I-Ag7 ß56/57 polymorphisms regulate non-cognate negative selection to CD4+ T cell orchestrators of type 1 diabetes.

Genetic susceptibility to type 1 diabetes is associated with homozygous expression of major histocompatibility complex class II alleles that carry specific beta chain polymorphisms. Why heterozygous expression of these major histocompatibility complex class II alleles does not confer a similar predisposition is unresolved. Using a nonobese diabetic mouse model, here we show that heterozygous expression of the type 1 diabetes-protective allele I-Ag7 ß56P/57D induces negative selection to the I-Ag7-restricted T cell repertoire, including beta-islet-specific CD4+ T cells. Surprisingly, negative selection occurs despite I-Ag7 ß56P/57D having a reduced ability to present beta-islet antigens to CD4+ T cells. Peripheral manifestations of non-cognate negative selection include a near complete loss of beta-islet-specific CXCR6+ CD4+ T cells, an inability to cross-prime islet-specific glucose-6-phosphatase catalytic subunit-related protein and insulin-specific CD8+ T cells and disease arrest at the insulitis stage. These data reveal that negative selection on non-cognate self-antigens in the thymus can promote T cell tolerance and protection from autoimmunity.

Direct Tumor Killing and Immunotherapy through Anti-SerpinB9 Therapy.

Cancer therapies kill tumors either directly or indirectly by evoking immune responses and have been combined with varying levels of success. Here, we describe a paradigm to control cancer growth that is based on both direct tumor killing and the triggering of protective immunity. Genetic ablation of serine protease inhibitor SerpinB9 (Sb9) results in the death of tumor cells in a granzyme B (GrB)-dependent manner. Sb9-deficient mice exhibited protective T cell-based host immunity to tumors in association with a decline in GrB-expressing immunosuppressive cells within the tumor microenvironment (TME). Maximal protection against tumor development was observed when the tumor and host were deficient in Sb9. The therapeutic utility of Sb9 inhibition was demonstrated by the control of tumor growth, resulting in increased survival times in mice. Our studies describe a molecular target that permits a combination of tumor ablation, interference within the TME, and immunotherapy in one potential modality.

Generation of functional human pancreatic ß cells in vitro.

The generation of insulin-producing pancreatic ß cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation therapy in diabetes. However, insulin-producing cells previously generated from human pluripotent stem cells (hPSC) lack many functional characteristics of bona fide ß cells. Here, we report a scalable differentiation protocol that can generate hundreds of millions of glucose-responsive ß cells from hPSC in vitro. These stem-cell-derived ß cells (SC-ß) express markers found in mature ß cells, flux Ca(2+) in response to glucose, package insulin into secretory granules, and secrete quantities of insulin comparable to adult ß cells in response to multiple sequential glucose challenges in vitro. Furthermore, these cells secrete human insulin into the serum of mice shortly after transplantation in a glucose-regulated manner, and transplantation of these cells ameliorates hyperglycemia in diabetic mice.

Genetic risk converges on regulatory networks mediating early type 2 diabetes.

Type 2 diabetes mellitus (T2D), a major cause of worldwide morbidity and mortality, is characterized by dysfunction of insulin-producing pancreatic islet ß cells1,2. T2D genome-wide association studies (GWAS) have identified hundreds of signals in non-coding and ß cell regulatory genomic regions, but deciphering their biological mechanisms remains challenging3-5. Here, to identify early disease-driving events, we performed traditional and multiplexed pancreatic tissue imaging, sorted-islet cell transcriptomics and islet functional analysis of early-stage T2D and control donors. By integrating diverse modalities, we show that early-stage T2D is characterized by ß cell-intrinsic defects that can be proportioned into gene regulatory modules with enrichment in signals of genetic risk. After identifying the ß cell hub gene and transcription factor RFX6 within one such module, we demonstrated multiple layers of genetic risk that converge on an RFX6-mediated network to reduce insulin secretion by ß cells. RFX6 perturbation in primary human islet cells alters ß cell chromatin architecture at regions enriched for T2D GWAS signals, and population-scale genetic analyses causally link genetically predicted reduced RFX6 expression with increased T2D risk. Understanding the molecular mechanisms of complex, systemic diseases necessitates integration of signals from multiple molecules, cells, organs and individuals, and thus we anticipate that this approach will be a useful template to identify and validate key regulatory networks and master hub genes for other diseases or traits using GWAS data.

