Ansuman Satpathy

All Publications

• Distinct epigenomic landscapes underlie tissue-specific memory T cell differentiation. Immunity Buquicchio, F. A., Fonseca, R., Yan, P. K., Wang, F., Evrard, M., Obers, A., Gutierrez, J. C., Raposo, C. J., Belk, J. A., Daniel, B., Zareie, P., Yost, K. E., Qi, Y., Yin, Y., Nico, K. F., Tierney, F. M., Howitt, M. R., Lareau, C. A., Satpathy, A. T., Mackay, L. K. 2024

  Abstract

  The memory CD8+ T cell pool contains phenotypically and transcriptionally heterogeneous subsets with specialized functions and recirculation patterns. Here, we examined the epigenetic landscape of CD8+ T cells isolated from seven non-lymphoid organs across four distinct infection models, alongside their circulating T cell counterparts. Using single-cell transposase-accessible chromatin sequencing (scATAC-seq), we found that tissue-resident memory T (TRM) cells and circulating memory T (TCIRC) cells develop along distinct epigenetic trajectories. We identified organ-specific transcriptional regulators of TRM cell development, including FOSB, FOS, FOSL1, and BACH2, and defined an epigenetic signature common to TRM cells across organs. Finally, we found that although terminal TEX cells share accessible regulatory elements with TRM cells, they are defined by TEX-specific epigenetic features absent from TRM cells. Together, this comprehensive data resource shows that TRM cell development is accompanied by dynamic transcriptome alterations and chromatin accessibility changes that direct tissue-adapted and functionally distinct T cell states.

  View details for DOI 10.1016/j.immuni.2024.06.014

  View details for PubMedID 39043184

• The temporal progression of lung immune remodeling during breast cancer metastasis. Cancer cell McGinnis, C. S., Miao, Z., Superville, D., Yao, W., Goga, A., Reticker-Flynn, N. E., Winkler, J., Satpathy, A. T. 2024

  Abstract

  Tumor metastasis requires systemic remodeling of distant organ microenvironments that impacts immune cell phenotypes, population structure, and intercellular communication. However, our understanding of immune phenotypic dynamics in the metastatic niche remains incomplete. Here, we longitudinally assayed lung immune transcriptional profiles in the polyomavirus middle T antigen (PyMT) and 4T1 metastatic breast cancer models from primary tumorigenesis, through pre-metastatic niche formation, to the final stages of metastatic outgrowth at single-cell resolution. Computational analyses of these data revealed a TLR-NFκB inflammatory program enacted by both peripherally derived and tissue-resident myeloid cells that correlated with pre-metastatic niche formation and mirrored CD14+ "activated" myeloid cells in the primary tumor. Moreover, we observed that primary tumor and metastatic niche natural killer (NK) cells are differentially regulated in mice and human patient samples, with the metastatic niche featuring elevated cytotoxic NK cell proportions. Finally, we identified cell-type-specific dynamic regulation of IGF1 and CCL6 signaling during metastatic progression that represents anti-metastatic immunotherapy candidate pathways.

  View details for DOI 10.1016/j.ccell.2024.05.004

  View details for PubMedID 38821060

• Latent human herpesvirus 6 is reactivated in CAR T cells. Nature Lareau, C. A., Yin, Y., Maurer, K., Sandor, K. D., Daniel, B., Yagnik, G., Peña, J., Crawford, J. C., Spanjaart, A. M., Gutierrez, J. C., Haradhvala, N. J., Riberdy, J. M., Abay, T., Stickels, R. R., Verboon, J. M., Liu, V., Buquicchio, F. A., Wang, F., Southard, J., Song, R., Li, W., Shrestha, A., Parida, L., Getz, G., Maus, M. V., Li, S., Moore, A., Roberts, Z. J., Ludwig, L. S., Talleur, A. C., Thomas, P. G., Dehghani, H., Pertel, T., Kundaje, A., Gottschalk, S., Roth, T. L., Kersten, M. J., Wu, C. J., Majzner, R. G., Satpathy, A. T. 2023

  Abstract

  Cell therapies have yielded durable clinical benefits for patients with cancer, but the risks associated with the development of therapies from manipulated human cells are understudied. For example, we lack a comprehensive understanding of the mechanisms of toxicities observed in patients receiving T cell therapies, including recent reports of encephalitis caused by reactivation of human herpesvirus 6 (HHV-6)1. Here, through petabase-scale viral genomics mining, we examine the landscape of human latent viral reactivation and demonstrate that HHV-6B can become reactivated in cultures of human CD4+ T cells. Using single-cell sequencing, we identify a rare population of HHV-6 'super-expressors' (about 1 in 300-10,000 cells) that possess high viral transcriptional activity, among research-grade allogeneic chimeric antigen receptor (CAR) T cells. By analysing single-cell sequencing data from patients receiving cell therapy products that are approved by the US Food and Drug Administration2 or are in clinical studies3-5, we identify the presence of HHV-6-super-expressor CAR T cells in patients in vivo. Together, the findings of our study demonstrate the utility of comprehensive genomics analyses in implicating cell therapy products as a potential source contributing to the lytic HHV-6 infection that has been reported in clinical trials1,6-8 and may influence the design and production of autologous and allogeneic cell therapies.

  View details for DOI 10.1038/s41586-023-06704-2

  View details for PubMedID 37938768

  View details for PubMedCentralID 9827115

• Lineage tracing reveals clonal progenitors and long-term persistence of tumor-specific T cells during immune checkpoint blockade. Cancer cell Pai, J. A., Hellmann, M. D., Sauter, J. L., Mattar, M., Rizvi, H., Woo, H. J., Shah, N., Nguyen, E. M., Uddin, F. Z., Quintanal-Villalonga, A., Chan, J. M., Manoj, P., Allaj, V., Baine, M. K., Bhanot, U. K., Jain, M., Linkov, I., Meng, F., Brown, D., Chaft, J. E., Plodkowski, A. J., Gigoux, M., Won, H. H., Sen, T., Wells, D. K., Donoghue, M. T., de Stanchina, E., Wolchok, J. D., Loomis, B., Merghoub, T., Rudin, C. M., Chow, A., Satpathy, A. T. 2023

  Abstract

  Paired single-cell RNA and T cell receptor sequencing (scRNA/TCR-seq) has allowed for enhanced resolution of clonal T cell dynamics in cancer. Here, we report a scRNA/TCR-seq analysis of 187,650 T cells from 31 tissue regions, including tumor, adjacent normal tissues, and lymph nodes (LN), from three patients with non-small cell lung cancer after immune checkpoint blockade (ICB). Regions with viable cancer cells are enriched for exhausted CD8+ T cells, regulatory CD4+ T cells (Treg), and follicular helper CD4+ T cells (TFH). Tracking T cell clonotypes across tissues, combined with neoantigen specificity assays, reveals that TFH and tumor-specific exhausted CD8+ T cells are clonally linked to TCF7+SELL+ progenitors in tumor draining LNs, and progressive exhaustion trajectories of CD8+ T, Treg, and TFH cells with proximity to the tumor microenvironment. Finally, longitudinal tracking of tumor-specific CD8+ and CD4+ T cell clones reveals persistence in the peripheral blood for years after ICB therapy.

  View details for DOI 10.1016/j.ccell.2023.03.009

  View details for PubMedID 37001526

• Macrophage inflammatory and regenerative response periodicity is programmed by cell cycle and chromatin state. Molecular cell Daniel, B., Belk, J. A., Meier, S. L., Chen, A. Y., Sandor, K., Czimmerer, Z., Varga, Z., Bene, K., Buquicchio, F. A., Qi, Y., Kitano, H., Wheeler, J. R., Foster, D. S., Januszyk, M., Longaker, M. T., Chang, H. Y., Satpathy, A. T. 2022

  Abstract

  Cell cycle (CC) facilitates cell division via robust, cyclical gene expression. Protective immunity requires the expansion of pathogen-responsive cell types, but whether CC confers unique gene expression programs that direct the subsequent immunological response remains unclear. Here, we demonstrate that single macrophages (MFs) adopt different plasticity states in CC, which leads to heterogeneous cytokine-induced polarization, priming, and repolarization programs. Specifically, MF plasticity to interferon gamma (IFNG) is substantially reduced during S-G2/M, whereas interleukin 4 (IL-4) induces S-G2/M-biased gene expression, mediated by CC-biased enhancers. Additionally, IL-4 polarization shifts the CC-phase distribution of MFs toward the G2/M phase, providing a subpopulation-specific mechanism for IL-4-induced, dampened IFNG responsiveness. Finally, we demonstrate CC-dependent MF responses in murine and human disease settings invivo, including Th2-driven airway inflammation and pulmonary fibrosis, where MFs express an S-G2/M-biased tissue remodeling gene program. Therefore, MF inflammatory and regenerative responses are gated by CC in a cyclical, phase-dependent manner.

  View details for DOI 10.1016/j.molcel.2022.11.017

  View details for PubMedID 36521490

• Enhanced T cell effector activity by targeting the Mediator kinase module. Science (New York, N.Y.) Freitas, K. A., Belk, J. A., Sotillo, E., Quinn, P. J., Ramello, M. C., Malipatlolla, M., Daniel, B., Sandor, K., Klysz, D., Bjelajac, J., Xu, P., Burdsall, K. A., Tieu, V., Duong, V. T., Donovan, M. G., Weber, E. W., Chang, H. Y., Majzner, R. G., Espinosa, J. M., Satpathy, A. T., Mackall, C. L. 2022; 378 (6620): eabn5647

  Abstract

  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.

  View details for DOI 10.1126/science.abn5647

  View details for PubMedID 36356142

• Divergent clonal differentiation trajectories of T cell exhaustion. Nature immunology Daniel, B., Yost, K. E., Hsiung, S., Sandor, K., Xia, Y., Qi, Y., Hiam-Galvez, K. J., Black, M., J Raposo, C., Shi, Q., Meier, S. L., Belk, J. A., Giles, J. R., Wherry, E. J., Chang, H. Y., Egawa, T., Satpathy, A. T. 2022

  Abstract

  Chronic antigen exposure during viral infection or cancer promotes an exhausted T cell (Tex) state with reduced effector function. However, whether all antigen-specific T cell clones follow the same Tex differentiation trajectory remains unclear. Here, we generate a single-cell multiomic atlas of T cell exhaustion in murine chronic viral infection that redefines Tex phenotypic diversity, including two late-stage Tex subsets with either a terminal exhaustion (Texterm) or a killer cell lectin-like receptor-expressing cytotoxic (TexKLR) phenotype. We use paired single-cell RNA and T cell receptor sequencing to uncover clonal differentiation trajectories of Texterm-biased, TexKLR-biased or divergent clones that acquire both phenotypes. We show that high T cell receptor signaling avidity correlates with Texterm, whereas low avidity correlates with effector-like TexKLR fate. Finally, we identify similar clonal differentiation trajectories in human tumor-infiltrating lymphocytes. These findings reveal clonal heterogeneity in the T cell response to chronic antigen that influences Tex fates and persistence.

  View details for DOI 10.1038/s41590-022-01337-5

  View details for PubMedID 36289450

• Genome-wide CRISPR screens of T cell exhaustion identify chromatin remodeling factors that limit T cell persistence. Cancer cell Belk, J. A., Yao, W., Ly, N., Freitas, K. A., Chen, Y. T., Shi, Q., Valencia, A. M., Shifrut, E., Kale, N., Yost, K. E., Duffy, C. V., Daniel, B., Hwee, M. A., Miao, Z., Ashworth, A., Mackall, C. L., Marson, A., Carnevale, J., Vardhana, S. A., Satpathy, A. T. 2022

  Abstract

  T cell exhaustion limits antitumor immunity, but the molecular determinants of this process remain poorly understood. Using a chronic stimulation assay, we performed genome-wide CRISPR-Cas9 screens to systematically discover regulators of T cell exhaustion, which identified an enrichment of epigenetic factors. In vivo CRISPR screens in murine and human tumor models demonstrated that perturbation of the INO80 and BAF chromatin remodeling complexes improved T cell persistence in tumors. In vivo Perturb-seq revealed distinct transcriptional roles of each complex and that depletion of canonical BAF complex members, including Arid1a, resulted in the maintenance of an effector program and downregulation of exhaustion-related genes in tumor-infiltrating T cells. Finally, Arid1a depletion limited the acquisition of exhaustion-associated chromatin accessibility and led to improved antitumor immunity. In summary, we provide an atlas of the genetic regulators of T cell exhaustion and demonstrate that modulation of epigenetic state can improve T cell responses in cancer immunotherapy.

  View details for DOI 10.1016/j.ccell.2022.06.001

  View details for PubMedID 35750052

• Recruiting T cells in cancer immunotherapy. Science (New York, N.Y.) Yost, K. E., Chang, H. Y., Satpathy, A. T. 2021; 372 (6538): 130–31

  View details for DOI 10.1126/science.abd1329

  View details for PubMedID 33833111

• Discovery and functional interrogation of SARS-CoV-2 RNA-host protein interactions. Cell Flynn, R. A., Belk, J. A., Qi, Y., Yasumoto, Y., Wei, J., Alfajaro, M. M., Shi, Q., Mumbach, M. R., Limaye, A., DeWeirdt, P. C., Schmitz, C. O., Parker, K. R., Woo, E., Chang, H. Y., Horvath, T. L., Carette, J. E., Bertozzi, C. R., Wilen, C. B., Satpathy, A. T. 2021

  Abstract

  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.

  View details for DOI 10.1016/j.cell.2021.03.012

  View details for PubMedID 33743211

• Single-Cell Analyses Identify Brain Mural Cells Expressing CD19 as Potential Off-Tumor Targets for CAR-T Immunotherapies. Cell Parker, K. R., Migliorini, D., Perkey, E., Yost, K. E., Bhaduri, A., Bagga, P., Haris, M., Wilson, N. E., Liu, F., Gabunia, K., Scholler, J., Montine, T. J., Bhoj, V. G., Reddy, R., Mohan, S., Maillard, I., Kriegstein, A. R., June, C. H., Chang, H. Y., Posey, A. D., Satpathy, A. T. 2020

  Abstract

  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.

  View details for DOI 10.1016/j.cell.2020.08.022

  View details for PubMedID 32961131

• Clonal replacement of tumor-specific T cells following PD-1 blockade. Nature medicine Yost, K. E., Satpathy, A. T., Wells, D. K., Qi, Y. n., Wang, C. n., Kageyama, R. n., McNamara, K. L., Granja, J. M., Sarin, K. Y., Brown, R. A., Gupta, R. K., Curtis, C. n., Bucktrout, S. L., Davis, M. M., Chang, A. L., Chang, H. Y. 2019

  Abstract

  Immunotherapies that block inhibitory checkpoint receptors on T cells have transformed the clinical care of patients with cancer1. However, whether the T cell response to checkpoint blockade relies on reinvigoration of pre-existing tumor-infiltrating lymphocytes or on recruitment of novel T cells remains unclear2-4. Here we performed paired single-cell RNA and T cell receptor sequencing on 79,046 cells from site-matched tumors from patients with basal or squamous cell carcinoma before and after anti-PD-1 therapy. Tracking T cell receptor clones and transcriptional phenotypes revealed coupling of tumor recognition, clonal expansion and T cell dysfunction marked by clonal expansion of CD8+CD39+ T cells, which co-expressed markers of chronic T cell activation and exhaustion. However, the expansion of T cell clones did not derive from pre-existing tumor-infiltrating T lymphocytes; instead, the expanded clones consisted of novel clonotypes that had not previously been observed in the same tumor. Clonal replacement of T cells was preferentially observed in exhausted CD8+ T cells and evident in patients with basal or squamous cell carcinoma. These results demonstrate that pre-existing tumor-specific T cells may have limited reinvigoration capacity, and that the T cell response to checkpoint blockade derives from a distinct repertoire of T cell clones that may have just recently entered the tumor.

  View details for DOI 10.1038/s41591-019-0522-3

  View details for PubMedID 31359002

• Massively parallel single-cell chromatin landscapes of human immune cell development and intratumoral T cell exhaustion. Nature biotechnology Satpathy, A. T., Granja, J. M., Yost, K. E., Qi, Y. n., Meschi, F. n., McDermott, G. P., Olsen, B. N., Mumbach, M. R., Pierce, S. E., Corces, M. R., Shah, P. n., Bell, J. C., Jhutty, D. n., Nemec, C. M., Wang, J. n., Wang, L. n., Yin, Y. n., Giresi, P. G., Chang, A. L., Zheng, G. X., Greenleaf, W. J., Chang, H. Y. 2019; 37 (8): 925–36

  Abstract

  Understanding complex tissues requires single-cell deconstruction of gene regulation with precision and scale. Here, we assess the performance of a massively parallel droplet-based method for mapping transposase-accessible chromatin in single cells using sequencing (scATAC-seq). We apply scATAC-seq to obtain chromatin profiles of more than 200,000 single cells in human blood and basal cell carcinoma. In blood, application of scATAC-seq enables marker-free identification of cell type-specific cis- and trans-regulatory elements, mapping of disease-associated enhancer activity and reconstruction of trajectories of cellular differentiation. In basal cell carcinoma, application of scATAC-seq reveals regulatory networks in malignant, stromal and immune cells in the tumor microenvironment. Analysis of scATAC-seq profiles from serial tumor biopsies before and after programmed cell death protein 1 blockade identifies chromatin regulators of therapy-responsive T cell subsets and reveals a shared regulatory program that governs intratumoral CD8+ T cell exhaustion and CD4+ T follicular helper cell development. We anticipate that scATAC-seq will enable the unbiased discovery of gene regulatory factors across diverse biological systems.

  View details for DOI 10.1038/s41587-019-0206-z

  View details for PubMedID 31375813

• Coupled Single-Cell CRISPR Screening and Epigenomic Profiling Reveals Causal Gene Regulatory Networks. Cell Rubin, A. J., Parker, K. R., Satpathy, A. T., Qi, Y., Wu, B., Ong, A. J., Mumbach, M. R., Ji, A. L., Kim, D. S., Cho, S. W., Zarnegar, B. J., Greenleaf, W. J., Chang, H. Y., Khavari, P. A. 2018

  Abstract

  Here, we present Perturb-ATAC, a method that 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 genotype-phenotype relationships. Perturb-ATAC in human Blymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning and identified a hierarchy of TFs that govern B cell state, variation, and disease-associated cis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules of cis-elements that specify keratinocyte fate. Combinatorial deletion of all pairs of these TFsuncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful strategy to dissect gene regulatory networks in development and disease.

  View details for PubMedID 30580963

• Transcript-indexed ATAC-seq for precision immune profiling. Nature medicine Satpathy, A. T., Saligrama, N. n., Buenrostro, J. D., Wei, Y. n., Wu, B. n., Rubin, A. J., Granja, J. M., Lareau, C. A., Li, R. n., Qi, Y. n., Parker, K. R., Mumbach, M. R., Serratelli, W. S., Gennert, D. G., Schep, A. N., Corces, M. R., Khodadoust, M. S., Kim, Y. H., Khavari, P. A., Greenleaf, W. J., Davis, M. M., Chang, H. Y. 2018

  Abstract

  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.

  View details for PubMedID 29686426

• Enhancer connectome in primary human cells identifies target genes of disease-associated DNA elements. Nature genetics Mumbach, M. R., Satpathy, A. T., Boyle, E. A., Dai, C. n., Gowen, B. G., Cho, S. W., Nguyen, M. L., Rubin, A. J., Granja, J. M., Kazane, K. R., Wei, Y. n., Nguyen, T. n., Greenside, P. G., Corces, M. R., Tycko, J. n., Simeonov, D. R., Suliman, N. n., Li, R. n., Xu, J. n., Flynn, R. A., Kundaje, A. n., Khavari, P. A., Marson, A. n., Corn, J. E., Quertermous, T. n., Greenleaf, W. J., Chang, H. Y. 2017

  Abstract

  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.

  View details for PubMedID 28945252

• Notch2-dependent classical dendritic cells orchestrate intestinal immunity to attaching-and-effacing bacterial pathogens NATURE IMMUNOLOGY Satpathy, A. T., Briseno, C. G., Lee, J. S., Ng, D., Manieri, N. A., Wumesh, K. C., Wu, X., Thomas, S. R., Lee, W., Turkoz, M., McDonald, K. G., Meredith, M. M., Song, C., Guidos, C. J., Newberry, R. D., Ouyang, W., Murphy, T. L., Stappenbeck, T. S., Gommerman, J. L., Nussenzweig, M. C., Colonna, M., Kopan, R., Murphy, K. M. 2013; 14 (9): 937-?

  View details for DOI 10.1038/ni.2679

  View details for Web of Science ID 000323377700011

• Re(de)fining the dendritic cell lineage NATURE IMMUNOLOGY Satpathy, A. T., Wu, X., Albring, J. C., Murphy, K. M. 2012; 13 (12): 1145-1154

  Abstract

  Dendritic cells (DCs) are essential mediators of innate and adaptive immune responses. Study of these critical cells has been complicated by their similarity to other hematopoietic lineages, particularly monocytes and macrophages. Progress has been made in three critical areas of DC biology: the characterization of lineage-restricted progenitors in the bone marrow, the identification of cytokines and transcription factors required during differentiation, and the development of genetic tools for the visualization and depletion of DCs in vivo. Collectively, these advances have clarified the nature of the DC lineage and have provided novel insights into their function during health and disease.

  View details for DOI 10.1038/ni.2467

  View details for Web of Science ID 000311217900007

  View details for PubMedID 23160217

  View details for PubMedCentralID PMC3644874

• Zbtb46 expression distinguishes classical dendritic cells and their committed progenitors from other immune lineages JOURNAL OF EXPERIMENTAL MEDICINE Satpathy, A. T., Wumesh, K. C., Albring, J. C., Edelson, B. T., Kretzer, N. M., Bhattacharya, D., Murphy, T. L., Murphy, K. M. 2012; 209 (6): 1135-1152

  Abstract

  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 Zbtb46(gfp/+) 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 Zbtb46(gfp/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.

