Biology subjects

Satpathy, A. T.

Publications and source records attributed to Satpathy, A. T..

At least 19 recordsLinked to original sources

bioRxiv2026

Systematic identification of seed-driven off-target effects in Perturb-seq experiments

Genome-wide Perturb-seq (GWPS) has emerged as a powerful approach for unbiased mapping of gene regulatory networks. A key assumption underlying many Perturb-seq analyses is that each guide RNA exclusively perturbs a single target locus. Without methods to identify and filter off-target events, erroneous gene-pathway associations driven by off-target activity can propagate into downstream analyses. Here, we present a workflow for the systematic identification of candidate off-target events in CRISPRi Perturb-seq experiments. Our approach exploits the observation that cells harboring a guide which represses an off-target gene display transcriptional similarity to cells in which that gene is directly targeted by an on-target guide. We apply our workflow to multiple GWPS datasets and nominate off-target events in which a guide nominally targeting one gene also represses a distinct gene producing a phenotype likely attributable to the off-target perturbation. We use both off-target gene repression and guide seed sequence alignments at the off-target promoter locus as evidence for off-target effects and find independent evidence of putative off-target events in separate GWPS datasets. Together, these results establish a principled framework for the identification and filtering of off-target guide effects in Perturb-seq experiments.

genomics↗

bioRxiv2026

Comparing bulk and single-cell methodologies and models to profile gene expression, chromatin accessibility and regulatory links in endothelial cells treated with TNFα

Genome-wide association studies (GWAS) have identified thousands of non-coding variants associated with complex traits and diseases. However, it remains challenging to pinpoint the causal genes that are regulated by associated genetic variants. Connecting causal non-coding variants with genes can rely on methods that identify direct physical interactions (e.g. chromosome conformation capture) or on probabilistic models that predict regulatory links. These statistical models take advantage of gene expression and chromatin accessibility profiles generated in cells and tissues by bulk or single-cell (sc) methodologies. Here, we tested whether using bulk or sc RNAseq/ATACseq data and corresponding predictive enhancer-to-gene models impact the prioritization of causal GWAS genes. Using non-treated and TNF-treated human endothelial cells in vitro as a well-controlled experimental system, we show that bulk and sc RNAseq/ATACseq profiles are similar and highlight the same biology (e.g. biological pathways). Despite these similarities, we show using GWAS results for coronary artery disease (CAD) and diastolic blood pressure that applying enhancer-to-gene models designed for bulk or sc methodologies can yield differences in terms of captured heritability, fine-mapped variants and linked genes. For instance, at one CAD locus, the bulk-based ABC model predicts a regulatory link with BCAR1, whereas the sc-based model scE2G prioritizes a different gene (CFDP1). On the same experimental model, our results indicate that choosing between a bulk or sc approach will influence regulatory link model predictions; this should be considered when planning functional experiments to characterize GWAS discoveries.

genomics↗

bioRxiv2026

Molecular pixelation of the CAR T cell surface proteome

Immunotherapies using CAR T cells are revolutionizing B-cell acute lymphoblastic leukemia treatments. However, the majority of patients remain unresponsive, and chronic stimulation of T cells is a common contributor that reduces effector function and persistence. We apply Molecular Pixelation, a recently developed single-cell technology for characterizing cellular surface proteomes, to determine characteristic topological surface-based proteomic signatures of CAR T cell exhaustion. We analyze 76 surface proteins on 8504 CAR T cells at a single-cell level, collected from three donors and either stimulated once or repeatedly, six times over two weeks. The abundances, polarizations, and colocalizations of surface proteins can each distinguish CAR T cells that were stimulated acutely or chronically, and all but one marker with polarization changes increased in polarization. These data also reveal disrupted adhesion signatures of protein colocalization in the peripheral supramolecular activation complex (pSMAC) and increased CD37/CD82 colocalization after chronic stimulation. These Molecular Pixelation results convey new spatial signatures for proteomic polarization and colocalization on the cell surface that represent new cell-state axes for immunology and systems biology.

systems biology↗

bioRxiv2025

Bispecific T cell engagers control solid tumors through clonal replacement and IL2-driven effector differentiation of CD8 T-cells

