During an immune response, T cell activation is mirrored by a dramatic metabolic shift from oxidative phosphorylation to glycolysis. The upregulation of glycolysis allows the cell to generate the molecules needed to rapidly proliferate and to synthesize effector molecules. The resolution of the T cell response is characterized by equally fast death of most effector T cells. The remaining T cells shift back to oxidative phosphorylation, allowing the cell to survive as a memory T cell. The upregulation of glycolysis and proliferation during the effector phase is paralleled by an increased sensitivity to T cell receptor restimulation-induced cell death (RICD). Whereas cellular metabolism and cell death are important in the proper function and response of T cells, it is not clear how metabolism regulates susceptibility to cell death, nor whether T cell proliferation and contraction are directly connected. The work presented in this dissertation provides a mechanistic link between T cell proliferation and contraction by demonstrating the regulation of caspase-3 activity by the metabolic state of T cells. In effector T cells, the cytokine interleukin (IL)-2 mediates the upregulation of glycolysis, while IL-15 induces oxidative phosphorylation and a memory-like state. IL-2 is known to sensitize T cells to RICD, while IL-15 reduces RICD and increases survival. This results from the ability of IL-2 and glycolysis to increase caspase-3 activity, whereas IL-15 induces the opposite phenotype. Activation of caspase-3 during glycolysis is mediated through clustering in lipid rafts in the plasma membrane. IL-15 is shown to inactivate caspase-3 through the posttranslational modification of protein glutathionylation, which is mediated by ROS generation in the mitochondria as a by-product of oxidative phosphorylation. We further observe that glycolysis parallels the reduced activity of the electron transport chain and oxidative phosphorylation, further increasing caspase-3 activity. This is mediated by the decreased expression of electron transport chain complexes and an increase in expression of the negative regulator of complex I, methylation-controlled J protein (MCJ). IL-15 promotes reduced expression of MCJ by its methylation. Similar to IL-15-cultured T cells, MCJ-deficient T cells manifest reduced glycolysis, caspase-3 activity, and RICD. Collectively, these findings demonstrate an adaptation that links metabolism to both cell proliferation and cell death to safeguard that proliferating cells do not escape regulation that could result in autoimmune disease or lymphomas.

The increasing threat of epidemic and pandemic influenza underscore the need to better-understand the immune response to influenza virus infections and to better understand the factors that contribute to the clearance of virus without complications of immunopathology. A hallmark of the adaptive immune response to primary influenza virus infections is the induction of influenza-specific CD8+ T cell responses. These T cells target and kill influenza-infected epithelial cells in the airway, thereby clearing the virus and allowing recovery of the infected host. Recent reports demonstrated that CD8+ T cells express TNF-related apoptosis-inducing ligand (TRAIL) after influenza virus infection. While roles for perforin/granzyme and Fas:FasL interactions in clearing influenza virus infections had been established, little was known about the role of TRAIL in the CD8+ T cell responses to influenza virus infection. We hypothesized that influenza-specific CD8+ T cells would express TRAIL after influenza infection and could utilize TRAIL to induce the apoptosis of virally-infected cells. We discovered that CD8+ T cells do express TRAIL after influenza infection, and that this expression occurs in an influenza-specific fashion. Further, we demonstrated that these influenza-specific CD8+ T cells utilize this TRAIL to kill virally infected cells and protect the host from death, while T cells lacking TRAIL were unable to kill targets as efficiently and provided reduced protection. These data supported our hypothesis that CD8+ T cells utilize TRAIL to kill infected cells. Unexpectedly, when we increased the initial viral inoculum, the pulmonary cytotoxicity of T cells in TRAIL-/- mice was increased compared to those in TRAIL+/+ mice. Investigation of this phenomenon revealed that changes in cytotoxicity correlated not with changes in effector molecule expression on the T cells, but with increased recruitment of T cells to the lung. T cell recruitment to the lungs of TRAIL-/- mice was dependent on CCR5 and CXCR3, and likely the result of aberrant expression of MIG and MIP-1α in the lungs. Together, these data suggest that TRAIL expression contributes not only to T cell cytotoxicity, but also to the regulation of chemokine expression and associated cell recruitment after influenza virus infections. To confirm the relevance of our animal model to the study of human disease, we examined the potential role for TRAIL in the human immune response to infection. We determined that in vitro influenza infection stimulates upregulation of functional TRAIL on the surface of CD3+, CD14+, CD19+, and CD56+ PBMC populations. This expression was not caused by infection of the cells, but by interferon produced as a result of the infection. Infected (TRAIL-expressing) PBMCs killed influenza-infected lung epithelial cells, revealing that influenza infection sensitizes epithelial cells to TRAIL-induced apoptosis. Surprisingly, blocking TRAIL signaling, but not FasL signaling, was able to abrogate this killing of infected epithelial cells. Together, these data support a role for TRAIL in the human immune response to influenza virus infections. Considered as a whole, the data from these studies suggest an additional, previously-unappreciated mechanism by which CD8+ T cells can kill virally infected cells, TRAIL. They also suggest additional, previously-unappreciated roles for TRAIL in immune responses: in helping clear virally infected cells after infections and in helping control cytokine/chemokine expression, and thus the immune response, after virus infection.

