DNA can be represented abstrzctly as a language with only four nucleotides represented by the letters A, C, G, and T, yet the arrangement of those four letters plays a major role in determining the development of an organism. Understanding the signi cance of certain arrangements of nucleotides can unlock the secrets of how the genome achieves its essential functionality. Regions of DNA particularly enriched with cytosine (C nucleotides) and guanine (G nucleotides), especially the CpG di-nucleotide, are frequently associated with biological function related to gene expression, and concentrations of CpGs referred to as \CpG islands" are known to collocate with regions upstream from gene coding sequences within the promoter region. The pattern of occurrence of these nucleotides, relative to adenine (A nucleotides) and thymine (T nucleotides), lends itself to analysis by machine-learning techniques such as Hidden Markov Models (HMMs) to predict the areas of greater enrichment. HMMs have been applied to CpG island prediction before, but often without an awareness of how the outcomes are a ected by the manner in which the HMM is applied. Two main ndings of this study are: 1. The outcome of a HMM is highly sensitive to the setting of the initial probability estimates. 2. Without the appropriate software techniques, HMMs cannot be applied e ectively to large data such as whole eukaryotic chromosomes. Both of these factors are rarely considered by users of HMMs, but are critical to a successful application of HMMs to large DNA sequences. In fact, these shortcomings were discovered through a close examination of published results of CpG island prediction using HMMs, and without being addressed, can lead to an incorrect implementation and application of HMM theory. A rst-order HMM is developed and its performance compared to two other historical methods, the Takai and Jones method and the UCSC method from the University of California Santa Cruz. The HMM is then extended to a second-order to acknowledge that pairs of nucleotides de ne CpG islands rather than single nucleotides alone, and the second-order HMM is evaluated in comparison to the other methods. The UCSC method is found to be based on properties that are not related to CpG islands, and thus is not a fair comparison to the other methods. Of the other methods, the rst-order HMM method and the Takai and Jones method are comparable in the tests conducted, but the second-order HMM method demonstrates superior predictive capabilities. However, these results are valid only when taking into consideration the highly sensitive outcomes based on initial estimates, and nding a suitable set of estimates that provide the most appropriate results. The rst-order HMM is applied to the problem of producing synthetic data that simulates the characteristics of a DNA sequence, including the speci ed presence of CpG islands, based on the model parameters of a trained HMM. HMM analysis is applied to the synthetic data to explore its delity in generating data with similar characteristics, as well as to validate the predictive ability of an HMM. Although this test fails to i meet expectations, a second test using a second-order HMM to produce simulated DNA data using frequency distributions of CpG island pro les exhibits highly accurate predictions of the pre-speci ed CpG islands, con- rming that when the synthetic data are appropriately structured, an HMM can be an accurate predictive tool. One outcome of this thesis is a set of software components (CpGID 2.0 and TrackMap) capable of ef- cient and accurate application of an HMM to genomic sequences, together with visualization that allows quantitative CpG island results to be viewed in conjunction with other genomic data. CpGID 2.0 is an adaptation of a previously published software component that has been extensively revised, and TrackMap is a companion product that works with the results produced by the CpGID 2.0 program. Executing these components allows one to monitor output aspects of the computational model such as number and size of the predicted CpG islands, including their CG content percentage and level of CpG frequency. These outcomes can then be related to the input values used to parameterize the HMM.

