Coordinated lymphocyte adhesion and migration is a hallmark of the adaptive immune response in both physiological and pathological conditions. Therefore, understanding the mechanisms underlying lymphocyte trafficking at the molecular level may provide novel targets for the treatment of immune-mediated diseases. The controlled migratory pattern of lymphocytes, commonly referred to as homing, is critically dependent on specialized microvasculature and is facilitated by the expression of adhesion molecules and signaling chemokines. During steady state conditions, homing of immune cells occurs continuously in the lymph nodes due to the constitutive expression of homing molecules, whereas during an inflammatory condition, a reactive up regulation of these adhesion molecules is necessary for immune cell trafficking to take place. The dynamics of immune cell recruitment, demonstrated by intravital microscopy, showed that lymphocyte adhesion in the target tissue's microvascular bed is mostly restricted to the post-capillary and collecting venules, whereas arterioles and capillaries can not support this interaction. High shear stress exerted by fluid dynamics in the lumen of venules requires intravascular lymphocytes to anchor using receptor molecules that form mechanically stable bonds with counter receptors in the vascular wall (von Andrian & Mackay, 2000). These molecules play a key role in lymphocyte binding and facilitate the directed migration of lymphocytes by functioning as a tissue specific recognition molecule differentially expressed on the surface of lymph node ECs (von Andrian & Mackay, 2000). Specialized venular ECs found in lymph nodes and Peyer's Patches (PPs) called high endothelial venules (HEVs) constitutively express on their surface a specialized type of this homing molecule called addressins, allowing for the continuous recruitment of lymphocytes during steady state conditions. Elsewhere in the body, ECs must be activated by exposure to inflammatory mediators in order to allow transendothelial migration to the inflamed tissue. There is strong evidence to show the homing signature of a lymphocyte is dependent on the expression of adhesion molecules and chemokine receptors on the cell surface as well as their ligands expressed by the venular endothelium. Here, we hypothesize that the cytokine signaling receptor subunit glycoprotein 130 (gp130) is a functional requirement for eliciting an effective recruitment of lymphocytes to secondary lymphoid organs during steady states. Glycoprotein 130 is a signaling subunit involved with the interleukin-6 (IL-6) family of cytokines. Previous studies done in the lab have shown that this glycoprotein is over expressed in the venular versus non-venular endothelial cells, indicating a potential role of gp130 in lymphocyte homing during steady state conditions. This hypothesis was tested by analyzing short term homing assays using donor β-actin-GFP splenocytes, with recipient litter-mate controls and recipient conditional or inducible conditional knockout mouse models that lack gp130 expression on ECs. By comparing the homing abilities of GFP+ splenocytes to various secondary lymphoid organs in wild type versus knockout mouse models, we were able to determine that gp130 expression on the endothelial cell compartment does have a role in lymphocyte homing, demonstrated by impaired homing capabilities evident in only the knockout mice. Identifying the function of gp130 expressed by venular ECs and its role in lymphocyte recruitment during steady state conditions may lead to a better understanding of the immune system and it's complexity during the dynamic maintenance of homeostatic health.

BK virus, a virus of the polyomaviridae family, is a latent infection in up to 70% of the general population. However, it has been an emerging cause of renal allograft dysfunction in kidney transplant recipients. Existing research has elucidated the risk factors that may precipitate BK viremia and BK Virus Associated Nephropathy. Research into the humoral and adaptive immune response to BK reactivation is still ongoing. This thesis is part of a larger study that aims to further elucidate the various aspects of the cellular response to BK reactivation. It focuses on the methods of quantitation of BK virus pre-transplant, 1 month, 3 months, 6 months and 12 months post-transplant. Laboratory qPCR and clinical PCR assays were conducted and analyzed. Various aspects of the cellular and humoral response were also examined and analyzed. As there exists no standard protocol for BK surveillance in kidney transplant recipients, analysis of these data will undoubtedly pave the way for future research in this field. It was concluded that BKV levels in plasma and urine should be more stringently monitored and the required assays perfected for a higher degree of sensitivity.

