Abstract The spatial organization of malignant and non-malignant cells within the tumor microenvironment (TME) critically influences tumor evolution and therapeutic response. However, the architecture of micro-niches remains incompletely understood. Leveraging Xenium-based spatial transcriptomics, we comprehensively mapped the spatial ecosystem of an orthotopic murine lung cancer model, identifying distinct spatial domains that form unique, organized cellular neighborhoods. These domains cluster into three major communities: (1) non-tumoral regions that recapitulate canonical normal lung structures; (2) a heterogeneous peri-tumoral region composed of spatial domains characterized by mesenchymal remodeling, active immune checkpoint signaling, and immunosuppressive myeloid populations; and (3) intra-tumoral regions that reveal marked tumor nodule heterogeneity, with unique tumor-specific domains exhibiting hallmark cancer pathways. Furthermore, our analytic approach was applicable to human lung cancer tissue. Notably, spatial domain analysis allowed us to resolve tumor nodules into multiple biologically distinct subtypes, defined by domain composition, hallmark cancer programs, and intercellular communication patterns within the TME. One Sentence Summary Spatial transcriptomics reveal diverse multicellular niches that organize intratumoral, peri-tumoral, and internodular heterogeneity in lung cancer.

The draining lymph node (LN) is the most frequent and often first site of cancer metastasis. Although endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is frequently performed as a standard practice in lung cancer diagnosis and staging, its diagnostic accuracy remains modest, primarily due to the minuscule sample size of needle aspirates. With the advent of single-cell technologies, we comprehensively analyzed the immune cell repertoire in a series of EBUS-TBNA samples. Intrathoracic LN samples from 18 subjects with pathologically confirmed metastasis and four controls without evidence of metastasis were compared using single-cell RNA sequencing and mass cytometry analyses. We found that immune cell composition and gene expression patterns differed markedly between metastatic and control LNs. In particular, metastatic LNs contained relatively more APOE-high myeloid cells, with the latter exhibiting significant transcriptional derangement and a powerful intercellular interaction signature. Additionally, CD8 T cells in metastatic LNs demonstrated a unique exhausted phenotype. In conclusion, immune cell phenotypes and gene expression patterns from EBUS-TBNA samples can be leveraged to advance our understanding of cancer immunology and may have independent diagnostic value when malignant cells fail to be identified on histopathology.

RATIONALE Asthma is a complex inflammatory airway disease in which a wide array of immune cells interact to induce symptomatic bronchoconstriction. To fully understand the global cellular characteristics of this network, a comprehensive analytic approach is needed. Spectral flow cytometry offers the speed and reliability of conventional flow cytometry while providing the high-dimensional capacity of mass cytometry, with increased signal sensitivity over both methods. Here we examined the utility of high-dimensional spectral flow in a mouse model of allergic airway inflammation. METHODS For this study, we adopted the well-established DRA triple allergen model, in which C57BL/6J mice were sensitized and challenged intranasally with a mix of dust mite, ragweed, and aspergillus crude extracts, as previously described. Cells were collected from BAL, lung parenchyma, and mediastinal lymph nodes of allergen-treated animals at days 3 and 7 post-challenge, as well as untreated animals. After staining with a panel consisting of 41 different fluorescent markers designed to elicit viability, cell identity, functional receptors, and secreted cytokines, data was collected on a 5-laser Aurora flow cytometer (Cytek) and analyzed in Flowjo (Becton Dickinson). Immune cells and their subsets were identified after unsupervised clustering, and functional markers were evaluated either by MFI or staining above FMO. RESULTS Our staining panel clearly identified all immune cells and their subsets, and additionally demonstrated a variety of functional changes induced through allergen exposure. As expected in a model of asthma, expansion of eosinophils, CD4+ T-cells, and innate lymphoid cells was observed in conjunction with increased Th2 cytokines IL-4, IL-5, and IL-13. CD4+ T-cells also upregulated exhaustion markers PD-1 and CTLA-4. In parallel with Th2 cells, allergen challenge increased Treg, Th17, and IL-17-secreting double-negative T-cells, largely γ/δ. Unlike T-cells, NK and B-cell populations contracted. We previously reported that type-2 conventional dendritic cells (cDC2) play a key role in allergen presentation, and here found expansion of this population along with increased expression of activation and trafficking markers CD86 and CCR7, respectively, along with reduced regulatory markers PD-L1, TIM-3, CD163, and CD200. This inflammatory phenotype was largely mirrored by interstitial macrophages, but alveolar macrophages took on a suppressive phenotype, downregulating CD83 and upregulating PD-L1, TIM-3, and CD200. CONCLUSIONS High-dimensional spectral flow cytometry provides a comprehensive analysis of immune cells in allergic lung inflammation. Our data readily demonstrates many expected findings in the DRA model, but also illustrates marked phenotypic diversity in pulmonary macrophages and identifies unique double-negative T-cells producing IL-17.

