CD4+ regulatory T (Treg) cells have a profound ability to suppress host immune responses, yet little is understood about how these cells are regulated. We describe a mechanism linking Toll-like receptor (TLR) 8 signaling to the control of Treg cell function, in which synthetic and natural ligands for human TLR8 can reverse Treg cell function. This effect was independent of dendritic cells but required functional TLR8-MyD88-IRAK4 signaling in Treg cells. Adoptive transfer of TLR8 ligand-stimulated Treg cells into tumor-bearing mice enhanced anti-tumor immunity. These results suggest that TLR8 signaling could play a critical role in controlling immune responses to cancer and other diseases.

Regulatory T cells play an important role in the maintenance of immunological self-tolerance by suppressing immune responses against autoimmune diseases and cancer. Little is known, however, about the nature of the physiological target antigens for CD4(+) regulatory T (Treg) cells. Here we report the identification of the LAGE1 protein as a ligand for tumor-specific CD4(+) Treg cell clones generated from the tumor-infiltrating lymphocytes (TILs) of cancer patients. Phenotypic and functional analyses demonstrated that they were antigen-specific CD4(+) Treg cells expressing CD25 and GITR molecules and possessing suppressive activity on the proliferative response of naive CD4(+) T cells to anti-CD3 antibody stimulation. Ligand-specific activation and cell-cell contact were required for TIL102 Treg cells to exert suppressive activity on CD4(+) effector cells. These findings suggest that the presence of tumor-specific CD4(+) Treg cells at tumor sites may have a profound effect on the inhibition of T cell responses against cancer.

Purpose: By hypomethylating genes, decitabine may up-regulate factors required for chemotherapeutic cytotoxicity. Platinum-resistant cells may have reduced expression of the copper/platinum transporter CTR1. Experimental Design: Thirty-one patients with refractory malignancies received decitabine 2.5 to 10 mg/m 2 on days 1 to 5, and 8 to 12 or 15 to 20 mg/m 2 on days 1 to 5. Tumor was assessed for DNA methylation (by LINE assays), apoptosis, necrosis, mitoses, Ki67, DNA methyltransferase (DNMT1), CTR1, and p16. Results: Febrile neutropenia was dose limiting. One thymoma patient responded. Decitabine decreased tumor DNA methylation (from median 51.2% predecitabine to 43.7% postdecitabine; P = 0.01, with effects at all doses) and in peripheral blood mononuclear cells (from 65.3-56.0%). There was no correlation between tumor and peripheral blood mononuclear cells. Patients starting decitabine ≤3 versus >3 months after last prior cytotoxic or targeted therapy had lower predecitabine tumor CTR1 scores (P = 0.02), higher p16 (P = 0.04), and trends (P = 0.07) toward higher tumor methylation and apoptosis. Decitabine decreased tumor DNMT1 for scores initially >0 (P = 0.04). Decitabine increased tumor apoptosis (P < 0.05), mitoses (if initially low, P = 0.02), and CTR1 (if initially low, P = 0.025, or if ≤3 months from last prior therapy, P = 0.04). Tumor CTR1 scores correlated inversely with methylation (r = −0.41, P = 0.005), but CTR1 promoter was not hypermethylated. Only three patients had tumor p16 promoter hypermethylation. P16 scores did not increase. Higher blood pressure correlated with lower tumor necrosis (P = 0.03) and a trend toward greater DNA demethylation (P = 0.10). Conclusions: Exposure to various cytotoxic and targeted agents might generate broad pleiotropic resistance by reducing CTR1 and other transporters. Decitabine decreases DNA methylation and augments CTR1 expression through methylation-independent mechanisms.

CD4+ regulatory T (Treg) cells play an important role in the maintenance of immunological self-tolerance by suppressing immune responses against autoimmune diseases and cancer. Yet very little is known about the natural antigenic ligands that preferentially activate CD4+ Treg cells. Here we report the establishment of tumor-specific CD4+ Treg cell clones from tumor-infiltrating lymphocytes (TILs) of cancer patients, and the identification of an Ag recognized by Treg cells (ARTC1) gene encoding a peptide ligand recognized by tumor-specific TIL164 CD4+ Treg cells. The mutations in a gene encoding an ARTC1 in 164mel tumor cells resulted in the translation of a gene product containing the peptide ligand recognized by CD4+ Treg cells. ARTC1 peptide-activated CD4+ Treg cells suppress the physiological function (proliferation and IL-2 secretion) of melanoma-reactive T cells. Furthermore, 164mel tumor cells, but not tumor lysates pulsed on B cells, were capable of activating TIL164 CD4+ Treg cells. These results suggest that tumor cells may uniquely present an array of peptide ligands that preferentially recruit and activate CD4+ Treg cells in sites where tumor-specific self-peptide is expressed, leading to the induction of local and tumor-specific immune suppression.