Les protéines à " Methyl-CpG-binding domain " (MBD) jouent un rôle important dans l'interprétationde la méthylation de l'ADN conduisant à la répression transcriptionnelle via le recrutement decomplexes remodelant la chromatine. Dans les cancers, MBD2 jouerait un rôle essentiel dans la perted'expression des gènes hyperméthylés. Ainsi, MBD2 serait une cible potentielle pour rétablir, enpartie au moins, leur expression. Caractériser, à l'échelle du génome, la distribution de MBD2 et sesconséquences sur la répression transcriptionnelle au cours de la cancérogenèse est donc une étapeincontournable. (1) L'impact sur l'expression génique de l'inhibition de MBD2 par interférence àl'ARN, a été étudié en utilisant des puces, dans des cellules normales MRC5. La perte de MBD2n'induit pas de surexpression génique globale et la densité en CpG des promoteurs méthylés sembleêtre une composante importante dans la force de répression par MBD2. (2) Les profils de méthylationde l'ADN, de liaisons de MBD2 et de l'ARN polymérase II dans les cellules HeLa ont été analysés parChIP-on-chip avec des puces promoteurs. Ces mêmes approches couplées à l'analyse de l'acétylationdes histones H3 ont été réalisées dans un modèle cellulaire syngénique de progression tumoralemammaire humain. Dans les modèles étudiés, une forte proportion de gènes silencieux et méthylés estliée par MBD2. Les comparaisons entre cellules immortalisées et transformées ne montrent pas dechangements majeurs de la méthylation de l'ADN ou de la répression transcriptionnelle, par contreune redistribution de MBD2 parmi ces sites est observée, suggérant une redondance entre les protéinesliant l'ADN méthylé.

This Ph.D. dissertation examined the effects of bacterial DNA on immune surveillance in ovine lymph nodes (LN). The first study identified long-term changes in lymph nodes following exposure to plasmid DNA. Treated lymph nodes were heavier and had larger medullary areas. Furthermore, medullary cord thickness and medullary sinus width as well as germinal center size and number were increased in plasmid treated lymph nodes.<p>To determine whether bacterial DNA altered cell trafficking through lymph nodes, the efferent lymphatic of the prescapular LN of sheep was cannulated and cells were collected. Intradermal injection of as little as 4 ìg of plasmid DNA expressing the green fluorescent protein of jellyfish (eGFP) caused a marked increase in the cell trafficking through the prescapular lymph node. A dose-dependent facet existed for this response, as the increase in cell trafficking response persisted longer with 40 ìg or 400 ìg of plasmid-eGFP than with 4 ìg. This increased cell trafficking was independent of green fluorescent protein expression as both pCAN1-eGFP and pCAN1 induced similar responses.<p>Increased cell traffic induced by bacterial DNA was further characterized by determining whether bacterial DNA form was critical for this response. Treatment with intact plasmid (circular) DNA induced the greatest increase in cell traffic. In contrast, when plasmid DNA was digested with restriction enzymes into linear fragments of DNA, then cell trafficking was not significantly increased. <p>Numerous studies have shown that immunostimulatory guanosine-cytosine (CpG) motifs activate cells in vitro and can improve protective immune responses in vivo . Therefore we tested the hypothesis that the presence of immunostimulatory CpG motifs within plasmids was responsible for altering cell trafficking. It was shown that plasmids with added immunostimulatory CpG sequences affected cell trafficking in a dose-dependent manner. The injection of 40 ìg of pBISIA-88 caused an increase in cell trafficking while injecting 400 ìg pBISIA-88 failed to increase cell flow above control levels.<p>Analysis of cell populations collected in efferent lymph gives insight into treatment effects on immune surveillance. This is well-studied following treatment with antigen and in general, antigen induces a selective movement of cells into lymph that occurs 24 hours post-treatment. In contrast, plasmid DNA induced a rapid, non-selective movement of lymphocytes through the lymph node, suggesting that bacterial DNA and antigen may affect cell trafficking by different mechanisms and thereby have distinct effects on immune surveillance.<p>Short-term effects of bacterial DNA on lymph node architecture and cellular composition were also investigated to determine whether an increase in cell trafficking was associated with structural changes within the lymph node. While it was shown that bacterial DNA induced changes within a lymph node, including and increased number of germinal centers and an increased frequency of CD72*CD21* B cells, these changes were not correlated to increased cell trafficking.<p>In summary, bacterial DNA altered immune surveillance in sheep lymph nodes by changing cell traffic and lymph node architecture and composition. Many of these responses differed from responses observed by others following antigen treatment. The present observations suggest that bacterial DNA can function as an important signal to modulate immune surveillance and host responses to infections by pathogens.

