SCCs are heavily infiltrated by FOXP3⁺ T reg cells recruited from blood. (A) T cells isolated from SCCs developing in normal individuals and transplant recipients contained many CD25^hiFOXP3⁺ T reg cells. (B) T reg cells isolated from SCCs were CD4⁺ central memory T cells (L-selectin/CCR7⁺) and were distinct from cutaneous T reg cells found in normal skin as shown by their lack of expression of key skin-homing addressins (CLA, CCR4). The last two graphs are gated to show only CD3⁺FOXP3⁺ T cells. (C) Direct study of FOXP3⁺ T reg cells in areas of invasive SCCs using immunofluorescence staining of frozen sections. SCCs were stained for CD3 (red) and FOXP3 (green). Two FOXP3⁺ T reg cells are shown at the top of the left image, and a FOXP3⁻ nonregulatory T cell is shown on the bottom. A larger field is shown in the right image. (D) A lower magnification image of another SCC, demonstrating that large numbers of FOXP3⁺ T reg cells (red cells with green nuclei) surround nodules of invasive tumor, which appear as pools of green secondary to nonspecific staining of tumor keratin. (E) Enumeration of T reg cells in frozen sections of SCCs. The number of T reg cells and nonregulatory T cells were counted in 10 high power (40X) fields in SCCs from normal patients (Immunocompetent) and transplant recipients (Transplant rcp) and the results were compared with that of normal skin. Shown are the mean and SD of counts from 10 fields. (F) FOXP3⁺ T reg cells are not locally expanded within SCCs. SCC sections were costained for FOXP3 and Ki-67, a marker of cell proliferation. Proliferative and nonproliferative FOXP3⁺ T reg cells were counted in 5 hpf for each donor; the mean and SD for each tumor are shown. SCC9 and 10 are from immunocompetent individuals; SCC11 is from a transplant recipient. Bars: (C, left) 10 μm; (C, right, and D) 100 μm.