Canine Hemophagocytic Histiocytic Sarcoma (CHHS) is an aggressive neoplasm of macrophages with local lymphocytic reaction. Similarities exist between CHHS and Familial Hemophagocytic Lymphohistiocytosis (FHL), a complex of histiocytic diseases in children, which is attributable to various defects in granule dependent killing (GDK). This led to the hypothesis that defective GDK compromises lymphocyte homeostasis and anti-tumor immunity which results in CHHS. The sequence of canine perforin, a key effector molecule of GDK, was determined by RT-PCR and RACE. Genomic DNA from healthy and CHHS-affected dogs was sequenced and analyzed, but mutations with functional implications were not identified. Subsequently, tumor infiltrating lymphocytes (TIL) of CHHS were examined for GDK functionality. CHHS-TIL were compared to their functional counterparts in canine cutaneous histiocytoma (CCH), a benign histiocytic tumor in dogs, known to regress via lymphocytic reaction. To facilitate such comparison, functionality of CCH-TIL was studied by immunohistochemistry and confocal microscopy and quantified by image analysis applications. This provided novel insights regarding the physiology of TIL in tumor microenvironment and further characterizing CCH as a model for anti-tumor immunity. The comparison revealed a clear, and highly significant structural difference in polarization and degranulation of CHHS-TIL which likely hampers GDK. This defect is similar to several variants of FHL, an association further supported by comparison of clinical and laboratory manifestations of CHHS and FHL. This study suggests that CHHS is a promising natural model for investigating the pathogenesis of FHL, for studying granule polarization and degranulation and assessing the role of TIL in anti-cancer immunity. / Pet Trust foundation

Thesis (Ph. D.)--University of Nevada, Reno, 1999. / Includes bibliographical references. Online version available on the World Wide Web.

Thesis (M.S.)--University of Nevada, Reno, 2001. / Includes bibliographical references. Online version available on the World Wide Web.

The Tim-3+ T cells in HIV infection are dysfunctional in proliferation or cytokine production. Here, we evaluated the effects of Tim-3 expression on the cytotoxicity of CD8+ T cells in HIV infection by examining 1) the ability of Tim-3+ CD8+ T cells to make perforin and 2) the direct ability of Tim-3+ CD8+ T cells to kill HIV infected CD4+ target cells. Tim-3+ CD8+ T cells maintained higher levels of perforin. However, these cells were defective in their ability to degranulate. Blocking the Tim-3 signalling pathway enhanced the cytotoxic capabilities of HIV specific CD8+ T cells by increasing: their degranulation capacity, their ability to release perforin, their ability to target activated granzyme B to HIV antigen expressing CD4+ T cells and their ability to suppress HIV infection of CD4+ T cells. Thus, the Tim-3 receptor can down-regulate the CD8+ T cell cytotoxicity through inhibition of degranulation and perforin/granzyme secretion.

The Tim-3+ T cells in HIV infection are dysfunctional in proliferation or cytokine production. Here, we evaluated the effects of Tim-3 expression on the cytotoxicity of CD8+ T cells in HIV infection by examining 1) the ability of Tim-3+ CD8+ T cells to make perforin and 2) the direct ability of Tim-3+ CD8+ T cells to kill HIV infected CD4+ target cells. Tim-3+ CD8+ T cells maintained higher levels of perforin. However, these cells were defective in their ability to degranulate. Blocking the Tim-3 signalling pathway enhanced the cytotoxic capabilities of HIV specific CD8+ T cells by increasing: their degranulation capacity, their ability to release perforin, their ability to target activated granzyme B to HIV antigen expressing CD4+ T cells and their ability to suppress HIV infection of CD4+ T cells. Thus, the Tim-3 receptor can down-regulate the CD8+ T cell cytotoxicity through inhibition of degranulation and perforin/granzyme secretion.

Two important mechanisms of lymphocyte-mediated cytotoxicity, one perforin based and the other Fas ligand (FasL) based, have been characterized recently. It has also been shown that CD44, an adhesion molecule, can participate in signaling cytotoxic activity of cytotoxic T lymphocytes (CTLs). In the current study we tested the hypothesis that activation of natural killer (NK) or lymphokine activated killer (LAK) cells induces the expression of FasL, perforin, and CD44 which together contribute towards increased cytolytic activity. To this effect, we used wild-type mice, perforin-knockout mice, and mice lacking a functional FasL. We observed that both interleukin-2 (IL-2) and Poly I:C triggered NK/LAK cells to lyse targets through the perforin- and FasL- pathways. In addition, Fas+ tumor targets were more susceptible to lysis by poly I:C and IL-2 activated NK/LAK cells when compared to Fas- targets. Furthermore, Fas- tumor cells injected subcutaneously into syngeneic mice could grow and induce tumors, whereas, Fas+ tumors were rejected. IL-2 treatment increased the CD44 expression on NK cells, which was responsible for the lysis of endothelial cells through its ligand, hyaluronate. Upregulation of perforin and FasL in activated NK/LAK cells may explain why such cells can kill a wide variety of tumor cells efficiently. On the other hand, activated NK/LAK cells express increase increased levels of CD44 and use this molecule to mediate cytotoxicity of endothelial cells, which may account for the vascular leak seen during IL-2 therapy. / Master of Science

