RNA interference targeting programmed death receptor-1 improves immune functions of tumor-specific T cells

Adoptive cell transfer (ACT), either using rapidly expanded tumor infiltrating lymphocytes or T-cell receptor transduced peripheral blood lymphocytes, can be considered one of the most promising approaches in cancer immunotherapy. ACT results in the repopulation of the host with high frequencies of tumor-specific T cells; however, optimal function of these cells within the tumor micro-environment is required to reach long-term tumor clearance. We and others have shown that ongoing anti-tumor immune responses can be impaired by the expression of ligands, such as PD-L1 (B7-H1) on tumor cells. Such inhibitory molecules can affect T cells at the effector phase via their receptor PD-1. PD-L1/PD-1 interaction has indeed been shown crucial in inducing T-cell anergy and maintaining peripheral tolerance. In order to maximize anti-tumor responses, antibodies that target the PD-1/PD-L1 axis are currently in phase I/II trials. Alternatively, a more refined approach could be the selective targeting of PD-1 in tumor-specific T cells to obtain long-term resistance against PD-1-mediated inhibition. We addressed whether this goal could be achieved by means of retroviral siRNA delivery. Effective siRNA sequences resulting in the reduction of surface PD-1 expression led to improved murine as well as human T-cell immune functions in response to PD-L1 expressing melanoma cells. These data suggest that blockade of PD-1-mediated T-cell inhibition through siRNA forms a promising approach to achieve long-lasting enhancement of tumor-specific T-cell function in adoptive T-cell therapy protocols.     

Blockade of the B7-H1/PD-1 pathway for cancer immunotherapy

The aim of cancer immunotherapy is to treat malignant disease by inducing or enhancing cancer specific immune responses. With the identification of tumor-associated antigens (TAAs) in the 1990s, cancer immunotherapy research largely focused on inducing immune responses against TAAs but achieved limited success. More recently, the underlying mechanisms and molecular pathways that cancers manipulate to subvert immune-mediated destruction have been identified, including a set of molecules with potent coinhibitory functions. Coinhibitory molecules are expressed on the surface of immune cells, cancer cells, and stromal cells and negatively regulate immune responses to cancer. In particular, one of these ligand-receptor coinhibitory interactions, B7-H1/PD-1, is critical for modulating immune responses to cancer. This knowledge led to the design of revolutionary new immunotherapeutics based on the manipulation of these molecular pathways. Monoclonal antibodies (mAbs) are the primary immunotherapeutic modality used to promote immune function via antagonism or agonism of inhibitory or stimulatory molecular pathways, respectively. Here, we review current knowledge on the function of the B7-H1/PD-1 pathway in mice and humans, its role in the subversion of immune responses in cancer, and clinical evidence that mAb targeting of this pathway results in profound immune anti-cancer effects.     

PD-1/PD-L1与Treg细胞相关性的研究新进展

PD-1和PD-L属于B7家族的共刺激分子,介导免疫反应的负性调节信号。Treg细胞是一个具有免疫调节作用的T细胞亚群,在机体的免疫耐受和免疫稳定中具有重要作用。本文就PD-1/PD-L1与Treg细胞的免疫调节作用及相关性研究进展作简要综述。     

