title: Differential LncRNA expression of 5 primary ovarian tumors and matched metastasis

city: Beijing

first: Pengfei

last: Zhao

address: No.17, Panjiayuan Nanli, South Huawei Road

institute: National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College

summary: To identify LncRNAs that exert essential functions in ovarian cancer metastasis, we carried out unbiased high-throughput screening by comparing the LncRNA expression profile of ovarian cancer primary and metastasis tissues

overall_design: RNA-seq was performed of 5 primary ovarian tumors and matched metastases from ovarian cancer patients

first: Xiao

last: Yu

first: Yabing

first: Shi

last: Liu

first: Yating

last: Wang

first: Bin

first: Zhihua


title: Rational design of immune gene therapy combinations via in vivo CRISPR activation screening of tumor microenvironment modulators [APCM_Screen_CLCG4I]

status: Public on Feb 02 2026

city: New Haven

first: Chuanpeng

last: Dong

state: CT

address: 850 West Campus Dr

department: Genetics

country: USA

institute: Yale University School of Medicine

summary: The hostile tumor microenvironment (TME) is major challenge for cancer immunotherapies. Here, we design and perform TME-targeted in vivo CRISPR activation (CRISPRa) screens to uncover factors that promote anti-tumor immunity, culminating in rationally designed immune gene therapy combinations. Through adeno-associated virus (AAV) delivery, we find that multiplexed activation of pooled immunoregulatory genes encoding antigen presentation, cytokine, and co-stimulation molecules (APCM) leads to enhanced anti-tumor immunity. A CRISPRa screen targeting APCM genes in metastatic tumors identifies CD80, TNFSF14, CXCL10, TNFSF18, TNFSF9, and IFNG as the top immunostimulatory candidates. Further optimization pinpoints 4-1BBL(TNFSF9) + IFNG + IL12B (4II) as a potent streamlined therapeutic combination. AAV delivered 4II has potent in vivo anti-tumor efficacy in multiple tumor models, by enhancing tumor antigen presentation while simultaneously promoting the infiltration, activation, and expansion of anti-tumor T cells. We further demonstrate that APCM therapy synergizes with CAR-T therapy against human solid tumors in vivo. CRISPRa screens and gene activation systems targeting APCM factors thus represent a powerful approach for rapid development of off-the-shelf immune gene therapies against solid tumors.

overall_design:
To generate metastatic lung tumors and perform in vivo screening under immune pressure, E0771-CLCG4I library cells were injected into 8-12 weeks old female C57BL/6J mice intravenously.
E0771-1 and E0771-2 are cells before selection, E0771-Lung1-5 are lung tumors after selection; 6 sgRNA to profile

first: Feifei

last: Zhang

first: Ryan D

last: Chow

first: Shan

last: Xin

first: Emily

last: He

first: Lvyun

last: Zhu

first: Daniyal

last: Mirza

first: Yanzhi

last: Feng

first: Xinyu

last: Ling

first: Qin

last: Han

first: Guangchuan

first: Sidi

last: Chen


title: Rational design of immune gene therapy combinations via in vivo CRISPR activation screening of tumor microenvironment modulators [APCM_scRNASeq_mouseCD45]

type: Expression profiling by high throughput sequencing

overall_design: B16F10‑OVA cells were injected intravenously to establish lung metastases. Mice received intravenous AAV‑4II (IFNG, IL12B, and 4‑1BBL) or AAV‑Vector control on day 26 post‑tumor inoculation. OT‑I T cells were infused intravenously 10 days. Five days after OT‑I transfer, CD45⁺ cells were sorted for downstream single‑cell RNA‑sequencing (scRNA‑seq).


title: RNA transcriptome sequencing analysis of SNU-668 Erastin-resistant cells transfected with shRNA-SPIN1 or control shRNA

city: shanghai

first: Ming

middle: zhe

last: Ma

email: mmz666@163.com, mmz666@shca.org.cn

address: Dongan Road 270

department: Department of gastric surgery

country: China

institute: Fudan university shanghai cancer center

summary: An RNA transcriptome sequencing analysis was performed in SNU-668 erastin-resistant cells to investigate their molecular profile. To assess the specific role of the SPIN1 protein, a comparative approach was undertaken. This involved transfecting the cells with either a targeted shRNA construct against SPIN1 or, for baseline comparison, a non-targeting control shRNA. The subsequent sequencing data were then generated to enable a detailed comparison of gene expression patterns between these two experimental conditions, aiming to identify key transcriptional changes associated with SPIN1 knockdown in the context of ferroptosis resistance.

overall_design: mRNA profiles of  SNU-668 Erastin-resistant cells transfected with shRNA-ALKBH3 or control shRNA

first: Mingzhe

first: Hui

last: Yang


title: TOX drives TH1 CD4+ T cell effector function, anti-tumor immunity, and pathogenicity in autoimmune diseases [RNA-Seq]

city: New York

first: Andrea

last: Schietinger

state: NY

address: 1275 York Avenue

department: Immunology

institute: Memorial Sloan Kettering Cancer Center

summary: RNA-seq analysis of TOX WT and TOX KO transgenic (TCRSMARTA) CD4 T cells from tdLN of tumor-bearing mice day 11 post transfer

overall_design: Naïve TOX WT and TOX KO TCRSMARTA CD4 T cells were transferred into B16-GP61 tumor-bearing hosts, and 11 days post transfer, sorted from tdLN and subjected to RNA-sequencing.

first: Brianna

last: Naizir

first: Andrew

last: Scott

first: Doron

last: Betel

first: Paul

last: Zumbo


title: TOX drives TH1 CD4+ T cell effector function, anti-tumor immunity, and pathogenicity in autoimmune diseases

type: Genome binding/occupancy profiling by high throughput sequencing

summary: ATAC-seq analysis of TOX WT and TOX KO  transgenic (TCRSMARTA) CD4 T cells from tdLN of tumor-bearing mice at day 11 post transfer

overall_design: Naïve TOX WT and KO TCRSMARTA CD4 T cells were transferred into B16-GP61 tumor-bearing hosts, and at 11 days post transfer, sorted from tdLN, and subjected to ATAC-sequencing


title: Occupancy-based Mechanism Is the Chief Mode of ROS1 Function in Preventing DNA Hypermethylation

status: Public on Feb 03 2026

city: Wuhan

first: Wenying

last: Zhong

address: Lion Rock Street

institute: Huazhong Agricultural University

type: Methylation profiling by high throughput sequencing

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

sample_organism:
Arabidopsis thaliana
Brassica napus

platform_organism:
Arabidopsis thaliana
Brassica napus

overall_design: Refer to individual Series


summary: This study presents a comprehensive ribosome profiling (Ribo-seq) analysis conducted on SNU-668 gastric cancer cells with acquired resistance to Erastin. These cells were genetically modified through stable transfection with either a short hairpin RNA (shRNA) construct specifically targeting and knocking down the expression of the ALKBH3 gene, or a corresponding non-targeting control shRNA, to systematically investigate the consequent alterations in global translational activity.

overall_design: Ribo-seq analysis analysis of  SNU-668 Erastin-resistant cells transfected with shRNA-ALKBH3 or control shRNA and related RNA-seq


title: Ribonuclease 4 Functions in Nociceptor-Mediated Nerve Homeostasis

city: Solna

first: Saida

last: Hadjab

address: Solnavägen 9

department: Neuroscience

country: Sweden

institute: Karolinska Institutet

summary: The regulation of nociceptor identity and function is essential, as disruptions can significantly influence pain sensation, yet our understanding of the molecular mechanisms involved remains incomplete. In this study, we identified ribonuclease 4 (RNase4) as selectively expressed in the unmyelinated nociceptor lineage. Analysis of Rnase4-deficient mice and single-cell transcriptomic data revealed a cell-autonomous role for RNase4 in regulating nociceptor function. Moreover, in a neuropathic pain model, Rnase4 expression was upregulated in nociceptors during the pain and recovery phases, and its deletion altered mechanical sensation. Additionally, RNase4 exerted non-cell autonomous effects on the structural organization of adjacent myelinated axons. These findings suggest that RNase4 contributes to nociceptor biology and point to a pathway for further investigation of pain regulation.

overall_design: Following tamoxifen injection DAPI+NeuN+ stained nuclei from dorsal root ganglia were isolated using fluoresence activate cell sorting (FACS) and analyzed using snRNA-seq.

first: Kaiwen

last: Zhang

first: Prach

last: Techameena


title: H3K115 marks fragile nucleosomes at regulatory sites [ATAC-seq]

city: Edinburgh

first: Yatendra

last: Kumar

email: yaten2020@gmail.com, ykumar@ed.ac.uk

address: Crewe Road south

department: MRC HGU

country: United Kingdom

institute: MRC Human Genetics Unit, The University Of Edinburgh

summary: Acetylation of lysine residues in the tail domain of histone H3 at active regulatory elements are well characterised, but the less studied acetylations of histone globular domain lysines also hold regulatory potential based on their potential impact on nucleosome dynamics and stability. Here we have mapped the genome-wide distribution of acetylated H3 lysine 115 (H3K115ac), a residue on the lateral surface of the nucleosomes, in mouse embryonic stem cells. We find that H3K115ac is associated with highly active promoters, especially those associated with CpG islands, and with enhancers. H3K115ac is dynamic, changing in line with gene activation during differentiation and correlated with changes in local chromatin accessibilityas assyed by ATAC-seq. Distinct from other commony studied histone acetylation marks, H3K115ac is enriched on non-canonical “fragile” nucleosomes precisely at the transcription start sites of active gene promoters and within the binding sites of pluripotency transcription factors. Surprisingly we also detect H3K115ac-marked fragile nucleosomes at sites most strongly occupied by CTCF, within the CTCF footprint and oriented relative to the CTCF motif. This unique enrichment and genomic distribution of modified fragile nucleosomes suggests a previously unappreciated role for H3K115ac in gene regulation and chromatin structure. We propose that H3K115ac is a very precise marker for identifying regulatory elements in mammalian genomes.

overall_design: Dynamics of chromatin accessiblility during ESCs differentiating into NPCs using ATAC-seq


title: Profiling of CSDE1-bound RNAs under MKRN2-mediated ubiquitination

city: Shanghai

first: Zi

last: Wang

address: 600 South Wanping Road, Xuhui District, Shanghai, China

institute: Shanghai Mental Health Center

summary: This study aims to elucidate how MKRN2-mediated ubiquitination regulates the RNA-binding capacity of CSDE1. By overexpressing CSDE1 in MKRN2 knockout (KO) SHSY5Y cells and complementing with MKRN2 for comparison, we systematically analyzed the alterations in the RNA-binding repertoire of CSDE1 upon its interaction with MKRN2.

overall_design:
Objective: Identify RNAs whose binding to CSDE1 depends on MKRN2.
Approach: RNA immunoprecipitation from MKRN2 KO cells subjected to:
1. Control: Empty vector transfection.
2. CSDE1-OE: CSDE1 overexpression alone.
3. Rescue: Co-expression of CSDE1 and MKRN2.
Outcome: Comparison of bound RNAs across conditions will reveal MKRN2-dependent targets.

first: Min

last: Zhu

first: Ronggui

last: Hu


title: TOX drives TH1 CD4+ T cell effector function, anti-tumor immunity, and pathogenicity in autoimmune diseases [CUT&RUN]

summary: CUT&RUN analysis of CD4 and CD8 T cells (confirmed to express TOX)

overall_design: WT congenic (Ly5.1) CD4+ TCRSMARTA (transferred into B16-GP61 tumor hosts) or CD8+ (Ly5.1) TCRTAG (transferred into liver tumor-bearing ASTxAlbCre hosts) were isolated and sorted from tdLN and liver tumors, respectively, and subjected to CUT&RUN

first: Simon

last: Grassmann


title: RNA transcriptome sequencing analysis of SNU-668 Erastin-resistant cells transfected with shRNA-ALKBH3 or control shRNA

first: Ming

summary: a RNA transcriptome sequencing analysis was performed in SNU-668 erastin-resistant cells that were transfected with  shRNA-ALKBH3 or control shRNA


title: H3K115 marks fragile nucleosomes at regulatory sites


title: Rational design of immune gene therapy combinations via in vivo CRISPR activation screening of tumor microenvironment modulators [APCM_BulkRNASeq_OT1]

first: Chuanpeng

last: Dong

overall_design: B16F10‑OVA cells were injected subcutaneously. On day 14, established tumors received an intratumoral injection of AAV-IFNG, IL12B, 4‑1BBL, andAAV‑4II or empty vector control. On day 21, mice received OT‑I T cells by intravenous infusion. Five days after T cell transfer (day 26), OT‑I T cells were identified from the tumor and sorted as CD45⁺ TCR Vα2⁺ Vβ5⁺.

