Abstract PD-L1 (CD274) contributes to functional exhaustion of T cells and limits immune responses in patients with cancer. In this study, we report the identification of an human leukocyte antigen (HLA)-A2–restricted epitope from PD-L1, and we describe natural, cytolytic T-cell reactivity against PD-L1 in the peripheral blood of patients with cancer and healthy individuals. Notably, PD-L1–specific T cells were able not only to recognize and kill tumor cells but also PD-L1–expressing dendritic cells in a PD-L1–dependent manner, insofar as PD-L1 ablation rescued dendritic cells from killing. Furthermore, by incubating nonprofessional antigen-presenting cells with long peptides from PD-L1, we found that PD-L1 was rapidly internalized, processed, and cross-presented by HLA-A2 on the cell surface. Apparently, this cross-presentation was TAP-independent, as it was conducted not only by B cells but in addition by TAP-deficient T2-cells. This is intriguing, as soluble PD-L1 has been detected in the sera from patients with cancer. PD-L1–specific CTL may boost immunity by the killing of immunosuppressive tumor cells as well as regulatory cells. However, PD-L1–specific CTLs may as well suppress immunity by the elimination of normal immune cells especially PD-L1 expressing mature dendritic cells. Cancer Res; 73(6); 1764–76. ©2012 AACR.