De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids.

Hematopoietic humanized (hu) mice are powerful tools for modeling the action of human immune system and are widely used for preclinical studies and drug discovery. However, generating a functional human T cell compartment in hu mice remains challenging, primarily due to the species-related differences between human and mouse thymus. While engrafting human fetal thymic tissues can support robust T cell development in hu mice, tissue scarcity and ethical concerns limit their wide use. Here, we describe the tissue engineering of human thymus organoids from inducible pluripotent stem cells (iPSC-thymus) that can support the de novo generation of a diverse population of functional human T cells. T cells of iPSC-thymus-engrafted hu mice could mediate both cellular and humoral immune responses, including mounting robust proinflammatory responses on T cell receptor engagement, inhibiting allogeneic tumor graft growth and facilitating efficient Ig class switching. Our findings indicate that hu mice engrafted with iPSC-thymus can serve as a new animal model to study human T cell-mediated immunity and accelerate the translation of findings from animal studies into the clinic.

Arrest in the Progression of Type 1 Diabetes at the Mid-Stage of Insulitic Autoimmunity Using an Autoantigen-Decorated All-trans Retinoic Acid and Transforming Growth Factor Beta-1 Single Microparticle Formulation.

Type 1 diabetes (T1D) is a disorder of impaired glucoregulation due to lymphocyte-driven pancreatic autoimmunity. Mobilizing dendritic cells (DC) in vivo to acquire tolerogenic activity is an attractive therapeutic approach as it results in multiple and overlapping immunosuppressive mechanisms. Delivery of agents that can achieve this, in the form of micro/nanoparticles, has successfully prevented a number of autoimmune conditions in vivo. Most of these formulations, however, do not establish multiple layers of immunoregulation. all-trans retinoic acid (RA) together with transforming growth factor beta 1 (TGFß1), in contrast, has been shown to promote such mechanisms. When delivered in separate nanoparticle vehicles, they successfully prevent the progression of early-onset T1D autoimmunity in vivo. Herein, we show that the approach can be simplified into a single microparticle formulation of RA + TGFß1 with surface decoration with the T1D-relevant insulin autoantigen. We show that the onset of hyperglycemia is prevented when administered into non-obese diabetic mice that are at the mid-stage of active islet-selective autoimmunity. Unexpectedly, the preventive effects do not seem to be mediated by increased numbers of regulatory T-lymphocytes inside the pancreatic lymph nodes, at least following acute administration of microparticles. Instead, we observed a mild increase in the frequency of regulatory B-lymphocytes inside the mesenteric lymph nodes. These data suggest additional and potentially-novel mechanisms that RA and TGFß1 could be modulating to prevent progression of mid-stage autoimmunity to overt T1D. Our data further strengthen the rationale to develop RA+TGFß1-based micro/nanoparticle "vaccines" as possible treatments of pre-symptomatic and new-onset T1D autoimmunity.

Interleukin-7 is a survival factor for CD4+ CD25+ T-cells and is expressed by diabetes-suppressive dendritic cells.

Dendritic cells can facilitate allograft survival and prevent autoimmunity via direct and indirect cell-mediated mechanisms. Recent studies demonstrate that immunoregulatory dendritic cells (iDCs) confer immune hyporesponsiveness in part through CD4(+) CD25(+) T regulatory cells (Tregs). Herein, we provide evidence to support the hypothesis that dendritic cells derived from NOD mice and engineered ex vivo to exhibit suppressed expression of the CD40, CD80, and CD86 costimulatory molecules motivate an increase in the prevalence of regulatory CD4(+) CD25(+) T-cells via interleukin (IL)-7. Unlike control dendritic cells, these dendritic cells expressed significant levels of IL-7. Exogenous addition of IL-7 to NOD T-cells did not promote expansion or proliferation, but instead selectively maintained the number of CD4(+) CD25(+) T-cells by inhibiting activation of apoptosis in these cells. In vitro, IL-7 receptor alpha-chain (IL-7Ralpha) was expressed at significantly higher levels on CD4(+) CD25(+) T-cells compared with CD4(+) CD25(-) T-cells irrespective of resting or stimulated state. In vivo, CD4(+) CD25(+) T-cells obtained from NOD-scid mice reconstituted with ex vivo engineered iDCs and NOD splenocytes expressed significantly higher levels of IL-7Ralpha compared with levels in the CD4(+) CD25(-) subset, especially in diabetes-suppressive dendritic cell-administered NOD-scid recipients. Taken together, our data suggest a novel mechanism by which iDCs delay autoimmunity through the CD4(+) CD25(+) Treg pathway and suggest IL-7 as a survival factor for these putative Tregs, which express the alpha-chain of its receptor at considerably higher levels than CD4(+) CD25(-) T-cells.

