Innate lymphoid cells (ILCs) fulfill critical roles in maintenance of tissue-specific homeostasis but have also been implicated in disease pathology when dysregulated. Although they are broadly classified into three core subsets, it is increasingly apparent that ILCs exhibit plasticity in response to microenvironmental factors. Accurate and holistic evaluation of the ILC landscape is critical to understanding the contribution of ILCs to disease pathology. Using high-parameter flow cytometry, we comprehensively interrogated the phenotypic and functional diversity of ILCs in healthy volunteers and patients with severe asthma (SA), assessing the reciprocity between peripheral blood and airway compartments and dissecting the impact of anti-IL-5/5Rα biologics on these responses. We identified substantial heterogeneity and putative plasticity in human ILC responses, highlighting inherent limitations of conventional enumeration strategies. Deep phenotypic and functional profiling demonstrated a distinct sexual dimorphism in ILC responses in patients with SA. Females displayed an elevated abundance of circulating ILC progenitors, ILC1s, and ILC3s, whereas males presented with diminished ILC2s compared with respective healthy controls. Circulating ILC progenitors inversely correlated with testosterone concentrations. Moreover, we identified a reciprocal influx of all core ILC subsets into the airways of patients with SA, with unbiased multisource clustering identifying a relationship between elevated airway ILC2s and reduced lung function. Last, we showed that anti-IL-5/5Rα biologics largely ablated airway ILC type 2 cytokine production without affecting core ILC subset abundance in the peripheral blood or airways, identifying a potential mechanism whereby anti-IL-5/5Rα biologics alleviate clinical disease in patients with SA.

A better understanding of the mechanisms governing virus-induced exacerbations of chronic obstructive pulmonary disease (COPD) is urgently needed to develop more effective therapeutic approaches. Animal models are invaluable tools for gaining mechanistic insight into innate immunity. Here, we describe the methods required to establish a short-term mouse model comprising elastase administration to induce features of COPD, combined with infection using the minor group rhinovirus serotype A1 (RV-A1). This model recapitulates the hallmark features of human virus-induced COPD exacerbation and facilitates the study of mechanisms that drive these episodes.

RATIONALE Mounting evidence demonstrates a role for extracellular vesicles (EVs) in driving lung disorders, such as COPD. While cigarette smoke (CS) is the primary risk factor for COPD, a link between CS and the EVs that could lead to COPD is unknown. OBJECTIVE To ascertain whether exposure to CS elicits a proteolytic EV signature capable of driving disease pathogenesis. METHODS Protease expression and enzymatic activity was measured in EVs harvested from the BAL fluid of smoke-exposed mice and otherwise healthy human smokers. Pathogenicity of EVs was examined using pathological tissue scoring following EV transfer into naïve recipient mice. MEASUREMENTS & MAIN RESULTS Our analyses reveal a unique EV profile defined by neutrophil and macrophage-derived EVs. These EVs are characterized by abundant surface expression of neutrophil elastase (NE) and matrix metalloproteinase (MMP-12), respectively. CS-induced mouse or human-derived airway EVs had a robust capacity to elicit rapid lung damage in naïve recipient mice, with an additive effect of NE and MMP-12-expressing EVs. CONCLUSIONS These studies demonstrate the capacity of CS to drive the generation of unique EV populations containing NE and MMP-12. The coordinated action of these EVs is completely sufficient to drive emphysematous disease, and their presence could operate as a prognostic indicator for COPD development. Furthermore, given the robust capacity of these EVs to elicit emphysema in naïve mice, they provide a novel model to facilitate pre-clinical COPD research. Indeed, the development of this model has led to the discovery of a previously unrecognized CS-induced protective mechanism against EV-mediated damage.

Leukotriene A4 hydrolase (LTA4H) is a bifunctional enzyme, with dual activities critical in defining the scale of tissue inflammation and pathology. LTA4H classically operates intracellularly, primarily within myeloid cells, to generate pro-inflammatory leukotriene B4. However, LTA4H also operates extracellularly to degrade the bioactive collagen fragment proline-glycine-proline to limit neutrophilic inflammation and pathological tissue remodeling. While the dichotomous functions of LTA4H are dictated by location, the cellular source of extracellular enzyme remains unknown. We demonstrate that airway extracellular LTA4H concentrations are governed by the level of pulmonary vascular permeability and influx of an abundant repository of blood-borne enzyme. In turn, blood LTA4H originates from liver hepatocytes, being released constitutively but further upregulated during an acute phase response. These findings have implications for our understanding of how inflammation and repair are regulated and how perturbations to the LTA4H axis may manifest in pathologies of chronic diseases.

