The endoplasmic reticulum (ER) is a critical regulator of cellular homeostasis, primarily responsible for handling calcium storage and signaling, lipid synthesis, and the proper glycosylation and folding of nascent transmembrane and secreted proteins. Numerous stimuli such as dysregulated oxidative stress, depletion of calcium stores, hypo- and hyperglycemia, hypoxia, inefficient cellular degradation pathways, and inflammation can disrupt the protein folding capacity of the ER, leading to a condition known as ER stress. The canonical unfolded protein response (UPR) is a three-pronged signaling axis charged with ameliorating ER stress, and is particularly important for the development of highly secretory tissues such as plasma cells, pancreatic acinar cells, and Paneth cells. Here we examine the requirement for the ER stress response transcription factor XBP1 in hematopoiesis, focusing specifically on granulocyte development. In this study we found that XBP1 is selectively required for the development of the eosinophil lineage, but not for other granulocyte lineages. Targeted hematopoietic ablation of XBP1 or its upstream activator IRE1α using Vav1-Cre conditional knockout mice resulted in complete loss of mature eosinophils and dramatic decrease in Lin-, Sca1-, CD34+, c-Kitlo, IL-5Rα+ eosinophil progenitors without altering neighboring hematopoietic lineages such as basophils and neutrophils. Myeloid and eosinophil progenitors selectively activated XBP1 without induction of parallel canonical ER stress signaling pathways. Through the use of mixed bone marrow chimeras, in vitro bone marrow-derived eosinophil cultures, and eosinophil-selective Cre mouse models, we demonstrated that XBP1 is required after eosinophil lineage commitment in a cell-intrinsic manner to sustain cell viability. RNA-sequencing and bioinformatic analyses of hematopoietic progenitors at different stages of eosinophil differentiation revealed that Xbp1 deficiency reduced the adaptive protein folding capacity of the ER. Upon eosinophil commitment, this vulnerability led to massive defects in post-translational maturation of key granule proteins required for survival, and these unresolvable structural defects fed back to suppress critical aspects of the transcriptional developmental program. Taken together, we present the first evidence that granulocyte subsets can be distinguished by their differential sensitivities to perturbations in XBP1-mediated secretory pathway functions. Furthermore, this work implicates the IRE1α/XBP1 signaling axis as a potential therapeutic target for eosinophil-mediated diseases. / Medical Sciences

Abstract Sialic acid acetylesterase (SIAE) removes 9-O-acetyl moieties from acetylated sialic acids. The B cell receptor (BCR) inhibitory receptor CD22 cannot bind 9-O-acetylated α2-6-linked sialic acid-containing ligands. Therefore, the removal of these moieties by SIAE is important for inhibition of signaling through the BCR by CD22. Previous studies on Siae-deficient mice revealed a role for SIAE in the maintenance of B cell tolerance. Deep sequencing of the SIAE exons in patients from several autoimmune cohorts revealed numerous single nucleotide polymorphisms (SNPs). Fluorometric enzymatic assays revealed that about half of these encode catalytically dead proteins while others have reduced activity. Coimmunoprecipitation studies indicated that mutant SIAE associates with wildtype SIAE in a multimer and acts in a dominant-interfering manner to decrease activity of the wildtype protein. We used monoclonal antibodies against different human SIAE epitopes in quantitative western blotting assays to assess whether variant-encoded SIAE proteins are misfolded and if activity and folding correlate. We found that most catalytically dead, disease-associated variants of SIAE are partially misfolded. Circular Dichroism studies were used to further investigate misfolding of mutant SIAE. Preliminary CD data indicate that mutant SIAE proteins are partially misfolded but retain significant structural integrity. These data are consistent with the finding that mutant SIAE proteins are able to multimerize with wildtype SIAE. We used FPLC to investigate the oligomeric structure of SIAE and found that SIAE exists as a dimer. We compared the results of the enzymatic assays of SIAE variants to the predictions generated by three commonly used algorithms; Polyphen-2, SIFT, and Provean. We found that the predictions of the algorithms were erroneous for between 11% (PolyPhen-2) and 28% (SIFT) of SIAE variants erroneous predictions for a given variant were often made by more than one algorithm, pointing to a need for non-computational predictive methods for the investigation of the effects of SNPs. / Medical Sciences

