ABSTRACT
Malaria is caused by Plasmodium species transmitted by Anopheles mosquitoes. Following a mosquito bite, Plasmodium sporozoites migrate from skin to liver, where extensive replication occurs, emerging later as merozoites that can infect red blood cells and cause symptoms of disease. As liver tissue-resident memory T cells (Trm cells) have recently been shown to control liver-stage infections, we embarked on a messenger RNA (mRNA)-based vaccine strategy to induce liver Trm cells to prevent malaria. Although a standard mRNA vaccine was unable to generate liver Trm or protect against challenge with Plasmodium berghei sporozoites in mice, addition of an agonist that recruits T cell help from type I natural killer T cells under mRNA-vaccination conditions resulted in significant generation of liver Trm cells and effective protection. Moreover, whereas previous exposure of mice to blood-stage infection impaired traditional vaccines based on attenuated sporozoites, mRNA vaccination was unaffected, underlining the potential for such a rational mRNA-based strategy in malaria-endemic regions.
Subject(s)
Malaria Vaccines , Malaria , Animals , Mice , Memory T Cells , Malaria/prevention & control , Liver , Plasmodium berghei/genetics , CD8-Positive T-LymphocytesABSTRACT
The sympathetic nervous system (SNS) controls various physiological functions via the neurotransmitter noradrenaline. Activation of the SNS in response to psychological or physical stress is frequently associated with weakened immunity. Here, we investigated how adrenoceptor signaling influences leukocyte behavior. Intravital two-photon imaging after injection of noradrenaline revealed transient inhibition of CD8+ and CD4+ T cell locomotion in tissues. Expression of ß-adrenergic receptor in hematopoietic cells was not required for NA-mediated inhibition of motility. Rather, chemogenetic activation of the SNS or treatment with adrenergic receptor agonists induced vasoconstriction and decreased local blood flow, resulting in abrupt hypoxia that triggered rapid calcium signaling in leukocytes and halted cell motility. Oxygen supplementation reversed these effects. Treatment with adrenergic receptor agonists impaired T cell responses induced in response to viral and parasitic infections, as well as anti-tumor responses. Thus, stimulation of the SNS impairs leukocyte mobility, providing a mechanistic understanding of the link between adrenergic receptors and compromised immunity.
Subject(s)
Adrenergic Agents/immunology , Cell Movement/immunology , Immunity/immunology , Leukocytes/immunology , Sympathetic Nervous System/immunology , Animals , Calcium Signaling/immunology , Cell Line, Tumor , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Receptors, Adrenergic/immunology , Signal Transduction/immunology , T-Lymphocytes/immunologyABSTRACT
The liver is positioned at the interface between two routes traversed by pathogens in disseminating infection. Whereas blood-borne pathogens are efficiently cleared in hepatic sinusoids by Kupffer cells (KCs), it is unknown how the liver prevents dissemination of peritoneal pathogens accessing its outer membrane. We report here that the hepatic capsule harbors a contiguous cellular network of liver-resident macrophages phenotypically distinct from KCs. These liver capsular macrophages (LCMs) were replenished in the steady state from blood monocytes, unlike KCs that are embryonically derived and self-renewing. LCM numbers increased after weaning in a microbiota-dependent process. LCMs sensed peritoneal bacteria and promoted neutrophil recruitment to the capsule, and their specific ablation resulted in decreased neutrophil recruitment and increased intrahepatic bacterial burden. Thus, the liver contains two separate and non-overlapping niches occupied by distinct resident macrophage populations mediating immunosurveillance at these two pathogen entry points to the liver.
