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Triple negative breast cancer (TNBC) is known for its heterogeneous nature and aggressive onset, limited unresponsiveness to hormone therapies and immunotherapy as well as high likelihood of metastasis and recurrence. Since no targeted standard treatment options are available for TNBC, novel and effective therapeutic targets are urgently needed. Ion channels have emerged as possible novel therapeutic candidates for cancer therapy. We previously showed that GABAA β3 subunit are expressed at higher levels in TNBC cell lines than non-tumorigenic MCF10A cells. GABAA β3 subunit knockdown causes cell cycle arrest in TNBC cell lines via decreased cyclin D1 and increased p21 expression. However, it is not known if the upregulated GABAAR express at the cell-surface in TNBC and mediate Cl- flux. Cl- ions are known to play a role in cell-cycle progression in other cancers such as gastric cancer. Here, using surface biotinylation and (N-(Ethoxycarbonylmethyl)-6-Methoxyquinolinium Bromide) MQAE-dye based fluorescence quenching, we show that upregulated GABAAR are on the cell-surface in TNBC cell lines and mediate significantly higher chloride (Cl-) flux as compared to non-tumorigenic MCF10A cells. Moreover, this GABAAR mediated Cl- flux can be modulated by pharmacological agents and is decreased in TNBC cells with GABAA β3 subunit knockdown. Further, treatment of TNBC cells with bicuculline, a GABAAR antagonist reduced cell viability in TNBC cells Overall, these results point to an unexplored role of GABAAR mediated Cl- flux in TNBC. [ABSTRACT FROM AUTHOR]

Triple negative breast cancer (TNBC) is known for its heterogeneous nature and aggressive onset. The unresponsiveness to hormone therapies and immunotherapy and the toxicity of chemotherapeutics account for the limited treatment options for TNBC. Ion channels have emerged as possible therapeutic candidates for cancer therapy, but little is known about how ligand gated ion channels, specifically, GABA type A ligand-gated ion channel receptors (GABAAR), affect cancer pathogenesis. Our results show that the GABAA β3 subunit is expressed at higher levels in TNBC cell lines than non-tumorigenic cells, therefore contributing to the idea that limiting the GABAAR via knockdown of the GABAA β3 subunit is a potential strategy for decreasing the proliferation and migration of TNBC cells. We employed pharmacological and genetic approaches to investigate the role of the GABAA β3 subunit in TNBC proliferation, migration, and cell cycle progression. The results suggest that pharmacological antagonism or genetic knockdown of GABAA β3 subunit decreases TNBC proliferation and migration. In addition, GABAA β3 subunit knockdown causes cell cycle arrest in TNBC cell lines via decreased cyclin D1 and increased p21 expression. Our findings suggest that membrane bound GABAA receptors containing the β3 subunit can be further developed as a potential novel target for the treatment of TNBC. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor of "Cell Death and Differentiation" journal regarding the apoptosis repressor with a caspase recruitment domain, an apoptosis suppressor limited to terminally differentiated cells, and its induction in human breast cancer and confers chemo- and radiation-resistance.

Abstract Background Carotenoids are isoprenoid pigments, essential for photosynthesis and photoprotection in plants. The enzyme phytoene synthase (PSY) plays an essential role in mediating condensation of two geranylgeranyl diphosphate molecules, the first committed step in carotenogenesis. PSY are nuclear enzymes encoded by a small gene family consisting of three paralogous genes (PSY1-3) that have been widely characterized in rice, maize and sorghum. Results In wheat, for which yellow pigment content is extremely important for flour colour, only PSY1 has been extensively studied because of its association with QTLs reported for yellow pigment whereas PSY2 has been partially characterized. Here, we report the isolation of bread wheat PSY3 genes from a Renan BAC library using Brachypodium as a model genome for the Triticeae to develop Conserved Orthologous Set markers prior to gene cloning and sequencing. Wheat PSY3 homoeologous genes were sequenced and annotated, unravelling their novel structure associated with intron-loss events and consequent exonic fusions. A wheat PSY3 promoter region was also investigated for the presence of cis-acting elements involved in the response to abscisic acid (ABA), since carotenoids also play an important role as precursors of signalling molecules devoted to plant development and biotic/abiotic stress responses. Expression of wheat PSYs in leaves and roots was investigated during ABA treatment to confirm the up-regulation of PSY3 during abiotic stress. Conclusions We investigated the structural and functional determinisms of PSY genes in wheat. More generally, among eudicots and monocots, the PSY gene family was found to be associated with differences in gene copy numbers, allowing us to propose an evolutionary model for the entire PSY gene family in Grasses.

The primary CD8 T cell immune response constitutes a major mechanism to fight an infection by intra-cellular pathogens. We aim at assessing whether pathogen-specific dynamical parameters of the CD8 T cell response can be identified, based on measurements of CD8 T cell counts, using a modeling approach. We generated experimental data consisting in CD8 T cell counts kinetics during the response to three different live intra-cellular pathogens: two viruses (influenza, vaccinia) injected intranasally, and one bacteria ( Listeria monocytogenes ) injected intravenously. All pathogens harbor the same antigen (NP68), but differ in their interaction with the host. In parallel, we developed a mathematical model describing the evolution of CD8 T cell counts and pathogen amount during an immune response. This model is characterized by 9 parameters and includes relevant feedback controls. The model outputs were compared with the three data series and an exhaustive estimation of the parameter values was performed. By focusing on the ability of the model to fit experimental data and to produce a CD8 T cell population mainly composed of memory cells at the end of the response, critical parameters were identified. We show that a small number of parameters (2–4) define the main features of the CD8 T cell immune response and are characteristic of a given pathogen. Among these parameters, two are related to the effector CD8 T cell mediated control of cell and pathogen death. The parameter associated with memory cell death is shown to play no relevant role during the main phases of the CD8 T cell response, yet it becomes essential when looking at the predictions of the model several months after the infection. [ABSTRACT FROM AUTHOR]

