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The ability of the CD8+HLA-DR+ regulatory T lymphocytes population to regulate the immune response was first described several years ago. It is known that the suppressive effects of these cells depend on intercellular interactions and are mediated by the expression of checkpoint inhibitor molecules such as CTLA-4, TIM-3, PD-1 and LAG-3. The CD8+HLA-DR+ regulatory T cells also share some properties with conventional CD4+ regulatory T lymphocytes. Nevertheless, the characteristics and function of this subpopulation remain poorly understood. Furthermore, studying the properties of CD8+HLA-DR+ regulatory T cells becomes relevant in the light of general age-associated changes in the human immune system and the increased sensitivity of CD8+T lymphocytes to these changes. Therefore, the aim of this study was to investigate the age dynamics and search for transcriptional signatures of the CD8+HLA-DR+ regulatory T cells. For this purpose, flow cytometric analysis of peripheral blood mononuclear cells from 18 donors aged 21 to 85 years was performed. Bioinformatic analysis of single-cell RNA sequencing data was carried out to search for signatures. It was found that CD8+HLA-DR+ regulatory T cells accumulate with age. The transcriptional signatures of this population consist of genes involved in antigen presentation and cytotoxicity, along with a decrease in the expression of genes encoding proteins of activating protein 1 complex. These data suggest mechanisms of suppressor function of CD8+HLA-DR+ regulatory T lymphocytes associated with the ability of these cells to present antigens and perform cytotoxic activity against effector T lymphocytes. The accumulation of the studied cells may imply a potential influence of CD8+HLA-DR+ regulatory T lymphocytes on the efficiency of adaptive immune response in the aging. Further studies of this population may provide insights into its role in age-related changes in the immune system and develop strategies to improve the immune response in the elderly.

IntroductionInflammatory epidermolysis bullosa acquisita (EBA) is characterized by a neutrophilic response to anti-type VII collagen (COL7) antibodies resulting in the development of skin inflammation and blistering. The antibody transfer model of EBA closely mirrors this EBA phenotype.MethodsTo better understand the changes induced in neutrophils upon recruitment from peripheral blood into lesional skin in EBA, we performed single-cell RNA-sequencing of whole blood and skin dissociate to capture minimally perturbed neutrophils and characterize their transcriptome.ResultsThrough this approach, we identified clear distinctions between circulating activated neutrophils and intradermal neutrophils. Most strikingly, the gene expression of multiple C-type lectin receptors, which have previously been reported to orchestrate host defense against fungi and select bacteria, were markedly dysregulated. After confirming the upregulation of Clec4n, Clec4d, and Clec4e in experimental EBA as well as in lesional skin from patients with inflammatory EBA, we performed functional studies in globally deficient Clec4e−/− and Clec4d−/− mice as well as in neutrophil-specific Clec4n−/− mice. Deficiency in these genes did not reduce disease in the EBA model.DiscussionCollectively, our results suggest that while the upregulation of Clec4n, Clec4d, and Clec4e is a hallmark of activated dermal neutrophil populations, their individual contribution to the pathogenesis of EBA is dispensable.

BackgroundT cell lymphomas (TCL) are a group of heterogeneous diseases with over 40 subtypes. In this study, we identified a novel TCL subtype which was featured by a unique T cell receptor (TCR) presentation, α, β and γ chains were co-existing in a single malignant T cell.Case presentationA 45-year-old male patient was diagnosed T cell lymphoma after 2-month of abdominal distension and liver enlargement. Combining histology review, PET-CT scanning and immunophenotyes, the patient was not classified to any existing TCL subtypes. To better understand this unclassified TCL case, we performed single cell RNA sequencing paired with TCR sequencing on the patient’s PBMC and bone marrow samples. To our surprise, we identified that the malignant T cells had a very rare TCR combination, by expressing two α chains, one β chain and one γ chain simultaneously. We further studied the molecular pathogenesis and tumor cell heterogeneity of this rare TCL subtype. A set of potential therapeutic targets were identified from the transcriptome data, such as CCL5, KLRG1 and CD38.ConclusionsWe identified the first TCL case co-expressing α, β and γ chains and dissected its molecular pathogenesis, providing valuable information for precision medicine options for this novel TCL subtype.

Translationally relevant animal models are essential for the successful translation of basic science findings into clinical medicine. While rodent models are widely accessible, there are numerous limitations that prevent the extrapolation of findings to human medicine. One approach to overcome these limitations is to use animal models that are genetically diverse and naturally develop disease. For example, pet dogs spontaneously develop diseases that recapitulate the natural progression seen in humans and live in similar environments alongside humans. Thus, dogs represent a useful animal model for many areas of research. Despite the value of the canine model, species specific reagent limitations have hampered in depth characterization of canine immune cells, which constrains the conclusions that can be drawn from canine immunotherapy studies. To address this need, we used single-cell RNA sequencing to characterize the heterogeneity of circulating leukocytes in healthy dogs (n = 7) and osteosarcoma (OS) affected dogs (n = 10). We present a cellular atlas of leukocytes in healthy dogs, then employ the dataset to investigate the impact of primary OS tumors on the transcriptome of circulating leukocytes. We identified 36 unique cell populations amongst dog circulating leukocytes, with a remarkable amount of heterogeneity in CD4 T cell subtypes. In our comparison of healthy dogs and dogs with OS, we identified relative increases in the abundances of polymorphonuclear (PMN-) and monocytic (M-) myeloid-derived suppressor cells (MDSCs), as well as aberrations in gene expression within myeloid cells. Overall, this study provides a detailed atlas of canine leukocytes and investigates how the presence of osteosarcoma alters the transcriptional profiles of circulating immune cells.