Human insulin as both antigen and protector in type 1 diabetes.

Type 1 diabetes (T1D) is characterized by T-cell responses to islet antigens. Investigations in humans and the nonobese diabetic (NOD) mouse model of T1D have revealed that T-cell reactivity to insulin plays a central role in the autoimmune response. As there is no convenient NOD-based model to study human insulin (hIns) or its T-cell epitopes in the context of spontaneous T1D, we developed a NOD mouse strain transgenically expressing hIns in islets under the control of the human regulatory region. Female NOD.hIns mice developed T1D at approximately the same rate and overall incidence as NOD mice. Islet-infiltrating T cells from NOD.hIns mice recognized hIns peptides; both CD8 and CD4 T-cell epitopes were identified. We also demonstrate that islet-infiltrating T cells from HLA-transgenic NOD.hIns mice can be used to identify potentially patient-relevant hIns T-cell epitopes. Besides serving as an antigen, hIns was expressed in the thymus of NOD.hIns mice and could serve as a protector against T1D under certain circumstances, as previously suggested by genetic studies in humans. NOD.hIns mice and related strains facilitate human-relevant epitope discovery efforts and the investigation of fundamental questions that cannot be readily addressed in humans.

Cell-Extrinsic MHC Class I Molecule Engagement Augments Human NK Cell Education Programmed by Cell-Intrinsic MHC Class I.

The effector potential of NK cells is counterbalanced by their sensitivity to inhibition by "self" MHC class I molecules in a process called "education." In humans, interactions between inhibitory killer immunoglobulin-like receptors (KIR) and human MHC (HLA) mediate NK cell education. In HLA-B(∗)27:05(+) transgenic mice and in patients undergoing HLA-mismatched hematopoietic cell transplantation (HCT), NK cells derived from human CD34(+) stem cells were educated by HLA from both donor hematopoietic cells and host stromal cells. Furthermore, mature human KIR3DL1(+) NK cells gained reactivity after adoptive transfer to HLA-B(∗)27:05(+) mice or bone marrow chimeric mice where HLA-B(∗)27:05 was restricted to either the hematopoietic or stromal compartment. Silencing of HLA in primary NK cells diminished NK cell reactivity, while acquisition of HLA from neighboring cells increased NK cell reactivity. Altogether, these findings reveal roles for cell-extrinsic HLA in driving NK cell reactivity upward, and cell-intrinsic HLA in maintaining NK cell education.

Simultaneous evaluation of treatment efficacy and toxicity for bispecific T-cell engager therapeutics in a humanized mouse model.

Immuno-oncology (IO)-based therapies such as checkpoint inhibitors, bi-specific antibodies, and CAR-T-cell therapies have shown significant success in the treatment of several cancer indications. However, these therapies can result in the development of severe adverse events, including cytokine release syndrome (CRS). Currently, there is a paucity of in vivo models that can evaluate dose-response relationships for both tumor control and CRS-related safety issues. We tested an in vivo PBMC humanized mouse model to assess both treatment efficacy against specific tumors and the concurrent cytokine release profiles for individual human donors after treatment with a CD19xCD3 bispecific T-cell engager (BiTE). Using this model, we evaluated tumor burden, T-cell activation, and cytokine release in response to bispecific T-cell-engaging antibody in humanized mice generated with different PBMC donors. The results show that PBMC engrafted NOD-scid Il2rgnull mice lacking expression of mouse MHC class I and II (NSG-MHC-DKO mice) and implanted with a tumor xenograft predict both efficacy for tumor control by CD19xCD3 BiTE and stimulated cytokine release. Moreover, our findings indicate that this PBMC-engrafted model captures variability among donors for tumor control and cytokine release following treatment. Tumor control and cytokine release were reproducible for the same PBMC donor in separate experiments. The PBMC humanized mouse model described here is a sensitive and reproducible platform that identifies specific patient/cancer/therapy combinations for treatment efficacy and development of complications.

A therapeutic convection-enhanced macroencapsulation device for enhancing ß cell viability and insulin secretion.

