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In metastatic urothelial cancer (mUC), cisplatin versus carboplatin leads to durable disease control in a subset of patients. The IMvigor130 trial reveals more favorable effects with atezolizumab combined with gemcitabine and cisplatin (GemCis) versus gemcitabine and carboplatin (GemCarbo). This study investigates the immunomodulatory effects of cisplatin as a potential explanation for these observations. Our findings indicate that improved outcomes with GemCis versus GemCarbo are primarily observed in patients with pretreatment tumors exhibiting features of restrained adaptive immunity. In addition, GemCis versus GemCarbo ± atezolizumab induces transcriptional changes in circulating immune cells, including upregulation of antigen presentation and T cell activation programs. In vitro experiments demonstrate that cisplatin, compared with carboplatin, exerts direct immunomodulatory effects on cancer cells, promoting dendritic cell activation and antigen-specific T cell killing. These results underscore the key role of immune modulation in cisplatin's efficacy in mUC and highlight the importance of specific chemotherapy backbones in immunotherapy combination regimens., Competing Interests: Declaration of interests M.D.G. has received grants or contracts from Bristol Myers Squibb, Novartis, Dendreon, AstraZeneca, and Merck; and has received consulting fees from Bristol Myers Squibb, Merck, Genentech Inc., AstraZeneca, Pfizer, EMD Serono, Seagen, Janssen, Numab, Dragonfly, GlaxoSmithKline, Basilea, UroGen, RapptaTherapeutics, Alligator, Silverback, Fujifilm, and Curis. X.G., D.R., A.S.R., H.M.S., R.B., K.Y., E.V., R.H., C.-J.H., Y.L., D.V., P.W., I.M., S.S., R.J., and S.M. are employees of Genentech Inc., and hold stocks or stock options in Roche/Genentech Inc. H.L. is an employee of Hoffmann-La Roche Ltd, Canada. L.W., J.Z., and H.Y. are employees of Sema4. E.H. was employed at Immunai during study conduct. R.H.H. and E. Kiner are employees of Immunai. A.B. has served as a consultant or advisor to Roche, Bristol Myers Squibb, MSD, and Pfizer; has received honoraria from Bristol Myers Squibb, MSD, and Pfizer; and has received educational and research grants from Pfizer, Pierre-Fabre, and Bristol Myers Squibb. M.D.S. has received consulting fees from AAA, Accord, Amgen, Astellas, AstraZeneca, Basilea, Bayer, Bioclin, Bristol Myers Squibb, Eisai, Ferring, Immunomedics, Ipsen, Janssen, MSD, Merck, Novartis, Pfizer, Pierre Fabre Oncology, Roche, Sandoz, Sanofi, and Seagen; has received payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from AAA, Accord, Amgen, Astellas, AstraZeneca, Basilea, Bayer, Bioclin, Bristol Myers Squibb, Eisai, Ferring, Immunomedics, Ipsen, Janssen, MSD, Merck, Novartis, Pfizer, Pierre Fabre Oncology, Roche, Sandoz, Sanofi, and Seagen; has received support for attending meetings and/or travel from AAA, Accord, Amgen, Astellas, AstraZeneca, Basilea, Bayer, Bioclin, Bristol Myers Squibb, Eisai, Ferring, Immunomedics, Ipsen, Janssen, MSD, Merck, Novartis, Pfizer, Pierre Fabre Oncology, Roche, Sandoz, Sanofi, and Seagen; has participated in a Data Safety Monitoring Board or Advisory Board for Roche, Orion, and CR-UK; and has other financial or non-financial interests by having worked on the ESMO guidelines on bladder cancer and the German S3 Leitlinie Blasenkarzinom. I.D.D. has participated in a Data Safety Monitoring Board or Advisory Board for Roche/Genentech Inc. (WO30070 [IMvigor130] international steering committee; unpaid) and Merck/Pfizer (APAC GU Advisory Board; unpaid: honoraria are invoiced by and paid directly to ANZUP Cancer Trials Group); and has a leadership or fiduciary role in the ANZUP Cancer Trials Group (as a Director and Board Chair; unpaid). J.A.A. has received third-party service as collaboration in the selection, and management of administrative requirements for the participation of SOGUG centers in the study from SOGUG during the conduct of the study; speaking or consulting fees from Bristol Myers Squibb, MSD, Roche, Astellas, Janssen Cilag, Pfizer, Novartis, and Bayer outside the submitted work; travel support from Bristol Myers Squibb, MSD, Roche, and Janssen Cilag outside the submitted work; research funding (from SOGUG) outside the submitted work from Bristol Myers Squibb, Novartis, and Pierre