Serum acid sphingomyelinase is upregulated in chronic hepatitis C infection and non alcoholic fatty liver disease

Sphingolipids constitute bioactive molecules with functional implications in homeostasis and pathogenesis of various diseases. However, the role of sphingolipids as possible disease biomarkers in chronic liver disease remains largely unexplored. In the present study we used mass spectrometry and spectrofluorometry methods in order to quantify various sphingolipid metabolites and also assess the activity of an important corresponding regulating enzyme in the serum of 72 healthy volunteers as compared to 69 patients with non-alcoholic fatty liver disease and 69 patients with chronic hepatitis C virus infection. Our results reveal a significant upregulation of acid sphingomyelinase in the serum of patients with chronic liver disease as compared to healthy individuals (p < 0.001). Especially in chronic hepatitis C infection acid sphingomyelinase activity correlated significantly with markers of hepatic injury (r = 0.312, p = 0.009) and showed a high discriminative power. Accumulation of various (dihydro-) ceramide species was identified in the serum of patients with non-alcoholic fatty liver disease (p < 0.001) and correlated significantly to cholesterol (r = 0.448, p < 0.001) but showed a significant accumulation in patients with normal cholesterol values as well (p < 0.001). Sphingosine, a further bioactive metabolite, was also upregulated in chronic liver disease (p < 0.001). However, no significant correlation to markers of hepatic injury was identified. Conclusion: Chronic hepatitis C virus infection and non-alcoholic fatty liver disease induce a significant upregulation of serum acid sphingomyelinase which appears as a novel biomarker in chronic hepatopathies. Further studies are required to elucidate the potential of the sphingolipid signaling pathway as putative therapeutic target in chronic liver disease.     

Interleukin-10 is upregulated by nanomolar rosiglitazone treatment of mature dendritic cells and human CD4+ T cells

Activators of peroxisome proliferator-activated receptor (PPAR)-gamma are anti-inflammatory and have been proposed as therapeutic agents for the treatment of Th1-type inflammatory diseases. We report that nanomolar concentrations of rosiglitazone enhance the production of IL-10 from activated human mature monocyte-derived dendritic cells. Also, rosiglitazone specifically induces the production of IL-10 from TCR-activated human CD4+ T cells and that this effect is PPAR-gamma-dependent. We also demonstrate for the first time the presence of a functional PPAR response element (PPRE) in the human IL-10 promoter region. Finally we show that rosiglitazone can induce IL-10 in combination with 1,25 alpha-dihydroxyvitamin D3 to a greater extent than each treatment alone. In summary our findings demonstrate that IL-10 is upregulated by nanomolar TZDs in immune cells, and this may, in part, be responsible for the potential anti-inflammatory effects of PPAR-gamma in humans.     

Kynurenine production mediated by indoleamine 2,3-dioxygenase aggravates liver injury in HBV-specific CTL-induced fulminant hepatitis

Indoleamine 2,3-dioxygenase (IDO), an enzyme that is ubiquitously distributed in mammalian tissues and cells, converts tryptophan to kynurenine, and is also known as a key molecule that promotes apoptosis in lymphocytes and neurons. In this study, we established hepatitis B virus (HBV)-transgenic (Tg)/IDO-knockout (KO) mice and examined the influence of IDO in a murine fulminant hepatitis model induced by HBV-specific cytotoxic T lymphocytes (CTL). An increase of IDO expression in the livers of HBV-Tg/IDO-wild-type (WT) mice administered HBV-specific CTL was confirmed by real-time polymerase chain reaction, western blotting, and evaluating IDO activity. Plasma alanine aminotransferase (ALT) levels in HBV-Tg/IDO-KO mice after HBV-specific CTL injection significantly decreased compared with those in HBV-Tg/IDO-WT mice. An inhibitor of IDO, 1-methyl-d-tryptophan (1-MT), could also attenuated the observed liver injury induced by this HBV-specific CTL. The expression levels of cytokine and chemokine mRNAs in the livers of HBV-Tg/IDO-WT mice were higher than those in the livers of HBV-Tg/IDO-KO mice. The administration of kynurenine aggravated the liver injury in HBV-Tg/IDO-KO mice injected with HBV-specific CTL. Simultaneous injection of recombinant murine interferon (IFN-γ) and kynurenine also increased the ALT levels in HBV-Tg/IDO-KO mice. The liver injury induced by IFN-γ and kynurenine was improved in HBV-Tg/tumor necrosis factor-α-KO mice. Conclusion: Kynurenine and IFN-γ induced by the administration with HBV-specific CTL are cooperatively involved in the progression of liver injury in acute hepatitis model. Our results may lead to a new therapy for the acute liver injury caused by HBV infection.     

