CD45 is a protein tyrosine phosphatase that is expressed on all nucleated hematopoietic cells. The major substrates of CD45 in thymocytes and T cells are the Src family kinases Lck and Fyn. The role of CD45 in thymocyte development and T cell activation via its regulation of Src family kinases in T cell receptor signaling has been studied extensively. However, the role of CD45 in processes that affect thymocyte development prior to the expression of the T cell receptor has not been explored. The overall hypothesis of this study was that CD45 is a regulator of spreading, migration, proliferation, and differentiation of early thymocytes during development in the thymus and the absence of CD45 would alter the outcome of thymocyte development. The first aim was to determine how CD45 regulates CD44-mediated signaling leading to cell spreading. The interaction between CD44 and Lck was first examined. CD44 associated with Lck in a zinc-dependent and a zinc-independent manner. Mutation analysis localized the zinc-dependent interaction to the membrane proximal region of CD44, but did not involve individual cysteine residues on CD44. CD44 and Lck co-localized in microclusters upon CD44-mediated cell spreading. CD45 co-localized with Lck and CD44 in microclusters and with F-actin in ring structures. The recruitment of CD45 to microclusters may be a mechanism of how CD45 negatively regulates CD44-mediated spreading. The second specific aim was to determine the role of CD45 in migration, proliferation, and progression and differentiation of early thymocytes. CD45 negatively regulated CXCL12-mediated migration, and positively regulated the proliferation and progression of CD117- DN1 thymocytes. Absence of CD45 led to an altered composition of thymic subsets. The CD45-/- thymus contained decreased numbers of ETPs and an aberrant CD117- DN1 population that lacked CD24, TCRbeta, and CCR7 expression. There were also increased thymic NK and gamma/delta T cells, but decreased NKT cells. In addition, a novel intermediate between DN1 and DN2 that required Notch for progression was identified. Overall, this study identified new roles for CD45 in early thymocytes and provided a better picture of how the development of T cells, a central component of the immune system, is regulated.

The clinicopathological feature of primary cutaneous lymphomas according to WHO/EORTC classification and their relationship to EBV in Taiwan has never been reported. This retrospective study collected the patients with cutaneous lymphomas from 1990 and 2006. The morphology, EBER in situ hybridization and immunohistochemistry of primary cutaneous lymphomas were studied to reclassify based on the WHO/EORTC classification. A total of 54 patients were included. Twenty-nine were primary cutaneous lymphomas and 25 were secondary cutaneous lymphomas. The age ranged from 21 to 86 years old (mean 62 years old). Twenty-one (72.4%) were primary cutaneous T-cell and NK-cell lymphoma, including 5 extranodal NK/T-cell lymphoma, nasal type (17.2%), 5 primary cutaneous peripheral T-cell lymphoma, unspecified (17.2%), 4 mycosis fungoides (13.8%), 1 Sezary syndrome, 3 primary cutaneous anaplastic large cell lymphoma, 2 primary cutaneous small-medium CD4+ T-cell lymphoma and 1 subcutaneous panniculitis-like T-cell lymphoma. Eight cases were primary cutaneous B-cell lymphoma (27.6%) including 3 cutaneous marginal zone B-cell lymphoma (10.3%), 3 cutaneous follicle center B-cell lymphoma (10.3%), and 2 diffuse large B-cell lymphoma, leg type (6.9%). Seventeen cases were secondary cutaneous T-cell and NK -cell lymphoma. Eight cases were secondary cutaneous B-cell lymphoma. All primary and secondary extranodal NK/T-cell lymphoma, nasal type, were positive for EBER, however, one of them (10%) without both angiocentric growth pattern and necrosis in histomorphological examination. This is the first clinicopathological study of cutaneous lymphoma according to recent WHO/EORTC classification in Taiwan. In comparison with the Western countries, mycosis fungoides is less common whereas primary extranodal NK/T-cell lymphoma, nasal type, and peripheral T-cell lymphoma, unspecified, is more common in Taiwan. EBER in situ hybridization study is helpful in the diagnosis of extranodal NK/T-cell lymphoma, nasal type, especially in tumor without both angiocentric growth pattern and necrosis.

