Deciphering the Regulatory Landscape of γδ T Cell Development by Single-Cell RNA-Sequencing

SUMMARYRecent studies have established γδ T cells as critical players in a broad range of infections, antitumor surveillance, autoimmune diseases and tissue homeostasis. However, differentiation of γδ T cells in the adult thymus remains poorly understood, due to the rare frequency of this lineage. Here, we infer high-resolution developmental trajectories of this rare population by single-cell RNA-sequencing. We reveal previously unknown subtypes and identify the transcription factor c-MAF as a novel key regulator of IL-17-producing γδ T cell (γδT17) differentiation. c-MAF knockout mice exhibit a complete block in γδT17 differentiation, absence of these cells from peripheral organs, and protection from an autoimmune phenotype in a psoriasis model. Single-cell RNA-sequencing of Sox13 and Rorc knockout mice pinpoints c-MAF as an essential missing link between these lineage-specifying factors. These findings significantly enhance our understanding of γδ T cell ontogeny. Our experimental strategy provides a blueprint for deciphering differentiation of rare cell types.

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Heterogeneity of T Cells in Atherosclerosis Defined by Single-Cell RNA-Sequencing and Cytometry by Time of Flight

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.120.312137 ◽

2020 ◽

Author(s):

Holger Winkels ◽

Dennis Wolf

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Rna Sequencing ◽

T Regulatory Cells ◽

Foam Cell ◽

Regulatory Cells ◽

T Helper ◽

Antigen Specificity ◽

Single Cell Rna Sequencing

The infiltration and accumulation of pro- and anti-inflammatory leukocytes within the intimal layer of the arterial wall is a hallmark of developing and progressing atherosclerosis. While traditionally perceived as macrophage- and foam cell-dominated disease, it is now established that atherosclerosis is a partial autoimmune disease that involves the recognition of peptides from ApoB (apolipoprotein B), the core protein of LDL (low-density lipoprotein) cholesterol particles, by CD4 + T-helper cells and autoantibodies against LDL and ApoB. Autoimmunity in the atherosclerotic plaque has long been understood as a pathogenic T-helper type-1 driven response with proinflammatory cytokine secretion. Recent developments in high-parametric cell immunophenotyping by mass cytometry, single-cell RNA-sequencing, and in tools exploring antigen-specificity have established the existence of several unforeseen layers of T cell diversity with mixed T H 1 and T regulatory cells transcriptional programs and unpredicted fates. These findings suggest that pathogenic ApoB-reactive T cells evolve from atheroprotective and immunosuppressive CD4 + T regulatory cells that lose their protective properties over time. Here, we discuss T cell heterogeneity in atherosclerosis with a focus on plasticity, antigen-specificity, exhaustion, maturation, tissue residency, and its potential use in clinical prediction.

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Reclassification of classic T cell subsets and identification of novel cell types by single-cell RNA sequencing

10.21203/rs.3.rs-79282/v1 ◽

2020 ◽

Author(s):

Xiangru Shen ◽

Xuefei Wang ◽

Shan Chen ◽

Hongyi Liu ◽

Ni Hong ◽

...

Keyword(s):

T Cell ◽

Single Cell ◽

Rna Sequencing ◽

T Cell Activation ◽

Cell Activation ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Cell Types ◽

T Cell Subsets ◽

Single Cell Rna Sequencing

Abstract Single cell RNA sequencing (scRNA-seq) clusters cells using genome-wide gene expression profiles. The relationship between scRNA-seq Clustered-Populations (scCPops) and cell surface marker-defined classic T cell subsets is unclear. Here, we interrogated 6 bead-enriched T cell subsets with 62,235 single cell transcriptomes and re-grouped them into 9 scCPops. Bead-enriched CD4 Naïve, CD8 Naïve and CD4 memory were mainly clustered into their scCPop counterparts, while the other T cell subsets were clustered into multiple scCPops including unexpected mucosal-associated invariant T cell and natural killer T cell. Most interestingly, we discovered a new T cell type that highly expressed Interferon Signaling Associated Genes (ISAGs), namely IFNhi T. We further enriched IFNhi T for scRNA-seq analyses. IFNhi T cluster disappeared on tSNE after removing ISAGs, and IFNhi T cluster showed up by tSNE analyses of ISAGs alone, indicating ISAGs are the major contributor of IFNhi T cluster. BST2+ cells and BST2- cells showing different efficiencies of T cell activation indicates high ISAGs may contribute to quick immune responses.

