Immune Cell Subpopulations Research Articles

Nuclear Methods for Immune Cell Imaging: Bridging Molecular Imaging and Individualized Medicine.

Inflammation is a key mechanistic contributor to the progression of cardiovascular disease, from atherosclerosis through ischemic injury and overt heart failure. Recent evidence has identified specific roles of immune cell subpopulations in cardiac pathogenesis that diverges between individual patients. Nuclear imaging approaches facilitate noninvasive and serial quantification of inflammation severity, offering the opportunity to predict eventual outcome, stratify patient risk, and guide novel targeted molecular therapies against specific leukocyte subpopulations. Here, we will discuss the established and emerging nuclear imaging methods to label and track exogenous and endogenous immune cells, with a particular focus on clinical situations in which targeted molecular inflammation imaging would be advantageous. The expanding options for imaging inflammation provide the foundation to bridge between molecular imaging and individual therapy.

It Runs in the Bromodomain Family: Speckled Proteins (SP) Play a Role in the Antitumor Immune Response in Solid Tumors

Cells and immune cells in the extracellular matrix: Depending on the tumor type and variety of TAAs (tumor-associated antigens), immune infiltrates are composed of many different subpopulations of immune cells. Epigenetic changes are also considered to be characteristic of cancer. Epigenetic factors taking part in the regulation of gene expression include the VII group of bromodomain proteins (BrD)-SP-family proteins. Here, we used transcriptomic data from the TCGA database, as well as immunological evidence from ESTIMATE, TIP, and TIMER2.0 databases for various solid tumor types and harnessed several publicly available bioinformatic tools (such as GSEA and GSCA) to demonstrate mechanisms and interactions between BrD proteins and immune infiltrates in cancer. We present a consistently positive correlation between the SP-family genes and immune score regardless of the tumor type. The SP-family proteins correlate positively with T cells' trafficking and infiltration into tumor. Our results also show an association between the high expression of SP family genes and enriched transcriptome profiles of inflammatory response and TNF-α signaling via NF-κβ. We also show that the SP-family proteins could be considered good predictors of high immune infiltration phenotypes.

Associations Of Fatty Acid Composition In Leukocyte Membranes With Systemic Inflammation In Chronic Obstructive Pulmonary Disease Progression

Background — The development of systemic inflammation is a key pathogenetic mechanism in progression of chronic obstructive pulmonary disease (COPD). Fatty acids (FAs) and their oxidized derivatives serve as essential regulators of inflammation. The relationship between systemic inflammation and FA metabolism in COPD is poorly understood. In our research, we focused on examining the FA composition of the leukocyte membrane in COPD and the FA metabolism in association with systemic inflammation. Objective — We examined 137 patients with mild, moderate, or severe COPD. The control group comprised 32 healthy non-smokers. Methods — Blood cytokines and immune cell subpopulations were evaluated by flow cytometry. The FA composition of the leukocyte membranes was analyzed by gas chromatography. The concentrations of eicosanoids (thromboxane B2 (TXB2), leukotriene B4 (LTB4)) in plasma were measured by ELISA. Results — Our results implied systemic inflammation in all patients with COPD. The analysis of the FA composition of leukocyte membrane demonstrated increased level of saturated FAs and n-6 polyunsaturated fatty acids (PUFAs), along with reduced levels of monounsaturated FAs and n-3 PUFAs, in patients with COPD. The TXB2 and LTB4 content was increasing in COPD patients. We established a significant correlation with n-6 PUFAs, immune cells, and cytokines, which was indicative of an important role of FAs in the progress of systemic inflammation in COPD. Conclusion — Thus, FA modification of immune cells in patients with chronic pathologies of the bronchopulmonary system leads not only to disruption of the cell membrane structure, but also to the pathology of immune response regulation, and contributes to the development of the inflammatory process. The latter is a decisive factor in the pathogenesis of COPD.

Integrative stemness characteristics associated with prognosis and the immune microenvironment in lung adenocarcinoma

BackgroundTo comprehensively analyze the stemness characteristics related to prognosis and the immune microenvironment in lung adenocarcinoma (LUAD).MethodsThe OCLR machine learning method was used to calculate the stemness index (mRNAsi) of the LUAD samples. DEGs common between the low mRNAsi, normal, and high mRNAsi groups were screened and the immune-stemness genes were obtained. Then the PPI network was created and enrichment analyses were performed. Moreover, different subtypes based on immune-stemness genes associated with prognosis were identified, and the relationships between LUAD stemness and TIME variables were systematically analyzed, followed by TMB analysis.ResultsPatients in the high mRNAsi groups with poor prognosis were screened along with 144 immune-stemness genes. IL-6, FPR2, and RLN3 showed a higher degree in the PPI network. A total of 26 immune-stemness genes associated with prognosis were screened. Two clusters were obtained (cluster 1 and cluster 2). Survival analysis revealed that patients in cluster 2 had a poor prognosis. A total of 12 immune cell subpopulations exhibited significant differences between cluster 1 and cluster 2 (P < 0.05). A total of 10 immune checkpoint genes exhibited significantly higher expression in cluster 1 (P < 0.05) than in cluster 2. Further, the TMB value in cluster 2 was higher than that in cluster 1 (P < 0.05).ConclusionImmune-stemness genes, including L-6, FPR2, and RLN3, might play significant roles in LUAD development via cytokine–cytokine receptor interaction, neuroactive ligand‒receptor interaction, and the JAK‒STAT pathway. Immune-stemness genes were related to tumor-infiltrating immune cells, TMB, and expression of immune checkpoint gene.

