Up-Regulation of PD-L1, IDO, and T regs in the Melanoma Tumor Microenvironment Is Driven by CD8 + T Cells

Background: The expression of phosphatidylserine (PS) on cell surfaces drives immunosuppressive mechanisms associated with tolerogenic cell death. In the tumor microenvironment, PS is exposed on tumor cells and tumor vascular endothelial cells treated with conventional therapies. PS signals through multiple immune cell signaling receptors where it drives the expansion of myeloid-derived suppressor cells (MDSCs), regulatory T cells, M2 macrophages, and stimulates their production of immunosuppressive cytokines (e.g., TGFβ and IL-10), rendering the tumor microenvironment non-responsive to immune activation. PS targeting antibodies have significant anti-tumor effects in multiple tumor xenografts, syngeneic tumors, and genetically engineered preclinical tumor models through mechanisms that re-program the tumor microenvironment from immunosuppressive to immune potentiating. Methods: The combination of PS-targeting and anti-PD1 antibodies was compared to single agent therapy in multiple pre-clinical mouse models, including syngeneic melanoma and breast tumors, and a genetically engineered mouse model (GEMM) of pancreatic cancer. Mice were treated weekly IP at 5 mg/kg with a PS targeting antibody, anti-PD1, or the combination for the duration of each experiment; tumor and spleen tissue microenvironments were immune profiled by FACS, ELISPOT, and immunohistochemistry. Results: In all tumor models examined, the anti-tumor effect of combination therapy was significantly superior to single agent therapy. In B16 and K1735 melanoma tumors, the combination more than doubled the anti-tumor effect of anti-PD-1 alone. Combination therapy significantly inhibited tumor growth by over 90% in E077.1 breast tumors and prolonged animal survival by 30% (n>25 each group) in the GEMM of pancreatic cancer despite the fact that anti-PD1 therapy alone was ineffective in each of these models. Analysis of immune cell subsets in the tumor microenvironment indicated that the combination of antibody-mediated blockade of PS and PD-1 significantly enhanced the effector function of tumor infiltrating CD8+ T cells and increased the ratio of T effector to T regulatory cells, as demonstrated by significant increases in TILs producing IFNγ, TNFα, IL-2, granzyme B, and Ki-67. Combination therapy re-conditioned the tumor microenvironment to favor immune potentiation, as demonstrated by significant decreases in the frequency of MDSC, the ratio of M2 to M1 macrophages, the expression of surface PD-L1, and reductions in TGFβ and IL-10. Furthermore, combination treatment induced systemic tumor specific CD8 T cell immunity, as mice demonstrating complete responses (in the K1735 and E077.1 models) rejected tumor cells upon re-challenge. Splenocytes from these mice had significantly higher numbers of tumor-specific IFNγ-producing cells in ELISPOT assay. Studies were completed without toxicity in any setting. Conclusions: Antibody-mediated PS blockade inhibits PS-mediated immune suppression and stimulates FcRγ activation. This results in immune activation that enables an otherwise non-responsive tumor microenvironment to respond to checkpoint inhibition. This abstract is also presented as Poster B08. Citation Format: Xianming Huang, Jian Gong, Michael Gray, Van Nguyen, Ryan Parks, Chris Hughes, Jeff Hutchins, Rolf Brekken, Bruce Freimark. Antibody-mediated blockade of phosphatidylserine synergizes with immune checkpoint blockade by inhibiting multiple immune suppressive mechanisms. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR09.

Elevated Metabolite Secretion Facilitates a Treg-Mediated Immunosuppressive Microenvironment in the Solid Tumor

Simple SummaryThe different components surrounding a tumor are collectively known as the tumor microenvironment (TME). The transforming growth factor-beta (TGF-β) signaling pathway is activated in the TME and the tumor, leading to alteration in the composition of the TME that favors tumor growth and aggressiveness. A major component of the TME, called Cancer-Associated Fibroblasts (CAFs) help the tumor grow and escape destruction by the host immune system. TGF-β signaling and CAF-associated alterations in the TME may also predict the response to cancer immunotherapy. Whether these changes in the TME are targetable alone or in combination with TGF-β inhibition is now being tested in the clinic.Various components of the tumor microenvironment (TME) play a critical role in promoting tumorigenesis, progression, and metastasis. One of the primary functions of the TME is to stimulate an immunosuppressive environment around the tumor through multiple mechanisms including the activation of the transforming growth factor-beta (TGF-β) signaling pathway. Cancer-associated fibroblasts (CAFs) are key cells in the TME that regulate the secretion of extracellular matrix (ECM) components under the influence of TGF-β. Recent reports from our group and others have described an ECM-related and CAF-associated novel gene signature that can predict resistance to immune checkpoint blockade (ICB). Importantly, studies have begun to test whether targeting some of these CAF-associated components can be used as a combinatorial approach with ICB. This perspective summarizes recent advances in our understanding of CAF and TGF-β-regulated immunosuppressive mechanisms and ways to target such signaling in cancer.