The antifouling herbicide Irgarol 1051 has been detected in recent years in numerous estuaries, marinas, harbors and coastal areas, and in some harbors on Lake Geneva, but so far only a few studies have investigated the ecotoxicological effects of this compound on microalgae. The purpose of this study was to assess the ecotoxicological impact of Irgarol 1051 on the algal communities of Lake Geneva, and to compare its phytotoxicity to that of the common triazine herbicide, atrazine. We investigated the response of phytoplanktonic and periphytonic algal communities and single-species isolates collected from the lake, to the PS II inhibitor Irgarol 1051 (growth, proxy of photosynthetic activity and community structure). A short-term bioassay was developed based on in vivo fluorescence, together with nanocosm experiments with natural algal communities, and single-species tests on algal strains isolated from the lake. The toxicity of Irgarol 1051 towards periphyton and phytoplankton was shown to be higher than that of atrazine. Indications of the tolerance induced by this triazine in the algal communities of Lake Geneva, suggests that even at the levels of contamination reported in some parts of the lake, Irgarol 1051 is already exerting selection pressure. Information about sensitivities, selection and tolerance from laboratory experiments are used to explain the observations in natural microalgal communities from the lake. [Copyright &y& Elsevier]

Cell therapies such as tumor-infiltrating lymphocyte (TIL) therapy have shown promise in the treatment of patients with refractory solid tumors, with improvement in response rates and durability of responses nevertheless sought. To identify targets capable of enhancing the antitumor activity of T cell therapies, large-scale in vitro and in vivo clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens were performed, with the SOCS1 gene identified as a top T cell-enhancing target. In murine CD8+ T cell-therapy models, SOCS1 served as a critical checkpoint in restraining the accumulation of central memory T cells in lymphoid organs as well as intermediate (Texint) and effector (Texeff) exhausted T cell subsets derived from progenitor exhausted T cells (Texprog) in tumors. A comprehensive CRISPR tiling screen of the SOCS1-coding region identified sgRNAs targeting the SH2 domain of SOCS1 as the most potent, with an sgRNA with minimal off-target cut sites used to manufacture KSQ-001, an engineered TIL therapy with SOCS1 inactivated by CRISPR/Cas9. KSQ-001 possessed increased responsiveness to cytokine signals and enhanced in vivo antitumor function in mouse models. These data demonstrate the use of CRISPR/Cas9 screens in the rational design of T cell therapies.

Nuclear envelope proteins play an important role in regulating nuclear size and structure in cancer. Altered expression of nuclear lamins are found in many cancers and its expression is correlated with better clinical outcomes. The nucleus is the largest organelle in the cell with a diameter between 10 and 20 μm. Nuclear size significantly impacts cell migration. Nuclear structural changes are predicted to impact cancer metastasis by regulating cancer cell migration. Here we show emerin regulates nuclear structure in invasive breast cancer cells to impact cancer metastasis. Invasive breast cancer cells had 40% to 50% less emerin than control cells, which resulted in decreased nuclear size. Overexpression of GFP-emerin in invasive breast cancer cells rescued nuclear size and inhibited migration through 3.0 and 8.0 μm pores. Mutational analysis showed emerin binding to nucleoskeletal proteins was important for its regulation of nuclear structure, migration, and invasion. Importantly, emerin expression inhibited lung metastasis by 91% in orthotopic mouse models of breast cancer. Emerin nucleoskeleton-binding mutants failed to inhibit metastasis. These results support a model whereby emerin binding to the nucleoskeleton regulates nuclear structure to impact metastasis. In this model, emerin plays a central role in metastatic transformation, because decreased emerin expression during transformation causes the nuclear structural defects required for increased cell migration, intravasation, and extravasation. IMPLICATIONS: Modulating emerin expression and function represents new targets for therapeutic interventions of metastasis, because increased emerin expression rescued cancer metastasis., (©2021 American Association for Cancer Research.)

Ductal carcinoma in situ (DCIS) is one of the earliest stages of breast cancer (BCa). The mechanisms by which DCIS lesions progress to an invasive state while others remain indolent are yet to be fully characterized and both diagnosis and treatment of this pre-invasive disease could benefit from better understanding the pathways involved. While a decreased expression of Caveolin-1 (Cav-1) in the tumor microenvironment of patients with DCIS breast cancer was linked to progression to invasive breast cancer (IBC), the downstream effector(s) contributing to this process remain elusive. The current report shows elevated expression of Signal Transducer and Activator of Transcription 5a (STAT5a) within the DCIS-like lesions in Cav-1 KO mice following estrogen treatment and inhibition of STAT5a expression prevented the formation of these mammary lesions. In addition, STAT5a overexpression in a human DCIS cell line (MCF10DCIS.com) promoted their invasion, a process accelerated by estrogen treatment and associated with increased levels of the matrix metalloproteinase-9 (MMP-9) precursor. In sum, our results demonstrate a novel regulatory axis (Cav-1♦STAT5a♦MMP-9) in DCIS that is fully activated by the presence of estrogen. Our sudies suggest to further study phosphorylated STAT5a (Y694) as a potential biomarker to guide and predict outcome of DCIS patient population.

