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

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.

scholarly journals Single cell RNA sequencing reveals regional heterogeneity of hepatobiliary innate lymphoid cells in a tissue-enriched fashion

PLoS ONE ◽  
2019 ◽  
Vol 14 (4) ◽  
pp. e0215481 ◽  
Author(s):  
Anna L. Peters ◽  
Zhenhua Luo ◽  
Jun Li ◽  
Reena Mourya ◽  
Yunguan Wang ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Innate Lymphoid Cells ◽  
Lymphoid Cells ◽  
Regional Heterogeneity ◽  
Single Cell Rna Sequencing

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

2016 ◽  
Vol 17 (4) ◽  
pp. 451-460 ◽  
Author(s):  
Åsa K Björklund ◽  
Marianne Forkel ◽  
Simone Picelli ◽  
Viktoria Konya ◽  
Jakob Theorell ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Innate Lymphoid Cells ◽  
Lymphoid Cells ◽  
Single Cell Rna Sequencing

scholarly journals SAT0302 INNATE LYMPHOID CELLS INDUCE A FIBROTIC PHENOTYPE OF FIBROBLASTS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1096.2-1096
Author(s):  
S. Weber ◽  
C. G. Anchang ◽  
S. Rauber ◽  
M. Luber ◽  
M. G. Raimondo ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Tissue Remodeling ◽  
Innate Lymphoid Cells ◽  
Lymphoid Cells ◽  
Research Support ◽  
Fibroblast Activation ◽  
Fibrotic Tissue ◽  
Single Cell Rna Sequencing ◽  
Fibrotic Diseases

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

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

Gut ◽  
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.

scholarly journals Single-Cell RNA Sequencing Reveals the Immunological Profiles of Renal Allograft Rejection in Mice

2021 ◽  
Vol 12 ◽  
Author(s):  
Qixia Shen ◽  
Yucheng Wang ◽  
Jiaoyi Chen ◽  
Lifeng Ma ◽  
Xiaoru Huang ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Allograft Rejection ◽  
Renal Allograft ◽  
Immune Cell ◽  
Fold Increase ◽  
Renal Allograft Rejection ◽  
Single Cell Rna Sequencing

Allograft rejection is a common immunological feature in renal transplantation and is associated with reduced graft survival. A mouse renal allograft rejection model was induced and single-cell RNA sequencing (scRNA-seq) data of CD45+ leukocytes in kidney allografts on days 7 (D7) and 15 (D15) after operation was analyzed to reveal a full immunological profiling. We identified 20 immune cell types among 10,921 leukocytes. Macrophages and CD8+ T cells constituted the main populations on both timepoints. In the process from acute rejection (AR) towards chronic rejection (CR), the proportion of proliferating and naïve CD8+ T cells dropped significantly. Both B cells and neutrophils decreased by about 3 folds. On the contrary, the proportion of macrophages and dendritic cells (DCs) increased significantly, especially by about a 4.5-fold increase in Ly6cloMrc1+ macrophages and 2.6 folds increase in Ly6cloEar2+ macrophages. Moreover, myeloid cells harbored the richest ligand and receptor (LR) pairs with other cells, particularly for chemokine ligands such as Cxcl9, Cxcl10, Cxcl16 and Yars. However, macrophages with weak response to interferon gamma (IFNg) contributed to rejection chronicization. To conclude, reduction in CD8 T cells, B cells and neutrophils while increasing in Ly6cloMrc1+ macrophages and Ly6cloEar2+ macrophages, may contribute significantly to the progress from AR towards CR.

scholarly journals Site to Site Comparison of Follicular Lymphoma Biopsies By Single Cell RNA Sequencing

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 297-297 ◽  
Author(s):  
Sarah Haebe ◽  
Tanaya Shree ◽  
Anuja Sathe ◽  
Grady Day ◽  
HoJoon Lee ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Board Of Directors ◽  
Rna Sequencing ◽  
Peripheral Blood ◽  
Advisory Committees ◽  
Blood Samples ◽  
Single Cell Rna Sequencing

