We previously showed that CD4(+) T cells acquired peptide/major histocompatibility complex (pMHC) I and costimulatory molecules by dendritic cell (DC) activation. However, the molecular mechanism for pMHC I acquisition is unclear. In this study, by using a panel of engineered DC2.4 cells or incubation of these cells with Con A-stimulated CD4(+) T cells, we conducted capping and synapse formation assay and examined them by confocal fluorescence microscopy. We demonstrated that (i) CD54 and CD80 colocalized with pMHC I/II in the same lipid rafts, whereas CD40, OX40L, and 41BBL localized in the lipid rafts but separately from pMHC I/II, and (ii) MHC I/II colocalized with the costimulatory molecules in the same synapse formed between a DC and a CD4(+) T cell, leading to expression of the acquired bystander pMHC I on CD4(+) T cells via internalization/recycling pathway. These results provide some useful information in composition and dynamics of immunological synapses. (c) 2007 Elsevier Inc. All rights reserved.