One of the hallmarks of the autoimmune disease systemic lupus erythematosus (SLE) is the production of pathogenic anti-nuclear antibodies (ANA). These autoantibodies are the result of a break in tolerance that allows for the activation and differentiation of autoreactive B cells into antibody-secreting plasma cells. ANA can form immune complexes with self nucleic acids, which can bind to and deposit in small blood vessels and instigate immunopathology. The CD45E613R knock-in model of SLE causes different phenotypic consequences on several murine genetic backgrounds, indicating that this signaling mutation is sensitive to genetic modifiers. Despite similar dysregulation of phosphatase activity, CD45E613R mice on a C57BL/6 (B6) background are resistant to SLE phenotypes, while CD45E613R.BALB/c are sensitive to ANA but not end organ damage. In an unbiased screen for genetic modifiers of ANA, we identify TLR9 as a putative modifier for the production of a specific ANA subtype, anti-dsDNA, in the context of CD45E613R.

Here, we examine whether TLR9 modifies autoantibody production in the CD45E613R model between the resistant CD45E613R.B6 and sensitive CD45E613R.BALB/c model. Since CD45E613R alters Src family kinase and immunoreceptor signaling in several immune lineages, we generated a series of mixed bone marrow chimeras in the sensitive BALB/c genetic background to test which lineages require CD45E613R. TLR9 is also broadly expressed, so we examined which cell lineages require TLR9 and whether CD45E613R and TLR9 are necessary in cis or trans for anti-dsDNA IgG production in the BALB/c background. Upon finding that both CD45E613R and TLR9 are necessary in B cells for autoreactivity, we examined B cells from both genetic backgrounds. TLR9 stimulation induces elevated signaling in B cells from B6 mice compared to BALB/c, and these signals are not mediated by CD45E613R or co-stimulation via the BCR. We find that TLR9 negatively regulates ANA production in resistant CD45E613R.B6 mice by altering central B cell tolerance. In contrast, TLR9 positively regulates ANA production in BALB/c mice, likely because the strength of signal downstream of TLR9 is not sufficient for tolerance. These results indicate that TLR9 is a genetic modifier of autoreactivity in the context of dysregulated CD45 signaling.

The mammalian immune system can mount several different types of innate and adaptive responses. The choice of the type of immune response depends on a variety of factors, including which innate immune receptors recognize conserved pathogen molecules on the invading pathogen. How allergens and other type 2 stimuli, such as parasitic worm infections, induce type 2 immunity have been intensely studied, but it is still unclear which pathways are critical for driving this type of immune response. Interestingly, although Toll-like receptor (TLR) stimulation often induces type 1 immune responses during bacterial or viral infections, it can also promote type 2 immune responses in the lung in some circumstances, and this may be important for development of asthma. The work presented in this dissertation investigate the mechanisms by which two different TLR ligands induce divergent immune responses in the lungs.

In Chapter 3, I describe experiments using an intranasal (i.n.) sensitization and challenge model to characterize and understand how a synthetic TLR9 ligand, unmethylated cytosine followed by guanosine (CpG) oligodeoxyribonucleotide (ODN), which has great promise of being a potent adjuvant in vaccines and an immunomodulator clinically, induces an immune response in the lungs. I demonstrated that the sensitization i.n. with the model antigen ovalbumin (OVA) plus CpG ODN as adjuvant, and subsequent rechallenge with OVA led to a Th1 response in the lungs and increased serum OVA-specific IgG2c. Early after CpG ODN and OVA i.n. administration, increased levels mRNA encoding IL-12 p40, and increased numbers of dendritic cells (DCs) and monocytes were present in the lungs. CpG ODN treatment led to the elevation of costimulatory molecules on DCs and monocytes in the lungs, and on the migratory DCs in the draining mediastinal lymph node (LN). I.n. CpG ODN was also able to induce early IFN-gamma production in both innate-like lymphocytes such as gamma delta T cells, natural killer (NK) T cells, and NK cells, and also in adaptive CD4 T cells and CD8 T cells. Strikingly, these innate and adaptive immune responses were dependent on myeloid differentiation primary response 88 (MyD88) signaling in DCs, and most production of IFN-gamma was dependent on IL-12, which was produced during the innate phase of the response. Based on these results, we suggest that DCs directly sense CpG ODN in the lungs via TLR9 and that this recognition both induces their maturation and their production of IL-12, which promotes innate production of IFN-gamma and the Th1 response.