Approximately one-third of the world’s population is currently infected with Mycobacterium tuberculosis (Mtb), the bacillus that causes tuberculosis. Globally, it is the second leading cause of death by a single infectious agent. An effective vaccine is needed to stop this ongoing pandemic, but efforts to design one are hampered by our limited understanding of host immunity to this pathogen. CD8+ T cells are elicited during tuberculosis and are required for optimum host resistance. They produce cytokines such as IFN-γ and can directly lyse infected cells. During infection, the expansion and differentiation of effector CD8+ T cells is a dynamically regulated process that is influenced by the inflammatory milieu of the infected host. Currently, the signals governing CD8+ T cell responses during tuberculosis are not well characterized. Utilizing a mouse model of disease, we address the effects of key cytokines on CD8+ T cells, beginning with IL-12, type 1 interferons (IFN), and IL-27. All three of these cytokines are produced by innate immune cells during tuberculosis and have profound effects on host resistance. IL-12 proves most essential for robust CD8+ T cell expansion and IFN-γ production and also drives the terminal differentiation of short-lived effector cells. However, IL-12 is not acting alone, and type 1 IFN and IL-27 each have non-redundant roles supporting expansion in infected lungs. Thus, CD8+ T cells reflect the inflammatory environment of the host, responding in different degrees to each cytokine present. We next examine the role of IL-21, a cytokine produced by activated CD4+ T cells. In the absence of IL-21 signaling, CD8+ T cell expansion and effector functions are severely compromised. IL-21 is also essential to prevent CD8+ T cell exhaustion at later time points during disease. These observations are the first to describe an essential role for IL-21 in the host immune response to Mtb. Together, these studies establish IL-12 and IL-21 as essential regulators of CD8+ T cells during tuberculosis, and indicate type 1 IFN and IL-27 support expansion in the lungs. We believe these observations have implications for future immunotherapies and rational vaccine design. / Medical Sciences

CD48 is an adhesion and costimulatory molecule expressed constitutively on nearly all hematopoietic cells. Via interactions with its ligand CD2, it contributes to synapse organization between T cells and APCs, and can enhance TCR signaling; via interactions with its higher affinity ligand CD244, CD48 mediates interactions with T cells and NK cells. In addition to its roles in T cell activation and NK-mediated lysis, CD48 deficiency is associated with development of spontaneous lupus-like disease on a mixed 129 and B6 genetic background, but not on a mixed 129 and Balb/c background. Despite these cellular and clinical observations, the mechanisms by which CD48 might contribute to autoimmunity and tolerance in vivo were not well defined. In this thesis we examined the immunoregulatory roles of CD48 in spontaneous and induced models of autoimmune disease, using CD48 deficient mouse strains and anti-CD48 antibodies. We found that CD48 deficiency did not precipitate spontaneous lupus-like disease in mice on a pure B6 background, but resulted in a spontaneous increase in lymphocyte activation within both young and aged mice. This implicated neighboring immunoreceptor genes in development of lupus-like disease. CD48 deficient mice had modestly attenuated disease in a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), including reduced severity and accelerated resolution. At the peak of disease, CNS-infiltrating CD4+ T cells in CD48 deficient mice produced less GM-CSF, a cytokine that contributes to encephalitogenicity of T cells in EAE. When we examined CD48 expression in wild type mice during EAE, we found that CD48 expression was increased on activated CD4+ T cells, and that CD48++ CD4+ T cells were enriched for GM-CSF, IL-17A and IFNγ-producing cells. Administration of anti-CD48 antibody during EAE in wild type mice dramatically reduced the number of these cytokine-producing cells, and could significantly attenuate or even prevent disease. Anti-CD48-mediated attenuation of EAE was partially dependent on Fc receptors, suggesting a mechanism of depletion of activated CD48++ CD4+ T effectors. Collectively, our data support a critical role for CD48 in regulating the generation of activated lymphocytes and suggest that CD48 may be used to identify pathogenic self-reactive T cells in autoimmune disease. / Medical Sciences

Defined by the transcription factor Foxp3, regulatory T cells (Tregs) are a lineage of CD4+ T lymphocytes critical for the maintenance of immune homeostasis and tolerance. A lack of functional Tregs in both mice and humans leads to a fatal systemic autoimmune disease, underscoring their importance as mediators of tolerance to self antigen. One notable distinction between conventional T cells (Tconv) and Tregs is their differential control of the phosphatidylinositol 3-kinase (PI3K) pathway: PTEN, the primary negative regulator of PI3K, is expressed at high levels constitutively in Tregs, preventing the downstream activation of PI3K targets. Regulation of signaling through PI3K has previously been described to play an important role in the maintenance of homeostasis in Tconv, but has not been well characterized in Tregs. Here, we show that control of PI3K in Tregs is essential for the maintenance of in vivo lineage homeostasis and stability. Mice lacking expression of Pten specifically in Foxp3+ Tregs developed an autoimmune/lymphoproliferative disease characterized by excessive TH1 responses, B cell activation and renal failure. Diminished control of PI3K activity in Tregs led to reduced expression of the high-affinity interleukin-2 (IL-2) receptor subunit CD25 and accumulation of Foxp3+CD25- cells in vivo. The downregulation of CD25 expression on PTEN- deficient Tregs preceded the eventual loss of Foxp3 expression in these cells, representing the total destabilization of the Treg lineage and accumulation of “exFoxp3 cells” in vivo. Collectively, these data demonstrate that control of PI3K signaling by PTEN is critical to maintain in vivo Treg homeostasis, function and stability. / Medical Sciences

