A unique subset of CD4+CD25high Foxp3+ T cells secreting interleukin-10 and transforming growth factor-β1 mediates suppression in the tumor microenvironment

Purpose: Immunosuppression, including that mediated by CD4(+)CD25 (high) Foxp3(+) regulatory T cells (Treg), is a characteristic feature of head and neck squamous cell carcinoma (HNSCC). Tregswith a distinct phenotype in tumor- infiltrating lymphocytes (TIL) contribute to local immune suppression. Experimental Design: The frequency and phenotype of Treg in TIL and/or peripheral blood mononuclear cells (PBMC) in 15 HNSCC patients and PBMC in 15 normal controls were compared. Single-cell sorted CD4(+)CD25(high) T cells were tested for regulatory function by coculture with carboxyfluorescein diacetate succinimidyl ester -labeled and activated autologous CD4(+)CD25(-) responder T cells.Transwell inserts separating Treg from responders and neutralizing interleukin-10 (IL-10) or transforming growth factor- beta 1 (TGF-beta 1) antibodies were used to evaluate the mechanisms used by Treg to suppress responder cell proliferation. Results: In TIL, CD25(+) cells were enriched in the CD3(+)CD4(+) subset (13 +/- 3%) relative to circulating CD3(+)CD4(+) T cells (3 +/- 0.7%) in HNSCC patients (P <= 0.01) or normal controls (2 +/- 1.5%; P <= 0.001). Among the CD3(+)CD4(+) subset, CD25(high) Treg represented 3 +/- 0.5% in TIL, 1 +/- 0.3% in PBMC, and 0.4 +/- 0.2% in normal controls. Tregs in TIL were GITR(+), IL-10(-), and TGF- beta 1. although circulating Treg up-regulated CD62L and CCR7 but not GITR, IL-10, orTGF-beta 1. Treg in TIL mediated stronger suppression (P:! 0.001) than Treg in PBMC of HNSCC patients.The addition of neutralizing IL-10 and TGF- antibodies almost completely abrogated suppression (5 +/- 2.51%).Transwell inserts partly prevented suppression (60 +/- 5% versus 95 +/- 5%). Conclusions: Suppression in the tumor microenvironment is mediated by a unique subset of Treg, which produce IL-10 and TGF- beta 1 and do not require cell-to-cell contact between Treg and responder cells for inhibition.