A shared B-cell clonotype in patients with Interstitial Cystitis/Bladder Pain Syndrome presenting with Hunner lesions

Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS) is characterized by bladder pain and lower urinary tract symptoms without obvious causes. A subset of patients with IC/BPS present with Hunner lesions (HL), which are focal regions of gross inflammation on the bladder wall. It has been previously proposed that cell populations within HL are enriched in B- and T-cells, suggesting that this form of IC/BPS may be caused by reaction to a specific antigen within the lesions. Alternatively, B-cell enrichment in the HL might be caused by generalized inflammatory processes. Here, we distinguished between these hypotheses by using single-cell sequencing to identify B-cell clonotypes in the HL and the bladder of IC/BPS patients. We identified a clonotype that is shared in two patients with IC/BPS and that represented a significant subpopulation of total immune cells within the lesions. This finding is strong evidence that B-cells in the patients’ bladders are reacting to a specific antigen. Further studies of this specific B-cell clonotype can identify the antigen, helping to define the pathophysiology for IC/BPS with HL.

MTOR inhibitor, gemcitabine and PD-L1 antibody blockade combination therapy suppresses pancreatic cancer progression via metabolic reprogramming and immune microenvironment remodeling in Trp53flox/+LSL-KrasG12D/+Pdx-1-Cre murine models

ObjectiveResistance to immunotherapy and chemotherapy hinders the prognosis of pancreatic cancer(PC). We hypothesized that the combination of mTOR inhibitor sirolimus and gemcitabine would change the metabolic landscape of PC and enhance the anti-PD-L1 therapy. MethodsIn KPC mice, the following regimens were administered and tumor growth inhibition rates(TGI%) were calculated: sirolimus(S), PD-L1 antibody(P), gemcitabine(G), sirolimus + PD-L1 antibody(SP), sirolimus + gemcitabine(SG), PD-L1 + gemcitabine(PG) and sirolimus + PD-L1 antibody + gemcitabine(SPG). The metabolic changes of tumors were identified by LC-MS and subpopulations of immune cells were measured by flow cytometry. Sirolimus treated macrophages were co-cultured with PC cells in vitro, and the metabolic changes of macrophages and tumor cells as well as tumor cells’ viability were detected. ResultsThe monotherapy of S, P and G didn't inhibit tumor growth significantly. The combination of SP, PG and SG didn't improve the TGI% significantly compared with monotherapy. However, the TGI% of SPG combination was higher than other groups. The proportion of CD68+ macrophages increased in the peripheral blood and CD8+ T cells decreased in the tumor tissues after SPG treatment. LC-MS identified 42 differential metabolites caused by sirolimus in SPG group, among which 10 metabolites had potential effects on macrophages. Sirolimus treated M1 and M2 macrophages inhibited the proliferation of tumor cells and decreased tumor cells' glycolysis. The glycolysis of M2 macrophages was increased by sirolimus. ConclusionsmTOR inhibitor can change the immune microenvironment of PC via metabolic reprogramming, thus promoting the efficacy of PD-L1 blockade when combined with gemcitabine.

Dickkopf-3: An Update on a Potential Regulator of the Tumor Microenvironment

Dickkopf-3 (Dkk-3) is a member of the Dickkopf family protein of secreted Wingless-related integration site (Wnt) antagonists that appears to modulate regulators of the host microenvironment. In contrast to the clear anti-tumorigenic effects of Dkk-3-based gene therapies, the role of endogenous Dkk-3 in cancer is context-dependent, with elevated expression associated with tumor promotion and suppression in different settings. The receptors and effectors that mediate the diverse effects of Dkk-3 have not been characterized in detail, contributing to an ongoing mystery of its mechanism of action. This review compares the various functions of Dkk-3 in the tumor microenvironment, where Dkk-3 has been found to be expressed by subpopulations of fibroblasts, endothelial, and immune cells, in addition to epithelial cells. We also discuss how the activation or inhibition of Dkk-3, depending on tumor type and context, might be used to treat different types of cancers.

