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Histopathology analysis of Hematoxylin-Eosin stained slides from HNC tumor and normal tissue

Quantitation of Kv1.3 protein expression in CD8+ TIL

Methods for Supplementary Figure S6

Competent antitumor immune cells are fundamental for tumor surveillance and combating active cancers. Once established, tumors generate a tumor microenvironment (TME) consisting of complex cellular and metabolic elements that serve to suppress the function of antitumor immune cells. T lymphocytes are key cellular elements of the TME. In this review, we explore the role of ion channels, particularly K+ channels, in mediating the suppressive effects of the TME on T cells. First, we will review the complex network of ion channels that mediate Ca2+ influx and control effector functions in T cells. Then, we will discuss how multiple features of the TME influence the antitumor capabilities of T cells via ion channels. We will focus on hypoxia, adenosine, and ionic imbalances in the TME, as well as overexpression of programmed cell death ligand 1 by cancer cells that either suppress K+ channels in T cells and/or benefit from regulating these channels’ activity, ultimately shaping the immune response. Finally, we will review some of the cancer treatment implications related to ion channels. A better understanding of the effects of the TME on ion channels in T lymphocytes could promote the development of more effective immunotherapies, especially for resistant solid malignancies.

In solid malignancies, including head and neck squamous cell carcinoma (HNSCC), the immunosuppressive molecule adenosine, which accumulates in the tumor, suppresses cytotoxic CD8+ T cell functions including chemotaxis and tumor infiltration. Adenosine functions through binding to the adenosine A2A receptor (A2AR) present on T cells. In order to increase T cell migration into the tumor, the negative effect of adenosine must be abrogated. Systemic drug treatments targeting A2AR are available; however, they could lead to negative toxicities due to the broad expression of this receptor. Herein, we developed a lipid nanoparticle (NP)-based targeted delivery approach to knock down A2AR in T cells in order to increase their chemotaxis in the presence of adenosine. By using flow cytometry, immunofluorescence, qRT-PCR, and 3D-chemotaxis, we demonstrated that CD45RO-labeled nanoparticles delivering ADORA2A gene-silencing-RNAs decreased ADORA2A mRNA expression and rescued the chemotaxis of HNSCC CD8+ memory T cells. Overall, the data indicate that targeting the adenosine signaling pathway with lipid NPs is successful at suppressing the inhibitory effect of adenosine on the chemotaxis of HNSCC memory T cells, which could ultimately help increase T cell infiltration into the tumor.

More than half of children who have autism spectrum disorder (ASD) suffer from motor impairment. In a retrospective study, the authors investigated the effect of a body-mediated workshop with dance movement therapy (DMT) on the motor skills and social skills of children with ASD by comparing 10 autistic children aged 7 to 10 years who benefited from DMT with 10 autistic children in a control group. Scores on the Movement Assessment Battery for Children and the Vineland Adaptive Behavior Scale were compared. The body-mediated workshop had significant benefits for motricity, especially manual dexterity, and for relational skills. A body-mediated workshop may have a multimodal effect and requires transmodal training. Regarding the mechanisms that explain the benefits and the cascading effect, the roles of imitation and multimodal connections are important.

Severe COVID-19 is characterized by cytokine storm, an excessive production of pro-inflammatory cytokines, which results in acute lung damage and death. Dexamethasone (Dex), a corticosteroid administered as a standard of care for severe COVID-19, has been shown to reduce the onset of severe complications in these patients. However, the mechanisms underlying the beneficial effects of Dex are poorly understood. We conducted a transcriptomic analysis (NanoString) of peripheral blood mononuclear cells (PBMCs) from COVID-19 patients with mild (no hospitalization) and severe (on ventilator in the ICU) disease. The latter group included patients ± Dex. Treatment of severe COVID-19 patients with Dex inhibited pro-inflammatory pathways, circulating cytotoxic and Th1 cells, interferon (IFN) signaling and the genes involved in cytokine storm (IL-1, IL-6 and IFN-γ), which were altered in severe vs mild disease. Pro-inflammatory immune cell functions are regulated by calcium signaling, which in turn is under the control of Kv1.3 potassium channels, but their role in the complications associated with COVID-19 remains elusive. RT-qPCR experiments in PBMCs from COVID-19 patient cohorts showed an increase in Kv1.3 expression in severe patients that was significantly reduced by Dex treatment. In agreement with these findings, in vitro treatment with Dex reduced Kv1.3 protein abundance in CD3+, CD4+, CD8+ T cell and CD56dim NK cells in healthy donor (HD) PBMC, while NanoString showed inhibition of pro-inflammatory genes and cytokines. Functional studies showed that Dex significantly reduced Kv1.3 activity and IFN-γ in HD T cells. Thus, our findings suggest that Kv1.3 inhibition contributes to the immunosuppression by Dex. This work was supported by Senior Pilot Grant, University of Cincinnati, Department of Internal Medicine to LC, NIH (R38HL155775 to MC) and Biospecimens and associated data were provided by the Cincinnati COVID-19 Repository at the University of Cincinnati and Cincinnati Children’s Hospital Medical Center.

