Identification of C18:1-phytoceramide as the candidate lipid mediator for hydroxyurea resistance in yeast.

Recent studies showed that deletion of ISC1, the yeast homologue of the mammalian neutral sphingomyelinase, resulted in an increased sensitivity to hydroxyurea (HU). This raised an intriguing question as to whether sphingolipids are involved in pathways initiated by HU. In this study, we show that HU treatment led to a significant increase in Isc1 activity. Analysis of sphingolipid deletion mutants and pharmacological analysis pointed to a role for ceramide in mediating HU resistance. Lipid analysis revealed that HU induced increases in phytoceramides in WT cells but not in isc1Δ cells. To probe functions of specific ceramides, we developed an approach to supplement the medium with fatty acids. Oleate (C18:1) was the only fatty acid protecting isc1Δ cells from HU toxicity in a ceramide-dependent manner. Because phytoceramide activates protein phosphatases in yeast, we evaluated the role of CDC55, the regulatory subunit of ceramide-activated protein phosphatase PP2A. Overexpression of CDC55 overcame the sensitivity to HU in isc1Δ cells. However, addition of oleate did not protect the isc1Δ,cdc55Δ double mutant from HU toxicity. These results demonstrate that HU launches a lipid pathway mediated by a specific sphingolipid, C18:1-phytoceramide, produced by Isc1, which provides protection from HU by modulating Swe1 levels through the PP2A subunit Cdc55.

Developmental analysis of a Medicago truncatula smooth leaf margin1 mutant reveals context-dependent effects on compound leaf development.

Compound leaf development requires highly regulated cell proliferation, differentiation, and expansion patterns. We identified loss-of-function alleles at the SMOOTH LEAF MARGIN1 (SLM1) locus in Medicago truncatula, a model legume species with trifoliate adult leaves. SLM1 encodes an auxin efflux carrier protein and is the ortholog of Arabidopsis thaliana PIN-FORMED1 (PIN1). Auxin distribution is impaired in the slm1 mutant, resulting in pleiotropic phenotypes in different organs. The most striking change in slm1 is the increase in the number of terminal leaflets and a simultaneous reduction in the number of lateral leaflets, accompanied by reduced expression of SINGLE LEAFLET1 (SGL1), an ortholog of LEAFY. Characterization of the mutant indicates that distinct developmental domains exist in the formation of terminal and lateral leaflets. In contrast with the pinnate compound leaves in the wild type, the slm1 sgl1 double mutant shows nonpeltately palmate leaves, suggesting that the terminal leaflet primordium in M. truncatula has a unique developmental mechanism. Further investigations on the development of leaf serrations reveal different ontogenies between distal serration and marginal serration formation as well as between serration and leaflet formation. These data suggest that regulation of the elaboration of compound leaves and serrations is context dependent and tightly correlated with the auxin/SLM1 module in M. truncatula.

Thrombin contributes to cancer immune evasion via proteolysis of platelet-bound GARP to activate LTGF-ß.

Cancer-associated thrombocytosis and high concentrations of circulating transforming growth factor-ß1 (TGF-ß1) are frequently observed in patients with progressive cancers. Using genetic and pharmacological approaches, we show a direct link between thrombin catalytic activity and release of mature TGF-ß1 from platelets. We found that thrombin cleaves glycoprotein A repetitions predominant (GARP), a cell surface docking receptor for latent TGF-ß1 (LTGF-ß1) on platelets, resulting in liberation of active TGF-ß1 from the GARP-LTGF-ß1 complex. Furthermore, systemic inhibition of thrombin obliterates TGF-ß1 maturation in platelet releasate and rewires the tumor microenvironment toward favorable antitumor immunity, which translates into efficient cancer control either alone or in combination with programmed cell death 1-based immune checkpoint blockade therapy. Last, we demonstrate that soluble GARP and GARP-LTGF-ß1 complex are present in the circulation of patients with cancer. Together, our data reveal a mechanism of cancer immune evasion that involves thrombin-mediated GARP cleavage and the subsequent TGF-ß1 release from platelets. We propose that blockade of GARP cleavage is a valuable therapeutic strategy to overcome cancer's resistance to immunotherapy.

GARP Dampens Cancer Immunity by Sustaining Function and Accumulation of Regulatory T Cells in the Colon.

