Selective proliferation of human γδ T cells in vitro

The effect of monoethylphosphate (MEP,commercial available or synthesized) together with IL-2 on the selective proliferation of human γδT cells in vitro from peripheral blood mononuclear cells (PBMC) of healthy donors and of cancer patients was investigated.The γδT cells were stimulated by MEP to proliferate in a dose-dependent manner.The effect of synthesized MEP was 10 times greater than that of commercial MEP.When the PBMCs of healthy donors were cultured for 25 d in the medium containing different concentrations of MEP,the total cell number increased about 1000-3000 fold;and the ratio of γδT cells reached to 70-80%.The selective expansion of γδT cells depended on the synergic action of MEP and IL-2.The bulk cultured γδT cells exhibited obvious cytotoxic activities against allogenic tumor cell lines (SQ-5,K562 and Daudi) and autologous tumor cells.The culture system described here not only offers a simple method for obtaining a large number of γδT cells which may become a new effector in the adoptive immunotherapy,but also provides a useful model for the further studies of the structure and function of γδT cells in vitro.     

Wnt pathway activator TWS119 enhances the proliferation and cytolytic activity of human γδT cells against colon cancer

γδT cells are a distinct T-cell subset that display unique characteristics regarding T-cell receptor gene usage, tissue tropism and antigen recognition. Adoptive γδT cell transfer therapy has recently been gaining importance as an efficient approach in cancer immunotherapy. However, exploiting γδT cell response for tumour immunotherapy is a challenge due to cell numbers, activities and differentiation states that minimize the clinical therapeutic effects. Previous studies have indicated that the wnt/β-catenin signalling pathway plays a crucial role in the differentiation, survival and enhancement of the immune response of T lymphocytes. In this study, we sought to evaluate whether the activation of the wnt/β-catenin pathway through inhibition of glycogen synthase kinase-3β (GSK-3β) using 4,6-disubstituted pyrrolopyrimidine (TWS119) could be an efficient strategy to improve the proliferation, differentiation and cytolytic activity of γδT cells against colon cancer cells. Remarkably, we found that TWS119 significantly enhanced the proliferation and survival of γδT cells via activation of the mammalian target of rapamycin (mTOR) pathway, upregulation of the expression of the anti-apoptotic protein Bcl-2 and inhibition of cleaved caspase-3 in addition to the Wnt pathway. Our results also showed that enhancement of the cytolytic activity of γδT cells against human colon cancer cells by TWS119 was chiefly associated with upregulation of the expression of perforin and granzyme B in vitro and in vivo. Additionally, TWS119 can induce the expression of CD62L or CCR5 to generate a population of CD62L⁺γδT or CCR5⁺γδT cells in a dose-dependent manner. These findings suggested that TWS119 could be a useful complementary agent for improving γδT cell-based immunotherapy.     

Abnormal activation of the Akt-GSK3β signaling pathway in peripheral blood T cells from patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease accompanied by the activation and proliferation of T cells and B cells. In this study, we found that the distributions of lymphocytes obtained from patients with SLE or SLE with renal disease (RSLE) were reduced in the G₀/G₁ phase and were elevated in the S phase after phytohemagglutinin treatment. Increased expression of CDK2 and decreased expression of cyclin-dependent kinase inhibitors p27^Kip1 and p21^WAF1/CIP1 were observed in RSLE and SLE lymphocytes. The phosphorylation levels of Akt473 and GSK3β (ser9) were increased in lymphocytes from the patients. Moreover, inhibition of GSK3β with lithium chloride or SB216763 induced T cell proliferation, and the most significant effects were observed in RSLE lymphocytes. These results indicate that upregulation of CDKs and downregulation of p27^Kip1 and p21^WAF1/CIP1 increased the proliferation of T lymphocytes in SLE patients. Abnormal activation of the Akt–GSK3β signaling pathway increased the proliferation of lupus lymphocytes.     

