Α4β2 Receptors Research Articles

Pyrrolidinyl benzofurans and benzodioxanes: Selective α4β2 nicotinic acetylcholine receptor ligands with different activity profiles at the two receptor stoichiometries

A series of racemic benzofurans bearing N-methyl-2-pyrrolidinyl residue at C(2) or C(3) has been synthesized and tested for affinity at the α4β2 and α3β4 nicotine acetylcholine receptors (nAChRs). As previously reported for the benzodioxane based analogues, hydroxylation at proper position of benzene ring results in high α4β2 nAChR affinity and α4β2 vs. α3β4 nAChR selectivity. 7-Hydroxy-N-methyl-2-pyrrolidinyl-1,4-benzodioxane (2) and its 7- and 5-amino benzodioxane analogues 3 and 4, which are all α4β2 nAChR partial agonists, and 2-(N-methyl-2-pyrrolidinyl)-6-hydroxybenzofuran (12) were selected for functional characterization at the two α4β2 stoichiometries, the high sensitivity (α4)2(β2)3 and the low sensitivity (α4)3(β2)2. The benzene pattern substitution, which had previously been found to control α4β2 partial agonist activity and α4β2 vs. α3β4 selectivity, proved to be also involved in stoichiometry-selectivity. The 7-hydroxybenzodioxane derivative 2 selectively activates (α4)2(β2)3 nAChR, which cannot be activated by its 5-amino analogue 4. A marginal structural modification, not altering the base pyrrolidinyl benzodioxane scaffold, resulted in opposite activity profiles at the two α4β2 nAChR isoforms providing an interesting novel case study.

Diazepam binding inhibitor positively modulates α5β3 GABA-A receptors

Benzodiazepines (BZDs) exert their antiepileptic, anxiogenic, and somnogenic effects by binding to GABA-A receptors (GABARs). GABARs are inhibitory pentameric ligand-gated channels commonly formed by 2α, 2β and 1γ subunits, though some GABARs are formed by only α and β subunits. The high-affinity binding site for BZDs is located in the extracellular domain between the α and γ subunits and is absent in αβ receptors.

Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination.

Antigen recognition through the T cell receptor (TCR) αβ heterodimer is one of the primary determinants of the adaptive immune response. Vaccines activate naïve T cells with high specificity to expand and differentiate into memory T cells. However, antigen-specific memory CD4 T cells exist in unexposed antigen-naïve hosts. In this study, we use high-throughput sequencing of memory CD4 TCRβ repertoire and machine learning to show that individuals with preexisting vaccine-reactive memory CD4 T cell clonotypes elicited earlier and higher antibody titers and mounted a more robust CD4 T cell response to hepatitis B vaccine. In addition, integration of TCRβ sequence patterns into a hepatitis B epitope-specific annotation model can predict which individuals will have an early and more vigorous vaccine-elicited immunity. Thus, the presence of preexisting memory T cell clonotypes has a significant impact on immunity and can be used to predict immune responses to vaccination.

Genomic landscape of TCRαβ and TCRγδ T-large granular lymphocyte leukemia

AbstractLarge granular lymphocyte (LGL) leukemia comprises a group of rare lymphoproliferative disorders whose molecular landscape is incompletely defined. We leveraged paired whole-exome and transcriptome sequencing in the largest LGL leukemia cohort to date, which included 105 patients (93 T-cell receptor αβ [TCRαβ] T-LGL and 12 TCRγδ T-LGL). Seventy-six mutations were observed in 3 or more patients in the cohort, and out of those, STAT3, KMT2D, PIK3R1, TTN, EYS, and SULF1 mutations were shared between both subtypes. We identified ARHGAP25, ABCC9, PCDHA11, SULF1, SLC6A15, DDX59, DNMT3A, FAS, KDM6A, KMT2D, PIK3R1, STAT3, STAT5B, TET2, and TNFAIP3 as recurrently mutated putative drivers using an unbiased driver analysis approach leveraging our whole-exome cohort. Hotspot mutations in STAT3, PIK3R1, and FAS were detected, whereas truncating mutations in epigenetic modifying enzymes such as KMT2D and TET2 were observed. Moreover, STAT3 mutations co-occurred with mutations in chromatin and epigenetic modifying genes, especially KMT2D and SETD1B (P < .01 and P < .05, respectively). STAT3 was mutated in 50.5% of the patients. Most common Y640F STAT3 mutation was associated with lower absolute neutrophil count values, and N647I mutation was associated with lower hemoglobin values. Somatic activating mutations (Q160P, D170Y, L287F) in the STAT3 coiled-coil domain were characterized. STAT3-mutant patients exhibited increased mutational burden and enrichment of a mutational signature associated with increased spontaneous deamination of 5-methylcytosine. Finally, gene expression analysis revealed enrichment of interferon-γ signaling and decreased phosphatidylinositol 3-kinase–Akt signaling for STAT3-mutant patients. These findings highlight the clinical and molecular heterogeneity of this rare disorder.