Islet transplantation for type 1 diabetes treatment has been limited by the need for lifelong immunosuppression regimens. This challenge has prompted the development of macroencapsulation devices (MEDs) to immunoprotect the transplanted islets. While promising, conventional MEDs are faced with insufficient transport of oxygen, glucose, and insulin because of the reliance on passive diffusion. Hence, these devices are constrained to two-dimensional, wafer-like geometries with limited loading capacity to maintain cells within a distance of passive diffusion. We hypothesized that convective nutrient transport could extend the loading capacity while also promoting cell viability, rapid glucose equilibration, and the physiological levels of insulin secretion. Here, we showed that convective transport improves nutrient delivery throughout the device and affords a three-dimensional capsule geometry that encapsulates 9.7-fold-more cells than conventional MEDs. Transplantation of a convection-enhanced MED (ceMED) containing insulin-secreting ß cells into immunocompetent, hyperglycemic rats demonstrated a rapid, vascular-independent, and glucose-stimulated insulin response, resulting in early amelioration of hyperglycemia, improved glucose tolerance, and reduced fibrosis. Finally, to address potential translational barriers, we outlined future steps necessary to optimize the ceMED design for long-term efficacy and clinical utility.

Prostaglandin E2 stimulates cAMP signaling and resensitizes human leukemia cells to glucocorticoid-induced cell death.

Glucocorticoid (GC) resistance remains a clinical challenge in pediatric acute lymphoblastic leukemia where response to GC is a reliable prognostic indicator. To identify GC resistance pathways, we conducted a genome-wide, survival-based, short hairpin RNA screen in murine T-cell acute lymphoblastic leukemia (T-ALL) cells. Genes identified in the screen interfere with cyclic adenosine monophosphate (cAMP) signaling and are underexpressed in GC-resistant or relapsed ALL patients. Silencing of the cAMP-activating Gnas gene interfered with GC-induced gene expression, resulting in dexamethasone resistance in vitro and in vivo. We demonstrate that cAMP signaling synergizes with dexamethasone to enhance cell death in GC-resistant human T-ALL cells. We find the E prostanoid receptor 4 expressed in T-ALL samples and demonstrate that prostaglandin E2 (PGE2) increases intracellular cAMP, potentiates GC-induced gene expression, and sensitizes human T-ALL samples to dexamethasone in vitro and in vivo. These findings identify PGE2 as a target for GC resensitization in relapsed pediatric T-ALL.

Enhanced development of functional human NK cells in NOD-scid-IL2rgnull mice expressing human IL15.

Human innate immunity plays a critical role in tumor surveillance and in immunoregulation within the tumor microenvironment. Natural killer (NK) cells are innate lymphoid cells that have opposing roles in the tumor microenvironment, including NK cell subsets that mediate tumor cell cytotoxicity and subsets with regulatory function that contribute to the tumor immune suppressive environment. The balance between effector and regulatory NK cell subsets has been studied extensively in murine models of cancer, but there is a paucity of models to study human NK cell function in tumorigenesis. Humanized mice are a powerful alternative to syngeneic mouse tumor models for the study of human immuno-oncology and have proven effective tools to test immunotherapies targeting T cells. However, human NK cell development and survival in humanized NOD-scid-IL2rgnull (NSG) mice are severely limited. To enhance NK cell development, we have developed NSG mice that constitutively expresses human Interleukin 15 (IL15), NSG-Tg(Hu-IL15). Following hematopoietic stem cell engraftment of NSG-Tg(Hu-IL15) mice, significantly higher levels of functional human CD56+ NK cells are detectable in blood and spleen, as compared to NSG mice. Hematopoietic stem cell (HSC)-engrafted NSG-Tg(Hu-IL15) mice also supported the development of human CD3+ T cells, CD20+ B cells, and CD33+ myeloid cells. Moreover, the growth kinetics of a patient-derived xenograft (PDX) melanoma were significantly delayed in HSC-engrafted NSG-Tg(Hu-IL15) mice as compared to HSC-engrafted NSG mice demonstrating that human NK cells have a key role in limiting the tumor growth. Together, these data demonstrate that HSC-engrafted NSG-Tg(Hu-IL15) mice support enhanced development of functional human NK cells, which limit the growth of PDX tumors.

