IDENTIFICATION OF A DYNAMIC INTRACELLULAR RESERVOIR OF CD86 PROTEIN IN PERIPHERAL-BLOOD MONOCYTES THAT IS NOT ASSOCIATED WITH THE GOLGI-COMPLEX

In the process of developing a cancer immunotherapy strategy, we have identified and characterized a novel intracellular reservoir of CD86 p rotein in peripheral blood monocytes. This observation emerged from st udies aimed at using retrovirus vectors to genetically modify tumor ce lls to express the costimulatory proteins CD80 and CD86, Retrovirus-me diated expression of CD80 and CD86 in T lymphoblastoid CEM cells resul ted in an unexpected intracellular focal concentration of both protein s in the genetically modified cells. By extending these studies to an analysis of CD80 and CD86 expression in PBMC, we observed that endogen ous CD86 expression in peripheral blood monocytes also involved a simi lar intracellular focal concentration of the protein, The intracellula r concentration of CD86 in monocytes was not due to storage within the Golgi apparatus, and required intact microtubules to retain structura l integrity. Furthermore, as the intensity of CD86 fluorescence increa sed on monocytes as a function of time in vitro, the intracellular foc al concentration correspondingly decreased. These results are consiste nt with antegrade CD86 transport from an intracellular reservoir to th e cell surface membrane. Ire 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 t rafficking of CD86 to the cell surface membrane in peripheral blood mo nocytes. We hypothesize that this intracellular reservoir allows rapid and sustained deployment of an important costimulatory molecule to th e monocyte surface membrane during initiation and maturation of the ce ll-mediated immune response.