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Background: Glioma is the predominant malignant brain tumor that lacks effective treatment options due to its shielding by the blood-brain barrier (BBB). Astrocytes play a role in the development of glioma, yet the diverse cellular composition of astrocytoma has not been thoroughly researched. Methods: We examined the internal diversity of seven distinct astrocytoma subgroups through single-cell RNA sequencing (scRNA-seq), pinpointed crucial subgroups using CytoTRACE, monocle2 pseudotime analysis, and slingshot pseudotime analysis, employed various techniques to identify critical subgroups, and delved into cellular communication analysis. Then, we combined the clinical information of GBM patients and used bulk RNA sequencing (bulk RNA-seq) to analyze the prognostic impact of the relevant molecules on GBM patients, and we performed in vitro experiments for validation. Results: The analysis of the current study revealed that C0 IGFBP7+ Glioma cells were a noteworthy subpopulation of astrocytoma, influencing the differentiation and progression of astrocytoma. A predictive model was developed to categorize patients into high- and low-scoring groups based on the IGFBP7 Risk Score (IGRS), with survival analysis revealing a poorer prognosis for the high-IGRS group. Analysis of immune cell infiltration, identification of genes with differential expression, various enrichment analyses, assessment of copy number variations, and evaluation of drug susceptibility were conducted, all of which highlighted their significant influence on the prognosis of astrocytoma. Conclusion: This research enhances comprehension of the diverse cell composition of astrocytoma, delves into the various factors impacting the prognosis of astrocytoma, and offers fresh perspectives on treating glioma. [ABSTRACT FROM AUTHOR]

Background: Severe acute pancreatitis (SAP) is characterized by inflammation, with inflammatory immune cells playing a pivotal role in disease progression. This study aims to understand variations in specific immune cell subtypes in SAP, uncover their mechanisms of action, and identify potential biological markers for predicting Acute Pancreatitis (AP) severity. Methods: We collected peripheral blood from 7 untreated SAP patients and employed single-cell RNA sequencing for the first time to construct a transcriptome atlas of peripheral blood mononuclear cells (PBMCs) in SAP. Integrating SAP transcriptomic data with 6 healthy controls from the GEO database facilitated the analysis of immune cell roles in SAP. We obtained comprehensive transcriptomic datasets from AP samples in the GEO database and identified potential biomarkers associated with AP severity using the "Scissor" tool in single-cell transcriptomic data. Results: This study presents the inaugural construction of a peripheral blood single-cell atlas for SAP patients, identifying 20 cell subtypes. Notably, there was a significant decrease in effector T cell subsets and a noteworthy increase in monocytes compared to healthy controls. Moreover, we identified a novel monocyte subpopulation expressing high levels of PPBP and PF4 which was significantly elevated in SAP. The proportion of monocyte subpopulations with high CCL3 expression was also markedly increased compared to healthy controls, as verified by flow cytometry. Additionally, cell communication analysis revealed insights into immune and inflammation-related signaling pathways in SAP patient monocytes. Finally, our findings suggest that the subpopulation with high CCL3 expression, along with upregulated proinflammatory genes such as S100A12, IL1B, and CCL3, holds promise as biomarkers for predicting AP severity. Conclusion: This study reveals monocytes' crucial role in SAP initiation and progression, characterized by distinct pro-inflammatory features intricately linked to AP severity. A monocyte subpopulation with elevated PPBP and CCL3 levels emerges as a potential biomarker and therapeutic target. [ABSTRACT FROM AUTHOR]

In a fuel cell, flow channels are crucial components responsible for various essential functions that enable the system to operate effectively. The design of a directly coupled flow channel in a Proton Exchange Membrane Fuel Cell (PEMFC) system, assuming deterministic parameters, has been extensively studied. However, this deterministic approach neglects the inherent uncertainties in system performance during real-life operation, resulting in potentially unreliable and suboptimal performance. To address this issue, we propose a reliability-based design optimization (RBDO) of the PEMFC's channel structure, considering uncertainties in operating parameters. This paper presents a numerical model of the PEMFC in COMSOL, deterministic designs, reliability-based designs and a global sensitivity analysis on the PEMFC cell's potential output and average water activity on the membrane. Although the RBDO approach shows a reduction in cell efficiency compared to the deterministic design, it significantly improves reliability, with increases from 60.92% to 95.10% for cell potential and from 79.31% to 96.85% for water activity. [ABSTRACT FROM AUTHOR]

Food components suppressing small intestinal tumorigenesis are not well-defined partly because of the rarity of this tumor type compared to colorectal tumors. Using Apcmin/+ mice, a mouse model for intestinal tumorigenesis, and antigen-free diet, we report here that food antigens serve this function in the small intestine. By depleting Peyer's patches (PPs), immune inductive sites in the small intestine, we found that PPs have a role in the suppression of small intestinal tumors and are important for the induction of small intestinal T cells by food antigens. On the follicle-associated epithelium (FAE) of PPs, microfold (M) cells pass food antigens from lumen to the dendritic cells to induce T cells. Single-cell RNA-seq (scRNA-seq) analysis of immune cells in PPs revealed a significant impact of food antigens on the induction of the PP T cells and the antigen presentation capacity of dendritic cells. These data demonstrate the role of food antigens in the suppression of small intestinal tumorigenesis by PP-mediated immune cell induction. [ABSTRACT FROM AUTHOR]

Objective: The involvement of immune cells in colorectal cancer (CRC) and their interplay with metabolic disorders are yet to be fully elucidated. This study examines how peripheral immune cells, inferred genetically, affect CRC and investigates the intermediary roles of metabolites. Methods: We employed a two-sample bidirectional Mendelian randomization (MR) approach to assess the causal influence of immune cells on CRC. Additionally, a two-step MR strategy was utilized to pinpoint potential metabolites that mediate this effect. Our analysis incorporated data from genome-wide association studies (GWAS), involving 731 immune cell types, 1,400 metabolites, and CRC outcomes. The primary method of analysis was randomized inverse variance weighting (IVW), supported by MR-Egger, weighted median, simple mode, and weighted mode analyses. Sensitivity checks were conducted using Cochran's Q test, MR-PRESSO test, MR-Egger regression intercept, and leave-one-out analysis. Results: The study identified 23 immune cell types and 17 metabolites that are causally linked to CRC. Our mediation analysis highlighted that nine metabolites act as intermediaries in the relationship between nine specific immune cells and CRC risk. Notably, The ratios of Adenosine 5'-monophosphate (AMP) to aspartate and Retinol (Vitamin A) to linoleoyl-arachidonoyl-glycerol (18:2 to 20:4) were found to concurrently mediate the promoting effects of Myeloid DC %DC and BAFF-R on B cells in colorectal cancer (CRC). Moreover, iminodiacetate (IDA) was found to mediate the protective effect of CD14+ CD16- monocytes on CRC, contributing 11.8% to this mediation. In contrast, IDA was also seen to decrease the protective effect of IgD+ CD38br %B cells on CRC risk, with a mediation effect proportion of -10.4%. Conclusion: This study delineates a complex network involving immune cells, metabolites, and CRC, suggesting a multifaceted pathophysiological interaction. The identified causal links and mediation pathways underscore potential therapeutic targets, providing a foundation for interventions aimed at modulating immune responses to manage CRC. [ABSTRACT FROM AUTHOR]

Background: Increasing evidence indicates a close relationship between alterations in human immune cells and plasma metabolites with Rheumatoid Arthritis (RA). However, limited studies have left the causal relationships behind these links unclear. Methods: A bidirectional Mendelian Randomization (MR) study was conducted, combined with mediation analysis, using data from genome-wide association study database covering 731 immune cell phenotypes and 1,400 plasma metabolite traits to explore their causal relationships with RA and potential mediating effects. The primary method used for MR analysis was inverse-variance weighted and False Discovery Rate (FDR) correction was applied to verify the robustness of our results. Results: HLA DR on CD33- HLA DR+ (myeloid cell group) (OR, 1.422; 95% CI, 1.194-1.694; P < 0.001; PFDR = 0.012) increased the risk of developing RA. CD19 on IgD+ CD38- naive (B cell group) (OR, 0.969; 95% CI, 0.954-0.985; P < 0.001; PFDR = 0.021) reduced the risk of developing RA. RA was a risk factor for HLA DR on CD14- CD16+ monocytes (monocyte group) (OR, 1.242; 95% CI, 1.102-1.401; P < 0.001; PFDR = 0.047). RA was a protective factor for memory B cell % lymphocyte (B cell group) (OR, 0.861; 95% CI, 0.795-0.933; P < 0.001; PFDR = 0.050), CD4+ CD8dim T cell %lymphocyte (TBNK group) (OR, 0.802; 95% CI, 0.711-0.904; P < 0.001; PFDR = 0.043), CD4+ CD8dim T cell %leukocyte (TBNK group) (OR, 0.814; 95% CI, 0.726-0.913; P < 0.001; PFDR = 0.046), CD24 on IgD+ CD24+ B cells (B cell group) (OR, 0.857; 95% CI, 0.793-0.927; P < 0.001; PFDR = 0.038), and CD24 on unswitched memory B cells (B cell group) (OR, 0.867; 95% CI, 0.797-0.942; P < 0.001; PFDR = 0.050). Increasing levels of docosatrienoate (22:3n3) (OR, 0.886; 95% CI, 0.838-0.936; P < 0.001; PFDR = 0.023) significantly reduced the risk of developing RA. The mediating effect of plasma metabolites in this context was not established. Conclusion: This study provides genetic evidence for the intricate relationships between immune cells, plasma metabolites, and RA, highlighting the potential mechanisms involved. This will contribute to future directions in precision medicine and research. [ABSTRACT FROM AUTHOR]