T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice.

Evaluation of the therapeutic potential of RNAi for HIV infection has been hampered by the challenges of siRNA delivery and lack of suitable animal models. Using a delivery method for T cells, we show that siRNA treatment can dramatically suppress HIV infection. A CD7-specific single-chain antibody was conjugated to oligo-9-arginine peptide (scFvCD7-9R) for T cell-specific siRNA delivery in NOD/SCIDIL2rgamma-/- mice reconstituted with human lymphocytes (Hu-PBL) or CD34+ hematopoietic stem cells (Hu-HSC). In HIV-infected Hu-PBL mice, treatment with anti-CCR5 (viral coreceptor) and antiviral siRNAs complexed to scFvCD7-9R controlled viral replication and prevented the disease-associated CD4 T cell loss. This treatment also suppressed endogenous virus and restored CD4 T cell counts in mice reconstituted with HIV+ peripheral blood mononuclear cells. Moreover, scFvCD7-9R could deliver antiviral siRNAs to naive T cells in Hu-HSC mice and effectively suppress viremia in infected mice. Thus, siRNA therapy for HIV infection appears to be feasible in a preclinical animal model.

A laminin 511 matrix is regulated by TAZ and functions as the ligand for the α6Bß1 integrin to sustain breast cancer stem cells.

Understanding how the extracellular matrix impacts the function of cancer stem cells (CSCs) is a significant but poorly understood problem. We report that breast CSCs produce a laminin (LM) 511 matrix that promotes self-renewal and tumor initiation by engaging the α6Bß1 integrin and activating the Hippo transducer TAZ. Although TAZ is important for the function of breast CSCs, the mechanism is unknown. We observed that TAZ regulates the transcription of the α5 subunit of LM511 and the formation of a LM511 matrix. These data establish a positive feedback loop involving TAZ and LM511 that contributes to stemness in breast cancer.

A rapid, sensitive, and reproducible in vivo PBMC humanized murine model for determining therapeutic-related cytokine release syndrome.

Immunotherapy is a powerful treatment strategy being applied to cancer, autoimmune diseases, allergies, and transplantation. Although therapeutic monoclonal antibodies (mAbs) have demonstrated significant clinical efficacy, there is also the potential for severe adverse events, including cytokine release syndrome (CRS). CRS is characterized by the rapid production of inflammatory cytokines following delivery of therapy, with symptoms ranging from mild fever to life-threating pathology and multi-organ failure. Overall there is a paucity of models to reliably and accurately predict the induction of CRS by immune therapeutics. Here, we describe the development of a humanized mouse model based on the NOD-scid IL2rgnull (NSG) mouse to study CRS in vivo. PBMC-engrafted NSG, NSG-MHC-DKO, and NSG-SGM3 mice were used to study cytokine release in response to treatment with mAb immunotherapies. Our data show that therapeutic-stimulated cytokine release in these PBMC-based NSG models captures the variation in cytokine release between individual donors, is drug dependent, occurs in the absence of acute xeno-GVHD, highlighting the specificity of the assay, and shows a robust response following treatment with a TGN1412 analog, a CD28 superagonist. Overall our results demonstrate that PBMC-engrafted NSG models are rapid, sensitive, and reproducible platforms to screen novel therapeutics for CRS.

Recovery of viable endocrine-specific cells and transcriptomes from human pancreatic islet-engrafted mice.

Human pancreatic islets engrafted into immunodeficient mice serve as an important model for in vivo human diabetes studies. Following engraftment, islet function can be monitored in vivo by measuring circulating glucose and human insulin; however, it will be important to recover viable cells for more complex graft analyses. Moreover, RNA analyses of dissected grafts have not distinguished which hormone-specific cell types contribute to gene expression. We developed a method for recovering live cells suitable for fluorescence-activated cell sorting from human islets engrafted in mice. Although yields of recovered islet cells were relatively low, the ratios of bulk-sorted ß, α, and δ cells and their respective hormone-specific RNA-Seq transcriptomes are comparable pretransplant and posttransplant, suggesting that the cellular characteristics of islet grafts posttransplant closely mirror the original donor islets. Single-cell RNA-Seq transcriptome analysis confirms the presence of appropriate ß, α, and δ cell subsets. In addition, ex vivo perifusion of recovered human islet grafts demonstrated glucose-stimulated insulin secretion. Viable cells suitable for patch-clamp analysis were recovered from transplanted human embryonic stem cell-derived ß cells. Together, our functional and hormone-specific transcriptome analyses document the broad applicability of this system for longitudinal examination of human islet cells undergoing developmental/metabolic/pharmacogenetic manipulation in vivo and may facilitate the discovery of treatments for diabetes.

