Hepatic CD4 Research Articles

Murine autoimmune cholangitis requires two hits: cytotoxic KLRG1+ CD8 effector cells and defective T regulatory cells (BA13P.122)

Abstract Primary biliary cirrhosis (PBC) is an enigmatic disease mediated by autoimmune destruction of cholangiocytes in hepatic bile ducts. Early immunological events in PBC are poorly understood; clinical signs of disease occur very late in the pathological process. We have used our unique murine model of PBC in dominant-negative TGF-β receptor type II transgenic mice to delineate critical early immunopathological pathways, and previously showed that dnTGFβRII CD8 T cells alone transfer biliary disease. Herein we report significantly increased numbers of hepatic terminally differentiated (KLRG1+) CD8 T cells in dnTGFβRII compared to B6; these cells are the major cytotoxic CD8 subset. Unexpectedly, the disease is not simply CD8 T cell intrinsic since mixed bone marrow chimeric mice were protected from biliary disease. Adoptive transfer studies showed that co-transfer of B6 but not dnTGFβRII Tregs prevented dnTGFβRII CD8 T cell mediated cholangitis; the protection was associated with decreased numbers of hepatic KLRG1+ CD8 T cells and with elimination of hepatic dnTGFβRII CD8 mediated cholangiocyte cytotoxicity. DnTGFβRII Treg cells were inferior in suppressing effector CD8 T cells in vitro compared to wild type B6 Tregs. In conclusion, these results emphasize that autoimmune cholangitis requires defects in both the T effector and regulatory compartments, which has important implications for understanding the early pathogenesis of human PBC.

Myeloid dendritic cells maintain Th17 lymphocyte responses during the obstructive phase of experimental biliary atresia (IRC10P.471)

Abstract Biliary atresia (BA) is a fibro-inflammatory obstruction of the extrahepatic biliary tree in newborns. It leads to rapid biliary fibrosis and is the most common indication for pediatric liver transplantation. In a model of rhesus rotavirus (RRV)-induced murine BA, plasmacytoid dendritic cells (DC) initiate NK-cell mediated hepatobiliary injury at 3 days post infection (dpi). We hypothesize that myeloid (m)DC coordinate T-cell driven injury during the later obstructive phase of BA. Following RRV injection into CD11c-DTR GFP pups on day 1 of life, frequency of hepatic DC increased 6fold at 12dpi compared with non-infected controls. Expansion of mDCs was accompanied by accumulation of activated CD4 cells in the liver. Plasma IL17A/F levels were increased at 8 and 12dpi (p<0.01) and hepatic IL17A mRNA increased 9fold between 8 and 12dpi. In vitro coculture of DC from 12dpi pups with naïve CD4 cells induced IL17A/6 production. DC-depletion following RRV infection with diphtheria toxin reduced the number of hepatic CD4 cells by 3fold which was associated with 2fold decrease in hepatic IL17A mRNA expression and plasma Th17-associated cytokine levels (IL-17F/6) at 12dpi. Importantly, diminished Th17 responses correlated with reduced plasma total bilirubin levels (6.1 vs 11.3mg/dL in control; p<0.01), a biomarker for biliary obstruction. We conclude that mDC maintain hepatic Th17 responses and regulate cholestatic liver injury during the obstructive phase of experimental BA.

Distinct from its canonical effects, deletion of IL-12p40 induces cholangitis and fibrosis in interleukin-2Rα−/− mice

