Intra-tumoral injection of caerin 1.1 and 1.9 peptides in vaccinated TC-1 tumour bearing mice with PD-1 blockade modulates macrophage heterogeneity and the activation of CD8+ T cells in the tumour microenvironment

Abstract Development of a vaccine formula that alters the tumour-infiltrating lymphocytes to be more immune active against a tumour is key to the improvement of clinical responses to immunotherapy. Here, we demonstrate that, in conjunction with E7 antigen specific immunotherapy, and IL-10 and PD-1 blockade, intra-tumoral administration of caerin 1.1 and 1.9 peptides further improves the tumour microenvironment (TME) when compared with injection of a control peptide. We used single cell transcriptomics and mass spectrometry-based proteomics to quantify changes in cellular activity across different cell types within the TME. We show that the injection of caerin 1.1/1.9 increases immune activating macrophages and NK cells, while reducing immunosuppressive macrophages with M2 phenotype, and increased numbers of activated CD8+ T cells with higher populations of memory and effector-memory CD8+ T subsets. Proteomic profiling demonstrated activation of Stat1 modulated apoptosis and production of nitric oxide. Further, computational integration of the proteome with the single cell transcriptome was consistent with deactivation of immune suppressive B cell function following caerin 1.1 and 1.9 treatment.

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Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers

Nature ◽

10.1038/s41586-021-03752-4 ◽

2021 ◽

Author(s):

Justina X. Caushi ◽

Jiajia Zhang ◽

Zhicheng Ji ◽

Ajay Vaghasia ◽

Boyang Zhang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

T Cell Activation ◽

Cd8 T Cells ◽

T Cell Receptors ◽

Tumour Microenvironment ◽

Lung Cancers ◽

Cell Receptors ◽

Interleukin 7

AbstractPD-1 blockade unleashes CD8 T cells1, including those specific for mutation-associated neoantigens (MANA), but factors in the tumour microenvironment can inhibit these T cell responses. Single-cell transcriptomics have revealed global T cell dysfunction programs in tumour-infiltrating lymphocytes (TIL). However, the majority of TIL do not recognize tumour antigens2, and little is known about transcriptional programs of MANA-specific TIL. Here, we identify MANA-specific T cell clones using the MANA functional expansion of specific T cells assay3 in neoadjuvant anti-PD-1-treated non-small cell lung cancers (NSCLC). We use their T cell receptors as a ‘barcode’ to track and analyse their transcriptional programs in the tumour microenvironment using coupled single-cell RNA sequencing and T cell receptor sequencing. We find both MANA- and virus-specific clones in TIL, regardless of response, and MANA-, influenza- and Epstein–Barr virus-specific TIL each have unique transcriptional programs. Despite exposure to cognate antigen, MANA-specific TIL express an incompletely activated cytolytic program. MANA-specific CD8 T cells have hallmark transcriptional programs of tissue-resident memory (TRM) cells, but low levels of interleukin-7 receptor (IL-7R) and are functionally less responsive to interleukin-7 (IL-7) compared with influenza-specific TRM cells. Compared with those from responding tumours, MANA-specific clones from non-responding tumours express T cell receptors with markedly lower ligand-dependent signalling, are largely confined to HOBIThigh TRM subsets, and coordinately upregulate checkpoints, killer inhibitory receptors and inhibitors of T cell activation. These findings provide important insights for overcoming resistance to PD-1 blockade.

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Knowledge-based classification of fine-grained immune cell types in single-cell RNA-Seq data with ImmClassifier

10.1101/2020.03.23.002758 ◽

2020 ◽

Author(s):

Xuan Liu ◽

Sara J.C. Gosline ◽

Lance T. Pflieger ◽

Pierre Wallet ◽

Archana Iyer ◽

...

