Toshiyuki Mera

High-mobility group box 1 is involved in the initial events of early loss of transplanted islets in mice

Islet transplantation for the treatment of type 1 diabetes mellitus is limited in its clinical application mainly due to early loss of the transplanted islets, resulting in low transplantation efficiency. NKT cell-dependent IFN-gamma production by Gr-1(+)CD11b(+) cells is essential for this loss, but the upstream events in the process remain undetermined. Here, we have demonstrated that high-mobility group box 1 (HMGB1) plays a crucial role in the initial events of early loss of transplanted islets in a mouse model of diabetes. Pancreatic islets contained abundant HMGB1, which was released into the circulation soon after islet transplantation into the liver. Treatment with an HMGB1-specific antibody prevented the early islet graft loss and inhibited IFN-gamma production by NKT cells and Gr-1(+)CD11b(+) cells. Moreover, mice lacking either of the known HMGB1 receptors TLR2 or receptor for advanced glycation end products (RAGE), but not the known HMGB1 receptor TLR4, failed to exhibit early islet graft loss. Mechanistically, HMGB1 stimulated hepatic mononuclear cells (MNCs) in vivo and in vitro; in particular, it upregulated CD40 expression and enhanced IL-12 production by DCs, leading to NKT cell activation and subsequent NKT cell-dependent augmented IFN-gamma production by Gr-1(+)CD11b(+) cells. Thus, treatment with either IL-12- or CD40L-specific antibody prevented the early islet graft loss. These findings indicate that the HMGB1-mediated pathway eliciting early islet loss is a potential target for intervention to improve the efficiency of islet transplantation.

Homogeneous Expansion of Human T-Regulatory Cells Via Tumor Necrosis Factor Receptor 2

T-regulatory cells (Tregs) are a rare lymphocyte subtype that shows promise for treating infectious disease, allergy, graft-versus-host disease, autoimmunity, and asthma. Clinical applications of Tregs have not been fully realized because standard methods of expansion ex vivo produce heterogeneous progeny consisting of mixed populations of CD4 + T cells. Heterogeneous progeny are risky for human clinical trials and face significant regulatory hurdles. With the goal of producing homogeneous Tregs, we developed a novel expansion protocol targeting tumor necrosis factor receptors (TNFR) on Tregs. In in vitro studies, a TNFR2 agonist was found superior to standard methods in proliferating human Tregs into a phenotypically homogeneous population consisting of 14 cell surface markers. The TNFR2 agonist-expanded Tregs also were functionally superior in suppressing a key Treg target cell, cytotoxic T-lymphocytes. Targeting the TNFR2 receptor during ex vivo expansion is a new means for producing homogeneous and potent human Tregs for clinical opportunities.

Targeting TNFR2 with antagonistic antibodies inhibits proliferation of ovarian cancer cells and tumor-associated Tregs

