NKT Cells Research Articles

Understanding and Using Animal Models of Hepatotoxicity.

Hepatotoxicity is a critical health hazard, primarily contributing to the increased incidence of deaths globally. The liver is one of the major and extremely vital organs of the human body. Autoimmune diseases, viruses, exposure to toxicants such as carcinogens, and changes in eating habits can all cause liver problems, among other things. Free radical generation, together with raised enzyme levels including SGOT, SGPT, and total bilirubin, are among the pathological changes set off by liver injury. Hepatotoxicity causes changes in cells, such as eosinophilic cytoplasm, nuclear pyknosis, fatty degeneration, too many liver lesions, and hepatic centrilobular necrosis due to lipid peroxidation. Researchers have used animal models to investigate liver diseases and toxicities. Drugs such as azathioprine, alcoholism, paracetamol intoxication, and anti-tuberculosis drugs are some of the most common causes of liver toxicity. These toxins cause calcium ions (Ca2+), reactive oxygen species (ROS), and inflammatory mediators to be released inside cells. This activates immune cells like NK cells, NKT cells, and Kupffer cells. These signaling pathways also play roles in hepatotoxicity. Due to its pathogenesis, no effective drug is currently available for hepatotoxicity due to a lack of understanding related to the signaling factors involved in it. The paper primarily examines different experimental models of hepatotoxicity, including non-invasive and invasive methods, as well as genetic models. As such, these models are crucial tools in advancing our understanding of hepatotoxicity, thus paving the way for new therapeutic interventions.

Diverse immune cell profiles in ASFV-associated lymphopenia

Pathogenic African swine fever virus (ASFV) remains a lethal causative agent in the domestic pig industry, which poses a burden on the swine market and causes substantial socioeconomic losses worldwide. Currently, there are no commercially efficacious vaccines or specific treatments available for ASF prevention and control. Unfortunately, little is known about the swine immune response upon ASFV infection. Here, we investigated the host immune response discrepancy induced by the field moderately virulent strain ASFV HB-2208 among healthy, diseased and asymptomatic pigs. In the peripheral blood of diseased swine, lymphopenia is caused by the massive loss of bystander lymphocytes, such as γδ T cells, B cells and CD4+ T cells. Conversely, ASFV has a strong tropism for the mononuclear phagocyte system (MPS) and partial dendritic cells (DCs), whose antigen-presenting ability is impeded by the downregulation of CD80 and MHC I. However, no significant difference in the number of CD8αhigh T cells was detected, whereas the frequencies of NK cells, NKT cells, and regulatory T cells (Tregs) were significantly increased. Additionally, an in vitro model was established with a coculture of primary pulmonary alveolar macrophages (PAMs) and peripheral blood mononuclear cells (PBMCs), which significantly reduced γδ T cells, B cells and CD4+ T cells and increased Tregs. The differentiated immune response might aid in enhancing the understanding of ASFV pathogenesis in suids and provide insights into the mechanism of ASFV-induced lymphopenia for further studies.

Enhancing COVID-19 Vaccine Efficacy: Dual Adjuvant Strategies with TLR7/8 Agonists and Glycolipids.

The controlled release of immunostimulatory agents represents a promising strategy to enhance vaccine efficacy while minimizing side effects. This study aimed to improve the efficacy of the RBD-Fc-based COVID-19 vaccine through combining of an iNKT cell agonist and a TLR7/8 agonist using covalent conjugation and temporal delivery. We hypothesized that these combinations would yield a more balanced Th1/Th2 immune response. For covalent conjugation, we employed an uncleavable linker and a self-immolative disulfide linker to conjugate α-galactosylceramide (αGC) to imidazoquinoline (IMDQ). The αGC-SS-IMDQ-Ac conjugate, designed with a prodrug strategy for controlled TLR7/8 agonist release, elicited a higher IFN-γ/IL-4 T cell response ratio than individual adjuvants or their admixture. In the temporal delivery approach, administering IMDQ followed by αGC after 2 h resulted in the highest IgG2a/IgG1 ratio, significantly surpassing other groups. A 6 h delay between glycolipid and IMDQ injections yielded balanced IgG responses, enhancing IgG, IgG1, and IgG2a levels synergistically.

Diverse NKT cells regulate early inflammation and neurological outcomes after cardiac arrest and resuscitation.