Resistance to Checkpoint Inhibition in Cancer Immunotherapy

The interaction of the host immune system with tumor cells in the tissue microenvironment is essential in understanding tumor immunity and development of successful cancer immunotherapy. The presence of lymphocytes in tumors is highly correlated with an improved outcome. T cells have a set of cell surface receptors termed immune checkpoints that when activated suppress T cell function. Upregulation of immune checkpoint receptors such as programmed cell death 1 (PD-1) and cytotoxic T lymphocyte associated protein 4 (CTLA-4) occurs during T cell activation in an effort to prevent damage from an excessive immune response. Immune checkpoint inhibitors allow the adaptive immune system to respond to tumors more effectively. There has been clinical success in different types of cancer blocking immune checkpoint receptors such as PD-1 and CTLA. However, relapse has occurred. The innate and acquired/therapy induced resistance to treatment has been encountered. Aberrant cellular signal transduction is a major contributing factor to resistance to immunotherapy. Combination therapies with other co-inhibitory immune checkpoints such as TIM-3, LAG3 and VISTA are currently being tested to overcome resistance to cancer immunotherapy. Expression of TIM-3 has been associated with resistance to PD-1 blockade and combined blockade of TIM-3 and PD-1 has demonstrated improved responses in preclinical models. LAG3 blockade has the potential to increase the responsiveness of cytotoxic T-cells to tumors. Furthermore, tumors that were found to express VISTA had an increased rate of growth due to the T cell suppression. The growing understanding of the inhibitory immune checkpoints’ ligand biology, signaling mechanisms, and T-cell suppression in the tumor microenvironment continues to fuel preclinical and clinical advancements in design, testing, and approval of agents that block checkpoint molecules. Our review seeks to bridge fundamental regulatory mechanisms across inhibitory immune checkpoint receptors that are of great importance in resistance to cancer immunotherapy. We will summarize the biology of different checkpoint molecules, highlight the effect of individual checkpoint inhibition as anti-tumor therapies, and outline the literatures that explore mechanisms of resistance to individual checkpoint inhibition pathways.     

PD-1/PD-L1-dependent immune response in colorectal cancer

Colorectal cancer (CRC) is still considered as the third most frequent cancer in the world. Microsatellite instability (MSI), inflammation, and microRNAs have been demonstrated as the main contributing factors in CRC. Subtype 1 CRC is defined by NK cells infiltration, induction of Th1 lymphocyte and cytotoxic T cell responses as well as upregulation of immune checkpoint proteins including programmed cell death-1 (PD-1). Based on the diverse features of CRC, such as the stage and localization of the tumor, several treatment approaches are available. However, the efficiency of these treatments may be decreased due to the development of diverse resistance mechanisms. It has been proven that monoclonal antibodies (mAbs) can increase the effectiveness of CRC treatments. Nowadays, several mAbs including nivolumab and pembrolizumab have been approved for the treatment of CRC. Immune checkpoint receptors including PD-1 can be inhibited by these antibodies. Combination therapy gives an opportunity for advanced treatment for CRC patients. In this review, an update has been provided on the molecular mechanisms involved in MSI colorectal cancer immune microenvironment by focusing on PD-ligand 1 (PD-L1) and treatment of patients with advanced immunotherapy, which were examined in the different clinical trial phases. Considering induced expression of PD-L1 by conventional chemotherapeutics, we have summarized the role of PD-L1 in CRC, the chemotherapy effects on the PD-1/PD-L1 axis and novel combined approaches to enhance immunotherapy of CRC by focusing on PD-L1.     

Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion

B7-H1, a recently described member of the B7 family of costimulatory molecules, is thought to be involved in the regulation of cellular and humoral immune responses through the PD-1 receptor on activated T and B cells. We report here that, except for cells of the macrophage lineage, normal human tissues do not express B7-H1. In contrast, B7-H1 is abundant in human carcinomas of lung, ovary and colon and in melanomas. The pro-inflammatory cytokine interferon-gamma upregulates B7-H1 on the surface of tumor cell lines. Cancer cell-associated B7-H1 increases apoptosis of antigen-specific human T-cell clones in vitro, and the apoptotic effect of B7-H1 is mediated largely by one or more receptors other than PD-1. In addition, expression of B7-H1 on mouse P815 tumor increases apoptosis of activated tumor-reactive T cells and promotes the growth of highly immunogenic B7-1(+) tumors in vivo. These findings have implications for the design of T cell-based cancer immunotherapy.     

Is it true that gut microbiota is considered as panacea in cancer therapy?

Recent studies demonstrated that a combination of the gut microbiome has the vital effect on the efficacy of anticancer immune therapies. Regulatory effects of microbiota have been shown in different types of cancer therapies such as chemotherapy and immunotherapy. Immune-checkpoint-blocked therapies are the recent efficient cancer immunotherapy strategies. The target of immune-checkpoint blocking is cytotoxic T lymphocyte protein-4 (CTLA-4) or blockade of programmed death-1 (PD-1) protein and its ligand programmed death ligand 1 (PD-L1) that they have been considered as cancer immunotherapy in recent years. In the latest studies, it have been demonstrated that several gut bacteria such as Akkermansia muciniphila, Bifidobacterium spp., Faecalibacterium spp., and Bacteroides fragilis have the regulatory effects on PD-1, PD-L1, and CTLA-4 blocked anticancer therapy outcome.     