  View details for DOI 10.1084/jem.20120030

  View details for Web of Science ID 000304907800009

  View details for PubMedID 22615127

  View details for PubMedCentralID PMC3371733

• Retinoic acid and TGF-β orchestrate organ-specific programs of tissue residency. Immunity Obers, A., Poch, T., Rodrigues, G., Christo, S. N., Gandolfo, L. C., Fonseca, R., Zaid, A., Kuai, J. E., Lai, H., Zareie, P., Yakou, M. H., Dryburgh, L., Burn, T. N., Dosser, J., Buquicchio, F. A., Lareau, C. A., Walsh, C., Judd, L., Theodorou, M. R., Gutbrod, K., Dörmann, P., Kingham, J., Stinear, T., Kallies, A., Schroeder, J., Mueller, S. N., Park, S. L., Speed, T. P., Satpathy, A. T., Phan, T. G., Wilhelm, C., Zaph, C., Evrard, M., Mackay, L. K. 2024

  Abstract

  Tissue-resident memory T (TRM) cells are integral to tissue immunity, persisting in diverse anatomical sites where they adhere to a common transcriptional framework. How these cells integrate distinct local cues to adopt the common TRM cell fate remains poorly understood. Here, we show that whereas skin TRM cells strictly require transforming growth factor β (TGF-β) for tissue residency, those in other locations utilize the metabolite retinoic acid (RA) to drive an alternative differentiation pathway, directing a TGF-β-independent tissue residency program in the liver and synergizing with TGF-β to drive TRM cells in the small intestine. We found that RA was required for the long-term maintenance of intestinal TRM populations, in part by impeding their retrograde migration. Moreover, enhanced RA signaling modulated TRM cell phenotype and function, a phenomenon mirrored in mice with increased microbial diversity. Together, our findings reveal RA as a fundamental component of the host-environment interaction that directs immunosurveillance in tissues.

  View details for DOI 10.1016/j.immuni.2024.09.015

  View details for PubMedID 39406245

• Mechanism-guided engineering of a minimal biological particle for genome editing. bioRxiv : the preprint server for biology Ngo, W., Peukes, J. T., Baldwin, A., Xue, Z. W., Hwang, S., Stickels, R. R., Lin, Z., Satpathy, A. T., Wells, J. A., Schekman, R., Nogales, E., Doudna, J. A. 2024

  Abstract

  The widespread application of genome editing to treat or even cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped Delivery Vehicles (EDVs) are engineered virally-derived particles capable of packaging and delivering CRISPR-Cas9 ribonucleoproteins (RNPs). However, the presence of lentiviral genome encapsulation and replication components in EDVs has obscured the underlying delivery mechanism and precluded particle optimization. Here we show that Cas9 RNP nuclear delivery is independent of the native lentiviral capsid structure. Instead, EDV-mediated genome editing activity corresponds directly to the number of nuclear localization sequences on the Cas9 enzyme. EDV structural analysis using cryo-electron tomography and small molecule inhibitors guided the removal of ~80% of viral residues, creating a minimal EDV (miniEDV) that retains full RNP delivery capability. MiniEDVs are 25% smaller yet package equivalent amounts of Cas9 RNPs relative to the original EDVs, and demonstrated increased editing in cell lines and therapeutically-relevant primary human T cells. These results show that virally-derived particles can be streamlined to create efficacious genome editing delivery vehicles that could simplify production and manufacturing.

  View details for DOI 10.1101/2024.07.23.604809

  View details for PubMedID 39091760

  View details for PubMedCentralID PMC11291128

• Systematic decoding of cis gene regulation defines context-dependent control of the multi-gene costimulatory receptor locus in human T cells. Nature genetics Mowery, C. T., Freimer, J. W., Chen, Z., Casaní-Galdón, S., Umhoefer, J. M., Arce, M. M., Gjoni, K., Daniel, B., Sandor, K., Gowen, B. G., Nguyen, V., Simeonov, D. R., Garrido, C. M., Curie, G. L., Schmidt, R., Steinhart, Z., Satpathy, A. T., Pollard, K. S., Corn, J. E., Bernstein, B. E., Ye, C. J., Marson, A. 2024

  Abstract

  Cis-regulatory elements (CREs) interact with trans regulators to orchestrate gene expression, but how transcriptional regulation is coordinated in multi-gene loci has not been experimentally defined. We sought to characterize the CREs controlling dynamic expression of the adjacent costimulatory genes CD28, CTLA4 and ICOS, encoding regulators of T cell-mediated immunity. Tiling CRISPR interference (CRISPRi) screens in primary human T cells, both conventional and regulatory subsets, uncovered gene-, cell subset- and stimulation-specific CREs. Integration with CRISPR knockout screens and assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling identified trans regulators influencing chromatin states at specific CRISPRi-responsive elements to control costimulatory gene expression. We then discovered a critical CCCTC-binding factor (CTCF) boundary that reinforces CRE interaction with CTLA4 while also preventing promiscuous activation of CD28. By systematically mapping CREs and associated trans regulators directly in primary human T cell subsets, this work overcomes longstanding experimental limitations to decode context-dependent gene regulatory programs in a complex, multi-gene locus critical to immune homeostasis.

  View details for DOI 10.1038/s41588-024-01743-5

  View details for PubMedID 38811842

  View details for PubMedCentralID 8577129

• Direct transposition of native DNA for sensitive multimodal single-molecule sequencing. Nature genetics Nanda, A. S., Wu, K., Irkliyenko, I., Woo, B., Ostrowski, M. S., Clugston, A. S., Sayles, L. C., Xu, L., Satpathy, A. T., Nguyen, H. G., Alejandro Sweet-Cordero, E., Goodarzi, H., Kasinathan, S., Ramani, V. 2024

  Abstract

  Concurrent readout of sequence and base modifications from long unamplified DNA templates by Pacific Biosciences of California (PacBio) single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90-99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000-50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.

  View details for DOI 10.1038/s41588-024-01748-0

  View details for PubMedID 38724748

  View details for PubMedCentralID 7877196

• Convergent epigenetic evolution drives relapse in acute myeloid leukemia. eLife Nuno, K., Azizi, A., Koehnke, T., Lareau, C., Ediriwickrema, A., Corces, M. R., Satpathy, A. T., Majeti, R. 2024; 13

  Abstract

  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.

  View details for DOI 10.7554/eLife.93019

  View details for PubMedID 38647535

  View details for PubMedCentralID PMC11034943

• FOXO1 is a master regulator of memory programming in CAR T cells. Nature Doan, A. E., Mueller, K. P., Chen, A. Y., Rouin, G. T., Chen, Y., Daniel, B., Lattin, J., Markovska, M., Mozarsky, B., Arias-Umana, J., Hapke, R., Jung, I. Y., Wang, A., Xu, P., Klysz, D., Zuern, G., Bashti, M., Quinn, P. J., Miao, Z., Sandor, K., Zhang, W., Chen, G. M., Ryu, F., Logun, M., Hall, J., Tan, K., Grupp, S. A., McClory, S. E., Lareau, C. A., Fraietta, J. A., Sotillo, E., Satpathy, A. T., Mackall, C. L., Weber, E. W. 2024

  Abstract

  A major limitation of chimeric antigen receptor (CAR) T cell therapies is the poor persistence of these cells in vivo1. The expression of memory-associated genes in CAR T cells is linked to their long-term persistence in patients and clinical efficacy2-6, suggesting that memory programs may underpin durable CAR T cell function. Here we show that the transcription factor FOXO1 is responsible for promoting memory and restraining exhaustion in human CAR T cells. Pharmacological inhibition or gene editing of endogenous FOXO1 diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype and impaired the antitumour activity of CAR T cells. Overexpression of FOXO1 induced a gene-expression program consistent with T cell memory and increased chromatin accessibility at FOXO1-binding motifs. CAR T cells that overexpressed FOXO1 retained their function, memory potential and metabolic fitness in settings of chronic stimulation, and exhibited enhanced persistence and tumour control in vivo. By contrast, overexpression of TCF1 (encoded by TCF7) did not enforce canonical memory programs or enhance the potency of CAR T cells. Notably, FOXO1 activity correlated with positive clinical outcomes of patients treated with CAR T cells or tumour-infiltrating lymphocytes, underscoring the clinical relevance of FOXO1 in cancer immunotherapy. Our results show that overexpressing FOXO1 can increase the antitumour activity of human CAR T cells, and highlight memory reprogramming as a broadly applicable approach for optimizing therapeutic T cell states.

  View details for DOI 10.1038/s41586-024-07300-8

  View details for PubMedID 38600391

  View details for PubMedCentralID 8900215

• Transcript-specific enrichment enables profiling rare cell states via scRNA-seq. bioRxiv : the preprint server for biology Abay, T., Stickels, R. R., Takizawa, M. T., Nalbant, B. N., Hsieh, Y. H., Hwang, S., Snopkowski, C., Yu, K. K., Abou-Mrad, Z., Tabar, V., Ludwig, L. S., Chaligné, R., Satpathy, A. T., Lareau, C. A. 2024

  Abstract

  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.

  View details for DOI 10.1101/2024.03.27.587039

  View details for PubMedID 38586040

  View details for PubMedCentralID PMC10996707

• Regulation of immune signal integration and memory by inflammation-induced chromosome conformation. bioRxiv : the preprint server for biology Daniel, B., Chen, A. Y., Sandor, K., Zhang, W., Miao, Z., Lareau, C. A., Yost, K. E., Chang, H. Y., Satpathy, A. T. 2024

  Abstract

  3-dimensional (3D) genome conformation is central to gene expression regulation, yet our understanding of its contribution to rapid transcriptional responses, signal integration, and memory in immune cells is limited. Here, we study the molecular regulation of the inflammatory response in primary macrophages using integrated transcriptomic, epigenomic, and chromosome conformation data, including base pair-resolution Micro-Capture C. We demonstrate that interleukin-4 (IL-4) primes the inflammatory response in macrophages by stably rewiring 3D genome conformation, juxtaposing endotoxin-, interferon-gamma-, and dexamethasone-responsive enhancers in close proximity to their cognate gene promoters. CRISPR-based perturbations of enhancer-promoter contacts or CCCTC-binding factor (CTCF) boundary elements demonstrated that IL-4-driven conformation changes are indispensable for enhanced and synergistic endotoxin-induced transcriptional responses, as well as transcriptional memory following stimulus removal. Moreover, transcriptional memory mediated by changes in chromosome conformation often occurred in the absence of changes in chromatin accessibility or histone modifications. Collectively, these findings demonstrate that rapid and memory transcriptional responses to immunological stimuli are encoded in the 3D genome.

  View details for DOI 10.1101/2024.02.29.582872

  View details for PubMedID 38496446

  View details for PubMedCentralID PMC10942375

• Inosine induces stemness features in CAR-T cells and enhances potency. Cancer cell Klysz, D. D., Fowler, C., Malipatlolla, M., Stuani, L., Freitas, K. A., Chen, Y., Meier, S., Daniel, B., Sandor, K., Xu, P., Huang, J., Labanieh, L., Keerthi, V., Leruste, A., Bashti, M., Mata-Alcazar, J., Gkitsas, N., Guerrero, J. A., Fisher, C., Patel, S., Asano, K., Patel, S., Davis, K. L., Satpathy, A. T., Feldman, S. A., Sotillo, E., Mackall, C. L. 2024

  Abstract

  Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8+ CAR-T cells express CD39 and CD73, which mediate proximal steps in Ado generation. Here, we sought to enhance CAR-T cell potency by knocking out CD39, CD73, or adenosine receptor 2a (A2aR) but observed only modest effects. In contrast, overexpression of Ado deaminase (ADA-OE), which metabolizes Ado to inosine (INO), induced stemness and enhanced CAR-T functionality. Similarly, CAR-T cell exposure to INO augmented function and induced features of stemness. INO induced profound metabolic reprogramming, diminishing glycolysis, increasing mitochondrial and glycolytic capacity, glutaminolysis and polyamine synthesis, and reprogrammed the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR-T cell products meeting criteria for clinical dosing. These results identify INO as a potent modulator of CAR-T cell metabolism and epigenetic stemness programming and deliver an enhanced potency platform for cell manufacturing.

  View details for DOI 10.1016/j.ccell.2024.01.002

  View details for PubMedID 38278150

• Temporal genomic analysis of melanoma rejection identifies regulators of tumor immune evasion. bioRxiv : the preprint server for biology Cohen Shvefel, S., Pai, J. A., Cao, Y., Pal, L. R., Levy, R., Yao, W., Cheng, K., Zemanek, M., Bartok, O., Weller, C., Yin, Y., Du, P. P., Yakubovich, E., Orr, I., Ben-Dor, S., Oren, R., Fellus-Alyagor, L., Golani, O., Goliand, I., Ranmar, D., Savchenko, I., Ketrarou, N., Schäffer, A. A., Ruppin, E., Satpathy, A. T., Samuels, Y. 2023

  Abstract

  Decreased intra-tumor heterogeneity (ITH) correlates with increased patient survival and immunotherapy response. However, even highly homogenous tumors may display variability in their aggressiveness, and how immunologic-factors impinge on their aggressiveness remains understudied. Here we studied the mechanisms responsible for the immune-escape of murine tumors with low ITH. We compared the temporal growth of homogeneous, genetically-similar single-cell clones that are rejected vs. those that are not-rejected after transplantation in-vivo using single-cell RNA sequencing and immunophenotyping. Non-rejected clones showed high infiltration of tumor-associated-macrophages (TAMs), lower T-cell infiltration, and increased T-cell exhaustion compared to rejected clones. Comparative analysis of rejection-associated gene expression programs, combined with in-vivo CRISPR knockout screens of candidate mediators, identified Mif (macrophage migration inhibitory factor) as a regulator of immune rejection. Mif knockout led to smaller tumors and reversed non-rejection-associated immune composition, particularly, leading to the reduction of immunosuppressive macrophage infiltration. Finally, we validated these results in melanoma patient data.

  View details for DOI 10.1101/2023.11.29.569032

  View details for PubMedID 38077050

  View details for PubMedCentralID PMC10705560

• FOXO1 is a master regulator of CAR T memory programming. Research square Doan, A., Mueller, K. P., Chen, A., Rouin, G. T., Daniel, B., Lattin, J., Chen, Y., Mozarsky, B., Markovska, M., Arias-Umana, J., Hapke, R., Jung, I., Xu, P., Klysz, D., Bashti, M., Quinn, P. J., Sandor, K., Zhang, W., Hall, J., Lareau, C., Grupp, S. A., Fraietta, J. A., Sotillo, E., Satpathy, A. T., Mackall, C. L., Weber, E. W. 2023

  Abstract

  Poor CAR T persistence limits CAR T cell therapies for B cell malignancies and solid tumors1,2. The expression of memory-associated genes such as TCF7 (protein name TCF1) is linked to response and long-term persistence in patients3-7, thereby implicating memory programs in therapeutic efficacy. Here, we demonstrate that the pioneer transcription factor, FOXO1, is responsible for promoting memory programs and restraining exhaustion in human CAR T cells. Pharmacologic inhibition or gene editing of endogenous FOXO1 in human CAR T cells diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype, and impaired antitumor activity in vitro and in vivo. FOXO1 overexpression induced a gene expression program consistent with T cell memory and increased chromatin accessibility at FOXO1 binding motifs. FOXO1-overexpressing cells retained function, memory potential, and metabolic fitness during settings of chronic stimulation and exhibited enhanced persistence and antitumor activity in vivo. In contrast, TCF1 overexpression failed to enforce canonical memory programs or enhance CAR T cell potency. Importantly, endogenous FOXO1 activity correlated with CAR T and TIL responses in patients, underscoring its clinical relevance in cancer immunotherapy. Our results demonstrate that memory reprogramming through FOXO1 can enhance the persistence and potency of human CAR T cells and highlights the utility of pioneer factors, which bind condensed chromatin and induce local epigenetic remodeling, for optimizing therapeutic T cell states.

  View details for DOI 10.21203/rs.3.rs-2802998/v1

  View details for PubMedID 37986944

  View details for PubMedCentralID PMC10659532

• Convergent Epigenetic Evolution Drives Relapse in Acute Myeloid Leukemia. bioRxiv : the preprint server for biology Nuno, K. A., Azizi, A., Kohnke, T., Lareau, C. A., Ediwirickrema, A., Ryan Corces, M., Satpathy, A. T., Majeti, R. 2023

  Abstract

  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.

  View details for DOI 10.1101/2023.10.10.561642

  View details for PubMedID 37873452

• Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells. Cell Tsuchida, C. A., Brandes, N., Bueno, R., Trinidad, M., Mazumder, T., Yu, B., Hwang, B., Chang, C., Liu, J., Sun, Y., Hopkins, C. R., Parker, K. R., Qi, Y., Hofman, L., Satpathy, A. T., Stadtmauer, E. A., Cate, J. H., Eyquem, J., Fraietta, J. A., June, C. H., Chang, H. Y., Ye, C. J., Doudna, J. A. 2023

  Abstract

  CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the targeted chromosome, including in preclinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells (NCT03399448), reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.

  View details for DOI 10.1016/j.cell.2023.08.041

  View details for PubMedID 37794590

• Modular pooled discovery of synthetic knockin sequences to program durable cell therapies. Cell Blaeschke, F., Chen, Y. Y., Apathy, R., Daniel, B., Chen, A. Y., Chen, P. A., Sandor, K., Zhang, W., Li, Z., Mowery, C. T., Yamamoto, T. N., Nyberg, W. A., To, A., Yu, R., Bueno, R., Kim, M. C., Schmidt, R., Goodman, D. B., Feuchtinger, T., Eyquem, J., Jimmie Ye, C., Carnevale, J., Satpathy, A. T., Shifrut, E., Roth, T. L., Marson, A. 2023; 186 (19): 4216-4234.e33

  Abstract

  Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.

  View details for DOI 10.1016/j.cell.2023.08.013

  View details for PubMedID 37714135

• Single-cell multi-omics of mitochondrial DNA disorders reveals dynamics of purifying selection across human immune cells. Nature genetics Lareau, C. A., Dubois, S. M., Buquicchio, F. A., Hsieh, Y. H., Garg, K., Kautz, P., Nitsch, L., Praktiknjo, S. D., Maschmeyer, P., Verboon, J. M., Gutierrez, J. C., Yin, Y., Fiskin, E., Luo, W., Mimitou, E. P., Muus, C., Malhotra, R., Parikh, S., Fleming, M. D., Oevermann, L., Schulte, J., Eckert, C., Kundaje, A., Smibert, P., Vardhana, S. A., Satpathy, A. T., Regev, A., Sankaran, V. G., Agarwal, S., Ludwig, L. S. 2023

  Abstract

  Pathogenic mutations in mitochondrial DNA (mtDNA) compromise cellular metabolism, contributing to cellular heterogeneity and disease. Diverse mutations are associated with diverse clinical phenotypes, suggesting distinct organ- and cell-type-specific metabolic vulnerabilities. Here we establish a multi-omics approach to quantify deletions in mtDNA alongside cell state features in single cells derived from six patients across the phenotypic spectrum of single large-scale mtDNA deletions (SLSMDs). By profiling 206,663 cells, we reveal the dynamics of pathogenic mtDNA deletion heteroplasmy consistent with purifying selection and distinct metabolic vulnerabilities across T-cell states in vivo and validate these observations in vitro. By extending analyses to hematopoietic and erythroid progenitors, we reveal mtDNA dynamics and cell-type-specific gene regulatory adaptations, demonstrating the context-dependence of perturbing mitochondrial genomic integrity. Collectively, we report pathogenic mtDNA heteroplasmy dynamics of individual blood and immune cells across lineages, demonstrating the power of single-cell multi-omics for revealing fundamental properties of mitochondrial genetics.

  View details for DOI 10.1038/s41588-023-01433-8

  View details for PubMedID 37386249

  View details for PubMedCentralID 3809581

• Clonal hematopoiesis is associated with protection from Alzheimer's disease. Nature medicine Bouzid, H., Belk, J. A., Jan, M., Qi, Y., Sarnowski, C., Wirth, S., Ma, L., Chrostek, M. R., Ahmad, H., Nachun, D., Yao, W., Beiser, A., Bick, A. G., Bis, J. C., Fornage, M., Longstreth, W. T., Lopez, O. L., Natarajan, P., Psaty, B. M., Satizabal, C. L., Weinstock, J., Larson, E. B., Crane, P. K., Keene, C. D., Seshadri, S., Satpathy, A. T., Montine, T. J., Jaiswal, S. 2023

  Abstract

  Clonal hematopoiesis of indeterminate potential (CHIP) is a premalignant expansion of mutated hematopoietic stem cells. As CHIP-associated mutations are known to alter the development and function of myeloid cells, we hypothesized that CHIP may also be associated with the risk of Alzheimer's disease (AD), a disease in which brain-resident myeloid cells are thought to have a major role. To perform association tests between CHIP and AD dementia, we analyzed blood DNA sequencing data from 1,362 individuals with AD and 4,368 individuals without AD. Individuals with CHIP had a lower risk of AD dementia (meta-analysis odds ratio (OR) = 0.64, P = 3.8 × 10-5), and Mendelian randomization analyses supported a potential causal association. We observed that the same mutations found in blood were also detected in microglia-enriched fraction of the brain in seven of eight CHIP carriers. Single-nucleus chromatin accessibility profiling of brain-derived nuclei in six CHIP carriers revealed that the mutated cells comprised a large proportion of the microglial pool in the samples examined. While additional studies are required to validate the mechanistic findings, these results suggest that CHIP may have a role in attenuating the risk of AD.

