An 11 year old, hispanic girl with a history of B-cell acute lymphoblastic leukemia was admitted to the hospital for symptoms compatible with Bartonella henselae infection. The first molecularly diagnosed case of disseminated Bartonella henselae infection was reported in an immunocompromised patient in Lima, Peru. The analysis was confirmed by Polymerase Chain Reaction and automated sequencing of a liver biopsy sample, even though the serologic tests were negative. In conclusion, Bartonella spp. infection should have a particular diagnostic consideration in immunocompromised patients with fever of unknown origin and further investigation regarding the patient's past exposures with cats should also be elicited.

Thesis advisor: Thomas C. Chiles / For ten weeks during the summer of 2005, I was a Summer Undergraduate Research Fellow in an immunology laboratory at the Mayo Clinic. My research focused on the BLyS/APRIL system and the receptor TACI on the surface of B cells. Going into my summer research, I had very little experience in immunology. Throughout the process of writing this thesis, I have sought to improve upon my knowledge of immunology, building a cohesive story that begins with basic biology and ends with the results of the summer's experiments. The first part of this thesis covers topics in general immunology, and narrow down in focus to cover the function and development of lymphocytes and B cell maturation and activation. In the second part of the thesis, the background for my research is described in more detail, and topics such as autoimmunity and cancer, lipid rafts, cell polarization, the BLyS/APRIL system for B cell survival, and TACI are covered extensively. The final portion of this thesis discusses the experimental logic, a background on materials and methods, and the results of the experiments I conducted over the summer. By reading this thesis, anyone with a background in biology should become familiar with basic subjects in immunology, advanced concepts in the study of lymphocytes, the ligands BLyS and APRIL, and the receptor TACI in B cells. / Thesis (BS) — Boston College, 2006. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program.

The proper regulation of inducible costimulator (ICOS) and its ligand (ICOSL) have been shown to be essential for maintaining immune homeostasis. Loss of either protein results in defective humoral immunity, and overexpression of ICOS results in aberrant antibody production resembling lupus. How ICOSL is regulated in response to ICOS interaction is still unclear. We demonstrate that ADAM10 is the primary physiological sheddase of ICOSL in both mouse and human. Using an in vivo system in which ADAM10 is deleted only on B cells (ADAM10B-/-), elevated levels of ICOSL were seen. This increase is also seen when ADAM10 is deleted from human B cell lines. Identification of the primary sheddase has allowed the characterization of a novel mechanism of ICOS regulation. In wildtype (WT) mice, interaction of ICOSL/ICOS results in ADAM10 induced shedding of ICOSL on B cells and moderate ICOS internalization on T cells. When this shedding is blocked, excessive ICOS internalization occurs. This results in severe defects in T follicular helper (TFH) development and Th2 polarization, seen in a house dust mite exposure model. In addition, enhanced Th1 and Th1 immune responses are seen in experimental allergic encephalomyelitis. Blockade of ICOSL rescues T cell ICOS surface expression and at least partially rescues both TFH numbers and the abnormal antibody production previously reported in these mice. Overall, we propose a novel regulation of the ICOS:ICOSL axis, with ADAM10 playing a direct role in regulating ICOSL as well as indirectly regulating ICOS, thus controlling ICOS:ICOSL-dependent responses. Additionally, we report a specific role for the metalloprotease ADAM10 on B cells in regulating both ICOSL and ICOS in a mouse model of increased humoral immunity using mir146a-/- mice and a model of lymphoproliferative disease using the well characterized lpr model. B6lpr mice lacking ADAM10 on B cells (A10Blpr) have decreased nodal proliferation and T cell accumulation compared to control B6lpr mice. Additionally, A10Blpr mice have a drastic reduction in autoimmune anti-dsDNA antibody production. In line with this, we found a significant reduction in follicular helper T cells (TFH) and germinal center (GC) B cells in these mice. We also show that lymphoproliferation in this model is closely tied to elevated ICOS levels and decreased ICOSL levels. Overall, our data not only shows a role of B cell ADAM10 in controlling autoimmunity, but also increases our understanding of the regulation of ICOS and ICOSL in the context of autoimmunity. Additionally, we found that ADAM17 is important for marginal zone (MZ) B cell development as well as responses to T-independent type 2 (TI2) immunizations. Mice which lack ADAM17 on B cells (A17B) have decreased MZ B cell numbers but have increased levels of antigen specific antibodies in response to TI2 Immunizations. ADAM17 also regulates the level of several surface molecules on plasma cells and MZ B cells necessary for their function and survival. We also show a role for ADAM17 in ILC2 responsiveness to IL-33. In vivo, mice that lack ADAM17 specifically on ILC2s (ADAM17ILC2-/-) exhibit decreased ILC2 expansion in response to intranasal IL-33 as well as Nippostrongylus brasiliensis (Nb) infection. However, ADAM17ILC2-/- mice have normal ILC2 numbers in a naïve state, suggesting this defect in ILC2 function is limited to cell activation. In vitro, ADAM17 inhibited ILC2s have an increased level of apoptosis and less IL-13 production in response to IL-33 compared to vehicle treated ILC2s. The defect in cytokine production following ADAM17 inhibition is not observed in response to IL-25 stimulation, suggesting this defect is limited to IL-33 stimulation Mechanistically, ADAM17 inhibition in ILC2s specifically causes a defect in IL-33 mediated ERK activation, potentially explaining the defective survival and IL-13 production following ADAM17 inhibition in these cells. Additionally, ADAM17 regulates the level of surface IL1R2 which may affect IL-33 signaling in ILC2s.

