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.

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.

BackgroundLymphodepletion enhances adoptive T cell transfer (ACT) therapy by activating the innate immune system via microbes released from the radiation-injured gut. Microbial components, such as LPS, are key mediators of total body irradiation (TBI) enhancement, but our ability to strategically use these toll-like receptor (TLR) agonists to bolster the potency of T cell-based therapies for cancer remains elusive. Herein, we used TLR4 agonist LPS as a tool to address how and when to use TLR agonists to effectively improve cancer immunotherapy.MethodsTo determine the mechanisms of how innate immune activation via lymphodepletion potentiated antitumor T cell immunity, we utilized the pmel-1 melanoma mouse model. B16F10-bearing mice were preconditioned with 5Gy TBI and given a tripartite ACT therapy (consisting of transferred pmel-1 CD8+ T cells, vaccination with fowlpox encoding gp100, and IL-2) along with TLR4 agonist LPS. The timing of LPS administration and the requirement of individual components of the tripartite therapy were evaluated based on tumor growth and the phenotype of recovered splenocytes by flow cytometry. We also evaluated the role of non-toxic and clinically used TLR4 and TLR9 agonists—monophosphoryl lipid A (MPL) and CpG Oligodeoxynucleotide (CpG ODN), respectively— for ACT therapy.ResultsHere we report that while exogenous administration of LPS was able to enhance adoptively transferred CD8+ T cells’ tumor destruction, LPS treatment alone did not replace individual components of the tripartite ACT regimen, or obviate TBI. Moreover, we found that sequentially administering LPS during or one day prior to ACT therapy compromised tumor regression. In contrast, administering LPS after ACT potentiated the antitumor effectiveness of the regimen, thereby supporting the expansion of transferred tumor-specific CD8+ T cells over host CD4+ T cells. We also found that non-toxic TLR agonists MPL and CpG potentiated the antitumor activity of infused CD8+ T cells. Finally, TBI was no longer needed to regress tumors in mice who were depleted of host CD4+ T cells, given a tripartite ACT regimen and then treated with low dose LPS.ConclusionsCollectively, our results identify how and when to administer TLR agonists to augment T cell-based immunotherapy in the absence or presence of host preconditioning for treatment of advanced malignancies. Our findings have clinical implications for the design of next generation immune-based therapies for patients with cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-016-0110-8) contains supplementary material, which is available to authorized users.

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CD24 expression has been implicated in the oncogenesis of multiple types of cancer and high tumor expression is considered a poor prognosis factor; however, the role of CD24 in oral cancer progression is unknown. Unlike other cancer types, we found that higher CD24 levels in human oral cancers are correlated to lower clinical stage and better overall survival. We then dissected the role of CD24 and mechanisms in oral cancer pathogenesis in mice using a genetic strategy and demonstrated that CD24 deficiency increased the oral cavity tumor burden in response to the carcinogen 4-nitroquioline 1-oxide (4-NQO). Immune profile analysis showed a significant expansion as well as increased suppressive function of myeloid-derived suppressor cells (MDSCs) in CD24 ¡/¡ mice, but no apparent impairment in T cells, B cells, or dendritic cells. Further, studies with an orthotopically transplanted syngeneic squamous carcinoma model in the tongue of CD24 ¡/¡ and CD24 C/¡ mice confirmed the protective roles of CD24 against cancer. Moreover, the difference in tumor growth between CD24 ¡/¡ and CD24 C/¡ mice was blunted by immunodepletion of MDSCs. We conclude that CD24 expression impedes MDSC expansion and function, and thus slows oral cancer oncogenesis. This study is the first to examine the role of CD24 in a de novo oral cancer model, and it highlights the need to consider the immune regulatory roles of CD24 in the development of CD24-targeted therapy for cancer.

Platelet-induced cancer progression is a well-known process driven by mitogenic factors released by activated platelets, among them the most pro-oncogenic is Latent Transforming Growth Factor Beta (LTGFβ). Glycoprotein-A Repetitions Predominant Protein (GARP), a receptor for LTGFβ that enhances its bioactivation, is expressed by regulatory T cells, platelets, and several human cancers. Upon activation platelets dramatically upregulate GARP, suggesting that GARP might play a role in activating LTGFβ released upon platelet degranulation and thus in platelet-induced cancer progression. To address this point we employed a novel mouse model based on the platelet-specific knock-out of the GARP gene. We show that in activated platelets GARP is critical for the expression of surface LTGFβ and for its conversion to the bioactive form. Lack of GARP on platelets, indeed, resulted in the complete absence of serum active TGFβ. To investigate whether GARP plays a role in platelet-induced cancer progression we tested multiple tumor models and observed that genetic deletion of GARP enhanced adoptive T cell therapy of B16 melanoma. We also found that in MC38 colon tumor model GARP on platelets contributes to cancer progression by increasing TGFβ bioavailability, promoting regulatory T lymphocytes and myeloid cells, and favoring the immune evasion of cancer cells. These results demonstrate that platelet-specific deletion of GARP blunted TGFβ activity in the tumor microenvironment and boosted protective immunity against pre-established cancers. We conclude that platelets constrain anti-tumor immunity via a GARP-TGFβ axis and we propose the combination of immunotherapy and platelet inhibitors as a novel treatment strategy against cancer.

Lymphodepletion enhances adoptive T cell transfer (ACT) therapy by activating the innate immune system via microbes released from the radiation-injured gut. Microbial LPS is a key mediator of lymphodepletion enhancement, but our ability to use these TLR agonists to bolster the potency of T cell-based cancer therapies remains elusive. Herein, we used LPS as a tool to address how and when to use TLR agonists to improve cancer immunotherapy. We utilized the pmel-1 melanoma mouse model. B16F10-bearing mice were lymphodepleted with 5Gy total body irradiation (TBI) and given a tripartite ACT therapy (consisting of transferred pmel-1 CD8+ T cells, vaccination with fowlpox encoding gp100, and IL-2) along with TLR4 agonist LPS. The timing of LPS administration and the requirement of individual components of the tripartite therapy were evaluated. We discovered that while exogenous administration of LPS was able to enhance CD8+ T cells’ tumor destruction, LPS treatment alone did not replace individual components of the tripartite regimen. Interestingly, administering LPS one day before ACT compromised tumor regression. Conversely, administering LPS after ACT potentiated the antitumor effectiveness of the regimen, thereby supporting the expansion of transferred CD8+ T cells over host Treg cells. Non-toxic TLR agonists MPL and CpG also improved ACT therapy. Finally, TBI preconditioning was no longer needed to regress tumors in mice depleted of host CD4+ T cells, given a tripartite ACT regimen and then treated with a TLR agonist. Collectively, our results identify how and when to administer TLR agonists to augment ACT in the absence of host preconditioning. These findings have implications for the design of next generation T cell therapies.