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CD4+CD25hiCD127lo/−FOXP3+ regulatory T cells (Tregs) play a key role in preventing autoimmunity. In autoimmune type 1 diabetes (T1D), adoptive transfer of autologous polyclonal Tregs has been shown to be safe in adults in phase 1 clinical trials. We explored factors contributing to efficacy of autologous polyclonal expanded Tregs (expTregs) in a randomized phase 2 multi-center, double-blind, clinical trial (Sanford/Lisata Therapeutics T-Rex phase 2 trial, ClinicalTrials.gov NCT02691247). One hundred ten treated children and adolescents with new-onset T1D were randomized 1:1:1 to high-dose (20 × 106 cells/kilogram) or low-dose (1 × 106 cells/kilogram) treatments or to matching placebo. Cytometry as well as bulk and single-cell RNA sequencing were performed on selected expTregs and peripheral blood samples from participants. The single doses of expTregs were safe but did not prevent decline in residual β cell function over 1 year compared to placebo (P = 0.94 low dose, P = 0.21 high dose), regardless of age or baseline C-peptide. ExpTregs were highly activated and suppressive in vitro. A transient increase of activated memory Tregs was detectable 1 week after infusion in the high-dose cohort, suggesting effective transfer of expTregs. However, the in vitro fold expansion of expTregs varied across participants, even when accounting for age, and lower fold expansion and its associated gene signature were linked with better C-peptide preservation regardless of Treg dose. These results suggest that a single dose of polyclonal expTregs does not alter progression in T1D; instead, Treg quality may be an important factor. Editor's summary: Regulatory T cells (Tregs) are important in immune tolerance. Infusion of autologous polyclonal Tregs, first expanded in vitro to increase their numbers, has been investigated for safety in small clinical studies, however usefulness of this therapy for type 1 diabetes (T1D) remains unclear. Bender et al. report that a phase 2 randomized trial of a single dose of expanded Tregs showed no efficacy in preserving C-peptide, an indicator of β cell function, in early-onset T1D. This negative in vivo result comes despite the suppressive capacity of the expanded Tregs in vitro, and will inform future studies of the role of polyclonal Tregs in T1D. —Catherine Charneski [ABSTRACT FROM AUTHOR]

Intercellular communication between different cell types in solid tumors contributes to tumor growth and metastatic dissemination. The secretome of cancer-associated fibroblasts (CAFs) plays major roles in these processes. Using human mammary CAFs, we showed that CAFs with a myofibroblast phenotype released extracellular vesicles that transferred proteins to endothelial cells (ECs) that affected their interaction with immune cells. Mass spectrometry–based proteomics identified proteins transferred from CAFs to ECs, which included plasma membrane receptors. Using THY1 as an example of a transferred plasma membrane–bound protein, we showed that CAF-derived proteins increased the adhesion of a monocyte cell line to ECs. CAFs produced high amounts of matrix-bound EVs, which were the primary vehicles of protein transfer. Hence, our work paves the way for future studies that investigate how CAF-derived matrix-bound EVs influence tumor pathology by regulating the function of neighboring cancer, stromal, and immune cells. Editor's summary: Cancer-associated fibroblasts promote tumor growth, in part, by releasing extracellular vesicles, which can carry proteins to cells in the tumor microenvironment. Santi et al. investigated intercellular communication between endothelial cells in blood vessels and cancer-associated fibroblasts isolated from patients with breast cancer. Endothelial cells in vitro and in vivo took up proteins from extracellular vesicles, specifically matrix-bound vesicles, released by cancer-associated fibroblasts. Uptake of the membrane glycoprotein THY1 from cancer-associated fibroblasts increased the adhesion of monocytes to endothelial cells. Cancer-associated fibroblasts that released the most matrix-bound vesicles resembled myofibroblasts. Thus, identifying the proteins released by myofibroblast-like cancer-associated fibroblasts that alter endothelial cell function could yield potential targets for disrupting this intercellular communication. —Wei Wong [ABSTRACT FROM AUTHOR]

Stimulator of interferon genes (STING) is an immune adaptor protein that senses cyclic GMP-AMP in response to self or microbial cytosolic DNA as a danger signal. STING is ubiquitously expressed in diverse cell populations, including cancer cells, with distinct cellular functions, such as activation of type I interferons, autophagy induction, or triggering apoptosis. It is not well understood whether and which subsets of immune cells, stromal cells, or cancer cells are particularly important for STING-mediated antitumor immunity. Here, using a polymeric STING-activating nanoparticle (PolySTING) with a shock-and-lock dual activation mechanism, we show that conventional type 1 dendritic cells (cDC1s) are essential for STING-mediated rejection of multiple established and metastatic murine tumors. STING status in the host but not in the cancer cells (Tmem173−/−) is important for antitumor efficacy. Specific depletion of cDC1 (Batf3−/−) or STING deficiency in cDC1 (XCR1creSTINGfl/fl) abolished PolySTING efficacy, whereas depletion of other myeloid cells had little effect. Adoptive transfer of wild-type cDC1 in Batf3−/− mice restored antitumor efficacy, whereas transfer of cDC1 with STING or IRF3 deficiency failed to rescue. PolySTING induced a specific chemokine signature in wild-type but not Batf3−/− mice. Multiplexed immunohistochemistry analysis of STING-activating cDC1s in resected tumors correlates with patient survival. Furthermore, STING-cDC1 signature was increased after neoadjuvant pembrolizumab therapy in patients with non–small cell lung cancer. Therefore, we have defined that a subset of myeloid cells is essential for STING-mediated antitumor immunity with associated biomarkers for prognosis. Editor's summary: Activation of the DNA sensing cGAS/STING pathway in tumors has opposing host-protective or tumor-promoting responses, depending on the tissue context. STING is broadly expressed by host cells, making its role in tumor control complex. To investigate the role of immune cell subsets in STING-mediated antitumor immunity, Wang et al. generated a polymeric STING-activating nanoparticle (PolySTING) with a "shock-and-lock" mode of dual STING signaling. Activation of STING in host conventional type 1 dendritic cells (cDC1s) was crucial for STING-mediated tumor rejection in mice. Furthermore, the presence of STING-activated cDC1s in human tumors correlated with better patient survival. These findings suggest that targeting STING in cDC1s may be an effective strategy to promote antitumor immunity. —Hannah Isles [ABSTRACT FROM AUTHOR]

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that recognize bacterial riboflavin–based metabolites as activating antigens. Although MAIT cells are found in tissues, it is unknown whether any host tissue–derived antigens exist. Here, we report that a sulfated bile acid, cholic acid 7-sulfate (CA7S), binds the nonclassical MHC class I protein MR1 and is recognized by MAIT cells. CA7S is a host-derived metabolite whose levels were reduced by more than 98% in germ-free mice. Deletion of the sulfotransferase 2a family of enzymes (Sult2a1-8) responsible for CA7S synthesis reduced the number of thymic MAIT cells in mice. Moreover, recognition of CA7S induced MAIT cell survival and the expression of a homeostatic gene signature. By contrast, recognition of a previously described foreign antigen, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), drove MAIT cell proliferation and the expression of inflammatory genes. Thus, CA7S is an endogenous antigen for MAIT cells, which promotes their development and function. Editor's summary: Mucosal-associated invariant T (MAIT) cells are an innate-like T cell subset important for mucosal homeostasis, which recognize microbiota-derived vitamin B metabolites presented by the MHC-Ib molecule MR1. It is unclear, however, whether any host-derived ligands are also important for MAIT cell function and survival. Ito et al. report that the host-derived bile acid metabolite cholic acid 7-sulfate (CA7S) binds MR1 and is recognized by MAIT cells in mice. Moreover, genetic deletion of sulfotransferase 2a enzymes (Sult2a1-8) needed for its synthesis in mice reduced thymic MAIT cell numbers. A previously characterized vitamin B metabolite, 5-OP-RU, triggered human MAIT cell proliferation and the expression of inflammatory genes. By contrast, CA7S promoted human MAIT cell survival and a gene program associated with wound healing and immunoregulation. —Seth Thomas Scanlon [ABSTRACT FROM AUTHOR]

The ubiquitination-dependent processing of NF-κB2 (also known as p100) is a critical step in the activation of the noncanonical NF-κB pathway. We investigated the molecular mechanisms regulating this process and showed that TRIM55 was the E3 ubiquitin ligase that mediated the ubiquitination of p100 and coordinated its processing. TRIM55 deficiency impaired noncanonical NF-κB activation and B cell function. Mice with a B cell–specific Trim55 deficiency exhibited reduced germinal center formation and antibody production. These mice showed less severe symptoms than those of control mice upon the induction of a systemic lupus–like disease, suggesting B cell–intrinsic functions of TRIM55 in humoral immune responses and autoimmunity. Mechanistically, the ubiquitination of p100 mediated by TRIM55 was crucial for p100 processing by VCP, an ATPase that mediates ubiquitin-dependent protein degradation by the proteasome. Furthermore, we found that TRIM55 facilitated the interaction between TRIM21 and VCP as well as TRIM21-mediated K63-ubiquitination of VCP, both of which were indispensable for the formation of the VCP-UFD1-NPL4 complex and p100 processing. Together, our results reveal a mechanism by which TRIM55 fine-tunes p100 processing and regulates B cell–dependent immune responses in vivo, highlighting TRIM55 as a potential therapeutic target for lupus-like disease. Editor's summary: The noncanonical NF-κB pathway is important for antibody-based immune responses mediated by B cells. Lin et al. found that the E3 ubiquitin ligase TRIM55 was critical for the generation of the transcription factor p52 from its precursor p100, an essential step in the activation of noncanonical NF-κB signaling. TRIM55 mediated the ubiquitination of p100 and facilitated the formation of protein complexes that processed ubiquitinated p100 into p52. B cell–specific deficiency of TRIM55 impaired antibody production and germinal center formation but also protected mice from a systemic lupus-like disease. Thus, TRIM55 promotes the activity of the noncanonical NK-κB pathway and could be a potential target for the treatment of autoimmune diseases. —Wei Wong [ABSTRACT FROM AUTHOR]

