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 herein show that genetic targeting of platelets significantly enhances adoptive T cell therapy of cancer. An unbiased biochemical and structural biology approach established transforming growth factor β (TGFβ) and lactate as the major platelet-derived soluble factors to obliterate CD4+ and CD8+ T cell functions. Moreover, we found that platelets are the dominant source of functional TGFβ systemically as well as in the tumor microenvironment through constitutive expression of TGFβ-docking receptor Glycoprotein A Repetitions Predominant (GARP) rather than secretion of TGFβ per se. Indeed, platelet-specific deletion of GARP-encoding gene Lrrc32 blunted TGFβ activity at the tumor site and potentiated protective immunity against both melanoma and colon cancer. Finally, we found that T cell therapy of cancer can be substantially improved by concurrent treatment with readily available anti-platelet agents. We conclude that platelets constrain T cell immunity though a GARP-TGFβ axis and suggest a combination of immunotherapy and platelet inhibitors as a therapeutic strategy against cancer.

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.

GARP encoded by the Lrrc32 gene is the cell surface docking receptor for latent TGF-β which is expressed naturally by platelets and regulatory T cells. 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 TGF-β 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 TGF-β bioactivity and promoted malignant transformation in immune deficient mice. In breast carcinoma-bearing mice that were immune competent, GARP overexpression promoted Foxp3+ regulatory T cell activity, which in turn contributed to enhancing cancer progression and metastasis. Notably, administration of a panel of GARP-specific monoclonal antibodies limited metastasis in an orthotopic model of human breast cancer. Overall, these results define the oncogenic effects of the GARP-TGF-β axis in the tumor microenvironment and suggest mechanisms that might be exploited for diagnostic and therapeutic purposes.

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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 co-receptor, Low Density Lipoprotein Receptor-Related Protein 6 (LRP6), the latent TGFβ 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.

Cancer-associated thrombocytosis and high concentrations of circulating transforming growth factor–β1 (TGF-β1) 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-β1 from platelets. We found that thrombin cleaves glycoprotein A repetitions predominant (GARP), a cell surface docking receptor for latent TGF-β1 (LTGF-β1) on platelets, resulting in liberation of active TGF-β1 from the GARP–LTGF-β1 complex. Furthermore, systemic inhibition of thrombin obliterates TGF-β1 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-β1 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-β1 release from platelets. We propose that blockade of GARP cleavage is a valuable therapeutic strategy to overcome cancer’s resistance to immunotherapy.

GARP (glycoprotein-A repetitions predominant) is a type I transmembrane cell surface docking receptor for latent transforming growth factor-β (TGF-β) that is abundantly expressed on regulatory T lymphocytes and platelets. GARP regulates the availability of membrane-bound latent TGF-β 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-β, 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.

Activated regulatory T (Treg) cells express the surface receptor glycoprotein-A repetitions predominant (GARP), which binds and activates latent TGFb. 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 þ and CD8 þ 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 À/À Treg cells were significantly reduced in the gut and exhibited a reduction in CD103 expression, a colon-specific migratory marker. In the coli-tis-associated colon cancer model, GARP on Treg cells dampened immune surveillance, and mice with GARP À/À Treg cells exhibited improved antitumor immunity. Thus, GARP empowers the functionality of Treg cells and their tissuespecific accumulation, highlighting the importance of cell surface TGFb in Treg function and GARP as a potential therapeutic target for colorectal cancer therapy. Significance: These findings uncover functions of membrane-bound TGFb and GARP that tune the activity of Treg cells, highlighting a potential treatment strategy in autoimmune diseases and cancer.