Endogenous IL-27 during toxoplasmosis limits early monocyte responses and their inflammatory activation by pathological T cells.

Mice that lack the genes for IL-27, or the IL-27 receptor, and infected with Toxoplasma gondii develop T cell-mediated pathology. Here, studies were performed to determine the impact of endogenous IL-27 on the immune response to T. gondii in wild-type (WT) mice. Analysis of infected mice revealed the early production of IL-27p28 by a subset of Ly6Chi, inflammatory monocytes, and sustained IL-27p28 production at sites of acute and chronic infection. Administration of anti-IL-27p28 prior to infection resulted in an early (day 5) increase in levels of macrophage and granulocyte activation, as well as enhanced effector T cell responses, as measured by both cellularity, cytokine production, and transcriptional profiling. This enhanced acute response led to immune pathology, while blockade during the chronic phase of infection resulted in enhanced T cell responses but no systemic pathology. In the absence of IL-27, the enhanced monocyte responses observed at day 10 were a secondary consequence of activated CD4+ T cells. Thus, in WT mice, IL-27 has distinct suppressive effects that impact innate and adaptive immunity during different phases of this infection. IMPORTANCE: The molecule IL-27 is critical in limiting the immune response to the parasite Toxoplasma gondii. In the absence of IL-27, a lethal, overactive immune response develops during infection. However, when exactly in the course of infection this molecule is needed was unclear. By selectively inhibiting IL-27 during this parasitic infection, we discovered that IL-27 was only needed during, but not prior to, infection. Additionally, IL-27 is only needed in the active areas in which the parasite is replicating. Finally, our work found that a previously unstudied cell type, monocytes, was regulated by IL-27, which contributes further to our understanding of the regulatory networks established by this molecule.

SRF617 Is a Potent Inhibitor of CD39 with Immunomodulatory and Antitumor Properties.

CD39 (ENTPD1) is a key enzyme responsible for degradation of extracellular ATP and is upregulated in the tumor microenvironment (TME). Extracellular ATP accumulates in the TME from tissue damage and immunogenic cell death, potentially initiating proinflammatory responses that are reduced by the enzymatic activity of CD39. Degradation of ATP by CD39 and other ectonucleotidases (e.g., CD73) results in extracellular adenosine accumulation, constituting an important mechanism for tumor immune escape, angiogenesis induction, and metastasis. Thus, inhibiting CD39 enzymatic activity can inhibit tumor growth by converting a suppressive TME to a proinflammatory environment. SRF617 is an investigational, anti-CD39, fully human IgG4 Ab that binds to human CD39 with nanomolar affinity and potently inhibits its ATPase activity. In vitro functional assays using primary human immune cells demonstrate that inhibiting CD39 enhances T-cell proliferation, dendritic cell maturation/activation, and release of IL-1ß and IL-18 from macrophages. In vivo, SRF617 has significant single-agent antitumor activity in human cell line-derived xenograft models that express CD39. Pharmacodynamic studies demonstrate that target engagement of CD39 by SRF617 in the TME inhibits ATPase activity, inducing proinflammatory mechanistic changes in tumor-infiltrating leukocytes. Syngeneic tumor studies using human CD39 knock-in mice show that SRF617 can modulate CD39 levels on immune cells in vivo and can penetrate the TME of an orthotopic tumor, leading to increased CD8+ T-cell infiltration. Targeting CD39 is an attractive approach for treating cancer, and, as such, the properties of SRF617 make it an excellent drug development candidate.

IL27 Signaling Serves as an Immunologic Checkpoint for Innate Cytotoxic Cells to Promote Hepatocellular Carcinoma.

