The aim of this study was to compare the immune responses of C57BL/6 mice immunized with two pathogens, foot-and-mouth disease virus (FMDV) and Senecavirus A (SVA), and to provide clues for revealing the regulatory mechanisms of acquired immunity. Inactivated and purified FMDV and SVA antigens were used to immunize C57BL/6 mice respectively, and the mice immunized with PBS were taken as the control. The percentages of Th1 and Th2 cells in the spleen lymphocytes of mice in each group were analyzed by flow cytometry at 14 and 28 days after immunization. RNA-Seq was performed for the spleen. Mouse macrophages were stimulated with the antigens in vitro to examine the expression of the differentially expressed genes (DEGs) screened out. The results showed that 14 days after immunization, there was no significant difference in the magnitude of the Th1/Th2 immune response elicited by the FMDV and SVA antigens. After 28 days, the magnitudes of the Th1 and Th2 immune responses elicited by the SVA antigen were higher than those elicited by the FMDV antigen. RNA-Seq revealed two common DEGs, Rsad2 and Tspan8, between the two immunization groups, which indicated that the two genes may be involved in the activation of the Th1/Th2 immune responses by FMDV and SVA antigens. FMDV and SVA antigens stimulated macrophages to secrete interleukin (IL)-12 and IL-33 in vitro, and the expression of Tspan8 and Rsad2 was consistent with the RNA-Seq results. The expression of Rsad2 was regulated by type I interferons (IFNα, IFNβ). In this study, we obtained the DEGs involved in the immune responses to the two antigens in mouse spleen, which provides a molecular basis for investigating the immune response mechanisms induced by FMDV and SVA.
Animals ; Foot-and-Mouth Disease Virus/genetics* ; Mice ; Spleen/cytology* ; Mice, Inbred C57BL ; Antigens, Viral/genetics* ; Transcriptome ; Th1 Cells/immunology* ; Immunization ; Viral Vaccines/immunology* ; Th2 Cells/immunology* ; Foot-and-Mouth Disease/immunology* ; Interleukin-33/genetics* ; Female ; Macrophages/immunology* ; Picornaviridae

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 explore the change in the expression of extracellular heat shock protein 70 (eHSP70) and interleukin 2 (IL-2) and their correlation in intestine of rats with severe scald injury, and to observe the effects of eHSP70 on CD3(+) T lymphocytes in Peyer's patch of intestine in rats with severe scald injury in vitro.

METHODS(1) Sixty male SD rats were divided into normal control group (NC, n=10, only anesthetized) and scald group (S, n=50) according to the random number table. Rats in scald group were inflicted with 30% total body surface area full-thickness scald on the back. Ten rats from group NC immediately after anesthetization and 10 rats from group S at post injury hour (PIH) 3, 6, 12, 24, 48 were sacrificed to harvest their small intestines. The expressions of eHSP70 and IL-2 were determined with enzyme-linked immunosorbent assay (ELISA), and their correlation was analyzed. (2) Another 2 male SD rats were inflicted with the same injury as above. At PIH 12, CD3(+) T lymphocytes in Peyer's patch of small intestine were isolated and cultured with RPMI 1640 nutrient solution containing 10% fetal bovine serum. Cells were divided into blank control group (BC) and 5, 10, 20 μg/mL eHSP70 groups according to the random number table, with 6 wells in each group. Cells in group BC didn't receive any other treatment, while cells in the latter three groups were treated with corresponding mass concentration of recombinant rat eHSP70. After being cultured for 48 hours, the proportions of Th1 and Th2 in CD3(+) T lymphocytes, and the apoptosis rate of CD3(+) T lymphocytes were detected with flow cytometer, while the expressions of IL-2 and IL-10 in culture supernatant of cells were determined with ELISA. The cell experiments were repeated for 10 times. Data were processed with one-way analysis of variance, Kruskal-Wallis rank sum test, SNK-q test, and Pearson correlation analysis.

