title: Large-scale discovery of chromatin dysregulation induced by oncofusions and other protein-coding variants

city: Madison

first: Max

last: Frenkel

address: 433 babcock drive

institute: UW Madison

summary: Although population-scale databases have expanded to millions of genetic variants, mechanistic insight into variant function has not kept pace. We present PROD-ATAC, a high-throughput method for discovering the effects of protein-coding variants on chromatin regulation. A pooled library of variants is expressed in a disease-agnostic biosensor cell line, and single-cell ATAC resolves each variant’s effect on chromatin. Using PROD-ATAC, we characterized the impacts of >100 oncogenic fusions and controls. We showed that pioneer activity is a common feature of fusions spanning an enormous range of frequencies. Further, fusion-induced dysregulation can be context-agnostic as observed mechanisms overlapped with cancer and cell-type specific prior knowledge. We also showed that gain-of-function pioneering is a common feature among oncofusions. This work provides a global view of fusion-induced chromatin disruption across many fusion frequencies and cancer types. Systematically dissecting this landscape revealed convergent mechanisms among seemingly disparate oncofusions and shared modes of chromatin dysregulation across different cancers.

overall_design: A pooled library of >100 oncofusions and controls was expressed in HEK293T cells where each cell contained one protein-coding perturbation. 10X Genomics' droplet platform was used to capture ~120,000 nuclei (~10,000 nuclei per sample for 12 samples) and Spear-ATAC was used to resolve the effects of each variant on chromatin regulation. Note in this case the 12 samples all dervied from the same biological library of cells and are in this way replicates used to increase the number of nuclei captured (but they are not different biological conditions).

first: Margaux

last: Hujoel

first: Zachary

last: Morris

first: Srivatsan

last: Raman


title: Pre-existing skin-resident CD8 and γδ T cell circuits mediate immune response in Merkel cell carcinoma and Predict immunotherapy efficacy

status: Public on Jul 24 2024

city: Chicago

first: Jaehyuk

last: Choi

state: Illinois

address: 303 E. Superior St. 5-220

department: Dermatology

country: USA

institute: Northwestern University

type: Expression profiling by high throughput sequencing

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Single nuclei RNAseq of the human periosteum from patients with congenital pseudarthrosis of the tibia

city: Créteil

first: Céline

last: Colnot

address: 8 rue du Général Sarrail

country: France

institute: INSERM U955

summary: Congenital pseudarthrosis of the tibia (CPT) is a severe pediatric disorder affecting children. CPT is characterized by tibial bowing at birth leading to spontaneous fracture and fibrous non-union. Our study showed the presence of biallelic inactivation of the NF1 gene in the periosteum at the pseudarthrosis site. To understand the impact of NF1 mutation on the periosteum, we performed single nuclei RNAseq of pathological periosteum collected from the pseudarthrosis site and of non-pathological periosteum collected from the iliac crest of 3 CPT patients.

overall_design: Snap frozen periosteum samples were defrosted and mechanically disrupted to extract nuclei. Isolated nuclei were sorted and processed using 10X genomics technology.

first: Simon

last: Perrin


title: Pre-existing skin-resident CD8 and γδ T cell circuits mediate immune response in Merkel cell carcinoma and Predict immunotherapy efficacy [bulkRNA-seq]

summary: Merkel Cell Carcinoma (MCC) is an aggressive skin cancer with limiterd treatment and low response rates to Immune Checkpoint Blockade (ICB) therapy. We compiled the largest sc database of MCC tumor samples and matched blood to identify tumor gener modules that Predict ICB response.

overall_design:
MCC tumor and matched blood samples were used for bulk and single-cell RNA-seq.

**Raw data is being deposited in dbGaP**

middle: Z

last: Reinstein

first: Yue

last: Zhang

first: Oscar

last: Ospina

first: Matt

last: Nichols

first: Victoria

last: Chu

first: Alvaro

last: de Mingo Pulido

first: Karol

last: Prieto

first: Johnathan

last: Ngyuen

first: Rui

last: Yin

first: Carlos

last: de Moran Segura

first: Ahmed

last: Usman

first: Brittney

last: Sell

first: Spencer

last: Ng

first: Janis

last: de la Iglesia

first: Sunandana

last: Chandra

first: Jeffery

last: Sosman

first: Raymond

middle: J

last: Cho

middle: B

last: Cheng

first: Ellie

middle: N

last: Ivanova

first: Sergei

last: Koralov

first: Robbert

last: Slebos

first: Christine

middle: H

last: Chung

first: Nikhill

middle: I

last: Khushalani

first: Jane

middle: L

last: Messina

first: Amod

middle: A

last: Sarnaik

middle: S

last: Zager

first: Vernon

middle: K

last: Sondak

first: Charles

last: Vaske

first: Sungjune

last: Kim

first: Andrew

last: Brohl

first: Xinlei

last: Min

first: Brian

middle: G

last: Pierce

first: Brooke

last: Fridley

first: Kenneth

middle: Y

last: Tsai


title: Microglia response upon demyelinating injury [snRNA-seq]

city: Munich

first: Vini

last: Tiwari

address: Feodor Lynen Straße 17

country: Germany

institute: Technical University Munich, (TUM), Deutsches Zentrum für Neurodegenerative Erkrankungen

summary: Following demyelinating injury, aged mice have poor recovery compared to young mice. We perforned snRNA seq on the white matter isolated from young and old mice after lesion induction to characterize the transcriptomic signature of glial cells with particular focus on microglia.

overall_design: Single nucleus RNA sequencing (snRNA seq) was performed on nuclei isolated from the white matter of young and old mice after lesion induction.

first: Bharat

last: Prajapati

first: Qing

last: Wang

first: Riki

last: Kawaguchi

first: Vivek

last: Swarup

first: Mikael

last: Simons


title: Single nuclei RNAseq of the bone fracture callus of Prss56-Nf1 KO mice

first: Céline

last: Colnot

summary: Periosteum deficiency affects bone regeneration, especially in the case of the bone disorder congenital pseudarthrosis of the tibia (CPT). We investigated a new mouse model of CPT caused by Nf1 inactivation in boundary cap-derivatives, the Prss56Cre; R26tdTom; Nf1fl/fl model. To investigate altered healing in this CPT model, we generated a dataset of the injured periosteum and fracture callus at day-7 post fracture from Prss56Cre; R26tdTom; Nf1fl/fl mice.

overall_design: Nuclei were extracted from uninjured periosteum, or injured periosteum and hematoma/callus at day 5 and 7 post-fracture. Nuclei were FACS-sorted and processed for single nuclei transcriptomics.

first: Simon

last: Perrin


title: Microglia response upon demyelinating injury [ATAC-seq]

type: Genome binding/occupancy profiling by high throughput sequencing

summary:
To understand the impaired transcriptional regulation of microglia/macropahges in aging following demyelinating injury.
Transcriptional regulation requires the repositioning of the nucleosomes around promoters and enhancers, thereby making chromatin accessible to transcription factors.

overall_design: ATAC (Assay for Transposase -Accessible Chromatin) sequencing was performed on CD11b+ cells isolated from the white matter of young and old mice at 4dpi after lesion induction.

first: Katarzyna

last: Leszczyńska

first: Jakub

last: Mieczkowski


title: Immune training in aged microglia [ChIP-seq]

summary: Aged mice were treated intravenously (i.v) with BCG or saline to study an effect  in microglia  for key histone modififications H3K4me3, H3K27ac involved in trained immune response.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for the histone modifications H3K4me3 and H3K27ac in Cd11b+ cells, isolated from mice treated with BCG or Control (Saline)


title: Effect of trained immune response upon demyelinating injury in brain [RNA-seq]

summary: To investigate the transcriptomic profile of cells involved in lesion recovery, we performed RNA seq on lesion tissue sections islaoted from mice treated with BCG or Saline

overall_design: RNA-seq of lesion tissue isolated from mice treated with BCG or saline 14 days post demyelinating injury.

first: Hao

last: Ji


title: Gene expression level of HEI-286 Schwann cells and MiaPaCa-2 pancreatic cancer cells

city: New York

first: Bic

last: MSKCC

state: NY

address: 1275 York Ave.

institute: Memorial SLoan-Kettering Cancer Center

summary:
We report the transcriptome analysis of HEI-286 human Schwann cells and MiaPaCa-2 pancreatic cancer cells
Nerves stimulate cancer progression for different cancer types, promoting both tumor growth and perineural invasion, a prominent process in pancreatic ductal adenocarcinoma. Yet little is known about how cells in the nerves contribute to cancer progression. Here we demonstrate that Schwann cells, the most abundant cell type in nerves, collectively function as tumor activated Schwann cell tracks (TASTs). Schwann cells in a 3D matrix organize into stellate tracks which serve as a conduit for cancer cell migration. TASTs actively promote pancreatic cancer dissemination during perineural invasion and neoneurogenesis. This key function of TASTs is induced by cancer cells triggering c-Jun activation of Schwann cells, analogous to the reprogramming that promotes nerve repair. In TASTs, dynamic Schwann cell processes wrap around and apply forces directly on cancer cells to enhance cancer motility. We define a model of cancer progression that relies upon collective Schwann cell behavior driven by c-Jun transcriptional reprogramming.

overall_design: Each samples are in quadruplate

first: Sylvie

last: Deborde

first: Richard

last: Wong


title: Chromatin-associated LTR RNA m5C oxidation by TET2 regulates leukemogenesis

city: Shanghai

first: Xiaoyang

last: Dou

address: 320 Yue Yang Road

country: China

institute: CAS Center for Excellence in Molecular Cell Science, CAS

type: Methylation profiling by high throughput sequencing

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

first: Zhongyu

last: Zou

first: Chuan

last: He


title: The IFITM5 mutation in osteogenesis imperfecta type V is associated with an ERK/SOX9-dependent osteoprogenitor differentiation defect

status: Public on Jul 23 2024

city: HOUSTON

first: Emily

last: Busse

state: TX

address: 1 Baylor Plaza

institute: Baylor College of Medicine

summary: Osteogenesis imperfecta (OI) type V is the second most common form of OI, distinguished by hyperplastic callus formation and calcification of the interosseous membranes in addition to bone fragility. All patients carry a dominant pathogenic variant (c.-14C>T) in IFITM5. Here, we generated a conditional Rosa26 knock-in mouse model to study the mechanistic consequences of the recurrent mutation. Expression of the mutant Ifitm5 in osteo-chondroprogenitor or chondrogenic cells resulted in low bone mass and growth retardation. Mutant limbs showed impaired endochondral ossification, cartilage overgrowth, and abnormal growth plate architecture. The cartilage phenotype correlates with the pathology reported in OI type V patients.  Surprisingly, expression of mutant Ifitm5 in matrix-synthesizing or mature osteoblasts caused no obvious skeletal abnormalities. In contrast, earlier expression in osteo-chondroprogenitors was associated with increase in the skeletal progenitor population within the periosteum. Mutant IFITM5 disrupts early skeletal homeostasis in part by activating ERK signaling and downstream SOX9 protein, and inhibition of these pathways partially rescued the phenotype in mutant animals.  .  Tracing of chondrogenic cells expressing the mutant Ifitm5 using the Ai9 reporter showed retention of Ai9+ cells in the growth plate and delayed migration to the bone shaft. RNA sequencing demonstrated enrichment for chondrogenic markers in the bone. These data identify the contribution of a defect in osteo-chondroprogenitor differentiation and not osteoblast function as a driver in the pathogenesis of OI type V.

overall_design: We performed bulk RNA sequencing in Rosa26mIfitm5;Acan-Cre ERT2 mouse femurs to search for downstream transcriptomic changes in the long bones following activation of the mutant Ifitm5 allele in chondrocytes from P10-15. 3 mutant mice and 3 control mice were analyzed.

