Investigation of regulatory B-cell responses during Mycobacterium tuberculosis exposure

Moore, Dannielle Keagen (2019-03)

Thesis (MMed)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Tuberculosis (TB) remains a global health challenge due to limited understanding of the complex host immune responses required for the successful control and eradication of invading Mycobacterium tuberculosis (M.tb) bacilli. To date, T-cells and macrophages have been regarded as the principle immune cells responsibly for protective anti-TB immunity. However, emerging evidence has revealed a fundamental role of B-lymphocytes (B-cells) during M.tb infection; although the function of B-cells in TB disease incidence and progression remains ill-defined. We hypothesize that B-cells influence anti-TB immune responses through modulation of T-cell activation, and that the microenvironment in which these interactions occur greatly impacts immune cell function, with an emphasis on B-cells. Healthy participants that were either pre-exposed or unexposed to M.tb were recruited for the study. Within the pilot study investigating the effect of B-cells on T-cell function: peripheral blood was collected, and B- or T-cell enriched using magnetic-activated cell sorting (MACS) bead technology for downstream analysis. Following sample collection, autologous T-cells were co-cultured with H37Rv/BCG-stimulated B-cells, pulsed with or without cluster of differentiation 40 Ligand (CD40L) and interleukin-5 (IL5). The resulting B- and T-cell phenotypic frequencies and cytokine profiles were subsequently evaluated. To determine the effect of the microenvironment complexity on cell function, peripheral blood was collected, and B-cell frequency and function (immunoglobulin isotype profile) determined in whole blood, peripheral blood mononuclear cells (PBMCs) and untouched, isolated Bcells following 24-hour stimulation with toll-like receptor 9 agonist (TLR9a) or H37Rv. Our results demonstrate that B-cells are able to modulate T-cell activation and function. We found that Bacillus Calmette-Guérin (BCG) and H37RV alike are able to induce killer/regulatory B-cell phenotypes in QuantiFERON positive and negative participants. Furthermore, the microenvironment was found to have a substantial effect on B-cell function with noticeable changes in B-cell development, shown by a decrease in mature B-cell frequencies following cellular isolation. Likewise, impairment of humoral responses, indicated by hampered ability to produce certain immunoglobulin isotypes in response to antigenic stimulation, were observed for isolated B-cells. Collectively, these findings indicate the potential influence B-cells have in anti-TB immune responses through modulation of T-cell behaviour, underscoring the role B-cells may play in initiating and guiding the immune response against M.tb. Our study highlights the need to further study B-cells as therapeutic targets in novel TB prevention strategies. Additionally, our study identifies significant limitations associated with the use of cell isolation studies for inference of cell function during health and disease. Findings from these studies, while informative, should be interpreted carefully, as the in vitro microenvironment may have considerably influenced the observed results.

AFRIKAANSE OPSOMMING: Tuberkulose (TB) bly 'n wêreldwye gesondheidsuitdaging as gevolg van die beperkte begrip van die komplekse gasheerimmuniteits reaksies wat benodig word vir die suksesvolle beheer en uitwissing van indringer Mycobacterium tuberculosis (M.tb) bakteriee. Tot op datum is T-selle en makrofages as die primêre immuunselle verantwoordelik vir beskermende anti-TB immuniteit beskou. Opkomende bewyse het egter 'n fundamentele rol van B-limfosiete (B-selle) tydens M.tb infeksie geopenbaar; alhoewel die funksie van B-selle in TB-siekte voorkoms en progressie steeds ondefinieerbaar is. Ons vermoed dat B-selle anti-TB immuunresponse beïnvloed deur modulasie van T-sel aktivering, en dat die mikro-omgewing waarin hierdie interaksies voorkom grootliks die immuunsel funksie beïnvloed, met die klem op B-selle. Gesonde deelnemers wie blootgestel was aan M.tb is vir die studie gewerf. Binne die loodsstudie wat die effek van B-selle op T-selfunksie ondersoek het, is perifere bloed ingesamel, en B- of T-sel verryk met behulp van magnetiese-geaktiveerde sel sortering (MACS) kraal tegnologie vir stroomaf analise. Na volgende monsterversameling, is autoloë T-selle gekweek met H37Rv/BCG-gestimuleerde B-selle, gepulseer met of sonder groepering van differensiasie 40 Ligand (CD40L) en Interleukine-5 (IL5). Die gevolglike B- en T-sel fenotipiese frekwensies en sitokienprofiele is vervolgens geëvalueer. Om die effek van die mikroomgewingskompleksiteit op selfunksie te bepaal, is perifere bloed ingesamel, en B-sel frekwensie en funksie (immunoglobulien isotipe profiel) is bepaal in volbloed, perifere bloedmononukleêre selle (PBMCs) en onaangeraakte geïsoleerde B-selle na 24 -uur stimulasie met tollike receptor 9 agonist (TLR9a) of H37Rv. Ons resultate toon dat B-selle T-sel aktivering en funksie kan moduleer. Ons het bevind dat Bacillus Calmette- Guérin (BCG) en H37Rv gelyk is aan regulatoriese B-sel fenotipes in QuantiFERON positiewe en negatiewe deelnemers. Verder is gevind dat die mikro-omgewing 'n wesenlike effek op B-selfunksie het met merkbare veranderinge in B-selontwikkeling, getoon deur 'n afname in volwasse B-sel frekwensies na sellulêre isolasie. Net so is waargeneem vir die geïsoleerde B-selle waardedaling van humorale response, aangedui deur die belemmerde vermoë om sekere immunoglobulien isotipes te produseer in reaksie op antigeen stimulasie. Gesamentlik dui hierdie bevindinge op die moontlike invloed wat B-selle in anti-TB immuunresponse het deur modulasie van T-selgedrag, wat die rol wat B-selle kan speel in die inisiatief en leiding van die immuunrespons teen M.tb beklemtoon. Ons studie beklemtoon die noodsaaklikheid om B-selle verder as terapeutiese teikens in nuwe TB-voorkomingstrategieë te bestudeer. Daarbenewens identifiseer ons studie beduidende beperkings wat verband hou met die gebruik van sel isolasie studies vir inferensie van selfunksie tydens gesondheid en siekte. Bevindings uit hierdie studies, terwyl insiggewend, moet versigtig geïnterpreteer word, aangesien die in vitro mikro-omgewing die waargenome resultate aansienlik beïnvloed het.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/105669
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