Functionally Distinct Subsets of CD1d-restricted Natural Killer T Cells Revealed by CD1d Tetramer Staining

CD1d-restricted natural killer (NK)T cells are known to potently secrete T helper (Th)1 and Th2 cytokines and to mediate cytolysis, but it is unclear how these contrasting functional activities are regulated. Using lipid antigen–loaded CD1d tetramers, we have distinguished two subsets of CD1d-restricted T cells in fresh peripheral blood that differ in cytokine production and cytotoxic activation. One subset, which was CD4−, selectively produced the Th1 cytokines interferon γ and tumor necrosis factor α, and expressed NKG2d, a marker associated with cytolysis of microbially infected and neoplastic cells. This subset up-regulated perforin after exposure to interleukin (IL)-2 or IL-12. In contrast, CD4+ CD1d-restricted NKT cells potently produced both Th1 and Th2 cytokines, up-regulated perforin in response to stimulation by phorbol myristate acetate and ionomycin but not IL-2 or IL-12, and could be induced to express CD95L. Further, for both CD1d-restricted NKT cell subsets, we found that antigenic stimulation induced cytokine production but not perforin expression, whereas exposure to inflammatory factors enhanced perforin expression but did not stimulate cytokine production. These results show that the various activities of CD1d-restricted T cells in tumor rejection, autoimmune disease, and microbial infections could result from activation of functionally distinct subsets, and that inflammatory and antigenic stimuli may influence different effector functions

CD94 and a Novel Associated Protein (94AP) Form a NK Cell Receptor Involved in the Recognition of HLA-A, HLA-B, and HLA-C Allotypes

AbstractWhereas the human killer cell inhibitory receptors (KIRs) for HLA class I are immunoglobulin-like monomeric type I glycoproteins, the murine Ly49 receptors for H-2 are type II homodimers of the C-type lectin superfamily. Here, we demonstrate that human NK cells also express C-type lectin receptors that influence recognition of polymorphic HLA-A, HLA-B, and HLA-C molecules. These receptors are heterodimers composed of CD94 chains covalently associated with novel tyrosine-phosphorylated glycoproteins (94AP). Some NK clones recognize a common HLA-C ligand using both KIRs and CD94–94AP receptors. These findings suggest the existence of human inhibitory MHC class I receptors of the immunoglobulin and C-type lectin superfamilies and indicate overlap in ligand specificity

Murine CD1d-Restricted T Cell Recognition of Cellular Lipids

AbstractNKT cells are associated with immunological control of autoimmune disease and cancer and can recognize cell surface mCD1d without addition of exogenous antigens. Cellular antigens presented by mCD1d have not been identified, although NKT cells can recognize a synthetic glycolipid, α-GalCer. Here we show that after addition of a lipid extract from a tumor cell line, plate-bound mCD1d molecules stimulated an NKT cell hybridoma. This hybridoma also responded strongly to three purified phospholipids, but failed to recognize α-GalCer. Seven of sixteen other mCD1d restricted hybridomas also showed a response to certain purified phospholipids. These findings suggest NKT cells can recognize cellular antigens distinct from α-GalCer and identify phospholipids as potential self-antigens presented by mCD1d

Mice Engrafted with Human Fetal Thymic Tissue and Hematopoietic Stem Cells Develop Pathology Resembling Chronic Graft-versus-Host Disease

AbstractChronic graft-versus-host disease (cGVHD) is a significant roadblock to long-term hematopoietic stem cell (HSC) transplantation success. Effective treatments for cGVHD have been difficult to develop, in part because of a paucity of animal models that recapitulate the multiorgan pathologies observed in clinical cGVHD. Here we present an analysis of the pathology that occurs in immunodeficient mice engrafted with human fetal HSCs and implanted with fragments of human fetal thymus and liver. Starting at time points generally later than 100 days post-transplantation, the mice developed signs of illness, including multiorgan cellular infiltrates containing human T cells, B cells, and macrophages; fibrosis in sites such as lungs and liver; and thickened skin with alopecia. Experimental manipulations that delayed or reduced the efficiency of the HSC engraftment did not affect the timing or progression of disease manifestations, suggesting that pathology in this model is driven more by factors associated with the engrafted human thymic organoid. Disease progression was typically accompanied by extensive fibrosis and degradation of the thymic organoid, and there was an inverse correlation of disease severity with the frequency of FoxP3+ thymocytes. Hence, the human thymic tissue may contribute T cells with pathogenic potential, but the generation of regulatory T cells in the thymic organoid may help to control these cells before pathology resembling cGVHD eventually develops. This model thus provides a new system to investigate disease pathophysiology relating to human thymic events and to evaluate treatment strategies to combat multiorgan fibrotic pathology produced by human immune cells

Recognition of Lyso-Phospholipids by Human Natural Killer T Lymphocytes

Natural killer T (NKT) cells are a subset of T lymphocytes with potent immunoregulatory properties. Recognition of self-antigens presented by CD1d molecules is an important route of NKT cell activation; however, the molecular identity of specific autoantigens that stimulate human NKT cells remains unclear. Here, we have analyzed human NKT cell recognition of CD1d cellular ligands. The most clearly antigenic species was lyso-phosphatidylcholine (LPC). Diacylated phosphatidylcholine and lyso-phosphoglycerols differing in the chemistry of the head group stimulated only weak responses from human NKT cells. However, lyso-sphingomyelin, which shares the phosphocholine head group of LPC, also activated NKT cells. Antigen-presenting cells pulsed with LPC were capable of stimulating increased cytokine responses by NKT cell clones and by freshly isolated peripheral blood lymphocytes. These results demonstrate that human NKT cells recognize cholinated lyso-phospholipids as antigens presented by CD1d. Since these lyso-phospholipids serve as lipid messengers in normal physiological processes and are present at elevated levels during inflammatory responses, these findings point to a novel link between NKT cells and cellular signaling pathways that are associated with human disease pathophysiology.</p