Subject(s)
Kupffer Cells/physiology , Listeria monocytogenes/immunology , Listeriosis/immunology , Liver/immunology , Macrophages/immunology , Neutrophils/immunology , Peritoneum/microbiology , Animals , Cell Communication , Cell Self Renewal , Host-Pathogen Interactions , Humans , Immunity, Innate , Kupffer Cells/microbiology , Liver/microbiology , Liver/pathology , Macrophages/microbiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Monocytes/immunology , Neutrophil Infiltration , Peritoneum/pathologyABSTRACT
In recent years, various intervention strategies have reduced malaria morbidity and mortality, but further improvements probably depend upon development of a broadly protective vaccine. To better understand immune requirement for protection, we examined liver-stage immunity after vaccination with irradiated sporozoites, an effective though logistically difficult vaccine. We identified a population of memory CD8+ T cells that expressed the gene signature of tissue-resident memory T (Trm) cells and remained permanently within the liver, where they patrolled the sinusoids. Exploring the requirements for liver Trm cell induction, we showed that by combining dendritic cell-targeted priming with liver inflammation and antigen recognition on hepatocytes, high frequencies of Trm cells could be induced and these cells were essential for protection against malaria sporozoite challenge. Our study highlights the immune potential of liver Trm cells and provides approaches for their selective transfer, expansion, or depletion, which may be harnessed to control liver infections or autoimmunity.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Immunologic Memory/immunology , Liver/immunology , Malaria/immunology , Animals , CD8-Positive T-Lymphocytes/parasitology , Culicidae , Dendritic Cells/immunology , Dendritic Cells/parasitology , Hepatocytes/immunology , Hepatocytes/parasitology , Liver/parasitology , Liver Diseases/immunology , Liver Diseases/parasitology , Malaria Vaccines/immunology , Mice , Plasmodium berghei/immunology , Sporozoites/immunology , Sporozoites/parasitology , Vaccination/methodsABSTRACT
Malaria remains a major cause of mortality in the world and an efficient vaccine is the best chance of reducing the disease burden. Vaccination strategies for the liver stage of disease that utilise injection of live radiation-attenuated sporozoites (RAS) confer sterile immunity, which is mediated by CD8+ memory T cells, with liver-resident memory T cells (TRM ) being particularly important. We have previously described a TCR transgenic mouse, termed PbT-I, where all CD8+ T cells recognize a specific peptide from Plasmodium. PbT-I form liver TRM cells upon RAS injection and are capable of protecting mice against challenge infection. Here, we utilize this transgenic system to examine whether nonliving sporozoites, killed by heat treatment (HKS), could trigger the development of Plasmodium-specific liver TRM cells. We found that HKS vaccination induced the formation of memory CD8+ T cells in the spleen and liver, and importantly, liver TRM cells were fewer in number than that induced by RAS. Crucially, we showed the number of TRM cells was significantly higher when HKS were combined with the glycolipid α-galactosylceramide as an adjuvant. In the future, this work could lead to development of an antimalaria vaccination strategy that does not require live sporozoites, providing greater utility.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Immunologic Memory , Liver/immunology , Malaria Vaccines/immunology , Malaria/immunology , Malaria/parasitology , Plasmodium/immunology , Animals , CD8-Positive T-Lymphocytes/metabolism , Disease Models, Animal , Host-Parasite Interactions/immunology , Hot Temperature , Immunization , Malaria Vaccines/administration & dosage , Mice , Mice, Transgenic , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/immunologyABSTRACT
Portal tracts are key intrahepatic structures where leukocytes accumulate during immune responses. They contain the blood inflow, which includes portal blood from the gut, and lymphatic and biliary outflow of the liver, and as such represent a key interface for potential pathogen entry to the liver. Myeloid cells residing in the interstitium of the portal tract might play an important role in the surveillance or prevention of pathogen dissemination; however, the exact composition and localization of this population has not been explored fully. Our in-depth characterization of portal tract myeloid cells revealed that in addition to T lymphocytes, portal tracts contain a heterogeneous population of MHCIIhigh myeloid cells with potential antigen presenting cell (APC) function. These include a previously unreported subset of CSF1R-dependent CX3CR1+ macrophages that phenotypically and morphologically resemble liver capsular macrophages, as well as the two main dendritic cell subsets (cDC1 and cDC2). These cells are not randomly distributed, but each subset forms interconnected networks intertwined with specific components of the portal tract. The CX3CR1+ cells were preferentially detected along the outer border of the portal tracts, and also in the portal interstitium adjacent to the portal vein, bile duct, lymphatic vessels and hepatic artery. cDC1s abounded along the lymphatic vessels, while cDC2s mostly surrounded the biliary tree. The specific distributions of these discrete subsets predict that they may serve distinct functions in this compartment. Overall, our findings suggest that portal tracts and their embedded cellular networks of myeloid cells form a distinctive lymphoid compartment in the liver that has the potential to orchestrate immune responses in this organ.
