Enhanced apoptosis of peripheral CD5-negative B lymphocytes from chronically hepatitis C virus-infected patients: reversal after antiviral treatment

Whereas enhanced peripheral T-cell apoptosis and its association with autoimmunity have recently been reported, the apoptotic status of peripheral B cells in chronic hepatitis C virus (HCV) infection remains ambiguous. We therefore sought to investigate the sensitivity of peripheral B cells to apoptosis and to assess the possible benefits of antiviral treatment in mitigating these effects. Spontaneous apoptosis, the extent of apoptosis rescue, and NF-kappaB expression in peripheral B cells were studied in patients with chronic HCV infections (group 1), in sustained responders after antiviral treatment (group 2), and in healthy controls (group 3). For group 1, spontaneous B-cell apoptosis was increased (26% +/- 4.6%) and apoptosis rescue was altered (39%) compared to group 3 (18% +/- 5% and 50%, respectively; P = 0.001). In contrast, apoptosis and apoptosis rescue were similar for groups 2 and 3. Enhanced B-cell apoptosis was associated with decreased NF-kappaB expression and was found only in CD5-negative (CD5(neg)) B cells, whereas CD5(pos) cells were apoptosis resistant. Chronic HCV infection is associated with enhanced peripheral B-cell apoptosis and decreased apoptosis rescue. Successful antiviral treatment reverses these abnormalities to the levels seen in healthy individuals. The relative resistance of the CD5(pos) B-cell subpopulation to apoptosis may play a role in HCV-related autoimmunity and lymphoproliferation.     

Expansion of functionally anergic CD21-/low marginal zone-like B cell clones in hepatitis C virus infection-related autoimmunity

Homeostasis of peripheral B cell subsets is disturbed during chronic hepatitis C virus (HCV) infection, leading to the occurrence of autoimmunity and B cell lymphoproliferation. However, mechanisms by which HCV causes lymphoproliferation remain controversial. We report in this article on the elevated number of clonal CD21(-/low)IgM(+)CD27(+) marginal zone (MZ)-like B cells, which correlates with autoimmunity and lymphoproliferation in HCV patients. We found an increase in autoreactive BCRs using V(H)1-69 and V(H)4-34 genes in CD21(-/low) MZ B cells. CD21(-/low) MZ B cells showed impaired calcium-mediated signaling, did not upregulate activation markers, and did not proliferate in response to BCR triggering. CD21(-/low) MZ B cells also were prone to dying faster than their CD21(+) counterparts, suggesting that these B cells were anergic. CD21(-/low) MZ B cells, in contrast, remained responsive to TLR9 stimulation. Gene array analyses revealed the critical role of Early growth response 2 and Cbl-b in the induction of anergy. Therefore, HCV patients who display high frequencies of unresponsive CD21(-/low) MZ B cells are more susceptible to developing autoimmunity and/or lymphoproliferation. These cells remain in peripheral blood controlled by functional anergy instead of being eliminated, and chronic antigenic stimulation through TLR stimulation may create a favorable environment for breaking tolerance and activating these cells.     

Identification and in vitro expansion of functional antigen-specific CD25+ FoxP3+ regulatory T cells in hepatitis C virus infection

CD4(+)CD25(+) regulatory T cells (CD25(+) Tregs) play a key role in immune regulation. Since hepatitis C virus (HCV) persists with increased circulating CD4(+)CD25(+) T cells and virus-specific effector T-cell dysfunction, we asked if CD4(+)CD25(+) T cells in HCV-infected individuals are similar to natural Tregs in uninfected individuals and if they include HCV-specific Tregs using the specific Treg marker FoxP3 at the single-cell level. We report that HCV-infected patients display increased circulating FoxP3(+) Tregs that are phenotypically and functionally indistinguishable from FoxP3(+) Tregs in uninfected subjects. Furthermore, HCV-specific FoxP3(+) Tregs were detected in HCV-seropositive persons with antigen-specific expansion, major histocompatibility complex class II/peptide tetramer binding affinity, and preferential suppression of HCV-specific CD8 T cells. Transforming growth factor beta contributed to antigen-specific Treg expansion in vitro, suggesting that it may contribute to antigen-specific Treg expansion in vivo. Interestingly, FoxP3 expression was also detected in influenza virus-specific CD4 T cells. In conclusion, functionally active and virus-specific FoxP3(+) Tregs are induced in HCV infection, thus providing targeted immune regulation in vivo. Detection of FoxP3 expression in non-HCV-specific CD4 T cells suggests that immune regulation through antigen-specific Treg induction extends beyond HCV.     