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OMIC-11. SINGLE CELL RNA SEQUENCING FROM THE CSF OF SUBJECTS WITH H3K27M+ DIPG/DMG TREATED WITH GD2 CAR T-CELLULAR THERAPY

Neuro-Oncology ◽

10.1093/neuonc/noab090.158 ◽

2021 ◽

Vol 23 (Supplement_1) ◽

pp. i39-i39

Author(s):

Aaron Mochizuki ◽

Sneha Ramakrishna ◽

Zina Good ◽

Shabnum Patel ◽

Harshini Chinnasamy ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Rna Sequencing ◽

Cell Product ◽

Myeloid Suppressor Cells ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Single Cell Rna Sequencing

Abstract Introduction We are conducting a Phase I clinical trial utilizing chimeric antigen receptor (CAR) T-cells targeting GD2 (NCT04196413) for H3K27M-mutant diffuse intrinsic pontine glioma (DIPG) and spinal cord diffuse midline glioma (DMG). Cerebrospinal fluid (CSF) is collected for correlative studies at the time of routine intracranial pressure monitoring via Ommaya catheter. Here we present single cell RNA-sequencing results from the first 3 subjects. Methods Single cell RNA-sequencing was performed utilizing 10X Genomics on cells isolated from CSF at various time points before and after CAR T-cell administration and on the CAR T-cell product. Output was aligned with Cell Ranger and analyzed in R. Results As detailed in the Majzner et al. abstract presented at this meeting, three of four subjects treated at dose-level one exhibited clear radiographic and/or clinical benefit. We have to date completed single cell RNA-sequencing for three of these four subjects (two with benefit, one without). After filtering out low-quality signals and doublets, 89,604 cells across 3 subjects were analyzed. Of these, 4,122 cells represent cells isolated from CSF and 85,482 cells represent CAR T-cell product. Two subjects who demonstrated clear clinical and radiographic improvement exhibited fewer S100A8+S100A9+ myeloid suppressor-cells and CD25+FOXP3+ regulatory T-cells in the CSF pre-infusion compared to the subject who did not derive a therapeutic response. In one subject with DIPG who demonstrated improvement, polyclonal CAR T-cells detectable in CSF at Day +14 demonstrated enrichment of CD8A, GZMA, GNLY and PDCD1 compared to the pre-infusion CAR T-cells by trajectory analysis, suggesting differentiation toward a cytotoxic phenotype; the same subject exhibited increasing numbers of S100A8+S100A9+ myeloid cells and CX3CR1+P2RY12+ microglia over time. Further analyses will be presented as data become available. Conclusions The presence of immunosuppressive myeloid populations, detectable in CSF, may correlate to clinical response in CAR T cell therapy for DIPG/DMG.

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Re-investigation of classic T cell subsets and identification of novel cell subpopulations by single-cell RNA sequencing

10.1101/2021.02.11.430754 ◽

2021 ◽

Author(s):

Xuefei Wang ◽

Xiangru Shen ◽

Shan Chen ◽

Hongyi Liu ◽

Ni Hong ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Surface ◽

Single Cell ◽

Rna Sequencing ◽

T Cell Activation ◽

Expression Profiles ◽

T Cell Subsets ◽

Cell Subpopulation ◽

Single Cell Rna Sequencing

AbstractClassic T cell subsets are defined by a small set of cell surface markers, while single cell RNA sequencing (scRNA-seq) clusters cells using genome-wide gene expression profiles. The relationship between scRNA-seq Clustered-Populations (scCPops) and cell surface marker-defined classic T cell subsets remain unclear. Here, we interrogated 6 bead-enriched T cell subsets with 62,235 single cell transcriptomes and re-grouped them into 9 scCPops. Bead-enriched CD4 Naïve and CD8 Naïve were mainly clustered into their scCPop counterparts, while cells from the other T cell subsets were assigned to multiple scCPops including mucosal-associated invariant T cells and natural killer T cells. The multiple T cell subsets that form a single scCPop exhibited similar expression pattern, but not vice versa, indicating scCPops are much homogeneous cell populations with similar cell states. Interestingly, we discovered and named IFNhi T, a new T cell subpopulation that highly expressed Interferon Signaling Associated Genes (ISAGs). We further enriched IFNhi T by FACS sorting of BST2 for scRNA-seq analyses. IFNhi T cluster disappeared on tSNE plot after removing ISAGs, while IFNhi T cluster showed up by tSNE analyses of ISAGs alone, indicating ISAGs are the major contributor of IFNhi T cluster. BST2+ T cells and BST2− T cells showing different efficiencies of T cell activation indicates high level of ISAGs may contribute to quick immune responses.

