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The relationship between plasma protease inhibitor (PI) trough concentrations and hyperlipidemic effects were evaluated retrospectively using data from 2 pilot clinical trials of a double-boosted PI regimen (saquinavir/lopinavir/ritonavir) in 25 HIV patients. The patients' median age was 39 years (range, 25-60). At baseline, PI-naive patients had a median viral load of 53 500 copies/mL and median CD4 of 296 cells/mm(3), while PI-experienced patients had 37 750 copies/mL and 214 cells/mm(3). Plasma PI trough concentrations of saquinavir, lopinavir, and ritonavir at week 12 were 520, 4482, and 153 ng/mL, respectively. At week 12, median fasting lipids increased significantly from baseline: total cholesterol increased from 165 to 189 mg/dL (P = .0005) and the triglyceride increased from 113 to 159 mg/dL (P = .001). There were no associations between PI trough concentrations at week 12 and the percent total cholesterol change at week 12. No associations were found between PI trough concentrations and lipid changes in HIV patients on a double-boosted PI regimen (saquinavir/lopinavir/ritonavir). Factors other than systemic exposure to PIs (such as host or genetic factors) may modulate the hyperlipidemic effect of PIs. [ABSTRACT FROM AUTHOR]

Background: Despite declines in morbidity and mortality with the use of combination antiretroviral therapy, its effectiveness is limited by adverse events, problems with adherence, and resistance of the human immunodeficiency virus (HIV).Methods: We randomly assigned persons infected with HIV who had a CD4+ cell count of more than 350 per cubic millimeter to the continuous use of antiretroviral therapy (the viral suppression group) or the episodic use of antiretroviral therapy (the drug conservation group). Episodic use involved the deferral of therapy until the CD4+ count decreased to less than 250 per cubic millimeter and then the use of therapy until the CD4+ count increased to more than 350 per cubic millimeter. The primary end point was the development of an opportunistic disease or death from any cause. An important secondary end point was major cardiovascular, renal, or hepatic disease.Results: A total of 5472 participants (2720 assigned to drug conservation and 2752 to viral suppression) were followed for an average of 16 months before the protocol was modified for the drug conservation group. At baseline, the median and nadir CD4+ counts were 597 per cubic millimeter and 250 per cubic millimeter, respectively, and 71.7% of participants had plasma HIV RNA levels of 400 copies or less per milliliter. Opportunistic disease or death from any cause occurred in 120 participants (3.3 events per 100 person-years) in the drug conservation group and 47 participants (1.3 per 100 person-years) in the viral suppression group (hazard ratio for the drug conservation group vs. the viral suppression group, 2.6; 95% confidence interval [CI], 1.9 to 3.7; P<0.001). Hazard ratios for death from any cause and for major cardiovascular, renal, and hepatic disease were 1.8 (95% CI, 1.2 to 2.9; P=0.007) and 1.7 (95% CI, 1.1 to 2.5; P=0.009), respectively. Adjustment for the latest CD4+ count and HIV RNA level (as time-updated covariates) reduced the hazard ratio for the primary end point from 2.6 to 1.5 (95% CI, 1.0 to 2.1).Conclusions: Episodic antiretroviral therapy guided by the CD4+ count, as used in our study, significantly increased the risk of opportunistic disease or death from any cause, as compared with continuous antiretroviral therapy, largely as a consequence of lowering the CD4+ cell count and increasing the viral load. Episodic antiretroviral therapy does not reduce the risk of adverse events that have been associated with antiretroviral therapy. (ClinicalTrials.gov number, NCT00027352 [ClinicalTrials.gov].). [ABSTRACT FROM AUTHOR]

Background and Purpose: Inhibitors of voltage-gated sodium channels (Na V s) are important anti-epileptic drugs, but the contribution of specific channel isoforms is unknown since available inhibitors are non-selective. We aimed to create novel, isoform selective inhibitors of Na v channels as a means of informing the development of improved antiseizure drugs., Experimental Approach: We created a series of compounds with diverse selectivity profiles enabling block of Na V 1.6 alone or together with Na V 1.2. These novel Na V inhibitors were evaluated for their ability to inhibit electrically evoked seizures in mice with a heterozygous gain-of-function mutation (N1768D/+) in Scn8a (encoding Na V 1.6) and in