Triple-negative breast cancer (TNBC) displays an aggressive clinical course, heightened metastatic potential, and is linked to poor survival rates. Through its lack of expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), this subtype remains unresponsive to traditional targeted therapies. Undesirable and sometimes life-threatening side effects associated with current chemotherapeutic agents warrant the development of more targeted treatment options. Targeting signal transducer and activator of transcription 3 (STAT3), a transcription factor implicated in breast cancer (BCa) progression, has proven to be an efficient approach to halt cancer growth in vitro and in vivo . Currently, there are no FDA-approved STAT3 inhibitors for TNBC. Although pimozide, a FDA-approved antipsychotic drug, has been attributed a role as a STAT3 inhibitor in several cancers, its role on this pathway remains unexplored in TNBC. As a "one size fits all" approach cannot be applied to TNBC therapies due to the heterogeneous nature of this aggressive cancer, we hypothesized that STAT3 could be a novel biomarker of response to guide pimozide therapy. Using human cell lines representative of four TNBC subtypes (basal-like 1, basal-like 2, mesenchymal-like, mesenchymal stem-like), our current report demonstrates that pimozide significantly reduced their invasion and migration, an effect that was predicted by STAT3 phosphorylation on tyrosine residue 705 (Tyr705). Mechanistically, phosphorylated STAT3 (Tyr705) inhibition resulting from pimozide treatment caused a downregulation of downstream transcriptional targets such as matrix metalloproteinase-9 (MMP-9) and vimentin, both implicated in invasion and migration. The identification of biomarkers of response to TNBC treatments is an active area of research in the field of precision medicine and our results propose phosphorylated STAT3 (Tyr705) as a novel biomarker to guide pimozide treatment as an inhibitor of invasion and migration.

Triple negative breast cancer (TNBC) is a heterogeneous disease, which lacks expression of the estrogen receptor (ER), progesterone receptor (PR) and the human epidermal growth factor 2 receptor (HER2). This subtype of breast cancer has the poorest prognosis with limited therapies currently available, and hence additional options are needed. CAPER is a coactivator of the activator protein-1 (AP-1) (interacting specifically with the c-Jun component) and the ER and is known to be involved in human breast cancer pathogenesis. Recent published data have demonstrated a role for CAPER in TNBC and, as such, disrupting the function of CAPER with c-Jun could be a novel approach to treat TNBC patients. The data presented here shows the development and in vitro testing of CAPER-derived peptides that inhibit the coactivator activity of CAPER with c-Jun. These CAPER peptides result in a decrease in cell number and an increase in apoptosis in two TNBC cell lines, MDA-MB-231 and BT-549, while having no effect on the non-tumorigenic cell line MCF 10A. Additionally, two modes of action were demonstrated which appear to be cell line dependent: 1) a modulation of phosphorylated c-Jun leading to a decrease in Bcl-2 in MDA-MB-231 cells and a decrease in p21 in BT-549 cells and 2) a decrease in DNA repair proteins, leading to impaired DNA repair function in MDA-MB-231 cells. The data presented here supports further development of CAPER-derived peptides for the treatment of TNBC.

Negative checkpoint regulators (NCRs) temper the T cell immune response to self-antigens and limit the development of autoimmunity. Unlike all other NCRs that are expressed on activated T lymphocytes, V-type immunoglobulin domain-containing suppressor of T cell activation (VISTA) is expressed on naïve T cells. We report an unexpected heterogeneity within the naïve T cell compartment in mice, where loss of VISTA disrupted the major quiescent naïve T cell subset and enhanced self-reactivity. Agonistic VISTA engagement increased T cell tolerance by promoting antigen-induced peripheral T cell deletion. Although a critical player in naïve T cell homeostasis, the ability of VISTA to restrain naïve T cell responses was lost under inflammatory conditions. VISTA is therefore a distinctive NCR of naïve T cells that is critical for steady-state maintenance of quiescence and peripheral tolerance., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Tumor hypoxia is a negative prognostic factor that is implicated in oncogenic signal activation, immune escape, and resistance to treatment. Identifying the mechanistic role of hypoxia in immune escape and resistance to immune-checkpoint inhibitors may aid the identification of therapeutic targets. We and others have shown that V-domain Ig suppressor of T-cell activation (VISTA), a negative checkpoint regulator in the B7 family, is highly expressed in the tumor microenvironment in tumor models and primary human cancers. In this study, we show that VISTA and HIF1α activity are correlated in a cohort of colorectal cancer patients. High VISTA expression was associated with worse overall survival. We used the CT26 colon cancer model to investigate the regulation of VISTA by hypoxia. Compared with less hypoxic tumor regions or draining lymph nodes, regions of profound hypoxia in the tumor microenvironment were associated with increased VISTA expression on tumor-infiltrating myeloid-derived suppressor cells (MDSC). Using chromatin immunoprecipitation and genetic silencing, we show that hypoxia-inducible factor (HIF)-1α binding to a conserved hypoxia response element in the VISTA promoter upregulated VISTA on myeloid cells. Further, antibody targeting or genetic ablation of VISTA under hypoxia relieved MDSC-mediated T-cell suppression, revealing VISTA as a mediator of MDSC function. Collectively, these data suggest that targeting VISTA may mitigate the deleterious effects of hypoxia on antitumor immunity., (©2019 American Association for Cancer Research.)