Neurological injury drives most deaths and morbidity among patients hospitalized for out-of-hospital cardiac arrest (OHCA). Despite its clinical importance, there are no effective pharmacological therapies targeting post-cardiac arrest (CA) neurological injury. Here, we analyzed circulating immune cells from a large cohort of patients with OHCA, finding that lymphopenia independently associated with poor neurological outcomes. Single-cell RNA sequencing of immune cells showed that T cells with features of both innate T cells and natural killer (NK) cells were increased in patients with favorable neurological outcomes. We more specifically identified an early increase in circulating diverse NKT (dNKT) cells in a separate cohort of patients with OHCA who had good neurological outcomes. These cells harbored a diverse T cell receptor repertoire but were consistently specific for sulfatide antigen. In mice, we found that sulfatide-specific dNKT cells trafficked to the brain after CA and resuscitation. In the brains of mice lacking NKT cells (Cd1d-/-), we observed increased inflammatory chemokine and cytokine expression and accumulation of macrophages when compared with wild-type mice. Cd1d-/- mice also had increased neuronal injury, neurological dysfunction, and worse mortality after CA. To therapeutically enhance dNKT cell activity, we treated mice with sulfatide lipid after CA, showing that it improved neurological function. Together, these data show that sulfatide-specific dNKT cells are associated with good neurological outcomes after clinical OHCA and are neuroprotective in mice after CA. Strategies to enhance the number or function of dNKT cells may thus represent a treatment approach for CA.

Analysis of the Blood Levels of NK and NKT Cells in Patients with Severe SARS-CoV-2 Infection.

Clinical features of SARS-CoV-2 infection vary, ranging from asymptomatic cases to pneumonia, and other serious complications. Some populations have been observed to be at higher risk for severe disease and death compared to other ethnical groups. To evaluate two parameters of the innate immune system, that play a significant role in viral immunity. In samples of peripheral blood from sixteen patients with severe COVID-19, ten with asymptomatic to mild illness, and fifteen healthy subjects, the percentage of NK and NKT cells, the expression of different NK cell receptors and the blood levels of pro-inflammatory cytokines were tested. We observed that patients with severe COVID-19 showed significantly lower frequencies of both CD56dim and CD56bright NK cells compared to patients with mild illness or healthy controls. Furthermore, patients with severe manifestation of COVID-19 exhibited an aberrant expression of the natural cytotoxicity receptors NKp30, NKp44 and NKp46. Similarly, NK cells from these patients showed statistically significant differences in the expression of various killer immunoglobulin-like receptors (KIRs) in the two main cell subsets (CD56bright, CD56dim) compared to controls or patients with mild disease. Moreover, patients with severe illness displayed decreased frequency of NKT cells (defined as CD3+CD56+) and elevated blood levels of the cytokines IL-6 and IL-8. This study suggests that the abnormal features of NK and NKT cells observed in patients with severe SARS-CoV-2 infection may play an important role in the outcome of this infectious disease in various population groups.

Development of a deep learning-based 1D convolutional neural network model for cross-species natural killer T cell identification using peripheral blood mononuclear cell single-cell RNA sequencing data

Background and Aim: Natural killer T (NKT) cells exhibit the traits of both T and NK cells. Although their roles have been well studied in humans and mice, limited knowledge is available regarding their roles in dogs and pigs, which serve as models for human immunology. Single-cell RNA sequencing (scRNA-Seq) can elucidate NKT cell functions. However, identifying cells in mixed populations, like peripheral blood mononuclear cells (PBMCs) is challenging using this technique. This study presented the application of one-dimensional convolutional neural network (1DCNN) for the identification of NKT cells within scRNA-seq data derived from PBMCs. Materials and Methods: We used human scRNA-Seq data to train a 1DCNN model for cross-species identification of NKT cells in canine and porcine PBMC datasets. K-means clustering was used to isolate human NKT cells for training the 1DCNN model. The trained model predicted NKT cell subpopulations in PBMCs from all species. We performed Differential gene expression and Gene Ontology (GO) enrichment analyses to assess shared gene functions across species. Results: We successfully trained the 1DCNN model on human scRNA-Seq data, achieving 99.3% accuracy, and successfully identified NKT cell candidates in human, canine, and porcine PBMC datasets using the model. Across species, these NKT cells shared 344 genes with significantly elevated expression (FDR ≤ 0.001). GO term enrichment analyses confirmed the association of these genes with the immunoactivity of NKT cells. Conclusion: This study developed a 1DCNN model for cross-species NKT cell identification and identified conserved immune function genes. The approach has broad implications for identifying other cell types in comparative immunology, and future studies are needed to validate these findings. Keywords: 1D convolutional neural network, K-means clustering, natural killer T cell, peripheral blood mononuclear cells, single-cell RNA sequencing.