Les cellules de mélanome humain expriment différents antigènes tumoraux qui sont reconnus par les lymphocytes T cytotoxiques CD8 + (CTL) induisant des réponses spécifiques de la tumeur in vivo. Cependant, chez les patients atteints de mélanome l'efficacité de la réponse naturelle des CTL ou stimulée par thérapie est limitée. Les mécanismes sous-jacents de l'échec de la phase effectrice des CTL contre les mélanomes sont encore largement méconnus. Notre hypothèse est que l'efficacité limitée des CTL dans leur combat contre les tumeurs est le résultat d'une balance défavorable entre la capacité des CTL à tuer les tumeurs et une résistance tumorale intrinsèque à l'activité cytolytique des CTL. Au cours de ma thèse je me suis concentrée sur la dynamique moléculaire qui se produit à la synapse lytique afin de pouvoir identifier un mécanisme précoce mis en place par les cellules de mélanome face à l'attaque des CTL. En combinant l'utilisation d'approches de microscopie de pointe et des outils moléculaires, j'ai pu montrer que, lors de l'interaction avec les CTL, les cellules de mélanome humain subissent une activation de leur trafic vésiculaire endosomal et lysosomal, lequel est intensifié à la synapse lytique et corrèle avec la dégradation par la cathepsine de la perforine et un défaut de pénétration d'entrée du granzyme B. De plus, j'ai démontré que le blocage du trafic lysosomal dépendant de SNAP23, la modification du pH (intra-vésiculaire) et l'inhibition de l'activité lysosomale protéotlytique des cellules de mélanome permet de restaurer leur sensibilité à l'attaque des CTL. Nos résultats révèlent une stratégie sans précédent d' " auto-défense " des cellules de mélanome à la synapse immunologique basée sur une sécrétion lysosomale massive et sur la dégradation de la perforine sécrétée par les CTL. Ainsi pouvoir interférer avec cette stratégie synaptique d'auto-défense des cellules de mélanome pourrait contribuer à potentialiser les réponses des CTL et les immunothérapies chez les patients atteints de mélanome. / Human melanoma cells express various tumor antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTL) and elicit tumor-specific responses in vivo. However, natural and therapeutically enhanced CTL responses in melanoma patients are of limited efficacy. The mechanisms underlying the failure of CTL effector phase against melanomas are still largely elusive. Our hypothesis is that the limited efficacy of CTL in their fight against tumors is the result of an unfavorable balance between CTL ability to kill tumors and an intrinsic tumor resistance to CTL cytolytic activity. During my thesis I focused on the molecular dynamics occurring at the lytic synapse in order to identify possible "early response-mechanism" of melanoma cells to CTL attack. Using a combination of cutting edge microscopy approaches and molecular tools, I showed that upon conjugation with CTL, human melanoma cells undergo an exacerbated late endosome/lysosome trafficking, which is intensified at the lytic synapse and is paralleled by cathepsin-mediated perforin degradation and deficient granzyme B penetration. Abortion of SNAP-23-dependent lysosomal trafficking, pH perturbation or impairment of lysosomal proteolytic activity restores susceptibility to CTL attack. Our results reveal an unprecedented strategy of melanoma cell "self-defense" at the immunologic synapse based on a lysosome secretory burst and perforin degradation at the lytic synapse. Interfering with this synaptic self-defense strategy might be instrumental to potentiate CTL-mediated therapies in melanoma patients.

