Naturally-aged microglia exhibit phagocytic dysfunction accompanied by gene expression changes reflective of underlying neurologic disease.

Age-associated microglial dysfunction contributes to the accumulation of amyloid-ß (Aß) plaques in Alzheimer's disease. Although several studies have shown age-related declines in the phagocytic capacity of myeloid cells, relatively few have examined phagocytosis of normally aged microglia. Furthermore, much of the existing data on aging microglial function have been generated in accelerated genetic models of Alzheimer's disease. Here we found that naturally aged microglia phagocytosed less Aß over time. To gain a better understanding of such dysfunction, we assessed differences in gene expression between young and old microglia that either did or did not phagocytose Aß. Young microglia had both phagocytic and neuronal maintenance signatures indicative of normal microglial responses, whereas, old microglia, regardless of phagocytic status, exhibit signs of broad dysfunction reflective of underlying neurologic disease states. We also found downregulation of many phagocytic receptors on old microglia, including TREM2, an Aß phagocytic receptor. TREM2 protein expression was diminished in old microglia and loss of TREM2+ microglia was correlated with impaired Aß uptake, suggesting a mechanism for phagocytic dysfunction in old microglia. Combined, our work reveals that normally aged microglia have broad changes in gene expression, including defects in Aß phagocytosis that likely underlies the progression to neurologic disease.

Immune Checkpoint Inhibitors Regulate K+ Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients.

Programmed death receptor-1 (PD-1) and its ligand (PD-L1) interaction negatively regulates T cell function in head and neck squamous cell carcinoma (HNSCC). Overexpression of PD-1 reduces intracellular Ca2+ fluxes, and thereby T cell effector functions. In HNSCC patients, PD-1 blockade increases KCa3.1 and Kv1.3 activity along with Ca2+ signaling and mobility in CD8+ peripheral blood T cells (PBTs). The mechanism by which PD-L1/PD-1 interaction regulates ion channel function is not known. We investigated the effects of blocking PD-1 and PD-L1 on ion channel functions and intracellular Ca2+ signaling in CD8+ PBTs of HNSCC patients and healthy donors (HDs) using single-cell electrophysiology and live microscopy. Anti-PD-1 and anti-PD-L1 antibodies increase KCa3.1 and Kv1.3 function in CD8+ PBTs of HNSCC patients. Anti-PD-1 treatment increases Ca2+ fluxes in a subset of HSNCC patients. In CD8+ PBTs of HDs, exposure to PD-L1 reduces KCa3.1 activity and Ca2+ signaling, which were restored by anti-PD-1 treatment. The PD-L1-induced inhibition of KCa3.1 channels was rescued by the intracellular application of the PI3 kinase modulator phosphatidylinositol 3-phosphate (PI3P) in patch-clamp experiments. In HNSCC CD8+ PBTs, anti-PD-1 treatment did not affect the expression of KCa3.1, Kv1.3, Ca2+ release activated Ca2+ (CRAC) channels, and markers of cell activation (CD69) and exhaustion (LAG-3 and TIM-3). Our data show that immune checkpoint blockade improves T cell function by increasing KCa3.1 and Kv1.3 channel activity in HNSCC patients.

Promotion of Anti-Tuberculosis Macrophage Activity by L-Arginine in the Absence of Nitric Oxide.

Macrophages are indispensable immune cells tasked at eliminating intracellular pathogens. Mycobacterium tuberculosis (Mtb), one of the most virulent intracellular bacterial pathogens known to man, infects and resides within macrophages. While macrophages can be provoked by extracellular stimuli to inhibit and kill Mtb bacilli, these host defense mechanisms can be blocked by limiting nutritional metabolites, such as amino acids. The amino acid L-arginine has been well described to enhance immune function, especially in the context of driving macrophage nitric oxide (NO) production in mice. In this study, we aimed to establish the necessity of L-arginine on anti-Mtb macrophage function independent of NO. Utilizing an in vitro system, we identified that macrophages relied on NO for only half of their L-arginine-mediated host defenses and this L-arginine-mediated defense in the absence of NO was associated with enhanced macrophage numbers and viability. Additionally, we observed macrophage glycolysis to be driven by both L-arginine and mechanistic target of rapamycin (mTOR), and inhibition of glycolysis or mTOR reduced macrophage control of Mtb as well as macrophage number and viability in the presence of L-arginine. Our data underscore L-arginine as an essential nutrient for macrophage function, not only by fueling anti-mycobacterial NO production, but also as a central regulator of macrophage metabolism and additional host defense mechanisms.