Although inflammatory mechanisms driving hepatocellular carcinoma (HCC) have been proposed, the regulators of anticancer immunity in HCC remain poorly understood. We found that IL27 receptor (IL27R) signaling promotes HCC development in vivo. High IL27EBI3 cytokine or IL27RA expression correlated with poor prognosis for patients with HCC. Loss of IL27R suppressed HCC in vivo in two different models of hepatocarcinogenesis. Mechanistically, IL27R sig-naling within the tumor microenvironment restrains the cytotoxicity of innate cytotoxic lymphocytes. IL27R ablation enhanced their accumulation and activation, whereas depletion or functional impairment of innate cytotoxic cells abrogated the effect of IL27R disruption. Pharmacologic neutralization of IL27 signaling increased infiltration of innate cytotoxic lymphocytes with upregulated cytotoxic molecules and reduced HCC development. Our data reveal an unexpected role of IL27R signaling as an immunologic checkpoint regulating innate cytotoxic lymphocytes and promoting HCC of different etiologies, thus indicating a therapeutic potential for IL27 pathway blockade in HCC. SIGNIFICANCE: HCC, the most common form of liver cancer, is characterized by a poor survival rate and limited treatment options. The discovery of a novel IL27-dependent mechanism controlling anticancer cytotoxic immune response will pave the road for new treatment options for this devastating disease. This article is highlighted in the In This Issue feature, p. 1825.

GILT in Thymic Epithelial Cells Facilitates Central CD4 T Cell Tolerance to a Tissue-Restricted, Melanoma-Associated Self-Antigen.

Central tolerance prevents autoimmunity, but also limits T cell responses to potentially immunodominant tumor epitopes with limited expression in healthy tissues. In peripheral APCs, γ-IFN-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of disulfide bond-containing proteins, including the self-antigen and melanoma Ag tyrosinase-related protein 1 (TRP1). The role of GILT in thymic Ag processing and generation of central tolerance has not been investigated. We found that GILT enhanced the negative selection of TRP1-specific thymocytes in mice. GILT expression was enriched in thymic APCs capable of mediating deletion, namely medullary thymic epithelial cells (mTECs) and dendritic cells, whereas TRP1 expression was restricted solely to mTECs. GILT facilitated MHC class II-restricted presentation of endogenous TRP1 by pooled thymic APCs. Using bone marrow chimeras, GILT expression in thymic epithelial cells (TECs), but not hematopoietic cells, was sufficient for complete deletion of TRP1-specific thymocytes. An increased frequency of TRP1-specific regulatory T (Treg) cells was present in chimeras with increased deletion of TRP1-specific thymocytes. Only chimeras that lacked GILT in both TECs and hematopoietic cells had a high conventional T/Treg cell ratio and were protected from melanoma challenge. Thus, GILT expression in thymic APCs, and mTECs in particular, preferentially facilitates MHC class II-restricted presentation, negative selection, and increased Treg cells, resulting in a diminished antitumor response to a tissue-restricted, melanoma-associated self-antigen.

Smartphone virtual reality to increase clinical balance assessment responsiveness.

OBJECTIVE: To determine if a low cost smartphone based, clinically applicable virtual reality (VR) modification to the standard Balance Error Scoring System (BESS) can challenge postural stability beyond the traditional BESS. DESIGN: Cross-sectional study. SETTING: University research laboratory. PARTICIPANTS: 28 adults (mean age 23.36 ±â€¯2.38 years, mean height 1.74 m ±â€¯0.13, mean weight 77.95 kg ±â€¯16.63). MAIN OUTCOME MEASURES: BESS postural control errors and center of pressure (CoP) velocity were recorded during the BESS test and a VR modified BESS (VR-BESS). The VR-BESS used a headset and phone to display a rollercoaster ride to induce a visual and vestibular challenge to postural stability. RESULTS: The VR-BESS significantly increased total errors (20.93 vs. 11.42, p < 0.05) and CoP velocity summed across all stances and surfaces (52.96 cm/s vs. 37.73 cm/s, p < 0.05) beyond the traditional BESS. CONCLUSION: The VR-BESS provides a standardized, and effective way to increase postural stability challenge in the clinical setting. The VR-BESS can use any smartphone technology to induce postural stability deficits that may otherwise normalize with traditional testing. Thus, providing a unique relatively inexpensive and simple to operate clinical assessment tool and∖or training stimulus.

Nab2 maintains thymus cellularity with aging and stress.

