Intracellular Galectin-9 Enhances Proximal TCR Signaling and Potentiates Autoimmune Diseases.

Galectin-9 is a risk gene in inflammatory bowel disease. By transcriptomic analyses of ileal biopsies and PBMCs from inflammatory bowel disease patients, we identified a positive correlation between galectin-9 expression and colitis severity. We observed that galectin-9-deficient T cells were less able to induce T cell-mediated colitis. However, several mouse-based studies reported that galectin-9 treatment induces T cell apoptosis and ameliorates autoimmune diseases in an exogenously modulated manner, indicating a complicated regulation of galectin-9 in T cells. We found that galectin-9 is expressed mainly inside T cells, and its secreted form is barely detected under physiological conditions. Endogenous galectin-9 was recruited to immune synapses upon T cell activation. Moreover, proximal TCR signaling was impaired in galectin-9-deficient T cells, and proliferation of these cells was decreased through an intracellularly modulated manner. Th17 cell differentiation was downregulated in galectin-9-deficient T cells, and this impairment can be rescued by strong TCR signaling. Taken together, these findings suggest that intracellular galectin-9 is a positive regulator of T cell activation and modulates the pathogenesis of autoimmune diseases.

Non-clinical assessment of cell therapy products: the perspective from five Asian countries/regions based on regulatory guidelines and the underpinning rationales.

BACKGROUND AIMS: Cell-based regenerative medicine is an innovative field that can potentially alter the overall survival and quality of life of patients with devastating diseases. Several cell therapy products (CTPs) have been approved within the last two decades, and more are under development. The establishment of an effective developmental strategy in accordance with the regulatory bodies of each country/region is crucial for fast delivery of each respective CTP. In particular, facilitating investigational new drug (IND) approval is important for accelerating the transition from non-clinical to clinical research/trial phases. METHODS: Here the authors compared the non-clinical prerequisites for initiating clinical studies in five Asian countries/regions (India, China, Korea, Taiwan and Japan) from an industry viewpoint. The authors first identified the differences and tried to clarify the perspectives/considerations underpinning the different requirements. RESULTS: The authors' findings revealed that differences in regulations and development experiences, especially with CTPs, have led to clear differences in the non-clinical study package and its corresponding study design. CONCLUSIONS: By sharing experiences of the research and development of CTPs among Asian countries/regions and including not only industry but also regulatory authorities, we will be able to expedite cross-border IND approval and eventually contribute to the early delivery of innovative CTPs to many Asian patients.

Role of Galectins in Tumors and in Clinical Immunotherapy.

Galectins are glycan-binding proteins that contain one or two carbohydrate domains and mediate multiple biological functions. By analyzing clinical tumor samples, the abnormal expression of galectins is known to be linked to the development, progression and metastasis of cancers. Galectins also have diverse functions on different immune cells that either promote inflammation or dampen T cell-mediated immune responses, depending on cognate receptors on target cells. Thus, tumor-derived galectins can have bifunctional effects on tumor and immune cells. This review focuses on the biological effects of galectin-1, galectin-3 and galectin-9 in various cancers and discusses anticancer therapies that target these molecules.

Differential modulation of IL-12 family cytokines in autoimmune islet graft failure in mice.

AIMS/HYPOTHESIS: The relative contribution of T helper (Th)1 and Th17 cells in graft rejection is inconclusive, on the basis of evidence provided by different T cell-related cytokine-deficient animal models and graft types. METHODS: We used novel antigen-presenting-cell-specific Il-12p35 (also known as Il12a)-knockout (KO), IL-23p19-knockdown (KD) and IL-27p28-KD strategies to investigate T cell differentiation in islet graft rejection. RESULTS: In vitro dendritic cell-T cell coculture experiments revealed that dendritic cells from Il-12p35-KO and IL-23p19-KD mice showed reduced ability to stimulate IFN-γ and IL-17 production in T cells, respectively. To further explore the T cell responses in islet graft rejection, we transplanted islets into streptozotocin-induced diabetic NOD/severe combined immunodeficiency (SCID) recipient mice with IL-12-, IL-23-, or IL-27-deficient backgrounds and then challenged them with NOD.BDC2.5 T cells. The survival of islet grafts was significantly prolonged in Il-12p35-KO and IL-23p19-KD recipients compared with the control recipients. T cell infiltrations and Th1 cell populations were also decreased in the grafts, correlating with prolonged graft survival. CONCLUSIONS/INTERPRETATION: Our results suggest that IL-12 and IL-23 promote and/or maintain Th1 cell-mediated islet graft rejection. Thus, blockade of IL-12 and IL-23 might act as therapeutic strategies for reducing rejection responses.

