LCMV induced downregulation of HVEM on antiviral T cells is critical for an efficient effector response.

T-cell responses against tumors and pathogens are critically shaped by cosignaling molecules providing a second signal. Interaction of herpes virus entry mediator (HVEM, CD270, TNFRSF14) with multiple ligands has been proposed to promote or inhibit T-cell responses and inflammation, dependent on the context. In this study, we show that absence of HVEM did neither affect generation of effector nor maintenance of memory antiviral T cells and accordingly viral clearance upon acute and chronic lymphocytic choriomeningitis virus (LCMV) infection, due to potent HVEM downregulation during infection. Notably, overexpression of HVEM on virus-specific CD8+ T cells resulted in a reduction of effector cells, whereas numbers of memory cells were increased. Overall, this study indicates that downregulation of HVEM driven by LCMV infection ensures an efficient acute response at the price of impaired formation of T-cell memory.

PPARγ is essential for the development of bone marrow erythroblastic island macrophages and splenic red pulp macrophages.

Tissue-resident macrophages play a crucial role in maintaining homeostasis. Macrophage progenitors migrate to tissues perinatally, where environmental cues shape their identity and unique functions. Here, we show that the absence of PPARγ affects neonatal development and VCAM-1 expression of splenic iron-recycling red pulp macrophages (RPMs) and bone marrow erythroblastic island macrophages (EIMs). Transcriptome analysis of the few remaining Pparg-deficient RPM-like and EIM-like cells suggests that PPARγ is required for RPM and EIM identity, cell cycling, migration, and localization, but not function in mature RPMs. Notably, Spi-C, another transcription factor implicated in RPM development, was not essential for neonatal expansion of RPMs, even though the transcriptome of Spic-deficient RPMs was strongly affected and indicated a loss of identity. Similarities shared by Pparg- and Spic-deficient RPM-like cells allowed us to identify pathways that rely on both factors. PPARγ and Spi-C collaborate in inducing transcriptional changes, including VCAM-1 and integrin αD expression, which could be required for progenitor retention in the tissue, allowing access to niche-related signals that finalize differentiation.

Preclinical Development of 18F-OF-NB1 for Imaging GluN2B-Containing N-Methyl-d-Aspartate Receptors and Its Utility as a Biomarker for Amyotrophic Lateral Sclerosis.

As part of our continuous efforts to develop a suitable 18F-labeled PET radioligand with improved characteristics for imaging the N-methyl-d-aspartate receptors (NMDARs) subtype 2B (GluN1/2B), we investigated in the current work ortho-fluorinated (OF) and meta-fluorinated (MF) analogs of 18F-para-fluorinated (PF)-NB1, a 3-benzazepine-based radiofluorinated probe. Methods: OF-NB1 and MF-NB1 were prepared using a multistep synthesis, and their binding affinities toward GluN2B subunits and selectivity over σ1 receptors (σ1Rs) were determined via competitive binding assays. 18F-OF-NB1 was synthesized via copper-mediated radiofluorination and was evaluated in Wistar rats by in vitro autoradiography, PET imaging, ex vivo biodistribution, metabolite experiments, and receptor occupancy studies using CP-101,606, an established GluN2B antagonist. To determine in vivo selectivity, 18F-OF-NB1 was validated in wild-type and σ1R knock-out mice. Translational relevance was assessed in autoradiographic studies using postmortem human brain tissues from healthy individuals and ALS patients, the results of which were corroborated by immunohistochemistry. Results: The binding affinity values for OF-NB1 and MF-NB1 toward the GluN2B subunits were 10.4 ± 4.7 and 590 ± 36 nM, respectively. For σ1R binding, OF-NB1 and MF-NB1 exhibited inhibition constants of 410 and 2,700 nM, respectively. OF-NB1, which outperformed MF-NB1, was radiolabeled with 18F to afford 18F-OF-NB1 in more than 95% radiochemical purity and molar activities of 192 ± 33 GBq/µmol. In autoradiography experiments, 18F-OF-NB1 displayed a heterogeneous and specific binding in GluN2B subunit-rich brain regions such as the cortex, striatum, hypothalamus, and hippocampus. PET imaging studies in Wistar rats showed a similar heterogeneous uptake, and no brain radiometabolites were detected. A dose-dependent blocking effect was observed with CP-101,606 (0.5-15 mg/kg) and resulted in a 50% receptor occupancy of 8.1 µmol/kg. Postmortem autoradiography results revealed lower expression of the GluN2B subunits in ALS brain tissue sections than in healthy controls, in line with immunohistochemistry results. Conclusion:18F-OF-NB1 is a highly promising PET probe for imaging the GluN2B subunits of the N-methyl-d-aspartate receptor. It possesses utility for receptor occupancy studies and has potential for PET imaging studies in ALS patients and possibly other brain disorders.