B lymphocytes play important roles in antibody production, cytokine production, and antigen presentation to T cells. Ligation of Toll-like receptors (TLRs) on B cells stimulates cellular activation and B cell effector functions. Synergistic activation of other receptors such as CD40 or the B cell receptor (BCR) with TLR ligation further enhances B cell activation and effector functions. The tumor necrosis factor receptor associated factor (TRAF) family of proteins act as cytoplasmic signaling adaptor molecules and moderate downstream signaling from both the tumor necrosis factor receptor (TNFR) superfamily of proteins, including CD40, and the IL-1R/TLR superfamily of proteins. To date, only TRAFs 3 and 6 have been shown to be involved in TLR signaling, with TRAF6 providing positive regulation and TRAF3 providing negative regulation of TLR signaling in B cells. Deficiency in another TRAF family member, TRAF5, has been implicated in the development of atherosclerosis, a disease developed in part due to TLR dysregulation. Here, we addressed the hypothesis that TRAF5 is a negative regulator of TLR signaling. We found that TRAF5 negatively regulated TLR-mediated cytokine and antibody production in B lymphocytes. The enhanced cytokine production seen in TLR-stimulated TRAF5 KO B cells was not attributable to altered cellular survival or proliferation, but instead more cytokine was produced on a per-cell basis, likely due to enhanced MAPK pathways after TLR ligation. Additionally, TRAF5 deficiency did not dramatically affect cytokine production in TLR-stimulated bone marrow-derived macrophages or dendritic cells, suggesting that TRAF5 plays a greater role in TLR signaling in lymphoid versus myeloid cells. TRAF5 associated with the TLR signaling proteins MyD88 and TAB2, and negatively regulated the association of TAB2 with its binding partner TRAF6. Furthermore, we manipulated B cell activation via ligation of various TLRs, CD40, and/or the BCR in order to activate the cells to effectively present antigen. Activated B cells pulsed with antigen served as an effective cellular vaccine and offered protection against both an infectious pathogen (Listeria monocytogenes) and a model of murine melanoma. We identified two candidate activation criteria for B cell vaccines (Bvacs): stimulation through the BCR and TLR7, and stimulation through CD40 and TLR4. Additionally, we found that high IL-6 production by the activated Bvac was essential for inducing optimal CD8+ T cell memory. These B cell activation protocols offer significant advantages over those currently being tested for clinical use. Understanding B cell activation through TLRs is a critical step in developing new therapies against cancer and infectious disease.

We have recently demonstrated in a model of influenza A virus (IAV) infection that the absence of specific pulmonary DC subsets, including plasmacytoid DC (pDC) and CD8a+ DC, from the lungs leads to a significant decrease in the number of virus-specific CD8 T cells. Reconstitution of the lungs with physiologic numbers of pDC or CD8a+ DC is able to restore the pulmonary IAV-specific CD8 T cell response to near normal levels via a mechanism that is dependent upon direct DC:T cell interactions, DC-expressed MHC I and the presence of viral antigen. Interestingly, however, this rescue is DC subset specific, as reconstitution with purified alveolar and airway DC or alveolar macrophages was unable to rescue the virus-specific CD8 T cell response. Following IAV infection there is an abundance of IAV antigen and MHC I expressing cells present in the lungs, including infected epithelial cells. Given this fact and the inability of all DC subsets to rescue the virus-specific CD8 T cell response, it suggested that there were additional, undefined requirements for pDC- and CD8a+ DC-mediated rescue of the T cell response in the lungs. Further, although it was known that the reduction in virus-specific CD8 T cells in the lungs was a result of increased T cell apoptosis, it remained unclear what pathways of apoptosis were contributing to the increased cell death, and what mechanism pulmonary DC subsets were utilizing to rescue this defect. Here, we demonstrate that in the absence of lung-resident DC subsets, virus-specific CD8 T cells undergo significantly increased levels of apoptosis via both extrinsic activation induced cell death and intrinsic activated cell-autonomous death pathways. Reconstitution of aDC depleted lungs with pulmonary pDC and CD8a+ DC promotes increased T cell expression of the pro-survival molecule Bcl-2 and hence, increased T cell survival and accumulation in the lungs. Our studies herein demonstrate that pulmonary DC subsets utilize a variety of mechanisms to promote the rescue of virus-specific CD8 T cells in the lungs. Blockade of the costimulatory molecules CD70, and in some cases, 4-1BBL and OX40L, ablates the pulmonary DC mediated rescue of CD8 T cell numbers in the lungs, suggesting that late costimulation is one essential mechanism that pulmonary DC use to regulate CD8 T cell immunity following IAV infection. Further, we demonstrate that the absence of DC following IAV infection results in significantly reduced levels of IL-15 in the lungs and that pulmonary DC-mediated rescue of virus-specific CD8 T cell responses in the lungs requires the trans-presentation of IL-15 via DC-expressed IL-15Ra. In addition to the role of pulmonary DC mediated costimulation and IL-15 trans-presentation, we further demonstrate a previously unrecognized role for viral antigen in regulating the accumulation of both pulmonary DC and virus-specific CD8 T cells in the lungs, suggesting that viral load can dictate the nature of the inflammatory environment in the lungs and thus, regulate the character of the ensuing IAV-specific immune response. Collectively, the results detailed here demonstrate a previously unrecognized role for pulmonary DC in regulating primary IAV-specific CD8 T cell immunity, and hence, promoting enhanced viral clearance and recovery from disease.