An antibody against TNFR2 may treat ovarian cancer by killing tumor cells while limiting the activity of suppressive immune cells. Killing ovarian cancer by trapping TNFR2 Signaling by tumor necrosis factor (TNF) family promotes tumor growth and progression. The TNF receptor 2 (TNFR2) is present on the surface of immunosuppressive regulatory T cells and some tumor cells. Torrey et al. (see coverage by Chen and Oppenheim) developed antibodies that bind and lock TNFR2 in an inactive conformation. The antibodies killed ovarian cancer cells in culture. Additionally, the antibodies inhibited the proliferation of regulatory T cells while promoting the proliferation of effector T cells isolated from metastatic sites (ascites) in ovarian cancer patients. The antibodies had less of an effect on T cells isolated from the peripheral blood of normal donors. Thus, these antibodies may be more specific and less toxic than current TNFR antibodies, providing a new path for treating ovarian cancer. Major barriers to cancer therapy include the lack of selective inhibitors of regulatory T cells (Tregs) and the lack of broadly applicable ways to directly target tumors through frequently expressed surface oncogenes. Tumor necrosis factor receptor 2 (TNFR2) is an attractive target protein because of its restricted abundance to highly immunosuppressive Tregs and oncogenic presence on human tumors. We characterized the effect of TNFR2 inhibition using antagonistic antibodies. In culture-based assays, we found that two TNFR2 antagonists inhibited Treg proliferation, reduced soluble TNFR2 secretion from normal cells, and enabled T effector cell expansion. The antagonistic activity occurred in the presence of added TNF, a natural TNFR2 agonist. These TNFR2 antibodies killed Tregs isolated from ovarian cancer ascites more potently than it killed Tregs from healthy donor samples, suggesting that these antibodies may have specificity for the tumor microenvironment. The TNFR2 antagonists also killed OVCAR3 ovarian cancer cells, which have abundant surface TNFR2. The antibodies stabilized antiparallel dimers in cell surface TNFR2 that rendered the receptor unable to activate the nuclear factor κB pathway and trigger cell proliferation. Our data suggest that, by targeting tumor cells and immunosuppressive tumor-associated Tregs, antagonistic TNFR2 antibodies may be an effective treatment for cancers positive for TNFR2.

The Hippo signaling pathway is required for salivary gland development and its dysregulation is associated with Sjogren's-like disease

Sjogren’s syndrome (SS) is a complex autoimmune disease that primarily affects salivary and lacrimal glands and is associated with high morbidity. Although the prevailing dogma is that immune system pathology drives SS, increasing evidence points to structural defects, including defective E-cadherin adhesion, to be involved in its etiology. We have shown that E-cadherin has pivotal roles in the development of the mouse salivary submandibular gland (SMG) by organizing apical-basal polarity in acinar and ductal progenitors and by signaling survival for differentiating duct cells. Recently, E-cadherin junctions have been shown to interact with effectors of the Hippo signaling pathway, a core pathway regulating the organ size, cell proliferation, and differentiation. We now show that Hippo signaling is required for SMG-branching morphogenesis and is involved in the pathophysiology of SS. During SMG development, a Hippo pathway effector, TAZ, becomes increasingly phosphorylated and associated with E-cadherin and α-catenin, consistent with the activation of Hippo signaling. Inhibition of Lats2, an upstream kinase that promotes TAZ phosphorylation, results in dysmorphogenesis of the SMG and impaired duct formation. SMGs from non-obese diabetic mice, a mouse model for SS, phenocopy the Lats2-inhibited SMGs and exhibit a reduction in E-cadherin junctional components, including TAZ. Importantly, labial specimens from human SS patients display mislocalization of TAZ from junctional regions to the nucleus, coincident with accumulation of extracellular matrix components, fibronectin and connective tissue growth factor, known downstream targets of TAZ. Our studies show that Hippo signaling has a crucial role in SMG-branching morphogenesis and provide evidence that defects in this pathway are associated with SS in humans.

Prevention of early loss of transplanted islets in the liver of mice by adenosine.

Background. The low efficiency of islet transplantation necessitating sequential transplantations with the use of 2 to 3 donors for a recipient has been a major obstacle facing clinical islet transplantation. We determined whether adenosine has any beneficial effects on preventing early loss of transplanted islets in the liver, thereby facilitating successful islet transplantation from one donor to one recipient in mice. Methods. Two hundred islets, the number of islets from a single mouse pancreas, were grafted into the liver of streptozotocin-induced diabetic C57BL/6 mice. Adenosine was administered once at the time of islet transplantation. Mononuclear cells in the liver of mice receiving islets were isolated and examined by flow cytometry. Results. A single injection of adenosine at the time of transplantation ameliorated hyperglycemia of diabetic mice receiving 200 syngenic islets with suppression of interferon (IFN)-&ggr; production of hepatic NKT cells and neutrophils, while that of control did not. The IFN-&ggr; production of NKT cells and neutrophils in the liver of mice treated with &agr;-galactosylceramide, a synthetic ligand of NKT cells was suppressed by adenosine. The beneficial effect of adenosine was also observed for BALB/c islet allografts when alloimmune rejection was prevented by anti-CD4 antibody. Conclusions. Adenosine suppresses the NKT cell-mediated IFN-&ggr; production of neutrophils in the liver of mice receiving islets, thus leading to prevention of early loss of transplanted syngenic and allogenic islets. The findings indicate that adenosine may improve efficiency of clinical islet transplantation.