first: Feifei

first: Ryan D

last: Chow

first: Shan

last: Xin

first: Emily

last: He

first: Lvyun

first: Daniyal

last: Mirza

first: Yanzhi

last: Feng

first: Xinyu

last: Ling

first: Qin

last: Han

first: Guangchuan

first: Sidi

last: Chen


title: Liver-specific loss of the transcriptional coregulator ARGLU1 protects against diet-induced obesity in mice through decreased lipid absorption.

city: Toronto

first: Carolyn

middle: L

last: Cummins

state: ON

address: 144 College St Room 1101

department: Pharmaceutical Sciences

country: Canada

institute: University of Toronto

summary: New medications that decrease food intake show promise in addressing the global obesity epidemic; however, additional therapies are needed for patients who are intolerant or unresponsive to these drugs. ARGLU1 is a transcriptional coregulator of several nuclear receptors. Herein, we detail the discovery that mice lacking ARGLU1 in hepatocytes (LKO) are resistant to diet-induced obesity, with no difference in food intake, locomotion, or energy expenditure compared to wildtype mice. LKO mice have lower plasma and liver cholesterol levels, attributed to impaired lipid absorption secondary to changes in bile acid composition. The decrease in CYP8B1 in LKO mice corresponded to fewer 12α-hydroxylated bile acids, increasing the relative hydrophobicity of bile, which decreased lipid (triglyceride and cholesterol) absorption. LKO mice further showed preferential use of fatty acids as their fuel source, thereby establishing a mechanistic basis for resistance of LKO mice to diet-induced obesity and identifying a new molecular target for obesity.

overall_design: RNA-seq of livers collected from WT and Arglu1 hepatocyte KO mice fed either a chow or a high-fat diet (TD. 88137) for 12-weeks.

first: Sarah

middle: B

last: Cash

first: Michael

middle: F

last: Saikali

first: Fatemeh

last: Molaei


title: Age-related molecular and functional remodeling of the choroid plexus in the mouse lemur

city: Sherbrooke

first: Mariano

last: Avino

address: 3201 Jean Mignault

department: Biochemistry and Functional Genomics

institute: Sherbrooke University

summary: Aging is accompanied by complex alterations in brain structure and functions, with growing evidence implicating the choroid plexus (CP) as a key regulator of cognitive and motor function. Here, we investigated age-related changes in the CP of the gray mouse lemur (Microcebus murinus), a non-human primate model that recapitulates human brain aging features. Lemurs of different ages underwent behavioral testing, followed by transcriptomic profiling and immunofluorescence analyses of lateral ventricle CP tissue. Behavioral assessments revealed age-related declines in motor coordination and exploratory drive, whereas working memory and visual discrimination remained largely preserved. Histological analysis showed no significant structural alterations in CP architecture. Transcriptomic profiling identified 1,519 upregulated and 1,682 downregulated genes with aging, highlighting increased interindividual heterogeneity, upregulation of immune- and transport-related pathways, and downregulation of signaling and intercellular communication processes. Functionally, AQP1 protein expression decreased with age without changes in mRNA levels, suggesting post-transcriptional regulation, whereas NKCC1 and TTR expression were largely maintained. Notably, reduced AQP1 expression correlated with age-related motor decline. These findings indicate that CP aging in the mouse lemur is characterized by selective functional vulnerability, immune activation, and transcriptional remodeling, yet essential barrier and secretory functions are largely preserved. Our results highlight a primate-like aging profile of the CP, providing insights into mechanisms by which CP dysfunction may contribute to age-related motor decline and altered brain homeostasis.

overall_design: RNA-seq profiling of the choroid plexus extracted from gray mouse lemurs of different age groups (young: Y; adult: A; old: O).

first: Benoit

last: Laurent

first: Fabien

last: Pifferi


title: Diverging routes of oligodendrocyte recruitment in adaptive and regenerative myelination

first: Tim

last: Czopka

address: 49 Little France Crescent

department: Institute of Neuroscience and Cardiovascular Research

institute: The University of Edinburgh

summary: Myelination by oligodendrocytes is dynamically regulated throughout life, supporting axon and circuit function during development, activity-dependent plasticity and repair after injury. How new oligodendrocytes are recruited from their lifelong pool of oligodendrocyte precursor cells (OPCs) in these distinct contexts remains unclear. We directly compared activity-induced and demyelination-induced oligodendrogenesis under otherwise matched conditions and found that OPCs activated different transcriptional programmes in these different circumstances.

overall_design: RNA sequencing was performed on RNA extracted from sorted oligodendrocyte precursor cells from zebrafish treated for 16 hours in the following conditions: Control, 4-aminopyridine, or capsaicin-TRPV1-induced demyelination.

first: Laura

middle: J

last: Hoodless


first: Ming

last: Ma

summary: An m1A enrichment analysis was conducted to investigate the epigenetic landscape and RNA modification profiles within SNU-668 Erastin-resistant gastric cancer cells following the experimental manipulation of ALKBH3 expression. Specifically, this analysis compared cells that had been stably transfected with a targeted shRNA construct designed to knock down ALKBH3 against control cells transfected with a non-targeting scramble shRNA sequence. The overarching goal was to assess and quantify the differential enrichment patterns of N1-methyladenosine (m1A) modifications across the transcriptome, thereby elucidating the potential regulatory role of the ALKBH3 demethylase in modulating the epitranscriptomic state underlying the acquired Erastin-resistant phenotype in this cellular model.

overall_design: MeRIP m1A enrichment analysis of  SNU-668 Erastin-resistant cells transfected with shRNA-ALKBH3 or control shRNA

first: Mingzhe

first: Hui

last: Yang


title: Integrative Epigenomic and Transcriptomic Analysis of Peripheral Blood Across Different SARS-CoV-2 Vaccine Recipients Reveals TGF-β-Mediated Exhaustion Mechanisms

city: Seoul

first: Minseok

last: Kim

address: 103 Daehak-ro, Jongno-gu, Seoul

country: South Korea

institute: Seoul National University College of Medicine

summary: While distinct SARS-CoV-2 vaccine platforms have demonstrated variable clinical efficacy, the molecular and epigenetic mechanisms driving these immunological disparities remain incompletely understood. Here, we present a high-resolution single-cell multi-omics atlas profiling the transcriptomic and epigenomic landscapes of peripheral immune cells across mRNA, viral vector, and protein subunit vaccine recipients. Our integrative analysis reveals that viral vector and protein subunit vaccines preferentially expand atypical B cell (atBC) subsets characterized by "exhausted" epigenetic signatures and regulatory potential. We identify these cells as a primary source of TGF-β, establishing a systemic negative feedback loop that acts as a homeostatic "brake" on immune activation. Mechanistically, this regulatory axis profoundly impacts the T cell compartment, particularly in protein subunit vaccine recipients. In this group, adjuvant-mediated innate priming drives robust MAIT cell activation, which is paradoxically counterbalanced by the downregulation of the TGF-β decoy receptor TGFBR3 and upregulation of signaling receptors—rendering these cells more sensitive to suppression. Consequently, unlike mRNA vaccines that appear to bypass this extensive feedback to sustain maximal immunogenicity, viral vector and protein subunit platforms induce a self-regulated immune response via the TGF-β axis. These findings highlight a fundamental trade-off between robust immunogenicity and homeostatic control, offering a novel cellular rationale for the differential waning kinetics and protection durability observed across vaccine platforms.

overall_design: Peripheral blood mononuclear cells were obtained and isolated from bloods of vaccine recipients, and they were frozen in CELLBANKER for liquid nitrogen storage until library preparation. Nuclei were isolated using the 10x Genomics workflow for single-cell RNA and ATAC sequencing, and super-loaded onto the Chromium Next GEM Chip J to generate single-cell libraries. Final libraries were sequenced by Illumina NovaSeq 6000.

first: Hyo Jeong

last: Nam

first: Sunyoung

last: Jung

first: Nari

last: Byun

first: Jeong-Ryeol

last: Gong


title: Comprehensive Characterization of the Human Neural Stem Cell Line HNSC.100 as a Versatile Model for Neurobiological Research

city: Ulm

first: Estera

middle: Anna

last: Jeruzalska

address: James-Franck-Ring N27

department: Institute of Pharmaceutical Biotechnology

country: Germany

institute: Ulm University

summary: Studying neural-related questions is inherently challenging due to the limited number of suitable cell models. Here, we characterize a previously reported immortalized human neural stem cell line, HNSC.100, serving as a robust model for a wide range of neurobiological research questions. The cell line expresses key neural stem cell markers, including SOX2, vimentin, nestin, and allows for efficient genetic manipulation. Furthermore, HNSC.100 cells can be differentiated into neurons, astrocytes and oligodendrocytes, thereby covering a wide spectrum of major neural cell types. We established a comprehensive panel of molecular markers to validate successful differentiation, enabling precise characterization of the resulting cell population. In addition, we provide a complete dataset of RNA expression levels for all detectable genes in HSNC.100 cells. Based on this dataset, we assembled a list of expressed genes implicated in neural disorders that can be studied with this cell line. Together, we present a detailed characterization of the HNSC.100 cell line and provide new tools and reference data to facilitate its use. This resource enables researchers to evaluate the line's suitability for specific applications and to rapidly integrate HNSC.100 cells into their experimental workflows.

overall_design: RNA sequencing of untreated HNSC.100 cells for expression analysis in this cell line

first: Carolin

last: Ketteler

first: Elisa

last: Stuetzenberger

first: Sandra

last: Burczyk

first: Larissa

last: Moeller

first: Dierk

last: Niessing


title: MHC Class I on Target Cells Regulates CD4+ T cell-mediated Immunity

city: Houston

first: Akira

last: Yamamoto

state: Texas

address: One Baylor Plaza

country: Japan

institute: Baylor College of Medicine

summary: The central tenet of T cell immune surveillance is that MHC class I and class II molecules present antigens to CD8+ and CD4+ T cells, respectively. Here, we uncover a surprising and previously unrecognized role for MHC class I in modulating CD4+ T cell-mediated immunity. Using multiple in vivo models of allogeneic graft-versus-host disease (GVHD) and tumor immunity, we demonstrate that the absence of MHC class I on target cells significantly increases their susceptibility to CD4+ T cell cytotoxicity. Transcriptomic and functional studies indicate this heightened sensitivity to enhanced ferroptosis of the target cells without affecting the degree of CD4+ T cell activation or inflammatory responses. The experimental results are corroborated utilizing multiple large human transcriptomic and sequencing datasets that suggest a role for CD4+ T cells in enhancing immune checkpoint blocker-mediated responses in patients with melanoma and mismatch repair-deficient (MMRd) colon cancers that have downregulated MHC class I. These findings revise and expand the known role of MHC class I in CD8+ T cell and NK cell immunity and demonstrate a previously unrecognized role in CD4+ T cell-mediated cancer and alloimmunity.

overall_design: Libraries were prepared from a single cell suspension of intestinal epithelial cells using 10x Chromium Next GEM Single Cell 3' GEM v3.1 Library Prep. Libraries were sequenced on NovaSeq X Plus 25B 2x150. At day 6 post-transplant, four biological replicates each of Cre– and Cre+ mice were collected for analysis.