Keyword(s):

T Cells ◽

Single Cell ◽

Immune Cells ◽

Immune Cell ◽

Cell Types ◽

Effector Memory ◽

Immune Cell Population ◽

Fine Grained ◽

Knowledge Based ◽

Comparable Performance

AbstractSingle-cell RNA sequencing is an emerging strategy for characterizing the immune cell population in diverse environments including blood, tumor or healthy tissues. While this has traditionally been done with flow or mass cytometry targeting protein expression, scRNA-Seq has several established and potential advantages in that it can profile immune cells and non-immune cells (e.g. cancer cells) in the same sample, identify cell types that lack precise markers for flow cytometry, or identify a potentially larger number of immune cell types and activation states than is achievable in a single flow assay. However, scRNA-Seq is currently limited due to the need to identify the types of each immune cell from its transcriptional profile, which is not only time-consuming but also requires a significant knowledge of immunology. While recently developed algorithms accurately annotate coarse cell types (e.g. T cells vs macrophages), making fine distinctions has turned out to be a difficult challenge. To address this, we developed a machine learning classifier called ImmClassifier that leverages a hierarchical ontology of cell type. We demonstrate that ImmClassifier outperforms other tools (+20% recall, +14% precision) in distinguishing fine-grained cell types (e.g. CD8+ effector memory T cells) with comparable performance on coarse ones. Thus, ImmClassifier can be used to explore more deeply the heterogeneity of the immune system in scRNA-Seq experiments.

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Single-cell RNA-Seq reveals the link between CD45 isoforms and tumor-infiltrating T cells heterogeneity in liver cancer

10.1101/2020.03.22.002824 ◽

2020 ◽

Author(s):

Biaofeng Zhou ◽

Shang Liu ◽

Liang Wu ◽

Yan Sun ◽

Jie Chen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Lymphocytes ◽

Single Cell ◽

Cell Function ◽

Effector Memory ◽

Single Cell Level ◽

Cell Level ◽

Cd45 Isoforms ◽

And Function

AbstractCD45 isoforms play a major role in characterizing T cell function, phenotype, and development. However, there is lacking comprehensive interrogation about the relationship between CD45 isoforms and T lymphocytes from cancer patients at the single-cell level yet. Here, we investigated the CD45 isoforms component of published 5,063 T cells of hepatocellular carcinoma (HCC), which has been assigned functional states. We found that the distribution of CD45 isoforms in T lymphocytes cells depended on tissue resource, cell type, and functional state. Further, we demonstrated that CD45RO and CD45RA dominate in characterizing the phenotype and function of T cell though multiple CD45 isoforms coexist in T cells, through a novel alternative splicing pattern analysis. We identified a novel development trajectory of tumor-infiltrating T cells from Tcm to Temra (effector memory T cells re-expresses CD45RA) after detecting two subpopulations in state of transition, Tcm (central memory T) and Tem (effector memory T). Temra, capable of high cytotoxic characteristics, was discovered to be associated with the stage of HCC and may be a target of immunotherapy. Our study presents a comprehension of the connection between CD45 isoforms and the function, states, sources of T lymphocytes cells in HCC patients at the single-cell level, providing novel insight for the effect of CD45 isoforms on T cell heterogeneity.

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Macrophage-Mediated Subversion of Anti-Tumour Immunity

Cells ◽

10.3390/cells8070747 ◽

2019 ◽

Vol 8 (7) ◽

pp. 747 ◽

Cited By ~ 20

Author(s):

Valeria Quaranta ◽

Michael C. Schmid

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Tumour Progression ◽

Immune Surveillance ◽

Cell Types ◽

Tumour Microenvironment ◽

Tumour Immunity ◽

Cell Recruitment ◽

Clinical Benefits

Despite the incredible clinical benefits obtained by the use of immune checkpoint blockers (ICBs), resistance is still common for many types of cancer. Central for ICBs to work is activation and infiltration of cytotoxic CD8+ T cells following tumour-antigen recognition. However, it is now accepted that even in the case of immunogenic tumours, the effector functions of CD8+ T cells are highly compromised by the presence of an immunosuppressive tumour microenvironment (TME) at the tumour site. Tumour-associated macrophages (TAMs) are among the most abundant non-malignant stromal cell types within the TME and they are crucial drivers of tumour progression, metastasis and resistance to therapy. TAMs are able to regulate either directly or indirectly various aspects of tumour immunity, including T cell recruitment and functions. In this review we discuss the mechanisms by which TAMs subvert CD8+ T cell immune surveillance and how their targeting in combination with ICBs represents a very powerful therapeutic strategy.

