DRAK2 is a serine/threonine kinase that is differentially expressed during lymphoid development. In vivo mouse models for multiple sclerosis and type 1 diabetes have shown that DRAK2-/- mice display diminished autoimmune susceptibility. Given that DRAK2 acts as a negative regulator of T cell activation, research was been geared toward the mechanism through which it alters autoimmunity. The work in this dissertation was proposed to investigate the mechanism by which DRAK2 alters T cell function and development.

Understanding the mechanisms that control T cell differentiation is crucial to develop novel therapeutic strategies to prevent autoimmune disease. Upon activation, T cells undergo metabolic reprogramming and different T cell subsets utilize selective metabolic pathways. The reason for this reprogramming is that unlike a naïve cell, activated T cells need to generate the necessary biomass and intermediates needed for rapid cell division. Hitherto, the regulating mechanisms promoting the switch from oxidative to glycolytic metabolism have remained elusive. We found that upon SEB stimulation, DRAK2 induces mitochondrial permeability transition pore activity, which allows T cell proliferation and subsequent glycolytic differentiation. This work provides important mechanistic insight into the transient regulation of the mitochondrial permeability transition pore and is the first to identify that this activity can be modulated during metabolic differentiation. Importantly, we show that loss of DRAK2 prevents the expansion of glycolytic cells and that appropriate signal strength, in the context of CD28 costimulation, is sufficient to overcome the characteristic survival defect. These data suggest that CD28 seems to be an important parallel pathway in terms of survival but not necessarily in terms of metabolism. Overall, this work examines the metabolic dysregulation that occurs following loss of DRAK2 in T cells and provides insight into how proliferation and glycolytic differentiation are initiated following activation.

Multiple Sclerosis (MS) is a chronic, autoimmune disease of the central nervous system (CNS), for which there is no cure. Deficits in neurological function occur as a result of widespread demyelination and axonal loss caused by infiltrating immune cells, mainly lymphocytes (T cells) and myeloid cells. Regulatory T cells (Tregs), characterized by expression of CD4 and FoxP3, are a specialized subset of T cells that are essential for the maintenance of T cell homeostasis and prevention of autoimmunity. Thus, dysregulation of Tregs has been linked to MS disease pathogenesis. Current therapies for MS are only focused on immunosuppression and fail to address the need for sustained remyelination and axonal protection. Transplantation of neural stem cells (NSCs) or neural precursor/ progenitor cells (NPCs) is a promising therapeutic strategy to treat conditions and diseases affecting white matter integrity, such as MS. NSCs and NPCs are a mixed population of self-renewing stem cells and cells that are poised to differentiate into the three types of neural cells found in the CNS; neurons, astrocytes, and myelin-producing oligodendrocytes. Early studies employing animal models of demyelination have demonstrated that surgical implantation of NSCs into the CNS reduces neuroinflammation and enhances remyelination. However, the mechanism by which these cells promote tissue repair remains poorly understood.

