Ly-5: A new T-lymphocyte antigen system

Ly-5 is a third genetic locus of the type so far represented in the mouse byLy-1 andLy-2/Ly-3; it specifies antithetical alloantigens, one of which is present exclusively on T lymphocytes of every mouse. The chromosomal locus ofLy-5 has not been established, but it is not closely linked toLy-1 orLy-2/Ly-3. Like other T-lymphocyte surface markers, expression of Ly-5 antigens on T-lymphocyte precursor cells can be initiated in vitro by inducers of T-cell differentiation.Recipient of a fellowship from the New York Cancer Research Institute, Inc.     

Further description of theLy-5 system

TheLy-5 system of alloantigens was reevaluated with the aid of an Ly-5 congenic mouse strain and an additional serological technique, the PA-SRBC rosette assay, not previously applied to Ly-5. In the PA-SRBC assay, SRBC to which PA (Staphylococcal Protein A) has been coupled react with antibodycoated cells to form rosettes. The PA-SRBC assay was more sensitive than the cytotoxicity assay in detecting Ly-5 on all cell types tested, with the exception of thymocytes, on which Ly-5 was more efficiently demonstrable by the latter test. Thus, the use of both assays gave a more complete picture of theLy-5 system. Evidently Ly-5 is expressed on most cells of the hematopoietic lineage, but on no other cells so far tested. The Ly-5⁺ cell population includes all known sets of T cells, prothymocytes, sets of B cells, cells of myeloid and monocyte-macrophage types and natural killer cells. Erythrocytes and proerythroblasts are Ly-5^?, but the Ly-5 phenotype of less differentiated erythrocytic cells is uncertain. By means of radiation chimeras, made by restoring lethally irradiated mice with Ly-5 congenic bone marrow, the weakly Ly-5⁺ phenotype of liver and of kidney was shown to be due to immigrant circulating cells. The Ly-5 phenotypes of tumors conform to this scheme of Ly-5 tissue representation. The Ly-5⁺ tumors included more than 40 leukemias tested, plasmacytomas, putative transformed equivalents of slg^?: Lyb-2⁺ early B cells, a mastocytoma, a monocytic line, and three lines related to the macrophage. The other tumors tested -a sarcoma, a spontaneous mammary carcinoma, a teratocarcinoma and a neuroblastoma — were Ly-5^?.     

Studies of the mouse Ly-6 alloantigen system

Three monoclonal antibodies were produced by fusing mouse myeloma cell line NS-1 with spleen cells from C3H/An mice hyperimmunized with B6-H-2^k spleen cells. These antibodies recognized an alloantigen displaying a similar strain distribution pattern to the Ly-6.2 and Ala-1.2 alloantigens. Analysis of C×B and B×H recombinant inbred mice revealed close linkage of genes controlling Ly-m6 and Ly-6. The monoclonal antibodies lysed 70 percent of cells in lymph nodes and 60 percent in spleen in direct cytotoxicity assays, but did not lyse significant numbers of cells of thymus and bone marrow. Separated T and B cells were reactive with the antibodies, but T cells were more sensitive to the antibody and complement than B cells. Virtually all cells in cultures of cells activated in the mixed lymphocyte reaction or by Concanavalin A were reactive with the monoclonal antibodies. Direct plaque-forming cells were completely eliminated by the monoclonal antibody and complement. By absorption tests, cells from all organs tested so far (thymus, lymph node, spleen, bone marrow, brain, kidney and liver) were shown to express the Ly-m6 determinant. Tumor cell lines with T, B or stem cell characteristics were reactive with the monoclonal antibody by direct cytotoxicity and absorption assays.     

Studies of the mouse Ly-6 alloantigen system

The discovery of several monoclonal antibodies provided the impetus to revisit the Ly-6 group of antigens. Our serological data point to the existence of at least five separate Ly-6 antigens. They are distinguished by the patterns of their tissue expression as (1) the classical Ly-6 alloantigen of peripheral lymphocytes (Ly-m6.2A), (2) a bone marrow cell-restricted antigen (Ly-m6.2B), (3) an antigen shared by bone marrow cells and peripheral lymphocytes (Lym6.2C, possibly identical with H9/25),(4) an antigen expressed on bone marrow cells, thymocytes, and peripheral lymphocytes (Ly-m6.2D), and (5) an antigen occurring exclusively on lymphoblasts (Ly-m6.IE, similar to Ala-1). ThB is a sixth distinct antigen of the group. The assumption that separate antigens exist is supported by distinctive distribution patterns in normal and neoplastic tissues. The genes controlling Ly-6 antigens are closely linked, as they are transmitted as two haplotypes only. One incidence of a crossover within the Ly-6 region was observed: the Ly-6B.2 alloantigen was expressed in NZB mice, which type Ly-6.1 for other Ly-6 specificities.     