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Aberrant peripheral immune responses in acute Kawasaki disease with single-cell sequencing

10.1101/2020.11.05.369348 ◽

2020 ◽

Author(s):

Zhen Wang ◽

Lijian Xie ◽

Sirui Song ◽

Liqin Chen ◽

Guang Li ◽

...

Keyword(s):

T Cells ◽

Kawasaki Disease ◽

Immune Responses ◽

Single Cell ◽

Cd8 T Cells ◽

Developed Countries ◽

Interferon Response ◽

Effector Memory ◽

Intravenous Immunoglobulin Therapy ◽

Single Cell Sequencing

AbstractKawasaki disease (KD) is the most common cause of acquired heart disease in children in developed countries. Although diverse immune aberrance was reported, a global understanding of immune responses underlying acute KD was lacking. Based on single-cell sequencing, we profiled peripheral blood mononuclear cells from patients with acute KD before and after intravenous immunoglobulin therapy and from healthy controls. Most differentially expressed genes were derived from monocytes, with upregulation of immunoglobulin receptors, complement and receptors and downregulation of MHC class II receptors before therapy. The percentage of B cells was significantly increased before therapy and rapidly returned to normal after therapy. There was also an increased abundance of B-cell receptors with IGHA and IGHG after therapy, accompanied by massive oligoclonal expansion. The percentage of CD8 T cells was remarkably decreased during acute KD, especially the subset of effector memory CD8 T cells. All lymphocyte compartments were characterized by underexpressed interferon response pathways before therapy. The identification of unique innate and adaptive immune responses suggests potential mechanisms underlying pathogenesis and progression of KD.

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Single-cell transcriptional atlas of the Chinese horseshoe bat (Rhinolophus sinicus) provides insight into the cellular mechanisms which enable bats to be viral reservoirs

10.1101/2020.06.30.175778 ◽

2020 ◽

Cited By ~ 2

Author(s):

Lili Ren ◽

Chao Wu ◽

Li Guo ◽

Jiacheng Yao ◽

Conghui Wang ◽

...

Keyword(s):

T Cells ◽

Single Cell ◽

Cell Biology ◽

Expression Patterns ◽

Cell Types ◽

Transcriptional Analysis ◽

Effector Memory ◽

Viral Reservoirs ◽

Isolated Lung ◽

Horseshoe Bat

AbstractBats are a major “viral reservoir” in nature and there is a great interest in not only the cell biology of their innate and adaptive immune systems, but also in the expression patterns of receptors used for cellular entry by viruses with potential cross-species transmission. To address this and other questions, we created a single-cell transcriptomic atlas of the Chinese horseshoe bat (Rhinolophus sinicus) which comprises 82,924 cells from 19 organs and tissues. This atlas provides a molecular characterization of numerous cell types from a variety of anatomical sites, and we used it to identify clusters of transcription features that define cell types across all of the surveyed organs. Analysis of viral entry receptor genes for known zoonotic viruses showed cell distribution patterns similar to that of humans, with higher expression levels in bat intestine epithelial cells. In terms of the immune system, CD8+ T cells are in high proportion with tissue-resident memory T cells, and long-lived effector memory nature killer (NK) T-like cells (KLRG1, GZMA and ITGA4 genes) are broadly distributed across the organs. Isolated lung primary bat pulmonary fibroblast (BPF) cells were used to evaluate innate immunity, and they showed a weak response to interferon β and tumor necrosis factor-α compared to their human counterparts, consistent with our transcriptional analysis. This compendium of transcriptome data provides a molecular foundation for understanding the cell identities, functions and cellular receptor characteristics for viral reservoirs and zoonotic transmission.