Chapter 4 describes studies in which CpG ODN and the TLR5 and TLR11 ligand, flagellin, were compared to examine how TLRs can promote different adaptive immune responses in the lungs. In contrast to mice i.n. sensitized with CpG ODN plus OVA, those sensitized with flagellin plus OVA exhibited an innate inflammatory response dominated by neutrophils and monocytes, developed a predominant Th2 response to OVA, and made substantial amounts of IgE anti-OVA antibodies. Antigenic re-challenge of the mice sensitized with flagellin plus OVA, but not the mice sensitized with CpG ODN plus OVA, led to a vigorous lung inflammation dominated by eosinophils, as expected for a Th2 response. Strikingly, the early cytokine responses in the lung to the two different TLR ligands showed a number of important differences. CpG ODN induced much higher levels of mRNA encoding IL-12 p40, whereas, flagellin preferentially induced TSLP mRNA and secretion of the mature form of IL-33, and also induced elevated levels of mRNAs encoding IL-1-alpha and IL-1-beta. In both cases, migratory DCs in the draining LN after stimulation with ligands for either TLR5/TLR11 or TLR9 had strong induction of the costimulators CD80 and CD86, but only CpG ODN also induced upregulation of CD40. Interestingly, MyD88 signaling in DCs was partially required for the flagellin-induced upregulation of CD80 on migratory DCs, and for the IL-4 production by CD4 T cells in the draining LN on d6. These results indicate that although TLR5, TLR11, and TLR9 all signal via the signaling adaptor MyD88, the innate cytokine response to these TLRs is quite different, and the distinctive innate cytokine production results in distinctive polarization of the adaptive humoral and cell-mediated immune responses.

These studies have provided insight into how TLR stimulation can drive Th1 and Th2 responses in the lungs, and how complex immune responses occur in the lungs, which have important implications for understanding infectious diseases and asthma, and for vaccine design.

Studies of genetic factors associated with human autoimmune disease suggest a multigenic origin of susceptibility, however, how these factors interact and through which tolerance pathways they operate generally remain to be defined. One key checkpoint occurs through the activity of the Aire gene which promotes central T cell tolerance. Recently, a variety of dominant-negative human AIRE mutations have been described which likely contribute to human autoimmunity more so than previously thought. The penetrance of autoimmunity is incomplete in these families suggesting that other checkpoints are playing a role in preventing autoimmunity. In Chapter two of this thesis, I describe studies testing if a defect in Lyn, an inhibitory protein tyrosine kinase implicated in systemic autoimmunity, could combine with an Aire mutation to provoke organ-specific autoimmunity. Indeed, mice with a dominant-negative allele of Aire and deficiency in Lyn spontaneously developed organ-specific autoimmunity in the eye. I further show that a small pool of retinal protein-specific T cells escaped thymic deletion due to the hypomorphic Aire function and escaped peripheral tolerance in the presence of Lyn-deficient dendritic cells, leading to highly destructive autoimmune attack. These findings demonstrate how two distinct tolerance pathways can synergize to unleash autoimmunity and have implications for the determinism of autoimmune disease.