Allogeneic natural killer (NK) cell adoptive transfer has shown the potential to induce remissions in relapsed or refractory leukemias and lymphomas, but strategies to enhance NK cell survival and function are needed to improve clinical efficacy. Here, we demonstrated that NK cells cultured ex vivo with interleukin-15 (IL-15) and nicotinamide (NAM) exhibited stable induction of l-selectin (CD62L), a lymphocyte adhesion molecule important for lymph node homing. High frequencies of CD62L were associated with elevated transcription factor forkhead box O1 (FOXO1), and NAM promoted the stability of FOXO1 by preventing proteasomal degradation. NK cells cultured with NAM exhibited metabolic changes associated with elevated glucose flux and protection against oxidative stress. NK cells incubated with NAM also displayed enhanced cytotoxicity and inflammatory cytokine production and preferentially persisted in xenogeneic adoptive transfer experiments. We also conducted a first-in-human phase 1 clinical trial testing adoptive transfer of NK cells expanded ex vivo with IL-15 and NAM (GDA-201) combined with monoclonal antibodies in patients with relapsed or refractory non-Hodgkin lymphoma (NHL) and multiple myeloma (MM) (NCT03019666). Cellular therapy with GDA-201 and rituximab was well tolerated and yielded an overall response rate of 74% in 19 patients with advanced NHL. Thirteen patients had a complete response, and 1 patient had a partial response. GDA-201 cells were detected for up to 14 days in blood, bone marrow, and tumor tissues and maintained a favorable metabolic profile. The safety and efficacy of GDA-201 in this study support further development as a cancer therapy. Editor's summary: Administration of haploidentical donor natural killer (NK) cells, rituximab, and IL-2 to patients with non-Hodgkin lymphoma (NHL) has been shown to be safe, but the objective response rate was low, suggesting that further strategies to improve NK cell engraftment and function are needed. Here, Cichocki and colleagues showed that supplementation with nicotinamide during ex vivo NK cell culture led to up-regulation of the lymphocyte homing molecule CD26L, leading to improved persistence and antitumor functions. In a phase 1 trial of nicotinamide-expanded allogeneic NK cells, monoclonal antibodies, and IL2, 11 of 19 patients with NHL demonstrated complete response, suggesting that supplementation with nicotinamide during ex vivo NK cell culture should be further investigated for the treatment of NHL. —Melissa Norton [ABSTRACT FROM AUTHOR]

Although blocking the binding of vascular endothelial growth factor (VEGF) to neuropilin-2 (NRP2) on tumor cells is a potential strategy to treat aggressive carcinomas, a lack of effective reagents that can be used clinically has hampered this potential therapy. Here, we describe the generation of a fully humanized, high-affinity monoclonal antibody (aNRP2-10) that specifically inhibits the binding of VEGF to NRP2, conferring antitumor activity without causing toxicity. Using triple-negative breast cancer as a model, we demonstrated that aNRP2-10 could be used to isolate cancer stem cells (CSCs) from heterogeneous tumor populations and inhibit CSC function and epithelial-to-mesenchymal transition. aNRP2-10 sensitized cell lines, organoids, and xenografts to chemotherapy and inhibited metastasis by promoting the differentiation of CSCs to a state that is more responsive to chemotherapy and less prone to metastasis. These data provide justification for the initiation of clinical trials designed to improve the response of patients with aggressive tumors to chemotherapy using this monoclonal antibody. Creating cancer chemosensitivity: Therapies targeting vascular endothelial growth factor (VEGF) have been developed for multiple types of tumors with varying degrees of success. This incomplete success may partially be due to the number of receptors that bind VEGF. Here, Xu et al. developed and characterized a monoclonal antibody that blocks the interaction between VEGF and neuropilin-2 (NRP2). This antibody, aNRP2-10, inhibited breast cancer stem cell function and sensitized breast cancer cell lines and organoids to chemotherapy. In vivo, aNRP2-10 inhibited metastasis and augmented the efficacy of chemotherapy. These results support further development of aNRP2-10 and similar antibodies. —CM [ABSTRACT FROM AUTHOR]

Sleep loss is associated with cognitive decline in the aging population and is a risk factor for Alzheimer's disease (AD). Considering the crucial role of immunomodulating genes such as that encoding the triggering receptor expressed on myeloid cells type 2 (TREM2) in removing pathogenic amyloid-β (Aβ) plaques and regulating neurodegeneration in the brain, our aim was to investigate whether and how sleep loss influences microglial function in mice. We chronically sleep-deprived wild-type mice and the 5xFAD mouse model of cerebral amyloidosis, expressing either the humanized TREM2 common variant, the loss-of-function R47H AD-associated risk variant, or without TREM2 expression. Sleep deprivation not only enhanced TREM2-dependent Aβ plaque deposition compared with 5xFAD mice with normal sleeping patterns but also induced microglial reactivity that was independent of the presence of parenchymal Aβ plaques. We investigated lysosomal morphology using transmission electron microscopy and found abnormalities particularly in mice without Aβ plaques and also observed lysosomal maturation impairments in a TREM2-dependent manner in both microglia and neurons, suggesting that changes in sleep modified neuro-immune cross-talk. Unbiased transcriptome and proteome profiling provided mechanistic insights into functional pathways triggered by sleep deprivation that were unique to TREM2 and Aβ pathology and that converged on metabolic dyshomeostasis. Our findings highlight that sleep deprivation directly affects microglial reactivity, for which TREM2 is required, by altering the metabolic ability to cope with the energy demands of prolonged wakefulness, leading to further Aβ deposition, and underlines the importance of sleep modulation as a promising future therapeutic approach. The benefits of sleep: Sleep loss is associated with cognitive decline in older adults and is thought to be a risk factor for Alzheimer's disease (AD), but the underlying mechanisms remain unclear. In a new study, Parhizkar et al. used different genetic mouse models that overproduce amyloid deposits in the brain to investigate the effects of sleep loss. The authors report that sleep loss reduces the ability of immune cells called microglia to remove brain amyloid deposits (a hallmark of AD). These findings suggest that sleep loss could contribute to cognitive decline by reducing immune cell function in the brain. —OMS [ABSTRACT FROM AUTHOR]

Cytokine release syndrome (CRS) is a systemic inflammatory syndrome associated with infection- or drug-induced T cell activation and can cause multiple organ failure and even death. Because current treatments are ineffective in some patients with severe CRS, we set out to identify risk factors and mechanisms behind severe CRS that might lead to preventive therapies and better clinical outcomes in patients. In mice, we found that deficiency in the adrenal stress response—with similarities to such in patients called relative adrenal insufficiency (RAI)—conferred a high risk for lethal CRS. Mice treated with CD3 antibodies were protected against lethal CRS by the production of glucocorticoids (GC) induced by the adrenal stress response in a manner dependent on the scavenger receptor B1 (SR-BI), a receptor for high-density lipoprotein (HDL). Mice with whole-body or adrenal gland-specific SR-BI deficiency exhibited impaired GC production, more severe CRS, and increased mortality in response to CD3 antibodies. Pretreatment with a low dose of GC effectively suppressed the development of CRS and rescued survival in SR-BI–deficient mice without compromising T cell function through apoptosis. Our findings suggest that RAI may be a risk factor for therapy-induced CRS and that pretreating RAI patients with GC may prevent lethal CRS. Glucocorticoids calm cytokine storms: T cell–stimulating therapies can inadvertently induce a rapid, systemic release of proinflammatory cytokines (called a cytokine storm) that can cause tissue damage and death. Guo et al. found that the release of glucocorticoids by the adrenal stress response was critical to preventing lethal cytokine storm in mice receiving an immunotherapeutic antibody. Wild-type mice and those unable to produce a glucocorticoid response to CD3 antibody treatment exhibited an initially similar, systemic release of cytokines, but only wild-type mice showed rapid resolution of cytokine production and long-term survival. Administration of low-dose glucocorticoids before antibody therapy rescued cytokine resolution and survival in mice lacking the adrenal response. The findings suggest that the adrenal stress response restrains immunotherapy-induced cytokine storm and that mimicking it might prevent severe outcomes in patients. —LKF [ABSTRACT FROM AUTHOR]

Myeloid cells can restrain antitumor immunity by metabolic pathways, such as the degradation of l-arginine, whose concentrations are regulated by the arginase 1 (ARG1) enzyme. Results from preclinical studies indicate the important role of arginine metabolism in pancreatic ductal adenocarcinoma (PDAC) progression, suggesting a potential for clinical application; however, divergent evolution in ARG1 expression and function in rodents and humans has restricted clinical translation. To overcome this dichotomy, here, we show that neutrophil extracellular traps (NETs), released by spontaneously activated neutrophils isolated from patients with PDAC, create a microdomain where cathepsin S (CTSS) cleaves human (h)ARG1 into different molecular forms endowed with enhanced enzymatic activity at physiological pH. NET-associated hARG1 suppresses T lymphocytes whose proliferation is restored by either adding a hARG1-specific monoclonal antibody (mAb) or preventing CTSS-mediated cleavage, whereas small-molecule inhibitors are not effective. We show that ARG1 blockade, combined with immune checkpoint inhibitors, can restore CD8+ T cell function in ex vivo PDAC tumors. Furthermore, anti-hARG1 mAbs increase the frequency of adoptively transferred tumor-specific CD8+ T cells in tumor and enhance the effectiveness of immune checkpoint therapy in humanized mice. Thus, this study shows that extracellular ARG1, released by activated myeloid cells, localizes in NETs, where it interacts with CTSS that in turn cleaves ARG1, producing major molecular forms endowed with different enzymatic activity at physiological pH. Once exocytosed, ARG1 activity can be targeted by mAbs, which bear potential for clinical application for the treatment of PDAC and require further exploration. Nothing but NET for PDAC: Pancreatic ductal adenocarcinoma (PDAC) is known for its immunosuppressive tumor microenvironment, in part due to arginine metabolism where the enzyme arginase 1 (ARG1) affects T cell proliferation. ARG1 is an enzyme that is evolutionarily different in mice and humans, which has so far created a translational hurdle. To overcome this, Canè et al. have shown that neutrophil extracellular traps (NETs) cleave human ARG1 (hARG1), suppressing T lymphocyte proliferation that can be restored with an hARG1 monoclonal antibody (mAb). hARG1 mAb combined with immune checkpoint inhibitors restored T cell proliferation in humanized mice and extended survival, representing a target that warrants further study. —DH [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are potent immune effectors that can be activated via antibody-mediated Fc receptor engagement. Using multiparameter flow cytometry, we found that NK cells degranulate and release IFN-γ upon stimulation with antibody-opsonized Plasmodium falciparum merozoites. Antibody-dependent NK (Ab-NK) activity was largely strain transcending and enhanced invasion inhibition into erythrocytes. Ab-NK was associated with the successful control of parasitemia after experimental malaria challenge in African adults. In an independent cohort study in children, Ab-NK increased with age, was boosted by concurrent P. falciparum infections, and was associated with a lower risk of clinical episodes of malaria. Nine of the 14 vaccine candidates tested induced Ab-NK, including some less well-characterized antigens: P41, P113, MSP11, RHOPH3, and Pf_11363200. These data highlight an important role of Ab-NK activity in immunity against malaria and provide a potential mechanism for evaluating vaccine candidates. A different antibody angle: Natural killer (NK) cells have been shown to mediate immune responses against Plasmodium falciparum using multiple effector functions. Odera et al. now show that antibody-opsonized P. falciparum merozoites can induce NK cell degranulation and IFN-γ production. This response was not strain specific and reduced invasion of merozoites into uninfected red blood cells. Using a controlled human malaria infection study in adults, they observed that antibody-dependent NK (Ab-NK) cell activity correlated directly with the control of parasitemia. In addition, in a cohort of children living in an endemic malaria setting, Ab-NK frequency increased with age, was boosted during P. falciparum infection, and was linked to a reduced risk of clinical malaria. These findings highlight a key role for antibody-mediated NK cell responses during malaria. —CF [ABSTRACT FROM AUTHOR]