RESULTS(1) Compared with those in group NC [(1 278±135) and (48.6±4.9) ng/mg], the levels of eHSP70 [(728±93), (412±31), (314±21), (528±40), (1 028±97) ng/mg] and IL-2 [(38.6±2.3), (32.3±1.0), (25.3±3.6), (33.9±4.1), (44.3±2.6) ng/mg] in intestine of rats in group S obviously decreased at PIH 3, 6, 12, 24, 48 (with q values from 3.48 to 5.32, P values below 0.05), reaching the nadir both at PIH 12, with a significantly positive correlation between the level of IL-2 and the level of eHSP70 (r=0.920, P<0.01). (2) Compared with those in group BC [(8.6±1.1)% and (3.75±0.45)%], the proportion of Th1 obviously increased [(11.3±2.1)%, (15.7±1.8)%, (10.8±1.5)%, with q values from 2.97 to 4.57, P values below 0.05], while the proportion of Th2 obviously decreased [(2.39±0.38)%, (1.05±0.23)%, (2.67±0.26)%, with q values from 2.48 to 4.32, P values below 0.05] in CD3(+) T lymphocytes of rats in 5, 10, 20 μg/mL eHSP70 groups. Compared with those in group BC [(34.3±2.2)% and (254±16) pg/mL], the apoptosis rate of CD3(+) T lymphocytes obviously decreased [(26.1±2.6)%, (20.7±1.5)%, (31.5±2.4)%, with q values from 3.47 to 4.95, P values below 0.05], while the level of IL-2 obviously increased [(417±22), (587±19), (307±27) pg/mL, with q values from 3.02 to 4.98, P values below 0.05] in culture supernatant of CD3(+) T lymphocytes of rats in 5, 10, 20 μg/mL eHSP70 groups. There was no significant difference in the level of IL-10 in culture supernatant of CD3(+) T lymphocytes of rats among the four groups (F=2.12, P>0.05).

CONCLUSIONSThe expressions of eHSP70 and IL-2 in intestine of rats are decreased after severe scald, with a obviously positive correlation between them. eHSP70 can promote the differentiation of CD3(+) T lymphocytes in Th1 orientation, decrease the apoptosis rate of the cells, and promote the release of IL-2 of cells in Peyer's patch of intestine in rats with severe scald injury in vitro.

Animals ; Burns ; metabolism ; CD3 Complex ; metabolism ; Enzyme-Linked Immunosorbent Assay ; HSP70 Heat-Shock Proteins ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-2 ; metabolism ; Intestine, Small ; metabolism ; Male ; Peyer's Patches ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Th1 Cells ; cytology

OBJECTIVETo investigate the effects of Th1/Th17 cell imbalance on the pathogenesis of acute graft-versus-host disease (GVHD) in mice.

METHODSIn a murine GVHD model of C57BL/6 (H-2(b)), a low dose of halofuginone (HF) was applied for treating the recipients in order to result in Th1/Th17 imbalance. Rechipient mice were divided into GVHD group (without HF intervention) and GVHD plus HF group (treated by HF). The recipients were monitored for survival rate, clinical scores of acute GVHD, contents of circulatory Th1 and Th17 cells, Th1/Th17 ratio and serum level of IFN-γ and IL-17A. Expression levels of IFN-γ and IL-17A in target organs were analyzed by using real-time PCR, and the target organs were delivered for histological examinations.

RESULTSRecipients treated with HF showed that all the mortality, circulatory Th1/Th17 ratio and clinical score were higher than those in the mice without HF intervention (P < 0.05). Circulatory Th1/Th17 ratio positively correlates with clinical score (P < 0.001). HF administration reduces the expression level of intestinal IL-17A and increases intrahepatic and intestinal IFN-γ level (P < 0.05), HF treatment aggravates GVHD in liver and small intestine with augmented hepatic and intestinal inflammation.

CONCLUSIONTh1/Th17 imbalance contributes to the pathogenesis of acute GVHD.

Animals ; Disease Models, Animal ; Graft vs Host Disease ; immunology ; Interferon-gamma ; blood ; Interleukin-17 ; blood ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Piperidines ; Quinazolinones ; Th1 Cells ; cytology ; Th17 Cells ; cytology

OBJECTIVETo investigate the effects of intragastric administration of human interferon-α (hIFN-α)-transformed Bifidobacterium on immune functions of mice.