AIM:Treatment options for viral lung infections are currently limited. We aimed to explore the safety and efficacy of inhaled ethanol in an influenza-infection mouse model.MATERIALS AND METHODS:In a safety and tolerability experiment, 80 healthy female BALB/c mice (20 per group) were exposed to nebulized saline (control) or three concentrations of ethanol (40/60/80% ethanol v/v in water) for 3x30-minute periods, with a two-hour break between exposures. In a separate subsequent experiment, 40 Female BALB/c mice were nasally inoculated with 104.5 plaque-forming units of immediate virulence "Mem71" influenza. Infection was established for 48-h before commencing treatment in 4 groups of 10 mice with either nebulized saline (control) or one of 3 different concentrations of ethanol (40/60/80% ethanol v/v in water) for 3x30-minute periods daily over three consecutive days. In both experiments, mouse behavior, clinical scores, weight change, bronchoalveolar lavage cell viability, cellular composition, and cytokine levels, were assessed 24-h following the final exposure, with viral load also assessed after the second experiment.RESULTS:In uninfected BALB/c mice, 3x30-minute exposures to nebulized 40%, 60%, and 80% ethanol resulted in no significant differences in mouse weights, cell counts/viability, cytokines, or morphometry measures. In Mem71-influenza infected mice, we observed a dose-dependent reduction in viral load in the 80%-treated group and potentiation of macrophage numbers in the 60%- and 80%-treated groups, with no safety concerns.CONCLUSIONS:Our data provides support for inhaled ethanol as a candidate treatment for respiratory infections.

Respiratory viruses are a major trigger of exacerbations in chronic obstructive pulmonary disease (COPD). Airway neutrophilia is a hallmark feature of stable and exacerbated COPD but roles played by neutrophil extracellular traps (NETS) in driving disease pathogenesis are unclear. Here, using human studies of experimentally-induced and naturally-occurring exacerbations we identify that rhinovirus infection induces airway NET formation which is amplified in COPD and correlates with magnitude of inflammation and clinical exacerbation severity. We show that inhibiting NETosis protects mice from immunopathology in a model of virus-exacerbated COPD. NETs drive inflammation during exacerbations through release of double stranded DNA (dsDNA) and administration of DNAse in mice has similar protective effects. Thus, NETosis, through release of dsDNA, has a functional role in the pathogenesis of COPD exacerbations. These studies open up the potential for therapeutic targeting of NETs or dsDNA as a strategy for treating virus-exacerbated COPD.

Leukotriene A4 4 hydrolase (LTA4H) 4 H) is a bifunctional enzyme, with dual activities critical in defining the scale of tissue inflammation and pathology. LTA4H 4 H classically operates intracellularly, primarily within myeloid cells, to generate pro-inflammatory leukotriene B4. 4 . However, LTA4H 4 H also operates extracellularly to degrade the bioactive collagen fragment proline-glycine-proline to limit neutrophilic inflammation and pathological tissue remodeling. While the dichotomous functions of LTA4H 4 H are dictated by location, the cellular source of extra- cellular enzyme remains unknown. We demonstrate that airway extracellular LTA4H 4 H concentrations are governed by the level of pulmonary vascular permeability and influx of an abundant repository of blood-borne enzyme. In turn, blood LTA4H 4 H originates from liver hepatocytes, being released constitutively but further up- regulated during an acute phase response. These findings have implications for our understanding of how inflammation and repair are regulated and how perturbations to the LTA4H 4 H axis may manifest in pathologies of chronic diseases.

The bone marrow is a specialised niche responsible for the maintenance of hematopoietic stem and progenitor cells during homeostasis and inflammation. Recent studies however have extended this essential role to the extramedullary and extravascular lung microenvironment. Here, we provide further evidence for a reservoir of hematopoietic stem and progenitor cells within the lung from embryonic day 18.5 until adulthood. These lung progenitors display distinct microenvironment-specific developmental kinetics compared to their bone marrow counterparts, exemplified by a rapid shift from a common myeloid to megakaryocyte-erythrocyte progenitor dominated niche with increasing age. In adult mice, Influenza A viral infection results in a transient reduction in multipotent progenitors within the lungs, with a parallel increase in downstream granulocyte-macrophage progenitors and dendritic cell populations associated with acute viral infections. Our findings suggest lung hematopoietic progenitors play a role in re-establishing immunological homeostasis in the respiratory mucosa, which may have significant clinical implications for maintaining pulmonary health following inflammatory perturbation.

