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Background: CAR T-cells targeting solid tumor antigens have not yet demonstrated similar success as seen for CD19, BCMA and CD22 in B-cell malignancies. Truly tumor-specific antigens are rare, yet pediatric solid tumors manifest stalled fetal developmental programs and often overexpress oncofetal antigens, normally restricted to prenatal tissues. Antigen density is a major factor determining CAR potency and whether such nonmutant antigens can be effectively and safely targeted remains unknown. Here, we engineered CAR T-cells targeting GPC2 to recognize clinically relevant antigen thresholds and assess safety. Methods: Expression of GPC2 was assessed during organ development (Cardoso-Moreira et al. 2019), by IHC in prenatal, infant and pediatric brain and in ssRNAseq datasets of fetal (La Manno et. al 2016) and adult brain (Allen Brain Atlas). To measure the molecules/cell of GPC2 and other immunotherapy-relevant targets on bone-marrow infiltrating neuroblastoma (NB) cells of high-risk patients, we developed a Flow Cytometric quantification assay and engineered GPC2-CAR T-cells to recognize relevant antigen thresholds. To assess binding specificity towards GPC2, a membrane proteome array encompassing >5,300 human proteins was utilized. Taking advantage of the murine cross-reactivity of our lead GPC2-CAR, toxicity was assessed in a relevant xenograft model. Results: During organ development, we found GPC2 expression restricted to the developing brain and a gradual decrease until silenced after birth. IHC of prenatal brain revealed an inverse correlation between GPC2 and gestational age (r = -0.775, p = 0.008) and very low staining in infant and pediatric brain (H-scores Conclusion: Our results demonstrate safety and efficacy of a lead GPC2-CAR and illuminate oncofetal antigens as a promising class of targets for CAR T cell therapy of solid tumors. This work establishes a strong basis for testing GPC2 CAR T-cells in early phase clinical trials. Citation Format: Sabine Heitzeneder, Kristopher R. Bosse, Zhongyu Zhu, Dontcho Jelev, Shaurya Dhingra, Robbie Majzner, Elena Sotillo-Pineiro, Samantha Buongervino, Peng Xu, Jing Huang, Alberto Delaidelli, Martin Hasselblatt, Kevin Parker, Hima Anbunathan, Anya Alag, Jennifer Hwang, Min Huang, Dorota D. Klysz, Johanna L. Theruvath, Jose G. Vilches-Moure, Ansuman T. Satpathy, Poul H. Sorensen, Dimiter S. Dimitrov, John M. Maris, Crystal L. Mackall. Potent activity of CAR T cells targeting the oncofetal protein GPC2 engineered to recognize low antigen density in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1548.

Understanding the cellular regulation of tumor-specific CD8+ T-cell responses is critical to designing improved clinical strategies for cancer immunotherapy. In this issue, Aoki and colleagues deepen our knowledge of this topic by demonstrating that transient depletion of CD4+ T cells in patients with gastrointestinal cancer induces remodeling of the T-cell repertoire, including clonal replacement and expansion of CD8+ T-cell clones shared between the blood and tumor. See article by Aoki et al., p. 624

SARS-CoV-2 is the cause of a pandemic with growing global mortality. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with ChIRP-MS data from three other RNA viruses defined viral specificity of RNA-host protein interactions. Targeted CRISPR screens revealed that the majority of functional RNA-binding proteins protect the host from virus-induced cell death, and comparative CRISPR screens across seven RNA viruses revealed shared and SARS-specific antiviral factors. Finally, by combining the RNA-centric approach and functional CRISPR screens, we demonstrated a physical and functional connection between SARS-CoV-2 and mitochondria, highlighting this organelle as a general platform for antiviral activity. Altogether, these data provide a comprehensive catalog of functional SARS-CoV-2 RNA-host protein interactions, which may inform studies to understand the host-virus interface and nominate host pathways that could be targeted for therapeutic benefit.

Long noncoding RNAs (lncRNAs) are emerging as critical regulators of gene expression in the immune system. Studies have shown that lncRNAs are expressed in a highly lineage-specific manner and control the differentiation and function of innate and adaptive cell types. In this Review, we focus on mechanisms used by lncRNAs to regulate genes encoding products involved in the immune response, including direct interactions with chromatin, RNA and proteins. In addition, we address new areas of lncRNA biology, such as the functions of enhancer RNAs, circular RNAs and chemical modifications to RNA in cellular processes. We emphasize critical gaps in knowledge and future prospects for the roles of lncRNAs in the immune system and autoimmune disease.

Ciita was discovered for its role in regulating transcription of major histocompatibility complex class II (MHCII) genes. Subsequently, CIITA was predicted to control many other genes based on reporter and ChIP-seq analysis but few such predictions have been verified in vivo using Ciita-/- mice. Testing these predictions for classical dendritic cells (cDCs) has been particularly difficult, since Ciita-/- mice lack MHCII expression required to identify cDCs. However, recent identification of the cDC-specific transcription factor Zbtb46 allows the identification of cDCs independently of MHCII expression. We crossed Zbtb46gfp mice onto the Ciita-/- background and found that all cDC lineages developed in vivo in the absence of Ciita. We then compared the complete transcriptional profile of wild-type and Ciita-/- cDCs to define the physiological footprint of CIITA for both immature and activated cDCs. We find that CIITA exerts a highly restricted control over only the MHCII, H2-DO and H2-DM genes, in DC1 and DC2 cDC subsets, but not over other proposed targets, including Ii. These findings emphasize the caveats needed in interpreting transcription factor binding sites identified by in-vitro reporter analysis, or by ChIP-seq, which may not necessarily indicate their functional activity in vivo.

Two studies reveal a role for the transcriptional regulator BATF3 as a T cell–intrinsic factor mediating effective memory responses. This finding opens future avenues of investigation and opportunities to enhance cellular immunotherapy.

SUMMARYCAR T cells mediate antitumor effects in a small subset of cancer patients, but dysfunction due to T cell exhaustion is an important barrier to progress. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system employing a tonically signaling CAR, which induces hallmarks of exhaustion described in other settings. Exhaustion was associated with a profound defect in IL-2 production alongside increased chromatin accessibility of AP-1 transcription factor motifs, and overexpression of numerous bZIP and IRF transcription factors that have been implicated in inhibitory activity. Here we demonstrate that engineering CAR T cells to overexpress c-Jun, a canonical AP-1 factor, enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved antitumor potency in numerous in vivo tumor models. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells and that engineering CAR T cells to overexpress c-Jun renders them exhaustion-resistant, thereby addressing a major barrier to progress for this emerging class of therapeutics.