Thesis (Ph.D.)--Boston University. Bibliography: p. 116-125. / It has been observed by many investigators that most persons seemed to possess an increased resistance to bacillary dysentery following recovery from this disease. However, shigella vaccines made from in vitro-grown organisms did not increase resistance to dysentery. The purpose of the present investigation was to determine whether or not Shigella flexneri produced antigens in the in vivo environment formed by culture-grown organisms which might resistance to reinfection. Immunological comparison of animal- and broth-grown Shigella flexneri types 1b and 3 involved comparing the agglutination reactions of animal- and broth-grown organisms and comparing their antigenic structures by means of the agar-diffusion precipitin technique. Antisera were prepared in rabbits by immunisation with either acetone-killed broth-grown organisms or viable organisms grown intra-abdominally in mice for three serial passages without culture. Antisera were absorbed with broth-grown Shigella flexneri of various types. Animal-grown Shigella flexneri were prepared by inoculating mice and guinea pigs with organisms suspended in neutralized streile 5 per cent mucin solution. The organisms were recovered by injecting intra-abdominally 0.15 M sodium chloride and aspirating the organisms with a needle and syringe. The resulting organisms were identified both serologically and culturally. Guinea pig-grown organisms were used for test antigens, whereas the mouse-grown organisms were used only for antiserum production in order to eliminate the possibility of reactions between normal guinea pig protein and antibody to mouse proteins. Saline solutions of water extracts of acetone-dried organisms were employed for the agar-diffusion precipitate stdies. [TRUNCATED]

BACKGROUND: The human immune system is comprised of various cells that function to fight infection while also avoiding harmful inflammatory and autoimmune responses. The immune system consists of the innate and adaptive immune responses. While the adaptive immune response is involved in the late phase of infection and plays a role in generating classic T and B-cell based immunological memory, the innate immune response is the first line of defense and plays a role in early recognition of foreign substances and induction of an inflammatory response. Although the innate immune response is a natural and inborn response, it varies with age. Human newborns in particular are immunologically distinct due to their polarized T helper type 2 (Th2) cell response and increased anti-inflammatory cytokine production. While these adaptations protect the fetus from rejection by the mother, they lead to increased susceptibility of newborns to infection. While immunization is the most promising strategy to combat this increased susceptibility of newborns, the responses of newborns to different vaccines are impaired as a result of their functionally distinct immune system. For this reason, there have been efforts to develop and incorporate adjuvants into vaccines in order to enhance the immune response of newborns and young infants, who suffer the greatest burden of infectious diseases. OBJECTIVE: The objective of this thesis is to determine which compound(s) in a small molecule library (compound 037 family) that had been identified based on a high throughput TNF-α screen of human primary mononuclear cells activates immune cells and has the potential to act as effective vaccine adjuvants. METHODS: Human cord blood was collected from 7 healthy term newborns after Cesarean section and peripheral blood was collected from the arms of 15 healthy adult volunteers between the ages of 18 and 60. The blood was processed and the compounds of the small molecule library were tested via whole blood assays and enzyme-linked immunosorbent assays (ELISAs) to measure production of pro-inflammatory cytokines, specifically tumor necrosis factor (TNF) and interleukin-1β (IL-1β). The responses of the compounds were further characterized by measuring additional cytokines and chemokines via a 41-plex cytokine multiplex assay. RESULTS: It was determined that at the top two concentrations that were tested (3.7 μM and 11.0 μM), certain compounds of the 037 family induced TNF and IL-1β production comparable to that produced by R848, an already established adjuvant. The compounds with the most activity depended on which cytokine was being produced as well as the age group (newborn vs. adult). Results of the 41-plex cytokine multiplex showed that while there is no clear indication of which group of cytokines are produced after stimulation with these compounds, there was increased production of certain chemokines, especially in adults. CONCLUSIONS: The difference in activity of the compounds in the 037 family suggests that the functional groups might play a role in enhancing activity of certain compounds. The increased production of chemokines after stimulation with these compounds suggests that the compounds might activate pathways different from TLR7/8 but still results in an inflammatory response. More work needs to be done in order to identify the receptors and pathways that these compounds activate.

