The 50 most-cited articles in The Journal of Immunology, 2023

Myeloid-derived suppressor cells (MDSCs) were initially identified in humans and mice with cancer where they profoundly suppress T cell- and NK cell-mediated antitumor immunity. Inflammation is a central feature of many pathologies and normal physiological conditions and is the dominant driving force for the accumulation and function of MDSCs. Therefore, MDSCs are present in conditions where inflammation is present. Although MDSCs are detrimental in cancer and conditions where cellular immunity is desirable, they are beneficial in settings where cellular immunity is hyperactive. Because MDSCs can be generated ex vivo, they are being exploited as therapeutic agents to reduce damaging cellular immunity. In this review, we discuss the detrimental and beneficial roles of MDSCs in disease settings such as bacterial, viral, and parasitic infections, sepsis, obesity, trauma, stress, autoimmunity, transplantation and graft-versus-host disease, and normal physiological settings, including pregnancy and neonates as well as aging. The impact of MDSCs on vaccination is also discussed.

NK cells and CD8 T cells use cytotoxic molecules to kill virally infected and tumor cell targets. While perforin and granzyme B (GzmB) are the most commonly studied lytic molecules, less is known about granzyme K (GzmK). However, this granzyme has been recently associated with improved prognosis in solid tumors. In this study, we show that, in humans, GzmK is predominantly expressed by innate-like lymphocytes, as well as a newly identified population of GzmK+CD8+ non- mucosal-associated invariant T cells with innate-like characteristics. We found that GzmK+ T cells are KLRG1+EOMES+IL-7R+CD62L-Tcf7int, suggesting that they are central memory T and effector memory T cells. Furthermore, GzmK+ cells are absent/low in cord blood, suggesting that GzmK is upregulated with immune experience. Surprisingly, GzmK+ cells respond to cytokine stimuli alone, whereas TCR stimulation downregulates GzmK expression, coinciding with GzmB upregulation. GzmK+ cells have reduced IFN-γ production compared with GzmB+ cells in each T cell lineage. Collectively, this suggests that GzmK+ cells are not naive,…

IL-4 has long been established as a key regulator of Th cells and for promoting effective B cell survival and isotype class switching. Yet, despite having been extensively studied, the specific role of IL-4 in generating humoral memory in vivo is unclear. In this review, we explore the recent studies that unravel the cellular sources and spatiotemporal production of IL-4, the relationship between IL-4 and IL-21 during germinal center responses and the formation of Ab-secreting cells, and the current understanding of whether IL-4 promotes or suppresses memory B cell generation in vitro and in vivo.

Growth differentiation factor 15 (GDF-15) is a cytokine that is widely used as a biomarker for the severity of diverse disease states. It also has been shown to play a protective role after tissue injury and to promote a negative energy balance during obesity and diabetes. In addition to its metabolic effects, GDF-15 also regulates the host's immune responses to infectious and noninfectious diseases. GDF-15 can suppress a type 1 and, in contrast, promote a type 2 inflammatory response. In this brief review, we discuss how GDF-15 affects the effector function and recruitment of immune cells, the pathways that induce its expression, and the diverse mechanisms by which it is regulated during inflammation and infection. We further highlight outstanding questions that should be the focus of future investigations in this emerging field.

The immunological hallmarks of sepsis include the inflammation-mediated cytokine storm, apoptosis-driven lymphopenia, and prolonged immunoparalysis. Although early clinical efforts were focused on increasing the survival of patients through the first phase, studies are now shifting attention to the long-term effects of sepsis on immune fitness in survivors. In particular, the most pertinent task is deciphering how the immune system becomes suppressed, leading to increased incidence of secondary infections. In this review, we introduce the contribution of numerical changes and functional reprogramming within innate (NK cells, dendritic cells) and adaptive (T cells, B cells) immune cells on the chronic immune dysregulation in the septic murine and human host. We briefly discuss how prior immunological experience in murine models impacts sepsis severity, immune dysfunction, and clinical relevance. Finally, we dive into how comorbidities, specifically autoimmunity and cancer, can influence host susceptibility to sepsis and the associated immune dysfunction.

The complement field has recently experienced a strong resurgence of interest because of the unexpected discovery of new complement functions extending complement's role beyond immunity and pathogen clearance, a growing list of diseases in which complement plays a role, and the proliferation of complement therapeutics. Importantly, although the majority of complement components in the circulation are generated by the liver and activated extracellularly, complement activation unexpectedly also occurs intracellularly across a broad range of cells. Such cell-autonomous complement activation can engage intracellular complement receptors, which then drive noncanonical cell-specific effector functions. Thus, much remains to be discovered about complement biology. In this brief review, we focus on novel noncanonical activities of complement in its "classic areas of operation" (kidney and brain biology, infection, and autoimmunity), with an outlook on the next generation of complement-targeted therapeutics.

Complement activation via the classical pathway is initiated when oligomeric Igs on target surfaces are recognized by C1 of the complement cascade. The strength of this interaction and activation of the complement system are influenced by structural variation of the Ab, including Ab isotype, subclass, and glycosylation profile. Polymorphic variants of IgG have also been described to influence Fc-dependent effector functions. Therefore, we assessed complement binding, deposition, and complement-dependent cytotoxicity (CDC) of 27 known IgG allotypes with anti-trinitrophenyl specificity. Differences between allotypes within subclasses were minor for IgG1, IgG3, and IgG4 allotypes, and more substantial for IgG2. Allelic variant IGHG2*06, containing a unique serine at position 378 in the CH3 domain, showed less efficient complement activation and CDC compared with other IgG2 polymorphisms. We also observed variable cell lysis between IgG1 and IgG3, with IgG3 being superior in lysis of human RBCs and Ramos cells, and IgG1 being superior in lysis of Raji and Wien133 cells, demonstrating…