Allogeneic CD20-targeted γδ T cells exhibit innate and adaptive antitumor activities in preclinical B-cell lymphoma models.

ObjectivesAutologous chimeric antigen receptor (CAR) αβ T‐cell therapies have demonstrated remarkable antitumor efficacy in patients with haematological malignancies; however, not all eligible cancer patients receive clinical benefit. Emerging strategies to improve patient access and clinical responses include using premanufactured products from healthy donors and alternative cytotoxic effectors possessing intrinsic tumoricidal activity as sources of CAR cell therapies. γδ T cells, which combine innate and adaptive mechanisms to recognise and kill malignant cells, are an attractive candidate platform for allogeneic CAR T‐cell therapy. Here, we evaluated the manufacturability and functionality of allogeneic peripheral blood‐derived CAR+ Vδ1 γδ T cells expressing a second‐generation CAR targeting the B‐cell‐restricted CD20 antigen.MethodsDonor‐derived Vδ1 γδ T cells from peripheral blood were ex vivo‐activated, expanded and engineered to express a novel anti‐CD20 CAR. In vitro and in vivo assays were used to evaluate CAR‐dependent and CAR‐independent antitumor activities of CD20 CAR+ Vδ1 γδ T cells against B‐cell tumors.ResultsAnti‐CD20 CAR+ Vδ1 γδ T cells exhibited innate and adaptive antitumor activities, such as in vitro tumor cell killing and proinflammatory cytokine production, in addition to in vivo tumor growth inhibition of B‐cell lymphoma xenografts in immunodeficient mice. Furthermore, CD20 CAR+ Vδ1 γδ T cells did not induce xenogeneic graft‐versus‐host disease in immunodeficient mice.ConclusionThese preclinical data support the clinical evaluation of ADI‐001, an allogeneic CD20 CAR+ Vδ1 γδ T cell, and a phase 1 study has been initiated in patients with B‐cell malignancies (NCT04735471).

Nanomicelles-in-coaxial nanofibers with exit channels as a transdermal delivery platform for smoking cessation.

Smoking is a life-threatening habit; that is why many nicotine-replacement therapies (NRTs), which include chewing gums, nicotine patches, lozenges, mouth sprays, inhalers and nasal sprays that are usually administered for 8-12 weeks, have been reported for smoking cessation. We report the fabrication of patches comprising nanomicelles-in-coaxial nanofibers (NFs) for the transdermal delivery of varenicline (VAR) tartrate, a partial agonist of the α4β2 receptor subtype, for smoking cessation. The cores of the fabricated coaxial NF structures are composed of polyethylene oxide, VAR-loaded Pluronic F127 nanomicelles (NPs) and free VAR, while the shell consists of a blend of cellulose acetate (CA) and polycaprolactone (PCL) in a ratio of 1 : 9 (w/w) that incorporates 50% (wt%) free VAR. The morphology and the coaxial structure of the NFs were investigated using TEM, SEM and fluorescent microscopy. The physicochemical and mechanical properties of the scaffolds were analyzed using FTIR, DSC, DLS, TGA and a universal testing machine. SEM micrographs depict NFs with a size ranging from 793.7 ± 518.9 to 324.5 ± 144.1 nm. In vitro release of VAR reaches almost 100% after 3, 9 and 28 days for free VAR, VAR-loaded NPs and the NPs-in-NFs patches, respectively, while the ex vivo release tested using albino rat skin, over a period of 60 days, showed up to 94% sustained release of VAR. Besides, skin permeation, in vivo release and plasma concentrations of VAR from the NF transdermal patches were monitored via cyclic voltammetric measurements during the course of treatment. DFT calculations as well as mathematical release kinetic models were performed in order to study the release mechanism. The cell viability of human skin fibroblast (HSF) cells in the case of plain and VAR-loaded NFs was 75.09 and 32.11%, respectively. The in vivo results showed that VAR was being continuously released from the transdermal patch over a period of 14 days. Besides, the treatment with VAR-loaded patches did not cause any severe conditions in the studied animal model. The new fabricated NPs-in-NFs transdermal patch for VAR tartrate delivery is considered as an effective, economic, safe and long-acting NRT for smoking cessation.

Cross Talk between α7 and α3β4 Nicotinic Receptors Prevents Their Desensitization in Human Chromaffin Cells.

