The role and clinical significance of programmed cell death- ligand 1 expressed on CD19+B-cells and subsets in systemic lupus erythematosus.

BACKGROUND: Programmed cell death-1 (PD-1) and programmed death-ligand 1 (PD-L1)-targeted therapies have enhanced T-cell response and demonstrated efficacy in the treatment of multiple cancers. However, the role and clinical significance of PD-L1 expression on CD19+ B-cells and their subsets, with particular reference to systemic lupus erythematosus (SLE), have not yet been studied in detail. OBJECTIVE: The present study aimed to investigate PD-L1 expression on CD19+ B-cells and their subsets, in addition to exploring its possible role in Tfh-cell activation and B-cell differentiation in SLE. METHODS: Frequencies of CD19+ B-cells, their subsets, PD-L1 and Tfh cells in the peripheral blood of SLE patients and healthy controls (HCs) were determined using cytometry. The clinical data of SLE patients were recorded in detail, and the correlation between their laboratory parameters, clinical parameters and disease activity indices was statistically analyzed. CD19+PD-L1+B-cells and CD19+PD-L1- B-cells were sorted and cultured with a stimulant, following which the supernatants were collected for immunoglobulin G and anti-double stranded DNA detection via enzyme-linked immunosorbent assay. RESULTS: In SLE patients, CD19+B-cells and partial subgroups were enriched in peripheral blood. Also, the observed increase in the frequency of CD19+PD-L1+B-cells was significantly associated with a higher disease activity index. An in vitro culture test demonstrated that the amounts of anti-dsDNA and immunoglobulin G secreted by the CD19+PD-L1+B-cells of SLE patients and HCs were vastly different. In addition, a strong correlation existed between the frequencies of CD19+PD-L1+B-cells and defined Tfh cells of SLE patients. CONCLUSION: This study demonstrated that the expression of CD19+PD-L1+B-cells in the peripheral blood of SLE patients was abnormal, and that disease-related laboratory parameters and clinical indicators were correlated. CD19+PD-L1+B-cells were enriched and played a critical role in activating the pathogenic T-cell and B-cell responses in patients with SLE.

Formation and aggregation of lipid rafts in γδ T cells following stimulation with Mycobacterium tuberculosis antigens.

Lipid rafts are plasma membrane microdomains that are implicated in diverse signaling pathways in immune cells. Based on the distinct types of T-cell receptors, two T-cell subpopulations have been identified: αß and γδ T cells. In humans, γδ T cells represent a relatively rare T lymphocyte population but play a critical role in the immune response to infection by Mycobacterium tuberculosis. It has been demonstrated that Mycobacterium tuberculosis antigens (Mtb-Ag) preferentially activate γδ T cells. Thus, we investigated whether lipid rafts are involved in the Mtb-Ag-mediated activation of γδ T cells. Human peripheral blood mononuclear cells (PBMCs) were stimulated with Mtb-Ag, and expression of a lipid raft marker ganglioside GM1 (GM1) was determined by flow cytometry. The aggregation of lipid rafts was evaluated by laser confocal microscopy. Non-stimulated fresh PBMCs minimally expressed GM1 (6.55 ± 2.01%) and had no aggregated rafts in γδ T cells. Mtb-Ag stimulation gradually increased the expression of GM1 in a time-dependent manner. At 72 h, the majority of γδ T cells expressed GM1 (88.69 ± 7.55%). Furthermore, accompanied with the increased expression of GM1, aggregation of lipid rafts became gradually visible in γδ T cells. The aggregated rafts, however, were not evenly distributed and only occurred over a small portion of GM1-positive cells. Pretreatment with methyl-ß-cyclodextrin, a cholesterol-depleting reagent, completely inhibited the Mtb-Ag-mediated aggregation of lipid rafts. These results demonstrate that lipid raft aggregation occurs in Mtb-Ag-activated γδ T cells, suggesting that lipid rafts are involved in activation of γδ T cells.

Effect of HMG-CoA reductase inhibitors on activation of human gammadeltaT cells induced by Mycobacterium tuberculosis antigens.

