID-19 patients was negatively correlated with the expression level of PD-1 on CD8^(+)T lymphocytes and NK cells(P<0.05),and positively correlated with the expression levels of NKG2D(P<0.05).Conclusion:The concentration of Mg^(2+) in the serum and PBMCs of COVID-19 patients is significantly reduced,which may cause the function of CD8^(+)T lymphocytes and NK cells to be inhibited.