first: Emma

last: Lauder

first: Pavan

last: Reddy


title: Ontogeny and transcriptional regulation of Thetis cells

first: Chrysothemis

last: Brown

address: 417 East 68th Street

department: Immunology Program, Sloan Kettering Institute


title: Stretching inhibits tumor growth in MMTV-PYMT via a direct mechanical effect

city: Bethesda

first: Lisbeth

last: Berrueta

state: MD

address: 30 Convent drive, 5th floor room 532

institute: NIDCR/NIH

summary: Background: Tumor-associated architecture and emerging mechanical properties (forces, pressure, tension, stiffness) affect the growth and invasiveness of cancer cells. Collagen fibers aligned perpendicular to the boundaries of tumors promote local tumor invasiveness in mouse mammary tumor models and are associated with a poor prognosis in human breast cancer. Our previous study revealed that daily gentle stretching (~25% strain) for 10 min reduced the growth of P53/PTEN-/- orthotopic mouse mammary tumors by 50%. Results: In this study, we further investigated the mechanism of stretching in a more aggressive MMTV-PYMT (mammary tumor virus- polyomavirus middle T antigen) tumor model in vivo and in vitro and analyzed its impact on collagen reorganization at both the tumor-stromal interface and the tumor microenvironment composition at single cell level. Stretching reduced the average tumor size by 30–50% in orthotopic (Active and Passive Stretch) and transgenic (Passive Stretch) models. In the orthotopic model, the ratio of parallel vs. perpendicular collagen fibers relative to the tumor boundary, was greater in the Stretch group compared with the No Stretch group. Finally, stretching reduced the cell migration of collagen-embedded tumor spheroids in vitro. Conclusions: These results show that short-duration, moderate-amplitude stretching reduces tumor growth in several different animal models.  We also provide evidence that this beneficial effect may be a direct mechanical effect on local matrix properties and tumor cell invasiveness

overall_design: A tissue microarray (TMA) paraffin block was prepared containing 1mm sections from each of the tumors (N= 40 ).  Then, a 5 mm FFPE tissue section was obtained from the TMA block and  applied onto the Xenium slide,  withing the imaging area;  this section was  deparaffinized and permeabilized to make the mRNA accessible. The mRNAs were targeted by a panel of custom designed probes (n=100). Probe hybridization was performed overnight at 50 °C with a probe concentration of 10 nM. After a stringency wash to remove un-hybridized probes, the probes were ligated at 37 °C for two hours, during which a rolling circle amplification  was performed. The circularized probes were then enzymatically amplified, producing multiple copies of the gene-specific barcode for each RNA binding event. After washing, background fluorescence was chemically quenched. The xenium slide was then placed into an imaging cassette for loading onto the xenium analyzer instrument. We used a custom designed panel containing  100 genes, which were chosen based on previous studies using single cell transcriptomics on the MMTV PYMT model to study tumor microenvironment cell composition and distribution,  including the use of conserved markers to identify cell populations such as: epithelial cells (luminal/basal), macrophages, T cells, NK cells, endothelial cells and fibroblasts

first: Chandra

last: Bhan

first: Yi

last: He

first: Rebecca

middle: D

last: Thompson

first: Oscar

last: Safairad

first: Andrew

last: Doyle

first: Paola

last: Perez

first: Daniel

last: Martin

first: Mazen

last: Mezher

first: Alexander

last: Cartagena-Rivera

first: Gary

last: Badger

first: Helene

middle: M

last: Langevin


title: Reduced expression of mitochondrial transcription factor A (TFAM) in CD8⁺ T cells drives senescence-associated inflammatory lung injury during respiratory viral infection

city: Grand Forks

first: Kai

last: Guo

state: ND

address: 1301 N Columbia Rd Stop 9037

institute: University of North Dakota

summary: Aging leads to dysregulated immune responses that worsen the severity of respiratory viral infections. As part of this dysfunction, CD8⁺ T cells exhibit altered inflammatory regulation, contributing both to viral clearance and to adverse lung injury. However, the cell-intrinsic mechanisms driving age-associated shifts in CD8⁺ T cell function remain poorly understood. Analysis of mouse and human single-cell datasets reveals that reduced expression of mitochondrial transcription factor A (TFAM) is a conserved feature of aging and acute viral illness, correlating with heightened inflammatory programs in CD8⁺ T cells. To define the functional consequences of this decline, we generated a CD8⁺ T cell–specific TFAM haploinsufficient mouse model (TFAM⁺/⁻) that recapitulates the physiological reduction of TFAM observed in aged human CD8⁺ T cells. We show that reduced TFAM expression is sufficient to disrupt mitochondrial integrity, activate mitochondrial DNA stress pathways, and prime CD8⁺ T cells toward a senescence-like state at homeostasis. During influenza infection, TFAM-insufficient CD8⁺ T cells mount an exaggerated early hyper-cytotoxic effector response and rapidly acquire exhaustion features. Despite adequate viral clearance, TFAM⁺/⁻ mice exhibit sustained inflammatory lung injury. Together, these findings demonstrate that aging-like TFAM insufficiency reprograms CD8⁺ T cell behavior through mitochondrial dysfunction, driving early inflammatory hyperactivation, premature exhaustion, and persistent lung immunopathology.

overall_design: Wild-type (WT) C57BL/6, TFAMfl/fl, and CD8α-Cre mice purchased from the Jackson Laboratory and bred in-house. In this Cre-expressing strain, unlike the proximal Cd8a promoter, the E8I enhancer is not active during the double-positive (DP) stage of thymocyte development. Its activity is restricted until the cells have committed to the CD8 single-positive (SP) lineage, allowing Cre-mediated deletion to occur only in mature CD8 T cells and avoids any 'off-target' recombination in the CD4 lineage. An equal number of male and female mice, aged 8-10 weeks, were included in the study. All animal experiments were performed in accordance with the guidelines of the University of Florida Institutional Animal Care and Use Committee. Mice were provided ad libitum access to food and water. CD8α-Cre mice were crossed with TFAMfl/fl mice to achieve a heterozygous TFAM deletion in CD8α-expressing cells, resulting in TFAM+/− mice. For Influenza A virus (strain A/Puerto Rico/8/1934 H1N1; PR8), Mice were anesthetized with a 4% (v/v) isoflurane and intranasally inoculated with 250 PFUs of IAV in 100 μL PBS. Mock-infected mice received 100 μL PBS. Following euthanasia by CO2 exposure and cervical dislocation (D7 and D14), the lungs were perfused, and BALF was collected at by instillation and aspiration of the lung with 1 ml of sterile ice-cold PBS through a 20-gauge tracheal catheter (BD Biosciences), and lungs were aseptically collected and processed for downstream analyses.

first: Nadeem

last: Khan

first: Zahrasadat

last: Navaeiseddighi

first: Zhihan

last: Wang


title: ATPase activity is essential to the silencing activity of SMCHD1

city: Heidelberg

first: Quentin

last: Gouil

state: VIC

address: Studley Road

country: Australia

institute: Olivia Newton-John Cancer Research Institute

summary: We characterise the E147A ATPase mutant of SMCHD1, including its effect on  gene expression in murine neural stem cells

overall_design: RNA-seq of male and female neural stem cells, wt or heterozygous for the SMCHD1 E147A mutation

first: Iromi

last: Wanigasuriya

first: Kelsey

last: Breslin

first: Tamara

last: Cameron

first: Alexandra

last: Gurzau

first: Marnie

last: Blewitt


title: NanoString PanCancer IO 360 expression profiling of histologically normal breast lobules from NHBW and NHWW donors (IO360)

city: Jacksonville

first: Derek

middle: Charles

last: Radisky

state: Florida

address: 4500 San Pablo Road

department: Cancer Biology

institute: Mayo Clinic

summary: Histologically normal breast lobules were obtained from non-Hispanic Black women (NHBW) and non-Hispanic White women (NHWW) (n=10 per group) matched by age, BMI, and parity. RNA extracted from FFPE sections was profiled using the NanoString PanCancer IO 360 CodeSet to characterize transcriptional programs associated with group differences in benign breast epithelium.

overall_design: Twenty histologically normal breast lobule samples (10 NHBW, 10 NHWW) were profiled on the NanoString PanCancer IO 360 panel. Raw RCC files are provided along with normalized expression values.

first: Joshua

middle: W

last: Ogony

middle: C


title: Effects of Aerobic Exercise on Depression-Like Behaviors and Hippocampal Transcriptomics in CSDS-Induced Adolescent Mice

city: Chengdu

first: Xia

last: Jiang

state: Sichuan province

address: West China School of Public Health and West China Fourth Hospital, Sichuan University, No.16, Section 3, South Renmin Road, Wuhou District, Chengdu, 610041, China

department: Department of Nutrition and Food Hygiene, West China School of Public Health and West China Fourth Hospital, Sichuan University, Chengdu, China.

institute: Sichuan University

summary: Background: Adolescence is a critical period of vulnerability to depression, yet most evidence for the antidepressant effects of exercise derives from adult models. This study aims to investigate the effects of aerobic exercise and identify its associated molecular changes in the adolescent depression mouse model. Methods: A chronic social defeat stress (CSDS) model was established in adolescent male C57BL/6J mice to induce depression-like behaviors. Thirty-six mice were randomly assigned to three groups: control (CG), model (MG), and model plus exercise (ME). MG and ME mice were subjected to CSDS for two weeks (days 7–20), while ME mice additionally received three weeks of aerobic treadmill training covering the whole CSDS period (days 0–20). Behavioral tests were conducted on days 21–26, followed by the collection of serum and hippocampal tissues for molecular, histological, and transcriptomic analyses. Results: CSDS induced significant depression-like behaviors in adolescent male mice, including social avoidance, anhedonia, and behavioral despair, all of which were effectively alleviated by aerobic exercise. Aerobic exercise appeared to mitigate CSDS-induced neural damage and maintain hippocampal tissue integrity. Moreover, aerobic exercise increased serum levels of serotonin. Transcriptomic analysis identified 587 differentially expressed genes (DEGs). Among these genes, 59 overlapping DEGs were regulated by both CSDS and exercise, and were enriched in carbohydrate metabolism and cholinergic signaling pathways. Conclusions: Aerobic exercise alleviates depression-like behaviors in adolescent male mice, possibly by modulating hippocampal gene expression – especially in cholinergic and carbohydrate metabolism pathways – which provides potential clues to how exercise could influence peripheral monoamine levels and hippocampal structural integrity. These putative mechanisms require further investigation.

overall_design: This study used RNA-seq to characterize hippocampal transcriptomic alterations induced by chronic social defeat stress (CSDS) and their modulation by aerobic exercise in adolescent male C57BL/6J mice. Animals were randomly assigned to three experimental groups: control (CG), CSDS model (MG), and CSDS with aerobic exercise intervention (ME), with six independent biological replicates per group. Following completion of behavioral testing, hippocampal tissues were collected from each mouse, and total RNA was extracted for RNA-seq analysis. Gene-level expression profiles were generated and used for differential gene expression analysis to identify CSDS-associated transcriptional changes and exercise-responsive molecular pathways.

first: Linna

last: Sha

first: Rong

last: Xiang

first: Ting

last: Yu

first: Jinyu

last: Zhou

first: Tao

last: Han

first: Yangdan

last: Zhong

first: Qin

last: Deng

first: Jiaojiao

last: Hou

first: Bin

last: Yang

first: Xiaomeng

last: Li

first: Lishi

last: Zhang

first: Jinyao

last: Chen


title: PTP1B inhibition promotes microglial phagocytosis in Alzheimer's disease models by enhancing SYK signaling

city: Woodbury

first: Jonathan

last: Preall

state: NEW YORK

address: 500 Sunnyside Blvd

institute: Cold Spring Harbor Laboratory

summary: Amyloid-β (Aβ) accumulation is a hallmark of Alzheimer’s disease (AD). Emerging evidence suggests that impaired microglial Aβ phagocytosis is a key feature in AD, highlighting the therapeutic potential of enhancing this innate immune function. Here, we demonstrate that genetic deletion or pharmacological inhibition of protein tyrosine phosphatase 1B (PTP1B) ameliorated memory deficits and reduced Aβ burden in APP/PS1 mice. Moreover, we show that PTP1B was highly expressed in microglia, and its deficiency promoted a transcriptional shift toward immune activation and phagocytosis. Consistently, PTP1B deletion in microglia enhanced phagocytosis and metabolic fitness, supported by increased AKT-mTOR signaling, a pathway essential for meeting the energy demands of activation. Mechanistically, we identified spleen tyrosine kinase (SYK), a key regulator of microglial phagocytosis, as a direct substrate of PTP1B. Inhibition of SYK showed that PTP1B modulates microglial activation in a SYK-dependent manner. These findings established PTP1B as a critical modulator of microglial activation and a potential therapeutic target for AD.

