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Communication between immune cells through direct contact is a critical feature of immune responses. Here, we developed a novel high-throughput method to study the transcriptome and adaptive immune receptor repertoire of single cells forming complexes without needing bioinformatic deconvolution. We found that T cells and monocytes forming complexes in blood during active tuberculosis (TB) and dengue hold unique transcriptomic signatures indicative of TCR/MCH-II immune synapses. Additionally, T cells in complexes showed enrichment for effector phenotypes, imaging and transcriptomic features of active TCR signaling, and increased immune activity at diagnosis compared to after anti-TB therapy. We also found evidence for bidirectional RNA exchange between T cells and monocytes, since complexes were markedly enriched for "dual-expressing" cells (i.e., co-expressing T cell and monocyte genes). Thus, studying immune cell complexes at a single-cell resolution offers novel perspectives on immune synaptic interactions occurring in blood during infection., Competing Interests: Conflict of interest DW is a consultant for Moderna.

E-cigarette use has become widespread, and its effects on airway inflammation and disease are not fully delineated. E-cigarette vapor extract (EVE) profoundly affects neutrophil function. We hypothesized that EVE also alters eosinophil function and thus could impact allergic airways disease. We employed RNA-sequencing to measure the ex vivo effect of EVE components on human eosinophil transcription. Blood eosinophils from 9 non-vaping subjects without asthma were isolated by negative selection. Cells were incubated for 48 hours with EVE consisting of glycerin, propylene glycol and nicotine (EVE+), EVE without nicotine ("EVE-"), air-exposed media termed Extract Buffer (EB), or untreated media. Bulk RNA-sequencing was performed. Transcriptomic analysis revealed that the EB, EVE-, and EVE+ conditions showed highly variable gene expression with respect to No Treatment and each other. Differential gene expression analysis comparing a combination of EVE+, EVE-, and EB revealed 3,030 differentially expressed genes (DEG) with adjusted p value < 0.05 and log2 fold change >0.5 or <0.5. There were 645 DEG between EB and EVE-, 1,713 between EB and EVE+, and 404 between EVE- and EVE+. Gene set enrichment analysis demonstrated that DEG between both EVE+ and EVE- and the EB control were positively enriched for heme metabolism and apoptosis and negatively enriched TNFα signaling, IFNγ signaling, and inflammatory response. Thus, EVE significantly alters eosinophil metabolic and inflammatory pathways, mediated by propylene glycol and glycerin with both enhancing and unique effects of nicotine. This study motivates further research into the pathogenic effects of vaping on airway eosinophils and allergic airways disease., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Objective: Rheumatoid arthritis (RA) is an autoimmune disease in which the joint lining or synovium becomes highly inflamed and majorly contributes to disease progression. Understanding pathogenic processes in RA synovium is critical for identifying therapeutic targets. We performed laser capture microscopy (LCM) followed by RNA sequencing (LCM-RNAseq) to study regional transcriptomes throughout RA synovium., Methods: Synovial lining, sublining, and vessel samples were captured by LCM from seven patients with RA and seven patients with osteoarthritis (OA). RNAseq was performed on RNA extracted from captured tissue. Principal component analysis was performed on the sample set by disease state. Differential expression analysis was performed between disease states based on log 2 fold change and q value parameters. Pathway analysis was performed using the Reactome Pathway Database on differentially expressed genes among disease states. Significantly enriched pathways in each synovial region were selected based on the false discovery rate., Results: RA and OA transcriptomes were distinguishable by principal component analysis. Pairwise comparisons of synovial lining, sublining, and vessel samples between RA and OA revealed substantial differences in transcriptional patterns throughout the synovium. Hierarchical clustering of pathways based on significance revealed a pattern of association between biologic function and synovial topology. Analysis of pathways uniquely enriched in each region revealed distinct phenotypic abnormalities. As examples, RA lining samples were marked by anomalous immune cell signaling, RA sublining samples were marked by aberrant cell cycle, and RA vessel samples were marked by alterations in heme scavenging., Conclusion: LCM-RNAseq confirms reported transcriptional differences between the RA synovium and the OA synovium and provides evidence supporting a relationship between synovial topology and molecular anomalies in RA., (© 2024 American College of Rheumatology.)

To understand the role of T cells in the pathogenesis of ulcerative colitis (UC), we analyzed colonic T cells isolated from patients with UC and controls. Here we identified colonic CD4 + and CD8 + T lymphocyte subsets with gene expression profiles resembling stem-like progenitors, previously reported in several mouse models of autoimmune disease. Stem-like T cells were increased in inflamed areas compared to non-inflamed regions from the same patients. Furthermore, TCR sequence analysis indicated stem-like T cells were clonally related to proinflammatory T cells, suggesting their involvement in sustaining effectors that drive inflammation. Using an adoptive transfer colitis model in mice, we demonstrated that CD4 + T cells deficient in either BCL-6 or TCF1, transcription factors that promote T cell stemness, had decreased colon T cells and diminished pathogenicity. Our results establish a strong association between stem-like T cell populations and UC pathogenesis, highlighting the potential of targeting this population to improve clinical outcomes., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

How human genetic variation contributes to vaccine effectiveness in infants is unclear, and data are limited on these relationships in populations with African ancestries. We undertook genetic analyses of vaccine antibody responses in infants from Uganda (n = 1391), Burkina Faso (n = 353) and South Africa (n = 755), identifying associations between human leukocyte antigen (HLA) and antibody response for five of eight tested antigens spanning pertussis, diphtheria and hepatitis B vaccines. In addition, through HLA typing 1,702 individuals from 11 populations of African ancestry derived predominantly from the 1000 Genomes Project, we constructed an imputation resource, fine-mapping class II HLA-DR and DQ associations explaining up to 10% of antibody response variance in our infant cohorts. We observed differences in the genetic architecture of pertussis antibody response between the cohorts with African ancestries and an independent cohort with European ancestry, but found no in silico evidence of differences in HLA peptide binding affinity or breadth. Using immune cell expression quantitative trait loci datasets derived from African-ancestry samples from the 1000 Genomes Project, we found evidence of differential HLA-DRB1 expression correlating with inferred protection from pertussis following vaccination. This work suggests that HLA-DRB1 expression may play a role in vaccine response and should be considered alongside peptide selection to improve vaccine design., (© 2024. The Author(s).)

