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

OBJECTIVETo study the influence of Yanyankang powder on Th1/Th2 in rats with experimental autoimmune uveitis (EAU).

METHODSThe EAU models were induced in Lewis rats by immunization with interphotoreceptor retinoid binding protein (IRBP) 1177-1191 in complete Freund's adjuvant. The rats were randomly divided into 3 groups: a model control group, a Yanyankang group, and a prednisone group, 9 rats in each group. The model control group was intervened with saline solution by gavage. The Yanyankang group was intervened with Yanyankang powder 4 g/(kg day) by gavage. The prednisone group were intervened with prednisone acetate tablets 5 mg/(kg d) by gavage. All groups were intervened after immunization once every 2 days for 18 days and monitored by slit-lamp biomicroscopy daily until day 18. The levels of gamma interferon (INF-γ) and interleukin-10 (IL-10) in the supernatants of T cells were determined by enzyme-linked immunosorbent assay. Polymerase chain reaction (PCR) technology was used for measuring Th1 and Th2 related cytokine mRNA expressions.

RESULTSSlighter intraocular inflammation was found in the Yanyankang group and the prednisone group than the control group. The levels of the IFN-γ and IL-10 in the supernatants of the spleen lymph node cells were 382.33±6.30, 155.87±4.46 μg/L in the Yanyankang group and 270.93±7.76, 265.32±11.88 μg/L in the prednisone group. Both had significant differences compared with the control group (941.53±8.59, 20.67±4.65 μg/L; =0.01). The PCR results showed the same tendency.

CONCLUSIONYanyankang powder showed favorable effects in the rats with EAU by influencing the function of Th1 and Th2 cells.

Animals ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Eye ; pathology ; Female ; Immunization ; Inflammation ; pathology ; Interferon-gamma ; genetics ; metabolism ; Interleukin-10 ; genetics ; metabolism ; Lymph Nodes ; drug effects ; metabolism ; Powders ; RNA, Messenger ; genetics ; metabolism ; Rats, Inbred Lew ; Spleen ; metabolism ; Th1 Cells ; drug effects ; immunology ; Th2 Cells ; drug effects ; immunology ; Uveitis ; drug therapy ; genetics ; immunology

OBJECTIVE: The aim of this study was to determine the expression of GATA-3 and the level of Th1 and Th2 cytokines upon repeated exposure to staphylococcal enterotoxin B(SEB) of different concentrations in the maxillary sinus mucosa of rabbits. METHOD: The rabbits were randomly divided into 2 groups (24 rabbits per group): low-dose SEB group and high-dose SEB group. The low-dose SEB group and high-dose SEB group received daily injections of 0.6 ng of SEB (2 ml) and 60 ng of SEB (2 ml) into the left maxillary sinus of rabbits for 28 days, respectively. Concurrent treatment of the right maxillary sinus with normal saline was used as a control. Six rabbits chosen randomly in two groups were killed on days 3, 7, 14, and 28, and to obtain the sinus mucosa from the two-side maxillary sinuses for measurement. Mucosal levels of IL-2, IL-4, IL-5, and IFN-γ were measured using ABC-ELISA. Tissue expression of GATA-3 were examined using Real-time PCR and immunohistochemistry. RESULT: IFN-γ and IL-2 levels were significantly elevated in the high-dose SEB group compared with the low-dose SEB and control groups on days 7, 14, and 28 (P < 0.05). However, IL-4 and IL-5 levels were markedly enhanced in the low-dose SEB group compared with the high-dose SEB and control groups on days 14 and 28 (P < 0.05). Real-time PCR showed that the expression of GATA-3 mRNA in the low-dose SEB group was markedly enhanced, and immunohistochemical staining illustrated that the number of GATA-3 positive cells was markedly increased in the low-dose SEB group as compared with the high-dose SEB group (P < 0.05). No significant differences were observed in GATA-3 expression between the high-dose SEB and the control groups (P > 0.05). CONCLUSION SEB promoted Th1 cytokines production at high concentrations, and enhanced Th2 cytokines expression and Th2 immune response at low concentrations.
Animals ; Cytokines ; metabolism ; Enterotoxins ; administration & dosage ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; Interferon-gamma ; metabolism ; Interleukin-2 ; metabolism ; Interleukin-4 ; metabolism ; Interleukin-5 ; metabolism ; Male ; Maxillary Sinus ; drug effects ; metabolism ; Nasal Mucosa ; metabolism ; RNA, Messenger ; metabolism ; Rabbits ; Th1 Cells ; Th2 Cells ; Transcription Factors ; metabolism

