Figure 1. Differentiation pathway of the tumor-infiltrating CD8⁺ T cells in metastatic cancer. In situations where CD8⁺ T cells encounter persistent, chronic antigenic stimulation such as metastatic cancer or uncontrolled chronic viral infections, CD8⁺ effector-memory T (T_EM) cells fail to coordinate downregulation of CD27 with upregulation of an end-stage CTL marker, CD57 and acquire a more cytolytic phenotype. Thus T_EM fail to transition from a granzyme B (GB⁺) perforin^lo (Perf^lo) cells into Perf^hi, highly cytotoxic end-stage CTL. This resulted in accumulation of CD8⁺ T cells at a transitional stage where markers for early T_EM (CD27, CD28) are co-expressed with CD57, even though the cells remain Perf^lo. We also found that TGF-β1, an immunosuppressive cytokine frequently found in the microenvironment of metastatic cancer, could also contribute to the arrested differentiation and accumulation of CD27⁺CD57^- precursor T cells and CD27⁺CD57⁺ T cells. We found that PD-1 was expressed more in the CD27⁺CD57⁺ T cells, which implied that they may exhibit a higher level of effector activity. When these tumor-infiltrating lymphocytes (TIL) are expanded with IL-2, a minor fraction (~30%) of CD27⁺CD57^- subset differentiated into CD27⁺CD57⁺ T cells. After TCR stimulation ex vivo, the CD27⁺CD28⁺CD57^- subset directly differentiated to become CD27^-CD57^- T cells. On the other hand, CD27⁺CD57⁺ differentiated to become CD27^-CD57⁺, and, in some patients, CD27^-CD57^-. These phenotypic changes were accompanied by increased perforin expression and acquisition of potent cytotoxicity against tumor cells. We think that CD57 is not a marker for T-cell senescence, but rather marks highly differentiated T cells that are in the process of transitioning into a truly end-stage, effector CTL. Currently it is not known which set of markers define truly senescent, end-stage, highly cytolytic CTL. We also propose that ultimately, both the CD8⁺CD57⁺ and CD8⁺CD57^- subsets may ultimately differentiate into a subset of CD8⁺CD27^-CD28^-CD57^- cells that may be more NK-like, expressing higher levels of CD122, KLRG1, NKG2D and other NK receptors.