CD8αα intestinal intraepithelial lymphocytes derived from two thymic precursors seed the intestine in early life

TCRαβ CD4 CD8αα intestinal intraepithelial lymphocytes (CD8αα IEL) descend from thymic precursors. To better define this IEL precursor (IELp) population, we analyzed their maturation, localization, and emigration. Using rigorous lineage exclusion criteria, we defined two precursors among DN TCRβ thymocytes: a nascent PD-1 population and a T-bet population that accumulates with age. Both gave rise to intestinal CD8αα IEL upon adoptive transfer. In adult mice, PD-1 cells contained more self-reactive clones, localized to the cortex, and were dominant in S1PR1-dependent thymic egress. Gut homing α4β7 was already expressed by these IELp at a thymic stage. To understand the kinetics of CD8αα IEL seeding the intestine, we performed "timestamp" experiments: We crossed Cd4 with Rosa26 (stop-floxed tdTomato) mice. In these mice, tamoxifen or its metabolite 4-OHT permanently labels every CD4 expressing cell. As TCRαβ T cells (including CD8αα IEL) go through a CD4 CD8 stage during thymic development, a single dose of tamoxifen or 4-OHT will label thymic IEL precursors permanently, so that they can be tracked when seeding the gut. Our results indicate that these cells enter the intestine during a narrow time window in early life and that this influx is almost completely shut down by the age of 3 weeks. These data provide an important foundation for understanding the biology of this abundant population of barrier surface T cells

The genetically engineered minipig as a preclinical disease model for Neurofibromatosis type 1 syndrome

University of Minnesota Ph.D. dissertation. 2020. Major: Comparative and Molecular Biosciences. Advisor: David Largaespada. 1 computer file (PDF); 136 pages.Neurofibromatosis type 1 (NF1) syndrome is one of the most common inherited neurological disorders, affecting about one in every three thousand individuals. The disease typically manifests in childhood and can result in significant morbidity and a shortened lifespan. Despite decades of research, there is still no cure for NF1, and treatment is largely symptomatic. This is due in part to the multisystemic nature of the disease and a lack of representative, translational animal models. Murine models of NF1 mimic individual aspects of the syndrome, but none fully represents the complexity of disease seen in human patients. There is a need for new models of NF1 to complement the mouse and improve success in clinical trials. The objective of this dissertation was to fill this need by developing and characterizing the first genetically engineered NF1+/- minipig and determining its utility as a preclinical disease model for human NF1. Using targeted gene editing and somatic cell nuclear transfer cloning, we generated NF1+/- minipigs harboring a specific human disease allele associated with severe phenotypes in NF1 patients. We enrolled cohorts of NF1+/- minipigs and wild-type litter mate controls in a longitudinal phenotyping study and assessed for manifestations of NF1 syndrome. We performed gross assessments by physical examination, radiography, and magnetic resonance imaging over time. We also assessed the histological, molecular and biochemical defects associated with NF1 using tissues and primary cells isolated from lesions in NF1+/- minipigs. We evaluated several targeted therapies currently in clinical trials for NF1-associated neoplasia using pharmacokinetic and pharmacodynamic analyses in blood and clinically relevant tissues from NF1+/- minipigs. The results of these studies show that our NF1 minipig model offers a predictive preclinical disease model that will be crucial for developing and validating new NF1-targeted therapies as well as improved imaging and surgical modalities for early detection of nervous system tumors. Furthermore, the methods presented provide a blueprint for using minipig tissues to evaluate pharmacodynamics of new targeted therapies and using primary cells from minipigs to uncover novel targets and cellular interactions within the neurofibroma microenvironment

Spontaneous and Engineered Large Animal Models of Neurofibromatosis Type 1

Animal models are crucial to understanding human disease biology and developing new therapies. By far the most common animal used to investigate prevailing questions about human disease is the mouse. Mouse models are powerful tools for research as their small size, limited lifespan, and defined genetic background allow researchers to easily manipulate their genome and maintain large numbers of animals in general laboratory spaces. However, it is precisely these attributes that make them so different from humans and explains, in part, why these models do not accurately predict drug responses in human patients. This is particularly true of the neurofibromatoses (NFs), a group of genetic diseases that predispose individuals to tumors of the nervous system, the most common of which is Neurofibromatosis type 1 (NF1). Despite years of research, there are still many unanswered questions and few effective treatments for NF1. Genetically engineered mice have drastically improved our understanding of many aspects of NF1, but they do not exemplify the overall complexity of the disease and some findings do not translate well to humans due to differences in body size and physiology. Moreover, NF1 mouse models are heavily reliant on the Cre-Lox system, which does not accurately reflect the molecular mechanism of spontaneous loss of heterozygosity that accompanies human tumor development. Spontaneous and genetically engineered large animal models may provide a valuable supplement to rodent studies for NF1. Naturally occurring comparative models of disease are an attractive prospect because they occur on heterogeneous genetic backgrounds and are due to spontaneous rather than engineered mutations. The use of animals with naturally occurring disease has been effective for studying osteosarcoma, lymphoma, and diabetes. Spontaneous NF-like symptoms including neurofibromas and malignant peripheral nerve sheath tumors (MPNST) have been documented in several large animal species and share biological and clinical similarities with human NF1. These animals could provide additional insight into the complex biology of NF1 and potentially provide a platform for pre-clinical trials. Additionally, genetically engineered porcine models of NF1 have recently been developed and display a variety of clinical features similar to those seen in NF1 patients. Their large size and relatively long lifespan allow for longitudinal imaging studies and evaluation of innovative surgical techniques using human equipment. Greater genetic, anatomic, and physiologic similarities to humans enable the engineering of precise disease alleles found in human patients and make them ideal for preclinical pharmacokinetic and pharmacodynamic studies of small molecule, cellular, and gene therapies prior to clinical trials in patients. Comparative genomic studies between humans and animals with naturally occurring disease, as well as preclinical studies in large animal disease models, may help identify new targets for therapeutic intervention and expedite the translation of new therapies. In this review, we discuss new genetically engineered large animal models of NF1 and cases of spontaneous NF-like manifestations in large animals, with a special emphasis on how these comparative models could act as a crucial translational intermediary between specialized murine models and NF1 patients

