Margrit P. Scheid

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−.

Lymphocyte differentiation from precursor cells in vitro.

Much of the recent work in our laboratory has centered about an induction assay developed by Komuro and Boyse in which one of the early maturational steps in the T-cell development, the differentiation from prothymocyte to early thymocyte, can be induced in vitro within two hours. This assay is based on a decade of work concerned with the characterization of cell-surface markers, especially the cytotypic differentiation alloantigens of murine T-cells. As a result of this work on the cell-surface composition of murine lymphocytes, we can summarize the antigenic phenotype of the cortical thymocyte as shown in FIGURE 1. The maturational step in the pathway of T-cell differentiation that can be induced in vitro involves the appearance of this characteristic antigen profile on the surface of the precursor cell; it can be seen that in this differentiation event the products of at least six unlinked genes are expressed. This rapid in vitro T-cell induction assay clearly had potential as a possible bioassay for the identification of the thymic hormone responsible for inducing T-cell differentiation in vivo. Our further studies were designed to answer pertinent questions with respect to this possible application of the assay.

Chemical Synthesis of a Peptide Fragment of Thymopoietin II that Induces Selective T cell Differentiation

Thymopoietin is a polypeptide hormone of the thymus that consists of a 49 amino acid polypeptide chain of 5562 daltons. A peptide corresponding to positions 29-41 of bovine thymopoietin II was synthesized by the Merrifield solid-phase technique. This peptide was shown to have a purity (correct sequence) of 96% by amino acid and C terminal analyses and by a complete determination of the amino acid sequence by manual Edman degradations. It displayed a selectivity of action similar to that of thymopoietin itself, inducing the differentiation of T lymphocytes but not of complement receptor (CR+) B lymphocytes. Although a number of substances induce the differentiation of both T cells and CR+B cells under the conditions of assay in vitro, only thymopoietin and the synthetic peptide described in this report have been shown to induce the differentiation of T cells selectively. Our data establish that the key residues involved in the active site of thymopoietin are present within a synthetic polypeptide which constitutes a minor portion of the amino acid sequence of thymopoietin. Since this peptide had 3% activity by comparison with thymopoietin, the tertiary structure of thymopoietin may be required for optimal configuration of the active site to produce full biological activity.

Biochemical features of Ly-5 alloantigen

Alloantigen of the Ly-5 system was originally thought to be confined to T lymphocytes (Komuro et al. 1975) but further analysis, aided by the use of Ly-5congenic mice and additional serological methods, revealed that Ly-5 is expressed on most or all hematopoietic cells at some stage of differentiation (Scheid and Triglia, unpublished data). We report here some biochemical features of the Ly-5 system, with emphasis on the isolation of Ly-5 from different Ly-5 + cell populations. Viable cells were radio-iodinated as described elsewhere (Baur et al. 1971) and lysates made by solubilization with NP-40. Cell-surface lg was removed with rabbit anti-mouse lg followed by goat anti-rabbit Ig. Antigen was precipitated from the cleared lysates by reaction with alloantiserum followed by goat anti-mouse Ig. These precipitates were solubilized in SDS buffer solution and run on either SDS polyacrylamide gel electrophoresis (SDS PAGE) cylindrical gels of 10, 8.5, 7.5 and 5~o acrylamide, or on slab gels of 7.5 and 5% acrylamide (Laemmli 1970). The cylindrical gels were sliced into I mm fractions and counted in a Packard Gamma Counter. The slab gels were stained, dried on filter paper, and autoradiographed (Kodak type B film; Dupont Cronex Screen, Exposure at -70~ Molecular weight (MW) was determined by comparison with the following standard proteins of known MW run on parallel lanes of the same slab gel:/~-galactosidase 116K, rat albumin 67K, ovalbumin 43K and carbonic anhydrase 29K. (Sigma Chemicals). Repeated estimates of MW by this method showed variation of the order of _+107o. With thymocytes, material from both c~-Ly-5.1and e-Ly-5.2 precipitates (antisera defined in Table 1) ran on SDS-PAGE as a molecule of 175K with a shoulder of lower MW (Fig. 2). But with spleen cells, both e-Ly-5.1 and c~-Ly-5.2 yielded three peaks of 175K, 185K and 220K (Fig. 1). The 175K and 185K species were difficult to separate on cylindrical gels but were clearly resolved on slab gels. Seen on slab gels, the 175K thymocyte peak is broad (Fig. 2), as expected of heavily glycosylated proteins, (though we have no evidence that Ly-5 has such a structure).