Structure-Affinity Relationships of 2,3,4,5-Tetrahydro-1H-3-benzazepine and 6,7,8,9-Tetrahydro-5H-benzo[7]annulen-7-amine Analogues and the Discovery of a Radiofluorinated 2,3,4,5-Tetrahydro-1H-3-benzazepine Congener for Imaging GluN2B Subunit-Containing N-Methyl-d-aspartate Receptors.

Aspiring to develop a positron emission tomography (PET) imaging agent for the GluN2B subunits of the N-methyl-d-aspartate receptor (NMDAR), a key therapeutic target for drug development toward several neurological disorders, we synthesized a series of 2,3,4,5-tetrahydro-1H-3-benzazepine and 6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-amine analogues. After in vitro testing via competition binding assay and autoradiography, [18F]PF-NB1 emerged as the best performing tracer with respect to specificity and selectivity over σ1 and σ2 receptors and was thus selected for further in vivo evaluation. Copper-mediated radiofluorination was accomplished in good radiochemical yields and high molar activities. Extensive in vivo characterization was performed in Wistar rats comprising PET imaging, biodistribution, receptor occupancy, and metabolites studies. [18F]PF-NB1 binding was selective to GluN2B-rich forebrain regions and was specifically blocked by the GluN2B antagonist, CP-101,606, in a dose-dependent manner with no brain radiometabolites. [18F]PF-NB1 is a promising fluorine-18 PET tracer for imaging the GluN2B subunits of the NMDAR and has utility for receptor occupancy studies.

Identification and Preclinical Evaluation of a Radiofluorinated Benzazepine Derivative for Imaging the GluN2B Subunit of the Ionotropic NMDA Receptor.

The previously reported carbon-11 labeled GluN2B PET radioligand 11C-Me-NB1 served as a starting point for derivatization and led to the successful development of a radiofluorinated analogue designated (R)-18F-OF-Me-NB1. Given the short physical half-life of 20.3 min for carbon-11, (R)-18F-OF-Me-NB1 with a physical half-life of 109.8 min would allow satellite distribution to nuclear medicine facilities without an on-site cyclotron. Methods: Two fluorinated Me-NB1 derivatives, OF-Me-NB1 and PF-Me-NB1, were synthesized. Upon chiral resolution, the respective enantiomers were radiolabeled with carbon-11 and assessed in a proof-of-concept study by applying in vitro autoradiography on rodent brain sections. Based on the autoradiograms, (R)-OF-Me-NB1 was selected for radiofluorination and preclinical evaluation by ex vivo autoradiography, PET imaging, biodistribution and metabolite studies in Wistar rats. To rule out off-target binding to the σ1 receptor, the brain uptake of (R)-18F-OF-Me-NB1 in wild-type mice was compared with σ1 receptor knock-out mice. Results: Autoradiographic assessment revealed that both enantiomers of 11C-PF-Me-NB1 distributed homogenously across all brain regions on rodent brain sections. In contrast, the two enantiomers of 11C-OF-Me-NB1 exhibited an entirely different behaviour. While (S)-11C-OF-Me-NB1 bound virtually to all brain regions with considerable σ1 receptor binding, (R)-11C-OF-Me-NB1 exhibited high selectivity and specificity for the GluN2B-rich rat forebrain. These findings were confirmed for the radiofluorinated analogue (R)-11C-OF-Me-NB1, which was obtained via copper-mediated radiofluorination in radiochemical yields of 13-25% and molar activities ranging from 61-168 GBq/µmol. PET imaging and biodistribution studies in Wistar rats indicated appropriate pharmacokinetic profile and high in vivo specific binding of (R)-18F-OF-Me-NB1 as revealed by blocking studies with GluN2B-antagonist CP101,606. Off-target binding to the σ1 receptor was excluded by PET imaging with σ1 receptor knock-out mice. Receptor occupancy experiments with CP101,606 revealed a D50-value of 8.3 µmol/kg (intravenous). Conclusion: (R)-18F-OF-Me-NB1 is a promising radiofluorinated probe that exhibits specificity and selectivity for the GluN2B-containing N-methyl-D-aspartate (NMDA) complex and enables in vivo target occupancy studies in rodents.