European Journal of Immunology最新文献

The stimulator of interferon genes (STING) pathway plays a pivotal role in innate immunity, acting as a key sensor of cytosolic DNA to initiate type-I Interferon (IFN) and pro-inflammatory cytokine production. This pathway is essential for host defence against bacterial, viral and other pathogenic threats and has emerged as a promising therapeutic target in cancer immunotherapy. However, conventional techniques such as immunoblotting and qPCR are limited in their capacity to study STING pathway activation in complex and heterogeneous biological systems, such as tumour masses or large cell populations. Here, we describe the application of mass cytometry (CyTOF) as a cutting-edge approach to characterize the STING pathway at the sub-population level. Using a high-dimensional panel of metal-labelled antibodies targeting key STING signalling components, we achieved resolution of pathway activation across diverse immune cell populations. This approach promises novel insights into cellular heterogeneity, pathway dynamics and the interplay between STING signalling and other immune pathways and underscores the power of high-dimensional analysis to overcome the limitations of traditional methods to enable a more comprehensive exploration of immune signalling pathways.

Regulatory T cells (Tregs), defined by the lineage-specific transcription factor FoxP3, are crucial for immune regulation and have been studied extensively in mammals. However, avian Tregs remain poorly characterized, leaving gaps in our understanding of their evolutionary conservation and unique features. In this study, we investigated the phenotype of chicken Tregs to define reliable markers for their identification and characterization. We analyzed CD4⁺ splenocytes sorted into CD25^negative, CD25^low, and CD25^high subpopulations using RNA sequencing. FOXP3 and other Treg-associated genes were expressed in both CD25^low and CD25^high populations, showing that CD25 expression alone is insufficient to distinguish chicken Tregs. To refine the marker profile, we evaluated additional markers, including CTLA-4 and GITR. Notably, we describe for the first time a chicken-specific CTLA-4 antibody, which uniquely stains CTLA-4 exclusively in intracellular (ic) compartments, distinguishing it from mammalian counterparts. Single-cell RNA sequencing further confirmed distinct FOXP3⁺ clusters enriched for expression of CTLA4 and TNFRSF18 (encoding GITR). While CTLA-4's ic expression limits usability in functional assays, the combination of CD4⁺/CD25⁺/CTLA-4⁺/GITR⁺ represents the most accurate characterization of putative chicken Tregs to date. These findings highlight evolutionary conservation and species-specific differences in Treg markers, providing the foundation for future studies on chicken Treg functionality.

The widespread translation of cryptic proteins derived from the non-coding genome expands the complexity of the human proteome. A vast majority of cryptic proteins are expressed at low levels, rapidly degraded and efficiently presented on class I major histocompatibility complexes (MHC-I). On the other hand, some cryptic proteins are stable and functional and may integrate into the proteome through ongoing selective pressures. Herein, we propose a model in which the translation of cryptic proteins increases the diversity of functional proteins on which evolution can act and, during this trial-and-error process, provides a valuable source of antigens for immunosurveillance.

Severe COVID-19 is an immunological disorder characterized by excessive immune activation following infection with SARS-CoV-2, which typically occurs around the time of seroconversion. Anti-spike IgG of critically ill COVID-19 patients induces excessive inflammation by activation of Fc gamma receptors (FcγRs) on human alveolar macrophages, leading to tissue damage, pulmonary edema, and coagulopathy. While metabolic reprogramming of immune cells is critical for the induction of inflammatory responses, still little is known about the metabolic pathways that are involved in COVID-19-specific hyperinflammation. In this study, we identified that anti-spike IgG immune complexes (ICs) induce rapid metabolic reprogramming of alveolar macrophages, which is essential for the induction of inflammation. Through functional inhibition, we identified that glycolysis, fatty acid synthesis, and pentose phosphate pathway (PPP) activation are critical for anti-spike IgG-induced hyperinflammation. Remarkably, while excessive proinflammatory cytokine production by macrophages is critically dependent on simultaneous stimulation with viral stimuli and anti-spike IgG complexes, we show that the required metabolic reprogramming is specifically driven by anti-spike IgG complexes. These findings provide new insights into the metabolic pathways driving hyperinflammation by macrophages in the context of severe COVID-19. Targeting of these pathways may reveal new possibilities to counteract pathological inflammatory responses in severe COVID-19 and related diseases.

