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1.  Increased frequency of Th17 cells in systemic sclerosis is related to disease activity and collagen overproduction 
Introduction
Although immune dysfunction plays a role in the pathogenesis of systemic sclerosis (SSc), involvement of T helper 17 (Th17) and T regulatory (Treg) cells remains unclear. We aimed to investigate the presence of Th17 and Treg cells in SSc patients and the role of Th17 cells in collagen production in SSc fibroblasts.
Methods
We analyzed inflammatory cell profiles in the skin of 13 SSc patients by immunohistochemistry, the percentage of Th17 and Treg cells in peripheral blood mononuclear cells (PBMCs) of 45 SSc patients and 24 healthy controls by flow cytometry, gene expression in PBMCs by real-time reverse transcription-polymerase chain reaction and interleukin-17 (IL-17) in sera and culture supernatants by enzyme-linked immunosorbent assay. We also investigated the effect of Th17 cell-derived IL-17 on fibroblast growth and collagen production.
Results
Infiltration of inflammatory cells including IL-17+ and Foxp3+ lymphocytes was detected in the skin of patients with early SSc. The percentages of circulating Th17 cells and IL-17 production were elevated in samples from patients with active SSc, whereas the percentage of circulating Treg cells was not affected. The number of Th17 cells was closely related to disease activity. IL-17 from SSc patients promoted fibroblast growth and collagen production, whereas IL-17 neutralizing antibody effectively blocked collagen production.
Conclusion
SSc progression might be linked to expansion of circulating Th17 cells and increased infiltration of IL-17+ cells in skin. Th17-derived IL-17 is involved in fibroblast growth and collagen production. IL-17 blocking antibody may be a useful tool for intervention in the fibrotic course of SSc.
doi:10.1186/ar4430
PMCID: PMC3979142  PMID: 24398084
2.  Melanoma Induces Immunosuppression by Upregulating FOXP3+ Regulatory T Cells1 
The Journal of surgical research  2007;141(1):72-77.
Background
The immune response to melanoma is rarely curative suggesting the emergence of immunosuppression. FOXP3-expressing regulatory T cells (Treg cells) function to suppress immune responses. The objective of this study was to determine if melanoma evades immune surveillance, in part, by inducing Treg cells.
Material and methods
Peripheral blood mononuclear cells (PBMCs) were isolated and exposed to melanoma-conditioned media (MCM) or control media for one week. The induction of Treg cells in these PBMCs was determined by measuring the proportion of CD25+FOXP3+ T cells in all CD4+ T cells by flow cytometry. FOXP3 expression was determined by mean fluorescence intensity (MFI) and Western blot. Supernatant cytokines were determined by ELISA.
Results
Normal PBMCs exposed to MCM revealed higher proportions of Treg cells than those exposed to control media after six days (3.4% v. 1.3%, respectively, P < 0.02). The expression of FOXP3 in Treg cells from PBMCs exposed to MCM increased over time by MFI and Western blot but was not significantly different than those exposed to control media. The level of IL-10 and TGF-β in supernatants after six days growth was higher in MCM than control media but this did not reach statistical significance.
Conclusion
Exposure of PBMCs to melanoma results in induction of FOXP3+ Treg cells.
doi:10.1016/j.jss.2007.03.053
PMCID: PMC2043471  PMID: 17574040
melanoma; regulatory T cells; FOXP3; cytokines
3.  Increased Expression of IL-22 Is Associated with Disease Activity in Behcet’s Disease 
PLoS ONE  2013;8(3):e59009.
Objective
Interleukin (IL)-22 has been reported to be involved in the development of autoimmune diseases. This study aimed to analyze the expression and potential role of IL-22 in the pathogenesis of Behcet’s disease (BD).
Methods
The levels of IL-22 in patient sera or supernatants of cultured peripheral blood mononuclear cells (PBMCs) and CD4+T cells were detected by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used to evaluate the frequency of IL-22–producing CD4+ T cells. IL-22 mRNA from erythema nodosum skin lesions was examined using real time quantitative RT-PCR.
Results
BD patients with active uveitis showed a significantly higher expression of IL-22 in the supernatants of stimulated PBMCs and CD4+T cells compared with BD patients without active uveitis and normal controls. An increased frequency of IL-22-producing CD4+T cells was also found in BD patients with active uveitis. IL-22 mRNA expression was elevated in erythema nodosum skin lesions. In BD patients, a high IL-22 level in the supernatant of stimulated PBMCs correlated with the presence of retinal vasculitis and erythema nodosum.
Conclusions
IL-22 was associated with disease activity in BD and correlated with the presence of small vessel inflammation, suggesting that it may be involved in its pathogenesis.
doi:10.1371/journal.pone.0059009
PMCID: PMC3602549  PMID: 23527071
4.  Evaluation of cytolytic activity and phenotypic changes of circulating blood immune cells in patients with colorectal cancer by a simple preparation of peripheral blood mononuclear cells 
Purpose
This study aimed to assess the cytolytic activity and the phenotype of circulating blood immune cells in cancer patients by using a simple preparation of peripheral blood mononuclear cells (PBMCs).
Methods
Peripheral blood was obtained from 94 diagnosed colorectal cancer (CRC) patients and 112 healthy donors. PBMCs were cocultured with K562 cells for 2 hours and lactate dehydrogenase released from the dead K562 cells was measured by using a spectrophotometer. Meanwhile, PBMCs were stained with fluorescence conjugated monoclonal antibodies (mAbs) and analyzed by flow cytometry.
