OBJECTIVE: To investigate the molecular alterations of splenocytes associated with anti-factor Ⅷ (FⅧ) immune response and the underlying mechanisms based on hemophilia A (HA) murine model via RNA sequencing (RNA-seq) technology. METHODS: Severe HA mice were immunized with recombinant human factor Ⅷ (rhF8) weekly for 4 weeks to establish an FⅧ inhibitor model. High quality raw data were obtained by using bulk RNA-seq and CASAVA base identification technology, and the differentially expressed genes (DEGs) were identified. The DEGs were statistically classified by gene ontology (GO) annotation to obtain information on the major signaling pathways and biological processes involved in anti-FⅧ immune response in HA mouse splenocytes. The cell clusters, genes, and signaling pathway datasets were comprehensively analyzed by GO, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and single cell RNA-seq (ScRNA-seq) analysis, respectively. Flow cytometry analysis was used to verify the changes in T follicular helper cells (Tfh) and regulatory T cells (Treg). RESULTS: A total of 3731 DEGs was identified, including 2275 genes with up-regulated expression and 1456 genes with down-regulated expression. The DEGs were enriched in helper T cell differentiation, cytokine receptor, T cell receptor signaling pathway, ferroptosis, etc. Uniform Manifold Approximation and Project (UMAP) downscaling and visualization analysis yielded a total number of 11 T/NK cell subsets, visualizing the overall expression distribution of C-X-C chemokine-specific receptor gene cxcr5 among these T/NK cell subsets. Higher expression of cxcr5 was found in activated Tfh from FⅧ inhibitor mice, in comparison to the control group. The visualization using Upset plot R language showed a close interaction between Tfh and Treg. Moreover, the increased frequencies of Tfh and the decreased frequencies of Treg in inhibitor mouse splenocytes were further verified by flow cytometry analysis. CONCLUSION Multiple immune cell subsets, signaling pathways, and characteristic genes may be involved in the process of anti-FⅧ immune response in HA mouse splenocytes. The molecules involved in the regulation of Tfh/Treg may play key roles, which provide potential biological targets and therapeutic strategies for HA patients with inhibitors in the future.
Animals ; Hemophilia A/genetics* ; Mice ; Sequence Analysis, RNA ; Disease Models, Animal ; Spleen/cytology* ; T-Lymphocytes, Regulatory/immunology* ; Humans ; Signal Transduction ; Factor VIII/immunology* ; T-Lymphocytes, Helper-Inducer/immunology*

Immune thrombocytopenia (ITP) is a hemorrhagic autoimmune disease characterized by antibody-mediated platelet injury. ITP has complicated immunopathological mechanisms that need further elucidation. It is well known that the costimulatory molecules OX40 ligand (OX40L) and OX40 play essential roles in the immunological mechanisms of autoimmune diseases. Previously, we discovered that the expression of OX40L and OX40 is significantly increased in the peripheral blood mononuclear cells (PBMCs) of ITP patients. In our present study, OX40L-induced follicular helper T (Tfh) cells exhibited an activated phenotype with elevated expression of inducible T-cell costimulator (ICOS), programmed cell death protein-1 (PD-1), and cluster of differentiation 40 ligand (CD40L) in vitro. Moreover, aberrant OX40L‒OX40 expression might promote the Tfh1-to-Tfh2 shift in vivo, inducing the generation of autoantibodies by enhancing the helper function of Tfh cells for B lymphocytes in a mouse model, which might accelerate the progression of ITP. Additionally, signal transduction through the OX40L‒OX40 axis might be related to the activation of tumor necrosis factor receptor-associated factor (TRAF)‒nuclear factor-κB (NF-κB) and Janus kinase (JAK)‒signal transducer and activator of transcription (STAT) signaling pathways. Overall, OX40L‒OX40 signaling is proposed as a potential novel therapeutic target for ITP.
Animals ; OX40 Ligand/physiology* ; Purpura, Thrombocytopenic, Idiopathic/immunology* ; Cell Differentiation ; Mice ; T-Lymphocytes, Helper-Inducer/cytology* ; T Follicular Helper Cells/cytology* ; Signal Transduction ; Receptors, OX40 ; Mice, Inbred C57BL ; Humans ; Female

