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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.

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.

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.

BackgroundIL-27 is a heterodimeric cytokine consisting of IL 27p28 and Epstein-Barr virus-induced gene 3 (EBI3) that binds the IL-27 receptor subunit alpha and glycoprotein 130. IL-27 is produced by activated macrophages and dendritic cells and limits the intensity and duration of immune responses in the tumor microenvironment by inducing the expression of immunoregulatory receptors (PD-L1, TIM3, LAG-3, TIGIT) and inhibiting production of proinflammatory cytokines (IFNγ, IL-17, TNFα). The IL-27 subunit EBI3 is elevated in plasma from patients with certain cancers including renal cell carcinoma, where it correlates with poor outcome. Based on high expression of IL-27 transcript in tumors from patients with hepatocellular carcinoma (HCC), the role of IL-27 was further explored in patient samples and a mouse model of HCC.MethodsGene expression profiles from the Cancer Genome Atlas (TCGA) were analyzed to identify tumors with elevated IL-27 transcripts. Serum from patients with HCC was analyzed for levels of the IL-27 subunit EBI3. The ability of SRF388, a first-in-class IL-27-blocking antibody that binds to IL-27p28, to reverse IL-27-induced inhibition of cytokine production in human immune cell cultures from patients with HCC was assessed in vitro. Finally, the anti-tumor activity of SRF388 was assessed in an orthotopic murine model of HCC.ResultsTCGA expression data revealed that IL-27p28 transcripts were elevated in tumors from patients with HCC relative to other indications. Serum levels of EBI3 were: 1) elevated in a subset of HCC patients; 2) inversely correlated with survival; 3) independent of serum alpha-fetoprotein levels; and 4) elevated in both hepatitis B/C virus positive and negative patients. Treatment with SRF388 stimulated increased cytokine production in activated peripheral blood mononuclear cells from patients with HCC that was further enhanced when combined with PD-1 blockade. Furthermore, SRF388 inhibited the growth of orthotopic Hepa1-6 liver tumors. mRNA transcriptional profiling of treated tumors revealed that SRF388 profoundly altered the transcriptional landscape in this model. In particular, treatment with SRF388 inhibited expression of immunoregulatory receptors PD-L1 and TIGIT, repressed transcripts associated with TGF-β signaling, and altered myeloid and natural killer cell transcripts.ConclusionsThese data indicate that elevated IL-27 subunit EBI3 is a hallmark of HCC and is associated with poor outcomes in these patients. Blockade of IL-27 with SRF388, currently being evaluated in a Phase 1 clinical trial in patients with advanced solid tumors (NCT04374877), may represent a promising therapy for patients with HCC where it can potentiate anti-tumor immune responses.

Targeting tumour-infiltrating suppressive myeloid cells with a selective PI3Kγ inhibitor overcomes resistance to checkpoint blockade therapy in various mouse myeloid-rich tumour models. Causes of checkpoint blockade resistance Therapeutic blockade of immune checkpoints with antibodies against CTLA-4 and PD-1 has proved effective against some cancer types, but clinical benefit has been limited to a subset of patients. Here Olivier De Henau et al . show that resistance to checkpoint blockade is associated with a high level of infiltration by suppressive myeloid cells in various mouse tumour models. In addition, targeting the myeloid-derived suppressor cells with a selective inhibitor of the γ isoform of phosphoinositide 3-kinase (PI3Kγ) increases sensitivity to checkpoint blockade therapy in a melanoma mouse model. 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 1 . 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 2 , 3 , 4 . Growing evidence suggests that high infiltration of immune-suppressive myeloid cells correlates with poor prognosis and ICB resistance 5 , 6 . 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.

Modulation of PI3Kγ activity regulates macrophage polarization during inflammation and cancer, whilst combining PI3Kγ inhibition with immune checkpoint inhibitors leads to synergistic tumour-inhibitory effects. Macrophage PI 3-kinase γ controls immunosuppression Macrophages are specialized immune cells that can either stimulate or inhibit inflammation. Here Judith Varner and colleagues provide evidence that the activation state of a single macrophage kinase, the PI 3-kinase γ (PI3Kγ) isoform, can control the switch between immune suppression and immune stimulation in cancer and inflammatory disease. PI3Kγ inhibition abates the immunosuppressive functions of tumour-associated macrophages to promote T-cell cytotoxicity. Combining PI3Kγ inhibition with immune checkpoint blockade leads to additive or synergistic tumour-inhibitory effects. This work suggests that that inhibitory targeting of macrophage signalling pathways may provide a novel approach to improving long-term survival of cancer patients. Macrophages play critical, but opposite, roles in acute and chronic inflammation and cancer 1 , 2 , 3 , 4 , 5 . 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 1 , 2 , 3 , 4 , 5 , 6 , 7 . 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/EBPβ 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/EBPβ 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.

