"Humanized" mouse models created by engraftment of immunodeficient mice with human hematolymphoid cells or tissues are an emerging technology with broad appeal across multiple biomedical disciplines. However, investigators wishing to utilize humanized mice with engrafted functional human immune systems are faced with a myriad of variables to consider. In this study, we analyze HSC engraftment methodologies using three immunodeficient mouse strains harboring the IL2rgamma(null) mutation; NOD-scid IL2rgamma(null), NOD-Rag1(null) IL2rgamma(null), and BALB/c-Rag1(null) IL2rgamma(null) mice. Strategies compared engraftment of human HSC derived from umbilical cord blood following intravenous injection into adult mice and intracardiac and intrahepatic injection into newborn mice. We observed that newborn recipients exhibited enhanced engraftment as compared to adult recipients. Irrespective of the protocol or age of recipient, both immunodeficient NOD strains support enhanced hematopoietic cell engraftment as compared to the BALB/c strain. Our data define key parameters for establishing humanized mouse models to study human immunity.

The pathogenesis of human type 1 diabetes, characterized by immune-mediated damage of insulin-producing β-cells of pancreatic islets, may involve viral infection. Essential components of the innate immune antiviral response, including type I interferon (IFN) and IFN receptor-mediated signaling pathways, are candidates for determining susceptibility to human type 1 diabetes. Numerous aspects of human type 1 diabetes pathogenesis are recapitulated in the LEW.1WR1 rat model. Diabetes can be induced in LEW.1WR1 weanling rats challenged with virus or with the viral mimetic polyinosinic:polycytidylic acid (poly I:C). We hypothesized that disrupting the cognate type I IFN receptor (type I IFN α/β receptor [IFNAR]) to interrupt IFN signaling would prevent or delay the development of virus-induced diabetes. We generated IFNAR1 subunit-deficient LEW.1WR1 rats using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9) genome editing and confirmed functional disruption of the Ifnar1 gene. IFNAR1 deficiency significantly delayed the onset and frequency of diabetes and greatly reduced the intensity of insulitis after poly I:C treatment. The occurrence of Kilham rat virus-induced diabetes was also diminished in IFNAR1-deficient animals. These findings firmly establish that alterations in innate immunity influence the course of autoimmune diabetes and support the use of targeted strategies to limit or prevent the development of type 1 diabetes.

Adoptive cell immunotherapy for human diseases, including the use of T cells modified to express an anti-tumour T-cell receptor (TCR) or chimeric antigen receptor, is showing promise as an effective treatment modality. Further advances would be accelerated by the availability of a mouse model that would permit human T-cell engineering protocols and proposed genetic modifications to be evaluated in vivo. NOD-scid IL2rγ(null) (NSG) mice accept the engraftment of mature human T cells; however, long-term evaluation of transferred cells has been hampered by the xenogeneic graft-versus-host disease (GVHD) that occurs soon after cell transfer. We modified human primary CD4(+) T cells by lentiviral transduction to express a human TCR that recognizes a pancreatic beta cell-derived peptide in the context of HLA-DR4. The TCR-transduced cells were transferred to NSG mice engineered to express HLA-DR4 and to be deficient for murine class II MHC molecules. CD4(+) T-cell-depleted peripheral blood mononuclear cells were also transferred to facilitate engraftment. The transduced cells exhibited long-term survival (up to 3 months post-transfer) and lethal GVHD was not observed. This favourable outcome was dependent upon the pre-transfer T-cell transduction and culture conditions, which influenced both the kinetics of engraftment and the development of GVHD. This approach should now permit human T-cell transduction protocols and genetic modifications to be evaluated in vivo, and it should also facilitate the development of human disease models that incorporate human T cells.