Background: Natural killer (NK) cells are proposed to participate in coronary artery disease (CAD) development. However, little is known about how CAD patients' NK cells respond to different stimulatory factors in terms of proliferation capability. Methods and results: Twenty-nine CAD patients' peripheral blood NK cells were isolated and individually treated with IL-2, IL-12, IL-15, IL-18, IL-21, cortisone acetate, hydrocortisone, or ascorbic acid for 36 hours, followed by cell cycle analysis using flow cytometry. The ratio of S and G2/M phase cell number to total cell number was defined as a proliferation index (PrI) and used for proliferative capability indication. The results showed that these eight factors resulted in different life cycle changes in the 29 NK cell samples. Remarkably, 28 out of 29 NK cell samples showed an obvious increase in PrI upon ascorbic acid treatment. The serum lactate dehydrogenase (LDH) level of the 29 CAD patients was measured. The results showed a negative correlation between serum LDH level and the CAD patients' NK cell PrI upon stimulation of interleukins, but not the non-interleukin stimulators. Consistently, a retrospective analysis of 46 CAD patients and 32 healthy donors showed that the circulating NK cell number negatively correlated with the serum LDH level in CAD patients. Unexpectedly, addition of LDH to NK cells significantly enhanced the production of IFN-γ, IL-10 and TNF-α, suggesting a strong regulatory role on NK cell's function. Conclusion: Ascorbic acid could promote the proliferation of the CAD patients' NK cells; LDH serum level may function as an indicator for NK cell proliferation capability and an immune-regulatory factor. [ABSTRACT FROM AUTHOR]

Cats have the highest incidence of lymphoma among all animal species. Lymphoma accounts for 41% of all malignant tumors in cats and is responsible for 90% of hematopoietic tumors in felines. Biopsies are considered the gold standard for diagnosis. Polymerase chain reaction (PCR)-based clonality assessment of antigen receptor gene rearrangements can be a valuable complementary tool for identifying infiltrating B-and T-lymphocyte clones. Many studies have focused on intestinal cases but few have addressed mediastinal lymphoma. This study aims to: (1) investigate the clonality patterns of lymphoma samples from various anatomical sites, with a particular focus on mediastinal lymphoma, and (2) evaluate the sensitivity and specificity of the clonality analysis of pleural effusion samples in comparison with cytology, histology, immunohistochemistry, and immunocytochemistry for diagnosing mediastinal lymphoma. There were 82 cases, divided into 49 formalin-fixed and paraffin-embedded biopsy specimens (FFPE), 22 cell pellets, and 11 fresh tissue. This study examined the sensitivity and specificity of PCR for antigen receptor rearrangement (PARR) compared to immunohistochemistry (IHC) and immunocytochemistry. For T-cell receptor gamma chain genes, PARR demonstrated a sensitivity of 58.33% for both fresh tissue and FFPE samples, with a specificity of 100%. Cell pellet analysis exhibited a sensitivity of 64.71% and maintained 100% specificity. A combined analysis of fresh tissue and FFPE with cell pellets showed a sensitivity of 62.07%. For IGH, the sensitivity for fresh tissue and FFPE samples was 56.25%, while cell pellet analysis showed a sensitivity of 62.50%. When considering fresh tissue and FFPE samples, the sensitivity was 57.14%. In conclusion, molecular techniques have emerged as valuable tools for detecting lymphoma, especially in cases where traditional diagnostic methods yield inconclusive results, such as mediastinal lymphoma. While biopsy may not always be feasible, cytology and cell pellets obtained from pleural effusion offer alternative immunocytochemistry and molecular analysis samples, provided they are of sufficient quality and quantity. All sample types considered in this study were suitable for PARR to aid in cases with inconclusive results. Therefore, the sample selection should be tailored to the clinical situation. [ABSTRACT FROM AUTHOR]

Toxoplasmosis is a worldwide parasitosis that is usually asymptomatic; cell-mediated immunity, particularly T cells, is a crucial mediator of the immune response against this parasite. Membrane protein expression has been studied for a long time in T lymphocytes, providing vital information to determine functional checkpoints. However, less is known about the role of post-translational modifications in T cell function. Glycosylation plays essential roles during maturation and function; particularly, sialic acid modulation is determinant for accurate T cell regulation of processes like adhesion, cell-cell communication, and apoptosis induction. Despite its importance, the role of T cell sialylation during infection remains unclear. Herein, we aimed to evaluate whether different membrane sialylation motifs are modified in T cells during acute Toxoplasma gondii infection using different lectins. To this end, BALB/c Foxp3EGFP mice were infected with T. gondii, and on days 3, 7, and 10 post-infection, splenocytes were obtained to analyze conventional (Foxp3-) CD4+ and CD8+ populations by flow cytometry. Among the different lectins used for analysis, only Sambucus nigra lectin, which detects sialic acid a2,6 linkages, revealed two distinctive populations (SNBright and SN-/Dim) after infection. Further characterization of CD4+ and CD8+ SN-/Dim lymphocytes showed that these are highly activated cells, with a TEf/EM or TCM phenotype that produce high IFN-γ levels, a previously undescribed cell state. This work demonstrates that glycan membrane analysis in T cells reveals previously overlooked functional states by evaluating only protein expression. [ABSTRACT FROM AUTHOR]

Background: Major depression disorder (MDD) and anxiety are common mental disorders that significantly affect the quality of life of those who suffer from them, altering the person's normal functioning. From the biological perspective, the most classical hypothesis explaining their occurrence relies on neurotransmission and hippocampal excitability alterations. However, around 30% of MDD patients do not respond to medication targeting these processes. Over the last decade, the involvement of inflammatory responses in depression and anxiety pathogenesis has been strongly acknowledged, opening the possibility of tackling these disorders from an immunological point of view. In this context, regulatory T cells (Treg cells), which naturally maintain immune homeostasis by suppressing inflammation could be promising candidates for their therapeutic use in mental disorders. Methods: To test this hypothesis, C57BL/6 adult male mice were submitted to classical stress protocols to induce depressive and anxiety-like behavior; chronic restriction stress (CRS), and chronic unpredictable stress (CUS). Some of the stressed mice received a single adoptive transfer of Treg cells during stress protocols. Mouse behavior was analyzed through the open field (OFT) and forced swim test (FST). Blood and spleen samples were collected for T cell analysis using cell cytometry, while brains were collected to study changes in microglia by immunohistochemistry. Results: Mice submitted to CRS and CUS develop anxiety and depressivelike behavior, and only CRS mice exhibit lower frequencies of circulating Treg cells. Adoptive transfer of Treg cells decreased anxiety-like behavior in the OFT only in CRS model, but not depressive behavior in FST in neither of the two models. In CRS mice, Treg cells administration lowered the number of microglia in the hippocampus, which increased due this stress paradigm, and restored its arborization. However, in CUS mice, Treg cells administration increased microglia number with no significant effect on their arborization. Conclusion: Our results for effector CD4+ T cells in the spleen and microglia number and morphology in the hippocampus add new evidence in favor of the participation of inflammatory responses in the development of depressive and anxiety-like behavior and suggest that the modulation of key immune cells such as Treg cells, could have beneficial effects on these disorders. [ABSTRACT FROM AUTHOR]

Introduction: Gliomas are the most common primary intracranial tumors, known for their high invasiveness and destructiveness. Sialic acid-binding immunoglobulin-like lectin 7 (SIGLEC7) is present in various immune cells, especially macrophages, and significantly affects immune homeostasis and cancer cell response. However, research on the role and prognostic impact of SIGLEC7 in glioma patients is currently limited. Methods: We utilized transcriptomic data from 702 glioma patients in The Cancer Genome Atlas (TCGA) and 693 glioma patients in the Chinese Glioma Genome Atlas (CGGA), along with clinical samples we collected, to comprehensively investigate the impact of SIGLEC7 on glioma expression patterns, biological functions, and prognostic value. We focused on its role in glioma-related immune responses and immune cell infiltration and analyzed its expression at the single-cell level. Finally, we validated the role of SIGLEC7 in gliomas through tissue and cell experiments. Results: SIGLEC7 expression was significantly increased in glioma patients with malignant characteristics. Survival analysis indicated that glioma patients with high SIGLEC7 expression had significantly lower survival rates. Gene function analysis revealed that SIGLEC7 is primarily involved in immune and inflammatory responses and is strongly negatively correlated with tumor-associated immune regulation. Additionally, the expression of most immune checkpoints was positively correlated with SIGLEC7, and immune cell infiltration analysis clearly demonstrated a significant positive correlation between SIGLEC7 expression and M2 macrophage infiltration levels. Single-cell analysis, along with tissue and cell experiments, confirmed that SIGLEC7 enhances macrophage polarization towards the M2 phenotype, thereby promoting glioma invasiveness through the immunosuppressive effects of M2 macrophages. Cox regression analysis and the establishment of survival prediction models indicated that high SIGLEC7 expression is an unfavorable prognostic factor for glioma patients. Discussion: High SIGLEC7 expression predicts poor prognosis in glioma patients and is closely associated with M2 macrophages in the tumor environment. In the future, SIGLEC7 may become a promising target for glioma immunotherapy. [ABSTRACT FROM AUTHOR]