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Targeted reconstruction of T cell receptor sequence from single cell RNA-sequencing links CDR3 length to T cell differentiation state

10.1101/072744 ◽

2016 ◽

Cited By ~ 4

Author(s):

Shaked Afik ◽

Kathleen B. Yates ◽

Kevin Bi ◽

Samuel Darko ◽

Jernej Godec ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Rna Sequencing ◽

Cell Response ◽

T Cell Response ◽

Effector Memory ◽

Cell State ◽

Tcr Repertoire ◽

Single Cell Rna Sequencing

ABSTRACTThe T cell compartment must contain diversity in both TCR repertoire and cell state to provide effective immunity against pathogens1,2. However, it remains unclear how differences in the TCR contribute to heterogeneity in T cell state at the single cell level because most analysis of the TCR repertoire has, to date, aggregated information from populations of cells. Single cell RNA-sequencing (scRNA-seq) can allow simultaneous measurement of TCR sequence and global transcriptional profile from single cells. However, current protocols to directly sequence the TCR require the use of long sequencing reads, increasing the cost and decreasing the number of cells that can be feasibly analyzed. Here we present a tool that can efficiently extract TCR sequence information from standard, short-read scRNA-seq libraries of T cells: TCR Reconstruction Algorithm for Paired-End Single cell (TRAPeS). We apply it to investigate heterogeneity in the CD8+T cell response in humans and mice, and show that it is accurate and more sensitive than previous approaches3,4. We applied TRAPeS to single cell RNA-seq of CD8+T cells specific for a single epitope from Yellow Fever Virus5. We show that the recently-described "naive-like" memory population of YFV-specific CD8+T cells have significantly longer CDR3 regions and greater divergence from germline sequence than do effector-memory phenotype CD8+T cells specific for YFV. This suggests that TCR usage contributes to heterogeneity in the differentiation state of the CD8+T cell response to YFV. TRAPeS is publicly available, and can be readily used to investigate the relationship between the TCR repertoire and cellular phenotype.

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Single-cell RNA sequencing reveals heterogeneous tumor and immune cell populations in early-stage lung adenocarcinomas harboring EGFR mutations

Oncogene ◽

10.1038/s41388-020-01528-0 ◽

2020 ◽

Cited By ~ 1

Author(s):

Di He ◽

Di Wang ◽

Ping Lu ◽

Nan Yang ◽

Zhigang Xue ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

Single Cell ◽

Tumor Cells ◽

Rna Sequencing ◽

Early Stage ◽

Cell Types ◽

Egfr Mutations ◽

Advanced Tumor ◽

Single Cell Rna Sequencing

Abstract Lung adenocarcinoma (LUAD) harboring EGFR mutations prevails in Asian population. However, the inter-patient and intra-tumor heterogeneity has not been addressed at single-cell resolution. Here we performed single-cell RNA sequencing (scRNA-seq) of total 125,674 cells from seven stage-I/II LUAD samples harboring EGFR mutations and five tumor-adjacent lung tissues. We identified diverse cell types within the tumor microenvironment (TME) in which myeloid cells and T cells were the most abundant stromal cell types in tumors and adjacent lung tissues. Within tumors, accompanied by an increase in CD1C+ dendritic cells, the tumor-associated macrophages (TAMs) showed pro-tumoral functions without signature gene expression of defined M1 or M2 polarization. Tumor-infiltrating T cells mainly displayed exhausted and regulatory T-cell features. The adenocarcinoma cells can be categorized into different subtypes based on their gene expression signatures in distinct pathways such as hypoxia, glycolysis, cell metabolism, translation initiation, cell cycle, and antigen presentation. By performing pseudotime trajectory, we found that ELF3 was among the most upregulated genes in more advanced tumor cells. In response to secretion of inflammatory cytokines (e.g., IL1B) from immune infiltrates, ELF3 in tumor cells was upregulated to trigger the activation of PI3K/Akt/NF-κB pathway and elevated expression of proliferation and anti-apoptosis genes such as BCL2L1 and CCND1. Taken together, our study revealed substantial heterogeneity within early-stage LUAD harboring EGFR mutations, implicating complex interactions among tumor cells, stromal cells and immune infiltrates in the TME.