Rationale: Mounting evidence demonstrates a role for extracellular vesicles (EVs) in driving lung disorders, such as chronic obstructive pulmonary disease (COPD). Although cigarette smoke (CS) is the primary risk factor for COPD, a link between CS and the EVs that could lead to COPD is unknown. Objective: To ascertain whether exposure to CS elicits a proteolytic EV signature capable of driving disease pathogenesis. Methods: Protease expression and enzymatic activity were measured in EVs harvested from the BAL fluid of smoke-exposed mice and otherwise healthy human smokers. Pathogenicity of EVs was examined using pathological tissue scoring after EV transfer into naive recipient mice. Measurements and Main Results: The analyses revealed a unique EV profile defined by neutrophil- and macrophage-derived EVs. These EVs are characterized by abundant surface expression of neutrophil elastase (NE) and matrix metalloproteinase 12 (MMP12), respectively. CS-induced mouse or human-derived airway EVs had a robust capacity to elicit rapid lung damage in naive recipient mice, with an additive effect of NE- and MMP12-expressing EVs. Conclusions: These studies demonstrate the capacity of CS to drive the generation of unique EV populations containing NE and MMP12. The coordinated action of these EVs is completely sufficient to drive emphysematous disease, and their presence could operate as a prognostic indicator for COPD development. Furthermore, given the robust capacity of these EVs to elicit emphysema in naive mice, they provide a novel model to facilitate preclinical COPD research. Indeed, the development of this model has led to the discovery of a previously unrecognized CS-induced protective mechanism against EV-mediated damage.

Immunotherapy treatment strategies have proven effective in a limited portion of patients, where identifying responders from non-responders to treatment remains a challenge. While some indications can be drawn from invasive biopsies, we need more accessible methods for predicting response and better correlates of response prior to starting therapy. Recent work has identified differences in immune composition at baseline in peripheral blood from melanoma patients responding to PD-1 blockade treatment. Through flow cytometric analysis of T cell receptors, phenotypical features of CD8+ and CD4+ T cells and Tregs could allow for the stratification of treatment response. Analysing T cells within peripheral blood could potentially allow for the stratification of PD-1 treatment response prior to therapy in different cancer settings.

Introduction:CXCL17 is a mucosally secreted protein, and the most recently identified human chemokine, an assignment based on protein fold prediction and chemotactic activity for leukocytes. However, these credentials have been the subject of much recent discussion and no experimental evidence has been presented regarding the definitive structure of CXCL17. In this study, we evaluated the structural and chemoattractant credentials of CXCL17 to better characterize this molecule, and gain deeper insights into its functional role as a glycosaminoglycan (GAG) binding protein.Methods:In the absence of structural information, in silico modeling techniques assessed the likelihood of CXCL17 adopting a chemokine fold. Recombinant CXCL17 was synthesized in mammalian and prokaryotic systems. Modified Boyden chamber and real-time chemotaxis assays assessed the ability of CXCL17 to promote chemotaxis of murine splenocytes, human neutrophils, and CXCR1 transfectants. The efficacy of CXCL17 binding to GAGs was quantified with solid-phase assays and bio-layer interferometry techniques.Results:All modeling efforts failed to support classification of CXCL17 as a chemokine based on its predicted conformation. Recombinant CXCL17 was observed to dimerize as a function of concentration, a characteristic of several chemokines. Contrary to a previous report, CXCL17 was not chemotactic for murine splenocytes, although it was a low-potency chemoattractant for human neutrophils at micromolar concentrations, several orders of magnitude higher than those required for CXCL8. As anticipated owing to its highly basic nature, CXCL17 bound to GAGs robustly, with key C-terminal motifs implicated in this process. While inactive via CXCR1, CXCL17 was found to inhibit CXCR1-mediated chemotaxis of transfectants to CXCL8 in a dose-dependent manner.Discussion:In summary, despite finding little evidence for chemokine-like structure and function, CXCL17 readily bound GAGs, and could modulate chemotactic responses to another chemokine in vitro. We postulate that such modulation is a consequence of superior GAG binding, and that C-terminal fragments of CXCL17 may serve as prototypic inhibitors of chemokine function.