Different modulation of Ptpn22 in effector and regulatory T cells leads to attenuation of autoimmune diabetes in transgenic nonobese diabetic mice.

Ptpn22 encodes PEST domain-enriched tyrosine phosphatase (Pep), which negatively regulates TCR proximal signaling and is strongly associated with a variety of autoimmune diseases in humans. The net effect of Pep on the balance of immunity and tolerance is uncertain because of the simultaneous inhibition of TCR-mediated signaling of effector and regulatory T cells (T(regs)). In this study, we generated transgenic NOD mice that overexpressed Pep in T cells. The transgenic mice had a significantly lower incidence of spontaneous autoimmune diabetes, which was accompanied by fewer IFN-γ-producing T cells, and an increased ratio of CD4(+)Foxp3(+) T(regs)to CD4(+)IFN-γ(+) or to CD8(+)IFN-γ(+) T cells, respectively, in pancreatic islets. Transgenic T cells showed markedly decreased TCR-mediated effector cell responses such as proliferation and Th1 differentiation. By contrast, the inhibitory effect of transgenic Pep on TCR signaling did not affect the differentiation of T(regs) or their suppressive activity. Adoptive transfer experiments showed that transgenic splenocytes exhibited attenuated diabetogenic ability. To examine further the pathogenic features of transgenic T cells, we generated Ptpn22/BDC2.5 doubly transgenic mice and found reduced proliferation and Th1 differentiation in CD4(+) T lymphocytes with additional Pep in pancreatic lymph nodes but not in inguinal lymph nodes of NOD/SCID recipients. This finding indicates that transgenic Pep attenuates T cell functions in an islet Ag-driven manner. Taken together, our results demonstrate that Pep overexpression in T cells attenuates autoimmune diabetes in NOD mice by preferentially modulating TCR signaling-mediated functions in diabetogenic T cells but not in T(regs).

Targeting pre-ligand assembly domain of TNFR1 ameliorates autoimmune diseases - an unrevealed role in downregulation of Th17 cells.

The pre-ligand assembly domain (PLAD) of tumor necrosis factor receptors mediates specific ligand-independent receptor assembly and subsequent signaling. However, the physiological role of PLAD in the regulation of TNFR-mediated immune responses in autoimmunity is still unclear. By using the recombinant PLAD.Fc protein to block TNFR1 assembly, we demonstrated that PLAD.Fc treatment significantly reduced the TNFR1-driving proinflammatory cytokines and protected NOD mice from diabetes. Strikingly, Th17 differentiation was significantly inhibited in PLAD.Fc-treated NOD and TNFR1-deficient mice, indicating a TNFR1-dependent Th17 development. PLAD.Fc-modulated effects on DCs, in terms of the downregulation of Th17-inducing cytokines, IL-6 and TGF-ß, explained the potential mechanism for Th17 suppression. Finally, we provided an additional result that PLAD.Fc administration diminished the infiltration of Th17 cells in the central nervous system and ameliorated the experimental autoimmune encephalomyelitis in mice. Collectively, these data demonstrated that targeting PLAD of TNFR1 provides protection from autoimmune diseases through the downregulation of Th17 and suggested a therapeutic potential of PLAD-modulation in TNF-involved inflammatory diseases.

Transgenic expression of single-chain anti-CTLA-4 Fv on beta cells protects nonobese diabetic mice from autoimmune diabetes.