The IL-12 family modulates T cell mediated autoimmune diseases and GWAS in PBC have suggested a critical role of IL-12 and its subunits in modulating portal inflammation. We have taken advantage of an aggressive model of portal inflammation and colitis in IL-2Rα−/− mice to study the specific role of IL-12 and, in particular, the immunobiology of p40−/−IL-2Rα−/− mice. Colonies of IL-2Rα+/−, IL-2Rα−/− and p40−/−IL-2Rα−/− mice were studied for the natural history of immunopathology in liver and colon using histology and immunohistochemistry. Further, to focus on mechanisms, liver, spleen and mesenteric lymph node flow cytometry was employed to identify specific phenotypes; cytokine analysis on inflammatory cell populations was compared between groups. Finally, Real-Time PCR was used to focus on the genes involved in hepatic fibrosis. Surprisingly, p40−/−IL-2Rα−/− mice manifest more severe portal inflammation and bile duct damage, including signs of portal hypertension and liver fibrosis, but a significant reduction in colitis. Indeed, p40−/−IL-2Rα−/− mice reveal a profound hepatic CD8+ T cell infiltrate, whose major component are effector memory cells as well as enhanced hepatic Th1 but reduced Th17 responses. These observations were confirmed by Real-Time PCR analysis of fibrosis-related genes in the liver. Distinct from its canonical effects, IL-12p40 plays a critical role in autoimmune cholangitis, including hepatic fibrosis. These data take on striking significance for any proposed human trials that modulate the IL-12p40 pathway in human PBC.

Murine autoimmune cholangitis requires two hits: Cytotoxic KLRG1+ CD8 effector cells and defective T regulatory cells

Primary biliary cirrhosis (PBC) is an enigmatic disease mediated by autoimmune destruction of cholangiocytes in hepatic bile ducts. The early immunological events leading to PBC are poorly understood; clinical signs of disease occur very late in the pathological process. We have used our unique murine model of PBC in dominant-negative TGF-β receptor type II transgenic mice to delineate critical early immunopathological pathways, and previously showed that dnTGFβRII CD8 T cells transfer biliary disease. Herein we report significantly increased numbers of hepatic dnTGFβRII terminally differentiated (KLRG1+) CD8 T cells, a CD8 subset previously shown to be enriched in antigen specific cells during hepatic immune response to viral infections. We performed bone marrow chimera studies to assess whether dnTGFβRII CD8 mediated disease was cell intrinsic or extrinsic. Unexpectedly, mixed (dnTGFβRII and B6) bone marrow chimeric (BMC) mice were protected from biliary disease compared to dnTGFβRII single bone marrow chimerics. To define the protective B6 cell subset, we performed adoptive transfer studies, which showed that co-transfer of B6 Tregs prevented dnTGFβRII CD8 T cell mediated cholangitis. Treg mediated disease protection was associated with significantly decreased numbers of hepatic KLRG1+ CD8 T cells. In contrast, co-transfer of dnTGFβRII Tregs offered no protection, and dnTGFβRII Treg cells were functionally defective in suppressing effector CD8 T cells in vitro compared to wild type B6 Tregs. In vitro cholangiocyte cytotoxicity assays demonstrated significantly increased numbers of cytotoxic hepatic dnTGFβRII KLRG1+ CD8 cells compared to B6. Protection from disease by B6 Tregs was associated with elimination of hepatic dnTGFβRII CD8 mediated cholangiocyte cytotoxicity. These results emphasize that autoimmune cholangitis requires defects in both the T effector and regulatory compartments, and that an intrinsic T cell effector defect is not sufficient to mediate autoimmune biliary disease in the setting of intact immune regulation. These results have important implications for understanding the early pathogenesis of human PBC.

CD49a promotes T‐cell‐mediated hepatitis by driving T helper 1 cytokine and interleukin‐17 production

It is becoming increasingly clear that the T-cell-mediated immune response is important in many diseases. In this study, we used concanavalin A (Con A) -induced hepatitis to investigate the role of CD49a in the molecular and cellular mechanism of the T-cell-mediated immune response. We found that CD49a(-/-) mice had significantly reduced levels of serum alanine aminotransferase and were protected from Con A-induced hepatitis. CD49a deficiency led to decreased production of interferon-γ (IFN-γ) and interleukin-17A (IL-17A) after Con A injection. Furthermore, we found that hepatic CD4(+) T cells and invariant natural killer T cells up-regulated CD49a expression, along with enhanced activation after Con A injection, leading to production of inflammatory cytokines by these T cells. Blockade of CD49a in vivo ameliorated Con A-induced hepatitis with reduced production of IFN-γ and IL-17A. Hence, CD49a promoted Con A-induced hepatitis through enhancing inflammatory cytokine production (IFN-γ and IL-17A) by CD4(+) T and invariant natural killer T cells. The protective effect of CD49a blockade antibody suggested a new target therapeutic molecule for intervention of T-cell-mediated liver injury.

