Natural killer (NK) cells and cytotoxic T lymphocytes (CTL) are critical for the protection of organisms against pathogens and cancer. The process by which these cells eliminate infected or transformed cells are through two basic mechanisms, receptor-mediated interactions, or delivery of contents from intracellular cytotoxic granules. Granules are comprised of perforin and a family of serine proteases, called granzymes. Upon entry into target cells, these proteins work together to initiate cellular death pathways. Previous and extensive biochemical studies had already established that granzyme B (GrB) was a powerful inducer of apoptosis, but sensitive assays to confirm its release from cytotoxic cells were lacking. We hypothesized that GrB release, measured by ELISPOT, directly assessed the lytic potential of antigen-specific cytotoxic cells. Indeed, data provided in this thesis established a strong correlation between GrB release and target cell lysis. Our results imply that GrB could be a promising tool to assess cell-mediated immunity during vaccine development. However, several other independent studies in grB-/- mice demonstrated that additional granzymes were capable of clearing viruses and tumorigenic cells. Granzyme H (GrH) is highly and constitutively expressed in human NK cells, and therefore, we hypothesized that it was also an effective cytotoxic molecule. Our experiments established that GrH-induced cell death by a mechanism distinct from those of GrB and Fas. We identified a GrH substrate, DFF45/ICAD, and showed that GrH induced mitochondrial damage through a Bid-independent mechanism. Furthermore, cell death was dependent on Bax and/or Bak, but independent of caspase activation. Hence, we have elucidated an alternative cytotoxic pathway that could be employed to eliminate target cells with immune evasion strategies targeted to GrB or Fas. Finally, control of serine proteases by endogenous inhibitors is important to numerous biological processes, including apoptosis. We hypothesized that as GrH displayed chymase activity, the serine protease inhibitor anti-chymotrypsin (ACT) would impair GrH function. Our data established that ACT effectively attenuated GrH cytotoxicity and prevented proteolysis of a GrH substrate. Collectively, this thesis describes a novel GrH inhibitor, provides a new tool to evaluate cell-mediated immunity, and provides evidence of an alternative mechanism of cytotoxicity.

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The magnitude and effectiveness of CD8 response against intracellular pathogens is directed by survival and apoptotic signals that govern the fate of T cells. XIAP is a bona fide endogenous inhibitor of apoptotic signals. In this thesis, I have investigated the role of XIAP at various stages of CD8 T cell response. I used both in vivo and in vitro models to show that XIAP acts in a CD8 T cell extrinsic and intrinsic manner to regulate the expansion and contraction phases of the CD8 T cell response, respectively. During the expansion phase, XIAP prevents the cell death of APCs to promote APC-T cell interaction and cytokine release, which facilitates the proliferation and survival of activated T cells. During the contraction phase, XIAP functions in a cell-intrinsic fashion to inhibit the proapoptotic signals in the activated CD8 T cells to prolong the immune response. Finally, I also demonstrate that the expression of XIAP in T cells is critical for their differentiation in to memory subsets. Overall, I present that XIAP plays a critical role in generating an effective CD8 T cell immune response.

