Lianfu Wang

Inhibition of T‐cell responses by hepatic stellate cells via B7‐H1–mediated T‐cell apoptosis in mice

In the injured liver, hepatic stellate cells (HSCs) secrete many different cytokines, recruit lymphocytes, and thus participate actively in the pathogenesis of liver disease. Little is known of the role of HSCs in immune responses. In this study, HSCs isolated from C57BL/10 (H2b) mice were found to express scant key surface molecules in the quiescent stage. Activated HSCs express major histocompatibility complex class I, costimulatory molecules, and produce a variety of cytokines. Stimulation by interferon γ (IFN‐γ) or activated T cells enhanced expression of these molecules. Interestingly, addition of the activated (but not quiescent) HSCs suppressed thymidine uptake by T cells that were stimulated by alloantigens or by anti‐CD3–mediated T‐cell receptor ligation in a dose‐dependent manner. High cytokine production by the T cells suggests that the inhibition was probably not a result of suppression of their activation. T‐cell division was also found to be normal in a CFSE dilution assay. The HSC‐induced T‐cell hyporesponsiveness was associated with enhanced T‐cell apoptosis. Activation of HSCs was associated with markedly enhanced expression of B7‐H1. Blockade of B7‐H1/PD‐1 ligation significantly reduced HSC immunomodulatory activity, suggesting an important role of B7‐H1. In conclusion, the bidirectional interactions between HSCs and immune cells may contribute to hepatic immune tolerance. (HEPATOLOGY 2004;40:1312–1321.)

Outcome of liver transplantation in hepatitis C virus–infected patients who received hepatitis C virus–infected grafts

BACKGROUND & AIMS The present organ shortage has brought into question the suitability of hepatitis C virus (HCV)-positive grafts. This study reviewed the outcome of such transplantations in our institution. METHODS Twenty-three HCV-positive patients who underwent orthotopic liver transplantation (OLT) for end-stage liver disease with HCV-positive grafts in 1992-1995 were studied. Only patients who survived more than 30 days were included in the analysis. Control group included 169 patients who underwent transplantation for HCV-related cirrhosis and received HCV-negative organs. RESULTS Patients who received HCV-infected organs had a cumulative survival rate of 89% and 72% at 1 and 5 years, respectively, vs. 88% and 73% for the control group (NS). There was no difference in graft survival, incidence of cirrhosis, mean hepatitis activity index score, fibrosis, or mean activity of serum transaminases. There was a trend toward lower incidence of recurrent hepatitis C in the study group compared with control (21% vs. 23% at 1 year and 47% vs. 64% at 5 years; NS). Patients in whom the donor strain became predominant after transplantation had significantly longer disease-free survival than patients who retained their own HCV strain (P < 0.003). CONCLUSIONS HCV-infected livers transplanted into HCV-infected recipients do not appear to convey a worse outcome in the initial years after OLT than HCV-negative grafts.

Marked Prolongation of Cardiac Allograft Survival by Dendritic Cells Genetically Engineered with NF-κB Oligodeoxyribonucleotide Decoys and Adenoviral Vectors Encoding CTLA4-Ig

Bone marrow-derived dendritic cells (DCs) can be genetically engineered using adenoviral (Ad) vectors to express immunosuppressive molecules that promote T cell unresponsiveness. The success of these DCs for therapy of allograft rejection has been limited in part by the potential of the adenovirus to promote DC maturation and the inherent ability of the DC to undergo maturation following in vivo administration. DC maturation occurs via NF-κB-dependent mechanisms, which can be blocked by double-stranded “decoy” oligodeoxyribonucleotides (ODNs) containing binding sites for NF-κB. Herein, we describe the combined use of NF-κB ODNs and rAd vectors encoding CTLA4-Ig (Ad CTLA4-Ig) to generate stably immature murine myeloid DCs that secrete the potent costimulation blocking agent. These Ad CTLA4-Ig-transduced ODN DCs exhibit markedly impaired allostimulatory ability and promote apoptosis of activated T cells. Furthermore, administration of Ad CTLA4-Ig ODN-treated donor DCs (C57BL10; B10(H-2b)) before transplant significantly prolongs MHC-mismatched (C3HHeJ; C3H(H-2k)) vascularized heart allograft survival, with long-term (>100 days) donor-specific graft survival in 40% of recipients. The mechanism(s) responsible for DC tolerogenicity, which may involve activation-induced apoptosis of alloreactive T cells, do not lead to skewing of intragraft Th cytokine responses. Use of NF-κB antisense decoys in conjunction with rAd encoding a potent costimulation blocking agent offers promise for therapy of allograft rejection or autoimmune disease with minimization of systemic immunosuppression.

