The vector-borne protozoan Leishmania spp. cause the spectrum of disease known as leishmaniasis in human and animal hosts. The most common manifestations of leishmaniasis are the chronic, ulcerative skin disease cutaneous leishmaniasis (CL), and the more serious visceral leishmaniasis (VL) in which parasites take up residence in internal organs, causing death if not treated. The role of neutrophils (PMNs) in the immune response to CL and VL is unclear. It is s generally thought that PMNs are only a short-lived effector cell, and have been disregarded as playing a role in chronic Leishmania spp. infection. As both CL and VL are diseases characterized by increased inflammatory immune mediators, we hypothesized that PMNs from human or animal models of chronic leishmaniasis would display different properties from PMNs from healthy controls. We found in a subset of CL and VL patients circulating PMNs expressing HLA-DR, the human form of MHC class II, a molecule thought to be restricted primarily to professional antigen cells. When we examined PMNs recruited to CL skin lesions in human patients, or similar lesions in experimental murine model of CL, we found significantly increased MHC class II+ PMNs. Circulating HLA-DR+ PMNs also expressed the co-stimulatory molecules CD80, CD86 and CD40. While this suggested an antigen-presenting cell-like phenotype by these HLA-DR+ PMNs, compared to conventional HLA-DR- PMNs, HLA-DR+ PMNs showed not only a neutrophil-like appearance and function, but in fact increased activation, degranulation, intracellular MPO and phagocytosis of parasites and zymosan particles. Incubation of healthy control whole blood with inflammatory cytokines resulted in increased HLA-DR+ PMNs and the presence of hladrb1 mRNA, suggesting a connection between neutrophil “priming” and upregulation of HLA-DR. In addition to HLA-DR+ PMNs in CL patients, we also identified the presence of so-called “low-density” neutrophils (LD-PMNs). These neutrophils, which are enriched in low-density fractions following centrifugation of blood over a density gradient, are reported in numerous disease states, including cancer, HIV, and systemic lupus erythematosus. In some disease states, LD-PMN are reported to be immunosuppressive toward T cell activation and proliferation. However, LD-PMNs from leishmaniasis patients showed no evidence of immunosuppression. Additionally, we found that LD-PMNs show significantly increased surface expression of MHC class II, suggesting a heretofore unappreciated connection between these atypical neutrophil phenotypes. We also investigated the in vitro interactions with different Leishmania infantum life-stages, both those that cause acute infection (promastigotes) and amastigotes, which are found during chronic stages of the disease. We found that PMNs are readily infected by all L. infantum life-stages, but that amastigotes may have different methods of interacting with PMN surface receptors and are better equipped to avoid PMN anti-microbial responses. These data suggest that circulating PMNs in chronic leishmaniasis may have unique phenotypes and interact differently with the Leishmania spp. life-cycle present during chronic infection. Further investigation of the role of PMNs and atypical PMN phenotypes in chronic disease may help identify new immunomodulatory roles for this cell type.

