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

OBJECTIVE: To investigate the protective effect of Zengye Decoction (, ZYD) on the submandibular glands (SMGs) in nonobese diabetic (NOD) mice. METHODS: Twenty-seven female NOD mice were randomly equally divided into 3 groups: the model group, the hydroxychloroquine (HCQ) group, and the ZYD group. Nine C57/B6 mice served as the normal group. After 1-week acclimation, the HCQ and ZYD groups were intragastrically administered with HCQ and ZYD, respectively, and the normal and model groups were administered with normal saline. Changes in the salivary flow rate were observed. Mice from all 4 groups were sacrificed at the age of 20 weeks. The serum and SMGs were collected. Serum cytokines gamma-interferon (IFN-γ), interleukin-10 (IL-10) were detected by enzyme-linked immunosorbent assay. Histological changes in the submandibular glands were examined by hematoxylin and eosin staining. The mRNA expression of IFN-γ, IL-10 and vasoactive intestinal peptide (VIP) in the submandibular glands were measured by real-time polymerase chain reaction. RESULTS: Compared with the model group, the salivary flow of the ZYD group significantly increased (P<0.05), the extent of the histological changes was ameliorated (P<0.05), and the Th1/Th2 cytokine imbalance was remedied (P<0.05). In the ZYD-treated mice, the VIP mRNA was up-regulated (P<0.05). CONCLUSIONS ZYD is beneficial in protecting structure and function of SMGs in NOD mice. The mechanism may be associated with the correction of the Th1/Th2 cytokine imbalance, and with the prevention of a progressive decline of the VIP level.
Animals ; Cytokines ; blood ; Drugs, Chinese Herbal ; pharmacology ; Female ; Mice ; Mice, Inbred C57BL ; Mice, Inbred NOD ; Salivation ; drug effects ; Sjogren's Syndrome ; drug therapy ; immunology ; Submandibular Gland ; drug effects ; pathology ; Th1 Cells ; immunology ; Th2 Cells ; immunology ; Vasoactive Intestinal Peptide ; genetics

OBJECTIVETo explore the changes in T helper lymphocytes and their subsets in children with tic disorders (TD) and their clinical significance.

METHODSFlow cytometry was used to measure the percentages of T helper lymphocytes and their subsets in the peripheral blood of children with TD and healthy children (controls).

RESULTSThe percentage of T helper lymphocytes was significantly lower in the TD group than in the control group (P<0.001). The abnormal rate of T helper lymphocytes in the TD group was significantly higher than that in the control group (68.7% vs 18.8%; P<0.001). The percentage of T helper lymphocytes was negatively correlated with Yale Global Tic Severity Scale score (r=-0.3945, P<0.001). As for the subsets of T helper lymphocytes, the TD group had a significantly higher percentage of Th1 cells and a significantly lower percentage of Th2 cells compared with the control group (P<0.001).

CONCLUSIONSThe abnormality of T helper lymphocytes and the imbalance of their subsets may be associated with the pathogenesis of TD in children. The percentage of T helper lymphocytes can be used as an indicator for assessing the severity of TD.

Child ; Child, Preschool ; Female ; Flow Cytometry ; Humans ; Lymphocyte Count ; Male ; T-Lymphocyte Subsets ; immunology ; T-Lymphocytes, Helper-Inducer ; immunology ; Th1 Cells ; immunology ; Th2 Cells ; immunology ; Tic Disorders ; genetics ; immunology