Genome-wide association studies identifying hundreds of susceptibility loci for autoimmune diseases indicate that genes active in immune cells predominantly mediate risk. However, identification and functional characterization of causal variants remain challenging. Here, we focused on the immunomodulatory role of a protective variant of histone deacetylase 7 (HDAC7). This variant (rs148755202, HDAC7.p.R166H) was identified in a study of low-frequency coding variation in multiple sclerosis (MS). Through transcriptomic analyses, we demonstrate that wild-type HDAC7 regulates genes essential for the function of Foxp3+ regulatory T cells (Tregs), an immunosuppressive subset of CD4 T cells that is generally dysfunctional in patients with MS. Moreover, Treg-specific conditional hemizygous deletion of HDAC7 increased the severity of experimental autoimmune encephalitis (EAE), a mouse model of neuroinflammation. In contrast, Tregs transduced with the protective HDAC7 R166H variant exhibited higher suppressive capacity in an in vitro functional assay, mirroring phenotypes previously observed in patient samples. In vivo modeling of the human HDAC7 R166H variant by generation of a knock-in mouse model bearing an orthologous R150H substitution demonstrated decreased EAE severity linked to transcriptomic alterations of brain-infiltrating Tregs, as assessed by single-cell RNA sequencing. Our data suggest that dysregulation of epigenetic modifiers, a distinct molecular class associated with disease risk, may influence disease onset. Last, our approach provides a template for the translation of genetic susceptibility loci to detailed functional characterization, using in vitro and in vivo modeling. Regulating neuroinflammation: Regulating neuroinflammationA variant in histone deacetylase 7 (HDAC7), R166H, was previously associated with reduced risk of developing multiple sclerosis. Here, Axisa et al. aimed to understand why this was the case. Through mechanistic studies, the authors found that HDAC7 was essential for regulating proper regulatory T (Treg) cell function. Mice with Treg cells lacking HDAC7 developed more severe disease in the context of experimental autoimmune encephalitis (EAE). In contrast, when Treg cells expressed an orthologous substitution to the human R166H variant (R150H), mice developed less severe EAE. These data highlight a role for HDAC7 in Treg cells and may explain why the R116H variant reduces risk of developing multiple sclerosis. -CM [ABSTRACT FROM AUTHOR]

Cytokines interact with their receptors in the extracellular space to control immune responses. How the physicochemical properties of the extracellular space influence cytokine signaling is incompletely elucidated. Here, we show that the activity of interleukin-2 (IL-2), a cytokine critical to T cell immunity, is profoundly affected by pH, limiting IL-2 signaling within the acidic environment of tumors. Generation of lactic acid by tumors limits STAT5 activation, effector differentiation, and antitumor immunity by CD8+ T cells and renders high-dose IL-2 therapy poorly effective. Directed evolution enabled selection of a pH-selective IL-2 mutein (Switch-2). Switch-2 binds the IL-2 receptor subunit IL-2Rα with higher affinity, triggers STAT5 activation, and drives CD8+ T cell effector function more potently at acidic pH than at neutral pH. Consequently, high-dose Switch-2 therapy induces potent immune activation and tumor rejection with reduced on-target toxicity in normal tissues. Last, we show that sensitivity to pH is a generalizable property of a diverse range of cytokines with broad relevance to immunity and immunotherapy in healthy and diseased tissues. Acid-tolerant IL-2 targets tumors: Hypoxic and acidic conditions prevalent in the tumor microenvironment present a formidable challenge for host lymphocytes trying to eradicate cancer cells. IL-2, a cytokine supporting CD8 T cell and NK cell functions, exhibits markedly compromised binding to the IL-2Rα receptor chain at pH 6 and below. Gaggero et al. used directed evolution to screen for IL-2 variants with mutations at the receptor interface that enhanced receptor binding at low pH. A mutated "Switch-2" variant of IL-2 with strong receptor binding at pH 6 but minimal interaction at neutral pH exhibited enhanced in vivo antitumor activity in mice. Recognition of the pH sensitivity of IL-2 and other cytokines will spur engineering of cytokine variants capable of enhanced performance in the acidic tumor microenvironment. [ABSTRACT FROM AUTHOR]

Signals that determine the differentiation of naïve CD4+ T helper (TH) cells into specific effector cell subsets are primarily stimulated by cytokines, but additional signals are required to adjust the magnitude of TH cell responses and set the balance between effective immunity and immunological tolerance. By inducing the post-thymic deletion of the T cell lineage signaling protein THEMIS, we showed that THEMIS promoted the development of optimal type 1 immune responses to foreign antigens but stimulated signals that favored encephalitogenic responses to self-neuroantigens. THEMIS was required to stimulate the expression of the gene encoding the transcriptional regulator T-BET and the production of the cytokine interferon-γ (IFN-γ), and it enhanced the ability of encephalitogenic CD4+ T cells to migrate into the central nervous system. Consistently, analysis of THEMIS expression in polarized CD4+ T cells showed that THEMIS was selectively increased in abundance in TH1 cells. The stimulation of predifferentiated effector CD4+ T cells with antigen-presenting cells revealed a stimulatory function for THEMIS on type 1 cytokine responses, similar to those observed ex vivo after immunization. In contrast, THEMIS exerted opposing effects on naïve CD4+ T cells in vitro by inhibiting the T cell receptor (TCR)–mediated signals that lead to TH1 cell responses. These data suggest that THEMIS exerts TCR-independent functions in effector T cells, which increase the magnitude of normal and pathogenic TH1 cell–mediated responses. THEMIS in T cells: Naïve T cells integrate antigen-dependent signals through their T cell receptor (TCR) and cytokine signals to differentiate into distinct T helper (TH) cell subsets with specialized immune functions. The T cell signaling protein THEMIS is important for T cell development. Yang et al. disrupted Themis in mice after thymic selection to investigate the function of THEMIS in peripheral T cells. Among different TH cell subsets, THEMIS promoted the function of only TH1 cells, and it enhanced the encephalitogenic responses of T cells in a mouse model of autoimmunity. However, THEMIS had opposing effects on TCR-dependent signaling in naïve T cells in vitro, suggesting that THEMIS signaling is context dependent and that these inhibitory effects are overcome during immune responses in vivo by TCR-independent signaling by THEMIS that promotes T cell effector function. [ABSTRACT FROM AUTHOR]

Gammaherpesviruses (γHVs) have coevolved with their host, leading to a remarkably high infection prevalence and establishment of latency. The lifelong persistence of γHVs in hosts appears to broadly shape host immunity, and we show here that pulmonary infection with Murid herpesvirus 4 (MuHV-4), a mouse γHV, drives the recruitment of Ly6Chi monocytes (MOs) into the airway, thereby modulating the host immune response. The absence of Ly6Chi MOs is associated with severe virus-induced immunopathology and the systemic release of inflammatory mediators. Mechanistically, MuHV-4–imprinted MOs recruit CD4 T cells to the airways and trigger immunosuppressive signaling pathways through the PD-L1/PD-1 axis, thereby dampening the deleterious activation of cytotoxic CD4 T cells. These results uncover a role for Ly6Chi MOs in modulating CD4 T cell functions and reveal pathways that could be targeted therapeutically to reduce detrimental immunopathological responses associated with respiratory viral infections. Monocyte mediator: Gammaherpesviruses (γHVs) coevolved with their hosts in a manner that allows for the lifelong persistence of these viruses in immunocompetent individuals. The presence of latent γHVs in host organisms has been shown to shape adaptive immunity, and here, Maquet et al. observed that pulmonary infection of mice with a murine γHV (MuHV-4) leads to recruitment of Ly6Chi monocytes (MOs) to airways. These γHV-imprinted Ly6Chi MOs recruit CD4 T cells to airways followed by engagement of the immunosuppressive PD-1/PD-L1 signaling pathway, which limits pathological cytotoxic CD4 T cell responses. These findings highlight a role for γHV-imprinted Ly6Chi MOs in shaping CD4 T cell functions and suggest a potential pathway for the development of therapeutics that target respiratory viral infections. [ABSTRACT FROM AUTHOR]