Obesity is a known risk factor for asthma development, progression, and exacerbation. Nevertheless, the underlying pathophysiological mechanisms explaining how obesity contributes to the development and progression of asthma have yet to be established. Here, we review human studies examining the associations between asthma and obesity, focusing on the literature from the past 10 years. Overall, current evidence suggests that while both asthma and obesity are complex diseases with significant heterogeneity, they both share various features of chronic inflammation. Furthermore, the interactions between asthma and obesity likely involve allergen-specific T helper type 2 (type 2) immune responses, as well as diverse non-type 2 inflammatory pathways. However, despite considerable progress, studies to date have not definitively elucidated the mechanisms that account for the observed association. A large-scale population-based study combined with translational immunological research, including targeted asthma therapies and pharmacological weight loss therapies, may be required to properly dissect the details of obesity-related asthma pathophysiology.

Human lung cancer carries high genetic alterations, expressing high tumor-specific neoantigens. Although orthotopic murine lung cancer models recapitulate many characteristics of human lung cancers, genetically engineered mouse models have fewer somatic mutations than human lung cancer, resulting in scarce immune cell infiltration and deficient immune responses. The endogenous mouse lung cancer model driven by Kras mutation and Trp53 deletion (KP model) has minimal immune infiltration because of a scarcity of neoantigens. Fine-tuning tumor antigenicity to trigger the appropriate level of antitumor immunity would be key to investigating immune responses against human lung cancer. We engineered the KP model to express antigens of OVA peptides (minOVA) as neoantigens along with ZsGreen, a traceable fluorescent conjugate. The KP model expressing minOVA exhibited stronger immunogenicity with higher immune cell infiltration comprised of CD8+ T cells and CD11c+ dendritic cells (DCs). Consequently, the KP model expressing minOVA exhibits suppressed tumor growth compared to its origin. We further analyzed tumor-infiltrated DCs. The majority of ZsGreen conjugated with minOVA was observed in the conventional type 2 DCs (cDC2), whereas cDC1 has minimal. These data indicate that tumor immunogenicity regulates host immune responses, and tumor neoantigen is mostly recognized by cDC2 cells, which may play a critical role in initiating antitumor immune responses in an orthotopic murine lung cancer model.

BACKGROUND:Dendritic cells (DCs) are heterogeneous, comprising multiple subsets with unique functional specifications. Our previous work has demonstrated that the specific conventional type 2 DC subset, CSF1R+cDC2s, plays a critical role in sensing aeroallergens.OBJECTIVE:It remains to be understood how CSF1R+cDC2s recognize inhaled allergens. We sought to elucidate the transcriptomic programs and receptor-ligand interactions essential for function of this subset in allergen sensitization.METHODS:We applied single-cell RNA sequencing to mouse lung DCs. Conventional DC-selective knockout mouse models were employed, and mice were subjected to inhaled allergen sensitization with multiple readouts of asthma pathology. Under the clinical arm of this work, human lung transcriptomic data were integrated with mouse data, and bronchoalveolar lavage (BAL) specimens were collected from subjects undergoing allergen provocation, with samples assayed for C1q.RESULTS:We found that C1q is selectively enriched in lung CSF1R+cDC2s, but not in other lung cDC2 or cDC1 subsets. Depletion of C1q in conventional DCs significantly attenuates allergen sensing and features of asthma. Additionally, we found that C1q binds directly to human dust mite allergen, and the C1q receptor CD91 (LRP1) is required for lung CSF1R+cDC2s to recognize the C1q-allergen complex and induce allergic lung inflammation. Lastly, C1q is enriched in human BAL samples following subsegmental allergen challenge, and human RNA sequencing data demonstrate close homology between lung IGSF21+DCs and mouse CSF1R+cDC2s.CONCLUSIONS:C1q is secreted from the CSF1R+cDC2 subset among conventional DCs. Our data indicate that the C1q-LRP1 axis represents a candidate for translational therapeutics in the prevention and suppression of allergic lung inflammation.

