published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

In my dissertation, I have worked on two distinct projects related to the immune system. The abstracts for the two projects that make up my dissertation work are below. In the first project (presented in Chapter 2), we study regulatory T (Treg) cells in chronic liver disease. Current dogma holds that Treg cells preserve tissue function in settings of inflammation and damage. Consistent with this, herein we observe that Treg cells – in particular those producing the epidermal growth factor receptor (EGFR)-ligand amphiregulin (Areg) – are enriched in the livers of mice and humans with non-alcoholic steatohepatitis (NASH). Mouse and human Treg cells undergo substantial transcriptional changes in chronic liver damage, reflecting their increased activation; however, rather than playing a protective role, we find that Treg cell–derived Areg promotes NASH-induced liver fibrosis, the key prognostic factor for patients – through the direct activation of EGFR on hepatic stellate cells. Clinically, NASH is closely linked to insulin resistance, but the mechanistic contributions of NASH-induced disease processes to insulin resistance has hitherto been unclear. We further observe that Treg cell–derived Areg promotes glucose intolerance in a NASH-dependent manner, also mediated through EGFR signaling on hepatic stellate cells. Mechanistically, in the setting of NASH, we find that Areg from Treg cells promotes hepatocyte gluconeogenesis – through hepatocyte detection of fibrosis development and soluble mediators, including IL-6, produced by activated hepatic stellate cells – offering new insight into the cellular interplay of how chronic liver disease promotes insulin resistance. Taken together, we provide the first evidence that Treg cells mediate a maladaptive role in tissue injury, finding that their production of a growth factor plays a central role in liver disease and promotes liver fibrosis and glucose intolerance in NASH. In the second project (presented in Chapter 3), we use engineered bacteria to produce chemokines in the tumor to promote anti-tumor immunity. Tumors employ multiple mechanisms to actively exclude immune cells involved in anti-tumor immunity. Strategies to overcome these exclusion signals remain limited due to an inability to target therapeutics specifically to the tumor. Synthetic biology enables engineering of cells and microbes for tumor-localized delivery of therapeutic candidates previously unavailable using conventional systemic administration techniques. Here, we engineer bacteria to intratumorally release chemokines to attract adaptive immune cells into the tumor environment. Bacteria expressing an activating mutant of the human chemokine CXCL16 (hCXCL16K42A) offer therapeutic benefit in multiple mouse tumor models – an effect mediated via recruitment of CD8+ T cells. Furthermore, we target the presentation of tumor-derived antigens by dendritic cells – using a second engineered bacterial strain expressing CCL20. This led to type 1 conventional dendritic cell recruitment and synergized with hCXCL16K42A-induced T cell recruitment to provide additional therapeutic benefit. In summary, we engineer bacteria to recruit and activate innate and adaptive anti-tumor immune responses, offering a new cancer immunotherapy strategy.

Thesis (MScMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Important steps towards the global control of Tuberculosis include the improvement of diagnosis, the development of effective vaccines and the identification of correlates of protection/protective immunity to Mycobacterium tuberculosis. This study has of three objectives: 1. To validate the findings of a previous study that showed increased levels of IL-1β and decreased levels of IL-17 in children who are exposed to tuberculosis but remain uninfected compared to those who are exposed/infected and unexposed/uninfected. 2. To define the protective immunological phenotype in children with negative IGRA’s and TST following exposure to Mycobacterium tuberculosis. 