T cells with a CD4+CD25+ regulatory phenotype suppress in vitro proliferation of virus-specific CD8+ T cells during chronic hepatitis C virus infection

Chronic hepatitis C virus (HCV) infection is associated with impaired proliferative, cytokine, and cytotoxic effector functions of HCV-specific CD8(+) T cells that probably contribute significantly to viral persistence. Here, we investigated the potential role of T cells with a CD4(+)CD25(+) regulatory phenotype in suppressing virus-specific CD8(+) T-cell proliferation during chronic HCV infection. In vitro depletion studies and coculture experiments revealed that peptide specific proliferation as well as gamma interferon production of HCV-specific CD8(+) T cells were inhibited by CD4(+)CD25(+) T cells. This inhibition was dose dependent, required direct cell-cell contact, and was independent of interleukin-10 and transforming growth factor beta. Interestingly, the T-cell-mediated suppression in chronically HCV-infected patients was not restricted to HCV-specific CD8(+) T cells but also to influenza virus-specific CD8(+) T cells. Importantly, CD4(+)CD25(+) T cells from persons recovered from HCV infection and from healthy blood donors exhibited significantly less suppressor activity. Thus, the inhibition of virus-specific CD8(+) T-cell proliferation was enhanced in chronically HCV-infected patients. This was associated with a higher frequency of circulating CD4(+)CD25(+) cells observed in this patient group. Taken together, our results suggest that chronic HCV infection leads to the expansion of CD4(+)CD25(+) T cells that are able to suppress CD8(+) T-cell responses to different viral antigens. Our results further suggest that CD4(+)CD25(+) T cells may contribute to viral persistence in chronically HCV-infected patients and may be a target for immunotherapy of chronic hepatitis C.     

CD27(+) peripheral blood B-cells are a useful biodosimetric marker in vitro

The CD8(+) natural killer (NK) subpopulation has recently been identified as a fast and reliable biodosimetric indicator within human peripheral blood mononuclear cells (PBMC) in vitro. In irradiated and subsequently cultivated PBMC, a decrease of the relative number of intact CD3(-)CD8(+) lymphocytes 16 and 48 h after treatment has allowed for estimating the received dose in the range of 0 - 10 Gy and lethal/sublethal dose discrimination, respectively. Here we show that suitable biodosimeters can also be found in the peripheral blood B-cell compartment. Multiparameter flow cytometric analysis of irradiated and subsequently cultivated human PBMC revealed that both the CD27(+) and CD21(-) B-cell subpopulations can be used as biodosimeters and the CD19(+)CD27(+) lymphocytes have proved useful for retrospective determination of the received dose in the range of 0 - 6 Gy. In addition, several CD19(+) lymphocyte subsets characterized by co expression of CD21, CD27 and CD38 have been shown to bear biodosimetric potential, too. However, when important parameters like the original size within the CD19(+) compartment, its radiation-induced changes and data variation had been taken into account, the CD27(+) subpopulation proved superior to the other B-cell subpopulations and subsets. It appears that, in the dose range of 0 - 6 Gy, the relative decrease of CD27(+) B lymphocytes provides more sensitive and reliable data than that of CD8(+) NK-cells due mainly to lower data variation. In contrast to CD27(+) B cells, the proportions of CD27(+) subpopulations of T-cells were not affected by irradiation. We have also proposed a simple experimental protocol based on full blood cultivation and three-color CD27/CD3/CD19 immuno-phenotyping as a time-saving and inexpensive approach for practical biodosimetric evaluations on simple, three-to-four color flow cytometers.     

