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

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

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

Dendritic cells (DC) are now recognized as the most potent professional antigen presenting cells (APC). Several studies on cancer immunotherapy using different approaches to induce cytotoxic T lymphocytes (CTL) in vivo recognizing tumor-associated antigens have been reported. However, the efficacy of immunotherapy in vivo may be limited by the local or systemic suppression of CTL generation or function. To explore the ability of lipopolysaccharide (LPS) stimulated human monocyte-derived DC involved in activity of autologous CD4(+)CD25(+) T cells, HLA-A2 restricted p53(264 - 272) peptide was used as tumor antigen, DC generated with LPS (DC-LPS(+)) or without LPS (DC-LPS(-)) were co-cultured with autologous T cells respectively. The results showed that CD4(+)CD25(+) T cell population in the DC-LPS(+) activated T cells was lower than that in the DC-LPS(-) activated T cells. This finding suggest that the relationship between DC-LPS(+) and population of CD4(+)CD25(+) T cells exists and this property may contribute to regulation of T cell responses to tumor-associated antigens.
CD4-Positive T-Lymphocytes ; cytology ; immunology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Coculture Techniques ; Dendritic Cells ; cytology ; immunology ; Humans ; Interleukin-2 Receptor alpha Subunit ; immunology ; Lipopolysaccharides ; pharmacology ; Lymphocyte Activation ; Monocytes ; cytology ; T-Lymphocyte Subsets ; cytology ; immunology

This study was purposed to investigate the effects of thalidomide on proliferation of peripheral blood mononuclear cells (PBMNCs), levels of lymphocyte subsets, secretion of cytokines and its killing activity, and to elucidate the immunoregulation mechanisms in treatment of multiple myeloma with thalidomide. The method of MTT was used to detect the effects of thalidomide on the proliferations and the cytotoxic activity of PBMNC; the flow cytometer was used to analyze the lymphocyte subsets; the ELISA was used to measure the concentrations of cytokines in culture supernatants. The results showed that thalidomide enhanced the proliferations of the CD8+ T, NK cells in PHA-stimulated PBMNC from healthy volunteers, increased the secretion of IL-6 significantly, and decreased the secretion of IFN-gamma, and the secretions of IL-2 and IL-10 were not affected. Compared with control group, at the same ratio of effectors to targets the thalidomide (5 microg/ml) could enhance the cytotoxic activity of PBMNC (P < 0.01), the cytotoxic activity was maximal when the ratio of effectors to targets was 40:1. It is concluded that thalidomide preferentially enhances the proliferations of CD8+ T, NK cells in PHA-stimulated PBMNC from healthy volunteers, and enhances the cytotoxic activity of PBMNC by increasing the secretion of IL-6 significantly, in short, thalidomide can exert anti-myeloma effects by increasing cellular immune function.
Adjuvants, Immunologic ; pharmacology ; Adult ; CD8-Positive T-Lymphocytes ; cytology ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cytokines ; biosynthesis ; Female ; Humans ; Immunity, Cellular ; drug effects ; Killer Cells, Natural ; cytology ; immunology ; Leukocytes, Mononuclear ; cytology ; drug effects ; immunology ; Lymphocyte Subsets ; drug effects ; immunology ; Male ; Middle Aged ; Multiple Myeloma ; pathology ; T-Lymphocytes, Cytotoxic ; drug effects ; Thalidomide ; pharmacology ; Tumor Cells, Cultured

This study was designed to detect the effects of Bazhen decoction on the proliferation and activation of T lymphocytes. T lymphocytes were isolated. The effects of Bazhen decoction on the prolifertion and activation of T lymphocytes, and on the secretion of IFN-gamma IL-2 from T lymphocytes, were detected by MTT, Flow cytometry and ELISA. Results showed that proliferation of T lymphocytes was promoted significantly by different concentration of Bazhen decoction; and after different time, the relationships of "the longer the time, the higher the concentration, and the more enhanced the proliferation" came to be apparent. After 72h, T lymphocytes were activated with different concentration of Bazhen decoction, the rate of CD69+ T cells increased signficantly, the secretion of IFN-gamma and IL-2 increased signficicantly, and the effect exhibited a dose-dependent manner. The results of this epxeriment indicated that Bazhen decoction could promote the proliferation and activation of T lymphocytes.
Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Flow Cytometry ; Humans ; Lymphocyte Activation ; drug effects ; immunology ; T-Lymphocytes ; cytology ; immunology

OBJECTIVETo investigate the pathogenesis of cytotoxic T cell (CTL) dysfunction in patients with HCV infection.