  View details for DOI 10.1038/s41591-023-02397-2

  View details for PubMedID 37322115

  View details for PubMedCentralID 4306669

• CD22 CAR T-cell associated hematologic toxicities, endothelial activation and relationship to neurotoxicity. Journal for immunotherapy of cancer Jess, J., Yates, B., Dulau-Florea, A., Parker, K., Inglefield, J., Lichtenstein, D., Schischlik, F., Ongkeko, M., Wang, Y., Shahani, S., Cullinane, A., Smith, H., Kane, E., Little, L., Chen, D., Fry, T. J., Shalabi, H., Wang, H. W., Satpathy, A., Lozier, J., Shah, N. N. 2023; 11 (6)

  Abstract

  Hematologic toxicities, including coagulopathy, endothelial activation, and cytopenias, with CD19-targeted chimeric antigen receptor (CAR) T-cell therapies correlate with cytokine release syndrome (CRS) and neurotoxicity severity, but little is known about the extended toxicity profiles of CAR T-cells targeting alternative antigens. This report characterizes hematologic toxicities seen following CD22 CAR T-cells and their relationship to CRS and neurotoxicity.We retrospectively characterized hematologic toxicities associated with CRS seen on a phase 1 study of anti-CD22 CAR T-cells for children and young adults with relapsed/refractory CD22+ hematologic malignancies. Additional analyses included correlation of hematologic toxicities with neurotoxicity and exploring effects of hemophagocytic lymphohistiocytosis-like toxicities (HLH) on bone marrow recovery and cytopenias. Coagulopathy was defined as evidence of bleeding or abnormal coagulation parameters. Hematologic toxicities were graded by Common Terminology Criteria for Adverse Events V.4.0.Across 53 patients receiving CD22 CAR T-cells who experienced CRS, 43 (81.1%) patients achieved complete remission. Eighteen (34.0%) patients experienced coagulopathy, of whom 16 had clinical manifestations of mild bleeding (typically mucosal bleeding) which generally subsided following CRS resolution. Three had manifestations of thrombotic microangiopathy. Patients with coagulopathy had higher peak ferritin, D-dimer, prothrombin time, international normalized ratio (INR), lactate dehydrogenase (LDH), tissue factor, prothrombin fragment F1+2 and soluble vascular cell adhesion molecule-1 (s-VCAM-1). Despite a relatively higher incidence of HLH-like toxicities and endothelial activation, overall neurotoxicity was generally less severe than reported with CD19 CAR T-cells, prompting additional analysis to explore CD22 expression in the central nervous system (CNS). Single-cell analysis revealed that in contrast to CD19 expression, CD22 is not on oligodendrocyte precursor cells or on neurovascular cells but is seen on mature oligodendrocytes. Lastly, among those attaining CR, grade 3-4 neutropenia and thrombocytopenia were seen in 65% of patients at D28.With rising incidence of CD19 negative relapse, CD22 CAR T-cells are increasingly important for the treatment of B-cell malignancies. In characterizing hematologic toxicities on CD22 CAR T-cells, we demonstrate that despite endothelial activation, coagulopathy, and cytopenias, neurotoxicity was relatively mild and that CD22 and CD19 expression in the CNS differed, providing one potential hypothesis for divergent neurotoxicity profiles. Systematic characterization of on-target off-tumor toxicities of novel CAR T-cell constructs will be vital as new antigens are targeted.NCT02315612.

  View details for DOI 10.1136/jitc-2022-005898

  View details for PubMedID 37295816

• Skin basal cell carcinomas assemble a pro-tumorigenic spatially organized and self-propagating Trem2+ myeloid niche. Nature communications Haensel, D., Daniel, B., Gaddam, S., Pan, C., Fabo, T., Bjelajac, J., Jussila, A. R., Gonzalez, F., Li, N. Y., Chen, Y., Hou, J., Patel, T., Aasi, S., Satpathy, A. T., Oro, A. E. 2023; 14 (1): 2685

  Abstract

  Cancer immunotherapies have revolutionized treatment but have shown limited success as single-agent therapies highlighting the need to understand the origin, assembly, and dynamics of heterogeneous tumor immune niches. Here, we use single-cell and imaging-based spatial analysis to elucidate three microenvironmental neighborhoods surrounding the heterogeneous basal cell carcinoma tumor epithelia. Within the highly proliferative neighborhood, we find that TREM2+ skin cancer-associated macrophages (SCAMs) support the proliferation of a distinct tumor epithelial population through an immunosuppression-independent manner via oncostatin-M/JAK-STAT3 signaling. SCAMs represent a unique tumor-specific TREM2+ population defined by VCAM1 surface expression that is not found in normal homeostatic skin or during wound healing. Furthermore, SCAMs actively proliferate and self-propagate through multiple serial tumor passages, indicating long-term potential. The tumor rapidly drives SCAM differentiation, with intratumoral injections sufficient to instruct naive bone marrow-derived monocytes to polarize within days. This work provides mechanistic insights into direct tumor-immune niche dynamics independent of immunosuppression, providing the basis for potential combination tumor therapies.

  View details for DOI 10.1038/s41467-023-37993-w

  View details for PubMedID 37164949

• Plasticity of clonal memory CD8 T cell differentiation Hiam-Galvez, K., Black, M., Raposo, C. J., Satpathy, A. T. AMER ASSOC IMMUNOLOGISTS. 2023

  View details for Web of Science ID 001106506503140

• Inosine Induces Stemness Features in CAR T cells and Enhances Potency. bioRxiv : the preprint server for biology Klysz, D. D., Fowler, C., Malipatlolla, M., Stuani, L., Freitas, K. A., Meier, S., Daniel, B., Sandor, K., Xu, P., Huang, J., Labanieh, L., Leruste, A., Bashti, M., Keerthi, V., Mata-Alcazar, J., Gkitsas, N., Guerrero, J. A., Fisher, C., Patel, S., Asano, K., Patel, S., Davis, K. L., Satpathy, A. T., Feldman, S. A., Sotillo, E., Mackall, C. L. 2023

  Abstract

  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.

  View details for DOI 10.1101/2023.04.21.537859

  View details for PubMedID 37162847

  View details for PubMedCentralID PMC10168291

• Aberrant activation of TCL1A promotes stem cell expansion in clonal haematopoiesis. Nature Weinstock, J. S., Gopakumar, J., Burugula, B. B., Uddin, M. M., Jahn, N., Belk, J. A., Bouzid, H., Daniel, B., Miao, Z., Ly, N., Mack, T. M., Luna, S. E., Prothro, K. P., Mitchell, S. R., Laurie, C. A., Broome, J. G., Taylor, K. D., Guo, X., Sinner, M. F., von Falkenhausen, A. S., Kääb, S., Shuldiner, A. R., O'Connell, J. R., Lewis, J. P., Boerwinkle, E., Barnes, K. C., Chami, N., Kenny, E. E., Loos, R. J., Fornage, M., Hou, L., Lloyd-Jones, D. M., Redline, S., Cade, B. E., Psaty, B. M., Bis, J. C., Brody, J. A., Silverman, E. K., Yun, J. H., Qiao, D., Palmer, N. D., Freedman, B. I., Bowden, D. W., Cho, M. H., DeMeo, D. L., Vasan, R. S., Yanek, L. R., Becker, L. C., Kardia, S. L., Peyser, P. A., He, J., Rienstra, M., Van der Harst, P., Kaplan, R., Heckbert, S. R., Smith, N. L., Wiggins, K. L., Arnett, D. K., Irvin, M. R., Tiwari, H., Cutler, M. J., Knight, S., Muhlestein, J. B., Correa, A., Raffield, L. M., Gao, Y., de Andrade, M., Rotter, J. I., Rich, S. S., Tracy, R. P., Konkle, B. A., Johnsen, J. M., Wheeler, M. M., Smith, J. G., Melander, O., Nilsson, P. M., Custer, B. S., Duggirala, R., Curran, J. E., Blangero, J., McGarvey, S., Williams, L. K., Xiao, S., Yang, M., Gu, C. C., Chen, Y. I., Lee, W. J., Marcus, G. M., Kane, J. P., Pullinger, C. R., Shoemaker, M. B., Darbar, D., Roden, D. M., Albert, C., Kooperberg, C., Zhou, Y., Manson, J. E., Desai, P., Johnson, A. D., Mathias, R. A., Blackwell, T. W., Abecasis, G. R., Smith, A. V., Kang, H. M., Satpathy, A. T., Natarajan, P., Kitzman, J. O., Whitsel, E. A., Reiner, A. P., Bick, A. G., Jaiswal, S. 2023

  Abstract

  Mutations in a diverse set of driver genes increase the fitness of haematopoietic stem cells (HSCs), leading to clonal haematopoiesis1. These lesions are precursors for blood cancers2-6, but the basis of their fitness advantage remains largely unknown, partly owing to a paucity of large cohorts in which the clonal expansion rate has been assessed by longitudinal sampling. Here, to circumvent this limitation, we developed a method to infer the expansion rate from data from a single time point. We applied this method to 5,071 people with clonal haematopoiesis. A genome-wide association study revealed that a common inherited polymorphism in the TCL1A promoter was associated with a slower expansion rate in clonal haematopoiesis overall, but the effect varied by driver gene. Those carrying this protective allele exhibited markedly reduced growth rates or prevalence of clones with driver mutations in TET2, ASXL1, SF3B1 and SRSF2, but this effect was not seen in clones with driver mutations in DNMT3A. TCL1A was not expressed in normal or DNMT3A-mutated HSCs, but the introduction of mutations in TET2 or ASXL1 led to the expression of TCL1A protein and the expansion of HSCs in vitro. The protective allele restricted TCL1A expression and expansion of mutant HSCs, as did experimental knockdown of TCL1A expression. Forced expression of TCL1A promoted the expansion of human HSCs in vitro and mouse HSCs in vivo. Our results indicate that the fitness advantage of several commonly mutated driver genes in clonal haematopoiesis may be mediated by TCL1A activation.

  View details for DOI 10.1038/s41586-023-05806-1

  View details for PubMedID 37046083

  View details for PubMedCentralID 4624443

• Cis interactions in the Irf8 locus regulate stage-dependent enhancer activation. Genes & development Liu, T. T., Ou, F., Belk, J. A., Bagadia, P., Anderson, D. A., Durai, V., Yao, W., Satpathy, A. T., Murphy, T. L., Murphy, K. M. 2023

  Abstract

  Individual elements within a superenhancer can act in a cooperative or temporal manner, but the underlying mechanisms remain obscure. We recently identified an Irf8 superenhancer, within which different elements act at distinct stages of type 1 classical dendritic cell (cDC1) development. The +41-kb Irf8 enhancer is required for pre-cDC1 specification, while the +32-kb Irf8 enhancer acts to support subsequent cDC1 maturation. Here, we found that compound heterozygous Δ32/Δ41 mice, lacking the +32- and +41-kb enhancers on different chromosomes, show normal pre-cDC1 specification but, surprisingly, completely lack mature cDC1 development, suggesting cis dependence of the +32-kb enhancer on the +41-kb enhancer. Transcription of the +32-kb Irf8 enhancer-associated long noncoding RNA (lncRNA) Gm39266 is also dependent on the +41-kb enhancer. However, cDC1 development in mice remained intact when Gm39266 transcripts were eliminated by CRISPR/Cas9-mediated deletion of lncRNA promoters and when transcription across the +32-kb enhancer was blocked by premature polyadenylation. We showed that chromatin accessibility and BATF3 binding at the +32-kb enhancer were dependent on a functional +41-kb enhancer located in cis Thus, the +41-kb Irf8 enhancer controls the subsequent activation of the +32-kb Irf8 enhancer in a manner that is independent of associated lncRNA transcription.

  View details for DOI 10.1101/gad.350339.122

  View details for PubMedID 36990511

• Co-opting signalling molecules enables logic-gated control of CAR T cells. Nature Tousley, A. M., Rotiroti, M. C., Labanieh, L., Rysavy, L. W., Kim, W. J., Lareau, C., Sotillo, E., Weber, E. W., Rietberg, S. P., Dalton, G. N., Yin, Y., Klysz, D., Xu, P., de la Serna, E. L., Dunn, A. R., Satpathy, A. T., Mackall, C. L., Majzner, R. G. 2023

  Abstract

  Although chimeric antigen receptor (CAR) T cells have altered the treatment landscape for B cell malignancies, the risk of on-target, off-tumour toxicity has hampered their development for solid tumours because most target antigens are shared with normal cells1,2. Researchers have attempted to apply Boolean-logic gating to CAR T cells to prevent toxicity3-5; however, a truly safe and effective logic-gated CAR has remained elusive6. Here we describe an approach to CAR engineering in which we replace traditional CD3ζ domains with intracellular proximal T cell signalling molecules. We show that certain proximal signalling CARs, such as a ZAP-70 CAR, can activate T cells and eradicate tumours in vivo while bypassing upstream signalling proteins, including CD3ζ. The primary role of ZAP-70 is to phosphorylate LAT and SLP-76, which form a scaffold for signal propagation. We exploited the cooperative role of LAT and SLP-76 to engineer logic-gated intracellular network (LINK) CAR, a rapid and reversible Boolean-logic AND-gated CAR T cell platform that outperforms other systems in both efficacy and prevention of on-target, off-tumour toxicity. LINK CAR will expand the range of molecules that can be targeted with CAR T cells, and will enable these powerful therapeutic agents to be used for solid tumours and diverse diseases such as autoimmunity7 and fibrosis8. In addition, this work shows that the internal signalling machinery of cells can be repurposed into surface receptors, which could open new avenues for cellular engineering.

  View details for DOI 10.1038/s41586-023-05778-2

  View details for PubMedID 36890224

  View details for PubMedCentralID 7433347

• Mitochondrial single-cell ATAC-seq for high-throughput multi-omic detection of mitochondrial genotypes and chromatin accessibility. Nature protocols Lareau, C. A., Liu, V., Muus, C., Praktiknjo, S. D., Nitsch, L., Kautz, P., Sandor, K., Yin, Y., Gutierrez, J. C., Pelka, K., Satpathy, A. T., Regev, A., Sankaran, V. G., Ludwig, L. S. 2023

  Abstract

  Natural sequence variation within mitochondrial DNA (mtDNA) contributes to human phenotypes and may serve as natural genetic markers in human cells for clonal and lineage tracing. We recently developed a single-cell multi-omic approach, called 'mitochondrial single-cell assay for transposase-accessible chromatin with sequencing' (mtscATAC-seq), enabling concomitant high-throughput mtDNA genotyping and accessible chromatin profiling. Specifically, our technique allows the mitochondrial genome-wide inference of mtDNA variant heteroplasmy along with information on cell state and accessible chromatin variation in individual cells. Leveraging somatic mtDNA mutations, our method further enables inference of clonal relationships among native ex vivo-derived human cells not amenable to genetic engineering-based clonal tracing approaches. Here, we provide a step-by-step protocol for the use of mtscATAC-seq, including various cell-processing and flow cytometry workflows, by using primary hematopoietic cells, subsequent single-cell genomic library preparation and sequencing that collectively take ~3-4 days to complete. We discuss experimental and computational data quality control metrics and considerations for the extension to other mammalian tissues. Overall, mtscATAC-seq provides a broadly applicable platform to map clonal relationships between cells in human tissues, investigate fundamental aspects of mitochondrial genetics and enable additional modes of multi-omic discovery.

  View details for DOI 10.1038/s41596-022-00795-3

  View details for PubMedID 36792778

  View details for PubMedCentralID 7307462

• A Cre-deleter specific for embryo-derived brain macrophages reveals distinct features of microglia and border macrophages. Immunity Brioschi, S., Belk, J. A., Peng, V., Molgora, M., Rodrigues, P. F., Nguyen, K. M., Wang, S., Du, S., Wang, W. L., Grajales-Reyes, G. E., Ponce, J. M., Yuede, C. M., Li, Q., Baer, J. M., DeNardo, D. G., Gilfillan, S., Cella, M., Satpathy, A. T., Colonna, M. 2023

  Abstract

  Genetic tools to target microglia specifically and efficiently from the early stages of embryonic development are lacking. We generated a constitutive Cre line controlled by the microglia signature gene Crybb1 that produced nearly complete recombination in embryonic brain macrophages (microglia and border-associated macrophages [BAMs]) by the perinatal period, with limited recombination in peripheral myeloid cells. Using this tool in combination with Flt3-Cre lineage tracer, single-cell RNA-sequencing analysis, and confocal imaging, we resolved embryonic-derived versus monocyte-derived BAMs in the mouse cortex. Deletion of the transcription factor SMAD4 in microglia and embryonic-derived BAMs using Crybb1-Cre caused a developmental arrest of microglia, which instead acquired a BAM specification signature. By contrast, the development of genuine BAMs remained unaffected. Our results reveal that SMAD4 drives a transcriptional and epigenetic program that is indispensable for the commitment of brain macrophages to the microglia fate and highlight Crybb1-Cre as a tool for targeting embryonic brain macrophages.

  View details for DOI 10.1016/j.immuni.2023.01.028

  View details for PubMedID 36791722

• TCR-BERT: learning the grammar of T-cell receptors for flexible antigen-binding analyses Wu, K., Yost, K. E., Daniel, B., Belk, J. A., Xia, Y., Egawa, T., Satpathy, A., Chang, H. Y., Zou, J., Knowles, D. A., Mostafavi, S. JMLR-JOURNAL MACHINE LEARNING RESEARCH. 2023

  View details for Web of Science ID 001221072500010

• Reinfection with SARS-CoV-2 and Waning Humoral Immunity: A Case Report. Vaccines Goldman, J. D., Wang, K., Röltgen, K., Nielsen, S. C., Roach, J. C., Naccache, S. N., Yang, F., Wirz, O. F., Yost, K. E., Lee, J. Y., Chun, K., Wrin, T., Petropoulos, C. J., Lee, I., Fallen, S., Manner, P. M., Wallick, J. A., Algren, H. A., Murray, K. M., Hadlock, J., Chen, D., Dai, C. L., Yuan, D., Su, Y., Jeharajah, J., Berrington, W. R., Pappas, G. P., Nyatsatsang, S. T., Greninger, A. L., Satpathy, A. T., Pauk, J. S., Boyd, S. D., Heath, J. R. 2022; 11 (1)

  Abstract

  Recovery from COVID-19 is associated with production of anti-SARS-CoV-2 antibodies, but it is uncertain whether these confer immunity. We describe viral RNA shedding duration in hospitalized patients and identify patients with recurrent shedding. We sequenced viruses from two distinct episodes of symptomatic COVID-19 separated by 144 days in a single patient, to conclusively describe reinfection with a different strain harboring the spike variant D614G. This case of reinfection was one of the first cases of reinfection reported in 2020. With antibody, B cell and T cell analytics, we show correlates of adaptive immunity at reinfection, including a differential response in neutralizing antibodies to a D614G pseudovirus. Finally, we discuss implications for vaccine programs and begin to define benchmarks for protection against reinfection from SARS-CoV-2.

  View details for DOI 10.3390/vaccines11010005

  View details for PubMedID 36679852

• Engineered cell entry links receptor biology with single-cell genomics. Cell Yu, B., Shi, Q., Belk, J. A., Yost, K. E., Parker, K. R., Li, R., Liu, B. B., Huang, H., Lingwood, D., Greenleaf, W. J., Davis, M. M., Satpathy, A. T., Chang, H. Y. 2022

  Abstract

  Cells communicate with each other via receptor-ligand interactions. Here, we describe lentiviral-mediated cell entry by engineered receptor-ligand interaction (ENTER) to display ligand proteins, deliver payloads, and record receptor specificity. We optimize ENTER to decode interactions between Tcell receptor (TCR)-MHC peptides, antibody-antigen, and other receptor-ligand pairs. A viral presentation strategy allows ENTER to capture interactions between B cell receptor and any antigen. We engineer ENTER to deliver genetic payloads to antigen-specific T or B cells to selectively modulate cellular behavior in mixed populations. Single-cell readout of ENTER by RNA sequencing (ENTER-seq) enables multiplexed enumeration of antigen specificities, TCR clonality, cell type, and states of individual Tcells. ENTER-seq of CMV-seropositive patient blood samples reveals the viral epitopes that drive effector memory Tcell differentiation and inter-clonal vs. intra-clonal phenotypic diversity targeting the same epitope. ENTER technology enables systematic discovery of receptor specificity, linkage to cell fates, and antigen-specific cargo delivery.

  View details for DOI 10.1016/j.cell.2022.11.016

  View details for PubMedID 36516854

• Lineage plasticity dictates responsiveness to anti-GD2 therapy in neuroblastoma. Mabe, N. W., Huang, M., Schaefer, D. A., Dalton, G. N., Digiovanni, G., Alexe, G., Geraghty, A. C., Khalid, D., Mader, M. M., Sheffer, M., Linde, M. H., Ly, N., Rotiroti, M., Smith, B. H., Wernig, M., Bertozzi, C. R., Monje, M., Mitsiades, C., Majeti, R., Satpathy, A. T., Stegmaier, K., Majzner, R. G. AMER ASSOC CANCER RESEARCH. 2022

  View details for Web of Science ID 000924848300026

• MARCH1 Controls an Exhaustion-like Program of Effector CD4+ T Cells Promoting Allergic Airway Inflammation. ImmunoHorizons Castellanos, C. A., Hiam-Galvez, K. J., Ishido, S., Satpathy, A. T., Shin, J. 2022; 6 (9): 684-692

  Abstract

  Persistent antigenic signaling leads to T cell exhaustion, a dysfunctional state arising in many chronic infections and cancers. Little is known concerning mechanisms limiting exhaustion in immune-stimulatory diseases such as asthma. We report that membrane-associated RING-CH1 (MARCH1), the ubiquitin ligase that mediates surface turnover of MHC class II (MHCII) and CD86 in professional APCs, plays an essential role in restraining an exhaustion-like program of effector CD4+ T cells in a mouse model of asthma. Mice lacking MARCH1 or the ubiquitin acceptor sites of MHCII and CD86 exhibited increased MHCII and CD86 surface expression on lung APCs, and this increase promoted enhanced expression of immune-inhibitory receptors by effector CD4+ T cells and inhibited their proliferation. Remarkably, ablation of MARCH1 in mice with established asthma reduced airway infiltration of eosinophils and Th2 cells. Thus, MARCH1 controls an exhaustion-like program of effector CD4+ T cells during allergic airway inflammation and may serve as a therapeutic target for asthma.