Introduction: The epididymis is important for sperm transport, maturation, and storage. Methods: The head and tail of the epididymis of 5-week-old and 10-week-old C57 BL/6J male mice were used for single-cell sequencing. Results: 10 cell types including main, basal, and narrow/clear cells are identified. Next, we performed cell subgroup analysis, functional enrichment analysis, and differentiation potential prediction on principal cells, clear cells, and basal cells. Our study indicates that the principal cells are significantly involved in sperm maturation, as well as in antiviral and anti-tumor immune responses. Clear cells are likely to play a crucial role in safeguarding sperm and maintaining epididymal pH levels. Basal cells are implicated in the regulation of inflammatory and stress responses. The composition and functions of the various cell types within the epididymis undergo significant changes before and after sexual maturity. Furthermore, pseudo-temporal analysis elucidates the protective and supportive roles of epididymal cells in sperm maturation during sexual maturation. Discussion: This study offers a theoretical framework and forecasts for the investigation of epididymal sperm maturation and epididymal immunity. [ABSTRACT FROM AUTHOR]

Delayed repair is a serious public health concern for diabetic populations. Intercellular adhesion molecule 1 (ICAM-1) and Lymphocyte function-associated antigen 1 (LFA-1) play important roles in orchestrating the repair process. However, little is known about their effects on endothelial cell (EC) proliferation and neutrophil activity in subjects with hyperglycemia (HG). We cultured ECs and performed a scratch-closure assay to determine the relationship between ICAM-1 and EC proliferation. Specific internally labeled bacteria were used to clarify the effects of ICAM-1 and LFA-1 on neutrophil phagocytosis. Transwell assay and fluorescence-activated cell sorting analysis evaluated the roles of ICAM-1 and LFA-1 in neutrophil recruitment. ICAM-1C/C and ICAM-1+/+ mice were used to confirm the findings in vivo. The results demonstrated that HG decreased the expression of ICAM-1, which lead to the low proliferation of ECs. HG also attenuated neutrophil recruitment and phagocytosis by reducing the expression of ICAM-1 and LFA-1, which were strongly associated with the delayed repair. [ABSTRACT FROM AUTHOR]

Objectives: Osteosarcoma (OS) is the primary bone tumor originating from transformed mesenchymal cells. It is unclear whether associations between specific circulating immune cells and OS are causal or due to bias. To clarify whether predicted genetically altered circulating immune cells are associated with OS development, we performed a two-sample Mendelian randomization (MR) analysis. Methods: The genetic variants strongly associated with immune cell traits as instrumental variables (IVs) were used to perform MR analyses. The effect of specific immune cells on OS risk was measured using the summary statistics from the genome-wide association studies (GWAS). Results: Our findings indicate that CD80 on CD62L+ myeloid dendritic cell and CD28-CD4-CD8-T-cell absolute count are positively associated with OS (CD80 on CD62L+ myeloid dendritic cell, OR: 3.41 [95% CI: 1.40 to 8.31], p = 0.007; CD28-CD4-CD8-T-cell absolute count, OR: 4.49 [95% CI: 1.29 to 15.62], p = 0.018). It is also found that CD20 has a negative effect on CD24+CD27+ B cell on OS (OR: 0.32 [95% CI: 0.14 to 0.72], p = 0.006) and a similar impact on IgD+ CD38-B cell on OS (OR: 0.19 [95% CI: 0.05 to 0.68], p = 0.011). Conclusions: These findings illustrate that the genetic predisposition to specific immune cells can exert a causal effect on OS risk, which confirms the crucial role played by immunity in OS development. Particularly, the causal association between immune cells and OS underscores the evidence for exploring the new treatment strategy for OS in the future. [ABSTRACT FROM AUTHOR]

Introduction: Innate lymphoid cells (ILCs) are enriched at mucosal surfaces where they respond rapidly to environmental stimuli and contribute to both tissue inflammation and healing. Methods: To gain insight into the role of ILCs in the pathology and recovery from COVID-19 infection, we employed a multi-omics approach consisting of Abseq and targeted mRNA sequencing to respectively probe the surface marker expression, transcriptional profile and heterogeneity of ILCs in peripheral blood of patients with COVID-19 compared with healthy controls. Results: We found that the frequency of ILC1 and ILC2 cells was significantly increased in COVID-19 patients. Moreover, all ILC subsets displayed a significantly higher frequency of CD69-expressing cells, indicating a heightened state of activation. ILC2s from COVID-19 patients had the highest number of significantly differentially expressed (DE) genes. The most notable genes DE in COVID-19 vs healthy participants included a) genes associated with responses to virus infections and b) genes that support ILC self-proliferation, activation and homeostasis. In addition, differential gene regulatory network analysis revealed ILC-specific regulons and their interactions driving the differential gene expression in each ILC. Discussion: Overall, this study provides mechanistic insights into the characteristics of ILC subsets activated during COVID-19 infection. [ABSTRACT FROM AUTHOR]

In this review, we consider the multipotency of neural crest cells (NCCs), Schwann cell precursors (SCPs), and their role in embryogenesis base on genetic tracing and knock out model animals and single cell transcriptomic analysis. In particular, we summarize and analyze data on the contribution of NCCs and SCPs to the gland development and functions. [ABSTRACT FROM AUTHOR]

Background: A high-fat diet (HFD) contributes to various metabolic disorders and obesity, which are major contributors to cardiovascular disease. As an essential regulator for heart homeostasis, cardiac resident macrophages may go awry and contribute to cardiac pathophysiology upon HFD. Thus, to better understand how HFD induced cardiac dysfunction, this study intends to explore the transcriptional and functional changes in cardiac resident macrophages of HFD mice. Methods: C57BL/6J female mice that were 6 weeks old were fed with HFD or normal chow diet (NCD) for 16 weeks. After an evaluation of cardiac functions by echocardiography, mouse hearts were harvested and cardiac resident CCR2- macrophages were sorted, followed by Smart sequencing. Bioinformatics analysis including GO, KEGG, and GSEA analyses were employed to elucidate transcriptional and functional changes. Results: Hyperlipidemia and obesity were observed easily upon HFD. The mouse hearts also displayed more severe fibrosis and diastolic dysfunction in HFD mice. Smart sequencing and functional analysis revealed metabolic dysfunctions, especially lipid-related genes and pathways. Besides this, antigenpresentation-related gene such as Ctsf and inflammation, particularly for NFkB signaling and complement cascades, underwent drastic changes in cardiac resident macrophages. GO cellular compartment analysis was also performed and showed specific organelle enrichment trends of the involved genes. Conclusion: Dysregulated metabolism intertwines with inflammation in cardiac resident macrophages upon HFD feeding in mice, and further research on crosstalk among organelles could shed more light on potential mechanisms. [ABSTRACT FROM AUTHOR]

The response of N2 fixation to projected future conditions in the ocean cannot be reliably predicted to date. We conducted a minicosm experiment with preacclimated cultures of the globally significant diazotroph Crocosphaera watsonii strain WH8501 ("Crocosphaera"). PH and temperature were altered simultaneously to match the RCP scenarios 4.5 and 6 and investigate a more realistic future scenario compared to studies that focus on changes of a single stressor only. The cell abundance and nitrogen metabolism of Crocosphaera was monitored over 5 days. Our results imply that Crocosphaera is able to simultaneously perform N2 fixation and assimilate dissolved inorganic nitrogen (DIN, i.e., nitrate and ammonium) under all the conditions tested and implies a competition with non-diazotrophic phytoplankton for DIN, which should be further investigated. Using NanoSIMS analysis of single cells, our results point towards a preference for DIN assimilation over N2 fixation under more acidic and warmer conditions. Overall, our results show that while the combined alteration of pH and temperature had a negative effect on the diazotroph's growth and N2 fixation, Crocosphaera is likely to cope well with conditions in the future ocean. The high intra-population variability in nitrogen assimilation pathways may give this species the flexibility to quickly react to environmental changes. [ABSTRACT FROM AUTHOR]

Background: Anoikis is a form of programmed cell death essential for preventing cancer metastasis. In some solid cancer, anoikis resistance can facilitate tumor progression. However, this phenomenon is underexplored in clear-cell renal cell carcinoma (ccRCC). Methods: Using SVM machine learning, we identified core anoikis-related genes (ARGs) from ccRCC patient transcriptomic data. A LASSO Cox regression model stratified patients into risk groups, informing a prognostic model. GSVA and ssGSEA assessed immune infiltration, and single-cell analysis examined ARG expression across immune cells. Quantitative PCR and immunohistochemistry validated ARG expression differences between immune therapy responders and non-responders in ccRCC. Results: ARGs such as CCND1, CDKN3, PLK1, and BID were key in predicting ccRCC outcomes, linking higher risk with increased Treg infiltration and reduced M1 macrophage presence, indicating an immunosuppressive environment facilitated by anoikis resistance. Single-cell insights showed ARG enrichment in Tregs and dendritic cells, affecting immune checkpoints. Immunohistochemical analysis reveals that ARGs protein expression is markedly elevated in ccRCC tissues responsive to immunotherapy. Conclusion: This study establishes a novel anoikis resistance gene signature that predicts survival and immunotherapy response in ccRCC, suggesting that manipulating the immune environment through these ARGs could improve therapeutic strategies and prognostication in ccRCC. [ABSTRACT FROM AUTHOR]