Induction of autoimmunity by expansion of autoreactive CD4+CD62Llow cells in vivo

The prerequisites of peripheral activation of self-specific CD4(+) T cells that determine the development of autoimmunity are incompletely understood. SJL mice immunized with myelin proteolipid protein (PLP) 139-151 developed experimental autoimmune encephalomyelitis (EAE) when pertussis toxin (PT) was injected at the time of immunization but not when injected 6 days later, indicating that PT-induced alterations of the peripheral immune response lead to the development of autoimmunity. Further analysis using IA(s)/PLP(139-151) tetramers revealed that PT did not change effector T cell activation or regulatory T cell numbers but enhanced IFN-gamma production by self-specific CD4(+) T cells. In addition, PT promoted the generation of CD4(+)CD62L(low) effector T cells in vivo. Upon adoptive transfer, these cells were more potent than CD4(+)CD62L(high) cells in inducing autoimmunity in recipient mice. The generation of this population was paralleled by higher expression of the costimulatory molecules CD80, CD86, and B7-DC, but not B7-RP, PD-1, and B7-H1 on CD11c(+)CD4(+) dendritic cells whereas CD11c(+)CD8alpha(+) dendritic cells were not altered. Collectively, these data demonstrate the induction of autoimmunity by specific in vivo expansion of CD4(+)CD62L(low) cells and indicate that CD4(+)CD62L(low) effector T cells and CD11c(+)CD4(+) dendritic cells may be attractive targets for immune interventions to treat autoimmune diseases.     

Tim-3 pathway controls regulatory and effector T cell balance during hepatitis C virus infection

Hepatitis C virus (HCV) is remarkable at disrupting human immunity to establish chronic infection. Upregulation of inhibitory signaling pathways (such as T cell Ig and mucin domain protein-3 [Tim-3]) and accumulation of regulatory T cells (Tregs) play pivotal roles in suppressing antiviral effector T cell (Teff) responses that are essential for viral clearance. Although the Tim-3 pathway has been shown to negatively regulate Teffs, its role in regulating Foxp3(+) Tregs is poorly explored. In this study, we investigated whether and how the Tim-3 pathway alters Foxp3(+) Treg development and function in patients with chronic HCV infection. We found that Tim-3 was upregulated, not only on IL-2-producing CD4(+)CD25(+)Foxp3(-) Teffs, but also on CD4(+)CD25(+)Foxp3(+) Tregs, which accumulate in the peripheral blood of chronically HCV-infected individuals when compared with healthy subjects. Tim-3 expression on Foxp3(+) Tregs positively correlated with expression of the proliferation marker Ki67 on Tregs, but it was inversely associated with proliferation of IL-2-producing Teffs. Moreover, Foxp3(+) Tregs were found to be more resistant to, and Foxp3(-) Teffs more sensitive to, TCR activation-induced cell apoptosis, which was reversible by blocking Tim-3 signaling. Consistent with its role in T cell proliferation and apoptosis, blockade of Tim-3 on CD4(+)CD25(+) T cells promoted expansion of Teffs more substantially than Tregs through improving STAT-5 signaling, thus correcting the imbalance of Foxp3(+) Tregs/Foxp3(-) Teffs that was induced by HCV infection. Taken together, the Tim-3 pathway appears to control Treg and Teff balance through altering cell proliferation and apoptosis during HCV infection.     

Elevated numbers of Fc gamma RIIIA+ (CD16+) effector CD8 T cells with NK cell-like function in chronic hepatitis C virus infection

CTL are crucial in the defense against viral infections. In the course of investigating peripheral blood and intrahepatic CD8 T cells in patients with chronic hepatitis C virus (HCV) infection, we observed a significant population of CD8 T cells expressing the FcgammaRIIIA (CD16) receptor. This observation led us to characterize these cells with respect to their phenotype and function in a cohort of patients with chronic HCV infection as well as in healthy blood donors. On average, 10% of peripheral blood CD8 T cells from HCV-infected patients expressed CD16 compared with only a few percent in healthy donors. CD16(+) CD8 T cells displayed a late-stage effector phenotype with high levels of perforin. These cells exhibited a restricted TCR profile suggesting underlying clonal expansion. Stimulation of CD16 on CD8 T cells evoked a vigorous response similar to that of CD16 stimulation in NK cells. Our data suggest that CD8 T cells, during chronic HCV infection in humans, continue to differentiate beyond defined stages of terminal effector cells, acquiring CD16 and NK cell-like functional properties.     

