OBJECTIVETo study the effect of rapamycin in inducing naïve murine effector T cell (Teff) convert to regulatory T cell (Treg) in vitro.

METHODSThe forkhead box protein 3 (FoxP3) negative Teff were isolated and purified from the spleen and lymph node of C57 BL/6 murines aged 6-8 weeks, then Teff were cultured in three groups with mature dendritic cells (mDC), B cells, and plate coated Anti-CD3. In addition, the control wells and the test wells were prepared in each group, rapamycin were not added in the control wells but added in the test wells with concentrations of 1, 10, 50, and 100 nmol/L. Percentages of FoxP3 positive Treg were examined by flow cytometry after 4 days in Anti-CD3 group and after 6 days in the other two groups.

RESULTSAs shown by the flow cytometry, the percentages of FoxP3 positive Treg were as follows in three group: in the mDC group, it was 0.01% in the control well and 0.39%, 0.47%, 0.34%, and 0.26% in test wells; in B cell group, it was 0.01% in the control wells and 5.56%, 5.89%, 7.15%, and 4.72% in the test wells; in Anti-CD3 group, it was 0.93% in the control wells and 1.35%, 1.07%, 1.02%, and 1.19% in test wells. No significant difference was found between the test wells and control wells in the mDC group and Anti-CD3 group; however, the percentages of FoxP3 positive Treg was significantly different between the test wells and control wells in the B cell group (P < 0.01).

CONCLUSIONWhen B cell is acted as the antigen-presenting cell, rapamycin can effectively induce Teff convert to Treg in vitro.

Animals ; B-Lymphocytes ; cytology ; drug effects ; immunology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; immunology ; Flow Cytometry ; Forkhead Transcription Factors ; immunology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred DBA ; Precursor Cells, T-Lymphoid ; cytology ; drug effects ; immunology ; Sirolimus ; pharmacology ; T-Lymphocyte Subsets ; cytology ; drug effects ; immunology ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; immunology

OBJECTIVETo explore an alternative method for easier and more effective establishment of allergen-specific T-cell clones (TCC) from peripheral blood monouclear cells (PBMCs) of allergic asthma patients with allogeneic feeding cells.

METHODSTo determine the optimal condition for T cell growth and effective dose and time of mitomycin-C (MMC) treatment of the feeding cells to prevent their proliferation, the PBMCs were treated with PHA, IL-2 or MMC at different concentrations, and the proliferation rate of the treated cells was analyzed by MTT assay. The effect of IL-4 on the growth and subset selection of TCC was also analyzed. Allergen-specific TCC was established by limiting dilution method with allogeneic PBMCs as the feeding cells, and the proliferation of the allergen-specific TCC was observed to evaluate the feasibility of the feeding cells.

RESULTSPHA at 25 microg/ml and IL-2 at 27 U/ml achieved optimal growth of the T cells, while MMC treatment at the dose of 60 microg/ml for 80 min effectively enriched the non-proliferative feeding cell from the PBMCs. IL-4 could not promote the survival of the TCC, but promoted the formation of CD(4)(+) TCC. Allergen-specific TCC obtained using allogeneic feeding cells required the presence of PHA, but the allergen reactivity of the TCC remained unpredictable.

CONCLUSIONIL-4 can promote the formation of CD(4)(+) TCC, but allogeneic feeding cells may fail to produce TCC with high allergen specificity.

Allergens ; immunology ; Asthma ; blood ; Cell Culture Techniques ; Cell Proliferation ; drug effects ; Cells, Cultured ; Clone Cells ; cytology ; drug effects ; immunology ; Humans ; Interleukin-2 ; pharmacology ; Leukocytes, Mononuclear ; cytology ; drug effects ; immunology ; Mitomycin ; pharmacology ; T-Lymphocyte Subsets ; cytology ; drug effects ; immunology ; T-Lymphocytes ; cytology ; drug effects ; immunology ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; immunology