Natural killer (NK) cells are a critical component of the immune response against viral infections. NK cell depletion prior to murine cytomegalovirus (MCMV) infections results in increased susceptibility to infection in several mouse strains. The mechanism of protection in C57Bl/6 mice is dependent on the activation of NK cells by Ly49H recognition of m157. Our previous studies have examined important residues of m157 for Ly49H recognition, as well as the contribution of m157 glycosylation to NK cell activation. However, what role the glycophosphatidyl inositol (GPI) anchor of m157 plays in Ly49H activation was unknown. Here we demonstrate that the GPI anchor of m157 regulates the surface expression of the protein. While the GPI anchor was not required for recognition of m157 by the activating or inhibitory Ly49 receptors, expression of GPI-anchored m157 resulted in greater receptor downregulation on NK cells, as well as increased NK cell cytotoxicity compared to transmembrane m157. In addition to MCMV infections, NK cells have been shown to participate in the immune response to influenza A virus (IAV). However the exact role of NK cells in IAV infection is less clear, as some studies have found NK cells to be protective, while others have shown that NK cells cause lethal immunopathology. It is likely that the severity of IAV infection may dictate the NK cell response to IAV infection (i.e. protective vs. immunopathogenic). Herein we show that NK cell accumulation in IAV-infected lungs and lung-draining lymph nodes (DLN) is regulated by the severity of IAV infection, where there is increased NK cell accumulation in the lungs during high dose IAV infection, and greater NK cell accumulation in the DLN in low dose IAV infections. Despite significant NK cell recruitment to the lung during IAV infection, as well as previously published studies demonstrating the importance of NK cells to IAV immunity, NK cell depletion prior to IAV infection did not result in a significant change in morbidity or mortality. Interestingly, NK cell depletion resulted in a significantly greater number of CD4 T cells in the IAV infected lung. Further, both CD4 and CD8 T cells in NK-depleted mice showed increased IFN-Γ production. Finally, while not statistically significant, NK cell depletion resulted in a trend toward greater protection from heterosubtypic IAV challenge infections. Taken together these results suggest that NK cells may either regulate the adaptive immune response to IAV infection through suppression of CD4 and CD8 T cells, or that the T cell response to IAV infection is able to compensate for the loss of NK cells. Moreover, while NK cell suppression of T cell function during a primary IAV infection does not result in increased susceptibility to primary IAV infections, NK cell regulation of adaptive immune responses may suppress the memory T cell response, and therefore leave the host more susceptible to secondary infections. Overall the studies presented herein demonstrate a complex role for NK cells in the immune response against viral infections. Ly49H+ NK cells directly kill MCMV-infected cells and m157-bearing targets, but NK cell activation is regulated by ligand density, as well as the ligand membrane anchor. Additionally, NK cells suppress adaptive immune responses during a primary IAV infection, resulting in changes to the T cell response during both primary and memory responses.

Germinal center (GC) reactions are central features of T cell-driven B cell responses, and the site where antibody (Ab) producing cells and memory B cells are generated. Within GCs, a range of complex cellular and molecular events occur which are critical for the generation of high affinity Abs. These processes require exquisite regulation not only to ensure the production of desired Abs, but to minimize unwanted autoreactive or low affinity Abs. To assess whether T regulatory cells (Treg) participate in the control of GC responses, immunized mice were treated with either an anti-glucocorticoid-induced TNFR-related protein (GITR) mAb or an anti-CD25 mAb to disrupt Treg activity. In both groups of treated mice, the GC B cell pool was significantly larger compared with control treated animals, with switched GC B cells composing an abnormally high proportion of the response. With these results indicating Tregs influence on GC dynamics, experiments were conducted to determine if Tregs were located in the GC, which subset of Treg was involved and by which mechanisms were their functions being effected. Within the spleens of immunized mice, CXCR5+ and CCR7- Tregs were documented by flow cytometry and Foxp3+ cells were found within GCs using immunohistology. Studies demonstrated administration of either anti-TGF-β or anti-IL-10R blocking mAb to likewise result in dysregulated GCs, suggesting that generation of inducible Tregs is important in controlling the GC response. Blockade of two Treg methods of suppression, PD-1/PD-L1 pathway and CTLA-4, also resulted in disrupted GCs, indicating the possible use of them for suppression by Treg. Collectively, these findings indicate that Tregs contribute to the overall size and quality of the humoral response by controlling homeostasis within GCs.