Distinct CD4+ CD8+ double-positive T cells in the blood and liver of patients during chronic hepatitis B and C

CD4(+) and CD8(+) T cells, the main effectors of adaptive cellular immune responses, differentiate from immature, non-functional CD4(+)CD8(+) double-positive T (DPT) cells in the thymus. Increased proportions of circulating DPT lymphocytes have been observed during acute viral infections; in chronic viral diseases, the role and repartition of extra-thymic DPT cells remain largely uncharacterized. We performed a phenotypic analysis of DPT cells in blood and liver from patients chronically infected by hepatitis C (HCV) or B (HBV) viruses. The highest percentages of DPT cells, predominantly CD4(high)CD8(low), were observed in patients infected by HCV, while HBV-infected patients mostly displayed CD4(low)CD8(high) and CD4(high)CD8(high) DPT cells. All proportions of DPT cells were higher in liver than in blood with, for each subpopulation referred to above, a correlation between their frequencies in these two compartments. In HCV patients, intra-hepatic DPT cells displayed more heterogeneous activation, differentiation and memory phenotypes than in the blood; most of them expressed CD1a, a marker of T cell development in the thymus. Ex vivo, the inoculation of liver slices with HCV produced in cell culture was accompanied by a disappearance of CD8(high) cells, suggesting a direct effect of the virus on the phenotype of DPT cells in the liver. Our results suggest that, in half of the patients, chronic HCV infection promotes the production of DPT cells, perhaps by their re-induction in the thymus and selection in the liver.     

Zyxin is upregulated in the nucleus by thymosin beta4 in SiHa cells

Thymosin beta4 is a 43-amino acid actin-binding protein that promotes cell migration and is important in angiogenesis, wound healing, and tumor metastasis. We searched for genes upregulated by thymosin beta4 and identified zyxin as increased in SiHa cells in the presence of exogenously added thymosin beta4 and when thymosin beta4 is overexpressed using adenoviral vectors. Both zyxin and thymosin beta4 show increased localization in the nucleus. We conclude that thymosin beta4 may exert some of its migration promoting activity via increased zyxin expression.     

Lack of Cyclin E1 in hepatocytes aggravates ethanol-induced liver injury and hepatic steatosis in experimental murine model of acute and chronic alcohol-associated liver disease

BackgroundCyclin E1 is the regulatory subunit of cyclin-dependent kinase 2 (Cdk2) and one of the central players in cell cycle progression. We recently showed its crucial role for initiation of liver fibrosis and hepatocarcinogenesis. In the present study, we investigated the role of Cyclin E1 in the development of alcohol-associated liver disease (ALD).MethodsMice with constitutive (E1^?/?), hepatocyte-specific (Cyclin E1^Δhepa), or intestinal-epithelial-cell-specific (Cyclin E1^ΔIEC) inactivation of Cyclin E1 and corresponding wild type littermate controls (WT) were administered either a Lieber-DeCarli ethanol diet (LDE) for 3 weeks or acute ethanol binges (6 g/kg) through oral gavage. Serum parameters of liver functionality were measured; hepatic tissues were collected for biochemical and histological analyses.ResultsThe administration of acute EtOH binge and chronic LDE diet to E1^?/? mice enhanced hepatic steatosis, worsened liver damage and triggered body weight loss. Similarly, in the acute EtOH binge model, Cyclin E1^Δhepa mice revealed a significantly worsened liver phenotype. In contrast, inactivation of Cyclin E1 only in intestinal epithelial cell (IECs)did not lead to any significant changes in comparison to WT mice after acute EtOH challenge. Remarkably, both acute and chronic EtOH administration in E1^?/? animals resulted in increased levels of ADH and decreased expression of ALDH1/2. The additional application of a pan-Cdk inhibitor (S-CR8) further promoted liver damage in EtOH-treated WT mice.ConclusionOur data point to a novel unexpected role of Cyclin E1 in hepatocytes for alcohol metabolism, which seems to be independent of the canonical Cyclin E1/Cdk2 function as a cell cycle regulator.     