Brain tumors in children are a devastating disease in a high proportion of patients. Owing to inconsistent results in clinical trials in unstratified patients, the role of immunotherapy remains unclear. We performed an in-depth survey of the single-cell transcriptomes and clonal relationship of intra-tumoral T cells from children with brain tumors. Our results demonstrate that a large fraction of T cells in the tumor tissue are clonally expanded with the potential to recognize tumor antigens. Such clonally expanded T cells display enrichment of transcripts linked to effector function, tissue residency, immune checkpoints and signatures of neoantigen-specific T cells and immunotherapy response. We identify neoantigens in pediatric brain tumors and show that neoantigen-specific T cell gene signatures are linked to better survival outcomes. Notably, among the patients in our cohort, we observe substantial heterogeneity in the degree of clonal expansion and magnitude of T cell response. Our findings suggest that characterization of intra-tumoral T cell responses may enable selection of patients for immunotherapy, an approach that requires prospective validation in clinical trials., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Conventional antiviral memory CD4 T cells typically arise during the first two weeks of acute infection. Unlike most viruses, cytomegalovirus (CMV) exhibits an extended persistent replication phase followed by lifelong latency accompanied with some gene expression. We show that during mouse CMV (MCMV) infection, CD4 T cells recognizing an epitope derived from the viral M09 protein only develop after conventional memory T cells have already peaked and contracted. Ablating these CD4 T cells by mutating the M09 genomic epitope in the MCMV Smith strain, or inducing them by introducing the epitope into the K181 strain, resulted in delayed or enhanced control of viral persistence, respectively. These cells were shown to be unique compared to their conventional memory counterparts; producing higher IFNγ and IL-2 and lower IL-10 levels. RNAseq analyses revealed them to express distinct subsets of effector genes as compared to classical CD4 T cells. Additionally, when M09 cells were induced by epitope vaccination they significantly enhanced protection when compared to conventional CD4 T cells alone. These data show that late-rising CD4 T cells are a unique memory subset with excellent protective capacities that display a development program strongly differing from the majority of memory T cells., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Brunel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Despite most of the asthma population having mild disease, the mild asthma phenotype is poorly understood. Here, we aim to address this gap in knowledge by extensively characterising the mild asthma phenotype and comparing this with difficult-to-treat asthma., Methods: We assessed two real-world adult cohorts from the South of England using an identical methodology: the Wessex AsThma CoHort of difficult asthma (WATCH) (n=498) and a mild asthma cohort from the comparator arm of the Epigenetics Of Severe Asthma (EOSA) study (n=67). Data acquisition included detailed clinical, health and disease-related questionnaires, anthropometry, allergy and lung function testing, plus biological samples (blood and sputum) in a subset., Results: Mild asthma is predominantly early-onset and is associated with type-2 (T2) inflammation (atopy, raised fractional exhaled nitric oxide (FeNO), blood/sputum eosinophilia) plus preserved lung function. A high prevalence of comorbidities and multimorbidity was observed in mild asthma, particularly depression (58.2%) and anxiety (56.7%). In comparison to difficult asthma, mild disease showed similar female predominance (>60%), T2-high inflammation and atopy prevalence, but lower peripheral blood/airway neutrophil counts and preserved lung function. Mild asthma was also associated with a greater prevalence of current smokers (20.9%). A multi-component T2-high inflammatory measure was comparable between the cohorts; T2-high status 88.1% in mild asthma and 93.5% in difficult asthma., Conclusion: Phenotypic characterisation of mild asthma identified early-onset disease with high prevalence of current smokers, T2-high inflammation and significant multimorbidity burden. Early comprehensive assessment of mild asthma patients could help prevent potential later progression to more complex severe disease., Competing Interests: The authors, JN, HM, FM, JB, MAK, CB, HMH, PD, RD, GS, PV, SHA, RJK, declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Since completion of this work, CB now works for Aztra Zeneca. Professor Ratko Djukanovic reports personal fees, has shares in the company and is a consultant to Synairgen, and personal fees from GlaxoSmithKline and Kymab, outside the submitted work., (© 2023 Naftel et al.)

Background: Patients with severe uncontrolled asthma represent a distinct endotype with persistent airway inflammation and remodeling that is refractory to corticosteroid treatment. CD4 + T H 2 cells play a central role in orchestrating asthma pathogenesis, and biologic therapies targeting their cytokine pathways have had promising outcomes. However, not all patients respond well to such treatment, and their effects are not always durable nor reverse airway remodeling. This observation raises the possibility that other CD4 + T cell subsets and their effector molecules may drive airway inflammation and remodeling., Methods: We performed single-cell transcriptome analysis of >50,000 airway CD4 + T cells isolated from bronchoalveolar lavage samples from 30 patients with mild and severe asthma., Findings: We observed striking heterogeneity in the nature of CD4 + T cells present in asthmatics' airways, with tissue-resident memory T (T RM ) cells making a dominant contribution. Notably, in severe asthmatics, a subset of CD4 + T RM cells (CD103-expressing) was significantly increased, comprising nearly 65% of all CD4 + T cells in the airways of male patients with severe asthma when compared to mild asthma (13%). This subset was enriched for transcripts linked to T cell receptor activation (HLA-DRB1, HLA-DPA1) and cytotoxicity (GZMB, GZMA) and, following stimulation, expressed high levels of transcripts encoding for pro-inflammatory non-T H 2 cytokines (CCL3, CCL4, CCL5, TNF, LIGHT) that could fuel persistent airway inflammation and remodeling., Conclusions: Our findings indicate the need to look beyond the traditional T2 model of severe asthma to better understand the heterogeneity of this disease., Funding: This research was funded by the NIH., Competing Interests: Declaration of interests S.H.A. received research funding unrelated to this work from Dyson Inc., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Mucosal-associated invariant T (MAIT) cells are a subset of T lymphocytes that respond to microbial metabolites. We defined MAIT cell populations in different organs and characterized the developmental pathway of mouse and human MAIT cells in the thymus using single-cell RNA sequencing and phenotypic and metabolic analyses. We showed that the predominant mouse subset, which produced IL-17 (MAIT17), and the subset that produced IFN-γ (MAIT1) had not only greatly different transcriptomes but also different metabolic states. MAIT17 cells in different organs exhibited increased lipid uptake, lipid storage, and mitochondrial potential compared with MAIT1 cells. All these properties were similar in the thymus and likely acquired there. Human MAIT cells in lung and blood were more homogeneous but still differed between tissues. Human MAIT cells had increased fatty acid uptake and lipid storage in blood and lung, similar to human CD8 T resident memory cells, but unlike mouse MAIT17 cells, they lacked increased mitochondrial potential. Although mouse and human MAIT cell transcriptomes showed similarities for immature cells in the thymus, they diverged more strikingly in the periphery. Analysis of pet store mice demonstrated decreased lung MAIT17 cells in these so-called "dirty" mice, indicative of an environmental influence on MAIT cell subsets and function.

The IL-2 receptor α chain (IL-2Rα/CD25) is constitutively expressed on double-negative (DN2/DN3 thymocytes and regulatory T cells (T regs ) but induced by IL-2 on T and natural killer (NK) cells, with Il2ra expression regulated by a STAT5-dependent super-enhancer. We investigated CD25 regulation and function using a series of mice with deletions spanning STAT5-binding elements. Deleting the upstream super-enhancer region mainly affected constitutive CD25 expression on DN2/DN3 thymocytes and T regs , with these mice developing autoimmune alopecia, whereas deleting an intronic region decreased IL-2-induced CD25 on peripheral T and NK cells. Thus, distinct super-enhancer elements preferentially control constitutive versus inducible expression in a cell type-specific manner. The mediator-1 coactivator colocalized with specific STAT5-binding sites. Moreover, both upstream and intronic regions had extensive chromatin interactions, and deletion of either region altered the super-enhancer structure in mature T cells. These results demonstrate differential functions for distinct super-enhancer elements, thereby indicating previously unknown ways to manipulate CD25 expression in a cell type-specific fashion.