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.

BackgroundImmunotherapy 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.MethodsFunctional 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).ResultsPembrolizumab 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.ConclusionsPembrolizumab 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.

BackgroundDeveloping 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).MethodsUsing 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.ResultsCompared 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.ConclusionsTogether 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.

The limited ability of cytotoxic CD8+ T cells to infiltrate solid tumors and function within the tumor microenvironment presents a major roadblock to effective immunotherapy. Ion channels and Ca2+-dependent signaling events control the activity of T cells and are implicated in the failure of immune surveillance in cancer. Reduced KCa3.1 channel activity mediates the heightened inhibitory effect of adenosine on the chemotaxis of circulating T cells from head and neck squamous cell carcinoma (HNSCC) patients. Herein, we conducted experiments that elucidate the mechanisms of KCa3.1 dysfunction and impaired chemotaxis in HNSCC CD8+ T cells. The Ca2+ sensor calmodulin (CaM) controls multiple cellular functions including KCa3.1 activation. Our data showed that CaM expression is lower in HNSCC than healthy donor (HD) T cells. This reduction was due to an intrinsic decrease in the genes encoding CaM combined to the failure of HNSCC T cells to upregulate CaM upon activation. Furthermore, the reduction in CaM was confined to the plasma membrane and resulted in decreased CaM-KCa3.1 association and KCa3.1 activity (which was rescued by the delivery of CaM). IFNγ production, also Ca2+- and CaM-dependent, was instead not reduced in HNSCC T cells, which maintained intact cytoplasmic CaM and Ca2+ fluxing ability. Knockdown of CaM in HD T cells decreased KCa3.1 activity, but not IFNγ production, and reduced their chemotaxis in the presence of adenosine, thus recapitulating HNSCC T cell dysfunction. Activation of KCa3.1 with 1-EBIO restored the ability of CaM knockdown HD T cells to chemotax in the presence of adenosine. Additionally, 1-EBIO enhanced INFγ production. Our data showed a localized downregulation of membrane-proximal CaM that suppressed KCa3.1 activity in HNSCC circulating T cells and limited their ability to infiltrate adenosine-rich tumor-like microenvironments. Furthermore, they indicate that KCa3.1 activators could be used as positive CD8+ T cell modulators in cancers.

The limited ability of cytotoxic CD8+ T cells to infiltrate solid tumors and function within the tumor microenvironment presents a major roadblock to effective immunotherapy. Ion channels and Ca2+-dependent signaling events control the activity of T cells and are implicated in the failure of immune surveillance in cancer. Reduced KCa3.1 channel activity mediates the heightened inhibitory effect of adenosine on the chemotaxis of circulating T cells from head and neck squamous cell carcinoma (HNSCC) patients. Herein, we conducted experiments that elucidate the mechanisms of KCa3.1 dysfunction and impaired chemotaxis in HNSCC CD8+ T cells. The Ca2+ sensor calmodulin (CaM) controls multiple cellular functions including KCa3.1 activation. Our data showed that CaM expression is lower in HNSCC than healthy donor (HD) T cells. This reduction was due to an intrinsic decrease in the genes encoding CaM combined to the failure of HNSCC T cells to upregulate CaM upon activation. Furthermore, the reduction in CaM was confined to the plasma membrane and resulted in decreased CaM-KCa3.1 association and KCa3.1 activity (which was rescued by the delivery of CaM). IFNγ production, also Ca2+- and CaM-dependent, was instead not reduced in HNSCC T cells, which maintained intact cytoplasmic CaM and Ca2+ fluxing ability. Knockdown of CaM in HD T cells decreased KCa3.1 activity, but not IFNγ production, and reduced their chemotaxis in the presence of adenosine, thus recapitulating HNSCC T cell dysfunction. Activation of KCa3.1 with 1-EBIO restored the ability of CaM knockdown HD T cells to chemotax in the presence of adenosine. Additionally, 1-EBIO enhanced INFγ production. Our data showed a localized downregulation of membrane-proximal CaM that suppressed KCa3.1 activity in HNSCC circulating T cells and limited their ability to infiltrate adenosine-rich tumor-like microenvironments. Furthermore, they indicate that KCa3.1 activators could be used as positive CD8+ T cell modulators in cancers.

