Single-cell RNA Sequencing Datasets Research Articles

P39 Using single-cell transcriptomics to investigate the potential role of the human hair follicle dermal papilla in controlling hair fibre pigmentation

Abstract Introduction and aims The human dermal papilla (DP) is a population of cells located at the base of the hair follicle, which signals to surrounding matrix cells facilitating their incorporation into the layers of the hair fibre and the hair follicle. The DP controls the process of hair follicle induction, reflecting its critical role in orchestrating processes integral in hair growth. We hypothesize that distinct functional subgroups exist within the structure, which would mean that there are different types of DP cells within each follicle. Methods To begin testing this, we reanalysed the single-cell RNA sequencing (RNA-Seq) dataset of the human anagen hair follicle used in the 2022 study by Shim et al. To expand on this, we are now generating single-cell RNA-Seq datasets of patient-matched pigmented and nonpigmented hair follicles (n = 4 patients). These datasets will be used to explore heterogeneity in the human DP, and to investigate which subpopulations are potentially changed in the case of pigmentation loss. This will allow us to explore a potentially undiscovered role of the DP in controlling hair fibre pigmentation. Results We found four distinct subpopulations in the DP with statistically significant marker genes in the Shim dataset. This confirms transcriptional heterogeneity in the human DP, hinting at clusters of DP cells with specialized functions within each hair follicle. One subcluster, which expressed S100B, endothelin-3 and bone morphogenetic protein (BMP)4, was of particular interest and gene ontology analysis of the genes in this cluster revealed terms associated with skeletal development and BMP signalling. We hypothesize that this DP cluster regulates melanin transfer from bulbar melanocytes to neighbouring keratinocytes, owing to the known involvement of the mentioned genes in both melanogenesis and melanin transfer. Conclusions The human DP is a heterogeneic structure, with a potentially unexplored role in controlling hair fibre pigmentation.

P14 Characterizing the hair follicle–nervous system connection

Abstract Introduction and aims The human hair follicle (HF) is a miniorgan in the skin that possesses a rich microenvironment composed of a complex assembly of sensory nerves that surround it. Recent studies have explored the effects of this perineural niche on the HF, but the influence of HF on the nervous system from a folliculocentric and skin-centric perspective remains underexplored. To explore the potential bidirectionality of the HF–nervous system connection in the context of nerve development, we asked whether the HF influences the differentiation and maturation of sensory neuron subtypes around it through the release of distinct neurotransmitters and neurotrophic factors. Methods To first identify which cell types might be involved in directing sensory neuron development, we reanalysed available skin and HF single-cell RNA sequencing (scRNA-Seq) datasets. Next, to evaluate neurotransmitter release from these cell types, we used fast-scan cyclic voltammetry (FSCV). Lastly, to interrogate the role of the HF on directing neuronal development in vitro, we used induced pluripotent stem cell-derived sensory neurons as a model for developing nerves. Results We found that a higher percentage of KRT14- (epithelial) and PDGFRA-expressing (mesenchymal) cells in the HF coexpressed genes encoding specific neurotrophic factors and enzymatic machinery for neurotransmitter production compared with equivalent cells in the skin, suggesting that the HF possesses a greater capacity to signal to the nervous system compared with interfollicular skin. Using FSCV, we then found that follicular outer root sheath and directionally selective cells are capable of releasing neurotransmitters such as serotonin and histamine. Conclusions We postulate that HF-derived neurotransmitters are important for aspects of sensory neuron development such as axonal growth, as serotonin has previously been shown to be an attractive guidance cue for neuronal growth cones. Overall, our work sheds light on the HF–nervous system connection and how the HF may regulate its microenvironment in the skin.