The physical interaction and functional cross talk among the different subtypes of neuronal nicotinic acetylcholine receptors (nAChRs) expressed in the various tissues is unknown. Here, we have investigated this issue between the only two nAChRs subtypes expressed, the α7 and α3β4 subtypes, in a human native neuroendocrine cell (the chromaffin cell) using electrophysiological patch-clamp, fluorescence, and Förster resonance energy transfer (FRET) techniques. Our data show that α7 and α3β4 receptor subtypes require their mutual and maximal efficacy of activation to increase their expression, to avoid their desensitization, and therefore, to increase their activity. In this way, after repetitive stimulation with acetylcholine (ACh), α7 and α3β4 receptor subtypes do not desensitize, but they do with choline. The nicotinic current increase associated with the α3β4 subtype is dependent on Ca2+ In addition, both receptor subtypes physically interact. Interaction and expression of both subtypes are reversibly reduced by tyrosine and serine/threonine phosphatases inhibition, not by Ca2+ In addition, expression is greater in human chromaffin cells from men compared to women, but FRET efficiency is not affected. Together, our findings indicate that human α7 and α3β4 subtypes mutually modulate their expression and activity, providing a promising line of research to pharmacologically regulate their activity.SIGNIFICANCE STATEMENT Desensitization of nicotinic receptors is accepted to occur with repetitive agonist stimulation. However, here we show that human native α3β4 and α7 nicotinic acetylcholine receptor (nAChR) subtypes do not desensitize, and instead, increase their activity when they are activated by the physiological agonist acetylcholine (ACh). An indispensable requirement is the activation of the other receptor subtype with maximal efficacy, and the presence of Ca2+ to cooperate in the case of the α3β4 current increase. Because choline is an α3β4 partial agonist, it will act as a limiting factor of nicotinic currents enhancement in the absence of ACh, but in its presence, it will further potentiate α7 currents.

Off-the-shelf Vδ1 gamma delta T cells engineered with glypican-3 (GPC-3)-specific chimeric antigen receptor (CAR) and soluble IL-15 display robust antitumor efficacy against hepatocellular carcinoma

BackgroundGlypican-3 (GPC-3) is an oncofetal protein that is highly expressed in various solid tumors, but rarely expressed in healthy adult tissues and represents a rational target of particular relevance in...

Ex Vivo T-Cell Receptor αβ +/CD19 +depletion of Peripheral Stem Cell Grafts for Pediatric Patients with Bone Marrow Failure (BMF) Undergoing Unrelated Donor Transplantation