Background: Immune disorder is an important feature of patients with out-of-hospital cardiac arrest (OHCA) after the return of spontaneous circulation (ROSC). We investigated the expression of circulatory T helper type (Th) 1, Th2, and Th17 cells to explore the early immune alteration in OHCA patients after ROSC. Methods: During July-September 2016 and March-September 2017, 65 consecutive OHCA patients with ROSC >12 h and 30 healthy individuals were enrolled in this study. Clinical and 28-day survival data were collected. Peripheral blood samples were analyzed to evaluate the expression of Th1/Th2/Th17 cells by flow cytometry from OHCA patients after ROSC on days 1 and 3 and from healthy individuals. Results: Compared with healthy individuals, T lymphocyte counts and Th1 cell counts decreased on days 1 and 3 after ROSC (1464 [1198, 2152] vs. 779 [481, 1140] vs. 581 [324, 1118]/μl, χ = 30.342, P < 0.001; 154 [90, 246] vs. 39 [19, 78] vs. 24 [12, 53]/μl, χ = 42.880, P < 0.001), and Th2 and Th17 cell counts decreased on day 3 (17.0 [10.8, 24.0] vs. 9.0 [3.0, 15.5]/μl, Z = -3.228, P = 0.001; 4.7 [2.7, 9.1] vs. 2.7 [1.0, 6.5]/μl, Z = -2.294, P = 0.022). No change in CD4+/CD3+ lymphocyte ratio was seen on day 1 or day 3 (57.9 [49.4, 63.0] vs. 55.4 [46.5, 66.5] vs. 55.4 [50.2, 67.0]%, χ = 0.171, P = 0.918). Th1/CD4+ lymphocyte ratio decreased on days 1 and 3 (19.0 [14.0, 24.9] vs. 9.3 [4.6, 13.9] vs. 9.5 [4.9, 13.6]%, χ = 25.754, P < 0.001), and Th2/CD4+ lymphocyte ratio increased on day 1 and decreased on day 3 (1.9 [1.2, 2.5] vs. 2.5 [1.6, 4.0] vs. 1.9 [1.6, 3.8]%, χ = 6.913, P = 0.032). Th1/Th2 cell ratio also decreased on both days (9.4 [7.3, 13.5] vs. 3.1 [1.9, 5.6] vs. 4.2 [2.8, 5.9], χ = 44.262, P < 0.001). Despite an upward trend in the median of Th17/CD4+ lymphocyte ratio in OHCA patients, there was no significant difference compared with healthy individuals (0.9 [0.4, 1.2] vs. 0.7 [0.4, 1.2] vs. 0.6 [0.3, 1.0]%, χ = 2.620, P = 0.270). The dynamic expression of Th1/Th2/Th17 cells on days 1 and 3 were simultaneously analyzed in 28/53 OHCA patients who survived >3 days; patients were divided into survivors (n = 10) and nonsurvivors (n = 18) based on 28-day survival. No significant differences in Th1/Th2/Th17 cell counts, ratios in CD4+ lymphocytes, and Th1/Th2 cell ratio were seen between survivors and nonsurvivors on both days (all P > 0.05). There was no difference over time in both survivors and nonsurvivors (all P > 0.05). Conclusion Downregulated T lymphocyte counts, including Th1/Th2/Th17 subsets and Th1/Th2 cell ratio imbalance, occur in the early period after ROSC, that may be involved in immune dysfunction in OHCA patients.
Aged ; Cardiopulmonary Resuscitation ; Female ; Humans ; Male ; Middle Aged ; Out-of-Hospital Cardiac Arrest ; immunology ; therapy ; Th1 Cells ; Th17 Cells ; Th2 Cells

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

OBJECTIVEThe aim of the study was to investigate whether Orai1 antibody intraperitoneal injection could improve the condition of allergic rhinitis (AR) in mice.

METHODSTwenty-four BALB/C mice (SPF grade) were classified into 4 groups (AR group, Control group, Experimental group 1 and experimental group 2) according to a random number table. A mouse model of AR was established (Control group was established by phosphate buffered solution), and experimental group 1 and Experimental group 2 were established through intraperitoneal injection of 100 μg and 150 μg Orai1 antibody respectively. The number of sneezing and rubbing and eosinophilia in mice were assessed after different doses of Orai1 antibody intraperitoneal injection were applied. Then Orai1 protein and its mRNA in nasal mucosa, histomine, eosionphil cation protein (ECP), interlukin (IL)-1β, IL-4, IL-5 and IL-6 and their mRNA in nasal lavage fluid (NLF) and nasal mucosa were evaluated using enzyme linked immunosorbent assay (ELISA) and real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). Furthermore, Orai1 protein and its mRNA in Th2 cells in peripheral blood, IL-4 and IL-5 in peripheral serum and their mRNAs in Th2 cells were also examined through ELISA and real-time RT-PCR. The data were analyzed by a statistical software of Graph Pad Prism 5.