免疫检查点程序性细胞死亡蛋白配体-1翻译后修饰及其在免疫治疗中的应用前景

免疫检查点程序性细胞死亡蛋白配体-1（programmed cell death 1 ligand 1,PD-L1）是一种主要表达于肿瘤细胞表面的免疫抑制性分子,其可与T淋巴细胞表面的程序性细胞死亡蛋白-1（programmed cell death protein 1,PD-1）结合,抑制T淋巴细胞的激活,发挥免疫抑制性功能。基于这一原理所开发的PD-1/PD-L1免疫阻断疗法,已在临床广泛应用于多种实体瘤的治疗,使诸多病人受益。与此同时,随着对PD-L1调控机制研究的深入,PD-L1的多种翻译后修饰形式陆续得到了鉴定,包括糖基化、磷酸化、泛素化和棕榈酰化等。研究表明,这些翻译后修饰过程可影响PD-L1的蛋白质稳定性与生理功能。因此,翻译后修饰成了PD-L1研究新的切入点。目前,PD-L1翻译后修饰靶向药物已在免疫治疗中展现出良好的应用前景。通过靶向PD-L1翻译后修饰过程,进而调控由PD-L1介导的肿瘤免疫逃逸,成了提高免疫治疗应答率的新思路和新策略。本文将对PD-L1翻译后修饰的研究进行系统总结,并陈述其在免疫治疗领域中的应用前景,希望为未来针对PD-L1翻译后修饰的研究提供理论支持。     

Constitutive and acquired mechanisms of resistance to immune checkpoint blockade in human cancer

Cancer immunotherapy with monoclonal antibodies directed against regulatory pathways in T lymphocytes has been revolutionizing medical oncology, and the clinical success of monoclonal antibodies targeting either cytotoxic T lymphocyte antigen-4 (CTLA-4) or program death-1 (PD-1) in patients affected by melanoma, Hodgkin’s lymphoma, Merkel cell carcinoma, and head and neck, bladder, renal cell or non-small cell lung cancer is way beyond the most optimistic expectation. However, immune checkpoint blockade (ICB) has failed to arrest progression in a consistent amount of patients affected by those tumors, and various histological types, including breast, colon and prostate cancer, are less sensitive to this therapeutic approach. Such clinical findings have fueled massive research efforts in the attempt to identify pre-existing and acquired mechanisms of resistance to ICB. Here we focus on evidences emerging from studies in humans on how tumor cells and the tumor microenvironment contribute to the heterogeneous clinical responses, and we propose strategies stemming from pre-clinical models that might improve clinical outcomes for patients.     

Macrophage polarity in cancer: A review

Macrophages are the most abundant cells within the tumor stroma displaying noticeable plasticity, which allows them to perform several functions within the tumor microenvironment. Tumor-associated macrophages commonly refer to an alternative M2 phenotype, exhibiting anti-inflammatory and pro-tumoral effects. M2 cells are highly versatile and multi-tasking cells that directly influence multiple steps in tumor development, including cancer cell survival, proliferation, stemness, and invasiveness along with angiogenesis and immunosuppression. M2 cells perform these functions through critical interactions with cells related to tumor progression, including Th2 cells, cancer-associated fibroblasts, cancer cells, regulatory T cells (Tregs), and myeloid-derived suppressor cells. M2 cells also have negative cross-talks with tumor suppressor cells, including cytotoxic T cells and natural killer cells. Programed death-1 (PD-1) is one of the key receptors expressed in M2 cells that, upon interaction with its ligand PD-L1, plays cardinal roles for induction of immune evasion in cancer cells. In addition, M2 cells can neutralize the effects of the pro-inflammatory and anti-tumor M1 phenotype. Classically activated M1 cells express high levels of major histocompatibility complex molecules, and the cells are strong killers of cancer cells. Therefore, orchestrating M2 reprogramming toward an M1 phenotype would offer a promising approach for reversing the fate of tumor and promoting cancer regression. Macrophage switching toward an anti-inflammatory M1 phenotype could be used as an adjuvant with other approaches, including radiotherapy and immune checkpoint blockades, such as anti-PD-L1/PD-1 strategies.     

