Transforming growth factor β (TGF-β) represents a well-established signal required for tissue-resident memory T cell (TRM) formation at intestinal surfaces, regulating the expression of a large collection of genes coordinately promoting intestinal TRM 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 TRM differentiation. Importantly, T-bet deficiency significantly rescues intestinal TRM formation in the absence of the TGF-β receptor. Hic1 induction further strengthens TRM 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 TRMs, which allows the functional dissection of TGF-β-induced transcriptional targets and molecular mechanisms for TRM differentiation.

Transforming growth factor (3 (TGF-(3) 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-(3-controlled transcription factor is not entirely known. Here, we find that TGF-(3-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-(3 receptor. Hic1 induction further strengthens T RM maturation in the absence of TGF-(3 and T-bet. Our results reveal that provision of certain TGF-(3-induced molecular events can partially replace TGF-(3 signaling to promote the establishment of intestinal T RM s, which allows the functional dissection of TGF-(3-induced transcriptional targets and molecular mechanisms for T RM differentiation.

The differentiation of naïve CD8+ cytotoxic T lymphocytes (CTLs) into effector and memory states results in large scale changes in transcriptional and phenotypic profiles. Little is known about how large-scale changes in genome organisation reflect or underpin these transcriptional programs. We utilised Hi-C to map changes in the spatial organisation of long-range genome contacts within naïve, effector and memory virus-specific CD8+ T cells. We observed that the architecture of the naive CD8+ T cell genome was distinct from effector and memory genome configurations with extensive changes within discrete functional chromatin domains. However, deletion of the BACH2 or SATB1 transcription factors was sufficient to remodel the naïve chromatin architecture and engage transcriptional programs characteristic of differentiated cells. This suggests that the chromatin architecture within naïve CD8+ T cells is preconfigured to undergo autonomous remodelling upon activation, with key transcription factors restraining differentiation by actively enforcing the unique naïve chromatin state.

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.

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.

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 chromosome, including in pre-clinical 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, 1 dramatically 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.

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 ge-netic 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 discov-ery of receptor specificity, linkage to cell fates, and antigen-specific cargo delivery.

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.

ABSTRACTCells communicate with each other via receptor-ligand interactions on the cell surface. Here we describe a technology for lentiviral-mediated cell entry byengineeredreceptor-ligand interaction (ENTER) to decode receptor specificity. Engineered lentiviral particles displaying specific ligands deliver fluorescent proteins into target cells upon cognate receptor-ligand interaction, without genome integration or transgene transcription. We optimize ENTER to decode interactions between T cell receptor (TCR)-MHC peptides, antibody-antigen, and other receptor-ligand pairs. We develop an effective presentation strategy to capture interactions between B cell receptor (BCR) and intracellular antigen epitopes. Single-cell readout of ENTER by RNA sequencing (ENTER-seq) enables multiplexed enumeration of TCR-antigen specificities, clonality, cell type, and cell states of individual T cells. ENTER-seq of patient blood samples after CMV infection reveals the viral epitopes that drive human effector memory T cell differentiation and inter-clonal phenotypic diversity that targets the same epitope. ENTER enables systematic discovery of receptor specificity, linkage to cell fates, and cell-specific delivery of gene or protein payloads.HIGHLIGHTSENTER displays ligands, deliver cargos, and records receptor specificity.ENTER deorphanizes antigen recognition of TCR and BCR.ENTER-seq maps TCR specificity, clonality and cell state in single cells.ENTER-seq of patient sample decodes antiviral T cell memory.

In response to infection, pathogen-specific CD8 T cells differentiate into functionally diverse effector and memory T cell populations critical for resolving disease and providing durable immunity. Through small-molecule inhibition, RNAi studies, and induced genetic deletion, we reveal an essential role for the chromatin modifier and BET family member BRD4 in supporting the differentiation and maintenance of terminally fated effector CD8 T cells during infection. BRD4 bound diverse regulatory regions critical to effector T cell differentiation and controlled transcriptional activity of terminal effector-specific super-enhancers in vivo. Consequentially, induced deletion of Brd4 or small molecule-mediated BET inhibition impaired maintenance of a terminal effector T cell phenotype. BRD4 was also required for terminal differentiation of CD8 T cells in the tumor microenvironment in murine models, which we show has implications for immunotherapies. Taken together, these data reveal an unappreciated requirement for BRD4 in coordinating activity of cis regulatory elements to control CD8 T cell fate and lineage stability.