CD40 is a TNF receptor superfamily (TNFRSF) member central to the development of many aspects of the adaptive immune response. CD40 signaling promotes adaptive immunity in part by inducing the expression of cytokines, chemokines, and various adhesion and co-stimulatory molecules. The family of cytoplasmic adapter proteins, the TNFR-associated factors (TRAFs), serve as major mediators of TNFRSF pathways. CD40 regulates itself in part via the signaling induced degradation of TRAF2 and TRAF3. However, the effect of CD40-induced TRAF degradation on other TRAF dependent pathways is unknown. Here I provide evidence that CD40-mediated degradation of TRAFs 2 and 3 also influences the responsiveness of immune cells to CD40-independent, TRAF2- and 3-dependent pathways. LMP1 is a functional mimic of CD40, but signals to B lymphocytes in an amplified and sustained manner. LMP1 contributes to the development of B cell lymphoma in immunosuppressed patients, and may exacerbate flares of certain autoimmune diseases. The cytoplasmic (CY) domain of LMP1 binds TRAF2 with lower avidity than the CY domain of CD40, and TRAF2 is needed for CD40-mediated degradation of TRAFs 2 and 3. LMP1 doesn't induce TRAF degradation, and employs TRAF3 as a positive mediator of cell signaling, whereas CD40 signals are inhibited by TRAF3. Here, I tested the hypothesis that relative affinity for TRAF2, and/or distinct sequence differences in the TRAF2/3 binding sites of CD40 vs. LMP1, controls the disparate ways in which CD40 and LMP1 use TRAFs 2 and 3. The results revealed that TRAF binding affinity and TRAF binding site sequence dictate a distinct subset of CD40 vs. LMP1 signaling properties. The E3 ubiquitin ligases, cIAP1 and cIAP2, have been reported to play a crucial role in CD40 signaling. Because LMP1 is a mimic of CD40 signals, I hypothesized that LMP1 requires the cIAPs for signaling. To elucidate the role of the cIAPs in CD40 and LMP1 signaling, I specifically depleted the cIAPs and found that the cIAPs are differentially utilized in CD40 and LMP1 signaling. I also sought to further the understanding of the molecular underpinnings of how CD40, but not LMP1 signaling induces TRAF2 and TRAF3 degradation upon signaling. To do this, I investigated the ability of various CD40 and LMP1 mutants to induce TRAF degradation in distinct TRAF or cIAP deficient models. I found that neither a high TRAF2 binding potential nor the presence of the cIAP molecules are required for this process. Thus, this work reveals important insights into the molecular mechanisms of and role of CD40-mediated TRAF degradation in the immune system.

Antigen-specific CD8 T cells play a critical role in protecting the host from infection by intracellular pathogens including viruses, bacteria and parasites. During the course of an infection, antigen-specific CD8 T cells undergo proliferative expansion to increase in number, which is followed by contraction and generation of a stable pool of long-lived memory cells. Importantly, memory CD8 T cells provide enhanced resistance to re-infection by the same pathogen. Moreover, the number of memory CD8 T cells correlates strongly with the level of protection against re-infection. Therefore, vaccines designed to promote cellular immunity should logically focus on achieving sufficiently high number of these memory cells for protection. Most current vaccines have relied on inducing antibodies to protect the host by neutralizing pathogens or blocking pathogen entry into the cells. However, there is a recognized need to design vaccines that also stimulate a strong CD8 T cell component of the adaptive immune response in addition to antibodies. Importantly, inflammatory cytokines induced by infection or vaccination with adjuvant act directly or indirectly on CD8 T cells to modulate their expansion, contraction and acquisition of memory characteristics. Thus, an understanding of how inflammatory cytokines regulate CD8 T cell memory differentiation may help guide the strategies for rational vaccine design. My studies examine the roles of inflammatory cytokines in regulating CD8 T cell memory differentiation. Specifically, my studies investigate the timing of inflammatory cytokine exposure and the role of type I IFNs and IL-12 in regulating effector/memory CD8 T cell differentiation, and exploiting the cross-presentation pathway to rapidly generate protective CD8 T cell immunity. Specifically, my results indicate that (i) encounter with inflammatory cytokines during the rapid proliferative phase deflects CD8 T cell differentiation away from memory towards a sustained effector program, (ii) that direct signaling by either type I IFN or IL-12 to the responding CD8 T cells promotes maximal expansion, but neither of these cytokines is essential to regulate the effector/memory differentiation program, and (iii) cross-priming with both cell-associated antigen and antigen-coated, biodegradable microspheres, accelerates CD8 T cell memory development that can be exploited to rapidly generate protective CD8 T cell immunity.

