Identification of a novel mitochondria-localized LKB1 variant required for the regulation of the oxidative stress response.

The tumor suppressor Liver Kinase B1 (LKB1) is a multifunctional serine/threonine protein kinase that regulates cell metabolism, polarity, and growth and is associated with Peutz-Jeghers Syndrome and cancer predisposition. The LKB1 gene comprises 10 exons and 9 introns. Three spliced LKB1 variants have been documented, and they reside mainly in the cytoplasm, although two possess a nuclear-localization sequence (NLS) and are able to shuttle into the nucleus. Here, we report the identification of a fourth and novel LKB1 isoform that is, interestingly, targeted to the mitochondria. We show that this mitochondria-localized LKB1 (mLKB1) is generated from alternative splicing in the 5' region of the transcript and translated from an alternative initiation codon encoded by a previously unknown exon 1b (131 bp) hidden within the long intron 1 of LKB1 gene. We found by replacing the N-terminal NLS of the canonical LKB1 isoform, the N-terminus of the alternatively spliced mLKB1 variant encodes a mitochondrial transit peptide that allows it to localize to the mitochondria. We further demonstrate that mLKB1 colocalizes histologically with mitochondria-resident ATP Synthase and NAD-dependent deacetylase sirtuin-3, mitochondrial (SIRT3) and that its expression is rapidly and transiently upregulated by oxidative stress. We conclude that this novel LKB1 isoform, mLKB1, plays a critical role in regulating mitochondrial metabolic activity and oxidative stress response.

Bruton's tyrosine kinase phosphorylates scaffolding and RNA-binding protein G3BP1 to induce stress granule aggregation during host sensing of foreign ribonucleic acids.

The Ras-GTPase activating protein SH3 domain-binding protein 1 (G3BP1) plays a critical role in the formation of classical and antiviral stress granules in stressed and virus-infected eukaryotic cells, respectively. While G3BP1 is known to be phosphorylated at serine residues which could affect stress granule assembly, whether G3BP1 is phosphorylated at tyrosine residues and how this posttranslational modification might affect its functions is less clear. Here, we show using immunoprecipitation and immunoblotting studies with 4G10 antibody that G3BP1 is tyrosine-phosphorylated when cells are stimulated with the synthetic double-stranded RNA analog polyinosinic:polycytidylic acid to mimic viral infection. We further demonstrate via co-immunoprecipitation and inhibitor studies that Bruton's tyrosine kinase (BTK) binds and phosphorylates G3BP1. The nuclear transport factor 2-like domain of G3BP1 was previously shown to be critical for its self-association to form stress granules. Our mass spectrometry, mutational and biochemical cross-linking analyses indicate that the tyrosine-40 residue in this domain is phosphorylated by BTK and critical for G3BP1 oligomerization. Furthermore, as visualized via confocal microscopy, pretreatment of cells with the BTK inhibitor LFM-A13 or genetic deletion of the btk gene or mutation of G3BP1-Y40 residue to alanine or phenylalanine all significantly attenuated the formation of antiviral stress granule aggregates upon polyinosinic:polycytidylic acid treatment. Taken together, our data indicate that BTK phosphorylation of G3BP1 induces G3BP1 oligomerization and facilitates the condensation of ribonucleoprotein complexes into macromolecular aggregates.

MSC-sEV Treatment Polarizes Pro-Fibrotic M2 Macrophages without Exacerbating Liver Fibrosis in NASH.

