Hepatic Stellate Cells Directly Inhibit B Cells via Programmed Death-Ligand 1.

We demonstrated previously that mouse hepatic stellate cells (HSCs) suppress T cells via programmed death-ligand 1 (PD-L1), but it remains unknown whether they exert any effects on B cells, the other component of the adaptive immune system. In this study, we found that mouse HSCs directly inhibited B cells and that PD-L1 was also integrally involved. We found that HSCs inhibited the upregulation of activation markers on activated B cells, as well as the proliferation of activated B cells and their cytokine/Ig production in vitro, and that pharmaceutically or genetically blocking the interaction of PD-L1 with programmed cell death protein 1 impaired the ability of HSCs to inhibit B cells. To test the newly discovered B cell-inhibitory activity of HSCs in vivo, we developed a protocol of intrasplenic artery injection to directly deliver HSCs into the spleen. We found that local delivery of wild-type HSCs into the spleens of mice that had been immunized with 4-hydroxy-3-nitrophenylacetyl-Ficoll, a T cell-independent Ag, significantly suppressed Ag-specific IgM and IgG production in vivo, whereas splenic artery delivery of PD-L1-deficient HSCs failed to do so. In conclusion, in addition to inhibiting T cells, mouse HSCs concurrently inhibit B cells via PD-L1. This direct B cell-inhibitory activity of HSCs should contribute to the mechanism by which HSCs maintain the liver's immune homeostasis.

Hepatic Stellate Cells Inhibit T Cells through Active TGF-ß1 from a Cell Surface-Bound Latent TGF-ß1/GARP Complex.

Hepatic stellate cells (HSCs) inhibit T cells, a process that could help the liver to maintain its immunoprivileged status. HSCs secrete latent TGF-ß1, but the detailed mechanisms by which latent TGF-ß1 is activated and whether it plays any role in HSC-mediated T cell suppression remain unclear. Glycoprotein A repetitions predominant (GARP) is a surface marker of activated regulatory T cells. GARP binds latent TGF-ß1 for its activation, which is critical for regulatory T cells to suppress effector T cells; however, it is still unclear whether GARP is present on HSCs and whether it has any impact on HSC function. In this study, we found that TGF-ß1(+/-) HSCs, which produce reduced levels of TGF-ß1, showed decreased potency in inhibiting T cells. We also found that pharmaceutical or genetic inhibition of the TGF-ß1 signaling pathway reduced the T cell-inhibiting activity of HSCs. Additionally, using isolated primary HSCs, we demonstrated that GARP was constitutively expressed on HSCs. Blocking GARP function or knocking down GARP expression significantly impaired the potency of HSCs to suppress the proliferation of and IFN-γ production from activated T cells, suggesting that GARP is important for HSCs to inhibit T cells. These results demonstrate the unexpected presence of GARP on HSCs and its significance in regard to the ability of HSCs to activate latent TGF-ß1 and thereby inhibit T cells. Our study reveals a new mechanism for HSC-mediated immune regulation and potentially for other conditions, such as liver fibrosis, that involve HSC-secreted TGF-ß1.

Mechanisms of liver-induced tolerance.

PURPOSE OF REVIEW: To highlight the results of the ongoing research on the mechanisms of liver-induced tolerance focusing on results from the last year. RECENT FINDINGS: The liver is exposed to a massive antigenic burden of dietary and commensal products from the gastrointestinal tract via portal vein, most of which are necessary for survival. To prevent the immune system from destroying these foreign yet beneficial elements, the liver has developed unique mechanisms to suppress immune responses. It is thought that these mechanisms of acquired tolerance may also underlie the spontaneous acceptance of liver allografts observed after transplantation in many species. The fact that isolated hepatocyte transplants are acutely rejected, suggests that nonparenchymal liver cells play a critical role in spontaneous liver allograft acceptance. IFN-γ, a key inflammatory cytokine produced by T effector (Tef) cells, is paradoxically compulsory for spontaneous liver allograft acceptance. Analysis of IFN-γ signaling points to liver mesenchymal nonparenchymal liver cell that eliminate infiltrating Tef cells via expression of B7-H1, IL-10, and tumor growth factor-ß, as well as the enhancement of Tregs and MDSCs. Thus, liver mesenchymal cells are thought to promote tolerance by eliminating alloreactive Tef cells and enhancing suppressor cells (T and B). SUMMARY: The research during last year offered some key insights into the mechanisms of liver-induced tolerance. Through interactions with activated T cells and B cells via IFN-γ/B7-H1 pathways, liver mesenchymal cells have been shown to be critical components of liver-specific tolerance induction.

