Browsing by Subject "Health Sciences, Immunology"

Concurrent with the global escalation of the AIDS pandemic, cryptococcal infections are increasing and are of significant medical importance. Although improvements in antifungal therapy have advanced the treatment of cryptococcosis, the mortality rate is approximately 12% in medically advanced countries, and approaches 50% in less developed regions. Additionally, C. neoformans can cause infection in seemingly healthy individuals, elevating its status as a primary human pathogen. Although numerous studies have examined virulence properties, less is understood regarding host immune factors in the lungs during early stages of fungal infection. In the present thesis studies, I examined the roles played by pulmonary surfactant proteins in response to C. neoformans in vitro and in vivo. We demonstrate that SP-D, but not SP-A, binds to the yeast and increases phagocytosis of poorly encapsulated yeast cells by macrophages, yet concomitantly protects the pathogenic microbes from macrophage-mediated defense mechanisms. Furthermore, we show that SP-D functions as risk factor in vivo by protecting the yeast cells against oxidant species and thus facilitating disease progression. The results of these studies provide a new paradigm on the role played by surfactant protein D during host responses to C. neoformans and, consequently, impart insight into potential future treatment strategies for cryptococcosis.

Mast cells, best known for their pathologic role in allergy, have recently been shown to have key roles in the initiation of adaptive immune responses. These cells are located throughout the body just beneath barriers separating host from environment, possess multiple pathogen recognition systems, and store large quantities of fully active inflammatory mediators. These key features make them uniquely situated to act as sentinels of immunity, releasing the very earliest alarm signals when a pathogen is present. As a testament to the importance of these cells, mast cell-deficient mice have suboptimal immune responses, and mast cell activators can act as potent adjuvants for experimental immunizations. Specifically, mast cells have been shown to enhance the number of naive lymphocytes in infection site-draining lymph nodes, and to encourage the migration of dendritic cells to responding lymph nodes.

Although infections usually occur at peripheral sites, adaptive immune responses are initiated in distant lymph nodes. Despite the distance, signals from the site of infection result in dramatic, rapid reorganization of the node, including massive recruitment of naive lymphocytes from the circulation and extensive vascular restructuring to accommodate the increase in size. How such signals reach the lymph node is not well understood.

When mast cells degranulate, in addition to releasing soluble mediators such as histamine, they expel large, stable, insoluble particles composed primarily of heparin and cationic proteins. The work presented herein demonstrates that these particles act as extracellular chaperones for inflammatory mediators, protecting them from dilution into the interstitial space, degradation, and interaction with non-target host cells and molecules. The data show clearly that mast cells release such particles, that they are highly stable, that they contain tumor necrosis factor (a critically important immunomodulator), and that they can traffic from peripheral sites to draining lymph nodes via lymphatic vessels. Furthermore, extensive biochemical characterization of purified mast cell-derived particles was performed. Finally, evidence is presented that such particles can elicit lymph node enlargement, an infection-associated phenomenon that favors the development of adaptive immunity, by delivering peripheral TNF to draining lymph nodes.

This signaling concept, that particles may chaperone signals between distant sites, also has important implications for adjuvant design. The evidence presented here shows that encapsulation of TNF into synthetic particles similar to mast cell-derived particles greatly enhances its potency for eliciting lymph node enlargement, an indication that adaptive immunity may be improved. This delivery system should ensure that more adjuvant arrives in the draining lymph node intact, where it would lead to changes favorable to the development of the immune response. Such a system would also facilitate the delivery of multi-component adjuvants that would act synergistically at the level of the lymph node when gradually released from microparticle carriers. An additional advantage of microparticle encapsulation is that vaccine formulations of this type may require much lower doses of expensive antigen and adjuvants.

The delivery of inflammatory mediators to lymph nodes during immune responses may be an important general feature of host defense. Although the action of mediators of peripheral origin on draining lymph nodes has been described before, this is the first demonstration of a specific adaptation to deliver such mediators. Not only is the characterization of mast cell-derived particles important to basic immunology, but mimicking this adaptation may also lead to improved therapeutics.

