The Journal of Immunology

Antibodies engage both activating Fc Receptors and the inhibitory receptor Fc{gamma}RIIB. Why macrophages need a dedicated inhibitory receptor rather than simply tuning activating receptor signaling is unclear. Using DNA-based chimeric receptors and in silico modeling, we independently controlled activating and inhibitory Fc Receptors. We found that Fc{gamma}RIIB imposed a time limit on macrophage phagocytosis and ERK signaling. The time limit is due to activating Fc Receptors converting PI(4,5)P2 to PI(3,4,5)P3, which is subsequently converted to PI(3,4)P2 by Fc{gamma}RIIB. This leads to a pulse of active signaling, which is sufficient for phagocytosis of small bacteria-sized targets but not phagocytosis of large targets and TNF secretion. Unlike engaging Fc{gamma}RIIB, reducing activating Fc Receptor signaling decreased initiation of phagocytosis, the speed of PI(3,4,5)P3 generation, and the amplitude of ERK signaling. Our results demonstrate that Fc{gamma}RIIB controls the duration of IgG signaling, while the activating Fc Receptors control sensitivity.

Control of Mycobacterium tuberculosis (Mtb) infection requires CD4+ T cell recognition of infected macrophages. However, T cells also colocalize with non-infected macrophages in granulomas. We investigated whether these bystander macrophages present Mtb antigens and shape human CD4+ T cell responses. Using ex vivo co-culture systems, non-infected monocyte-derived macrophages (MDMs) were exposed to Mtb-infected MDMs or infection-conditioned supernatants before co-incubation with autologous memory CD4+ T cells from individuals with latent Mtb infection (LTBI). Bystander macrophages activated memory CD4+ T cells through MHC-II-dependent antigen presentation. Single-cell T cell receptor (TCR) sequencing and TCR-transduced reporter cell lines identified Mtb-specific clonotypes recognizing both infected and bystander macrophages, as well as clonotypes preferentially recognizing infected cells. Strikingly, a subset of TCRs recognized infected but not bystander macrophages. Antigen transfer occurred through soluble Mtb proteins rather than extracellular vesicles. Compared with responses to infected macrophages, bystander macrophages induced attenuated effector responses. These findings reveal antigen-specific recognition of bystander macrophages and suggest that antigens preferentially presented by infected cells may inform TB vaccine design.

Dendritic cells (DCs) are central orchestrators of antiviral immunity but can be exploited by HIV-1 to facilitate systemic dissemination. HIV-1 recognition by CD169 triggers massive endocytosis (MEND)-related internalization of viral particles into sac-like compartments in monocyte-derived dendritic cells (MDDCs), thereby promoting trans-infection of CD4+ T cells. However, MDDCS represent only one DC subpopulation, and whether other physiologically relevant DC subsets exhibit similar behavior remains unclear. Moreover, endogenous CD169 expression and viral capture have not been fully characterized in cDC1, DC2, or pDCs, while remain unknown in DC3. We therefore investigated the contribution of these DC subsets to CD169-mediated HIV-1 capture and subsequent viral dissemination. We used flow cytometry to assess CD169 expression and viral capture across DC subsets differentiated from CD34 hematopoietic stem and progenitor cells (HSPC) obtained from bone marrow (BM) and cord blood (CB), two sources that differ in cellular maturity. Confocal microscopy was used to evaluate sac-like compartment formation following immune activation with IFN- or LPS and treatment with MEND inhibitors. Trans-infection capacity was quantified by measuring luciferase activity in TZM-bl reporter cells. We identified cDC1, DC2, DC3, moDC (similar to MDDCs), and pDC subsets following the differentiation of CD34 HSPC from both CB and BM. Among these populations, DC2, DC3, and moDC displayed high CD169 expression, which was significantly upregulated by IFN- or LPS stimulation. BM-derived DCs exhibited higher basal CD169 expression than CB-derived DCs, whereas stimulation induced greater CD169 upregulation in CB-derived DCs. DC2, DC3, and moDC efficiently captured HIV-1 particles in cultures derived from both sources, and viral uptake was further enhanced by immune activation. Viral internalization into sac-like compartments depended on CD169 binding and MEND-related mechanisms, ultimately facilitating HIV-1 transfer to target cells. DC2 and DC3 are key dendritic cell subsets that support CD169 dependent HIV-1 capture and are associated with MEND-driven internalization into sac-like compartments and subsequent viral transfer to target cells, as was previously described for moDCs. We further observed distinct basal and inducible CD169 expression profiles in BM- and CB-derived DCs, consistent with source-dependent maturation states. These results identify DC2 and DC3 as key cellular contributors to HIV-1 dissemination.