Results
The cytolytic activity of PBMCs were significantly different between CRC patient and healthy groups (8.82% ± 3.84% vs. 17.51% ± 8.57%; P < 0.001). However, no significant difference in the cytolytic activity was observed after surgery in the CRC patient group (before surgery, 8.82% ± 3.84% vs. after surgery, 9.95% ± 4.94%; P = 0.326). In addition, the percentage of peripheral blood natural killer cells was significantly higher in the preoperative patient group than in the healthy group (19.97% ± 11.51% vs. 15.60% ± 5.77%, P = 0.041). In contrast, the percentage of peripheral blood lymphocytes was lower in the preoperative patient group than in the healthy group (28.41% ± 8.31% vs. 36.4% ± 8.6%, P < 0.001).
Conclusion
These results demonstrate that circulating blood immune cells of CRC patients are functionally impaired and undergo an immunophenotypic perturbation, and show that a simple preparation of PBMCs can be useful to evaluate cellular immunity in cancer.
doi:10.4174/jkss.2013.85.5.230
PMCID: PMC3834022  PMID: 24266014
Blood cells; Natural killer cells; Immunologic cytotoxicity; Colorectal neoplasms
5.  CD4+CD25+FOXP3+ Regulatory T Cells Suppress Anti-Tumor Immune Responses in Patients with Colorectal Cancer 
PLoS ONE  2006;1(1):e129.
Background
A wealth of evidence obtained using mouse models indicates that CD4+CD25+FOXP3+ regulatory T cells (Treg) maintain peripheral tolerance to self-antigens and also inhibit anti-tumor immune responses. To date there is limited information about CD4+ T cell responses in patients with colorectal cancer (CRC). We set out to measure T cell responses to a tumor-associated antigen and examine whether Treg impinge on those anti-tumor immune responses in CRC patients.
Methodology and Principal Findings
Treg were identified and characterized as CD4+CD25+FOXP3+ using flow cytometry. An increased frequency of Treg was demonstrated in both peripheral blood and mesenteric lymph nodes of patients with colorectal cancer (CRC) compared with either healthy controls or patients with inflammatory bowel disease (IBD). Depletion of Treg from peripheral blood mononuclear cells (PBMC) of CRC patients unmasked CD4+ T cell responses, as observed by IFNγ release, to the tumor associated antigen 5T4, whereas no effect was observed in a healthy age-matched control group.
Conclusions/Significance
Collectively, these data demonstrate that Treg capable of inhibiting tumor associated antigen-specific immune responses are enriched in patients with CRC. These results support a rationale for manipulating Treg to enhance cancer immunotherapy.
doi:10.1371/journal.pone.0000129
PMCID: PMC1762416  PMID: 17205133
6.  Regulatory T cells and Atherosclerosis 
Atherosclerosis is a chronic autoimmune inflammatory disease. The involvement of both innate and adaptive immune responses in the pathogenesis of the disease has been well recognized. Tregs are an essential part of the immune system and have indispensable functions in maintaining immune system homeostasis, mediating peripheral tolerance, preventing autoimmune diseases, and suppressing inflammatory and proatherogenic immune response. Tregs carry out their immunosuppressive functions via several mechansims. One of the well-documented suppressive mechanisms of Tregs is the secretion of anti-inflammatory cytokines including IL-10, TGF-β, and IL-35. Studies have found that IL-10 and TGF-β have atheroprotective properties.
In addition, Tregs can suppress the activity of proatherogenic effector T cells, suggesting an atheroprotective role. In fact, fewer Tregs are found in atherogenic ApoE−/− mice comparing to wild-type mice, suggesting an uncontrolled balance between weakened Tregs and effector T cells in atherogenesis. Some clinical studies of autoimmune diseases also suggest that decreased Tregs numbers are associated with increased disease activity. The importance of Tregs in many autoimmune diseases and experimental atherosclerosis has been established in in vivo and in vitro studies. However, the roles of Tregs in atherosclerosis in the clinical setting remains to be further characterized.
doi:10.4172/2155-9880.S12-002
PMCID: PMC3757946  PMID: 23997979
regulatory T cells; vascular inflammation; atherosclerosis; immune suppression
7.  Peripheral blood but not synovial fluid natural killer T cells are biased towards a Th1-like phenotype in rheumatoid arthritis 
Arthritis Research & Therapy  2005;7(3):R493-R502.
Natural killer T (NKT) cells have been implicated in the regulatory immune mechanisms that control autoimmunity. However, their precise role in the pathogenesis of rheumatoid arthritis (RA) remains unclear. The frequency, cytokine profile and heterogeneity of NKT cells were studied in peripheral blood mononuclear cells (PBMCs) from 23 RA patients and 22 healthy control individuals, including paired PBMC–synovial fluid samples from seven and paired PBMC–synovial tissue samples from four RA patients. Flow cytometry revealed a decreased frequency of NKT cells in PBMCs from RA patients. NKT cells were present in paired synovial fluid and synovial tissue samples. Based on the reactivity of PBMC-derived NKT cells toward α-galactosylceramide, RA patients could be divided into responders (53.8%) and nonresponders (46.2%). However, NKT cells isolated from synovial fluid from both responders and nonresponders expanded upon stimulation with α-galactosylceramide. Analysis of the cytokine profile of CD4+ and CD4- PBMC derived NKT cell lines from RA patients revealed a significantly reduced number of IL-4 producing cells. In contrast, synovial fluid derived NKT cell lines exhibited a Th0-like phenotype, which was comparable to that in healthy control individuals. This suggests that synovial fluid NKT cells are functional, even in patients with nonresponding NKT cells in their blood. We conclude that, because the number of Vα24+Vβ11+CD3+ NKT cells is decreased and the cytokine profile of blood-derived NKT cells is biased toward a Th1-like phenotype in RA patients, NKT cells might be functionally related to resistance or progression of RA. Providing a local boost to the regulatory potential of NKT cells might represent a useful candidate therapy for RA.