m6A methylation potentiates cytosolic dsDNA recognition in a sequence-specific manner.

Nucleic acid sensing through pattern recognition receptors is critical for immune recognition of microbial infections. Microbial DNA is frequently methylated at the N6 position of adenines (m6A), a modification that is rare in mammalian host DNA. We show here how that m6A methylation of 5'-GATC-3' motifs augments the immunogenicity of synthetic double-stranded (ds)DNA in murine macrophages and dendritic cells. Transfection with m6A-methylated DNA increased the expression of the activation markers CD69 and CD86, and of Ifnß, iNos and Cxcl10 mRNA. Similar to unmethylated cytosolic dsDNA, recognition of m6A DNA occurs independently of TLR and RIG-I signalling, but requires the two key mediators of cytosolic DNA sensing, STING and cGAS. Intriguingly, the response to m6A DNA is sequence-specific. m6A is immunostimulatory in some motifs, but immunosuppressive in others, a feature that is conserved between mouse and human macrophages. In conclusion, epigenetic alterations of DNA depend on the context of the sequence and are differentially perceived by innate cells, a feature that could potentially be used for the design of immune-modulating therapeutics.

PD1 blockade enhances K+ channel activity, Ca2+ signaling, and migratory ability in cytotoxic T lymphocytes of patients with head and neck cancer.

BACKGROUND: Immunotherapy has emerged as a promising treatment modality for head and neck squamous cell carcinoma (HNSCC). Pembrolizumab, an anti-programmed death 1 antibody, is an immunotherapy agent currently approved for metastatic HNSCC and curative intent clinical trials. Although clinical responses to pembrolizumab are promising, many patients fail to respond. However, it is well known that T cell cytotoxicity and chemotaxis are critically important in the elimination of HNSCC tumors. These functions depend on ion channel activity and downstream Ca2+ fluxing abilities, which are defective in patients with HNSCC. The purpose of this study was to elucidate the effects of pembrolizumab on potassium (K+) channel (KCa3.1 and Kv1.3) activity, Ca2+ fluxes, and chemotaxis in the cytotoxic T cells of patients with HNSCC and to determine their correlation with treatment response. METHODS: Functional studies were conducted in CD8+ peripheral blood T cells (PBTs) and tumor infiltrating lymphocytes (TILs) from patients with HNSCC treated with pembrolizumab. Untreated patients with HNSCC were used as controls. The ion channel activity of CD8+ T cells was measured by patch-clamp electrophysiology; single-cell Ca2+ fluxing abilities were measured by live microscopy. Chemotaxis experiments were conducted in a three-dimensional collagen matrix. Pembrolizumab patients were stratified as responders or non-responders based on pathological response (percent of viable tumor remaining at resection; responders: ≤80% viable tumor; non-responders: >80% viable tumor). RESULTS: Pembrolizumab increased K+ channel activity and Ca2+ fluxes in TILs independently of treatment response. However, in PBTs from responder patients there was an increased KCa3.1 activity immediately after pembrolizumab treatment that was accompanied by a characteristic increase in Kv1.3 and Ca2+ fluxes as compared with PBTs from non-responder patients. The effects on Kv1.3 and Ca2+ were prolonged and persisted after tumor resection. Chemotaxis was also improved in responder patients' PBTs. Unlike non-responders' PBTs, pembrolizumab increased their ability to chemotax in a tumor-like, adenosine-rich microenvironment immediately after treatment, and additionally they maintained an efficient chemotaxis after tumor resection. CONCLUSIONS: Pembrolizumab enhanced K+ channel activity, Ca2+ fluxes and chemotaxis of CD8+ T cells in patients with HNSCC, with a unique pattern of response in responder patients that is conducive to the heightened functionality of their cytotoxic T cells.