The MRTFs (myocardin-related transcription factors) are essential for effective cytoskeletal function during differentiation and development. To investigate the role played by the MRTFs in T cell development and function we inactivated Mrtfb in Mrtfa-null mice at the DN2 stage (MrtfabKOT animals). Positive selection, and development of MrtfabKOT regulatory (Treg), {gamma}{delta}, and NK T cells proceeded normally. Thymic egress was impaired, however, with mature T cells accumulating at the cortico-medullary junction, and in vitro MrtfabKOT thymocytes exhibited impaired cytoskeletal responses to S1P and migratory cytokines. MrtfabKOT mice were lymphopenic, with defective homeostatic proliferation and T cell homing to peripheral lymphoid organs. Although thymocyte negative selection was unaffected, MrtfabKOT animals develop autoimmune phenotypes in lacrimal gland and intestinal mucosa. Using the experimental colitis model, we show that these phenotypes reflect functional deficits in MrtfabKOT Treg cells. Our findings show that MRTF-SRF signalling plays an unanticipated role in thymocyte egress and immune tolerance, and reveal a role of the MRTFs in Treg function.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by T-cell mediated destruction of pancreatic islet {beta}-cells with genetic, environmental, and molecular triggers involved in disease pathogenesis. Patients with T1D have elevated serum levels of microRNA146a (miR146a). Polymorphisms in the miR146a gene that result in reduced expression of miR146a are associated with protection from T1D. We studied physiological regulators of miR146a expression and found that both hyperglycemia and elevated O-GlcNAcylation increased miR146a expression in T cells. Peripheral blood mononuclear cells (PBMCs) from T1D patients showed increased miR146a and O-GlcNAc transferase (OGT) expression, suggesting increased O-GlcNAcylation may promote miR146a expression in T1D patients. To determine the genetic and developmental role of miR146a in T1D, we generated miR146a-knockout (KO) non-obese diabetic (NOD) mice and found that absence of miR146a significantly protected NOD mice from spontaneous autoimmune diabetes. While we found no impact of miR146a knockout on general hematopoietic parameters and immune cell populations, remarkably, immune cell infiltration into the pancreas was significantly attenuated. Protection from autoimmune diabetes in miR146a-KO NOD mice was associated with increased regulatory T (Treg) cells in the spleen and pancreatic lymph node. Mechanistically, absence of miR146a increased NF-{kappa}B c-Rel expression in Treg cells and enhanced c-Rel binding at the Forkhead box protein P3 (FOXP3) promoter, which positively regulated Treg cell development and suppressor function, offering protection from T1D in miR146a-KO NOD mice. Our findings reveal miR146a as a key regulator of Treg cell-mediated immune tolerance through controlling NF-{kappa}B c-Rel-dependent FOXP3 expression and suggest targeting miR146a as a potential strategy to restore peripheral tolerance in T1D.