Systemic lupus erythematosus (SLE) is a highly heterogeneous and prevalent autoimmune disease. The development of anti-nuclear autoantibodies (ANAs) is a hallmark of SLE and many patients will go on to develop glomerulonephritis (GN), a potentially fatal destruction of the kidneys. In the CD45E613R murine model of SLE, a single point mutation in the juxtamembrane wedge region of the protein tyrosine phosphatase CD45, results in hyperactivated immunoreceptor tyrosine-based activation motif (ITAM) signaling. Interestingly, despite the mutation functioning biochemically identically, different strains expressing the mutation result in different phenotypic consequences. CD45E613R B6 and 129 mice develop normally, while CD45E613R B6-129 F1 mice develop double stranded (ds)DNA autoantibodies, lymphoproliferative disease (LPD) and GN. CD45E613R BALB/c mice develop dsDNA autoantibodies, slight LPD, and no GN. This indicated genetic modifiers on these backgrounds modulated disease phenotype, and a single nucleotide polymorphism (SNP) screen performed between B6 and BALB/c mice for the development of ANAs identified two susceptibility loci. One locus, termed Wedge Associated Modifier (Wam) 1 on the BALB/c background identified tlr9 as the putative modifier gene. The second locus identified on the B6 background corresponded to the MHC H2 region.

Here we examine the role of these two susceptibility regions in the CD45E613R BALB/c and B6 models of SLE. Previous studies from our lab identified two alleles of TLR9, the B6 (TLR9B6) and BALB/c (TLR9Ba) alleles, hypothesized to result in the signaling difference observed between the two backgrounds. In CD45E613R BALB/c TLR9-/- mice, B cell expression of CD45E613R and TLR9 is required for ANA development. Interestingly, CD45E613R B6 TLR9-/- mice acquired ANA development, suggesting the two alleles are regulating ANA and B cell tolerance in opposing ways. In Chapter II, we developed TLR9 congenic mice on the B6 and BALB/c backgrounds with the CD45E613R polymorphisms to determine if signaling difference and autoantibody development observed in intact mice were intrinsic to the TLR9 alleles. We show here that the TLR9Ba allele positively regulates autoreactive B cell development and is required for the development of ANAs, GN, and results in hyperresponsive B cell activation upon stimulation resistant CD45E613R B6 mice. Furthermore, the TLR9B6 allele negatively regulates ANA production, resulting in tolerance in susceptible CD45E613R BALB/c mice. The TLR9 alleles also dictate the signaling differences, such that the TLR9B6 allele results in hyperresponsive signaling and the TLR9Ba allele results in hyporesponsive signaling, regardless of genetic background, potentiating the differences in disease phenotypes. Together, these data demonstrate the dual role two allelic variants of TLR9 plays in regulating B cell tolerance.

In Chapter III, we determine the role of the B6 susceptibility loci, MHC H2b (MHCB6), in the development of pathogenesis in the CD45E613R BALB/c model. We hypothesized the protective endogenous BALB/c MHC allele was preventing development of the severe GN observed in the CD45E613R B6-129 F1 model. In order to test this, we developed congenic CD45E613R BALB/c mice congenic for the MHC H2 region. CD45E613R BALB/c MHCB6 mice developed accelerated autoantibody production compared to CD45E613R BALB/c mice and eventually GN upon aging. B cells from CD45E613R BALB/c MHCB6 mice became dysregulated during development and were hyperactivated upon stimulation compared to CD45E613R BALB/c B cells. Further investigation revealed the B6 MHC H2b allele resulted in increased MHC class II expression on the surface of B cells compared to the BALB/c MHC H2d allele, but not other cell populations prior to disease onset. These data demonstrate one mechanism by which MHC susceptibility alleles identified in autoimmune patient populations are required for disease progression from autoreactivity to autoimmunity. Taken together with the TLR9 studies, we hypothesize the MHCB6 allele, in cooperation with the TLR9Ba allele and the sensitizing CD45E613R mutation results in the severe progression of autoimmunity in the CD45E613R B6 TLR9Ba model.

