Therapeutic antibodies targeting programmed cell death 1 (PD-1) activate tumor-specific immunity and have shown remarkable efficacy in the treatment of melanoma. Yet, little is known about tumor ...cell-intrinsic PD-1 pathway effects. Here, we show that murine and human melanomas contain PD-1-expressing cancer subpopulations and demonstrate that melanoma cell-intrinsic PD-1 promotes tumorigenesis, even in mice lacking adaptive immunity. PD-1 inhibition on melanoma cells by RNAi, blocking antibodies, or mutagenesis of melanoma-PD-1 signaling motifs suppresses tumor growth in immunocompetent, immunocompromised, and PD-1-deficient tumor graft recipient mice. Conversely, melanoma-specific PD-1 overexpression enhances tumorigenicity, as does engagement of melanoma-PD-1 by its ligand, PD-L1, whereas melanoma-PD-L1 inhibition or knockout of host-PD-L1 attenuate growth of PD-1-positive melanomas. Mechanistically, the melanoma-PD-1 receptor modulates downstream effectors of mTOR signaling. Our results identify melanoma cell-intrinsic functions of the PD-1:PD-L1 axis in tumor growth and suggest that blocking melanoma-PD-1 might contribute to the striking clinical efficacy of anti-PD-1 therapy.
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•Human melanomas frequently contain PD-1-expressing cancer cell subpopulations•Inhibition of melanoma-PD-1 reduces tumor growth, independently of adaptive immunity•PD-1 overexpression and melanoma-PD-1:PD-L1 interactions promote tumor growth•Activation of the melanoma-PD-1 receptor modulates downstream mTOR signaling
PD-1/PD-L1 signaling has cell-intrinsic functions in certain types of mouse and human tumors, boosting cancer growth and promoting tumorigenesis. This suggests that immunotherapy with PD-1 blockers may produce an effect on tumor growth that is separate from their effect on the immune response.
Advances in cancer immunotherapy have offered new hope for patients with metastatic disease. This unfolding success story has been exemplified by a growing arsenal of novel immunotherapeutics, ...including blocking antibodies targeting immune checkpoint pathways, cancer vaccines, and adoptive cell therapy (ACT). Nonetheless, clinical benefit remains highly variable and patient-specific, in part, because all immunotherapeutic regimens vitally hinge on the capacity of endogenous and/or adoptively transferred T-effector (Teff) cells, including chimeric antigen receptor (CAR) T cells, to home efficiently into tumor target tissue. Thus, defects intrinsic to the multi-step T-cell homing cascade have become an obvious, though significantly underappreciated contributor to immunotherapy resistance. Conspicuous have been low intralesional frequencies of tumor-infiltrating T-lymphocytes (TILs) below clinically beneficial threshold levels, and peripheral rather than deep lesional TIL infiltration. Therefore, a Teff cell ‘homing deficit' may arguably represent a dominant factor responsible for ineffective immunotherapeutic outcomes, as tumors resistant to immune-targeted killing thrive in such permissive, immune-vacuous microenvironments. Fortunately, emerging data is shedding light into the diverse mechanisms of immune escape by which tumors restrict Teff cell trafficking and lesional penetrance. In this review, we scrutinize evolving knowledge on the molecular determinants of Teff cell navigation into tumors. By integrating recently described, though sporadic information of pivotal adhesive and chemokine homing signatures within the tumor microenvironment with better established paradigms of T-cell trafficking under homeostatic or infectious disease scenarios, we seek to refine currently incomplete models of Teff cell entry into tumor tissue. We further summarize how cancers thwart homing to escape immune-mediated destruction and raise awareness of the potential impact of immune checkpoint blockers on Teff cell homing. Finally, we speculate on innovative therapeutic opportunities for augmenting Teff cell homing capabilities to improve immunotherapy-based tumor eradication in cancer patients, with special focus on malignant melanoma.
