Ly-6 Family Research Articles

Structural similarity between uPAR, other proteins of the Ly-6 family and snake venom α-neurotoxins

Structural similarity between uPAR, other proteins of the Ly-6 family and snake venom α-neurotoxins

Isolation of a cDNA encoding thymic shared antigen-1. A new member of the Ly6 family with a possible role in T cell development.

We have previously characterized a novel mouse thymocyte marker, defined as thymic shared Ag-1 (TSA-1), present on both immature thymocytes and a subset of thymic medullary epithelial cells. MTS 35, a mAb specific for TSA-1, alters T cell differentiation when added to fetal thymic organ cultures, suggesting TSA-1 may be important for T cell development in the thymus. In this study, we describe the isolation of a cDNA encoding TSA-1 using transient expression of COS-7 cells and selection with MTS 35. The predicted amino acid sequence of this cDNA encodes a 15 to 17-kDa protein and the expressed protein is linked to the membrane via a phosphatidylinositol moiety. TSA-1 is transcriptionally active at various levels in all organs examined, suggesting that its role is not solely intrathymic. TSA-1 shares amino acid sequence homology to the mouse Ly6 multigene family, epidermal growth factor-like receptors, and to cobra venom neurotoxin. The Tsa-1 locus is located on chromosome 15 linked to Ly6 on the mouse genome. We also examined the effects of MTS 35 in fetal thymic organ cultures repopulated with two subsets of thymocytes representing defined stages of T cell development. Our results suggest that TSA-1 may play a role during positive selection and the transition from CD4+CD8+ thymocytes to the mature CD4+CD8- and CD4-CD8+ subsets.

Selective expression of Ly-6G on myeloid lineage cells in mouse bone marrow. RB6-8C5 mAb to granulocyte-differentiation antigen (Gr-1) detects members of the Ly-6 family.

Mouse Ly-6 proteins are characterized by lineage-restricted patterns of expression on lymphoid cells. A mAb (1A8) was produced to Ly-6G, a newly described member of the Ly-6 locus. Based on selective reactivity to cloned Ly-6 gene products expressed in EL4J cells, 1A8 was determined to be specific for Ly-6G. Furthermore, mAb to other Ly-6 specificities did not bind to Ly-6G-transfected EL4J cells, indicating that Ly-6G is distinct from other serologically defined Ly-6 specificities. FACS analysis using 1A8 demonstrated that Ly-6G was expressed in bone marrow but not substantially on other lymphoid tissues, including activated T and B cells. In the bone marrow, Ly-6G expression was primarily restricted to the cells with more forward angle light scatter, which are mostly granulocytes. The RB6-8C5 mAb, previously described to detect a myeloid-restricted Ag (Gr-1) on more differentiated granulocytes, also reacted with Ly-6G- and Ly-6C-transfected EL4J cells. Both 1A8 and RB6-8C5 selectively precipitate a M(r) 21 to 25 kDa, glycosylphosphatidylinositol-anchored protein. Collectively, these data indicate that the Gr-1 Ag is a member of the Ly-6 family and further link expression of individual Ly-6 genes with distinct lineages in mouse bone marrow cells.

CD59 functions as a signal-transducing molecule for human T cell activation.

The CD59 Ag is a 20-kDa protein that is widely expressed on most leukocytes and RBC, is coupled to the membrane by a phosphatidylinositol-glycan anchoring structure, plays a role in cell interaction between monocytes and T cells, and also functions as an inhibitor of cytolysis by the terminal C components C5b-9. Because this molecule is structurally related to the murine Ly-6 family of Ag, we have investigated whether anti-CD59 mAb might be capable of activating human T lymphocytes in a manner similar to that described for antibodies to the murine Ly-6 Ag. In the presence of the appropriate co-stimulators, mAb to one of the two epitopes on CD59 were capable of inducing both a rise in intracytoplasmic free Ca2+, inositol phosphate production, IL-2 production, and T cell proliferation. Anti-CD59-induced inositol phosphate turnover and IL-2 production were dependent on co-expression of the CD3/TCR complex. CD59-loss mutants of the Jurkat cell line were completely responsive to stimulation by anti-CD3 thereby demonstrating that CD59 does not play a role as a signal transducer downstream from the TCR. Taken together, these results demonstrate that the CD59 Ag can play multiple distinct roles in the regulation of the immune response.