wild-type mice., Key Results: Pharmacologic inhibition of Na V 1.6 in Scn8a N1768D/+ mice prevented seizures evoked by a 6-Hz shock. Inhibitors were also effective in a direct current maximal electroshock seizure assay in wild-type mice. Na V 1.6 inhibition correlated with efficacy in both models, even without inhibition of other CNS Na V isoforms., Conclusions and Implications: Our data suggest Na V 1.6 inhibition is a driver of efficacy for Na V inhibitor anti-seizure medicines. Sparing the Na V 1.1 channels of inhibitory interneurons did not compromise efficacy. Selective Na V 1.6 inhibitors may provide targeted therapies for human Scn8a developmental and epileptic encephalopathies and improved treatments for idiopathic epilepsies., (© 2024 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Cancer exploits different mechanisms to escape T-cell immunosurveillance, including overexpression of checkpoint ligands, secretion of immunosuppressive molecules, and aberrant glycosylation. Herein, we report that IFNγ, a potent immunomodulator secreted in the tumor microenvironment, can induce α2,6 hypersialylation in cancer cell lines derived from various histologies. We focused on Siglec-9, a receptor for sialic acid moieties, and demonstrated that the Siglec-9+ T-cell population displayed reduced effector function. We speculated that Siglec-9 in primary human T cells can act as a checkpoint molecule and demonstrated that knocking out Siglec-9 using a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system enhanced the functionality of primary human T cells. Finally, we aimed to augment cancer-specific T-cell activity by taking advantage of tumor hypersialylation. Thus, we designed several Siglec-9-based chimeric switch receptors (CSR), which included an intracellular moiety derived from costimulatory molecules (CD28/41BB) and different hinge regions. In an antigen-specific context, T cells transduced with Siglec-9 CSRs demonstrated increased cytokine secretions and upregulation of activation markers. Moreover, T cells equipped with specific Siglec-9 CSRs mediated robust antitumor activity in a xenograft model of human tumors. Overall, this work sheds light on tumor evasion mechanisms mediated by sialylated residues and exemplifies an approach to improve engineered T cell-based cancer treatment. See related Spotlight by Abken, p. 1310., (©2024 American Association for Cancer Research.)

Abstract: HBI0101 is an academic chimeric antigen receptor T-cell (CART)-targeted to B-cell maturation antigen (BCMA) for the treatment of relapsed and refractory multiple myeloma (R/RMM) and light chain amyloidosis. Herein, we present the phase 1b/2 results of 50 heavily pretreated patients with R/RMM dosed with 800 × 106 CART cells. Inclusion criteria were relatively permissive (i.e., performance status and baseline organ function) and consequently, approximately half of the enrolled patients would have been ineligible for pivotal clinical trials. The median time elapsed from patient enrollment until CART delivery was 25 days (range, 14-65). HBI0101-related toxicities included grade 1 to 3 cytokine release syndrome, grade 3 to 4 hematologic toxicities, and grade 1 to 2 immune effector cell-associated neurotoxicity syndrome. Responses were achieved in 90% of the patients, 56% achieved stringent and complete response, and 70% reached a minimal residual disease negativity. Within a median follow-up of 12.3 months, the median progression-free survival (PFS) was 11.0 months (95% confidence interval [CI], 6.2-14.6), and the overall survival was not reached (95% CI, 13.3 to not reached). Multivariable analysis on patient/disease and CART-related characteristics revealed that high-risk cytogenetic, extramedullary disease, and increased number of effector-memory T cells in CART products were independently associated with inferior PFS. In conclusion, comprehensive analyses of the parameters affecting the response to CART therapy are essential for improving patients' outcome. This trial was registered at www.ClinicalTrials.gov as #NCT04720313., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: T cells play a central role in the antitumor response. However, they often face numerous hurdles in the tumor microenvironment, including the scarcity of available essential metabolites such as glucose and amino acids. Moreover, cancer cells can monopolize these resources to thrive and proliferate by upregulating metabolite transporters and maintaining a high metabolic rate, thereby outcompeting T cells., Methods: Herein, we sought to improve T-cell antitumor