first: Marom

last: Ronit

first: Lee

last: Brendan


title: ARID1A-BAF coordinates ZIC2 genomic occupancy for epithelial to mesenchymal transition in cranial neural crest lineage commitment.

city: London

first: Marco

last: Trizzino

state: UK

address: Imperial College Road

department: Life Sciences

country: United Kingdom

institute: Imperial College London

summary: The BAF chromatin remodeler regulates lineage commitment including cranial neural crest cell (CNCC) specification. BAF subunit mutations cause Coffin-Siris Syndrome (CSS), a congenital disorder characterized by distinct craniofacial features and intellectual disability. Approximately 50% of CSS patients carry mutations in one of the mutually exclusive BAF subunits, ARID1A/ARID1B. While Arid1a deletion in mouse neural crest causes severe craniofacial phenotypes, little is known about the role of ARID1A in CNCC specification. Using CSS patient-derived ARID1A+/- iPSCs to model CNCC specification, we discovered that ARID1A-haploinsufficency impairs epithelial to mesenchymal transition (EMT), a process necessary for CNCC delamination and migration from the neural tube. Furthermore, wild-type ARID1A-BAF regulates enhancers associated with EMT genes. ARID1A-BAF binding at these enhancers is impaired in heterozygotes, while binding at promoters is unaffected. At the sequence level, these EMT enhancers contain binding motifs for ZIC2, and ZIC2 binding at these sites is ARID1A-dependent. When excluded from EMT enhancers, ZIC2 relocates to neuronal enhancers, triggering aberrant neuronal gene activation. In mice, deletion of Zic2 impairs NCC delamination, while ZIC2 overexpression in chick embryos at pre-migratory neural crest stages elicits abnormal cell delamination from the neural tube. These findings reveal a novel ARID1A-ZIC2 axis essential for EMT and CNCC delamination.

overall_design:
RNA-seq of two Coffin Siris Syndrome patient-derived ARID1A-haploinsufficient iPSC lines and two control iPSC lines  (3 replicates per line)
RNA-seq of two Coffin Siris Syndrome patient-derived ARID1A-haploinsufficient cell lines and two control cell lines at day 10 of in vitro cranial neural crest cell specification (3 replicates per line)
RNA-seq of two Coffin Siris Syndrome patient-derived ARID1A-haploinsufficient cell lines and two control cell lines at day 14 of in vitro cranial neural crest cell specification in early maintenance media (3 replicates per line)
RNA-seq of two Coffin Siris Syndrome patient-derived ARID1A-haploinsufficient cell lines and two control cell lines at day 14 of in vitro cranial neural crest cell specification in late maintenance media (at least 2 replicates per line)
ChIP-seq of ZIC2 in two Coffin Siris Syndrome patient-derived ARID1A-haploinsufficient cell lines and two control cell lines at day 10 of in vitro cranial neural crest cell specification
ChIP-seq of H3K27ac in two Coffin Siris Syndrome patient-derived ARID1A-haploinsufficient cell lines and two control cell lines at day 10 of in vitro cranial neural crest cell specification
ChIP-seq of ARID1A in two Coffin Siris Syndrome patient-derived ARID1A-haploinsufficient cell lines and two control cell lines at day 10 of in vitro cranial neural crest cell specification
ATAC-seq of two Coffin Siris Syndrome patient-derived ARID1A-haploinsufficient cell lines and two control cell lines at day 10 of in vitro cranial neural crest cell specification (2 replicates per line)

first: Samantha

middle: M

last: Barnada

first: Aida

last: Giner de Gracia

first: Cruz

last: Morenilla-Palao

first: María

middle: T

last: López-Cascales

first: Chiara

last: Scopa

first: Francis

last: Waltrich

first: Maria

middle: E

last: Cicardi

first: Jonathan

last: Karlin

first: Davide

last: Trotti

first: Kevin

last: Peterson

last: Brugmann

first: Gijs

last: Santen

first: Steven

last: McMahon

first: Eloísa

last: Herrera


title: External magnetic-field driven delivery of exosomes modulates immune and metabolic changes of dystrophic muscle I

city: Milano

first: Yvan

last: Torrente

address: Via Festa del Perdono, 7

country: Italy

institute: Università degli Studi di Milano

summary: Exosomes are nanosized extracellular vesicles which have been recently demonstrated as promising agents for tissue repair/regeneration by inducing and guiding appropriate immune responses in dystrophic pathologies. Unfortunately, the accurate manipulation of exosomes by controlling their biodistribution still poses significant challenges. Here we overcome this limitation by developing an externally controlled delivery system for primed ANXA1 myo-exosomes (Exomyo). Effective nanocarriers are realized by immobilizing the Exomyo onto ferromagnetic nanotubes (NT-MAG) to achieve a controlled delivery and localization of Exomyo into skeletal muscles by an applied external magnetic field. Quantitative muscle-level analyses revealed that macrophages dominate the uptake of Exomyo from NT-MAG in vivo, to synergistically promote beneficial muscle responses in a murine animal model of Duchenne Muscular Dystrophy (DMD) thanks to the successful localization of therapeutic Exomyo upon systemic injection. These findings provide valuable insights into the development of exosome-based therapies for muscle diseases and in general highlight the formulation of effective functional nanocarriers aimed at optimizing exosome biodistribution.

overall_design: To characterize the molecular traits induced by repetitive magnetic delivery of anti-inflammatory exosomes Exomyo, we performed two main experiments. The first experiment involved three groups of mice that received intravenous magnetic NT-MAG-CFSE-ExoMyo infusion once (NT-MAG-Exomyo I), twice, one a week (NT-MAG-Exomyo II) or three times, once a week (NT-MAG-Exomyo III). In the second experiment, NT-MAG-Exomyo III was compared to a group receiving intravenous non-magnetic NT-CFSE-ExoMyo delivery three times, once a week (NT-ExoMyo III). A control group of mdx mice, receiving intravenous administration of saline for three weeks (vehicle), was included in each of the main experiments. After 30 days from the first injection, mice were sacrificed, and hind limb muscle tissues prepared for RNA sequencing analyses of single muscle fibers (MS) and freshly FACS-isolated circulating macrophages that infiltrate muscles and entrapped CFSE positive exosomes (Ly6ChiCFSEhi; iMAC).

last: Villa

first: Mirco

last: Macchi


title: External magnetic-field driven delivery of exosomes modulates immune and metabolic changes of dystrophic muscle II


title: Two-factor authentication underpins the precision of piRNA-directed LINE1 DNA methylatio

city: Edinburgh

first: Shaun

middle: Michael

last: Webb

address: 2.21 Swann Building, Kings Buildings

department: Wellcome Trust Centre for Cell Biology

institute: University of Edinburgh

summary: The PIWI-interacting RNA (piRNA) pathway guides the DNA methylation of young active transposons during male mouse germline development. piRNAs tether the PIWI protein MIWI2 (PIWIL4) to the nascent transposon transcript that results in DNA methylation through SPOCD1. Transposon methylation requires exacting precision: all copies need to be methylated yet, at the same time, off-target methylation must be avoided. However, the underlying mechanisms that ensure this precision remain unknown. Here, we show that SPOCD1 directly interacts with SPIN1 (SPINDLIN1), a chromatin reader that primarily binds H3K4me3 K9me3. The prevailing assumption is that all molecular events required for piRNA-directed DNA methylation occur after the engagement of MIWI2. Interestingly, we find that SPIN1 expression precedes that of both SPOCD1 and MIWI2. Furthermore, we demonstrate that young LINE1s, but not old copies are marked by H3K4me3, H3K9me3 and SPIN1 prior to the initiation of piRNA-directed DNA methylation. We generated a Spocd1 separation-of-function allele in the mouse that encodes a SPOCD1 variant that no longer interacts with SPIN1. We found that the SPOCD1-SPIN1 interaction is essential for spermatogenesis and piRNA-directed DNA methylation of young LINE1 elements. We propose that young LINE1 elements require a two-factor authentication process for DNA methylation, the first being the recruitment of SPIN1-SPOCD1 to licence the locus and the second is MIWI2 engagement with the nascent transcript, which is also the trigger for methylation. In summary, independent events that licence, and trigger methylation underpin the basis of precision.

overall_design: CUT&TAG sequencing of histone modifications H3K4me3 and H3K9me3 as well as the protein SPIN1 in E14.5 foetal germ cells with corresponding IgG controls.

first: Madeleine

last: Heep

first: Ansgar

last: Zoch

first: Jessica

last: Leismann

first: Rebecca

middle: V

last: Berrens

first: Devisree

last: Valsakumar

first: Yuka

last: Kabayama

first: Martina

last: Schito

first: Tamoghna

last: Chowdhury

first: David

last: MacLeod

first: Xinyu

last: Xiang

first: Juan

last: Zou

first: Juri

last: Rappsilber

first: Robin

middle: C

last: Allshire

first: Philipp

last: Voigt

first: Atlanta

last: Cook

first: Joan

last: Barau

first: Dónal

last: O’Carroll


title: External magnetic-field driven delivery of exosomes modulates immune and metabolic changes of dystrophic muscle

first: Yvan

last: Torrente


title: Administration of anti-HIV-1 broadly neutralizing monoclonal antibodies with increased binding affinity to Fcg receptors during acute SHIV infection shapes innate and adaptive cellular immunity [set1]

first: Slim

last: Fourati

address: 300 E Superior St

department: Medicine

summary: Anti-HIV-1 broadly neutralizing antibodies (bNAbs) have the dual potential of mediating virus neutralization and antiviral effector functions through their Fab and Fc domains, respectively. So far, bNAbs with enhanced Fc effector functions in vitro have only been tested in NHPs during chronic simian-HIV (SHIV) infection. Here, we investigated the effects of administering in acute SHIVAD8-EO infection either wild-type (WT) bNAbs or bNAbs carrying the S239D/I332E/A330L (DEL) mutation, which increases binding to FcgRs. Emergence of plasma and lymph node (LN) virus was delayed in bNAb-treated monkeys and occurred earlier in monkeys given DEL bNAbs than in those given WT bNAbs, consistent with faster clearance of DEL bNAbs from plasma. DEL bNAb-treated monkeys had higher levels of circulating virus-specific IFNg single-producing and IFNg/MIP-1b double-producing CD8+ CD69+ T cells than the other groups. In LNs, WT bNAbs were evenly distributed between follicular and extrafollicular areas, but DEL bNAbs predominated in the latter. At week 8 post-challenge, LN monocytes and NK cells from DEL bNAb-treated monkeys upregulated proinflammatory signaling pathways and LN T cells downregulated TNF signaling via NF-kB. Overall, bNAbs with increased binding affinity to FcgRs shaped innate and adaptive cellular immunity, which may be important to consider in future strategies of passive bNAb therapy.

overall_design: Bulk mRNA-Seq of 175 samples collected from 17 rhesus macaques infected with simian-HIV and then treated with a wild-type broadly neutralizing antibody (bNAb; VRC07-523-LS, n=6), a bNAb with the DEL mutation (n=6) or kept untreated (n=5). Lymph nodes were collected at three timepoints (pre-infusion, day 14 and week 8) and immune cells from five subsets were sorted and profilled by mRNA-Seq.