Activated regulatory T (Treg) cells express the surface receptor glycoprotein-A repetitions predominant (GARP), which binds and activates latent TGFß. How GARP modulates Treg function in inflammation and cancer remains unclear. Here we demonstrate that loss of GARP in Treg cells leads to spontaneous inflammation with highly activated CD4+ and CD8+ T cells and development of enteritis. Treg cells lacking GARP were unable to suppress pathogenic T-cell responses in multiple models of inflammation, including T-cell transfer colitis. GARP-/- Treg cells were significantly reduced in the gut and exhibited a reduction in CD103 expression, a colon-specific migratory marker. In the colitis-associated colon cancer model, GARP on Treg cells dampened immune surveillance, and mice with GARP-/- Treg cells exhibited improved antitumor immunity. Thus, GARP empowers the functionality of Treg cells and their tissue-specific accumulation, highlighting the importance of cell surface TGFß in Treg function and GARP as a potential therapeutic target for colorectal cancer therapy.Significance: These findings uncover functions of membrane-bound TGFß and GARP that tune the activity of Treg cells, highlighting a potential treatment strategy in autoimmune diseases and cancer.

Immunoregulatory functions and the therapeutic implications of GARP-TGF-ß in inflammation and cancer.

GARP (glycoprotein-A repetitions predominant) is a type I transmembrane cell surface docking receptor for latent transforming growth factor-ß (TGF-ß) that is abundantly expressed on regulatory T lymphocytes and platelets. GARP regulates the availability of membrane-bound latent TGF-ß and modulates its activation. For this reason, GARP expression on immune and non-immune cells is involved in maintaining peripheral tolerance. It plays an important role in preventing inflammatory diseases such as allergy and graft versus host disease (GvHD). GARP is also frequently hijacked by cancer cells to promote oncogenesis. This review summarizes the most important features of GARP biology described to date including gene regulation, protein expression and mechanism in activating latent TGF-ß, and the function of GARP in regulatory T cell biology and peripheral tolerance, as well as GARP's increasingly recognized roles in platelet-mediated cancer immune evasion. The promise for GARP-targeted strategy as a novel immunotherapy of cancer is also highlighted.

B lymphocytes confer immune tolerance via cell surface GARP-TGF-ß complex.

GARP, a cell surface docking receptor for binding and activating latent TGF-ß, is highly expressed by platelets and activated Tregs. While GARP is implicated in immune invasion in cancer, the roles of the GARP-TGF-ß axis in systemic autoimmune diseases are unknown. Although B cells do not express GARP at baseline, we found that the GARP-TGF-ß complex is induced on activated human and mouse B cells by ligands for multiple TLRs, including TLR4, TLR7, and TLR9. GARP overexpression on B cells inhibited their proliferation, induced IgA class-switching, and dampened T cell-independent antibody production. In contrast, B cell-specific deletion of GARP-encoding gene Lrrc32 in mice led to development of systemic autoimmune diseases spontaneously as well as worsening of pristane-induced lupus-like disease. Canonical TGF-ß signaling more readily upregulates GARP in Peyer patch B cells than in splenic B cells. Furthermore, we demonstrated that B cells are required for the induction of oral tolerance of T cell-dependent antigens via GARP. Our studies reveal for the first time to our knowledge that cell surface GARP-TGF-ß is an important checkpoint for regulating B cell peripheral tolerance, highlighting a mechanism of autoimmune disease pathogenesis.

Platelets subvert T cell immunity against cancer via GARP-TGFß axis.

Cancer-associated thrombocytosis has long been linked to poor clinical outcome, but the underlying mechanism is enigmatic. We hypothesized that platelets promote malignancy and resistance to therapy by dampening host immunity. We show that genetic targeting of platelets enhances adoptive T cell therapy of cancer. An unbiased biochemical and structural biology approach established transforming growth factor ß (TGFß) and lactate as major platelet-derived soluble factors to obliterate CD4+ and CD8+ T cell functions. Moreover, we found that platelets are the dominant source of functional TGFß systemically as well as in the tumor microenvironment through constitutive expression of the TGFß-docking receptor glycoprotein A repetitions predominant (GARP) rather than secretion of TGFß per se. Platelet-specific deletion of the GARP-encoding gene Lrrc32 blunted TGFß activity at the tumor site and potentiated protective immunity against both melanoma and colon cancer. Last, this study shows that T cell therapy of cancer can be substantially improved by concurrent treatment with readily available antiplatelet agents. We conclude that platelets constrain T cell immunity through a GARP-TGFß axis and suggest a combination of immunotherapy and platelet inhibitors as a therapeutic strategy against cancer.

Clients and Oncogenic Roles of Molecular Chaperone gp96/grp94.