Monoclonal antibodies (mAbs) have become an important part of therapy for a number of cancers. The first mAb to be approved for clinical use is rituximab, which is currently used for the treatment of various B cell malignancies. Despite its clinical value, the mechanisms in which rituximab induces tumor regression are unclear. Growing evidence suggests that multiple mechanisms involving complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) are involved. However, the direct interactions between CDC and ADCC have yet to be investigated. My studies examine the relationship between complement fixation and the activation of NK cells by utilizing in vitro assays, a syngeneic murine lymphoma model, and clinical samples from patients. Using these systems, I demonstrate that the initiation of the complement cascade inhibits NK cell activation and ADCC induced by rituximab in vitro. I also show that depletion of complement enhances the activation of NK cells and improves the efficacy of mAb therapy in a murine model. Lastly, I demonstrate that NK cell activation correlates with decreased complement activity in patients after rituximab treatment. The studies described in this dissertation have furthered the understanding of the mechanisms involved in antibody therapy. These results have described a novel inhibitory role for complement activity in the anti-tumor responses of mAbs. Furthermore, these findings suggest that strategies to circumvent the inhibitory effect of complement may improve how current mAbs are used and the how mAbs are designed in the future.

Zika virus (ZIKV) has emerged in many regions of the world, with infection outcomes spanning from no apparent illness to crippling nervous system disease. ZIKV and its close relatives, West Nile virus, Japanese encephalitis virus, dengue virus, and yellow fever virus are primarily transmitted by mosquitoes. Three ZIKVs were selected: MR-766 (Uganda, 1947), P6-740 (Malaysia, 1966), and PRVABC-59 (Puerto Rico, 2015), whose place of origin and time of isolation differ substantially. Stable, complementary DNA (cDNA) copies of the three ZIKV RNA genomes were cloned to examine the significance of viral and host genetic variations in directing ZIKV infection outcomes. Using a new toolbox for ZIKV genome engineering and protein analysis, combined with various cell culture and mouse infection model systems, the following were determined: (1) Genome-wide landscape of viral gene products and their related species, with several immuno-reactive gene products identified in the case of all three cloned ZIKVs. (2) Viral replicability in cultured cells, varied significantly depending on the virus strain and host cell type, with one cow cell line being resistant to ZIKV infection. (3) Virus induced neurological disease in mice, differed dramatically depending on the virus dose and strain, mouse age and strain, route of infection, and presence or absence of immune system components. Overall, the findings demonstrate the impact of the viral and host genetic backgrounds on the ability of ZIKV to replicate and cause disease. The ZIKV strain-specific characterizations and molecular instruments described will provide multiple avenues for developing and testing medical countermeasures.

