Increased EZH2 function in regulatory T cells promotes their capacity to suppress autoimmunity by driving effector differentiation prior to activation.

The immunosuppressive function of regulatory T (Treg) cells is essential for maintaining immune homeostasis. Enhancer of zeste homolog 2 (EZH2), a histone H3 lysine 27 (H3K27) methyltransferase, plays a key role in maintaining Treg cell function upon CD28 co-stimulation, and Ezh2 deletion in Treg cells causes autoimmunity. Here we assessed whether increased EZH2 activity in Treg cells would improve Treg cell function. Using an Ezh2 gain-of-function mutation, Ezh2 Y641F , we found that Treg cells expressing Ezh2 Y641F displayed an increased effector Treg phenotype and were poised for improved homing to organ tissues. Expression of Ezh2 Y641F in Treg cells led to more rapid remission from autoimmunity. H3K27me3 profiling and transcriptomic analysis revealed a redistribution of H3K27me3, which prompted a gene expression profile in naïve Ezh2 Y641F Treg cells that recapitulated aspects of CD28-activated Ezh2 WT Treg cells. Altogether, increased EZH2 activity promotes the differentiation of effector Treg cells that can better suppress autoimmunity. Highlights: EZH2 function promotes effector differentiation of Treg cells.EZH2 function promotes Treg cell migration to organ tissues.EZH2 function in Treg cells improves remission from autoimmunity.EZH2 function poises naïve Treg cells to adopt a CD28-activated phenotype.

A mass cytometry approach to track the evolution of T cell responses during infection and immunotherapy by paired T cell receptor repertoire and T cell differentiation state analysis.

T cell receptor (TCR) recognition followed by clonal expansion is a fundamental feature of adaptive immune responses. Here, we developed a mass cytometric (CyTOF) approach combining antibodies specific for different TCR Vα- and Vß-chains with antibodies against T cell activation and differentiation proteins to identify antigen-specific expansions of T cell subsets and assess aspects of cellular function. This strategy allowed for the identification of expansions of specific Vß and Vα chain expressing CD8+ and CD4+ T cells with varying differentiation states in response to Listeria monocytogenes, tumors, and respiratory influenza infection. Expanded Vß chain expressing T cells could be directly linked to the recognition of specific antigens from Listeria, tumor cells, or influenza. In the setting of influenza infection, we showed that the common therapeutic approaches of intramuscular vaccination or convalescent serum transfer altered the clonal diversity and differentiation state of responding T cells. Thus, we present a new method to monitor broad changes in TCR specificity paired with T cell differentiation during adaptive immune responses.

Separating the Good from the Bad: Tumor-Infiltrating Tregs Have Increased Fucosylation.

Regulatory T cells (Treg) can suppress antitumor immune responses, and their presence in tumors is associated with worse prognoses in most cancers. Strategies to neutralize Treg-mediated suppression in tumors without immune-related adverse events, however, are challenging due to the essential role of Tregs in maintaining immune homeostasis. In this issue, Pinioti and colleagues identify fucosylation as a critical regulator of Treg function in tumors that can be targeted therapeutically without impacting immune homeostasis. See related article by Pinioti et al., p. 1611 (3) .

CXCR3 expression in regulatory T cells drives interactions with type I dendritic cells in tumors to restrict CD8+ T cell antitumor immunity.

Infiltration of regulatory T (Treg) cells, an immunosuppressive population of CD4+ T cells, into solid cancers represents a barrier to cancer immunotherapy. Chemokine receptors are critical for Treg cell recruitment and cell-cell interactions in inflamed tissues, including cancer, and thus are an ideal therapeutic target. Here, we show in multiple cancer models that CXCR3+ Treg cells were increased in tumors compared with lymphoid tissues, exhibited an activated phenotype, and interacted preferentially with CXCL9-producing BATF3+ dendritic cells (DCs). Genetic ablation of CXCR3 in Treg cells disrupted DC1-Treg cell interactions and concomitantly increased DC-CD8+ T cell interactions. Mechanistically, CXCR3 ablation in Treg cells increased tumor antigen-specific cross-presentation by DC1s, increasing CD8+ T cell priming and reactivation in tumors. This ultimately impaired tumor progression, especially in combination with anti-PD-1 checkpoint blockade immunotherapy. Overall, CXCR3 is shown to be a critical chemokine receptor for Treg cell accumulation and immune suppression in tumors.

