Direct CD1d-mediated stimulation of APC IL-12 production and protective immune response to virus infection in vivo.

CD1d-restricted NKT cells rapidly stimulate innate and adaptive immunity through production of Th1 and/or Th2 cytokines and induction of CD1d(+) APC maturation. However, therapeutic exploitation of NKT cells has been hampered by their paucity and defects in human disease. NKT cell-APC interactions can be modeled by direct stimulation of human APCs through CD1d in vitro. We have now found that direct ligation with multiple CD1d mAbs also stimulated bioactive IL-12 release from CD1d(+) but not CD1d knockout murine splenocytes in vitro. Moreover, all of the CD1d mAbs tested also induced IL-12 as well as both IFN-gamma and IFN-alpha in vivo from CD1d(+) but not CD1d-deficient recipients. Unlike IFN-gamma, CD1d-induced IFN-alpha was at least partially dependent on invariant NKT cells. Optimal resistance to infection with picornavirus encephalomyocarditis virus is known to require CD1d-dependent APC IL-12-induced IFN-gamma as well as IFN-alpha. CD1d ligation in vivo enhanced systemic IL-12, IFN-gamma, and IFN-alpha and was protective against infection by encephalomyocarditis virus, suggesting an alternative interpretation for previous results involving CD1d "blocking" in other systems. Such protective responses, including elevations in Th1 cytokines, were also seen with CD1d F(ab')(2)s in vivo, whereas an IgM mAb (with presumably minimal tissue penetration) was comparably effective at protection in vivo as well as cytokine induction both in vivo and in vitro. Although presumably acting immediately "downstream," CD1d mAbs were protective later during infection than the invariant NKT cell agonist alpha-galactosylceramide. These data indicate that NKT cells can be bypassed with CD1d-mediated induction of robust Th1 immunity, which may have therapeutic potential both directly and as an adjuvant.

[Preparation of monoclonal antibody against E domain III (ED3) protein of duck Tembusu virus with neutralizing activity].

Objective To prepare the specific monoclonal antibody (mAb) against E domain III (ED3) of duck Tembusu virus (DTMUV) and explore its neutralization activity. Methods The ED3 gene was amplified by using reverse transcription PCR according to the genome of the DTMUV AH-F10 strain. Then, the recombinant expression vector pET32a-ED3 was constructed and transformed into E.coli Rosetta. The ED3 protein was expressed and purified by nickel column affinity chromatography. After the recombinant ED3 protein was identified by SDS-PAGE and Western blot analysis, the BALB/c mice were immunized subcutaneously three times. The splenocytes of the immunized mice were hybridized with Sp2/0 myeloma cells, and the hybridization was screened by the limiting dilution method. The specificity and sensitivity of the antibody were identified by indirect immuno-fluorescent assay and Western blot analysis. Subsequently, antibody titers were determined by ELISA. Finally, this study titrated the neutralization titers of the antibodies on DTMUV-infected BHK-21 cells. Results The ED3 protein was successfully prepared and purified using the prokaryotic expression system. Three strains of monoclonal antibodies named B9D10C7, B9D7B8G10 and B9D7B8F11 were prepared. Their subtypes were IgG1, IgG2a and IgG2b, respectively. The titers of monoclonal antibody ascites can reach 1:51 200, and they could specifically recognize the E protein of DTMUV. Neutralization test showed that they had a certain neutralizing activities. Conclusion The monoclonal antibodies against ED3 protein of DTMUV are successfully prepared.

Anemia and Diabetic Kidney Disease Had Joint Effect on Diabetic Retinopathy Among Patients With Type 2 Diabetes.