Immunotherapies that block inhibitory checkpoint receptors on T cells have transformed the clinical care of cancer patients. However, which tumor-specific T cells are mobilized following checkpoint blockade remains unclear. Here, we performed paired single-cell RNA- and T cell receptor (TCR)-sequencing on 79,046 cells from site-matched tumors from patients with basal cell carcinoma (BCC) or squamous cell carcinoma (SCC) pre- and post-anti-PD-1 therapy. Tracking TCR clones and transcriptional phenotypes revealed a coupling of tumor-recognition, clonal expansion, and T cell dysfunction: the T cell response to treatment was accompanied by clonal expansions of CD8+CD39+T cells, which co-expressed markers of chronic T cell activation and exhaustion. However, this expansion did not derive from pre-existing tumor infiltrating T cell clones; rather, it comprised novel clonotypes, which were not previously observed in the same tumor. Clonal replacement of T cells was preferentially observed in exhausted CD8+T cells, compared to other distinct T cell phenotypes, and was evident in BCC and SCC patients. These results, enabled by single-cell multi-omic profiling of clinical samples, demonstrate that pre-existing tumor-specific T cells may be limited in their capacity for re-invigoration, and that the T cell response to checkpoint blockade relies on the expansion of a distinct repertoire of T cell clones that may have just recently entered the tumor.

Immunotherapies that block inhibitory checkpoint receptors on T cells have transformed the clinical care of patients with cancer

Understanding complex tissues requires single-cell deconstruction of gene regulation with precision and scale. Here we present a massively parallel droplet-based platform for mapping transposase-accessible chromatin in tens of thousands of single cells per sample (scATAC-seq). We obtain and analyze chromatin profiles of over 200,000 single cells in two primary human systems. In blood, scATAC-seq allows marker-free identification of cell type-specificcis- andtrans-regulatory elements, mapping of disease-associated enhancer activity, and reconstruction of trajectories of differentiation from progenitors to diverse and rare immune cell types. In basal cell carcinoma, scATAC-seq reveals regulatory landscapes of malignant, stromal, and immune cell types in the tumor microenvironment. Moreover, scATAC-seq of serial tumor biopsies before and after PD-1 blockade allows identification of chromatin regulators and differentiation trajectories of therapy-responsive intratumoral T cell subsets, revealing a shared regulatory program driving CD8+T cell exhaustion and CD4+T follicular helper cell development. We anticipate that droplet-based single-cell chromatin accessibility will provide a broadly applicable means of identifying regulatory factors and elements that underlie cell type and function.

Neoantigens are now recognized drivers of the antitumor immune response. Recurrent neoantigens, shared among groups of patients, have thus become increasingly coveted therapeutic targets. Here, we report on the data-driven identification of a robustly presented, immunogenic neoantigen that is derived from the combination of HLA-A*01:01 and RAS.Q61K. Analysis of large patient cohorts indicated that this combination applies to 3% of patients with melanoma. Using HLA peptidomics, we were able to demonstrate robust endogenous presentation of the neoantigen in 10 tumor samples. We detected specific reactivity to the mutated peptide within tumor-infiltrating lymphocytes (TILs) from 2 unrelated patients, thus confirming its natural immunogenicity. We further investigated the neoantigen-specific clones and their T cell receptors (TCRs) via a combination of TCR sequencing, TCR overexpression, functional assays, and single-cell transcriptomics. Our analysis revealed a diverse repertoire of neoantigen-specific clones with both intra- and interpatient TCR similarities. Moreover, 1 dominant clone proved to cross-react with the highly prevalent RAS.Q61R variant. Transcriptome analysis revealed a high association of TCR clones with specific T cell phenotypes in response to cognate melanoma, with neoantigen-specific cells showing an activated and dysfunctional phenotype. Identification of recurrent neoantigens and their reactive TCRs can promote "off-the-shelf" precision immunotherapies, alleviating limitations of personalized treatments.

Skin conventional dendritic cells (cDCs) exist as two distinct subsets, cDC1s and cDC2s, which maintain the balance of immunity to pathogens and tolerance to self and microbiota. Here, we examined the roles of dermal cDC1s and cDC2s during bacterial infection, notably Propionibacterium acnes (P. acnes). cDC1s, but not cDC2s, regulated the magnitude of the immune response to P. acnes in the murine dermis by controlling neutrophil recruitment to the inflamed site and survival and function therein. Single-cell mRNA sequencing revealed that this regulation relied on secretion of the cytokine vascular endothelial growth factor alpha (VEGF-alpha) by a minor subset of activated EpCAM(+)CD59(+) Ly-6D(+) cDC1s. Neutrophil recruitment by dermal cDC1s was also observed during S. aureus, bacillus Calmette-Guerin (BCG), or E. coli infection, as well as in a model of bacterial insult in human skin. Thus, skin cDC1s are essential regulators of the innate response in cutaneous immunity and have roles beyond classical antigen presentation.

Classical type 1 dendritic cells (cDC1s) are required for antiviral and antitumor immunity, which necessitates an understanding of their development. Development of the cDC1 progenitor requires an E-protein-dependent enhancer located 41 kilobases downstream of the transcription start site of the transcription factor Irf8 (+41-kb Irf8 enhancer), but its maturation instead requires the Batf3-dependent +32-kb Irf8 enhancer. To understand this switch, we performed single-cell RNA sequencing of the common dendritic cell progenitor (CDP) and identified a cluster of cells that expressed transcription factors that influence cDC1 development, such as Nfil3, Id2 and Zeb2. Genetic epistasis among these factors revealed that Nfil3 expression is required for the transition from Zeb2hi and Id2lo CDPs to Zeb2lo and Id2hi CDPs, which represent the earliest committed cDC1 progenitors. This genetic circuit blocks E-protein activity to exclude plasmacytoid dendritic cell potential and explains the switch in Irf8 enhancer usage during cDC1 development. Classical type 1 dendritic cells (cDC1s) are essential for activation of antiviral and anticancer T cell responses. Murphy and colleagues describe a genetic circuit that involves the transcription factors Nfil3, Id2 and Zeb2. This circuit imposes a molecular switch that allows cDC1 specification and development.