Inflammation and cancer metastasis are associated with extravasation of leukocytes or tumor cells from blood into tissue. Such movement is believed to follow a coordinated and sequential molecular ...cascade initiated, in part, by the three members of the selectin family of carbohydrate-binding proteins: E-selectin (CD62E), L-selectin (CD62L) and P-selectin (CD62P). E-selectin is particularly noteworthy in disease by virtue of its expression on activated endothelium and on bone-skin microvascular linings and for its role in cell rolling, cell signaling and chemotaxis. E-selectin, along with L- or P-selectin, mediates cell tethering and rolling interactions through the recognition of sialo-fucosylated Lewis carbohydrates expressed on structurally diverse protein-lipid ligands on circulating leukocytes or tumor cells. Major advances in understanding the role of E-selectin in inflammation and cancer have been advanced by experiments assaying E-selectin-mediated rolling of leukocytes and tumor cells under hydrodynamic shear flow, by clinical models of E-selectin-dependent inflammation, by mice deficient in E-selectin and by mice deficient in glycosyltransferases that regulate the binding activity of E-selectin ligands. Here, the authors elaborate on how E-selectin and its ligands may facilitate leukocyte or tumor cell recruitment in inflammatory and metastatic settings. Antagonists that target cellular interactions with E-selectin and other members of the selectin family, including neutralizing monoclonal antibodies, competitive ligand inhibitors or metabolic carbohydrate mimetics, exemplify a growing arsenal of potentially effective therapeutics in controlling inflammation and the metastatic behavior of cancer.
P-selectin glycoprotein ligand-1 (PSGL-1) and its glycostructural determinants facilitate responses to infection and cancer by promoting immune effector-cell trafficking into inflamed tissue. In this ...issue of Immunity, Tinoco et al. (2016) report homing-independent functions of PSGL-1 in immune checkpoint regulation and T cell effector activity, in models of chronic viral infection and melanoma.
P-selectin glycoprotein ligand-1 (PSGL-1) and its glycostructural determinants facilitate responses to infection and cancer by promoting immune effector-cell trafficking into inflamed tissue. In this issue of Immunity, Tinoco et al. (2016) report homing-independent functions of PSGL-1 in immune checkpoint regulation and T cell effector activity, in models of chronic viral infection and melanoma.
Eosinophilic inflammation is a characteristic feature of asthma. Integrins are highly versatile cellular receptors that regulate extravasation of eosinophils from the postcapillary segment of the ...bronchial circulation to the airway wall and airspace. Such movement into the asthmatic lung is described as a sequential, multistep paradigm, whereby integrins on circulating eosinophils become activated, eosinophils tether in flow and roll on bronchial endothelial cells, integrins on rolling eosinophils become further activated as a result of exposure to cytokines, eosinophils arrest firmly to adhesive ligands on activated endothelium, and eosinophils transmigrate to the airway in response to chemoattractants. Eosinophils express seven integrin heterodimeric adhesion molecules: α4β1 (CD49d/29), α6β1 (CD49f/29), αMβ2 (CD11b/18), αLβ2 (CD11a/18), αXβ2 (CD11c/18), αDβ2 (CD11d/18), and α4β7 (CD49d/β7). The role of these integrins in eosinophil recruitment has been elucidated by major advances in the understanding of integrin structure, integrin function, and modulators of integrins. Such findings have been facilitated by cellular experiments of eosinophils in vitro, studies of allergic asthma in humans and animal models in vivo, and crystal structures of integrins. Here, we elaborate on how integrins cooperate to mediate eosinophil movement to the asthmatic airway. Antagonists that target integrins represent potentially promising therapies in the treatment of asthma.
