Porunelloor A. Mathew

Cutting edge: Lectin like transcript-1 is a ligand for the inhibitory human NKR-P1A receptor

Increasingly, roles are emerging for C-type lectin receptors in immune regulation. One receptor whose function has remained largely enigmatic is human NKR-P1A (CD161), present on NK cells and subsets of T cells. In this study, we demonstrate that the lectin-like transcript-1 (LLT1) is a physiologic ligand for NKR-P1A. LLT1-containing liposomes bind to NKR-P1A+ cells, and binding is inhibited by anti-NKR-P1A mAb. Additionally, LLT1 activates NFAT-GFP reporter cells expressing a CD3ζ-NKR-P1A chimeric receptor; reciprocally, reporter cells with a CD3ζ-LLT1 chimeric receptor are stimulated by NKR-P1A. Moreover, LLT1 on target cells can inhibit NK cytotoxicity via interactions with NKR-P1A.

2B4 Acts As a Non-Major Histocompatibility Complex Binding Inhibitory Receptor on Mouse Natural Killer Cells

Natural killer (NK) cells are critical in the immune response to tumor cells, virally infected cells, and bone marrow allografts. 2B4 (CD244) is expressed on all NK cells and the ligand for 2B4, CD48, is expressed on hematopoietic cells. Cross-linking 2B4 on NK cells with anti-2B4 monoclonal antibody leads to NK cell activation in vitro. Therefore, 2B4 is considered to be an activating receptor. Surprisingly, we have found, using antibody-blocking and 2B4-deficient NK cells, that NK lysis of CD48+ tumor and allogeneic targets is inhibited by 2B4 ligation. Interferon γ production by NK cells is also inhibited. Using a peritoneal tumor clearance assay, it was found that 2B4−/− mice have increased clearance of CD48+ tumor cells in vivo. Retroviral transduction of 2B4 was sufficient to restore inhibition in 2B4−/− primary NK cells. It was found that although mature NK cells express SH2D1A, in vitro–derived NK cells do not. However, both populations are inhibited by 2B4 ligation. This indicates that 2B4 inhibitory signaling occurs regardless of the presence of SH2D1A. These findings reveal a novel role for 2B4 as a non–major histocompatibility complex binding negative regulator of NK cells.

2B4 (CD244) and CS1: novel members of the CD2 subset of the immunoglobulin superfamily molecules expressed on natural killer cells and other leukocytes

Summary: 2B4 is a member of the CD2 subset of the immunoglobulin superfamily molecules expressed on natural killer (NK) cells and other leukocytes. It is the high affinity ligand for CD48. Engagement of 2B4 on NK‐cell surfaces with specific antibodies or CD48 can trigger cell‐mediated cytotoxicity, interferon‐γ secretion, phosphoinositol turnover and NK‐cell invasiveness. The function of 2B4 in CD8+ T cells and myeloid cells remains unknown. The cytoplasmic domain of 2B4 contains unique tyrosine motifs (TxYxxV/I) that associate with src homology 2 domain‐containing protein or signaling lymphocyte activation molecule (SLAM)‐associated protein, whose mutation is the underlying genetic defect in the X‐linked lymphoproliferative disease (XLPD). Impaired signaling via 2B4 and SLAM is implicated in the immunopathogenesis of XLPD. CS1 is a novel member of the CD2 subset that contains two of the unique tyrosine motifs present in 2B4 and SLAM. Signaling through 2B4, CS1 and other members of the CD2 subset may play a major role in the regulation of NK cells and other leukocyte functions.

Perforin: more than just an effector molecule

Abstract Perforin mediates the destruction of virus-infected or transformed cells. However, the finding that perforin deficiency is the underlying cause of familial hemophagocytic lymphohistiocytosis indicates that it also regulates the immune response.