Bispecific T cell engagers (TCEs) often exhibit limited efficacy in solid tumors, in part due to immunosuppressive cues in the tumor microenvironment and low expression of targetable tumor antigens. Therapeutic strategies to improve TCE target sensitivity and enhance T cell effector functions therefore have significant translational potential. Here, we engineered TCEs that induce T cell activation in vitro against the low-abundance target antigens, TRP2/Kb and DLL3. Despite in vitro activity in these models, TCE monotherapy showed limited control of tumor growth in immunocompetent mice. Leveraging this in vivo model of TCE treatment failure, we discovered that co-treatment with TCE and a CD25-biased Interleukin-2 (IL2) rescues anti-tumor activity. Further, multimodal single-cell transcriptomic and immune repertoire analyses revealed that TCE-IL2 combination therapy controlled tumors by recruiting and activating new CD8+ T cells into the tumor microenvironment. These findings demonstrate that TCE-mediated anti-tumor responses function through a CD8+ T cell clonal replacement mechanism that can be augmented by cytokine therapy. One Sentence SummaryCombining TCE therapy with IL-2 enhances TCE efficacy in aggressive small-cell lung cancer and melanoma models with low target antigen density through T cell clonal replacement and CD8+ effector T cell differentiation.

cancer biology↗

bioRxiv2025

A unified genetic perturbation language for human cellular programming

Evolution simultaneously and combinatorially explores complex genetic changes across perturbation classes, including gene knockouts, knockdowns, overexpression, and the creation of new genes from existing domains. Separate technologies are capable of genetic perturbations at scale in human cells, but these methods are largely mutually incompatible. Here we present CRISPR-All, a unified genetic perturbation language for programming of any major type of genetic perturbation simultaneously, in any combination, at genome scale, in primary human cells. This is enabled by a standardized molecular architecture for each major perturbation class, development of a functional syntax for combining arbitrary numbers of elements across classes, and linkage to unique single cell compatible barcodes. To facilitate use, CRISPR-All converts high level descriptions of desired complex genetic changes into a single DNA sequence that can rewire genomic programs within a cell. Using the CRISPR-All language allowed for head-to-head functional comparisons across perturbation types in a comprehensive analysis of all previously identified genetic enhancements of human CAR-T cells. Combining CRISPR-All programs with single cell RNA sequencing revealed a greater diversity of phenotypic states, including improved functional performance, only accessible through distinct perturbation classes. Finally, CRISPR-All combinatorial genome scale screening of up to four distinct perturbations simultaneously revealed additive functional improvements in human T cells accessible only through iterative multiplexing of modifications across perturbation classes. CRISPR-All enables exploration of a combinatorial genetic perturbation space, which may be impactful for biological and clinical applications.

genetics↗

bioRxiv2025

Scalable transcription factor mapping uncovers the regulatory dynamics of natural and synthetic transcription factors in human T cell states

Heterogeneous T cell states are critical in immune responses and have been explored by CRISPR-based and synthetic domain-swapped transcription factor (TF) screens, yielding novel insights and immunotherapeutics. However, a scalable strategy to map TFs in primary human T cells is lacking, which limits our understanding of the functions of critical TFs. We therefore adapted a transposon-based TF mapping strategy termed Calling Cards for primary human CD8 T cells, applying it to five key TFs with undefined binding sites in this cell type: TOX, TOX2, TCF7, SOX4, and RBPJ. To derive biological insights from these data, we developed an analytical framework to integrate TF binding with multi-omic sequencing data, revealing convergence of TOX and TCF7 binding at dynamic enhancers of memory CD8 T cells. We then identified TF co-bound gene programs related to memory and exhaustion states in addition to putative gene targets of known and unappreciated TF roles, including TOX binding at critical genes of both exhaustion and terminal effector memory differentiation. To further scale our TF analysis platform, we modified Calling Cards to create TFlex: a method uniquely suited for multiplexed mapping of paralogous TFs. We applied TFlex to simultaneously map eight natural and domain-swapped TFs in primary human CD8 T cells, which demonstrated that domain-swapped TFs display emergent behavior in binding site selection and transcriptional effects on target genes that cannot be estimated as the sum of their constituent domains. Collectively, our data highlight the importance of scalable TF mapping in primary human T cells to elucidate TF function and the transcriptional regulation of cell states.

