Liver-draining Lymph Nodes Research Articles

Abstract 624: The role of draining lymph nodes in the post-transplant recurrence of liver cancer: Examining immune response using spatial proteomics

Abstract Hepatocellular Carcinoma (HCC) and Cholangiocarcinoma (CCA) are the predominant forms of liver cancer, with a devastating combined incidence of 40,000 cases per year in the United States. Despite many differences between the cancers, liver transplant has found great success in treating both over traditional methods. However, up to 35% of patients’ cancer still recur within five years. Given the risk and resource intensiveness of this treatment, it is critical to identify factors that influence recurrence in these cancers. Previous studies have found the micro-environments of HCC tumors tend to be immune-active while those of CCA tumors tend to be immunosuppressive. Increased immune activity at the tumor level is associated with better patient outcomes, but the role and impact of the regional immune response in liver cancer is not well understood. Liver draining lymph node tissue was collected from patients undergoing orthotopic liver transplant (OLT) for HCC, CCA, or other reasons (control). Nanostring GeoMx Digital Spatial Profiler (DSP) was used to measure immune marker levelsWe found increased immune activity in the LNs of patients with HCC compared to CCA, showing significantly higher levels of lymphocytes and immune activity markers, matching previous tumor microenvironment findings. Additionally, comparing recurrent and non-recurrent HCC or CCA samples, we see generally increased immune activity in non-recurrent lymph nodes, also identifying specific immune markers in both cancer types that correlate with cancer recurrence or non-recurrence. These also reveal patterns of activity specific to both cancer type and recurrence status. Overall, this study provides novel insights into the regional immune response in liver cancers and identifies candidate proteins to serve as predictors of recurrence in liver cancer. We suggest CD163, CD66b, CD8, and CD56 as markers to predict post-OLT HCC recurrence and CD163, CD66b, ID01, and OX40L as candidate markers to predict post-OLT CCA recurrence using LN samples. Further spatial analysis of this data may further characterize immune activity in these lymph nodes, and future studies focusing on identified differences in immune activity may reveal factors affecting liver cancer aggressiveness and recurrence. Citation Format: Peyton Hickman, Aubrey E. Thompson, Justin Nguyen. The role of draining lymph nodes in the post-transplant recurrence of liver cancer: Examining immune response using spatial proteomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 624.

Oxidized low density lipoprotein in the liver causes decreased permeability of liver lymphatic- but not liver sinusoidal-endothelial cells via VEGFR-3 regulation of VE-Cadherin.

The lymphatic vasculature of the liver is vital for liver function as it maintains fluid and protein homeostasis and is important for immune cell transport to the lymph node. Chronic liver disease is associated with increased expression of inflammatory mediators including oxidized low-density lipoprotein (oxLDL). Intrahepatic levels of oxLDL are elevated in nonalcoholic fatty liver disease (NAFLD), chronic hepatitis C infection (HCV), alcohol-associated liver disease (ALD), and cholestatic liver diseases. To determine if liver lymphatic function is impaired in chronic liver diseases, in which increased oxLDL has been documented, we measured liver lymphatic function in murine models of NAFLD, ALD and primary sclerosing cholangitis (PSC). We found that Mdr2-/- (PSC), Lieber-DeCarli ethanol fed (ALD) and high fat and high cholesterol diet fed (NAFLD) mice all had a significant impairment in the ability to traffic FITC labeled dextran from the liver parenchyma to the liver draining lymph nodes. Utilizing an in vitro permeability assay, we found that oxLDL decreased the permeability of lymphatic endothelial cells (LEC)s, but not liver sinusoidal endothelial cells (LSEC)s. Here we demonstrate that LECs and LSECs differentially regulate SRC-family kinases, MAPK kinase and VE-Cadherin in response to oxLDL. Furthermore, Vascular Endothelial Growth Factor (VEGF)C or D (VEGFR-3 ligands) appear to regulate VE-Cadherin expression as well as decrease cellular permeability of LECs in vitro and in vivo after oxLDL treatment. These findings suggest that oxLDL acts to impede protein transport through the lymphatics through tightening of the cell-cell junctions. Importantly, engagement of VEGFR-3 by its ligands prevents VE-Cadherin upregulation and improves lymphatic permeability. These studies provide a potential therapeutic target to restore liver lymphatic function and improve liver function.