Lipid rafts are cholesterol-enriched microdomains which act as a platform for the initiation of T-cell activation. To investigate effect of endogenous cholesterol on lipid rafts formation and activation of gammadeltaT cells, human peripheral blood mononuclear cells were stimulated in vitro with Mycobacterium tuberculosis antigens (Mtb-Ag). Lovastatin and fluvastatin, two 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR) inhibitors, were used to block endogenous cholesterol biosynthesis. The expression of ganglioside GM1 (GM1), a lipid rafts marker, and CD69, an activation marker, and the level of tyrosine phosphorylation in gammadeltaT cells were measured by flow cytometry. The expression and aggregation of GM1 were also detected with laser confocal microscopy. We found that lovastatin and fluvastatin could obviously inhibit tyrosine phosphorylation and expression of GM1 and CD69 in gammadeltaT cells induced by Mtb-Ag. These results collectively indicated that HMGCR inhibitors might interfere with the formation of lipid rafts and inhibit the activation of gammadeltaT cells induced by Mtb-Ag.

Interleukin 17-producing gamma delta T cells increased in patients with active pulmonary tuberculosis.

Although it has been known that gammadelta T cells may play an important role in the immune response to infection of Mycobacterium tuberculosis (M. tb), the mechanisms by which the gammadelta T cells participate in the innate and/or acquired immunity to tuberculosis (TB) have not been full elucidated. In the present study, 27 patients with active pulmonary TB and 16 healthy donors (HD) were performed. We found that proportion of IL-17-producing cells among lymphocyte was similar between TB patients and HD, whereas the proportions of gammadelta T cells in IL-17-producing cells (59.2%) and IL-17-producing cells in gammadelta T cells (19.4%) in peripheral blood were markedly increased in TB patients when compared to those in HD (43.9% and 7.7%, respectively). In addition, the proportions of IFN-gamma-producing gammadelta T cells in TB patients were obviously lower than that in HD. Upon re-stimulated with M. tb heat-treated antigen (M. tb-HAg) in vitro, fewer IL-17-producing gammadelta T cells were generated from HD and TB patients, whereas IFN-gamma-producing gammadelta T cells were increased in TB patients compared to that in HD. Our findings in TB patients and healthy human were consistent with other murine investigation that the IL-17-producing gammadelta T cells were main source of IL-17 in mouse model of BCG infection, suggesting that gammadelta T cells might be involved in the formation of tubercular granuloma in pulmonary TB patients, but need further identification.

[Peripheral blood T cell TNF-α and IFN-γ production stimulated by low molecular peptide of Mycobacterium tuberculosis heat-resistant antigen for differential diagnosis between pulmonary tuberculosis and latent tuberculosis infection].

OBJECTIVE: To investigate the effects of low molecular peptide of Mycobacterium tuberculosis heat-resistant antigen (Mtb-HAg-10k) on the production of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in peripheral blood T cells and test the feasibility of differential diagnosis between pulmonary tuberculosis (PTB) and latent tuberculosis infection (LTBI) by assessing the number of Mtb-HAg-10k-stimulated IFN-γ-producing T cells. METHODS: Peripheral blood mononuclear cells (PBMCs) were separated from the peripheral blood of 10 healthy adults, 6 individuals with LTBI and 13 patients with PTB. The PBMCs were cultured in the presence of Mtb-HAg-10k obtained by ultrafiltration centrifugation, with Mtb-HAg and phytohaemagglutinin (PHA) as the controls. The proportions of TNF-α- and IFN-γ-producing cells in the T cell subsets were detected by flow cytometry (FCM), and the number of IFN-γ-producing cells from patients with PTB and LTBI was detected with ELISPOT. RESULTS: Flow cytometry showed that Mtb-HAg-10k exposure resulted in a significantly higher proportion of TNF-α-producing γδT cells than that of IFN-γ-producing γδT cells in the PBMCs (P<0.01). Compared with the PBMCs exposed to PHA, the PBMCs exposed to Mtb-HAg-10k exhibited a significantly greater proportion of γδT cells that produced both TNF-α and IFN-γ (P<0.01) but a significantly lower proportion of αßT cells producing both TNF-α and IFN-γ (P<0.01). Mtb-HAg-10k exposure of the PBMCs caused a significant reduction in the number of IFN-γ-producing cells as compared with Mtb-HAg and PHA treatments (P<0.01), and this reduction was more obvious in PBMCs from patients with PTB than in those from individuals with LTBI (P<0.01). CONCLUSION: Mtb-HAg-10k can markedly induce γδT cells in the PBMCs to produce TNF-α and IFN-γ, and detection of the number of IFN-γ-producing cells in the PBMCs following Mtb-HAg-10k stimulation helps in the differential diagnosis between pulmonary tuberculosis and latent tuberculosis infection.

