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.

Natural killer T cells (NKT cells) expressing a semiinvariant CD1d-reactive T cell receptor (invariant NKT, iNKT) can be rapidly activated by monocytes or immature dendritic cells (iDCs) bearing aCD1d-presented glycolipid antigen and can in turn stimulate these myeloid cells to mature and produce IL-12. Previous studies have shown that iNKT-produced IFN␥ 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. IFN␥ alone had no effect, but it markedly enhanced CD1d-stimulated IL-12 production. Monocyte differentiation, as assessed by CD40 and CD1a upregulation, 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 I B, a critical step in NF-B activation. Selective NF-B 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.antigen-presenting cell ͉ innate immunity ͉ natural killer T cell C D1d is a nonpolymorphic MHC class I-like protein constitutively expressed by antigen-presenting cells (APC) and by some epithelia, whereas CD1a-c are induced upon dendritic cell (DC) maturation. CD1d is recognized by a subpopulation of T cells, many of which express markers typical of natural killer cells and have been termed natural killer T cells (NKT cells) (1-3). A major fraction of CD1d-restricted T cells recognize CD1d through an invariant T cell receptor (TCR)␣ chain (V␣24-J␣18 in humans). CD1d-reactive NKT cells using this invariant TCR (iNKT) are largely primed in vivo for the rapid production of T helper 1 and T helper 2 cytokines (IFN␥ and IL-4) and have regulatory functions in innate and adaptive immune responses (3, 4). A glycolipid antigen isolated from marine sponge (␣-galactosylceramide, ␣galcer) is recognized by iNKT in the context of CD1d (5-8). In vivo, ␣galcer causes activation of iNKT, rapid cytokine production, and subsequent systemic activation of innate and adaptive immune cells (9, 10).Data from many groups indicate that iNKT functions can be mediated through interactions with CD1d ϩ APC including . Murine studies have shown that iNKT activation can be enhanced by B7 ligation of CD28 and that IL-12 produced by DCs can further enhance iNKT IFN␥ production (11,12,20). Conversely, murine and human studies have shown that iNKT can stimulate the maturation of ␣galcer-pulsed DCs (11,12,18,20). This DC stimulation appears to be mediated by CD40 ligand on the iNKT through ligation of CD40 ...

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.

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 iNKTcells may provide a novel strategy for the immunotherapy in multiple myeloma. Experimental Design: We activated and expanded iNKT cells from multiple myeloma patients with a-galactosylceramide (a-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 iNKTcells.We also investigated the expression of CD1d by primary multiple myeloma cells and its function to activate iNKTcells. 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 a-GalCer-pulsed primary multiple myeloma cells, CD1d-transfected MM1S cell line, and dendritic cells. Moreover, iNKT cell lines displayed strong cytotoxicity against a-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 iNKTcell 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.

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

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 γ-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. [Cancer Res 2009;69 (23):8949-57]

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.

OBJECTIVE-Bone marrow (BM) Th1 populations can contribute to graft-versus-leukemia (GvL) responses. G/GM-CSF-mobilized peripheral blood progenitor cells (PBPC) have become widely accepted alternatives to BM transplantation (BMT). T cells co-expressing NK proteins (NKT) include a CD1d-reactive subset which influence 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 TCR-α. Other "non-invariant" CD1d-reactive NKT from BM and liver produce large amounts of IL-4 or IFN-γ respectively, and within the intestine can be biased in either direction. Recent data suggests that NKT might contribute to clinical benefits of PBPC.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. G-CSF treatment of BM to yield PBPC in vivo as well as in vitro Th2-polarizes conventional T cells and iNKT. However, G-CSF treatment of BM in vitro produced Th1-biased NKT, providing a mechanism for opposite polarization of NKT from BM versus PBPC.CONCLUSIONS-These results suggest distinct Th1 CD1d-reactive NKT cells could stimulate anti-tumor responses from those previously described, which can suppress GvHD.