Characterisation of activation markers to assess suitability of THP-1 as an alternative cell source for the monocyte monolayer assay 9165

Abstract Description The functional in vitro monocyte monolayer assay (MMA) is used to predict whether antigen-positive red blood cells (RBCs) are safe to transfuse to alloimmunised patients. Monocyte-mediated RBC clearance is an active process involving receptor binding, cell activation, cytoskeletal rearrangement and cytokine production. The MMA requires peripheral blood mononuclear cell (PBMC) derived monocytes, limiting accessibility and consistency in assay outcomes, the alternative use of immortalised monocytic cell lines might overcome these limitations. The standard MMA protocol was adapted to evaluate the suitability of THP-1 cells following exposure to lipopolysaccharide (LPS), or RhD-positive RBCs opsonised with anti-D antibodies. Cellular activation assessment used flow cytometry markers (CD9, CD38, CD40, CD80, CD83, CD86, HLA-DR), and cytokine secretion was quantified via cytometric bead array. Parametric paired two-tailed T tests were used to compare untreated (UT) with LPS and saline RBCs with anti-D RBCs at 1- and 4-hours. LPS-treated THP-1 cells exhibited increased CD83 and decreased CD38 and CD9 expression. Exposure to anti-D sensitised RBCs resulted in reduced CD40 at 1-hour and downregulated CD38 and HLA-DR at 4-hours. Increased cytokine production evident following 4-hour exposure to LPS, no significant cytokine response following exposure to anti-D opsonised RBC. THP-1 cells exhibited limited immune activation and suboptimal mimicry of PBMC responses in the modified MMA. Funding Sources This work was supported by the University of the Sunshine Coast and the Australian Red Cross Lifeblood. Topic Categories Translational and Interventional Immunology (TI)