XCR1+ type 1 conventional dendritic cells drive liver pathology in non-alcoholic steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are prevalent liver conditions that underlie the development of life-threatening cirrhosis, liver failure and liver cancer. Chronic necro-inflammation is a critical factor in development of NASH, yet the cellular and molecular mechanisms of immune dysregulation in this disease are poorly understood. Here, using single-cell transcriptomic analysis, we comprehensively profiled the immune composition of the mouse liver during NASH. We identified a significant pathology-associated increase in hepatic conventional dendritic cells (cDCs) and further defined their source as NASH-induced boost in cycling of cDC progenitors in the bone marrow. Analysis of blood and liver from patients on the NAFLD/NASH spectrum showed that type 1 cDCs (cDC1) were more abundant and activated in disease. Sequencing of physically interacting cDC-T cell pairs from liver-draining lymph nodes revealed that cDCs in NASH promote inflammatory T cell reprogramming, previously associated with NASH worsening. Finally, depletion of cDC1 in XCR1DTA mice or using anti-XCL1-blocking antibody attenuated liver pathology in NASH mouse models. Overall, our study provides a comprehensive characterization of cDC biology in NASH and identifies XCR1+ cDC1 as an important driver of liver pathology.

Oxidized Low-Density Lipoprotein Drives Dysfunction of the Liver Lymphatic System.

Background and AimsAs the incidence of nonalcoholic steatohepatitis (NASH) continues to rise, understanding how normal liver functions are affected during disease is required before developing novel therapeutics which could reduce morbidity and mortality. However, very little is understood about how the transport of proteins and cells from the liver by the lymphatic vasculature is affected by inflammatory mediators or during disease.MethodsTo answer these questions, we utilized a well-validated mouse model of NASH and exposure to highly oxidized low density lipoprotein (oxLDL). In addition to single cell sequencing, multiplexed immunofluorescence and metabolomic analysis of liver lymphatic endothelial cells (LEC)s we evaluated lymphatic permeability and transport both in vitro and in vivo.ResultsConfirming similarities between human and mouse liver lymphatic vasculature in NASH, we found that the lymphatic vasculature expands as disease progresses and results in the downregulation of genes important to lymphatic identity and function. We also demonstrate, in mice with NASH, that fluorescein isothiocyanate (FITC) dextran does not accumulate in the liver draining lymph node upon intrahepatic injection, a defect that was rescued with therapeutic administration of the lymphatic growth factor, recombinant vascular endothelial growth factor C (rVEGFC). Similarly, exposure to oxLDL reduced the amount of FITC-dextran in the portal draining lymph node and through an LEC monolayer. We provide evidence that the mechanism by which oxLDL impacts lymphatic permeability is via a reduction in Prox1 expression which decreases lymphatic specific gene expression, impedes LEC metabolism and reorganizes the highly permeable lymphatic cell-cell junctions which are a defining feature of lymphatic capillaries.ConclusionsWe identify oxLDL as a major contributor to decreased lymphatic permeability in the liver, a change which is consistent with decreased protein homeostasis and increased inflammation during chronic liver disease.

Specific Commensal Bacterium Critically Regulates Gut Microbiota Osteoimmunomodulatory Actions During Normal Postpubertal Skeletal Growth and Maturation.

