(A) The most common method used to generate DCs for clinical trials is to culture CD14⁺ monocytes in serum-free media in the presence of GM-CSF and IL-4. Following 5–7 days in culture, the monocytes differentiate into immature DCs, which lose CD14 expression and express moderate to low levels of CD40 and the costimulatory ligands B7-1 and B7-2. DC maturation is accomplished by culturing the immature DCs for an additional 24–48 hours in the presence of several biological agents, the most popular combination being TNF, IL-6, IL-1β, and PGE₂ (41). Mature DCs further upregulate CD40, B7-1, and B7-2 and induce the de novo expression of the lymph node homing receptor CC chemokine receptor 7 (CCR7). Antigen loading occurs at either the immature or mature DC stage. (B) Mature antigen-loaded DCs are injected into patients subcutaneously, intradermally, or intravenously. They migrate to the draining lymph node, where they encounter and present antigen (not shown) to cognate CD4⁺ T cells. Cross-linking CD40 on the DCs by CD40L, which is expressed on the antigen-activated CD4⁺ T cell, induces the mature DCs to differentiate further, a process known as licensing. Licensed DCs upregulate additional cell surface products, notably the ligands for OX40 and 4-1BB (OX40L and 4-1BBL, respectively). The licensed DCs present antigen to cognate CD8⁺ T cells. 4-1BBL–mediated costimulation through 4-1BB on the antigen-activated CD8⁺ T cells enhances the survival and proliferative capacity of the activated CD8⁺ T cells. Likewise, OX40L-mediated costimulation enhances the survival and proliferation of the activated CD4⁺ T cells (not shown).