European Journal of Immunology

Ummarino, Aldo; Pensado‐López, Alba; Migliore, Roberta; Alcaide‐Ruggiero, Lourdes; Calà, Nicholas; Caputo, Michele; Gambaro, Francesco M.; Anfray, Clément; Ronzoni, Flavio L.; Kon, Elizaveta; Allavena, Paola; Torres Andón, Fernando

doi: 10.1002/eji.202350507pmid: 37713238

Osteoarthritis (OA) is characterized by an abundance of inflammatory M1‐like macrophages damaging local tissues. The search for new potential drugs for OA suffers from the lack of appropriate methods of long‐lasting inflammation. Here we developed and characterized an in vitro protocol of long‐lasting culture of primary human monocyte‐derived macrophages differentiated with a combination of M‐CSF+GM‐CSF that optimally supported long‐cultured macrophages (LC‐Mϕs) for up to 15 days, unlike their single use. Macrophages repeatedly stimulated for 15 days with the TLR2 ligand Pam3CSK4 (LCS‐Mϕs), showed sustained levels over time of IL‐6, CCL2, and CXCL8, inflammatory mediators that were also detected in the synovial fluids of OA patients. Furthermore, macrophages isolated from the synovia of two OA patients showed an expression profile of inflammation‐related genes similar to that of LCS‐Mϕs, validating our protocol as a model of chronically activated inflammatory macrophages. Next, to confirm that these LCS‐Mϕs could be modulated by anti‐inflammatory compounds, we employed dexamethasone and/or celecoxib, two drugs widely used in OA treatment, that significantly inhibited the production of inflammatory mediators. This easy‐to‐use in vitro protocol of long‐lasting inflammation with primary human macrophages could be useful for the screening of new compounds to improve the therapy of inflammatory disorders.

Fiore, Alessandra; Sala, Eleonora; Laura, Chiara; Riba, Michela; Nelli, Maria; Fumagalli, Valeria; Oberrauch, Federico; Mangione, Marta; Cristofani, Claudia; Provero, Paolo; Iannacone, Matteo; Kuka, Mirela

doi: 10.1002/eji.202350529pmid: 37741290

TDC are hematopoietic cells that combine dendritic cell (DC) and conventional T‐cell markers and functional properties. They were identified in secondary lymphoid organs (SLOs) of naïve mice as cells expressing CD11c, major histocompatibility molecules (MHC)‐II, and the T‐cell receptor (TCR). Despite thorough characterization, a physiological role for TDC remains to be determined. Unfortunately, using CD11c as a marker for TDC has the caveat of its upregulation on different cells, including T cells, upon activation. Here, we took advantage of Zbtb46‐GFP reporter mice to explore the frequency and localization of TDC in different tissues at steady state and upon viral infection. RNA sequencing analysis confirmed that TDC sorted from Zbtb46‐GFP mice have a gene signature that is distinct from conventional T cells and DC. In addition, this reporter model allowed for identification of TDC in situ not only in SLOs but also in the liver and lung of naïve mice. Interestingly, we found that TDC numbers in the SLOs increased upon viral infection, suggesting that TDC might play a role during viral infections. In conclusion, we propose a visualization strategy that might shed light on the physiological role of TDC in several pathological contexts, including infection and cancer.

Heger, Lukas; Dudziak, Diana; Amon, Lukas; Hatscher, Lukas; Kaszubowski, Tomasz; Lehmann, Christian H. K.

doi: 10.1002/eji.202249917pmid: 36563130

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state‐of‐the‐art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non‐lymphoid tissues. Within this article, detailed protocols are presented that allow for the generation of single cell suspensions from human lymphohematopoietic tissues including blood, spleen, thymus, and tonsils with a focus on the subsequent analysis of DC via flow cytometry, as well as flow cytometric cell sorting of primary human DC. Further, prepared single cell suspensions as well as cell sorter‐purified DC can be subjected to other applications including cellular enrichment procedures, RNA sequencing, functional assays, and many more. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer‐reviewed by leading experts and approved by all co‐authors, making it an essential resource for basic and clinical DC immunologists.

Chawda, Mira M.; Ross, Susan; Lau, Ching‐In; Yánez, Diana C.; Rowell, Jasmine; Kilbride, Peter; Crompton, Tessa

doi: 10.1002/eji.202350546pmid: 37751619

Yeoh, Wen Jie; Krebs, Philippe

doi: 10.1002/eji.202350446pmid: 37742135

Phosphoinositide‐3‐kinase/AKT (PI3K/AKT) signaling plays key roles in the regulation of cellular activity in both health and disease. In immune cells, this PI3K/AKT pathway is critically regulated by the phosphoinositide phosphatase SHIP1, which has been reported to modulate the function of most immune subsets. In this review, we summarize our current knowledge of SHIP1 with a focus on innate immune cells, where we reflect on the most pertinent aspects described in the current literature. We also present several small‐molecule agonists and antagonists of SHIP1 developed over the last two decades, which have led to improved outcomes in several preclinical models of disease. We outline these promising findings and put them in relation to human diseases with unmet medical needs, where we discuss the most attractive targets for immune therapies based on SHIP1 modulation.