IFN-γ-dependent regulation of intestinal epithelial homeostasis by NKT cells

Intestinal homeostasis is maintained through the combined functions of epithelial and immune cells that collaborate to preserve the integrity of the intestinal barrier. However, the mechanisms by which immune cell populations regulate intestinal epithelial cell (IEC) homeostasis remain unclear. Here, we use a multi-omics approach to study the immune-epithelial crosstalk and identify CD1d-restricted natural killer T (NKT) cells as key regulators of IEC biology. We find that NKT cells are abundant in the proximal small intestine and show hallmarks of activation at steady state. Subsequently, NKT cells regulate the survival and the transcriptional and cellular composition landscapes of IECs in intestinal organoids, through interferon-γ (IFN-γ) and interleukin-4 secretion. Invivo, lack of NKT cells results in an increase in IEC turnover, while NKT cell activation leads to IFN-γ-dependent epithelial apoptosis. Our findings propose NKT cells as potent producers of cytokines that contribute to the regulation of IEC homeostasis.

Effects of Psychological Stress on Multiple Sclerosis via HPA Axis-mediated Modulation of Natural Killer T Cell Activity.

The involvement of psychological stress and Natural Killer T (NKT) cells in the pathophysiology of multiple sclerosis has been identified in the progression of this disease. Psychological stress can impact disease occurrence, relapse, and severity through its effects on the Hypothalamic- Pituitary-Adrenal (HPA) axis and immune responses. NKT cells are believed to play a pivotal role in the pathogenesis of multiple sclerosis, with recent evidence suggesting their distinct functional alterations following activation of the HPA axis under conditions of psychological stress. This review summarizes the associations between psychological stress, NKT cells, and multiple sclerosis while discussing the potential mechanism for how NKT cells mediate the effects of psychological stress on this disease.

Analysis of the Potential Link Between Dermatomyositis and Cancer.

Dermatomyositis (DM) is an inflammatory muscle disease that increases the risk of cancer, although the precise connection is not fully understood. The aim of this study was to investigate the mechanisms linking DM to cancer and identify potential therapeutic targets. We conducted differential gene expression analysis on the GSE128470 dataset and employed WGCNA to pinpoint key genes related to DM. Central genes were identified with the LASSO and SVM-RFE methods. The expression levels and diagnostic relevance of these genes were confirmed via the GSE1551 dataset. Immune cell infiltration was analyzed in relation to central genes, and RT‒qPCR was utilized to evaluate the expression of key genes across various cancers. In total, differentially expressed genes (DEGs), involved mainly in innate immunity, cytokine responses, and autoimmune diseases, were identified. In the WGCNA, 399 significant genes related to DM were identified, with central genes including MIF, C1QA, and CDKN1A. Immune infiltration analysis revealed diverse immune cell populations in DM patients, with significant correlations between central genes and these immune cells. MIF levels were notably elevated in various tumors and correlated with the prognosis of specific cancers. Furthermore, MIF was negatively associated with most immune cells but positively correlated with CD4+ Th1 cells, NKT cells, and MDSCs. Factors such as immune regulatory elements, TMB, and MSI indicated that MIF may affect immunotherapy outcomes. The increased expression of MIF mRNA was confirmed via RT‒qPCR. The findings demonstrate that MIF, C1QA, and CDKN1A are differentially expressed in DM patients, with MIF showing significant alterations in DM patients with cancer. MIF may serve as a crucial prognostic biomarker and therapeutic target for various cancers, playing a pivotal role in linking DM to cancer through the modulation of CD4+ Th1 cells, NKT cells, and MDSCs.