Purpose: Whether the association between diabetic kidney disease (DKD) and diabetic retinopathy (DR) in patients with type 2 diabetes mellitus (T2DM) is leveraged by anemia remains unclear. This study is to evaluate the joint effect of DKD and anemia on DR. Methods: Data were collected from electronic medical records of 1389 patients with T2DM in the Yiwu Central Hospital of Zhejiang Province from 2018 to 2019. Based on retinal examination findings, patients were classified as without diabetic retinopathy (non-DR), non-proliferative diabetic retinopathy (NPDR), and proliferative diabetic retinopathy (PDR). Odds ratio (OR) from multinomial logistic regression models adjusting for potential risk factors of DR were used to evaluate associations of DKD, renal function measures, and anemia with risk of NPDR and PDR. Path analysis was performed to help understand the association of DKD and hemoglobin (Hb) with DR. Results: The study included 901 patients with non-DR, 367 patients with NPDR and 121 patients with PDR. Both high DKD risk and abnormal renal function measures were significantly associated with PDR. Anemia was associated with increased risk of NPDR (OR = 1.75, 95% confidence interval [CI] = 1.18-2.58) and PDR (OR = 3.71, 95% CI = 2.23-6.18). DKD severity and anemia had joint effect on NPDR (OR = 2.29, 95% CI = 1.32-3.96) and PDR (OR = 11.31, 95% CI = 5.95-21.51). These associations were supported by path analysis. Conclusions: DKD severity, abnormal estimated glomerular filtration rate (eGFR), and urinary albumin/creatinine ratio (UACR) were associated with increased risk of DR in patients with T2DM, and anemia had joint effect on these associations. Improving Hb level may decrease the risk of DR in patients with T2DM.

Generation of antitumor invariant natural killer T cell lines in multiple myeloma and promotion of their functions via lenalidomide: a strategy for immunotherapy.

PURPOSE: CD1d-restricted invariant natural killer T (iNKT) cells are important immunoregulatory cells in antitumor immune responses. However, the quantitative and qualitative defects of iNKT cells in advanced multiple myeloma hamper their antitumor effects. Therefore, the development of functional iNKT cells may provide a novel strategy for the immunotherapy in multiple myeloma. EXPERIMENTAL DESIGN: We activated and expanded iNKT cells from multiple myeloma patients with alpha-galactosylceramide (alpha-GalCer)-pulsed dendritic cells, characterized their antitumor effects by the cytokine production profile and cytotoxicity against multiple myeloma cells, and explored the effects of immunomodulatory drug lenalidomide on these iNKT cells. We also investigated the expression of CD1d by primary multiple myeloma cells and its function to activate iNKT cells. RESULTS: We established highly purified functional iNKT cell lines from newly diagnosed and advanced multiple myeloma patients. These CD1d-restricted iNKT cell lines produced high level of antitumor Th1 cytokine in response to alpha-GalCer-pulsed primary multiple myeloma cells, CD1d-transfected MM1S cell line, and dendritic cells. Moreover, iNKT cell lines displayed strong cytotoxicity against alpha-GalCer-pulsed primary multiple myeloma cells. Importantly, lenalidomide further augmented the Th1 polarization by iNKT cell lines via increased Th1 cytokine production and reduced Th2 cytokine production. We also showed that CD1d was expressed in primary multiple myeloma cells at mRNA and protein levels from the majority of multiple myeloma patients, but not in normal plasma cells and multiple myeloma cell lines, and CD1d(+) primary multiple myeloma cells presented antigens to activate iNKT cell lines. CONCLUSIONS: Taken together, our results provide the preclinical evidence for the iNKT cell-mediated immunotherapy and a rationale for their use in combination with lenalidomide in multiple myeloma treatment.

Peripheral blood progenitor cell product contains Th1-biased noninvariant CD1d-reactive natural killer T cells: implications for posttransplant survival.

OBJECTIVE: Bone marrow (BM) Th1 populations can contribute to graft-vs-leukemia responses. Granulocyte/granulocyte macrophage colony-stimulating factor (CSF)-mobilized peripheral blood progenitor cells (PBPC) have become widely accepted alternatives to BM transplantation. T cells coexpressing natural killer cell proteins (NKT) include a CD1d-reactive subset that influences immunity by rapidly producing large amounts of Th1 and/or Th2 cytokines dependent upon microenvironment and disease. There are two types of CD1d-reactive NKT. iNKT express a semi-invariant T-cell receptor-alpha. Other noninvariant CD1d-reactive NKT from BM and liver produce large amounts of interleukin-4 or interferon-gamma, respectively, and within the intestine can be biased in either direction. Recent data suggests that NKT might contribute to clinical benefits of PBPC. MATERIALS AND METHODS: To address these issues, we phenotypically and functionally studied PBPC NKT. RESULTS: Similarly to BM, NKT-like cells were common in allogeneic and autologous PBPC, there were relatively few classical iNKT, but high CD1d-reactivity concentrated in NKT fractions. Significantly, PBPC CD1d-reactive cells were relatively Th1-biased and their presence was associated with better prognosis. Granulocyte CSF treatment of BM to yield PBPC in vivo as well as in vitro Th2-polarizes conventional T cells and iNKT. However, granulocyte CSF treatment of BM in vitro produced Th1-biased NKT, providing a mechanism for opposite polarization of NKT from BM vs PBPC. CONCLUSIONS: These results suggest distinct Th1 CD1d-reactive NKT cells could stimulate anti-tumor responses from those previously described, which can suppress graft-vs-host disease.