In this study, OVA-induced asthma mice was taken as the model, and orally administered with different concentration of ethanol extracts of crude and processed Stemona tuberosa, in order to determine the cytokine level released from Th1 and Th2 in splenocytes. RT-PCR was carried out to determine the genetic expression of T-bet/GATA-3 in lung, and compare the differentiation between ethanol extracts of crude and processed S. tuberosa in therapeutic effect on asthma in mice. According to the results, compared with the crude samples, processed samples significantly increased the levels of inflammatory factor INF-gamma (P < 0.05) and decreased IL-5 (P < 0.05) in splenocytes. According to the RT-PCR results, the administration of processed samples could increase the ratio of T-bet/GATA-3 (P < 0.05). The experiment showed that ethanol extracts of both crude and processed S. tuberosa could treat asthma by regulating Th1/Th2 ratio, but processed samples showed more notable effect. This indicated that crude and processed S. tuberosa had significant pharmacological difference. Therefore, it was more rational to apply processed S. tuberosa in clinical treatment of asthma and chronic cough, which layed a foundation for further revealing the processing mechanism of S. tuberosa.
Administration, Oral ; Animals ; Asthma ; drug therapy ; immunology ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; therapeutic use ; GATA3 Transcription Factor ; metabolism ; Gene Expression Regulation ; drug effects ; Mice ; Mice, Inbred BALB C ; Stemonaceae ; chemistry ; T-Box Domain Proteins ; metabolism ; Th1 Cells ; drug effects ; secretion ; Th2 Cells ; drug effects ; secretion

OBJECTIVEAnti-asthma herbal medicine intervention (ASHMI(TM)), a combination of three traditional Chinese medicinal herbs developed in our laboratory, has demonstrated efficacy in both mouse models of allergic asthma, and a double-blind placebo-controlled clinical trial in patients with asthma. This study was designed to determine if the anti-inflammatory effects of individual herbal constituents of ASHMI(TM) exhibited synergy.

METHODSEffects of ASHMI and its components aqueous extracts of Lingzhi (Ganoderma lucidum), Kushen (Sophora flavescens) and Gancao (Glycyrrhiza uralensis), on Th2 cytokine secretion by murine memory Th2 cells (D10.G4.1) and eotaxin-1 secretion by human lung fibroblast (HLF-1) cells were determined by measuring levels in culture supernatants by enzyme-linked immunosorbent assay. Potential synergistic effects were determined by computing interaction indices from concentration-effect curve parameters.

RESULTSIndividual Lingzhi, Kushen and Gancao extracts and ASHMI (the combination of individual extracts) inhibited production of interleukin (IL)-4 and IL-5 by murine memory Th2 cells and eotaxin-1 production by HLF-1 cells. The mean 25%-inhibitory-concentration (IC25) values (mg/mL) for ASHMI, Lingzhi, Kushen and Gancao for IL-4 production were 30.9, 79.4, 123, and 64.6, respectively; for IL-5 production were 30.2, 263, 123.2 and 100, respectively; for eotaxin-1 were 13.2, 16.2, 30.2, and 25.1, respectively. The IC50 values (mg/mL) for ASHMI, Lingzhi, Kushen and Gancao for IL-4 production were 158.5, 239.9, 446.7, and 281.8, respectively; for eotaxin-1 were 38.1, 33.1, 100, and 158.5, respectively. The interaction indices of ASHMI constituents at IC25 were 0.35 for IL-4, 0.21 for IL-5 and 0.59 for eotaxin-1. The interaction indices at IC50 values were 0.50 for IL-4 and 0.62 for eotaxin-1 inhibition. Inhibition of IL-5 did not reach IC50 values. All interaction indices were below 1 which indicated synergy.

CONCLUSIONBy comparing the interaction index values, we find that constituents in ASHMI(TM) synergistically inhibited eotaxin-1 production as well as Th2 cytokine production.