Myeloid-derived suppressor cells are associated with viral persistence and downregulation of TCR ζ chain expression on CD8(+) T cells in chronic hepatitis C patients.

Myeloid-derived suppressor cells (MDSCs) play an important role in impairing the function of T cells. We characterized MDSCs in two chronic hepatitis C (CHC) cohorts: a cross-sectional group that included 61 treatment-naive patients with CHC, 14 rapid virologic response (RVR) cases and 22 early virologic response (EVR) cases; and a longitudinal group of 13 cases of RVR and 10 cases of EVR after pegylated-interferon-α/ribavirin treatment for genotype 1b HCV infection. Liver samples from 32 CHC patients and six healthy controls were subjected to immunohistochemical analysis. MDSCs frequency in treatment-naive CHC was significantly higher than in RVR, EVR, or healthy subjects and was positively correlated with HCV RNA. Patients infected with HCV genotype 2a had a significantly higher frequency of MDSCs than those infected with genotype 1b. Decreased T cell receptor (TCR) ζ expression on CD8+ T cells was significantly associated with an increased frequency of MDSCs in treatment-naive CHC patients and was restored by L-arginine treatment in vitro. Increased numbers of liver arginase-1+ cells were closely associated with the histological activity index in CHC. The TCR ζ chain was significantly downregulated on hepatic CD8+ T cells in CHC. During antiviral follow up, MDSCs frequency in peripheral blood mononuclear cells was directly correlated with the HCV RNA load in the plasma and inversely correlated with TCR ζ chain expression in CD8+ T cells in both RVR and EVR cases. Notably, the RVR group had a higher frequency of MDSCs at baseline than the EVR group. Collectively, this study provides evidence that MDSCs might be associated with HCV persistence and downregulation of CD8 ζ chain expression.

Unique T Cells with Unconventional Cytokine Profiles Induced in the Livers of Mice during Schistosoma mansoni Infection

During infection with Schistosoma, serious hepatic disorders are induced in the host. The liver possesses unique immune systems composed of specialized cells that differ from those of other immune competent organs or tissues. Host immune responses change dramatically during Schistosoma mansoni infection; in the early phase, Th1-related responses are induced, whereas during the late phase Th2 reactions dominate. Here, we describe unique T cell populations induced in the liver of mice during the period between Th1- and Th2-phases, which we term the transition phase. During this phase, varieties of immune cells including T lymphocytes increase in the liver. Subsets of CD4+ T cells exhibit unique cytokine production profiles, simultaneously producing both IFN-γ and IL-13 or both IFN-γ and IL-4. Furthermore, cells triply positive for IFN-γ, IL-13 and IL-4 also expand in the S. mansoni-infected liver. The induction of these unique cell populations does not occur in the spleen, indicating it is a phenomenon specific to the liver. In single hepatic CD4+ T cells showing the unique cytokine profiles, both T-bet and GATA-3 are expressed. Thus, our studies show that S. mansoni infection triggers the induction of hepatic T cell subsets with unique cytokine profiles.

The Tim-3/galectin-9 pathway involves in the homeostasis of hepatic Tregs in a mouse model of concanavalin A-induced hepatitis

T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) is a negative regulator of interferon (IFN)-γ-secreting CD4+ Th1 cells and plays a key role in autoimmune diseases. Here, we report that galectin-9 expression was increased in hepatic CD4+CD25+ T cells in a mouse model of concanavalin A (Con A)-induced hepatitis. Moreover, Tim-3 showed increased levels in CD4+CD25+ Foxp3+ regulatory T cells (Tregs). Further analyses showed that blocking the Tim-3/galectin-9 pathway resulted in the suppression of Tregs in vitro, thereby significantly increasing interferon (IFN)-γ production from hepatic Teffs. Moreover, blockade of Tim-3 in vivo with an anti-Tim-3 antibody exacerbated the acute hepatitis, possibly by increased IFN-γ production. Furthermore, we found that in vitro activation of CD4+CD25− T cells with the T cell receptor (TCR) plus interleukin 2 (IL-2) up-regulated Tim-3 expression. And the induced Tim-3 interacted with galectin-9 to induce CD4+ T cell apoptosis which could be partly reversed by blocking Tim-3 signaling. Our results suggested that the Tim-3/galectin-9 pathway plays a critical role in the homeostasis of hepatic Tregs through the elimination induction in Teffs and the inhibition of IFN-γ release, which contributes to the pathogenesis of liver damage and constitutes at least part of the mechanism underlying the induction of hepatitis by Con A.