Role of adhesion molecules in recruitment of Vδ1 T cells from the peripheral blood to the tumor tissue of esophageal cancer patients

 The mechanism responsible for tissue specific localization of γδ T cell subsets is not well understood. In order to explain the sequestration of specific γδ T cell subsets in the peripheral blood and tumor tissue of patients with esophageal cancer, we examined the function and expression of adhesion molecules on these cells. A hierarchy in the expression of adhesion molecules was observed. In vitro activated γδ T cells showed dominant expression of LFA-1 (CD11a), VLA-α4 (CD49d), intermediate expression of VLA-α5 (CD49e) and L-selectin (CD62L), but low expression of CD44v6 and α_Eβ₇ (CD103). It was observed that the γδ T cells use LFA-1, L-selectin and CD44v6 to bind to squamous cell carcinoma (SCC) cells, whereas they adhere to fibroblast cells using LFA-1, VLA-α4 and VLA-α5. Vδ1 T cell subsets from the peripheral blood γδ T cells utilize a larger array of adhesion molecules, namely LFA-1, VLA-α4, VLA-α5, L-selectin and α_Eβ_7, to bind to SCC cells compared to the restricted usage of LFA-1, L-selectin and CD44v6 by the Vδ2 T cells. Flow cytometric analysis of tumor infiltrating lymphocytes from the esophageal tumors confirmed the selective accumulation of Vδ1⁺γδ T cells in the tumor compartment. It thus appears that adhesion molecules expressed on these lymphocytes play an important role in the recruitment and retention of Vδ1 T cells in the tumor milieu. Received: 27 November 2000 / Accepted: 1 March 2001     

Impaired activation of IFN-γCD4 T cells in peripheral blood of patients with dilated cardiomyopathy

Viral persistence and autoantibodies are pathogenic components in patients with idiopathic dilated cardiomyopathy (DCM). The aim was to evaluate T-cell function in DCM using different flow cytometry based detection techniques. Following stimulation, the frequency of IFN-γ-producing CD4⁺ T cells was significantly lower in patients compared with controls. In contrast, the frequency of IL-4 producing CD4⁺ T cells was no different. In supernatants of cultured PBMC, IFN-γ and IL-10 were significantly lower in patients. In addition, lymphocyte proliferation was significantly lower in patients compared with controls, whereas major lymphocyte subsets were not different. IFN-γ and IL-10 are key cytokines in the ability to mount protective immune responses and to maintain self-tolerance. A reduced activation of T-helper 1 (IFN-γ producing) cells and a decreased capacity to produce IL-10, found in the present study, could explain parts of the autoimmune features seen in patients with DCM.     

Chemical analysis and antioxidant activity in vitro of a β-D-glucan isolated from Dictyophora indusiata

In this study, a water-soluble polysaccharide (PPS) from Dictyophora indusiata was purified and investigated through a combination of gel chromatography (Sephadex G-200), infrared (IR) spectroscopy, and gas chromatography-mass spectrometry (GC-MS). The results indicated that PPS has a backbone of β-conformation, mainly consist of glucose (98.58%). And the antioxidant activities of PPS were investigated in vitro including reducing power, hydroxyl assay, superoxide radical assay and DPPH scavenge activity. The results showed that PPS has antioxidant activities in all these assay systems, suggesting it may be explored as a novel natural antioxidant with potential therapeutic properties in mammalian systems.     

Tumor necrosis factor α release in peripheral blood mononuclear cells of cutaneous lupus and dermatomyositis patients

Introduction  

Several studies have reported that TNFα is substantially increased within skin lesions of patients with discoid lupus erythematosus (DLE), subacute cutaneous lupus erythematosus (SCLE) and dermatomyositis (DM) compared to controls. Elevated TNFα has been reported in the sera of some patients with systemic lupus erythematosus, DLE and SCLE, but not in the sera of patients with DM. Because of the key pathogenic role of autoimmunity in these diseases, in this study we sought to evaluate TNFα production by a readily available source of immune cells (namely, peripheral blood mononuclear cells (PBMCs)) taken from controls and from patients with cutaneous lupus or DM.     

IFN-γ upregulates survivin and Ifi202 expression to induce survival and proliferation of tumor-specific T cells

Background

A common procedure in human cytotoxic T lymphocyte (CTL) adoptive transfer immunotherapy is to expand tumor-specific CTLs ex vivo using CD3 mAb prior to transfer. One of the major obstacles of CTL adoptive immunotherapy is a lack of CTL persistence in the tumor-bearing host after transfer. The aim of this study is to elucidate the molecular mechanisms underlying the effects of stimulation conditions on proliferation and survival of tumor-specific CTLs.