Nonpathogenic commensal microbiota and their metabolites and components are essential to maintain a tolerogenic environment and promote beneficial health effects. The metabolic environment critically impacts the outcome of immune responses and likely impacts autoimmune and allergic responses. Short-chain fatty acids (SCFAs) are the main metabolites produced by microbial fermentation in the gut. Given the high concentration of SCFAs in the gut and portal vein and their broad immune regulatory functions, SCFAs significantly influence immune tolerance and gut-liver immunity. Alterations of SCFA-producing bacteria and SCFAs have been identified in a multitude of inflammatory diseases. These data have particular significance in primary biliary cholangitis, primary sclerosing cholangitis, and autoimmune hepatitis because of the close proximity of the liver to the gut. In this focused review, we provide an update on the immunologic consequences of SCFA-producing microbiota and in particular on three dominant SCFAs in autoimmune liver diseases.

The braking mechanisms to protect the host from tissue damage and inflammatory disease caused by an overexuberant immune response are common in many T cell subsets. However, the negative regulation of T cell responses and detailed mechanisms are not well understood in early vertebrates. In the current study, using a Nile tilapia (Oreochromis niloticus) model, we investigated the suppression of T cell immunity by IL-10. Tilapia encodes an evolutionarily conserved IL-10, whose expression in lymphocytes is markedly induced during the primary adaptive immune response against Aeromonas hydrophila infection. Activated T cells of tilapia produce IL-10, which in turn inhibits proinflammatory cytokine expression and suppresses PHA-induced T cell activation. Moreover, administration of IL-10 impairs the proliferation of tilapia T cells, reduces their potential to differentiate into Th subsets, and cripples the cytotoxic function, rendering the animals more vulnerable to pathogen attack. After binding to its receptor IL-10Ra, IL-10 activates the JAK1/STAT3 axis by phosphorylation and enhances…

Cyclic GMP-AMP synthase (cGAS), as a cytosolic DNA sensor, plays a crucial role in antiviral immunity, and its overactivation induces excess inflammation and tissue damage. Macrophage polarization is critically involved in inflammation; however, the role of cGAS in macrophage polarization during inflammation remains unclear. In this study, we demonstrated that cGAS was upregulated in the LPS-induced inflammatory response via the TLR4 pathway, and cGAS signaling was activated by mitochondria DNA in macrophages isolated from C57BL/6J mice. We further demonstrated that cGAS mediated inflammation by acting as a macrophage polarization switch, which promoted peritoneal macrophages and the bone marrow-derived macrophages to the inflammatory phenotype (M1) via the mitochondrial DNA-mTORC1 pathway. In vivo studies verified that deletion of Cgas alleviated sepsis-induced acute lung injury by promoting macrophages to shift from the M1 phenotype to the M2 phenotype. In conclusion, our study demonstrated that cGAS mediated inflammation by regulating macrophage polarization through the mTORC1…

Being able to accurately predict the three-dimensional structure of an Ab can facilitate Ab characterization and epitope prediction, with important diagnostic and clinical implications. In this study, we evaluated the ability of AlphaFold to predict the structures of 222 recently published, high-resolution Fab H and L chain structures of Abs from different species directed against different Ags. We show that although the overall Ab prediction quality is in line with the results of CASP14, regions such as the complementarity-determining regions (CDRs) of the H chain, which are prone to higher variation, are predicted less accurately. Moreover, we discovered that AlphaFold mispredicts the bending angles between the variable and constant domains. To evaluate the ability of AlphaFold to model Ab-Ag interactions based only on sequence, we used AlphaFold-Multimer in combination with ZDOCK to predict the structures of 26 known Ab-Ag complexes. ZDOCK, which was applied on bound components of both the Ab and the Ag, succeeded in assembling 11 complexes, whereas AlphaFold succeeded in…

Among the cytokines regulating immune cells, IL-9 has gained considerable attention for its ability to act on multiple cell types as a regulator of beneficial and pathologic immune responses. Yet, it is still not clearly defined how IL-9 impacts immune responses. IL-9 demonstrates a remarkable degree of tissue-specific functionality and has cellular sources that vary by tissue site and the context of the inflammatory milieu. Here, we provide perspective to summarize the biological activities of IL-9 and highlight cell type-specific roles in the immune pathogenesis of diseases. This perspective will be important in defining the diseases where targeting IL-9 as a therapeutic strategy would be beneficial and where it has the potential to complicate clinical outcomes.

Macrophages are sentinels of the innate immune system that maintain tissue homeostasis and contribute to inflammatory responses. Their broad scope of action depends on both functional heterogeneity and plasticity. Small noncoding RNAs called microRNAs (miRNAs) contribute to macrophage function as post-transcriptional inhibitors of target gene networks. Genetic and pharmacologic studies have uncovered genes regulated by miRNAs that control macrophage cellular programming and macrophage-driven pathology. miRNAs control proinflammatory M1-like activation, immunoregulatory M2-like macrophage activation, and emerging macrophage functions in metabolic disease and innate immune memory. Understanding the gene networks regulated by individual miRNAs enhances our understanding of the spectrum of macrophage function at steady state and during responses to injury or pathogen invasion, with the potential to develop miRNA-based therapies. This review aims to consolidate past and current studies investigating the complexity of the miRNA interactome to provide the reader with a mechanistic view of…

