Granzyme F: Exhaustion Marker and Modulator of Chimeric Antigen Receptor T Cell-Mediated Cytotoxicity.

Granzymes are a family of proteases used by CD8 T cells to mediate cytotoxicity and other less-defined activities. The substrate and mechanism of action of many granzymes are unknown, although they diverge among the family members. In this study, we show that mouse CD8+ tumor-infiltrating lymphocytes (TILs) express a unique array of granzymes relative to CD8 T cells outside the tumor microenvironment in multiple tumor models. Granzyme F was one of the most highly upregulated genes in TILs and was exclusively detected in PD1/TIM3 double-positive CD8 TILs. To determine the function of granzyme F and to improve the cytotoxic response to leukemia, we constructed chimeric Ag receptor T cells to overexpress a single granzyme, granzyme F or the better-characterized granzyme A or B. Using these doubly recombinant T cells, we demonstrated that granzyme F expression improved T cell-mediated cytotoxicity against target leukemia cells and induced a form of cell death other than chimeric Ag receptor T cells expressing only endogenous granzymes or exogenous granzyme A or B. However, increasing expression of granzyme F also had a detrimental impact on the viability of the host T cells, decreasing their persistence in circulation in vivo. These results suggest a unique role for granzyme F as a marker of terminally differentiated CD8 T cells with increased cytotoxicity, but also increased self-directed cytotoxicity, suggesting a potential mechanism for the end of the terminal exhaustion pathway.

Managing With Atrial Fibrillation: An Exploratory Model-Based Cluster Analysis of Clinical and Personal Patient Characteristics.

Background: Examining characteristics of patients with atrial fibrillation (AF) has the potential to help in identifying groups of patients who might benefit from different management approaches. Methods: Secondary analysis of online survey data was combined with clinic referral data abstraction from 196 patients with AF attending an AF specialty clinic. Cluster analyses were performed to identify distinct, homogeneous clusters of AF patients defined by 11 relevant variables: CHA2DS2-VASc score, age, AF symptoms, overall health, mental health, AF knowledge, perceived stress, household and recreation activity, overall AF quality of life, and AF symptom treatment satisfaction. Follow-up analyses examined differences between the cluster groups in additional clinical variables. Results: Evidence emerged for both 2- and 4-cluster solutions. The 2-cluster solution involved a contrast between patients who were doing well on all variables (n = 129; 66%) vs those doing less well (n = 67; 34%). The 4-cluster solution provided a closer-up view of the data, showing that the group doing less well was split into 3 meaningfully different subgroups of patients who were managing in different ways. The final 4 clusters produced were as follows: (i) doing well; (ii) stressed and discontented; (iii) struggling and dissatisfied; and (iv) satisfied and complacent. Conclusions: Patients with AF can be accurately classified into distinct, natural groupings that vary in clinically important ways. Among the patients who were not managing well with AF, we found 3 distinct subgroups of patients who may benefit from tailored approaches to AF management and support. The tailoring of treatment approaches to specific personal and/or behavioural patterns, alongside clinical patterns, holds potential to improve patient outcomes (eg, treatment satisfaction).