INTRODUCTION Cancer is one of the leading causes of death worldwide. Although the 2% of the human genome that encodes proteins has been extensively studied, much remains to be learned about the noncoding genome and gene regulation in cancer. Genes are turned on and off in the proper cell types and cell states by transcription factor (TF) proteins acting on DNA regulatory elements that are scattered over the vast noncoding genome and exert long-range influences. The Cancer Genome Atlas (TCGA) is a global consortium that aims to accelerate the understanding of the molecular basis of cancer. TCGA has systematically collected DNA mutation, methylation, RNA expression, and other comprehensive datasets from primary human cancer tissue. TCGA has served as an invaluable resource for the identification of genomic aberrations, altered transcriptional networks, and cancer subtypes. Nonetheless, the gene regulatory landscapes of these tumors have largely been inferred through indirect means. RATIONALE A hallmark of active DNA regulatory elements is chromatin accessibility. Eukaryotic genomes are compacted in chromatin, a complex of DNA and proteins, and only the active regulatory elements are accessible by the cell’s machinery such as TFs. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) quantifies DNA accessibility through the use of transposase enzymes that insert sequencing adapters at these accessible chromatin sites. ATAC-seq enables the genome-wide profiling of TF binding events that orchestrate gene expression programs and give a cell its identity. RESULTS We generated high-quality ATAC-seq data in 410 tumor samples from TCGA, identifying diverse regulatory landscapes across 23 cancer types. These chromatin accessibility profiles identify cancer- and tissue-specific DNA regulatory elements that enable classification of tumor subtypes with newly recognized prognostic importance. We identify distinct TF activities in cancer based on differences in the inferred patterns of TF-DNA interaction and gene expression. Genome-wide correlation of gene expression and chromatin accessibility predicts tens of thousands of putative interactions between distal regulatory elements and gene promoters, including key oncogenes and targets in cancer immunotherapy, such as MYC , SRC , BCL2 , and PDL1 . Moreover, these regulatory interactions inform known genetic risk loci linked to cancer predisposition, nominating biochemical mechanisms and target genes for many cancer-linked genetic variants. Lastly, integration with mutation profiling by whole-genome sequencing identifies cancer-relevant noncoding mutations that are associated with altered gene expression. A single-base mutation located 12 kilobases upstream of the FGD4 gene, a regulator of the actin cytoskeleton, generates a putative de novo binding site for an NKX TF and is associated with an increase in chromatin accessibility and a concomitant increase in FGD4 gene expression. CONCLUSION The accessible genome of primary human cancers provides a wealth of information on the susceptibility, mechanisms, prognosis, and potential therapeutic strategies of diverse cancer types. Prediction of interactions between DNA regulatory elements and gene promoters sets the stage for future integrative gene regulatory network analyses. The discovery of hundreds of noncoding somatic mutations that exhibit allele-specific regulatory effects suggests a pervasive mechanism for cancer cells to manipulate gene expression and increase cellular fitness. These data may serve as a foundational resource for the cancer research community.

Modular domains of long non-coding RNAs can serve as scaffolds to bring distant regions of the linear genome into spatial proximity. Here, we present HiChIRP, a method leveraging bio-orthogonal chemistry and optimized chromosome conformation capture conditions, which enables interrogation of chromatin architecture focused around a specific RNA of interest down to approximately ten copies per cell. HiChIRP of three nuclear RNAs reveals insights into promoter interactions (7SK), telomere biology (telomerase RNA component) and inflammatory gene regulation (lincRNA-EPS). HiChIRP combines a modified chromosome conformation capture protocol with enrichment of RNA-associated chromosome conformation to visualize genome-wide looping linked to an RNA of interest.

CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase 1 clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the endogenous T cell receptor (TCR) chains, TCRα (TRAC) and TCRβ (TRBC), were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding programmed cell death protein 1 (PD-1; PDCD1), was performed to improve antitumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Although chromosomal translocations were detected, the frequency decreased over time. Modified T cells persisted for up to 9 months, suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of CRISPR gene editing for cancer immunotherapy. Citation Format: Edward Stadtmauer, Joseph Fraietta, Ansuman Satpathy, Carl H. June. Single cell analysis of CRISPR T cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA01.

CD19-directed immunotherapies are clinically effective for treating B cell malignancies but also cause a high incidence of neurotoxicity. A subset of patients treated with chimeric antigen receptor (CAR) Tcells or bispecific Tcell engager (BiTE) antibodies display severe neurotoxicity, including fatal cerebral edema associated with Tcell infiltration into the brain. Here, we report that mural cells, which surround the endothelium and are critical for blood-brain-barrier integrity, express CD19. We identify CD19 expression in brain mural cells using single-cell RNA sequencing data and confirm perivascular staining at the protein level. CD19 expression in the brain begins early in development alongside the emergence of mural cell lineages and persists throughout adulthood across brain regions. Mouse mural cells demonstrate lower levels of Cd19 expression, suggesting limitations in preclinical animal models of neurotoxicity. These data suggest an on-target mechanism for neurotoxicity in CD19-directed therapies and highlight the utility of human single-cell atlases for designing immunotherapies.

