Adaptive immune responses are highlyco-ordinated and rely upon efficient intracellular communication to orchestratecell function. Phosphoinositide 3-kinase (PI3K) signalling is a well-studiedand important positive mediator in T lymphocyte function; however the role for SH2-domaincontaining inositol phosphatase 1 (SHIP-1), a negative regulator of PI3Ksignalling, has not been so thoroughly investigated. The use of knockout mousemodels has given an insight into the role of SHIP-1 in murine T cells, butthese are compromised by loss of function during development which impingesupon mature T cell function and by the loss of non-catalytic functions ofSHIP-1. Recent work has indicated a clear rolefor reactive oxygen species (ROS), specifically hydrogen peroxide (H2O2),in immune cell signalling and functional responses including migration. Howeverlike SHIP-1, the functional roles of ROS are poorly understood in human Tlymphocytes, particularly the mechanisms by which ROS signals to alter Tlymphocyte biology. ROS has been previously shown to activate PI3K, Mitogenactivate protein kinase (MAPK) and Src family tyrosine kinase (SFK) signallingin a number of different cell types. In addition, ROS have been shown to inactivatephosphatase and tensin homology (PTEN), another negative regulator of PI3K, andare postulated to inactive SHIP-1 signalling. A pharmacological approach wasutilised to manipulate the catalytic activity of SHIP-1 and the cellularaccumulation of ROS in primary human T lymphocytes. Remarkably, it wasdetermined that both SHIP-1 activation and inhibition reduced the ligand-mediatedfunctions of human T lymphocytes, including signalling, proliferation, adhesionand migration. Furthermore, H2O2 selectively inhibited directionalmigration to chemokine CXCL11, enhanced F-actin polymerisation and decreasedactin polarisation to CXCL11. H2O2 required SFKsignalling to induce the phosphorylation/catalytic activation of SHIP-1 and todecrease the surface expression of CXCR3, both of which could be mechanismsunderlying the deficiency in migration observed with H2O2.

Measures of temperament have been shown to influence physiological responses. Exit velocity (EV) has been identified as an objective, robust measure of temperament that can be used to predict subsequent performance of cattle. Additionally, previous studies from our lab indicate this measure of temperament may be related to production of interferon-γ (IFN-γ), a cytokine associated with cell-mediated immunity (CMI). Whereas research has investigated effects of EV upon immune responses, the overall goal of these studies was to examine this relationship under a variety of scenarios including human handling, transportation, and exposure to endophyte-infected tall fescue (E+) for determination of its ability to influence CMI in cattle. In each of 5 experiments, calves were classified as either high or low EV animals, based upon measurements obtained prior to initiation of experimental periods. The hypothesis for these studies was that calves with high exit velocities would have lower systemic immune responses to applied treatments. Two experiments were designed to examine the relationship between exit velocity and lymphocyte IFN-γ production during and following a period of exposure to E+ seed and increased temperature humidity index conditions. Preliminary measures of this cytokine indicated a positive relationship with EV. During application of heat and E+ treatment application, no differences in IFN-γ production were detected between EV or endophyte treatment groups. However, in both experiments, after temperatures were returned to thermoneutral and E+ heifers were placed on the endophyte-free treatment, the positive relationship between exit velocity and total lymphocyte production of IFN-γ observed in baseline samples was reestablished. Similarly, during an experiment examining IFN-γ production by lymphocytes in steers during the 4 weeks following a 10h, 805 km transport study, average lymphocyte production of IFN-γ was higher and lymphocyte proportions producing IFN-γ lower in low EV steers, but total lymphocyte production of this cytokine did not differ between exit velocity treatments. In a grazing and finishing study, cattle were placed on E+ or novel endophyte pastures, with balanced representation of low and high EV treatments within each pasture. During the subsequent finishing period, blood samples for lymphocyte IFN-γ production were collected from a single high EV calf from each pasture group. Neither endophyte nor exit velocity was detected to be related with lymphocyte production of IFN-γ. In an experiment examining changes in cytokine gene expression changes during acclimation to human handling, IFN-γ, Il-6, IL-10, and IL-12 were observed to increase linearly over the experimental period in all calves, irrespective of exit velocity designation. In the same experiment, whole period pro-inflammatory tumor necrosis factor-α expression was higher for high EV calves, but interferon-γ (IFN-γ) was lower in this same treatment group. These studies, cumulatively, indicate EV may be related to systemic production of IFN-γ, but abrupt changes to an animal’s environment may serve to mask this relationship.

