CD27 expression by melanoma antigen-specific CD8⁺ T cells increases in an indolent manner and identifies a subset of memory cell precursors within the effector cell population. (A) The increase in CD27 by tumor-specific CD8⁺ T cells occurs in an indolent fashion. Dot plot analysis shows that the MART-1:26-35(27L) peptide-specific lymphocyte population from the peripheral blood of patient 9 is composed of Vβ12⁺ cells 55 days after cell transfer (left). Representative histograms show the steady temporal increase in CD27 expression level and frequency by Vβ12⁺ CD8⁺ T cells from patient 9. (B) The frequency of CD27-expressing tumor-reactive T cells decreases in the early aftermath of cell transfer in select patients. Dot plot analysis of CD8⁺ T cells from patient 10 TIL and PBLs demonstrates the early reduction in CD27 expression by the Vβ7⁺ tumor-reactive population at day 7 after transfer, compared with TIL, and the uniformity of CD27 expression 524 days after ACT. (C) The frequency of CD27-expressing melanoma-reactive CD8⁺ T cells increases with continued persistence in vivo. Tumor antigen-specific CD8⁺ T cells from patients 9 (x), 10 (▧), 20 (⋄), 21 (□), 23 (○), and 28 (▵) were assayed for CD27 expression in the TIL or peripheral blood 1, 4, and 8 or more weeks after ACT. The average of all values is shown (•). (D) CD27 identifies a stable subset of tumor antigen-specific CD8⁺ effector T cells that give rise to the memory T-cell population in most patients. Longitudinal examination of the absolute number of CD28- (⋄), CD27- (□), or IL-7Rα- (▵) expressing melanoma antigen-specific CD8⁺ T cells compared with the total number of melanoma antigen-specific CD8⁺ T cells (•) in the blood of all 6 patients is shown. Values represent the number of tumor antigen-specific CD8⁺ T cells with the indicated phenotype in the peripheral blood as cells/mm³.