Head and neck cancer (HNC) is the sixth most common cancer worldwide with a five-year survival of 50%. Immunotherapy is emerging as a promising treatment modality in HNC. Pembrolizumab, an anti-PD-1 antibody, is an immunotherapy currently approved in metastatic HNC and in clinical trials for curative intent. Although there are promising responses to Pembrolizumab, its effect on cytotoxic CD8+ T cell function is not understood. T cell cytotoxicity is critically important in the elimination of tumors and in the tumor microenvironment, cytotoxicity depends on ion channel activity which is defective in HNC patients. Therefore, to elucidate mechanisms of Pembrolizumab, we studied potassium channel (KCa3.1 and Kv1.3) functionality and Ca2+ fluxes in CD8+ peripheral blood T cells (PBTs) of naïve HNC patients before/after Pembrolizumab treatment (n=22) as well as tumor infiltrating lymphocytes (TILs) functionality. Untreated HNC patients were controls (n= 13). Pembrolizumab-treated patients were categorized as responders (R) or non-responders (NR) based on pathological response. We then performed electrophysiological and Ca2+ flux experiments. We observed that Pembrolizumab increased KCa3.1 currents in PBTs (29.8%, p=0.026). Moreover, after treatment, R PBTs also had increases in Kv1.3 currents and Ca2+ fluxes (22.1%, p=0.047; 16.5%, p

Highlights • First investigation of the role of NMNAT1 in the early stage of tauopathies in mice. • htau mice display a robust deficit in food burrowing prior to onset of cognitive symptoms. • NMNAT1 overexpression rescues this behavioral abnormality., NAD metabolism and the NAD biosynthetic enzymes nicotinamide nucleotide adenylyltransferases (NMNATs) are thought to play a key neuroprotective role in tauopathies, including Alzheimer’s disease. Here, we investigated whether modulating the expression of the NMNAT nuclear isoform NMNAT1, which is important for neuronal maintenance, influences the development of behavioral and neuropathological abnormalities in htau mice, which express non-mutant human tau isoforms and represent a model of tauopathy relevant to Alzheimer’s disease. Prior to the development of cognitive symptoms, htau mice exhibit tau hyperphosphorylation associated with a selective deficit in food burrowing, a behavior reminiscent to activities of daily living which are impaired early in Alzheimer’s disease. We crossed htau mice with Nmnat1 transgenic and knockout mice and tested the resulting offspring until the age of 6 months. We show that overexpression of NMNAT1 ameliorates the early deficit in food burrowing characteristic of htau mice. At 6 months of age, htau mice did not show neurodegenerative changes in both the cortex and hippocampus, and these were not induced by downregulating NMNAT1 levels. Modulating NMNAT1 levels produced a corresponding effect on NMNAT enzymatic activity but did not alter NAD levels in htau mice. Although changes in local NAD levels and subsequent modulation of NAD-dependent enzymes cannot be ruled out, this suggests that the effects seen on behavior may be due to changes in tau phosphorylation. Our results suggest that increasing NMNAT1 levels can slow the progression of symptoms and neuropathological features of tauopathy, but the underlying mechanisms remain to be established.

The limited ability of cytotoxic T cells to infiltrate solid tumors hampers immune surveillance and the efficacy of immunotherapies in cancer. Adenosine accumulates in solid tumors and inhibits tumor-specific T cells. Adenosine inhibits T cell motility through the A 2A receptor (A 2A R) and suppression of KCa3.1 channels. We conducted three-dimensional chemotaxis experiments to elucidate the effect of adenosine on the migration of peripheral blood CD8 + T cells from head and neck squamous cell carcinoma (HNSCC) patients. The chemotaxis of HNSCC CD8 + T cells was reduced in the presence of adenosine, and the effect was greater on HNSCC CD8 + T cells than on healthy donor (HD) CD8 + T cells. This response correlated with the inability of CD8 + T cells to infiltrate tumors. The effect of adenosine was mimicked by an A 2A R agonist and prevented by an A 2A R antagonist. We found no differences in A 2A R expression, 3′,5′-cyclic adenosine monophosphate abundance, or protein kinase A type 1 activity between HNSCC and HD CD8 + T cells. We instead detected a decrease in KCa3.1 channel activity, but not expression, in HNSCC CD8 + T cells. Activation of KCa3.1 channels by 1-EBIO restored the ability of HNSCC CD8 + T cells to chemotax in the presence of adenosine. Our data highlight the mechanism underlying the increased sensitivity of HNSCC CD8 + T cells to adenosine and the potential therapeutic benefit of KCa3.1 channel activators, which could increase infiltration of these T cells into tumors.

Axon injury leads to rapid depletion of NAD-biosynthetic enzyme NMNAT2 and high levels of its substrate, NMN. We proposed a key role for NMN in Wallerian degeneration but downstream events and their relationship to other mediators remain unclear. Here, we show, in vitro and in vivo, that axotomy leads to a late increase in intra-axonal Ca(2+), abolished by pharmacological or genetic reduction of NMN levels. NMN requires the pro-degenerative protein SARM1 to stimulate Ca(2+) influx and axon degeneration. While inhibition of NMN synthesis and SARM1 deletion block Ca(2+) rise and preserve axonal integrity, they fail to prevent early mitochondrial dynamic changes. Furthermore, depolarizing mitochondria does not alter the rate of Wallerian degeneration. These data reveal that NMN and SARM1 act in a common pathway culminating in intra-axonal Ca(2+) increase and fragmentation and dissociate mitochondrial dysfunctions from this pathway, elucidating which steps may be most effective as targets for therapy.

