Molecular Immunology

Naturally occurring variation in the bovine mannose receptor C-type 1 gene (MRC1) may shape macrophage responses to Mycobacterium (M.) bovis, a key driver of bovine tuberculosis (bTB). We identified four coding region SNPs in MRC1 across Bos taurus (Holstein Friesian, Brown Swiss) and Bos indicus (Boran, Sahiwal) cattle breeds, including a non-synonymous variant, rs380943118 (c.2963G>A; Ser988Asn) in C-type lectin-like domain (CTLD) 6, most prevalent in Sahiwal cattle. Structural modelling suggested that the S988N substitution, which is spatially separated from the monosaccharide binding site of CTLD4, might indirectly affect glycan binding, perhaps through a conformational change in the receptor. Monocyte-derived macrophages upregulated MR expression during differentiation, with heterozygous (G/A) animals showing higher MR expression and increased uptake of GFP-M. bovis BCG, although differences were not statistically significant. Anti-CD206 blockade did not inhibit BCG internalization, either indicating that this specific antibody did not bind to a CTLD involved in ligand binding or that MR is not the sole entry receptor. These results highlight naturally occurring MRC1 polymorphisms that may influence MR structure and macrophage function, providing a foundation for future studies to assess their role in bTB susceptibility.

The IFN-I (type I interferon) signaling pathway is the first line of defence against foreign pathogens. Stringent control of signalling pathways is necessary to maintain host immune responses and homeostasis. However, the underlying mechanism for its tight regulation is yet completely understood. In this study, we demonstrated that the TRIM family protein tripartite motif-containing 52 (TRIM52) is a novel negative regulator of IFN-{beta} production. Ectopically expressed TRIM52 markedly inhibited the activation of the IFN-{beta} promoter by ectopic expression of cGAS/STING, RIG-IN, or TRIF, MAVS, STING, and TBK1 but not by IRF3/5D, indicating that TRIM52 targets TBK1. TRIM52 also significantly inhibited the IFN-{beta}, ISG54, and ISG56 production, the dimerization of IRF3 and the nuclear localization of IRF3-YFP induced by ectopic expression of TBK1. Co-immunoprecipitation experiment revealed that TRIM52 specifically interacted with TBK1. Furthermore, the TBK1 protein, but not its mRNA, decreased considerably with increasing expression of TRIM52, and TRIM52 did not decrease the expression of the cGAS, STING, or IRF3 proteins. In addition, proteasome inhibitor MG-132 blocked the reduced TBK1 induced by TRIM52, indicating that TRIM52 caused TBK1 degradation via the proteasome pathway. Co-IP and ubiquitination assays demonstrated that TRIM52 promotion of K48-linked ubiquitination of TBK1, which depends on its E3 ubiquitin ligase. Collectively, our findings identify a previously unrecognized role of TRIM52 in regulating the IFN-I signalling pathway through targeting TBK1 for polyubiquitination and degradation.

Turbot (Scophthalmus maximus) is an important aquaculture species, but the genomic organization and expressed diversity of its antibody repertoire remain incompletely characterized. In this study, we annotated the immunoglobulin heavy chain (IGH) locus using the haplotype resolved fScoMax1.1 genome assembly, and we used this as a reference to profile the expressed turbot IgM, IgD and IgT repertoires in skin and spleen. The primary IGH locus was located on chromosome 19, spanned approximately 72 kb, and contained 25 IGHV genes, including 24 functional genes and one pseudogene, together with three IGHD, seven IGHJ and three IGHC genes corresponding to IgT, IgM and IgD. Comparison with the alternate fScoMax1.1 haplotype and a second turbot genome assembly showed conserved IGHD, IGHJ and IGHC content, whereas IGHV gene number differed among assemblies. High throughput 5RACE repertoire sequencing revealed isotype and tissue associated differences in expressed IGH diversity. IgM represented the dominant productive repertoire in both skin and spleen and showed the highest clonotypic diversity, particularly in spleen. IgD displayed an intermediate profile, whereas IgT was more enriched in skin and exhibited the strongest clonal restriction. IGHV subgroup usage was dominated by IGHV3 in IgM and IgD, whereas IgT showed a distinct profile characterized by preferential use of IGHV4, especially in skin. Gene level analysis further showed broad IGHV-IGHJ pairing in IgM and IgD, with preferential use IGHJ3 segment, while IgT sequences paired exclusively with IGHJT. Clonotype sharing between skin and spleen was isotype dependent, being strongest for IgT, intermediate for IgM, and negligible for IgD, suggesting that clonal expansion did not necessarily predict inter tissue trafficking. Together, these results provide a curated genomic and expressed repertoire framework for turbot IGH genes and reveal isotype specific organization of antibody diversity, with IgT displaying a particular repertoire pattern.