Associations between blood markers of glucose metabolism and characteristics of circulating lymphocytes

AimsThe pathophysiology of diabetes is not fully understood; recent research indicates close relations with immunological alterations. Therefore, the aim of this study was to investigate the associations between markers of glucose metabolism and characteristics of blood lymphocytes in a population-based cohort. MethodsThe analysis was based on data from 219 non-diabetic participants of the MEGA study in Augsburg, Germany, who were recruited between 2018 and 2021. The majority of participants were examined two different times with a time lag of 9 months. Fasting venous blood samples were taken and oral glucose tolerance tests (OGTT) were performed at both visits. Immune cells were analyzed from fresh blood using flow cytometry. The associations between fasting blood glucose levels, glucose levels at 2 hours after oral glucose bolus and glycated hemoglobin (HbA1c) concentrations and the quantity of different lymphocyte subsets were analyzed using linear mixed regression models with random intercept. P values were FDR-adjusted. ResultsHbA1c was negatively associated with the marginal zone B cells (IgD+ CD27+ B cells). Fasting glucose was positively associated with natural killer (NK) cells and 2-hour OGTT glucose was positively associated with NKT cells. Finally, HbA1c showed significantly negative associations with the CD57-PD1-NKT cell subset. ConclusionMarkers of glucose metabolism showed significant associations with B cell, NK cell and NKT cell subsets, which clearly indicates a relation between glucose metabolism and the adaptive immune system.

Clinical Manifestations, Prognostic Factors, and Outcomes of Extranodal Natural Killer T-Cell Lymphoma: A Single-Center Experience in Thailand

Background/Objectives: The primary objective of this study was to investigate clinical manifestations, time to diagnosis, and number of biopsies in patients with extranodal natural killer T-cell lymphoma (ENKTL). The secondary objectives were to determine response rates, survival outcomes, prognostic factor for overall survival (OS), and validation of the Prognostic Index of Natural Killer Lymphoma (PINK), Ann Arbor staging system (AASS), and the CA system. Methods: This retrospective study included data pertaining to patients with newly diagnosed ENKTL in Chiang-Mai University Hospital from 2004 to 2020. Comparisons between the areas under the receiver operating characteristic curve (AUC) of prognostic models (PINK, AASS, and CA system) were made. Results: Sixty patients were enrolled (n = 60) with a mean age of 49.1 ± 13.4 years. The most frequent symptom of ENKTL was nasal obstruction (66%). The median time to diagnosis was 22 days (ranging from 3 to 84 days), with 36.7% requiring more than one biopsy for diagnosis. Most patients presented with limited stage disease (75%). The median OS was 49 months. Factors associated with increased mortality were advanced stage, bone marrow involvement, gastrointestinal tract involvement, and receiving chemotherapy. Following prognostic model validation, the CA system model scored the highest level of accuracy (AUC 0.61), followed by AASS (AUC 0.58) and PINK (AUC 0.54). Conclusions: Patients with ENKTL commonly presented with nasal obstruction, with 36.7% requiring more than one biopsy for diagnosis. An advanced stage, bone marrow involvement, or gastrointestinal tract involvement were associated with poor OS. The CA system model has the highest level of accuracy for prognostic determination.

IFITM1 aggravates ConA-Induced autoimmune hepatitis by promoting NKT cell activation through increased AMPK-Dependent mitochondrial function

Although interferon-induced transmembrane 1 (IFITM1) is known for its crucial role in antiviral immunity, its involvement in autoimmune hepatitis (AIH) remains largely unexplored. In this study, we observed that IFITM1 expression is markedly upregulated in a Concanavalin A (ConA)-induced AIH model, with particularly high and markedly elevated expression in natural killer T (NKT) cells. To further understand the role of IFITM1, we examined the responses of IFITM1−/− mice in a model of ConA-induced liver injury. In comparison to wild-type mice, IFITM1−/− mice exhibited reduced sensitivity in this model, as evidenced by significantly ameliorated necrosis areas, lower serum aminotransferase levels, a reduced number of intrahepatic NKT cells, and decreased expression of inflammatory factors, such as IL-1β, IL-6, IFN-γ and TNF-α. Notably, by using IFITM1-GFP mice and IFITM1−/− mice, we demonstrated that IFITM1 expression in NKT cells is crucial for their proliferation, proinflammatory cytokine production, and cytotoxic functions. Furthermore, analysis of single-cell RNA sequencingdata revealed that IFITM1 is essential for mitochondrial function, which is mediated by the AMP-activated protein kinase (AMPK) pathway. We also validated the importance of IFITM1 for the AMPK pathway and mitochondrial ATP synthesis in vivo. Together, our findings elucidate that IFITM1 could regulate NKT cell activation and survival by promoting mitochondrial function during AIH.