<p>The humoral immune response is dependent on the formation of antibodies. Antibodies are produced by terminally differentiated B cells, plasma cells. Plasma cells are generated either directly from antigen challenged B cells, memory cells or from cells that have undergone the germinal center (GC) reaction. The GC is the main site for class switch, somatic hypermutation and generation of memory cells.</p><p>Different factors, both internal and external, shape the outcome of the immune response. In this thesis, we have studied a few factors that influence the maturation of the humoral response. We have studied how age affects the response, and we show that responses against thymus dependent antigens (TD) are more affected than responses to thymus independent (TI) antigens, in concordance with the view that the T cell compartment is more affected by age than the B cell compartment. Furthermore, we demonstrate that priming early in life have a big influence on the immune response in the aged individual. Priming with a TI form of the carbohydrate dextran B512 (Dx) induces a reduction of IgG levels in later TD responses against Dx. We have evaluated possible mechanisms for this reduction. The reduction does not seem to be caused by clonal exhaustion or antibody mediated mechanisms. We also showed that the reduced TD response after TI priming can be induced against another molecule than Dx. With the hypothesis that TI antigens induce a plasma cell biased maturation of the responding B cells, we examined the presence of Blimp-1, a master regulator of plasma cell differentiation, in GCs induced by TD and TI antigen. Blimp-1 was found earlier in GCs induced by TI antigen and the staining intensity in these GCs was stronger than in TD antigen induced GCs, indicating that plasma cells might be continuously recruited from these GCs.</p><p>B cells undergoing the GC reaction are thought to be under a strict selection pressure that removes cells with low affinity for the antigen and also cells that have acquired self-reactivity. We investigated the effect of apoptotic deficiencies on the accumulation of somatic mutations in GC B cells. In mice lacking the death receptor Fas, <i>lpr</i> mice, the frequency of mutations was increased but the pattern of the mutations did not differ from wild type mice. In contrast, mice over-expressing the anti-apoptotic protein Bcl-2, had a lowered frequency of mutations and the mutations introduced had other characteristics.</p>

