Further definition of the Ly-5 system

Ly-5 is expressed by cells of the hematopoietic branch of development. Further serological analysis of the Ly-5 system, aided by Ly-5 monoclonal antibodies and by two Ly-5 congenic mouse strains, reveals two new Ly-5 alloantigens, Ly-5. 3 and Ly-5.4. The data define three thymocyte phenotypes, Ly-5.1,3, Ly-5.2,4, and Ly-5.2,3, and three corresponding genotypes, Ly-5 ^a, Ly-5 ^b, and Ly-5 ^c, respectively. Ly-5 ^ais by far the most common allele. The Ly-5 ^callele is found only in the ST/bJ strain, a finding that accords with the presently unique pattern of restriction fragments previously observed in Southern blotting of ST/bJ DNA with an Ly-5 cDNA probe. Present serological and biochemical data favor the interpretation that the compound Ly-5 phenotype of thymocytes is attributable to two separate Ly-5 molecular isoforms that exhibit a discrete difference in protein composition, bear different Ly-5 antigens, and are produced jointly by thymocytes, unlike other Ly-5 isoforms previously shown to distinguish different hematopoietic cell lineages.     

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.     

Ly-6 region regulates expression of multiple allospecificities

The relationship between two alloantigens on mouse lymphocytes, that is Ly-6.2 and H9/25, which have previously been shown to have identical strain distribution patterns, was further investigated. Analysis of 39 (AKR × CBA) × CBA backcross progeny showed no segregation between these two antigens, indicating a close genetic linkage between them. Serological analysis showed that Ly-6.2 and H9/25 are differentially expressed on T-cell hybrid lines. Furthermore, cross-absorption of anti-Ly-6.2 serum with two cell lines revealed a heterogeneity among Ly-6 specificities. Semipurified H9/25 antigen failed to block anti-Ly-6.2 serum while anti-Ly-6.2 serum did not significantly block monoclonal antibody H9/25. These results suggest the presence of multiple allospecificities encoded for by the Ly-6 region.     

Definition of new alloantigens encoded by genes in the Ly-6 complex

Three alloantigens encoded by Ly-6-linked genes are defined by monoclonal antibodies. The Ly-27.2 antigen is defined by antibody 5075-19.1, Ly-28.2 by 5075-3.6, -12.1, -16.10 and by 5095-16.6. The strain distribution pattern of these antibodies is the same and identical with Ly-6.2. However the tissue distribution of these antigens is unique and distinguishes these antigens from the Ly-6.2 antigen or any known antigen encoded by Ly-6-linked genes. Ly-27.2 is present on all thymocytes, T cells, and B cells but is absent from bone marrow cells, whereas Ly-28.2 is absent from most thymocytes and is present on a subpopulation of T cells and B cells but is found on 60–70% of bone marrow cells. No recombination between the Ly-6/Ly-27/Ly-28 loci was found in linkage studies using 41 recombinant inbred strains and 57 backcross mice and indicates very close linkage of these genes. In addition, close linkage to 24 minor histocompatibility genes was excluded using the Bailey HW bilineal congenic mice. The data presented indicate that either the Ly-6 complex is composed of a family of tightly linked genes or the antigens are the products of a single gene that undergoes extensive modification during differentiation.     

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.     

Differential regulation by Ly-5 and Lyb-2 of IgG production induced by lipopolysaccharide and B cell stimulatory factor-1 (IL-4)

We have previously shown that mAb Ly-5 which on B cells recognizes a 220,000-Da (B220) molecule, inhibits LPS-induced IgG responses without affecting IgM or proliferative responses, whereas mAb Lyb-2 which modulates B cell activation processes induced by B cell stimulatory factor-1 (BSF-1) or IL-4, has no effect on LPS-induced B cell responses. In this report we further examined the cellular mechanisms of Ly-5 antibody action and the effect of Lyb-2 antibody in IgG responses induced by LPS and BSF-1. The results presented demonstrated that the inhibitory effect of Ly-5 antibody seems to be restricted to the IgG class and is observed in all IgG subclasses induced by LPS. Limiting dilution analysis showed that the Ly-5 antibody reduces primarily the precursor frequency of IgG-secreting cells and that the effect on the clone size is partial. Lyb-2 antibody, on the other hand, greatly inhibited IgG1 induction initiated by LPS and BSF-1 by the action on processes triggered by BSF-1, although it could not reverse the reduced IgG2b or IgG3 responses. Limiting dilution analysis revealed that Lyb-2 antibody reduces the precursor frequency but not the clone size of BSF-1-induced IgG1-producing cells, supporting our previous proposition that Lyb-2 plays a critical role in the B cell differentiation mediated by BSF-1. Taken together, these results indicate that both Ly-5 and Lyb-2 are important molecules in IgG subclass regulation, each acting on a distinct activation step.     