LMP1-deficient Epstein-Barr virus mutant requires T cells for lymphomagenesis

Epstein-Barr virus (EBV) infection transforms B cells in vitro and is associated with human B cell lymphomas. The major EBV oncoprotein, latent membrane protein 1 (LMP1), mimics constitutively active CD40 and is essential for outgrowth of EBV-transformed B cells in vitro; however, EBV-positive diffuse large B cell lymphomas and Burkitt lymphomas often express little or no LMP1. Thus, EBV may contribute to the development and maintenance of human lymphomas even in the absence of LMP1. Here, we found that i.p. injection of human cord blood mononuclear cells infected with a LMP1-deficient EBV into immunodeficient mice induces B cell lymphomas. In this model, lymphoma development required the presence of CD4+ T cells in cord blood and was inhibited by CD40-blocking Abs. In contrast, LMP1-deficient EBV established persistent latency but did not induce lymphomas when directly injected into mice engrafted with human fetal CD34+ cells and human thymus. WT EBV induced lymphomas in both mouse models and did not require coinjected T cells in the cord blood model. Together, these results demonstrate that LMP1 is not essential for EBV-induced lymphomas in vivo and suggest that T cells supply signals that substitute for LMP1 in EBV-positive B cell lymphomagenesis

Analysis of the CD1 Antigen Presenting System in Humanized SCID Mice

CD1 molecules are glycoproteins that present lipids and glycolipids for recognition by T cells. CD1-dependent immune activation has been implicated in a wide range of immune responses, however, our understanding of the role of this pathway in human disease remains limited because of species differences between humans and other mammals: whereas humans express five different CD1 gene products (CD1a, CD1b, CD1c, CD1d, and CD1e), muroid rodents express only one CD1 isoform (CD1d). Here we report that immune deficient mice engrafted with human fetal thymus, liver, and CD34+ hematopoietic stem cells develop a functional human CD1 compartment. CD1a, b, c, and d isoforms were highly expressed by human thymocytes, and CD1a+ cells with a dendritic morphology were present in the thymic medulla. CD1+ cells were also detected in spleen, liver, and lungs. APCs from spleen and liver were capable of presenting bacterial glycolipids to human CD1-restricted T cells. ELISpot analyses of splenocytes demonstrated the presence of CD1-reactive IFN-γ producing cells. CD1d tetramer staining directly identified human iNKT cells in spleen and liver samples from engrafted mice, and injection of the glycolipid antigen α-GalCer resulted in rapid elevation of human IFN-γ and IL-4 levels in the blood indicating that the human iNKT cells are biologically active in vivo. Together, these results demonstrate that the human CD1 system is present and functionally competent in this humanized mouse model. Thus, this system provides a new opportunity to study the role of CD1-related immune activation in infections to human-specific pathogens

Determination of Cellular Lipids Bound to Human CD1d Molecules

CD1 molecules are glycoproteins that present lipid antigens at the cell surface for immunological recognition by specialized populations of T lymphocytes. Prior experimental data suggest a wide variety of lipid species can bind to CD1 molecules, but little is known about the characteristics of cellular ligands that are selected for presentation. Here we have molecularly characterized lipids bound to the human CD1d isoform. Ligands were eluted from secreted CD1d molecules and separated by normal phase HPLC, then characterized by mass spectroscopy. A total of 177 lipid species were molecularly identified, comprising glycerophospholipids and sphingolipids. The glycerophospholipids included common diacylglycerol species, reduced forms known as plasmalogens, lyso-phospholipids (monoacyl species), and cardiolipins (tetraacyl species). The sphingolipids included sphingomyelins and glycosylated forms, such as the ganglioside GM3. These results demonstrate that human CD1d molecules bind a surprising diversity of lipid structures within the secretory pathway, including compounds that have been reported to play roles in cancer, autoimmune diseases, lipid signaling, and cell death

Modeling Human Antitumor Responses In Vivo Using Umbilical Cord Blood-Engrafted Mice

Mice engrafted with human immune cells offer powerful in vivo model systems to investigate molecular and cellular processes of tumorigenesis, as well as to test therapeutic approaches to treat the resulting cancer. The use of umbilical cord blood mononuclear cells as a source of human immune cells for engraftment is technically straightforward, and provides T lymphocytes and autologous antigen-presenting cells (including B cells, monocytes, and DCs) that bear cognate antigen presenting molecules. By using a human-specific oncogenic virus, such as Epstein-Barr virus, de novo neoplastic transformation of the human B cells can be induced in vivo in a manner that models progressive stages of tumorigenesis from nascent neoplasia to the establishment of vascularized tumor masses with an immunosuppressive environment. Moreover, since tumorigenesis occurs in the presence of autologous T cells, this type of system can be used to investigate how T cells become suppressed during tumorigenesis, and how immunotherapies counteract immunosuppression. This minireview will provide a brief overview of the use of human umbilical cord blood transplanted into immunodeficient murine hosts to model antitumor responses