2B4 (CD244)-Mediated Activation of Cytotoxicity and IFN-γ Release in Human NK Cells Involves Distinct Pathways

2B4 (CD244), a member of the CD2 subset of the Ig superfamily receptors, is expressed on all human NK cells, a subpopulation of T cells, basophils and monocytes. 2B4 activates NK cell mediated cytotoxicity, induces secretion of IFN-γ and matrix metalloproteinases, and NK cell invasiveness. Although there has been several molecules shown to interact with 2B4, the signaling mechanism of 2B4-mediated activation of NK cells is still unknown. In this study, we found cross-linking of 2B4 on YT cells, a human NK cell line, results in the increased DNA binding activity of activator protein-1 (AP-1), an important regulator of nuclear gene expression in leukocytes. We investigated the possible role of various signaling molecules that may be involved in the activation of lytic function of YT cells via 2B4. Treatment of YT cells with various specific inhibitors indicate that 2B4-stimulation of YT cells in spontaneous and Ab-dependent cytotoxicity is Ras/Raf dependent and involves multiple MAPK signaling pathways (ERK1/2 and p38). However, only inhibitors of transcription and p38 inhibited 2B4-mediated IFN-γ release indicating distinct pathways are involved in cytotoxicity and cytokine release. In this study we also show that 2B4 constitutively associates with the linker for activation of T cells (LAT) and that 2B4 may mediate NK cell activation via a LAT-dependent signaling pathway. These results indicate that 2B4-mediated activation of NK cells involves complex interactions involving LAT, Ras, Raf, ERK and p38 and that cytolytic function and cytokine production may be regulated by distinct pathways.

CS1, a novel member of the CD2 family, is homophilic and regulates NK cell function

CS1 is a novel member of the CD2 subset of immunoglobulin superfamily (IgSF) expressed on NK, T and stimulated B cells. The cytoplasmic domain of CS1 contains immunoreceptor tyrosine-based switch motif (ITSM) which is present in 2B4, SLAM and CD84. The signaling adaptor molecule SAP/SH2D1A, the defective gene in X-linked lymphoproliferative disease (XLPD), binds to ITSM and regulates immune cell function. However, recent studies indicate that CS1 may be regulated by a SAP-independent mechanism. In this study, we have examined the ligand specificity of CS1 and the effect of CS1 interaction with its ligand on the cytolytic activity of YT, a human NK cell line. Recombinant fusion protein, CS1-Ig, containing the CS1 extracellular domain and Fc portion of the human IgG bound cells transfected with CS1. CS1-Ig did not show any binding to cells expressing other members of the CD2 family. The cytolytic activity of YT was enhanced in presence of soluble CS1-Ig fusion protein. These results demonstrate that CS1 is a self-ligand and homophilic interaction of CS1 regulates NK cell cytolytic activity.

Absence of mouse 2B4 promotes NK cell–mediated killing of activated CD8 + T cells, leading to prolonged viral persistence and altered pathogenesis

Persistent viral infections are often associated with inefficient T cell responses and sustained high-level expression of inhibitory receptors, such as the NK cell receptor 2B4 (also known as CD244), on virus-specific T cells. However, the role of 2B4 in T cell dysfunction is undefined, and it is unknown whether NK cells contribute to regulation of these processes. We show here that persistent lymphocytic choriomeningitis virus (LCMV) infection of mice lacking 2B4 resulted in diminished LCMV-specific CD8+ T cell responses, prolonged viral persistence, and spleen and thymic pathologies that differed from those observed in infected wild-type mice. Surprisingly, these altered phenotypes were not caused by 2B4 deficiency in T cells. Rather, the entire and long-lasting pathology and viral persistence were regulated by 2B4-deficient NK cells acting early in infection. In the absence of 2B4, NK cells lysed activated (defined as CD44hi) but not naive (defined as CD44lo) CD8+ T cells in a perforin-dependent manner in vitro and in vivo. These results illustrate the importance of NK cell self-tolerance to activated CD8+ T cells and demonstrate how an apparent T cell-associated persistent infection can actually be regulated by NK cells.