[Molecular mechanism of anti-apoptotic action of survivin in NCI-H446 lung cancer cells].

OBJECTIVE: To investigate cell apoptosis induced by survivin ASODN and clarify the precise mechanism of anti-apoptotic action of survivin. METHODS: Cells of lung cancer cell line NCI-H446 were treated with survivin ASODN at different concentrations. The changes of survivin mRNA and protein expression were assessed by RT-PCR and Western blot assay. The apoptosis index (AI) and proliferation index (PI) were determined by flow cytometry (FCM). After 500 mmol/L survivin ASODN treatment, cells were stained with Rh123 to detect changes of mitochondrial membrane potential (deltapsim) by FCM. The concentration of cytoplasmic cytochrome c (cyt-c) was continuously determined by ELISA. Relative activities of caspase-9 and caspase-3 were assessed by colorimetric assay. The expression of caspase-8 protein was measured by Western blot assay. The apoptotic rates of lung cancer cells induced by survivin ASODN with or without mitochondrial permeability transition pole (MPTP) inhibitor CsA treatment were assessed by FCM. RESULTS: Down-regulated survivin mRNA was shown to be in dose-dependent and time-dependent manners. Its maximal effect was achieved at a concentration of 500 nmol/L for 72 h, at which mRNA was down-regulated by 62.7%, the expression of survivin protein in NCI-H446 cells was also obviously decreased. After treatment with survivin ASODN at concentration of 500 mmol/L for 72 h, AI was 48.35%, higher than that of control, lipofectin, NSODN, survivin ASODN 100 mmol/L and 300 mmol/L groups (3.75%, 3.41%, 4.69%, 19.85% and 34.39%, respectively). PI was 24.38%, lower than that of control, lipofectin, NSODN, survivin ASODN100 and 300 mmol/L groups (75.74%, 73.12%, 71.76%, 51.03% and 38.94%, respectively). Deltapsim was decreased in 9.54% of NCI-H446 cells treated with survivin ASODN for 3 h and 97.06% for 24 h. Following it, release of cyt-c from mitochondria to cytosol and activation of caspase-9 and caspase-3 increased significantly. The above mentioned indicators changed with a time-dependent and time diversity relationship. In the presence of CsA, the apoptotic rate of lung cancer cells induced by survivin ASODN was decreased significantly. No up-regrulation and activation in caspase-8 protein was observed. CONCLUSION: Survivin inhibits apoptosis via regulation of mitochondrial-dependent pathway. survivin ASODN can not only induce apoptosis but also inhibit cell proliferation through blocking the expression of survivin mRNA and protein.

[Frequency Distribution Features of Innate-like Lymphocytes in Peripheral Blood of Normal Adults].

OBJECTIVE: To investigate the frequency distribution features of innate-like lymphocytes (iNKT cells, γΔT cells and B1 cells) in peripheral blood of normal adults. METHODS: The flow cytometry with 6 fluorescence staining was used to detect the percentages of iNKT lymphocytes, γΔT lymphocytes, B1 lymphocytes and adaptive T lymphocyte, B2 lymphocytes in peripheral blood lymphocytes of 50 normal adults. The difference and correlation between these lymphocyte subsets were analyzed by statistical software. RESULTS: The percentage of iNKT cells in peripheral blood of 50 normal adults was 0.18% (0.01%-2.01%), the percentage of γΔT cells was 4.90% (1.45%-20.14%), the percentage of B1 lymphocytes was 1.62% (0.20%-3.77%), the percentage of adaptive T cells was 63.52% (33.20%-83.22%), the percentage of B2 cells was 6.64% (3.07%-13.80%). B1 and B2 were two subsets of B lymphocyte, the percentage of B2 in B lymphocyte was 81.43% (57.90%-94.12%) and more than that of B1 lymphocyte; the percentage of B1 lymphocytes was 17.28% (5.28%-41.13%). In T lymphocyte group the percentage of iNKT cell was 0.32% (0.01%-3.6%), the percentages of γΔT cells and adaptive T cells were 7.55% (3.04%-27.66%) and 91.98% (72.22%-96.86%) respectively. Spearman correlation analysis was used to analyze the correlation between the percentages of several lymphocyte subsets. There was a positive correlation between iNK T cells and γΔT cells, γΔT cells and adaptive T cells, B1 cells and B2 cells (r=0.39, P=0.0056; r=0.6028, P<0.0001; r=0.4791, P=0.0004). It was also found that the percentage of iNKT cells in female peripheral blood lymphocytes was 0.29% (0.06%-2.01%), and significantly higher than that in male peripheral blood lymphocytes 0.12% (0.01%-1.37%) (P<0.05). CONCLUSION: The percentages of γΔT cells, B1 cells and iNKT cells in peripheral blood lymphocytes of normal adults are significantly lower than that of adaptive lymphocytes, and their contents in peripheral blood decrease in turn. There are no sex differences in the percentages of these lymphocyte subsets except iNKT cells.