Swine coronavirus-porcine epidemic diarrhea virus (PEDV) with specific susceptibility to pigs has existed for decades, and recurrent epidemics caused by mutant strains have swept the world again since 2010. In this study, single-cell RNA sequencing was used to perform for the first time, to our knowledge, a systematic analysis of pig jejunum infected with PEDV. Pig intestinal cell types were identified by representative markers and identified a new tuft cell marker, DNAH11. Excepting enterocyte cells, the goblet and tuft cells confirmed susceptibility to PEDV. Enrichment analyses showed that PEDV infection resulted in upregulation of cell apoptosis, junctions, and the MAPK signaling pathway and downregulation of oxidative phosphorylation in intestinal epithelial cell types. The T cell differentiation and IgA production were decreased in T and B cells, respectively. Cytokine gene analyses revealed that PEDV infection downregulated CXCL8, CXCL16, and IL34 in tuft cells and upregulated IL22 in Th17 cells. Further studies found that infection of goblet cells with PEDV decreased the…

C-reactive protein (CRP) is a highly conserved pentraxin with pattern recognition receptor-like activities. However, despite being used widely as a clinical marker of inflammation, the in vivo functions of CRP and its roles in health and disease remain largely unestablished. This is, to certain extent, due to the drastically different expression patterns of CRP in mice and rats, raising concerns about whether the functions of CRP are essential and conserved across species and how these model animals should be manipulated to examine the in vivo actions of human CRP. In this review, we discuss recent advances highlighting the essential and conserved functions of CRP across species, and propose that appropriately designed animal models can be used to understand the origin-, conformation-, and localization-dependent actions of human CRP in vivo. The improved model design will contribute to establishing the pathophysiological roles of CRP and facilitate the development of novel CRP-targeting strategies.

African swine fever is one of the most serious viral diseases that affects domestic and wild pigs. The causative agent, African swine fever virus (ASFV), has evolved sophisticated immune evasion mechanisms that target both innate and adaptive immune responses. However, the underlying molecular mechanisms have not been fully understood. Here, we report that ASFV E184L protein inhibits host innate immune response via targeting the stimulator of IFN genes (STING)-mediated signaling pathway in both human embryonic kidney HEK-293T cells and porcine pulmonary alveolar macrophages. E184L interacts with STING, impairing dimerization and oligomerization of STING but not affecting its puncta formation at the perinuclear region. Furthermore, E184L disrupts STING-TBK1-IRF3 complex formation, leading to inhibition of STING phosphorylation, and IRF3 dimerization and nuclear translocation. The 1-20 aa region in E184L is essential for E184L-STING interaction and blocking IL-1β and type I IFN production. Deletion of E184L in ASFV considerably impairs antagonistic function of the virus in suppression…

Antibody secretion into sera, selection for higher affinity BCR, and the generation of higher Ab affinities are important elements of immune response optimization, and a core function of germinal center reactions. B cell proliferation requires nutrients to support the anabolism inherent in clonal expansion. Glucose usage by GC B cells has been reported to contribute little to their energy needs, with questions raised as to whether or not glucose uptake or glycolysis increases in GC B cells compared to their naïve precursors. Indeed, metabolism can be highly flexible, such that supply shortage along one pathway may be compensated by increased flux on others. We now show that elimination of the glucose transporter GLUT1 after establishment of a pre-immune B cell repertoire, even after initiation of the GC B cell gene expression program, decreased initial GC B cell population numbers, affinity maturation, and PC outputs. Glucose oxidation was heightened in GC B cells, but this hexose flowed more into the pentose phosphate pathway (PPP), whose activity was important in controlling…

Type I IFNs with strong positive charges exhibit robust bactericidal activity and a protective effect against bacterial infections. However, the antibacterial mechanism in vivo remains unknown. In this study, Ab blockade of IFN1, a member of type I IFNs in grass carp (Ctenopharyngodon idella), resulted in high mortality, tissue bacterial loads, and low expression of immune factors after bacterial challenge, which indicates that the antibacterial activity of IFN1 has physiological significance. Meanwhile, we injected grass carp with the recombinant and purified intact IFN1 protein after bacterial injection, and the result demonstrated a remarkable therapeutic effect. Furthermore, we found that IFN1 expression was remarkably induced in blood cells after bacterial challenge, and prophagocytosis via IFN1 mostly increased in thrombocytes. Then, we isolated peripheral blood thrombocytes by polyclonal Ab of CD41 and stimulated thrombocytes with recombinant IFN1, and the results indicated that immune factors and complement components (especially C3.3) were induced. Unexpectedly, complements…

Mucosal-associated invariant T (MAIT) cells are unconventional T cells with innate-like antimicrobial responsiveness. MAIT cells are known for MR1 (MHC class I-related protein 1)-restricted recognition of microbial riboflavin metabolites giving them the capacity to respond to a broad range of microbes. However, recent progress has shown that MAIT cells can also respond to several viral infections in humans and in mouse models, ranging from HIV-1 and hepatitis viruses to influenza virus and SARS-CoV-2, in a primarily cognate Ag-independent manner. Depending on the disease context MAIT cells can provide direct or indirect antiviral protection for the host and may help recruit other immune cells, but they may also in some circumstances amplify inflammation and aggravate immunopathology. Furthermore, chronic viral infections are associated with varying degrees of functional and numerical MAIT cell impairment, suggesting secondary consequences for host defense. In this review, we summarize recent progress and highlight outstanding questions regarding the emerging role of MAIT cells in…

Intestinal inflammatory diseases affect millions of people worldwide, and one class of drugs showing promise toward treatment of several inflammatory diseases is probiotics. Numerous studies have been performed using probiotics to prevent and treat intestinal inflammatory diseases. Most of these studies used intact bacteria, and neither the active molecule nor the molecular mechanisms by which they affect immune responses are known. We have shown that the probiotic Bacillus subtilis is anti-inflammatory and can protect mice from acute colitis induced by the enteric pathogen Citrobacter rodentium. We identified and purified the active molecule, exopolysaccharide (EPS), and showed that it protects mice from C. rodentium-induced colitis by inducing anti-inflammatory M2 macrophages or inhibitory dendritic cells (DCs), both of which inhibit excessive T cell responses. We showed previously that EPS affects macrophages and DCs in a TLR4-dependent manner, and in the current study we asked how EPS induces these anti-inflammatory cells and how they function to inhibit T cells. By…

