Effect of gene dosage on cell-surface expression of Thy-1 antigen in somatic cell hybrids between Thy-1− abelson-leukemia-virus induced lymphomas and Thy-1+ mouse lymphomas

Hybrids between pseudodiploid Thy-1.1⁺ lymphomas and Thy 1.2^– pseudodiploid Abelson-leukemia-virus-induced (ALV-induced) lymphomas express Thy-1 glycoprotein on their cell surface. These Thy-1⁺ hybrids invariably express the Thy 1.1 allelic form of the glycoprotein and may be either Thy 1.2⁺ or Thy 1.2^–. Sublines expressing both Thy 1.1 and Thy 1.2 can be isolated from Thy 1.1⁺, Thy 1.2^– hybrids by cell sorting. In contrast to hybrids with pseudodiploid ALV-induced lymphomas, hybrids between Thy 1.1⁺ lymphomas and pseudotetraploid Thy 1.2^– Abelson-leukemia-virus-induced lymphomas do not express Thy-1 glycoprotein on their cell surface and Thy-1 glycoprotein cannot be detected in detergent extracts of these cells. Thy-1⁺ revertants were isolated from one of the Thy-1^– hybrids by cell sorting. — These results demonstrate a gene dosage effect for the expression of the Thy-1 glycoprotein in somatic cell hybrids. They are consistent with the idea that diffusable gene products regulate Thy-1-glycoprotein expression in these hybrids. They also suggest that there may be additional, apparentlycis-active, regulatory mechanisms which determine the ability of theThy-1 structural genes of the Abelson-leukemia-virus-induced lymphoma parent to be expressed in somatic cell hybrids.     

A Thy-1 − mutant defining a gene acting in trans position to regulate cell-surface Thy-1 glycoprotein expression and Thy-1 messenger RNA content

The Thy-1 glycoprotein is a differentiation antigen which exhibits tissue-specific regulation. A mutant of a Thy-1.1⁺ T-cell lymphoma has been isolated which does not express Thy-1 glycoprotein on the cell surface and does not accumulate Thy-1 mRNA in the cytoplasm. Hybrids between the mutant and a Thy-1.2⁺ T-cell lymphoma express 20–30-fold lower levels of Thy-1 glycoprotein on their cell surface compared to wild-type T-cell lymphomas, and they have correspondingly low levels of cytoplasmic Thy-1 mRNA. A revertant of one hybrid was isolated which expressed wild-type levels of both Thy-1 alleles on its surface and contained correspondingly increased levels of Thy-1 mRNA. A Thy-1⁺ revertant of the Thy-1 ^– mutant was isolated by cell sorting. A second generation Thy-1 ^– mutant could be isolated from this revertant which also did not accumulate Thy-1 mRNA and which behaved in a way similar to the first generation mutant when hybridized to a Thy-1.2⁺ lymphoma. No changes in the structure or copy number of the Thy-1 structural gene could be detected in this series of mutants and revertants. These properties are consistent with a mutation in one (or more) gene(s) which acts in trans position to regulate Thy-1 glycoprotein expression.     

Sequential activation and loss of the pre-B cell Thy-1 gene in T-cell x pre-B cell somatic hybrids

In somatic cell hybrids between the pseudodiploid Thy-1^– Abelson-leukemia-virus-induced pre-B cell lymphoma RAW 253.1 and the Thy-1⁺ T-cell lymphoma, AKR1 (Thy-1⁺), all cells express the Thy-1 allele of the T-cell parent but most hybrid cells do not express the Thy-1 allele of the pre-B cell lymphoma parent. The Thy-1 allele of the pre-B cell parent, however, is spontaneously activated in a minor proportion of hybrid cells. By sorting for cells expressing the Thy-1 allele of the pre-B cell parent, derivative clones in which 100% of cells express both parental Thy-1 alleles can be isolated. Revertants with a phenotype identical with that of the original hybrid cell line can be isolated from these derivatives by sorting for nonexpression of the Thy-1 allele of the pre-B cell parent. These first-generation revertant cell lines have lost one copy of the Thy-1 gene derived from the pre-B cell lymphoma parent. By a further cycle of sorting, derivatives in which 100% of cells express both parental Thy-1 alleles can again be obtained. Second-generation revertants isolated by sorting these Thy-1⁺ hybrid cells for nonexpression of the Thy-1 allele of the pre-B cell parent no longer contain a normal copy of the pre-B cell Thy-1 allele and this surface antigen is no longer expressed by any cells in the population. These results are consistent with a mechanism that sequentially activates each copy of the Thy-1 gene derived from the pre-B cell lymphoma parent. Hybrids between the class D Thy-1 ^– mutant, AKR1(Thy-1^–d), in which the 5 region of the Thy-1 structural gene has been deleted, and RAW 253.1 cannot be activated to express either Thy-1 allele. This result indicates that a sequence upstream of exon 2 of the active Thy-1 allele is critical for the initial activation event.     