Background: Cow milk (CM) allergy is the most prevalent food allergy in young children in the United States and Great Britain. Current diagnostic tests are either unreliable (IgE test and skin prick test) or resource-intensive with risks (food challenges)., Objective: We sought to determine whether allergen-specific T cells in CM-allergic (CMA) patients have a distinct quality and/or quantity that could potentially be used as a diagnostic marker., Methods: Using PBMCs from 147 food-allergic pediatric subjects, we mapped T-cell responses to a set of reactive epitopes in CM that we compiled in a peptide pool. This pool induced cytokine responses in in vitro cultured cells distinguishing subjects with CMA from subjects without CMA. We further used the pool to isolate and characterize antigen-specific CD4 memory T cells using flow cytometry and single-cell RNA/TCR sequencing assays., Results: We detected significant changes in the transcriptional program and clonality of CM antigen-specific (CM+) T cells elicited by the pool in subjects with CMA versus subjects without CMA ex vivo. CM+ T cells from subjects with CMA had increased percentages of FOXP3 + cells over FOXP3 - cells. FOXP3 + cells are often equated with regulatory T cells that have suppressive activity, but CM+ FOXP3 + cells from subjects with CMA showed significant expression of interferon-responsive genes and dysregulated chemokine receptor expression compared with subjects without CMA, suggesting that these are not conventional regulatory T cells. The CM+ FOXP3 + cells were also more clonally expanded than the FOXP3 - population. We were further able to use surface markers (CD25, CD127, and CCR7) in combination with our peptide pool stimulation to quantify these CM+ FOXP3 + cells by a simple flow-cytometry assay. We show increased percentages of CM+ CD127 - CD25 + cells from subjects with CMA in an independent cohort, which could be used for diagnostic purposes. Looking specifically for T H 2 cells normally associated with allergic diseases, we found a small population of clonally expanded CM+ cells that were significantly increased in subjects with CMA and that had high expression of T H 2 cytokines and pathogenic T H 2/T follicular helper markers., Conclusions: Overall, these findings suggest that there are several differences in the phenotypes of CM+ T cells with CM allergy and that the increase in CM+ FOXP3 + cells is a potential diagnostic marker of an allergic state. Such markers have promising applications in monitoring natural disease outgrowth and/or the efficacy of immunotherapy that will need to be validated in future studies., (Copyright © 2023 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Purpose: Due to their abundance in the blood, low RNA content, and short lifespan, neutrophils have been classically considered to be one homogenous pool. However, recent work has found that mature neutrophils and neutrophil progenitors are composed of unique subsets exhibiting context-dependent functions. In this study, we ask if neutrophil heterogeneity is associated with melanoma incidence and/or disease stage., Experimental Design: Using mass cytometry, we profiled melanoma patient blood for unique cell surface markers among neutrophils. Markers were tested for their predictiveness using flow cytometry data and random forest machine learning., Results: We identified CD79b + neutrophils (CD3 - CD56 - CD19 - Siglec8 - CD203c - CD86 Lo CD66b + CD79b + ) that are normally restricted to the bone marrow in healthy humans but appear in the blood of subjects with early-stage melanoma. Further, we found CD79b + neutrophils present in tumors of subjects with head and neck cancer. AI-mediated machine learning analysis of neutrophils from subjects with melanoma confirmed that CD79b expression among peripheral blood neutrophils is highly important in identifying melanoma incidence. We noted that CD79b + neutrophils possessed a neutrophilic appearance but have transcriptional and surface-marker phenotypes reminiscent of B cells. Compared to remaining blood neutrophils, CD79b + neutrophils are primed for NETosis, express higher levels of antigen presentation-related proteins, and have an increased capacity for phagocytosis., Conclusion: Our work suggests that CD79b + neutrophils are associated with early-stage melanoma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AR declared a shared affiliation with the authors YZ and CO to the handling editor at the time of review., (Copyright © 2023 Meyer, Dinh, Alimadadi, Araujo, Chatterjee, Gutierrez, Zhu, Hunter, Liang, Seumois, Kiosses, Catz, Vijayanand, Ottensmeier and Hedrick.)

Background: Asthma is conventionally stratified as type 2 inflammation (T2)-high or T2-low disease. Identifying T2 status has therapeutic implications for patient management, but a real-world understanding of this T2 paradigm in difficult-to-treat and severe asthma remains limited., Objectives: To identify the prevalence of T2-high status in difficult-to-treat asthma patients using a multicomponent definition and compare clinical and pathophysiologic characteristics between patients classified as T2-high and T2-low., Methods: We evaluated 388 biologic-naive patients from the Wessex Asthma Cohort of difficult asthma (WATCH) study in the United Kingdom. Type 2-high asthma was defined as 20 parts per billion or greater FeNO , 150 cells/μL or greater peripheral blood eosinophils, the need for maintenance oral corticosteroids, and/or clinically allergy-driven asthma., Results: This multicomponent assessment identified T2-high asthma in 93% of patients (360 of 388). Body mass index, inhaled corticosteroid dose, asthma exacerbations, and common comorbidities did not differ by T2 status. Significantly worse airflow limitation was found in T2-high compared with T2-low patients (FEV 1 /FVC 65.9% vs 74.6%). Moreover, 75% of patients defined as having T2-low asthma had raised peripheral blood eosinophils within the preceding 10 years, which left only seven patients (1.8%) who had never had T2 signals. Incorporation of sputum eosinophilia 2% or greater into the multicomponent definition in a subset of 117 patients with induced sputum data similarly found that 96% (112 of 117) met criteria for T2-high asthma, 50% of whom (56 of 112) had sputum eosinophils 2% or greater., Conclusions: Almost all patients with difficult-to-treat asthma have T2-high disease; less than 2% of patients never display T2-defining criteria. This highlights a need to assess T2 status comprehensively in clinical practice before labeling a patient with difficult-to-treat asthma as T2-low., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Although mucosal-associated invariant T (MAIT) cells provide rapid, innate-like responses, they are not pre-set, and memory-like responses have been described for MAIT cells following infections. The importance of metabolism for controlling these responses, however, is unknown. Here, following pulmonary immunization with a Salmonella vaccine strain, mouse MAIT cells expanded as separate CD127 - Klrg1 + and CD127 + Klrg1 - antigen-adapted populations that differed in terms of their transcriptome, function and localization in lung tissue. These populations remained altered from steady state for months as stable, separate MAIT cell lineages with enhanced effector programmes and divergent metabolism. CD127 + MAIT cells engaged in an energetic, mitochondrial metabolic programme, which was critical for their maintenance and IL-17A synthesis. This programme was supported by high fatty acid uptake and mitochondrial oxidation and relied on highly polarized mitochondria and autophagy. After vaccination, CD127 + MAIT cells protected mice against Streptococcus pneumoniae infection. In contrast, Klrg1 + MAIT cells had dormant but ready-to-respond mitochondria and depended instead on Hif1a-driven glycolysis to survive and produce IFN-γ. They responded antigen independently and participated in protection from influenza virus. These metabolic dependencies may enable tuning of memory-like MAIT cell responses for vaccination and immunotherapies., (© 2023. The Author(s).)

With the continued promise of immunotherapy for treating cancer, understanding how host genetics contributes to the tumor immune microenvironment (TIME) is essential to tailoring cancer screening and treatment strategies. Here, we study 1084 eQTLs affecting the TIME found through analysis of The Cancer Genome Atlas and literature curation. These TIME eQTLs are enriched in areas of active transcription, and associate with gene expression in specific immune cell subsets, such as macrophages and dendritic cells. Polygenic score models built with TIME eQTLs reproducibly stratify cancer risk, survival and immune checkpoint blockade (ICB) response across independent cohorts. To assess whether an eQTL-informed approach could reveal potential cancer immunotherapy targets, we inhibit CTSS, a gene implicated by cancer risk and ICB response-associated polygenic models; CTSS inhibition results in slowed tumor growth and extended survival in vivo. These results validate the potential of integrating germline variation and TIME characteristics for uncovering potential targets for immunotherapy., (© 2023. The Author(s).)