[Expression and clinical significance of PD-L1 on CD14⁺ monocyte in the peripheral blood of patients with systemic lupus erythematosus].

AIM: To detect expression of (programmed death ligand 1, PD-L1)on CD14(+) Monocyte(Mo)in the patients with systemic lupus erythematosus(SLE)and their clinical significance is analyzed. METHODS: The expression of PD-L1 on the CD14(+) Mo was examined in patients with 51 active SLE and 38 healthy controls(HC) by flow cytometry. The proportions of expression of PD-L1 on CD14(+) Mo were compared between not only inactive or active SLE patients and healthy controls(HC), but also between patients with lupus nephritis and without lupus nephritis. Correlation with clinical manifestations and laboratory findings was analyzed. RESULTS: The Proportions of CD14(+) PD-L1(+) Mo were significantly increased in active and inactive SLE patients as compared with HC(all P<0.05). The proportions of CD14(+) PD-L1(+) Mo in SLE patients with nephritis were significantly higher than those in patients without nephritis. A positive correlation was observed for proportions of CD14(+) PD-L1(+) Mo with SLEDAI score, and amounts of proteinuria. Proportions of CD14(+) PD-L1(+) Mo in SLE patients with anti-dsDNA, anti-Sm, anti-U1RNP and anti-nucleosome were higher than those in SLE patients without those antibody(P<0.05). CONCLUSION: The aberrations of proportions of CD14(+) PD-L1(+) Mo were observed in patients with SLE. Proportions of CD14(+) PD-L1(+) Mo were correlated with disease activity and production of antibody.

[Eukaryotic expression of recombinant porcine single-chain IL-12 and its biologic activity].

AIM: To express the recombinant porcine single-chain interleukin-12 (pscIL-12) gene in CHO-K1 cells, and identify biological activity of pscIL-12 fusion protein. METHODS: The recombinant pcDNA3.1(+)-pscIL-12 plasmid was transfected into the CHO-K1 cells using Sofast(TM); reagent. The levels of pscIL-12 fusion protein and IFN-γ produced by human peripheral blood mononuclear cells (PBMCs) stimulated with the supernatant of CHO-K1 cells were detected by ELISA. NK cell cytotoxicity was tested by MTT assay. The lymphocyte proliferation was examined by flow cytometry. RESULTS: ELISA showed that the transfected CHO-K1 cells had a relatively higher expression of pscIL-12 fusion protein. And the pscIL-12 fusion protein possessed strong bioactivities in enhancing the NK cell cytotoxicity, increasing the IFN-γ production, and stimulating the lymphocyte proliferation. CONCLUSION: CHO-K1 cells transfected with pcDNA3.1(+)-pscIL-12 recombinant plasmid can successfully express pscIL-12 fusion protein, which has the expected biological activities.

Lower concentrations of methyl-ß-cyclodextrin combined with interleukin-2 can preferentially induce activation and proliferation of natural killer cells in human peripheral blood.

