The aged T cell is characterized by decreased responsiveness to stimulation. Aging is associated with reduced membrane glycerophospholipid (GPL) to cholesterol ratios so it is interesting that deletion of mitochondrial glycerol-3-phosphate acyltransferase-1 which catalyzes the first step in de novo GPL synthesis induces an aged T cell phenotype in otherwise healthy mice. GPAT-1 could regulate T cell function through three possible mechanisms: maintenance of membrane GPL ratios and membrane based signaling, providing a specific substrate for downstream signaling, or direct regulation of cellular metabolism. Therefore, the goal of this project was to determine whether these mechanisms contribute to the dysfunctional T cell phenotype observed with decreased GPAT-1 activity. T cell stimulation requires significant upregulation of metabolic processes to drive clonal expansion and cytokine production. T cell dysfunction in GPAT-1 knockout mice may be partially explained by altered metabolic function. We found that GPAT-1 KO T cells have significantly reduced basal respiration rates and spare respiratory capacity which is not compensated for by increased glycolytic metabolism suggesting an inherent metabolic defect in GPAT-1 KO T cells. To better understand mechanistically how GPAT-1 regulates T cell function we moved into the Jurkat T cell line and found that shRNA mediated knockdown of the human isoform of GPAT-1 (GPAM) recapitulated key aspects of the dysfunctional T cell phenotype we observed in the mouse including highly significant reductions in IL-2 production and altered membrane GPL to cholesterol ratios. Phosphatidic acid addition was not capable of rescuing these deficiencies suggesting that GPAT-1/GPAM activity is required for proper T cell function. This was the first time that GPAT-1 activity has been shown to be important for T cell function in a non-murine model system and strongly suggests that GPAT-1/GPAM deficiency regulates T cell function at the cellular level. We further demonstrate that phosphorylation of ZAP-70 a proximal effector of T cell activation is significantly reduced in GPAM knock down Jurkat T cells, suggesting that membrane based signaling is dysfunctional. Taken together these data suggest that GPAT-1 is necessary for regulating cellular energy demands in T cells and essential for optimal T cell activation following stimulation. / text