Background: Success of alternative donor stem cell transplantation (SCT) for acquired and inherited bone marrow failure (BMF) syndromes has previously been limited by significant risks of severe graft versus host disease (GvHD) and graft failure, particularly for patients who lack fully HLA-matched donors. The development of post-transplant cyclophosphamide (ptCy)-based haploidentical donor SCT has mitigated but not eliminated these risks, and the relatively slow engraftment seen with ptCy bone marrow grafts may not be ideal given high rates of infectious disease and bleeding concerns in patients with BMF. Partial T cell depletion of mobilized peripheral stem cell (PSC) grafts can greatly reduce risks of graft versus host disease while facilitating rapid and robust engraftment by providing a high stem cell dose. We previously reported excellent outcomes with minimal GVHD and no graft rejection using peripheral stem cell transplant (PSCT) combined with ex vivo CD3 +/CD19 +depletion and low dose CD3+ T cell addback from matched unrelated donors (MUD) and mismatched unrelated donors (MMUD) in pediatric patients with BMF. Here, we describe the use of selective ex vivoT cell receptor (TCR)αβ +/CD19 +depletion of mobilized PSC from MUD and MMUD for pediatric patients with BMF, which has the advantage of retaining TCRgd +T cells which may help facilitate engraftment and decrease infections.Methods: We report the outcomes of 26 pediatric patients with BMF (excluding MDS-defining clonal evolution) who underwent MUD/MMUD PSCT with TCRαβ +T cell/CD19 +depletion using CliniMACS at The Children's Hospital of Philadelphia from 2017 to 2021. Patients were enrolled on a prospective clinical trial for patients with BMF (NCT03047746, n=21)or on an expanded access study (NCT03145545, n=5). Conditioning regimens consisted of thymoglobulin (9mg/kg), cyclophosphamide (100mg/kg), fludarabine (150mg/m 2), and low dose TBI (200-300 cGy) for patients with acquired BMF disorders, thymoglobulin (9mg/kg), busulfan (PK-adjusted), fludarabine (150mg/m 2), and thiotepa (10mg/kg) for patients with single lineage BMF or thymoglobulin. One patient with Fanconi Anemia received thymoglobulin and fludarabine, with reduced dosing of busulfan and cyclophosphamide. Patients undergoing MSD-BMT for BMF over a similar time period served as a comparison group for engraftment kinetics, rates of GVHD, donor chimerism, immune reconstitution, and overall survival.Results: Subjects included 18 with severe acquired aplastic anemia (SAAA), 4 with SAAA and concurrent paroxysmal nocturnal hemoglobinuria (PNH), 2 patients with acquired BMF not otherwise specified, 1 patient with DBA, and 1 patient with Fanconi Anemia. 11 patients with SAAA underwent SCT as initial therapy while 11 patients had SCT after failing previous medical therapies. Median age at diagnosis was 10.3 years (0.1-20.6) and at transplant 11.1 years (0.9-21). HLA match of unrelated donors was either 10/10 (n=15), or 9/10 (n=11). Median CD34 +and TCR αβ +T cell dose was 12.0x10 6cells/kg (3-22.6) and 0.1x10 5cells/kg (0.0-4.2). Median times to neutrophil and platelet engraftment per CIBMTR criteria were 15 days (10-22) and 15 days (13-19), respectively, both significantly earlier than engraftment following MSD-BMT (Fig 1a). At a median follow-up of 727 days (39-1498), 25 of 26 patients are alive with resolved hematologic disease (Fig 1b). One patient with SAAA+PNH who failed prior IST achieved trilinear engraftment without GVHD, but died on Day+95 due to acute disseminated toxoplasmosis. No patients exhibited immunologic graft rejection. 2/26 patients had grade II acute GVHD that responded to steroids and none developed Grade III-IV acute or chronic extensive GVHD (Fig 1c) CMV viremia/reactivation occurred in 5 subjects, all responding to antiviral pharmacotherapy, and none developed end-organ CMV disease (Fig 1d). One patient developed recipient-derived EBV post-transplant lymphoproliferative disorder requiring multimodal treatment. Only one patient developed BK cystitis. Total peripheral chimerism exceeded 90% in all patients. Immune reconstitution kinetics were similar to that seen in MSD-BMT.Conclusion: MUD/MMUD PSCT with TCRαβ +T cell/CD19 +depletion in patients with BMF enables rapid, durable engraftment with minimal risk of GVHD and immunologic graft rejection. [Display omitted] DisclosuresMonos: Omixon: Consultancy, Patents & Royalties. Grupp: Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Novartis, Kite, Vertex, and Servier: Research Funding; Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy.

Expansion and Enrichment of Gamma-Delta (&amp;#947;&amp;#948;) T Cells from Apheresed Human Product

Although Vγ9Vδ2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major histocompatibility complex (MHC)-independent manner and develop strong cytolytic effector function that makes it an ideal candidate for cancer immunotherapy. Due to the low frequency of Gamma-Delta (γδ) T cells in the peripheral blood, we developed an effective protocol to greatly expand a highly pure γδ T cells drug product for first-in-human use of allogeneic γδ T cells in patients with acute myeloid leukemia (AML). Using healthy donor apheresis as an allogenic cell source, the lymphocytes are isolated using a validated device for a counterflow centrifugation method of separating cells by size and density. The lymphocyte-rich fraction is utilized, and the γδ T cells are preferentially activated with zoledronic acid (FDA-approved) and interleukin (IL)-2 for 7 days. Following the preferential expansion of γδ T cells, a clinical-grade magnetic cell-separation device and TCRαβ beads are used to deplete contaminating T-cell receptor (TCR)αβ T cells. The highly enriched γδ T cells then undergo a second expansion using engineered artificial antigen-presenting cells (aAPCs) derived from K562 cells-genetically engineered to express single-chain variable fragment (scFv) for CD3 and CD28, 41BBL (CD137L) and IL15-RA-together with zoledronic acid and IL-2. Seeding all day-7 enriched γδ T cells in co-culture with the aAPCs facilitates the manufacture of highly pure γδ T cells with an average fold expansion of >229,000-fold from healthy donor blood.

Expansion and Enrichment of Gamma-Delta (γδ) T Cells from Apheresed Human Product.