Cancer immunotherapy including immune checkpoint inhibitors and adoptive cell therapy has gained revolutionary success in the treatment of hematologic tumors; however, it only gains limited success in solid tumors. For example, chimeric antigen receptor T (CAR-T) cell therapy has shown significant effects and potential for curing patients with B-cell malignancies. In contrast, it remains a challenge for CAR-T cell therapy to gain similar success in solid tumors. The anti-tumor effect of endogenous or adoptively transferred tumor-specific T cells depends largely on their differentiation status. T cells at early differentiation stage show better anti-tumor therapeutic effects than fully differentiated effector T cells. In cancer patients, the persistence of tumor-specific T cells with the stem cell memory or precursor phenotype is significantly associated with improved therapeutic outcomes; therefore, adoptively transfered CAR-T cells with stem cell memory and/or central memory is expected to gain better anti-tumor effects. Herein we focused on the in vitro optimized culture and expansion system to obtain CAR-T cells with stem cell memory or central memory phenotype for the review.
Humans ; Immunotherapy, Adoptive/methods* ; Receptors, Chimeric Antigen/genetics* ; Neoplasms/immunology* ; Immunologic Memory ; T-Lymphocytes/immunology* ; Memory T Cells/immunology* ; Animals ; Stem Cells/cytology* ; Cell Differentiation

Age-associated changes in immune cells have been linked to an increased risk for infection. However, a global and detailed characterization of the changes that human circulating immune cells undergo with age is lacking. Here, we combined scRNA-seq, mass cytometry and scATAC-seq to compare immune cell types in peripheral blood collected from young and old subjects and patients with COVID-19. We found that the immune cell landscape was reprogrammed with age and was characterized by T cell polarization from naive and memory cells to effector, cytotoxic, exhausted and regulatory cells, along with increased late natural killer cells, age-associated B cells, inflammatory monocytes and age-associated dendritic cells. In addition, the expression of genes, which were implicated in coronavirus susceptibility, was upregulated in a cell subtype-specific manner with age. Notably, COVID-19 promoted age-induced immune cell polarization and gene expression related to inflammation and cellular senescence. Therefore, these findings suggest that a dysregulated immune system and increased gene expression associated with SARS-CoV-2 susceptibility may at least partially account for COVID-19 vulnerability in the elderly.
Adult ; Aged ; Aged, 80 and over ; Aging ; genetics ; immunology ; Betacoronavirus ; CD4-Positive T-Lymphocytes ; metabolism ; Cell Lineage ; Chromatin Assembly and Disassembly ; Coronavirus Infections ; immunology ; Cytokine Release Syndrome ; etiology ; immunology ; Cytokines ; biosynthesis ; genetics ; Disease Susceptibility ; Flow Cytometry ; methods ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Gene Rearrangement ; Humans ; Immune System ; cytology ; growth & development ; immunology ; Immunocompetence ; genetics ; Inflammation ; genetics ; immunology ; Mass Spectrometry ; methods ; Middle Aged ; Pandemics ; Pneumonia, Viral ; immunology ; Sequence Analysis, RNA ; Single-Cell Analysis ; Transcriptome ; Young Adult

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. This malignancy is associated with poor prognosis and high mortality. Novel approaches for prolonging the overall survival of patients with advanced HCC are urgently needed. The antitumor activities of adoptive cell transfer therapy (ACT), such as strategies based on tumor-infiltrating lymphocytes and cytokine-induced killer cells, are more effective than those of traditional strategies. Currently, chimeric antigen receptor T-cell (CAR-T) immunotherapy has achieved numerous breakthroughs in the treatment of hematological malignancies, including relapsed or refractory lymphoblastic leukemia and refractory large B-cell lymphoma. Nevertheless, this approach only provides a modest benefit in the treatment of solid tumors. The clinical results of CAR-T immunotherapy for HCC that could be obtained at present are limited. Some published studies have demonstrated that CAR-T could inhibit tumor growth and cause severe side effects. In this review, we summarized the current application of ACT, the challenges encountered by CAR-T technology in HCC treatment, and some possible strategies for the future direction of immunotherapeutic research.
Adoptive Transfer ; methods ; Carcinoma, Hepatocellular ; immunology ; therapy ; Humans ; Immunotherapy, Adoptive ; methods ; Liver Neoplasms ; immunology ; therapy ; Lymphocytes, Tumor-Infiltrating ; cytology ; Randomized Controlled Trials as Topic ; Receptors, Chimeric Antigen ; T-Lymphocytes ; cytology