B lymphocytes play important roles in antibody production, cytokine production, and antigen presentation to T cells. Ligation of Toll-like receptors (TLRs) on B cells stimulates cellular activation and B cell effector functions. Synergistic activation of other receptors such as CD40 or the B cell receptor (BCR) with TLR ligation further enhances B cell activation and effector functions. The tumor necrosis factor receptor associated factor (TRAF) family of proteins act as cytoplasmic signaling adaptor molecules and moderate downstream signaling from both the tumor necrosis factor receptor (TNFR) superfamily of proteins, including CD40, and the IL-1R/TLR superfamily of proteins. To date, only TRAFs 3 and 6 have been shown to be involved in TLR signaling, with TRAF6 providing positive regulation and TRAF3 providing negative regulation of TLR signaling in B cells. Deficiency in another TRAF family member, TRAF5, has been implicated in the development of atherosclerosis, a disease developed in part due to TLR dysregulation. Here, we addressed the hypothesis that TRAF5 is a negative regulator of TLR signaling. We found that TRAF5 negatively regulated TLR-mediated cytokine and antibody production in B lymphocytes. The enhanced cytokine production seen in TLR-stimulated TRAF5 KO B cells was not attributable to altered cellular survival or proliferation, but instead more cytokine was produced on a per-cell basis, likely due to enhanced MAPK pathways after TLR ligation. Additionally, TRAF5 deficiency did not dramatically affect cytokine production in TLR-stimulated bone marrow-derived macrophages or dendritic cells, suggesting that TRAF5 plays a greater role in TLR signaling in lymphoid versus myeloid cells. TRAF5 associated with the TLR signaling proteins MyD88 and TAB2, and negatively regulated the association of TAB2 with its binding partner TRAF6. Furthermore, we manipulated B cell activation via ligation of various TLRs, CD40, and/or the BCR in order to activate the cells to effectively present antigen. Activated B cells pulsed with antigen served as an effective cellular vaccine and offered protection against both an infectious pathogen (Listeria monocytogenes) and a model of murine melanoma. We identified two candidate activation criteria for B cell vaccines (Bvacs): stimulation through the BCR and TLR7, and stimulation through CD40 and TLR4. Additionally, we found that high IL-6 production by the activated Bvac was essential for inducing optimal CD8+ T cell memory. These B cell activation protocols offer significant advantages over those currently being tested for clinical use. Understanding B cell activation through TLRs is a critical step in developing new therapies against cancer and infectious disease.

Abnormal T lymphocyte reactions in both autism and Alzheimer's disease (AD) have been reported. This research investigated the possibility that these abnormalities may involve circulating antithymic antibodies. Plasma samples from autistic patients, AD patients, and normal-matched controls were tested for reactivity against murine thymocytes. In the first of 3 studies results of the enzyme-linked immunosorbent assay (ELISA) were statistically significant for binding (P < 0.001) between antithymic antibodies in plasmas of AD patients and murine thymocytes. Binding (P < 0.05) in low dilutions (1/2.5 and 1/5} of autistic patient plasmas was also observed. In the second study, plasmas of neither autistic nor AD patients significantly inhibited DNA synthesis of thymic cells in the presence of interleukin-1 (IL-l} and phytohemagglutinin (PHA). In the third study, no significant increases (P > 0.05) in cytotoxic activities were detected using AD patient plasmas and both untreated and heat-treated autistic patient plasmas. After further testing, these heat-treated plasmas diluted 1/64 and 1/128 had increased cytotoxicities (P Therefore, circulating antithymic antibodies may be involved in abnormal T lymphocyte reactions in autism and AD. Since they probably do not act alone, future research should study these complex abnormalities using human thymocytes.