RESULTSThere were significant differences in sneezing, nasal rubbing and local invading eosinophils in nasal mucosa after the treatment (t100 μg=7.88, t100 μg=9.92, t100 μg=4.30, respectively; t150 μg=16.43, t150 μg=16.31, t150 μg=9.35, respectively, all P-values<0.01). The Orai1 antibody intervention decreased contents of Orai1 in nasal mucosa, histomine, ECP, IL-1β, IL-4, IL-5 and IL-6. The contents of experimental group 1 were (0.186±0.015) μg/ml, (6.618±0.180) ng/ml, (2.555±0.031) ng/ml, (85.26±2.94) pg/ml, (55.12±1.21) pg/ml, (58.45±2.11) pg/ml and (77.12±2.13) pg/ml, respectively. The contents of experimental group 2 were (0.089±0.003) μg/ml, (4.501±0.310) ng/ml, (1.260±0.017) ng/ml, (48.49±2.12) pg/ml, (33.15±0.87) pg/ml, (38.24±0.95) pg/ml and (51.72±0.81) pg/ml, respectively. The differences were siginificant between group 1, group 2 and AR group(t value was 3.29, 10.44, 9.45, 17.53, 74.53, 87.06, 3.98; 8.54, 13.32, 23.00, 20.89, 80.73, 103.70, 13.34, all P<0.01). However, there were no significant differences in Orai1 protein and its mRNA in peripheral Th2 cells, IL-4 and IL-5 in peripheral serum and their mRNAs in Th2 cells (all P-values>0.05). In addition, the effect of 150 μg Orai1 antibody treatment was better than 100 μg one (all P-values<0.05).

CONCLUSIONOrai1 antibody intraperitoneal injection can improve the symptoms of AR mice, and alleviate the condition of allergic inflammation. Orai1 may become a novel aim in the AR study.

Animals ; Antibodies ; pharmacology ; Calcium Channels ; immunology ; metabolism ; Cytokines ; immunology ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Eosinophilia ; therapy ; Eosinophils ; immunology ; Immunotherapy ; Inflammation ; Injections, Intraperitoneal ; Mice ; Mice, Inbred BALB C ; Nasal Mucosa ; metabolism ; ORAI1 Protein ; RNA, Messenger ; Rhinitis, Allergic ; therapy ; Th2 Cells ; immunology