T cell-mediated immunodestruction of pancreatic beta cells is the key process responsible for both the development of autoimmune diabetes and the induction of rejection during islet transplantation. In this study, we investigate the hypothesis that transgenic expression of an agonistic, membrane-bound single-chain anti-CTLA-4 Fv (anti-CTLA-4 scFv) on pancreatic beta cells can inhibit autoimmune processes by selectively targeting CTLA-4 on pathogenic T cells. Strikingly, transgenic expression of anti-CTLA-4 scFv on pancreatic beta cells significantly protected NOD mice from spontaneous autoimmune diabetes. Interestingly, local expression of this CTLA-4 agonist did not alter the diabetogenic properties of systemic lymphocytes, because splenocytes from transgenic mice or their nontransgenic littermates equally transferred diabetes in NOD/SCID recipients. By analyzing the T cell development in anti-CTLA-4 scFv/Th1/Th2 triple transgenic mice, we found that beta cell-specific expression of CTLA-4 agonist did not affect the development of Th1/Th2 or CD4(+)CD25(+) regulatory T cells. Most strikingly, islets from transgenic mice inhibited T cell response to immobilized anti-CD3 in a T cell-islet coculture system, suggesting a trans-mediated inhibition provided by transgenic islets. Finally, transgenic islets implanted in diabetic recipients survived much longer than did wild-type islets, indicating a therapeutic potential of this genetically modified islet graft in autoimmune diabetes.

Attenuation of Th1 response through galectin-9 and T-cell Ig mucin 3 interaction inhibits autoimmune diabetes in NOD mice.

Galectin-9 (gal-9), widely expressed in many tissues, regulates Th1 cells and induces their apoptosis through its receptor, T-cell Ig mucin 3, which is mainly expressed on terminally differentiated Th1 cells. Type 1 diabetes is a Th1-dominant autoimmune disease that specifically destroys insulin-producing beta cells. To suppress the Th1 immune response in the development of autoimmune diabetes, we overexpressed gal-9 in NOD mice by injection of a plasmid encoding gal-9. Mice treated with gal-9 plasmid were significantly protected from diabetes and showed less severe insulitis compared with controls. Flow cytometric analyses in NOD-T1/2 double transgenic mice showed that Th1-cell population in spleen, pancreatic lymph node and pancreas was markedly decreased in gal-9 plasmid-treated mice, indicating a negative regulatory role of gal-9 in the development of pathogenic Th1 cells. Splenocytes from gal-9 plasmid-treated mice were less responsive to mitogenic stimulation than splenocytes from the control group. However, adoptive transfer of splenocytes from gal-9-treated or control mice caused diabetes in NOD/SCID recipients with similar kinetics, suggesting that gal-9 treatment does not induce active tolerance in NOD mice. We conclude that gal-9 may downregulate Th1 immune response in NOD mice and could be used as a therapeutic target in autoimmune diabetes.

Overexpression of thioredoxin in islets transduced by a lentiviral vector prolongs graft survival in autoimmune diabetic NOD mice.

UNLABELLED: Pancreatic islet transplantation is considered an appropriate treatment to achieve insulin independence in type I diabetic patients. However, islet isolation and transplantation-induced oxidative stress and autoimmune-mediated destruction are still the major obstacles to the long-term survival of graft islets in this potential therapy. To protect islet grafts from inflammatory damage and prolong their survival, we transduced islets with an antioxidative gene thioredoxin (TRX) using a lentiviral vector before transplantation. We hypothesized that the overexpression of TRX in islets would prolong islet graft survival when transplanted into diabetic non-obese diabetic (NOD) mice. METHODS: Islets were isolated from NOD mice and transduced with lentivirus carrying TRX (Lt-TRX) or enhanced green fluorescence protein (Lt-eGFP), respectively. Transduced islets were transplanted under the left kidney capsule of female diabetic NOD mice, and blood glucose concentration was monitored daily after transplantation. The histology of the islet graft was assessed at the end of the study. The protective effect of TRX on islets was investigated. RESULTS: The lentiviral vector effectively transduced islets without altering the glucose-stimulating insulin-secretory function of islets. Overexpression of TRX in islets reduced hydrogen peroxide-induced cytotoxicity in vitro. After transplantation into diabetic NOD mice, euglycemia was maintained for significantly longer in Lt-TRX-transduced islets than in Lt-eGFP-transduced islets; the mean graft survival was 18 vs. 6.5 days (n = 9 and 10, respectively, p < 0.05). CONCLUSION: We successfully transduced the TRX gene into islets and demonstrated that these genetically modified grafts are resistant to inflammatory insult and survived longer in diabetic recipients. Our results further support the concept that the reactive oxygen species (ROS) scavenger and antiapoptotic functions of TRX are critical to islet survival after transplantation.

Peripheral Autoimmune Regulator Induces Exhaustion of CD4+ and CD8+ Effector T Cells to Attenuate Autoimmune Diabetes in Non-Obese Diabetic Mice.