BET-bromodomain and EZH2 inhibitor-treated chronic GVHD mice have blunted germinal centers with distinct transcriptomes.

Despite advances in the field, chronic graft-versus-host-disease (cGVHD) remains a leading cause of morbidity and mortality following allogenic hematopoietic stem cell transplant. Because treatment options remain limited, we tested efficacy of anticancer, chromatin-modifying enzyme inhibitors in a clinically relevant murine model of cGVHD with bronchiolitis obliterans (BO). We observed that the novel enhancer of zeste homolog 2 (EZH2) inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 each improved pulmonary function; impaired the germinal center (GC) reaction, a prerequisite in cGVHD/BO pathogenesis; and JQ5 reduced EZH2-mediated H3K27me3 in donor T cells. Using conditional EZH2 knockout donor cells, we demonstrated that EZH2 is obligatory for the initiation of cGVHD/BO. In a sclerodermatous cGVHD model, JQ5 reduced the severity of cutaneous lesions. To determine how the 2 drugs could lead to the same physiological improvements while targeting unique epigenetic processes, we analyzed the transcriptomes of splenic GCB cells (GCBs) from transplanted mice treated with either drug. Multiple inflammatory and signaling pathways enriched in cGVHD/BO GCBs were reduced by each drug. GCBs from JQ5- but not JQ1-treated mice were enriched for proproliferative pathways also seen in GCBs from bone marrow-only transplanted mice, likely reflecting their underlying biology in the unperturbed state. In conjunction with in vivo data, these insights led us to conclude that epigenetic targeting of the GC is a viable clinical approach for the treatment of cGVHD, and that the EZH2 inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 demonstrated clinical potential for EZH2i and BETi in patients with cGVHD/BO.

MHC-II neoantigens shape tumour immunity and response to immunotherapy.

The ability of the immune system to eliminate and shape the immunogenicity of tumours defines the process of cancer immunoediting1. Immunotherapies such as those that target immune checkpoint molecules can be used to augment immune-mediated elimination of tumours and have resulted in durable responses in patients with cancer that did not respond to previous treatments. However, only a subset of patients benefit from immunotherapy and more knowledge about what is required for successful treatment is needed2-4. Although the role of tumour neoantigen-specific CD8+ T cells in tumour rejection is well established5-9, the roles of other subsets of T cells have received less attention. Here we show that spontaneous and immunotherapy-induced anti-tumour responses require the activity of both tumour-antigen-specific CD8+ and CD4+ T cells, even in tumours that do not express major histocompatibility complex (MHC) class II molecules. In addition, the expression of MHC class II-restricted antigens by tumour cells is required at the site of successful rejection, indicating that activation of CD4+ T cells must also occur in the tumour microenvironment. These findings suggest that MHC class II-restricted neoantigens have a key function in the anti-tumour response that is nonoverlapping with that of MHC class I-restricted neoantigens and therefore needs to be considered when identifying patients who will most benefit from immunotherapy.

Enhanced adaptive immune responses in lung adenocarcinoma through natural killer cell stimulation.

Natural killer (NK) cells inhibit tumor development in mouse models and their presence in tumors correlates with patient survival. However, tumor-associated NK cells become dysfunctional; thus, stimulation of NK cells in cancer is emerging as an attractive immunotherapeutic strategy. In a mouse model of lung adenocarcinoma, NK cells localized to tumor stroma with immature phenotypes and low functional capacity. To test their responsiveness within established disease, we engineered a system for inducible expression of activating ligands in tumors. After stimulation, NK cells localized inside tumors, with increased cytokine production capacity. Strikingly, T cells were also recruited to tumors in an NK cell-dependent manner, and exhibited higher functionality. In neoantigen-expressing tumors, NK cell stimulation enhanced the number and function of tumor-specific T cells and, in long-term settings, reduced tumor growth. Thus, even in established disease NK cells can be activated to contribute to antitumor immunity, supporting their potential as an important target in cancer immunotherapy.

