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Unlike many other hematologic malignancies, Richter syndrome (RS), an aggressive B cell lymphoma originating from indolent chronic lymphocytic leukemia, is responsive to PD-1 blockade. To discover the determinants of response, we analyze single-cell transcriptome data generated from 17 bone marrow samples longitudinally collected from 6 patients with RS. Response is associated with intermediate exhausted CD8 effector/effector memory T cells marked by high expression of the transcription factor ZNF683, determined to be evolving from stem-like memory cells and divergent from terminally exhausted cells. This signature overlaps with that of tumor-infiltrating populations from anti-PD-1 responsive solid tumors. ZNF683 is found to directly target key T cell genes (TCF7, LMO2, CD69) and impact pathways of T cell cytotoxicity and activation. Analysis of pre-treatment peripheral blood from 10 independent patients with RS treated with anti-PD-1, as well as patients with solid tumors treated with anti-PD-1, supports an association of ZNF683 high T cells with response., Competing Interests: Declaration of interests C.J.W. receives funding support from: Pharmacyclics; holds equity in: BioNTech, Inc; G.G. is a founder, consultant and holds privately held equity in Scorpion Therapeutics, receives funding support from: IBM and Pharmacyclics, is an inventor on patent applications related to: MuTect, ABSOLUTE, MutSig, MSMuTect, MSMutSig, MSIDetect, POLYSOLVER, and TensorQTL; R.G. receives funding support from: Abbvie, Janssen, Gilead, AstraZeneca, and Roche; N.J. receives research funding from: Pharmacyclics, AbbVie, Genentech, AstraZeneca, BMS, Pfizer, Servier, ADC Therapeutics, Cellectis, Precision BioSciences, Adaptive Biotechnologies, Incyte, Aprea Therapeutics, Fate Therapeutics, Mingsight, Takeda, Medisix, Loxo Oncology, Novalgen and serves on Advisory Board/Honoraria: Pharmacyclics, Janssen, AbbVie, Genentech, AstraZeneca, BMS, Adaptive Biotechnologies, Precision BioSciences, Servier, Beigene, Cellectis, TG Therapeutics, ADC Therapeutics, MEI Pharma; W.G.W. reports funding from GSK/Novartis, Abbvie, Genentech, Pharmacyclics LLC, AstraZeneca/Acerta Pharma, Gilead Sciences, Juno Therapeutics, KITE Pharma, Sunesis, Miragen, Oncternal Therapeutics, Inc., Cyclacel, Loxo Oncology, Inc., Janssen, Xencor. B.A.K., C.J.W. and G.G. are inventors on patent: “Compositions, panels, and methods for characterizing chronic lymphocytic leukemia” (PCT/US21/45144); S.A.S. reports nonfinancial support from Bristol-Myers Squibb, and equity in Agenus Inc., Agios Pharmaceuticals, Breakbio Corp., Bristol-Myers Squibb and Lumos Pharma. N.P. is currently an employee of Bristol Myers Squibb. K.J.L. holds equity in Standard BioTools Inc. (formerly Fluidigm Corporation). C.J.W. and E.M.P are inventors on a patent, US Utility Application No. US-2022-0298580-A1 filed on 02/10/2022, International Application No. WO/2021/041669 filed on 9/15/2022, “Immune Signatures Predictive of Response to PD-1 Blockade in Richter’s transformation.” M.D., J.D.K. and B.T. have no relevant conflict of interest. C.T. reports honorarium from Beigene, Janssen, Abbvie, AZ and LOXO and research funding from Beigene, Janssen, and AbbVie., (Copyright © 2023 Elsevier Inc. All rights reserved.)