Human rhinoviruses cause the common cold and exacerbate chronic respiratory diseases. Although infection elicits neutralizing antibodies, these do not persist or cross-protect across multiple rhinovirus strains. To analyze rhinovirus-specific B cell responses in humans, we developed techniques using intact RV-A16 and RV-A39 for high-throughput high-dimensional single-cell analysis, with parallel assessment of antibody isotypes in an experimental infection model. Our approach identified T-bet+ B cells binding both viruses that account for similar to 5% of CXCR5- memory B cells. These B cells infiltrate nasal tissue and expand in the blood after infection. Their rapid secretion of heterotypic immunoglobulin G (IgG) in vitro, but not IgA, matches the nasal antibody profile post-infection. By contrast, CXCR5+ memory B cells binding a single virus are clonally distinct, absent in nasal tissue, and secrete homotypic IgG and IgA, mirroring the systemic response. Temporal and spatial functions of dichotomous memory B cells might explain the ability to resolve infection while rendering the host susceptible to re-infection.

Background: Allergic asthmatic subjects are uniquely susceptible to acute wheezing episodes provoked by rhinovirus. However, the underlying immune mechanisms and interaction between rhinovirus and allergy remain enigmatic, and current paradigms are controversial. Objective: We sought to perform a comprehensive analysis of type 1 and type 2 innate and adaptive responses in allergic asthmatic subjects infected with rhinovirus. Methods: Circulating virus-specific T(H)1 cells and allergenspecific T(H)2 cells were precisely monitored before and after rhinovirus challenge in allergic asthmatic subjects (total IgE, 133-4692 IU/mL; n = 28) and healthy nonallergic controls (n = 12) using peptide/MHCII tetramers. T cells were sampled for up to 11 weeks to capture steady-state and postinfection phases. T-cell responses were analyzed in parallel with 18 cytokines in the nose, upper and lower airway symptoms, and lung function. The influence of in vivo IgE blockade was also examined. Results: In uninfected asthmatic subjects, higher numbers of circulating virus-specific PD-1(+) T(H)1 cells, but not allergenspecific T(H)2 cells, were linked to worse lung function. Rhinovirus infection induced an amplified antiviral T(H)1 response in asthmatic subjects versus controls, with synchronized allergen-specific T(H)2 expansion, and production of type 1 and 2 cytokines in the nose. In contrast, T(H)2 responses were absent in infected asthmatic subjects who had normal lung function, and in those receiving anti-IgE. Across all subjects, early induction of a minimal set of nasal cytokines that discriminated high responders (G-CSF, IFN-gamma, TNF-alpha) correlated with both egress of circulating virus-specific T(H)1 cells and worse symptoms. Conclusions: Rhinovirus induces robust T(H)1 responses in allergic asthmatic subjects that may promote disease, even after the infection resolves.

