Malignant pleural effusion (MPE) provides a liquid tumor microenvironment model that includes cancer cells and immune cells. However, the characteristics of tumor antigen-specific CD8 T cells have not been investigated in detail. Here, we analyzed MPE samples taken from a patient with pancreatic cancer who received a dendritic cell vaccine targeting Wilms' Tumor 1 (WT1) antigen over the disease course (two points at MPE and 2, two months after MPE1). Epithelial cell adhesion molecule (EpCAM) cancer cells (PD-L1 or T cell immunoglobulin mucin-3, TIM-3), both PD-1 or TIM-3 positive CD8 T cells, and CD14CD68CD163TIM-3 macrophages increased from the MPE to MPE. The ratio of WT1-specific cytotoxic lymphocytes (WT1-CTLs) to MPE CD8 T cells and IFN-γ secretion of WT1-CTLs were reduced with disease progression. Coincidentally, the fraction of central memory T (T) of WT1-CTLs was decreased. On the other hand, CD8 T cells in response to SMAD4, which is homogeneously expressed in EpCAM cancer cells, were detected using in vitro expansion with the HLA-A*11:01 restrictive SVCVNLYH neoantigen. Furthermore, the CD8 T cell response to SMAD4 was diminished following remarkably decreased numbers of CD8 T in MPE samples. In conclusion, CD8 T cells responding to WT1 or SMAD4 neoantigen expressed in EpCAM pancreatic cancer cells were detected in MPE. A tumor antigen-specific immune response would provide novel insight into the MPE microenvironment.