Extracellular adenosine 5′-triphosphate (ATP) is an agonist for the P2Z receptor of human leukaemic lymphocytes and opens a Ca ²⁺-selective ion channel, which also conducts Ba²⁺, Sr²⁺ and the small fluorescent dye, ethidium⁺. A wide range of receptor agonists, many of which raise cytosolic [Ca²⁺] activate phospholipase D (PLD). In the present study, it was shown that both ATP and 3′-O-(4-benzoylbenzoyl)-ATP (BzATP) stimulated PLD activity in a concentration-dependent manner, and the inhibitory effects of suramin, oxidised ATP, extracellular Na⁺ and Mg²⁺ suggested that the effect of these agonists is mediated by P2Z receptors. The role of divalent cations in ATP-stimulated PLD activity was investigated. Several agonists (eg ATP, thapsigargin, ionomycin) stimulated a rise in cytosolic [Ca²⁺] in human lymphocytes, but only ATP and ionomycin stimulated PLD activity. When Ca²⁺ influx was prevented by EGTA, the majority of ATP-stimulated and all of ionomycin-stimulated PLD activity was inhibited. Preloading cells with the Ca²⁺ chelator, BAPTA, reduced cytosolic [Ca²⁺] and, paradoxically, ATP-stimulated PLD activity was potentiated. ATP-stimulated PLD activity was supported by both Ba²⁺ and Sr²⁺ when they were substituted for extracellular Ca²⁺. Furthermore, both ATP-stimulated PLD activity and ATP-stimulated ¹³³Ba²⁺ influx showed a linear dependence on extracellular [Ba²⁺]. Thus it was concluded that ATP stimulated PLD activity in direct proportion to the influx of divalent cations through the P2Z ion channel and this PLD activity was insensitive to changes in bulk cytosolic [Ca²⁺]. The calmodulin (Ca²⁺/CaM) inhibitor, trifluoperazine (TFP) inhibited ionomycin- and ATP-stimulated PLD activity and ATP-stimulated apoptosis, but had no effect on PLD activity already activated by ATP. However, TFP inhibited ATP-stimulated Ca²⁺, Ba²⁺ and ethidium⁺ fluxes, at concentrations below those which inhibit Ca²⁺/CaM, suggesting that TFP inhibits the P2Z receptor. Similarly, the isoquinolinesulphonamide, KN-62, a selective inhibitor of Ca²⁺/CaM-dependent protein kinase II (CaMKII), also prevented ATP-stimulated apoptosis, but had no effect on pre-activated PLD. In addition, KN-62, and an analogue, KN-04, which has no effect on CaMKII, potently inhibited ATP-stimulated Ba²⁺ influx (IC₅₀ 12.7 ± 1.5 and 17.3 ± 2.7 nM, respectively), ATP-stimulated ethidium⁺ uptake (IC₅₀ 13.1 ± 2.6 and 37.2 ± 8.9 nM, respectively), ATP-stimulated phospholipase D activity (50% inhibition 5.9 ± 1.2 and 9.7 ± 2.8 nM, respectively) and ATP-induced shedding of the surface adhesion molecule, L-selectin (IC₅₀ 31.5 ± 4.5 and 78.7 ± 10.8 nM, respectively). They did not inhibit phorbol ester- or ionomycin-stimulated PLD activity or phorbol ester-induced L-selectin shedding. Neither KN-62 nor KN-04 (both 500 nM) have any effect on UTP-stimulated Ca²⁺ transients in fura-2-loaded human neutrophils, a response which is mediated by the P2Y₂ receptor, neither did they inhibit ATP-stimulated contractile responses mediated by the P2X₁ receptor of guinea pig urinary bladder. Thus, KN-62 and KN-04 are almost equipotent as P2Z inhibitors with IC₅₀s in the nanomolar, indicating that their actions cannot be due to CaMKII inhibition, but rather that they are potent and direct inhibitors of the P2Z receptor. Extracellular ATP-induced shedding of L-selectin from lymphocytes into the medium is a Ca²⁺-independent response. L-selectin is either cleaved by a metalloproteinase or a PLD with specificity for glycosylphosphatidylinositol (GPI). The novel hydroxamic acid-based zinc chelator, Ro-31-9790 blocks ATP-induced L-selectin shedding, but was without effect on ATP-induced Ba²⁺ influx or ATP-stimulated PLD activity. Furthermore, another zinc chelator, 1,10-phenanthroline, an inhibitor of a GPI-PLD, potentiated rather than inhibited ATP-stimulated PLD activity, suggesting that ATP-induced L-selectin shedding and ATP-stimulated PLD activity are independent of each other. Although extracellular ATP is the natural ligand for the lymphocyte P2Z receptor, it is less potent than BzATP in stimulating Ba²⁺ influx. Concentration-response curves for BzATP- and ATP-stimulated ethidium⁺ influx gave EC₅₀s 15.4 ± 1.4 µM and 85.6 ± 8.8 µM, respectively. The maximal response to ATP was only 69.8 ± 1.9% of that for BzATP. Hill coefficients were 3.17 ± 0.24 and 2.09 ± 0.45 for BzATP and ATP respectively, suggesting greater positive cooperativity for BzATP than for ATP in opening the P2Z-operated ion channel. A rank order of agonist potency of BzATP > ATP = 2MeSATP > ATPγS was observed for agonist-stimulated ethidium⁺ influx, while maximal influxes followed a rank order of BzATP > ATP > 2MeSATP > ATPγS. When ATP (300 -1000 µM) was added simultaneously with 30 µM BzATP (EC₉₀), it reduced both ethidium⁺ and Ba²⁺ fluxes by 30 - 40% relative to values observed with BzATP alone. KN-62, previously shown to be a specific inhibitor of the lymphocyte P2Z receptor, was a less potent antagonist of BzATP-induced fluxes than