Fabre; and has participated in industry-sponsored clinical trials for Bristol Myers Squibb, MSD, Roche, Astellas, Janssen Cilag, Pfizer, and Novartis outside the submitted work. E. Kikuchi has received grants or contracts (through his institution) from Takeda, Nippon Kayaku, and Taiho; has received consulting fees from Chugai, Nippon Kayaku, MSD, Takeda, Pfizer, Astellas, Bristol Myers Squibb, and Yansen; has received payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from Takeda, Nippon Kayaku, and Taiho; and has received consulting fees from Chugai, Nippon Kayaku, MSD, Takeda, Taiho, AstraZeneca, Pfizer, Astellas, Bristol Myers Squibb, and Yansen. S.B. is an employee of Roche Products Ltd, UK, holds stocks or stock options in Roche, and has other financial or non-financial interests in Roche. C.L. is an employee of Roche Products Ltd, UK, holds stocks or stock options in Roche, and has other financial or non-financial interests in Roche. P.C.B. has participated in advisory boards for Janssen, Merck, Roche/Genentech Inc., Bristol Myers Squibb, Urogen, EMD Serono, Bayer, Astellas, AbbVie, AstraZeneca, Ferring, H3-Biomedicine, Sanofi, Pfizer, Prokarium, Protara Therapeutics, Stimit, and Verity; has received payment or honoraria as a speaker from Janssen, Bayer, Ferring, H3-Biomedicine, and Pfizer; has participated in clinical trials for Roche/Genentech Inc., Bristol Myers Squibb, and AstraZeneca; and has received non-financial support (scientific collaboration on bladder cancer genomics/transcriptomics) from Decipher Biosciences/Veracyte. E.G. has received grants or contracts from Astellas, AstraZeneca, Ipsen, Merck KGaA, and Pfizer; has received payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from Astellas, AstraZeneca, Bayer, Bristol Myers Squibb, Eisai, Esteve, Ipsen, Janssen, Lilly, Merck KGaA, MSD, Pfizer, Raffo, and Roche; and has received support for attending meetings and/or travel from Roche, AstraZeneca, Bristol Myers Squibb, Pfizer, and Merck KGaA., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In tumors, a subset of CD8 + T cells expressing the transcription factor TCF-1 drives the response to immune checkpoint blockade. We examined the mechanisms that maintain these cells in an autochthonous model of lung adenocarcinoma. Longitudinal sampling and single-cell sequencing of tumor-antigen specific TCF-1 + CD8 + T cells revealed that while intratumoral TCF-1 + CD8 + T cells acquired dysfunctional features and decreased in number as tumors progressed, TCF-1 + CD8 + T cell frequency in the tumor draining LN (dLN) remained stable. Two discrete intratumoral TCF-1 + CD8 + T cell subsets developed over time-a proliferative SlamF6 + subset and a non-cycling SlamF6 - subset. Blocking dLN egress decreased the frequency of intratumoral SlamF6 + TCF-1 + CD8 + T cells. Conventional type I dendritic cell (cDC1) in dLN decreased in number with tumor progression, and Flt3L+anti-CD40 treatment recovered SlamF6 + T cell frequencies and decreased tumor burden. Thus, cDC1s in tumor dLN maintain a reservoir of TCF-1 + CD8 + T cells and their decrease contributes to failed anti-tumor immunity., Competing Interests: Declaration of interests T.J. is a member of the Board of Directors of Amgen and Thermo Fisher Scientific. He is also a co-founder of Dragonfly Therapeutics and T2 Biosystems. T.J. serves on the Scientific Advisory Board of Dragonfly Therapeutics, SQZ Biotech, and Skyhawk Therapeutics. He is the President of Break Through Cancer. None of these affiliations represent a conflict of interest with respect to the design or execution of this study or interpretation of data presented in this manuscript. T.J. laboratory currently also receives funding from the Johnson & Johnson Lung Cancer Initiative and The Lustgarten Foundation for Pancreatic Cancer Research, but this funding did not support the research described in this manuscript. A.R. is a co-founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas, and was an SAB member of ThermoFisher Scientific, Syros Pharmaceuticals, Neogene Therapeutics and Asimov until July 31, 2020. From August 1, 2020, A.R. is an employee of Genentech., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The choroid plexus (ChP) in each brain ventricle produces cerebrospinal fluid (CSF) and forms the blood-CSF barrier. Here, we construct a single-cell and