scholarly journals ECOA-2. Immuno-sensitization of Glioblastoma to NY-ESO-1 Targeting via Promoter Demethylation

2021 ◽  
Vol 3 (Supplement_2) ◽  
pp. ii1-ii1
Author(s):  
Thomas Lai ◽  
Janet Treger ◽  
Jingyou Rao ◽  
Tie Li ◽  
Albert Lai ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Promoter Methylation ◽  
Bisulfite Sequencing ◽  
Malignant Gliomas ◽  
Cancer Testis Antigens ◽  
Single Cell Rna Sequencing ◽  
Cancer Testis

Abstract Introduction The immunotherapeutic targeting of New York-esophageal squamous cell carcinoma (NY-ESO-1) and other cancer/testis antigens (CTA) is an appealing strategy for the treatment of malignant gliomas because CTA are not expressed in most normal adult tissues and their expression can be induced in tumors for targeting by T-cells. Basally, NY-ESO-1 is often poorly expressed in glioblastoma (GBM), presumably through promoter methylation. Mechanisms governing the expression of CTA have been explored in other cancers; however, neither the prevalence of NY-ESO-1 downregulation in GBM patient tumors nor the presumed mechanism of downregulation by promoter methylation in GBM has been formally established. Methods We characterized baseline CpG methylation of NY-ESO-1 in 30 bulk patient GBM samples, 10 patient-derived gliomaspheres, and three established tumor cell lines using bisulfite sequencing. We induced NY-ESO-1 expression in vitro in U251 human GBM cells using the hypomethylating agent decitabine (DAC). We investigated the epigenetic response of DAC-treated U251 with bisulfite sequencing and NY-ESO-1 expression with quantitative real-time PCR. Lastly, we performed single-cell RNA sequencing on DAC-treated GBM U251 to evaluate tumor subpopulations that upregulate NY-ESO-1 and other co-expressed CTA after DAC treatment. Results Baseline NY-ESO-1 expression is associated with promoter methylation in the majority of GBM. Treatment of cells with 1 µM DAC every day for 4 days explicitly demethylated the promoter region of NY-ESO-1 and resulted in a 1000-fold increase in mRNA expression. DAC treatment upregulates NY-ESO-1 coordinately with other cancer/testis antigens CTAG2 and MAGEA4 as demonstrated by single-cell RNA sequencing. Conclusion Exposure of U251 to DAC results in promoter demethylation in NY-ESO-1 and increased expression of CTA. DAC treatment may therefore render GBM susceptible to targeting of these antigens by T-cells, revealing a feasible strategy of NY-ESO-1 and co-expressed CTA promoter demethylation to sensitize GBM to immunotherapy.

scholarly journals Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David S. Fischer ◽  
Meshal Ansari ◽  
Karolin I. Wagner ◽  
Sebastian Jarosch ◽  
Yiqi Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Ex Vivo ◽  
Severe Disease ◽  
Human Leukocyte ◽  
Cell Receptor ◽  
Single Cell Rna Sequencing

AbstractThe in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

IMMU-30. IDENTIFICATION OF COORDINATELY REGULATED IMMUNO-MODULATORY GENES USING SINGLE-CELL RNA SEQUENCING OF DECITABINE TREATED GLIOMA CELLS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi99-vi99
Author(s):  
Thomas Lai ◽  
Janet Treger ◽  
Jingyou Rao ◽  
Robert Prins ◽  
Richard Everson
Keyword(s):  
New York ◽  
T Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Bisulfite Sequencing ◽  
Malignant Gliomas ◽  
Glioma Cells ◽  
Sequencing Data ◽  
Single Cell Rna Sequencing ◽  
Cell Data

Abstract INTRODUCTION The immunotherapeutic targeting of New York-esophageal squamous cell carcinoma (NY-ESO-1) and other cancer/testis antigens (CTA) is an appealing strategy for the treatment of malignant gliomas because CTA are not expressed in most normal adult tissues and their expression can be induced in tumors for targeting by T-cells. Basally, NY-ESO-1 is often poorly expressed in glioblastoma (GBM), presumably through promoter methylation. Our previous data has shown that the hypomethylating agent decitabine (DAC) is a strong sensitizer of GBM to NY-ESO-1 specific adoptive T-cell induced cytotoxicity. However, we hypothesized that DAC alters expression of other immuno-modulatory genes in addition to NY-ESO-1 that may also increase T-cell mediated killing in GBM. The extent of regulation of other immuno-modulatory genes to DAC demethylation has not yet been thoroughly investigated. METHODS We performed single-cell RNA sequencing on DAC and non-DAC treated glioma cells. We confirmed DAC treatment induced NY-ESO-1 expression with quantitative real-time PCR and demethylation using bisulfite sequencing. We analyzed our single-cell RNA sequencing data with Seurat and our customized R-based pipelines to identify coordinate differential expression of immuno-modulatory genes and their tumor subpopulations. RESULTS Using our single-cell data, we identified tumor subpopulations with coordinate differentially expressed immuno-modulatory genes including cancer testes antigens, antigen presentation proteins, and apoptosis regulators. Amongst these candidate genes, we validated their expression with qPCR and promoter demethylation with bisulfite sequencing and TA bisulfite cloning. CONCLUSION Exposure of glioma cells to DAC results in promoter demethylation of NY-ESO-1, with increased expression of CTA and other immunomodulatory genes. DAC treatment may therefore sensitize GBM to the immunotherapeutic targeting of these antigens and reveals a feasible strategy for clinical translation.

