A broadly protective licensed vaccine against enterotoxigenic Escherichia coli (ETEC) remains unavailable. To inform rational ETEC vaccine design, we characterized immune responses to the noncanonical antigens EatA mucinase, EtpA adhesin, and YghJ metalloprotease following experimental ETEC infection. We analyzed longitudinal cellular and humoral responses to EatA, EtpA, and YghJ in 30 volunteers infected with ETEC. Antigen-specific CD4+ T cell responses were assessed using an activation-induced marker (AIM) assay detecting CD69, CD134, CD137, and CD154 expression after antigen stimulation. Antigen-specific IgA and IgG responses in serum and IgA responses in intestinal lavage were quantified using bead-based multiplex flow cytometry. Antigen-reactive CD69+CD134+ CD4+ T cells increased on Day 10 for all three antigens, with EtpA also inducing CD134+CD137+ responses. Serum anti-EatA IgA and IgG increased on Days 10 and 28, accompanied by increased intestinal lavage anti-EatA IgA on Day 10. Mucosal IgA responses to EatA and EtpA correlated strongly with systemic IgG. Immune responses did not seem to be associated with ETEC colonization or host blood group. This first integrated characterization of human immune responses against the ETEC antigens EatA, EtpA, and YghJ demonstrates their immunogenicity in both cellular and humoral compartments, and supports their relevance as potential vaccine targets.
Vaccine-induced transcriptional responses have been extensively characterized at the gene level, but whether vaccination also alters transcript isoform usage remains largely unexplored. Here, we reanalyzed longitudinal whole-blood RNA-seq data from a discovery cohort of mRNA COVID-19 vaccine recipients using the IsoformSwitchAnalyzeR framework and validated the findings in an independent cohort. Key findings were validated by full-length RNA long-read sequencing and extended to four additional vaccine cohorts covering distinct platforms and pathogens. mRNA vaccination induced a rapid and transient wave of differential transcript usage, peaking at 24 h post-vaccination with 131 isoforms significantly altered across 107 genes, before largely resolving by Day 14. Isoform switching events were reproducible across independent cohorts and confirmed by full-length RNA long-read sequencing. Structural annotation of switching transcripts, including RMI2, WARS1, and NT5C3A, revealed changes affecting predicted protein domains and signal peptides. Notably, highly concordant isoform switching patterns were observed across MVA-based SARS-CoV-2, influenza, and Ebola vaccine cohorts and showed dose-dependent modulation. Overall, differential transcript isoform usage is a rapid and transient feature of the early human immune response to vaccination that was observed across multiple vaccine platforms. These findings reveal an underappreciated layer of transcriptional regulation that complements conventional gene-level analyses and warrants integration into future vaccine immunogenicity studies.
The Global North is increasingly exposed to a Western diet characterized by high fat, sugar, and salt content. Excess dietary salt has been linked to cardiovascular disease and hypertension and can accumulate in multiple tissues, exerting local immunomodulatory effects. Beyond these systemic consequences, a high-salt diet (HSD) is associated with gut dysbiosis, which alters the production of microbial metabolites, such as short-chain fatty acids (SCFAs), and compromises intestinal barrier integrity, thereby facilitating bacterial translocation and contributing to liver and kidney injury. These alterations are associated with inflammatory responses, although their direction and magnitude depend on dietary duration, microbial baseline composition, and experimental models. While most studies have focused on HSD-induced modulation of T cell responses, emerging data highlight macrophages as underexplored mediators of HSD-driven immune and metabolic effects. In this review, we summarize current knowledge on HSD-induced alterations of the intestinal microbiota, microbial metabolites, gut barrier function and macrophage function, and discuss their potential interplay along the gut-liver axis. In addition, we highlight key gaps and challenges that must be addressed to improve translational relevance.
Inflammaging, defined as the persistent, low-grade sterile inflammation accompanying aging, represents a central driver of age-related pathology, including cardiovascular dysfunction, neurodegeneration, metabolic disorders, and frailty. This review discusses the most recent advances in understanding its mechanistic basis, encompassing cellular senescence, the senescence-associated secretory phenotype (SASP), mitochondrial dysfunction, immune cell senescence, innate immune hyperactivation, defective inflammatory resolution, and nutrient-sensing dysregulation. Single-cell and spatial transcriptomics reveal tissue-specific and context-dependent patterns, highlighting the systemic complexity of inflammaging. Preclinical interventions demonstrate that inflammaging is modifiable through senolytics, which selectively eliminate senescent cells, and senomorphics, which suppress SASP without inducing cell death. Metabolic modulators such as metformin and rapamycin attenuate inflammatory signaling, while immune-directed therapies and microbiome-targeted interventions provide synergistic benefits through combinatorial approaches. Early-phase clinical trials in frail older adults show feasibility, safety, and preliminary efficacy, including reductions in circulating inflammatory markers, improved physical function, and enhanced immune responsiveness. Inflammaging trajectories are shaped by lifestyle, environmental exposures, and evolutionary factors, underscoring the need for personalized interventions. Remaining challenges include biomarker development, long-term safety evaluation, and heterogeneity across aging populations. Addressing these through interdisciplinary research supports a precision geroscience paradigm, where multimodal targeting of inflammaging can extend healthspan and reduce chronic disease burden.