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis-induced hospitalization in young children. A natural RSV infection fails to elicit long-lasting immunity, further increasing the need for an effective vaccine. Despite the significant healthcare burden, there is no licensed RSV vaccine currently available. While most RSV vaccine strategies focus on the induction of humoral immunity, high antibody titers do not prevent RSV infection. It remains unclear if protective immunity can be achieved through robust cellular immunity. Previous work has indicated that a relatively low frequency of virus-specific CD8 T cells is induced following an RSV infection in human infants. In addition, RSV-specific memory CD8 T cells diminish to almost undetectable frequencies in the blood of the elderly. The lack of long-lasting immunity against RSV may be explained by an absence or low frequency of memory CD8 T cells within the lung following infection. However, I determined that the majority of effector CD8 T cells reside within the lung tissue following infection with either RSV or influenza A virus (IAV), both of which replicate primarily in the airways. In addition, approximately 70% of antigen-experienced memory CD8 T cells persist in the lung tissue at day 30 following RSV infection. In contrast, the majority of CD8 T cells remain in the pulmonary vasculature following intranasal infection with either of the systemically replicating viruses lymphocytic choriomeningitis virus or vaccinia virus. Therefore, the tissue tropism of a virus will determine if CD8 T cells preferentially accumulate in the lung tissue following infection of the respiratory tract. An experimental formalin-inactivated RSV (FI-RSV) vaccine caused enhanced respiratory disease in vaccinated children following a natural RSV infection. Incomplete knowledge of the underlying immunological mechanisms that were responsible for mediating the enhanced disease has greatly hampered vaccine development. Previous studies have indicated that eosinophils, non-neutralizing antibodies, and CD4 T cells may be required to elicit FI-RSV vaccine-enhanced disease. I determined that distinct CD4 T cell subsets mediate individual disease parameters. The Th2-biased immune response, but not eosinophils specifically, was responsible for induction of airway hyperresponsiveness and mucus hypersecretion. On the other hand, the Th1-associated pro-inflammatory cytokine TNF-α was required to mediate baseline pulmonary dysfunction and weight loss. Lastly, while depletion of CD4 T cells ameliorated all disease parameters evaluated, the antibody titers remained unaltered in depleted mice. Thus, antibodies induced by FI-RSV immunization were not required for vaccine-enhanced disease. My data demonstrate that discrete disease manifestations associated with FI-RSV immunization are orchestrated by distinct subsets of CD4 T cells. The CD8 T cell response is believed to contribute to both pathogen clearance and immunopathology following an acute RSV infection. However, it is unclear if robust memory CD8 T cell responses will protect against an RSV infection. I determined that induction of a high-magnitude, epitope-specific memory CD8 T cell pool mediated increased viral clearance following RSV challenge. However, mice with robust secondary CD8 T cell responses exhibit increased airway dysfunction, weight loss, and mortality as compared to mock-immunized mice undergoing an acute RSV infection. The enhanced disease severity was unique to the context of an RSV infection as similarly immunized mice were protected from chge with a lethal dose of a recombinant IAV engineered to express an RSV-derived epitope. In addition, the increased morbidity and mortality was associated with an elevated amount of both IFN-γ and TNF-α in the serum of immunized mice. Neutralization of either IFN-γ or TNF-α led to a significant reduction in disease severity and survival of all mice. These results demonstrate that robust memory CD8 T cell responses enhance viral clearance, but also lead to severe pulmonary immunopathology following RSV infection. Overall, I establish that the majority of effector CD8 T cells are localized within the lung tissue following a respiratory infection, and determine that either memory CD4 or CD8 T cell responses elicits severe immunopathology following a RSV infection.

