Persisting melanoma antigen-specific CD8⁺ T cells express both CD27 and CD28 in vivo. (A) Expression of CD27 and CD28 by the reconstituting endogenous CD8⁺ T-cell repertoire. Dot plot analysis of gated CD8⁺ MART-1/A2 tetramer-negative T cells from patient 28's PBLs obtained prior to and after ACT of TIL reveals that the phenotype of pretreatment CD8 T cells is restored in reconstituting CD8 T-cell population. The frequency of CD28⁺ CD27^- is low in PBLs prior to lymphodepletion and at day 63 after cell infusion. (B) Transfer of predominantly CD27^- CD28^- TIL results in the generation of a persisting population of CD27⁺ CD28⁺ tumor antigen-specific CD8⁺ T cells. Representative dot plots show the expression of CD27 and CD28 on tumor antigen-specific CD8⁺ T cells from TIL and posttreatment PBL samples from patients 28 (MART-1⁺), 23 (MART-1⁺), 10 (Vβ7⁺), and 21 (Vβ22⁺). The tumor antigen-reactive T-cell population transitioned through a CD28⁺ CD27^- 1 week after transfer before becoming CD27⁺ CD28⁺ memory T cells. (C) CD28-expressing melanoma-reactive CD8⁺ T cells that concomitantly express CD27 persist after ACT in vivo. The absolute number of transferred tumor antigen-specific CD8⁺ T cells expressing both CD27 and CD28 was assessed in PBLs from patients with objective clinical responses (10, 21, and 28) beginning 1 week after infusion and is represented as cells/mm³, depicted in log scale.