doi:10.1186/ar1695
PMCID: PMC1174940  PMID: 15899036
8.  Hepatitis E rORF2p Stimulated and Unstimulated Peripheral Expression Profiling in Patients with Self-Limiting Hepatitis E Infection 
Journal of Immunology Research  2014;2014:565284.
To improve the current knowledge on the involvement of peripheral lymphocytes in hepatitis E virus (HEV) associated pathogenesis, we analyzed alterations in (1) immunophenotypic expressions (by flow cytometry) and (2) gene expression patterns (by TaqMan Low Density Array) of activatory, inhibitory, integrin, homing, ectonucleotidase machinery, costimulatory, inflammatory markers, and T regulatory cells (Treg) associated cytokines on HEV rORF2p stimulated and unstimulated PBMCs of 43 acute HEV patients, 30 recovered individuals, and 43 controls. The phenotypic expressions of key molecules CTLA-4, GITR, CD103, CD25, CD69, IL10 and TGF-β1 in the acute patients and TGF-β1 in the recovered individuals were significantly elevated on both unstimulated and stimulated PBMCs. Gene expression array data revealed upregulations of CD25, PD1, CD103, CCR4, IL10, and TGF-β1 on both unstimulated and HEV rORF2p stimulated PBMCs of acute patients. The observed upregulations of inhibitory, integrin, activatory, and Treg-associated cytokine genes on the PBMCs of acute HEV patients complemented by their frequency data suggest them as the major players in the fine-tuning of immune response in self-limiting hepatitis E infection.
doi:10.1155/2014/565284
PMCID: PMC4052084  PMID: 24963498
9.  Pattern of activation of human antigen presenting cells by genotype GII.4 norovirus virus-like particles 
Background
Virus-like particles (VLPs) from an Italian GII.4 norovirus strain were used to investigate activation and maturation of circulating antigen presenting cells (APCs) of human origin.
Methods
Peripheral blood mononuclear cells (PBMCs) isolated from five healthy subjects were pulsed ex vivo with VLPs, and stained with a set of monoclonal antibodies (MAbs) for phenotypic analysis by flow cytometry. Cytokine release in cell supernatants was investigated by ELISA.
Results
Norovirus VLPs induced activation and maturation of circulating APCs derived from the five donors, as well as production of IL-6, IFN-γ and TNF-α cytokines.
Conclusions
The present results suggest that VLPs can activate antigen presenting cells for an efficient induction of the adaptive immune response.
doi:10.1186/1479-5876-11-127
PMCID: PMC3671189  PMID: 23705987
Norovirus; Immunology; PBMCs; VLPs
10.  The Effects of Isoproterenol and Propranolol on Cytokine Profile Secretion by Cultured Tumor-infiltrating Lymphocytes Derived from Colorectal Cancer Patients 
Cell Journal (Yakhteh)  2011;13(4):281-289.
Objective:
Anti-tumor immunity and cytokine profiles have important roles in the development of cancer. Norepinephrine (NE) release due to sympathetic activation leads to a Th2 deviation via the beta-2 adrenergic receptor Beta-2 adrenergic receptor (β-2AR) and could increase cancer progression. This study intends to determine the effects of isoproterenol (ISO; beta-agonist) and propranolol (PRO; beta-antagonist) on the production of IFN-γ, IL-4, and IL-17. Cytokine levels have been examined in tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs) of patients with colorectal cancer (CRC). The β-2AR expression on lymphocyte subsets was also assessed.
Materials and Methods:
In this experimental study, TILs were isolated from fresh CRC tissue and patient PBMCs were obtained just prior to surgery. The cells were cultured in medium for 72 hours. Concomitantly, cells were stimulated with 10 µg/ml phytohemagglutinin (PHA) alone or in the presence of either 1 µmol/L of PRO or 1 µmol/L ISO. The concentration of cytokines in the supernatants was measured by ELISA. Three-color flow cytometry was used to determine the expression of β-2AR on the lymphocyte subsets. Statistical analyses were performed via paired or independent t-test.
Results:
Levels of IFN-γ, IL-4 and IL-17 were elevated after PHA-stimulation of PBMCs and TILs. However, the elevation of IFN-γ and IL-17 production by TILs in response to PHA was significantly lower than PBMCs. In the presence of ISO, the IFN-γ/IL-4 ratio reduced in all groups, but this reduction was very low in TILs. Interestingly, the effects of PRO on cytokine production were, at least partially, comparable to those of ISO. Depressed levels of β-2AR expression were demonstrated on CD4+IFN-γ+ and CD4+IL-17+ lymphocytes in patients' PBMCs and TILs.
Conclusion:
This study has demonstrated the effects of ISO and PRO on cytokine production by TILs and determined β-2AR expression on these cells. ISO failed to induce a shift toward the expected Th2 cytokine profile in CRC patients' TILs, which might be due to the downregulation of β-2AR expression on TILs. Additionally, in this study, PRO induced a shift to a Th2 profile in PBMCs.