It is clear that dendritic cells (DCs) are essential for priming of T cell responses against tumors. However, the distinct roles DC subsets play in regulation of T cell responses in vivo are largely undefined. In this study, we investigated the capacity of ovalbumin (OVA)-presenting CD48, CD4+8, or CD48+ DCs (OVA-pulsed DC (DCOVA)) from mouse spleen in stimulation of OVA-specific T cell responses. Our data show that each DC subset stimulated proliferation of allogeneic and autologous OVA-specific CD4+ and CD8+ T cells in vitro, but that the CD48 DCs did so only weakly. Both CD4+8 and CD48+ DCOVA induced strong tumor-specific CD4+ Th1 responses and fully protective CD8+ cytotoxic T lymphocyte (CTL)-mediated antitumor immunity, whereas CD48 DCOVA, which were less mature and secreted substantial transforming growth factor (TGF- ) upon coculture with T cell receptor (TCR)-transgenic OT II CD4+ T cells, induced the development of interleukin-10 (IL-10)-secreting CD4+ T regulatory 1 (Tr1) cells. Transfer of these Tr1 cells, but not T cells from cocultures of CD48 DCOVA and IL-10/ OT II CD4+ T cells, into CD48+ DCOVA-immunized animals abrogated otherwise inevitable development of antitumor immunity. Taken together, CD48 DCs stimulate development of IL-10-secreting CD4+ Tr1 cells that mediated immune suppression, whereas both CD4+8 and CD48+ DCs effectively primed animals for protective CD8+ CTL-mediated antitumor immunity. <p> Different DC subsets play distinct roles in immune responses. CD4-8- DCs secreting TGF-â stimulate CD4+ regulatory T type 1 (Trl) cell responses leading to inhibition of CD8 CTL responses and antitumor immunity. In this study, we explored the potential effect of three stimuli CpG, lipopolysaccharide (LPS) and anti-CD40 antibody in conversion of CD4-8- DC-induced tolerance. We demonstrated that when CD4-8- DCs were isolated from overnight culture and cultured for another 8 hrs in AIM-V plus recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) (15-20 ng/ml) and OVA (0.1 mg/ml) with CpG (5 ug/ml), LPS (2 ug/ml) and anti-CD40 antibody (10 ug/ml), their phenotype became more mature compared with the freshly isolated ones. CpG is the only agent that stimulates the DCs to secrete significant level of interleukin-6 (IL-6) and interleukin-15 (IL-15); DNA array analyses also indicate that CpG stimulates higher expression of IL-6 and IL-15 mRNA. CpG treatment most efficiently converts the tolerogenic DCs into immunogenic ones which stimulated the OTII CD4+ T cell to become T helper type 1 (Th1) and T helper type 17 (Th17) rather Tr1, while the other two stimulator-treated DCs could not induce Th17 response. Their vaccination also induced the strongest antitumor CTL responses and protective immunity against tumor cell challenge. When CD4-8- DCs were isolated from IL-6 knock out (IL-6-/-) mice, CpG-treated DCOVA vaccination almost completely lost their animal protection capacity. Wild type B6 DCOVA-vaccinated IL-15 receptor knock out (IL-15R-/-) mice can only provide up to 30% protection against tumor challenge. Those results indicate that IL-6/ IL-l5-induced Th17 plays a critical role in their conversion. Taken together, our findings indicate that CpG treatment is the most efficient agent that can convert tolerogenic DCs into immunogenic ones and induce long-lasting antitumor immunity. We previously demonstrated that the nonspecific CD4+ T cells can acquire antigen-specific DC-released exosomes (EXO) and these CD4+ T cells with acquired exosomal MHC I peptide complex (pMHC I) can stimulate antigen-specific CD8+ CTL responses. In my project we have found that CD4-8-DCs could induce regulatory T cell type 1(Tr1) response, thus it would be very necessary to know whether regulatory T cells would change their antigen specificity if they got the membrane complex from DC through coculture or DC-derived exosome pulsing. During the beginning of my regulatory T cell project, we found that CD8+CD25+ Tr were much more easily expanded, while CD4+CD25+ Tr usually began to die just after 3 days in vitro culture and its very hard to get enough cells for further research. Therefore, CD8+CD25+ were used as a model Tr cells in the following project. To assess whether the nonspecific CD8+CD25+ Tr cells can acquire antigen-specificity via acquired exosomal pMHC I, we purified CD8+CD25+ Tr cells from wild-type C57BL/6 mice and OVA-pulsed DCOVA-released EXOOVA expressing pMHC I complexes. We demonstrated that the nonspecific CD8+CD25+ Tr cells expressing forkhead box P3 (Foxp3), cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor (GITR), perforin and granzyme B inhibited in vitro T cell proliferation and in vivo OVA-specific CD4+ T cell-dependent and independent CD8+ CTL responses and antitumor immunity. CD8+CD25+ Tr cells suppressive effect is possibly mediated through its inhibition of DC maturation, down-regulation of secretion of Th1 polarization cytokines by DCs and its induction of T cell anergy via cell-to-cell contact. The nonspecific CD8+CD25+ Tr cells acquired antigen specificity by uptake of DCOVA-released EXOOVA expressing pMHC I and enhanced its effect on inhibition of OVA-specific CD8+ T cell responses and antitumor immunity by 10-folds. The principles elucidated in this study may have significant implications not only in antitumor immunity, but also in other sectors of immunology (e.g, autoimmunity and transplantation).