Previous studies have demonstrated that high concentrations of methyl-ß-cyclodextrin (MßCD, 10-15 mM) can interfere with the formation of lipid rafts and inhibit activation of lymphocytes. In this report, we determined that lower concentrations of MßCD (1-4 mM) could accelerate the proliferation of lymphocytes in human peripheral blood mononuclear cells (PBMCs). In the expanded cells, CD3(-)CD56(+) natural killer (NK) cells were the dominant subpopulation, and a significant dose-effect relationship existed between the proportion of NK cells and the concentration of MßCD. In the groups treated with 3-4 mM MßCD, the proportions of NK cells reached a level of more than 60%. When PBMCs were treated with MßCD, CD69 was more preferentially expressed on CD3(-)CD56(+) cells than on CD3(+) cells at 48 and 72 hours. The expression of CD25 had no distinct difference at 48 hours, but when recombinant human interleukin-2 (IL-2) was added for a further 24 hours, it was also preferentially expressed on NK cells. MßCD and IL-2 synergistically could also induce interferon-γ (IFN-γ) production in CD56(+) human PBMCs. Mechanistic studies revealed that IFN-γ production in response to MßCD plus IL-2 was IL-12 independent but depended on endogenous IL-18 and IL-1ß, and CD56(+)CD14(+) dendritic cell-like cells and B cells might mediate the ability of MßCD to activate NK cells. The MßCD-activated NK cells also had high cytotoxicity against the natural killer cell-sensitive K562 cells or lymphokine-activated killer cell-sensitive DAUDI cells in vitro. These studies indicated that lower concentrations of MßCD combined with IL-2 can preferentially induce activation and proliferation of NK cells in PBMCs.

[Construction and eukaryotic expression of recombinant porcine single-chain interleukin-12].

OBJECTIVE: To clone the p40 and p35 subunit cDNA of porcine IL-12(pIL-12) and construct the fusion gene of recombinant porcine single-chain interleukin-12 (pscIL-12). METHODS: The total RNAs were extracted from porcine peripheral blood mononuclear cells (PBMCs) and porcine splenic lymphocytes for cloning pIL-12 p35 and p40 cDNA by RT-PCR. A hydrophobic polypeptide linker (Gly4Ser)3 was used for splicing two different gene fragments (pIL-12) p40+linker+p35 (pscIL-12) by recombinant PCR to construct pscIL-12 fusion gene. The pscIL-12 fusion gene was then inserted into pcDNA3.1(+) eukaryotic expression plasmid, and the resulted pcDNA3.1(+)-pscIL-12 was transfected into CHO-K1 cells via lipofectin. The expression of pscIL-12 mRNA in the transfected cells was identified by RT-PCR. RESULTS: The sequence of the cloned porcine IL-12 p40 and p35 cDNA and the constructed pscIL-12 fusion gene were verified by PCR, restriction enzyme digestion and sequencing. The mRNA of pscIL-12 fusion gene was detected in the transfected CHO-K1 cells by RT-PCR. CONCLUSION: The constructed pcDNA3.1(+)-pscIL-12 eukaryotic expression plasmid allows expression of pscIL-12 in CHO-K1 cells, thus facilitating further study of the biological activity and adjuvant effect of pscIL-12 fusion protein.

[The Enhanceing effect of IL-12 on phagocytosis and killing of Mycobacterium tuberculosis by neutrophils in tuberculosis patients].

AIM: To explore the effects of IL-12 on phagocytosis and killing of Mycobacterium tuberculosis by neutrophils or polymorphonuclear cells (PMNs) in tuberculosis patients. METHODS: The fresh peripheral blood samples from TB patients and healthy adults were incubated with M.tb labeled with FITC, and the percentages of phagocytosis of M.tb by PMNs was measured by flow cytometry (FCM). The fresh peripheral blood samples were incubated with DCFH-DA, and with or without M.tb for different times, the percentage of activation and the ROS production of PMNs were measured by FCM. Whole blood samples were pretreated with IL-12, the changes of phagocytosis, activation and ROS production of PMNs were measured by FCM. RESULTS: The percentages of phagocytosis by PMNs, activation and ROS production of PMNs in both TB patients and healthy adults increased dependent on the time of incubation with M.tb. Only the phagocytosis of M.tb by PMNs at 5 min in TB patients of tuberculosis patients (51.82±6.93)% was obviously higher than that in healthy adults (47.20±4.26)%, (P<0.05). Pretreatment of whole blood with IL-12 before incubation with M.tb, the percentages of phagocytosis, activation and ROS production of PMNs in both TB patients and healthy adults increased in dose dependent manner, but no significant difference was found between both groups. CONCLUSION: The results indicated that the phagocytosis of M.tb and ROS production by PMNs in TB patients were almost the same as that in healthy controls, except for phagocytosis is higher at early stage. Furthermore, IL-12 can enhance the responsiveness to the phagocytosis and ROS production of PMNs.

