Differential expression of the human Thy-1 gene in rodent-human somatic cell hybrids [corrected

Thy-1 is a cell surface differentiation marker which shows distinct patterns of tissue-specific expression in different species. In man, the Thy-1 antigen is encoded by chromosome 11. We have examined the regulatory signals determining human Thy-1 expression through serologic analysis of rodent-human somatic cell hybrids retaining human chromosome 11 in which the fusion partners belong to distinct differentiation lineages. Cell surface expression of human Thy-1 was determined by mixed hemadsorption assays with two monoclonal antibodies (mAb), K117 and L127, shown to detect authentic human Thy-1 through analysis of COS-7 monkey kidney cells transfected with a cloned human Thy-1 gene. Three different patterns of human Thy-1 expression were observed when hybrid cells, constructed with different human and rodent cell types, were tested with mAb K117 and L127. Hybrids formed between Thy-1+ human neuroblastoma cells and Thy-1- mouse neuroblastoma cells, or hybrids between Thy-1+ human fibroblasts and the Thy-1- mouse kidney carcinoma, RAG, retain human Thy-1 expression. In contrast, hybrids formed between either Thy-1+ human neuroblastoma cells or Thy-1+ human fibroblasts and Thy-1- mouse L cells lose expression of human Thy-1 even though chromosome 11 is retained. Finally, hybrids formed between Thy-1- human peripheral lymphocytes or a Thy-1- lymphoblastoid B cell line and Thy-1- Chinese hamster fibroblasts begin to express human Thy-1. These studies suggest that both positive and negative trans-acting signals may play a role in the tissue-specific regulation of the human Thy-1 gene.     

Identification of a novel bone marrow-derived B-cell progenitor population that coexpresses B220 and Thy-1 and is highly enriched for Abelson leukemia virus targets.

A novel stage in early B-lymphocyte differentiation has been identified in normal mouse bone marrow cells. Earlier work had demonstrated that bone marrow cells characterized by low levels of Thy-1 and lack of a panel of lineage markers (Thy-1lo Lin- cells) were highly enriched for pluripotent hematopoietic stem cells. In this paper, we present evidence that another bone marrow population, which expressed low levels of Thy-1 and coexpressed B220, a B-lineage-specific form of the leukocyte common antigen, contained early and potent precursors for B lymphocytes upon in vivo transfer to irradiated hosts. These Thy-1lo B220+ cells, comprising 1 to 2% of bone marrow cells, were enriched for large cells in the mitotic cycle; the population lacked significant pluripotent hematopoietic stem cell activity and myeloid-erythroid progenitors. Most strikingly, Thy-1lo B220+ cells represented a highly enriched population of bone marrow cells that could be targets of Abelson murine leukemia virus transformation. We propose that Thy-1lo B220+ bone marrow cells represent the earliest stage of committed lymphocyte progenitors, intermediate in differentiation between Thy-1lo Lin- pluripotent stem cells and, in the B lineage, Thy-1- B220+ pre-B cells.     

Thy-1 is a differentiation antigen that characterizes immature murine erythroid and myeloid hematopoietic progenitors

To determine the role of Thy-1 antigen in murine hematopoietic differentiation, bone marrow was treated with anti-Thy-1.2 antibody and complement or complement alone. Growth of immature hematopoietic progenitors, erythroid burst-forming units (BFU-E), and granulocyte/macrophage colony-forming units (CFU-GM) was greatly reduced following antibody and complement treatment and was not restored by mitogen-stimulated spleen cell supernatants. In contrast, more mature erythroid and myeloid progenitors, the erythroid colony-forming unit (CFU-E) and the macrophage progenitor stimulated by L-cell-conditioned media (LCM), were spared by anti-Thy-1.2 antibody and complement treatment. Here, to separate the effects of anti-Thy-1.2 antibody treatment on accessory cells from those on progenitors, splenic T cells and thymocytes were added to treated marrow at ratios of up to 200%. Growth of BFU-E and CFU-GM was not restored. To more precisely replace required accessory cells, male complement-treated marrow was cocultured with female anti-Thy-1.2 antibody and complement-treated marrow. Even marrow cells failed to restore female BFU-E and CFU-GM growth. Fluorescent-activated cell sorting (FACS) and immune sheep red cell rosetting with anti-Thy-1.2-labeled marrow were then performed to determine if immature hematopoietic progenitors bear Thy-1.2. These techniques revealed enrichment of BFU-E and CFU-GM in the Thy-1.2-positive fraction, demonstrating the presence of Thy-1.2 on early murine hematopoietic progenitors. CFU-E and CFU-M were present in the Thy-1.2-negative fraction following FACS separation. These data demonstrate that Thy-1.2 is a differentiation antigen, present on at least some murine BFU-E and CFU-GM and lost as they mature to CFU-E and CFU-M.     