Treg programming and therapeutic reprogramming in cancer.

Overcoming the immunosuppressive tumour microenvironment is the major challenge impeding cancer immunotherapy today. Regulatory T-cells (Tregs) are prevalent in nearly all cancers and, as immunosuppressive regulators of immune responses, they are the principal opponents of cancer immunotherapy. However, disabling Tregs systemically causes severe autoimmune toxicity, hastening the need for more selective methods to target intratumoural Tregs. In this review, we discuss a burgeoning new modality to specifically target tumour-infiltrating Tregs (TI-Tregs) by reprogramming their functionality from immunosuppressive to immune stimulatory within tumours. As the basis for therapeutic selectivity of TI-Tregs, we will focus on the defining features of Tregs within cancer: their highly activated state controlled by the engagement of key surface receptors, their distinct metabolic programme, and their unique transcriptional programme. By identifying proteins and pathways that distinguish TI-Tregs from other Tregs in the body, as well as from the beneficial antitumour effector T-cells within tumours, we highlight mechanisms to selectively reprogramme TI-Tregs for the treatment of cancer.

Clonal Deletion of Tumor-Specific T Cells by Interferon-γ Confers Therapeutic Resistance to Combination Immune Checkpoint Blockade.

Resistance to checkpoint-blockade treatments is a challenge in the clinic. We found that although treatment with combined anti-CTLA-4 and anti-PD-1 improved control of established tumors, this combination compromised anti-tumor immunity in the low tumor burden (LTB) state in pre-clinical models as well as in melanoma patients. Activated tumor-specific T cells expressed higher amounts of interferon-γ (IFN-γ) receptor and were more susceptible to apoptosis than naive T cells. Combination treatment induced deletion of tumor-specific T cells and altered the T cell repertoire landscape, skewing the distribution of T cells toward lower-frequency clonotypes. Additionally, combination therapy induced higher IFN-γ production in the LTB state than in the high tumor burden (HTB) state on a per-cell basis, reflecting a less exhausted immune status in the LTB state. Thus, elevated IFN-γ secretion in the LTB state contributes to the development of an immune-intrinsic mechanism of resistance to combination checkpoint blockade, highlighting the importance of achieving the optimal magnitude of immune stimulation for successful combination immunotherapy strategies.

Targeting EZH2 Reprograms Intratumoral Regulatory T Cells to Enhance Cancer Immunity.

Regulatory 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.

Harnessing the plasticity of CD4(+) T cells to treat immune-mediated disease.

CD4(+) T cells differentiate and acquire distinct functions to combat specific pathogens but can also adapt their functions in response to changing circumstances. Although this phenotypic plasticity can be potentially deleterious, driving immune pathology, it also provides important benefits that have led to its evolutionary preservation. Here, we review CD4(+) T cell plasticity by examining the molecular mechanisms that regulate it - from the extracellular cues that initiate and drive cells towards varying phenotypes, to the cytosolic signalling cascades that decipher these cues and transmit them into the cell and to the nucleus, where these signals imprint specific gene expression programmes. By understanding how this functional flexibility is achieved, we may open doors to new therapeutic approaches that harness this property of T cells.

Regulatory T Cells in Tumor-Associated Tertiary Lymphoid Structures Suppress Anti-tumor T Cell Responses.

Infiltration of regulatory T (Treg) cells into many tumor types correlates with poor patient prognoses. However, mechanisms of intratumoral Treg cell function remain to be elucidated. We investigated Treg cell function in a genetically engineered mouse model of lung adenocarcinoma and found that Treg cells suppressed anti-tumor responses in tumor-associated tertiary lymphoid structures (TA-TLSs). TA-TLSs have been described in human lung cancers, but their function remains to be determined. TLSs in this model were spatially associated with >90% of tumors and facilitated interactions between T cells and tumor-antigen-presenting dendritic cells (DCs). Costimulatory ligand expression by DCs and T cell proliferation rates increased in TA-TLSs upon Treg cell depletion, leading to tumor destruction. Thus, we propose that Treg cells in TA-TLSs can inhibit endogenous immune responses against tumors, and targeting these cells might provide therapeutic benefit for cancer patients.