CXCR4-mediated neutrophil dynamics in periodontitis

Background and objectivePeriodontitis is a common oral disease closely related to immune response and this study is aimed to identify the key immune-related pathogenic genes and analyze the infiltration and function of immune cells in the disease using bioinformatics methods. MethodsTranscriptome datasets and single-cell RNA sequencing (scRNA-seq) datasets were downloaded from the GEO database. We utilized weighted correlation network analysis and least absolute selection and shrinkage operator, protein–protein interaction network construction to screen out key pathogenic genes as well as conducted the cell-type identification by estimating relative subsets of RNA transcripts algorithm to analyze and characterize immune cell types in periodontal tissues. In addition to bioinformatics validations, clinical and cell samples were collected and mouse periodontitis models were constructed to validate the important role of key genes in periodontitis. ResultsBioinformatics analysis pointed out the positive correlation between CXCR4 expression and periodontitis, and revealed the increased infiltration of neutrophils in periodontal inflammatory. Similar results were obtained from clinical samples and animal models. In addition, the clustering and functional enrichment results based on CXCR4 expression levels included activation of immune response and cell migration, implying the possible function of CXCR4 on regulating neutrophil dynamics, which might contribute to periodontitis. Subsequent validation experiments confirmed that the increased expression of CXCR4 in neutrophils under periodontitis, where cell migration-related pathways also were activated. ConclusionCXCR4 could be the key pathogenic gene of periodontitis and CXCR4/CXCL12 signal axial might contribute to the development of periodontitis by mediating neutrophil dynamics, suggesting that CXCR4 could be a potential target to help identify novel strategies for the clinical diagnosis and treatment of periodontitis.

Monocyte bioenergetics: An immunometabolic perspective in metabolic dysfunction-associated steatohepatitis

Monocytes (Mos) are crucial in the evolution of metabolic dysfunction-associated steatotic liver disease (MASLD) to metabolic dysfunction-associated steatohepatitis (MASH), and immunometabolism studies have recently suggested targeting leukocyte bioenergetics in inflammatory diseases. Here, we reveal a peculiar bioenergetic phenotype in circulating Mos of patients with MASH, characterized by high levels of glycolysis and mitochondrial (mt) respiration. The enhancement of mt respiratory chain activity, especially complex II (succinate dehydrogenase [SDH]), is unbalanced toward the production of reactive oxygen species (ROS) and is sustained at the transcriptional level with the involvement of the AMPK-mTOR-PGC-1α axis. The modulation of mt activity with dimethyl malonate (DMM), an SDH inhibitor, restores the metabolic profile and almost abrogates cytokine production. Analysis of a public single-cell RNA sequencing (scRNA-seq) dataset confirms that in murine models of MASH, liver Mo-derived macrophages exhibit an upregulation of mt and glycolytic energy pathways. Accordingly, the DMM injection in MASH mice contrasts Mo infiltration and macrophagic enrichment, suggesting immunometabolism as a potential target in MASH.

Comprehensive analysis of gene signatures associated with aging in human aortic dissection

BackgroundAortic dissection (AD) is a lethal aortic disease with limited effective therapeutic strategies. Aging increases the risk of AD, yet the underlying mechanisms remain unclear. This study aims to analyze the association of aging-related genes (Args) and AD using bioinformatic analysis. This helps provide novel insights into AD pathogenesis and contributes to developing novel therapeutic strategies. MethodsmRNA (GSE52093, GSE153434), miRNA (GSE98770) and single-cell RNA-sequencing (scRNA-seq, GSE213740) datasets of AD were downloaded from GEO database. Args were downloaded from Aging Atlas database. Differentially-expressed Args were determined by intersecting Args and differentially-expressed mRNAs of two mRNA datasets. Cytoscape was used to identify hub genes and construct hub gene regulatory networks related to miRNAs. Seurat and clusterProfiler R package were used for investigating expression patterns of hub genes at single-cell level, and functional analysis, respectively. To validate the cellular expression pattern of hub genes, the same analysis was applied to our own scRNA-seq data. Drugs targeting hub Args were determined using the DGIdb database. ResultsHGF, CXCL8, SERPINE1, HIF1A, TIMP1, ESR1 and PLAUR were identified as aging-related hub genes in AD. miR-221-3p was predicted to interact with ESR1. A decreased ESR1 expression in smooth muscle cell subpopulation 4 (SMC4) was observed in AD versus normal aortic tissues, which was validated by sequencing 197,605 aortic cells from 13 AD patients. Additionally, upregulated genes of SMC4 in AD tissues were enriched in the “cellular senescence” pathway. These data indicated that decreased ESR1 might promote SMC4 aging during AD formation. Eleven existing drugs targeting hub genes were identified, including ruxolitinib and filgrastim, which are associated with AD. ConclusionsBy sequencing transcriptomic data, this study revealed aging-related hub genes and regulatory network involved in AD formation. Additionally, this study proposed a noteworthy hypothesis that downregulated ESR1 may exacerbate AD by promoting SMC aging, which requires further investigation.