The humoral immune response is dependent on the formation of antibodies. Antibodies are produced by terminally differentiated B cells, plasma cells. Plasma cells are generated either directly from antigen challenged B cells, memory cells or from cells that have undergone the germinal center (GC) reaction. The GC is the main site for class switch, somatic hypermutation and generation of memory cells. Different factors, both internal and external, shape the outcome of the immune response. In this thesis, we have studied a few factors that influence the maturation of the humoral response. We have studied how age affects the response, and we show that responses against thymus dependent antigens (TD) are more affected than responses to thymus independent (TI) antigens, in concordance with the view that the T cell compartment is more affected by age than the B cell compartment. Furthermore, we demonstrate that priming early in life have a big influence on the immune response in the aged individual. Priming with a TI form of the carbohydrate dextran B512 (Dx) induces a reduction of IgG levels in later TD responses against Dx. We have evaluated possible mechanisms for this reduction. The reduction does not seem to be caused by clonal exhaustion or antibody mediated mechanisms. We also showed that the reduced TD response after TI priming can be induced against another molecule than Dx. With the hypothesis that TI antigens induce a plasma cell biased maturation of the responding B cells, we examined the presence of Blimp-1, a master regulator of plasma cell differentiation, in GCs induced by TD and TI antigen. Blimp-1 was found earlier in GCs induced by TI antigen and the staining intensity in these GCs was stronger than in TD antigen induced GCs, indicating that plasma cells might be continuously recruited from these GCs. B cells undergoing the GC reaction are thought to be under a strict selection pressure that removes cells with low affinity for the antigen and also cells that have acquired self-reactivity. We investigated the effect of apoptotic deficiencies on the accumulation of somatic mutations in GC B cells. In mice lacking the death receptor Fas, lpr mice, the frequency of mutations was increased but the pattern of the mutations did not differ from wild type mice. In contrast, mice over-expressing the anti-apoptotic protein Bcl-2, had a lowered frequency of mutations and the mutations introduced had other characteristics.

The presence of autoantibodies in systemic lupus erythematosus (SLE) suggests a breach of tolerance. Recently, the IgM-IgD+ naïve B cell population has been shown to be enriched for self-reactive cells that are anergic in healthy subjects. Therefore, to determine whether there is altered selection of self-reactive cells in SLE, this population was examined using multiparameter flow cytometry. SLE patients had increased proportions of IgM-IgD+ cells in mature and transitional B cell compartments that were activated as compared to controls. Comparison of mature and transitional IgM-IgD+ B cell proportions suggested altered selection between the transitional to mature stages in SLE. There was no correlation between altered B cell function or genetic polymorphisms in B cell signalling molecules and the expansion or activation of IgM-IgD+ cells. Thus, selection of self-reactive B cells appears to be abnormal in SLE, but this does not appear to result from altered responses to Ig crosslinking.

The presence of autoantibodies in systemic lupus erythematosus (SLE) suggests a breach of tolerance. Recently, the IgM-IgD+ naïve B cell population has been shown to be enriched for self-reactive cells that are anergic in healthy subjects. Therefore, to determine whether there is altered selection of self-reactive cells in SLE, this population was examined using multiparameter flow cytometry. SLE patients had increased proportions of IgM-IgD+ cells in mature and transitional B cell compartments that were activated as compared to controls. Comparison of mature and transitional IgM-IgD+ B cell proportions suggested altered selection between the transitional to mature stages in SLE. There was no correlation between altered B cell function or genetic polymorphisms in B cell signalling molecules and the expansion or activation of IgM-IgD+ cells. Thus, selection of self-reactive B cells appears to be abnormal in SLE, but this does not appear to result from altered responses to Ig crosslinking.

Specific and sensitive recognition of foreign agents is a critical attribute of the overall effective immune system required for maintaining host protection against challenge from pathogenic cells. In the humoral arm of the immune system, this recognition attribute is carried out by the cell surface bound immunoglobulin-like receptors (BCR) and its soluble forms i.e. antibodies. Over several million years of evolution, the immune system has adopted several strategies for diversifying the antibody sequence and thus its ability to recognize an astronomical variety of molecules through the combinatorial assembly of a small number of DNA segments or genes. Among these immunoglobulin gene diversification strategies, antibody somatic VDJ recombination and junctional diversity are the fundamental mechanisms in generating a broad range of antibody specificities. Understanding how the genetic diversity of antibodies is affected in health and disease is critical for a wide range of medical applications, from vaccine evaluation to diagnostics and therapeutics discovery. Because of the very large number of distinct antibodies encoded by the more than 100 billion B cells in humans, it is essential to use high throughput next generation sequencing technologies in order to obtain an adequate sampling of the sequences and relative abundance of different antibodies expressed by B cells in clinical samples. The process requires rigorous methods for first, experimentally determining the sequences of antibodies in a sample and for second, informatics tools designed for distilling this information for practical purposes. This dissertation describes a variety of experimental approaches and informatics tools developed for the determination and mining of the antibody repertoire. The information from this work has led to major conclusions regarding the nature of the antibody repertoire in healthy individuals, in volunteers following vaccination, and in HIV-1 patients. / text