Human Anti-HIV-1 gp120 Monoclonal Antibodies with Neutralizing Activity Cloned from Humanized Mice Infected with HIV-1.

Broadly neutralizing, anti-HIV-1 gp120 mAbs have been isolated from infected individuals, and there is considerable interest in developing these reagents for Ab-based immunoprophylaxis and treatment. As a means to identify potentially new anti-HIV Abs, we exploited humanized NOD-scid IL2rγnull mice systemically infected with HIV-1 to generate a wide variety of Ag-specific human mAbs. The Abs were encoded by a diverse range of variable gene families and Ig classes, including IgA, and several showed significant levels of somatic mutation. Moreover, the isolated Abs not only bound target Ags with similar affinity as broadly neutralizing Abs, they also demonstrated neutralizing ability against multiple HIV-1 clades. The use of humanized mice will allow us to use our knowledge of HIV-1 gp120 structure and function, and the immune response targeting this protein, to generate native human prophylactic Abs to reduce the infection and spread of HIV-1.

Long-term implant fibrosis prevention in rodents and non-human primates using crystallized drug formulations.

Implantable medical devices have revolutionized modern medicine. However, immune-mediated foreign body response (FBR) to the materials of these devices can limit their function or even induce failure. Here we describe long-term controlled-release formulations for local anti-inflammatory release through the development of compact, solvent-free crystals. The compact lattice structure of these crystals allows for very slow, surface dissolution and high drug density. These formulations suppress FBR in both rodents and non-human primates for at least 1.3 years and 6 months, respectively. Formulations inhibited fibrosis across multiple implant sites-subcutaneous, intraperitoneal and intramuscular. In particular, incorporation of GW2580, a colony stimulating factor 1 receptor inhibitor, into a range of devices, including human islet microencapsulation systems, electrode-based continuous glucose-sensing monitors and muscle-stimulating devices, inhibits fibrosis, thereby allowing for extended function. We believe that local, long-term controlled release with the crystal formulations described here enhances and extends function in a range of medical devices and provides a generalized solution to the local immune response to implanted biomaterials.

Lack of acute xenogeneic graft- versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression.

Immunodeficient mice engrafted with human peripheral blood mononuclear cells (PBMCs) support preclinical studies of human pathogens, allograft rejection, and human T-cell function. However, a major limitation of PBMC engraftment is development of acute xenogeneic graft- versus-host disease (GVHD) due to human T-cell recognition of murine major histocompatibility complex (MHC). To address this, we created 2 NOD- scid IL-2 receptor subunit γ ( IL2rg) null (NSG) strains that lack murine MHC class I and II [NSG-ß-2-microglobulin ( B2M) null ( IA IE)null and NSG -( Kb Db) null ( IAnull)]. We observed rapid human IgG clearance in NSG- B2Mnull ( IA IE) null mice whereas clearance in NSG -( Kb Db) null ( IAnull) mice and NSG mice was comparable. Injection of human PBMCs into both strains enabled long-term engraftment of human CD4+ and CD8+ T cells without acute GVHD. Engrafted human T-cell function was documented by rejection of human islet allografts. Administration of human IL-2 to NSG -( Kb Db) null ( IAnull) mice via adeno-associated virus vector increased human CD45+ cell engraftment, including an increase in human regulatory T cells. However, high IL-2 levels also induced the development of GVHD. These data document that NSG mice deficient in murine MHC support studies of human immunity in the absence of acute GVHD and enable evaluation of human antibody therapeutics targeting human T cells.-Brehm, M. A., Kenney, L. L., Wiles, M. V., Low, B. E., Tisch, R. M., Burzenski, L., Mueller, C., Greiner, D. L., Shultz, L. D. Lack of acute xenogeneic graft- versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression.

