Antigens, CD ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; metabolism ; CD3 Complex ; metabolism ; Cells, Cultured ; Humans ; Kinetics ; Lectins, C-Type ; metabolism ; Lymphocyte Activation ; T-Lymphocyte Subsets ; drug effects ; immunology ; metabolism

OBJECTIVE: To explore the role of DNAM-1 in the activation, proliferation and function of type Ⅰ regulatory T cells (Tr1 cells). METHODS: Anti-CD3/CD28 antibodies were used to stimulate mouse T cells derived from the spleen of wild-type (WT) mice, and the expression level of DNAM-1 in resting and activated Tr1 cells was evaluated with flow cytometry. Na?ve CD4⁺ T cells isolated by magnetic cell sorting from the spleens of WT mice and DNAM-1 knockout (KO) mice were cultured in Tr1 polarizing conditions for 3 days, after which CD25 and CD69 expressions were measured using flow cytometry. The induced Tr1 cells were labelled with CFSE and cultured in the presence of anti-CD/CD28 antibodies for 3 days, and their proliferative activity was analyzed. The expressions of IL-10 and p-STAT5 in DNAM-1-deficient Tr1 cells were detected before and after IL-2 stimulation. RESULTS: The expression level of DNAM-1 was significantly upregulated in CD4⁺ T cells and Tr1 cells after stimulation with anti-CD3/CD28 antibodies (P < 0.05). DNAM-1 knockout did not cause significant changes in the number or proportion of Tr1 cells, but but significantly increased the expression levels of the activation markers CD69 and CD25 (P < 0.05). Compared with WT Tr1 cells, DNAM-1-deficient Tr1 cells exhibited reduced proliferative activity in vitro (P < 0.05) with downregulated IL-10 production (P < 0.05) and decreased expressions of Il-10 and Gzmb mRNA (P < 0.05). In DNAM-1-deficient Tr1 cells, IL-2 stimulation significantly reduced IL-10 secretion level and the expression of p-STAT5 as compared with WT Tr1 cells. CONCLUSION DNAM-1 participate in the activation and proliferation of Tr1 cells and affect the biological functions of Tr1 cells through the IL-2/STAT5 pathway.
Animals ; Antigens, Differentiation, T-Lymphocyte ; CD28 Antigens/metabolism* ; Cell Proliferation ; Cells, Cultured ; Interleukin-10 ; Interleukin-2/metabolism* ; Mice ; RNA, Messenger ; STAT5 Transcription Factor/metabolism* ; T-Lymphocytes, Regulatory

In order to study the possible mechanism of CD226 monoclonal antibody (mAb)-mediated intracellular message transduction in human umbilical vein endothelial cells (HUVECs), the influence of CD226 mAb and its cross-linking by secondary antibody (II Ab) on the concentration changes in [Ca(2+)](i) in the HUVECs under different conditions were determined by confocal laser scanning microscopy. The main results are as follows. (1) When the culture medium was balanced by Hanks Buffer, [Ca(2+)](i) in HUVECs increased slowly after stimulation by CD226 mAb, whereas [Ca(2+)](i) increase was accompanied by [Ca(2+)](o) decrease after the mAb was cross-linked by goat anti-mouse IgG. Then [Ca(2+)](i) and [Ca(2+)](o) all returned to the normal level. (2) When the culture medium was balanced by D-Hanks buffer, [Ca(2+)](i) in HUVECs showed little variation when the cells were stimulated by CD226 mAb, but [Ca(2+)](i) decreased markedly after cross-linking. (3) When HUVECs were pretreated with EGTA, there was no variation in [Ca(2+)](i) of HUVECs after CD226 mAb stimulation alone or cross-linking of the mAb. Our results suggest that stimulation by CD226 mAb and cross-linking by goat anti-mouse IgG induce the variation of [Ca(2+)](i) in HUVECs under different conditions and the variation of [Ca(2+)](i) in HUVECs may play an important role in many physiological and pathological processes.
Antibodies, Monoclonal ; pharmacology ; Antigens, Differentiation, T-Lymphocyte ; immunology ; Calcium ; metabolism ; Cells, Cultured ; Human Umbilical Vein Endothelial Cells ; metabolism ; physiology ; Humans