To better define the control of immune system regulation, we generated an atlas of microRNA (miRNA) expression from 63 mouse immune cell populations and connected these signatures with assay for transposase-accessible chromatin using sequencing (ATAC–seq), chromatin immunoprecipitation followed by sequencing (ChIP–seq) and nascent RNA profiles to establish a map of miRNA promoter and enhancer usage in immune cells. miRNA complexity was relatively low, with >90% of the miRNA compartment of each population comprising <75 miRNAs; however, each cell type had a unique miRNA signature. Integration of miRNA expression with chromatin accessibility revealed putative regulatory elements for differentially expressed miRNAs, including miR-21a, miR-146a and miR-223. The integrated maps suggest that many miRNAs utilize multiple promoters to reach high abundance and identified dominant and divergent miRNA regulatory elements between lineages and during development that may be used by clustered miRNAs, such as miR-99a/let-7c/miR-125b, to achieve distinct expression. These studies, with web-accessible data, help delineate the cis-regulatory elements controlling miRNA signatures of the immune system. Brown and colleagues generated an atlas of miRNA expression profiles from primary mouse immune cell populations and connected these signatures with ATAC–seq, ChIP–seq and nascent RNA profiles to establish a map of miRNA promoter and enhancer usage in immune cells.

Inflammatory bowel disease (IBD) encompasses a spectrum of gastrointestinal disorders driven by dysregulated immune responses against gut microbiota. We integrated single-cell RNA and antigen receptor sequencing to elucidate key components, cellular states, and clonal relationships of the peripheral and gastrointestinal mucosal immune systems in health and ulcerative colitis (UC). UC was associated with an increase in IgG1+ plasma cells in colonic tissue, increased colonic regulatory T cells characterized by elevated expression of the transcription factor ZEB2, and an enrichment of a γδ T cell subset in the peripheral blood. Moreover, we observed heterogeneity in CD8+ tissue-resident memory T (TRM) cells in colonic tissue, with four transcriptionally distinct states of differentiation observed across health and disease. In the setting of UC, there was a marked shift of clonally related CD8+ TRM cells toward an inflammatory state, mediated, in part, by increased expression of the T-box transcription factor Eomesodermin. Together, these results provide a detailed atlas of transcriptional changes occurring in adaptive immune cells in the context of UC and suggest a role for CD8+ TRM cells in IBD.

T follicular helper (TFH) cells are a distinct type of CD4+ T cells that are essential for most antibody and B lymphocyte responses. TFH cell regulation and dysregulation is involved in a range of diseases. Bcl-6 is the lineage-defining transcription factor of TFH cells and its activity is essential for TFH cell differentiation and function. However, how Bcl-6 controls TFH biology has largely remained unclear, at least in part due to the intrinsic challenges of connecting repressors to gene upregulation in complex cell types with multiple possible differentiation fates. Multiple competing models were tested here by a series of experimental approaches to determine that Bcl-6 exhibits negative autoregulation and controls pleiotropic attributes of TFH differentiation and function, including migration, costimulation, inhibitory receptors and cytokines, via multiple repressor-of-repressor gene circuits. Bcl-6 is the signature transcription factor for TFH cells. Crotty and colleagues provide a comprehensive transcriptional map depicting the regulatory circuitry controlled by Bcl-6 in determining TFH cell fate and function.