Background: Autologous T cells genetically modified with a lentiviral vector to express affinity-enhanced T cell receptors (TCR) or chimeric antigen receptors have shown great promise for the treatment of cancer. NY-ESO-1 is a cancer testis antigen with little normal tissue expression but with aberrant expression in MM, sarcomas, and melanomas. An HLA-A201 restricted TCR recognizing the NY-ESO-1/LAGE-1 157-165 epitope (SLLMWITQC) kills NY-ESO positive cell lines and has been used to treat 25 patients with MM after ASCT with expansion, persistence, antigen-directed functionality and long-term safety and antitumor activity (Nat Med 2015, Blood Adv 2019). We hypothesized removal of the genes encoding the endogenous TCR, TCRα (TRAC) and TCRβ (TRBC), would enhance NY-ESO TCR expression and reduce TCR mispairing and with removal of PD-1 (PDCD1) would enhance activity and persistence. We previously demonstrated CRISPR/Cas9 and TCRα, TCRβ and PDCD1 targeting gRNAs could be successfully introduced via electroporation in preclinical models to disrupt gene expression (Clin Cancer Res 2017). We therefore began a phase 1 pilot clinical trial for pts with advanced MM and sarcoma of NY-ESO-1 TCR-expressing T cells with CRISPR/Cas9 TCRα, TCRβ and PDCD1 edited genes to assess safety, feasibility and activity (NCT03399448). Methods: Adults with HLA-A*0201 and expressing NY-ESO-1 and/or LAGE-1 antigen with advanced MM, synovial sarcoma, and myxoid/round cell liposarcoma (MRCL) with adequate performance and organ function and, for MM relapsed or refractory to at least 3 prior regimens and, for MRCL, proven metastatic disease or surgically inoperable local recurrence, were enrolled. Autologous T cells were transfected with Cas9 protein complexed with single guide RNAs against TRAC, TRBC and PDCD1 and subsequently transduced to express NY-ESO-1-specific TCR at the University of Pennsylvania. Frequency of NYCE T cells in final product was measured by flow cytometric dextramer analysis. Once cells were successfully manufactured and released, pts received fludarabine 30mg/m2 and cyclophosphamide 300mg/m2 daily on day -4,-3,-2. On Day 0 pts received a single infusion of thawed NYCE T cells as an out-patient. Pts were monitored closely for the first 28 days, monthly till 6 mo and then followed every 3 mo for adverse events, antitumor response and survival, NYCE T cell expansion, persistence, trafficking, phenotype and function, and immunogenicity. An assessment after accrual of the first 3 subjects in this ongoing trial was planned and is reported here. Results: 3 pts, 2 with MM and 1 with MRCL, have received NYCE T cells. Pt 1 is a 67 y/o F with IgG kappa MM with lytic bone lesions, and a +17q after 8 lines of therapy including 3 ASCTs, lenalidomide, pomalidomide, bortezomib, carfilzomib, daratumumab, and panobinostat. Pt 2 is a 65 y/o M with a recurrent MRCL manifested by abdominal and pelvic involvement after neo-adjuvant doxorubicin, multiple resections and radiation treatments with progression at time of enrollment. Pt 3 is a 62 y/o F with kappa light chain MM with lytic bone lesions and plasmacytomas and a +1q after 7 lines of therapy including lenalidomide, pomalidomide, bortezomib, carfilzomib, daratumumab, 2 ASCTs and an immunoconjugate . Manufacturing for these pts resulted in satisfactory products with 89.4 to 96% viability, transduction efficiency by qPCR of 0.04 to 0.2 copies/cell , residual Cas9 concentration 0 to 0.37 ng/ml, dextramer 0.4 to 1.8% NY-ESO-1 expression. TRAC, TRBC, and PDCD1 disruption efficiency was 44.3 to 49.4, 3.61 to 15.7 and 15.6 to 20.2% respectively. Pts tolerated treatment well without neurotoxicity or CRS. By day +60 pt 1 progressed by IMWG. Pt 2 received 1 U PRBC. By day +90 he remained with stable disease by serial CT scans. Pt 3 is too early to evaluate. Serial qPCR for copies of lentiviral transcripts in peripheral blood and tumor biopsies for pts 1+2 showed in vivo expansion, stable persistence and tumor targeting (Figure). Conclusion: Early results of a phase 1 trial of NYCE T cells infused in 3 pts with advanced MM and MRCL show safety and feasibility and viable, expanding, and persisting CRISPR/Cas9 gene edited T cells that trafficked to tumor. The persistence of the NYCE T cells suggests that immunogenicity from multiplexed gene-editing using Cas9 is minimal under these conditions. Further characterization of phenotype and function of these cells and clinical outcomes will be presented. Figure Disclosures Stadtmauer: Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Novartis: Consultancy, Research Funding; Tmunity: Research Funding; Abbvie: Research Funding. Cohen:Poseida Therapeutics, Inc.: Research Funding. Lacey:Novartis: Patents & Royalties: Patents related to CAR T cell biomarkers; Tmunity: Research Funding; Novartis: Research Funding. Melenhorst:Incyte: Research Funding; Novartis: Research Funding, Speakers Bureau; Parker Institute for Cancer Immunotherapy: Research Funding; Genentech: Speakers Bureau; Stand Up to Cancer: Research Funding; IASO Biotherapeutics, Co: Consultancy; Simcere of America, Inc: Consultancy; Shanghai Unicar Therapy, Co: Consultancy; Colorado Clinical and Translational Sciences Institute: Membership on an entity's Board of Directors or advisory committees; National Institutes of Health: Research Funding. Fraietta:Tmunity: Research Funding; Cabaletta: Research Funding; LEK Consulting: Consultancy. Mangan:amgen: Speakers Bureau; takeda: Speakers Bureau; celgene: Speakers Bureau; janssen: Speakers Bureau. Lancaster:novartis: Research Funding. Suhoski:novartis: Research Funding. Fesnak:Novartis: Research Funding. Young:novartis: Research Funding. Chew:tmunity: Other: Scientific Founder, Research Funding; novartis: Research Funding. Zhao:Tmunity: Membership on an entity's Board of Directors or advisory committees, Research Funding; novartis: Research Funding. Hwang:Novartis: Research Funding; Tmunity: Research Funding. Hexner:novartis: Research Funding. June:Novartis: Research Funding; Tmunity: Other: scientific founder, for which he has founders stock but no income, Patents & Royalties.

The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues1–3. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption4–6, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa)7 to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (TH17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs.

When different types of functional genomics data are generated on single cells from different samples of cells from the same heterogeneous population, the clustering of cells in the different samples should be coupled. We formulate this “coupled clustering” problem as an optimization problem, and propose the method of coupled nonnegative matrix factorizations (coupled NMF) for its solution. The method is illustrated by the integrative analysis of single cell RNA-seq and single cell ATAC-seq data.Significance StatementsBiological samples are often heterogeneous mixtures of different types of cells. Suppose we have two single cell data sets, each providing information on a different cellular feature and generated on a different sample from this mixture. Then, the clustering of cells in the two samples should be coupled as both clusterings are reflecting the underlying cell types in the same mixture. This “coupled clustering” problem is a new problem not covered by existing clustering methods. In this paper we develop an approach for its solution based the coupling of two nonnegative matrix factorizations. The method should be useful for integrative single cell genomics analysis tasks such as the joint analysis of single cell RNA-seq and single cell ATAC-seq data.

Incomplete annotation of cell-to-cell state variance and widespread linkage disequilibrium in the human genome represent significant challenges to elucidating mechanisms of trait-associated genetic variation. Here, using data from the UK Biobank, we perform genetic fine-mapping for 16 blood cell traits to quantify posterior probabilities of association while allowing for multiple independent signals per region. We observe an enrichment of fine-mapped variants in accessible chromatin of lineage-committed hematopoietic progenitor cells. Further, we develop a novel analytic framework that identifies “core gene” cell type enrichments and show that this approach uniquely resolves relevant cell types within closely related populations. Applying our approach to single cell chromatin accessibility data, we discover significant heterogeneity within classically defined multipotential progenitor populations. Finally, using several lines of empirical evidence, we identify relevant cell types, predict target genes, and propose putative causal mechanisms for fine-mapped variants. In total, our study provides an analytic framework for single-variant and single-cell analyses to elucidate putative causal variants and cell types from GWAS and high-resolution epigenomic assays.

