Søren Egedal Degn

TRAF3 (TNF Receptor Associated Factor 3) is a regulator of NF-κB signaling, acting mainly as an inhibitor of the alternative NF-κB pathway. While TRAF3 has a well-established role in immune function, mainly via B- and T-lymphocyte regulation, its roles in cancer remain unclear. Breast cancer is the most common malignancy in women and a neoplasm displaying high levels of intratumoral heterogeneity. Identifying and understanding key molecules at the interface of breast cancer cells and the immune system is crucial for advancing therapeutic strategies for breast cancer patients. Here, by employing publicly available breast cancer datasets, breast cancer cell lines stably expressing TRAF3, mass spectrometry analysis in combination with functional assays, co-culture systems, and signal pathway characterization, we sought to assess the specific role of TRAF3 in breast cancer cells and how TRAF3-expressing breast cancer cells affect their immune microenvironment. Our results indicate that TRAF3 protein overexpression inhibits colony formation through apoptosis regulation. Proteome analysis for TRAF3 interactors and over-representation analysis identified multiple protein complexes related to cell cycle, apoptosis, and immune responses. Furthermore, TRAF3-expressing breast cancer cells displayed reduced levels of PD-L1 and when co-cultured with PBMCs induced a pro-inflammatory profile with increased CD16-NK cells and higher levels of IFN-γ and TNF-α and lower IL-10 and Tregs in the culture. These findings further expand the role of TRAF3 in breast cancer, not only as a regulator of EMT and survival of cancer cells, but also as a modulator of the tumor-immune microenvironment.

Activation-induced cytidine deaminase (AID) is essential for B cell affinity maturation. We investigated why AID deficiency gives rise to giant germinal centers (GCs) 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 upregulate 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 was unable to restore plasma cell differentiation of AIDR112H B cells, while ectopic expression of catalytically active AID rescued plasma cell generation. AID and ten-eleven translocation 2 (TET2) synergistically facilitated demethylation of the Irf4 promoter/enhancer, and this was impeded in AIDR112H cells. These data reveal a B cell–intrinsic mechanism that governs the plasma cell fate decision through epigenetic remodeling mediated by AID in cooperation with TET2.

Hyper-IgE syndrome (HIES) is characterized by elevated serum IgE levels, eczema, and recurrent skin and pulmonary infections, classically caused by autosomal dominant (AD) STAT3 loss-of-function variants. Here we describe a patient presenting with elevated IgE levels, atopic eczema, chronic pulmonary aspergillosis, and bone fractures, homozygous for a rare missense variant in the oncostatin M receptor (OSMR) gene. We demonstrate that the patient OSMR V436D variant is deleterious and leads to decreased OSMR surface expression and impaired OSM-induced STAT3 phosphorylation. Blood profiling showed elevated IgE-expressing plasmablasts and peripheral T follicular helper cells and atypical memory B cells. A germinal center model revealed a B cell–extrinsic defect in concordance with a largely fibroblast-confined phenotype. Finally, reconstitution of patient fibroblasts led to functional complementation of the cellular phenotype. We propose OSMR deficiency as a novel genetic etiology of AR-HIES, resembling the clinical and immunological phenotype of STAT3 AD-HIES, but mediating its phenotypic profile mainly in the stromal rather than hematopoietic compartment.

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.

Background: Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide, with chronic liver inflammation being a major risk factor. Tertiary lymphoid structures (TLS) have been associated with improved survival and better response to immune checkpoint inhibitors in several cancers. However, their role in HCC remains unclear, and findings are often contradictory. Methods: The study investigated the presence and composition of TLSs across multiple liver disease models, including livers containing HCC. TLSs were characterized to identify the essential cellular components for functional lymphoid activity. The functional role of age-associated B cells (ABCs) was further explored by blocking T-bet expression in B cells, thereby preventing their differentiation into ABCs. Results: TLS frequency was increased in livers containing HCC. Characterization confirmed the presence of key immune cell subsets supporting lymphoid function. Blocking T-bet in B cells led to a reduction in ABCs, which resulted in smaller TLS, a specific decrease in hepatic B cells, and reduced tumor lesion size in HCC-bearing mice. Conclusions: These findings demonstrate that ABCs promote TLS formation and growth, facilitating B-cell recruitment to the liver and supporting HCC progression. Targeting T-bet–dependent ABC differentiation may represent a novel strategy to limit TLS formation and slow HCC progression.

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.

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 optimised 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.