The PMN-MDSC – A key player in glucocorticoid resistance following combined physical and psychosocial trauma

Stress-associated somatic and psychiatric disorders are often linked to non-resolving low-grade inflammation, which is promoted at least in part by glucocorticoid (GC) resistance of distinct immune cell subpopulations. While the monocyte/macrophage compartment was in the focus of many clinical and preclinical studies, the role of myeloid-derived suppressor cells (MDSCs) in stress-associated pathologies and GC resistance is less understood. As GC resistance is a clear risk factor for posttraumatic complications in patients on intensive care, the exact interplay of physical and psychosocial traumatization in the development of GC resistance needs to be further clarified.In the current study we employ the chronic subordinate colony housing (CSC) paradigm, a well-characterized mouse model of chronic psychosocial stress, to study the role of myeloid cells, in particular of MDSCs, in innate immune activation and GC resistance following combined psychosocial and physical (e.g., bite wounds) trauma. Our findings support the hypothesis that stress-induced neutrophils, polymorphonuclear (PMN)-MDSCs and monocytes/monocyte-like (MO)-MDSCs get primed and activated locally in the bone marrow as determined by toll-like receptor (TLR)2 upregulation and increased basal and lipopolysaccharide (LPS)-induced in vitro cell viability. These primed and activated myeloid cells emigrate into the peripheral circulation and subsequently, if CSC is accompanied by significant bite wounding, accumulate in the spleen. Here, PMN-MDSCs and monocytes/MO-MDSCs upregulate TLR4 expression, which exclusively in PMN-MDSCs promotes NF-κB hyperactivation upon LPS-stimulation, thereby exceeding the anti-inflammatory capacities of GCs and resulting in GC resistance.

Identification of fatty acid metabolism-related molecular subtype biomarkers and their correlation with immune checkpoints in cutaneous melanoma.

Fatty acid metabolism (FAM) affects the immune phenotype in a metabolically dynamic tumor microenvironment (TME), but the use of FAM-related genes (FAMGs) to predict the prognosis and immunotherapy response of cutaneous melanoma (CM) patients has not been investigated. In this study, we aimed to construct FAM molecular subtypes and identify key prognostic biomarkers in CM. We used a CM dataset in The Cancer Genome Atlas (TCGA) to construct FAM molecular subtypes. We performed Kaplan-Meier (K-M) analysis, gene set enrichment analysis (GSEA), and TME analysis to assess differences in the prognosis and immune phenotype between subtypes. We used weighted gene co-expression network analysis (WGCNA) to identify key biomarkers that regulate tumor metabolism and immunity between the subtypes. We compared overall survival (OS), progression-free survival (PFS), and disease-specific survival (DSS) between CM patients with high or low biomarker expression. We applied univariable and multivariable Cox analyses to verify the independent prognostic value of the FAM biomarkers. We used GSEA and TME analysis to investigate the immune-related regulation mechanism of the FAM subtype biomarker. We evaluated the immune checkpoint inhibition (ICI) response and chemotherapy sensitivity between CM patients with high or low biomarker expression. We performed real-time fluorescent quantitative PCR (qRT-PCR) and semi-quantitative analysis of the immunohistochemical (IHC) data from the Human Protein Atlas to evaluate the mRNA and protein expression levels of the FAM biomarkers in CM. We identified 2 FAM molecular subtypes (cluster 1 and cluster 2). K-M analysis showed that cluster 2 had better OS and PFS than cluster 1 did. GSEA showed that, compared with cluster 1, cluster 2 had significantly upregulated immune response pathways. The TME analysis indicated that immune cell subpopulations and immune functions were highly enriched in cluster 2 as compared with cluster 1. WGCNA identified 6 hub genes (ACSL5, ALOX5AP, CD1D, CD74, IL4I1, and TBXAS1) as FAM biomarkers. CM patients with high expression levels of the six biomarkers had better OS, PFS, and DSS than those with low expression levels of the biomarkers. The Cox regression analyses verified that the 6 FAM biomarkers can be independent prognostic factors for CM patients. The single-gene GSEA showed that the high expression levels of the 6 genes were mainly enriched in T-cell antigen presentation, the PD-1 signaling pathway, and tumor escape. The TME analysis confirmed that the FAM subtype biomarkers were not only related to immune infiltration but also highly correlated with immune checkpoints such as PD-1, PD-L1, and CTLA-4. TIDE scores confirmed that patients with high expression levels of the 6 biomarkers had worse immunotherapy responses. The 6 genes conveyed significant sensitivity to some chemotherapy drugs. qRT-PCR and IHC analyses verified the expression levels of the 6 biomarkers in CM cells. Our FAM subtypes verify that different FAM reprogramming affects the function and phenotype of infiltrating immune cells in the CM TME. The FAM molecular subtype biomarkers can be independent predictors of prognosis and immunotherapy response in CM patients.