ABSTRACTThe commensal gut microbiota critically regulates immunomodulatory processes that influence normal skeletal growth and maturation. However, the influence of specific microbes on commensal gut microbiota osteoimmunoregulatory actions is unknown. We have shown previously that the commensal gut microbiota enhances TH17/IL17A immune response effects in marrow and liver that have procatabolic/antianabolic actions in the skeleton. Segmented filamentous bacteria (SFB), a specific commensal gut bacterium within phylum Firmicutes, potently induces TH17/IL17A‐mediated immunity. The study purpose was to delineate the influence of SFB on commensal gut microbiota immunomodulatory actions regulating normal postpubertal skeletal development. Two murine models were utilized: SFB‐monoassociated mice versus germ‐free (GF) mice and specific‐pathogen‐free (SPF) mice +/− SFB. SFB colonization was validated by 16S rDNA analysis, and SFB‐induced TH17/IL17A immunity was confirmed by upregulation of Il17a in ileum and IL17A in serum. SFB‐colonized mice had an osteopenic trabecular bone phenotype, which was attributed to SFB actions suppressing osteoblastogenesis and enhancing osteoclastogenesis. Intriguingly, SFB‐colonized mice had increased expression of proinflammatory chemokines and acute‐phase reactants in the liver. Lipocalin‐2 (LCN2), an acute‐phase reactant and antimicrobial peptide, was substantially elevated in the liver and serum of SFB‐colonized mice, which supports the notion that SFB regulation of commensal gut microbiota osteoimmunomodulatory actions are mediated in part through a gut–liver–bone axis. Proinflammatory TH17 and TH1 cells were increased in liver‐draining lymph nodes of SFB‐colonized mice, which further substantiates that SFB osteoimmune‐response effects may be mediated through the liver. SFB‐induction of Il17a in the gut and Lcn2 in the liver resulted in increased circulating levels of IL17A and LCN2. Recognizing that IL17A and LCN2 support osteoclastogenesis/suppress osteoblastogenesis, SFB actions impairing postpubertal skeletal development appear to be mediated through immunomodulatory effects in both the gut and liver. This research reveals that specific microbes critically impact commensal gut microbiota immunomodulatory actions regulating normal postpubertal skeletal growth and maturation. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Chronic Liver Disease in Humans Causes Expansion and Differentiation of Liver Lymphatic Endothelial Cells.

Liver lymphatic vessels support liver function by draining interstitial fluid, cholesterol, fat, and immune cells for surveillance in the liver draining lymph node. Chronic liver disease is associated with increased inflammation and immune cell infiltrate. However, it is currently unknown if or how lymphatic vessels respond to increased inflammation and immune cell infiltrate in the liver during chronic disease. Here we demonstrate that lymphatic vessel abundance increases in patients with chronic liver disease and is associated with areas of fibrosis and immune cell infiltration. Using single-cell mRNA sequencing and multi-spectral immunofluorescence analysis we identified liver lymphatic endothelial cells and found that chronic liver disease results in lymphatic endothelial cells (LECs) that are in active cell cycle with increased expression of CCL21. Additionally, we found that LECs from patients with NASH adopt a transcriptional program associated with increased IL13 signaling. Moreover, we found that oxidized low density lipoprotein, associated with NASH pathogenesis, induced the transcription and protein production of IL13 in LECs both in vitro and in a mouse model. Finally, we show that oxidized low density lipoprotein reduced the transcription of PROX1 and decreased lymphatic stability. Together these data indicate that LECs are active participants in the liver, expanding in an attempt to maintain tissue homeostasis. However, when inflammatory signals, such as oxidized low density lipoprotein are increased, as in NASH, lymphatic function declines and liver homeostasis is impeded.

Malaria Immunity: The Education of an Unnatural Response

In this issue of Cell Host & Microbe, Kurup etal. report that infection of the liver by Plasmodium parasites promotes the recruitment of dendritic cells that acquire and present parasite antigen from infected hepatocytes. These cells then prime parasite-specific CD8 Tcells in liver-draining lymph nodes.

Monocyte-Derived CD11c+ Cells Acquire Plasmodium from Hepatocytes to Prime CD8 T Cell Immunity to Liver-Stage Malaria

Plasmodium sporozoites inoculated by mosquitoes migrate to the liver and infect hepatocytes prior to release of merozoites that initiate symptomatic blood-stage malaria. Plasmodium parasites are thought to be restricted to hepatocytes throughout this obligate liver stage of development, and how liver-stage-expressed antigens prime productive CD8 Tcell responses remains unknown. We found that a subset of liver-infiltrating monocyte-derived CD11c+ cells co-expressing F4/80, CD103, CD207, and CSF1R acquired parasites during the liver stage of malaria, but only after initial hepatocyte infection. These CD11c+ cells found in the infected liver and liver-draining lymph nodes exhibited transcriptionally and phenotypically enhanced antigen-presentation functions and primed protective CD8 Tcell responses against Plasmodium liver-stage-restricted antigens. Our findings highlight a previously unrecognized aspect of Plasmodium biology and uncover the fundamental mechanism by which CD8 Tcell responses are primed against liver-stage malaria antigens.