Subject(s)
Liver , Macrophages , Dendritic CellsABSTRACT
Human noroviruses are highly infectious single-stranded RNA (ssRNA) viruses and the major cause of nonbacterial gastroenteritis worldwide. With the discovery of murine norovirus (MNV) and the introduction of an effective model for norovirus infection and replication, knowledge about infection mechanisms and their impact on the host immune response has progressed. A major player in the immune response against viral infections is the group of major histocompatibility complex (MHC) class I proteins, which present viral antigen to immune cells. We have observed that MNV interferes with the antigen presentation pathway in infected cells by reducing the surface expression of MHC class I proteins. We have shown that MNV-infected dendritic cells or macrophages have lower levels of surface expression of MHC class I proteins than uninfected and bystander cells. Transcriptional analysis revealed that this defect is not due to a decreased amount of mRNA but is reflected at the protein level. We have determined that this defect is mediated via the MNV NS3 protein. Significantly, treatment of MNV-infected cells with the endocytic recycling inhibitor dynasore completely restored the surface expression of MHC class I proteins, whereas treatment with the proteasome inhibitor MG132 partly restored such expression. These observations indicate a role for endocytic recycling and proteasome-mediated degradation of these proteins. Importantly, we show that due to the reduced surface expression of MHC class I proteins, antigen presentation is inhibited, resulting in the inability of CD8+ T cells to become activated in the presence of MNV-infected cells.IMPORTANCE Human noroviruses (HuNoVs) are the major cause of nonbacterial gastroenteritis worldwide and impose a great burden on patients and health systems every year. So far, no antiviral treatment or vaccine is available. We show that MNV evades the host immune response by reducing the amount of MHC class I proteins displayed on the cell surface. This reduction leads to a decrease in viral antigen presentation and interferes with the CD8+ T cell response. CD8+ T cells respond to foreign antigen by activating cytotoxic pathways and inducing immune memory to the infection. By evading this immune response, MNV is able to replicate efficiently in the host, and the ability of cells to respond to consecutive infections is impaired. These findings have a major impact on our understanding of the ways in which noroviruses interact with the host immune response and manipulate immune memory.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Caliciviridae Infections/immunology , Histocompatibility Antigens Class I/metabolism , Norovirus/pathogenicity , Animals , Antigen Presentation , Caliciviridae Infections/virology , Dendritic Cells/immunology , Lymphocyte Activation , Macrophages/immunology , Mice , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Viral Nonstructural Proteins/metabolismABSTRACT
We describe an MHC class II (I-Ab)-restricted TCR transgenic mouse line that produces CD4+ T cells specific for Plasmodium species. This line, termed PbT-II, was derived from a CD4+ T cell hybridoma generated to blood-stage Plasmodium berghei ANKA (PbA). PbT-II cells responded to all Plasmodium species and stages tested so far, including rodent (PbA, P. berghei NK65, Plasmodium chabaudi AS, and Plasmodium yoelii 17XNL) and human (Plasmodium falciparum) blood-stage parasites as well as irradiated PbA sporozoites. PbT-II cells can provide help for generation of Ab to P. chabaudi infection and can control this otherwise lethal infection in CD40L-deficient mice. PbT-II cells can also provide help for development of CD8+ T cell-mediated experimental cerebral malaria (ECM) during PbA infection. Using PbT-II CD4+ T cells and the previously described PbT-I CD8+ T cells, we determined the dendritic cell (DC) subsets responsible for immunity to PbA blood-stage infection. CD8+ DC (a subset of XCR1+ DC) were the major APC responsible for activation of both T cell subsets, although other DC also contributed to CD4+ T cell responses. Depletion of CD8+ DC at the beginning of infection prevented ECM development and impaired both Th1 and follicular Th cell responses; in contrast, late depletion did not affect ECM. This study describes a novel and versatile tool for examining CD4+ T cell immunity during malaria and provides evidence that CD4+ T cell help, acting via CD40L signaling, can promote immunity or pathology to blood-stage malaria largely through Ag presentation by CD8+ DC.