Cytokine storm syndromes (CSSs) are potentially fatal hyperinflammatory states that share the underpinnings of persistent immune cell activation and uninhibited cytokine production. CSSs can be genetically determined by inborn errors of immunity (i.e., familial hemophagocytic lymphohistiocytosis) or develop as a complication of infections, chronic inflammatory diseases (e.g., Still disease), or malignancies (e.g., T cell lymphoma). Therapeutic interventions that activate the immune system such as chimeric Ag receptor T cell therapy and immune checkpoint inhibition can also trigger CSSs in the setting of cancer treatment. In this review, the biology of different types of CSSs is explored, and the current knowledge on the involvement of immune pathways and the contribution of host genetics is discussed. The use of animal models to study CSSs is reviewed, and their relevance for human diseases is discussed. Lastly, treatment approaches for CSSs are discussed with a focus on interventions that target immune cells and cytokines.

Immune cell-derived IL-17A is one of the key pathogenic cytokines in psoriasis, an immunometabolic disorder. Although IL-17A is an established regulator of cutaneous immune cell biology, its functional and metabolic effects on nonimmune cells of the skin, particularly keratinocytes, have not been comprehensively explored. Using multiomics profiling and systems biology-based approaches, we systematically uncover significant roles for IL-17A in the metabolic reprogramming of human primary keratinocytes (HPKs). High-throughput liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance spectroscopy revealed IL-17A-dependent regulation of multiple HPK proteins and metabolites of carbohydrate and lipid metabolism. Systems-level MitoCore modeling using flux-balance analysis identified IL-17A-mediated increases in HPK glycolysis, glutaminolysis, and lipid uptake, which were validated using biochemical cell-based assays and stable isotope-resolved metabolomics. IL-17A treatment triggered downstream mitochondrial reactive oxygen species and HIF1α expression and resultant…

African swine fever is a fatal infectious disease caused by African swine fever virus (ASFV). The high mortality caused by this infectious disease is a significant challenge to the swine industry worldwide. ASFV virulence is related to its ability to antagonize IFN response, yet the mechanism of antagonism is not understood. Recently, a less virulent recombinant virus has emerged that has a EP402R gene deletion within the parental ASFV HLJ/18 (ASFV-ΔEP402R) strain. EP402R gene encodes CD2v. Hence we hypothesized that ASFV uses CD2v protein to evade type I IFN-mediated innate immune response. We found that ASFV-ΔEP402R infection induced higher type I IFN response and increased the expression of IFN-stimulated genes in porcine alveolar macrophages when compared with parental ASFV HLJ/18. Consistent with these results, CD2v overexpression inhibited type I IFN production and IFN-stimulated gene expression. Mechanistically, CD2v, by interacting with the transmembrane domain of stimulator of IFN genes (STING), prevented the transport of STING to the Golgi apparatus, and thereby inhibited…

C4b-binding protein (C4BP) is a fluid-phase complement inhibitor that prevents uncontrolled activation of the classical and lectin complement pathways. As a complement inhibitor, C4BP also promotes apoptotic cell death and is hijacked by microbes and tumors for complement evasion. Although initially characterized for its role in complement inhibition, there is an emerging recognition that C4BP functions in a complement-independent manner to promote cell survival, protect against autoimmune damage, and modulate the virulence of microbial pathogens. In this Brief Review, we summarize the structure and functions of human C4BP, with a special focus on activities that extend beyond the canonical role of C4BP in complement inhibition.

Current Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using short-read sequencing strategies resolve expressed Ab transcripts with limited resolution of the C region. In this article, we present the near-full-length AIRR-seq (FLAIRR-seq) method that uses targeted amplification by 5' RACE, combined with single-molecule, real-time sequencing to generate highly accurate (99.99%) human Ab H chain transcripts. FLAIRR-seq was benchmarked by comparing H chain V (IGHV), D (IGHD), and J (IGHJ) gene usage, complementarity-determining region 3 length, and somatic hypermutation to matched datasets generated with standard 5' RACE AIRR-seq using short-read sequencing and full-length isoform sequencing. Together, these data demonstrate robust FLAIRR-seq performance using RNA samples derived from PBMCs, purified B cells, and whole blood, which recapitulated results generated by commonly used methods, while additionally resolving H chain gene features not documented in IMGT at the time of submission. FLAIRR-seq data provide, for the first time, to our knowledge, simultaneous…

HLA class I proteins, a critical component in adaptive immunity, bind and present intracellular Ags to CD8+ T cells. The extreme polymorphism of HLA genes and associated peptide binding specificities leads to challenges in various endeavors, including neoantigen vaccine development, disease association studies, and HLA typing. Supertype classification, defined by clustering functionally similar HLA alleles, has proven helpful in reducing the complexity of distinguishing alleles. However, determining supertypes via experiments is impractical, and current in silico classification methods exhibit limitations in stability and functional relevance. In this study, by incorporating three-dimensional structures we present a method for classifying HLA class I molecules with improved breadth, accuracy, stability, and flexibility. Critical for these advances is our finding that structural similarity highly correlates with peptide binding specificity. The new classification should be broadly useful in peptide-based vaccine development and HLA-disease association studies.