ATP, when maximal concentrations of both agonists (50 and 500 µM respectively) were used. However, when BzATP (18 µM) was used at a concentration equiactive with a maximally effective ATP concentration, KN-62 showed the same inhibitory potency for both agonists. The ecto-ATPase antagonist, ARL-67156, inhibited both ATP- and BzATP-stimulated Ba²⁺ influx, suggesting that the lower efficacy of ATP compared with BzATP was not due to preferential hydrolysis of ATP. Thus, the natural ligand, ATP, is a partial agonist for the P2Z receptor while BzATP is a full agonist. Moreover the competitive studies show that only a single class of P2-receptor (P2Z class) is expressed on human leukaemic lymphocytes. Both ATP- and BzATP-stimulated PLD activity were significantly inhibited (P < 0.05) when cells were suspended in iso-osmotic choline Cl medium. Choline⁺ was found to be a permeant for the P2Z ion channel, since ATP induced a large uptake of [¹⁴C]choline⁺ (60 to 150 µmol/ml intracellular water) during a 5 min incubation, which remained in the cells for several hours, and ATP was used to load cells with these levels of choline⁺. Intracellular choline⁺ inhibited ATP-, BzATP-, PMA- and ionomycin-stimulated PLD activity. Brief exposure of lymphocytes to ATP increased the subsequent basal rate of ethidium⁺ uptake, and this was prevented by intracellular choline⁺. It is proposed that P2Z-mediated Ca²⁺ influx in lymphocytes activates PLD leading to significantly changes of the phospholipid composition of the plasma membrane, which subsequently produces a permeability lesion, which in turn contributes to cell death.

Although melanoma accounts for only 4% of skin cancers, it is responsible for 80% of deaths from skin cancers and its incidence in Caucasians has been increasing steadily during the last 30 years. So far, no treatment - except surgery at the earliest stages of the disease - has been shown to significantly improve survival. New treatments are thus clearly needed. The interest of immunologists for melanoma is based on particular features of this tumor. Rare spontaneous regressions have been described, which are possibly mediated by immune responses. Moreover, melanoma cell lines are relatively easy to obtain, providing essential tools for laboratory studies. The first melanoma vaccines involved inoculations of patients with autologous or allogeneic melanoma cells, as well as a variety of immunological adjuvants. Since the beginning of the nineties, the identification of antigens recognized on human tumors by autologous T lymphocytes has opened the way for new vaccination strategies involving molecularly defined tumor antigens. An important group of antigens recognized by T lymphocytes is encoded by “cancer-germ line genes”, which are expressed in tumors of various histological types, but are silent in normal tissues, with the exception of testis germinal cells and placental trophoblast. Since the latter do not express the HLA molecules required to present these antigens to the T lymphocytes, cancer-germ line genes encoded antigens are only present on tumors, which should limit the risk of generating autoimmune diseases as a consequence of vaccination. Therefore, these widely shared tumor specific antigens should represent good targets for the development of cancer vaccines. Our clinical research program of therapeutic vaccinations focuses on antigens encoded by the MAGE family of cancer-germ line genes. Most of the patients included in our phase I/II immunization trials had measurable metastatic melanoma. Several MAGE peptides as well as a recombinant MAGE-3 protein have been tested, while several additional trials are ongoing, including immunizations with a recombinant poxvirus coding for 2 MAGE epitopes. No major toxicity was reported. Tumor regressions have been observed in a minority of patients, mainly those who had regional or distant metastases without visceral involvement. Some of these regressions have been complete and long lasting. Although the rate of objective tumor response observed is low, it is clearly higher than the rate of spontaneous tumor regression observed in melanoma. Other immunization modes against T-cell defined epitopes are currently being explored by several groups in human clinical trials. Vaccines include peptides presented by class I or class II HLA molecules, proteins given alone or mixed with immunological adjuvants or cytokines, recombinant viral or bacterial vectors, dendritic cells and DNA encoding the antigen. Adoptive transfer of T lymphocytes selected for their capacity to recognize defined epitopes presented by the tumor represents another type of approach aiming at the destruction of the tumor by the immune system. It is difficult to predict whether and when therapeutic vaccination against cancer will reach an efficacy that will be sufficient for a standard cancer treatment. Provided their low toxicity, these vaccines should be tested in an adjuvant setting, at earlier stages of the disease.