  View details for DOI 10.4049/immunohorizons.2200056

  View details for PubMedID 36100368

• A RORgammat+ cell instructs gut microbiota-specific Treg cell differentiation. Nature Kedmi, R., Najar, T. A., Mesa, K. R., Grayson, A., Kroehling, L., Hao, Y., Hao, S., Pokrovskii, M., Xu, M., Talbot, J., Wang, J., Germino, J., Lareau, C. A., Satpathy, A. T., Anderson, M. S., Laufer, T. M., Aifantis, I., Bartleson, J. M., Allen, P. M., Paidassi, H., Gardner, J. M., Stoeckius, M., Littman, D. R. 2022

  Abstract

  The mutualistic relationship of gut-resident microbiota and the host immune system promotes homeostasis that ensures maintenance of the microbial community and of a largely non-aggressive immune cell compartment1,2. The consequences of disturbing this balance include proximal inflammatory conditions, such as Crohn's disease, and systemic illnesses. This equilibrium is achieved in part through the induction of both effector and suppressor arms of the adaptive immune system. Helicobacter species induce T regulatory (Treg) and T follicular helper (TFH) cells under homeostatic conditions, but induce inflammatory T helper 17 (TH17) cells when induced Treg (iTreg) cells are compromised3,4. How Helicobacter and other gut bacteria direct T cells to adopt distinct functions remains poorly understood. Here we investigated the cells and molecular components required for iTreg cell differentiation. We found that antigen presentation by cells expressing RORgammat, rather than by classical dendritic cells, was required and sufficient for induction of Treg cells. These RORgammat+ cells-probably type 3 innate lymphoid cells and/or Janus cells5-require the antigen-presentation machinery, the chemokine receptor CCR7 and the TGFbeta activator alphav integrin. In the absence of any of these factors, there was expansion of pathogenic TH17 cells instead of iTreg cells, induced by CCR7-independent antigen-presenting cells. Thus, intestinal commensal microbes and their products target multiple antigen-presenting cells with pre-determined features suited to directing appropriate T cell differentiation programmes, rather than a common antigen-presenting cell that they endow with appropriate functions.

  View details for DOI 10.1038/s41586-022-05089-y

  View details for PubMedID 36071167

• RASA2 ablation in T cells boosts antigen sensitivity and long-term function. Nature Carnevale, J., Shifrut, E., Kale, N., Nyberg, W. A., Blaeschke, F., Chen, Y. Y., Li, Z., Bapat, S. P., Diolaiti, M. E., O'Leary, P., Vedova, S., Belk, J., Daniel, B., Roth, T. L., Bachl, S., Anido, A. A., Prinzing, B., Ibanez-Vega, J., Lange, S., Haydar, D., Luetke-Eversloh, M., Born-Bony, M., Hegde, B., Kogan, S., Feuchtinger, T., Okada, H., Satpathy, A. T., Shannon, K., Gottschalk, S., Eyquem, J., Krenciute, G., Ashworth, A., Marson, A. 2022

  Abstract

  The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints1,2. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function3-10. Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulationandcan increase gradually with chronic antigen exposure. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in thebone marrow in a mouse model of leukaemia. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.

  View details for DOI 10.1038/s41586-022-05126-w

  View details for PubMedID 36002574

• Runx3 drives a CD8+ T cell tissue residency program that is absent in CD4+ T cells. Nature immunology Fonseca, R., Burn, T. N., Gandolfo, L. C., Devi, S., Park, S. L., Obers, A., Evrard, M., Christo, S. N., Buquicchio, F. A., Lareau, C. A., McDonald, K. M., Sandford, S. K., Zamudio, N. M., Zanluqui, N. G., Zaid, A., Speed, T. P., Satpathy, A. T., Mueller, S. N., Carbone, F. R., Mackay, L. K. 2022

  Abstract

  Tissue-resident memory T cells (TRM cells) provide rapid and superior control of localized infections. While the transcription factor Runx3 is a critical regulator of CD8+ T cell tissue residency, its expression is repressed in CD4+ T cells. Here, we show that, as a direct consequence of this Runx3-deficiency, CD4+ TRM cells lacked the transforming growth factor (TGF)-beta-responsive transcriptional network that underpins the tissue residency of epithelial CD8+ TRM cells. While CD4+ TRM cell formation required Runx1, this, along with the modest expression of Runx3 in CD4+ TRM cells, was insufficient to engage the TGF-beta-driven residency program. Ectopic expression of Runx3 in CD4+ T cells incited this TGF-beta-transcriptional network to promote prolonged survival, decreased tissue egress, a microanatomical redistribution towards epithelial layers and enhanced effector functionality. Thus, our results reveal distinct programming of tissue residency in CD8+ and CD4+ TRM cell subsets that is attributable to divergent Runx3 activity.

  View details for DOI 10.1038/s41590-022-01273-4

  View details for PubMedID 35882933

• Transition to a mesenchymal state in neuroblastoma confers resistance to anti-GD2 antibody via reduced expression of ST8SIA1. Nature cancer Mabe, N. W., Huang, M., Dalton, G. N., Alexe, G., Schaefer, D. A., Geraghty, A. C., Robichaud, A. L., Conway, A. S., Khalid, D., Mader, M. M., Belk, J. A., Ross, K. N., Sheffer, M., Linde, M. H., Ly, N., Yao, W., Rotiroti, M. C., Smith, B. A., Wernig, M., Bertozzi, C. R., Monje, M., Mitsiades, C. S., Majeti, R., Satpathy, A. T., Stegmaier, K., Majzner, R. G. 2022

  Abstract

  Immunotherapy with anti-GD2 antibodies has advanced the treatment of children with high-risk neuroblastoma, but nearly half of patients relapse, and little is known about mechanisms of resistance to anti-GD2 therapy. Here, we show that reduced GD2 expression was significantly correlated with the mesenchymal cell state in neuroblastoma and that a forced adrenergic-to-mesenchymal transition (AMT) conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2-expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells and re-expression of ST8SIA1, restoring surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential EZH2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma.

  View details for DOI 10.1038/s43018-022-00405-x

  View details for PubMedID 35817829

• Single-cell multi-omic profiling and clonal tracing of the human gynecological tumor microenvironment Liu, V., Sandor, K., Daniel, B., Berthoin, L., Sabri, S., Panagiotopoulou, S., Yin, Y., Hiam-Galvez, K., Sit, R., Fan, Z., Galvin, B., Khan, O., Bezman, N., Grogan, J., Howitt, B., Zheng, G., Lareau, C., Satpathy, A. AMER ASSOC CANCER RESEARCH. 2022

  View details for Web of Science ID 000892509506622

• Enhanced effector activity of mediator CDK8 kinase module deficient CAR-T Cells Freitas, K. A., Belk, J. A., Sotillo, E., Daniel, B., Sandor, K., Klysz, D., Duong, V. T., Xu, P., Malipatlolla, M., Weber, E. W., Majzner, R. G., Chang, H. Y., Satpathy, A. T., Mackall, C. AMER ASSOC CANCER RESEARCH. 2022

  View details for Web of Science ID 000892509507415

• Epigenetic regulation of T cell exhaustion. Nature immunology Belk, J. A., Daniel, B., Satpathy, A. T. 2022

  Abstract

  Chronic antigen stimulation during viral infections and cancer can lead to T cell exhaustion, which is characterized by reduced effector function and proliferation, and the expression of inhibitory immune checkpoint receptors. Recent studies have demonstrated that T cell exhaustion results in wholescale epigenetic remodeling that confers phenotypic stability to these cells and prevents T cell reinvigoration by checkpoint blockade. Here, we review foundational technologies to profile the epigenome at multiple scales, including mapping the locations of transcription factors and histone modifications, DNA methylation and three-dimensional genome conformation. We discuss how these technologies have elucidated the development and epigenetic regulation of exhausted T cells and functional implications across viral infection, cancer, autoimmunity and engineered T cell therapies. Finally, we cover emerging multi-omic and genome engineering technologies, current and upcoming opportunities to apply these to T cell exhaustion, and therapeutic opportunities for T cell engineering in the clinic.

  View details for DOI 10.1038/s41590-022-01224-z

  View details for PubMedID 35624210

• BCL6-dependent TCF-1+ progenitor cells maintain effector and helper CD4+ Tcell responses to persistent antigen. Immunity Xia, Y., Sandor, K., Pai, J. A., Daniel, B., Raju, S., Wu, R., Hsiung, S., Qi, Y., Yangdon, T., Okamoto, M., Chou, C., Hiam-Galvez, K. J., Schreiber, R. D., Murphy, K. M., Satpathy, A. T., Egawa, T. 2022

  Abstract

  Soon after activation, CD4+ Tcells are segregated into BCL6+ follicular helper (Tfh) and BCL6- effector (Teff) Tcells. Here, we explored how these subsets are maintained during chronic antigen stimulation using the mouse chronic LCMV infection model. Using single cell-transcriptomic and epigenomic analyses, we identified a population of PD-1+ TCF-1+ CD4+ Tcells with memory-like features. TCR clonal tracing and adoptive transfer experiments demonstrated that these cells have self-renewal capacity and continue to give rise to both Teff and Tfh cells, thus functioning as progenitor cells. Conditional deletion experiments showed Bcl6-dependent development of these progenitors, which were essential for sustaining antigen-specific CD4+ Tcell responses to chronic infection. An analogous CD4+ Tcell population developed in draining lymph nodes in response to tumors. Our study reveals the heterogeneity and plasticity of CD4+ Tcells during persistent antigen exposure and highlights their population dynamics through a stable, bipotent intermediate state.

  View details for DOI 10.1016/j.immuni.2022.05.003

  View details for PubMedID 35637103

• Spatiotemporal co-dependency between macrophages and exhausted CD8+ T cells in cancer. Cancer cell Kersten, K., Hu, K. H., Combes, A. J., Samad, B., Harwin, T., Ray, A., Rao, A. A., Cai, E., Marchuk, K., Artichoker, J., Courau, T., Shi, Q., Belk, J., Satpathy, A. T., Krummel, M. F. 2022

  Abstract

  T cell exhaustion is a major impediment to antitumor immunity. However, it remains elusive how other immune cells in the tumor microenvironment (TME) contribute to this dysfunctional state. Here, we show that the biology of tumor-associated macrophages (TAMs) and exhausted T cells (Tex) in the TME is extensively linked. We demonstrate that in vivo depletion of TAMs reduces exhaustion programs in tumor-infiltrating CD8+ T cells and reinvigorates their effector potential. Reciprocally, transcriptional and epigenetic profiling reveals that Tex express factors that actively recruit monocytes to the TME and shape their differentiation. Using lattice light sheet microscopy, we show that TAM and CD8+ T cells engage in unique, long-lasting, antigen-specific synaptic interactions that fail to activate T cells but prime them for exhaustion, which is then accelerated in hypoxic conditions. Spatially resolved sequencing supports a spatiotemporal self-enforcing positive feedback circuit that is aligned to protect rather than destroy a tumor.

  View details for DOI 10.1016/j.ccell.2022.05.004

  View details for PubMedID 35623342

• BCL6-dependent TCF-1+progenitor cells maintain effector and helper CD4 T cell responses to persistent antigen Xia, Y., Sandor, K., Pai, J. A., Daniel, B., Raju, S., Wu, R., Hsiung, S., Qi, Y., Yangdon, T., Okamoto, M., Schreiber, R. D., Murphy, K. M., Satpathy, A., Egawa, T. AMER ASSOC IMMUNOLOGISTS. 2022

  View details for DOI 10.4049/jimmunol.208.Supp.169.08

  View details for Web of Science ID 000892531602100

• Lymph node colonization induces tumor-immune tolerance to promote distant metastasis. Cell Reticker-Flynn, N. E., Zhang, W., Belk, J. A., Basto, P. A., Escalante, N. K., Pilarowski, G. O., Bejnood, A., Martins, M. M., Kenkel, J. A., Linde, I. L., Bagchi, S., Yuan, R., Chang, S., Spitzer, M. H., Carmi, Y., Cheng, J., Tolentino, L. L., Choi, O., Wu, N., Kong, C. S., Gentles, A. J., Sunwoo, J. B., Satpathy, A. T., Plevritis, S. K., Engleman, E. G. 2022

  Abstract

  For many solid malignancies, lymph node (LN) involvement represents a harbinger of distant metastatic disease and, therefore, an important prognostic factor. Beyond its utility as a biomarker, whether and how LN metastasis plays an active role in shaping distant metastasis remains an open question. Here, we develop a syngeneic melanoma mouse model of LN metastasis to investigate how tumors spread to LNs and whether LN colonization influences metastasis to distant tissues. We show that an epigenetically instilled tumor-intrinsic interferon response program confers enhanced LN metastatic potential by enabling the evasion of NK cells and promoting LN colonization. LN metastases resist T cell-mediated cytotoxicity, induce antigen-specific regulatory T cells, and generate tumor-specific immune tolerance that subsequently facilitates distant tumor colonization. These effects extend to human cancers and other murine cancer models, implicating a conserved systemic mechanism by which malignancies spread to distant organs.

  View details for DOI 10.1016/j.cell.2022.04.019

  View details for PubMedID 35525247

• Enhanced safety and efficacy of protease-regulated CAR-T cell receptors. Cell Labanieh, L., Majzner, R. G., Klysz, D., Sotillo, E., Fisher, C. J., Vilches-Moure, J. G., Pacheco, K. Z., Malipatlolla, M., Xu, P., Hui, J. H., Murty, T., Theruvath, J., Mehta, N., Yamada-Hunter, S. A., Weber, E. W., Heitzeneder, S., Parker, K. R., Satpathy, A. T., Chang, H. Y., Lin, M. Z., Cochran, J. R., Mackall, C. L. 2022

  Abstract

  Regulatable CAR platforms could circumvent toxicities associated with CAR-T therapy, but existing systems have shortcomings including leakiness and attenuated activity. Here, we present SNIP CARs, a protease-based platform for regulating CAR activity using an FDA-approved small molecule. Design iterations yielded CAR-T cells that manifest full functional capacity with drug and no leaky activity in the absence of drug. In numerous models, SNIP CAR-T cells were more potent than constitutive CAR-T cells and showed diminished T cell exhaustion and greater stemness. In a ROR1-based CAR lethality model, drug cessation following toxicity onset reversed toxicity, thereby credentialing the platform as a safety switch. In the same model, reduced drug dosing opened a therapeutic window that resulted in tumor eradication in the absence of toxicity. SNIP CARs enable remote tuning of CAR activity, which provides solutions to safety and efficacy barriers that are currently limiting progress in using CAR-T cells to treat solid tumors.

  View details for DOI 10.1016/j.cell.2022.03.041

  View details for PubMedID 35483375

• KIR+CD8+ T cells suppress pathogenic T cells and are active in autoimmune diseases and COVID-19. Science (New York, N.Y.) Li, J., Zaslavsky, M., Su, Y., Guo, J., Sikora, M. J., van Unen, V., Christophersen, A., Chiou, S., Chen, L., Li, J., Ji, X., Wilhelmy, J., McSween, A. M., Palanski, B. A., Mallajosyula, V. V., Bracey, N. A., Dhondalay, G. K., Bhamidipati, K., Pai, J., Kipp, L. B., Dunn, J. E., Hauser, S. L., Oksenberg, J. R., Satpathy, A. T., Robinson, W. H., Dekker, C. L., Steinmetz, L. M., Khosla, C., Utz, P. J., Sollid, L. M., Chien, Y., Heath, J. R., Fernandez-Becker, N. Q., Nadeau, K. C., Saligrama, N., Davis, M. M. 2022: eabi9591

  Abstract

  Here we find that CD8+ T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) are the human equivalent of Ly49+CD8+ regulatory T cells in mice and are increased in the blood and inflamed tissues of patients with a variety of autoimmune diseases. Moreover, these CD8+ T cells efficiently eliminated pathogenic gliadin-specific CD4+ T cells from celiac disease patients' leukocytes in vitro. We also find elevated levels of KIR+CD8+ T cells, but not CD4+ regulatory T cells, in COVID-19 patients, which correlated with disease severity and vasculitis. Selective ablation of Ly49+CD8+ T cells in virus-infected mice led to autoimmunity post infection. Our results indicate that in both species, these regulatory CD8+ T cells act uniquely to suppress pathogenic T cells in autoimmune and infectious diseases.

  View details for DOI 10.1126/science.abi9591

  View details for PubMedID 35258337

• Mitochondrial variant enrichment from high-throughput single-cell RNA sequencing resolves clonal populations. Nature biotechnology Miller, T. E., Lareau, C. A., Verga, J. A., DePasquale, E. A., Liu, V., Ssozi, D., Sandor, K., Yin, Y., Ludwig, L. S., El Farran, C. A., Morgan, D. M., Satpathy, A. T., Griffin, G. K., Lane, A. A., Love, J. C., Bernstein, B. E., Sankaran, V. G., van Galen, P. 2022

  Abstract

  The combination of single-cell transcriptomics with mitochondrial DNA variant detection can be used to establish lineage relationships in primary human cells, but current methods are not scalable to interrogate complex tissues. Here, we combine common 3' single-cell RNA-sequencing protocols with mitochondrial transcriptome enrichment to increase coverage by more than 50-fold, enabling high-confidence mutation detection. The method successfully identifies skewed immune-cell expansions in primary human clonal hematopoiesis.

  View details for DOI 10.1038/s41587-022-01210-8

  View details for PubMedID 35210612

• Bystander T cells in cancer immunology and therapy. Nature cancer Meier, S. L., Satpathy, A. T., Wells, D. K. 2022; 3 (2): 143-155

  Abstract

  Cancer-specific T cells are required for effective anti-cancer immunity and have a central role in cancer immunotherapy. However, emerging evidence suggests that only a small fraction of tumor-infiltrating T cells are cancer specific, and T cells that recognize cancer-unrelated antigens (so-called 'bystanders') are abundant. Although the role of cancer-specific T cells in anti-cancer immunity has been well established, the implications of bystander T cells in tumors are only beginning to be understood. It is becoming increasingly clear that bystander T cells are not a homogeneous group of cells but, instead, they differ in their specificities, their activation states and effector functions. In this Perspective, we discuss recent studies of bystander T cells in tumors, including experimental and computational approaches that enable their identification and functional analysis and viewpoints on how these insights could be used to develop new therapeutic approaches for cancer immunotherapy.

  View details for DOI 10.1038/s43018-022-00335-8

  View details for PubMedID 35228747

• GPC2-CAR T cells tuned for low antigen density mediate potent activity against neuroblastoma without toxicity CANCER CELL Heitzeneder, S., Bosse, K. R., Zhu, Z., Zhelev, D., Majzner, R. G., Radosevich, M. T., Dhingra, S., Sotillo, E., Buongervino, S., Pascual-Pasto, G., Garrigan, E., Xu, P., Huang, J., Salzer, B., Delaidelli, A., Raman, S., Cui, H., Martinez, B., Bornheimer, S. J., Sahaf, B., Alag, A., Fetahu, I. S., Hasselblatt, M., Parker, K. R., Anbunathan, H., Hwang, J., Huang, M., Sakamoto, K., Lacayo, N. J., Klysz, D. D., Theruvath, J., Vilches-Moure, J. G., Satpathy, A. T., Chang, H. Y., Lehner, M., Taschner-Mandl, S., Julien, J., Sorensen, P. H., Dimitrov, D. S., Maris, J. M., Mackall, C. L. 2022; 40 (1): 53-+

  View details for DOI 10.1016/j.ccell.2021.12-005

  View details for Web of Science ID 000744691500012

• GPC2-CAR Tcells tuned for low antigen density mediate potent activity against neuroblastoma without toxicity. Cancer cell Heitzeneder, S., Bosse, K. R., Zhu, Z., Zhelev, D., Majzner, R. G., Radosevich, M. T., Dhingra, S., Sotillo, E., Buongervino, S., Pascual-Pasto, G., Garrigan, E., Xu, P., Huang, J., Salzer, B., Delaidelli, A., Raman, S., Cui, H., Martinez, B., Bornheimer, S. J., Sahaf, B., Alag, A., Fetahu, I. S., Hasselblatt, M., Parker, K. R., Anbunathan, H., Hwang, J., Huang, M., Sakamoto, K., Lacayo, N. J., Klysz, D. D., Theruvath, J., Vilches-Moure, J. G., Satpathy, A. T., Chang, H. Y., Lehner, M., Taschner-Mandl, S., Julien, J., Sorensen, P. H., Dimitrov, D. S., Maris, J. M., Mackall, C. L. 1800

  Abstract

  Pediatric cancers often mimic fetal tissues and express proteins normally silenced postnatally that could serve as immune targets. We developed Tcells expressing chimeric antigen receptors (CARs) targeting glypican-2 (GPC2), a fetal antigen expressed on neuroblastoma (NB) and several other solid tumors. CARs engineered using standard designs control NBs with transgenic GPC2 overexpression, but not those expressing clinically relevant GPC2 site density (5,000 molecules/cell, range 1-6* 103). Iterative engineering of transmembrane (TM) and co-stimulatory domains plus overexpression of c-Jun lowered the GPC2-CAR antigen density threshold, enabling potent and durable eradication of NBs expressing clinically relevant GPC2 antigen density, without toxicity. These studies highlight the critical interplay between CAR design and antigen density threshold, demonstrate potent efficacy and safety of a lead GPC2-CAR candidate suitable for clinical testing, and credential oncofetal antigens as a promising class of targets for CAR Tcell therapy of solid tumors.

  View details for DOI 10.1016/j.ccell.2021.12.005

  View details for PubMedID 34971569

• Charting the tumor antigen maps drawn by single-cell genomics. Cancer cell Lareau, C. A., Parker, K. R., Satpathy, A. T. 1800; 39 (12): 1553-1557

  Abstract

  The remarkable specificity of antibodies has enabled precision cancer immunotherapies, including chimeric antigen receptor Tcells and antibody-drug conjugates. In parallel, single-cell genomics technologies present the possibility of a comprehensive annotation of antigen expression throughout tissues of the human body and on cancer cells. We reflect on the rationale for antigen targets currently used in immunotherapies, their adverse effects revealed in the clinic, and the opportunity to utilize large genomics datasets to de-risk potential targets and nominate optimal antigens for therapy.

