Chronic viral infections afflict hundreds of millions of people worldwide, representing a huge financial and health burden. The interleukin-6 family cytokines are potent immune modulators, which signal through a common gp130 co-receptor. IL-27, a novel member of the IL-6 family, is a pleotropic cytokine with both activating and immunosuppressing roles in antiviral immunity. Previous research from our lab showed immune response defects to LCMV viral infection in IL27R-/- mice including decreased type-I interferon, reduced antibody production, and loss of viral control. To assess the temporal and therapeutic importance of IL-27, I treated mice with IL-27 or blocked its receptor and monitored host responses to chronic LCMV infection. Neither IL-27 administration nor IL-27R blockade altered the immune responses to LCMV. In contrast, studies with IL27R-/-IL6R-/- mice recapitulated the IL27R-/- immune defects. Comparisons between IL27R-/-IL6R-/- mice, and previously published L6R-/- mice and IL27R-/- mice showed regulation of several immune responses against LCMV to be under control of Interleukin-27 including T cell accumulation, viral specific T cell maintenance and IgG production. Taken together, our work demonstrates an important role for IL-27 in viral control.

During chronic infections, sustained cell adaptation has been mostly studied in the adaptive immune compartment but much less is known on how innate immune cells adjust to a persistently infectious milieu. Particularly, dendritic cells (DCs), which are central players in immune responses, adapt during chronic infections. However, the underlying mechanisms remain largely unknown. Thus, to understand how short-lived innate cells adapt to lifelong persistent infections, we first studied plasmacytoid DCs (pDCs), which specialize in Type I Interferon (IFN-I) production and often become functionally exhausted in chronic settings. Using a murine chronic viral infection model of lymphocytic choriomeningitis virus (LCMV), we found that bone marrow pDC progenitors exhibited quantitative and qualitative defects and failed to generate functional pDCs ex vivo in an IFN-I-dependent manner. Exhausted pDC numbers were, however, maintained by peripheral self-renewal via sustained proliferation of CD4- pDC subsets that was induced by IFN-I receptor and Toll-like receptor 7 (TLR7) signaling in a cell-intrinsic manner. In contrast, functional impairment of exhausted pDCs was independent of IFN-I receptor signaling. We further studied mechanisms underlying adaptation of progenitors, that can give rise to conventional DCs (cDCs) as well as pDCs, by determining the transcriptional and chromatin landscapes of bone marrow DC progenitors from LCMV-infected mice and applying these datasets to Taiji algorithm to predict the activity of transcription factors (TFs). We found that, Glucocorticoid Modulatory Element Binding Protein 1 (Gmeb1), which was predicted to exhibit increased activity in progenitors during LCMV infection, suppressed pDC development in a glucocorticoid-dependent manner. Further studies revealed that glucocorticoid suppressed pDC development during LCMV infection. Gmeb1 also promoted development but suppressed maturation of cDC1s in a glucocorticoid-independent manner. Overall, our work provides a framework to understand how innate immune adaptation can be triggered and sustained during chronic viral infection. Moreover, by highlighting novel TF regulators of DC progenitors and their progeny, our work enhances our understanding of DC biology and unveils potential therapeutic targets to harness DCs.

Viral infections elicit host adaptations that can enable host-pathogen equilibrium. Here, we investigated adaptations of the gastrointestinal tract elicited by a persistent virus, their underlying mechanisms and their implications for the infected host. By using murine infection with lymphocytic choriomeningitis virus (LCMV) we demonstrated that infection with a persistent viral isolate led to long-term viral replication in hematopoietic and mesenchymal, but not epithelial cells (IEC) in the intestine. Viral persistence drove sustained intestinal epithelial barrier leakage, which was characterized by increased paracellular flux of small molecules and was associated with enhanced colitis susceptibility. IFN-I signaling caused tight junction dysregulation in IEC, promoted gut microbiome shifts and enhanced intestinal CD8 T cell responses. Notably, both IFN-I receptor blockade and CD8 T cell depletion prevented infection-induced barrier leakage. Our results demonstrated that infection with a virus that persistently replicated in intestinal mucosa increased epithelial barrier permeability and revealed IFN-I and CD8 T cells as causative factors of intestinal leakage during chronic infections. We further discovered that mouse infection with the fast-spreading and persistent (but not the slow-spreading acute) isolate of LCMV induced large-scale microbiome shifts characterized by increased Verrucomicrobia and reduced Firmicute/Bacteroidetes ratio. Remarkably, the most profound microbiome changes occurred transiently after infection with the fast-spreading persistent isolate, were uncoupled from sustained viral loads and were instead largely caused by CD8 T cell responses and/or CD8-T-cell-induced anorexia. Among the taxa enriched by infection with the fast-spreading virus, Akkermansia muciniphila, broadly regarded as a beneficial commensal, bloomed upon starvation and in a CD8-T-cell-dependent manner. Strikingly, oral administration of Akkermansia muciniphila suppressed selected effector features of CD8 T cells in the context of both infections. Our findings define unique microbiome differences after chronic versus acute viral infections and identify CD8 T cell responses and downstream anorexia as driver mechanisms of microbial dysbiosis after infection with a fast-spreading virus. Our data also highlight potential context-dependent effects of probiotics and suggest a model in which changes in host behavior and downstream microbiome dysbiosis may constitute a previously unrecognized negative feedback loop that contributes to CD8 T cell adaptations after infections with fast-spreading and/or persistent pathogens.