published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

To study the pathogenesis and preventive strategies of acute and chronic graft-versus-host disease (aGVHD, cGVHD) after hematopoietic stem cell transplantation (HSCT), murine models of aGVHD and cGVHD were constructed. In addition, the role of CD4+CD25+ regulatory T cells (Tregs) in GVHD was also investigated in these models. My project consisted of three parts, including MHCI,II mismatched (part 1) and haploidentical BMT(part2) based aGVHD, and MHC matched, minor histocompatibility antigen (miHA)mismatched cGVHD(part 3). In the first model, aGVHD resulting from an MHC I, II mismatched aGVHD (B6(H-2b)→BALB/c(H-2d)) HSCT was studied, particularly with respect to the role that CD4+CD25+Tregs played. The results showed that CD4+CD25-T-cells induced more severe aGVHD than CD4+ T-cells, resulting in more extensive target organ lesions, especially in colon. The possible mechanism might be due to the enhanced proliferation and differentiation towards pathogenic Th1 cells. In the second model, haploidentical (B6(H-2b)→[C57BL/6×CBA/Ca]F1(H-2b×k)) HSCT was used to studied the role of CD4+or CD8+T-cells in aGVHD.Both high dose of donor CD4+-and CD8+-T-cells have the ability to induce lethal aGVHD in the hosts. However, the clinical and histological features of aGVHD induced by CD4+T-cells were significantly different from that induced by CD8+T-cells. Both donor CD4+-and CD8+-T-cells showed marked proliferation and differentiation towards CD4+IFN-γ+Th1and CD8+IFN-γ+cells, respectively. Polyclonalexpanded freshly isolatednTregs (exp-nTregs) showed obvious proliferation, increased apoptosis, and rapid loss of Foxp3 expression with impaired suppressive function. Exp-nTregs were further investigatedfor their preventive function in aGVHD in this haploidentical HSCT model. The results showed that exp-nTregs were capable of attenuating either CD4+-or CD8+-T-cell-induced aGVHD with significantly prolonged survival rate. In the third model, cGVHD was investigatedin DBA/2 (H-2d)→BALB/c (H-2d) HSCT, where the biologic readout was proteinuria and skin fibrosis. The results showed that donor CD4+T-and B220+B-cells were the main effectors in the pathogenesis of cGVHD. Notably, a more active germinal center (GC) reaction existed in the cGVHD cohorts compared with the control syngeneic cohort. Furthermore, Tfh and GC B-cells were shown to have originated from donor CD4+T-and B220+B-cells, respectively. Importantly, Tfh and GC B cells were mutually stimulatory and inter-dependent. In conclusion, three murine models were used to investigate aGVHD and cGVHD. The results showed that Tregs played a significant suppressive role in aGVHD complicating haploidentical HSCT. Furthermore, a hyperactive germinal center reaction might be the main cause of cGVHD. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Leptin, a protein hormone encoded by obese (ob) gene, is mainly produced by adipocytes. Leptin plays an important role in regulating neuroendocrine function and energy metabolism. As a cytokine, leptin is involved in modulating the hematopoiesis and lymphopoiesis. Although leptin has been found to promote T cells activation, it is largely unclear whether and how leptin regulates T cell differentiation and function. Leptin has been associated with disease severity in rheumatoid arthritis (RA). Elevated leptin levels have been detected in the sera and synovial fluid of active RA patients. Th17 cells play key roles in synovitis and joint damage during arthritis development. However, the role of leptin on Th17 cells has not been investigated so far. In culture, leptin promoted Th17 cells differentiationfrom naïve 〖CD4〗^+ T cells via upregulating ROR-γt expression. Moreover, Th17 cells and IL-17 levels were significantly increased in leptin-treated naïve CD4+ T cells. Moreover, this study found that synoviocytes and chondrocytes produced large amounts of leptin especially during acute and chronic stages of mice with collagen-induced arthritis (CIA). Furthermore, leptin levels, Th17 cells in the joint and IL-17 levels in the synovial fluid were closely related to disease activity. Leptin intraarticular injection led to earlier onset of disease, higher clinical score, and more severe joint destruction compared with PBS-treated control mice. Importantly, leptin-injected mice had higher percentages of Th17 cells and cell numbers, elevated IL-17 levels in the synovial fluid, and increased infiltration of Th17 cells in the inflamed joint tissues compared with PBS-treated mice. T follicular helper (Tfh) cells are indispensible for pathogenic autoantibodies production. However, whether leptin receptor (ObR) signaling has a role on Tfh cells and its implication in CIA remain elusive. Upon a T cell-dependent antigen TNP-KLH immunization, germinal center (GC) response, plasma cells (PCs) and memory B cells formation were impaired in db/db mice compared with wild-type (WT) controls. In coculture of Tfh cells from db/db and WT mice with WT GC B cells, anti-TNP IgGs titers in supernatants of db/db Tfh cells were significantly reduced. Intravenously transfer of naïve CD4+ T cells from db/db and WT mice to BoyJ recipient mice, donor CD4+ T cells from db/db mice showed impaired Tfh cells generation in spleens of BoyJ recipient mice compared with mice received with WT CD4+ T cells. These data indicated that ObR-mediated signaling intrinsically modulate Tfh cells generation. In culture, leptin promoted Tfh cells differentiation via inducing Bcl6 expression, and increased IL-21 production in Tfh cells in a dose-dependent manner. Leptin significantly enhanced the phosphorylation of STAT3, upregulated Bcl6 expression, and increased p-STAT3 binding to the Il21 promoter in CD4+ T cells with leptin receptor b (Ob-Rb) overexpression. Upon CIA induction, db/db mice exhibited ameliorated disease severity with impaired Tfh cells response. However, WT Tfh cells transfer to db/db mice restored GC responses, PCs formation, antibody production, and exacerbated synovium inflammation and joint damage in db/db recipient mice. Together, these findings demonstrate that leptin modulates arthritis development via enhancement of Th17 and Tfh cells responses. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

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The aging process is strongly associated with decreased activity in the immune system. Dysregulation of T-lymphocyte function, such as reduced proliferation, is one problem faced by most elder people, which prevents them from successfully dealing with exogenous pathogens. Effective regulation of T-lymphocyte activity depends on the proper and prompt transduction of both positive and negative signals within Tlymphocytes and reflects the balance between positive and negative effects. Decline of positive signaling in aging has been studied and reported, while mechanisms concerning up-regulation of negative signaling with age and its role in immune senescence are still unclear. Cbl-b, an E3 ubiquitin ligase, was studied by our lab since it regulates the ubiquitin process, a protein modification process that has suppressive effects on signaling pathways. We first determined the reaction of Cbl-b to different stimuli in young rat splenic T-lymphocytes, and showed that there is a decrease in Cbl-b protein expression upon CD28 stimulation and such protein degradation is proteasome-dependent only. We also showed the mechanism of Cbl-b expression regulation involves the intracellular movement of Nedd4 toward Cbl-b and an up-regulation of Nedd4 expression. Then we proved that in old splenic T-lymphocytes, decreased proteasome activity was unable to down-regulate the Cbl-b protein. High levels of Cbl-b in old T-lymphocytes are functional in preventing PI3K activity and are associated with reduced T-lymphocyte proliferation upon regular stimulation. T-lymphocytes from old Cbl-b knock-out mice show similar proliferative reaction to CD3 stimulation as T-lymphocytes from young wild-type, which establishes the causeeffect relationship between sustained Cbl-b expression and decreased T-lymphocyte proliferation. In summary, these data suggest a unique role of Cbl-b in regulating Tlymphocyte signal transduction and provide critical preliminary data for extending Cbl-b studies into other fields, such as carcinogenesis.

