Abstract 17074: Methadone Potently Blocks Cardiac IK1 Leading to Membrane Instability

Introduction: Methadone is the second most frequently reported cause of drug-induced cardiac arrest in pharmacovigilance databases, yet the mechanism of its pro-arrhythmia is unclear. Methadone-induced QTU wave prolongation has been repeatedly observed and attributed to inhibition of the delayed rectifier hERG current, but QTU fusion suggests the inwardly rectifying K + current (IK1) might also be affected by methadone. Hypothesis: Methadone pro-arrhythmia is associated with potent block of the IK1 current responsible for rapid terminal repolarization of the cardiac action potential (AP). Methods: Human Kir2.2, encoding the IK1 current, was transiently expressed in COS cells. hERG1a was stably expressed in CHO cells. Cardiac myocytes from swine were obtained by ventricular enzymatic dissociation. Ionic current and APs were measured using patch-clamp methods. Methadone HCl (R+S racemates) was dissolved in Tyrode solution. Results: Methadone suppressed IK1 current with an IC 50 of 1.47 uM (Fig 1A). Methadone also suppressed outward IK1 (-60 mV) measured in swine myocytes (Ba 2+ -sensitive current) with an IC 50 of 1.52 μM. Methadone suppressed hERG currents with an IC 50 of 2.1 μM. APs measured in swine myocytes exhibited significant prolongation (13 ± 4 % increase of APD 90 , p<0.029, n=7) as well as slowing of the rate of terminal repolarization (a specific marker of IK1 blockade) in the presence of 1 μM methadone (Fig. 1B). Fluctuations of diastolic voltage increased by 30 ± 12 and 151 ± 27 % (n=3; p<0.04) in 0.1 and 1 μM methadone, respectively, consistent with a reduction in membrane stability. Conclusions: Methadone is an equipotent blocker of IK1 and hERG. The effect of IK1 block coupled with modest hERG block has a synergistic effect on terminal repolarization that may partially explain the pro-arrhythmic impact of methadone. Moreover, this observation may be generalized to other drugs where unsuspected IK1 blockade may contribute to pro-arrhythmia and torsade de pointes.

Fast scanned widefield scheme provides tunable and uniform illumination for optimized SMLM on large fields of view

Quantitative analyses in classical fluorescence microscopy and Single Molecule Localization Microscopy (SMLM) require uniform illumination over the field of view; ideally coupled with optical sectioning techniques such as Total Internal Reflection Fluorescence (TIRF) to remove out of focus background. In SMLM, high irradiances (several kW/cm²) are crucial to drive the densely labeled sample into the single molecule regime, and conventional gaussian-shaped lasers will typically restrain the usable field of view to around 40 µm x 40 µm. Here we present Adaptable Scanning for Tunable Excitation Regions (ASTER), a novel and versatile illumination technique that generates uniform illumination over adaptable fields of view and is compatible with illumination schemes from epifluorescence to speckle-free TIRF. For SMLM, ASTER delivers homogeneous blinking kinetics at reasonable laser power, providing constant precision and higher throughput over fields of view 25 times larger than typical. This allows improved clustering analysis and uniform size measurements on sub-100 nm objects, as we demonstrate by imaging nanorulers, microtubules and clathrin-coated pits in COS cells, as well as periodic β2-spectrin along the axons of neurons. ASTER’s sharp, quantitative TIRF and SMLM images up to 200 µm x 200 µm in size pave the way for high-throughput quantification of cellular structures and processes.

SUN-263 In Vitro Modeling of Brain-Bone Communication: Factors Secreted from Immortalized Female Kisspeptin Neurons Modulate Gene Expression in Osteoblasts

