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Major structural differences occur between the thymic and peripheral T cell forms of the Ly-2/3 antigen. Thymus Ly-2/3 consists of similar amounts of two types of disulfide-linked heterodimer, alpha beta and alpha' beta (Mr alpha = 38000, Mr alpha' = 35000, Mr beta = 30000). In contrast material from peripheral T cells consists almost exclusively of alpha beta dimers. The alpha chains of thymus and peripheral T cells differ also in isoelectric point with the thymic alpha chain being the more acidic. Based on peptide mapping experiments the alpha and alpha' chains of thymus are likely to be alternatively modified forms of the same polypeptide backbone. Individual T cell clones or T cell tumors propagated in vitro exhibit either a typical thymus or a typical peripheral T cell Ly-2/3 polypeptide pattern indicating that the synthesis of both alpha and alpha' chains can occur in the same cell. The heterogeneity of thymic Ly-2/3 can be considerably reduced by removal of sialic acid residues, and after desialylation the alpha chains of thymus and a cloned cytotoxic T lymphocyte (CTL) line cannot be electrophoretically distinguished. If Ly-2 structures affected the antigen specificity of CTL, a different structural variant would be expected in individual clones. The electrophoretic identity of desialylated thymus and CTL alpha chains suggests that Ly-2 does not exhibit clonal variation in polypeptide structure and, therefore, cannot contribute to antigen specificity.

Six monoclonal anti-Ly-2.1 antibodies are described that arose from a single fusion using the spleen from a 129/ReJ mouse immunized with CBA/H thymus cells. The specificity was determined from the strain distribution, and testing of congenic strains where all six monoclonal antibodies detected the Ly-2.1 alloantigen. Furthermore, the antibodies fall into two groups: Group I (four antibodies) detected the expected number of Ly-2+ thymocytes and peripheral T cells, whereas Group II (two antibodies) detected 10% fewer peripheral T cells. Functional studies demonstrated that the Ly-2.1 determinant detected by a Group I antibody (49-31.1) was presented on cytotoxic T cells (Ly-1+2+), on concanavalin A (Con A)-induced suppressor T cells (Ly-1-2+), but absent from helper T cells (Ly-1+2-). One antibody (49-11.1) precipitated a 68,000-75,000 mol.wt glycoprotein, which on reduction yielded 30,000 and 35,000 mol.wt moieties. Thus, by functional, genetic and biochemical criteria these antibodies detected the Ly-2.1 specificity. In addition, a monoclonal, noncytotoxic, IgGl, anti-Thy-1.2 antibody is described.

The bifunctional cross-linking reagent dithiobis(succinimidyl propionate) (DSP) was used to cross-link 125I surface-labelled glycoproteins from viable thymocytes. The cells were solubilized, and the cross-linked material immunoprecipitated and analysed by SDS-PAGE. When DSP cross-linked thymocyte material was immunoprecipitated with either anti-ThB or anti-Ly 5 monoclonal antibodies, and then cleaved, molecules with masses identical to Ly 5 (Mr 180 kD) and ThB (Mr 16-18 kD) were obtained. However, if the cross-linker was not cleaved, the intact product had a molecular mass of greater than 200 kD. The identity of these co-precipitated, cross-linked moieties was formally proved by limited proteolysis peptide map analysis. The data indicated that the ThB and Ly 5 antigens were associated on the thymocyte cell surface but no such association could be found on peripheral lymphocytes. The ThB-Ly 5 interaction may indicate an association relevant to the differentiation of thymocytes.

Competitive binding studies and immunoprecipitation experiments define at least five distinct epitopes encoded by Ly-6-linked genes--Ly-6A.2, Ly-6B.2, Ly-6C.2, Ly-6D.2, and ThB. Ly-6A.2, a 33 kd protein, and Ly-6D.2 are closely overlapping epitopes that can be distinguished by their unique thymus reactions of 10-20% or greater than 90%, respectively. Similarly, the Ly-6C.2 antigen present on a 14 kd moiety loosely overlaps the Ly-6B.2 antigen. Ly-6C.2 and Ly-6B.2 antigens are distinct from Ly-6A.2 and Ly-6D.2, however. ThB is a 16-18 kd antigen which is not associated on the cell surface with any other "Ly-6" antigens. In addition, independently derived antibodies made to the Ly-6C.2 antigen detect an identical epitope, as do antibodies to Ly-6A.2 and Ly-6B.2. These results imply the existence of a single antigenic site on each of these molecules.