Osteopontin neutralization increases vitamin D receptors on NKT cells and ameliorates liver fibrosis by promoting their activity.

Vitamin D has an immunomodulatory property influencing the activity of NKT cells. We aimed to study the impact of osteopontin (OPN), a key driver of fibrosis, on NKT cells' vitamin D receptor (VDR) and activity alterations. Liver fibrosis was induced in BALB/C mice with carbon-tetrachloride (CCl4) for 8weeks with either vitamin D [100ng/kg] or InVivoMAb anti-mouse OPN [100μg/kg] 2X/week started at week-4 of CCl4. The liver injury profile of serum ALT, AST, and inflammatory cytokines were evaluated. Histopathological findings were assessed via H&E staining and Sirius-Red staining. Fibrotic genes of αSMA, CREBP, and collagen III were assessed using RT-PCR. Fast blood sugar, insulin, liver cholesterol, and triglyceride were evaluated. Liver tissue-resident (tr)-NKT cells were obtained for VDR expressions, molecular pathways of p-STAT1 and P-STAT-5, and activation markers of CD107a and NKp46 using flow cytometry. Following vitamin D treatment, H&E staining revealed reduced microvascular and macrovascular steatosis, while Sirius-Red staining showed less fibrosis accumulation in liver fibrosis mice than in untreated counterparts. Results were associated with a significant decrease in serum cytokines of IL-β/IL-6/IL-4/OPN/TNF-α and serum AST and ALT by 2-fold and 3-fold, respectively. Fibrotic markers showed an average 1.3-fold decrease in αSMA, CREB, and Col-III in liver fibrosis mice following vitamin D treatment. Quantitated liver cholesterol and triglycerides, serum insulin, and fasting blood sugar ameliorated their levels following vitamin D treatment in liver fibrosis mice. OPN-neutralizing antibody over-expressed VDR on trNKT cells and increased CD107a and NKp46 activities of 3.1 and 3.5 folds, respectively, associated with increasing in p-STAT1 and p-STAT5 phosphorylation. These results were accompanied with a decrease in hepatic-stellate-cell activation markers of αSMA, Col-III, and desmin. VDR expressions affect trNKT cells activity and could modulate progressions of liver fibrosis. Using an OPN-neutralizing antibody exhibited an antifibrotic effect by alleviating the liver injury profile through NKT cells. It is also suggested as an immunomodulatory target of liver fibrosis.

Large granular lymphocyte leukemia: a clonal disorder with autoimmune manifestations.

Large granular lymphocyte (LGL) leukemia is a rare lymphoproliferative disorder characterized by an expansion of clonal T or natural killer lymphocytes. Neutropenia-related infections and anemia represent the main manifestations. LGL leukemia is frequently associated with autoimmune disorders such as rheumatoid arthritis, Sjögren's syndrome, autoimmune endocrinopathies, vasculitis, or autoimmune cytopenia. Recent advances in the phenotypic and molecular characterization of LGL clones have underscored the pivotal role of a chronic antigenic stimulation and a dysregulation of the Jak/STAT signaling pathway in the pathophysiology linking leukemic-cell expansion and autoimmunity. In more than half of patients, there is a somatic STAT3 mutation. The disease is characterized by an indolent course, but approximately half of all patients will eventually require therapy. The first-line treatment for LGL leukemia is historically based on immunosuppressive agents (methotrexate, cyclophosphamide, or cyclosporine). However, cytokines blocking molecules or Jak/STAT inhibitors represent a new conceptual therapeutic approach for LGL leukemia. In this review, we present an overview of the spectrum of LGL proliferations, potential links between LGL expansion and autoimmunity, and therapeutic approaches.

Relationship between NKT cell phenotype and liver fibrosis degree in patients with chronic hepatitis C before and after treatment

Relationship between NKT cell phenotype and liver fibrosis degree in patients with chronic hepatitis C before and after treatment

Selective regulation of IFN-γ and IL-4 co-producing unconventional T cells by purinergic signaling.