spatial atlas of each ChP in the developing, adult, and aged mouse brain. We delineate diverse cell types, subtypes, cell states, and expression programs in epithelial and mesenchymal cells across ages and ventricles. In the developing ChP, we predict a common progenitor pool for epithelial and neuronal cells, validated by lineage tracing. Epithelial and fibroblast cells show regionalized expression by ventricle, starting at embryonic stages and persisting with age, with a dramatic transcriptional shift with maturation, and a smaller shift in each aged cell type. With aging, epithelial cells upregulate host-defense programs, and resident macrophages upregulate interleukin-1β (IL-1β) signaling genes. Our atlas reveals cellular diversity, architecture and signaling across ventricles during development, maturation, and aging of the ChP-brain barrier., Competing Interests: Declaration of interests A.R. is a co-founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas, and was an SAB member of ThermoFisher Scientific, Syros Pharmaceuticals, Neogene Therapeutics, and Asimov until July 31, 2020. A.R. has been an employee of Genentech since August 1, 2020. O.R.-R. is an employee of Genentech and co-inventor on patent applications filed by the Broad related to single cell genomics. R.H.H. has been an employee of Immunai since August 16, 2020. A.R., R.H.H., N.H., M.K.L., N.D., and A.J. are co-inventors on a provisional patent application filed by the Broad Institute relating to this manuscript., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Regulatory T cells (T regs ) can impair anti-tumor immune responses and are associated with poor prognosis in multiple cancer types. T regs in human tumors span diverse transcriptional states distinct from those of peripheral T regs , but their contribution to tumor development remains unknown. Here, we use single-cell RNA sequencing (RNA-seq) to longitudinally profile dynamic shifts in the distribution of T regs in a genetically engineered mouse model of lung adenocarcinoma. In this model, interferon-responsive T regs are more prevalent early in tumor development, whereas a specialized effector phenotype characterized by enhanced expression of the interleukin-33 receptor ST2 is predominant in advanced disease. T reg -specific deletion of ST2 alters the evolution of effector T reg diversity, increases infiltration of CD8 + T cells into tumors, and decreases tumor burden. Our study shows that ST2 plays a critical role in T reg -mediated immunosuppression in cancer, highlighting potential paths for therapeutic intervention., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Neuroimmune interactions have emerged as critical modulators of allergic inflammation, and type 2 innate lymphoid cells (ILC2s) are an important cell type for mediating these interactions. Here, we show that ILC2s expressed both the neuropeptide calcitonin gene-related peptide (CGRP) and its receptor. CGRP potently inhibited alarmin-driven type 2 cytokine production and proliferation by lung ILC2s both in vitro and in vivo. CGRP induced marked changes in ILC2 expression programs in vivo and in vitro, attenuating alarmin-driven proliferative and effector responses. A distinct subset of ILCs scored highly for a CGRP-specific gene signature after in vivo alarmin stimulation, suggesting CGRP regulated this response. Finally, we observed increased ILC2 proliferation and type 2 cytokine production as well as exaggerated responses to alarmins in mice lacking the CGRP receptor. Together, these data indicate that endogenous CGRP is a critical negative regulator of ILC2 responses in vivo., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Genome-wide association studies (GWAS) have revealed risk alleles for ulcerative colitis (UC). To understand their cell type specificities and pathways of action, we generate an atlas of 366,650 cells from the colon mucosa of 18 UC patients and 12 healthy individuals, revealing 51 epithelial, stromal, and immune cell subsets, including BEST4 + enterocytes, microfold-like cells, and IL13RA2 + IL11 + inflammatory fibroblasts, which we associate with resistance to anti-TNF treatment. Inflammatory fibroblasts, inflammatory monocytes, microfold-like cells, and T cells that co-express CD8 and IL-17 expand with disease, forming intercellular interaction hubs. Many UC