Thymus atrophy is highly inducible by stress and prolonged thymus atrophy can contribute to T cell deficiency or inhibit immune recovery after acute peripheral T cell depletion. Little is known regarding the mechanisms driving thymic involution or thymic reconstitution after acute stress. Leptin deficiency in mice results in chronic thymic atrophy, suppressed cell-mediated immunity, and decreased numbers of total lymphocytes, suggesting a role for leptin in regulating thymopoiesis and overall immune homeostasis. Exogenous leptin administration during stress has been shown to protect against thymic damage, yet the mechanisms governing these thymostimulatory effects are currently undefined. Studies herein define the impact of endotoxin-induced thymic damage in the stromal and lymphoid compartment of the thymus and systemic glucocorticoid and cytokine responses in the animal. We report here the novel finding that leptin receptor expression is restricted to medullary thymic epithelial cells in the normal thymus. Using a model of endotoxin-induced acute thymic involution and recovery, we have demonstrated a role for the metabolic hormone leptin in protection of medullary thymic epithelial cells from acute endotoxin-induced damage. We also demonstrated that systemic leptin treatment decreased endotoxin-induced apoptosis of double positive thymocytes and promoted proliferation of double negative thymocytes in vivo through a leptin receptor isoform b-specific mechanism. Leptin treatment increased thymic expression of IL-7, an important soluble thymocyte growth factor produced by medullary thymic epithelial cells. We also found leptin to inhibit systemic glucocorticoid and pro-inflammatory cytokine responses. Using leptin-deficient and leptin receptor-deficient mice in our stress model, we found that endotoxin-induced thymic atrophy was exacerbated in the absence of leptin, despite an inability to mount a proper pro-inflammatory cytokine response. Together, these data support a model in which leptin can function to protect the thymus gland from stress-induced acute damage in part by reduction of systemic corticosteroid and pro-inflammatory cytokine responses, and intrathymically through a mechanism orchestrated by medullary thymic epithelial cells and their soluble mediators (e.g. IL-7). Taken together, these studies suggest a physiological role for leptin signaling in the thymus for maintaining healthy thymic epithelium and promoting thymopoiesis, which is revealed when thymus homeostasis is perturbed by stress.

Extracellular GRP94 can elicit both innate and adaptive immune responses by interacting with endocytic and signaling receptors on professional antigen presenting cells (pAPCs). CD91 was the first receptor proposed to facilitate GRP94-mediated immune responses. Using a GRP94 affinity matrix, a CD91 fragment was isolated from the detergent-solubilized membranes of a pAPC cell line. It was then demonstrated that CD91 ligands could inhibit GRP94-mediated peptide cross-presentation, suggesting that CD91 played a critical role in this process. While these studies implied that CD91 could function as a GRP94 endocytic receptor, later works suggested that CD91 may not recognize GRP94 at the cell surface. These opposing observations have lead to a significant controversy surrounding the identity of CD91 as an endocytic receptor for GRP94. Because the ability of CD91 to directly mediate GRP94 surface binding and uptake has not been established, the studies included in this dissertation have focused on evaluating the ability of CD91 to facilitate three processes that are necessary for GRP94-mediated peptide cross-presentation: surface binding, internalization, and processing.

These studies utilized a recombinantly-expressed N-terminal domain of GRP94 (GRP94.NTD), which was previously shown to have nearly identical biological activity to full length GRP94. The ability of CD91 to directly bind and internalize GRP94.NTD was examined using murine embryonic fibroblast (MEF) cell lines whose expression of CD91 was either reduced via siRNA, or eliminated by genetic disruption of the CD91 locus. Binding competition experiments were also conducted. Together, these studies reveal that CD91 does not directly interact with GRP94 at the cell surface. The ability of CD91 to directly facilitate GRP94 internalization was examined using various internalization and internalization competition assays. These studies demonstrated that GRP94.NTD and the CD91 ligand RAP were internalized through spatially and kinetically distinct pathways, that CD91 was not necessary for GRP94.NTD internalization, and that RAP did not inhibit GRP94 endocytosis. Together, these studies strongly suggest that CD91 does not directly facilitate GRP94 internalization. When these studies were extended to DC2.4 mouse dendritic cells, the CD91 ligand RAP reduced GRP94.NTD internalization/process by ~15%. This suggests that CD91 may indirectly facilitate GRP94 internalization in pAPC cell lines. Lastly, cross-presentation studies were utilized to examine the ability of various CD91 ligands to influence GRP94.NTD-mediated peptide cross-presentation through a post-uptake mechanism using the DC2.4/OT-1 system. Although it was discovered that DC2.4 cells can internalize and process GRP94.NTD/peptide complexes through fluid-phase endocytosis, CD91 ligands did not significantly inhibit GRP94-mediated peptide cross-presentation by DC2.4 cells. These studies demonstrate that CD91 does not play a primary role in GRP94-mediated peptide cross-presentation.