Contexte: L'examen des caractéristiques des patients atteints de fibrillation auriculaire (FA) pourrait permettre de mieux cerner les groupes qui pourraient bénéficier de différentes approches de prise en charge. Méthodologie: Nous avons combiné une analyse secondaire de données issues d'un sondage en ligne et les données issues de l'orientation clinique de 196 patients atteints de FA d'une clinique spécialisée en FA. Des analyses par grappes ont été réalisées pour cerner des groupes homogènes et distincts de patients atteints de FA, définis grâce à 11 variables pertinentes : score CHA2DS2-VASc, âge, symptômes de FA, état de santé général, état de santé mentale, niveau de connaissances sur la FA, niveau de stress perçu, activités récréatives et domestiques, qualité de vie générale avec la FA, et satisfaction concernant le traitement des symptômes de FA. Des ana-lyses ultérieures ont permis de se pencher sur les différences entre les groupes pour d'autres variables cliniques. Résultats: Deux solutions de regroupement des patients sont apparues possibles à l'analyse : en 2 groupes ou en 4 groupes. Le regroupement en 2 groupes mettait en relief le contraste entre les patients qui avaient des résultats favorables pour tous les paramètres (n = 129; 66 %) et ceux qui avaient des résultats moins favorables (n = 67; 34 %). Le regroupement en 4 groupes permettait d'observer les données plus en détail, et démontrait que le groupe avec des résultats moins favorables se subdivisait en 3 sous-groupes avec des distinctions pertinentes, qui vivaient leur maladie de façon différente. Les 4 groupes finaux étaient les suivants : (i) patients avec une expérience positive; (ii) patients vivant du stress et du mécontentement; (iii) patients vivant des difficultés et une insatisfaction; et (iv) patients vivant une satisfaction complaisante. Conclusions: Les patients atteints de FA peuvent être classés avec exactitude dans des groupes naturels distincts dont les différences sont d'intérêt clinique. Parmi les patients chez qui la prise en charge de la FA n'est pas optimale, il existe 3 sous-groupes différents qui pourraient tirer profit d'une approche de soutien et de prise en charge adaptée à leur profil. La personnalisation des approches thérapeutiques selon le type de comportements et de traits de personnalité, en plus du tableau clinique, pourrait permettre d'améliorer les résultats des patients (p. ex. la satisfaction par rapport au traitement).

Quality of life among patients with atrial fibrillation: A theoretically-guided cross-sectional study.

BACKGROUND: Patients with atrial fibrillation (AF) have significantly lower health-related quality of life (HRQoL) compared to the general population and patients with other heart diseases. The research emphasis on the influence of AF symptoms on HRQoL overshadows the role of individual characteristics. To address this gap, this study's purpose was to test an incremental predictive model for AF-related HRQoL following an adapted HRQoL conceptual model that incorporates both symptoms and individual characteristics. METHODS: Patients attending an AF specialty clinic were invited to complete an online survey. Hierarchical regression analyses were conducted to examine whether individual characteristics (overall mental health, perceived stress, sex, age, AF knowledge, household and recreational physical activity) incremented prediction of HRQoL and AF treatment satisfaction beyond AF symptom recency and overall health. RESULTS: Of 196 participants (mean age 65.3 years), 63% were male and 90% were Caucasian. Most reported 'excellent' or 'good' overall and mental health, had high overall AF knowledge scores, had low perceived stress scores, and had high household and recreation physical activity. The mean overall AF Effect On Quality-Of-Life Questionnaire (AFEQT) and AF treatment satisfaction scores were 70.62 and 73.84, respectively. Recency of AF symptoms and overall health accounted for 29.6% of the variance in overall HRQoL and 20.2% of the variance in AF treatment satisfaction. Individual characteristics explained an additional 13.6% of the variance in overall HRQoL and 7.6% of the variance in AF treatment satisfaction. Perceived stress and household physical activity were the largest contributors to overall HRQoL, whereas age and AF knowledge made significant contributions to AF treatment satisfaction. CONCLUSIONS: Along with AF symptoms and overall health, individual characteristics are important predictors of HRQoL and AF treatment satisfaction in AF patients. In particular, perceived stress and household physical activity could further be targeted as potential areas to improve HRQoL.

Usability and Feasibility Testing of an Atrial Fibrillation Educational Website with Patients Referred to an Atrial Fibrillation Specialty Clinic.

BACKGROUND: The purpose of this study was to design, usability test, and explore the feasibility of a web-based educational platform/intervention for patients with atrial fibrillation (AF) as part of their virtual AF care. METHODS: Participants were patients attending a specialized AF clinic. The multiple mixed-methods design included website design, think-aloud usability test, 1-month unstructured pre-testing analysis using Google Analytics, follow-up interviews, and a non-randomized one-group feasibility test using pre/post online surveys and Google Analytics. RESULTS: Usability testing participants (n = 2) guided adjustments for improving navigation. Pre-testing participants' (n = 9) website activity averaged four sessions (SD = 2.6) at 10 (SD 8) minutes per session during a 1-month study period. In the feasibility test, 30 patients referred to AF specialty clinic care completed the baseline survey, and 20 of these completed the 6-month follow-up survey. A total of 19 patients accessed the website over the 6 months, and all 30 participants were sent email prompts containing information from the website. Health-related quality of life, treatment satisfaction, household activity, and AF knowledge scores were higher at follow-up than baseline. There was an overall downward trend in self-reported healthcare utilization at follow-up. CONCLUSIONS: Access to a credible education website for patients with AF has great potential to complement virtual and hybrid models of care.