Tumor-infiltrating lymphocytes (TIL) are potent mediators of an antitumor response. However, their function is attenuated in solid tumors. CD8+ T-cell effector functions, such as cytokine and granzyme production, depend on cytoplasmic Ca2+, which is controlled by ion channels. In particular, Kv1.3 channels regulate the membrane potential and Ca2+ influx in human effector memory T (TEM) cells. In this study, we assessed the contribution of reduced Kv1.3 and Ca2+ flux on TIL effector function in head and neck cancer (HNC). We obtained tumor samples and matched peripheral blood from 14 patients with HNC. CD3+ TILs were composed of 57% CD4+ (82% TEM and 20% Tregs) and 36% CD8+ cells. Electrophysiology revealed a 70% reduction in functional Kv1.3 channels in TILs as compared with peripheral blood T cells from paired patients, which was accompanied by a decrease in Ca2+ influx. Immunofluorescence analysis showed that CD8+ TILs expressing high Kv1.3 preferentially localized in the stroma. Importantly, high expression of Kv1.3 correlated with high Ki-67 and granzyme B expression. Overall, these data indicate that defective Kv1.3 channels and Ca2+ fluxes in TILs may contribute to reduced immune surveillance in HNC. Cancer Res; 77(1); 53–61. ©2016 AACR.

Axon degeneration precedes cell body death in many age-related neurodegenerative disorders, often determining symptom onset and progression. A sensitive method for revealing axon pathology could indicate whether this is the case also in Huntington's disease (HD), a fatal, devastating neurodegenerative disorder causing progressive deterioration of both physical and mental abilities, and which brain region is affected first. We studied the spatio-temporal relationship between axon pathology, neuronal loss, and mutant Huntingtin aggregate formation in HD mouse models by crossing R6/2 transgenic and HdhQ140 knock-in mice with YFP-H mice expressing the yellow fluorescent protein in a subset of neurons. We found large axonal swellings developing age-dependently first in stria terminalis and then in corticostriatal axons of HdhQ140 mice, whereas alterations of other neuronal compartments could not be detected. Although mutant Huntingtin accumulated with age in several brain areas, inclusions in the soma did not correlate with swelling of the corresponding axons. Axon abnormalities were not a prominent feature of the rapid progressive pathology of R6/2 mice. Our findings in mice genetically similar to HD patients suggest that axon pathology is an early event in HD and indicate the importance of further studies of stria terminalis axons in man.

Wallerian degeneration of physically injured axons involves a well-defined molecular pathway linking loss of axonal survival factor NMNAT2 to activation of pro-degenerative protein SARM1. Manipulating the pathway through these proteins led to the identification of non-axotomy insults causing axon degeneration by a Wallerian-like mechanism, including several involving mitochondrial impairment. Mitochondrial dysfunction is heavily implicated in Parkinson’s disease, Charcot-Marie-Tooth disease, hereditary spastic paraplegia and other axonal disorders. However, whether and how mitochondrial impairment activates Wallerian degeneration has remained unclear. Here, we show that disruption of mitochondrial membrane potential leads to axonal NMNAT2 depletion in mouse sympathetic neurons, increasing the substrate-to-product ratio (NMN/NAD) of this NAD-synthesising enzyme, a metabolic fingerprint of Wallerian degeneration. The mechanism appears to involve both impaired NMNAT2 synthesis and reduced axonal transport. Expression of WLDS and Sarm1 deletion both protect axons after mitochondrial uncoupling. Blocking the pathway also confers neuroprotection and increases the lifespan of flies with Pink1 loss-of-function mutation, which causes severe mitochondrial defects. These data indicate that mitochondrial impairment replicates all the major steps of Wallerian degeneration, placing it upstream of NMNAT2 loss, with the potential to contribute to axon pathology in mitochondrial disorders.

Immunotherapy offers promising results in head and neck cancer (HNC) albeit it faces the problem of high resistance. PD1 (programmed death receptor-1) and its ligand (PD-L1/PD-L2) interaction reduces T cell receptor (TCR)-stimulated Ca2+ fluxes. Ion channels regulate Ca2+ entry in T cells downstream to the TCR (store-operated Ca2+ entry, SOCE). We hypothesized that anti-PD1 antibody (Pembrolizumab, Pembro) increases SOCE. We studied the effect of Pembro and PD-L1 on SOCE in CD8+ peripheral blood T cells (PBTs) of HNC patients and healthy donors (HD) using Thapsigargin (TG). Additionally, we tested the effect of Pembro on KCa3.1, Kv1.3 and Ca2+ release activated Ca2+ (CRAC) channels in PBTs of HNC patients using patch-clamp electrophysiology. TCR-mediated Ca2+ fluxes were assessed in PD1-expressing NFAT-reporter Jurkat cells. In these cells, Pembro increased the number of cells that responded to TCR stimulation with a sustained increase in Ca2+ (30%, p