Expression of functional bovine cholesterol side chain cleavage cytochrome P450 (P450scc) in Escherichia coli

Escherichia coli expression vectors containing the trc promoter and the complete DNA sequence of either the precursor or the mature form of bovine adrenocortical cholesterol side chain cleavage cytochrome P450 (P450scc) were transformed into E. coli strain JM109 and transcription induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). Immunoreactive cytochrome P450scc was produced using the plasmid containing the mature P450scc sequence but not with the plasmid containing the sequence of the precursor form of P450scc, even though P450scc RNA was detectable in both cases. The mature form of P450scc was detected spectrophotometrically in a reduced CO-difference spectrum in E. coli (40-60 nmol/liter culture). Cholesterol and hydroxylated derivatives (22-hydroxycholesterol and 25-hydroxycholesterol) produce a type 1 substrate-binding spectrum in IPTG-induced, transformed E. coli. The P450scc was found to be associated with the E. coli membrane fraction and the enzymatic activity of side chain cleavage of 25-hydroxycholesterol was reconstituted using solubilized membranes, in the presence of purified bovine adrenocortical adrenodoxin and NADPH-adrenodoxin reductase (turnover number; 15.4 nmol/min/nmol P450). This bacterial expression system provides functional P450scc, in the absence of other forms of P450, which can be used for evaluation of enzymatic and spectral properties of this mitochondrial P450 by site-directed mutagenesis.

Gene structure of the murine NK cell receptor 2B4: presence of two alternatively spliced isoforms with distinct cytoplasmic domains

The NK cell receptor 2B4 is expressed on the surface of all murine NK cells and a subset of T cells. Ligation of 2B4 with monoclonal antibodies increases target cell lysis and IFN‐γ production. 2B4 is the high‐affinity counter‐receptor for CD48 in mice and humans. 2B4‐L is a member of the CD2 subgroup of the immunoglobulin supergene family, which includes CD48, LFA‐3, CD84, Ly9 and SLAM. Here we describe 2B4‐S, a second 2B4 isoform, and the genomic structure of the 2B4 gene. 2B4‐S is identical to the 5′ end of 2B4‐L, differing only at the 3′ end, corresponding to a portion of the cytoplasmic domain and the 3′ untranslated sequence. Both 2B4‐L and 2B4‐S are expressed on IL‐2‐activated NK cells. The genomic clone of 2B4 reveals that the two cDNA clones are products of alternative splicing. Since they differ only in a portion of the cytoplasmic domain, it is likely that they transduce different signals.

Allorecognition by murine natural killer cells: lysis of T-lymphoblasts and rejection of bone-marrow grafts

Summary: Natural killer (NK) cells of inbred mice reject allogeneic bone‐marrow cells, and NK cells of F1 hybrid mice can reject parental bone‐marrow cells (hybrid resistance). In some cases these patterns of rejection can be mimicked in vitro by utilizing IL‐2 cultured NK effector cells and allogeneic or parental T‐lymphoblasts as target cells. Lysis of allogeneic parental targets in vitro can be explained on the basis of the missing self hypothesis. Subsets of NK cells that bear non‐overlapping MHC class I inhibitory receptors belonging to the Ly49 family lyse allogeneic targets because they do not express self class I molectiles of the NK cell donor. Parental strain targets are lysed because they do not express all of the self class I antigens of the Fl hybrid, and hence fail to deliver inhibitory signals to all subsets of Fl NK cells. The expression of Ly49 receptors on NK cells is regulated by liost MHC to ensure maximal sensitivity to alterations in self class I molecules and to prevent autoreactivity. In many instances, however, the rejection of allogeneic bone marrow cells in vivo cannot be readily explained by the missing self hypothesis. In these instances, it appears that rejection is initiated by class 1 MHC receptors on NK ceils Out recognize allogeneic class I molecules as non‐self, and activate rather than inhibit NK cell function.