Animals ; Asthma ; drug therapy ; metabolism ; Cell Line ; Chemokine CCL11 ; metabolism ; Down-Regulation ; drug effects ; Drug Synergism ; Drugs, Chinese Herbal ; analysis ; pharmacology ; Fibroblasts ; drug effects ; metabolism ; Humans ; Interleukin-4 ; metabolism ; Interleukin-5 ; genetics ; immunology ; Mice ; Plants, Medicinal ; chemistry ; Th2 Cells ; drug effects ; metabolism

OBJECTIVETo explore the regulatory mechanism of Xiaoyin Recipe () on the T helper 1/T helper 2 (Th1/Th2) immune balance.

METHODSThirty-six experimental animals were divided into three groups, 12 rats in each group: blank control group (B group), negative control group (N group), and Xiaoyin Recipe treatment group (T group). The latter two groups received immunization of experimental autoimmune thyroiditis (EAT), and T group were treated with Xiaoyin Recipe for a month. Then, the expression of Th1-Th2-related genes in peripheral blood mononuclear cells (PBMCs) were screened with Oligo GEArray Rat Th1-Th2-Th3 Microarray. The expressions of tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), T-box expressed in T-cells (T-bet), and GATA-binding protein-3 (GATA-3) were detected by real-time polymerase chain reaction (RT-PCR).

RESULTSGene array screening showed that compared to N group, in T group after Xiaoyin Recipe treatment, 3 genes were upregulated in EAT rats, including interleukin-27 receptor alpha (IL-27rα), glomulin (Glmn), and GATA-3, while 38 genes were downregulated, such as CD28, IL-18, signal transducer, and activator of transcription 1 (STAT1), T-bet, TNF receptor superfamily member 4 (TNFRSF4), TNF ligand superfamily member 5 (TNFSF5), and TNF receptor superfamily member 5 (TNFRSF5). While RT-PCR showed that there was an increased level of TNF-α mRNA (P<0.01), an elevated ratio of T-bet/GATA-3, and a decreased level of IL-10 mRNA in PBMC of N and T group compared to B group (P <0.01); and after treatment with Xiaoyin Recipe, IL-10 mRNA level increased (P <0.01), while TNF-α mRNA level and T-bet/GATA-3 ratio decreased in T group compared to N group (P <0.01).

CONCLUSIONXiaoyin Recipe for psoriasis could induce a Th1/Th2 balance drift toward Th2 in PBMC of EAT rats and thus improve the conditions.

Animals ; Autoantibodies ; blood ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; GATA3 Transcription Factor ; genetics ; metabolism ; Gene Expression Regulation ; drug effects ; Interleukin-10 ; genetics ; metabolism ; Psoriasis ; blood ; drug therapy ; immunology ; pathology ; Rats ; Rats, Wistar ; T-Box Domain Proteins ; genetics ; metabolism ; Th1-Th2 Balance ; drug effects ; Th2 Cells ; drug effects ; immunology ; Thyroiditis, Autoimmune ; blood ; drug therapy ; immunology ; pathology ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

OBJECTIVETo study the regulatory effect of San'ao Tang on Thl/Th2 cells and its interference on transcription factor GATA-3 and T-bet-mRNA in asthmatic rats.

METHODRat asthma model was established by abdominal injection and aerosol inhalation of OVA. The content of cytokines IL-4 and IFN-gamma in alveolus lavage fluid of asthmatic rats was detected before and after the oral administration with large, medium, low dose of San'ao Tang and dexamethasone. T-bet and GATA-3 mRNA expressions in lung tissues were determined by RT-PCR.

RESULTCompared with other groups, the medium-dose group showed a notable decrease in IL-4 in rat BALF and an increasing IFN-gamma. Its IL-4/IFN-gamma is significant lower than the asthma group, with expressions of lung tissue GATA-3 and transcription factor T-bet-mRNA in line with changes in IL-4 and IFN-gamma.

CONCLUSIONSan'ao Tang may have a inhibitory effect on airway inflammation of asthmatic rats by regulating Th1/Th2 transfer factors.