Lupus nephritis (LN) occurs in up to 60% of patients with systemic lupus erythematosus (SLE) and it remains the predominant cause of morbidity and mortality. Current available therapies for SLE have serious side effects and limited efficacy. Our research aims to develop a novel therapy for SLE with improved efficacy and minimal side effects. SLE is in large part driven by hyperactive T cells, which present with multiple defects including increased INFγ, chemokine receptors and CD40L that exacerbate tissue infiltration, inflammation and B cell stimulation. Kv1.3 channels regulate the Ca2+ signaling necessary for these effector functions. We have developed lipid nanoparticles that selectively target memory T (TM) cells and reduce their hyper-functionality by downregulating Kv1.3 via siRNAs (Kv1.3-NPs). Our in-vitro data showed that Kv1.3-NPs suppressed Ca2+ signaling, CD40L expression and IFNγ release selectively in TM cells. To demonstrate therapeutic efficacy of the Kv1.3-NPs we have developed a humanized mouse model of LN by engrafting peripheral blood mononuclear cells (PBMCs) from LN patients into immune-deficient NSG mice. Kidneys of these mice were highly infiltrated with human CD3+ T cells and these cells produced elevated IFNγ. Furthermore, these mice demonstrated a lower survival compared to control mice engrafted with PBMCs from healthy donors. Lastly, we tested the efficacy of the Kv1.3-NPs. We found that LN mice engrafted with PBMCs pretreated in-vitro with Kv1.3-NPs survived longer than mice engrafted with PBMCs pretreated with scramble RNA-NPs. We also found that Kv1.3-NPs were selectively delivered in-vivo into TM cells of humanized mice. Overall, these data support the potential therapeutic value of Kv1.3-NPs.

Axons require the axonal NAD-synthesizing enzyme NMNAT2 to survive. Injury or genetically induced depletion of NMNAT2 triggers axonal degeneration or defective axon growth. We have previously proposed that axonal NMNAT2 primarily promotes axon survival by maintaining low levels of its substrate NMN rather than generating NAD; however, this is still debated. NMN deamidase, a bacterial enzyme, shares NMN-consuming activity with NMNAT2, but not NAD-synthesizing activity, and it delays axon degeneration in primary neuronal cultures. Here we show that NMN deamidase can also delay axon degeneration in zebrafish larvae and in transgenic mice. Like overexpressed NMNATs, NMN deamidase reduces NMN accumulation in injured mouse sciatic nerves and preserves some axons for up to three weeks, even when expressed at a low level. Remarkably, NMN deamidase also rescues axonal outgrowth and perinatal lethality in a dose-dependent manner in mice lacking NMNAT2. These data further support a pro-degenerative effect of accumulating NMN in axons in vivo. The NMN deamidase mouse will be an important tool to further probe the mechanisms underlying Wallerian degeneration and its prevention.

Ca(2+) signaling controls activation and effector functions of T lymphocytes. Ca(2+) levels also regulate NFAT activation and CD40 ligand (CD40L) expression in T cells. CD40L in activated memory T cells binds to its cognate receptor, CD40, on other cell types resulting in the production of antibodies and pro-inflammatory mediators. The CD40L/CD40 interaction is implicated in the pathogenesis of autoimmune disorders and CD40L is widely recognized as a therapeutic target. Ca(2+) signaling in T cells is regulated by Kv1.3 channels. We have developed lipid nanoparticles that deliver Kv1.3 siRNAs (Kv1.3-NPs) selectively to CD45RO(+) memory T cells and reduce the activation-induced Ca(2+) influx. Herein we report that Kv1.3-NPs reduced NFAT activation and CD40L expression exclusively in CD45RO(+) T cells. Furthermore, Kv1.3-NPs suppressed cytokine release and induced a phenotype switch of T cells from predominantly memory to naive. These findings indicate that Kv1.3-NPs operate as targeted immune suppressive agents with promising therapeutic potentials.

The NH2 terminus of the sodium-bicarbonate cotransporter 1 (NBCe1) plays an important role in its targeting to the plasma membrane. To identify the amino acid residues that contribute to the targeting of NBCe1 to the plasma membrane, polarized MDCK cells were transfected with expression constructs coding for green fluorescent protein (GFP)-tagged NBCe1 NH2-terminal deletion mutants, and the localization of GFP-tagged proteins was analyzed by confocal microscopy. Our results indicate that the amino acids between residues 399 and 424 of NBCe1A contain important sequences that contribute to its localization to the plasma membrane. Site-directed mutagenesis studies showed that GFP-NBCe1A mutants D405A and D416A are retained in the cytoplasm of the polarized MDCK epithelial cells. Examination of functional activities of D405A and D416A reveals that their activities are reduced compared with the wild-type NBCe1A. Similarly, aspartic acid residues 449 and 460 of pancreatic NBCe1 (NBCe1B), which correspond to residues 405 and 416 of NBCe1A, are also required for its full functional activity and accurate targeting to the plasma membrane. In addition, while replacement of D416 with glutamic acid did not affect the targeting or functional activity of NBCe1A, substitution of D405 with glutamic acid led to the retention of the mutated protein in the intracellular compartment and impaired functional activity. These studies demonstrate that aspartic acid residues 405 and 416 in the NH2 terminus of NBCe1A are important in its accurate targeting to the plasma membrane.

