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Tetraspanins are four-transmembrane proteins that play fundamental roles in the immune system by enabling processes like migration, proliferation, signaling and protein trafficking. While the importance of cell surface tetraspanins has been established, the function of intracellular tetraspanins is less well understood. Here, we investigated the role of tetraspanin 3 (Tspan3) in lymphocytes. Tspan3 expression was low in T cells and high in B cells which increased during B cell activation. Tspan3 localized to late endosomal structures where it colocalized with major histocompatibility complex II (MHCII) in the MHCII compartment. There, inhibiting the formation of intraluminal vesicles (ILVs) showed that Tspan3 localization was not affected in contrast to its homologue CD63. Using a peptide-pulldown approach, we identified that Tspan3 interacts with AP2 via its C-terminus that harbors a YXXΦ-based sorting motif. Interestingly, mutating this motif did not impair Tspan3 localization. Instead, leucine 246 was required for its intracellular localization, identifying an unrecognized leucine-based sorting motif responsible for Tspan3 localization to MHCII compartment in B cells. Taken together, we report a new sorting motif for Tspan3 to MHCII compartments in human B cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The invention of nanosized biomaterials has paved the way for novel therapeutics that can manipulate cells on a nanoscale. Nanosized immunofilaments (IFs) are synthetic filamentous polymers consisting out of polyisocyanopeptides, which have been recently established as a powerful platform to activate specific immune cells in vivo such that they raise an antitumor immune response. However, toxicological effects or immunogenicity toward the IFs have not yet been investigated. In this study, we evaluated potential toxic or immunogenic effects in C57BL/6 mice upon intravenous or subcutaneous injection of nonfunctionalized IFs or immunostimulatory IFs over 30 days. We here present a detailed analysis of the gross pathology, hematological parameters, blood biochemistry, histology, and antibody-response against the IF backbone. Our results demonstrate that IFs do not induce severe acute or chronic toxicity in mice. After 30 days, we only found elevated IgG-titers in intravenously injected but not subcutaneously injected mice. In summary, we demonstrate that IFs can be administered into a living organism without adverse side effects, thereby establishing the safety of IFs as a therapeutic intervention., Competing Interests: The authors declare the following competing financial interest(s): C.F. is the chief scientific officer and co-founder of Simmunext biotherapeutics develops novel immunotherapies by mimicking immune cell function through its proprietary polymer platform technology. C.F. is an inventor on patent WO2012004369 (2012); C.F., and R.H. are inventors on patent WO2019154865 (2019); C.F. and R.H. are inventors on patent WO2020174041. The other authors declare no conflict of interest., (© 2024 The Authors. Published by American Chemical Society.)

Diffuse large B-cell lymphoma (DLBCL) represents the most common form of non-Hodgkin lymphoma (NHL) that is still incurable in a large fraction of patients. Tetraspanin CD37 is highly expressed on mature B lymphocytes, and multiple CD37-targeting therapies are under clinical development for NHL. However, CD37 expression is nondetectable in ∼50% of DLBCL patients, which correlates with inferior treatment outcome, but the underlying mechanisms for differential CD37 expression in DLBCL are still unknown. Here, we investigated the regulation of the CD37 gene in human DLBCL at the (epi-)genetic and transcriptional level. No differences were observed in DNA methylation within the CD37 promoter region between CD37-positive and CD37-negative primary DLBCL patient samples. On the contrary, CD37-negative DLBCL cells specifically lacked CD37 promoter activity, suggesting differential regulation of CD37 gene expression. Using an unbiased quantitative proteomic approach, we identified transcription factor IRF8 to be significantly higher expressed in nuclear extracts of CD37-positive as compared with CD37-negative DLBCL. Direct binding of IRF8 to the CD37 promoter region was confirmed by DNA pulldown assay combined with mass spectrometry and targeted chromatin immunoprecipitation (ChIP). Functional analysis indicated that IRF8 overexpression enhanced CD37 protein expression, while CRISPR/Cas9 knockout of IRF8 decreased CD37 levels in DLBCL cell lines. Immunohistochemical analysis in a large cohort of primary DLBCL (n = 206) revealed a significant correlation of IRF8 expression with detectable CD37 levels. Together, this study provides new insight into the molecular mechanisms underlying differential CD37 expression in human DLBCL and reveals IRF8 as a transcriptional regulator of CD37 in B-cell lymphoma., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Mast cells (MCs) are long-living immune cells highly specialized in the storage and release of different biologically active compounds and are involved in the regulation of innate and adaptive immunity. MC degranulation and replacement of MC granules are accompanied by active membrane remodelling. Tetraspanins represent an evolutionary conserved family of transmembrane proteins. By interacting with lipids and other membrane and intracellular proteins, they are involved in organisation of membrane protein complexes and act as "molecular facilitators" connecting extracellular and cytoplasmic signaling elements. MCs express different tetraspanins and MC degranulation is accompanied by changes in membrane organisation. Therefore, tetraspanins are very likely involved in the regulation of MC exocytosis and membrane reorganisation after degranulation. Antiviral response and production of exosomes are further aspects of MC function characterized by dynamic changes of membrane organization. In this review, we pay a particular attention to tetraspanin gene expression in different human and murine MC populations, discuss tetraspanin involvement in regulation of key MC signaling complexes, and analyze the potential contribution of tetraspanins to MC antiviral response and exosome production. In-depth knowledge of tetraspanin-mediated molecular mechanisms involved in different aspects of the regulation of MC response will be beneficial for patients with allergies, characterized by overwhelming MC reactions.

Mast cells (MCs) have long been mainly regarded as effector cells in IgE-associated allergic disorders with potential immunoregulatory roles. Located close to the allergen entry sites in the skin and mucosa, MCs can capture foreign substances such as allergens, toxins, or noxious substances and are exposed to the danger signals produced by epithelial cells. MC reactivity shaped by tissue-specific factors is crucial for allergic responses ranging from local skin reactions to anaphylactic shock. Development of Th2 response leading to allergen-specific IgE production is a prerequisite for MC sensitization and induction of FcεRI-mediated MC degranulation. Up to now, IgE production has been mainly associated with proteins, whereas lipids present in plant pollen grains, mite fecal particles, insect venoms, or food have been largely overlooked regarding their immunostimulatory and immunomodulatory properties. Recent studies, however, have now demonstrated that lipids affect the sensitization process by modulating innate immune responses of epithelial cells, dendritic cells, and NK-T cells and thus crucially contribute to the outcome of sensitization. Whether and how lipids affect also MC effector functions in allergic reactions has not yet been fully clarified. Here, we discuss how lipids can affect MC responses in the context of allergic inflammation. Direct effects of immunomodulatory lipids on MC degranulation, changes in local lipid composition induced by allergens themselves and changes in lipid transport affecting MC reactivity are possible mechanisms by which the function of MC might be modulated.