Changes in immune cell populations in the periphery and liver of GBV-B-infected and convalescent tamarins (Saguinus labiatus)

Flaviviruses related to hepatitis C virus (HCV) in suitable animal models may provide further insight into the role that cellular immunity contributes to spontaneous clearance of HCV. We characterised changes in lymphocyte populations in tamarins with an acute GBV-B infection, a hepatitis virus of the flaviviridae. Major immune cell populations were monitored in peripheral and intra-hepatic lymphocytes at high viraemia or following a period when peripheral virus was no longer detected. Limited changes in major lymphocyte populations were apparent during high viraemia; however, the proportions of CD3+ lymphocytes decreased and CD20+ lymphocytes increased once peripheral viraemia became undetectable. Intrahepatic lymphocyte populations increased at both time points post-infection. Distinct expression patterns of PD-1, a marker of T-cell activation, were observed on peripheral and hepatic lymphocytes; notably there was elevated PD-1 expression on hepatic CD4+ T-cells during high viraemia, suggesting an activated phenotype, which decreased following clearance of peripheral viraemia. At times when peripheral vRNA was not detected, suggesting viral clearance, we were able to readily detect GBV-B RNA in the liver, indicative of long-term virus replication. This study is the first description of changes in lymphocyte populations during GBV-B infection of tamarins and provides a foundation for more detailed investigations of the responses that contribute to the control of GBV-B infection.

WSX-1 Signalling Inhibits CD4+ T Cell Migration to the Liver during Malaria Infection by Repressing Chemokine-Independent Pathways

IL-27 is an important and non-redundant regulator of effector T cell accumulation in non-lymphoid tissues during infection. Using malaria as a model systemic pro-inflammatory infection, we demonstrate that the aberrant accumulation of CD4+ T cells in the liver of infected IL27R−/− (WSX-1−/−) mice is a result of differences in cellular recruitment, rather than changes in T cell proliferation or cell death. We show that IL-27 both inhibits the migratory capacity of infection-derived CD4+ T cells towards infection-derived liver cells, but also suppresses the production of soluble liver-derived mediator(s) that direct CD4+ T cell movement towards the inflamed tissue. Although CCL4 and CCL5 expression was higher in livers of infected WSX-1−/− mice than infected WT mice, and hepatic CD4+ T cells from WSX-1−/− mice expressed higher levels of CCR5 than cells from WT mice, migration of CD4+ T cells to the liver of WSX-1−/− mice during infection was not controlled by chemokine (R) signalling. However, anti-IL-12p40 treatment reduced migration of CD4+ T cells towards infection-derived liver cells, primarily by abrogating the hepatotropic migratory capacity of T cells, rather than diminishing soluble tissue-derived migratory signals. These results indicate that IL-27R signalling restricts CD4+ T cell accumulation within the liver during infection primarily by suppressing T cell chemotaxis, which may be linked to its capacity to repress Th1 differentiation, as well as by inhibiting the production of soluble, tissue-derived chemotaxins.