Self-tolerance in T cells is a vital self-defense strategy for mammals to specifically respond to invading pathogens. During T cell development in the thymus, thymocytes migrate from the cortex to the medulla to sequentially acquire non-self-reactivity and self-tolerance. This cortex-to-medulla migration is regulated by CCR7-mediated chemokine signaling. Previous studies have identified CCL21 but not CCL19 as a functional ligand for this CCR7-dependent migration. CCL21 in the mouse is encoded by multiple genes, including CCL21Ser-encoding Ccl21a and several CCL21Leu-encoding genes, including Ccl21b. The importance of Ccl21a in thymocyte migration has been demonstrated, whereas the role of CCL21Leu-encoding genes remains unclear. By producing mice specifically deficient in Ccl21b, we show that Ccl21b plays little to no role in the cortex-to-medulla migration of developing thymocytes. CCL21Leu-encoding gene transcripts remain detectable even in the absence of Ccl21b, suggesting that Ccl21b is not a major source of CCL21Leu. We further show that the copy number of CCL21Leu-encoding genes is smaller than the currently estimated copy number in a public database. These findings underscore the predominant role of Ccl21a over Ccl21b in the mouse thymus.

Graft-versus-host disease is mediated by donor-derived T cells reactive against the recipient's broadly expressed minor histocompatibility antigens (mHA). Regulatory T cells (Treg) have been explored as a therapeutic approach for chronic GVHD (cGVHD). The promising results from polyclonal Treg trials in this setting have led us to develop a Treg product specific for mismatched minor antigens between patient and donor (mTreg), circumventing broad immune suppression risks. HLA-matched siblings of opposite sexes were used to obtain the sister's CD4⁺CD25^hiCD127^low Treg for co-culture with the respective brother's dendritic cells as a source of mismatched mHA. We have established the optimal culture conditions resulting in the highest mTreg proliferation and viability. Comprehensive phenotyping during the ex vivo selection shows PD-1, CTLA-4, CD39 and HLA-DR expression. Transcriptomic analysis revealed a switch in metabolic process, and up-regulation of functional Treg genes. Furthermore, mTreg possess specific and potent suppressive activity, in which there is a dependency on cell-to-cell contact and a role for HLA class II expression on mTreg. This protocol would allow the generation of Treg specific to an array of mHA from the recipient's healthy tissues, likely providing a directed and strong suppression of cGVHD.

Mast cells are evolutionarily ancient immune cells located at strategic entry points for pathogens and allergens. Allergen exposure activates signal transduction pathways resembling those downstream of antigen receptors in T and B lymphocytes, leading to mast cell degranulation and cytokine secretion. The paralogous RNA-binding proteins ROQUIN-1 and ROQUIN-2 prevent aberrant T cell activation and differentiation and are cleaved upon antigen receptor engagement. Here, we investigated their roles in connective tissue mast cells using conditional gene knockout in mice. We show that ROQUIN-1 and ROQUIN-2 are dispensable for skin mast cell development and maintenance, while they are essential for serosal mast cells residing in the peritoneal and pleural cavities. Concurrent ablation of both paralogs did not affect mast cell degranulation in vitro and in vivo, nor did it alter activation-induced secretion of TNF and IL-6, cytokines that are regulated by ROQUIN proteins in other cell types. Furthermore, we globally define ROQUIN-regulated mRNAs in mast cells, and validate Runx1t1 and Ebi3 as indirect and Lfng as direct ROQUIN targets. Collectively, our results highlight the essential function of ROQUIN in connective tissue mast cells in serosal cavities.

