Biology subjects

Stender, J. D.

Publications and source records attributed to Stender, J. D..

5 recordsLinked to original sources

bioRxiv2026

A genome-wide CRISPR Screening identifies targets that drive Tolerogenic Dendritic Cells

Tolerogenic dendritic cells (TolDCs) are essential for immune tolerance and offer promise for treating autoimmune diseases. Despite the clinical evidence of their therapeutic potential, the key molecular pathways guiding their differentiation and tolerogenic phenotype remain elusive due to complex interactions identified in functional assays. Here we investigated the molecular profiles and regulatory programs underlying the functional status of tolerogenic dendritic cell populations in response to known tolerizing agents. We identified CD86 as a consistent and robust marker downregulated in tolerogenic state. Using CD86 blocking antibodies or CRISPR-mediated gene inactivation we demonstrated that CD86 is functionally required for TolDC-mediated suppression of T cell proliferation and cytokine secretion, establishing CD86 as both a consensus phenotypic and functional screening marker and a mechanistic regulator of tolerance. Leveraging CD86 as a scalable readout, we performed a pooled genome-wide CRISPR-Cas9 knockout screen to identify regulators of TolDC function. This approach uncovered UBE2L6 as a novel modulator that promotes the tolerogenic phenotype and restricts TolDC-mediated T cell activation. Mechanistically, UBE2L6 deficiency leads to coordinated upregulation of ISG15 and USP18, indicating a possible ISGylation-dependent pathway regulating CD86 expression and tolerogenic function. Together, this study identifies pathways that can be targeted to promote immune tolerance in immune-mediated inflammatory diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/713621v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@4c3636org.highwire.dtl.DTLVardef@17b27e5org.highwire.dtl.DTLVardef@783d95org.highwire.dtl.DTLVardef@1337ce_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Optimization of Genome-wide CRISPR Screens using dual-guide RNA Infection with Cas9 Electroporation (DICE)

Single-guide RNA lentiviral infection with Cas9 protein electroporation (SLICE) enables CRISPR screening in primary cell types that require transient Cas9 expression yet is limited by scalability and robustness. Here, we introduce dual-guide RNA infection with Cas9 electroporation (DICE), which expresses two guides from the same lentiviral construct that target the same gene. In genome-wide screens, DICE outperformed SLICE in defining essential genes and modulators of PD-L1 expression in IFN gamma activated THP1 cells. Collectively, these data demonstrate that DICE can be utilized for reduced-scale CRISPR screens in cell types with transient Cas9 protein expression without sacrificing screening quality.

genomics↗

bioRxiv2023

A genome-wide CRISPR screen supported by human genetics identifies the TNRC18 gene locus as a novel regulator of inflammatory signaling

Interleukin-1{beta} (IL-1{beta}) is dysregulated in many chronic inflammatory diseases, yet the genetic factors influencing IL-1{beta} production and signaling remain largely unknown. Myeloid-derived cells are the primary producers of IL-1{beta}, prompting a genome-wide CRISPR knockout screen in the human myeloid-derived U937 cell model, treated with lipopolysaccharide (LPS) to mimic inflammatory conditions, and sorted for high and low intracellular IL-1{beta} levels. A total of 295 genes were identified as regulators of IL-1{beta} production, including known mediators, such as TLR4, JAK-STAT, IL-10 receptor, and the Cullin ring finger ligase complex. Notably, 57 out of the 295 genes overlapped with loci associated with human inflammatory diseases, including the TNRC18 gene on chromosome 7p22.1 associated with multiple diseases in the Finnish population. U937 cells engineered with the homozygous rs748670681 risk allele associated with inflammatory bowel disease, demonstrated decreased levels of mRNA for TNRC18 and an adjacent gene WIPI2, reduction in LPS-dependent gene activation and cytokine production, but elevation of interferon-responsive gene programs. Transcriptomic profiles for individual knockouts of TNRC18 and WIPI2 attributed the loss of LPS-dependent signaling primarily to TNRC18 while the exacerbation of interferon signaling is a hallmark of loss of WIPI2. Collectively, these findings delineate the global regulatory mechanisms of IL-1{beta} production and provide molecular insights to the role of the rs748670681 variant as a pleiotropic risk factor for inflammatory diseases.

genomics↗

bioRxiv2019

PHLPP1 Counter-regulates STAT1-mediated Inflammatory Signaling

Inflammation is an essential aspect of innate immunity but also contributes to diverse human diseases. Although much is known about the kinases that control inflammatory signaling, less is known about the opposing phosphatases. Here we report that deletion of the gene encoding PH domain Leucine-rich repeat Protein Phosphatase 1 (PHLPP1) protects mice from lethal lipopolysaccharide (LPS) challenge and live Escherichia coli infection. Investigation of PHLPP1 function in macrophages reveals that it controls the magnitude and duration of inflammatory signaling by dephosphorylating the transcription factor STAT1 on Ser727 to inhibit its activity, reduce its promoter residency, and reduce the expression of target genes involved in innate immunity and cytokine signaling. This previously undescribed function of PHLPP1 depends on a bipartite nuclear localization signal in its unique N-terminal extension. Our data support a model in which nuclear PHLPP1 dephosphorylates STAT1 to control the magnitude and duration of inflammatory signaling in macrophages.\n\nHIGHLIGHTSO_LIPHLPP1 controls the transcription of genes involved in inflammatory signaling\nC_LIO_LIPHLPP1 dephosphorylates STAT1 on Ser727 to reduce its transcriptional activity\nC_LIO_LIPHLPP1 has a nuclear localization signal and a nuclear exclusion signal\nC_LIO_LILoss of PHLPP1 protects mice from sepsis-induced death\nC_LI

biochemistry↗

bioRxiv2018

Diverse motif ensembles specify non-redundant DNA binding activities of AP-1 family members in macrophages

Mechanisms by which members of the AP-1 family of transcription factors play both redundant and non-redundant biological roles despite recognizing the same DNA sequence remain poorly understood. To address this question, we investigated the molecular functions and genome-wide DNA binding patterns of AP-1 family members in macrophages. ChIP-sequencing showed overlapping and distinct binding profiles for each factor that were remodeled following TLR4 ligation. Development of a machine learning approach that jointly weighs hundreds of DNA recognition elements yielded dozens of motifs predicted to drive factor-specific binding profiles. Machine learning-based predictions were confirmed by analysis of the effects of mutations in genetically diverse mice and by loss of function experiments. These findings provide evidence that non-redundant genomic locations of different AP-1 family members in macrophages largely result from collaborative interactions with diverse, locus-specific ensembles of transcription factors and suggest a general mechanism for encoding functional specificities of their common recognition motif.

genomics↗