Cytokines as potential combination agents with PD-1/PD-L1 blockade for cancer treatment

Immunotherapy has caused a paradigm shift in the treatment of several malignancies, particularly the blockade of programmed death-1 (PD-1) and its specific receptor/ligand PD-L1 that have revolutionized the treatment of a variety of malignancies, but significant durable responses only occur in a small percentage of patients, and other patients failed to respond to the treatment. Even those who initially respond can ultimately relapse despite maintenance treatment, there is considerable potential for synergistic combinations of immunotherapy and chemotherapy agents with immune checkpoint inhibitors into conventional cancer treatments. The clinical experience in the use of cytokines in the clinical setting indicated the efficiency of cytokine therapy in cancer immunotherapy. Combinational approaches to enhancing PD-L1/PD-1 pathways blockade efficacy with several cytokines such as interleukin (IL)-2, IL-15, IL-21, IL-12, IL-10, and interferon-α (IFN-α) may result in additional benefits. In this review, the current state of knowledge about PD-1/PD-L1 inhibitors, the date in the literature to ascertain the combination of anti-PD-1/PD-L1 antibodies with cytokines is discussed. Finally, it is noteworthy that novel therapeutic approaches based on the efficient combination of recombinant cytokines with the PD-L1/PD-1 blockade therapy can enhance antitumor immune responses against various malignancies.     

Blocking of the PD-1/PD-L1 interaction by a novel cyclic peptide inhibitor for cancer immunotherapy

The interaction of PD-1/PD-L1 allows tumor cells to escape from immune surveillance. Clinical success of the antibody drugs has proven that blockade of PD-1/PD-L1 pathway is a promising strategy for cancer immunotherapy. Here, we developed a cyclic peptide C8 by using Ph.D.-C7 C phage display technology. C8 showed high binding affinity with h PD-1 and could effectively interfere the interaction of PD-1/PD-L1. Furthermore, C8 could stimulate CD8+T cell activation in human peripheral blood mononuclear cells(PBMCs). We also observed that C8 could suppress tumor growth in CT26 and B16-OVA, as well as anti-PD-1 antibody resistant B16 mouse model. CD8+T cells infiltration significantly increased in tumor microenvironment, and IFN-γ secretion by CD8+T cells in draining lymph nodes also increased. Simultaneously, we exploited T cells depletion models and confirmed that C8 exerted anti-tumor effects via activating CD8+T cells dependent manner. The interaction model of C8 with h PD-1 was simulated and confirmed by alanine scanning. In conclusion, C8 shows anti-tumor capability by blockade of PD-1/PD-L1 interaction, and C8 may provide an alternative candidate for cancer immunotherapy.     

Deubiquitinating enzyme OTUB1 promotes cancer cell immunosuppression via preventing ER-associated degradation of immune checkpoint protein PD-L1

Upregulation of programmed death ligand 1 (PD-L1) helps tumor cells escape from immune surveillance, and therapeutic antibodies targeting PD-1/PD-L1 have shown better patient outcomes only in several types of malignancies. Recent studies suggest that the clinical efficacy of anti-PD-1/PD-L1 treatments is associated with PD-L1 levels; however, the underlying mechanism of high PD-L1 protein levels in cancers is not well defined. Here, we report that the deubiquitinase OTUB1 positively regulates PD-L1 stability and mediates cancer immune responses through the PD-1/PD-L1 axis. Mechanistically, we demonstrate that OTUB1 interacts with and removes K48-linked ubiquitin chains from the PD-L1 intracellular domain in a manner dependent on its deubiquitinase activity to hinder the degradation of PD-L1 through the ERAD pathway. Functionally, depletion of OTUB1 markedly decreases PD-L1 abundance, reduces PD-1 protein binding to the tumor cell surface, and causes increased tumor cell sensitivity to human peripheral blood mononuclear cells (PBMCs)-mediated cytotoxicity. Meanwhile, OTUB1 ablation-induced PD-L1 destabilization facilitates more CD8⁺ T cells infiltration and increases the level of IFN-γ in serum to enhance antitumor immunity in mice, and the tumor growth suppression by OTUB1 silencing could be reversed by PD-L1 overexpression. Furthermore, we observe a significant correlation between PD-L1 abundance and OTUB1 expression in human breast carcinoma. Our study reveals OTUB1 as a deubiquitinating enzyme that influences cancer immunosuppression via regulation of PD-L1 stability and may be a potential therapeutic target for cancer immunotherapy.Subject terms: Proteins, Immune evasion     