There is great interest in the potential of the human endocrine pancreas for regeneration by β-cell replication or neogenesis. Our aim was to explore this potential in adult human pancreases and in both islet and exocrine tissue transplanted into mice. The design was to examine pancreases obtained from cadaver donors, autopsies, and fresh surgical specimens and compare these findings with those obtained from islet and duct tissue grafted into the kidney. Islets and exocrine tissue were transplanted into normoglycemic ICR-SCID mice and studied 4 and 14 weeks later. β-Cell replication, as assessed by double staining for insulin and Ki67, was 0.22 ± 0.03% at 4 weeks and 0.13 ± 0.03% at 14 weeks. In contrast, no evidence of β-cell replication could be found in 11 cadaver donor and 10 autopsy pancreases. However, Ki67 staining of β-cells in frozen sections obtained at surgery was comparable to that found in transplanted islets. Evidence for neogenesis in transplanted pancreatic exocrine tissue was supported by finding β-cells within the duct epithelium and the presence of cells double stained for insulin and cytokeratin 19 (CK19). However, β-cells within the ducts never constituted more than 1% of the CK19-positive cells. With confocal microscopy, 7 of 12 examined cells expressed both markers, consistent with a neogeneic process. Mice with grafts containing islet or exocrine tissue were treated with various combinations of exendin-4, gastrin, and epidermal growth factor; none increased β-cell replication or stimulated neogenesis. In summary, human β-cells replicate at a low level in islets transplanted into mice and in surgical pancreatic frozen sections, but rarely in cadaver donor or autopsy pancreases. The absence of β-cell replication in many adult cadaver or autopsy pancreases could, in part, be an artifact of the postmortem state. Thus, it appears that adult human β-cells maintain a low level of turnover through replication and neogenesis.

Neonatal β cells do not secrete glucose-responsive insulin and are considered immature. We previously showed the transcription factor MAFA is key for the functional maturation of β cells, but the physiological regulators of this process are unknown. Here we show that postnatal rat β cells express thyroid hormone (TH) receptor isoforms and deiodinases in an age-dependent pattern as glucose responsiveness develops. In vivo neonatal triiodothyronine supplementation and TH inhibition, respectively, accelerated and delayed metabolic development. In vitro exposure of immature islets to triiodothyronine enhanced the expression of Mafa, the secretion of glucose-responsive insulin, and the proportion of responsive cells, all of which are effects that were abolished in the presence of dominant-negative Mafa. Using chromatin immunoprecipitation and electrophoretic mobility shift assay, we show that TH has a direct receptor-ligand interaction with the Mafa promoter and, using a luciferase reporter, that this interaction was functional. Thus, TH can be considered a physiological regulator of functional maturation of β cells via its induction of Mafa.

Poor bone quality, increased fracture risks, and impaired bone healing are orthopedic comorbidities of type 1 diabetes (T1DM). Standard osteogenic growth factor treatments are inadequate in fully rescuing retarded healing of traumatic T1DM long bone injuries where both periosteal and bone marrow niches are disrupted. We test the hypotheses that osteogenesis of bone marrow-derived stromal cells (BMSCs) and periosteum-derived cells (PDCs), two critical skeletal progenitors in long bone healing, are both impaired in T1DM and that they respond differentially to osteogenic bone morphogenetic proteins (BMPs) and/or insulin-like growth factor-1 (IGF-1) rescue.

Salmonella enterica serovar Typhi causes typhoid fever only in humans. Murine infection with S. Typhimurium is used as a typhoid model, but its relevance to human typhoid is limited. Non-obese diabetic-scid IL2rγnull mice engrafted with human hematopoietic stem cells (hu-SRC-SCID) are susceptible to lethal S. Typhi infection. In this study, we use a high-density S. Typhi transposon library in hu-SRC-SCID mice to identify virulence loci using transposon-directed insertion site sequencing (TraDIS). Vi capsule, lipopolysaccharide (LPS), and aromatic amino acid biosynthesis were essential for virulence, along with the siderophore salmochelin. However, in contrast to the murine S. Typhimurium model, neither the PhoPQ two-component system nor the SPI-2 pathogenicity island was required for lethal S. Typhi infection, nor was the CdtB typhoid toxin. These observations highlight major differences in the pathogenesis of typhoid and non-typhoidal Salmonella infections and demonstrate the utility of humanized mice for understanding the pathogenesis of a human-specific pathogen.