In the course of these studies, cell surface heparan sulfate proteoglycans (HSPGs) were identified as novel cell surface binding sites for GRP94.NTD on MEF cells. This conclusion was established using three distinct experimental approaches. GRP94.NTD surface binding was significantly decreased following heparin pre-treatment, following incubation with the sulfation inhibitor sodium chlorate, and following digestion with extracellular heparinase II. Conversely, these treatments did not significantly influence GRP94.NTD binding to RAW264.7 mouse macrophage-like cells. This suggested that GRP94.NTD-HSPG cell surface interactions may require the expression of a specific type of cell surface HSPG that is not expressed by RAW264.7 cells. However, additional studies strongly suggested that GRP94.NTD-HSPG cell surface interactions were mediated by the heparan sulfate-containing side chains rather than the presence of a specific cell surface HSPG core protein.

This dissertation focuses on the critical re-examination of CD91 functions in GRP94 surface binding, uptake, and cross-presentation. Together, these results clarify conflicting data on CD91 function in GRP94 surface binding and endocytosis. This dissertation also describes the identification of cell surface HSPGs as GRP94 binding sites on MEF cells. These studies extend the diversity of surface receptors that recognize of GRP94, and suggest that cell surface HSPG-dependent interactions may contribute to the biology of GRP94-elicited immune responses.

The receptor-recognized form of α₂-macroglobulin (α₂M*) targets antigens (Ag) to professional Ag-presenting cells (APCs) for rapid internalization, processing, and presentation. When employed as an Ag delivery vehicle, α₂M* amplifies major histocompatibility complex (MHC) class II presentation as demonstrated by increased antibody (Ab) titers. Recent evidence, however, suggests that α₂M*-encapsulation may also enhance Ag-specific cytotoxic T lymphocyte (CTL) immunity. In these studies, we demonstrate that α₂M*-delivered Ag (ovalbumin, OVA) enhances the production of specific in vitro and in vivo CTL responses.

Murine splenocytes expressing a transgenic T cell receptor (TCR) specific for CTL peptide OVA_257-264 (SIINFEKL) demonstrated up to 25-fold greater IFN-γ and IL-2 secretion when treated in vitro with α₂M*-OVA compared to soluble OVA. The frequency of IFN-γ -producing cells was increased ~15-fold as measured by ELISPOT. Expansion of the OVA-specific CD8+ T cells, as assayed by tetramer binding and [3H]thymidine incorporation, and cell-mediated cytotoxicity, as determined by a flow cytometric assay, were also significantly enhanced by α₂M*-OVA. Furthermore, CTL responses were observed at Ag doses tenfold lower than those required with OVA alone.

We also observed enhanced humoral and CTL responses by naïve mice following intradermal immunization with α₂M*-OVA. These α₂M*-OVA-immunized mice displayed increased protection against a subcutaneously implanted OVA-expressing tumor, as demonstrated by delayed tumor growth and prolonged animal survival. The anti-tumor response observed with α₂M*-mediated Ag delivery was comparable to that of an accepted vaccine adjuvant (CpG 1826) and appeared superior to a cell-based vaccine technique.

To further understand the mechanism underlying this enhanced CTL immunity, the subsets of professional APCs capable of cross-presenting α₂M*-encapsulated Ag were investigated. Although both dendritic cells (DCs) and macrophages appear to stimulate some degree of cross-priming in response to α₂M*-encapsulated Ag, CD8+CD4- and CD8-CD4+ DCs appear to do so with the greatest efficiency. The implications of this finding to the ongoing debate regarding the relative contributions of APC subsets to Ag cross-presentation and the determinants of which cells cross-present with high efficiency are discussed.

These observations demonstrate that α₂M*-mediated Ag delivery promotes cross-presentation resulting in enhanced Ag-specific CTL immunity. Considered in the context of previous work, these results support α₂M* as an effective Ag delivery system that may be particularly useful for vaccines based on weakly immunogenic subunits or requiring dose sparing.

Normally, neutrophil pools are maintained by "steady-state" granulopoiesis. Infections and inflammation, however, trigger neutrophilias that are supported by a hematopoietic program of accelerated granulopoiesis known as "emergency" granulopoiesis. Steady-state and emergency granulopoiesis are thought to depend on distinct members of the CCAAT enhancer binding protein (C/EBP) family of transcription factors, yet the extracellular cues that determine these developmental pathways are unclear. I hypothesize that inflammation elicits IL-1 which acts directly on hematopoietic progenitor cells for the induction of emergency granulopoiesis. Indeed, IL-1RI-/- mice fail to mount reactive neutrophilias in response to adjuvant-induced inflammation. Analysis of this specific impairment revealed an unanticipated role for IL-1RI in supporting increased proliferation by granulocyte/macrophage progenitors (GMP) and, surprisingly, more primitive multipotent progenitors (MPP) and hematopoietic stem cells (HSC). Whereas IL-1 drives HSC proliferation directly in vitro, inflammation induces comparable rates of proliferation in IL-1RI deficient and -sufficient HSC, MPP, and GMP in mixed chimeric mice. Thus, IL-1RI signals play a necessary, but indirect role in the support of alum-induced neutrophilias by expanding both pluripotent and myeloid progenitor compartments to accelerate granulopoiesis.