[Truncated abstract] The design and development of effective anti-viral immunotherapies requires a comprehensive understanding of the cellular and molecular processes that are involved in the generation and regulation of immune responses. The fundamental objective of the immune system is to successfully complete the task of eliminating/controlling the invading pathogen without causing overt pathology. Cytomegaloviruses (CMVs) are large DNA viruses that are able to evade immune attack and persist lifelong within the host. In a healthy host, CMV causes an asymptomatic infection, but in instances of decreased immune functions, such as in newborns, acquired immunodeficiency syndrome (AIDS) patients and transplant recipients, the infection can result in serious morbidity and mortality. Thus, human CMV (HCMV) is a clinically important pathogen and an understanding of the pathogenesis, mechanisms of immune subversion and, importantly the cascade of immune events that ensue following infection is highly relevant. The studies presented in this thesis have provided useful insight into various aspects of viral immunity and it is hoped that they will assist in the design of more effective therapies against viruses of clinical importance. Genetic variability in humans can greatly influence anti-viral immune responses and the outcome of viral infection. ... Furthermore, these studies provide novel evidence that NK cells are also crucial for the control of virus in some organs of susceptible mice during early acute infection. The data reveals that both NK cells and CD8+ T cells utilise perforin- and IFN-? dependent control of MCMV. Furthermore, these studies provide novel evidence that protection mediated by Ly49H+ NK cells in resistant mice is dependent on perforin. Chapter 3 focuses on the biological relevance of Grz during MCMV infection. These studies found that GrzA and GrzB are essential components of the machinery involved in limiting MCMV during acute infection. These analyses also provide the first evidence suggesting that GrzM plays a role, albeit minor, in controlling MCMV replication. Furthermore, the current studies suggest that Grz can mediate direct antiviral activities independent of the induction of cell death in conjunction with perforin. Interestingly, in the absence of both GrzA and GrzB (GrzAB), mice were as susceptible to MCMV infection as perforin-deficient mice. However, unlike perforin-deficient mice, GrzAB-deficient mice controlled and survived the infection. In Chapter 4 the roles of perforin, GrzA and GrzB in anti-viral immunity and immunopathology during MCMV infection were examined. These studies show that NK cell-derived perforin is required to eliminate infected targets as well as activated effector cells, suggesting that NK cells are crucial not only in defensive immunity but also in limiting the immune activation that follows MCMV infection. In summary, the studies presented in this thesis define the significant role played by specific effector molecules in limiting MCMV replication during different stages of this viral infection. Furthermore, these studies provide novel evidence that perforin, GrzA and GrzB play distinct roles in defensive immunity and limiting immunopathology during MCMV infection.

A major objective in Theileria parva subunit vaccine development is to induce a vaccine antigen specific response mediated by cytotoxic CD8+ T cells (CTL). Therefore it is essential to be able to measure the frequency of the responding CD8+ T cells after vaccination and correlate it with a clinical outcome on challenge. Recently concluded immunogenicity and efficacy studies of T. parva specific CTL antigens showed successful induction of CTL responses in some animals, which correlated with reduced disease severity after challenge. To provide correlates of immunity antigen-specific CD8+ T cell mediated IFN-γ responses and CTL lytic responses were measured over the course of the experiments. Several challenges presented in these trials aimed at optimising vaccine efficacy. While the IFN-γ ELISPOT is a sensitive and reliable assay widely used in vaccine research, the use of chromium/indium release assay remains to be the only assay in use that measures T. parva-specific CTL activity. Hence the overall goal of the study was to develop novel reagents and novel assays to identify parasite-specific CD8+ T lymphocytes with lytic potential. To address this objective, bovine perforin, granzymes A and B, as specific effector proteins expressed in activated CTL were cloned and expressed using a baculovirus expression system. Sequence analysis of the cloned cDNAs showed the isolated cDNA belonged to the perforin and granzyme sub-families respectively. Perforin cDNA demonstrated 85% homology to human perforin with presence of conserved regions resembling calcium binding motif, membrane attack complex component as well complement protein. The sequences encoded by the cloned granzyme A and B cDNAs have the features of a trypsin like serine protease and demonstrates over 70% homology to the human cDNA over the active enzyme region as well catalytic residues characteristic of serine proteases. The expressed polypeptides of all three proteins were used to produce specific antibodies for use as reagents in immunoassays including ELISpot and intracellular staining for flow cytometric analysis. While the antibodies showed reactivity to the recombinant proteins, these reagents displayed different functionality in the recognition of the native protein. Peptide-major histocompatibility complexes (MHC) class I tetrameric complexes (tetramers) are proving invaluable as fluorescent reagents for enumeration, characterisation and isolation of peptide-specific CD8+ T cells and have afforded advantages to phenotype antigen-specific T cells with minimal in vitro manipulation. Fluorescent bovine tetramers were shown to specifically stain antigen-specific CTL by directly binding the T cell receptor (TCR). Analyses of CD8 T-cell responses in live-vaccine immunised cattle also showed that this method is robust and demonstrates changes in the kinetics and specificity of the CD8+ T cell response in primary and secondary infections with T. parva. On average, results of functional assays and tetramer staining followed parallel trends, measured roughly the same populations and allowed for surface and intracellular staining for CD8 T cell marker and perforin, respectively, demonstrating a method that reliably quantifies the frequency, phenotype and function of specific CD8+ T cells. The technical simplicity, rapidity and ability of the flow cytometric technique described in this thesis to measure low frequency antigen-specific responses suggests that tetramer staining, combined with functional assays could be broadly applicable to the valuation of vaccination efficacy to determine which protocols are most successful in inducing CTL responses.

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, PKCθ 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.