Type 17 immune response promotes oral epithelial cell proliferation in periodontitis

ObjectivesThis study aims to investigate the effects of type 17 immune response on the proliferation of oral epithelial cells in periodontitis. DesignA time-dependent ligature induced periodontitis mouse model was utilized to explore gingival hyperplasia and the infiltration of interleukin 17A (IL-17A) positive cells. Immunohistochemistry and flow cytometry were employed to determine the localization and expression of IL-17A in the ligature induced periodontitis model. A pre-existing single-cell RNA sequencing dataset, comparing individuals affected by periodontitis with healthy counterparts, was reanalyzed to evaluate IL-17A expression levels. We examined proliferation markers, including proliferating cell nuclear antigen (PCNA), signal transducer and activator of transcription (STAT3), Yes-associated protein (YAP), and c-JUN, in the gingival and tongue epithelium of the periodontitis model. An anti-IL-17A agent was administered daily to observe proliferative changes in the oral mucosa within the periodontitis model. Cell number quantification, immunofluorescence, and western blot analyses were performed to assess the proliferative responses of human normal oral keratinocytes to IL-17A treatment in vitro. ResultsThe ligature induced periodontitis model exhibited a marked infiltration of IL-17A-positive cells, alongside significant increase in thickness of the gingival and tongue epithelium. IL-17A triggers the proliferation of human normal oral keratinocytes, accompanied by upregulation of PCNA, STAT3, YAP, and c-JUN. The administration of an anti-IL-17A agent attenuated the proliferation in oral mucosa. ConclusionsThese findings indicate that type 17 immune response, in response to periodontitis, facilitates the proliferation of oral epithelial cells, thus highlighting its crucial role in maintaining the oral epithelial barrier.

IFI30 as a key regulator of PDL1 immunotherapy prognosis in breast cancer

BackgroundIFI30 is a lysosomal thiol reductase involved in antigen presentation and immune regulation in various cancers, including breast cancer. Despite its known involvement, the precise mechanism, function, and relationship with the PD-L1 axis and immune response remain unclear. MethodsWe conducted an extensive investigation into IFI30 mRNA expression in breast cancer utilizing data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Furthermore, we characterized IFI30 mRNA expression across various cell types using publicly available single-cell RNA sequencing datasets, and assessed protein expression through immunohistochemistry using an in-house breast cancer tissue microarray. Functional experiments were performed to elucidate the effects of IFI30 overexpression on PD-L1 expression and inhibitory efficacy in both macrophages and breast tumor cells. ResultsOur study unveiled a marked upregulation of IFI30 expression in breast cancer tissues compared to their normal counterparts, with notable associations identified with tumor stage and prognosis. Additionally, IFI30 expression demonstrated significant correlations with various immune-related signaling pathways, encompassing peptide antigen binding, cytokine binding, and MHC class II presentation. Notably, breast cancer samples exhibiting high IFI30 expression in tumor cells displayed high PD-L1 expression on corresponding cells, alongside a diminished ratio of CD8 + T cell infiltration within the tumor microenvironment. Furthermore, ectopic knockdown of IFI30 in both tumor cells and macrophages resulted in a reduction of PD-L1 expression, while conversely, overexpression of IFI30 led to an increase in PD-L1 expression. ConclusionsThis study offers new insights into the involvement of IFI30 in breast cancer, elucidating its interplay with the PD-L1 axis and immune response dynamics. Our findings suggest that modulation of the IFI30-PD-L1 axis could serve as a promising strategy for regulating T cells infiltration in breast cancer thus treating breast cancer.

Re-analysis of single-cell RNA-seq data reveals the origin and roles of cycling myeloid cells.

Cycling myeloid cells (CMCs) are often detected from various tissues using single-cell RNA sequencing (scRNA-seq) datasets, however, their research value was not noticed before. For the first time, our study preliminarily revealed the origin, differentiation, and roles of CMCs in physiological processes. Particularly, subgroup a of cycling myeloid cells (aCMCs) were conclusively identified as belonging to a specific cell type. In an active state, aCMCs rapidly proliferate during the early stages of an embryonic development. With an individual maturing, most aCMCs differentiate into specialized cells, while a small portion of them enter an inactive or dormant state. Under pathological conditions, aCMCs restore their proliferative and differentiation capacities via activation or revival. The present study has set the stage for future research on CMCs by linking them with progenitors of immune cells, and provided a crucial starting point to understand the origin, differentiation, and roles of CMCs in various physiological and pathological processes, particularly those related to traumatic injury, cancer, and pathogen infection, leading to develop targeted therapies or interventions.