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.

The in vivo microenvironment of tissues provides myriad unique signals to cells. Thus, following isolation, many cell types change in culture, often preserving some but not all of their in vivo characteristics in culture. At least some of the in vivo microenvironment may be mimicked by providing specific cues to cultured cells. Here, we show that after isolation and during maintenance in culture, adherent rat islets reduce expression of key β-cell transcription factors necessary for β-cell function and that soluble pancreatic decellularized matrix (DCM) can enhance β-cell gene expression. Following chromatographic fractionation of pancreatic DCM, we performed proteomics to identify soluble factors that can maintain β-cell stability and function. We identified Apolipoprotein E (ApoE) as an extracellular protein that significantly increased the expression of key β-cell genes. The ApoE effect on beta cells was mediated at least in part through the JAK/STAT signaling pathway. Together, these results reveal a role for ApoE as an extracellular factor that can maintain the mature β-cell gene expression profile.

The transplantation of pancreatic islet cells could restore glycaemic control in patients with type-I diabetes. Microspheres for islet encapsulation have enabled long-term glycaemic control in diabetic rodent models; yet human patients transplanted with equivalent microsphere formulations have experienced only transient islet-graft function, owing to a vigorous foreign-body reaction (FBR), to pericapsular fibrotic overgrowth (PFO) and, in upright bipedal species, to the sedimentation of the microspheres within the peritoneal cavity. Here, we report the results of the testing, in non-human primate (NHP) models, of seven alginate formulations that were efficacious in rodents, including three that led to transient islet-graft function in clinical trials. Although one month post-implantation all formulations elicited significant FBR and PFO, three chemically modified, immune-modulating alginate formulations elicited reduced FBR. In conjunction with a minimally invasive transplantation technique into the bursa omentalis of NHPs, the most promising chemically modified alginate derivative (Z1-Y15) protected viable and glucose-responsive allogeneic islets for 4 months without the need for immunosuppression. Chemically modified alginate formulations may enable the long-term transplantation of islets for the correction of insulin deficiency.

Type 1 diabetes (T1D) is caused by the autoimmune destruction of pancreatic beta cells. Pluripotent stem cells can now be differentiated into beta cells, thus raising the prospect of a cell replacement therapy for T1D. However, autoimmunity would rapidly destroy newly transplanted beta cells. Using a genome-scale CRISPR screen in a mouse model for T1D, we show that deleting RNLS, a genome-wide association study candidate gene for T1D, made beta cells resistant to autoimmune killing. Structure-based modelling identified the U.S. Food and Drug Administration-approved drug pargyline as a potential RNLS inhibitor. Oral pargyline treatment protected transplanted beta cells in diabetic mice, thus leading to disease reversal. Furthermore, pargyline prevented or delayed diabetes onset in several mouse models for T1D. Our results identify RNLS as a modifier of beta cell vulnerability and as a potential therapeutic target to avert beta cell loss in T1D.

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.

Targeted drug delivery systems hold the remarkable potential to improve the therapeutic index of anticancer medications markedly. Here, we report a targeted delivery platform for cancer treatment using clathrin light chain (CLC)-conjugated drugs. We conjugated CLC to paclitaxel (PTX) through a glutaric anhydride at high efficiency. Labeled CLCs localized to 4T1 tumors implanted in mice, and conjugation of PTX to CLC enhanced its delivery to these tumors. Treatment of three different mouse models of cancer-melanoma, breast cancer, and lung cancer-with CLC-PTX resulted in significant growth inhibition of both the primary tumor and metastatic lesions, as compared to treatment with free PTX. CLC-PTX treatment caused a marked increase in apoptosis of tumor cells and reduction of tumor angiogenesis. Our data suggested HSP70 as a binding partner for CLC. Our study demonstrates that CLC-based drug-conjugates constitute a novel drug delivery platform that can augment the effects of chemotherapeutics in treating a variety of cancers. Moreover, conjugation of therapeutics with CLC may be used as means by which drugs are delivered specifically to primary tumors and metastatic lesions, thereby prolonging the survival of cancer patients.