Telehealth Satisfaction in Patients Receiving Virtual Atrial Fibrillation Care: Quantitative Exploratory Study.

BACKGROUND: Telehealth can optimize access to specialty care for patients with atrial fibrillation (AF). Virtual AF care, however, may not fit with the complex needs of patients with AF. OBJECTIVE: This study aims to explore the correlation among attitudes toward health care technologies, self-efficacy, and telehealth satisfaction as part of the future planning of virtual AF clinic care. METHODS: Patients with AF older than 18 years from an urban-based, highly specialized AF clinic who had an upcoming telehealth visit were invited to participate in a web-based survey. The survey asked about demographic characteristics; use of technology; general, computer, and health care technology self-efficacy (HTSE) and health care technology attitudes, using a validated 30-item tool; and telehealth satisfaction questionnaire using a validated 14-item questionnaire. Data were analyzed with descriptive statistics, correlational analyses, and linear regression modeling. RESULTS: Participants (n=195 of 579 invited, for a 34% response rate) were primarily older, male, and White, had postsecondary schooling or more, and had high self-reported overall and mental health ratings. A variety of technologies were used in their daily lives and for health care, with the majority of technologies comprising desktop and laptop computers, smartphones, and tablets. Self-efficacy and telehealth satisfaction questionnaire scores were high overall, with male participants having higher general self-efficacy, computer self-efficacy, HTSE, and technology attitude scores. After controlling for age and sex, only HTSE was significantly related to individuals' attitudes toward health care technology. Both general self-efficacy and attitude toward health care technology were positively related to telehealth satisfaction. CONCLUSIONS: Consistent with a previous study, only HTSE significantly influenced attitudes toward health care technology. This finding confirms that, in this regard, self-efficacy is not a general perception but is domain specific. Considering participants' predominant use of the telephone for virtual care, it follows that general self-efficacy and attitude toward health care technology were significant contributors to telehealth satisfaction. Given our patients' frequent use of technology and high computer self-efficacy and HTSE scores, the use of video for telehealth appointments could be supported.

Myelodysplastic neoplasm-associated U2AF1 mutations induce host defense defects by compromising neutrophil chemotaxis.

Myelodysplastic neoplasm (MDS) is a hematopoietic stem cell disorder that may evolve into acute myeloid leukemia. Fatal infection is among the most common cause of death in MDS patients, likely due to myeloid cell cytopenia and dysfunction in these patients. Mutations in genes that encode components of the spliceosome represent the most common class of somatically acquired mutations in MDS patients. To determine the molecular underpinnings of the host defense defects in MDS patients, we investigated the MDS-associated spliceosome mutation U2AF1-S34F using a transgenic mouse model that expresses this mutant gene. We found that U2AF1-S34F causes a profound host defense defect in these mice, likely by inducing a significant neutrophil chemotaxis defect. Studies in human neutrophils suggest that this effect of U2AF1-S34F likely extends to MDS patients as well. RNA-seq analysis suggests that the expression of multiple genes that mediate cell migration are affected by this spliceosome mutation and therefore are likely drivers of this neutrophil dysfunction.

Standardization of flow cytometry and cell sorting to enable a transcriptomic analysis in a multi-site sarcoidosis study.

The contribution and regulation of various CD4+ T cell lineages that occur with remitting vs progressive courses in sarcoidosis are poorly understood. We developed a multiparameter flow cytometry panel to sort these CD4+ T cell lineages followed by measurement of their functional potential using RNA-sequencing analysis at six-month intervals across multiple study sites. To obtain good quality RNA for sequencing, we relied on chemokine receptor expression to identify and sort lineages. To minimize gene expression changes induced by perturbations of T cells and avoid protein denaturation caused by freeze/thaw cycles, we optimized our protocols using freshly isolated samples at each study site. To accomplish this study, we had to overcome significant standardization challenges across multiple sites. Here, we detail standardization considerations for cell processing, flow staining, data acquisition, sorting parameters, and RNA quality control analysis that were performed as part of the NIH-sponsored, multi-center study, BRonchoscopy at Initial sarcoidosis diagnosis Targeting longitudinal Endpoints (BRITE). After several rounds of iterative optimization, we identified the following aspects as critical for successful standardization: 1) alignment of PMT voltages across sites using CS&T/rainbow bead technology; 2) a single template created in the cytometer program that was used by all sites to gate cell populations during data acquisition and cell sorting; 3) use of standardized lyophilized flow cytometry staining cocktails to reduce technical error during processing; 4) development and implementation of a standardized Manual of Procedures. After standardization of cell sorting, we were able to determine the minimum number of sorted cells necessary for next generation sequencing through analysis of RNA quality and quantity from sorted T cell populations. Overall, we found that implementing a multi-parameter cell sorting with RNA-seq analysis clinical study across multiple study sites requires iteratively tested standardized procedures to ensure comparable and high-quality results.