scholarly journals Single-cell RNA sequencing reveals in vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

2020 ◽  
Author(s):  
David S. Fischer ◽  
Meshal Ansari ◽  
Karolin I. Wagner ◽  
Sebastian Jarosch ◽  
Yiqi Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Severe Disease ◽  
Human Leukocyte ◽  
Cell Receptor ◽  
Phenotypic Profile ◽  
Single Cell Rna Sequencing

ABSTRACTThe in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we used single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induced transcriptional shifts by antigenic stimulation in vitro and took advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allowed identification of SARS-CoV-2-reactive TCRs and revealed phenotypic effects introduced by antigen-specific stimulation. We characterized transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and showed correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

scholarly journals 903 Human cancer-associated fibroblast subsets can predict immune checkpoint response in head and neck cancer patients

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A947-A947
Author(s):  
Diana Graves ◽  
Aleksandar Obradovic ◽  
Michael Korrer ◽  
Yu Wang ◽  
Sohini Roy ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
T Cells ◽  
Head And Neck Cancer ◽  
Cell Carcinoma ◽  
Head And Neck ◽  
Single Cell ◽  
Rna Sequencing ◽  
Neck Cancer ◽  
Squamous Cell ◽  
Single Cell Rna Sequencing

BackgroundUse of anti-PD-1 immune checkpoint inhibitors (ICI) is currently the first line therapy for recurrent/metastatic head and neck squamous cell carcinoma (HNSCC), but critical work remains in identifying factors guiding resistance mechanisms.1 2 While recent studies have specifically implicated cancer-associated fibroblasts (CAFs) as potential mediators of immunotherapy response, the immunoregulatory role of CAFs in head and neck cancer has not been thoroughly explored.3–5MethodsTo determine if there are changes in cell populations associated with anti-PD-1 therapy in head and neck cancer patients, we performed high dimensional single-cell RNA sequencing (scRNA-SEQ) from a neoadjuvant trial of 50 advanced-stage head and neck squamous cell carcinoma (HNSCC) patients that were treated with the anti-PD-1 therapy, nivolumab, for the duration of one month. Tumor specimens were analyzed pre- and post-treatment with single-cell RNA sequencing performed on 4 patients as well as bulk RNA sequencing on 40 patients. Matched scRNA-SEQ data was analyzed using the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) and Virtual Inference of Protein-activity by Enriched Regulon (VIPER) bioinformatic analysis platform to determine TME cells that correlated with response and resistance to nivolumab.6 For CAF functional studies, surgical tumor specimens were processed and enriched for CAF subtypes, and these were co-cultured with T cells from peripheral blood and tumor infiltrating lymphocytes.ResultsWe identified 14 distinct cell types present in HNSCC patients. Of these 14 cell types, the fibroblast subtype showed significant changes in abundance following nivolumab treatment. We identified 5 distinct clusters of cancer-associated fibroblast subsets (HNCAF-0, 1, 2, 3, and 4) of which, two clusters, HNCAF-0 and HNCAF-3 were predictive of patient response to anti-PD-1 therapy. To determine the significance of these CAF subsets’ function, we isolated HNCAF-0/3 cells from primary HNSCC tumor specimens and co-cultured with primary human T cells. Analysis by flow cytometry showed that HNCAF-0/3 reduced TGFβ-dependent PD-1+TIM-3+ exhaustion of T cells and increased CD103+NKG2A+ resident memory phenotype and cytotoxicity to enhance overall function.ConclusionsTo our knowledge, we are the first to characterize CAF heterogeneity within the head and neck TME and show direct immunostimulatory activity of CAFs. Our findings demonstrate the functional importance of CAF subsets in modulating the immunoregulatory milieu of the human HNSCC, and we have identified clinically actionable CAF subtypes that can be used as a biomarker of response and resistance in future clinical trials.Trial RegistrationNCT03238365ReferencesFerris RL, Blumenschein Jr G, Fayette J, Guigay J, Colevas AD, Licitra L, Harrington K, Kasper S, Vokes EE, Even C, et al. Nivolumab for recurrent squamous-cell carcinoma of the head and neck. N Engl J Med 2016;375:1856–1867.Seiwert TY, Burtness B, Mehra R, Weiss J, Berger R, Eder JP, Heath K, McClanahan T, Lunceford J, Gause C, et al. Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial. Lancet Oncol 2016;17:956–965.Dominguez CX, Muller S, Keerthivasan S, Koeppen H, Hung J, Gierke S, Breart B, Foreman O, Bainbridge TW, Castiglioni A, et al. Single-cell RNA sequencing reveals stromal evolution into LRRC15(+) myofibroblasts as a determinant of patient response to cancer immunotherapy. Cancer Discov 2020;10:232–253.Feig C, Jones JO, Kraman M, Wells RJ, Deonarine A, Chan DS, Connell CM, Roberts EW, Zhao Q, Caballero OL, et al. Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer. Proc Natl Acad Sci U S A 2013;110:20212–20217.Kieffer Y, Hocine HR, Gentric G, Pelon F, Bernard C, Bourachot B, Lameiras S, Albergante L, Bonneau C, Guyard A, et al. Single-cell analysis reveals fibroblast clusters linked to immunotherapy resistance in cancer. Cancer Discov 2020;10:1330–1351.Obradovic A, Chowdhury N, Haake SM, Ager C, Wang V, Vlahos L, Guo XV, Aggen DH, Rathmell WK, Jonasch E, et al. Single-cell protein activity analysis identifies recurrence-associated renal tumor macrophages. Cell 2021;184:2988–3005.Ethics ApprovalPatients provided informed consent for this work. All experimental procedures were approved by the Institutional Review Board of Vanderbilt University Medical Center (IRB: 171883).

