Journal of immunology最新文献

The extracellular fibrinogen-binding protein (Efb) is one of nearly a dozen proteins secreted by Staphylococcus aureus to inhibit complement activation or amplification. The C-terminal domain of Efb (Efb-C) forms a high-affinity interaction with the thioester-containing domain of C3b (TED/C3d), thereby blocking formation of the C3 proconvertase complex through an allosteric mechanism. However, further functional consequences of Efb-C binding to C3b remain unexplored. Here, we identified a previously unknown interaction between Efb-C, C3b, and the complement regulatory molecule FHR2 (factor H-related protein 2). Since the FHR2/C3b interaction is centered upon the 2 C-terminal-most domains of FHR2 (FHR2[3-4]) and the TED/C3d domain of C3b, we tested whether Efb-C could influence the FHR2(3-4)/C3d interaction. We observed a significant enhancement of FHR2(3-4)/C3d binding in the presence of Efb-C. We studied the FHR2(3-4)/C3d/Efb-C complex by X-ray crystallography and found that Efb-C forms few direct interactions with FHR2(3-4). Yet, the presence of Efb-C also enhanced binding of FHR2(3-4) and full-length FHR2 to C3b, suggesting that the effect of Efb-C on the FHR2/C3b interaction arises from increased accessibility of the FHR2-binding site. We found that enhanced FHR2 binding did not impact the rate of C3 convertase formation more than Efb-C alone, nor did it impart decay acceleration or cofactor activity. However, we observed potent, synergistic inhibition of complement downstream of C5 activation by Efb-C and FHR2 but not by Efb-C and FHR2(3-4). Our results show that Efb-C binding to C3b exerts additional inhibitory effects on the central complement components beyond blocking formation of the C3 proconvertase alone.

Peptide-based therapeutic vaccines exploit cross-presentation by dendritic cells for the induction of effective T cell responses. Their clinical success, however, has been limited due to incomplete understanding of antigen processing and presentation (APP). Bioorthogonal chemistry (BOC) uses chemical "click" reactions that can be performed selectively without interference of the cellular environment. A "click handle" can be incorporated into a biomolecule with only minor effects on its characteristics, allowing selective visualization and tracking of the biomolecule. We generated 10 analogues of the HLA-A2-restricted, immunogenic hepatitis B virus-derived epitope HBcAg18-27 by replacing each amino acid with the click handle homopropargylglycine. Each analogue was tested for (1) peptide binding affinity to HLA-A2, (2) preservation of immunogenicity, and (3) accessibility for on-cell click reactions. Lastly, we performed a proof-of-concept experiment in which we demonstrate the feasibility of BOC for APP studies. All amino acids could be modified with the click handle without loss of HLA binding. Furthermore, 7 out of 10 analogues retained cognate T cell recognition. Two of the most promising analogues were tested and demonstrated to be accessible for on-cell click as well as suitable for long peptide processing and presentation studies. To our knowledge, we are the first to show the feasibility of BOC to study APP in the human setting. With BOC techniques, we may now be able to sensitively follow antigen routing by visualizing the intracellular location of (long) peptides. Furthermore, this tool enables direct and quantitative epitope studies in a T cell-independent manner.

The Mycobacterium tuberculosis complex (MTBC), comprising species such as M. tuberculosis, M. africanum, and M. canettii, is the causative agent of tuberculosis (TB), one of the deadliest infectious diseases worldwide. MTBC strains exhibit genetic diversity that influences host-pathogen interactions, immune evasion, and disease outcomes. The complement system, a crucial component of innate immunity, plays a dual role in pathogen detection and potential immune evasion, yet its interactions with MTBC strains remain underexplored. We investigated the roles of C1q and mannose-binding lectin (MBL), key pattern recognition molecules (PRMs) of the classical and lectin pathways, respectively, in complement activation against diverse clinical MTBC strains. We observed that both C1q and MBL recognize mycobacteria directly as PRMs but that the degree of binding was strain dependent. Both molecules facilitated complement cascade activation, leading to the deposition of C4b and C3b and the formation of the membrane attack complex (MAC) on bacterial surfaces. However, inhibition experiments revealed that C1q is the primary driver of complement activation in nonimmune serum, while MBL plays a supportive but nonredundant role. Despite robust complement activation, MAC formation did not significantly impact the viability of MTBC strains. Nevertheless, these findings highlight a nuanced interplay between the complement system and MTBC lineage diversity. Our results provide novel insights into early host-pathogen dynamics in TB, emphasizing the importance of considering MTBC lineage diversity in understanding the immune response against mycobacteria.