Identification and separation of Thy-1 positive mouse spleen cells active in natural cytotoxicity and antibody-dependent cell-mediated cytotoxicity.

The expression of the Thy-1 antigen on mouse spleen cells responsible for NK activity and ADCC was investigated by using a monoclonal IgM anti-Thy-1.2 antibody. Both C-mediated cytotoxicity and the fluorescence-activated cell sorter were used to fractionate cells. The effector cells were found to be heterogeneous in their expression of Thy-1. Effector cells from nude BALB/c mice were predominantly Thy-1 positive; some of the NK cells in CBA spleens appeared to be Thy-1 positive, but at least one-third of the lytic activity was due to Thy-1 negative cells. The effects of treatments on NK cytotoxicity and ADCC were very similar, supporting the hypothesis that the same cells mediate both activities.     

Pluripotential hematopoietic stem cells in post-5-fluorouracil murine bone marrow express the Thy-1 antigen

Expression of the Thy-1 alloantigen by hematopoietic stem and progenitor cells in post-5-fluorouracil (5-FU) murine bone marrow was investigated. FACS analysis of BDF1 bone marrow stained for Thy-1.2 with a triple-layer amplified labeling technique demonstrated that 35% of the total bone marrow population expressed Thy-1.2 (Thy-1.2+). Two distinct size subpopulations were observed in post-5-FU BDF1 marrow. Thy-1.2+ cells were present in both the large and the small subpopulations. FACS-separated bone marrow cells were also plated in methylcellulose cultures. Ninety percent of all colony-forming cells surviving in vivo administration of 5-FU were Thy-1.2+. Replating of primary hemopoietic colonies and morphologic examination of primary and secondary colonies demonstrated that the most primitive stem cells including "stem" (S) cells were Thy-1.2+. These cells (Thy-1.2+) were capable of self-renewal in vitro and exhibited multiple differentiation potentials in comparison to Thy-1.2-cells, which lacked significant self-renewal capability and were mono- or bipotent progenitor cells. Separation of Thy-1.2+ cells into large or small Thy-1.2+ subpopulations showed that only the large Thy-1.2+ colony-forming cells possessed significant self-renewal capacity. Treatment of BDF1 bone marrow with anti-Thy-1.2 plus complement reduced primary colony formation by 67% and eliminated those colony-forming cells which had extensive self-renewal properties. In the presence of PWMSCM, depletion and reconstitution of T lymphocytes had no effect on primary or secondary colony formation. These data demonstrate that Thy-1 is present on primitive hematopoietic stem cells in post-5-FU bone marrow. In addition, they show that the murine S cell is Thy-1+.     

Regulation of Thy-1 gene expression in transgenic mice

Genomic DNA fragments encompassing the human Thy-1 or mouse Thy-1.1 gene have been microinjected into pronuclei of mouse embryos homozygous for the Thy-1.2 allele. In the resulting transgenic mice, the human gene is expressed in a pattern characteristic of normal human tissues, and is not influenced by the pattern of endogenous mouse Thy-1 expression. The mouse Thy-1.1 gene fragment is expressed in a pattern typical of mouse Thy-1, although it is more limited in its distribution. The results indicate the presence of multiple cis-acting regulators of Thy-1 gene expression that have changed in both their character and arrangement over the course of Thy-1 gene evolution.     

Carbohydrate differentiation antigens of murine T cells: expression on intestinal lymphocytes and intestinal epithelium