While the sex steroid hormone estrogen (E2) is essential for maximum reproductive fitness, circulating E2 is also involved in maintaining skeletal homeostasis in females. E2 in male and female animals has been shown to stimulate cancellous bone formation via activation of estrogen receptor alpha (ERα), and it was widely thought that this effect was mediated directly at the level of bone. A recent study, however, demonstrated a large increase in bone density in female mice in which ERα was deleted from specific neuroendocrine neurons in the arcuate nucleus of the hypothalamus, specifically those expressing kiss1, a population required for fertility and pubertal progression. Bone from transgenic Kiss1-cre X ERα floxed females showed higher osteoblast functioning, accompanied by increases in the expression of sp7 and runx2, positing the existence of a direct neural-bone regulatory axis that is altered by circulating E2 at the level of the brain (Herber et al., Nat Commun 2019). Our laboratory recently published a study demonstrating that GnRH and Kisspeptin, typically thought to act primarily within the neuroendocrine reproductive axis, are synthesized and secreted in an autocrine fashion by canine osteosarcoma cells in vitro, and that these neuropeptides can stimulate tumor cell proliferation (BMC Cancer 2019). Separately, our lab has also recently generated two immortalized Kiss1-expressing and –secreting cell lines, KTaR-1 (representative of female arcuate Kiss-1 neurons) and KTaV-3 cells (representative of female AVPV Kiss-1 neurons) (Endocrinology 2017). In the current study, we have combined these two in vitro models to explore if factors secreted by female ARC-derived KTaR-1 cells may affect multiple parameters of osteoblast function, including sp7 and runx2 expression (evaluated by qPCR), and ability to form bone matrix (evaluated by Alizarin Red assay). Preliminary results suggest that exposure canine osteosarcoma (COS) cells to conditioned media from KTaR-1 cells leads to increases in sp7 expression in an E2-dependent manner, such that 24h E2-deprivation of these neurons stimulates secretion of osteogenic factors. Additionally, media from both KTaR-1 and KTaV-3 cells stimulated Ca2+ production from cultured osteoblasts, as evaluated by Alizarin Red. We are continuing to explore these in vitro interactions using COS cells, as well as normal osteoblasts (cNOB) and immortalized cNOBs, and are evaluating media and exosomal proteomics to further characterize putative factors. While further study is required, these initial results suggest that our immortalized neuronal KP cell models may provide useful molecular tools to explore the regulation of this newly-proposed neural-bone axis.

PD-1 Inhibits TCR Proximal Signaling By Sequestering SHP-2 Phosphatase and Facilitating Csk-Mediated Inhibitory Phosphorylation of Lck

Programmed death (PD)-1 is a negative regulator of T cell responses, which controls peripheral tolerance but also suppresses anti-viral and anti-tumor immunity. The biochemical mechanisms via which PD-1 inhibits T cell activation remain poorly understood. The cytoplamsic tail of PD-1 contains two structural motifs, an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). It has been reported that SHP-2 tyrosine phosphatase constitutively interacts with the ITSM and has a critical role in PD-1-mediated inhibitory function but the mechanism remains unclear. Although phosphatases are generally considered negative regulators of activation, somatic mutations of SHP-2 leading to gain of function have been identified in MDS and AML, where they induce activation of Ras-MAPK cascade. In contrast, fibroblasts and T cells from SHP-2 deficient mice display impaired activation of MAPK in response to growth factor-mediated activation and TCR/CD3-mediated activation, respectively. These studies suggest that SHP-2 has a role in activating some signaling pathways. In the present study we sought to determine the mechanism via which PD-1: SHP-2 interaction leads to inhibition of T cell activation. SHP-2 has two src homology domains, (N-SH2 and C-SH2) and one PTP domain. To identify the region of SHP-2 that interacts with PD-1, we generated five different GST-fusion proteins in which GST was fused with either SHP-2 full length (FL), SHP-2-N-SH2, SHP-2-C-SH2, SHP-2-ΔN-SH2 (lacking the N-terminus SH2 domain) or SHP-2-PTP. Pull-down assays using lysates from naive or activated primary human T cells revealed that PD-1 interacted with full length GST-SHP-2 fusion protein only after TCR/CD3-mediated activation and simultaneous PD-1 ligation. Pull-down assays with each GST-fusion protein showed that this interaction was mediated selectively via the SH2 domains of SHP-2, indicating that PD-1 requires prior tyrosine phosphorylation for interaction with SHP-2. The TCR itself lacks kinase activity but constitutively interacts with Fyn, the most TCR-proximal tyrosine kinase, which induces TCR phosphorylation upon oligomerization of the TCR/CD3 chains. To determine whether Fyn can also induce PD-1 phosphorylation during simultaneous ligation with TCR/CD3, we used COS cells to express PD-1 together with empty vector, Fyn, or a kinase inactive mutant of Fyn, followed by pull-down with GST-SHP-2 fusion protein. No interaction between PD-1 and GST-SHP-2 fusion protein was detected in lysates from COS cells expressing empty vector or kinase inactive Fyn mutant. In contrast, in the presence of active Fyn, PD-1 underwent phosphorylation and association with SHP-2. A key event required for the activation of the TCR/CD3 downstream signaling cascade is the downregulation of Csk-mediated inactivating C-terminal phosphorylation of Lck on Tyr-505. In epithelial cells, upon growth factor stimulation, SHP-2 is recruited to the plasma membrane and abrogates Csk recruitment and Csk-mediated inactivating C-terminal phosphorylation of Src kinase. We examined whether SHP-2 might be recruited to the plasma membrane during TCR/CD3-mediated stimulation and whether this event might be affected by PD-1: SHP-2 interaction. Using nitrogen cavitation to fractionate cytoplasmic and membranous compartments, we determined that stimulation of primary human T cells by TCR/CD3 resulted in robust membrane translocation of SHP-2. This event correlated with downregulation of the inactivating phosphorylation of Lck on Tyr-505. In contrast, stimulation of T cells with simultaneous ligation of PD-1 resulted in PD-1: SHP-2 association and sequestration, and prevented the translocation of SHP-2 to the membranous fraction and the downregulation of the inactivating Lck phosphorylation on Tyr-505. Moreover, phosphorylation of Lck substrates including PLC-γ1 was impaired. Consistent with the key role of PLC-γ1 in the activation of Ras/MAPK via RasGRP1, activation of Ras and MAPK was also defective. Our results unravel a previously unidentified mechanism via which PD-1 inhibits T cell activation by sequestering SHP-2 and preventing its translocation to the plasma membrane where SHP-2 plays a key role in activating the TCR signaling cascade by reversing Csk-mediated inactivating phosphorylation of Lck. Disclosures No relevant conflicts of interest to declare.

