Head and neck cancer (HNC) is the sixth most common cancer worldwide with a five‐year survival rate of 50%. Immunotherapy has been emerging as a promising treatment modality as conventional treatment strategies (surgery, chemotherapy and radiation) have limited efficacy and cause debilitating co‐morbidities. Immunotherapy harnesses the patient’s own immune system to fight disease. Targeting the immune system is crucial because immune cell (specifically cytotoxic T cell) infiltration into the tumor microenvironment (TME) and subsequent cytotoxicity is critical for tumor elimination. Pembrolizumab, an anti‐PD‐1 (programmed cell death‐1 receptor) antibody, is an immunotherapy currently approved for metastatic or recurrent unresectable HNC and is in clinical trials for curative intent. While there have been promising long‐lasting clinical responses to Pembrolizumab, many patients still do not respond. Thus, it is critical to understand the mechanism of action of Pembrolizumab in order to reduce treatment resistance. Moreover, it has been shown that Pembrolizumab increases cytotoxic T cell density in the TME; nevertheless, the underlying mechanism is not fully understood. However, it is known that there is an increased accumulation of adenosine (Ado), an anti‐inflammatory purine nucleoside, in the TME of HNC patients, which inhibits T cell chemotaxis by blocking the function of the potassium ion channel, KCa3.1. Therefore, to elucidate possible effects of Pembrolizumab on T cell chemotaxis, we studied KCa3.1 activity and subsequent chemotaxis in CD8 + peripheral blood T cells (PBTs) of naïve (never treated) HNC patients before/after Pembrolizumab treatment (n=25) via electrophysiological and 3D chemotaxis experiments. Furthermore, Pembrolizumab treated patients were categorized as responders (R) or non‐responders (NR) based on treatment response (i.e., reduction in tumor viability with a cutoff of ≥20% for R). We observed that Pembrolizumab increased the KCa3.1 activity in post‐treatment patient PBTs by 29.8% (p=0.026). Because we observed an increase in KCa3.1 activity in treated patients, we next assessed whether there was an effect on the chemotaxis of PBTs in presence of Ado. While Ado inhibited the chemotactic ability in treatment naïve PBTs by 53.9% (p<0.001, n=22), we determined that post‐treatment R PBTs were not inhibited by Ado (p = 0.142, n=11) whereas post‐treatment NR PBTs were inhibited by Ado (p = 0.005, n=10). These data support a role of ion channels and chemotaxis in patient response to Pembrolizumab and could lead to better understanding of the effect of Pembrolizumab in immune cell infiltration into the tumor and, ultimately, its mechanism of action/resistance. Support or Funding Information DoD CA160714; T32CA117846; 2R01 CA95286
T lymphocyte and antigen presenting cell contact triggers the formation of the immunological synapse (IS), a signalosome important for T cell activation. Kv1.3 channels participate in T cell activation by modulating Ca2+ influx in resting but not in activated (pre-exposed to antigen) T cells. Further, Kv1.3 channels have been shown to localize in the IS (Panyi et al., PNAS 2004). Despite the key role of these channels in T cell activation, their behavior during IS formation is not fully understood. Herein we characterized the kinetics of Kv1.3 channel recruitment into the IS in resting and activated human T cells. IS formation was induced by 1–30min exposure to either anti-CD3/CD28 antibody coated beads or EBV infected B cells. Cells were immunostained for Kv1.3 and F-actin and imaged with fluorescence and confocal microscopy. We found that binding of the CD3/CD28 beads to both T cell types leads to colocalization of Kv1.3 with F-actin (marker of IS) at the bead/cell contact area. Kv1.3 redistribution depends on IS formation, as it was not observed with control beads. Further, recruitment of Kv1.3 channels in the IS in resting T cells builds up progressively, with maximal recruitment at 30min. In contrast, activated T cells show maximal Kv1.3 recruitment at 1min and by 15min the channels move out of the IS. These results were confirmed using B cells. In conclusion Kv1.3 channel redistribution in the IS follows a characteristic time course depending on the T cell activation state. This difference may reflect the different role that Kv1.3 channels play in these cells. (NIH-CA95286 to LC).
Abstract In rat white adipocytes histamine is oxidized by a semicarbazide-sensitive amine oxidase which has benzylamine or preferential substrate (Bz-SSAO). To determine whether Bz-SSAO could control the extracellular levels of histamine and other histamine-related compounds active in lipid mobilization, a series of histaminergic compounds was screened as possible substrates or inhibitors of Bz-SSAO activity. Histaminergic compounds with imidazolo or thiazolo groups are oxidized by rat white-adipocyte Bz-SSAO whereas S-isothiourea derivatives, with two- or three-carbon-atom alkyl chains between the isothiourea and the N,N-dimethyl residue are, instead, inhibitors of the enzyme. Amtamine has been identified as a selective, high affinity substrate for rat white adipocyte Bz-SSAO. This enzymatic degradation might represent a catabolic pathway for the drug. These results show that the histaminase property of the rat white-adipocyte enzyme Bz-SSAO also extends to other histamine derivatives active at histamine receptors.
Abstract Glutamate excitotoxicity is implicated in the aetiology of amyotrophic lateral sclerosis (ALS) with impairment of glutamate transport into astrocytes a possible cause of glutamate‐induced injury to motor neurons. It is possible that mutations of Cu/Zn superoxide dismutase (SOD1), responsible for about 20% of familial ALS, down‐regulates glutamate transporters via oxidative stress. We transfected primary mouse astrocytes to investigate the effect of the FALS‐linked mutant hSOD1 G93A and wild‐type SOD1 (hSOD1 wt ) on the glutamate uptake system. Using western blotting, immunocytochemistry and RT‐PCR it was shown that expression of either hSOD1 G93A or hSOD1 wt in astrocytes produced down‐regulation of the levels of a glutamate transporter GLT‐1, without alterations in its mRNA level. hSOD1 G93A or hSOD1 wt expression caused a decrease of the monomeric form of GLT‐1 without increasing oxidative multimers of GLT‐1. The effects were selective to GLT‐1, since another glutamate transporter GLAST protein and mRNA levels were not altered. Reflecting the decrease in GLT‐1 protein, [ 3 H] d ‐aspartate uptake was reduced in cultures expressing hSOD1 G93A or hSOD1 wt . The hSOD1‐induced decline in GLT‐1 protein and [ 3 H] d ‐aspartate uptake was not blocked by the antioxidant Trolox nor potentiated by antioxidant depletion using catalase and glutathione peroxidase inhibitors. Measurement of 2′,7′‐dichlorofluorescein (DCF)‐induced fluorescence revealed that expression of hSOD1 G93A or hSOD1 wt in astrocytes does not lead to detectable increase of intracellular reactive oxygen species. This study suggests that levels of GLT‐1 protein in astrocytes are reduced rapidly by overexpression of hSOD1, and is due to a property shared between the wild‐type and G93A mutant form, but does not involve the production of intracellular oxidative stress.
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