A theoretical framework of immune cell phenotypic classification and discovery

Immune cells are highly heterogeneous and show diverse phenotypes, but the underlying mechanism remains to be elucidated. In this study, we proposed a theoretical framework for immune cell phenotypic classification based on gene plasticity, which herein refers to expressional change or variability in response to conditions. The system contains two core points. One is that the functional subsets of immune cells can be further divided into subdivisions based on their highly plastic genes, and the other is that loss of phenotype accompanies gain of phenotype during phenotypic conversion. The first point suggests phenotypic stratification or layerability according to gene plasticity, while the second point reveals expressional compatibility and mutual exclusion during the change in gene plasticity states. Abundant transcriptome data analysis in this study from both microarray and RNA sequencing in human CD4 and CD8 single-positive T cells, B cells, natural killer cells and monocytes supports the logical rationality and generality, as well as expansibility, across immune cells. A collection of thousands of known immunophenotypes reported in the literature further supports that highly plastic genes play an important role in maintaining immune cell phenotypes and reveals that the current classification model is compatible with the traditionally defined functional subsets. The system provides a new perspective to understand the characteristics of dynamic, diversified immune cell phenotypes and intrinsic regulation in the immune system. Moreover, the current substantial results based on plasticitomics analysis of bulk and single-cell sequencing data provide a useful resource for big-data–driven experimental studies and knowledge discoveries

Cytokine-like 1 Chemoattracts Monocytes/Macrophages via CCR2

Abstract Cytokine-like 1 (CYTL1) is a novel potential cytokine that was first identified in CD34+ cells derived from bone marrow and cord blood, and it was also found using our immunogenomics strategy. The immunobiological functions of CYTL1 remain largely unknown, and its potential receptor(s) has not been identified. A previous proposed hypothesis suggested that CYTL1 had structural similarities with CCL2 and that CCR2 was a potential receptor of CYTL1. In this study, we verify that CYTL1 possesses chemotactic activity and demonstrate that its functional receptor is CCR2B using a series of experiments performed in HEK293 cells expressing CCR2B or CCR2B-EGFP, including chemotaxis, receptor internalization, and radioactive binding assays. CYTL1 chemoattracts human monocytes but not PBLs, and its chemotactic activity toward monocytes is dependent on the CCR2B-ERK pathway. Furthermore, both human and mouse recombinant CYTL1 protein have chemotactic effects on macrophages from wild-type mice but not from Ccr2−/− mice. Furthermore, the chemotactic activity of CYTL1 is sensitive to pertussis toxin. All of the above data confirm that CCR2B is a functional receptor of CYTL1.</jats:p

A Comparative Analysis of the Immunoglobulin Repertoire in Leukemia Cells and B Cells in Chinese Acute Myeloid Leukemia by High-Throughput Sequencing

It is common knowledge that immunoglobulin (Ig) is produced by B lymphocytes and mainly functions as an antibody. However, it has been shown recently that myeloblasts from acute myeloid leukemia (AML) could also express Ig and that AML-Ig played a role in leukemogenesis and AML progression. The difference between Ig from myeloblasts and B cells has not been explored. Studying the characteristics of the Ig repertoire in myeloblasts and B cells will be helpful to understand the function and significance of AML-Ig. We performed 5\u27 RACE-related PCR coupled with PacBio sequencing to analyze the Ig repertoire in myeloblasts and B cells from Chinese AML patients. Myeloblasts expressed all five classes of IgH, especially Igγ, with a high expression frequency. Compared with B-Ig in the same patient, AML-Ig showed different biased V(D)J usages and mutation patterns. In addition, the CDR3 length distribution of AML-Ig was significantly different from those of B-Ig. More importantly, mutations of AML-IgH, especially Igμ, Igα, and Igδ, were different from that of B-IgH in each AML patient, and the mutations frequently occurred at the sites of post-translational modification. AML-Ig has distinct characteristics of variable regions and mutations, which may have implications for disease monitoring and personalized therapy

Identification of FAM3D as a novel endogenous chemotaxis agonist for the FPRs (formyl peptide receptors)

