Wayne C. Lai

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.

Ly49I NK Cell Receptor Transgene Inhibition of Rejection of H2b Mouse Bone Marrow Transplants

The Ly49 family of genes encode NK cell receptors that bind class I MHC Ags and transmit negative signals if the cytoplasmic domains have immunoregulatory tyrosine-based inhibitory motifs (ITIMs). 5E6 mAbs recognize Ly49C and Ly49I receptors and depletion of 5E6+ NK cells prevents rejection of allogeneic or parental-strain H2d bone marrow cell (BMC) grafts. To determine the function of the Ly49I gene in the rejection of BMC grafts, we transfected fertilized eggs of FVB mice with a vector containing DNA for B6 strain Ly49I (Ly49IB6). Ly49IB6 is ITIM+ and is recognized by 5E6 as well as Ly49I-specific 8H7 mAbs. Normal FVB H2q mice reject H2b but not H2d BMC allografts, and the rejection of H2b BMC was inhibited partially by anti-NK1.1 and completely by anti-asialo GM1, but not by anti-CD8, Abs. In FVB mice, NK1.1 is expressed on only 60% NK cells. FVB.Ly49IB6 hosts failed to reject H2d or H2b BMC, but did reject class I-deficient TAP-1−/− BMC, indicating that NK cells were functional. Nondepleting doses of anti-Ly49I Abs reversed the acceptance of H2b BMC by FVB.Ly49IB6 mice. FVB.Ly49IB6+/− mice were crossed and back-crossed with 129 mice—H2b, 5E6−, poor responders to H2d BMC grafts. While transgene-negative H2b/q F1 or first-generation back-crossed mice rejected H2b marrow grafts (hybrid resistance), transgene-positive mice did not. Thus B6 strain Ly49I receptors transmit inhibitory signals from H2b MHC class I molecules. Moreover, Ly49IB6 has no positive influence on the rejection of H2d allografts.

Potential subunit vaccine against Mycoplasma pulmonis purified by a protective monoclonal antibody

Monoclonal antibodies (mAbs) were produced against Mycoplasma pulmonis (MP); some were highly protective in the treatment of experimental infections of BALB/c mice. The mAbs inhibited MP growth in vitro and prevented the attachment of MP to fibroblasts or to red blood cells. Three separate mAbs recognized 54-76% of 54 clinical isolates of MP, and the three together detected all 54 isolates. We used the mAbs to purify the antigens by affinity column chromatography. The purified antigen used to vaccinate mice and to immunize rabbits produced antibodies capable of significant growth inhibition in sera and tracheolung lavage fluids. The vaccinated mice were challenged with various doses of a highly virulent T2 strain of MP. Assays for viable MP organisms and for histopathological changes in the lungs of infected mice indicated that mice were protected if the challenge dose was 10(3)-10(5), but not 10(7), c.f.u. The sera of immunized rabbits were used to passively transfer immunity to mice. The sera provided complete protection against 1 x 10(6) c.f.u. T2 MP. We conclude that MP antigens purified by this protocol can provide a safe vaccine against this disease, at least in mice.

Protection of mice against experimental murine mycoplasmosis by a Mycoplasma pulmonis immunogen in lysogenized Escherichia coli

A construct of the Mycoplasma pulmonis (MP) genomic library, using randomly sheared DNA, was cloned in lambda gt11 and transfected into C600 Escherichia coli organisms. Clones of E. coli expressing a fusion protein reactive with anti-MP and monospecific serum were transferred orally or intravenously into Balb/c mice. Expression of the fusion protein was induced by adding isopropyl-beta-D-thiogalactopyranoside to the drinking water. This vaccination protocol led to local and systemic antibody formation, to generation of immune lymphocytes and to protection against large numbers of virulent MP organisms. This approach might be generally successful in preventing infectious disease.

Molecular characterization of the rat NK cell receptor 2B4

2B4 (CD244) is a cell surface glycoprotein of the immunoglobulin superfamily involved in the regulation of natural killer and T lymphocyte function. It is the high affinity counter-receptor for CD48. In mouse and human NK cells, crosslinking of 2B4 with a specific monoclonal antibody or with CD48 can trigger cell-mediated cytotoxicity, IFN-gamma secretion, phosphoinositol turnover and NK cell invasiveness. Recent reports of defective 2B4 signaling and NK cell function in X-linked lymphoproliferative syndrome suggest that this may contribute to the progression of this human disease. Here we describe the molecular characterization of the rat 2B4 gene. The cDNA encodes a protein of 395 amino acid residues that contain two Ig domains in the extracellular region and three unique tyrosine motifs (TxYxxV/I/A) in the cytoplasmic region. The predicted protein has 81 and 68% similarity with mouse 2B4 and human 2B4, respectively. Additionally, it has 94 and 89% similarity at the protein level with the recently reported rat 2B4 related genes, r2B4R-tm and r2B4R-se respectively. Northern blot analysis indicated the presence of multiple transcripts in rat LAK cells and RNK-16 cells. Immunoprecipitation and deglycosylation studies showed that rat 2B4 is glycosylated to similar extent as that of mouse and human 2B4. The cloning of r2B4 in the light of the availability of rat NK cell lines should facilitate in vitro and in vivo experiments to decipher the functional role of 2B4 in NK cell biology.