Mesenchymal stem/stromal cell small extracellular vesicles (MSC-sEVs) have shown promise in treating a wide range of animal models of various human diseases, which has led to their consideration for clinical translation. However, the possibility of contraindication for MSC-sEV use is an important consideration. One concern is that MSC-sEVs have been shown to induce M2 macrophage polarization, which is known to be pro-fibrotic, potentially indicating contraindication in fibrotic diseases such as liver fibrosis. Despite this concern, previous studies have shown that MSC-sEVs alleviate high-fat diet (HFD)-induced non-alcoholic steatohepatitis (NASH). To assess whether the pro-fibrotic M2 macrophage polarization induced by MSC-sEVs could worsen liver fibrosis, we first verified that our MSC-sEV preparations could promote M2 polarization in vitro prior to their administration in a mouse model of NASH. Our results showed that treatment with MSC-sEVs reduced or had comparable NAFLD Activity Scores and liver fibrosis compared to vehicle- and Telmisartan-treated animals, respectively. Although CD163+ M2 macrophages were increased in the liver, and serum IL-6 levels were reduced in MSC-sEV treated animals, our data suggests that MSC-sEV treatment was efficacious in reducing liver fibrosis in a mouse model of NASH despite an increase in pro-fibrotic M2 macrophage polarization.

Mechanism for the attenuation of neutrophil and complement hyperactivity by MSC exosomes.

Complements and neutrophils are two key players of the innate immune system that are widely implicated as drivers of severe COVID-19 pathogenesis, as evident by the direct correlation of respiratory failure and mortality with elevated levels of terminal complement complex C5b-9 and neutrophils. In this study, we identified a feed-forward loop between complements and neutrophils that could amplify and perpetuate the cytokine storm seen in severe SARS-CoV-2-infected patients. We observed for the first time that the terminal complement activation complex C5b-9 directly triggered neutrophil extracellular trap (NET) release and interleukin (IL)-17 production by neutrophils. This is also the first report that the production of NETs and IL-17 induced by C5b-9 assembly on neutrophils could be abrogated by mesenchymal stem cell (MSC) exosomes. Neutralizing anti-CD59 antibodies abolished this abrogation. Based on our findings, we hypothesize that MSC exosomes could alleviate the immune dysregulation in acute respiratory failure, such as that observed in severe COVID-19 patients, by inhibiting complement activation through exosomal CD59, thereby disrupting the feed-forward loop between complements and neutrophils to inhibit the amplification and perpetuation of inflammation during SARS-CoV-2 infection.

Development and validation of a serum microRNA biomarker panel for detecting gastric cancer in a high-risk population.

OBJECTIVE: An unmet need exists for a non-invasive biomarker assay to aid gastric cancer diagnosis. We aimed to develop a serum microRNA (miRNA) panel for identifying patients with all stages of gastric cancer from a high-risk population. DESIGN: We conducted a three-phase, multicentre study comprising 5248 subjects from Singapore and Korea. Biomarker discovery and verification phases were done through comprehensive serum miRNA profiling and multivariant analysis of 578 miRNA candidates in retrospective cohorts of 682 subjects. A clinical assay was developed and validated in a prospective cohort of 4566 symptomatic subjects who underwent endoscopy. Assay performance was confirmed with histological diagnosis and compared with Helicobacter pylori (HP) serology, serum pepsinogens (PGs), 'ABC' method, carcinoembryonic antigen (CEA) and cancer antigen 19-9 (CA19-9). Cost-effectiveness was analysed using a Markov decision model. RESULTS: We developed a clinical assay for detection of gastric cancer based on a 12-miRNA biomarker panel. The 12-miRNA panel had area under the curve (AUC)=0.93 (95% CI 0.90 to 0.95) and AUC=0.92 (95% CI 0.88 to 0.96) in the discovery and verification cohorts, respectively. In the prospective study, overall sensitivity was 87.0% (95% CI 79.4% to 92.5%) at specificity of 68.4% (95% CI 67.0% to 69.8%). AUC was 0.848 (95% CI 0.81 to 0.88), higher than HP serology (0.635), PG 1/2 ratio (0.641), PG index (0.576), ABC method (0.647), CEA (0.576) and CA19-9 (0.595). The number needed to screen is 489 annually. It is cost-effective for mass screening relative to current practice (incremental cost-effectiveness ratio=US$44 531/quality-of-life year). CONCLUSION: We developed and validated a serum 12-miRNA biomarker assay, which may be a cost-effective risk assessment for gastric cancer. TRIAL REGISTRATION NUMBER: This study is registered with ClinicalTrials.gov (Registration number: NCT04329299).