The interrogation of T cell signaling over the past fifty years has led to the discovery of amazingly intricate cascade networks and elaborate descriptions of individual proteins' domains and functions. A complex landscape has been rendered in which proteins relay messages from the extracellular ligation of the TCR by a cognate peptide loaded MHC via changes in sub-cellular location, phosphorylation, and binding affinities and partners to enact nuclear localization of three key transcription factors required for cellular effector function and proliferation: AP-1, NF-AT, and NF-κB. Dogma has favored activation of each of these transcription regulating elements to be a linear and parallel activity, thus very little interaction between pathways has been highlighted by previous findings in the molecular immunology community. The focus of this dissertation explores the role of NF-κB in T cell signaling with emphasis on subunits p50, cRel, IκBα, and IKKβ, and with respect to NF-κB’s ability to modulate calcium and NF-AT signaling, proximal TCR phosphorylation, and CRAC and purinergic calcium channel proteins. The role of NF-κB in T cells can be a difficult thing to establish, as this thirteen member family innervates almost every cellular process from homeostasis to activation, and even functions in the opposing processes of survival and apoptosis. To convolute the investigation further, many family members also fulfill redundant tasks, as a result of their high evolutionarily conserved sequence homology. To this end, we discovered the best way to evaluate the function of NF-κB in the activation of T cells was to knockdown two family members: p50 and cRel. In doing this, we rendered mice that were viable (unlike knockdown of RelA) and fertile, but possessed T cells that were highly unresponsive to strong stimulation (anti CD3/CD28) or foreign antigen (OVA) presented to mice bearing the correct transgenic TCRs (OT-1) by professional antigen presenting cells (APC). Through in vitro assays, we discovered that in addition to the specific defects in NF-κB activation, NF-AT signaling was also greatly disrupted in these cells, sequela to retarded calcium influx and signaling. This was of great interest, as while several studies have shown that calcium signaling has the ability to amplify and fine tune NF-κB activation, there is a dearth of studies and publications highlighting the effect of an activated NF-κB pathway on calcium influx and signaling leading to the activation of NF- AT. Another fascinating discovery, that explicated the calcium reduction and NF-AT inhibition, was that ablation of p50 and cRel led to decreases in mRNA and protein levels of two additional NF-κB family members: IKKβ and IKKγ. The results presented here suggest that it is the reduction in IKKβ and IKKγ that leads to impaired phosphorylation of the key TCR proximal proteins: Zap70 and PLC&gamma1, and it is the decrease in activated PLCγ1 that renders less IP3 and ultimately abrogates calcium signaling. Overall, this thesis highlights the ability of IKKβ to enhance general proximal TCR protein phosphorylation (and specifically Zap70) leading to a greater influx of calcium (perhaps aided by IKKβ also augmenting the function of the CRAC protein, STIM1) which leads to superior activation of NF- AT, and amplifies downstream cellular effector functions such as IL-2 production and proliferation. Moreover, this work demonstrates that NF-κB subunits likely form supermolecular clusters, and ablation of certain subunits (i.e. p50 and cRel) can lead to instability and decreased levels of other family members (i.e. IKKβ and IKKγ.)

ABSTRACT Alzheimer's disease (AD) is the most common progressive age related dementia and the fourth major cause of mortality in the elderly in the United States. AD is pathologically characterized by deposition of amyloid beta (Aβ) plaques in the brain parenchyma and neurofibrillary tangles (NFTs) within the neuronal soma. While pharmacological targets have been discovered, current strategies for the symptomatic or disease-modifying treatment of AD do not significantly slow or halt the underlying pathological progression of the disease. Consequently, more effective treatment is needed. One possibility for amelioration is using human umbilical cord blood cell (HUCBC) therapy. HUCBCs comprise a population of hematopoietic stem and progenitor cells. During recent years, functional recovery has been observed from the use of HUCBCs in pre-clinical animal models of brain and spinal cord injuries. Thus, modulation by cell therapy, specifically HUCBCs, may be a suitable treatment for AD and other models because of the observed cognitive and behavioral improvements. The studies presented in this dissertation centers on the suitability of using HUBCs as a potential treatment for AD. Expanding on this, the aims of the study sought to: (I) Investigate bio-distribution of HUCBC transplantation in PSAPP mice, (II) Characterize efficacy and determine therapeutic outcome of HUCBC following short and long term multi injections at early and late disease stages in PSAPP mice and (III) Determine AD-like pathological and cognitive changes associated with multiple HUCBC-derived monocyte (CD14) injections in PSAPP mice. Thus the findings of this work evolved from experiments that characterized the effects of low-dose infusions of HUCBC and HUCBC-derived monocytes into 6 month old Presenilin 1/Amyloid Precursor Protein (PSAPP) plaque-developing transgenic AD mice. Treated mice were studied using standard behavioral tests to determine the effects of infusion on the multiple cognitive domains affected by AD, followed by biochemical and histological analyses that included Aβ load and amyloid precursor protein (APP) processing. Specifically, PSAPP mice and their wild-type (WT) littermates were treated monthly with a peripheral HUCBC infusion over a period of 6 and 10 months, followed by cognitive and motor evaluation. Additionally, based on reports that tumor cells can originate from stem cells present in HUCB, we further examined whether monocytes purified from HUCBCs would have a similar significant effect on the reduction of AD-like pathology in PSAPP mice. HUCB cells homed into tissues including the brain. The principal finding was significant reduction in Aβ levels and β–amyloid plaques following low-dose infusions of both HUCBC– derived mononuclear cells as well as HUCBC-derived monocytes, with the monocytes providing a stronger effect. Results further demonstrated that HUCBC and HUCBC– derived monocyte infusion could improve memory function and locomotor ability in treated PSAPP mice. A possible reason for behavioral improvements in these animals may be the significant reduction in both Aβ levels and plaque load. This study also identified significant reduction in microglial activation and astrocytosis, both of which can contribute to AD pathology. In conclusion, our data suggest that it might be the HUCBC–derived monocytic population rather than stem cells that are responsible for the reduction in AD pathology.