B cell development is a highly organized process, which commences in the fetal liver during embryogenesis and in the bone marrow (BM) after birth. Surface IgM+ immature B cells emigrate from the BM via the blood stream to the spleen and finally differentiate into conventional mature follicular B (FoB) cells and marginal zone (MZ) B cells. Conversely, some sIgM+ immature B cells can also mature into IgD+ FoB cells in the BM. The ubiquitously expressed cell surface glycoprotein CD47 and its receptor signal regulatory protein α (SIRPα) are members of the immunoglobulin superfamily. Both individually and upon their interaction, CD47 and SIRPα have been found to play important role in the homeostasis of T lymphocytes or CD8­ conventional dendritic cells (cDCs) in secondary lymphoid organs. However, their role in regulating B cell homeostasis has remained unknown. The present study describes important roles of CD47 and SIRPα in B cell homeostasis. Lack of SIRPα signaling in adult SIRPα mutant (MT - cytoplasmic domain deletion) mice resulted in an impaired B cell maturation in the BM and spleen, which was also reflected in the blood. In the BM and spleen of SIRPα MT mice, reduced numbers of semi-mature IgD+IgMhi follicular type-II (F-II) and mature IgD+IgMlo follicular type-I (F-I) B cells were observed, while earlier BM B cell progenitors or splenic transitional B cells remained unaltered. In SIRPα MT mice, maturing B cells in BM and spleen were found to express higher levels of the pro-apoptotic protein BIM and contained an increased level of apoptotic cells. In contrast to that for FoB cells, the splenic MZ B cell population was increased with age in SIRPα MT mice without showing an increased level of activation markers. Immunohistochemical analysis revealed an increased follicular localization of MZ B cells in the spleens of SIRPα MT mice. In addition, MZ macrophages and marginal metallophilic macrophages were not restricted to their normal position in SIRPα MT spleens. Interestingly, CD47-deficient (CD47-/-) mice mimicked the FoB cell phenotype observed in SIRPα MT mice and had a reduced number of  FoB cells in the BM, blood and the spleen at 5­6 months of age, but not in younger mice. Similar to SIRPα MT mice, CD47-/- mice also displayed an increased number of splenic MZ B cells. Sera form both mouse strains did not show any signs of an increased production of autoantibodies or antinuclear antigens. BM reconstitution experiments identified a requirement for non-hematopoietic SIRPα signaling for normal B cell maturation in the BM and to maintain normal numbers and retention of MZ B cells in the splenic MZ. On the contrary, hematopoietic SIRPα signaling appeared to be important for FoB cell maturation in the spleen. Interestingly, hematopoietic SIRPα was required for normal MZ retention of MZ macrophages while normal distribution of metallophilic macrophages required non­hematopoietic SIRPα signaling.  Collectively, these findings revealed an important role of CD47 and of SIRPα signaling in B cell homeostasis in different lymphoid organs.

Allergens are complex mixes of proteins and other compounds that have innate signaling capacity leading to Th2 inflammation. Understanding the role of each of these signals is essential to determining what separates allergens from innocuous proteins. Here, we examine two models for Th2 inflammation: infection with the helminth Trichinella spiralis and footpad immunization with papain, a cysteine protease structurally similar to proteases found in many common allergens including grass pollen and dust mites and helminth-secreted proteases secreted. Together, these studies highlight previously unappreciated effector roles of accessory cells during Th2 inflammation.