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Single-cell transcriptome analysis of the immunosuppressive effect of differential expression of tumor PD-L1 on responding TCR-T cells

10.1101/2020.07.23.217059 ◽

2020 ◽

Author(s):

Renpeng Ding ◽

Shang Liu ◽

Shanshan Wang ◽

Huanyi Chen ◽

Fei Wang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Tumor Cells ◽

Cell Function ◽

Immune Cell ◽

Gene Clusters ◽

Cellular Assays ◽

Cell Transcriptome ◽

Single Cell Transcriptome

AbstractPD-L1 expression levels in tumors do not consistently predict cancer patients’ response to PD-(L)1 inhibitors. We therefore evaluated how tumor PD-L1 levels affect the anti-PD-(L)1 efficacy and T cell function. We used MART-1-specific TCR-T cells (TCR-TMART-1) stimulated with MART-127-35 peptide-loaded MEL-526 tumor cells with different proportions of them expressing PD-L1 to perform cellular assays and high-throughput single-cell RNA sequencing. Compared to control T cells, TCR-TMART-1 were more sensitive to exhaustion and secreted lower pro-inflammatory but higher anti-inflammatory cytokines with increasing proportions of PD-L1+ tumor cells. The colocalization of T cells and tumor cells in gene clusters correlated negatively with the proportion of PD-L1+ tumor cells and positively with immune cell cytotoxicity. Moreover, elevated proportion of PD-L1+ tumor cells increased PD-L1 expression and decreased PD-1 expression on T cells and enhanced T cell death. The expression of PD-1 and PD-L1 in T cells and macrophages also correlated positively with COVID-19 severity.

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Knowledge-based classification of fine-grained immune cell types in single-cell RNA-Seq data

Briefings in Bioinformatics ◽

10.1093/bib/bbab039 ◽

2021 ◽

Author(s):

Xuan Liu ◽

Sara J C Gosline ◽

Lance T Pflieger ◽

Pierre Wallet ◽

Archana Iyer ◽

...

Keyword(s):

T Cells ◽

Single Cell ◽

Immune Cell ◽

Cell Types ◽

Effector Memory ◽

Fine Grained ◽

Knowledge Based ◽

Learning Classifier ◽

Comparable Performance ◽

Difficult Challenge

Abstract Single-cell RNA sequencing (scRNA-Seq) is an emerging strategy for characterizing immune cell populations. Compared to flow or mass cytometry, scRNA-Seq could potentially identify cell types and activation states that lack precise cell surface markers. However, scRNA-Seq is currently limited due to the need to manually classify each immune cell from its transcriptional profile. While recently developed algorithms accurately annotate coarse cell types (e.g. T cells versus macrophages), making fine distinctions (e.g. CD8+ effector memory T cells) remains a difficult challenge. To address this, we developed a machine learning classifier called ImmClassifier that leverages a hierarchical ontology of cell type. We demonstrate that its predictions are highly concordant with flow-based markers from CITE-seq and outperforms other tools (+15% recall, +14% precision) in distinguishing fine-grained cell types with comparable performance on coarse ones. Thus, ImmClassifier can be used to explore more deeply the heterogeneity of the immune system in scRNA-Seq experiments.