HMGB1-Mediated Early Loss of Transplanted Islets Is Prevented by Anti-IL-6R Antibody in Mice.

Objectives The limited success in achieving insulin independence of patients with type 1 diabetes mellitus after islet transplantation from a single donor, mainly due to early loss of transplanted islets, hampers clinical application of islet transplantation. Previously, we have shown in mice that the early loss of transplanted islets in the liver, the site of islet transplantation, is caused by innate immune rejection triggered by high-mobility group box 1 (HMGB1) protein released from transplanted islets. We herein determined whether the HMGB1-mediated early loss of transplanted mouse islets is prevented by anti–interleukin-6 receptor (IL-6R) antibody. Methods The effect of anti–IL-6R antibody on amelioration of hyperglycemia in streptozocin-induced diabetic mice receiving 200 islets into the liver from a single donor was evaluated in association with HMGB1-stimulated interferon-&ggr; production of hepatic mononuclear cells. Results Hyperglycemia of diabetic mice receiving 200 syngeneic islets was ameliorated with down-regulation of interferon-&ggr; production of hepatic natural killer T cells and neutrophils when anti–IL-6R was administered at the time of transplantation. This beneficial effect was also seen in allografts when alloimmune rejection was prevented by anti-CD4 antibody. Conclusions These findings demonstrate that anti–IL-6R antibody prevented the early loss of intrahepatic islet grafts with inhibiting HMGB1-induced immune activation after islet transplantation.

Pretreatment of donor islets with the Na(+) /Ca(2+) exchanger inhibitor improves the efficiency of islet transplantation.

Pancreatic islet transplantation is an attractive therapy for the treatment of insulin‐dependent diabetes mellitus. However, the low efficiency of this procedure necessitating sequential transplantations of islets with the use of 2–3 donors for a single recipient, mainly due to the early loss of transplanted islets, hampers its clinical application. Previously, we have shown in mice that a large amount of HMGB1 is released from islets soon after their transplantation and that this triggers innate immune rejection with activation of DC, NKT cells and neutrophils to produce IFN‐γ, ultimately leading to the early loss of transplanted islets. Thus, HMGB1 release plays an initial pivotal role in this process; however, its mechanism remains unclear. Here we demonstrate that release of HMGB1 from transplanted islets is due to hypoxic damage resulting from Ca2+ influx into β cells through the Na+/Ca2+ exchanger (NCX). Moreover, the hypoxia‐induced β cell damage was prevented by pretreatment with an NCX‐specific inhibitor prior to transplantation, resulting in protection and long‐term survival of transplanted mouse and human islets when grafted into mice. These findings suggest a novel strategy with potentially great impact to improve the efficiency of islet transplantation in clinical settings by targeting donor islets rather than recipients.