The lack of alum-induced neutrophilia in IL-1RI-/- mice is due to defective mobilization of bone marrow (BM) neutrophils and impaired proliferation of hematopoietic stem and progenitor cells (HSPC). Coincident defects in neutrophil mobilization and HSPC proliferation suggest that the trigger for emergency granulopoiesis might be the exhaustion of neutrophil compartments rather than inflammatory inductions of growth factors. Consistent with this hypothesis, non-inflammatory reductions in BM neutrophil numbers elicit granulopoietic responses similar to those induced by adjuvant. Alum mobilizes BM neutrophils via G-CSF, but increased HSPC proliferation results from a density-dependent mechanism that is only partially dependent on G-CSF. Notably, C/EBPβ, thought to be necessary for enhanced generative capacity of BM, is dispensable for increased proliferation of HSPC, but plays a role in the terminal differentiation of neutrophils. These observations indicate that the draining of BM neutrophil pools is sufficient to activate a latent, homeostatic mechanism of accelerated granulopoiesis. I propose a common model for the regulation of neutrophil production that explains both steady-state and emergency granulopoiesis through negative feedback.

The E-protein transcription factors E2A and HEB regulate various cell processes during the development of B and T lymphocytes, including cell differentiation, lineage commitment, recombination of immune receptor genes, proliferation, and survival. B cell development is dependent on E2A from the earliest stages whereas T cell development relies on the cooperative efforts of both E2A and HEB. Established work demonstrates that the timing and dosage of E-protein expression is critical for mediating these diverse functions. The goal of this dissertation is to develop and utilize new genetic tools to manipulate the timing and dosage of E2A and HEB expression in order to enhance our understanding of E-protein function. Here we develop two new mouse models to identify novel lineage and stage specific roles of E-proteins during B lineage commitment and thymocyte selection.

First, we have generated an E2A inducible mouse model to allow reversible regulation of E2A function and precise timing of induction at the protein level. This system was created by inserting a tamoxifen responsive region of the estrogen receptor ligand binding domain (ER) at the carboxyl end of the tcfe2a gene, encoding E2A, to generate E2AER fusion proteins. To our knowledge, the ER fusion system has not yet been tested from an endogenous locus in live animals. Using the E2AER system, we have demonstrated rapidly induced E2AER activity upon tamoxifen treatment that is capable of supporting B cell development in an ex vivo culture system. In addition to characterizing the kinetics and reversibility of this inducible system, we have utilized tamoxifen treatment of E2AER B cell progenitors to identify potential novel E2A target genes driving B lineage commitment.

Second, we have analyzed E-protein function during the double positive (DP) stage of alpha beta T cell development by using a Cre-loxp conditional deletion system. Here, E-protein dosage was manipulated by removal of both E2A and HEB, and the timing of deletion was controlled by using a CD4Cre transgene. During development, survival through the DP stage and initiation of differentiation to the subsequent single positive (SP) stage for generation mature alpha beta T cells is dependent on the production of a functional alpha beta T cell receptor (TCR). The mechanism that maintains cells at the DP stage prior to expression of a mature TCR remains unclear. In this study, we have shown that E2A and HEB together are required to maintain DP fate and regulate the transition to the SP stage. Loss of E2A and HEB in DP thymocytes was sufficient to trigger DP to SP differentiation, even in the absence of a TCR. Deletion of E2A and HEB allowed cells to bypass the requirement for a TCR-mediated positive selection signal. These findings identify E2A and HEB as key regulators enforcing thymocyte positive selection to ensure maturing T cells express a functional receptor.