Gold nanoparticles, a class of plasmonic nanoparticle, have increasingly been explored as an imaging and therapeutic agent to treat cancer due to their characteristic surface plasmon resonance phenomenon and penchant for tumor accumulation. Photothermal therapy has been shown as a promising cancer treatment by delivering heat specifically to the tumor site via gold nanoparticles. In this study, we demonstrate that gold nanorod (GNR)-mediated photothermal therapy can be more effective through the understanding of cell death mechanisms. By targeting GNRs to various cellular localizations, we explored the association of GNR localization with cell death pathway response to photothermal therapy. Furthermore, we compared the 2D monolayer experiments with 3D in vitro tumor models, multicellular tumor spheroids (MCTS), to mimic the structure of in vivo tumors. With MCTS, we evaluated the cell death response with GNRs distributed only on the periphery, as seen in typical in vivo studies, and distributed evenly throughout the tumor. We demonstrated that GNR localization influences the cell death response to photothermal therapy by showing the power threshold necessary to induce significant apoptotic and necrotic increases was lower for internalized GNRs than membrane-bound GNRs. Furthermore, apoptosis was found to increase with increasing laser power until the necrotic threshold and decreased above it, as necrosis became the dominant cell death pathway response. A similar trend was revealed with the 3D MCTS; however, the overall cell death percentages were lower, most likely due to the upregulated cell repair response and varied GNR distributions due to the presence of cell-cell and cell-matrix interactions. Furthermore, the uniformly distributed GNRs induced more apoptosis and necrosis than GNRs located in the MCTS periphery. In conclusion, we quantitatively analyzed the cell death pathway response to GNR-mediated photothermal therapy to establish that it has some dependence on GNR localization and distribution to gain a more thorough understanding of this response for photothermal therapy optimization. / text

BACKGROUND: Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis (SJS/TEN) is the most severe form of cutaneous adverse drug reaction and is characterized by extensive epidermal destruction of the skin and mucosal surfaces. Controversy remains regarding the immunopathogenesis of the disease. It has long been assumed that CD8 cytotoxic T cells mediate cell death by releasing cytotoxic granules and soluble granulysin that trigger keratinocyte apoptosis. However, this does not explain the massive cell death or inflammation that is observed clinically. We have preliminary evidence from transcriptional profiling of patient skin samples suggesting that the cell death pathways necroptosis and pyroptosis may mediate SJS/TEN. Herein we utilize retrospectively and prospectively collected patient samples to investigate these cell death pathways. OBJECTIVE: The goals of this study are two-fold: (i) to investigate cell death pathways in retrospectively-collected (SJS/TEN) patient skin samples and (ii) to directly test the cell death mediators and pathways mediating SJS/TEN using a novel in vitro model. METHODS: Clinically and histopathologically confirmed SJS/TEN skin specimens and control skin specimens from non-blistering T cell mediated drug reactions and healthy skin were obtained following retrospective analysis from a multi-centered patient database. Gene expression profiling is being performed using the NanoString nCounter® System on these samples as a second patient cohort to confirm and expand on preliminary study findings. In parallel, we have optimized the use of a novel human skin platform for an in vitro model of SJS/TEN. We also collected human serum from a prospective study of SJS/TEN and control patients and have optimized and are actively collecting blister fluid from SJS/TEN and control patients in an ongoing prospective study for use in this model. RESULTS: Through an extensive pathology database and medical record search of potential cases at Brigham and Women's Hospital, we identified a second patient cohort of SJS/TEN, non-blistering delayed-type drug hypersensitivity reactions and healthy controls. We identified and are collecting thorough demographic, clinical and laboratory data on 61 potential candidates for SJS/TEN, 4 for Drug Reaction with Eosinophilia Syndrome (DRESS), and 200 for Morbilliform Drug Eruptions (MDE). This second cohort is in the final step of analysis with review by an expert clinician to confirm cases. In parallel, we have designed an expansive gene panel to confirm cell death mediator and marker transcription in our bank of skin samples. This 815 gene panel uses the pre-designed panel from Nanostring®, spiked with an additional 30 genes specific to apoptosis, pyroptosis, and necroptosis. We reviewed multiple potential in vitro skin models and identified GenoSkin® as the most suitable human skin platform for our in vitro model. We collected serum from 6 SJS/TEN patients and 6 non-blistering drug reaction patients and 3 healthy controls, and are actively collecting blister fluid from SJS/TEN and thermal burn control patients for analysis in this model. CONCLUSIONS: Our preliminary data suggest necroptosis and pyroptosis induced by soluble death mediators tumor necrosis factor (TNF) alpha and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as the main cell death pathways responsible for SJS/TEN. We have successfully identified a large number of potential patient samples of both cases and controls to perform transcriptional profiling using a self-designed gene panel to confirm and expand upon our preliminary data. We have successfully collected prospectively patient serum and are actively collecting patient blister fluid for analysis in an optimized in vitro model using GenoSkin®. SJS/TEN is severely understudied and lacks a standard protocol for care. This stems from uncertainty surrounding disease pathobiology. It is critical that we use innovative approaches to interrogate the mechanism mediating disease to advance the field, and, most importantly, to improve the quality of care for these patients.