During progression of type 2 diabetes, pancreatic β cells are subjected to sustained metabolic overload. We postulated that this state mediates a hypoxic phenotype driven by hypoxia-inducible factor–1α (HIF-1α) and that treatment with the HIF-1α inhibitor PX-478 would improve β cell function. Our studies showed that the HIF-1α protein was present in pancreatic β cells of diabetic mouse models. In mouse islets with high glucose metabolism, the emergence of intracellular Ca2+ oscillations at low glucose concentration and the abnormally high basal release of insulin were suppressed by treatment with the HIF-1α inhibitor PX-478, indicating improvement of β cell function. Treatment of db/db mice with PX-478 prevented the rise of glycemia and diabetes progression by maintenance of elevated plasma insulin concentration. In streptozotocin-induced diabetic mice, PX-478 improved the recovery of glucose homeostasis. Islets isolated from these mice showed hallmarks of improved β cell function including elevation of insulin content, increased expression of genes involved in β cell function and maturity, inhibition of dedifferentiation markers, and formation of mature insulin granules. In response to PX-478 treatment, human islet organoids chronically exposed to high glucose presented improved stimulation index of glucose-induced insulin secretion. These results suggest that the HIF-1α inhibitor PX-478 has the potential to act as an antidiabetic therapeutic agent that preserves β cell function under metabolic overload. Inhibiting HIF-1α improves glucose homeostasis: Type 2 diabetes (T2D) places insulin-secreting beta cells under sustained metabolic stress. Ilegems et al. reasoned that such stress might result in a hypoxic state and thereby impair β cell function. The authors confirmed a glucose metabolism–mediated increase in hypoxia in multiple mouse models with metabolic dysfunction and showed that a HIF-1α inhibitor improved β cell function in db/db and streptozotocin-induced mouse models of diabetes. Human islet organoids cultured under high-glucose conditions also showed improved β cell function in response to HIF-1α inhibitor treatment, suggesting that these findings may translate to humans. [ABSTRACT FROM AUTHOR]

To uncover underlying mechanisms associated with failure of indoleamine 2,3-dioxygenase 1 (IDO1) blockade in clinical trials, we conducted a pilot, window-of-opportunity clinical study in 17 patients with newly diagnosed advanced high-grade serous ovarian cancer before their standard tumor debulking surgery. Patients were treated with the IDO1 inhibitor epacadostat, and immunologic, transcriptomic, and metabolomic characterization of the tumor microenvironment was undertaken in baseline and posttreatment tumor biopsies. IDO1 inhibition resulted in efficient blockade of the kynurenine pathway of tryptophan degradation and was accompanied by a metabolic adaptation that shunted tryptophan catabolism toward the serotonin pathway. This resulted in elevated nicotinamide adenine dinucleotide (NAD+), which reduced T cell proliferation and function. Because NAD+ metabolites could be ligands for purinergic receptors, we investigated the impact of blocking purinergic receptors in the presence or absence of NAD+ on T cell proliferation and function in our mouse model. We demonstrated that A2a and A2b purinergic receptor antagonists, SCH58261 or PSB1115, respectively, rescued NAD+-mediated suppression of T cell proliferation and function. Combining IDO1 inhibition and A2a/A2b receptor blockade improved survival and boosted the antitumor immune signature in mice with IDO1 overexpressing ovarian cancer. These findings elucidate the downstream adaptive metabolic consequences of IDO1 blockade in ovarian cancers that may undermine antitumor T cell responses in the tumor microenvironment. Saying "I DO" to combination therapy: Indoleamine 2,3-dioxygenase 1 (IDO1) drives immunosuppression in high-grade serous ovarian cancer. Inhibition of IDO1 in combination with chemotherapy has shown limited efficacy in clinical trials. Here, Odunsi et al. treated patients with the IDO1 inhibitor epacadostat and studied the effect on the tumor microenvironment (TME) in tumor biopsies. IDO1 inhibition increased nicotinamide adenine dinucleotide (NAD+), which reduced T cell function in the TME. Combining epacadostat with purinergic receptor antagonists rescued T cell proliferation in a mouse model of ovarian cancer. These findings highlight the potential downside of IDO1 inhibition and suggest that IDO1 inhibitor therapy will require combination with NAD+ signaling blockade. [ABSTRACT FROM AUTHOR]

Dysregulation of innate immune signaling pathways is implicated in various hematologic malignancies. However, these pathways have not been systematically examined in acute myeloid leukemia (AML). We report that AML hematopoietic stem and progenitor cells (HSPCs) exhibit a high frequency of dysregulated innate immune-related and inflammatory pathways, referred to as oncogenic immune signaling states. Through gene expression analyses and functional studies in human AML cell lines and patient-derived samples, we found that the ubiquitin-conjugating enzyme UBE2N is required for leukemic cell function in vitro and in vivo by maintaining oncogenic immune signaling states. It is known that the enzyme function of UBE2N can be inhibited by interfering with thioester formation between ubiquitin and the active site. We performed in silico structure-based and cellular-based screens and identified two related small-molecule inhibitors UC-764864/65 that targeted UBE2N at its active site. Using these small-molecule inhibitors as chemical probes, we further revealed the therapeutic efficacy of interfering with UBE2N function. This resulted in the blocking of ubiquitination of innate immune- and inflammatory-related substrates in human AML cell lines. Inhibition of UBE2N function disrupted oncogenic immune signaling by promoting cell death of leukemic HSPCs while sparing normal HSPCs in vitro. Moreover, baseline oncogenic immune signaling states in leukemic cells derived from discrete subsets of patients with AML exhibited a selective dependency on UBE2N function in vitro and in vivo. Our study reveals that interfering with UBE2N abrogates leukemic HSPC function and underscores the dependency of AML cells on UBE2N-dependent oncogenic immune signaling states. Stemming AML with UBE2N inhibition: Relapse in acute myeloid leukemia (AML) is often due to the inability to fully eradicate leukemic hematopoietic stem and progenitor cells (HSPCs). These leukemic HSPCs co-opt innate immune signaling pathways for tumorigenesis and represent a potential target of therapeutic intervention. Barreyro et al. have identified that dysregulated innate immune pathway activation depends on ubiquitin-conjugating enzyme E2 N (UBE2N). Pharmacologic targeting of UBE2N in mice carrying patient-derived xenografts suppressed AML and inhibited the expansion of leukemic HSPCs. UBE2N represents an actionable target for AML and other malignant hematopoietic cell disorders. [ABSTRACT FROM AUTHOR]

T cell receptor (TCR)–based therapy has the potential to induce durable clinical responses in patients with cancer by targeting intracellular tumor antigens with high sensitivity and by promoting T cell survival. However, the need for TCRs specific for shared oncogenic antigens and the need for manufacturing protocols able to redirect T cell specificity while preserving T cell fitness remain limiting factors. By longitudinal monitoring of T cell functionality and dynamics in 15 healthy donors, we isolated 19 TCRs specific for Wilms' tumor antigen 1 (WT1), which is overexpressed by several tumor types. TCRs recognized several peptides restricted by common human leukocyte antigen (HLA) alleles and displayed a wide range of functional avidities. We selected five high-avidity HLA-A*02:01–restricted TCRs, three that were specific to the less explored immunodominant WT137–45 and two that were specific to the noncanonical WT1−78–64 epitopes, both naturally processed by primary acute myeloid leukemia (AML) blasts. With CRISPR-Cas9 genome editing tools, we combined TCR-targeted integration into the TCR α constant (TRAC) locus with TCR β constant (TRBC) knockout, thus avoiding TCRαβ mispairing and maximizing TCR expression and function. The engineered lymphocytes were enriched in memory stem T cells. A unique WT137–45-specific TCR showed antigen-specific responses and efficiently killed AML blasts, acute lymphoblastic leukemia blasts, and glioblastoma cells in vitro and in vivo in the absence of off-tumor toxicity. T cells engineered to express this receptor are being advanced into clinical development for AML immunotherapy and represent a candidate therapy for other WT1-expressing tumors. Alternative WT1 targets: Adoptive cell therapy using T cell receptor (TCR)–modified T cells has shown clinical success. However, it remains a challenge to find TCRs that respond to shared tumor antigens presented in common human leukocyte antigen molecules. To that end, Ruggiero et al. and Lahman et al. identified and tested TCR constructs targeting alternative epitopes in the Wilms' tumor antigen 1 (WT1) protein. Ruggiero and colleagues identified WT137–45 as a candidate peptide for TCR-based therapies and used CRISPR-Cas9 editing to target WT137–45-specific TCR sequences to the TRAC locus. Lahman and colleagues showed that, whereas a TCR-targeted WT1 epitope was lost because of immunoproteasome down-regulation, WT137–45 retains its expression and could be targeted by T cells expressing TCRs specific to that epitope, even in cancer cells that lose immunoproteasome activity. In both cases, T cells expressing WT137–45-specific TCRs controlled tumor burden in murine models. Thus, WT137–45 represents a candidate target for TCR-based immunotherapies that is resistant to immunoproteasome down-regulation. [ABSTRACT FROM AUTHOR]

The Wolfram syndrome is a rare autosomal recessive disease affecting many organs with life-threatening consequences; currently, no treatment is available. The disease is caused by mutations in the WSF1 gene, coding for the protein wolframin, an endoplasmic reticulum (ER) transmembrane protein involved in contacts between ER and mitochondria termed as mitochondria-associated ER membranes (MAMs). Inherited mutations usually reduce the protein's stability, altering its homeostasis and ultimately reducing ER to mitochondria calcium ion transfer, leading to mitochondrial dysfunction and cell death. In this study, we found that activation of the sigma-1 receptor (S1R), an ER-resident protein involved in calcium ion transfer, could counteract the functional alterations of MAMs due to wolframin deficiency. The S1R agonist PRE-084 restored calcium ion transfer and mitochondrial respiration in vitro, corrected the associated increased autophagy and mitophagy, and was able to alleviate the behavioral symptoms observed in zebrafish and mouse models of the disease. Our findings provide a potential therapeutic strategy for treating Wolfram syndrome by efficiently boosting MAM function using the ligand-operated S1R chaperone. Moreover, such strategy might also be relevant for other degenerative and mitochondrial diseases involving MAM dysfunction. Treating Wolfram syndrome in the ER: Wolfram syndrome (WS) is a genetic disease characterized by early diabetes, optic atrophy, and deafness caused by mutations in the WSF1 gene. Pathologic mutations lead to altered communication between the endoplasmic reticulum (ER) and the mitochondria within the mitochondria-associated ER membranes (MAMs), ultimately resulting in impaired mitochondrial function and cell death. Here, Crouzier et al. used preclinical models of wolframin deficiency and showed that activation of the sigma-1 receptor in the ER restored MAM alterations, boosted mitochondrial function, and improved behavioral symptoms in zebrafish and rodents. The study identified a potential therapeutic target that could be targeted for alleviating symptoms in patients with WS and other MAM-related diseases. [ABSTRACT FROM AUTHOR]