Rejection triggers liver transplant tolerance: Involvement of mesenchyme-mediated immune control mechanisms in mice.

UNLABELLED: Liver tolerance was initially recognized by the spontaneous acceptance of liver allografts in many species. The underlying mechanisms are not completely understood. However, liver transplant (LT) tolerance absolutely requires interferon (IFN)-γ, a rejection-associated inflammatory cytokine. In this study, we investigated the rejection of liver allografts deficient in the IFN-γ receptor and reveal that the liver graft is equipped with machineries capable of counterattacking the host immune response through a mesenchyme-mediated immune control (MMIC) mechanism. MMIC is triggered by T effector (Tef) cell-derived IFN-γ that drives expression of B7-H1 on graft mesenchymal cells leading to Tef cell apoptosis. We describe the negative feedback loop between graft mesenchymal and Tef cells that ultimately results in LT tolerance. Comparable elevations of T-regulatory cells and myeloid-derived suppressor cells were observed in both rejection and tolerance groups and were not dependent on IFN-γ stimulation, suggesting a critical role of Tef cell elimination in tolerance induction. We identify potent MMIC activity in hepatic stellate cells and liver sinusoidal endothelial cells. MMIC is unlikely exclusive to the liver, given that spontaneous acceptance of kidney allografts has been reported, although less commonly, probably reflecting variance in MMIC activity. CONCLUSION: MMIC may represent an important homeostatic mechanism that supports peripheral tolerance and could be a target for the prevention and treatment of transplant rejection. This study highlights that the graft is an active participant in the equipoise between tolerance and rejection and warrants more attention in the search for tolerance biomarkers.

All-trans retinoic acid induces arginase-1 and inducible nitric oxide synthase-producing dendritic cells with T cell inhibitory function.

Hepatic stellate cells (HSC) are a major source of the immunoregulatory metabolite all-trans retinoic acid (ATRA), which may contribute to the generation of tolerogenic dendritic cells (DCs) in the liver. The present study seeks to clarify the mechanism(s) through which ATRA promotes the development of tolerogenic DCs. Although bone marrow-derived ATRA-treated DCs (RA-DCs) and conventional DCs had comparable surface phenotype, RA-DCs had diminished stimulatory capacity and could directly inhibit the expansion of DC/OVA-stimulated OT-II T cells. Arginase-1 (Arg-1) was found promote suppression because 1) ATRA was a potent inducer of Arg-1 protein and activity, 2) the Arg-1 inhibitor N(w)-hydroxy nor-l-arginine partially reversed suppression, and 3) the suppressive function of RA-DCs was partially compromised using OT-II T cells from GCN2(-/-) mice, which are insensitive to Arg-1. Inducible NO synthase (iNOS), however, was found to be a more significant contributor to RA-DC function because 1) ATRA potentiated the expression of IFN-γ-induced iNOS, 2) suppressive function in RA-DCs was blocked by the iNOS inhibitor N(G)-monomethyl-l-arginine, monoacetate salt, and 3) RA-DCs derived from iNOS(-/-) mice exhibited near complete loss of tolerogenic function, despite sustained Arg-1 activity. The expression of iNOS and the suppressive function of RA-DCs were dependent on both IFN-γ and ATRA. Furthermore, the in vivo behavior of RA-DCs proved to be consistent with their in vitro behavior. Thus, we conclude that ATRA enhances both Arg-1 and iNOS expression in IFN-γ-treated DCs, resulting in a tolerogenic phenotype. These findings elucidate mechanisms through which ATRA may contribute to liver immune tolerance.