Conditional disruption of AMP kinase in dopaminergic neurons promotes Parkinson's disease-associated phenotypes in vivo.

Emerging studies implicate energy dysregulation as an underlying trigger for Parkinson's disease (PD), suggesting that a better understanding of the molecular pathways governing energy homeostasis could help elucidate therapeutic targets for the disease. A critical cellular energy regulator is AMP kinase (AMPK), which we have previously shown to be protective in PD models. However, precisely how AMPK function impacts on dopaminergic neuronal survival and disease pathogenesis remains elusive. Here, we showed that Drosophila deficient in AMPK function exhibits PD-like features, including dopaminergic neuronal loss and climbing impairment that progress with age. We also created a tissue-specific AMPK-knockout mouse model where the catalytic subunits of AMPK are ablated in nigral dopaminergic neurons. Using this model, we demonstrated that loss of AMPK function promotes dopaminergic neurodegeneration and associated locomotor aberrations. Accompanying this is an apparent reduction in the number of mitochondria in the surviving AMPK-deficient nigral dopaminergic neurons, suggesting that an impairment in mitochondrial biogenesis may underlie the observed PD-associated phenotypes. Importantly, the loss of AMPK function enhances the susceptibility of nigral dopaminergic neurons in these mice to 6-hydroxydopamine-induced toxicity. Notably, we also found that AMPK activation is reduced in post-mortem PD brain samples. Taken together, these findings highlight the importance of neuronal energy homeostasis by AMPK in PD and position AMPK pathway as an attractive target for future therapeutic exploitation.

Faim knockout leads to gliosis and late-onset neurodegeneration of photoreceptors in the mouse retina.

Fas Apoptotic Inhibitory Molecule protein (FAIM) is a death receptor antagonist and an apoptosis regulator. It encodes two isoforms, namely FAIM-S (short) and FAIM-L (long), both with significant neuronal functions. FAIM-S, which is ubiquitously expressed, is involved in neurite outgrowth. In contrast, FAIM-L is expressed only in neurons and it protects them from cell death. Interestingly, FAIM-L is downregulated in patients and mouse models of Alzheimer's disease before the onset of neurodegeneration, and Faim transcript levels are decreased in mouse models of retinal degeneration. However, few studies have addressed the role of FAIM in the central nervous system, yet alone the retina. The retina is a highly specialized tissue, and its degeneration has proved to precede pathological mechanisms of neurodegenerative diseases. Here we describe that Faim depletion in mice damages the retina persistently and leads to late-onset photoreceptor death in older mice. Immunohistochemical analyses showed that Faim knockout (Faim-/- ) mice present ubiquitinated aggregates throughout the retina from early ages. Moreover, retinal cells released stress signals that can signal to Müller cells, as shown by immunofluorescence and qRT-PCR. Müller cells monitor retinal homeostasis and trigger a gliotic response in Faim-/- mice that becomes pathogenic when sustained. In this regard, we observed pronounced vascular leakage at later ages, which may be caused by persistent inflammation. These results suggest that FAIM is an important player in the maintenance of retinal homeostasis, and they support the premise that FAIM is a plausible early marker for late photoreceptor and neuronal degeneration.

The stress granule protein G3BP1 binds viral dsRNA and RIG-I to enhance interferon-ß response.

RIG-I senses viral RNA in the cytosol and initiates host innate immune response by triggering the production of type 1 interferon. A recent RNAi knockdown screen yielded close to hundred host genes whose products affected viral RNA-induced IFN-ß production and highlighted the complexity of the antiviral response. The stress granule protein G3BP1, known to arrest mRNA translation, was identified as a regulator of RIG-I-induced IFN-ß production. How G3BP1 functions in RIG-I signaling is not known, however. Here, we overexpress G3BP1 with RIG-I in HEK293T cells and found that G3BP1 significantly enhances RIG-I-induced ifn-b mRNA synthesis. More importantly, we demonstrate that G3BP1 binds RIG-I and that this interaction involves the C-terminal RGG domain of G3BP1. Confocal microscopy studies also show G3BP1 co-localization with RIG-I and with infecting vesicular stomatitis virus in Cos-7 cells. Interestingly, immunoprecipitation studies using biotin-labeled viral dsRNA or poly(I·C) and cell lysate-derived or in vitro translated G3BP1 indicated that G3BP1 could directly bind these substrates and again via its RGG domain. Computational modeling further revealed a juxtaposed interaction between G3BP1 RGG and RIG-I RNA-binding domains. Together, our data reveal G3BP1 as a critical component of RIG-I signaling and possibly acting as a co-sensor to promote RIG-I recognition of pathogenic RNA.

