Miniaturized CAR knocked onto CD3ε extends TCR function with CAR specificity under control of endogenous TCR signaling cascade.

Immunotherapy using TCR and especially CAR transgenic T cells is a rapidly advancing field with the potential to become standard of care for the treatment of multiple diseases. While all current FDA approved CAR T cell products are generated using lentiviral gene transfer, extensive work is put into CRISPR/Cas mediated gene delivery to develop the next generation of safer and more potent cell products. One limitation of all editing systems is the size restriction of the knock-in cargo. Targeted integration under control of an endogenous promotor and/or signaling cascades opens the possibility to reduce CAR gene size to absolute minimum. Here we demonstrate that a first-generation CAR payload can be reduced to its minimum component - the antigen-binding domain - by targeted integration under control of the CD3ε promoter generating a CAR-CD3ε fusion protein that exploits the endogenous TCR signaling cascade. Miniaturizing CAR payload in this way results in potent CAR activity while simultaneously retaining the primary antigen recognition function of the TCR. Introducing CAR-specificity using a CAR binder only while maintaining endogenous TCR function may be an appealing design for future autologous CAR T cell therapies.

Next generation automated traceless cell chromatography platform for GMP-compliant cell isolation and activation.

Large-scale target cell isolation from patient blood preparations is one of the critical operations during drug product manufacturing for personalized cell therapy in immuno-oncology. Use of high-affinity murine antibody coated magnetic nanoparticles that remain on isolated cells is the current standard applied for this purpose. Here, we present the transformation of previously described technology - non-magnetic immunoaffinity column chromatography-based cell selection with reversible reagents into a new clinical-grade cell isolation platform called Automated Traceless Cell affinity chromatography (ATC). ATC is a fully closed and GMP-compliant cell selection and manufacturing system. Reversibility of reagents enables (sequential) positive cell selection, optionally in combination with depletion columns, enabling capture of highly specific cell subsets. Moreover, synergy with other Streptamer-based technologies allows novel uses beyond cell isolation including integrated and automated on-column target cell activation. In conclusion, ATC technology is an innovative as well as versatile platform to select, stimulate and modify cells for clinical manufacturing and downstream therapies.

Interleukin-12/23 deficiency differentially affects pathology in male and female Alzheimer's disease-like mice.

Pathological aggregation of amyloid-ß (Aß) is a main hallmark of Alzheimer's disease (AD). Recent genetic association studies have linked innate immune system actions to AD development, and current evidence suggests profound gender differences in AD pathogenesis. Here, we characterise gender-specific pathologies in the APP23 AD-like mouse model and find that female mice show stronger amyloidosis and astrogliosis compared with male mice. We tested the gender-specific effect of lack of IL12p40, the shared subunit of interleukin (IL)-12 and IL-23, that we previously reported to ameliorate pathology in APPPS1 mice. IL12p40 deficiency gender specifically reduces Aß plaque burden in male APP23 mice, while in female mice, a significant reduction in soluble Aß1-40 without changes in Aß plaque burden is seen. Similarly, plasma and brain cytokine levels are altered differently in female versus male APP23 mice lacking IL12p40, while glial properties are unchanged. These data corroborate the therapeutic potential of targeting IL-12/IL-23 signalling in AD, but also highlight the importance of gender considerations when studying the role of the immune system and AD.

Arabidopsis FORGETTER1 mediates stress-induced chromatin memory through nucleosome remodeling.

Plants as sessile organisms can adapt to environmental stress to mitigate its adverse effects. As part of such adaptation they maintain an active memory of heat stress for several days that promotes a more efficient response to recurring stress. We show that this heat stress memory requires the activity of the FORGETTER1 (FGT1) locus, with fgt1 mutants displaying reduced maintenance of heat-induced gene expression. FGT1 encodes the Arabidopsis thaliana orthologue of Strawberry notch (Sno), and the protein globally associates with the promoter regions of actively expressed genes in a heat-dependent fashion. FGT1 interacts with chromatin remodelers of the SWI/SNF and ISWI families, which also display reduced heat stress memory. Genomic targets of the BRM remodeler overlap significantly with FGT1 targets. Accordingly, nucleosome dynamics at loci with altered maintenance of heat-induced expression are affected in fgt1. Together, our results suggest that by modulating nucleosome occupancy, FGT1 mediates stress-induced chromatin memory.

eQTL mapping of transposon silencing reveals a position-dependent stable escape from epigenetic silencing and transposition of AtMu1 in the Arabidopsis lineage.

Transposons are massively abundant in all eukaryotic genomes and are suppressed by epigenetic silencing. Transposon activity contributes to the evolution of species; however, it is unclear how much transposition-induced variation exists at a smaller scale and how transposons are targeted for silencing. Here, we exploited differential silencing of the AtMu1c transposon in the Arabidopsis thaliana accessions Columbia (Col) and Landsberg erecta (Ler). The difference persisted in hybrids and recombinant inbred lines and was mapped to a single expression quantitative trait locus within a 20-kb interval. In Ler only, this interval contained a previously unidentified copy of AtMu1c, which was inserted at the 3' end of a protein-coding gene and showed features of expressed genes. By contrast, AtMu1c(Col) was intergenic and associated with heterochromatic features. Furthermore, we identified widespread natural AtMu1c transposition from the analysis of over 200 accessions, which was not evident from alignments to the reference genome. AtMu1c expression was highest for insertions within 3' untranslated regions, suggesting that this location provides protection from silencing. Taken together, our results provide a species-wide view of the activity of one transposable element at unprecedented resolution, showing that AtMu1c transposed in the Arabidopsis lineage and that transposons can escape epigenetic silencing by inserting into specific genomic locations, such as the 3' end of genes.

Distinct signal transduction pathways downstream of the (P)RR revealed by microarray and ChIP-chip analyses.

The (pro)renin receptor ((P)RR) signaling is involved in different pathophysiologies ranging from cardiorenal end-organ damage via diabetic retinopathy to tumorigenesis. We have previously shown that the transcription factor promyelocytic leukemia zinc finger (PLZF) is an adaptor protein of the (P)RR. Furthermore, recent publications suggest that major functions of the (P)RR are mediated ligand-independently by its transmembrane and intracellular part, which acts as an accessory protein of V-ATPases. The transcriptome and recruitmentome downstream of the V-ATPase function and PLZF in the context of the (P)RR are currently unknown. Therefore, we performed a set of microarray and chromatin-immunoprecipitation (ChIP)-chip experiments using siRNA against the (P)RR, stable overexpression of PLZF, the PLZF translocation inhibitor genistein and the specific V-ATPase inhibitor bafilomycin to dissect transcriptional pathways downstream of the (P)RR. We were able to identify distinct and overlapping genetic signatures as well as novel real-time PCR-validated target genes of the different molecular functions of the (P)RR. Moreover, bioinformatic analyses of our data confirm the role of (P)RRs signal transduction pathways in cardiovascular disease and tumorigenesis.