Low TCR Binding Strength Results in Increased Progenitor-like CD8+ Tumor-Infiltrating Lymphocytes.

T-cell receptor (TCR) binding strength to peptide-MHC antigen complex influences numerous T-cell functions. However, the vast diversity of a polyclonal T-cell repertoire for even a single antigen greatly increases the complexity of studying the impact of TCR affinity on T-cell function. Here, we determined how TCR binding strength affected the protein and transcriptional profile of an endogenous, polyclonal T-cell response to a known tumor-associated antigen (TAA) within the tumor microenvironment (TME). We confirmed that the staining intensity by flow cytometry and the counts by sequencing from MHC-tetramer labeling were reliable surrogates for the TCR-peptide-MHC steady-state binding affinity. We further demonstrated by single-cell RNA sequencing that tumor-infiltrating lymphocytes (TIL) with high and low binding affinity for a TAA can differentiate into cells with many antigen-specific transcriptional profiles within an established TME. However, more progenitor-like phenotypes were significantly biased towards lower affinity T cells, and proliferating phenotypes showed significant bias towards high-affinity TILs. In addition, we found that higher affinity T cells advanced more rapidly to terminal phases of T-cell exhaustion and exhibited better tumor control. We confirmed the polyclonal TIL results using a TCR transgenic mouse possessing a single low-affinity TCR targeting the same TAA. These T cells maintained a progenitor-exhausted phenotype and exhibited impaired tumor control. We propose that high-affinity TCR interactions drive T-cell fate decisions more rapidly than low-affinity interactions and that these cells differentiate faster. These findings illustrate divergent forms of T-cell dysfunction based on TCR affinity which may impact TIL therapies and antitumor responses.

Dimensionality, Item Response Theory, Effect Size Attenuation, and Test Bias Analyses of the Self-Importance of Moral Identity Scale (SIMIS).

The extent to which morality and being a moral person are important to one's identity is most commonly assessed using Aquino and Reed's (2002) Self-Importance of Moral Identity Scale (SIMIS). This study provided detailed psychometric examinations of the structure and discrimination levels of the SIMIS in a large (N = 2108) and heterogeneous sample. Results indicated that the SIMIS is clearly 2-dimensional, as expected. The Internalization and Symbolization subscales provided sufficient, and sometimes high levels of test information across the latent trait continuums. There were no redundant items and no bias based on gender. The most notable, albeit minor, shortcomings were that there are too many response options and that test information (discrimination power) was diminished at high levels of the Internalization latent trait continuum, apparently due to skewness. The fluctuating levels of measurement precision resulted in slightly greater attenuations in effect sizes for Internalization than for Symbolization across data for 31 other measures. The present findings from a large dataset and a variety of modern, revealing statistical methods provided relatively consistent, favorable findings for the measure.

Bigger isn't always better: an exploration of social perception bias against high levels of muscularity in women.

Bodybuilding is often considered more appropriate for men than for women. Previous research has shown that knowledge of a target's involvement in bodybuilding influences interpersonal judgments. The present study examined whether this is also the case for women with competitive-type bodybuilder physiques. Participants (N = 263) were shown photographs of women who competed professionally in different categories of bodybuilding (i.e., bikini, figure, and bodybuilding/physique). Participants were then asked to make a series of judgments on the basis of the photos alone. It was found that the degree of muscularity of the targets shaped participants' estimates of perceived outcomes and of the targets' traits in non-bodybuilding domains (e.g., expected positive life outcomes, gender role traits, and sexual orientation). Both men and women apparently considered moderate to high levels of muscularity in women targets as violations of gender norms.