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel.

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

Treg cells in pancreatic lymph nodes: the possible role in diabetogenesis and ß cell regeneration in a T1D model.

Previously, we established a model in which physiologically adequate function of the autologous ß cells was recovered in non-obese diabetic (NOD) mice after the onset of hyperglycemia by rendering them hemopoietic chimera. These mice were termed antea-diabetic. In the current study, we addressed the role of T regulatory (Treg) cells in the mechanisms mediating the restoration of euglycemia in the antea-diabetic NOD model. The data generated in this study demonstrated that the numbers of Treg cells were decreased in unmanipulated NOD mice, with the most profound deficiency detected in the pancreatic lymph nodes (PLNs). The impaired retention of the Treg cells in the PLNs correlated with the locally compromised profile of the chemokines involved in their trafficking, with the most prominent decrease observed in SDF-1. The amelioration of autoimmunity and restoration of euglycemia observed in the antea-diabetic mice was associated with restoration of the Treg cell population in the PLNs. These data indicate that the function of the SDF-1/CXCR4 axis and the retention of Treg cells in the PLNs have a potential role in diabetogenesis and in the amelioration of autoimmunity and ß cell regeneration in the antea-diabetic model. We have demonstrated in the antea-diabetic mouse model that lifelong recovery of the ß cells has a strong correlation with normalization of the Treg cell population in the PLNs. This finding offers new opportunities for testing the immunomodulatory regimens that promote accumulation of Treg cells in the PLNs as a therapeutic approach for type 1 diabetes (T1D).

An improved intracellular staining protocol for efficient detection of nuclear proteins in YFP-expressing cells.

Intracellular staining is a widely used flow cytometry (FCM)-based technique to detect the expression of cytoslio nucleic antigens. However, intracellular staining of cells expressing cytosolic fluorescent protein (FP) markers was proven to be problematic as significant loss of the FP-signal was routinely observed. Using splenocytes harvested from mice constitutively expressing the enhanced yellow fluorescent proteins (YFP) as a model, we modified the widely used intracellular staining protocol and successfully achieved simultaneous detection of both the nuclear proteins and YFP in T-regulatory cells. The improved protocol can be used to perform antibody-based intracellular characterization of FP-labeled target cells, while maintaining their fluorescent reporter signals for easy tracing and identification.

A multivalent vaccine for type 1 diabetes skews T cell subsets to Th2 phenotype in NOD mice.

Previous studies by our group, using an experimental autoimmune thyroiditis (EAT) model in Strain 13 inbred guinea pigs, resulted in T cell-mediated delayed hypersensitivity; however, autoantibodies proved not to be cytotoxic to thyroid epithelial cells in the presence or absence of complement proteins. Albeit, T cell-mediated lymphocyte cytotoxicity began to diminish sharply concomitantly with increasing titers of circulating autoantibodies, indicating a skewing of the self-reactive response and amelioration of the EAT. Furthermore, immunization of guinea pigs with thyroglobulin in incomplete Freund's adjuvant (IFA) generated a high titer of antithyroglobulin antibodies and proved to inhibit thyroiditis. These observations indicated that the shift in the immune response from Th1 to Th2 and the production of antibodies were likely responsible for ameliorating EAT. Based upon these results, we extrapolated our studies to design a multivalent vaccine, which shows promise in preventing/reversing T1D in NOD mice. A small pilot study was conducted in which a total of 34 mice, 20 non-immunized controls and 14 immunized with syngeneic islet lysate, were monitored for mean day to diabetes for a total of 28 weeks. Immunization of NOD animals with syngeneic islet lysates resulted in a significant delay in diabetes onset (P < 0.001) as compared to non-immunized controls. To further assess the vaccine's efficacy, robustness, and delay of disease, a large-scale experiment was conducted and monitored for 32 weeks using 106 mice, 64 non-immunized controls and 42 immunized with syngeneic islet lysate. At the end of the study, 90% of the non-immunized group developed diabetes, while less than 25% of the immunized group became diabetic (P < 0.0001). The protective effect, as a result of vaccination, correlated with an increase in the levels of IL-10 and IL-4 cytokines as well as a skewing to Th2-dependent isotype antibodies in serum. Strikingly, adoptive transfer of spleen cells from immunized animals into NOD.scid recipients provided protection against transfer of diabetes by diabetogenic spleen cells. The results of this study provide evidence that vaccination with islet lysate leads to a Th2-dependent skewing of the immune response to islet beta cells as a possible mechanism of protection. This strategy may be implemented as a possible vaccination protocol for arresting and/or preventing T1D in patients.