The family with sequence similarity 3 (FAM3) gene family is a cytokine-like gene family with four members FAM3A, FAM3B, FAM3C, and FAM3D. In this study, we found that FAM3D strongly chemoattracted human peripheral blood neutrophils and monocytes. To identify FAM3D receptor, we used chemotaxis, receptor internalization, calcium flux and radioligand-binding assays in FAM3D-stimulated HEK293 cells that transiently expressed FPR1 or FPR2 to show that FAM3D was a high affinity ligand of formyl peptide receptors (FPR1 and FPR2), both of which were highly expressed on the surface of neutrophils and monocytes/macrophages. After being injected into the mouse peritoneal cavity, FAM3D chemoattracted CD11b+Ly6G+ neutrophils in a short time. In response to FAM3D stimulation, p-ERK and p-p38 were up-regulated in the mouse neutrophils, which could be inhibited by an inhibitor of FPR1 or FPR2. FAM3D was reported to be constitutively expressed in the gastrointestinal tract. We found that FAM3D expression increased significantly in dextran sulfate sodium-induced colitis. Taken together, we propose that FAM3D plays a role in gastrointestinal homeostasis and inflammation through its receptors FPR1 and FPR2.</jats:p

Electronic sorting of immune cell subpopulations based on highly plastic genes

Abstract The publicly available omic big data provide a great and valuable resource to drive big-data-based discoveries. Previously, we used immunologically relevant transcriptome data from microarrays to analyze gene co-expression, gene plasticity (GPL) and biomarker evaluation for human and mouse immune cells, and have built two immunological databases, ImmuCo (http://immuco.bjmu.edu.cn/) and ImmuSort (http://immusort.bjmu.edu.cn/), for gene co-expression and gene plasticity analyses, respectively. In the current study, we addressed a brand new method for the first time, electronic sorting, which surveys big data samples to do virtual analysis and to sort immune cell subpopulations based on highly plastic genes at mRNA level. We also called this process either ‘virtual sorting’ or ‘in silico sorting’, because no actual immune cells were sorted as they are in the fluorescence-activated cell sorting (FACS) process. Through virtual sorting of human Tregs and cytokine-producing T helper cells, our results strongly supported the feasibility, effectiveness and predictability of this method. For example, we found that human CD4+FOXP3hi T cells were highly expressive of certain known molecules such as CD25 and CTLA4, and that this process of investigation did not require isolating or inducing these immune cells in vitro. We made a systematic virtual analysis of all of the potential human CD4+ T cell subpopulations, and potential signature genes or marker molecules for these subpopulations were identified. Besides CD4+ T cells, the methods in this study are also suitable for other immune cells, non-immune cells and even for cancers for studies on gene expression and regulation.</jats:p

ImmuMethy, a database of DNA methylation plasticity at a single cytosine resolution in human blood and immune cells

Abstract Differential DNA methylation is a feature of numerous physiological and pathological processes. However, the extent to which single-base cytosine methylation modifies cellular responses to various stimuli has not been well characterized. In this study, we carried out a systematic analysis of methylome data derived from human blood and immune cells and constructed the ImmuMethy database. ImmuMethy allows interrogation of DNA methylation plasticity (MPL) at the single cytosine level. MPL, which refers to the variability of DNA methylation, is quantitatively measured in multiple ways, such as quartiles and standard deviations. ImmuMethy comprises over 36 000 samples from the Human Methylation450 and MethylationEPIC BeadChips platforms and provides multiple applications, such as an overview of methylation status and plasticity, differential methylation analysis, identification of methylation markers and sample stratification. An analysis of all datasets revealed that DNA methylation is generally stable, with minimal changes in beta values. This further supports the characteristics of DNA methylation homeostasis. Based on the beta value distribution, we identified three types of methylation sites: methylation tendency sites, unmethylation tendency sites and dual tendency or nonbiased methylation sites. These sites represent different methylation tendentiousness of DNA methylation across samples. The occurrence of multiple methylation tendencies in a site means split methylation, which generally corresponds to high MPL. Inverted methylation tendencies from methylation tendency sites to unmethylation tendency sites, or vice versa, represent strong differential methylation in response to conditions. All these sites can be identified in ImmuMethy, making it a useful tool for omics-based data-driven knowledge discovery. Database URL: http://immudb.bjmu.edu.cn/immumethy/</jats:p