[Role of transcription factor T-bet and Eomes in IFN-gamma secretion of different human T cell subsets].

AIM: To investigate the effect of siRNAs specific for T-bet and Eomesodermin (Eomes) in interferon-gamma (IFN-gamma) production of different human T cell subsets. METHODS: Double-stranded small interfering RNA (siRNA) sequences specific for genes of T-bet and Eomes were chemically synthesized and transfected into anti-CD3 mAb activated alphabeta T cells and Mtb-Ag activated gammadelta T cells. CD4(+), CD8(+) T and gammadelta T cells were sorted by flow cytometry and the expressions of T-bet and Eomes gene mRNA were detected by RT-PCR technique. The changes of IFN-gamma production were determined by flow cytometry. RESULTS: After two series of transfection, the siRNA-FAM(+) cells were about 50% in the transfected cells. The IFN-gamma(+) cells in CD4(+) T cells (50.20%) decreased in the cells transfected with T-bet siRNA (18.46%) but no obvious decrease was observed in the cells transfected with Eomes siRNA, whereas the IFN-gamma(+) cells in CD8(+) T cells (76.51%) decreased in the cells transfected with Eomes siRNA (25.37%) and no obvious decrease was observed in the cells transfected with T-bet siRNA. However, the IFN-gamma producing cells in gammadelta T cells (76.52%) decreased in the cells transfected with T-bet siRNA (56.57%) or with Eomes siRNA (42.53%). CONCLUSION: At the level of transcription factors, T-bet and Eomes are important transcription factors for the regulation of IFN-gamma production in CD4(+) and CD8(+) T cells, respectively. Both T-bet and Eomes may simultaneously be involved in the regulation of the IFN-gamma production of gammadeltaT cells.

[IL-2 stimulated responses of CD3(+) CD56(+) NKT cells in pulmonary tuberculosis patients].

AIM: To observe the activation and proliferation characteristics of IL-2 stimulated CD3(+);CD56(+); NKT cells in pulmonary tuberculosis (PTB) patients. METHODS: Peripheral blood mononuclear cells (PBMCs) from PTB patients and normal subjects were stimulated with IL-2 and cultured for different time points. The CD69 expression on and amount of the CD3(+);CD56(+); NKT cells were detected by multi fluorescence staining and flow cytometry at different time of stimulation and culture. RESULTS: There was no significant difference in percentage of NKT cells between PTB patients and normal healthy controls before culture. When IL-2 was used to stimulate for 0 h, 8 h, 16 h, 40 h and 64 h, the expression of CD69 on NKT cells in normal controls and PTB patients increased significantly, but the CD69 expression level of NKT cells in PTB patients was significantly higher than that in normal persons(P<0.05). In PTB patients group, PBMCs were expanded significantly after stimulated by IL-2, the absolute number of NKT cells increased from (3.44+/-1.20)x10(4); to (323.23+/-75.98) x10(4); (P<0.01), expanded by 108.69+/-59.22 fold, In normal control group the absolute number of NKT cells increased from (5.57+/-5.16)x10(4); to (1475.05+/-868.98)x10(4); (P<0.01), expanded by 246.26+/-134.06 fold, the expanding efficiency in PTB group was significantly lower than that in normal control group (P<0.05). CONCLUSION: NKT cells in PTB patients present with high activation but low proliferation after stimulated by IL-2.

Noise-induced anomalous diffusion over a periodically modulated saddle.

We study analytically and numerically the anomalous diffusion across periodically modulated parabolic potential within Langevin and Fokker-Planck descriptions. We find that the probability of particles passing over the saddle is affected strikingly by the periodical modulation with average zero bias. Particularly, the initial phase plays an important role in the modulation effect. The effect of the correlation time of external Ornstein-Uhlenbeck noise on dynamical process is also discussed. A reduction in overpassing probability is observed due to finite correlation time.