Monoclonal antibodies (abs) targeting the programmed cell death 1 (PD-1) immune checkpoint pathway have revolutionized tumor therapy. Because T-cell-directed PD-1 blockade boosts tumor immunity, ...anti-PD-1 abs have been developed for examining T-cell-PD-1 functions. More recently, PD-1 expression has also been reported directly on cancer cells of various etiology, including in melanoma. Nevertheless, there is a paucity of studies validating anti-PD-1 ab clone utility in specific assay types for characterizing tumor cell-intrinsic PD-1. Here, we demonstrate reactivity of several anti-murine PD-1 ab clones and recombinant PD-L1 with live B16-F10 melanoma cells and YUMM lines using multiple independent methodologies, positive and negative PD-1-specific controls, including PD-1-overexpressing and PD-1 knockout cells. Flow cytometric analyses with two separate anti-PD-1 ab clones, 29F.1A12 and RMP1-30, revealed PD-1 surface protein expression on live murine melanoma cells, which was corroborated by marked enrichment in PD-1 gene (Pdcd1) expression. Immunoblotting, immunoprecipitation, and mass spectrometric sequencing confirmed PD-1 protein expression by B16-F10 cells. Recombinant PD-L1 also recognized melanoma cell-expressed PD-1, the blockade of which by 29F.1A12 fully abrogated PD-1:PD-L1 binding. Together, our data provides multiple lines of evidence establishing PD-1 expression by live murine melanoma cells and validates ab clones and assay systems for tumor cell-directed PD-1 pathway investigations.
How cancer cells bind to vascular surfaces and extravasate into target organs is an underappreciated, yet essential step in metastasis. We postulate that the metastatic process involves discrete ...adhesive interactions between circulating cancer cells and microvascular endothelial cells. Sialyl Lewis X (sLeX) on prostate cancer (PCa) cells is thought to promote metastasis by mediating PCa cell binding to microvascular endothelial (E)-selectin. Yet, regulation of sLeX and related E-selectin ligand expression in PCa cells is a poorly understood factor in PCa metastasis. Here, we describe a glycobiological mechanism regulating E-selectin-mediated adhesion and metastatic potential of PCa cells. We demonstrate that α1,3 fucosyltransferases (FT) 3, 6, and 7 are markedly elevated in bone- and liver-metastatic PCa and dictate synthesis of sLeX and E-selectin ligands on metastatic PCa cells. Upregulated FT3, FT6, or FT7 expression induced robust PCa PC-3 cell adhesion to bone marrow (BM) endothelium and to inflamed postcapillary venules in an E-selectin-dependent manner. Membrane proteins, CD44, carcinoembryonic antigen (CEA), podocalyxin-like protein (PCLP), and melanoma cell adhesion molecule (MCAM) were major scaffolds presenting E-selectin-binding determinants on FT-upregulated PC-3 cells. Furthermore, elevated FT7 expression promoted PC-3 cell trafficking to and retention in BM through an E-selectin dependent event. These results indicate that α1,3 FTs could enhance metastatic efficiency of PCa by triggering an E-selectin-dependent trafficking mechanism.
Germinal centers (GC) are microanatomical niches where B cells proliferate, undergo antibody affinity maturation, and differentiate to long-lived memory B cells and antibody-secreting plasma cells. ...For decades, GC B cells have been defined by their reactivity to the plant lectin peanut agglutinin (PNA), which binds serine/threonine (O-linked) glycans containing the asialylated disaccharide Gal-β1,3-GalNAc-Ser/Thr (also called T-antigen). In T cells, acquisition of PNA binding by activated T cells and thymocytes has been linked with altered tissue homing patterns, cell signaling, and survival. Yet, in GC B cells, the glycobiological basis and significance of PNA binding remains surprisingly unresolved. Here, we investigated the basis for PNA reactivity of GC B cells. We found that GC B cell binding to PNA is associated with downregulation of the α2,3 sialyltransferase,
(ST3Gal1), and overexpression of ST3Gal1 was sufficient to reverse PNA binding in B cell lines. Moreover, we found that the primary scaffold for PNA-reactive O-glycans in B cells is the B cell receptor-associated receptor-type tyrosine phosphatase CD45, suggesting a role for altered O-glycosylation in antigen receptor signaling. Consistent with similar reports in T cells, ST3Gal1 overexpression in B cells
induced drastic shortening in O-glycans, which we confirmed by both antibody staining and mass spectrometric O-glycomic analysis. Unexpectedly, ST3Gal1-induced changes in O-glycan length also correlated with altered binding of two glycosylation-sensitive CD45 antibodies, RA3-6B2 (more commonly called B220) and MEM55, which (in humans) have previously been reported to favor binding to naïve/GC subsets and memory/plasmablast subsets, respectively. Analysis of primary B cell binding to B220, MEM55, and several plant lectins suggested that B cell differentiation is accompanied by significant loss of O-glycan complexity, including loss of extended Core 2 O-glycans. To our surprise, decreased O-glycan length from naïve to post-GC fates best correlated not with ST3Gal1, but rather downregulation of the Core 2 branching enzyme GCNT1. Thus, our data suggest that O-glycan remodeling is a feature of B cell differentiation, dually regulated by ST3Gal1 and GCNT1, that ultimately results in expression of distinct O-glycosylation states/CD45 glycoforms at each stage of B cell differentiation.