[Formulae and special characters can only be approximated here. Please see the pdf version of the abstract for an accurate reproduction.] Apoptosis is a morphologically distinct form of cell death that is involved in the regulation of normal and aberrant cell systems. The complexities of the apoptotic cell death pathway arise from variation in both the cellular specialisation and initial stimulus. The corpus luteum (CL) is an endocrine gland that whilst critical to the maintenance of pregnancy in the rat, regresses at the completion of each oestrous cycle and pregnancy. This regression is facilitated through apoptosis; though, the stimulus and factors involved in the apoptotic pathway are poorly understood. Previous studies suggest that CL regression is not initiated through failure of luteotrophic support, but rather the active production of a luteolytic factor, of which tumour necrosis factor -alpha (TNFα) is one possible candidate. Several publications have reported the participation of the immune system in ovarian events. There is evidence that TNFα expression within the ovary is coordinated between cells of the immune system and the hormonal regulation of the CL. This study has focussed on the role of TNFα in CL apoptosis and the factors involved in this apoptotic pathway. TNFα-induced cell death is governed by the presence of the two TNFα receptors (TNFR) and several second messenger systems that include; the sphingolipids, mitogen-activated protein (MAP) kinases, nitric oxide (NO), nuclear factor-kappaB (NF-κB) and the caspases. These factors and their interactions were assessed in the rat CL during pregnancy and post-partum, and in vitro. Apoptosis was measured through the analysis of DNA fragmentation using DNA 3’ end labelling and single cell electrophoresis (COMET assay). Assessment of mRNA and protein expression was through Real-time RT-PCR and Western blot analysis; proteins were localised within the CL by immunocytochemistry. In addition, specific measurement of sphingolipid expression and nitric oxide (NO) production was by high performance liquid chromatography (HPLC) and NO assay respectively. Following parturition, TNFα mRNA and protein expression increased corresponding to the onset of CL apoptosis and increased expression of the chemotactic factor monocyte chemoattractant protein -1 (MCP-1). Furthermore, CL apoptosis was induced by treatment with recombinant TNFα in a time- and dose-dependent manner. A similar effect was observed in isolated luteal cells. Simultaneously, the functional regression of the CL was assessed by measurement of both progesterone synthesis and steroidogenic acute regulatory (StAR) protein expression. StAR mRNA and protein expression declined toward parturition in vivo. Immunocytochemical studies revealed the presence of TNFα receptors 1 (TNFR1) and 2 (TNFR2) in luteal cells. Furthermore, TNFR mRNA was isolated from CL throughout pregnancy and post-partum. Subsequently, the role of the sphingolipids ceramide and sphingosine was examined during CL apoptosis in vitro. Ceramide and sphingosine were found to be potent apoptotic agents when administered in vitro (50µM). The downstream signal transduction of TNFα and ceramide was assessed through MAP kinase expression. Both TNFα and ceramide increased expression of the pro-apoptotic p38 MAP kinase with no change to the non-apoptotic extracellular signal-related kinase (ERK1&2). Despite previous reports of c-Jun NH2 terminal kinase (JNK) involvement in the cell death pathway, JNK expression was not evident in the rat CL. The caspases are a family of cysteine proteases central to the regulation and execution of apoptosis. General inhibition of the caspase cascade in vitro was effective in preventing apoptosis regardless of the apoptotic stimulus (TNFα, ceramide and sphingosine), suggesting that this pathway is central to CL apoptosis. Specific inhibition of several caspases produced a varying effect; inhibition of caspases 3, 6 and 8 significantly reduced the level of TNFα-induced apoptosis, thus supporting their classification as either regulatory or effector caspases. NO is endowed with the unique ability to initiate and to block apoptosis and this dichotomy extends to the cytotoxic actions of TNFα. Inhibition of NO production by treating CL with L-NAME prevented the onset of apoptosis, whilst NO production increased in response to increasing levels of apoptosis following trophic withdrawal. However, this effect was not seen during TNFα-induced apoptosis, suggesting that the actions of NO are independent of TNFα. The data presented within this study examine multiple elements of the TNFα cell death pathway in a single system. The results suggest that these elements are involved in TNFα signal transduction and furthermore, in rat CL apoptosis. It can be said that TNFα plays an active role in CL regression through the activation of the caspases, the sphingolipids and the MAP kinases.