Tuberculosis (TB) has been a leading cause of death from a single infectious agent for decades. Bacille Calmette-Guerin (BCG) remains both the primary TB vaccine strategy and the oldest vaccine in circulation, with severe limitations in adult populations. Recently, live attenuated vaccine strategies, or generating safe strains of Mycobacterium tuberculosis (Mtb) through genetic engineering, have shown considerable promise. We previously developed an attenuated strain of Mtb lacking the nucleoid-associated protein Lsr2 ({Delta}lsr2) which is also phthiocerol dimycocerosates (PDIM) deficient and induces an immune response that represents an intermediate stage between the parental Mtb strain and BCG. In this study we examined the immune response of {Delta}lsr2 vaccinated mice in comparison to BCG and found a substantially stronger CD4 and CD8 T cell responses from {Delta}lsr2 vaccinated mice. Complementary, we conducted Mtb protection studies in {Delta}lsr2 and BCG vaccinated mice and guinea pigs, where we found that {Delta}lsr2 provided superior protection in both animals. This improved protection is shown with reduced bacterial burden and improved organ pathology in the lungs and spleen. Taken together, our work shows {Delta}lsr2 serves as a promising vaccine candidate for continued preclinical development.

Autoimmune diseases, such as systemic lupus erythematosus (SLE), are underscored by dysregulated B cell function including the production of autoantibodies, skewed population ratios, and aberrant signaling. Given that the family of nuclear factor kappa B (NF-{kappa}B) transcription factors govern responses to stimuli, survival, differentiation, and so forth understanding the intricate regulatory network of NF-{kappa}B in B cell biology is paramount for unraveling treatments for B cell-linked autoimmune diseases. Here, we focus on a negative regulator of NF-{kappa}B signaling, A20 (TNFAIP3), that deactivates NF-{kappa}B transcription factor translocation through the ubiquitination and deubiquitination of target proteins. Haploinsufficiency in A20 results in an autoimmune phenotype and mutations to A20 have been associated with SLE, suggesting implications to B cell function. To investigate the role of A20 in NF-{kappa}B in human B cells, we generated a TNFAIP3 knockout (KO) Raji cell line. Cells were stimulated with either anti-IgM or Resiquimod (R848) to activate distinct NF-{kappa}B signaling pathways. Using qRT-PCR, western blotting, and flow cytometry, we assessed differences in gene expression, protein production, and NF-{kappa}B activation. We observed key limitations in using Epstein-Barr virus transformed B cell lines to model inducible NF-{kappa}B signaling.

Abstract Introduction: IL-15 is one of the most promising candidate cytokines in cancer immunotherapy due to its ability to promote the activity of different cytotoxic innate immune cell subsets such as NK, ILC1 and gammadelta T cells. During biosynthesis, IL-15 associates with IL-15alpha and is transported to the cell surface where IL-15Ralpha trans-presents IL-15 to target neighboring cells expressing the beta chain (IL-2Rbeta) and the common gamma chain. Our group previously showed that in autoimmune type 1 diabetes and early innate immune responses to infections trans-presentation by IL-15Ralpha is dispensable. Here we addressed the relative roles of IL-15 and trans-presented IL-15 in the control of established tumors and spontaneous tumor development. Methodology: Growth kinetics of tumor cell lines were monitored in WT, Il15-/- and Il15ra-/- mice. Spontaneous fibrosarcoma was induced with Methylcholanthrene (MCA) in WT, Il15-/- and Il15ra-/- mice. Cell lines were established from MCA-induced tumors to characterize their immunogenicity. Results: Growth of established tumor cell lines were comparable in the three genotypes. MCA-induced tumor incidence was reduced in Il15ra-/- mice when compared to WT and Il15-/- mice. In vitro, MCA tumor-derived cell lines expressed MHC-I and PD-L1 and had comparable proliferation rates. In vivo, MCA tumor-derived cell lines established from the 3 genotypes showed comparative growth in WT mice suggesting that IL-15 does not impact immunoediting. Nonetheless, NLRC5 expressing B16-F10 tumors were contained in WT and Il15ra-/- mice but not in Il15-/- mice. Conclusions: Taken together, these results show that in the absence of trans-presentation by IL-15Ralpha, IL-15 can better control spontaneous tumor development and that IL-15 signaling plays a minor role in immunosurveillance in this model. IL-15 signaling, independent of IL-15Ralpha has a significant role in the control of solid tumors.