G protein–coupled receptors (GPCRs) that couple to the Gαi family of G proteins are key regulators of cell and tissue physiology. Our previous work has revealed new roles for Gαi in regulating the migration of neutrophils and fibrosarcoma cells downstream of activated chemoattractant receptors. Here, we used an intact cell proximity–based labeling coupled to tandem mass tag (TMT)–based quantitative proteomics analysis to identify proteins that selectively interacted with the GTP-bound form of Gαi1. Multiple targets were identified and validated with a BioID2-tagged, constitutively active Gαi1 mutant, suggesting a network of interactions for activated GαI proteins in intact cells. We showed that active Gαi1, but not Gαi2, stimulated one candidate protein, PDZ-RhoGEF (PRG), despite more than 85% sequence identity between the G proteins. We also demonstrated in primary human neutrophils that active Gαi likely regulated the polarization of phosphorylated myosin light chain, a process critical for migration, through the activation of PRG. The identification and characterization of new targets directly or indirectly regulated by Gαi will aid in the investigation of the functional roles of Gαi-coupled GPCRs in multiple biological processes. Expanding the Gαi interactome: Activated chemoattractant receptors stimulate the G protein αi subunit to induce de-adhesion in migrating neutrophils and fibrosarcoma cells. To identify effectors for active Gαi, Chandan et al. performed proximity labeling and mass spectrometry analysis in cells expressing inactive and constitutively active Gαi. Of the candidates identified by the authors, the RhoGEF PRG was of particular interest because of its previously identified role in neutrophil chemotaxis induced by the activation of Gαi-coupled chemoattractant receptors. In human neutrophils, Gαi was required for PRG-mediated stimulation of RhoA activity upon activation of the chemoattractant receptor FPR1. Thus, the authors' approach could be used to identify binding partners that interact with other G protein subunits in intact cells. [ABSTRACT FROM AUTHOR]

keyimage.jpg Another G for GABAB receptor: Activation of GABABR, the G protein–coupled receptor for the inhibitory neurotransmitter GABA, is thought to be neuroprotective through exclusive engagement of Gi/o. However, Wang et al. found that GABABR also engaged G13 to differentially activate the MAPK pathway kinase JNK. In cultured cerebellar granule neurons, this G13-mediated pathway increased the abundance of the postsynaptic scaffolding protein PSD95 and enhanced neuronal survival under low-potassium conditions. The authors further uncovered biological synergy between the two G protein–mediated pathways, with different kinetics in agonist responses. The findings reveal how GABA can mediate neuroprotection through multiple synergistic pathways that depend on distinct G proteins. G protein–coupled receptors (GPCRs) activate various mitogen-activated protein kinase (MAPK) pathways to regulate critical cell functions. β-Arrestins mediate this mechanism for most GPCRs but not the GABAB receptor (GABABR). When coupled to the G protein Gi/o, GABABR phosphorylates the kinases ERK1 and ERK2. Here, we uncovered a distinct β-arrestin–independent mechanism of MAPK pathway activation by GABABR. We found that GABABR also phosphorylated the kinase JNK downstream of activation of the small guanosine triphosphatases (GTPases) RhoA and Rac1 in primary mouse neurons. However, instead of Gi/o proteins, activation of this RhoA/Rac1-JNK pathway was mediated by G13. This pathway promoted the phosphorylation and accumulation of the postsynaptic scaffolding protein PSD95 and GABABR-mediated neuroprotection in granule neurons. In addition, this pathway synergized with a previously reported GABABR-mediated neuroprotection mediated by a Gi/o-dependent mechanism. GABABR agonists activated G13 with slower kinetics and lower potency than with which they activated Gi/o. Our findings reveal distinct, β-arrestin–independent, context-specific synergistic mechanisms of MAPK activation by G protein–mediated GPCR signaling. [ABSTRACT FROM AUTHOR]

P-selectin as a treatment for lupus: Dysfunctional regulatory T (Treg) cells are known to be involved in the pathogenesis of systemic lupus erythematosus (SLE), but the mechanisms mediating Treg cell dysfunction are not clear. Here, Scherlinger et al. demonstrated that platelets can impair Treg cell function after forming aggregates mediated by platelet (P)–selectin expression on platelets and P-selectin glycoprotein ligand-1 (PSGL-1) expression on Treg cells. Treg cells aggregated more frequently with platelets from individuals with active SLE as compared to healthy donors. In addition, blocking the interaction between P-selectin and PSGL-1 reduced disease severity in a mouse model of SLE. Thus, the P-selectin/PSGL-1 axis may represent a therapeutic target for SLE. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a loss of tolerance toward self-nucleic acids, autoantibody production, interferon expression and signaling, and a defect in the regulatory T (Treg) cell compartment. In this work, we identified that platelets from patients with active SLE preferentially interacted with Treg cells via the P-selectin/P-selectin glycoprotein ligand-1 (PSGL-1) axis. Selectin interaction with PSGL-1 blocked the regulatory and suppressive properties of Treg cells and particularly follicular Treg cells by triggering Syk phosphorylation and an increase in intracytosolic calcium. Mechanistically, P-selectin engagement on Treg cells induced a down-regulation of the transforming growth factor–β axis, altering the phenotype of Treg cells and limiting their immunosuppressive responses. In patients with SLE, we found an up-regulation of P- and E-selectin both on microparticles and in their soluble forms that correlated with disease activity. Last, blocking P-selectin in a mouse model of SLE improved cardinal features of the disease, such as anti-dsDNA antibody concentrations and kidney pathology. Overall, our results identify a P-selectin–dependent pathway that is active in patients with SLE and validate it as a potential therapeutic avenue. [ABSTRACT FROM AUTHOR]

S1P signaling CLICs into place: The chloride intracellular channels CLIC1 and CLIC4 are implicated in vascular development, particularly in response to oxidative stress. Mao et al. found that these CLICs mediate the selective activation of small GTPases in response to the lipid second messenger S1P. In cultured endothelial cells, CLIC1 and CLIC4 together mediated S1P-induced Rac activation and downstream endothelial cell adhesion and sprouting, whereas CLIC1 alone mediated S1P-induced Rho activation and downstream endothelial stress fiber formation. The findings establish mechanisms in which these CLICs regulate endothelial cell function through both coordinated and distinct manners. Chloride intracellular channels 1 (CLIC1) and 4 (CLIC4) are expressed in endothelial cells and regulate angiogenic behaviors in vitro, and the expression of Clic4 is important for vascular development and function in mice. Here, we found that CLIC1 and CLIC4 in endothelial cells regulate critical G protein–coupled receptor (GPCR) pathways associated with vascular development and disease. In cultured endothelial cells, we found that CLIC1 and CLIC4 transiently translocated to the plasma membrane in response to sphingosine 1-phosphate (S1P). Both CLIC1 and CLIC4 were essential for mediating S1P-induced activation of the small guanosine triphosphatase (GTPase) Rac1 downstream of S1P receptor 1 (S1PR1). In contrast, only CLIC1 was essential for S1P-induced activation of the small GTPase RhoA downstream of S1PR2 and S1PR3. Neither were required for other S1P-S1PR signaling outputs. Rescue experiments revealed that CLIC1 and CLIC4 were not functionally interchangeable, suggesting distinct and specific functions for CLICs in transducing GPCR signaling. These CLIC-mediated mechanisms were critical for S1P-induced stimulation of the barrier function in endothelial cell monolayers. Our results define CLICs as previously unknown players in the pathways linking GPCRs to small GTPases and vascular endothelial function. [ABSTRACT FROM AUTHOR]

The article discusses two research papers that further understanding of the molecular basis for cellular mechanisms. The first of these is mechanosensitive adhesions that are mediated by membrane proteins known as integrins and are believed to underlie many areas relevant to human physiology and disease. The second is the strengthening of these adhesions through forces that are applied by either the cells' cytoskeleton or from external sources and trigger a variety of intracellular signals. One paper provides evidence that the fibronectin-binding integrin undergoes a conformational transition. The other focuses on the properties of talin.