first: Rafick-Pierre

last: Sekaly

first: Daniel

last: Douek

first: Joana

last: Dias

first: Richard

last: Koup


title: RNA sequencing analysis of 5637 cells with D-xyl@epsilonPLCDs

city: Tianjin

first: Chunhui

last: Miao

address: 22 Qixiangtai Road, Heping District

department: Immunology

institute: Tianjin Medical University

summary: We designed D-xylose-modified, epsilon-poly-L-lysine-based carbon dots (D-xyl@epsilonPLCDs) to effectively inhibit intracellular UPEC. D-xyl@εPLCDs effectively entered cells, reduced bacterial adhesion and promoted intracellular UPEC clearance. To investigate the regulation of immune responses in bladder epithelial 5637 cells after treatment with D-xyl@epsilonPLCDs, RNA sequencing analysis were performed.

overall_design: Human bladder epithelial 5637 cells were treated with either epsilonPLCDs, D-xyl@epsilonPLCDs or sterilized water for 3 hours. The cells were harvested and subjected to RNA extraction using TRIzol methods followed by deep RNA sequencing. Subsequently, we performed gene expression profile analysis based on RNA sequencing data obtained from 5637 cells in three groups (Control, epsilonPLCDs and D-xyl@epsilonPLCDs).

first: Quan

last: Wang

first: Xiaojuan

first: Yajie

last: Zhang


title: Cancer cell stiffening via CoQ10 and UBIAD1 regulates ECM signaling and ferroptosis resistance in Breast Cancer

city: Padova

first: Giovanni

last: Tosi

state: Padova

address: Via Ugo Bassi 58/B

institute: University of Padova

summary: Here, we discovered that CoQ10 and its biosynthetic enzyme UBIAD1 play a critical role in plasma membrane mechanical properties that are of interest for breast cancer (BC) progression and treatment. In detail, CoQ10 and UBIAD1 increased membrane fluidity leading to increased cell stiffness in BC. High UBIAD1/CoQ10 states impair lipid rafts and ECM-mediated oncogenic signaling and reduce the ferroptosis resistance of BC cells. Furthermore, analyses on BC patients and BC mouse models reveal that UBIAD1 loss is associated with BC development and progression. UBIAD1 expression in aggressive BC limits CTCs survival and lung metastasis formation in experimental xenograft assays.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for MDA-MB-231 Scramble (alias "553") and UBIAD1-overexpressing cells (alias "561"). One week after cell transduction, 4 biological replicates were analyzed per condition. Stable overexpression was obtain with CRISPR-dCas9 technology (PMID: 28333914) using lentiviral vectors.

first: Massimo

last: Santoro


title: RNA sequencing analysis of bladder tissue from CFT073-infected C57B/6J mice with or without treatments of D-xyl@epsilonPLCDs and epsilonPLCDs

summary: We designed D-xylose-modified, epsilon-poly-L-lysine-based carbon dots (D-xyl@epsilonPLCDs) to effectively inhibit intracellular UPEC. Treatment with D-xyl@epsilonPLCDs ultimately resulted in a reduction of UPEC in the bladders of mouse UTIs model. To investigate the regulation of immune responses in bladders of CFT073-infected mice post-D-xyl@epsilonPLCDs treatment, RNA sequencing analysis were performed.

overall_design: RNA sequencing was conducted to investigate the regulation of immune response in the bladder of CFT073-infected mice following treatment with D-xyl@epsilonPLCDs. Eight-week-old female C576B/J mice were infected with CFT073 for 6 hours and subsequently treated overnight with either epsilonPLCDs, D-xyl@epsilonPLCDs or sterilized water. The bladders were harvested, rinsed with 75% ethanol, and subjected to RNA extraction using TRIzol methods followed by deep RNA sequencing. Subsequently, we performed gene expression profile analysis based on RNA sequencing data obtained from the bladders of three groups of mice (Control, epsilonPLCDs and D-xyl@epsilonPLCDs), with five mice per group.


title: Administration of anti-HIV-1 broadly neutralizing monoclonal antibodies with increased binding affinity to Fcg receptors during acute SHIV infection shapes innate and adaptive cellular immunity [set2]

overall_design: Bulk mRNA-Seq of 391 samples collected from 12 rhesus macaques infected with simian-HIV and then treated with a wild-type broadly neutralizing antibody (bNAb; VRC07-523-LS, n=4), a bNAb with the DEL mutation (n=4) or kept untreated (n=4). Lymph nodes were collected at three timepoints (pre-infusion, day 14 and week 8) and 11 subsets of T cells were sorted and profilled by mRNA-Seq.


title: Cancer-cell-secreted miR-204-5p induces leptin signaling pathway in white adipose tissue to promote cancer-associated cachexia

city: Wuhan

first: Yan

last: Wei

address: Bayi road 229

institute: wuhan university

type: Non-coding RNA profiling by high throughput sequencing

summary: Cachexia is a wasting disorder of adipose tissues that leads to profound weight loss and frailty. One key characteristic of cachexia is elevated resting energy expenditure, which has been linked to increased fat lipolysis and thermogenesis.

overall_design: Comparative miRNA expression profiling analysis of small RNA deep data sequencing for EVs

first: Yong

last: Hu

first: Wei

last: Yan


title: The impact of the embryonic DNA methylation program on CTCF-mediated genome regulation

city: Paris

first: Julien

last: Richard Albert

address: 15 Rue Hélène Brion

institute: Institut Jacques Monod, CNRS, Université de Paris

summary: During mammalian embryogenesis, both the 5-cytosine DNA methylation (5meC) landscape and three dimensional (3D) chromatin architecture are profoundly remodeled during a process known as 'epigenetic reprogramming.' An understudied aspect of epigenetic reprogramming is how the 5meC flux, per se, affects the 3D genome. This is pertinent given the 5meC-sensitivity of DNA binding for a key regulator of chromosome folding: CTCF. We profiled the CTCF binding landscape using a mouse embryonic stem cell (ESC) differentiation protocol that models embryonic 5meC dynamics. Mouse ESCs lacking DNA methylation machinery are able to exit naive pluripotency, thus allowing for dissection of subtle effects of CTCF on gene expression. We performed CTCF HiChIP in both wild-type and mutant conditions to assess gained CTCF-CTCF contacts in the absence of 5meC. We performed H3K27ac HiChIP to determine the impact that ectopic CTCF binding has on cis-regulatory contacts. Using 5meC epigenome editing, we demonstrated that the methyl-mark is able to impair CTCF binding at select loci. Finally, a detailed dissection of the imprinted Zdbf2 locus showed how 5meC-antagonism of CTCF allows for proper gene regulation during differentiation. This work provides a comprehensive overview of how 5meC impacts the 3D genome in a relevant model for early embryonic events.

overall_design: HiChIP, CUT&RUN and 4C was performed on mouse embryonic stem cells and epiblast-like cells for CTCF. H3K27ac HiChIP was also performed on the same cells. 4C-seq was conducted on TET-1 epigenome-edited epiblast-like cells.

first: Ana

last: Monteagudo-Sánchez

first: Margherita

last: Scarpa

first: Daan

last: Noordermeer

first: Maxim

last: Greenberg


title: Engineered T cell therapy for central nervous system injury [CD45+ scRNA-seq]

city: Saint Louis

first: Jonathan

last: Kipnis

state: MO

address: 4515 McKinley Ave

department: Pathology and Immunology

institute: Washington University in Saint Louis

summary: Traumatic injuries to the central nervous system (CNS) affect millions of people worldwide yet lack an effective treatment. These injuries contain infiltrating immune cells that can promote tissue repair and could be exploited for therapeutic benefit. Here, using single-cell RNA-sequencing of T cells infiltrating the injured CNS we demonstrate their clonal expansion and antigen specificity towards CNS derived self-peptides. We confirm the beneficial effect of these injury-associated autoimmune CD4+ T cells in murine models of optic nerve and spinal cord injury. Subsequently, using mRNA-based transient T cell receptor (TCR) reconstitution, we demonstrate a therapeutic T cell strategy to alleviate CNS injury. Treatment of CNS-injured mice with this therapy improved locomotion and alleviated histological signs of damage, through regulation of myeloid cells, without detrimental autoimmune side effects. This strategy provides a means of developing custom-designed T cell therapies for CNS injury, and possibly for other neurodegenerative disorders.

overall_design: Mice were induced with spinal cord injury followed immediately by injection of Cp-T cells or PBS as control. The injured portion of spinal cord (injury site ±0.5 mm) was collected for cell isolation 7 days after injury. Mice were injected with a lethal dose of Euthasol (10% vol/vol) and transcardially perfused with PBS containing 0.025% heparin. For spinal cord isolation, the spine was cut on both sides and the spinal cord was flushed out using a PBS filled syringe with 18-gauge needle. Spinal cord was chopped using a surgical blade and digested in digestion buffer (RPMI-1640 medium supplemented with 2% FBS, 1 mg mL-1 Collagenase VIII, and 0.5 mg mL-1 of DNase I) at 37 °C for 30 minutes, triturated with a 1 mL pipette, and digested for another 15 minutes. After digestion, enzymes were neutralized with RPMI with 10% FBS and tissue samples were mechanically homogenized and filtered through 70 μm cell strainer. Cells were then centrifuged at 450 g for 4 minutes and the supernatant was removed. Myelin was removed by resuspending cells with 30% percoll or 10-15% BSA in PBS and centrifuged at 800 g for 10 minutes with a slow brake. After centrifugation, the upper myelin-containing layer and supernatant were removed, and the cell pellet was resuspended with RPMI containing 10 % FBS. Red blood cells were lysed with ammonium-chloridepotassium (ACK) lysis buffer for 1 minute at room temperature and neutralized with the same volume of RPMI containing 10% of FBS. Cells were then centrifuged and cell pellets were resuspended with RPMI containing 10% FBS and kept on ice until use. CD45 Microbeads UltraPure (130-052-301, Miltenyi Biotec) and MACS LS columns (Miltenyi Biotec) were used as per manufacturer’s instructions to isolate the CD45+ population. Isolated CD45+ cells were resuspended in PBS containing 0.04% BSA. Cell number and viability were determined using Viastain AOPI Staining Solution (CS2-0106, Nexcelom Bioscience) and CellDrop FL Fluorescence Cell Counter (DeNovix). Sample loading and library construction were performed using the 10X Genomics Chromium platform and Chromium Single Cell 3’ Library & Gel Bead Kit version 3.

first: Wenqing

last: Gao

first: Taitea

last: Dykstra


title: Engineered T cell therapy for central nervous system injury [spinal cord TCR scRNA-seq]

overall_design: The injured portion of spinal cord (injury site ±0.5 mm) was collected for T cell isolation 2 weeks after injury. Mice were injected with a lethal dose of Euthasol (10% vol/vol) and transcardially perfused with PBS containing 0.025% heparin. For spinal cord isolation, the spine was cut on both sides and the spinal cord was flushed out using a PBS filled syringe with 18-gauge needle. Spinal cord was chopped using a surgical blade and digested in digestion buffer (RPMI-1640 medium supplemented with 2% FBS, 1 mg mL-1 Collagenase VIII, and 0.5 mg mL-1 of DNase I) at 37 °C for 30 minutes, triturated with a 1 mL pipette, and digested for another 15 minutes. After digestion, enzymes were neutralized with RPMI with 10% FBS and tissue samples were mechanically homogenized and filtered through 70 μm cell strainer. Cells were then centrifuged at 450 g for 4 minutes and the supernatant was removed. Myelin was removed by resuspending cells with 30% percoll or 10-15% BSA in PBS and centrifuged at 800 g for 10 minutes with a slow brake. After centrifugation, the upper myelin-containing layer and supernatant were removed, and the cell pellet was resuspended with RPMI containing 10% FBS. Red blood cells were lysed with ammonium-chloridepotassium (ACK) lysis buffer for 1 minute at room temperature and neutralized with the same volume of RPMI containing 10% of FBS. Cells were then centrifuged and cell pellets were resuspended with RPMI containing 10% FBS and kept on ice until use. Single-cell suspensions were washed with PBS. Fc receptors were blocked for 5 min with the anti-CD16/CD32 antibody cocktail and cells were incubated with primary antibody cocktails for 10 minutes at room temperature for surface staining. Antibodies were all diluted in FACS buffer (2% BSA, 1 mM EDTA, 20 mM HEPES in PBS). After staining, samples were washed, resuspended with FACS buffer (with DAPI if needed), filtered with 70 μm cell strainer and sorted using a BD Biosciences FACSAria II or BD Influx cell sorter. Viable T cells (DAPI- CD45+ Thy1.2+ CD4+ or CD8+) were sorted into a 1.5mL Eppendorf tube with RPMI medium containing 10% FBS. CD4+ and CD8+ T cells were combined. T cells were washed, counted, and resuspended with PBS containing 0.04% BSA. T cell samples were loaded onto a 10X Genomics Chromium platform for Gel Bead-in Emulsions (GEMs) and Mouse T cell Chromium Single Cell V(D)J Enrichment Kit and Chromium Single Cell 5’ Library & Gel Bead Kit were used separately to generate a cDNA library targeting the TCR and for transcriptome expression.