Prostaglandins (PG) are ubiquitous lipid mediators that play key roles in pathophysiological responses to infections. They are considered to have both pro and anti-inflammatory roles depending upon the time of inflammation, the receptors that they bind to and the tissues that they act upon. Hence given their pleiotropic effects, a perfect balance between the pro and anti-inflammatory functions of PGs are required to ensure that a controlled timely immune response is elicited to mediate protection and to avoid immunopathology. PGD2 is one such PG that was reported to increase with age in the lungs of mice and to mediate an anti-inflammatory effect thereby blunting the immune response following Severe Acute Respiratory Syndrome – Coronavirus (SARS-CoV). Increase in PGD2 with age incapacitates respiratory dendritic cells (rDC) to migrate from lungs to the draining lymph node following SARS-CoV infection due to down regulation of CCR7 (a receptor for chemokines CCL19/21). Migration of rDCs to draining lymph nodes requires high expression of CCR7 and it's binding to CCL19/21, a chemokine that mediates migration of dendritic cells along its gradient. Although increase in levels of PGD2 might prove beneficial in high inflammatory conditions, it should be noted that high levels of such a potent anti-inflammatory mediator during the initiation of an immune response could prove detrimental. In chapter II of this thesis I show that age-related increases in oxidative stress result in the upregulation of a single phospholipase (PLA2) group II D (G2D) (PLA2G2D) with anti-inflammatory roles. PLA2G2D functions by releasing Arachidonic acid (AA) from the lipid membrane, which will be further metabolized to other pro-resolving/ anti-inflammatory lipid mediators including PGD2. I show that inducing oxidative stress in young mice as well as in human peripheral blood macrophages, results in the upregulation of PLA2G2D (probably as a counter mechanism against oxidative stress). Also increase in the expression levels of this gene during the course of SARS-CoV infection results in the upregulation of PGD2, which is completely abrogated in Pla2g2d-/- mice. I also show Pla2g2d/- middle-aged mice have low levels of PGD2 and that they are capable of mounting a strong immune response and survive the otherwise lethal SARS-CoV infection. PGD2 is also a major PG in the brain and its role has been investigated in many non-infectious setting such as stroke and Alzheimer' disease. The PGD2 binding to one of its receptors DP1 has been shown to have primarily a neuro-protective role. In chapter III, I show that PGD2/DP1 signaling has beneficial effects in the brain of mice infected with a neurotropic strain of murine hepatitis virus (MHV) (rj2.2). In agreement with the neuro-protective role of PGD2, at least 60% of DP1-/- mice succumb to a sublethal dose of rj2.2. rj2.2 infection in these mice is characterized by a delay in the induction of IFN I response and lower activation status of microglia and macrophages in the brain. I also show that abrogation of DP1 signaling results in global defects in the immune system response to infection. Notably, a genome wide expression analysis using microarray, shows that a gene, Pydc3 with putative inflammasome inhibiting function is upregulated in WT mice compared to DP1-/- mice in the CD11b population of cells which primarily comprises microglia and macrophages. In line with the predicted function of Pydc3, DP1-/- mice have higher frequency and number of IL-1β+ producing microglia in the brain. Studies are underway to determine the exact role of DP1 signaling in Pydc3 expression as well as the role of this gene in inflammasome function. Overall these studies emphasize the immuno-modulatory roles of PGs in the context of a viral infection. Thus, altering the levels of these lipid mediators at appropriate times during the course of infection might prove useful as an effective therapeutic strategy to decide the fate of an infection.

Cancer immunotherapy has proven to be challenging as it depends on overcoming multiple mechanisms that mediate immune tolerance to self-antigens. In situ immunization is based on the concept that it is possible to break immune tolerance by inducing tumor cell death in situ in a manner that provides antigen presenting cells such as dendritic cells (DCs) with a wide selection of tumor antigens that can then be presented to the immune system and result in a therapeutic anticancer immune response. Based on recent advances in the understanding of antitumor immunity, we designed a three-step approach to in situ immunization to lymphoma: (1) Inducing immunogenic tumor cell death with the chemotherapeutic drug Doxorubicin (Dox). Dox enhances the expression of "eat-me" signals by dying tumor cells, facilitating their phagocytosis by dendritic cells (DCs). Due to the vesicant activity of Dox, microparticles (MPs) made of PLGA (a biodegradable polymer) can safely deliver Dox intratumorally and are effective vaccine adjuvants; (2) Enhancing antigen presentation and T cell activation using anti-OX40; (3) Sustaining T cell responses by checkpoint blockade using anti-CTLA-4. In vitro, Dox MPs were less cytotoxic to DCs than to B lymphoma cells, did not require internalization by the lymphoma cells, and significantly enhanced phagocytosis of tumor cells by DCs as compared to soluble Dox. In mice, this three-step therapy induced CD4- and CD8-dependent systemic immune responses that enhanced T cell infiltration into distant lymphoma tumors leading to their eradication and significantly improving survival. Our findings demonstrate that systemic antitumor immune responses can be generated locally by three-step therapy and merit further investigation of three-step therapy for immunotherapy of lymphoma patients. Furthermore, we designed another in situ immunization approach using PLGA MPs loaded with both Dox and CpG oligodeoxynucleotides (CpG). The addition of CpG was to further enhance the Dox MP design by including an agent that addresses Step Two in situ, by enhancing tumor antigen presentation by DCs. In vitro, we show that Dox/CpG MPs can kill B and T lymphoma cells and are less toxic to DCs than soluble Dox. In vivo, Dox/CpG MPs combined with anti-CTLA-4 and anti-OX40 generated systemic immune responses that suppressed injected and distant tumors in a murine B lymphoma model, leading to tumor-free mice. The combination regimen was also effective at reducing T cell lymphoma and melanoma tumor burdens. In conclusion, Dox/CpG MPs represent a versatile, efficient and safe tool for in situ immunization that could provide a promising component of immunotherapy for patients with a variety of types of cancer.

