AbstractCD40 engagement is the major signal that induces B cells to efficiently present antigen to T cells. We previously demonstrated that human peripheral blood–derived CD40-activated B cells (CD40–B cells) function as antigen-presenting cells (APCs). Here, we have established a culture system to generate these APCs under clinically applicable conditions using guanylic acid–grade soluble trimeric CD40 ligand. To monitor APC function and antigen loading for these cells, simple and efficient quality control assays have been developed. Using this approach, we demonstrate that CD40–B cells from healthy donors and cancer patients are fully functional and equally expanded in long-term cultures. These B cells boost robust memory T-cell responses, but more importantly, they also prime naive T-cell responses against neoantigens ex vivo. CD40–B cells overcome current obstacles, such as the difficulty of isolation, generation, and long-term expansion observed with other APCs. Therefore, they are an excellent source of professional APCs for immune assessment, antigen discovery, and antigen-specific immunotherapy.