The chromatin-modifying enzyme Ezh2 is critical for the maintenance of regulatory T cell identity after activation.

Regulatory T cells (Treg cells) are required for immune homeostasis. Chromatin remodeling is essential for establishing diverse cellular identities, but how the epigenetic program in Treg cells is maintained throughout the dynamic activation process remains unclear. Here we have shown that CD28 co-stimulation, an extracellular cue intrinsically required for Treg cell maintenance, induced the chromatin-modifying enzyme, Ezh2. Treg-specific ablation of Ezh2 resulted in spontaneous autoimmunity with reduced Foxp3(+) cells in non-lymphoid tissues and impaired resolution of experimental autoimmune encephalomyelitis. Utilizing a model designed to selectively deplete wild-type Treg cells in adult mice co-populated with Ezh2-deficient Treg cells, Ezh2-deficient cells were destabilized and failed to prevent autoimmunity. After activation, the transcriptome of Ezh2-deficient Treg cells was disrupted, with altered expression of Treg cell lineage genes in a pattern similar to Foxp3-deficient Treg cells. These studies reveal a critical role for Ezh2 in the maintenance of Treg cell identity during cellular activation.

Control of PI(3) kinase in Treg cells maintains homeostasis and lineage stability.

Foxp3(+) regulatory T cells (Treg cells) are required for immunological homeostasis. One notable distinction between conventional T cells (Tconv cells) and Treg cells is differences in the activity of phosphatidylinositol-3-OH kinase (PI(3)K); only Tconv cells downregulate PTEN, the main negative regulator of PI(3)K, upon activation. Here we found that control of PI(3)K in Treg cells was essential for lineage homeostasis and stability. Mice lacking Pten in Treg cells developed an autoimmune-lymphoproliferative disease characterized by excessive T helper type 1 (TH1) responses and B cell activation. Diminished control of PI(3)K activity in Treg cells led to reduced expression of the interleukin-2 (IL-2) receptor α subunit CD25, accumulation of Foxp3(+)CD25(-) cells and, ultimately, loss of expression of the transcription factor Foxp3 in these cells. Collectively, our data demonstrate that control of PI(3)K signaling by PTEN in Treg cells is critical for maintaining their homeostasis, function and stability.

Genetically engineered mouse models of cancer reveal new insights about the antitumor immune response.

Cancer is a complex disease that can originate in virtually all the tissues of the body, and tumors progress through many different stages during their development. While genetic mutations in the emerging cancer cells drive this disease, it has become increasingly clear that cancer development is strongly influenced by the surrounding microenvironment. Cells of the immune system are critical components of this extrinsic network of cancer regulators, contributing significantly to the microenvironment of most cancers and either promoting or inhibiting the initiation and progression of this disease. Genetically engineered mouse (GEM) mouse models of spontaneous cancer are starting to shape our understanding of how antitumor T cells may act to prevent or inhibit cancer progression in some settings and not others. Lessons learned from investigating spontaneous mouse cancer models have important implications for directing clinical efforts that attempt to direct a cancer patient's immune system to eradicate their disease.

Expression of tumour-specific antigens underlies cancer immunoediting.

Cancer immunoediting is a process by which immune cells, particularly lymphocytes of the adaptive immune system, protect the host from the development of cancer and alter tumour progression by driving the outgrowth of tumour cells with decreased sensitivity to immune attack. Carcinogen-induced mouse models of cancer have shown that primary tumour susceptibility is thereby enhanced in immune-compromised mice, whereas the capacity for such tumours to grow after transplantation into wild-type mice is reduced. However, many questions about the process of cancer immunoediting remain unanswered, in part because of the known antigenic complexity and heterogeneity of carcinogen-induced tumours. Here we adapted a genetically engineered, autochthonous mouse model of sarcomagenesis to investigate the process of cancer immunoediting. This system allows us to monitor the onset and growth of immunogenic and non-immunogenic tumours induced in situ that harbour identical genetic and histopathological characteristics. By comparing the development of such tumours in immune-competent mice with their development in mice with broad immunodeficiency or specific antigenic tolerance, we show that recognition of tumour-specific antigens by lymphocytes is critical for immunoediting against sarcomas. Furthermore, primary sarcomas were edited to become less immunogenic through the selective outgrowth of cells that were able to escape T lymphocyte attack. Loss of tumour antigen expression or presentation on major histocompatibility complex I was necessary and sufficient for this immunoediting process to occur. These results highlight the importance of tumour-specific-antigen expression in immune surveillance, and potentially, immunotherapy.