Effects of fractionated x-irradiation on the Ly-6--Ril-1--Pol-5 region

Our laboratory has focused on defining, localizing, and understanding the mode of action of genes involved in fractionated x-irradiation (FXI) leukemia in susceptible and resistant mouse strains. We have described the genetic and molecular evidence suggesting the existence of multiple independent loci involved in FXI-induced leukemogenesis. These studies indicated that one of these, Ril-1, a locus on the distal portion of chromosome 15, is the major locus influencing susceptibility to the disease. Our data unequivocally place Ril-1 in the gene complex Ly-6--Ril-1--Sis--H-30--Pol-5. Ril-1 appears to be closest to Ly-6 and Sis. We report that in FXI-induced leukemias there are hypomethylation changes in the Ly-6 region as compared to normal thymocytes. In contrast, Sis was found to be hypermethylated and not expressed. In addition, we have noted DNA rearrangements in the Ly-6--Pol-5 region in the majority of tumors examined using the Ly-6 and spleen focus-forming virus (SFFLV) molecular probes. Increased expression of Ly-6 and other surface markers encoded in this region has been noted in FXI-induced thymomas. Address correspondence and offprint request to: N. M. B. Amari.     

Active tyrosine phosphatase in immunoprecipitates of multiple isoforms of Ly-5

Using tumour cell lines expressing specific isoforms of murine Ly-5 (molecular weights of 180,000, 200,000 and 240,000) we find that all forms of Ly-5 and immuno-affinity purified forms of Ly-5 contain tyrosyl phosphatase activity. These results demonstrate that these isoforms of Ly-5 belong to the same family of functional receptor-linked tyrosine phosphatases as the human leukocyte common antigen. CD45.     

Allelic variants of Ly-5 in inbred and natural populations of mice

Allelic variants of Ly-5 in inbred commensal and other natural populations of mice were analyzed by patterns of restriction fragment length polymorphisms (RFLP) and Southern hybridization using an Ly-5 cDNA probe and by cell-surface staining with a panel of antibodies directed against polymorphic and nonpolymorphic Ly-5 determinants. New Ly-5 alleles were defined by RFLPs generated by both Eco RI and Bam HI restriction enzyme digests. The Mus musculus subspecies and other species within the genus Mus showed a strong correlation between allelic variants defined by restriction enzymes and serologic specificities. The data also suggest the conservation of the Ly-5 gene throughout the genus Mus.     

Structural features of Ly-5 glycoproteins of the mouse and counterparts in other mammals

The Ly-5 system of the mouse defines a set of transmembrane glycoprotein isoforms (T200, B220, etc) that hallmark various lineages and stages of hematopoietic differentiation. These isoforms are the products of a single Ly-5 gene comprising 34 exons, 32 of them (Exs-3-34) protein-coding and three (Exs-5-7) selectively represented in different isoforms (e.g., all three in isoform B220 but none in isoform T200). Probable structural features of Ly-5 glycoproteins, largely inferred from Ly-5 gene composition, are presented and compared with the rat L-CA and human LCA/T200 systems, which are phylogenetic counterparts of Ly-5 as an index of the extent and nature of structural conservation. The outer (N-terminal) region of the Ly-5 T200 isoform comprises three broadly similar domains (Exs-4, 8, 9) with salient features that jointly favor free interaction with the aqueous environment and are shared by the L-CA and human LCA/T200 systems despite an overall interspecies protein sequence similarity in this region of only about 50 %. In the larger B220 isoform this region includes epitopes dictated by the selective exons Exs-5, 6, 7, these being more conserved than the shared exons Exs-4, 8, 9 and no doubt sustaining the differential functions of the respective isoforms. Comparison of the genomic sequences of Ex-5 in the Ly-5 and human systems suggests that a shift in splice donor site accounts for an extra 23 amino acids in the human Ex-5-coding domain, which is the only salient structural difference between the mouse Ly-5 and human systems. The inner extracellular region (Exs-10-16) includes subregions of high variability, but again there are shared salient interspecies similarities such as sites and numbers of Cys residues that imply a conserved, tightly-folded conformation, in contrast to the more open conformation predicted for the outer extracellular region. The transmembrane region (Ex-17) is highly conserved, as is the very large cytoplasmic region (Exs-17-34) which may interact with the plasma membrane but probably does not traverse it.     

Selective inhibition of lipopolysaccharide-induced polyclonal IgG response by monoclonal Ly-5 antibody

We have identified two important molecules involved in the regulation of B cell differentiation, namely Lyb-2 and Ly-5. To gain further insight into the function of these two molecules, we examined the effect of monoclonal Lyb-2 and Ly-5 antibodies on lipopolysaccharide (LPS)-induced B cell growth and maturation. We found that Lyb-2 antibody does not have any effect on LPS-induced proliferation and on polyclonal IgM or total IgG responses. On the other hand, although Ly-5 antibody did not affect proliferation and polyclonal IgM responses, it strongly inhibited polyclonal IgG responses, presumably by direct action on B cells. This inhibition was not caused by direct suppressive effect of Ly-5 antibody or Fc receptor-mediated negative signaling. To exert maximal inhibitory effect, Ly-5 antibody had to be added to the culture during the initial 48 hr. However, the presence of Ly-5 antibody during the first 2 days did not cause a significant inhibition. It is thus likely that Ly-5 plays a critical role in the regulation of LPS-induced B cell maturation into IgG-secreting cells at a phase starting within 48 hr after LPS stimulation and continuing thereafter.     

Ly-15.2 and Ly-21.2 are distinct polymorphisms of the LFA-1 heavy chain

Address correspondence and offprint requests to: I. F. C. McKenzie.