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Although the importance of genome organization for transcriptional regulation of cell-fate decisions and function is clear, the changes in chromatin architecture and how these impact effector and memory CD8+ T cell differentiation remain unknown. Using Hi-C, we studied how genome configuration is integrated with CD8+ T cell differentiation during infection and investigated the role of CTCF, a key chromatin remodeler, in modulating CD8+ T cell fates through CTCF knockdown approaches and perturbation of specific CTCF-binding sites. We observed subset-specific changes in chromatin organization and CTCF binding and revealed that weak-affinity CTCF binding promotes terminal differentiation of CD8+ T cells through the regulation of transcriptional programs. Further, patients with de novo CTCF mutations had reduced expression of the terminal-effector genes in peripheral blood lymphocytes. Therefore, in addition to establishing genome architecture, CTCF regulates effector CD8+ T cell heterogeneity through altering interactions that regulate the transcription factor landscape and transcriptome. [Display omitted] • Genome organization changes with effector CD8+ T cell differentiation • CTCF-binding patterns are altered with effector CD8+ T cell differentiation • CTCF regulates subset-specific transcriptional programs and chromatin accessibility • CTCF controls optimal CD8+ T cell-fate decisions in response to infection How changes in spatial chromatin organization are integrated into the network of molecular mechanisms mediating CD8+ T cell effector functions and memory formation is not well understood. Quon et al. characterize genome interactions accompanying the CD8+ T cell response to infection and find that the DNA architectural protein, CTCF, regulates the balance of terminally differentiated-effector cells and memory-fated cells. [ABSTRACT FROM AUTHOR]

Cells communicate with each other via receptor-ligand interactions. Here, we describe lentiviral-mediated cell entry by engineered receptor-ligand interaction (ENTER) to display ligand proteins, deliver payloads, and record receptor specificity. We optimize ENTER to decode interactions between T cell receptor (TCR)-MHC peptides, antibody-antigen, and other receptor-ligand pairs. A viral presentation strategy allows ENTER to capture interactions between B cell receptor and any antigen. We engineer ENTER to deliver genetic payloads to antigen-specific T or B cells to selectively modulate cellular behavior in mixed populations. Single-cell readout of ENTER by RNA sequencing (ENTER-seq) enables multiplexed enumeration of antigen specificities, TCR clonality, cell type, and states of individual T cells. ENTER-seq of CMV-seropositive patient blood samples reveals the viral epitopes that drive effector memory T cell differentiation and inter-clonal vs. intra-clonal phenotypic diversity targeting the same epitope. ENTER technology enables systematic discovery of receptor specificity, linkage to cell fates, and antigen-specific cargo delivery. [Display omitted] • ENTER displays ligands, delivers payloads, and records receptor specificity • ENTER enables antigen-specific expansion or depletion of T or B cells • ENTER-seq maps TCR specificity, clonality, and cell state in single cells • ENTER-seq of patient sample decodes antiviral T cell memory A modular, lentiviral-based display and delivery platform enables decoding of ligand-receptor interactions, receptor-specific genetic cargo delivery, and mapping of receptor specificity and cell states of single cells. [ABSTRACT FROM AUTHOR]

The long non-coding RNA (lncRNA) XIST establishes X chromosome inactivation (XCI) in female cells in early development and thereafter is thought to be largely dispensable. Here, we show XIST is continually required in adult human B cells to silence a subset of X-linked immune genes such as TLR7. XIST-dependent genes lack promoter DNA methylation and require continual XIST-dependent histone deacetylation. XIST RNA-directed proteomics and CRISPRi screen reveal distinctive somatic cell-type-specific XIST complexes and identify TRIM28 that mediates Pol II pausing at promoters of X-linked genes in B cells. Single-cell transcriptome data of female patients with either systemic lupus erythematosus or COVID-19 infection revealed XIST dysregulation, reflected by escape of XIST-dependent genes, in CD11c+ atypical memory B cells (ABCs). XIST inactivation with TLR7 agonism suffices to promote isotype-switched ABCs. These results indicate cell-type-specific diversification and function for lncRNA-protein complexes and suggest expanded roles for XIST in sex-differences in biology and medicine. [Display omitted] • XIST prevents escape of X-linked genes with DNA hypomethylated promoters in B cells • XIST maintains X-inactivation through continuous deacetylation of H3K27ac • XIST ChIRP-MS and CRISPRi screen reveal a B cell-specific XIST cofactor TRIM28 • XIST loss and TLR7 stimulation promotes CD11c+ atypical B cell formation XIST restrains human atypical B cell development. A distinct XIST-associated protein complex contributes to XIST-mediated gene silencing in B cells. [ABSTRACT FROM AUTHOR]