LAT (linker for activation of T cells) is an important transmembrane adaptor protein in TCR-mediated signaling. Upon TCR engagement, LAT associates with multiple proteins which allows for the activation of downstream signaling pathways. The interaction between LAT with phospholipase C (PLC-gamma1) is especially critical for T cell receptor (TCR)-mediated Ca2+ signaling and MAPK activation. Knock-in mice harboring a mutation at the PLC-gamma1 binding site (Y136) of LAT develop a severe lymphoproliferative syndrome. These mice have defective thymic development and selection and lack natural regulatory T cells, implicating a breakdown of both central and peripheral tolerance. The phenotype observed in LAT-/- mice is even more severe.T cells are absent in the periphery of these mice due to a complete block in thymocyte development at the DN3 stage thereby making it difficult to study the physiological role of LAT in the activation and function of mature T cells. In order to bypass the developmental defects exhibited by LAT-/- and LATY136F mice, we developed conditional knock-in lines in which only a nonfunctional (ERCreLATf/-) or a LATY136F-mutated allele (ERCreLATf/m) of LAT is expressed in mature T cells after deletion of the wildtype LAT allele.

Analysis of ERCreLATf/m T cells after LAT deletion indicated that the interaction between LAT and PLC-gamma1 plays an important role in TCR-mediated signaling, proliferation, and IL-2 production. Furthermore, the deletion of LAT induced the development of the LATY136F lymphoproliferative syndrome in these mice. Although Foxp3+ natural Treg cells were present in these mice after deletion, they were unable to suppress the proliferation of conventional T cells. Our data indicated that the binding of LAT to PLC-gamma1 is essential for the suppressive function of CD4+CD25+ regulatory T cells.

We have also performed studies using ERCreLATf/- T cells to demonstrate that total LAT deficiency reduced the expression of Foxp3, CTLA4, and CD25 in peripheral Treg cells. Interestingly, mice with LAT deleted in peripheral T cells developed a lymphoproliferative syndrome similar to that observed in LATY136F mice although the disease caused by the LATY136F mutation was more severe. These data implicate LAT in both the positive and the negative regulation of mature T cells. Moreover, our findings indicate that LAT is essential in the maintenance of the regulatory T cell profile in the periphery, thereby aiding in the prevention of lymphoproliferative autoimmune disease.

Several HIV-1 neutralizing antibodies (e.g. 2F5, 4E10) have been shown to react with self-antigens, suggesting that effective humoral responses to HIV-1 may be constrained by the tolerization of HIV-reactive B cells that also recognize self-antigens. I have tracked the development of 2F5-like HIV-1 gp41 membrane proximal external region (MPER)-reactive B cells throughout ontogeny using B-cell tetramer reagents. In BL/6 mice, MPER-binding populations are lost during normal B-cell development and immunization with HIV-1 MPER antigen does not elicit robust humoral responses. I have identified Kynureninase as a self-antigen that is recognized by 2F5 antibody and, therefore, is a molecule that could mediate the developmental loss of B cells reactive to an epitope shared by HIV gp41 and Kynureninase. To recover these MPER-reactive cells, I describe and characterize a stromal-cell independent culture system that efficiently supports pro-B cell to IgM+ B-cell development with near normal levels of IgH and Igkappa diversity. B-cell development in vitro closely follows the patterns of development in vivo with culture derived (CD) B cells demonstrating characteristic patterns of surface antigen expression and gene activation. Immature and transitional B-cell compartments are reduced, due to the induction of tolerance, in the bone marrow of 3H9 IgH knockin mice ; however, cultures of 3H9 IgH knockin pro-B cells yields high frequencies of "forbidden", autoreactive IgM+ B cells. Furthermore, serum IgG autoantibody exceeded that present in autoimmune, C4-/- animals following the reconstitution of RAG-1-/- mice with IgM+ CD cells derived from BL/6 mice. I show that HIV-1 MPER-reactive B cells are recovered from both BL/6 and 2F5 IgH knockin bone marrow using this in vitro culture system. RAG-1-/- mice reconstituted with these culture-derived B and T cells generate strong germinal center and antibody responses to HIV-1 MPER antigens. These data demonstrate that the humoral immune response to this HIV-1 gp41 MPER antigen can be restored in mice when the constraints of B-cell tolerance have been relaxed.