Distinct Immune Homeostasis Remodeling Patterns after HLA-Matched and Haploidentical Transplantation

The homeostatic immune system in a healthy state represents a dynamic balance between immunogenicity and immune tolerance. Allogenic hematopoietic stem cell transplantation (allo-HSCT) is not only a curative therapy for hematological malignancies, but also has been used to remodel the immune system to a homeostatic state, such as inducing allograft tolerance in solid organ transplantation and treating autoimmune diseases. Therefore, the homeostatic immune system after allo-HSCT represents donor-derived hematopoietic cells that reconstitute the immune system in recipients and establish tolerance to recipients. However, little is known about the responsible genes or relevant cell types that remodel immune homeostasis after allo-HSCT. Moreover, whether haploidentical stem cell transplantation (haplo-SCT) shares a similar pattern of immune homeostasis remodeling with HLA-matched sibling donor transplantation (MSDT) is also unclear. In this study, we used a comprehensive single-cell RNA-seq (scRNA-seq) and single-cell TCR sequencing (scTCR-seq) approach coupled with bulk transcriptome (RNA-seq) and epigenome (ATAC-seq) analysis to dissect the landscape of the rebalanced homeostatic immune system in recipients after allo-HSCT (Fig. 1A). We identified 35 subpopulations in the 5 lineages based on the expression of cell type-associated genes, including the hematopoietic progenitor compartment with highly proliferative ability, T/NK lineage cells, B lineage cells, myeloid lineage cells and platelets (Fig. 1B). All cell subsets were present in both donors and recipients, which indicated that the immune system was successfully reconstituted in the recipients after allo-HSCT (Fig. 1B). Moreover, we found HSPC compartments adequately reconstitute in those recipients who have rebalanced to homeostasis after allo-HSCT. Although all immune cell subpopulations from donors were reconstituted in the paired recipients, there were different patterns of immune subpopulation distributions in recipients after allo-HSCT. In MSDT, donor-derived immune cell reconstitution in recipients as similar to that in their paired donors, while in haplo-SCT, there were low levels of CD4+ and CD8+ naïve T cells, Tcm cells, CD4+ Tem cells and CD4 Treg cells and high levels of CD4+ Teff cells, CD8+ Teff cells, CD8+ Tem cells and CD8+ Trp cells in recipients compared with their paired donors (Fig. 1C). We observed that memory-like CD8 T cells with immunoregulatory functions (CD8 regulatory precursors, CD8 Trp) were expanded in haplo-SCT recipients. CD8 Trp showed similar transcriptional profiling compared with CD4 Treg indicated that increased CD8 Trp subpopulations might act as regulatory cells and contribute to maintaining the tolerance of the donor-derived immune system in recipients of haplo-SCT. To investigate the dynamic changes in TCR clonotype after allo-HSCT, we integrated the analyzed single-cell transcriptomes and TCR sequences. We found that the dominant clonotype of CD8 Teff cells in MSDT recipients is the clonotype consistent with that in their paired donors, while the dominant clonotype of CD8 Teff cells in haplo-SCT recipients is the recipient's unique clonotype and educated by recipient thymic with high ZNF683 expression (Fig. 1D-E). Combined analysis of bulk CD8 T cell RNA-seq and ATAC-seq, we found that transcription factor ZNF683 was specifically activated in CD8+ T cells from haplo-SCT recipients compared with their paired donors (Fig. 1F). In vitro experiments indicated that a high expression level of ZNF683 in human primary T cells promoted inactivation of CD8+ T cells, maintained CD8+ T-cell replicative capacity and inhibited CD8+ T-cell apoptosis, which might suppress CD8 T cell senescence and corelated with CD8 T cell persistence. Moreover, ZNF683 could be downregulated when CD8+ T cells were activated, which suggests that ZNF683 might be a checkpoint of long-lived quiescent CD8+ T cells. In summary, our study provides a rich dataset of cellular states, gene programs, and T-cell repertoires in recipients whose donor-derived immune system rebalanced toward homeostasis after allo-HSCT. Understanding the molecular mechanisms underlying different patterns of immune homeostasis remodeling in different transplantation models will help us provide more precise medical care for patients who receive allo-HSCT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Profiling Immune Cells in DLBCL Patients with Mass Cytometry By Time-of-Flight (CyTOF)