METHODSBALB/c mice were immunized by subcutaneous injection of polypeptides from HCV core region, and the CTL activity of mouse spleen cells was detected by the LDH release test. Two polypeptides which can enhance CTL function and two polypeptides which can inhibit CTL function were selected and cross-combined. BALB/c mice were immunized using the combined polypeptides and the CTL activities were detected afterwards.

RESULTSCTL activity was inhibited by CPA9 (39-74 amino acids), CPB7 (67-76 amino acids) and CPB8 (71-80 amino acids), and promoted by CPA10 (5-23 amino acids), CPB6 (63-72 amino acids) and CPB2 (131-140 amino acids). Using single factor analysis of variance, the CTL activity in the mice could be enhanced by polypeptides from the HCV core region, CPB2+CPB8, CPB6+CPB8, respectively. There was no obvious difference between CPB2+CPB7, CPB6+CPB7 and negative control.

CONCLUSIONSCPA9, CPB7, and CPB8, the 3 polypeptides from HCV core region play an inhibition role and CPA10, CPB6, and CPB2 play an enhancement role in CTL activity in mice. The inhibition and enhancement functions of the polypeptides from HCV core region interact each other.

Animals ; Cytotoxicity Tests, Immunologic ; Cytotoxicity, Immunologic ; drug effects ; Mice ; Mice, Inbred BALB C ; Peptide Fragments ; administration & dosage ; immunology ; Spleen ; cytology ; drug effects ; immunology ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Viral Core Proteins ; administration & dosage ; chemistry ; immunology

Purpose of this study was to establish an effective method in vitro to proliferate natural killer T (NKT) cells from umbilical cord blood (UCB) and peripheral blood (PB), and to study their different phenotype. Mononuclear cells (MNC) from UCB and PB were cultured in the presence of IL-2 (100 U/ml), with or without alpha-Galcer. TCR Valpha24 Vbeta11 double positive natural killer T-cells (NKT cells) and their other phenotypes were determined by flow cytometry. The results showed that after expansion for 7 days, TCRValphabeta(+) NKT cells from UCB-MNCs increased by (8.74 +/- 4.37) x 10(2) times as much, but most of them did not express NK1.1 and its TCR Vbeta11(+) was higher than TCR Valpha24(+). After expansion for 14 days, TCR Valphabeta(+) NKT cells from PB-MNCs increased by (3.72 +/- 2.01) x 10(2) times, the expression of NK1.1 was high and its TCR Vbeta11(+) was almost equal to TCR Valpha24(+). It is concluded that human TCR Valpha24 Vbeta11 double positive NKT cells can expand by addition of alpha-Galcer. The proliferation efficiency in UCB-MNCs is greater than that in PB-MNCs. Most of the UCB-NKT is NK1.1(-), while the PB-NKT is NK1.1(+), a new subset of NKT cells.
Cell Proliferation ; Cells, Cultured ; Fetal Blood ; cytology ; Galactosylceramides ; pharmacology ; Humans ; Interleukin-2 ; pharmacology ; Killer Cells, Natural ; cytology ; drug effects ; immunology ; Leukocytes, Mononuclear ; cytology ; Phenotype ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; immunology

OBJECTIVETo observe the effect of the blood cooling and detoxification formula and its dismantled formulae on activated T lymphocytes-induced keratinocyte proliferation and cytokine secretion.

METHODRat drug-containing serum was prepared. PDB-stimulated T lymphocytes and Colo-16 cells were co-cultured, then added with the drug-containing serum, and laid aside for 24 h. The cell proliferation was detected by MTT assay. Cytokines TNF-alpha, sICAM-1 and IFN-gamma were detected by ELISA.

RESULTThe blood cooling and detoxification formula has a notable inhibitory effect on activated T lymphocyte-induced Colo-16 keratinocyte proliferation, with a significant difference between the whole formula group and the blood cooling group (P < 0.01). After activated T lymphocytes had added into Colo-16 cells for 24 hour, sICAM-1, TNF-alpha and IFN-gamma significantly increased. Though the whole blood cooling and detoxification formula, the blood cooling formula and the detoxification formula could notably reduce sICAM-1, TNF-alpha, IFN-gamma, the whole formula group showed the most significant effect.

CONCLUSIONThe blood cooling and detoxification formula, as an effective traditional Chinese medicine compound for clinical treatment of psoriasis, can significantly inhibit activated T lymphocyte-induced keratinocyte proliferation and cytokine secretion.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cytokines ; immunology ; Drugs, Chinese Herbal ; administration & dosage ; Keratinocytes ; cytology ; drug effects ; immunology ; Lymphocyte Activation ; drug effects ; Rats ; Rats, Wistar ; T-Lymphocytes ; drug effects ; immunology