PPARγ attenuates cellular senescence of alveolar macrophages in asthma-COPD overlap

BackgroundAsthma-chronic obstructive pulmonary disease (COPD) overlap (ACO) represents a complex condition characterized by shared clinical and pathophysiological features of asthma and COPD in older individuals. However, the pathophysiology of ACO remains unexplored. We aimed to identify the major inflammatory cells in ACO, examine senescence within these cells, and elucidate the genes responsible for regulating senescence.MethodsBioinformatic analyses were performed to investigate major cell types and cellular senescence signatures in a public single-cell RNA sequencing (scRNA-Seq) dataset derived from the lung tissues of patients with ACO. Similar analyses were carried out in an independent cohort study Immune Mechanisms Severe Asthma (IMSA), which included bulk RNA-Seq and CyTOF data from bronchoalveolar lavage fluid (BALF) samples.ResultsThe analysis of the scRNA-Seq data revealed that monocytes/ macrophages were the predominant cell type in the lung tissues of ACO patients, constituting more than 50% of the cells analyzed. Lung monocytes/macrophages from patients with ACO exhibited a lower prevalence of senescence as defined by lower enrichment scores of SenMayo and expression levels of cellular senescence markers. Intriguingly, analysis of the IMSA dataset showed similar results in patients with severe asthma. They also exhibited a lower prevalence of senescence, particularly in airway CD206 + macrophages, along with increased cytokine expression (e.g., IL-4, IL-13, and IL-22). Further exploration identified alveolar macrophages as a major subtype of monocytes/macrophages driving cellular senescence in ACO. Differentially expressed genes related to oxidation-reduction, cytokines, and growth factors were implicated in regulating senescence in alveolar macrophages. PPARγ (Peroxisome Proliferator-Activated Receptor Gamma) emerged as one of the predominant regulators modulating the senescent signature of alveolar macrophages in ACO.ConclusionThe findings suggest that senescence in macrophages, particularly alveolar macrophages, plays a crucial role in the pathophysiology of ACO. Furthermore, PPARγ may represent a potential therapeutic target for interventions aimed at modulating senescence-associated processes in ACO.Key words ACO, Asthma, COPD, Macrophages, Senescence, PPARγ.

Resident Memory T Cells in the Atherosclerotic Lesion Associate With Reduced Macrophage Content and Increased Lesion Stability.

Tissue resident memory T (TRM) cells are a T-cell subset that resides at the site of prior antigen recognition to protect the body against reoccurring encounters. Besides their protective function, TRM cells have also been implicated in inflammatory disorders. TRM cells are characterized by the expression of CD69 and transcription factors Hobit (homolog of Blimp-1 [B lymphocyte-induced maturation protein 1] in T cells) and Blimp-1. As the majority of T cells in the arterial intima expresses CD69, TRM cells may contribute to the pathogenesis of atherosclerosis as well. Here, we aimed to assess the presence and potential role of TRM cells in atherosclerosis. To identify TRM cells in human atherosclerotic lesions, a single-cell RNA-sequencing data set was interrogated, and T-cell phenotypes were compared with that of integrated predefined TRM cells. The presence and phenotype of TRM in atherosclerotic lesions was corroborated using a mouse model that enabled tracking of Hobit-expressing TRM cells. To explore the function of TRM cells during atherogenesis, RAG1-/- (RAG1 deficient) LDLr-/- (low-density lipoprotein receptor knockout) mice received a bone marrow transplant from HobitKO/CREBlimp-1flox/flox mice, which exhibit abrogated TRM cell formation, whereafter the mice were fed a Western-type diet for 10 weeks. Human atherosclerotic lesions contained T cells that exhibited a TRM cell-associated gene signature. Moreover, a fraction of these T cells clustered together with predefined TRM cells upon integration. The presence of Hobit-expressing TRM cells in the atherosclerotic lesion was confirmed in mice. These lesion-derived TRM cells were characterized by the expression of CD69 and CD49α. Moreover, we demonstrated that this small T-cell subset significantly affects lesion composition, by reducing the amount of intralesional macrophages and increasing collagen content. TRM cells, characterized by the expression of CD69 and CD49α, constitute a minor population in atherosclerotic lesions and are associated with increased lesion stability in a Hobit and Blimp-1 knockout mouse model.