[Effects of Shen-song-yang-xin capsule on ventricular arrhythmias in ischemia/reperfusion: experiment with rats].

OBJECTIVE: To investigate the effects of Shen-song-yang-xin capsule containing ginseng, ophiopogon root, red sage root, etc, on the ventricular arrhythmias caused by ischemic/reperfusion. METHODS: Thirty SD rats were randomly divided into 2 equal groups: Shen-song-yang-xin group undergoing gastric infusion of Shen-song-yang-xin 4 g x kg(-1) x d(-1) for 2 weeks and then subjected to left coronary artery occlusion for 30 minutes followed by reperfusion for 60 minutes, and control group undergoing ischemia/reperfusion only. Electrocardiography was conducted to record the ventricular arrhythmias, and after 60 minutes of reperfusion the rats were killed with their hearts removed to measure the infarction size. RESULTS: (1) During the 30 minutes' ischemia, the average number of episode of ischemia-induced premature ventricular contraction of the Shen-song-yang-xin group was (6.7 +/- 0.3) beats/30 min, significantly lower than that of the control group [(15.0 +/- 1.1) beats/30 min, P = 0.007]; the average number of episode of ischemia-induced ventricular tachycardia of the Shen-song-yang-xin group was (3.5 +/- 0.2) episodes/30 min, significantly lower than that of the control group [(14.7 +/- 0.6) episodes/30 min, P = 0.003]; the duration of ventricular tachycardia of the Shen-song-yang-xin group was (10.3 +/- 2.0) s, significantly shorter than that of the control group [(28.3 +/- 4.6) s, P = 0.018]; and the duration of ventricular fibrillation of the Shen-song-yang-xin group was (7.0 +/- 1.1) s, significantly shorter than that of the control group [(15.0 +/- 1.7) s, P = 0.031]. (2) 60 minutes after the reperfusion, the number of episode of premature ventricular contraction of the Shen-song-yang-xin group was (5.3 +/- 0.8) beats/60 min, significantly lower than that of the control group [(15.6 +/- 1.8) beats/60 min, P = 0.002], and the duration of reperfusion-induced ventricular tachycardia was (4.6 +/- 0.5) s, significantly shorter than that of the control group [(13.5 +/- 0.6) s, P = 0.001]. CONCLUSION: Shen-song-yang-xin capsule significantly reduces the ischemia-induced ventricular arrhythmia, average episode number of reperfusion-induced premature ventricular contraction, and duration of reperfusion-induced ventricular tachycardia.

[The effects after withdrawal of simvastatin on brachial artery endothelial function in patient with coronary heart disease or risk factors].