function in the tumor vicinity by enhancing their glycolytic capacity to better compete with tumor cells. To achieve this, we engineered human T cells to express a key glycolysis enzyme, phosphofructokinase, in conjunction with Glucose transporter 3, a glucose transporter. We co-expressed these, along with tumor-specific chimeric antigen or T-cell receptors., Results: Engineered cells demonstrated an increased cytokine secretion and upregulation of T-cell activation markers compared with control cells. Moreover, they displayed superior glycolytic capacity, which translated into an improved in vivo therapeutic potential in a xenograft model of human tumors., Conclusion: In summary, these findings support the implementation of T-cell metabolic engineering to enhance the efficacy of cellular immunotherapies for cancer., Competing Interests: Competing interests: We declare that CJC, RTZ and TB are inventors on a submitted PCT Application No. PCT/IL2023/050248 related to this study. All other authors declare that they have no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The molecular and cellular basis of health in human tendons remains poorly understood. Among human tendons, hamstring tendon has markedly low pathology and can provide a prototypic healthy tendon reference. The aim of this study was to determine the transcriptomes and location of all cell types in healthy hamstring tendon. Using single nucleus RNA sequencing, we profiled the transcriptomes of 10 533 nuclei from four healthy donors and identified 12 distinct cell types. We confirmed the presence of two fibroblast cell types, endothelial cells, mural cells, and immune cells, and identified cell types previously unreported in tendons, including different skeletal muscle cell types, satellite cells, adipocytes, and undefined nervous system cells. The location of these cell types within tendon was defined using spatial transcriptomics and imaging, and potential transcriptional networks and cell-cell interactions were analyzed. We demonstrate that fibroblasts have the highest number of potential cell-cell interactions in our dataset, are present throughout the tendon, and play an important role in the production and organization of extracellular matrix, thus confirming their role as key regulators of hamstring tendon homeostasis. Overall, our findings underscore the complexity of the cellular networks that underpin healthy human tendon function and the central role of fibroblasts as key regulators of hamstring tendon tissue homeostasis., (© 2024 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Voltage-gated sodium channel (Na V ) inhibitors are used to treat neurological disorders of hyperexcitability such as epilepsy. These drugs act by attenuating neuronal action potential firing to reduce excitability in the brain. However, all currently available Na V -targeting antiseizure medications nonselectively inhibit the brain channels Na V 1.1, Na V 1.2, and Na V 1.6, which potentially limits the efficacy and therapeutic safety margins of these drugs. Here, we report on XPC-7724 and XPC-5462, which represent a new class of small molecule Na V -targeting compounds. These compounds specifically target inhibition of the Na V 1.6 and Na V 1.2 channels, which are abundantly expressed in excitatory pyramidal neurons. They have a > 100-fold molecular selectivity against Na V 1.1 channels, which are predominantly expressed in inhibitory neurons. Sparing Na V 1.1 preserves the inhibitory activity in the brain. These compounds bind to and stabilize the inactivated state of the channels thereby reducing the activity of excitatory neurons. They have higher potency, with longer residency times and slower off-rates, than the clinically used antiseizure medications carbamazepine and phenytoin. The neuronal selectivity of these compounds is demonstrated in brain slices by inhibition of firing in cortical excitatory pyramidal neurons, without impacting fast spiking inhibitory interneurons. XPC-5462 also suppresses epileptiform activity in an ex vivo brain slice seizure model, whereas XPC-7224 does not, suggesting a possible requirement of Nav1.2 inhibition in 0-Mg 2+ - or 4-AP-induced brain slice seizure models. The profiles of these compounds will facilitate pharmacological dissection of the physiological roles of Na V 1.2 and Na V 1.6 in neurons and help define the role of specific channels in disease states. This unique selectivity profile provides a new approach to potentially treat disorders of neuronal hyperexcitability by selectively downregulating excitatory circuits.