Quantitative and Label-Free Detection of Protein Kinase A Activity Based on Surface-Enhanced Raman Spectroscopy with Gold Nanostars.

The activity of extracellular protein kinase A (PKA) is known to be a biomarker for cancer. However, conventional PKA assays based on colorimetric, radioactive, and fluorometric techniques suffer from intensive labeling-related preparations, background interference, photobleaching, and safety concerns. While surface-enhanced Raman spectroscopy (SERS)-based assays have been developed for various enzymes to address these limitations, their use in probing PKA activity is limited due to subtle changes in the Raman spectrum with phosphorylation. Here, we developed a robust colloidal SERS-based scheme for label-free quantitative measurement of PKA activity using gold nanostars (AuNS) as a SERS substrate functionalized with bovine serum albumin (BSA)-kemptide (Kem) bioconjugate (AuNS-BSA-Kem), where BSA conferred colloidal stability and Kem is a high-affinity peptide substrate for PKA. By performing principle component analysis (PCA) on the SERS spectrum, we identified two Raman peaks at 725 and 1395 cm-1, whose ratiometric intensity change provided a quantitative measure of Kem phosphorylation by PKA in vitro and allowed us to distinguish MDA-MB-231 and MCF-7 breast cancer cells known to overexpress extracellular PKA catalytic subunits from noncancerous human umbilical vein endothelial cells (HUVEC) based on their PKA activity in cell culture supernatant. The outcome demonstrated potential application of AuNS-BSA-Kem as a SERS probe for cancer screening based on PKA activity.

Excessive interferon-α signaling in autoimmunity alters glycosphingolipid processing in B cells.

Excessive interferon-α (IFN-α) production by innate immune cells is a hallmark of autoimmune diseases. What other cell type secretes IFN-α and how IFN-α affects immune cell metabolism and homeostasis in autoimmunity are largely unclear. Here, we report that autoimmune B cells, arising from two different B cell-specific genetic lesions in mice, secrete IFN-α. In addition, IFN-α, found in abundance in autoimmunity, elicited profound changes in the B cell lipidome, increasing their expression of glycosphingolipids (GSLs) and leading to their CD1d-mediated depletion of iNKT cells in vitro and in vivo. IFN-α receptor blockade could reverse the loss of iNKT cells. Excessive stimulation of B cells with IFN-α altered the expression of enzymes that catalyze critical steps in GSL processing, increasing the expressions of glucosylceramide synthase (GCS) and globotrihexosylceramide synthase (Gb3S) but decreasing that of α-galactosidase A (α-galA). Inhibiting GCS or restoring α-galA expression prevented iNKT depletion by IFN-α-activated B cells. Taken together, our work indicated that excessive IFN-α perturbs GSL metabolism in B cells which in turn adversely affects iNKT homeostasis.

Adaptor protein DOK3 promotes plasma cell differentiation by regulating the expression of programmed cell death 1 ligands.