RNA-binding protein LARP4 plays an important role in T cell activation and differentiation, but its role in B cell biology and the pathogenesis of systemic lupus erythematosus (SLE) remains unclear. This study found that LARP4 was specifically highly expressed in B cells of SLE patients and was positively correlated with disease activity. By constructing T cell-specific and B cell-specific conditional knockout mice, we found that deletion of LARP4 in B cells, but not in T cells, significantly alleviated pristane-induced and Bm12-induced lupus nephritis. Further analysis showed that LARP4 deletion selectively inhibited B cell differentiation into plasma cells, but did not affect germinal center B cell formation. Integrated transcriptomic and metabolomics analyses revealed that this effect is due to reduced phosphatidic acid synthesis and decreased mTORC1 activity caused by mitochondrial oxidative phosphorylation dysfunction. Furthermore, we used LIPEP, a LARP4 inhibitory peptide that effectively mimicked the therapeutic effects of LARP4 gene knockout in the MRL/lpr spontaneous lupus model and outperformed cyclophosphamide in reducing glomerular immune complex deposition and improving extrarenal dermatitis. These results indicates that LARP4 is a key metabolic checkpoint regulating B cell differentiation into Plasma cells and suggest that it may be a potential therapeutic target for SLE.

Fc receptor-like protein 5 (FCRL5) is a low-affinity IgG receptor expressed on B cells, with emerging therapeutic relevance due to its expression on multiple myeloma cells, and a potential role in regulating B cell responses. Previous reports on the FCRL5-IgG interaction vary widely in reported affinities, binding differences across IgG subclasses, and molecular requirements for maximal binding. Furthermore, the impact of Fc-engineering strategies, as used in (therapeutic) monoclonal antibodies, remains poorly understood. Here, we provide a comprehensive biochemical analysis of the FCRL5-IgG interaction. We demonstrate that FCRL5 is a true IgG Fc-receptor, binding with very low affinity (60-80 M). FCRL5 binds IgG in a manner involving primarily the two N-terminal domains of FCRL5, and the third domain for maximal binding, but with distinct essential residues in the IgG Fc-tail. Surface plasmon resonance analysis of the binding of FCRL5 to the various IgG subclasses revealed a preference for IgG1 and IgG4. Interestingly, various Fc-engineered IgG variants commonly used for silencing or enhancing of Fc receptor binding do not impact FCRL5 binding. Screening the binding of a set of IgG antibodies carrying defined sets of Fc-mutations to FCRL5 revealed E293 as a key binding determinant and led to the discovery of E293R as a mutation that selectively abrogates FCRL5 binding while preserving binding to other classical Fc{gamma}Rs. Lastly, we show that FCRL5 has considerable preference for binding afucosylated IgG. Together, our results define the essential characteristics of the IgG-FCRL5 interaction and demonstrate the potential of both naturally occurring IgG variants as well as therapeutically explored bioengineered IgG formats to differentially engage FCRL5.

