scholarly journals Single-Cell Proteomics Study of Immune Cell Diversity by Quantitating 465 Proteins

2022 ◽  
Author(s):  
Liwei Yang ◽  
Jesse Liu ◽  
Revanth Reddy ◽  
Jun Wang
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Single Cell ◽  
Immune Cell ◽  
Single Cells ◽  
Additional Information ◽  
Sequencing Technologies ◽  
Cell Diversity ◽  
Cell Subpopulations ◽  
T Cell Subpopulations

The identification and characterization of T cell subpopulations is critical to reveal cell development throughout life and immune responses to environmental factors. Next-generation sequencing technologies have dramatically advanced the single-cell genomics and transcriptomics for T cell classification. However, gene expression is often not correlated with protein expression, and immunotyping is mostly accepted in the protein format. Current single-cell proteomics technologies are either limited in multiplex capacity or not sensitive enough to detect the critical functional proteins. Herein we present a cyclic multiplex in situ tagging (Cyclic MIST) technology to simultaneously measure 465 proteins, a scale of >10 times than similar technologies, in single cells. Such a high multiplexity is achieved by reiterative staining of the single cells coupled with a MIST array for detection. This technology has been thoroughly validated through comparison with flow cytometry and fluorescence immunostaining techniques. Both THP1 and CD4+ T cells are analyzed by the Cyclic MIST technology, and over 300 surface markers have been profiled to classify the subpopulations. This represents the most comprehensive mapping of the diversity of immune cells at the protein level. With additional information from intracellular proteins of the same single cells, our technology can potentially facilitate mechanistic studies of immune responses, particularly cytokine storm that results in sepsis.

scholarly journals 641 Spatially organized multicellular immune hubs in MMRd and MMRp colorectal cancer

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A670-A670
Author(s):  
Jonathan Chen ◽  
Karin Pelka ◽  
Matan Hofree ◽  
Marios Giannakis ◽  
Genevieve Boland ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Mismatch Repair ◽  
Molecular Mechanisms ◽  
Immune Cell ◽  
Single Cells ◽  
Large Set ◽  
Colon Tissue

BackgroundImmune responses to cancer are highly variable, with DNA mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. Almost all tumors are infiltrated with immune cells, but the types of immune responses and their effects on tumor growth, metastasis and death, vary greatly between different cancers and individual tumors. Which of the numerous cell subsets in a tumor contribute to the response, how their interactions are regulated, and how they are spatially organized within tumors remains poorly understood.MethodsTo understand the rules governing these varied responses, we transcriptionally profiled 371,223 single cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd treatment-naive patients. We developed a systematic approach to discover cell types, their underlying gene programs, and cellular communities based on single cell RNA-seq (scRNAseq) profiles and applied it to study the distinguishing features of human MMRd and MMRp colorectal cancer. Cellular communities discovered from this analysis were spatially mapped in tissue sections using multiplex RNA in situ hybridization microscopy.ResultsTo understand the basis for differential immune responses in CRC, we first determined and compared the immune cell composition of MMRd and MMRp CRC and normal colon tissue, finding dramatic remodeling between tumor and normal tissue and between MMRd and MMRp tumors, particularly within the myeloid, T cell, and stromal compartments. Among the clusters enriched in MMRd tumors were activated CXCL13+ CD8 T cells. Importantly, gene program co-variation analysis revealed multicellular networks. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage, and an MMRd-enriched immune hub within the tumor, with activated IFNG+ and CXCL13+ T cells together with malignant and myeloid cells expressing T-cell-attracting chemokines (figure 1).ConclusionsOur study provides a rich dataset of cellular states, gene programs and their transformations in tumors across a relatively large cohort of patients with colorectal cancer. Our predictions of several multicellular hubs based on co-variation of gene expression programs, and subsequent spatial localization of two major immune-malignant hubs, organizes a large set of cell states and programs into a smaller number of coordinated networks of cells and processes. Understanding the molecular mechanisms underlying these hubs, and studying their temporal and spatial regulation upon treatment will be critical for advancing cancer therapy.Ethics ApprovalThis study was approved by the DF-HCC institutional review board (protocols 03-189 and 02-240).Abstract 641 Figure 1A coordinated network of CXCL13+ T cells with myeloid and malignant cells expressing ISGs. Image shows a portion of formalin-fixed paraffin-embedded tissue from an MMRd CRC specimen stained with multiplex RNA ISH / IF for PanCK-IF, CD3E-ISH, CXCL10/CXCL11-ISH, CXCL13-ISH, and IFNG-ISH. Note IFNG+ and CXCL13+ cells in proximity to cells expressing the chemokines CXCL10/CXCL11

scholarly journals Single cell transcriptomics reveals the effect of PD-L1/TGF-β blockade on the tumor microenvironment

2020 ◽  
Author(s):  
Yoong Wearn Lim ◽  
Garry L. Coles ◽  
Savreet K. Sandhu ◽  
David S. Johnson ◽  
Adam S. Adler ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Microenvironment ◽  
Single Cell ◽  
Immune Cell ◽  
Single Cells ◽  
Cell Infiltration ◽  
Cytotoxic Lymphocytes ◽  
T Cell Infiltration ◽  
Anti Tumor Activity