Current understanding of the structure, function, and molecular dynamics of the alternative pathway C3-convertase has been shaped by the clinical impact of genetic variants in complement genes. Characterizing the functional impact of different mutations not only provides patient-specific insights into the pathogenic mechanisms underlying a number of complement-mediated diseases but, at a broader level, allows us to dissect the molecular underpinnings of protein-protein interactions that drive complement amplification and its control. In this review, we contextualize over 100 variants in C3, CFB, CFD, CFH, and CFI to illustrate how this collective knowledge informs known mechanisms of complement biology and illuminates gaps that remain to be defined.
The development of unconventional αβ T cells, including invariant natural killer T (iNKT) and mucosal-associated invariant T (MAIT) cells, in the thymus is distinct from conventional T cells. Unconventional αβ T cells adopt a memory phenotype, acquire effector functions, and can reside in the thymus long-term. It is well-established that positive selection of these unconventional T cells from CD4+CD8+ double-positive (DP) precursors depends on interaction with other DP cells. However, postselection, the regulation of their maturation and effector differentiation is less well understood. Professional antigen-presenting cells (APCs) are thought to have a role, but their roles have only been partially investigated previously. In this study, we investigate the impact of perturbing thymic dendritic cell (DC) and macrophage populations on intrathymic iNKT and MAIT effector subsets in C57BL/6 mice. We show that conventional type 1 DCs (cDC1s) support iNKT1 cells, while CX3CR1- and Mgl2-expressing cDC2s and macrophages support MAIT17 cells. Lastly, we show that disrupting the XCR1-XCL1 axis, which was previously shown to control cDC1 localization to the thymic cortex, alters the balance between iNKT and MAIT cells in the thymus, where there is augmentation of the iNKT cell compartment at the expense of MAIT cells. These findings further highlight the roles of hematopoietic APCs in supporting intrathymic unconventional αβ T cells.
Antiphospholipid antibodies (aPL) are drivers of inflammation and thrombosis in antiphospholipid syndrome (APS). However, the molecular mechanisms underlying infection-induced exacerbation of APS remain incompletely understood. To identify novel pathways contributing to the pro-inflammatory and pro-coagulant activation of monocytes in APS, we screened fourteen Toll-like receptor (TLR) ligands for their ability to induce tissue factor (TF) expression in a monocyte-like cell line (THP-1). Among these, Pam3CSK4, a synthetic TLR1/2 ligand, emerged as a potent inducer of TF in the presence of aPL. Subsequent experiments revealed that Pam3CSK4 synergized with aPL to amplify both TF expression and IL-1β secretion. This synergistic effect was mediated via NF-κB signaling and required activation of the NLRP3 inflammasome. aPL priming promoted ATP release, providing the second signal for NLRP3 activation. Inhibition of NLRP3 markedly reduced Pam3CSK4-induced TF expression, suggesting a critical role for inflammasome signaling in monocyte procoagulant responses. These in vitro findings were confirmed in a mouse model of APS, supporting the pathogenic relevance of the TLR1/2-NLRP3 axis in vivo. Taken together, our results validate the "second hit" paradigm in APS and suggest a novel TLR1/2-NLRP3 axis as a key mediator of infection-driven inflammatory and thrombotic responses in aPL-primed monocytes.
The complement system is a key component of innate immunity, modulating immunological processes to maintain proper homeostasis. However, dysregulation of the complement can lead to severe pathologies. Complement therapeutics strive to reduce and lower the risk of breakthrough complement activation, and the field has been growing steadily since the first complement-regulating compound was introduced to the clinic in 2007, with many complement-regulating strategies currently under development. This review focuses on therapeutic developments surrounding factor H (FH), which is a critical and native regulator of complement. FH is a complement inhibitor that controls the self-amplifying alternative pathway (AP). It functions by destabilizing C3 and C5 AP convertases through decay-accelerating activity, mainly via competition with factor B for binding to C3b. Additionally, FH acts as a cofactor for factor I, which cleaves C3b into iC3b. The classical and lectin pathways converge at the C3 level, triggering the AP self-amplification loop. Independently, the AP is triggered by a tick-over mechanism that is continuously activated. Both the amplification loop and the tick-over mechanism of the AP are controlled by FH. Consequently, enhancing FH function in a dysregulated system represents a promising therapeutic approach. FH-driven therapeutic strategies include replenishing FH via plasma-derived or recombinantly produced full-length FH. On top of that, protein-engineered constructs that contain FH fragments both with and without targeting mechanisms are under development, as well as modulating moieties such as antibodies and peptides that bind and potentiate FH. In this review, we provide an extensive overview of these developments, discuss the underlying rationale of each strategy, and evaluate their status in the therapeutic pipeline.