Black-footed ferrets (Mustela nigripes) are highly susceptible to sylvatic plague caused by Yersinia pestis, but the genetic basis of this vulnerability remains poorly understood. Here, comparative immunogenomic analyses across Carnivora species identified a conserved class of immunoglobulin lambda variable (IGLV) genes with unusually long antigen-binding sites (CDRL1) that are common among Caniformia species but absent in Feliformia species. First discovered in the domestic ferret (Mustela putorius furo), these genes encode tyrosine-rich and anionic motifs resembling the chemokine receptor CCR5 and contain experimentally validated sulfotyrosines previously associated with pathogen-interacting interfaces. Evolutionary analyses revealed distinct selective pressures across Caniformia lineages and showed strong purifying selection acting on long-CDRL1 IGLV genes in mustelids and bears. Antibody repertoire sequencing demonstrated that these genes are actively utilized in expressed repertoires and that their usage correlates with evolutionary conservation. Functional analyses of monoclonal antibodies derived from the long-CDRL1 IGLV gene identified an antibody that significantly reduced intracellular Y. pestis survival in macrophages and revealed a positive correlation between anti-plague activity and sulfotyrosine signal. Notably, all analyzed black-footed ferrets carried a frameshifting deletion in the long-CDRL1 IGLV gene resulting in loss of its expression in antibody repertoires. Together, these findings uncover a germline-encoded immunoglobulin feature conserved across dog-like carnivores and suggest a potential link between antibody germline variation and immune responses to plague.

Detection of viral infection leads to both cell-intrinsic and cell-extrinsic responses. In mammals, cell-intrinsic detection of viral infection leads to cell-extrinsic activation of STAT (Signal Transduction and Activators of Transcription) proteins, a family of transcription factors that promote anti-viral defense. In the nematode C. elegans, STA-1/STAT is a negative regulator of anti-viral defense, but it is not known if it acts cell-intrinsically or cell-extrinsically and whether it has functional domains conserved with mammalian STATs. Here we show that C. elegans STA-1 protein disappears from nuclei of cells infected with the natural viral pathogen, Orsay virus, but remains nuclear in uninfected cells, indicating a cell-intrinsic site of action. During viral infection, STA-1 forms cytoplasmic puncta that interact with the RNA viral sensor DRH-1, suggesting that DRH-1 helps restrain this immune-repressive factor. STA-1 overexpression causes increased susceptibility to viral infection, in a manner dependent on conserved residues important for DNA binding, nuclear localization and phosphorylation. Structural predictions indicate that STA-1 is most similar to STAT5 proteins in mammals, which have known immune-repressive roles. Our transcriptomic analysis demonstrates that C. elegans STA-1 regulates a general anti-pathogen program, including genes upregulated later during viral infection. Altogether, our findings provide insight into conserved and distinct features of STA-1 in C. elegans, indicating an ancient role for cell-intrinsic, immune-repressive STATs. Author SummaryAll living organisms must detect viral infections and mount a defense to survive. One major antiviral defense pathway in mammals is the interferon response, which involves sensing viral infection in one cell, and delivering an interferon message to neighboring cells. These neighboring cells then turn on anti-viral defense gene expression using proteins called STAT transcription factors. We study anti-viral defense in the roundworm C. elegans, and in this study show that viral infected cells themselves use a STAT protein called STA-1, with perhaps a lesser role for STA-1 in neighboring cells, in contrast to mammals. We also extend on previous findings that STA-1 turns off anti-viral gene expression, and we analyze regions in the protein to demonstrate that STA-1 is bona fide transcription factor with an immune-repressive role. Structural prediction analysis of STA-1 indicates it is most similar to STAT5 in mammals, suggesting an ancient role for this protein as an immune-repressive factor acting directly in virally infected cells.

BackgroundTuberculosis remains a leading global infectious killer, with BCG offering inconsistent adult protection and rising drug-resistant strains demanding novel vaccine strategies. We report the first multi-epitope vaccine construct simultaneously targeting three previously unexplored Mycobacterium tuberculosis virulence proteins; EccB3, MycP, and polyketide synthase which collectively govern nutrient acquisition, ESX secretion integrity, and innate immune evasion. MethodsUsing a reverse vaccinology pipeline, B-cell, CTL, and HTL epitopes were predicted, filtered for allergenicity, toxicity, and IFN-{gamma} induction, then assembled into an 823-residue chimeric construct incorporating beta-defensin and PADRE adjuvants with AAY/GPGPG linkers, covering [~]90% global HLA diversity. The construct underwent AlphaFold structure prediction, 3DRefine refinement, disulfide engineering, PROCHECK/ProSA validation, ClusPro 2.0 docking against TLR1/TLR2, and C-IMMSIM immune simulation. ResultsThe construct (82.3 kDa, instability index 32.48) showed strong structural quality (94.7% favoured Ramachandran residues), stable TLR1/TLR2 binding (weighted energy: -1,371.0 kcal/mol), and robust in silico immune responses and durable memory cell formation following booster simulation. ConclusionThis computationally validated construct represents a promising multi-target TB vaccine candidate warranting experimental advancement.