<p> Elucidating how multipotent memory precursor (MP) cells maintain developmental plasticity and longevity to provide long-term immunity while most effector cells develop into terminally differentiated effector (TE) cells with limited survival is necessary for understanding immunological memory formation. We profiled active (H3K27Ac) and repressed (H3K27me3) chromatin states in na&iuml;ve, MP and TE CD8⁺ T cells during viral infection, and observed increased H3K27me3 at numerous pro-memory and pro-survival genes in TE relative to MP cells, indicative of fate restriction, but permissive chromatin at both pro-memory and pro-effector genes in MP cells, indicative of multipotency. Deficiency in PRC2-mediated H3K27me3 deposition impaired clonal expansion and TE cell differentiation, but minimally impacted memory cell maturation. Abundant H3K27me3 deposition at pro-memory genes occurred relatively late during TE cell development in a FOXO1-regulated manner. These results outline a detailed temporal model for how effector T cells lose memory cell potential through selective epigenetic-silencing of pro-memory genes.</p>

JMV-1 cell-line, a Marek's disease virus transformed lymphoblastoid cell-line, could be adapted to grow in serum free medium conditions. The cell-line exudes into its growth medium a complex array of proteins ranging from high molecular weight to low molecular weight. JMV-1 cell-line inspite of its helper T-cell phenotype with the presence of Ia antigen on its surface did not produce any interleukin-2, interferon or macrophage activating factor. Marek's disease virus could not be isolated form the JMV-1 cell-line. An analysis of the genome of the JMV-1 cell-line indicated the presence of the entire Marek's disease virus genome. The genome had few copies of Bam HI fragment C, -E and -G while multiple copies of Bam HI fragments -D, -H and I$\sb2$ were observed. The ultrastructural study of JMV-1 did not reveal the presence of any Marek's disease virus particles in the nucleus. A viral inclusion like particle was observed in an isolated cell. JMV-1 cell-line produced Marek's disease virus specific antigens. These antigens could be precipitated by convalescent serum from Marek's disease virus infected birds. At least three species of antigens could be precipitated. The molecular weight of the proteins were 100000 d, 60,000 d and 50,000 d. JMV-1 cell-line supernatant imparts immunity against JMV lethal lethal lymphoblastoid transplant. The immunity is lost when JMV-1 supernatant is passed through anti-Marek's disease affinity column. The precipitated antigens failed to impart immunity against JMV. JMV-1 supernatant failed to induce antibodies in chicken nor did the supernatant induce cell-proliferation in-vitro.

Over the past decades, information has accumulated concerning the mechanism how an exterior signal induced by ligand on neighboring cells is transmitted to the nucleus through the Notch receptor and the cellular effects of Notch signaling on the regulation of differentiation, proliferation and apoptosis in many cell types. However, the function and the mechanism of Notch signaling in peripheral T cells still remains to be addressed. Therefore, we asked whether Notch1 is involved in CD8+ cytolytic effector T cell (CTLs) maturation and effector functions and how Notch1 exerts its cellular function in the nucleus and in the cytoplasm. The maturation of naïve CD8+ T cells into CTLs is a critical feature of a functional adaptive immune system. Development of CTLs depends, in part, upon the expression of the transcriptional regulator, Eomesodermin (EOMES), which is thought to regulate the expression of two key effector molecules, perforin and granzyme B. In addition, the data from previous studies in our lab showed that Notch signaling results in the activation of NF-κB, IFN-γ secretion and cell proliferation both in CD4 + and CD8+ T cells. Therefore, we hypothesized that Notch1 may be involved in CD8+ T cell maturation and effector function. We observed that Notch1 regulates the expression of EOMES, perforin and granzyme B through direct binding to the promoters of these crucial effector molecules. By abrogating Notch signaling, both biochemically as well as genetically, we conclude that Notch activity mediates CTL development through direct regulation of EOMES, perforin and granzyme B. We further investigated the molecular steps leading to the formation of intracellular Notch1 (N1ICD)/CSL (also known as CBF1/RBP-Jκ in mammals; Suppressor of Hairless in Drosophila ; and Lag-1 in C. elegans) with other co-factors in target promoters of Notch1 signaling. We proposed that the association of two nuclear complexes with N1ICD controls the transcription of genes, allowing the development of effector CTL in the immune system. Recent studies proposed a model where Notch1 colocalizes with CD4, a component of the immune synapse, upon T cell stimulation and directly associates with p56Lck and CD28, as well as PI3K. However, the link between Notch and the TCR signalosome needed further investigation. We found that Notch1 functions as a scaffold, associated with the cytosolic components, Carma1, Bcl10, PKC&thetas; and the IKK complex upon TCR stimulation, leading to the activation of NF-κB and IL-2 production. We further showed that the N-terminal region of N1ICD is essential for interaction with Carma1 and that deficiency of Notch1 abolishes the nuclear binding of NF-κB on the il-2 promoter, leading to reduced IL-2 production.