A pollen/food-associated syndrome (PFAS) has been described between peach and cypress pollen. Cross-reactive allergens were characterized which belong to the Gibberellin-regulated protein (GRP) family, BP14 in cypress pollen and Pru p 7 in peach. GRP are small cationic protein with anti-microbial properties. A patient suffering from a peach/cypress syndrome was explored clinically and biologically using 2 types of immunoglobulin E (IgE) multiarray microchip, immunoblots and a basophil activation test to assess the clinical relevance of various extracts and purified allergens from fruits or cypress pollen. In addition to PR10 sensitization, the patient showed specific IgE to Pru p 7, BP14 and allergen from pomegranate. These last 3 allergens and allergenic sources are able to induce ex vivo basophil activation characterized by the monitoring of the expression of CD63 and CD203c, both cell surface markers correlated with a basophil mediator release. Up to 100% of cells expressed CD203c at 50 ng/mL of BP14 protein. In contrast, snakin-1, a GRP from potato sharing 82% sequence identity with Pru p 7 did not activate patient's basophils. These results strongly suggest that, like Pru p 7, BP14 is a clinically relevant allergenic GRP from pollen. Allergen members of this newly described protein family are good candidates for PFAS where no cross-reactive allergens have been characterized., Competing Interests: There are no financial or other issues that might lead to conflict of interest., (Copyright © 2019 The Korean Academy of Asthma, Allergy and Clinical Immunology · The Korean Academy of Pediatric Allergy and Respiratory Disease.)

Breast cancers (BCas) that lack expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) are referred to as triple negative breast cancers (TNBCs) and have the poorest clinical outcome. Once these aggressive tumors progress to distant organs, the median survival decreases to 12 months. With endocrine therapies being ineffective in this BCa subtype, highly toxic chemo- and radiation therapies are the only options. A better understanding of the functional role(s) of molecular targets contributing to TNBC progression could help in the design and development of new treatments that are more targeted with less toxicity. CAPER (Co-activator of AP-1 and ER) is a nuclear transcriptional co-activator that was recently involved in ER-positive BCa progression, however its role in hormone-independent cancers remains unknown. Our current report demonstrates that CAPER expression is upregulated in human TNBC specimens compared to normal breast tissue and that its selective downregulation through a lentiviral-mediated shRNA knockdown approach resulted in decreased cell numbers in MDA-MB-231 and BT549 TNBC cell lines without affecting the growth of non-tumorigenic cell line MCF-10A. Concordant with these observations, CAPER knockdown was also associated with a decrease in DNA repair proteins leading to a marked increase in apoptosis, through caspase-3/7 activation without any changes in cell cycle. Collectively, we propose CAPER as an important signaling molecule in the development of TNBC linked to DNA repair mechanisms, which could lead to new therapeutic modalities for the treatment of this aggressive cancer., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

DC-SIGN (CD209/CLEC4L) is a C-type lectin receptor (CLR) that serves as a reliable cell-surface marker of interleukin 4 (IL-4)-activated human macrophages [M(IL-4)], which historically represent the most studied subset within the M2 spectrum of macrophage activation. Although DC-SIGN plays important roles in Mycobacterium tuberculosis (Mtb) interactions with dendritic cells, its contribution to the Mtb-macrophage interaction remains poorly understood. Since high levels of IL-4 are correlated with tuberculosis (TB) susceptibility and progression, we investigated the role of DC-SIGN in M(IL-4) macrophages in the TB context. First, we demonstrate that DC-SIGN expression is present both in CD68 + macrophages found in tuberculous pulmonary lesions of non-human primates, and in the CD14 + cell population isolated from pleural effusions obtained from TB patients (TB-PE). Likewise, we show that DC-SIGN expression is accentuated in M(IL-4) macrophages derived from peripheral blood CD14 + monocytes isolated from TB patients, or in macrophages stimulated with acellular TB-PE, arguing for the pertinence of DC-SIGN-expressing macrophages in TB. Second, using a siRNA-mediated gene silencing approach, we performed a transcriptomic analysis of DC-SIGN-depleted M(IL-4) macrophages and revealed the upregulation of pro-inflammatory signals in response to challenge with Mtb, as compared to control cells. This pro-inflammatory gene signature was confirmed by RT-qPCR, cytokine/chemokine-based protein array, and ELISA analyses. We also found that inactivation of DC-SIGN renders M(IL-4) macrophages less permissive to Mtb intracellular growth compared to control cells, despite the equal level of bacteria uptake. Last, at the molecular level, we show that DC-SIGN interferes negatively with the pro-inflammatory response and control of Mtb intracellular growth mediated by another CLR, Dectin-1 (CLEC7A). Collectively, this study highlights a dual role for DC-SIGN as, on the one hand, being a host factor granting advantage for Mtb to parasitize macrophages and, on the other hand, representing a molecular switch to turn off the pro-inflammatory response in these cells to prevent potential immunopathology associated to TB.

Rationale: In addition to their well-known function as antibody-producing cells, B lymphocytes can markedly influence the course of infectious or noninfectious diseases via antibody-independent mechanisms. In tuberculosis (TB), B cells accumulate in lungs, yet their functional contribution to the host response remains poorly understood., Objectives: To document the role of B cells in TB in an unbiased manner., Methods: We generated the transcriptome of B cells isolated from Mycobacterium tuberculosis (Mtb)-infected mice and validated the identified key pathways using in vitro and in vivo assays. The obtained data were substantiated using B cells from pleural effusion of patients with TB., Measurements and Main Results: B cells isolated from Mtb-infected mice displayed a STAT1 (signal transducer and activator of transcription 1)-centered signature, suggesting a role for IFNs in B-cell response to infection. B cells stimulated in vitro with Mtb produced type I IFN, via a mechanism involving the innate sensor STING (stimulator of interferon genes), and antagonized by MyD88 (myeloid differentiation primary response 88) signaling. In vivo, B cells expressed type I IFN in the lungs of Mtb-infected mice and, of clinical relevance, in pleural fluid from patients with TB. Type I IFN expression by B cells induced an altered polarization of macrophages toward a regulatory/antiinflammatory profile in vitro. In vivo, increased provision of type I IFN by B cells in a murine model of B cell-restricted Myd88 deficiency correlated with an enhanced accumulation of regulatory/antiinflammatory macrophages in Mtb-infected lungs., Conclusions: Type I IFN produced by Mtb-stimulated B cells favors macrophage polarization toward a regulatory/antiinflammatory phenotype during Mtb infection.

