CD4+CD25+ Regulatory T Cells Inhibit Natural Killer Cell Hepatocytotoxicity of Hepatitis B Virus Transgenic Mice via Membrane-Bound TGF-? and OX40.
ABSTRACT: CD4+CD25+ regulatory T cells (Tregs) are involved in the regulation of physiological and pathological hepatic immune responses, but the roles are not well explored in natural killer (NK) cell-mediated liver diseases. In this study, using the NK cell-mediated oversensitive liver injury model of hepatitis B virus transgenic (HBs-Tg) mice triggered by a low dose of concanavalin A, it was observed that an increased number of CD4+CD25+Foxp3+ Tregs were accumulated in the liver, along with the recovery of liver injury. Adoptive transfer of hepatic Tregs from HBs-Tg mice but not wild B6 mice could significantly attenuate the oversensitive liver injury via inhibiting liver accumulation and decreasing NK cell group 2D-mediated activation of NK cells in the recipient HBs-Tg mice. Furthermore, upregulated expression of membrane-bound TGF-? (mTGF-?) and OX40 on hepatic Tregs were demonstrated to account for inhibiting the NK cell-mediated hepatic injury in HBs-Tg mice through cell-cell contact, confirmed by antibody blockade and cell Transwell experiments in vivo and in vitro. Our findings for the first time indicated that CD4+CD25+ Tregs directly suppressed NK cell-mediated hepatocytotoxicity through mTGF-? and OX40/OX40L interaction in a cell-cell contact manner in HBV-associated liver disease.
Project description:Gut-derived bacterial products contribute to liver inflammation and injury during chronic hepatitis B virus infection; however, the underlying mechanisms remain obscure. In this study, hepatitis B surface antigen transgenic (HBs-Tg) mice and their wild-type (WT) control C57BL/6 mice were injected with CpG-oligodeoxynucleotides (ODNs) to mimic the translocation of gut microbial products into the systemic circulation. We found that, compared with the WT mice, the HBs-Tg mice were oversensitive to CpG-ODN-induced liver injury, which was dependent on natural killer T (NKT) cells. CpG-ODN injection enhanced the expression of Fas ligand (FasL) on NKT cells. In addition, hepatocytes from the HBs-Tg mice expressed higher levels of Fas than did those from the WT mice, which was further augmented by CpG-ODN. Interaction of Fas and FasL was involved in the cytotoxicity of NKT cells against hepatocytes in the HBs-Tg mice. Moreover, Kupffer cells in the HBs-Tg mice expressed higher levels of CD205 and produced greater amounts of interleukin (IL)-12 than did those in the WT mice. Finally, the depletion of Kupffer cells, neutralization of IL-12 or specific silencing of CD205 on Kupffer cells significantly inhibited CpG-ODN-induced liver injury and NKT activation in the HBs-Tg mice. Our data suggest that CD205-expressing Kupffer cells respond to CpG-ODNs and subsequently release IL-12 to promote NKT cell activation. Activated NKT cells induce liver damage through the Fas signaling pathway in HBs-Tg mice.
Project description:Although recent studies have identified important roles for T and NK cells in the pathogenesis of biliary atresia (BA), the mechanisms by which susceptibility to bile duct injury is restricted to the neonatal period are unknown.We characterised hepatic regulatory T cells (Tregs) by flow cytometry in two groups of neonatal mice challenged with rhesus rotavirus (RRV) at day 7 (no ductal injury) or day 1 of life (resulting in BA), determined the functional interaction with effector cells in co-culture assays, and examined the effect of adoptive transfer of CD4+ cells on the BA phenotype.While day 7 RRV infection increased hepatic Tregs (Foxp3+ CD4+ CD25+) by 10-fold within 3 days, no increase in Tregs occurred at this time point following infection on day 1. In vitro, Tregs effectively suppressed NK cell activation by hepatic dendritic cells and decreased the production of pro-inflammatory cytokines, including TNFalpha and IL-15, following RRV infection. In vivo, adoptive transfer of CD4+ cells prior to RRV inoculation led to increased survival, improved weight gain, decreased population of hepatic NK cells, and persistence of donor Tregs in the liver.(1) The liver is devoid of Tregs early after perinatal RRV infection; (2) Tregs suppress DC-dependent activation of naive NK cells in vitro, and Treg-containing CD4+ cells inhibit hepatic NK cell expansion in vivo. Thus, the post-natal absence of Tregs may be a key factor that allows hepatic DCs to act unopposed in NK cell activation during the initiation of neonatal bile duct injury.
