Pneumocystis (PC) pneumonia is a life-threatening opportunistic fungal infection observed in individuals with severe immunodeficiencies, such as AIDS. This dissertation evaluates functions of antibodies and conserved fungal cell wall carbohydrate antigens in host defense against PC. We demonstrate that a novel recombinant protein consisting of the extracellular domain of the beta-glucan receptor dectin-1 fused to the constant portion of murine IgG1, binds beta-glucan and recognizes Fc-γ receptors; and functionally, impairs growth of PC in the lungs of immunocompromised mice. As an antibody-like molecule targeting a conserved fungal cell wall carbohydrate enhanced host defense against PC, we questioned whether the host produces similar antibodies. We identified natural IgM antibodies, conserved across species and not requiring microbial stimulation for production, that recognize fungal cell wall carbohydrates beta-glucan and chitosan/chitin. In mice, naïve serum containing natural antibodies impairs the growth of PC organisms in the lungs at intermediate stages of infection, while at earliest stages limits pulmonary neutrophil recruitment. Mice unable to secrete IgM, sIgM(-/-), manifest similar impairments in pathogen clearance at intermediate stages of infection. Additionally, sIgM(-/-) mice demonstrate diminished trafficking of fungal cell wall carbohydrate antigen by CD11c+ cells to draining lymph nodes, impaired production of Th2 and Th17 cytokines in lymph nodes after PC challenge, and altered adaptive antibody responses, with diminished anti-PC IgG1 (Th2 associated) while enhanced IgG2a (Th1 associated) adaptive antibody responses. Thus, sIgM, of which a significant component is natural antibody, influences PC Ag presentation at earliest stages of infection, enhancing host defense and biasing the host towards Th2 adaptive responses. Additionaly, we observe that beta-glucan and chitosan/chitin are targets of induced antibody responses. PC challenge leads to the induction of specific serum IgG, and increased quantities of multiple isotypes at the lung mucosa against these carbohydrates. Mucosal responses against beta-glucan and chitosan/chitin after PC challenge are regulated by CD4+ T cells, and we provide evidence that functional memory B cell responses against fungal wall carbohydrates are generated as a consequence of PC challenge. Collectively, these studies demonstrate the importance of conserved fungal cell wall carbohydrate antigens, and primitive antibody isotypes, in host defense responses against PC.

We previously identified the aberrantly expressed cell cycle regulator cyclin B1 as a tumor antigen that can elicit both humoral and cellular immune responses in cancer patients. While cyclin B1 is only transiently expressed in normal cells, cancers of many tissue origins constitutively overexpress the cell cyclin in the cytoplasm, which correlates with poorer patient prognosis. We propose that this tumor-specific overabundance and cytoplasmic location of cyclin B1 leads to the presentation of highand therefore immunogenicconcentrations of cyclin B1 peptides to the immune system. Our studies focused on the source of immune responses against cyclin B1 and the significance of these immune responses in the setting of cancer. To study the significance of the anti-cyclin B1 immune response in human cancer, we tested plasma from patients with non-small cell lung cancer (NSCLC) for anti-cyclin B1 IgG and demonstrated that a longer overall survival in patients with stage IB NSCLC is correlated with high levels of anti-cyclin B1 IgG. We also demonstrated that cyclin B1-specific antibody and T cell responses exist in healthy individuals who have no history of cancer. Further, the cyclin B1-specific T cells in healthy individuals are antigen experienced and in both CD4+ and CD8+ T cell compartments. We then sought to determine the potential significance of a preexisting anti-cyclin B1 immune response in the setting of cyclin B1+ tumor development. Using both transplantable and spontaneous mouse models of cyclin B1+ tumors, we demonstrated that vaccination against cyclin B1 prior to the administration or spontaneous development of cyclin B1+ tumors inhibits tumor growth. Finally, given that viral infection has been shown to lead to overexpression of cyclin B1, we proposed that the anti-abnormal self protein, anti-tumor antigen immune responses we observed in healthy people were a result of a virus infection. The extension of that hypothesis is that infections with viruses can train the immune system to recognize abnormal expression of self proteins and therefore protect from cancers that abnormally express those proteins as well. We demonstrated that infection with ectromelia, a mouse pox virus, protects from tumor challenge.

Reactivation of herpes simplex virus (HSV) from neuronal latency is a common and potentially devastating cause of disease worldwide. CD8 T cells can completely inhibit HSV reactivation, and IFN-gamma affords a portion of this protection. We now show that CD8 T cell lytic granules are also required for the maintenance of HSV type 1 (HSV-1) neuronal latency both in vivo and in ex vivo cultures, and that their directed release into junctions with neurons in latently infected ganglia does not induce neuronal apoptosis. Our findings support a non-lethal mechanism of viral inactivation by demonstrating that the lytic granule component, granzyme B, degrades the HSV-1 immediate early protein, ICP4, which is essential for further viral gene expression. These findings reveal a novel non-apoptotic function of granzyme B in the context of inhibiting viral reactivation from latency.