Utilization of negative checkpoint regulators (NCRs) for cancer immunotherapy has garnered significant interest with the completion of clinical trials demonstrating efficacy. While the results of monotherapy treatments are compelling, there is increasing emphasis on combination treatments in an effort to increase response rates to treatment. One of the most recently discovered NCRs is VISTA (V-domain Ig-containing Suppressor of T cell Activation). In this review, we describe the functions of this molecule in the context of cancer immunotherapy. We also discuss factors that may influence the use of anti-VISTA antibody in combination therapy and how genomic analysis may assist in providing indications for treatment., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Immune response against pathogens is a tightly regulated process that must ensure microbial control while preserving integrity of the infected organs. Tuberculosis (TB) is a paramount example of a chronic infection in which antimicrobial immunity is protective in the vast majority of infected individuals but can become detrimental if not finely tuned. Here, we report that C-type lectin dendritic cell (DC) immunoreceptor (DCIR), a key component in DC homeostasis, is required to modulate lung inflammation and bacterial burden in TB. DCIR is abundantly expressed in pulmonary lesions in Mycobacterium tuberculosis-infected nonhuman primates during both latent and active disease. In mice, we found that DCIR deficiency impairs STAT1-mediated type I IFN signaling in DCs, leading to increased production of IL-12 and increased differentiation of T lymphocytes toward Th1 during infection. As a consequence, DCIR-deficient mice control M. tuberculosis better than WT animals but also develop more inflammation characterized by an increased production of TNF and inducible NOS (iNOS) in the lungs. Altogether, our results reveal a pathway by which a C-type lectin modulates the equilibrium between infection-driven inflammation and pathogen's control through sustaining type I IFN signaling in DCs., Competing Interests: The authors declare no conflict of interest.

Negative checkpoint regulators function to restrain T cell responses to maintain tolerance and limit immunopathology. However, in the setting of malignancy, these pathways work in concert to promote immune-mediate escape leading to the development of a clinically overt cancer. In the recent years, clinical trials demonstrating the efficacy of blocking antibodies against these molecules have invigorated the field of immunotherapy. In this review, we discuss the current understanding on established NCR blockade and how strategic combination therapy with anti-VISTA antibody can be used to target multiple non-redundant NCR pathways.

In the last two years, clinical trials with blocking antibodies to the negative checkpoint regulators CTLA-4 and PD-1 have rekindled the hope for cancer immunotherapy. Multiple negative checkpoint regulators protect the host against autoimmune reactions but also restrict the ability of T cells to effectively attack tumors. Releasing these brakes has emerged as an exciting strategy for cancer treatment. Conversely, these pathways can be manipulated to achieve durable tolerance for treatment of autoimmune diseases and transplantation. In the future, treatment may involve combination therapy to target multiple cell types and stages of the adaptive immune responses. In this review, we describe the current knowledge on the recently discovered negative checkpoint regulators, future targets for immunotherapy.

Understanding the molecular components of immune recognition of the tuberculosis (TB) bacillus, Mycobacterium tuberculosis, can help designing novel strategies to combat TB. Here, we identify collectin CL-LK as a novel soluble C-type lectin able to bind M. tuberculosis, and characterize mycobacterial mannose-capped lipoarabinomannan as a primary ligand for CL-LK. Mice deficient in CL-K1, one of the CL-LK subunits, do not display altered susceptibility to M. tuberculosis. However, we found that the amount of CL-LK in the serum of patients with active TB is reduced, compared to that in controls, and correlates inversely to the magnitude of the immune response to the pathogen. These findings indicate that CL-LK might be of interest for future diagnostic and treatment monitoring purposes.

Mast cells (MCs) play an important role in both inflammatory and immunosuppressive responses [1]. The importance of MCs in maintaining peripheral tolerance was discovered in a FoxP3(+) regulatory T-cell (Treg)-mediated skin transplant model [2]. MCs can directly mediate tolerance by releasing anti-inflammatory mediators (reviewed in ref. 3) or by interacting with other immune cells in the graft. Here we will present protocols used to study the role of MCs in peripheral tolerance with the emphasis on how MCs can regulate T-cell functionality. First we will introduce the skin transplant model followed by reconstitution of mast cell-deficient mice (B6.Cg-Kit (W-sh) ). This includes the preparation of MCs from the bone marrow. Finally the methods used to study the influence of MCs on T-cell responses and Treg functionality will be presented by modulating the balance between tolerance and inflammation.