Advanced prostate cancer commonly metastasizes to bone, but transit of malignant cells across the bone marrow endothelium (BMEC) remains a poorly understood step in metastasis. Prostate cancer cells ...roll on E-selectin(+) BMEC through E-selectin ligand-binding interactions under shear flow, and prostate cancer cells exhibit firm adhesion to BMEC via β1, β4, and αVβ3 integrins in static assays. However, whether these discrete prostate cancer cell-BMEC adhesive contacts culminate in cooperative, step-wise transendothelial migration into bone is not known. Here, we describe how metastatic prostate cancer cells breach BMEC monolayers in a step-wise fashion under physiologic hemodynamic flow. Prostate cancer cells tethered and rolled on BMEC and then firmly adhered to and traversed BMEC via sequential dependence on E-selectin ligands and β1 and αVβ3 integrins. Expression analysis in human metastatic prostate cancer tissue revealed that β1 was markedly upregulated compared with expression of other β subunits. Prostate cancer cell breaching was regulated by Rac1 and Rap1 GTPases and, notably, did not require exogenous chemokines as β1, αVβ3, Rac1, and Rap1 were constitutively active. In homing studies, prostate cancer cell trafficking to murine femurs was dependent on E-selectin ligand, β1 integrin, and Rac1. Moreover, eliminating E-selectin ligand-synthesizing α1,3 fucosyltransferases in transgenic adenoma of mouse prostate mice dramatically reduced prostate cancer incidence. These results unify the requirement for E-selectin ligands, α1,3 fucosyltransferases, β1 and αVβ3 integrins, and Rac/Rap1 GTPases in mediating prostate cancer cell homing and entry into bone and offer new insight into the role of α1,3 fucosylation in prostate cancer development.
Galectin-1 (Gal-1), a β-galactoside-binding lectin, plays a profound role in modulating adaptive immune responses by altering the phenotype and fate of T cells. Experimental data showing recombinant ...Gal-1 (rGal-1) efficacy on T cell viability and cytokine production, nevertheless, is controversial due to the necessity of using stabilizing chemicals to help retain Gal-1 structure and function. To address this drawback, we developed a mouse Gal-1 human Ig chimera (Gal-1hFc) that did not need chemical stabilization for Gal-1 ligand recognition, apoptosis induction, and cytokine modulation in a variety of leukocyte models. At high concentrations, Gal-1hFc induced apoptosis in Gal-1 ligand(+) Th1 and Th17 cells, leukemic cells, and granulocytes from synovial fluids of patients with rheumatoid arthritis. Importantly, at low, more physiologic concentrations, Gal-1hFc retained its homodimeric form without losing functionality. Not only did Gal-1hFc-binding trigger IL-10 and Th2 cytokine expression in activated T cells, but members of the CD28 family and several other immunomodulatory molecules were upregulated. In a mouse model of contact hypersensitivity, we found that a non-Fc receptor-binding isoform of Gal-1hFc, Gal-1hFc2, alleviated T cell-dependent inflammation by increasing IL-4(+), IL-10(+), TGF-β(+), and CD25(high)/FoxP3(+) T cells, and by decreasing IFN-γ(+) and IL-17(+) T cells. Moreover, in human skin-resident T cell cultures, Gal-1hFc diminished IL-17(+) T cells and increased IL-4(+) and IL-10(+) T cells. Gal-1hFc will not only be a useful new tool for investigating the role of Gal-1 ligands in leukocyte death and cytokine stimulation, but for studying how Gal-1-Gal-1 ligand binding shapes the intensity of immune responses.
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