risk genes are cell type specific and co-regulated within relatively few gene modules, suggesting convergence onto limited sets of cell types and pathways. Using this observation, we nominate and infer functions for specific risk genes across GWAS loci. Our work provides a framework for interrogating complex human diseases and mapping risk variants to cell types and pathways., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In the small intestine, a niche of accessory cell types supports the generation of mature epithelial cell types from intestinal stem cells (ISCs). It is unclear, however, if and how immune cells in the niche affect ISC fate or the balance between self-renewal and differentiation. Here, we use single-cell RNA sequencing (scRNA-seq) to identify MHC class II (MHCII) machinery enrichment in two subsets of Lgr5 + ISCs. We show that MHCII + Lgr5 + ISCs are non-conventional antigen-presenting cells in co-cultures with CD4 + T helper (Th) cells. Stimulation of intestinal organoids with key Th cytokines affects Lgr5 + ISC renewal and differentiation in opposing ways: pro-inflammatory signals promote differentiation, while regulatory cells and cytokines reduce it. In vivo genetic perturbation of Th cells or MHCII expression on Lgr5 + ISCs impacts epithelial cell differentiation and IEC fate during infection. These interactions between Th cells and Lgr5 + ISCs, thus, orchestrate tissue-wide responses to external signals., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Intestinal epithelial cells absorb nutrients, respond to microbes, function as a barrier and help to coordinate immune responses. Here we report profiling of 53,193 individual epithelial cells from the small intestine and organoids of mice, which enabled the identification and characterization of previously unknown subtypes of intestinal epithelial cell and their gene signatures. We found unexpected diversity in hormone-secreting enteroendocrine cells and constructed the taxonomy of newly identified subtypes, and distinguished between two subtypes of tuft cell, one of which expresses the epithelial cytokine Tslp and the pan-immune marker CD45, which was not previously associated with non-haematopoietic cells. We also characterized the ways in which cell-intrinsic states and the proportions of different cell types respond to bacterial and helminth infections: Salmonella infection caused an increase in the abundance of Paneth cells and enterocytes, and broad activation of an antimicrobial program; Heligmosomoides polygyrus caused an increase in the abundance of goblet and tuft cells. Our survey highlights previously unidentified markers and programs, associates sensory molecules with cell types, and uncovers principles of gut homeostasis and response to pathogens.

Background: The merging of genomes in inter-specific hybrids can result in novel phenotypes, including increased growth rate and biomass yield, a phenomenon known as heterosis. Heterosis is typically viewed as the opposite of hybrid incompatibility. In this view, the superior performance of the hybrid is attributed to heterozygote combinations that compensate for deleterious mutations accumulating in each individual genome, or lead to new, over-dominating interactions with improved performance. Still, only fragmented knowledge is available on genes and processes contributing to heterosis., Results: We describe a budding yeast hybrid that grows faster than both its parents under different environments. Phenotypically, the hybrid progresses more rapidly through cell cycle checkpoints, relieves the repression of respiration in fast growing conditions, does not slow down its growth when presented with ethanol stress, and shows increased signs of DNA damage. A systematic genetic screen identified hundreds of S. cerevisiae alleles whose deletion reduced growth of the hybrid. These growth-affecting alleles were condition-dependent, and differed greatly from alleles that reduced the growth of the S. cerevisiae parent., Conclusions: Our results define a budding yeast hybrid that is perturbed in multiple regulatory processes but still shows a clear growth heterosis. We propose that heterosis results from incompatibilities that perturb regulatory mechanisms, which evolved to protect cells against damage or prepare them for future challenges by limiting cell growth.