Innate immune cells, including macrophages and DCs, are critical for controlling infection by Cryptococcus neoformans. Although C. neoformans is phagocytosed equally by each pulmonary phagocyte subset, CD11b+ DCs support fungal growth, whereas Ly6c- monocyte-like macrophages are antifungal. The metabolic programs underlying these contrasting responses remain incompletely understood, but our previous work points to increased glycolysis in antifungal cells. Therefore, we hypothesized that metabolic reprogramming in phagocytes following cryptococcal exposure leads to different outcomes. We examined ten metabolic pathways using flow cytometry, metabolic inhibition, antifungal assays, and pulmonary infection. Cryptococcal exposure increased GLUT-1, HK, HIF-1, and lactate production in J774A.1 macrophages, consistent with enhanced glycolytic metabolism in antifungal cells. In contrast, ATP5a and IDH2 were reduced, suggesting diminished mitochondrial metabolism. Cryptococcus also decreased G6PD, ACAC, ASS1, and PRDX2, indicating alterations in PPP activity, fatty acid synthesis, arginine metabolism, and antioxidant defense. Pharmacologic inhibition of PPP further reduced G6PD expression and significantly decreased intracellular cryptococcal burden, supporting a role for glucose-derived metabolic pathways in regulating fungal survival. In vivo, infection increased GLUT-1 and HK expression in antifungal female Ly6c- monocyte-like macrophages but not in permissive CD11b+ DCs, whereas other metabolic markers showed limited or subset-specific changes. Collectively, these findings demonstrate that antifungal cells use a predominantly glycolytic program accompanied by reduced mitochondrial, anabolic, and antioxidant-associated protein expression following interaction with C. neoformans. This phagocyte subset-dependent metabolic response during pulmonary infection may influence antifungal activity and identify metabolic pathways that could be targeted to strengthen host defense.

Core fucosylation (alpha1,6-fucosylation) of N-glycans, catalyzed solely by fucosyltransferase 8 (FUT8), plays important roles in development, immunity, and antibody effector functions. Here we report the generation of Fut8 floxed mice using a CRISPR/Cas9-based Extreme Genome Editing (EGE) system, in which loxP sites flank exon 8 of the Fut8 gene and a LoxP-STOP-LoxP-RFP cassette was knocked in downstream of the promoter, rendering the targeted allele functionally null before Cre-mediated recombination. Crossing floxed mice with CMV-Cre transgenic mice yielded complete knockout mice carrying the Cre-recombined allele. A PCR-based genotyping method was established that unambiguously distinguishes the wild-type, heterozygous, and homozygous genotypes (+/+, delta/+, and delta/delta). Intercrossing of heterozygous mice revealed that homozygous knockout mice die during the early postnatal period, consistent with previous reports, whereas heterozygous mice are viable. Flow cytometric analysis of AAL binding to splenic B cells and AAL lectin blotting both demonstrated markedly reduced core fucosylation in heterozygous mice (60.0 +/- 6.8% and approximately 48% of wild-type levels, respectively). These results show that disruption of a single Fut8 allele substantially reduces core fucosylation, and the mouse model described here provides a practical tool for functional studies of core fucosylation.

Chronic HIV infection can leave persistent immune dysregulation despite effective antiretroviral therapy (ART), but how this altered immune landscape affects antibody induction and durability after antigen exposure remains unclear. We used SARS-CoV-2 infection and vaccination as a model of humoral immunity to a novel antigen, evaluating anti-RBD IgG titers and immune cell phenotypes in people with HIV (PWH) and without HIV (PWoH) with hybrid immunity, stratified by early (<90 days) or late ([&ge;]90 days) post-exposure time points. PWH who failed to mount protective anti-RBD IgG titers early had a B cell compartment that was distinct from PWoH and high-titer PWH. Specifically, low-titer PWH had the highest expansion of activated naive, CD11c age-associated, and IgM-skewed B cells with reduced regulatory marker expression, even after controlling for age, sex, BMI and days since infection. PWH who had low-titer responses during the late phase of the response had persistent CD11c-associated features with expansion of double-negative and altered class-switched memory B cell compartments. These B cell features correlated with CD4 and CD8 T cell activation and senescence-associated phenotypes, suggesting that failure to mount high titer antibody response and maintenance in a subset of PWH is shaped by coordinated immune dysregulation.