In precision medicine, there is much interest in estimating the expected-to-benefit (EB) subset, i.e. the subset of patients who are expected to benefit from a new treatment based on a collection of baseline characteristics. There are many statistical methods for estimating the EB subset, most of which produce a 'point estimate' without a confidence statement to address uncertainty. Confidence intervals for the EB subset have been defined only recently, and their construction is a new area for methodological research. This article proposes a pseudo-response approach to EB subset estimation and confidence interval construction. Compared to existing methods, the pseudo-response approach allows us to focus on modelling a conditional treatment effect function (as opposed to the conditional mean outcome given treatment and baseline covariates) and is able to incorporate information from baseline covariates that are not involved in defining the EB subset. Simulation results show that incorporating such covariates can improve estimation efficiency and reduce the size of the confidence interval for the EB subset. The methodology is applied to a randomized clinical trial comparing two drugs for treating HIV infection., Competing Interests: Conflict of interest: Zhiwei Zhang and Calvin J. Cohen are employed by Gilead Sciences. Ping Xu is employed by Janssen. This article expresses the authors’ personal opinions and does not represent the official position of Janssen, Gilead or the National Institutes of Health., (© The Royal Statistical Society 2024. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Divalent metal transporter 1 (DMT1) cotransports ferrous iron and protons and is the primary mechanism for uptake of nonheme iron by enterocytes. Inhibitors are potentially useful as therapeutic agents to treat iron overload disorders such as hereditary hemochromatosis or β -thalassemia intermedia, provided that inhibition can be restricted to the duodenum. We used a calcein quench assay to identify human DMT1 inhibitors. Dimeric compounds were made to generate more potent compounds with low systemic exposure. Direct block of DMT1 was confirmed by voltage clamp measurements. The lead compound, XEN602, strongly inhibits dietary nonheme iron uptake in both rats and pigs yet has negligible systemic exposure. Efficacy is maintained for >2 weeks in a rat subchronic dosing assay. Doses that lowered iron content in the spleen and liver by >50% had no effect on the tissue content of other divalent cations except for cobalt. XEN602 represents a powerful pharmacological tool for understanding the physiologic function of DMT1 in the gut. SIGNIFICANCE STATEMENT: This report introduces methodology to develop potent, gut-restricted inhibitors of divalent metal transporter 1 (DMT1) and identifies XEN602 as a suitable compound for in vivo studies. We also report novel animal models to quantify the inhibition of dietary uptake of iron in both rodents and pigs. This research shows that inhibition of DMT1 is a promising means to treat iron overload disorders., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

Ankylosing spondylitis (AS) is a common, highly heritable inflammatory arthritis characterized by enthesitis of the spine and sacroiliac joints. Genome-wide association studies (GWASs) have revealed more than 100 genetic associations whose functional effects remain largely unresolved. Here, we present a comprehensive transcriptomic and epigenomic map of disease-relevant blood immune cell subsets from AS patients and healthy controls. We find that, while CD14 + monocytes and CD4 + and CD8 + T cells show disease-specific differences at the RNA level, epigenomic differences are only apparent upon multi-omics integration. The latter reveals enrichment at disease-associated loci in monocytes. We link putative functional SNPs to genes using high-resolution Capture-C at 10 loci, including PTGER4 and ETS1 , and show how disease-specific functional genomic data can be integrated with GWASs to enhance therapeutic target discovery. This study combines epigenetic and transcriptional analysis with GWASs to identify disease-relevant cell types and gene regulation of likely pathogenic relevance and prioritize drug targets., Competing Interests: J.M., T.H., D.T., S.L., and M.H.B. are full-time employees of Janssen R&D and own shares of stock in Johnson & Johnson. K.S.E. and A.C.B. are supported by Janssen Oxford Translational Fellowships., (© 2023 The Author(s).)

Ankylosing Spondylitis (AS) is a chronic inflammatory arthritis of the spine exhibiting a strong genetic background. The mechanistic and functional understanding of the AS-associated genomic loci, identified with Genome Wide Association Studies (GWAS), remains challenging. Chromosome conformation capture (3C) and derivatives are recent techniques which are of great help in elucidating the spatial genome organization and of enormous support in uncover a mechanistic explanation for disease-associated genetic variants. The perturbation of three-dimensional (3D) genome hierarchy may lead to a plethora of human diseases, including rheumatological disorders. Here we illustrate the latest approaches and related findings on the field of genome organization, highlighting how the instability of 3D genome conformation may be among the causes of rheumatological disease phenotypes. We suggest a new perspective on the inclusive potential of a 3C approach to inform GWAS results in rheumatic diseases. 3D genome organization may ultimately lead to a more precise and comprehensive functional interpretation of AS association, which is the starting point for emerging and more specific therapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Davidson, Wordsworth, Cohen, Knight and Vecellio.)