Interferon-gamma drives programmed death-ligand 1 expression on islet β cells to limit T cell function during autoimmune diabetes

Type 1 diabetes is caused by autoreactive T cell-mediated beta cell destruction. Even though co-inhibitory receptor programmed death-1 (PD-1) restrains autoimmunity, the expression and regulation of its cognate ligands on beta cell remains unknown. Here, we interrogated beta cell-intrinsic programmed death ligand-1 (PD-L1) expression in mouse and human islets. We measured a significant increase in the level of PD-L1 surface expression and the frequency of PD-L1+beta cells as non-obese diabetic (NOD) mice aged and developed diabetes. Increased beta cell PD-L1 expression was dependent on T cell infiltration, as beta cells from Rag1-deficient mice lacked PD-L1. Using Rag1-deficient NOD mouse islets, we determined that IFN-gamma promotes beta cell PD-L1 expression. We performed analogous experiments using human samples, and found a significant increase in beta cell PD-L1 expression in type 1 diabetic samples compared to type 2 diabetic, autoantibody positive, and non-diabetic samples. Among type 1 diabetic samples, beta cell PD-L1 expression correlated with insulitis. In vitro experiments with human islets from nondiabetic individuals showed that IFN-gamma promoted beta cell PD-L1 expression. These results suggest that insulin-producing beta cells respond to pancreatic inflammation and IFN-gamma roduction by upregulating PD-L1 expression to limit self-reactive T cells.NIH [R01 AI106791, P01 AI35296, U24 U24-AI118635, T32DK007203]; Juvenile Diabetes Research Foundation [2-2011-662, 3-2014-215]; Regenerative Medicine of Minnesota RMM [11215 TR002]; Frieda Martha Kunze Fellowship; University of Minnesota Foundation [11724]; Network for Pancreatic Organ Donors with Diabetes (nPOD); Juvenile Diabetes Research Foundation International (JDRF)Open access journal.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

T cell progenitor therapy–facilitated thymopoiesis depends upon thymic input and continued thymic microenvironment interaction

Infusion of in vitro–derived T cell progenitor (proT) therapy with hematopoietic stem cell transplant aids the recovery of the thymus damaged by total body irradiation. To understand the interaction between proTs and the thymic microenvironment, WT mice were lethally irradiated and given T cell–deficient (Rag1-/-) marrow with WT in vitro–generated proTs, limiting mature T cell development to infused proTs. ProTs within the host thymus led to a significant increase in thymic epithelial cells (TECs) by day 21 after transplant, increasing actively cycling TECs. Upon thymus egress (day 28), proT TEC effects were lost, suggesting that continued signaling from proTs is required to sustain TEC cycling and cellularity. Thymocytes increased significantly by day 21, followed by a significant improvement in mature T cell numbers in the periphery by day 35. This protective surge was temporary, receding by day 60. Double-negative 2 (DN2) proTs selectively increased thymocyte number, while DN3 proTs preferentially increased TECs and T cells in the spleen that persisted at day 60. These findings highlight the importance of the interaction between proTs and TECs in the proliferation and survival of TECs and that the maturation stage of proTs has unique effects on thymopoiesis and peripheral T cell recovery

T cell progenitor therapy–facilitated thymopoiesis depends upon thymic input and continued thymic microenvironment interaction

Infusion of in vitro–derived T cell progenitor (proT) therapy with hematopoietic stem cell transplant aids the recovery of the thymus damaged by total body irradiation. To understand the interaction between proTs and the thymic microenvironment, WT mice were lethally irradiated and given T cell–deficient (Rag1-/-) marrow with WT in vitro–generated proTs, limiting mature T cell development to infused proTs. ProTs within the host thymus led to a significant increase in thymic epithelial cells (TECs) by day 21 after transplant, increasing actively cycling TECs. Upon thymus egress (day 28), proT TEC effects were lost, suggesting that continued signaling from proTs is required to sustain TEC cycling and cellularity. Thymocytes increased significantly by day 21, followed by a significant improvement in mature T cell numbers in the periphery by day 35. This protective surge was temporary, receding by day 60. Double-negative 2 (DN2) proTs selectively increased thymocyte number, while DN3 proTs preferentially increased TECs and T cells in the spleen that persisted at day 60. These findings highlight the importance of the interaction between proTs and TECs in the proliferation and survival of TECs and that the maturation stage of proTs has unique effects on thymopoiesis and peripheral T cell recovery