Fc function in focus: There are multiple mechanisms by which broadly neutralizing antibodies (bnAbs) promote protection against HIV, including neutralization of virions and Fc receptor–mediated induction of effector functions on immune cells. Here, Hangartner et al. used variants of the potent HIV bnAb, PGT121, to investigate whether Fc receptor function is required for the protective effects elicited by antibody treatment. The authors showed that variants of PGT121 that could not bind to Fc receptors still protected female macaques against intravaginal simian-HIV infection. These findings underpin the importance of neutralization in determining effectiveness of bnAb treatment for HIV. Protection from immunodeficiency virus challenge in nonhuman primates (NHPs) by a first-generation HIV broadly neutralizing antibody (bnAb) b12 has previously been shown to benefit from interaction between the bnAb and Fcγ receptors (FcγRs) on immune cells. To investigate the mechanism of protection for a more potent second-generation bnAb currently in clinical trials, PGT121, we carried out a series of NHP studies. These studies included treating with PGT121 at a concentration at which only half of the animals were protected to avoid potential masking of FcγR effector function benefits by dominant neutralization and using a new variant that more completely eliminated all rhesus FcγR binding than earlier variants. In contrast to b12, which required FcγR binding for optimal protection, we concluded that PGT121-mediated protection is not augmented by FcγR interaction. Thus, for HIV-passive antibody prophylaxis, these results, together with existing literature, emphasize the importance of neutralization potency for clinical antibodies, with effector function requiring evaluation for individual antibodies. [ABSTRACT FROM AUTHOR]

Prolonged prevention of autoimmune diabetes: Teplizumab was previously shown in a clinical trial to delay onset of type 1 diabetes (T1D) in high-risk relatives of individuals with T1D. Now, Sims et al. extend the follow-up analysis of this trial by 12 months, finding that efficacy of the initial 2-week treatment course persisted, with an extended time to T1D diagnosis in the teplizumab-treated group. Clinical benefits associated with reversed C-peptide decline improved beta cell function and partial exhaustion in CD8+ T cells in the treated patients. HbA1c did not differ between placebo and treatment groups. This follow-up study further supports the use of anti-CD3 treatment for the prevention of T1D. We analyzed the effects of a single 14-day course of teplizumab treatment on metabolic function and immune cells among participants in a previously reported randomized controlled trial of nondiabetic relatives at high risk for type 1 diabetes (T1D). In an extended follow-up (923-day median) of a previous report of teplizumab treatment, we found that the median times to diagnosis were 59.6 and 27.1 months for teplizumab- and placebo-treated participants, respectively (HR = 0.457, P = 0.01). Fifty percent of teplizumab-treated but only 22% of the placebo-treated remained diabetes-free. Glucose tolerance, C-peptide area under the curve (AUC), and insulin secretory rates were calculated, and relationships to T cell subsets and function were analyzed. Teplizumab treatment improved beta cell function, reflected by average on-study C-peptide AUC (1.94 versus 1.72 pmol/ml; P = 0.006). Drug treatment reversed a decline in insulin secretion before enrollment, followed by stabilization of the declining C-peptide AUC seen with placebo treatment. Proinsulin:C-peptide ratios after drug treatment were similar between the treatment groups. The changes in C-peptide with teplizumab treatment were associated with increases in partially exhausted memory KLRG1+TIGIT+CD8+ T cells (r = 0.44, P = 0.014) that showed reduced secretion of IFNγ and TNFα. A single course of teplizumab had lasting effects on delay of T1D diagnosis and improved beta cell function in high-risk individuals. Changes in CD8+ T cell subsets indicated that partially exhausted effector cells were associated with clinical response. Thus, this trial showed improvement in metabolic responses and delay of diabetes with immune therapy. [ABSTRACT FROM AUTHOR]

CD100 mutation primes PSC: Primary sclerosing cholangitis (PSC) is an inflammatory liver disease without effective treatments. Although genetic factors are thought to predispose to PSC, no causative mutation has yet been identified. Jiang et al. performed whole-exome sequencing on a family with autosomal dominant inheritance of PSC and identified a germline mutation in SEMA4D, encoding CD100. The mutation resulted in T cell functional defects, including impaired interferon-γ production. Mutation knock-in mice were more prone to develop severe cholangitis after toxin exposure, and adoptive transfer of wild-type T cells into knock-in mice resulted in less severe disease. This protective role of T cells in PSC might be exploited therapeutically. Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disease without clear etiology or effective treatment. Genetic factors contribute to PSC pathogenesis, but so far, no causative mutation has been found. We performed whole-exome sequencing in a family with autosomal dominant inheritance of PSC and identified a heterozygous germline missense mutation in SEMA4D, encoding a K849T variant of CD100. The mutation was located in an evolutionarily conserved, unstructured cytosolic region of CD100 affecting downstream signaling. It was found to alter the function of CD100-expressing cells with a bias toward the T cell compartment that caused increased proliferation and impaired interferon-γ (IFN-γ) production after stimulation. Homologous mutation knock-in mice developed similar IFN-γ impairment in T cells and were more prone to develop severe cholangitis when exposed to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet. Transfer of wild-type T cells to knock-in mice before and during DDC exposure attenuated cholangitis. Taken together, we identified an inherited mutation in the disordered cytosolic region of CD100 resulting in T cell functional defects. Our findings suggest a protective role for T cells in PSC that might be used therapeutically. [ABSTRACT FROM AUTHOR]

A shared Toll for innate immunity: Toll-like receptor 2 (TLR2) is found in various tissues but is best known for its role in the innate immune system of activating sentinel immune cells in response to infection. Using conditional knockout mice, McCoy et al. found that TLR2 also mediated innate immune signaling within the endothelium. TLR2 in endothelial cells activated proinflammatory signaling that promoted angiogenesis and immune cell recruitment in response to various "danger" signals, such as those produced during infection or tissue damage. Endothelial TLR2 also supported tumor growth in a mouse model of prostate cancer. These findings show that the endothelium contributes to innate immune responses and that TLR2 may be a therapeutic target in cancer (see also the Focus by Mahfoud and Petrova). Toll-like receptor 2 (TLR2) is implicated in various pathologies, mainly in terms of its function within innate immune cells. However, TLR2 is also present in endothelial cells. Here, we explored the physiological and pathophysiological roles of endothelial TLR2 signaling. We found that TLR2 was highly abundant in the endothelium within various tissues using TLR2-IRES-EGFP reporter mice and was required for proinflammatory endothelial cell function. Endothelial cells lacking TLR2 exhibited reduced proinflammatory potential at the protein, cell, and tissue levels. Loss of endothelial TLR2 blunted the inflammatory response to both exogenous and endogenous danger signals in endothelial cells in culture and in vivo. Endothelial TLR2 promoted tumor growth, angiogenesis, and protumorigenic immune cell recruitment in a mouse model of prostate cancer. Furthermore, the cell surface localization of P-selectin and the subsequent production of other critical cell adhesion molecules (such as E-selectin, ICAM-1, and VCAM-1) that recruit immune cells required endothelial TLR2. Our findings demonstrate that endothelial cells actively contribute to innate immune pathways and propose that endothelial TLR2 has a pathological role in proinflammatory conditions. [ABSTRACT FROM AUTHOR]

Rethinking metabolic manipulations in MS: The metabolic state of TH17 cells determines whether they drive experimental autoimmune encephalitis (EAE), a mouse model of multiple sclerosis (MS). Activation of the metabolic enzyme PKM2, which catalyzes a critical step in glycolysis, has been proposed as a potential treatment for MS. Seki et al. found that although PKM2 activators did limit TH17 cell differentiation by suppressing the production of IL-17, they enhanced the production of the encephalitogenic cytokine GM-CSF, which was associated with disease pathology in the brain rather than the spinal cord. PKM2 activators also blocked the generation of Tregs, an anti-inflammatory T cell subset that suppresses T cell activation. Genetic ablation of Pkm2 led to a compensatory increase in the related enzyme PKM1, which suggests that inhibiting PKM2 may lead to similar outcomes as activating PKM2. These results suggest that the therapeutic potential of PKM2 activators for MS must be reassessed. Small molecules that promote the metabolic activity of the pyruvate kinase isoform PKM2, such as TEPP-46 and DASA-58, limit tumorigenesis and inflammation. To understand how these compounds alter T cell function, we assessed their therapeutic activity in a mouse model of T cell–mediated autoimmunity that mimics multiple sclerosis (MS). TH17 cells are believed to orchestrate MS pathology, in part, through the production of two proinflammatory cytokines: interleukin-17 (IL-17) and GM-CSF. We found that both TEPP-46 and DASA-58 suppressed the development of IL-17–producing TH17 cells but increased the generation of those producing GM-CSF. This switch redirected disease pathology from the spinal cord to the brain. In addition, we found that activation of PKM2 interfered with TGF-β1 signaling, which is necessary for the development of TH17 and regulatory T cells. Collectively, our data clarify the therapeutic potential of PKM2 activators in MS-like disease and how these agents alter T cell function. [ABSTRACT FROM AUTHOR]

Polarized by purinergic signaling: Migration enables T cells to carry out many of their functions. Ledderose et al. found that T cell migration requires the redistribution of the purinergic receptor P2Y11 to the rear of cells. In this subcellular location, P2Y11 receptors suppress mitochondrial metabolism and promote uropod retraction. P2Y11 receptor redistribution was also found necessary to restrict signaling mediated by P2X4 purinergic receptors to the front of migrating cells to stimulate pseudopod formation and mitochondrial metabolism that sustain migration. These results indicate that P2Y11 signaling at the rear of cells confers the polarization in morphology, mitochondrial metabolism, and signaling necessary to ensure efficient T cell migration. T cells must migrate to encounter antigen-presenting cells and perform their roles in host defense. Here, we found that autocrine stimulation of the purinergic receptor P2Y11 regulates the migration of human CD4 T cells. P2Y11 receptors redistributed from the front to the back of polarized cells where they triggered intracellular cAMP/PKA signals that attenuated mitochondrial metabolism at the back. The absence of P2Y11 receptors at the front of cells resulted in hotspots of mitochondrial metabolism and localized ATP production that stimulated P2X4 receptors, Ca2+ influx, and pseudopod protrusion at the front. This regulatory function of P2Y11 receptors depended on their subcellular redistribution and autocrine stimulation by cellular ATP release and was perturbed by indiscriminate global stimulation. We conclude that excessive extracellular ATP—such as in response to inflammation, sepsis, and cancer—disrupts this autocrine feedback mechanism, which results in defective T cell migration, impaired T cell function, and loss of host immune defense. [ABSTRACT FROM AUTHOR]