The focus of this dissertation is to elucidate the mechanisms by which NSCs promote remyelination following transplantation in murine models of MS. Viral infection has long been considered a potential triggering mechanism involved in immune-mediated demyelination, therefore viral models of demyelination are relevant for modeling human demyelinating diseases, such as MS. Intracranial inoculation of susceptible mice with the neurotropic JHM variant of mouse hepatitis virus (JHMV) leads to immune-mediated demyelination and axonopathy. We have demonstrated that intraspinal injection of syngeneic mouse NSCs into the CNS of JHMV-infected mice results in remyelination associated with axonal sparing due to cell replacement. In addition, JHMV-infected mice that received transplants of xenogeneic human NPCs also displayed sustained remyelination and decreased neuroinflammation, which was associated with an increase in CD4+CD25+FoxP3+Tregs. Importantly, recovery was not a result of cell replacement, hNPCs underwent xenograft rejection, rather due to immune modulation (Chapter 1). Chapter 1 is a re-print of a review article published in Developmental Dynamics that summarizes our recent findings transplanting mNPCs and hNPCs into the spinal cord of JHMV infected mice, and serves as an introduction to the dissertation. Immunization of mice with myelin oligodendrocyte glycoprotein is the prototypic murine model of MS, experimental autoimmune encephalomyelitis (EAE), which is characterized by ascending immune-mediated inflammation and demyelination of axonal tracks, as well as neuronal death resulting in motor deficits. In support of our previous findings, engraftment of syngeneic mNSCs facilitated remyelination in EAE mice with no effect upon the immune system. Transplantation of xenogeneic hNSCs into EAE mice resulted in dampened neuroinflammation and remyelination due to expansion of neural antigen-specific Tregs. Ablation of Tregs abrogated remyelination. Additionally, hNSCs promoted expansion of neural antigen-specific Tregs (hNSC-Tregs) in vitro, from the ‘exTreg’ pool, a population of thymically derived Tregs that lost FoxP3 expression in the periphery. hNSCs reinvigorated FoxP3 expression in ‘exTregs’ following co-culture with hNSCs, supporting the hypothesis that self-peptide/MHC is important for maintenance of FoxP3 expression and Treg function. hNSC-Tregs display a unique gene expression signature, upregulating molecules known to be involved in suppression of neuroinflammation, Dickkopf 3, and Transglutaminase 2 which facilitates oligodendrocyte progenitor cell differentiation to mature, myelin producing oligodendrocytes (Chapter 2). Utilizing two-photon microscopy of spinal cord explants, we discovered that Tregs localize to the sites of hNSC transplantion and interact with cells, most likely oligodendrocytes, that produce a component of the myelin sheath, proteolipid protein (Chapter 3). These findings support the concept that Tregs not only function in suppressing inflammatory responses, but also support their role as potentiators of tissue repair. Therefore, there has been an increasing interest utilizing Tregs as immunotherapies to treat autoimmune diseases. Therapies to promote expansion of Tregs and adoptive T cell therapy are currently under investigation in clinical trials for autoimmune diseases including, graft versus host disease and type I diabetes myelitis, although the ability of Tregs to modulate tissue repair is not well studied in the clinic (Chapter 4). Thus, hNSC expanded Tregs may provide new therapeutic strategies for treatment of autoimmune, neurodegenerative diseases, such as MS.

Development of functional adaptive and innate immune responses requires strict regulation of programmed cell death signaling pathways. These signaling pathways are essential for shaping and maintaining the immune system, and ensuring functional immune responses. Programmed death is required for clonal deletion of lymphocytes during an infection, and dysregulation results in defective clearance of autoreactive T cells and autoimmune disease.

Death Receptor (DR) pathways regulate apoptotic signaling and are essential for immune homeostasis and tolerance. Ligation of DR, CD95/Fas/Apo-1, triggers formation of a cytosolic complex known as the "death-inducing signaling complex" (DISC), which includes adaptor molecule FADD (Fas associated with death domain), Receptor Interacting Protein (RIP) Kinase 1, and caspase-8. Caspase 8 is required for extrinsic apoptosis upon DR ligation, while promoting clonal expansion in T cells following T cell receptor stimulation. In the absence of caspase 8, cells succumb to a programmed necrosis-like death process facilitated by the generation of RIPK1-RIPK3 "necrosomes". The aim of this dissertation is to elucidate the crosstalk between necroptosis and apoptosis in T lymphocytes in the context of caspase 8. By generating RIPK3-/- x FADDdd double mutant mice, we show in Chapter 2 that FADD, caspase 8 and RIP kinases are all essential for clonal expansion, contraction, and antiviral responses.

FADDdd T cells also display hyper-autophagy upon activation; thus, another aim of this dissertation is to uncover the role of autophagic signaling in T lymphocytes. Using mice genetically deficient in autophagy protein, Atg5, we demonstrate in Chapter 3 the differential roles for autophagy in naïve and proliferating T cells. Autophagy is required for clearing excess mitochondria in naïve T cells, while activated cells are capable of diluting mitochondria through rapid division. In Chapter 4, we characterize the platform(s) that promote recruitment of RIPK1-RIPK3 complex in T cells by identifying the kinetics, constituency, and subcellular localization of the necrosome. Understanding the paradigms of necroptosis and autophagy and defining the switch between apoptosis and necrosis will allow us to manipulate death pathways to modulate desired immune responses to various diseases and infections.

Multiple sclerosis (MS) is a chronic autoimmune demyelinating disease for which there is no cure. Compelling evidence from animal models of immune-mediated demyelination suggests transplanted neural precursor cells (NPCs) dampen neuroinflammation and promote remyelination, resulting in improved clinical outcome. However, much of the work evaluating NPC transplantation has been conducted using grafts from genetically identical donors or immune suppressed subjects. From a clinical perspective, NPCs are most likely to be genetically mismatched (allogeneic), and systemic immune suppression increases the risk of peripheral infection for the host. Therefore, when considering NPC transplantation for MS, the potential for engrafted cells to elicit an immune response, the consequences of engraftment in an environment with ongoing inflammation, and the susceptibility of NPCs to infection must be evaluated.