The cytokine IFN-γ is important in mediating immune responses, while the Notch family of transmembrane receptors has been implicated in modulating T cell differentiation at several stages of development. Stimulation of peripheral T cells through the T cell receptor (TCR) increases Notch-1 expression, and here we show that Notch-1 specifically upregulates interferon-γ (IFN-γ) through nuclear factor-κB (NF-κB) activation. Overexpressing the active form of Notch-1 in T cell lines or upregulating Notch-1 by TCR stimulation in peripheral T cells leads to phosphorylation of inhibitor of kBα (IκBα), increased NF-κB DNA binding activity, and subsequent IFN-γ secretion. Inhibiting Notch-1 or NF-κB activation abrogated IFN-γ secretion, indicating that Notch-1 plays an important role in modulating IFN-γ secretion through NF-κB. Furthermore, using the protein kinase C-&thetas; (PKC-&thetas;) inhibitor, rottlerin, as well as peripheral T cells from PKC-knockout (KO) mice, we show that inhibiting PKC-&thetas; signaling prevents Notch-1 upregulation, NF-κB activation, and IFN-γ production. Additionally, we also show that Notch-1 activates ERK1/2 MAP kinase and that inhibition of ERK1/2 with MAP kinase inhibitor PD98059 prevents Notch-1-upregulated NF-κB activation and IFN-γ secretion. These data suggest that Notch may modulate peripheral immune responses by regulating IFN-γ secretion.

We sought to evaluate the individual contributions of Notch1 and PKC-Theta to disease progression in a mouse model of immune-mediated bone marrow failure and to define a mechanism for their potential cellular cooperation. We transferred parental bulk splenocytes into F1-hybrid recipients to induce a robust immune-mediated bone marrow failure (BMF) that we could partially rescue by administering a pharmacological inhibitor of Notch activation. Transferring splenocytes from PKC-Theta deficent animals did not induce disease, and treating animals with a pharmacological inhibitor of PKC-Theta also provided full protection from disease. We found that inhibiting Notch1 resulted in PKC-Theta down-regulation, and blocking PKC-Theta reduced Notch1 activation. We further evaluated this cross-talk using in vitro studies that demonstrated direct interaction of Notch1 and PKC-Theta following stimulation through the T cell receptor together with co-stimulation through CD28. Our data suggest that both Notch1 and PKC-Theta contribute to disease progression in our mouse model of immune-mediated bone marrow failure. Furthermore, we can demonstrate physical interactions between Notch1, members of the T cell signalosome and PKC-Theta that are essential to mediating full activation of T cells following signaling through the TCR and CD28. Notch1 and/or PKC-Theta may represent novel therapeutic targets in the treatment of bone marrow failure.

Th17 cells are pro-inflammatory cells that are characterized by the production of their signature cytokine, IL-17. Although they are thought to have arisen to protect against extracellular bacteria and fungi they have been shown to mediate autoimmune diseases such as EAE and psoriasis. Notch protein is a cell-surface receptor that has been widely conserved among species. It plays an essential role in determining multiple cell fates. More recently, it has been implicated in regulating peripheral CD4+ T-cell responses. In these studies, we report that blockade of Notch signaling significantly down-regulates the production of IL-17 and associated cytokines in both mouse and human in-vitro polarized Th17 cells, suggesting an intrinsic requirement for Notch during Th17 differentiation in both species. We also present evidence, using promoter reporter assays, knockdown studies as well as chromatin immunoprecipitation, that IL-17 and ROR-γt are direct transcriptional targets of Notch signaling in Th17 cells, with Notch 1 being the responsible Notch family member important in regulating the differentiation of human Th17 cells. In-vivo inhibition of Notch signaling reduced IL-17 production and Th17 mediated disease progression in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. In addition, by using Notch1 and Notch3 knockout mice, we have shown that Notch 3 is the Notch family member that is essential for murine Th17 differentiation. We have also investigated non-canonical Notch signaling in Th17 cells by using CD4+ T-cells from CSL/RBP-Jk knockout mice. Based on data obtained, we have concluded that canonical Notch signaling is dispensable in Th17 responses. Thus, this study highlights the importance of different Notch family members in Th17 differentiation and indicates that selective targeted therapy against Notch may be an important tool to treat autoimmune disorders, including multiple sclerosis.