Chronic inflammatory enteropathy (CIE) is a common condition in dogs causing recurrent or persistent gastrointestinal clinical signs. Pathogenesis is thought to involve intestinal mucosal inflammatory infiltrates, but histopathological evaluation of intestinal biopsies from dogs with CIE fails to guide treatment, inform prognosis, or correlate with clinical remission. We employed single-cell RNA sequencing to catalog and compare the diversity of cells present in duodenal mucosal endoscopic biopsies from 3 healthy dogs and 4 dogs with CIE. Through characterization of 35,668 cells, we identified 31 transcriptomically distinct cell populations, including T cells, epithelial cells, and myeloid cells. Both healthy and CIE samples contributed to each cell population. T cells were broadly subdivided into GZMAhigh (putatively annotated as tissue resident) and IL7Rhigh (putatively annotated as non-resident) T cell categories, with evidence of a skewed proportion favoring an increase in the relative proportion of IL7Rhigh T cells in CIE dogs. Among the myeloid cells, neutrophils from CIE samples exhibited inflammatory (SOD2 and IL1A) gene expression signatures. Numerous differentially expressed genes were identified in epithelial cells, with gene set enrichment analysis suggesting enterocytes from CIE dogs may be undergoing stress responses and have altered metabolic properties. Overall, this work reveals the previously unappreciated cellular heterogeneity in canine duodenal mucosa and provides new insights into molecular mechanisms which may contribute to intestinal dysfunction in CIE. The cell type gene signatures developed through this study may also be used to better understand the subtleties of canine intestinal physiology in health and disease. [ABSTRACT FROM AUTHOR]

Cellular movement is essential for many vital biological functions where it plays a pivotal role both at the single cell level, such as during division or differentiation, and at the macroscopic level within tissues, where coordinated migration is crucial for proper morphogenesis. It also has an impact on various pathological processes, one for all, cancer spreading. Cell migration is a complex phenomenon and diverse experimental methods have been developed aimed at dissecting and analysing its distinct facets independently. In parallel, corresponding analytical procedures and tools have been devised to gain deep insight and interpret experimental results. Here we review established experimental techniques designed to investigate specific aspects of cell migration and present a broad collection of historical as well as cutting-edge computational tools used in quantitative analysis of cell motion. [ABSTRACT FROM AUTHOR]

Hypoxia exerts a profound influence on the tumor microenvironment and immune response, shaping treatment outcomes and prognosis. Utilizing consistency clustering, we discerned two hypoxia subtypes in OPSCC bulk sequencing data from GEO. Key modules within OPSCC were identified through weighted gene correlation network analysis (WGCNA). Core modules underwent CIBERSORT immune infiltration analysis and GSEA functional enrichment. Univariate Cox and LASSO analyses were employed to construct prognostic models for seven hypoxia-related genes. Further investigation into clinical characteristics, the immune microenvironment, and TIDE algorithm prediction for immunotherapy response was conducted in high- and low-risk groups. scRNA-seq data were visually represented through TSNE clustering, employing the scissors algorithm to map hypoxia phenotypes. Interactions among cellular subpopulations were explored using the Cellchat package, with additional assessments of metabolic and transcriptional activities. Integration with clinical data unveiled a prevalence of HPV-positive patients in the low hypoxia and low-risk groups. Immunohistochemical validation demonstrated low TDO2 expression in HPV-positive (P16-positive) patients. Our prediction suggested that HPV16 E7 promotes HIF-1α inhibition, leading to reduced glycolytic activity, ultimately contributing to better prognosis and treatment sensitivity. The scissors algorithm effectively segregated epithelial cells and fibroblasts into distinct clusters based on hypoxia characteristics. Cellular communication analysis illuminated significant crosstalk among hypoxia-associated epithelial, fibroblast, and endothelial cells, potentially fostering tumor proliferation and metastasis. [ABSTRACT FROM AUTHOR]

Background: Persistent radiological lung abnormalities are evident in many survivors of acute coronavirus disease 2019 (COVID-19). Consolidation and ground glass opacities are interpreted to indicate subacute inflammation whereas reticulation is thought to reflect fibrosis. We sought to identify differences at molecular and cellular level, in the local immunopathology of post-COVID inflammation and fibrosis. Methods: We compared single-cell transcriptomic profiles and T cell receptor (TCR) repertoires of bronchoalveolar cells obtained from convalescent individuals with each radiological pattern, targeting lung segments affected by the predominant abnormality. Results: CD4 central memory T cells and CD8 effector memory T cells were significantly more abundant in those with inflammatory radiology. Clustering of similar TCRs from multiple donors was a striking feature of both phenotypes, consistent with tissue localised antigen-specific immune responses. There was no enrichment for known SARS-CoV-2-reactive TCRs, raising the possibility of T cell-mediated immunopathology driven by failure in immune self-tolerance. Conclusions: Post-COVID radiological inflammation and fibrosis show evidence of shared antigen-specific T cell responses, suggesting a role for therapies targeting T cells in limiting post-COVID lung damage. [ABSTRACT FROM AUTHOR]

Dromedary camel is an important livestock species with special economic value in arid and semi-arid regions of the world. Given the limited data on detailed immune cell composition and cell marker expression in the dromedary camel lymph node tissue, the present study was undertaken to investigate the immune cell composition of bronchial and mesenteric lymph nodes from healthy dromedary camels using flow cytometry. In this study, we applied flow cytometry and multicolor immuno-fluorescence to phenotype the main populations of immune cells in the bronchial and mesenteric camel lymph nodes and compared them with separated peripheral blood mononuclear cells and granulocytes. We used antibodies to detect several cell surface molecules associated with camel T cells (CD4, WC1), B cells (MHCII, BAQ44A), monocytes/macrophages (CD172a, CD14, CD163), in addition to the pan-leukocyte marker CD45 and the cell adhesion molecules CD44 and CD18. Compared to blood mononuclear cells, camel lymph node cells contained a higher percentage of lymphoid cells with only a minor fraction of myeloid cells. In addition, the lower expression of CD44 and CD18 on lymph node lymphocytes compared to lymphocytes from peripheral blood indicates higher frequency of naïve lymphocytes in the lymph nodes. The frequency of CD4+ T cells, B cells and γδ T cells within camel lymph node lymphocytes compared to blood indicates a similar tissue distribution pattern of lymphocyte subsets in camel and bovine and supports previous reports on the similarity between the camel immune system and the immune system of other ruminants. Lymph node neutrophils were identified as CD45++ CD172a++, CD14+, MHCIIlow, BAQ44A+, CD44++, CD18++ cells. In conclusion, the present study is describing the employment of flow cytometric single-cell analysis and immunostaining for the analysis of the immune cell composition in the camel lymph node. [ABSTRACT FROM AUTHOR]

Introduction: Hypoxia is a common pathological driver contributing to various forms of pulmonary vascular diseases leading to pulmonary hypertension (PH). Pulmonary interstitialmacrophages (IMs) play pivotal roles in immune and vascular dysfunction, leading to inflammation, abnormal remodeling, and fibrosis in PH. However, IMs' response to hypoxia and their role in PH progression remain largely unknown. We utilized a murine model of hypoxia-induced PH to investigate the repertoire and functional profiles of IMs in response to acute and prolonged hypoxia, aiming to elucidate their contributions to PH development. Methods: We conducted single-cell transcriptomic analyses to characterize the repertoire and functional profiles of murine pulmonary IMs following exposure to hypobaric hypoxia for varying durations (0, 1, 3, 7, and 21 days). Hallmark pathways from the mouse Molecular Signatures Database were utilized to characterize the molecular function of the IM subpopulation in response to hypoxia. Results: Our analysis revealed an early acute inflammatory phase during acute hypoxia exposure (Days 1-3), which was resolved by Day 7, followed by a proremodeling phase during prolonged hypoxia (Days 7-21). These phases were marked by distinct subpopulations of IMs: MHCIIhiCCR2+EAR2+ cells characterized the acute inflammatory phase, while TLF+VCAM1hi cells dominated the pro-remodeling phase. The acute inflammatory phase exhibited enrichment in interferon-gamma, IL-2, and IL-6 pathways, while the proremodeling phase showed dysregulated chemokine production, hemoglobin clearance, and tissue repair profiles, along with activation of distinct complement pathways. Discussion: Our findings demonstrate the existence of distinct populations of pulmonary interstitial macrophages corresponding to acute and prolonged hypoxia exposure, pivotal in regulating the inflammatory and remodeling phases of PH pathogenesis. This understanding offers potential avenues for targeted interventions, tailored to specific populations and distinct phases of the disease. Moreover, further identification of triggers for pro-remodeling IMs holds promise in unveiling novel therapeutic strategies for pulmonary hypertension. [ABSTRACT FROM AUTHOR]

Background: During aging, chronic inflammation can promote tumor development and metastasis. Patients with chronic inflammatory bowel diseases (IBD) are at an increased risk of developing colorectal cancer (CRC). However, the molecular mechanism underlying is still unclear. Methods: We conducted a large-scale single-cell sequencing analysis comprising 432,314 single cells from 92 CRC and 24 IBD patients. The analysis focused on the heterogeneity and commonality of CRC and IBD with respect to immune cell landscape, cellular communication, aging and inflammatory response, and Meta programs. Results: The CRC and IBD had significantly different propensities in terms of cell proportions, differential genes and their functions, and cellular communication. The progression of CRC was mainly associated with epithelial cells, fibroblasts, and monocyte-macrophages, which displayed pronounced metabolic functions. In particular, monocyte-macrophages were enriched for the aging and inflammation-associated NF-kB pathway. And IBD was enriched in immunerelated functions with B cells and T cells. Cellular communication analysis in CRC samples displayed an increase in MIF signaling from epithelial cells to T cells, and an increase in the efferent signal of senescence-associated SPP1 signaling from monocyte-macrophages. Notably, we also found some commonalities between CRC and IBD. The efferent and afferent signals showed that the pro-inflammatory cytokine played an important role. And the activity of aging and inflammatory response with AUCell analysis also showed a high degree of commonality. Furthermore, using the Meta programs (MPs) with the NMF algorithm, we found that the CRC non-malignant cells shared a substantial proportion of the MP genes with CRC malignant cells (68% overlap) and IBD epithelial cells (52% overlap), respectively. And it was extensively involved in functions of cell cycle and immune response, revealing its dual properties of inflammation and cancer. In addition, CRC malignant and non-malignant cells were enriched for the senescence-related cell cycle G2M phase transition and the p53 signaling pathway. Conclusion: Our study highlights the characteristics of aging, inflammation and tumor in CRC and IBD at the single-cell level, and the dual property of inflammation-cancer in CRC non-malignant cells may provide a more up-todate understanding of disease transformation. [ABSTRACT FROM AUTHOR]