PMCID: PMC3584474  PMID: 23507624
Isoproterenol; Propranolol; Beta-2 Adrenergic Receptor (β-2AR); Tumor-infiltrating Lymphocytes; Colorectal Cancer
11.  HLA Class I Expressions on Peripheral Blood Mononuclear Cells in Colorectal Cancer Patients 
Objective
To investigate the expression change of human leukocyte antigen (HLA) class I on human peripheral blood mononuclear cells (PBMCs) at both mRNA and protein levels, and to evaluate its roles in the development of colorectal cancer (CRC).
Methods
In the present study, 50 patients with CRC, 35 patients with benign colorectal lesion and 42 healthy volunteers were enrolled. Expression levels of HLA class I mRNA and protein were determined using real-time quantitative reverse transcription PCR (RT-PCR) and flow cytometry analysis, respectively.
Results
The expression levels of HLA class I mRNA and proteins were not influenced by age and gender. The relative ratios of HLA class I mRNA were 0.99±0.27 in healthy controls, 0.76±0.19 in benign patients, and 0.48±0.21 in CRC patients. Mean fluorescence intensities of HLA class I were 145.58±38.14 in healthy controls, 102.05±35.98 in benign patients and 87.44±34.01 in CRC patients. HLA class I on PBMCs was significantly down-regulated at both mRNA and protein levels in patients with stage III and IV CRC. CRC patients with lymph node metastasis also showed a decreased HLA class I expression at protein level.
Conclusion
HLA class I expressions on PBMCs are associated with staging of CRC and lymph node metastasis. Monitoring the expression of HLA class I on PBMCs may provide useful information for diagnosis and metastasis judgement of CRC.
doi:10.1007/s11670-012-0077-z
PMCID: PMC3555254  PMID: 23359566
HLA class I; Peripheral blood mononuclear cells; RT-PCR; Flow cytometry; Colorectal cancer
12.  Probiotic Bio-Three induces Th1 and anti-inflammatory effects in PBMC and dendritic cells 
AIM: To investigate the immune response of peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) that were stimulated by probiotic preparations.
METHODS: PBMCs were isolated, cultured, and stimulated with Bio-Three (a mixture of Bacillus mesentericus, Clostridium butyricum and Enterococcus faecalis; 105, 106 and 107 CFU/mL for 24 h). Cytokine production of (1) circulating PBMCs; (2) PBMCs stimulated by probiotic preparation; (3) monocyte-derived DCs; and (4) DC and T cell co-culture was determined by enzyme-linked immunosorbent assay. Phenotypic analysis of circulating PBMCs was also investigated by flow cytometry. Blood was obtained from individuals who consumed Bio-Three (109 CFU/d B. mesentericus, C. butyricum and E. faecalis) for 2 wk, or those who did not take probiotics orally.
RESULTS: In culture supernatants, interferon-γ (IFN-γ) and interleukin (IL)-10 production increased, but IL-4 and tumor necrosis factor-α (TNF-α) production by PBMCs decreased after 1 and 2 wk of probiotic treatment. Flow cytometry was also performed on day 14 and detected enhanced expression of CD11b, HLA-DR, CD4, CD45RA, CD25, CD44 and CD69 in response to Bio-Three. Furthermore, IL-10 and IL-12 were upregulated in supernatants of monocyte-derived DCs, and IFN-γ and IL-10 were enhanced in supernatants of CD4+ T cells co-cultured with DCs.
CONCLUSION: Bio-Three appeared to stimulate the Th1 immune response, downregulate pro-inflammatory cytokines (TNF-α) and upregulate anti-inflammatory cytokine (IL-10). Probiotics could be effective in activation of PBMCs and DCs.
doi:10.3748/wjg.v16.i28.3529
PMCID: PMC2909552  PMID: 20653061
Probiotics; Bio-Three; Peripheral blood mononuclear cells; Dendritic cells
13.  Overexpression of Evi-1 Oncoprotein Represses TGF-β Signaling in Colorectal Cancer 
Molecular carcinogenesis  2011;52(4):255-264.
Human colorectal cancer (CRC) cells are resistant to the anti-proliferative effect of transforming growth factor-β (TGF-β), suggesting that disruption of TGF-β signaling plays an important role in colorectal carcinogenesis. Ecotropic virus integration site-1 (Evi-1) oncoprotein represses TGF-β signaling by interacting with Smads, but its role in CRC has not been established. The purpose of this study is to determine whether Evi-1 plays role(s) in CRCs and to characterize Evi-1 transcript(s) in CRCs. Evi-1 was overexpressed in 53% of human CRC samples, 100% of colon adenoma samples, and 100% of human colon cancer cell lines tested. Using 5′ RACE, we cloned a novel Evi-1 transcript (Evi-1e) from a human CRC tissue and found that this novel transcript was expressed at a higher level in CRC tissues than in normal tissues and was the major Evi-1 transcript in CRCs. Transient Evi-1 transfection inhibited TGF-β-induced transcriptional activity and reversed the growth inhibitory effect of TGF-β in MC-26 mouse colon cancer cells. In conclusion, we have identified overexpression of Evi-1 oncoprotein as a novel mechanism by which a subset of human CRCs may escape TGF-β regulation. We have also identified a novel Evi-1 transcript, Evi-1e, as the major Evi-1 transcript expressed in human CRCs.
doi:10.1002/mc.21852
PMCID: PMC3922648  PMID: 22161860
colorectal cancer; ecotropic virus integration site-1; transforming growth factor-β; Smad proteins; rapid amplification of cDNA ends; growth inhibition
14.  IL-27-Induced Gene Expression Is Downregulated in HIV-Infected Subjects 
PLoS ONE  2012;7(9):e45706.