IL-12–Based Vaccination Therapy Reverses Liver-Induced Systemic Tolerance in a Mouse Model of Hepatitis B Virus Carrier

Liver-induced systemic immune tolerance that occurs during chronic hepadnavirus infection is the biggest obstacle for effective viral clearance. Immunotherapeutic reversal of this tolerance is a promising strategy in the clinic but remains to be explored. In this study, using a hepatitis B virus (HBV)-carrier mouse model, we report that IL-12-based vaccination therapy can efficiently reverse systemic tolerance toward HBV. HBV-carrier mice lost responsiveness to hepatitis B surface Ag (HBsAg) vaccination, and IL-12 alone could not reverse this liver-induced immune tolerance. However, after IL-12-based vaccination therapy, the majority of treated mice became HBsAg(-) in serum; hepatitis B core Ag was also undetectable in hepatocytes. HBV clearance was dependent on HBsAg vaccine-induced anti-HBV immunity. Further results showed that IL-12-based vaccination therapy strongly enhanced hepatic HBV-specific CD8(+) T cell responses, including proliferation and IFN-γ secretion. Systemic HBV-specific CD4(+) T cell responses were also restored in HBV-carrier mice, leading to the arousal of HBsAg-specific follicular Th-germinal center B cell responses and anti-hepatitis B surface Ag Ab production. Recovery of HBsAg-specific responses also correlated with both reduced CD4(+)Foxp3(+) regulatory T cell frequency and an enhanced capacity of effector T cells to overcome inhibition by regulatory T cells. In conclusion, IL-12-based vaccination therapy may reverse liver-induced immune tolerance toward HBV by restoring systemic HBV-specific CD4(+) T cell responses, eliciting robust hepatic HBV-specific CD8(+) T cell responses, and facilitating the generation of HBsAg-specific humoral immunity; thus, this therapy may become a viable approach to treating patients with chronic hepatitis B.

Perforin and granzymes work in synergy to mediate cholangiocyte injury in experimental biliary atresia

Biliary atresia represents obstructive cholangiopathy in infants progressing rapidly to cirrhosis and end-stage liver disease. Activated NK cells expressing Nkg2d have been linked to bile duct injury and obstruction by establishing contact with cholangiocytes. To define the mechanisms used by cytotoxic cells, we investigated the role of perforin and granzymes in a neonatal mouse model of rotavirus (RRV)-induced biliary atresia. We used complementary cell lysis assays, flow cytometric analyses, quantitative PCRs and in vivo systems to determine the mechanisms of bile duct epithelial injury and the control of the tissue phenotype in experimental biliary atresia. RRV-infected hepatic NK and CD8 T cells increased the expression of perforin and injured cholangiocytes in short-term culture in a perforin-dependent fashion. However, the loss of perforin in vivo delayed but did not prevent the obstruction of bile ducts. Based on the increased expression of granzymes by perforin-deficient cytotoxic cells in long-term cytolytic assays, we found that the inhibition of granzymes by nafamostat mesilate (FUT-175) blocked cholangiocyte lysis. Administration of FUT-175 to perforin-deficient mice after RRV infection decreased the development of jaundice, minimized epithelial injury, and improved long-term survival. However, the inhibition of granzymes alone in wild-type mice was not sufficient to prevent the atresia phenotype in newborn mice. In infants with biliary atresia, hepatic Granzymes A and B mRNA, but not Perforin, increased at the time of portoenterostomy. Perforin and granzymes have complementary roles mediating epithelial injury by NK and CD8 T cells. The prevention of experimental biliary atresia can only be achieved by inhibiting both granules.

Adipose tissue derived stromal stem cell therapy in murine ConA‐derived hepatitis is dependent on myeloid‐lineage and CD4+T‐cell suppression

Mesenchymal stromal stem cells (MSCs) are an attractive therapeutic model for regenerative medicine due to their pluripotency. MSCs are used as a treatment for several inflammatory diseases, including hepatitis. However, the detailed immunopathological impact of MSC treatment on liver disease, particularly for adipose tissue derived stromal stem cells (ADSCs), has not been described. Here, we investigated the immuno-modulatory effect of ADSCs on hepatitis using an acute ConA C57BL/6 murine hepatitis model. i.v. administration of ADSCs simultaneously or 3 h post injection prevented and treated ConA-induced hepatitis. Immunohistochemical analysis revealed higher numbers of CD11b(+), Gr-1(+), and F4/80(+) cells in the liver of ConA-induced hepatitis mice was ameliorated after the administration of ADSCs. Hepatic expression of genes affected by ADSC administration indicated tissue regeneration-related biological processes, affecting myeloid-lineage immune-mediating Gr-1(+) and CD11b(+) cells. Pathway analysis of the genes expressed in ADSC-treated hepatic inflammatory cells revealed the possible involvement of T cells and macrophages. TNF-α and IFN-γ expression was downregulated in hepatic CD4(+) T cells isolated from hepatitis livers co-cultured with ADSCs. Thus, the immunosuppressive effect of ADSCs in a C57BL/6 murine ConA hepatitis model was dependent primarily on the suppression of myeloid-lineage cells and, in part, of CD4(+) T cells.

