Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2008. / Vita. Bibliography: p.138-150

With the development of Highly Active Anti-Retroviral Therapy (HAART), Human Immunodeficiency Virus (HIV) infection has evolved from a fatal disease to a chronic condition with increased risk for non-infectious comorbidities, including reduced bone density. Bone density is maintained through the coupled activities of osteoblast matrix deposition and osteoclast resorption; while uncoupling this process can result in bone loss and increased fracture risk. CD4+ T cells are critical in regulating the activity of these cells. Relevant to this thesis, studies have shown that HAART treated patients experience higher levels of immune activation; possibly contributing to the observed bone loss. If osteoimmune dysregulation does occur, there is a need to develop therapeutics that target this process, especially in the context of HIV infection. To evaluate osteoblast differentiation, we developed a high-throughput screening method to identify osteo-regulatory compounds. By screening over 5,000 compounds, we identified 18 that robustly induced osteoblast differentiation, using a mouse mesenchymal stem cell. We validated two of these compounds, rapamycin and FK-506, which are known immunosuppressants. Secondly, we addressed the role of activated CD4+T cells and HIV-infected T cells in osteogenesis. We found that supernatants from activated T cells potently inhibit osteoblast differentiation. However, when osteoblasts were co-cultured with HIV-infected T cells, differentiation was inhibited regardless of activation status, suggesting intrinsic differences between HIV infected and uninfected T cells. Finally, to prevent the inhibition of osteogenesis by activated T cells, we evaluated rapamycin, our pro-osteogenic and T cell activation antagonist, as well as the novel compound JQ1, an inflammatory inhibitor that targets bromodomain-containing proteins. Both rapamycin and JQ1 efficiently blocked the cytotoxic effects of supernatants from non-infected activated T cells on osteoblasts, whereas only rapamycin prevented inhibition in the co-culture model. In contrast, neither rapamycin nor JQ1 were able to prevent inhibition by HIV infected, activated T cells. This suggests that HIV exacerbates the negative effects of T cell activation on osteoblastogenesis. These data support a mechanism for HIV infection and T cell activation mediating bone loss.

The Notch receptor is an evolutionarily highly conserved transmembrane protein essential to a wide spectrum of cellular systems. Notch is especially important to T-cell development, and its deregulation leads to leukemia. Although not well characterized, Notch signaling continues to play an integral role in peripheral T-cells, in which a unique mode of Notch activation can occur. In contrast to canonical Notch activation initiated by adjacent ligand-expressing cells, T-cell receptor (TCR)-stimulation is sufficient to induce robust Notch signaling. However, the interactions between these two pathways have not been defined. In this dissertation, we show that Notch activation occurs in peripheral T-cells within a few hours post TCR-stimulation and is required for optimal T-cell activation. Utilizing a panel of inhibitors against components of the TCR signaling cascade, we demonstrate that Notch activation is facilitated through initiation of protein kinase C-induced ADAM-metalloprotease activity. Moreover, internalization of Notch via endocytosis is indispensible for this process. Whereas ligand-mediated Notch stimulation relies on mechanical pulling forces that disrupt the autoinhibitory domain of Notch, we hypothesized that in T-cells in the absence of ligands, these conformational changes are induced through chemical adjustments in the endosome, causing alleviation of autoinhibition and receptor activation. Our data show that endocytosis is not only a prerequisite for TCR-induced Notch processing during normal T-cell function, but is essential even in Notch-mutated T-leukemia cells exhibiting constitutively active Notch signaling. Our work has also focused on signaling mechanisms of Notch following receptor activation. The Notch signal is transduced via cleavage of the intracellular portion of the receptor that subsequently translocates to the nucleus where it regulates gene transcription via interactions with its DNA-binding partner, RBPJκ. Utilizing RBPJκ-deficient T-cells, we show that, although Notch signaling is required, RBPJκ-dependent signaling is dispensable for peripheral T-cell proliferation and activation. Using retroviral constructs that encode modified, active forms of Notch restricted to the nucleus or cytoplasm, we provide evidence that Notch signaling may utilize RBPJκ-independent pathways for signal transduction. In conclusion, T-cells have evolved a unique method of Notch receptor activation, described for the first time in this dissertation, as well as novel mechanisms that facilitate downstream signaling.