METHODSThe E.coli-Bifidobacterium shuttle expression vector containing hIFN-α gene was constructed and transformed into Bifidobacterium. The hIFN-α-transformed Bifidobacterium suspension (1010 /ml) was prepared after induction with 0.2% L-arabinose for hIFN-α expression and administered intragastrically in male Balb/C mice at the dose of 0.1 ml every other day for 2 weeks, with the mice receiving empty vector-transformed Bifidobacteria as the negative control and those having an equal volume of saline as the blank control. The percentages of mononuclear cell subsets in the thymus, spleen and blood were detected in the mice by flow cytometry, and the serum levels of IL-4, IL-12, IFN-γ and TNF-α were assayed using mouse cytokine FlowCytomix Kit.

RESULTSThe percentages of CD3⁺CD8⁺ and CD4⁺CD8⁺ cells in the thymus, CD3⁺CD4⁺, CD3⁺CD8⁺ and CD4⁺CD8⁺ cells in the spleen, and CD3⁺CD8⁺ cells in the blood all increased significantly in IFN group as compared with those in the negative and blank control groups (P<0.01 or 0.05). The serum level of IFN-γ also increased significantly (P<0.05) while IL-4 level remained unchanged in IFN group compared with those in the two groups.

CONCLUSIONIntragastric administration of hIFN-α-transformed Bifidobacterium promotes lymphocyte proliferation and maturation and increases the serum levels of Th1 cytokines in mice.

Animals ; Bifidobacterium ; Cell Proliferation ; Genetic Vectors ; Humans ; Interferon-alpha ; pharmacology ; Interferon-gamma ; blood ; Interleukin-12 ; blood ; Interleukin-4 ; blood ; Lymphocyte Activation ; drug effects ; Male ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; pharmacology ; Spleen ; cytology ; Th1 Cells ; cytology ; Thymus Gland ; cytology ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVETo investigate the influence of maternal atopy on cord blood effector T cells and to identify these biologic markers as predictors of atopic dermatitis (AD).

METHODSSeventy mother-infant pairs were recruited in this prospective birth cohort study. Suspected factors for allergy, including maternal allergic history, total serum IgE, and maternal age at birth, were collected. Mother peripheral blood samples and cord blood were obtained and assayed for the percentage of interferon-γ (IFN-γ) and interleukin 4 (IL-4) producing T cells(Th1 and Th2 respectively) using flow cytometry. Their offspring at the age of 2 years old were evaluated by their dermatologist whether they had AD. Statistical analysis was performed using multiple logistic regression models and receiver-operating characteristic curve was employed to predict atopic dermatitis.

RESULTSTwenty-one allergic and 49 nonallergic mothers were recruited in this study. During the first two years of life, 15.7% children (n = 11) developed a physician-diagnosed AD (all children were the only child in the family). In group with maternal allergic rhinitis, a significantly increased percentage of Th2 was observed in peripheral blood of mother (7.10[1.18;16.1]% vs. 0.37[0.25;0.72]%, U = 10.0, P < 0.05) and cord blood of newborns (1.02[0.57;1.34]% vs. 0.21[0.15;0.42]%, U = 127.5, P < 0.05), respectively. Maternal atopic history did not affect the percentage of Th1 cells in cord blood (0.69[0.40;1.12]% vs.0.50[0.31;0.66]%, U = 361.0, P > 0.05). Children with reduced Th1/Th2 ratio in cord blood had a higher risk to develop AD (OR = 1.72, P = 0.001) . The model including Th1/Th2, maternal allergy, maternal age at birth and maternal total IgE showed high ability to discriminate children with and without AD. AUC was 0.907 (95% CI: 0.804-1.011, P < 0.001).

CONCLUSIONSElevated IL-4⁺CD4⁺ T cells in cord blood were of relevance with maternal allergic history. Imbalance between Th1 cell and Th2 cell at birth are associated with maternal allergy and promoted subsequent AD development.

Adult ; Child, Preschool ; Dermatitis, Atopic ; immunology ; Female ; Fetal Blood ; cytology ; Flow Cytometry ; Humans ; Immunoglobulin E ; blood ; Infant ; Infant, Newborn ; Mothers ; Prospective Studies ; Rhinitis, Allergic ; blood ; immunology ; Th1 Cells ; cytology ; Th1-Th2 Balance ; Th2 Cells ; cytology

OBJECTIVEParkinson's disease (PD), a neurodegenerative disorder, has been reported to be associated with brain neuroinflammation in its pathogenesis. Herein, changes in peripheral immune system were determined to better understand PD pathogenesis and provide possible target for treatment of PD through improvement of immune disorder.