A cDNA clone encoding the mouse Ly-17+ Fc receptor for IgG, isolated from a myelomonocytic cell line, was sequenced and expression of mRNA and the functional Fc gamma R investigated. The receptor is a 301 amino acid transmembrane glycoprotein with two homologous extracellular domains that are also homologous to members of the Ig superfamily. The receptor has four sites of N-linked glycosylation and a long 94 amino acid cytoplasmic tail. Northern analysis, immune complex binding, and serological studies demonstrate that the receptor encoded by the cDNA clone binds mouse IgG gamma 1/2b and rabbit IgG complexes.

Using monoclonal antibodies, mouse peritoneal neutrophils were typed for the presence of 23 cell surface alloantigens, the expression of which was quantitated by flow cytofluorometry and compared with that of lymphocytes. The H-2K and H-2D alloantigens and beta 2-microglobulin were present on all neutrophils, but Ia and Qa antigens were not detected. It was found that Ly-5.1, Ly-15.2, Ly-21.2, Ly-24.2 (Pgp-1) and Ly-25.1 were present on greater than 90% of neutropils; Ly-6.2 and Ly-27.2 were absent, but Ly-28.2 (encoded by an Ly-6 linked gene), was present on greater than 90% of neutrophils. As expected, the lymphocyte-specific antigens Ly-1.1, Ly-2.2, Ly-3.1, Ly-7.2, Ly-12.1 and Thy-1.2 were absent from the neutrophils. When compared with lymphocytes, marked differences in alloantigen expression on neutrophils were seen for Ly-5.1, Ly-24.2 and Ly-28.2. These studies should be of value in the study of neutrophil structure and function.

The mouse Ly-17.2 alloantigen has recently been defined with both conventional and monoclonal antibodies; it identifies a locus, sited on chromosome 1, the products of which were considered to be specific for B cells. Using another Ly-17.2-specific monoclonal antibody (described herein), the tissue distribution of the Ly-17.2 antigen was shown to extend to a subpopulation of T lymphocytes and to neutrophils. This distribution is remarkably similar to that of the Fc receptor for immunoglobulin. Indeed, we now demonstrate that the Ly-17 locus codes for a polymorphism of the Fc receptor, a conclusion based upon (a) an identical tissue distribution of Ly-17.2 and FcR on both normal and tumor tissue; (b) specific inhibition of EA rosette formation by F(ab')2 fragments of anti-Ly-17.2; (c) inhibition of the binding of the 2.4G2 monoclonal rat antimouse Fc receptor antibody by Ly-17.2 antibody; (d) precipitation of an identical series of molecules by our Ly-17.2-specific antibody and by the recognized Fc receptor-specific antibody (2.4G2); and (e) the demonstration by coprecipitation that the Ly-17.2 specificity is present on Fc receptor molecules. The studies suggest that the xenogeneic monoclonal antibody (2.4G2) which recognizes an invariant site on the FcR molecule and the polymorphic site are closely associated. In addition, the studies firmly map a gene coding for or regulating the expression of the FcR to chromosome 1.

Recent studies have demonstrated that lymphocytes express an array of cell surface receptors for sulphated polysaccharides (SP). Experiments were undertaken to determine the binding characteristics of these receptors and establish whether any known lymphocyte cell surface antigens interact with sulphated carbohydrates. It was found that murine thymocytes lack receptors for chondroitin-4-sulphate but express saturable, high affinity binding sites for heparin, fucoidan and dextran sulphate, with an apparent affinity constant range of 0.03-2.6 x 10(-9) mol/l. Binding inhibition experiments revealed one class of binding sites on murine thymocytes that is shared by heparin, fucoidan and dextran sulphate and another class of sites that is dextran sulphate-specific. The cell surface receptors for the SP were affinity-purified by applying detergent lysates of 125I-labelled thymocyte membranes to SP-coupled solid supports. It was found that the Thy-1 and Ly-5 (T-200 or leucocyte common antigen) molecules of murine thymocytes bind to sulphated carbohydrates, although the two molecules differed substantially in their reactivity with the four different SP tested. Furthermore, only subpopulations of the Thy-1 and Ly-5 molecules interacted with sulphated sugars. Four additional sulphated carbohydrate-binding molecules were also detected. It is suggested that the SP-binding molecules are involved in the interaction of lymphocytes with glycosaminoglycans on other cells and in the interstitial space.

