Erratum: Corrigendum: The heterogeneity of human CD127+ innate lymphoid cells revealed by single-cell RNA sequencing

Background:Fibrotic diseases are characterized by excessive extracellular matrix production as a result of immune-mediated permanent fibroblast activation. Innate lymphoid cells type II (ILC2) are an only recently discovered cell type involved in barrier integrity and tissue homeostasis. There is upcoming evidence that ILC2s play a central role in mediating fibrotic diseases.Objectives:The aim of the study was to further elucidate the role of ILC2s in fibrotic tissue remodeling and fibroblast activation.Methods:Skin biopsies of patients with systemic sclerosis (SSc) or sclerodermatous chronic graft versus host disease (scGvHD) as well as lung biopsies of patients with idiopathic pulmonary fibrosis (IPF) were analyzed by immunofluorescence (IF) staining. Single cell RNA-sequencing (scRNA-seq) was performed on ILCs from fibrotic skin and lung of bleomycin-challenged mice. Further characterization of ILC2 phenotypes in fibrosis models was done by flow cytometry.In vitroculture of fibroblasts and ILC2s was used to study cellular interaction and fibrotic activation. Quantitative realtime-PCR, western blot, IF staining and ELISA were used as readouts.Results:Two different subtypes of ILC2s were found in skin of SSc and scGvHD patients as well as in lungs of IPF patients with one subpopulation being particularly increased in fibrotic tissue. Single cell RNA-sequencing confirmed the existence of two major populations of ILC2s in experimental fibrosis. One subtype showed features of immature ILC2 progenitors and was actively recruited from the bone marrow during fibrotic tissue remodeling. The other ILC2 subset was highly activated and expressed pro-fibrotic cytokines. These profibrotic ILC2s directly interacted with fibroblasts in a cell contact dependent manner. Semaphorin 4A (SEMA4A) expressed by ILC2s bound to Plexin D1 (PLXND1) on fibroblasts. This interaction resulted into fibrotic imprinting with high expression levels of the transcription factor PU.1 which was recently described as central regulator of the pro-fibrotic gene expression program (Wohlfahrt et al. 2019). Signaling through Jagged 1 (JAG1) and Notch receptor 2 (NOTCH2) was identified as a second mechanism of interaction between fibroblasts and ILC2s. JAG1 expressed by fibroblasts activated NOTCH2 signaling in ILC2s which emphazised the secretion of pro-fibrotic cytokines.Conclusion:We identified a bidirectional interaction between ILCs and fibroblasts incorporating a vicious circle of fibrotic tissue remodelling. As ILCs are still not accessible as therapeutic targets these results might contribute to the development of new strategies for anti-fibrotic therapies.References:[1]Wohlfahrt, Thomas, Simon Rauber, Steffen Uebe, Markus Luber, Alina Soare, Arif Ekici, Stefanie Weber, Alexandru-Emil Matei, Chih-Wei Chen, Christiane Maier, Emmanuel Karouzakis, Hans P. Kiener, Elena Pachera, Clara Dees, Christian Beyer, Christoph Daniel, Kolja Gelse, Andreas E. Kremer, Elisabeth Naschberger, Michael Stürzl, Falk Butter, Michael Sticherling, Susetta Finotto, Alexander Kreuter, Mark H. Kaplan, Astrid Jüngel, Steffen Gay, Stephen L. Nutt, David W. Boykin, Gregory M. K. Poon, Oliver Distler, Georg Schett, Jörg H. W. Distler, and Andreas Ramming. 2019. ‘PU.1 controls fibroblast polarization and tissue fibrosis’,Nature, 566: 344-49.Disclosure of Interests:Stefanie Weber: None declared, Charles Gwellem Anchang: None declared, Simon Rauber: None declared, Markus Luber: None declared, Maria Gabriella Raimondo Grant/research support from: Celgene, Partner Fellowship, Yuko Ariza Employee of: Ono Pharmaceutical Co., Ltd., Aleix Rius Rigau: None declared, Alexander Kreuter: None declared, Georg Schett Speakers bureau: AbbVie, BMS, Celgene, Janssen, Eli Lilly, Novartis, Roche and UCB, Jörg Distler Grant/research support from: Boehringer Ingelheim, Consultant of: Boehringer Ingelheim, Paid instructor for: Boehringer Ingelheim, Speakers bureau: Boehringer Ingelheim, Andreas Ramming Grant/research support from: Pfizer, Novartis, Consultant of: Boehringer Ingelheim, Novartis, Gilead, Pfizer, Speakers bureau: Boehringer Ingelheim, Roche, Janssen

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The tumour microenvironment shapes innate lymphoid cells in patients with hepatocellular carcinoma

Gut ◽

10.1136/gutjnl-2021-325288 ◽

2021 ◽

pp. gutjnl-2021-325288

Author(s):

Bernd Heinrich ◽

E Michael Gertz ◽

Alejandro A Schäffer ◽

Amanda Craig ◽

Benjamin Ruf ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Flow Cytometry ◽