Vaccinia virus (VV) is the most thoroughly studied member of the Poxviridae family and the vaccine used to achieve the only successful eradication of a human disease. Over the years, it has proven itself as a useful tool for the study of antiviral immunity, vaccine development, and potentially cancer immunotherapy. VV is capable of eliciting a robust immune response; however the mechanisms by which VV accomplishes this task remain unknown. The overall goal of this thesis project is to determine how VV activates the innate immune system, and how this activation contributes to viral clearance in vivo. We determined that VV or VV-DNA activated the TLR8-MyD88 pathway in plasmacytoid dendritic cells (pDC), resulting in the production of type I interferons (IFN). We also demonstrated that TLR8-mediated production of type I IFN by pDC was crucial to efficient VV control and clearance in vivo. Moreover, we identified the polyA- and polyT-rich sequences in VV-DNA was the possible motif recognize by TLR8. Type I IFN, known for ability to establish the "antiviral state", are also critical mediators of NK cell activation. In the setting of VV infection, we demonstrated that direct action of type I IFN on NK cells, but not accessory cells such as DC, was necessary for NK cell activation in vivo. We further demonstrated that type I IFN-dependent activation of NK cells was required for optimal VV clearance in vivo. Given the importance of NK cells in anti-VV innate immunity, we next examined what role the TLR2-MyD88 pathway, critical for activation of cDC, played in the activation of NK cells. NK cells from TLR2-/- or MyD88-/- mice displayed a reduction in activation and cytolytic function, and this defect was independent of pro-inflammatory cytokine signaling. We were able to demonstrate that direct TLR2 signaling on NK cells was required for their optimal activation and function in response to VV infection. Moreover, we were able to demonstrate that TLR2-MyD88 signaling resulted in the activation of the PI3K-ERK pathway, which was necessary for NK cell cytotoxicity. In addition, we identified the NKG2D pathway as critical for efficient NK cell activation and function in response to VV infection, independent of the TLR2 pathway. Both the NKG2D and TLR2 pathways were crucial for optimal VV clearance and control in vivo. Collectively, this project illuminates the roles and mechanisms of the innate immune system in the control of VV in vivo.

The role of CD4 T cell help in primary and secondary CD8 T cell responses to infectious pathogens remains incompletely defined. The primary CD8 T response to infections was initially thought to be largely independent of CD4 T cells, but it is not clear why some primary, pathogen-specific CD8 T cell responses are CD4 T cell-dependent. Furthermore, although the generation of functional memory CD8 T cells is CD4 T cell help-dependent, it remains controversial when the "help" is needed. The goal of this thesis project is to determine requirement and mechanisms of CD4 help during the CD8 response to vaccinia viral (VV) infection.

The first aim of this project was to determine when CD4 T cell help is required during the CD8 response to VV infection. Using both CD4-deficient mice and mice with transient depletion of CD4 T cells, we demonstrated that CD4 T cell help was not needed for the activation and effector differentiation of CD8 T cells during the primary response to VV infection. However, the activated CD8 T cells showed poor survival without CD4 T cell help, leading to a reduction in clonal expansion and a diminished, but stable CD8 memory pool. In addition, we observed that CD4 T cell help provided during both the primary and secondary responses was required for the survival of memory CD8 T cells during recall expansion. Our study indicates that CD4 T cells play a crucial role in multiple stages of CD8 T cell response to VV infection and may help to design effective vaccine strategies.

Given that CD4 T cell help is critical for the survival of activated CD8 T cells during both the primary and memory recall responses, it is still unclear how CD4 T cell help promotes CD8 T cell survival. The second aim of this project was to determine the mechanism of CD4 help for the survival of activated CD8 cells. We first showed that CD4 help in vitro was mediated by IL-21, a cytokine produced predominantly by activated CD4 T cells. We then demonstrated direct action of IL-21 on CD8 T cells was critical for the VV-specific CD8 T cell response in vivo. This intrinsic IL-21 signaling was essential for the survival of activated CD8 T cells and the generation of long-lived memory cells. We further revealed that IL-21 promoted CD8 T cell survival in a mechanism dependent on activation of the STAT1 and STAT3 pathways and subsequent upregulation of the pro-survival molecules Bcl-2 and Bcl-xL. Collectively, these results identify a critical role for CD4-derived IL-21 signaling in CD8 T cell responses to acute VV infection in vivo and may help design effective vaccine strategies in situations where CD4 cells are not fully functional.