Subject(s)
Antigen Presentation , CD4-Positive T-Lymphocytes/immunology , CD40 Antigens/immunology , Dendritic Cells/immunology , Malaria/immunology , Mice, Transgenic/immunology , Parasitemia/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Antigens, Protozoan/immunology , CD40 Antigens/deficiency , CD40 Ligand/immunology , Cells, Cultured , Crosses, Genetic , Hybridomas , Lymphocyte Activation , Malaria, Cerebral/immunology , Malaria, Cerebral/prevention & control , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic/genetics , Plasmodium berghei/immunology , Radiation ChimeraABSTRACT
Liver fibrosis is a progressive pathological process involving inflammation and extracellular matrix deposition. Dipeptidyl peptidase 4 (DPP4), also known as CD26, is a cell surface glycoprotein and serine protease. DPP4 binds to fibronectin, can inactivate specific chemokines, incretin hormone and neuropeptides, and influences cell adhesion and migration. Such properties suggest a pro-fibrotic role for this peptidase but this hypothesis needs in vivo examination. Experimental liver injury was induced with carbon tetrachloride (CCl4) in DPP4 gene knockout (gko) mice. DPP4 gko had less liver fibrosis and inflammation and fewer B cell clusters than wild type mice in the fibrosis model. DPP4 inhibitor-treated mice also developed less liver fibrosis. DNA microarray and PCR showed that many immunoglobulin (Ig) genes and some metabolism-associated transcripts were differentially expressed in the gko strain compared with wild type. CCl4-treated DPP4 gko livers had more IgM+ and IgG+ intrahepatic lymphocytes, and fewer CD4+, IgD+ and CD21+ intrahepatic lymphocytes. These data suggest that DPP4 is pro-fibrotic in CCl4-induced liver fibrosis and that the mechanisms of DPP4 pro-fibrotic action include energy metabolism, B cells, NK cells and CD4+ cells.
Subject(s)
Dipeptidyl Peptidase 4/metabolism , Liver Cirrhosis/enzymology , Liver Cirrhosis/pathology , Liver/enzymology , Liver/injuries , Animals , Carbon Tetrachloride , Cell Line , Dipeptidyl-Peptidase IV Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Hepatic Stellate Cells/drug effects , Hepatic Stellate Cells/metabolism , Hepatic Stellate Cells/pathology , Humans , Leukocytes/drug effects , Leukocytes/metabolism , Leukocytes/pathology , Liver/pathology , Liver Cirrhosis/genetics , Mice , Mice, Knockout , Phenotype , Spleen/pathology , Up-RegulationABSTRACT
WC1 proteins are uniquely expressed on γδ T cells and belong to the scavenger receptor cysteine-rich (SRCR) superfamily. While present in variable, and sometimes high, numbers in the genomes of mammals and birds, in cattle there are 13 distinct genes (WC1-1 to WC1-13). All bovine WC1 proteins can serve as coreceptors for the TCR in a tyrosine phosphorylation dependent manner, and some are required for the γδ T cell response to Leptospira. We hypothesized that individual WC1 receptors encode Ag specificity via coligation of bacteria with the γδ TCR. SRCR domain binding was directly correlated with γδ T cell response, as WC1-3 SRCR domains from Leptospira-responsive cells, but not WC1-4 SRCR domains from Leptospira-nonresponsive cells, bound to multiple serovars of two Leptospira species, L. borgpetersenii, and L. interrogans. Three to five of eleven WC1-3 SRCR domains, but none of the eleven WC1-4 SRCR domains, interacted with Leptospira spp. and Borrelia burgdorferi, but not with Escherichia coli or Staphylococcus aureus. Mutational analysis indicated that the active site for bacterial binding in one of the SRCR domains is composed of amino acids in three discontinuous regions. Recombinant WC1 SRCR domains with the ability to bind leptospires inhibited Leptospira growth. Our data suggest that WC1 gene arrays play a multifaceted role in the γδ T cell response to bacteria, including acting as hybrid pattern recognition receptors and TCR coreceptors, and they may function as antimicrobials.