Defining the immunological landscape of human tissue is an important area of research, but challenges include the impact of tissue disaggregation on cell phenotypes and the low abundance of immune cells in many tissues. Here, we describe methods to troubleshoot and standardize Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) for studies involving enzymatic digestion of human tissue. We tested epitope susceptibility of 92 antibodies commonly used to differentiate immune lineages and cell states on human peripheral blood mononuclear cells following treatment with an enzymatic digestion cocktail used to isolate islets. We observed CD4, CD8a, CD25, CD27, CD120b, CCR4, CCR6, and PD1 display significant sensitivity to enzymatic treatment, effects that often could not be overcome with alternate antibodies. Comparison of flow cytometry-based CITE-seq antibody titrations and sequencing data supports that for the majority of antibodies, flow cytometry accurately predicts optimal antibody concentrations for CITE-seq. Comparison by CITE-seq of immune cells in enzymatically digested islet tissue and donor-matched spleen not treated with enzymes revealed little digestion-induced epitope cleavage, suggesting increased sensitivity of CITE-seq and/or that the islet structure may protect resident immune cells from enzymes. Within islets, CITE-seq identified immune cells difficult to identify by transcriptional signatures alone, such as distinct tissue-resident T cell subsets, mast cells, and innate lymphoid cells (ILCs). Collectively this study identifies strategies for the rational design and testing of CITE-seq antibodies for single-cell studies of immune cells within islets and other tissues.

IntroductionHigh stress in the compartment surrounded by the deep fascia can cause acute compartment syndrome (ACS) that may result in necrosis of the limbs. The study aims to investigate the cellular heterogeneity of the deep fascia in ACS patients by single-cell RNA sequencing (scRNA-seq).MethodsWe collected deep fascia samples from patients with ACS (high-stress group, HG, n=3) and patients receiving thigh amputation due to osteosarcoma (normal-stress group, NG, n=3). We utilized ultrasound and scanning electron microscopy to observe the morphologic change of the deep fascia, used multiplex staining and multispectral imaging to explore immune cell infiltration, and applied scRNA-seq to investigate the cellular heterogeneity of the deep fascia and to identify differentially expressed genes.ResultsNotably, we identified GZMK+interferon-act CD4 central memory T cells as a specific high-stress compartment subcluster expressing interferon-related genes. Additionally, the changes in the proportions of inflammation-related subclusters, such as the increased proportion of M2 macrophages and decreased proportion of M1 macrophages, may play crucial roles in the balance of pro-inflammatory and anti-inflammatory in the development of ACS. Furthermore, we found that heat shock protein genes were highly expressed but metal ion-related genes (S100 family and metallothionein family) were down-regulated in various subpopulations under high stress.ConclusionsWe identified a high stress-specific subcluster and variations in immune cells and fibroblast subclusters, as well as their differentially expressed genes, in ACS patients. Our findings reveal the functions of the deep fascia in the pathophysiology of ACS, providing new approaches for its treatment and prevention.

Necroptosis is a programmed cell death playing a significant role in cancer. Although necroptosis has been related to tumor immune environment (TIME) remodeling and cancer prognosis, however, the role of necroptosis-related genes (NRGs) in glioma is still elusive. In this study, a total of 159 NRGs were obtained, and parameters such as mutation rate, copy number variation (CNV), and relative expression level were assessed. Then, we constructed an 18-NRGs-based necroptosis-related signature (NRS) in the TCGA dataset, which could predict the patient’s prognosis and was validated in two external CGGA datasets. We also explored the correlation between NRS and glioma TIME, chemotherapy sensitivity, and certain immunotherapy-related factors. The two necroptosis-related subtypes were discovered and could also distinguish the patients' prognosis. Through the glioblastoma (GBM) scRNA-seq data analysis, NRGs’ expression levels in different GBM patient tissue cell subsets were investigated and the relative necroptosis status of different cell subsets was assessed, with the microglia score culminating among all. Moreover, we found a high infiltration level of immunosuppressive cells in glioma TIME, which was associated with poor prognosis in the high-NRS glioma patient group. Finally, the necroptosis suppressor CASP8 exhibited a high expression in glioma and was associated with poor prognosis. Subsequent experiments were performed in human glioma cell lines and patients' tissue specimens to verify the bioinformatic analytic findings about CASP8. Altogether, this study provides comprehensive evidence revealing a prognostic value of NRGs in glioma, which is associated with TIME regulation.

