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.

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.

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.

Isolation and Characterization of T Lymphocyte-Exosomes Using Mass Spectrometry.

Exosomes are cell-derived vesicles that have been implicated in the pathogenesis of many inflammatory diseases. In the immune system, it has been shown that T lymphocyte-derived exosomes are able to induce diverse cellular responses. There are several methods to isolate and to characterize exosomes, each with their own advantages and disadvantages. Here, we describe a centrifugation approach, combined with mass spectrometry characterization, as a means to study exosomes derived from primary human T lymphocytes. This method is sensitive and therefore can be applied when a limited amount of sample is available.

Proteomic analysis of human T cell-derived exosomes reveals differential RAS/MAPK signaling.

Exosomes are cell-derived vesicles that have been implicated in the pathogenesis of many inflammatory diseases. More specifically, it has been shown that T cell-derived exosomes can induce immunological responses; however, little is known about the mechanism and the molecular content of these vesicles. Here, we used a proteomic approach to characterize human T cell-derived exosomes. We found that specific proteins of the RAS signaling pathway were enriched in exosomes derived from activated T cells, and that these vesicles induced ERK phosphorylation in recipient immune cells. Our findings support a mechanistic role of exosomes in cellular activation, and further studies should consider exosomes as a biomarker for inflammatory diseases.

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.

The tyrosine phosphatase SHP-1 promotes T cell adhesion by activating the adaptor protein CrkII in the immunological synapse.

The adaptor protein CrkII regulates T cell adhesion by recruiting the guanine nucleotide exchange factor C3G, an activator of Rap1. Subsequently, Rap1 stimulates the integrin LFA-1, which leads to T cell adhesion and interaction with antigen-presenting cells (APCs). The adhesion of T cells to APCs is critical for their proper function and education. The interface between the T cell and the APC is known as the immunological synapse. It is characterized by the specific organization of proteins that can be divided into central supramolecular activation clusters (c-SMACs) and peripheral SMACs (p-SMACs). Through total internal reflection fluorescence (TIRF) microscopy and experiments with supported lipid bilayers, we determined that activated Rap1 was recruited to the immunological synapse and localized to the p-SMAC. C3G and the active (dephosphorylated) form of CrkII also localized to the same compartment. In contrast, inactive (phosphorylated) CrkII was confined to the c-SMAC. Activation of CrkII and its subsequent movement from the c-SMAC to the p-SMAC depended on the phosphatase SHP-1, which acted downstream of the T cell receptor. In the p-SMAC, CrkII recruited C3G, which led to Rap1 activation and LFA-1-mediated adhesion of T cells to APCs. Functionally, SHP-1 was necessary for both the adhesion and migration of T cells. Together, these data highlight a signaling pathway in which SHP-1 acts through CrkII to reshape the pattern of Rap1 activation in the immunological synapse.

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.

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions.

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.(1,2) These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

T cell chemokine receptor patterns as pathogenic signatures in autoimmunity.

Autoimmune diseases arise from aberrant activation of immune cells directed against endogenous autoantigens expressed throughout the human body. While the initiating triggers remain poorly understood, the self-perpetuating phase of these diseases is directly linked to the ongoing recruitment of inflammatory cells that traffic to the affected anatomical sites. T lymphocytes are prominent drivers of many autoimmune diseases and the targeted trafficking of these cells to infiltrate the affected organs is often a common denominator. The regulation of T cell trafficking involves the coordinated expression of specific patterns of chemokines and the reciprocal expression of cognate chemokine receptors on T cell membranes. Thereby, chemokines direct the specific trafficking of a wide array of responsive activated immune cells. Specific patterns of chemokine receptor expression can correlate with disease activity in an autoimmune disease, confirming the importance of further characterizing the T cells that infiltrate specific sites of autoimmunity. Herein, we will review our current understanding of the roles of chemokines in two common autoimmune diseases: rheumatoid arthritis and multiple sclerosis. We also discuss the implications for chemokine receptor signatures in autoimmune pathogenesis, and how these may provide novel targets for therapeutic intervention.

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.

Analysis of Programmed Death-1 in Patients with Psoriatic Arthritis.

