Thesis (MScMed (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2006. / The ESAT-6 gene cluster regions are duplicated 5 times in the genome of Mycobacterium tuberculosis. ESAT-6 gene cluster region 1 is the most frequently studied region as it contains RD1 (region of difference 1). RD1 is a 9.5 Kb deletion region confirmed to be involved in mycobacterial virulence and pathogenesis, and is present in virulent M. bovis strains, yet absent in all attenuated M. bovis BCG vaccine strains. The antigens CFP-10 and ESAT-6, which both evoke strong T-cell responses in experimental animals and humans, are situated in the RD1 region, and are thought to be key antigens in mycobacterial virulence. The absence of this region from the genomes of all BCG vaccine strains, led to the conclusion that the mechanism of attenuation of M. bovis BCG was due to the loss of RD1. Studies have shown that this attenuation is attributed to the loss of cytolytic activity mediated by secreted ESAT-6 (and some of the genes responsible for its secretion), which in turn results in reduced tissue invasiveness. The potent T-cell antigens ESAT-6 and CFP-10 are secreted without ordinary sec-dependent secretion signals. A study of the potential functions of the proteins encoded by the ESAT-6 gene clusters shows that most of these proteins have a potential to function in a protein-dependent ATP-binding cassette active transport system. It has been shown that ESAT-6 gene cluster region 1 is responsible for the secretion of the ESAT-6 and CFP-10 genes contained in this region, explaining the absence of any ordinary sec-dependent secretion signals in the amino acid sequences of members of this family. In order to elucidate the regulation of expression of the ESAT-6 gene cluster region 1, shown to encode for a secretion system for ESAT-6 and CFP-10 and to be involved in virulence, an operon analysis and promoter identification experiments were carried out in this study. The analysis of the ESAT-6 gene cluster region 1 showed the existence of more than one operon in this region and three constitutively-expressed promoters driving the expression of the genes in the operons. These results provide insight into the functional relationship (regulatory and secretory mechanisms) between the genes contained within ESAT-6 gene cluster region 1.None of the other four ESAT-6 gene cluster regions have been proven to also encode secretion systems. Preliminary studies indicated that the ESAT-6 gene cluster region 3 is expressed in its entirety as one single operon and a strong promoter involved in the expression of this region was identified. Mtb9.9A (the ESAT-6 antigen of the ESAT-6 gene cluster region 5) have also been shown to evoke strong T cell responses and to be secreted without any ordinary secretion signal. During the present study, we thus aimed to investigate the secretion of Mtb9.9A in order to determine whether it is also secreted by a dedicated secretion system encoded by ESAT-6 gene cluster region 5. The fact that region 5 was shown to be the last of the four duplications is important, as a positive result with this region would indicate whether the other four gene clusters share a similar secretion function. ESAT-6 gene cluster regions 2, 4 and 5 were isolated in the present study to form part of subsequent ESAT-6 gene cluster region secretion studies. Mtb9.9A was cloned, expressed and purified for antibody-generation, Resulting antibodies were used in an antigen secretion analysis. The secretion analysis entailed the integration of the isolated ESAT-6 gene cluster region 5 into the genome of M. smegmatis and investigation of the influence of the genes (contained in region 5) on the secretion of a heterologously expressed Mtb9.9A-HA-tagged fusion protein. We therefore attempted to show whether the proteins encoded by the ESAT-6 gene cluster region 5 also function together as a mycobacterial membrane-bound complex involved in protein-dependent transport and if so, whether this transport system is responsible for the active secretion of the native ESAT-6 antigen (designated Mtb9.9A) of region 5. This study opens the way for the understanding of the regulation, transport- and secretion mechanisms of important T-cell antigens of the mycobacteria, thereby giving insight into and building onto our understanding of the pathogenicity of Mycobacterium tuberculosis. A better understanding of these mechanisms could lead to the development of efficient strategies to either terminate or enhance secretion of antigens, which in turn will have an impact on drug and vaccine design and development.