Background. The cholinergic anti-inflammatory pathway regulates inflammation through vagus signaling that activates the 7 nicotinic acetylcholine receptor (7nAChR) on leukocytes. Suppression of macrophage inflammatory cytokine production through 7nAChR-mediated inhibition of NF-{kappa}B activation is considered a central mechanism of this pathway. However, the effects of cholinergic activation of 7nAChR on neutrophils remain poorly understood. Here, we investigated whether and how activation of 7nAChR in neutrophils regulates neutrophil migration, defined the underlying molecular mechanism, and evaluated its contribution to anti-inflammatory protection during endotoxemia. Results. The ex vivo swarming assays revealed that activation of 7nAChR with the selective agonists PNU-282987 and AR-R17779 significantly inhibited human and mouse neutrophil migration. The inhibitory effect of PNU-282987 was abolished in 7nAChR-deficient neutrophils, confirming receptor specificity. Mechanistically, 7nAChR activation reduced integrin M{beta}2 activation, decreased F-actin polymerization, and impaired neutrophil polarization. These effects required M{beta}2, as PNU-282987 failed to inhibit actin remodeling in M-deficient neutrophils. Modulation of M{beta}2 activation with an activating antibody (MEM48) demonstrated that 7nAChR regulates neutrophil migration by limiting excessive M{beta}2 activation and maintaining an optimal adhesive state. In vivo, adoptively transferred fluorescently labeled PNU-282987-treated neutrophils exhibited reduced recruitment to the lungs compared with vehicle-treated neutrophils during LPS-induced endotoxemia. Furthermore, neutrophil depletion markedly diminished the protective effect of PNU-282987 on survival, demonstrating that neutrophils contribute to 7nAChR-mediated protection during systemic inflammation. Conclusions. Our findings identify neutrophils as an important cellular target of the cholinergic anti-inflammatory pathway and demonstrate that 7nAChR activation limits neutrophil migration by suppressing integrin M{beta}2 activation and actin cytoskeletal remodeling. Together, these findings establish M{beta}2 as a critical downstream effector of 7nAChR signaling in neutrophils and identify regulation of integrin activation as a previously unrecognized mechanism of the cholinergic anti-inflammatory pathway.

The skull bone marrow axis has recently emerged as an important source of immune cells during neuroinflammation, including in the experimental autoimmune encephalomyelitis (EAE) , the mouse model of multiple sclerosis. The cribriform plate bone marrow (cpBM), is a portion of the ethmoid bone of the skull of mice, and is positioned next to nasal regions. At the cribriform plate olfactory nerves pass through the skull and recent evidence supports that it represents an important immune niche with links to CSF outflow and connections to cpBM. Previous data supports cellular composition of the ethmoid bone marrow in mice is altered in neuroinflammation suggesting it senses inflammatory cues, but the exact nature of these changes is still unclear. Here using scRNAseq and flow cytometry of the ethmoid bone marrow region we test how the region responds to EAE. In addition we also compare the cpBM region with systemic bone marrow in the femur to understand region specific differences. We report that neuroinflammation creates a regulatory response at these sites and implicates the cpBM in controlling these effects. A better understanding of the cribriform niche may offer new treatments for neuroinflammatory disease

Antiviral monoclonal antibodies (mAbs) rely on Fc-mediated effector functions, which depend on interactions with Fc receptors (FcRs) expressed on immune cells such as monocytes and dendritic cells (DCs). While combinations of antiviral mAbs with type I interferons (IFN-I) or IFN inducers are being explored to enhance therapeutic efficacy, the impact of these immunomodulators on FcR expression remains poorly understood. In this study, we investigated the effects of IFN-2, IFN-{beta}, and the IFN-inducer TAK-981 (a SUMOylation inhibitor) on FcR modulation and functional activation in human monocyte subsets and monocyte-derived DCs (moDCs). Using flow cytometry and imaging techniques, we mapped the expression of activating Fc{gamma}Rs (CD64, CD32A, CD16A) and neonatal FcR (FcRn) under steady-state and stimulated conditions. We found that IFN-I selectively downregulates CD16A expression in classical monocytes while preserving other FcRs, whereas TAK-981 reduces FcRn expression in classical monocytes without affecting CD16A. Additionally, IFN-{beta} and TAK-981 but not IFN-2, selectively enhanced Fc{gamma}RI expression in moDC. IFN-{beta} also induced a stronger maturation and functional activation program in monocytes and moDCs compared with IFN-2, whereas TAK-981 largely reproduced the IFN-{beta}-driven phenotype and secretory profile. Furthermore, we demonstrated that IFN-I signaling alters the spatial distribution of FcRs, with IFN-{beta} and TAK-981 promoting distinct redistribution patterns of Fc{gamma}Rs in monocytes and moDCs. These findings reveal a novel layer of FcR regulation, highlighting that IFN-I and IFN inducers differentially modulate FcR expression, localization, and functional activation in a cell-type- and stimulus-specific manner. Overall, this work provides a framework for understanding how IFN-I-based immunotherapies may influence Fc-mediated immune functions while preserving FcRn-dependent antibody persistence.