overall_design: 13-month-old female mice from APP/PS1;PTP1B+/+ and APP/PS1;PTP1B-/- groups were sacrificed for the sample preparation. Mice were perfused with cold HBSS, and hippocampal tissues were dissociated in dissection buffer (DPBS Gibco, 14-287-080). Tissues dissociation was performed using the Adult Brain Dissociation Kit (Miltenyi Biotech, 130-107-677) according to the manufacturer’s instructions, with manual trituration. Briefly, the chopped tissues were incubated in digestion buffer at 37 °C in a water bath for 15 min, with gently tapping of the tubes 3-4 times during the digestion. Following digestion, the tissues were triturated with fire-polished glass pipettes to obtain a single cell preparation, which was then filtered through a 70 μm cell strainer. To minimize ex vivo microglial activation, transcriptional and translational inhibitors were added in perfusion, dissection and digestion buffer as described (Marsh et al, doi: 10.1038/s41593-022-01022-8). In each sample, cells from two animals were pooled together.

first: Yuxin

last: Cen

first: Steven

middle: R

last: Alves

first: Christy

last: Felice

first: Dongyan

last: Song

first: Linda

last: Van Aelst

first: Nicholas

middle: K

last: Tonks


title: Effects of trichostatin A and phenamil methanesulfonate treatment on gene expression during osteogenic differentiation of senescent rMSCs

city: Cheonan

first: Cheng Ji

address: 119 Dandea-ro, Dongnam-gu

institute: ITREN

summary: Cell fate transitions are governed by coordinated programs involving epigenetic regulators and lineage-specific transcription factors. RNA sequencing was performed to profile transcriptomic responses of senescent rat mesenchymal stem cells (rMSCs) subjected to a sequential small-molecule treatment paradigm designed to enhance osteogenic competence. TSA treatment increased expression of the epigenetic regulator KDM1B and was associated with activation of early epigenetic/lineage priming programs. Subsequent phenamil treatment increased BMP2 expression and enrichment of BMP-responsive signaling, consistent with activation of downstream osteogenic gene networks. Collectively, this dataset provides a molecular framework for how epigenetic modulation followed by pro-osteogenic signaling can cooperate to restore osteogenic differentiation capacity in senescent rMSCs.

overall_design: Senescent rat mesenchymal stem cells (rMSCs) were allocated to experimental groups cultured in osteogenic induction medium, with or without addition of the indicated extracts (TSA and benzyl ether). After 24 hours of treatment to capture early transcriptional responses, total RNA was isolated, sequencing libraries were prepared using standard RNA-seq workflows, and samples were sequenced to generate gene-level expression profiles. Each condition was represented by a single sample (no biological replicates).

first: Jung-Hwan

last: Lee


title: NanoString expression profiling of histologically normal breast lobules from NHBW and NHWW donors (BC360)

first: Derek

last: Radisky

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

overall_design: Refer to individual Series


title: Targeting Modulated Vascular Smooth Muscle Cells in Atherosclerosis via FAP-Directed Immunotherapy [Xenium in situ]

city: St Louis

first: Junedh

last: Amrute

state: MO

address: 660 S Euclid Ave

institute: Washington University School of Medicine

summary: Vascular smooth muscle cell (VSMCs) cell diversification drives atherosclerotic coronary artery disease (CAD). Mechanisms governing these cell state transitions remain unclear. We applied multi-omic single-cell profiling, epitope mapping, and spatial transcriptomics across 27 human coronary arteries, identifying fibroblast activation protein (FAP) as a marker of modulated VSMCs. Lineage tracing in mice indicated that FAP⁺ cells originate from Myh11⁺ VSMCs, and FAP PET imaging in CAD patients showed plaque uptake. FAP⁺ cells states resided in the macrophage-rich neo-intima. Therapeutically, we developed an anti-FAP bispecific T-cell engager, which reduced plaque burden and remodeled the stromal–immune microenvironment through T-cell clonal expansion. Our study delivers a single-cell and spatial atlas of human CAD, establishes FAP as a marker of modulated VSMCs, and highlights immunotherapy for lipid-independent targets.

overall_design: Xenium in situ: A custom gene panel was designed (Supplementary Table 4) based on the CITE-seq reference map and combined with the Xenium Human Multi-Tissue and Cancer Panel (#1000626, 10x Genomics). FFPE blocks and sectioned and the 10x Genomics Xenium In Situ Gene Expression with Cell Segmentation Staining User Guide protocol (10x Genomics, Protocol number CG000749) used for sample (#1000661; #1000460; #1000487. 10x Genomics). 17 regions were analyzed.

first: In-Hyuk

last: Jung

first: Tracy

last: Yamawaki

first: Wen-Ling

last: Lin

first: Andrea

last: Bredemeyer

first: Johanna

last: Diekmann

first: Sikander

last: Hayat

first: Xianglong

first: Devin

last: Wakefield

first: Xin

last: Luo

first: Sidrah

last: Maryam

first: Gyu Seong

last: Heo

first: Chang Jie Mick

first: Chen

last: Wang

first: Caroline

last: Chou

first: Christoph

last: Kuppe

first: Kevin

last: Cook

first: Atilla

last: Kovacs

first: Vishnu

last: Chintalgattu

first: Danielle

last: Pruitt

first: Jose

last: Barreda

first: Nathan

last: Stitziel

first: Paul

last: Cheng

first: Yongjian

last: Liu

first: Rafael

last: Kramann

first: Daniel

last: Kreisel

first: Roger

last: Foo

first: Ingrid

last: Rulifson

first: Scott

last: Martin

first: David

last: Grunert

first: Melissa

last: Thomas

first: Jixin

last: Cui

first: Thomas

last: Quertermous

first: Frank

last: Bengel

first: Simon

last: Jackson

first: Chi-Ming

first: Brandon

last: Ason

first: Kory

last: Lavine


title: Glycosylated GM-CSF expands B-1b cells and B-1b plasma cells and programs them for immunosuppression

city: Würzburg

first: Haisam

last: Alattar

state: Bayern

address: Versbacherstr. 7

department: Inistitute of Virology and Immunobiology

institute: Wuerzburg University

summary: The myeloid growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) exhibits paradoxical pro- and anti-inflammatory functions, but the factors determining these divergent outcomes remain unclear. Here, we report that this functional divergence is controlled by its glycosylation. Murine recombinant fully glycosylated GM-CSF (rgGM-CSF) specifically induces immunosuppressive cell types, whereas its recombinant non-glycosylated counterpart (rngGM-CSF) promotes effector cells. Using single-cell ATAC-sequencing and flow cytometry, we show that rgGM-CSF has a previously unrecognized ability to effectively expand IL-10+ LAG-3+ PD-L1+ B-1b plasma cells (PCs) with immunosuppressive properties. Although rgGM-CSF also promotes the expansion of hematopoietic stem and progenitor cells (HSPCs) and monocytic myeloid-derived suppressor cells (M-MDSCs), adoptive transfer experiments clearly demonstrate that only the rgGM-CSF-induced B-1b PCs are primarily responsible for an IL-10-dependent long-term protection in mice from experimental autoimmune-encephalomyelitis (EAE). Our data suggest that glycosylation enhances the systemic bioavailability and activity of GM-CSF and promotes the expansion of immunoregulatory cells rather than pro-inflammatory myeloid effector cells. Together, these results demonstrate that the 'dual activity' of GM-CSF is controlled by its glycosylation, resulting in opposing immune functions. These findings support a re-evaluation of human rgGM-CSF (regramostim) as a potential therapeutic strategy for immunosuppression in transplantation and autoimmune diseases.

overall_design: Single-cell ATAC-sequencing (scATAC-seq) was performed to characterize chromatin accessibility changes in splenic hematopoietic stem and progenitor cells (HSPCs) and associated immune cell populations following in vivo administration of glycosylated GM-CSF. C57BL/6 mice were injected intraperitoneally once daily for 10 days with recombinant fully glycosylated GM-CSF, while untreated mice served as controls. On day 11, spleens were harvested and single-cell suspensions were prepared. HSPCs were enriched by fluorescence-activated cell sorting as lineage-negative (Ter119⁻, B220⁻, CD4⁻, CD8⁻), CD11b⁻, c-Kit⁺ cells. To enable biological replication within a single sequencing run, cells from one wild-type and one luciferase-transgenic mouse per condition were pooled at a 1:1 ratio prior to library preparation. The luciferase transgene sequence was used as a genetic barcode to assign individual cells back to their respective animals during downstream bioinformatic analysis.

first: Arpa

last: Aintablian

first: Ina

middle: N

last: Eckert

first: Haroon

last: Shaikh

first: Laura

last: Cyran

first: Zheng

first: Bhupesh

last: Prusty

first: Fabian

last: Imdahl

first: Elisabeth

last: Zinser

first: Andreas

last: Beilhack

first: Nadine

last: Hövelmeyer

first: Björn

middle: E

last: Clausen

first: Tom

last: Gräfenhan

first: Florian

last: Erhard

first: Manfred

last: Lutz


title: Targeting Modulated Vascular Smooth Muscle Cells in Atherosclerosis via FAP-Directed Immunotherapy [Visium]

first: Junedh

last: Amrute

overall_design: Visium FFPE: Fresh coronary tissue was fixed in 4% PFA overnight, washed with PBS, and embedded in FFPE blocks. FFPE blocks were sectioned onto Visium slides and underwent H&E staining before imaging.  Libraries were generated using the Visium FFPE v1 kit (#1000188; #1000362; #1000364) and sequenced on a NextSeq2000 according to manufacturer’s recommendations. Libraries were sequenced on a NovaSeq 6000. Xenium in situ: A custom gene panel was designed (Supplementary Table 4) based on the CITE-seq reference map and combined with the Xenium Human Multi-Tissue and Cancer Panel (#1000626, 10x Genomics). FFPE blocks and sectioned and the 10x Genomics Xenium In Situ Gene Expression with Cell Segmentation Staining User Guide protocol (10x Genomics, Protocol number CG000749) used for sample (#1000661; #1000460; #1000487. 10x Genomics,).

first: In-Hyuk

last: Jung

first: Tracy

last: Yamawaki

first: Wen-Ling

last: Lin

first: Andrea

last: Bredemeyer

first: Johanna

last: Diekmann

first: Sikander

last: Hayat

first: Xianglong

last: Zhang

first: Devin

last: Wakefield

first: Xin

last: Luo

first: Sidrah

last: Maryam

first: Gyu Seong

last: Heo

first: Steven

last: Yang

first: Chang Jie Mick

last: Lee

first: Chen

last: Wang

first: Caroline

last: Chou

first: Christoph

last: Kuppe

first: Kevin

last: Cook

first: Atilla

last: Kovacs

first: Vishnu

last: Chintalgattu

first: Danielle

last: Pruitt

first: Jose

last: Barreda

first: Nathan

last: Stitziel

first: Paul

last: Cheng

first: Yongjian

last: Liu

first: Rafael

last: Kramann

first: Daniel

last: Kreisel

first: Roger

last: Foo

first: Ingrid

last: Rulifson

first: Scott

last: Martin

first: David

last: Grunert

first: Melissa

last: Thomas

first: Jixin

last: Cui

first: Thomas

last: Quertermous

first: Frank

last: Bengel

first: Simon

last: Jackson

first: Chi-Ming

last: Li

first: Brandon


title: Cis-regulation of IL-22 gene in type 3 immune cells [RNA-Seq]

city: Columbus

first: Ankita

last: Saini

state: Ohio

address: 460 W 12th Ave

department: Microbial Infection and Immunity

institute: The Ohio State University Wexner Medical Center

summary: IL-22 is a signature cytokine of type 3 immune cells which include ILC3s and Th17/22. Despite its critical role in barrier tissue integrity, the field lacks the understanding of cis mechanisms regulating IL-22. We took initiative to study and reveal the regulatory mechanisms controlling IL-22 expression in ILC3s and Th17/22 cells.