Atherosclerosis is a complex disease initiated by the vascular accumulation of lipoproteins in the sub-endothelial space, followed by the infiltration of monocytes into the arterial intima. Caveolin-1 (Cav-1) plays an essential role in the regulation of cellular cholesterol metabolism and of various signaling pathways. In order to study specifically the role of macrophage Cav-1 in atherosclerosis, we used Cav-1 (-/-) Apoe (-/-) mice and transplanted them with bone marrow (BM) cells obtained from Cav-1 (+/+) Apoe (-/-) or Cav-1 (-/-) Apoe (-/-) mice and vice versa. We found that Cav-1 (+/+) mice harboring Cav-1 (-/-) BM-derived macrophages developed significantly larger lesions than Cav-1 (+/+) mice harboring Cav-1 (+/+) BM-derived macrophages. Cav-1 (-/-) macrophages were more susceptible to apoptosis and more prone to induce inflammation. The present study provides clear evidence that the absence of Cav-1 in macrophage is pro-atherogenic, whereas its absence in endothelial cells protects against atherosclerotic lesion formation. These findings demonstrate the cell-specific role of Cav-1 during the development of this disease.

V domain-containing Ig suppressor of T-cell activation (VISTA) is a negative checkpoint regulator that suppresses T cell-mediated immune responses. Previous studies using a VISTA-neutralizing monoclonal antibody show that VISTA blockade enhances T-cell activation. The current study describes a comprehensive characterization of mice in which the gene for VISTA has been deleted. Despite the apparent normal hematopoietic development in young mice, VISTA genetic deficiency leads to a gradual accumulation of spontaneously activated T cells, accompanied by the production of a spectrum of inflammatory cytokines and chemokines. Enhanced T-cell responsiveness was also observed upon immunization with neoantigen. Despite the presence of multiorgan chronic inflammation, aged VISTA-deficient mice did not develop systemic or organ-specific autoimmune disease. Interbreeding of the VISTA-deficient mice with 2D2 T-cell receptor transgenic mice, which are predisposed to the development of experimental autoimmune encephalomyelitis, drastically enhanced disease incidence and intensity. Disease development is correlated with the increase in the activation of encephalitogenic T cells in the periphery and enhanced infiltration into the CNS. Taken together, our data suggest that VISTA is a negative checkpoint regulator whose loss of function lowers the threshold for T-cell activation, allowing for an enhanced proinflammatory phenotype and an increase in the frequency and intensity of autoimmunity under susceptible conditions.

V-domain Ig suppressor of T-cell activation (VISTA) is a novel negative checkpoint ligand that is homologous to PD-L1 and suppresses T-cell activation. This study demonstrates the multiple mechanisms whereby VISTA relieves negative regulation by hematopoietic cells and enhances protective antitumor immunity. VISTA is highly expressed on myeloid cells and Foxp3(+)CD4(+) regulatory cells, but not on tumor cells within the tumor microenvironment (TME). VISTA monoclonal antibody (mAb) treatment increased the number of tumor-specific T cells in the periphery and enhanced the infiltration, proliferation, and effector function of tumor-reactive T cells within the TME. VISTA blockade altered the suppressive feature of the TME by decreasing the presence of monocytic myeloid-derived suppressor cells and increasing the presence of activated dendritic cells within the tumor microenvironment. In addition, VISTA blockade impaired the suppressive function and reduced the emergence of tumor-specific Foxp3(+)CD4(+) regulatory T cells. Consequently, VISTA mAb administration as a monotherapy significantly suppressed the growth of both transplantable and inducible melanoma. Initial studies explored a combinatorial regimen using VISTA blockade and a peptide-based cancer vaccine with TLR agonists as adjuvants. VISTA blockade synergized with the vaccine to effectively impair the growth of established tumors. Our study therefore establishes a foundation for designing VISTA-targeted approaches either as a monotherapy or in combination with additional immune-targeted strategies for cancer immunotherapy., (©2014 AACR.)

Several specific lipids of the cell envelope are implicated in the pathogenesis of M. tuberculosis (Mtb), including phthiocerol dimycocerosates (DIM) that have clearly been identified as virulence factors. Others, such as trehalose-derived lipids, sulfolipids (SL), diacyltrehaloses (DAT) and polyacyltrehaloses (PAT), are believed to be essential for Mtb virulence, but the details of their role remain unclear. We therefore investigated the respective contribution of DIM, DAT/PAT and SL to tuberculosis by studying a collection of mutants, each with impaired production of one or several lipids. We confirmed that among those with a single lipid deficiency, only strains lacking DIM were affected in their replication in lungs and spleen of mice in comparison to the WT Mtb strain. We found also that the additional loss of DAT/PAT, and to a lesser extent of SL, increased the attenuated phenotype of the DIM-less mutant. Importantly, the loss of DAT/PAT and SL in a DIM-less background also affected Mtb growth in human monocyte-derived macrophages (hMDMs). Fluorescence microscopy revealed that mutants lacking DIM or DAT/PAT were localized in an acid compartment and that bafilomycin A1, an inhibitor of phagosome acidification, rescued the growth defect of these mutants. These findings provide evidence for DIM being dominant virulence factors that mask the functions of lipids of other families, notably DAT/PAT and to a lesser extent of SL, which we showed for the first time to contribute to Mtb virulence., (© 2013 John Wiley & Sons Ltd.)