Human rhinoviruses (RV) account for half a billion colds annually in the US, and exacerbate chronic respiratory diseases. Repeated infections continue into adulthood, owing to the lack of a durable protective antibody response that remains ill-understood. Here, we sought to rigorously define B-cell responses to RV using novel serologic and flow cytometric immunoassays. Serum antibodies were assayed in 16 subjects before (day 0) and after (day 21) experimental infection with RV-A16 using bead-bound capsid proteins and purified whole virus. RV-specific B-cells were isolated from 6 subjects with a history of natural RV infection by labeling with fluorescent virus. Their capacity to secrete antibodies was assessed in vitro by stimulating FACS-sorted cells with soluble anti-CD40 and CpG-DNA, and analyzing supernatants on days 2, 4, 6, and 8. After infection, significant increases in multiple serum antibody isotypes were restricted to whole virus (IgG: p<0.01; IgA: p<0.01; and IgM: p<0.05), and all isotypes cross-reacted with a second RV-A strain. Virus-labeled B-cells were enriched for "Age-Associated B-cells" (ABC, ∼20%) based on expression of T-bet, CD11c, CD21, CXCR5, and IgG. Additionally, 5.0+/-2.7% of total ABCs were RV-specific, and RV-specific ABCs bound multiple RV-A strains. The capacity of RV-specific ABCs to secrete antibodies was reduced compared with other RV-specific memory cells. RV infection induces cross-reactive antibodies directed against intact capsid, and disproportionately expands hypofunctional ABCs. Given that ABCs may be exhausted in chronic viral infections, their expansion suggests the induction of anergy in broadly-reactive B-cells, as a consequence of repeated RV infections.

Despite the recognition of various endotypes in allergic disease, surprisingly little is known regarding the complexity of T helper (Th) cells that respond to house dust mite (HDM), especially given the IgE dominance of HDM in patients with asthma and atopic dermatitis (AD). Thus, we rigorously analyzed dust mite-reactive Th cells using a novel 22-marker immunophenotyping panel for spectral flow cytometry in patients with different diseases. Peripheral blood mononuclear cells isolated from allergic adults with asthma and/or AD (ImmunoCAP class ≥3 for HDM and Der p 1) were labeled with cell trace dye (CellTraceTM Violet), and cultured for 7 days with HDM or Der p 1. Cells were then stained for surface (CD3, CD4, CD27, CRTH2, CD45RO, CD45RA, CD25) and intracellular (IL-4, IL-5, IL-9, IL-10, IL-13, IL-22, TNF-α, IFN-γ, IL-17A) markers. Dust mite-reactive cells (cell trace dye low) were analyzed by spectral flow cytometry. As described previously, Th2 signatures were enriched within CD27-CRTH2+ cells and Th1/Th17 signatures dominated CD27+/-CRTH2- subsets. However, HDM- and Der p 1-reactive T cells encompassed a broad spectrum of cytokine profiles (Th1, Th2, Th17, Th1/Th17, Th9, Th22), and generally lacked CRTH2 expression. The extent of heterogeneity and dominance of specific Th types varied among patients with the same or different disease. Moreover, addition of IL-33 and endotoxin augmented Der p 1-specific Th1 responses. Multi-dimensional analysis by spectral flow cytometry provides a valuable tool to classify dust mite-specific Th cells and assess their mechanisms of induction in the context of discrete clinical diseases.

Sepsis is an often deadly complication of infection in which systemic inflammation damages the vasculature, leading to tissue hypoperfusion and multiple organ failure. Currently, the standard of care for sepsis is predominantly supportive, with few therapeutic options available. Because of increased sepsis incidence worldwide, there is an urgent need for discovery of novel therapeutic targets and development of new treatments. The recently discovered function of the endoplasmic reticulum (ER) in regulation of inflammation offers a potential avenue for sepsis control. Here, we identify the ER-resident protein sigma-1 receptor (S1R) as an essential inhibitor of cytokine production in a preclinical model of septic shock. Mice lacking S1R succumb quickly to hypercytokinemia induced by a sublethal challenge in two models of acute inflammation. Mechanistically, we find that S1R restricts the endonuclease activity of the ER stress sensor IRE1 and cytokine expression but does not inhibit the classical inflammatory signaling pathways. These findings could have substantial clinical implications, as we further find that fluvoxamine, an antidepressant therapeutic with high affinity for S1R, protects mice from lethal septic shock and dampens the inflammatory response in human blood leukocytes. Our data reveal the contribution of S1R to the restraint of the inflammatory response and place S1R as a possible therapeutic target to treat bacterial-derived inflammatory pathology.