Reversing the dysfunctional T cell state that arises in cancer and chronic viral infections is the focus of therapeutic interventions; however, current therapies are effective in only some patients and some tumor types. To gain a deeper molecular understanding of the dysfunctional T cell state, we analyzed population and single-cell RNA profiles of CD8(+) tumor-infiltrating lymphocytes (TILs) and used genetic perturbations to identify a distinct gene module for T cell dysfunction that can be uncoupled from T cell activation. This distinct dysfunction module is downstream of intracellular metallothioneins that regulate zinc metabolism and can be identified at single-cell resolution. We further identify Gata-3, a zinc-finger transcription factor in the dysfunctional module, as a regulator of dysfunction, and we use CRISPR-Cas9 genome editing to show that it drives a dysfunctional phenotype in CD8(+) TILs. Our results open novel avenues for targeting dysfunctional T cell states while leaving activation programs intact., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Finding the components of cellular circuits and determining their functions systematically remains a major challenge in mammalian cells. Here, we introduced genome-wide pooled CRISPR-Cas9 libraries into dendritic cells (DCs) to identify genes that control the induction of tumor necrosis factor (Tnf) by bacterial lipopolysaccharide (LPS), a key process in the host response to pathogens, mediated by the Tlr4 pathway. We found many of the known regulators of Tlr4 signaling, as well as dozens of previously unknown candidates that we validated. By measuring protein markers and mRNA profiles in DCs that are deficient in known or candidate genes, we classified the genes into three functional modules with distinct effects on the canonical responses to LPS and highlighted functions for the PAF complex and oligosaccharyltransferase (OST) complex. Our findings uncover new facets of innate immune circuits in primary cells and provide a genetic approach for dissection of mammalian cell circuits., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Ψ-seq for transcriptome-wide quantitative mapping of pseudouridine. We validate Ψ-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of unique sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUS) uncovers which pseudouridine synthase modifies each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided pseudouridine synthases. Upon heat shock in yeast, Pus7p-mediated pseudouridylation is induced at >200 sites, and PUS7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcriptome-wide scope for pseudouridine and methods to dissect its underlying mechanisms and function., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Aneuploidy, an abnormal number of chromosomes, is a widespread phenomenon found in unicellulars such as yeast, as well as in plants and in mammalians, especially in cancer. Aneuploidy is a genome-scale aberration that imposes a severe burden on the cell, yet under stressful conditions specific aneuploidies confer a selective advantage. This dual nature of aneuploidy raises the question of whether it can serve as a stable and sustainable evolutionary adaptation. To clarify this, we conducted a set of laboratory evolution experiments in yeast and followed the long-term dynamics of aneuploidy under diverse conditions. Here we show that chromosomal duplications are first acquired as a crude solution to stress, yet only as transient solutions that are eliminated and replaced by more efficient solutions obtained at the individual gene level. These transient dynamics of aneuploidy were repeatedly observed in our laboratory evolution experiments; chromosomal duplications gained under stress were eliminated not only when the stress was relieved, but even if it persisted. Furthermore, when stress was applied gradually rather than abruptly, alternative solutions appear to have emerged, but not aneuploidy. Our findings indicate that chromosomal duplication is a first evolutionary line of defense, that retains survivability under strong and abrupt selective pressures, yet it merely serves as a "quick fix," whereas more refined and sustainable solutions take over. Thus, in the perspective of genome evolution trajectory, aneuploidy is a useful yet short-lived intermediate that facilitates further adaptation.

Protein localization within cells can be achieved by the targeting and localized translation of mRNA. Yet, our understanding of the dynamics of mRNA targeting and protein localization, and of how general this phenomenon is, is not clear. Plasmid-based expression systems have been used to visualize exogenously expressed mRNAs and proteins; however, these methods typically produce them at levels greater than endogenous and can result in mislocalization. Hence, a method that allows for the simultaneous visualization of endogenous mRNAs and their translation products in living cells is needed. We previously developed a method (m-TAG) to localize endogenously expressed mRNAs in yeast by chromosomal insertion of the MS2 aptamer sequence between the open-reading frame (ORF) and 3' UTR of any gene. Upon coexpression with the MS2 RNA-binding coat protein (MS2-CP) fused with GFP, the aptamer-tagged mRNAs bearing their 3' UTRs are localized using fluorescence microscopy. Here we describe an advanced method (mp-TAG) that allows for the simultaneous visualization of both endogenously expressed mRNAs and their translation products in living yeast for the first time. Homologous recombination is used to insert the mCherry gene and MS2-CP binding sites downstream from any ORF, in order to localize protein and mRNA, respectively. As proof of the concept, we tagged ATP2 as a representative gene and demonstrated that endogenous ATP2 mRNA and protein localize to mitochondria, as shown previously. In addition, we demonstrate that tagged proteins like Hhf2, Vph1, and Yef3 localize to their expected subcellular location, while the localization of their mRNAs is revealed for the first time.