CD8 T cell exhaustion during chronic infection and cancer is marked by impaired effector function and limited responsiveness to stimulation. While memory CD8 T cells can be activated TCR-independently by IL-12 and IL-18, it remains unclear whether exhausted cells, despite diminished TCR responsiveness, retain this cytokine-driven effector capacity. Here, we show that IL-12 and IL-18 stimulation induce robust IFN{gamma} production in exhausted CD8 T cells from chronic LCMV Cl13 infection, exceeding responses to antigen-specific stimulation. This activation does not result in upregulation of Tox, suggesting engagement of transcriptional circuits distinct from those promoting exhaustion. Single-cell transcriptomics revealed an innate acting T cell (TIA)-specific gene signature enriched within exhausted populations in chronic infection and tumor-infiltrating lymphocytes, particularly at the interface of intermediate and terminal exhaustion. Using heterologous Legionella pneumophila infection, we confirmed in vivo bystander responsiveness of exhausted CD8 T cells, reducing bacterial burden, highlighting their functional capacity. Our findings demonstrate that exhausted CD8 T cells retain inducible effector capacity through IL-12 and IL-18 stimulation, providing new insights into possible routes to activate exhausted T cells.

Autoreactive B cells must escape immune tolerance mechanisms to drive autoimmune disease, potentially through alternative paths to memory and antibody-secreting cell formation. "Atypical" B cells represent one such alternative pathway and are expanded in several human autoimmune diseases. Atypical B cells display phenotypic heterogeneity, but how this connects with differences in transcriptional programming, isotype switching and somatic hypermutation is incompletely understood in type 1 diabetes (T1D). Screening efforts to detect T1D prior to clinical diabetes onset affords an opportunity to study B cells early in immunopathogenesis, in which they primarily function as antigen-presenting cells. Prior evidence points to atypical B cell populations as reservoirs for islet autoreactivity in human T1D. We therefore developed sorting strategies to enrich for atypical and other autoreactive-prone B cell populations from the peripheral blood of early-stage T1D individuals and performed single-cell RNA/BCR-sequencing. We identified four transcriptionally defined atypical B cell clusters, each showing differential upregulation of atypical B cell defining genes. Additional gene expression changes were noted in broadly transcriptionally similar, but phenotypically distinct atypical B cell populations, with differences noted in the average extent of isotype switching and somatic hypermutation. Some atypical B cell populations showed extensive clonal sharing with classical memory or plasmablast subsets, consistent with relationships amongst these subsets. B cells reactive against a key islet autoantigen, insulin, were enriched in select atypical B cell populations. These findings highlight specific atypical B cell populations and features with potential value as biomarkers of disease progression or response to immunotherapy in T1D, and potentially other autoimmune diseases.

Innate immune responses must be tightly controlled to maintain immuno-homeostasis. However, the regulatory mechanisms underlying the activation and early stages of the innate immune response remain poorly defined. Here, we found the soluble C-type lectin Clec18a is up-regulated in multiple inflammatory diseases, including sepsis and influenza, and secreted within minutes upon activation in multiple immune cells, including macrophages, dendritic cells (DCs), and T cells. Clec18a-myeloid-knockout mice exhibited reduced survival and exacerbated inflammation than the wild-type. However, exogenous recombinant Clec18a administration alleviated inflammation both in vitro and in vivo. Mechanistically, we identified macrophage-inducible C-type lectin (Mincle) as the receptor of Clec18a. Clec18a-Mincle binding triggers Trpm7-dependent calcium influx, which activates the ubiquitin E3 ligase Cbl and leads to Mincle-Cbl-MyD88-Trpm7 complex formation and degradation. Cbl deficiency, calcium inhibition and Trpm7 knockdown abrogated Clec18a-mediated immune suppression. Thus, our study revealed Clec18a as a critical checkpoint on the initiation of innate immune response through an intrinsic crosstalk between CLR and TLR signaling.