Myeloid-derived suppressor cells as a potential therapy for experimental autoimmune myasthenia gravis.

We recently demonstrated that hepatic stellate cells induce the differentiation of myeloid-derived suppressor cells (MDSCs) from myeloid progenitors. In this study, we found that adoptive transfer of these MDSCs effectively reversed disease progression in experimental autoimmune myasthenia gravis (EAMG), a T cell-dependent and B cell-mediated model for myasthenia gravis. In addition to ameliorated disease severity, MDSC-treated EAMG mice showed suppressed acetylcholine receptor (AChR)-specific T cell responses, decreased levels of serum anti-AChR IgGs, and reduced complement activation at the neuromuscular junctions. Incubating MDSCs with B cells activated by anti-IgM or anti-CD40 Abs inhibited the proliferation of these in vitro-activated B cells. Administering MDSCs into mice immunized with a T cell-independent Ag inhibited the Ag-specific Ab production in vivo. MDSCs directly inhibit B cells through multiple mechanisms, including PGE2, inducible NO synthase, and arginase. Interestingly, MDSC treatment in EAMG mice does not appear to significantly inhibit their immune response to a nonrelevant Ag, OVA. These results demonstrated that hepatic stellate cell-induced MDSCs concurrently suppress both T and B cell autoimmunity, leading to effective treatment of established EAMG, and that the MDSCs inhibit AChR-specific immune responses at least partially in an Ag-specific manner. These data suggest that MDSCs could be further developed as a novel approach to treating myasthenia gravis and, even more broadly, other diseases in which T and B cells are involved in pathogenesis.

The role of complement component 3 (C3) in differentiation of myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) play an important role in the regulation of the immune response. MDSC expansion occurs in many circumstances, including cancer, inflammation, stresses, and transplant tolerance. Liver transplants in mice are spontaneously accepted, but hepatocyte transplants are acutely rejected, suggesting the immunoregulatory activities of liver nonparenchymal cells. We have reported that hepatic stellate cells (HpSCs), the stromal cells in the liver, are immensely immunosuppressive and can effectively protect islet transplants via induction of MDSCs. The present study shows that the addition of HpSCs into dendritic cell (DC) culture promoted development of MDSCs, instead of DCs, which was highly dependent on complement component 3 (C3) from HpSCs. The C3(-/-) HpSCs lost their ability to induce MDSCs and, consequently, failed to protect the cotransplanted islet allografts. HpSCs produced complement activation factor B and factor D which then enhanced C3 cleavage to activation products iC3b and C3d. Addition of exogenous iC3b, but not C3d, into the DC culture led to the differentiation of MDSCs with potent immune-inhibitory function. These findings provide novel mechanistic insights into the differentiation of myeloid cells mediated by local tissue cells, and may assist in the development of MDSC-based therapy in clinical settings.

Interferon- γ triggers hepatic stellate cell-mediated immune regulation through MEK/ERK signaling pathway.

Hepatic stellate cells (HSCs) interact with immune cells to actively participate in regulating immune response in the liver which is mediated by the effector molecules, including B7-H1. We demonstrated here that expression of B7-H1 on HSCs was markedly enhanced by interferon-(IFN-) γ stimulation. IFN- γ stimulated HSCs inhibited T-cell proliferation via induction of T-cell apoptosis (22.1% ± 1.6%). This immunosuppressive effect was inhibited by preincubation with an anti-B7-H1 antibody, or inhibitor of the MEK/ERK pathway inhibited IFN- γ mediated expression of B7-H1. Thus, regulation of B7-H1 expression on HSCs by IFN- γ represents an important mechanism that regulates immune responses in the liver favoring tolerogenicity rather than immunogenicity. Involvement of MEK/ERK pathway provides a novel target for therapeutic approaches.

A selective and potent CXCR3 antagonist SCH 546738 attenuates the development of autoimmune diseases and delays graft rejection.