Gamma interferon (gamma-IFN), a lymphokine produced by activated T lymphocytes, has a variety of effects on target cell. It induces class II antigens of the major histocompatibility complex not only in immunocompetent cells but also in non-immunocompetent cells. gamma-IFN also can exert, in addition to anti-viral activity, a series of anticellular effects on a variety of cell types. The effects of gamma-IFN on the proliferation of cultured epidermal cell (EC) and induction of HLA-DR antigen expression by EC (HLA-DR+KC) were studied. Furthermore, the immunologic role of HLA-DR+KC in the mixed epidermal cell-lymphocyte reaction (MECLR) was studied. The antiproliferative effect of gamma-IFN on the cultured EC was seen 3 days after treatment of gamma-IFN and the effect was dose-dependent. Number of HLA-DR+KC was increased dose-dependently with treatment of gamma-IFN. In MECLR, HLA-DR+KC had been found to exert stimulatory role on allogenic lymphocytes. However, there was no significant role of HLA-DR+KC on autologous lymphocytes.
Adolescent ; Adult ; Cell Division/*drug effects ; Female ; HLA-DR Antigens/*immunology ; Humans ; Interferon-gamma/*pharmacology ; Lymphocytes/cytology/drug effects/*immunology ; Male ; Skin/*cytology/drug effects

The aim of this study was to explore the regulatory function of interleukin-6(IL-6) on human Th17 cells. Human peripheral blood CD4(+) T cells were purified from healthy donors by anti-CD4 monoclonal antibody (mAb) conjugated microbeads. The experiment was divided into 2 groups. Test group in which CD4(+) T cells (1 × 10(6)/ml) were stimulated by human recombined IL-6 (20 ng/ml) for 4 days; control group in which CD4(+) T cells did not stimulated by IL-6. The concentrations of IL-17 protein in the supernatants were assayed by enzyme-linked immunosorbent assay (ELISA), and quantity of Th17 cells were detected by flow cytometry. The results showed that as compared to control group, IL-17 protein level in the supernatants of CD4(+) T cells significantly increased in IL-6 stimulated group: (337.05 ± 189.09 pg/ml; vs 15.07 ± 12.70 pg/ml) (p < 0.05). Furthermore, the percentage of Th17 cells in cultures of CD4(+) T cells stimulated by IL-6 was significantly higher than that in control group (4.05% ± 0.30% vs. 2.81% ± 0.44%)(p < 0.01). It is concluded that IL-6 promotes the expansion of Th17 cells in vitro.
CD4-Positive T-Lymphocytes ; cytology ; immunology ; Cells, Cultured ; Humans ; Interleukin-6 ; pharmacology ; Lymphocyte Activation ; immunology ; Th17 Cells ; drug effects ; immunology

OBJECTIVETo explore the immunoregulatory effects of human amniotic mesenchymal cells (hAMCs) on allogeneic peripheral blood lymphocytes.

METHODSThe hAMCs were isolated from abandoned human amnion. Peripheral blood mononuclear lymphocytes (PBMLs) were separated from healthy donors by density gradient centrifugation. Then, PBMLs were treated with phytohemagglutinin (PHA) and different concentrations of hAMCs. Proliferation effect of PBMLs was tested using MTS assay, and production of IFN-gamma and TNF-alpha by PBMLs was detected by ELISA.

RESULTShAMCs could remarkably inhibit the lymphocytes proliferation. When the ratios of hAMCs to PBMLs were 0.05: 1, 0.10 :1, 0.20: 1, the inhibitory rates of PBMLs proliferation were 16.91%, 20.83% and 28.19%, respectively. HAMCs also decreased the production of IFN-gamma and TNF-alpha by PBMLs in a dose-dependent manner (P<0.01).

CONCLUSIONSHAMCs could inhibit the proliferation of allogeneic lymphocytes and reduce secretion of IFN-gamma and TNF-alpha, which might be one of the mechanism for prevention and remission of transplant rejection.

Amnion ; cytology ; Cell Proliferation ; Humans ; Immune Tolerance ; Interferon-gamma ; biosynthesis ; Lymphocyte Activation ; immunology ; Lymphocytes ; cytology ; drug effects ; immunology ; Mesoderm ; cytology ; Phytohemagglutinins ; immunology ; Tumor Necrosis Factor-alpha ; biosynthesis

