Spatial localization is a key determinant of cellular fate and behavior, but methods for spatially resolved, transcriptome-wide gene expression profiling across complex tissues are lacking. RNA ...staining methods assay only a small number of transcripts, whereas single-cell RNA-seq, which measures global gene expression, separates cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos and generated a transcriptome-wide map of spatial patterning. We confirmed Seurat's accuracy using several experimental approaches, then used the strategy to identify a set of archetypal expression patterns and spatial markers. Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems.
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IJS, NUK, SBMB, UL, UM, UPUK
Pluripotent stem cells (PSCs) are capable of dynamic interconversion between distinct substates; however, the regulatory circuits specifying these states and enabling transitions between them are not ...well understood. Here we set out to characterize transcriptional heterogeneity in mouse PSCs by single-cell expression profiling under different chemical and genetic perturbations. Signalling factors and developmental regulators show highly variable expression, with expression states for some variable genes heritable through multiple cell divisions. Expression variability and population heterogeneity can be influenced by perturbation of signalling pathways and chromatin regulators. Notably, either removal of mature microRNAs or pharmacological blockage of signalling pathways drives PSCs into a low-noise ground state characterized by a reconfigured pluripotency network, enhanced self-renewal and a distinct chromatin state, an effect mediated by opposing microRNA families acting on the Myc/Lin28/let-7 axis. These data provide insight into the nature of transcriptional heterogeneity in PSCs.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
T cells create vast amounts of diversity in the genes that encode their T cell receptors (TCRs), which enables individual clones to recognize specific peptide-major histocompatibility complex (MHC) ...ligands. Here we combined sequencing of the TCR-encoding genes with assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis at the single-cell level to provide information on the TCR specificity and epigenomic state of individual T cells. By using this approach, termed transcript-indexed ATAC-seq (T-ATAC-seq), we identified epigenomic signatures in immortalized leukemic T cells, primary human T cells from healthy volunteers and primary leukemic T cells from patient samples. In peripheral blood CD4
T cells from healthy individuals, we identified cis and trans regulators of naive and memory T cell states and found substantial heterogeneity in surface-marker-defined T cell populations. In patients with a leukemic form of cutaneous T cell lymphoma, T-ATAC-seq enabled identification of leukemic and nonleukemic regulatory pathways in T cells from the same individual by allowing separation of the signals that arose from the malignant clone from the background T cell noise. Thus, T-ATAC-seq is a new tool that enables analysis of epigenomic landscapes in clonal T cells and should be valuable for studies of T cell malignancy, immunity and immunotherapy.
Dysfunction in T cells limits the efficacy of cancer immunotherapy. We profiled the epigenome, transcriptome, and enhancer connectome of exhaustion-prone GD2-targeting HA-28z chimeric antigen ...receptor (CAR) T cells and control CD19-targeting CAR T cells, which present less exhaustion-inducing tonic signaling, at multiple points during their ex vivo expansion. We found widespread, dynamic changes in chromatin accessibility and three-dimensional (3D) chromosome conformation preceding changes in gene expression, notably at loci proximal to exhaustion-associated genes such as
,
, and
, and increased DNA motif access for AP-1 family transcription factors, which are known to promote exhaustion. Although T cell exhaustion has been studied in detail in mice, we find that the regulatory networks of T cell exhaustion differ between species and involve distinct loci of accessible chromatin and cis-regulated target genes in human CAR T cell exhaustion. Deletion of exhaustion-specific candidate enhancers of
suppress the expression of PD-1 in an in vitro model of T cell dysfunction and in HA-28z CAR T cells, suggesting enhancer editing as a path forward in improving cancer immunotherapy.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
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).
For the past several decades, due to technical limitations, the field of transcriptomics has focused on population-level measurements that can mask significant differences between individual cells. ...With the advent of single-cell RNA-Seq, it is now possible to profile the responses of individual cells at unprecedented depth and thereby uncover, transcriptome-wide, the heterogeneity that exists within these populations. This unit describes a method that merges several important technologies to produce, in high-throughput, single-cell RNA-Seq libraries. Complementary DNA (cDNA) is made from full-length mRNA transcripts using a reverse transcriptase that has terminal transferase activity. This, when combined with a second "template-switch" primer, allows for cDNAs to be constructed that have two universal priming sequences. Following preamplification from these common sequences, Nextera XT is used to prepare a pool of 96 uniquely indexed samples ready for Illumina sequencing.
Chimeric antigen receptor (CAR) T cells mediate anti-tumour effects in a small subset of patients with cancer
, 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 with a tonically signaling CAR, which induces hallmark features of exhaustion
. Exhaustion was associated with a profound defect in the production of IL-2, along with increased chromatin accessibility of AP-1 transcription factor motifs and overexpression of the bZIP and IRF transcription factors that have been implicated in mediating dysfunction in exhausted T cells
. Here we show that CAR T cells engineered to overexpress the canonical AP-1 factor c-Jun have enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved anti-tumour potency in five different mouse tumour models in vivo. 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 resistant to exhaustion, thereby addressing a major barrier to progress for this emerging class of therapeutic agents.
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IJS, KISLJ, NUK, UL, UM, UPUK
Reversing the dysfunctional T cell state that arises in cancer and chronic viral infections is the focus of therapeutic interventions; however, current therapies are effective in only some patients ...and some tumor types. To gain a deeper molecular understanding of the dysfunctional T cell state, we analyzed population and single-cell RNA profiles of CD8+ tumor-infiltrating lymphocytes (TILs) and used genetic perturbations to identify a distinct gene module for T cell dysfunction that can be uncoupled from T cell activation. This distinct dysfunction module is downstream of intracellular metallothioneins that regulate zinc metabolism and can be identified at single-cell resolution. We further identify Gata-3, a zinc-finger transcription factor in the dysfunctional module, as a regulator of dysfunction, and we use CRISPR-Cas9 genome editing to show that it drives a dysfunctional phenotype in CD8+ TILs. Our results open novel avenues for targeting dysfunctional T cell states while leaving activation programs intact.
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•Distinct gene modules for T cell dysfunction and activation can be uncoupled•Single-cell profiling of CD8 TILs shows that these modules are exclusive•Metallothioneins, zinc regulators, promote T cell dysfunction•CRISPR-Cas9 targeting shows Gata-3, a zinc-finger TF, promotes dysfunction
Single-cell profiling of tumor-infiltrating lymphocytes identifies critical regulators of T cell dysfunction in cancer, opening new avenues for targeting dysfunctional T cell states while leaving activation programs intact.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
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.
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