技术资料

How the microbiome regulates responses of systemic innate immune cells is unclear. In the present study,our purpose was to document a novel mechanism by which the microbiome mediates crosstalk with the systemic innate immune system. We have identified a family of microbiome Bacteroidota-derived lipopeptides—the serine-glycine (S/G) lipids,which are TLR2 ligands,access the systemic circulation,and regulate proinflammatory responses of splenic monocytes. To document the role of these lipids in regulating systemic immunity,we used oral gavage with an antibiotic to decrease the production of these lipids and administered exogenously purified lipids to increase the systemic level of these lipids. We found that decreasing systemic S/G lipids by decreasing microbiome Bacteroidota significantly enhanced splenic monocyte proinflammatory responses. Replenishing systemic levels of S/G lipids via exogenous administration returned splenic monocyte responses to control levels. Transcriptomic analysis demonstrated that S/G lipids regulate monocyte proinflammatory responses at the level of gene expression of a small set of upstream inhibitors of TLR and NF-κB pathways that include Trem2 and Irf4. Consistent with enhancement in proinflammatory cytokine responses,decreasing S/G lipids lowered gene expression of specific pathway inhibitors. Replenishing S/G lipids normalized gene expression of these inhibitors. In conclusion,our results suggest that microbiome-derived S/G lipids normally establish a level of buffered signaling activation necessary for well-regulated innate immune responses in systemic monocytes. By regulating gene expression of inflammatory pathway inhibitors such as Trem2,S/G lipids merit broader investigation into the potential dysfunction of other innate immune cells,such as microglia,in diseases such as Alzheimer’s disease. View Publication

Human regulatory T cells (Treg) suppress other immune cells. Their dysfunction contributes to the pathophysiology of autoimmune diseases,including type 1 diabetes (T1D). Infusion of Tregs is being clinically evaluated as a novel way to prevent or treat T1D. Genetic modification of Tregs,most notably through the introduction of a chimeric antigen receptor (CAR) targeting Tregs to pancreatic islets,may improve their efficacy. We evaluated CAR targeting of human Tregs to monocytes,a human β cell line and human islet β cells in vitro. Targeting of HLA-A2-CAR (A2-CAR) bulk Tregs to HLA-A2+ cells resulted in dichotomous cytotoxic killing of human monocytes and islet β cells. In exploring subsets and mechanisms that may explain this pattern,we found that CD39 expression segregated CAR Treg cytotoxicity. CAR Tregs from individuals with more CD39low/- Tregs and from individuals with genetic polymorphism associated with lower CD39 expression (rs10748643) had more cytotoxicity. Isolated CD39− CAR Tregs had elevated granzyme B expression and cytotoxicity compared to the CD39+ CAR Treg subset. Genetic overexpression of CD39 in CD39low CAR Tregs reduced their cytotoxicity. Importantly,β cells upregulated protein surface expression of PD-L1 and PD-L2 in response to A2-CAR Tregs. Blockade of PD-L1/PD-L2 increased β cell death in A2-CAR Treg co-cultures suggesting that the PD-1/PD-L1 pathway is important in protecting islet β cells in the setting of CAR immunotherapy. In summary,introduction of CAR can enhance biological differences in subsets of Tregs. CD39+ Tregs represent a safer choice for CAR Treg therapies targeting tissues for tolerance induction. View Publication

Background & AimsThe immune tolerance induced by hepatitis B virus (HBV) is a major challenge for achieving effective viral clearance,and the mechanisms involved are not well-understood. One potential factor involved in modulating immune responses is mesencephalic astrocyte-derived neurotrophic factor (MANF),which has been reported to be increased in patients with chronic hepatitis B. In this study,our objective is to examine the role of MANF in regulating immune responses to HBV.MethodsWe utilized a commonly used HBV-harboring mouse model,where mice were hydrodynamically injected with the pAAV/HBV1.2 plasmid. We assessed the HBV load by measuring the levels of various markers including hepatitis B surface antigen,hepatitis B envelope antigen,hepatitis B core antigen,HBV DNA,and HBV RNA.ResultsOur study revealed that following HBV infection,both myeloid cells and hepatocytes exhibited increased expression of MANF. Moreover,we observed that mice with myeloid-specific MANF knockout (ManfMye-/-) displayed reduced HBV load and improved HBV-specific T cell responses. The decreased HBV-induced tolerance in ManfMye-/- mice was associated with reduced accumulation of myeloid-derived suppressor cells (MDSCs) in the liver. Restoring MDSC levels in ManfMye-/- mice through MDSC adoptive transfer reinstated HBV-induced tolerance. Mechanistically,we found that MANF promoted MDSC expansion by activating the IL-6/STAT3 pathway. Importantly,our study demonstrated the effectiveness of a combination therapy involving an hepatitis B surface antigen vaccine and nanoparticle-encapsulated MANF siRNA in effectively clearing HBV in HBV-carrier mice.ConclusionThe current study reveals that MANF plays a previously unrecognized regulatory role in liver tolerance by expanding MDSCs in the liver through IL-6/STAT3 signaling,leading to MDSC-mediated CD8+ T cell exhaustion. Graphical abstract View Publication