title: An RdDM-independent function of Pol V transcripts in gene regulation and plant defense

city: Beiing

first: Yijun

last: Qi

address: NO.1 Qinghuayuan

department: School of Life Sciences

institute: Tsinghua University

summary: RNA polymerase V (Pol V) and Pol IV are known to be specialized for RNA-directed DNA methylation (RdDM). Here, we report that Pol V, but not Pol IV, regulates hundreds of genes including JA-responsive genes and confers plant defense to Botrytis cinerea and Spodoptera exigua. About half of the Pol V-regulated genes are associated with Pol V transcripts (PVTs). We thus hypothesized that some PVTs could regulate gene expression in an RdDM-independent manner. To test this hypothesis, we studied three PVTs, PVT-ERF5a/b and PVT-ERF6, as models. PVT-ERF5a/b and PVT-ERF6 are transcribed from the upstream regions of ERF5 and ERF6 and positively regulate their transcription, thereby regulating plant defense. We further show that such regulation involves PVT-dependent H3K4me3 deposition and requires the DRD1-DMS3-RDM1 complex that mediates Pol V recruitment to the target loci. Our findings highlight an unprecedented role for PVTs in regulating gene transcription, apart from serving as scaffold RNAs to direct DNA methylation.

overall_design: RNA-seq of 2-week-old seedlings of Col-0, nrpd1-3, nrpe1-11

first: Yuxiang

last: Yuan

first: Yujie

last: Liu

first: Lu

last: Han

last: Li


title: Engineered T cell therapy for central nervous system injury [control tissues TCR scRNA-seq]

overall_design: To FACS sort T cells, mice were given a lethal dose of anesthetics by i.p. euthasol (10% v/v) and transcardially perfused with ice-cold 0.025% (w/v) heparin in PBS. Before perfusion, peripheral blood was collected by retro-orbital bleeding (200-300ul) and diluted 1:1 with PBS containing heparin. Red blood cells were lysed twice by resuspension in ACK lysis buffer for 5 min at RT. Cells were washed with FACS buffer (2 mM EDTA, 25 mM HEPES, 1% BSA in 1X PBS) before being re-suspended for immunostaining. After perfusion, meninges and diaphragm were digested for 20 and 30 min, respectively, at 37 °C with 1.4 U/mL of Collagenase VIII (Sigma Aldrich) and 35 U/mL of DNAse I (Sigma Aldrich) in IMDM (Sigma Aldrich) media. Following the digestion step, the tissues were gently pressed through 70 μm nylon mesh cell strainers with a sterile plastic plunger. Cells were then centrifuged at 450g at 4°C for 4:30 min. The cell pellets were resuspended in ice-cold FACS buffer and stained at 1:300 dilution. The single-cell suspensions were immunostained with Live/Dead Fixable Aqua Dead (Biolegend; #Cat: 423102), anti-CD45 APC (BD Bioscience; Clone: 30-F11; #Cat: 559864), anti-TCR FITC (Biolegend; Clone: H57-597; #Cat: 109206), anti-CD4 PE (BD Bioscience; Clone: RM4-5; #Cat: 553049), and anti-CD8a PE (eBioscience; Clone: 53-6.7; #Cat: 12-0081-81) using the BD Influx Cell Sorter (BD Biosciences). CD4+ and CD8+ T cells were combined into a single tube (CD45+TCR +CD4+ and CD45+TCR +CD8+, respectively). Cells were sequenced in 4 separate runs to improve sequencing depth via 5' chemistry.


title: Engineered T cell therapy for central nervous system injury [FACS-Tcells]

overall_design: CD4+ T cells with retrovirus-based TCR-Cp and GFP expression were injected intravenously into the mice after injury. The injured part of spinal cord (injury site ±0.5 mm) and spleen was collected for T cell isolation 2 weeks after injury. Mice were injected with a lethal dose of Euthasol (10% vol/vol) and transcardially perfused with PBS containing 0.025% heparin. Spleen were directly collected using forceps. For spinal cord isolation, the spine was cut on both sides and the spinal cord was flushed out using a PBS filled syringe with 18-gauge needle. Spinal cord was chopped using a surgical blade and digested in digestion buffer (RPMI-1640 medium supplemented with 2% FBS, 1 mg mL-1 Collagenase VIII, and 0.5 mg mL-1 of DNase I) at 37 °C for 30 minutes, triturated with a 1 mL pipette, and digested for another 15 minutes. After digestion, enzymes were neutralized with RPMI with 10% FBS and tissue samples were mechanically homogenized and filtered through 70 μm cell strainer. Cells were then centrifuged at 450 g for 4 minutes and the supernatant was removed. Myelin was removed by resuspending cells with 30% percoll or 10-15% BSA in PBS and centrifuged at 800 g for 10 minutes with a slow brake. After centrifugation, the upper myelin-containing layer and supernatant were removed, and the cell pellet was resuspended with RPMI containing 10 % FBS. Spleen was directly minced to get single lymphocyte. Red blood cells were lysed with ammonium-chloridepotassium (ACK) lysis buffer for 1 minute at room temperature and neutralized with the same volume of RPMI containing 10% of FBS. Cells were then centrifuged and cell pellets were resuspended with RPMI containing 10% FBS and kept on ice until use. Single-cell suspensions were washed with PBS. Fc receptors were blocked for 5 min with the anti-CD16/CD32 antibody cocktail and cells were incubated with primary antibody cocktails for 10 minutes at room temperature for surface staining. Antibodies were all diluted in FACS buffer (2% BSA, 1 mM EDTA, 20 mM HEPES in PBS). After staining, samples were washed, resuspended with FACS buffer (with DAPI if needed), filtered with 70 μm cell strainer and sorted using a BD Biosciences FACSAria II or BD Influx cell sorter. Cp-T cells (DAPI- CD45+ CD11b- TCRβ+ CD4+ CD8- GFP+) from the injured spinal cord and spleen were sorted into individual wells of a 96-well plate with 10X lysis buffer (635013, TaKaRa) and 5% RNase inhibitor (03335399001, Millipore Sigma). Plates was sealed with adhesive plate seal, frozen rapidly over dry ice after sorting, and stored at -80 °C until sequencing.


title: A developmental mechanism to Regulate Alternative Polyadenylation in an Adult Stem Cell Lineage [RNA-seq]

status: Public on Jul 22 2024

city: Stanford

first: Lorenzo

last: Gallicchio

state: california

address: Campus Drive W

department: developmental Biology

institute: Stanford University

summary: Alternative Cleavage and Polyadenylation (APA) often results in production of mRNA isoforms with either longer or shorter 3’UTRs from the same genetic locus, potentially impacting mRNA translation, localization and stability. Developmentally regulated APA can thus make major contributions to cell-type-specific gene expression programs as cells differentiate. During Drosophila spermatogenesis, approximately 500 genes undergo APA when proliferating spermatogonia differentiate into spermatocytes, producing transcripts with shortened 3’ UTRs, leading to profound stage-specific changes in the proteins expressed. The molecular mechanisms that specify usage of upstream polyadenylation sites in spermatocytes are thus key to understanding the changes in cell state. Here, we show that upregulation of PCF11 and Cbc, the two components of Cleavage Factor II (CFII), orchestrates APA during Drosophila spermatogenesis. Knock down of PCF11 or cbc in spermatocytes caused dysregulation of APA, with many transcripts normally cleaved at a proximal site in spermatocytes now cleaved at their distal site, as in spermatogonia. Forced overexpression of CFII components in spermatogonia switched cleavage of some transcripts to the proximal site normally used in spermatocytes. Our findings reveal a developmental mechanism where changes in expression of specific cleavage factors can direct cell-type-specific APA at selected genes.

overall_design: RNAseq was performed on Control testes (bamGal4) and in testes in which PCF11 or cbc were knocked down in spermatocytes using bamGa4. Libraries were performed in duplictaes. Per each library roughly 150 pairs of testes were used.

first: Neruza

middle: R

last: Matias


title: Transcriptomic profiling of Schlemm’s canal cells reveals a lymphatic-biased identity and three major cell states

first: Revathi

last: Balasubramanian

address: 635 West 165th street

department: Ophthalmology

institute: Columbia University

summary: Schlemm’s canal (SC) is central in intraocular pressure regulation but requires much characterization. It has distinct inner and outer walls, each composed of Schlemm’s canal endothelial cells (SECs) with different morphologies and functions. Recent transcriptomic studies of the anterior segment added important knowledge, but were limited in power by SEC numbers or did not focus on SC. To gain a more comprehensive understanding of SC biology, we performed bulk RNA sequencing on C57BL/6J SC, blood vessel and lymphatic endothelial cells from limbal tissue (∼4500 SECs). We also analyzed mouse limbal tissues by single-cell and single-nucleus RNA sequencing (C57BL/6J and 129/Sj strains), successfully sequencing 903 individual SECs. Together, these datasets confirm that SC has molecular characteristics of both blood and lymphatic endothelia with a lymphatic phenotype predominating. SECs are enriched in pathways that regulate cell-cell junction formation pointing to the importance of junctions in determining SC fluid permeability. Importantly, and for the first time, our analyses characterize 3 molecular classes of SECs, molecularly distinguishing inner wall from outer wall SECs and discovering two inner wall cell states that likely result from local environmental differences. Further, and based on ligand and receptor expression patterns, we document key interactions between SECs and cells of the adjacent trabecular meshwork (TM) drainage tissue. Also, we present cell type expression for a collection of human glaucoma genes. These data provide a new molecular foundation that will enable the functional dissection of key homeostatic processes mediated by SECs as well as the development of new glaucoma therapeutics.

overall_design: The anterior segment of the eye including the limbus was dissected from each of ten 3-month-old Prox1-GFP mice to make 1 biological replicate of each cell type. The iris, cornea, and ciliary body were gently and carefully removed using forceps to prevent damage to the delicate aqueous humor drainage structures. A strip of limbal tissue 1-2 mm wide consisting of the aqueous humor outflow tissues SC and TM, limbal blood vessels, and lymphatics was dissected from each anterior segment. Limbal strips were dissected in ice-cold PBS. Strips from 20 eyes were pooled and dissociated paying strict attention to osmolarity (290-300 mOSm) and mechanical force, using 1mg/ml Collagenase Type 4 (Worthington Biochemical Cat. no. LS004188) to preserve membrane proteins for the staining cell surface marker for flow cytometry.  Single-cell suspension was stained with Lyve1 conjugated to eFluor 660 (ALY7 eBioscience Cat. no. 14-0443-82), Endomucin conjugated to phycoerythrin (eBio V.7.C7 Cat. no.14-5851-82), and propidium iodide to mark dead cells. Forward and side scatter gates were first used to eliminate events with low scatter which include derbies, cell fragments and pyknotic cells. Then propidium iodide positive dead cells were gated out. Further gating on the viable cells was applied such that distinct population of cells were isolated a) SECs -GFP+Lvye1-collected as, b) lymphatic endothelial cells (LECs)-GFP+ Lyve1+, c) GFP- blood endothelial cells (BECs)-Endomucin+. A total of 1500-1700 cells for SEC, 200-300 cells for LEC and 2000-2500 cells for BEC were collected. Three biological replicates were processed for each of SECs, BECs, and LECs.