Thymic cellularity is influenced by a variety of biological and environmental factors, such as age and stress; however, little is known about the molecular genetic mechanisms that regulate this process. Immediate early genes of the Early growth response (Egr) family have critical roles in immune function and response to environmental stress. The transcription factors, Egr1, Egr2 and Egr3, play roles in the thymus and in peripheral T-cell activation. Nab2, which binds Egrs 1, 2, and 3 as a co-regulator of transcription, also regulates peripheral T-cell activation. However, a role for Nab2 in the thymus has not been reported. Using Nab2-deficient (KO) mice we found that male Nab2KO mice have reduced thymus size and decreased numbers of thymocytes, compared with age-matched wildtype (WT) mice. Furthermore, the number of thymocytes in Nab2KO males decreases more rapidly with age. This effect is sex-dependent as female Nab2KO mice show neither reduced thymocyte numbers nor accelerated thymocyte loss with age, compared to female WT littermates. Since stress induces expression of Nab2 and the Egrs, we examined whether loss of Nab2 alters stress-induced decrease in thymic cellularity. Restraint stress induced a significant decrease in thymic cellularity in Nab2KO and WT mice, with significant changes in the thymocyte subset populations only in the Nab2KO mice. Stress reduced the percentage of DP cells by half and increased the percentage of CD4SP and CD8SP cells by roughly three-fold in Nab2KO mice. These findings indicate a requirement for Nab2 in maintaining thymocyte number in male mice with age and in response to stress.

PI3Kγ is a molecular switch that controls immune suppression.

Macrophages play critical, but opposite, roles in acute and chronic inflammation and cancer. In response to pathogens or injury, inflammatory macrophages express cytokines that stimulate cytotoxic T cells, whereas macrophages in neoplastic and parasitic diseases express anti-inflammatory cytokines that induce immune suppression and may promote resistance to T cell checkpoint inhibitors. Here we show that macrophage PI 3-kinase γ controls a critical switch between immune stimulation and suppression during inflammation and cancer. PI3Kγ signalling through Akt and mTor inhibits NFκB activation while stimulating C/EBPß activation, thereby inducing a transcriptional program that promotes immune suppression during inflammation and tumour growth. By contrast, selective inactivation of macrophage PI3Kγ stimulates and prolongs NFκB activation and inhibits C/EBPß activation, thus promoting an immunostimulatory transcriptional program that restores CD8+ T cell activation and cytotoxicity. PI3Kγ synergizes with checkpoint inhibitor therapy to promote tumour regression and increased survival in mouse models of cancer. In addition, PI3Kγ-directed, anti-inflammatory gene expression can predict survival probability in cancer patients. Our work thus demonstrates that therapeutic targeting of intracellular signalling pathways that regulate the switch between macrophage polarization states can control immune suppression in cancer and other disorders.

Overcoming resistance to checkpoint blockade therapy by targeting PI3Kγ in myeloid cells.

Recent clinical trials using immunotherapy have demonstrated its potential to control cancer by disinhibiting the immune system. Immune checkpoint blocking (ICB) antibodies against cytotoxic-T-lymphocyte-associated protein 4 or programmed cell death protein 1/programmed death-ligand 1 have displayed durable clinical responses in various cancers. Although these new immunotherapies have had a notable effect on cancer treatment, multiple mechanisms of immune resistance exist in tumours. Among the key mechanisms, myeloid cells have a major role in limiting effective tumour immunity. Growing evidence suggests that high infiltration of immune-suppressive myeloid cells correlates with poor prognosis and ICB resistance. These observations suggest a need for a precision medicine approach in which the design of the immunotherapeutic combination is modified on the basis of the tumour immune landscape to overcome such resistance mechanisms. Here we employ a pre-clinical mouse model system and show that resistance to ICB is directly mediated by the suppressive activity of infiltrating myeloid cells in various tumours. Furthermore, selective pharmacologic targeting of the gamma isoform of phosphoinositide 3-kinase (PI3Kγ), highly expressed in myeloid cells, restores sensitivity to ICB. We demonstrate that targeting PI3Kγ with a selective inhibitor, currently being evaluated in a phase 1 clinical trial (NCT02637531), can reshape the tumour immune microenvironment and promote cytotoxic-T-cell-mediated tumour regression without targeting cancer cells directly. Our results introduce opportunities for new combination strategies using a selective small molecule PI3Kγ inhibitor, such as IPI-549, to overcome resistance to ICB in patients with high levels of suppressive myeloid cell infiltration in tumours.

Diverse cellular and organismal functions of the lysosomal thiol reductase GILT.