Liver-derived DEC205+B220+CD19- dendritic cells regulate T cell responses

Leukocytes resident in the liver may play a role in immune responses. We describe a cell population propagated from mouse liver nonparenchymal cells in IL-3 and anti-CD40 mAb that exhibits a distinct surface immunophenotype and function in directing differentiation of naive allogeneic T cells. After culture, such cells are DEC-205brightB220+CD11c−CD19−, and negative for T (CD3, CD4, CD8α), NK (NK 1.1) cell markers, and myeloid Ags (CD11b, CD13, CD14). These liver-derived DEC205+B220+ CD19− cells have a morphology and migratory capacity similar to dendritic cells. Interestingly, they possess Ig gene rearrangements, but lack Ig molecule expression on the cell surface. They induce low thymidine uptake of allogeneic T cells in MLR due to extensive apoptosis of activated T cells. T cell proliferation is restored by addition of the common caspase inhibitor peptide, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk). T cells stimulated by liver-derived DEC205+B220+D19− cells release both IL-10 and IFN-γ, small amounts of TGF-β, and no IL-2 or IL-4, a cytokine profile resembling T regulatory type 1 cells. Expression of IL-10 and IFN-γ, but not bioactive IL-12 in liver DEC205+B220+CD19− cells was demonstrated by RNase protection assay. In vivo administration of liver DEC205+B220+CD19− cells significantly prolonged the survival of vascularized cardiac allografts in an alloantigen-specific manner.

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

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. (HEPATOLOGY 2011;)

Myeloid-derived suppressor cells protect islet transplants by B7-H1 mediated enhancement of T regulatory cells.

Background. Side effects of lifetime immunosuppression for cell transplants often outweigh the benefits; therefore, induction of transplant tolerance is needed. We have shown that cotransplantation with myeloid-derived suppressor cells (MDSC) effectively protect islet allografts from rejection without requirement of immunosuppression. This study was to investigate the underlying mechanisms. Methods. MDSC were generated by addition of hepatic stellate cells from various stain mice into dendritic cell (DC) culture. The quality of MDSC was monitored by phenotype and function analyses. MDSC mixed with islet allografts were transplanted into diabetic recipients. T-cell response was analyzed after transplantation by using flow and histochemical analyses, and was compared with islet alone and islet/DC transplant groups. B7-H1 knockout mice were used to determine the role of B7-H1 on MDSC in regulation of T-cell response. Results. Cotransplantation with MDSC (not DC) effectively protected islet allografts without requirement of immunosuppression. This is associated with attenuation of CD8 T cells in the grafts and marked expansion of regulatory T (Treg) cells, which contributed to MDSC-induced T-cell hyporesponsiveness. Antigen-specific Treg cells were prone to accumulate in lymphoid organs close to the grafts. Both in vitro and in vivo data demonstrated that B7-H1 was absolutely required for MDSC to exert immune regulatory activity and induction of Treg cells. Conclusion. The described approach holds great clinical application potential and may overcome the limitation of requiring chronic administration of immunosuppression in cell transplants. Understanding the underlying mechanisms will facilitate the development of this novel therapeutic strategy.

Hepatic Stellate Cells Preferentially Expand Allogeneic CD4 + CD25 + FoxP3 + Regulatory T Cells in an IL-2 Dependent Manner

Background. Organ transplantation has been successfully practiced for decades, but the outcome of cell transplantation remains disappointing. This is the case in animal models; liver allografts in mice are spontaneously accepted without requirement of immunosuppression, whereas hepatocyte transplants in the same combination are acutely rejected, apparently resulting from immune attacks because syngeneic hepatocyte transplants survive indefinitely. This suggests that liver nonparenchymal cells play an important role in protecting parenchymal cell from rejection. We have shown that hepatic stellate cells (HpSC), well known to participate in liver repairing and fibrosis, mediate potent immunomodulatory functions through induction of activated T-cell death. Methods and Results. Here, we report that HpSC acquired antigen presenting capacity after activated by interferon-&ggr;. In contrast to professional antigen-presenting cells dendritic cells that predominantly stimulated CD4+ T cells to generate CD25+ forkhead box P3 (Foxp3)− effector cells, HpSC selectively expanded CD4+CD25+Foxp3+ cells in an interleukin-2-dependent manner. These expanded CD4+CD25+Foxp3+ cells showed T regulatory cell (Treg) activity in effectively inhibiting T-cell proliferation in responses to anti-CD3 monoclonal antibody or alloantigens in a major histocompatibility complex nonspecific fashion. The Treg cells were expanded from the CD4+CD25+ population with the help of interleukin-2, independent of B7-H1 and transforming growth factor-&bgr;. Administration of HpSC into allogeneic recipients resulted in expansion of CD4+CD25+FoxP3+ cells in vivo. Conclusion. Liver stromal HpSC acted as nonprofessional antigen-presenting cells, and preferentially expanded CD25+FoxP3+ Treg cells, which may contribute to immune regulation in the liver.