Animals ; Asthma ; drug therapy ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; therapeutic use ; Male ; Rats ; Rats, Sprague-Dawley ; Th1 Cells ; drug effects ; metabolism ; Th2 Cells ; drug effects ; metabolism

BACKGROUNDThe signal transducer and activator of transcription 6 (STAT6) expression in lung epithelial cells plays a pivotal role in asthma pathogenesis. Activation of STAT6 expression results in T helper cell type 2 (Th2) cell differentiation leading to Th2-mediated IgE production, development of allergic airway inflammation and hyperreactivity. Therefore, antagonizing the expression and/or the function of STAT6 could be used as a mode of therapy for allergic airway inflammation.

METHODSIn this study, we synthesized a 20-mer phosphorothioate antisense oligonucleotide (ASODN) overlapping the translation starting site of STAT6 and constructed STAT6 antisense RNA (pANTI-STAT6), then transfected them into murine spleen lymphocytes and analyzed the effects of antagonizing STAT6 function in vitro and in a murine model of asthma.

RESULTSIn vitro, we showed suppression of STAT6 expression and interleukin (IL)-4 production of lymphocytes by STAT6 ASODN. This effect was more prominent when cells were cultured with pANTI-STAT6. In a murine model of asthma associated with allergic pulmonary inflammation in ovalbumin (OVA)-sensitized mice, local intranasal administration of fluorescein isothiocyanate (FITC)-labeled STAT6 ASODN to DNA uptake in lung cells was accompanied by a reduction of intracellular STAT6 expression. Such intrapulmonary blockade of STAT6 expression abrogated signs of lung inflammation, infiltration of eosinophils and Th2 cytokine production.

CONCLUSIONThese data suggest a critical role of STAT6 in the pathogenesis of asthma and the use of local delivery of STAT6 ASODN as a novel approach for the treatment of allergic airway inflammation such as in asthma.

Animals ; Asthma ; drug therapy ; metabolism ; Blotting, Western ; Cell Differentiation ; drug effects ; Cells, Cultured ; Female ; Interleukin-4 ; metabolism ; Lymphocytes ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Oligonucleotides, Antisense ; chemistry ; pharmacology ; Phosphates ; pharmacology ; RNA, Antisense ; chemistry ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; STAT6 Transcription Factor ; genetics ; metabolism ; Th2 Cells ; drug effects ; metabolism

BACKGROUNDEffects of icariin on airway inflammation in asthmatic rats and the intervention of LPS induced inflammation are interfered with the machanism of icariin. Our study aimed to observe the effect of icariin on ovalbumin-induced imbalance of Th1/Th2 cytokine expression and its mechanism.

METHODSSixty male SD rats were randomly divided into control group (PBS), asthma group (ovalbumin (OVA)-induced), dexamethasone group, and OVA+icariin low, medium and high dose groups (5, 10, 20 mg/kg, respectively). Each group had ten rats. The model of OVA sensitization was a rat asthma model. Enzyme-linked immunosorbent assay (ELISA) method was used to observe the effects of icariin on interleukin-4 (IL-4) and inerferon γ (IFN-γ) in rats' lung tissue. Immunohistochemical staining was applied to detect the intervention effects of icariin on T cells (T-bet) and gatabinding protein 3 (GATA-3) in rat pulmonary tissue. Realtime RT-PCR was used to observe the intervention effects of icariin on T-bet and GATA-3 mRNA expression in rat pulmonary tissue and spleen lymphocytes. Western blotting was used to observe the icariin intervention effects on T-bet, GATA-3 and nuclear factor-Kappa B (NF-κB) p65 protein expressions in rat pulmonary tissue.