The study of brain development in humans is limited by the lack of tissue samples and suitable in vitro models. Here, we model early human neural tube development using human embryonic stem cells cultured in a microfluidic device. The approach, named microfluidic-controlled stem cell regionalization (MiSTR), exposes pluripotent stem cells to signaling gradients that mimic developmental patterning. Using a WNT-activating gradient, we generated a neural tissue exhibiting progressive caudalization from forebrain to midbrain to hindbrain, including formation of isthmic organizer characteristics. Single-cell transcriptomics revealed that rostro-caudal organization was already established at 24 h of differentiation, and that the first markers of a neural-specific transcription program emerged in the rostral cells at 48 h. The transcriptomic hallmarks of rostro-caudal organization recapitulated gene expression patterns of the early rostro-caudal neural plate in mouse embryos. Thus, MiSTR will facilitate research on the factors and processes underlying rostro-caudal neural tube patterning. Patterning of the human neural tube is modeled in a microfluidic device.

BackgroundB7-H7 is a unique immunomodulatory receptor that can suppress T and NK cell activation through KIR3DL3 and stimulate T and NK cells through TMIGD2. NextPoint has developed T cell engagers that bind CD3 and B7-H7 with differential ability to allow for TMIGD2 interaction. T cell engagers that spared TMIGD2 binding to B7-H7 demonstrated enhanced T cell activation and potency against B7-H7+ tumors compared to those that blocked this interaction. Additional in vitro assays demonstrated the specific role of TMIGD2 co-stimulation as the source of increased T cell engager activity. As a follow-on, NextPoint Therapeutics has generated trispecific constructs with separate domains targeting the tumor antigen B7-H7, CD3, and TMIGD2 for potent anti-tumor T cell activity. The TMIGD2 binding arm co-stimulates T cells independent of the natural B7-H7-TMIGD2 interaction, expanding the potential applicability of the antibody beyond B7-H7 expressing tumor cells to other tumor targets.MethodsBispecific CD3/B7-H7 T cell engagers were generated with and without TMIGD2 binding capabilities. T cell engagers were evaluated for T cell anti-tumor activity in vitro and in vivo. Upon confirmation of the specific role of TMIGD2, CD3/TMIGD2/B7-H7 trispecific antibodies were generated and compared to the parental CD3/B7-H7 bispecific antibody for binding and anti-tumor T cell activation using a Jurkat reporter system with or without overexpression of TMIGD2. The role of TMIGD2 co-stimulation in primary T cell activation studies was further explored.ResultsCD3/B7-H7 T cell engagers with TMIGD2 co-stimulation induced substantial T cell activation in vitro and had potent anti-tumor activity in B7-H7+ tumor models in vivo. Antibody blockade of TMIGD2 co-stimulation confirmed the specific role of TMIGD2 in the observed potency. CD3/TMIGD2/B7-H7 trispecific antibodies demonstrated even stronger tumor-specific immune cell activation as compared to the parental bispecific in Jurkat reporter cells lines and primary cell in vitro assays.ConclusionsCo-stimulation via TMIGD2 in a T cell engager demonstrated significant target dependent activation in vitro and in vivo. Trispecific antibodies targeting CD3, TMIGD2, and B7-H7 demonstrated potent anti-tumor immune activation. A trispecific T cell engager with a TMIGD2 co-stimulatory domain has the potential to induce particularly potent anti-tumor activity and potentially improve the potency of bispecific T cell engagers. As such, NextPoint has begun characterizing T cell engagers for additional tumor targets, confirming the broad utility of TMIGD2 co-stimulation.

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. Cross-sectional study. University research laboratory. 28 adults (mean age 23.36 ± 2.38 years, mean height 1.74 m ± 0.13, mean weight 77.95 kg ± 16.63). 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. 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. 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. •Clinical balance tests such as the BESS may be unable to detect deficits in recovering patients.•Research grade virtual reality can increase balance test responsiveness but can be challenging to administer.•This work demonstrates that a smartphone app can similarly increase balance assessment responsiveness.•Clinicians can use this technique to more easily bring virtual reality into their clinical practice.

This research thesis suggests that Complete Streets principles, and other related current street design principles and best practices, can complement the National Main Street Center’s design approach for use in rural small towns. The addition of these principles would strengthen the design component for the Main Street approach. Since the scope of current street design guidelines and principles are so broad, targeting the key principles of these programs, provides an appropriate level of detail to add to the Main Street design process. The resultant design proposals, using an improved design framework, could be scalable in both cost and implementation to accommodate the needs and the means of rural small towns. Sleepy Eye, Minnesota is used as a case study to demonstrate how the principles and elements of this approach can be scalable and flexible to improve a selected small-town main street. Complete Street principles and related street design best practices are a useful first step for rural small-town Main Street designs.