Dynamic gene expression during cellular differentiation is tightly coordinated by transcriptional and post-transcriptional mechanisms. An emerging theme is the central role of long noncoding RNAs (lncRNAs) in the regulation of this specificity. Recent advances demonstrate that lncRNAs are expressed in a lineage-specific manner and control the development of several cell types in the hematopoietic system. Moreover, specific lncRNAs are induced to modulate innate and adaptive immune responses. lncRNAs can function via RNA-DNA, RNA-RNA, and RNA-protein target interactions. As a result, they affect several stages of gene regulation, including chromatin modification, mRNA biogenesis, and protein signaling. We discuss recent advances, future prospects, and challenges in understanding the roles of lncRNAs in immunity and immune-mediated diseases.

The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer-promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases.

The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa) to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (TH17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs.

The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here, we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naïve T cells to T helper 17 cells or regulatory T cells creates subtype-specific enhancer-promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a four-fold increase of potential target genes for autoimmune and cardiovascular diseases.

In NOD mice, CD11c+ cells increase greatly with islet inflammation and contribute to autoimmune destruction of pancreatic β cells. In this study, we investigated their origin and mechanism of recruitment. CD11c+ cells in inflamed islets resembled classical dendritic cells based on their transcriptional profile. However, the majority of these cells were not from the Zbtb46-dependent dendritic-cell lineage. Instead, monocyte precursors could give rise to CD11c+ cells in inflamed islets. Chemokines Ccl5 and Ccl8 were persistently elevated in inflamed islets and the influx of CD11c+ cells was partially dependent on their receptor Ccr5. Treatment with islet Ag-specific regulatory T cells led to a marked decrease of Ccl5 and Ccl8, and a reduction of monocyte recruitment. These results implicate a monocytic origin of CD11c+ cells in inflamed islets and suggest that therapeutic regulatory T cells directly or indirectly regulate their influx by altering the chemotactic milieu in the islets.

We present Omni-ATAC, an improved ATAC-seq protocol for chromatin accessibility profiling that works across multiple applications with substantial improvement of signal-to-background ratio and information content. The Omni-ATAC protocol enables chromatin accessibility profiling from archival frozen tissue samples and 50 μm sections, revealing the activities of disease-associated DNA elements in distinct human brain structures. The Omni-ATAC protocol enables the interrogation of personal regulomes in tissue context and translational studies.

Here, we define the landscape and dynamics of active regulatory DNA in cutaneous T cell lymphoma (CTCL) by ATAC-seq. Analysis of 111 human CTCL and control samples revealed extensive chromatin signatures that distinguished leukemic, host, and normal CD4+ T cells. We identify three dominant patterns of transcription factor (TF) activation that drive leukemia regulomes, as well as TF deactivations that alter host T cells in CTCL patients. Clinical response to histone deacetylase inhibitors (HDACi) is strongly associated with a concurrent gain in chromatin accessibility. HDACi causes distinct chromatin responses in leukemic and host CD4+ T cells, reprogramming host T cells toward normalcy. These results provide a foundational framework to study personal regulomes in human cancer and epigenetic therapy.

Summary Regulatory T (Treg) cells maintain immune tolerance through the master transcription factor forkhead box P3 (FOXP3), which is crucial for Treg cell function and homeostasis. We identified an IPEX (immune dysregulation polyendocrinopathy enteropathy X-linked) syndrome patient with a FOXP3 mutation in the domain swap interface of the protein. Recapitulation of this Foxp3 variant in mice led to the development of an autoimmune syndrome consistent with an unrestrained T helper type 2 (Th2) immune response. Genomic analysis of Treg cells by RNA-sequencing, Foxp3 chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-sequencing), and H3K27ac-HiChIP revealed a specific de-repression of the Th2 transcriptional program leading to the generation of Th2-like Treg cells that were unable to suppress extrinsic Th2 cells. Th2-like Treg cells showed increased intra-chromosomal interactions in the Th2 locus, leading to type 2 cytokine production. These findings identify a direct role for Foxp3 in suppressing Th2-like Treg cells and implicate additional pathways that could be targeted to restrain Th2 trans-differentiated Treg cells.

SummaryHere we present Perturb-ATAC, a method which combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells, based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in ∼4,300 single cells, encompassing more than 63 unique genotype-phenotype relationships. Perturb-ATAC in human B lymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning, and identified a hierarchical organization of TFs that govern B cell state, variation, and disease-associatedcis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules ofcis-elements that specify keratinocyte fate, orchestrated by the TFs JUNB, KLF4, ZNF750, CEBPA, and EHF. Combinatorial deletion of all pairs of these TFs uncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful and general strategy to dissect gene regulatory networks in development and disease.HighlightsA new method for simultaneous measurement of CRISPR perturbations and chromatin state in single cells.Perturb-ATAC reveals regulatory factors that controlcis-element accessibility,trans-factor occupancy, and nucleosome positioning.Perturb-ATAC reveals regulatory modules of coordinatedtrans-factor activity in B lymphoblasts.Keratinocyte differentiation is orchestrated by synergistic activities of co-binding TFs oncis-elements.

