OBJECTIVETo establish a method for isolating exosomes from dendritic cells (DC), and to analyse its biological characteristics and function in antitumor immunity.

METHODSImmature DCs (im-DC) from human peripheral blood mononuclear cells were loaded with the antigen of K562 tumor cells, then exosomes were secreted from imDC and lipopolysaccharide (LPS) induced mature DC (mDC). The exosomes from imDC and mDC were isolated separately by ultracentrifugation and ultrafiltration. The exosomes diameter was determined, their profile was observed by electron microscope, and the surface molecules were detected by Western blot. To analyse the effect of exosomes on antitumor immunity, the proliferation, IFN-gamma expression, CD69 up-regulation and cytotoxicity of antigen-specific T cells were measured.

RESULTSExosomes were small flattened sphere vesicles with an average diameter of 72.3 nm and expressed CD80, CD86, HLA-DR, FasL, CD54 and MFG-E8 molecules. As compared to immature exosomes, exosomes from mDC were proved to express more CD80 and less MFG-E8, to be more potent for inducing antigen-specific T cells proliferation and immunity respond in vitro: at its optimum concentration, the absorption value of T cell proliferation test was 0.50 +/- 0.01, CD69 was up-regulated and (13.4 +/- 5.8)% of T cells was in proliferating, (22.8 +/-2.4)% of T cells expressed IFN-gamma, and (21.3 +/-8.6)% of tumor cells were killed.

CONCLUSIONA simple and quick method to isolate and analyse exosomes is established. The exosomes can induce antitumor immunity respond.

Cells, Cultured ; Dendritic Cells ; immunology ; secretion ; Exosomes ; immunology ; Humans ; Lymphocyte Activation ; drug effects ; T-Lymphocytes ; drug effects ; immunology

Anemia, Aplastic ; drug therapy ; immunology ; Animals ; Antilymphocyte Serum ; immunology ; therapeutic use ; Calcineurin Inhibitors ; immunology ; therapeutic use ; Humans ; Immunoglobulins ; immunology ; therapeutic use ; Lymphocytes ; drug effects ; immunology ; Swine

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 induce anti-B-cell acute lymphoblastic leukemia (B-ALL) cytotoxic T lymphocyte response against immunoglobulin heavy chain frame-derived nonapeptide.

METHODSThe peptide, QLVQSGAEV, containing IgHV1 frame region 3rd-11th amino acids (IgHV1(3-11)), was synthesized. IgHV1(3-11)-T2 binding tests were performed. HLA-A * 0201-positive normal peripheral blood mononuclear cells (PBMNC) were stimulated by IgHV1(3-11)-loaded antigen presenting cells three times at weekly intervals. HLA-A * 0201/IgHV1(3-11) tetramers were used to detect the proliferation of IgHV1(3-11)-specific CD8(+) T cells in the culture. Seven IgHV gene families of B-ALL patients were respectively amplified by PCR and the PCR products were sequenced to select IgHV1 and IgHV3 family monoallelic functional rearrangements. Among them, HLA-A * 0201 positive individuals were subsequently identified. Cytotoxicity of IgHV1(3-11)-specific CD8(+) T cells against HLA-A * 0201-positive IgHV1/IgHV3 family B-ALL cells was measured by MTT assay.

RESULTSThe synthesized IgHV1(3-11) up-regulated HLA-A * 0201 expression on T2 cell surface by 1.63-folds. The percentage of IgHV1(3-11)-specific CD8(+) T cells in HLA-A * 0201-positive normal PBMNC was increased from 1.64% after second stimulation to 82.57% after third stimulation. At effector: target ratio of 20:1, the killing rate of IgHV1(3-11)-specific CD8(+) T cells against IgHV1 family B-ALL cells was 18.24%, being 1.8-folds as that against IgHV3 family B-ALL cells (P = 0.01).

CONCLUSIONCytolytic T lymphocytes generated in vitro against immunoglobulin heavy chain frame-derived nonapeptides can kill B-ALL cells expressing these peptides.

Cells, Cultured ; Humans ; Immunoglobulin Heavy Chains ; immunology ; pharmacology ; Leukemia, B-Cell ; immunology ; pathology ; T-Lymphocytes, Cytotoxic ; drug effects ; immunology

OBJECTIVETo investigate the enhancing effect of Chinese medicine-Xuebijing injection on lipopolysaccharide (LPS) -induced apoptosis of CD4+ CD25+ regulatory T cells (Tregs) and polarization of helper T cells (Th).

METHODSCD4+ CD25+ Tregs collected from rat spleen in vitro by immunomagnetic beads assay were divided into the control group, anti-CD3/CD28 group, anti-CD3/CD28 + LPS group, anti-CD3/CD28 + "Xuebijing injection" group and anti-CD3/CD28 + LPS + "Xuebijing injection" group. Tregs apoptosis rate and expression of winged helix transcription factor (Foxp3) in Tregs were detected by flow cytometry on 3rd post culture day. CD4+ CD25- T cells were co-cultured with CD4+ CD25- Tregs (1:1) for 68 hours with canavalin A stimulation. Interferon gamma (gamma-IFN), interleukin (IL)-4 and IL-17 in supernatants, which respectively was secreted by Th1, Th2 and Th17, were measured by ELISA.

