Tolerogenic Model Research Articles

Immunoregulatory role of acid sphingomyelinase in allergic asthma.

Acid sphingomyelinase (ASM) is one of the enzymes that catalyzes the breakdown of sphingomyelin to ceramide and phosphorylcholine. In this study, we aimed at elucidating the role of ASM in allergic asthma. We used an ovalbumin-induced murine model of asthma where we compared wild-type and ASM-deficient mice. In wild-type mice, secretory ASM activity in the bronchoalveolar lavage fluid was increased in the acute ovalbumin model, but not in a tolerogenic model. Furthermore, in the absence of ASM, the serum IgE level was reduced, compared with wild-type mice, while an accumulation of interstitial macrophages and foreign antigen-induced regulatory T cells along with exhausted CD4+ PD1+ T cells was observed in the lungs of ASM-/- mice. In conclusion, in the absence of ASM, we observed an accumulation of immunosuppressive antigen-induced regulatory T cells expressing Foxp3 and CTLA4 in the lung as well as multinucleated interstitial macrophages and exhausted CD4+ PD1+ T cells associated with inhibition of serum IgE in asthma.

Virus-Like Vesicle-Based Therapeutic Vaccine Vectors for Chronic Hepatitis B Virus Infection.

More than 500,000 people die each year from the liver diseases that result from chronic hepatitis B virus (HBV) infection. Therapeutic vaccines, which aim to elicit an immune response capable of controlling the virus, offer a potential new treatment strategy for chronic hepatitis B. Recently, an evolved, high-titer vaccine platform consisting of Semliki Forest virus RNA replicons that express the vesicular stomatitis virus glycoprotein (VSV G) has been described. This platform generates virus-like vesicles (VLVs) that contain VSV G but no other viral structural proteins. We report here that the evolved VLV vector engineered to additionally express the HBV middle surface envelope glycoprotein (MHBs) induces functional CD8 T cell responses in mice. These responses were greater in magnitude and broader in specificity than those obtained with other immunization strategies, including recombinant protein and DNA. Additionally, a single immunization with VLV-MHBs protected mice from HBV hydrodynamic challenge, and this protection correlated with the elicitation of a CD8 T cell recall response. In contrast to MHBs, a VLV expressing HBV core protein (HBcAg) neither induced a CD8 T cell response in mice nor protected against challenge. Finally, combining DNA and VLV-MHBs immunization led to induction of HBV-specific CD8 T cell responses in a transgenic mouse model of chronic HBV infection. The ability of VLV-MHBs to induce a multispecific T cell response capable of controlling HBV replication, and to generate immune responses in a tolerogenic model of chronic infection, indicates that VLV vaccine platforms may offer a unique strategy for HBV therapeutic vaccination. HBV infection is associated with significant morbidity and mortality. Furthermore, treatments for chronic infection are suboptimal and rarely result in complete elimination of the virus. Therapeutic vaccines represent a unique approach to HBV treatment and have the potential to induce long-term control of infection. Recently, a virus-based vector system that combines the nonstructural proteins of Semliki Forest virus with the VSV glycoprotein has been described. In this study, we used this system to construct a novel HBV vaccine and demonstrated that the vaccine is capable of inducing virus-specific immune responses in mouse models of acute and chronic HBV replication. These findings highlight the potential of this new vaccine system and support the idea that highly immunogenic vaccines, such as viral vectors, may be useful in the treatment of chronic hepatitis B.

Antigen –specific immunotherapy inhibited Th2 via GATA-3 and Th17 cells in the absence of T-bet in an allergic asthma model (140.6)

Abstract Asthma is caused by T-helper 2 (Th2)-driven immune-responses in which upregulation of GATA-3 has been shown to play a crucial role in both in humans and in murine models of this disease. T-cell tolerance, induced by respiratory exposure to allergen prior to allergen sensitization, can inhibit the development of airway hyperractivity (AHR), a cardinal feature of asthma. However, the subset of T cells that mediates this protective function in allergic asthma has not been clearly identified yet. We recently set up a tolerogenic model of allergic asthma. In this model, peripheral T cell tolerance is induced by respiratory exposure to allergen and is characterized by decreased Th2 (IL-4,IL-5 and IL-13)-based responses via downregulation of the main Th2 transcription factor GATA-3 in the lung which results in decreased allergen induced AHR and lung eosinophilia. In addition, we found that our tolerogenic model is characterized also by decreased differentiation of Th17 cells. Moreover respiratory exposure to allergen prior to sensitization protected also airway hyperreactivity induced in the absence of T-bet. Thus this model suppressed effector Th2 and Th17 mediated immune responses also in the absence of IFN-gamma and could therefore provide a new avenue also for genetically determined allergic diseases. This work is supported by a SFB 548-B8 project in Mainz, Germany