Colorectal cancer (CRC) progression depends on the close interaction of tumour cells and the tumour microenvironment (TME). Although the TME contributes to poor therapy responses and immune evasion, immune cells within the TME can be therapeutically leveraged, as exemplified by immune checkpoint blockade (ICB). Unfortunately, only a small subset of patients with CRC benefit from ICB therapy; those with immune-activated, microsatellite unstable CRC respond, whereas the predominant group of patients with CRC, those with microsatellite-stable tumours, do not. Although challenging, modulating the TME of CRC to convert these lowly immunogenic and immunosuppressed tumours into immune-activated tumours holds tremendous therapeutic potential. In this Review we provide an overview of the cellular and molecular components of immunity in the TME of CRCs at various stages of disease as well as the mechanisms of immunosuppression and immune evasion. We further describe how systemic and local therapies for CRC impact the tumour and systemic immune microenvironments, and how immunity could serve as a therapeutic and prognostic biomarker. Lastly, we highlight novel immunotherapeutic strategies and approaches that modulate the TME of CRCs to make them amenable to immunotherapy.

The tumor microenvironment is composed of multiple cell types, including malignant cancer cells and tumor-infiltrating leukocytes (TIL). A possible mechanism of immunosuppression in the tumor microenvironment (TME) is cancer cells outcompeting anti-cancer TIL for nutrients such as glucose. 18F-deoxyglucose (FDG) positron emission tomography (PET) imaging is a staple of diagnosing and monitoring many types of cancer and is based on the Warburg model in which cancer cells within the tumor utilize glucose for growth and proliferation. Here, we use magnetic bead sorting to fractionate FDG-avid murine tumors and measure tumor cell-specific glucose uptake. We find that CD45+ immune cells are more FDG-avid than CD45− cancer cells. We further fractionate immune cell subsets into CD4/8+ T cells and CD11b+ myeloid cells to demonstrate that TIL T cells take up more glucose than CD45− cancer cells and resting splenic T cells. Strikingly, CD11b+ myeloid cells are the most FDG-avid cells in the TME. In MC38 colorectal cancer tumors, we show that CD11b+ F4/80 hi macrophages have high glucose uptake, and by extracellular flux analysis demonstrate higher metabolic activity than tumor T cells and CD45− cancer cells. Intriguingly, while glucose uptake is low in CD45− cancer cells, 18F-glutamine uptake is higher in cancer cells than immune cells. Our results illustrate a novel approach to measuring nutrient uptake in the TME and suggest that TIL are not starved of nutrients in the TME. Future work will determine the effects of immunotherapy and metabolism-targeted therapeutics on cell-specific glucose and glutamine uptake in the TME, as well as determine the immune contribution to cancer PET scans in the context of immunotherapy response.

BackgroundChimeric antigen receptor (CAR)-based T cell therapy and other forms of adoptive cell therapies (ACTs) have shown remarkable success in the treatment of hematologic malignancies; however, reports of clinical activity...

The tumor microenvironment (TME) is a multifaceted and ever-changing assemblage of cells and extracellular constituents. These components are closely linked to the onset and progression of malignancies, as well as their treatment. The TME is characterized by aberrant vasculature, altered extracellular matrix, immune cells, secreted factors, and cancer-associated fibroblasts and macrophages. The importance of the tumor microenvironment (TME) in understanding the course of cancer and resistance to treatment has been highlighted. The TME can suppress immune responses and promote tumor survival by inducing immunosuppressive mechanisms, such as regulatory T cells, myeloid-derived suppressor cells (MDSCs), and checkpoint molecule expression (e.g., PD-L1). Recent research has focused on understanding the interactions between immune cells within the TME to develop strategies that can remodel this environment and increase the effectiveness of immunotherapy. However, the efficacy of immunotherapeutic strategies is frequently hindered by the immunosuppressive nature of the TME. This abstract explores how these dynamics have led to the development of novel immunotherapeutic strategies aimed at reprogramming the TME to enhance antitumor immune responses. Novel approaches targeting TME therapy, such as immune checkpoint blockade (ICB), metabolic inhibitors, and key enzymes of immune metabolism, have been used to treat of cancer immunotherapy. Additionally, new and promising treatments including CAR-T cell therapy, oncolytic viruses, and cytokine-mediated TME modulation have shown promising results. This review provides a general overview of the TME, its components, its impact on immunotherapy outcomes, and emerging approaches to enhance therapeutic efficacy by remodeling the TME.