T cells mediate antigen-specific immune responses to disease through the specificity and diversity of their clonotypic T cell receptors (TCRs). Determining the spatial distributions of T cell clonotypes in tissues is essential to understanding T cell behavior, but spatial sequencing methods remain unable to profile the TCR repertoire. Here, we developed Slide-TCR-seq, a 10-μm-resolution method, to sequence whole transcriptomes and TCRs within intact tissues. We confirmed the ability of Slide-TCR-seq to map the characteristic locations of T cells and their receptors in mouse spleen. In human lymphoid germinal centers, we identified spatially distinct TCR repertoires. Profiling T cells in renal cell carcinoma and melanoma specimens revealed heterogeneous immune responses: T cell states and infiltration differed intra- and inter-clonally, and adjacent tumor and immune cells exhibited distinct gene expression. Altogether, our method yields insights into the spatial relationships between clonality, neighboring cell types, and gene expression that drive T cell responses., Competing Interests: Declaration of interests F.C., C.J.W., S.L., J.B.I., K.J.L., and S. Li have filed a patent on this work. D.A.B. reports non-financial support from Bristol Myers Squibb; honoraria from LM Education/Exchange Services; advisory board fees from Exelixis and AVEO; personal fees from Charles River Associates, Schlesinger Associates, Imprint Science, Insight Strategy, Trinity Group, Cancer Expert Now, Adnovate Strategies, MDedge, CancerNetwork, Catenion, OncLive, Cello Health BioConsulting, PWW Consulting, Haymarket Medical Network, Aptitude Health, and AbbVie; and research support from Exelixis, outside of the submitted work. C.J.W. holds equity in BioNTech, Inc. K.J.L. holds equity in Standard BioTools Inc. (formerly Fluidigm Corporation). F.C. is a paid consultant for Atlas Bio., (Copyright © 2022 Elsevier Inc. All rights reserved.)

To elucidate mechanisms by which T cells eliminate leukemia, we study donor lymphocyte infusion (DLI), an established immunotherapy for relapsed leukemia. We model T cell dynamics by integrating longitudinal, multimodal data from 94,517 bone marrow-derived single T cell transcriptomes in addition to chromatin accessibility and single T cell receptor sequencing from patients undergoing DLI. We find that responsive tumors are defined by enrichment of late-differentiated T cells before DLI and rapid, durable expansion of early differentiated T cells after treatment, highly similar to "terminal" and "precursor" exhausted subsets, respectively. Resistance, in contrast, is defined by heterogeneous T cell dysfunction. Surprisingly, early differentiated T cells in responders mainly originate from pre-existing and novel clonotypes recruited to the leukemic microenvironment, rather than the infusion. Our work provides a paradigm for analyzing longitudinal single-cell profiling of scenarios beyond adoptive cell therapy and introduces Symphony, a Bayesian approach to infer regulatory circuitry underlying T cell subsets, with broad relevance to exhaustion antagonists across cancers., Competing Interests: Declaration of interests C.J.W. is an equity holder of BioNTech. P.B. reports equity in Agenus, Amgen, Breakbio Corp., Johnson & Johnson, Exelixis, and BioNTech. C.J.W. and D.N. receive research funding from Pharmacyclics. D.S.N. reports stock ownership in Madrigal Pharmaceuticals. V.N.N. is an employee of Bluebird Bio. D.B.K. has previously advised Neon Therapeutics and has received consulting fees from Neon Therapeutics, and owns equity in Aduro Biotech, Agenus, Armata Pharmaceuticals, Breakbio, BioMarin Pharmaceutical, Bristol-Myers Squibb, Celldex Therapeutics, Editas Medicine, Exelixis, Gilead Sciences, IMV, Lexicon Pharmaceuticals, Moderna, and Regeneron Pharmaceuticals. BeiGene, a Chinese biotech company, supports unrelated research at the DFCI Translational Immunogenomics Laboratory (TIGL). J.R. receives research funding from Amgen, Equillium, Novartis, and Kite/Gilead and serves on Data Safety Monitoring Committees for AvroBio and Scientific Advisory Boards for Akron Biotech, Clade Therapeutics, Garuda Therapeutics, Immunitas Therapeutics, LifeVault Bio, Novartis, Rheos Medicines, Talaris Therapeutics, and TScan Therapeutics. R.J.S. serves on the Board of Directors for Kiadis and Be The Match/National Marrow Donor Program; provided consulting