Neutrophils are also known to acquire the characteristics of dendritic cells (DCs) under the appropriate conditions. In this study, neutrophils were cultivated in vitro in the presence or absence of compounds modulating their survival in an attempt to characterize the expression profile of the DC markers. Higher MHC-II, CD80, CD86, CD83, and CD40 expression levels were detected on the surface of the cultured neutrophils for 24 h than on the freshly isolated cells. The annexin V-positive cells showed a higher expression level of the DC markers than the annexin V-negative cells. The population of neutrophils double stained with annexin V and the DC markers increased after being incubated with agonistic anti-Fas Ab. LPS, the anti-apoptotic compound, decreased the CD86 and MHC-II expression levels but 50-60% of the DC marker-positive cells were detected in the annexin V-positive cells. In contrast, CD80, CD86, CD83, and HLA-DR mRNA levels increased in the GM-CSF-treated neutrophils but not in the anti-Fas Ab-treated neutrophils. T cell proliferation was inhibited by co-culturing them with anti-Fas Ab- or LPS-treated neutrophils at a high neutrophil:T cell ratio. However, the superantigen-mediated T cell proliferation was increased by the LPS-treated neutrophils but decreased by the anti-Fas Ab-treated neutrophils. There was a lower level of interferon-gamma production in the T cells co-cultured with anti-Fas Ab-treated neutrophils than with the LPS-treated neutrophils. This suggests that apoptotic neutrophils express DC markers on their surface and the differential expression of DC markers might have a detrimental effect on the immune reaction.
Antigen Presentation ; Antigens, CD/biosynthesis ; Antigens, CD95/pharmacology ; Antigens, Differentiation/*biosynthesis ; *Apoptosis ; Cells, Cultured ; Dendritic Cells/*metabolism ; Humans ; Lipopolysaccharides/pharmacology ; Lymphocyte Activation ; Neutrophils/*metabolism/physiology ; T-Lymphocytes/immunology

Asthma is a chronic disease of airway inflammation due to excessive T helper cell type 2 (Th2) response. Present treatment based on inhalation of synthetic glucocorticoids can only control Th2-driven chronic eosinophilic inflammation, but cannot change the immune tolerance of the body to external allergens. Regulatory T cells (Tregs) are the main negative regulatory cells of the immune response. Tregs play a great role in regulating allergic, autoimmune, graft-versus-host responses, and other immune responses. In this review, we will discuss the classification and biological characteristics, the established immunomodulatory mechanisms, and the characteristics of induced differentiation of Tregs. We will also discuss the progress of Tregs in the field of asthma. We believe that further studies on the regulatory mechanisms of Tregs will provide better treatments and control strategies for asthma.
Antigens, CD/analysis* ; Apyrase/analysis* ; Asthma/immunology* ; Cell Differentiation ; Cytokines/metabolism* ; Humans ; Lymphocyte Transfusion ; T-Lymphocytes, Regulatory/immunology*

OBJECTIVETo observe the change of activation and proliferation ability of rat T-lymphocytes after suppress ICOS gene expression by RNA interference.

METHODSFour interference sites targeting at rat ICOS gene were designed and four pairs of oligonucleotide fragments were cloned into the pSilencer 4.1-CMV neo plasmid vectors then transfected into rat lymphocytes with cationic liposome. The expression of mRNA and protein of ICOS was detected by RT-PCR and flow cytometry. The alteration of lymphocyte proliferation ability was evaluated by mix lymphocyte reaction, and the secretion levels of IFN-gamma and IL-4 were measured by ELISA procedure.

RESULTSAfter transfection, the expression of mRNA and protein of ICOS in test groups were lower than that in control groups (P < 0.05). The ability of T-lymphocytes in proliferation was poor and the levels of IFN-gamma and IL-4 were reduced with ICOS gene shut down.