Binding of the antigen receptor on T cells initiates a rapid series of signaling events leading to an immune response. To fully understand T cell mediated immunity, underlying regulatory properties of the receptor network must be understood. Monitoring dynamic protein signaling events allows for network analysis. Unfortunately, dynamic data acquisition is often extremely time-consuming and expensive with conventional methods; the number of proteins monitored at the same time on the same sample is limited. Furthermore, with conventional, multi-well plate assays it is difficult to achieve adequate resolution at sub-minute timescales. Microfluidics is a capable alternative, providing uniformity in sample handling to reduce error between experiments and precision in timing, an important factor in monitoring phosphorylation events that occur within minutes of stimulation. We used a two-module microfluidic platform for simultaneous multi-time point stimulation and lysis of T cells to investigate early signaling events with a resolution down to 20 seconds using only small amounts of cells and reagents. The device did not elicit adverse cellular stress in Jurkat cells. The activation of 6 important proteins in the signaling cascade upon stimulation with a soluble form of α-CD3 in the device was quantified and compared under a variety of conditions. First, in comparison to manual pipetting, the microdevice exhibits significantly less error between experiments. Secondly, a comparison between Jurkat cells and primary T cells shows similar dynamic trends across the 6 proteins. Finally, we have used the device to compare properties of long-term vs, short-term cultured primary T cells. As expected, older cells present a much weakened response to antigenic cues, as measured with TCR response markers. This modular microdevice provides a flexible format for investigating cell signaling properties through the use of soluble cue stimuli.

Approximately two-thirds of US adults are overweight or obese, and obesity is also becoming more prevalent in children and adolescents. Similar to adults, obese children are at a higher risk of developing health problems due in part to dysfunctional immune surveillance. Obesity has been shown reduce the generation of new T-cells by accelerating thymic aging in an adult mouse. This study therefore aimed at determining whether similar diet induced obesity (DIO) changes can be induced in a young mouse. Comparisons made between lean and DIO C57Bl/6 mice showed a significant increase in thymic weight, decrease in thymic cellularity and thymic output, and impaired T-cell development at the double negative stage. We associate these alterations with changes in thymic architecture and accumulation of lipid droplets within the thymic cortex and medulla of the obese mice. The above observations indicate that DIO can induce fat accumulation and reduce thymic function at a young age. Resveratrol, a natural polyphenolic compound, was then used to regulate fat metabolism in an attempt to reduce these DIO changes we observed. Resveratrol induces fat oxidation via 5' adenosine monophosphate-activated protein kinase (AMPK), and its reciprocal regulation of glycerol-3-phosphate acyltransferase-1 (GPAT-1) and carnitine palmitoyltransferase-1 (CPT-1), the rate-limiting enzymes required for glycerophospholipid biosynthesis and oxidation, respectively. Through resveratrol feeding, we were able to prevent the effects of DIO on thymic architecture and thymic T-cell proliferation. This was achieved by manipulating AMPK into inhibiting GPAT-1 and enhancing CPT-1 activity. Since the expression of GPAT-1 was upregulated in the obese mice, we investigated whether deleting GPAT-1 altogether might prevent the thymic involution, by inhibiting synthesis of glycerophospholipids and triacylglycerol. Instead, we found that GPAT-1 deletion slowed thymic growth and reduced cellularity in young mice, which we associated with impaired thymic T-cell function and development, suggesting that the deleterious effects of GPAT-1 deficiency may be due to perturbations in thymic T-cell activation and signaling. These data provide a novel link between lipid metabolism and T-cell development, and identify the use of the naturally-occurring resveratrol to reduce lipid accumulation within the involution-prone thymus, thus providing a useful approach to preventing a decline in thymic function in childhood. / text