Purpose: AL amyloidosis (AL) treatments are generally based on those employed for multiple myeloma. Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor T (CART)-cell therapy, already approved for multiple myeloma, might be too toxic for patients with AL., Experimental Design: Here we describe the ex vivo applicability of a novel in-house, academic anti-BCMA CAR construct on AL primary cells, as well as the safety and efficacy in 4 patients with relapsed/refractory (RR) primary AL, treated in a phase I clinical trial (NCT04720313)., Results: Three had MAYO stage IIIa cardiac involvement at enrollment. The treatment proved relatively safe, with a short and manageable grade 3 cytokine release syndrome evident in 2 patients and no neurotoxicity in any. Cardiac decompensations, observed in 2 patients, were also short and manageable. The overall hematologic response and complete response rates were observed in all patients with an organ response evident in all four. Within a median follow-up period of 5.2 (2.5-9.5) months, all 4 patients maintained their responses., Conclusions: BCMA-CART cells provide a first proof-of-concept that this therapy is safe enough and highly efficacious for the treatment of patients with advanced, RR AL., (©2022 American Association for Cancer Research.)

Genetically engineered T cells are a powerful new modality for cancer immunotherapy. However, their clinical application for solid tumors is challenging, and crucial knowledge on cell functionality in vivo is lacking. Here, we fabricated a nanoprobe composed of dendrimers incorporating a calcium sensor and gold nanoparticles, for dual-modal monitoring of engineered T cells within a solid tumor. T cells engineered to express a melanoma-specific T-cell receptor and loaded with the nanoprobe were longitudinally monitored within melanoma xenografts in mice. Fluorescent imaging of the nanoprobe's calcium sensor revealed increased intra-tumoral activation of the T cells over time, up to 24 h. Computed tomography imaging of the nanoprobe's gold nanoparticles revealed the cells' intra-tumoral distribution pattern. Quantitative analysis revealed the intra-tumoral T cell quantities. Thus, this nanoprobe reveals intra-tumoral persistence, penetration and functional status of genetically engineered T cells, which can advance T cell-based immunotherapy and promote next-generation live cell imaging., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Chimeric antigen receptor (CAR) T-cell based immunotherapy has become a promising treatment mainly for hematological malignancies. Following the major success of CD19-targeted CAR, new potential targets for other malignancies are required. As such, B-cell maturation antigen (BCMA) is an attractive tumor-associated antigen to be targeted in multiple myeloma (MM). Herein, we aimed at assessing the function and optimal configuration of different BCMA-specific CAR, based on the same targeting moiety but with a different hinge and co-stimulatory domain. We compared their function to that of a previously characterized BCMA-CAR used in clinical trials. All constructs were expressed at high levels by primary human T cells and could trigger cytokine production and cytotoxicity upon co-culture with multiple myeloma targets. Nonetheless, critical differences were observed in off-target activation, exhaustion, and activation marker expression and in vivo antitumoral activity mediated by these different constructs. Interestingly, we noted that CD8-based hinge, combined with a 4-1BB intracellular domain, proved superior compared to IgG4-connecting regions, and/or a CD28-signaling moiety respectively. Overall, this study emphasizes the influence of CAR primary structure on its function and led to the identification of a highly efficient BCMA-specific CAR, namely H8BB, which displayed superior anti-tumoral activity both in vitro and long-term in vivo efficacy.