It is clear that dendritic cells (DCs) are essential for priming of T cell responses against tumors. However, the distinct roles DC subsets play in regulation of T cell responses in vivo are largely undefined. In this study, we investigated the capacity of ovalbumin (OVA)-presenting CD48, CD4+8, or CD48+ DCs (OVA-pulsed DC (DCOVA)) from mouse spleen in stimulation of OVA-specific T cell responses. Our data show that each DC subset stimulated proliferation of allogeneic and autologous OVA-specific CD4+ and CD8+ T cells in vitro, but that the CD48 DCs did so only weakly. Both CD4+8 and CD48+ DCOVA induced strong tumor-specific CD4+ Th1 responses and fully protective CD8+ cytotoxic T lymphocyte (CTL)-mediated antitumor immunity, whereas CD48 DCOVA, which were less mature and secreted substantial transforming growth factor (TGF- ) upon coculture with T cell receptor (TCR)-transgenic OT II CD4+ T cells, induced the development of interleukin-10 (IL-10)-secreting CD4+ T regulatory 1 (Tr1) cells. Transfer of these Tr1 cells, but not T cells from cocultures of CD48 DCOVA and IL-10/ OT II CD4+ T cells, into CD48+ DCOVA-immunized animals abrogated otherwise inevitable development of antitumor immunity. Taken together, CD48 DCs stimulate development of IL-10-secreting CD4+ Tr1 cells that mediated immune suppression, whereas both CD4+8 and CD48+ DCs effectively primed animals for protective CD8+ CTL-mediated antitumor immunity. <p> Different DC subsets play distinct roles in immune responses. CD4-8- DCs secreting TGF-â stimulate CD4+ regulatory T type 1 (Trl) cell responses leading to inhibition of CD8 CTL responses and antitumor immunity. In this study, we explored the potential effect of three stimuli CpG, lipopolysaccharide (LPS) and anti-CD40 antibody in conversion of CD4-8- DC-induced tolerance. We demonstrated that when CD4-8- DCs were isolated from overnight culture and cultured for another 8 hrs in AIM-V plus recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) (15-20 ng/ml) and OVA (0.1 mg/ml) with CpG (5 ug/ml), LPS (2 ug/ml) and anti-CD40 antibody (10 ug/ml), their phenotype became more mature compared with the freshly isolated ones. CpG is the only agent that stimulates the DCs to secrete significant level of interleukin-6 (IL-6) and interleukin-15 (IL-15); DNA array analyses also indicate that CpG stimulates higher expression of IL-6 and IL-15 mRNA. CpG treatment most efficiently converts the tolerogenic DCs into immunogenic ones which stimulated the OTII CD4+ T cell to become T helper type 1 (Th1) and T helper type 17 (Th17) rather Tr1, while the other two stimulator-treated DCs could not induce Th17 response. Their vaccination also induced the strongest antitumor CTL responses and protective immunity against tumor cell challenge. When CD4-8- DCs were isolated from IL-6 knock out (IL-6-/-) mice, CpG-treated DCOVA vaccination almost completely lost their animal protection capacity. Wild type B6 DCOVA-vaccinated IL-15 receptor knock out (IL-15R-/-) mice can only provide up to 30% protection against tumor challenge. Those results indicate that IL-6/ IL-l5-induced Th17 plays a critical role in their conversion. Taken together, our findings indicate that CpG treatment is the most efficient agent that can convert tolerogenic DCs into immunogenic ones and induce long-lasting antitumor immunity. We previously demonstrated that the nonspecific CD4+ T cells can acquire antigen-specific DC-released exosomes (EXO) and these CD4+ T cells with acquired exosomal MHC I peptide complex (pMHC I) can stimulate antigen-specific CD8+ CTL responses. In my project we have found that CD4-8-DCs could induce regulatory T cell type 1(Tr1) response, thus it would be very necessary to know whether regulatory T cells would change their antigen specificity if they got the membrane complex from DC through coculture or DC-derived exosome pulsing. During the beginning of my regulatory T cell project, we found that CD8+CD25+ Tr were much more easily expanded, while CD4+CD25+ Tr