A microsphere-based vaccine prevents and reverses new-onset autoimmune diabetes.

OBJECTIVE: This study was aimed at ascertaining the efficacy of antisense oligonucleotide-formulated microspheres to prevent type 1 diabetes and to reverse new-onset disease. RESEARCH DESIGN AND METHODS: Microspheres carrying antisense oligonucleotides to CD40, CD80, and CD86 were delivered into NOD mice. Glycemia was monitored to determine disease prevention and reversal. In recipients that remained and/or became diabetes free, spleen and lymph node T-cells were enriched to determine the prevalence of Foxp3(+) putative regulatory T-cells (Treg cells). Splenocytes from diabetes-free microsphere-treated recipients were adoptively cotransferred with splenocytes from diabetic NOD mice into NOD-scid recipients. Live-animal in vivo imaging measured the microsphere accumulation pattern. To rule out nonspecific systemic immunosuppression, splenocytes from successfully treated recipients were pulsed with beta-cell antigen or ovalbumin or cocultured with allogeneic splenocytes. RESULTS: The microspheres prevented type 1 diabetes and, most importantly, exhibited a capacity to reverse clinical hyperglycemia, suggesting reversal of new-onset disease. The microspheres augmented Foxp3(+) Treg cells and induced hyporesponsiveness to NOD-derived pancreatic beta-cell antigen, without compromising global immune responses to alloantigens and nominal antigens. T-cells from successfully treated mice suppressed adoptive transfer of disease by diabetogenic splenocytes into secondary immunodeficient recipients. Finally, microspheres accumulated within the pancreas and the spleen after either intraperitoneal or subcutaneous injection. Dendritic cells from spleen of the microsphere-treated mice exhibit decreased cell surface CD40, CD80, and CD86. CONCLUSIONS: This novel microsphere formulation represents the first diabetes-suppressive and reversing nucleic acid vaccine that confers an immunoregulatory phenotype to endogenous dendritic cells.

Antisense oligonucleotides down-regulating costimulation confer diabetes-preventive properties to nonobese diabetic mouse dendritic cells.

Phenotypically "immature" dendritic cells (DCs), defined by low cell surface CD40, CD80, and CD86 can elicit host immune suppression in allotransplantation and autoimmunity. Herein, we report the most direct means of achieving phenotypic immaturity in NOD bone marrow-derived DCs aiming at preventing diabetes in syngeneic recipients. CD40, CD80, and CD86 cell surface molecules were specifically down-regulated by treating NOD DCs ex vivo with a mixture of antisense oligonucleotides targeting the CD40, CD80, and CD86 primary transcripts. The incidence of diabetes was significantly delayed by a single injection of the engineered NOD DCs into syngeneic recipients. Insulitis was absent in diabetes-free recipients and their splenic T cells proliferated in response to alloantigen. Engineered DC promoted an increased prevalence of CD4(+)CD25(+) T cells in NOD recipients at all ages examined and diabetes-free recipients exhibited significantly greater numbers of CD4(+)CD25(+) T cells compared with untreated NOD mice. In NOD-scid recipients, antisense-treated NOD DC promoted an increased prevalence of these putative regulatory T cells. Collectively, these data demonstrate that direct interference of cell surface expression of the major costimulatory molecules at the transcriptional level confers diabetes protection by promoting, in part, the proliferation and/or survival of regulatory T cells. This approach is a useful tool by which DC-mediated activation of regulatory T cells can be studied as well as a potential therapeutic option for type 1 diabetes.

Immunosuppressive effects of glucosamine.

Glucosamine is a naturally occurring derivative of glucose and is an essential component of glycoproteins and proteoglycans, important constituents of many eukaryotic proteins. In cells, glucosamine is produced enzymatically by the amidation of glucose 6-phosphate and can then be further modified by acetylation to result in N-acetylglucosamine. Commercially, glucosamine is sold over-the-counter to relieve arthritis. Although there is evidence in favor of the beneficial effects of glucosamine, the mechanism is unknown. Our data demonstrate that glucosamine suppresses the activation of T-lymphoblasts and dendritic cells in vitro as well as allogeneic mixed leukocyte reactivity in a dose-dependent manner. There was no inherent cellular toxicity involved in the inhibition, and the activity was not reproducible with other amine sugars. More importantly, glucosamine administration prolonged allogeneic cardiac allograft survival in vivo. We conclude that, despite its documented effects on insulin sensitivity, glucosamine possesses immunosuppressive activity and could be beneficial as an immunosuppressive agent.