Differentiation of human peripheral blood Vδ1+ T cells expressing the natural cytotoxicity receptor NKp30 for recognition of lymphoid leukemia cells

The success of cancer immunotherapy depends on productive tumor cell recognition by killer lymphocytes. γδ T cells are a population of innate-like lymphocytes endowed with strong, MHC-unrestricted cytotoxicity against tumor cells. This notwithstanding, we recently showed that a large proportion of human hematologic tumors is resistant to γδ peripheral blood lymphocytes (PBLs) activated with specific agonists to the highly prevalent Vγ9Vδ2 TCR. Although this probably constitutes an important limitation to current γδ T cell-mediated immunotherapy strategies, we describe here the differentiation of a novel subset of Vδ2(-) Vδ1(+) PBLs expressing natural cytotoxicity receptors (NCRs) that directly mediate killing of leukemia cell lines and chronic lymphocytic leukemia patient neoplastic cells. We show that Vδ1(+) T cells can be selectively induced to express NKp30, NKp44 and NKp46, through a process that requires functional phosphatidylinositol 3-kinase (PI-3K)/AKT signaling on stimulation with γ(c) cytokines and TCR agonists. The stable expression of NCRs is associated with high levels of granzyme B and enhanced cytotoxicity against lymphoid leukemia cells. Specific gain-of-function and loss-of-function experiments demonstrated that NKp30 makes the most important contribution to TCR-independent leukemia cell recognition. Thus, NKp30(+) Vδ1(+) T cells constitute a novel, inducible and specialized killer lymphocyte population with high potential for immunotherapy of human cancer.     

Age-associated alteration of γδ T-cell repertoire and different profiles of activation-induced death of Vδ1 and Vδ2 T cells

It has been suggested that γδ T cells are involved in certain autoimmune disorders. To establish reference data for clinical studies to explore the role of γδ T cells in autoimmune bone marrow failure syndrome, we examined the γδ T-cell repertoire in 120 healthy Japanese individuals by flow cytometry. The average numbers of T lymphocytes in blood were as follows: 1,084 ± 369 (SD) αβ T cells, 68 ± 44 γδ T cells, 16 ± 12 Vδ1 T cells, and 43 ± 36 Vδ2 T cells (/μl). Absolute numbers of γδ T cells decreased with aging (R = -0.378, P < 0.001). The decrease of γδ T cells was the result of reduction of Vδ2, but not of Vδ1, T cells. Numbers of Vδ2 T cells were significantly higher in male than in female donors (P = 0.007). The Vδ2 T cells but not Vδ1 T cells showed a rapid reduction in cell numbers on mitogen stimulation, which was accompanied by modest down-regulation of Bcl-2 protein expression. These results indicate that age and gender have a major impact on γδ T-cell repertoire in Japanese donors, as well as European and American donors. The age-related decrease of Vδ2 T cells may be explained by their susceptibility to activation-induced cell death.     

Gastric cancer cells induce human CD4^＋ Foxp3^＋ regulatory T cells through the production of TGF-β1

AIM: To elucidate the molecular and cellular features responsible for the increase of regulatory T cells (Tregs) in gastric cancer.METHODS: The frequencies of CD4⁺Foxp3⁺ Tregs and the level of transforming growth factor-β1 (TGF-β1) were analyzed from 56 patients with gastric cancer by flow cytometry and enzyme-linked immunosorbent assay respectively. Foxp3 gene expression was analyzed by real-time polymerase chain reaction. The gastric cancer microenvironment was modeled by establishing the co-culture of gastric cancer cell line, MGC-803, with sorting CD4⁺ T cells. The normal gastric mucosa cell line, GES-1, was used as the control. The production of TGF-β1 was detected in supernatant of MGC and GES-1. The carboxyfluorescein diacetatesuccinimidyl ester (CFSE) dilution assay was performed to evaluate the proliferation characteristics of induced Tregs. Neutralizing anti-TGF-β1 antibody was added to the co-culture system for neutralization experiments.RESULTS: The level of serum TGF-β1 in gastric cancer patients (15.1 ± 5.5 ng/mL) was significantly higher than that of the gender- and age-matched healthy controls (10.3 ± 3.4 ng/mL) (P < 0.05). Furthermore, the higher TGF-β1 level correlated with the increased population of CD4⁺Foxp3⁺ Tregs in advanced gastric cancer (r = 0.576, P < 0.05). A significant higher frequency of CD4⁺Foxp3⁺ Tregs was observed in PBMCs cultured with the supernatant of MGC than GES-1 (10.6% ± 0.6% vs 8.7% ± 0.7%, P < 0.05). Moreover, using the purified CD4⁺CD25^- T cells, we confirmed that the increased Tregs were mainly induced from the conversation of CD4⁺CD25^- naive T cells, and induced Tregs were functional and able to suppress the proliferation of effector T cells. Finally, we demonstrated that gastric cancer cells induced the increased CD4⁺Foxp3⁺ Tregs via producing TGF-β1. Gastric cancer cells upregulated the production of TGF-β1 and blockade of TGF-β1 partly abrogated Tregs phenotype.CONCLUSION: Gastric cancer cell can induce Tregs development via producing TGF-β1, by which the existence of cross-talk between the tumor and immune cells might regulate anti-tumor immune responses.     