[Phenotype expression and function of antigen presenting cells in huaman gammadelta T cells activated by peptide antigen from Mycobacterium tuberculosis].

AIM: To explore whether the human peripheral gammadelta T cells stimulated by polypeptide heat resistant antigen from Mycobacterium tuberculosis (Mtb-HAg) express phenotype and have the fuction of antigen presentation. METHODS: The monocytes isolated by adhesion method from peripheral blood mononuclear cells (PBMCs) were cultured with GM-CSF, IL-4 and induced into the mature dendritic cells by adding LPS. PBMCs were stimulated with Mtb-HAg and IL-2 to expand predominantally gammadelta T cells that were further purified by flow sorting. The pure T cells were isolated from PBMCs using adhesion revomal and nylon column method, labeled with CFSE, and cultured in alone with Mtb secretory antigen (Mtb-SAg), in monocytes with Mtb-SAg, or with Mtb-SAg pre-pulsed dendritic cells or Mtb-HAg activated gammadelta T cells for 11 days. The proliferation of the CD4(+) T cells were measured by flowcytometry. The Mtb-HAg activated gammadelta T cells were incubated with FITC labelled Mtb-SAg for different time, and the ingestion of Mtb-SAg by gammadelta T cells was measured by flowcytometry and observed with laser confocal microscopye. RESULTS: The expressions of MHC-II and co-stimulatory molecules CD80 and CD86 on gammadelta T cells that activated by Mtb-HAg marketly incrased in comparison with that on the resting gammadelta T cells. The expanded cell counts and proliferation index of the pure naïve T cells that induced by Mtb-SAg pretreated activated gammadelta T cells were similar to that induced by mature DC. By using flowcytometer and laser confocal microscope, FITC labellaed Mtb-SAg was ingested by the activated gammadelta T cells. CONCLUSION: gammadelta T cells stimulated by polypeptide Mtb-HAg express the phenotype of professional antigen-presenting cells and they are able to present Mtb-SAg to naïve T cells to induce the proliferation of CD4(+) T cells.

[Expression of Foxp3 in CD4+CD25high Treg cells of neonatal cord blood].

AIM: To investigate the amount of CD4+CD25high Treg cells and the expression of Foxp3 in neonatal cord blood, and to analyze the expression of Treg cells in neonates. METHODS: The mononuclear cells from cord blood or peripheral blood were isolated from neonatal cord blood (n=15) or healthy adult's peripheral blood (n=12) by density gradient centrifugation. Then they were stained with fluorescence labeled monoclonal antibodies for cell surface and intracellular protein. The amount of CD4+CD25high Treg cells and intercellular transcription factor Foxp3 were measured by flow cytometry. RESULTS: The amount of CD4+CD25high Treg cells in CD3+CD4+ T cells in cord blood was significantly higher than that in adult's peripheral blood (3.86%+/-1.63% vs 0.87%+/-0.74%, P<0.01), whereas the expression of Foxp3 in CD4+CD25high Treg cells in cord blood was markedly lower than in adult's blood (23.21%+/-8.9% vs 71.3%+/-11.6%, P<0.01). CONCLUSION: The low expression of Foxp3 in CD4+CD25high Treg cells in cord blood suggests Treg cells in neonates are not mature in suppression.

[Construction and expression of eukaryotic expression vector of human 4-1BB ligand gene in tumor cells and its antitumor activity in vitro].

