Published OnlineFirst February 26, 2014; DOI: 10.1158/0008-5472.CAN-13-2928 Cancer Microenvironment and Immunology Research Foxp3þ T Cells Inhibit Antitumor Immune Memory Modulated by mTOR Inhibition Yanping Wang1, Tim Sparwasser3, Robert Figlin2, and Hyung L. Kim1 Abstract Inhibition of mTOR signaling enhances antitumor memory lymphocytes. However, pharmacologic mTOR inhibition also enhances regulatory T-cell (Treg) activity. To counter this effect, Treg control was added to mTOR inhibition in preclinical models. Tregs were controlled with CD4-depleting antibodies because CD4 depletion has high translational potential and already has a well-established safety profile in patients. The antitumor activity of the combination therapy was CD8 dependent and controlled growth of syngeneic tumors even when an adoptive immunotherapy was not used. Lymphocytes resulting from the combination therapy could be transferred into na€ve mice to inhibit aggressive growth of lung metastases. The combination therapy enhanced CD8 memory formation as determined by memory markers and functional studies of immune recall. Removal of FoxP3- expressing T lymphocytes was the mechanism underlying immunologic memory formation following CD4 depletion. This was confirmed using transgenic DEREG (depletion of regulatory T cells) mice to specifically þ remove Foxp3 T cells. It was further confirmed with reciprocal studies where stimulation of immunologic þ memory because of CD4 depletion was completely neutralized by adoptively transferring tumor-specific Foxp3 T cells. Also contributing to tumor control, Tregs that eventually recovered following CD4 depletion were less immunosuppressive. These results provide a rationale for further study of mTOR inhibition and CD4 depletion in patients. Cancer Res; 74(8); 1–12. Ó2014 AACR. Introduction are already approved by the U.S. Food and Drug Administra- The immune system can provide protection against cancers. tion (FDA) for clinical use, are a promising adjunct for use with Effective immune stimulation produces long-lasting memory cancer vaccines. lymphocytes, capable of rapidly responding to repeat antigen Strategies to limit immune suppression by mTOR inhibitors challenge. The mTOR pathway is an important checkpoint that may make this class of drugs even more useful with cancer governs the formation of CD8 memory cells (1–3). In mouse vaccines. Pharmacologic mTOR inhibition suppresses the models, decreased mTOR signaling promotes formation of immune system at least in part by enhancing CD4 regulatory CD8 memory cells that provide protection against bacteria T-cell (Treg) activity (2, 5). Therefore, we explored a combi- (4), virus (1) or cancer (2, 3). This is surprising because nation therapy targeting the mTOR pathway and Tregs. The rapamycin, which is the prototypic mTOR inhibitor, is con- most reliable Treg marker is forkhead box transcription factor fi sidered an immunosuppressant and is widely used to prevent (FoxP3), which is speci c for Tregs and is required for its rejection of solid organ transplants. In murine models of renal function (6). Unfortunately, there is no clinical strategy for cell carcinoma (RCC) and melanoma, pharmacologic mTOR targeting FoxP3-expressing cells in patients. Therefore, an inhibition had both immune stimulating and immune sup- alternative strategy is to target CD25, which is expressed by pressing effects (2). However, the net effect resulted in the majority of Tregs. However, this strategy has limitations decreased tumor growth. Therefore, mTOR inhibitors, which because some Tregs are CD25 negative. Furthermore, activated CD8 lymphocytes express CD25 and can be depleted by CD25- targeting strategies. In murine models, depleting CD25-expres- Authors' Affiliations: 1Department of Surgery, Division of Urology; sing cells with aCD25 antibodies was effective in preventing 2Department of Medicine, Division of Hematology and Oncology, 3 tumor growth, but was not effective in treating established Cedars-Sinai Medical Center, Los Angeles, California; and Institute of – Infection Immunology, TWINCORE, Centre for Experimental and Clinical tumors (7 9) and has been shown to restrict adoptive immu- Infection Research, Hannover, Germany notherapy (10, 11). Another strategy uses an engineered pro- Note: Supplementary data for this article are available at Cancer Research tein that combines interleukin-2 (IL-2) and diphtheria toxin Online (http://cancerres.aacrjournals.org/). (denileukin diftitox, trade name Ontax) to target CD25-expres- Corresponding Author: Hyung Kim, Cedars-Sinai Medical Center, 8635 sing cells. The approach has been tested in patients with RCC W. Third Street, Suite 1070, Los Angeles, CA 90048. Phone: 310-423-4700; or melanoma (12, 13), however clinical effectiveness was Fax: 310-423-4711; E-mail: [email protected] limited, possibly because of depletion of CD8 effector cells. doi: 10.1158/0008-5472.CAN-13-2928 Using preclinical models, we explored a combination of Ó2014 American Association for Cancer Research. pharmacologic mTOR inhibition and Treg