AbstractBackgroundThe anti-tumor activity of anti-PD-1/PD-L1 therapies correlates with T cell infiltration in tumors. Thus, a major goal in oncology is to find strategies that enhance T cell infiltration and efficacy of anti-PD-1/PD-L1 therapy. TGF-β has been shown to contribute to T cell exclusion and anti-TGF-β improves anti-PD-L1 efficacy in vivo. However, TGF-β inhibition has frequently been shown to induce toxicity in the clinic, and the clinical efficacy of combination PD-L1 and TGF-β blockade has not yet been proven. To identify strategies to overcome resistance to PD-L1 blockade, the transcriptional programs associated with PD-L1 and/or TGF-β blockade in the tumor microenvironment should be further elucidated.ResultsFor the first time, we used single-cell RNA sequencing to characterize the transcriptomic effects of PD-L1 and/or TGF-β blockade on nearly 30,000 single cells in the tumor and surrounding microenvironment. Combination treatment led to upregulation of immune response genes, including multiple chemokine genes such as CCL5, in CD45+ cells, and down-regulation of extracellular matrix genes in CD45-cells. Analysis of publicly available tumor transcriptome profiles showed that the chemokine CCL5 was strongly associated with immune cell infiltration in various human cancers. Further investigation with in vivo models showed that intratumorally administered CCL5 enhanced cytotoxic lymphocytes and the anti-tumor activity of anti-PD-L1.ConclusionsTaken together, our data could be leveraged translationally to improve anti-PD-L1 plus anti-TGF-β combination therapy, for example through companion biomarkers, and/or to identify novel targets that could be modulated to overcome resistance.

Role of T cell subpopulations in mice infected with Angiostrongylus cantonensis

1996 ◽  
Vol 70 (3) ◽  
pp. 211-214 ◽  
Author(s):  
J.D. Lee ◽  
J.J. Wang ◽  
J.H. Chang ◽  
L.Y. Chung ◽  
E.R. Chen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Angiostrongylus Cantonensis ◽  
T Helper ◽  
The Third ◽  
Cell Subpopulations ◽  
T Cell Subpopulations

AbstractWhen C57BL/6 mice were infected with Angiostrongylus cantonensis, the percentage of T helper (CD4+) cells and T supressor (CD8+) cells in peripheral blood increased weekly until the third and seventh week respectively, and then gradually decreased. C57BL/6 mice were depleted of CD4+ and CD8+ T cells by in vivo injection of anti-CD4 and anti-CD8 monoclonal antibodies, respectively, and then infected with A. cantonensis. There were significantly more and less worms recovered in the mice depleted of CD4+ and CD8+ T cells respectively than in undepleted mice. Discrete subpopulations of T cells from mice exposed to A. cantonensis for 3 weeks or 7 weeks were adoptively transferred to syngeneic recipients which were then given a challenge infection. Protection was mediated by a CD4+ T cell population present in mice after 3 weeks of infection but was not demonstrable with cells taken 7 weeks after infection. When CD4+ T cells obtained from 3-week infected mice were mixed with 5% CD8+ T cells obtained from mice infected for 7 weeks, no significant transfer of resistance was observed. Thus, immune responses to A. cantonensis in mice were regulated by discrete subpopulations of T lymphocytes.

scholarly journals Single-Cell TCR and Transcriptome Analysis: An Indispensable Tool for Studying T-Cell Biology and Cancer Immunotherapy

2021 ◽  
Vol 12 ◽  
Author(s):  
Anna Pasetto ◽  
Yong-Chen Lu
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cancer Immunotherapy ◽  
Transcriptome Analysis ◽  
Cell Biology ◽  
Cell Receptor ◽  
Cell Subpopulations ◽  
Indispensable Tool ◽  
T Cell Subpopulations

T cells have been known to be the driving force for immune response and cancer immunotherapy. Recent advances on single-cell sequencing techniques have empowered scientists to discover new biology at the single-cell level. Here, we review the single-cell techniques used for T-cell studies, including T-cell receptor (TCR) and transcriptome analysis. In addition, we summarize the approaches used for the identification of T-cell neoantigens, an important aspect for T-cell mediated cancer immunotherapy. More importantly, we discuss the applications of single-cell techniques for T-cell studies, including T-cell development and differentiation, as well as the role of T cells in autoimmunity, infectious disease and cancer immunotherapy. Taken together, this powerful tool not only can validate previous observation by conventional approaches, but also can pave the way for new discovery, such as previous unidentified T-cell subpopulations that potentially responsible for clinical outcomes in patients with autoimmunity or cancer.

scholarly journals Massively parallel single-cell chromatin landscapes of human immune cell development and intratumoral T cell exhaustion