Identity of murine lymphocyte alloantigens DAG,ALA-1, Ly-8, and Ly-6?

The DAG, Ala-1, Ly-8 and Ly-6 lymphocyte membrane alloantigens, identified by immune sera raised in different strain combinations, were originally thought to be distinct specificities. With the use of a congenic strain, BALB-DAG, we have now further evidence for antigenic identity based on serological and genetic criteria.     

Biochemical identification of rat ART-1 and Ly-1 alloantigens

The results of this study indicate that the ART-1 and Ly-1 rat alloantigens are synonymous with each other and also with the leukocyte-common (L-C) antigen which has been previously identified as a major glycoprotein of rat thymocytes and T and B lymphocytes. This conclusion is supported by the following observations: (i) when labeling of rat lymphoid cells was studied with a fluorescence-activated cell sorter, the profiles obtained were similar for labeling with ART-1 and Ly-1 alloantibodies and a monoclonal antibody to L-C antigen: (ii) this labeling was almost completely inhibited by purified L-C antigen: (iii) preincubation with L-C antigen completely inhibited binding of the alloantibodies in a cellular radioimmunoassay; (iv) the cytotoxic effect of the alloantibodies was completely abolished by preincubation with purified L-C antigen; (v) the strain distribution of the ART-1 and Ly-1 alloantigens was identical for 11 rat strains and in linkage analysis the ART-1 and Ly-1 alloantigens were found to cosegregate.Genetic linkage studies have shown that the L-C antigen locus is unlinked to the major histocompatibility antigen (RT1), the immunoglobulin light chain (1^k) and to the coat color gene (C) loci.Abbreviations used in this paper BSA Bovine serum albumin - DAB Dulbecco's salt solution - FCS Fetal calf serum - L-C antigen Leucocyte-common antigen - LN Lymph node - TDL Thoracic duct lymphocytes     

Studies of murine lymphocytes and alloantigens: I. Ly-6 mitogen and MLR responses

Antisera to the mouse lymphocyte surface alloantigens Ly-6.1 and Ly-6.2 were used to further study the functional distribution of these antigens. After selective depletion with antiserum + rabbit complement (RC), lymph node or spleen cells from Ly-6 congenic (C3H and C3H.B6-Ly-6^b) and noncongenic strains of mice were tested for: (a) their proliferative responses to T- and B-cell mitogens; and (b) their proliferative responses to alloantigens, or ability to stimulate in the MLR. Lymphoid cells required in the proliferative responses to the mitogens leucoagglutinin, concanavalin A (Con A), lipopolysaccharide (LPS), and pokeweed mitogen (PWM) were Ly-6⁺. Lymph node responder cells in the mixed lymphocyte reaction (MLR) were also Ly-6⁺, whereas spleen stimulator cells were Ly-6^?. Treatment of lymph node cells with anti-Ly-6 sera in the absence of RC had no specific blocking effect on the response to any of these mitogens. The studies indicate that the Ly-6 antigen is a potentially valuable marker for distinguishing between functionally distinct Ly-1⁺ T-cell subsets.     

Expression of Ly-6A/E alloantigens in thymocyte and T-lymphocyte subsets: variability related to the Ly-6 a and Ly-6 b haplotypes