The role of two cell extrinsic signals, T cell receptor (TCR) ligation and interleukin-7, in promoting glucose uptake and survival of T lymphocytes is examined in this work. Both of these signals are capable of regulating the uptake and fate of glucose, but the requirement of this regulation for T cell homeostasis and functionality remains unclear. To examine the role of TCR mediated increases of glucose metabolism and the signals involved, primary murine T cells were activated in vitro and the role and regulation of glucose uptake was examined. We show that glucose uptake is limiting in T cell activation and that CD28 costimulation is required for maximal glucose uptake following TCR stimulation by upregulating expression and promoting the cell surface trafficking of the glucose transporter Glut1. Regulation of T cell glucose uptake and Glut1 was critical, as low glucose prevented appropriate T cell responses. Additionally, transgenic expression of Glut1 augmented T cell activation, and led to accumulation of readily activated memory-phenotype T cells with signs of autoimmunity in aged mice. To further examine the regulation of glucose uptake, we analyzed CD28 activation of Akt, which appeared necessary for maximal glucose uptake of stimulated cells and which we have shown can promote Glut1 cell surface trafficking. Consistent with a role for Akt in Glut1 trafficking, transgenic expression of constitutively active Akt (mAkt) increased glucose uptake of resting T cells, but did not alter Glut1 protein levels. Therefore, CD28 appeared to promote Akt-independent upregulation of Glut1 protein and Akt-dependent Glut1 cell surface trafficking. In support of this model, co-expression of Glut1 and mAkt transgenes resulted in a synergistic increase in glucose uptake and accumulation of activated T cells in vivo that were largely independent of CD28. Induction of Glut1 protein and Akt regulation of Glut1 trafficking are therefore separable functions of CD28 costimulation that cooperate to promote glucose metabolism necessary for T cell activation and proliferation.

Glucose uptake is dramatically increased in response to TCR and costimulation signaling, however, glucose uptake must be maintained at a low level in naive T cells to promote survival and homeostasis. Interleukin-7 (IL-7) plays a central role in maintaining naive T cell homeostasis, and mediates this effect in vivo at least in part through control of homeostatic proliferation and inhibition of apoptosis. IL-7 can promote glucose uptake and glycolysis in vitro and may also promote glucose metabolism in vivo to maintain T cell survival. To determine if IL-7 regulates T cell metabolism in vivo, we generated a transgenic model for conditional IL-7 receptor (IL 7R) expression on IL-7R-/- T cells. T cells in this model developed normally and, consistent with previous work, deletion of the IL-7R transgene in vivo led to cell death even in an otherwise normal lymphoid compartment. Importantly, in vivo deletion of IL 7R also led to decreased cell size and glycolytic flux. However, glucose uptake was not altered following deletion of the IL-7R indicating that while not essential for glucose uptake, IL-7 is required for maintenance of glycolysis. These data are the first to identify a signal required in vivo to regulate lymphocyte metabolism and demonstrate that in addition to its well-defined roles in homeostatic proliferation and cell survival, IL-7 plays a key and non-redundant role to maintain T cell glycolysis. Together, these data concerning the role of TCR, costimulation, and IL-7 in the regulation of glucose uptake and metabolism exemplify the importance of cell extrinsic signals and the regulation of glucose utilization.

Recombination of Tcrb gene segments in DN thymocytes is subject to allelic exclusion, such that only a single functional V_β - DJ_β rearrangement is generated per T cell. For Tcrb to be allelically excluded the two alleles must initiate recombination asynchronously and once a β-protein is selected, feedback signals must suppress further recombination. Earlier studies of antigen-receptor loci implicated directed monoallelic association with pericentromeric heterochromatin in the initiation or maintenance of allelic exclusion. In this study we used three-dimensional fluorescent in situ hybridization to directly visualize the nuclear localization of Tcra and Tcrb, pericentromeric heterochromatin, and the nuclear lamina. Here we provide evidence for a fundamentally different basis for Tcrb allelic exclusion. We demonstrate that Tcrb is highly associated with pericentromeric heterochromatin and the nuclear lamina in pro-B cells and in DN and DP thymocytes. We also find that Tcrb does not associate with peri-centromeric heterochromatin and the nuclear lamina in a strict monoallelic fashion. Rather, Tcrb alleles independently associate with the two compartments, leading to a stochastic distribution of nuclei containing both, one, or neither allele associated. In the subset of DN thymocyte nuclei with monoallelically associated Tcrb alleles, the non-rearranged allele is most often associated with repressive compartments. This suggests that association with these compartments inhibits recombination prior to β-selection. This inhibition occurs without altering the conformation of the locus. Moreover, the introduction of an ectopic enhancer into Tcrb, led to both a repositioning of Tcrb away from these repressive compartments. This repositioning was correlated with an increase in the frequency of recombination and a break in allelic exclusion. These data lead us to propose that stochastic rather than directed interactions of Tcrb alleles with repressive nuclear compartments bias initial Tcrb recombination to be monoallelic in developing thymocytes and that such interactions are essential for Tcrb allelic exclusion.