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Chicken peripheral blood lymphocyte response to ALV-J infection assessed by single-cell RNA sequencing

10.1101/2021.01.12.426350 ◽

2021 ◽

Author(s):

Manman Dai ◽

Min Feng ◽

Ziwei Li ◽

Weisan Chen ◽

Ming Liao

Keyword(s):

T Cells ◽

T Cell ◽

Viral Infection ◽

Single Cell ◽

Rna Sequencing ◽

Peripheral Blood ◽

Cell Populations ◽

Control Group ◽

Marker Genes ◽

Single Cell Rna Sequencing

ABSTRACTChicken peripheral blood lymphocytes (PBLs) exhibit wide-ranging cell types, but current understanding of their subclasses, immune cell classification, and function is limited and incomplete. Previously, we found that viremia caused by avian leukosis virus subgroup J (ALV‐J) was eliminated by 21 days post infection (DPI), accompanied by increased CD8+ T cell ratio in PBLs and low antibody levels. Here we performed single-cell RNA sequencing (scRNA-seq) of PBLs in ALV-J infected and control chickens at 21 DPI to determine chicken PBL subsets and their specific molecular and cellular characteristics, before and after viral infection. Eight cell clusters and their potential marker genes were identified in chicken PBLs. T cell populations (clusters 6 and 7) had the strongest response to ALV-J infection at 21 DPI, based on detection of the largest number of differentially expressed genes (DEGs). T cell populations of clusters 6 and 7 could be further divided into four subsets: activated CD4+ T cells (cluster A0), Th1-like cells (cluster A2), Th2-like cells (cluster A1), and cytotoxic CD8+ T cells. Hallmark genes for each T cell subset response to viral infection were initially identified. Furthermore, pseudotime analysis results suggested that chicken CD4+ T cells could potentially differentiate into Th1-like and Th2-like cells. Moreover, ALV-J infection probably induced CD4+ T cell differentiation into Th1-like cells in which the most immune related DEGs were detected. With respect to the control group, ALV-J infection also had an obvious impact on PBL cell composition. B cells showed inconspicuous response and their numbers decreased in PBLs of the ALV-J infected chickens at 21 DPI. Percentages of cytotoxic Th1-like cells and CD8+ T cells were increased in the T cell population of PBLs from ALV-J infected chicken, which were potentially key mitigating factors against ALV-J infection. More importantly, our results provided a rich resource of gene expression profiles of chicken PBL subsets for a systems-level understanding of their function in homeostatic condition as well as in response to viral infection.

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Single-cell RNA-sequencing identifies the developmental trajectory of C-Myc-dependent NK1.1− T-bet+ intraepithelial lymphocyte precursors

Mucosal Immunology ◽

10.1038/s41385-019-0220-y ◽

2019 ◽

Vol 13 (2) ◽

pp. 257-270 ◽

Cited By ~ 2

Author(s):

Jonas F. Hummel ◽

Patrice Zeis ◽

Karolina Ebert ◽

Jonas Fixemer ◽

Philip Konrad ◽

...

Keyword(s):

T Cells ◽

Single Cell ◽

Rna Sequencing ◽

Intraepithelial Lymphocytes ◽

Developmental Trajectory ◽

High Avidity ◽

Clonal Deletion ◽

Adaptive Immune Cells ◽

Single Cell Rna Sequencing ◽

Factor C

Abstract Natural intraepithelial lymphocytes (IELs) are thymus-derived adaptive immune cells, which are important contributors to intestinal immune homeostasis. Similar to other innate-like T cells, they are induced in the thymus through high-avidity interaction that would otherwise lead to clonal deletion in conventional CD4 and CD8 T cells. By applying single-cell RNA-sequencing (scRNA-seq) on a heterogeneous population of thymic CD4−CD8αβ−TCRαβ+NK1.1− IEL precursors (NK1.1− IELPs), we define a developmental trajectory that can be tracked based on the sequential expression of CD122 and T-bet. Moreover, we identify the Id proteins Id2 and Id3 as a novel regulator of IELP development and show that all NK1.1− IELPs progress through a PD-1 stage that precedes the induction of T-bet. The transition from PD-1 to T-bet is regulated by the transcription factor C-Myc, which has far reaching effects on cell cycle, energy metabolism, and the translational machinery during IELP development. In summary, our results provide a high-resolution molecular framework for thymic IEL development of NK1.1− IELPs and deepen our understanding of this still elusive cell type.