Myeloid Cell-Derived IL-1 Signaling Damps Neuregulin-1 from Fibroblasts to Suppress Colitis-Induced Early Repair of the Intestinal Epithelium.

Neuregulin-1 (Nrg1, gene symbol: Nrg1), a ligand of the ErbB receptor family, promotes intestinal epithelial cell proliferation and repair. However, the dynamics and accurate derivation of Nrg1 expression during colitis remain unclear. By analyzing the public single-cell RNA-sequencing datasets and employing a dextran sulfate sodium (DSS)-induced colitis model, we investigated the cell source of Nrg1 expression and its potential regulator in the process of epithelial healing. Nrg1 was majorly expressed in stem-like fibroblasts arising early in mouse colon after DSS administration, and Nrg1-Erbb3 signaling was identified as a potential mediator of interaction between stem-like fibroblasts and colonic epithelial cells. During the ongoing colitis phase, a significant infiltration of macrophages and neutrophils secreting IL-1β emerged, accompanied by the rise in stem-like fibroblasts that co-expressed Nrg1 and IL-1 receptor 1. By stimulating intestinal or lung fibroblasts with IL-1β in the context of inflammation, we observed a downregulation of Nrg1 expression. Patients with inflammatory bowel disease also exhibited an increase in NRG1+IL1R1+ fibroblasts and an interaction of NRG1-ERBB between IL1R1+ fibroblasts and colonic epithelial cells. This study reveals a novel potential mechanism for mucosal healing after inflammation-induced epithelial injury, in which inflammatory myeloid cell-derived IL-1β suppresses the early regeneration of intestinal tissue by interfering with the secretion of reparative neuregulin-1 by stem-like fibroblasts.

Tertiary lymphoid structures associated with enhanced anti-tumor immunity and favorable prognosis in cervical squamous carcinoma.

Cervical squamous carcinoma (CESC) is the main subtype of cervical cancer. Unfortunately, there are presently no effective treatment options for advanced and recurrent CESC. Tertiary lymphoid structures (TLSs) are clusters of lymphoid cells that resemble secondary lymphoid organs; nevertheless, there is no summary of the clinical importance of TLS in CESC. A large set of transcriptomic and single-cell RNA-sequencing (scRNA-seq) datasets were used to analyze the pattern of TLS and its immuno-correlations in CESC. Additionally, an independent in-house cohort was collected to validate the correlation between TLS and TME features. In the current study, we found that the presence of TLS could predict better prognosis in CESC and was correlated with the activation of immunological signaling pathways and enrichment of immune cell subpopulations. In addition, TLS was associated with reduced proliferation activity in tumor cells, indicating the negative correlation between TLS and the degree of malignancy. Last but not least, in two independent immunotherapy cohorts, tumors with the presence of TLS were more sensitive to immunotherapy. Overall, TLS is related to an inflamed TME and identified immune-hot tumors, which could be an indicator for the identification of immunological features in CESC.

Molecular heterogeneity of quiescent melanocyte stem cells revealed by single-cell RNA-sequencing.

Melanocyte stem cells (McSCs) of the hair follicle are a rare cell population within the skin and are notably underrepresented in whole-skin, single-cell RNA sequencing (scRNA-seq) datasets. Using a cell enrichment strategy to isolate KIT+/CD45- cells from the telogen skin of adult female C57BL/6J mice, we evaluated the transcriptional landscape of quiescent McSCs (qMcSCs) at high resolution. Through this evaluation, we confirmed existing molecular signatures for qMcCS subpopulations (e.g., Kit+, Cd34+/-, Plp1+, Cd274+/-, Thy1+, Cdh3+/-) and identified novel qMcSC subpopulations, including two that differentially regulate their immune privilege status. Within qMcSC subpopulations, we also predicted melanocyte differentiation potential, neural crest potential, and quiescence depth. Taken together, the results demonstrate that the qMcSC population is heterogeneous and future studies focused on investigating changes in qMcSCs should consider changes in subpopulation composition.