Junctional adhesion molecule-like protein (JAML) serves as a co-stimulatory molecule in γδ T cells. While it has recently been described as a cancer immunotherapy target in mice, its potential to cause toxicity, specific mode of action with regard to its cellular targets, and whether it can be targeted in humans remain unknown. Here, we show that JAML is induced by T cell receptor engagement, reveal that this induction is linked to cis-regulatory interactions between the CD3D and JAML gene loci. When compared with other immunotherapy targets plagued by low target specificity and end-organ toxicity, we find JAML to be mostly restricted to and highly expressed by tissue-resident memory CD8 + T cells in multiple cancer types. By delineating the key cellular targets and functional consequences of agonistic anti-JAML therapy in a murine melanoma model, we show its specific mode of action and the reason for its synergistic effects with anti-PD-1., Competing Interests: Declaration of interests The La Jolla Institute of Immunology has filed a patent “Methods for modulating an immune response to cancer or tumor cells” related to this work, and S.E., P.V., and C.H.O. are co-inventors on this patent., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Previous studies suggest that monocytes are an important contributor to tuberculosis (TB)-specific immune signatures in blood., Methods: Here, we carried out comprehensive single-cell profiling of monocytes in paired blood samples of active TB (ATB) patients at diagnosis and mid-treatment, and healthy controls., Results: At diagnosis, ATB patients displayed increased monocyte-to-lymphocyte ratio, increased frequency of CD14+CD16- and intermediate CD14+CD16+ monocytes, and upregulation of interferon signaling genes that significantly overlapped with previously reported blood TB signatures in both CD14+ subsets. In this cohort, we identified additional transcriptomic and functional changes in intermediate CD14+CD16+ monocytes, such as the upregulation of inflammatory and MHC-II genes, and increased capacity to activate T cells, reflecting overall increased activation in this population. Single-cell transcriptomics revealed that distinct subsets of intermediate CD14+CD16+ monocytes were responsible for each gene signature, indicating significant functional heterogeneity within this population. Finally, we observed that changes in CD14+ monocytes were transient, as they were no longer observed in the same ATB patients mid-treatment, suggesting they are associated with disease resolution., Discussion: Together, our study demonstrates for the first time that both intermediate and classical monocytes individually contribute to blood immune signatures of ATB and identifies novel subsets and associated gene signatures that may hold disease relevance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hillman, Khan, Singhania, Dubelko, Soldevila, Tippalagama, DeSilva, Gunasena, Perera, Scriba, Ontong, Fisher, Luabeya, Taplitz, Seumois, Vijayanand, Hedrick, Peters and Burel.)

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can result in severe immune dysfunction, hospitalization, and death. Many patients also develop long-COVID-19, experiencing symptoms months after infection. Although significant progress has been made in understanding the immune response to acute SARS-CoV-2 infection, gaps remain in our knowledge of how innate immunity influences disease kinetics and severity. We hypothesized that cytometry by time-of-flight analysis of PBMCs from healthy and infected subjects would identify novel cell surface markers and innate immune cell subsets associated with COVID-19 severity. In this pursuit, we identified monocyte and dendritic cell subsets that changed in frequency during acute SARS-CoV-2 infection and correlated with clinical parameters of disease severity. Subsets of nonclassical monocytes decreased in frequency in hospitalized subjects, yet increased in the most severe patients and positively correlated with clinical values associated with worse disease severity. CD9, CD163, PDL1, and PDL2 expression significantly increased in hospitalized subjects, and CD9 and 6-Sulfo LacNac emerged as the markers that best distinguished monocyte subsets amongst all subjects. CD9 + monocytes remained elevated, whereas nonclassical monocytes remained decreased, in the blood of hospitalized subjects at 3-4 months postinfection. Finally, we found that CD9 + monocytes functionally released more IL-8 and MCP-1 after LPS stimulation. This study identifies new monocyte subsets present in the blood of COVID-19 patients that correlate with disease severity, and links CD9 + monocytes to COVID-19 progression., (©2022 Society for Leukocyte Biology.)

Phosphoinositide 3-kinase δ (PI3Kδ) has a key role in lymphocytes, and inhibitors that target this PI3K have been approved for treatment of B cell malignancies 1-3 . Although studies in mouse models of solid tumours have demonstrated that PI3Kδ inhibitors (PI3Kδi) can induce anti-tumour immunity 4,5 , its effect on solid tumours in humans remains unclear. Here we assessed the effects of the PI3Kδi AMG319 in human patients with head and neck cancer in a neoadjuvant, double-blind, placebo-controlled randomized phase II trial (EudraCT no. 2014-004388-20). PI3Kδ inhibition decreased the number of tumour-infiltrating regulatory T (T reg ) cells and enhanced the cytotoxic potential of tumour-infiltrating T cells. At the tested doses of AMG319, immune-related adverse events (irAEs) required treatment to be discontinued in 12 out of 21 of patients treated with AMG319, suggestive of systemic effects on T reg cells. Accordingly, in mouse models, PI3Kδi decreased the number of T reg cells systemically and caused colitis. Single-cell RNA-sequencing analysis revealed a PI3Kδi-driven loss of tissue-resident colonic ST2 T reg cells, accompanied by expansion of pathogenic T helper 17 (T H 17) and type 17 CD8 + T (T C 17) cells, which probably contributed to toxicity; this points towards a specific mode of action for the emergence of irAEs. A modified treatment regimen with intermittent dosing of PI3Kδi in mouse models led to a significant decrease in tumour growth without inducing pathogenic T cells in colonic tissue, indicating that alternative dosing regimens might limit toxicity., (© 2022. The Author(s).)

Background: Cancer is characterized by an accumulation of somatic mutations, of which a significant subset can generate cancer-specific neoepitopes that are recognized by autologous T cells. Such neoepitopes are emerging as important targets for cancer immunotherapy, including personalized cancer vaccination strategies., Methods: We used whole-exome and RNA sequencing analysis to identify potential neoantigens for a patient with non-small cell lung cancer. Thereafter, we assessed the autologous T-cell reactivity to the candidate neoantigens using a long peptide approach in a cultured interferon gamma ELISpot and tracked the neoantigen-specific T-cells in the tumor by T-cell receptor (TCR) sequencing. In parallel, identified gene variants were incorporated into a Modified Vaccinia Ankara-based vaccine, which was evaluated in the human leucocyte antigen A*0201 transgenic mouse model (HHD)., Results: Sequencing revealed a tumor with a low mutational burden: 2219 sequence variants were identified from the primary tumor, of which 23 were expressed in the transcriptome, involving 18 gene products. We could demonstrate spontaneous T-cell responses to 5/18 (28%) mutated gene variants, and further analysis of the TCR repertoire of neoantigen-specific CD4 + and CD8 + T cells revealed TCR clonotypes that were expanded in both blood and tumor tissue. Following vaccination of HHD mice, de novo T-cell responses were generated to 4/18 (22%) mutated gene variants; T cells reactive against two variants were also evident in the autologous setting. Subsequently, we determined the major histocompatibility complex restriction of the T-cell responses and used in silico prediction tools to determine the likely neoepitopes., Conclusions: Our study demonstrates the feasibility of efficiently identifying tumor-specific neoantigens that can be targeted by vaccination in tumors with a low mutational burden, promising successful clinical exploitation, with trials currently underway., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

The impact of genetic variants on cells challenged in biologically relevant contexts has not been fully explored. Here, we activated CD4 + T cells from 89 healthy donors and performed a single-cell RNA sequencing assay with >1 million cells to examine cell type-specific and activation-dependent effects of genetic variants. Single-cell expression quantitative trait loci (sc-eQTL) analysis of 19 distinct CD4 + T cell subsets showed that the expression of over 4000 genes is significantly associated with common genetic polymorphisms and that most of these genes show their most prominent effects in specific cell types. These genes included many that encode for molecules important for activation, differentiation, and effector functions of T cells. We also found new gene associations for disease-risk variants identified from genome-wide association studies and highlighted the cell types in which their effects are most prominent. We found that biological sex has a major influence on activation-dependent gene expression in CD4 + T cell subsets. Sex-biased transcripts were significantly enriched in several pathways that are essential for the initiation and execution of effector functions by CD4 + T cells like TCR signaling, cytokines, cytokine receptors, costimulatory, apoptosis, and cell-cell adhesion pathways. Overall, this DICE (Database of Immune Cell Expression, eQTLs, and Epigenomics) subproject highlights the power of sc-eQTL studies for simultaneously exploring the activation and cell type-dependent effects of common genetic variants on gene expression (https://dice-database.org).