CONCLUSIONSRNA interference plasmid vector can suppress ICOS expression in rat T-lymphocytes significantly, and it may be useful for further study on transplantation immunity tolerance.

Animals ; Antigens, Differentiation, T-Lymphocyte ; genetics ; metabolism ; Cell Proliferation ; Cells, Cultured ; Female ; Genetic Vectors ; Inducible T-Cell Co-Stimulator Protein ; Interferon-gamma ; metabolism ; Interleukin-4 ; metabolism ; Lymphocyte Activation ; Male ; RNA Interference ; Rats ; Rats, Sprague-Dawley ; T-Lymphocytes ; immunology ; metabolism ; Transfection

OBJECTIVETo investigate the infiltration of T lymphocytes with CD3 expression as surface marker and the activation of T lymphocytes with CD69 expression as activation marker.

METHODSNasal polyp tissue samples and peripheral blood were obtained from 21 patients. The normal inferior turbinate mucosa and peripheral blood were obtained as comparison. Flow cytometry was adopted to detect the expression of CD3 and CD69 of T lymphocytes.

RESULTSNasal polyp tissue consisted of abundant T lymphocytes. Activation marker CD69 was expressed in T lymphocytes of nasal polyps (36.96 +/- 2.50)% and peripheral blood (4.66 +/- 0.18)% from the same patient. The expression rates of CD69 after a short-term stimulation (5 h) in response to PDB were (59.88 +/- 2.59)% and (92.76 +/- 0.55)% respectively. While T lymphocytes was rarely detected in normal inferior turbinate and the expression of CD69 was low in peripheral blood from normal human but almost all T lymphocytes were activated after stimulation.

CONCLUSIONSThere were generous of T lymphocytes infiltrating in nasal polyps. The expression of CD69 in T lymphocytes was abnormally high, which indicated that T lymphocytes infiltrating in nasal polyps were in activated state immunologically.

Adolescent ; Adult ; Aged ; Antigens, CD ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; metabolism ; CD3 Complex ; metabolism ; Case-Control Studies ; Female ; Humans ; Lectins, C-Type ; metabolism ; Male ; Middle Aged ; Nasal Polyps ; immunology ; metabolism ; T-Lymphocytes ; metabolism ; Young Adult

This study was purposed to explore the effect of bone marrow derived mesenchymal stem cells (MSCs) on the expression of CD69 on cytokine-induced killer (CIK)/natural killer (NK) cells derived from cord blood and on the quantity ratio of CD4+CD25+ T regulatory cells in CIK/NK cell culture system using Transwell non-contact cell culture system. The experiments were divided into two groups: Transwell non-contact culture and mixture culture. The ratio of MSC to CIK/ NK cells was 1:20, 1:50 and 1:100. In mixture culture groups, MSC and CIK/NK cells were co-cultured by together contact as the same ratio of Transwell non-contact culture groups. The expression of CD69 on CIK/NK cells, as well as the quantity ratio of CD4+CD25+ T regulatory cells in CIK/NK cell culture were evaluated by flow cytometry. The results showed that the expression of CD69 on CIK/NK cells in experimental groups were significantly lower than that in control group (p<0.001). As to Transwell groups, CD69 expression on the CIK/NK cells at 1:20 ratio of MSC and CIK/NK was significantly lower than that at 1:50 and 1:100 ratio. There were no differences in the expression of CD69 on CIK cells in mixture groups with various MSC ratios, whereas the expression of CD69 on NK cells at 1:20 ratio was significantly lower than that at 1:50 and 1:100. The quantity ratio of CD4+CD25+ cells in CIK/NK cell culture system of experimental groups with MSC co-culture was significantly higher than that in control. As to Transwell groups, the ratio of CD4+CD25+ cells in CIK/NK cell culture system at 1:20 and 1:50 was significantly higher than that at 1:100. The quantity ratio of CD4+CD25+ cells in CIK/NK cell culture system showed significant differences in various mixture groups. As to 1:20 ratio the amount of CD4+CD25+ cells in CIK/NK cell culture system of mixture groups was significantly higher than that in Transwell groups, while there were no differences of the quantity ratio of CD4+CD25+ cells in CIK/NK cell culture at 1:50 and 1:100. It is concluded that either by non-contact Transwell or mixed co-culture, the MSC can suppress the activation of allogeneic CB-CIK/NK cells, which maybe relate to up-regulating the ratio of CD4+CD25+ T regulatory cells in CIK/NK cell culture system in dose-dependent manner.
Antigens, CD ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; metabolism ; Cell Culture Techniques ; Cells, Cultured ; Cytokine-Induced Killer Cells ; immunology ; metabolism ; Fetal Blood ; cytology ; immunology ; Humans ; Lectins, C-Type ; metabolism ; Mesenchymal Stromal Cells ; cytology ; T-Lymphocytes, Regulatory ; cytology ; metabolism