The gastrointestinal tract, home to a vast number of bacteria, requires finely-tuned regulatory and effector immune mechanisms to maintain homeostasis and tolerance. In a large-scale screen, we studied the impacts of single microbes on major immune populations, whole intestinal tissue homeostasis and metabolism. Bacteria interacted with the host at multiple levels including cytokine responses, accumulation of various T cells, alterations in composition of mononuclear phagocytes and induction of epithelial cell genes as measured by transcriptome analysis of whole intestinal tissue. Interestingly, taxonomically unrelated bacteria elicited similar immune phenotypes and metabolic effects. A more focused analysis of the induction of regulatory mechanisms revealed a microbiota-dependent, context-specific transcriptional control of Foxp3+ regulatory T cells and of IL17 producing T cells. These facets were both regulated by Rorγ, a transcription factor known for its antagonistic effects on Foxp3. Paradoxically, Rorγ expression induced by bacteria in colonic Foxp3+ regulatory T cells was necessary for function of these cells especially in the context of IL17 and IFNγ-mediated colitis. Overall, this large-scale screen provides a comprehensive study of how individual bacterial species shape many aspects of the host immunity and metabolism, and exemplifies a microbiota-dependent, context-specific mechanism that potentiates function in Foxp3+ regulatory T cells. / Medical Sciences

During priming, CD8+ T cells integrate a plethora of signals that affect their differentiation into subsets of CD8+ T cells with distinct migratory properties and functions. Given that CD8+ T cells exert their protective function via cell-cell contacts, the migratory patterns and spatial distribution of CD8+ T cell subsets induced by primary challenge are of critical importance to the host. Dendritic cells (DCs), as the primary initiators of these responses, play a pivotal role in shaping the size and differentiation status of CD8+ T cells that emerge. However, inadequate markers for CD8+ T cell subsets have hindered study of their lineage relationships, as well as their migratory behaviors. Here, we use a novel marker for identification of CD8+ T cell subsets to interrogate whether subsets of DCs skew CD8+ T cell fate decisions, the differentiation pattern of CD8+ T cell subsets, and the migratory behavior of these CD8+ T cell subsets. Within secondary lymphoid organs (SLOs), CD8+ T cells encounter subsets of DCs that may differentially impact subsequent T cell fate decisions. While distinct DC subsets were found to influence CD8+ T cell priming and subsequent differentiation in vitro, these differences were masked when priming occurred in vivo. This prompted us to delve deeper into how CD8+ T cell subsets are defined in vivo. Classically, memory CD8+ T cells are divided into two subsets: central memory T cells (TCM) and effector memory T cells (TEM). Based on the variable expression of the chemokine receptor, CX3CR1, we define TCM as CX3CR1- and TEM as CX3CR1high. Additionally, a previously undefined subset of T cells was identified that express intermediate levels of CX3CR1. Flow cytometric analysis of the subsets migrating through murine peripheral tissues in the memory phase established CX3CR1int cells as the dominant subset, thus these cells have been termed peripheral memory T cells (TPM). Lineage tracing of these three subsets established a uni-directional relationship where increasing levels of CX3CR1 marked further terminal differentiation: TCM (CX3CR1-) to TPM (CX3CR1int) to TEM (CX3CR1hi). The finding that TPM, and not TEM, migrated through peripheral tissues was intriguing because it contradicted previous studies suggesting that TEM had this migratory pattern. To resolve this contradiction, we visualized the migration of TEM precursors, CX3CR1hi effector T cells (TEff), by intravital multi-photon microscopy (IV-MPM) in real-time. Surprisingly, CX3CR1hi TEff adhered to and patrolled along the dermal endothelium of mice. Specifically, migration was enriched along arteriolar endothelium and tended to be against blood flow. Patrolling occurred for both CX3CR1hi TEff and TEM and was limited to CX3CR1hi CD8+ T cells and CX3CR1hi monocytes, but was not dependent on functional CX3CR1. Moreover, addition of cognate antigen (Ag) resulted in rapid stopping of Ag-specific T cells, suggesting that patrolling T cells scan arteriolar endothelium for cognate Ag. Together, these results challenge the paradigm that TEM function by migration through peripheral tissues and establish a new migratory behavior by TEM and their effector precursors that promotes intravascular scanning of arteriolar endothelium. / Medical Sciences