Prognostic accuracy of a peripheral blood transcriptome signature in chronic hypersensitivity pneumonitis.

The prognostic value of peripheral blood mononuclear cell (PBMC) expression profiles, when used in patients with chronic hypersensitivity pneumonitis (CHP), as an adjunct to traditional clinical assessment is unknown. RNA-seq analysis on PBMC from 37 patients with CHP at initial presentation determined that (1) 74 differentially expressed transcripts at a 10% false discovery rate distinguished those with (n=10) and without (n=27) disease progression, defined as absolute FVC and/or diffusing capacity of the lungs for carbon monoxide (DLCO) decline of ≥10% and increased fibrosis on chest CT images within 24 months, and (2) classification models based on gene expression and clinical factors strongly outperform models based solely on clinical factors (baseline FVC%, DLCO% and chest CT fibrosis).

Chromodomain helicase DNA-binding 4 (CHD4) regulates early B cell identity and V(D)J recombination.

B lymphocytes develop from uncommitted precursors into immunoglobulin (antibody)-producing B cells, a major arm of adaptive immunity. Progression of early progenitors to antibody-expressing cells in the bone marrow is orchestrated by the temporal regulation of different gene programs at discrete developmental stages. A major question concerns how B cells control the accessibility of these genes to transcription factors. Research has implicated nucleosome remodeling ATPases as mediators of chromatin accessibility. Here, we describe studies of chromodomain helicase DNA-binding 4 (CHD4; also known as Mi-2ß) in early B cell development. CHD4 comprises multiple domains that function in nucleosome mobilization and histone binding. CHD4 is a key component of Nucleosome Remodeling and Deacetylase, or NuRD (Mi-2) complexes, which assemble with other proteins that mediate transcriptional repression. We review data demonstrating that CHD4 is necessary for B lineage identity: early B lineage progression, proliferation in response to interleukin-7, responses to DNA damage, and cell survival in vivo. CHD4-NuRD is also required for the Ig heavy-chain repertoire by promoting utilization of distal variable (VH ) gene segments in V(D)J recombination. In conclusion, the regulation of chromatin accessibility by CHD4 is essential for production of antibodies by B cells, which in turn mediate humoral immune responses to pathogens and disease.

Mapping Macrophage Polarization and Origin during the Progression of the Foreign Body Response to a Poly(ethylene glycol) Hydrogel Implant.

Poly(ethylene glycol) (PEG) hydrogels hold promise for in vivo applications but induce a foreign body response (FBR). While macrophages are key in the FBR, many questions remain. This study investigates temporal changes in the transcriptome of implant-associated monocytes and macrophages. Proinflammatory pathways are upregulated in monocytes compared to control monocytes but subside by day 28. Macrophages are initially proinflammatory but shift to a profibrotic state by day 14, coinciding with fibrous capsule emergence. Next, this study assesses the origin of macrophages responsible for fibrous encapsulation using wildtype, C-C Motif Chemokine Receptor 2 (CCR2)-/- mice that lack recruited macrophages, and Macrophage Fas-Induced Apoptosis (MaFIA) mice that enable macrophage ablation. Subpopulations of recruited and tissue-resident macrophages are identified. Fibrous encapsulation proceeds in CCR2-/- mice similar to wildtype mice. However, studies in MaFIA mice indicate that macrophages are necessary for fibrous capsule formation. These findings suggest that macrophage origin impacts the FBR progression and provides evidence that tissue-resident macrophages and not the recruited macrophages may drive fibrosis in the FBR to PEG hydrogels. This study demonstrates that implant-associated monocytes and macrophages have temporally distinct transcriptomes in the FBR and that profibrotic pathways associated with macrophages may be enriched in tissue-resident macrophages.

Defining CD4 T helper and T regulatory cell endotypes of progressive and remitting pulmonary sarcoidosis (BRITE): protocol for a US-based, multicentre, longitudinal observational bronchoscopy study.