immunology↗

bioRxiv2025

Clonal lineage tracing of innate immune cells in human cancer

Innate immune cells constitute the majority of the tumor microenvironment (TME), where they mediate both natural anti-tumor immunity and immunotherapy responses. While single-cell T- and B-cell receptor sequencing has provided fundamental insights into the clonal dynamics of human adaptive immunity, the lack of appropriate tools has precluded similar analysis of innate immune cells. Here, we describe a method that leverages somatic mitochondrial DNA (mtDNA) mutations to reconstruct clonal lineage relationships between single cells across cell types in native human tissues. We jointly sequenced single-cell transposase-accessible chromatin and mtDNA to profile n=124,958 cells from matched tumor, non-involved lung tissue (NILT), and peripheral blood of early-stage non-small cell lung cancer (NSCLC) patients, as well as n=93,757 cells from matched tumor and peripheral blood of ovarian cancer patients. Single-cell concomitant profiling of lineage and cell states of thousands of immune cells resolved clonality across cell types, tissue sites, and malignancies. Clonal tracing of innate immune cells demonstrates that TME-resident myeloid subsets, including macrophages and type 3 dendritic cells (DC3), are clonally linked to both circulating and tissue-infiltrating monocytes. Further, we identify distinct DC-biased and macrophage-biased myeloid clones, enriched in the tumor and NILT, respectively, and find that their circulating monocyte precursors exhibit distinct epigenetic profiles, suggesting that myeloid differentiation fate may be predetermined before TME infiltration. These results delineate the clonal pathways of intratumoral myeloid cell recruitment and differentiation in human cancer and suggest that remodeling of the tumor myeloid compartment may be peripherally programmed.

genomics↗

bioRxiv2025

Cell cycle-coupled transcriptional network orchestrates human B cell fate bifurcation

Bifurcation of activated human B cells into plasmablast (PB) and germinal center precursor (preGC) fates underlies protective and autoimmune antibody responses, yet gene regulatory networks (GRNs) governing the alternative trajectories remain poorly defined. Using temporal single-cell multiomics, we assembled state-specific human B cell GRNs spanning four scales: transcription factor (TF)-to-fate, TF-to-gene, cis-regulatory element (CRE)-to-gene and nucleotide-to-CRE. Applying the framework to in vitro differentiated and tonsil B cells revealed concordant regulatory architectures and impacts of in silico TF perturbations. CRISPR perturbations validated many TF-to-fate and TF-to-gene predictions and revealed a mutually repressive BATF-IRF4/PRDM1 network module. The GRNs were used to predict and interpret effects of autoimmune disease variants, uncovering partitioning of disease risk at distinct TF motifs and B cell states. Predicted functional variants were independently supported by chromatin and expression QTLs and reporter assays. A web application enables exploration of B cell regulatory determinants of vaccine responses and autoimmune diseases.

immunology↗

bioRxiv2025

Functional memory T cells are derived from exhausted clones and expanded by checkpoint blockade

Immune checkpoint blockade can facilitate tumor clearance by T cells, resulting in long term patient survival. However, the capacity of exhausted CD8+ T cells (Tex), present during chronic antigen exposure, to form memory after antigen clearance remains unclear. Here, we performed longitudinal single cell RNA/T cell receptor sequencing and ATAC-sequencing on antigen-specific T cells after the clearance of chronic lymphocytic choriomeningitis virus (LCMV) infection. These data revealed the formation of a robust population of memory CD8+ T cells that transcriptionally, epigenetically, and functionally resemble central memory T cells (Tcm) that form after clearance of acute infection. To lineage trace the origin and memory recall response of Tex-derived memory clones, we utilized T cell receptor sequencing over the course of primary infection and rechallenge. We show that chronic Tcm are a clonally distinct lineage of Tex derived from progenitor exhausted cells, persist long-term in the absence of antigen, and undergo rapid clonal expansion during rechallenge. Finally, we demonstrate that PD-L1 immune checkpoint blockade after chronic LCMV infection preferentially expands clones which form Tcm after clearance. Together, these data support the concept that chronically stimulated T cells form bona fide functional memory T cells through an analogous differentiation pathway to acutely stimulated T cells, which may have significant implications for enhancing immune memory to cancer through checkpoint blockade and vaccination.