OBJECTIVE: Landmark trials have demonstrated that statins can reduce the risk of coronary events. Despite the widespread use of statins in the settings of primary and secondary prevention of CHD, withdrawal of statins is a frequent problem in clinical practice. Several recent clinical studies have suggested that withdrawal of statin therapy might be associated with an increase in thrombotic vascular events and the onset of acute coronary syndromes. However, the effects of discontinuing of statins treatment on endothelial function and underlying mechanism are unknown. Objectives We investigated the effects after withdrawal of simvastatin on brachial artery endothelial function in patients unreached cholesterol target with coronary heart disease (CHD) or CHD risk factors. METHODS: We included 33 patients with established CHD or CHD risk factors, whose serum cholesterol did not achieve NCEP target level. They were administered simvastatin (20 mg) for 4 weeks. Endothelial dependent flow-mediated vasodilation (FMD) was assessed in the brachial artery using high-resolution ultrasound at baseline, after 4 weeks of simvastatin and after termination of therapy 1 week. We evaluated fasting serum lipid profiles and vasoactive substances simultaneously, included nitric oxide (NO), endothelin (ET), 6-keto-PGF1(alpha) and thromboxane B(2) (TXB(2)), which were measured as plasma prostacyclin and TXA(2) respectively. RESULTS: Simvastatin treatment reduced low density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) levels and improved endothelial-dependent vasodilation in patients after 4 weeks. Withdrawal of simvastatin, however, FMD showed a significant reduction [(4.82 +/- 0.71)% vs (11.51 +/- 0.87)%, P < 0.01], that remained in low level after 1 week, and the FMD were even lower than the baseline values [(4.82 +/- 0.71)% vs (5.89 +/- 0.65)%, P < 0.01]. After terminating simvastatin treatment, serum NO and plasma 6-keto-PGF1(alpha) levels decreased, as well as plasma ET and serum LDL-C levels increased. But there was no significant difference between plasma TXB(2) levels before and after withdrawal of simvastatin (P > 0.05). Overall, there were significant positive correlations between withdrawal-induced changes in FMD and serum NO level (r = 0.674, P = 0.004), whereas no correlations were shown between the changes in FMD and serum LDL-C level (r = -0.414, P = 0.083). CONCLUSIONS: Abrupt withdrawal of simvastatin therapy resulted in the significant adverse impact on brachial artery endothelial function in patients unreached cholesterol target with CHD or CHD risk factors. Termination of therapy may suppress endothelial NO production and impair endothelial function that is independent of lipid-lowering effect.

Posttransplant lymphoproliferative disorder associated with an Epstein-Barr-related virus in cynomolgus monkeys.

BACKGROUND: Human posttransplant lymphoproliferative disorder (PTLD) has been shown to be associated with Epstein-Barr virus (EBV) infection. Primate animal models of PTLD and the use of molecular markers in its diagnosis have not been reported. This study was designed to evaluate the frequency, pathology, and molecular characteristics of PTLD in cynomolgus kidney allograft recipients. METHODS: Over a 5-year period (January 1995 to November 2000), 160 primate renal transplants were performed at the Massachusetts General Hospital (MGH). Of these, all cases (n=9) that developed PTLD were included. H&E stained paraffin sections of all available tissue samples from the cases were evaluated for the presence of PTLD. Immunoperoxidase staining for T cells (CD3), B cells (CD20), kappa and lambda light chains as well as EBV nuclear antigens (EBNA2) and latent membrane proteins (EBV LMP-1) was done on paraffin sections using standard immunohistochemical (IHC) methods. In situ hybridization for EBV encoded RNA (EBER) was performed in all tissue samples with atypical lymphoid proliferations, using a novel EBER nucleotide probe based on consensus gene sequences from EBV and the related herpes lymphocryptoviruses (LCV) infecting baboons and rhesus macaques. RESULTS: Of 160 consecutive primate renal transplants performed at MGH, 5.6% developed PTLD 28-103 days after transplantation. In all cases, the lymph nodes were involved and effaced by an atypical polymorphous lymphoid proliferation of EBER+ B cells, diagnostic for PTLD. Focal staining for EBNA-2 was noted in tumor cells. In 67% (six of nine) the PTLD infiltrates were present in extra nodal sites, notably liver (56%), lung (44%), heart (44%), renal allograft (44%), and native kidney (22%). The spleen was involved by PTLD in all four animals that had not undergone a pretransplant splenectomy. The PTLD morphology was similar in all cases and predominantly of the polymorphous type, however, some of these showed areas that appeared minimally polymorphous. No cases of monomorphic PTLD were seen. CONCLUSIONS: By in situ hybridization, expression of the RNA product, homologous for EBV-encoded RNA (EBER) was identified in the PTLD tumor cells of all cases, indicating latent primate EBV- related infection. This report identifies a novel animal model of EBV associated PTLD in the setting of kidney transplantation, with valuable implications for managing and understanding human PTLD and oncogenesis.

