Ruojie Wang

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-γ 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 α-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-γ 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.

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 α-galactosylceramide (α-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 α-GalCer-pulsed primary multiple myeloma cells, CD1d-transfected MM1S cell line, and dendritic cells. Moreover, iNKT cell lines displayed strong cytotoxicity against α-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.

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-β, but it was further reduced by the neutralization of IL-10 in vitro. Additional experiments revealed discordant roles for TGF-β and IL-10 on human iNKT cells, because TGF-β suppressed iNKT cell activation and proliferation and IFN-γ 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

ABSTRACT 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-γ) 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-γ and alpha interferon (IFN-α) 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-α. 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-α production. These results point to the existence of a previously unrecognized mechanism of rapid CD1d-dependent stimulation of the antiviral adaptive cellular immune response.

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.

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-γ and IFN-α in vivo from CD1d+ but not CD1d-deficient recipients. Unlike IFN-γ, CD1d-induced IFN-α 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-γ as well as IFN-α. CD1d ligation in vivo enhanced systemic IL-12, IFN-γ, and IFN-α 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(abʹ)2s 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 α-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.