RATIONALE Mounting evidence demonstrates a role for extracellular vesicles (EVs) in driving lung disorders, such as COPD. While cigarette smoke (CS) is the primary risk factor for COPD, a link between CS and the EVs that could lead to COPD is unknown. OBJECTIVE To ascertain whether exposure to CS elicits a proteolytic EV signature capable of driving disease pathogenesis. METHODS Protease expression and enzymatic activity was measured in EVs harvested from the BAL fluid of smoke-exposed mice and otherwise healthy human smokers. Pathogenicity of EVs was examined using pathological tissue scoring following EV transfer into naïve recipient mice. MEASUREMENTS & MAIN RESULTS Our analyses reveal a unique EV profile defined by neutrophil and macrophage-derived EVs. These EVs are characterized by abundant surface expression of neutrophil elastase (NE) and matrix metalloproteinase (MMP-12), respectively. CS-induced mouse or human-derived airway EVs had a robust capacity to elicit rapid lung damage in naïve recipient mice, with an additive effect of NE and MMP-12-expressing EVs. CONCLUSIONS These studies demonstrate the capacity of CS to drive the generation of unique EV populations containing NE and MMP-12. The coordinated action of these EVs is completely sufficient to drive emphysematous disease, and their presence could operate as a prognostic indicator for COPD development. Furthermore, given the robust capacity of these EVs to elicit emphysema in naïve mice, they provide a novel model to facilitate pre-clinical COPD research. Indeed, the development of this model has led to the discovery of a previously unrecognized CS-induced protective mechanism against EV-mediated damage.

BACKGROUND:Results from recent clinical studies suggest potential efficacy of immune training (IT)-based approaches for protection against severe lower respiratory tract infections in infants, but underlying mechanisms are unclear. OBJECTIVE:We used systems-level analyses to elucidate IT mechanisms in infants in a clinical trial setting. METHODS:Pre- and posttreatment peripheral blood mononuclear cells from a placebo-controlled trial in which winter treatment with the IT agent OM85 reduced infant respiratory infection frequency and/or duration were stimulated for 24 hours with the virus/bacteria mimics polyinosinic:polycytidylic acid/lipopolysaccharide. Transcriptomic profiling via RNA sequencing, pathway and upstream regulator analyses, and systems-level gene coexpression network analyses were used sequentially to elucidate and compare responses in treatment and placebo groups. RESULTS:In contrast to subtle changes in antivirus-associated polyinosinic:polycytidylic acid response profiles, the bacterial lipopolysaccharide-triggered gene coexpression network responses exhibited OM85 treatment-associated upregulation of IFN signaling. This was accompanied by network rewiring resulting in increased coordination of TLR4 expression with IFN pathway-associated genes (especially master regulator IRF7); segregation of TNF and IFN-γ (which potentially synergize to exaggerate inflammatory sequelae) into separate expression modules; and reduced size/complexity of the main proinflammatory network module (containing, eg, IL-1,IL-6, and CCL3). Finally, we observed a reduced capacity for lipopolysaccharide-induced inflammatory cytokine (eg, IL-6 and TNF) production in the OM85 group. CONCLUSION:These changes are consistent with treatment-induced enhancement of bacterial pathogen detection/clearance capabilities concomitant with enhanced capacity to regulate ensuing inflammatory response intensity and duration. We posit that IT agents exemplified by OM85 potentially protect against severe lower respiratory tract infections in infants principally by effects on innate immune responses targeting the bacterial components of the mixed respiratory viral/bacterial infections that are characteristic of this age group.

Background: Proline-Glycine-Proline (PGP) is a bioactive collagen-derived fragment proteolytically liberated during inflammation, and subsequently able to promote both neutrophil recruitment and epithelial repair. During acute, self-resolving inflammation, PGP is readily degraded by LTA4H to limit its bioavailability. In the chronic lung diseases (CLDs) asthma and COPD, a failure to degrade PGP results in chronic neutrophilia and pathological epithelial remodelling. However, the PGP-LTA4H axis has not been investigated in idiopathic pulmonary fibrosis (IPF) - a CLD characterised by aberrant epithelial repair and ensuing fibrosis. Methods: The PGP-LTA4H axis was characterised in bronchoalveolar lavage of 69 IPF and 17 control patients, and in a mouse bleomycin model of pulmonary fibrosis. The role of PGP in driving the pathology of bleomycin-induced fibrosis was defined by preventing its degradation (lta4h−/− mice) or via administration of PGP. Results: Unlike in other CLDs, PGP was undetectable in IPF and in bleomycin treated mice due to overzealous degradation by high levels of LTA4H. Studies in bleomycin treated lta4h−/− mice suggested distinct biphasic roles of the enzyme – limiting neutrophilic inflammation early after bleomycin treatment but enabling aberrant epithelial repair in the fibrotic phase. Accordingly, administration of PGP to bleomycin treated wild type mice during the early phase augmented neutrophilia, while delayed administration of PGP during the late fibrotic phase alleviated pathology. Conclusions: Overzealous degradation of PGP by LTA4H enables aberrant epithelial repair responses and ensuing pulmonary fibrosis in IPF patients.

