T cell exhaustion limits immune responses against cancer and is a major cause of resistance to chimeric antigen receptor (CAR)–T cell therapeutics. Using murine xenograft models and an in vitro model wherein tonic CAR signaling induces hallmark features of exhaustion, we tested the effect of transient cessation of receptor signaling, or rest, on the development and maintenance of exhaustion. Induction of rest through enforced down-regulation of the CAR protein using a drug-regulatable system or treatment with the multikinase inhibitor dasatinib resulted in the acquisition of a memory-like phenotype, global transcriptional and epigenetic reprogramming, and restored antitumor functionality in exhausted CAR-T cells. This work demonstrates that rest can enhance CAR-T cell efficacy by preventing or reversing exhaustion, and it challenges the notion that exhaustion is an epigenetically fixed state.

No abstract

Reconstructing lineage relationships in complex tissues can reveal mechanisms underlying development and disease. Recent methods combine single-cell transcriptomics with mitochondrial DNA variant detection to establish lineage relationships in primary human cells, but are not scalable to interrogate complex tissues. To overcome this limitation, here we develop a technology for highconfidence detection of mitochondrial mutations from high-throughput single-cell RNA-sequencing. We use the new method to identify skewed immune cell expansions in primary human clonal hematopoiesis. Main textSingle-cell RNA-sequencing (scRNA-seq) enables the unbiased assessment of cell states in health and disease 1,2 . Combined acquisition of cell state and genetic information can provide additional insight, such as targeted enrichment of cancer driver mutations from single-cell transcriptomes 3,4 . Separately, combining scRNA-seq with genetic cell barcodes is a powerful method to reveal clonal relationships and evolutionary dynamics of cells within organisms 5,6 . However, this has largely been limited to experimental model systems that can be genetically manipulated to insert cell barcodes. To infer clonal dynamics in primary human cells, recent methods have detected and utilized mitochondrial DNA (mtDNA) mutations as naturally occurring genetic cell barcodes [7][8][9] . The combination of scRNA-seq with mtDNA mutation detection can inform clonal relationships with high confidence, but is currently restricted to expensive, low-throughput, full-length transcript sequencing technologies like SmartSeq2 7,10 . To enable the reconstruction of clonal relationships in complex human tissues, we developed a method that captures genetic variants from high-throughput scRNA-seq platforms: MAESTER, or Mitochondrial Alteration Enrichment from Singlecell Transcriptomes to Establish Relatedness (Figure 1A). MAESTER is compatible with the most common high-throughput scRNA-seq platforms, including 10x Genomics 3' protocols, Seq-Well S 3 , and Drop-seq (Supplemental Figures 1-3) 11,12 . An intermediate step in each of these platforms yields full length cDNA transcripts, from which we enrich all 15 mitochondrial transcripts using pools of primers, while maintaining cell-identifying barcodes (Figure 1B, Supplemental Figure 4). Standard next-generation sequencing with 250 bp reads is then used to obtain the sequence of the amplified mitochondrial transcripts (Figure 1A). We developed a computational toolkit to call mtDNA variants from MAESTER data, the Mitochondrial Alteration Enrichment and Genome Analysis Toolkit (maegatk, Supplemental Figure 5, Methods). Building on previous tools that we developed 8 for mtDNA variant detection from single-cell ATAC or SmartSeq2, maegatk specifically handles technical biases implicit in high-throughput transcriptomic libraries. Critically, maegatk leverages unique molecular identifiers (UMIs) to collapse multiple sequencing reads of the same starting transcript, creating a consensus call for every

SUMMARY Transcriptional and chromatin regulations mediate the liver response to nutrient availability. The role of chromatin factors involved in hormonal regulation in response to fasting is not fully understood. We have identified SETDB2, a glucocorticoid-induced putative epigenetic modifier, as a positive regulator of GR-mediated gene activation in liver. Insig2a increases during fasting to limit lipid synthesis, but the mechanism of induction is unknown. We show Insig2a induction is GR-SETDB2-dependent. SETDB2 facilitates GR chromatin enrichment and is key to glucocorticoid dependent enhancer-promoter interactions. INSIG2 is a negative regulator of SREBP and acute glucocorticoid treatment decreased active SREBP during refeeding or in livers of Ob/Ob mice; both systems of elevated SREBP-1c driven lipogenesis. Knockdown of SETDB2 or INSIG2 reversed the inhibition on active SREBPs. Overall, these studies identify a GR-SETDB2 regulatory axis of hepatic transcriptional reprogramming and identify SETDB2 as a potential target for metabolic disorders with aberrant glucocorticoid actions.

Extrathymic Aire-expressing cells are hematopoietic migratory DC–like populations with homology to medullary thymic epithelium.

T cells are the major arm of the immune system responsible for controlling and regressing cancers. To identify genes limiting T cell function, we conducted genome-wide CRISPR knockout screens in human chimeric antigen receptor (CAR) T cells. Top hits were MED12 and CCNC , components of the Mediator kinase module. Targeted MED12 deletion enhanced antitumor activity and sustained the effector phenotype in CAR- and T cell receptor–engineered T cells, and inhibition of CDK8/19 kinase activity increased expansion of nonengineered T cells. MED12 -deficient T cells manifested increased core Meditator chromatin occupancy at transcriptionally active enhancers—most notably for STAT and AP-1 transcription factors—and increased IL2RA expression and interleukin-2 sensitivity. These results implicate Mediator in T cell effector programming and identify the kinase module as a target for enhancing potency of antitumor T cell responses.

No abstract

The Autoimmune Regulator (Aire) gene, well defined for its role in medullary thymic epithelial cells (mTECs) and immune self-tolerance, is also expressed in extrathymic Aire-expressing cells (eTACs) in the secondary lymphoid organs. eTACs have been shown to be hematopoietic antigen presenting cells (APCs) and potent inducers of immune tolerance (1–3). However, the precise identity and function of these cells remain unclear. Here, we use high-dimensional single-cell multiomics and functional approaches to define eTACs at the transcriptional, genomic, and proteomic level. We find that eTACs consist of two similar cell types: CCR7+ Aire-expressing migratory dendritic cells (AmDCs) and a unique Aire-hi population co-expressing Aire and RAR-related orphan receptor gamma-t (RORγt). The latter, which have significant transcriptional and genomic homology to migratory dendritic cells (migDCs) and mTECs, we term Janus cells (JCs). All eTACs, and JCs in particular, have a highly accessible chromatin structure and high levels of broad gene expression, including tissue-specific antigens, as well as remarkable transcriptional and genomic homology to thymic medullary epithelium. As in the thymus, Aire expression in eTACs is also dependent on RANK-RANK-ligand interactions. Furthermore, lineage-tracing shows that JCs are not precursors to the majority of AmDCs. Finally, self-antigen expression by eTACs is sufficient to mediate negative selection of T cells escaping thymic selection and can prevent autoimmune diabetes in non-obese diabetic mice. This transcriptional, genomic, and functional symmetry between a hematopoietic Aire-expressing population in the periphery and an epithelial Aire-expressing population in the thymus suggests that a core biological program may influence self-tolerance and self-representation across the spectrum of immune development.