The tumor microenvironment (TME) is a significant contributor to cancer progression containing complex connections between cellular and chemical components and provides a suitable substrate for tumor growth and development. Growing evidence shows targeting tumor cells while ignoring the surrounding TME is not effective enough to overcome the cancer disease. Fibroblasts are essential sentinels of the stroma that due to certain conditions in TME, such as oxidative stress and local hypoxia, become activated, and play the prominent role in the physical support of tumor cells and the enhancement of tumorigenesis. Activated fibroblasts in TME, defined as cancer-associated fibroblasts (CAFs), play a crucial role in regulating the biological behavior of tumors, such as tumor metastasis and drug resistance. CAFs are highly heterogeneous populations that have different origins and, in addition to their role in supporting stromal cells, have multiple immunosuppressive functions via a membrane and secretory patterns. The secretion of different cytokines/chemokines, interactions that mediate the recruitment of regulatory immune cells and the reprogramming of an immunosuppressive function in immature myeloid cells are just a few examples of how CAFs contribute to the immune escape of tumors through various direct and indirect mechanisms on specific immune cell populations. Moreover, CAFs directly abolish the role of cytotoxic lymphocytes. The activation and overexpression of inhibitory immune checkpoints (iICPs) or their ligands in TME compartments are one of the main regulatory mechanisms that inactivate tumor-infiltrating lymphocytes in cancer lesions. CAFs are also essential players in the induction or expression of iICPs and the suppression of immune response in TME. Based on available studies, CAF subsets could modulate immune cell function in TME through iICPs in two ways; direct expression of iICPs by activated CAFs and indirect induction by production soluble and then upregulation of iICPs in TME. With a focus on CAFs’ direct and indirect roles in the induction of iICPs in TME as well as their use in immunotherapy and diagnostics, we present the evolving understanding of the immunosuppressive mechanism of CAFs in TME in this review. Understanding the complete picture of CAFs will help develop new strategies to improve precision cancer therapy.

The treatment of cancer has been revolutionized by the development of immune-based therapies designed to boost the number and function of cytotoxic T cells that kill tumor cells. However, it is already apparent that this strategy alone will not benefit all patients, as the majority of cancers generate a highly immunosuppressive tumor microenvironment (TME) that shuts down even the most potent T cells. Furthermore, as a consequence of ramping up the immune system, these immune stimulating drugs instigate significant autoimmune toxicity. We aim to develop a radical new strategy to circumvent both challenges by identifying immunosuppressive regulatory mechanisms unique to cancerous tissues, thereby uncovering pathways active only in tumors that can be targeted to generate precision cancer immunotherapies that preserve immune tolerance in healthy tissues. The focus will be on Regulatory T cells (Tregs), a subset of immunosuppressive CD4+T cells that lie at the fulcrum of immunity, directing cells of the immune system to go or stop. In cancer, Tregsinfiltrate tumors and dampen anti-tumor immune responses, but Tregs also play an essential role in preventing autoimmunity. This proposal will test the hypothesis that human cancers generate an immunosuppressive TME by enforcing a unique epigenetic program in intratumoral Tregsthat, if identified, could provide new targets to selectively modulate tumor-infiltrating Tregsand limit autoimmunity. To do this, the research will employ genetically engineered mouse (GEM) cancers, an accurate and flexible platform to test the full complement of factors that make up the TME of human cancers, combined with sophisticated genetic tools to track Treg entry and activity in the TME. Our investigations thus far have led us to the investigation of Ezh2 activity, a H3K27 methyltransferase, in defining Treg behavior selectively in the TME. Citation Format: Michel DuPage. Engineering precision cancer immunotherapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr IA012.