OBJECTIVETo construct a plasmid carrying granulysin (GLS) and to study the effect of the GLS on apoptosis, mitochondrial transmembrane potential and cytochrome C release of SMMC-7721 cells.

METHODSThe coding sequence of the GLS was amplified from the total RNA of human CTL cells, and it was inserted into pBudCE4.1 plasmid and then it was used to transfect SMMC-7721 cells. The expression of GLS was detected by RT-PCR and confirmed by immunocytochemistry method. Cell apoptosis was ascertained by Hoechst staining and electron microscopy; mitochondrial transmembrane potential was detected using Mitocapture and cytochrome C release was studied using Western blot.

RESULTSRecombinant pBudCE4.1/GLS plasmid was successfully constructed. GLS protein was successfully expressed in the SMMC-7721 cells and it induced apoptosis of the SMMC-7721 cells, and at the same time, mitochondrial transmembrane potential was reduced and cytochrome C was released from mitochondria into the cytosol.

CONCLUSIONSGLS gene carried by recombinant plasmid could express in SMMC-7721 cells and induce cells apoptosis. The change of mitochondrial transmembrane potential and the release of cytochrome C might be one of the key factors of apoptosis induced by GLS.

Antigens, Differentiation, T-Lymphocyte ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cytochromes c ; metabolism ; Humans ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; metabolism ; physiology

Cytomegalovirus (CMV) infection is a dangerous complication in patients with chronic graft versus host disease (cGVHD). CMV-specific immunity depends on the activity of T cells. This study was aimed to investigate the effect of CMV pp65 gene modified dendritic cells (DCs) on activation of autologous T cells. Lentivirus system was utilized to introduce the CMV full-length pp65 gene into mouse DCs; CpG-DNA was used to induce mature DCs; flow cytometry and immunofluorescence were used to determine the expression of antigen and IFNgamma in T lymphocytes. The results showed that the DCs were infected with lentivirus at a multiplicity of infection (MOI) of 50 with optimal infectious efficiency of 30%-40%; mature DCs expressing pp65 gene could stimulate autologous naive T cells to express CD69 specifically; mature DCs expressing PP65 could stimulate autologous CD4+ or CD8+ T cells to produce IFNgamma. It is concluded that CMV pp65-modified and CpG-DNA-induced mature DCs can activate CMV-specific T lymphocytes in vitro.
Animals ; Antigens, CD ; genetics ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; genetics ; metabolism ; Antigens, Viral ; immunology ; CD4-Positive T-Lymphocytes ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; CpG Islands ; genetics ; Cytomegalovirus ; immunology ; DNA ; genetics ; Dendritic Cells ; cytology ; immunology ; metabolism ; Humans ; Interferon-gamma ; genetics ; metabolism ; Lectins, C-Type ; Lentivirus ; genetics ; metabolism ; Mice ; Phosphoproteins ; genetics ; metabolism ; Viral Matrix Proteins ; genetics ; metabolism