Objectives Diffuse large B-cell lymphoma (DLBCL) is a disease with highly heterogeneous molecular characteristics and clinical course. Refractory/relapsed DLBCL shows poor prognosis, and new therapeutic approaches are urgently needed to improve its clinical outcomes. It is important to investigate the mechanism of development of DLBCL, in order to discover new molecular therapeutic targets. In this study, Cytometry by Time-Of-Flight mass spectrometry (CyTOF) was used to profile the immune cells in peripheral blood of DLBCL patients, to investigate the changes of immune cell subpopulation, and to find possible new targets for immune therapy of DLBCL. Methods Two CyTOF antibody panels targeting innate and adaptive immune cells, respectively, were designed to detect the characteristics of immune cell subpopulation. The antibodies include cell surface, intracellular and nuclear markers, targeting immune-related functions such as cell phenotyping, activation, proliferation, immune checkpoints, and chemokine receptors. Peripheral blood was collected from 50 DLBCL patients and 14 healthy controls. After staining and data collection, dimensional reduction such as viSNE and clustering such as SPADE and PARC were performed in Cytobank, OMIQ and R to comprehensively compare the differences in cell subset composition and function between patients and healthy controls. Results CD8+ T cells were more differentiated into effector phenotype with senescence and exhausted characteristics in DLBCL patients CD8+ T cells decreased significantly in DLBCL patients. Percentages of effector memory (EM) and CD45RA+ effector memory cells (EMRA) in CD8+ T cells were elevated in DLBCL patients. The higher expression intensities of CD45RO, KLRG1, PD-1, CTLA-4, and TIGIT in CD8+T cells imply the senescence and exhausted characteristics of CD8+ T cells in patients. After clustering analysis by SPADE, CD8+ T cells could be divided into 4 clusters, among which the proportion of CD45RO+KLRG1+ cluster was significantly higher in patients. This subset highly expressed PD-1, CTLA-4, TIGIT, GITR, CD25, and FoxP3, suggesting that they are exhausted effector CD8+ T cells with immunosuppressive phenotype. Proportions of the other 3 clusters, which were CD27+CCR7+ naïve CD8+ T cells, Granzyme B+HLA-DR+Ki-67+ central memory CD8+ T cells, and Granzyme B+HLA-DR+ central memory CD8+ T cells respectively, did not change significantly. CD56brightNK cell subset decreased significantly in DLBCL patients NK cells decreased significantly in DLBCL patients. By PARC clustering analysis, NK cells were divided into 3 clusters. The patient had a significantly reduced CD56bright subset. While, CD56brightKi-67+ cluster as well as CD56dimCD16+ cluster did not significantly change. Two subsets of monocytes with immunosuppressive phenotype emerged in DLBCL patients Compared with healthy controls, monocytes showed a significant elevation in DLBCL patients. However, the proportion of CD14+HLA-DR+ monocytes decreased in patients. 2 subsets of CD33+CD11b+ and BDCA3+CXCR3+ monocytes appeared, which showed higher expression of immunosuppression-related markers CD163, PD-1, and LAG3, and relatively lower expression of HLA-DR. (Figure 1) CD33+CD11b+ cells were consistent with the MDSC phenotype; whereas the proportion of BDCA3+CXCR3+ subset in total leukocytes correlated with patients' treatment response for chemotherapy. The level of BDCA3+CXCR3+ monocytes was significantly higher in patients who obtained CR after 6 cycles of chemotherapy than in patients who did not. (Figure 2) Conclusions By a multidimensional CyTOF staining approach, we depicted the immune cell profile of peripheral blood in DLBCL patients, analyzed the changes of the proportion of immune cell subset, and explored the changes in differentiation and function of different immune cell subsets in depth. For the first time, we found an increase in BDCA3+CXCR3+ monocyte subpopulation in DLBCL patients, and its proportion may be closely related to the prognosis of DLBCL patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Parallel Detection of Single-Cells and Extracellular Vesicles from Bone Marrow and Peripheral Blood Reveals Asymmetric Immunometabolic Profile in Acute Myeloid Leukemia