Although Vγ9Vδ2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major histocompatibility complex (MHC)-independent manner and develop strong cytolytic effector function that makes it an ideal candidate for cancer immunotherapy. Due to the low frequency of Gamma-Delta (γδ) T cells in the peripheral blood, we developed an effective protocol to greatly expand a highly pure γδ T cells drug product for first-in-human use of allogeneic γδ T cells in patients with acute myeloid leukemia (AML). Using healthy donor apheresis as an allogenic cell source, the lymphocytes are isolated using a validated device for a counterflow centrifugation method of separating cells by size and density. The lymphocyte-rich fraction is utilized, and the γδ T cells are preferentially activated with zoledronic acid (FDA-approved) and interleukin (IL)-2 for 7 days. Following the preferential expansion of γδ T cells, a clinical-grade magnetic cell-separation device and TCRαβ beads are used to deplete contaminating T-cell receptor (TCR)αβ T cells. The highly enriched γδ T cells then undergo a second expansion using engineered artificial antigen-presenting cells (aAPCs) derived from K562 cells-genetically engineered to express single-chain variable fragment (scFv) for CD3 and CD28, 41BBL (CD137L) and IL15-RA-together with zoledronic acid and IL-2. Seeding all day-7 enriched γδ T cells in co-culture with the aAPCs facilitates the manufacture of highly pure γδ T cells with an average fold expansion of >229,000-fold from healthy donor blood.

Presence of ethanol-sensitive and ethanol-insensitive glycine receptors in the ventral tegmental area and prefrontal cortex in mice.

Background and PurposeGlycine receptors composed of α1 and β subunits are primarily found in the spinal cord and brainstem and are potentiated by ethanol (10–100 mM). However, much less is known about the presence, composition and ethanol sensitivity of these receptors in higher CNS regions. Here, we examined two regions of the brain reward system, the ventral tegmental area (VTA) and the prefrontal cortex (PFC), to determine their glycine receptor subunit composition and sensitivity to ethanol.Experimental ApproachWe used Western blot, immunohistochemistry and electrophysiological techniques in three different models: wild‐type C57BL/6, glycine receptor subunit α1 knock‐in and glycine receptor subunit α2 knockout mice.Key ResultsSimilar levels of α and β receptor subunits were detected in both brain regions, and electrophysiological recordings demonstrated the presence of glycine‐activated currents in both areas. Sensitivity of glycine receptors to glycine was lower in the PFC compared with VTA. Picrotoxin only partly blocked the glycine‐activated current in the PFC and VTA, indicating that both regions express heteromeric αβ receptors. Glycine receptors in VTA neurons, but not in PFC neurons, were potentiated by ethanol.Conclusion and ImplicationsGlycine receptors in VTA neurons from WT and α2 KO mice were potentiated by ethanol, but not in neurons from the α1 KI mice, supporting the conclusion that α1 glycine receptors are predominantly expressed in the VTA. By contrast, glycine receptors in PFC neurons were not potentiated in any of the mouse models studied, suggesting the presence of α2/α3/α4, rather than α1 glycine receptor subunits.

Effects of nicotinic antagonists on working memory performance in young rhesus monkeys

Acetylcholine plays a pivotal neuromodulatory role in the brain, influencing neuronal activity and cognitive function. Nicotinic receptors, particularly α7 and α4β2 receptors, modulate firing of dorsolateral prefrontal (dlPFC) excitatory networks that underlie successful working memory function. Minimal work however has been done examining working memory following systemic blockade of nicotinic receptor systems in nonhuman primates, limiting the ability to explore interactions of other neuromodulatory influences with working memory impairment caused by nicotinic antagonism. In this study, we investigated working memory performance after administering three nicotinic antagonists, mecamylamine, methyllycaconitine, and dihydro-β-erythroidine, in rhesus macaques tested in a spatial delayed response task. Surprisingly, we found that no nicotinic antagonist significantly impaired delayed response performance compared to vehicle. In contrast, the muscarinic antagonist scopolamine reliably impaired delayed response performance in all monkeys tested. These findings suggest there are some limitations on using systemic nicotinic antagonists to probe the involvement of nicotinic receptors in aspects of dlPFC-dependent working memory function, necessitating alternative strategies to understand the role of this system in cognitive deficits seen in aging and neurodegenerative disease.

Dissecting the Molecular Determinants of GABAA Receptors Current Rundown, a Hallmark of Refractory Human Epilepsy.

GABAA receptors-(Rs) are fundamental for the maintenance of an efficient inhibitory function in the central nervous system (CNS). Their dysfunction is associated with a wide range of CNS disorders, many of which characterized by seizures and epilepsy. Recently, an increased use-dependent desensitization due to a repetitive GABA stimulation (GABAA current rundown) of GABAARs has been associated with drug-resistant temporal lobe epilepsy (TLE). Here, we aimed to investigate the molecular determinants of GABAA current rundown with two different heterologous expression systems (Xenopus oocytes and human embryonic kidney cells; HEK) which allowed us to manipulate receptor stoichiometry and to study the GABAA current rundown on different GABAAR configurations. To this purpose, we performed electrophysiology experiments using two-electrode voltage clamp in oocytes and confirming part of our results in HEK. We found that different degrees of GABAA current rundown can be associated with the expression of different GABAAR β-subunits reaching the maximum current decrease when functional α1β2 receptors are expressed. Furthermore, the blockade of phosphatases can prevent the current rundown observed in α1β2 GABAARs. Since GABAAR represents one important therapeutic target in the treatment of human epilepsy, our results could open new perspectives on the therapeutic management of drug-resistant patients showing a GABAergic impairment.