RESULTSThe ELISA results from pulmonary tissue showed that IL-4 expression was significantly reduced (P < 0.05), while the IFN-γ expression increased but not significantly when we compared OVA+icariin medium and high dose groups with the asthma group. Immunohistochemical staining of pulmonary tissue showed that the GATA-3 decreased significantly while the T-bet staining did not change in the OVA+icariin high dose group. In pulmonary tissue and spleen lymphocytes T-bet and GATA-3 mRNA expressions were significantly reduced (P < 0.05) in icariin treatment groups compared with the asthma model group. GATA-3 and T-bet mRNA in rat spleen lymphocytes in the asthma group were higher than in the control group. GATA-3 mRNA expression in pulmonary tissue significantly decreased (P < 0.05) while T-bet mRNA expression decreased but not significantly in the icariin treatment group compared with the asthma group. T-bet and GATA-3 protein expressions in pulmonary tissue increased significantly compared with the asthma group, which meant that icariin could inhibit the increase of GATA-3 protein, but not of T-bet. The bronchus, blood vessels and periphery pulmonary tissue had infiltration of inflammatory cells in the OVA+icariin high dose group while NF-κB p65 cells were reduced, and expression of NF-κB p65 in this group was less than in the asthma group. The expression of total p65 protein decreased with icariin treatment while the expression of cytoplasmic p65 protein increased.

CONCLUSIONSIcariin could regulate the imbalance of Th1/Th2 cytokines in asthmatic rat pulmonary tissue. Icariin could regulate the imbalance of Th1/Th2 associated transcription factors T-bet and GATA-3 in asthmatic rat pulmonary tissue and spleen lymphocytes. Icariin could inhibit the activation of NF-κB p65 protein in asthmatic rat pulmonary tissue.

Animals ; Asthma ; drug therapy ; immunology ; metabolism ; Blotting, Western ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Flavonoids ; therapeutic use ; GATA3 Transcription Factor ; metabolism ; Immunohistochemistry ; Interferon-gamma ; metabolism ; Interleukin-4 ; metabolism ; Lung ; metabolism ; Male ; Ovalbumin ; metabolism ; Polymerase Chain Reaction ; Rats ; Rats, Sprague-Dawley ; T-Box Domain Proteins ; metabolism ; Th1 Cells ; drug effects ; metabolism ; Th2 Cells ; drug effects ; metabolism ; Transcription Factor RelA ; metabolism

Anti-cancer drug bleomycin (BLM) can cause acute lung injury (ALI) which often results in pulmonary fibrosis due to a failure of resolving acute inflammatory response. The aim of this study is to investigate whether toll-like receptor (TLR) 2 mediates BLM-induced ALI, inflammation and fibrosis. BLM-induced dendritic cells (DCs) maturation was analyzed by flow cytometry and cytokine secretion was detected by the ELISA method. The expression and activity of p38 and ERK MAPK were determined with Western blotting. The roles of TLR2 in ALI, inflammation and fibrosis were investigated in C57BL/6 mice administered intratracheally with BLM. The results demonstrated that BLM-administered mice had higher expression of TLR2 (P<0.001) and its signaling molecules. Blocking TLR2 significantly inhibited the maturation of DCs and reversed BLM-stimulated secretion of cytokines in DCs, such as IL-6 (P<0.001), IL-17 (P<0.05) and IL-23 (P<0.05). TLR2 inhibition attenuated BLM-induced increase of inflammatory cells in bronchoalveolar lavage fluid (BALF), and reversed the immunosuppressive microenvironment by enhancing TH1 response (P<0.05) and inhibiting TH2 (P<0.001), Treg (P<0.01) and TH17 (P<0.01) responses. Importantly, blocking TLR2 in vivo significantly protected BLM-administered mice from pulmonary injury, inflammation and fibrosis and subsequently increased BLM-induced animal survival (from 50% to 92%). Therefore, TLR2 is a novel potential target for ALI and pulmonary fibrosis.
Acute Lung Injury ; chemically induced ; metabolism ; pathology ; Animals ; Bleomycin ; toxicity ; Bronchoalveolar Lavage Fluid ; Cells, Cultured ; Cytokines ; secretion ; Dendritic Cells ; cytology ; metabolism ; Inflammation ; chemically induced ; metabolism ; pathology ; Interleukin-17 ; secretion ; Interleukin-23 ; secretion ; Interleukin-6 ; secretion ; Lung ; metabolism ; MAP Kinase Signaling System ; Male ; Mice ; Mice, Inbred C57BL ; Pulmonary Fibrosis ; chemically induced ; metabolism ; pathology ; T-Lymphocytes, Regulatory ; drug effects ; Th1 Cells ; drug effects ; Th2 Cells ; drug effects ; Toll-Like Receptor 2 ; metabolism ; physiology