The peripheral naïve T cell pool is comprised of a heterogeneous population of cells at various stages of development, which is a process that begins in the thymus and is completed after a post-thymic maturation phase in the periphery. One hallmark of naïve T cells in secondary lymphoid organs is their unique ability to produce TNF rapidly after activation and prior to acquiring other effector functions. To determine how maturation influences the licensing of naïve T cells to produce TNF, we compared cytokine profiles of CD4(+) and CD8(+) single positive (SP) thymocytes, recent thymic emigrants (RTEs) and mature-naïve (MN) T cells during TCR activation. SP thymocytes exhibited a poor ability to produce TNF when compared to splenic T cells despite expressing similar TCR levels and possessing comparable activation kinetics (upregulation of CD25 and CD69). Provision of optimal antigen presenting cells from the spleen did not fully enable SP thymocytes to produce TNF, suggesting an intrinsic defect in their ability to produce TNF efficiently. Using a thymocyte adoptive transfer model, we demonstrate that the ability of T cells to produce TNF increases progressively with time in the periphery as a function of their maturation state. RTEs that were identified in NG-BAC transgenic mice by the expression of GFP showed a significantly enhanced ability to express TNF relative to SP thymocytes but not to the extent of fully MN T cells. Together, these findings suggest that TNF expression by naïve T cells is regulated via a gradual licensing process that requires functional maturation in peripheral lymphoid organs.

Type 1 diabetes studies consistently generate data showing islet β-cell dysfunction and T cell-mediated anti-β-cell-specific autoimmunity. To explore the pathogenesis, we interrogated the β-cell transcriptomes from donors with and without type 1 diabetes using both bulk-sorted and single β-cells. Consistent with immunohistological studies, β-cells from donors with type 1 diabetes displayed increased Class I transcripts and associated mRNA species. These β-cells also expressed mRNA for Class II and Class II antigen presentation pathway components, but lacked the macrophage marker CD68. Immunohistological study of three independent cohorts of donors with recent-onset type 1 diabetes showed Class II protein and its transcriptional regulator Class II MHC trans-activator protein expressed by a subset of insulin+CD68- β-cells, specifically found in islets with lymphocytic infiltrates. β-Cell surface expression of HLA Class II was detected on a portion of CD45-insulin+ β-cells from donors with type 1 diabetes by immunofluorescence and flow cytometry. Our data demonstrate that pancreatic β-cells from donors with type 1 diabetes express Class II molecules on selected cells with other key genes in those pathways and inflammation-associated genes. β-Cell expression of Class II molecules suggests that β-cells may interact directly with islet-infiltrating CD4+ T cells and may play an immunopathogenic role.

The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in individuals with diabetes. Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically, but these approaches are limited by the adverse effects of immunosuppressive therapy over the lifetime of the recipient and the limited supply of donor tissue. The latter concern may be addressed by recently described glucose-responsive mature beta cells that are derived from human embryonic stem cells (referred to as SC-β cells), which may represent an unlimited source of human cells for pancreas replacement therapy. Strategies to address the immunosuppression concerns include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier. However, clinical implementation has been challenging because of host immune responses to the implant materials. Here we report the first long-term glycemic correction of a diabetic, immunocompetent animal model using human SC-β cells. SC-β cells were encapsulated with alginate derivatives capable of mitigating foreign-body responses in vivo and implanted into the intraperitoneal space of C57BL/6J mice treated with streptozotocin, which is an animal model for chemically induced type 1 diabetes. These implants induced glycemic correction without any immunosuppression until their removal at 174 d after implantation. Human C-peptide concentrations and in vivo glucose responsiveness demonstrated therapeutically relevant glycemic control. Implants retrieved after 174 d contained viable insulin-producing cells.