3. To evaluate a number of cytokines in both serum and saliva samples of identified tuberculosis cases and controls for their diagnostic potential and to evaluate saliva as a possible new diagnostic sample type. The study designs were as follows: Objectives1, and 2: Children with documented tuberculosis exposure and with Mycobacterium tuberculosis infection as assessed through interferon gamma release assays, children with exposure but no infection and a control group with no exposure nor infection were investigated. These participants were selected according to their exposure and infection phenotypes from a larger TB household contact study that was conducted in communities in Cape Town. Whole blood was stimulated in QuantiFeron tubes overnight and ten cytokines were measured in antigen stimulated and unstimulated supernatants by Luminex multiplex Immunoassay. Differential production of cytokines in the three groups was evaluated. Objective 3. Saliva and serum samples were collected from thirty eight adults with suspected tuberculosis who were recruited from a community health centre in Cape Town, after which the levels of thirty three host markers were evaluated in the samples using the Luminex platform. The main findings of the studies included: 1. Increased levels of IL-1β and decreased levels of IL-17 in children who are tuberculosis exposed but remain uninfected compared to those who are exposed/infected and unexposed/uninfected could not be confirmed. 2. Immune responses other than IFN-γ are different in children with different exposure and infection phenotypes. Higher IL-23 and IL-33 levels in children with tuberculosis exposure without subsequent Mycobacterium tuberculosis infection compared to children with no exposure were shown. 3. In both the tuberculosis cases and controls, the levels of most markers were above the minimum detectable limit in both serum and saliva, but marker levels were not consistently higher in one sample type. The levels of fractalkine , IL-17, IL-6, IL-9, MIP-1β, CRP, VEGF and IL-5 in saliva, and those of IL-6, IL-2, SAP and SAA in serum, were significantly higher in tuberculosis patients, in comparison to the levels obtained in those without active tuberculosis (p<0.05). The area under the ROC curve was ≥ 0.70 for most of these markers, thereby confirming their diagnostic potential for TB disease. The work presented in this thesis has identified markers that may grant an improved understanding on the mechanisms that are associated with protection against Mycobacterium tuberculosis in children. The preliminary results presented show that the identification of host markers in saliva is possible and the utility of saliva for the development of rapid immune-based tests for active tuberculosis is promising. / AFRIKAANSE OPSOMMING: Noemenswaardige vooruitgang in die globale beheer van Tuberkulose is onderworpe aan verbeterde diagnose, die ontwikkeling van doeltreffende vaksienes en die identifikasie van aanwysers van immuniteit teen Mycobacterium tuberculosis. Die doel van hierdie studie is: 1. Om die bevindinge van ‘n vorige studie te bevestig, waar verhoogde vlakke van IL-1β en verlaagde vlakke van IL-17 waargeneem is in kinders wat aan tuberkulose blootgestel is, maar nie geïnfekteer is nie. Hierdie bevindinge was in vergelyking met geïnfekteerde en nie-blootgestelde kinders. 2. Om ‘n beskermende immunologiese fenotipe te definieer in kinders met negatiewe IGRA’s en TST, na blootstelling aan Mycobacterium tuberculosis. 3. Om sekere sitokines, in beide serum en speeksel monsters van tuberkulose gevalle en kontroles, te evalueer as potensiële diagnosemiddels, asook die moontlikheid dat speeksel kan dien as ‘n nuwe diagnostiese monstertipe. Die studieraamwerk was as volg: Doel 1 &2:Die volgende groepe was onder meer ondersoek – Kinders blootgestel aan tuberkulose en wat gevolglik geïnfekteer is, soos vasgestel deur interferon gamma vrystellingstoetse; kinders wat wel blootgestel is maar nie geïnfekteer is nie en ‘n kontrolegroep wat geen blootstelling aan Mycobacterium tuberculosis gehad het nie. Hierdie individue is geselekteer volgens hul blootstellingsprofiel en infeksiefenotipes, uit ‘n groter blootstellingstudie op Kaapse huishoudings. Heelbloed is oornag gestimuleer en tien sitokiene is gemeet in antigeen-gestimuleerde en ongestimuleerde supernatante, deur middel van Luminex multipleks Immunotoetse. Differensiële produksie van sitokienes in hierdie groepe is gevolglik geëvalueer Doel 3: Speeksel en serummonsters van 38 volwassenes met vermeende tuberkulose, is versamel en die vlakke van drie en dertig gasheermerkers is gemeet deur middel van die Luminex platvorm. Die hoof bevindinge van hierdie studie sluit in: 1.Vehoogde vlakke van IL-1β en verlaagde vlakke van IL-17 kon nie bevestig word in die verskeie kindergroepe (Sien doel 1) nie. 2. Die immuunrespons, uitsluitend die IFN- γ respons, is veskillend in kinders met uiteenlopende blootstelling en infeksiefenotipes. Hoër vlakke van IL-23 en IL-33 is gevind in kinders wat blootgestel is aan tuberkulose, maar nie geïnfekteer is nie, in teenstelling met nie-blootgestelde kinders.. 