Autoimmune diseases disproportionately affect females more than males. The XX sex chromosome complement is strongly associated with susceptibility to autoimmunity. Xist long non-coding RNA (lncRNA) is expressed only in females to randomly inactivate one of the two X chromosomes to achieve gene dosage compensation. Here, we show that the Xist ribonucleoprotein (RNP) complex comprising numerous autoantigenic components is an important driver of sex-biased autoimmunity. Inducible transgenic expression of a non-silencing form of Xist in male mice introduced Xist RNP complexes and sufficed to produce autoantibodies. Male SJL/J mice expressing transgenic Xist developed more severe multi-organ pathology in a pristane-induced lupus model than wild-type males. Xist expression in males reprogrammed T and B cell populations and chromatin states to more resemble wild-type females. Human patients with autoimmune diseases displayed significant autoantibodies to multiple components of XIST RNP. Thus, a sex-specific lncRNA scaffolds ubiquitous RNP components to drive sex-biased immunity. [Display omitted] • Transgenic mouse models inducibly express Xist in male animals • Xist expression in males induces autoantibodies and autoimmune pathology • Xist in males reprograms T and B cell populations to female-like patterns • Autoantibodies to Xist RNP characterize female-biased autoimmune diseases in patients The Xist RNA protein complex, present only in females, is immunogenic and may underlie female-biased autoimmunity. [ABSTRACT FROM AUTHOR]

CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the targeted chromosome, including in preclinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells (NCT03399448), reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic. [Display omitted] • Cas9 genome editing in T cells results in unintended but targeted chromosome loss • Chromosome loss from T cell genome editing is generalizable across target sites • Cas9-induced chromosome loss persists for weeks in cultured T cells • A modified protocol mitigates chromosome loss in T cells used for a clinical trial CRISPR-Cas9 genome editing induces persistent chromosome loss in human primary T cells and engineered CAR T cells for clinical use, a phenomenon that can be mitigated by modifying T cell preparation protocols prior to clinical application. [ABSTRACT FROM AUTHOR]

Although viral infections elicit robust interferon-γ (IFN-γ) and long-lived antibody-secreting cell (ASC) responses, the roles for IFN-γ and IFN-γ-induced transcription factors (TFs) in ASC development are unclear. We showed that B cell intrinsic expression of IFN-γR and the IFN-γ-induced TF T-bet were required for T-helper 1 cell-induced differentiation of B cells into ASCs. IFN-γR signaling induced Blimp1 expression in B cells but also initiated an inflammatory gene program that, if not restrained, prevented ASC formation. T-bet did not affect Blimp1 upregulation in IFN-γ-activated B cells but instead regulated chromatin accessibility within the Ifng and Ifngr2 loci and repressed the IFN-γ-induced inflammatory gene program. Consistent with this, B cell intrinsic T-bet was required for formation of long-lived ASCs and secondary ASCs following viral, but not nematode, infection. Therefore, T-bet facilitates differentiation of IFN-γ-activated inflammatory effector B cells into ASCs in the setting of IFN-γ-, but not IL-4-, induced inflammatory responses. • Differentiation of Th1-activated B (Be1) cells requires B intrinsic T-bet and IFN-γR • Be1 differentiation requires T-bet repression of NF-κB, TLR, and STAT pathways • Plasma cell formation in Be2 cultures and nematode infection is T-bet independent • T-bet controls formation of long-lived and secondary plasma cells in flu infection T-bet-expressing B cells are expanded in autoimmunity and viral infection; however, whether these cells represent early plasma cells was unknown. Stone et al. identify T-bet-repressed pathways that permit plasma cell differentiation in the presence of IFN-γ. They show that T-bet-expressing B cells are required for long-lived immunity to flu. [ABSTRACT FROM AUTHOR]