T cell-derived cancers are hallmarked by heterogeneity,aggressiveness,and poor clinical outcomes. Available targeted therapies are severely limited due to a lack of target antigens that allow discrimination of malignant from healthy T cells. Here,we report a novel approach for the treatment of T cell diseases based on targeting the clonally rearranged T cell receptor displayed by the cancerous T cell population. As a proof of concept,we identified an antibody with unique specificity toward a distinct T cell receptor (TCR) and developed antibody-drug conjugates,precisely recognizing and eliminating target T cells while preserving overall T cell repertoire integrity and cellular immunity. Our anti-TCR antibody-drug conjugates demonstrated effective receptor-mediated cell internalization,associated with induction of cancer cell death with strong signs of apoptosis. Furthermore,cell proliferation-inhibiting bystander effects observed on target-negative cells may contribute to the molecules’ anti-tumor properties precluding potential tumor escape mechanisms. To our knowledge,this represents the first anti-TCR antibody-drug conjugate designed as custom-tailored immunotherapy for T cell-driven pathologies. Graphical abstract Harald Kolmar and colleagues report a novel approach for the treatment of the difficult-to-treat T cell lymphoma/leukemia based on targeting the clonally rearranged T cell receptor expressed by the malignant T cell population. The developed antibody-drug conjugates precisely eliminate target T cells while preserving the integrity of the T cell repertoire and cellular immunity. View Publication

The differentiation and specificity of human CD4+ T follicular helper cells (TFH cells) after influenza vaccination have been poorly defined. Here we profiled blood and draining lymph node (LN) samples from human volunteers for over 2 years after two influenza vaccines were administered 1 year apart to define the evolution of the CD4+ TFH cell response. The first vaccination induced an increase in the frequency of circulating TFH (cTFH) and LN TFH cells at week 1 postvaccination. This increase was transient for cTFH cells,whereas the LN TFH cells further expanded during week 2 and remained elevated in frequency for at least 3 months. We observed several distinct subsets of TFH cells in the LN,including pre-TFH cells,memory TFH cells,germinal center (GC) TFH cells and interleukin-10+ TFH cell subsets beginning at baseline and at all time points postvaccination. The shift toward a GC TFH cell phenotype occurred with faster kinetics after the second vaccine compared to the first vaccine. We identified several influenza-specific TFH cell clonal lineages,including multiple responses targeting internal influenza virus proteins,and found that each TFH cell state was attainable within a clonal lineage. Thus,human TFH cells form a durable and dynamic multitissue network. Schattgen et al. profiled the subsets and clonality of CD4+ TFH cells in the blood and lymph nodes of human volunteers who received two influenza vaccines 1 year apart to characterize their dynamics and clonal evolution over 2 years. View Publication

Dendritic cells (DCs) are pivotal in regulating allergic asthma. Our research has shown that the absence of Sema3E worsens asthma symptoms in acute and chronic asthma models. However,the specific role of PlexinD1 in these processes,particularly in DCs,remains unclear. This study investigates the role of PlexinD1 in CD11c+ DCs using a house dust mite (HDM) model of asthma. We generated CD11c+ DC-specific PlexinD1 knockout (CD11cPLXND1 KO) mice and subjected them,alongside wild-type controls (PLXND1fl/fl),to an HDM allergen protocol. Airway hyperresponsiveness (AHR) was measured using FlexiVent,and immune cell populations were analyzed via flow cytometry. Cytokine levels and immunoglobulin concentrations were assessed using mesoscale and ELISA,while collagen deposition and mucus production were examined through Sirius-red and periodic acid Schiff (PAS) staining respectively. Our results indicate that CD11cPLXND1 KO mice exhibit significantly exacerbated AHR,characterized by increased airway resistance and tissue elastance. Enhanced mucus production and collagen gene expression were observed in these mice compared to wild-type counterparts. Flow cytometry revealed higher CD11c+ MHCIIhigh CD11b+ cell recruitment into the lungs,and elevated total and HDM-specific serum IgE levels in CD11cPLXND1 KO mice. Mechanistically,co-cultures of B cells with DCs from CD11cPLXND1 KO mice showed significantly increased IgE production compared to wild-type mice.These findings highlight the critical regulatory role of the plexinD1 signaling pathway in CD11c+ DCs in modulating asthma features. View Publication