Recognition of cognate antigen by the B cell antigen receptor (BCR) is one of the primary events that ultimately facilitate the production of pathogen-specific antibodies that are critical for host immunity. The process of B cell activation by antigen must be tightly regulated to ensure robust responses against pathogens while limiting responses directed at our own tissues. Much of this regulation is controlled at the level of signal transduction downstream of the BCR which requires a precise balance between signal amplification and feedback inhibition of pathways activated downstream of this receptor. The work presented in this thesis explores two distinct mechanisms by which signaling downstream of the BCR is regulated to govern the response of cells to cognate antigen stimulation.

Chapter 2 describes studies addressing the intracellular sources and physiological function of reactive oxygen species (ROS) produced in primary B cells in response to BCR stimulation. It was found that BCR stimulation of primary resting murine B cells induced the rapid production of ROS that occurred within minutes, and was maintained for at least 24 h following receptor stimulation. While the early production of ROS (0-2 h) was dependent on the Nox2 isoform of NADPH oxidase, at later stages of B cell activation (6-24 h) ROS were generated by a second pathway, which appeared to be dependent on mitochondrial respiration. B cells from mice deficient in the Nox2 NADPH oxidase complex lacked detectible early production of extracellular and intracellular ROS following BCR stimulation, but had normal proximal BCR signaling and BCR-induced activation and proliferation in vitro, and mounted normal or somewhat elevated antibody responses in vivo. In contrast, neutralizing both pathways of BCR-derived ROS with the scavenger N-acetylcysteine resulted in impaired in vitro BCR-induced activation and proliferation, and attenuated BCR signaling through the phosphatidylinositol 3-kinase pathway at later times. These results indicate that the production of ROS downstream of the BCR is derived from at least two distinct cellular sources and plays a critical role at the later stages of B cell activation by promoting sustained BCR signaling via the phosphatidylinositol 3-kinase signaling pathway, which is needed for effective B cell responses to antigen.

Chapter 3 describes studies addressing the role of metabolism of the second messenger diacylglycerol (DAG) by diacylglycerol kinase enzymes (DGKs), in modulating the magnitude of signaling by this second messenger downstream of the BCR. In the absence of DGKζ, the threshold for BCR signaling through the Ras-ERK MAP kinase pathway was markedly reduced in mature follicular B cells, resulting in exaggerated responses to antigen in vitro and in vivo. Inhibition of DAG signaling by DGKζ was especially important for limiting the number of antibody-secreting cells generated early in response to both T-independent type 2 antigens and T cell-dependent antigens. Furthermore, deficiency in DGKζ closely resembled the effects of increasing antigen affinity for the BCR during the T cell-dependent antibody response, strongly indicating that the magnitude of DAG signaling, likely through the degree of ERK activation, is important for translating the affinity of the BCR for antigen into the amount of antibody produced during early stages of an immune response.

Together, these studies provide novel insight into the mechanisms involved in fine-tuning BCR signaling to promote high quality humoral immune responses while avoiding immunopathological consequences such as autoimmunity or lymphoma.

Innate lymphoid cells (ILCs) are a family of immune cells involved in development, homeostasis, and the host response to pathogens. In order to identify subsets of these cells in situ and genetically target them, additional markers for ILCs need to be established. Here, I demonstrate that arginase-I (Arg1), a urea cycle enzyme induced in alternatively activated macrophages, is expressed by multiple ILC lineages in the fetal and adult mouse. In the adult lung, group 2 ILCs (ILC2s) express Arg1 at rest and during type 2 inflammation induced by the parasitic helminth Nippostrongylus brasiliensis. While macrophages induce Arg1 through STAT6 activation, ILC2s express Arg1 in a STAT6-independent manner. Total numbers of Arg1+ cells in the lung are regulated by IL-33, which elevates ILC2 numbers and indirectly induces Arg1 expression in macrophages through activation of STAT6. Arg1 expression in ILC2s may provide a way to target these cells without altering T cell responses.

