Development of an in vitro cell culture model to investigate HCMV priming of CD8+ T cells

Abstract

Abstract Human cytomegalovirus (HCMV)-specific CD8+ T cells are characterized by an unique, non-exhausting, effector memory phenotype. How HCMV induces this phenotype is poorly characterized. We hypothesized that HCMV may skew T cell priming producing functionally distinct CD8+ T cells. To investigate this question, we have established an autologous 3-cell culture system using human umbilical tissue in which naïve CD8+ T cells are isolated from cord blood, myeloid DCs (mDCs) are generated from cord blood CD34+ precursor cells and fibroblasts, which are a primary target of HCMV infection, are generated from matching umbilical cord (UC-F). Previous studies have reported IFN-γ treatment of fibroblasts induces MHC-II expression, leading to suggestions that fibroblasts can acquire APC-like activity. To investigate this, we used flow cytometry to examine the effects of HCMV infection +/− IFN-γ on MHC-II, CD40, CD80 and CD86 surface expression on fibroblasts. HCMV alone did not induce MHC-II or costimulatory molecules. HCMV infection +/− IFN-γ induced MHC-II but costimulatory molecules remained undetectable. This suggests HCMV-infected fibroblasts do not acquire APC-like phenotype and that fibroblasts mediate T cell priming through cross-presentation. Fibroblasts release extracellular vesicles (EVs), which are important mediators of cell signaling, including antigen cross-presentation. We used electron microscopy to confirm that fibroblasts release EVs. Following AD169-GFP infection, 5.5–8% of EV collected were GFP-positive by ImageStream. Future studies will investigate whether UC-F modulate mDC priming of CD8+ T cells through direct cell-cell contact and/or indirectly, including through the release of EVs.