overall_design: Mnk3 cells were either treated for ~16 hours with IL-1b+IL-23 or left untreated.

first: Eugene

last: Oltz


title: Targeting Modulated Vascular Smooth Muscle Cells in Atherosclerosis via FAP-Directed Immunotherapy [Foam_Cell]

first: Junedh

last: Amrute

overall_design: THP-1 cells (#TIB-202, ATCC;) were cultivated in RPMI-1640 medium (#10-041-CV, Corning;) with 10% FBS. They were then treated with PMA (#5.00582.0001, Millipore Sigma) to reach 15 nM concentration and plated in a 12-well plate (#150628, Thermo Fisher Scientific) at a density of 5 x 105 cells per well. The plate was incubated for a duration of 3 days to promote differentiation. On the fourth day, the cells were gently washed with 1 mL of fresh medium to remove any residual PMA and other soluble factors. Following this, oxLDL (#L34357, Invitrogen) was introduced to each well to reach a final concentration of 50 µg mL-1, ensuring thorough mixing for uniform exposure. The cells were incubated for an additional four days to allow for foam cell formation. Upon completion of the incubation period, the medium was carefully aspirated from each well. The cells were detached by adding 200 µL of trypsin-EDTA (#25-053-CI, Corning) per well and incubating at 37°C for 5 min to facilitate cell detachment. To neutralize the trypsin activity, 1 mL of medium was added per well and the mixture was gently pipetted to ensure complete suspension of the cells. Finally, the cell solution was filtered through a 100 µm nylon cell strainer (#352360, Falcon) to remove any cell clumps, thereby obtaining a uniform single-cell suspension ready for further analysis.

first: In-Hyuk

last: Jung

first: Tracy

last: Yamawaki

first: Wen-Ling

last: Lin

first: Andrea

last: Bredemeyer

first: Johanna

last: Diekmann

first: Sikander

last: Hayat

first: Xianglong

last: Zhang

first: Devin

last: Wakefield

first: Xin

last: Luo

first: Sidrah

last: Maryam

first: Gyu Seong

last: Heo

first: Steven

last: Yang

first: Chang Jie Mick

last: Lee

first: Chen

last: Wang

first: Caroline

last: Chou

first: Christoph

last: Kuppe

first: Kevin

last: Cook

first: Atilla

last: Kovacs

first: Vishnu

last: Chintalgattu

first: Danielle

last: Pruitt

first: Jose

last: Barreda

first: Nathan

last: Stitziel

first: Paul

last: Ason

first: Kory

last: Lavine


title: Targeting Modulated Vascular Smooth Muscle Cells in Atherosclerosis via FAP-Directed Immunotherapy [SMC_Foam_Cell

first: Junedh

last: Amrute

overall_design: Human primary coronary artery smooth muscle cells (SMC) (Lot# 20TL266549, Cat# CC-2583, Lonza) were cultured in completed SmGM-2 growth medium (#CC-3182, Lonza). Foam cell conditioned medium was collected from cultures of THP-1-derived foam cells as previously described, with supernatant retrieval following centrifugation at 150 g for 5 minutes and filtration through a 0.22 μm membrane filter (#GSWP02500, Millipore). On day 0, the medium swap group was plated in 6-well plates (#140675, Thermo Fisher Scientific) at a density of 1 x 105 cells per well in SmGM basal medium containing 5% FBS (BM-FBS medium). On day 4 and day 8, the medium was replaced with fresh BM-FBS medium containing 50% foam cell conditioned medium. The control group was plated at the same density in completed SmGM-2 growth medium three days before harvest. On day 12, SMCs were detached by adding 1 mL of 0.05% trypsin-EDTA (#25300-054, Gibco) per well and incubating at 37°C for 3 minutes, then neutralized with 10 mL of BM-FBS medium. Viable cells were counted using trypan blue solution (#15250061, Gibco). The cell suspension was subsequently filtered through a 100 µm nylon cell strainer (#352360, Falcon) to remove clumps, resulting in a uniform single-cell suspension for further analysis.

first: In-Hyuk

last: Jung

first: Tracy

last: Yamawaki

first: Wen-Ling

last: Lin

first: Andrea

last: Bredemeyer

first: Johanna

last: Diekmann

first: Sikander

last: Hayat

first: Xianglong

last: Zhang

first: Devin

last: Wakefield

last: Cheng

first: Yongjian

last: Liu

first: Rafael

last: Kramann

first: Daniel

last: Kreisel

first: Roger

last: Foo

first: Ingrid

last: Rulifson

first: Scott

last: Martin

first: David

last: Grunert

first: Melissa

last: Thomas

first: Jixin

last: Cui

first: Thomas

last: Quertermous

first: Frank

last: Bengel

first: Simon

last: Jackson

first: Chi-Ming

last: Li

first: Brandon


title: Cis-regulation of IL-22 gene in type 3 immune cells [ATAC-seq_Mnk3]

first: Ankita

last: Saini

first: Eugene

last: Oltz


title: Targeting Modulated Vascular Smooth Muscle Cells in Atherosclerosis via FAP-Directed Immunotherapy

first: Junedh

last: Amrute

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Expansion of outer cortical layer Cux2+ neurons required selective adaptations for DNA repair

city: Los Angeles

first: Riki

last: Kawaguchi

state: CA

address: 760 Westwood Plaza, Room 37-420

department: Department of Psychiatry and Neurology Department of Psychiatry

institute: University of California, Los Angeles

summary: During mammalian evolution, upper cortical layer 2/3 excitatory neurons have shown a disproportionate expansion compared to other layers. Replicative expansion of cortical neural progenitors is associated with significant oxidative DNA damage. Here we show that Activating Transcription Factor 4 (Atf4) has new roles as a critical regulator of the DNA Damage Response, directly activating components of double stranded DNA repair, including CIRBP, Uba52 and Ebf1. Strikingly, pan-cortical knockout (Emx1cre, Atf4fl/fl) demonstrates that Atf4 is specifically required for development of upper layer 2/3 neurons, marked by expression of CUT-homeodomain protein, Cux2.  Atf4 functions to repair DNA damage and attenuate cell death of embryonic radial glial progenitors in a p53-dependent manner.  In particular, we show that Cold-Inducible RNA-Binding Protein (Cirbp) is a transcriptional target of Atf4 that is required for the normal phosphorylation of the key double strand DNA repair factor Ataxia-Telangiectasia Mutated (ATM). These findings establish that Atf4 is an essential regulator of the DNA Damage Response. They further indicate extraordinary requirements for DNA repair post-replicative stress in Cux2+ neurons during mammalian brain development.

overall_design: We collected the E11.5 embryonic cortex from timed-pregnant mice, and after the genotyping been done, three same genotype cortex been pool together as one to get enough sample for snRNA sequencing, Atf4 fl/fl as control and Emx1-Cre, Atf4 fl/fl as knockout group


title: Cis-regulation of IL-22 gene in type 3 immune cells [ATAC-seq]

overall_design: E22-1 or E22-2 cis-elements were deleted from Mnk3 cells using Cas9-RNP system. Single-cell-derived subclones lacking either E22-1 or E22-2 were obtained, along with their corresponding sister subclones. The cells were treated for ~16 hours with IL-1b+IL-23 or left untreated.


title: Cis-regulation of IL-22 gene in type 3 immune cells

overall_design: Mnk3 cells (a mouse ILC3 cell line model) were treated for ~16 hours with IL-1b+IL-23 or left untreated. The cells were then harvested and CUT&RUN was performed using following antibodies: Runx3 (Clone 2B3; Invitrogen, MA5-17169), mouse IgG control (Diagenode, C15400001-100), RORγt (Abcam, ab207082), and rabbit IgG control (Diagenode, C15410206). The CUTANA™ ChIC/CUT&RUN (EpiCypher, 14-1048) and the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher, 14-1001) were used for the experiments. The libraries were sequenced on Illumina NovaSeq X Plus platform to obtain > 10 million paired-end reads per sample.


title: Generation of persistent HIV CAR-TSCM cells by treatment with a novel common-gamma-chain cytokine-based fusion molecule

city: Bronx

first: Harris

last: Goldstein

address: 1300 Morris Park Avenue

department: Pediatrics

institute: Albert Einstein College of Medicine

summary: Anti-CD3/CD28 (αCD3/28) activation efficiently enables T cells to be transformed by transduction with CAR-expressing lentiviral vectors (LV) into chimeric antigen receptor (CAR)-T cells. However, the long-term effectiveness of this approach is compromised by the more differentiated phenotype of these CAR-Ts, associated with reduced in vivo functional persistence. Treatment with IL-7, IL-15, and/or IL-21 expands CAR-Ts with a T-stem-cell (TSCM) phenotype. We investigated whether we could use an alternative strategy to generate CAR-Ts, by using a scaffold-linked IL-7, IL-15, and IL-21 (HCW9206) to render T cells permissive for LV transduction and thereby promote the generation of HIV-specific duoCAR-T cells (duoCAR-T) enriched in a TSCM phenotype to enhance in vivo persistence. Compared to αCD3/28-generated duoCAR-Ts, HCW9206-generated duoCAR-Ts were markedly enriched for TSCM (> 50%), displayed potent in vitro HIV-1 viral suppression, and antigen-induced TNFα and IFNγ production. RNA sequencing analysis of the transcriptome of duoCAR-T cells generated by IL-7, IL-15, and IL-21 treatment delivered by HCW9206, compared to αCD3/αCD28 activation, identified multiple uniquely expressed genes. These results suggested that duoCAR-T-HCW9206 cells are enriched in both a TSCM phenotype, as indicated by pro-survival and TSCM-like gene signatures, as well as more differentiated effector-like phenotypes, likely contributing to the potent HIV suppression observed both in vitro and in vivo, despite primarily displaying a TSCM phenotype. Our results indicate that scaffold-linked IL-7, IL-15, and IL-21 fusion-treatment represents an alternative method for CAR-T cell generation, more effective than standard αCD3/28 activation for producing functionally persistent CAR-Ts.

overall_design: To evaluate the transcriptional differences between anti-HIV duoCAR-T cells generated by standard anti-CD3/CD28 stimulation (duoCAR-T-αCD3/28) and treatment with the novel human cytokine-based scaffold HCW9206 (duoCAR-T-HCW9206). The CAR-T transcriptomes were compared with or without stimulation with 293T cells expressing HIV-1 envelope protein (293T-gp120). CD8+ duoCAR-T-αCD3/28 cells and duoCAR-T-HCW9206 cells were cultured with 293T cells (unactivated) or cultured with 293T-gp120 cells (activated) for 16 hours. CD8+ duoCAR-T cells were purified by FACS, and total RNA was isolated. 2 biological replicates underwent treatment, cell sorting, RNA isolation, and sequencing independently.

first: Erin

last: Cole

first: Hong Beom

last: Hur

first: Christopher

last: Hiner

first: Manoj

last: Kandpal


title: DNA damage burden causes selective CUX2 neuron loss in neuroinflammation

summary: Neurodegeneration shows regional and cell-type-specific patterns in ageing and disease1, but the underlying mechanisms for cell type-specific neuronal losses remain poorly understood. Previous studies have shown upper cortical layer thinning in progressive human multiple sclerosis (MS) and that cortical layer 2/3 excitatory neurons (L2/3 ENs) that express CUT like homeobox 2 (CUX2) are selectively vulnerable to degeneration2. Here, we report that L2/3 ENs within MS cortical lesions have elevated DNA damage. DNA damage and selective loss of L2/3 ENs was recapitulated in diverse mouse models of demyelination and pan-cortical inflammation, confirming their intrinsic vulnerability. Functions of Cux2 and Activating transcription factor 4 (Atf4) were essential for resilience of L2/3 ENs during postnatal neuroinflammation, acting in neurons to enhance DNA double strand break repair. Interferon-γ, a cytokine implicated in MS pathogenesis3,4, was sufficient to elevate reactive oxygen species leading to DNA damage-mediated neuronal death in vitro and caused selective depletion of L2/3 neurons in mice. These findings indicate that DNA damage burden and inadequate repair in CUX2+ L2/3 ENs contributes to selective vulnerability in neuroinflammatory injury.