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ATIM-34. SINGLE-CELL RNA SEQUENCING REVEALS DYNAMIC IMMUNE RESPONSE CHANGES IN GLIOBLASTOMA PATIENT WITH DURABLE COMPLETE RESPONSE TO CMV PP65-LAMP RNA-PULSED DENDRITIC VACCINES

Neuro-Oncology ◽

10.1093/neuonc/noz175.033 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi9-vi9

Author(s):

Oleg Yegorov ◽

Changlin Yang ◽

Maryam Rahman ◽

Ashley Ghiaseddin ◽

Anjelika Dechkovskaia ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Rna Sequencing ◽

Cytotoxic T Cells ◽

Complete Response ◽

Nkt Cells ◽

Single Cell Sequencing ◽

Single Cell Rna Sequencing ◽

Dc Vaccines

Abstract BACKGROUND The application of single cell sequencing as a novel immune monitoring platform can enhance the ability to interrogate at unprecedented depth the single-cell level dynamic phenotypic and functional attributes of immune cells in patients undergoing cancer immunotherapy treatment. We applied single-cell sequencing analysis of PBMCs in a patient with newly-diagnosed GBM enrolled on the ATTAC II clinical trial (FDA IND BB-16530) who experienced a sustained complete radiographic response to autologous CMV pp65-LAMP RNA-pulsed DC vaccines plus GM-CSF and tetanus-diphtheria booster administered during adjuvant cycles of dose-intensified TMZ. METHODS We constructed 5’ gene expression libraries and T cell receptor enriched libraries for 10x Genomics single-cell sequencing, generated from PBMCs collected prior to and during patient immunization using dendritic cells loaded with RNA encoding the CMV matrix protein pp65 conjugated with the lysosomal associated membrane protein (LAMP) sequence. RESULTS RNA-Seq revealed dynamic changes in immune cell subsets over course of first three vaccines, including increases in cytotoxic T cells and memory T cell subsets. Increased markers of T cell activation were observed during vaccination including enhanced T cell signaling pathways, proliferative signals, and cytokine production. We found that proportion of cytotoxic T cells increased from 3.42% to 11.74% in whole PBMCs after immunization. Surprisingly, we observed very high level of frequency natural killer T (NKT) cells comprising 4.75 % of this patient’s PBMCs at baseline. After three DC vaccines, the level of NKT cells in PBMC increased up to 10%. CONCLUSIONS Single cell RNA sequencing of a patient with sustained complete response to DC vaccination during cycles of dose-intensified TMZ reveals dynamic immune activation and expansion within the cytotoxic T cells and NKT compartments. These results emphasize the importance of subset specific profiling to achieve higher resolution in monitoring immune responses compared with bulk expression profiling in patients receiving immunotherapeutic treatment.

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Single-Cell RNA Sequencing Reveals Tissue Compartment-Specific Plasticity of Mycosis Fungoides Tumor Cells

Frontiers in Immunology ◽

10.3389/fimmu.2021.666935 ◽

2021 ◽

Vol 12 ◽

Author(s):

Katharina Rindler ◽

Wolfgang M. Bauer ◽

Constanze Jonak ◽

Matthias Wielscher ◽

Lisa E. Shaw ◽

...

Keyword(s):

T Cells ◽

Lymph Node ◽

T Cell ◽

Single Cell ◽

Tumor Cells ◽

Rna Sequencing ◽

Mycosis Fungoides ◽

Cell Receptor ◽

Skin Tumor ◽

Single Cell Rna Sequencing

Mycosis fungoides (MF) is the most common primary cutaneous T-cell lymphoma. While initially restricted to the skin, malignant cells can appear in blood, bone marrow and secondary lymphoid organs in later disease stages. However, only little is known about phenotypic and functional properties of malignant T cells in relationship to tissue environments over the course of disease progression. We thus profiled the tumor micromilieu in skin, blood and lymph node in a patient with advanced MF using single-cell RNA sequencing combined with V-D-J T-cell receptor sequencing. In skin, we identified clonally expanded T-cells with characteristic features of tissue-resident memory T-cells (TRM, CD69+CD27-NR4A1+RGS1+AHR+). In blood and lymph node, the malignant clones displayed a transcriptional program reminiscent of a more central memory-like phenotype (KLF2+TCF7+S1PR1+SELL+CCR7+), while retaining tissue-homing receptors (CLA, CCR10). The skin tumor microenvironment contained potentially tumor-permissive myeloid cells producing regulatory (IDO1) and Th2-associated mediators (CCL13, CCL17, CCL22). Given their expression of PVR, TNFRSF14 and CD80/CD86, they might be under direct control by TIGIT+CTLA4+CSF2+TNFSF14+ tumor cells. In sum, this study highlights the adaptive phenotypic and functional plasticity of MF tumor cell clones. Thus, the TRM-like phenotype enables long-term skin residence of MF cells. Their switch to a TCM-like phenotype with persistent skin homing molecule expression in the circulation might explain the multi-focal nature of MF.

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