Double-positive CD4 + CD8αβ + (DP) cells are thought to reside as T cell progenitors exclusively within the thymus. We recently discovered an unexpected CD4 + and CD8αβ + immune cell population in healthy and atherosclerotic mice by single-cell RNA sequencing. Transcriptomically, these cells resembled thymic DPs. Flow cytometry and three-dimensional whole-mount imaging confirmed DPs in thymus, mediastinal adipose tissue, and aortic adventitia, but nowhere else. Deep transcriptional profiling revealed differences between DP cells isolated from the three locations. All DPs were dependent on RAG2 expression and the presence of the thymus. Mediastinal adipose tissue DPs resided in close vicinity to invariant NKT cells, which they could activate in vitro. Thymus transplantation failed to reconstitute extrathymic DPs, and frequencies of extrathymic DPs were unaltered by pharmacologic inhibition of S1P1, suggesting that their migration may be locally confined. Our results define two new, transcriptionally distinct subsets of extrathymic DPs that may play a role in aortic vascular homeostasis., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Background: The century-old Mycobacterium bovis Bacillus Calmette-Guerin (BCG) remains the only licensed vaccine against tuberculosis (TB). Despite this, there is still a lot to learn about the immune response induced by BCG, both in terms of phenotype and specificity., Methods: We investigated immune responses in adult individuals pre and 8 months post BCG vaccination. We specifically determined changes in gene expression, cell subset composition, DNA methylome, and the TCR repertoire induced in PBMCs and CD4 memory T cells associated with antigen stimulation by either BCG or a Mycobacterium tuberculosis (Mtb)-derived peptide pool., Findings: Following BCG vaccination, we observed increased frequencies of CCR6+ CD4 T cells, which includes both Th1* (CXCR3+CCR6+) and Th17 subsets, and mucosal associated invariant T cells (MAITs). A large number of immune response genes and pathways were upregulated post BCG vaccination with similar patterns observed in both PBMCs and memory CD4 T cells, thus suggesting a substantial role for CD4 T cells in the cellular response to BCG. These upregulated genes and associated pathways were also reflected in the DNA methylome. We described both qualitative and quantitative changes in the BCG-specific TCR repertoire post vaccination, and importantly found evidence for similar TCR repertoires across different subjects., Interpretation: The immune signatures defined herein can be used to track and further characterize immune responses induced by BCG, and can serve as reference for benchmarking novel vaccination strategies., Competing Interests: Declaration of Competing Interest The authors declare no competing interests, except for Dr. Phillips who reports grants from NHMRC Australia, grants from NIH, personal fees from Uptodate/Lexicomp, personal fees from Biocryst, from Patent for HLA-B*57:01 testing for abacavir HSR, personal fees and other from Biocryst, personal fees from Janssen, personal fees from Vertex, personal fees from Verve, other from Provisional patent pending for HLA-A*32:01 testing for Vancomycin hypersensitivity, personal fees from Regeneron, outside the submitted work; In addition, Dr. Phillips has a patent issued for HLA-B*57:01 testing for abacavir hypersensitivity to IIID Pty Ltd, where she acts as the co-director., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Fungal sensitivity has been associated with severe asthma outcomes. However, the clinical implication of Aspergillus fumigatus sensitization in difficult-to-treat (or difficult) asthma is unclear., Objectives: To characterize the clinical implications of A fumigatus sensitization in a large difficult asthma cohort., Methods: Participants who underwent both skin prick and specific IgE testing to A fumigatus (n = 318) from the longitudinal real-life Wessex AsThma CoHort of difficult asthma, United Kingdom, were characterized by A fumigatus sensitization (either positive skin prick test result or specific IgE) and allergic bronchopulmonary aspergillosis status using clinical/pathophysiological disease measures., Results: A fumigatus sensitization was found in 23.9% (76 of 318) of patients with difficult asthma. Compared with A fumigatus nonsensitized subjects, those with sensitization were significantly more often male (50% vs 31%), older (58 years) with longer asthma duration (33 years), higher maintenance oral corticosteroid (39.7%) and asthma biologic use (27.6%), raised current/maximum log 10 total IgE+1 (2.43/2.72 IU/L), worse prebronchodilator airflow obstruction (FEV 1 62.2% predicted, FEV 1 /forced vital capacity 61.2%, forced expiratory flow between 25% and 75% exhalation 30.9% predicted), and frequent radiological bronchiectasis (40%), but had less psychophysiologic comorbidities. Allergic bronchopulmonary aspergillosis diagnosis was associated with higher treatment needs and stronger eosinophilic signals. Factors independently associated with A fumigatus sensitization in difficult asthma included maintenance oral corticosteroid use (odds ratio [OR], 3.34) and maximum log 10 total IgE+1 (OR, 4.30), whereas for allergic bronchopulmonary aspergillosis included maintenance oral corticosteroid use (OR, 6.98), maximum log 10 total IgE+1 (OR, 4.65), and radiological bronchiectasis (OR, 4.08)., Conclusions: A fumigatus sensitization in difficult asthma identifies a more severe form of airways disease associated with greater morbidity, treatment need, and airways dysfunction/damage, but fewer psychophysiologic comorbidities. Screening of A fumigatus status should be an early element in the comprehensive assessment of patients with difficult asthma., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Common genetic polymorphisms associated with COVID-19 illness can be utilized for discovering molecular pathways and cell types driving disease pathogenesis. Given the importance of immune cells in the pathogenesis of COVID-19 illness, here we assessed the effects of COVID-19-risk variants on gene expression in a wide range of immune cell types. Transcriptome-wide association study and colocalization analysis revealed putative causal genes and the specific immune cell types where gene expression is most influenced by COVID-19-risk variants. Notable examples include OAS1 in non-classical monocytes, DTX1 in B cells, IL10RB in NK cells, CXCR6 in follicular helper T cells, CCR9 in regulatory T cells and ARL17A in T H 2 cells. By analysis of transposase accessible chromatin and H3K27ac-based chromatin-interaction maps of immune cell types, we prioritized potentially functional COVID-19-risk variants. Our study highlights the potential of COVID-19 genetic risk variants to impact the function of diverse immune cell types and influence severe disease manifestations., (© 2021. The Author(s).)