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A Single-Cell Transcriptome Profiling of Anterior Kidney Leukocytes From Nile Tilapia (Oreochromis niloticus)

Frontiers in Immunology ◽

10.3389/fimmu.2021.783196 ◽

2021 ◽

Vol 12 ◽

Author(s):

Liting Wu ◽

Along Gao ◽

Lan Li ◽

Jianlin Chen ◽

Jun Li ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Single Cell ◽

Nile Tilapia ◽

Immune Cell ◽

Cell Types ◽

Cell Transcriptome ◽

B Cell Subsets ◽

Single Cell Transcriptome

Teleost fish anterior kidney (AK) is an important hematopoietic organ with multifarious immune cells, which have immune functions comparable to mammalian bone marrow. Myeloid and lymphoid cells locate in the AK, but the lack of useful specific gene markers and antibody-based reagents for the cell subsets makes the identification of the different cell types difficult. Single-cell transcriptome sequencing enables single-cell capture and individual library construction, making the study on the immune cell heterogeneity of teleost fish AK possible. In this study, we examined the transcriptional patterns of 11,388 AK leukocytes using 10× Genomics single-cell RNA sequencing (scRNA-seq). A total of 22 clusters corresponding to five distinct immune cell subsets were identified, which included B cells, T cells, granulocytes, macrophages, and dendritic cells (DCs). However, the subsets of myeloid cells (granulocytes, macrophages, and DCs) were not identified in more detail according to the known specific markers, even though significant differences existed among the clusters. Thereafter, we highlighted the B-cell subsets and identified them as pro/pre B cells, immature/mature B cells, activated B/plasmablasts, or plasma cells based on the different expressions of the transcription factors (TFs) and cytokines. Clustering of the differentially modulated genes by pseudo-temporal trajectory analysis of the B-cell subsets showed the distinct kinetics of the responses of TFs to cell conversion. Moreover, we classified the T cells and discovered that CD3+CD4−CD8−, CD3+CD4+CD8+, CD4+CD8−, and CD4−CD8+ T cells existed in AK, but neither CD4+CD8− nor CD4−CD8+ T cells can be further classified into subsets based on the known TFs and cytokines. Pseudotemporal analysis demonstrated that CD4+CD8− and CD4−CD8+ T cells belonged to different states with various TFs that might control their differentiation. The data obtained above provide a valuable and detailed resource for uncovering the leukocyte subsets in Nile tilapia AK, as well as more potential markers for identifying the myeloid and lymphoid cell types.

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Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Peripheral Mononuclear Cells in Patients With Ankylosing Spondylitis

Frontiers in Immunology ◽

10.3389/fimmu.2021.760381 ◽

2021 ◽

Vol 12 ◽

Author(s):

Huixuan Xu ◽

Haiyan Yu ◽

Lixiong Liu ◽

Hongwei Wu ◽

Cantong Zhang ◽

...

Keyword(s):

T Cells ◽

Ankylosing Spondylitis ◽

Single Cell ◽

Cd8 T Cells ◽

Mononuclear Cells ◽

Cell Types ◽

Magnetic Bead ◽

Point Of View ◽

Pathogenic Genes ◽

Peripheral Mononuclear Cells

ObjectiveGenetic studies on ankylosing spondylitis (AS) have identified more than 100 pathogenic genes. Building a bridge between these genes and biologically targeted therapies is the current research hotspot.MethodsWe integrated single-cell assaying transposase-accessible chromatin sequencing (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) to explore the key genes and related mechanisms associated with AS pathogenesis.ResultsWe identified 18 cell types in peripheral mononuclear cells from patients with AS and normal controls and summarized the cell-type-specific abnormal genes by scRNA-seq. Interestingly, we found that the pathogenic gene NFKB involved in AS progression originated from CD8+ T cells. Moreover, we observed an abnormal tumor TNF pathway mediated by abnormal expression of TNF, NFKB, FOS, JUN, and JUNB, and scATAC-seq results confirmed the abnormal accessible binding sites of transcriptional factors FOS, JUN, and JUNB. The final magnetic bead sorting and quantitative real-time PCR(RT-qPCR) confirmed that NFKB, FOS, JUN, and JUNB in CD8+ T cells differed in the AS group.ConclusionsOur results revealed a possible mechanism by which NFKB abnormally regulates FOS, JUN, and JUNB and drives AS progression, providing a novel perspective from a single cell point of view in AS.

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