BACKGROUND: The CXCR3 receptor and its three interferon-inducible ligands (CXCL9, CXCL10 and CXCL11) have been implicated as playing a central role in directing a Th1 inflammatory response. Recent studies strongly support that the CXCR3 receptor is a very attractive therapeutic target for treating autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis and psoriasis, and to prevent transplant rejection. We describe here the in vitro and in vivo pharmacological characterizations of a novel and potent small molecule CXCR3 antagonist, SCH 546738. RESULTS: In this study, we evaluated in vitro pharmacological properties of SCH 546738 by radioligand receptor binding and human activated T cell chemotaxis assays. In vivo efficacy of SCH 546738 was determined by mouse collagen-induced arthritis, rat and mouse experimental autoimmune encephalomyelitis, and rat cardiac transplantation models. We show that SCH 546738 binds to human CXCR3 with a high affinity of 0.4 nM. In addition, SCH 546738 displaces radiolabeled CXCL10 and CXCL11 from human CXCR3 with IC50 ranging from 0.8 to 2.2 nM in a non-competitive manner. SCH 546738 potently and specifically inhibits CXCR3-mediated chemotaxis in human activated T cells with IC90 about 10 nM. SCH 546738 attenuates the disease development in mouse collagen-induced arthritis model. SCH 546738 also significantly reduces disease severity in rat and mouse experimental autoimmune encephalomyelitis models. Furthermore, SCH 546738 alone achieves dose-dependent prolongation of rat cardiac allograft survival. Most significantly, SCH 546738 in combination with CsA supports permanent engraftment. CONCLUSIONS: SCH 546738 is a novel, potent and non-competitive small molecule CXCR3 antagonist. It is efficacious in multiple preclinical disease models. These results demonstrate that therapy with CXCR3 antagonists may serve as a new strategy for treatment of autoimmune diseases, including rheumatoid arthritis and multiple sclerosis, and to prevent transplant rejection.

Hepatic stellate cells regulate immune response by way of induction of myeloid suppressor cells in mice.

UNLABELLED: Although organ transplants have been applied for decades, outcomes of somatic cell transplants remain disappointing, presumably due to lack of appropriate supporting stromal cells. Thus, cotransplantation with liver stromal cells, hepatic stellate cells (HSC), achieves long-term survival of islet allografts in mice by way of induction of effector T cell apoptosis and generation of regulatory T (Treg) cells. In this study we provide evidence both in vitro and in vivo that HSC can promote generation of myeloid-derived suppressor cells (MDSC). HSC-induced MDSC demonstrate potent immune inhibitory activity. Induction of MDSC is dependent on an intact interferon gamma signaling pathway in HSC and is mediated by soluble factors, suggesting that the specific tissue stromal cells, such as HSC, play a crucial role in regulating immune response by way of inflammation-induced generation of MDSC. Large amounts of MDSC can be propagated in vitro from bone marrow-derived myeloid precursor cells under the influence of HSC. CONCLUSION: Cotransplantation with in vitro generated MDSC can effectively protect islet allografts from host immune attack. Local delivery of potent immune suppressor cells for cell transplants holds great clinical application potential.

Gα13 is closely related to hematopoiesis in zebrafish.

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) function as signal transducers and control many different physiologic processes. G proteins can be grouped into four families: Gs, Gi, Gq and G12. Gα13 belongs to the G12 family. In zebrafish, there are two isoforms of Gα13: Gα13a and Gα13b. We show here that knockdown of Gα13b in zebrafish results in hematopoietic and angiogenic defects. The Gα13b morphants don't show complete loss of expression of gata1, pu.1 or flk until 35 hpf suggests that Gα13b is closely related to the development of hematopoietic cells. Further studies reveal that blood cells and vascular endothelial cells have undergone apoptosis through a p53-dependent pathway in Gα13b-depleted embryos. Injection of p53 morpholino could partially rescue the phenotype of Gα13b morphants. These data possibly demonstrate a new role for Gα13 in cell survival.

Mechanistic insights into immunomodulation by hepatic stellate cells in mice: a critical role of interferon-gamma signaling.