Immune-checkpoint blockade (ICB) has shown remarkable clinical success in boosting antitumor immunity. However, the breadth of its cellular targets and specific mode of action remain elusive. We find that tumor-infiltrating follicular regulatory T (T FR ) cells are prevalent in tumor tissues of several cancer types. They are primarily located within tertiary lymphoid structures and exhibit superior suppressive capacity and in vivo persistence as compared with regulatory T cells, with which they share a clonal and developmental relationship. In syngeneic tumor models, anti-PD-1 treatment increases the number of tumor-infiltrating T FR cells. Both T FR cell deficiency and the depletion of T FR cells with anti-CTLA-4 before anti-PD-1 treatment improve tumor control in mice. Notably, in a cohort of 271 patients with melanoma, treatment with anti-CTLA-4 followed by anti-PD-1 at progression was associated with better a survival outcome than monotherapy with anti-PD-1 or anti-CTLA-4, anti-PD-1 followed by anti-CTLA-4 at progression or concomitant combination therapy., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Upon Ag encounter, T cells can rapidly divide and form an effector population, which plays an important role in fighting acute infections. In humans, little is known about the molecular markers that distinguish such effector cells from other T cell populations. To address this, we investigated the molecular profile of T cells present in individuals with active tuberculosis (ATB), where we expect Ag encounter and expansion of effector cells to occur at higher frequency in contrast to Mycobacterium tuberculosis -sensitized healthy IGRA + individuals. We found that the frequency of HLA-DR + cells was increased in circulating CD4 T cells of ATB patients, and was dominantly expressed in M. tuberculosis Ag-specific CD4 T cells. We tested and confirmed that HLA-DR is a marker of recently divided CD4 T cells upon M. tuberculosis Ag exposure using an in vitro model examining the response of resting memory T cells from healthy IGRA + to Ags. Thus, HLA-DR marks a CD4 T cell population that can be directly detected ex vivo in human peripheral blood, whose frequency is increased during ATB disease and contains recently divided Ag-specific effector T cells. These findings will facilitate the monitoring and study of disease-specific effector T cell responses in the context of ATB and other infections., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Severe asthma accounts for almost half the cost associated with asthma. Severe asthma is driven by heterogeneous molecular mechanisms. Conventional clinical trial design often lacks the power and efficiency to target subgroups with specific pathobiological mechanisms. Furthermore, the validation and approval of new asthma therapies is a lengthy process. A large proportion of that time is taken by clinical trials to validate asthma interventions. The National Institutes of Health Precision Medicine in Severe and/or Exacerbation Prone Asthma (PrecISE) program was established with the goal of designing and executing a trial that uses adaptive design techniques to rapidly evaluate novel interventions in biomarker-defined subgroups of severe asthma, while seeking to refine these biomarker subgroups, and to identify early markers of response to therapy. The novel trial design is an adaptive platform trial conducted under a single master protocol that incorporates precision medicine components. Furthermore, it includes innovative applications of futility analysis, cross-over design with use of shared placebo groups, and early futility analysis to permit more rapid identification of effective interventions. The development and rationale behind the study design are described. The interventions chosen for the initial investigation and the criteria used to identify these interventions are enumerated. The biomarker-based adaptive design and analytic scheme are detailed as well as special considerations involved in the final trial design., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Systemic lupus erythematosus (SLE) is an incurable autoimmune disease disproportionately affecting women. A major obstacle in finding targeted therapies for SLE is its remarkable heterogeneity in clinical manifestations as well as in the involvement of distinct cell types. To identify cell-specific targets as well as cross-correlation relationships among expression programs of different cell types, we here analyze six major circulating immune cell types from SLE patient blood. Our results show that presence of an interferon response signature stratifies patients into two distinct groups (IFNneg vs. IFNpos). Comparing these two groups using differential gene expression and differential gene coexpression analysis, we prioritize a relatively small list of genes from classical monocytes including two known immune modulators: TNFSF13B/BAFF (target of belimumab , an approved therapeutic for SLE) and IL1RN (the basis of anakinra, a therapeutic for rheumatoid arthritis). We then develop a multi-cell type extension of the weighted gene coexpression network analysis (WGCNA) framework, termed mWGCNA. Applying mWGCNA to RNA-seq data from six sorted immune cell populations (15 SLE, 10 healthy donors), we identify a coexpression module with interferon-stimulated genes (ISGs) among all cell types and a cross-cell type correlation linking expression of specific T helper cell markers to B cell response as well as to TNFSF13B expression from myeloid cells, all of which in turn correlates with disease severity of IFNpos patients. Our results demonstrate the power of a hypothesis-free and data-driven approach to discover drug targets and to reveal novel cross-correlation across cell types in SLE with implications for other autoimmune diseases., (© 2021 Panwar et al.; Published by Cold Spring Harbor Laboratory Press.)

Invariant natural killer T cells (iNKT cells) differentiate into thymic and peripheral NKT1, NKT2 and NKT17 subsets. Here we use RNA-seq and ATAC-seq analyses and show iNKT subsets are similar, regardless of tissue location. Lung iNKT cell subsets possess the most distinct location-specific features, shared with other innate lymphocytes in the lung, possibly consistent with increased activation. Following antigenic stimulation, iNKT cells undergo chromatin and transcriptional changes delineating two populations: one similar to follicular helper T cells and the other NK or effector like. Phenotypic analysis indicates these changes are observed long-term, suggesting that iNKT cells gene programs are not fixed, but they are capable of chromatin remodeling after antigen to give rise to additional subsets.

Immunodominance to nonneutralizing epitopes is a roadblock in designing vaccines against several diseases of high interest. One hypothetical possibility is that limited CD4 T cell help to B cells in a normal germinal center (GC) response results in selective recruitment of abundant, immunodominant B cells. This is a central issue in HIV envelope glycoprotein (Env) vaccine designs, because precursors to broadly neutralizing epitopes are rare. Here, we sought to elucidate whether modulating the quantity of T cell help can influence recruitment and competition of broadly neutralizing antibody precursor B cells at a physiological precursor frequency in response to Env trimer immunization. To do so, two new Env-specific CD4 transgenic (Tg) T cell receptor (TCR) mouse lines were generated, carrying TCR pairs derived from Env-protein immunization. Our results suggest that CD4 T cell help quantitatively regulates early recruitment of rare B cells to GCs., Competing Interests: Disclosures: M.K. Jenkins reported a patent to US2019/044605 issued. W.R. Schief reported grants from Compuvax, Inc outside the submitted work; in addition, W.R. Schief had a patent to BG505 GT3.1 pending and a patent to BG505 MD39 pending. No other disclosures were reported., (© 2020 Lee et al.)

The molecular properties of CD8 + T cells that respond to SARS-CoV-2 infection are not fully known. Here, we report on the single-cell transcriptomes of >80,000 virus-reactive CD8 + T cells, obtained using a modified Antigen-Reactive T cell Enrichment (ARTE) assay, from 39 COVID-19 patients and 10 healthy subjects. COVID-19 patients segregated into two groups based on whether the dominant CD8 + T cell response to SARS-CoV-2 was 'exhausted' or not. SARS-CoV-2-reactive cells in the exhausted subset were increased in frequency and displayed lesser cytotoxicity and inflammatory features in COVID-19 patients with mild compared to severe illness. In contrast, SARS-CoV-2-reactive cells in the dominant non-exhausted subset from patients with severe disease showed enrichment of transcripts linked to co-stimulation, pro-survival NF-κB signaling, and anti-apoptotic pathways, suggesting the generation of robust CD8 + T cell memory responses in patients with severe COVID-19 illness. CD8 + T cells reactive to influenza and respiratory syncytial virus from healthy subjects displayed polyfunctional features and enhanced glycolysis. Cells with such features were largely absent in SARS-CoV-2-reactive cells from both COVID-19 patients and healthy controls non-exposed to SARS-CoV-2. Overall, our single-cell analysis revealed substantial diversity in the nature of CD8 + T cells responding to SARS-CoV-2., (Copyright © 2021, American Association for the Advancement of Science.)