Equine influenza virus causes a mild, self-limiting upper respiratory disease in its natural host. In stark contrast, equine influenza viruses of the H7N7 subtype produce lethal infection in BALB/c mice. This dissertation explored the mechanism underlying the differential pathogenicity of the equine H7N7 influenza virus observed in horses and BALB/c mice. Initially, a comparative study of the pathogenesis was conducted in BALB/c mice inoculated intranasally with a representative isolate of either H7N7 or H3N8 subtype equine influenza virus. All H3N8 virus-infected mice survived the infection whereas 100% mortality was documented for the mice receiving the H7N7 virus by day 8 post infection. Both viruses replicated to a similar degree in the lungs at the early stages of infection. However, after day 2 post infection until the death of the mice, the pulmonary viral loads of the H7N7 group were significantly higher than those of the control, whereas the H3N8 virus was eventually eradicated from the mice at day 7 p.i. Correspondingly, a vigorous pro-inflammatory cytokine response in the lung was induced by the H7N7 virus but not the H3N8 virus, which reflected a desperate attempt by the host immune responses to restrain the overwhelming infection. The H7N7 virus was poorly sensitive to the innate immune containment, resulting in a significant higher cumulative mortality rate than that of the control virus in chicken embryos aged 9 days and older. On the contrary, in horses, replication of the paired viruses was completely cleared by the host immune responses at day 7 p.i. and the infections produced an acute yet non-lethal illness, albeit the H3N8 virus induced generally more pronounced clinical manifestations than the H7N7 virus. The clinical severity correlated to the difference in cytokine-inducing capacity between the two viruses in horses, as evidenced by the finding that the H3N8 virus triggered significantly higher levels of gene transcription of multiple key inflammatory cytokines in the circulation than those seen for the H7N7 virus. In addition, equine peripheral monocyte-derived macrophages were found to be a target of equine influenza virus and can support the productive replication of the virus in vitro.

The mammalian immune system is equipped to both eliminate pathogenic microorganisms and tumors, while remaining in homeostasis with commensal species at mucosal surfaces and tolerant towards self. Suppressor regulatory T cells (Tregs) are a major sentinel of this immunological tolerance. Induced Tregs (iTregs) arise in the periphery following the integration of cues from the metabolites, cytokines, etc. which make up its milieu. Dysregulation of iTreg development, function or homing underlies the etiology of many autoimmune diseases and immunopathologies. The amelioration or prevention of multiple murine disease models by boosting Treg cell numbers foreshadows clinical efficacy of iTreg therapy, but an incomplete understanding of Treg development has thus far prevented successful translation. Therefore, we considered the basic biology of T cell fate decision making from two unique, but integrated angles. First, we show that the stimulation of PPARγ in human T cells upregulates RDH10, a molecule which catalyzes the rate limiting step in the oxidation of retinol to transcriptionally active all-trans retinoic acid (ATRA), a positive regulator of iTreg development. This functionally intact pathway endows T cells the ability to autonomously sense and respond to retinoid signals present during Treg development and at tissue sites. Next, we asked questions about how T cells sense nutrient and oxygen availability as they differentiate. Tregs lacking the serine/threonine kinase PINK1 have limited activation-induced phosphorylation of Akt and oxidative phosphorylation rates, and reduced suppressor function. Notably, the uncoupling of iTreg function from normal FoxP3 expression reinforces the recent hypothesis that the PI3K/Akt/mTORC1 axis and metabolic checkpoints are decisive players in the acquisition of suppressor activity. Ultimately, the studies described herein converge on Akt and metabolism, and contribute to our understanding of how T cells integrate diverse signals present during fate determinism, provoking future Treg based therapeutics.