EPSAH is an exopolysaccharide from Aphanothece halophytica GR02. The present study was designed to evaluate its toxicity and adjuvant potential in the specific cellular and humoral immune responses in ovalbumin (OVA) in mice. EPSAH did not cause any mortality and side effects when the mice were administered subcutaneously twice at the dose of 50 mg·kg(-1). Hemolytic activity in vitro indicated that EPSAH was non-hemolytic. Splenocyte proliferation in vitro was assayed with different concentrations of EPSAH. The mice were immunized subcutaneously with OVA 0.1 mg alone or with OVA 0.1 mg dissolved in saline containing Alum (0.2 mg) or EPSAH (0.2, 0.4, or 0.8 mg) on Day 1 and 15. Two weeks later, splenocyte proliferation, natural killer (NK) cell activity, production of cytokines IL-2 from splenocytes, and serum OVA-specific antibody titers were measured. Phagocytic activity, production of pro-inflammatory cytokines IL-1 and IL-12 in mice peritoneal macrophages were also determined. EPSAH showed a dose-dependent stimulating effect on mitogen-induced proliferation. The Con A-, LPS-, and OVA-induced splenocyte proliferation and the serum OVA-specific IgG, IgG1, and IgG2a antibody titers in the immunized mice were significantly enhanced. EPSAH also significantly promoted the production of Th1 cytokine IL-2. Besides, EPSAH remarkably increased the killing activities of NK cells from splenocytes in the immunized mice. In addition, EPSAH enhanced phagocytic activity and the generation of pro-inflammatory cytokines IL-1 and IL-12 in macrophages. These results indicated that EPSAH had a strong potential to increase both cellular and humoral immune responses, particularly promoting the development of Th1 polarization.
Adjuvants, Immunologic ; administration & dosage ; Animals ; Cyanobacteria ; chemistry ; Female ; Immunity, Cellular ; Immunity, Humoral ; Immunization ; Interleukin-12 ; immunology ; Interleukin-2 ; immunology ; Killer Cells, Natural ; immunology ; Mice ; Mice, Inbred ICR ; Ovalbumin ; immunology ; Polysaccharides ; administration & dosage ; immunology ; Rabbits ; Th1 Cells ; immunology ; Th2 Cells ; immunology

OBJECTIVETo investigate the effect of Qiguiyin Decoction, QGYD) on multidrug-resistant Pseudomonas aeruginosa infection in Sprague-Dawley (SD) rats.

METHODSA pseudomonal infection model in SD rats was established by injecting multidrug-resistant P. aeruginosa intraperitoneally. Infected rats were randomized into four groups treated with Pure water, QGYD, ceftazidime, or combined QGYD and ceftazidime. Blood samples were obtained from the abdominal aorta. Serum was then collected and analyzed by peptide array for immune responsiveness to multidrug-resistant beta-lactamase proteins, including Verona integronen-coded metallo-beta-lactamase 1 (VIM-1), Sao Paulo metallo-beta-lactamase 1 (SPM-1), and Temoniera (TEMs). Blood levels of interleukin-1β (IL-1β), interleukin-4 (IL-4), and interferon-γ (IFN-γ) were assessed by enzyme-linked immunosorbent assay.

RESULTSQGYD enhanced antibody reactivity against VIM-1 [epitopes 7-11 and 36-40] and TEM-1 [epitopes 26-27, 52-55, and 66-70]. QGYD treatment restored the compromised antibody reactivity against VIM-1 [epitopes 53-54 and 56-58] and SPM-1 [epitopes 16-19 and 82-85] following pseudomonal infection. Serum levels of IL-1β and Th1/Th2 in the rats were significantly elevated following pseudomonal infection (P<0.05 orP<0.01). In contrast, QGYD and combination QGYD and ceftazidime treatment restored the elevated serum IL-1β and Th1/Th2 levels to normal (P>0.05).

CONCLUSIONSQGYD improves the immune response to pseudomonal infection in rats by stimulating the production of protective antibodies against drug-resistant proteins VIM-1, SPM-1, and TEM-1. In addition, it protects the immune system and maintains immune responsiveness by restoring IL-1β and Th1/Th2 levels.

Animals ; Antibodies, Bacterial ; blood ; Drug Resistance, Multiple, Bacterial ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Interleukin-1beta ; blood ; Male ; Pseudomonas Infections ; drug therapy ; Pseudomonas aeruginosa ; Rats ; Rats, Sprague-Dawley ; Th1 Cells ; immunology ; Th2 Cells ; immunology ; beta-Lactamases ; immunology

OBJECTIVETo evaluate the roles of various cytokines, histone acetyltransferase (HAT) and histone deacetylase (HDAC) in the pathogenesis of bronchial asthma.

