ARTICLE ABSTRACTPurpose: Tumor‐specific T cells are frequently induced naturally in melanoma patients and infiltrate tumors. It is enigmatic why these patients fail to experience tumor regression. Given that CD8+ T cells mediate antigen‐specific killing of tumor cells, the focus of this study was to identify alterations in the differentiation of CD8+ residing at the tumor site, with emphasis on a population expressing CD57, a marker for terminal differentiation.Experimental Design: We conducted flow cytometric analysis of CD8+ tumor‐infiltrating lymphocytes (TIL) isolated from 44 resected melanoma metastases with known T‐cell differentiation markers. For comparison, peripheral blood mononuclear cells were isolated from matched melanoma patients. We sorted different CD8+ subsets found in TIL and determined their effector functions. In addition, we carried out Vβ clonotype expression analysis of T‐cell receptors to determine lineage relationship between the CD8+ TIL subsets.Results: The majority of CD8+ TIL was in the early-effector memory stage of differentiation. A significant population consisted of an oligoclonal subset of cells coexpressing CD27, CD28, CD57, and Granzyme B, with little or no perforin. These cells could be induced to proliferate, produce a high level of IFN-γ, and differentiate into CD27−CD57+, perforinhigh mature CTL in vitro. Addition of TGF‐β1 prevented further differentiation.Conclusions: Our studies identified a novel subset of incompletely differentiated CD8+ CTL coexpressing early effector memory and late CTL markers. This population resembles that found in patients with uncontrolled chronic viral infections. TGF-β1, frequently produced by melanoma tumors, may be a key cytokine inhibiting further maturation of this subset. Clin Cancer Res; 18(9); 2465–77. ©2012 AACR.