overall_design: We collected E18.5 Wildtype, Cux2-Cre/Cre, Cux2-Cre/Wt;Atf4 fl/fl, Cux2-Cre/Cre;Atf4 fl/fl whole cortex to do the snRNA sequencing to compare the gene expression changes in between the different genotypes


title: Targeting Modulated Vascular Smooth Muscle Cells in Atherosclerosis via FAP-Directed Immunotherapy [Mouse_Aorta_BiTE]

overall_design: In Vivo BiTE Treatment Studies Animal studies were performed in compliance with guidelines set forth by the National Institutes of Health Office of Laboratory Animal Welfare and approved by the Washington University institutional animal care and use committee. Animals were housed in a controlled environment with a 12 h light–dark cycle, with free access to water and a standard chow diet.  All experimental mice were fed a high fat diet containing 21% fat by weight (42% kcal from fat) and 0.2% cholesterol (#TD.88137, Envigo Teklad) for 4, 16, or 20 weeks (For Apoe-/-) starting at 8 or 9 weeks of age. For all studies an isotype BiTE was used for the control group and an anti-FAP BiTE for the treatment group. Animals were injected intraperitoneally and dosed at 400 μg/kg. For the acute model animals were treated with BiTE once a week starting week 1 for a total of 4 injections. For chronic studies mice were treated once a week in the same manner starting at 12 weeks of HFD feeding.  Single cell RNA-seq in murine atherosclerosis The ascending aorta (from beginning of aorta up to aortic arch including first intercostal artery) were isolated and transferred to an enzymatic dissociation cocktail with 250 U ml-1 Collagenase IV (#C5138, Sigma Aldrich), 60 U ml-1 Hyaluronidase (#H3506), and 60 U ml-1 DNase I (#D4527, all purchased from Sigma Aldrich) in Dulbecco’s Modified Eagle Medium (DMEM) and slightly minced. After incubation at 37 °C for 60 min with agitation, the digestion reaction was quenched with 6 mL of HBB buffer (2% Fetal Bovine Serum (FBS) and 0.2% Bovine Serum Albumin (BSA) in Hank’s Balanced Salt Solution (HBSS)), then filtered through 70 μm filters. Samples were pelleted by centrifugation at 4 °C, 1,400 rpm for 5 min and the supernatant was discarded. Cells were resuspended in FACS buffer (2% FBS and 2mM Ethylenediaminetetraacetic acid (EDTA) in calcium/magnesium free PBS) and centrifugation was repeated in above conditions and supernatant aspirated. Cell pellet was resuspended in 100 µL FACS buffer with the addition of 1 µL of DRAQ5 (#564907, Thermo Fisher Scientific) and incubated on ice for 30 min. Solution was washed with FACS buffer three times following same centrifugation as above and then resuspended in 500 µL of FACS buffer and 1 µL DAPI (#564907, BD Biosciences,) and filtered into filter-top FACS tubes. First singlets were gated and subsequent DRAQ5+/DAPI- events were collected in 300 µL cell resuspension buffer (0.04% BSA in PBS) – collected cells were centrifuged as above and resuspended in collection buffer to a target concentration of 1,000 cells µL-1. Cells were counted on a hemocytometer before proceeding with the 10x protocol. Collected cells were processed using the single Cell 3’ Kit v 3.1 (#1000268, 10x Genomics). 10,000 cells were loaded onto ChipG (#1000121) for GEM generation. Reverse transcription, barcoding, and complementary DNA amplification of the RNA was performed as recommended in the 3’ v3.1 chromium protocol. Libraries were sequenced on a NovaSeq 6000.  Isolation of T-cells for scRNA-seq and TCR-seq in murine atherosclerosis The ascending aorta (from beginning of aorta up to aortic arch including first intercostal artery) were isolated and transferred to an enzymatic dissociation cocktail with 2 U ml-1 Liberase (#5401127001, Sigma Aldrich), and 2 U ml-1 Elastase (#LS002279, Worthington) in DMEM and slightly minced. After incubation at 37 °C for 45 min with agitation, the digestion reaction was quenched with 6 mL of HBB buffer, then filtered through 70 μm filters. Samples were pelleted by centrifugation at 4 °C, 1,400 rpm for 5 min and the supernatant was discarded. Cells were resuspended in FACS buffer and centrifugation was repeated in above conditions and supernatant aspirated. Cell pellet was resuspended in 100 µL FACS buffer with the addition of 1 µL of CD45, CD11b, and CD3 and incubated on ice for 30 min. Solution was washed with FACS buffer three times following same centrifugation as above and then resuspended in 500 µL of FACS buffer and filtered into filter-top FACS tubes. First singlets were gated and subsequent CD45+/CD11b-/CD3+ events were collected in 300 µL cell resuspension buffer (0.04% BSA in PBS) – collected cells were centrifuged as above and resuspended in collection buffer to a target concentration of 1,000 cells µL-1. Cells were counted on a hemocytometer before proceeding with the 10x protocol. Collected cells were processed using the single Cell 5’ Kit v 2 (#1000244, 10x Genomics). 10,000 cells were loaded onto ChipK (#2000182) for GEM generation. Reverse transcription, barcoding, and complementary DNA amplification of the RNA were performed as recommended in the 5’ v2 chromium protocol. V(D)J amplification and library construction was performed per protocol (#1000252, 10x Genomics). Libraries were sequenced on a NovaSeq 6000.

first: In-Hyuk

last: Jung

first: Tracy

last: Yamawaki

first: Wen-Ling

last: Lin

first: Andrea

last: Bredemeyer

first: Johanna

last: Diekmann

first: Sikander

last: Hayat

first: Xianglong

last: Zhang

first: Devin

last: Wakefield

first: Xin

last: Luo

first: Sidrah

last: Maryam

first: Gyu Seong

last: Heo

first: Steven

last: Yang

first: Chang Jie Mick

last: Lee

first: Chen

last: Wang

first: Caroline

last: Chou

first: Christoph

last: Kuppe

first: Kevin

last: Cook

first: Atilla

last: Kovacs

first: Vishnu

last: Chintalgattu

first: Danielle

last: Pruitt

first: Jose

last: Barreda

first: Nathan

last: Stitziel

first: Paul

last: Cheng

first: Yongjian

last: Liu

first: Rafael

last: Kramann

first: Daniel

last: Kreisel

first: Roger

last: Foo

first: Ingrid

last: Rulifson

first: Scott

last: Martin

first: David

last: Ason

first: Kory

last: Lavine


title: Targeting Modulated Vascular Smooth Muscle Cells in Atherosclerosis via FAP-Directed Immunotherapy [Human_CITEseq]

first: Junedh

last: Amrute

overall_design: Ethical Approval for Human Specimens The study is compliant with all relevant ethical regulations and has been approved by the Washington University School of Medicine Institutional Review Board (IRB #201104172). Informed consent was obtained from each patient prior to tissue collection by Washington University School of Medicine, and no compensation was provided in exchange for subject participation in the study. All demographic and clinical data have been de-identified and provided in Supplementary Table 1. Patients included in this study span diverse races, ages, and sexes to provide an inclusive trans-ethnic study population.  Inclusion Criteria Prior to tissue collection, specific inclusion criteria were applied to ensure well-controlled study groups. Any patients with HIV or hepatitis and known genetic cardiomyopathies were excluded from this study. The left anterior descending coronary artery was isolated and flash frozen from donor hearts: patients with stable ejection fractions and no known history of cardiac disease who experienced a non-cardiac cause of death/transplant and patients with chronic heart failure. For all samples, the proximal left anterior descending coronary artery was used.  Human single cell isolation for CITE-seq Fresh human coronary arteries from explanted hearts at the time of transplantation or donors declared dead after cardiac death (DCD) or brain death (DBD) at Washington University School of Medicine and Mid America Transplant Service were harvested, perfused with cardioplegia, and transported on ice. Starting at the coronary sinus, the left main, left anterior descending, and left circumflex arteries were dissected removing any epicardial fat while preserving the adventitia. Sections were partitioned for histopathology, spatial transcriptomics, single cell isolation, and flash frozen. Cells were isolated as before (17). Briefly: Coronaries were minced using a razor blade on ice and transferred to a 15 mL conical tube containing 3 mL DMEM with 170 µL Collagenase IV (250 U mL-1 final concentration), 35 µL DNAse1 (60 U mL-1), and 75 µL Hyaluronidase (60 U mL-1, all purchased from Sigma Aldrich) and incubated at 37 °C for 45 min with agitation. After 45 min, the digestion reaction was quenched with 6 mL of HBB buffer (2% FBS and 0.2% BSA in HBSS), filtered through 40 µm filters into a 50 mL conical tube and transferred back into a 15 mL conical tube to obtain tighter pellets. Samples were then centrifuged at 4°C, 1,200 rpm for 5 min, and the supernatant was discarded. The pellets were resuspended in 1 mL ACK Lysis buffer (#A10492-01, Gibco) and incubated at room temperature for 5 min followed by the addition of 9 mL DMEM and centrifugation at 4°C, 1,200 rpm for  5 min. Supernatant was discarded and the pellet was resuspended in 2 mL FACS buffer (2% FBS and 2 mM EDTA in calcium/magnesium free PBS), and centrifugation was repeated in above conditions and supernatant aspirated. The TotalSeq A 277 panel (BioLegend) antibody cocktail was resuspended in 100 µL of FACS buffer and 1 µL each of custom oligo-labeled FAP (Amgen) and LRRC15 (Amgen) were added.  The combined 102 µL were used to resuspend the pellet with the addition of 1 µL of DRAQ5 (#564907, Thermo Fisher Scientific) and incubated on ice for 30 min. Solution was washed with FACS buffer three times following same centrifugation as above and then resuspended in 500 µL of FACS buffer and 1 µL DAPI (#564907, BD Biosciences), then filtered into filter-top FACS tubes. First singlets were gated and subsequent DRAQ5+/DAPI- events were collected in 300 µL cell resuspension buffer (0.04% BSA in PBS). The collected cells were centrifuged as above and resuspended in collection buffer to a target concentration of 1,000 cells µL-1. Cells were counted on a hemocytometer before proceeding with the 10x protocol.