NBI-921352 (formerly XEN901) is a novel sodium channel inhibitor designed to specifically target Na V 1.6 channels. Such a molecule provides a precision-medicine approach to target SCN8A -related epilepsy syndromes ( SCN8A -RES), where gain-of-function (GoF) mutations lead to excess Na V 1.6 sodium current, or other indications where Na V 1.6 mediated hyper-excitability contributes to disease (Gardella and Møller, 2019; Johannesen et al., 2019; Veeramah et al., 2012). NBI-921352 is a potent inhibitor of Na V 1.6 (IC 50 0.051 µM), with exquisite selectivity over other sodium channel isoforms (selectivity ratios of 756 X for Na V 1.1, 134 X for Na V 1.2, 276 X for Na V 1.7, and >583 Xfor Na V 1.3, Na V 1.4, and Na V 1.5). NBI-921352is a state-dependent inhibitor, preferentially inhibiting inactivatedchannels. The state dependence leads to potent stabilization of inactivation, inhibiting Na V 1.6 currents, including resurgent and persistent Na V 1.6 currents, while sparing the closed/rested channels. The isoform-selective profile of NBI-921352 led to a robust inhibition of action-potential firing in glutamatergic excitatory pyramidal neurons, while sparing fast-spiking inhibitory interneurons, where Na V 1.1 predominates. Oral administration of NBI-921352 prevented electrically induced seizures in a Scn8a GoF mouse,as well as in wild-type mouse and ratseizure models. NBI-921352 was effective in preventing seizures at lower brain and plasma concentrations than commonly prescribed sodium channel inhibitor anti-seizure medicines (ASMs) carbamazepine, phenytoin, and lacosamide. NBI-921352 waswell tolerated at higher multiples of the effective plasma and brain concentrations than those ASMs. NBI-921352 is entering phase II proof-of-concept trials for the treatment of SCN8A- developmental epileptic encephalopathy ( SCN8A -DEE) and adult focal-onset seizures., Competing Interests: JJ, TF, KK, CD, SG, JA, GB, KB, EC, SC, RD, Gd, SD, CD, MF, WG, MG, AH, AH, QJ, SL, JL, SL, AL, VL, JM, RN, KN, NS, LS, SS, MT, MW, DW, SW, AW, MW, ZX, RY, CY, AZ, WZ, AC, RS, SP, RW, CC, JE is employed by Xenon Pharmaceuticals, Inc and may hold equity in Xenon Pharmaceuticals, Inc, PT, DB was previously employed by Xenon Pharmaceuticals, Inc and may hold equity in Xenon Pharmaceuticals, Inc, (© 2022, Johnson et al.)

Background: Ankylosing Spondylitis (AS) is a common form of inflammatory spinal arthritis with a complex aetiology and high heritability, involving more than 100 genetic associations. These include several AS-associated single nucleotide polymorphisms (SNPs) upstream of RUNX3, which encodes the multifunctional RUNT-related transcription factor (TF) 3. The lead associated SNP rs6600247 ( p = 2.6 × 10 -15 ) lies ∼13kb upstream of the RUNX3 promoter adjacent to a c-MYC TF binding-site. The effect of rs6600247 genotype on DNA binding and chromosome looping were investigated by electrophoretic mobility gel shift assays (EMSA), Western blotting-EMSA (WEMSA) and Chromosome Conformation Capture (3C). Results: Interrogation of ENCODE published data showed open chromatin in the region overlapping rs6600247 in primary human CD14 + monocytes, in contrast to the Jurkat T cell line or primary human T-cells. The rs6600247 AS-risk allele is predicted to specifically disrupt a c-MYC binding-site. Using a 50bp DNA probe spanning rs6600247 we consistently observed reduced binding to the AS-risk "C" allele of both purified c-MYC protein and nuclear extracts (NE) from monocyte-like U937 cells. WEMSA on U937 NE and purified c-MYC protein confirmed these differences ( n = 3; p < 0.05). 3C experiments demonstrated negligible interaction between the region encompassing rs6600247 and the RUNX3 promoter. A stronger interaction frequency was demonstrated between the RUNX3 promoter and the previously characterised AS-associated SNP rs4648889 . Conclusion: The lead SNP rs6600247, located in an enhancer-like region upstream of the RUNX3 promoter, modulates c-MYC binding. However, the region encompassing rs6600247 has rather limited physical interaction with the promoter of RUNX3 . In contrast a clear chromatin looping event between the region encompassing rs4648889 and the RUNX3 promoter was observed. These data provide further evidence for complexity in the regulatory elements upstream of the RUNX3 promoter and the involvement of RUNX3 transcriptional regulation in AS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cohen, Davidson, Selmi, Bowness, Knight, Wordsworth and Vecellio.)