Gastric cancer cells induce human CD4~+ Foxp3~+ regulatory T cells through the production of TGF-β1

AIM: To elucidate the molecular and cellular features responsible for the increase of regulatory T cells (Tregs) in gastric cancer. METHODS: The frequencies of CD4 + Foxp3 + Tregs and the level of transforming growth factor-β1 (TGF-β1) were analyzed from 56 patients with gastric cancer byflow cytometry and enzyme-linked immunosorbent assay respectively. Foxp3 gene expression was analyzed by real-time polymerase chain reaction. The gastric cancer microenvironment was modeled by establishing the coculture of ...     

Chlamydia muridarum respiratory tract infection induces the proliferation of γδT cells and the secretion of IFN-γ and IL-17

<正>Objective To investigate the proliferation,activation and cytokine production ofγδT cells during different periods of Chlamydia muridarum(Cm)respiratory tract infection.Methods C57BL/6 mice were inoculated intranasally with 3×103inclusion-forming units(IFU)of Cm strains to induce the murine model of chlamydial     

(−)-Epigallocatechin-3-gallate induces apoptosis and suppresses proliferation by inhibiting the human Indian Hedgehog pathway in human chondrosarcoma cells

Purpose  

Chondrosarcoma is a soft tissue sarcoma with a poor prognosis that is unresponsive to conventional chemotherapy. The regulatory mechanisms for the rapid proliferation of chondrosarcoma cells and the particular aggressiveness of this sarcoma remain poorly understood. In this study, we investigate the effect of epigallocatechin-3-gallate (EGCG) on growth and apoptosis of chondrosarcoma cells.     

Cooperative effects of gamma-interferon and 1α, 25-dihydroxyvitamin D3 on in vitro differentiation of the blast cells of RAEB and RAEB-T

Summary We added recombinant human gammainterferon (-IFN) and 1 , 25-dihydroxyvitamin D₃ (1 , 25 (OH)₂D₃) to the bone marrow cells from six patients with RAEB or RAEB-T in liquid suspension cultures. After cultivation for 7 to 9 days, numerical, morphological and functional changes of the cells were assessed. -IFN and 1 , 25 (OH)₂D₃ additively suppressed cell growth, especially the number of blast cells decreased. The expression of -naphthylbutyrate esterase (NBE) activity appeared to be promoted but that of naphthol AS-D chloroacetate esterase (NAE) activity was apparently suppressed by the addition of -IFN and/or 1 , 25 (OH)₂D₃. The percentage of NBT reduction-positive cells and latex-phagocytizing cells was only slightly increased by both agents. These results indicate that -IFN and 1 , 25 (OH)₂D₃ cooperate to induce monocytoid differentiation of the patients' blast cells. Combination therapy with both agents merits further study.     

Epigallocatechin-3-gallate suppresses TNF-α -induced production of MMP-1 and -3 in rheumatoid arthritis synovial fibroblasts

Rheumatoid arthritis (RA) synovial fibroblasts produce matrix metaloproteinases (MMPs), which destruct cartilage and bone in RA joint. Tumor necrosis factor-α (TNF-α) is one of the most important mediator leading to MMP production in RA synovial fibroblasts. Here we show that epigallocatechin-3-Gallate (EGCG) suppresses TNF-α-induced production of MMP-1 and MMP-3 in RA synovial fibroblasts, which was accompanied by inhibition of mitogen activated protein kinase (MAPK) and activator protein-1 (AP-1) pathways. EGCG treatment resulted in dose-dependent inhibition of TNF-α-induced production of MMP-1 and MMP-3 at the protein and mRNA levels in RA synovial fibroblast. EGCG treatment also inhibited TNF-α-induced phosphorylation of MAPKs, such as ERK1/2, p38, JNK. Electrophoretic mobility shift assay revealed that EGCG inhibits binding of AP-1 proteins to its response elements in synovial fibroblast treated. Thus, EGCG may play a role in regulating inflammation and bone destruction in RA patients.     

