Identification of a Dynamic Intracellular Reservoir of CD86 Protein in Peripheral Blood Monocytes That Is Not Associated with the Golgi Complex

Abstract

In the process of developing a cancer immunotherapy strategy, we have identified and characterized a novel intracellular reservoir of CD86 protein in peripheral blood monocytes. This observation emerged from studies aimed at using retrovirus vectors to genetically modify tumor cells to express the costimulatory proteins CD80 and CD86. Retrovirus-mediated expression of CD80 and CD86 in T lymphoblastoid CEM cells resulted in an unexpected intracellular focal concentration of both proteins in the genetically modified cells. By extending these studies to an analysis of CD80 and CD86 expression in PBMC, we observed that endogenous CD86 expression in peripheral blood monocytes also involved a similar intracellular focal concentration of the protein. The intracellular concentration of CD86 in monocytes was not due to storage within the Golgi apparatus, and required intact microtubules to retain structural integrity. Furthermore, as the intensity of CD86 fluorescence increased on monocytes as a function of time in vitro, the intracellular focal concentration correspondingly decreased. These results are consistent with antegrade CD86 transport from an intracellular reservoir to the cell surface membrane. In this report, we detail the intracellular and membrane localization studies with tumor cell lines and PBMC, and describe the temporal relationship between intracellular storage and trafficking of CD86 to the cell surface membrane in peripheral blood monocytes. We hypothesize that this intracellular reservoir allows rapid and sustained deployment of an important costimulatory molecule to the monocyte surface membrane during initiation and maturation of the cell-mediated immune response.