Biology subjects

Degn, S. E.

Publications and source records attributed to Degn, S. E..

9 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↗

bioRxiv2026

Thymic Treg-derived T follicular regulatory cells prevent catastrophic humoral autoimmunity in response to TLR7-driven inflammation

T follicular regulatory cells (TFR) are a follicle-resident subset of regulatory T cells (TREG) that limit germinal center (GC) responses and enforce humoral tolerance. Although TFR are known to focus antibody responses to foreign antigens, how GC-specific regulation is maintained during inflammation remains unclear. Here, TLR7-driven inflammation unmasked a critical, non-redundant role for TFR in preserving immune tolerance. Selective loss of TFR caused severe autoimmunity and increased mortality, driven by inflammation-induced expansion of autoreactive B and T cell clones and epitope spreading. Autoreactivity resolved upon cessation of inflammation in TFR-sucicient mice but persisted in their absence. Mechanistically, TFR limited the establishment and expansion of spontaneous GCs in response to inflammation, and responding TFR displayed transcriptional, phenotypic, and clonal features of thymic TREG. This suggests that whereas TFH upregulate FoxP3 to shut down end-stage GCs, it is thymic TREG-derived TFR that safeguard GC integrity under inflammatory stress to prevent lethal autoimmunity.

immunology↗

bioRxiv2025

Display of clustered antigen by follicular dendritic cells tunes B-cell receptor activation

To generate high-affinity antibodies against pathogens, B cells engage antigens displayed on the surface of follicular dendritic cells (FDCs) in secondary lymphoid tissues. Binding of cognate antigen by the B-cell receptor (BCR) causes Src kinase activation and phosphorylation of SYK (pSYK), serving as a platform for downstream signaling. However, how presentation of antigen by FDCs influence antigen binding and intracellular signaling in B cells remains unclear. Here, we show that antigen occurs in submicron pre-clusters on FDCs. We use nanoscale ligand patterning to present clustered antigens on tension force tethers in relevant cluster geometries in vitro. Clustering of antigens greatly increases BCR activation in a pattern-size-dependent manner, correlating to exclusion of the tyrosine phosphatase CD45 from BCR-antigen complexes. Increased height of antigen placement reduces CD45 exclusion with concomitant reduction in pSYK. Super-resolution imaging reveals reduced levels of pSYK at the edges of antigen patterns where CD45 interactions are greater, indicating a geometric control of BCR triggering.

immunology↗

bioRxiv2025

AID facilitates TET2 demethylation of Irf4 for plasma cell fate in germinal center B cells

Activation-induced cytidine deaminase (AID) is essential for B cell affinity maturation. We investigated why AID deficiency gives rise to giant germinal centers (GC) using the AIDR112H mouse model that is devoid of AID activity. The increased GC response was associated with accumulation of GC B cells in the light zone in immunized AIDR112H mice. AIDR112H GC B cells had reduced capacity to up-regulate IRF4 to initiate plasma cell differentiation, leading to accumulation of a transitional GC population with reduced GL7 expression. Genetic introduction of a high affinity B cell receptor (BCR) was unable to restore plasma cell differentiation of AIDR112H B cells while ectopic expression of catalytic active AID rescued plasma cell generation. AIDR112H impaired recruitment of AID to the Irf4 promoter/enhancer and disrupted the interaction with Ten-eleven Translocation 2 (TET2). Consequently, DNA demethylation at the Irf4 promoter/enhancer was reduced in AIDR112H GC B cells and impeded high Irf4 expression for transition into plasma cells. This data reveals a B cell-intrinsic mechanism that governs the plasma cell fate decision through epigenetic remodeling mediated by AID in cooperation with TET2. Key messagesO_LIAID deficiency leads to accumulation of a transitional (t)GC population of B cells with low expression of GL7 and failure to up-regulate IRF4 C_LIO_LIAID deamination activity is needed to promote up-regulation of IRF4 for plasma cell differentiation. C_LIO_LICo-operation between TET2 and AID facilitates demethylation of the Irf4 enhancer/promoter. C_LI

immunology↗

bioRxiv2025

LRP2 expression in melanoma is associated with a transitory cell state, increased T cell infiltration, and is upregulated by IFNγ signaling

Low density lipoprotein receptor-related protein 2 (LRP2) is a 600 kilodalton multi-ligand endocytic membrane receptor expressed in several cell types during fetal development, including neuroepithelial cells, and in select absorptive epithelial cells in the adult. In epithelial cancers, LRP2 expression is associated with a differentiated tumor cell state and better prognosis. In previous work, we found that while LRP2 is not expressed in benign naevi, it is frequently acquired in melanoma. However, the molecular drivers of LRP2 expression in melanoma and characteristics of LRP2-expressing melanoma have yet to be described. Here, we show that LRP2 expression is related to a transitory melanoma cell state defined by co-expression of melanocyte lineage and neural crest transcriptional programs. Further, we reveal that melanoma LRP2 expression is increased in T cell-inflamed tumors, and is directly upregulated through interferon-gamma signaling. Correlation of melanoma LRP2 expression with clinicopathological variables demonstrates that LRP2 expression is associated with low Breslow thickness and low clinical stage in primary melanomas. Taken together, the present study describes the characteristics of LRP2-expressing melanoma and reveals interferon gamma signaling as a novel strong positive regulator of LRP2 expression in melanoma. SignificanceMelanoma cells often acquire LRP2 expression but the drivers of LRP2 expression in this setting and characteristics of LRP2-expressing melanoma remain unclear. Here, we show that LRP2 expression is related to a transitory melanoma differentiation cell state. Further, LRP2 expression in melanoma correlates with a T cell-inflamed tumor microenvironment and LRP2 expression in melanoma cells can be directly increased by interferon-gamma. In addition, LRP2 expression is associated with less advanced histopathological characteristics of melanoma. These findings encourage future studies on LRP2 in settings with increased interferon signaling, in particular in melanoma metastases following immunotherapy.