Background: In classical Hodgkin lymphoma (cHL), the rare malignant Hodgkin Reed-Sternberg (HRS) cells are surrounded by an inflammatory infiltrate. Yet, the host anti-tumor immune response is ineffective. HRS cells have multifaceted mechanisms to evade the immune system including 9p24.1/PD-L1/PD-L2 genetic alterations leading to overexpression of PD-1 ligands and subsequent T cell exhaustion, aberrant antigen presentation and modulation of the tumor microenvironment (TME). The clinical success of PD-1 blockade in cHL suggests the TME contains reversibly exhausted T-effectors (Teff). Paradoxically, durable responses are observed in patients with β2M/MHC class I loss on HRS cells, raising the possibility of non-CD8+ mediated mechanisms of efficacy of PD-1 blockade. For this reason, we sought to characterize HRS cells and the surrounding TME. Methods: Using CyTOF technology, we evaluated 7 primary cHL suspensions and 10 reactive lymphoid tissue (RLT) samples at the single-cell protein level. We designed a custom panel of 39 isotope-conjugated antibodies. A combination of surface and intracellular markers distinguish T cell subsets according to lineage, differentiation, polarization, activation and exhaustion. Additional markers were incorporated to identify B cells, NK cells and macrophages. HRS cells were defined by CD15/CD30/Pax5 positivity. Inclusion of β2M and MHC class I allowed assessment of antigen presentation on HRS cells. The data was acquired on a Helios CyTOF and analyzed using a fast k-weighted nearest neighbor algorithm, X-shift. X-shift clustered cells with phenotypic similarities together. Then, samples were separated into cHL and RLT and the contribution of a sample to a given cluster was quantified. Results: Comparison of viable cell suspensions from RLT and cHL revealed loss of naïve T-cells and skewing towards differentiation of Teff in both CD4+ and CD8+ subsets in cHLs. This prompted a second X-shift analysis focused on CD3+ cells, which highlighted salient differences between cHL and RLT within the CD4+ subset. In cHL, we found expansion of Teff and regulatory T cells (Treg) with a reduction of follicular helper T cells. Furthermore, both Treg and Teff populations were largely Th1 (T-bet+/CCR5+) polarized. Evaluation of PD-1 expression showed Tregs had little/no PD-1 while Teff had intermediate/high expression. Hence, Tregs retain functionality in contrast to Teff, which are exhausted, providing two mechanisms of immunosuppression. Manual gating identified HRS cells with a characteristic phenotype: CD15, CD30, Pax5, rosetted by CD4+ T cells. Importantly, we found loss or decrease of β2M and MHC class I in 5/7 cases. Conclusions: The TME in cHL is CD4+ T cell rich with frequent loss of MHC class I on HRS cells. Differential PD-1 expression results in functional CD4+ Tregs and exhausted Teff, a synergistic bases for the observed immunosuppression in cHL. Citation Format: Fathima Z. Cader, Ron C. Schackmann, Xihao Hu, Kirsty Wienand, Robert A. Redd, Bjoern Chapuy, Jing Ouyang, Nicole E. Paul, Evisa Gjini, Mikel Lipschitz, Laura M. Selfors, Philippe Armand, David Wu, Jonathan R. Fromm, Donna Neuberg, Xiaole S. Liu, Scott J. Rodig, Margaret A. Shipp. Single-cell mass cytometry of classical Hodgkin lymphoma defines an exhausted and immunosuppressive microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5675.

Redundant tumor microenvironment (TME) immunosuppressive mechanisms and epigenetic maintenance of terminal T cell exhaustion greatly hinder functional antitumor immune responses in chronic lymphocytic leukemia (CLL). Bromodomain and extraterminal (BET) proteins regulate key pathways contributing to CLL pathogenesis and TME interactions, including T cell function and differentiation. Herein, we report that blocking BET protein function alleviates immunosuppressive networks in the CLL TME and repairs inherent CLL T cell defects. The pan-BET inhibitor OPN-51107 reduced exhaustion-associated cell signatures resulting in improved T cell proliferation and effector function in the Eμ-TCL1 splenic TME. Following BET inhibition (BET-i), TME T cells coexpressed significantly fewer inhibitory receptors (IRs) (e.g., PD-1, CD160, CD244, LAG3, VISTA). Complementary results were witnessed in primary CLL cultures, wherein OPN-51107 exerted proinflammatory effects on T cells, regardless of leukemic cell burden. BET-i additionally promotes a progenitor T cell phenotype through reduced expression of transcription factors that maintain terminal differentiation and increased expression of TCF-1, at least in part through altered chromatin accessibility. Moreover, direct T cell effects of BET-i were unmatched by common targeted therapies in CLL. This study demonstrates the immunomodulatory action of BET-i on CLL T cells and supports the inclusion of BET inhibitors in the management of CLL to alleviate terminal T cell dysfunction and potentially enhance tumoricidal T cell activity.