The cAMP/PKA signaling system constitutes an inhibitory pathway in T cells and, although its biochemistry has been thoroughly investigated, its possible effects on ion channels are still not fully understood. KV1.3 channels play an important role in T-cell activation, and their inhibition suppresses T-cell function. It has been reported that PKA modulates KV1.3 activity. Two PKA isoforms are expressed in human T cells: PKAI and PKAII. PKAI has been shown to inhibit T-cell activation via suppression of the tyrosine kinase Lck. The aim of this study was to determine the PKA isoform modulating KV1.3 and the signaling pathway underneath. 8-Bromoadenosine 3′,5′-cyclic monophosphate (8-BrcAMP), a nonselective activator of PKA, inhibited KV1.3 currents both in primary human T and in Jurkat cells. This inhibition was prevented by the PKA blocker PKI6–22. Selective knockdown of PKAI, but not PKAII, with siRNAs abolished the response to 8-BrcAMP. Additional studies were performed to determine the signaling pathway mediating PKAI effect on KV1.3. Overexpression of a constitutively active mutant of Lck reduced the response of KV1.3 to 8-Br-cAMP. Moreover, knockdown of the scaffolding protein disc large 1 (Dlg1), which binds KV1.3 to Lck, abolished PKA modulation of KV1.3 channels. Immunohistochemistry studies showed that PKAI, but not PKAII, colocalizes with KV1.3 and Dlg1 indicating a close proximity between these proteins. These results indicate that PKAI selectively regulates KV1.3 channels in human T lymphocytes. This effect is mediated by Lck and Dlg1. We thus propose that the KV1.3/Dlg1/Lck complex is part of the membrane pathway that cAMP utilizes to regulate T-cell function.

The response of T cells to antigens (T cell activation) is marked by an increase in intracellular Ca2+ levels. Voltage-gated and Ca2+-dependent K+ channels control the membrane potential of human T cells and regulate Ca2+influx. This regulation is dependent on proper accumulation of K+ channels at the immunological synapse (IS) a signaling zone that forms between a T cell and antigen presenting cell. It is believed that the IS provides a site for regulation of the activation response and that K+ channel inhibition occurs at the IS, but the underlying mechanisms are unknown. A mathematical model was developed to test whether K+ efflux through K+ channels leads to an accumulation of K+ in the IS cleft, ultimately reducing K+ channel function and intracellular Ca2+ concentration ([Ca2+]i). Simulations were conducted in models of resting and activated T cell subsets, which express different levels of K+ channels, by varying the K+ diffusion constant and the spatial localization of K+ channels at the IS. K+ accumulation in the IS cleft was calculated to increase K+ concentration ([K+]) from its normal value of 5.0 mM to 5.2–10.0 mM. Including K+ accumulation in the model of the IS reduced calculated K+ current by 1–12% and consequently, reduced calculated [Ca2+]i by 1–28%. Significant reductions in K+ current and [Ca2+]i only occurred in activated T cell simulations when most K+ channels were centrally clustered at the IS. The results presented show that the localization of K+ channels at the IS can produce a rise in [K+] in the IS cleft and lead to a substantial decrease in K+ currents and [Ca2+]i in activated T cells thus providing a feedback inhibitory mechanism during T cell activation.

Hypoxia in solid tumors contributes to decreased immunosurveillance via down-regulation of Kv1.3 channels in T lymphocytes and associated T cell function inhibition. However, the mechanisms responsible for Kv1.3 down-regulation are not understood. We hypothesized that chronic hypoxia reduces Kv1.3 surface expression via alterations in membrane trafficking. Chronic hypoxia decreased Kv1.3 surface expression and current density in Jurkat T cells. Inhibition of either protein synthesis or degradation and endocytosis did not prevent this effect. Instead, blockade of clathrin-coated vesicle formation and forward trafficking prevented the Kv1.3 surface expression decrease in hypoxia. Confocal microscopy revealed an increased retention of Kv1.3 in the trans-Golgi during hypoxia. Expression of adaptor protein-1 (AP1), responsible for clathrin-coated vesicle formation at the trans-Golgi, was selectively down-regulated by hypoxia. Furthermore, AP1 down-regulation increased Kv1.3 retention in the trans-Golgi and reduced Kv1.3 currents. Our results indicate that hypoxia disrupts AP1/clathrin-mediated forward trafficking of Kv1.3 from the trans-Golgi to the plasma membrane thus contributing to decreased Kv1.3 surface expression in T lymphocytes.