Project description:The role of hepatic NK cells in the pathogenesis of HCV-associated hepatic failure is incompletely understood. In this study, we investigated the effect of HCV on ConA-induced immunological hepatic injury and the influence of HCV on hepatic NK cell activation in the liver after ConA administration. An immunocompetent HCV mouse model that encodes the entire viral polyprotein in a liver-specific manner based on hydrodynamic injection and ?C31o integrase was used to study the role of hepatic NK cells. Interestingly, the frequency of hepatic NK cells was reduced in HCV mice, whereas the levels of other intrahepatic lymphocytes remained unaltered. Next, we investigated whether the reduction in NK cells within HCV mouse livers might elicit an effect on immune-mediated liver injury. HCV mice were subjected to acute liver injury models upon ConA administration. We observed that HCV mice developed more severe ConA-induced immune-mediated hepatitis, which was dependent on the accumulated intrahepatic NK cells. Our results indicated that after the administration of ConA, NK cells not only mediated liver injury through the production of immunoregulatory cytokines (IFN-?, TNF-? and perforin) with direct antiviral activity, but they also killed target cells directly through the TRAIL/DR5 and NKG2D/NKG2D ligand signaling pathway in HCV mice. Our findings suggest a critical role for NK cells in oversensitive liver injury during chronic HCV infection.
Project description:Hepatic macrophages play a central role in disease pathogenesis during hepatitis B virus (HBV) infection. Our previous study found that CD205<sup>+</sup> macrophages in the liver of hepatitis B surface antigen transgenic (HBs-Tg) mice increased significantly compared with those in wild-type mice, and these increased CD205<sup>+</sup> macrophages were involved in CpG-oligodeoxynucleotide-induced liver injury in HBs-Tg mice. Here, we analysed the phenotype and function of CD205<sup>+</sup> macrophages derived from the liver of HBs-Tg mice and patients with chronic hepatitis B (CHB). We found that HBs-Tg mice-derived hepatic macrophages produced larger amounts of pro-inflammatory cytokines, including IL-6, IL-12, TNF-?, and of the anti-inflammatory cytokine IL-10 after stimulation with CpG-oligodeoxynucleotides or commensal bacteria DNA than B6 mice-derived hepatic macrophages. Furthermore, hepatic CD205<sup>+</sup> macrophages from HBs-Tg mice showed an activated phenotype and expressed higher levels of inflammatory cytokine genes, chemokine genes, and phagocytosis-related genes than hepatic CD205<sup>-</sup> macrophages. In addition, CD205<sup>+</sup> macrophages displayed an inflammatory phenotype and were increased in the liver of patients with CHB compared with those in healthy controls. Our data suggest that hepatic CD205<sup>+</sup> macrophages are a unique pro-inflammatory subset observed during HBV infection. Thus, development of intervention targeting these cells is warranted for immunotherapy of HBV-induced liver diseases.