Methodology/Principal Findings

Tumor-specific CTLs were stimulated with either CD3 mAb or cognate Ag and analyzed for their proliferation and survival ex vivo and persistence in tumor-bearing mice. Although both Ag and CD3 mAb effectively induced the cytotoxic effecter molecules of the CTLs, we observed that Ag stimulation is essential for sustained CTL proliferation and survival. Further analysis revealed that Ag stimulation leads to greater proliferation rates and less apoptosis than CD3 mAb stimulation. Re-stimulation of the CD3 mAb-stimulated CTLs with Ag resulted in restored CTL proliferative potential, suggesting that CD3 mAb-induced loss of proliferative potential is reversible. Using DNA microarray technology, we identified that survivin and ifi202, two genes with known functions in T cell apoptosis and proliferation, are differentially induced between Ag- and CD3 mAb-stimulated CTLs. Analysis of the IFN-γ signaling pathway activation revealed that Ag stimulation resulted in rapid phosphorylation of STAT1 (pSTAT1), whereas CD3 mAb stimulation failed to activate STAT1. Chromatin immunoprecipitation revealed that pSTAT1 is associated with the promoters of both survivin and ifi202 in T cells and electrophoresis mobility shift assay indicated that pSTAT1 directly binds to the gamma activation sequence element in the survivin and ifi202 promoters. Finally, silencing ifi202 expression significantly decreased T cell proliferation.

Conclusions/Significance

Our findings delineate a new role of the IFN-γ signaling pathway in regulating T cell proliferation and apoptosis through upregulating survivin and ifi202 expression.     

Induction of bacillus-Calmette-Guérin-activated killer cells from human peripheral blood mononuclear cells against human bladder carcinoma cell lines in vitro

Cytotoxicity against two human bladder carcinoma cell lines (BT-A and BT-B) was investigated using human peripheral blood mononuclear cells (PBMC) stimulated with viable bacillus Calmette-Guérin (BCG) or sonicated BCG (s-BCG). We applied a cytotoxicity assay based on radioactive labelling of tumour cells by incorporation ofl[³H]methionine. The results were compared with the cytotoxicity exerted by lymphokine-activated killer (LAK) cells generated by interleukin-2 (IL-2) and interferon (IFN). BCG-stimulated PBMC showed a cytotoxic potential against BT-A and BT-B comparable to that of IFN-generated LAK cells, but this did not reach the level of IL-2-generated LAK cells. We termed these cytotoxic effectors BCG-activated killer (BAK) cells. In contrast to their cytotoxicity against bladder tumour cells. BAK cells did not differ from unstimulated PBMC in the killing of K562 cells. Only viable but not sonicated BCG was able to induce cytotoxicity against BT-A and BT-B. We could demonstrate the presence of the cytokines IFN, IL-2, tumour necrosis factor (TNF) and TNFß in the supernatants harvested during the generation of BAK cells. Monoclonal antibodies neutralizing IFN were able to inhibit BCG-mediated cytotoxicity, giving evidence of the involvement of IFN in the induction of BAK cells. Furthermore, we performed experiments to investigate the cytotoxic potential of distinct cell populations. The cells effective in BCG-activated killing of bladder tumour cells could be localized within the CD8+/CD56+ lymphocyte subset. CD4+ cells and macrophages did not exhibit cytolytic activity. Our findings imply that the activation by BCG of CD8+/CD56+ killer cells might be an important antitumoral mechanism during BCG therapy against superficial urothelial bladder cancer.     

Genetic variations at the T cell receptor γ locus in circulating peripheral blood mononuclear cells of clinically categorised leprosy patients

The allelic polymorphisms at exon 3 and exon 2 of the T cell receptor (TCR) Cγ2 (TRGC2) gene, generating 18-kb and 5.4-kb HindIII fragments, respectively, were found to be more frequent in multibacillary leprosy patients than in the controls (P < 0.005 and P < 0.001, respectively) when screened with the IDP2.11 probe. The frequencies of heterozygotes for the 18-kb allele and homozygotes for the 5.4-kb allele were found to be significantly higher in the multibacillary patients than in the controls (P < 0.001). Interestingly, the 8.0-kb allele, originating from the triplication of exon 2 of Cγ2, was observed exclusively in the paucibacillary leprosy patients. Further, when DNA samples were screened with the pH60 probe for the HindIII RFLP at the TCR Jγ2 (TRGJ2) gene segment, the 2.1-kb allele was again more prevalent in leprosy patients with the multibacillary form of the disease than in the paucibacillary patients and the controls (P < 0.025). The frequency of homozygotes for the 2.1-kb allele was also significantly higher in the multibacillary patients than in the paucibacillary patients (P < 0.010) and the controls (P < 0.025). A significant difference was observed in the frequencies of detectable rearrangements involving the Vγ7/8 and Vγ9 gene segments at the γ locus between circulating peripheral blood mononuclear cells of the multibacillary leprosy patients and the controls. These rearrangements were detected less frequently in the multibacillary patients (P < 0.001 for Vγ7/8 and P < 0.005 for Vγ9). Received: 28 October 1996 / Accepted: 13 February 1997     