Unconventional T cells, including mucosal-associated invariant T (MAIT), natural killer T (NKT), and gamma-delta T (γδT) cells, comprise distinct T-bet+, IFN-γ+ and RORγt+, IL-17+ subsets which play differential roles in health and disease. NKT1 cells are susceptible to ARTC2-mediated P2X7 receptor (P2RX7) activation, but the effects on other unconventional T-cell types are unknown. Here, we show that MAIT, γδT, and NKT cells express P2RX7 and are sensitive to P2RX7-mediated cell death. Mouse peripheral T-bet+ MAIT1, γδT1, and NKT1 cells, especially in liver, co-express ARTC2 and P2RX7. These markers could be further upregulated upon exposure to retinoic acid. Blocking ARTC2 or inhibiting P2RX7 protected MAIT1, γδT1, and NKT1 cells from cell death, enhanced their survival in vivo, and increased the number of IFN-γ-secreting cells without affecting IL-17 production. Importantly, this revealed the existence of IFN-γ and IL-4 co-producing unconventional T-cell populations normally lost upon isolation due to ARTC2/P2RX7-induced death. Administering extracellular NAD in vivo activated this pathway, depleting P2RX7-sensitive unconventional T cells. Our study reveals ARTC2/P2RX7 as a common regulatory axis modulating the unconventional T-cell compartment, affecting the viability of IFN-γ- and IL-4-producing T cells, offering important insights to facilitate future studies into how these cells can be regulated in health and disease.

CD2 expressing innate lymphoid and T cells are critical effectors of immunopathogenesis in hidradenitis suppurativa

Hidradenitis suppurativa (HS) is a chronic, debilitating inflammatory skin disease with a poorly understood immunopathogenesis. Here, we report that HS lesional skin is characterized by the expansion of innate lymphocytes and T cells expressing CD2, an essential activation receptor and adhesion molecule. Lymphocytes expressing elevated CD2 predominated with unique spatial distribution throughout the epidermis and hypodermis in the HS lesion. CD2+ cells were mainly innate lymphocytes expressing the NK cell marker, CD56, and CD4+ T cells. Importantly, these CD2+ cells interacted with CD58 (LFA3) expressing epidermal keratinocytes and fibroblasts in the hypodermis. Granzyme Abright NKT cells (CD2+CD3+CD56bright) clustered with α-SMA expressing fibroblasts juxtaposed to epithelialized tunnels and fibrotic regions of the hypodermis. Whereas NK cells (CD2+CD56dim) were perforin+, granzymes A+ and B+, and enriched adjacent to hyperplastic follicular epidermis and tunnels of HS showing presence of apoptotic cells. The cytokines IL-12, IL-15, and IL-18, which enhance NK cell maturation and function were significantly elevated in HS. Ex vivo HS skin explant cultures treated with CD2:CD58 interaction-blocking anti-CD2 monoclonal antibody attenuated secretion of inflammatory cytokines/chemokines and suppressed inflammatory gene signature. Additionally, CD2:CD58 blockade altered miRNAs involved in NK/NKT differentiation and/or function. In summary, we show that a cellular network of heterogenous NKT and NK cell populations drives inflammation and is critical in the pathobiology of HS, including tunnel formation and fibrosis. Finally, CD2 blockade is a viable immunotherapeutic approach for the effective management of HS.

Identification of Functional Immune Biomarkers in Breast Cancer Patients.

Cancer immunotherapy has emerged as an effective, personalized treatment for certain patients, particularly for those with hematological malignancies. However, its efficacy in breast cancer has been marginal-perhaps due to cold, immune-excluded, or immune-desert tumors. Natural killer T (NKT) cells play a critical role in cancer immune surveillance and are reduced in cancer patients. Thus, we hypothesized that NKT cells could serve as a surrogate marker for immune function. In order to assess which breast cancer patients would likely benefit from immune cell-based therapies, we have developed a quantitative method to rapidly assess NKT function using stimulation with artificial antigen presenting cells followed by quantitative real-time PCR for IFN-γ. We observed a significant reduction in the percentage of circulating NKT cells in breast cancer patients, compared to healthy donors; however, the majority of patients had functional NKT cells. When we compared BC patients with highly functional NKT cells, as indicated by high IFN-γ induction, to those with little to no induction, following stimulation of NKT cells, there was no significant difference in NKT cell number between the groups, suggesting functional loss has more impact than physical loss of this subpopulation of T cells. In addition, we assessed the percentage of tumor-infiltrating lymphocytes and PD-L1 expression within the tumor microenvironment in the low and high responders. Further characterization of immune gene signatures in these groups identified a concomitant decrease in the induction of TNFα, LAG3, and LIGHT in the low responders. We next investigated the mechanisms by which breast cancers suppress NKT-mediated anti-tumor immune responses. We found that breast cancers secrete immunosuppressive lipids, and treatment with commonly prescribed medications that modulate lipid metabolism, can reduce tumor growth and restore NKT cell responses.