NAD(+) synthesizing enzyme NMNAT1 constitutes most of the sequence of neuroprotective protein Wld(S), which delays axon degeneration by 10-fold. NMNAT1 activity is necessary but not sufficient for Wld(S) neuroprotection in mice and 70 amino acids at the N-terminus of Wld(S), derived from polyubiquitination factor Ube4b, enhance axon protection by NMNAT1. NMNAT1 activity can confer neuroprotection when redistributed outside the nucleus or when highly overexpressed in vitro and partially in Drosophila. However, the role of endogenous NMNAT1 in normal axon maintenance and in Wallerian degeneration has not been elucidated yet. To address this question we disrupted the Nmnat1 locus by gene targeting. Homozygous Nmnat1 knockout mice do not survive to birth, indicating that extranuclear NMNAT isoforms cannot compensate for its loss. Heterozygous Nmnat1 knockout mice develop normally and do not show spontaneous neurodegeneration or axon pathology. Wallerian degeneration after sciatic nerve lesion is neither accelerated nor delayed in these mice, consistent with the proposal that other endogenous NMNAT isoforms play a principal role in Wallerian degeneration.

6059Background: Immune checkpoint inhibitors have been shown to induce durable tumor response in a subset of recurrent and/or metastatic HNSCC. Higher expression of PD-L1, INF-γ, and composite sign...

Adenosine accumulates in the tumor microenvironment and contributes to the inhibition of T cell function via the A2A receptor (A2AR). Blockade of the A2AR has been shown to rescue T cell function and decrease tumor burden. However, A2AR is present in a variety of cells and thus a limitation of non-selective A2AR pharmacological blockers is the development of side effects. Our goal is to selectively knockdown the A2AR in cytotoxic CD8+ T cells through lipid nanoparticles (NPs) decorated with anti-CD8 antibody and containing A2AR siRNAs. The A2AR knockdown by A2AR siRNAs was verified in human peripheral blood T cells (PBT). Nucleofection of PBTs with A2AR or scramble siRNAs downregulated mRNA and protein by 61% and 14–42%, respectively (as determined by RT-qPCR and flow cytometry). A functional assay for IL-2 production (a cytokine produced by activated PBTs and suppressed by the A2AR pathway) in PBTs treated with siRNAs and activated in the presence of CGS21680 (an A2AR agonist) was used to verify the A2AR knockdown. Treatment of PBTs with A2AR siRNAs prevents the CGS21680-induced inhibition of IL-2 production by 27%. Additionally, NPs were fabricated using biotinylated lipids as previously described (Hajdu et al., Biomaterials 2013) and labeled with anti-CD8 antibody. Their selectivity and internalization were determined. Flow cytometry revealed that CD8-labeled NPs are specific to CD4− PBTs and suggested that the NPs are subsequently internalized. Thus, these data indicate the feasibility of fabrication and potential efficacy of CD8-specific A2AR siRNAs-loaded NPs that should restore the functionality selectively in CD8+ tumor infiltrating lymphocytes—a potential targeted cancer therapy with limited side effects.

The limited ability of the cytotoxic CD8+ T cells to infiltrate solid tumors presents a major roadblock to developing effective immunotherapy. Adenosine (Ado) accumulates in high concentrations in solid tumors where it contributes to the suppression of T cell function. We have previously shown that Ado reduces the chemotactic ability of peripheral blood CD8+ T cells (PBTs) from head and neck squamous cell carcinoma (HNSCC) patients by suppressing KCa3.1 channel function. Herein, we conducted experiments to elucidate the mechanism of KCa3.1 dysregulation in HNSCC PBTs. KCa3.1 channels are calcium-activated and require binding of calmodulin (CAM). PBTs were isolated from HNSCC patients and healthy donors (HD), and activated with CD3/CD28 antibodies. CAM levels decreased post-activation in HNSCC PBTs (by ~24%, n=7) while they increased in HD PBTs (by ~37%, n=6, p=0.001). To study whether CAM downregulation contributes to KCa3.1 dysfunction, we transfected HD PBTs with siRNA against CAM (siCam). siCam transfection decreased CAM expression and KCa3.1 currents, but not KCa3.1 expression. We then studied whether CAM downregulation affects the T cell chemotactic response to Ado in HD PBTs. Control (scrRNA-transfected) HD PBTs migrated towards CXCL12 in the presence of Ado, but downregulation of CAM abrogated their chemotactic ability in the presence of Ado (~71% inhibition, n=8, p