Regulation of B lymphocyte responses to IL-4 and IFN-gamma by activation through Ly-6A/E molecules.

The Ly-6 family of cell surface molecules has previously been shown to participate in T cell activation. We show that Ly-6A/E proteins also modulated the response of normal B lymphocytes in three separate in vitro assays. First, unfractionated or small resting B cells proliferated when cultured with IFN-gamma, IL-4, and an anti-Ly-6A/E mAb. Second, this anti-Ly-6A/E mAb restored B cell proliferation responses that were inhibited when coculturing the B cells in IFN-gamma, IL-4, and anti-IgM. Third, anti-Ly-6A/E specifically up-regulated the cell surface expression of its own Ag, and this response was dependent upon co-stimulation with IFN-gamma. Mixing of T and B cells in culture suggested that T cells did not contribute substantially to the B cell proliferative response. Moreover, up-regulation of Ly-6A/E was observed for one B cell lymphoma, WEHI-231. Therefore, it appeared that modulation of B cell function by anti-Ly-6A/E was due to a direct effect of the mAb binding to the B cells. Taken together, these data suggest Ly-6A/E proteins are functional on B cells and may play a regulatory role in B cell activation.

Ly‐6 A/E antigen of murine T cells is associated with a distinct pathway of activation. Requirements for interferon and exogenous interleukin 2

The Ly-6 pathway of T cell activation was analyzed to identify its essential requirements. Using a monospecific chicken antiserum to Ly-6E, fully cross-reactive to its allelic counterpart, Ly-6A, but unreactive with other members of the Ly-6 family, we have found that interferon (IFN)-alpha/beta or gamma, Ly-6 antibody and interleukin 2 (IL 2) act synergistically in inducing T cell proliferation. The action of IFN can be attributed to induction of Ly-6A/E antigen on T cells, as described previously, and this induction is transcriptionally controlled. Exposure of T cells with elevated Ly-6 concentrations to chicken anti-Ly-6 antibody leads to expression of IL 2 receptors. Consequently, the addition of IL 2 drives T cell proliferation. Thus, in BALB/c mice the minimum requirements for activation by the Ly-6 pathway are IFN (as a means of inducing Ly-6). Ly-6 antibody (as inducer of IL 2 receptors) and IL 2. In mice of the Ly-6.2 haplotype, IFN is not an absolute requirement. This may be related to the fact that these animals, in contrast to those of Ly-6.1 haplotype, express Ly-6 constitutively on a substantial proportion of resting T cells. Thus, T cells of C57BL/6 or DBA/2 mice can be induced to proliferate with Ly-6 antibody and IL 2 alone, although IFN pretreatment enhances this response. In BALB/c mice the IL 2-driven proliferative response induced through the Ly-6 pathway occurs selectively in the L3T4- population.

Demonstration of phosphatidylinositol anchors on Ly-6 molecules by specific phospholipase C digestion and gel electrophoresis in octylglucoside

Release by phosphatidylinositol-specific phospholipase C is frequently provided as evidence for membrane anchorage of a protein through phosphatidylinositol. Demonstration that the enzyme removes a lipophilic moiety from the protein is stronger evidence, and is presented here for members of the Ly-6 family of lymphocyte antigens: Ly-6A, C and E. Treatment of these molecules with the enzyme greatly increased their electrophoretic mobilities on polyacrylamide gels containing nonionic detergent. Furthermore, the mobilities of the digested, but not native Ly-6 molecules, were independent of detergent. This analytical method was applied to pure antigen, radiolabelled immunoprecipitate, or immunochemically detected Ly-6 antigens on blots.