Early Growth Response Genes (EGR) is a family of four transcription factors containing a unique zinc finger domain. EGR-2 and EGR-3 are important for hindbrain development and myelination. These transcription factors are also necessary for lymphocyte function however, the mechanisms are still unclear. Previous findings have shown that EGR-2cKO mice develop lupus-like autoimmune disease with high levels of pro-inflammatory cytokines despite showing normal T and B cell proliferation after mitogenic stimulation. Therefore we established the CD2-EGR-2-/-EGR-3-/- mouse model to explore the phenotype, susceptibility to autoimmune disease and relevant lymphocyte function. We discovered that CD2-EGR-2-/-EGR-3-/- mice developed severe systemic autoimmune disease and expressed high levels of inflammatory cytokines. More importantly we discovered a novel finding that CD2-EGR-2-/-EGR-3-/- T and B cells had impaired cell proliferation after mitogenic stimulation. Further investigations revealed that the molecular mechanism defected in the T cell receptor signalling pathway is due to a dysfunction in Activator Protein-1 (AP-1). AP-1 is a heterodimeric protein composed of AP-1 family members including Jun, Atf and Fos. Our data shows that EGR-2 and EGR-3 directly bind with the Atf family member Batf, which prevents Batf’s inhibitory function on AP-1 activation. This research demonstrates that EGR-2 and EGR-3 intrinsically regulate chronic inflammation and also positively regulate antigen receptor activation. In conclusion EGR-2 and EGR-3 are essential for providing optimal immune responses, whilst limiting inflammatory immunopathology. We propose that this new model could be used for studying autoimmune disease.

HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunnelling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown. In this dissertation, we developed and characterized a lentiviral vector-based system to express Nef in T-cell lines and primary human peripheral blood mononuclear cells, and then used this system to perform a proteomic screen to identify Nef-associated host cell factors and better understand how Nef hijacks the T-cell machinery to maximize HIV production and dissemination. Bioinformatic and cell-based analysis of the resulting host factors revealed a mechanism by which Nef enhances nanotube formation. To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3). Wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6), an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery. Together, our findings identify the exocyst complex as a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Furthermore, linkages revealed between Nef and the exocyst complex suggest a new paradigm of exocyst involvement in polarized targeting for intercellular transfer of viral proteins and viruses.

Immune protection wanes during aging. This is evidenced by increased morbidity and mortality from infectious disease in aged individuals. As the aging population continues to increase worldwide, it will become increasingly important to determine both causes and therapeutic strategies for defects in the aged immune response. In particular, CD8 T cells have been shown to be highly susceptible to age-related defects. Recently, metabolic pathways have been implicated as critical factors in T cell fate decisions during immune responses. Of note, metabolic pathways are also considered primary determinants of lifespan in mammals. Therefore, we hypothesized that metabolic manipulations to extend lifespan would have significant effects on the aging immune system and protection during infection. In particular, we investigated the impact of rapamycin (rapa), both acute and chronic treatment regimens, on adult and old mice. Specifically, we tested how T cell development, peripheral homeostasis, and effector immunity became altered during treatment. We made side-by-side comparisons in calorically restricted (CR) old mice as a gold standard model of longevity extension. Importantly, both of these interventions have been reported to benefit immune function and extend lifespan in mice. However, our data strongly indicate that both rapa and CR induce distinct but deleterious consequences to overall immunity in mice. We conclude that neither rapa nor CR may be ideal candidates for extending lifespan in humans.

CD45 is a protein tyrosine phosphatase that is expressed on all nucleated hematopoietic cells. The major substrates of CD45 in thymocytes and T cells are the Src family kinases Lck and Fyn. The role of CD45 in thymocyte development and T cell activation via its regulation of Src family kinases in T cell receptor signaling has been studied extensively. However, the role of CD45 in processes that affect thymocyte development prior to the expression of the T cell receptor has not been explored. The overall hypothesis of this study was that CD45 is a regulator of spreading, migration, proliferation, and differentiation of early thymocytes during development in the thymus and the absence of CD45 would alter the outcome of thymocyte development. The first aim was to determine how CD45 regulates CD44-mediated signaling leading to cell spreading. The interaction between CD44 and Lck was first examined. CD44 associated with Lck in a zinc-dependent and a zinc-independent manner. Mutation analysis localized the zinc-dependent interaction to the membrane proximal region of CD44, but did not involve individual cysteine residues on CD44. CD44 and Lck co-localized in microclusters upon CD44-mediated cell spreading. CD45 co-localized with Lck and CD44 in microclusters and with F-actin in ring structures. The recruitment of CD45 to microclusters may be a mechanism of how CD45 negatively regulates CD44-mediated spreading. The second specific aim was to determine the role of CD45 in migration, proliferation, and progression and differentiation of early thymocytes. CD45 negatively regulated CXCL12-mediated migration, and positively regulated the proliferation and progression of CD117- DN1 thymocytes. Absence of CD45 led to an altered composition of thymic subsets. The CD45-/- thymus contained decreased numbers of ETPs and an aberrant CD117- DN1 population that lacked CD24, TCRbeta, and CCR7 expression. There were also increased thymic NK and gamma/delta T cells, but decreased NKT cells. In addition, a novel intermediate between DN1 and DN2 that required Notch for progression was identified. Overall, this study identified new roles for CD45 in early thymocytes and provided a better picture of how the development of T cells, a central component of the immune system, is regulated.