Quantification of hepatic FOXP3+ T‐lymphocytes in HIV/hepatitis C coinfection

Coinfection with HIV adversely impacts every stage of hepatitis C (HCV) infection. Liver damage in HCV infection results from host antiviral responses rather than direct viral pathogenesis. Despite depressed cellular immunity, coinfected patients show accelerated hepatic fibrosis compared with HCV monoinfected patients. This paradox is poorly understood. T-regulatory (Treg) cells (CD4+ and FOXP3+) are hypothesized to limit hepatic damage in HCV. Our hypothesis was that reduced frequency of hepatic Treg in HIV/HCV coinfection compared with HCV monoinfection may explain poorer outcomes. We quantified FOXP3+, CD4+, CD8+ and CD20+ cells in liver biopsies of 35 male subjects matched by age and ISHAK fibrosis score, 12 HIV monoinfected, 11 HCV monoinfected and 12 HIV/HCV coinfected. Cell counts were performed using indirect immunohistochemical staining and light microscopy. HIV/HCV coinfected subjects had fewer hepatic FOXP3+ (P=0.031) and CD4+ cells (P=0.001) than HCV monoinfected subjects. Coinfected subjects had more hepatic CD8+ cells compared with HCV monoinfected (P=0.023), and a lower ratio of FOXP3+ to CD8+ cells (0.08 vs 0.27, P<0.001). Multivariate analysis showed number of CD4+ cells controlled for differences in number of FOXP3+ cells. Fewer hepatic FOXP3+ and CD4+ cells in HIV/HCV coinfection compared with HCV monoinfection suggests lower Treg activity, driven by an overall loss of CD4+ cells. Higher number of CD8+ cells in HIV/HCV coinfection suggests higher cytotoxic activity. This may explain poorer outcomes in HIV/HCV coinfected patients and suggests a potential mechanism by which highly active antiretroviral therapy may benefit these patients.

In vivo CD8+ T Cell Dynamics in the Liver of Plasmodium yoelii Immunized and Infected Mice

Plasmodium falciparum malaria remains one of the most serious health problems globally and a protective malaria vaccine is desperately needed. Vaccination with attenuated parasites elicits multiple cellular effector mechanisms that lead to Plasmodium liver stage elimination. While granule-mediated cytotoxicity requires contact between CD8+ effector T cells and infected hepatocytes, cytokine secretion should allow parasite killing over longer distances. To better understand the mechanism of parasite elimination in vivo, we monitored the dynamics of CD8+ T cells in the livers of naïve, immunized and sporozoite-infected mice by intravital microscopy. We found that immunization of BALB/c mice with attenuated P. yoelii 17XNL sporozoites significantly increases the velocity of CD8+ T cells patrolling the hepatic microvasculature from 2.69±0.34 μm/min in naïve mice to 5.74±0.66 μm/min, 9.26±0.92 μm/min, and 7.11±0.73 μm/min in mice immunized with irradiated, early genetically attenuated (Pyuis4-deficient), and late genetically attenuated (Pyfabb/f-deficient) parasites, respectively. Sporozoite infection of immunized mice revealed a 97% and 63% reduction in liver stage density and volume, respectively, compared to naïve controls. To examine cellular mechanisms of immunity in situ, naïve mice were passively immunized with hepatic or splenic CD8+ T cells. Unexpectedly, adoptive transfer rendered the motile CD8+ T cells from immunized mice immotile in the liver of P. yoelii infected mice. Similarly, when mice were simultaneously inoculated with viable sporozoites and CD8+ T cells, velocities 18 h later were also significantly reduced to 0.68±0.10 μm/min, 1.53±0.22 μm/min, and 1.06±0.26 μm/min for CD8+ T cells from mice immunized with irradiated wild type sporozoites, Pyfabb/f-deficient parasites, and P. yoelii CS280–288 peptide, respectively. Because immobilized CD8+ T cells are unable to make contact with infected hepatocytes, soluble mediators could potentially play a key role in parasite elimination under these experimental conditions.