Attenuation of acute and chronic liver injury in rats by iron-deficient diet

Oxidative stress due to iron deposition in hepatocytes or Kupffer cells contributes to the initiation and perpetuation of liver injury. The aim of this study was to clarify the association between dietary iron and liver injuries in rats. Liver injury was initiated by the administration of thioacetamide or ligation of the common bile duct in rats fed a control diet (CD) or iron-deficient diet (ID). In the acute liver injury model induced by thioacetamide, serum levels of aspartate aminotransferase and alanine aminotransferase, as well as hepatic levels of lipid peroxide and 4-hydroxynonenal, were significantly decreased in the ID group. The expression of 8-hydroxydeoxyguanosine and terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling positivity showed a similar tendency. The expression of interleukin-1beta and monocyte chemotactic protein-1 mRNA was suppressed in the ID group. In liver fibrosis induced by an 8-wk thioacetamide administration, ID suppressed collagen deposition and smooth muscle alpha-actin expression. The expressions of collagen 1A2, transforming growth factor beta, and platelet-derived growth factor receptor beta mRNA were all significantly decreased in the ID group. Liver fibrosis was additionally suppressed in the bile-duct ligation model by ID. In culture experiments, deferoxamine attenuated the activation process of rat hepatic stellate cells, a dominant producer of collagen in the liver. In conclusion, reduced dietary iron is considered to be beneficial in improving acute and chronic liver injuries by reducing oxidative stress. The results obtained in this study support the clinical usefulness of an iron-reduced diet for the improvement of liver disorders induced by chronic hepatitis C and alcoholic/nonalcoholic steatohepatitis.     

Rad9 is upregulated and plays protective roles in an acute lung injury model

The Rad9-Hus1-Rad1 protein complex is believed to respond to DNA damage and play important roles in the cell cycle. We studied the role of Rad9 protein in alveolar epithelial cells in the pathogenesis of acute lung injury. In a mouse model of lung injury induced by bleomycin or lipopolysaccharide, Rad9 expression is increased in type II alveolar epithelial cells from the early stage of lung injury. A549 cells and mouse primary alveolar epithelial cells also upregulated Rad9 expression after exposure to bleomycin. Gene silencing of Rad9 using siRNA decreased the G2/M arrest in A549 cells induced by bleomycin and also decreased the survival of A549 cells following exposure to bleomycin and hydrogen peroxide. In conclusion, Rad9 is a signal in the earlier stage of epithelial cell cycle regulation and plays protective roles in alveolar epithelial cells in the pathogenesis of acute lung injury.     

Divergent functions of CD4+ T lymphocytes in acute liver inflammation and injury after ischemia-reperfusion

Hepatic ischemia-reperfusion results in an acute inflammatory response culminating in the recruitment of activated neutrophils that directly injure hepatocytes. Recent evidence suggests that CD4+ lymphocytes may regulate this neutrophil-dependent injury, but the mechanisms by which this occurs remain to be elucidated. In the present study, we sought to determine the type of CD4+ lymphocytes recruited to the liver after ischemia-reperfusion and the manner in which these cells regulated neutrophil recruitment and tissue injury. Wild-type and CD4 knockout (CD4-/-) mice were subjected to hepatic ischemia-reperfusion. CD4+ lymphocytes were recruited in the liver within 1 h of reperfusion and remained for at least 4 h. These cells were comprised of conventional (alphabetaTCR-expressing), unconventional (gammadeltaTCR-expressing), and natural killer T cells. CD4-/- mice were then used to determine the functional role of CD4+ lymphocytes in hepatic ischemia-reperfusion injury. Compared with wild-type mice, CD4-/- mice had significantly greater liver injury, yet far less neutrophil accumulation. Adoptive transfer of CD4+ lymphocytes to CD4-/- mice recapitulated the wild-type response. In wild-type mice, neutralization of interleukin (IL)-17, a cytokine released by activated CD4+ lymphocytes, significantly reduced neutrophil recruitment in association with suppression of MIP-2 expression. Finally, oxidative burst activity of liver-recruited neutrophils was higher in CD4-/- mice compared with those from wild-type mice. These data suggest that CD4+ lymphocytes are rapidly recruited to the liver after ischemia-reperfusion and facilitate subsequent neutrophil recruitment via an IL-17-dependent mechanism. However, these cells also appear to attenuate neutrophil activation. Thus the data suggest that CD4+ lymphocytes have dual, opposing roles in the hepatic inflammatory response to ischemia-reperfusion.     