first: Balasubramanian

last: Revathi

first: Kizhatil

last: Krishnakumar

first: Li

last: Taibo

first: John

last: Simon


title: Transcription factor network dynamics during the commitment to oncogene-induced senescence [RNA-Seq]

city: Piscataway

first: Ricardo

middle: Iván

last: Martínez Zamudio

state: New Jersey

address: 675 Hoes Lane West

department: Pharmacology

institute: Rutgers Biomedical and Health Sciences | Rutgers-Robert Wood Johnson Medical School

summary: Aberrant oncogenic signaling causes cells to transition into oncogene-induced senescence (OIS) to limit uncontrolled proliferation. Despite being a potent tumor suppressor mechanism, OIS is an unstable cell state susceptible to reprogramming that can promote tumorigenesis. Therefore, elucidating the underlying gene regulatory mechanisms that commit cells to OIS is critical to identifying actionable targets to modulate the senescence state. We previously showed that timely execution of the OIS program is governed by hierarchical transcription factor (TF) networks. However, the gene regulatory mechanisms that prime cells to commit to the OIS fate early upon oncogene hyperactivation are currently not known. Here, we leveraged our time-resolved multi-omic profiling approach to generate TF networks during the first 24 h of oncogenic HRASG12V activation. Using this approach, we demonstrate that the commitment to OIS requires the rearrangement of the TF network on a pre-established epigenomic landscape, priming the cells for the substantial chromatin remodeling that underpins the transition to OIS. Our results provide a detailed map of the chromatin landscape before cells transition to OIS thus offering a platform for manipulation of senescence outcomes of potentially therapeutic value.

overall_design: RNA seq of WI38-ER:RAS(G12V) cells collected at indicated time-points post induction of oncogenic HRAS(G12V) with 4-hydroxy tamoxifen (4-OHT) treatment.

first: Themistoklis

last: Vasilopoulos


title: T cell receptor β-chains display abnormal shortening and repertoire sharing in type 1 diabetes

city: Hinxton

first: Anna

last: Lorenc

address: Genome Campus

department: Human Genetics

institute: Wellcome Trust Sanger Institute

summary: Defects in T cell receptor (TCR) repertoire are proposed to predispose to autoimmunity. Here we show, by analyzing >2×108 TCRB sequences of circulating naive, central memory, reg- ulatory and stem cell-like memory CD4+ T cell subsets from patients with type 1 diabetes and healthy donors, that patients have shorter TCRB complementarity-determining region 3s (CDR3), in all cell subsets, introduced by increased deletions/reduced insertions during VDJ rearrangement. High frequency of short CDR3s is also observed in unproductive TCRB sequences, which are not subjected to thymic culling, suggesting that the shorter CDR3s arise independently of positive/negative selection. Moreover, TCRB CDR3 clonotypes expressed by autoantigen-specific CD4+ T cells are shorter compared with anti-viral T cells, and with those from healthy donors. Thus, early events in thymic T cell development and repertoire generation are abnormal in type 1 diabetes, which suggest that short CDR3s increase the potential for self-recognition, conferring heightened risk of autoimmune disease.

overall_design:
We sorted  CD4+ T cell subsets  (central memory, naive cells)  from 14  individuals newly diagnosed with type 1 diabetes and 14 healthy individuals matched by sex and age. Additionaly, for 8 individuals from each  group we sorted  regulatory T cells and T-memory stem cells (Tscm).
We isolated total RNA from sorted cells and  Adaptive Biotechnologies performed TRB sequencing from it

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Raw data generated by Adaptive Biotechnologies. Submitter states that Adaptive Biotechnologies does not release raw data.
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first: Iria

last: Gomez Tourino

first: Mark

last: Peakman

first: Roman

last: Baptista

first: Yogesh

last: Kamra


title: A developmental mechanism to Regulate Alternative Polyadenylation in an Adult Stem Cell Lineage [3'seq_OEs]

overall_design: We performed 3'seq on control bam-/- flies (nosGal4/Y;+/+;bam86,bamGal4/bam1) and compared to bam mutant fies in which either cbc alone, or PCf11 alone or cbc plus PCF11 were overexpressed via the Gal4-UAS system. Libraries were performed in duplicates. Per each library roughly 150 pairs of testes were used.


title: Transcription factor network dynamics during the commitment to oncogene-induced senescence [CUT&Tag]

overall_design: We performed cleavage under targets and tagmentation (CUT&Tag)  followed by sequencing in WI38-ER:RAS(G12V) cells (human lung fibroblasts) collected at indicated time-points post induction of oncogenic HRAS(G12V) with 4-hydroxy tamoxifen (4-OHT) treatment. The assay was performed for histone modications H3K4me1, H3K27me3 and HK27ac.


title: Transcription factor network dynamics during the commitment to oncogene-induced senescence [ATAC-Seq]

overall_design: Assay for transposase accessible chromatin followed by sequencing (ATAC-seq) in WI38-ER:RAS(G12V) cells (human lung fibroblasts) collected at indicated time-points post induction of oncogenic HRAS(G12V) with 4-hydroxy tamoxifen (4-OHT) treatment.


title: Engineered T cell therapy for central nervous system injury

first: Jonathan

last: Kipnis

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Epithelial organoid supports resident memory CD8 T cell differentiation [RNA-seq: tcm_spln_cd103_trm_frt]

city: Providence

first: Geongoo

last: Han

address: 171 Meeting St.

institute: Brown University

summary: Resident Memory T cells (TRM) play a vital role in regional immune defense in barrier organs. Although laboratory rodents have been extensively used to study fundamental TRM biology, issues like poor isolation efficiency, sampling bias and low survival rates have limited our ability to conduct TRM-focused high-throughput assays. Here we engineered a murine vaginal epithelial organoid (VEO)-CD8 T cell coculture system that supports CD8 TRM differentiation in vitro. The three-dimensional VEOs established from adult stem cells resembled stratified squamous vaginal epithelium and induced gradual differentiation of activated CD8 T cells into epithelial TRM. These in vitro generated TRM were phenotypically and transcriptionally similar to in vivo TRM and their epithelial residence phenotype (CD69+ CD103+) was further enhanced with a second cognate-antigen exposure during coculture. TRM differentiation was not affected even when VEOs and CD8 T cells were separated by a semipermeable barrier, indicating soluble factors' involvement. Pharmacological and genetic approaches showed that TGF-β signaling played a crucial role in their differentiation. We found that the VEOs in our model remain susceptible to viral infections and the CD8 T cells were amenable to genetic manipulation; both of which will allow detailed interrogation of antiviral CD8 T cell biology in a reductionist setting. In summary, we established a robust model which captures bonafide TRM differentiation that is scalable, open to iterative sampling, and can be subjected to high throughput assays that will rapidly add to our fundamental understanding of TRM.

overall_design:
To investigate the gene expression of CD8 TCM and TRM, we infected B6 mice with HSV-2. At 30 dpi, we isolated gBT-I transgenic CD8 T cells and isolated CD8 TCM from the spleen and CD103+ CD8 TRM from the female reproductive tract.
And then we did transcriptome analysis to evaluate the differences between CD8 TCM and TRM

first: Max

last: Ulibarri

first: Ying

last: Lin

first: Julian

last: Ramprashad

first: Mohammad

last: Hasan

first: Farha

middle: J

last: Mithila

first: Smita

last: Gopinath

first: Nu

last: Zhang

first: Justin

last: Milner

first: Lalit

last: Beura


title: Informing Hazard Identification and Risk Characterization of Environmental Chemicals by Combining Transcriptomic and Functional Data from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

city: College Station

first: Ivan

last: Rusyn

address: 4458 TAMU

department: Department of Veterinary Integrative Biosciences

institute: Texas A&M University

summary: Cardiovascular disease remains a significant global health concern, yet the cardiotoxic effects of environmental chemicals are largely unexplored. Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-Cardiomyocytes) are well-established as valuable high-throughput cardiotoxicity testing model based on the analysis of various beating parameters; however, additional adverse effects that may not be directly related to ion channel activity are difficult to ascertain from traditional assays. Transcriptomic data may provide a more comprehensive assessment for additional molecular responses and serve as a basis for dose-response analysis. We hypothesized that transcriptomic data and functional phenotypic measurements in iPSC-Cardiomyocytes could be used to effectively screen for both hazard and risk associated with chemical exposures. To test this hypothesis, we performed concentration-response analysis of 464 chemicals from 12 diverse chemical classes. Functional effects (beat frequency, QT-prolongation, and asystole) and cytotoxicity were measured. In addition, whole transcriptome RNA-seq was used to investigate global gene expression profiles. Points of departure (PODs) were derived from both phenotypic endpoints and transcriptomic data. Margin of safety (MOS) for drugs and margin of exposure (MOE) for non-drug chemicals were calculated using PODs and the exposure estimates. The phenotypic assays revealed that a varying proportion (10% to 44%) of chemicals in each class had effects on iPSC-Cardiomyocytes; positive chronotrope was the most sensitive phenotype. Overall, 244 (53%) chemicals were active in at least one functional phenotype. As expected, drugs with known adverse effects on the heart were most active. Transcriptomic data showed 69 (15%) had significant effects on the biological pathways, the pathways that were significantly affected by some tested compounds were highly relevant to cardiomyocyte function. Specifically, the enriched pathways aligned well with key characteristics of known cardiovascular toxicants, with mitochondrial dysfunction being the most affected key characteristic. Using exposure and hazard (transcriptomic and phenotypic PODs from this study), we derived MOS and MOE and found that drugs tend to have lower MOS than MOE for the environmental chemicals. Thirteen and 10 drugs had MOS < 1 using phenotypic and transcriptomic data, respectively, suggesting that further investigation may be needed for these substances to test for potential cardiovascular risk. Overall, our findings demonstrate how the integrative use of in vitro transcriptomic data and phenotypic assays in iPSC-Cardiomyocytes not only offers a unique and complementary approach for hazard and risk prioritization, but also offers comprehensive mechanistic support for in vitro test results.

overall_design: Comparative gene expression profiling analysis and dose-response modeling of TempO-Seq data for 464 chemical treatments

first: Han-Hsuan

middle: D

last: Tsai

first: Lucie

last: Ford

first: Sarah

last: Burnett

first: Allison

middle: N

last: Dickey

first: Fred

last: Wright

first: Weihsueh

last: Chiu


title: Reprogramming of diabetic wounds by Sox2 triggers a pro-healing transcriptional signature

city: Bethesda

first: Stephen

last: Brooks

state: MD

address: 50 SOUTH DR, RM 1140

department: Biodata Mining and Discovery Section

institute: NIAMS/NIH

summary: We show that a combinatorial upregulation of SOX2 and hyperglycemia in cutaneous keratinocytes triggers a pro-healing signature and increased neutrophil chemoattraction, putatively by Ccr2, Ccr5, and Csf3r signaling. Neutrophil depletion and deregulation through the TREM1/FOXM1 pathway are directly implicated in diabetic foot ulcer (DFU) pathophysiology. Our results highlight the role of neutrophil recruitment in wound healing and can be used to explore potential therapeutic targets for DFUs in patients.