AIM: To construct eukaryotic expression vector of human 4-1BB ligand (4-1BBL) gene and express it in HT-29 cell line. To explore the effect on activation and cytotoxicity of human cytotoxic T lymphocytes(CTLs) induced by human 4-1BBL gene transfection into tumor cells in vitro. METHODS: RT-PCR was applied to amplify the full-length of human 4-1BBL gene from Raji cells. After sequencing, the cDNA was recombinated into the eukaryotic expression vector pcDNA3.1(-) and transfected into HT-29 cells through Lipofect AMINE 2000. Human 4-1BBL mRNA and protein expression of transfected cells was detected by RT-PCR and FACS respectively. Human peripheral blood mononuclear cells were stimulated with anti-CD3 mAb and incubated with non-transfected or transfected HT-29 cells, respectively. The MTT colorimetry was used to detect the proliferation and cytotoxic effect of T lymphocytes. Meanwhile, the expression of intracellular IFN-gamma was detected by FCM. RESULTS: The HT-29 cells transfected by pcDNA3.1(-)-h4-1BBL could express human 4-1BBL efficiently. As compared with wild type HT-29 cells, the transfected HT-29 cells had more effect for proliferation, IFN-gamma production and cytotoxic activity of lymphocytes. CONCLUSION: The recombinant eukaryotic expression vector of pcDNA3.1(-)-h4-1BBL is successfully constructed. The transfection of human 4-1BBL gene in HT-29 cells is effctive in enhancing its immunogenicity and inducing antitumor immune response in vitro.

[Exploration of ganglioside GM1 as an activation marker on T cells].

AIM: To compare and analyze the patterns of ganglioside GM1 and CD69 expressions on activated gammadeltaT cells and CD3(+) T cells from human peripheral blood. METHODS: PBMCs were stimulated in vitro with anti-CD3 mAb or Mycobacterium tuberculosis antigen (Mtb-Ag). In some experimental groups, PBMCs were pretreated with different inhibitors of signal transduction pathway before stimulation. The expressions of GM1 or CD69 on gammadeltaT and CD3(+) T cells at different time points after stimulation were measured by flow cytometry (FCM). RESULTS: GM1 appeared on gammadeltaT cells at 30 min after stimulation with Mtb-Ag and retained a higher level with the lapse of time; whereas CD69 expression appeared at 3 h after stimulation and reached peak at 24 h, and then decreased gradually to the resting level on day 6 after stimulation. The expressions of CD69 and GM1 on CD3(+) T cells stimulated with anti-CD3 mAb were similar to those on gammadeltaT cells. The expression of GM1 on gammadeltaT cells was inhibited by pretreatment with PD98059 or LY294002. PMA increased the expression of GM1. CONCLUSION: GM1 can serve as a novel marker of activated T cells. During the activation of T cells, the dynamic expression of GM1 was different from that of CD69, a very early activation antigen. The Ras-Erk signal transduction pathway and PI3K and PKC might be involved in the expression of GM1.

[The role of MAPK signal transduction pathway in killing tumor cells by Mtb-Ag activated gammadelta T cells].

AIM: To explore the role of MAPK signal transduction pathway in killing tumor cells by Mycobacteria tuberculosis antigen (Mtb-Ag) activated human gammadeltaT cells. METHODS: Normal human peripheral blood mononuclear cells (PBMCs) were stimulated with Mtb-Ag and expanded in rIL-2-containing medium to induce gammadeltaT cells. The highly purified gammadeltaT cells were isolated by positive selection with MACS separator. Proportion of gammadeltaT cells was detected by flow cytometry (FCM). The cytotoxicity and CD69 expression of gammadeltaT cells pre-treated with or without PD98059 (Erk inhibitor) or SB203580 (p38 inhibitor) were detected by MTT colorimetry assay and FCM, respectively. RESULTS: The percentages of gammadeltaT cells in freshly isolated PBMCs, Mtb-Ag-activated PBMCs and cells sorted by MACS were 3.56%, 74.63% and 98.20%, respectively. The cytotoxicities of gammadeltaT cells to tumor cell lines (K562 and Raji cells) were inhibited by PD98059. The inhibitory rate of cytotoxicity to K562 cells by gammadeltaT cells (39.27%) was higher than that to Raji cells(26.58%). CD69 expressions on gammadeltaT cells induced by K562 or Raji cells were decreased by pretreatment with 100 mumol/L of PD98059. But SB203580 had no effects on the cytotoxicity and CD69 expression on gammadeltaT cells induced by K562 and Raji cells. CONCLUSION: Erk MAPK pathway, not p38 MAPK, plays an important role in triggering cytotoxicity of gammadeltaT cells. The Erk pathway may affect on the granule exocytosis of gammadeltaT cells more than the Fas/FasL-mediated cytotoxicity of gammadeltaT cells. Moreover, Erk MAPK pathway is also involved in expression of activation molecule CD69 on gammadeltaT cells induced by tumor cells.