Characterization of Antigen-Presenting Cell Subsets in Human Liver-Draining Lymph Nodes.

T-cell immunity in the liver is tightly regulated to prevent chronic liver inflammation in response to antigens and toxins derived from food and intestinal bacterial flora. Since the main sites of T cell activation in response to foreign components entering solid tissues are the draining lymph nodes (LN), we aimed to study whether Antigen-Presenting Cell (APC) subsets in human liver lymph-draining LN show features that may contribute to the immunologically tolerant liver environment. Healthy liver LN, iliac LN, spleen and liver perfusates were obtained from multi-organ donors, while diseased liver LN were collected from explanted patient livers. Inguinal LN were obtained from kidney transplant recipients. Mononuclear cells were isolated from fresh tissues, and immunophenotypic and functional characteristics of APC subsets were studied using flowcytometry and in ex vivo cultures. Healthy liver-draining LN contained significantly lower relative numbers of CD1c+ conventional dendritic cells (cDC2), plasmacytoid DC (PDC), and CD14+CD163+DC-SIGN+ macrophages (MF) compared to inguinal LN. Compared to spleen, both types of LN contained low relative numbers of CD141hi cDC1. Both cDC subsets in liver LN showed a more activated/mature immunophenotype than those in inguinal LN, iliacal LN, spleen and liver tissue. Despite their more mature status, cDC2 isolated from hepatic LN displayed similar cytokine production capacity (IL-10, IL-12, and IL-6) and allogeneic T cell stimulatory capacity as their counterparts from spleen. Liver LN from patients with inflammatory liver diseases showed a further reduction of cDC1, but had increased relative numbers of PDC and MF. In steady state conditions human liver LN contain relatively low numbers of cDC2, PDC, and macrophages, and relative numbers of cDC1 in liver LN decline during liver inflammation. The paucity of cDC in liver LN may contribute to immune tolerance in the liver environment.

The differential organogenesis and functionality of two liver-draining lymph nodes in mice

The liver is an immunological organ. However, fundamental knowledge concerning liver-draining lymph nodes (LNs), which have been newly identified in mice as the portal and celiac LNs, is still lacking. Here, we revealed that the portal LN and celiac LN drain liver lymph through different lymphatic vessels. Although both the portal LN and celiac LN possess typical structures, they have different cell compositions. Interestingly, these two LNs form at different times during fetal development. Moreover, the organogenesis of the celiac LN, but not the portal LN, is controlled by the transcription factor NFIL3. Furthermore, the portal LN and celiac LN also perform different functions. The celiac LN is the predominant site of liver antiviral immune responses, whereas the portal LN functions in the in situ induction of dietary antigen-specific regulatory T cells. In conclusion, the portal LN and celiac LN are two independent liver-draining LNs with different organogenesis histories and separate functions in maintaining immune homeostasis in the liver.

Different-Sized Gold Nanoparticle Activator/Antigen Increases Dendritic Cells Accumulation in Liver-Draining Lymph Nodes and CD8+ T Cell Responses.

The lack of efficient antigen and activator delivery systems, as well as the restricted migration of dendritic cells (DCs) to secondary lymph organs, dramatically limits DC-based adoptive immunotherapy. We selected two spherical gold nanoparticle (AuNP)-based vehicles of optimal size for activator and antigen delivery. Their combination (termed the NanoAu-Cocktail) was associated with the dual targeting of CpG oligonucleotides (CpG-ODNs) and an OVA peptide (OVAp) to DC subcellular compartments, inducing enhanced antigen cross-presentation, upregulated expression of costimulatory molecules and elevated secretion of T helper1 cytokines. We demonstrated that the intravenously transfused NanoAu-Cocktail pulsed DCs showed dramatically improved in vivo homing ability to lymphoid tissues and were settled in T cell area. Especially, by tissue-distribution analysis, we found that more than 60% of lymphoid tissues-homing DCs accumulated in liver-draining lymph nodes (LLNs). The improved homing ability of NanoAu-Cocktail pulsed DCs was associated with the high expression of chemokine receptor 7 (CCR7) and rearrangement of the cytoskeletons. In addition, by antigen-specific tetramers detection, NanoAu-Cocktail pulsed DCs were proved able to elicit strong antigen-specific CD8+ T cell responses, which provided enhanced protection from viral invasions. This study highlights the importance of codelivering antigen/adjuvant using different sized gold nanoparticles to improve DC homing and therapy.