overall_design: Whole skin mRNA profiles in unwounded transgenic skin from mice treated with an ethanol vehicle, tamoxifen, vehicle and streptozotocin, or tamoxifen and streptozotocin were examined in triplicate or quadruplicate.

first: Christopher

last: O'Neill

first: Andrew

middle: P

last: Sawaya

first: Spencer

last: Mehdizadeh

first: Kowser

last: Hasneen

last: Overmiller

first: Maria

middle: I

last: Morasso


title: RNA-seq analysis of WT and STING knockout airway derived cell lines

first: Sebla

middle: Bulent

last: Kutluay

address: 660 S. Euclid Avenue, Campus Box 8230

institute: Washington University School of Medicine

summary: In a screen of airway-derived cell lines, we found several that appeared to resist SARS-CoV-2 replication despite expressing the viral receptor, ACE2. We found that innate immune activation mediated by cGAS-STING and subsequent upregulation of interferons and interferon-stimulated genes underlies this phenotype. RNA-seq was conducted on WT and STING knockout (via CRISPR) SCC25, H596, OE21 and Detroit 562 and WT Calu-3 cell lines

overall_design: Cells were grown in 6-well dishes to 90% confluency and total RNA extracted using Trizol following manufacturer's protocol. RNA was subsequently prepared for sequencing using Illumina Truseq stranded mRNA kit. Sequencing was done on Nextseq500 platform.

middle: B


title: Innate immune memory after brain injury drives inflammatory cardiac dysfunction [CUT&tag]

city: Munich

first: Arthur

last: Liesz

address: Feodor-Lynen-Straße 17

department: Institute for Stroke and Dementia (ISD)

institute: LMU Hospital

summary: The enormous medical burden of stroke is not only due to the brain injury itself and the acute systemic effects, but is largely determined by chronic comorbidities that develop secondarily after stroke. We hypothesized that the high rate of comorbidity developing after a stroke might have a shared immunological cause, however, the chronic effects of brain injury on systemic immunity have so far been barely investigated. Here, we identified myeloid innate immune memory as a cause of remote organ dysfunction after stroke. Using single-cell sequencing, we identified persistent pro-inflammatory transcriptomic changes in resident monocytes/macrophages in multiple organs one month after experimental ischemic brain injury, which was particularly abundant in the heart and associated with the development of cardiac fibrosis and diastolic dysfunction. A similar phenotype was seen in myocardial autopsy samples from stroke versus control patients. We observed chronic functional changes in myeloid hematopoiesis driven by post-stroke IL-1-mediated epigenetic changes. These alterations could be transplanted to naïve recipient mice and were sufficient to induce cardiac dysfunction. By effectively blocking the trafficking of pro-inflammatory monocytes from the bone marrow to the heart using a dual CCR2/5 inhibitor, we successfully prevented post-stroke cardiac dysfunction. This approach holds promising potential as a novel immune-targeted secondary prevention therapy. We anticipate that the epigenetic immune reprogramming mechanisms detailed here for the brain-heart axis could be generalized to provide a novel framework for explaining the development of various comorbidities after acute tissue injury in remote organs.

overall_design: CUT&tag sequencing of mouse BM monocytes and HSPC-enriched myeloid cells from mice one month after stroke or control conditions was performed

first: Alba

last: Simats

first: Sijia

last: Zhang

first: Denise

last: Messerer

first: Faye

last: Chong

first: Sude

last: Beskardes

first: Aparna Sharma

last: Chivukula

first: Jiayu

last: Cao

first: Simon

last: Besson-Girard

first: Felipe A.

last: Montellano

first: Caroline

last: Morbach

first: Olga

last: Carofiglio

first: Alessio

last: Ricci

first: Stefan

last: Roth

first: Gemma

last: Llovera

first: Rashween

last: Singh

first: Yiming

last: Chen

first: Severin

last: Filser

first: Nikolaus

last: Plesnila

first: Christian

last: Braun

first: Hannah

last: Spitzer

first: Ozgun

last: Gocke

first: Martin

last: Dichgans

first: Peter U.

last: Heuschmann

first: Kinta

last: Hatakyama

first: Eduardo

last: Beltrán

first: Sebastian

last: Clauss

first: Boyan

last: Bonev

last: Schulz


title: RPL22 is a tumor suppressor in MSI-high cancers and a key splicing regulator of MDM4

city: San Francisco

first: Hanbing

last: Song

state: CA

address: 513 Parnassus Ave, HSE

department: Department of Medicine

institute: UCSF

summary: Microsatellite instability high (MSI-H) tumors are malignant tumors that, despite harboring a high mutational burden, often have intact TP53. One of the most frequent mutations in MSI-H tumors is a frameshift mutation in RPL22, a ribosomal protein. Here, we identified RPL22 as a modulator of MDM4 splicing through an alternative splicing switch in exon 6. RPL22 loss increased MDM4 exon 6 inclusion, cell proliferation, and augmented resistance to the MDM inhibitor Nutlin-3A. RPL22 represses expression of its paralog, RPL22L1, by mediating the splicing of a cryptic exon corresponding to a truncated transcript. Therefore, RPL22 loss is a driver of oncogenic MDM4 induction and key to a common splicing circuit in MSI-H tumors that may inform therapeutic targeting of the MDM4-p53 axis and oncogenic RPL22L1 induction.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for ZR-75-1, NCI-H2110, CAL851, and LNCAP cells and their KD, KO, and OE derivatives.

last: Weinstein

first: Kevin

last: Hu

first: Lisa

last: Fish

first: Yih-An

first: Paul

last: Allegakoen

first: Keliana

middle: S

last: Hui

first: Julia

middle: H

last: Pham

first: Maria

last: Baco

first: Andrew

middle: O

last: Giacomelli

first: Francisca

last: Vazquez

first: Mahmoud

last: Ghandi

first: Hani

last: Goodarzi

first: Franklin

middle: W

last: Huang


title: Innate immune memory after brain injury drives inflammatory cardiac dysfunction [ATAC-Seq]

first: Arthur

last: Liesz

overall_design: Bulk ATAC sequencing of mouse HSPC-enriched BM cells treated with recombinant IL-1b or vehicle

first: Alba

last: Simats

first: Sijia

last: Zhang

first: Denise

last: Messerer

first: Faye

last: Chong

first: Sude

last: Beskardes

first: Aparna Sharma

last: Chivukula

first: Jiayu

last: Cao

first: Simon

last: Besson-Girard

first: Felipe A.

last: Montellano

first: Caroline

last: Morbach

first: Olga

last: Carofiglio

first: Alessio

last: Ricci

first: Stefan

last: Roth

first: Gemma

last: Llovera

first: Rashween

last: Singh

first: Yiming

last: Chen

first: Severin

last: Filser

first: Nikolaus

last: Plesnila

first: Christian

last: Braun

first: Hannah

last: Spitzer

first: Ozgun

last: Gocke

first: Martin

last: Dichgans

first: Peter U.

last: Heuschmann

first: Kinta

last: Hatakeyama

last: Beltrán


title: Innate immune memory after brain injury drives inflammatory cardiac dysfunction [bone marrow scRNA-Seq]

first: Arthur

last: Liesz

summary: Besides the local inflammatory immune response in the brain, stroke    also alters systemic immunity. Within the acute and sub-acute phase,    the systemic immune response to stroke has been described in detail    over last decades. The long-term systemic immunological consequences after stroke    are however still elusive. Therefore, a better understanding of these long-lasting chronic effects of    stroke on systemic immunity and its impact on remote organ function    and secondary comorbidities is needed. Here, we used single-cell RNA    sequencing (scRNA-Seq) to investigate the chronic effect of stroke on    the transcriptomic signatures of resident myeloid immune cells in various peripheral    organs remote from the brain, including the lung, heart, liver,    spleen, blood and bone marrow. We observed that monocytes    in peripheral organs and in the bone marrow adopt a pro-inflammatory    phenotype which persists chronically after stroke. Moreover, the    pro-inflammatory profile of monocytes in the bone marrow was transmissible    by BM transplantation to naïve recipients, indicating potentially epigenetically    imprinted chronic innate immune memory after stroke. Indeed, by performing single-nuclei ATAC sequencing, we found that post-stroke myeloid immune memory after stroke is likely mediated by IL-1b-driven mechanisms, and that neutralization of the post-stroke increase in circulating IL-1b prevented myeloid immune memory.

overall_design: A single-cell RNA experiment was performed on bone marrow myeloid progenitor and mature cells chronically after stroke

first: Alba

last: Simats

first: Sijia

last: Zhang

first: Denise

last: Messerer

first: Faye

last: Chong

first: Sude

last: Beskardes

first: Aparna Sharma

last: Chivukula

first: Jiayu

last: Cao

first: Simon

last: Besson-Girard

last: Montellanos

first: Petter U.

first: Eduardo

first: Sebastian

last: Clauss

first: Boyan

last: Bonev

last: Schulz


title: Innate immune memory after brain injury drives inflammatory cardiac dysfunction [BMT2]

overall_design: A single-cell RNA experiment was performed on blood and heart myeloid cells from BM-depleted recipient mice that were transplanted with bone marrow cells from stroke or control donors.

last: Besson-Giard

first: Felipe A.

first: Caroline

last: Morbach

first: Olga

last: Carofiglio

first: Alessio

last: Ricci

first: Stefan

last: Roth

first: Gemma

last: Llovera

first: Rashween

last: Singh

first: Yiming

last: Chen

first: Severin

last: Filser

first: Nikolaus

last: Plesnila

first: Christian

last: Braun

first: Hannah

last: Spitzer

first: Ozgun

last: Gocke

last: Dichgans


title: Single-nuclei gene expression profiling of human SMARCB1-deficient peripheral T cell lymphomas

city: Münster

first: Kornelius

last: Kerl

address: Albert-Schweitzer-Campus 1

department: Department of Pediatric Hematology and Oncology

institute: University Children’s Hospital Münster

summary: Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) is a heterogeneous group of malignancies with poor outcome. Here we identified a subgroup, PTCL-NOS SMARCB1-, which is characterized by the lack of SMARCB1 protein expression and is more common in young patients under 25 years of age. To investigate the cellular heterogeneity of PTCL-NOS SMARCB1- and the role of its tumor microenvironment, we performed single-nuclei RNA sequencing (snRNA-seq) of five patient samples.

overall_design: Gene expression profiles of PTCL-NOS SMARCB1- patients (n = 5) were generated by snRNA-seq of formalin-fixed, paraffin-embedded (FFPE) tissue samples using Chromium Fixed RNA Profiling for Multiplexed Samples technology (10X Genomics).

first: Thomas

last: Albert

first: Archana

last: Verma

first: Clara

last: Inserte

first: Anja

last: Fischer

first: Reiner

last: Siebert


title: Gene expression profile at single cell level of CD8+ T cells and NK cells as part of neonatal antiviral response in C57BL/6J and PrP KO mice infected with mouse cytomegalovirus (MCMV)

city: Rijeka

first: Tihana

last: Lenac Rovis

address: B. Branchetta 20

department: Center for Proteomics

country: Croatia

institute: University of Rijeka Faculty of Medicine

summary: Cytomegaloviral (CMV) infection poses risks to newborns, necessitating effective therapies. Given that the damage includes both viral infection of brain cells and immune system-related damage, we investigated the involvement of cellular prion protein (PrP), which plays vital roles in neuroprotection and immune regulation. We used single cell RNA sequencing (scRNA-seq) to analyze the differences in CD8+ T cell and NK cell response to congenital MCMV infection in wild type mice and strain lacking functional PrP protein (PrP KO).