Inhibition on the production of collagen type Ⅰ,Ⅲ of activated hepatic stellate cells by antisense TIMP-1 recombinant plasmid

AIM: To investigate the inhibition effects on the production of collagen type Ⅰ, Ⅲ secreted by activated rat hepatic stellate cells (rHSCs) by antisense tissue inhibitors of metalloproteinase 1 (TIMP-1) recombinant plasmid through elevating interstitial collagenase activity.METHODS: rHSCs were extracted from normal rat liver by pronase and collagenase digestion and purified by centrifugal elutriation, and were cultured on plastic dishes until they were activated to a myofibroblastic phenotype after 7-10 days. RT-Nest-PCR and gene recombinant techniques were used to construct the rat antisense TIMP1 recombinant plasmids which can express in eucaryotic cells. The recombinant plasmid and the pcDNA3 empty plasmid were transfected in rHSCs by Effectene (QIAGEN)separately. Cells were selected after growing in DMEM containing 400μg/ml G418 for 2-3 weeks. Expression of exogenous gene was assessed by Northern blot, and expression of TIMP-1 in rHSCs was determined by Northern blot and Western blot. We tested the interstitial collagenase activity with FITC-labled type I collagen as substrate.Ultimately, we quantified the type Ⅰ,Ⅲ collagen byWestern blot.RESULTS: The exogenous antisense TIMP-1 recombinant plasmid could be expressed in rHSCs well, which could block the expression of TIMP-1 greatly, the ratio of TIMP-1/GAPDH was 0.67, 2.41, and 2.97 separately at mRNA level (P&lt;0.05); the ratio of TIMP-1/β-actin was 0.31, 0.98 and 1.32 separately at protein level (P&lt;0.05), It might elevate active and latent interstitial collagenase activity,the collagenase activity was 0.3049, 0.1411 and 0.1196 respectively. (P&lt;0.05), which led to promotion the degradation of type Ⅰ, Ⅲ collagen, the ratio of collagen I/β-actin was 0.63, 1.78 and 1.92 separately (P&lt;0.05); and the ratio of collagen Ⅲ/β-actin was 0.59, 1.81 and 1.98separately (P&lt;0.05).CONCLUSION: These data shows that the antisense TIMP-1 recombinant plasmid has the inhibitory effects on the production of type Ⅰ,Ⅲ collagens secreted by activated rHSCs in vitro. It could be a novel method to reverse hepatic fibrosis in the future.     

Engagement of the insulin-sensitive pathway in the stimulation of glucose transport by α-lipoic acid in 3T3-L1 adipocytes

Aims/hypothesis. A natural cofactor of mitochondrial dehydrogenase complexes and a potent antioxidant, α-lipoic acid improves glucose metabolism in people with Type II (non-insulin-dependent) diabetes mellitus and in animal models of diabetes. In this study we investigated the cellular mechanism of action of α-lipoic acid in 3T3-L1 adipocytes.¶Methods. We treated 3T3-L1 adipocytes with 2.5 mmol/l R (+) α-lipoic acid for 2 to 60 min, followed by assays of: 2-deoxyglucose uptake; glucose transporter 1 and 4 (GLUT1 and GLUT4) subcellular localization; tyrosine phosphorylation of the insulin receptor or of the insulin receptor substrate-1 in cell lysates; association of phosphatidylinositol 3-kinase activity with immunoprecipitates of proteins containing phosphotyrosine or of insulin receptor substrate-1 using a in vitro kinase assay; association of the p85 subunit of phosphatidylinositol 3-kinase with phosphotyrosine proteins or with insulin receptor substrate-1; and in vitro activity of immunoprecipitated Akt1. The effect of R (+) α-lipoic acid was also compared with that of S(–) α-lipoic acid.¶Results. Short-term treatment of 3T3-L1 adipocytes with R (+) α-lipoic acid rapidly stimulated glucose uptake in a wortmannin-sensitive manner, induced a redistribution of GLUT1 and GLUT4 to the plasma membrane, caused tyrosine phosphorylation of insulin receptor substrate-1 and of the insulin receptor, increased the antiphosphotyrosine-associated and insulin receptor substrate-1 associated phosphatidylinositol 3-kinase activity and stimulated Akt activity.¶Conclusion/interpretation. These results indicate that R (+) α-lipoic acid directly activates lipid, tyrosine and serine/threonine kinases in target cells, which could lead to the stimulation of glucose uptake induced by this natural cofactor. These properties are unique among all agents currently used to lower glycaemia in animals and humans with diabetes. [Diabetologia (2000) 43: 294–303]     

Calcium channel blocker treatment of tumor cells induces alterations in the cytoskeleton,mobility of the integrin αIIbβ3 and tumor-cell-induced platelet aggregation

Summary Calcium channel blockers of the phenylalkylamine (i.e. verapamil), benzothiazepine (i.e. diltiazem) and dihydropyridine (i.e. nifedipine) classes were evaluated for effects on the tumor cell/platelet interactions using Walker 256 carcinosarcoma cells (W256 cells). When W256 cells were pretreated for 15 min with channel blockers at concentrations of 50–200 M, macroscopic tumor-cell-induced platelet aggregation was inhibited (order of potency; nifedipine>diltiazemverapamil). However, ultrastructural analysis revealed limited, focal platelet aggregates associated with tumor cell plasma membranes of verapamil- and diltiazem-treated cells. There was no evidence of platelet activation or platelet association with the tumor cell membrane in cells pretreated with nifedipine. Walker 256 cells possess the integrin _IIb3. Tumor cell _IIb3 was shown to mediate tumor cell/platelet interactions in vitro [Chopra et al. (1988) Cancer Res. 48:3787]. Patching and capping of surface _IIb3 were inhibited by nifedipine>diltiazemverapamil. The degree of inhibition of _IIb3 receptor mobility parallels the inhibition of tumor-cell-induced platelet aggregation. W256 cells are characterized by a well-developed microfilament and intermediate filament network and by the absence of a distinct microtubular network. Calcium channel blockers had no effect on the low polymerization level of tubulin. However, they induced rearrangement of microfilament stress fibers. Intermediate filaments were also rearranged but to varying degrees. The order of effectiveness for alteration of intermediate filament organization was nifedipine>diltiazem while verapamil was ineffective. We propose that the previously reported inhibition of tumor cell/platelet interaction and tumor cell metastasis by calcium channel blockers [Honn et al. (1984) Clin Exp Metastasis 1:61] is due not only to the effects of the Ca²⁺ channel blockers on platelets, but also to their effect on the tumor cell cytoskeleton resulting in an inhibition of the mobility and function of the _IIb3 receptor.Abbreviations CCB calcium channel blocker - _IIb3 platelet glycoprotein IIb/IIIa complex - TCIPA tumor cell induced platelet aggregation - W256 Walker 256 carcinosarcoma This work was supported by Public Health Service grant CA-47115, a grant from Harper Hospital and a grant from the Veterans Administration     

Alterations of mast cells and TGF-β1 on the silymarin treatment for CCl_4-induced hepatic fibrosis

AIM: Silymarin is a potent antioxidant, antiinflammatory and anti-fibrogenic agent in the liver, which is mediated by alteration of hepatic Kupffer cell function, lipid peroxidation, and collagen production. Especially, in hepatic fibrogenesis, mast cells are expressed in chronic inflammatory conditions, and promote fibroblast growth and stimulate production of the extracellular matrix by hepatic stellate cells. METHODS: We examined the inhibitory mechanism of silymarin on CCI4-induced hepatic cirrhosis in rats. At 4, 8, and 12 wk, liver tissues were examined histopathologically for fibrotic changes produced by silymarin treatment. RESULTS: In the silymarin with CCU-treated group, increase of hepatic stellate cells and TGF-β1 production were lower than in the CCI4-treated group at early stages. Additionally, at the late fibrogenic stage, expressions of TGF-β1 were weaker and especially not expressed in hepatocytes located in peripheral areas. Moreover, the number of mast cell in portal areas gradually increased and was dependent on the fibrogenic stage, but those of CCI4+silymarin-treated group decreased significantly. CONCLUSION: Anti-fibrotic and antiinflammatory effects of silymarin were associated with activation of hepatic stellate cells through the expression of TGF-β1 and stabilization of mast cells. These results suggest that silymarin prevent hepatic fibrosis through suppression of inflammation and hypoxia in the hepatic fibrogenesis.