usually began to die just after 3 days in vitro culture and its very hard to get enough cells for further research. Therefore, CD8+CD25+ were used as a model Tr cells in the following project. To assess whether the nonspecific CD8+CD25+ Tr cells can acquire antigen-specificity via acquired exosomal pMHC I, we purified CD8+CD25+ Tr cells from wild-type C57BL/6 mice and OVA-pulsed DCOVA-released EXOOVA expressing pMHC I complexes. We demonstrated that the nonspecific CD8+CD25+ Tr cells expressing forkhead box P3 (Foxp3), cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor (GITR), perforin and granzyme B inhibited in vitro T cell proliferation and in vivo OVA-specific CD4+ T cell-dependent and independent CD8+ CTL responses and antitumor immunity. CD8+CD25+ Tr cells suppressive effect is possibly mediated through its inhibition of DC maturation, down-regulation of secretion of Th1 polarization cytokines by DCs and its induction of T cell anergy via cell-to-cell contact. The nonspecific CD8+CD25+ Tr cells acquired antigen specificity by uptake of DCOVA-released EXOOVA expressing pMHC I and enhanced its effect on inhibition of OVA-specific CD8+ T cell responses and antitumor immunity by 10-folds. The principles elucidated in this study may have significant implications not only in antitumor immunity, but also in other sectors of immunology (e.g, autoimmunity and transplantation).

This Ph.D. dissertation examined the effects of bacterial DNA on immune surveillance in ovine lymph nodes (LN). The first study identified long-term changes in lymph nodes following exposure to plasmid DNA. Treated lymph nodes were heavier and had larger medullary areas. Furthermore, medullary cord thickness and medullary sinus width as well as germinal center size and number were increased in plasmid treated lymph nodes.<p>To determine whether bacterial DNA altered cell trafficking through lymph nodes, the efferent lymphatic of the prescapular LN of sheep was cannulated and cells were collected. Intradermal injection of as little as 4 ìg of plasmid DNA expressing the green fluorescent protein of jellyfish (eGFP) caused a marked increase in the cell trafficking through the prescapular lymph node. A dose-dependent facet existed for this response, as the increase in cell trafficking response persisted longer with 40 ìg or 400 ìg of plasmid-eGFP than with 4 ìg. This increased cell trafficking was independent of green fluorescent protein expression as both pCAN1-eGFP and pCAN1 induced similar responses.<p>Increased cell traffic induced by bacterial DNA was further characterized by determining whether bacterial DNA form was critical for this response. Treatment with intact plasmid (circular) DNA induced the greatest increase in cell traffic. In contrast, when plasmid DNA was digested with restriction enzymes into linear fragments of DNA, then cell trafficking was not significantly increased. <p>Numerous studies have shown that immunostimulatory guanosine-cytosine (CpG) motifs activate cells in vitro and can improve protective immune responses in vivo . Therefore we tested the hypothesis that the presence of immunostimulatory CpG motifs within plasmids was responsible for altering cell trafficking. It was shown that plasmids with added immunostimulatory CpG sequences affected cell trafficking in a dose-dependent manner. The injection of 40 ìg of pBISIA-88 caused an increase in cell trafficking while injecting 400 ìg pBISIA-88 failed to increase cell flow above control levels.<p>Analysis of cell populations collected in efferent lymph gives insight into treatment effects on immune surveillance. This is well-studied following treatment with antigen and in general, antigen induces a selective movement of cells into lymph that occurs 24 hours post-treatment. In contrast, plasmid DNA induced a rapid, non-selective movement of lymphocytes through the lymph node, suggesting that bacterial DNA and antigen may affect cell trafficking by different mechanisms and thereby have distinct effects on immune surveillance.