TLR ligand-dependent activation of naive CD4 T cells by plasmacytoid dendritic cells is impaired in hepatitis C virus infection

Chronic hepatitis C virus (HCV) infection is characterized by diminished numbers and function of HCV-reactive T cells and impaired responses to immunization. Because host response to viral infection likely involves TLR signaling, we examined whether chronic HCV infection impairs APC response to TLR ligand and contributes to the origin of dysfunctional T cells. Freshly purified myeloid dendritic cells (MDC) and plasmacytoid DC (PDC) obtained from subjects with chronic HCV infection and healthy controls were exposed to TLR ligands (poly(I:C), R-848, or CpG), in the presence or absence of cytokine (TNF-alpha or IL-3), and examined for indices of maturation and for their ability to activate allogeneic naive CD4 T cells to proliferate and secrete IFN-gamma. TLR ligand was observed to enhance both MDC and PDC activation of naive CD4 T cells. Although there was increased CD83 and CD86 expression on MDC from HCV-infected persons, the ability of MDC to activate naive CD4 T cells in the presence or absence of poly(I:C) or TNF-alpha did not differ between HCV-infected and healthy control subjects. In contrast, PDC from HCV-infected persons had reduced activation marker (HLA-DR) and cytokine (IFN-alpha) expression upon R-848 stimulation, and these were associated with impaired activation of naive CD4 T cells. These data indicate that an impaired PDC responsiveness to TLR ligation may play an important role in the fundamental and unexplained failure to induce new T cell responses to HCV Ags and to other new Ags as a consequence of HCV infection.     

Discordant response of CD4+ T cells to antiretroviral therapy in HIV-infected patients coinfected with hepatitis C virus is accompanied by increased liver damage

A study of HIV-infected patients coinfected with hepatitis C virus and receiving antiretroviral therapy (ART) but not treated with interferon was performed. Patients were divided into two groups—with standard and inefficient recovery of CD4⁺ T cells. It was found that patients with discordant response of CD4⁺ T cells to ART showed heavier destructive processes in the liver than the successfully recovered subjects. They had increased levels of ALT and AST. In these patients, the risk of development of liver cirrhosis is greater.     

Dietary amino acid taurine ameliorates liver injury in chronic hepatitis patients

The effect of dietary amino acid taurine on the liver function of chronic hepatitis patients was investigated. The 24 chronic hepatitis patients with 2-5 times over normal activities of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) were selected and equally divided into taurine treatment and control groups. In taurine treatment group, each patient took 2 g taurine 3 times a day for three months, and then stopped treatment for 1 month. Patients taking placebo without taurine for 4 months served as a control group. ALT and AST activities and levels of cholesterol, triglyceride and thiobarbituric acid relative substances of serum plasma in the taurine group were all decreased at the end of three month treatment. The study suggested that dietary amino acid taurine may ameliorate liver injury for chronic hepatitis patients.     

CD36 aggravates podocyte injury by activating NLRP3 inflammasome and inhibiting autophagy in lupus nephritis

A major cause of proteinuria in lupus nephritis (LN) is podocyte injury, and determining potential therapeutic targets to prevent podocyte injury is important from a clinical perspective in the treatment of LN. CD36 is involved in podocyte injury in several glomerulopathies and was reported to be a vital candidate gene in LN. Here, we determined the role of CD36 in the podocyte injury of LN and the underlying mechanisms. We observed that CD36 and NLRP3 (NLR family pyrin domain containing 3) were upregulated in the podocytes of lupus nephritis patients and MRL/lpr mice with renal impairment. In vitro, CD36, NLRP3 inflammasome, and autophagy were elevated accompanied with increased podocyte injury stimulated by IgG extracted from lupus nephritis patients compared that from healthy donors. Knocking out CD36 with the CRISPR/cas9 system decreased the NLRP3 inflammasome levels, increased the autophagy levels and alleviated podocyte injury. By enhancing autophagy, NLRP3 inflammasome was decreased and podocyte injury was alleviated. These results demonstrated that, in lupus nephritis, CD36 promoted podocyte injury by activating NLRP3 inflammasome and inhibiting autophagy by enhancing which could decrease NLRP3 inflammasome and alleviate podocyte injury.Subject terms: Mechanisms of disease, Inflammasome, Lupus nephritis, Autophagy     

