Changing the location of proteins on the cell surface is a promising strategy for modulating T cell functions.

Targeting immune receptors on T cells is a common strategy to treat cancer and autoimmunity. Frequently, this is accomplished through monoclonal antibodies targeting the ligand binding sites of stimulatory or inhibitory co-receptors. Blocking ligand binding prevents downstream signalling and modulates specific T cell functions. Since 1985, the FDA has approved over 100 monoclonal antibodies against immune receptors. This therapeutic approach significantly improved the care of patients with numerous immune-related conditions; however, many patients are unresponsive, and some develop immune-related adverse events. One reason for that is the lack of consideration for the localization of these receptors on the cell surface of the immune cells in the context of the immune synapse. In addition to blocking ligand binding, changing the location of these receptors on the cell surface within the different compartments of the immunological synapse could serve as an alternative, efficient, and safer approach to treating these patients. This review discusses the potential therapeutic advantages of altering proteins' localization within the immune synapse and summarizes published work in this field. It also discusses the novel use of bispecific antibodies to induce the clustering of receptors on the cell surface. It presents the rationale for developing novel antibodies, targeting the organization of signalling receptor complexes on the cell surface. This approach offers an innovative and emerging technology to treat cancer patients resistant to current immunotherapies.

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.

PD-1-induced proliferating T cells exhibit a distinct transcriptional signature.

Ligation of the inhibitory receptor PD-1 on T cells results in the inhibition of numerous cellular functions. Despite the overtly inhibitory outcome of PD-1 signalling, there are additionally a collection of functions that are activated. We have observed that CD4+ T cells stimulated through the T-cell receptor and PD-1 primarily do not proliferate; however, there is a population of cells that proliferates more than T-cell receptor stimulation alone. These highly proliferating cells could potentially be associated with PD-1-blockade unresponsiveness in patients. In this study, we have performed RNA sequencing and found that following PD-1 ligation proliferating and non-proliferating T cells have distinct transcriptional signatures. Remarkably, the proliferating cells showed an enrichment of genes associated with an activated state despite PD-1 signalling. Additionally, circulating follicular helper T cells were significantly more prevalent in the non-proliferating population, demonstrated by enrichment of the associated genes CXCR5, CCR7, TCF7, BCL6 and PRDM1 and validated at the protein level. Translationally, we also show that there are more follicular helper T cells in patients that respond favourably to PD-1 blockade. Overall, the presence of transcriptionally and functionally distinct T cell populations responsive to PD-1 ligation may provide insights into the clinical differences observed following therapeutic PD-1 blockade.

Affinity purification mass spectrometry analysis of PD-1 uncovers SAP as a new checkpoint inhibitor.

Programmed cell death-1 (PD-1) is an essential inhibitory receptor in T cells. Antibodies targeting PD-1 elicit durable clinical responses in patients with multiple tumor indications. Nevertheless, a significant proportion of patients do not respond to anti-PD-1 treatment, and a better understanding of the signaling pathways downstream of PD-1 could provide biomarkers for those whose tumors respond and new therapeutic approaches for those whose tumors do not. We used affinity purification mass spectrometry to uncover multiple proteins associated with PD-1. Among these proteins, signaling lymphocytic activation molecule-associated protein (SAP) was functionally and mechanistically analyzed for its contribution to PD-1 inhibitory responses. Silencing of SAP augmented and overexpression blocked PD-1 function. T cells from patients with X-linked lymphoproliferative disease (XLP), who lack functional SAP, were hyperresponsive to PD-1 signaling, confirming its inhibitory role downstream of PD-1. Strikingly, signaling downstream of PD-1 in purified T cell subsets did not correlate with PD-1 surface expression but was inversely correlated with intracellular SAP levels. Mechanistically, SAP opposed PD-1 function by acting as a molecular shield of key tyrosine residues that are targets for the tyrosine phosphatase SHP2, which mediates PD-1 inhibitory properties. Our results identify SAP as an inhibitor of PD-1 function and SHP2 as a potential therapeutic target in patients with XLP.

EF Hand Domain Family Member D2 Is Required for T Cell Cytotoxicity.