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

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

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Mucosal associated invariant T-cells (MAIT) are a phylogenetically conserved subset of alpha/beta T-cells with natural killer-like (NK) activity. MAIT are defined by the expression of an invariant T-cell receptor alpha (TCRα) chain; in mice and humans this chain uses the orthologous mVα19/hVα7.2-Jα33 genes respectively. Available evidence indicates that MAIT are restricted by MR1, a highly conserved MHC class I-related molecule, and that their development is dependent on B lymphocytes. They appear to constitute part of the innate immune response, but their precise functional role is poorly understood. This study aimed to characterise MAIT and MR1 in ruminants, and to further the knowledge and understanding of these unique cells. Using PCR primers based on partial database sequences, orthologous full-length TCRα chains were identified in circulating bovine and ovine T cells. The germline elements of the respective α chains were identified and their overall frequency of expression within the bovine TCRα repertoire determined. Experiments using the orthologous TCRα chain as a marker for MAIT cells to examine expression in bovine and ovine blood and various tissues showed that spleen and mesenteric lymph nodes contained the highest frequency of MAIT cells. Use of the same technique to study levels of this marker in cattle of different ages revealed very low numbers of MAIT cells in neonatal animals, followed by a marked increase in the first 3 weeks of life. Analyses of MAIT TCRα expression in different T cell subsets showed that, unlike mice and humans in which MAIT cells are predominantly within the CD4-/CD8- T-cell population, MAIT cells in bovine blood are predominantly CD8+. Full-length cDNAs were isolated for bovine and sheep MR1 and their sequences were found to display marked cross-species conservation. Using a specific PCR, MR1 was shown to be expressed in peripheral blood and by different lineages of Theileria-transformed cells. Alternatively-spliced transcripts of MR1 were detected in both cattle and sheep and several of these retained an intact open-reading frame. Constructs of bovine MR1 and an MR1/MHC chimera were prepared in a eukaryotic expression vector but these failed to give detectable cell surface expression following transfection into Cos-7, despite positive intracellular expression.

Myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) play an essential role in the immunosuppressive networks that contribute to tumor immune evasion. The mechanisms by which tumors promote the expansion and/or function of these suppressive cells and the cross-regulation between MDSC and Treg remain incompletely defined. The current work evaluates the influence of MDSC, expanded in two mouse cancer models, on immunosuppressive Treg. We demonstrate that tumor-induced MDSC endowed with the potential of suppressing conventional T lymphocytes surprisingly impair TGF-β1-mediated generation of induced Treg (iTreg) from naïve CD4⁺ T lymphocytes. Suppression of iTreg generation by MDSC occurs early in the differentiation process, and is cell contact dependent. This inhibition of FoxP3-expressing T lymphocyte differentiation by MDSC does not depend on arginase 1, cystine/cysteine depletion, iNOS/NO, or PD-1/PD-L1 signaling. These findings therefore indicate that MDSC from tumor-bearing hosts have the heretofore unreported ability to restrict some immunosuppressive Treg subpopulations.

TGFβ is an immunoregulatory cytokine that has a pivotal function in maintenance of immune tolerance via the control of lymphocyte proliferation, differentiation and survival. Defects in TGFβ1 expression or in its signaling in T cells correlate with the onset of several autoimmune diseases. However, the early effects of this cytokine on the innate immune system, particularly the dendritic cells (DCs) which play an equally important role in development of immune tolerance, are not well documented in vivo. In the current study, we developed conditional knockout mice with targeted deletion of Tgfbr2 specifically in dendritic cells. DC-Tgfbr2 KO mice developed spontaneous multi-organ autoimmune inflammation with T and B cell activation. Phenotypic analysis of dendritic cells revealed no significant differences in the expression of MHCII and co-stimulatory molecules between control and DC-Tgfbr2 KO mice. However, we found that DCs from DC-Tgfbr2 KO mice were more pro-inflammatory, which exacerbated the severity of disease in a T cell transfer model of colitis. Furthermore, increased IFNγ expression by Tgfbr2-deficient DCs inhibited antigen-specific regulatory T cells (Tregs) differentiation by DCs in the presence of TGFβ. Since DCs play an important role in Treg homeostasis in vivo, we also examined the phenotype of Tregs and observed a significant increase in the frequency and numbers of Foxp3⁺ T cells in both the spleen and MLNs of DC- Tgfbr2 KO mice. Further analysis of these Tregs revealed attenuated expression of Foxp3 and an expansion in the numbers of CD4⁺CD25⁻Foxp3⁺T cells suggesting that the Tregs from KO mice may not be fully immunosuppressive. Adoptive transfer of in vitro differentiated iTregs into 2-3 week old DC-Tgfbr2 KO mice partially rescued the autoimmune phenotype by reducing the frequency of activated T cells and severity of colitis but did not prevent inflammation in other organs. The phenotype of this novel mouse model clearly indicates the importance of TGFβ signaling in DCs in the maintenance of immune homeostasis and prevention of autoimmunity and provides an opportunity to study the pathogenesis of complex disorders such as autoimmune gastritis, pancreatitis, hepatitis and inflammatory bowel diseases.