Interleukin-38 (IL-38) is a cytokine of the IL-1 cytokine family that promotes the resolution of inflammation. Resolution mechanisms comprise the induction or recovery of immune tolerance that is lacking in various acute and chronic inflammatory pathologies, including Graft-versus-Host Disease (GvHD). The role of IL-38 in the context of immune tolerance, its primary immune cell targets and underlying molecular mechanisms are not defined. In this study, we investigated the impact of IL-38 on human alloreactivity and in a mouse model of acute GvHD. Our data suggests that monocytes differentiating into macrophages are the main cellular target of IL-38. Specifically, IL-38 reduces antigen presentation capacity in differentiating monocytes through an IL-1 family receptor-independent mechanism, which subsequently avoids T-cell activation. In parallel, IL-38 ameliorates inflammation in allogeneic settings in human and murine GvHD models by promoting the expansion of regulatory T-cells. Our findings indicate that IL-38 promotes immune tolerance during alloreactivity by affecting myeloid cells and T-cells.

T cells are important components of the adaptive immune system and develop through a selection process regulated by signaling through the T-cell receptor (TCR). Thymocyte-Expressed Molecule Expressed in Selection (THEMIS) is a TCR-proximal protein that modulates the activity of Shp1 phosphatase to influence TCR signaling during development. THEMIS has been shown to both activate and inhibit Shp1, but the molecular mechanisms of these functions are poorly understood. THEMIS contains two rare Cysteine All-Beta In THEMIS (CABIT) domains, the N-terminal of which interacts with Shp1 and is likely responsible for modulation of its phosphatase activity. Herein, we report the first crystal structure of the THEMIS CABIT1 domain. While a portion of the CABIT1 domain is poorly resolved, it appears to share the same overall fold observed in our recent CABIT2 crystal structure and AlphaFold predictions. We show that phosphorylation of the CABIT1 domain by LCK is required for association with SHP1 and that phosphorylated CABIT1 can protect Shp1 from oxidation and inhibition by reactive oxygen species (ROS), which may serve as a mechanism by which THEMIS enhances Shp1 activity.

EWSR1::FLI1, the oncogenic chimeric transcription factor driving Ewing sarcoma (EwS)induces expression of exquisitely EwS-specific neogenes (Ew_NGs) through neomorphic binding and transcription activation at GGAA microsatellites in genomic regions that are silent in normal tissues. We show that peptides encoded by Ew_NGs are presented on HLA-I complexes on EwS cells. The cytokine secretion of CD8+ T cells specific for Ew_NG-encoded HLA-I-bound peptides is activated by all HLA-I-matched EwS cells but not by non-EwS cells. These T cells kill EwS cells in an HLA-I restricted manner. This cytotoxicity is dependent on the expression of EWSR1::FLI1 and of the corresponding Ew_NG. It can be reproduced by transduction of the TCR into donor T cells (TCR-T) which kill EwS cells in vivo. Moreover, we show that neither off target nor allogeneic activation are observed with TCR-T thus paving the way for cell therapy in relapsed/resistant EwS patients for which therapeutic options are very limited. Statement of significanceThe chimeric transcription factor EWSR1::FLI1 generates tumor-specific neogenes encoding neoantigen presented by the HLA-I molecules of Ewing cells. Neoantigen-specific CD8+ T-cell clones and engineered TCR-T cells can selectively recognize and kill EwS tumor cells in vitro and in vivo.