Carbohydrate differentiation antigens (CT antigens) which previously had been shown to be associated with cytotoxic T cells were found at high levels on intestinal intraepithelial lymphocytes (IEL) and on the intestinal epithelium. Histological examination of intestinal sections demonstrated that the CT1 MAb defined epitopes on IEL and on epithelial cells located in the base of the villi crypts. The CT2 MAb reacted with IEL but also bound to the majority of cells in the intestinal epithelium. When isolated intestinal cell populations were analyzed by flow cytometry, two major size classes of cells were evident. The smaller cells, corresponding to lymphocytes, were primarily Lyt-2+, with a high proportion expressing CT antigens. Another differentiation antigen defined by the MAb J11d was absent from IEL, indicating that those IEL of T cell origin are likely to be mature because thymocytes, but not peripheral T cells, express the J11d antigen. Two-color fluorescence analysis indicated that the CT determinants were present on the Thy-1+, Lyt-2+, and the Thy-1-, Lyt-2+ subsets of IEL. However, the small percentage of L3T4+ IEL were CT-, further supporting our previous demonstration of a correlation between CT expression and Lyt-2 expression. Interesting phenotypic characteristics of IEL other than CT antigen expression were also detected. IEL did not express the MEL-14 lymphocyte homing receptor, and the cell surface level of LFA-1 was significantly lower than that of other peripheral lymphocytes. It was also shown that a small percentage of IEL express a T cell receptor allotypic marker, indicating that at least some of the cells are mature in terms of T cell receptor gene rearrangements. The large intestinal cells, although CT+, were not hematopoietic in origin because they were T200- and were shown by using chimeric mice not to be bone marrow-derived. In contrast to previously reported results, the cytotoxic activity of IEL was negligible with detectable lysis against NK-sensitive cells and other tumor cells, being observed in only one of seven experiments. Thus, the expression of the CT determinants was not indicative of cytotoxic ability, as previously suggested. The presence of specific carbohydrate residues on the cell surface of a subset of lymphocytes in an anatomically distinct immune compartment suggests that a unique differentiation pathway is followed by these cells.     

Differential expression of the mouse and human Thy-1 gene in embryonal carcinoma cells.

Mouse P19 embryonal carcinoma (EC) cells express on their surfaces a Thy-1 glycoprotein. The expression of Thy-1 at the mRNA and protein levels is down-regulated during differentiation induced by retinoic acid (RA). Thy-1 is also expressed in human NTERA-2 EC cells, but its expression is not down-regulated during RA-induced differentiation. As a first step towards understanding differential regulation of the mouse and human Thy-1 gene in EC cells, we have introduced genomic DNA fragments encompassing the mouse or human Thy-1 gene into NTERA-2 and P19-derived cells and analyzed surface properties of the transfectants. In the transient transfection assay, both mouse and human Thy-1 genes were expressed on cell surfaces at comparable levels. P19-derived stable transfectants exhibited great clonal variations in the expressions of the transfected Thy-1 gene products, which in part reflected copy numbers. There was no simple correlation between the expression of the transfected Thy-1 gene and two stem cell surface markers, TEC-1 and TEC-4. In the course of differentiation induced by RA several clones with a surface phenotype of EC cells exhibited a significant decrease in the expression of the transfected mouse Thy-1, whereas expression of the human Thy-1 was less efficiently down-regulated. The results suggest the presence of multiple cis- and trans-acting elements controlling expression of the mouse and human Thy-1 genes in P19 EC cells and their differentiated derivatives.     

Glycolipid markers of murine lymphocyte subpopulations.

We have shown previously that purified antibodies to ganglioside GM1 react with peripheral T cells and most thymocytes in several strains of mice, independent of Thy-1 phenotype. GM1 and the Thy-1.2 antigen cap independently on C3H thymocytes, which provides additional evidence that GM1 is not the Thy-1.2 antigen. In C3H and nude mice antibodies to GM1 also react with a population of cells, comprising about 25% of lymphocytes from lymph nodes or spleen, that bear surface immunoglobulin. After removal of immunoglobulin from these cells by digestion with proteolytic enzyme, the GM1+ cells regenerate their surface immunoglobulin during 18 hr in culture, which indicates that these double-labeled cells synthesize their surface immunoglobulin. Protease treatment of lymphocytes reveals receptors for antibodies to GM1 on most cells. These data indicate that T and B cells differ in the accessibility of GM1 to antibody, and not necessarily in their content of GM1. Purified antibodies to asialo GM1 react with mature T cells in all strains of mice tested. In contrast to anti-GM1, these antibodies do not react with most thymocytes, with immunoglobulin-bearing lymphocytes of C3H or nude mice, nor with pronase-treated B cells.     