Differential binding properties of B7-H1 and B7-DC to programmed death-1

Programmed death-1 (PD-1) is a negative regulatory receptor expressed on activated T and B cells. Two ligands for PD-1, B7-H1 (PD-L1) and B7-DC (PD-L2), have been identified, but their binding properties have not been characterized yet. In this study, we generated soluble Ig fusion proteins of these molecules and examined the kinetics and relative affinities of the interactions between B7-H1 or B7-DC and PD-1 by flow cytometry and surface plasmon resonance. The interaction of B7-DC/PD-1 exhibited a 2-6-fold higher affinity and had different association/dissociation kinetics compared with the interaction of B7-H1/PD-1. Our results suggest that the differential binding properties of B7-H1 and B7-DC may be responsible for differential contributions of these two PD-1 ligands to immune responses.     

CD8+ cytotoxic T lymphocytes in cancer immunotherapy: A review

CD8⁺ cytotoxic T lymphocytes (CTLs) are preferred immune cells for targeting cancer. During cancer progression, CTLs encounter dysfunction and exhaustion due to immunerelated tolerance and immunosuppression within the tumor microenvironment (TME), with all favor adaptive immune-resistance. Cancer-associated fibroblasts (CAFs), macrophage type 2 (M2) cells, and regulatory T cells (Tregs) could make immunologic barriers against CD8 ⁺ T cell-mediated antitumor immune responses. Thus, CD8 ⁺ T cells are needed to be primed and activated toward effector CTLs in a process called tumor immunity cycle for making durable and efficient antitumor immune responses. The CD8 ⁺ T cell priming is directed essentially as a corroboration work between cells of innate immunity including dendritic cells (DCs) and natural killer (NK) cells with CD4 ⁺ T cells in adoptive immunity. Upon activation, effector CTLs infiltrate to the core or invading site of the tumor (so-called infiltrated–inflamed [I–I] TME) and take essential roles for killing cancer cells. Exogenous reactivation and/or priming of CD8 ⁺ T cells can be possible using rational immunotherapy strategies. The increase of the ratio for costimulatory to coinhibitory mediators using immune checkpoint blockade (ICB) approach. Programmed death-1 receptor (PD-1)–ligand (PD-L1) and CTL-associated antigen 4 (CTLA-4) are checkpoint receptors that can be targeted for relieving exhaustion of CD8 ⁺ T cells and renewing their priming, respectively, and thereby eliminating antigen-expressing cancer cells. Due to a diverse relation between CTLs with Tregs, the Treg activity could be dampened for increasing the number and rescuing the functional potential of CTLs to induce immunosensitivity of cancer cells.     

Correlation between PD-1/PD-L1 expression and polarization in tumor-associated macrophages: A key player in tumor immunotherapy

Tumor immunotherapy, such as PD-1/PD-L1 blockade, has shown promising clinical efficacy in patients with various types of tumors. However, the response to PD-1/PD-L1 blockade in a majority of malignancies is limited, indicating an urgent need for a deeper understanding of the mechanisms of PD-1/PD-L1 axis-mediated tumor tolerance. As the most abundant immune cells in the tumor stroma, macrophages display multiple phenotypes and functions in response to the stimuli of the tumor microenvironment. PD-1/PD-L1 has been demonstrated to be highly expressed in tumor-associated macrophages (TAMs), and TAM polarization has been shown to be important during tumor progression. In this review, we outline the relationship between TAM PD-1/PD-L1 expression and polarizations, summarize the involvement of M2 TAMs in PD-1/PD-L1-mediated T-cell exhaustion, and discuss improved approaches for overcoming PD-1/PD-L1 blockade resistance by inducing M2/M1 switching of TAMs.     