  View details for DOI 10.1016/j.ccell.2021.11.005

  View details for PubMedID 34906314

• Toward a better understanding of T cells in cancer CANCER CELL Oh, D. Y., Fong, L., Newell, E. W., Turk, M., Chi, H., Chang, H. Y., Satpathy, A. T., Fairfax, B., Silva-Santos, B., Lantz, O. 2021; 39 (12): 1549-1552

  Abstract

  T cells mediate anti-tumor immune responses and are the key target of immune checkpoint therapy, but they can also promote immune tolerance. A clear understanding of the specific contributions and biology of different T cell subsets is required to fully harness the curative potential of immunotherapies. Experts discuss the state of the field and key challenges for moving forward.

  View details for Web of Science ID 000731472200001

  View details for PubMedID 34906313

• ecDNA hubs drive cooperative intermolecular oncogene expression. Nature Hung, K. L., Yost, K. E., Xie, L., Shi, Q., Helmsauer, K., Luebeck, J., Schopflin, R., Lange, J. T., Chamorro Gonzalez, R., Weiser, N. E., Chen, C., Valieva, M. E., Wong, I. T., Wu, S., Dehkordi, S. R., Duffy, C. V., Kraft, K., Tang, J., Belk, J. A., Rose, J. C., Corces, M. R., Granja, J. M., Li, R., Rajkumar, U., Friedlein, J., Bagchi, A., Satpathy, A. T., Tjian, R., Mundlos, S., Bafna, V., Henssen, A. G., Mischel, P. S., Liu, Z., Chang, H. Y. 2021

  Abstract

  Extrachromosomal DNA (ecDNA) is prevalent in human cancers and mediates high expression of oncogenes through gene amplification and altered gene regulation1. Gene induction typically involves cis-regulatory elements that contact and activate genes on the same chromosome2,3. Here we show that ecDNA hubs-clusters of around 10-100 ecDNAs within the nucleus-enable intermolecular enhancer-gene interactions to promote oncogene overexpression. ecDNAs that encode multiple distinct oncogenes form hubs in diverse cancer cell types and primary tumours. Each ecDNA is more likely to transcribe the oncogene when spatially clustered with additional ecDNAs. ecDNA hubs are tethered by the bromodomain and extraterminal domain (BET) protein BRD4 in a MYC-amplified colorectal cancer cell line. The BET inhibitor JQ1 disperses ecDNA hubs and preferentially inhibits ecDNA-derived-oncogene transcription. The BRD4-bound PVT1 promoter is ectopically fused to MYC and duplicated in ecDNA, receiving promiscuous enhancer input to drive potent expression of MYC. Furthermore, the PVT1 promoter on an exogenous episome suffices to mediate gene activation in trans by ecDNA hubs in a JQ1-sensitive manner. Systematic silencing of ecDNA enhancers by CRISPR interference reveals intermolecular enhancer-gene activation among multiple oncogene loci that are amplified on distinct ecDNAs. Thus, protein-tethered ecDNA hubs enable intermolecular transcriptional regulation and may serve as units of oncogene function and cooperative evolution and as potential targets for cancer therapy.

  View details for DOI 10.1038/s41586-021-04116-8

  View details for PubMedID 34819668

• Clonal Hematopoiesis is Associated with Reduced Risk of Alzheimer's Disease Bouzid, H., Belk, J., Jan, M., Qi, Y., Sarnowski, C., Wirth, S., Ma, L., Chrostek, M., Ahmad, H., Nachun, D., Yao, W., Beiser, A., Bick, A. G., Bis, J., Fornage, M., Longstreth, W. T., Lopez, O., Nataranjan, P., Psaty, B., Satizabal, C., Weinstock, J., Larson, E., Crane, P., Keene, C., Seshadri, S., Satpathy, A. T., Montine, T., Jaiswal, S. AMER SOC HEMATOLOGY. 2021

  View details for DOI 10.1182/blood-2021-151064

  View details for Web of Science ID 000736398800006

• Single-cell multiomics defines tolerogenic extrathymic Aire-expressing populations with unique homology to thymic epithelium. Science immunology Wang, J., Lareau, C. A., Bautista, J. L., Gupta, A. R., Sandor, K., Germino, J., Yin, Y., Arvedson, M. P., Reeder, G. C., Cramer, N. T., Xie, F., Ntranos, V., Satpathy, A. T., Anderson, M. S., Gardner, J. M. 2021; 6 (65): eabl5053

  Abstract

  [Figure: see text].

  View details for DOI 10.1126/sciimmunol.abl5053

  View details for PubMedID 34767455

• Combined presentation and immunogenicity analysis reveals a recurrent RAS.Q61K neoantigen in melanoma. The Journal of clinical investigation Peri, A., Greenstein, E., Alon, M., Pai, J. A., Dingjan, T., Reich-Zeliger, S., Barnea, E., Barbolin, C., Levy, R., Arnedo-Pac, C., Kalaora, S., Dassa, B., Feldmesser, E., Shang, P., Greenberg, P., Levin, Y., Benedek, G., Levesque, M. P., Adams, D. J., Lotem, M., Wilmott, J. S., Scolyer, R. A., Jonsson, G. B., Admon, A., Rosenberg, S. A., Cohen, C. J., Niv, M. Y., Lopez-Bigas, N., Satpathy, A. T., Friedman, N., Samuels, Y. 2021; 131 (20)

  Abstract

  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.

  View details for DOI 10.1172/JCI129466

  View details for PubMedID 34651586

• Archetypes of checkpoint-responsive immunity. Trends in immunology Im, K., Combes, A. J., Spitzer, M. H., Satpathy, A. T., Krummel, M. F. 2021

  Abstract

  Responsiveness to immune checkpoint blockade (ICB) therapy in cancer is currently predicted by disparate individual measures - with varying degrees of accuracy - including tumor mutation burden, tumor-infiltrating T cell densities, dendritic cell frequencies, and the expression of checkpoint ligands. We propose that many of these individual parameters are linked, forming two distinct 'reactive' immune archetypes - collections of cells and gene expression - in ICB-responsive patients. We hypothesize that these are 'seeds' of antitumor immunity and are supported by specific elements of the tumor microenvironment (TME) and by actions of the microbiome. Although removing 'immunosuppressive' factors in the TME is important, understanding and parsing reactive immunity is crucial for optimal prognosis and for engaging this biology with candidate therapies to increase tumor cure rates.

  View details for DOI 10.1016/j.it.2021.09.007

  View details for PubMedID 34642094

• A human mutation in STAT3 promotes type 1 diabetes through a defect in CD8+ T cell tolerance. The Journal of experimental medicine Warshauer, J. T., Belk, J. A., Chan, A. Y., Wang, J., Gupta, A. R., Shi, Q., Skartsis, N., Peng, Y., Phipps, J. D., Acenas, D., Smith, J. A., Tamaki, S. J., Tang, Q., Gardner, J. M., Satpathy, A. T., Anderson, M. S. 2021; 218 (8)

  Abstract

  Naturally occurring cases of monogenic type 1 diabetes (T1D) help establish direct mechanisms driving this complex autoimmune disease. A recently identified de novo germline gain-of-function (GOF) mutation in the transcriptional regulator STAT3 was found to cause neonatal T1D. We engineered a novel knock-in mouse incorporating this highly diabetogenic human STAT3 mutation (K392R) and found that these mice recapitulated the human autoimmune diabetes phenotype. Paired single-cell TCR and RNA sequencing revealed that STAT3-GOF drives proliferation and clonal expansion of effector CD8+ cells that resist terminal exhaustion. Single-cell ATAC-seq showed that these effector T cells are epigenetically distinct and have differential chromatin architecture induced by STAT3-GOF. Analysis of islet TCR clonotypes revealed a CD8+ cell reacting against known antigen IGRP, and STAT3-GOF in an IGRP-reactive TCR transgenic model demonstrated that STAT3-GOF intrinsic to CD8+ cells is sufficient to accelerate diabetes onset. Altogether, these findings reveal a diabetogenic CD8+ T cell response that is restrained in the presence of normal STAT3 activity and drives diabetes pathogenesis.

  View details for DOI 10.1084/jem.20210759

  View details for PubMedID 34115115

• Dynamic chromatin regulatory landscape of human CAR T cell exhaustion. Proceedings of the National Academy of Sciences of the United States of America Gennert, D. G., Lynn, R. C., Granja, J. M., Weber, E. W., Mumbach, M. R., Zhao, Y., Duren, Z., Sotillo, E., Greenleaf, W. J., Wong, W. H., Satpathy, A. T., Mackall, C. L., Chang, H. Y. 2021; 118 (30)

  Abstract

  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 PDCD1, CTLA4, and HAVCR2, 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 PDCD1 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.

  View details for DOI 10.1073/pnas.2104758118

  View details for PubMedID 34285077

• High-throughput and single-cell T cell receptor sequencing technologies. Nature methods Pai, J. A., Satpathy, A. T. 2021

  Abstract

  T cells express T cell receptors (TCRs) composed of somatically recombined TCRalpha and TCRbeta chains, which mediate recognition of major histocompatibility complex (MHC)-antigen complexes and drive the antigen-specific adaptive immune response to pathogens and cancer. The TCR repertoire in each individual is highly diverse, which allows for recognition of a wide array of foreign antigens, but also presents a challenge in analyzing this response using conventional methods. Recent studies have developed high-throughput sequencing technologies to identify TCR sequences, analyze their antigen specificities using experimental and computational tools, and pair TCRs with transcriptional and epigenetic cell state phenotypes in single cells. In this Review, we highlight these technological advances and describe how they have been applied to discover fundamental insights into T cell-mediated immunity.

  View details for DOI 10.1038/s41592-021-01201-8

  View details for PubMedID 34282327

• Potent activity of CAR T cells targeting the oncofetal protein GPC2 engineered to recognize low antigen density in neuroblastoma. Heitzeneder, S., Bosse, K. R., Zhu, Z., Jelev, D., Dhingra, S., Majzner, R., Sotillo-Pineiro, E., Buongervino, S., Xu, P., Huang, J., Delaidelli, A., Hasselblatt, M., Parker, K., Anbunathan, H., Alag, A., Hwang, J., Huang, M., Klysz, D. D., Theruvath, J. L., Vilches-Moure, J. G., Satpathy, A. T., Sorensen, P. H., Dimitrov, D. S., Maris, J. M., Mackall, C. L. AMER ASSOC CANCER RESEARCH. 2021

  View details for Web of Science ID 000680263502187

• Identification of a T-bethi Quiescent Exhausted CD8 T Cell Subpopulation That Can Differentiate into TIM3+CX3CR1+ Effectors and Memory-like Cells. Journal of immunology (Baltimore, Md. : 1950) Raju, S., Xia, Y., Daniel, B., Yost, K. E., Bradshaw, E., Tonc, E., Verbaro, D. J., Kometani, K., Yokoyama, W. M., Kurosaki, T., Satpathy, A. T., Egawa, T. 2021

  Abstract

  Persistent Ag induces a dysfunctional CD8 T cell state known as "exhaustion" characterized by PD-1 expression. Nevertheless, exhausted CD8 T cells retain functionality through continued differentiation of progenitor into effector cells. However, it remains ill-defined how CD8 T cell effector responses are sustained in situ. In this study, we show using the mouse chronic lymphocytic choriomeningitis virus infection model that CX3CR1+ CD8 T cells contain a T-bet-dependent TIM3-PD-1lo subpopulation that is distinct from the TIM3+CX3CR1+PD-1+ proliferative effector subset. The TIM3-CX3CR1+ cells are quiescent and express a low but significant level of the transcription factor TCF-1, demonstrating similarity to TCF-1hi progenitor CD8 T cells. Furthermore, following the resolution of lymphocytic choriomeningitis virus viremia, a substantial proportion of TCF-1+ memory-like CD8 T cells show evidence of CX3CR1 expression during the chronic phase of the infection. Our results suggest a subset of the CX3CR1+ exhausted population demonstrates progenitor-like features that support the generation of the CX3CR1+ effector pool from the TCF-1hi progenitors and contribute to the memory-like pool following the resolution of viremia.

  View details for DOI 10.4049/jimmunol.2001348

  View details for PubMedID 34088768

• Repertoire Remodeling through CD4+ T-cell Depletion. Cancer immunology research Yao, W., Satpathy, A. T. 2021; 9 (6): 601

  Abstract

  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.

  View details for DOI 10.1158/2326-6066.CIR-21-0301

  View details for PubMedID 34365414

• Repertoire Remodeling through CD4(+) T-cell Depletion CANCER IMMUNOLOGY RESEARCH Yao, W., Satpathy, A. T. 2021; 9 (6): 601

  View details for DOI 10.1158/2326-6066.CIR-21-0301

  View details for Web of Science ID 000659292000002

• Differential usage of transcriptional repressor Zeb2 enhancers distinguishes adult and embryonic hematopoiesis. Immunity Huang, X., Ferris, S. T., Kim, S., Choudhary, M. N., Belk, J. A., Fan, C., Qi, Y., Sudan, R., Xia, Y., Desai, P., Chen, J., Ly, N., Shi, Q., Bagadia, P., Liu, T., Guilliams, M., Egawa, T., Colonna, M., Diamond, M. S., Murphy, T. L., Satpathy, A. T., Wang, T., Murphy, K. M. 2021

  Abstract

  The transcriptional repressor ZEB2 regulates development of many cell fates among somatic, neural, and hematopoietic lineages, but the basis for its requirement in these diverse lineages is unclear. Here, we identified a 400-basepair (bp) region located 165 kilobases (kb) upstream of the Zeb2 transcriptional start site (TSS) that binds the E proteins at several E-box motifs and was active in hematopoietic lineages. Germline deletion of this 400-bp region (Zeb2Delta-165mice) specifically prevented Zeb2 expression in hematopoietic stem cell (HSC)-derived lineages. Zeb2Delta-165 mice lacked development of plasmacytoid dendritic cells (pDCs), monocytes, and B cells. All macrophages in Zeb2Delta-165 mice were exclusively of embryonic origin. Using single-cell chromatin profiling, we identified a second Zeb2 enhancer located at+164-kb that was selectively active in embryonically derived lineages, but not HSC-derived ones. Thus, Zeb2 expression in adult, but not embryonic, hematopoiesis is selectively controlled by the -165-kb Zeb2 enhancer.

  View details for DOI 10.1016/j.immuni.2021.04.015

  View details for PubMedID 34004142

• The Role of Aire in the selection of Regulatory T cells in Type 1 Diabetes Bridge, J., Balolong, J., Belk, J., Dong, S., Cramer, N., Mowery, C., Crawford, F., Ye, J., Satpathy, A., Kappler, J. W., Bluestone, J. A., Anderson, M. S. AMER ASSOC IMMUNOLOGISTS. 2021

  View details for Web of Science ID 000713665800398

• Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling. Science (New York, N.Y.) Weber, E. W., Parker, K. R., Sotillo, E., Lynn, R. C., Anbunathan, H., Lattin, J., Good, Z., Belk, J. A., Daniel, B., Klysz, D., Malipatlolla, M., Xu, P., Bashti, M., Heitzeneder, S., Labanieh, L., Vandris, P., Majzner, R. G., Qi, Y., Sandor, K., Chen, L., Prabhu, S., Gentles, A. J., Wandless, T. J., Satpathy, A. T., Chang, H. Y., Mackall, C. L. 2021; 372 (6537)

  Abstract

  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.

  View details for DOI 10.1126/science.aba1786

  View details for PubMedID 33795428

• Profiling Chromatin Accessibility at Single-cell Resolution GENOMICS PROTEOMICS & BIOINFORMATICS Sinha, S., Satpathy, A. T., Zhou, W., Ji, H., Stratton, J. A., Jaffer, A., Bahlis, N., Morrissy, S., Biernaskie, J. A. 2021; 19 (2): 172-190

  View details for DOI 10.1016/j.gpb.2020.06.0101672-0229

  View details for Web of Science ID 000719420900002

• Interrogating immune cells and cancer with CRISPR-Cas9. Trends in immunology Buquicchio, F. A., Satpathy, A. T. 2021

  Abstract

  CRISPR-Cas9 technologies have transformed the study of genetic pathways governing cellular differentiation and function. Recent advances have adapted these methods to immune cells, which has accelerated the pace of functional genomics in immunology and enabled new avenues for the design of cellular immunotherapies for cancer. In this review, we summarize recent developments in CRISPR-Cas9 technology and discuss how they have been leveraged to discover and manipulate novel genetic regulators of the immune system. We envision that these results will provide a valuable resource to aid in the design, implementation, and interpretation of CRISPR-Cas9-based screens in immunology and immuno-oncology.

  View details for DOI 10.1016/j.it.2021.03.003

  View details for PubMedID 33812776

• Surface proteomics reveals CD72 as a target for in vitro-evolved nanobody-based CAR-T cells in KMT2A/MLL1-rearranged B-ALL. Cancer discovery Nix, M. A., Mandal, K., Geng, H., Paranjape, N., Lin, Y. T., Rivera, J., Marcoulis, M., White, K. L., Whitman, J. D., Bapat, S. P., Parker, K. R., Ramirez, J., Deucher, A., Phojanokong, P., Steri, V., Fattahi, F., Hann, B., Satpathy, A. T., Manglik, A., Stieglitz, E., Wiita, A. P. 2021

  Abstract

  Alternate strategies are needed for B-cell malignancy patients relapsing after CD19-targeted immunotherapy. Here, cell surface proteomics revealed CD72 as an optimal target for poor-prognosis KMT2A/MLL1-rearranged (MLLr) B-ALL, which we further found to be expressed in other B-cell malignancies. Using a recently-described, fully-in vitro system we selected synthetic CD72-specific nanobodies, incorporated them into CARs, and demonstrated robust activity against B-cell malignancy models, including CD19 loss. Taking advantage of CD72's role in inhibiting B-cell receptor signaling, we found that pharmacologic SHIP1 inhibition increased CD72 surface density. We establish CD72-nanobody CAR T's as a promising therapy for MLLr B-ALL.

  View details for DOI 10.1158/2159-8290.CD-20-0242

  View details for PubMedID 33727310

• B cell-specific XIST complex enforces X-inactivation and restrains atypical B cells. Cell Yu, B., Qi, Y., Li, R., Shi, Q., Satpathy, A. T., Chang, H. Y. 2021

  Abstract

  The long non-coding RNA (lncRNA) XIST establishes X chromosome inactivation (XCI) in female cells in early development and thereafter is thought to be largely dispensable. Here, we show XIST is continually required in adult human B cells to silence a subset of X-linked immune genes such as TLR7. XIST-dependent genes lack promoter DNA methylation and require continual XIST-dependent histone deacetylation. XIST RNA-directed proteomics and CRISPRi screen reveal distinctive somatic cell-type-specific XIST complexes and identify TRIM28 that mediates Pol II pausing at promoters of X-linked genes in B cells. Single-cell transcriptome data of female patients with either systemic lupus erythematosus or COVID-19 infection revealed XIST dysregulation, reflected by escape of XIST-dependent genes, in CD11c+ atypical memory B cells (ABCs). XIST inactivation with TLR7 agonism suffices to promote isotype-switched ABCs. These results indicate cell-type-specific diversification and function for lncRNA-protein complexes and suggest expanded roles for XIST in sex-differences in biology and medicine.

  View details for DOI 10.1016/j.cell.2021.02.015

  View details for PubMedID 33735607

• Profiling Chromatin Accessibility at Single-cell Resolution. Genomics, proteomics & bioinformatics Sinha, S., Satpathy, A. T., Zhou, W., Ji, H., Stratton, J. A., Jaffer, A., Bahlis, N., Morrissy, S., Biernaskie, J. A. 2021

  Abstract

  How distinct transcriptional programs are enacted to generate cellular heterogeneity and plasticity, and enable complex fate decisions are important open questions. One key regulator is the cell's epigenome state that drives distinct transcriptional programs by regulating chromatin accessibility. Genome-wide chromatin accessibility measurements can impart insights into regulatory sequences (in)accessible to DNA-binding proteins at a single-cell resolution. This review outlines molecular methods and bioinformatic tools for capturing cell-to-cell chromatin variation using single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) in a scalable fashion. It also covers joint profiling of chromatin with transcriptome/proteome measurements, computational strategies to integrate multi-omic measurements, and predictive bioinformatic tools to infer chromatin accessibility from single-cell transcriptomic datasets. Methodological refinements that increase power for cell discovery through robust chromatin coverage and integrate measurements from multiple modalities will further expand our understanding of gene regulation during homeostasis and disease.

  View details for DOI 10.1016/j.gpb.2020.06.010

  View details for PubMedID 33581341

• Charting a shared epigenetic pathway to CD8+ T cell dysfunction in infection and cancer. Molecular cell Sandor, K., Daniel, B., Satpathy, A. T. 2021; 81 (11): 2272-2274

  Abstract

  Pritykin et al. (2021) establish a comprehensive chromatin atlas of CD8+ T cell dysfunction in chronic viral infection and cancer via analysis of bulk and single-cell ATAC-seq datasets across immune challenges. These results unify the classification scheme and molecular programs driving CD8+ T cell dysfunction across disease settings and will facilitate basic discovery and translational efforts in T cell immunity.