As an important part of the immune system, T lymphocytes exhibit undoubtedly an important role in targeting and eradicating cancer. However, despite these characteristics, their natural antitumor response may be insufficient. Numerous clinical trials in terminally ill cancer patients testing the design of novel and efficient immunotherapeutic approaches based on the adoptive transfer of autologous tumor-specific T lymphocytes have shown encouraging results. Moreover, this also led to the approval of engineered T-cell therapies in patients. Herein, we will expand on the development and the use of such strategies using tumor-infiltrating lymphocytes or genetically engineered T-cells. We will also comment on the requirements and potential hurdles encountered when elaborating and implementing such treatments as well as the exciting prospects for this kind of emerging personalized medicine therapy., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Natural killer (NK)-cell-based immunotherapy is emerging as an attractive approach for cancer treatment. However, to facilitate and expedite clinical implementation, important questions must be answered regarding the in vivo functionality and trafficking patterns of the transferred cells. We have recently developed a noninvasive cell-tracking technique, based on gold nanoparticles (GNPs) as cell-labeling and contrast agents for whole-body computed tomography (CT) imaging. Herein, we report the implementation of this technique for longitudinal and quantitative tracking of NK cell kinetics, the migration and biodistribution in tumor-bearing mice. NK cells were successfully labeled with GNPs, without impairing their biological function, as assessed both in vitro , by cytokine release and cytotoxicity assays, and in vivo , using a xenograft model of human tumors. Using CT, we longitudinally tracked the migration of intravenously injected NK cells and observed an accumulation of effector cell clusters at the tumor site, up to 72 h. Fluorescence imaging of the cells over time correlated with ex vivo quantitative analysis of gold content in the tumor, validating the accuracy and reliability of our technique. Our cell-tracking approach thus offers a valuable tool for preclinical studies, as well as for clinical applications, to elucidate the fate of NK cells and promote the implementation of NK-cell-based immunotherapy., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Neoantigens are now recognized drivers of the antitumor immune response. Recurrent neoantigens, shared among groups of patients, have thus become increasingly coveted therapeutic targets. Here, we report on the data-driven identification of a robustly presented, immunogenic neoantigen that is derived from the combination of HLA-A*01:01 and RAS.Q61K. Analysis of large patient cohorts indicated that this combination applies to 3% of patients with melanoma. Using HLA peptidomics, we were able to demonstrate robust endogenous presentation of the neoantigen in 10 tumor samples. We detected specific reactivity to the mutated peptide within tumor-infiltrating lymphocytes (TILs) from 2 unrelated patients, thus confirming its natural immunogenicity. We further investigated the neoantigen-specific clones and their T cell receptors (TCRs) via a combination of TCR sequencing, TCR overexpression, functional assays, and single-cell transcriptomics. Our analysis revealed a diverse repertoire of neoantigen-specific clones with both intra- and interpatient TCR similarities. Moreover, 1 dominant clone proved to cross-react with the highly prevalent RAS.Q61R variant. Transcriptome analysis revealed a high association of TCR clones with specific T cell phenotypes in response to cognate melanoma, with neoantigen-specific cells showing an activated and dysfunctional phenotype. Identification of recurrent neoantigens and their reactive TCRs can promote "off-the-shelf" precision immunotherapies, alleviating limitations of personalized treatments.

CD45, the predominant transmembrane tyrosine phosphatase in leukocytes, is required for the efficient induction of T cell receptor signaling and activation. We recently reported that the CD45-intracellular signals in peripheral blood mononuclear cells (PBMCs) of triple negative breast cancer (TNBC) patients are inhibited. We also reported that C24D, an immune modulating therapeutic peptide, binds to CD45 on immune-suppressed cells and resets the functionality of the immune system via the CD45 signaling pathway. Various studies have demonstrated that also viruses can interfere with the functions of CD45 and that patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are immune-suppressed. Given the similarity between the role of CD45 in viral immune suppression and our findings on TNBC, we hypothesized that the C24D peptide may have a similar "immune-resetting" effect on PBMCs from COVID-19 patients as it did on PBMCs from TNBC patients. We tested this hypothesis by comparing the CD45/TCR intracellular signaling in PBMCs from ten COVID-19 patients vs. PBMCs from ten healthy volunteers. Herein, we report our findings, demonstrating the immune reactivating effect of C24D via the phosphorylation of the tyrosine 505 and 394 in Lck, the tyrosine 493 in ZAP-70 and the tyrosine 172 in VAV-1 proteins in the CD45 signaling pathway. Despite the relatively small number of patients in this report, the results demonstrate that C24D rescued CD45 signaling. Given the central role played by CD45 in the immune system, we suggest CD45 as a potential therapeutic target., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer OZ declared a past collaboration with several of the authors JL, AR, and RY to the handling editor., (Copyright © 2021 Alon, Paitan, Robinson, Ganor, Lipovetsky, Yerushalmi, Cohen and Raiter.)

