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

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

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 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

OBJECTIVETo investigate the levels of T cell receptor rearrangement excision DNA circles (TRECs) within peripheral blood from workers exposed to lead, and thereby to evaluate the number of naive T cells and recent thymic output function.

METHODSQuantitative detection of TRECs in peripheral blood mononuclear cells (PBMNC) from 10 cases of workers exposed to lead was performed by real time PCR analysis. 11 workers without exposure to lead served as unexposed controls. In addition, the relationship between TRECs, age, length of service, blood lead, urea lead, blood ZPP and urea delta-ALA was investigated.

RESULTSThe mean value of TRECs in workers exposed to lead was (2.44 +/- 1.87)/1000 PBMC, significantly under (5.60 +/- 3.96)/1000 PBMC in unexposed controls. A significant negative correlation was found between the TRECs and urea-ALA. But there was no significant correlation between them after controlling for blood lead, urea lead.

CONCLUSIONLead exposure may damage thymic output naive T cells function. Furthermore, low-level exposure to lead may damage immune system and earlier than expected.

Adult ; DNA ; drug effects ; Gene Rearrangement, T-Lymphocyte ; drug effects ; Humans ; Lead ; toxicity ; Leukocytes, Mononuclear ; Male ; Occupational Exposure ; adverse effects ; Receptors, Antigen, T-Cell ; genetics ; immunology ; T-Lymphocytes ; drug effects ; immunology ; Thymus Gland ; immunology

OBJECTIVETo investigate the cellular immune regulation of the long-term intervention of moxa smoke.

METHODSThirty-two Wistar rats were randomly divided into a blank group, a low concentration group, a medium concentration group and a high concentration group, 8 cases in each group. In addition to the blank group, rats in the other groups were exposed to the corresponding concentration moxa smoke for 20 min every day, the T lymphocyte subsets and proportion of the CD4+ CD25+ Treg in CD4+ T cells in peripheral blood were tested by flow cytometry after 6 months.

RESULTSCompared with the blank group, the proportions of CD3+ CD4+, CD3+ CD8+ T cells and CD3+ CD4/CD3+ CD8+ in the other 3 moxa smoke groups were not significantly different (P > 0.05), while the proportions of the CD4+ CD25+ Treg in CD4+ T cells were significantly lower (P < 0.05), but no statistically significant differences among those 3 moxa smoke intervention groups (P > 0.05).

CONCLUSIONLong-term moxa smoke intervention has no significant effect on the proportions of CD3+ CD4+, CD3+ CD8+ T cells and CD3+ CD4+/CD3+ CD8+, but it can decrease the proportions of the CD4+ CD25+ Treg in CD4+ T cells in peripheral blood of rats. The way produced by pretreatment with moxa smoke may play immunomodulatory effect.

Animals ; Lymphocyte Count ; Male ; Moxibustion ; Rats ; Rats, Wistar ; Smoke ; analysis ; T-Lymphocyte Subsets ; drug effects ; immunology ; T-Lymphocytes, Regulatory ; drug effects ; immunology ; Time Factors

OBJECTIVETo observe effect of oral scorpio and scolopendra powder on T-cell subsets in peripheral blood and intestine from rats with collagen induced arthritis (CIA).

METHOD60 rats were randomly divided into 6 groups: normal control group, model control group, low-dose scorpio and scolopendra group, middle-dose scorpio and scolopendra group, high-dose scorpio and scolopendra group, and type II collagen group. Rat's rheumatoid arthritis was induced by collagen II (C II). Level of T-cell subsets from peripheral blood and intestine was measured by flow cytometry.

RESULTCD4+ T cellular level was obviously increased (P < 0.05 or P < 0.01) or kept increased tendency in peripheral blood and intestine from the model group compared with that of the normal group, while the ratio of CD4+/CD8+ in intestine was obviously descent but the contrary in peripheral blood (P < 0.05 or P < 0.01). CD4+, CD8+ T cellular level in intestine were obviously descent and the ratio of CD4+ /CD8+ increased in all treated groups when compared with in the model group (P < 0.05 or P < 0.01). However, CD4+ T cellular level and the ratio of CD4+/CD8+ in peripheral blood were remarkablely decreased.

CONCLUSIONThe mechanism that scorpio and scolopendra could treat rat's rheumatoid arthritis may be regulating balance of T-lymphocyte subsets in peripheral blood and intestine.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Arthritis, Experimental ; immunology ; Arthritis, Rheumatoid ; immunology ; CD4 Lymphocyte Count ; CD4-Positive T-Lymphocytes ; CD8-Positive T-Lymphocytes ; Female ; Intestinal Mucosa ; immunology ; Medicine, Chinese Traditional ; Rats ; Rats, Wistar ; Scorpions ; chemistry ; T-Lymphocyte Subsets ; drug effects ; immunology

BACKGROUNDLymphocyte subsets play important roles in rejection in liver transplant recipients, and the effect of splenic function on these roles remains unknown. The aim of this study was to explore the feasibility to adjust immunosuppressive agents based on splenic function status through detecting the lymphocyte subsets in liver transplantBeijing recipients.

METHODSThe lymphocyte subsets of 49 liver transplant recipients were assessed in the 309th Hospital of Chinese People's Liberation Army between June 2014 and August 2015. The patients were divided into splenectomy group (n = 9), normal splenic function group (n = 24), and hypersplenism group (n = 16). The percentages and counts of CD4+ T, CD8+ T, natural killer (NK) cell, B-cell, regulatory B-cell (Breg), and regulatory T-cell (Treg) were detected by flow cytometer. In addition, the immunosuppressive agents, histories of rejection and infection, and postoperative time of the patients were compared among the three groups.

RESULTSThere was no significant difference of clinical characteristics among the three groups. The percentage of CD19+CD24+CD38+ Breg was significantly higher in hypersplenism group than normal splenic function group and splenectomy group (3.29 ± 0.97% vs. 2.12 ± 1.08% and 1.90 ± 0.99%, P = 0.001). The same result was found in CD4+CD25+FoxP3+ Treg percentage (0.97 ± 0.39% vs. 0.54 ± 0.31% and 0.56 ± 0.28%, P = 0.001). The counts of CD8+ T-cell, CD4+ T-cell, and NK cell were significantly lower in hypersplenism group than normal splenic function group (254.25 ± 149.08 vs. 476.96 ± 225.52, P= 0.002; 301.69 ± 154.39 vs. 532.50 ± 194.42, P= 0.000; and 88.56 ± 63.15 vs. 188.33 ± 134.51, P = 0.048). Moreover, the counts of CD4+ T-cell and NK cell were significantly lower in hypersplenism group than splenectomy group (301.69 ± 154.39 vs. 491.89 ± 132.31, P= 0.033; and 88.56 ± 63.15 vs. 226.00 ± 168.85, P = 0.032).

CONCLUSIONSplenic function status might affect the immunity of liver transplant recipients, that should be considered when we make immunosuppressive protocols.

CD4-Positive T-Lymphocytes ; drug effects ; immunology ; Female ; Humans ; Hypersplenism ; immunology ; Immunosuppressive Agents ; administration & dosage ; therapeutic use ; Killer Cells, Natural ; drug effects ; immunology ; Liver Transplantation ; methods ; Lymphocyte Subsets ; drug effects ; immunology ; Male ; Middle Aged ; Retrospective Studies ; Sirolimus ; administration & dosage ; therapeutic use ; Spleen ; drug effects ; immunology ; T-Lymphocytes, Regulatory ; drug effects ; immunology