The severe disease associated with seasonal epidemics of influenza A virus (IAV), as well as pandemic outbreaks, have highlighted the necessity for novel, broadly cross-reactive vaccination and therapeutic strategies against IAV. Our studies have focused on the contribution of IAV-specific CD8 T cells to mediating protection following IAV vaccination and infection as IAV-specific CD8 T cells are required for clearance of IAV. Further, IAV-specific CD8 T cells are typically cross-protective as they are generally directed at highly conserved areas of IAV. Recently, influenza virus-like particles (VLPs) have been developed from recombinant baculoviruses containing influenza proteins hemagglutinin (HA) and/or neuraminidase (NA) on the surface and matrix (M1) in the VLP core. Influenza VLPs induce potent antibody responses and have been shown to provide protection from morbidity and mortality during lethal homo- and hetero-subtypic IAV challenge. This suggests that conserved, VLP-induced CD8 T cell responses may also contribute to the overall protective ability of VLPs. However, whether influenza VLPs can induce influenza-specific CD8 T cell responses and if these T cells are protective during IAV challenge remains unknown. Here, I demonstrate that a single, intranasal vaccination with VLPs containing HA and M1 leads to a significant increase in HA533-specific CD8 T cells in the lungs and lung-draining lymph nodes. Our results also indicate that HA533-specific CD8 T cells primed by influenza VLP vaccination are significantly increased in the lungs following lethal IAV challenge. These VLP-induced memory CD8 T cells are critical in providing protection from lethality following subsequent challenge infections, as depletion of CD8 T cells leads to increased mortality, even when total, but not VLP-induced memory, CD8 T cell numbers have been allowed to recover prior to lethal dose IAV challenge. In addition, my studies also importantly demonstrate that these VLP-induced, HA533-specific CD8 T cells aid in protection from high-dose, heterosubtypic IAV infections where CD8 T cell epitopes are conserved, but the targets of neutralizing antibodies have been destroyed. This dissertation further elucidates the requirements for the regulation of the IAV-specific CD8 T cell response in the periphery (i.e. lung) by pDC and CD8α+ DC. Our studies have previously demonstrated that pDC or CD8α+ DC must present viral antigen in the context of MHC class I along with trans-presentation of IL-15 to effector, IAV-specific CD8 T cells in the lungs to protect the T cells from apoptosis and allow generation of the full magnitude CD8 T cell response needed to clear IAV infection. Herein, I demonstrate that in addition to antigen presentation and IL-15, costimulatory molecules on the surface of pDC and CD8α+ DC are also required. However, the specific costimulatory molecules required depends upon both the mouse strain utilized for IAV infection as well as DC subset. In addition to costimulatory molecules, I also demonstrate that the requirement for pDC and CD8α+ DC to be infected differs in order for them to participate in this pulmonary rescue of the IAV-specific CD8 T cell response. While CD8α+ DC are able to efficiently cross-present exogenous antigen, pDC must be directly infected and utilize the endogenous, direct antigen presentation pathway to present viral antigen to IAV-specific CD8 T cells in the lungs during IAV infection. These data suggest there are distinct differences between pDC and CD8α+ DC in their mechanism of regulating the pulmonary IAV-specific CD8 T cell response, which had not been previously appreciated. Together, the results presented herein further detail the mechanism of regulation of effector IAV-specific CD8 T cells by DC as well as the contribution of IAV-specific CD8 T cells to a novel, IAV VLP vaccination strategy. These findings highlight the importance of IAV-specific CD8 T cells in mediating protection following IAV vaccination and infection.