Bien que la plupart des études se focalisent sur les lymphocytes T CD4+ régulateurs, il a été observé que les lymphocytes T CD8+ régulateurs peuvent jouer un rôle important dans l’induction et le maintien de la tolérance immunitaire. Le transfert adoptif chez la souris et l’induction chez l’homme de lymphocytes T CD8+ régulateurs peuvent inhiber le rejet d’allogreffes et le développement de pathologies autoimmunes. Ces observations suggèrent que l’induction de ces populations peut avoir un potentiel thérapeutique. Des études supplémentaires sont encore nécessaires pour définir les conditions optimales de leur induction. Tant les nouveaux nés humains que murins ont une plus forte capacité que l’adulte à développer des lymphocytes T CD4+ régulateurs induits par une reconnaissance antigénique. La période néonatale serait donc particulièrement appropriée à l’induction de circuits régulateurs. Dans le cadre de ce travail, nous avons étudié le rôle des lymphocytes T CD8+, induits à la naissance, dans le contrôle de la réponse des lymphocytes T CD4+ de type Th2. Des souris BALB/c sont immunisées à la naissance à l’aide de cellules spléniques semi-allogéniques hybrides F1 (AJAX x BALB/c). Ces cellules persistent dans l’animal, au sein des organes lymphoïdes et stimulent ainsi de manière chronique les lymphocytes T CD4+ et T CD8+ du receveur et induisent une réponse de type Th2. Suite à l’injection des cellules spléniques semi-allogéniques au nouveau né de souris, nous avons observé l’expansion d’une population de lymphocytes T CD8+CD25+, dont le phénotype se caractérise par l’expression de Foxp3 et la production conjointe d’IFN- et l’IL-10. Nous avons pu observer que ces cellules sont capables d’inhiber la production de cytokines Th2 produites par les lymphocytes T CD4+ allospécifiques activés. Par contre, ces cellules régulatrices aggravent des réponses Th2 non apparentées. En effet, suite à une sensibilisation à l’ovalbumine, à l’âge adulte, ces souris développent de plus fortes réponses asthmatiques. D’autre part, les nouveaux nés de souris BALB/c ont été immunisés à la naissance à l’aide de cellules dendritiques semi-allogéniques hybrides F1 (AJAX x BALB/c) qui activent de manière aigüe leurs lymphocytes T. Ces souris présentent une forte réponse Th1 et Tc1/Tc2 spécifique de l’alloantigène et sont protégées contre le développement d’un asthme induit. Il a aussi été montré dans ce travail que suite à l’immunisation néonatale à l’aide de cellules dendritiques semi-allogéniques, le nombre de lymphocytes T CD8+CD44high, CD8+CD62Lhigh et CD8+CD25+ producteurs d’IFN- augmente significativement. L’IFN- produit par ces cellules contrôle l’hyperproduction des IgE spécifiques de l’allergène et la synthèse d’ARNm de l’IL-4 et de l’IL-13 induite dans les poumons des souris allergiques. Des études supplémentaires sont nécessaires pour mieux comprendre comment des lymphocytes T CD8+ régulateurs induits lors de stimulations antigéniques aigües ou chroniques en début de vie peuvent être capables de contrôler l’apparition de pathologies allergiques. De plus, le développement de vaccins capables de cibler les lymphocytes T CD8+ chez le jeune enfant pourrait contribuer à la recherche de nouveaux traitements des allergies et de l’asthme.