The activation mechanism of chimeric antigen receptor (CAR)-engineered T cells may differ substantially from T cells carrying native T cell receptor, but this difference remains poorly understood. We present the first comprehensive portrait of single-cell level transcriptional and cytokine signatures of anti-CD19/4-1BB/CD28/CD3ζ CAR-T cells upon antigen-specific stimulation. Both CD4 helper T (T) cells and CD8 cytotoxic CAR-T cells are equally effective in directly killing target tumor cells and their cytotoxic activity is associated with the elevation of a range of T1 and T2 signature cytokines, e.g., interferon γ, tumor necrotic factor α, interleukin 5 (IL5), and IL13, as confirmed by the expression of master transcription factor genes TBX21 and GATA3. However, rather than conforming to stringent T1 or T2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed T1/T2 function in the same cell. The regulatory T cell activity, although observed in a small fraction of activated cells, emerges from this hybrid T1/T2 population. Granulocyte-macrophage colony stimulating factor (GM-CSF) is produced from the majority of cells regardless of the polarization states, further contrasting CAR-T to classic T cells. Surprisingly, the cytokine response is minimally associated with differentiation status, although all major differentiation subsets such as naïve, central memory, effector memory, and effector are detected. All these suggest that the activation of CAR-engineered T cells is a canonical process that leads to a highly mixed response combining both type 1 and type 2 cytokines together with GM-CSF, supporting the notion that polyfunctional CAR-T cells correlate with objective response of patients in clinical trials. This work provides new insights into the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients.
Antigens ; metabolism ; CTLA-4 Antigen ; metabolism ; Cell Differentiation ; drug effects ; Cell Line ; Cytokines ; metabolism ; Cytotoxicity, Immunologic ; drug effects ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Lymphocyte Activation ; drug effects ; immunology ; Lymphocyte Subsets ; drug effects ; metabolism ; Phenotype ; Proteomics ; Receptors, Chimeric Antigen ; metabolism ; Single-Cell Analysis ; methods ; T-Lymphocytes, Regulatory ; drug effects ; metabolism ; Th1 Cells ; cytology ; drug effects ; Th2 Cells ; cytology ; drug effects ; Transcription, Genetic ; drug effects ; Up-Regulation ; drug effects

Astragalus membranaceus may be a potential therapy for childhood asthma but its driving mechanism remains elusive. The main components of A. membranaceus were identified by HPLC. The children with asthma remission were divided into two combination group (control group, the combination of budesonide and terbutaline) and A. membranaceus group (treatment group, the combination of budesonide, terbutaline and A. membranaceus). The therapeutic results were compared between two groups after 3-month therapy. Porcine peripheral blood mononuclear cells (PBMCs) were isolated from venous blood by using density gradient centrifugation on percoll. The levels of FoxP3, EGF-β, IL-17 and IL-23 from PBMCs and serum IgE were measured. The relative percentage of Treg/Th17 cells was determined using flow cytometry. The main components of A. membranaceus were calycosin-7-O-glucoside, isoquercitrin, ononin, calycosin, quercetin, genistein, kaempferol, isorhamnetin and formononetin, all of which may contribute to asthma therapy. Lung function was significantly improved in the treatment group when compared with a control group (P < 0.05). The efficacy in preventing the occurrence of childhood asthma was higher in the treatment group than the control group (P < 0.05). The levels of IgE, IL-17 and IL-23 were reduced significantly in the treatment group when compared with the control group, while the levels of FoxP3 and TGF-β were increased in the treatment group when compared with the control group (P < 0.05). A. membranaceus increased the percentage of Treg cells and reduced the percentage of Th17 cells. A. membranaceus is potential natural product for improving the therapeutic efficacy of combination therapy of budesonide and terbutaline for the children with asthma remission by modulating the balance of Treg/Th17 cells.
Animals ; Asthma ; drug therapy ; immunology ; Astragalus propinquus ; chemistry ; Budesonide ; administration & dosage ; Cells, Cultured ; Child ; Child, Preschool ; Cytokines ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Female ; Humans ; Immunologic Factors ; administration & dosage ; pharmacology ; Leukocytes, Mononuclear ; drug effects ; metabolism ; Lung ; drug effects ; physiology ; Male ; Swine ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; Terbutaline ; administration & dosage ; Th17 Cells ; cytology ; drug effects ; Treatment Outcome

