Laser capture microscopy (LCM)-RNAseq for topological mapping of synovial pathology during rheumatoid arthritis.

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 vessels were captured by LCM from seven RA and seven osteoarthritis (OA) patients. RNAseq was performed on RNA extracted from captured tissue. Principal component analysis (PCA) was performed on the sample set by disease state. Differential expression analysis was performed between disease states based on log2 fold-change and q-value parameters. Pathway analysis was performed using Reactome Pathway Database on differentially expressed genes (DEGs) between disease states. Significantly enriched pathways in each synovial region were selected based on false discovery rate (FDR). RESULTS: RA and OA transcriptomes were distinguishable by PCA. Pairwise comparisons of synovial lining, sublining, and vessels 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 biological function and synovial topology. Analysis of pathways uniquely enriched in each region revealed distinct phenotypic abnormalities. As examples, RA lining was marked by anomalous immune cell signaling, RA sublining by aberrant cell cycle, and RA vessels by alterations in heme scavenging. CONCLUSION: LCM-RNAseq confirms reported transcriptional differences between RA and OA synovium and provides evidence supporting a relationship between synovial topology and molecular anomalies in RA.

Intra-tumoral T cells in pediatric brain tumors display clonal expansion and effector properties.

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.

Late-rising CD4 T cells resolve mouse cytomegalovirus persistent replication in the salivary gland.

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.

How Does Mild Asthma Differ Phenotypically from Difficult-to-Treat Asthma?

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.

Distinct use of super-enhancer elements controls cell type-specific CD25 transcription and function.

The IL-2 receptor α chain (IL-2Rα/CD25) is constitutively expressed on double-negative (DN2/DN3 thymocytes and regulatory T cells (Tregs) 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 Tregs, 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.

Transcriptomes and metabolism define mouse and human MAIT cell populations.

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.

Human CD79b+ neutrophils in the blood are associated with early-stage melanoma.

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-CD86LoCD66b+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.

Cytotoxic CD4+ tissue-resident memory T cells are associated with asthma severity.

BACKGROUND: Patients with severe uncontrolled asthma represent a distinct endotype with persistent airway inflammation and remodeling that is refractory to corticosteroid treatment. CD4+ TH2 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 (TRM) cells making a dominant contribution. Notably, in severe asthmatics, a subset of CD4+ TRM 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-TH2 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.

Identification of cow milk epitopes to characterize and quantify disease-specific T cells in allergic children.

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 TH2 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 TH2 cytokines and pathogenic TH2/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.

Germline modifiers of the tumor immune microenvironment implicate drivers of cancer risk and immunotherapy response.

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.

Comprehensive Characterization of Difficult-to-Treat Asthma Reveals Near Absence of T2-Low Status.

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 (FEV1/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.

Divergent metabolic programmes control two populations of MAIT cells that protect the lung.

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.

JAML immunotherapy targets recently activated tumor-infiltrating CD8+ T cells.

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.

Single-cell immune profiling reveals long-term changes in myeloid cells and identifies a novel subset of CD9+ monocytes associated with COVID-19 hospitalization.

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.

Intermittent PI3Kδ inhibition sustains anti-tumour immunity and curbs irAEs.

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 malignancies1-3. Although studies in mouse models of solid tumours have demonstrated that PI3Kδ inhibitors (PI3Kδi) can induce anti-tumour immunity4,5, 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 (Treg) 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 Treg cells. Accordingly, in mouse models, PI3Kδi decreased the number of Treg cells systemically and caused colitis. Single-cell RNA-sequencing analysis revealed a PI3Kδi-driven loss of tissue-resident colonic ST2 Treg cells, accompanied by expansion of pathogenic T helper 17 (TH17) and type 17 CD8+ T (TC17) 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.

Targeting the tumor mutanome for personalized vaccination in a TMB low non-small cell lung cancer.

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.

Single-cell eQTL analysis of activated T cell subsets reveals activation and cell type-dependent effects of disease-risk variants.

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).

Single-cell profiling reveals distinct subsets of CD14+ monocytes drive blood immune signatures of active tuberculosis.

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.

CD4+CCR6+ T cells dominate the BCG-induced transcriptional signature.

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.

Thymus-Derived CD4+CD8+ Cells Reside in Mediastinal Adipose Tissue and the Aortic Arch.

Double-positive CD4+CD8αß+ (DP) cells are thought to reside as T cell progenitors exclusively within the thymus. We recently discovered an unexpected CD4+ and CD8αß+ 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.