Gamma-interferon-inducible lysosomal thiol reductase (GILT) is the only enzyme known to catalyze disulfide bond reduction in the endocytic pathway. GILT facilitates the presentation of a subset of epitopes from disulfide bond-containing antigens. Enhanced presentation of MHC class II-restricted epitopes alters central tolerance and modulates CD4+ T cell-mediated autoimmunity. Improved cross-presentation of viral epitopes results in improved cross-priming of viral-specific CD8+ T cells. GILT regulates the cellular redox state. In GILT-/- cells, there is a shift from the reduced to the oxidized form of glutathione, resulting in mitochondrial autophagy, decreased superoxide dismutase 2, and elevated superoxide levels. GILT expression diminishes cellular activation, including decreased phosphorylated ERK1/2, and decreases cellular proliferation. GILT enhances the activity of bacterial hemolysins, such as listeriolysin O, and increases bacterial replication and infection. GILT expression in cancer cells is associated with improved patient survival. These diverse roles of GILT are discussed.

An exhaustion-like phenotype constrains the activity of CD4+ T cells specific for a self and melanoma antigen.

While the immune system has the capacity to recognize and destroy melanoma, tolerance mechanisms often hinder the development of effective anti-tumor immune responses. Since many melanoma antigens are self proteins expressed in normal melanocytes, self antigen exposure before tumor development can negatively impact the function of T cells specific for these self/tumor antigens. However, the contribution of self tolerance to anti-melanoma T cell dysfunction remains largely unexplored. We have previously described a TCR transgenic (Tg) mouse model in which T cells specific for the self/melanoma antigen, tyrosinase-related protein 1 (TRP1), develop in the presence of endogenous TRP1 expression (Ag+) and diminished antigen presentation due to the absence of gamma-interferon-inducible lysosomal thiol reductase (GILT-/-). We show that TRP1-specific T cells from these Ag+GILT-/-Tg mice do not protect from melanoma tumor growth, fail to induce autoimmune vitiligo, and undergo diminished proliferation compared to T cells from Ag-GILT+/+Tg mice. Despite an increased frequency of TRP1-specific Treg cells in Ag+GILT-/-Tg mice compared to Ag-GILT+/+Tg animals, Treg cell depletion only partially rescues the proliferative capacity of T cells from TRP1-expressing mice, suggesting the involvement of additional suppressive mechanisms. An increased percentage of melanoma-specific T cells from Ag+GILT-/-Tg animals express PD-1, an inhibitory receptor associated with the maintenance of T cell exhaustion. Antibody blockade of PD-1 partially improves the ability of TRP1-specific T cells from Ag+GILT-/-Tg mice to produce IL-2. These findings demonstrate that melanoma-specific T cells exposed to a self/melanoma antigen in healthy tissue develop an exhaustion-like phenotype characterized by PD-1-mediated immunosuppression prior to encounter with tumor.

A stressful microenvironment: opposing effects of the endoplasmic reticulum stress response in the suppression and enhancement of adaptive tumor immunity.

The recent clinical success of immunotherapy in the treatment of certain types of cancer has demonstrated the powerful ability of the immune system to control tumor growth, leading to significantly improved patient survival. However, despite these promising results current immunotherapeutic strategies are still limited and have not yet achieved broad acceptance outside the context of metastatic melanoma. The limitations of current immunotherapeutic approaches can be attributed in part to suppressive mechanisms present in the tumor microenvironment that hamper the generation of robust antitumor immune responses thus allowing tumor cells to escape immune-mediated destruction. The endoplasmic reticulum (ER) stress response has recently emerged as a potent regulator of tumor immunity. The ER stress response is an adaptive mechanism that allows tumor cells to survive in the harsh growth conditions inherent to the tumor milieu such as low oxygen (hypoxia), low pH and low levels of glucose. Activation of ER stress can also alter the cancer cell response to therapies. In addition, the ER stress response promotes tumor immune evasion by inducing the production of protumorigenic inflammatory cytokines and impairing tumor antigen presentation. However, the ER stress response can boost antitumor immunity in some situations by enhancing the processing and presentation of tumor antigens and by inducing the release of immunogenic factors from stressed tumor cells. Here, we discuss the dualistic role of the ER stress response in the modulation of tumor immunity and highlight how strategies to either induce or block ER stress can be employed to improve the clinical efficacy of tumor immunotherapy.

GILT modulates CD4+ T-cell tolerance to the melanocyte differentiation antigen tyrosinase-related protein 1.