Hematopoiesis is a well-characterized paradigm of cellular differentiation that is highly regulated to ensure balanced proportions of mature blood cells. However, many aspects of this process remain poorly understood in humans. For example, there is extensive variation in commonly measured blood cell traits, which can manifest as diseases at extreme ends of the spectrum, yet the vast majority of genetic loci responsible for driving these differences are currently unknown. Here, we integrate fine-mapped population genetics with high-resolution chromatin landscapes to gain novel insights into regulatory mechanisms critical for human blood cell production and disease. First, we conducted a genome-wide association study in 115,000 individuals from the UK Biobank, measuring the effects of genetic variation on 16 blood traits spanning 7 hematopoietic lineages (erythroid, platelet, lymphocyte, monocyte, neutrophil, eosinophil, basophil). Within each region of association (n = 2,056), we performed Bayesian fine-mapping on all common variants to resolve the most likely causal hits. Going further, we were interested in whether genetic variants predominantly act in terminal cell states or less differentiated progenitors. To this end, we overlapped fine-mapped variants with chromatin accessibility profiles (ATAC-seq) of 18 primary hematopoietic populations sorted from healthy donors. Across all lineages, 21% of regulatory variants were restricted to accessible chromatin (AC) peaks in terminal progenitors. Interestingly, 59% of variants fell in AC regions of one or more upstream progenitor states, suggesting that a significant amount of variation in blood traits stems from regulatory signaling in earlier stages of hematopoiesis. Motivated by this finding, we hypothesized that different branches of hematopoiesis (e.g., monocyte and red blood cell count) could be co-regulated by pleiotropic variants acting in common progenitor populations. Therefore, we investigated variants associated with 2 or more of the 7 blood cell types for which phenotypes were available. Remarkably, across 172 such variants, there was an average of 60% more open chromatin in progenitors than terminal cell types (mean 4.01 vs. 2.44 counts per million; p = 0.025). Examining the directional effects of these variants on distinct lineages, we discovered that 91% of pleiotropic variants exhibited a tune mechanism by changing the levels of different blood cells in the same direction. One such example was rs17758695 located in intron 1 of BCL2, an anti-apoptotic protein known to regulate cell death similarly across multiple hematopoietic cell types. In contrast, the remaining 9% of pleiotropic variants favored one lineage at the expense of others (switch mechanism), including novel variants near key myeloid-determining transcription factors CEBPA and MYC (rs78744187 and rs562240450). Together, these results suggest that pleiotropic variants 1) preferentially act in common progenitor rather than terminal cell types, and 2) predominantly tune multiple traits in the same direction, but may favor one at the expense of others when influencing lineage commitment. Finally, given the enrichment of fine-mapped variants in common progenitor states, we set out to determine whether classically defined hematopoietic populations could be divided into lineage-biased subpopulations based on differential genetic regulation of blood traits. To do so, we measured the enrichment of fine-mapped variants in the chromatin landscapes of 2,034 single cells isolated from 8 hematopoietic progenitor populations. Strikingly, we discovered significant heterogeneity within the common myeloid progenitor (CMP) population, in which one subset of cells exhibited greater open-chromatin enrichment for myeloid trait variants and relevant transcription factor (TF) binding (CEBPA, IRF8), whereas the other subset showed enrichment for erythroid trait variants and TFs (GATA1, KLF1). By integrating genetic fine-mapping with chromatin data, we identified hundreds of causal variants regulating 16 blood traits, characterized novel mechanisms of pleiotropic effects, and discovered cell states enriched for blood trait regulation. These findings provide new insights into the importance of genetic regulation in progenitor cell states and will contribute to knowledge of how these processes go awry in diseases of blood cell production. Disclosures No relevant conflicts of interest to declare.

Defense against attaching-and-effacing bacteria requires the sequential generation of interleukin 23 (IL-23) and IL-22 to induce protective mucosal responses. Although CD4(+) and NKp46(+) innate lymphoid cells (ILCs) are the critical source of IL-22 during infection, the precise source of IL-23 is unclear. We used genetic techniques to deplete mice of specific subsets of classical dendritic cells (cDCs) and analyzed immunity to the attaching-and-effacing pathogen Citrobacter rodentium. We found that the signaling receptor Notch2 controlled the terminal stage of cDC differentiation. Notch2-dependent intestinal CD11b(+) cDCs were an obligate source of IL-23 required for survival after infection with C. rodentium, but CD103(+) cDCs dependent on the transcription factor Batf3 were not. Our results demonstrate a nonredundant function for CD11b(+) cDCs in the response to pathogens in vivo.

Spatial organization of the genome plays a central role in gene expression, DNA replication, and repair. But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization (ATAC-see), a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ, cell sorting, and deep sequencing to reveal the identity of the imaged elements. ATAC-see revealed the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion, termed NETosis. Integration of ATAC-see with flow cytometry enables automated quantitation and prospective cell isolation as a function of chromatin accessibility, and it reveals a cell-cycle dependence of chromatin accessibility that is especially dynamic in G1 phase. The integration of imaging and epigenomics provides a general and scalable approach for deciphering the spatiotemporal architecture of gene control.

CD11c+ cells increase greatly with islet inflammation in non-obese diabetic mice and contribute to autoimmune destruction of pancreatic beta cells. In this study, we investigated their origin and mechanism of recruitment. CD11c+ cells in inflamed islets resembled classical dendritic cells (DC) based on their transcriptional profile. However, the majority of these cells were not from the Zbtb46-dependent DC lineage. Instead, monocyte precursors could give rise to CD11c+ cells in inflamed islets. Chemokines Ccl5 and Ccl8 were persistently elevated in inflamed islets and the influx of CD11c+ cells was partially dependent on their receptor Ccr5. Treatment with islet antigen-specific regulatory T cells (Tregs) led to a marked decrease of Ccl5 and Ccl8 and a reduction of monocyte recruitment. These results implicate a monocytic origin of CD11c+ cells in inflamed islets and suggest that therapeutic Tregs directly or indirectly regulate their influx by altering the chemotactic milieu in the islets.

International audience; Long intergenic noncoding RNAs (lincRNA) are important regulators of gene expression. Although lincRNAs are expressed in immune cells, their functions in immunity are largely unexplored. Here we identify an immunoregulatory lincRNA, lincRNA-EPS, that is precisely regulated in macrophages to control the expression of immune response genes (IRGs). Transcriptome analysis of macrophages from lincRNA-EPS-deficient mice, combined with gain-of-function and rescue experiments, revealed a specific role for this lincRNA in restraining IRG expression. Consistently, lincRNA-EPS-deficient mice manifest enhanced inflammation and lethality following endotoxin challenge in vivo. lincRNA-EPS associates with chromatin at regulatory regions of IRGs to control nucleosome positioning and repress transcription. Further, lincRNA-EPS mediates these effects by interacting with heterogeneous nuclear ribonucleoprotein L via a CANACA motif located in its 3′ end. Together, these findings identify lincRNA-EPS as a repressor of inflammatory responses highlighting the importance of lincRNAs in the immune system.

Activation of naïve cluster of differentiation (CD)8 + cytotoxic T lymphocytes (CTLs) is a tightly regulated process, and specific dendritic cell (DC) subsets are typically required to activate naive CTLs. Potential pathways for antigen presentation leading to CD8 + T-cell priming include direct presentation, cross-presentation, and cross-dressing. To distinguish between these pathways, we designed single-chain trimer (SCT) peptide–MHC class I complexes that can be recognized as intact molecules but cannot deliver antigen to MHC through conventional antigen processing. We demonstrate that cross-dressing is a robust pathway of antigen presentation following vaccination, capable of efficiently activating both naïve and memory CD8 + T cells and requires CD8α + /CD103 + DCs. Significantly, immune responses induced exclusively by cross-dressing were as strong as those induced exclusively through cross-presentation. Thus, cross-dressing is an important pathway of antigen presentation, with important implications for the study of CD8 + T-cell responses to viral infection, tumors, and vaccines.