The focus of this dissertation is to elucidate mechanisms of immune rejection and repair following transplantation of NPCs in a virus-induced model of neuroinflammatory demyelination. Inoculation of susceptible mice with the neuroattenuated JHM variant of mouse hepatitis virus (MHV) induces acute encephalomyelitis followed by chronic immune-mediated demyelination that recapitulates histopathological and clinical features of MS. Using this well-accepted model, we demonstrate that allogeneic NPCs are antigenic and are rejected following transplantation. Rejection is correlated with expression of major histocompatibility complex (MHC) and co-stimulatory molecules by NPCs, as well as increased CD4+ and CD8+ T cell activity in vitro and in vivo (Chapter 2). Additionally, allogeneic NPCs are lysed by natural killer (NK) cells following binding of retinoic acid early inducible-1 (RAE-1) to the NK-activating receptor NKG2D. NK cells localize with allografted NPCs and NK cell infiltration is correlated with reduced graft survival. Furthermore, NPCs can be infected by MHV, which elicits destruction via NK cell-mediated mechanisms (Chapter 3). We provide evidence that CD4+ and CD8+ T cells participate in suppression of virus replication in MHV-infected NPCs via secretion of interferon-gamma (IFN-γ) and direct cytolysis (Chapter 4). Lastly, induced pluripotent stem cells (iPSCs) represent an unlimited source of NPCs that are genetically identical to the host. We demonstrate that NPCs derived from human iPSCs promote focal remyelination concomitant with suppressed CD4+ T cell infiltration in the CNS dependent on increased regulatory T cell (Treg) activity (Chapter 5).

Multiple sclerosis (MS) is a chronic, inflammatory disease characterized by focal demyelinated lesions in central nervous system (CNS) caused by infiltrating autoreactive T cells that target components of the myelin sheath. These autoimmune attacks result in demyelination and lead to axonal loss, which results in decreased neurological function. Patients suffering with MS can experience a wide range of symptoms including vision problems, muscle weakness, fatigue, and paralysis. Currently, more than 2.8 million people suffer from MS worldwide. Available therapeutics rely on disease modifying therapies (DMTs) which suppress the immune system to limit disease progression. Currently there are no available therapeutics for MS which restore or repair damage in the CNS caused by the immune system. Human Neural Stem Cell (hNSC) transplantation offers a promising approach to restoring function to patients by replacing or restoring damaged cells in the CNS. Recent studies using hNSC transplantation in mouse models of MS have revealed that despite being rejected, hNSC transplantation results in immune suppression and endogenous remyelination. However, it remains unclear how this remyelination response is generated. In Chapter 2, we present direct evidence that hNSC transplantation in an experimental autoimmune encephalomyelitis (EAE) mouse model of MS drives the expansion of Tregs. This increase in Tregs is correlated with an increase in local remyelination. Treg dependent remyelination occurs, even in the absence of cellular reconstitution by transplanted hNSCs. Chapter 3 explores the mechanism by which hNSCs increase the Treg population. Co-cultures of hNSCs with splenocytes revealed that hNSCs drive the conversion of T conventional (Tconv) cells to peripheral Tregs (pTregs) through presentation of self-antigens expressed by hNSCs. Generation of pTregs is not driven by direct interactions with hNSCs as thymically presented self-antigens are sufficient to drive pTreg generation in vitro and in vivo. Chapter 4 further explores the importance of self-antigen recognition and Treg generation by examining a population of cells that at one time expressed the master Treg transcription factor Foxp3, but no longer express Foxp3, known as ‘Ex-Foxp3’ cells. These studies demonstrate that Ex-Foxp3 cells are generated when autoreactive Tconv cells encounter thymically presented self-antigen in the periphery. Furthermore, Ex-Foxp3 cells are present in mice which lack Tregs refuting the paradigm that Ex-Foxp3 cells are the result of loss of function by Tregs. Finally, Ex-Foxp3 cells were demonstrated to contribute significantly to the generation of Tregs when co-cultured with hNSCs suggesting that Ex-Foxp3 cells are poised to generate Tregs when stimulated with self-antigen. The work presented here contributes strongly to our understanding of how the immune system interacts with self-antigens in the periphery as well as the demonstrating the therapeutic potential of Tregs as mediators of remyelination.