The adaptor protein Shb has been implicated in the signaling of several tyrosine kinase receptors and previous studies have suggested a role for Shb in the signal transduction of T cells. Shb associates with the T cell receptor (TCR) and partakes in the signal propagation of activated T lymphocytes. In order to explore Shb’s influence on TCR signaling in vivo, T cell development and function was studied in a Shb knockout mouse. The loss of Shb led to aberrant TCR signaling in both thymocytes and peripheral CD4+ TH cells, with elevated basal phosphorylation of key components in the signal cascade. Shb was found to be dispensable for thymocyte development, but its absence resulted in a TH2 bias in in vitro stimulated peripheral CD4+ TH cells. As imbalances in TH2 responses are linked to allergic diseases, we further explored Shb’s role in immune regulation in a mouse model of atopic dermatitis. Shb knockout mice exhibit more aggravated signs of atopic dermatitis, including increased immune cell recruitment to the affected areas and elevated mRNA levels of typical TH2 cytokines. The effect of Shb on hematopoiesis in general was determined by examining populations of long-term hematopoietic stem cells (LT-HSCs) and hematopoietic progenitor cells in bone marrow of Shb knockout and wild type mice. Shb deficient bone marrow was found to contain significantly fewer relative numbers of LT-HSCs due to a proliferative defect. The reduced cell cycle activity of Shb LT-HSCs could further be linked to an abnormal regulation of the focal adhesion kinase/Rac1/p21-activated kinase pathway. Since alterations in LT-HSC proliferative abilities may have implications for leukemia development, BCR-Abl induced myeloid neoplasia was investigated in the absence of Shb. Shb deficiency confers a more aggressive progression of BCR-Abl induced myeloid neoplasia characterized by an increased peripheral blood neutrophilia and a deregulated cytokine profile. In addition, focal adhesion kinase and STAT3 signaling is hyperactivated in Shb knockout leukemic cells. In conclusion, Shb appears to be a multifunctional signaling mediator that controls several responses in hematopoietic cells, under homeostatic as well as disease conditions.