UNLABELLED: The liver is considered to be an immune-privileged organ that favors the induction of tolerance. The underlying mechanisms are not completely understood. Interestingly, liver transplants are spontaneously accepted in several animal models, but hepatocyte transplants are acutely rejected, suggesting that liver nonparenchymal cells may effectively protect the parenchymal cells from immune attack. We have shown the profound T cell inhibitory activity of hepatic stellate cells (HSCs). Thus, cotransplantation with HSCs effectively protects islet allografts from rejection in mice. In this study, using T cell receptor transgenic and gene knockout approaches, we provided definitive evidence that HSCs protected cotransplanted islet allografts by exerting comprehensive inhibitory effects on T cells, including apoptotic death in graft-infiltrating antigen-specific effector T cells and marked expansion of CD4(+) Forkhead box protein (Foxp)3(+) T regulatory (Treg) cells. All these effects required an intact interferon-gamma (IFN-gamma) signaling in HSCs, demonstrated by using HSCs isolated from IFN-gamma receptor 1 knockout mice. B7-H1 expression on HSCs, a product molecule of IFN-gamma signaling, was responsible for induction of T cells apoptosis, but had no effect on expansion of Treg cells, suggesting that undetermined effector molecules produced by IFN-gamma signaling is involved in this process. CONCLUSION: Upon inflammatory stimulation, specific organ stromal cells (such as HSCs in the liver) demonstrate potent immune regulatory activity. Understanding of the mechanisms involved may lead to development of novel strategies for clinical applications in transplantation and autoimmune diseases.

Endoplasmic reticulum stress-mediated apoptosis involved in indirect recognition pathway blockade induces long-term heart allograft survival.

Implementation of dendritic cell- (DC-) based therapies in organ transplantation can reduce dependency on nonspecific immunosuppression. Despite extensive research, mechanisms of equipped DCs inducing transplant tolerance remain incomplete. Here, we applied RNA interference technique to inhibit CD80 and CD86 expression in host bone marrow-derived DCs. This approach could specifically and effectively knock down CD80 and CD86 expression. T cells primed by these DCs inhibited allogeneic responses. Administration of recipient DCs loaded with alloantigen after CD80 and CD86 blockade prolonged cardiac allograft survival. We also found a higher percentage of apoptotic T cells in lymph tissues and grafts than that detected in control group. In addition, these T cells expressed high expression of GRP78 than controls, indicating activation of unfolded protein responses. Upregulation of CHOP expression among these cells suggested that the endoplasmic reticulum stress (ERS) response switched to a proapoptotic response. Our results indicated that ERS-induced apoptosis may be involved in allogeneic T-cell apoptosis, and the ERS-mediated apoptosis pathway may be a novel target in clinical prevention and therapy of allograft rejection.

A critical role of TRAIL expressed on cotransplanted hepatic stellate cells in prevention of islet allograft rejection.

Hepatic stellate cells (HSCs) have demonstrated a strong T-cell inhibitory activity. In a mouse islet transplantation model, cotransplanted HSCs can protect islet allografts from rejection. The involved mechanism is not fully understood. We showed in this study that expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), an important apoptosis-inducing ligand, on HSCs was crucial in protection of islet allografts, since HSCs derived from TRAIL knockout mice demonstrated less inhibitory activity towards T-cell proliferative responses, and substantially lost their capacity in protecting cotransplanted islet allografts from rejection, suggesting that TRAIL-mediated T cell apoptotic death is important in HSC-delivered immune regulation activity.

Distinct response of liver myeloid dendritic cells to endotoxin is mediated by IL-27.