The freshwater snail Biomphalaria glabrata is an intermediate host of Schistosoma mansoni, the agent of human intestinal schistosomiasis. However, much is to be discovered about its innate immune system that appears as a complex black box, in which the immune cells (called hemocytes) play a major role in both cellular and humoral response towards pathogens. Until now, hemocyte classification has been based exclusively on cell morphology and ultrastructural description and depending on the authors considered from 2 to 5 hemocyte populations have been described. In this study, we proposed to evaluate the hemocyte heterogeneity at the transcriptomic level. To accomplish this objective, we used single cell RNA sequencing (scRNAseq) technology coupled to a droplet-based system to separate hemocytes and analyze their transcriptome at a unique cell level in naive Biomphalaria glabrata snails. We were able to demonstrate the presence of 7 hemocyte transcriptomic populations defined by the expression of specific marker genes. As a result, scRNAseq approach showed a high heterogeneity within hemocytes, but provides a detailed description of the different hemocyte transcriptomic populations in B. glabrata supported by distinct cellular functions and lineage trajectory. As a main result, scRNAseq revealed the 3 main population as a super-group of hemocyte diversity but, on the contrary, a great hemocytes plasticity with a probable capacity of hemocytes to engage to different activation pathways. This work opens a new field of research to understand the role of hemocytes particularly in response to pathogens, and towards S. mansoni parasites.

CD11c+ T-bet+ atypical B cells (ABCs) have been identified in the context of vaccination, acute and chronic infections and autoimmune disease. However, the origins and functions of ABCs remain elusive. A major obstacle in the study of ABCs, and human MBCs more generally, has been the use of different phenotypic markers in different contexts to identify what appear to be phenotypically similar cells. Advances in single-cell RNA sequencing (scRNA-seq) technology have allowed researchers to accurately identify ABCs in different immune contexts such as diseases and tissues. Notably, recent studies utilizing single cell techniques have demonstrated ABCs are a highly conserved memory B cell lineage. This analysis has also revealed that ABCs are more abundant in ostensibly healthy donors than previously thought. Nonetheless, the normal function of these cells remains elusive. In this review, we will focus on scRNA-seq studies to discuss recent advances in our understanding about the development and functions of ABCs.

Plasmacytoid dendritic cells [pDCs] represent a rare innate immune subset uniquely endowed with the capacity to produce substantial amounts of type-I interferons. This function of pDCs is critical for effective antiviral defenses and has been implicated in autoimmunity. While IFN-I and select cytokines have been recognized as pDC secreted products, a comprehensive agnostic profiling of the pDC secretome in response to a physiologic stimulus has not been reported. We applied LC-MS/MS to catalogue the repertoire of proteins secreted by pDCs in the unperturbed condition and in response to challenge with influenza H1N1. We report the identification of a baseline pDC secretome, and the repertoire of virus-induced proteins including most type-I interferons, various cytokines, chemokines and granzyme B. Additionally, using single-cell RNA-seq [scRNA-seq], we perform multidimensional analyses of pDC transcriptional diversity immediately ex vivo and following stimulation. Our data evidence preexisting pDC heterogeneity, with subsequent highly specialized roles within the pDC population upon stimulation ranging from dedicated cytokine super-producers to cells with APC-like traits. Dynamic expression of transcription factors and surface markers characterize subclusters within activated pDCs. Integrating the proteomic and transcriptomic datasets confirms the pDC-subcluster origin of the proteins identified in the secretome. Our findings represent the most comprehensive molecular characterization of primary human pDCs at baseline, and in response to influenza virus, reported to date.

Solid organ transplant recipients require long-term immunosuppression for prevention of rejection. Calcineurin inhibitor (CNI)-based immunosuppressive regimens have remained the primary means for immunosuppression for four decades now, yet little is known about their effects on graft resident and infiltrating immune cell populations. Similarly, the understanding of rejection biology under specific types of immunosuppression remains to be defined. Furthermore, development of innovative, rationally designed targeted therapeutics for mitigating or preventing rejection requires a fundamental understanding of the immunobiology that underlies the rejection process. The established use of microarray technologies in transplantation has provided great insight into gene transcripts associated with allograft rejection but does not characterize rejection on a single cell level. Therefore, the development of novel genomics tools, such as single cell sequencing techniques, combined with powerful bioinformatics approaches, has enabled characterization of immune processes at the single cell level. This can provide profound insights into the rejection process, including identification of resident and infiltrating cell transcriptomes, cell-cell interactions, and T cell receptor α/β repertoires. In this review, we discuss genomic analysis techniques, including microarray, bulk RNAseq (bulkSeq), single-cell RNAseq (scRNAseq), and spatial transcriptomic (ST) techniques, including considerations of their benefits and limitations. Further, other techniques, such as chromatin analysis via assay for transposase-accessible chromatin sequencing (ATACseq), bioinformatic regulatory network analyses, and protein-based approaches are also examined. Application of these tools will play a crucial role in redefining transplant rejection with single cell resolution and likely aid in the development of future immunomodulatory therapies in solid organ transplantation.