Programmed cell death 1 (PD-1) is a major coinhibitory receptor and a member of the immunological synapse (IS). To uncover proteins that regulate PD-1 recruitment to the IS, we searched for cytoskeleton-related proteins that also interact with PD-1 using affinity purification mass spectrometry. Among these proteins, EF hand domain family member D2 (EFHD2), a calcium binding adaptor protein, was functionally and mechanistically analyzed for its contribution to PD-1 signaling. EFHD2 was required for PD-1 to inhibit cytokine secretion, proliferation, and adhesion of human T cells. Interestingly, EFHD2 was also required for human T cell-mediated cytotoxicity and for mounting an antitumor immune response in a syngeneic murine tumor model. Mechanistically, EFHD2 contributed to IS stability, lytic vesicles trafficking, and granzyme B secretion. Altogether, EFHD2 is an important regulator of T cell cytotoxicity and further studies should evaluate its role in T cell-mediated inflammation.

PLCε1 regulates SDF-1α-induced lymphocyte adhesion and migration to sites of inflammation.

Regulation of integrins is critical for lymphocyte adhesion to endothelium and migration throughout the body. Inside-out signaling to integrins is mediated by the small GTPase Ras-proximate-1 (Rap1). Using an RNA-mediated interference screen, we identified phospholipase Cε 1 (PLCε1) as a crucial regulator of stromal cell-derived factor 1 alpha (SDF-1α)-induced Rap1 activation. We have shown that SDF-1α-induced activation of Rap1 is transient in comparison with the sustained level following cross-linking of the antigen receptor. We identified that PLCε1 was necessary for SDF-1α-induced adhesion using shear stress, cell morphology alterations, and crawling on intercellular adhesion molecule 1 (ICAM-1)-expressing cells. Structure-function experiments to separate the dual-enzymatic function of PLCε1 uncover necessary contributions of the CDC25, Pleckstrin homology, and Ras-associating domains, but not phospholipase activity, to this pathway. In the mouse model of delayed type hypersensitivity, we have shown an essential role for PLCε1 in T-cell migration to inflamed skin, but not for cytokine secretion and proliferation in regional lymph nodes. Our results reveal a signaling pathway where SDF-1α induces T-cell adhesion through activation of PLCε1, suggesting that PLCε1 is a specific potential target in treating conditions involving migration of T cells to inflamed organs.

Chaperone-mediated specificity in Ras and Rap signaling.

Ras and Rap proteins are closely related small guanosine triphosphatase (GTPases) that share similar effector-binding domains but operate in a very different signaling networks; Ras has a dominant role in cell proliferation, while Rap mediates cell adhesion. Ras and Rap proteins are regulated by several shared processes such as post-translational modification, phosphorylation, activation by guanine exchange factors and inhibition by GTPase-activating proteins. Sub-cellular localization and trafficking of these proteins to and from the plasma membrane are additional important regulatory features that impact small GTPases function. Despite its importance, the trafficking mechanisms of Ras and Rap proteins are not completely understood. Chaperone proteins play a critical role in trafficking of GTPases and will be the focus of the discussion in this work. We will review several aspects of chaperone biology focusing on specificity toward particular members of the small GTPase family. Understanding this specificity should provide key insights into drug development targeting individual small GTPases.

Rap1 and its effector RIAM are required for lymphocyte trafficking.

Regulation of integrins is critical for lymphocyte adhesion to endothelium and trafficking through secondary lymphoid organs. Inside-out signaling to integrins is mediated by the small GTPase Rap1. Two effectors of Rap1 regulate integrins, RapL and Rap1 interacting adaptor molecule (RIAM). Using mice conditionally deficient in both Rap1a and Rap1b and mice null for RIAM, we show that the Rap1/RIAM module is not required for T- or B-cell development but is essential for efficient adhesion to intercellular adhesion molecule (ICAM) 1 and vascular cell adhesion molecule (VCAM) 1 and for proper trafficking of lymphocytes to secondary lymphoid organs. Interestingly, in RIAM-deficient mice, whereas peripheral lymph nodes (pLNs) were depleted of both B and T cells and recirculating B cells were diminished in the bone barrow (BM), the spleen was hypercellular, albeit with a relative deficiency of marginal zone B cells. The abnormality in lymphocyte trafficking was accompanied by defective humoral immunity to T-cell-dependent antigens. Platelet function was intact in RIAM-deficient animals. These in vivo results confirm a role for RIAM in the regulation of some, but not all, leukocyte integrins and suggest that RIAM-regulated integrin activation is required for trafficking of lymphocytes from blood into pLNs and BM, where relatively high shear forces exist in high endothelial venules and sinusoids, respectively.

CD28 inhibits T cell adhesion by recruiting CAPRI to the plasma membrane.