B-cell activation requires extensive metabolic reprogramming, yet how B cells acquire and utilize extracellular fatty acids and adapt to changes in lipid availability remains poorly understood. Here, we show that B-cell activation and the extracellular lipid environment dynamically regulate B cells. B-cell receptor (BCR) and Toll-like receptor 4 (TLR4) stimulation induced distinct mitochondrial and lipid-handling programs, differentially regulating fatty-acid uptake, cholesterol acquisition, lipid storage and mitochondrial respiration. Disrupting long-chain fatty-acid uptake, acyl-CoA activation, or mitochondrial utilisation impaired B-cell proliferation, demonstrating that multiple steps of fatty-acid metabolism are required for B-cell expansion. However, increasing extracellular fatty-acid availability alone did not enhance proliferation, indicating that fatty-acid acquisition and metabolic utilisation are functionally distinct. B cells also acquired fatty acids directly from adipocytes, identifying the local microenvironment as a potential source of metabolic substrates. In vivo, dietary fatty-acid chain length differentially shaped the germinal-centre response. A long-chain fatty-acid-rich diet increased germinal-centre B cells and T follicular helper cells and induced mitochondrial and transcriptional changes that partially converged with those observed following obesogenic feeding. Circulating lipidomics further revealed shared remodelling of lipid species between long-chain fatty-acid-rich and obesogenic diets, including altered partitioning of stearate-containing lipids. Exogenous fatty acids also modified membrane order in activated B cells, suggesting that lipid availability can influence both metabolic and biophysical properties. Together, these findings establish fatty-acid uptake, activation, and utilization as integral components of B-cell metabolic adaptation and demonstrate that dietary fatty-acid composition can remodel the lipid environment and germinal-centre response.

Lymphocyte activation gene-3 (Lag3) is an inhibitory receptor that limits effector T cell activation and function. We previously demonstrated that Lag3 supports regulatory T (Treg) cell function through Myc-dependent metabolic reprogramming. However, the intracellular mechanisms underlying this regulation remain incompletely understood. Here, we generated mice with Treg-specific deletion of the Lag3 KIEELE motif and demonstrated that this motif is required for Treg-mediated suppression of autoimmune inflammation. Loss of the Lag3 KIEELE motif led to enhanced TCR/PI3K-Akt/MEK-ERK signaling and Myc expression, accompanied by increased glycolytic activity and acquisition of effector T cell-like features. We also identified that hematopoietic progenitor kinase 1 (HPK1) and Cbl-interacting protein of 85 kDa (CIN85) associate with Lag3 through the KIEELE motif. HPK1 depletion partially recapitulated the phenotypic changes and impaired Treg suppressive function in vivo. Together, these findings identify the KIEELE motif as a critical link between Lag3-associated signaling machinery and metabolic regulation that sustains Treg suppressive function during autoimmune inflammation.

Aspergillus fumigatus is the most common cause of invasive aspergillosis (IA), an opportunistic pulmonary infection of immunocompromised patients that can be fatal, despite treatment with modern antifungal drugs. During infection, myeloid cells, such as neutrophils, monocytes, and monocyte-derived dendritic cells (Mo-DCs) are recruited to the lung and are critical for phagocytosing and killing A. fumigatus spores. Recent evidence has shown that intracellular crosstalk between these professional immune cells and other resident pulmonary cell types is required for an effective immune response to A. fumigatus, and that signaling via the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) is part of this intracellular communication. In this study, we sought to determine a role for GM-CSF signaling on monocytes and their progeny. We harnessed a mouse model of deletion of GM-CSF receptor {beta} chain (encoded by Csf2rb) in CCR2-expressing cells. Using this model, we found that GM-CSF signaling is required on CCR2+ cells for killing of A. fumigatus and for host survival. This observation expands the importance of GM-CSF in host defense during A. fumigatus pulmonary infection and extends its role in pathways of intracellular crosstalk that mediate effective pulmonary antifungal immunity.

