Agonist Anti-GITR Monoclonal Antibody Induces Melanoma Tumor Immunity in Mice by Altering Regulatory T Cell Stability and Intra-Tumor Accumulation Adam D. Cohen1,3¤. , David A. Schaer2., Cailian Liu2, Yanyun Li2, Daniel Hirschhorn-Cymmerman2, Soo Chong Kim2, Adi Diab 2 , Gabrielle Rizzuto2,3, Fei Duan2,3, Miguel A. Perales1,3, Taha Merghoub 2 , Alan N. Houghton1,2,3", Jedd D. Wolchok1,2,3*" 1 Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America, 2 Immunology Program, Sloan-Kettering Institute for Cancer Research, New York, New York, United States of America, 3 Weill Medical College of Cornell University, New York, New York, United States of America Abstract In vivo GITR ligation has previously been shown to augment T-cell-mediated anti-tumor immunity, yet the underlying mechanisms of this activity, particularly its in vivo effects on CD4+ foxp3+ regulatory T cells (Tregs), have not been fully elucidated. In order to translate this immunotherapeutic approach to the clinic it is important gain better understanding of its mechanism(s) of action. Utilizing the agonist anti-GITR monoclonal antibody DTA-1, we found that in vivo GITR ligation modulates regulatory T cells (Tregs) directly during induction of melanoma tumor immunity. As a monotherapy, DTA-1 induced regression of small established B16 melanoma tumors. Although DTA-1 did not alter systemic Treg frequencies nor abrogate the intrinsic suppressive activity of Tregs within the tumor-draining lymph node, intra-tumor Treg accumulation was significantly impaired. This resulted in a greater Teff:Treg ratio and enhanced tumor-specific CD8+ T-cell activity. The decreased intra-tumor Treg accumulation was due both to impaired infiltration, coupled with DTA-1-induced loss of foxp3 expression in intra-tumor Tregs. Histological analysis of B16 tumors grown in Foxp3-GFP mice showed that the majority of GFP+ cells had lost Foxp3 expression. These ‘‘unstable’’ Tregs were absent in IgG-treated tumors and in DTA-1 treated TDLN, demonstrating a tumor-specific effect. Impairment of Treg infiltration was lost if Tregs were GITR2/2, and the protective effects of DTA-1 were reduced in reconstituted RAG12/2 mice if either the Treg or Teff subset were GITR-negative and absent if both were negative. Our results demonstrate that DTA-1 modulates both Teffs and Tregs during effective tumor treatment. The data suggest that DTA-1 prevents intra-tumor Treg accumulation by altering their stability, and as a result of the loss of foxp3 expression, may modify their intra-tumor suppressive capacity. These findings provide further support for the continued development of agonist anti-GITR mAbs as an immunotherapeutic strategy for cancer. Citation: Cohen AD, Schaer DA, Liu C, Li Y, Hirschhorn-Cymmerman D, et al. (2010) Agonist Anti-GITR Monoclonal Antibody Induces Melanoma Tumor Immunity in Mice by Altering Regulatory T Cell Stability and Intra-Tumor Accumulation. PLoS ONE 5(5): e10436. doi:10.1371/journal.pone.0010436 Editor: Derya Unutmaz, New York University, United States of America Received March 15, 2010; Accepted April 8, 2010; Published May 3, 2010 Copyright: 2010 Cohen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: ADC (ASCO Young Investigator Award, Charles A. Dana Foundation, H-C Fellowship, and NIH K08CA127143-01). MAP (NIH K08CA10260 and MSKCC Byrne Fund). DAS (NIH Clinical Training for Scholar Grant K12 CA120121-01, NIH/NCI Immunology Training Grant T32 CA09149-30 and John D. Proctor Foundation: Margaret A. Cunningham Immune Mechanisms in Cancer Research Fellowship Award). JDW CA56821, CA33049 and CA59350 (Damon Runyon-Lilly Clinical Investigator Award). ANH (NIH/NCI (CA56821, CA47179, CA33049 and CA59350) and Damon Runyon-Lilly Foundation). Additional support provided by Swim Across America, Lita Annenberg Hazen Foundation, TJ Martell Foundation, Louis & Anne Abrons Foundation, Mr. and Mrs. Quentin J. Kennedy Fund, Mr. William H. Goodwin and Mrs. Alice Goodman Funds, and MSKCC Experimental Therapeutics Center. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: wolchokj@mskcc.org ¤ Current address: Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America . These authors contributed equally to this work. " These authors also contributed equally to this work. Introduction GITR (glucocorticoid-induced tumor necrosis factor (TNF) receptor, or TNFRSF18) is a type I transmembrane protein with homology to other TNF receptor family members such as OX40, CD27, and 4-1BB.[1] GITR is normally expressed at low levels on resting CD4+foxp3- and CD8+ T cells, but is constitutively expressed at high levels on CD4+CD25+foxp3+ regulatory T cells (Tregs). Expression increases on all 3 subpopulations following T- cell activation. GITR ligation provides a co-stimulatory signal that enhances both CD4+ and CD8+ T-cell proliferation and effector functions, particularly in the setting of suboptimal TCR stimulation.[2,3,4,5] In addition, co-stimulation through GITR has been shown to make na¨ıve or effector T cells (Teffs) resistant to the suppressive effects of Tregs in vitro, and can enhance auto- reactive, allo-reactive, and anti-viral T-cell responses in vivo.[2,6,7,8,9,10,11,12,13] This makes targeting GITR a poten- tial immunotherapeutic approach to cancer treatment. Recently, we and others have demonstrated that in vivo GITR ligation using an agonist anti-GITR mAb, DTA-1, can augment anti-tumor T-cell responses and induce tumor rejection in B16 melanoma and other murine models.[14,15,16,17,18,19] Howev- PLoS ONE | www.plosone.org 1 May 2010 | Volume 5 | Issue 5 | e10436