Apolipoprotein A-I gene expression is upregulated by polychlorinated biphenyls in rat liver

Xenobiotics such as polychlorinated biphenyls (PCB) increase serum cholesterol level (especially high density lipoprotein cholesterol) and apolipoprotein A-I (apo A-I) level in rats. The effect of PCB on serum apo A-I and hepatic apo A-I gene expression and the relationship between apo A-I and drug-metabolizing enzymes in rats were investigated. Serum levels of cholesterol and apo A-I were increased by dietary addition of PCB in a dose-dependent manner (0-500 mg/kg diet). Hepatic apo A-I mRNA level was also elevated by PCB in a similar fashion. Serum level of cholesterol gradually increased during feeding period of PCB (200 mg/kg diet, 105 days) and reached a two-fold higher level in PCB group than in controls. The levels of serum apo A-I and hepatic apo A-I mRNA linearly elevated during feeding period of PCB and were increased 3- or 4-fold, respectively, compared to controls. Although acute administration (16 hr) of PCB, 3-methylcholanthrene, and phenobarbital induced cytochrome P-450 gene expression in the liver, hepatic apo A-I gene expression was not increased by these xenobiotics. These results indicated that the serum levels of cholesterol and apo A-I had positive correlation with hepatic level of apo A-I mRNA in rats fed PCB, and that hepatic apo A-I gene expression was dependent upon intake of PCB but was not directly related to the induction of drug-metabolizing enzymes. This study demonstrated that xenobiotic-induced hyper-alpha-cholesterolemia would be caused by the increased apo A-I gene expression and cholesterol synthesis in the liver, coordinately.     

Hyper-activated pro-inflammatory CD16 monocytes correlate with the severity of liver injury and fibrosis in patients with chronic hepatitis B

Background

Extensive mononuclear cell infiltration is strongly correlated with liver damage in patients with chronic hepatitis B virus (CHB) infection. Macrophages and infiltrating monocytes also participate in the development of liver damage and fibrosis in animal models. However, little is known regarding the immunopathogenic role of peripheral blood monocytes and intrahepatic macrophages.

Methodology/Principal Findings

The frequencies, phenotypes, and functions of peripheral blood and intrahepatic monocyte/macrophage subsets were analyzed in 110 HBeAg positive CHB patients, including 32 immune tolerant (IT) carriers and 78 immune activated (IA) patients. Liver biopsies from 20 IA patients undergoing diagnosis were collected for immunohistochemical analysis. IA patients displayed significant increases in peripheral blood monocytes and intrahepatic macrophages as well as CD16⁺ subsets, which were closely associated with serum alanine aminotransferase (ALT) levels and the liver histological activity index (HAI) scores. In addition, the increased CD16⁺ monocytes/macrophages expressed higher levels of the activation marker HLA-DR compared with CD16⁻ monocytes/macrophages. Furthermore, peripheral blood CD16⁺ monocytes preferentially released inflammatory cytokines and hold higher potency in inducing the expansion of Th17 cells. Of note, hepatic neutrophils also positively correlated with HAI scores.

Conclusions

These distinct properties of monocyte/macrophage subpopulations participate in fostering the inflammatory microenvironment and liver damage in CHB patients and further represent a collaborative scenario among different cell types contributing to the pathogenesis of HBV-induced liver disease.     

Anergy in memory CD4+ T cells is induced by B cells

Induction of tolerance in memory T cells has profound implications in the treatment of autoimmune diseases and transplant rejection. Previously, we reported that the presentation of low densities of agonist peptide/MHC class II complexes induced anergy in memory CD4(+) T cells. In the present study, we address the specific interaction of different types of APCs with memory CD4(+) T cells. A novel ex vivo anergy assay first suggested that B cells induce anergy in memory T cells, and an in vivo cell transfer assay further confirmed those observations. We demonstrated that B cells pulsed with defined doses of Ag anergize memory CD4 cells in vivo. We established that CD11c(+) dendritic cells do not contribute to anergy induction to CD4 memory T cells, because diphtheria toxin receptor-transgenic mice that were conditionally depleted of dendritic cells optimally induced anergy in memory CD4(+) T cells. Moreover, B cell-deficient muMT mice did not induce anergy in memory T cells. We showed that B2 follicular B cells are the specific subpopulation of B cells that render memory T cells anergic. Furthermore, we present data showing that anergy in this system is mediated by CTLA-4 up-regulation on T cells. This is the first study to demonstrate formally that B cells are the APCs that induce anergy in memory CD4(+) T cells.     

