Biology subjects

Schumann, K.

Publications and source records attributed to Schumann, K..

5 recordsLinked to original sources

bioRxiv2026

SATB1 is a targetable modulator of JAK-STAT signaling and cytokines in human Treg and Tconv cells

The chromatin organizer SATB1 is indispensable for thymic regulatory T cell (Treg cell) development and T helper cell induction. Several gene loci have been described to be SATB1-controlled, including the transcription factor GATA3 and the cytokine loci IL-4 and IL-17. However, the global effects of SATB1 on fully differentiated human CD4 conventional T cells (Tconv cells) and Treg cells, and thus SATB1s potential as a target for T cell engineering, are poorly understood. We describe SATB1-regulated gene signatures as largely subset-specific, with broader effects on Treg cells. Despite of the distinct gene-regulatory patterns, we observe overarching dysregulated cytokine and JAK-STAT signaling after SATB1 ablation. Functionally, SATB1 KO reduces human Treg cell suppressive capacities but boosts tumor clearance via CD4 CAR T cells in a preclinical, humanized mouse model. Together, Treg destabilization and simultaneous increased activation of CD4 CAR T cells by SATB1 modulation may be an interesting strategy to boost the efficiency of CAR T cell therapies.

immunology↗

bioRxiv2024

Quantitative imaging of species-specific lipid transport in mammalian cells

Eukaryotic cells produce over 1000 different lipid species which tune organelle membrane properties, control signalling and store energy1,2. How lipid species are selectively sorted between organelles to maintain specific membrane identities is largely unknown due to the difficulty to image lipid transport in cells3. Here, we measured transport and metabolism of individual lipid species in mammalian cells using time-resolved fluorescence imaging of bifunctional lipid probes in combination with ultra-high resolution mass spectrometry and mathematical modelling. Quantification of lipid flux between organelles revealed that directional, non-vesicular lipid transport is responsible for fast, species-selective lipid sorting compared to slow, unspecific vesicular membrane trafficking. Using genetic perturbations, we found that coupling between active lipid flipping and passive non-vesicular transport is a mechanism for directional lipid transport. Comparison of metabolic conversion and transport rates showed that non-vesicular transport dominates the organelle distribution of lipids while species-specific phospholipid metabolism controls neutral lipid accumulation. Our results provide the first quantitative map of retrograde lipid flux in cells4. We anticipate that our pipeline for quantitative mapping of lipid flux through physical and chemical space in cells will boost our understanding of lipids in cell biology and disease.

cell biology↗

bioRxiv2017

A CRISPR-Cas9 Genome Engineering Platform in Primary CD4+ T Cells for the Interrogation of HIV Host Factors

CRISPR-Cas9 gene editing strategies have revolutionized our ability to engineer the human genome for robust functional interrogation of complex biological processes. We have recently adapted this technology to primary human T cells to generate a high-throughput platform for analyzing the role of host factors in pathogen infection and lifecycle. Here, we describe applications of this system to investigate HIV pathogenesis in CD4+ T cells. Briefly, CRISPR-Cas9 ribonucleoproteins (crRNPs) are synthesized in vitro and delivered to activated primary human CD4+ T cells by nucleofection. These edited cells are then validated and expanded for use in downstream cellular, genetic, or protein-based assays. Our platform supports the arrayed generation of several gene manipulations in only a few hours time and is widely adaptable across culture conditions, infection protocols, and downstream applications. We present detailed protocols for crRNP synthesis, primary T cell culture, 96-well nucleofection, molecular validation, and HIV infection with additional considerations for guide and screen design as well as crRNP multiplexing.

systems biology↗

bioRxiv2017

Correction of autoimmune IL2RA mutations in primary human T cells using non-viral genome targeting

Human T cells are central to physiological immune homeostasis, which protects us from pathogens without collateral autoimmune inflammation. They are also the main effectors in most current cancer immunotherapy strategies1. Several decades of work have aimed to genetically reprogram T cells for therapeutic purposes2-5, but as human T cells are resistant to most standard methods of large DNA insertion these approaches have relied on recombinant viral vectors, which do not target transgenes to specific genomic sites6, 7. In addition, the need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells through homology-directed repair (HDR), but to date in human T cells this still requires viral transduction8, 9. Here, we developed a non-viral, CRISPR-Cas9 genome targeting system that permits the rapid and efficient insertion of individual or multiplexed large (>1 kilobase) DNA sequences at specific sites in the genomes of primary human T cells while preserving cell viability and function. We successfully tested the potential therapeutic use of this approach in two settings. First, we corrected a pathogenic IL2RA mutation in primary T cells from multiple family members with monogenic autoimmune disease and demonstrated enhanced signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR redirecting T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized the tumour antigen, with concomitant cytokine release and tumour cell killing. Taken together, these studies provide preclinical evidence that non-viral genome targeting will enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

genetics↗

bioRxiv2016

Discovery of an autoimmunity-associated IL2RA enhancer by unbiased targeting of transcriptional activation

The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell type-specific transcriptional programs and responses to specific extracellular cues 1-3. In order to understand the mechanisms by which non-coding genetic variation contributes to disease, systematic mapping of functional enhancers and their biological contexts is required. Here, we develop an unbiased discovery platform that can identify enhancers for a target gene without prior knowledge of their native functional context. We used tiled CRISPR activation (CRISPRa) to synthetically recruit transcription factors to sites across large genomic regions (>100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA (interleukin-2 receptor alpha; CD25). We identified several CRISPRa responsive elements (CaREs) with stimulation-dependent enhancer activity, including an IL2RA enhancer that harbors an autoimmunity risk variant. Using engineered mouse models and genome editing of human primary T cells, we found that sequence perturbation of the disease-associated IL2RA enhancer does not block IL2RA expression, but rather delays the timing of gene activation in response to specific extracellular signals. This work develops an approach to rapidly identify functional enhancers within non-coding regions, decodes a key human autoimmunity association, and suggests a general mechanism by which genetic variation can cause immune dysfunction.

genetics↗