The urinary tract is one of the most intractable mucosal surfaces for pathogens to colonize. In addition to the natural barriers at this site, potential pathogens have to contend with the vigorous local innate immune response that is initiated by engagement of surveillance molecule TLRs. TLR4 appears to be not only exclusively expressed on superficial BECs but also critical to triggering robust local innate immune responses. TLR4 recognizes Gram-negative bacterial component LPS and initiates a series of intracellular NF-kappaB associated signaling events resulting in a cytokine response. We examined intracellular signaling events in human BECs leading to the production of IL-6, a major urinary cytokine, following activation by E. coli and isolated LPS, and observed that, in addition to the classical NF-kappaB associated pathway, BEC TLR4 triggers a distinct and more rapid signaling response involving, sequentially, Ca2+, AC3 generated cAMP, and the transcriptional factor CREB. This capacity of BECs to mobilize secondary messengers and evoke a more rapid IL-6 response might be critical in their role as first responders to microbial challenge in the urinary tract.

Here, we also report two additional distinct TLR4-mediated defense mechanisms in BECs. First, BEC TLR4 inhibits bacterial invasion, a necessary step for successful infection. TLR4-mediated suppression of bacterial invasion was linked to increased intracellular cAMP levels which negatively impacted Rac-1 mediated mobilization of the cytoskeleton. Additionally, we found that BECs continue to fight UPEC even after bacterial invasion by triggering bacterial exocytosis through a distinct TLR4-mediated mechanism following activation by LPS. In addition, we reveal that Caveolin-1, Rab27b, PKA, and MyRIP are components of the exocytic compartment and that they form a complex involved in the exocytosis of bacteria. The ability of TLR4 to mediate the rapid cytokine response, the inhibition of bacterial invasion, and the expulsion of intracellular bacteria from infected cells represents three previously unrecognized functions for this innate immune receptor.

Genetic variation is a natural occurrence in the genome that contributes to the phenotypic differences observed between individuals as well as the phenotypic outcomes of various diseases, including infectious disease and cancer. Single nucleotide polymorphisms (SNPs) have been identified as genetic factors influencing host susceptibility to infectious disease while the study of copy number variation (CNV) in various cancers has led to the identification of causal genetic factors influencing tumor formation and severity. In this work, we evaluated the association between genomic variation and disease phenotypes to identify SNPs contributing to host susceptibility in Staphylococcus aureus (S. aureus) infection and to characterize a nervous system brain tumor, known as oligodendroglioma (OD), using the CNV observed in tumors with varying degree of malignancy.

Using SNP data, we utilized a computational approach, known as in silico haplotype mapping (ISHM), to identify genomic regions significantly associated with susceptibility to S. aureus infection in inbred mouse strains. We conducted ISHM on four phenotypes collected from S. aureus infected mice and identified genes contained in the significant regions, which were considered to be potential candidate genes. Gene expression studies were then conducted on inbred mice considered to be resistant or susceptible to S. aureus infection to identify genes differentially expressed between the two groups, which provided biological validation of the genes identified in significant ISHM regions. Genes identified by both analyses were considered our top priority genes and known biological information about the genes was used to determine their function roles in susceptibility to S. aureus infection.

We then evaluated CNV in subtypes of ODs to characterize the tumors by their genomic aberrations. We conducted array-based comparative genomic hybridization (CGH) on 74 ODs to generate genomic profiles that were classified by tumor grade, providing insight about the genomic changes that typically occur in patients with OD ranging from the less to more severe tumor types. Additionally, smaller genomic intervals with substantial copy number differences between normal and OD DNA samples, known as minimal critical regions (MCRs), were identified among the tumors. The genomic regions with copy number changes were interrogated for genes and assessed for their biological roles in the tumors' phenotype and formation. This information was used to assess the validity of using genomic variation in these tumors to further classify these tumors in addition to standard classification techniques.

The studies described in this project demonstrate the utility of using genetic variation to study disease phenotypes and applying computational and experimental techniques to identify the underlying genetic factors contributing to disease pathogenesis. Moreover, the continued development of similar approaches could aid in the development of new diagnostic procedures as well as novel therapeutics for the generation of more personalized treatments. The genomic regions with copy number changes were interrogated for genes and assessed for their biological roles in the tumors' phenotype and formation. This information was used to assess the validity of using genomic variation in these tumors to further classify these tumors in addition to standard classification techniques.

The studies described in this project demonstrate the utility of using genetic variation to study disease phenotypes and applying computational and experimental techniques to identify the underlying genetic factors contributing to disease pathogenesis. Moreover, the continued development of similar approaches could aid in the development of new diagnostic procedures as well as novel therapeutics for the generation of more personalized treatments.