Protective effects of VMY-2-95 on corticosterone-induced injuries in mice and cellular models

Nicotinic α4β2 receptor antagonists have drawn increasing attention in the development of new antidepressants. In this study, we aimed to investigate the protective effect of VMY-2-95, the new selective antagonist of α4β2 nicotinic acetylcholine receptor (nAChR) on corticosterone (CORT) injured mice and cellular models. Fluoxetine was applied as a positive control, and the effects of VMY-2-95 were investigated with three different doses or concentrations (1, 3, 10 mg/kg in mice, and 0.003, 0.03, 0.1 μmol/L in cells). As a result, VMY-2-95 showed significant antidepressant-like effects in the CORT injured mice by improving neuromorphic function, promoting hippocampal nerve proliferation, and regulating the contents of monoamine transmitters. Meanwhile, VMY-2-95 exhibited protective effects on cell viability, cell oxidant, cell apoptosis, and mitochondrial energy metabolism on corticosterone-impaired SH-SY5Y cells. Also, the PKA–CREB–BDNF signaling pathway was up-regulated by VMY-2-95 both in vitro and in vivo, and pathway blockers were also combined with VMY-2-95 to verify the effects furtherly. Therefore, we preliminarily proved that VMY-2-95 had protective effects in depressed mice and SH-SY5Y cells against injuries induced by corticosterone. This work indicated that the application of VMY-2-95 is a potential pharmacological solution for depression. This study also supported the development of α4β2 nAChR antagonists towards neuropsychiatric dysfunctions.

HLA-haploidentical TCRαβ+/CD19+-depleted stem cell transplantation in children and young adults with Fanconi anemia

AbstractWe report on the outcome of 24 patients with Fanconi anemia (FA) lacking an HLA matched related or unrelated donor, given an HLA-haploidentical T-cell receptor αβ (TCRαβ+) and CD19+ cell-depleted hematopoietic stem cell transplantation (HSCT) in the context of a prospective, single-center phase 2 trial. Sustained primary engraftment was achieved in 22 (91.6%) of 24 patients, with median time to neutrophil recovery of 12 days (range, 9-15 days) and platelet recovery of 10 days (range, 7-14 days). Cumulative incidences of grade 1 to 2 acute graft-versus-host disease (GVHD) and chronic GVHD were 17.4% (95% confidence interval [CI], 5.5%-35.5%) and 5.5% (95% CI, 0.8%-33.4%), respectively. The conditioning regimen, which included fludarabine, low-dose cyclophosphamide and, in most patients, single-dose irradiation was well tolerated; no fatal transplant-related toxicity was observed. With a median follow-up of 5.2 years (range, 0.3-8.7 years), the overall and event-free survival probabilities were 100% and 86.3% (95% CI, 62.8%-95.4%), respectively (2 graft failures and 1 case of poor graft function were considered as events). The 2 patients who experienced primary graft failure underwent a subsequent successful HSCT from the other parent. This is the first report of FA patients given TCRαβ+/CD19+-depleted haplo-HSCT in the context of a prospective trial, and the largest series of T-cell–depleted haplo-HSCT in FA reported to date. This trial was registered at www.clinicaltrials.gov as #NCT01810120.

17 - Unrelated Donor Peripheral Stem Cell Transplantation (SCT) with Ex Vivo T Cell Receptor (TCR) αβ+/CD19+depletion for Pediatric Patients with Bone Marrow Failure (BMF)

17 - Unrelated Donor Peripheral Stem Cell Transplantation (SCT) with Ex Vivo T Cell Receptor (TCR) αβ+/CD19+depletion for Pediatric Patients with Bone Marrow Failure (BMF)

CALM-AF10 is a common fusion transcript in T-ALL and is specific to the TCRγδ lineage