A Novel Peptide Mimotope Identified As a Potential Immunosuppressive Vaccine for Organ Transplantation

We reported that anti-histone H1 autoantibody is one of the main immunosuppressive factors in serum that is induced after orthotopic liver transplantation in a rat tolerogenic model. We generated a novel anti-histone H1 IgM mAb produced by hybridoma 16G9 (16G9 mAb) that shows MLR-inhibitory activity. Identification of a functional epitope responsible for the immunosuppressive activity of 16G9 mAb may lead to the establishment of a novel therapeutic strategy. We used a combinatorial phage display peptide library to screen for peptides that bind to 16G9 mAb. Consequently, two peptides that bind to 16G9 mAb, SSV and LPQ, were selected from the library. The binding of 16G9 mAb to histone H1 was inhibited by SSV. SSV was recognized by rat tolerogenic post-orthotopic liver transplantation serum and the binding to SSV was inhibited by histone H1. Mice were immunized with keyhole limpet hemocyanin-conjugated SSV and LPQ. Abs induced by SSV immunization inhibited Con A-stimulated splenocyte proliferation, and the inhibition was neutralized by preincubation with SSV. Splenocytes stimulated by anti-CD3 Ab were inhibited by SSV-induced Abs using CFSE labeling. SSV immunization in rats before heterotopic heart transplantation resulted in significant prolonged allograft survival. These findings suggested that SSV is a functional histone H1-binding epitope for 16G9 mAb. SSV is capable of determining serum immunoreactivity against histone H1 as an index marker for tolerance. The inhibitory activity of SSV-induced Abs on blast cell proliferation and the prolonged graft survival that results from SSV immunization imply a potential for the development of an immunosuppressive vaccine.

Involvement of autoimmunity against nuclear histone H1 in liver transplantation tolerance

Background Our recent studies suggested that anti-histone H1 autoantibody (auto-Ab) plays an important role in experimental and clinical liver allograft tolerance as a natural immunosuppressive factor. The present study aimed to explore how the autoimmune response against histone H1 is involved in tolerance induction. Methods The measurement of anti-histone H1 auto-Ab and immunohistochemical analysis were performed in serum and liver allografts after orthotopic liver transplantation (OLT). To compare the auto-Ab response against histone H1 between the recipients of rejector (DA-LEW) and tolerogenic (DA-PVG) OLT models, naïve recipients were immunized with calf thymus histone H1. The immunosuppressive state of histone H1-immunized rats was assayed by mixed lymphocyte reaction (MLR). Results Anti-histone H1 Ab titer was transiently increased during the rejection phase after OLT (days 7–21) in the DA-PVG combination, while no such response was confirmed in the DA-LEW acute rejection model. Nuclear histone H1 antigens were found in the cytoplasm and the extracellular environment in liver allografts at the rejection phase in the tolerogenic model but not in the rejector model, resulting from the transient induction of anti-histone H1 auto-Ab in recipient PVG rats after OLT. Low dose and short-term immunization with histone H1 upregulated the anti-histone H1 Ab titer in naïve PVG rats, which exhibited a low allogeneic immune response, while no such response was found in naïve LEW rats. Conclusions These results suggest that the sensitivity to nuclear antigens such as histone H1 may be a key factor determining the acceptance or rejection of donor liver grafts, at least in DA-PVG and DA-LEW combinations.

Characterization of immunosuppressive factors expressed in serum by rat tolerogenic liver transplantation

Background In a rat tolerogenic model of orthotopic liver transplantation (OLT), recipient serum after OLT (post-OLT serum) possesses strong immunosuppressive activity. This study aimed to identify immunosuppressive factors present in early post-OLT serum. Methods Immunosuppressive activity was evaluated in vitro by inhibition of the mixed-lymphocyte reaction (MLR). Autoantigens recognized by MLR-inhibitory IgG were identified by the internal protein sequencing. Results Recipient post-OLT serum inhibited MLR, and OLT-inducible IgG was the major immunosuppressive factor. IgG from post-OLT sera (2 to 3 weeks) specifically reacted to 31; 34; and 73-kd autoantigens on spleen cells. The internal sequences of the 31- and 34-kd antigens coincided completely with those of histone H1 molecules. Immunodepletion of anti-histone H1 antibodies (Abs) from early post-OLT serum abolished the MLR-inhibitory activity. Furthermore, rabbit polyclonal Ab-directed histone H1 not only significantly suppressed rat and human MLR but also prolonged survival of heart allografts. Flow-cytometric analysis revealed that some live PVG splenocytes were stained with antihistone H1 Abs, and that these positive cells increased on Con A stimulation. Western blot analysis indicated that several cross-reactive antigens against anti-histone H1 Abs were found in their membrane fraction. Conclusions In this study we provide evidence that autoreactive Abs, against histone H1 are a major OLT-induced graft survival factor, and may play at least a part in overcoming the acute rejection phase to establish solid allograft tolerance.