Single-cell data revealed the regulatory mechanism of TNK cell heterogeneity in liver metastasis from gastric cancer

AimThe present work set out to classify cell subpopulations related to liver metastasis from gastric cancer (GC) and the mechanisms of their interactions with other immune cell subpopulations.BackgroundGC is characterized by a high degree of heterogeneity and liver metastasis. Exploring the mechanism of liver metastasis of GC from the perspective of heterogeneity of the tumor microenvironment (TME) might help improve the efficacy of GC treatment.ObjectiveBased on the cellular subpopulation characteristics of GC with liver metastasis, the regulatory mechanisms contributing to GC progression were analyzed, with special focuses on the roles of signaling pathways, transcription factors (TFs) and ligand–receptor pairs.MethodsThe GSE163558 dataset was downloaded from the Gene Expression Omnibus (GEO) database to collect single-cell transcriptomic data of GC patients and their metastasis groups for cell clustering and relevant analyses. Differentially expressed genes (DEGs) in the GC and GC liver metastasis groups were screened and subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. SCENIC analysis was used to mine TFs that affected cellular subpopulations during liver metastasis from GC. The relative expression levels of TFs in GC were determined using qRT-PCR. Transwell and wound healing assays were utilized to verify the regulation of the TFs on the migration and invasion of GC cells. Interaction network between the cellular subpopulations was developed applying CellChat.ResultsSingle-cell clustering was performed to group six major cell subpopulations, namely, Myeloid cells, B cells, Mast cells, Epithelial cells, Fibroblasts, and TNK cells, among which the number of TNK cells was significantly increased in the GC liver metastasis group. Differentially enriched pathways of TNK cells between GC and GC liver metastasis groups mainly included IL-17 and Pi3k–Akt signaling pathways. TNK cell subsets could be further categorized into CD8 T cells, Exhausted T cells, NK cells, NKT cells, and Treg cells, with the GC liver metastasis group showing significantly more CD8 T cells and NKT cells. FOS and JUNB were the TFs of TNK cell marker genes that contributed to liver metastasis from GC and the invasion and migration of GC cell lines. Significant differences in immune cell communication ligand–receptor pairs existed between the GC and GC liver metastasis groups.ConclusionThis study revealed the critical role of TNK cell subsets in GC with liver metastasis applying single-cell transcriptomics analysis. The findings provided an important theoretical basis for developing novel therapies to inhibit liver metastasis from GC.

IMMU-24. GLIOBLASTOMA ENRICHED GLYCOSPHINGOLIPIDS MODULATE THE FUNCTION OF HUMAN INKT CELLS

Abstract Glioblastoma is an aggressive primary brain tumor highly resistant to currently available immunotherapies. A deeper understanding of its underlying immunoregulatory mechanisms is paramount to developing future immunotherapeutic treatments. Invariant natural killer T (iNKT) cells are unconventional T cells that recognize lipid antigens presented by an MHC-like molecule called CD1d. Although iNKT cells have been shown to regulate tumor immunity in other cancer types, their role in glioblastoma is not well characterized. Given the lipid-rich nature of the brain and the unique metabolic activity of glioblastoma cells, we hypothesized that interactions between glioblastoma and iNKT cells through glioblastoma-produced lipids could contribute to the immunosuppressive nature of the disease. We report multiple lipid species enriched in aggressive human glioblastoma stem-like cell (GSC) lines that activate human iNKT cells and modulate their functions. Lipidomic LC-MS analysis of GSCs, low-grade glioma stem-like cell lines, and normal human astrocytes revealed various glycosphingolipid species, including sulfatides, highly enriched in the GSCs. Multiple enriched sulfatide species, when presented by CD1d, were recognized by and activated iNKT cells in a dose-dependent manner. Additionally, cytokine analysis of stimulated human PBMC-derived iNKT cells demonstrated that the enriched sulfatides did not induce Th1 cytokine production, but rather many non-Th1 cytokines that potentially counteract Th1-type immune responses. This modulation of iNKT cell function by glioblastoma-enriched glycosphingolipids may contribute to the immunosuppression of glioblastoma and could highlight sulfatide production as a potential therapeutic target for glioblastoma treatment.