Myeloid ecotropic viral integration site 1 (Meis1) forms a heterodimer with Pbx1 that augments Hox-dependent gene expression and is associated with leukemogenesis and HSC self-renewal. Here we identified 2 independent actions of Meis1 in hematopoietic development: one regulating cellular proliferation and the other involved in megakaryocyte lineage development. First, we found that endogenous Mesp1 indirectly induces Meis1 and Meis2 in endothelial cells derived from embryonic stem cells. Overexpression of Meis1 and Meis2 greatly enhanced the formation of hematopoietic colonies from embryonic stem cells, with the exception of erythroid colonies, by maintaining hematopoietic progenitor cells in a state of proliferation. Second, overexpression of Meis1 repressed the development of early erythroid progenitors, acting in vivo at the megakaryocyte-erythroid progenitor stage to skew development away from erythroid generation and toward megakaryocyte development. This previously unrecognized action of Meis1 may explain the embryonic lethality observed in Meis1−/− mice that arises from failure of lymphatic-venous separation and can result as a consequence of defective platelet generation. These results show that Meis1 exerts 2 independent functions, with its role in proliferation of hematopoietic progenitors acting earlier in development from its influence on the fate choice at the megakaryocyte-erythroid progenitor between megakaryocytic and erythroid development.

The zinc finger transcription factor Zbtb46 specifically marks cDCs and their committed precursors and, when overexpressed in BM progenitors, promotes cDC development at the expense of granulocytes., Distinguishing dendritic cells (DCs) from other cells of the mononuclear phagocyte system is complicated by the shared expression of cell surface markers such as CD11c. In this study, we identified Zbtb46 (BTBD4) as a transcription factor selectively expressed by classical DCs (cDCs) and their committed progenitors but not by plasmacytoid DCs (pDCs), monocytes, macrophages, or other lymphoid or myeloid lineages. Using homologous recombination, we replaced the first coding exon of Zbtb46 with GFP to inactivate the locus while allowing detection of Zbtb46 expression. GFP expression in Zbtb46gfp/+ mice recapitulated the cDC-specific expression of the native locus, being restricted to cDC precursors (pre-cDCs) and lymphoid organ– and tissue-resident cDCs. GFP+ pre-cDCs had restricted developmental potential, generating cDCs but not pDCs, monocytes, or macrophages. Outside the immune system, Zbtb46 was expressed in committed erythroid progenitors and endothelial cell populations. Zbtb46 overexpression in bone marrow progenitor cells inhibited granulocyte potential and promoted cDC development, and although cDCs developed in Zbtb46gfp/gfp (Zbtb46 deficient) mice, they maintained expression of granulocyte colony-stimulating factor and leukemia inhibitory factor receptors, which are normally down-regulated in cDCs. Thus, Zbtb46 may help enforce cDC identity by restricting responsiveness to non-DC growth factors and may serve as a useful marker to identify rare cDC progenitors and distinguish between cDCs and other mononuclear phagocyte lineages.

While most blood lineages are assumed to mature through a single cellular and developmental route downstream of HSCs, dendritic cells (DCs) can be derived from both myeloid and lymphoid progenitors in vivo. To determine how distinct progenitors can generate similar downstream lineages, we examined the transcriptional changes that accompany loss of in vivo myeloid potential as common myeloid progenitors differentiate into common DC progenitors (CDPs), and as lymphoid-primed multipotent progenitors (LMPPs) differentiate into all lymphoid progenitors (ALPs). Microarray studies revealed that IFN regulatory factor 8 (IRF-8) expression increased during each of these transitions. Competitive reconstitutions using Irf8−/− BM demonstrated cell-intrinsic defects in the formation of CDPs and all splenic DC subsets. Irf8−/− common myeloid progenitors and, unexpectedly, Irf8−/− ALPs produced more neutrophils in vivo than their wild-type counterparts at the expense of DCs. Retroviral expression of IRF-8 in multiple progenitors led to reduced neutrophil production and increased numbers of DCs, even in the granulocyte-macrophage progenitor (GMP), which does not normally possess conventional DC potential. These data suggest that IRF-8 represses a neutrophil module of development and promotes convergent DC development from multiple lymphoid and myeloid progenitors autonomously of cellular context.

Dendritic cells (DCs) are a heterogeneous population within the mononuclear phagocyte system (MPS) that derive from bone marrow precursors. Commitment and specification of hematopoietic progenitors to the DC lineage is critical for the proper induction of both immunity and tolerance. This review summarizes the important cytokines and transcription factors required for differentiation of the DC lineage as well as further diversification into specific DC subsets. We highlight recent advances in the characterization of immediate DC precursors arising from the common myeloid progenitor (CMP). Particular emphasis is placed on the corresponding temporal expression of relevant factors involved in regulating developmental options.

Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of B cell leukemia. However, application of CAR T cell therapy in solid tumors has been disappointing. We believe the development of T cell exhaustion, a phenomenon well described in chronic viral infection, is a major barrier to widespread success of CAR T cell therapy in other types of cancer. Therefore, understanding mechanisms of and engineering solutions to avoid the development of CAR T cell exhaustion is highly warranted. Here, we utilize a high affinity (HA) GD2-directed CAR model in which T cells undergo chronic activation via spontaneous clustering of surface CAR molecules. Tonically signaling HA-28Z CAR T cells demonstrate phenotypic and functional characteristics of exhaustion with high expression of inhibitory receptors (PD-1, TIM-3, LAG-3, and CD39), poor cytokine production, enhanced effector differentiation, and failure to eradicate disease upon transfer into tumor-bearing mice. To interrogate the transcriptional and epigenetic programs driving CAR T cell exhaustion, we performed total RNA-seq and ATAC-seq using HA-28Z or control CD19-28Z CAR T cells. The most significantly enriched transcription factor motifs in HA-specific open chromatin peaks belonged to the AP1 family, suggesting AP1 factors may play an integral role in mediating exhaustion. RNA-seq and western blot confirmed increased expression of AP1 factors in exhausted CAR T cells. Interestingly, exhausted HA-28Z CAR T cells demonstrate insufficient IL2 production, a gene classically regulated by AP1 Fos/cJun heterodimers, although Fos and cJun are present in CAR T cells. However, other potentially inhibitory AP1 family members (including JunB, BATF, ATF, and IRF family members) are overexpressed to a greater degree. We hypothesize that the relative balance of classical and inhibitory AP1 family members may contribute to exhaustion. To disrupt this balance, we constitutively overexpressed Fos and cJun in exhausted HA-28Z CAR T cells and observed that JUN+ CAR T cells demonstrated increased IL2 and IFNg production and reduced inhibitory receptor expression. Furthermore, adoptively transferred JUN-HA-28Z CAR T cells resulted in rapid tumor clearance and long term survival of NSG mice bearing GD2+ leukemia whereas control HA-28Z CAR T cells could not overcome tumor growth. Co-immunoprecipitation experiments show high levels of JunB/BATF3/IRF4 complexed with cJun in exhausted HA-28Z CAR T cells. Preliminary data using CRISPR-Cas9 gene disruption suggest that knocking out inhibitory AP1 factors in HA-28Z CAR T cells can also enhance functional activity. Together, our data highlight the AP1 family in regulating T cell dysfunction upon chronic antigen encounter. Finally, we demonstrate that AP1 factors can be engineered to produce superior T cells for adoptive cell therapy. Citation Format: Rachel C. Lynn, Evan W. Weber, David Gennert, Elena Sotillo, Robert Jones, Peng Xu, Ansuman Satpathy, Howard Y. Chang, Crystal L. Mackall. Engineering AP1 to combat CAR T cell exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-112.

Regulatory T cells (Tregs) are a specialized subset of CD4+ T cells that suppress inflammation to maintain homeostasis and prevent autoimmunity. Treg development and function depend on expression of the master transcription factor Foxp3. While Tregs have been thought to be irreversibly committed to suppressive functions, lineage tracing studies have challenged this by revealing that Tregs can exhibit plasticity. Tregs that lose Foxp3 expression, termed ‘exTregs’, have been shown to acquire cytokine production capabilities of pro-inflammatory effector T cells and exacerbate autoimmunity. However, the gene regulatory programs that promote or disrupt Foxp3 stability in Tregs under various physiological conditions are not well understood. Here we have leveraged improved functional genetic tools, including pooled CRISPR screens, to identify nuclear factors that regulate Treg plasticity. A Foxp3 lineage-tracing reporter mouse model was used to confidently distinguish Foxp3− exTregs from contaminating Foxp3−T effectors. Using HiChIP, we identified Treg and exTreg-specific chromatin loops and systematically assessed their contribution to Foxp3 stability. Furthermore, we investigated transcriptional signatures of distinct exTreg populations residing in the peripheral organs using single-cell RNA sequencing. Collectively, this work helps us better understand the genetic networks that control stable expression of Foxp3 in Tregs as they encounter changing inflammatory environments.

Regulatory T cells (Treg) play a fundamental role in maintaining immune tolerance. These cells are defined and characterized by expression of the master regulator Foxp3, a transcription factor that is crucial for Treg function and homeostasis. Natural occurring mutations in Foxp3 gene have been shown to be responsible for the immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, which is characterized by widespread autoimmune diseases. We recently identified a Foxp3 mutation in a boy that manifested a severe IPEX syndrome. This mutation, resulting in the substitution of methionine for isoleucine at amino acid 370 was determined to be part of a class of Foxp3 mutations that disrupt the dimerization motif in the domain swap interface of the protein. We have shown that, although dimerization mutants retain DNA binding, allows Treg development and certain Treg characteristics, it inevitably led to an IPEX syndrome in humans and mice. Genomic analysis of M370I mutant Treg by RNAseq, ChIPseq and HiChIP revealed a de-repression of the T helper type 2 (Th2) transcriptional program leading to the generation of Th2-like Treg. We have shown that, dimerization mutant proteins bind to multiple locations in the Th2 locus leading to increased intrachromosomal interactions and Th2 cytokines production. As expected, M370I Tregs were unable to suppress Th2 immune responses. Transgenic mice expressing M370I Foxp3 developed autoimmune diseases characterized by systemic Th2 cytokines production and skin and lung inflammation. In conclusion, disruption of the domain swap interface of Foxp3 by M370I mutation led to the development of Th2-like Treg which are functionally unable to suppress the development/function of Th2 cells.

FoxP3 + CD4 + regulatory T cells (Tregs) play a key role in the maintenance of peripheral self-tolerance, and it has been suggested that diabetes-susceptible nonobese diabetic (NOD) mice are defective in the generation and numbers of Tregs. We found thymic selection of Tregs to be under genetic control. Fetal thymic organ cultures on the NOD background required 3- to 10-fold more antigen than corresponding cultures on the B6 background for optimal induction of Tregs, but once the threshold for induction was reached the NOD background yielded close to 10-fold more Tregs. This increased selection of Tregs was also found in nontransgenic NOD mice in fetal through adult stages. This trait did not map to the MHC, idd3 , or the chromosome 3 ( Chr3 ) regions that control clonal deletion, but mainly to two regions on Chr1 and Chr11 . Thus, NOD mice do not have a global defect in the generation or maintenance of Tregs; if anything, they show the opposite.

Runx1 and Cbfβ are critical for the establishment of definitive hematopoiesis and are implicated in leukemic transformation. Despite the absolute requirements for these factors in the development of hematopoietic stem cells and lymphocytes, their roles in the development of bone marrow progenitor subsets have not been defined. Here, we demonstrate that Cbfβ is essential for the development of Flt3(+) macrophage-dendritic cell (DC) progenitors in the bone marrow and all DC subsets in the periphery. Besides the loss of DC progenitors, pan-hematopoietic Cbfb-deficient mice also lack CD105(+) erythroid progenitors, leading to severe anemia at 3 to 4 months of age. Instead, Cbfb deficiency results in aberrant progenitor differentiation toward granulocyte-macrophage progenitors (GMPs), resulting in a myeloproliferative phenotype with accumulation of GMPs in the periphery and cellular infiltration of the liver. Expression of the transcription factor Irf8 is severely reduced in Cbfb-deficient progenitors, and overexpression of Irf8 restors DC differentiation. These results demonstrate that Runx proteins and Cbfβ restrict granulocyte lineage commitment to facilitate multilineage hematopoietic differentiation and thus identify their novel tumor suppressor function in myeloid leukemia.