Browsing by Author "Van Coller, Ansia"

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    Development of functional immune readouts for the confirmation of mendelian susceptibility to mycobacterial disease and related primary immunodeficiencies
    (Stellenbosch : Stellenbosch University, 2019-04) Van Coller, Ansia; Glashoff, Richard; Esser, Monika; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Microbiology.
    Background: Mendelian Susceptibility to Mycobacterial Disease (MSMD) is a primary immunodeficiency (PID) characterised by a predisposition to infection by weakly-pathogenic mycobacteria. In countries with a high prevalence of tuberculosis, individuals with MSMD are also prone to severe, persistent, unusual or recurrent infections by pathogenic Mycobacterium tuberculosis. Several MSMD-associated genes have been described, including IFNGR1, IFNGR2, IL12RB1, IL12B, STAT1, NEMO, ISG15, IRF8, TYK2, and CYBB, many resulting in a disruption of IL-12 and IFN-γ cytokine axis, which is essential for control of mycobacterial infections. This genetic heterogeneity results in many distinct disorders, which vary in their mode of inheritance and clinical presentation. An accurate molecular diagnosis, confirmed by immune functional studies, is essential to ensure that the patient receives optimal treatment and prophylaxis for infections. The aim of this study was to implement and optimise a set of immune phenotyping and functional validation tests for the key pathway, the IFN-γ and IL-12 cytokine axis, involved in MSMD, and to use these assays to assess immune function in a cohort of suspected MSMD patients. Methodology: Blood was collected from 17 participants with MSMD-like clinical phenotypes. DNA was extracted and PBMCs were isolated from the patients’ blood. Whole exome sequencing (WES) was performed and the resulting data was processed using an in-house bioinformatics pipeline, TAPER™. A set of flow cytometry and ELISA-based functional assays were implemented and optimised to assess the integrity of the IL-12-IFN-γ pathway. IFN-γR1 and IL-12Rβ1 expression were assessed by means of standard surface flow cytometry, and IFN-γ and IL-12 signalling was assessed by the detection of pSTAT1 and pSTAT4 respectively through intracellular phospho-specific flow cytometry. IFN-γ-induced IL-12 production as well as IL-12-induced IFN-γ production was also assessed by ELISA after 48-hour in vitro stimulation. The functional and genetic data were then reconciled in order to confirm the extent of functional impairment associated with each genetic variant. Results: Plausible disease-causing variants were identified through genetic investigations for 11 of the 17 participants. Variants in MSMD-associated genes were found in 8 of these patients, although only one of the identified variants, IFNGR1 (c.818del4), has been described before. Variants in genes not previously associated with MSMD were also found, including variants in IKZF1, NOD2, IRAK1, IKBKB, and NFKB2. All the functional assays were optimised and the combination of the three assays for the assessment of the integrity of the IL-12-IFN-γ pathway was successful in identifying immune deficits in essentially all of the participants included in this study. Conclusions: The current study led to the implementation of functional immune readouts that allowed for the evaluation of the functional impact of both novel and previously described genetic variants on the IL-12-IFN-γ pathway. The results generated from the functional assays were highly variable and often defects within the same gene lead to different phenotypes, which emphasises the importance of in vitro functional confirmation of all PIDs. Hence it would be beneficial to apply these assays routinely for patients with suspected PID relating to mycobacterial susceptibility. A molecular diagnosis with confirmed functional impairment paves the way for targeted treatment and improved disease management options for these patients.
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    Phenotypic and functional immune cell profiling of patients with primary immunodeficiency associated with mycobacterial infections in a tuberculosis endemic region
    (Stellenbosch : Stellenbosch University, 2022-12) Van Coller, Ansia; Glashoff, Richard Helmuth; Glanzmann, Brigitte; Esser, Monika; Stellenbosch University. Faculty of Science. Dept. Department of Pathology. Medical Microbiology.
    ENGLISH ABSTRACT: Background: Inborn errors of immunity (IEI) relating to increased susceptibility to severe, persistent, unusual and/or recurrent (SPUR) mycobacterial infections are of particular concern in countries such as South Africa that are hyperendemic for tuberculosis (TB). Mendelian susceptibility to mycobacterial disease (MSMD), the principal IEI relating to SPUR mycobacterial infections, was originally defined to encompass only weakly pathogenic mycobacteria such as the Bacillus Calmette–Guérin (BCG) vaccine. However, more recent studies have shown that South African MSMD patients are also likely to present with SPUR TB. There are 15 known MSMD-associated genes, which all fall within the Interleukin-12-Interferon-γ (IL-12-IFN-γ) immunological pathway, and mutations in these genes have been described to result in either reduced IFN-γ production or a lack of immune response to IFN-γ. The aim of this study was to evaluate immune dysfunction in patients that present with suspected MSMD using genetic sequencing and in vitro functional profiling assays. Additionally, T-bet, a common transcription factor that is also integral to the IL-12-IFN-γ pathway, was investigated as a potential proxy marker for MSMD. Methodology: Blood was collected from 18 patients presenting with SPUR TB for genetic sequencing and functional immune profiling. Whole genome Sequencing (WGS) was performed to identify candidate disease-associated variants. The immune profiling assays included assessment of the IL-12-IFN-γ pathway through flow cytometric detection of cytokine receptors and intracellular signalling, as well as assessment of immune cell subset distributions and Luminex®-based detection of cytokine/chemokine readouts following stimulation of patient cells with Phytohemagglutinin (PHA), BCG, and IL-12/IFN-γ. T-bet expression was measured by means of intracellular flow cytometry. Results: Through WGS, likely disease-associated novel variants were identified in 82% of the 11 patients that were successfully sequenced and 89% of the identified variants were in known MSMD-associated genes. All patients had some degree of impairment in the IL-12-IFN-γ pathway, and these readouts corresponded with the WGS findings. Further immune investigations revealed that the overall patient group had significantly reduced levels of circulating CD16+ monocytes and lymphocytes as well as reduced levels of inflammatory cytokine/chemokine production following PHA or BCG stimulation. All patients also had aberrant T-bet expression, with reduced expression in CD16+ monocytes and natural killer (NK) cells being the most prominent. There were also very strong correlations between the components of the IL-12-IFN-γ pathway, T-bet expression, CD16-expressing cells, and various cytokines/chemokines. Conclusions: The in vitro functional assessment revealed disruptions in the IL-12-IFN-γ pathway of all patients, and a lack of key immune cell subsets and the cytokines/chemokines that are typically expressed by these cells, supporting the clinical diagnosis of MSMD in these patients. While there were some commonalities for the overall patient group, each individual had a very unique phenotype, emphasising the importance of in vitro assessment in all individuals with suspected MSMD – patients with the same or different variants in the same gene had different functional readouts. T-bet was demonstrated to be a promising proxy marker for MSMD as it correlated well with the immunological deficits observed in the patients and will allow for easier detection of potential MSMD in patients with SPUR TB, which may aid in the estimation of the true prevalence of MSMD in future studies.