Autoimmune regulator (Aire) is one of the most crucial genes expressed in the thymus, where it orchestrates the promiscuous expression and presentation of tissue-specific antigens during thymocyte selection. The presence of Aire-expressing cells outside the thymus points toward its plausible extrathymic functions; however, the relative contribution of Aire-expressing cells of hematopoietic origin and their role in the modulation of autoimmune diseases are still obscure. Here, we report that non-obese diabetic mice with transgenic Aire expression under the control of the CD11c (integrin alpha X) promoter were significantly protected from autoimmune diabetes compared with their non-transgenic littermates. The protective effect of Aire transgene was mediated primarily by an increase in the "exhausted" populations of CD4+ and CD8+ T cells, both demonstrating poor expressions of interferon-γ and tumor necrosis factor-α. Both CD4+ and CD8+ effector T cells in transgenic mice displayed distinctive and differential expression of T-bet and Eomesodermin, respectively, in conjunction with high expression of programmed cell death protein-1 and other exhaustion-associated markers. Importantly, transgenic Aire expression did not result in any detectable changes in the population of Foxp3+ regulatory T (Treg) cells. Co-transfer experiments also demonstrated that Aire transgenic dendritic cells, as a "stand-alone" cell population, had the potential to suppress effector T cells in vivo without the support of Treg cells, but eventually failed to prevent the diabetogenesis in recipient mice. In conclusion, our study suggests that apart from its role in clonal deletion of autoreactive T cells or clonal diversion to Treg lineage, Aire can also contribute to tolerance by forcing effector T cells into a state of exhaustion with poor effector functions, thereby effectively containing autoimmune diseases.

Rodent models for investigating the dysregulation of immune responses in type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease mediated by T cells that selectively destroy the insulin-producing ß cells. Previous reports based on epidemiological and animal studies have demonstrated that both genetic factors and environmental parameters can either promote or attenuate the progression of autoimmunity. In recent decades, several inbred rodent strains that spontaneously develop diabetes have been applied to the investigation of the pathogenesis of T1D. Because the genetic manipulation of mice is well developed (transgenic, knockout, and conditional knockout/transgenic), most studies are performed using the nonobese diabetic (NOD) mouse model. This paper will focus on the use of genetically manipulated NOD mice to explore the pathogenesis of T1D and to develop potential therapeutic approaches.

Overexpression of galectin-9 in islets prolongs grafts survival via downregulation of Th1 responses.

The differential activation of T helper (Th) cells and production of cytokines contribute to graft rejection or tolerance. In general, the Th1-type cytokines and cytotoxic T-cells are detected consistently in a host who is undergoing rejection, whereas Th2 responses are linked to a tolerance condition. Galectin-9 modulates Th1 cell immunity by binding to the T-cell immunoglobulin mucin-3 (Tim-3) molecule expressed on the Th1 cells. We investigate whether overexpression of galectin-9 in islets prolongs grafts survival in diabetic recipients. Islets were transduced with lentiviruses carrying galectin-9 and were then transplanted to streptozotocin-induced diabetic NOD/SCID recipients. The normoglycemic recipients then received splenocytes from diabetic NOD mice. Blood glucose concentration was monitored daily after adoptive transfer. The histology of the islet grafts and flow cytometric analyses were assessed at the end of the study. Overexpression of galectin-9 in islets prolonged graft survival in NOD/SCID mice after challenge with diabetogenic splenocytes (mean graft survival, 38.5 vs. 26.0 days, n=10, respectively; p=0.0096). The galectin-9-overexpressed grafts showed decreased infiltration of IFN-γ-producing CD4(+) and CD8(+) T-cells, but not of IL-17-producing CD4(+) T-cells. Strikingly, this islet-specific genetic manipulation did not affect the systemic lymphocyte composition, indicating that galectin-9 may regulate T-cell-mediated inflammation in situ. We demonstrate that galectin-9 protects grafts from Th1 and Tc1 cell-mediated rejections, suggesting that galectin-9 has preventive and/or therapeutic benefit in transplant therapy for autoimmune diabetes and may be applied further to the transplantation of other organs or tissues.

Genetically engineered islets and alternative sources of insulin-producing cells for treating autoimmune diabetes: quo vadis?