Background: Globally, esophageal squamous cell carcinoma (ESCC) stands out as a common cancer type, characterized by its notably high rates of occurrence and mortality. Recent advancements in treatment methods, including immunotherapy, have shown promise, yet the prognosis remains poor. In the context of tumor development and treatment outcomes, the tumor microenvironment (TME), especially the function of dendritic cells (DCs), is significantly influential. Our study aims to delve deeper into the heterogeneity of DCs in ESCC using single-cell RNA sequencing (scRNA-seq) and bulk RNA analysis. Methods: In the scRNA-seq analysis, we utilized the SCP package for result visualization and functional enrichment analysis of cell subpopulations. CellChat was employed to identify potential oncogenic mechanisms in DCs, while Monocle 2 traced the evolutionary trajectory of the three DC subtypes. CopyKAT assessed the benign or malignant nature of cells, and SCENIC conducted transcription factor regulatory network analysis, offering a preliminary exploration of DC heterogeneity. In Bulk-RNA analysis, we constructed a prognostic model for ESCC prognosis and immunotherapy response, based on DC marker genes. This model was validated through quantitative PCR (qPCR) and immunohistochemistry (IHC), confirming the gene expression levels. Results: In this study, through intercellular communication analysis, we identified GALECTIN and MHC-I signaling pathways as potential oncogenic mechanisms within dendritic cells. We categorized DCs into three subtypes: plasmacytoid (pDC), conventional (cDC), and tolerogenic (tDC). Our findings revealed that pDCs exhibited an increased proportion of cells in the G2/M and S phases, indicating enhanced cellular activity. Pseudotime trajectory analysis demonstrated that cDCs were in early stages of differentiation, whereas tDCs were in more advanced stages, with pDCs distributed across both early and late differentiation phases. Prognostic analysis highlighted a significant correlation between pDCs and tDCs with the prognosis of ESCC (P< 0.05), while no significant correlation was observed between cDCs and ESCC prognosis (P = 0.31). The analysis of cell malignancy showed the lowest proportion of malignant cells in cDCs (17%), followed by pDCs (29%), and the highest in tDCs (48%), with these results being statistically significant (P< 0.05). We developed a robust ESCC prognostic model based on marker genes of pDCs and tDCs in the GSE53624 cohort (n = 119), which was validated in the TCGA-ESCC cohort (n = 139) and the IMvigor210 immunotherapy cohort (n = 298) (P< 0.05). Additionally, we supplemented the study with a novel nomogram that integrates clinical features and risk assessments. Finally, the expression levels of genes involved in the model were validated using qPCR (n = 8) and IHC (n = 16), thereby confirming the accuracy of our analysis. Conclusion: This study enhances the understanding of dendritic cell heterogeneity in ESCC and its impact on patient prognosis. The insights gained from scRNA-seq and Bulk-RNA analysis contribute to the development of novel biomarkers and therapeutic targets. Our prognostic models based on DC-related gene signatures hold promise for improving ESCC patient stratification and guiding treatment decisions. [ABSTRACT FROM AUTHOR]

One of the main challenges in cancer treatment is the selection of treatment resistant clones which leads to the emergence of resistance to previously efficacious therapies. Identifying vulnerabilities in the form of cellular tradeoffs constraining the phenotypic possibility space could allow to avoid the emergence of resistance by simultaneously targeting cellular processes that are involved in different alternative phenotypic strategies linked by trade-offs. The Pareto optimality theory has been proposed as a framework allowing to identify such trade-offs in biological data from its prediction that it would lead to the presence of specific geometrical patterns (polytopes) in, e.g., gene expression space, with vertices representing specialized phenotypes. We tested this approach in diffuse large B-cell lymphoma (DLCBL) transcriptomic data. As predicted, there was highly statistically significant evidence for the data forming a tetrahedron in gene expression space, defining four specialized phenotypes (archetypes). These archetypes were significantly enriched in certain biological functions, and contained genes that formed a pattern of shared and unique elements among archetypes, as expected if trade-offs between essential functions underlie the observed structure. The results can be interpreted as reflecting trade-offs between aerobic energy production and protein synthesis, and between immunotolerant and immune escape strategies. Targeting genes on both sides of these trade-offs simultaneously represent potential promising avenues for therapeutic applications. [ABSTRACT FROM AUTHOR]

Background: Pre-neutrophils, while developing in the bone marrow, transcribe the Inhba gene and synthesize Activin-A protein, which they store and release at the earliest stage of their activation in the periphery. However, the role of neutrophil-derived Activin-A is not completely understood. Methods: To address this issue, we developed a neutrophil-specific Activin-Adeficient animal model (S100a8-Cre/Inhbafl/fl mice) and analyzed the immune response to Influenza A virus (IAV) infection. More specifically, evaluation of body weight and lung mechanics, molecular and cellular analyses of bronchoalveolar lavage fluids, flow cytometry and cell sorting of lung cells, as well as histopathological analysis of lung tissues, were performed in PBS-treated and IAV-infected transgenic animals. Results: We found that neutrophil-specific Activin-A deficiency led to exacerbated pulmonary inflammation and widespread hemorrhagic histopathology in the lungs of IAV-infected animals that was associated with an exuberant production of neutrophil extracellular traps (NETs). Moreover, deletion of the Activin-A receptor ALK4/ACVR1B in neutrophils exacerbated IAV-induced pathology as well, suggesting that neutrophils themselves are potential targets of Activin-A-mediated signaling. The pro-NETotic tendency of Activin-A-deficient neutrophils was further verified in the context of thioglycollate-induced peritonitis, a model characterized by robust peritoneal neutrophilia. Of importance, transcriptome analysis of Activin-A-deficient neutrophils revealed alterations consistent with a predisposition for NET release. Conclusion: Collectively, our data demonstrate that Activin-A, secreted by neutrophils upon their activation in the periphery, acts as a feedback mechanism to moderate their pro-NETotic tendency and limit the collateral tissue damage caused by neutrophil excess activation during the inflammatory response. [ABSTRACT FROM AUTHOR]

Counting and analyzing of blood cells, as well as their subcellular structures, are indispensable for understanding biological processes, studying cell functions, and diagnosing diseases. In this paper, we combine digital holographic microscopy with cell segmentation guided by the Sobel operator using Dice coefficients for automatic threshold selection and aimed to automatic counting and analysis of blood cells in flow and different kinds of cells in the static state. We demonstrate the proposed method with automatic counting and analyzing rat red blood cells (RBCS) flowing in a microfluidic device, extracting quickly and accurately the size, concentration, and dry mass of the sample in a label-free manner. The proposed technique was also demonstrated for automatic segmentation of different cell types, such as COS7 and Siha. This method can help us in blood inspection, providing pathological information in disease diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Introduction: The clinical efficacy of CAR-NK cells against CD19-expressing blood cancers has been demonstrated, and they have shown potential for treating solid tumors as well. However, the efficacy of CAR-NK cells for treating human oral tongue squamous cell carcinoma (OTSCC) has not been examined. Methods: We assessed MUC1 expression in human OTSCC tissue and a cell line using immunohistochemistry and immunofluorescence. We constructed NK cells that express CAR targeted to MUC1 from pluripotent stem cells (iPSCderived MUC1-targeted CAR-NK cells) and evaluated their effectiveness against OTSCC in vitro using the xCELLigence Real-Time Cell Analysis system and CCK8 assay, and in vivo by measuring xenograft growth daily in BNDG mice treated with MUC1-targeted CAR-NK cells. As controls, we used iPSC-derived NK cells and NK-free media, which were CAR-free and blank, respectively. Results: MUC1 expression was detected in 79.5% (66/83) of all OTSCC patients and 72.7% (24/33) of stage III and IV. In stage III and IV MUC1 positive OTSCC, 63.6% (21/33) and 48.5% (16/33) patients had a MUC1-positive cancer cell rate of more than 50% and 80%, respectively. The iPSC-derived MUC1-targeted CARNK cells exhibited significant cytotoxicity against MUC1-expressing OTSCC cells in vitro, in a time- and dose-dependent manner, and showed a significant inhibitory effect on xenograft growth compared to both the iPSC-derived NK cells and the blank controls. We observed no weight loss, severe hematological toxicity or NK cell-mediated death in the BNDG mice. Conclusion: The MUC1-targetedCAR-NK cells had significant efficacy against human OTSCC, and their promising therapeutic response warrants further clinical trials. [ABSTRACT FROM AUTHOR]

Rituximab is a commonly used chemotherapeutic drug for patients with aggressive lymphomas, such as non-Hodgkin's lymphoma (NHL). Currently, the combination of Rituximab and chemotherapy (R-CHOP) stands as the most prevalent first-line therapy for NHL. Nevertheless, the development of new therapeutic approaches remains imperative. An increasing body of evidence highlights a novel role for IBTK in tumorigenesis and cancer growth. In this study, we aim to broaden our understanding of IBTK's function in B-lymphoma, with a particular focus on its impact on the expression of the oncogene MYC. Here, we assessed the effects of combining Rituximab with IBTK silencing on cell viability through cell cycle analysis and Annexin V assays in vitro. Furthermore, we leveraged the transplantability of Eμ-myc lymphomas to investigate whether the inhibition of IBTK could elicit anti-tumor effects in the treatment of lymphomas in vivo. Our data suggests that IBTK silencing may serve as an effective anti-tumor agent for aggressive B-Lymphomas, underscoring its role in promoting apoptosis when used in combination with Rituximab, both in in vitro and in vivo settings. [ABSTRACT FROM AUTHOR]