Severe asthma in children is a debilitating condition that accounts for a disproportionately large health and economic burden of asthma. Reasons for the lack of a response to standard anti-inflammatory therapies remain enigmatic. Work in the last decade has shed new light on the heterogeneous nature of asthma, and the varied immunopathologies of severe disease, which are leading to new treatment approaches for the individual patient. However, most studies to date that explored the immune landscape of the inflamed lower airways have focused on adults. T cells are pivotal to the inception and persistence of inflammatory processes in the diseased lungs, despite a contemporary shift in focus to immune events at the epithelial barrier. This article outlines current knowledge on the types of T cells and related cell types that are implicated in severe asthma. The potential for environmental exposures and other inflammatory cues to condition the immune environment of the lung in early life to favour pathogenic T cells and steroid resistance is discussed. The contributions of T cells and their cytokines to inflammatory processes and treatment resistance are also considered, with an emphasis on new observations in children that argue against conventional type 1 and type 2 T cell paradigms. Finally, the ability for new technologies to revolutionize our understanding of T cells in severe childhood asthma, and to guide future treatment strategies that could mitigate this disease, is highlighted.

Human rhinoviruses (RV) cause roughly half of the one-billion colds experienced annually within the US, while simultaneously precipitating life-threatening respiratory distress in asthmatic populations. Meanwhile, for reasons that remain unclear, adaptive immunity does not promote long-term protection from infection despite each instance of viral clearance generally coinciding with the development of serum neutralizing antibodies. To better understand these responses at a cellular level, we have implemented methods to appreciate fluctuations in peripheral blood B-cells during RV infection. Six human subjects were nasally inoculated with RV strain A39, and peripheral blood mononuclear cells were drawn and isolated at days 0, 5, and 21 post-infection. Cells were barcoded with palladium-labeled anti-CD45 antibodies, and combined for subsequent processing steps. Next, B-cells were magnetically enriched by negative selection against CD3, CD14, and CD16, and then stained with a 40-marker mass cytometry antibody panel. After collection on a CyTOF2, multiplexed sample data were debarcoded and subjected to FlowSOM clustering analysis. At day 5, a variety of memory phenotype clusters exhibited modest decreases in frequency. In contrast, the expansion of a singular plasmablast phenotype was more pronounced (p < 0.05). These nascent cells demonstrated classical plasmablast markers (CD19low, CD20low, CD38hi), displayed an activated phenotype (CD27hi, CD95hi, CD80/6hi, CD40low), expressed inflamed airway homing receptors (CCR5+, CXCR3+, Itgα4β1+), and bore indicators of an early, short-lived response (IgM+, Bcl-2low, Ki-67+). Our high-dimensional approach highlights the early adaptive response to RV, suggesting that initial humoral contributions during acute infection are mediated by IgM-secreting plasmablasts acting at the site of infection.

Background: The pathogenesis of severe asthma in childhood remains poorly understood. Objective: We sought to construct the immunologic landscape in the airways of children with severe asthma. Methods: Comprehensive analysis of multiple cell types and mediators was performed by using flow cytometry and a multiplex assay with bronchoalveolar lavage (BAL) specimens (n = 68) from 52 highly characterized allergic and nonallergic children (0.5-17 years) with severe treatment-refractory asthma. Multiple relationships were tested by using linear mixed-effects modeling. Results: Memory CCR5(+) T(H)1 cells were enriched in BAL fluid versus blood, and pathogenic respiratory viruses and bacteria were readily detected. IFN-gamma(+) IL-17(+) and IFN-gamma(-) 2 IL-17(+) subsets constituted secondary T-H types, and BAL fluid CD8 1 T cells were almost exclusively IFN-gamma(+). The T(H)17-associated mediators IL-23 and macrophage inflammatory protein 3 alpha/CCL20 were highly expressed. Despite low T(H)2 numbers, T(H)2 cytokines were detected, and T(H)2 skewing correlated with total IgE levels. Type 2 innate lymphoid cells and basophils were scarce in BAL fluid. Levels of IL-5, IL-33, and IL-28A/IFN-lambda 2 were increased in multisensitized children and correlated with IgE levels to dust mite, ryegrass, and fungi but not cat, ragweed, or food sources. Additionally, levels of IL-5, but no other cytokine, increased with age and correlated with eosinophil numbers in BAL fluid and blood. Both plasmacytoid and IgE 1 FceRI 1 myeloid dendritic cells were present in BAL fluid. Conclusions: The lower airways of children with severe asthma display a dominant T(H)1 signature and atypical cytokine profiles that link to allergic status. Our findings deviate from established paradigms and warrant further assessment of the pathogenicity of T(H)1 cells in patients with severe asthma.