The adaptor Downstream-of-Kinase (DOK) 3 functions as a negative regulator and attenuates B-cell receptor-mediated calcium signaling. Although DOK3 is dispensable for early B-cell development, its role in plasma cell (PC) differentiation is unknown. Here, we show that Dok3(-/-) mice have increased populations of T follicular-helper (Tfh) and germinal center (GC) B cells upon immunization with a T-cell-dependent antigen. However, interestingly, they generate significantly fewer PCs. Bone marrow reconstitution experiments show that the PC defect is B-cell intrinsic and due to the inability of Dok3(-/-) B cells to sustain programmed cell death 1 (PD-1) ligand 1 (PDL1) and up-regulate PD-1 ligand 2 (PDL2) expressions that are critical for PC differentiation. Overexpression of PDL2 rectifies the PC differentiation defect in Dok3(-/-) B cells. We further demonstrate that calcium signaling suppresses the transcription of PD-1 ligands. Abrogation of calcium signaling in B cells by deleting BTK or PLCγ2 or inhibiting calcineurin with cyclosporine A leads to increased expression of PD-1 ligands. Thus, our study reveals DOK3 as a nonredundant regulator of PC differentiation by up-regulating PD-1 ligand expression through the attenuation of calcium signaling.

Loss of miR-182 affects B-cell extrafollicular antibody response.

MicroRNAs have been shown to play a role in B-cell differentiation and activation. Here, we found miR-182 to be highly induced in activated B cells. However, mice lacking miR-182 have normal B-cell and T-cell development. Interestingly, mutant mice exhibited a defective antibody response at early time-points in the immunization regimen when challenged with a T-cell-dependent antigen. Germinal centres were formed but the generation of extrafollicular plasma cells was defective in the spleens of immunized miR-182-deficient mice. Mutant mice were also not able to respond to a T-cell-independent type 2 antigen, which typically elicited an extrafollicular B-cell response. Taken together, the data indicated that miR-182 plays a critical role in driving extrafollicular B-cell antibody responses.

DOK3 is required for IFN-ß production by enabling TRAF3/TBK1 complex formation and IRF3 activation.

The downstream of kinase (DOK) family of adaptors is generally involved in the negative regulation of signaling pathways. DOK1, 2, and 3 were shown to attenuate TLR4 signaling by inhibiting Ras-ERK activation. In this study, we elucidated a novel role for DOK3 in IFN-ß production. Macrophages lacking DOK3 were impaired in IFN-ß synthesis upon influenza virus infection or polyinosinic-polyribocytidylic acid stimulation. In the absence of DOK3, the transcription factor IFN regulatory factor 3 was not phosphorylated and could not translocate to the nucleus to activate ifn-ß gene expression. Interestingly, polyinosinic-polyribocytidylic acid-induced formation of the upstream TNFR-associated factor (TRAF) 3/TANK-binding kinase (TBK) 1 complex was compromised in dok3(-/-) macrophages. DOK3 was shown to bind TBK1 and was required for its activation. Furthermore, we demonstrated that overexpression of DOK3 and TBK1 could significantly enhance ifn-ß promoter activity. DOK3 was also shown to bind TRAF3, and the binding of TRAF3 and TBK1 to DOK3 required the tyrosine-rich C-terminal domain of DOK3. We further revealed that DOK3 was phosphorylated by Bruton's tyrosine kinase. Hence, DOK3 plays a critical and positive role in TLR3 signaling by enabling TRAF3/TBK1 complex formation and facilitating TBK1 and IFN regulatory factor 3 activation and the induction of IFN-ß production.

Proteomic analysis of the SH2 domain-containing leukocyte protein of 76 kDa (SLP76) interactome in resting and activated primary mast cells [corrected].

