Biology subjects

Mikkelsen, J. G.

Publications and source records attributed to Mikkelsen, J. G..

5 recordsLinked to original sources

bioRxiv2026

A minimal two-residue C-terminal truncation of AID in adult-onset hyper-IgM syndrome type 2 (HIGM2)

Activation-induced cytidine deaminase (AID), encoded by AICDA, facilitates class-switch recombination (CSR) and somatic hypermutation (SHM) in activated B cells. Loss of AID function causes hyper-IgM syndrome type 2 (HIGM2). While typically autosomal recessive, three C-terminal truncation variants have been reported to act in autosomal dominant manner. We describe a novel heterozygous AICDA nonsense variant, c.589G>T (p.G197*), that truncates the final two C-terminal residues of AID and results in adult-onset HIGM2. Single-cell sequencing of primary patient B cells revealed a complete block in CSR and a [~]10-fold reduction in SHM. AICDA mRNA expression was balanced between wild-type and variant alleles, yet AID protein abundance was reduced to [~]56% with normal subcellular localization. Remarkably, inducible germinal center (iGB) cultures restored CSR in purified naive patient B cells. CRISPR-Cas9 editing of healthy donor B cells confirmed that the iGB platform can rescue B cells harboring heterozygous, but not homozygous, AICDA variants. These results demonstrate that even a two-amino-acid truncation at the AID C-terminus is sufficient to disrupt its function and cause HIGM2.

immunology↗

bioRxiv2025

Mechanical stretch regulates inflammatory signaling in human smooth muscle cells

Background and aimsVascular smooth muscle cells (SMCs) cultured under standard static conditions adopt a modulated phenotype that resembles the SMC states found in atherosclerotic plaques, but the mechanical determinants of this drift are not well defined. We investigated how mechanical loading shapes SMC phenotype and inflammatory signaling. MethodsHuman aortic SMCs were maintained under static conditions, physiological cyclic stretch (10% elongation), or pathological stretch (15% elongation) and analyzed by bulk and single-cell RNA sequencing. Mechanistic experiments included siRNA-mediated knockdown of IKBKB, p65 immunofluorescence, and regulon inference from single-cell transcriptomes. ResultsStretch regulated cell cycle, contractile, and inflammatory gene programs in an intensity-dependent manner: 10% stretch suppressed basal and TNF-induced inflammatory gene expression, whereas 15% stretch did not. The anti-inflammatory effect of physiological stretch required IKBKB, yet the proportion of cells with nuclear p65 was unchanged, indicating that stretch constrains NF-{kappa}B output downstream of p65 nuclear entry rather than by blocking translocation. Single-cell RNA sequencing resolved nine states for SMCs in culture whose transcriptomes overlapped substantially with the modulated mesenchymal populations of human coronary and carotid plaques, and physiological stretch attenuated pro-inflammatory gene expression across the major clusters. Regulatory network analysis identified inflammatory transcription factors (RELB, CEBPB/D, IRF1/2, STAT2) as less active under physiological stretch, whereas mechano-lineage regulators (MEF2C, TEAD1, SMAD6) were selectively induced, providing candidate mediators of the effect. ConclusionsStatic culture represents a disease-like SMC baseline that physiological stretch attenuates, in part by constraining NF-{kappa}B transcriptional output at a step downstream of p65 nuclear entry. Graphical abstractPhysiological stretch (10%) maintains a low-inflammatory, healthy-like state in human aortic smooth muscle cells, whereas its absence (static culture) or excess (15% stretch) favors an inflammatory, plaque-like state. NF-{kappa}B-p65 enters the nucleus under all conditions; physiological stretch constrains its inflammatory output downstream of nuclear entry. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/685276v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@bbcf58org.highwire.dtl.DTLVardef@29ba95org.highwire.dtl.DTLVardef@1989aa4org.highwire.dtl.DTLVardef@1d0c253_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Multiparametric optimization of human primary B-cell cultures using Design of Experiments

B cells are essential in the immune system, driving antibody production, cytokine secretion, and antigen presentation. Studies in mouse models have illuminated key mechanisms underlying B-cell activation, differentiation, class-switch recombination, and somatic hypermutation. However, the extent to which these findings translate to human biology remains unclear. To address this, we developed a human primary B-cell culture system using feeder cells engineered to express CD40L, supplemented with the cytokines BAFF, IL-4, and IL-21. Using a Design of Experiments (DOE) approach, we optimized critical parameters and dissected the individual contributions of each specific factor. Our results reveal that BAFF plays a negligible role, and IL-21 has more subtle effects, whereas CD40L and IL-4 are critical determinants of cell viability, proliferation and IgE class-switching. Furthermore, we find that engineered feeder cells can serve equally well as a source of cytokines, but providing these in purified form increases the flexibility of the system. This platform enables detailed investigation of human B-cell biology, offering insights into intrinsic and extrinsic regulators of antibody responses and providing a foundation for in vitro production of human primary antibodies.

immunology↗

bioRxiv2023

Resistance to vincristine in cancerous B-cells by disruption of p53-dependent mitotic surveillance

The frontline therapy R-CHOP for patients with diffuse large B-cell lymphoma (DLBCL) has remained unchanged for two decades despite numerous phase III clinical trials investigating new alternatives. Multiple large studies have uncovered genetic subtypes of DLBCL enabling a targeted approach. To further pave the way for precision oncology, we perform genome-wide CRISPR screening to uncover the cellular response to one of the components of R-CHOP, vincristine, in the DLBCL cell line SU-DHL-5. We discover important pathways and subnetworks using gene-set enrichment analysis and protein-protein interaction networks and identify genes related to mitotic spindle organization that are essential during vincristine treatment. Inhibition of KIF18A, a mediator of chromosome alignment, using the small molecule inhibitor BTB-1 causes complete cell death in a synergistic manner when administered together with vincristine. We also identify the genes KIF18B and USP28 for which CRISPR/Cas9-directed knockout induces vincristine resistance across two DLBCL cell lines. Mechanistic studies show that lack of KIF18B or USP28 counteracts a vincristine-induced p53 response involving the mitotic surveillance pathway (USP28-53BP1-p53). Collectively, our CRISPR screening data uncover potential drug targets and mechanisms behind vincristine resistance, which may support the development of future drug regimens. Key pointsO_LIInhibition of the mitotic surveillance pathway (USP28-53BP1-p53) and KIF18B induces resistance to vincristine C_LIO_LISubstantial synergistic effects observed when using the KIF18A-inhibitor BTB-1 with vincristine in eradicating GCB-subtype DLBCL cells C_LI

cancer biology↗

bioRxiv2021

A truncated reverse transcriptase enhances prime editing by split AAV vectors

Prime editing is a new CRISPR-based genome editing technology that relies on the prime editor (PE), a fusion protein of Cas9-nickase and M-MLV reverse transcriptase (RT), and a prime editing guide RNA (pegRNA) that serves both to target PE to the desired genomic locus and to carry the edit to be introduced. Here, we make advancements to the RT moiety to improve prime editing efficiencies and truncations to mitigate issues with AAV viral vector size limitations, which currently do not support efficient delivery of the large prime editing components. These efforts include RT variant screening, codon optimization, and PE truncation by removal of the RNase H domain and further trimming. This led to a codon-optimized and size-minimized PE that has an expression advantage (1.4x fold) and size advantage (621 bp shorter). In addition, we optimize the split intein PE system and identify Rma-based Cas9 split sites (573-574 and 673-674) that combined with the truncated PE delivered by dual AAVs result in superior AAV titer and prime editing efficiency. This novel minimized PE provides great value to AAV-based delivery applications in vivo.

genetics↗