[Clinical observation on effect of Chinese herbal medicine plus human chorionic gonadotropin and progesterone in treating anticardiolipin antibody-positive early recurrent spontaneous abortion].

OBJECTIVE: To find a method without corticosteroids, aspirin or heparin for treatment of anticardiolipin antibody-positive early recurrent spontaneous abortion (AARSA). METHODS: Twenty-three patients of AARSA in the treated group were treated with Chinese herbal medicine (CHM) plus human chorionic gonadotropin and progesterone, and 18 patiens in the control group were treated with multi-vitamin only. The change of anticardiolipin antibody was determined by enzyme linked immunoabsorbent assay (ELISA). RESULTS: After treatment, anticardiolipin antibody negative converted in 20 cases (86.9%) of the treated group. The cure rate of abortion in the treated group was 82.6% (19/23), which was raised to 95% (19/20) in those patients with antibody negative conversion, while in the control group, it was 16.7% (3/18) merely, comparison between the two groups in cure rate showed significant difference (P < 0.01). CONCLUSION: CHM plus human chorionic gonadotropin and progesterone could cure AARSA effectively.

Short-term withdrawal of simvastatin induces endothelial dysfunction in patients with coronary artery disease: a dose-response effect dependent on endothelial nitric oxide synthase.

BACKGROUND: Patients with coronary artery disease (CAD) have impaired endothelial function. Simvastatin therapy has been demonstrated to significantly improve endothelial function in these patients. Although withdrawal of statins is a frequent problem in clinical practice, the effects after discontinuation of statins treatment on endothelial function in patients with CAD are largely unknown. OBJECTIVE: This study investigated the effects after withdrawal of simvastatin on brachial artery endothelial function in patients with CAD and the underlying mechanisms. METHODS: We recruited 30 patients with established CAD. They were treated with 20 mg simvastatin for 4 weeks. Endothelial dependent flow-mediated vasodilation (FMD) was assessed in the brachial artery using high-resolution ultrasound at baseline, 4 weeks during simvastatin treatment, and 1 week after termination of therapy. 20 healthy subjects were also studied as a control group. Furthermore, we investigated underlying mechanisms on human umbilical vein endothelial cells (HUVECs) confluent monolayers at passages 2-3. HUVECs were exposed to simvastatin. After 24 h cells were repeatedly washed to remove the drugs, and the conditioned mediums were collected at the indicated time points. The nitric oxide (NO) production and levels of eNOS mRNA after 24 h of withdrawal of statins were examined. RESULTS: (1) Abrupt discontinuation of simvastatin treatment leads to a rebound of serum total cholesterol (21.3%) and LDL cholesterol (18.2%) in patients within 1 week, but they were still lower than the baseline values (P<0.05 for each parameter). (2) A significant decreased of FMD (-59.3%) was observed in patients after discontinuation of simvastatin in 1 week, and furthermore, the FMD was even lower than the baseline levels (4.6% vs. 5.6%, P<0.05). The reduction of FMD was not correlated with the change of LDL cholesterol (r=-0.343, P=0.081). In contrast to the unchanged LDL cholesterol level, abrupt discontinuation of therapy caused a rapid and significant decrease in FMD from 10.6% to 5.2% in healthy subjects at day 1, but it returned to baseline levels within 1 week. (3) In HUVECs, a maximum decrease of nitrite levels (-80%) was observed at 6 h after stopping simvastatin treatment, which was below the control levels. 24 h after stopping 10(-5) mmol/L and 10(-6) mmol/L simvastatin treatment, eNOS mRNA expression decreased to -71% and -42% (P<0.05), respectively. CONCLUSIONS: Abrupt withdrawal of simvastatin treatment not only acutely and completely abrogates its beneficial effects on endothelial function in patients with CAD, but also induced further vascular injury compared with pretreatment status, independent of cholesterol levels. The underlying mechanism of these negative effects may be related to the suppression of endothelial NO production, which are dose-dependent.

De novo discovery of a gamma-secretase inhibitor response signature using a novel in vivo breast tumor model.