first: In-Hyuk

last: Jung

first: Tracy

last: Yamawaki

first: Wen-Ling

last: Lin

first: Andrea

last: Bredemeyer

first: Johanna

last: Diekmann

first: Sikander

last: Hayat

first: Xianglong

last: Zhang

first: Devin

last: Wakefield

first: Xin

last: Luo

first: Sidrah

last: Maryam

first: Gyu Seong

last: Heo

first: Steven

last: Yang

first: Chang Jie Mick

last: Lee

first: Chen

last: Wang

first: Caroline

last: Chou

last: Grunert

first: Melissa

last: Thomas

first: Jixin

last: Cui

first: Thomas

last: Quertermous

first: Frank

last: Bengel

first: Simon

last: Jackson

first: Chi-Ming

last: Li

first: Brandon


title: Lack of phosphoenolpyruvate carboxykinase 1 in hepatic stellate cells causes liver fibrosis by fueling tricarboxylic acid cycle and increasing glycolysis

city: Santiago de Compostela

first: Rubén

last: Nogueiras Pozo

state: A Coruña

address: Campus Vida Avenida Barcelona, s/n, CIMUS

department: Fisiology

country: Spain

institute: CIMUS - USC

summary: Phosphoenolpyruvate carboxykinase (PCK1) is a key integrator of hepatic energy metabolism, but its role in hepatic stellate cells (HSCs), the main fibrogenic cells in the liver, remains unknown. We found that PCK1 is reduced in HSCs from fibrotic animals and people with fibrosis, correlating negatively correlated with fibrosis severity. Silencing PCK1 activates human HSCs and increases fibrotic markers, whereas ectopic PCK1 expression blunts transforming growth factor β1-induced activation. Activated HSCs show elevated glycolysis and tricarboxylic acid cycle activity, but PCK1 overexpression reduces acetyl-CoA, limiting tricarboxylic acid cycle intermediates and ameliorating HSCs activation. In mice, HSC-specific PCK1 loss accelerates diet-induced liver fibrosis. Notably, mice lacking PCK1 in HSCs also develop spontaneous fibrosis on a normal diet. These findings show that disrupted cataplerosis from PCK1 loss enhances glycolysis and activates HSCs, promoting liver fibrosis.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for liver tissue. The study compares liver transcriptomes from HSC-PCK1-KO mice versus Control (WT) mice (n=4 biological replicates per group). All mice were adult and fed a CDHFD for 8 weeks prior to tissue collection.

first: Eva

last: Novoa

first: Tamara

last: Parracho

first: Julica

last: Inderhees

first: Amaia

last: Rodriguez

first: Cristina

last: Riobello

first: Jose

last: Iglesias-Moure

first: Anabel

last: Fernández-Iglesias

first: Sara

last: Martinez-Martinez

first: Ana

last: Senra

first: Samuel

last: Seoane

first: Marcos

last: Fondevila

last: Iglesias

first: Alba

last: Cabaleiro

first: Natalia

last: da Silva Lima

first: Valentina

last: Dorta

first: Borja

last: López-Picallo

first: Lucia

last: Ramos-Lage

first: Ashwin

last: Woodhoo

first: Miguel

last: Fidalgo

first: Maria

last: Martínez-Chantar

first: Francisco Javier

last: Cubero

last: López

first: Carlos

last: Dieguez

first: Diana

last: Guallar

first: Vincent

last: Prevot

first: Robert

last: Schwabe

first: Gema

last: Frühbeck

first: Javier

last: Crespo

first: Marta

last: Varela-Rey

last: Gonzalez-Rellan

first: Jordi

last: Gracia-Sancho

first: Paula

last: Iruzubieta

first: Markus

last: Schwaninger

first: Ruben

last: Nogueiras


title: Hetereogenity of liver Treg in the CDAHFD model of MASH

city: Boston

first: CBDM

last: Lab

state: MA

address: 77 Avenue Louis Pasteur

department: Microbiology and Immunobiology

institute: Harvard Medical School

summary: Metabolic-dysfunction–associated steatohepatitis (MASH) is a chronic liver disease driven by the confluence of metabolic stress and destructive hepatic inflammation. The immunoregulatory mechanisms that temper this process remain poorly understood. Exploiting a complementary pair of murine models and published single-cell RNA-sequencing (scRNA-seq) datasets from human liver, we uncovered a critical protective role for Foxp3+CD4+ regulatory T cells (Tregs) in MASH. Tregs progressively accumulated in the diseased liver, adopting an activated, nonlymphoid-tissue phenotype marked by the expression of Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) and a reparative transcriptional program. Punctual ablation of Tregs during established MASH unleashed a catastrophic inflammatory cascade, including exaggerated T-helper (Th)1, Th2 and Th17 responses, expansion of a pathogenic CD8+ T cell population, and hepatocellular injury. Concomitantly, Treg deficiency disrupted key metabolic pathways in the liver, accelerating disease progression. These findings establish Tregs as non-redundant custodians of both immunologic and metabolic homeostasis in the liver, highlighting their promise as targets for temporally tuned immunoregulatory therapies in metabolic liver disease.

overall_design: We investigated the transcriptional heterogeneity of liver regulatory T cells (Tregs) 8 days after feeding with CDAHFD or control diet via single-cell RNA sequencing (scRNA-seq). Tregs from livers of Foxp3Gfp mice ere sorted by flow cytometry and encapsulated using the Chromium Single Cell 3′ v2 platform.

first: Bola

last: Hanna

first: Paul

last: Langston

first: Diane

last: Mathis


title: Cis-regulation of IL-22 gene in type 3 immune cells [Chip-seq]

first: Ankita

last: Saini

first: Eugene

last: Oltz


title: Gene silencing of ANGPTL3 suppresses α1-antitrypsin and lectin mannose-binding 1 expression in the hepatoma Huh7 cell line

city: Padova

first: Giulio

last: Ceolotto

address: Via Orus 2B

department: Medicine

country: Italy

institute: University of Padova

summary: Background: Angiopoietin-like 3 (ANGPTL3) is a hepatokine acting as negative regulator of hepatic and endothelial lipases. Vupanorsen, an antisense oligonucleotide targeting ANGPTL3, reduced triglycerides up to 60% in a phase 2b trial, but caused dose-dependent increases in hepatic fat fraction. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plasma levels also correlates with liver fat accumulation. Methods: To further elucidate the biological activity of ANGPTL3 on lipid metabolism in the liver we performed a RNAseq analysis from human hepatoma Huh7 cells transfected with siRNA-ANGPTL3, siRNA-PCSK9 and double siRNA-ANGPTL3/PCSK9. Results: RNA-seq analysis detected 192 genes out of 13,945 transcripts differential ex-pressed in response to ANGPTL3-siRNA compared to scramble-siRNA controls. In cells transfected with PCSK9-siRNA, 88 genes were differentially expressed, while dual transfection with both ANGPTL3- and PCSK9-siRNA resulted in 219 differentially ex-pressed genes. When ANGPTL3 gene expression was silenced we observed a compensa-tory induction of proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA and pro-tein expression. Moreover, PCSK9 silencing increased ANGPTL3 mRNA expression. Bi-oinformatic analysis revealed that gene silencing of ANGPTL3 or both ANGPTL3/PCSK9 suppresses SERPINA1, gene encoding α1-antitrypsin, and lectin mannose-binding 1 ex-pression (LMAN1). These data were confirmed by western blot and qRT-PCR analysis. Conclusions: Our data identified two key genes SERPINA1 and LMAN1, involved in endoplasmic reticulum stress, lipid accumulation and liver damage, that are downregu-lated in response to gene silencing of ANGPTL3 in Huh7 hepatoma cell line.

overall_design: Huh7 cells transfected with siRNA-ANGPTL3, siRNA-PCSK9 and double siRNA-ANGPTL3/PCSK9 for 48 hours

first: Ilaria

last: Rossi

first: Enidia

last: Hazizaj

first: Maria Giovanna

last: Lupo

first: Giorgia

last: Marodin

first: Stjin

last: Cos

first: Alessandra

last: Giannella

first: Nicola

last: Ferri


title: Chemotherapy-induced reactive myelopoiesis leads to expansion of immunosuppressive neutrophil-like monocytes in mice and humans [ATAC-seq]

city: Augusta

first: Huidong

last: Shi

state: GA

department: Georgia Cancer Center

institute: Augusta University

summary: Cytotoxic chemotherapy primarily targets rapidly proliferating cancer cells but also depletes normal myeloid cells. The resulting cell loss triggers reactive myelopoiesis, a compensatory process in which hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM) regenerate myeloid lineages. We previously showed that the alkylating agent cyclophosphamide (CTX) induces myelopoiesis leading to the expansion of immunosuppressive monocytes in mice. However, the molecular features and clinical relevance of these cells remain poorly understood. Here, we report the emergence of immunosuppressive monocytes in the peripheral blood of lymphoma patients receiving CTX-containing chemotherapy. To gain mechanistic insight into CTX-induced myelopoiesis, we performed single-cell RNA sequencing (scRNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) on BM monocytes from CTX-treated mice. These analyses revealed a heterogeneous monocyte population and demonstrated that CTX skews myelopoiesis toward the generation of neutrophil-like monocytes (NeuMo). Moreover, CTX-induced NeuMo cells, enriched within the CXCR4⁺CX3CR1⁻ monocyte subset, exhibited potent T-cell suppressive activity. Using the NeuMo gene signature, reanalysis of public scRNA-seq datasets identified a transcriptionally similar monocyte subset in chemotherapy-treated cancer patients. Collectively, our findings suggest that the expansion of NeuMo-like cells following chemotherapy represents a conserved immunoregulatory feedback mechanism with potential impact on tumor response to chemoimmunotherapy.

overall_design: Nuclei were prepared from freshly isolated monocytes (CD11b+Ly6Chi) by fluorescence-activated cell sorting (FACS) and used for the ATAC-seq library construction. The differential analysis was used to detect the changes in the chromatin accessibility after CTX treatment in monocytes.

first: Gang

last: Zhou

first: Zhichun

last: Ding


title: Chemotherapy-induced reactive myelopoiesis leads to expansion of immunosuppressive neutrophil-like monocytes in mice and humans [scRNA-Seq]

overall_design: For scRNA-seq of whole bone marrow cells, bone marrows were collected from cyclophosphamide-treated mice on day 2 (CTX_D2) and day 7 (CTX_D7). Bone marrows from untreated mice were used as the control (naïve). Live cells were enriched using the EasySep Dead Cell Removal kit (Stemcell Technologies). Equal numbers of cells from three individual mice under each condition were mixed and the pooled samples were used for downstream sequencing. For scRNA-seq of pre-enriched monocytes, bone marrow cells were stained with anti-CD11b and anti-Ly6C antibodies and sorted for CD11b+Ly6Chi monocytes by Bigfoot Spectral Cell Sorter. The purity of sorted monocytes was greater than 98%. Freshly isolated whole bone marrow cells or monocytes (CD11b+Ly6Chi) were processed for scRNA-seq libraries using the Chromium Controller (10X Genomics).


title: Characterization of esophageal squamous cell carcinoma cell lines sensitivity to Palbociclib

city: Brussels

first: Xavier

last: Bisteau

address: Route de Lennik 808

country: Belgium

institute: Université Libre de Bruxelles

summary:
Background: Esophageal squamous cell carcinoma (eSCC) is a highly aggressive malignancy with poor prognosis and limited therapeutic options. Although immune checkpoint inhibitors such as nivolumab, have shown clinical benefit, particularly in patients with high PD-L1 expression, this subgroup represents only a small fraction of eSCC cases. CDK4/6 inhibitors such as palbociclib have only been tested as second-line agents in eSCC, often in combination with EGFR inhibitors, with minimal benefit.
Methods: Our study evaluates palbociclib as a first-line therapy in treatment-naive eSCC models. Using a panel of 22 eSCC cell lines with integrated multi-omics and phenotypic assays.
Results: We identified three response subtypes, resistant, delayed and arrested, correlated to Rb-pathway status. Interestingly, in delayed responders, palbociclib induced replication stress, DNA damage, and unprotected micronuclei enriched for cGAS, triggering activation of interferon-stimulated genes. Consistent with this, palbociclib enhanced immune cell infiltration in delayed eSCC spheroids within a preclinical vascularized 3D microfluidic system.
Conclusions: Our study demonstrates that first-line palbociclib treatment unmasks intrinsic vulnerabilities in the CDK4/6-Rb axis and triggers innate immune activation in molecularly defined eSCC. Using a translationally relevant 3D vascularized microfluidic system, we provide evidence that early CDK4/6 inhibition not only stall cancer cell growth but also promotes immune cells recruitment. In conclusion, our study identifies palbociclib as a viable first-line therapeutic candidate in selected eSCC patients and uncover its immunomodulatory potential. These findings support further research into CDK4/6 inhibition as first-line treatment for eSCC and highlight its potential to influence the tumor immune microenvironment in ways that could improve responses to combination therapies.