CD28 is a coreceptor expressed on T lymphocytes. Signaling downstream of CD28 promotes multiple T cell functions such as proliferation, survival, and cytokine secretion. Adhesion to APCs is another function of T cells; however, little is known with regard to the role of CD28 in this process. Our previous studies have shown that CD28 inhibits T cell adhesion, but the underlying mechanism that mediates this process remains unknown. In the present study we discovered that signaling downstream of CD28 resulted in inhibition of Rap1 activity and decreased LFA-1-mediated adhesion. We showed that this was regulated by the recruitment of calcium-promoted Ras inactivator (CAPRI), a GTPase-activating protein, to the plasma membrane downstream of CD28 signaling. CAPRI trafficking to the plasma membrane was secondary to calcium influx and was mediated by its C2A and C2B domains. We conclude that CD28 inhibits Rap1-mediated adhesion by recruiting the protein CAPRI to the plasma membrane.

Prolonged Morphine Exposure Induces Increased Firm Adhesion in an in Vitro Model of the Blood-Brain Barrier.

The blood-brain barrier (BBB) has been defined as a critically important protective barrier that is involved in providing essential biologic, physiologic, and immunologic separation between the central nervous system (CNS) and the periphery. Insults to the BBB can cause overall barrier damage or deregulation of the careful homeostasis maintained between the periphery and the CNS. These insults can, therefore, yield numerous phenotypes including increased overall permeability, interendothelial gap formation, alterations in cytokine and chemokine secretion, and accelerated cellular passage. The current studies expose the human brain microvascular endothelial cell line, hCMEC/D3, to prolonged morphine exposure and aim to uncover the mechanisms underlying alterations in barrier function in vitro. These studies show alterations in the mRNA and protein levels of the cellular adhesion molecules (CAMs) intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and activated leukocyte cell adhesion molecule that correlate with an increased firm adhesion of the CD3⁺ subpopulation of peripheral blood mononuclear cells (PBMCs). Overall, these studies suggest that prolonged morphine exposure may result in increased cell migration into the CNS, which may accelerate pathological processes in many diseases that involve the BBB.

Programmed death-1 pathway in cancer and autoimmunity.

Programmed death-1 (PD-1) is a co-receptor that is expressed predominantly by T cells. The binding of PD-1 to its ligands, PD-L1 or PD-L2, is vital for the physiologic regulation of the immune system. A major functional role of the PD-1 signaling pathway is the inhibition of self-reactive T cells, which serve to protect against autoimmune diseases. Elimination of the PD-1 pathway can therefore result in the breakdown of immune tolerance that can ultimately lead to the development of pathogenic autoimmunity. Conversely, tumor cells can at times co-opt the PD-1 pathway to escape from immunosurveillance mechanisms. Therefore, blockade of the PD-1 pathway has become an attractive target in cancer therapy. Recent clinical trials have shown that anti-PD-1 agents have profound effects on solid tumor regression. Current approaches include six agents that are either PD-1 and PD-L1 targeted neutralizing antibodies or fusion proteins. More than forty clinical trials are underway to better define the role of PD-1 blockade in variety of tumor types. In this review we will highlight the basic biology of the PD-1 system and discuss its potential roles in both autoimmunity and cancer. We propose that future research on PD-1 may lead to the translation of fundamental regulatory pathways into the development of practical new approaches for the treatment of autoimmune diseases and cancer.

Combination Approaches to Target PD-1 Signaling in Cancer.

Cancer remains the second leading cause of death in the US, accounting for 25% of all deaths nationwide. Immunotherapy techniques bolster the immune cells' ability to target malignant cancer cells and have brought immense improvements in the field of cancer treatments. One important inhibitory protein in T cells, programmed cell death protein 1 (PD-1), has become an invaluable target for cancer immunotherapy. While anti-PD-1 antibody therapy is extremely successful in some patients, in others it fails or even causes further complications, including cancer hyper-progression and immune-related adverse events. Along with countless translational studies of the PD-1 signaling pathway, there are currently close to 5,000 clinical trials for antibodies against PD-1 and its ligand, PD-L1, around 80% of which investigate combinations with other therapies. Nevertheless, more work is needed to better understand the PD-1 signaling pathway and to facilitate new and improved evidence-based combination strategies. In this work, we consolidate recent discoveries of PD-1 signaling mediators and their therapeutic potential in combination with anti-PD-1/PD-L1 agents. We focus on the phosphatases SHP2 and PTPN2; the kinases ITK, VRK2, GSK-3, and CDK4/6; and the signaling adaptor protein PAG. We discuss their biology both in cancer cells and T cells, with a focus on their role in relation to PD-1 to determine their potential in therapeutic combinations. The literature discussed here was obtained from a search of the published literature and ClinicalTrials.gov with the following key terms: checkpoint inhibition, cancer immunotherapy, PD-1, PD-L1, SHP2, PTPN2, ITK, VRK2, CDK4/6, GSK-3, and PAG. Together, we find that all of these proteins are logical and promising targets for combination therapy, and that with a deeper mechanistic understanding they have potential to improve the response rate and decrease adverse events when thoughtfully used in combination with checkpoint inhibitors.

