Jwu-Sheng Tung

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-5a, Ly-5b, and Ly-5c, respectively. Ly-5ais by far the most common allele. The Ly-5callele 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.

Organization of the Ly-5 gene.

A single Ly-5 gene is known to generate a variety of transmembrane glycoprotein isoforms that distinguish various cell lineages and stages of differentiation within the hematopoietic developmental compartment of the mouse. Systems homologous to Ly-5 are known in rats and in humans. The complete exon-intron organization of the Ly-5 gene is described in this report. The Ly-5 gene occupies about 120 kilobases of chromosome 1 and comprises 34 exons, of which 32 (Ex-3 to Ex-34) are protein coding. Ex-1, Ex-2, and parts of Ex-3 and Ex-34 are untranslated. In all cDNA clones examined, either Ex-1 or Ex-2 was represented, but not both, implying that Ex-1 and Ex-2 in Ly-5 mRNA may be mutually exclusive. Primer extension and S1 nuclease protection mapping were used to identify initiation (cap) sites for transcription. The finding of putative cap sites for Ex-1 and Ex-2, and of corresponding TATA-like sequences, suggests the presence of two promoters. In both Ex-1+ and Ex-2+ cDNA clones the next exon is Ex-3, which has a translation-initiating codon. The intron between Ex-3 and Ex-4 is unusually long, about 50 kilobases. Evidence is given that Ex-5, like Ex-6 and Ex-7 (studied previously), is another alternative exon that is selectively programmed, alone or together with Ex-6 or Ex-7 or both, to generate actual or potential Ly-5 isoforms by alternative splicing.

Different forms of Ly-5 within the T-cell lineage

The Ly-5 system, defined both by alloantibodies (Komuro et al. 1975) and by xenoantibodies (Omary et al. 1980, Siadak and Nowinski 1980), is noted for molecular differences that distinguish various hematopoietic cell lineages (Michaelson et al. 1979). A 200K (Mr 200000) is expressed by T cells, a 205K form occurs on macrophages, and a 220K form [which can be separately identified by xenoantibody (Coffman and Weissman 1981, Dalchau and Fabre 1981, Kincade et al. 1981) and may therefore identify an epitope lacking in the smaller forms] is characteristic of B cells. This relation of molecular form to cell lineage has been confirmed by ascertaining the Ly-5 molecular phenotypes of cloned cell lines representing T, B, and macrophage lineages (Tung et al. 1981). Extending our survey to a further range of cloned T-cell lines, we now find that the T lineage itself is diverse in expression of distinguishable Ly-5 molecular forms. Each of the 10 cell lines, shown in Table 1, seven of which have the phenotype Ly-23 and three the phenotype Ly-1, was examined in the usual way by cell surface radioiodination, immunoprecipitation (with monoclonal Ly-5.1 alloantibody; Shen 1981), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). Figure 1 shows that all three Ly-1 lines express the familiar 200K form previously found to typify T cells, whereas all seven Ly-23 lines expressed two forms, 210K and 215K, neither of which has previously been seen in any lineage. These two newly identified forms bring the number of distinguishable Ly-5 forms to five, and they are shown in Figure 2 to be separable from the three other previously known forms. All five forms were recognizable in combined lysates of spleen cells and CTLL-R8 Ly-23 cells (Fig. 2). The reason why the 210K and 215K forms were not observed before is probably that Ly-23 is a small cell set that requires cloning or other enrichment to provide enough material.

Expression of the Q8/9d gene by T cells of the mouse.

TheQ genes, specifying Qa antigens and situated in the extended part of the major histocompatibility complex (MHC) of the mouse, comprise a subgroup of MHC class I genes whose significance and function are still largely unknown. In screening a cDNA library made from the BALB/c inducer T-cell line Cl.Ly1-T1, we isolated 11 clones representingQ8/9, but none representingQ6 orQ7. Confirmatory evidence is given that theQ8/9 gene originated from fusion of the 5′ region of theQ8 gene with the 3′ region of theQ9 gene at a recombination site or hot spot in the vicinity of intron 4. Contrary to previous impressions thatQ8/9 is an inert pseudogene, we find that theQ8/9 gene can be functional and encode a Qa-2,3 antigen. One variety of the 11 Q8/9 clones isolated lacked exon 5, which encodes the transmembrane domain of class I glycoproteins, and thus may account for secretion of a soluble form of Qa-2,3 antigen thought to be released by activated T cells.