Retention of CD4+ CD25+ FoxP3+ regulatory T cells in the liver after therapy-induced hepatitis C virus eradication in humans

Following infection with the hepatitis C virus (HCV), in most cases immunity fails to eradicate the virus, resulting in slowly progressing immunopathology in the HCV-infected liver. We are the first to examine intrahepatic T cells and CD4(+) CD25(+) FoxP3(+) regulatory T cells (Treg) in patients chronically infected with HCV (chronic HCV patients) during and after antiviral therapy by collecting multiple aspiration biopsy samples from the liver at different time points. We found that intrahepatic Treg frequencies were increased upon alpha interferon and ribavirin administration in about 50% of chronic HCV patients, suggesting stronger regulation of intrahepatic immunity by Treg during antiviral therapy. After cessation of antiviral therapy, the frequency of intrahepatic Treg remained above baseline in the large majority of livers of individuals who successfully cleared the virus. The phenotype of those Treg that were retained in the liver months after therapy-induced clearance of HCV RNA indicated a reduced contribution of effector memory cells. Our findings, gathered by multiple samplings of the liver, indicate that successful antiviral therapy of chronic HCV patients does not lead to normalization of the local immune response to a resting state comparable to that for healthy livers. The continuous presence of high numbers of Treg, with a phenotype reflecting a relatively weak suppressive activity, suggests ongoing residual regulation of immunopathology. These findings provide important insight into the dynamics of the immune response to HCV, as well as the effect of therapy on intrahepatic immunity.     

Enhanced RIG-I expression is mediated by interferon regulatory factor-2 in peripheral blood B cells from hepatitis C virus-infected patients

Chronic hepatitis C patients carry the risk of developing into B-cell non-Hodgkin’s lymphoma (B-NHL). To clarify the mechanisms underlying this association, we first investigated the molecular markers of B cells from hepatitis C virus (HCV)-infected patients. CD19-positive cells were isolated as B cells from the peripheral blood mononuclear cells of patients infected with the hepatitis C virus and IFN-related gene expression was analyzed. We found that RIG-I and IRF-2 expression were up-regulated in CD19-positive cells from the infected patients. In vitro luciferase reporter analysis using human cell lines indicated that IRF-2 activates the human RIG-I promoter. IRF-2 expression levels were enhanced by HCV cDNA transfection in Huh7 cells. In addition, we observed much less induction in the interferon stimulated gene 15 (ISG15) after Sendai virus (SenV) stimulation of CD19-positive cells from infected patients versus healthy controls, thereby suggesting an impairment of RIG-I downstream signaling in HCV-infected patients. Hence, we found that the failure of the anti-viral response with enhanced IRF-2 oncogenic protein expression in blood B cells from HCV-infected patients. Our results provide important information to better understand the role of IRFs in the cause of HCV chronic infection.     

Low doses of the novel caspase-inhibitor GS-9450 leads to lower caspase-3 and -8 expression on peripheral CD4+ and CD8+ T-cells