3. In beide die pasiënte en kontroles was die meeste sitokienvlakke hoër as die minimum meetbare limiet in beide speeksel en serummonsters, hoewel merkervlakke nie konstant hoër was in enige van die twee monstertipes nie. Die vlakke van fractalkine, IL-17, IL-6, IL-9, MIP-1β, CRP, VEGF en IL-5 in speeksel en IL-6, IL-2, SAP en SAA in serum, was merkbaar hoër in tuberkulosepasiënte, in vergelyking met vasgestelde vlakke in individue sonder aktiewe tuberkulose. (p<0.05). Die oppervlak onder die ROC kurwe was ≥ 0.70 vir die meerderheid van die merkers. Dit is ‘n sterk aanduiding dat hierdie merkers potensiaal het as diagnostiese merkers vir tuberkulose. Hierdie navorsing het merkers geïdentifiseer wat die begrip van die megansime waarmee beskerming teen Mycobacterium tuberculosis gebied word in kinders, verbreed. Hierdie voorlopige resultate dui aan dat die identifikasie van gasheermerkers in speeksel moontlik is en dat speeksel moontlik kan dien as ‘n proefkonyn vir die ontwikkeling van immuungebaseerde sneltoetse vir die diagnose van aktiewe tuberkulose. / The EDCTP through the African European Tuberculosis Consortium (AE-TBC, grant number IP_2009_32040) / Trials of Excellence in Southern Africa (TESA, project code CG_cb_07_41700)

Indiana University-Purdue University Indianapolis (IUPUI) / Immunostimulatory cytokines can enhance anti-tumor immunity and are part of the therapeutic armamentarium for cancer treatment. We previously reported that chemotherapy-treated lymphoma patients acquire a deficiency of Signal Transducer and Activator of Transcription 4 (STAT4), which results in defective IFNy production during clinical immunotherapy. With the goal of further improvement in cytokine-based immunotherapy, we examined the effects of a soybean peptide called lunasin that exhibits immunostimulatory effects on natural killer cells (NKCs). Peripheral blood mononucleated cells (PBMCs) from healthy donors and chemotherapy-treated lymphoma patients were stimulated with or without lunasin in the presence of IL-12 or IL-2. NK activation was evaluated, and its tumoricidal activity was assessed using in vitro and in vivo tumor models. Chromatin immunoprecipitation (ChIP) assay was performed to evaluate the histone modification of gene loci that are regulated by lunasin and cytokine. Adding lunasin to IL-12- or IL-2-cultuted NK cells demonstrated synergistic effects in the induction of IFNG and genes involved in cytotoxicity. The combination of lunasin and cytokines (IL-12 plus IL-2) was capable of restoring IFNy production by NK cells from post-transplant lymphoma patients. In addition, NK cells stimulated with lunasin plus cytokines have higher tumoricidal activity than those stimulated with cytokines alone using in vitro tumor models. The underlying mechanism responsible for the effects of lunasin on NK cells is likely due to epigenetic modulation at target gene loci. Lunasin represents a different class of immune modulating agent that may augment the therapeutic responses mediated by cytokine-based immunotherapy.

Indiana University-Purdue University Indianapolis (IUPUI) / CD4 T helper cells control immunity to pathogens and the development of inflammatory disease by acquiring the ability to secrete effector cytokines. Cytokine responsiveness is a critical component of the ability of cells to respond to the extracellular milieu by activating Signal Transducer and Activator of Transcription factors that induce the expression of other transcription factors important for cytokine production. STAT4 is a critical regulator of Th1 differentiation and inflammatory disease that attenuates the gene-repressing activity of Dnmt3a. In the absence of STAT4, genetic loss of Dnmt3a results in de-repression of a subset of Th1 genes, and a partial increase in expression that is sufficient to observe a modest recovery of STAT4-dependent inflammatory disease. STAT4 also induces expression of the transcription factors Twist1 and Etv5. We demonstrate that Twist1 negatively regulates Th1 cell differentiation through several mechanisms including physical interaction with Runx3 and impairing STAT4 activation. Following induction by STAT3-activating cytokines including IL-6, Twist1 represses Th17 and Tfh differentiation by directly binding to, and suppressing expression of, the Il6ra locus, subsequently reducing STAT3 activation. In contrast, Etv5 contributes only modestly to Th1 development but promotes Th differentiation by directly activating cytokine production in Th9 and Th17 cells, and Bcl6 expression in Tfh cells. Thus, the transcription factors Twist1 and Etv5 provide unique regulation of T helper cell identity, ultimately impacting the development of cell-mediated and humoral immunity.