T follicular helper (TFH) cells are a distinct type of CD4 T cells that are essential for most antibody and B lymphocyte responses. TFH regulation and dysregulation is involved in a range of diseases. Bcl6 is the lineage defining transcription factor of TFH cells and its activity is essential for TFH cell differentiation and function. However, how Bcl6 controls TFH biology has largely remained unclear, at least in part due to intrinsic challenges of connecting repressors to gene upregulation in complex cell types with multiple possible differentiation fates. Multiple competing models were tested here by a series of experimental approaches. We ruled out the model that TFH is a default pathway of CD4 T cell by utilizing Bcl6 and Blimp1 double deficient mice in acute virus infection and protein immunization. We showed that Bcl6 has a strong negative autoregulatory loop in TFH cells, by generating mice containing a deletion mutation of an 8-nucleotide Bcl6 binding site in the Bcl6 promoter. To determine putative Bcl6 target TFs, we developed an integrated analytic approach using RNA-seq, BCL6 ChIP-seq, and ATAC-seq data and Bcl6 and Blimp1 double KO mice. Using this approach, Runx2, Runx3, and Klf2 were identified as Bcl6-targeted repressors (Bcl6-r TFs) that regulate important TFH genes. Taken together, Bcl6 controls pleiotropic attributes of TFH differentiation and function (migration, costimulation, inhibitory receptors, and cytokines) via multiple repressor-of-repressor gene circuits.

Generation of T-cell memory is crucial in conferring vaccine-induced immunity, particularly against pathogens where neutralizing antibodies alone are insufficient at providing long-term protection. While great advances have been made in understanding the generation and maintenance of memory CD8+ T cells and B cells, mechanisms underlying the generation of memory CD4+ T cells have remained relatively elusive. This limitation is in part due to the multi-potency and lineage plasticity exhibited by CD4+ T helper (TH) cells. Using the LCMV viral infection model, we show that the T follicular helper (TFH) subset are the predominant CD4+ memory T cell type based on its relative abundance following pathogen elimination as well as its multi-potent potential upon antigen re-challenge. This multi-potency of TFH memory cells during secondary challenge has also been observed in influenza infection and acute bacterial infection, suggesting that the TFH memory subset is most capable of providing a comprehensive and robust secondary response. From advancements in CD8+ memory T cell studies, it has become clear that transcription factors (TF) serve as crucial arbiters for the cell-fate decisions between short-lived effector and memory. To investigate the transcriptional mechanisms underlying TFH formation, we employed a novel bioinformatics analysis utilizing the PageRank algorithm that combines both RNA-seq and ATAC-seq and have identified putative transcriptional regulators of memory TFH differentiation. Further understanding of TFH memory formation will undoubtedly unveil new insights into CD4+ T cell memory in hopes of improving vaccine-based immunity.

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.

Naive T cell differentiation into effector and memory subsets in response to infection requires the coordination of numerous factors that regulate changes in gene expression to support proliferation and cell function. Chromatin accessibility and DNA looping influence transcription factor binding, facilitate enhancer-promoter interactions, and insulate genes. CCCTC-Binding Factor (CTCF) is a highly conserved protein important for organizing the genome and partnering with transcription factors, but its role in CD8+ T cell differentiation has yet to be determined. We show that loss of CTCF in CD8+ T cells alters the expression of genes important for subset differentiation and these changes are reflected during Listeria infection where the formation of memory-precursor cells is favored while the formation of terminal effector cells is impaired. At a memory timepoint, CTCF knockdown enhances intestinal Trm accumulation despite impairing circulating memory maintenance. ChIP-Seq and HiC reveals that terminal effector cells gain CTCF binding within topologically associated domains, often overlapping with H3K27ac peaks which marks active enhancers and promoters. As genes near the overlap of CTCF and H3K27ac peaks have increased expression in terminal effector cells, we propose that one role of CTCF is to promote effector function by stabilizing promoter-enhancer interactions which influences the expression of fate-determining genes. Insights into the role of CTCF in CD8+ T cell differentiation during infection will yield valuable insight into dynamic chromatin changes regulating T cell differentiation and inform the development of therapies which better utilize the T cell response.

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.