Chronic hepatitis C virus (HCV) infection is characterized by increased rates of apoptotic hepatocytes and activated caspases have been shown in HCV-infected patients. GS-9450, a novel caspase-inhibitor has demonstrated hepatoprotective activity in fibrosis/apoptosis animal models. This study evaluated the effects of GS-9450 on peripheral T-cell apoptosis in chronic HCV-infected patients. As sub study of the GS-US-227-0102, a double-blind, placebo-controlled phase 2a trial evaluating the safety and tolerability of GS-9450, apoptosis of peripheral CD4+ and CD8+ T-cells was measured using activated caspase-3, activated caspase-8 and CD95 (Fas). Blood samples were drawn at baseline, day 14 after therapy and at 5 weeks off-treatment follow-up in the first cohort of 10 mg. In contrast to the placebo-treated patients, GS-9450 caused a median of 46% decrease in ALT-values from baseline to day 14 in all treated patients (median of 118–64 U/l) rising again to a median of 140 U/l (19%) at 5 weeks off-treatment follow-up. In GS9450-treated patients, during treatment and follow-up, percentages of activated caspase-3+ and caspase-8 expression tended to decrease, in contrast to placebo-treated patients. Interestingly, compared to healthy controls, higher percentages of caspase-3 and caspase-8 positive CD4+ and CD8+ T-cells were demonstrated in HCV-infected patients at baseline. Decreased ALT-values were observed in all HCV-infected patients during treatment with low dose of the caspase-inhibitor GS-9450 accompanied by a lower expression of caspase-3 and -8 on peripheral T-cells. Furthermore, at baseline percentages of activated caspase-3, activated caspase-8 and CD95+ T-cells were higher in chronic HCV-infected patients compared to healthy controls.     

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.     

Bone marrow of persistently hepatitis C virus-infected individuals accumulates memory CD8+ T cells specific for current and historical viral antigens: a study in patients with benign hematological disorders

The role of virus-specific T cells in hepatitis C virus (HCV) pathogenesis is not clear. Existing knowledge on the frequency, phenotype, and behavior of these cells comes from analyses of blood and liver, but other lymphoid compartments that may be important sites for functionally mature T cells have not yet been analyzed. We studied HCV-specific T cells from bone marrow, in comparison to those from peripheral blood and liver biopsy tissue, from 20 persistently HCV-infected patients with benign hematological disorders. Bone marrow contained a sizeable pool of CD8(+) T cells specific for epitopes from structural and nonstructural HCV proteins. These cells displayed the same effector memory phenotype as liver-derived equivalents and the same proliferative potential as blood-derived equivalents but had greater antiviral effector functions such as Ag-specific cytotoxicity and IFN-gamma production. These features were not shared by influenza virus-specific CD8(+) T cells in the same bone marrow samples. Despite their highly differentiated phenotype and activated status, some bone marrow-resident HCV-specific CD8(+) T cells were not directed against the infecting virus but, instead, against historical HCV Ags (i.e., viral species of a previous infection or minor viral species of the current infection). These findings provide a snapshot view of the distribution, differentiation, and functioning of virus-specific memory T cells in patients with persistent HCV infection.     

Malignant B cells induce the conversion of CD4+CD25- T cells to regulatory T cells in B-cell non-Hodgkin lymphoma

Recent evidence has demonstrated that regulatory T cells (Treg) were enriched in the tumor sites of patients with B-cell non-Hodgkin lymphoma (NHL). However, the causes of enrichment and suppressive mechanisms need to be further elucidated. Here we demonstrated that CD4(+)CD25(+)FoxP3(+)CD127(lo) Treg were markedly increased and their phenotypes were different in peripheral blood (PB) as well as bone marrow (BM) from newly diagnosed patients with B-cell NHL compared with those from healthy volunteers (HVs). Involved lymphatic tissues also showed higher frequencies of Treg than benign lymph nodes. Moreover, the frequencies of Treg were significantly higher in involved lymphatic tissues than those from PB as well as BM in the same patients. Suppression mediated by CD4(+)CD25(+) Treg co-cultured with allogeneic CFSE-labeled CD4(+)CD25(-) responder cells was also higher in involved lymphatic tissues from B-cell NHL than that mediated by Treg from HVs. In addition, we found that malignant B cells significantly induced FoxP3 expression and regulatory function in CD4(+)CD25(-) T cells in vitro. In contrast, normal B cells could not induce the conversion of CD4(+)CD25(-) T cells to Treg. We also showed that the PD-1/B7-H1 pathway might play an important role in Treg induction. Taken together, our results suggest that malignant B cells induce the conversion of CD4(+)CD25(-) T cells to Treg, which may play a role in the pathogenesis of B-cell NHL and represent a promising therapeutic target.     