Dynamics of antigen presentation to transgene product-specific CD4+ T cells and of Treg induction upon hepatic AAV gene transfer

The tolerogenic hepatic microenvironment impedes clearance of viral infections but is an advantage in viral vector gene transfer, which often results in immune tolerance induction to transgene products. Although the underlying tolerance mechanism has been extensively studied, our understanding of antigen presentation to transgene product-specific CD4+ T cells remains limited. To address this, we administered hepatotropic adeno-associated virus (AAV8) vector expressing cytoplasmic ovalbumin (OVA) into wt mice followed by adoptive transfer of transgenic OVA-specific T cells. We find that that the liver-draining lymph nodes (celiac and portal) are the major sites of MHC II presentation of the virally encoded antigen, as judged by in vivo proliferation of DO11.10 CD4+ T cells (requiring professional antigen-presenting cells, e.g., macrophages) and CD4+CD25+FoxP3+ Treg induction. Antigen presentation in the liver itself contributes to activation of CD4+ T cells egressing from the liver. Hepatic-induced Treg rapidly disseminate through the systemic circulation. By contrast, a secreted OVA transgene product is presented in multiple organs, and OVA-specific Treg emerge in both the thymus and periphery. In summary, liver draining lymph nodes play an integral role in hepatic antigen presentation and peripheral Treg induction, which results in systemic regulation of the response to viral gene products.

Distinct features of newly found liver-draining lymph nodes in mice (IRC4P.612)

Abstract Draining lymph nodes (LNs) are specialized sites where adaptive immune responses take place. Recently, the anatomical location of two LNs draining the mouse liver, named as portal LN (PLN) and celiac LN (CLN) respectively, has been characterized. However, the immune responses in the liver-draining LNs and their contribution to the establishment and maintenance of liver tolerance are still not fully understood. In our further study, we find that these two liver-draining LNs (PLN and CLN) have different lymphatic vessels, excluding the possibility that one LN is secondary to the other one. Interestingly, although both PLN and CLN can drain liver lymph directly, they are different in many aspects. The two liver-draining LNs have different cellular composition, with PLN being highly similar to mesenteric LN but different from periphery LNs, and CLN being special with unique composition of innate immune cells. Importantly, the development of these two liver-draining LNs might be controlled by different transcription factors. Moreover, our preliminary data shows that the immune responses of these two liver-draining LNs exhibit different features during exogenous pathogen infection. Overall, PLN and CLN are two liver-draining LNs with distinct characteristics and their contribution to liver tolerance remains to be defined.

AN ORAL-SALMONELLA CANCER IMMUNOTHERAPY INDUCES A TUMOR-SPECIFIC IMMUNE RESPONSE IN LIVER AND LIVER DRAINING LYMPH NODES

AN ORAL-SALMONELLA CANCER IMMUNOTHERAPY INDUCES A TUMOR-SPECIFIC IMMUNE RESPONSE IN LIVER AND LIVER DRAINING LYMPH NODES

Tissue Requirements for Establishing Long-Term CD4+ T Cell–Mediated Immunity following Leishmania donovani Infection

Organ-specific immunity is a feature of many infectious diseases, including visceral leishmaniasis caused by Leishmania donovani. Experimental visceral leishmaniasis in genetically susceptible mice is characterized by an acute, resolving infection in the liver and chronic infection in the spleen. CD4+ T cell responses are critical for the establishment and maintenance of hepatic immunity in this disease model, but their role in chronically infected spleens remains unclear. In this study, we show that dendritic cells are critical for CD4+ T cell activation and expansion in all tissue sites examined. We found that FTY720-mediated blockade of T cell trafficking early in infection prevented Ag-specific CD4+ T cells from appearing in lymph nodes, but not the spleen and liver, suggesting that early CD4+ T cell priming does not occur in liver-draining lymph nodes. Extended treatment with FTY720 over the first month of infection increased parasite burdens, although this associated with blockade of lymphocyte egress from secondary lymphoid tissue, as well as with more generalized splenic lymphopenia. Importantly, we demonstrate that CD4+ T cells are required for the establishment and maintenance of antiparasitic immunity in the liver, as well as for immune surveillance and suppression of parasite outgrowth in chronically infected spleens. Finally, although early CD4+ T cell priming appeared to occur most effectively in the spleen, we unexpectedly revealed that protective CD4+ T cell-mediated hepatic immunity could be generated in the complete absence of all secondary lymphoid tissues.