Few cancers arising in fluid organ systems can be cured with localized therapeutic modalities, such as radiation or surgical organ removal. Chemotherapy and hematopoietic stem cell transplants have long been employed as the standard of care for patients diagnosed with leukemias and lymphomas. Though research continues to propose new, more potent chemotherapeutic agents, a new paradigm of treating cancerous malignancies with tumor-specific monoclonal antibodies, adoptively transferred tumor-fighting cells, and other exogenously administered immunomodulatory agents, has emerged over the past decade. These immunotherapies have dramatically improved the outcomes of patients diagnosed with cancers of B lymphocytes, referred to as B-cell malignancies. Though curative FDA-approved therapies for patients diagnosed with B-cell malignancies have yet to be established, recent research in the field of adoptive T-cell therapy has produced promising results. Tumor infiltrating lymphocyte therapy (TIL therapy), T-cell Receptor Therapy (TCR therapy) and Chimeric Antigen Receptor T-cell Therapy (CAR T-cell therapy) are the three most extensively studied adoptive T-cell immunotherapies in the context of B-cell malignancies. TIL and TCR therapies, in which patients are provided with either the patient’s own tumor-specific T-cells or T-cells expressing engineered, tumor-specific TCRs, respectively, enhance the patient’s immune system to mount a more potent, anti-tumor response. However, these adoptive T-cell therapies do not change the mechanisms of the immune response. Cancerous cells can evade immune attack and dampen immune responses to survive and thrive in the body. By down-regulating their expression of human major histocompatibility complex I (MHC I), for example, cancer cells escape T-cell recognition, which is dependent on MHC expression. A chimeric antigen receptor (CAR), is composed of an antibody-derived (B-cell derived) extracellular, antigen-recognition domain, and T-cell derived intracellular domains. CAR T-cells, therefore, exploit the cytotoxic nature of CD8+ T-cells, and the MHC independent recognition of B-cell receptors, to identify and destroy all cells expressing a specific target. Consequently, many of the cancer cell’s mechanisms of immune evasion are less effective in the presence of CAR T-cells. Progressive generations of CAR T-cell designs couple these receptors with costimulatory molecules to amplify the activation, efficacy, and potency of these cells in-vivo. Over the past five years, phase I and IIa clinical trials have produced remarkable results in the treatment of advanced stage, high-risk B-cell malignancies, namely Acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), and Non-Hodgkin’s Lymphoma (NHL). However, the significant oncogenic risks and fatal adverse events associated with this therapy necessitate further research to improve safety and reliable clinical efficacy of CAR T-cell therapy. In spite of these risks, the adoptive transfer of CD19-targeting, CAR expressing, cytotoxic T-cells (anti-CD19 CAR-T-cells) has produced sustained, complete remissions in patients diagnosed with progressive, advanced-stage, B-cell malignancies, for whom alternative treatments were not available. The unprecedented results of early clinical trials, as well as ongoing preclinical studies aimed at improving the design and production of CAR T-cells suggest a promising future for CAR T-cell therapy as a cure for B-cell malignancies.

The incidence of colorectal cancer (CRC) is increasing and the prognosis for patients with advanced or metastatic disease is relatively poor. Immunotherapies hold great promise, but deploying them effectively in CRC patients will require further knowledge of the complex cellular and molecular interactions that occur between intestinal tumours and the host immune system. The objective of this study is to understand the mechanisms by which lack of immune cell regulation in the gut can drive the formation of colon adenocarcinomas. In addition, this work aims to identify new mechanisms involved in progression to metastatic disease. Using mouse model systems, we found that aberrant activity of Treg cells deficient in IL-10 can promote inflammation-driven CRC. IL-10 deficient Tregs have increased capacity to drive tumourigenesis compared to their CD4⁺ effector T cell counterparts. RNA sequencing revealed specific upregulation of several genes, including a newly-described cytokine, in tumour-promoting Tregs. We explored cytokine regulation and the tumour microenvironment, and show that the inflammatory cytokine IL-6 and TGFÎ2 are necessary for tumour formation in this model. Moreover, disease is associated with a marked stromal cell signature that is induced as a consequence of Treg deficiency in IL-10 production. Gp38⁺ stromal cells are dominant producers of IL-6, and potent ECM modellers. Furthermore, tumours driven by IL-10 deficient Tregs express high amounts of the pro-mesenchymal transcription factor Sox4. Using combined in vitro and in vivo analyses, we confirm that Sox4 is involved in tumour growth and characterise its expression in CRC patients. Collectively, our findings suggest that Tregs and stromal cells act together to foster a microenvironment that promotes disease progression, notably through the expression of Sox4 in tumour cells. These findings open an exciting avenue to explore the phenotype of tumour-promoting Tregs and to study Sox4 function in metastatic disease.