INTRODUCTION: Sarcoidosis is a multiorgan granulomatous disorder thought to be triggered and influenced by gene-environment interactions. Sarcoidosis affects 45-300/100 000 individuals in the USA and has an increasing mortality rate. The greatest gap in knowledge about sarcoidosis pathobiology is a lack of understanding about the underlying immunological mechanisms driving progressive pulmonary disease. The objective of this study is to define the lung-specific and blood-specific longitudinal changes in the adaptive immune response and their relationship to progressive and non-progressive pulmonary outcomes in patients with recently diagnosed sarcoidosis. METHODS AND ANALYSIS: The BRonchoscopy at Initial sarcoidosis diagnosis Targeting longitudinal Endpoints study is a US-based, NIH-sponsored longitudinal blood and bronchoscopy study. Enrolment will occur over four centres with a target sample size of 80 eligible participants within 18 months of tissue diagnosis. Participants will undergo six study visits over 18 months. In addition to serial measurement of lung function, symptom surveys and chest X-rays, participants will undergo collection of blood and two bronchoscopies with bronchoalveolar lavage separated by 6 months. Freshly processed samples will be stained and flow-sorted for isolation of CD4 +T helper (Th1, Th17.0 and Th17.1) and T regulatory cell immune populations, followed by next-generation RNA sequencing. We will construct bioinformatic tools using this gene expression to define sarcoidosis endotypes that associate with progressive and non-progressive pulmonary disease outcomes and validate the tools using an independent cohort. ETHICS AND DISSEMINATION: The study protocol has been approved by the Institutional Review Boards at National Jewish Hospital (IRB# HS-3118), University of Iowa (IRB# 201801750), Johns Hopkins University (IRB# 00149513) and University of California, San Francisco (IRB# 17-23432). All participants will be required to provide written informed consent. Findings will be disseminated via journal publications, scientific conferences, patient advocacy group online content and social media platforms.

Single cell analysis of host response to helminth infection reveals the clonal breadth, heterogeneity, and tissue-specific programming of the responding CD4+ T cell repertoire.

The CD4+ T cell response is critical to host protection against helminth infection. How this response varies across different hosts and tissues remains an important gap in our understanding. Using IL-4-reporter mice to identify responding CD4+ T cells to Nippostrongylus brasiliensis infection, T cell receptor sequencing paired with novel clustering algorithms revealed a broadly reactive and clonally diverse CD4+ T cell response. While the most prevalent clones and clonotypes exhibited some tissue selectivity, most were observed to reside in both the lung and lung-draining lymph nodes. Antigen-reactivity of the broader repertoires was predicted to be shared across both tissues and individual mice. Transcriptome, trajectory, and chromatin accessibility analysis of lung and lymph-node repertoires revealed three unique but related populations of responding IL-4+ CD4+ T cells consistent with T follicular helper, T helper 2, and a transitional population sharing similarity with both populations. The shared antigen reactivity of lymph node and lung repertoires combined with the adoption of tissue-specific gene programs allows for the pairing of cellular and humoral responses critical to the orchestration of anti-helminth immunity.

The molecular and epigenetic mechanisms of innate lymphoid cell (ILC) memory and its relevance for asthma.

Repetitive exposure of Rag1-/- mice to the Alternaria allergen extract generated a form of memory that elicited an asthma-like response upon a subthreshold recall challenge 3-15 wk later. This memory was associated with lung ICOS+ST2+ ILC2s. Genetic, pharmacologic, and antibody-mediated inhibition and adoptive transfer established an essential role for ILC2s in memory-driven asthma. ATAC-seq demonstrated a distinct epigenetic landscape of memory ILC2s and identified Bach2 and AP1 (JunD and Fosl2) motifs as major drivers of altered gene accessibility. scRNA-seq, gene knockout, and signaling studies suggest that repetitive allergenic stress induces a gene repression program involving Nr4a2, Zeb1, Bach2, and JunD and a preparedness program involving Fhl2, FosB, Stat6, Srebf2, and MPP7 in memory ILC2s. A mutually regulated balance between these two programs establishes and maintains memory. The preparedness program (e.g., Fhl2) can be activated with a subthreshold cognate stimulation, which down-regulates repressors and activates effector pathways to elicit the memory-driven phenotype.

GATA2 regulates mast cell identity and responsiveness to antigenic stimulation by promoting chromatin remodeling at super-enhancers.