<p><p>LAT is a transmembrane adaptor protein that is critical for the emanation of signals downstream of the TCR. Following TCR engagement, LAT is phosphorylated on multiple tyrosine residues, allowing it to serve as a scaffold for a multi-protein signaling complex. Mutation of tyrosine 136 on LAT abrogates binding of PLC-&#947;1. The disruption of this interaction has severe consequences on TCR-mediated calcium signaling and MAPK activation. Mice harboring a mutation at this tyrosine, LATY136F (LAT<super>m/m</super>) mice, have drastically impaired thymocyte development; however, CD4<super>+</super> T cells in the periphery rapidly expand and instigate a fatal lymphoproliferative syndrome. In order to bypass the severe developmental defects exhibited in LAT<super>m/m</super> mice, our laboratory previously developed a conditional knock-in mouse line in which the mutated LAT allele is expressed in mature T cells following deletion of a floxed wildtype LAT allele (ERCre<super>+</super>LAT<super>f/m</super> mice). LAT<super>f/m</super> mice develop a similar lymphoproliferative syndrome as LAT<super>m/m</super> mice. We used both of these mouse models to analyze the contribution of two other proteins that are essential for TCR-mediated signaling, RasGRP1 and Gads, in LAT-mediated autoimmunity. </p><p><p>Analysis of LAT<super>m/m</super>RasGRP1<super>-/-</super> mice demonstrated that the additional deletion of RasGRP1 increased the thymocyte development block and, as a result, young mice contained markedly reduced T cell populations. However, by four months of age, a lymphoproliferative disease had developed in these mice. To bypass the severe developmental block, we analyzed LAT<super>f/m</super>RasGRP1<super>-/-</super> mice and observed that they developed disease similarly to LAT<super>f/m</super> mice. We also assessed the effect of Gads deletion in both mouse models of LAT disease. LAT<super>m/m</super>Gads<super>-/-</super> mice had an even more dramatic block in the DN stage of thymocyte development compared to LAT<super>m/m</super> controls, although by four months of age CD4<super>+</super> T cells had expanded. Following deletion of the wildtype LAT allele, LAT<super>f/m</super>Gads<super>-/-</super> mice also developed disease. Our results indicated that LAT-mediated autoimmunity can occur independently of the critical T cell signaling components RasGRP1 and Gads. </p><p><p>In addition, we more closely examined RasGRP1-mediated Erk activation in T cells. RasGRP1 is a Ras-guanyl nucleotide exchange factor that is required for positive selection of thymocytes, activation of T cells, and control of T cell mediated-autoimmunity. While the importance of various RasGRP1 structural domains has previously been explored, RasGRP1 also contains a tail domain of unknown function. To elucidate the physiological role of this domain, we generated knock-in mice expressing RasGRP1 without the tail domain, RasGRP1<super>d/d</super> mice. Analysis of these mice demonstrated that deletion of the tail domain led to impaired T cell development but, with age, CD4<super>+</super> T cells expanded and auto-antibodies were produced. RasGRP1<super>d/d</super> thymocytes were unable to activate Erk and underwent aberrant thymic selection processes. Mechanistically, the tail-deleted form of RasGRP1 was not able to traffic to the cell membrane following stimulation, indicating a potential reason for its inability to activate Erk. While the DAG-binding C1 domain of RasGRP1 has long been recognized as an important factor mediating Erk activation, our data revealed the physiological relevance of the tail domain of RasGRP1 in the control of Erk signaling.</p> / Dissertation

The use of genome wide ENU mutagenesis screening has uncovered vast numbers of novel genes involved in the control of the immune system. This thesis describes the characterization of a novel mutant, Captain Morgan (CPM), originally identified in an immunization screen designed to evaluate both the initial antibody response to antigen and the ability to sustain antibody production. Mapping of this mutant lead to the identification of a single base pair mutation in a novel guanine nucleotide exchange factor, dedicator of cytokinesis 8 (DOCK8). The mutation was found to result in altered gene splicing of the DOCK8 protein leading to the truncation of the protein and loss of catalytic function. The importance of understanding the role of DOCK8 in host immunity has been recently underlined by the discovery that cohorts of patients suffering from autosomal recessive forms of hyper-IgE syndrome have loss-of-function or deletions in this novel guanine nucleotide exchange factor. Disease in these patients is characterised by recurrent viral and bacterial infections mainly of the skin and lungs, with reduced levels of peripheral CD4⁺ and CD8⁺ T cells in the blood of patients. Patients also have high levels of IgE and eosinophilia in the blood and are highly atopic with increased prevalence of allergic diseases including asthma. Loss of DOCK8 function results in a number of phenotypes in CPM mice, which may help understand the immunodeficiency syndrome experienced by DOCK8 deficient patients. CPM mice, like DOCK8 deficient patients, are lymphopenic with losses of both CD4⁺ and CD8⁺ T cells in the blood and secondary lymphoid organs. Challenge of CPM mice with modified vaccina virus (MVA) and influenza strain X31 demonstrated normal primary anti-viral responses. However, similar to the loss of germinal centre B cells previously described in these mice, memory T cell responses were diminished, which may explain the susceptibility of DOCK8 deficient patients to recurrent infections. In addition to the loss of peripheral T cells, rare populations of lymphocytes such as invariant natural killer T cells (iNKT) were also reduced in the liver and thymus. Due to their roles in bacterial and viral responses and cancer immunosurveillance it is expected that loss of these cells will contribute to disease severity. Together these findings illustrate the importance of the ENU mutagenesis model for generating new mutants, which can enhance our understanding of mammalian genes and create disease models of human disease. Further characterization of DOCK8 deficiency and the molecular mechanisms of DOCK8 function will have important implications for disease diagnosis and ongoing treatment for patients.