AbstractThe t(10;11)(p13-14;q14-21) associated with CALM-AF10 is considered to be rare and associated with a variety of acute lymphoid and myeloid leukemias. Twelve (9%) of 131 unselected T-cell acute lymphoid leukemias (T-ALLs) expressed CALM-AF10 by reverse transcription–polymerase chain reaction or fluorescence in situ hybridization (or both), including 8% of children and 10% of adults, of whom only half demonstrated a t(10;11) by classical cytogenetics. CALM-AF10 was not found in T-cell–receptor αβ (TCRαβ) lineage T-ALLs, as defined by expression of TCRαβ, cytoplasmic TCRβ, or TCRβVDJ rearrangement in immature cytoplasmic TCRβ- cases, compared with 19% of TCRγδ T-ALLs and 33% of immature δ/γ T-ALLs. The latter differed from their CALM-AF10- immature counterparts by a CD5+/CD2-phenotype, as found in TCRγδ but not TCRαβ T-ALLs and in their TCRγ and TCRδ configurations, altogether suggesting that CALM-AF10+ immature δ/γT-ALLs are TCRγδ precursors and that, within T-ALL, CALM-AF10 is specific for this lineage. Nine of 12 immature CALM-AF10 T-ALLs demonstrated 3′ fusion transcripts, whereas 6 of 7 TCRγδ T-ALLs demonstrated 5′ fusion transcripts. The latter retain the AF10 extended LAP/PHD domain necessary for homo-oligomerization. All 8 patients with CALM-AF10+TCRγδ T-ALLs are alive, compared with only 3 of 12 with immature CALM-AF10+ T-ALLs. Six CALM-AF10+ non-T acute leukemias all expressed CD7 and demonstrated T-restricted TCRδ rearrangements, suggesting that they may also be related to the TCRγδ lineage. CALM-AF10 is therefore the most common fusion protein in T-ALL. It requires molecular and immunophenotypic characterization for appropriate prognostic evaluation and should be included in diagnostic screening panels of T-ALL and immature acute leukemias. Analysis of immature CALM-AF10+ leukemias will also facilitate analysis of the early stages of development of the TCRγδ lineage.

Single-Cell Roadmap of Immune Cell Response in Chronic Myeloid Leukemia

Only half of chronic myeloid leukemia (CML) patients in deep molecular remission are able to maintain treatment free remission (TFR) after tyrosine kinase inhibitor (TKI) discontinuation. Identifying predictive markers for TFR remains a key issue. The immunological control or eradication of TKI insensitive leukemic stem cells may contribute to successful cessation, as recent work has demonstrated that differences in immune cell compositions can be associated with better probability of TFR. In order to understand the precise immunological changes in CML, we profiled over 170,000 cells from 25 CML samples from different clinical phases (untreated, during TKI therapy and after TKI cessation) using single-cell RNA and T cell receptor (TCR) αβ sequencing (scRNA+TCRab-seq). We profiled both CD45+ sorted peripheral blood samples (n=20) and CD34+ sorted bone marrow samples (n=5). To understand antigen-specific responses, we profiled TCRs specific to tumor-antigen PR1 (n=12) and compared these to unsorted TCRβ-sequenced samples from CML (n=137) and healthy donors (n=786). To understand the distinctive immunological features in CML, we compared the immune system in CML (n=20) to those in other hematological and solid malignancies profiled with scRNA+TCRab-seq (n=9). We discovered NK cells to be the most unique feature in CML, as NK CD56dim cells were more abundant in CML patients (Padj&amp;lt;0.001) and the NK cell repertoire was more transcriptionally heterogenous and included otherwise rarely seen exhausted NK population with upregulated HAVCR2 and TIGIT expression (Padj &amp;lt;0.0001). Further, the NK cell maturation changed during TKI-treatment from an effector CD56dim state towards an adaptive NK-cell state. We sought to understand whether the immunological activity differs before and after TKI discontinuation in different clinical outcomes following discontinuation. Therefore, we reclustered the discontinuation samples from three different outcomes: TFR, early relapse (&amp;lt;6 months after cessation) or late relapse (&amp;gt;6 months). In all clinical outcomes, TKI cessation invigorated NK cell exhaustion and the most upregulated pathways included IFNg and TNFa via NFkB pathways. Patients with successful TFR had less differences in their immune system after cessation in comparison to relapsing patients, which could hint that their immune subsets were better prepared for the cessation. Unlike the early relapse patients' quiescent immune cells, the late relapse patients' immune subsets were active, but under pronounced inhibitory Treg signals, providing a clinically interesting approach to alter the outcome of these patients. Next, to understand how malignant CML cells evade or interact with the immune system, we analyzed CD34+ cells and calculated a BCR-ABL1 activity score. The most primitive CD34+ cells had lower BCR-ABL1 score and not as many immunological ligand-receptor pairs as their less primitive, highly BCR-ABL1 pathway expressing, counterparts (P&amp;lt;0.0001). LGALS9, that is expressed on both BCR-ABL1 high and low CD34+ cells, arose as one of the most interesting ligands mediating tumor-immune cell interactions and it has many potential receptors expressed by NK cells, including HAVCR2. Finally, we found that tumor-antigen PR1 is preferentially expressed on CD34+ cells with high BCR-ABL1 score. Most TCRs recognizing PR1 are restricted to individuals, but they share short amino-acid motifs that are shared across patients. Thus, we were able to generate an in silico tool to recognize PR1-specific TCRs from unsorted TCR-seq samples with high accuracy (AUC 0.91 from 10-fold cross validation). With our tool, we were able to show that anti-PR1 responses were more frequent in CML than in healthy donors (P&amp;lt;0.0001), are more expanded in bone-marrow than in blood (P&amp;lt;0.0001) and diversified during TKI treatment (P&amp;lt;0.0001). In the scRNA+TCRab-seq data, the anti-PR1 response differed between TFR and early relapse patient significantly at phenotype level, where the TFR patient had cytotoxic anti-leukemic response before TKI cessation, in comparison to the early relapse patient's exhausted and less cytotoxic PR1-specific cells. In conclusion, our results provide high-resolution insights into anti-leukemic immune responses in CML and how we could harness and monitor them to enable successful TKI cessations. Figure Disclosures Olsson-Strömberg: Pfizer: Research Funding. Hjorth-Hansen:Pfizer: Honoraria, Research Funding; Austrian Orphan Pharma: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding. Mustjoki:Pfizer: Research Funding; Novartis: Research Funding; BMS: Honoraria, Research Funding.