Leukemia Escape From HLA-Specific T Lymphocyte Pressure in a Recipient of HLA One Locus-Mismatched Bone Marrow Transplantation

Abstract 1899 Allogeneic HSCT is curative for leukemia by virtue of the immune reaction mediated by donor T lymphocytes, but unfortunately, some patients relapse after transplantation. It is reasonable to assume that the selective pressure exerted by donor T lymphocytes can lead to the outgrowth of pre-existing leukemia variants that have lost expression of gene products such as HLA molecules. However, the mechanisms of leukemia relapse in HLA-mismatched HSCT recipients remain largely uninvestigated. A case of leukemia escape from an HLA-specific cytotoxic T lymphocyte (CTL) response in a recipient of HLA one locus-mismatched BMT is presented. A 24-year-old man with primary refractory T lymphoblastic leukemia/lymphoma received BMT from his HLA-B*51:01-mismatched mother with a T lymphocyte-repleted graft. The preparative regimen consisted of L-PAM and TBI. GVHD prophylaxis consisted of tacrolimus and methotrexate. The patient developed acute GVHD involving skin, gut, and liver on day 46 (maximum grade: III on day 53). Acute GVHD was incurable and transitioned to chronic GVHD. The patient relapsed with ascites, a hydrocele, and a subpapillary tumor on day 261. Immunosuppressant therapy was required to control GVHD until his death on day 279. Flow cytometric analysis for each HLA-A, B, or DR locus in the leukemia blasts was performed, and it showed that only the expression of B51 was down-regulated in post-transplant leukemia blasts compared with that in pre-transplant blasts. Ten CTL clones were isolated from the patient's blood on day 56. In a Cr release assay, all isolated CTL clones lysed recipient B-LCL but failed to lyse donor B-LCL. The nucleotide sequences of the uniquely rearranged TCR Vβ gene of each clone indicated that 10 clones had been derived from six independent clones. COS cells transfected with the HLA-B*51:01 cDNA construct clearly stimulated IFN-γ production by six independent CTL clones (ELISA), and the donor B-LCL transfected with HLA-B*51:01 cDNA were lysed by six CTL clones (Cr release assay), indicating that all clones recognized the B*51:01 molecule as an alloantigen. On the other hand, these data suggest that the CTL response toward the B*51:01 molecule accounted for a majority of the recipient's CTL alloresponse during acute GVHD. The recipient B*51:01 and the donor B*52:01 differed in two amino acids at positions 63 and 67, both of which constitute peptide-binding pockets. We generated four mutated B*51:01 cDNA constructs, Asn63Glu and Phe67Ser, in which individual amino acids were substituted with the corresponding amino acid in B*52:01, and Val194Ile and Ala199Val, in which both amino acids localize outside the positions constituting peptide binding pockets. Stimulation by COS cells transfected with Asn63Glu or Phe67Ser, but not with Val194Ile or Ala199Val, significantly decreased IFN-γ production by all CTL clones, suggesting that recognition of the HLA-B*51:01 molecule by CTL clones was peptide-dependent. Additionally, CTL clones should recognize certain peptides other than leukemia antigens, because B*51:01-transfected “COS cells” stimulated IFN-γ production of CTLs, and B*51:01-transfected “donor B-LCL” were lysed by CTLs. We then attempted to determine that the leukemia blasts escaped from the cytotoxicity of B*51:01-specific CTL clones. The pre-transplant leukemia blasts (purity, ∼62%) were weakly but clearly lysed by CTL clone, whereas the post-transplant leukemia blasts (∼99%) were not lysed by any CTL clones at all. In addition, the IFN-γ ELISPOT assay was performed to detect the B*51:01-reactive T lymphocytes in patient blood on day 232, one month before clinical leukemia relapse. IFN-γ-producing B*51:01-reactive T lymphocytes were detected at a level nearly equal to the level of recipient B-LCL-reactive T lymphocytes, that is, the total of the T lymphocyte alloresponse. The mechanism of leukemia relapse in this patient can be explained as follows: CTLs specific for HLA-B*51:01 molecule/non-leukemia peptide complexes were generated in the patient blood during acute GVHD, and these CTLs continued to produce immunological pressure on leukemia blasts for at least 8 months after transplantation, but B*51:01-down-regulated leukemia blasts escaped from the pressure of B*51:01-specific CTLs, and then the leukemia clinically relapsed. These findings can explain, at least in part, the mechanism of how leukemia relapse occurs during persistent GVHD after HSCT. Disclosures: Naoe: Zenyaku-Kogyo: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Chugai Pharma.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Kyowa-Hakko Kirin.: Research Funding; Otsuka Pharma.: Research Funding.