Single Cell ◽

Rna Sequencing ◽

Mononuclear Cells ◽

Innate Lymphoid Cells ◽

Lymphoid Cells ◽

Cancer Tissue ◽

Single Cell Rna Sequencing ◽

Cytokine Milieu

ObjectiveHepatocellular carcinoma (HCC) represents a typical inflammation-associated cancer. Tissue resident innate lymphoid cells (ILCs) have been suggested to control tumour surveillance. Here, we studied how the local cytokine milieu controls ILCs in HCC.DesignWe performed bulk RNA sequencing of HCC tissue as well as flow cytometry and single-cell RNA sequencing of enriched ILCs from non-tumour liver, margin and tumour core derived from 48 patients with HCC. Simultaneous measurement of protein and RNA expression at the single-cell level (AbSeq) identified precise signatures of ILC subgroups. In vitro culturing of ILCs was used to validate findings from in silico analysis. Analysis of RNA-sequencing data from large HCC cohorts allowed stratification and survival analysis based on transcriptomic signatures.ResultsRNA sequencing of tumour, non-tumour and margin identified tumour-dependent gradients, which were associated with poor survival and control of ILC plasticity. Single-cell RNA sequencing and flow cytometry of ILCs from HCC livers identified natural killer (NK)-like cells in the non-tumour tissue, losing their cytotoxic profile as they transitioned into tumour ILC1 and NK-like-ILC3 cells. Tumour ILC composition was mediated by cytokine gradients that directed ILC plasticity towards activated tumour ILC2s. This was liver-specific and not seen in ILCs from peripheral blood mononuclear cells. Patients with high ILC2/ILC1 ratio expressed interleukin-33 in the tumour that promoted ILC2 generation, which was associated with better survival.ConclusionOur results suggest that the tumour cytokine milieu controls ILC composition and HCC outcome. Specific changes of cytokines modify ILC composition in the tumour by inducing plasticity and alter ILC function.

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Porcine intestinal innate lymphoid cells and lymphocyte spatial context revealed through single-cell RNA sequencing

10.1101/2022.01.09.475571 ◽

2022 ◽

Author(s):

Jayne E Wiarda ◽

Julian M Trachsel ◽

Sathesh K Sivasankaran ◽

Christopher K Tuggle ◽

Crystal L Loving

Keyword(s):

T Cells ◽

B Cells ◽

Single Cell ◽

Rna Sequencing ◽

Innate Lymphoid Cells ◽

Lymphoid Cells ◽

Spatial Context ◽

Intestinal Lymphocytes ◽

Single Cell Rna Sequencing ◽

Group 1

Intestinal lymphocytes are crucial members of the mucosal immune system with impact over outcomes of intestinal health versus dysbiosis. Resolving intestinal lymphocyte complexity and function is a challenge, as the intestine provides cellular snapshots of a diverse spectrum of immune states. In pigs, intestinal lymphocytes are poorly described relative to humans or traditional model species. Enhanced understanding of porcine intestinal lymphocytes will promote food security and improve utility of pigs as a biomedical model for intestinal research. Single-cell RNA sequencing (scRNA-seq) was performed to provide transcriptomic profiles of lymphocytes in the porcine ileum, with 31,983 cells annotated into 26 cell types. Deeper interrogation revealed previously undescribed cells in porcine ileum, including SELLhi γδ T cells, group 1 and group 3 innate lymphoid cells (ILCs), and four subsets of B cells. Single-cell transcriptomes in ileum were compared to those in porcine blood, and subsets of activated lymphocytes were detected in ileum but not periphery. Comparison to scRNA-seq human and murine ileum data revealed a general consensus of ileal lymphocytes across species. Lymphocyte spatial context in porcine ileum was conferred through differential tissue dissection prior to scRNA-seq. Antibody-secreting cells, B cells, follicular αβ T cells, and cycling T/ILCs were enriched in ileum with Peyer's patches, while non-cycling γδ T, CD8 αβ T, and group 1 ILCs were enriched in ileum without Peyer's patches. scRNA-seq findings were leverages to develop advanced toolsets for further identification of ILCs in porcine ileum via flow cytometry and in situ staining. Porcine ileal ILCs identified via scRNA-seq did not transcriptionally mirror peripheral ILCs (corresponding to natural killer cells) but instead had gene signatures indicative of tissue- and activation-specific functions, indicating potentially similar roles to intestinal ILCs identified in humans. Overall, the data serve as a highly-resolved transcriptomic atlas of the porcine intestinal immune landscape and will be useful in further understanding intestinal immune cell function.

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Tissue-specific transcriptional imprinting and heterogeneity in human innate lymphoid cells revealed by full-length single-cell RNA-sequencing

Cell Research ◽

10.1038/s41422-020-00445-x ◽

2021 ◽

Author(s):

Luca Mazzurana ◽

Paulo Czarnewski ◽

Viktor Jonsson ◽

Leif Wigge ◽

Markus Ringnér ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Trajectory Analysis ◽

Lymphoid Cells ◽

Full Length ◽

Tissue Specific ◽

Single Cell Rna Sequencing ◽

Gene Modules ◽

The Impact ◽

Differentiation Pathways

AbstractThe impact of the microenvironment on innate lymphoid cell (ILC)-mediated immunity in humans remains largely unknown. Here we used full-length Smart-seq2 single-cell RNA-sequencing to unravel tissue-specific transcriptional profiles and heterogeneity of CD127+ ILCs across four human tissues. Correlation analysis identified gene modules characterizing the migratory properties of tonsil and blood ILCs, and signatures of tissue-residency, activation and modified metabolism in colon and lung ILCs. Trajectory analysis revealed potential differentiation pathways from circulating and tissue-resident naïve ILCs to a spectrum of mature ILC subsets. In the lung we identified both CRTH2+ and CRTH2− ILC2 with lung-specific signatures, which could be recapitulated by alarmin-exposure of circulating ILC2. Finally, we describe unique TCR-V(D)J-rearrangement patterns of blood ILC1-like cells, revealing a subset of potentially immature ILCs with TCR-δ rearrangement. Our study provides a useful resource for in-depth understanding of ILC-mediated immunity in humans, with implications for disease.

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