Expression of clusterin in a rat tolerogenic OLT model

IN RAT orthotopic liver transplantation (OLT), PVG recipients of DA livers (tolerogenic model) survive without immunosuppression and become tolerant of subsequent grafts of other DA organs such as skin, heart, and kidney. Many aspects of the mechanism regarding this naturally achieved tolerance have been discussed in our previous reports. However, the mechanism of liver allograft tolerance from the aspect of the complement system and its regulatory proteins has not yet been investigated. The complement system comprises a group of proteins that interact with other immune system molecules to provide many of the effector functions of humoral immunity and inflammation. Clusterin, which is a plasma glycoprotein, regulates the complement system by inhibiting the membrane attack complex (MAC) formation. Here, we studied the kinetics of clusterin expression after transplantation in the tolerogenic model (DA-PVG) compared with syngenic model (DA-DA) and acute rejector model (DA-LEW) by immunoblotting and Northern blotting.

Clusterin may be involved in rat liver allograft tolerance

Little is known about the possible role of complement inhibitors on tolerance induced by liver allografts. Clusterin, which is a plasma glycoprotein, inhibits cytolytic membrane attack complex (MAC) of complement by binding to soluble C5b-7 complex. The role of clusterin in relation to the naturally achieved tolerance in a rat orthotopic liver transplantation (OLT) has not been investigated before. Here we determined the kinetics of clusterin expression at different post-transplantation time points in a tolerogenic model (DA-PVG) where rejection was naturally overcome without any immunosuppressive drugs in comparison with the syngenic OLT model (DA-DA). Peripheral blood and liver tissues were taken from OLT at various post-operative time points. A strong expression of soluble clusterin was observed on post-transplantation day 7, which occurred at the peak of the rejection in this tolerogenic OLT model. The expression of clusterin remained strong even after tolerance was achieved. The intensity of clusterin expression was much stronger when compared with the syngenic OLT (DA-DA) model after OLT. A strong expression of clusterin mRNA was also observed in the tolerogenic model on post-OLT day (POD) 7 and the expression persisted when compared with the syngenic model on post-OLT day 60. Our data have shown that the strongest levels of clusterin during the reaction phase in tolerogenic OLT may be involved in tolerance induction.

Allograft acceptance and rejection, mediated by a liver suppressor factor, LSF-1, purified from serum of liver transplanted rats

In certain rat strain combinations liver allografts are spontaneously accepted without immunosuppression and induce donor-specific tolerance to further skin and heart grafts in the recipient. Such an effect is also transferrable using serum from orthotopically liver transplanted rats (OLT serum). In the OLT serum of one such combination. DA (RT1 a) donor into PVG (RT1 c) recipient, a 40 kDa protein (liver suppressor factor, LSF-1) has been identified and shown to be immunosupressive in vitro. The aim of the present study is to investigate the immunological effect of LSF-1 and a polyclonal antibody (anti-LSF-1) against this molecule, in a rat heterotopic heart transplant (HHT) model and OLT model, respectively. Intramuscular injection of 300 μg of LSF-1, 1 h postoperatively, into a PVG recipient of either a DA or BN (RT1 n) cardiac allograft caused significant prolongation of graft survival. Intravenous injection of polyclonal rabbit sera raised against an N-terminal peptide of LSF-1 (anti-LSF-1), within 1 h postoperatively, had variable effects on the survival of DA liver grafts in PVG recipients. In 5 6 cases injection of between 1 and 2 ml of anti-LSF-1 resulted in death of the recipient. Histological examination of the liver showed severe rejection with lymphoid cell infiltration of the portal tract and sinusoids and extensive damage to the parenchyma. All control rats survived for more than 60 days without any signs of rejection. The anti-LSF-1 polyclonal antibody prevented the induction of tolerance in the normally tolerogenic model (DA into PVG). This, together with the in vivo results, suggests a role for LSF-1 in the induction of tolerance.