A Unique Subset of CD4+CD25highFoxp3+ T Cells Secreting Interleukin-10 and Transforming Growth Factor-β1 Mediates Suppression in the Tumor Microenvironment

Abstract

Purpose: Immunosuppression, including that mediated by CD4⁺CD25^highFoxp3⁺ regulatory T cells (Treg), is a characteristic feature of head and neck squamous cell carcinoma (HNSCC). Tregs with 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-β1 (TGF-β1) 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-β1⁺, although circulating Treg up-regulated CD62L and CCR7 but not GITR, IL-10, or TGF-β1. Treg in TIL mediated stronger suppression (P ≤ 0.001) than Treg in PBMC of HNSCC patients. The addition of neutralizing IL-10 and TGF-β 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-β1 and do not require cell-to-cell contact between Treg and responder cells for inhibition.