Marmosets (MAR, Callithrix jacchus) are nonhuman primates extensively studied in a broad array of preclinical biomedical research areas. However, the limited number of available immunological reagents remains a critical shortcoming of these models. We successfully developed monoclonal antibodies (mAbs) pairs that can be used in immunoassays for the identification and quantification of the biomarkers of inflammation C-reactive protein (CRP), CXCL-10 (IP-10), interleukin (IL)-4, IL-6, and IL-10. Recombinant MAR protein variants were produced in mammalian cells, purified and used for mice immunization. Hybridoma clones were selected first for their capacity to bind the recombinant proteins. All possible pair combinations of these mAbs were then tested for their capacity to identify their targets in MAR-derived samples. These MAR samples consisted of supernatant of PBMCs exposed ex vivo to different stimulants, and plasma samples from animals stimulated in vivo with a low-dose intravenous LPS inoculation. The majority of the mAbs that recognized the recombinant proteins failed to bind to the MAR-derived homologs. However, we found MAR-derived reactive mAbs and selected pairs with optimal sensitivity and signal-to-noise ratio for immunoassays that included Luminex multiplexing assays, ELISA, and ELISpot assays. These immunoreagents and assays will improve the translational value of different MAR biomedical models.

Interleukin-15 (IL-15) trans-presentation (TP) by B cells is an important mechanism of T-cell activation; however, the spatial organisation of interleukin-15 receptor (IL-15R) relative to major histocompatibility complex (MHC) molecules on B-cell microvilli remains poorly understood. As microvilli protrude from the B-cell surface and may serve as sites of initial B cell-T-cell contact, the distribution of IL-15R and MHC molecules within these structures may be important during the earliest stages of T-cell recognition and activation. Here, we investigated the spatial association and molecular proximity of IL-15R with MHC class I and class II molecules on B-cell microvilli before immunological synapse formation, using confocal microscopy, stimulated emission depletion (STED) microscopy, stochastic optical reconstruction microscopy (STORM), and fluorescence lifetime imaging microscopy-based Forster resonance energy transfer (FLIM-FRET). Both MHC class I and class II molecules showed significant spatial association with IL-15R; however, the extent of colocalisation decreased as spatial resolution increased. STED microscopy revealed significant colocalisation between IL-15R and MHC class I, whereas STORM did not detect this association. In contrast, IL-15R and MHC class II remained significantly colocalised at both resolutions. FLIM-FRET further demonstrated molecular proximity between IL-15R and both MHC class I and class II molecules, with higher FRET efficiency observed for MHC class II. Collectively, these findings indicate that IL-15R is spatially organised in proximity to both MHC class I and class II molecules on B-cell microvilli before immunological synapse formation. This arrangement at potential sites of initial B-cell-T-cell contact may facilitate the coordination of IL-15 trans-presentation and antigen presentation during the earliest stages of B-cell-T-cell interactions.

Serious pertussis infections disproportionately affect infants but the biological basis for this age-dependent susceptibility remains unclear. Infant mouse models recapitulate features of severe human infant pertussis. We investigated the role of interferon lambda (IFN-{lambda}), a key regulator of mucosal immunity, in Bordetella pertussis infection of infant mice. While infected adult mice upregulate lung IFN-{lambda}, infant mice inoculated at P7 fail to upregulate IFN-{lambda} and succumb to infection. We hypothesized that failure to produce IFN-{lambda} during infection represents a critical immunological deficit in infant mice, and that restoring IFN-{lambda} signaling would improve survival outcomes. Whereas wild-type mice gained complete protection from lethal B. pertussis infection by P10, mice lacking the IFN-{lambda} receptor component IFNLR1 did not achieve full protection until P21. Loss of IFNLR1 was associated with enhanced bacterial dissemination to systemic organs. Infant mice possessed a functional IFN-{lambda} receptor in the lungs but failed to upregulate IFN-{lambda} during infection, and supplementing IFN-{lambda} exogenously extended survival. RNA sequencing of lung tissue from infected and uninfected wild-type and IFNLR1-deficient mice inoculated at different ages identified an immune transcriptional framework distinguishing susceptible from resistant animals at a systems level and revealed IFN-{lambda} signaling as a critical driver of immunological maturation in the infant lung. Infected infant IFNLR1-deficient mice had dysregulated immune cell recruitment to the lungs, indicating a quantitatively expanded but qualitatively impaired response. These findings demonstrate that IFN-{lambda} affects immune maturation accounting for a critical window of age-dependent resistance to lethal pertussis with novel therapeutic possibilities for human infants with this disease.