In mice, splenic conventional dendritic cells (cDCs) can be separated, based on their expression of CD8 alpha into CD8(-) and CD8(+) cDCs. Although previous experiments demonstrated that injection of antigen (Ag)-pulsed CD8(-) cDCs into mice induced CD4 T cell differentiation toward Th2 cells, the mechanism involved is unclear. In the current study, we investigated whether OX40 ligand (OX4OL) on CD8(-) cDCs contributes to the induction of Th2 responses by Ag-pulsed CD8(-) cDCs in vivo, because OX40-OX4OL interactions may play a preferential role in Th2 cell development. When unseparated Ag-pulsed OX40L-deficient cDCs were injected into syngeneic BALB/c mice, Th2 cytokine (IL-4, IL-5, and IL-10) production in lymph node cells was significantly reduced. Splenic cDCs were separated to CD8(-) and CD8(+) cDCs. OX4OL expression was not observed on freshly isolated CD8(-) cDCs, but was induced by anti-CD40 mAb stimulation for 24 h. Administration of neutralizing anti-OX4OL mAb significantly inhibited IL-4, IL-5, and IL-10 production induced by Ag-pulsed CD8(-) cDC injection. Moreover, administration of anti-OX4OL mAb with Ag-pulsed CD8(-) cDCs during a secondary response also significantly inhibited Th2 cytokine production. Thus, OX4OL on CD8(-) cDCs physiologically contributes to the development of Th2 cells and secondary Th2 responses induced by Ag-pulsed CD8(-) cDCs in vivo. (C) 2014 Elsevier Inc. All rights reserved.