Human monoclonal antibodies that inhibit binding of hepatitis C virus E2 protein to CD81 and recognize conserved conformational epitopes

The intrinsic variability of hepatitis C virus (HCV) envelope proteins E1 and E2 complicates the identification of protective antibodies. In an attempt to identify antibodies to E2 proteins from divergent HCV isolates, we produced HCV E2 recombinant proteins from individuals infected with HCV genotypes 1a, 1b, 2a, and 2b. These proteins were then used to characterize 10 human monoclonal antibodies (HMAbs) produced from peripheral B cells isolated from an individual infected with HCV genotype 1b. Nine of the antibodies recognize conformational epitopes within HCV E2. Six HMAbs identify epitopes shared among HCV genotypes 1a, 1b, 2a, and 2b. Six, including five broadly reactive HMAbs, could inhibit binding of HCV E2 of genotypes 1a, 1b, 2a, and 2b to human CD81 when E2 and the antibody were simultaneously exposed to CD81. Surprisingly, all of the antibodies that inhibited the binding of E2 to CD81 retained the ability to recognize preformed CD81-E2 complexes generated with some of the same recombinant E2 proteins. Two antibodies that did not recognize preformed complexes of HCV 1a E2 and CD81 also inhibited binding of HCV 1a virions to CD81. Thus, HCV-infected individuals can produce antibodies that recognize conserved conformational epitopes and inhibit the binding of HCV to CD81. The inhibition is mediated via antibody binding to epitopes outside of the CD81 binding site in E2, possibly by preventing conformational changes in E2 that are required for CD81 binding.     

CD5 is dissociated from the B-cell receptor in B cells from bovine leukemia virus-infected, persistently lymphocytotic cattle: consequences to B-cell receptor-mediated apoptosis

Bovine leukemia virus (BLV), a retrovirus related to human T-cell leukemia virus types 1 and 2, can induce persistent nonneoplastic expansion of the CD5(+) B-cell population, termed persistent lymphocytosis (PL). As in human CD5(+) B cells, we report here that CD5 was physically associated with the B-cell receptor (BCR) in normal bovine CD5(+) B cells. In contrast, in CD5(+) B cells from BLV-infected PL cattle, CD5 was dissociated from the BCR. In B cells from PL cattle, apoptosis decreased when cells were stimulated with antibody to surface immunoglobulin M (sIgM), while in B cells from uninfected cattle, apoptosis increased after sIgM stimulation. The functional significance of the CD5-BCR association was suggested by experimental dissociation of the CD5-BCR interaction by cross-linking of CD5. This caused CD5(+) B cells from uninfected animals to decrease apoptosis when stimulated with anti-sIgM. In contrast, in CD5(+) B cells from PL animals, in which CD5 was already dissociated from the BCR, there was no statistically significant change in apoptosis when CD5 was cross-linked and the cells were stimulated with anti-sIgM. Disruption of CD5-BCR interactions and subsequent decreased apoptosis and increased survival in antigenically stimulated B cells may be a mechanism of BLV-induced PL.     

Pervasive influence of hepatitis C virus on the phenotype of antiviral CD8+ T cells

Recent studies using MHC class I tetramers have shown that CD8(+) T cell responses against different persistent viruses vary considerably in magnitude and phenotype. At one extreme, hepatitis C virus (HCV)-specific CD8(+) T cell responses in blood are generally weak and have a phenotype that is perforin low and CCR7 high (early memory). At the other, specific responses to CMV are strong, perforin high, and CCR7 low (mature or effector memory). To examine the potential mechanisms behind this diversity, we compared CMV-specific responses in HCV-infected and healthy individuals. We find a striking difference in the phenotype of CMV-specific CD8(+) T cells between these groups. In the HCV-infected cohort, CMV-specific CD8(+) T cells lost markers associated with maturity; they had increased expression of CCR7 and reduced expression of Fas and perforin. They nevertheless responded to Ag in vitro in a manner similar to controls, with strong proliferation and appropriate acquisition of effector memory markers. The reduction in mature CD8 T cells in HCV-infected individuals may arise through either impairment or regulation of T cell stimulation, or through the early loss of mature T cells. Whatever the mechanism, HCV has a pervasive influence on the circulating CD8(+) T cell population, a novel feature that may be a hallmark of this infection.     