Background: Neutrophil extracellular traps (NETs) play a key role in thrombus formation in patients with coronavirus disease 2019 (COVID-19). However, the existing detection and observation methods for NETs are limited in their ability to provide quantitative, convenient, and accurate descriptions of in situ NETs. Therefore, establishing a quantitative description of the relationship between NETs and thrombosis remains a challenge. Objective: We employed morphological observations of blood cells and statistical analyses to investigate the correlation between the NETs/neutrophilic segmented granulocyte ratio and mortality risk in patients with COVID-19. Methods: Peripheral blood samples were collected from 117 hospitalized patients with COVID-19 between November 2022 and February 2023, and various blood cell parameters were measured. Two types of smudge cells were observed in the blood and counted: lymphatic and neutral smudge cells. Statistical data analysis was used to establish COVID-19 mortality risk assessment indicators. Results: Morphological observations of neutrophilic smudge cells revealed swelling, eruption, and NETs formation in the neutrophil nuclei. Subsequently, the NETs/neutrophilic segmented granulocyte ratio (NNSR) was calculated. A high concentration of NETs poses a fatal risk for thrombus formation in patients. Statistical analysis indicated that a high NNSR was more suitable for evaluating the risk of death in patients with COVID-19 compared to elevated fibrinogen (FIB) and D-dimer (DD) levels. Conclusion: Observing blood cell morphology is an effective method for the detection of NETs, NNSR are important markers for revealing the mortality risk of patients with COVID-19. [ABSTRACT FROM AUTHOR]

Background: Lung cancer is the leading cause of cancer deaths globally, with lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) being major subtypes. Immunotherapy has emerged as a promising approach for the treatment of lung cancer, but understanding the underlying mechanisms of immune dysregulation is crucial for the development of effective therapies. This study aimed to investigate the distinctive cellular features of LUAD and LUSC and identify potential biomarkers associated with the pathogenesis and clinical outcomes of each subtype. Methods: We used digital cytometry techniques to analyze the RNA-Seq data of 1128 lung cancer patients from The Cancer Genome Atlas (TCGA) database. The abundance of cell subtypes and ecotypes in LUAD and LUSC patients was quantified. Univariate survival analysis was used to investigate their associations with patient overall survival (OS). Differential gene expression analysis and gene co-expression network construction were carried out to explore the gene expression patterns of LUSC patients with distinct survival outcomes. Scratch wound-healing assay, colony formation assay, and transwell assay were used to validate the candidate drugs for LUSC treatment. Results: We found differential expression of cell subtypes between LUAD and LUSC, with certain cell subtypes being prognostic for survival in both subtypes. We also identified differential gene expression and gene co-expression modules associated with macrophages.3/PCs.2 ratio in LUSC patients with distinct survival outcomes. Furthermore, ecotype ratios were found to be prognostic in both subtypes and machine learning models showed that certain cell subtypes, such as epithelial.cells.1, epithelial.cells.5, and endothelial.cells.2 are important for predicting LUSC. Ginkgolide B and triamterene can inhibit the proliferation, invasion, and migration of LUSC cell lines. Conclusion: We provide insight into the distinctive cellular features of LUAD and LUSC, and identify potential biomarkers associated with the pathogenesis and clinical outcomes of each subtype. Ginkgolide B and triamterene could be promising drugs for LUSC treatment. [ABSTRACT FROM AUTHOR]

Objectives: It is important to accurately discriminate between clinical Clostridioides difficile infection (CDI) and colonization (CDC) for effective antimicrobial treatment. Methods: In this study, 37 stool samples were collected from 17 CDC and 20 CDI cases, and each sample were tested in parallel through the real-time cell analysis (RTCA) system, real-time PCR assay (PCR), and enzyme-linked immunosorbent assay (ELISA). Results: RTCA-measured functional and toxical C. difficile toxin B (TcdB) concentrations in the CDI group (302.58 ± 119.15 ng/mL) were significantly higher than those in the CDC group (18.15 ± 11.81 ng/mL) (p = 0.0008). Conversely, ELISA results revealed no significant disparities in TcdB concentrations between the CDC (26.21 ± 3.57 ng/mL) and the CDI group (17.07 ± 3.10 ng/mL) (p = 0.064). PCR results indicated no significant differences in tcdB gene copies between the CDC (774.54 ± 357.89 copies/μL) and the CDI group (4,667.69 ± 3,069.87 copies/μL) (p = 0.407). Additionally, the functional and toxical TcdB concentrations secreted from C. difficile isolates were measured by the RTCA. The results from the CDC (490.00 ± 133.29 ng/mL) and the CDI group (439.82 ± 114.66 ng/mL) showed no significant difference (p = 0.448). Notably, RTCA-measured functional and toxical TcdB concentration was significantly decreased when mixed with pooled CDC samples supernatant (p = 0.030). Conclusion: This study explored the novel application of the RTCA assay in effectively discerning clinical CDI from CDC cases. [ABSTRACT FROM AUTHOR]

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading threat to public health as it is resistant to most currently available antibiotics. Prodigiosin is a secondary metabolite of microorganisms with broad-spectrum antibacterial activity. This study identified a significant antibacterial effect of prodigiosin against MRSA with a minimum inhibitory concentration as low as 2.5 mg/L. The results of scanning electron microscopy, crystal violet staining, and confocal laser scanning microscopy indicated that prodigiosin inhibited biofilm formation in S. aureus USA300, while also destroying the structure of the cell wall and cell membrane, which was confirmed by transmission electron microscopy. At a prodigiosin concentration of 1.25 mg/L, biofilm formation was inhibited by 76.24%, while 2.5 mg/L prodigiosin significantly reduced the vitality of MRSA cells in the biofilm. Furthermore, the transcriptomic results obtained at 1/8 MIC of prodigiosin indicated that 235and 387 genes of S. aureus USA300 were significantly up- and downregulated, respectively. The downregulated genes were related to two-component systems, including the transcriptional regulator LytS, quorum sensing histidine kinases SrrB, NreA and NreB, peptidoglycan biosynthesis enzymes (MurQ and GlmU), iron-sulfur cluster repair protein ScdA, microbial surface components recognizing adaptive matrix molecules, as well as the key arginine synthesis enzymes ArcC and ArgF. The upregulated genes were mainly related to cell wall biosynthesis, as well as two-component systems including vancomycin resistance-associated regulator, lipoteichoic acid biosynthesis related proteins DltD and DltB, as well as the 9 capsular polysaccharide biosynthesis proteins. This study elucidated the molecular mechanisms through which prodigiosin affects the cell envelope of MRSA from the perspectives of cell wall synthesis, cell membrane and biofilm formation, providing new potential targets for the development of antimicrobials for the treatment of MRSA. [ABSTRACT FROM AUTHOR]

Ca2+ microdomains play a key role in intracellular signaling processes. For instance, they mediate the activation of T cells and, thus, the initial adaptive immune system. They are, however, also of utmost importance for activation of other cells, and a detailed understanding of the dynamics of these spatially localized Ca2+ signals is crucial for a better understanding of the underlying signaling processes. A typical approach to analyze Ca2+ microdomain dynamics is live cell fluorescence microscopy imaging. Experiments usually involve imaging a larger number of cells of different groups (for instance, wild type and knockout cells), followed by a time consuming image and data analysis. With DARTS, we present a modular Python pipeline for efficient Ca2+ microdomain analysis in live cell imaging data. DARTS (Deconvolution, Analysis, Registration, Tracking, and Shape normalization) provides state-of-the-art image postprocessing options like deep learning-based cell detection and tracking, spatio-temporal image deconvolution, and bleaching correction. An integrated automated Ca2+ microdomain detection offers direct access to global statistics like the number of microdomains for cell groups, corresponding signal intensity levels, and the temporal evolution of the measures. With a focus on bead stimulation experiments, DARTS provides a so-called dartboard projection analysis and visualization approach. A dartboard projection covers spatiotemporal normalization of the bead contact areas and cell shape normalization onto a circular template that enables aggregation of the spatiotemporal information of the microdomain detection results for the individual cells of the cell groups of interest. The dartboard visualization allows intuitive interpretation of the spatio-temporal microdomain dynamics at the group level. The application of DARTS is illustrated by three use cases in the context of the formation of initial Ca2+ microdomains after cell stimulation. DARTS is provided as an open-source solution and will be continuously extended upon the feedback of the community. [ABSTRACT FROM AUTHOR]

Introduction: Heat ablation is one of the key modalities in treating liver cancer, yet the residual cancer tissues suffering sublethal heat treatment possess a potential for increased malignancy. This study conducts a comprehensive analysis of cellular dynamics, metabolic shifts, and macrophage polarization within the tumor microenvironment following sublethal heat treatment. Methods: We observed significant acidification in tumor cell supernatants, attributed to increased lactic acid production. The study focused on how this pH shift, crucial in tumor progression and resistance, influences macrophage polarization, especially towards the M2 phenotype known for tumor-promoting functions. We also examined the upregulation of MCT1 expression post sublethal heat treatment and its primary role in lactic acid transport. Results: Notably, the study found minimal disparity in MCT1 expression between hepatocellular carcinoma patients and healthy liver tissues, highlighting the complexity of cancer biology. The research further revealed an intricate relationship between lactic acid, MCT1, and the inhibition of macrophage pyroptosis, offering significant insights for therapeutic strategies targeting the tumor immune environment. Post sublethal heat treatment, a reduction in paraspeckle under lactic acid exposure was observed, indicating diverse cellular impacts. Additionally, PKM2 was identified as a key molecule in this context, with decreased levels after sublethal heat treatment in the presence of lactic acid. Discussion: Collectively, these findings illuminate the intertwined mechanisms of sublethal heat treatments, metabolic alterations, and immune modulation in the tumor milieu, providing a deeper understanding of the complex interplay in cancer biology and treatment. [ABSTRACT FROM AUTHOR]