Introduction. Acute myeloid leukemia (AML) is an aggressive disease with a high relapse rate. Alteration in cell metabolism and metabolic adaptation are crucial hallmarks in AML. Although several studies have addressed the metabolic profile of leukemic cells, the metabolic cross-modulation of immune cells according to their resident (bone marrow, BM) and circulating (peripheral blood, PB) status has been poorly investigated. Growing evidence has highlighted a key role for extracellular vesicles (EVs), shed from a broad variety of cells and cargo of crucial factors, in modulating several functions of leukemic cells, including bioenergetic metabolism. Again, few studies have addressed the impact of EVs in regulating the metabolism of immune cells from leukemic microenvironment. Therefore, in our work, we dissected the immunometabolic profile of immune cells and EV signature profile in parallel from BM versus PB from AML patients. Methods. BM/PB samples and plasma were collected from AML patients at diagnosis (n=40). Leukemic EVs (herein referred to as AML EVs) were purified from platelet-poor plasma BM/PB by size exclusion chromatography and ultrafiltration. MACSPlex Exosome Kit was used for screening surface EV markers. Flow cytometry-based Single Cell ENergetic metabolism by profilIng Translation inhibition (SCENITH) method was applied in fresh whole blood. In addition, BM/PB mononuclear cells from AML patients were characterized by SCENITH after co-culture with circulating AML EVs. Results. To explore the metabolic reprogramming within the leukemic immune microenvironment, we firstly applied the SCENITH method in multiple immune cell subpopulations from AML patients. In comparison with the paired PB, we observed a significant increase in glucose dependence of AML BM CD8+ T cells. By contrast, the capacity to use fatty acids and amino acids as sources for ATP production when glucose oxidation is inhibited was prominently reduced in AML BM CD8+ T cells and increased in AML PB CD8+ T cells. Also, mitochondrial dependence of CD8+ T cells was higher in the BM compartment, suggesting a switch from oxidative phosphorylation to glycolysis in circulation. Interestingly, in comparison to paired PB, only BM CD3+ and CD4+ T cells from female AML patients revealed a marked increase in their glucose dependence reporting gender-dependent metabolic differences. Towards clinically relevant implication, according to European LeukemiaNet (ELN) 2017 risk classification, SCENITH analysis showed a critical reduction in mitochondrial dependence at the expense of glycolytic capacity in circulating CD4+ T cells from favorable-risk AML compared to intermediate/adverse-risk patients. Given the immunometabolic profile between BM and PB cell compartments, we hypothesized that EVs may differentially contribute to immunometabolism according to their BM versus PB localization. Preliminarily, we explored the immune profiling of plasma-derived EVs from AML BM versus PB. We found that BM EVs exhibited higher expressions of specific markers including CD4, CD40, ROR-1 and HLA-II/-ABC compared to PB equivalents. Of note, we observed a novel link between metabolic regulation and EV profiling. Indeed, the glucose dependence of PB CD8+ T cells was negatively correlated to the expression of CD133-1 (stem cell marker) and CD209 (dendritic marker) depicted on circulating (PB) EVs. Finally, we co-cultured AML circulating EVs with BM/PB mononuclear cells from AML patients in pairs to investigate the EV's role in metabolic regulation. Notably, BM CD8+ T cells in monocultures showed a reduction in glucose dependence compared to their PB equivalents. In parallel, mitochondrial dependence was more pronounced in PB CD4+ T cells compared to other circulating immune subsets. However, PB EVs caused a metabolic shift in the PB compartment towards high glycolytic capacity in CD8+ T cells and high mitochondrial dependency in CD34+ cells suggesting an EV-driven metabolic rewiring. Apparently, the co-cultures with PB EVs did not influence the energy profile of resident (BM) immune subsets. Conclusions. Overall, this study shows the immunometabolic asymmetry orchestrated in AML at diagnosis, driven by distinct immune cell subsets according to their location (BM versus PB). We also describe the putative role of AML EVs in subverting the immune cell metabolism revealing critical implications for immunotherapy in AML.

TMIC-58. PATTERNS OF CELLULAR SUBPOPULATION COHABITATION DEFINE GLIOBLASTOMA STATES

Abstract Characterizing intra- and inter-tumoral heterogeneity of glioblastoma has historically relied on discrete classifications of malignant cell populations leaving immune and other cell populations, known to exist admixed with the malignant tumor cells, relatively neglected. Manifold learning algorithms can manage deconvolving multiple cell populations and are often used to track cell state transitions in single cell transcriptomics. We applied a manifold learning approach to TCGA microarray data (Nf525) and bulk transcriptomics of 134 image localized biopsies across 30 patients with primary and 9 with recurrent glioblastoma to further elucidate how to organize biopsies across a spectrum of possible tissue states. The algorithm revealed a low-dimensional manifold graph for which each biopsy lives across 3 polarizing tissue states - one that is associated with diffusely invaded brain, one that is enriched in mesenchymal genes, and one that is enriched in classical proliferative tumor signatures. We deconvolved the bulk transcriptomics of the image-localized biopsies to reveal the relative abundance of 18 malignant, immune, and other cell subpopulations in each biopsy. Overlaying the cellular decomposition onto the manifold graph visualizing the tissue state distributions revealed that transitions between states correlate with changes in cellular cohabitation composition. The tumor cellular cohabitation ecologies have the lowest diversity, as inferred by ecological measures such as Shannon entropy and evenness, at the distal poles of the graph when compared to the transitional arms. Further, we found that the relationship between imaging appearance of contrast enhancement on T1-weighted MRI and the biopsy cellular composition varies with sex and primary vs recurrent biopsy status. The limited spectrum of possible tissue states revealed by the manifold learning is suggestive of a limited continuum along which tumor and non-tumoral cell populations can cohabitate. Such a limited low-dimensional biological space may constrain the dynamics of tumor biology in a predictable manner.