Objective
To characterize the effect of HIV infection on IL-27-induced gene expression.
Design
During HIV infection, cytokine expression and function become deregulated. IL-27 is an important modulator of inflammatory responses. Interestingly, IL-27 can inhibit HIV replication in T cells and monocytes, implicating IL-27 as a potential adjunct to anti-viral treatment. Our previous work demonstrated that circulating HIV may suppress IL-27 expression, therefore, this study, in continuation of our previous work, aimed to understand how HIV affects expression levels of the IL-27 receptor and downstream functions of IL-27.
Methods
Peripheral blood mononuclear cells (PBMC) were isolated from whole blood of HIV negative and HIV positive (viremic) individuals to assess IL-27-induced gene expression by flow cytometry and ELISA. PBMC were also processed for monocyte enrichment to assess IL-27 receptor expression by flow cytometry and real-time PCR.
Results
Expression of the IL-27 receptor subunit, gp130, was upregulated in response to IL-27 in HIV negative individuals, however, in HIV positive individuals, this IL-27 response was diminished. Furthermore, we observed downregulation of IL-27-induced IL-6, TNF-α, and IL-10 expression in HIV positive subjects.
Conclusion
In HIV infection, IL-27-induced gene expression was impaired, indicating HIV-mediated dysregulation of IL-27 functions occurs during HIV infection. This study provides evidence for new viral pathogenic mechanisms contributing to the widespread impairment of immune responses observed in HIV pathogenesis.
doi:10.1371/journal.pone.0045706
PMCID: PMC3458084  PMID: 23049843
15.  Reduced Cellular Susceptibility to In Vitro HIV Infection Is Associated with CD4+ T Cell Quiescence 
PLoS ONE  2012;7(9):e45911.
Background
HIV preferentially establishes productive infection in activated CD4+ T cells. Since proportions of activated CD4+ T cells vary between individuals, this study aimed to determine if individuals with a greater proportion of activated CD4+ T cells would be more susceptible to in vitro HIV infection.
Methodology/Principal Findings
Unstimulated peripheral blood mononuclear cells (PBMC) from various donors were inoculated with HIVML1956 in vitro. HIV replication was evaluated by HIV p24 ELISA of culture supernatants and intracellular staining for HIV p24, which was detected by flow cytometry. Baseline T cell phenotypes and infected cell phenotypes were also evaluated by flow cytometry. Ex vivo phenotyping at the time of blood draw showed that elevated T cell activation and reduced Tregs were associated with increased cellular susceptibility to in vitro infection. Furthermore, the infected CD4+ T cell population was enriched for activated cells.
Conclusion/Significance
These data suggest that CD4+ T cell quiescence provides an environment less conducive to the establishment of HIV infection by limiting the pool of activated target cells.
doi:10.1371/journal.pone.0045911
PMCID: PMC3448692  PMID: 23029309
16.  BAG-1 suppresses expression of the key regulatory cytokine transforming growth factor beta [TGF-β1] in colorectal tumour cells 
Oncogene  2012;32(38):10.1038/onc.2012.480.
As colorectal cancer remains the second highest cause of cancer-related deaths in much of the industrialised world, identifying novel strategies to prevent colorectal tumour development remains an important challenge. BAG-1 is a multi-functional protein, the expression of which is up-regulated at relatively early stages in colorectal tumorigenesis. Importantly, BAG-1 is thought to enhance colorectal tumour progression through promoting tumour cell survival. Here we report for the first time a novel role for BAG-1, establishing it as a suppressor of transforming growth factor beta [TGF-β1] expression in colorectal tumour cells. Microarray analysis first highlighted the possibility that BAG-1 may regulate TGF-β1 expression, a key cytokine in normal colonic tissue homeostasis. Q-RT-PCR and ELISA demonstrated TGFB1 mRNA and protein expression to be significantly increased when BAG1 levels were reduced by siRNA; additionally, induction of BAG-1L caused suppression of TGFB1 mRNA in colorectal tumour cells. Using reporter and ChIP assays, a direct association of BAG-1 with the TGFB1 gene regulatory region was identified. Immunohistochemistry and Weiser fraction data indicated levels of BAG-1 and TGF-β1 are inversely correlated in the normal colonic epithelium in vivo, consistent with a role for BAG-1-mediated repression of TGF-β1 production. In vitro studies showed that the change in TGF-β1 production following manipulation of BAG-1 is functionally relevant; through induction of anchorage-independent growth in TGF-β1 dependent NRK fibroblasts and regulation of SMAD2 phosphorylation in TGF-β1 sensitive adenoma cells. Taken together, this study identifies the anti-apoptotic protein BAG-1 as a suppressor of the inhibitory growth factor TGF-β1, suggesting that high expression of BAG-1 can impact on a number of the hallmarks of cancer, of potential importance in promoting the early stages of colorectal tumorigenesis. Establishing BAG-1 as a repressor of TGF-β1 has important biological implications, and highlights a new role for BAG-1 in colorectal tumorigenesis.
doi:10.1038/onc.2012.480
PMCID: PMC3880928  PMID: 23108401
BAG-1; TGF-β1; colorectal cancer; adenoma; transcriptional repression
17.  An Increased Abundance of Tumor-Infiltrating Regulatory T Cells Is Correlated with the Progression and Prognosis of Pancreatic Ductal Adenocarcinoma 
PLoS ONE  2014;9(3):e91551.