Colorectal cancer (CRC) resulting from chronic inflammation is a crucial issue in patients with inflammatory bowel disease (IBD). Although many reports established that intestinal resident CX3CR1high macrophages play an essential role in suppressing intestinal inflammation,their function in colitis-related CRC remains unclear. In this study,we found that colonic CX3CR1high macrophages,which were positive for MHC-II,F4/80 and CD319,promoted colitis-associated CRC. They highly expressed Col1a1,Tgfb,II10,and II4,and were considered to be fibrocytes with an immunosuppressive M2-like phenotype. CX3CR1 deficiency led to reductions in the absolute numbers of CX3CR1high fibrocytes through increased apoptosis,thereby preventing the development of colitis-associated CRC. We next focused statins as drugs targeting CX3CR1high fibrocytes. Statins have been actively discussed for patients with IBD and reported to suppress the CX3CL1/CX3CR1 axis. Statin treatment after azoxymethane/dextran sulfate sodium-induced inflammation reduced CX3CR1high fibrocyte counts and suppressed colitis-associated CRC. Therefore,CX3CR1high fibrocytes represent a potential target for carcinogenesis-preventing therapy,and statins could be safe therapeutic candidates for IBD. View Publication

NKG2D is a central activating receptor involved in target recognition and killing by Natural Killer and CD8+ T cells. The known role of NKG2D is to recognize a family of self-induced stress ligands that are upregulated on stressed cells such as cancerous or virally infected cells. Fungal pathogens are a major threat to human health,infecting more than a billion patients yearly and becoming more common and drug resistant. Here we show that NKG2D plays a critical role in the immune response against fungal infections. NKG2D can recognize fungal pathogens from most major families including Candida,Cryptococcus and Aspergillus species,and mice lacking NKG2D are extremely sensitive to fungal infections in models of both invasive and mucosal infections,making NKG2D an anti-fungal pattern recognition receptor. NKG2D is a central activating receptor know to recognise stress ligangs upregulated during cancer or infection. Here,Charpak-Amikam et al show that NKG2D also recognises fungal pathogens and plays a critical role in mounting an appropriate immune response to them. View Publication

BackgroundMonocytes comprise subsets of classical,intermediate and non-classical monocytes with distinct anti- or pro-tumor effects in breast cancer (BC). They are modulated by estrogen,and can contribute to BC control by endocrine therapy in preclinical models.MethodsTo elucidate whether changes in monocyte subsets are associated with treatment and response,we investigated peripheral blood samples of 73 postmenopausal women with estrogen receptor (ER) positive BC,who received aromatase inhibitor therapy with or without the mucin-1 vaccine tecemotide in the ABCSG34 trial. Blood was retrieved at baseline,midterm and end of therapy,and was analyzed for the distribution and ER expression of monocyte subsets by flow cytometry.ResultsWhen 40 healthy,age-matched women were compared with BC patients before treatment start,ER levels of monocytes did not differ,yet patients presented with a higher frequency of classical and fewer non-classical monocytes. Endocrine therapy triggered a significant increase in ER levels in all monocyte subsets,without affecting subset distribution. Vaccination had no overall impact on subset frequency and ER expression. Yet,a shift from intermediate to classical monocytes during therapy correlated with changes in plasma cytokines and chemokines and was significantly associated with low residual cancer burden in vaccinated patients. Without tecemotide,baseline ER levels in classical monocytes were significantly higher in women with good response to endocrine therapy.ConclusionsThis study identified classical monocytes to be associated with ER positive BC and with patient response to neoadjuvant endocrine treatment and cancer vaccination.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-024-05659-w. View Publication