CAPER is an estrogen receptor (ER) co-activator that was recently shown to be involved in human breast cancer pathogenesis. Indeed, we reported increased expression of CAPER in human breast cancer specimens. We demonstrated that CAPER was undetectable or expressed at relatively low levels in normal breast tissue and assumed a cytoplasmic distribution. In contrast, CAPER was expressed at higher levels in ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) specimens, where it assumed a predominantly nuclear distribution. However, the functional role of CAPER in human breast cancer initiation and progression remained unknown. Here, we used a lentiviral-mediated gene silencing approach to reduce the expression of CAPER in the ER-positive human breast cancer cell line MCF-7. The proliferation and tumorigenicity of MCF-7 cells stably expressing control or human CAPER shRNAs was then determined via both in vitro and in vivo experiments. Knockdown of CAPER expression significantly reduced the proliferation of MCF-7 cells in vitro. Importantly, nude mice injected with MCF-7 cells harboring CAPER shRNAs developed smaller tumors than mice injected with MCF-7 cells harboring control shRNAs. Mechanistically, tumors derived from mice injected with MCF-7 cells harboring CAPER shRNAs displayed reduced expression of the cell cycle regulators PCNA, MCM7, and cyclin D1, and the protein synthesis marker 4EBP1. In conclusion, knockdown of CAPER expression markedly reduced human breast cancer cell proliferation in both in vitro and in vivo settings. Mechanistically, knockdown of CAPER abrogated the activity of proliferative and protein synthesis pathways.

Plasmacytoid dendritic cells (pDC) are key regulators of antiviral immunity. In previous studies, we reported that pDC-infiltrating human primary breast tumors represent an independent prognostic factor associated with poor outcome. To understand this negative impact of tumor-associated pDC (TApDC), we developed an orthotopic murine mammary tumor model that closely mimics the human pathology, including pDC and regulatory T cell (Treg) infiltration. We showed that TApDC are mostly immature and maintain their ability to internalize antigens in vivo and to activate CD4(+) T cells. Most importantly, TApDC were specifically altered for cytokine production in response to Toll-like receptor (TLR)-9 ligands in vitro while preserving unaltered response to TLR7 ligands (TLR7L). In vivo pDC depletion delayed tumor growth, showing that TApDC provide an immune-subversive environment, most likely through Treg activation, thus favoring tumor progression. However, in vivo intratumoral administration of TLR7L led to TApDC activation and displayed a potent curative effect. Depletion of pDC and type I IFN neutralization prevented TLR7L antitumoral effect. Our results establish a direct contribution of TApDC to primary breast tumor progression and rationalize the application of TLR7 ligands to restore TApDC activation in breast cancer. Cancer Res; 73(15); 4629-40. ©2013 AACR.

Caveolin-1 (Cav-1) is a critical regulator of tumor progression in a variety of cancers where it has been shown to act as either a tumor suppressor or tumor promoter. In glioblastoma multiforme, it has been previously demonstrated to function as a putative tumor suppressor. Our studies here, using the human glioblastoma-derived cell line U-87MG, further support the role of Cav-1 as a negative regulator of tumor growth. Using a lentiviral transduction approach, we were able to stably overexpress Cav-1 in U-87MG cells. Gene expression microarray analyses demonstrated significant enrichment in gene signatures corresponding to downregulation of MAPK, PI3K/AKT and mTOR signaling, as well as activation of apoptotic pathways in Cav-1-overexpressing U-87MG cells. These same gene signatures were later confirmed at the protein level in vitro. To explore the ability of Cav-1 to regulate tumor growth in vivo, we further show that Cav-1-overexpressing U-87MG cells display reduced tumorigenicity in an ectopic xenograft mouse model, with marked hypoactivation of MAPK and PI3K/mTOR pathways. Finally, we demonstrate that Cav-1 overexpression confers sensitivity to the most commonly used chemotherapy for glioblastoma, temozolomide. In conclusion, Cav-1 negatively regulates key cell growth and survival pathways and may be an effective biomarker for predicting response to chemotherapy in glioblastoma.

Introduction: Previous studies have identified cholesterol as an important regulator of breast cancer development. High-density lipoprotein (HDL) and its cellular receptor, the scavenger receptor class B type I (SR-BI) have both been implicated in the regulation of cellular cholesterol homeostasis, but their functions in cancer remain to be established., Methods: In the present study, we have examined the role of HDL and SR-BI in the regulation of cellular signaling pathways in breast cancer cell lines and in the development of tumor in a mouse xenograft model., Results: Our data show that HDL is capable of stimulating migration and can activate signal transduction pathways in the two human breast cancer cell lines, MDA-MB-231 and MCF7. Furthermore, we also show that knockdown of the HDL receptor, SR-BI, attenuates HDL-induced activation of the phosphatidylinositol 3-kinase (PI3K)/protein Kinase B (Akt) pathway in both cell lines. Additional investigations show that inhibition of the PI3K pathway, but not that of the mitogen-activated protein kinase (MAPK) pathway, could lead to a reduction in cellular proliferation in the absence of SR-BI. Importantly, whereas the knockdown of SR-BI led to decreased proliferation and migration in vitro, it also led to a significant reduction in tumor growth in vivo. Most important, we also show that pharmacological inhibition of SR-BI can attenuate signaling and lead to decreased cellular proliferation in vitro. Taken together, our data indicate that both cholesteryl ester entry via HDL-SR-BI and Akt signaling play an essential role in the regulation of cellular proliferation and migration, and, eventually, tumor growth., Conclusions: These results identify SR-BI as a potential target for the treatment of breast cancer.

Nutrient deprivation based on the loss of essential amino acids by catabolic enzymes in the microenvironment is a critical means to control inflammatory responses and immune tolerance. Here we report the novel finding that Tph-1 (tryptophan hydroxylase-1), a synthase which catalyses the conversion of tryptophan to serotonin and exhausts tryptophan, is a potent regulator of immunity. In models of skin allograft tolerance, tumor growth, and experimental autoimmune encephalomyelitis, Tph-1 deficiency breaks allograft tolerance, induces tumor remission, and intensifies neuroinflammation, respectively. All of these effects of Tph-1 deficiency are independent of its downstream product serotonin. Because mast cells (MCs) appear to be the major source of Tph-1 and restoration of Tph-1 in the MC compartment in vivo compensates for the defect, these experiments introduce a fundamentally new mechanism of MC-mediated immune suppression that broadly impacts multiple arms of immunity.