Most aspects of physiology, including immunity, present 24-h variations called circadian rhythms. In this review, we examine the literature on the circadian regulation of CD8+ T cells, which are important to fight intracellular infections and tumors. CD8+ T cells express circadian clock genes, and ∼6% of their transcriptome presents circadian oscillations. CD8+ T cell counts present 24-h rhythms in the blood and in secondary lymphoid organs, which depend on the clock in these cells as well as on hormonal rhythms. Moreover, the strength of the response of these cells to Ag presentation varies according to time of day, a rhythm dependent on the CD8+ T cell clock. The relevance of CD8+ T cell circadian rhythms is shown by the daily variations in the fight of intracellular infections. Such a circadian regulation also has implications for cancer, as well as the optimization of vaccination and immunotherapy.

Immunometabolism is an interdisciplinary field that focuses on the relationship between metabolic pathways and immune responses. Dysregulated immunometabolism contributes to many pathological settings, such as cytokine storm or immune tolerance. Aconitate decarboxylase 1 (ACOD1, also known as immunoresponsive gene 1), the mitochondrial enzyme responsible for catalyzing itaconate production, was originally identified as a bacterial LPS-inducible gene involved in innate immunity in mouse macrophages. We now know that the upregulation of ACOD1 expression in immune or nonimmune cells plays a context-dependent role in metabolic reprogramming, signal transduction, inflammasome regulation, and protein modification. The emerging function of ACOD1 in inflammation and infection is a double-edged sword. In this review, we discuss how ACOD1 regulates anti-inflammatory or proinflammatory responses in an itaconate-dependent or -independent manner. Further understanding of ACOD1 expression and function may pave the way for the development of precision therapies for inflammatory diseases.

The endothelial lining of blood vessels is covered with a thin polysaccharide coat called the glycocalyx. This layer of polysaccharides contains hyaluronan that forms a protective coat on the endothelial surface. Upon inflammation, leukocytes leave the circulation and enter inflamed tissue by crossing inflamed endothelial cells, mediated by adhesion molecules such as ICAM-1/CD54. To what extent the glycocalyx participates in the regulation of leukocyte transmigration is not clear. During extravasation, leukocyte integrins cluster ICAM-1, resulting in the recruitment of a number of intracellular proteins and subsequent downstream effects in the endothelial cells. For our studies, we used primary human endothelial and immune cells. With an unbiased proteomics approach, we identified the full ICAM-1 adhesome and identified 93 (to our knowledge) new subunits of the ICAM-1 adhesome. Interestingly, we found the glycoprotein CD44 as part of the glycocalyx to be recruited to clustered ICAM-1 specifically. Our data demonstrate that CD44 binds hyaluronan to the endothelial surface, where it…

Lipid accumulation in macrophages (Mφs) is a hallmark of atherosclerosis, yet how lipid accumulation affects inflammatory responses through rewiring of Mφ metabolism is poorly understood. We modeled lipid accumulation in cultured wild-type mouse thioglycolate-elicited peritoneal Mφs and bone marrow-derived Mφs with conditional (Lyz2-Cre) or complete genetic deficiency of Vhl, Hif1a, Nos2, and Nfe2l2. Transfection studies employed RAW264.7 cells. Mφs were cultured for 24 h with oxidized low-density lipoprotein (oxLDL) or cholesterol and then were stimulated with LPS. Transcriptomics revealed that oxLDL accumulation in Mφs downregulated inflammatory, hypoxia, and cholesterol metabolism pathways, whereas the antioxidant pathway, fatty acid oxidation, and ABC family proteins were upregulated. Metabolomics and extracellular metabolic flux assays showed that oxLDL accumulation suppressed LPS-induced glycolysis. Intracellular lipid accumulation in Mφs impaired LPS-induced inflammation by reducing both hypoxia-inducible factor 1-α (HIF-1α) stability and transactivation capacity; thus, the…

Activated T cells undergo metabolic reprogramming to meet anabolic, differentiation, and functional demands. Glutamine supports many processes in activated T cells, and inhibition of glutamine metabolism alters T cell function in autoimmune disease and cancer. Multiple glutamine-targeting molecules are under investigation, yet the precise mechanisms of glutamine-dependent CD8 T cell differentiation remain unclear. We show that distinct strategies of glutamine inhibition by glutaminase-specific inhibition with small molecule CB-839, pan-glutamine inhibition with 6-diazo-5-oxo-l-norleucine (DON), or by glutamine-depleted conditions (No Q) produce distinct metabolic differentiation trajectories in murine CD8 T cells. T cell activation with CB-839 treatment had a milder effect than did DON or No Q treatment. A key difference was that CB-839-treated cells compensated with increased glycolytic metabolism, whereas DON and No Q-treated cells increased oxidative metabolism. However, all glutamine treatment strategies elevated CD8 T cell dependence on glucose metabolism, and No Q treatment…

To precisely identify mouse resident alveolar macrophages (AMs) and bone marrow (BM)-derived macrophages, we developed a technique to separately label AMs and BM-derived macrophages with a fluorescent lipophilic dye followed by FACS. We showed that this technique overcomes issues in cell identification related to dynamic shifts in cell surface markers that occurs during lung inflammation. We then used this approach to track macrophage subsets at different time points after intratracheal (i.t.) instillation of Escherichia coli LPS. By isolating BM-derived macrophages and AMs, we demonstrated that BM-derived macrophages were enriched in expression of genes in signal transduction and immune system activation pathways whereas resident AMs were enriched in cellular processes, such as lysosome/phagosome pathways, efferocytosis, and metabolic pathways related to fatty acids and peroxisomes. Taken together, these data indicate that more accurate identification of macrophage origin can result in improved understanding of differential phenotypes and functions between AMs and BM-derived…