To establish chronic infection in the vasculature of their infected host, schistosomes have developed multifaceted strategies of immune subversion. Extracellular parasite proteins are believed to play immunomodulatory functions, but their mode of action remains largely elusive. To investigate whether proteins secreted by the Schistosoma mansoni parasite have the potential to directly interact with host immune receptors, we performed a large-scale protein:protein interaction study between selected parasite proteins sharing structural similarities with known host immune effectors and a protein array of over 750 full-length human ectodomains mostly expressed by immune cells. We identified CD177 as a neutrophil receptor for S. mansoni Granulin (SmGrn). SmGrn exclusively bound the surface of CD177+ human neutrophils and led to cellular hyporesponsiveness following stimulation with LPS as evidenced by decreases in surface markers of activation, delayed reactive oxygen species production and reduced IL-8 release. In addition, human neutrophils exposed to SmGrn showed delayed apoptosis and morphological changes compatible with a more quiescent state as well as transcriptional upregulation of negative regulators of interferon signalling. These data suggest that SmGrn dampens human neutrophil response to stimulation and may lead to suboptimal function during schistosome infection.

Ubiquitin signalling is mediated by structurally distinct polyubiquitin chains that encode discrete cellular functions. Progress in deciphering this ubiquitin code, particularly for the less abundant atypical chain types, has been hindered by limited availability of versatile chain type-specific affinity reagents. Here, we demonstrate that human single-domain antibodies (sdAbs) provide a versatile scaffold for the generation of ubiquitin linkage-specific binders. Using phage display and synthetic human sdAb libraries, we identified 2A6, an sdAb that specifically recognises methionine-1 (M1)-linked ubiquitin chains. To our knowledge, 2A6 represents the first reported sdAb with specificity for a defined homotypic ubiquitin chain linkage. 2A6 bound M1-linked ubiquitin chains with nanomolar affinity and was specific for M1-linked chains at the level of both diubiquitin and long polyubiquitin chains. AlphaFold3 modelling, supported by saturation mutagenesis, predicted that 2A6 recognises the proximal and distal ubiquitin moieties together with the region near the M1 linkage. Functionally, 2A6 enabled specific detection and enrichment of M1-linked ubiquitin across multiple applications, including ELISA, immunoblotting, immunoprecipitation under semi-denaturing conditions, substrate ubiquitination analysis, and immunofluorescence microscopy. The sdAb can be readily produced in E. coli from a single expression plasmid, providing a tractable, cost-effective and versatile reagent for investigating M1-linked ubiquitin signalling. Our work establishes sdAbs as a versatile scaffold for ubiquitin linkage-specific affinity reagents, providing a framework for the development of analogous binders specifically targeting additional ubiquitin linkages or architectures.

Background: Abdominal aortic aneurysms (AAA) are characterized by dilation of the aorta that can lead to aortic rupture and death. The transcriptional co-activators Yes-Associated Protein (YAP) and WW-domain-containing transcriptional co-activator with PDZ-binding motif (TAZ) are mechanosensitive effectors of the highly conserved Hippo signaling pathway. It is hypothesized that cell-specific YAP/TAZ signaling in endothelial cells (EC) plays a pivotal role in mediating AAA formation and rupture. Methods: Single-cell RNA-sequencing in human AAAs was performed and differentially expressed genes (DEGs) were identified in the endothelial cell cluster. YAP/TAZ mRNA and protein expression were also assessed in human AAA and control aortic tissue. Two established murine AAA models were used with male C57BL/6 and EC-CreERT2-YAPfl/fl/TAZfl/fl mice with/without Verteporfin (VPF, YAP/TAZ inhibitor) and XMU-MP-1 (YAP/TAZ activator) treatments. On postoperative days 14 and 28, aortic diameter, histology, cytokine, and MMP2 expressions were evaluated. Results: A significant alteration in EC-specific differentially expressed YAP/TAZ-related genes was observed in which 242 genes were upregulated and 71 genes were downregulated in AAA compared to controls. Human AAA tissue showed a significant increase in YAP and TAZ protein expressions compared to controls. Elastase-treated EC-YAP/TAZ-/- mice showed a significant decrease in AAA diameter compared to littermate controls. Histological quantification revealed preservation of -smooth muscle actin, reduced elastin fiber breaks, and decreased macrophage infiltration in EC-YAP/TAZ-/- mice compared to littermate controls. Importantly, pharmacological inhibition of YAP/TAZ using VPF significantly attenuated AAAs in two experimental murine models. In vitro data demonstrates that VPF inhibits endothelial cell YAP expression, downregulating pathways associated with pathogenic angiogenesis and vascular inflammation. Conclusions: These data suggest that EC-specific YAP/TAZ signaling mediates AAA formation. Pharmacological inhibition of the Hippo pathway can significantly mitigate aortic inflammation and vascular remodeling to decrease the progression of AAAs and prevent aortic rupture.