End Sequence Analysis Toolkit (ESAT) expands the extractable information from single-cell RNA-seq data.

RNA-seq protocols that focus on transcript termini are well suited for applications in which template quantity is limiting. Here we show that, when applied to end-sequencing data, analytical methods designed for global RNA-seq produce computational artifacts. To remedy this, we created the End Sequence Analysis Toolkit (ESAT). As a test, we first compared end-sequencing and bulk RNA-seq using RNA from dendritic cells stimulated with lipopolysaccharide (LPS). As predicted by the telescripting model for transcriptional bursts, ESAT detected an LPS-stimulated shift to shorter 3'-isoforms that was not evident by conventional computational methods. Then, droplet-based microfluidics was used to generate 1000 cDNA libraries, each from an individual pancreatic islet cell. ESAT identified nine distinct cell types, three distinct ß-cell types, and a complex interplay between hormone secretion and vascularization. ESAT, then, offers a much-needed and generally applicable computational pipeline for either bulk or single-cell RNA end-sequencing.

Humanized mouse models of immunological diseases and precision medicine.

With the increase in knowledge resulting from the sequencing of the human genome, the genetic basis for the underlying differences in individuals, their diseases, and how they respond to therapies is starting to be understood. This has formed the foundation for the era of precision medicine in many human diseases that is beginning to be implemented in the clinic, particularly in cancer. However, preclinical testing of therapeutic approaches based on individual biology will need to be validated in animal models prior to translation into patients. Although animal models, particularly murine models, have provided significant information on the basic biology underlying immune responses in various diseases and the response to therapy, murine and human immune systems differ markedly. These fundamental differences may be the underlying reason why many of the positive therapeutic responses observed in mice have not translated directly into the clinic. There is a critical need for preclinical animal models in which human immune responses can be investigated. For this, many investigators are using humanized mice, i.e., immunodeficient mice engrafted with functional human cells, tissues, and immune systems. We will briefly review the history of humanized mice, the remaining limitations, approaches to overcome them and how humanized mouse models are being used as a preclinical bridge in precision medicine for evaluation of human therapies prior to their implementation in the clinic.

AK002, a Humanized Sialic Acid-Binding Immunoglobulin-Like Lectin-8 Antibody that Induces Antibody-Dependent Cell-Mediated Cytotoxicity against Human Eosinophils and Inhibits Mast Cell-Mediated Anaphylaxis in Mice.

INTRODUCTION: Pathologic accumulation and activation of mast cells and eosinophils are implicated in allergic and inflammatory diseases. Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is an inhibitory receptor selectively expressed on mast cells, eosinophils and, at a lower extent, basophils. When engaged with an antibody, Siglec-8 can induce apoptosis of activated eosinophils and inhibit mast cell activation. AK002 is a humanized, non-fucosylated IgG1 anti-Siglec-8 antibody undergoing clinical investigation for treatment of allergic, inflammatory, and proliferative diseases. Here we examine the human tissue selectivity of AK002 and evaluate the in vitro, ex vivo, and in vivo activity of AK002 on eosinophils and mast cells. METHODS: The affinity of AK002 for Siglec-8 and CD16 was determined by biolayer interferometry. Ex vivo activity of AK002 on human eosinophils from blood and dissociated human tissue was tested in apoptosis and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. The in vivo activity of a murine precursor of AK002 (mAK002) was tested in a passive systemic anaphylaxis (PSA) humanized mouse model. RESULTS: AK002 bound selectively to mast cells, eosinophils and, at a lower level, to basophils in human blood and tissue and not to other cell types examined. AK002 induced apoptosis of interleukin-5-activated blood eosinophils and demonstrated potent ADCC activity against blood eosinophils in the presence of natural killer cells. AK002 also significantly reduced eosinophils in dissociated human lung tissue. Furthermore, mAK002 prevented PSA in humanized mice through mast cell inhibition. CONCLUSION: AK002 selectively evokes potent apoptotic and ADCC activity against eosinophils and prevents systemic anaphylaxis through mast cell inhibition.