Islet transplantation is a promising therapy for patients with type 1 diabetes that can provide moment-to-moment metabolic control of glucose and allow them to achieve insulin independence. However, two major problems need to be overcome: (1) detrimental immune responses, including inflammation induced by the islet isolation/transplantation procedure, recurrence autoimmunity, and allorejection, can cause graft loss and (2) inadequate numbers of organ donors. Several gene therapy approaches and pharmaceutical treatments have been demonstrated to prolong the survival of pancreatic islet grafts in animal models; however, the clinical applications need to be investigated further. In addition, for an alternative source of pancreatic ß-cell replacement therapy, the ex vivo generation of insulin-secreting cells from diverse origins of stem/progenitor cells has become an attractive option in regenerative medicine. This paper focuses on the genetic manipulation of islets during transplantation therapy and summarizes current strategies to obtain functional insulin-secreting cells from stem/progenitor cells.

Decoy receptor 3 protects non-obese diabetic mice from autoimmune diabetes by regulating dendritic cell maturation and function.

Decoy receptor 3 (DcR3), a member of the tumor necrosis factor receptor superfamily, regulates immune responses through competing with receptors of Fas ligand (FasL), LIGHT and TNF-like molecule 1A (TL1A). We have previously demonstrated that transgenic expression of DcR3 in a ß cell-specific manner significantly protects non-obese diabetic (NOD) mice from autoimmune diabetes. In this study, we further investigated the systemic effect of DcR3 in regulating lymphocytes and dendritic cells in NOD mice. Our results demonstrated that both DcR3 plasmid and protein treatments significantly inhibited insulitis and diabetes. Lymphocytes from DcR3.Fc-treated mice revealed less proliferative potential and transferred ameliorated diabetes. By administration of DcR3.Fc in T1 and T2 double transgenic NOD mice expressing human Thy1 or murine Thy1.1 surface marker under IFN-γ or IL-4 promoter control respectively, we observed a remarkable reduction of Th1 and an increase of Th2 immune responses in vivo. Strikingly, in vitro polarization experiments exhibited that not only Th1 but also Th17 cell differentiation was significantly inhibited in splenocytes treated with DcR3.Fc protein. However, this phenomenon was only observed in splenocytes, not in purified CD4(+) T cells, suggesting that DcR3-mediated inhibition of Th1 and Th17 differentiation is not T cell-autonomous and maybe through other cell types such as dendritic cells. Finally, our results demonstrated that DcR3 directly modulates the differentiation and maturation of dendritic cells and subsequently regulates the differentiation and effector function of T cells.

Protective role of programmed death 1 ligand 1 (PD-L1)in nonobese diabetic mice: the paradox in transgenic models.

OBJECTIVE: Coinhibitory signals mediated via programmed death 1 (PD-1) receptor play a critical role in downregulating immune responses and in maintaining peripheral tolerance. Programmed death 1 ligand 1 (PD-L1), the interacting ligand for PD-1, widely expressed in many cell types, acts as a tissue-specific negative regulator of pathogenic T-cell responses. We investigated the protective potential of PD-L1 on autoimmune diabetes by transgenically overexpressing PD-L1 in pancreatic beta-cells in nonobese diabetic (NOD) mice. RESEARCH DESIGN AND METHODS: We established an insulin promoter-driven murine PD-L1 transgenic NOD mouse model to directly evaluate the protective effect of an organ-specific PD-L1 transgene against autoimmune diabetes. Transgene expression, insulitis, and diabetic incidence were characterized in these transgenic NOD mice. Lymphocyte development, Th1 cells, and regulatory T-cells were analyzed in these transgenic mice; and T-cell proliferation, adoptive transfer, and islet transplantation were performed to evaluate the PD-L1 transgene-mediated immune-protective mechanisms. RESULTS: The severity of insulitis in these transgenic mice is significantly decreased, disease onset is delayed, and the incidence of diabetes is markedly decreased compared with littermate controls. NOD/SCID mice that received lymphocytes from transgenic mice became diabetic at a slower rate than mice receiving control lymphocytes. Moreover, lymphocytes collected from recipients transferred by lymphocytes from transgenic mice revealed less proliferative potential than lymphocytes obtained from control recipients. Transgenic islets transplanted in diabetic recipients survived moderately longer than control islets. CONCLUSIONS: Our results demonstrate the protective potential of transgenic PD-L1 in autoimmune diabetes and illustrate its role in downregulating diabetogenic T-cells in NOD mice.