We have studied the cellular basis for differential expression of the Ly-6A/E alloantigen on T cells obtained from mice of the Ly-6 ^a (10–20% Ly-6A/E ⁺) and Ly-6 ^b (50–60% Ly-6A/E ⁺) haplotypes. During T-cell ontogeny only a small fraction (< 12 %) of thymocytes expressed Ly-6A/E. By 4 weeks of age adult levels of Ly-6A/E bearing lymphocytes were seen in peripheral lymphoid tissue. Immunohistochemical studies of the thymus revealed that Ly-6A/E⁺ cells were located predominantly in the medulla with small clusters of Ly-6A/E⁺ cells throughout the cortex. Consistent with this result, phenotypic studies showed that in the adult thymus the majority of Ly-6A/E expression was on mature CD4⁺ CD8^– and CD4^– CD8⁺ cortisone-resistant and precursor CD4^– CD8^– thymocytes. However, a much higher percentage of CD4⁺ CD8^– and CD4^– CD8^– thymocytes as well as CD4⁺ CD8^– peripheral T cells expressed Ly-6A/E from Ly-6 ^b mice. Furthermore, although gamma interferon induced increased Ly-6A/E expression in certain thymocyte and T-cell subsets, this induction functioned preferentially for cells obtained from Ly-6 ^b mice. Studies using F₁ hybrid mice (Ly-6 ^a × Ly-6 ^b) indicated that the basal level of Ly-6A/E expression on these subsets appeared to be under codominant genetic control, whereas gamma interferon-induced regulation of Ly-6A/E expression appeared to be under dominant genetic control. Collectively, these results suggest that the expression of Ly-6A/E on a particular T-cell subset is established in the thymus and is a stable characteristic of each haplotype. In addition, the low levels of Ly-6A/E expression for the Ly-6 ^a haplotype appear to be partially due to the inability of the majority of resting CD4⁺ T cells to express Ly-6A/E and to the relatively poor induction of this protein by gamma interferon.     

Mouse Ly-31.1 is an alloantigenic determinant of alkaline phosphatase predominantly expressed in the kidney and bone

The mouse lymphocyte surface alloantigen, Ly-31, defined by monoclonal antibody N1.10 (IgG2b, k) and controlled by a gene locus closely linked to theAkp-2 locus on chromosome 4, was biochemically investigated. By employing a quantitative immunoassay system, it was found that the Ly-31.1-specific antibody detected an allotypic determinant of mouse alkaline phosphatase. Ly-31.1, i. e., mouse alkaline phosphatase, was expressed predominantly in kidney and bone and was also detected in placenta, lung, and testis. Concerning tumor cell lines, they varied in the amount of antigen present, with both T and B lymphoid lineages selectively possessing the antigen. In normal lymphoid tissues, lesser amounts of antigen were detected. The binding of mouse alkaline phosphatase to Ly-31.1-specific monoclonal antibodies was specific in nature. The Ly-31.1 antigen was immunoprecipitated from the lysates of surface-radiolabeled YAC-1 moloney leukemia cells, and appeared as a single band of about 78 000 under both reduced and nonreduced conditions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, treatment of tumor cell lines with phosphatidylinositol-specific-phospholipase C resulted in the removal of Ly-31 antigen from the cell surface. These results suggest that a gene cluster containing theLy-31 andAkp-2 loci which control the alkaline phosphatase is formed on mouse chromosome 4. The Ly-31 antigen is the first enzyme demonstrated to be a lymphocyte surface alloantigen.     

The Ly-10 antigen is a marker of mouse-activated T lymphocytes

Ly-10.1 is a lymphocyte surface antigen controlled by a gene linked to the Ly-1.1 locus and expressed on activated T helper, T suppressor (Ts), and cytotoxic T lymphocytes (CTL). In this report, we describe the following:

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.     

Identification of new murine lymphocyte alloantigens: Antisera prepared between C57L, 129 and related strains define new loci

Antisera prepared by immunizing between the strains 129 and C57L and other related strains identified new antigens expressed on lymphocytes and in particular on thymocytes. Absorption analysis demonstrated that the antisera were complex, and contained several new antibodies including some which were not cytotoxic, but could be detected by rosetting. The loci defined by these antibodies are referred to asLy-9, Ly-11, Ly-12, Ly-13, andLy-14, although several of the antigens were not confined to lymphocytes. In addition, the Ea-7 specificities, previously considered to be purely red-cell alloantigens, were also found on thymocytes.     

