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Phenotypic and functional immune cell profiling of patients with primary immunodeficiency associated with mycobacterial infections in a tuberculosis endemic region
Thesis (PhD)--Stellenbosch University, 2022.
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| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2022
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| _version_ | 1867613813353742336 |
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| access_status_str | Open Access |
| author | Van Coller, Ansia |
| author2 | Glashoff, Richard Helmuth |
| author_browse | Glashoff, Richard Helmuth Van Coller, Ansia |
| author_facet | Glashoff, Richard Helmuth Van Coller, Ansia |
| author_sort | Van Coller, Ansia |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhD)--Stellenbosch University, 2022. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/126295 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:42:06.574Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2022 |
| publishDateRange | 2022 |
| publishDateSort | 2022 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/126295 Phenotypic and functional immune cell profiling of patients with primary immunodeficiency associated with mycobacterial infections in a tuberculosis endemic region Van Coller, Ansia Glashoff, Richard Helmuth Glanzmann, Brigitte Esser, Monika Stellenbosch University. Faculty of Science. Dept. Department of Pathology. Medical Microbiology. Mycobacterial diseases Tuberculosis -- Susceptibility Interleukin-12 Interferon UCTD Thesis (PhD)--Stellenbosch University, 2022. 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. AFRIKAANSE OPSOMMING: Agtergrond: Ingebore immuniteitsfoute (IEI) wat verband hou met vatbaarheid vir ernstige, aanhoudende, ongewone en/of herhalende (SPUR) mikobakteriële infeksies is kommerwekkend in lande soos Suid-Afrika waar tuberkulose (TB) hiperendemies is. Mendeliese vatbaarheid vir mikobakteriële infeksies (MSMD), die primêre IEI wat verband hou met SPUR-mikobakteriële infeksies, was oorspronklik net geassosieer met vatbaarheid vir infeksies deur opportunistiese mikobakterieë soos die Bacillus Calmette-Guérin (BCG)-entstof. Onlangse studies het egter getoon dat Suid-Afrikaanse MSMD-pasiënte ook vatbaar is vir SPUR TB. Daar is 15 MSMD-geassosieerde gene beskryf wat almal binne die Interleukin-12-Interferon-γ (IL-12-IFN-γ) immunologiese padweë val, en mutasies in hierdie gene kan lei tot óf verminderde IFN-γ-produksie óf ‘n gebrekkige immuunrespons tot IFN-γ. Die doel van hierdie studie was om immuunversteurings te evalueer in pasiënte wat vermoedelik MSMD het, deur gebruik te maak van genetiese volgordebepaling en in vitro funksionele fenotipering. Daarbenewens is T-bet, 'n algemene transkripsiefaktor, ondersoek as 'n potensiële instaanmerker vir MSMD. Metodes: Bloedmonsters van 18 pasiënte met SPUR TB is vir genetiese volgordebepaling en immuun-fenotipering geneem. Heelgenoomvolgordebepaling (WGS) is uitgevoer om moontlike siekte-geassosieerde variante te identifiseer. Die immuun-fenotipering het assessering van die IL 12-IFN-γ padweë deur vloeisitometriese opsporing van sitokien reseptore en intrasellulêre sein-oordag ingesluit, asook bepaling van immuunselsamestelling en Luminex®-gebaseerde opsporing van sitokiene/chemokine na stimulasie van pasiënt selle met Phytohemagglutinin (PHA), BCG en IL-12/IFN-γ. T-bet uitdrukking is ondersoek deur intrasellulêre vloeisitometrie. Resultate: Nuwe moontlike siekte-geassosieerde variante is in 82% van pasiënte deur WGS geïdentifiseer (in die 11 pasiënte waar WGS suksesvol was), en 89% van die geïdentifiseerde variante was in voorafbekende MSMD gene. Alle pasiënte het 'n mate van versteuring in die IL-12-IFN-γ- padweë getoon, en hierdie versteuring het ooreengestem met die WGS-bevindinge. Verdere immuunondersoeke het aan die lig gebring dat die pasiëntgroep aansienlik verlaagde vlakke van CD16+ monosiete en limfosiete in hul sirkulasie gehad het, asook verminderde vlakke van inflammatoriese sitokien- en chemokienproduksie na PHA/BCG stimulasie. Alle pasiënte het ook abnormale T-bet vlakke gehad, met aansienlik verminderde uitdrukking in CD16+ monosiete en natuurlike doder (NK) selle. Daar was boonop baie sterk korrelasies tussen die komponente van die IL-12-IFN-γ padweë, T-bet uitdrukking, immuunselle met CD16 uitdrukking en verskeie sitokiene/chemokiene. Gevolgtrekkings: Die immuun-fenotipering het ontwrigtings in die IL-12-IFN-γ padweë van alle pasiënte onthul, asook 'n tekort van belangrike immuunselle en die sitokiene/chemokiene wat tipies deur hierdie selle uitgedruk word, wat hul diagnose van MSMD verder ondersteun. Alhoewel daar hierdie algemene kenmerke vir die pasiëntgroep was, het elke individu 'n baie unieke fenotipe gehad, wat die belangrikheid van in vitro assessering in individue met MSMD beklemtoon - pasiënte met dieselfde of verskillende variante in dieselfde geen het verskillende funksionele fenotipes gehad. Dit is ook gedemonstreer dat T-bet 'n goeie instaanmerker vir MSMD kan wees, aangesien dit goed ooreenstem met die immunologiese tekorte wat in die pasiënte waargeneem is. Hierdie instaanmerker sal dit in die toekoms makliker sal maak om MSMD in pasiënte met SPUR TB op te spoor, wat sal help om die ware voorkoms van MSMD te bepaal. Doctoral 2022-11-15T09:30:46Z 2023-01-23T06:50:53Z 2023-05-15T03:00:08Z 2022-12 Thesis http://hdl.handle.net/10019.1/126295 en_ZA Stellenbosch University xx, 233 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Mycobacterial diseases Tuberculosis -- Susceptibility Interleukin-12 Interferon UCTD Van Coller, Ansia Phenotypic and functional immune cell profiling of patients with primary immunodeficiency associated with mycobacterial infections in a tuberculosis endemic region |
| title | Phenotypic and functional immune cell profiling of patients with primary immunodeficiency associated with mycobacterial infections in a tuberculosis endemic region |
| title_full | Phenotypic and functional immune cell profiling of patients with primary immunodeficiency associated with mycobacterial infections in a tuberculosis endemic region |
| title_fullStr | Phenotypic and functional immune cell profiling of patients with primary immunodeficiency associated with mycobacterial infections in a tuberculosis endemic region |
| title_full_unstemmed | Phenotypic and functional immune cell profiling of patients with primary immunodeficiency associated with mycobacterial infections in a tuberculosis endemic region |
| title_short | Phenotypic and functional immune cell profiling of patients with primary immunodeficiency associated with mycobacterial infections in a tuberculosis endemic region |
| title_sort | phenotypic and functional immune cell profiling of patients with primary immunodeficiency associated with mycobacterial infections in a tuberculosis endemic region |
| topic | Mycobacterial diseases Tuberculosis -- Susceptibility Interleukin-12 Interferon UCTD |
| url | http://hdl.handle.net/10019.1/126295 |
| work_keys_str_mv | AT vancolleransia phenotypicandfunctionalimmunecellprofilingofpatientswithprimaryimmunodeficiencyassociatedwithmycobacterialinfectionsinatuberculosisendemicregion |