Numerous studies have demonstrated that tumour cells have the ability to alter immune function to create an immune suppressed environment. This allows tumour cells to escape immune surveillance and consequently the tumour can progress. Dendritic and T cells have critical roles in immune activation and tolerance and are thus major targets of tumour-mediated immune suppression. Understanding the mechanism(s) by which tumour cells modulate the immune system will facilitate the development of immune system-based therapies for cancer treatments. In this study we sought to determine the nature of, and cellular and molecular mechanisms underlying, changes in immune status during tumour progression using mouse models of prostate cancer. Detailed analysis of the immunological status in a mouse prostate dysplasia model (12T-7slow) revealed that immune suppression accompanied tumour progression. We found that T cells isolated from tumour-bearing hosts were hypo-responsive to antigen stimulation. Furthermore, we demonstrated that CD4+CD25+ regulatory T cells were responsible, at least in part, for this alteration. Anti-CD25 antibody treatment reduced, but did not prevent, tumour growth in either a transplanted prostate tumour model or a spontaneously developing prostate tumour model. In addition, an altered dendritic cell phenotype and an elevated frequency of CD4+CD25+ regulatory T cells were observed within the tumour mass. Similar alterations were observed in the prostate-specific Pten knockout mice which develop advanced prostate adenocarcinoma. Interestingly, evidence of immune activation, such as an increased frequency of activated T cells, was detected in the tumour microenvironment in both mouse prostate tumour models. To identify factors that may play critical roles in the altered immune cell phenotype observed in the tumour microenvironment, a global gene expression profiling analysis was carried out to evaluate the changes in immune-related gene expression patterns. This analysis provided additional evidence for the co-existence of immune suppression and immune activation. Moreover, subsequent analyses suggested that one differentially expressed transcript, interferon regulatory factor 7, and its target genes might be involved in modulating immune cells and/or tumour progression. Taken together, these studies have important implications for designing specific and effective anti-tumour immune therapy strategies that involve manipulation of tumour cells, dendritic cells and regulatory T cells.

Piperine, a pungent alkaloid found in the fruits of long and black pepper plants, has diverse physiological effects, including anti-inflammatory and anti-cancer activities. The effect of piperine on the function of T cells and endothelial cells, two important elements of inflammation, have not been examined previously and were the focus of this study. Piperine inhibited the proliferation of human endothelial cells, murine T cells, and IL-2-dependent CTLL-2 T cells, without affecting cell viability. Progression into the S phase of the cell cycle was inhibited in all three cell types. In T cells, piperine inhibited expression of the early activation marker CD25, production of IFN-?, IL-2, IL-4, and IL-17A, and the generation of cytotoxic effector cells. In endothelial cells, piperine inhibited migration and tubule formation in vitro and ex vivo, as well as breast cancer cell-induced angiogenesis in chick embryos. Piperine inhibited Akt phosphorylation in signaling pathways associated with growth factor receptors on endothelial cells, T cell receptor and CD28 on T cells, and IL-2 receptor on CTLL-2 cells. Additionally, piperine inhibited ERK1/2 and I?B phosphorylation in activated T cells, as well as STAT3, STAT5, and ERK1/2 phosphorylation in IL-2-stimulated CTLL-2 cells. However, piperine is not a broad-spectrum inhibitor of phosphorylation as it did not inhibit ZAP-70 phosphorylation in activated T cells or phosphorylation of JAK1 and JAK3 in IL-2-stimulated CTLL-2 cells. Piperine-mediated inhibition of T cell activation and IL-2 receptor signaling suppresses T cell proliferation and effector cell differentiation, suggesting possible utility in treating T cell-mediated autoimmune and chronic inflammatory conditions. Additionally, the potent anti-angiogenic activity of piperine warrants further study for the prevention of inflammation- and cancer-promoting angiogenesis.