Subject(s)
Membrane Glycoproteins/immunology , Receptors, Antigen, T-Cell, gamma-delta/immunology , T-Lymphocyte Subsets/immunology , Amino Acid Sequence , Animals , Binding Sites , Borrelia burgdorferi/immunology , Borrelia burgdorferi/metabolism , Borrelia burgdorferi/pathogenicity , Cattle , Escherichia coli/genetics , Escherichia coli/immunology , Escherichia coli/metabolism , Escherichia coli/pathogenicity , Gene Expression , HEK293 Cells , Humans , Leptospira/immunology , Leptospira/metabolism , Leptospira/pathogenicity , Leptospira interrogans/immunology , Leptospira interrogans/metabolism , Leptospira interrogans/pathogenicity , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/genetics , Models, Molecular , Molecular Sequence Data , Mutation , Protein Binding , Protein Structure, Tertiary , Receptors, Antigen, T-Cell, gamma-delta/chemistry , Receptors, Antigen, T-Cell, gamma-delta/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Sequence Alignment , Staphylococcus aureus/immunology , Staphylococcus aureus/metabolism , Staphylococcus aureus/pathogenicity , T-Lymphocyte Subsets/metabolism , T-Lymphocyte Subsets/microbiologyABSTRACT
BACKGROUND & AIMS: Acute hepatitis is often mediated by cytotoxic T lymphocytes (CTLs); however, the intrinsic parameters that limit CTL-mediated liver injury are not well understood. METHODS: To investigate whether acute liver damage is limited by molecules that decrease the lifespan or effector function of CTLs, we used a well-characterized transgenic (Tg) mouse model in which acute liver damage develops upon transfer of T cell receptor (TCR) Tg CD8 T cells. Recipient Tg mice received donor TCR Tg T cells deficient for either the pro-apoptotic molecule Bim, which regulates CTL survival, or suppressor of cytokine signaling-1 (SOCS-1), which controls expression of common gamma chain cytokines; the effects of anti-PD-L1 neutralizing antibodies were also assessed. RESULTS: Use of Bim-deficient donor T cells and/or PD-L1 blockade increased the number of intrahepatic T cells without affecting the degree and kinetic of acute hepatitis. In contrast, SOCS-1-deficient T cells induced a heightened, prolonged acute hepatitis caused by their enhanced cytotoxic function and increased expansion. Although they inflicted more severe acute liver damage, SOCS-1-deficient T cells never precipitated chronic hepatitis and became exhausted. CONCLUSIONS: The degree of acute hepatitis is regulated by the function of CD8 T cells, but is not affected by changes in CTL lifespan. Although manipulation of the examined parameters affected acute hepatitis, persistent hepatitis did not ensue, indicating that, in the presence of high intrahepatic antigen load, changes in these factors in isolation were not sufficient to prevent T cell exhaustion and mediate progression to chronic hepatitis.
Subject(s)
Hepatitis/etiology , T-Lymphocytes, Cytotoxic/immunology , Acute Disease , Animals , B7-H1 Antigen/antagonists & inhibitors , B7-H1 Antigen/physiology , Bcl-2-Like Protein 11/physiology , Cell Survival , Hepatitis/immunology , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Transgenic , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/physiology , Receptors, Antigen, T-Cell/physiology , Suppressor of Cytokine Signaling 1 Protein/physiology , T-Lymphocytes, Cytotoxic/physiologyABSTRACT
