Checkpoint Blockade Immunotherapy Relies on T-betbutNotEomes to InduceEffectorFunction in Tumor-In fi ltrating CD 8 þ T Cells

Coinhibitory receptor blockade is a promising strategy to boost T-cell immunity against a variety of human cancers. However, many patients still do not benefit from this treatment, and responders often experience immune-related toxicities. These issues highlight the need for advanced mechanistic understanding to improve patient outcomes and uncover clinically relevant biomarkers of treatment efficacy. However, the T-cell–intrinsic signaling pathways engaged during checkpoint blockade treatment are not well defined, particularly for combination approaches. Using a murine model to study how effector CD8þ T-cell responses to tumors may be enhanced in a tolerizing environment, we identified a critical role for the T-box transcription factor T-bet. Combination blockade of CTLA-4, PD-1, and LAG-3 induced T-bet expression in responding tumor/self-reactive CD8þ T cells. Eradication of established leukemia using this immunotherapy regimen depended on T-bet induction, which was required for IFNg production and cytotoxicity by tumor-infiltrating T cells, and for efficient trafficking to disseminated tumor sites. These data provide new insight into the success of checkpoint blockade for cancer immunotherapy, revealing T-bet as a key transcriptional regulator of tumor-reactive CD8þ T-cell effector differentiation under otherwise tolerizing conditions. Cancer Immunol Res; 3(2); 116–24. 2014 AACR. Introduction CD8þ T cells are important for tumor surveillance and play a major role in controlling tumor growth (1, 2). However, mechanisms of immune evasion undermine T-cell activity and promote disease (3). The quality of CD8þ T-cell responses to cancer is often dictated by environmental cues received during priming and after encounter with tumor cells. These signals determine the amplitude of the CD8þ T-cell response, and can be either stimulatory or inhibitory in nature. Although costimulatory signals induce T-cell differentiation and cytokine production associated with effective immunity, coinhibitory signals subvert costimulation to limit CD8þ T-cell function (4–6). These coinhibitory pathways are essential to maintain tolerance toward self-tissues and prevent autoimmunity, but also impede immune responses against cancer (7). Studies in animal models have demonstrated that attenuation of such tolerizing signals using blocking antibodies (i.e., checkpoint blockade) canboost antitumor immunity (8–10), and these strategies have been translated into clinical successes in human patients with cancer (11, 12). Although checkpoint blockade is reshaping the landscape of cancer immunotherapy, little is known about the CD8þ T-cell–intrinsic pathways required to achieve therapeutic benefits. Additional unknowns surround the cellular mechanisms that lead to adverse immune-related toxicities in patients treated with checkpoint blockade antibodies, which may intriguingly coincide with therapeutic efficacy (13). The T-box transcription factors Eomesodermin (Eomes) and T-bet have important and well-described roles in CD8þ T-cell activation, differentiation, and memory formation (14–17). These T-box–mediated operations are undeniably valuable for the generation of antitumor immunity, which has been directly demonstrated in mouse models of cancer (18). More importantly, the induction of T-bet in tumor-infiltrating lymphocytes (TIL) has been correlated with positive outcomes in patients with colorectal cancer (19). However, there is only sparse evidence that coinhibitory receptor pathways influence T-bet and Eomes expression in T cells (20, 21), leaving the exact relationship between these transcriptional regulators and checkpoint blockade immunotherapy largely undefined. We recently demonstrated that combination antibody blockade of CTLA-4, PD-1, and LAG-3 improved IFNg production and cytotoxicity by transferred tumor/self-reactive CD8þT cells during cancer immunotherapy (8). These effector responses hint at a possible link to T-box transcription factor–mediated CD8þ T-cell differentiation. We now report that low expression of T-bet and Eomes defines dysfunctional T cells rendered tolerant in vivo by encounterwith tumor/self-antigen. Therapeutic interventionwith combination checkpoint blockade (i.e., anti–CTLA-4, PD-1, and LAG-3) induced both T-bet andEomes expression in responding T cells under these same tolerizing conditions, but only T-bet was Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, Missouri. Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri. Saint Louis University Cancer Center, St. Louis, Missouri. Note: Supplementary data for this article are available at Cancer Immunology Research Online (http://cancerimmunolres.aacrjournals.org/). CorrespondingAuthor:RyanM. Teague, Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1100 South Grand Boulevard, Doisy Research Center Room 705, St. Louis, MO 63104. Phone: 314-977-8871; Fax: 314-977-8717; E-mail: rteague@slu.edu doi: 10.1158/2326-6066.CIR-14-0159 2014 American Association for Cancer Research. Cancer Immunology Research Cancer Immunol Res; 3(2) February 2015 116 on June 19, 2017. © 2015 American Association for Cancer Research. cancerimmunolres.aacrjournals. org Downloaded from Published OnlineFirst December 16, 2014; DOI: 10.1158/2326-6066.CIR-14-0159