Follicular lymphoma (FL) originates from a single B cell that has rearranged one copy of its BCL2 gene on chromosome 18 to the Ig locus on chromosome 14 and in addition has acquired a mutation in a histone modifying gene such as CREBBP or KMTD2. By the time the disease is diagnosed the progeny of this original cell harbors additional mutations and is usually found at multiple lymphoid sites throughout the body. At each of these sites the malignant cells are accompanied by a rich network of follicular dendritic cells, T cells and other immune cells. This tumor microenvironment (TME) is clearly an important feature of the biology of FL and can impact the clinical behavior of the disease (Dave et al., NEJM, 2004). It remains unknown whether tumor clonal heterogeneity and the composition of the TME differ between various lymphoma sites within the same patient. Single cell RNA sequencing facilitates a detailed and unbiased view of both the tumor clone and the complex TME. To profile the TME and explore FL tumor evolution, we obtained fine needle aspirates (FNAs) at 2 different sites in the body and peripheral blood specimens all on the same day and subjected these samples to single cell RNA sequencing and immune repertoire analysis. These biopsies were taken prior to therapy from patients entering immunotherapy clinical trials (NCT02927964, NCT03410901). Single cell RNA sequencing of FNA and blood samples was performed using the 10X Genomics platform to an average targeted depth of 50,000 reads/cell. We have prepared sequencing libraries from 15 tumor FNA and peripheral blood samples from 5 patients thus far. Typically, 3,000-10,000 cells have been sequenced per sample, with excellent sequencing quality metrics. By applying Uniform Manifold Approximation and Projection (UMAP), a dimensionality reduction algorithm, we found the TME of these FL patients to be richly populated by many phenotypically discrete non-malignant cells, including many subpopulations of T cells, B-cells, myeloid cells, NK cells and dendritic cells. Evaluating the combined dataset containing all tumor samples for all 5 patients, we found that malignant B cells from different patients clearly clustered apart from each other, a feature not dependent on immunoglobulin clonality or HLA type. Each patient's tumor population contained 3-5 distinct subpopulations, presumably a result of multiclonal tumor evolution. Nonetheless, we were able to define several malignant B-cell sub-phenotypes common to all patients. Intriguingly, compared to malignant B cells, infiltrating non-malignant B cells showed higher MHC I expression, activation markers, and an enrichment in interferon-induced genes. Of note, we could also detect circulating tumor cells in peripheral blood samples of several patients, and these exhibited a distinct gene expression profile compared to their counterparts within lymph nodes. Analysis of the diverse T cell subpopulations within tumors revealed distinct functional states. For example, in regulatory and T follicular helper cells, we identified activated clusters (CD27, BATF, TNFRSF4) and putative resting clusters (SELL, KLF2, IL7R), while effector T cells resided in separate cytotoxic (GZMA, GZMB, GNLY) and exhausted (TIGIT, CXCL13, LAG3) clusters. Tumor B cell gene expression and composition of the TME from site to site within the same patient were similar in some cases and remarkably divergent in others. For example, we detected a significant upregulation of interferon signaling pathways in the tumor B cells and an enrichment of effector T cells in only one of the two sites within one patient. Analysis of B cell and T cell antigen receptor sequences to evaluate tumor subclonality and TCR clonotype diversity are ongoing. To the best of our knowledge, this is the first study to compare different sites of FL in the same patients at the single cell level. Our analyses characterize inter- and intra-patient heterogeneity in malignant and immune cell subsets and provide a baseline for eventual comparison of alterations occurring over time as these patients receive experimental immunotherapy interventions. Disclosures Levy: XCella: Membership on an entity's Board of Directors or advisory committees; Immunocore: Membership on an entity's Board of Directors or advisory committees; Walking Fish: Membership on an entity's Board of Directors or advisory committees; Five Prime: Membership on an entity's Board of Directors or advisory committees; Corvus: Membership on an entity's Board of Directors or advisory committees; Quadriga: Membership on an entity's Board of Directors or advisory committees; BeiGene: Membership on an entity's Board of Directors or advisory committees; GigaGen: Membership on an entity's Board of Directors or advisory committees; Teneobio: Membership on an entity's Board of Directors or advisory committees; Sutro: Membership on an entity's Board of Directors or advisory committees; Checkmate: Membership on an entity's Board of Directors or advisory committees; Nurix: Membership on an entity's Board of Directors or advisory committees; Dragonfly: Membership on an entity's Board of Directors or advisory committees; Innate Pharma: Membership on an entity's Board of Directors or advisory committees; Abpro: Membership on an entity's Board of Directors or advisory committees; Apexigen: Membership on an entity's Board of Directors or advisory committees; Nohla: Membership on an entity's Board of Directors or advisory committees; Spotlight: Membership on an entity's Board of Directors or advisory committees; 47 Inc: Membership on an entity's Board of Directors or advisory committees.

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

2016 ◽  
Vol 17 (6) ◽  
pp. 740-740 ◽  
Author(s):  
Åsa K Björklund ◽  
Marianne Forkel ◽  
Simone Picelli ◽  
Viktoria Konya ◽  
Jakob Theorell ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Innate Lymphoid Cells ◽  
Lymphoid Cells ◽  
Single Cell Rna Sequencing

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.

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).

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