depletion using www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst February 26, 2014; DOI: 10.1158/0008-5472.CAN-13-2928 Wang et al. aCD4 antibody. This is an attractive approach because CD4- CD32 (9.3), anti-CD90.1 (OX-7), anti-CD11c (N418), anti-Bcl2 depleting antibodies have been studies in patients with periph- (BCL/10C4), anti-T-bet (4B10), anti-CD62L (MEL-14), anti- eral T-cell lymphoma (14, 15), Crohn disease (16), and multiple CD279 (PD-1,29F.1A12), anti-FoxP3 (FJK-16s), anti-IFN-g sclerosis (17, 18), and have a well-established safety profile. (XMG1.2), anti-IL-2 (JES6.5H4), anti-IL-4 (11B11), IL-17A However, CD4 depletion removes CD4 effector cells, which are (eBio1787). CellTrace 5-(and 6-)carboxyfluorescein diacetate required for initiation of an immune response. Therefore, CD4 succinimidyl ester (CFSE) Cell Proliferation Kit was purchased depletion was timed to occur after immune priming has taken from Invitrogen. CD4 (GK1.5) and CD8 (2.43) antibodies for T place. In murine models for RCC and melanoma, mTOR cells depletion were purchased from BioXcell. Temsirolimus inhibition and CD4 depletion produced a robust cellular was purchased from LC Laboratory. immune response that was transferable and effective in con- trolling subcutaneous tumors as well as lung metastases. The T-cell enrichment and Treg sorting combination treatment produced highly effective memory Mouse spleen and lymph nodes were collected and processed lymphocytes with robust recall responses. The stimulation of into single-cell suspensions. CD8 and CD4 T cells were nega- immunological memory because of CD4 depletion was attrib- tively enriched using mouse CD8 or CD4 recovery column kits uted to Treg depletion based on experiments using transgenic (Cedarlane Labs). Purity of CD8 and CD4 cells after negative fi selection was greater than 85%. FoxP3-GFP cells or antibody models to speci cally deplete Tregs ("Treg knock-out") or þ þ replace tumor-specific Tregs ("Treg knock-in") following CD4 stained CD4 CD25 cells were sorted by MoFlo Cell Sorter. depletion. Another mechanism contributing to the antitumor Preparation of dendritic cells and T-cell stimulation response was that Tregs that returned after CD4 depletion were Dendritic cell (DC) preparation has been described (2). To less immunosuppressive than Tregs from mice without CD4 prepare DC vaccine for treatment of mice, DCs were pulsed manipulation. with tumor cell lysate and activated with 10 mg/mL CpG. DCs Materials and Methods were subcutaneously injected into mouse. For in vitro activa- tion of Pmel-1 cells, DC was pulsed with 10 ng/mL mouse gp100 Mice and tumor cells peptide (amino acids 25–33, which is presented by H2-Db class Female C57BL/6J, Balb/c mice and Pmel-1 mice, 6- to 8-week I molecules; Alpha Diagnostic International) and activated old, were purchased from Jackson Laboratory and housed with 10 mg/mL CpG for 2 hours. DC was washed with PBS, under pathogen-free conditions. FoxP3-GFP mice were a gen- and cocultured with CFSE-labeled Pmel-1 cells. Pmel-1 cells erous gift from Dr. V. Kuchroo (Harvard University, Boston, proliferation was analyzed by FACscan. MA). DEREG [depletion of regulatory T cells; Tg(Foxp3-DTR/ EGFP)23.2Spar] transgenic mice was generated and described Adoptive transfer, CD4 cells depletion, and mTOR by T. Sparwasser (19). All experiments involving animals were inhibition in compliance with federal and state standards, which include Pmel-1 lymphocytes were isolated from lymph nodes and the federal Animal Welfare Act and the NIH guide for the care spleen of na€ve Pmel-1 mice. CD8 lymphocytes were enriched and use of laboratory animals. by negative selection using Cedarlane purification column. At þ Human gp100-transduced B 16 cells (B16-gp100) were kindly least 85% of the resulting cells were CD8 . A total of 5 Â 105 provided by Dr. A. Rakhmilevich from University of Wisconsin- cells were transferred into B57BL/6 mice. The day after adop- Madison. RENCA, a murine RCC line, was a gift from Dr. A. tive transfer, mice received tumor lysate–pulsed DC vaccine. Belldegrum (University of California, Los Angeles, CA). All cells To deplete CD4 cells, aCD4 was administered approximately were periodically authenticated by morphologic and histologic 7 and 9 days later; mice were inject intraperitoneally with inspection, and animal grafting for assessing their ability to 250 mg of CD4 mAb (clone GK1.5). To deplete CD8 cells, mice grow and metastasize. Cells were annually tested for myco- received 250 mg of CD8 mAb (clone 2.43). To deplete FoxP3 cells plasma using Myco Alert Kit (Lonza). The cells were main- in DEREG mice, 5 mg DT was injected. Flow cytometry was used tained in Dulbecco's modified Eagle medium or RPMI 1640 to confirm depletion of target cells. For mTor inhibitor treat- medium supplemented with 10% heat-inactivated FBS (Life ment, 15 mg temsirolimus was injected intraperitoneally each Technologies), 2 mmol/L of L-glutamine, 100 units/mL of day for 2 weeks. Flow cytometry was used to analyze memory penicillin, and 100 mg/mL of streptomycin.