Although most self-reactive T cells are eliminated in the thymus, mechanisms to inactivate or control T cells specific for extrathymic antigens are required and exist in the periphery. By investigating the site in which autoreactive T cells are tolerized, we identify a unique mechanism of peripheral deletion in which naïve autoreactive CD8 T cells are rapidly eliminated in the liver after intrahepatic activation. T cells actively invade hepatocytes, enter endosomal/lysosomal compartments, and are degraded. Blockade of this process leads to accumulation of autoreactive CD8 T cells in the liver and breach of tolerance, with the development of autoimmune hepatitis. Cell into cell invasion, or emperipolesis, is a long-observed phenomenon for which a physiological role has not been previously demonstrated. We propose that this "suicidal emperipolesis" is a unique mechanism of autoreactive T-cell deletion, a process critical for the maintenance of tolerance.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cell Death/immunology , Emperipolesis/immunology , Hepatocytes/immunology , Peripheral Tolerance/immunology , Animals , Apoptosis Regulatory Proteins/genetics , Bcl-2-Like Protein 11 , Homeodomain Proteins/genetics , Membrane Proteins/genetics , Mice , Mice, Knockout , Microscopy, Confocal , Microscopy, Electron , Proto-Oncogene Proteins/geneticsABSTRACT
While CD4+ T cells are a prerequisite for CD8+ T cell-mediated protection against intracellular hepatotropic pathogens, the mechanisms facilitating the transfer of CD4-help to intrahepatic CD8+ T cells are unknown. Here, we developed an experimental system to investigate cognate CD4+ and CD8+ T cell responses to a model-antigen expressed de novo in hepatocytes and reveal that after initial priming, effector CD4+ and CD8+ T cells migrate into portal tracts and peri-central vein regions of the liver where they cluster with type-1 conventional dendritic cells. These dendritic cells are locally licensed by CD4+ T cells and expand the number of CD8+ T cells in situ, resulting in larger effector and memory CD8+ T cell pools. These findings reveal that CD4+ T cells promote intrahepatic immunity by amplifying the CD8+ T cell response via peripheral licensing of hepatic type-1 conventional dendritic cells and identify intrahepatic perivascular compartments specialized in facilitating effector T cell-dendritic cell interactions.
Subject(s)
CD4-Positive T-Lymphocytes , Liver , Lymphoid Tissue , Antigens , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Dendritic Cells/immunology , Liver/immunology , Humans , Lymphoid Tissue/immunologyABSTRACT
UNLABELLED: Mixed cryoglobulinemia (MC) is the most common extrahepatic manifestation of chronic hepatitis C virus (HCV) infection. Although the formation of inflammation-triggering immune complexes is driven by clonal expansions of autoreactive B cells, we found total B cell numbers paradoxically reduced in HCV-infected patients with MC. HCV patients with MC (n = 17) also displayed a reduced number and a reduced frequency of naïve B cells compared with HCV-infected patients without MC (n = 19), hepatitis B virus-infected patients (n = 10), and uninfected controls (n = 50). This was due to an increased sensitivity of naïve B cells to apoptosis resulting in a reduction in the size of the naïve B cell subset. In addition, 4-fold expansion and skewing (lower T1/T2-ratio) of the immature B cell subset was noted in MC patients, suggesting that apoptosis of naïve B cells triggered the release of B cell precursors from bone marrow in an attempt to maintain normal B cell numbers. Following treatment of MC with the B cell-depleting antibody rituximab, the size of all B cell subsets, the T1/T2-ratio, and the cyroglobulin levels all normalized. Cryoglobulin levels correlated with in vivo proliferation of T2 B cells, suggesting a link between the skewing of the T1/T2 ratio and the formation of immune complexes. CONCLUSION: This study provides insight into the mechanisms maintaining B cell homeostasis in HCV-induced MC and the ability of rituximab therapy to restore normal B cell compartments. (HEPATOLOGY 2012;56:1602-1610).