cancer 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↗

bioRxiv2025

Serial intravital microscopy reveals temporal dynamics of autoreactive germinal centers in the spleen

The spleen plays a key role in clearing blood-borne infections and is involved in autoimmune and hematological disorders. It undergoes extensive remodeling during inflammation and immune reactions, but its localization in the peritoneal cavity has hampered studies of these dynamic changes. Here, we establish and validate a protocol for serial 2-photon microscopy of the murine spleen to capture dynamic processes in the living animal. As a proof-of-principle, we elucidate the expansion and contraction of autoreactive germinal centers (GCs) induced by epicutaneous application of the small-molecule TLR7 agonist resiquimod (R848). Leveraging a biocompatible abdominal imaging window, intravital labeling techniques, and fluorescent reporters, we follow GCs up to 180 {micro}m below the capsule for more than 2 weeks by tracking follicular dendritic cell (FDC) networks. This was accomplished without appreciable perturbation of normal physiology, paving the way for a deeper understanding of the biology of the spleen and its associated disease states. HighlightAn abdominal imaging window allowing the study of dynamic processes in the spleen of live mice over the course of several weeks.

immunology↗

bioRxiv2023

STING activation counters glioblastoma by vascular alteration and immune surveillance

Glioblastoma (GBM) is an aggressive brain tumor with a median survival of 15 months and has limited treatment options. Immunotherapy with checkpoint inhibitors has shown minimal efficacy in combating GBM, and large clinical trials have failed. New immunotherapy approaches and a deeper understanding of immune surveillance of GBM are needed to advance treatment options for this devastating disease. In this study, we used two preclinical models of GBM: orthotopically delivering either GBM stem cells or employing CRISPR-mediated tumorigenesis by adeno-associated virus, to establish immunologically proficient and non-inflamed tumors, respectively. After tumor development, the innate immune system was activated through long-term STING activation by a pharmacological agonist, which reduced tumor progression and prolonged survival. Recruitment and activation of cytotoxic T-cells were detected in the tumors, and T-cell specificity towards the cancer cells was observed. Interestingly, prolonged STING activation altered the tumor vasculature, inducing hypoxia and activation of VEGFR, as measured by a kinome array and VEGF expression. Combination treatment with anti-PD1 did not provide a synergistic effect, indicating that STING activation alone is sufficient to activate immune surveillance and hinder tumor development through vascular disruption. These results guide future studies to refine innate immune activation as a treatment approach for GBM, in combination with anti-VEGF to impede tumor progression and induce an immunological response against the tumor.

cancer biology↗

bioRxiv2022

Germinal center block exacerbates extrafollicular responses and accelerates autoimmune disease progression in a murine lupus model

Many autoimmune diseases are characterized by germinal center (GC) derived affinity-matured, class-switched autoantibodies. Strategies to block GC formation and progression are currently being explored clinically, however, extrafollicular responses may also contribute to early events in autoimmunity. To investigate the relative contribution of these two pathways in autoimmune disease development, we leveraged a transgenic strategy to genetically block the GC pathway. Surprisingly, this accelerated extrafollicular responses and failed to curb autoimmune progression in two lupus models. In vitro, loss of the GC transcription factor Bcl-6 prevented cellular expansion and accelerated plasma cell differentiation, suggesting the in vivo phenotype was caused by rewiring of B cell intrinsic transcriptional programming. In a competitive scenario in vivo, B cells harboring the genetic GC block contributed disproportionately to the plasma cell output. Taken together, this emphasizes the extrafollicular pathway as a key contributor to autoimmune progression, and suggests that strategies aimed at blocking GCs should simultaneously target this pathway to avoid rerouting the pathogenic response. Highlights- A genetic GC block fails to prevent autoimmune progression in two lupus models - An intrinsic GC block drives B cell differentiation into terminally differentiated plasma cells in vitro - B cells harboring a GC block competitively contribute to the plasma cell compartment in an autoreactive setting in vivo - Lupus mice with a GC block display immune complex deposition in kidney glomeruli that is indistinguishable from their wild-type counterparts SummaryAffinity-matured autoantibodies generated in germinal centers are a hallmark of autoimmune diseases. Voss et al. block germinal centers in two autoimmune models, but surprisingly find that disease progresses unimpeded. They identify the extrafollicular pathway as a backdoor to autoimmunity.

immunology↗