CD9 expression on T cells and IL-10 production in B cells

Dissertation - University of Veterinary Medicine Vienna - 2022

A comprehensive study of the porcine immune system will contribute to our understanding of immune memory in swine and enable us to develop better vaccines; to this end, we endeavour to expand the repertoire of monoclonal antibodies (mAbs) available for characterising the cellular components of it. In this work we phenotype porcine immune cells using novel monoclonal antibodies specific for the CD9 molecule, alongside established differentiation markers. CD9 is ubiquitously expressed on monocytes and variably expressed on lymphocytes. We found that CD9 can be expressed de novo on T cells following in vitro activation using concanavalin A, linking CD9 to activation status. Moreover, we found that CD9 is expressed on the majority of CD4 T cells that respond to virus antigen, in an in vitro antigen specific recall assay, by expressing intracellular interferon gamma (IFNg) and tumour necrosis factor alpha (TNFa) cytokines, we therefore propose that CD9+ T cells play a role in immune memory. I discuss these findings in comparison with the expression of the human and mouse paralogue, to consider the possible functional roles of CD9 on immune cells. Whether CD9 is a marker of long-lived immune memory warrants further investigation. Next, we questioned whether CD9 is a marker of interleukin-10 (IL-10) competent B cells, based on reported observations in mice. Our data indicated that this is not the case in pigs, so we interrogated the phenotype of IL-10 competent B cells in swine further. To do this, we created flow cytometry panels suitable for identifying porcine B cell subsets based on recent advances for discerning plasma cells and B1 cells, as well as isotype switched B cells. Porcine B cells could be induced to express IL-10 in vitro following 18 h culture with CpG containing oligodeoxynucleotides (CpG-ODN) and 4 h phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation, similar to methods used for mouse and human B10 cells. Our data indicates that porcine IL-10 competent B cells belong to multiple B cell subsets in pigs. Both the characterisation of CD9 expressing T cells and identification of IL-10 competent B cells lays a foundation for future study of these immune cells when evaluating porcine immune responses following vaccination.

Dissertation - Veterinärmedizinische Universität Wien - 2022

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