IL-12 Antagonism Enhances Apoptotic Death of T Cells Within Hepatic Allografts from Flt3 Ligand-Treated Donors and Promotes Graft Acceptance

Mouse livers are accepted across MHC barriers and induce donor-specific tolerance without immunosuppressive therapy. By contrast, livers from donors treated with Flt3 ligand, which dramatically increases hepatic interstitial dendritic cells, are rejected acutely (median survival time 5 days). This switch from tolerance to rejection is associated with a marked reduction in apoptotic activity of graft-infiltrating cells. We hypothesized that IL-12 production by enhanced numbers of donor APC might inhibit apoptosis, promote expansion of Th1 cells, and play a key role in liver rejection. Therefore, C3H (H2k) recipients of liver grafts from Flt3 ligand-treated B10 donors were given neutralizing anti-IL-12 mAb (200 or 500 μg) on days 0 and 2 after transplant. Graft survival was markedly prolonged at the higher mAb dose, with 50% of grafts surviving >100 days. This effect was associated with reductions in IFN-γ gene transcripts within the graft-infiltrating cell population and with reductions in circulating IFN-γ and IL-10 levels, donor-specific CTL and NK cell activities, and circulating alloantibody levels. At the same time, there were marked increases in apoptotic (TUNEL+) CD4+ and especially CD8+ cells, both within the grafts and in spleens of anti-IL-12 mAb-treated mice. In vitro, exogenous IL-12 inhibited apoptotic death induced in naive allogeneic T cells by liver nonparenchymal cells. These findings suggest that suppression of rejection by IL-12 antagonism, linked to restoration of apoptotic activity within the peripheral alloreactive T cell population, is important for liver allograft survival and tolerance induction.

Mechanistic Insights into Immunomodulation by Hepatic Stellate Cells in Mice: A Critical Role of Interferon-γ Signaling

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‐γ (IFN‐γ) signaling in HSCs, demonstrated by using HSCs isolated from IFN‐γ receptor 1 knockout mice. B7‐H1 expression on HSCs, a product molecule of IFN‐γ 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‐γ 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. (HEPATOLOGY 2009.)

Prolongation of cardiac allograft survival by systemic administration of immature recipient dendritic cells deficient in NF-κB activity

Objective:To develop a more applicable approach that uses recipient-derived dendritic cells (DC) for organ transplantation. Summary Background Data:Systemic administration of immature donor DC that are deficient in costimulatory molecules delays the onset of allograft rejection. However, this approach requires in vitro DC propagation and would not be applicable to deceased donor organ transplantation. Methods:DC were propagated from C3H (H2k) mouse bone marrow with GM-CSF; their maturation was arrested by treatment with oligodeoxyribonucleotides (ODN) specifically against nuclear factor (NF)-κB. The DC were pulsed with B10 (H2b) splenocyte lysate. DC phenotype was examined by flow cytometry. Their allostimulatory activity was assessed in vitro by MLR and CTL assays and in vivo by the influence on B10 cardiac allograft survival. Cytokine profiles were analyzed by ELISA and RNase protection assay. NF-κB activity in DC nuclear protein was detected by gel shifting assay. Results:Compared with mature DC, NF-κB ODN-treated immature DC pulsed with B10 (H2b) spleen cell lysate elicited markedly lower proliferative responses and correlated with reduced IFN-γ and increased IL-10 production. In contrast to administration of mature C3H DC pulsed with B10 antigen that accelerated rejection of B10 cardiac allografts, a single injection of NF-κB ODN DC pulsed with donor antigens significantly prolonged allograft survival in an antigen-specific manner. This was associated with induction of T-cell hyporesponsiveness and enhanced T-cell apoptosis. Conclusions:An approach to use recipient DC as a “vaccine” strategy provides a more feasible approach for deceased-donor organ transplantation.