Abstract LB338: Targeted deletion of CD244 on monocytes promotes differentiation into anti-tumorigenic macrophages and potentiates PD-L1 blockade in melanoma

Abstract Background: In the myeloid compartment of the tumor microenvironment, CD244 signaling has been implicated in immunosuppressive phenotype of monocytes. However, the precise molecular mechanism and contribution of CD244 to tumor immunity in monocytes/macrophages remains elusive due to the co-existing lymphoid cells expressing CD244. Methods: To directly assess the role of CD244 in tumor-associated macrophages, monocyte-lineage-specific CD244-deficient mice were generated using cre-lox recombination and challenged with B16F10 melanoma. The phenotype and function of tumor-infiltrating macrophages along with antigen-specific CD8 T cells were analyzed by flow cytometry and single cell RNA sequencing data analysis, and the molecular mechanism underlying anti-tumorigenic macrophage differentiation, antigen presentation, phagocytosis was investigated ex vivo. Finally, the clinical feasibility of CD244-negative monocytes as a therapeutic modality in melanoma was confirmed by adoptive transfer experiments. Results: CD244fl/flLysMcre mice demonstrated a significant reduction in tumor volume (61% relative to that of the CD244fl/fl control group) 14 days after tumor implantation. Within tumor mass, CD244fl/flLysMcre mice also showed higher percentages of Ly6Clow macrophages, along with elevated gp100+IFN-γ+ CD8 T cells. Flow cytometry and RNA sequencing data demonstrated that ER stress resulted in increased CD244 expression on monocytes. This, in turn, impeded the generation of anti-tumorigenic Ly6Clow macrophages, phagocytosis and MHC-I antigen presentation by suppressing autophagy pathways. Combining anti-PD-L1 antibody with CD244-/- bone marrow-derived macrophages markedly improved tumor rejection compared to the anti-PD-L1 antibody alone or in combination with wild-type macrophages. Consistent with the murine data, transcriptome analysis of human melanoma tissue single-cell RNA-sequencing dataset revealed close association between CD244 and the inhibition of macrophage maturation and function. Furthermore, the presence of CD244-negative monocytes/macrophages significantly increased patient survival in primary and metastatic tumors. Conclusion: Our study highlights the novel role of CD244 on monocytes/macrophages in restraining anti-tumorigenic macrophage generation and tumor antigen-specific T cell response in melanoma. Importantly, our findings suggest that CD244-deficient macrophages could potentially be used as a therapeutic agent in combination with immune checkpoint inhibitors. Furthermore, CD244 expression in monocyte-lineage cells serve as a prognostic marker in cancer patients. Citation Format: Jeongsoo Kim, Tae-Jin Kim, Sehyun Chae, Hyojeong Ha, Yejin Park, Seon Ah Lim, Hyemin Lee, Younji Sim, Seyoung Oh, Geon Mo Koo, Minkyung Jo, Daehee Hwang, Kyung-Mi Lee. Targeted deletion of CD244 on monocytes promotes differentiation into anti-tumorigenic macrophages and potentiates PD-L1 blockade in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB338.

Abstract SY39-03: Illuminating the tumor immune GPCRome: Novel immunotherapy targets

Abstract Immune checkpoint blockade (ICB) targeting PD-1 and CTLA-4 has revolutionized cancer treatment. However, many cancers do not respond to ICB, prompting the search for additional strategies to achieve durable cancer remission. In this regard, G-protein-coupled receptors (GPCRs) represent the largest family of cell surface receptors involved in signal transduction and are the target of >30% of all approved drugs. GPCRs are the most pharmacologically favorable targets due to their druggability and direct relevance to many key physiological processes and a myriad of human diseases. However, GPCRs have been largely underexploited in oncology, and specifically understudied in cancer immunotherapy. GPCRs are primarily coupled to 4 distinct families of G proteins α subunit (Gαi, Gαs, Gαq, and Gα12), which dictate the signaling events controlled by each GPCR. Recently, we cross-integrated large single-cell RNA-sequencing datasets from CD8+ T cells covering 19 distinct cancer types and identified an enrichment of Gαs-coupled GPCRs on exhausted CD8+ T cells. These include GPCRs activated by inflammatory mediators, protons, neurotransmitters, and metabolites that accumulate in the tumor microenvironment, thereby promoting T cell dysfunction. We also developed transgenic mice expressing a chemogenetic CD8-restricted Gαs-GPCR and showed that a Gαs-PKA signaling axis promotes CD8+ T cell exhaustion and immunotherapy failure. These studies revealed that tumor cells may avoid immune elimination by stimulating Gαs-GPCRs on infiltrating CD8 T cells, thereby exposing novel druggable immune checkpoints that can be exploited therapeutically. We have recently extended these studies and mapped the expression patterns of all human GPCRs in nearly a million-tumor infiltrating immune cells. The emerging tumor-immune GPCR atlas may provide a valuable resource to explore the role of GPCRs in cancer immunology. Ultimately, our study may reveal new and exciting GPCR therapeutic targets to enhance the response to ICB, and help identify potential co-medications that may limit the success of ICB therapies. Citation Format: J. Silvio Gutkind. Illuminating the tumor immune GPCRome: Novel immunotherapy targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY39-03.