The role of B7 costimulation in CD4/CD8 T cell homeostasis

The effect of B7-mediated costimulation on T cell homeostasis was examined in studies of B7-1 (CD80) and B7-2 (CD86) transgenic as well as B7-deficient mice. B7 overexpression in transgenic mice resulted in marked polyclonal peripheral T cell hyperplasia accompanied by skewing toward an increased proportion of CD8 single-positive cells and a decreased proportion of CD4 single-positive cells in thymus and more markedly in peripheral T cells. B7-induced T cell expansion was dependent on both CD28 and TCR expression. Transgenic overexpression of B7-1 or B7-2 resulted in down-regulation of cell surface CD28 on thymocytes and peripheral T cells through a mechanism mediated by intercellular interaction. Mice deficient in B7-1 and B7-2 exhibited changes that were the reciprocal of those observed in B7-overexpressing transgenics: a marked increase in the CD4/CD8 ratio in peripheral T cells and an increase in cell surface CD28 in thymus and peripheral T cells. These reciprocal effects of genetically engineered increase or decrease in B7 expression indicate that B7 costimulation plays a physiological role in the regulation of CD4+ and CD8+ T cell homeostasis.     

Accumulation of B lymphocytes with a naive,resting phenotype in a subset of hepatitis C patients

Chronic infection with hepatitis C virus (HCV) is associated with disturbances of B lymphocyte activation and function: autoantibody production, mixed cryoglobulinemia, and B cell lymphomas. It has been proposed that these abnormalities reflect chronic antigenic stimulation or aberrant signaling through the B cell coreceptor, the latter mediated by binding of the HCV E2 glycoprotein to CD81. To test this hypothesis, we measured expression of activation and differentiation markers on peripheral blood B cells from patients with chronic HCV infection. Thirty-six HCV patients with and without mixed cryoglobulinemia were compared with 18 healthy control volunteers and 17 sustained virologic responders who had cleared HCV infection. Ten of the 36 HCV patient samples showed increased B cell frequencies; B cell frequency was higher in patients with more severe hepatic fibrosis. However, these samples lacked evidence of Ag-driven activation or proliferation. The expanded cells were low in the activation markers CD25, CD69, CD71, CD80, and CD86. Proliferation of circulating B cells was unchanged in HCV patients. These cells did not express the differentiation marker CD27, suggesting that they were not enriched in memory B cells. Furthermore, the expanded B cells expressed both IgD and IgM, suggesting that they were antigenically naive. Together, these results indicate that B cell expansion in the peripheral blood of HCV patients is not associated with Ag-mediated activation and differentiation. Instead, factors other than antigenic stimulation may promote the accumulation of peripheral blood B cells with a naive phenotype in a subset of HCV patients.     

Deficient CD4+CD25high T regulatory cell function in patients with active systemic lupus erythematosus

CD4(+)CD25(+) T regulatory cells (Tregs) play an essential role in maintaining immunologic homeostasis and preventing autoimmunity. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a loss of tolerance to nuclear components. We hypothesized that altered function of CD4(+)CD25(high) Tregs might play a role in the breakdown of immunologic self-tolerance in patients with SLE. In this study, we report a significant decrease in the suppressive function of CD4(+)CD25(high) Tregs from peripheral blood of patients with active SLE as compared with normal donors and patients with inactive SLE. Notably, CD4(+)CD25(high) Tregs isolated from patients with active SLE expressed reduced levels of FoxP3 mRNA and protein and poorly suppressed the proliferation and cytokine secretion of CD4(+) effector T cells in vitro. In contrast, the expression of FoxP3 mRNA and protein and in vitro suppression of the proliferation of CD4(+) effector T cells by Tregs isolated from inactive SLE patients, was comparable to that of normal individuals. In vitro activation of CD4(+)CD25(high) Tregs from patients with active SLE increased FoxP3 mRNA and protein expression and restored their suppressive function. These data are the first to demonstrate a reversible defect in CD4(+)CD25(high) Treg function in patients with active SLE, and suggest that strategies to enhance the function of these cells might benefit patients with this autoimmune disease.