overall_design: 10 000 cells per sample were used for analysis in CD8+ T cell: NK cell ratio 9:1. For spleen samples we FACS sorted cells from congenitally MCMV infected and naïve PrP KO and C57BL/6J mice 14 days post infection. Since there are no infiltrating immune cells in brains of healthy animals, naïve samples have been excluded, thus the two brain samples consisted of cells isolated from infected mice.

first: Dubravka

last: Karner

first: Vanda

last: Juranic Lisnic

first: Daria

last: Kvestak

first: Valentina

last: Libri

first: Ilija

last: Brizic

first: Milena

last: Hasan

first: Sebastien

last: Mella


title: Innate immune memory after brain injury drives inflammatory cardiac dysfunction II

first: Arthur

last: Liesz

summary: Besides the local inflammatory immune response in the brain, stroke  also alters systemic immunity. Within the acute and sub-acute phase,  the systemic immune response to stroke has been described in detail over last decades. The long-term systemic immunological consequences after stroke  are however still elusive. Therefore, a better understanding of these long-lasting chronic effects of stroke on systemic immunity and its impact on remote organ function     and secondary comorbidities is needed. Here, we investigated the long-term consequences of stroke on the heart. By performing serial cardiac ultrasound imaging in mice, we found that stroke leads to chronic cardiac dysfunction, which was associated with increased cardiac fibrosis. We found that this phenomenon was driven by an increased infiltration of bone marrow-derived monocytes into the heart after stroke, which results in increased expression of metalloproteinase-9 (Mmp9) by cardiac monocytes/macrophages. To further investigate the translational relevance of this post-stroke cardiac phenotype, we analyzed heart samples from patients who died due to stroke and control patients and performed bulk mRNA sequencing.

overall_design: Bulk mRNA sequencing of human heart paraffin embeded samples from stroke patients and control patients

first: Alba

last: Simats

first: Sijia

last: Zhang

first: Denise

last: Messerer

first: Faye

last: Chong

first: Sude

last: Beskardes

first: Aparna Sharma

last: Chivukula

first: Jiayu

last: Cao

first: Simon

last: Besson-Girard

first: Felipe A.

last: Montellano

first: Caroline

last: Morback

first: Olga

last: Carofiglio

first: Alessio

last: Ricci

first: Stefan

last: Roth

first: Gemma

last: Llovera

first: Rashween

last: Singh

first: Yiming

last: Chen

first: Severin

last: Filser

first: Nikolaus

last: Plensila

first: Christian

last: Braun

last: Spitzer

last: Gokce

first: Martin

first: Peter U.

last: Heuschmann

first: Kinta

last: Hatakeyama

first: Eduardo

last: Beltrán

first: Sebastian

last: Clauss

first: Boyan

last: Bonev

last: Schulz


title: Epithelial organoid supports resident memory CD8 T cell differentiation

first: Geongoo

last: Han

overall_design:
To investigate CD8 TRM differentiation in vitro, we established vaginal epithelial organoid system and VEO-CD8 T cell coculture system.
And then we did transcriptome analysis to evaluate the similarity of in vitro generated TRM and in vivo TRM

first: Max

last: Ulibarri

first: Ying

last: Lin

first: Julian

last: Ramprashad

first: Mohammad

last: Hasan

first: Farha

last: Mithila

first: Smita

last: Gopinath

first: Nu

last: Zhang

first: Justin

last: Milner

first: Lalit

middle: K

last: Beura


title: Innate immune memory after brain injury drives inflammatory cardiac dysfunction

first: Arthur

last: Liesz

summary: Besides the local inflammatory immune response in the brain, stroke     also alters systemic immunity. Within the acute and sub-acute phase,     the systemic immune response to stroke has been described in detail     over last decades. The long-term systemic immunological consequences after stroke     are however still elusive. Therefore, a better understanding of these long-lasting chronic effects of     stroke on systemic immunity and its impact on remote organ function     and secondary comorbidities is needed. Here, we used single-cell RNA     sequencing (scRNA-Seq) to investigate the chronic effect of stroke on     the transcriptomic signatures of resident myeloid immune cells in various peripheral     organs remote from the brain, including the lung, heart, liver,     spleen, blood and bone marrow. We observed that monocytes     in peripheral organs and in the bone marrow adopt a pro-inflammatory     phenotype which persists chronically after stroke. Moreover, the     pro-inflammatory profile of monocytes in the bone marrow was transmissible     by BM transplantation to naïve recipients, indicating potentially epigenetically     imprinted chronic innate immune memory after stroke. Indeed, by performing single-nuclei ATAC sequencing, we found that post-stroke myeloid immune memory after stroke is likely mediated by IL-1b-driven mechanisms, and that neutralization of the post-stroke increase in circulating IL-1b prevented myeloid immune memory.

overall_design: Three independent mouse single-cell RNA experiments were performed on myeloid cells from different peripheral organs (blood, spleen, heart, lung, liver) and bone marrow, chronically after stroke and control conditions.

first: Alba

last: Simats

first: Sijia

first: Denise

last: Messerer

first: Faye

last: Chong

first: Sude

last: Beskardes

first: Aparna Sharma

last: Chivukula

first: Jiayu

last: Cao

first: Simon

last: Besson-Girard

first: Felipe A.

last: Montellanos

first: Caroline

last: Morbach

first: Olga

last: Carofiglio

first: Alessio

last: Ricci

first: Stefan

last: Roth

first: Gemma

last: Llovera

first: Rashween

last: Singh

first: Yiming

last: Chen

first: Severin

last: Filser

first: Nikolaus

last: Plesnila

first: Christian

last: Braun

first: Hannah

last: Spitzer

first: Ozgun

last: Gocke

last: Dichgans

first: Petter U.


title: Single, off-the-shelf treatment of Neuropathic Mucopolysaccharidoses with iPSC-derived microglia [scRNA-seq]

city: New York

last: Irion

state: New York

address: 415 E 29th St

institute: BlueRock Therapeutics

summary: Lysosomal storage disorders (LSDs) are a group of inherited metabolic disorders . A subgroup of LSDs is called Mucopolysaccharidoses (MPS), where accumulation of glycosaminoglycans causes a progressive degenerative disorder. The central nervous system (CNS) is particularly impacted with developmental delays, neurological regression, and early mortality. Current treatments are often insufficient to fully address the clinical need. Here we report the use of microglia derived from healthy human induced pluripotent stem cells (hiPSCs) as a potential one-time, allogeneic off-the-shelf cell therapy for several MPS. iPSC-derived microglia, termed MG01   , are replete with wild-type levels of lysosomal enzymes and we show that MG01 can deliver the functional enzyme into four different MPS knockout cell lines via mannose-6-phosphate receptor-mediated endocytosis in vitro. We then show that a single administration of MG01 provides sufficient enzyme to prevent behavioral deficits in two different animal models of MPS, efficacious to at least eight months after transplantation.

overall_design: Single cell RNAseq of cryopreserved hiPSCs or MG01 after thaw using 10x Genomics platform.

first: Panagiotis

last: Douvaras

first: Diego

last: Buenaventura

first: Bruce

last: Sun

first: Ashley

last: Lepack

first: Elizabeth

last: Baker

last: Simpson

first: Mark

last: Ebel

first: Gregory

last: Lallos

first: Deven

last: LoSchiavo

first: Nick

last: Stitt

first: Nathaniel

last: Adams

first: Conor

last: McAuliffe

first: Ana

last: Forton-Juarez

first: Brian

last: Kosmyna

last: Pereira

first: David

last: Dilworth

first: Stephanie

last: Fisher

first: Jing

last: Wang

first: Peter

last: Tonge

last: Tomishima

first: Carlos

last: Paladini

first: Dan

last: Wilkinson

first: Chew-Li

last: Soh

first: Maya

last: Srinivas

first: Christoph

last: Patsch


title: Dynamic landscapes and protective immunity coordinated by influenza-specific lung-resident memory CD8+ T cells revealed by intravital imaging

city: Iowa City

last: van de Wall

address: 500 Newton Road

institute: University of Iowa

summary: Lung tissue-resident memory CD8+ T-cells (Trm) are critical for heterosubtypic immunity against influenza virus-(IAV)-reinfection. How these cells surveil the lung, respond to infection and interact with other cells remains unresolved. Here, we used mouse models to identify and enrich lung Trm in combination with intravital and static imaging to assess spatiotemporal dynamics of IAV-induced lung TRM before and after recall-infection. CD69+CD103+ Trm localize to sites of prior influenza infection where they exhibit substantially slower movement properties than non-Trm that also surveil the lung. After rechallenge, lung Trm form tight clusters in an antigen-dependent manner. IAV-specific Trm express several factors that regulate myeloid cell biology and their protective immune responses are accompanied by activation and migration of dendritic cells to draining lymph nodes and recruitment of inflammatory monocytes. Overall, these data reveal the dynamic landscapes of lung Trm cells associated with early protective immunity against IAV infection.

overall_design:
The goal of this study is to assess the spatial, transcriptomic and functional properties of lung CD103+CD69+ tissue-resident CD8+ T cells.
C57BL/6 mice were adoptively transferred with Thy1.1 P14 transgenic T cells (TCR-specific for LCMV GP33-41) and subsequently infected with influenza (PR8-GP33). Memory P14 cells were isolated from spleen (CD103-CD69- P14s) and lungs (CD103+CD69+ P14s) at day 26-30 post infection and sort purified directly in TRIzol.
Comparative gene expression profiling analysis of RNA-seq data for lung CD103+CD69+ P14s and splenic CD103-CD69- cells

first: Vladimir

last: Badovinac

first: John

middle: T

last: Harty


title: Innate immune memory after brain injury drives inflammatory cardiac dysfunction [snATAC-seq]

first: Arthur

last: Liesz

summary: Besides the local inflammatory immune response in the brain, stroke also alters systemic immunity. Within the acute and sub-acute phase, the systemic immune response to stroke has been described in detail over last decades. The long-term systemic immunological consequences after stroke are however still elusive. Therefore, a better understanding of these long-lasting chronic effects of stroke on systemic immunity and its impact on remote organ function and secondary comorbidities is needed. Here, we used single-cell RNA sequencing (scRNA-Seq) to investigate the chronic effect of stroke on the transcriptomic signatures of resident myeloid immune cells in various peripheral organs remote from the brain, including the lung, heart, liver, spleen, blood and bone marrow. We observed that monocytes in peripheral organs and in the bone marrow adopt a pro-inflammatory phenotype which persists chronically after stroke. Moreover, the pro-inflammatory profile of monocytes in the bone marrow was transmissible by BM transplantation to naïve recipients, indicating potentially epigenetically imprinted chronic innate immune memory after stroke. Indeed, by performing single-nuclei ATAC sequencing, we found that post-stroke myeloid immune memory after stroke is likely mediated by IL-1b-driven mechanisms, and that neutralization of the post-stroke increase in circulating IL-1b prevented myeloid immune memory.

overall_design: Mouse single-nuclei ATAC experiment performed on myeloid cells from bone marrow chronically after stroke and control conditions. An additional group of bone marrow cells from stroke mice treated with neutralizing antibodies against IL-1b was also included (stroke-treated group).

first: Alba

last: Simats

first: Sijia

last: Zhang

first: Denise

last: Messerer

first: Faye

last: Chong

first: Sude

last: Beskardes

first: Aparna Sharma

last: Chivukula

first: Jiayu

last: Cao

first: Simon

last: Besson-Girard

first: Felipe A.