Inducing Humoral and Cellular Responses to Multiple Sporozoite and Liver-Stage Malaria Antigens Using Exogenous Plasmid DNA

A vaccine candidate that elicits humoral and cellular responses to multiple sporozoite and liver-stage antigens may be able to confer protection against Plasmodium falciparum malaria; however, a technology for formulating and delivering such a vaccine has remained elusive. Here, we report the preclinical assessment of an optimized DNA vaccine approach that targets four P. falciparum antigens: circumsporozoite protein (CSP), liver stage antigen 1 (LSA1), thrombospondin-related anonymous protein (TRAP), and cell-traversal protein for ookinetes and sporozoites (CelTOS). Synthetic DNA sequences were designed for each antigen with modifications to improve expression and were delivered using in vivo electroporation (EP). Immunogenicity was evaluated in mice and nonhuman primates (NHPs) and assessed by enzyme-linked immunosorbent assay (ELISA), gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay, and flow cytometry. In mice, DNA with EP delivery induced antigen-specific IFN-γ production, as measured by ELISpot assay and IgG seroconversion against all antigens. Sustained production of IFN-γ, interleukin-2, and tumor necrosis factor alpha was elicited in both the CD4(+) and CD8(+) T cell compartments. Furthermore, hepatic CD8(+) lymphocytes produced LSA1-specific IFN-γ. The immune responses conferred to mice by this approach translated to the NHP model, which showed cellular responses by ELISpot assay and intracellular cytokine staining. Notably, antigen-specific CD8(+) granzyme B(+) T cells were observed in NHPs. Collectively, the data demonstrate that delivery of gene sequences by DNA/EP encoding malaria parasite antigens is immunogenic in animal models and can harness both the humoral and cellular arms of the immune system.

The inhibitory receptor NKG2A on NK cells controls CD8+ T cell responses to hepatic viral infection (P6109)

Abstract NKG2A is an inhibitory receptor expressed on a variety of liver lymphocytes, including NK and activated CD8+ T cells. NKG2A recruits the phosphatase SHP1 and inhibits signaling cascades necessary for immune cell activation when bound to its ligand, the nonclassical MHC-I Qa1b/Qdm complex. Elevated NKG2A expression on circulating NK cells is associated with chronic hepatitis C virus infections in humans; however, the impact of NKG2A+ NK cells in host immunity and chronic infections has not been elucidated. Using NKG2A-/- mice infected with adenovirus-ova, we have observed that loss of NKG2A signaling on NK cells leads to an increase of antigen-specific CD8+ T cell numbers with enhanced IFNγ production in the liver, but not the spleen, from days 6 through 40 post infection. Importantly, intrinsic NKG2A signaling on CD8+ T cells does not appear to control their proliferation or IFNγ expression. Furthermore, blockade of NKG2A-Qa-1b interaction on NK cells by the administration of Qa1b neutralization antibody or NK cell depletion prior to infection leads to increased hepatic CD8+ T cell numbers, suggesting NK cells are actively dampening CD8+ T cell cellularity and functions. Taken together, these results indicate that NK cell-mediated NKG2A signaling plays a pivotal role in controlling CD8+ T cell responses to hepatic viral infection.

