PD-1 is predominantly expressed by tumor-infiltrating T cells compared with peripheral blood T cells. Cryopreserved tumor digests from patients with metastatic melanoma (Pt) and PBL from the same patients and healthy donors were thawed and immediately stained with CD3, CD8, and PD-1 mAbs. (A) Dot plots were gated on CD3⁺ lymphocytes. The percentage for each quadrant represents the fraction of PD-1⁺ T cells in CD8 T cells (top right quadrant) or in CD4 T cells (top left quadrant). These were representative of 14 patients and 7 healthy donors. (B) The percentage of PD-1⁺ CD8 T cells per total CD8 T-cell population and (C) the percentage of PD-1⁺ CD4 T cells per total CD4 T-cell population were quantified in PBL and tumor digests from the same patients (n = 14). P value was calculated using the paired t test. (D) In 3 patients who had detectable circulating MART-1 tetramer CD8 T cells in their peripheral blood, we compared PD-1 expression on MART-1 tetramer⁺ CD8 T cells in tumor digest samples versus those in peripheral blood in the same patients as described in Figure 2.