FOXP3 expression accurately defines the population of intratumoral regulatory T cells that selectively accumulate in metastatic melanoma lesions

Regulatory T(T-reg) cells are often found in human tumors; however, their functional characteristics have been difficult to evaluate due to low cell numbers and the inability to adequately distinguish between activated and T-reg cell populations. Using a novel approach, we examined the intracellular cytokine production capacity of tumor-infiltrating T cells in the single-cell suspensions of enzymatically digested tumors to differentiate T-reg cells from effector T cells. Similar to T-reg cells in the peripheral blood of healthy individuals, tumor-infiltrating FOXP3 + CD4 T cells, unlike FOXP3 + T cells, were unable to produce IL-2 and IFN-gamma upon ex vivo stimulation, indicating that FOXP3 expression is a valid biological marker for human T-reg cells even in the tumor microenvironment. Accordingly, we enumerated FOXP3 + CD4 T-reg cells in intratumoral and peritumoral sections of metastatic melanoma tumors and found a significant increase in proportion of FOXP3 + CD4 T-reg cells in the intratumoral compared with peritumoral areas. Moreover, their frequencies were 3-to 5-fold higher in tumors than in peripheral blood from the same patients or healthy donors, respectively. These findings demonstrate that the tumor-infiltrating CD4 T-reg cell population is accurately depicted by FOXP3 expression, they selectively accumulate in tumors, and their frequency in peripheral blood does not properly reflect tumor microenvironment. (Blood. 2008; 112: 4953-4960)