Quantitative phosphoproteomic analysis reveals involvement of PD-1 in multiple T cell functions

Programmed cell death protein 1 (PD-1) is a critical inhibitory receptor that limits excessive T cell responses. Cancer cells have evolved to evade these immunoregulatory mechanisms by upregulating PD-1 ligands and preventing T cell–mediated anti-tumor responses. Consequently, therapeutic blockade of PD-1 enhances T cell–mediated anti-tumor immunity, but many patients do not respond and a significant proportion develop inflammatory toxicities. To improve anti-cancer therapy, it is critical to reveal the mechanisms by which PD-1 regulates T cell responses. We performed global quantitative phosphoproteomic interrogation of PD-1 signaling in T cells. By complementing our analysis with functional validation assays, we show that PD-1 targets tyrosine phosphosites that mediate proximal T cell receptor signaling, cytoskeletal organization, and immune synapse formation. PD-1 ligation also led to differential phosphorylation of serine and threonine sites within proteins regulating T cell activation, gene expression, and protein translation. In silico predictions revealed that kinase/substrate relationships engaged downstream of PD-1 ligation. These insights uncover the phosphoproteomic landscape of PD-1–triggered pathways and reveal novel PD-1 substrates that modulate diverse T cell functions and may serve as future therapeutic targets. These data are a useful resource in the design of future PD-1–targeting therapeutic approaches.     

Tumor-infiltrating programmed death receptor-1+ dendritic cells mediate immune suppression in ovarian cancer

Within the ovarian cancer microenvironment, there are several mechanisms that suppress the actions of antitumor immune effectors. Delineating the complex immune microenvironment is an important goal toward developing effective immune-based therapies. A dominant pathway of immune suppression in ovarian cancer involves tumor-associated and dendritic cell (DC)-associated B7-H1. The interaction of B7-H1 with PD-1 on tumor-infiltrating T cells is a widely cited theory of immune suppression involving B7-H1 in ovarian cancer. Recent studies suggest that the B7-H1 ligand, programmed death receptor-1 (PD-1), is also expressed on myeloid cells, complicating interpretations of how B7-H1 regulates DC function in the tumor. In this study, we found that ovarian cancer-infiltrating DCs progressively expressed increased levels of PD-1 over time in addition to B7-H1. These dual-positive PD-1(+) B7-H1(+) DCs have a classical DC phenotype (i.e., CD11c(+)CD11b(+)CD8(-)), but are immature, suppressive, and respond poorly to danger signals. Accumulation of PD-1(+)B7-H1(+) DCs in the tumor was associated with suppression of T cell activity and decreased infiltrating T cells in advancing tumors. T cell suppressor function of these DCs appeared to be mediated by T cell-associated PD-1. In contrast, ligation of PD-1 expressed on the tumor-associated DCs suppressed NF-κB activation, release of immune regulatory cytokines, and upregulation of costimulatory molecules. PD-1 blockade in mice bearing ovarian cancer substantially reduced tumor burden and increased effector Ag-specific T cell responses. Our results reveal a novel role of tumor infiltrating PD-1(+)B7-H1(+) DCs in mediating immune suppression in ovarian cancer.     

Clinical Perspectives to Overcome Acquired Resistance to Anti–Programmed Death-1 and Anti–Programmed Death Ligand-1 Therapy in Non-Small Cell Lung Cancer

Immune checkpoint inhibitors have changed the paradigm of treatment options for non-small cell lung cancer (NSCLC). Monoclonal antibodies targeting programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) have gained wide attention for their application, which has been shown to result in prolonged survival. Nevertheless, only a limited subset of patients show partial or complete response to PD-1 therapy, and patients who show a response eventually develop resistance to immunotherapy. This article aims to provide an overview of the mechanisms of acquired resistance to anti–PD-1/PD-L1 therapy from the perspective of tumor cells and the surrounding microenvironment. In addition, we address the potential therapeutic targets and ongoing clinical trials, focusing mainly on NSCLC.