Monoclonal anti-Lyt-1.1 alloantibody was produced as tissue culture supernatant and administered to mice. The antibody, given intraperitoneally, resulted in the suppression of all T cell functions studied, but was without direct effect on B cells. Thus, skin and tumour allograft survival was prolonged and there was suppression of the delayed-type hypersensitivity response; T cell help inthe anti-sheep red blood cell antibody response, responder cells in the mixed lymphocyte reaction (MLR), leucoagglutinin-responsive cells, cytotoxic T cell (Tc) function and the induction of Tc were either totally or partially suppressed, all these responses being mediated by Lyt-1+2- or Lyt-1+2+ cells in CBA/H mice. By contrast, there was no inhibitory effect on the MLR-stimulating or lipopolysaccharide-responsive cells. The administration of the anti-Lyt-1.1 antibody was accompanied by a depletion of Lyt-1.1+ T cells from both spleen and lymph node. These studies indicate that the monoclonal anti-Lyt-1.1 antibody is active in vivo with a selective effect on T cells. The results also have important implications for studies of T cell interactions in the mouse in vivo, and for similar studies in man.

The Ly-6 locus is now regarded as a gene complex consisting of at least five closely linked loci (Ly-6A-Ly-6E) whose polymorphic products are identified by monoclonal antibodies and distinguished by different tissue distributions. Ly-6 has been assigned by other investigators to chromosome (Chr) 9 (linked to Thy-1) or to Chr 2. We report that the Ly-6 gene complex, together with the Xp-14 and Gdc-1 loci, is situated on Chr 15 linked to Gpt-1. These new linkage data are derived from four sources: (1) three separate crosses that failed to demonstrate linkage of Ly-6 to either Thy-1 on Chr 9 or to any of five genes present on Chr 2; (2) the NXSM recombinant inbred strains, which suggested the linkage of Ly-6 and Xp-14 to Gpt-1 on Chr 15; (3) several Gpt-1 and Gdc-1 congenic strains that confirmed the assignment of Ly-6 and Xp-14 to Chr 15; and (4) backcrosses that further confirmed the linkage of Ly-6, Gpt-1, Gdc-1, and Xp-14, the probable gene order being Gpt-1/Ly-6-Xp-14-Gdc-1.

Three allele-specific monoclonal antibodies to Pgp-1 (Ly-24) were used to biochemically characterize the cell surface structures with which they reacted and to map the gene(s) coding for these antigens. The targets of these three monoclonal antibodies (mAb) were shown to be encoded by a gene situated on chromosome 2 close to beta 2m [gene order (Pgp-1-beta 2m-a)] and no recombination between the loci detected by the three antibodies was revealed by genetic analysis. The genetic mapping of loci and tissue distribution of these antigens suggested that they might all correspond to a particular allelic form of the mouse phagocyte glycoprotein-1 (Pgp-1) antigen. Biochemical and serological analysis confirmed that this was indeed the case and revealed that all three mAbs were directed to one epitope. It is surprising that the tissue distribution defined by one mAb (Ly-24A) was different from that for the two other (Ly-24B) antibodies, despite the serological and biochemical identity of their respective targets. The possible reason for this unusual finding is discussed.

In this study, the relative mass of the Ly-6A.2 antigen was shown to be 12,000-14,000, in contrast to initial studies which showed the relative mass to be 33,000. Using polymorphic Ly-6-specific antibodies, the 33,000 molecules could be immunoprecipitated from surface-iodinated thymocytes of Ly-6A.2+, Ly-6A.2- strains and a Ly-6A.2- mutant cell line BW(Thy-1-e). This clearly demonstrated that 33,000 molecules were not associated with the Ly-6 polymorphism. By contrast, when biosynthetically labeled Ly-6A.2+ spleen cell lysates were analyzed, the major species immunoprecipitated by the polymorphic Ly-6A.2-specific antibody was 12,000-14,000, although a minor 33,000 species were also evident. The Ly-6A-specific antibody D7 which detects a monomorphic epitope on the Ly-6A molecule could immunoprecipitate the 12,000-14,000 molecules from surface-labeled cells. By contrast, the Ly-6A.2-specific antibodies detecting the polymorphic Ly-6A.2 determinant could not, though the reasons for this difference are not clear. Thus 12,000-14,000 molecules were only immunoprecipitated from Ly-6A.2+ cells, whereas 33,000 molecules were precipitated from both Ly-6A.2+ cells and Ly-6A.2- cells. These findings suggest that the 33,000 molecules immunoprecipitated by 5041-24.2 are most likely to be an unrelated protein, possibly cross-reactive with some Ly-6A.2 antibodies.

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.