In fetal development, specialized members of group 3 ILCs (ILC3s), lymphoid tissue inducer (LTi) cells, are required for lymph node and Peyer's patch organogenesis, but how LTi cells develop at these sites remains unclear. Here, we identify an Arg1+, Id2+ ILC population in the fetal intestine that can differentiate into IL-7Rα+NK1.1+T-bet+ group 1 ILCs (ILC1s), GATA3hi ILC2s, and RORγt+ group ILC3s in vitro. Based on transcription factor expression, ILC precursors in the fetal intestine represent an intermediate developmental stage between T-bet RORγt- ILC precursors and differentiated ILC lineages. These ILC precursors outnumber other innate lymphoid populations in the intestine at embryonic day (E) 13.5. At E16.5, after the initiation of Peyer's patch organogenesis, intestinal ILC precursors accumulate at the Peyer's patch anlage in a manner that is dependent on lymphotoxin-α (LTα). Thus, during development, ILC precursors accumulate in the intestine, where they aggregate at sites of lymphoid tissue organogenesis and differentiate into mature ILC lineages.

The ultimate goal of the immune system is to eliminate pathogens and establish an immunological memory for rapid response upon reinfection. It can be broadly divided into innate and adaptive compartments. Immediately or shortly after infection occurs, the innate immune system uses pattern recognition receptors (PRRs) to identify ancient conserved features of pathogens, for example, DNA, RNA and carbohydrate, termed pathogen associated molecular patters (PAMPs). Stimulation of PRRs by PAMPs triggers signaling events that both limit the spread of infection as well as inform the adaptive immune system to generate the appropriate type of immunological memory. For example, the adaptive system uses cellular immunity to eliminate pathogens such as Mycobacterium tuberculosis that reside within cells while it generates antibodies (humoral immunity) to neutralize and remove extracellular pathogens such as the bacteria Pseudomonas aeruginosa that causes pneumonia. Thus, innate responses provide the first wave of defense by containing infection while the slower acting adaptive response eliminates pathogens and programs the ability to respond more efficiently upon subsequent exposures.

How innate signals sculpt adaptive responses is not well understood, and therefore, this thesis focuses on the ability of innate signaling by a family of PRRs known as toll-like receptors (TLRs) to program antibody responses. Ten and twelve TLRs have been identified in humans and mice, respectively, and they recognize a variety of PAMPs expressed by bacteria, viruses, fungi, and parasites including lipopolysaccharide (TLR4), flagellin (TLR5 and TLR11), and unmethylated CpG motifs in DNA (TLR9). Briefly, antibody responses may be broadly divided into those that require T cell help (T-dependent) and those that are independent of T cell help (T-independent). T-dependent antibody responses can occur either outside the B cell follicle (extrafollicular) or within the follicle in specialized transient structures termed germinal centers (GCs) that generate high affinity, long-lived humoral memory. To dissect how TLR signaling impacts GC antibody responses, I immunized mice with an oligovalent T-dependent protein antigen that was linked to either oligonucleotides containing (CpG) or lacking (nonCpG) a TLR9 ligand. In chapter two, I focus on the cellular contribution of TLR signaling to GC magnitude and quality by using a Cre recombinase system to selectively delete the TLR signaling adaptor MyD88 in either dendritic cells or B cells. This series of experiments revealed a division of labor for TLR signaling in DCs and B cells that controls GC magnitude and quality, respectively. In chapter three, I address the role of costimulation by the inducible costimulator (ICOS) on T cells and ICOS ligand (ICOSL) on B cells to direct the GC response against antigen linked to a TLR9 ligand. These experiments revealed that ICOSL acted B cell-extrinsically to impact GC quality and also unveiled a surprising B cell-intrinsic role for TLR9 signaling in affinity maturation. Collectively, these studies suggest that innate signals imprint the quality of T cell help that subsequently defines the antibody response and that they also act on B cells receiving TLR9 stimuli to directly enhance their affinity maturation. These results have implications in both human disease and rational vaccine design.