Structural features and selective expression of three Ly-5+ cell-surface molecules

Conventional Ly-5 alloantisera precipitate cell-surface molecules of three sizes: 200K, 205K, and 220K. In SDS-PAGE, the rat monoclonal antibody 74/8′ precipitates the same three molecules from both Ly-5.1 and Ly-5.2 cells. Cleveland mapping of the three molecules, precipitated by reaction of conventional Ly-5 alloantisera or 74/8′ monoclonal antibody with lysates of 125I-labeled cells, disclosed no differences among the three molecular forms, but markedly distinguished all three Ly-5.1 molecules from all three Ly-5.2 molecules. Each of the three molecular forms can be expressed independently of the other two by cloned culture lines of Ly-5+ cells of different hematopoietic lineage. All of the seven cloned lines tested expressed only one form. However, two of the seven uncloned culture lines tested, plasmacytoma MOPC-70A and the X.1 putative macrophage line which originated from an SJL tumor, yielded both 200K and 205K forms.

Immune function in fully allogeneic mouse bone marrow chimeras

Abstract Stable, long-lived, immunologically functional H-2 allogeneic chimeras, free of graft vs host disease, were established by reconstituting irradiated CBA J (H-2k) mice with C57BL 6 (H-2b) bone marrow, previously depleted of Thy 1+ cells with monoclonal anti-Thy 1.2 (mc-α-Thy 1.2) serum and complement (C). All lymph node cells of these chimeras expressed the donor H-2 phenotype, while a small, significant number of cells expressing host H-2 determinants were detectable in the spleens of the chimeras throughout the period of investigation. Skin graft rejection pattern, MLC, and CML responses of chimeric mice were normal against third-party targets, but reflected complete tolerance against donor and host determinants. NK activity against tumor cell targets was also normal. The host-like pattern of chimeric ADCC response against chicken red blood cells suggested the persistent activity of host macrophages. In contrast to the reduced primary PFC response against SRBC, the vigorous secondary response of the chimeras suggested that haplotype restrictions are not absolutely binding when there is an opportunity for prior learning.

Effect of the TP5 analogue of thymopoietin on the rejection of male skin by aged and thymectomized female mice

Although young adult C3H/HeJ (OH) females do not reject C3H male skin grafts, OH females older than 1 year commonly do so, as also do many thymectomized, young adult C3H females. Therapy With TP5, a synthetic pentapeptide analogue of thymopoietin which has biological properties of the parent molecule, substantially reduced the capacity of aged OH females and of thymectomized, young OH females to reject OH male skin.

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.

Immunological studies of mouse decidual cells. II. Studies of cells in artificially induced decidua

Cells from artificially induced decidual tissue (deciduoma) in the mouse were examined for Thy-1 surface antigen and receptors for the Fc portion of immunoglobulin G (FcR) and compared with cells of the normal decidua from 6 to day 9 of pregnancy. It was shown that (1) Thy-1 antigen is present on the same proportion of cells in decidua and deciduoma on day 6 and day 7, (2) FcR-bearing cells can be detected in similar numbers on day 6 and day 7 but this does not increase on day 8 in deciduoma as it does in decidua, and (3) progesterone treatment after induction of decidualization allowed further increase of FcR-bearing cells in deciduoma. These results present further evidence of the similarity between deciduoma and decidua in the mouse. They indicate that these two membrane markers are present in the early decidua, regardless of the presence of an embryo, and suggest that progesterone may play a part in the increase of FcR-bearing cells in the decidua during pregnancy.

Characterization of interleukin 2-dependent cytotoxic T-cell clones: IV. Production of α, β and γ interferons and interleukin 2 by Lyt-2+ T cells

Abstract The production of α, β and γ interferons (IFN) and interleukin 2 (IL-2) by Lyt-2 + -dependent cytotoxic T-cell lines/clones was investigated. Cloned and uncloned T-cell lines specific for H-2D d or the unique RL♂1 leukemia antigen were studied. After infection with Sendai virus (SV) or Newcastle disease virus (NDV) all cell lines produced IFN-α and -β. Induction of IFN-γ was attempted with the mitogens Con A, PHA, PWM, SEA, and SEB, with poly(I:C), with antibodies Lyt-1.2, -2.2, and Thy-1.2, or with the target cells Meth A (H-2D d+ ) and RL♂1. All mitogens were effective inducers. However, the antibodies and poly(I:C) were not. One uncloned RL♂1-specific cell line CTLL-RP, produced IFN-γ after induction with RL♂1. Production of IFN-α, β depended on IL-2, whereas production of IFN-γ did not, although addition of highly purified IL-2 increased IFN-γ production even in the absence of other inducers. Crude IL-2 inhibited the production of IFN-γ but not IFN-α, β. In response to mitogens, some T-cell clones also produced IL-2. The results demonstrate that Lyt-2 + cells can produce a broad spectrum of lymphokine activities after appropriate stimulation. Their availability now affords us the opportunity to study the regulation of lymphokine production at the clonal level.