Alcohol abuse has been commonly associated with enhanced susceptibility to pathogens. Studies on the effects of ethanol on the immune system are complicated by a lack of consensus on whether ethanol activates, inhibits or has no effect on immune cells. We present data showing that acute exposure of T cells to ethanol elicits responses that broadly parallel responses seen in normally stimulated T cells such as the formation of the immune synapse, polarization of the microtubule organizing center (MTOC) to the synapse and tyrosine phosphorylation of signaling proteins as seen when the T cell Receptor (TcR) engages antigen-MHC. However, incomplete activation of the T cell signaling program leads to unresponsive or anergic T cells. Our data suggests the hypothesis that ethanol can activate T cells in a manner that leads to anergy. We have found that ethanol triggers calcium signaling and this has provided one of the primary tools for analyzing the effects of ethanol on T cells. Ethanol induced calcium transients are dose-dependent and are comparable to those triggered by low doses of anti-TcR antibody. This is important because it allows us to compare ethanol dependent signaling to that normally triggered through stimulation of the T cell receptors. Analysis of the calcium signaling pathway indicates that ethanol-stimulated calcium transients depend on calcium entry and are likely due to opening of CRAC type calcium channels. The observed calcium transients go a long way towards explaining how ethanol may stimulate T cells and provides a mechanism for immune suppression through the observed translocation of NF-AT in ethanol pulsed cells. The translocation of NF-AT is particularly important because of reports that it plays a crucial role in triggering anergy and immunosuppression. Taken together, these data can help explain how ethanol can both activate T cells and cause immunosuppression.

In recent years, great advances have been made in the field of stem cell differentiation. Seminal insights in the area of developmental biology and tissue regeneration have made ex vivo differentiated cells a realistic alternative for transplantation applications. The recent application of these murine-based insights to human systems has paved new paths in autoimmune disease, chemotherapy, and immuno-deficiency research. Such strides would eliminate the hurdles associated with adoptive transfer including limited availability of transplantable cells, site morbidity, difficulties in cell isolation and expansion lag time. Current approaches in ex vivo hematopoiesis and T cell differentiation have begun to explore the effects of biomaterials on differentiation efficiency. These approaches, however, have not fully studied the quantitative effects of biomaterials and their properties on hematopoietic and T cell differentiation generation. Our goal was to design biomaterials whose properties could be tailored to improve differentiation efficiencies in T cell differentiation. Our work is dedicated to fabricating and characterizing Notch ligand functionalized microbeads for T cell differentiation applications. Our work has shown stable functionalization of Notch ligands on microbeads that can be quantitatively varied to achieve optimal Notch signaling. We have also demonstrated limited cellular toxicity and effective Notch signaling upon exposure to Notch ligand functionalized beads. Finally, we have successfully differentiated T cell progenitors from hematopoietic stem cells using the functionalized microbeads. As a side study, we have fabricated and characterized polymeric PLA scaffolds that were systematically varied and studied for their effects on hematopoietic differentiation efficiency. Insights gained from these studies should provide a better understanding of the microenvironmental signals in hematopoiesis and aid in the development of efficient technologies for the production of hematopoietic progenitors and T cells for therapeutic applications.

Leishmaniasis is a spectrum of diseases caused by several species of protozoan parasites belonging to the genus, Leishmania. The disease, which is transmitted by Sandflies, ranges from self-healing cutaneous lesions to the life-threatening visceral leishmaniasis. Cutaneous leishmaniasis, caused by L. major, is the most common form of the disease. With no vaccine available for use in humans, cutaneous leishmaniasis remains a global public health problem. Since understanding the factors that regulate effective immunity to cutaneous leishmaniasis is critical for the development of an affective vaccine and treatment strategies, the overall aim of my thesis was to decipher the host and pathogen factors that regulate immunity in cutaneous leishmaniasis. Firstly, I show that parasite dose affects the expansion of different T cell subsets following L. major infection; with low dose infection inducing more CD8+ T cells while high dose infection induced more CD4+ T cells. However, although CD8+ T cells were important for optimal primary immunity following low dose infection, they where dispensable during secondary immunity. Secondly, I found that blockade of LIGHT, (lymphotoxin like, exhibits inducible expression and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes) significantly impaired DC maturation, expression of co-stimulatory molecules, and early cytokine production (IL-12 and IFN-γ) following L. major infection. Interestingly, LIGHT was completely dispensable during secondary immunity in wild type mice but was critical for effective secondary immunity in CD40 deficient mice. Thirdly,I compared disease progression and immune response in CD40 and CD40L deficient mice infected with L. major under identical experimental conditions. I found significant differences in disease progression and immune response between CD40KO and CD40L KO mice infected with virulent L. major and treated with recombinant IL-12. My data revealed a novel pathway (CD40L-Mac-1 interaction) for IL-12 production and resistance to Leishmania major. Collectively, this thesis provides novel insights into the mechanisms involved in the development and maintenance of protective immunity against cutaneous leishmaniasis, which could lead to the development of a more efficient and effective immunotherapeutic and/or vaccination strategies against the disease. / October 2015