Infiltration and dysfunction of immune cells have been documented in many types of cancers. We previously reported that plasmacytoid dendritic cells (pDC) within primary breast tumors correlate with an unfavorable prognosis for patients. The role of pDC in cancer remains unclear but they have been shown to mediate immune tolerance in other pathophysiologic contexts. We postulated that pDC may interfere with antitumor immune response and favor tolerance in breast cancer. The present study was designed to decipher the mechanistic basis for the deleterious impact of pDC on the clinical outcome. Using fresh human breast tumor biopsies (N = 60 patients), we observed through multiparametric flow cytometry increased tumor-associated (TA) pDC (TApDC) rates in aggressive breast tumors, i.e., those with high mitotic index and the so-called triple-negative breast tumors (TNBT). Furthermore, TApDC expressed a partially activated phenotype and produced very low amounts of IFN-α following toll-like receptor activation in vitro compared with patients' blood pDC. Within breast tumors, TApDC colocalized and strongly correlated with TA regulatory T cells (TATreg), especially in TNBT. Of most importance, the selective suppression of IFN-α production endowed TApDC with the unique capacity to sustain FoxP3(+) Treg expansion, a capacity that was reverted by the addition of exogenous IFN-α. These findings indicate that IFN-α-deficient TApDC accumulating in aggressive tumors are involved in the expansion of TATreg in vivo, contributing to tumor immune tolerance and poor clinical outcome. Thus, targeting pDC to restore their IFN-α production may represent an attractive therapeutic strategy to overcome immune tolerance in breast cancer.

Increasing chronological age is the most significant risk factor for human cancer development. To examine the effects of host aging on mammary tumor growth, we used caveolin (Cav)-1 knockout mice as a bona fide model of accelerated host aging. Mammary tumor cells were orthotopically implanted into these distinct microenvironments (Cav-1(+/+) versus Cav-1(-/-) age-matched young female mice). Mammary tumors grown in a Cav-1-deficient tumor microenvironment have an increased stromal content, with vimentin-positive myofibroblasts (a marker associated with oxidative stress) that are also positive for S6-kinase activation (a marker associated with aging). Mammary tumors grown in a Cav-1-deficient tumor microenvironment were more than fivefold larger than tumors grown in a wild-type microenvironment. Thus, a Cav-1-deficient tumor microenvironment provides a fertile soil for breast cancer tumor growth. Interestingly, the mammary tumor-promoting effects of a Cav-1-deficient microenvironment were estrogen and progesterone independent. In this context, chemoprevention was achieved by using the mammalian target of rapamycin (mTOR) inhibitor and anti-aging drug, rapamycin. Systemic rapamycin treatment of mammary tumors grown in a Cav-1-deficient microenvironment significantly inhibited their tumor growth, decreased their stromal content, and reduced the levels of both vimentin and phospho-S6 in Cav-1-deficient cancer-associated fibroblasts. Since stromal loss of Cav-1 is a marker of a lethal tumor microenvironment in breast tumors, these high-risk patients might benefit from treatment with mTOR inhibitors, such as rapamycin or other rapamycin-related compounds (rapalogues)., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Significance: Here, we review certain recent advances in oxidative stress and tumor metabolism, which are related to understanding the contributions of the microenvironment in promoting tumor growth and metastasis. In the early 1920s, Otto Warburg, a Nobel Laureate, formulated a hypothesis to explain the "fundamental basis" of cancer, based on his observations that tumors displayed a metabolic shift toward glycolysis. In 1963, Christian de Duve, another Nobel Laureate, first coined the phrase auto-phagy, derived from the Greek words "auto" and "phagy," meaning "self" and "eating.", Recent Advances: Now, we see that these two ideas (autophagy and aerobic glycolysis) physically converge in the tumor stroma. First, cancer cells secrete hydrogen peroxide. Then, as a consequence, oxidative stress in cancer-associated fibroblasts drives autophagy, mitophagy, and aerobic glycolysis., Critical Issues: This "parasitic" metabolic coupling converts the stroma into a "factory" for the local production of recycled and high-energy nutrients (such as L-lactate)-to fuel oxidative mitochondrial metabolism in cancer cells. We believe that Warburg and de Duve would be pleased with this new two-compartment model for understanding tumor metabolism. It adds a novel stromal twist to two very well-established cancer paradigms: aerobic glycolysis and autophagy., Future Directions: Undoubtedly, these new metabolic models will foster the development of novel biomarkers, and corresponding therapies, to achieve the goal of personalized cancer medicine. Given the central role that oxidative stress plays in this process, new powerful antioxidants should be developed in the fight against cancer.

The current chapter focuses on the role of Caveolin-1 (Cav-1) in cellular growth with an emphasis on its implication in breast cancer initiation, progression, clinical prognosis and as a potential therapeutic target. The role of Cav-1 as a tumor suppressor in breast cancer has emerged in the past few years, with dual functions on both cancer epithelium as well as the cancer stroma. Its multiple functions as a regulator of estrogen signaling and kinase activity and its newly found role as an important factor controlling the dynamic relationship between cancer epithelia and stroma position Cav-1 as a new therapeutic target for the treatment of breast cancer.