Glioma is the most devastating high-grade tumor of the central nervous system, with dismal prognosis. Existing treatment modality does not provide substantial benefit to patients and demands novel strategies. One of the first-line treatments for glioma, temozolomide, provides marginal benefit to glioma patients. Repurposing of existing non-cancer drugs to treat oncology patients is gaining momentum in recent years. In this study, we investigated the therapeutic benefits of combining three repurposed drugs, namely, metformin (anti-diabetic) and epigallocatechin gallate (green tea-derived antioxidant) together with temozolomide in a glioma-induced xenograft rat model. Our triple-drug combination therapy significantly inhibited tumor growth in vivo and increased the survival rate (50%) of rats when compared with individual or dual treatments. Molecular and cellular analyses revealed that our triple-drug cocktail treatment inhibited glioma tumor growth in rat model through ROS-mediated inactivation of PI3K/AKT/mTOR pathway, arrest of the cell cycle at G1 phase and induction of molecular mechanisms of caspases-dependent apoptosis. In addition, the docking analysis and quantum mechanics studies performed here hypothesize that the effect of triple-drug combination could have been attributed by their difference in molecular interactions, that maybe due to varying electrostatic potential. Thus, repurposing metformin and epigallocatechin gallate and concurrent administration with temozolomide would serve as a prospective therapy in glioma patients. [ABSTRACT FROM AUTHOR]

Background: Cholangiocarcinoma is characterized by significant cellular heterogeneity and complex intercellular communication, which contribute to its progression and therapeutic resistance. Therefore, unraveling this complexity is essential for the development of effective treatments. Methods: We employed single-cell RNA sequencing (scRNA-seq) to investigate cellular heterogeneity and intercellular communication in cholangiocarcinoma and adjacent normal tissues from two patients. Distinct cell types were identified, and gene ontology analyses were conducted to determine enriched pathways. Moreover, cell-cell communications were analyzed using CellChat, a computational framework. Additionally, we performed sub-clustering analysis of T cells and fibroblasts. Results: The scRNA-seq analysis revealed distinct cell clusters and diverse cellular compositions of cholangiocarcinoma. CellChat analysis underscored an amplified outgoing signal from fibroblasts within the tumor, suggesting their pivotal role in the tumor microenvironment. Furthermore, T cell sub-clustering analysis revealed an active immune response within the tumor and new tumor-specific T cell clonotypes, suggesting scope for targeted immunotherapies. Moreover, fibroblast sub-clustering analysis indicated distinct functional states and highlighted the role of activated fibroblasts in shaping intercellular communication, particularly via CD99 and FN1 signaling. Conclusion: Our findings reveal the intricate cellular heterogeneity and dynamic intercellular communication in cholangiocarcinoma, providing valuable insights into disease progression and potential therapeutic strategies. [ABSTRACT FROM AUTHOR]

Background: Insects have specialized cell types that participate in the elimination of parasites, for instance, the lamellocytes of the broadly studied species Drosophila melanogaster. Other drosophilids, such as Drosophila ananassae and the invasive Zaprionus indianus, have multinucleated giant hemocytes, a syncytium of blood cells that participate in the encapsulation of the eggs or larvae of parasitoid wasps. These cells can be formed by the fusion of hemocytes in circulation or originate from the lymph gland. Their ultrastructure highly resembles that of the mammalian megakaryocytes. Methods: Morphological, protein expressional, and functional features of blood cells were revealed using epifluorescence and confocal microscopy. The respective hemocyte subpopulations were identified using monoclonal antibodies in indirect immunofluorescence assays. Fluorescein isothiocyanate (FITC)-labeled Escherichia coli bacteria were used in phagocytosis tests. Gene expression analysis was performed following mRNA sequencing of blood cells. Results: D. ananassae and Z. indianus encapsulate foreign particles with the involvement of multinucleated giant hemocytes and mount a highly efficient immune response against parasitoid wasps. Morphological, protein expressional, and functional assays of Z. indianus blood cells suggested that these cells could be derived from large plasmatocytes, a unique cell type developing specifically after parasitoid wasp infection. Transcriptomic analysis of blood cells, isolated from naïve and wasp-infected Z. indianus larvae, revealed several differentially expressed genes involved in signal transduction, cell movements, encapsulation of foreign targets, energy production, and melanization, suggesting their role in the anti-parasitoid response. A large number of genes that encode proteins associated with coagulation and wound healing, such as phenoloxidase activity factor-like proteins, fibrinogen-related proteins, lectins, and proteins involved in the differentiation and function of platelets, were constitutively expressed. The remarkable ultrastructural similarities between giant hemocytes and mammalian megakaryocytes, and presence of platelets, and giant cellderived anucleated fragments at wound sites hint at the involvement of this cell subpopulation in wound healing processes, in addition to participation in the encapsulation reaction. Conclusion: Our observations provide insights into the broad repertoire of blood cell functions required for efficient defense reactions to maintain the homeostasis of the organism. The analysis of the differentiation and function of multinucleated giant hemocytes gives an insight into the diversification of the immune mechanisms. [ABSTRACT FROM AUTHOR]

Introduction: Tumor-infiltrating myeloid cells (TIMs) are key regulators in tumor progression, but the similarity and distinction of their fundamental properties in pancreatic ductal adenocarcinoma (PDAC) remain elusive. Method: In this study, we conducted scRNA-seq data analysis of cells from 12 primary tumor (PT) tissues, 4 metastatic (Met) tumor tissues, 3 adjacent normal pancreas tissues (Para), and PBMC samples across 16 PDAC patients, and revealed a heterogeneous TIMs environment in PDAC. Result: Systematic comparisons between tumor and non-tumor samples of myeloid lineages identified 10 necroptosis-associated genes upregulated in PDAC tumors compared to 5 upregulated in paratumor or healthy peripheral blood. A novel RTM (resident tissue macrophages), GLUL-SQSTM1- RTM, was found to act as a positive regulator of immunity. Additionally, HSP90AA1+HSP90AB1+ mast cells exhibited pro-immune characteristics, and JAK3+TLR4+ CD16 monocytes were found to be anti-immune. The findings were validated through clinical outcomes and cytokines analyses. Lastly, intercellular network reconstruction supported the associations between the identified novel clusters, cancer cells, and immune cell populations. Conclusion: Our analysis comprehensively characterized major myeloid cell lineages and identified three subsets of myeloid-derived cells associated with necroptosis. These findings not only provide a valuable resource for understanding the multi-dimensional characterization of the tumor microenvironment in PDAC but also offer valuable mechanistic insights that can guide the design of effective immuno-oncology treatment strategies. [ABSTRACT FROM AUTHOR]

Background: There is growing evidence that the lung is a target organ for injury in diabetes and hypertension. There are no studies on the status of the lungs, especially cellular subpopulations, and related functions in patients with diabetes, hypertension, and hypertension-diabetes after combined SARS-CoV-2 infection. Method: Using single-cell meta-analysis in combination with bulk-RNA analysis, we identified three drug targets and potential receptors for SARS-CoV-2 infection in lung tissues from patients with diabetes, hypertension, and hypertension-diabetes, referred to as "co-morbid" patients. Using single-cell meta-analysis analysis in combination with bulk-RNA, we identified drug targets and potential receptors for SARS-CoV-2 infection in the three co-morbidities. Results: The single-cell meta-analysis of lung samples from SARS-CoV-2-infected individuals with diabetes, hypertension, and hypertension-diabetes comorbidity revealed an upregulation of fibroblast subpopulations in these disease conditions associated with a predictive decrease in lung function. To further investigate the response of fibroblasts to therapeutic targets in hypertension and diabetes, we analyzed 35 upregulated targets in both diabetes and hypertension. Interestingly, among these targets, five specific genes were upregulated in fibroblasts, suggesting their potential association with enhanced activation of endothelial cells. Furthermore, our investigation into the underlying mechanisms driving fibroblast upregulation indicated that KREMEN1, rather than ACE2, could be the receptor responsible for fibroblast activation. This finding adds novel insights into the molecular processes involved in fibroblast modulation in the context of SARS-CoV-2 infection within these comorbid conditions. Lastly, we compared the efficacy of Pirfenidone and Nintedanib as therapeutic interventions targeting fibroblasts prone to pulmonary fibrosis. Our findings suggest that Nintedanib may be a more suitable treatment option for COVID-19 patients with diabetes and hypertension who exhibit fibrotic lung lesions. Conclusion: In the context of SARS-CoV-2 infections, diabetes, hypertension, and their coexistence predominantly lead to myofibroblast proliferation. This phenomenon could be attributed to the upregulation of activated endothelial cells. Moreover, it is noteworthy that therapeutic interventions targeting hypertension-diabetes demonstrate superior efficacy. Regarding treating fibrotic lung conditions, Nintedanib is a more compelling therapeutic option. [ABSTRACT FROM AUTHOR]