RESULTSTregs apoptosis rate of anti-CD3/CD28 + LPS + "Xuebijing injection" group (45.1 +/- 2.7%) was significantly higher than that of anti-CD3/CD28 + LPS group (29.4 +/- 1.6%, P < 0.01). Meanwhile, Foxp3 expressions in Tregs in above 2 groups were 95 +/- 9 and 140 +/- 18 respectively, showing statistically significant difference between them (P < 0.01). Gamma-IFN levels secreted in anti-CD3/CD28 + LPS + "Xuebijing injection" group were significantly higher than those in anti-CD3/CD28 + LPS group (P < 0.01), while IL-4 levels had an opposite tendency compared with gamma-IFN (P < 0.05), resulting in a marked increase in the ra- tio of gamma-IFN/IL-4 in anti-CD3/CD28 + LPS + "Xuebijing injection" group (P < 0.01). In anti-CD3/ CD28 + "Xuebijing injection" group, IL-17 secretion levels were significantly decreased compared with anti-CD3/CD28 group (P < 0.05).

CONCLUSIONSActivation of CD4+ CD25+ Tregs induced by LPS may mediate Th1 shift to Th2 response. "Xuebijing injection" can effectively regulate immune function of T cells, increase the LPS-induced apoptosis of CD4+ CD25+ Tregs as well as enhance the polarization of Th2 to Th1, thereby abating the suppressive state of cell-mediated immunity.

Animals ; Apoptosis ; Drugs, Chinese Herbal ; pharmacology ; Endotoxins ; Lipopolysaccharides ; Male ; Rats ; Rats, Wistar ; T-Lymphocytes, Helper-Inducer ; drug effects ; immunology ; T-Lymphocytes, Regulatory ; drug effects ; immunology

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

This study was purposed to investigate the effect of prostaglandin E2 (PGE2) on proliferation of peripheral blood T lymphocytes, and to evaluate the regulatory role of PGE2 on immunological balance between Th1/Th2 and Tc1/Tc2 lymphocytes. The peripheral blood mononuclear cells (PBMNC) were stimulated by anti-human CD3 monoclonal antibody (mAb) and anti-human CD28 mAb, and were cultured in the presence of different concentration of PGE2 for 120 hours. The proliferation of peripheral blood T lymphocytes was assayed according to the manufacture protocol of BrdU Kit; the IFN-gamma and IL-4 levels in supernatants cultured for 24, 48, 72 and 120 hours were detected by ELISA; the ratios of CD4+IL-4+ T cells/CD4+ IFN-gamma+ T cells and CD8+IL-4+ T cell/CD8+IFN-gamma+ T cells were determined by flow cytometry. The cells cultured without PGE2 were used as control. The results indicated that (1) with the raising of concentration of PGE2, the inhibitory rate of T cell proliferation in vitro significantly increased (p=0.001). There was significant positive correlation between inhibitory rate of T cells and PGE2 concentration (correlation coefficient=0.889, p=0.000). (2) the difference between the IFN-gamma concentrations in supernatant cultured for 120 and 72 hours in test groups had no statistical significance (p=0.917). The IFN-gamma concentration increased continually with prolonging of culture time in control group (p=0.046). The IFN-gamma concentrations produced at different times in test group were significantly lower compared with those in control group (p<0.05). The IL-4 concentrations produced at different time had no significant change in test groups (p=0.400). The IL-4 concentration in 24 hours in control group was significantly higher than that at 48, 72 and 120 hours in control group (p=0.007, 0.003 and 0.002). After cultured for 24 hours the IL-4 concentration in test group was significantly lower than that in control group (p=0.037), but after cultured for 48, 72 and 120 hours, the IL-4 concentration in test group did not show statistical difference in comparison with control group (p>0.05). (3) the proportions of CD4+IFN-gamma+T cells in test group and in control group had no significant difference (p=0.767). The proportion of CD4+IL-4+T cells in test group was slightly higher than that in control group (p=0.051). The ratio of CD4+IL-4+T cells to CD4+IFN-gamma+ T cells in test group was significantly higher than that in control group (p=0.011). The proportions of CD8+IFN-gamma+ T cells in test group and in control group had no statistical difference (p=0.441). The proportion of CD8+IL-4+T cells in test group was significantly higher than that in control group (p=0.015). The ratio of CD8+IL-4+ T cells to CD8+IFN-gamma+ T cells in test group were obviously higher than that in control group(p=0.038). It is concluded that the PGE2 inhibits the proliferation of T lymphocytes in vitro. PGE2 influences the production of IFN-gamma and IL-4, and significantly influences peak appearance of IFN-gamma produced by T lymphocyte. PGE2 can continuously inhibit the production of IFN-gamma, but its continuous effect on IL-4 is no significant. PGE2 enhances the ratio of CD4+IL-4+T lymphocytes to CD4+IFN-gamma+T lymphocytes and the ratio of CD8+IL-4+T lymphocytes to CD8+IFN-gamma+T lymphocytes, and regulates development of T cells toward Th2/Tc2 cells.
Cell Proliferation ; drug effects ; Dinoprostone ; pharmacology ; Flow Cytometry ; Humans ; Lymphocyte Activation ; drug effects ; Lymphocyte Count ; T-Lymphocytes, Cytotoxic ; drug effects ; immunology ; Th1 Cells ; drug effects ; immunology ; Th2 Cells ; drug effects ; 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