To confirm the mechanism of exosomes as tumor vaccines inducing immunity response, dendritic cells (DCs) were induced from human peripheral blood mononuclear cells, while exosomes were isolated from DC loaded tumor antigen. The effect of exosomes on priming T cell proliferation was analysed under conditions with or without DCs, or DCs at different mature stages. The function of exosomes in immunity was detected through block test after blocking some molecules (CD11a, CD11b, CD11c, CD54, MFG-E8 and CD83). The effect of DCs on embedded exosomes was observed by confocal microscopy, the effect of blocking surface molecules on exosomes on DC-embedding exosomes was assayed by flow cytometry. The results indicated that both exosomes derived from imDC (imDex) and exosomes derived from mDC (mDex) could not prime T cells without DC or with imDC. The exosomes derived from mDC induced with different cytokines (LPS, TNF-alpha, CpG, CD40L) were no significant difference in concentrations but were different in effect. The immunity function of exosomes depended on CD11a, CD11b, CD11c, CD54, MFG-E8 and CD83 molecules, the effect of priming T cells is reduced when these molecules were blocked. Confocal microscopy and FACS assay showed that blocking CD11a and CD54 could inhibit exosome-targeted DC and DC-embedded exosomes. It is concluded that the exosomes target DCs through their surface molecules, therefore results in immune response of T cells.
Antigens, Neoplasm ; immunology ; Cells, Cultured ; Dendritic Cells ; cytology ; immunology ; secretion ; Exosomes ; immunology ; Humans ; K562 Cells ; Lymphocyte Activation ; drug effects ; T-Lymphocytes ; cytology ; immunology

AIM AND METHODSThe effect of intrahippocampal microinjection of noradrenaline (NA) and its receptors antagonists and agonists on cellular immune functions were investigated in normal and adrenalectomy rat by determine the proliferative activity of Con A-stimulated splenic lymphocytes in MTT method and natural killer (NK) cell activity.

RESULTS(1) In normal group, the proliferative activity of Con A-Stimulated splenic lymphocytes were inhibited and the activity of NK cell were reduced with microinjection NA and beta1-, beta2-adrenergic receptor agonists Dobutamine (Dob, 4 microl, 6.0 x 10(-3) moL/L), Metaproterenol (Met, 4 microl, 8.0 x 10(-3) mol/L), compared with their intensity of effect, NA > Met > Dob; the immunosuppression effect induced by NA was partly hindered by alpha- and beta-receptor antagonists, phentolamine (Phen, 2 microl, 1.6 x 10(-2) mol/L) and propranolol (Prop, 2 microl, 1.6 x 10(-3) mol/L), and the action of Prop was more evident. (2) In adrenalectomy group, immunosuppression effect induced by NA was unconspicuous.

CONCLUSIONThe results suggested that NA in hippocampus could inhibit distinctly cellular immune functions, which was predominantly mediated by beta2- adrenergic receptor with a minor contribution of beta1- and alpha- adrenergic receptors. Moreover, keeping intact construction and function of adrenal gland have an important role in the effect of NA on cellular immune function.

Adrenergic Agonists ; pharmacology ; Animals ; Hippocampus ; drug effects ; Immunity, Cellular ; drug effects ; Killer Cells, Natural ; immunology ; Lymphocytes ; immunology ; Microinjections ; Norepinephrine ; pharmacology ; Rats ; Rats, Wistar ; Spleen ; cytology ; immunology

AIMTo investigate effects of hsBAFF synthesized in Escherichia coli on spleen B lymphocyte immune response and its intracellular free Ca2+ ([Ca2]i]) signaling in mice.

METHODSTwenty ICR mice, half males-half females, were chosen and randomly divided into a normal control group (n=10) and a hsBAFF treatment group (n-10). The mice in hsBAFF treatment group were given abdominal cavity injection of hsBAFF solution which was diluted with phosphate buffered saline (PBS) at dosage of 0.1 mg/kg body weight once each day for over eight days. The mice in control group were received abdominal injection of PBS at the same dose and frequency. Spleen B lymphocyte proliferation and its immune response to LPS stimulation in mice were evaluated using an MTT assay, and change of spleen B lymphocyte [Ca2+]i was assayed under a laser scanning confocal microscope.

RESULTSB lymphocyte proliferation and its immune response to LPS stimulation were significantly higher in hsBAFF-treated mice than in control mice (P < 0.05). The B lymphocyte [Ca2+]i fluorescence intensity in hsBAFF-treated mice maintained at a relatively high level fluctuation, and its average intensity was significantly higher to that of control mice (P < 0.01), but change rate of the intensity was lower compared to that of control group.