Thymopentin down-regulates both activity and expression of iNOS in blood cells of Sézary syndrome patients

While thymopentin has been used for many years in the experimental treatment of Sézary syndrome (SS), a rare and very aggressive lymphoma, its mechanism of action is still not known. Herein we show that this peptide acts as an inhibitor of isolated iNOS and nNOS isoforms, and reduces iNOS protein/mRNA levels and iNOS activity in blood cells obtained from both healthy donors and SS patients. Similar results were obtained with TPN-2, the N(ω)-nitro analogue of the Arg-Lys motif present in thymopentin. Additional investigations are necessary to confirm the role and the relative importance of the two mechanisms of iNOS down-regulation in the therapeutic action of these peptides against SS.     

Interleukin-1β regulates cell proliferation and activity of extracellular matrix remodelling enzymes in cultured primary pig heart cells

After myocardial infarction, elevated levels of interleukins (ILs) are found within the myocardial tissue and IL-1β is considered to play a major role in tissue remodelling events throughout the body. In the study presented, we have established a cell culture model of primary pig heart cells to evaluate the effects of different concentrations of IL-1β on cell proliferation as well as expression and activity of enzymes typically involved in tissue remodelling.Primary pig heart cell cultures were derived from three different animals and stimulated with recombinant pig IL-1β. RNA expression was detected by RT-PCR, protein levels were evaluated by Western blotting, activity of matrix metalloproteinases (MMPs) was quantified by gelatine zymography and cell proliferation was measured using colorimetric MTS assays.Pig heart cells express receptors for IL-1 and application of IL-1β resulted in a dose-dependent increase of cell proliferation (P < 0.05 vs. control; 100 ng/ml; 24 h). Gene expression of caspase-3 was increased by IL-1β (P < 0.05 vs. control; 100 ng/ml; 3 h), and pro-caspase-3 but not active caspase was detected in lysates of pig heart cells by Western blotting. MMP-2 gene expression as well as enzymatic activities of MMP-2 and MMP-9 were increased by IL-1β (P < 0.05 vs. control; 100 ng/ml; 3 h for gene expression, 48 and 72 h for enzymatic activities of MMP-2 and MMP-9, respectively).Our in vitro data suggest that IL-1β plays a major role in the events of tissue remodelling in the heart. Combined with our recently published in vivo data (Meybohm et al., PLoS One, 2009), the results presented here strongly suggest IL-1β as a key molecule guiding tissue remodelling events after myocardial infarction.     

Expression of TNFα membrane-bound receptors in the peripheral blood mononuclear cells (PMBC) in rheumatoid arthritis patients

ObjectiveTo investigate the expression of TNFα membrane-bound receptors: the percentage of cells expressing these receptors and the number of molecules expressed on different immune cell subsets, and to evaluate serum concentrations of soluble TNFα and its receptors (sTNFRI and sTNFRII) in patients with rheumatoid arthritis in acute stage and after response to treatment compared to healthy donors.MethodsThe objects of the study are peripheral blood mononuclear cells (PBMC) of healthy donors (n = 150) and RA patients (n = 40) subjected to hospital treatment with either biological agents (Rituximab) or glucocorticosteroids (methylprednisolone). To determine PBMC phenotype antibodies anti-hCD3-APC, anti-hCD19 PECy7, anti-hCD14 FITC (eBioscience), as well as anti-hTNFRI-PE and anti-hTNFRII-PE (R&D Systems) were used. To determine receptor number on the cells Quantibrite PE Beads (BD) were used.ResultsCells obtained from patients who responded to therapy and achieved disease remission exhibited either an increase in the percentage of TNFRI+ cells or elevated expression density of this receptor type.ConclusionSubsets of immunocompetent cells from RA patients show variation in the percentage of membrane-bound receptor positive cells and receptor expression density, which influences the development and progression of the pathological processes in RA. Response to therapy and achievement of disease remission are associated with an increase of TNFRI expression.     

Influence of β-endorphin on the proliferative and phagocytic activities of peripheral blood cells of men and women from different age groups

It has been found that β-endorphin modulation of lymphocyte proliferative activity in male donors is mainly observed at a relatively young age (in groups aged 20–29 and 30–39 years), it gradually becomes lower with age, and disappears in donors at aged 50–60 years. At the same time, women have a prolonged modulating effect of peptide on proliferation. In women aged 50–59 years, the peptide has a marked promotional effect on spontaneous proliferation at concentrations of 10^?7, 10^?8, and 10^?10 M induced by a suboptimal concentration of phytohemagglutinin (PHA) at 10^?10 M, while in women aged 30–39 years, β-endorphin suppresses PHA-induced proliferative response. In men aged 20–29 years, β-endorphin stimulates the uptake capacity of neutrophils, whereas in those aged 50–59 years, this capacity is suppressed by β-endorphin. In female donors from any age groups, β-endorphin was not found to influence the activity of neurophils.     