A molecular genetic linkage map of mouse chromosome 19, including thelpr,Ly-44, andTdt genes

The mouselpr gene, which is an autosomal recessive gene causing autoimmune disease with features of human systemic lupus erythematosus and eventually death from severe immune-complex glomerulonephritis, has been mapped on chromosome 19. To determine its exact chromosomal location, a three-point backcross was carried out by mating (MRL/MpJ-lpr/lpr × MOL-MIT)F₁ × MRL/MpJ-lpr/lpr using the genesLy-44 (lymphocyte differentiation antigen-44) andTdt (terminal deoxynucleotidyl transferase) as markers. The following order of genes is proposed, with the distances between genes given in parentheses: centromere-Ly-44 (19.3 cM)-lpr (6.1 cM)-Tdt-telomere. TheLy-44 ^a andTdt ^a alleles are found in all laboratory strains and in the wild Western European subspecies,domesticus andbrevirostris. In contrast, theLy-44 ^b andTdt ^b alleles are found in some Asian subspecies, Chinese mice of wild origin,yamashinai andmolossinus. Furthermore the thirdTdt allele,Tdt ^c , is detected incastaneus.Some of the data in this study were previously presented at the 4th Mouse Gene Mapping Workshop, Annapolis, Maryland, in November 1990.     

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.     

The polypeptide structure and assembly of Ly-2/3 heterodimers

Mild reduction of mature, thymic Ly-2/3 heterodimers of M _r 67 000 resulted in dissociation into three individual polypeptide chains, , , and , of respective M _r values 38000, 35000, and 30000. The and chains were both immunoprecipitated by a monoclonal antibody directed to the Ly-2.1 epitope whereas the Ly-3.1 antibody bound only the chain. The possibility that the and chains of each heterodimer established their interchain links within a labile precursor protein in which a and segments were fused was considered but discounted by the finding that in mice heterozygous for both Ly-2 and Ly-3 loci, the Ly-2 product of one chromosome was not exclusively joined to Ly-3 structures coded by the same chromosome. By utilizing ionic detergents which selectively alter the charge of intrinsic membrane proteins, both Ly-2 and Ly-3 polypeptides were shown to have membrane insertion sites. It is suggested that as a consequence of their likely synthesis on membrane-bound polysomes, newly synthesized Ly-2 and Ly-3 structures accumulate within the same subcellular compartment — the membranes of the rough endoplasmic reticulum. Their elevated concentration within this space may facilitate a low affinity binding interaction between Ly-2 and Ly-3 which is later stabilized by interchain disulfide bond formation.Abbreviations used in this paper BSA bovine serum albumin - DOC sodium deoxycholate - DTT dithiothreitol - HA hemagglutinin - HTAB hexadecyltrimethylammonium bromide - RER rough endoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TX100 Triton X-100     

Biochemical identification of rat ART-1 and Ly-1 alloantigens

The results of this study indicate that the ART-1 and Ly-1 rat alloantigens are synonymous with each other and also with the leukocyte-common (L-C) antigen which has been previously identified as a major glycoprotein of rat thymocytes and T and B lymphocytes. This conclusion is supported by the following observations: (i) when labeling of rat lymphoid cells was studied with a fluorescence-activated cell sorter, the profiles obtained were similar for labeling with ART-1 and Ly-1 alloantibodies and a monoclonal antibody to L-C antigen: (ii) this labeling was almost completely inhibited by purified L-C antigen: (iii) preincubation with L-C antigen completely inhibited binding of the alloantibodies in a cellular radioimmunoassay; (iv) the cytotoxic effect of the alloantibodies was completely abolished by preincubation with purified L-C antigen; (v) the strain distribution of the ART-1 and Ly-1 alloantigens was identical for 11 rat strains and in linkage analysis the ART-1 and Ly-1 alloantigens were found to cosegregate. Genetic linkage studies have shown that the L-C antigen locus is unlinked to the major histocompatibility antigen (RT1), the immunoglobulin light chain (1k) and to the coat color gene (C) loci.     

A monoclonal antibody detecting the Ly-9.2 (Lgp 100) cell-membrane alloantigen

A mouse monoclonal cell line (20-1.5) was produced by the cell fusion method and the antibody secreted by this line defined the Ly-9.2 specificity — the reciprocal specificity to that previously identified as the Lgp 100 or the T100 molecule. Although most concentrated on lymph-node cells, the antigen is also found on thymocytes, spleen and bone-marrow cells as well as liver and brain tissue. The monoclonal antibody precipitates a 100000 molecular weight moiety from thymocytes. The antigenic specificities appear to be highly immunogeneic and antibodies to these specificities contaminate many antisera. These sera are noncytotoxic as is the case with the monoclonal antibody even though it is of the IgG2a subclass. As with T100 or Lgp 100, theLy-9 locus appears to be linked to theH-25 locus.