Memory CD8+ T cells afford long-lived, durable protection against infection and malignancy. Despite the critical relevance of memory T cells to vaccination and immunotherapies, the transcriptional and epigenetic signals instructing memory T cell fate remain unclear. Here, we utilized a pooled RNA interference screen to evaluate the activity of transcription factors and chromatin modifiers governing memory T cell differentiation in vivo. The epigenetic ‘reader,’ Brd4 of the BET protein family, emerged as a top regulator of memory T cell differentiation. Through RNA interference, small molecule inhibition and inducible genetic deletion, we established a central role for Brd4 in mediating CD8+ T cell proliferation, differentiation and function during acute viral infection. Brd4 was required for optimal expression of fate-specifying transcription factors, and a deficiency of Brd4 activity resulted in diminished formation of effector memory and CD103+ tissue-resident memory CD8+ T cells. Given that BET inhibition has emerged as a powerful approach for suppressing tumor growth, we also evaluated how BET inhibition and Brd4-deficiency influence T cell activity during cancer. Brd4 was required for intratumoral accumulation and function of T cells in a mouse model of melanoma, and BET inhibition suppressed T cell-mediated control of tumor growth. However, epigenetic targeting of Brd4 constrained terminal differentiation of T cells within tumors, biasing tumor-resident T cells towards a reprogrammable TCF1-expressing phenotype. These studies establish a novel role for Brd4 in CD8+ T cell biology and provide insight for immunotherapy approaches designed to leverage the dynamic activity of CD8+ T cells in tumors.

Author(s): Yu, Bingfei | Advisor(s): Goldrath, Ananda W | Abstract: CD8 + T cells are a central component of the adaptive immune system. Upon infection, a naive CD8 + T cell will differentiate into a heterogeneous population of effector T cells composed of terminal-effector and memory-precursor CD8 + T cells. Terminal-effector T cells rapidly decay after pathogens are eradicated while memory-precursor T cells survive during the contraction phase to become memory T cells, providing long-term protection from reinfection. Similar to the heterogeneity of effector T cells, memory T cells can also be divided into central-memory, effector-memory and tissue-resident memory subsets based on trafficking, location, proliferation potential and cytotoxic function. These memory subsets collaborate together to enhance the pathogen clearance and vaccine efficacy. The differentiation of a naive CD8 + T cell into a specific effector or memory subset is influenced by cell-extrinsic environmental signals and cell-intrinsic factors including transcription factors (TFs), epigenetic modification and chromatin organization. Considerable advances have been made to identify key TFs that regulate these T cell fate decisions. However, the TF-mediated transcriptional network responsible for specific subset differentiation and how epigenetic modifications and chromatin configuration modulate CD8 + T cell fate determination is still largely unknown. To address these questions, we deciphered epigenetic landscapes in effector and memory CD8 + T cells in response to bacterial infection by characterizing the genome-wide histone modification, chromatin accessibility and transcriptional program. Integrative analysis of epigenomics data showed that subset-specific enhancers established by key TFs foreshadow the specific lineage differentiation. To better identify cruicial TFs from multilayered epigenetic landscapes, we developed a webpage ranking-based algorithm (PageRank) to rank the importance of TFs from transcriptional network and identified a novel function of two TFs: YY1 and Nr3c1 to regulate terminal-effector and memory-precursor subset differentiation, respectively. By leveraging the PageRank analysis and chromatin accessibility data, we developed a computational screen to predict key TFs for tissue-resident memory T cell differentiation. Combining this approach with shRNA functional screen, we identified the role of Runx3 in programming tissue-residency signatures in non-lymphoid tissues and tumors. Finally, we discovered a novel role for the genome organizer CTCF in CD8 + T cell fate decisions illustrating the impact of chromatin organization on effector and memory T cell differentiation. Taken together, we uncovered a multi-layered regulation of chromatin state, accessibility and organization, that influences T cell fate decisions by fine-tuning transcriptional circuits. We further constructed a computational framework that integrates these high-dimensional data, facilitating identification of key transcriptional regulators and providing valuable biological insights.