Characterization of the liver-draining lymph nodes in mice and their role in mounting regional immunity to HBV

The lymphatic system is important in mounting an immune response to foreign antigens and tumors in humans and animal models. The liver produces a large amount of lymph, and its lymphatic system is divided into three major components: the portal, sublobular and superficial lymphatic vessels. Despite the fact that mice are the most commonly used laboratory animals, detailed descriptions of the anatomical location and function of the lymph nodes (LNs) that drain the liver are surprisingly absent. In this study, we found that the portal and celiac LNs adjacent to mouse liver were stained with Evans blue within 5-8min. Enhanced green fluorescence protein (EGFP)-positive cells from the liver also drained into the two aforementioned LNs. These data indicate that the portal and celiac LNs drain the mouse liver. Lymphadenectomy of the identified liver-draining LNs resulted in hepatitis B virus (HBV) persistence in immunocompetent mice compared with the sham group. In addition, the frequencies of CD8(+) T cells and dendritic cells (DCs) increased significantly in the liver-draining LNs after hydrodynamic injection of HBV plasmid. Liver-draining LN cells in HBV plasmid-injected mice also showed significant antigen-specific proliferation in response to stimulation with recombinant hepatitis B core antigen in vitro. Adoptive transfer of these cells into Rag1(-/-) mice induced a reduction in the serum concentration of hepatitis B surface antigen (HBsAg) compared to liver-draining LN cells in uninjected mice. Altogether our data characterize the liver-draining LNs and provide evidence that the liver-draining LNs induce an anti-HBV-specific immune response responsible for HBV clearance.

Failure of CD4 T-Cells to Respond to Liver-Derived Antigen and to Provide Help to CD8 T-Cells

CD4 T-cell help is required for the induction of efficient CD8 T-cells responses and the generation of memory cells. Lack of CD4 T-cell help may contribute to an exhausted CD8 phenotype and viral persistence. Little is known about priming of CD4 T-cells by liver-derived antigen. We used TF-OVA mice expressing ovalbumin in hepatocytes to investigate CD4 T-cell priming by liver-derived antigen and the impact of CD4 T-cell help on CD8 T-cell function. Naïve and effector CD4 T-cells specific for ovalbumin were transferred into TF-OVA mice alone or together with naïve ovalbumin-specific CD8 T-cells. T-cell activation and function were analyzed. CD4 T-cells ignored antigen presented by liver antigen-presenting cells (APCs) in vitro and in vivo but were primed in the liver-draining lymph node and the spleen. No priming occurred in the absence of bone-marrow derived APCs capable of presenting ovalbumin in vivo. CD4 T-cells primed in TF-OVA mice displayed defective Th1-effector function and caused no liver damage. CD4 T-cells were not required for the induction of hepatitis by CD8 T-cells. Th1-effector but not naïve CD4 T-cells augmented the severity of liver injury caused by CD8 T-cells. Our data demonstrate that CD4 T-cells fail to respond to liver-derived antigen presented by liver APCs and develop defective effector function after priming in lymph nodes and spleen. The lack of CD4 T-cell help may be responsible for insufficient CD8 T-cell function against hepatic antigens.

