Phenotypic Characteristics Of Hepatic γδT17 Cells And Their Role In Defense Against Listeria Monocytogenes Infection

The liver serves not only as a metabolic and detoxification organ,but also as a crucial component of the immune system.It possesses a robust innate immune response and is abundant in innate immune cells such as macrophages,NK cells,NKT cells,etc.,which effectively and rapidly defend against invading microorganisms and exhibit antitumor functions.Research has revealed the presence of diverse tissue-resident lymphocytes in the liver including innate lymphocytes(such as NK cells or ILC1,ILC2,and ILC3),NKT cells,and tissue-resident memory T cells(such as CD8+TRM cells).These liver-resident lymphocytes act as sentinels by participating in immune surveillance and defense mechanisms against infectious and non-infectious injuries while maintaining liver homeostasis.In pathological conditions,these resident lymphocytes play distinct roles in various liver diseases.A comprehensive understanding of the characteristics and functions of liver-resident lymphocytes will provide both theoretical foundations for further research on hepatic diseases along with novel therapeutic strategies.Notably,γδ T cells have been found to be relatively abundant within the liver exhibiting tissue-residency features primarily characterized by IL-17A secretion.As an exceptional subset of innate T lymphocytes that recognize antigens independently from MHC molecules without requiring antigen-presenting cell processing or presentation capabilities;they contribute to rapid immune responses garnering increasing attention.However,compared to other types of resident lymphocytes within the liver there remains a lack of systematic comprehension regarding gene expression characteristics alongside resident features specifically pertaining to hepatic γδT17 cell involvement in different forms of hepatic disease.Listeria monocytogenes is an intracellular foodborne pathogen capable of breaching the intestinal barrier,entering the bloodstream via lymph nodes,and causing gastroenteritis and bacteremia.It can also disseminate to the liver and spleen,cross the blood-brain barrier and placental barrier,leading to meningitis and fetal death.Although the incidence of Listeria monocytogenes infection is low,its mortality rate remains high.Therefore,it is crucial to comprehensively investigate the immune defense mechanisms,immune surveillance,and immune clearance responses against Listeria monocytogenes by the immune system.γδT cells are positioned at the forefront of immune defense as they promptly recognize various antigens expressed during infections,inflammation or tumors.They play a pivotal role in immune surveillance while maintaining tissue homeostasis.Recent studies have revealed that γδ T cells(particularly γδT17 cells)contribute to resistance against Listeria monocytogenes infection in intestinal tissues and establish memory γδ T cell populations residing in mesenteric lymph nodes.Upon rechallenge with secondary infection,these memory cells exhibit enhanced clearance capabilities.IL-17A produced by γδ T cells has been reported to play a critical role in clearing Listeria monocytogenes infection in both liver and testis tissues.However,there still exists limited understanding regarding tissueresident characteristics of hepatic γδT17 cells involved in defending against Listeria monocytogenes infection within liver tissues as well as phenotypic properties and dynamic changes exhibited by different subsets of γδT cells during resistance against this pathogenic organism’s invasion.The rapid accumulation process of hepatic γδT17 cells at sites of infection along with their early-stage involvement remain incompletely elucidated.This study extensively investigates the distinct gene expression properties,tissueresident characteristics,and functions of hepatic γδT17 cells in defense against Listeria monocytogenes infection.The main research content and results are as follows:1.scRNA-seq revealed the gene expression profiles of hepatic γδT17 cellsIn this study,10 × Genomics single-cell RNA sequencing(scRNA-seq)was used to investigate the gene expression profiles of hepatic γδT17 cells.Principal component analysis(PCA),k-means unsupervised clustering analysis and t-distribution random neighbor embedding(t-SNE)were utilized for dimensionality reduction analysis and data visualization.The differentially expressed genes(DEGs)in γδ T cells from liver,thymus,lymph nodes,spleen,as well as different subsets of hepatic γδ T cells were analyzed.The gene expression characteristics of hepatic Il17a+γδ T cell subset were compared with Ifng+γδ T cell subset as a control.Gene Onotology(GO)enrichment analysis and KEGG pathway analysis were performed to elucidate the biological functions of γδT17 cells.The following results were obtained:scRNA-seq analysis identified six subpopulations within hepatic γδT cells,among which Cluster 6 represented the γδT17 population.Compared to other liver-derivedy δT subpopulations and those derived from thymus,lymph nodes,and spleen;hepaticy δT17cells displayed unique gene expression patterns characterized by differential cytokine/effector molecule genes’ expressions along with transcriptional regulationrelated genes and cytokine receptor-encoding genes’ expressions when compared toy δTlsubpopulation’s profile.This reflects distinctive gene expression features specific to hepaticy δ T17cell subset." 1.2 Hepaticy δ T17cells may contribute towards immunosurveillance mechanisms.GO enrichment analysis indicated that this particular subgroup was enriched with genes associated with positive regulation of immune response and lymphocyte activation.1.1 Hepatic γδT17 cells exhibited unique gene expression characteristicsThe proportion of γδ T cells in the liver was higher than that in the spleen,thymus and inguinal lymph node and was dominated by the γδT17 subpopulation.scRNA-seq analysis identified six subpopulations within hepatic γδT cells,among which Cluster 6 represented the γδT17 population.Compared to other liver-derived γδ T subpopulations and γδT17 cells derived from thymus,lymph nodes and spleen,hepatic γδT17 cells displayed unique gene expression patterns.Compared to the γδT1 subpopulation,hepatic γδT17 cells showed significant differences in the expression of cytokine/effector molecule genes,transcriptional regulation-related genes,and cytokine receptor-encoding genes.This reflects distinctive gene expression features specific to hepatic γδT17cell subset.1.2 Hepatic γδT17 cells may play a crucial role in immunosurveillanceGO enrichment analysis revealed that the hepatic γδT17 cell subpopulation exhibited specific enrichment of genes associated with positive regulation of immune response,lymphocyte activation and response to stimulation.KEGG pathway analysis suggested that the hepatic γδT17 cell subpopulation might significantly contribute to inflammatory bowel disease,amoeba-induced infectious diseases,viral infections and tumors.In summary,this study identified unique gene expression characteristics of hepaticγδT17 cells distinct from other subpopulations and highlighted their potential importance in immune surveillance.2.Phenotypic characteristics and tissue-resident properties of hepatic γδT17 cellsPhenotypic characteristics and tissue-resident properties of hepatic γδT17 cells were investigated in this study by analyzing the expression of relevant molecules and establishing a parabiosis model.To explore and validate the phenotypic characteristics of hepatic γδT17 cells,we employed scRNA-seq to analyze Cd44 and Cd27 expression in hepatic γδ T cells while using FACS to detect CD44,CD27,Vγ chains expression levels as well as IL-17A and IFN-γ secretion levels.Additionally,to verify the residency of hepatic γδT17 cells and Vγ4+T and Vγ6+T cells,we utilized scRNA-seq to examine tissue residency-related gene expression levels of γδT17 cells in liver,lymph node,spleen and thymus followed by establishment of a parabiosis model.The following results were obtained through these approaches:2.1 The phenotype of hepatic γδT17cells was characterized by CD44hiCD27profileCD44 and CD27 can be used to distinguish hepatic γδT17 and γδT1 cell subsets.The liver demonstrated an abundance of IL-17A-producing CD44hiCD27-γδ T cells followed by IFN-γ-producing CD44+CD27+γδT cells.2.2 Hepatic CD44hiCD27-γδT cells exhibited heterogeneityHepatic CD44hiCD27-γδ T cells exhibited heterogeneity,encompassing both Vγδ+and Vγδ+subsets,with Vγδ+T cells accounted for approximately 67.27%±4.82%and Vγδ+T cells accounted for approximately 25.57%±4.82%.2.3 Hepatic CD44hiCD27-γδ T cells resided in the liverCompared to other tissue-derived γδT17 cells and hepatic γδT1 cells,hepaticγδT17 cells displayed higher gene expression levels of molecules associated with hepatic chemotaxis and adhesion,while exhibiting lower gene expression levels of molecules related to lymphocyte homing and egress.The parabiosis model further confirmed the tissue-resident properties of hepatic CD44hiCD27-γδT cells.Compared with Vγ4+T cells,Vγ6+T cells had more obvious retention characteristics.In summary,this study validated the single-cell sequencing-based gene expression profiles