Summary Responses to anti-PD-1 immunotherapy occur but are infrequent in bladder cancer. The specific T cells that mediate tumor rejection are unknown. T cells from human bladder tumors and non-malignant tissue were assessed with single-cell RNA and paired T cell receptor (TCR) sequencing of 30,604 T cells from 7 patients. We find that the states and repertoires of CD8 + T cells are not distinct in tumors compared with non-malignant tissues. In contrast, single-cell analysis of CD4 + T cells demonstrates several tumor-specific states, including multiple distinct states of regulatory T cells. Surprisingly, we also find multiple cytotoxic CD4 + T cell states that are clonally expanded. These CD4 + T cells can kill autologous tumors in an MHC class II-dependent fashion and are suppressed by regulatory T cells. Further, a gene signature of cytotoxic CD4 + T cells in tumors predicts a clinical response in 244 metastatic bladder cancer patients treated with anti-PD-L1.

SUMMARYRegulatory T cells (Tregs) are critical for maintaining immune homeostasis, but their presence in tumor tissues impairs anti-tumor immunity and portends poor prognoses in cancer patients. Here, we reveal a mechanism to selectively target and reprogram the function of tumor-infiltrating Tregs (TI-Tregs) by exploiting their dependency on the histone H3K27 methyltransferase enhancer of zeste homolog 2 (EZH2) in tumors. Disruption of EZH2 activity in Tregs, either pharmacologically or genetically, drove the acquisition of pro-inflammatory functions in TI-Tregs, remodeling the tumor microenvironment and enhancing the recruitment and function of CD8+ and CD4+ effector T cells that eliminate tumors. Moreover, abolishing EZH2 function in Tregs was mechanistically distinct from, more potent than, and less toxic than a generalized Treg depletion approach. This study reveals a strategy to target Tregs in cancer that mitigates autoimmunity by reprogramming their function in tumors to enhance anti-cancer immunity.In BriefEZH2 plays an intrinsic role in neoplastic cells as an oncogene, prompting the development of EZH2 inhibitors for cancer therapy. Wang et al. show that disrupting EZH2 function also has immunomodulatory activities and, when blocked in Tregs, promotes potent cancer immunity.

Hematopoietic stem cell transplantation, which was first successfully performed in the 1950s, remains a critical therapeutic modality for treatment of a diverse array of diseases, including a multitude of hematological malignancies, autoimmune disorders, amyloidosis and inherited genetic hematological disorders. Although great advances have been made in understanding and application of this therapy, significant complications still exist, warranting further investigation. Of critical importance, graft‐versus‐host disease (GVHD), in both acute and chronic forms, remains a major complication of hematopoietic stem cell transplantation, responsible for both the development of chronic illness and morbidity, as well as mortality. Use of an appropriate preclinical model may provide significant insight into the mechanistic pathways leading to the development and progression of graft‐versus‐host disease, as well as cancer in general. However, existing preclinical modeling systems exhibit significant limitations, and development of models that recapitulate the complex and comprehensive clinical scenario and provide a tool by which therapeutic intervention may be developed and assessed is of utmost importance. Here, we review the present status of the field of graft‐versus‐host disease research. We discuss and summarize the preclinical models currently in use, as well as their advantages and limitations.

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Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a potential curative option for treating a variety of hematologic diseases, but acute and chronic graft-versus-host disease (GVHD) remain major barriers limiting efficacy. Acute gut GVHD occurs with marked increases in pro-inflammatory cytokines (including TNF and IL-6), which we recently demonstrated was exacerbated in obesity resulting in severe gastrointestinal pathology. Given the pleiotropic and overlapping effects of these two cytokines, we assessed the impact of dual TNF and IL-6R blockade on GVHD as well as graft-versus tumor (GVT) effects in different mouse GVHD models. Early administration of combined blockade resulted in greater protection and survival from acute gut GVHD compared to single blockade regimens and even development of later chronic skin GVHD. Importantly, double cytokine blockade preserved GVT effects reinforcing that GVT and GVHD can be delineated and may result in greater efficacy in allo-HSCT.

The use of immunotherapy (IT) in cancer has recently resulted in impressive responses. Yet, their usages, especially those involving cytokine therapies, have also resulted in the induction of severe systemic toxicities often not previously characterized in their preclinical animal studies. Our laboratory has demonstrated that treatment of EL4 lymphoma with a combination immunotherapy consisting of monoclonal agonistic antibody CD40 and IL-2 resulted in significant anti-tumor responses leading to overall increases in survival using young inbred mice (2-6 months of age). Yet, the majority of hematological cancers occur in aged individuals, with most diagnoses occurring past the age of 60. We now demonstrate that systemic immunotherapy administration in aged (>16 months of age), but not young, mice resulted in the induction of a rapid cytokine release syndrome, also known as “cytokine storm”, culminating in multi-organ damage (liver, lung, and gut) leading to rapid lethality by day 2 of treatment. Aging is accompanied by an overall redistribution of body mass, with a decrease in lean muscle mass and increase in adiposity. Similarly, we found that normal aging in inbred mice housed under specific pathogen free conditions was accompanied by increases in visceral fat that was similar to young obese (ob/ob or diet-induced obese [DIO]) mice. We therefore assessed the impact of aging and obesity on inflammatory responses to cancer immunotherapeutics. We determined the effects of increased body fat on systemic immunotherapy tolerance in aged mice compared to young obese mice. Both young ob/ob and DIO generated pro-inflammatory cytokine (ie TNFa and IL6) levels and organ pathologies comparable to aged WT mice following immunotherapy, culminating in rapid lethality after several days of therapy. We observed that young obese mice exhibited greater ratios of M1/M2 macrophages within the peritoneal and visceral adipose tissues and higher percentages of TNFa+ macrophages in comparison to young lean mice with immunotherapy. Administration of aCD40/IL-2 with macrophage depletion or TNF-blockade prevented the development of cytokine storms within young obese mice, providing protection from lethality, suggesting that the toxicity was macrophage mediated through increases in TNFa. Calorie-restricted aged mice contain less visceral fat and upon aCD40/IL-2 administration displayed reduced cytokine levels, protection from organ pathology, and protection from lethality. Our data demonstrates adiposity as a critical factor in the age-associated inflammatory pathologic responses to systemic anti-cancer immunotherapy and may have a significant impact when immunotherapy is used clinically within cancer therapy regimens. These data also underscore the critical importance of taking aging and body fat into consideration with preclinical assessments. Disclosures No relevant conflicts of interest to declare.