HBcAg-specific CD4+CD25+regulatory T cells modulate immune tolerance and acute exacerbation on the natural history of chronic hepatitis B virus infection

Acute exacerbations (AEs) of chronic hepatitis B (CH-B) are accompanied by increased T cell responses to hepatitis B core and e antigens (HBcAg/HBeAg). Why patients are immunotolerant (IT) to the virus and why AEs occur spontaneously on the immunoactive phase remain unclear. The role of HBcAg-specific CD4(+)CD25(+) regulatory T (T(reg)) cells in AE and IT phases was investigated in this study. The SYFPEITHI scoring system was employed to predict MHC class II-restricted epitope peptides on HBcAg overlapping with HBeAg that were used for T(reg)-cell cloning and for the construction of MHC class II tetramers to measure T(reg) cell frequencies (T(reg) f). The results showed that HBcAg-specific T(reg) f declined during AE accompanied by increased HBcAg peptide-specific cytotoxic T lymphocyte frequencies. Predominant Foxp3-expressing T(reg) cell clones were generated from patients on the immune tolerance phase, while the majority of Th1 clones were obtained from patients on the immunoactive phase. T(reg) cells from liver and peripheral blood of CH-B patients express CD152 and PD1 antigens that exhibit suppression on PBMCs proliferation to HBcAg. These data suggest that HBcAg peptide-specific T(reg) cells modulate the IT phase, and that their decline may account for the spontaneous AEs on the natural history of chronic hepatitis B virus infection.     

Nephronectin expression is regulated by SMAD signaling in osteoblast-like MC3T3-E1 cells

Nephronectin (Npnt) is an extracellular matrix protein known to be a ligand for the integrin α8β1. We previously demonstrated that Npnt expression was suppressed by TGF-β through ERK1/2 and JNK in osteoblasts. In this study, we found that inhibition of a TGF-β type I receptor (TGF-β R1, Alk5) by a specific inhibitor {2-[3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine} strongly induced Npnt expression in osteoblast-like MC3T3-E1 cells. The Alk5 inhibitor-induced increase of Npnt expression occurred in both time- and dose-dependent manners, while that expression was also induced by introduction of an siRNA for Smad2, a central intracellular mediator of TGF-β signaling. These results suggest that the expression of Npnt is regulated by the Alk5-SMAD signaling pathway in osteoblasts.     

TGF-beta-mediated suppression by CD4+CD25+ T cells is facilitated by CTLA-4 signaling

CD4+CD25+ T cells play a pivotal role in immunological homeostasis by their capacity to exert immunosuppressive activity. However, the mechanism by which these cells function is still a subject for debate. We previously reported that surface (membrane) TGF-beta produced by CD4+CD25+ T cells was an effector molecule mediating suppressor function. We now support this finding by imaging surface TGF-beta on Foxp3+CD4+CD25+ T cells in confocal fluorescence microscopy. Then, using a TGF-beta-sensitive mink lung epithelial cell (luciferase) reporter system, we show that surface TGF-beta can be activated to signal upon cell-cell contact. Moreover, if such TGF-beta signaling is blocked in an in vitro assay of CD4+CD25+ T cell suppression by a specific inhibitor of TGF-betaRI, suppressor function is also blocked. Finally, we address the role of CTLA-4 in CD4+CD25+ T cell suppression, showing first that whereas anti-CTLA-4 does not block in vitro suppressor function, it does complement the blocking activity of anti-TGF-beta. We then show with confocal fluorescence microscopy that incubation of CD4+CD25+ T cells with anti-CTLA-4- and rB7-1/Fc-coated beads results in accumulation of TGF-beta at the cell-bead contact site. This suggests that CTLA-4 signaling facilitates TGF-beta-mediated suppression by intensifying the TGF-beta signal at the point of suppressor cell-target cell interaction.