Gräbnitz, Fabienne; Oxenius, Annette

doi: 10.1002/eji.202250225pmid: 36788705

Establishment of cellular diversity is a basic requirement for the development of multicellular organisms. Cellular diversification can be induced by asymmetric cell division (ACD), during which the emerging two daughter cells unequally inherit lineage specific cargo (including transcription factors, receptors for specific signaling inputs, metabolic platforms, and possibly different epigenetic landscapes), resulting in two daughter cells endowed with different fates. While ACD is strongly involved in lineage choices in mammalian stem cells, its role in fate diversification in lineage committed cell subsets that still exhibit plastic potential, such as T‐cells, is currently investigated. In this review, we focus predominantly on the role of ACD in fate diversification of CD8 T‐cells. Further, we discuss the impact of differential T‐cell receptor stimulation strengths and differentiation history on ACD‐mediated fate diversification and highlight a particular importance of ACD in the development of memory CD8 T‐cells upon high‐affinity stimulation conditions.

Wu, Sihan; Zhang, Xuan; Hu, Cuiping; Zhong, Yajie; Chen, Jun; Chong, Wai Po

doi: 10.1002/eji.202350574pmid: 37689974

Various regulatory CD8+ T‐cell subsets have been proposed for immune tolerance and have been implicated in controlling autoimmune diseases. However, their phenotypic identities and suppression mechanisms are not yet understood. This study found that coculture of T‐cell receptor (TCR)‐ or interferon (IFN)‐β‐activated CD8+ T cells significantly suppressed the cytokine production of Th1 and Th17 cells. By experimenting with the experimental autoimmune uveitis (EAU), we found that adoptive transfer of TCR or IFN‐β‐activated CD8+ T cells significantly lessened disease development in an IFN‐γ‐dependent manner with a decreased uveitogenic Th1 and Th17 response. Interestingly, after adoptive transfer into the EAU mice, the IFN‐γ+CD8+ T cells were recruited more efficiently into the secondary lymphoid organs during the disease‐priming phase. This recruitment depends on the IFN‐γ‐inducible chemokine receptor CXCR3; knocking out CXCR3 abolishes the protective effect of CD8+ T cells in EAU. In conclusion, we identified the critical role of IFN‐γ for CD8+ T cells to inhibit Th1 and Th17 responses and ameliorate EAU. CXCR3 is necessary to recruit IFN‐γ+CD8+ T cells to the secondary lymphoid organ for the regulation of autoreactive Th1 and Th17 cells.

Clausen, Björn E.; Amon, Lukas; Backer, Ronald A.; Berod, Luciana; Bopp, Tobias; Brand, Anna; Burgdorf, Sven; Chen, Luxia; Da, Meihong; Distler, Ute; Dress, Regine J.; Dudziak, Diana; Dutertre, Charles‐Antoine; Eich, Christina; Gabele, Anna; Geiger, Melanie; Ginhoux, Florent; Giusiano, Lucila; Godoy, Gloria J.; Hamouda, Ahmed E.I.;

Zhao, Lu; Song, Renjie; Liu, Yang

doi: 10.1002/eji.202350536pmid: 37724936

Viral infections can result in metabolism rewiring of host cells, which in turn affects the viral lifecycle. Phosphoenolpyruvate (PEP), a metabolic intermediate in the glycolytic pathway, plays important roles in several biological processes including anti‐tumor T cell immunity. However, whether PEP might participate in modulating viral infection remains largely unknown. Here, we demonstrate that PEP generally inhibits viral replication via upregulation of apoptosis‐associated tyrosine kinase (AATK) expression. Targeted metabolomic analyses have shown that the intracellular level of PEP was increased upon viral infection. PEP treatment significantly restricted viral infection and hence declined subsequent inflammatory response both in vitro and in vivo. Besides, PEP took inhibitory effect on the stage of viral replication and also decreased the mortality of mice with viral infection. Mechanistically, PEP significantly promoted the expression of AATK. Knockdown of AATK led to enhanced viral replication and consequent increased levels of cytokines. Moreover, AATK deficiency disabled the antiviral effect of PEP. Together, our study reveals a previously unknown role of PEP in broadly inhibiting viral replication by promoting AATK expression, highlighting the potential application of activation or upregulation of the PEP‐AATK axis in controlling viral infections.

Waterhölter, Alex; Wunderlich, Malte; Turner, Jan‐Eric

doi: 10.1002/eji.202350483pmid: 37740567

Mucosal‐associated invariant T (MAIT) cells are T cells that express a semi‐invariant αβ T‐cell receptor (TCR), recognizing non‐peptide antigens, such as microbial‐derived vitamin B2 metabolites, presented by the nonpolymorphic MHC class I related‐1 molecule. Like NKT cells and γδT cells, MAIT cells belong to the group of innate‐like T cells that combine properties of the innate and adaptive immune systems. They account for up to 10% of the blood T‐cell population in humans and are particularly abundant at mucosal sites. Beyond the emerging role of MAIT cells in antibacterial and antiviral defenses, increasing evidence suggests additional functions in noninfectious settings, including immune‐mediated inflammatory diseases and tissue repair. Here, we discuss recent advances in the understanding of MAIT cell functions in sterile tissue inflammation, with a particular focus on autoimmunity, chronic inflammatory diseases, and tissue repair.