Expression quantitative trait loci (eQTLs) studies provide associations of genetic variants with gene expression but fall short of pinpointing functionally important eQTLs. Here, using H3K27ac HiChIP assays, we mapped eQTLs overlapping active cis-regulatory elements that interact with their target gene promoters (promoter-interacting eQTLs, pieQTLs) in five common immune cell types (Database of Immune Cell Expression, Expression quantitative trait loci and Epigenomics (DICE) cis-interactome project). This approach allowed us to identify functionally important eQTLs and show mechanisms that explain their cell-type restriction. We also devised an approach to eQTL discovery that relies on HiChIP-based promoter interaction maps as a structural framework for deciding which SNPs to test for association with gene expression, and observe ultra-long-distance pieQTLs (>1 megabase away), including several disease-risk variants. We validated the functional role of pieQTLs using reporter assays, CRISPRi, dCas9-tiling guides and Cas9-mediated base-pair editing. In this article we present a method for functional eQTL discovery and provide insights into relevance of noncoding variants for cell-specific gene regulation and for disease association beyond conventional eQTL mapping.

The contribution of CD4 + T cells to protective or pathogenic immune responses to SARS-CoV-2 infection remains unknown. Here, we present single-cell transcriptomic analysis of >100,000 viral antigen-reactive CD4 + T cells from 40 COVID-19 patients. In hospitalized patients compared to non-hospitalized patients, we found increased proportions of cytotoxic follicular helper cells and cytotoxic T helper (T H ) cells (CD4-CTLs) responding to SARS-CoV-2 and reduced proportion of SARS-CoV-2-reactive regulatory T cells (T REG ). Importantly, in hospitalized COVID-19 patients, a strong cytotoxic T FH response was observed early in the illness, which correlated negatively with antibody levels to SARS-CoV-2 spike protein. Polyfunctional T H 1 and T H 17 cell subsets were underrepresented in the repertoire of SARS-CoV-2-reactive CD4 + T cells compared to influenza-reactive CD4 + T cells. Together, our analyses provide insights into the gene expression patterns of SARS-CoV-2-reactive CD4 + T cells in distinct disease severities., Competing Interests: Declaration of Interests The authors declare no competing interests., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

Our recent work has highlighted that care needs to be taken when interpreting single cell data originating from flow cytometry acquisition or cell sorting: We found that doublets of T cells bound to other immune cells are often present in the live singlet gate of human peripheral blood samples acquired by flow cytometry. This hidden "contamination" generates atypical gene signatures of mixed cell lineage in what is assumed to be single cells, which can lead to data misinterpretation, such as the description of novel immune cell types. Here, based on the example of T cell-monocyte complexes, we identify experimental and data analysis strategies to help distinguishing between singlets and cell-cell complexes in non-imaging flow cytometry and single-cell sorting. We found robust molecular signatures in both T cell-monocyte and T cell-B cell complexes that can distinguish them from singlets at both protein and mRNA levels. Imaging flow cytometry with appropriate gating strategy (matching the one used in cell sorting) and direct microscopy imaging after cell sorting were the two methods of choice to detect the presence of cell-cell complexes in suspicious dual-expressing cells. We finally applied this knowledge to highlight the likely presence of T cell-B cell complexes in a recently published dataset describing a novel cell population with mixed T cell and B cell lineage properties. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry., (© 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.)

Background: Understanding neutrophil heterogeneity and its relationship to disease progression has become a recent focus of cancer research. Indeed, several studies have identified neutrophil subpopulations associated with protumoral or antitumoral functions. However, this work has been hindered by a lack of widely accepted markers with which to define neutrophil subpopulations., Methods: To identify markers of neutrophil heterogeneity in cancer, we used single-cell cytometry by time-of-flight (CyTOF) coupled with high-dimensional analysis on blood samples from treatment-naïve patients with melanoma., Results: Our efforts allowed us to identify seven blood neutrophil clusters, including two previously identified individual populations. Interrogation of these neutrophil subpopulations revealed a positive trend between specific clusters and disease stage. Finally, we recapitulated these seven blood neutrophil populations via flow cytometry and found that they exhibited diverse capacities for phagocytosis and reactive oxygen species production in vitro., Conclusions: Our data provide a refined consensus on neutrophil heterogeneity markers, enabling a prospective functional evaluation in patients with solid tumors., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific histone modifications, such as H3K27ac, to help identify cis-regulatory DNA elements. The manual process to complete a ChIP-Seq is labor intensive, technically challenging, and often requires large-cell numbers (>100,000 cells). The method described here helps to overcome those challenges. A complete semiautomated, microscaled H3K27ac ChIP-Seq procedure including cell fixation, chromatin shearing, immunoprecipitation, and sequencing library preparation, for batch of 48 samples for cell number inputs less than 100,000 cells is described in detail. The semiautonomous platform reduces technical variability, improves signal-to-noise ratios, and drastically reduces labor. The system can thereby reduce costs by allowing for reduced reaction volumes, limiting the number of expensive reagents such as enzymes, magnetic beads, antibodies, and hands-on time required. These improvements to the ChIP-Seq method suit perfectly for large-scale epigenetic studies of clinical samples with limited cell numbers in a highly reproducible manner.

Background: IgE is the least abundant immunoglobulin and tightly regulated, and IgE-producing B cells are rare. The cellular origin and evolution of IgE responses are poorly understood., Objective: The cellular and clonal origin of IgE memory responses following mucosal allergen exposure by sublingual immunotherapy (SLIT) were investigated., Methods: In a randomized double-blind, placebo-controlled, time course SLIT study, PBMCs and nasal biopsy samples were collected from 40 adults with seasonal allergic rhinitis at baseline and at 4, 8, 16, 28, and 52 weeks. RNA was extracted from PBMCs, sorted B cells, and nasal biopsy samples for heavy chain variable gene repertoire sequencing. Moreover, mAbs were derived from single B-cell transcriptomes., Results: Combining heavy chain variable gene repertoire sequencing and single-cell transcriptomics yielded direct evidence of a parallel boost of 2 clonally and functionally related B-cell subsets of short-lived IgE + plasmablasts and IgG + memory B cells. Mucosal grass pollen allergen exposure by SLIT resulted in highly diverse IgE and IgG E repertoires. These were extensively mutated and appeared relatively stable as per heavy chain isotype, somatic hypermutations, and clonal composition. Single IgG E + memory B-cell and IgE + preplasmablast transcriptomes encoded antibodies that were specific for major grass pollen allergens and able to elicit basophil activation at very low allergen concentrations., Conclusion: For the first time, we have shown that on mucosal allergen exposure, human IgE memory resides in allergen-specific IgG + memory B cells. These cells rapidly switch isotype, expand into short-lived IgE + plasmablasts, and serve as a potential target for therapeutic intervention., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