immunology↗

bioRxiv2024

Mapping enhancer-gene regulatory interactions from single-cell data

Mapping enhancers and their target genes in specific cell types is crucial for understanding gene regulation and human disease genetics. However, accurately predicting enhancer-gene regulatory interactions from single-cell datasets has been challenging. Here, we introduce a new family of classification models, scE2G, to predict enhancer-gene regulation. These models use features from single-cell ATAC-seq or multiomic RNA and ATAC-seq data and are trained on a CRISPR perturbation dataset including >10,000 evaluated element-gene pairs. We benchmark scE2G models against CRISPR perturbations, fine-mapped eQTLs, and GWAS variant-gene associations and demonstrate state-of-the-art performance at prediction tasks across multiple cell types and categories of perturbations. We apply scE2G to build maps of enhancer-gene regulatory interactions in heterogeneous tissues and interpret noncoding variants associated with complex traits, nominating regulatory interactions linking INPP4B and IL15 to lymphocyte counts. The scE2G models will enable accurate mapping of enhancer-gene regulatory interactions across thousands of diverse human cell types.

genetics↗

bioRxiv2024

Evolutionarily guided transcription factor design programs novel T cell states

Protein-coding genes in the human genome evolved via modular rearrangement of domains from ancestral genes1. Here, we develop a scalable, evolutionarily guided method to assemble novel protein-coding genes from constituent domains within a protein family, termed DESynR (Domain Engineered via Synthesis and Recombination) genes. Using primary human chimeric antigen receptor T cells as a model system, we find that the expression of DESynR Activator Protein-1 (AP-1) transcription factors (TFs) significantly outperforms the overexpression of natural AP-1 TFs in multiple functional assays in vitro and in vivo. Top DESynR AP-1 TFs exhibit non-intuitive architectures of constituent domains, including from TFs that are not canonically expressed in T cells. DESynR AP-1 TFs induce broad transcriptional and epigenetic reprogramming of T cells and, in some cases, lead to the development of non-natural T cell states, engaging gene expression modules from disparate human cell types. Taken together, we demonstrate that novel configurations of existing protein domains may uncover non-evolved genes that program cell states with therapeutically relevant functions.

genetics↗

bioRxiv2024

Transcript-specific enrichment enables profiling rare cell states via scRNA-seq

Single-cell genomics technologies have accelerated our understanding of cell-state heterogeneity in diverse contexts. Although single-cell RNA sequencing (scRNA-seq) identifies many rare populations of interest that express specific marker transcript combinations, traditional flow sorting limits our ability to enrich these populations for further profiling, including requiring cell surface markers with high-fidelity antibodies. Additionally, many single-cell studies require the isolation of nuclei from tissue, eliminating the ability to enrich learned rare cell states based on extranuclear protein markers. To address these limitations, we describe Programmable Enrichment via RNA Flow-FISH by sequencing (PERFF-seq), a scalable assay that enables scRNA-seq profiling of subpopulations from complex cellular mixtures defined by the presence or absence of specific RNA transcripts. Across immune populations (n = 141,227 cells) and fresh-frozen and formalin-fixed paraffin-embedded brain tissue (n = 29,522 nuclei), we demonstrate the sorting logic that can be used to enrich for cell populations via RNA-based cytometry followed by high-throughput scRNA-seq. Our approach provides a rational, programmable method for studying rare populations identified by one or more marker transcripts.

genomics↗

bioRxiv2023

Convergent Epigenetic Evolution Drives Relapse in Acute Myeloid Leukemia

Relapse of acute myeloid leukemia (AML) is highly aggressive and often treatment refractory. We analyzed previously published AML relapse cohorts and found that 40% of relapses occur without changes in driver mutations, suggesting that non-genetic mechanisms drive relapse in a large proportion of cases. We therefore characterized epigenetic patterns of AML relapse using 26 matched diagnosis-relapse samples with ATAC-seq. This analysis identified a relapse-specific chromatin accessibility signature for mutationally stable AML, suggesting that AML undergoes epigenetic evolution at relapse independent of mutational changes. Analysis of leukemia stem cell (LSC) chromatin changes at relapse indicated that this leukemic compartment underwent significantly less epigenetic evolution than non-LSCs, while epigenetic changes in non-LSCs reflected overall evolution of the bulk leukemia. Finally, we used single-cell ATAC-seq paired with mitochondrial sequencing (mtscATAC) to map clones from diagnosis into relapse along with their epigenetic features. We found that distinct mitochondrially-defined clones exhibit more similar chromatin accessibility at relapse relative to diagnosis, demonstrating convergent epigenetic evolution in relapsed AML. These results demonstrate that epigenetic evolution is a feature of relapsed AML and that convergent epigenetic evolution can occur following treatment with induction chemotherapy.

cancer biology↗

bioRxiv2023

The temporal progression of immune remodeling during metastasis.