Congenital hyperinsulinism of infancy (CHI) can be caused by inactivating mutations in the gene encoding short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD), a ubiquitously expressed enzyme involved in fatty acid oxidation. The hypersecretion of insulin may be explained by a loss of interaction between SCHAD and glutamate dehydrogenase in the pancreatic β-cells. However, there is also a general accumulation of metabolites specific for the enzymatic defect in affected individuals. It remains to be explored whether hypoglycemia in SCHAD CHI can be uncoupled from the systemic effect on fatty acid oxidation. We therefore transplanted islets from global SCHAD knockout (SCHADKO) mice into mice with streptozotocin-induced diabetes. After transplantation, SCHADKO islet recipients exhibited significantly lower random and fasting blood glucose compared with mice transplanted with normal islets or nondiabetic, nontransplanted controls. Furthermore, intraperitoneal glucose tolerance was improved in animals receiving SCHADKO islets compared with those receiving normal islets. Graft β-cell proliferation and apoptosis rates were similar in the two transplantation groups. We conclude that hypoglycemia in SCHAD-CHI is islet cell-autonomous.

Host recognition and immune-mediated foreign body response to biomaterials can compromise the performance of implanted medical devices. To identify key cell and cytokine targets, here we perform in-depth systems analysis of innate and adaptive immune system responses to implanted biomaterials in rodents and non-human primates. While macrophages are indispensable to the fibrotic cascade, surprisingly neutrophils and complement are not. Macrophages, via CXCL13, lead to downstream B cell recruitment, which further potentiated fibrosis, as confirmed by B cell knockout and CXCL13 neutralization. Interestingly, colony stimulating factor-1 receptor (CSF1R) is significantly increased following implantation of multiple biomaterial classes: ceramic, polymer and hydrogel. Its inhibition, like macrophage depletion, leads to complete loss of fibrosis, but spares other macrophage functions such as wound healing, reactive oxygen species production and phagocytosis. Our results indicate that targeting CSF1R may allow for a more selective method of fibrosis inhibition, and improve biomaterial biocompatibility without the need for broad immunosuppression.

Many patients with type 1 diabetes (T1D) have residual β cells producing small amounts of C-peptide long after disease onset but develop an inadequate glucagon response to hypoglycemia following T1D diagnosis. The features of these residual β cells and α cells in the islet endocrine compartment are largely unknown, due to the difficulty of comprehensive investigation. By studying the T1D pancreas and isolated islets, we show that remnant β cells appeared to maintain several aspects of regulated insulin secretion. However, the function of T1D α cells was markedly reduced, and these cells had alterations in transcription factors constituting α and β cell identity. In the native pancreas and after placing the T1D islets into a non-autoimmune, normoglycemic in vivo environment, there was no evidence of α-to-β cell conversion. These results suggest an explanation for the disordered T1D counterregulatory glucagon response to hypoglycemia.

Hepatocytes represent an important target for gene therapy and editing of single-gene disorders. In α-1 antitrypsin (AAT) deficiency, one missense mutation results in impaired secretion of AAT. In most patients, lung damage occurs due to a lack of AAT-mediated protection of lung elastin from neutrophil elastase. In some patients, accumulation of misfolded PiZ mutant AAT protein triggers hepatocyte injury, leading to inflammation and cirrhosis. We hypothesized that correcting the Z mutant defect in hepatocytes would confer a selective advantage for repopulation of hepatocytes within an intact liver. A human PiZ allele was crossed onto an immune-deficient (NSG) strain to create a recipient strain (NSG-PiZ) for human hepatocyte xenotransplantation. Results indicate that NSG-PiZ recipients support heightened engraftment of normal human primary hepatocytes as compared with NSG recipients. This model can therefore be used to test hepatocyte cell therapies for AATD, but more broadly it serves as a simple, highly reproducible liver xenograft model. Finally, a promoterless adeno-associated virus (AAV) vector, expressing a wild-type AAT and a synthetic miRNA to silence the endogenous allele, was integrated into the albumin locus. This gene-editing approach leads to a selective advantage of edited hepatocytes, by silencing the mutant protein and augmenting normal AAT production, and improvement of the liver pathology.