This 25-parameter, 22-color full spectrum flow cytometry panel was designed and optimized for the comprehensive enumeration and functional characterization of innate lymphoid cell (ILC) subsets in mouse tissues. The panel presented here allows the discrimination of ILC progenitors (ILCP), ILC1, ILC2, NCR+ ILC3, NCR- ILC3, CCR6+ lymphoid tissue-inducer (LTi)-like ILC3 and mature natural killer (NK) cell populations. Further characterization of ILC and NK cell functional profiles in response to stimulation is provided by the inclusion of subset-specific cytokine markers, and proliferation markers. Development and optimization of this panel was performed on freshly isolated cells from adult BALB/c lungs and small intestine lamina propria, and ex vivo stimulation with phorbol 12-myrisate 13-acetate, ionomycin, and pro-ILC activating cytokines.

Abstract Objectives Incomplete maturation of immune regulatory functions at birth is antecedent to the heightened risk for severe respiratory infections during infancy. Our forerunner animal model studies demonstrated that maternal treatment with the microbial‐derived immune training agent OM‐85 during pregnancy promotes accelerated postnatal maturation of mechanisms that regulate inflammatory processes in the offspring airways. Here, we aimed to provide proof of concept for a novel solution to reduce the burden and potential long‐term sequelae of severe early‐life respiratory viral infection through maternal oral treatment during pregnancy with OM‐85, already in widespread human clinical use. Methods In this study, we performed flow cytometry and targeted gene expression (RT‐qPCR) analysis on lungs from neonatal offspring whose mothers received oral OM‐85 treatment during pregnancy. We next determined whether neonatal offspring from OM‐85 treated mothers demonstrate enhanced protection against lethal lower respiratory infection with mouse‐adapted rhinovirus (vMC0), and associated lung immune changes. Results Offspring from mothers treated with OM‐85 during pregnancy display accelerated postnatal seeding of lung myeloid populations demonstrating upregulation of function‐associated markers. Offspring from OM‐85 mothers additionally exhibit enhanced expression of TLR4/7 and the IL‐1β/NLRP3 inflammasome complex within the lung. These treatment effects were associated with enhanced capacity to clear an otherwise lethal respiratory viral infection during the neonatal period, with concomitant regulation of viral‐induced IFN response intensity. Conclusion These results demonstrate that maternal OM‐85 treatment protects offspring against lethal neonatal respiratory viral infection by accelerating development of innate immune mechanisms crucial for maintenance of local immune homeostasis in the face of pathogen challenge.