CONCLUSIONhsBAFF synthesized in Escherichia coli can enhance immune function in the body by increasing B lymphocyte proliferation and its immune response. hsBAFF-activated B lymphocyte function may be associated with increasing B lymphocytes [Ca2+]i.

Animals ; B-Cell Activating Factor ; immunology ; pharmacology ; B-Lymphocytes ; cytology ; drug effects ; immunology ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; Cell Proliferation ; drug effects ; Female ; Male ; Mice ; Mice, Inbred ICR ; Spleen ; cytology ; drug effects ; immunology

The purpose of this paper is to study the effect of kinetin (Kn) on immunity and splenic lymphocyte proliferation in vitro of aging rats induced by D-galactose (D-gal). Fifty SD rats were randomly divided into five groups: control group, aging model group, Kn low dose group, Kn middle dose group and Kn high dose group. The aging model group was proposed by napes subcutaneous injection of D-gal (125 mg/kg) for 45 d, and anti-aging groups were intragastrically administered with 5, 10, 20 mg/kg of Kn respectively from day 11. IgG, IgA, IgM contents of serum, the apoptosis percentage, stimulation index (SI) and proliferation index (PI) of splenic lymphocyte in vitro were evaluated. The results showed that the apoptosis percentage of splenic lymphocyte in aging model rats was higher, the serum IgG, IgA and IgM contents, SI and PI were lower than control group. Kn significantly decreased the apoptosis percentage of splenic lymphocyte, while increased the serum IgG, IgA and IgM contents, SI and PI in aging model group. These results suggest that Kn could inhibit the apoptosis, while promote the proliferation of splenic lymphocyte, and then effectively enhance the immune power of the aging rats and slow down the aging process.
Aging ; drug effects ; immunology ; Animals ; Antibodies ; blood ; Apoptosis ; Cell Proliferation ; drug effects ; Galactose ; adverse effects ; Kinetin ; pharmacology ; Lymphocytes ; cytology ; Rats ; Rats, Sprague-Dawley ; Spleen ; cytology

OBJECTIVETo explore the in vitro anti-tumor effect and mechanism of dendritic cell (DC) tumor vaccine induced by astragalus polysacharin (APS).

METHODSPeripheral blood mononuclear cells (PBMCs) isolated from human peripheral blood. DCs obtained from human peripheral blood were cultivated and added with culture solution for in vitro inducing them to immature DCs. On the 5th day of culture, 100 microg/mL (as the final concentration) APS was added to cells in the APS group. DCs were induced to mature in the cytokine groups by adding 20 ng/mL rhTNF-alpha (as the final concentration). Changes of morphology and phenotype of DCs were observed. Mature DCs were sensitized with tumor antigen SGC-7901 and co-cultured with allogeneic T cells. The proliferative function of T lymphocytes was detected by MTT assay. Levels of IL-12 and IFN-gamma in co-cultured supernatant were detected by ELISA. Cytotoxic lymphocytes (CTL) activated by DC were co-cultured with tumor cell SGC-7901. The specific killing capacity of CTL to target cells was detected by LDH release assay.

RESULTSThe morphological observation and phenotypic identification of APS induced DCs were in accordance with the characteristics of mature DCs. APS induced mature DCs could stimulate the proliferation of allogeneic T lymphocytes. The proliferation index of T cells increased with increased ratio of stimulator cells to effector cells (P < 0.05). Levels of IL-12 and IFN-gamma in co-culture supernatant significantly increased in a time-dependent manner (P < 0.05). CTL cells activated by sensitization of DCs could significantly kill tumor cells, and the killing effect increased along with increased effector-to-target ratio.

CONCLUSIONAPS could in vitro induce DCs to mature, promote its antigen-presenting capacity, effectively activate CTLs, and enhance anti-tumor function of the organism.

Antigen-Presenting Cells ; cytology ; drug effects ; immunology ; Cancer Vaccines ; immunology ; Cell Line ; Cell Proliferation ; drug effects ; Coculture Techniques ; Dendritic Cells ; cytology ; drug effects ; immunology ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Interferon-gamma ; immunology ; Interleukin-12 ; immunology ; Leukocytes, Mononuclear ; cytology ; immunology ; Lymphocyte Activation ; T-Lymphocytes, Cytotoxic ; cytology ; drug effects