<p>Short-term effects of bacterial DNA on lymph node architecture and cellular composition were also investigated to determine whether an increase in cell trafficking was associated with structural changes within the lymph node. While it was shown that bacterial DNA induced changes within a lymph node, including and increased number of germinal centers and an increased frequency of CD72*CD21* B cells, these changes were not correlated to increased cell trafficking.<p>In summary, bacterial DNA altered immune surveillance in sheep lymph nodes by changing cell traffic and lymph node architecture and composition. Many of these responses differed from responses observed by others following antigen treatment. The present observations suggest that bacterial DNA can function as an important signal to modulate immune surveillance and host responses to infections by pathogens.

Galectins are a family of secreted animal lectins with a high affinity to betagalactosides commonly involved in cellular functions such as apoptosis, adhesion and migration. Galectin 15 (LGALS15), a newest member of the galectin superfamily, has a unique C-terminal RGD sequence and participates in integrin-mediated ovine trophectoderm cell attachment and migration. In the ovine uterus, LGALS15 is expressed only by the endometrial luminal (LE) and superficial glandular (sGE) epithelia, induced by progesterone between Days 10 and 12 of the cycle and pregnancy, and then stimulated by interferon tau (IFNT) from the conceptus after Day 14 of pregnancy. During early pregnancy, the canonical janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway is not active in the endometrial LE/sGE. Therefore, IFNT may utilizes a non-canonical signaling pathway to increase transcription of genes, including CST3, CTSL, HIF2A, LGALS15, and WNT7A, specifically in the endometrial LE/sGE. Alternatively, IFNT and progesterone could indirectly affect epithelial gene expression by influencing gene expression in the stroma, which then communicates with the epithelium. Although the LGALS15 gene is present in ovine, caprine and bovine species, it is only expressed in uteri of sheep and goats. Available data shows a tissue- and speciesspecific expression pattern for LGALS15, likely involving multiple layers of transcription regulation in the ruminant endometrium. Further analysis of the LGALS15 5? promoter/enhancer region revealed similar predicted transcription factor binding sites in all three species, including; PU.1, Ets-1, AP1, Sp1, and GRE or PRE sites. Interestingly, the proximal promoter region of the LGALS15 gene in all three species exhibited a conserved Sp1 binding site upstream of an AP1 binding site on both sense and antisense strands, and with similar spacing between binding sites. Sequence analysis revealed key differences in LGALS15 gene structure between ruminant species including the proximity of repetitive DNA sequences to the transcription start site (+1). Bovine LGALS15 has repetitive DNA sequences start at - 145 whereas in ovine or caprine LGALS15 it starts at about -300. The length of the repetitive DNA sequence is similar (~1.2 kb) in the 5' promoter/enhancer region of LGALS15 in all three species. Transient transfection analyses found that repetitive DNA sequences reduced basal promoter activity and responsiveness to treatments. None of the promoter construct showed responsiveness to interferon tau (IFNT). The bovine LGALS15 gene promoter showed no activity under any experimental conditions. The current studies indicate that uterine LGALS15 is expressed in ovine and caprine but not bovine species. Additionally, repetitive DNA sequences found in the promoter region may contribute to modulating the LGALS15 gene expression. Therefore, the ruminant LGALS15 gene, like other galectins, is under tight transcriptional control involving hormones, requisite transcription factors and potentially chromatin remodeling complexes working synergistically for LGALS15 promoter transactivation.