Project description:RTS,S/AS01, a vaccine targeting pre-erythrocytic stages of Plasmodium falciparum, is undergoing clinical trials. We report an analysis of cellular immune response to component Ags of RTS,S-hepatitis B surface Ag (HBs) and P. falciparum circumsporozoite (CS) protein-among Tanzanian children in a phase IIb RTS,S/AS01(E) trial. RTS,S/AS01 (E) vaccinees make stronger T cell IFN-?, CD69, and CD25 responses to HBs peptides than do controls, indicating that RTS,S boosts pre-existing HBs responses. T cell CD69 and CD25 responses to CS and CS-specific secreted IL-2 were augmented by RTS,S vaccination. Importantly, more than 50% of peptide-induced IFN-?(+) lymphocytes were NK cells, and the magnitude of the NK cell CD69 response to HBs peptides correlated with secreted IL-2 concentration. CD69 and CD25 expression and IL-2 secretion may represent sensitive markers of RTS,S-induced, CS-specific T cells. The potential for T cell-derived IL-2 to augment NK cell activation in RTS,S-vaccinated individuals, and the relevance of this for protection, needs to be explored further.
Project description:Overexpression of a constitutively active form of Stat5b (Stat5b-CA) increases regulatory T cells (Tregs). We show that Stat5b-CA transgenic (TG) CD4(+) T cells had a markedly reduced graft-versus-host disease (GVHD) capacity versus wild-type (WT) T cells. Stat5b-CA TG versus WT CD4(+) T cells had a higher proportion of Tregs, which were superior in suppressing alloresponses mediated by CD4(+)CD25(-) effector T cells (Teffs). By day 5 after transplantation, Stat5b-CA TG Tregs had expanded approximately 3-fold more than WT Tregs. Purified Stat5b-CA TG Tregs added to WT CD4(+)CD25(-) Teffs were superior on a per-cell basis for inhibiting GVHD versus WT Tregs. Surprisingly, rigorously Treg-depleted Stat5b-CA TG versus WT CD4(+)CD25(-) Teffs caused less GVHD lethality associated with diminished Teff proinflammatory and increased Th2 anti-inflammatory cytokine responses. Reduced GVHD by Stat5b-CA TG versus WT Teffs could not be explained by conversion into Tregs in day 10 posttransplantation spleen or small intestine. In addition, Stat5b-CA TG Teffs retained a graft-versus-leukemia response. These results indicate a major role for Stat5 in Treg expansion and potency along with a lesser but significant role in Teff activation and suggest a strategy of pharmacologic Stat5b up-regulation as a means of decreasing GVHD while retaining a graft-versus-leukemia effect.
Project description:Increasing evidence indicates a role for regulatory T cells (Tregs) in the immune response and in autoimmune diseases, but the role of Tregs and cytokines in autoimmune hepatic diseases remains largely unclear and controversial, especially in patients with primary biliary cirrhosis (PBC). This study was undertaken to investigate Tregs and different cytokines in the liver and peripheral blood of PBC patients. We found that these patients demonstrated a reduction of CD4(+)CD25(+) T cells but elevated CD4(+)Foxp3(+) T cells in peripheral blood mononuclear cells (PBMCs) and CD4(+) T cells. The percentage of CD4(+)CD25(+) T cells in PBMCs was negatively correlated with elevated plasma interferon (IFN)-γ levels. A liver-specific analysis showed that the frequency of Foxp3(+) Tregs, transforming growth factor (TGF)-β1 and IFN-γ were increased in PBC patients. Our findings suggest that an imbalance between CD4(+)CD25(+) Tregs and cytotoxic cytokines plays a crucial role in the pathogenesis of PBC while the role of Foxp3 needs further investigation.