for Gilead, Rheos Therapeutics, Cugene, Precision Bioscience, Mana Therapeutics, VOR Biopharma, and Novartis; and has served on the Data Safety Monitoring Board for Juno/Celgene. D.P. serves on the Board of Insitro. The remaining authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The tumor immune microenvironment plays a critical role in cancer progression and response to immunotherapy in clear cell renal cell carcinoma (ccRCC), yet the composition and phenotypic states of immune cells in this tumor are incompletely characterized. We performed single-cell RNA and T cell receptor sequencing on 164,722 individual cells from tumor and adjacent non-tumor tissue in patients with ccRCC across disease stages: early, locally advanced, and advanced/metastatic. Terminally exhausted CD8 + T cells were enriched in metastatic disease and were restricted in T cell receptor diversity. Within the myeloid compartment, pro-inflammatory macrophages were decreased, and suppressive M2-like macrophages were increased in advanced disease. Terminally exhausted CD8 + T cells and M2-like macrophages co-occurred in advanced disease and expressed ligands and receptors that support T cell dysfunction and M2-like polarization. This immune dysfunction circuit is associated with a worse prognosis in external cohorts and identifies potentially targetable immune inhibitory pathways in ccRCC., Competing Interests: Declaration of interests D.A.B. reports nonfinancial support from Bristol Myers Squibb, honoraria from LM Education/Exchange Services, and personal fees from Octane Global, Defined Health, Dedham Group, Adept Field Solutions, Slingshot Insights, Blueprint Partnerships, Charles River Associates, Trinity Group, and Insight Strategy, outside of the submitted work. S.H.G. has patents licensed to Novalgen Inc, outside of the submitted work. Z.B. reports nonfinancial support from Bristol Myers Squibb and Genentech/imCore. D.B.K. has acted as an advisor to and has received consulting fees from Neon Therapeutics and owns equity in Agenus, Armata pharmaceuticals, Breakbio, Biomarin Pharmaceutical, Bristol Myers Squibb, Celldex Therapeutics, Chinook Therapeutics, Editas Medicine, Exelixis, Gilead Sciences, IMV, Lexicon Pharmaceuticals, Moderna, and Regeneron Pharmaceuticals. K.M.M. reports research support from Bristol Myers Squibb. B.A.M. reports consulting fees from Bayer, Astellas, Astra Zeneca, Seattle Genetics, Dendreon, Calithera, Exelixis, Nektar, Pfizer, Janssen, Genentech, Eisai, and EMD Serono, and research support to DFCI from Bristol Myers Squibb, Calithera, Exelixis, Seattle Genetics. D.F.M. has received consulting fees from Bristol Myers Squibb, Pfizer, Merck, Alkermes, EMD Serono, Eli Lilly, Iovance, and Eisai, and research support from Bristol Myers Squibb, Merck, Genentech, Pfizer, Exelixis, X4 Pharma, and Alkermes. S.A.S. reports nonfinancial support from Bristol Myers Squibb outside the submitted work. S.A.S. previously advised and has received consulting fees from Neon Therapeutics. S.A.S. reports nonfinancial support from Bristol Myers Squibb, and equity in Agenus Inc., Agios Pharmaceuticals, Breakbio Corp., Bristol Myers Squibb, and Lumos Pharma, outside the submitted work. S.S. reported personal fees from Merck, AstraZeneca, Bristol Myers Squibb, CRISPR Therapeutics AG, AACR, and NCI, grants from Bristol Myers Squibb, AstraZeneca, Novartis, and Exelixis, and royalties from Biogenex. A.H.S. has patents/pending royalties on the PD-1 pathway from Roche and Novartis. A.H.S. is on advisory boards for Bicara, Janssen Immunology, Surface Oncology, Elstar, SQZ Biotechnologies, Elpiscience, Selecta, and Monopteros, and consults for Novartis. A.H.S. has received research funding from AbbVie, Novartis, Roche, UCB, Ipsen, Quark, and Merck. D.F.M. reports personal fees from Bristol Myers Squibb, Pfizer, Merck, Novartis, Exelixis, Array BioPharm, Genentech, Alkermes, Jounce Therapeutics, X4 Pharma, Peloton, EMD Serono, and Eli Lilly, and research support from Bristol Myers Squibb, Prometheus Laboratories, Merck, Genentech, Pfizer, Exelixis, Novartis, X4 Pharma, Alkermes, and Peloton. T.K.C. reports grants and personal fees from Astra Zeneca; personal fees from Bayer; grants and personal fees from Bristol Myers Squibb; personal fees from Cerulean; grants and personal fees from Eisai; personal fees from Foundation Medicine Inc.; grants and personal fees from Exelixis; grants