CD4+CD25+Foxp3+ regulatory T cells (Tregs) can inhibit cytotoxic responses. Though several studies have analyzed Treg frequency in the peripheral blood mononuclear cells (PBMCs) of pancreatic ductal adenocarcinoma (PDA) patients using flow cytometry (FCM), few studies have examined how intratumoral Tregs might contribute to immunosuppression in the tumor microenvironment. Thus, the potential role of intratumoral Tregs in PDA patients remains to be elucidated. In this study, we found that the percentages of Tregs, CD4+ T cells and CD8+ T cells were all increased significantly in tumor tissue compared to control pancreatic tissue, as assessed via FCM, whereas the percentages of these cell types in PBMCs did not differ between PDA patients and healthy volunteers. The percentages of CD8+ T cells in tumors were significantly lower than in PDA patient PBMCs. In addition, the relative numbers of CD4+CD25+Foxp3+ Tregs and CD8+ T cells were negatively correlated in the tissue of PDA patients, and the abundance of Tregs was significantly correlated with tumor differentiation. Additionally, Foxp3+ T cells were observed more frequently in juxtatumoral stroma (immediately adjacent to the tumor epithelial cells). Patients showing an increased prevalence of Foxp3+ T cells had a poorer prognosis, which was an independent factor for patient survival. These results suggest that Tregs may promote PDA progression by inhibiting the antitumor immunity of CD8+ T cells at local intratumoral sites. Moreover, a high proportion of Tregs in tumor tissues may reflect suppressed antitumor immunity.
doi:10.1371/journal.pone.0091551
PMCID: PMC3956642  PMID: 24637664
18.  T-regulatory lymphocytes in peripheral blood of gastric and colorectal cancer patients 
AIM: To assess the absolute number of T-regulatory cells (Tregs; CD4+CD25+Foxp3+) in the peripheral blood of gastric and colorectal cancer patients.
METHODS: We enrolled 70 cancer patients (33 gastric cancer, 37 colorectal cancer) and 17 healthy volunteers. The CD3+CD4+ lymphocytes and CD4+CD25+Foxp3+ Tregs in the peripheral blood were analyzed with flow cytometry. The absolute numbers of Tregs were calculated based on the CD4+CD25+Foxp3+ cells percentage of CD3+CD4+ cells and the absolute numbers of CD3+CD4+ cells per microliter.
RESULTS: The mean number of CD4+CD25+Foxp3+ cells per microliter in colorectal cancer patients was 15.7 (SD: 21.8), for gastric cancer patients 12.2 (SD: 14.3), and for controls 17.5 (SD: 11.4). The absolute number of Tregs was significantly lower in gastric cancer patients than in controls (P = 0.026). There was no statistically significant difference for gastric vs colorectal cancer or colorectal cancer vs controls. The absolute number of Tregs was also significantly depressed in N+ vs N- cancer patients [22.0 (27.7) vs 10.1 (9.0), P = 0.013], and in the subgroup of gastric cancer patients [30.3 (27.6) vs 9.6 (8.0), P = 0.003]. No statistical difference was observed in the proportion of Tregs in the CD4+ population between the groups.
CONCLUSION: The absolute number of Tregs in peripheral blood of gastric cancer but not colorectal cancer patients was significantly decreased in comparison with that in healthy controls.
doi:10.3748/wjg.v17.i3.343
PMCID: PMC3022294  PMID: 21253393
CD4+CD25+Foxp3+ cells; T regulatory cells; Peripheral blood; Gastric cancer; Colorectal cancer
19.  Transforming growth factor-β1 in plaque morphea 
Introduction
Morphea (localized scleroderma) is a rare cutaneous disease characterized by skin fibrosis of unknown pathogenesis. Transforming growth factor-β (TGF-β) is a potent profibrotic factor. The role of TGF-β in morphea remains unclear.
Aim
The goal of this study was to estimate the expression level of TGF-β1 in skin and peripheral blood mononuclear cells as well as the plasma levels of TGF-β1 in plaque morphea (MEP).
Material and methods
The study involved 20 MEP patients. Three control groups were involved: 1 – plasma: 36 healthy volunteers; 2 – PBMC: 47 healthy volunteers; 3 – skin biopsies: 13 samples collected during mastectomy (breast cancer was not skin involved). The analysis of TGF-β1 plasma levels was performed with the use an adequate ELISA kit, while real-time polymerase chain reaction was employed for the expression of TGF-β1 in peripheral blood mononuclear cells (PBMC) and skin.
Results
In our study we have not detected differences in TGF-β 1 expression in PBMC, skin, nor in plasma levels of TGF-β1 between MEP patients and healthy controls, regardless of disease activity and its duration.
Conclusions
The results of our study contradict the claim of the substantial role of TGF-β1 in the most common morphea subtype – MEP.
doi:10.5114/pdia.2013.39431
PMCID: PMC3907897  PMID: 24493995
morphea; scleroderma; transforming growth factor-β; transforming growth factor
20.  In vitro synthesis of primary specific anti-breast cancer antibodies by normal human peripheral blood mononuclear cells 
Cancer immunology, immunotherapy : CII  2011;60(12):1707-1720.
