PBMC and fibroblast cocultures to mimic the in vivo effect of BCG on trained immunity

Abstract

BCG greatly stimulates innate immune cells. Previous studies demonstrated that BCG-stimulated monocytes develop trained immunity whereby they respond to homologous and heterologous antigens. Previous studies used isolated monocytes or animal models to study BCG-induced trained immunity, which have benefits and limitations. To approximate in vivo conditions, we stimulated peripheral blood mononuclear cells (PBMCs) with BCG-treated human fibroblasts. We found that compared with BCG stimulation, the addition of fibroblasts increased the expression of IFN-γ in NK and γδ T cells and of TNF-α and IL-10 in monocytes. We conclude that BCG-treated fibroblasts offer advantages over BCG alone for studying trained immunity.

Figure 1. PBMC and Fibroblast responses to BCG stimulation

Fibroblasts were stimulated with BCG overnight prior to addition of PBMCs and compared to unstimulated Fibroblasts. A) Cytokine expression in Fibroblasts the following day prior to addition of PBMCs. PBMCs were added to stimulated fibroblasts and compared via flow cytometry to unstimulated controls. B) Expression of IFN-γ on NK Cells or 482 TCRγδ cells in response to BCG stimulation of fibroblasts. C-E) Expression of C)IL-1β, D)TNF, or E)IL-10 on Monocytes or myeloid Dendritic Cell subsets. F) PBMCs were then rested for 3 days and restimulated with R848, and myeloid cells were assessed for expression of IL-1β and compared with BCG unstimulated controls or BCG stimulated without fibroblast controls.