Natural clearance of hepatitis C virus (HCV) infection occurs in about 25% of primary infections, but offers only partial protective immunity against re-infections. This study hypothesised that long-lived polyfunctional HCV-specific CD8+ memory stem T cells (T_SCM) contribute to protective immunity in rare super-clearer subjects who repeatedly clear viraemia. Six super-clearers and four clearer-chronic subjects who resolved a primary infection but subsequently developed chronic infection were studied at multiple timepoints. The T_SCM population (CCR7+CD45RA+CD95+) was bulk sorted, labelled with CellTrace Violet (CTV), and stimulated in vitro for five days with cognate HCV peptide, IL-2/IL-15, and autologous PBMCs. Functionality of the expanded HCV-specific T_SCM was assessed via the proliferation, multi-potency, and stemness indices. Total HCV-specific CD8+ T cells from super-clearers exhibited enhanced proliferative recall capability compared with clearer-chronics. Furthermore, super-clearers exhibited higher HCV-T_SCM frequencies post-expansion (22.35 ± 34.35 vs. 2.41 ± 9.83; p = 0.0066). Notably, HCV-T_SCM in clearer-chronics had 'stemness' indices of zero in samples before the re-infection (i.e., no ability to generate T_SCM as progeny), whereas super-clearers consistently retained this key functional property. These findings suggest that the maintenance of self-renewing HCV-specific T_SCM may underpin long-term protective immunity against re-infection and could inform vaccine design strategies targeting durable cellular memory.

Monoclonal antibodies (mAbs) targeting cell-surface molecules are pivotal in biomedical research, diagnostic applications, and biotechnology. Over the past four decades, the CD nomenclature system, established by the Human Leukocyte Differentiation Antigens Workshops and endorsed by the International Union of Immunological Societies (IUIS), has provided a standardized naming convention for both mAbs and the cell surface molecules they target. G protein-coupled receptors (GPCRs) represent the largest family of cell-surface receptors, playing essential roles in both innate and adaptive immune responses. Despite their significance, GPCRs are underrepresented in terms of well-validated mAbs available for flow cytometry and therapeutic applications. At the Eleventh HLDA Workshop (HLDA11), new CD nomenclature has been assigned to thirteen GPCR cell-surface molecules expressed on immune cells: CD198 (CCR8), CD199 (CCR9), CD372 (CCR10), CD373 (CX3CR1), CD374 (XCR1), CD375 (GPR15), CDw376 (GPR26), CD377 (SSTR3), CD378 (C3AR1), CDw379 (FPR2), CD380 (LTB4R), CDw381 (GPR183), and CDw382 (F2RL1). In this article, we introduce the newly established CD nomenclature for mAbs targeting the GPCR family. We detail the quantitative expression profiles of these molecules on various subsets of leukocytes and provide validation data for these mAbs. The implications of these expression profiles are discussed for the potential therapeutic targeting of immune-mediated diseases and cancer.

Secretory IgM plays a pivotal role in promoting robust antigen-specific IgG responses, yet the mechanisms underlying its immune-enhancing effects remain incompletely understood. IgM functions through two distinct pathways: engagement of the IgM Fc receptor (FcµR) and activation of the classical complement pathway. However, the extent of redundancy between these pathways and their roles at different stages of B cell differentiation remains unclear. To address this, we utilized FcµR-deficient mice and Cµ13 mice, which express mutant IgM incapable of activating complement. Both strains exhibited impaired T-dependent immune responses to low-dose 4-hydroxy-3-nitrophenyl-chicken γ globulin. Remarkably, FcµR^-/-Cµ13 double-mutant mice showed profound defects in antigen-specific IgG production compared with either single mutant, revealing nonredundant, synergistic roles for FcµR and complement. Mechanistically, both pathways are required for early B cell activation and expansion, promoting efficient class switch recombination, germinal center (GC) formation, and plasma cell differentiation. During the GC response, IgM BCR-mediated complement activation, but not FcµR, is required for GC B-cell proliferation, survival, and affinity maturation. In contrast, FcµR primarily enhances BCR signaling in naïve B cells through downstream PI3K-AKT and MAPK pathways. These findings define two cooperative yet distinct IgM-mediated mechanisms that promote humoral immunity and regulate B cell differentiation in vivo.