Gamma-IFN-inducible lysosomal thiol reductase (GILT) facilitates major histocompatibility complex class II-restricted processing through endocytic reduction of protein disulfide bonds and is necessary for efficient class II-restricted processing of melanocyte differentiation antigen, tyrosinase-related protein 1 (TRP1). Using class II-restricted, TRP1-specific T-cell receptor transgenic mice, we identify a role, to our knowledge, previously unreported, for GILT in the maintenance of tolerance to TRP1. TRP1-specific thymocytes are centrally deleted in the presence of GILT and TRP1. In contrast, CD4 single-positive thymocytes and peripheral T cells develop in the absence of GILT or TRP1, demonstrating that GILT is required for negative selection of TRP1-specific thymocytes. Although TRP1-specific T cells escape thymic deletion in the absence of GILT, they are tolerant to TRP1 and do not induce vilitigo. TRP1-specific T cells that develop in the absence of GILT have diminished IL-2 and IFN-γ production. Furthermore, GILT-deficient mice have a 4-fold increase in the percentage of TRP1-specific regulatory T (Treg) cells compared with TRP1-deficient mice, and depletion of Treg cells partially restores the ability of GILT-deficient TRP1-specific CD4(+) T cells to induce vitiligo. Thus, GILT has a critical role in regulating CD4(+) T-cell tolerance to an endogenous skin-restricted antigen relevant to controlling autoimmunity and generating effective immunotherapy for melanoma.

GILT accelerates autoimmunity to the melanoma antigen tyrosinase-related protein 1.

Melanocyte differentiation Ags, including tyrosinase-related protein (TRP) 1, are relevant to both autoimmune skin depigmentation (vitiligo) and tumor immunity, because they are expressed by both benign melanocytes and many malignant melanomas. Melanoma patients generate CD4(+) T cells that specifically recognize these proteins. TRP1 contains internal disulfide bonds and is presented by MHC class II molecules. Gamma-IFN-inducible lysosomal thiol reductase (GILT) facilitates the generation of class II-binding peptides by the endocytic reduction of protein disulfide bonds. We show in this study that GILT is required for efficient MHC class II-restricted processing of a TRP1 epitope in vitro and accelerates the onset of vitiligo in TRP1-specific TCR transgenic mice. The presence of GILT confers a small increase in the percentage of autoreactive T cells with an effector memory phenotype that may contribute to earlier disease onset. The onset of vitiligo is associated with a greater increase in the percentage of autoreactive T cells with an effector memory phenotype. Given that many self and tumor Ags have disulfide bonds and are presented on MHC class II, GILT is likely to be important in the pathogenesis of other CD4(+) T cell-mediated autoimmune diseases and for the development of effective cancer immunotherapy.

An orally active small molecule TGF-beta receptor I antagonist inhibits the growth of metastatic murine breast cancer.

BACKGROUND: Transforming growth factor beta (TGF-beta) plays a complex role in breast carcinogenesis. Initially functioning as a tumor suppressor, this cytokine later contributes to the progression of malignant cells by enhancing their invasive and metastatic potential as well as suppressing antitumor immunity. The purpose of this study was to investigate the efficacy of SM16, a novel small molecule ALK5 kinase inhibitor, to treat a highly metastatic, TGF-beta-producing murine mammary carcinoma (4T1). MATERIALS AND METHODS: Mice bearing established 4T1 tumors were treated with SM16 intraperitoneally (i.p.) or orally, and primary and metastatic tumor growth was assessed. RESULTS: SM16 inhibited Smad2 phosphorylation in cultured 4T1 tumor cells as well as primary and metastatic 4T1 tumor tissue. Blockade of TGF-beta signal transduction in 4T1 tumor cells by SM16 prevented TGF-beta-induced morphological changes and inhibited TGF-beta-induced invasion in vitro. When delivered via daily i.p. injection or orally through mouse chow, SM16 inhibited the growth of primary and metastatic 4T1 tumors. Splenocytes isolated from mice on the SM16 diet displayed enhanced IFN-gamma production and antitumor CTL activity. Furthermore, SM16 failed to inhibit the growth and metastasis of established 4T1 tumors in immunodeficient SCID mice. CONCLUSION: Taken together, the data indicate that the antitumor efficacy of SM16 is dependent on an immune-mediated mechanism and that SM16 may represent a safe and effective treatment for metastatic breast cancer.