The Spleen Contributes Stem Cells to Peripheral Blood Stem Cell Transplants

Treatment of malignancies with Peripheral Blood Stem Cell Transplants (PBSCTs) from donors given Granulocyte- Colony-Stimulating-Factor (G-CSF) has improved survival relative to bone marrow transplants. G-CSF mobilizes CD34+ hematopoietic stem cells from bone marrow into the blood. Enrichment of PBSCT by purification of CD34+ stem cells fails to produce superior clinical benefits. We hypothesize that the reason why CD34+-enriched PBSCTs are not more effective is because the enrichment and purification process leaves out G-CSF-mobilized stem cells from another source, the spleen, which holds a unique reservoir of Hox11+ stem cells. Quantitative mRNA analysis was used to determine whether G-CSF mobilizes Hox11+ stem cells and whether expression occurs in a cell population distinct from CD34+ cells. Samples of peripheral blood lymphocytes (PBLs) were obtained from ten normal untreated donors and 18 normal donors treated with G-CSF. G-CSF was found to mobilize both CD34+ stem cells (p=0.02) and even more dramatically mobilize Hox11+ splenic stem cells (p=0.000013) into the peripheral blood. The findings support the hypothesis that G-CSF mobilizes two distinct stem cell populations, one from the bone marrow and the other from the spleen. The inferior clinical performance of CD34+-enriched and purified PBSCTs compared to unenriched PBSCTs may be explained by the omission of Hox11+ stem cells. These findings suggest that PBSCTs without enrichment and purification of CD34+ may improve treatment of cancer and potentially other diseases in tissues derived from Hox11+ stem cells.

Prevention of high-mobility group box 1-mediated early loss of transplanted mouse islets in the liver by antithrombin III.

Background The low efficiency of pancreatic islet transplantation mainly because of the early loss of transplanted islets hampers its clinical application. Previously, we have shown in mice that the early loss of transplanted islets in the liver is caused by innate immune rejection in concert with dendritic cells, natural killer T cells, and neutrophils to produce interferon (IFN)-&ggr;, which is triggered by high-mobility group box 1 (HMGB1) released from transplanted islets. We herein determined whether the HMGB1-mediated early loss of transplanted mouse islets is prevented by antithrombin (ATIII). Methods The effect of ATIII on in vitro and in vivo HMGB1-stimulated IFN-&ggr; production of hepatic mononuclear cells was examined. Then, the effect of ATIII on amelioration of hyperglycemia in streptozotocin-induced diabetic mice receiving 200 syngeneic islets from a single donor was determined. Results In vitro and in vivo IFN-&ggr; production of mononuclear cells in the liver of mice in response to HMGB1 was suppressed by ATIII. Hyperglycemia of streptozotocin-induced diabetic mice receiving 200 syngeneic islets into the liver from a single donor was ameliorated with down-regulation of IFN-&ggr; production of natural killer T cells and neutrophils in the liver when ATIII but not vehicle was administered once at the time of islet transplantation. The favorable effect of ATIII was similarly achieved in mice receiving islet allografts when rejection was prevented with anti-CD4 antibody treatment. Conclusions These findings demonstrate that ATIII prevents HMGB1-mediated early loss of transplanted islets caused by innate immune rejection, suggesting a potential application of ATIII to improve efficiency of clinical islet transplantation.

Removal of donor human leukocyte antigen class I proteins with papain: translation for possible whole organ practices.

Background Papain is a protease with potential use in transplantation because of its targeted capacity to selectively remove human leukocyte antigen (HLA) class I proteins from donor human cells. However, its proteolytic activity has not been studied under conditions suitable for use in perfusing donor organs, namely, under a temperature of 4°C and dissolution in Belzer-UW solution. Methods We test papain’s HLA class I removing activity under recognized whole organ transplant conditions of lowered temperature. The activity of papain’s substrate selectivity was tested using both a test substrate (casein) and fresh peripheral blood lymphocytes (PBLs). The activity of papain was also tested at 4°C, the temperature of whole organ storage. Results We found that papain at a range of concentrations is nearly as active in cleaving the test substrate in Belzer-UW solution as in distilled water. In distilled water, papain is as active in cleaving a test substrate at a temperature of 4°C as compared to its optimal temperature of 37°C, if the incubation time is extended from 10 min to 3 hr. This finding also holds true if papain is dissolved in Belzer-UW solution. In peripheral blood lymphocytes, papain cleaved off HLA class I proteins as effectively at 4°C as at 37°C, provided the incubation time was also extended to 3 hr. Conclusion These findings suggest that papain’s targeted enzymatic cleavage of donor HLA class I has potential use in the whole organ transplant setting with retained activity at lower temperatures and when activated and dissolved in Belzer-UW solution.