METHODS1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was intraperitoneally injected into mice to prepare PD model. Expression levels of pro-inflammatory and anti-inflammatory cytokines and transcription factors of CD4+ T lymphocyte subsets in spleen and mesenteric lymph nodes and concentrations of the cytokines in serum were examined on day 7 after MPTP injection. Percentages of CD4+ T lymphocyte subsets were measured by flow cytometry.

RESULTSMPTP induced PD-like changes such as motor and behavioral deficits and nigrostriatal impairment. Expression levels of the pro-inflammatory cytokines including interferon (IFN)-γ, interleukin (IL)-2, IL-17 and IL-22, in spleen and mesenteric lymph nodes were upregulated and their concentrations in serum were elevated in PD progression. But, the concentrations of the anti-inflammatory cytokines including IL-4, IL-10 and transforming growth factor (TGF)-β were not altered in the two lymphoid tissues or serum of PD mice. In addition, expression of T-box in T cells (T-bet), the specific transcription factor of helper T (Th) 1 cells, was downregulated, but expression of transcription factor forkhead box p3 (Foxp3), the transcription factor of regulatory T (Treg) cells, was upregulated. In support of the results, the numbers of IFN-γ-producing CD4+ cells (Th1 cells) were reduced but CD4+CD25+ cells (Treg cells) were elevated in both the lymphoid tissues of PD mice.

CONCLUSIONPD has a dysfunction of peripheral immune system. It manifests enhancement of proinflammatory response and CD4+ T cell differentiation bias towards Treg cells away from Th1 cells.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Animals ; CD4-Positive T-Lymphocytes ; pathology ; Cell Differentiation ; Cytokines ; blood ; Disease Models, Animal ; Flow Cytometry ; Forkhead Transcription Factors ; metabolism ; Interferon-gamma ; blood ; Interleukin-10 ; blood ; Interleukin-17 ; blood ; Interleukin-2 ; blood ; Interleukin-4 ; blood ; Interleukins ; blood ; Lymph Nodes ; cytology ; Lymphocyte Activation ; Mice ; Parkinson Disease ; immunology ; physiopathology ; Spleen ; cytology ; T-Box Domain Proteins ; metabolism ; T-Lymphocytes, Regulatory ; Th1 Cells ; Transforming Growth Factor beta ; blood

This study was aimed to detect the level of the peripheral blood Breg and CD4(+) T cell subgroups in patients with chronic idiopathic thrombocytopenic purpura (CITP) before and after therapy, and to analyse the charge of related cytokines and their correlation, to explore their roles in the pathogenesis of CITP. A total of 35 CITP cases were taken as the research group and 35 healthy persons were served as the control group. The peripheral blood mononuclear cells (PBMNC) were separated, the percentages of Th1, Th17, Th22 and Breg cells were detected by flow cytometry before and after treatment of glucocorticoid, and the IFN-γ, IL-17, IL-22 and IL-10 levels from PBMNC culture supernatant also were determined by ELISA. The results showed that there was significant difference as compared with the healthy controls, the proportion of peripheral blood Th1, Th17, Th22 cell subgroups all increased in CITP patients before treatment with glucocorticoid, the regulatory B cells (Breg) ratio was reduced, the differences had statistical significance (P < 0.05), but the differences were no statistically significant after treatment with glucocorticoid (P > 0.05). The levels of IFN-γ, IL-17, IL-22 from culture supernatant all increased in CITP patients before treatment, the level of IL-10 was lower than that of the healthy control, the difference was statistically significant (P < 0.05), but the there were no statistically significant differences after treatment (P > 0.05). There were positive correlation between the Breg cells and IL-10 expression in CITP patients (P < 0.05), the Breg cells and Th1, Th17, Th22 cells showed a negative correlation, IL-10 and IFN-γ, IL-17, IL-22 levels also showed a negative correlation. It is concluded that the down-regulation of regulatory B cells proportion and the IL-10 level may participate in the mechanism of CD4(+) T cell immunity disorder in CITP, which can provide new targets and ideas for the clinical immune regulation therapy.
Adolescent ; Adult ; Aged ; B-Lymphocytes, Regulatory ; cytology ; immunology ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; Case-Control Studies ; Cells, Cultured ; Female ; Humans ; Interleukin-10 ; immunology ; Male ; Middle Aged ; Purpura, Thrombocytopenic, Idiopathic ; blood ; immunology ; Th1 Cells ; Th17 Cells ; Young Adult

