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Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T cells have shown promising clinical responses in patients with relapsed/refractory multiple myeloma. Lenalidomide, an immunomodulatory drug, potentiates T cell functionality, drives antimyeloma activity, and alters the suppressive microenvironment; these properties may effectively combine with anti-BCMA CAR T cells to enhance function. Using an anti-BCMA CAR T, we demonstrated that lenalidomide enhances CAR T cell function in a concentration-dependent manner. Lenalidomide increased CAR T effector cytokine production, particularly under low CAR stimulation or in the presence of inhibitory ligand programmed cell death 1 ligand 1. Notably, lenalidomide also enhanced CAR T cytokine production, cytolytic activity, and activation profile relative to untreated CAR T cells in chronic stimulation assays. This unique potentiation of both short-term CAR T activity and long-term functionality during chronic stimulation prompted investigation of the molecular profile of lenalidomide-treated CAR T cells. Signatures from RNA sequencing and assay for transposase-accessible chromatin using sequencing indicated that pathways associated with T-helper 1 response, cytokine production, T cell activation, cell-cycle control, and cytoskeletal remodeling were altered with lenalidomide. Finally, study of lenalidomide and anti-BCMA CAR T cells in a murine, disseminated, multiple myeloma model indicated that lenalidomide increased CAR T cell counts in blood and significantly prolonged animal survival. In summary, preclinical studies demonstrated that lenalidomide potentiated CAR T activity in vivo in low-antigen or suppressive environments and delayed onset of functional exhaustion. These results support further investigation of lenalidomide and anti-BCMA CAR T cells in the clinic.

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Duchenne muscular dystrophy (DMD) is a devastating muscle-wasting disease caused by mutations in the dystrophin gene. Utrophin is a homologue of dystrophin that can compensate for its absence when overexpressed in DMD animal models. Utrophin upregulation is therefore a promising therapeutic approach for DMD. Utrophin is regulated at both transcriptional and posttranscriptional levels. Transcriptional regulation has been studied extensively, and assays have been described for the identification of utrophin promoter-targeting molecules. However, despite the profound impact that posttranscriptional regulation has on utrophin expression, screening assays have not yet been described that could be used to discover pharmaceuticals targeting this key phase of regulation. We describe the development and validation of a muscle cell linebased assay in which a stably expressed luciferase coding sequence is flanked by the utrophin 5′-and 3′-untranslated regions (UTRs). The assay was validated using the posttranscriptional regulation of utrophin by miR-206. The assay has a Z′ of 0.7, indicating robust performance in high-throughput format. This assay can be used to study utrophin regulatory mechanisms or to screen chemical libraries for compounds that upregulate utrophin posttranscriptionally via its UTRs. Compounds identified via this assay, used alone or in a synergistic combination with utrophin promoter-targeting molecules, would be predicted to have therapeutic potential for DMD.

BackgroundDiffuse traumatic brain injury (TBI) is known to lead to microstructural changes within both white and grey matter detected in vivo with diffusion tensor imaging (DTI). Numerous studies have shown alterations in fractional anisotropy (FA) and mean diffusivity (MD) within prominent white matter tracts, but few have linked these to changes within the grey matter with confirmation via histological assessment. This is especially important as alterations in the grey matter may be predictive of long-term functional deficits.MethodsA total of 33 male Sprague Dawley rats underwent severe closed-head TBI. Eight animals underwent tensor-based morphometry (TBM) and DTI at baseline (pre-TBI), 24 hours (24 h), 7, 14, and 30 days post-TBI. Immunohistochemical analysis for the detection of ionised calcium-binding adaptor molecule 1 (IBA1) to assess microglia number and percentage of activated cells, β-amyloid precursor protein (APP) as a marker of axonal injury, and myelin basic protein (MBP) to investigate myelination was performed at each time-point.ResultsDTI showed significant alterations in FA and RD in numerous white matter tracts including the corpus callosum, internal and external capsule, and optic tract and in the grey-matter in the cortex, thalamus, and hippocampus, with the most significant effects observed at 14 D post-TBI. TBM confirmed volumetric changes within the hippocampus and thalamus. Changes in DTI were in line with significant axonal injury noted at 24 h post-injury via immunohistochemical analysis of APP, with widespread microglial activation seen within prominent white matter tracts and the grey matter, which persisted to 30 D within the hippocampus and thalamus. Microstructural alterations in MBP+ve fibres were also noted within the hippocampus and thalamus, as well as the cortex.ConclusionThis study confirms the widespread effects of diffuse TBI on white matter tracts which could be detected via DTI and extends these findings to key grey matter regions, with a comprehensive investigation of the whole brain. In particular, the hippocampus and thalamus appear to be vulnerable to ongoing pathology post-TBI, with DTI able to detect these alterations supporting the clinical utility in evaluating these regions post-TBI.