According to the World Health Organization (WHO), approximately 6% of COVID-19 cases develop serious long-term sequelae referred to as post-COVID-19 condition (PCC). Immunological disturbances such as persistent activation of immune cells and reduced cytotoxicity by natural killer (NK) cells are reported as key aspects in PCC. Recently, electrophysiological studies by our group demonstrated impairment of transient receptor potential melastatin 3 (TRPM3) ion channels in NK cells from PCC patients. The significant reduction in TRPM3 channel function and reduced functional activity by NK cells warrants further investigation. Hence, using live cell calcium (Ca2+) imaging ex vivo, we examined the downstream impact of TRPM3 ion channel dysfunction on intracellular and mitochondrial Ca2+ mobilization in NK cells from N = 8 PCC patients, age and sex matched to N = 8 PCC healthy controls (HC). Our findings provide new evidence of altered passive and TRPM3-mediated Ca2+ influx, significantly impacting cytoplasmic and mitochondrial Ca2+ mobilization in PCC. Passive cytosolic Ca2+ influx amplitude (p < 0.0001) was significantly reduced in PCC; however, passive mitochondrial Ca2+ mobilization (p < 0.0001) was significantly increased. Importantly, cytoplasmic and mitochondrial response rates (slope, p < 0.001) to pregnenolone sulphate stimulation were significantly reduced in PCC. Consequently, TRPM3-dependent cytosolic (p < 0.001) and mitochondrial (p < 0.0005) Ca2+ mobilization were significantly reduced in PCC compared with HC. Altered ion channel Ca2+ signalling can severely impact both the immune system and bioenergetic processes, potentially leading to broader systemic dysregulations underpinning the pathomechanism of the PCC condition, and warrants further investigations.
The pancreatic tissue is composite and plastic. The epigenetic makeup of different cell types is poorly characterized due to the lack of protocols that enable efficient recovery of epithelial cells. Here, we improve the canonical pipeline for single-cell analysis, optimizing the harvesting of nuclei from hard-to-dissociate tissues. We provide an in-depth mapping of the murine pancreas through paired RNA/ATAC multiomic single-nucleus sequencing, documenting the transcriptomic and chromatin accessibility profiles of every cell in the parenchyma. This enables a superior examination of the exocrine fraction of the pancreas. We described endocrine-exocrine interaction in silico and validated pro-proliferative signals in vitro. At the same time, we assessed pancreatic cellular heterogeneity in a holistic manner. We showed that pancreatic acinar cells can acquire multiple phenotypic states that lead to the functional diversification of pancreatic acini. In particular, we identified ultra-specialized ZG16HIGH cells in vivo and associated them with superior protein synthesis by tracking the incorporation of a synthetic amino acid. Leveraging the pairing of gene expression and chromatin opening, we studied the epigenetic elements that dictate acinar cell specialization and how they are affected by tissue repair after inflammation. We found that a subset of acinar cells shows enhanced sensitivity to inflammatory cues that determine extensive and persistent chromatin opening. The dataset is an open-source framework to interrogate the molecular histology of the murine pancreas.
Discovered in the late 19th century as a heat-sensitive plasma factor called "alexin", complement was first identified for its ability to work with antibodies to destroy microorganisms. Over the past two centuries, research advances have firmly established the complement system as a fundamental component of the immune system, with broader roles in immune surveillance, inflammation, and clearing immune complexes and apoptotic debris, while also bridging innate and adaptive immunity. Due to complement playing a central role in modulating biological processes on a system-wide scale, dysregulation or excessive activation can drive harmful inflammation and self-tissue damage. Despite some initial safety concerns and biological complexity, therapeutic targeting of the complement system has, over the past decade, emerged as a key strategy for controlling disorders in which its unregulated activation becomes pathogenic. However, inhibition of complement, particularly at the level of C3 or C5, predisposes patients to infections, most notably by encapsulated bacteria. These include a markedly increased risk of invasive infections caused by Neisseria meningitidis, as well as susceptibility to Streptococcus pneumoniae, Haemophilus influenzae, and other opportunistic viral and fungal pathogens. In this review, we aim to describe the infection risks associated with therapeutic complement inhibition and outline emerging approaches to mitigate their complications. These include optimised vaccination protocols, antimicrobial prophylaxis, patient education, and surveillance programs, as well as next-generation approaches such as pathway-selective inhibitors, personalised risk stratification, and adjunctive immune support. Enhancing these protective measures will be vital to optimising the therapeutic benefit of complement inhibition while reducing infectious morbidity and mortality.