Enhancing cardiac recovery with exosomes: Cell transplant has been an attractive potential therapy for cardiovascular disease; however, poor cell engraftment limits efficacy of the approach. Gao and colleagues compared transplanting a mixture of human induced pluripotent stem cell–derived cardiomyocytes, endothelial cells, and smooth muscle cells to transplant of exosomes produced by these cells in a pig model of myocardial infarction. They saw similar improvements in cardiac function in cell, cell fragment, and exosome transplant groups without evidence of increased arrhythmogenicity. This suggests that exosomes could have potential as an acellular therapy for cardiovascular disease. Cell therapy treatment of myocardial infarction (MI) is mediated, in part, by exosomes secreted from transplanted cells. Thus, we compared the efficacy of treatment with a mixture of cardiomyocytes (CMs; 10 million), endothelial cells (ECs; 5 million), and smooth muscle cells (SMCs; 5 million) derived from human induced pluripotent stem cells (hiPSCs), or with exosomes extracted from the three cell types, in pigs after MI. Female pigs received sham surgery; infarction without treatment (MI group); or infarction and treatment with hiPSC-CMs, hiPSC-ECs, and hiPSC-SMCs (MI + Cell group); with homogenized fragments from the same dose of cells administered to the MI + Cell group (MI + Fra group); or with exosomes (7.5 mg) extracted from a 2:1:1 mixture of hiPSC-CMs:hiPSC-ECs:hiPSC-SMCs (MI + Exo group). Cells and exosomes were injected into the injured myocardium. In vitro, exosomes promoted EC tube formation and microvessel sprouting from mouse aortic rings and protected hiPSC-CMs by reducing apoptosis, maintaining intracellular calcium homeostasis, and increasing adenosine 5′-triphosphate. In vivo, measurements of left ventricular ejection fraction, wall stress, myocardial bioenergetics, cardiac hypertrophy, scar size, cell apoptosis, and angiogenesis in the infarcted region were better in the MI + Cell, MI + Fra, and MI + Exo groups than in the MI group 4 weeks after infarction. The frequencies of arrhythmic events in animals from the MI, MI + Cell, and MI + Exo groups were similar. Thus, exosomes secreted by hiPSC-derived cardiac cells improved myocardial recovery without increasing the frequency of arrhythmogenic complications and may provide an acellular therapeutic option for myocardial injury. [ABSTRACT FROM AUTHOR]

Feeding with benefits: In addition to providing health benefits for infants, breastfeeding has also been reported to offer metabolic benefits for mothers, but the underlying biology explaining this observation has remained elusive. By studying both human and murine mothers over time, Moon et al. identified long-term improvements in pancreatic β cell mass and function, as well as glucose tolerance, all associated with lactation. The authors linked the hormone prolactin to serotonin production and proliferation of the β cells, providing a mechanistic explanation for the beneficial effects of lactation. Pregnancy imposes a substantial metabolic burden on women through weight gain and insulin resistance. Lactation reduces the risk of maternal postpartum diabetes, but the mechanisms underlying this benefit are unknown. Here, we identified long-term beneficial effects of lactation on β cell function, which last for years after the cessation of lactation. We analyzed metabolic phenotypes including β cell characteristics in lactating and non-lactating humans and mice. Lactating and non-lactating women showed comparable glucose tolerance at 2 months after delivery, but after a mean of 3.6 years, glucose tolerance in lactated women had improved compared to non-lactated women. In humans, the disposition index, a measure of insulin secretory function of β cells considering the degree of insulin sensitivity, was higher in lactated women at 3.6 years after delivery. In mice, lactation improved glucose tolerance and increased β cell mass at 3 weeks after delivery. Amelioration of glucose tolerance and insulin secretion were maintained up to 4 months after delivery in lactated mice. During lactation, prolactin induced serotonin production in β cells. Secreted serotonin stimulated β cell proliferation through serotonin receptor 2B in an autocrine and paracrine manner. In addition, intracellular serotonin acted as an antioxidant to mitigate oxidative stress and improved β cell survival. Together, our results suggest that serotonin mediates the long-term beneficial effects of lactation on female metabolic health by increasing β cell proliferation and reducing oxidative stress in β cells. [ABSTRACT FROM AUTHOR]

Balancing B cell signals: Diacylglycerol kinases (DGKs) catalyze the conversion of the lipid signaling molecule DAG into phosphatidic acid (PA), both of which have distinct effects. Merino-Cortés et al. found that the DGKζ isoform was critical in promoting signaling required for the optimal function of B cells. Compared with wild-type B cells, DGKζ-deficient B cells exhibited decreased mechanical forces at the plasma membrane, impairing the ability of these cells to form stable contacts with antigen-presenting cells, extract antigen, and present it to T cells. As a result, mice with DGKζ-deficient B cells showed diminished antibody responses in germinal centers. These data suggest that DGKζ regulates the balance in signaling between DAG and PA that is required for optimal B cell function. Diacylglycerol kinases (DGKs) limit antigen receptor signaling in immune cells by consuming the second messenger diacylglycerol (DAG) to generate phosphatidic acid (PA). Here, we showed that DGKζ promotes lymphocyte function–associated antigen 1 (LFA-1)–mediated adhesion and F-actin generation at the immune synapse of B cells with antigen-presenting cells (APCs), mostly in a PA-dependent manner. Measurement of single-cell mechanical force generation indicated that DGKζ-deficient B cells exerted lower forces at the immune synapse than did wild-type B cells. Nonmuscle myosin activation and translocation of the microtubule-organizing center (MTOC) to the immune synapse were also impaired in DGKζ-deficient B cells. These functional defects correlated with the decreased ability of B cells to present antigen and activate T cells in vitro. The in vivo germinal center response of DGKζ-deficient B cells was also reduced compared with that of wild-type B cells, indicating that loss of DGKζ in B cells impaired T cell help. Together, our data suggest that DGKζ shapes B cell responses by regulating actin remodeling, force generation, and antigen uptake–related events at the immune synapse. Hence, an appropriate balance in the amounts of DAG and PA is required for optimal B cell function. [ABSTRACT FROM AUTHOR]

Checkpoint musical chairs: Anti–PD-1 or PD-L1 antibodies can reinvigorate antitumor immunity in a subset of patients with cancer. To better understand the mechanisms behind successful therapy, Mayoux et al. characterized various ligands on the surface of dendritic cells (DCs). PD-L1 on DCs can bind B7.1 on the same cell, potentially preventing PD-1 ligation on T cells or B7.1 ligation of its partner CD28. They saw that PD-L1 was expressed in excess of B7.1, likely preventing T cell stimulation through these two pathways. Patients with a high DC signature before treatment were more likely to respond to PD-L1 blockade. These results reveal that in cis interactions on DCs have immunological and likely clinical consequences for checkpoint blockade therapy. PD-L1/PD-1 blocking antibodies have demonstrated therapeutic efficacy across a range of human cancers. Extending this benefit to a greater number of patients, however, will require a better understanding of how these therapies instigate anticancer immunity. Although the PD-L1/PD-1 axis is typically associated with T cell function, we demonstrate here that dendritic cells (DCs) are an important target of PD-L1 blocking antibody. PD-L1 binds two receptors, PD-1 and B7.1 (CD80). PD-L1 is expressed much more abundantly than B7.1 on peripheral and tumor-associated DCs in patients with cancer. Blocking PD-L1 on DCs relieves B7.1 sequestration in cis by PD-L1, which allows the B7.1/CD28 interaction to enhance T cell priming. In line with this, in patients with renal cell carcinoma or non–small cell lung cancer treated with atezolizumab (PD-L1 blockade), a DC gene signature is strongly associated with improved overall survival. These data suggest that PD-L1 blockade reinvigorates DC function to generate potent anticancer T cell immunity. [ABSTRACT FROM AUTHOR]

How too much fluoride derails dentition: Excessive fluoride ingestion during childhood results in defective tooth enamel mineralization, which can lead to dental problems later in life. Aulestia et al. investigated the molecular mechanisms underlying fluorosis in enamel-forming cells isolated from rats and in an enamel cell line. Exposure of enamel cells to fluoride resulted in decreases in ER Ca2+ content and store-operated Ca2+ entry into the ER, reduced the expression of genes encoding ER stress–response proteins, and resulted in mitochondrial dysfunction. These effects were not seen in HEK-293 cells (which are derived from kidney epithelium). These data may explain how fluorosis affects Ca2+ homeostasis in enamel-forming cells and highlight cell type–specific stress responses. Fluoride ions are highly reactive, and their incorporation in forming dental enamel at low concentrations promotes mineralization. In contrast, excessive fluoride intake causes dental fluorosis, visually recognizable enamel defects that can increase the risk of caries. To investigate the molecular bases of dental fluorosis, we analyzed the effects of fluoride exposure in enamel cells to assess its impact on Ca2+ signaling. Primary enamel cells and an enamel cell line (LS8) exposed to fluoride showed decreased internal Ca2+ stores and store-operated Ca2+ entry (SOCE). RNA-sequencing analysis revealed changes in gene expression suggestive of endoplasmic reticulum (ER) stress in fluoride-treated LS8 cells. Fluoride exposure did not alter Ca2+ homeostasis or increase the expression of ER stress–associated genes in HEK-293 cells. In enamel cells, fluoride exposure affected the functioning of the ER-localized Ca2+ channel IP3R and the activity of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump during Ca2+ refilling of the ER. Fluoride negatively affected mitochondrial respiration, elicited mitochondrial membrane depolarization, and disrupted mitochondrial morphology. Together, these data provide a potential mechanism underlying dental fluorosis. [ABSTRACT FROM AUTHOR]

Diversity starts at the gene level: Natural killer (NK) cells are immune cells with potent antiviral and antitumor activities. These cells express several germline-encoded inhibitory receptors (KIRs) that prevent NK cells from killing healthy cells but enable the targeting of infected and transformed cells. Using various imaging techniques and functional assays, Kennedy et al. investigated the effects of genetic diversity in KIR-encoding genes on receptor organization and activity. They found that KIRs encoded by distinct genes were present at varying abundances and in clusters of varying sizes, which led to differences in downstream signaling that affected NK cell function. Together, these data suggest that genetic diversity in KIR-encoding genes affects receptor organization, signaling, and functional outcomes. Genetic diversity in human natural killer (NK) cell receptors is linked to resistance and susceptibility to many diseases. Here, we tested the effect of this diversity on the nanoscale organization of killer cell immunoglobulin-like receptors (KIRs). Using superresolution microscopy, we found that inhibitory KIRs encoded by different genes and alleles were organized differently at the surface of primary human NK cells. KIRs that were found at low abundance assembled into smaller clusters than those formed by KIRs that were more highly abundant, and at low abundance, there was a greater proportion of KIRs in clusters. Upon receptor triggering, a structured interface called the immune synapse assembles, which facilitates signal integration and controls NK cell responses. Here, triggering of low-abundance receptors resulted in less phosphorylation of the downstream phosphatase SHP-1 but more phosphorylation of the adaptor protein Crk than did triggering of high-abundance receptors. In cells with greater KIR abundance, SHP-1 dephosphorylated Crk, which potentiated NK cell spreading during activation. Thus, genetic variation modulates both the abundance and nanoscale organization of inhibitory KIRs. That is, as well as the number of receptors at the cell surface varying with genotype, the way in which these receptors are organized in the membrane also varies. Essentially, a change in the average surface abundance of a protein at the cell surface is a coarse descriptor entwined with changes in local nanoscale clustering. Together, our data indicate that genetic diversity in inhibitory KIRs affects membrane-proximal signaling and, unexpectedly, the formation of activating immune synapses. [ABSTRACT FROM AUTHOR]