This 19-parameter, 18-color flow cytometry panel was designed and optimized to enable the comprehensive and simultaneous immunophenotyping of distinct T-cell, B-cell, and antibody secreting cell (ASC) subsets within murine tissues (Table 1). Cellular populations identified by using this OMIP include two major subsets of B-cells (memory and activated), two ASC subsets (plasma cells and plasmablasts), and seven major subsets of CD4+ T-cells (naïve, central memory, effector memory, helper, regulatory, follicular helper, and follicular regulatory). Staining was performed on freshly isolated splenocytes from 21-day-old BALB/c mice, however, due to the omission of mouse strain-specific markers, this OMIP can be implemented across a range of murine models where in-depth immunophenotyping of the diverse repertoire of T-cell, B-cell, and ASC populations is required. There is now considerable evidence demonstrating that both prenatal and postnatal exposure to particular classes of microbial stimuli can provide beneficial signals during early life immune development, resulting in the protection against future inflammatory disease [1-3]. The principal target of this beneficial immunostimulation appears to be the innate immune system [4, 5], and the mechanisms driving protection underlay the paradigm of innate immune training, whereby certain classes of microbial stimuli can alter the functional state of innate immune cells, leading to the optimization of immunocompetence [6]. Immune training focuses on the phenotypic and transcriptional profiles of several prototypical innate populations [6, 7], however, the characterization of downstream adaptive responses associated with protection via innate immune training are of critical importance for understanding disease pathogenesis, and the potential for therapeutic mitigation. Due to this gap in our current understanding, the broader protective mechanisms remain incompletely understood. To address this requirement, we have developed and optimized a novel 19-parameter flow cytometry panel to comprehensively and simultaneously characterize distinct T-cell, B-cell, and ASC subsets localized within tissues of BALB/c mice in response to immune training during early life. The developmental phase of this flow cytometry panel involved the prioritization of T-cell, B-cell, and ASC subsets central to the maintenance of immunological homeostasis, as based on the current literature and forerunner studies. As such, a degree of emphasis was placed on effector, regulatory, and memory subsets within T-cell and B-cell populations. In regard to T-cells, the conversion of peripheral naïve CD4+ T-cells to effector T (Teff) cells is denoted by upregulation of the activation marker CD25, while concomitant upregulation of both CD25 and intracellular Foxp3 expression is essential for the peripheral induction of regulatory T-cells (Treg) [8], a process previously recognized in the protection against allergic airways inflammation following microbial-derived immunomodulation [9, 10]. Furthermore, the expression of CD44 on Treg has been implicated in promoting enhanced function [11, 12], while inducible costimulator (ICOS)+ Tregs are recognized to have superior suppressive capacity and interleukin (IL)-10 production compared to ICOS− Tregs [13, 14]. Following activation and contraction, CD4+ T-cells transition toward a memory phenotype via the gradual upregulation of CD44 expression in parallel with transient expression of CD62L, driving the establishment of a dynamic repository of central memory (TCM) and effector memory (TEM) T-cells [15-17]. In addition to establishing peripheral memory, activated CD4+ T-cells have the capacity to upregulate extracellular expression of CXCR5, ICOS, and programmed cell death protein 1 (PD-1) [18, 19], resulting in the generation of a highly specialized population of T follicular helper (TFH) cells required for the formation of germinal centers within secondary lymphoid organs, while also providing crucial survival signals to support high-affinity B-cells during affinity maturation and proliferation [20, 21]. A separate subset of thymic-derived cells that share homology with the TFH phenotype in addition to Foxp3 and bimodal CD25 expression, termed follicular regulatory T (TFR) cells, have also been identified, however, this subset has been attributed to the inhibition of TFH activity and subsequent generation of humoral immunity [22, 23]. The immunophenotypic characterization of B-cell and ASC subsets for this OMIP was centered around the classic expression of CD19 and B220. To maximize the capacity of a 5-laser BD LSRFortessa™, CD19 (B-cell and ASC subsets) and CD4 (T-cell) antibodies were conjugated to the same fluorochrome, since co-expression is essentially absent in single-cell analysis. Within secondary lymphoid tissues, the antigen-specific activation of B-cells involves the constitutive upregulation of major histocompatibility complex class-II (MHC class II; mouse I-A/I-E) and CD80 expression, in conjunction with the membrane-bound expression of both immunoglobulin (Ig) M and IgD [24-26]. Following antigen-specific activation, B-cells upregulate Synd-1 expression and