T-cell-activating monoclonal antibodies, reacting with both leukocytes and erythrocytes, recognize the guinea pig Thy-1 differentiation antigen: characterization and cloning of guinea pig CD90

A glycophosphatidylinositol (GPI)-linked differentiation antigen expressed on guinea pig T and B lymphocytes was identified by several monoclonal antibodies; it has been shown previously that this membrane protein induced strong polyclonal T cell proliferation upon antibody binding and costimulation by PMA. Purification by immunoadsorption and microsequencing revealed that this T-cell-activating protein is the homologue of Thy-1 or CD90. In contrast to the Thy-1 antigen of most other species, guinea pig Thy-1 has a much higher molecular weight, which is due to a more extensive N-linked glycosylation, bringing the molecular weight of the total antigen up to 36 kDa. Molecular cloning of guinea pig Thy-1 indicated that the deduced molecular weight of the protein backbone is 12,777 after removal of an N-terminal 19-amino-acid leader peptide and cleavage of the 31 amino acids for GPI anchoring the C-terminal end. Sequence comparison showed that guinea pig Thy-1 has an 82% homology to human and a 72% homology to mouse Thy-1 on the amino acid level. Immunohistological staining of cryostat sections revealed intensive staining with the monoclonal antibody H154 on fibroblasts, fibrocytes, Kupffer cells, alveolar macrophages, and mesangial cells. As observed in the human, mouse, and rat, Thy-1 is abundant in the guinea pig brain. Unlike Thy-1 expression in other species, guinea pig Thy-1 is strongly expressed on most resting, nonactivated B cells and, to a lesser extent, on erythrocytes. While treatment of erythrocytes and lymphocytes with GPI-specific phospholipase C largely decreased reactivity with mAb H154, T cells retained the proliferative response to antibody and phorbol esters.     

Production of a monoclonal antibody strongly reacting with immature thymic T lymphocytes and its immunohistological application

A monoclonal antibody Th-5 has been produced against mouse immature thymic lymphocytes and employed to study the process of T cell differentiation in the thymus. Immunohistologically, Th-5 positive thymic T lymphocytes were first found at Day 12 of gestation. They increased in number as well as staining intensity until Day 18 of gestation and decreased thereafter. Th-5 antigen expression was not seen in lymphoid cells in the fetal liver. In the newborn thymus, lymphocytes in the subcapsular layer were still strongly positive, while other cortical lymphocytes became moderately positive for Th-5. Th-5 positiveness was more pronounced in the medulla than in the cortex in the thymus of young adult mice. The staining pattern of Th-5 in the thymus was apparently different from those with other T cell markers (Thy-1, CD3, CD4, CD5, CD8) including J11d, Pgp-1, IL-2R, and 3A10 (TCR gamma delta). Flow cytometric analyses showed that the expression of Th-5 was mostly associated with the Thy-1 antigen. However, the fluorescent intensity of Th-5 gradually declined with ontogenic development of the thymus, and the molecular size of the antigen was approximately 100 kDa, which is different from Thy-1 antigen (25-30 kDa). Considering these findings, the strong expression of Th-5 could be one of the markers of immature thymic T lymphocytes in the early phase of the ontogenic development.     

High-efficiency transduction of human lymphoid progenitor cells and expression in differentiated T cells.

Gene therapy strategies for humans have been limited by low transduction efficiencies and poor expression of retroviral vectors in differentiated progeny cells carrying the transduced vector. Here we describe a strategy utilizing a cell surface reporter gene, murine thy-1.2, selectable by fluorescence-activated cell sorting (FACS), to achieve higher gene marking efficiencies. Human CD34-positive cells were transduced by a murine retroviral vector bearing the thy-1.2 marker and pseudotyped with vesicular stomatitis virus G protein, followed by FACS to enrich for CD34-positive cells that express Thy-1.2 on the cell surface. Gene marking and expression after differentiation into thymocytes were assessed in a SCID-hu Thy/Liv mouse model for human lymphoid progenitor cell gene therapy. We found that virtually all of the differentiated T-cell progeny were marked with vector sequences. It is of particular importance that reconstitution with the selected cells resulted in expression of Thy-1.2 in up to 71% of donor-derived thymocytes. It is of note that the donor-derived thymocytes that did not express Thy-1.2 still harbored vector thy-1.2 sequences, suggesting repression of transgene expression in some cells during progenitor cell differentiation into thymocytes. These studies provide a proof of concept for efficient expression of transgenes through T-lymphoid differentiation and a potential basis for utilizing similar strategies in human gene therapy clinical trials.     

T cell independent Thy-1 allo-antibody response with the use of transgenic mice

We have introduced a mouse Thy-1.1 gene into the germline of Thy-1.2 mice. The introduced gene was shown to be expressed at very high levels in thymocytes when compared with the endogenous gene. Transgenic thymocytes were shown to evoke a higher than normal primary anti-Thy-1.1 antibody response in plaque-forming cell (PFC) assays. This result suggests that a direct quantitative interaction of the Thy-1 antigen activates the B cell response.     