Axon degeneration is an early event in many neurodegenerative disorders. In some, the mechanism is related to injury-induced Wallerian degeneration, a proactive death program that can be strongly delayed by the neuroprotective slow Wallerian degeneration protein (Wld(S)) protein. Thus, it is important to understand the Wallerian degeneration mechanism and how Wld(S) blocks it. Wld(S) location is influenced by binding to valosin-containing protein (VCP), an essential protein for many cellular processes including membrane fusion and endoplasmic reticulum-associated degradation. In mice, the N-terminal 16 amino acids (N16), which mediate VCP binding, are essential for Wld(S) to protect axons, a role which another VCP binding sequence can substitute. In Drosophila, the Wld(S) phenotype is weakened by a similar N-terminal truncation and by knocking down the VCP homologue ter94. Neither null nor floxed VCP mice are viable so it is difficult to confirm the requirement for VCP binding in mammals in vivo. However, the hypothesis can be tested further by introducing a Wld(S) missense mutation, altering its affinity for VCP but minimizing the risk of disturbing other aspects of its structure or function. We introduced the R10A mutation, which weakens VCP binding in vitro, and expressed it in transgenic mice. R10AWld(S) fails to co-immunoprecipitate VCP from mouse brain, and only occasionally and faintly accumulates in nuclear foci for which VCP binding is necessary but not sufficient. Surprisingly however, axon protection remains robust and indistinguishable from that in spontaneous Wld(S) mice. We suggest that either N16 has an additional, VCP-independent function in mammals, or that the phenotype requires only weak VCP binding which may be driven forwards in vivo by the high VCP concentration.

The immunological synapse (IS), a highly organized structure that forms at the point of contact between a T cell and an APC, is essential for the proper development of signaling events, including the Ca2+ response. Kv1.3 channels control Ca2+ homeostasis in human T cells and move into the IS upon Ag presentation. However, the process involved in channel accumulation in the IS and the functional implications of this localization are not yet known. Here we define the movement of Kv1.3 into the IS and study whether Kv1.3 localization into the IS influences Ca2+ signaling in Jurkat T cells. Crosslinking of the channel protein with an extracellular Ab limits Kv1.3 mobility and accumulation at the IS. Moreover, Kv1.3 recruitment to the IS does not involve the transport of newly synthesized channels and it does not occur through recycling of membrane channels. Kv1.3 localization in the IS modulates the Ca2+ response. Blockade of Kv1.3 movement into the IS by crosslinking significantly increases the amplitude of the Ca2+ response triggered by anti-CD3/anti-CD28-coated beads, which induce the formation of the IS. On the contrary, the Ca2+ response induced by TCR stimulation without the formation of the IS with soluble anti-CD3/anti-CD28 Abs is unaltered. The results presented herein indicate that, upon Ag presentation, membrane-incorporated Kv1.3 channels move along the plasma membrane to localize in the IS. This localization is important to control the amplitude of the Ca2+ response, and disruption of this process can account for alterations of downstream Ca2+-dependent signaling events.

Tiny medicine refers to the development of small easy to use devices that can help in the early diagnosis and treatment of disease. Early diagnosis is the key to successfully treating many diseases. Nanomaterial-based biosensors utilize the unique properties of biological and physical nanomaterials to recognize a target molecule and effect transduction of an electronic signal. In general, the advantages of nanomaterial-based biosensors are fast response, small size, high sensitivity, and portability compared to existing large electrodes and sensors. Systems integration is the core technology that enables tiny medicine. Integration of nanomaterials, microfluidics, automatic samplers, and transduction devices on a single chip provides many advantages for point of care devices such as biosensors. Biosensors are also being used as new analytical tools to study medicine. Thus this paper reviews how nanomaterials can be used to build biosensors and how these biosensors can help now and in the future to detect disease and monitor therapies.

Motor neuron degeneration in amyotrophic lateral sclerosis involves oxidative damage. Glutathione (GSH) is critical as an antioxidant and a redox modulator. We used a motor neuronal cell line (NSC-34) to investigate whether wild-type and familial amyotrophic lateral sclerosis-linked G93A mutant Cu,Zn superoxide dismutase (wt/G93ASOD1) modified the GSH pool and glutamate cysteine ligase (GCL), the rate-limiting enzyme for GSH synthesis. We studied the effect of various G93ASOD1 levels and exposure times. Mutant Cu,Zn superoxide dismutase induced an adaptive process involving the upregulation of GSH synthesis, even at very low expression levels. However, cells with a high level of G93ASOD1 cultured for 10 weeks showed GSH depletion and a decrease in expression of the modulatory subunit of GCL. These cells also had lower levels of GSH and GCL activity was not induced after treatment with the pro-oxidant tert-butylhydroquinone. Cells with a low level of G93ASOD1 maintained higher GSH levels and GCL activity, showing that the exposure time and the level of the mutant protein modulate GSH synthesis. We conclude that failure of the regulation of the GSH pathway caused by G93ASOD1 may contribute to motor neuron vulnerability and we identify this pathway as a target for therapeutic intervention.