IκBα is a critical protein that inhibits NF-κB nuclear translocation and impairs NF-κB-mediated signaling. The abundance of IκBα determines the activation and restoration of the inflammatory response. However, posttranslational regulation of IκBα remains to be fully understood. In this study, we identified ubiquitin-specific protease 39 (USP39) as a negative regulator in the NF-κB inflammatory response by stabilizing basal IκBα. The expression of USP39 in macrophages was reduced under LPS-induced inflammation. Knockdown or knockout of USP39 in macrophages significantly increased the expression and secretion of proinflammatory cytokines upon exposure to LPS or Escherichia coli, whereas reexpression of exogenous USP39 in USP39-deficient macrophages rescued the effect. Moreover, USP39-defective mice were more sensitive to LPS or E. coli-induced systemic sepsis. Mechanistically, USP39 interacted with and stabilized IκBα by reducing K48-linked polyubiquination of IκBα. Taken together, to our knowledge, our study for the first time revealed the inhibitory function of USP39 in the NF-κB…

Regulatory T cells (Tregs) contribute to the formation of a tumor-immunosuppressive microenvironment. CCR8 is reportedly selectively expressed in tumor Tregs, and an anti-CCR8 Ab can exert potent antitumor effects by eliminating intratumor Tregs in murine tumor models. In this study, we analyzed changes to intratumor immunity after anti-CCR8 Ab administration, especially in CD8+ T cells, which are involved in cancer cell killing, using the CT26 colorectal carcinoma mouse model. Immunophenotyping of tumor-infiltrating cells by mass cytometry after Ab administration on day 5 of tumor inoculation revealed that CD8+ T cell subsets were dramatically altered in the CCR8 Ab-treated group, with an increase in naive cells and nonexhausted effector cells and a decrease in exhausted cells with high expression levels of TOX. These results were corroborated with flow cytometry analysis. Delayed administration of the anti-CCR8 Ab on day 9 or 12, when the amount of CCR8+ Tregs and CD8+ T cell exhaustion were more progressed, also resulted in a decrease in exhausted CD8+ T cells, leading to tumor…

The mammalian heart is characterized by the presence of striated myocytes, which allow continuous rhythmic contraction from early embryonic development until the last moments of life. However, the myocardium contains a significant contingent of leukocytes from every major class. This leukocyte pool includes both resident and nonresident immune cells. Over recent decades, it has become increasingly apparent that the heart is intimately sensitive to immune signaling and that myocardial leukocytes exhibit an array of critical functions, both in homeostasis and in the context of cardiac adaptation to injury. Here, we systematically review current knowledge of all major leukocyte classes in the heart, discussing their functions in health and disease. We also highlight the connection between the myocardium, immune cells, lymphoid organs, and both local and systemic immune responses.

NK cells are best known for their killing of virus-infected cells and tumor cells via release of cytotoxic factors. However, NK cells can also produce growth factors and cytokines, and thus have the potential to influence physiological processes such as wound healing. In this study, we test the hypothesis that NK cells play a physiological role in skin wound healing of C57BL/6J mice. Immunohistochemical and flow cytometry assays showed that NK cells accumulate in excisional skin wounds, peaking on day 5 postinjury. We also found that NK cells proliferate locally in wounds, and blocking IL-15 activity locally reduces NK cell proliferation and accumulation in wounds. Wound NK cells exhibit primarily a mature CD11b+CD27- and NKG2A+NKG2D- phenotype and express LY49I and proinflammatory cytokines such as IFN-γ, Tnf-a, and Il-1β. Systemic depletion of NK cells resulted in enhanced re-epithelization and collagen deposition, suggesting a negative role for these cells in skin wound healing. Depletion of NK cells did not influence accumulation of neutrophils or monocytes/macrophages in wounds…

Cytotoxic lymphocytes eliminate cancer cells through the release of lytic granules, a specialized form of secretory lysosomes. This compartment is part of the pleomorphic endolysosomal system and is distinguished by its highly dynamic Ca2+ signaling machinery. Several transient receptor potential (TRP) calcium channels play essential roles in endolysosomal Ca2+ signaling and ensure the proper function of these organelles. In this study, we examined the role of TRPML1 (TRP cation channel, mucolipin subfamily, member 1) in regulating the homeostasis of secretory lysosomes and their cross-talk with mitochondria in human NK cells. We found that genetic deletion of TRPML1, which localizes to lysosomes in NK cells, led to mitochondrial fragmentation with evidence of collapsed mitochondrial cristae. Consequently, TRPML1-/- NK92 (NK92ML1-/-) displayed loss of mitochondrial membrane potential, increased reactive oxygen species stress, reduced ATP production, and compromised respiratory capacity. Using sensitive organelle-specific probes, we observed that mitochondria in NK92ML1-/- cells…

CTLs play important roles in host immune responses to tumors. CD4 CTLs are characterized by their ability to secrete cytotoxic effector molecules, such as granzyme B and perforin, and kill target cells in a MHC class II-restricted manner. However, the cell surface markers of CD4 CTLs remain unknown, which hinders their separation and research on their function. In this study, we performed a bioinformatics analysis and experimental validation that revealed that G protein-coupled receptor 56 (GPR56) is a cell surface marker that can be used to characterize CD4 CTLs. We found that GPR56 and granzyme B were coexpressed in extremely high levels in human peripheral blood T cells, and that anti-GPR56 stimulation significantly upregulated the expression of granzyme B in both CD4+GPR56+ and CD8+GPR56+ T cells. These findings suggest that GPR56 expression and the GPR56 signaling pathway could contribute directly to the toxic function of either CD4+ or CD8+ T cells. We also used GPR56 as a biomarker to investigate the clinical significance of CD4 CTLs. GPR56+ T cell levels were increased in…