Objective: This study amied to investigate the prognostic characteristics of triple negative breast cancer (TNBC) patients by analyzing B cell marker genes based on single-cell and bulk RNA sequencing. Methods: Utilizing single-cell sequencing data from TNBC patients, we examined tumor-associated B cell marker genes. Transcriptomic data from The Cancer Genome Atlas (TCGA) database were used as the foundation for predictive modeling. Independent validation set was conducted using the GSE58812 dataset. Immune cell infiltration into the tumor was assessed through various, including XCELL, TIMER, QUANTISEQ, CIBERSORT, CIBERSORT-ABS, and ssGSEA. The TIDE score was utilized to predict immunotherapy outcomes. Additional investigations were conducted on the immune checkpoint blockade gene, tumor mutational load, and the GSEA enrichment analysis. Results: Our analysis encompassed 22,106 cells and 20,556 genes in cancerous tissue samples from four TNBC patients, resulting in the identification of 116 B cell marker genes. A B cell marker gene score (BCMG score) involving nine B cell marker genes (ZBP1, SEL1L3, CCND2, TNFRSF13C, HSPA6, PLPP5, CXCR4, GZMB, and CCDC50) was developed using TCGA transcriptomic data, revealing statistically significant differences in survival analysis (P<0.05). Functional analysis demonstrated that marker genes were predominantly associated with immune-related pathways. Notably, substantial differences between the higher and lower-BCMG score groups were observed in terms of immune cell infiltration, immune cell activity, tumor mutational burden, TIDE score, and the expression of immune checkpoint blockade genes. Conclusion: This study has established a robust model based on B-cell marker genes in TNBC, which holds significant potential for predicting prognosis and response to immunotherapy in TNBC patients. [ABSTRACT FROM AUTHOR]

Macrophages are highly heterogeneous immune cells with a role in maintaining tissue homeostasis, especially in activating the defense response to bacterial infection. Using flow cytometric and single-cell RNA-sequencing analyses of peritoneal cells, we here show that small peritoneal macrophage and immature macrophage populations are enriched in histamine-deficient (Hdc-/-) mice, characterized by a CD11bmiF4/80loCCR2+MHCIIhi and CD11bloF4/ 80miTHBS1+IL-1α+ phenotype, respectively. Molecular characterization revealed that immature macrophages represent an abnormally differentiated form of large peritoneal macrophages with strong inflammatory properties. Furthermore, deficiency in histamine signaling resulted in significant impairment of the phagocytic activity of peritoneal macrophage populations, conferring high susceptibility to bacterial infection. Collectively, this study reveals the importance of histamine signaling in macrophage differentiation at the molecular level to maintain tissue homeostasis, offering a potential therapeutic target for bacterial infection-mediated diseases. [ABSTRACT FROM AUTHOR]

Mast cell tumor (MCT) is a common skin cancer in dogs that has a wide range of clinical behaviors. The purpose of this study was to develop a novel multicolor flow cytometry (FC) panel that will enable the quantification of candidate prognostic markers (Ki-67 and pKIT) in fine needle aspirate (FNA) samples prior to surgical removal of the tumors. FNA of canine MCTs and the NI-1 cell line were utilized to develop a FC panel that includes a viability dye (FVS620, BD Biosciences; 7-AAD, Invitrogen) and the following primary conjugated antibodies: CD117-PE (ACK45, BD Biosciences), pKIT-A647 (polyclonal bs-3242R, BIOSS) and Ki-67- FITC (20Raj1, eBioscience; MIB-1, DAKO). A total of nine FNA samples of canine MCTs were collected, seven out which produced sufficient cells for FC analysis. The Ki-67 antibody clone 20Raj1 produced a positive signal when applied to blood leukocytes but failed to provide robust labeling of neoplastic mast cells. The Ki-67 antibody clone MIB-1 delivered a superior staining quality in both the NI-1 cells and primary MCT cells. CD117-PE signal was adequate post fixation and permeabilization and in the combination of 7-AAD. pKIT produced non-specific staining and was not suitable for this multicolor FC panel. In conclusion, FNA samples of canine MCTs can often yield adequate cell numbers for FC analysis, and a multicolor FC panel was developed that can detect Ki-67 in canine mast cells. This would permit further studies into the potential use of this panel for canine cutaneous and subcutaneous MCT prognostication purposes. [ABSTRACT FROM AUTHOR]

Rationale: While the immune system plays a crucial role in the development of hypertension, the specific contributions of distinct immune cell populations remain incompletely understood. The emergence of single-cell RNAsequencing (scRNA-seq) technology enables us to analyze the transcriptomes of individual immune cells and to assess the significance of each immune cell type in hypertension development. Objective: We aimed to investigate the hypothesis that B cells play a crucial role in the development of fructose-induced hypertension. Methods and Results: Eight-week-old Dahl salt-sensitive (SS) male rats were divided into two groups and given either tap water (TW) or a 20% fructose solution (HFS) for 4 weeks. Systolic blood pressure was measured using the tailcuff method. ScRNA-seq analysis was performed on lamina propria cells (LPs) and peripheral blood mononuclear cells (PBMCs) obtained from SS rats subjected to either TW or HFS. The HFS treatment induced hypertension in the SS rats. The analysis revealed 27 clusters in LPs and 28 clusters in PBMCs, allowing for the identification and characterization of various immune cell types within each cluster. Specifically, B cells and follicular helper T (Tfh) cells were prominent in LPs, while B cells and M1 macrophages dominated PBMCs in the HFS group. Moreover, the HFS treatment triggered an increase in the number of B cells in both LPs and PBMCs, accompanied by activation of the interferon pathway. Conclusions: The significant involvement of B cells in intestinal and PBMC responses indicates their pivotal contribution to the development of hypertension. This finding suggests that targeting B cells could be a potential strategy to mitigate high blood pressure in fructose-induced hypertension. Moreover, the simultaneous increase in follicular B cells and Tfh cells in LPs, along with the upregulation of interferon pathway genes in B cells, underscores a potential autoimmune factor contributing to the pathogenesis of fructoseinduced hypertension in the intestine. [ABSTRACT FROM AUTHOR]

Macrophages are highly heterogeneous immune cells with a role in maintaining tissue homeostasis, especially in activating the defense response to bacterial infection. Using flow cytometric and single-cell RNA-sequencing analyses of peritoneal cells, we here show that small peritoneal macrophage and immature macrophage populations are enriched in histamine-deficient (Hdc-/-) mice, characterized by a CD11bmiF4/80loCCR2+MHCIIhi and CD11bloF4/80miTHBS1+IL-1α+ phenotype, respectively. Molecular characterization revealed that immature macrophages represent an abnormally differentiated form of large peritoneal macrophages with strong inflammatory properties. Furthermore, deficiency in histamine signaling resulted in significant impairment of the phagocytic activity of peritoneal macrophage populations, conferring high susceptibility to bacterial infection. Collectively, this study reveals the importance of histamine signaling in macrophage differentiation at the molecular level to maintain tissue homeostasis, offering a potential therapeutic target for bacterial infection-mediated diseases. [ABSTRACT FROM AUTHOR]

Single cell computational analysis has emerged as a powerful tool in the field of oncology, enabling researchers to decipher the complex cellular heterogeneity that characterizes cancer. By leveraging computational algorithms and bioinformatics approaches, this methodology provides insights into the underlying genetic, epigenetic and transcriptomic variations among individual cancer cells. In this paper, we present a comprehensive overview of single cell computational analysis in oncology, discussing the key computational techniques employed for data processing, analysis, and interpretation. We explore the challenges associated with single cell data, including data quality control, normalization, dimensionality reduction, clustering, and trajectory inference. Furthermore, we highlight the applications of single cell computational analysis, including the identification of novel cell states, the characterization of tumor subtypes, the discovery of biomarkers, and the prediction of therapy response. Finally, we address the future directions and potential advancements in the field, including the development of machine learning and deep learning approaches for single cell analysis. Overall, this paper aims to provide a roadmap for researchers interested in leveraging computational methods to unlock the full potential of single cell analysis in understanding cancer biology with the goal of advancing precision oncology. For this purpose, we also include a notebook that instructs on how to apply the recommended tools in the Preprocessing and Quality Control section. [ABSTRACT FROM AUTHOR]

Oral administration of antigen induces regulatory T cells (Treg) that can not only control local immune responses in the small intestine, but also traffic to the central immune system to deliver systemic suppression. Employing murine models of the inherited bleeding disorder hemophilia, we find that oral antigen administration induces three CD4+ Treg subsets, namely FoxP3+LAP-, FoxP3+LAP+, and FoxP3- LAP+. These T cells act in concert to suppress systemic antibody production induced by therapeutic protein administration. Whilst both FoxP3+LAP+ and FoxP3-LAP+ CD4+ T cells express membrane-bound TGF-β (latency associated peptide, LAP), phenotypic, functional, and single cell transcriptomic analyses reveal distinct characteristics in the two subsets. As judged by an increase in IL-2Rα and TCR signaling, elevated expression of co-inhibitory receptor molecules and upregulation of the TGFβ and IL-10 signaling pathways, FoxP3+LAP+ cells are an activated form of FoxP3+LAP-Treg. Whereas FoxP3-LAP+ cells express low levels of genes involved in TCR signaling or co-stimulation, engagement of the AP-1 complexmembers Jun/Fos and Atf3 is most prominent, consistent with potent IL-10 production. Single cell transcriptomic analysis further reveals that engagement of the Jun/Fos transcription factors is requisite for mediating TGFb expression. This can occur via an Il2ra dependent or independent process in FoxP3+LAP+ or FoxP3-LAP+ cells respectively. Surprisingly, both FoxP3+LAP+ and FoxP3-LAP+ cells potently suppress and induce FoxP3 expression in CD4+ conventional T cells. In this process, FoxP3-LAP+ cells may themselves convert to FoxP3+ Treg. We conclude that orally induced suppression is dependent on multiple regulatory cell types with complementary and interconnected roles. [ABSTRACT FROM AUTHOR]