Notch pathway signaling plays a fundamental role in normal biological processes and is frequently deregulated in many cancers. Although several hypotheses regarding cancer subpopulations most likely to respond to therapies targeting the Notch pathway have been proposed, clinical utility of these predictive markers has not been shown. To understand the molecular basis of gamma-secretase inhibitor (GSI) sensitivity in breast cancer, we undertook an unbiased, de novo responder identification study using a novel genetically engineered in vivo breast cancer model. We show that tumors arising from this model are heterogeneous on the levels of gene expression, histopathology, growth rate, expression of Notch pathway markers, and response to GSI treatment. In addition, GSI treatment of this model was associated with inhibition of Hes1 and proliferation markers, indicating that GSI treatment inhibits Notch signaling. We then identified a pretreatment gene expression signature comprising 768 genes that is significantly associated with in vivo GSI efficacy across 99 tumor lines. Pathway analysis showed that the GSI responder signature is enriched for Notch pathway components and inflammation/immune-related genes. These data show the power of this novel in vivo model system for the discovery of biomarkers predictive of response to targeted therapies, and provide a basis for the identification of human breast cancers most likely to be sensitive to GSI treatment.

Circulating myeloid dendritic cells of advanced cancer patients result in reduced activation and a biased cytokine profile in invariant NKT cells.

CD1d-restricted invariant NKT (iNKT) cells play important regulatory roles in various immune responses, including antitumor immune responses. Previous studies have demonstrated quantitative and qualitative defects in iNKT cells of cancer patients, and these defects are clinically relevant as they are associated with poor prognosis. In this study we demonstrate that defects in the iNKT cell population can, at least in part, be attributed to defective interactions between iNKT cells and CD1d-expressing circulating myeloid dendritic cells (mDC), as mDC of patients with advanced melanoma and renal cell cancer reduced the activation and Th1 cytokine production of healthy donor-derived iNKT cells. Interestingly, this reduced activation of iNKT cells was restricted to patients with low circulating iNKT cell numbers and could be reversed by IL-12 and in part by the neutralization of TGF-beta, but it was further reduced by the neutralization of IL-10 in vitro. Additional experiments revealed discordant roles for TGF-beta and IL-10 on human iNKT cells, because TGF-beta suppressed iNKT cell activation and proliferation and IFN-gamma production while IL-10 was identified as a cytokine involved in stimulating the activation and expansion of iNKT cells that could subsequently suppress NK cell and T cell responses.

CD1d mediates T-cell-dependent resistance to secondary infection with encephalomyocarditis virus (EMCV) in vitro and immune response to EMCV infection in vivo.

The innate and adaptive immune responses have evolved distinct strategies for controlling different viral pathogens. Encephalomyocarditis virus (EMCV) is a picornavirus that can cause paralysis, diabetes, and myocarditis within days of infection. The optimal innate immune response against EMCV in vivo requires CD1d. Interaction of antigen-presenting cell CD1d with distinct natural killer T-cell ("NKT") populations can induce rapid gamma interferon (IFN-gamma) production and NK-cell activation. The T-cell response of CD1d-deficient mice (lacking all NKT cells) against acute EMCV infection was further studied in vitro and in vivo. EMCV persisted at higher levels in CD1d-knockout (KO) splenocyte cultures infected in vitro. Furthermore, optimal resistance to repeat cycles of EMCV infection in vitro was also shown to depend on CD1d. However, this was not reflected in the relative levels of NK-cell activation but rather by the responses of both CD4(+) and CD8(+) T-cell populations. Repeated EMCV infection in vitro induced less IFN-gamma and alpha interferon (IFN-alpha) from CD1d-deficient splenocytes than with the wild type. Furthermore, the level of EMCV replication in wild-type splenocytes was markedly and specifically increased by addition of blocking anti-CD1d antibody. Depletion experiments demonstrated that dendritic cells contributed less than the combination of NK and NKT cells to anti-EMCV responses and that none of these cell types was the main source of IFN-alpha. Finally, EMCV infection in vivo produced higher levels of viremia in CD1d-KO mice than in wild-type animals, coupled with significantly less lymphocyte activation and IFN-alpha production. These results point to the existence of a previously unrecognized mechanism of rapid CD1d-dependent stimulation of the antiviral adaptive cellular immune response.