abstract: Vaccination remains one of the most effective means for preventing infectious diseases. During viral infection, activated CD8 T cells differentiate into cytotoxic effector cells that directly kill infected cells and produce anti-viral cytokines. Further T cell differentiation results in a population of memory CD8 T cells that have the ability to self-renew and rapidly proliferate into effector cells during secondary infections. However during persistent viral infection, T cell differentiation is disrupted due to sustained antigen stimulation resulting in a loss of T cell effector function. Despite the development of vaccines for a wide range of viral diseases, efficacious vaccines for persistent viral infections have been challenging to design. Immunization against virus T cell epitopes has been proposed as an alternative vaccination strategy for persistent viral infections, such as HIV. However, vaccines that selectively engage T cell responses can result in inappropriate immune responses that increase, rather than prevent, disease. Quantitative models of virus infection and immune response were used to investigate how virus and immune system variables influence pathogenic versus protective T cell responses generated during persistent viral infection. It was determined that an intermediate precursor frequency of virus-specific memory CD8 T cells prior to LCMV infection resulted in maximum T cell mediated pathology. Increased pathology was independent of antigen sensitivity or the diversity of TCR in the CD8 T cell response, but was dependent on CD8 T cell production of TNF and the magnitude of initial virus exposure. The threshold for exhaustion of responding CD8 T cells ultimately influences the precursor frequency that causes enhanced disease.In addition, viral infection can occur in the context of co-infection by heterologous pathogens that modulate immune responses and/or disease. Co-infection of two unrelated viruses in their natural host, Ectromelia virus (ECTV) and Lymphocytic Choriomeningitis virus (LCMV) infection in mice, were studied. ECTV infection can be a lethal infection in mice due in part to the blockade of antiviral cytokines, including Type I Interferons (IFN-I). It was determined that ECTV/LCMV co-infection results in decreased ECTV viral load and amelioration of ECTV-induced disease, presumably due to IFN-I induction by LCMV. However, immune responses to LCMV in ECTV co-infected mice were also lower compared to mice infected with LCMV alone and biased toward effector-memory cell generation. Thus, providing evidence for bi-directional effects of viral co-infection that modulate disease and immunity. Together the results suggest heterogeneity in T cell responses during vaccination with viral vectors may be in part due to heterologous virus infection or vaccine usage and that TNF-blockade may be useful for minimizing pathology while maintaining protection during virus infection. Lastly, quantitative mathematical models of virus and T cell immunity can be useful to generate predictions regarding which molecular and cellular pathways mediate T cell protection versus pathology. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2015

Previous research has provided evidence that CD8 T cells mediate immunity against infection with Theileria parva. However, the immunity induced by one parasite strain doesn‟t give complete protection against other strains and this is associated with parasite strain specificity of the CD8 T cell responses. There is evidence that such strain specificity is a consequence of the CD8 T cell responses of individual animals being focused on a limited number of immunodominant polymorphic peptide-MHC determinants. Dominant responses to the Tp2 antigen have been demonstrated in animals homozygous for the A10 MHC haplotype. Three Tp2 epitopes recognised by A10+ animals (Tp249-59, Tp250-59 and Tp298-106) have been defined. This project set out to investigate the dominance of these epitopes and to examine the T cell receptor (TCR) repertoires of the responding T cells. The specific objectives were to: (i) Determine the dominance hierarchies of the three defined Tp2 epitopes in both A10-homozygous and -heterozygous cattle. (ii) Examine the clonal repertoires of epitope-specific responses by analysis of TCR gene expression. (iii) Isolate full-length cDNAs encoding TCR α and β chain pairs from T cell clones of defined epitope specificity and use them to generate cells expressing the functional TCRs. Using MHC class I tetramers the relative dominance of CD8 T cell responses were found to differ between A10-homozygous and heterozygous cattle. All A10-homozygous cattle examined had detectable responses to all 3 Tp2 epitopes, the Tp249-59 epitope consistently being the most dominant. By contrast, only some A10-heterozygous cattle had detectable responses to Tp2 and when present the response was specific only for the Tp298-106 epitope. Analyses of the sequences of expressed TCR β chains showed that the responses in individual animals were clonotypically diverse, but often contained a few large expanded clonotypes. The TCRs of Tp298-106–specific T cells showed preferential usage of the Vβ13.5 gene and the frequent presence of a “LGG” motif within the CDR3 of the B chain. A conserved (public) TCRβ clonotype shared by the Tp250-59-specific CD8 T cells from all A10-homozygous cattle was identified. The TCRα chains co-expressed with this public TCRβ clonotype were identified for a number of T cell clones. Lentivirus transduction of Jurkat cells with three full-length TCR α and β chain pairs resulted in successful expression of one of the α/β chain pairs as a functional TCR, thus providing the basis for future work to generate bovine T cells expressing defined TCRs in vitro.