overall_design:
We analyzed 22 human esophageal squamous cell carcinoma cell lines to investigate transcriptional responses to CDK4/6 inhibition. The cell lines used in this study were: TE-1, TE-4, TE-5, TE-6, TE-8, TE-9, TE-10, TE-11, TE-14, TE-15, KYSE-30, KYSE-70, KYSE-140, KYSE-150, KYSE-180, KYSE-270, KYSE-410, KYSE-450, KYSE-510, KYSE-520, COLO-680N, ECGI10
Each cell line was cultured under standard conditions and treated with either DMSO (vehicle control) or 1 μM palbociclib for 6 days. Total RNA was extracted at the end of treatment, and bulk RNA-seq was performed. For each condition, experiments were conducted in biological duplicate.

first: Fabiana

last: Moresi


title: Chemotherapy-induced reactive myelopoiesis leads to expansion of immunosuppressive neutrophil-like monocytes in mice and humans

overall_design: Refer to individual Series


title: Haldane’s rule in the developing brain: X-linked and imprinted genes contribute to male-biased transgressive expression in hybrid mice

city: Madrid

first: Lena

last: Arévalo

state: Madrid

address: Avda. Puerta de Hierro, 18

department: Animal Reproduction

institute: INIA-CSIC

summary: The placenta and developing brains of eutherian mammals are intimately connected and enriched for imprinted gene (IG) expression. In hybrids, the placenta is a hotspot for genetic and epigenetic incompatibilities that preferentially affect males. Whether the hybrid brain is similarly affected is an open question. We evaluated the effects of hybridization and placental dysregulation on neural gene expression (embryonic, juvenile, adult) and behavior (juvenile, adult) in a cross between the house mouse (Mus m. domesticus) and Algerian mouse (M. spretus), in which transgressive expression of imprinted and X-linked genes is a prominent feature of undersized F1 male placentas. In hybrid hypothalamus, transgressive expression was mainly embryonic and highly male-biased. Although fewer genes were dysregulated in hybrid male embryonic hypothalamus relative to placenta, we infer a similar genetic architecture involving X-IG incompatibilities, and incompatibilities between IGs and autosomal interaction partners. Hybrids had heightened anxiety-like behaviors and reduced activity relative to parental species, with a tendency towards larger behavioral differences in adult males. As the first study to characterize the effects of interspecific hybridization on neural gene expression and behavior in a mammal, this work extends the scope of phenotypes impacted by intrinsic incompatibilities and demonstrates that the brain obeys Haldane’s rule.

sample_organism:
Mus musculus domesticus
Mus spretus
Mus musculus domesticus x Mus spretus

overall_design: hypothalamic transcriptomes of M. m. domesticus, M. spretus and M. m. domesticus/M. spretus hybrids at three developmental time points (embryo, juvenile, adult)

first: Polly

last: Campbell


title: Heterozygous NFKB1 variant causes inflammatory dysregulation shaped by broader genetic context in common variable immunodeficiency

first: Pushpinder

last: Bawa

state: Massachusetts

address: 670 Albany St

department: Boston University Medical Campus

institute: Center for Regenerative Medicine

summary: Common variable immunodeficiency (CVID) is the most prevalent symptomatic primary antibody deficiency. For unclear reasons, inflammatory complications, like gastrointestinal (GI) disease, occur in ~50% of CVID cases, worsening morbidity and mortality. NFKB1 variants are among the most frequent genetic variants in CVID. While impact of NFKB1 variants is not well understood, we previously found frameshift heterozygous NFKB1 variants to increase cytokines, monocytes, and inflammatory complications in CVID. In this report, we used induced pluripotent stem cell (iPSC)-derived monocytes (iMONOs) with CRISPR/Cas9-mediated gene editing to study a heterozygous NFKB1 frameshift found in a CVID patient with severe GI disease. The heterozygous NFKB1 variant similarly reduced NFKB1 protein in CVID patient and healthy donor derived iMONOs, but elevated lipopolysaccharide-induced IL-1β release and expression of inflammatory genes, including IL1B, IL6, TNF, and neutrophil chemoattractants only in CVID patient iMONOs. CVID patient iMONOs also had elevations of IL-12, CCL4, and CCL12 unaffected by presence or absence of the NFKB1 variant. TNF antagonism improved the patient’s GI disease, diminishing neutrophilic gastritis, circulating neutrophils, and the neutrophil chemoattractant CXCL1 in the blood. While the biology remains complex, our approach found heterozygous NFKB1 variant-induced inflammatory changes intensified in CVID iMONOs, corresponding with clinical response to TNF antagonism.

overall_design: Isogenic comparisons are made between iPSC-derived monocytes from a CVID patient with a heterozygous NFKB1 variant that has or has not been corrected by gene editing as well as iPSC-derived monocytes from a healthy donor in which the heterozygous NFKB1 variant has or has not been introduced by gene editing.

first: Kayes

last: Kevin M

first: Bawa

last: Pushpinder

first: Maglione

last: Paul J.


title: Analysis of mRNA expression in piRNA FERPIR (DQ69304) knocked down mouse cardiomyocytes

city: Qingdao

first: Kun

last: Wang

state: Shandong

address: 308 Ningxia Road

institute: Qingdao University

summary: To elucidate the molecular mechanism by which piRNA FERPIR (DQ69304) regulates gene expression, RNA-seq was performed on control or FERPIR-knockdown mouse neonatal cardiomyocytes. Based on RNA-seq data, 2513 differentially expressed genes were shown in  FERPIR-knockdown cardiomyocyte relative to NC control, including 1132 upregulated and 1391 downregulated genes.

overall_design: FERPIR Antagomir or Negative control (NC) were transfected into mouse neonatal cardiomyocytes. Total RNAs were extracted from Antagomir or Negative control knockdown cardiomyocytes. Then, high throughput RNA sequencing was performed to identify differences in the gene expression profile.

first: Fang

last: Liu


title: Prenatal low-dose MeHg exposure leads to proteomic and transcriptomic alterations consistent with neurodegenerative disease in the cerebellum of C57BL/6 mice

city: Ottawa

first: Jing

state: Ontario

address: 501 smyth road

institute: University of Ottawa

summary: Methylmercury (MeHg) is a global pollutant that readily crosses the blood–brain barrier and placenta, posing significant risks to fetal neurodevelopment. While the cerebellum is a recognized target of MeHg toxicity in adults, the mechanisms underlying fetal exposure remain poorly defined. In this study, we investigated the neurotoxic effects of low-dose MeHg exposure (0.2 ppm via drinking water) on the cerebellums of prenatal C57BL/6 mice using integrated transcriptomic and proteomic analyses. Cerebellar tissues collected from postnatal day 90–120 (P90–120) mice (n = 3/group) were processed for RNA sequencing and proteomics analysis. Differentially expressed genes (DEGs) and proteins (DEPs) revealed significant changes in multiple pathways associated with neurodegeneration, including Huntington’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Overlapping transcriptomic and proteomic findings identified potential underlying mechanisms such as chemical carcinogenesis driven by reactive oxygen species and retrograde endocannabinoid signaling, underscoring the central role of oxidative stress in MeHg-induced neurotoxicity. Collectively, these results indicate that prenatal MeHg exposure induces persistent molecular alterations consistent with neurodegenerative processes and synaptic dysfunction, despite the absence of overt behavioral changes at the time of sacrifice. The long-term consequences for delayed symptom onset and the potential contribution of these changes to the etiology of neurodevelopmental disorders warrant further investigation.

overall_design: Pregnant C57BL/6 mice were dosed with 0 ppm or 0.2 ppm MeHg via drinking water starting from the first day of their pregnancy (E0) until birth (P0). The 0.2 ppm MeHg solution was prepared by a 1:5000 dilution of a stock solution of 4mM MeHg(II)Cl (cat#33553, Alfa Aesar) in drinking water. At sacrifice, the brains were dissected, and the cerebellum was isolated and immediately frozen with liquid nitrogen and stored at -80°C for analysis.

first: Allison

last: Loan

first: Rochelle

last: D’Mello

first: Yingxi

last: Li

first: Tuana

last: Nurkan

first: Zoran

last: Minic

first: Hing Man

last: Chan


title: Genome-wide profiling of histone modification H3K27ac and CTCF transcription factor binding in Rett syndrome iPS cells

city: Tokyo

first: Yutaka

last: Saito

department: Artificial Intelligence Research Center (AIRC)

institute: National Institute of Advanced Industrial Science and Technology (AIST)

summary: Rett syndrome (RTT) is a rare neurodevelopmental disorder that primarily affects females. It is caused by mutations in the MECP2 gene located on the X chromosome, which plays a critical role in normal brain development and function. The mutation induces epigenetic changes and chromatin re-arrangement which lead to alteration to H3K27ac and CTCF binding in the genome. To determine the epigenetic changes in RTT cells, we performed ChIP-seq on healthy and Rett syndrome iPS cells. RTT increased the H3K27ac marks and reduce CTCF binding, which supports the evidence of MeCP2 role in chromatin alterations.

overall_design: We performed chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for histone modification H3K27ac and transcription factor CCCTC-binding factor (CTCF) in both wild-type and MeCP2-mutated (RTT) iPS cells. Two biological replicates were used and the experiments were performed at most three replicates. Peak calling and differential ChIP-seq peak analysis was performed to elucidate the genomic changes occurred in RTT cells.

first: Wang

last: Yicheng

first: Ab Ghani

last: Nur Syatila

first: Dutta

last: Munmee

first: Adachi

last: Shungo

first: Katoh

last: Kaoru

first: Namihira

last: Masakazu

first: Mitsuyama

last: Toutai

first: Saito

last: Yutaka


title: Cryptic redundancy between PAR1b and PAR1a, two members of the PAR1 kinase family, in the survival of PAR1b-knockout mice

city: Sapporo

first: Naoko

last: Murata-Kamiya

state: Hokkaido

address: Kita 15, Nishi 7, Kita-ku

department: Institute for Genetic Medicine

institute: Hokkaido University

summary: The PAR1/MARK family of serine/threonine kinases (Par1a-d) redundantly regulates cell polarity. Furthermore, PAR1b facilitates nuclear translocation of BRCA1 through phosphorylation. Within the nucleus, BRCA1 protects stalled replication forks and repairs DNA double-strand breaks through homologous recombination. Consequently, BRCA1-knockout mice are embryonically lethal. In contrast, conventional PAR1b-knockout mice reach adulthood, suggesting a compensatory mechanism for PAR1b in regulating BRCA1 in these knockout mice. Here we find that while PAR1b knockout leads to the death of wild-type mouse embryonic fibroblasts (MEFs), PAR1b-null MEFs can be readily established from conventional PAR1b-knockout mice. In PAR1b-null MEFs from PAR1b-knockout mice, PAR1a, but not PAR1c or PAR1d, is essential for cell survival. Thus, PAR1a-mediated cryptic redundancy is unleashed only when functional PAR1b is absent from the onset of embryogenesis, which induces epigenetic changes in gene regulation. This study presents the first evidence of cryptic redundancy driven by epigenetic modifications in the regulation of gene expression during early embryogenesis.

overall_design: ATAC-seq of mouse embryonic fibroblasts (MEFs) prepared from wild-type and conventional PAR1b-knockout mice

first: Adriana

middle: A

last: Del Valle Lazarte

first: Ippei

last: Kikuchi

first: Masanori

last: Hatakeyama


title: Mesenchymal stem cells receive adaptive islet-derived exosomal miR-151 to promote β-cell compensation in obesity

city: Nanjing

first: Xinwei

last: Guo

state: -- None selected --

institute: China Pharmaceutical university

type: Non-coding RNA profiling by high throughput sequencing

summary: C57/B6J mice were fed with NCD or HFD for 9 weeks. The isolated islets were resuspended and cultured in a serum-sEVs-free medium for 48 hours. The culture medium was collected for sEVs isolation. Total RNA from sEVs were extracted using the miRNeasy Mini Kit (Qiagen, USA). A total of 5 ng RNA was used for miRNA library construction with the NEBNexR UltraTM small RNA Sample Library Prep Kit for IlluminaR (NEB, USA). The library was sequenced using the Illumina NovaSeq 6000 platform.

overall_design: miRNA-seq of sEVs derived from NCD and HFD islets.