Programmed death-1 (PD-1) is an inhibitory co-receptor that is highly expressed in T lymphocytes that has been shown to downregulate inflammatory responses in several inflammatory diseases including systemic lupus erythematosus and rheumatoid arthritis. Yet, the role of PD-1 in psoriatic arthritis (PsA) has not been studied. In order to fill this gap, we measured the expression levels of PD-1 in peripheral T cells from patients with active disease. Twenty patients and fifteen age-matched healthy control subjects were recruited. The percentage of CD3(+)PD-1(+) T cells was measured by flow cytometry. Despite normal concentration of peripheral T cells, the expression levels of PD-1 were significantly higher in patients compared to healthy controls. Interestingly, among the patients, the expression levels inversely correlated with disease activity measured by disease activity scores (DAS28). PD-1 expression levels strongly correlated with the number of tender and swollen joints, but not with C-reactive protein (CRP) levels or psoriasis area and severity index (PASI). Functionally, in vitro ligation of PD-1 receptor in PsA T cells inhibited interleukin-2 (IL-2) secretion, Akt phosphorylation, and Rap1 activation. These findings suggest that PD-1 might serve as a biomarker for disease activity in PsA and highlight the need for additional studies in order to establish the role of PD-1 in PsA pathogenesis.

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.

Static adhesion assay for the study of integrin activation in T lymphocytes.

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits αL and ß2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g. VLA-4 - fibronectin).

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.

Distinct molecular regulation of glycogen synthase kinase-3alpha isozyme controlled by its N-terminal region: functional role in calcium/calpain signaling.

Glycogen synthase kinase-3 (GSK-3) is expressed as two isozymes α and ß. They share high similarity in their catalytic domains but differ in their N- and C-terminal regions, with GSK-3α having an extended glycine-rich N terminus. Here, we undertook live cell imaging combined with molecular and bioinformatic studies to understand the distinct functions of the GSK-3 isozymes focusing on GSK-3α N-terminal region. We found that unlike GSK-3ß, which shuttles between the nucleus and cytoplasm, GSK-3α was excluded from the nucleus. Deletion of the N-terminal region of GSK-3α resulted in nuclear localization, and treatment with leptomycin B resulted in GSK-3α accumulation in the nucleus. GSK-3α rapidly accumulated in the nucleus in response to calcium or serum deprivation, and accumulation was strongly inhibited by the calpain inhibitor calpeptin. This nuclear accumulation was not mediated by cleavage of the N-terminal region or phosphorylation of GSK-3α. Rather, we show that calcium-induced GSK-3α nuclear accumulation was governed by GSK-3α binding with as yet unknown calpain-sensitive protein or proteins; this binding was mediated by the N-terminal region. Bioinformatic and experimental analyses indicated that nuclear exclusion of GSK-3α was likely an exclusive characteristic of mammalian GSK-3α. Finally, we show that nuclear localization of GSK-3α reduced the nuclear pool of ß-catenin and its target cyclin D1. Taken together, these data suggest that the N-terminal region of GSK-3α is responsible for its nuclear exclusion and that binding with a calcium/calpain-sensitive product enables GSK-3α nuclear retention. We further uncovered a novel link between calcium and nuclear GSK-3α-mediated inhibition of the canonical Wnt/ß-catenin pathway.

Rifampin inhibits prostaglandin E2 production and arachidonic acid release in human alveolar epithelial cells.

Rifampin, a potent antimicrobial agent, is a major drug in the treatment of tuberculosis. There is evidence that rifampin also serves as an immunomodulator. Based on findings that arachidonic acid and its metabolites are involved in the pathogeneses of Mycobacterium tuberculosis infections, we investigated whether rifampin affects prostaglandin E(2) (PGE(2)) production in human alveolar epithelial cells stimulated with interleukin-1beta. Rifampin caused a dose-dependent inhibition of PGE(2) production. At doses of 100, 50, and 25 microg/ml, it inhibited PGE(2) production by 75%, 59%, and 45%, respectively (P < 0.001). Regarding the mechanism involved, rifampin caused a time- and dose-dependent inhibition of arachidonic acid release from the alveolar cells. At doses of 100, 50, 25, and 10 mug/ml, it significantly inhibited the release of arachidonic acid by 93%, 64%, 58%, and 35%, respectively (P < 0.001). Rifampin did not affect the phosphorylation of cytosolic phospholipase A(2) or the expression of cyclooxygenase-2. The inhibition of PGE(2), and presumably other arachidonic acid products, probably contributes to the efficacy of rifampin in the treatment of tuberculosis and may explain some of its adverse effects.