Suppression of lung adenocarcinoma progression by Nkx2-1.

Despite the high prevalence and poor outcome of patients with metastatic lung cancer the mechanisms of tumour progression and metastasis remain largely uncharacterized. Here we modelled human lung adenocarcinoma, which frequently harbours activating point mutations in KRAS and inactivation of the p53 pathway, using conditional alleles in mice. Lentiviral-mediated somatic activation of oncogenic Kras and deletion of p53 in the lung epithelial cells of Kras(LSL-G12D/+);p53(flox/flox) mice initiates lung adenocarcinoma development. Although tumours are initiated synchronously by defined genetic alterations, only a subset becomes malignant, indicating that disease progression requires additional alterations. Identification of the lentiviral integration sites allowed us to distinguish metastatic from non-metastatic tumours and determine the gene expression alterations that distinguish these tumour types. Cross-species analysis identified the NK2-related homeobox transcription factor Nkx2-1 (also called Ttf-1 or Titf1) as a candidate suppressor of malignant progression. In this mouse model, Nkx2-1 negativity is pathognomonic of high-grade poorly differentiated tumours. Gain- and loss-of-function experiments in cells derived from metastatic and non-metastatic tumours demonstrated that Nkx2-1 controls tumour differentiation and limits metastatic potential in vivo. Interrogation of Nkx2-1-regulated genes, analysis of tumours at defined developmental stages, and functional complementation experiments indicate that Nkx2-1 constrains tumours in part by repressing the embryonically restricted chromatin regulator Hmga2. Whereas focal amplification of NKX2-1 in a fraction of human lung adenocarcinomas has focused attention on its oncogenic function, our data specifically link Nkx2-1 downregulation to loss of differentiation, enhanced tumour seeding ability and increased metastatic proclivity. Thus, the oncogenic and suppressive functions of Nkx2-1 in the same tumour type substantiate its role as a dual function lineage factor.

Endogenous T cell responses to antigens expressed in lung adenocarcinomas delay malignant tumor progression.

Neoantigens derived from somatic mutations in tumors may provide a critical link between the adaptive immune system and cancer. Here, we describe a system to introduce exogenous antigens into genetically engineered mouse lung cancers to mimic tumor neoantigens. We show that endogenous T cells respond to and infiltrate tumors, significantly delaying malignant progression. Despite continued antigen expression, T cell infiltration does not persist and tumors ultimately escape immune attack. Transplantation of cell lines derived from these lung tumors or prophylactic vaccination against the autochthonous tumors, however, results in rapid tumor eradication or selection of tumors that lose antigen expression. These results provide insight into the dynamic nature of the immune response to naturally arising tumors.

Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase.

The development of animal models of lung cancer is critical to our understanding and treatment of the human disease. Conditional mouse models provide new opportunities for testing novel chemopreventatives, therapeutics and screening methods that are not possible with cultured cell lines or xenograft models. This protocol describes how to initiate tumors in two conditional genetic models of human non-small cell lung cancer (NSCLC) using the activation of oncogenic K-ras alone or in combination with the loss of function of p53. We discuss methods for sporadic expression of Cre in the lungs through engineered adenovirus or lentivirus, and provide a detailed protocol for the administration of the virus by intranasal inhalation or intratracheal intubation. The protocol requires 1-5 min per mouse with an additional 30-45 min to set-up and allow for the recovery of mice from anesthesia. Mice may be analyzed for tumor formation and progression starting 2-3 weeks after infection.