Notch signals are required to promote T lineage commitment and development and suppress alternative cell fates in the thymus. Although the Notch activating ligand(s) in the thymus is(are) not known, studies have shown that hematopoietic progenitors are sensitive to Delta-like (DL), but not Jagged (Jag)-type ligands. In Chapter 3, I show that DL-expressing bone marrow stromal cell lines exhibit Notch ligand-independent functional heterogeneity in their capacity to support T cell development in vitro. These findings thus suggest the existence of stromal cell-derived signals that work with Notch to support T cell development. In Chapters 4 and 5, I investigated the ability of Fringe proteins to modulate Notch ligand-receptor interactions and the developmental consequences of these interactions for hematopoetic progenitors. Fringe proteins are glycosyl-transferases that enhance Notch activation by DL ligands and inhibit Notch activation by Jag ligands. In Chapter 4 I show that Lunatic Fringe (Lfng) enhances the strength of DL-mediated Notch activation to drive proliferation and expansion of early thymocytes and that DL4 and DL1 display different potencies to induce Notch-dependent outcomes. In Chapter 5, I demonstrate for the first time in a mammalian system that Lfng and Manic Fringe (Mfng) co-operate to enhance DL-Notch interactions and inhibit Jag-Notch interactions in hematopoietic stem cells. Thus, Lfng and Mfng function together to induce T cell development and inhibit B cell, myeloid and NK cell development. Collectively, these data highlight the importance of Fringe proteins in modulating the strength and specificity of Notch signaling levels during hematopoieisis.

The pathology of multiple sclerosis (MS) is characterized by an inflammatory mononuclear infiltration in the white matter. There has been converging evidence of the oxidative stress playing a role in the onset and progression of MS. We postulated that the decreasing oxidative stress might help in the management of MS. We know that the induction of phase 2 enzymes decreases the oxidative stress. The experimental allergic encephalomyelitis (EAE) induced in the Lewis rats were used to test this hypothesis. The 24 animals were placed into two groups: 1) those on normal rat chow, 2) those on rat chow containing 7.5 g/kg of tetra-butylhydroxyanisole (BHA), a food preservative. All the animals were administered 100 µg of guinea pig myelin basic protein in their tails to induce EAE and examined daily in a double blinded fashion. On 29th day of the induction, the animals were sacrificed, blood collected for glutathione (GSH) measurements and tissues collected for histology. All the animals, regardless of their diet status, developed symptoms of EAE on different days ranging from tail weakness to hind limb paralysis and all of them reached remission of acute EAE before the 28th day of induction. The non-BHA fed animals developed hind limb weakness in 8 animals and hind limb paralysis in 4 cases, while that of BHA fed group developed tail paralysis in 2, hind limb weakness in 2 and hind limb paralysis in 8 cases. The histology of the non-BHA group correlated well with the clinical symptoms of perivascular mononuclear infiltration. However, the BHA group revealed complete pathological recovery. Animals with BHA in the diet had significantly raised GSH, indicating the induction of phase 2 enzymes. We conclude that dietary phase 2 enzyme inducers show potential therapeutic benefits in EAE and should be examined for this role in MS.

Notch signals are required to promote T lineage commitment and development and suppress alternative cell fates in the thymus. Although the Notch activating ligand(s) in the thymus is(are) not known, studies have shown that hematopoietic progenitors are sensitive to Delta-like (DL), but not Jagged (Jag)-type ligands. In Chapter 3, I show that DL-expressing bone marrow stromal cell lines exhibit Notch ligand-independent functional heterogeneity in their capacity to support T cell development in vitro. These findings thus suggest the existence of stromal cell-derived signals that work with Notch to support T cell development. In Chapters 4 and 5, I investigated the ability of Fringe proteins to modulate Notch ligand-receptor interactions and the developmental consequences of these interactions for hematopoetic progenitors. Fringe proteins are glycosyl-transferases that enhance Notch activation by DL ligands and inhibit Notch activation by Jag ligands. In Chapter 4 I show that Lunatic Fringe (Lfng) enhances the strength of DL-mediated Notch activation to drive proliferation and expansion of early thymocytes and that DL4 and DL1 display different potencies to induce Notch-dependent outcomes. In Chapter 5, I demonstrate for the first time in a mammalian system that Lfng and Manic Fringe (Mfng) co-operate to enhance DL-Notch interactions and inhibit Jag-Notch interactions in hematopoietic stem cells. Thus, Lfng and Mfng function together to induce T cell development and inhibit B cell, myeloid and NK cell development. Collectively, these data highlight the importance of Fringe proteins in modulating the strength and specificity of Notch signaling levels during hematopoieisis.