BACKGROUND/AIMS: The liver lies downstream of the gut, and is constantly exposed to bacteria. Liver dendritic cells (DC) are known to possess properties of tolerance, and respond to LPS differently when compared to conventional DC, but the underlying mechanisms are not completely understood. The aim of this study was to evaluate liver DC response to LPS stimulation. METHODS: Liver or spleen derived DC were isoloated from mice treated with plasmid-GM-CSF hydrodynamic injection. The surface molecules and TLR4 expression on DC and cytokine productions of LPS stimulated DC were determinded by FACS analysis, ELISA and qPCR. The ability of DC to elicit T cell responses and differentiation were examined by MLR/CTL assay and qPCR for molecular markers related to Th1/Th2/Treg. RESULTS: In this study, we demonstrated that the threshold of LPS stimulation for liver DC was markedly higher than spleen DC, even though the expression of TLR4 on both DCs was comparable. In contrast to spleen DC that produced high levels of IL-12 and induced Th1 response upon LPS stimulation, LPS-liver DC preferentially produced IL-10 and IL-27, instead of IL-12. In addition, liver DC induced T cell hyporesponsiveness, associated with selective expansion of CD4(+)Foxp3(+)T regulatory cells. Addition of exogenous IL-12 only slightly enhanced liver DC-induced T cell response. Interestingly, abrogation of IL-27 ligation by using IL-27R(-/-) T cells synergistically augmented the effect of IL-12, suggesting that IL-27 produced by liver DC plays a crucial role in induction of T cell hyporesponsiveness. CONCLUSIONS: Liver DC respond distinctly to LPS stimulation by secreting IL-27 which synergizes with silencing of bioactive IL-12 activity leading to profound T cell inhibition.

Induced pluripotent stem cells-derived myeloid-derived suppressor cells regulate the CD8+ T cell response.

Myeloid-derived suppressor cells (MDSCs) are markedly increased in cancer patients and tumor-bearing mice and promote tumor growth and survival by inhibiting host innate and adaptive immunity. In this study, we generated and characterized MDSCs from murine-induced pluripotent stem cells (iPSCs). The iPSCs were co-cultured with OP9 cells, stimulated with GM-CSF, and became morphologically heterologous under co-culturing with hepatic stellate cells. Allogeneic and OVA-specific antigen stimulation demonstrated that iPS-MDSCs have a T-cell regulatory function. Furthermore, a popliteal lymph node assay and autoimmune hepatitis model showed that iPS-MDSCs also regulate immune responsiveness in vivo and have a therapeutic effect against hepatitis. Taken together, our results demonstrated a method of generating functional MDSCs from iPSCs and highlighted the potential of iPS-MDSCs as a key cell therapy resource for transplantation and autoimmune diseases.

[Inducing liver-derived dendritic cell proliferation by plasmid-IL-3 and CD40L genes in mice in vivo].

OBJECTIVES: Try to induce liver-derived dendritic cells proliferation by plasmid-IL-3 and CD40L genes in mice in vivo. METHODS: Rapid tail-vein injection of large amounts of plasmid-carrying genes was performed to transfect genes in mice livers. Liver nonparenchymal cells were isolated by Percoll gradient centrifugation. Dendritic cell markers were detected and dendritic cells were sorted out by flow cytometry. Morphology of dendritic cells was studied microscopically (with Giemsa staining) and under scanning electron microscopy. RESULTS: Liver nonparenchymal cells dramatically increased in the liver lobules, portal and periportal areas in the treated group, but not in the control group. B220+/DEC205+ dendritic cells were detected and sorted by flow cytometry. There were more B220+/DEC205+ dendritic cells (16.0%) in the experiment group than those in the control group (1.1%). Morphologically, the sorted B220+/DEC205+ cells showed irregular shaped nuclei, paucity of cytoplasmic granules and extensive cytoplasmic processes. CONCLUSION: B220+/DEC205+ dendritic cells were expanded in vivo in mice livers by rapid tail-vein injection of plasmid-carrying genes encoding IL-3 and CD40L in a large amount. Inducing liver dendritic cell proliferation in vivo provides a more convenient way for studying the biology of these cells.

C-C Chemokine Receptor Type 2-Dependent Migration of Myeloid-Derived Suppressor Cells in Protection of Islet Transplants.

