Generally, APCs activate CD4 T cells against peptides derived from exogenous Ag in the context of MHC II molecules. In this study, using transgenic B lymphocytes as model APCs, we demonstrate CD4 T cell priming in vivo against peptides derived from endogenously synthesized Ag targeted either to the cytosol or to the endoplasmic reticulum (ER). Surprisingly, priming by Ag containing the KDEL-retention motif yielded higher levels of two important proinflammatory cytokines, IFN-gamma and TNF-alpha, in responding CD4 T cells. Importantly, we found that KDEL-mediated retention of Ag up-regulates ER-stress responsive genes in primary B lymphocytes. We also found that thapsigargin treatment of A20 lymphoma cells up-regulates transcription of ER stress and proinflammatory genes along with IL-23p19. Induction of ER stress by thapsigargin also up-regulated IL-23p19 in primary B lymphocytes, macrophages, and bone marrow-derived dendritic cells. We conclude that perturbation of the secretory pathway and/or ER stress play an important role in modulating the gene program in professional APCs and in shaping CD4 T cell responses in vivo. These findings are relevant to a better understanding of the immune response after infection by viral and bacterial pathogens and the pathogenesis of certain autoimmune diseases.