Background: The role of tumor-associated macrophages (TAMs) in determining the outcome between the antitumor effects of the adaptive immune system and the tumor's anti-immunity stratagems, is controversial. Macrophages modulate their activities and phenotypes by integration of signals in the tumor microenvironment. Depending on how macrophages are activated, they may adopt so-called M1-like, antitumor or M2-like, protumor profiles. In many solid tumors, a dominance of M2-like macrophages is associated with poor outcomes but in some tumor types, strong M1-like profiles are linked to better outcomes. We aimed to investigate the interrelationship of these TAM populations to establish how they modulate the efficacy of the adaptive immune system in early lung cancer., Methods: Macrophages from matched lung (non-tumor-associated macrophages (NTAMs)) and tumor samples (TAMs) from resected lung cancers were assessed by bulk and single-cell transcriptomic analysis. Protein expression of genes characteristic of M1-like (chemokine (C-X-C motif) ligand 9) or M2-like (matrix metallopeptidase 12) functions was confirmed by confocal microscopy. Immunohistochemistry related the distribution of TAM transcriptomic signatures to density of CD8 + tissue-resident memory T cells (T RM ) in tumors and survival data from an independent cohort of 393 patients with lung cancer., Results: TAMs have significantly different transcriptomic profiles from NTAMs with >1000 differentially expressed genes. TAMs displayed a strong M2-like signature with no significant variation between patients. However, single-cell RNA-sequencing supported by immuno-stained cells revealed that additionally, in 25% of patients the M2-like TAMs also co-expressed a strong/hot M1-like signature (M1 hot ). Importantly, there was a strong association between the density of M1 hot TAMs and T RM cells in tumors that was in turn linked to better survival. Our data suggest a mechanism by which M1 hot TAMs may recruit T RM cells via CXCL9 expression and sustain them by making available more of the essential fatty acids on which T RM depend., Conclusions: We showed that in early lung cancer, expression of M1-like and M2-like gene signatures are not mutually exclusive since the same TAMs can simultaneously display both gene-expression profiles. The presence of M1 hot TAMs was associated with a strong T RM tumor-infiltrate and better outcomes. Thus, therapeutic approaches to re-program TAMs to an M1 hot phenotype are likely to augment the adaptive antitumor responses., Competing Interests: Competing interests: PV reports grants and personal fees from Pfizer, from null, outside the submitted work., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

CD4 + T helper (T H ) cells and regulatory T (T reg ) cells that respond to common allergens play an important role in driving and dampening airway inflammation in patients with asthma. Until recently, direct, unbiased molecular analysis of allergen-reactive T H and T reg cells has not been possible. To better understand the diversity of these T cell subsets in allergy and asthma, we analyzed the single-cell transcriptome of ~50,000 house dust mite (HDM) allergen-reactive T H cells and T reg cells from asthmatics with HDM allergy and from three control groups: asthmatics without HDM allergy and nonasthmatics with and without HDM allergy. Our analyses show that HDM allergen-reactive T H and T reg cells are highly heterogeneous and certain subsets are quantitatively and qualitatively different in individuals with HDM-reactive asthma. The number of interleukin-9 (IL-9)-expressing HDM-reactive T H cells is greater in asthmatics with HDM allergy compared with nonasthmatics with HDM allergy, and this IL-9-expressing T H subset displays enhanced pathogenic properties. More HDM-reactive T H and T reg cells expressing the interferon response signature (T H IFNR and T reg IFNR) are present in asthmatics without HDM allergy compared with those with HDM allergy. In cells from these subsets (T H IFNR and T reg IFNR), expression of TNFSF10 was enriched; its product, tumor necrosis factor-related apoptosis-inducing ligand, dampens activation of T H cells. These findings suggest that the T H IFNR and T reg IFNR subsets may dampen allergic responses, which may help explain why only some people develop T H 2 responses to nearly ubiquitous allergens., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

CD8 T cells are considered important contributors to the immune response against Mycobacterium tuberculosis , yet limited information is currently known regarding their specific immune signature and phenotype. In this study, we applied a cell population transcriptomics strategy to define immune signatures of human latent tuberculosis infection (LTBI) in memory CD8 T cells. We found a 41-gene signature that discriminates between memory CD8 T cells from healthy LTBI subjects and uninfected controls. The gene signature was dominated by genes associated with mucosal-associated invariant T cells (MAITs) and reflected the lower frequency of MAITs observed in individuals with LTBI. There was no evidence for a conventional CD8 T cell-specific signature between the two cohorts. We, therefore, investigated MAITs in more detail based on Vα7.2 and CD161 expression and staining with an MHC-related protein 1 (MR1) tetramer. This revealed two distinct populations of CD8 + Vα7.2 + CD161 + MAITs: MR1 tetramer + and MR1 tetramer - , which both had distinct gene expression compared with memory CD8 T cells. Transcriptomic analysis of LTBI versus noninfected individuals did not reveal significant differences for MR1 tetramer + MAITs. However, gene expression of MR1 tetramer - MAITs showed large interindividual diversity and a tuberculosis-specific signature. This was further strengthened by a more diverse TCR-α and -β repertoire of MR1 tetramer - cells as compared with MR1 tetramer + Thus, circulating memory CD8 T cells in subjects with latent tuberculosis have a reduced number of conventional MR1 tetramer + MAITs as well as a difference in phenotype in the rare population of MR1 tetramer - MAITs compared with uninfected controls., (Copyright © 2020 The Authors.)

Determining mechanisms of resistance to αPD-1/PD-L1 immune-checkpoint immunotherapy is key to developing new treatment strategies. Cancer-associated fibroblasts (CAF) have many tumor-promoting functions and promote immune evasion through multiple mechanisms, but as yet, no CAF-specific inhibitors are clinically available. Here we generated CAF-rich murine tumor models (TC1, MC38, and 4T1) to investigate how CAFs influence the immune microenvironment and affect response to different immunotherapy modalities [anticancer vaccination, TC1 (HPV E7 DNA vaccine), αPD-1, and MC38] and found that CAFs broadly suppressed response by specifically excluding CD8 + T cells from tumors (not CD4 + T cells or macrophages); CD8 + T-cell exclusion was similarly present in CAF-rich human tumors. RNA sequencing of CD8 + T cells from CAF-rich murine tumors and immunochemistry analysis of human tumors identified significant upregulation of CTLA-4 in the absence of other exhaustion markers; inhibiting CTLA-4 with a nondepleting antibody overcame the CD8 + T-cell exclusion effect without affecting Tregs. We then examined the potential for CAF targeting, focusing on the ROS-producing enzyme NOX4, which is upregulated by CAF in many human cancers, and compared this with TGFβ1 inhibition, a key regulator of the CAF phenotype. siRNA knockdown or pharmacologic inhibition [GKT137831 (Setanaxib)] of NOX4 "normalized" CAF to a quiescent phenotype and promoted intratumoral CD8 + T-cell infiltration, overcoming the exclusion effect; TGFβ1 inhibition could prevent, but not reverse, CAF differentiation. Finally, NOX4 inhibition restored immunotherapy response in CAF-rich tumors. These findings demonstrate that CAF-mediated immunotherapy resistance can be effectively overcome through NOX4 inhibition and could improve outcome in a broad range of cancers. SIGNIFICANCE: NOX4 is critical for maintaining the immune-suppressive CAF phenotype in tumors. Pharmacologic inhibition of NOX4 potentiates immunotherapy by overcoming CAF-mediated CD8 + T-cell exclusion. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/9/1846/F1.large.jpg. See related commentary by Hayward, p. 1799 ., (©2020 American Association for Cancer Research.)

Dengue virus (DENV) can cause diseases ranging from dengue fever (DF) to more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Whether antiviral T cells contribute to the protection against or pathogenesis of severe disease is not well defined. Here, we identified antigen-specific IL-10 + IFN-γ + double-positive (DP) CD4 T cells during acute DENV infection. While the transcriptomic signatures of DP cells partially overlapped with those of cytotoxic and type 1 regulatory CD4 T cells, the majority of them were non-cytotoxic/Tr1 and included IL21, IL22, CD109, and CCR1. Although we observed a higher frequency of DP cells in DHF, the transcriptomic profile of DP cells was similar in DF and DHF, suggesting that DHF is not associated with the altered phenotypic or functional attributes of DP cells. Overall, this study revealed a DENV-specific DP cell subset in patients with acute dengue disease and argues against altered DP cells as a determinant of DHF., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)