differentiate into the two major classes of ASC; the rapidly produced and short-lived plasmablasts and the short-lived peripheral plasma cells, both of which have the capacity to secrete IgM [27-30]. A major difference between these two antibody-secreting subsets, however, is the absence of classic mature B-cell markers CD19, B220, and MHC-II on plasma cells [28, 31]. The eventual transition of B-cells toward a memory phenotype results in the loss of Synd-1 expression with parallel upregulation of programmed cell death protein 1 ligand 2 (PD-L2), generating a long-lived secondary lymphoid population expressing IgM +/− IgD that can rapidly differentiate into ASC upon re-stimulation [32-36]. Panel optimization was performed on a BD LSRFortessa™, with all fluorochrome-conjugated antibodies (Table 2) titrated during the optimization phase (Figure S1). Prior to multicolor extracellular staining, splenocytes were incubated in Fc Block™ (Purified recombinant CD16/32) to inhibit non-antigen-specific binding of fluorochrome-conjugated antibodies to the nonpolymorphic epitope of FcγIII (CD16) and FcγII (CD32) receptors expressed on multiple myeloid populations and B-cells. A representative gating strategy to delineate the T-cell, B-cell, and ASC subsets described above is detailed in Figure 1. Briefly, splenocytes were first gated on side-scatter (SSC) and forward-scatter (FSC) parameters (Figure 1A) to remove sample debris, followed by single-cell gating (Figure 1B) to remove doublets. Gating was then performed on viable CD45+ cells (Figure 1C) to remove dead/dying cells and stromal cells from the analysis. The primary T-cell/B-cell/ASC separation involved delineation of TCRβ and CD4/CD19 expression (Figure 1D). Double positive cells were classified as CD4+ T-cells, as CD19+ B-cells and ASC subsets will be present within the TCRβ− population (Figure 1D) due to the absence of TCRβ/CD19 co-expression (Figure S2A). An additional TCRβ−CD4/CD19− gate was included to enable the characterization of B220−Synd-1+MHC class II−IgM+ plasma cells (PC; Figure 1E). CD19+ B-cells and ASC subsets were then defined as B220lo/+Synd-1+MHC class II+IgM+ plasmablasts (PB; Figure 1F), B220+Synd-1−CD80+PD-L2−MHC class II+IgM+IgD+ activated B-cells (Figure 1G) and B220+Synd-1−CD80+PD-L2+IgM+IgD+/− memory B-cells (Figure 1H). CD4+ T-cells were defined as CD62L+CD44lo/− naïve T-cells (Figure 1I), CD62L+CD44hi TCM (Figure 1I), CD62L−CD44hi TEM (Figure 1I), CD25+Foxp3− Teff (Figure 1J), CD25+Foxp3+ Treg (Figure 1J) ICOS+CD44+ Treg (Figure 1K), CXCR5+ICOS+PD-1+ TFH (Figure 1L), and CXCR5+ICOS+PD-1+CD25+/-Foxp3+ TFR (Figure 1M). To perform high-dimensional analysis on 21-day-old naïve splenocytes, viable CD45+ cells (Figure 1C) underwent high-resolution FlowSOM clustering to define cell populations, followed by metaclustering for visualization with Uniform Manifold Approximation and Projection (UMAP) [37] using the Cytometry Data Analysis Tool (CATALYST) pipeline [38, 39]. Primary unsupervised analysis was performed to identify CD4+ T-cell and B-cell/ASC clusters based on extracellular receptor co-expression (Figure S3A). CD4+ T-cell (Figure S3B), and B-cell/ASC (Figure S3C) clusters were then isolated for secondary subset analysis. The OMIP described here shares a small degree of marker similarity (TCRβ, CD4, CD44, CD62L, PD-1, CD19, B220) with OMIP-031 [40], OMIP-032 [41], and OMIP-061 [42], which are focused on immunologic checkpoint expression on murine T-cell subsets, the characterization of innate and adaptive populations within the murine mammary gland and murine antigen-presenting cells, respectively. While both OMIP-031 and OMIP-032 characterize TCRβ+CD4+ effector and memory T-cell subsets based on a combination of CD44 and/or CD62L expression, OMIP-032 employs an additional CD19+ gate to delineate B-cells. OMIP-061 utilized B220 to identify B-cells. A distinct difference between these OMIPs and the OMIP described here is that our panel was developed for the sole purpose of comprehensively immunophenotyping T-cell, B-cell, and ASC subsets simultaneously, and we therefore include an additional 12 markers to allow the characterization of two major B-cell, two ASC and seven major T-cell populations within a single sample. The OMIP described here also exhibits minor overlap with OMIP-054 [43], however, our panel was developed to maximize the potential of a 5-laser BD LSRFortessa™ in facilities without the capacity to perform mass cytometry. The authors would like to thank Steven Roberts and Dr. Andrew Lim of BD Biosciences (Australia) for their valuable advice during the initial design of this OMIP. Kyle Mincham: Conceptualization; data curation; formal analysis; funding acquisition; investigation; methodology; validation; visualization; writing-original draft; writing-review & editing. Jacob Young: Data curation; formal analysis; investigation; validation; writing-original draft. Deborah Strickland: Conceptualization; formal analysis; funding acquisition; investigation; methodology; project administration; writing-original draft; writing-review & editing. The authors declare no conflict of interest exists. Table S1 Instrument Optical Configuration Table