This study presents an in vitro model of regulatory T cell induction and explores the impact of eosinophils on developing naïve T cell populations. The research is relevant to the type 2 helper T cell mediated allergic response. As regulatory T cells are normally responsible for the suppression of other helper T cells, including type 2 cells, it serves an essential role in preventing allergic responses. The polarization between helper and regulatory T cell is only one part of allergy, which first needs an antigen presenting cell to process and present allergens to naïve T cells. In either case, eosinophils seem to have a limited scope in allergy compared to T cells. However, eosinophils have a unique feature, in that they can be induced to present antigen to CD4 T cells. Aspergillus fumigatus, a common fungal allergen, is a prime candidate to study the effects of an allergic response. Along with its ubiquitous nature, it has been known to generate varying effects on eosinophils and regulatory T cells. Eosinophils themselves have been established as mere indicators of allergy, but the actual influence that eosinophils have on other T cells in an allergic response is still unclear.

Dendritic cells are professional antigen presenting cells that activate naïve T cell to promote specific T cell responses. Until recently, the DCs’ recognition of microbial or inflammatory stimuli through PRRs was solely recognized to initiate DCs’ maturation. On the other hand, the role of PRR-independent activation of DCs has remained largely unexplored. This thesis will focus on a recent discovery of cAMP dependent DCs’ maturation. We treated various types of DCs with GPCR ligands and other regulators affecting GPCR/cAMP pathway. Here, we show that DCs’ varying levels of cAMP determine DCs’ ability to promote either Th2 responses or Th17 responses. We discovered that the increased cAMP level reduced the expression of DC’s transcription factors like IRF4 and KLF4. Subsequently, DC’s increased cAMP level reduced Th2 responses and increased Th17 responses. In fact, this cAMP dependent DC maturation is so robust that inducing cAMP signaling alone allowed Th2-promoting DC to switch into Th17-promoting DC. Surprisingly, we discovered that DC’s expression of IRF5 and inhibition of IRF4 are essential in Th17 polarization. In addition, we also described the implication of GPCR/cAMP pathway’s role in the onset of neutrophilic asthma. Th2 response in the airway is known to promote eosinophilic asthma while additional Th17 response induce neutrophilic asthma. Here, we show that a long-term

exposure to asthma’s common bronchodilator, LABA, also switches Th2 mediated eosinophilic asthma into Th17 mediated neutrophilic asthma by elevating DC’s cAMP level. These findings delineate an unforeseen contribution of cAMP signaling in the regulation of innate and adaptive immunity.

Little is understood about the genesis of autoimmune diseases caused by deficiencies in TAM receptors (Tyro3, Axl, Mer). One of the causes of autoimmunity could be defective thymic education. Our goal was to elucidate the localization and role of TAM receptors and their ligands within the thymus. We show that Axl and Mer, but not Tyro3, are expressed within the thymus. Through immunohistochemistry, we identify Axl and Mer expression primarily on macrophages in both the medulla and cortex. Using knockout mice, we show that Axl and Mer perform necessary but redundant roles in the elimination of basally accumulated apoptotic thymocytes. The absence of both receptors leads to immense apoptotic cell accumulation within thymic cortex and medulla.These changes result in minor morphological defects as the weight but not the ratio of medulla to cortex was affected in the Axl-/-Mertk-/- mice. We further use this model to instigate Axl dependence on Gas6 as a ligand in thymus. Additionally, we show that during acute thymocyte apoptosis, Mer but not Axl is critical in cellular clearance.

B cells are an important facet of the immune response against chronic viral infection. In the first chapter of this thesis, we studied the role of gut microbiome composition on B cell responses to chronic lymphocytic choriomeningitis virus clone 13 (LCMV Cl13) infection in mice. We used genetically similar mice from two different vendors, Taconic Biosciences and The Jackson Laboratory, that have been established to contain dramatically different intestinal microbiome compositions. We found that mice from Taconic Biosciences differed from mice from The Jackson Laboratory in the accumulation of B cells in the blood and spleen at certain timepoints during persistent LCMV Cl13 infection. Co-housing mice from different vendors resulted in the partial transfer of B cell accumulation in the blood and spleen and also resulted in impaired viral clearance. In the second chapter, we investigated the role of late B cell-derived IL-27 on B cell responses to persistent LCMV infection in mice. We used genetic systems to modulate the availability of the IL-27p28 subunit in an inducible manner specifically in B cells. Late deletion of B cell-derived IL-27p28 resulted in subtle increases in the accumulation of IgD-IgM+ non-naïve B cells and IgD-IgM- class-switched B cells, which peaked by 60 days post-infection. At even later timepoints post-infection, B cell-derived IL-27p28 deletion resulted in increases in the accumulation of total B cells. Overall, our results suggest that B cell responses to persistent viral infection are modulated by gut microbiome composition and may be downregulated by B cell-derived IL-27.

The transition from naïve to effector CD8+ T cells is marked by metabolic reprograming that requires the interplay of different organelles to maintain cellular homeostasis amid changing intracellular demands and extracellular conditions. Previous studies have demonstrated that peroxisomes have important functions in fatty acid oxidation, regulating reactive oxygen species, and propagating type I interferon signals. However, up to this point, little is known about the importance of this organelle in CD8+ T cells. Thus, this thesis addresses this gap in our understanding of peroxisomes in CD8+ T cell development and function.viii To address this, we generated a mouse model of peroxisome deficient CD8+ T cells and analyzed their development both in-vitro and during acute and chronic lymphocytic chronic meningitis virus (LCMV) infection. Peroxisome-deficient CD8+ T cells behaved identically to wild-type controls in-vitro. However, in-vivo, the lack of this organelle resulted in a significant impairment of their ability to survive beyond day four post-infection. Subsisting peroxisome- deficient CD8+ T cells retained their ability to undergo normal proliferation, function, and differentiation into effector and memory lineages. These results show that peroxisomes are required for viability in-vivo. Further research needs to be conducted to uncover the mechanisms by which peroxisome deficiency is detrimental to CD8+ T cell survival.