Subject(s)
B-Lymphocyte Subsets/pathology , Cryoglobulinemia/immunology , Hepatitis C, Chronic/immunology , Homeostasis/drug effects , Precursor Cells, B-Lymphoid/pathology , Adult , Antibodies, Monoclonal, Murine-Derived/pharmacology , Antibodies, Monoclonal, Murine-Derived/therapeutic use , Antigens, CD19/metabolism , Apoptosis , B-Lymphocyte Subsets/drug effects , B-Lymphocyte Subsets/metabolism , Caspase 3/metabolism , Caspase 8/metabolism , Cryoglobulinemia/drug therapy , Cryoglobulinemia/virology , Cryoglobulins/metabolism , Female , Hepatitis C, Chronic/complications , Humans , Immunologic Factors/pharmacology , Immunologic Factors/therapeutic use , Lymphocyte Count , Male , Middle Aged , Neprilysin/metabolism , Precursor Cells, B-Lymphoid/drug effects , Precursor Cells, B-Lymphoid/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Rituximab , Statistics, NonparametricABSTRACT
BACKGROUND & AIMS: The occurrence of primary CD8 T cell activation within the liver, unique among the non-lymphoid organs, is now well accepted. However, the outcome of intrahepatic T cell activation remains controversial. We have previously reported that activation initiated by hepatocytes results in a tolerogenic phenotype characterized by low expression of CD25 and IL-2, poor cytotoxic T lymphocyte (CTL) function, and excessive expression of the pro-apoptotic protein Bim. METHODS: To investigate whether this phenotype was due to activation in the absence of co-stimulation, we generated bone marrow (bm) radiation chimeras in which adoptively transferred naïve transgenic CD8 T cells were activated in the presence of co-stimulation by liver bm-derived cells. RESULTS: Despite expressing pro-inflammatory cytokines, high levels of CD25 and CD54, donor T cells activated by liver bm-derived cells did not produce detectable IL-2 and displayed poor CTL function, suggesting incomplete acquisition of effector function. Simultaneously, these cells expressed high levels of Bim and died by neglect. Transfer of Bim-deficient T cells resulted in increased T cell numbers. CONCLUSIONS: These results imply that expression of CD25 and CD54 is co-stimulation dependent and distinguishes T cell activated by hepatocytes and liver bm-derived cells. In contrast, low expression of IL-2, poor CTL function and excess Bim production represent a more universal phenotype defining T cells undergoing primary activation by both types of hepatic antigen presenting cells (APC). These results have important implications for transplantation, in which all liver antigen presenting cells contribute to activation of T cells specific for the allograft.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , CD8-Positive T-Lymphocytes/immunology , Interleukin-2/metabolism , Liver/cytology , Lymphocyte Activation/immunology , Membrane Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Adoptive Transfer , Animals , Apoptosis , Bcl-2-Like Protein 11 , Bone Marrow Cells/cytology , Bone Marrow Cells/immunology , Cytotoxicity Tests, Immunologic , H-2 Antigens/immunology , H-2 Antigens/metabolism , Hepatocytes/immunology , Liver/immunology , Lymph Nodes/cytology , Mice , Mice, Transgenic , Phenotype , Radiation ChimeraABSTRACT
BACKGROUND & AIMS: Mathematical modeling of hepatitis C virus (HCV) kinetics indicated that cellular immune responses contribute to interferon (IFN)-induced clearance of HCV. We investigated a potential role of natural killer (NK) cells in this process. METHODS: Phenotype and function of blood and liver NK cells were studied during the first 12 weeks of treatment with pegylated IFN-alfa and ribavirin, the time period used to define the early virological response. RESULTS: Within hours of treatment initiation, NK cells of patients that had an early virological response increased expression of activating receptors NKG2D, NKp30, and CD16 and decreased expression of NKG2C and 2B4, along with inhibitory receptors SIGLEC7 and NKG2A, resulting in NK cell activation. NK cell cytotoxicity, measured by degranulation and tumor necrosis factor-related apoptosis-inducing ligand production, peaked after 24 hours (P<.01), concomitant with an increase in alanine aminotransferase levels (P<.05), whereas IFN-γ production decreased within 6 hours and did not recover for more than 4 weeks (P<.05). NK cells from liver biopsies taken 6 hours after treatment initiation had increased numbers of cytotoxic CD16+NK cells (P<.05) and a trend toward increased production of tumor necrosis factor-related apoptosis-inducing ligand. Degranulation of peripheral blood NK cells correlated with treatment-induced, first-phase decreases in viral load (P<.05) and remained higher in early virological responders than in nonresponders for weeks. CONCLUSIONS: IFN activates NK cells early after treatment is initiated. Their cytotoxic function, in particular, is strongly induced, which correlates to virologic response. Therefore, NK cell activation indicates responsiveness to IFN-α-based treatment and suggests the involvement of the innate immune cells in viral clearance.