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. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Epigenetic regulation of gene expression plays a pivotal role in the orchestration of immune responses. In particular, they have been implicated in the generation of in vitro cytokine-driven T cell polarization and therefore may determine the vigor, quality and/or longevity of such responses in vivo. Chemical allergens form two categories: skin sensitizing chemicals associated with allergic contact dermatitis, such as 2,4-dinitrochlorobenzene (DNCB) that result in type 1/type 17 responses in mice, and chemicals that cause sensitization of the respiratory tract and occupational asthma, for example trimellitic anhydride (TMA) that induces preferential type 2 responses in mice. To explore the regulation and maintenance of these divergent responses generated by polarised T cell populations in vivo, BALB/c strain mice were exposed topically DNCB and TMA. DNA from draining lymph nodes (LN) was processed for methylated DNA (5mC) immunoprecipitation (MeDIP) followed by hybridization to a whole-genome DNA promoter array. A higher number of DNCB-associated differently methylated regions (DMR) were identified and there was significant crossover between allergen treatments. Promoter-associated DMR, unique to either DNCB or TMA, were generally hypomethylated. Pathway analyses highlighted a number of immune related pathways, including chemokine and cytokine signalling. A number of these DMR were hypothesised to be candidate biomarkers of chemical allergy. To confirm this, novel analysis of hydroxymethylated (5hmC) DNA in the in vivo allergen-activated LN was compared to analysis of 5mC to identify LN specific DMR. The Gmpr DMR is suggested as a possible biomarker for contact allergen-induced immune responses and the Nwc DMR was characteristic of TMA treatment, highlighting its possible utility as a biomarker for responses induced by chemical respiratory allergens. These data not only represent novel analysis of 5hmC in response to chemical allergy in vivo, but also provide a possible basis for differentiation between classes of chemical allergens. Finally, a combined population of effector/effector memory T cells (TEff/TEM) was isolated from the CD4+ and CD8+ populations of allergen-activated draining lymph nodes (LN). Levels of 5mC and 5hmC at T cell lineage cytokine prompters was determined and analysed by comparison with concurrently sorted naïve T cells. In CD8+ TEff/TEM from DNCB-stimulated LN, increased expression of Ifng and Gzmb correlated with a reduction 5mC at their respective promoters. There were also reduced levels of 5mC at an Ifng enhancer. In contrast, TMA-simulated CD4+ TEff/TEM were characterised by high levels of Il4 expression which were associated with a decrease in promoter 5mC and an increase in 5hmC, as well as increased 5hmC at an Il4 enhancer region. These data demonstrate that exposure to chemical allergens results in characteristic DNA methylation patterns indicative of epigenetic regulation of divergent T cell populations in vivo. Furthermore, it highlights a particularly important role for DNA hydroxymethylation at the Th2 locus. In conclusion, exposure to chemical allergens results in divergent patterns of 5mC and 5hmC. These provide possible biomarkers for the different classes of chemical allergens and represent an insight into the importance of 5mC and 5hmC in the control of polarised T cell responses in vivo.

Endosomal trafficking can influence the composition of the plasma membrane and the ability of cells to polarize their membranes. Here, we examined whether trafficking through clathrin-independent endocytosis (CIE) affects the ability of T cells to form a cell-cell conjugate with antigen-presenting cells (APCs). We show that CIE occurs in both the Jurkat T cell line and primary human T cells. In Jurkat cells, the activities of two guanine nucleotide binding proteins, Arf6 and Rab22 (also known as Rab22a), influence CIE and conjugate formation. Expression of the constitutively active form of Arf6, Arf6Q67L, inhibits CIE and conjugate formation, and results in the accumulation of vacuoles containing lymphocyte function-associated antigen 1 (LFA-1) and CD4, molecules important for T cell interaction with the APC. Moreover, expression of the GTP-binding defective mutant of Rab22, Rab22S19N, inhibits CIE and conjugate formation, suggesting that Rab22 function is required for these activities. Furthermore, Jurkat cells expressing Rab22S19N were impaired in spreading onto coverslips coated with T cell receptor-activating antibodies. These observations support a role for CIE, Arf6 and Rab22 in conjugate formation between T cells and APCs.