CD1d ligation on human monocytes directly signals rapid NF-kappaB activation and production of bioactive IL-12.

Natural killer T cells (NKT cells) expressing a semi-invariant CD1d-reactive T cell receptor (invariant NKT, iNKT) can be rapidly activated by monocytes or immature dendritic cells (iDCs) bearing a CD1d-presented glycolipid antigen and can in turn stimulate these myeloid cells to mature and produce IL-12. Previous studies have shown that iNKT-produced IFNgamma and CD40 ligand contribute to this dendritic cell maturation. This study demonstrates that CD1d ligation alone, in the absence of iNKT, could rapidly (within 24 h) stimulate production of bioactive IL-12p70 by CD1d+ human peripheral blood monocytes as well as iDCs. IFNgamma alone had no effect, but it markedly enhanced CD1d-stimulated IL-12 production. Monocyte differentiation, as assessed by CD40 and CD1a up-regulation, was also accelerated by CD1d stimulation, consistent with this representing a physiological response. CD1d ligation on the human monocytic cell line THP-1 similarly specifically stimulated IL-12 production. Biochemical studies showed that IL-12 release correlated with rapid phosphorylation of IkappaB, a critical step in NF-kappaB activation. Selective NF-kappaB inhibition blocked this CD1d-stimulated IL-12 production. Finally, CD1d ligation could also enhance IL-12 production in the presence of suboptimal LPS or CD40 stimulation. These findings demonstrate an innate immune signaling function for CD1d and provide a mechanism for the rapid activation of monocytes and iDCs by CD1d-reactive T cells.

Hepatic CD1d expression in hepatitis C virus infection and recognition by resident proinflammatory CD1d-reactive T cells.

A subset of CD161(+)CD56(+/-) NKT cells can recognize glycolipids presented by CD1d and positively or negatively regulate inflammatory responses, including those implicated in several models of hepatitis. CD1d is expressed at very low levels in the healthy liver, but there is a large fraction of CD161(+)CD56(+) NKT cells. There are high levels of nonclassical proinflammatory hepatic CD1d-reactive T cells in hepatitis C virus (HCV) infection. Hepatic inflammatory cells and biliary cells adjacent to portal tract fibrotic areas of HCV-infected donors specifically up-regulated CD1d. A hepatocyte cell line expressing minimal CD1d was efficiently recognized by hepatic CD1d-reactive T cells, suggesting a role for these cells in disease. Hepatic CD1d-reactive T cells from HCV-positive as well as negative donors produced large amounts of IFN-gamma with some IL-13, but only rarely detectable IL-4. We confirmed large numbers of hepatic CD161(+) T cells, lower levels of CD56(+) T cells, and small numbers of classic invariant NKT cells. However, hepatic CD1d-reactivity was not restricted to any of these populations. We suggest virally infected hepatic cells can process potent CD1d-presented liver Ag(s), for surveillance by resident Th1 hepatic CD1d-reactive T cells. This process may be beneficial in acute viral clearance, but in chronic infection could contribute to liver injury.

Cutting edge: Compartmentalization of Th1-like noninvariant CD1d-reactive T cells in hepatitis C virus-infected liver.

Murine intrahepatic lymphocytes (IHL) are dominated by invariant TCR alpha-chain expressing CD1d-reactive NKT cells, which can cause model hepatitis. Invariant NKT (CD56(+/-)CD161(+)) and recently identified noninvariant CD1d-reactive T cells rapidly produce large amounts of IL-4 and/or IFN-gamma and can regulate Th1/Th2 responses. Human liver contains large numbers of CD56(+) NKT cells but few invariant NKT. Compared with matched peripheral blood T cell lines, primary IHL lines from patients with chronic hepatitis C had high levels of CD161 and CD1d reactivity, but the invariant TCR was rare. CD1d-reactive IHL were strikingly Th1 biased. IHL also demonstrated CD1d-specific cytotoxic activity. Hepatocytes and other liver cells express CD1d. These results identify a novel population of human T cells that could contribute to destructive as well as protective immune responses in the liver. CD1d-reactive T cells may have distinct roles in different tissues.