Author Correction: Ablation of CD8α+ dendritic cell mediated cross-presentation does not impact atherosclerosis in hyperlipidemic mice.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

CD244 represents a new therapeutic target in head and neck squamous cell carcinoma.

BACKGROUND: Developing novel strategies to overcome the immunosuppressive tumor microenvironment is a critically important area of cancer therapy research. Here, we assess the therapeutic potential of CD244 (2B4/signaling lymphocyte activation molecule family 4), an immunoregulatory receptor found on a variety of immune cells, including exhausted CD8+ T cells, dendritic cells (DCs), and myeloid-derived suppressor cells (MDSCs). METHODS: Using de-identified human tumor and blood samples from patients with head and neck squamous cell carcinoma (HNSCC) and HNSCC models in WT and CD244-/- mice, we assessed the therapeutic potential of CD244 using flow cytometry, RT-PCR, Luminex immunoassays and histopathological analyses. RESULTS: Compared with healthy tissues, tumor infiltrating CD8+ T cells from HNSCC patients and a HNSCC mouse model showed significant increased expression of CD244 expression that correlated with PD1 expression. Moreover, CD244 was increased on intratumoral DC and MDSC and high CD244 expression correlated with PD-L1 expression and increased spontaneous expression of immune-suppressive mediators. In addition, CD244 activation inhibited production of proinflammatory cytokines in human DC in vitro. Importantly, CD244-/- mice showed significantly impaired tumor growth of HNSCC and interventional treatment of WT mice with anti-CD244 monoclonal antibody significantly impaired the growth of established HNSCC tumors and increased tumor-infiltrating CD8+ T cells. CONCLUSIONS: Together these data suggest that CD244 contributes to the overall immune-suppressive environment and therefore has potential as a new immunotherapy target in the treatment of malignancies.

Type I IFN Drives Experimental Systemic Lupus Erythematosus by Distinct Mechanisms in CD4 T Cells and B Cells.

Myriad studies have linked type I IFN to the pathogenesis of autoimmune diseases, including systemic lupus erythematosus (SLE). Although increased levels of type I IFN are found in patients with SLE, and IFN blockade ameliorates disease in many mouse models of lupus, its precise roles in driving SLE pathogenesis remain largely unknown. In this study, we dissected the effect of type I IFN sensing by CD4 T cells and B cells on the development of T follicular helper cells (TFH), germinal center (GC) B cells, plasmablasts, and antinuclear dsDNA IgG levels using the bm12 chronic graft-versus-host disease model of SLE-like disease. Type I IFN sensing by B cells decreased their threshold for BCR signaling and increased their expression of MHC class II, CD40, and Bcl-6, requirements for optimal GC B cell functions. In line with these data, ablation of type I IFN sensing in B cells significantly reduced the accumulation of GC B cells, plasmablasts, and autoantibodies. Ablation of type I IFN sensing in T cells significantly inhibited TFH expansion and subsequent B cell responses. In contrast to the effect in B cells, type I IFN did not promote proliferation in the T cells but protected them from NK cell-mediated killing. Consequently, ablation of either perforin or NK cells completely restored TFH expansion of IFNAR-/- TFH and, subsequently, restored the B cell responses. Together, our data provide evidence for novel roles of type I IFN and immunoregulatory NK cells in the context of sterile inflammation and SLE-like disease.

Gab3 is required for IL-2- and IL-15-induced NK cell expansion and limits trophoblast invasion during pregnancy.

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.

Thrombin Signaling Promotes Pancreatic Adenocarcinoma through PAR-1-Dependent Immune Evasion.