Interleukin-28A promotes IFN-γ production by peripheral blood mononuclear cells from patients with Behçet’s disease

Behçet’s disease (BD) is an autoimmune disease of unknown etiology. Interleukin-28A (IL-28A) promotes immune responses and may participate in the pathogenesis of autoimmune diseases. To examine the role of IL-28A in the pathogenesis of BD, we measured the expression of IFN-γ and IL-17 by IL-28A-stimulated peripheral blood mononuclear cells (PBMCs) from 19 patients with BD and 16 healthy individuals. We found that IFN-γ and IL-17 were undetectable in the sera from BD patients and control subjects. The mRNA expression and protein production of IFN-γ by IL-28A-stimulated PBMCs from BD patients were significantly increased compared to healthy individuals. No significant difference was observed in the mRNA expression and protein production of IL-17 by IL-28A-stimulated PBMCs between BD patients and normal individuals.     

Analysis of T cell receptor β and γ genes from peripheral blood,regional lymph node and tumor-infiltrating lymphocyte clones from melanoma patients

Summary A total of 199 T cell clones from two melanoma patients were derived from progenitor T cells from recurrent melanoma, regional lymph nodes (either involved or uninvolved with malignancy) and peripheral blood by inoculating single cells directly into the wells of microtiter plates before in vitro expansion. The surface marker phenotype of most clones was CD4⁺CD8^–, although some were CD4^–CD8⁺. Genomic DNA prepared from all clones was analyzed by Southern blot hybridization using T cell receptor (TCR) and gene probes, seeking clones with identical TCR gene rearrangement patterns as direct evidence for in vivo progenitor T cell clonal amplification. ProbingHindIII-digested DNA with TCR and TCR probes revealed several clones with identical TCR gene rearrangement patterns. These clones had subsequent probing ofBamHI-digested DNA with TCR and TCR probes, which showed all but 2 clones to have distinct rearrangement patterns. These analyses provide clear molecular evidence for in vivo polyclonal CD4⁺ T cell populations in each of several separate immune compartments in these patients.This investigation was supported by National Institutes of Health, National Research Service Award CA-08 397 from the National Cancer Institute as well as NIH CA-32 685, CA-30 688, DOE FG028 760 502 and American Cancer Society Grant ACS CH-237     

TNF-α is upregulated in T2DM patients with fracture and promotes the apoptosis of osteoblast cells in vitro in the presence of high glucose

Fracture healing is regulated by proinflammatory mediators such as tumor necrosis factor-α (TNF-α), which poses influence on the balance between bone formation and remodeling. And the diabetes is thought to contribute to the delayed diabetic fracture healing. In the present study, we examined the promotion to proinflammatory cytokines and chemokines in type 2 diabetes mellitus (T2DM) patients with bone fractures, and then evaluated the promotion to TNF-α by the high glucose treatment in human osteoblast-like MG-63 cells and the regulatory role of the promoted TNF-α on the MG-63 cell apoptosis. It was demonstrated that there were significantly-upregulated high-sensitivity C-reactive protein (hsCRP) TNF-α, IL-1β, IL-6, IFN-γ-inducible protein 10 (IP-10) and RANTES in T2DM patients with bone fracture. And the promotion to TNF-α and IL-1β was confirmed in vitro in both mRNA and protein levels in high glucose-treated MG-63 cells. And either TNF-α or high glucose reduced the viability of MG-63 cells, promoted apoptosis and upregulated apoptosis-associated markers, such as released cytochrome c, cleaved caspase 3 and lyzed PARP. Moreover, there was a synergistic effect between TNF-α and high glucose. The viability reduction and the apoptosis induction of MG-63 cells were significantly higher in the group with both TNF-α and high glucose treatments, than in the group with singular TNF-α treatment. In conclusion, our study demonstrated that proinflammatory cytokines and chemokines were promoted in T2DM patients with bone fracture or in osteoblasts by the high glucose stimulation. TNF-α and high glucose synergistically reduced the viability and induced the apoptosis in the osteoblast-like MG-63 cells in vitro. It implies the significant regulatory role of TNF-α in the delayed fracture healing in T2DM.