OBJECTIVETo investigate the effects of down-regulating Tim-3 gene in the peripheral blood mononuclear cells (PBMCs) of an asthmatic mouse model by short hairpin RNA (shRNA) and to explore the effect of Tim-3 on Th1 and Th17 cell differentiation.

METHODSAn asthmatic murine model was established by ovalbumin sensitization and challenge. PBMCs were isolated from asthmatic mice and transfected by shRNA targeting Tim-3 gene. The mRNA and protein expressions of Tim-3 were detected by quantitative PCR and Western blot. Flow cytometry analysis was performed to determine the levels of Th1 and Th17, and ELISA was performed to determine concentrations of IFN-γ, IL-4 and IL-17 in the supernatant.

RESULTSTim-3 mRNA expression in PBMCs was significantly increased in asthmatic mice. The mRNA and protein expression of Tim-3 decreased significantly in the shRNA group. Compared with the negative groups, Th1 cell levels increased and Th17 cell levels decreased significantly in the asthmatic groups after Tim-3 shRNA interference. In the Tim-3 shRNA interference groups concentrations of IFN-γ increased significantly while IL-17 decreased significantly.

CONCLUSIONSSpecific Tim-3 shRNA effectively silences the expression of Tim-3 and change in Tim-3 expression could affect T cell differentiation.

Animals ; Asthma ; immunology ; therapy ; Cell Differentiation ; Female ; Gene Silencing ; Hepatitis A Virus Cellular Receptor 2 ; Mice ; Mice, Inbred BALB C ; RNA, Small Interfering ; genetics ; Receptors, Virus ; antagonists & inhibitors ; genetics ; Th1 Cells ; cytology ; Th17 Cells ; cytology

BACKGROUND: Interleukin-17 (IL-17)-producing T helper (Th) 17 cells are considered as a new subset of cells critical to the development of rheumatoid arthritis (RA). We aimed to investigate the distribution of Th1 and Th17 cells and their association with disease activity, and determine the Th17-related cytokine levels in the peripheral blood of RA patients. METHODS: Peripheral blood mononuclear cells from 55 RA and 20 osteoarthritis (OA) patients were stimulated with mitogen, and the distributions of CD4+Interferon (INF)+IL-17- (Th1 cells) and CD4+INF-IL-17+ (Th17 cells) were examined by flow cytometry. Serum levels of IL-6, IL-17, IL-21, IL-23, and tumor necrosis factor (TNF)-alpha were measured by ELISA. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were recorded. The 28-joint disease activity score (DAS28) was also assessed. RESULTS: The median percentage of Th17 cells was higher in RA patients than in OA patients (P=0.04), and in active than in inactive RA (P=0.03), whereas that of Th1 cells was similar in both groups. Similarly, the levels of IL-17, IL-21, and IL-23 were detected in a significantly higher proportion of RA patients than OA patients and the frequencies of detectable IL-6, IL-17, and IL-21 were higher in active RA than in inactive RA group. The percentage of Th17 cells positively correlated with the DAS28, ESR, and CRP levels. CONCLUSIONS: These observations suggest that Th17 cells and Th17-related cytokines play an important role in RA pathogenesis and that the level of Th17 cells in peripheral blood is associated with disease activity in RA.
Adult ; Aged ; Arthritis, Rheumatoid/blood/metabolism/*pathology ; Blood Sedimentation ; C-Reactive Protein/analysis ; Cytokines/blood ; Female ; Humans ; Male ; Middle Aged ; Osteoarthritis/blood/metabolism/pathology ; Severity of Illness Index ; Th1 Cells/cytology/immunology/metabolism ; Th17 Cells/*cytology/immunology/metabolism