1396 THERAPEUTIC EFFICACY OF D-PENICILLAMINE ENCAPSULATED ALGINATE/CHITOSAN NANOPARTICLES IN RAT MODEL OF COPPER TOXICITY WITH NEUROBEHAVIORAL IMPAIRMENTS

higher prevalence of bile duct obstruction in these mice (number of mice with obstructed/patent bile ducts: 9/2 in ABCB4 vs 3/11 in wt, p = 0.005). Furthermore, frequencies of hepatic CD8 and of NK lymphocytes were both higher in ABCB4 compared with wt mice (%CD8+CD3+/lymphocytes: 10.3 vs 6.2, p = 0.03; %NK+CD3−/lymphocytes: 12.7 vs 7.3%, p = 0.002), as determined by flow cytometry. Interestingly, enhanced lymphocyte activation was accompanied by 25-fold up-regulation of hepatic iNOS mRNA expression in these mice. Conclusion: An infant with EHBA had anatomical and molecular features of ABCB4/MDR3 deficiency. In a murine model, ABCB4 heterozygosity enhances hepatic CD8 and NK lymphocyte responses to virus infection and increases the frequency of ductal obstruction. Whether this activation is causally linked to hepatic up-regulation of iNOS and low phospholipid “toxic bile” requires further investigation.

Liver grafts from CD39-overexpressing rodents are protected from ischemia reperfusion injury due to reduced numbers of resident CD4+ T cells

Ischemia-reperfusion injury (IRI) is a major limiting event for successful liver transplantation, and CD4+ T cells and invariant natural killer T (iNKT) cells have been implicated in promoting IRI. We hypothesized that hepatic overexpression of CD39, an ectonucleotidase with antiinflammatory functions, will protect liver grafts after prolonged cold ischemia. CD39-transgenic (CD39tg) and wildtype (WT) mouse livers were transplanted into WT recipients after 18 hours cold storage and pathological analysis was performed 6 hours after transplantation. Serum levels of alanine aminotransferase and interleukin (IL)-6 were significantly reduced in recipients of CD39tg livers compared to recipients of WT livers. Furthermore, less severe histopathological injury was demonstrated in the CD39tg grafts. Immune analysis revealed that CD4+ T cells and iNKT cells were significantly decreased in number in the livers of untreated CD39tg mice. This was associated with a peripheral CD4+ T cell lymphopenia due to defective thymocyte maturation. To assess the relative importance of liver-resident CD4+ T cells and iNKT cells in mediating liver injury following extended cold preservation and transplantation, WT mice depleted of CD4+ T cells or mice genetically deficient in iNKT cells were used as donors. The absence of CD4+ T cells, but not iNKT cells, protected liver grafts from early IRI. Hepatic CD4+ T cells, but not iNKT cells, play a critical role in early IRI following extended cold preservation in a liver transplant model.

Overexpression of microRNA-21 is associated with elevated pro-inflammatory cytokines in dominant-negative TGF-β receptor type II mouse

Dominant-negative TGF-β receptor II (dnTGF-βRII) mice spontaneously develop an autoimmune cholangitis resembling human primary biliary cirrhosis (PBC). Interestingly, the dominant-negative TGF-β receptor is expressed by both CD4+ and CD8+ T cells and leads to greatly reduced (but not absent) TGF-β signaling resulting in T cell intrinsic cell mediated autoimmunity. However, the mechanisms of the T cell dysregulation remain unclear. Recently it has been shown that TGF-β signaling is intimately involved with miRNA biogenesis and control. Herein we show that lack of T cell TGF-β signaling leads to down regulation of T cell miRNAs but up-regulation of the key inflammatory miRNA 21. Furthermore, the expression of miR-21 from hepatic effector CD8+ T cells is significantly higher than in the same subsets isolated from spleen and mesenteric lymph nodes of the dnTGF-βRII mice. Previous studies indicate that miR-21 increases the synthesis of IFN-γ and IL-17A by T cells and suppresses apoptosis via programmed cell death protein 4 (PDCD4). Data presented herein demonstrate that transfecting w.t. B6 T cell subsets with miR-21 resulted in up-regulation of the inflammatory cytokines TNF-α and IFN-γ, thus partly replicating the dnTGF-βRII T cell phenotype. In conclusion, these data suggest miR-21 plays a critical role in the production of pro-inflammatory cytokines in dnTGF-βRII mice, which could be a contributing factor for the development of the organ-specific autoimmune cholangitis and colitis in this murine model of human PBC.