IntroductionHuman bone marrow stromal cells (MSCs), more recently referred to as mesenchymal stem cells, are capable of differentiating along multiple mesenchymal lineages in addition to supporting hematopoiesis. 1,2 Due to their potential for differentiation into osteocytes, chondrocytes, myocytes, and adipocytes, MSCs have emerged as a promising tool for clinical applications such as tissue engineering and cell and gene therapy. 3,4 MSCs are of inherently low immunogenicity and, more importantly, are capable of inhibiting allogeneic T-cell responses. 5-8 These intriguing observations have prompted clinical studies to investigate cotransplantation of MSCs in allogeneic hematopoietic stem cell transplantation (HSCT) in order to promote hematopoietic engraftment by preventing host-versusgraft reactivity and to suppress graft-versus-host reactions. 9,10 As of yet, the molecular mechanisms responsible for the immunosuppressive effects of MSCs have not been unequivocally identified. The reports describing a potential role of transforming growth factor-␤1 and hepatocyte growth factor as mediators of T-cell inhibition remain controversial, but most studies agree that soluble factors are involved. [6][7][8]11 In professional antigen-presenting cells (APCs), expression of indoleamine 2,3-dioxygenase (IDO) induced by interferon-␥ (IFN-␥) and other proinflammatory cytokines catalyzes conversion from tryptophan to kynurenine and has recently been identified as a major immunosuppressive effector pathway that inhibits T-cell responses to autoantigens and fetal alloantigens in vivo. [12][13][14][15][16] Based on these findings, we investigated whether MSCs exhibit IFN-␥-inducible IDO activity and whether this mechanism contributes to T-cell inhibition mediated by MSCs. Study design Culture of human bone marrow-derived MSCsBone marrow aspirates were harvested from volunteer donors who had provided informed consent; the study was approved by the institutional review board of the University Clinic, Düsseldorf, Germany. Primary human MSCs were generated as previously described 17 except that culture medium was supplemented with 3 ng/mL basic fibroblast growth factor (R&D Systems, Minneapolis, MN). Mixed lymphocyte reactions (MLRs)Standard 5-day MLR cultures were set up with 5 ϫ 10 4 mitomycin C-treated human peripheral blood mononuclear cells (PBMCs) as stimulators and 2 ϫ 10 5 human T cells purified using sheep red blood cell rosetting as responder cells. 5,11 In MSC/MLR coculture experiments, MLRs were performed on a layer of either 5 ϫ 10 3 or 2 ϫ 10 4 MSCs seeded one day before. IFN-␥ concentration was determined in MSC/MLR coculture supernatants using a commercially available enzyme-linked immunosorbent assay (ELISA; R&D Systems) according to manufacturer's instructions. Detection of IDO expression and activityMSCs were stimulated with IFN-␥ (R&D Systems) and assayed for IDO expression and function. Standard Western blot analysis for IDO protein expression was performed. 18 IDO enzyme activity following IFN-␥ stimulation of ...

It has been postulated that antibodies specific to the hypervariable region 1 (HVR1) within the putative envelop protein E2 of hepatitis C virus (HCV) can neutralize virus. We studied such antibodies in sera of patients who were infected in a single-source outbreak by a contaminated anti-D immunoglobulin preparation (HCV-AD78). The nucleotide sequences of cDNAs encoding HVR1 of HCV-AD78 were determined. The four major variants (HVR1.A, B, C, and D) were expressed as fusion proteins in Escherichia coli. Sixty-seven percent of sera contained antibodies to HVR1.A. Sera unrelated to infection of the outbreak also recognized HVR1.A but to a lesser extent (15%), suggesting that not all HVR1-specific antibodies are absolutely isolate-specific. Antibodies directed against individual variants of HVR1 were found in sera obtained early postinfection (p.i.) (< or = 1 year) but also in sera obtained several years later. An in vitro binding assay of HCV to tissue culture cells was employed to further characterize these sera. Five of seven sera that were obtained early p.i. prevented binding of HCV to cells. Preincubation of such sera with HVR1-specific fusion proteins restored binding of HCV to cells in four of five sera. These findings suggest that the majority of neutralizing antibodies are directed against HVR1.

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Hepatitis C virus (HCV) is a major cause of parenterally Antibodies directed to hypervariable region 1 (HVR1) transmitted acute and chronic hepatitis. 1 Upon infection, paof hepatitis C virus (HCV) have recently been shown to tients develop a chronic persistent infection in more than neutralize the corresponding HCV isolate in vitro. We 50% of these cases. 2 Antibody responses 3 and T-cell-medianalyzed the appearance of antibodies directed to HVR1 ated immune responses to various regions of the HCV polyduring the course of infection in a large group of paprotein 4,5 have been studied, but so far a prognostic marker tients who have been infected by the same isolate of a associated with acute self-limited or persistent infections has HCV contaminated anti-D immunoglobulin (HCV-AD78).not been found. An enzyme-linked immunosorbent assay (ELISA) was esVaccination studies of chimpanzees have shown the importablished using a synthetic peptide to detect antibodies tance of the immune response against the putative HCV enagainst the main HVR1 variant of HCV-AD78. 207 sera velope proteins E1/E2. 6 However, protection of animals deobtained at different time points post infection (p.i.) of pends on the HCV isolate used for challenge of immunized 51 patients having either acute self-limiting (n Å 28) or animals. In addition, reinfection occurred in chimpanzees 7 chronic infection (n Å 23) were studied. Antibodies diand was suggested for patients. [8][9][10] Isolate-specific, neutralizrected to HVR1 were found at least at one time point ing antibodies directed against E1 and E2 may be involved during the infection course in 15 of 28 patients (53%) in determination of the course of infection. Antibodies present having acute self-limiting infections and in 17 of 23 pain chronically infected patients after several years postinfectients (74%) with chronic disease. The time of appeartion (p.i.) fail to neutralize the viral variant present in the ance of anti-HVR1 was significantly different between infectious source 11 most probably because of an immune esthese two patient groups (P õ .025) although appearance cape of newly arising viral variants of hypervariable region and titers of other HCV-specific antibodies were found 1 (HVR1). 12 Sequences of HVR1 found in earlier time points to be similar at early time points p.i. In acute self-limof infection are rapidly mutated during chronic infection and iting infections 9 of 21 sera (43%) of respective patients followed by respective, HVR1-specific antibody response. 13,14with sera available within the first 6 months p.i. were A monoclonal antibody directed against HVR1 was capable anti-HVR1 positive. The highest prevalence of antiof HCV in vitro neutralization. 15 Antibodies that neutralize HVR1 in this group of patients was within month 6 to HCV have also been observed in sera of HCV-infected pa-12 p.i. (64%). None of the sera available after 24 months tients. 11,16 Such neutralizing antibodies in patient sera are p.i. had such antibodies. In contrast, only 2 of 15 sera mainly direc...