  View details for DOI 10.1016/j.molcel.2021.05.020

  View details for PubMedID 34087178

• NOT-Gated CD93 CAR T Cells Effectively Target AML with Minimized Endothelial Cross-Reactivity. Blood cancer discovery Richards, R. M., Zhao, F., Freitas, K. A., Parker, K. R., Xu, P., Fan, A., Sotillo, E., Daugaard, M., Oo, H. Z., Liu, J., Hong, W. J., Sorensen, P. H., Chang, H. Y., Satpathy, A. T., Majzner, R. G., Majeti, R., Mackall, C. L. 2021; 2 (6): 648-665

  Abstract

  Chimeric antigen receptor (CAR) T cells hold promise for the treatment of acute myeloid leukemia (AML), but optimal targets remain to be defined. We demonstrate that CD93 CAR T cells engineered from a novel humanized CD93-specific binder potently kill AML in vitro and in vivo but spare hematopoietic stem and progenitor cells (HSPC). No toxicity is seen in murine models, but CD93 is expressed on human endothelial cells, and CD93 CAR T cells recognize and kill endothelial cell lines. We identify other AML CAR T-cell targets with overlapping expression on endothelial cells, especially in the context of proinflammatory cytokines. To address the challenge of endothelial-specific cross-reactivity, we provide proof of concept for NOT-gated CD93 CAR T cells that circumvent endothelial cell toxicity in a relevant model system. We also identify candidates for combinatorial targeting by profiling the transcriptome of AML and endothelial cells at baseline and after exposure to proinflammatory cytokines.CD93 CAR T cells eliminate AML and spare HSPCs but exert on-target, off-tumor toxicity to endothelial cells. We show coexpression of other AML targets on endothelial cells, introduce a novel NOT-gated strategy to mitigate endothelial toxicity, and demonstrate use of high-dimensional transcriptomic profiling for rational design of combinatorial immunotherapies.See related commentary by Velasquez and Gottschalk, p. 559. This article is highlighted in the In This Issue feature, p. 549.

  View details for DOI 10.1158/2643-3230.BCD-20-0208

  View details for PubMedID 34778803

  View details for PubMedCentralID PMC8580619

• Identification of presented SARS-CoV-2 HLA class I and HLA class II peptides using HLA peptidomics. Cell reports Nagler, A., Kalaora, S., Barbolin, C., Gangaev, A., Ketelaars, S. L., Alon, M., Pai, J., Benedek, G., Yahalom-Ronen, Y., Erez, N., Greenberg, P., Yagel, G., Peri, A., Levin, Y., Satpathy, A. T., Bar-Haim, E., Paran, N., Kvistborg, P., Samuels, Y. 2021: 109305

  Abstract

  The human leukocyte antigen (HLA)-bound viral antigens serve as an immunological signature that can be selectively recognized by T cells. As viruses evolve by acquiring mutations, it is essential to identify a range of presented viral antigens. Using HLA peptidomics, we are able to identify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-derived peptides presented by highly prevalent HLA class I (HLA-I) molecules by using infected cells as well as overexpression of SARS-CoV-2 genes. We find 26 HLA-I peptides and 36 HLA class II (HLA-II) peptides. Among the identified peptides, some are shared between different cells and some are derived from out-of-frame open reading frames (ORFs). Seven of these peptides were previously shown to be immunogenic, and we identify two additional immunoreactive peptides by using HLA multimer staining. These results may aid the development of the next generation of SARS-CoV-2 vaccines based on presented viral-specific antigens that span several of the viral genes.

  View details for DOI 10.1016/j.celrep.2021.109305

  View details for PubMedID 34166618

• Single cell analysis of CRISPR T cells Stadtmauer, E., Fraietta, J., Satpathy, A., June, C. H. AMER ASSOC CANCER RESEARCH. 2020

  View details for Web of Science ID 000587913100002

• The Power of Single Cell Technologies; from T Cell Receptor to Antigen(s) in Multiple Sclerosis Saligrama, N., Fernandes, R. A., Pai, J., Oksenberg, J., Satpathy, A., Davis, M. M. WILEY. 2020: S198

  View details for Web of Science ID 000572509100353

• Affinity-Restricted Memory B Cells Dominate Recall Responses to Heterologous Flaviviruses. Immunity Wong, R., Belk, J. A., Govero, J., Uhrlaub, J. L., Reinartz, D., Zhao, H., Errico, J. M., D'Souza, L., Ripperger, T. J., Nikolich-Zugich, J., Shlomchik, M. J., Satpathy, A. T., Fremont, D. H., Diamond, M. S., Bhattacharya, D. 2020

  Abstract

  Memory B cells (MBCs) can respond to heterologous antigens either by molding new specificities through secondary germinal centers (GCs) or by selecting preexisting clones without further affinity maturation. To distinguish these mechanisms in flavivirus infections and immunizations, we studied recall responses to envelope protein domain III (DIII). Conditional deletion of activation-induced cytidine deaminase (AID) between heterologous challenges of West Nile, Japanese encephalitis, Zika, and dengue viruses did not affect recall responses. DIII-specific MBCs were contained mostly within the plasma-cell-biased CD80+ subset, and few GCs arose following heterologous boosters, demonstrating that recall responses are confined by preexisting clonal diversity. Measurement of monoclonal antibody (mAb) binding affinity to DIII proteins, timed AID deletion, single-cell RNA sequencing, and lineage tracing experiments point to selection of relatively low-affinity MBCs as a mechanism to promote diversity. Engineering immunogens to avoid this MBC diversity may facilitate flavivirus-type-specific vaccines with minimized potential for infection enhancement.

  View details for DOI 10.1016/j.immuni.2020.09.001

  View details for PubMedID 33010224

• Impaired mitochondrial oxidative phosphorylation limits the self-renewal of T cells exposed to persistent antigen. Nature immunology Vardhana, S. A., Hwee, M. A., Berisa, M., Wells, D. K., Yost, K. E., King, B., Smith, M., Herrera, P. S., Chang, H. Y., Satpathy, A. T., van den Brink, M. R., Cross, J. R., Thompson, C. B. 2020

  Abstract

  The majority of tumor-infiltrating T cells exhibit a terminally exhausted phenotype, marked by a loss of self-renewal capacity. How repetitive antigenic stimulation impairs T cell self-renewal remains poorly defined. Here, we show that persistent antigenic stimulation impaired ADP-coupled oxidative phosphorylation. The resultant bioenergetic compromise blocked proliferation by limiting nucleotide triphosphate synthesis. Inhibition of mitochondrial oxidative phosphorylation in activated T cells was sufficient to suppress proliferation and upregulate genes linked to T cell exhaustion. Conversely, prevention of mitochondrial oxidative stress during chronic T cell stimulation allowed sustained T cell proliferation and induced genes associated with stem-like progenitor T cells. As a result, antioxidant treatment enhanced the anti-tumor efficacy of chronically stimulated T cells. These data reveal that loss of ATP production through oxidative phosphorylation limits T cell proliferation and effector function during chronic antigenic stimulation. Furthermore, treatments that maintain redox balance promote T cell self-renewal and enhance anti-tumor immunity.

  View details for DOI 10.1038/s41590-020-0725-2

  View details for PubMedID 32661364

• Curated, multi-omic, ML-driven single-cell atlas for characterizing the human immune system across disease states Kiner, E., Sharim, H., Montalbano, A., Raveh-Sadka, T., Yanover, U., Wells, D. K., Satpathy, A. AMER ASSOC IMMUNOLOGISTS. 2020

  View details for Web of Science ID 000589972401533

• E protein support of Zeb2 expression regulates development of multiple hematopoietic lineages Huang, X., Ferris, S., Satpathy, A., Murphy, T. L., Murphy, K. M. AMER ASSOC IMMUNOLOGISTS. 2020

  View details for Web of Science ID 000589972402318

• CD19-Positive Brain Pericytes as Targets of Immunotherapy-Associated Neurotoxicity Migliorini, D., Parker, K., Perkey, E., Bagga, P., Liu, F., Maillard, I., June, C. H., Chang, H. Y., Posey, A. D., Satpathy, A. CELL PRESS. 2020: 210

  View details for Web of Science ID 000530089301033

• Chromatin accessibility landscapes of skin cells in systemic sclerosis nominate dendritic cells in disease pathogenesis. Nature communications Liu, Q., Zaba, L., Satpathy, A. T., Longmire, M., Zhang, W., Li, K., Granja, J., Guo, C., Lin, J., Li, R., Tolentino, K., Kania, G., Distler, O., Fiorentino, D., Chung, L., Qu, K., Chang, H. Y. 2020; 11 (1): 5843

  Abstract

  Systemic sclerosis (SSc) is a disease at the intersection of autoimmunity and fibrosis. However, the epigenetic regulation and the contributions of diverse cell types to SSc remain unclear. Here we survey, using ATAC-seq, the active DNA regulatory elements of eight types of primary cells in normal skin from healthy controls, as well as clinically affected and unaffected skin from SSc patients. We find that accessible DNA elements in skin-resident dendritic cells (DCs) exhibit the highest enrichment of SSc-associated single-nucleotide polymorphisms (SNPs) and predict the degrees of skin fibrosis in patients. DCs also have the greatest disease-associated changes in chromatin accessibility and the strongest alteration of cell-cell interactions in SSc lesions. Lastly, data from an independent cohort of patients with SSc confirm a significant increase of DCs in lesioned skin. Thus, the DCs epigenome links inherited susceptibility and clinically apparent fibrosis in SSc skin, and can be an important driver of SSc pathogenesis.

  View details for DOI 10.1038/s41467-020-19702-z

  View details for PubMedID 33203843

• Systematic discovery and functional interrogation of SARS-CoV-2 viral RNA-host protein interactions during infection. bioRxiv : the preprint server for biology Flynn, R. A., Belk, J. A., Qi, Y. n., Yasumoto, Y. n., Schmitz, C. O., Mumbach, M. R., Limaye, A. n., Wei, J. n., Alfajaro, M. M., Parker, K. R., Chang, H. Y., Horvath, T. L., Carette, J. E., Bertozzi, C. n., Wilen, C. B., Satpathy, A. T. 2020

  Abstract

  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a pandemic with growing global mortality. There is an urgent need to understand the molecular pathways required for host infection and anti-viral immunity. 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 viral ChIRP-MS data from three other positive-sense RNA viruses defined pan-viral and SARS-CoV-2-specific host interactions. Functional interrogation of these factors with a genome-wide CRISPR screen revealed that the vast majority of viral RNA-binding proteins protect the host from virus-induced cell death, and we identified known and novel anti-viral proteins that regulate SARS-CoV-2 pathogenicity. Finally, our RNA-centric approach demonstrated a physical connection between SARS-CoV-2 RNA and host mitochondria, which we validated with functional and electron microscopy data, providing new insights into a more general virus-specific protein logic for mitochondrial interactions. Altogether, these data provide a comprehensive catalogue of SARS-CoV-2 RNA-host protein interactions, which may inform future studies to understand the mechanisms of viral pathogenesis, as well as nominate host pathways that could be targeted for therapeutic benefit.· ChIRP-MS of SARS-CoV-2 RNA identifies a comprehensive viral RNA-host protein interaction network during infection across two species· Comparison to RNA-protein interaction networks with Zika virus, dengue virus, and rhinovirus identify SARS-CoV-2-specific and pan-viral RNA protein complexes and highlights distinct intracellular trafficking pathways· Intersection of ChIRP-MS and genome-wide CRISPR screens identify novel SARS-CoV-2-binding proteins with pro- and anti-viral function· Viral RNA-RNA and RNA-protein interactions reveal specific SARS-CoV-2-mediated mitochondrial dysfunction during infection.

  View details for DOI 10.1101/2020.10.06.327445

  View details for PubMedID 33052334

  View details for PubMedCentralID PMC7553159

• An old BATF's new T-ricks. Nature immunology Lareau, C. A., Satpathy, A. T. 2020

  View details for DOI 10.1038/s41590-020-0796-0

  View details for PubMedID 32989330

• Chromatin Landscape Underpinning Human Dendritic Cell Heterogeneity. Cell reports Leylek, R. n., Alcántara-Hernández, M. n., Granja, J. M., Chavez, M. n., Perez, K. n., Diaz, O. R., Li, R. n., Satpathy, A. T., Chang, H. Y., Idoyaga, J. n. 2020; 32 (12): 108180

  Abstract

  Human dendritic cells (DCs) comprise subsets with distinct phenotypic and functional characteristics, but the transcriptional programs that dictate their identity remain elusive. Here, we analyze global chromatin accessibility profiles across resting and stimulated human DC subsets by means of the assay for transposase-accessible chromatin using sequencing (ATAC-seq). We uncover specific regions of chromatin accessibility for each subset and transcriptional regulators of DC function. By comparing plasmacytoid DC responses to IFN-I-producing and non-IFN-I-producing conditions, we identify genetic programs related to their function. Finally, by intersecting chromatin accessibility with genome-wide association studies, we recognize DC subset-specific enrichment of heritability in autoimmune diseases. Our results unravel the basis of human DC subset heterogeneity and provide a framework for their analysis in disease pathogenesis.

  View details for DOI 10.1016/j.celrep.2020.108180

  View details for PubMedID 32966789

• Human B Cell Clonal Expansion and Convergent Antibody Responses to SARS-CoV-2. Cell host & microbe Nielsen, S. C., Yang, F. n., Jackson, K. J., Hoh, R. A., Röltgen, K. n., Jean, G. H., Stevens, B. A., Lee, J. Y., Rustagi, A. n., Rogers, A. J., Powell, A. E., Hunter, M. n., Najeeb, J. n., Otrelo-Cardoso, A. R., Yost, K. E., Daniel, B. n., Nadeau, K. C., Chang, H. Y., Satpathy, A. T., Jardetzky, T. S., Kim, P. S., Wang, T. T., Pinsky, B. A., Blish, C. A., Boyd, S. D. 2020

  Abstract

  B cells are critical for the production of antibodies and protective immunity to viruses. Here we show that patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) who develop coronavirus disease 2019 (COVID-19) display early recruitment of B cells expressing a limited subset of IGHV genes, progressing to a highly polyclonal response of B cells with broader IGHV gene usage and extensive class switching to IgG and IgA subclasses with limited somatic hypermutation in the initial weeks of infection. We identify convergence of antibody sequences across SARS-CoV-2-infected patients, highlighting stereotyped naive responses to this virus. Notably, sequence-based detection in COVID-19 patients of convergent B cell clonotypes previously reported in SARS-CoV infection predicts the presence of SARS-CoV/SARS-CoV-2 cross-reactive antibody titers specific for the receptor-binding domain. These findings offer molecular insights into shared features of human B cell responses to SARS-CoV-2 and SARS-CoV.

  View details for DOI 10.1016/j.chom.2020.09.002

  View details for PubMedID 32941787

• Human B cell clonal expansion and convergent antibody responses to SARS-CoV-2. bioRxiv : the preprint server for biology Nielsen, S. C., Yang, F. n., Jackson, K. J., Hoh, R. A., Röltgen, K. n., Stevens, B. n., Lee, J. Y., Rustagi, A. n., Rogers, A. J., Powell, A. E., Najeeb, J. n., Otrelo-Cardoso, A. R., Yost, K. E., Daniel, B. n., Chang, H. Y., Satpathy, A. T., Jardetzky, T. S., Kim, P. S., Wang, T. T., Pinsky, B. A., Blish, C. A., Boyd, S. D. 2020

  Abstract

  During virus infection B cells are critical for the production of antibodies and protective immunity. Here we show that the human B cell compartment in patients with diagnostically confirmed SARS-CoV-2 and clinical COVID-19 is rapidly altered with the early recruitment of B cells expressing a limited subset of IGHV genes, progressing to a highly polyclonal response of B cells with broader IGHV gene usage and extensive class switching to IgG and IgA subclasses with limited somatic hypermutation in the initial weeks of infection. We identify extensive convergence of antibody sequences across SARS-CoV-2 patients, highlighting stereotyped naïve responses to this virus. Notably, sequence-based detection in COVID-19 patients of convergent B cell clonotypes previously reported in SARS-CoV infection predicts the presence of SARS-CoV/SARS-CoV-2 cross-reactive antibody titers specific for the receptor-binding domain. These findings offer molecular insights into shared features of human B cell responses to SARS-CoV-2 and other zoonotic spillover coronaviruses.

  View details for DOI 10.1101/2020.07.08.194456

  View details for PubMedID 32676593

  View details for PubMedCentralID PMC7359515

• CRISPR-engineered T cells in patients with refractory cancer. Science (New York, N.Y.) Stadtmauer, E. A., Fraietta, J. A., Davis, M. M., Cohen, A. D., Weber, K. L., Lancaster, E. n., Mangan, P. A., Kulikovskaya, I. n., Gupta, M. n., Chen, F. n., Tian, L. n., Gonzalez, V. E., Xu, J. n., Jung, I. Y., Melenhorst, J. J., Plesa, G. n., Shea, J. n., Matlawski, T. n., Cervini, A. n., Gaymon, A. L., Desjardins, S. n., Lamontagne, A. n., Salas-Mckee, J. n., Fesnak, A. n., Siegel, D. L., Levine, B. L., Jadlowsky, J. K., Young, R. M., Chew, A. n., Hwang, W. T., Hexner, E. O., Carreno, B. M., Nobles, C. L., Bushman, F. D., Parker, K. R., Qi, Y. n., Satpathy, A. T., Chang, H. Y., Zhao, Y. n., Lacey, S. F., June, C. H. 2020

  Abstract

  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 I 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 PD-1 (PDCD1), was performed to improve anti-tumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Though 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.

  View details for DOI 10.1126/science.aba7365

  View details for PubMedID 32029687

• First-in-Human Assessment of Feasibility and Safety of Multiplexed Genetic Engineering of Autologous T Cells Expressing NY-ESO -1 TCR and CRISPR/Cas9 Gene Edited to Eliminate Endogenous TCR and PD-1 (NYCE T cells) in Advanced Multiple Myeloma (MM) and Sarcoma. Blood Stadtmauer, E. A., Cohen, A. D., Weber, K., Lacey, S. F., Gonzalez, V. E., Melenhorst, J. J., Fraietta, J. A., Plesa, G., Shea, J., Matlawski, T., Cervini, A., Mangan, P., Gaymon, A., Desjardins, S., Lancaster, E., Salas-Mckee, J., Suhoski, M. M., Fesnak, A., O'Rourke, M., Lamontagne, A., Siegel, D. L., Young, R. M., Chew, A., Nobles, C. L., Bushman, F. D., Chang, H. Y., Satpathy, A. T., Zhao, Y., Hwang, W., Hexner, E. O., June, C. H. 2019; 134 (Supplement_1): 49

  Abstract

  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.

  View details for DOI 10.1182/blood-2019-122374

  View details for PubMedID 31724015

• HiChIRP reveals RNA-associated chromosome conformation. Nature methods Mumbach, M. R., Granja, J. M., Flynn, R. A., Roake, C. M., Satpathy, A. T., Rubin, A. J., Qi, Y., Jiang, Z., Shams, S., Louie, B. H., Guo, J. K., Gennert, D. G., Corces, M. R., Khavari, P. A., Atianand, M. K., Artandi, S. E., Fitzgerald, K. A., Greenleaf, W. J., Chang, H. Y. 2019

  Abstract

  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).

  View details for DOI 10.1038/s41592-019-0407-x

  View details for PubMedID 31133759

• A Subset of Type I Conventional Dendritic Cells Controls Cutaneous Bacterial Infections through VEGFalpha-Mediated Recruitment of Neutrophils. Immunity Janela, B., Patel, A. A., Lau, M. C., Goh, C. C., Msallam, R., Kong, W. T., Fehlings, M., Hubert, S., Lum, J., Simoni, Y., Malleret, B., Zolezzi, F., Chen, J., Poidinger, M., Satpathy, A. T., Briseno, C., Wohn, C., Malissen, B., Murphy, K. M., Maini, A. A., Vanhoutte, L., Guilliams, M., Vial, E., Hennequin, L., Newell, E., Ng, L. G., Musette, P., Yona, S., Hacini-Rachinel, F., Ginhoux, F. 2019; 50 (4): 1069

  Abstract

  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.

  View details for PubMedID 30926233

• A Subset of Type I Conventional Dendritic Cells Controls Cutaneous Bacterial Infections through VEGF alpha-Mediated Recruitment of Neutrophils IMMUNITY Janela, B., Patel, A. A., Lau, M., Goh, C., Msallam, R., Kong, W., Fehlings, M., Hubert, S., Lum, J., Simoni, Y., Malleret, B., Zolezzi, F., Chen, J., Poidinger, M., Satpathy, A. T., Briseno, C., Wohn, C., Malissen, B., Murphy, K. M., Maini, A. A., Vanhoutte, L., Guilliams, M., Vial, E., Hennequin, L., Newell, E., Ng, L., Musette, P., Yona, S., Hacini-Rachinel, F., Ginhoux, F. 2019; 50 (4): 1069–83

  View details for DOI 10.1016/j.immuni.2019.03.001

  View details for Web of Science ID 000464660800023

• Interrogation of human hematopoiesis at single-cell and single-variant resolution. Nature genetics Ulirsch, J. C., Lareau, C. A., Bao, E. L., Ludwig, L. S., Guo, M. H., Benner, C., Satpathy, A. T., Kartha, V. K., Salem, R. M., Hirschhorn, J. N., Finucane, H. K., Aryee, M. J., Buenrostro, J. D., Sankaran, V. G. 2019

  Abstract

  Widespread linkage disequilibrium and incomplete annotation of cell-to-cell state variation represent substantial challenges to elucidating mechanisms of trait-associated genetic variation. Here we perform genetic fine-mapping for blood cell traits in the UK Biobank to identify putative causal variants. These variants are enriched in genes encoding proteins in trait-relevant biological pathways and in accessible chromatin of hematopoietic progenitors. For regulatory variants, we explore patterns of developmental enhancer activity, predict molecular mechanisms, and identify likely target genes. In several instances, we localize multiple independent variants to the same regulatory element or gene. We further observe that variants with pleiotropic effects preferentially act in common progenitor populations to direct the production of distinct lineages. Finally, we leverage fine-mapped variants in conjunction with continuous epigenomic annotations to identify trait-cell type enrichments within closely related populations and in single cells. Our study provides a comprehensive framework for single-variant and single-cell analyses of genetic associations.

  View details for PubMedID 30858613

• A Mutation in the Transcription Factor Foxp3 Drives T Helper 2 Effector Function in Regulatory T Cells. Immunity Van Gool, F. n., Nguyen, M. L., Mumbach, M. R., Satpathy, A. T., Rosenthal, W. L., Giacometti, S. n., Le, D. T., Liu, W. n., Brusko, T. M., Anderson, M. S., Rudensky, A. Y., Marson, A. n., Chang, H. Y., Bluestone, J. A. 2019

  Abstract

  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.