BACKGROUND: Islet transplantation is a promising therapeutic approach to restore the physical response to blood glucose in type 1 diabetes. Current chronic use of immunosuppressive reagents for preventing islet allograft rejection is associated with severe complications. In addition, many of the immunosuppressive drugs are diabetogenic. The induction of transplant tolerance to eliminate the dependency on immunosuppression is ideal, but remains challenging. METHODS: Addition of hepatic stellate cells allowed generation of myeloid-derived suppressor cells (MDSC) from precursors in mouse bone marrow. Migration of MDSC was examined in an islet allograft transplant model by tracking the systemic administered MDSC from CD45.1 congenic mice. RESULTS: The generated MDSC were expressed C-C chemokine receptor type 2 (CCR2), which was enhanced by exposure to interferon-γ. A single systemic administration of MDSC markedly prolonged survival of islet allografts without requirement of immunosuppression. Tracking the administered MDSC showed that they promptly migrated to the islet graft sites, at which point they exerted potent immune suppressive activity by inhibiting CD8 T cells, enhancing regulatory T cell activity. MDSC generated from CCR2 mice failed to be mobilized and lost tolerogenic activity in vivo, but sustained suppressive activity in vitro. CONCLUSIONS: MDSC migration was dependent on expression of CCR2, whereas CCR2 does not directly participate in immune suppression. Expression of CCR2 needs to be closely monitored for quality control purpose when MDSC are generated in vitro for immune therapy.

Prevention of diabetes in NOD mice by administration of dendritic cells deficient in nuclear transcription factor-kappaB activity.

Abnormalities of dendritic cells (DCs) have been identified in type 1 diabetic patients and in nonobese diabetic (NOD) mice that are associated with augmented nuclear transcription factor (NF)-kappaB activity. An imbalance that favors development of the immunogenic DCs may predispose to the disease, and restoration of the balance by administration of DCs deficient in NF-kappaB activity may prevent diabetes. DCs propagated from NOD mouse bone marrow and treated with NF-kappaB-specific oligodeoxyribonucleotide (ODN) in vitro (NF-kappaB ODN DC) were assessed for efficacy in prevention of diabetes development in vivo. Gel shift assay with DC nuclear extracts confirmed specific inhibition of NF-kappaB DNA binding by NF-kappaB ODN. The costimulatory molecule expression, interleukin (IL)-12 production, and immunostimulatory capacity in presenting allo- and islet-associated antigens by NF-kappaB ODN DC were significantly suppressed. NF-kappaB ODN renders DCs resistant to lipopolysaccharide stimulation. Administration of 2 x 10(6) NF-kappaB ODN DCs into NOD mice aged 6-7 weeks effectively prevented the onset of diabetes. T-cells from pancreatic lymph nodes of NF-kappaB ODN DC-treated animals exhibited hyporesponsiveness to islet antigens with low production of interferon-gamma and IL-2. These findings provide novel insights into the mechanisms of autoimmune diabetes and may lead to development of novel preventive strategies.

Induction of tumor immunity and cytotoxic t lymphocyte responses using dendritic cells transduced by adenoviral vectors encoding HBsAg: comparison to protein immunization.

Dendritic cells (DC) are specialized antigen-presenting cells with powerful immunostimulatory properties. Their use for induction of anti-tumor immunity has been limited by several factors, including identification of appropriate tumor-associated antigens, delivery of antigens to DC, and maintaining DC in a highly activated state. Here, DC propagated in vitro were transduced with an adenoviral (Ad) vector to express hepatitis B surface antigen (HBsAg), an antigen present in hepatocellular carcinoma (HCC). Many patients with HCC demonstrate evidence of prior HBV exposure, suggesting that the presence of the virus in a quiescent state may promote tumorigenesis. Ad-HBsAg-transduced DC stimulated strong cytotoxic T lymphocyte (CTL) responses to HBsAg-expressing tumor cells, and protected mice from lethal tumor challenge. Immunity was antigen-specific, as wild-type tumor (HBsAg -) grew normally. Furthermore, DC transduced with an irrelevant vector had no effect. Vaccination with HBsAg protein, a clinically utilized preparation that confers immunity to HBV infection, did not protect against tumor challenge even though it induced a strong antibody response. These studies describe for the first time the contributions of humoral and cellular immune responses to tumor immunity induced by Ad-transduced DC compared to protein vaccination.