Project description:Virus infection, such as hepatitis B virus (HBV), occasionally causes endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) is counteractive machinery to ER stress, and the failure of UPR to cope with ER stress results in cell death. Mechanisms that regulate the balance between ER stress and UPR are poorly understood. Type 1 and type 2 interferons have been implicated in hepatic flares during chronic HBV infection. Here, we examined the interplay between ER stress, UPR, and IFNs using transgenic mice that express hepatitis B surface antigen (HBsAg) (HBs-Tg mice) and humanized-liver chimeric mice infected with HBV. IFNα causes severe and moderate liver injury in HBs-Tg mice and HBV infected chimeric mice, respectively. The degree of liver injury is directly correlated with HBsAg levels in the liver, and reduction of HBsAg in the transgenic mice alleviates IFNα mediated liver injury. Analyses of total gene expression and UPR biomarkers' protein expression in the liver revealed that UPR is induced in HBs-Tg mice and HBV infected chimeric mice, indicating that HBsAg accumulation causes ER stress. Notably, IFNα administration transiently suppressed UPR biomarkers before liver injury without affecting intrahepatic HBsAg levels. Furthermore, UPR upregulation by glucose-regulated protein 78 (GRP78) suppression or low dose tunicamycin alleviated IFNα mediated liver injury. These results suggest that IFNα induces ER stress-associated cell death by reducing UPR. IFNγ uses the same mechanism to exert cytotoxicity to HBsAg accumulating hepatocytes. Collectively, our data reveal a previously unknown mechanism of IFN-mediated cell death. This study also identifies UPR as a potential target for regulating ER stress-associated cell death.
Project description:For many years, high-affinity subunit of IL-2 receptor (CD25) has been considered as a promising therapeutic target for different pathologic conditions like allograft rejection, autoimmunity, and cancers. Although CD25 is transiently expressed by newly-activated T cells, it is the hallmark of regulatory T (Treg) cells which are the most important immunosuppressive elements in tumor microenvironment. Thus, Tregs can be considered as a potential target for chimeric antigen receptor (CAR)-based therapeutic approaches. On the other hand, due to some profound adverse effects pertaining to the use of CAR T cells, CAR NK cells have caught researchers' attention as a safer choice. Based on these, the aim of this study was to design and develop a CAR NK cell against CD25 as the most prominent biomarker of Tregs with the prospect of overcoming immune escape mechanism in solid and liquid cancers. In the current study, an anti-CD25 CAR was designed and evaluated by comprehensive <i>in silico</i> analyses. Then, using lentiviral transduction system, NK-92 cell line was engineered to express this anti-CD25 CAR construct. <i>In vitro</i> functional analyses of anti-CD25 CAR for its reactivity against CD25 antigen as well as for cytotoxicity and cytokine production assays against CD25 bearing Jurkat cell line were done. <i>In silico</i> analyses demonstrated that the anti-CD25 CAR transcript and scFv protein structures were stable and had proper interaction with the target. Also, <i>in vitro</i> analyses showed that the anti-CD25 CAR-engineered NK-92 cells were able to specifically detect and lyse target cells with an appropriate cytokine production and cytotoxic activity. To conclude, the results showed that this novel CAR NK cell is functional and warrant further investigations.
Project description:IL-12 and IL-18 synergize to promote T<sub>H</sub>1 responses and have been implicated as accelerators of autoimmune pathogenesis in type 1 diabetes (T1D). We investigated the influence of these cytokines on immune cells involved in human T1D progression: natural killer (NK) cells, regulatory T cells (Tregs), and cytotoxic T lymphocytes (CTL). NK cells from T1D patients exhibited higher surface CD226 versus controls and lower CD25 compared to first-degree relatives and controls. Changes in NK cell phenotype towards terminal differentiation were associated with cytomegalovirus (CMV) seropositivity, while possession of IL18RAP, IFIH1, and IL2RA T1D-risk variants impacted NK cell activation as evaluated by immuno-expression quantitative trait loci (eQTL) analyses. IL-12 and IL-18 stimulated NK cells from healthy donors exhibited enhanced specific killing of myelogenous K562 target cells. Moreover, activated NK cells increased expression of NKG2A, NKG2D, CD226, TIGIT and CD25, which enabled competition for IL-2 upon co-culture with Tregs, resulting in Treg downregulation of FOXP3, production of IFN?, and loss of suppressive function. We generated islet-autoreactive CTL "avatars", which upon exposure to IL-12 and IL-18, upregulated IFN? and Granzyme-B leading to increased lymphocytotoxicity of a human ?-cell line in vitro. These results support a model for T1D pathogenesis wherein IL-12 and IL-18 synergistically enhance CTL and NK cell cytotoxic activity and disrupt immunoregulation by Tregs.