  View details for PubMedID 30709738

• c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature Lynn, R. C., Weber, E. W., Sotillo, E. n., Gennert, D. n., Xu, P. n., Good, Z. n., Anbunathan, H. n., Lattin, J. n., Jones, R. n., Tieu, V. n., Nagaraja, S. n., Granja, J. n., de Bourcy, C. F., Majzner, R. n., Satpathy, A. T., Quake, S. R., Monje, M. n., Chang, H. Y., Mackall, C. L. 2019

  Abstract

  Chimeric antigen receptor (CAR) T cells mediate anti-tumour effects in a small subset of patients with cancer1-3, but dysfunction due to T cell exhaustion is an important barrier to progress4-6. 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 exhaustion6. 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 cells7-10. 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.

  View details for DOI 10.1038/s41586-019-1805-z

  View details for PubMedID 31802004

• GWAS for systemic sclerosis identifies multiple risk loci and highlights fibrotic and vasculopathy pathways. Nature communications López-Isac, E. n., Acosta-Herrera, M. n., Kerick, M. n., Assassi, S. n., Satpathy, A. T., Granja, J. n., Mumbach, M. R., Beretta, L. n., Simeón, C. P., Carreira, P. n., Ortego-Centeno, N. n., Castellvi, I. n., Bossini-Castillo, L. n., Carmona, F. D., Orozco, G. n., Hunzelmann, N. n., Distler, J. H., Franke, A. n., Lunardi, C. n., Moroncini, G. n., Gabrielli, A. n., de Vries-Bouwstra, J. n., Wijmenga, C. n., Koeleman, B. P., Nordin, A. n., Padyukov, L. n., Hoffmann-Vold, A. M., Lie, B. n., Proudman, S. n., Stevens, W. n., Nikpour, M. n., Vyse, T. n., Herrick, A. L., Worthington, J. n., Denton, C. P., Allanore, Y. n., Brown, M. A., Radstake, T. R., Fonseca, C. n., Chang, H. Y., Mayes, M. D., Martin, J. n. 2019; 10 (1): 4955

  Abstract

  Systemic sclerosis (SSc) is an autoimmune disease that shows one of the highest mortality rates among rheumatic diseases. We perform a large genome-wide association study (GWAS), and meta-analysis with previous GWASs, in 26,679 individuals and identify 27 independent genome-wide associated signals, including 13 new risk loci. The novel associations nearly double the number of genome-wide hits reported for SSc thus far. We define 95% credible sets of less than 5 likely causal variants in 12 loci. Additionally, we identify specific SSc subtype-associated signals. Functional analysis of high-priority variants shows the potential function of SSc signals, with the identification of 43 robust target genes through HiChIP. Our results point towards molecular pathways potentially involved in vasculopathy and fibrosis, two main hallmarks in SSc, and highlight the spectrum of critical cell types for the disease. This work supports a better understanding of the genetic basis of SSc and provides directions for future functional experiments.

  View details for DOI 10.1038/s41467-019-12760-y

  View details for PubMedID 31672989

• An Nfil3-Zeb2-Id2 pathway imposes Irf8 enhancer switching during cDC1 development. Nature immunology Bagadia, P. n., Huang, X. n., Liu, T. T., Durai, V. n., Grajales-Reyes, G. E., Nitschké, M. n., Modrusan, Z. n., Granja, J. M., Satpathy, A. T., Briseño, C. G., Gargaro, M. n., Iwata, A. n., Kim, S. n., Chang, H. Y., Shaw, A. S., Murphy, T. L., Murphy, K. M. 2019

  Abstract

  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.

  View details for DOI 10.1038/s41590-019-0449-3

  View details for PubMedID 31406377

• Cryptic activation of an Irf8 enhancer governs cDC1 fate specification. Nature immunology Durai, V. n., Bagadia, P. n., Granja, J. M., Satpathy, A. T., Kulkarni, D. H., Davidson, J. T., Wu, R. n., Patel, S. J., Iwata, A. n., Liu, T. T., Huang, X. n., Briseño, C. G., Grajales-Reyes, G. E., Wöhner, M. n., Tagoh, H. n., Kee, B. L., Newberry, R. D., Busslinger, M. n., Chang, H. Y., Murphy, T. L., Murphy, K. M. 2019

  Abstract

  Induction of the transcription factor Irf8 in the common dendritic cell progenitor (CDP) is required for classical type 1 dendritic cell (cDC1) fate specification, but the mechanisms controlling this induction are unclear. In the present study Irf8 enhancers were identified via chromatin profiling of dendritic cells and CRISPR/Cas9 genome editing was used to assess their roles in Irf8 regulation. An enhancer 32 kilobases (kb) downstream of the Irf8 transcriptional start site (+32-kb Irf8) that was active in mature cDC1s was required for the development of this lineage, but not for its specification. Instead, a +41-kb Irf8 enhancer, previously thought to be active only in plasmacytoid dendritic cells, was found to also be transiently accessible in cDC1 progenitors, and deleting this enhancer prevented the induction of Irf8 in CDPs and abolished cDC1 specification. Thus, cryptic activation of the +41-kb Irf8 enhancer in dendritic cell progenitors is responsible for cDC1 fate specification.

  View details for DOI 10.1038/s41590-019-0450-x

  View details for PubMedID 31406378

• Tracking the immune response with single-cell genomics. Vaccine Yost, K. E., Chang, H. Y., Satpathy, A. T. 2019

  Abstract

  The immune system is composed of a diverse array of cell types, each with a specialized role in orchestrating the immune response to pathogens or cancer. Even within a single cell 'type,' individual cells can access a wide spectrum of differentiation and activation states, which reflect the physiological response of each cell to the tissue environment and immune stimuli. Thus, the cellular diversity of the immune system is inherently quite complex and understanding this complexity has greatly benefited from technologies that measure immune responses at single-cell resolution, in addition to the systems-level response as a whole. In this Commentary, we focus on recent work at the interface of immunology and single-cell genomics and highlight advances in technologies and their application to immune cells. In particular, we highlight recent single-cell genomic profiling studies of T cells, since somatic rearrangements in the T cell receptor (TCR) loci enable the tracking of clonal T cell responses through space and time. Finally, we discuss opportunities for future use of these technologies in understanding vaccination and the basis for effective vaccine-induced immunity.

  View details for DOI 10.1016/j.vaccine.2019.11.035

  View details for PubMedID 31859202

• Enhancer connectome nominates target genes of inherited risk variants from inflammatory skin disorders. The Journal of investigative dermatology Jeng, M. Y., Mumbach, M. R., Granja, J. M., Satpathy, A. T., Chang, H. Y., Chang, A. L. 2018

  Abstract

  The vast majority of polymorphisms for human dermatologic diseases fall in non-coding DNA regions, leading to difficulty interpreting their functional significance. Recent work utilizing chromosome conformation capture (3C) technology in combination with chromatin immunoprecipitation (ChIP) has provided a systematic means of linking non-coding variants within active enhancer loci to putative gene targets. Here, we apply H3K27ac HiChIP high-resolution contact maps, generated from primary human T-cell subsets (CD4+ Naive, TH17, and Treg), to 21 dermatologic conditions associated with single nucleotide polymorphisms (SNPs) from 106 genome-wide association studies (GWAS). This "enhancer connectome" identified 1,492 HiChIP gene-targets from 542 non-coding SNPs (p<5.0x10-8). SNP-containing enhancers from inflammatory skin conditions were significantly enriched within the human leukocyte antigen (HLA)-locus, and also targeted several key factors from the JAK-STAT signaling pathway, while non-immune conditions did not. A focused profiling of systemic lupus erythematosus (SLE) HiChIP-genes identified enhancer interactions with factors important for effector CD4+ T-cell differentiation and function, including interferon regulatory factor 8 (IRF8) and members of the Ikaros family of zinc-finger proteins. Our results demonstrate the ability of the enhancer connectome to nominate functionally-relevant candidates from GWAS-identified variants, representing a powerful tool to guide future studies into the genomic regulatory mechanisms underlying dermatologic diseases.

  View details for PubMedID 30315781

• Notch2-dependent DC2s mediate splenic germinal center responses. Proceedings of the National Academy of Sciences of the United States of America Briseno, C. G., Satpathy, A. T., Davidson, J. T., Ferris, S. T., Durai, V., Bagadia, P., O'Connor, K. W., Theisen, D. J., Murphy, T. L., Murphy, K. M. 2018

  Abstract

  CD4+ T follicular helper (TFH) cells support germinal center (GC) reactions promoting humoral immunity. Dendritic cell (DC) diversification into genetically distinct subsets allows for specialization in promoting responses against several types of pathogens. Whether any classical DC (cDC) subset is required for humoral immunity is unknown, however. We tested several genetic models that selectively ablate distinct DC subsets in mice for their impact on splenic GC reactions. We identified a requirement for Notch2-dependent cDC2s, but not Batf3-dependent cDC1s or Klf4-dependent cDC2s, in promoting TFH and GC B cell formation in response to sheep red blood cells and inactivated Listeria monocytogenes This effect was mediated independent of Il2ra and several Notch2-dependent genes expressed in cDC2s, including Stat4 and Havcr2 Notch2 signaling during cDC2 development also substantially reduced the efficiency of cDC2s for presentation of MHC class II-restricted antigens, limiting the strength of CD4 T cell activation. Together, these results demonstrate a nonredundant role for the Notch2-dependent cDC2 subset in supporting humoral immune responses.

  View details for PubMedID 30279176

• Discovery of stimulation-responsive immune enhancers with CRISPR activation (vol 549, pg 111, 2017) NATURE Simeonov, D. R., Gowen, B. G., Boontanrart, M., Roth, T. L., Gagnon, J. D., Mumbach, M. R., Satpathy, A. T., Lee, Y., Bray, N. L., Chan, A. Y., Lituiev, D. S., Nguyen, M. L., Gate, R. E., Subramaniam, M., Li, Z., Woo, J. M., Mitros, T., Ray, G. J., Curie, G. L., Naddaf, N., Chu, J. S., Ma, H., Boyer, E., Van Gool, F., Huang, H., Liu, R., Tobin, V. R., Schumann, K., Daly, M. J., Farh, K. K., Ansel, K., Ye, C. J., Greenleaf, W. J., Anderson, M. S., Bluestone, J. A., Chang, H. Y., Corn, J. E., Marson, A. 2018; 559 (7715): E13

  Abstract

  In this Letter, analysis of steady-state regulatory T (Treg) cell percentages from Il2ra enhancer deletion (EDEL) and wild-type (WT) mice revealed no differences between them (Extended Data Fig. 9d). This analysis included two mice whose genotypes were incorrectly assigned. Even after correction of the genotypes, no significant differences in Treg cell percentages were seen when data across experimental cohorts were averaged (as was done in Extended Data Fig. 9d). However, if we normalize the corrected data to account for variation among experimental cohorts, a subtle decrease in EDEL Treg cell percentages is revealed and, using the corrected and normalized data, we have redrawn Extended Data Fig. 9d in Supplementary Fig. 1. The Supplementary Information to this Amendment contains the corrected and reanalysed Extended Data Fig. 9d. The sentence "This enhancer deletion (EDEL) strain also had no obvious T cell phenotypes at steady state (Extended Data Fig. 9)." should read: "This enhancer deletion (EDEL) strain had a small decrease in the percentage of Treg cells (Extended Data Fig. 9).". This error does not affect any of the main figures in the Letter or the data from mice with the human autoimmune-associated single nucleotide polymorphism (SNP) knocked in or with a 12-base-pair deletion at the site (12DEL). In addition, we stated in the Methods that we observed consistent immunophenotypes of EDEL mice across three founders, but in fact, we observed consistent phenotypes in mice from two founders. This does not change any of our conclusions and the original Letter has not been corrected.

  View details for PubMedID 29899441

• Integrative analysis of single-cell genomics data by coupled nonnegative matrix factorizations. Proceedings of the National Academy of Sciences of the United States of America Duren, Z., Chen, X., Zamanighomi, M., Zeng, W., Satpathy, A. T., Chang, H. Y., Wang, Y., Wong, W. H. 2018

  Abstract

  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-sequencing (RNA-seq) and single-cell ATAC-sequencing (ATAC-seq) data.

  View details for PubMedID 29987051

• Engineering AP1 to combat CAR T cell exhaustion Lynn, R. C., Weber, E. W., Gennert, D., Sotillo, E., Jones, R., Xu, P., Satpathy, A., Chang, H. Y., Mackall, C. L. AMER ASSOC CANCER RESEARCH. 2018

  View details for DOI 10.1158/1538-7445.AM2018-LB-112

  View details for Web of Science ID 000468818900275

• Foxp3 domain-swap interface is required to suppress T helper type 2 transcriptional program in Regulatory T cells. Van Gool, F., Nguyen, M. L., Mumbach, M. R., Satpathy, A. T., Anderson, M. S., Marson, A., Chang, H. Y., Bluestone, J. A. AMER ASSOC IMMUNOLOGISTS. 2018

  View details for Web of Science ID 000459977701025

• Dissecting the genetic networks that control regulatory T cell stability using pooled CRISPR screens Cortez, J. T., Shifrut, E., Lee, Y., Mumbach, M. R., Satpathy, A. T., Granja, J., Subramaniam, M., Roth, T., Simeonov, D., Ye, C. J., Chang, H. Y., Van Gool, F., Marson, A. AMER ASSOC IMMUNOLOGISTS. 2018

  View details for Web of Science ID 000459977701021

• The chromatin accessibility landscape of primary human cancers. Science (New York, N.Y.) Corces, M. R., Granja, J. M., Shams, S. n., Louie, B. H., Seoane, J. A., Zhou, W. n., Silva, T. C., Groeneveld, C. n., Wong, C. K., Cho, S. W., Satpathy, A. T., Mumbach, M. R., Hoadley, K. A., Robertson, A. G., Sheffield, N. C., Felau, I. n., Castro, M. A., Berman, B. P., Staudt, L. M., Zenklusen, J. C., Laird, P. W., Curtis, C. n., Greenleaf, W. J., Chang, H. Y. 2018; 362 (6413)

  Abstract

  We present the genome-wide chromatin accessibility profiles of 410 tumor samples spanning 23 cancer types from The Cancer Genome Atlas (TCGA). We identify 562,709 transposase-accessible DNA elements that substantially extend the compendium of known cis-regulatory elements. Integration of ATAC-seq (the assay for transposase-accessible chromatin using sequencing) with TCGA multi-omic data identifies a large number of putative distal enhancers that distinguish molecular subtypes of cancers, uncovers specific driving transcription factors via protein-DNA footprints, and nominates long-range gene-regulatory interactions in cancer. These data reveal genetic risk loci of cancer predisposition as active DNA regulatory elements in cancer, identify gene-regulatory interactions underlying cancer immune evasion, and pinpoint noncoding mutations that drive enhancer activation and may affect patient survival. These results suggest a systematic approach to understanding the noncoding genome in cancer to advance diagnosis and therapy.

  View details for DOI 10.1126/science.aav1898

  View details for PubMedID 30361341

• Pembrolizumab for advanced basal cell carcinoma: an investigator-initiated, proof-of-concept study. Journal of the American Academy of Dermatology Chang, A. L., Tran, D. C., Cannon, J. G., Li, S. n., Jeng, M. n., Patel, R. n., Van der Bokke, L. n., Pague, A. n., Brotherton, R. n., Rieger, K. E., Satpathy, A. T., Yost, K. E., Reddy, S. n., Sarin, K. n., Colevas, A. D. 2018

  View details for PubMedID 30145186

• Expression of the transcription factor ZBTB46 distinguishes human histiocytic disorders of classical dendritic cell origin. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc Satpathy, A. T., Brown, R. A., Gomulia, E. n., Briseño, C. G., Mumbach, M. R., Pan, Z. n., Murphy, K. M., Natkunam, Y. n., Chang, H. Y., Kim, J. n. 2018

  Abstract

  Distinguishing classical dendritic cells from other myeloid cell types is complicated by the shared expression of cell surface markers. ZBTB46 is a zinc finger and BTB domain-containing transcription factor, which is expressed by dendritic cells and committed dendritic cell precursors, but not by plasmacytoid dendritic cells, monocytes, macrophages, or other immune cell populations. In this study, we demonstrate that expression of ZBTB46 identifies human dendritic cell neoplasms. We examined ZBTB46 expression in a range of benign and malignant histiocytic disorders and found that ZBTB46 is able to clearly define the dendritic cell identity of many previously unclassified histiocytic disease subtypes. In particular, all examined cases of Langerhans cell histiocytosis and histiocytic sarcoma expressed ZBTB46, while all cases of blastic plasmacytoid dendritic cell neoplasm, chronic myelomonocytic leukemia, juvenile xanthogranuloma, Rosai-Dorfman disease, and Erdheim-Chester disease failed to demonstrate expression of ZBTB46. Moreover, ZBTB46 expression clarified the identity of diagnostically challenging neoplasms, such as cases of indeterminate cell histiocytosis, classifying a fraction of these entities as dendritic cell malignancies. These findings clarify the lineage origins of human histiocytic disorders and distinguish dendritic cell disorders from all other myeloid neoplasms.

  View details for PubMedID 29743654

• Transforming Growth Factor Beta 3 (TGFB3) - a Novel Systemic Sclerosis Susceptibility Locus Involved in Fibrosis and Th17 Cell Development Identified By Genome-Wide Association Study in African Americans from the Genome Research in African American Scleroderma Patients Consortium Gourh, P., Remmers, E. F., Satpathy, A., Boyden, S., Morgan, N. D., Shah, A. A., Adeyemo, A., Bentley, A., Carns, M. A., Chandrasekharappa, S. C., Chung, L., Criswell, L. A., Derk, C. T., Domsic, R. T., Doumatey, A., Gladue, H., Goldberg, A., Gordon, J. K., Hsu, V., Jan, R., Khanna, D., Mayes, M. D., Medsger, T. A., Mumbach, M., Ramos, P. S., Trojanowski, M., Saketkoo, L., Schiopu, E., Shanmugam, V. K., Shriner, D., Silver, R. M., Steen, V. D., Valenzuela, A., Varga, J., Chang, H., Rotimi, C., Wigley, F. M., Boin, F., Kastner, D. L. WILEY. 2017

  View details for Web of Science ID 000411824106434

• Cells in Inflamed Islets of Autoimmune Diabetes Mice. Journal of immunology Klementowicz, J. E., Mahne, A. E., Spence, A., Nguyen, V., Satpathy, A. T., Murphy, K. M., Tang, Q. 2017

  Abstract

  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.

  View details for DOI 10.4049/jimmunol.1601062

  View details for PubMedID 28550204

• Discovery of stimulation-responsive immune enhancers with CRISPR activation. Nature Simeonov, D. R., Gowen, B. G., Boontanrart, M. n., Roth, T. L., Gagnon, J. D., Mumbach, M. R., Satpathy, A. T., Lee, Y. n., Bray, N. L., Chan, A. Y., Lituiev, D. S., Nguyen, M. L., Gate, R. E., Subramaniam, M. n., Li, Z. n., Woo, J. M., Mitros, T. n., Ray, G. J., Curie, G. L., Naddaf, N. n., Chu, J. S., Ma, H. n., Boyer, E. n., Van Gool, F. n., Huang, H. n., Liu, R. n., Tobin, V. R., Schumann, K. n., Daly, M. J., Farh, K. K., Ansel, K. M., Ye, C. J., Greenleaf, W. J., Anderson, M. S., Bluestone, J. A., Chang, H. Y., Corn, J. E., Marson, A. n. 2017

  Abstract

  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.

  View details for PubMedID 28854172

• An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nature methods Corces, M. R., Trevino, A. E., Hamilton, E. G., Greenside, P. G., Sinnott-Armstrong, N. A., Vesuna, S. n., Satpathy, A. T., Rubin, A. J., Montine, K. S., Wu, B. n., Kathiria, A. n., Cho, S. W., Mumbach, M. R., Carter, A. C., Kasowski, M. n., Orloff, L. A., Risca, V. I., Kundaje, A. n., Khavari, P. A., Montine, T. J., Greenleaf, W. J., Chang, H. Y. 2017

  Abstract

  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 generates chromatin accessibility profiles 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.

  View details for PubMedID 28846090

• Gene regulation in the immune system by long noncoding RNAs. Nature immunology Chen, Y. G., Satpathy, A. T., Chang, H. Y. 2017; 18 (9): 962–72

  Abstract

  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.

  View details for PubMedID 28829444

• Chromatin Accessibility Landscape of Cutaneous T Cell Lymphoma and Dynamic Response to HDAC Inhibitors. Cancer cell Qu, K. n., Zaba, L. C., Satpathy, A. T., Giresi, P. G., Li, R. n., Jin, Y. n., Armstrong, R. n., Jin, C. n., Schmitt, N. n., Rahbar, Z. n., Ueno, H. n., Greenleaf, W. J., Kim, Y. H., Chang, H. Y. 2017

  Abstract

  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.

  View details for PubMedID 28625481

• Revisiting the specificity of the MHC class II transactivator CIITA in classical murine dendritic cells in vivo. European journal of immunology Anderson, D. A., Grajales-Reyes, G. E., Satpathy, A. T., Hueichucura, C. E., Murphy, T. L., Murphy, K. M. 2017

  Abstract

  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 identification of cDCs independently of MHCII expression. We crossed Zbtb46(gfp) 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. This article is protected by copyright. All rights reserved.

  View details for PubMedID 28608405

• ATAC-see reveals the accessible genome by transposase-mediated imaging and sequencing. Nature methods Chen, X., Shen, Y., Draper, W., Buenrostro, J. D., Litzenburger, U., Cho, S. W., Satpathy, A. T., Carter, A. C., Ghosh, R. P., East-Seletsky, A., Doudna, J. A., Greenleaf, W. J., Liphardt, J. T., Chang, H. Y. 2016

  Abstract

  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.