Glanzmann Mutations : New Allelic Variants and Role of the β3 Lys253 in the αIIb/β3 Interaction Highlighted by the Lys253Met Substitution.

Abstract 1312 Poster Board I-336 Introduction Glanzmann thrombasthenia (GT) is an autosomal recessive inherited bleeding disorder characterized by an impaired platelet aggregation. GT results from defects of the platelet fibrinogen receptor αIIbβ3. GT mutations provide useful tools for structure-function relationship studies of αIIbβ3. Patient and methods Genomic DNA from 6 patients has been amplified for αIIb and β3 promoters and exons sequences. PCR products were directly sequenced. Potential RNA processing alterations have been studied in silico by using Genscan, NNSPLICE and ESEFinder online tools. When no RNA splicing anomaly was predicted, the effect of single point mutation on αIIbβ3 expression has been studied by using transiently transfected Cos cells. Finally, structural consequences of amino acid substitutions has been studied using the published model of αIIbβ3 (code PDB 2VDL) and structural modelling. Results 7 new mutations have been characterized. 1 deletion / insertion, 2 single point mutations inducing stop codon and 1 resulting in splicing site disruption were identified. The 3 last identified single point mutations were not predicted to affect normal RNA processing but has been shown to prevent normal expression of mutant αIIbβ3 at the surface of Cos cells. The p.Meth118Arg and p.Gly221Asp substitutions that induce both important steric hindrance and charge modifications, are located inside the β-I domain of β3. So they should deeply alter the proper folding of the β-I domain, preventing the complex expression at the platelet surface. On the other hand, the p.Lys253Met protrudes from the β-I domain toward the αIIb β-propeller. A structural model of the Met253 β-I mutant has been done. An estimation of the direct electrostatic and desolvation free energies of interaction between the β-I domain surface and the αIIb β-propeller indicated that rather than the presence of a methionine, it is the lost of the Lys253 which is responsible for the complex expression defect. Conclusion Seven new GT mutations have been identified and the p.Lys253Met substitution helped to define a key role of the Lys253 in the αIIb β-propeller / β3 β-I domains interaction. Disclosures No relevant conflicts of interest to declare.