scholarly journals The immune-suppressive landscape in lepromatous leprosy revealed by single-cell RNA sequencing

2022 ◽  
Vol 8 (1) ◽  
Author(s):  
Zihao Mi ◽  
Zhenzhen Wang ◽  
Xiaotong Xue ◽  
Tingting Liu ◽  
Chuan Wang ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Lepromatous Leprosy ◽  
High Expression ◽  
Expression Level ◽  
Intracellular Bacteria ◽  
Monocyte Subset ◽  
Single Cell Rna Sequencing ◽  
High Expression Level

AbstractLepromatous leprosy (L-LEP), caused by the massive proliferation of Mycobacterium leprae primarily in macrophages, is an ideal disease model for investigating the molecular mechanism of intracellular bacteria evading or modulating host immune response. Here, we performed single-cell RNA sequencing of both skin biopsies and peripheral blood mononuclear cells (PBMCs) of L-LEP patients and healthy controls. In L-LEP lesions, we revealed remarkable upregulation of APOE expression that showed a negative correlation with the major histocompatibility complex II gene HLA-DQB2 and MIF, which encodes a pro-inflammatory and anti-microbial cytokine, in the subset of macrophages exhibiting a high expression level of LIPA. The exhaustion of CD8+ T cells featured by the high expression of TIGIT and LAG3 in L-LEP lesions was demonstrated. Moreover, remarkable enhancement of inhibitory immune receptors mediated crosstalk between skin immune cells was observed in L-LEP lesions. For PBMCs, a high expression level of APOE in the HLA-DRhighFBP1high monocyte subset and the expansion of regulatory T cells were found to be associated with L-LEP. These findings revealed the primary suppressive landscape in the L-LEP patients, providing potential targets for the intervention of intracellular bacteria caused persistent infections.

Abstract A10: Using single-cell RNA sequencing to assess the impact of pancreatic oncogenic Kras on macrophage gene expression in vitro

2019 ◽  
Author(s):  
Katelyn Donahue ◽  
Yaqing Zhang ◽  
Veerin Sirihorachai ◽  
Stephanie The ◽  
Arvind Rao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Rna Sequencing ◽  
Single Cell Rna Sequencing ◽  
The Impact

scholarly journals Single-Cell RNA Sequencing Reveals Renal Endothelium Heterogeneity and Metabolic Adaptation to Water Deprivation

2019 ◽  
Vol 31 (1) ◽  
pp. 118-138 ◽  
Author(s):  
Sébastien J. Dumas ◽  
Elda Meta ◽  
Mila Borri ◽  
Jermaine Goveia ◽  
Katerina Rohlenova ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Oxidative Phosphorylation ◽  
Single Cell ◽  
Rna Sequencing ◽  
Water Deprivation ◽  
Metabolic Adaptation ◽  
Single Cell Rna Sequencing