of hepatic γδT17 cells,identifying their phenotype as CD44hiCD27-.HepaticγδT17 cells were heterogeneous and resided in liver.3.Liver-resident γδT17 cells played a crucial role in defense against Listeria monocytogenes infectionTo investigate the function of liver-resident γδT17 cells during Listeria monocytogenes(Lm)infection,we examined dynamic changes in hepatic CD44hiCD27-γδT cells upon infection as well as their interactions with macrophages.We established an Lm infection model using TCRδ-/-mice by intravenous injection of Lm-RFP(DP-L5538).Intravital imaging experiments were conducted to observe dynamic changes and movement characteristics of γδT cells along with their interaction with macrophages in C57BL/6-TrdctmlMal/J mice.Using FACS analysis,we assessed proportions,absolute numbers,proliferation level,and cytokine secretion levels of hepatic γδT cells,CD44hiCD27-γδT cell subset and macrophages.Furthermore,adoptive transfer experiments were performed to elucidate the role played by liverresident CD44hiCD27-γδT cells in defense against Lm infection.We investigated the source of CD44hiCD27-γδ T cells at the infected site and the role of hepatic macrophages in recruiting liver-resident CD44hiCD27-γδT cells using a parabiosis model and transwell assay.The following results were obtained from these experiments:3.1 Hepatic γδ T cells played a crucial role in defense against Lm infectionCompared to WT mice,TCRδ-/-mice exhibited more severe Lm infection.The number of γδ T cells in the liver increased rapidly from day three onwards,actively migrating towards the infection foci.In infected mice,IL-17A production was primarily attributed to γδT cells rather than αβ T cells.3.2 Liver-resident CD44hiCD27-γδ T cells were essential for defense against Lm infectionDuring early-stage infection,there was a continuous increase in hepatic CD44hiCD27-γδT cell numbers with rapid elevation of Ki67 expression,indicating their expansion during this phase.The hepatic CD44hiCD27-γδT cells demonstrated functional plasticity by simultaneously producing IL-17A and IFN-y upon response to Lm infection.Adoptive transfer of hepatic CD44hiCD27-γδT cells into TCRδ-/-mice resulted in reduced infection severity at an early stage suggesting that hepatic CD44hiCD27-γδ T cells play an important antimicrobial effect by producing IL-17A and IFN-γ during the early stage of infection.Compared to Vγ4+T cells,the Vγ6+T cell subpopulation predominated in the liver of infected mice which may have a dominant role in resistance to Lm infection.3.3 Macrophages facilitate the accumulation of liver-resident CD44hiCD27-γδT cells in infection foci of the liverHepatic CD44hiCD27-γδT cells in infected mice maintained liver-resident characteristics,indicating that the increase in hepatic CD44hiCD27-γδT cell numbers resulted from rapid proliferation rather than recruitment of peripheral circulating γδT cells.Additionally,hepatic macrophages recruited liver-resident CD44hiCD27-γδT cells to infection sites and synergistically defended against Lm infection.In summary,this study demonstrates the functional plasticity of liver-resident CD44hiCD27-γδT cells,their ability to rapidly proliferate and accumulate at liver infection sites during Lm infection,and their simultaneous production of IL-17A and IFN-γ for defense.Furthermore,hepatic macrophages promote the accumulation of liver-resident CD44hiCD27-γδT cells at infected foci and synergize with them to resist Lm infection.Conclusions:This study reveals unique gene expression characteristics of hepaticγδT17 cells distinct from those found in other organs or other subsets of hepatic γδT cells.We identify IL-17A-producing γδT cells as CD44hiCD27-subset with liverresident characteristics and heterogeneity.Liver-resident CD44hiCD27-γδT cells exhibit functional plasticity by producing both IL-17A and IFN-γ upon Lm infection.Moreover,we demonstrate that hepatic macrophages facilitate migration and accumulation of liver-resident CD44hiCD27-γδT cells at infection sites while collaborating with them to combat Lm infection.Importantly,our findings highlight the tissue-residency properties of Vγ6+ subset within the liver which may play a dominant role in defense against Lm infection.Overall,this study provides comprehensive insights into phenotypic characteristics and tissue residency features of hepatic γδT17 cells while shedding light on mechanisms underlying protective immune responses against infections,and provides a theoretical basis and direction for the development of new therapeutic strategies based on γδ T cells against intracellular bacterial infection.