Subject(s)
Antiviral Agents/therapeutic use , Hepatitis C/drug therapy , Immunity, Innate/drug effects , Interferon-alpha/therapeutic use , Killer Cells, Natural/drug effects , Liver/drug effects , Polyethylene Glycols/therapeutic use , Ribavirin/therapeutic use , Adult , Alanine Transaminase/blood , Antigens, Differentiation, Myelomonocytic/metabolism , Biopsy , Cell Degranulation/drug effects , Cells, Cultured , Cytokines/metabolism , Drug Therapy, Combination , Female , Hepacivirus/genetics , Hepatitis C/diagnosis , Hepatitis C/immunology , Humans , Interferon-gamma/metabolism , Killer Cells, Natural/immunology , Killer Cells, Natural/virology , Lectins/metabolism , Liver/immunology , Liver/virology , Male , Maryland , Middle Aged , NK Cell Lectin-Like Receptor Subfamily C/metabolism , RNA, Viral/blood , Recombinant Proteins/therapeutic use , TNF-Related Apoptosis-Inducing Ligand/metabolism , Time Factors , Treatment Outcome , Viral LoadABSTRACT
Malaria parasites replicate within the liver shortly after infection. This stage can be controlled by CD8 T cells, but which subsets undertake this function is unclear. Lefebvre et al. now elegantly show that effector memory T (TEM) cells are avid participants, working as a dynamic duo with liver tissue-resident memory T (TRM) cells to combat infection.
Subject(s)
Malaria , Songbirds , Animals , CD8-Positive T-Lymphocytes/immunology , Humans , Immunologic Memory/immunology , Liver/parasitology , Malaria/parasitologyABSTRACT
Full-spectrum flow cytometry is now routinely used in many laboratories internationally, and the demand for this technology is rapidly increasing. With capacity to use larger and more complex staining panels, standardized protocols are required for optimal panel design and analysis. Importantly, for ex vivo analysis, tissue preparation methods also need to be optimized to ensure samples are truly representative of tissues in situ. This is particularly relevant given the recent interest in adaptive immune cells that form residency in specific organs. Here we provide optimized protocols for tissue processing and phenotyping of memory T cells and natural killer T (NKT) cell subsets from liver, lung, spleen, and lymph node using full-spectrum flow cytometry. We provide a 21-color antibody panel for identification of different memory subsets, including tissue-resident memory T (TRM ) cells, which are increasingly regarded as important effectors in adaptive immunity. We show that processing procedures can affect outcomes, with liver TRM cells particularly sensitive to heat, such that accurate evaluation requires fast processing at defined temperatures. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Processing mouse liver for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 2: Processing mouse spleen for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 3: Processing mouse lungs for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 4: Processing mouse lymph nodes for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 5: Staining and flow cytometric analysis of samples for memory T and NKT cell subsets Support Protocol: Obtaining cell counts from flow cytometry data.
Subject(s)
Natural Killer T-Cells , Animals , Flow Cytometry/methods , Mice , Phenotype , Spleen , Staining and LabelingABSTRACT
Self-adjuvanting vaccines consisting of peptide epitopes conjugated to immune adjuvants are a powerful way of generating antigen-specific immune responses. We previously showed that a Plasmodium-derived peptide conjugated to a rearranged form of α-galactosylceramide (α-GalCer) could stimulate liver-resident memory T (TRM) cells that were effective killers of liver-stage Plasmodium berghei ANKA (Pba)-infected cells. To investigate if similar or even superior TRM responses can be induced by modifying the α-GalCer adjuvant, we created new conjugate vaccine cadidates by attaching an immunogenic Plasmodium-derived peptide antigen to 6â³-substituted α-GalCer analogues. Vaccine synthesis involved developing an efficient route to α-galactosylphytosphingosine (α-GalPhs), from which the prototypical iNKT cell agonist, α-GalCer, and its 6â³-deoxy-6â³-thio and -amino analogues were derived. Attaching a cathepsin B-cleavable linker to the 6â³-modified α-GalCer created pro-adjuvants bearing a pendant ketone group available for peptide conjugation. Optimized reaction conditions were developed that allow for the efficient conjugation of peptide antigens to the pro-adjuvants via oxime ligation to create new glycolipid-peptide (GLP) conjugate vaccines. A single dose of the vaccine candidates induced acute NKT and Plasmodium-specific CD8+ T cell responses that generated potent hepatic TRM responses in mice. Our findings demonstrate that attaching antigenic peptides to 6â³-modifed α-GalCer generates powerful self-adjuvanting conjugate vaccine candidates that could potentially control hepatotropic infections such as liver-stage malaria.