Pancreatic ductal adenocarcinoma (PDAC) is associated with robust activity of the coagulation system. To determine mechanisms by which clotting factors influence PDAC tumor progression, we generated and characterized C57Bl/6-derived KPC (KRasG12D, TRP53R172H ) cell lines. Tissue factor (TF) and protease-activated receptor-1 (PAR-1) were highly expressed in primary KPC pancreatic lesions and KPC cell lines similar to expression profiles observed in biopsies of patients with PDAC. In allograft studies, tumor growth and metastatic potential were significantly diminished by depletion of TF or Par-1 in cancer cells or by genetic or pharmacologic reduction of the coagulation zymogen prothrombin in mice. Notably, PAR-1-deleted KPC cells (KPC-Par-1KO) failed to generate sizable tumors, a phenotype completely rescued by restoration of Par-1 expression. Expression profiling of KPC and KPC-Par-1KO cells indicated that thrombin-PAR-1 signaling significantly altered immune regulation pathways. Accordingly, KPC-Par-1KO cells failed to form tumors in immune-competent mice but displayed robust tumor growth comparable to that observed with control KPC cells in immune-compromised NSG mice. Immune cell depletion studies indicated that CD8 T cells, but not CD4 cells or natural killer cells, mediated elimination of KPC-Par-1KO tumor cells in C57Bl/6 mice. These results demonstrate that PDAC is driven by activation of the coagulation system through tumor cell-derived TF, circulating prothrombin, and tumor cell-derived PAR-1 and further indicate that one key mechanism of thrombin/PAR-1-mediated tumor growth is suppression of antitumor immunity in the tumor microenvironment. SIGNIFICANCE: The tissue factor-thrombin-PAR-1 signaling axis in tumor cells promotes PDAC growth and disease progression with one key mechanism being suppression of antitumor immunity in the microenvironment.

Loss of GTPase of immunity-associated protein 5 (Gimap5) promotes pathogenic CD4+ T-cell development and allergic airway disease.

BACKGROUND: GTPase of immunity-associated protein 5 (GIMAP5) is essential for lymphocyte homeostasis and survival. Recently, human GIMAP5 single nucleotide polymorphisms have been linked to an increased risk for asthma, whereas loss of Gimap5 in mice has been associated with severe CD4+ T cell-driven immune pathology. OBJECTIVE: We sought to identify the molecular and cellular mechanisms by which Gimap5 deficiency predisposes to allergic airway disease. METHODS: CD4+ T-cell polarization and development of pathogenic CD4+ T cells were assessed in Gimap5-deficient mice and a human patient with a GIMAP5 loss-of-function (LOF) mutation. House dust mite-induced airway inflammation was assessed by using a complete Gimap5 LOF (Gimap5sph/sph) and conditional Gimap5fl/flCd4Cre/ert2 mice. RESULTS: GIMAP5 LOF mutations in both mice and human subjects are associated with spontaneous polarization toward pathogenic TH17 and TH2 cells in vivo. Mechanistic studies in vitro reveal that impairment of Gimap5-deficient TH cell differentiation is associated with increased DNA damage, particularly during TH1-polarizing conditions. DNA damage in Gimap5-deficient CD4+ T cells could be controlled by TGF-ß, thereby promoting TH17 polarization. When challenged with house dust mite in vivo, Gimap5-deficient mice displayed an exacerbated asthma phenotype (inflammation and airway hyperresponsiveness), with increased development of TH2, TH17, and pathogenic TH17/TH2 cells. CONCLUSION: Activation of Gimap5-deficient CD4+ T cells is associated with increased DNA damage and reduced survival that can be overcome by TGF-ß. This leads to selective survival of pathogenic TH17 cells but also TH2 cells in human subjects and mice, ultimately promoting allergic airway disease.

STING, DCs and the link between innate and adaptive tumor immunity.