OBJECTIVEAging is associated with the development of diseases because of immunosuppression and altered functioning of the neuroendocrine system. The medicinal properties of Morinda citrifolia L. have been widely exploited for the treatment of age-associated diseases. This study aims to investigate the in vitro and in vivo effects of noni (M. citrifolia) fruit juice (NFJ) on neuro-immunomodulation in the lymph node lymphocytes of F344 rats.

METHODSLymphocytes isolated from axillary and inguinal lymph nodes of young (3-4 months) and old (18-21 months) rats were treated in vitro with different concentrations (0.0001%, 0.01%, and 1%) of NFJ for a period of 24 h. In the in vivo study, old (16-17 months) male F344 rats were treated with 5 mL/kg body weight of 5%, 10% and 20% of NFJ, twice a day, by oral gavage, and lymph node lymphocytes were isolated after 60 d. Concanavalin A (Con A)-induced lymphocyte proliferation, interleukin-2 (IL-2) and interferon-γ (IFN-γ) production and expression of intracellular markers, such as phospho-extracellular signal-regulated kinase (p-ERK1/2), phospho-cAMP response element-binding protein, phospho-protein kinase B (p-Akt), phospho-tyrosine hydroxylase (p-TH), phospho-nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor-α (p-IκB-α) and phospho-nuclear factor-κB (p-NF-κB p65 and p50) were examined in the lymphocytes of lymph nodes.

RESULTSNFJ increased Con A-induced lymphocyte proliferation, IL-2 and IFN-γ production, and p-ERK1/2 expression both in vitro and in vivo. In in vivo NFJ-treated old rats, lymph node lymphocytes showed increased expression of p-TH and Akt, nitric oxide production and decreased expression of p-NF-κB p65 and p50.

CONCLUSIONThese results suggest that the immunostimulatory properties of NFJ are facilitated through intracellular signaling pathways involving ERK1/2, Akt and NF-κB.

Adjuvants, Immunologic ; metabolism ; Aging ; immunology ; metabolism ; Animals ; Cell Proliferation ; Fruit ; chemistry ; metabolism ; Fruit and Vegetable Juices ; analysis ; Humans ; Interleukin-2 ; immunology ; Lymph Nodes ; cytology ; immunology ; Lymphocytes ; cytology ; immunology ; Male ; Morinda ; chemistry ; metabolism ; NF-kappa B ; immunology ; Plant Preparations ; metabolism ; Rats ; Rats, Inbred F344 ; Transcription Factor RelA ; immunology