Recently, the prediction of promoters has attracted many researchers' attention. Unfortunately, most previous prediction algorithms did not provide high enough sensitivity and specificity. The goal of this thesis is to develop an efficient prediction algorithm that can increase the detection power (power = 1 - false negative). We do not try to find more distinct features in promoters one by one, such as transcriptional elements. Our main idea is to use the computer power to calculate all possible patterns which are the possible features of promoters. Accordingly, we shall define some scoring methods for training a given set of sequences, which involve promoter sequences and non-promoter sequences. Then, we can obtain a threshold value for determining whether a testing sequence is a promoter or not. By the experimental results, our prediction has higher correct rate than other previous methods.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 126-144).

Toll-like receptors (TLRs) recognize microbial components as danger signals and induce immune responses. TLR’s are expressed in many tissues of the host that are involved in immune responses including the intestines where they are abundantly expressed. This situation presents a challenge in the gastrointestinal tract which is constantly exposed to a wide variety of commensal organisms. Therefore, innate immune recognition in the intestine must be tightly regulated to prevent unwanted inflammation against harmless commensal micro-organisms and yet allow for the induction of protective immunity to invading pathogens. A dysregulation of this balance can result in intestinal inflammation. Peyer’s patches (PP) are the primary site for the induction of immune responses in the intestine and abundantly express TLRs. It is not known how PP regulate microbial signals from commensal bacteria and yet mount vigorous immune responses against dangerous pathogens. CpG DNA, an agonist for TLR9, can strongly activate immune cells in blood, lymph nodes and spleen. However, CpG very poorly activates immune cells from Peyer’s patches, although these cells express TLR9 [1, 2]. Understanding how TLR responses are regulated in PP cells will unveil important information on how immune responses are regulated in the intestine. Investigations from our laboratory have revealed a B cell population (CD5-CD11c-CD21+) in PP that spontaneously secrete high levels of IL-10 which in turn down regulates TLR9 induced IFN and IL-12 production. These IL-10-secreting PP B cells represent a novel subset of the recently proposed regulatory B cells (Bregs) in the intestine [1, 3]. Bregs may have a role in maintaining tolerance to commensal bacteria thereby achieving intestinal homeostasis. The overall goal of the work described in this thesis was to improve our understanding of the immunobiology of Bregs. We performed several experiments to achieve this goal. First, we studied the development of regulatory B cells in lambs of different ages. Jejunal PP were collected from 3-4 month old, neonatal and fetal lambs and the production of IL-10 (the immunoregulatory cytokine secreted by Bregs) was assayed. We found that IL-10 was secreted by CD21+ B cells from the PP in all the three age groups, confirming that Bregs develop prior to birth. We then wondered whether our CD21+ B cells might be contaminated with other cells or activated when using MACS to enrich B cells. To address this issue, we prepared very highly purified CD21+ B cell population using high speed cell sorting to negatively enrich for B cells. We also sorted DCs and assayed IL-10 production in both cell populations. Only the PP B cells spontaneously secreted IL-10. In contrast, dendritic cells, T cells, macrophages, neutrophils and NK cells did not secrete detectable IL-10. Since B cells exist as regulatory and effector populations in mice, we wondered whether an effector B cell population also existed in ovine PP that secreted the pro-inflammatory cytokines IFN-, IFN- and IL-12. Therefore, ovine PP B cells were fractionated into CD72+CD21+and CD72+CD21- subpopulations to assess their capacity to secrete pro-inflammatory cytokines. Interestingly, the CD72+CD21- B cell population secreted the cytokines IFN-, IFN- and IL-12 suggesting there was an effector population. We then surveyed for Bregs in different mucosal and peripheral tissues in sheep. We observed the Bregs frequency varied among the different lymphoid tissues. Finally, we investigated whether Bregs were present in PP of other ruminant species. We identified Bregs exist in PP of neonatal calves. In conclusion, our investigations reveal that ovine Bregs develop in utero prior to antigen exposure, and are present in a variety of mucosal and peripheral tissues. We also report the novel observation that two distinct B cell sub-populations are present in ovine jejunal PP’s: Regulatory and effector B cells.