T cells provide antigen-specific immunity to pathogens, but are also capable of inducing destructive autoimmunity when responding to self antigens. In order to achieve full activation, T cells must receive two signals: one from the T cell antigen receptor, and a second from a costimulatory receptor, such as CD28. Expression of the ligands for CD28 activation is strongly induced on professional antigen presenting cells under conditions of inflammation or danger-sensing. Hence, CD28 costimulatory receptor acts as a gatekeeper to T cell activation and is essential in maintaining the balance between immunity and self-tolerance. CD28 function has been implicated in many animal models of T cell-dependent infection control and autoimmune disorders. Intensive study of CD28 signaling biology over more than two decades has elucidated key signaling pathways and transcriptional targets that help to explain the unique CD28-dependent functions in T cells. Some signaling pathways downstream of CD28 activation have been mapped to particular motifs within the CD28 cytoplasmic domain, although no consensus has emerged on the importance of these signaling motifs to CD28 function in vivo. This thesis describes the discovery of a novel motif within CD28 cytoplasmic domain that regulates receptor signal initiation through membrane binding and kinase recruitment, and discusses the new findings in light of existing literature. / Medical Sciences

Defective interfering particles of human parainfluenza virus 3 (HPIV3) were generated and/or amplified by serial undiluted passage of the standard virus. Analysis of the progeny virus titer at each cell passage revealed a cyclic pattern of virus production. Viruses produced from serial passages 8 and 9 interfered with replication of the standard virus. When cells were mixedly infected with standard virus and virus from either serial passages 5 or 8, three subgenomic RNA species, in addition to the standard virus genomic RNA, were detected in the progeny virions. Northern blot analysis revealed that all three subgenomic RNAs contained sequences from the 5$\sp\prime$-end of the standard virus genome. The data indicates that 5$\sp\prime$ defective interfering (DI) particles were generated and/or amplified during serial undiluted passage of HPIV3. Two independent HPIV3 persistent infections of LLC-MK$\sb2$ cells were established. One persistently infected culture was established by propagating the few cells which survived from an acute standard virus infection. The other persistently infected culture was readily established with virus from serial undiluted passage 8. This suggests that the DI particles present in serial passage 8 virus protected the cells from standard virus destruction. The two persistently infected cultures were propagated for nearly three years and the virus released from both cell lines at all times examined was noncytocidal. Subgenomic RNAs (putative DI particle genomes) were detected in virions released from both persistently infected cultures at various cell passages. No apparent changes in the size of the viral proteins were observed throughout the persistent infections. The DI particle-size genomes and the noncytocidal mutant virus are both likely to be involved in the maintenance of the persistent state. The degree of genetic change undergone by the viral genome during long-term persistence was examined. Nucleotide sequence analysis of the M gene and flanking regions of virus recovered after 147 cell passages (29 months of persistence) revealed that the viral genome did not undergo extensive genetic change. In the M protein coding region, only one conservative amino acid change was observed indicating that M protein mutation is not likely to be involved in the maintenance of the persistent infections. In contrast, the genomic 3$\sp\prime$-termini of viruses recovered after 146 cell passages was found to be highly mutated. The mutations observed were either U to C or A to G changes. Such exclusive substitution of one type of nucleotide for another is unlikely to be due to intrinsic polymerase errors but to an extrinsic biased hypermutational activity. An RNA unwinding/modifying activity that could give rise to the biased hypermutations is discussed.

Classification of cytokines into T helper type 1 (Th1) and T helper type 2 (Th2) has helped in the elucidation of the mechanisms of resistance or susceptibility to infections. HIV infection causes CD4$\sp+$ T cell dysfunction and depletion by indirect mechanisms; for example: inhibition of immunoregulatory cytokines. Interleukin (IL-10), the subject of this study, is secreted mostly by CD4$\sp+$ human Th2-like, but also by Th0,Th1-like, and by a proportion of CD8$\sp+$ T cell clones. HIV sero-positive patients exhibit depressed cell mediated immune responses, B cell hyperplasia, and hypergammaglobulinemia which may result from downregulation of Th1 and upregulation of Th2 class responses respectively. In this study, the expression of interferon$\gamma$ (IFN$\gamma$) and IL-10 which mediate Th1 and Th2 responses respectively, in unstimulated peripheral blood lymphocytes (PBLs) from HIV$\sp+$ patients were investigated. IL-10 expression as observed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis was significantly upregulated in patients with less than 400 CD4$\sp+$ T cells in comparison with HIV$\sp+$ patients with more than 400 CD4$\sp+$ T cells and normal controls. A semi-quantitative RT-PCR analysis of unstimulated PBLs further demonstrated IL-10 upregulation was inversely associated with IFN$\gamma$ downregulation in the same individuals. Similar results were observed as determined by measuring IL-10 and IFN$\gamma$ production in the supernatants of unstimulated PBLs by employing enzyme immunoassay techniques. These results suggest that HIV infected individuals express predominately Th2 type cytokines in their PBLs.