Neutralization of the adaptor protein PAG by monoclonal antibody limits murine tumor growth.

The transmembrane adaptor phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (PAG) is phosphorylated in T cells downstream of PD-1 signaling and contributes to the resulting functional inhibition of multiple cellular processes. Furthermore, PAG expression is negatively correlated with survival in multiple human tumors and is a driver of murine tumor growth and immune evasion. Here we develop an antibody that targets the extracellular domain of human PAG, with cross-reactivity to murine PAG. We demonstrate that this antibody binds to extracellular PAG on intact cells and affects T cell activation. Finally, we show that administration of anti-PAG monoclonal antibody in combination with anti-PD-1 antibody to mice bearing MC38 tumors limited tumor growth and enhanced T cell infiltration to tumors.

Role of mu-opioids as cofactors in human immunodeficiency virus type 1 disease progression and neuropathogenesis.

About one third of acquired immunodeficiency syndrome cases in the USA have been attributed to the use of injected addictive drugs, frequently involving opioids like heroin and morphine, establishing them as significant predisposing risk factors for contracting human immunodeficiency virus type 1 (HIV-1). Accumulating evidence from in vitro and in vivo experimental systems indicates that opioids act in concert with HIV-1 proteins to exacerbate dysregulation of neural and immune cell function and survival through diverse molecular mechanisms. In contrast, the impact of opioid exposure and withdrawal on the viral life cycle and HIV-1 disease progression itself is unclear, with conflicting reports emerging from the simian immunodeficiency virus and simian-human immunodeficiency virus infection models. However, these studies suggest a potential role of opioids in elevated viral production. Because human microglia, astrocytes, CD4+ T lymphocytes, and monocyte-derived macrophages express opioid receptors, it is likely that intracellular signaling events triggered by morphine facilitate enhancement of HIV-1 infection in these target cell populations. This review highlights the biochemical changes that accompany prolonged exposure to and withdrawal from morphine that synergize with HIV-1 proteins to disrupt normal cellular physiological functions especially within the central nervous system. More importantly, it collates evidence from epidemiological studies, animal models, and heterologous cell systems to propose a mechanistic link between such physiological adaptations and direct modulation of HIV-1 production. Understanding the opioid-HIV-1 interface at the molecular level is vitally important in designing better treatment strategies for HIV-1-infected patients who abuse opioids.

Bridging the Gap: Connecting the Mechanisms of Immune-Related Adverse Events and Autoimmunity Through PD-1.

The emergence of anti-cytotoxic T-lymphocyte antigen 4 (anti-CTLA-4), anti-programmed cell death 1 ligand (anti-PD-1), and anti-PD-L1 antibodies as immune checkpoint inhibitors (ICIs) revolutionized the treatment of numerous types of tumors. These antibodies, both alone and in combination, provide great clinical efficacy as evidenced by tumor regression and increased overall patients' survival. However, with this success comes multiple challenges. First, while patients who respond to ICIs have outstanding outcomes, there remains a large proportion of patients who do not respond at all. This all-or-none response has led to looking downstream of programmed cell death 1 (PD-1) for additional therapeutic targets and for new combination therapies. Second, a majority of patients who receive ICIs go on to develop immune-related adverse events (irAEs) characterized by end-organ inflammation with T-cell infiltrates. The hallmarks of these clinically observed irAEs share many similarities with primary autoimmune diseases. The contribution of PD-1 to peripheral tolerance is a major mechanism for protection against expansion of self-reactive T-cell clones and autoimmune disease. In this review, we aim to bridge the gaps between our cellular and molecular knowledge of PD-1 signaling in T cells, ICI-induced irAEs, and autoimmune diseases. We will highlight shared mechanisms and the potential for new therapeutic strategies.