High-dimensional single-cell cartography tracking of immune cells subpopulation of mice peripheral blood treated with gold nanorods and black phosphorus nanosheets

The gold nanorods (Au NRs) and black phosphorus nanosheets (BP NSs) are one of the commonly investigated nanomaterials (NMs) that substantially promoted biomedical nanotechnology. Recently, NMs-based immunotherapies have been a hot research focus in various fields, and some studies have revealed their immunotoxicity. However, their effects on the immune system are still overlooked, making it necessary to assess their impact on immune cells entirely. A challenge in evaluating the immunotoxicity of NMs is tracking the subpopulation of immune cells at single-cell level. Therefore, we applied the cytometry by time-of-flight (CyTOF) that exhibied an unparalleled ability to phenotypically and functionally profile complex cellular subpopulations using multiple panels (42 immune markers) to analyze the change of immune cells subpopulation in peripheral blood after treated with Au NRs and BP NSs. As a proof of concept, the analysis showed that both NMs induced significant changes of almost immune cell subpopulation in peripheral blood, which perturbates the innate and adaptive immune responses significantly. The work revealed not only the immunotoxicity of NMs but also guided the application of NM-based strategies and might facilitate further understanding of their toxicology.

Cuproptosis-related LncRNA signatures as a prognostic model for head and neck squamous cell carcinoma.

Cuproptosis is a novel, distinct form of regulated cell death. However, little is known about the role of cuproptosis-related lncRNAs (CRlncRNAs) in head and neck squamous cell carcinoma (HNSCC). This study aimed to identify a CRlncRNAs signature, explore its prognostic value in HNSCC. RNA-seq data and relevant clinical data were downloaded from The Cancer Genome Atlas (TCGA) database, and cuproptosis-related genes were identified from a search of the relevant candidate-gene literature. Analysis of differentially expressed lncRNAs (DElncRNAs) was performed using the R package "edgeR". The intersection of the lncRNAs between DElncRNAs and CRlncRNAs was obtained using the R package "Venn Diagram". Univariate Cox regression was used to identify cuproptosis-related prognostic lncRNAs. LASSO-Cox method was used to narrow these cuproptosis-related prognostic lncRNAs and construct a prognostic model. Multiple statistical methods were used to evaluate the predictive ability of the model. Moreover, the relationships between the model and immune cell subpopulations, related functions and pathways and drug sensitivity were explored. Then, two risk groups were established according to the risk score calculated by the CRlncRNAs signature included three lncRNAs. In HNSCC patients, the risk score was a better predictor of survival than traditional clinicopathological features. In addition, significant differences in immune cells such as B cells, T cells and macrophages were observed between the two groups. Finally, the high-risk group had a lower IC50 for certain chemotherapeutic agents, such as cisplatin and cetuximab. This 3 CRlncRNAs signature is a powerful prognostic biomarker for predicting clinical outcomes and therapeutic responses in HNSCC patients.

Immunohistochemical Characterization of Immune System Cells in Lymphoid Organs from Roe and Fallow Deer.

Roe and Fallow deer are common wild ruminants widely distributed in Italy. Infectious diseases of these species can potentially pose health risks to domestic animals and humans. However, few studies have been conducted in which immune system cells in these species were phenotyped. The aims of this study were to determine the cross-reactivity of a wide anti-human panel of commercial antibodies on formalin-fixed and paraffin-embedded (FFPE) samples and to describe the distribution of roe and fallow deer main immune cell subsets in the lymph nodes and spleen. Twenty retromandibular lymph nodes (RLNs) and spleen samples were collected from 10 roe deer and 10 fallow deer and were tested by a panel of 12 commercial anti-human antibodies. The CD79a, CD20, CD3, Iba-1, MAC387, and AM-3K antibodies were successfully labeled cells in cervine tissue, while the Foxp3 and the CD68 did not show suitable immunostaining. This study supplies the first immunohistochemical description of immune cell subpopulations in non-pathological spleen and RLNs from roe and fallow deer and provides an easily repeatable manual IHC protocol to immunolocalize cervine B-, T-cells, and macrophages subsets in FFPE tissue samples.

Single-cell and spatial transcriptomics reveal the fibrosis-related immune landscape of biliary atresia.