Tumor metastasis requires systemic remodeling of distant organ microenvironments which impacts immune cell phenotypes, population structure, and intercellular communication networks. However, our understanding of immune phenotypic dynamics in the metastatic niche remains incomplete. Here, we longitudinally assayed lung immune cell gene expression profiles in mice bearing PyMT-driven metastatic breast tumors from the onset of primary tumorigenesis, through formation of the pre-metastatic niche, to the final stages of metastatic outgrowth. Computational analysis of these data revealed an ordered series of immunological changes that correspond to metastatic progression. Specifically, we uncovered a TLR-NF{kappa}B myeloid inflammatory program which correlates with pre-metastatic niche formation and mirrors described signatures of CD14+ activated MDSCs in the primary tumor. Moreover, we observed that cytotoxic NK cell proportions increased over time which illustrates how the PyMT lung metastatic niche is both inflammatory and immunosuppressive. Finally, we predicted metastasis-associated immune intercellular signaling interactions involving Igf1 and Ccl6 which may organize the metastatic niche. In summary, this work identifies novel immunological signatures of metastasis and discovers new details about established mechanisms that drive metastatic progression. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/539153v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1333376org.highwire.dtl.DTLVardef@77c937org.highwire.dtl.DTLVardef@3b78b1org.highwire.dtl.DTLVardef@70d497_HPS_FORMAT_FIGEXP M_FIG C_FIG In briefMcGinnis et al. report a longitudinal scRNA-seq atlas of lung immune cells in mice bearing PyMT-driven metastatic breast tumors and identify immune cell transcriptional states, shifts in population structure, and rewiring of cell-cell signaling networks which correlate with metastatic progression. HighlightsO_LILongitudinal scRNA-seq reveals distinct stages of immune remodeling before, during, and after metastatic colonization in the lungs of PyMT mice. C_LIO_LITLR-NF{kappa}B inflammation correlates with pre-metastatic niche formation and involves both tissue-resident and bone marrow-derived myeloid cell populations. C_LIO_LIInflammatory lung myeloid cells mirror activated primary tumor MDSCs, suggesting that primary tumor-derived cues induce Cd14 expression and TLR-NF{kappa}B inflammation in the lung. C_LIO_LILymphocytes contribute to the inflammatory and immunosuppressive lung metastatic microenvironment, highlighted by enrichment of cytotoxic NK cells in the lung over time. C_LIO_LICell-cell signaling network modeling predicts cell type-specific Ccl6 regulation and IGF1-IGF1R signaling between neutrophils and interstitial macrophages. C_LI

immunology↗

bioRxiv2023

Inosine Induces Stemness Features in CAR T cells and Enhances Potency

Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8+ CAR T cells mediate Ado-induced immunosuppression through CD39/73-dependent Ado production. Knockout of CD39, CD73 or A2aR had modest effects on exhausted CAR T cells, whereas overexpression of Ado deaminase (ADA), which metabolizes Ado to inosine (INO), induced stemness features and potently enhanced functionality. Similarly, and to a greater extent, exposure of CAR T cells to INO augmented CAR T cell function and induced hallmark features of T cell stemness. INO induced a profound metabolic reprogramming, diminishing glycolysis and increasing oxidative phosphorylation, glutaminolysis and polyamine synthesis, and modulated the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR T cell products meeting criteria for clinical dosing. These data identify INO as a potent modulator of T cell metabolism and epigenetic stemness programming and deliver a new enhanced potency platform for immune cell manufacturing. Statement of SignificanceAdenosine is well known to inhibit T cell function and substantial effort has focused on inhibiting adenosine generation and signaling. Here, we show that exhausted T cells are suppressed by adenosine, which is only modestly impacted by inhibiting adenosine generation or signaling. In contrast, metabolism of adenosine to inosine augmented T cell function and culture of T cells with inosine induced multi-level reprogramming leading to stemness and improved anti-tumor potency. We demonstrate the feasibility of introducing inosine during GMP cell manufacturing as a novel strategy to generate enhanced CAR-T cells.