Cancer and the immune system are intimately related. Much of the bulk of tumors is comprised of stromal leukocytes with immune functions, which serve to both promote and inhibit tumor growth, invasion and metastasis. The T lymphocytes of the adaptive immune system are essential for tumor immunity, and these T cells are generated by cross-priming against tumor associated antigens. Dendritic cells (DCs) are essential in this process, serving as the cellular link between innate and adaptive immunity. As a prerequisite for priming of adaptive immune responses, DCs must take up tumor antigens, process them and present them in the context of the major histocompatibility complex (MHC). DCs also serve as sensors of innate activation signals from cancer that are necessary for their activation and effective priming of cancer specific T cells. Here we discuss the role of DCs in the sensing of cancer and in priming the adaptive response against tumors. Furthermore, we present the essential role of the Stimulator of Interferon Genes (STING) signaling pathway in producing type I interferons (IFNs) that are essential in this process.

The Emerging Role of CD244 Signaling in Immune Cells of the Tumor Microenvironment.

In cancer, immune exhaustion contributes to the immunosuppressive tumor microenvironment. Exhausted immune cells demonstrate poor effector function and sustained expression of certain immunomodulatory receptors, which can be therapeutically targeted. CD244 is a Signaling Lymphocyte Activation Molecule (SLAM) family immunoregulatory receptor found on many immune cell types-including NK cells, a subset of T cells, DCs, and MDSCs-that represents a potential therapeutic target. Here, we discuss the role of CD244 in tumor-mediated immune cell regulation.

Defective transcription elongation in a subset of cancers confers immunotherapy resistance.

The nature and role of global transcriptional deregulations in cancers are not fully understood. We report that a large proportion of cancers have widespread defects in mRNA transcription elongation (TE). Cancers with TE defects (TEdeff) display spurious transcription and defective mRNA processing of genes characterized by long genomic length, poised promoters and inducible expression. Signaling pathways regulated by such genes, such as pro-inflammatory response pathways, are consistently suppressed in TEdeff tumors. Remarkably, TEdeff correlates with the poor response and outcome in immunotherapy, but not chemo- or targeted therapy, -treated renal cell carcinoma and metastatic melanoma patients. Forced pharmacologic or genetic induction of TEdeff in tumor cells impairs pro-inflammatory response signaling, and imposes resistance to the innate and adaptive anti-tumor immune responses and checkpoint inhibitor therapy in vivo. Therefore, defective TE is a previously unknown mechanism of tumor immune resistance, and should be assessed in cancer patients undergoing immunotherapy.

Type I interferons regulate susceptibility to inflammation-induced preterm birth.

Preterm birth (PTB) is a leading worldwide cause of morbidity and mortality in infants. Maternal inflammation induced by microbial infection is a critical predisposing factor for PTB. However, biological processes associated with competency of pathogens, including viruses, to induce PTB or sensitize for secondary bacterial infection-driven PTB are unknown. We show that pathogen/pathogen-associated molecular pattern-driven activation of type I IFN/IFN receptor (IFNAR) was sufficient to prime for systemic and uterine proinflammatory chemokine and cytokine production and induction of PTB. Similarly, treatment with recombinant type I IFNs recapitulated such effects by exacerbating proinflammatory cytokine production and reducing the dose of secondary inflammatory challenge required for induction of PTB. Inflammatory challenge-driven induction of PTB was eliminated by defects in type I IFN, TLR, or IL-6 responsiveness, whereas the sequence of type I IFN sensing by IFNAR on hematopoietic cells was essential for regulation of proinflammatory cytokine production. Importantly, we also show that type I IFN priming effects are conserved from mice to nonhuman primates and humans, and expression of both type I IFNs and proinflammatory cytokines is upregulated in human PTB. Thus, activation of the type I IFN/IFNAR axis in pregnancy primes for inflammation-driven PTB and provides an actionable biomarker and therapeutic target for mitigating PTB risk.

Dendritic Cells in Systemic Lupus Erythematosus: From Pathogenic Players to Therapeutic Tools.