Summary A complete chart of cis -regulatory elements and their dynamic activity is necessary to understand the transcriptional basis of differentiation and function of an organ system. We generated matched epigenome and transcriptome measurements in 86 primary cell types that span the mouse immune system and its differentiation cascades. This breadth of data enable variance components analysis that suggests that genes fall into two distinct classes, controlled by either enhancer- or promoter-driven logic, and multiple regression that connects genes to the enhancers that regulate them. Relating transcription factor (TF) expression to the genome-wide accessibility of their binding motifs classifies them as predominantly openers or closers of local chromatin accessibility, pinpointing specific cis -regulatory elements where binding of given TFs is likely functionally relevant, validated by chromatin immunoprecipitation sequencing (ChIP-seq). Overall, this cis -regulatory atlas provides a trove of information on transcriptional regulation through immune differentiation and a foundational scaffold to define key regulatory events throughout the immunological genome. Graphical Abstract Highlights • Atlas of 512,595 cis -regulatory elements active in 86 immunologic cell types • Two classes of loci, controlled by either promoter- or enhancer-driven logic • Inference of enhancer elements that activate each gene across differentiation • Context-specificity of enhancer activation by transcription factors A cis -regulatory map of the mouse immune system illuminates gene expression patterns and regulatory logic across 86 primary cell types and pairs immune transcription factors with cell-type-specific regulatory elements. [ABSTRACT FROM AUTHOR]

Transforming growth factor β (TGF-β) represents a well-established signal required for tissue-resident memory T cell (T RM ) formation at intestinal surfaces, regulating the expression of a large collection of genes coordinately promoting intestinal T RM differentiation. The functional contribution from each TGF-β-controlled transcription factor is not entirely known. Here, we find that TGF-β-induced T-bet downregulation and Hic1 induction represent two critical events during intestinal T RM differentiation. Importantly, T-bet deficiency significantly rescues intestinal T RM formation in the absence of the TGF-β receptor. Hic1 induction further strengthens T RM maturation in the absence of TGF-β and T-bet. Our results reveal that provision of certain TGF-β-induced molecular events can partially replace TGF-β signaling to promote the establishment of intestinal T RM s, which allows the functional dissection of TGF-β-induced transcriptional targets and molecular mechanisms for T RM differentiation., Competing Interests: Declaration of interests A.W.G. is a cofounder of TCura Bioscience, Inc. and serves on the scientific advisory boards of ArsenalBio and Foundery Innovations., (Published by Elsevier Inc.)

The differentiation of naive CD8 + T lymphocytes into cytotoxic effector and memory CTL results in large-scale changes in transcriptional and phenotypic profiles. Little is known about how large-scale changes in genome organization underpin these transcriptional programs. We use Hi-C to map changes in the spatial organization of long-range genome contacts within naive, effector, and memory virus-specific CD8 + T cells. We observe that the architecture of the naive CD8 + T cell genome is distinct from effector and memory genome configurations, with extensive changes within discrete functional chromatin domains associated with effector/memory differentiation. Deletion of BACH2, or to a lesser extent, reducing SATB1 DNA binding, within naive CD8 + T cells results in a chromatin architecture more reminiscent of effector/memory states. This suggests that key transcription factors within naive CD8 + T cells act to restrain T cell differentiation by actively enforcing a unique naive chromatin state., Competing Interests: Declaration of interests A.W.G. is a member of the scientific advisory board of ArsenalBio. No funding from ArsenalBio was provided for this work., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

T cell receptor (TCR) stimulation of naive CD8 + T cells initiates reprogramming of cis-regulatory landscapes that specify effector and memory cytotoxic T lymphocyte (CTL) differentiation. We mapped regions of hyper-accessible chromatin in naive cells during TCR stimulation and discovered that the transcription factor (TF) Runx3 promoted accessibility to memory CTL-specific cis-regulatory regions before the first cell division and was essential for memory CTL differentiation. Runx3 was specifically required for accessibility to regions highly enriched with IRF, bZIP and Prdm1-like TF motifs, upregulation of TFs Irf4 and Blimp1, and activation of fundamental CTL attributes in early effector and memory precursor cells. Runx3 ensured that nascent CTLs differentiated into memory CTLs by preventing high expression of the TF T-bet, slowing effector cell proliferation, and repressing terminal CTL differentiation. Runx3 overexpression enhanced memory CTL differentiation during iterative infections. Thus, Runx3 governs chromatin accessibility during TCR stimulation and enforces the memory CTL developmental program., (Copyright © 2018 Elsevier Inc. All rights reserved.)