Nature 523, 337–341 (2015); doi:10.1038/nature14432 We would like to correct this Letter, which demonstrated the molecular characteristics and functional nature of lymphatics serving the central nervous system (CNS), by adding two reference citations, of which we became aware after publication. Foldi et al.

Human rhinoviruses (RV) account for 31 million estimated cases of the common cold per year within the US, and trigger disease exacerbations in allergic asthmatics. Adaptive immunity to RV does not provide durable protection owing partly to considerable diversity of RV strains. Here we describe circulating lymphocytes in the context of RV infection, exploring factors that determine the quality of the humoral response. Mass cytometry was used to capture fluctuations in discrete B-cell populations during an experimental RV-16 infection in allergic asthmatics. The findings were used to inform the design of an antigen-specific fluorescence-based flow cytometry panel in order to further interrogate the B-cell compartment in relation to RV infection. B-cell antigen-specificity was validated in blood from control subjects and atopics using fluorescent tetramers of tetanus toxoid and Der p 1, respectively. We readily identified naïve B-cells (IgD+), plasmablasts (CD20-, CD38+), and 2 discrete memory populations that differentially expressed the follicular-homing marker, CXCR5. Whereas CXCR5+ memory cells expressed CD27 and diverse isotypes, their CXCR5- counterparts were CD27lo, expressed the transcription factor T-bet, and lacked IgM. This latter phenotype disappeared from the blood in conjunction with memory T-cells during the acute phase of RV infection, while plasmablasts that primarily expressed IgA were expanded. Our findings are consistent with the presence of pre-existing memory B-cells that are poised for reactivation by cognate antigen stimulation, potentially with or without T-cell help. Further work is necessary to determine the B-cell mechanisms that promote virus-neutralizing activity, including how T-cells might instruct the protectiveness of antibodies produced.

Lymphatic endothelial cells (LECs) directly express peripheral tissue antigens and induce CD8 T-cell deletional tolerance. LECs express MHC-II molecules, suggesting they might also tolerize CD4 T cells. We demonstrate that when β-galactosidase (β-gal) is expressed in LECs, β-gal-specific CD8 T cells undergo deletion via the PD-1/PD-L1 and LAG-3/MHC-II pathways. In contrast, LECs do not present endogenous β-gal in the context of MHC-II molecules to β-gal-specific CD4 T cells. Lack of presentation is independent of antigen localization, as membrane-bound haemagglutinin and I-Eα are also not presented by MHC-II molecules. LECs express invariant chain and cathepsin L, but not H2-M, suggesting that they cannot load endogenous antigenic peptides onto MHC-II molecules. Importantly, LECs transfer β-gal to dendritic cells, which subsequently present it to induce CD4 T-cell anergy. Therefore, LECs serve as an antigen reservoir for CD4 T-cell tolerance, and MHC-II molecules on LECs are used to induce CD8 T-cell tolerance via LAG-3.

Lymphatic endothelial cells are most often thought of as structural cells that form the lymphatic vasculature, which transports fluid out of peripheral tissues and transports antigens and antigen presenting cells to lymph nodes. Recently, it has been shown that lymphatic endothelial cells also dynamically respond to and influence the immune response in several ways. Here, we describe how lymphatic endothelial cells induce peripheral T-cell tolerance and how this relates to tolerance induced by other types of antigen presenting cells. Furthermore, the ability of lymphatic endothelial cells to alter immune responses under steady-state or inflammatory conditions is explored, and the therapeutic potential of bypassing lymphatic endothelial cell-induced tolerance to enhance cancer immunotherapy is discussed.