The lung vascular filter as a site of immune induction for T cell responses to large embolic antigen

The bloodstream is an important route of dissemination of invading pathogens. Most of the small bloodborne pathogens, like bacteria or viruses, are filtered by the spleen or liver sinusoids and presented to the immune system by dendritic cells (DCs) that probe these filters for the presence of foreign antigen (Ag). However, larger pathogens, like helminths or infectious emboli, that exceed 20 µm are mostly trapped in the vasculature of the lung. To determine if Ag trapped here can be presented to cells of the immune system, we used a model of venous embolism of large particulate Ag (in the form of ovalbumin [OVA]-coated Sepharose beads) in the lung vascular bed. We found that large Ags were presented and cross-presented to CD4 and CD8 T cells in the mediastinal lymph nodes (LNs) but not in the spleen or liver-draining LNs. Dividing T cells returned to the lungs, and a short-lived infiltrate consisting of T cells and DCs formed around trapped Ag. This infiltrate was increased when the Toll-like receptor 4 was stimulated and full DC maturation was induced by CD40 triggering. Under these conditions, OVA-specific cytotoxic T lymphocyte responses, as well as humoral immunity, were induced. The T cell response to embolic Ag was severely reduced in mice depleted of CD11chi cells or Ly6C/G+ cells but restored upon adoptive transfer of Ly6Chi monocytes. We conclude that the lung vascular filter represents a largely unexplored site of immune induction that traps large bloodborne Ags for presentation by monocyte-derived DCs.

Differential priming of CD8 and CD4 T-cells in animal models of autoimmune hepatitis and cholangitis

The pathogenesis of autoimmune liver diseases is poorly understood. Animal models are necessary to investigate antigen presentation and priming of T-cells in the context of autoimmunity in the liver. Transgenic mouse models were generated in which the model antigen ovalbumin is expressed in hepatocytes (TF-OVA) or cholangiocytes (ASBT-OVA). Transgenic OT-I (CD8) or OT-II (CD4) T-cells specific for ovalbumin were adoptively transferred into TF-OVA and ASBT-OVA mice to induce in vivo priming of antigen-specific T-cells. T-cell migration and activation, as well as induction of liver inflammation, were studied. OT-I T-cells preferentially located to the liver of both mouse strains whereas no migration of OT-II T-cells to the liver was observed. OT-I T-cells proliferated in the liver of TF-OVA mice and the liver and liver draining lymph nodes of ASBT-OVA mice. OT-II CD4 T-cells were activated in spleen and liver draining lymph node of TF-OVA mice but not in ASBT-OVA mice. Transfer of OT-I T-cells led to histologically distinct inflammatory conditions in the liver of ASBT-OVA and TF-OVA mice and caused liver injury as determined by the elevation of serum alanine aminotransferase. An antigen expressed in hepatocytes is presented to CD8 and CD4 T-cells, whereas the same antigen expressed in cholangiocytes is presented to CD8 but not CD4 T-cells. In both models, activation of CD8 T-cells occurs within the liver and causes liver inflammation. The models presented here are valuable to investigate the priming of T-cells in the liver and their role in the development of autoimmune disease of the liver.

Plasmacytoid Dendritic Cells Do Not Migrate in Intestinal or Hepatic Lymph

Plasmacytoid dendritic cells (pDCs) recognize pathogen-associated molecules, particularly viral, and represent an important mechanism in innate defense. They may however, also have roles in steady-state tolerogenic responses at mucosal sites. pDCs can be isolated from blood, mucosa, and lymph nodes (LNs). Although pDCs can express peripherally derived Ags in LNs and at mucosal sites, it is not clear whether pDCs actually migrate from the periphery in lymph or whether LN pDCs acquire Ags by other mechanisms. To determine whether pDCs migrate in lymph, intestine or liver-draining LNs were removed and thoracic duct leukocytes (TDLs) were collected. TDLs expressing MHC-II and CD45R, but not TCRalphabeta or CD45RA, were then analyzed. These enriched TDLs neither transcribe type I IFNs nor secrete inflammatory cytokines in response to viral stimuli in vitro or after a TLR7/8 stimulus in vivo. In addition, these TDLs do not express CD5, CD90, CD200, or Siglec-H, but do express Ig, and therefore represent B cells, despite their lack of CD45RA expression. Intestinal and hepatic lymph are hence devoid of bona fide pDCs under both steady-state conditions and after TLR7/8 stimulation. This shows that any role for pDCs in Ag-specific T cell activation or tolerance must differ from the roles of classical dendritic cells, because it cannot result from peripheral Ag capture, followed by migration of pDCs via lymph to the LN.