We report the first proteomic analysis of the SLP76 interactome in resting and activated primary mouse mast cells. This was made possible by a novel genetic approach used for the first time here. It consists in generating knock-in mice that express signaling molecules bearing a C-terminal tag that has a high affinity for a streptavidin analog. Tagged molecules can be used as molecular baits to affinity-purify the molecular complex in which they are engaged, which can then be studied by mass spectrometry. We examined first SLP76 because, although this cytosolic adapter is critical for both T cell and mast cell activation, its role is well known in T cells but not in mast cells. Tagged SLP76 was expressed in physiological amounts and fully functional in mast cells. We unexpectedly found that SLP76 is exquisitely sensitive to mast cell granular proteases, that Zn(2+)-dependent metalloproteases are especially abundant in mast cells and that they were responsible for SLP76 degradation. Adding a Zn(2+) chelator fully protected SLP76 in mast cell lysates, thereby enabling an efficient affinity-purification of this adapter with its partners. Label-free quantitative mass spectrometry analysis of affinity-purified SLP76 interactomes uncovered both partners already described in T cells and novel partners seen in mast cells only. Noticeably, molecules inducibly recruited in both cell types primarily concur to activation signals, whereas molecules recruited in activated mast cells only are mostly associated with inhibition signals. The transmembrane adapter LAT2, and the serine/threonine kinase with an exchange factor activity Bcr were the most recruited molecules. Biochemical and functional validations established the unexpected finding that Bcr is recruited by SLP76 and positively regulates antigen-induced mast cell activation. Knock-in mice expressing tagged molecules with a normal tissue distribution and expression therefore provide potent novel tools to investigate signalosomes and to uncover novel signaling molecules in mast cells.

Deficiency in TNFRSF13B (TACI) expands T-follicular helper and germinal center B cells via increased ICOS-ligand expression but impairs plasma cell survival.

Mutations in TNFRSF13B, better known as transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), contribute to common variable immunodeficiency and autoimmunity in humans. How TACI regulates these two opposing conditions is unclear, however. TACI binds the cytokines BAFF and APRIL, and previous studies using gene KO mice indicated that loss of TACI affected only T-cell-independent antibody responses. Here we demonstrate that Taci(-/-) mice have expanded populations of T follicular helper (T(fh)) and germinal center (GC) B cells in their spleens when immunized with T-cell-dependent antigen. The increased numbers of T(fh) and GC B cells in Taci(-/-) mice are largely a result of up-regulation of inducible costimulator (ICOS) ligand on TACI-deficient B cells, given that ablation of one copy of the Icosl allele restores normal levels of T(fh) and GC B cells in Taci(-/-) mice. Interestingly, despite the presence of increased T(fh) and antigen-specific B cells, immunized Taci(-/-) mice demonstrate defective antigen-specific antibody responses resulting from significantly reduced numbers of antibody-secreting cells (ASCs). This effect is attributed to the failure to down-regulate the proapoptotic molecule BIM in Taci(-/-) plasma cells. Ablation of BIM could rescue ASC formation in Taci(-/-) mice, suggesting that TACI is more important for the survival of plasma cells than for the differentiation of these cells. Thus, our data reveal dual roles for TACI in B-cell terminal differentiation. On one hand, TACI modulates ICOS ligand expression and thereby limits the size of T(fh) and GC B-cell compartments and prevents autoimmunity. On the other hand, it regulates the survival of ASCs and plays an important role in humoral immunity.

Bruton's tyrosine kinase phosphorylates Toll-like receptor 3 to initiate antiviral response.

Toll-like receptor 3 (TLR3) mediates antiviral response by recognizing double-stranded RNA. Its cytoplasmic domain is tyrosine phosphorylated upon ligand binding and initiates downstream signaling via the adapter TIR-containing adaptor inducing interferon-ß (TRIF). However, the kinase responsible for TLR3 phosphorylation remains unknown. We show here that Bruton's tyrosine kinase (BTK)-deficient macrophages failed to secrete inflammatory cytokines and IFN-ß upon TLR3 stimulation and were impaired in clearing intracellular dengue virus infection. Mutant mice were also less susceptible to d-galactosamine/p(I:C)-induced sepsis. In the absence of BTK, TLR3-induced phosphoinositide 3-kinase (PI3K), AKT and MAPK signaling and activation of NFκB, IRF3, and AP-1 transcription factors were all defective. We demonstrate that BTK directly phosphorylates TLR3 and in particular the critical Tyr759 residue. BTK point mutations that abrogate or led to constitutive kinase activity have opposite effects on TLR3 phosphorylation. Loss of BTK also compromises the formation of the downstream TRIF/receptor-interacting protein 1 (RIP1)/TBK1 complex. Thus, BTK plays a critical role in initiating TLR3 signaling.

The RNase III enzyme Dicer is essential for germinal center B-cell formation.

MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression and are important for pre-B and follicular B lymphopoiesis as demonstrated, respectively, by mb-1-Cre- and cd19-Cre-mediated deletion of Dicer, the RNase III enzyme critical for generating mature miRNAs. To explore the role of miRNAs in B-cell terminal differentiation, we use Aicda-Cre to specifically delete Dicer in activated B cells where activation-induced cytidine deaminase is highly expressed. We demonstrate that mutant mice fail to produce high-affinity class-switched antibodies and generate memory B and long-lived plasma cells on immunization with a T cell-dependent antigen. More importantly, germinal center (GC) B-cell formation is drastically compromised in the absence of Dicer, as a result of defects in cell proliferation and survival. Dicer-deficient GC B cells express higher levels of cell cycle inhibitor genes and proapoptotic protein Bim. Ablation of Bim could partially rescue the defect in GC B-cell formation in Dicer-deficient mice. Taken together, our data suggest that Dicer and probably miRNAs are critical for GC B-cell formation during B-cell terminal differentiation.

Comprehensive analysis of CD4+ T cells in the decision between tolerance and immunity in vivo reveals a pivotal role for ICOS.

We have established a comprehensive in vivo mouse model for the CD4(+) T cell response to an "innocuous" versus "dangerous" exogenous Ag and developed an in vivo test for tolerance. In this model, specific gene-expression signatures, distinctive upregulation of early T cell-communication molecules, and differential expansion of effector T cells (Teff) and regulatory T cells (Treg) were identified as central correlates of T cell tolerance and T cell immunity. Different from essentially all other T cell-activation molecules, ICOS was found to be induced in the immunity response and not by T cells activated under tolerogenic conditions. If expressed, ICOS did not act as a general T cell costimulator but selectively caused a massive expansion of effector CD4(+) T cells, leaving the regulatory CD4(+) T cell compartment largely undisturbed. Thus, ICOS strongly contributed to the dramatic change in the balance between Ag-specific Teff and Treg from ∼1:1 at steady state to 21:1 at the height of the immune response. This newly defined role for the balance of Teff to Treg, together with its known key function in T cell help for B cells, establishes ICOS as a central mediator of immunity. Given its exceptionally selective induction on CD4(+) T cells under inflammatory, but not tolerogenic, conditions, ICOS emerges as a pivotal effector molecule in the early decision between tolerance and immunity to exogenous Ag.

Natural killer cells drive 4-1BBL positive uveal melanoma towards EMT and metastatic disease.

BACKGROUND: Inflammation in the eye is often associated with aggravated ocular diseases such as uveal melanoma (UM). Poor prognosis of UM is generally associated with high potential of metastatic liver dissemination. A strong driver of metastatic dissemination is the activation of the epithelial-mesenchymal transition (EMT) regulating transcription factor ZEB1, and high expression of ZEB1 is associated with aggressiveness of UM. While ZEB1 expression can be also associated with immune tolerance, the underlying drivers of ZEB1 activation remain unclear. METHODS: Transcriptomic, in vitro, ex vivo, and in vivo analyses were used to investigate the impact on clinical prognosis of immune infiltration in the ocular tumor microenvironment. A metastatic liver dissemination model of was developed to address the role of natural killer (NK) cells in driving the migration of UM. RESULTS: In a pan-cancer TCGA analysis, natural killer (NK) cells were associated with worse overall survival in uveal melanoma and more abundant in high-risk monosomy 3 tumors. Furthermore, uveal melanoma expressed high levels of the tumor necrosis factor superfamily member 4-1BB ligand, particularly in tumors with monosomy 3 and BAP1 mutations. Tumors expressing 4-1BB ligand induced CD73 expression on NK cells accompanied with the ability to promote tumor dissemination. Through ligation of 4-1BB, NK cells induced the expression of the ZEB1 transcription factor, leading to the formation of liver metastasis of uveal melanoma. CONCLUSIONS: Taken together, the present study demonstrates a role of NK cells in the aggravation of uveal melanoma towards metastatic disease.