Suppressing antitumor immunity: The cytokine TGF-β has both immune-suppressive and tumor-suppressive functions; thus, a better understanding of the cell-type specificity of the effects of TGF-β might improve therapeutic strategies that target it. Dimeloe et al. found that TGF-β from tumor effusions suppressed the antitumor activity of CD4+ T cells by inhibiting their production of the inflammatory cytokine IFN-γ. The effects of TGF-β were mediated by Smad proteins in the mitochondria, rather than in the nucleus, and led to decreased mitochondrial respiration. Indeed, direct inhibition of a mitochondrial electron transport chain complex in CD4+ T cells was sufficient to inhibit IFN-γ production. Thus, these data suggest that TGF-β targets T cell metabolism to suppress antitumor immunity. Transforming growth factor–β (TGF-β) is produced by tumors, and increased amounts of this cytokine in the tumor microenvironment and serum are associated with poor patient survival. TGF-β–mediated suppression of antitumor T cell responses contributes to tumor growth and survival. However, TGF-β also has tumor-suppressive activity; thus, dissecting cell type–specific molecular effects may inform therapeutic strategies targeting this cytokine. Here, using human peripheral and tumor-associated lymphocytes, we investigated how tumor-derived TGF-β suppresses a key antitumor function of CD4+ T cells, interferon-γ (IFN-γ) production. Suppression required the expression and phosphorylation of Smad proteins in the TGF-β signaling pathway, but not their nuclear translocation, and depended on oxygen availability, suggesting a metabolic basis for these effects. Smad proteins were detected in the mitochondria of CD4+ T cells, where they were phosphorylated upon treatment with TGF-β. Phosphorylated Smad proteins were also detected in the mitochondria of isolated tumor-associated lymphocytes. TGF-β substantially impaired the ATP-coupled respiration of CD4+ T cells and specifically inhibited mitochondrial complex V (ATP synthase) activity. Last, inhibition of ATP synthase alone was sufficient to impair IFN-γ production by CD4+ T cells. These results, which have implications for human antitumor immunity, suggest that TGF-β targets T cell metabolism directly, thus diminishing T cell function through metabolic paralysis. [ABSTRACT FROM AUTHOR]

Enhancing NK cells: Natural killer (NK) cells are innate immune cells with a critical role in antitumor immunity. In the tumor microenvironment, the cytokine transforming growth factor–β (TGF-β) acts through its receptor to promote the differentiation of NK cells into a less suppressive cell type, thus inhibiting antitumor immunity. Rautela et al. showed that activin-A, another member of the TGF-β family, had similar effects on both mouse and human NK cells, although in a TGF-β receptor–independent manner. Inhibition of activin-A reduced orthotopic melanoma growth in mice, suggesting that targeting this pathway could therapeutically enhance NK cell function and antitumor immunity. Natural killer (NK) cells are innate lymphocytes that play a major role in immunosurveillance against tumor initiation and metastatic spread. The signals and checkpoints that regulate NK cell fitness and function in the tumor microenvironment are not well defined. Transforming growth factor–β (TGF-β) is a suppressor of NK cells that inhibits interleukin-15 (IL-15)–dependent signaling events and increases the abundance of receptors that promote tissue residency. Here, we showed that NK cells express the type I activin receptor ALK4, which, upon binding to its ligand activin-A, phosphorylated SMAD2/3 to suppress IL-15–mediated NK cell metabolism. Activin-A impaired human and mouse NK cell proliferation and reduced the production of granzyme B to impair tumor killing. Similar to TGF-β, activin-A also induced SMAD2/3 phosphorylation and stimulated NK cells to increase their cell surface expression of several markers of ILC1 cells. Activin-A also induced these changes in TGF-β receptor–deficient NK cells, suggesting that activin-A and TGF-β stimulate independent pathways that drive SMAD2/3-mediated NK cell suppression. Last, inhibition of activin-A by follistatin substantially slowed orthotopic melanoma growth in mice. These data highlight the relevance of examining TGF-β–independent SMAD2/3 signaling mechanisms as a therapeutic axis to relieve NK cell suppression and promote antitumor immunity. [ABSTRACT FROM AUTHOR]

Gab3 adapter prevents NK cell dysfunction: Natural killer (NK) cells are innate effector cells that help defend mammals from viral infections and cancer while acting locally in the uterus to support successful pregnancy outcomes. Sliz et al. used a chemical mutagenesis screen in mice to identify Gab3 as a scaffolding protein required for NK cell priming and peripheral expansion in response to cytokines. Gab3 mutant mice exhibited defects in their ability to control tumors and successfully complete pregnancies because of impaired NK cell function. This study identifies an adaptor protein required to achieve full NK cell function and enhances our understanding of how subtle uterine NK cell perturbations can contribute to the demise of pregnancies. See related Focus by Colucci. The scaffolding protein Grb2-associated binding protein 3 (Gab3) is a member of the Gab family, whose functions have remained elusive. Here, we identify Gab3 as a key determinant of peripheral NK cell expansion. Loss of Gab3 resulted in impaired IL-2 and IL-15–induced NK cell priming and expansion due to a selective impairment in MAPK signaling but not STAT5 signaling. In vivo, we found that Gab3 is required for recognition and elimination of "missing-self" and tumor targets. Unexpectedly, our studies also revealed that Gab3 plays an important role during pregnancy. Gab3-deficient mice exhibited impaired uterine NK cell expansion associated with abnormal spiral artery remodeling and increased trophoblast invasion in the decidua basalis. This coincided with stillbirth, retained placenta, maternal hemorrhage, and undelivered fetoplacental units at term. Thus, Gab3 is a key component required for cytokine-mediated NK cell priming and expansion that is essential for antitumor responses and limits trophoblast cell invasion during pregnancy. [ABSTRACT FROM AUTHOR]

Licensing interleukin-9 production: Whereas the biological roles of T helper 1 (TH1), TH2, and TH17 cells are reasonably well established, the functions of interleukin-9 (IL-9)–secreting TH9 cells remains elusive. Several studies have documented the presence of TH9 cells in both humans and mice. Here, by studying human TH cells ex vivo, Micossé et al. propose that TH9 cells are a subpopulation of TH2 cells that transiently up-regulate IL-9 and report the transcription factor peroxisome proliferator–activated receptor–γ (PPAR-γ) to be a key regulator of IL-9 production. The results presented by Micossé et al. call for a closer examination of the ontogeny of IL-9–producing TH cells using cytokine reporter mouse strains. Although TH1, TH2, and TH17 cells are well-defined TH cell lineages in humans, it remains debated whether IL-9–producing TH cells represent a bona fide "TH9" lineage. Our understanding of the cellular characteristics and functions of IL-9–producing TH cells in humans is still nascent. Here, we report that human IL-9–producing TH cells express the chemokine receptors CCR4 and CCR8, produce high levels of IL-5 and IL-13, and express TH2 lineage–associated transcription factors. In these cells, IL-9 production is activation dependent, transient, and accompanied by down-regulation of TH2 cytokines, leading to an apparent "TH9" phenotype. IL-9+ TH2 cells can be distinguished from "conventional" TH2 cells based on their expression of the transcription factor PPAR-γ. Accordingly, PPAR-γ is induced in naïve TH cells by priming with IL-4 and TGF-β ("TH9" priming) and is required for IL-9 production. In line with their identity as early activated TH2 cells, IL-9+ TH2 cells are found in acute allergic skin inflammation in humans. We propose that IL-9–producing TH cells are a phenotypically and functionally distinct subpopulation of TH2 cells that depend on PPAR-γ for full effector functions. [ABSTRACT FROM AUTHOR]

Fluorescent Ca2+ indicators have been essential for the analysis of Ca2+ signaling events in various cell types. We showed that chemical Ca2+ indicators, but not a genetically encoded Ca2+ indicator, potently suppressed the activity of Na+- and K+-dependent adenosine triphosphatase (Na,K-ATPase), independently of their Ca2+ chelating activity. Loading of commonly used Ca2+ indicators, including Fluo-4 acetoxymethyl (AM), Rhod-2 AM, and Fura-2 AM, and of the Ca2+ chelator BAPTA AM into cultured mouse or human neurons, astrocytes, cardiomyocytes, or kidney proximal tubule epithelial cells suppressed Na,K-ATPase activity by 30 to 80%. Ca2+ indicators also suppressed the agonist-induced activation of the Na,K-ATPase, altered metabolic status, and caused a dose-dependent loss of cell viability. Loading of Ca2+ indicators into mice, which is carried out for two-photon imaging, markedly altered brain extracellular concentrations of K+ and ATP. These results suggest that a critical review of data obtained with chemical Ca2+ indicators may be necessary. [ABSTRACT FROM AUTHOR]

The article focuses findings of a study on application of reaction kinetics to explain complex patterns of antibiotic action. It states that chemical binding kinetics can be used to phenomena like post-antibiotic growth suppression, and density-dependent antibiotic effects using single cell data and time-kill curves of Escherichia coli and Vibrio cholerae exposed to different antibiotics, and mentions a model that considers binding kinetics for explaining for the phenomena.

The article notes a paper by Clemson and associates in a 2009 issue of "Molecular Cell" on "NEAT1," a non-coding RNA (ncRNA) compound and intranuclear bodies called paraspeckles. Research using mouse and human cells found that by forming paraspeckles, NEAT1 has a role in nuclear compartmentalization. Cells without NEAT1 did not contain paraspeckles.