Assigning antigen specificity to T cell receptor (TCR) sequences is challenging due to the TCR repertoire's diversity and the complexity of TCR-antigen recognition. We developed the peptide-driven identification of TCRs (PDI-TCR) assay that combines in vitro expansion of cells with peptide pools, bulk TCR sequencing, and statistical analysis to identify antigen-specific TCRs from human blood. A key feature of PDI-TCR is the ability to distinguish true antigen-specific TCR clonotypes from TCRs associated with unspecific bystander activation by comparing responses to nonoverlapping peptide pools. We applied PDI-TCR to tuberculosis (TB) patients, sampling blood at diagnosis and throughout treatment, and Mycobacterium tuberculosis (Mtb)-sensitized healthy individuals (IGRA+). We identified hundreds of Mtb-specific TCRs, as well as unspecific TCRs, and characterized their phenotype in each cohort by single-cell RNA sequencing ex vivo. Mtb-specific T cells were highly diverse, with short-lived effector phenotypes only present in TB at diagnosis, while memory phenotypes were maintained through treatment. In contrast, unspecific expanded T cells were more clonally restricted, had a cytotoxic phenotype, and were maintained throughout treatment. While the PDI-TCR parameters used in this study are specific to Mtb, the underlying approach is broadly applicable to the study of antigen-specific T cells and can be adapted as needed for other antigen systems. Thus, PDI-TCR is a powerful tool for identifying antigen-specific TCRs and enables direct ex vivo identification and monitoring of antigen-specific T cells.

Interferon regulatory factors 3 and 7 (IRF3 and IRF7, respectively), which serve as key transcription factors in interferon (IFN) activation, are tightly regulated by a variety of mechanisms, including multiple posttranslational modifications, to fulfill their normal physiological functions. Nevertheless, the methylation-mediated regulation of IRF3 and IRF7 in grass carp (Ctenopharyngodon idella) remains poorly understood. In this study, we found that SMYD3, a lysine methyltransferase, is conserved across species and is induced by grass carp reovirus (GCRV) infection in grass carp. Furthermore, overexpression of grass carp SMYD3 exerted a negative regulatory effect on antiviral innate immunity. Conversely, knocking down of SMYD3 in cells enhanced the GCRV-induced antiviral gene expression. Mechanistically, SMYD3 interacts with the transcription factors IRF3 and IRF7, as demonstrated by co-immunoprecipitation and immunofluorescence confocal microscopy assays. Moreover, SMYD3 was found to orchestrate the di- or tri-methylation of the fifth lysine of IRF3 and the 11th lysine of IRF7, as identified by mass spectrometry. Furthermore, treatment with the SMYD3-specific inhibitor BCI121 significantly enhanced resistance to GCRV infection in cells and grass carp. Our results reveal a novel function of the lysine methyltransferase SMYD3 in anti-GCRV immunity and identify SMYD3 as a potential target for breeding new grass carp strains with anti-GCRV ability. In addition, our findings suggest that BCI121, a SMYD3-specific small molecule inhibitor, can be developed as an effective anti-GCRV drug for the treatment of grass carp hemorrhagic disease caused by GCRV.

Memory stem CD8+ T cells (TSCM) are a long-lived T-cell subset with stem cell-like properties, playing a key role in antiviral immunity. Despite their importance, comprehensive single-cell transcriptomic profiling of antigen-specific TSCM has not been previously conducted. In this study, an in vitro single-cell colony expansion protocol was used to investigate human CD8+ T-cell clones specific for cytomegalovirus (CMV) epitopes. Clonal lineages from selected donors were generated by single-cell sorting of dextramer-positive CD8+ T cells with varied effector and memory phenotypes, all specific for one of 2 HLA-restricted CMV epitopes. Phenotypic and functional characterization of clonal lineages derived from antigen-specific TSCM revealed differentiated memory and effector subsets (TCM, TEM, TEMRA) as well as TSCM, with the latter subset featuring multi-potentiality and multi-cytokine production. Studying TSCM-derived progeny of these varied differentiation phenotypes in single-cell transcriptomic analysis revealed strong progenitor-to-progeny effects, whereby daughter cells from a shared progenitor clustered closely regardless of progeny differentiation phenotype, suggesting a dominant impact associated with heritable genes. TSCM-derived progeny that retained a TSCM phenotype expressed canonical early-memory genes such as IL7R, CCR7, SELL, and CD27, along with novel transcripts consistently shared across donors and epitopes. The transcriptional features of clonal lineages are strongly dependent on the progenitor cell, but TSCM have an additional transcriptomic signature, likely underpinning their unique differentiation and functional characteristics. These findings have important implications for identification of long-lived and multipotent CD8 T cells for immunotherapy and immunization against viral infections.

Antigen-specific responses to complex antigens encompass a range of cell states and reactivities to an array of epitopes, reflective of the heterogeneity in immune responses. Single-cell sequencing has created new opportunities when combined with flow cytometry for profiling of immune repertoire and cell phenotype. Rare B cells with fine specificities can be precisely isolated using flow cytometry, and with oligonucleotide-tagged antigen assemblies enable the isolation of a pool of cells reactive to different antigens in parallel from biological samples. Single-cell sequencing can then detect these tags to allow mapping of individual B cell reactivities to specific antigens, in addition to simultaneous profiling of cellular phenotypes. We establish workflows for these approaches with antigen reactivity frequencies typical of human peripheral blood, by conducting all staining prior to sorting to minimize loss of rare antigen-specific cells. We identify antigen:streptavidin ratio as a priority for optimization when using novel antigens to generate oligonucleotide-tagged assemblies for B cell staining, which is demonstrated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike antigens. We develop a novel approach of combining dual labelling using separately oligonucleotide- and fluor-tagged assemblies, to increase staining sensitivity sufficiently for exploratory detection of dengue-reactive cells in naturally exposed individuals. Together these findings support parallel profiling of cells with differing antigen-specificities, conferring new opportunities to advance understanding beyond characterization of individual clonotypes to their role in the broader humoral response. This can be important in pathogen, vaccine, malignancy, and autoreactivity responses, where both antigen-reactive cells at low frequencies and interactions between different epitope responses can shape clinical outcomes.

The sympathetic nervous system modulates immune responses through the release of norepinephrine (NE), yet the dynamics of this signaling differ across lymphoid organs. In this study, we investigated how NE release and α2-adrenergic receptor (α2AR) modulation influence neuroimmune interactions in the spleen and mesenteric lymph nodes (MLNs), 2 secondary lymphoid tissues with distinct innervation patterns. Using fast-scan cyclic voltammetry (FSCV), we found that the spleen exhibited more frequent and higher-amplitude NE events than the MLNs, consistent with its denser sympathetic innervation. Pharmacological manipulation of α2ARs revealed that yohimbine hydrochloride, an α2AR antagonist, increased NE release in both organs, while dexmedetomidine hydrochloride, an α2AR agonist, suppressed it, often below detection thresholds. Complementary 3D immunohistochemistry demonstrated tissue- and cell type-specific changes in immune cell proximity to neuronal structures following adrenergic modulation, with T cells and B cells displaying distinct spatial reorganization. These findings highlight that α2AR signaling fine-tunes NE release and immune cell localization in a context-dependent manner, influenced by organ-specific architecture and innervation. Our results underscore the importance of dynamic local neuroimmune interactions in immune regulation and suggest potential therapeutic opportunities for targeting adrenergic signaling in inflammatory and autoimmune diseases.

STAT3 is pivotal for governing myeloid responses to inflammatory stimuli to prevent hyperinflammation in vivo, yet whether STAT3 mediates pathogen control and clearance by myeloid cells remains unclear. In this study, we identified significant enrichment of IFN-stimulated transcriptional pathways in Stat3-deficient bone marrow-derived macrophages (BMDMs) at steady state. This was accompanied by activation of autocrine type I IFN (IFN-I) and aberrant autocrine IL-6 signaling associated with increased STAT1 activity. Despite exaggerated baseline IFN-STAT1 signaling, Stat3-deficient BMDMs were significantly impaired in their ability to induce expression of key pathogen defense genes and specific immune mediators upon LPS stimulation. STAT3 was also required in Citrobacter rodentium-infected BMDMs for expression of pathogen defense genes and effective bacterial killing. Moreover, bone marrow chimeric mice with 20% Stat3-deficient hematopoietic cells were more susceptible to infection with C. rodentium and showed increased bacterial dissemination and reduced production of specific cytokines. IL-6 blockade subdued the intrinsic IFN response and rescued expression of select pathogen defense genes in Stat3-deficient BMDMs upon LPS stimulation yet was unable to restore bacterial killing activity. Taken together, our results identify novel functions for STAT3 in orchestrating an optimal microbial defense response and suggest this is regulated by discrete mechanisms including modulation of autocrine IL-6 signaling in macrophages.

Mucosal immunoglobulin A (IgA) promotes the survival of commensal bacteria while it inhibits the invasion by pathogens. Bacterial coating may be mediated by antigen-specific IgA recognition, polyreactivity, and/or by the IgA-associated glycans. We compared human polyclonal secretory SIgA both in vitro and in vivo with polymeric (p) monoclonal myeloma IgA proteins of defined glycan structures to assess their protective activity against necrotoxigenic Escherichia coli O55. Specifically, we evaluated the adhesion and penetration of E. coli O55 into porcine intestinal IPEC-1 cells following preincubation of the bacteria with various pIgA1 or pIgA2 preparations. The preparation designated pIgA2(F2), which exhibited a unique N-glycan composition and demonstrated the highest level of protection in vitro, was further tested in vivo in an experimental intestinal infection model using antibody-free newborn piglets. In brief, pIgA2(F2) effectively reduced inflammatory activation of gut tissue and prevented pathological alterations in intestinal architecture, performing equally well as concurrently tested milk/colostrum-derived SIgA. Future studies would lead to the identification of the specific pIgA2-associated glycans responsible for mediating protection against targeted bacterial gut infections.