Ectopic expression of Thy-1 in the kidneys of transgenic mice induces functional and proliferative abnormalities

Hybrid human--mouse Thy-1.1 genes were injected into pronuclei of Thy-1.2 mice to produce transgenic animals. A hybrid gene composed of the 5' part of the mouse Thy-1.1 gene combined with the 3' human untranslated regions was expressed abnormally in the kidney podocytes, which resulted in severe protein-uria and subsequent death in several founder mice. A hybrid Thy-1 gene composed of the human coding region with the 5' and 3' flanking regions of the mouse gene was expressed abnormally in a different part of the kidney (the tubular epithelia), which resulted in a proliferative kidney disorder. In addition, a neoplasm was found in the brain of one of these mice. These results show that the Thy-1 protein can play an important role in the activation, proliferation, and differentiation of many different cell types.     

Cell surface antigens of clonal teratocarcinoma cells at various stages of differentiation

We have studied cell surface antigen expression of teratocarcinoma cells at various stages of differentiation. These cells can be maintained in the undifferentiated state or will differentiate in vitro in a manner which parallels the early development of the mouse embryo. Three antigens were studied: a stem cell antigen (C); the major histocompatibility alloantigens (H-2); and the alloantigen Thy-1.The stem cell antigen was recognized by an anti-serum raised against a pluripotent teratocarcinoma cell line. This antiserum was shown to label embryonal carcinoma cells and early mouse embryo cells. The activity of the antiserum against embryonal carcinoma cells could be adsorbed with brain, kidney, and sperm from adult mice.The phenotype of the undifferentiated embryonal carcinoma cells is C⁺, H-2⁻, Thy-1⁻ or C⁻, H-2⁻, Thy-1⁻. The first stage in the process of differentiation is the formation of simple embryoid bodies with a layer of endodermal cells surrounding an inner core of embryonal carcinoma cells. The endodermal cells are C⁻, H-2⁻, Thy-1⁻. Further differentiation of the embryoid bodies attached to a substratum is associated with the appearance of H-2⁺ and Thy-1⁺ cells in the cultures.     

Properties and applications of monoclonal antibodies directed against determinants of they Thy-1 locus.

Fusion of cells of the mouse myeloma line, P3/X63-Ag8 with spleen cells from AKR/J mice immunized against C3H thymocytes or from (BALB/c x BALB.K)F1 mice immunized against AKR/J thymocytes gave rise to hybrid cell lines that continuously secrete antibodies specific for the Thy-1.2 and Thy-1.1 antigens, respectively. Monoclonal antibodies from four such cell lines were analyzed in detail. All were 19S IgM, and, in the presence of complement (C), had high lytic titers on T cells of the appropriate antigenicity. Their specificity was shown by lysis of thymocytes from Thy-1 congenic mouse strains, A/J(Thy-1.2) and A. Thy 1.1. Furthermore, they lyse only 60 to 70% of lymph node cells, suggesting cytotoxicity for mature T cells and not B cells. Treatment of peripheral lymphocyte populations with monoclonal antibody plus C eliminated effector cytotoxic T lymphocytes, their precursors, and the mitogenic response to Con A, but did not affect the response to LPS. Purified, fluorescein-labeled monoclonal anti-Thy-1 antibody could be used to distinguish T and B cells. Purified antibody coupled to Sepharose 6MB was used to separate viable T and B cells. Two independently isolated anti-Thy-1.2 hybridomas are indistinguishable and bind the same determinant whereas a third is unique and may bind a separate site.     

Evidence for a gene extinguishing cell-surface expression of the Thy-1 antigen

Hybrids between the Thy-1^– murine myeloma S194 and the Thy-l⁺ lymphoma BW5147 (Thy-l⁺) express neither the Thy-1.1 nor the Thy-1.2 antigen on their cell-surface. Subclones isolated from Thy-1^– clones express both Thy-1.1 and Thy-1.2 antigens in amounts similar to those present on wild-type Thy-1⁺ lymphomas, demonstrating that the Thy-1^– hybrids retain all the genes necessary for Thy-1 expression. The results are consistent with the idea that myeloma cells have a functional gene which acts to extinguish cell-surface Thy-1 expression in hybrids. The exact mechanism by which the gene acts to produce the Thy-1^– phenotype remains to be determined.