Expression of macrophage receptor with collagenous structure (MARCO) by tumor-associated macrophages is associated with poor prognosis of multiple types of cancer. In this article, we report that cancer cells (e.g., breast cancer and glioblastoma cell lines) can upregulate surface MARCO expression on human macrophages not only via IL-6-induced STAT3 activation but also via sphingosine-1-phosphate receptor (S1PR)-mediated IL-6 and IL-10 expression followed by STAT3 activation. We further found that MARCO ligation induces activation of the MEK/ERK/p90RSK/CREB signaling cascade, leading to IL-10 expression followed by STAT3-dependent PD-L1 upregulation. Such MARCO-induced macrophage polarization is accompanied by increased expression of PPARG, IRF4, IDO1, CCL17, and CCL22. Ligation of surface MARCO can thus result in decreased T cell responses mainly by reduction of their proliferation. Taken together, cancer cell-induced MARCO expression and its intrinsic regulatory function within macrophages are, to our knowledge, new aspects of cancer immune evasion mechanisms that need to be…

Efferocytosis is a phagocytic process by which apoptotic cells are cleared by professional and nonprofessional phagocytic cells. In tumors, efferocytosis of apoptotic cancer cells by tumor-associated macrophages prevents Ag presentation and suppresses the host immune response against the tumor. Therefore, reactivating the immune response by blockade of tumor-associated macrophage-mediated efferocytosis is an attractive strategy for cancer immunotherapy. Even though several methods have been developed to monitor efferocytosis, an automated and high-throughput quantitative assay should offer highly desirable advantages for drug discovery. In this study, we describe a real-time efferocytosis assay with an imaging system for live-cell analysis. Using this assay, we successfully discovered potent anti-MerTK Abs that block tumor-associated macrophage-mediated efferocytosis in mice. Furthermore, we used primary human and cynomolgus monkey macrophages to identify and characterize anti-MerTK Abs for potential clinical development. By studying the phagocytic activities of different types of…

Abstract Background Previous studies have reported impaired humoral responses after SARS-CoV-2 mRNA vaccination in immunocompromised patients with immune-mediated inflammatory diseases (IMID), particularly those treated with anti-tumor necrosis factor (TNF) biologics. We previously reported that IMID patients exhibited greater waning of antibody and T cell responses compared to healthy controls after dose 2. Fewer data are available on the effects of third and fourth doses. Methods This observational cohort study collected plasma and peripheral blood mononuclear cells from healthy controls and untreated or treated IMID patients, pre-vaccination and after one to four doses of SARS-CoV-2 mRNA vaccine (BNT162b2 or mRNA-1273). SARS-CoV-2- specific antibody levels, neutralization, and T cell cytokine responses were measured against Wildtype (WT) and BA.1 and BA.5 variants of concern (VOCs). Results Third vaccine doses substantially restored and prolonged antibody and T cell responses in IMID patients and broadened responses against VOCs. Fourth dose effects were subtle but also prolonged…

Hematoimmunopoiesis takes place in the adult human bone marrow (BM), which is composed of heterogeneous niches with complex architecture that enables tight regulation of homeostatic and stress responses. There is a paucity of representative culture systems that recapitulate the heterogeneous three-dimensional (3D) human BM microenvironment and that can endogenously produce soluble factors and extracellular matrix that deliver culture fidelity for the study of both normal and abnormal hematopoiesis. Native BM lymphoid populations are also poorly represented in current in vitro and in vivo models, creating challenges for the study and treatment of BM immunopathology. BM organoid models leverage normal 3D organ structure to recreate functional niche microenvironments. Our focus herein is to review the current state of the art in the use of 3D BM organoids, focusing on their capacities to recreate critical quality attributes of the in vivo BM microenvironment for the study of human normal and abnormal hematopoiesis.

High CXCL16 levels during acute cardiovascular events increase long-term mortality. However, the mechanistic role of CXCL16 in myocardial infarction (MI) is unknown. Here we investigated the role of CXCL16 in mice with MI injury. CXCL16 deficiency increased the survival of mice after MI injury, and inactivation of CXCL16 resulted in improved cardiac function and decreased infarct size. Hearts from CXCL16 inactive mice exhibited decreased infiltration of Ly6Chigh monocytes. In addition, CXCL16 promoted the macrophage expression of CCL4 and CCL5. Both CCL4 and CCL5 stimulated Ly6Chigh monocyte migration, and CXCL16 inactive mice had a reduced expression of CCL4 and CCL5 in the heart after MI. Mechanistically, CXCL16 promoted CCL4 and CCL5 expression by activating the NF-κB and p38 MAPK signaling pathways. Anti-CXCL16 neutralizing Ab administration inhibited Ly6Chigh monocyte infiltration and improved cardiac function after MI. Additionally, anti-CCL4 and anti-CCL5 neutralizing Ab administration inhibited Ly6Chigh monocyte infiltration and improved cardiac function after MI. Thus,…

Abs can be glycosylated in both their Fc and Fab regions with marked effects on Ab function and binding. High levels of IgG Fab glycosylation are associated with malignant and autoimmune conditions, exemplified by rheumatoid arthritis and highly Fab-glycosylated (∼90%) anti-citrullinated protein Abs (ACPAs). Important properties of IgG, such as long half-life and placental transport, are facilitated by the human neonatal Fc receptor (hFcRn). Although it is known that glycosylation of Abs can affect binding to Fc receptors, little is known on the impact of IgG Fab glycosylation on hFcRn binding and transplacental transport. Therefore, we analyzed the interaction between hFcRn and IgG with and without Fab glycans in vitro with various methods as well as in vivo by studying placental transfer of Fab-glycosylated Abs from mothers to newborns. No effect of Fab glycosylation on IgG binding to hFcRn was found by surface plasmon resonance and hFcRn affinity chromatography. In contrast, studies in a cell membrane context revealed that Fab glycans negatively impacted IgG-hFcRn interaction. In…