We describe the synthesis and biological evaluation of a series of novel aryl sulfonamides that exhibit potent inhibition of Na V 1.5. Unlike local anesthetics that are currently used for treatment of Long QT Syndrome 3 (LQT-3), the most potent compound (-)-6 in this series shows high selectivity over hERG and other cardiac ion channels and has a low brain to plasma ratio to minimize CNS side effects. Compound (-)-6 is also effective inshortening prolonged action potential durations (APDs) in a pharmacological model of LQT-3 syndrome in pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Unlike most aryl sulfonamide Na V inhibitors that bind to the channel voltage sensors, these Na V 1.5 inhibitors bind to the local anesthetic binding site in the central pore of the channel., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objective: To investigate the functional consequences of the single-nucleotide polymorphism rs4648889 in a putative enhancer upstream of the RUNX3 promoter associated with susceptibility to ankylosing spondylitis (AS)., Methods: Using nuclear extracts from Jurkat cells and primary human CD8+ T cells, the effects of rs4648889 on allele-specific transcription factor (TF) binding were investigated by DNA pull-down assay and quantitative mass spectrometry (qMS), with validation by electrophoretic mobility shift assay (EMSA), Western blotting of the pulled-down eluates, and chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) analysis. Further functional effects were tested by small interfering RNA knockdown of the gene for interferon regulatory factor 5 (IRF5), followed by reverse transcription-qPCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) to measure the levels of IFNγ messenger RNA (mRNA) and protein, respectively., Results: In nuclear extracts from CD8+ T cells, results of qMS showed that relative TF binding to the AS-risk A allele of rs4648889 was increased 3.7-fold (P < 0.03) for Ikaros family zinc-finger protein 3 (IKZF3; Aiolos) and components of the NuRD complex, including chromodomain helicase DNA binding protein 4 (CHD4) (3.6-fold increase; P < 0.05) and retinoblastoma binding protein 4 (RBBP4) (4.1-fold increase; P < 0.03). In contrast, IRF5 bound significantly more to the AS-protective G allele compared to the AS-risk A allele (fold change 8.2; P = 0.003). Validation with Western blotting, EMSA, and ChIP-qPCR confirmed the differential allelic binding of IKZF3, CHD4, RBBP4, and IRF5. Silencing of IRF5 in CD8+ T cells increased the levels of IFNγ mRNA as measured by RT-qPCR (P = 0.03) and IFNγ protein as measured by ELISA (P = 0.02)., Conclusion: These findings suggest that the association of rs4648889 with AS reflects allele-specific binding of this enhancer-like region to certain TFs, including IRF5, IKZF3, and members of the NuRD complex. IRF5 may have crucial influences on the functions of CD8+ lymphocytes, a finding that could reveal new therapeutic targets for the management of AS., (© 2020 The Authors. Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

Breast cancer subtypes have not shown significant response to current immunomodulatory therapies. Although most subtypes are treatable, triple negative breast cancer (TNBC), an aggressive highly metastatic cancer, comprising 10-20% of breast cancers, remains an unmet medical need. New strategies are needed in order to overcome flaws in the responsiveness to current TNBC therapies. Our aims were: first, to determine the efficacy of a novel immunomodulatory peptide, C24D, on TNBC and second, to elucidate the molecular mechanism by which C24D induces immune-modulating tumor killing. Using mass spectrometry analysis, we identified CD45 as the C24D binding receptor. In vitro and in vivo TNBC models were used to assess the efficacy of C24D in reversing TNBC-induced immunosuppression and in triggering immune-modulated tumor cell killing. The CD45 signal transduction pathway was evaluated by western blot and FACS analyses. We revealed that addition of PBMCs from healthy female donors to TNBC cells results in a cascade of suppressive CD45 intracellular signals. On binding to CD45's extra-cellular domain on TNBC-suppressed leukocytes, the C24D peptide re-activates the Src family of tyrosine kinases, resulting in specific tumor immune response. In vitro , immune reactivation by C24D results in an increase of CD69+ T and CD69+ NK cells, triggering specific killing of TNBC cells. In vivo , C24D induced CD8+ and activated CD56+ tumor infiltrated cells, resulting in tumor apoptosis. Our results should renew interest in molecules targeting CD45, such as the C24D peptide, as a novel strategy for TNBC immunotherapy., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)