Removal of Fc-fragments from human IgG or Fc-fused proteins. (1) IgG or Fc-fusion proteins are incubated with IdeS-Fc; (2) Fc fragments are retained using protein G agarose beads; and (3) the F(ab')2 fragments or Fc-free proteins are collected by centrifugation in the original buffer without IdeS contamination.
Type 1 diabetes (T1D) results from T cell-mediated destruction of insulin-producing pancreatic beta-cells. Recently, the immune-provoking role of stress-related neoantigens has become evident. Neoantigens unlikely contribute to central tolerance and therefore hold strong immunogenic potential, but their role in peripheral immune regulation is unknown. Here, we sought proof of concept that Tregs can be generated against islet neoantigen INS-DRiP that results from stress-induced ribosomal misreads of insulin mRNA. Tregs were induced from naïve CD4 T cells isolated from a healthy donor and co-cultured with monocyte-derived tolerogenic DCs either pulsed with neoantigen INS-DRiP or native autoantigen (proinsulin-peptide C19A3) that can induce Tregs in T1D patients. Their phenotypes, cytokine profiles and suppressive capacity were compared. Tregs induced against neoantigen completely inhibited proliferation of naïve T cells upon cognate antigen-pulsed DC stimulation, which was indistinguishable from Tregs induced against C19A3. Phenotype and cytokine profiling showed co-clustering of native autoantigen- and neoantigen-specific Tregs, and distinction from T cells generated with antigen-pulsed proinflammatory instead of tolerogenic DCs. Naïve T cells exist against islet neoantigen that can be primed to become Tregs despite the high immunogenic potential of neoantigen. Induction of immune regulation to neoantigens may be useful as immune intervention or prevention of T1D.
The possibility of enhancing T cell function by deleting specific genes represents a long-sought goal in preclinical studies and ultimately for clinical applications. Using CRISPR/Cas9 genome editing, we report that, in human cytotoxic CD8 T cell clones, the cell cycle checkpoint gene CDKN2A, encoding p16INK4A, plays a nonredundant role in controlling T cell receptor (TCR)-dependent and independent cell expansion. Deletion of CDKN2A dramatically enhanced antigen-driven and homeostatic proliferation, while preserving effector functions. In contrast, the deletion of other cell cycle inhibitors (CDKN1B, CDKN2C, and CDKN2D), alone or in combination, had no impact on T cell proliferation. We also report that mediator complex subunit 12 (MED12) and the E3 ubiquitin ligase CBL-B deletions did not affect proliferative capacity of CD8 T cell clones. Interestingly, deletion of the negative regulator of Ras signaling, RASA2, increased antigen sensitivity and cytotoxic activity, while not improving in vitro expansion. Collectively, these findings reveal a unique and critical nonredundant role for p16INK4A in regulating CD8 T cells. Deletion of CDKN2A offers a promising strategy to enhance CD8 T cell expansion ex vivo, thereby improving TCR discovery pipelines and, potentially, therapeutic applications.
The continuous evolution of SARS-CoV-2 raises concerns about immune escape from preexisting immunity. The monovalent JN.1adapted mRNA vaccine was developed to better match circulating variants, yet data on its ability to induce and broaden humoral and cellular immunity in individuals with or without prior infection remain limited. We recruited 37 immunocompetent adults before and two weeks after JN.1 vaccination to assess vaccine-induced immunity. Spike-specific CD4 and CD8 T cells were quantified following stimulation with spike-derived peptides from the parental strain, XBB.1.5, and JN.1, and their CTLA-4 expression and cytokine profiles were analyzed by flow cytometry. Spike-specific IgG and neutralizing activity against authentic parental, XBB.1.5, JN.1, and KP.3.1.1 isolates were also measured. JN.1 vaccination significantly increased spike-specific CD4+ and CD8+ T-cell frequencies with comparable cytokine profiles across variants and enhanced CTLA-4 expression. IgG levels and neutralizing titers rose markedly, with the strongest relative increases against Omicron lineage variants. Prior infection was associated with higher neutralizing titers but did not influence T-cell responses. Influenza vaccine co-administration had no adverse effect on JN.1 immunogenicity. These findings indicate that hybrid immunity enhances antibody-mediated protection, while robust, cross-reactive T-cell responses may contribute to sustained protection against severe disease irrespective of infection history.