  View details for DOI 10.1038/nmeth.4031

  View details for PubMedID 27749837

• A Long Noncoding RNA lincRNA-EPS Acts as a Transcriptional Brake to Restrain Inflammation CELL Atianand, M. K., Hu, W., Satpathy, A. T., Shen, Y., Ricci, E. P., Alvarez-Dominguez, J. R., Bhatta, A., Schattgen, S. A., McGowan, J. D., Blin, J., Braun, J. E., Gandhi, P., Moore, M. J., Chang, H. Y., Lodish, H. F., Caffrey, D. R., Fitzgerald, K. A. 2016; 165 (7): 1672-1685

  Abstract

  Long intergenic noncoding RNAs (lincRNAs) 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 localizes 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.

  View details for DOI 10.1016/j.cell.2016.05.075

  View details for PubMedID 27315481

• Cellular morphology of BRAF V600E-positive Langerhans cell histiocytosis BLOOD Satpathy, A. T., Tan, B. T. 2015; 126 (15): 1857-1857

  View details for DOI 10.1182/blood-2015-07-656900

  View details for Web of Science ID 000365451500021

  View details for PubMedID 26734696

• Long Noncoding RNA in Hematopoiesis and Immunity IMMUNITY Satpathy, A. T., Chang, H. Y. 2015; 42 (5): 792-804

  Abstract

  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.

  View details for DOI 10.1016/j.immuni.2015.05.004

  View details for Web of Science ID 000354827400007

• Long noncoding RNA in hematopoiesis and immunity. Immunity Satpathy, A. T., Chang, H. Y. 2015; 42 (5): 792-804

  Abstract

  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.

  View details for DOI 10.1016/j.immuni.2015.05.004

  View details for PubMedID 25992856

• Runx1 and Cbf beta regulate the development of Flt3(+) dendritic cell progenitors and restrict myeloproliferative disorder BLOOD Satpathy, A. T., Briseno, C. G., Cai, X., Michael, D. G., Chou, C., Hsiung, S., Bhattacharya, D., Speck, N. A., Egawa, T. 2014; 123 (19): 2968-2977

  Abstract

  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.

  View details for DOI 10.1182/blood-2013-11-539643

  View details for Web of Science ID 000335897900015

  View details for PubMedID 24677539

  View details for PubMedCentralID PMC4014839

• Heme-Mediated SPI-C Induction Promotes Monocyte Differentiation into Iron-Recycling Macrophages CELL Haldar, M., Kohyama, M., So, A. Y., Wumesh, K. C., Wu, X., Briseno, C. G., Satpathy, A. T., Kretzer, N. M., Arase, H., Rajasekaran, N. S., Wang, L., Egawa, T., Igarashi, K., Baltimore, D., Murphy, T. L., Murphy, K. M. 2014; 156 (6): 1223-1234

  Abstract

  Splenic red pulp macrophages (RPM) degrade senescent erythrocytes and recycle heme-associated iron. The transcription factor SPI-C is selectively expressed by RPM and is required for their development, but the physiologic stimulus inducing Spic is unknown. Here, we report that Spic also regulated the development of F4/80(+)VCAM1(+) bone marrow macrophages (BMM) and that Spic expression in BMM and RPM development was induced by heme, a metabolite of erythrocyte degradation. Pathologic hemolysis induced loss of RPM and BMM due to excess heme but induced Spic in monocytes to generate new RPM and BMM. Spic expression in monocytes was constitutively inhibited by the transcriptional repressor BACH1. Heme induced proteasome-dependent BACH1 degradation and rapid Spic derepression. Furthermore, cysteine-proline dipeptide motifs in BACH1 that mediate heme-dependent degradation were necessary for Spic induction by heme. These findings are the first example of metabolite-driven differentiation of a tissue-resident macrophage subset and provide new insights into iron homeostasis.

  View details for DOI 10.1016/j.cell.2014.01.069

  View details for Web of Science ID 000332945100012

  View details for PubMedID 24630724

  View details for PubMedCentralID PMC4010949

• L-Myc expression by dendritic cells is required for optimal T-cell priming NATURE Wumesh, K. C., Satpathy, A. T., Rapaport, A. S., Briseno, C. G., Wu, X., Albring, J. C., Russler-Germain, E. V., Kretzer, N. M., Durai, V., Persaud, S. P., Edelson, B. T., Loschko, J., Cella, M., Allen, P. M., Nussenzweig, M. C., Colonna, M., Sleckman, B. P., Murphy, T. L., Murphy, K. M. 2014; 507 (7491): 243-?

  Abstract

  The transcription factors c-Myc and N-Myc--encoded by Myc and Mycn, respectively--regulate cellular growth and are required for embryonic development. A third paralogue, Mycl1, is dispensable for normal embryonic development but its biological function has remained unclear. To examine the in vivo function of Mycl1 in mice, we generated an inactivating Mycl1(gfp) allele that also reports Mycl1 expression. We find that Mycl1 is selectively expressed in dendritic cells (DCs) of the immune system and controlled by IRF8, and that during DC development, Mycl1 expression is initiated in the common DC progenitor concurrent with reduction in c-Myc expression. Mature DCs lack expression of c-Myc and N-Myc but maintain L-Myc expression even in the presence of inflammatory signals such as granulocyte-macrophage colony-stimulating factor. All DC subsets develop in Mycl1-deficient mice, but some subsets such as migratory CD103(+) conventional DCs in the lung and liver are greatly reduced at steady state. Importantly, loss of L-Myc by DCs causes a significant decrease in in vivo T-cell priming during infection by Listeria monocytogenes and vesicular stomatitis virus. The replacement of c-Myc by L-Myc in immature DCs may provide for Myc transcriptional activity in the setting of inflammation that is required for optimal T-cell priming.

  View details for DOI 10.1038/nature12967

  View details for Web of Science ID 000332651800041

  View details for PubMedID 24509714

  View details for PubMedCentralID PMC3954917

• Embryonic and Adult-Derived Resident Cardiac Macrophages Are Maintained through Distinct Mechanisms at Steady State and during Inflammation IMMUNITY Epelman, S., Lavine, K. J., Beaudin, A. E., Sojka, D. K., Carrero, J. A., Calderon, B., Brija, T., Gautier, E. L., Ivanov, S., Satpathy, A. T., Schilling, J. D., Schwendener, R., Sergin, I., Razani, B., Forsberg, E. C., Yokoyama, W. M., Unanue, E. R., Colonna, M., Randolph, G. J., Mann, D. L. 2014; 40 (1): 91-104

  Abstract

  Cardiac macrophages are crucial for tissue repair after cardiac injury but are not well characterized. Here we identify four populations of cardiac macrophages. At steady state, resident macrophages were primarily maintained through local proliferation. However, after macrophage depletion or during cardiac inflammation, Ly6c(hi) monocytes contributed to all four macrophage populations, whereas resident macrophages also expanded numerically through proliferation. Genetic fate mapping revealed that yolk-sac and fetal monocyte progenitors gave rise to the majority of cardiac macrophages, and the heart was among a minority of organs in which substantial numbers of yolk-sac macrophages persisted in adulthood. CCR2 expression and dependence distinguished cardiac macrophages of adult monocyte versus embryonic origin. Transcriptional and functional data revealed that monocyte-derived macrophages coordinate cardiac inflammation, while playing redundant but lesser roles in antigen sampling and efferocytosis. These data highlight the presence of multiple cardiac macrophage subsets, with different functions, origins, and strategies to regulate compartment size.

  View details for DOI 10.1016/j.immuni.2013.11.019

  View details for Web of Science ID 000331476900012

  View details for PubMedID 24439267

  View details for PubMedCentralID PMC3923301

• Extrathymic Aire-Expressing Cells Are a Distinct Bone Marrow-Derived Population that Induce Functional Inactivation of CD4(+) T Cells IMMUNITY Gardner, J. M., Metzger, T. C., McMahon, E. J., Au-Yeung, B. B., Krawisz, A. K., Lu, W., Price, J. D., Johannes, K. P., Satpathy, A. T., Murphy, K. M., Tarbell, K. V., Weiss, A., Anderson, M. S. 2013; 39 (3): 560-572

  Abstract

  The autoimmune regulator (Aire) is essential for prevention of autoimmunity; its role is best understood in the thymus, where it promotes self-tolerance through tissue-specific antigen (TSA) expression. Recently, extrathymic Aire-expressing cells (eTACs) have been described in murine secondary lymphoid organs, but the identity of such cells and their role in immune tolerance remains unclear. Here we have shown that eTACs are a discrete major histocompatibility complex class II (MHC II)(hi), CD80(lo), CD86(lo), epithelial cell adhesion molecule (EpCAM)(hi), CD45(lo) bone marrow-derived peripheral antigen-presenting cell (APC) population. We also have demonstrated that eTACs can functionally inactivate CD4⁺ T cells through a mechanism that does not require regulatory T cells (Treg) and is resistant to innate inflammatory stimuli. Together, these findings further define eTACs as a distinct tolerogenic cell population in secondary lymphoid organs.

  View details for DOI 10.1016/j.immuni.2013.08.005

  View details for Web of Science ID 000330949600018

  View details for PubMedID 23993652

• Bcl11a Controls Flt3 Expression in Early Hematopoietic Progenitors and Is Required for pDC Development In Vivo PLOS ONE Wu, X., Satpathy, A. T., Wumesh, K. C., Liu, P., Murphy, T. L., Murphy, K. M. 2013; 8 (5)

  Abstract

  Bcl11a is a transcription factor known to regulate lymphoid and erythroid development. Recent bioinformatic analysis of global gene expression patterns has suggested a role for Bcl11a in the development of dendritic cell (DC) lineages. We tested this hypothesis by analyzing the development of DC and other lineages in Bcl11a (-/-) mice. We found that Bcl11a was required for expression of IL-7 receptor (IL-7R) and Flt3 in early hematopoietic progenitor cells. In addition, we found severely decreased numbers of plasmacytoid dendritic cells (pDCs) in Bcl11a (-/-) fetal livers and in the bone marrow of Bcl11a (-/-) fetal liver chimeras. Moreover, Bcl11a (-/-) cells showed severely impaired in vitro development of Flt3L-derived pDCs and classical DCs (cDCs). In contrast, we found normal in vitro development of DCs from Bcl11a (-/-) fetal liver cells treated with GM-CSF. These results suggest that the persistent cDC development observed in Bcl11a (-/-) fetal liver chimeras reflects derivation from a Bcl11a- and Flt3-independent pathway in vivo.

  View details for DOI 10.1371/journal.pone.0064800

  View details for Web of Science ID 000319799900108

  View details for PubMedID 23741395

  View details for PubMedCentralID PMC3669380

• Ly6C(hi) Monocytes in the Inflamed Colon Give Rise to Proinflammatory Effector Cells and Migratory Antigen-Presenting Cells IMMUNITY Zigmond, E., Varol, C., Farache, J., Elmaliah, E., Satpathy, A. T., Friedlander, G., Mack, M., Shpigel, N., Boneca, I. G., Murphy, K. M., Shakhar, G., Halpern, Z., Jung, S. 2012; 37 (6): 1076-1090

  Abstract

  Ly6C(hi) monocytes seed the healthy intestinal lamina propria to give rise to resident CX(3)CR1(+) macrophages that contribute to the maintenance of gut homeostasis. Here we report on two alternative monocyte fates in the inflamed colon. We showed that CCR2 expression is essential to the recruitment of Ly6C(hi) monocytes to the inflamed gut to become the dominant mononuclear cell type in the lamina propria during settings of acute colitis. In the inflammatory microenvironment, monocytes upregulated TLR2 and NOD2, rendering them responsive to bacterial products to become proinflammatory effector cells. Ablation of Ly6C(hi) monocytes ameliorated acute gut inflammation. With time, monocytes differentiated into migratory antigen-presenting cells capable of priming naive T cells, thus acquiring hallmarks reminiscent of dendritic cells. Collectively, our results highlight cellular dynamics in the inflamed colon and the plasticity of Ly6C(hi) monocytes, marking them as potential targets for inflammatory bowel disease (IBD) therapy.

  View details for DOI 10.1016/j.immuni.2012.08.026

  View details for Web of Science ID 000312575000015

  View details for PubMedID 23219392

• Compensatory dendritic cell development mediated by BATF-IRF interactions NATURE Tussiwand, R., Lee, W., Murphy, T. L., Mashayekhi, M., Wumesh, K. C., Albring, J. C., Satpathy, A. T., Rotondo, J. A., Edelson, B. T., Kretzer, N. M., Wu, X., Weiss, L. A., Glasmacher, E., Li, P., Liao, W., Behnke, M., Lam, S. S., Aurthur, C. T., Leonard, W. J., Singh, H., Stallings, C. L., Sibley, L. D., Schreiber, R. D., Murphy, K. M. 2012; 490 (7421): 502-?

  Abstract

  The AP1 transcription factor Batf3 is required for homeostatic development of CD8α(+) classical dendritic cells that prime CD8 T-cell responses against intracellular pathogens. Here we identify an alternative, Batf3-independent pathway in mice for CD8α(+) dendritic cell development operating during infection with intracellular pathogens and mediated by the cytokines interleukin (IL)-12 and interferon-γ. This alternative pathway results from molecular compensation for Batf3 provided by the related AP1 factors Batf, which also functions in T and B cells, and Batf2 induced by cytokines in response to infection. Reciprocally, physiological compensation between Batf and Batf3 also occurs in T cells for expression of IL-10 and CTLA4. Compensation among BATF factors is based on the shared capacity of their leucine zipper domains to interact with non-AP1 factors such as IRF4 and IRF8 to mediate cooperative gene activation. Conceivably, manipulating this alternative pathway of dendritic cell development could be of value in augmenting immune responses to vaccines.

  View details for DOI 10.1038/nature11531

  View details for Web of Science ID 000310196200034

  View details for PubMedID 22992524

  View details for PubMedCentralID PMC3482832

• Cross-dressed CD8 alpha(+)/CD103(+) dendritic cells prime CD8(+) T cells following vaccination PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Li, L., Kim, S., Herndon, J. M., Goedegebuure, P., Belt, B. A., Satpathy, A. T., Fleming, T. P., Hansen, T. H., Murphy, K. M., Gillanders, W. E. 2012; 109 (31): 12716-12721

  Abstract

  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.

  View details for DOI 10.1073/pnas.1203468109

  View details for Web of Science ID 000307538200087

  View details for PubMedID 22802630

  View details for PubMedCentralID PMC3411977

• Dual actions of Meis1 inhibit erythroid progenitor development and sustain general hematopoietic cell proliferation BLOOD Cai, M., Langer, E. M., Gill, J. G., Satpathy, A. T., Albring, J. C., Wumesh, K. C., Murphy, T. L., Murphy, K. M. 2012; 120 (2): 335-346

  Abstract

  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.

  View details for DOI 10.1182/blood-2012-01-403139

  View details for Web of Science ID 000307412400018

  View details for PubMedCentralID PMC3628121

• IRF-8 extinguishes neutrophil production and promotes dendritic cell lineage commitment in both myeloid and lymphoid mouse progenitors BLOOD Becker, A. M., Michael, D. G., Satpathy, A. T., Sciammas, R., Singh, H., Bhattacharya, D. 2012; 119 (9): 2003-2012

  Abstract

  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.

  View details for DOI 10.1182/blood-2011-06-364976

  View details for PubMedID 22238324

  View details for PubMedCentralID PMC3311244

• Transcription factor networks in dendritic cell development SEMINARS IN IMMUNOLOGY Satpathy, A. T., Murphy, K. M., Wumesh, K. C. 2011; 23 (5): 388-397

  Abstract

  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.

  View details for DOI 10.1016/j.smim.2011.08.009

  View details for Web of Science ID 000297447300010

  View details for PubMedID 21924924

  View details for PubMedCentralID PMC4010935

• Targeting of B and T lymphocyte associated (BTLA) prevents graft-versus-host disease without global immunosuppression JOURNAL OF EXPERIMENTAL MEDICINE Albring, J. C., Sandau, M. M., Rapaport, A. S., Edelson, B. T., Satpathy, A., Mashayekhi, M., Lathrop, S. K., Hsieh, C., Stelljes, M., Colonna, M., Murphy, T. L., Murphy, K. M. 2010; 207 (12): 2551-2559

  Abstract

  Graft-versus-host disease (GVHD) causes significant morbidity and mortality in allogeneic hematopoietic stem cell transplantation (aHSCT), preventing its broader application to non-life-threatening diseases. We show that a single administration of a nondepleting monoclonal antibody specific for the coinhibitory immunoglobulin receptor, B and T lymphocyte associated (BTLA), permanently prevented GVHD when administered at the time of aHSCT. Once GVHD was established, anti-BTLA treatment was unable to reverse disease, suggesting that its mechanism occurs early after aHSCT. Anti-BTLA treatment prevented GVHD independently of its ligand, the costimulatory tumor necrosis factor receptor herpesvirus entry mediator (HVEM), and required BTLA expression by donor-derived T cells. Furthermore, anti-BTLA treatment led to the relative inhibition of CD4(+) forkhead box P3(-) (Foxp3(-)) effector T cell (T eff cell) expansion compared with precommitted naturally occurring donor-derived CD4(+) Foxp3(+) regulatory T cell (T reg cell) and allowed for graft-versus-tumor (GVT) effects as well as robust responses to pathogens. These results suggest that BTLA agonism rebalances T cell expansion in lymphopenic hosts after aHSCT, thereby preventing GVHD without global immunosuppression. Thus, targeting BTLA with a monoclonal antibody at the initiation of aHSCT therapy might reduce limitations imposed by histocompatibility and allow broader application to treatment of non-life-threatening diseases.

  View details for DOI 10.1084/jem.20102017

  View details for Web of Science ID 000285505000003

  View details for PubMedID 21078889

  View details for PubMedCentralID PMC2989771

• Commercially Available Outbred Mice for Genome-Wide Association Studies PLOS GENETICS Yalcin, B., Nicod, J., Bhomra, A., Davidson, S., Cleak, J., Farinelli, L., Osteras, M., Whitley, A., Yuan, W., Gan, X., Goodson, M., Klenerman, P., Satpathy, A., Mathis, D., Benoist, C., Adams, D. J., Mott, R., Flint, J. 2010; 6 (9)

  Abstract

  Genome-wide association studies using commercially available outbred mice can detect genes involved in phenotypes of biomedical interest. Useful populations need high-frequency alleles to ensure high power to detect quantitative trait loci (QTLs), low linkage disequilibrium between markers to obtain accurate mapping resolution, and an absence of population structure to prevent false positive associations. We surveyed 66 colonies for inbreeding, genetic diversity, and linkage disequilibrium, and we demonstrate that some have haplotype blocks of less than 100 Kb, enabling gene-level mapping resolution. The same alleles contribute to variation in different colonies, so that when mapping progress stalls in one, another can be used in its stead. Colonies are genetically diverse: 45% of the total genetic variation is attributable to differences between colonies. However, quantitative differences in allele frequencies, rather than the existence of private alleles, are responsible for these population differences. The colonies derive from a limited pool of ancestral haplotypes resembling those found in inbred strains: over 95% of sequence variants segregating in outbred populations are found in inbred strains. Consequently it is possible to impute the sequence of any mouse from a dense SNP map combined with inbred strain sequence data, which opens up the possibility of cataloguing and testing all variants for association, a situation that has so far eluded studies in completely outbred populations. We demonstrate the colonies' potential by identifying a deletion in the promoter of H2-Ea as the molecular change that strongly contributes to setting the ratio of CD4+ and CD8+ lymphocytes.

  View details for DOI 10.1371/journal.pgen.1001085

  View details for Web of Science ID 000282369200055

  View details for PubMedID 20838427

• Enhanced thymic selection of FoxP3(+) regulatory T cells in the NOD mouse model of autoimmune diabetes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Feuerer, M., Jiang, W., Holler, P. D., Satpathy, A., Campbell, C., Bogue, M., Mathis, D., Benoist, C. 2007; 104 (46): 18181-18186

  Abstract

  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.

  View details for DOI 10.1073/pnas.0708899104

  View details for Web of Science ID 000251077000052

  View details for PubMedID 17991775

• Cytokines in type 2 diabetes INTERLEUKINS Johnson, D. R., O'Connor, J. C., Satpathy, A., Freund, G. G. 2006; 74: 405-441

  View details for DOI 10.1016/S0083-6729(06)74017-1

  View details for Web of Science ID 000242541700017

  View details for PubMedID 17027525

• IL-1 beta-mediated innate immunity is amplified in the db/db mouse model of type 2 diabetes JOURNAL OF IMMUNOLOGY O'Connor, J. C., Satpathy, A., Hartman, M. E., Horvath, E. M., Kelley, K. W., Dantzer, R., Johnson, R. W., Freund, G. G. 2005; 174 (8): 4991-4997

  Abstract

  Chronic inflammation appears to play a critical role in type 2 diabetes and its complications. Here we tested the hypothesis that this inflammatory dysregulation affects the IL-1beta system and has functional consequences in the brain. Diabetic, db/db, and nondiabetic, db/+, mice were administered i.p. LPS, a potent cytokine inducer, at a dose of 100 microg/kg/mouse. db/db mouse innate immune-associated sickness behavior was 14.8, 33, 44.7, and 34% greater than that of db/+ mice at 2, 4, 8, and 12 h, respectively. When a fixed dose of LPS was used (5 microg/mouse), db/db mouse sickness was again enhanced 18.4, 22.2, and 14.5% at 4, 8, and 12 h as compared with db/+ mice. In diabetic mice, peritoneal macrophages produced more IL-1beta in response to LPS, and peritoneal levels of IL-1beta induced by LPS were increased. Importantly, IL-1R antagonist and type 2 IL-1 receptor (IL-1R2) failed to up-regulate in response to LPS in db/db mice. Finally, both peripheral and central administration of IL-1beta, itself, induced sickness in db/db mice that mimicked the effects of peripheral LPS and was significantly greater than that seen in db/+ mice. Taken together, these results indicate that IL-1beta-mediated innate immunity is augmented in db/db mice both at the periphery and in the brain, and the mechanism is due to diabetes-associated loss of IL-1beta counterregulation.

  View details for Web of Science ID 000228234600069

  View details for PubMedID 15814729