Influenza A virus (IAV) is a major cause of serious respiratory illness worldwide, leading to approximately 5 million severe cases and 500,000 deaths per year. Given the disease severity, associated economic costs, and recent appearance of novel IAV strains, there is a renewed interest in developing novel and efficacious “universal” IAV vaccination strategies as well as therapeutic remedies. Previous studies from our laboratory have concentrated on IAV-specific CD8 T cell-mediated protection against IAV infection as IAV-specific CD8 T cells are needed for efficient clearance of virus. Recent studies highlight that immunizations capable of generating local (i.e., nasal mucosa and lung) tissue-resident memory T and B cells in addition to systemic immunity offer the greatest protection against future IAV encounters. Current IAV vaccines are designed to largely stimulate IAV-specific antibodies, but do not generate the lung-resident memory T and B cells induced during IAV infections. In order to effectively generate lung-resident memory populations, it is believed a local antigen depot is needed as tissue-resident memory formation is enhanced by the presence of local antigen. Recently, polyanhydride nanoparticles have been demonstrated to slowly release their contents at the site of inoculation serving as an antigen depot. However, the ability of an intranasal vaccination with polyanhydride nanoparticles to induce IAV-specific lung-resident immune responses and provide protection against subsequent IAV infection has not been determined. Here, I report on the intranasal administration of a biocompatible polyanhydride nanoparticle-based IAV vaccine (IAV-nanovax). IAV-nanovax is capable of providing protection against subsequent homologous and heterologous IAV infections in both inbred and outbred populations. My findings demonstrate that vaccination with IAV-nanovax promotes the induction of germinal center B cells within the lungs that are associated with both systemic IAV-specific IgG as well as local lung IAV-specific IgG and IgA antibodies. Furthermore, intranasal IAV-nanovax vaccination leads to a significant increase in IAV-specific CD4 and CD8 T cells within the lung vasculature as well as in the lung tissue. Most importantly, my studies demonstrate that IAV-nanovax induced lung-resident IAV-specific CD4 and CD8 T cells express canonical tissue-resident memory markers. This dissertation further explores a novel regulation pathway previously identified by our laboratory where plasmacytoid dendritic cells (pDCs) eliminate IAV-specific CD8 T cells early during high-dose and high-pathogenic IAV infections in a FasL:Fas (pDCs:CD8 T cell) dependent manner. However, recent studies suggest that B cells are the predominate lymphocyte to express FasL in mice. Here, I demonstrate that FasLpos B cells greatly outnumber FasLpos pDC within the lung draining lymph nodes (dLNs) during IAV infections. Interestingly, my results demonstrate the presence of two subsets, CD11cpos and CD11cneg, of FasL-expressing B cells that differentially influence the IAV-specific CD8 T cell response during high-dose IAV infections. While CD11cneg B cells kill IAV-specific CD8 T cells, contributing to lethality during high-dose IAV infections, CD11cpos B cells may instead be protective. In addition to the negative impacts of high-dose IAV infections, I also demonstrate that chronic ethanol (EtOH) consumption detrimentally impacts existing IAV-specific CD8 T cell memory responses. Here, my results reveal that chronic EtOH consumption causes a numerical loss in existing IAV-specific CD8 T cell memory responses. This numerical loss in existing IAV-specific CD8 T cell memory is associated with a reduction in cytotoxic activity within the lungs as well as an increase in morbidity and mortality during a secondary IAV challenge. Together, the results presented herein demonstrate the ability of a novel polyanhydride nanovaccine to induce robust pulmonary IAV-specific T and B cell responses and further our understanding of factors that can negatively impact IAV-specific CD8 T cells as well as protection against IAV infection. Overall these findings highlight the importance of IAV-specific CD8 T cells, as well as CD4 T cells and B cells, in providing protection against IAV infections.