In this study, we developed a unique in vitro model to mimic the endogenous tumor microenvironment to understand the effect of immunotherapy with activated T-cells (ATC) armed with anti-CD3 × anti-Her2 bispecific antibody (aATC) on antibody response by naive immune cells. This model contained a co-culture of naïve peripheral blood mononuclear cells (PBMC), breast cancer cells (SK-BR-3), ATC or aATC and CpG ODNs. Culture supernatants were tested at various time points for anti-SK-BR-3 antibodies by ELISA, Western blot and flow cytometry. PBMC cocultured with non-irradiated aATC or irradiated (*) aATC showed significant increases in anti-tumor antibody production at day 14 (P < 0.0001) in the presence of CpG-ODN compared to unstimulated PBMC cultures (n = 9). Antibody specificity was confirmed by ELISA, Western blot and flow cytometry. Co-cultures containing *aATC and CpG showed significantly enhanced levels of IgG2 (P < 0.001) and cytokines that promote IgG2 synthesis including IL-13 (P < 0.02), IFNγ (P < 0.01) and GM-CSF (P < 0.05) compared to unstimulated PBMC control (n = 3). We show that aATC targeting and lysis of tumor cells induces an anti-tumor antibody response in our in vitro model. This model provides a unique opportunity to evaluate the interactions of T-cells, B-cells, and antigen-presenting cells leading to specific anti-tumor antibody responses.
doi:10.1007/s00262-011-1056-9
PMCID: PMC3792712  PMID: 21713642
In Vitro tumor-specific antibody synthesis; Peripheral blood mononuclear cells; Breast cancer; Bispecific antibody; Her2/neu; Immunotherapy
21.  Possible association of decreased NKG2D expression levels and suppression of the activity of natural killer cells in patients with colorectal cancer 
International Journal of Oncology  2011;40(4):1285-1290.
Natural-killer group 2 (NKG2), a natural killer (NK) cell receptor, plays a critical role in regulating NK cytotoxicity. In this study, we investigated the expression levels of natural killer group 2 member A (NKG2A) and natural killer group 2 member D (NKG2D) in NK cells as well as the regulatory function of NKG2D in patients with colorectal cancer (CRC). Sixty-two CRC patients and 32 healthy controls were enrolled in this study. The expression levels of NKG2A and NKG2D mRNA in peripheral blood mononuclear cells (PBMCs) were investigated using real-time PCR. Flow cytometry was performed to assay the levels of NKG2A and NKG2D proteins in NK cells. The levels of NKG2D mRNA in PBMCs in the patients were significantly lower than those in the controls [mean ± SD, 1.11±0.60 (CRC patients) vs. 1.65±0.71 (healthy controls); p<0.01], whereas the 2 groups showed no apparent difference in the levels of NKG2A mRNA (p>0.05). In addition, the patients showed significantly lower NKG2D levels in NK cells than the controls did (71.23%±8.31% [CRC patients] vs. 79.39%±5.58% [healthy controls]; p<0.01). However, we observed no distinct difference in the NKG2A expression levels in NK cells between the 2 groups (p>0.05). Notably, blockage of NKG2D signaling with anti-NKG2D antibodies ex vivo resulted in decreased cytotoxicity and CD107a degranulation. Our data revealed that the decrease in NKG2D expression levels may have been associated with suppression of NK cell activity in CRC patients.
doi:10.3892/ijo.2011.1315
PMCID: PMC3584522  PMID: 22200673
NKG2; natural killer cells; colorectal cancer; suppression
22.  Variations in T-helper 17 and Regulatory T Cells during The Menstrual Cycle in Peripheral Blood of Women with Recurrent Spontaneous Abortion 
Background:
Disorders in immune system regulation may result in pregnancy abnormalities such as recurrent spontaneous abortion (RSA). This study aims to determine the ratio of regulatory T (Treg) and T helper (Th) 17 cells in unexplained RSA (URSA) women during proliferative and secretory phases of their menstrual cycles compared to healthy non-pregnant women.
Materials and Methods:
In this case control study, 25 women with URSA and 35 healthy, non-pregnant women were enrolled. The percentage of Th17 and Treg cells in participants peripheral blood were determined by flow cytometry.
Results:
The percentage of Th17 cells and their related cytokines in serum (IL-17A) were higher in the proliferative and secretory phases of the menstrual cycles of URSA women compared to the control women. However, a lower percentage of Treg cells and their related cytokines in serum, transforming growth factor (TGF) β1 and interleukin (IL)-10 were detected in the proliferative but not the secretory phase of the URSA group. The ratio of Th17/CD4+ Treg was higher in the URSA group than the control group. We observed an increased ratio of Th17/CD4+ Treg during the proliferative and secretory phases in URSA women.
Conclusion:
The imbalance between Th17 and Treg cells during the proliferative phase of menstrual cycles in the URSA group may be considered a cause for spontaneous abortion.
PMCID: PMC3973173  PMID: 24695956
Regulatory T Cells; T Helper 17; Menstrual Cycle; Pregnancy
23.  Increased frequency and clinical significance of myeloid-derived suppressor cells in human colorectal carcinoma 
AIM: To investigate the frequency and clinical significance of the myeloid-derived suppressor cells (MDSC) in human colorectal carcinoma (CRC).
METHODS: Samples of peripheral blood and tumor tissue from 49 CRC patients were analyzed. Mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation and were subjected to a flow cytometry-based immunophenotypic analysis.