METHODSBALB/C mice were randomly assigned to control, untreated asthma, hormone treatment and TSA treatment groups. Bronchial asthma was induced by intraperitoneal injections and atomization inhalation of ovalbumin (OVA) in the asthma, hormone treatment and trichostatin (TSA) treatment groups. The mice in the hormone treatment and TSA treatment groups were administered with dexamethasone 1.0 mg/kg and TSA 1.0 mg/kg respectively by an intraperitoneal injection 30 minutes before challenge of asthma. At 24 hours after the last challenge, IL-4, IL-8 and IFN- levels in serum were measured using ELISA, and activities of HAT and HDAC in peripheral blood mononuclear cells (PBMC) were determined by the enzyme linked immunofluorescence assay.

RESULTSThe serum levels of IL-4 and IL-8 in the untreated asthma group were higher than in the control, hormone treatment and TSA treatment groups (P<0.05). There was no difference in the serum levels of IL-4 and IL-8 among the control, hormone treatment and TSA treatment groups (P>0.05). The activity of HDAC in the untreated asthma group was lower than in the control, hormone treatment and TSA treatment groups (P<0.05). Hormone treatment significantly decreased the activity of HAT compared with the untreated asthma group (P<0.05). There was no difference in the activities of HAT and HDAC among the control, hormone treatment and TSA treatment groups (P>0.05).

CONCLUSIONSThe decreased activity of HDAC leads to an increased secretion of inflammatory factors and thus induces asthma.

Animals ; Asthma ; enzymology ; etiology ; immunology ; Cytokines ; blood ; Histone Acetyltransferases ; physiology ; Histone Deacetylases ; physiology ; Male ; Mice ; Mice, Inbred BALB C ; Th1 Cells ; immunology ; Th2 Cells ; immunology

OBJECTIVETo observe effects of mild moxibustion and lovastatin on immunologic function in rabbits with chronic hyperlipidaemia and atherosclerosis (AS) to initially explain regulating rules of mild moxibustion on immunologic function.

METHODSAmong thirty-two Japanese male big-ear rabbits, 8 rabbits were randomly selec ted as a blank group, the rest 24 rabbits were fed with method of endothelial injury and high-fat diet to establish AS model. The blank group was raised with normal diet and free water. After ten weeks of model establishment, the rest 24 rabbits were randomly divided into a model group, a moxibustion group and a medicine group, eight rabbits in each one. Moxibustion was applied at "Shenque" (CV 8) and "Zusanli" (ST 36) for 10 min per acupoint per day in the moxibustion group, while intragastric administration of 3.6 mg/kg lovastatin capsule was applied in the medicine group. After treatment, serum was acquired. Spectrophotometry method was adapted to measure cholesterol (TC) and high-density lipoprotein (HDL-C) and evaluated atherosclerosis index (AI), while enzyme-linked immunosorbent assay method was used to measure interferon-gamma (IFN-gamma) and interleukin-4 (IL-4).

RESULTS(1) The serum TC and HDL-C in the model group were significantly higher than those in the blank group, moxibustion group and medicine group (all P < 0.01). The mean value of AI was 1.683 +/- 0.486 in the moxibustion group, which was obviously lower than 20.301 +/- 4.022 in the model group (P < 0.01). (2) The ratio of Th1/Th2 was 0.569 +/- 0.143 in the moxibustion group and 0.445 +/- 0.079 in the medicine group, which were significantly lower than 0.917 +/- 0.255 in the model group (both P < 0.01), but there was no statistically significant difference between the moxibustion group and the medicine group (P > 0.05).

CONCLUSIONThe moxibustion for AS could reduce atherosclerosis index, influence drift and bias of helper T cell and regulate balance between humoral immunity and cellular immunity. As a result, status of relative balance of immunity is acquired, which could slow down the development of atherosclerosis and process of thrombus burst.

Animals ; Atherosclerosis ; immunology ; therapy ; Humans ; Hyperlipidemias ; immunology ; therapy ; Interferon-gamma ; immunology ; Interleukin-4 ; immunology ; Male ; Moxibustion ; Rabbits ; Th1 Cells ; immunology ; Th2 Cells ; immunology