Cancer immunotherapy has revolutionized cancer treatment, and it relies heavily on the comprehensive understanding of the immune landscape of the tumor microenvironment (TME). Here, we obtain a detailed immune cell atlas of esophageal squamous cell carcinoma (ESCC) at single-cell resolution. Exhausted T and NK cells, regulatory T cells (Tregs), alternatively activated macrophages and tolerogenic dendritic cells are dominant in the TME. Transcriptional profiling coupled with T cell receptor (TCR) sequencing reveal lineage connections in T cell populations. CD8 T cells show continuous progression from pre-exhausted to exhausted T cells. While exhausted CD4, CD8 T and NK cells are major proliferative cell components in the TME, the crosstalk between macrophages and Tregs contributes to potential immunosuppression in the TME. Our results indicate several immunosuppressive mechanisms that may be simultaneously responsible for the failure of immuno-surveillance. Specific targeting of these immunosuppressive pathways may reactivate anti-tumor immune responses in ESCC.

Plasticity is a hallmark of aggressive tumors, including glioblastoma (GBM), enabling tumor cells and the tumor microenvironment (TME) to adapt to diverse niches and evade treatment. Here, we discuss how innate and adaptive immune players cooperate in time and space to create an immunosuppressive TME that supports GBM growth and confers resistance to conventional treatments and immunotherapies. We highlight how therapeutic interventions reshape the TME, underscoring the need for targeted approaches to overcome resistance. We introduce the concepts of local TME priming and TME rewiring as necessary foundations for achieving more effective and durable clinical responses in the future.

This review provides an in-depth examination of the tumor microenvironment, macrophages, and PD-1 checkpoint inhibitors. It delves into the fundamental aspects of the tumor microenvironment and macrophages, discussing the evolution of key concepts, the involvement of macrophages in tumor progression, and their interactions with tumor cells. The historical development of PD-1 checkpoint inhibitors is explored, detailing their discovery, early clinical trials, and initial challenges. Current insights into macrophages within the tumor microenvironment are presented, including their polarization, mechanisms of immunosuppression, and diagnostic techniques. The clinical application of PD-1 inhibitors is reviewed, encompassing therapeutic strategies, comparative efficacy across various cancers, and management of side effects. Additionally, the paper addresses technological advancements in tumor microenvironment research, future directions, and ongoing controversies, aiming to provide a comprehensive overview to inform further research and clinical applications.

T regulatory cells (Tregs) play different roles in the regulation of anti-tumor immunity in colorectal cancer (CRC), depending on the presence of different Treg subsets. We investigated correlations between different CD4+ Treg/T cell subsets in CRC patients with immune checkpoint-expressing CD4+ T cells. Positive correlations were observed between levels of different immune checkpoint-expressing CD4+ T cells, including PD-1, TIM-3, LAG-3, and CTLA-4 with FoxP3+ Tregs, Helios+ T cells, FoxP3+Helios+ Tregs, and FoxP3+Helios− Tregs in the tumor microenvironment (TME). However, negative correlations were observed between levels of these immune checkpoint-expressing CD4+ T with FoxP3−Helios− T cells in the TME. These correlations in the TME highlight the role of cancer cells in the upregulation of IC-expressing Tregs. Additionally, positive correlations were observed between levels of FoxP3+ Tregs, Helios+ T cells, FoxP3+Helios+ Tregs, and FoxP3+Helios− Tregs and levels of CD4+CTLA-4+ T cells and CD4+PD-1+ T cells in peripheral blood mononuclear cells (PBMCs) and normal tissue-infiltrating lymphocytes (NILs). These observations suggest that CTLA-4 and PD-1 expressions on CD4+ T cell subsets are not induced only by the TME. This is the first study to investigate the correlations of different FoxP3+/−Helios+/− T cell subsets with immune checkpoint-expressing CD4+ T cells in CRC patients. Our data demonstrated strong correlations between FoxP3+/Helios+/− Tregs but not FoxP3−Helios+/− non-Tregs and multiple immune checkpoints, especially in the TME, providing a rationale for targeting these cells with highly immunosuppressive characteristics. Understanding the correlations between different immune checkpoints and Treg/T cell subsets in cancer patients could improve our knowledge of the underlying mechanisms of Treg-mediated immunosuppression in cancer.