As an endoplasmic reticulum heat shock protein (HSP) 90 paralogue, glycoprotein (gp) 96 possesses immunological properties by chaperoning antigenic peptides for activation of T cells. Genetic studies in the last decade have unveiled that gp96 is also an essential master chaperone for multiple receptors and secreting proteins including Toll-like receptors (TLRs), integrins, the Wnt coreceptor, Low Density Lipoprotein Receptor-Related Protein 6 (LRP6), the latent TGFß docking receptor, Glycoprotein A Repetitions Predominant (GARP), Glycoprotein (GP) Ib and insulin-like growth factors (IGF). Clinically, elevated expression of gp96 in a variety of cancers correlates with the advanced stage and poor survival of cancer patients. Recent preclinical studies have also uncovered that gp96 expression is closely linked to cancer progression in multiple myeloma, hepatocellular carcinoma, breast cancer and inflammation-associated colon cancer. Thus, gp96 is an attractive therapeutic target for cancer treatment. The chaperone function of gp96 depends on its ATPase domain, which is structurally distinct from other HSP90 members, and thus favors the design of highly selective gp96-targeted inhibitors against cancer. We herein discuss the strategically important oncogenic clients of gp96 and their underlying biology. The roles of cell-intrinsic gp96 in T cell biology are also discussed, in part because it offers another opportunity of cancer therapy by manipulating levels of gp96 in T cells to enhance host immune defense.

Surface Expression of TGFß Docking Receptor GARP Promotes Oncogenesis and Immune Tolerance in Breast Cancer.

GARP encoded by the Lrrc32 gene is the cell surface docking receptor for latent TGFß, which is expressed naturally by platelets and regulatory T cells (Treg). Although Lrrc32 is amplified frequently in breast cancer, the expression and relevant functions of GARP in cancer have not been explored. Here, we report that GARP exerts oncogenic effects, promoting immune tolerance by enriching and activating latent TGFß in the tumor microenvironment. We found that human breast, lung, and colon cancers expressed GARP aberrantly. In genetic studies in normal mammary gland epithelial and carcinoma cells, GARP expression increased TGFß bioactivity and promoted malignant transformation in immunodeficient mice. In breast carcinoma-bearing mice that were immunocompetent, GARP overexpression promoted Foxp3+ Treg activity, which in turn contributed to enhancing cancer progression and metastasis. Notably, administration of a GARP-specific mAb limited metastasis in an orthotopic model of human breast cancer. Overall, these results define the oncogenic effects of the GARP-TGFß axis in the tumor microenvironment and suggest mechanisms that might be exploited for diagnostic and therapeutic purposes. Cancer Res; 76(24); 7106-17. ©2016 AACR.

Assessment of heat shock protein 70 induction by heat in alfalfa varieties and constitutive overexpression in transgenic plants.

Heat shock proteins (HSPs) are molecular chaperones involved in many cellular functions. It has been shown that mammalian cytosolic HSP70 binds antigenic peptides mediating the activation of the immune system, and that it plays a determining role in tumour immunogenicity. This suggests that HSP70 may be used for the production of conjugated vaccines. Human and plant HSPs share high sequence similarity and some important biological functions in vitro. In addition, plant HSPs have no endotoxic side effects. Extraction of HSP70 from plants for use as vaccine adjuvant requires enhancing its concentration in plant tissues. In this work, we explored the possibility to produce HSP70 in both transgenic and non-transgenic plants, using alfalfa as a model species. First, a transcriptional analysis of a constitutive and an inducible HSP70 genes was conducted in Arabidopsis thaliana. Then the coding sequence of the inducible form was cloned and introduced into alfalfa by Agrobacterium-mediated transformation, and the accumulation of the protein in leaf tissue of transgenic plants was demonstrated. We also tested diverse alfalfa varieties for heat-inducible expression of endogenous HSP70, revealing variety-specific responses to heat shock.

GP96 is a GARP chaperone and controls regulatory T cell functions.

Molecular chaperones control a multitude of cellular functions via folding chaperone-specific client proteins. CD4+FOXP3+ Tregs play key roles in maintaining peripheral tolerance, which is subject to regulation by multiple molecular switches, including mTOR and hypoxia-inducible factor. It is not clear whether GP96 (also known as GRP94), which is a master TLR and integrin chaperone, controls Treg function. Using murine genetic models, we demonstrated that GP96 is required for Treg maintenance and function, as loss of GP96 resulted in instability of the Treg lineage and impairment of suppressive functions in vivo. In the absence of GP96, Tregs were unable to maintain FOXP3 expression levels, resulting in systemic accumulation of pathogenic IFN-γ-producing and IL-17-producing T cells. We determined that GP96 serves as an essential chaperone for the cell-surface protein glycoprotein A repetitions predominant (GARP), which is a docking receptor for latent membrane-associated TGF-ß (mLTGF-ß). The loss of both GARP and integrins on GP96-deficient Tregs prevented expression of mLTGF-ß and resulted in inefficient production of active TGF-ß. Our work demonstrates that GP96 regulates multiple facets of Treg biology, thereby placing Treg stability and immunosuppressive functions strategically under the control of a major stress chaperone.