Mast cells are critical effectors of allergic inflammation and protection against parasitic infections. We previously demonstrated that transcription factors GATA2 and MITF are the mast cell lineage-determining factors. However, it is unclear whether these lineage-determining factors regulate chromatin accessibility at mast cell enhancer regions. In this study, we demonstrate that GATA2 promotes chromatin accessibility at the super-enhancers of mast cell identity genes and primes both typical and super-enhancers at genes that respond to antigenic stimulation. We find that the number and densities of GATA2- but not MITF-bound sites at the super-enhancers are several folds higher than that at the typical enhancers. Our studies reveal that GATA2 promotes robust gene transcription to maintain mast cell identity and respond to antigenic stimulation by binding to super-enhancer regions with dense GATA2 binding sites available at key mast cell genes.

MDS-associated SF3B1 mutations enhance proinflammatory gene expression in patient blast cells.

Two factors known to contribute to the development of myelodysplastic syndrome (MDS) and other blood cancers are (i) somatically acquired mutations in components of the spliceosome and (ii) increased inflammation. Spliceosome genes, including SF3B1, are mutated at high frequency in MDS and other blood cancers; these mutations are thought to be neomorphic or gain-of-function mutations that drive disease pathogenesis. Likewise, increased inflammation is thought to contribute to MDS pathogenesis; inflammatory cytokines are strongly elevated in these patients, with higher levels correlating with worsened patient outcome. In the current study, we used RNAseq to analyze pre-mRNA splicing and gene expression changes present in blast cells isolated from MDS patients with or without SF3B1 mutations. We determined that SF3B1 mutations lead to enhanced proinflammatory gene expression in these cells. Thus, these studies suggest that SF3B1 mutations could contribute to MDS pathogenesis by enhancing the proinflammatory milieu in these patients.

BATF acts as an essential regulator of IL-25-responsive migratory ILC2 cell fate and function.

A transitory, interleukin-25 (IL-25)-responsive, group 2 innate lymphoid cell (ILC2) subset induced during type 2 inflammation was recently identified as iILC2s. This study focuses on understanding the significance of this population in relation to tissue-resident nILC2s in the lung and intestine. RNA-sequencing and pathway analysis revealed the AP-1 superfamily transcription factor BATF (basic leucine zipper transcription factor, activating transcription factor-like) as a potential modulator of ILC2 cell fate. Infection of BATF-deficient mice with Nippostrongylus brasiliensis showed a selective defect in IL-25-mediated helminth clearance and a corresponding loss of iILC2s in the lung characterized as IL-17RBhigh, KLRG1high, BATFhigh, and Arg1low BATF deficiency selectively impaired iILC2s because it had no impact on tissue-resident nILC2 frequency or function. Pulmonary-associated iILC2s migrated to the lung after infection, where they represented an early source of IL-4 and IL-13. Although the composition of ILC2s in the small intestine was distinct from those in the lung, their frequency and IL-13 expression remained dependent on BATF, which was also required for optimal goblet and tuft cell hyperplasia. Findings support IL-25-responsive ILC2s as early sentinels of mucosal barrier integrity.

CHD4 is essential for transcriptional repression and lineage progression in B lymphopoiesis.

Cell lineage specification is a tightly regulated process that is dependent on appropriate expression of lineage and developmental stage-specific transcriptional programs. Here, we show that Chromodomain Helicase DNA-binding protein 4 (CHD4), a major ATPase/helicase subunit of Nucleosome Remodeling and Deacetylase Complexes (NuRD) in lymphocytes, is essential for specification of the early B cell lineage transcriptional program. In the absence of CHD4 in B cell progenitors in vivo, development of these cells is arrested at an early pro-B-like stage that is unresponsive to IL-7 receptor signaling and unable to efficiently complete V(D)J rearrangements at Igh loci. Our studies confirm that chromatin accessibility and transcription of thousands of gene loci are controlled dynamically by CHD4 during early B cell development. Strikingly, CHD4-deficient pro-B cells express transcripts of many non-B cell lineage genes, including genes that are characteristic of other hematopoietic lineages, neuronal cells, and the CNS, lung, pancreas, and other cell types. We conclude that CHD4 inhibits inappropriate transcription in pro-B cells. Together, our data demonstrate the importance of CHD4 in establishing and maintaining an appropriate transcriptome in early B lymphopoiesis via chromatin accessibility.