Transmembrane adaptor protein PAG is a mediator of PD-1 inhibitory signaling in human T cells.

The inhibitory receptor PD-1 is expressed on T cells to inhibit select functions when ligated. The complete signaling mechanism downstream of PD-1 has yet to be uncovered. Here, we discovered phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (PAG) is phosphorylated following PD-1 ligation and associate this with inhibitory T cell function. Clinical cohort analysis correlates low PAG expression with increased survival from numerous tumor types. PAG knockdown in T cells prevents PD-1-mediated inhibition of cytokine secretion, cell adhesion, CD69 expression, and ERK204/187 phosphorylation, and enhances phosphorylation of SRC527 following PD-1 ligation. PAG overexpression rescues these effects. In vivo, PAG contributes greatly to the growth of two murine tumors, MC38 and B16, and limits T cell presence within the tumor. Moreover, PAG deletion sensitizes tumors to PD-1 blockade. Here PAG is established as a critical mediator of PD-1 signaling and as a potential target to enhance T cell activation in tumors.

SHP2 Targets ITK Downstream of PD-1 to Inhibit T Cell Function.

PD-1 is a critical therapeutic target in cancer immunotherapy and antibodies blocking PD-1 are approved for multiple types of malignancies. The phosphatase SHP2 is the main effector mediating PD-1 downstream signaling and accordingly attempts have been made to target this enzyme as an alternative approach to treat immunogenic tumors. Unfortunately, small molecule inhibitors of SHP2 do not work as expected, suggesting that the role of SHP2 in T cells is more complex than initially hypothesized. To better understand the perplexing role of SHP2 in T cells, we performed interactome mapping of SAP, an adapter protein that is associated with SHP2 downstream signaling. Using genetic and pharmacological approaches, we discovered that SHP2 dephosphorylates ITK specifically downstream of PD-1 and that this event was associated with PD-1 inhibitory cellular functions. This study suggests that ITK is a unique target in this pathway, and since ITK is a SHP2-dependent specific mediator of PD-1 signaling, the combination of ITK inhibitors with PD-1 blockade may improve upon PD-1 monotherapy in the treatment of cancer.

The Complexity of Targeting Chemokines to Promote a Tumor Immune Response.

Immunotherapeutic treatment strategies greatly extend patient survival following malignant disease across a wide range of tumor types, including even those with metastatic disease. While diverse in approach, adoptive cell therapy, introduction of T cells that express chimeric antigen receptors, and checkpoint inhibitors all aim to re-invigorate the immune system to promote tumor cell identification and elimination. This review will focus on immune cell infiltration into tumors as well as a cellular organization within the tumor microenvironment as directed by the cell-specific expression patterns of chemokines and chemokine receptors. Through better understanding the chemokine network within tumors, we can uncover mechanisms to promote beneficial immune cell infiltration that can be combined with checkpoint inhibition. Conversely, chemokine expression is not limited to cells of the immune system, and it is understood that tumor cells also express chemokines and chemokine receptors. Tumor cells can hijack the chemokine networks to promote immune suppression and metastatic tumor cell trafficking. We will discuss the ways in which the chemokine network lies at the crossroad of immune evasion and tumor regression. Overall, this review will summarize key publications in the field of immune cell recruitment to tumors, highlight the dichotomous nature of chemokine interventions into cancer, and aims to identify therapeutic pathways forward.

Chemokines and Cancer: Friends or Foes?

Immune cell infiltration into tumors, intratumoral cellular organization, and the cell-specific expression patterns of chemokines and chemokine receptors greatly influence the efficacy of immunotherapeutic treatment strategies. In our recent review article, we shined a light on the deciding role of the chemokine network between immune mediated tumor regression or immune evasion of the tumor. Current T cell centric immunotherapeutic strategies primarily rely on increasing cellular activation and decreasing cellular inhibition, with the overall goal of enhancing effector cell function. These strategies neglect to account for the presence of the T cells within the tumor, hardly boosting immune cell infiltration. Chemokines and chemokine receptors are the regulators of recruitment, migration, and intratumoral compartmentalization. Yet, utilizing the chemokine network to recruit immune cells that will drive tumor regression is not a straightforward path, as tumor cells often hijack these pathways in the effort of immune evasion. Many novel therapeutic strategies involving chemokine targeting are under trial for many diverse tumor types. As a field, we can learn from both the successes and failures of these trials in order to push forward the next generation of immunotherapeutic strategies that include augmented T cell trafficking.