Hematopoietic stem cells (HSCs) play a pivotal role in the lifelong maintenance of hematopoiesis. However, heterogeneity and age-related alterations in HSC populations hinders accurate HSC analysis. Here, we show that bone marrow (BM) macrophage fragments that preferentially express F4/80 adhere to proliferative rather than dormant HSCs. The adhesion of macrophage fragments to proliferative HSCs occurred throughout the process of BM cell preparation in vitro. Consistently, proliferative HSCs express genes involved in the adhesion of macrophage fragments at higher levels than dormant HSCs. Notably, by using that as a benchmark, dormant HSCs can be easily identified as F4/80lowHSCs throughout their lifespan, thereby revealing that they retain considerable stemness and remain functional with aging. Collectively, we propose a novel and straightforward method for the rapid identification, isolation, and analysis of distinct HSC subpopulations, which will be helpful for a wide range of hematological studies and will provide insights into HSC biology.

Chitin is a highly abundant poly- N-acetyl-glucosamine (GlcNAc) and linked to immune recognition of fungal infections and asthma in humans. Ubiquitous in fungi and insects, in mammals and plants chitin represents a microbe-associated molecular pattern (MAMP) and whereas highly polymeric chitin is insoluble and immunologically inert, soluble chitin oligomers of 6 to 15 GlcNAc activate immediate pro-inflammatory cytokine release via TLR2 in human immune cells. However, TLR2 ligands do not the most typical activators of the NLRP3 inflammasome pathway or innate immune training, a phenomenon of long-term immunological remodeling. Here we show that especially 16-20 GlcNAc long chitin oligomers activate NLRP3-dependent IL-1{beta} and IL-18 release in human myeloid immune cells in an atypical, phagocytosis-dependent manner. Moreover, phagocytosis and methyl transferase activity were essential for innate immune training, by which the same chito-oligomer-training enhanced TNF release in primary murine and human immune cells. Collectively, this suggests that oligomer length impacts on the immune features of chitin which can be customized using glycan assembly.

Background: This study aimed to elucidate B cell subset pathology in COPD, a poorly characterized area, with a focus on its similarities to and differences from classical autoimmune disorders. Methods: Single-cell RNA-sequencing (scRNA-seq) data from COPD and autoimmune diseases were obtained from the Gene Expression Omnibus (GEO) for comparative analyses of B cell subsets and functions via differentially expressed genes (DEGs), KEGG, protein-protein interaction (PPI), and cell-cell communication analyses. Serum IgG4 was measured by ELISA and correlated with clinical parameters. Peripheral blood B cells were sorted by flow cytometry for single-cell B cell receptor (BCR) sequencing. A v-Abl-Bcl2 pro-B cell line was stimulated with cigarette smoke extract (CSE) to assess abnormal development in vitro. Results: In lung tissue, IgG4 plasma cells were enriched and expressed BCR activation and inflammatory genes and TNF-NF-kB-MAPK pathways. Serum IgG4 concentrations correlated negatively with pre- and post-bronchodilator FEV1-FVC. B cells interacted with monocytes, macrophages, fibroblasts, and endothelial cells via IL-1B-IL-6, integrin, and chemokine signaling, contributing to chronic inflammation and remodeling. In peripheral blood, transitional T1 B cells were increased, accompanied by lambda-chain enrichment and increased IGLV1-47 usage, as well as enrichment of autoimmune pathways. In the bone marrow, the numbers of pre-B I cells were increased while those of small pre-B III cells were reduced, with altered expression of BCR development genes. CSE stimulation of the pro-B cell line reduced lambda expression in a concentration-dependent manner. Conclusions: The autoimmune abnormalities in COPD appear more restricted, although IgG4 antibody generation may contribute to immune-mediated lung damage.