Human mucin 1 (MUC1) is a highly glycosylated transmembrane glycoprotein that is expressed on the luminal surface of ductal epithelial cells. Human adenocarcinomas overexpress MUC1 as a tumor-associated antigen (TAA) that presents to the immune system peptide epitopes and glycopeptide epitopes with tumor specific carbohydrates, such as mono- and disaccharides known as Tn, sialyl-Tn, and TF antigens. Studies in MUC1 transgenic (MUC1-Tg) mice have indicated that, compared to transgene negative wild-type (WT) mice, the MUC1-Tg immune system maintains a certain level of tolerance to the MUC1 peptide, reflected most notably in decreased CD4 T cell help. We made a novel observation that in contrast to suppressed responses to the MUC1-peptide vaccine, vaccination of MUC1-Tg mice with tumor-associated MUC1 glycopeptide resulted in effective anti-MUC1 immunity similar to that elicited in WT mice. We hypothesized that the tumor-associated glycopeptides were seen as foreign and therefore not subject to tolerance. To study CD4 T cell responses to MUC1 glycopeptides we generated glycopeptide GST(GalNAc;Tn)A specific, MHC-Class II restricted CD4 T cell hybridoma, RF6. We cloned the RF6 TCR (RFT; Vá4.1Já16-Vâ15Jâ1.3) and generated TCR transgenic mice RFT-Tg. Using the RFT-Tg mice and the previously generated MUC1 peptide-specific TCR transgenic VFT-Tg mice, we show that peptide-specific VFT CD4 T cells transferred to MUC1-Tg mice are suppressed through mechanisms of peripheral tolerance, which are not induced against MUC1-glycopeptide specific RFT CD4 T cells. We show that peptide-specific CD4 T cells are transiently activated upon transfer into MUC1-Tg mice, suggesting that MUC1-peptide epitope is presented in the periphery in healthy mice as well as in tumor bearing mice. In contrast, MUC1-glycopeptide epitopes are tumor specific and thus treated as foreign antigens in MUC1-Tg mice, resulting in effective activation of glycopeptide-specific CD4 T cells. Simultaneous activation of glycopeptide-specific T cells can break tolerance of peptide-specific T cells. Our findings with MUC1 apply to other TAA that contain some epitopes that are self and subject to self tolerance and other epitopes that are tumor specific (foreign) and not affected by tolerance. Understanding this distinction is very important in the development of effective and safe cancer vaccines.

Organ transplantation is a life-saving and increasingly common procedure, as it often serves as the only treatment available for end-stage organ disease. Although the constant development of new and more effective immunosuppressive drugs has revolutionized the prevention and treatment of acute graft rejection, these drugs have significant toxicity, greatly increase patient susceptibility to neoplasms and infection and exert little impact on chronic rejection. A major obstacle to developing improved therapeutics is a lack of understanding the mechanisms by which the adaptive immune response is initiated, and how cellular therapies impact this response. Previous research has provided a mechanistic scaffold, however numerous gaps, often filled with assumptions rather than data, remain. In this dissertation, I demonstrate that contrary to current dogma, dendritic cell (DC)-based therapies simply serve as a source of alloantigen, and therefore have comparable efficacy to alternative cellular therapies. Further, I show that contradictory to the current paradigm of direct pathway T cell priming, recipient antigen-presenting cells (APC) stimulated via CD40 ligation by indirect pathway CD4+ T cells is requisite for a direct pathway response and allograft rejection. Conversely, I did validate the assumption that donor passenger APC are required for the direct pathway T cell response, but further show that they are also required for the indirect pathway T cell response, indicating that donor APC serve as a source of alloantigen for presentation by recipient APC. Finally, through investigating the role of recipient APC in cardiac allograft rejection, I identified that the recently described population of inflammatory monocyte-derived DC play a crucial role as effector cells that mediate a DTH-like response within cardiac allografts during acute rejection, while at the same time, inhibiting T cell effector responses within the graft and systemically. Overall, our data provide essential puzzle pieces to understanding the processes of acute allograft rejection and insight into the utility of DC-based therapies for transplantation.