last: Montellano

first: Caroline

last: Morbach

first: Olga

last: Carofiglio

first: Alesio

last: Ricci

first: Stefan

last: Roth

first: Gemma

last: Llovera

first: Rashween

last: Singh

first: Yiming

last: Chen

first: Severin

last: Filser

first: Nikolaus

last: Plesnila

first: Christian

last: Braun

first: Hannah

last: Spitzer

first: Ozgun

last: Gocke

first: Martin

last: Dichgans

first: Petter U.

last: Heuschmann

first: Kinta

last: Hatakeyama

first: Eduardo

last: Beltrán

first: Sebastian

last: Clauss

first: Boyan

last: Bonev

last: Schulz


title: Single, off-the-shelf treatment of Neuropathic Mucopolysaccharidoses with iPSC-derived microglia

last: Irion

overall_design: Refer to individual Series


title: Cellular origin and clonal evolution of human dedifferentiated liposarcoma

first: Fabien

last: Quinquis

address: 26 rue d'Ulm

institute: Institut Curie

summary: Dedifferentiated liposarcoma (DDLPS) is the most frequent high-grade soft tissue sarcoma subtype. It is characterized by a component of undifferentiated tumor cells coexisting with a component of well-differentiated adipocytic tumor cells. Both dedifferentiated (DD) and well-differentiated (WD) components exhibit MDM2 amplification, however their cellular origin remains elusive. Using single-cell RNA sequencing and functional assays in paired WD and DD components from primary DDLPS tumors, we characterized the cellular heterogeneity of DDLPS tumor and micro-environment. We identified a population of tumor adipocyte stem cells (ASC) showing striking similarities with adipocyte stromal progenitors found in white adipose tissue. We show that tumor ASC harbor the ancestral genomic alterations of WD and DD components, suggesting that both derive from these progenitors following clonal evolution. Last, we show that DD tumor cells keep important biological properties of ASC including pluripotency and that their adipogenic properties are inhibited by a TGF-β high immunosuppressive tumor micro-environment.

overall_design: Primary DDLPS, WDLPS, liposarcoma and peritumoral fat tissue of untreated patients were isolated and analyzed using RNAseq.

first: Nadège

last: Gruel

first: Chloé

last: Quignot

first: Sophie

last: El Zein

first: Laëtitia

last: Lesage

first: Sylvie

last: Bonvalot

first: Dimitri

last: Tzanis

first: Julien

last: Vibert

first: Nadine

last: El Tannir

first: Didier

last: Decaudin

first: Héloïse

last: Derrien

first: Ahmed

last: Dahmani

first: Odette

last: Mariani

last: Linares

first: Sylvain

last: Baulande

first: Joshua

last: Waterfall

first: Olivier

last: Delattre

first: Sarah

last: Watson


title: Single, off-the-shelf treatment of Neuropathic Mucopolysaccharidoses with iPSC-derived microglia [bulk RNA-seq]

first: Stefan

last: Irion

overall_design: Bulk RNAseq of MG01 xenograft in the brain of NSG-Quad mouse. Tissue obtained from formalin fixed cryosections.

first: Panagiotis

last: Douvaras

first: Diego

last: Buenaventura

first: Bruce

last: Sun

first: Ashley

last: Lepack

first: Elizabeth

last: Baker

last: Simpson

first: Benjamin

last: Burnett

first: Mark

last: Ebel

first: Gregory

last: Lallos

first: Deven

last: LoSchiavo

first: Nick

last: Stitt

first: Nathaniel

last: Adams

first: Conor

last: McAuliffe

first: Ana

last: Forton-Juarez

first: Brian

last: Kosmyna

last: Pereira

first: David

last: Dilworth

first: stephanie

last: Fisher

first: Jing

last: Wang

first: Peter

last: Tonge

last: Tomishima

first: Carlos

last: Paladini

first: Dan

last: Wilkinson

first: Chew-Li

last: Soh

first: Maya

last: Srinivas

first: Christoph

last: Patsch


title: Lineage-Specific Mesenchymal Stromal Cells Derived from Human iPSCs Showed Distinct Patterns in Transcriptomic Profile and Extracellular Vesicle Production

status: Public on Jul 21 2024

city: Syracuse

first: Yuanhui

last: Song

address: 318 Bowne Hall

institute: Syracuse University

summary: Over the past decades, mesenchymal stromal cells (MSCs) have been extensively investigated as a potential therapeutic cell source for the treatment of various disorders. Differentiation of MSCs from human induced pluripotent stem cells (iMSCs) has provided a scalable approach for the biomanufacturing of MSCs and related biological products. Although iMSCs shared typical MSC markers and functions as primary MSCs (pMSCs), there is a lack of lineage specificity in many iMSC differentiation protocols. Here, we employed a stepwise hiPSC-to-iMSC differentiation method via intermediate cell stages of neural crest and cytotrophoblast to generate lineage-specific MSCs with varying differentiation efficiencies and gene expression. Through a comprehensive comparison between early developmental cell types (hiPSCs, neural crest, and cytotrophoblast), two lineage-specific iMSCs, and six source-specific pMSCs, we were able to not only distinguish the transcriptomic differences between MSCs and early developmental cells, but also determine the transcriptomic similarities of iMSC subtypes to postnatal or perinatal pMSCs. Additionally, we demonstrated that different iMSC subtypes and priming conditions affected EV production, exosomal protein expression, and cytokine cargo.

overall_design: we differentiated two iMSC subtypes via two intermediate cell types of neural crest (NC-iMSCs) and cytotrophoblast (CT-iMSCs) using serum-free chemical-defined media.

first: Tackla

last: Winston

first: Huaiyu

last: Shi

first: Junhui

last: Yang

first: Munther

last: Alsudais

first: Maria

last: Kontaridis

first: Yaoying

last: Wu

first: Thomas

last: Gaborski

first: Qinghe

last: Meng

first: Robert

last: Cooney

first: Zhen

last: Ma


title: Innate immune memory after brain injury drives inflammatory cardiac dysfunction (cardiac interstitial cells)

first: Arthur

last: Liesz

overall_design: A single-cell RNA experiment was performed on cardiac interstitial cells from mice one month after stroke or control conditions

first: Alba

last: Simats

first: Sijia

last: Zhang

first: Denise

last: Messerer

first: Faye

last: Chong

first: Sude

last: Beskardes

first: Aparna Sharma

last: Chivukula

first: Jiayu

last: Cao

first: Simon

last: Besson-Girard

first: Felipe A.

last: Montellanos

first: Caroline

last: Morbach

first: Olga

last: Carofiglio

first: Alessio

last: Ricci

first: Stefan

last: Roth

first: Gemma

last: Llovera

first: Rashween

last: Singh

first: Yiming

last: Chen

first: Severin

last: Filser

first: Nikolaus

last: Plesnila

first: Christian

last: Braun

first: Hannah

last: Spitzer

first: Ozgun

last: Gocke

first: Martin

last: Dichgans

first: Petter U.

last: Heuschmann

first: Kinta

last: Hatakeyama

first: Eduardo

last: Beltrán

first: Sebastian

last: Clauss

first: Boyan

last: Bonev

last: Schulz


title: The influence of CLEC5A on early macrophage-mediated inflammation in COPD progression.

city: Guangzhou

first: Erkang

last: Yi

state: Guangdong

address: 195 Dongfeng Xi Road

department: State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease

institute: The First Affiliated Hospital of Guangzhou Medical University

summary:
Methods: This study analyzed lung tissue sequencing data from early-stage COPD patients (GSE47460) and smoke-exposed mice. We employed Weighted Gene Co-Expression Network Analysis (WGCNA) and machine learning to identify potential pathogenic genes. Further analyses included single-cell sequencing from mice and COPD patients to identify gene expression in specific cell subgroups. Cell-chat and pseudo-temporal analyses were conducted, with findings validated in smoke-exposed mice. Additionally, Mendelian randomization (MR) was utilized to verify the relationship between candidate genes and lung function/COPD. Lastly, functional validation was performed in vitro in cell cultures.
Results: "Machine learning analysis of 30 differentially expressed genes pinpointed 8 key genes, with CLEC5A identified as a potential pathogenic factor in early COPD. Bioinformatics suggested CLEC5A's role in macrophage-mediated inflammation in COPD. Two-sample Mendelian randomization linked CLEC5A SNPs with FEV1, FEV1/FVC, and emphysema/chronic bronchitis. In vitro, CLEC5A knockdown reduced inflammatory markers in macrophages.
Conclusion: Our study identifies CLEC5A as a pivotal gene in early-stage COPD, contributing to its pathogenesis through pro-inflammatory mechanisms. This discovery offers insights for early diagnosis and treatment strategies in COPD, highlighting CLEC5A as a target for further research.

overall_design: To investigate the  function of CLEC5A in macrophages, we established THP-1 cell lines stimulated with  PMA and stimulated with  CSE in which CLECE5A gene has been knocked down by siRNA.

first: Qingyang

last: Li


city: Paris

first: Fabien

last: Quinquis

summary: This SuperSeries is composed of the SubSeries listed below.

first: Nadège

last: Gruel

first: Chloé

last: Quignot

first: Sophie

last: El Zein

first: Laëtitia

last: Lesage

first: Sylvie

last: Bonvalot

first: Dimitri

last: Tzanis

first: Julien

last: Vibert

first: Nadine

last: El Tannir

first: Didier

last: Decaudin

first: Héloïse

last: Derrien

first: Ahmed

last: Dahmani

first: Odette

last: Mariani

last: Linares

first: Sylvain

last: Baulande

first: Joshua

last: Waterfall

first: Olivier

last: Delattre

first: Sarah

last: Watson


title: Probiotic neoantigen delivery vectors for precision cancer immunotherapy

first: Nicholas

last: Arpaia

address: 701 West 168th Street

department: Microbiology & Immunology

institute: Columbia University Irving Medical Center

summary: Microbial systems have been synthetically engineered to deploy therapeutic payloads in vivo. With emerging evidence that bacteria naturally home to tumors and modulate anti-tumor immunity one promising application is the development of bacterial vectors as precision cancer vaccines. In this study, we engineered probiotic E. coli Nissle 1917 (EcN) as an anti-tumor vaccination platform optimized for enhanced production and cytosolic delivery of neoepitope-containing peptide arrays, with increased susceptibility to blood clearance and phagocytosis. These features enhance both safety and immunogenicity, achieving a system which drives potent and specific T cell–mediated anti‑cancer immunity that effectively controls or eliminates tumor growth and extends survival in advanced murine primary and metastatic solid tumors. We demonstrate that the elicited anti-tumor immune response involves extensive priming and activation of neoantigen-specific CD4+ and CD8+ T cells, broader activation of both T and NK cells, and a reduction of tumor-infiltrating immunosuppressive myeloid and regulatory T and B cell populations. Taken together, this work leverages the advantages of living medicines to deliver arrays of tumor‑specific neoantigen–derived epitopes within the optimal context to induce specific, effective, and durable systemic anti-tumor immunity.

overall_design: To predict neoantigens generated by tumor-specific mutations present in the murine CT26 colorectal carcinoma and the murine B16F10 melanoma, we grew each tumor line subcutaneously in the respective MHC-haplotype matched murine host, harvested and homogenized tumors, and extracted total RNA and DNA. We performed RNA-sequencing with gene expression analysis, and whole-exome sequencing with exonic mutation analysis.

first: Andrew

last: Redenti


title: Single-cell and In Situ Spatial Transcriptomics of Marjolin ulcers

city: Calgary

first: Jeff

last: Biernaskie

state: Alberta

address: 3330 Hospital Drive NW

department: Comparative Biology and Experimental Medicine

country: Canada

institute: University of Calgary