Biliary atresia (BA) is a devastating inflammatory and fibrosing cholangiopathy of neonates with unknown aetiology. We aim to investigate the relationship between these two main characteristics. Single-cell RNA sequencing and spatial transcriptomics were performed on liver samples from a cohort of 14 objects (BA: n = 6; control: n = 8). We conducted data integration and cell-type annotation based on gene expression profiling. Furthermore, we identified fibrosis-related immune cells according to their spatial locations, GO and KEGG analysis. Finally, SPOTlight and CIBERSORTx were used to deconvolute ST data and microarray data of the GSE46960 cohorts, respectively. Immune subpopulations inhabiting the 'fibrotic niche' (areas of scarring), comprising 'intermediate' CD14++ CD16+ monocytes, scar-associated macrophages, natural killer T cells, transitional B cells and FCN3+ neutrophils were identified. GO and KEGG analyses showed that pathways including 'positive regulation of smooth muscle cell/fibroblast proliferation' and 'positive regulation of/response to VEGFR/VEGF/EGFR/FGF' were enriched in these cell types. Interactions analysis showed that communication among 'FGF_FGFR', 'RPS19-C5AR1', 'CD74_COPA/MIF/APP' and 'TNFRSF1A/B_GRN' was extensive. Finally, the results of deconvolution for ST data and microarray data validated that the proportions of certain identified fibrosis-related cell types we identified were increased in BA. Fibrosis is an important feature of BA, in which the immune system plays an important role. Our work reveals the subpopulations of immune cells enriched in the fibrotic niche of BA liver, as well as key related pathways and molecules; some are highlighted for the first time in liver fibrosis. These newly identified interactions might partly explain why the rate of liver fibrosis occurs much faster in BA than in other liver diseases. Our study revealed the molecular, cellular and spatial immune microenvironment of the fibrotic niche of BA.

Single-cell mechanistic studies of radiation-mediated bystander effects.

Ionizing radiation (IR) has been widely used in the diagnosis and treatment of clinical diseases, with radiation therapy (RT) being particularly rapid, but it can induce "bystander effects" that lead to biological responses in non-target cells after their neighboring cells have been irradiated. To help clarify how radiotherapy induces these effects, To help clarify how radiotherapy induces these effects, we analyzed single-cell RNA sequencing data from irradiated intestinal tissues on day 1 (T1 state), day 3 (T3 state), day 7 (T7 state), and day 14 (T14 state) after irradiation, as well as from healthy intestinal tissues (T0 state), to reveal the cellular level, molecular level, and involvement of different time irradiated mouse intestinal tissues in biological signaling pathways. In addition, changes in immune cell subpopulations and myeloid cell subpopulations after different radiation times were further explored, and gene regulatory networks (GRNs) of these cell subpopulations were constructed. Cellular communication between radiation-specific immune cells was explored by cell-to-cell communication events. The results suggest that radiotherapy trigger changes in immune cell subsets, which then reprogram the immune ecosystem and mediate systemic bystander effects. These radiation-specific immune cells participate in a wide range of cell-to-cell communication events. In particular, radiation-specific CD8+T cells appear to be at the core of communication and appear to persist in the body after recovery from radiotherapy, with enrichment analysis showing that radiation-specific CD8+ T cells are associated with ferroptosis. Thus, radiation-specific CD8+ T cells may be involved in cellular ferroptosis-mediated adverse effects caused by RT.

ImmCluster: an ensemble resource for immunology cell type clustering and annotations in normal and cancerous tissues.

Single-cell transcriptome has enabled the transcriptional profiling of thousands of immune cells in complex tissues and cancers. However, subtle transcriptomic differences in immune cell subpopulations and the high dimensionality of transcriptomic data make the clustering and annotation of immune cells challenging. Herein, we introduce ImmCluster (http://bio-bigdata.hrbmu.edu.cn/ImmCluster) for immunology cell type clustering and annotation. We manually curated 346 well-known marker genes from 1163 studies. ImmCluster integrates over 420 000 immune cells from nine healthy tissues and over 648 000 cells from different tumour samples of 17 cancer types to generate stable marker-gene sets and develop context-specific immunology references. In addition, ImmCluster provides cell clustering using seven reference-based and four marker gene-based computational methods, and the ensemble method was developed to provide consistent cell clustering than individual methods. Five major analytic modules were provided for interactively exploring the annotations of immune cells, including clustering and annotating immune cell clusters, gene expression of markers, functional assignment in cancer hallmarks, cell states and immune pathways, cell-cell communications and the corresponding ligand-receptor interactions, as well as online tools. ImmCluster generates diverse plots and tables, enabling users to identify significant associations in immune cell clusters simultaneously. ImmCluster is a valuable resource for analysing cellular heterogeneity in cancer microenvironments.

A Systematic Review on the Impact of Hypofractionated and Stereotactic Radiotherapy on Immune Cell Subpopulations in Cancer Patients.

We investigated how hypofractionated radiotherapy (HFRT) and stereotactic body radiotherapy (SBRT) may impact immune cells in different type of tumors. A systematic review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The PubMed, Embase and Cochrane databases were searched. Overall, 11 studies met the inclusion criteria and were eligible for the present analysis. Both HFRT and SBRT have different impact on lymphocyte subpopulations, confirming their immunomodulatory effect which may have a crucial role in future combined treatment with new emergent therapies such as immunotherapy. Further studies are needed to shed more light on this emerging topic to ultimately improve patient care, treatment and clinical benefits for cancer patients.