immunology↗

bioRxiv2023

Codon affinity in mitochondrial DNA shapes evolutionary and somatic fitness

Summary ParagraphSomatic variation contributes to biological heterogeneity by modulating cellular proclivity to differentiate, expand, adapt, or die. While large-scale sequencing efforts have revealed the foundational role of somatic variants to drive human tumor evolution, our understanding of the contribution of mutations to modulate cellular fitness in non-malignant contexts remains understudied. Here, we identify a mosaic synonymous variant (m.7076A>G) in the mitochondrial DNA (mtDNA) encoded cytochrome c-oxidase subunit 1 gene (MT-CO1, p.Gly391=), which was present at homoplasmy in 47% of immune cells from a healthy donor. Using single-cell multi-omics, we discover highly specific selection against the m.7076G mutant allele in the CD8+ effector memory T cell compartment in vivo, reminiscent of selection observed for pathogenic mtDNA alleles1, 2 and indicative of lineage-specific metabolic requirements. While the wildtype m.7076A allele is translated via Watson-Crick-Franklin base-pairing, the anticodon diversity of the mitochondrial transfer RNA pool is limited, requiring wobble-dependent translation of the m.7076G mutant allele. Notably, mitochondrial ribosome profiling revealed altered codon-anticodon affinity at the wobble position as evidenced by stalled translation of the synonymous m.7076G mutant allele encoding for glycine. Generalizing this observation, we provide a new ontogeny of the 8,482 synonymous variants in the human mitochondrial genome that enables interpretation of functional mtDNA variation. Specifically, via inter- and intra-species evolutionary analyses, population-level complex trait associations, and the occurrence of germline and somatic mtDNA mutations from large-scale sequencing studies, we demonstrate that synonymous variation impacting codon:anticodon affinity is actively evolving across the entire mitochondrial genome and has broad functional and phenotypic effects. In summary, our results introduce a new ontogeny for mitochondrial genetic variation and support a model where organismal principles can be discerned from somatic evolution via single-cell genomics.

genetics↗

bioRxiv2022

Single-cell multi-omics reveals dynamics of purifying selection of pathogenic mitochondrial DNA across human immune cells

Cells experience intrinsic and extrinsic pressures that affect their proclivity to expand and persist in vivo. In congenital disorders caused by loss-of-function mutations in mitochondrial DNA (mtDNA), metabolic vulnerabilities may result in cell-type specific phenotypes and depletion of pathogenic alleles, contributing to purifying selection. However, the impact of pathogenic mtDNA mutations on the cellular hematopoietic landscape is not well understood. Here, we establish a multi-omics approach to quantify deletions in mtDNA alongside cell state features in single cells derived from Pearson syndrome patients. We resolve the interdependence between pathogenic mtDNA and lineage, including purifying selection against deletions in effector/memory CD8 T-cell populations and recent thymic emigrants and dynamics in other hematopoietic populations. Our mapping of lineage-specific purifying selection dynamics in primary cells from patients carrying pathogenic heteroplasmy provides a new perspective on recurrent clinical phenotypes in mitochondrial disorders, including cancer and infection, with potential broader relevance to age-related immune dysfunction.

genetics↗

bioRxiv2022

Enhanced Effector Activity of Mediator Kinase Module Deficient CAR-T Cells

Adoptive T cell immune therapies mediate impressive clinical benefit in a fraction of patients, but anti-tumor effects are often limited by inadequate T cell potency. To identify genes limiting T cell effector function, we conducted genome-wide CRISPR knock-out screens in human primary CAR-T cells. The top hits were MED12 and CCNC, components of the cyclin-dependent kinase (CDK) module of the Mediator complex, an evolutionarily conserved regulator of gene transcription. MED12 or CCNC deficient CAR-T cells manifest increased expansion, cytokine production, metabolic fitness, effector function, anti-tumor activity and reduced terminal effector differentiation. Chemical inhibition of CDK8/19 kinase activity recapitulated some features of genetic loss of MED12, including increased T cell expansion. MED12 deficient CAR-T cells showed widespread but selective increases in chromatin accessibility, MED1 chromatin occupancy, and H3K27 acetylation at enhancers used by transcription factors playing a critical role in T cell fate, including several STAT and AP1 family members. The most pronounced enhancement was observed for STAT5 which manifested as increased sensitivity to IL-2 in MED12 deficient T cells. These results link Mediator induced transcriptional coactivation with T cell effector programming and identify the CDK module as a target for enhancing the potency of anti-tumor T cell responses. One Sentence SummaryThe Mediator kinase module is a primary regulator of T cell differentiation, and genetic or small molecule-based inhibition of this module enhances effector T cell potency.

immunology↗