Single cell RNA-sequencing reveals the cellular senescence of placental mesenchymal stem/stromal cell in preeclampsia

IntroductionPreeclampsia (PE) is a severe obstetric complication closely associated with placental dysfunction. Placental mesenchymal stem/stromal cells (PMSCs) modulate placental development while PE PMSCs are excessively senescent to disturb placental function. Nevertheless, the senescence mechanism of PE PMSCs remains unclear. MethodsPE-related single-cell RNA sequencing datasets (GSE173193), data of chip detection (GSE99007) and bulk transcriptome RNA sequencing datasets (GSE75010) were extracted from the GEO database. Firstly, the functional enrichment analyses of the differentially expressed genes (DEGs) in PMSCs were conducted. Then, the clusters of PE PMSCs were distinguished according to the expressions of senescence-related genes (SRGs) by consensus clustering analysis. Cell cycle analysis, senescence β-galactosidase, Transwell, and tube formation were conducted. Next, the expressions of the senescence-associated secretory phenotype (SASPs) were displayed. The characteristic genes of PE were screened by the least absolute shrinkage and selection operator analysis. CTSZ was suppressed in PMSCs and the cellular senescence levels were evaluated. ResultsIn this study, The DEGs in PMSCs were closely associated with cellular senescence. The score of SRGs was significantly higher and most of the SASPs were abnormally expressed in the senescent group. Seven characteristic genes of PE were identified, thereinto, CTSZ reduction may accelerate the senescence in PMSCs in vitro. DiscussionCombined with bioinformatic analysis and lab experiments, our study emphatically revealed the abnormal cellular senescence in PE PMSCs, in which CTSZ, one of the PE characteristic genes, regulated the cellular senescence levels in PMSCs. These findings might help to deepen the understanding of the senescence mechanism of PMSCs in PE.

Single-cell RNA sequencing reveals the expansion of circulating tissue-homing B cell subsets in secondary acute dengue viral infection

The roles of antibodies secreted by subsets of B cells in dengue virus (DENV) infection have been extensively studied, yet, the contribution of tissue-homing B cells to antiviral immunity remains unclear. In this study, we performed a comprehensive analysis of B cell subpopulations in peripheral blood samples from DENV-infected patients using single-cell RNA-sequencing (scRNA-seq) datasets and flow cytometry. We showed that plasma cells (PCs) and plasmablasts (PBs) were the predominant B cell populations during the acute phase of secondary natural DENV infection, but not in convalescent phase nor in healthy controls. Interestingly, these cells expressed proliferation, adhesion, and tissue-homing genes, including SELPLG, a homing marker of the skin, the initial infected site of DENV. Flow cytometry analysis confirmed a significant upregulation of cell surface expression of a cutaneous lymphocyte-associated antigen (CLA) encoded by SELPLG in PCs and PBs, compared to naive and memory B cells from the same patients. The analysis of an independent single-cell B-cell receptor sequencing (scBCR-seq) dataset of DENV-infected patients revealed that the peripheral blood PCs and PBs exhibited the highest clonal expansion in secondary DENV infection compared to other B cell subsets. These clonally expanded cells also expressed the highest levels of tissue-homing genes, including SELPLG. In addition, by utilizing a public scRNA-seq dataset of SARS-CoV2 infection, we demonstrated the upregulation of several tissue-homing genes in PCs and PBs. Our study provides evidence for the potential roles of tissue-homing B cell subsets in the context of immune responses against viral infections in humans.

Comprehensive profiling of the human fecal proteome from IBD patients with DIA-MS enables evaluation of disease-relevant proteins.

Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD), is characterized by chronic gastrointestinal inflammation. A high unmet need exists for noninvasive biomarkers in IBD to monitor changes in disease activityand guide treatment decisions. Stool is an easily accessed, disease proximal matrix in IBD, however the composition of the IBD fecal proteome remains poorly characterized. A data-independent acquisition LC-MS/MS approach was used to profile the human fecal proteome in two independent cohorts (Cohort 1: healthy n=5, UC n=5, CD n=5, Cohort 2: healthy n=20, UC n=10, and CD n=10) to identify noninvasive biomarkers reflective of disease activity. 688 human proteins were quantified, with 523 measured in both cohorts. In UC stool 96 proteins were differentially abundant and in CD stool 126 proteins were differentially abundant compared to healthy stool (absolute log2 fold change>1, p-value<0.05). Many of these fecal proteins are associated with infiltrating immune cells and ulceration/rectal bleeding, which are hallmarks of IBD pathobiology. Mapping the identified fecal proteins to a whole blood single-cell RNA sequencing data set revealed the involvement of various immune cell subsets to the IBD fecal proteome. Findings from this study not only confirmed the presence of established fecal biomarkers for IBD, such as calprotectin and lactoferrin, but also revealed new fecal proteins from multiple pathways known to be dysregulated in IBD. These novel proteins could serve as potential noninvasive biomarkers to monitor specific aspects of IBD disease activity which could expedite clinical development of novel therapeutic targets.

High-dimensional deconstruction of ovarian cancer at single-cell precision reveals HEBP2 that reshape the TIME and drive carboplatin resistance

BackgroundSingle-cell sequencing was employed to analyze the tumor immune microenvironment in ovarian cancer (OC) patients, exploring the evolutionary roles of various macrophage subgroups in OC progression and their correlation with fatty acid metabolism-related genes in contributing to drug resistance. MethodsThis study aimed to decipher the mechanisms underlying OC chemoresistance (OC-CR) and carboplatin resistance by integrating and analyzing multiple single-cell RNA sequencing datasets from OC patients. The tumor immune microenvironment in OC-CR patients exhibited notable alterations in cellular interactions and the proportions of different immune cell populations, in contrast to the cohort sensitive to OC chemotherapy. ResultsThe study demonstrates that the fatty acid-associated gene HEBP2 not only accelerates OC progression but also modifies the immune landscape of OC, driving the polarization from M0_TAM to M2_TAM. This shift results in a diminished efficacy of chemotherapy in OC. Furthermore, both in vitro and in vivo experiments underscored HEBP2′s role in boosting the proliferation of OC-resistant cell lines and suppressing apoptosis, thereby facilitating carboplatin resistance. ConclusionIn conclusion, the immune microenvironments of OC-CR significantly differ from those sensitive to chemotherapy, underscoring HEBP2′s role in fostering OC resistance. This establishes HEBP2 as a promising prognostic marker and a novel target for therapeutic strategies against OC resistance.

Deconvolution of synovial myeloid cell subsets across pathotypes and role of COL3A1+ macrophages in rheumatoid arthritis remission.

Monocyte/macrophage (Mo/Mp) is a critical cell population involved in immune modulation of rheumatoid synovitis (RA) across different pathotypes. This study aims to investigate the contribution of Mo/Mp clusters to RA activity, and the biological function of particular subtypes in RA remission. We integrated single-cell RNA sequencing datasets from 4 published and 1 in-house studies using Liger selected by comparison. We estimated the abundance of Mo/Mp subtypes in bulk RNA-seq data from the 81 patients of the Pathobiology of Early Arthritis Cohort (PEAC) using deconvolution analysis. Correlations between Mo/Mp subtypes and RA clinical metrics were assessed. A particular cell type was identified using multicolor immunofluorescence and flow cytometry in vivo and successfully induced from a cell line in vitro. Potential immune modulation function of it was performed using immunohistochemical staining, adhesion assay, and RT-qPCR. We identified 8 Mo/Mp clusters. As a particular subtype among them, COL3A1+ Mp (CD68+, COL3A1+, ACTA2-) enriched in myeloid pathotype and negatively correlated with RA severity metrics in all pathotypes. Flow cytometry and multicolor immunofluorescence evidenced the enrichment and M2-like phenotype of COL3A1+ Mp in the myeloid pathotype. Further assays suggested that COL3A1+ Mp potentially attenuates RA severity via expressing anti-inflammatory cytokines, enhancing Mp adhesion, and forming a physical barrier at the synovial lining. This study reported unexplored associations between different pathologies and myeloid cell subtypes. We also identified a fibroblast-and-M2-like cluster named COL3A1+ Mp, which potentially contributes to synovial immune homeostasis. Targeting the development of COL3A1+ Mp may hold promise for inducing RA remission.