BackgroundRenal endothelial cells from glomerular, cortical, and medullary kidney compartments are exposed to different microenvironmental conditions and support specific kidney processes. However, the heterogeneous phenotypes of these cells remain incompletely inventoried. Osmotic homeostasis is vitally important for regulating cell volume and function, and in mammals, osmotic equilibrium is regulated through the countercurrent system in the renal medulla, where water exchange through endothelium occurs against an osmotic pressure gradient. Dehydration exposes medullary renal endothelial cells to extreme hyperosmolarity, and how these cells adapt to and survive in this hypertonic milieu is unknown.MethodsWe inventoried renal endothelial cell heterogeneity by single-cell RNA sequencing >40,000 mouse renal endothelial cells, and studied transcriptome changes during osmotic adaptation upon water deprivation. We validated our findings by immunostaining and functionally by targeting oxidative phosphorylation in a hyperosmolarity model in vitro and in dehydrated mice in vivo.ResultsWe identified 24 renal endothelial cell phenotypes (of which eight were novel), highlighting extensive heterogeneity of these cells between and within the cortex, glomeruli, and medulla. In response to dehydration and hypertonicity, medullary renal endothelial cells upregulated the expression of genes involved in the hypoxia response, glycolysis, and—surprisingly—oxidative phosphorylation. Endothelial cells increased oxygen consumption when exposed to hyperosmolarity, whereas blocking oxidative phosphorylation compromised endothelial cell viability during hyperosmotic stress and impaired urine concentration during dehydration.ConclusionsThis study provides a high-resolution atlas of the renal endothelium and highlights extensive renal endothelial cell phenotypic heterogeneity, as well as a previously unrecognized role of oxidative phosphorylation in the metabolic adaptation of medullary renal endothelial cells to water deprivation.

scholarly journals Pathogenic Autoimmunity in Atherosclerosis Evolves From Initially Protective Apolipoprotein B 100 –Reactive CD4 + T-Regulatory Cells

Circulation ◽  
2020 ◽  
Vol 142 (13) ◽  
pp. 1279-1293 ◽  
Author(s):  
Dennis Wolf ◽  
Teresa Gerhardt ◽  
Holger Winkels ◽  
Nathaly Anto Michel ◽  
Akula Bala Pramod ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Apolipoprotein B ◽  
T Helper Cell ◽  
Helper Cell ◽  
Cell Type ◽  
T Helper ◽  
Single Cell Rna Sequencing ◽  
Helper Cell Type

Background: Throughout the inflammatory response that accompanies atherosclerosis, autoreactive CD4 + T-helper cells accumulate in the atherosclerotic plaque. Apolipoprotein B 100 (apoB), the core protein of low-density lipoprotein, is an autoantigen that drives the generation of pathogenic T-helper type 1 (T H 1) cells with proinflammatory cytokine secretion. Clinical data suggest the existence of apoB-specific CD4 + T cells with an atheroprotective, regulatory T cell (T reg ) phenotype in healthy individuals. Yet, the function of apoB-reactive T regs and their relationship with pathogenic T H 1 cells remain unknown. Methods: To interrogate the function of autoreactive CD4 + T cells in atherosclerosis, we used a novel tetramer of major histocompatibility complex II to track T cells reactive to the mouse self-peptide apo B 978-993 (apoB + ) at the single-cell level. Results: We found that apoB + T cells build an oligoclonal population in lymph nodes of healthy mice that exhibit a T reg -like transcriptome, although only 21% of all apoB + T cells expressed the T reg transcription factor FoxP3 (Forkhead Box P3) protein as detected by flow cytometry. In single-cell RNA sequencing, apoB + T cells formed several clusters with mixed T H signatures that suggested overlapping multilineage phenotypes with pro- and anti-inflammatory transcripts of T H 1, T helper cell type 2 (T H 2), and T helper cell type 17 (T H 17), and of follicular-helper T cells. ApoB + T cells were increased in mice and humans with atherosclerosis and progressively converted into pathogenic T H 1/T H 17-like cells with proinflammatory properties and only a residual T reg transcriptome. Plaque T cells that expanded during progression of atherosclerosis consistently showed a mixed T H 1/T H 17 phenotype in single-cell RNA sequencing. In addition, we observed a loss of FoxP3 in a fraction of apoB + T regs in lineage tracing of hyperlipidemic Apoe –/– mice. In adoptive transfer experiments, converting apoB + T regs failed to protect from atherosclerosis. Conclusions: Our results demonstrate an unexpected mixed phenotype of apoB-reactive autoimmune T cells in atherosclerosis and suggest an initially protective autoimmune response against apoB with a progressive derangement in clinical disease. These findings identify apoB autoreactive T regs as a novel cellular target in atherosclerosis.

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