Epoxyeicosatrienoic acids (EETs) and other epoxy fatty acids are short-acting lipids involved in resolution of inflammation. Their short half-life, due to its metabolism by soluble epoxide hydrolase (sEH), limits their effects. Specialized proresolving mediators (SPMs) are endogenous regulatory lipids insufficiently synthesized in uncontrolled and chronic inflammation. Using an experimental periodontitis model, we pharmacologically inhibited sEH, examining its impact on T cell activation and systemic SPM production. In humans, we analyzed sEH in the gingival tissue of periodontitis patients. Mice were treated with sEH inhibitor (sEHi) and/or EETs before ligature placement and treated for 14 d. Bone parameters were assessed by microcomputed tomography and methylene blue staining. Blood plasma metabololipidomics were carried out to quantify SPM levels. We also determined T cell activation by reverse transcription-quantitative PCR and flow cytometry in cervical lymph nodes. Human gingival samples were collected to analyze sEH using ELISA and electrophoresis. Data reveal that…

Idiopathic pulmonary fibrosis (IPF) is a fibrotic age-related chronic lung disease characterized by the accumulation of senescent cells. Whether impaired immune response is responsible for the accumulation of senescent cells in the IPF lung remains unknown. In this study, we characterized the NK phenotype in IPF lungs via flow cytometry using 5-dodecanoylaminofluorescein di-β-d-galactopyranoside, markers of tissue residence, and chemokine receptors. The effect of the lung microenvironment was evaluated using lung fibroblast (LF) conditioned media (CM), and the bleomycin-induced pulmonary fibrosis mouse model was used to assess the in vivo relationship between NK cells and the accumulation of senescent cells. We found that NK cells from the lower lobe of IPF patients exhibited immune-senescent and impaired CD57-NKG2A+ phenotype. We also observed that culture of NK cells from healthy donors in CM from IPF lower lobe lung fibroblasts induced a senescent-like phenotype and impaired cytotoxic capacity. There is an impaired NK recruitment by LF, and NKs presented decreased migration…

Tumor-specific CD8+ T cells are critical components of antitumor immunity; however, factors that modulate their phenotype and function have not been completely elucidated. Cytokines IL-12 and IL-27 have recognized roles in promoting CD8+ T cells' effector function and mediated antitumor responses. Tumor-specific CD8+ tumor-infiltrating lymphocytes (TILs) can be identified based on surface expression of CD39, whereas bystander CD8+ TILs do not express this enzyme. It is currently unclear how and why tumor-specific CD8+ T cells uniquely express CD39. Given the important roles of IL-12 and IL-27 in promoting CD8+ T cell functionality, we investigated whether these cytokines could modulate CD39 expression on these cells. Using in vitro stimulation assays, we identified that murine splenic CD8+ T cells differentially upregulate CD39 in the presence of IL-12 and IL-27. Subsequently, we assessed the exhaustion profile of IL-12- and IL-27-induced CD39+CD8+ T cells. Despite the greatest frequency of exhausted CD39+CD8+ T cells after activation with IL-12, as demonstrated by the coexpression…

Retinoic acid-inducible gene I (RIG-I) is essential for activating host cell innate immunity to regulate the immune response against many RNA viruses. We previously identified that a small molecule compound, KIN1148, led to the activation of IFN regulatory factor 3 (IRF3) and served to enhance protection against influenza A virus (IAV) A/California/04/2009 infection. We have now determined direct binding of KIN1148 to RIG-I to drive expression of IFN regulatory factor 3 and NF-κB target genes, including specific immunomodulatory cytokines and chemokines. Intriguingly, KIN1148 does not lead to ATPase activity or compete with ATP for binding but activates RIG-I to induce antiviral gene expression programs distinct from type I IFN treatment. When administered in combination with a vaccine against IAV, KIN1148 induces both neutralizing Ab and IAV-specific T cell responses compared with vaccination alone, which induces comparatively poor responses. This robust KIN1148-adjuvanted immune response protects mice from lethal A/California/04/2009 and H5N1 IAV challenge. Importantly, KIN1148…

Fish possess a powerful IFN system to defend against aquatic virus infections. Nevertheless, spring viremia of carp virus (SVCV) causes large-scale mortality in common carp and significant economic losses to aquaculture. Therefore, it is necessary to investigate the strategies used by SVCV to escape the IFN response. In this study, we show that the SVCV nucleoprotein (N protein) negatively regulates cellular IFN production by degrading stimulator of IFN genes (STING) via the autophagy-lysosome-dependent pathway. First, overexpression of N protein inhibited the IFN promoter activation induced by polyinosinic-polycytidylic acid and STING. Second, the N protein associated with STING and experiments using a dominant-negative STING mutant demonstrated that the N-terminal transmembrane domains of STING were indispensable for this interaction. Then, the N protein degraded STING in a dose-dependent and autophagy-lysosome-dependent manner. Intriguingly, in the absence of STING, individual N proteins could not elicit host autophagic flow. Furthermore, the autophagy factor Beclin1 was found to…

Regulatory T cells (Tregs) are critical mediators of immune tolerance and play a diametric role in cancer and autoimmunity. Tumor-infiltrating Tregs are often associated with poor prognosis in solid tumors because their enrichment in the tumor microenvironment contributes to immunosuppression. Conversely, dysregulation in the Treg compartment can disrupt self-tolerance, leading to autoimmunity. In the present study, we describe what is, to our knowledge, a novel regulator of Tregs, the GTPase activator regulator of G protein 1 (RGS1), demonstrating that RGS1-deficient human Tregs show downregulation of Treg-associated genes and are less immunosuppressive. These RGS1-deficient Tregs exhibit perturbations to the FOXP3-c-MYC transcriptional axis and downstream metabolic and autophagy programs by shifting their energy demands toward glycolysis and rendering them less autophagic. Taken together, RGS1 may serve as an apical node of Treg function by regulating the FOXP3-c-MYC transcriptional axis, thereby providing a therapeutic rationale for targeting RGS1 for treatment of cancer and…