Interleukin-17 (IL-17) is a cytokine produced mainly by T cell lineages that plays a key role in regulation of neutrophil responses. Given the importance of neutrophils in the immune response directed against extracellular pathogens, it is no surprise that IL-17 is important in host defense against a multitude of pathogens. Importantly, however, neutrophils also have been shown to play a role in several immunopathological conditions, including acute lung injury. In this dissertation, we evaluate the role that IL-17 plays in the immunopathology of influenza infection. We show here that IL-17 is produced as early as day 2 following influenza challenge, and that this expression is sustained throughout the first week of infection. Further, we identify γδ T cells as at least one important source of IL-17 in response to influenza. We also demonstrate that loss of IL-17 receptor A (IL-17RA) signaling results in a profound decrease in neutrophil recruitment to the lung following influenza challenge. This decrease in neutrophils results in substantially less inflammation and lung injury, as well as higher survival rates. Additionally, there is only a moderate impact on viral clearance and T cell responses. Further, we detail similar findings in a non-infectious aspiration model of acute lung injury. Taken together, this data suggests that IL-17 signaling may be a key event, and intriguing therapeutic target, in the pathogenesis of acute lung injury.

Dendritic cells (DC) are professional antigen presenting cells that can prime naïve T cells to elicit immunity or tolerance. The ability to regulate immunity or tolerance is governed by the type of polarization state of these activated T cells. T-bet (T-box expressed in T cells) has been identified as the master regulator of Type 1 polarization in T cells, and its expression in T cells is essential for immunity. Conversely, Foxp3 expression in T cells (T regulatory cells) engenders a tolerogenic phenotype that can suppress immunity in Type-1, as well as the Type-2 and -17 subsets, and DC. Interestingly, T-bet is also expressed in DC and its abolishment resulted in impaired Type-1 T cell responses. Furthermore, Foxp3 expression in non-T cell subsets, such as adenocarinoma, has shown potent immunosuppressive characteristics in the tumor microenvironment and draining lymph nodes. Therefore, we examined the role of adenoviral transduced T-bet and Foxp3 in the myeloid cell lineage, specifically monocyte-derived DC. We evaluated the phenotypic expression of DC and T cell responses upon priming by T-bet expressing DC (DC.bet) or Foxp3 expressing DC (DC.Fox). DC.bet potently primed naïve T cells towards Type 1 immunity, inducing 2-3 fold increased levels of T-bet, IFNγ, CXCR3, and Granzyme-B. Interestingly, we found little-to-no changes in costimulatory molecule expression. However, DC were completely impaired in production of Type-1-inducing cytokines. We confirmed cytokine-independent Type 1 polarization by using neutralization antibodies. More surprisingly, we found that Foxp3 in DC induced tolerance. Mechanisms included direct suppression of CD8+ T cell subsets and indirect suppression of Type-1 responses by increased generation of T regulatory cell subsets. These generated DC.Fox induced-Tregs (XiTregs) expressed high levels of CTLA-4, CD25, and GITR and concomitantly suppressed naïve and memory CD8+ T cell proliferation and IFNγ production. Neutralizing agents confirmed that tryptophan catabolizing enzyme-IDO and TGFβ were important in suppressing generation of Tregs and effector functions of T cells. In summation, this work shows that T-bet and Foxp3 expression in DC play similar roles to expression in T cells by governing immunity or tolerance. Furthermore, this provides a basis for the usage of these DC in immunotherapies.

NF-êB is a family of transcription factors that play a pivotal role in inflammation, cell proliferation, cell survival, and apoptosis in response to endogenous and exogenous stress stimuli. NF-êB is implicated in numerous chronic inflammatory and degenerative diseases. In this thesis, the consequences of NF-êB suppression on pathologies associated with inflammatory bowel disease (IBD) and age-associated degeneration are explored. To test the hypothesis that NF-êB plays a causal role in driving the degenerative changes associated with the diseases we evaluated the efficacy of a pharmacologic peptide IKK/NF-êB activation inhibitor in the IL-10-/- model of colitis and genetic depletion and pharmacologic inhibition of NF-êB in a mouse model of accelerated aging (Ercc1-/∆ mice). Pathologic and immunologic markers of IBD were reduced in the presence of systemic pharmacologic NF-êB suppression. Furthermore, this study provides evidence of the efficacy of the NBD inhibitory peptide in vitro and in vivo experiments, and importantly provided possible therapeutic avenues for the treatment of IBD. Like naturally aged mice, NF-êB is activated in Ercc1-/∆ mice compared to wild type littermates. Ercc1-/∆ mice haploinsufficient for the p65/RelA subunit of NF-êB had a modest delay in the onset of age-related symptoms. This was recapitulated in mice chronically treated with the peptide inhibitor of NF-êB, which exhibited a significant delay in overall aging score and improved histopathological alterations. These implicate NF-êB as a major driver of degenerative changes associated with aging and set a precedent for therapeutic intervention. As activated macrophages are mediators of inflammatory and age-associated degenerative changes, we futher evaluated the role of NF-êB suppression in this cell type. Macropahges and monocyte derived DC cells underwent programmed cell death (PCD) in the presence of pharmacologic NF-êB inhibition. Unlike previous studies which implicated TNFá signaling in this pathway, the mechanisms behind this PCD is induction is induction of ROS formation. This observed macrophage NF-êB induced PCD may be one of the mechanisms by which inflammatory and age-associated disease pathologies are reduced in response to NF-êB suppression.