S2. Reagents used in the final OMIP Table S3. Reagents used Table S4. Extracellular multicolor antibody staining cocktail for 1x sample Figure S1. Titrations of each individual component used in the final OMIP. All antibodies were individually titrated on splenocytes from naïve 21-day-old BALB/c mice. Data are splenocytes pre-gated to remove debris (SSC/FSC) and doublets. Figure S2. Absence of TCRβ and CD19 coexpression. (A) TCRβ BB700 and CD19 BV786 staining in the absence of sentinel CD4 BV786 staining (CD4 BV786 FMO), demonstrating the absence of TCRβ and CD19 coexpression. (B) TCRβ BB700 and CD4 BV786 staining in the absence of sentinel CD19 BV786 staining (CD19 BV786 FMO), demonstrating the presence of a minor population of CD4+ non-T-cells within 21-day-old spleens. Population proportions downstream of TCRβ−CD4+ gate = % of TCRβ−CD4+ cells. Data are splenocytes stained for FVS575 BV605, CD45 APC-Cy7, TCRβ BB700 and CD19 BV786 or CD4 BV786. Figure S3. High-dimensional analysis of CD45+ splenocytes. Dimensionality reduction and clustering by UMAP demonstrating (A) distribution of TCRβ, CD4/19 and B220 expression on viable CD45+ splenocytes, (B) CD4+ T-cell and (C) B-cell/ASC clusters. Dimensionality reduction and UMAP visualization was performed using 12,000 total splenocytes from 8 individual 21-day-old naïve BALB/c mice (1500 cells per sample). Figure S4. Fluorescence Minus One (FMO) controls. Data are splenocytes showing terminal population gates and intermediate gates where required for (A) Synd-1 BB515, (B-D) IgM BV711, (E) CD80 BV650, (F) PD-L2 BUV395 (G) IgM BV711, (H) IgD BUV496, (I) CD44 BUV737, (J) CD62L APC-R700, (K) CXCR5 PE-Cy7 and (L) PD-1 BV480 FMO controls. Figure S5. Titration data for CD3ε PerCP and CD3ε BB700 antibodies. Titrations of (A) CD3ε PerCP and (B) CD3ε BB700 antibodies not used in the final panel. Data are splenocytes pre-gated to remove debris (SSC/FSC) and doublets. Figure S6. Initial panel staining with CD3ε. Splenocytes were initially stained with CD3ε BB700 at a dilution of 1:200 for the delineation of T-cells, prior to replacement with TCRβ BB700 in the final iteration of the OMIP. Data are splenocytes from naïve 21-day-old BALB/c mice. Figure S7. Titration data for Foxp3 PE clone MF23 antibody. Titration of (A) Foxp3 PE clone MF23 antibody not used in the final panel and (B) Foxp3 PE clone FJK-16 s antibody used in the final panel. Data are splenocytes pre-gated to remove debris (SSC/FSC) and doublets. Figure S8. Initial panel staining with CD62L AF700. (A) Titration of CD62L AF700. (B) Poor discrimination of CD45+TCRβ+CD4+CD62L+CD44lo/− naïve, of CD45+TCRβ+CD4+CD62L+CD44hi central memory and of CD45+TCRβ+CD4+CD62L−CD44hi effector memory T-cell subsets when using the AF700 fluorochrome. Data are splenocytes from naïve 21-day-old BALB/c mice. Figure S9. Compensation matrix. Based on data displayed in Figure 1. Acquisition-defined compensation matrix was manually generated post-acquisition. Figure S10. Comparison of CXCR5 expression on adolescent and adult splenocytes. Data are representative flow cytometry plots from 21-day-old and 20-week-old BALB/c mice demonstrating the age-dependent expression of CXCR5 PE-Cy7 against PD-1 BV480 on CD4+ T-cells. Figure S11. Initial panel staining with IgD BUV496 clone 217–170. (A) Titration of IgD BUV496 clone 217–170. (B) Suboptimal detection of IgD expression on CD45+TCRβ−CD19+B220+Synd-1−CD80+PD-L2−MHC class II+IgM+IgD+ activated and CD45+TCRβ−CD19+B220+Synd-1−CD80+PD-L2+IgM+IgD− memory B-cell subsets. Data are splenocytes from naïve 21-day-old BALB/c mice. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The gut microbiota is influenced by environmental factors such as food. Maternal diet during pregnancy modifies the gut microbiota composition and function, leading to the production of specific compounds that are transferred to the fetus and enhance the ontogeny and maturation of the immune system. Prebiotics are fermented by gut bacteria, leading to the release of short-chain fatty acids that can specifically interact with the immune system, inducing a switch toward tolerogenic populations and therefore conferring health benefits. In this study, pregnant BALB/cJRj mice were fed either a control diet or a diet enriched in prebiotics (Galacto-oligosaccharides/Inulin). We hypothesized that galacto-oligosaccharides/inulin supplementation during gestation could modify the maternal microbiota, favoring healthy immune imprinting in the fetus. Galacto-oligosaccharides/inulin supplementation during gestation increases the abundance of Bacteroidetes and decreases that of Firmicutes in the gut microbiota, leading to increased production of fecal acetate, which was found for the first time in amniotic fluid. Prebiotic supplementation increased the abundance of regulatory B and T cells in gestational tissues and in the fetus. Interestingly, these regulatory cells remained later in life. In conclusion, prebiotic supplementation during pregnancy leads to the transmission of specific microbial and immune factors from mother to child, allowing the establishment of tolerogenic immune imprinting in the fetus that may be beneficial for infant health outcomes.