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The introduction of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib had remarkable impact on the treatment of chronic lymphocytic leukemia (CLL), but responses are slow and rates of eventual relapse are high. To identify targetable vulnerabilities that may arise as the result of ibrutinib treatment, we investigated the chromatin accessibility landscape in patient CLL cells before and during therapy with ibrutinib. Differences in the accessibility of enhancers and promoters revealed ibrutinib-directed changes in cellular processes including signaling pathways and metabolic circuits. To determine how chromatin remodeling translates into drug sensitivity, we profiled a library of approved drugs and small molecules for their ability to specifically target the CLL clonal population using a next generation functional drug sensitivity assay on matched samples. Ibrutinib-dependent and independent ex vivo drug responses in individual patients and at the cohort level were observed. Integration of chromatin accessibility and differential chemosensitivity data revealed robust ibrutinib-induced signatures, which we exploited to prioritize drugs for synergistic treatment of CLL. Our study demonstrates a systematic and widely applicable approach to identify rationale drug combinations for clinical trial designs based on integrated profiling of epigenomes and cellular phenotypes.

Summary The role of intestinal eosinophils in immune homeostasis is enigmatic and the molecular signals that drive them from protective to tissue damaging are unknown. Most commonly associated with Th2 cell-mediated diseases, we describe a role for eosinophils as crucial effectors of the interleukin-23 (IL-23)-granulocyte macrophage colony-stimulating factor (GM-CSF) axis in colitis. Chronic intestinal inflammation was characterized by increased bone marrow eosinopoiesis and accumulation of activated intestinal eosinophils. IL-5 blockade or eosinophil depletion ameliorated colitis, implicating eosinophils in disease pathogenesis. GM-CSF was a potent activator of eosinophil effector functions and intestinal accumulation, and GM-CSF blockade inhibited chronic colitis. By contrast neutrophil accumulation was GM-CSF independent and dispensable for colitis. In addition to TNF secretion, release of eosinophil peroxidase promoted colitis identifying direct tissue-toxic mechanisms. Thus, eosinophils are key perpetrators of chronic inflammation and tissue damage in IL-23-mediated immune diseases and it suggests the GM-CSF-eosinophil axis as an attractive therapeutic target., Graphical Abstract, Highlights • GM-CSF synergizes with IL-5 to exacerbate eosinopoiesis during chronic colitis • GM-CSF-activated eosinophils promote IL-23 driven colitis • Depletion of eosinophils, but not of neutrophils, dampens colitis • GM-CSF increases eosinophil production of inflammatory cytokines TNF and IL-13, Although eosinophils are most commonly associated with Th2-cell-mediated diseases, Powrie and colleagues describe a crucial role for eosinophils in IL-23-mediated chronic colitis. GM-CSF was a potent activator of eosinopoiesis and concomitant accumulation of activated and tissue-toxic eosinophils in the inflamed intestine, as well as their secretion of inflammatory cytokines.

Non-lymphoid tissues (NLTs) harbor a pool of adaptive immune cells with largely unexplored phenotype and development. We used single-cell RNA-seq to characterize 35,000 CD4+ regulatory (Treg) and memory (Tmem) T cells in mouse skin and colon, their respective draining lymph nodes (LNs) and spleen. In these tissues, we identified Treg cell subpopulations with distinct degrees of NLT phenotype. Subpopulation pseudotime ordering and gene kinetics were consistent in recruitment to skin and colon, yet the initial NLT-priming in LNs and the final stages of NLT functional adaptation reflected tissue-specific differences. Predicted kinetics were recapitulated using an in vivo melanoma-induction model, validating key regulators and receptors. Finally, we profiled human blood and NLT Treg and Tmem cells, and identified cross-mammalian conserved tissue signatures. In summary, we describe the relationship between Treg cell heterogeneity and recruitment to NLTs through the combined use of computational prediction and in vivo validation.

Regulatory B cells (Bregs) play a critical role in the control of autoimmunity and inflammation. IL-10 production is the hallmark for the identification of Bregs. However, the molecular determinants that regulate the transcription of IL-10 and control the Breg developmental program remain unknown. Here, we demonstrate that aryl hydrocarbon receptor (AhR) regulates the differentiation and function of IL-10-producing CD19+CD21hiCD24hiBregs and limits their differentiation into B cells that contribute to inflammation. Chromatin profiling and transcriptome analyses show that loss of AhR in B cells reduces expression of IL-10 by skewing the differentiation of CD19+CD21hiCD24hiB cells into a pro-inflammatory program, under Breg-inducing conditions. B cell AhR-deficient mice develop exacerbated arthritis, show significant reductions in IL-10-producing Bregs and regulatory T cells, and show an increase in T helper (Th) 1 and Th17 cells compared with B cell AhR-sufficient mice. Thus, we identify AhR as a relevant contributor to the transcriptional regulation of Breg differentiation.

Chronic lymphocytic leukemia (CLL) is a genetically, epigenetically, and clinically heterogeneous disease. Despite this heterogeneity, the Bruton tyrosine kinase (BTK) inhibitor ibrutinib provides effective treatment for the vast majority of CLL patients. To define the underlining regulatory program, we analyzed high-resolution time courses of ibrutinib treatment in closely monitored patients, combining cellular phenotyping (flow cytometry), single-cell transcriptome profiling (scRNA-seq), and chromatin mapping (ATAC-seq). We identified a consistent regulatory program shared across all patients, which was further validated by an independent CLL cohort. In CLL cells, this program starts with a sharp decrease of NF-κB binding, followed by reduced regulatory activity of lineage-defining transcription factors (including PAX5 and IRF4) and erosion of CLL cell identity, finally leading to the acquisition of a quiescence-like gene signature which was shared across several immune cell types. Nevertheless, we observed patient-to-patient variation in the speed of its execution, which we exploited to predict patient-specific dynamics in the response to ibrutinib based on pre-treatment samples. In aggregate, our study describes the cellular, molec- ular, and regulatory effects of therapeutic B cell receptor inhibition in CLL at high temporal resolution, and it establishes a framework for epigenome/transcriptome-based treatment monitoring.