The preservation of reactive oxygen species (ROS) throughout evolution is evidence that ROS are critical components in immunity. These constituents ignite a multitude of redox-dependent pathways triggering pro-inflammatory cytokine production, dendritic cell maturation, and subsequent T cell activation in response to pathogen, autoimmune, and alloimmune insults. The aim of this thesis is to test the ability of redox modulation to suppress aberrant immune responses in islet transplantation. Presently, islet transplantation is only used in desperate cases of glucose dysregulation due to the high failure rate of the procedure, which forces the majority of recipients to resume exogenous insulin within a year of islet transplantation. Hurdles in islet transplantation include hypoxia during islet isolation and ischemia-reperfusion injury upon transplantation. These insults result in primary non-function of islets, while harsh immunosuppressive agents yield islet toxicity and a multitude of complications for the recipient. We tested a catalytic antioxidant (CA), FBC-007, in islet transplantation based on previous work demonstrating that CA impairs innate-immune ROS and pro-inflammatory cytokine production by inhibiting NFκB-DNA binding, hinders CD4 T cell activation, and prevents the transfer of diabetes into young NOD.scid mice. First, the effects of redox modulation on CD8 T cell effector function in allogeneic and transgenic responses were examined in vitro. Human islets were also used in vitro and murine experiments were performed in vivo to test the ability of CA to protect from streptozotocin-induced islet cell death. Additionally, murine islets were incubated with CA in in vivo models of ischemia-reperfusion injury (antigen-independent) or allogeneic (antigen-dependent) transplantation and separately, CA was used as a systemic therapy for allograft recipients. Further experiments were performed to elucidate in vivo protective mechanisms of CA-treatment. An additional approach of interest is the induction of antigen-specific hyporesponsiveness to replace nonspecific immunosuppression. A negative vaccination strategy delivering apoptotic donor alloantigen in a non-inflammatory adjuvant prior to allograft transplantation, was also tested. Collectively, this work demonstrates 1) CA is a non-toxic, islet-sparing, cytoprotective, and immunomodulatory agent capable of promoting islet-function, 2) CA impairs the alloimmune response to induce antigen-specific hyporesponsiveness, and 3) the negative vaccination protocol achieves long-term allograft tolerance.

Current immunotherapies designed to stimulate specific T cell-mediated immunity have thus far yielded modest objective clinical response rates, despite the increase of tumor-specific T cells have been observed in treated patient blood. Since the majority of tumor antigens being targeted in immunotherapies are non-mutated, self antigens, current clinical results may relate, in part, to the low-to-moderate avidity, negatively-selected T cell repertoire in patients that is being asked to regulate tumor progression. In the current thesis, I hypothesized that by conditionally enhancing the proteasomal degradation of tumor antigens, I could generate a synchronized pool of derivative peptides that could then be presented in a wave-like temporal fashion in MHC class I complexes on the tumor cell surface. For at least a transient period thereafter, I theorized that specific CD8+ T cell recognition and anti-tumor activities would be improved. I selected a family of tumor-associated antigens, receptor tyrosine kinases (RTK) for study, as their overexpression has been linked with poor clinical prognosis in many forms of cancer. In this thesis, I show that EphA2 agonists, as well as, HSP90 inhibitors effectively promote EphA2 degradation via a proteasome- dependent manner, providing the delivery of EphA2 peptides into the classical MHC class I presentation pathway. I also show that specific CD8+ T cell recognition of EphA2 peptides derived from both the extracellular and intracellular domains of this transmembrane protein was improved as a consequence of tumor cell treatment with these agents being in consistent with the use of TAP- and ER-associated degradation. Notably, the combination of both drugs further enhanced anti-EphA2 T cell recognition of tumor cells, suggesting these modalities work via complementary, but not identical mechanisms. Importantly, complete tumor eradication was achieved in vivo (in a Hu-SCID tumor model) using a combinational therapy consisting of agonist administration just prior to the adoptive transfer of human anti-EphA2 CD8+ T cells, where either single modality was minimally beneficial. Preliminary data from additional studies targeting the tumor cell-overexpressed RTKs, Her2/neu and EGFR, suggest that this core treatment paradigm may be generalizeable to many (if not all) RTKs.