Chimeric antigen receptors (CAR) incorporating single-chain variable fragments (scFv) exhibit much higher affinity for their cognate antigens than native T cell receptors (TCR). Reducing the affinity of the CAR has been shown to improve the function of effector CAR-T cells by limiting their activation-induced exhaustion while preserving their capacity for serial killing of antigen-rich tumor cells. CAR technologies are increasingly being applied to regulatory T cell (Treg)-based immunotherapies as well. However, the impact of CAR affinity on Treg biology and function remains poorly understood. To address this question, we transduced purified human Tregs with second-generation CAR constructs bearing scFvs with varying affinities toward HLA-A2 and compared their properties both in vitro and in vivo. High-affinity (HA) CAR-Tregs displayed higher avidity and more pronounced CAR downregulation upon antigen engagement. In contrast, low-affinity (LA) CAR-Tregs exhibited enhanced antigen-specific activation and superior suppressive capacity. These differences were confirmed using human and mouse precision-cut liver slices and a xenogeneic graft-versus-host disease (GVHD) murine model. LA CAR-Tregs exhibited greater accumulation/persistence, delayed GVHD onset, and improved survival than HA CAR-Tregs. Our findings, indicating that CAR affinity strongly influences CAR-Treg function, provide important considerations for the optimization of engineered Treg therapies and the benchmarking of existing cell products.
Plasmacytoid dendritic cells (pDCs) are specialized antiviral sentinels defined by rapid type I interferon (IFN‑I) production, yet their proteomic organization and metabolic requirements remain incompletely understood. We established the steady‑state proteome of murine splenic pDCs directly ex vivo using deep, absolute quantitative mass spectrometry and compared it with conventional dendritic cell subsets and human pDCs. pDCs exhibited a highly conserved proteomic architecture across species, with selective divergence in central carbon metabolism, amino‑acid utilization, and nutrient transporter expression. Notably, pDCs expressed exceptionally high levels of the transferrin receptor (TFRC) in both mice and humans and displayed robust transferrin‑mediated iron uptake relative to other splenic immune populations. Despite this, pDCs did not demonstrate increased total cellular iron or enhanced ferritin‑based storage. Instead, proteome‑wide iron mapping revealed preferential allocation of iron to functional iron-sulfur and heme‑containing proteins, particularly within mitochondrial pathways. Detection of the iron exporter ferroportin indicated coordinated iron import and efflux, establishing sustained iron flux rather than net accumulation. Functional assays showed that iron availability does not constrain TLR9‑induced IFN‑I or TNF production. Together, these data define a conserved iron‑handling program in pDCs characterized by high TFRC expression, balanced iron flux, and targeted redistribution into essential protein systems.
PMA/ionomycin and CD3/CD28 stimulation induce fundamentally distinct signaling programs in primary human T cells. While CD3/CD28 stimulation activates both ERK and IL-2/STAT5 signaling pathways, PMA/ionomycin induces predominantly ERK-dependent proliferation despite robust IL-2 production and CD25 upregulation. These findings highlight important mechanistic and methodological differences between pharmacological and receptor-mediated T-cell activation.
T cell exhaustion is now recognised as a structured, antigen-driven differentiation programme rather than a state of cellular fatigue. Under sustained antigen exposure, CD8+ T cells progress through distinct, hierarchically organised differentiation states, initiated by progenitor exhausted cells (TPEX), which retain self-renewal, multipotency, and responsiveness to immune checkpoint blockade. Continued stimulation drives differentiation into intermediate (TEXint) and terminally exhausted (TEX term) states, with TEXint retaining greater effector capacity than TEXterm despite both exhibiting restraint relative to functional effector T cells, alongside increasingly consolidated epigenetic architecture. Rather than reflecting immunological failure, exhaustion preserves long-term antigen surveillance while limiting tissue damage. Convergence between exhaustion-associated transcriptional modules and tissue-resident memory (TRM) programmes highlights shared mechanisms of adaptation to restrictive microenvironments. Yet TRM and TEX arise in distinct contexts and are not interchangeable. Recognising exhaustion as a context-dependent differentiation process reframes therapeutic strategies, in line with current evidence indicating that immune checkpoint blockade primarily acts by expanding and redirecting the TPEX pool rather than reversing terminal exhaustion. This framework integrates insights from chronic infection, tumour immunology, and tissue adaptation.