High numbers of tumor infiltrating regulatory T (Treg) cells are indicative of poor outcome in several malignancies, including ovarian cancer, colorectal cancer, and melanoma. Thus, selectively abrogating intratumoral Treg cell function while maintaining systemic immune tolerance remains an attractive, albeit elusive, strategy for cancer immunotherapy. We have identified the epigenetic enzyme Ezh2, an H3K27 methyltransferase, to be a critical mediator of lineage stability and function in activated Treg cells. Consequently, deficiency of Ezh2 in Treg cells strongly impairs their function in non-lymphoid tissues. We hypothesized that this phenomenon might translate to a similar defect that is restricted to the tumor microenvironment. Here we tested this hypothesis in three transplantable syngeneic tumor models in mice (MC38 colon carcinoma, TRAMP-C2 prostate cancer, and B16F10 melanoma). We observed that constitutive deletion of Ezh2 in Treg cells resulted in potent anti-tumor activity, significantly impaired tumor outgrowth, and in many instances, complete tumor rejection. These results were also recapitulated when Ezh2 was temporally deleted in the vast majority of Treg cells at the time of tumor inoculation. Functional analyses at early time points after Ezh2 deletion revealed enhanced functionality of effector CD4 and CD8 populations, as evidenced by increased IFN-γ production. These effects were evident without reduction of intratumoral Treg cell frequencies and despite presence of residual wild type Treg cells. Mechanistic studies showed that Ezh2-deficient Treg cells were destabilized and produced pro-inflammatory cytokines IL-2 and IFN-γ selectively within the tumor microenvironment, suggesting a direct contribution of Ezh2-deficient Treg cells to tumor clearance. To more directly address this hypothesis, we generated mice that harbored both Ezh2-deficient and wild type Treg cells and observed anti-tumor activity similar to mice that exclusively harbored Ezh2-deficient Treg cells. In stark contrast to Ezh2 deficiency, mice whose Treg cells were completely depleted, using a Foxp3-driven diphtheria toxin receptor allele, were unable to reject tumors, further supporting a dominant role of Ezh2-deficient Treg cells in driving anti-tumor immunity. Finally, tumor protection in the presence of Ezh2-deficient Treg cells occurred without significant morbidity due to autoimmune pathologies, which were prevalent in mice with systemic Treg cell depletion. These results suggest that targeted deletion of Ezh2 in Treg cells drives a unique phenotype among tumor infiltrating Treg cells, reprogramming the tumor microenvironment and selectively augmenting the anti-tumor immune response. Cancer therapies that pharmacologically inhibit Ezh2 to target tumor cells are under investigation, going forward it will also be important to assess the potential for beneficial effects via modulating the immune response. Citation Format: David Q. Wang, Jason R. Quiros, Chien-Chun S. Pai, Lawrence H. Fong, Jeffrey A. Bluestone, Michel J. DuPage. Selective impairment of intratumoral regulatory T cells by targeting Ezh2 enhances cancer immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1014. doi:10.1158/1538-7445.AM2017-1014

The obesity rate is increasing in the United States with more than a third of U.S. adults being obese. Obesity is a form of sterile chronic inflammation, termed “meta-inflammation”, which is local, chronic and triggered by metabolical pathways. Therefore, there is a need to uncover the potential role of obesity in the development of immune cell subsets post-HSCT. In this study, we wanted to assess the impact of obesity on immune reconstitution following congenic HSCT in lean and DIO (diet-induced obese) recipients. C57BL/6 mice received lethal radiation dose at 1100cGy and 5 million T cell-depleted bone marrow cells from H2-matched B6.SJL mice. Our data demonstrated that DIO recipients displayed a more rapid and robust reconstitution affecting both myeloid and lymphoid compartments. DIO recipients had increased neutrophils, platelets and total lymphocytes post-HSCT compared to control mice. Colony forming unit-granulocyte macrophage (CFU-GM) assays showed significantly higher numbers in DIO recipients at day 14 post-HSCT. Organ cellularity from thymus, spleen and bone marrow were increased in DIO recipients compared to lean mice at day 14 and 21 post-HSCT. Higher numbers of CD4+CD8+ thymocytes, as well as higher number of NK cells and B cells in the spleen and lymph nodes were also observed. Splenocytes from DIO recipients had greater proliferative ability either after stimulation with concanavalin A or in mixed lymphocyte reaction compared to lean controls. Taken together; these results indicated that obesity results in acceleration of engraftment and donor immune reconstitution.