and personal fees from Genentech; personal fees from Roche; grants and personal fees from GlaxoSmithKline; grants and personal fees from Merck, from Novartis, Peloton, and Pfizer; personal fees from Prometheus Labs; grants and personal fees from Corvus; personal fees from Ipsen; grants from Tracon; and grants from Astellas outside the submitted work. The other authors declare no potential conflicts of interest. C.J.W. is an equity holder of BioNTech., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Hotspot mutation of IKZF3 (IKZF3-L162R) has been identified as a putative driver of chronic lymphocytic leukemia (CLL), but its function remains unknown. Here, we demonstrate its driving role in CLL through a B cell-restricted conditional knockin mouse model. Mutant Ikzf3 alters DNA binding specificity and target selection, leading to hyperactivation of B cell receptor (BCR) signaling, overexpression of nuclear factor κB (NF-κB) target genes, and development of CLL-like disease in elderly mice with a penetrance of ~40%. Human CLL carrying either IKZF3 mutation or high IKZF3 expression was associated with overexpression of BCR/NF-κB pathway members and reduced sensitivity to BCR signaling inhibition by ibrutinib. Our results thus highlight IKZF3 oncogenic function in CLL via transcriptional dysregulation and demonstrate that this pro-survival function can be achieved by either somatic mutation or overexpression of this CLL driver. This emphasizes the need for combinatorial approaches to overcome IKZF3-mediated BCR inhibitor resistance., Competing Interests: Declaration of interests C.J.W. is an equity holder of Biontech, Inc. and receives research funding from Pharmacyclics. D.S.N. has been a consultant for H3 Biomedicine and received research funding from Celgene. J.A.B. reports receiving grant support and advisory board fees from Pharmacyclics, grant support, advisory board fees, and lecture fees from Gilead, advisory board fees from AstraZeneca, and lecture fees and travel support from Janssen. T.J.K. has received research funding and/or has served as an advisor to Ascerta/AstraZeneca, Celgene, Genentech/Roche, Gilead, Janssen, Loxo Oncology, Octernal Therapeutics, Pharmacyclics/AbbVie, TG Therapeutics, VelosBio, and Verastem. Cirmtuzumab was developed by T.J.K. and licensed by the University of California to Oncternal Therapeutics, Inc., which has provided stock/options to the university and T.J.K. All other authors do not have any relevant conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Mitochondrial apoptosis can be effectively targeted in lymphoid malignancies with the FDA-approved B cell lymphoma 2 (BCL-2) inhibitor venetoclax, but resistance to this agent is emerging. We show that venetoclax resistance in chronic lymphocytic leukemia is associated with complex clonal shifts. To identify determinants of resistance, we conducted parallel genome-scale screens of the BCL-2-driven OCI-Ly1 lymphoma cell line after venetoclax exposure along with integrated expression profiling and functional characterization of drug-resistant and engineered cell lines. We identified regulators of lymphoid transcription and cellular energy metabolism as drivers of venetoclax resistance in addition to the known involvement by BCL-2 family members, which were confirmed in patient samples. Our data support the implementation of combinatorial therapy with metabolic modulators to address venetoclax resistance., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Cellular reprogramming through manipulation of defined factors holds great promise for large-scale production of cell types needed for use in therapy and for revealing principles of gene regulation. However, most reprogramming systems are inefficient, converting only a fraction of cells to the desired state. Here, we analyze MYOD-mediated reprogramming of human fibroblasts to myotubes, a well-characterized model system for direct conversion by defined factors, at pseudotemporal resolution using single-cell RNA-seq. To expose barriers to efficient conversion, we introduce a novel analytic technique, trajectory alignment, which enables quantitative comparison of gene expression kinetics across two biological processes. Reprogrammed cells navigate a trajectory with branch points that correspond to two alternative decision points, with cells that select incorrect branches terminating at aberrant or incomplete