Clonally Expanded Bone Marrow T Cells Show Effector Differentiation and Rarely Recognize Disease-Associated Antigens in Multiple Myeloma

Multiple myeloma is a malignancy of monoclonal plasma cells accumulating in the bone marrow. The critical influence of tumor-infiltrating T cells on disease control and therapeutic responses has been shown in a variety of malignancies, however, the role of multiple myeloma bone marrow-infiltrating T cells is incompletely understood. Although it has been shown that multiple myeloma neo-antigen-specific T cells can be expanded in vitro, little is known about functions and specificities of clonally expanded multiple myeloma-infiltrating bone marrow T cells. Here we asked at the single cell level whether clonally expanded T cells i) were detectable in multiple myeloma bone marrow and peripheral blood, ii) showed characteristic immune phenotypes, and iii) recognized antigens selectively presented on multiple myeloma cells. A total of 6,744 single bone marrow T cells from 13 treatment-naïve patients were index-sorted and sequenced using our methodologies for determination of paired T cell receptor (TCR) αβ sequences along with immune phenotype, transcription factor and cytokine expression. Clonal T cell expansion occurred predominantly within the CD8+ compartment. Phenotypes of clonally expanded T cells were distinctive of cytolytic effector differentiation and significantly different from non-expanded CD8+ T cells. Less than 25% of expanded CD8+ T cell clones expressed the immune checkpoint molecules programmed death-1 (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), or T cell immunoglobulin and mucin-domain containing-3 (TIM-3), while B and T lymphocyte attenuator (BTLA) was expressed on more than half of the expanded clones. Clonal T cell expansion did not correlate with neo-antigen load as determined by whole exome and RNA sequencing of purified multiple myeloma cells. Furthermore, peripheral blood TCRβ repertoire sequencing from five selected patients with substantial bone marrow T cell expansion identified 90% of expanded bone marrow T cell clones overlapping with peripheral blood. To determine whether clonally expanded bone marrow T cells recognized antigens selectively presented on multiple myeloma cells, 71 dominant TCRs from five selected patients with substantial clonal T cell expansion were re-expressed in 58α-β- T-hybridoma reporter T cells and co-incubated with CD38-enriched multiple myeloma cells from the same patients. Only one of these TCRs recognized antigens selectively presented on multiple myeloma cells and this TCR was not neo-antigen-specific. Hypothesizing that the target antigen was a non-mutated self-antigen, we could show that this TCR also recognized the plasma cell leukemia cell line U-266 in an HLA-A*02:01-restricted manner. In summary, clonally expanded T cells in multiple myeloma bone marrow of newly diagnosed patients show cytolytic effector differentiation. In the majority of patients, clonally expanded bone marrow T cells do not recognize antigens presented on multiple myeloma cells and are not neo-antigen-specific. Our findings are relevant for the design of future therapeutics and clinical trials. The identified TCR, which recognizes a multiple myeloma antigen shared with U-266 in an HLA-A*02:01-restricted manner, could be a promising candidate for T cell therapy. Disclosures Bullinger: Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Hexal: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Menarini: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees.