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Conditioning murine B cells for immunoglobulin A production In vitro

Goh, Wil (2012) Conditioning murine B cells for immunoglobulin A production In vitro. Honours thesis, Murdoch University.

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The respiratory mucosa is continuously exposed to a myriad of pathogens and allergens that are inhaled during respiration. B cells of the immune system produce immunoglobulin A (IgA) that protects the respiratory mucosa from inhaled pathogens and allergens. IgA is produced after naïve B cells undergo activation, proliferation and differentiation, developing into antibody producing plasma cells. The process that facilitates IgA production as B cells develop into plasma cells is known as class switch recombination (CSR).

Factors favouring IgA have been determined by in vitro studies of CSR. These established two cytokines, IL-21 and TGF-β1, to be important factors for IgA production. Prior studies with human naïve B cells that were cultured with IL-21 and TGF-β1, in addition to anti-IgM, anti-CD40, IL-2 and CpG, developed into precursors of IgA producing plasma cells (plasmablasts) that possessed mucosal homing capabilities: a process termed B cell conditioning.

The aim of this project was to establish whether the same factors were active on murine naïve B cells in order to eventually test the in vivo therapeutic potential of B cell conditioning. A protocol was optimised for CD19+CD27- naïve B cell isolation from mouse spleen and cell division, which is crucial for IgA CSR, by CFSE labelling and flow cytometry. The number of divisions predicts the generation of IgA mucosal homing plasmablasts in vitro. Cells were found to have undergone 5 divisions after 7 days in culture suggesting that cells had undergone a sufficient number of divisions for CSR and IgA production. Attempts were made to quantitate IgA production in culture supernatants, however evidence for the generation of these cells by IgA production remains to be confirmed. The results of this project provide preliminary evidence that murine naïve B cells can be conditioned in vitro for IgA production, with potential for homing to, and protection of, mucosal surfaces in vivo.

Item Type: Thesis (Honours)
Murdoch Affiliation(s): School of Veterinary and Biomedical Sciences
Supervisor(s): Stumbles, Phil, Penhale, William, Etherington, Sarah and Strickland, Debbie
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