reprogramming outcomes. Analysis of these branch points revealed insulin and BMP signaling as crucial molecular determinants of reprogramming. Single-cell trajectory alignment enables rigorous quantitative comparisons between biological trajectories found in diverse processes in development, reprogramming, and other contexts., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Mutations in SF3B1, which encodes a spliceosome component, are associated with poor outcome in chronic lymphocytic leukemia (CLL), but how these contribute to CLL progression remains poorly understood. We undertook a transcriptomic characterization of primary human CLL cells to identify transcripts and pathways affected by SF3B1 mutation. Splicing alterations, identified in the analysis of bulk cells, were confirmed in single SF3B1-mutated CLL cells and also found in cell lines ectopically expressing mutant SF3B1. SF3B1 mutation was found to dysregulate multiple cellular functions including DNA damage response, telomere maintenance, and Notch signaling (mediated through KLF8 upregulation, increased TERC and TERT expression, or altered splicing of DVL2 transcript, respectively). SF3B1 mutation leads to diverse changes in CLL-related pathways., Competing Interests: Michael Seiler, Silvia Buonamici, Peter G. Smith are employees and shareholders of H3 Biomedicine. Catherine J. Wu is co-founder and scientific advisory board member of Neon Therapeutics, Inc. All other authors have no conflicts of interest., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Intratumoral heterogeneity plays a critical role in tumor evolution. To define the contribution of DNA methylation to heterogeneity within tumors, we performed genome-scale bisulfite sequencing of 104 primary chronic lymphocytic leukemias (CLLs). Compared with 26 normal B cell samples, CLLs consistently displayed higher intrasample variability of DNA methylation patterns across the genome, which appears to arise from stochastically disordered methylation in malignant cells. Transcriptome analysis of bulk and single CLL cells revealed that methylation disorder was linked to low-level expression. Disordered methylation was further associated with adverse clinical outcome. We therefore propose that disordered methylation plays a similar role to that of genetic instability, enhancing the ability of cancer cells to search for superior evolutionary trajectories., (Copyright © 2014 Elsevier Inc. All rights reserved.)

There has been great interest in the prospects of using single-nucleotide polymorphisms (SNPs) in the search for complex disease genes, and several initiatives devoted to the identification and mapping of SNPs throughout the human genome are currently underway. However, actual data investigating the use of SNPs for identification of complex disease genes are scarce. To begin to look at issues surrounding the use of SNPs in complex disease studies, we have initiated a collaborative SNP mapping study around APOE, the well-established susceptibility gene for late-onset Alzheimer disease (AD). Sixty SNPs in a 1.5-Mb region surrounding APOE were genotyped in samples of unrelated cases of AD, in controls, and in families with AD. Standard tests were conducted to look for association of SNP alleles with AD, in cases and controls. We also used family-based association analyses, including recently developed methods to look for haplotype association. Evidence of association (P

Many of the genes in the regulatory hierarchy controlling sex determination in Drosophila melanogaster are known. Here we examine how this regulatory hierarchy controls the expression of the structural genes encoding the female-specific yolk polypeptides. Temperature shift experiments with a temperature-sensitive allele of the sex determination regulatory gene transformer-2 (tra-2) showed that tra-2+ function is required in the adult for both the sex-specific initiation and maintenance of YP synthesis. Control of the YP genes by this regulatory hierarchy is at the level of transcription, or transcript stability. The results of temperature shift experiments with abdomens isolated from tra-2ts homozygotes support the notion that the tra-2+ function acts in a cell-autonomous manner to control YP synthesis. These results provide a paradigm for the way this regulatory hierarchy controls the terminal differentiation functions for sexually dimorphic development.