System lupus erythematosus (SLE) is a multifactorial systemic autoimmune disease with a wide variety of presenting features. SLE is believed to result from dysregulated immune responses, loss of tolerance of CD4 T cells and B cells to ubiquitous self-antigens, and the subsequent production of anti-nuclear and other autoreactive antibodies. Recent research has associated lupus development with changes in the dendritic cell (DC) compartment, including altered DC subset frequency and localization, overactivation of mDCs and pDCs, and functional defects in DCs. Here we discuss the current knowledge on the role of DC dysfunction in SLE pathogenesis, with the focus on DCs as targets for interventional therapies.

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

Ablation of CD8α(+) dendritic cell mediated cross-presentation does not impact atherosclerosis in hyperlipidemic mice.

Clinical complications of atherosclerosis are almost exclusively linked to destabilization of the atherosclerotic plaque. Batf3-dependent dendritic cells specialize in cross-presentation of necrotic tissue-derived epitopes to directly activate cytolytic CD8 Tcells. The mature plaque (necrotic, containing dendritic cells and CD8 Tcells) could offer the ideal environment for cross-presentation, resulting in cytotoxic immunity and plaque destabilization. Ldlr(-/-) mice were transplanted with batf3(-/-) or wt bone marrow and put on a western type diet. Hematopoietic batf3 deficiency sharply decreased CD8α(+) DC numbers in spleen and lymph nodes (>80%; P < 0,001). Concordantly, batf3(-/-) chimeras had a 75% reduction in OT-I cross-priming capacity in vivo. Batf3(-/-) chimeric mice did not show lower Tcell or other leukocyte subset numbers. Despite dampened cross-presentation capacity, batf3(-/-) chimeras had equal atherosclerosis burden in aortic arch and root. Likewise, batf3(-/-) chimeras and wt mice revealed no differences in parameters of plaque stability: plaque Tcell infiltration, cell death, collagen composition, and macrophage and vascular smooth muscle cell content were unchanged. These results show that CD8α(+) DC loss in hyperlipidemic mice profoundly reduces cross-priming ability, nevertheless it does not influence lesion development. Taken together, we clearly demonstrate that CD8α(+) DC-mediated cross-presentation does not significantly contribute to atherosclerotic plaque formation and stability.

Loss of Phagocytic and Antigen Cross-Presenting Capacity in Aging Dendritic Cells Is Associated with Mitochondrial Dysfunction.

Impaired functionality of dendritic cells (DCs) significantly contributes to decreased adaptive immune responses in aged hosts. The expression of MHC-peptide on the DC surface is the critical first step in T cell priming, but few studies have addressed the effect of aging on Ag acquisition, processing, and presentation by DCs. In this study, we show that aged murine DCs were less efficient in the cross-presentation of cell-associated Ag and subsequently in the cross-priming of CD8(+) T cells than were their young counterparts. The decreased cross-presentation was associated with a reduction in the frequency of CD8α DCs and merocytic (CD8α(-)CD11b(-))DCs that could endocytose cell-associated Ag, as well as the number and the size of the endocytosed particles in the DC that did internalize cell-associated materials. Mechanistically, phagocytic capacity has been associated with mitochondrial activity and membrane potential (Δψm). Aged DCs exhibited profound signs of mitochondrial dysfunction, illustrated by lower Δψm, reduced ATP turnover and coupling efficiency, decreased baseline oxidative phosphorylation, and greater proton leak and reactive oxygen species (ROS) production. Mimicking the aged metabolic phenotype in young DCs by pharmacologic manipulation indicated that the reductions in Δψm and ATP impeded the phagocytic capacity whereas ROS interfered with a later step in the cross-presentation process. Conversely, in vitro scavenging of ROS partially restored cross-presentation by aged DCs. Taken together, these data suggest that improvement of aged DC functionality might be feasible in the elderly by targeting metabolic dysfunction or its downstream sequelae, thereby opening new avenues for enhancing vaccine efficiency in this population.