2019 ◽  
Author(s):  
Ansuman T. Satpathy ◽  
Jeffrey M. Granja ◽  
Kathryn E. Yost ◽  
Yanyan Qi ◽  
Francesca Meschi ◽  
...  
Keyword(s):  
T Cell ◽  
Single Cell ◽  
Immune Cell ◽  
Single Cells ◽  
Cell Types ◽  
Cell Development ◽  
Massively Parallel ◽  
T Cell Exhaustion ◽  
Cell Type ◽  
Cell Exhaustion

AbstractUnderstanding complex tissues requires single-cell deconstruction of gene regulation with precision and scale. Here we present a massively parallel droplet-based platform for mapping transposase-accessible chromatin in tens of thousands of single cells per sample (scATAC-seq). We obtain and analyze chromatin profiles of over 200,000 single cells in two primary human systems. In blood, scATAC-seq allows marker-free identification of cell type-specificcis- andtrans-regulatory elements, mapping of disease-associated enhancer activity, and reconstruction of trajectories of differentiation from progenitors to diverse and rare immune cell types. In basal cell carcinoma, scATAC-seq reveals regulatory landscapes of malignant, stromal, and immune cell types in the tumor microenvironment. Moreover, scATAC-seq of serial tumor biopsies before and after PD-1 blockade allows identification of chromatin regulators and differentiation trajectories of therapy-responsive intratumoral T cell subsets, revealing a shared regulatory program driving CD8+T cell exhaustion and CD4+T follicular helper cell development. We anticipate that droplet-based single-cell chromatin accessibility will provide a broadly applicable means of identifying regulatory factors and elements that underlie cell type and function.

scholarly journals Interpretation of T cell states from single-cell transcriptomics data using reference atlases

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Massimo Andreatta ◽  
Jesus Corria-Osorio ◽  
Sören Müller ◽  
Rafael Cubas ◽  
George Coukos ◽  
...  
Keyword(s):  
T Cell ◽  
Single Cell ◽  
Immune Cell ◽  
Meta Analysis ◽  
Single Cell Rna Sequencing ◽  
Cell Diversity ◽  
Transcriptomics Data ◽  
Consistent Basis ◽  
Definition Of ◽  
Human And Mouse

AbstractSingle-cell RNA sequencing (scRNA-seq) has revealed an unprecedented degree of immune cell diversity. However, consistent definition of cell subtypes and cell states across studies and diseases remains a major challenge. Here we generate reference T cell atlases for cancer and viral infection by multi-study integration, and develop ProjecTILs, an algorithm for reference atlas projection. In contrast to other methods, ProjecTILs allows not only accurate embedding of new scRNA-seq data into a reference without altering its structure, but also characterizing previously unknown cell states that “deviate” from the reference. ProjecTILs accurately predicts the effects of cell perturbations and identifies gene programs that are altered in different conditions and tissues. A meta-analysis of tumor-infiltrating T cells from several cohorts reveals a strong conservation of T cell subtypes between human and mouse, providing a consistent basis to describe T cell heterogeneity across studies, diseases, and species.

scholarly journals Single-cell transcriptomics reveals the effect of PD-L1/TGF-β blockade on the tumor microenvironment

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Yoong Wearn Lim ◽  
Garry L. Coles ◽  
Savreet K. Sandhu ◽  
David S. Johnson ◽  
Adam S. Adler ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Microenvironment ◽  
Single Cell ◽  
Immune Cell ◽  
Single Cells ◽  
Cell Infiltration ◽  
In Vivo Models ◽  
T Cell Infiltration ◽  
Anti Tumor Activity

Abstract Background The anti-tumor activity of anti-PD-1/PD-L1 therapies correlates with T cell infiltration in tumors. Thus, a major goal in oncology is to find strategies that enhance T cell infiltration and efficacy of anti-PD-1/PD-L1 therapy. TGF-β has been shown to contribute to T cell exclusion, and anti-TGF-β improves anti-PD-L1 efficacy in vivo. However, TGF-β inhibition has frequently been shown to induce toxicity in the clinic, and the clinical efficacy of combination PD-L1 and TGF-β blockade has not yet been proven. To identify strategies to overcome resistance to PD-L1 blockade, the transcriptional programs associated with PD-L1 and/or TGF-β blockade in the tumor microenvironment should be further elucidated. Results We used single-cell RNA sequencing in a mouse model to characterize the transcriptomic effects of PD-L1 and/or TGF-β blockade on nearly 30,000 single cells in the tumor and surrounding microenvironment. Combination treatment led to upregulation of immune response genes, including multiple chemokine genes such as CCL5, in macrophages, and downregulation of extracellular matrix genes in fibroblasts. Analysis of publicly available tumor transcriptome profiles showed that the chemokine CCL5 was strongly associated with immune cell infiltration in various human cancers. Further investigation with in vivo models showed that intratumorally administered CCL5 enhanced cytotoxic lymphocytes and the anti-tumor activity of anti-PD-L1. Conclusions Taken together, our data could be leveraged translationally to complement or find alternatives to anti-PD-L1 plus anti-TGF-β combination therapy, for example through companion biomarkers, and/or to identify novel targets that could be modulated to overcome resistance.

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