BackgroundActivated fibroblast-like synoviocytes (FLS) are drivers of synovitis and structural joint damage in rheumatoid arthritis (RA). Despite the use of disease-modifying drugs,only about 50% of RA patients reach remission in real-world settings. We used an unbiased approach to investigate the effects of standard-of-care methotrexate (MTX) and a Janus kinase inhibitor,tofacitinib (TOFA),on gene expression in RA-FLS,in order to identify untargeted disease mediators.MethodsPrimary RA-FLS were activated by stimulation with interleukin-1β (IL-1β) or platelet-derived growth factor + IL-1β in the presence or absence of MTX or TOFA,with or without additional inhibitors. Co-cultures of synovial cells were performed in direct and indirect systems. Cells were collected for RNA sequencing or qPCR,and supernatants were analyzed for protein concentrations.ResultsSix thousand three hundred fifty genes were differentially expressed,the majority being upregulated,in MTX-treated activated RA-FLS and 970 genes,the majority being downregulated,in TOFA-treated samples. Pathway analysis showed that MTX had largest effects on ‘Molecular mechanisms of cancer’ and TOFA on ‘Interferon signaling’. Targeted analysis of disease-associated genes revealed that MTX increased the expression of cell cycle-regulating genes but also of pro-inflammatory mediators like IL-1α (IL1A) and granulocyte–macrophage colony-stimulating factor,GM-CSF (CSF2). The MTX-promoted expression of CSF2 in activated RA-FLS peaked at 48 h,could be mediated via either NF-κB or AP-1 transcription factors,and was abrogated by IL-1 inhibitors (IRAK4 inhibitor and anakinra). In a co-culture setting,MTX-treatment of activated RA-FLS induced IL1B expression in macrophages.ConclusionsMTX treatment induces secretion of IL-1 from activated RA-FLS which by autocrine signaling augments their release of GM-CSF. This unexpected effect of MTX might contribute to the persistence of synovitis. View Publication

In the pathophysiology of osteoarthritis and osteoporosis,articular cartilage and bone represent the target tissues,respectively,but muscle is also involved. Since many changes in energy metabolism occur in muscle with aging,the aim of the present work was to investigate the involvement of carnitine palmitoyl transferase 1b (Cpt1b) in the muscle pathophysiology of the two diseases. Healthy subjects (CTR,n = 5),osteoarthritic (OA,n = 10),and osteoporotic (OP,n = 10) patients were enrolled. Gene expression analysis conducted on muscle and myoblasts showed up-regulation of CPT1B in OA patients; this result was confirmed by immunohistochemical and immunofluorescence analyses and enzyme activity assay,which showed increased Cpt1b activity in OA muscle. In addition,CPT1B expression resulted down-regulated in cultured OP myoblasts. Given the potential involvement of Cpt1b in the modulation of oxidative stress,we investigated ROS levels,which were found to be lower in OA myoblasts,and gene expression of nicotinamide adenine dinucleotide phosphate hydrogen oxidase 4 (Nox4),which resulted up-regulated in OA cells. Finally,the immunofluorescence of BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3) showed a decreased expression in OP myoblasts,with respect to CTR and OA. Contextually,through an ultrastructural analysis conducted by Transmission Electron Microscopy (TEM),the presence of aberrant mitochondria was observed in OP muscle. This study highlights the potential role of Cpt1b in the regulation of muscle homeostasis in both osteoarthritis and osteoporosis,allowing for the expansion of the current knowledge of what are the molecular biological pathways involved in the regulation of muscle physiology in both diseases. View Publication

Natural adjuvants have recently garnered interest in the field of vaccinology as their immunostimulatory effects. In this study,we aimed to investigate the potential use of Peyssonnelia caulifera (PC),a marine alga,as a natural adjuvant for an inactivated split A/Puerto Rico/8/1934 H1N1 influenza vaccine (sPR8) in a murine model. We administered PC-adjuvanted vaccines to a murine model via intramuscular prime and boost vaccinations,and subsequently analyzed the induced immunological responses,particularly the production of antigen-specific IgG1 and IgG2a antibodies,memory T and B cell responses,and the protective efficacy against a lethal viral infection. PC extract significantly bolstered the vaccine efficacy,demonstrating balanced Th1/Th2 responses,increased memory T and B cell activities,and improved protection against viral infection. Notably,within 3 days post-vaccination,the PC adjuvant stimulated activation markers on dendritic cells (DCs) and macrophages at the inguinal lymph nodes (ILN),emphasizing its immunostimulatory capabilities. Furthermore,the safety profile of PC was confirmed,showing minimal local inflammation and no significant adverse effects post-vaccination. These findings contribute to our understanding of the immunomodulatory properties of natural adjuvants and suggest the promising roles of natural adjuvants in the development of more effective vaccines for infectious diseases.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-76736-9. View Publication