OBJECTIVE: To study the effect of electro- acupuncture at Zusanli acupoint in regulating perioperative cell immune functions in rats. METHODS: Forty-two SD rats were divided into blank control group (=6), model group (=18), and electroacupuncture group (=18). The rats in the latter two groups underwent thigh incision and femoral dissection under anesthesia; the rats in electro-acupuncture group received electro-acupuncture at bilateral Zusanli acupoint for 15 min before anesthesia and 1 h after the surgery. The rats in the model group and electro-acupuncture group were sacrificed at 6 h, 24 h, and 72 h after the operation and blood samples were taken from the ventricle for analyzing CD3, CD4, and CD8 T cell subpopulations and calculation of CD4/CD8 using flow cytometry. ELISA was used to detect the levels of interleukin-1 (IL-1) and IL-6. RESULTS: The CD3 T cell subpopulation was significantly lower in the model group and electro-acupuncture group than in the blank group at 6 h and 24 h after the operation. At 72 h after the operation, CD3 subpopulation levels still remained low in the model group, but recovered the control level in electro-acupuncture group. At each time point of measurement, CD3 level was significantly lower in the model group than in the electro-acupuncture group. CD4 level in the model group was significantly lowered at 6 h and 24 h after the operation, and recovered the control level at 72 h. In the electro-acupuncture group, CD4 level was significantly lowered at 6 h after the operation, but recovered the control level at 24 h. At 24 h and 72 h, the levels of CD4 were significantly lower in the model group than in the electro-acupuncture group. CD8 level underwent no significant changes after the operation in either the model group or electro-acupuncture group. CD4/CD8 was significantly lowered at 24 h and 72 h after the operation in the model group but showed no significant variation in the electro-acupuncture group. Compared with that in the control group, IL-1 level was significantly lowered in both the model group and electroacupuncture group at 6 h, 24 h, and 72 h after the operation, and was significantly lower in the model group than in the electroacupuncture group at these time points. IL-6 level increased significantly in the model group and the electro- acupuncture group at 6 h and 24 h. at 72 h, IL-6 level was obviously lowered in the electro-acupuncture group but remained elevated in the model group. CONCLUSIONS Electro-acupuncture alleviates postoperative immune suppression and promotes recovery of the immune function in rats, suggesting a protective effect of electro-acupuncture at Zusanli acupoint on cellular immune function after surgery.
Acupuncture Points ; Animals ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Electroacupuncture ; methods ; Femur ; surgery ; Flow Cytometry ; Humans ; Immunity, Cellular ; Perioperative Period ; Rats ; Rats, Sprague-Dawley ; T-Lymphocyte Subsets ; cytology ; immunology

BACKGROUND: Previous studies have shown that the neutrophil-to-lymphocyte ratio (NLR) has a significant impact on the prognosis of many malignant tumors such as gastric cancer, colorectal cancer and pancreatic cancer, but the study on the prognosis of patients with resectable lung adenocarcinoma is less. The aim of this study is to investigate the correlation between the NLR and the clinicopathologic features of adenocarcinoma of lung patients who underwent radical pneumonectomy. Furthermore, this study aimed to clarify the predictive and prognostic significance of NLR in patients who underwent pneumonectomy for lung adenocarcinoma. METHODS: This study reviewed the medical records of 163 patients with lung adenocarcinoma who underwent pneumonectomy. The receiver operating characteristic (ROC) curve and Youden index were used to determine the cut-off value of the NLR. Survival curves were described by Kaplan-Meier method and compared by Log-rank test. The univariate and multivariate analyses were performed with the Cox proportional hazard model to identify the prognostic factors. RESULTS: When the NLR value was 2.96, the Youden index was maximal, with a sensitivity of 77.5% and a specificity of 75.9%. The 5-year survival rate in the low NLR group was higher than that in the high NLR group (P<0.05). The univariate and multivariate analyses showed that TNM staging and NLR were independent factors in predicting survival rate. CONCLUSIONS The NLR value was a simple and useful tool to predict the prognosis of lung adenocarcinoma after radical pneumonectomy.
Adenocarcinoma ; diagnosis ; immunology ; pathology ; surgery ; Adenocarcinoma of Lung ; Aged ; Cell Count ; Female ; Follow-Up Studies ; Humans ; Kaplan-Meier Estimate ; Lung Neoplasms ; diagnosis ; immunology ; pathology ; surgery ; Lymphocytes ; cytology ; Male ; Middle Aged ; Neoplasm Staging ; Neutrophils ; cytology ; Pneumonectomy ; Prognosis ; ROC Curve ; Retrospective Studies