Single-Cell RNA Sequencing of Blood and Ileal T Cells From Patients With Crohn's Disease Reveals Tissue-Specific Characteristics and Drug Targets

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.

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

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.903 ◽

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).

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

Cell Discovery ◽

10.1038/s41421-021-00353-3 ◽

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.

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Single-Cell RNA Sequencing Unveils Unique Transcriptomic Signatures of Organ-Specific Endothelial Cells

Circulation ◽

10.1161/circulationaha.119.041433 ◽

2020 ◽

Vol 142 (19) ◽

pp. 1848-1862 ◽

Cited By ~ 1

Author(s):

David T. Paik ◽

Lei Tian ◽

Ian M. Williams ◽

Siyeon Rhee ◽

Hao Zhang ◽

...

Keyword(s):

Endothelial Cells ◽

Single Cell ◽

Rna Sequencing ◽

Expression Patterns ◽

Receptor Expression ◽

Transcriptional Networks ◽

Sequencing Data ◽

Tissue Specific ◽

Functional Relationships ◽

Single Cell Rna Sequencing

Background: Endothelial cells (ECs) display considerable functional heterogeneity depending on the vessel and tissue in which they are located. Whereas these functional differences are presumably imprinted in the transcriptome, the pathways and networks that sustain EC heterogeneity have not been fully delineated. Methods: To investigate the transcriptomic basis of EC specificity, we analyzed single-cell RNA sequencing data from tissue-specific mouse ECs generated by the Tabula Muris consortium. We used a number of bioinformatics tools to uncover markers and sources of EC heterogeneity from single-cell RNA sequencing data. Results: We found a strong correlation between tissue-specific EC transcriptomic measurements generated by either single-cell RNA sequencing or bulk RNA sequencing, thus validating the approach. Using a graph-based clustering algorithm, we found that certain tissue-specific ECs cluster strongly by tissue (eg, liver, brain), whereas others (ie, adipose, heart) have considerable transcriptomic overlap with ECs from other tissues. We identified novel markers of tissue-specific ECs and signaling pathways that may be involved in maintaining their identity. Sex was a considerable source of heterogeneity in the endothelial transcriptome and we discovered Lars2 to be a gene that is highly enriched in ECs from male mice. We found that markers of heart and lung ECs in mice were conserved in human fetal heart and lung ECs. We identified potential angiocrine interactions between tissue-specific ECs and other cell types by analyzing ligand and receptor expression patterns. Conclusions: We used single-cell RNA sequencing data generated by the Tabula Muris consortium to uncover transcriptional networks that maintain tissue-specific EC identity and to identify novel angiocrine and functional relationships between tissue-specific ECs.

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Unravelling the heterogeneity and dynamic relationships of tumor‐infiltrating T cells by single‐cell RNA sequencing analysis

Journal of Leukocyte Biology ◽

10.1002/jlb.6mr0320-234r ◽

2020 ◽

Vol 107 (6) ◽

pp. 917-932 ◽

Cited By ~ 3

Author(s):

Xin Yu ◽

Lei Zhang ◽

Ashutosh Chaudhry ◽

Aaron S. Rapaport ◽

Wenjun Ouyang

Keyword(s):

T Cells ◽

Single Cell ◽

Rna Sequencing ◽

Sequencing Analysis ◽

Single Cell Rna Sequencing ◽

Dynamic Relationships

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

Circulation ◽

10.1161/circulationaha.119.042863 ◽

2020 ◽

Vol 142 (13) ◽

pp. 1279-1293 ◽

Cited By ~ 5

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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