Biology subjects

Burdine, L.

Publications and source records attributed to Burdine, L..

4 recordsLinked to original sources

bioRxiv2026

Acellular normothermic spleen perfusion resolves transcriptional and non-transcriptional mechanisms of steroid immunosuppression

The limiting step in immune-active drug development is increasingly not candidate generation but testing whether a candidate therapy is effective in a system that preserves tissue architecture, vascular exposure, multicellular interaction, and repeated pharmacodynamic sampling without patient exposure. We developed an acellular normothermic machine-perfusion platform for intact porcine spleen designed as a translational immune-organ assay. Across independent acellular perfusions, the circuit maintained physiologic parameters, preserved red- and white-pulp histology, and yielded viable effluent cells suitable for serial flow cytometry and multiomics. High-dose methylprednisolone was used as a clinically familiar perturbation to determine whether the platform could resolve steroid immunosuppression at mechanistically distinct levels. Effluent RNA-seq identified canonical glucocorticoid-responsive transcriptional programs, including DUSP1, FKBP5, PER1, DDIT4, SGK1, KLF9, ANXA1, NF-{kappa}B feedback regulators, and JAK/STAT suppressor pathways. SOCS3 was a prominent early signal in the perfusion transcriptome and was validated orthogonally at the protein level in prednisone-treated, CD3/CD28-activated primary murine splenocytes, strengthening its role as a candidate pharmacodynamic marker. In parallel, data independent acquisition (DIA) proteomics of effluent cell pellets nominated a non-transcriptional protein-level response: a Sus scrofa LGALS13-annotated, CLC/Galectin-10-like galectin detected despite absence of the corresponding effluent-cell transcript. Because this porcine LGALS13-annotated protein group is treated here as an orthologous CLC/Galectin-10-like signal rather than as canonical human placental Galectin-13/PP13, we tested recombinant human Galectin-10 in vitro. Human Galectin-10 induced marked apoptosis of CD3/CD28-stimulated Jurkat cells, prioritizing this axis for future mechanistic testing without proving causality in the perfused spleen. These data establish acellular spleen perfusion as a serial multiomic platform for translational immunopharmacology and motivate deployment with otherwise-discarded human donor spleens. One sentence summaryAn acellular intact-spleen perfusion platform enables serial cellular, transcriptomic, proteomic, and functional pharmacodynamic sampling that identifies steroid-responsive transcriptional programs, validates SOCS3 protein induction, and nominates a CLC/Galectin-10-like non-transcriptional immunosuppressive axis for translation to discarded human donor spleens.

bioengineering↗

bioRxiv2025

DNA-PKcs governs LAT-dependent signaling in CD4+ and CD8+ T cells

Formation of the immune synapse (IS) following T cell antigen recognition includes recruitment of the Linker for Activation of T cells (LAT). Once at the IS, LAT tyrosines are phosphorylated allowing it to serve as a scaffold for formation of the "signalosome", a multiprotein complex that drives TCR signaling. Here, we show that upon T cell activation, DNA dependent protein kinase catalytic subunit (DNA-PKcs) interacts with LAT and localizes to the IS. Inhibition of DNA-PKcs diminishes LAT localization at the IS. We identified two LAT serines phosphorylated by DNA-PKcs, S224 and S241, that impact LAT tyrosine phosphorylation, protein binding, and cytokine production. Using our mouse model designed to delete DNA-PKcs expression within mature CD4+ or CD8+ T cells, we show loss of DNA-PKcs results in T cells unable to control tumor growth or induce allogeneic graft rejection. These data bring to the forefront DNA-PKcs as a pivotal protein in T cell function.

immunology↗

bioRxiv2022

Inhibition of DNA-PKcs impairs the activation and cytotoxicity of CD4+ helper and CD8+ effector T cells

Modulation of T cell activity is an effective strategy for the treatment of autoimmune diseases, immune-related disorders and cancer. This highlights a critical need for continued investigation of proteins that regulate T cell function. The kinase DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is emerging as a potent regulator of the immune system spurring interest in its use as a therapeutic target for immune-related diseases. In murine models of autoimmune disease including asthma and rheumatoid arthritis, treatment with small molecule DNA-PKcs inhibitors, which are in clinical trials for cancer therapy, decreased disease severity. Additionally, DNA-PKcs inhibitors reduced T cell-mediated graft rejection and extended graft survival in a murine allogenic skin graft rejection model. These in vivo studies suggest the therapeutic use of DNA-PKcs inhibitors for autoimmune and T cell-mediated disorders. In this study, we sought to further characterize the effects of DNA-PKcs inhibitors on T cells to better understand their clinical potential. We determined that pharmacological inhibition of DNA-PKcs abrogated activation of murine and human CD4+ and CD8+ T cells as evident by reduced expression of the activation markers CD69 and CD25. Furthermore, inhibition of DNA-PKcs impeded metabolic pathways and proliferation of anti-CD3/CD28 activated CD4+ and CD8+ T cells as well as peptide-stimulated OTI-CD8+ T cells. This reduced the ability of OTI-CD8+ T cells to kill cancer cells and the expression of IFN{gamma} and the cytotoxic genes eomes, perforin and granzyme B. These results suggest a novel role for DNA-PKcs in early T cell activation. Furthermore, our data support the therapeutic potential of DNA-PKcs inhibitors on diseases of immune dysregulation.

immunology↗

bioRxiv2021

DNA-PKcs kinase activity stabilizes the transcription factor Egr1 in activated immune cells

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is known primarily for its function in DNA double-stranded break repair and non-homologous end joining (NHEJ). However, DNA-PKcs also has a critical yet undefined role in immunity impacting both myeloid and lymphoid cell lineages spurring interest in targeting DNA-PKcs for therapeutic strategies in immune-related diseases. To gain insight into the function of DNA-PKcs within immune cells, we performed a quantitative phosphoproteomic screen in T cells to identify first order phosphorylation targets of DNA-PKcs. Results indicate that DNA-PKcs phosphorylates the transcription factor Egr1 (early growth response protein 1) at S301. Expression of Egr1 is induced early upon T cell activation and dictates T cell response by modulating expression of cytokines and key costimulatory molecules. Mutation of serine 301 to alanine via CRISPR-Cas9 resulted in increased proteasomal degradation of Egr1 and a decrease in Egr1-dependent transcription of IL2 (interleukin-2) in activated T cells. Our findings identify DNA-PKcs as a critical intermediary link between T cell activation and T cell fate and a novel phosphosite involved in regulating Egr1 activity.

molecular biology↗