Structure and biosynthesis of Lyt-1

Two-dimensional chymotryptic peptide maps of Lyt-1.1 and Lyt-1.2 from Lyt-1 congenic thymocytes labeled with 125I in the usual way before lysis did not significantly differ, but there was a characteristic difference between maps of Lyt-1.1 and Lyt-1.2 obtained from 125I-labeled solubilized membrane fragments. We conclude that the Lyt-1 locus of chromosome 19 includes the protein-structural gene for Lyt-1. This conclusion is further supported by evidence with 35S-cysteine-labeled thymocytes: Thus an early 62K intermediate form, and a 60K form from tunicamycin-treated cells devoid of N-linked and O-linked carbohydrates, were precipitated by Lyt-1 alloantibody, which implies that the allo-specificity of Lyt-1 glycoprotein (67K) resides partly or wholly in protein.

Some Compartments of B Cell Differentiation

The Lyb-2 system was defined by Sato & Boyse (1976) with an antiserum made by immunizing C3H,l-H-2^ congenic mice with 1,29 cells. The 1,29 tumor arose spontaneously in the ascites form in i/StBoy (H-20 mice. An account ofthe Ig products of L29 given by Sitia et al, (1981, 1982) includes reference to synthesis of IgM and IgA, and conversion from IgM production to IgA production. After absorption, to remove unidentified antibodies, this antiserum reacted exclusively with cells of the B lymphocyte lineage, according to the following criteria: The alloantigen now named Lyb-2,1 could be found only on lymphoid cells, and the proportion of positive cells in different lymphoid organs corresponded with the normal disposition of B cells: < 5 % among thymocytes, 50-60% in spleen, and 30-40% in lymph nodes and bone marrow. Counts of Thy-T and Lyb-2,1 cells were additive; the Lyb-2* population was enriched by eliminating Thy-l* cells and reduced by eliminating Ig* cells; elimination of Lyb-2 cells reduced the Ig* population; and elution from nylon gave reduced Lyb-2 counts and raised Thy-l* counts. In nude athymic mice, and in B-mice (thymectomized, lethally-irradiated mice restored with T-depleted syngeneic bone marrow cells), the proportion of

The same genetic locus directs differentiation-linked expression of endogenous retrovirus gp70 on thymocytes and spleen cells in the mouse

The major envelope protein of murine leukemia virus (MuLV), gp70, is known to be expressed on thymocytes of certain mouse strains which rarely, if ever, express complete virions (Tung et al. 1975a, b). The occurrence of this virally encoded protein was originally detected because in some strains it carries the antigen G~x (Tung et al. 1975a, Stockert et al. 1971). Classically G~x has been described as an antigen characteristic of the thymic stage of T-cell differentiation, analogous in this respect to TL antigens. In G~x + strains such as 129, G~x can be detected on the surface of thymocytes but not on spleen cells (Stockert et al. 1971). Some Glxmouse strains such as C57BL/6 (B6) express gp70 molecules lacking the G~x antigen on thymocytes (Tung et al. 1975b). A few strains exhibit no detectable gp70, G~x + or Glx -. An interesting example of this sort of mouse is the congenic 129-G~xstrain, in which the G~xphenotype of B6 has been crossed into the 129 strain (Stockert et al. 1975). This strain is also negative for the thymocyte-surface gp70 characteristic of B6 mice, thus demonstrating that the Gix + gp70 of 129 and the Gixgp70 of B6 are encoded at nonallelic genetic loci (Tung et al. 1975b). [In the 129 mouse the locus designated Gv-I probably contains the structural gene for Grx + gp70, since this locus is semidominant in genetic crosses (Stockert et al. 1971)]. As predicted by this model, the reciprocal congenic, B6-GIx ÷, expresses both species of gp70 on its thymocytes. By the use of broadly reactive hyperimmune anti-gp70 serum, we have found that spleen cells from 129, B6, and B6-GIx +, but not 129-G~x-, mice also express cell-surface gp70. The gp70 in spleen appears to be present on mature T cells and on at least one other cell type. The latter probably includes B cells, but conclusive evidence on this point is not yet available. In G~x + mice the occurrence of endogenously encoded gpT0 molecules on spleen cells seems to be controlled by the same genetic locus which determines the presence of Glx + gp70 on thymocytes. Our analysis of spleen-cell surfaces for expression of MuLV-gp70 molecules involved standard immunochemical methods. Spleen cells of 129, B6, and their congenic Gixand Glx + derivatives were surface-radioiodinated by the lactoperoxidase method and the cells lysed with detergents. A group-specific goat anti-