RESULTS: A considerable increase in the percentage of CD33+HLA-DR- MDSCs was observed in the peripheral blood (1.89% ± 0.75%) and tumor tissues (2.99% ± 1.29%) of CRC patients as compared with that in the peripheral blood of healthy controls (0.54% ± 0.35%). This expanded CD33+HLA-DR- subset exhibited immature myeloid cell markers, but not lineage markers, and showed up-regulation of CD18/CD11b expression as compared with the MDSCs from healthy donors. Further studies showed that the MDSC proportion in CRC peripheral blood was correlated with nodal metastasis
(P = 0.023), whereas that in tumor tissues was correlated with nodal/distant metastasis (P = 0.016/P = 0.047) and tumor stage (P = 0.028), suggesting the involvement of MDSCs in CRC tumor development.
CONCLUSION: Characterization of MDSCs in CRC suggests the clinical significance of circulating and tumor-infiltrating MDSCs and may provide new insights into the CRC immunotherapy targeting MDSCs.
doi:10.3748/wjg.v18.i25.3303
PMCID: PMC3391769  PMID: 22783056
Myeloid-derived suppressor cell; Colorectal carcinoma; Tumor metastasis
24.  Increased interleukin-23 receptor+ T cells in peripheral blood mononuclear cells of patients with systemic lupus erythematosus 
Arthritis Research & Therapy  2010;12(6):R215.
Introduction
Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by production of autoantibodies and immune complex deposition in various organs. Aberrations in the T lymphocyte compartment and dysregulated cytokine production are key features of SLE pathogenesis and disease progression. Recently, the role of the interleukin (IL)-17/IL-23 axis in the pathogenesis of SLE has been reported. IL-23 and IL-23R are essential for expansion of pathogenic IL-17-producing T lymphocytes and have been shown to be important in the pathogenesis of lupus in animal models.
Methods
In this study, the expression of IL-23R and IL-17 in CD4+ and CD8+ T lymphocytes in peripheral blood mononuclear cells (PBMCs) of SLE patients and control subjects were examined by flow cytometry. Twenty-nine SLE patients and 10 control subjects were recruited in this study. Patients were divided into active and inactive groups based on the SLE disease activity index (SLEDAI). As another disease control population, five psoriatic patients were recruited in this study.
Results
Percentages of both IL23R+ CD4+ and IL-23R+ CD8+ T cell subsets were significantly higher in freshly isolated PBMCs from both groups of SLE patients compared to control subjects (P = 0.0021 and P = 0.0006, respectively). In addition, this difference was maintained after ex vivo stimulation with plate-bound anti-CD3/CD28 antibodies (P = 0.007 and P = 0.0019, respectively). When the fold increase in IL-17+ T cells after ex vivo stimulation for three days was compared between patients and controls, SLE patients exhibited significantly higher increases in CD4+ IL-17+ and CD8+ IL-17+ T cells, suggesting that PBMCs from SLE patients promoted the expansion of IL-17-producing T cells upon stimulation more vigorously than control PBMCs. These trends were not observed in psoriasis patients. The correlations between IL-23R+ T cells and IL-17+ T cells and IL-23R+ CD8+ T cells and SLEDAI scores in patients were also found to be statistically significant.
Conclusions
The results of our study confirmed the relevance of the IL-23/IL-17 axis in the pathogenesis of SLE and further highlighted the importance of IL-23R+ T cell subsets in this autoimmune disease.
doi:10.1186/ar3194
PMCID: PMC3046525  PMID: 21110900
25.  Cytokine/chemokine profiles contribute to understanding the pathogenesis and diagnosis of primary Sjögren's syndrome 
To investigate the pathogenesis of localized autoimmune damage in Sjögren's syndrome (SS) by examining the expression patterns of cytokines, chemokines and chemokine receptors at sites of autoimmune damage. mRNA expression of these molecules in the labial salivary glands (LSGs) and peripheral blood mononuclear cells (PBMCs) from 36 SS patients was examined using a real-time polymerase chain reaction-based method. Subsets of the infiltrating lymphocytes and chemokines/chemokine receptors expression in the LSG specimens were examined by immunohistochemistry. Cytokines/chemokine concentrations in the saliva were analysed using flow cytometry or enzyme-linked immunosorbent assay. mRNA expression of T helper type 1 (Th1) cytokines, chemokines and chemokine receptors was higher in LSGs than in PBMCs. In contrast, mRNA expression of Th2 cytokines, chemokines [thymus and activation-regulated chemokine (TARC/CCL17), macrophage-derived chemokine (MDC/CCL22)] and chemokine receptor (CCR4) was associated closely with strong lymphocytic accumulation in LSGs. Furthermore, TARC and MDC were detected immunohistochemically in/around the ductal epithelial cells in LSGs, whereas CCR4 was detected on infiltrating lymphocytes. The concentrations of these cytokines/chemokines were significantly higher in the saliva from SS patients than those from controls, and the concentrations of Th2 cytokines/chemokines were associated closely with strong lymphocytic accumulation in LSGs. These results suggest that SS might be initiated and/or maintained by Th1 and Th17 cells and progress in association with Th2 cells via the interaction between particular chemokines/chemokine receptors. Furthermore, the measurement of cytokines/chemokines in saliva is suggested to be useful for diagnosis and also to reveal disease status.
doi:10.1111/j.1365-2249.2012.04587.x
PMCID: PMC3390469  PMID: 22670774
chemokines; cytokines; Sjögren's syndrome; T cells

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