A mutant lymphoma cell line with a defectiveThy-1 glycoprotein gene

We characterized a mutant T -cell lymphoma line selected for the inability to express the Thy-1 glycoprotein. This cell line is a member of the D complementation class of Thy-1^– somatic cell mutants, and it lacks detectable cell-surface Thy-1.1 glycoprotein and detectable cytoplasmic Thy-1 mRNA. Southern blot analysis using a number of probes isolated from the clonedThy-1.2 gene demonstrated that, in the mutant, one copy of theThy-1 gene is absent from the genome and the other has undergone rearrangement. This rearrangement results from a deletion of the 5 portion of the gene removing the first two alternate exons and promoters and a portion of the second intron. The deletion breakpoint within the mutantThy-1 gene was localized to within 400 nucleotides by Southern blot analysis. The breakpoint is near two classes of mouse repetitive elements-a mouse B1-family repetitive element and a simple repetitive sequence-suggesting a mechanism of rearrangement leading to the mutation. Southern blot analysis demonstrated that two closely linked molecular markers on chromosome 9 are unaltered, demonstrating that the deletion in this mutant cell line is subchromosomal.     

Genetic basis for Ly-6− defect: Complementation between Ly-6− and Thy-1− mutant cell lines

Selection of antigen loss variants from the BW5147 cell line after chemical mutagenesis, using antibodies to Thy-1.1, Ly-6.2 and H9/25 allospecificities, produced cell lines with a pleiotropic defect in cell surface antigen expression; selection against any antigen led to the loss of all three determinants. The genetic basis for the mutant phenotype has been analyzed by gene complementation by somatic cell hybridization between Ly-6 ^– or H9/25 ^– and Thy-1 ^– mutants, which comprise different Thy-1 gene complementation classes. The Ly-6 ^– and H9/25 ^– mutants can be classified as belonging to either the A or E Thy-1^– mutant class.     

Evidence for a genetic basis for the class A Thy-1− defect

When Thy-1^– cell lines derived from different Thy-1⁺ murine thymic lymphomas are analyzed by complementation analysis, most fall into the A complementation class. A possible explanation for this result is that the Class A phenotype is due to a mutation in a gene on the X chromosome. To test this idea, selection for 6-thioguanine resistance was carried out on Thy-1⁺ hybrid cell lines between complementary Class A and Class C Thy-1^– mutant cell lines. In some hybrid clones, there was complete concordance between 6-thioguanine resistance and a change of the phenotype of the hybrid from Thy-1⁺ to Thy-1^–. Detailed study of one of these hybrid clones showed that 6-thioguanine resistance was accompanied by loss of hypoxanthine guanine phosphoribosyltransferase activity and that the Thy-1^– phenotype was attributable to loss of the gene complementing the Class A Thy-1^– mutation.Other hybrid clones, however, had some thioguanine resistant lines which remained Thy-1⁺. The degree of concordance was a characteristic of the particular hybrid clone examined and subclones which showed complete concordance could be derived from clones showing incomplete concordance. The variability in the degree of concordance between 6-thioguanine resistance and the Thy-1^– phenotype in different hybrid cell lines was also seen among individual hybrid clones isolated from a fusion between a Class A mutant and normal spleen cell blasts.We conclude from these results that the basis of the Class A Thy-1^– phenotype is genetic, but given the variability in the degree of linkage observed, we cannot determine whether the gene determining the Class A mutant phenotype is X-linked in the normal situation.     

The Pgp-1 antigen is expressed on early fetal thymocytes

The Pgp-1 glycoprotein is expressed on the bone marrow prothymocyte and on a class of intrathymic progenitor cells in the adult animal. Only 5% of adult thymocytes are strongly Pgp-1⁺. When fetal thymocytes of day 13–14 of gestation are examined by flow cytometric analysis, 80–90% of thymocytes are Pgp-1⁺, while the bulk of thymocytes are Thy-1^–. By day 15–16, the percentage of Pgp-1⁺ cells begins to fall while nearly all cells become Thy-1⁺. Two-color immunofluorescence indicates that many Pgp-1⁺ cells are Thy-1⁺. The percentage of Pgp-1⁺ cells continues to fall over the next several days, reaching adult levels by day 19. These observations are consistent with the interpretation that at least some classes of thymocyte progenitors are Pgp-1⁺.     

Characteristics of the Cells-Producents of Thymic Chemotactic Factor for Bone Marrow Stem Elements

It was shown using complement-dependent cytolysis and monoclonal antibodies against CD4, CD8, and NK1.1 antigens that the cortisone-resistant CD3⁺4^–8^–NK1.1^–-thymocytes spontaneously secreted a chemotactic transmitter inducing the release and directed migration of bone marrow cells. When estimating the general profile of the cytokines of these thymocytes by PCR with revertase, it was demonstrated the cells in question did not express cytokines with colony stimulating activities (SCF, IL-3, or GM-CSF) or cytokines affecting the migration of bone marrow stem elements (IL-2, 4, or 7). In addition, an active expression of gene bcl-2 was detected. Thus, the chemotactic cytokine inducing the release of bone marrow stem elements is a product of the cortisone-resistant long-living CD3⁺4^–8^–NK1.1^–-T-cells of the thymus.     

Characterization of surface alloantigens of murine neutrophils

Serological analysis of highly purified (>97%) mouse peritoneal exudate neutrophils using a protein-A rosetting technique, showed that these cells possessed the surface phenotype: Ig^–, Thy-1^–, Ly-1^–, Ly-2^–, Ly-3^–, Ly-4⁺, Ly-5⁺, Ly-6⁺, Ly-7^–, Ia^–, FcR⁺ and C3R⁺.     

Stimulation of lymphocyte receptor capping by the ionophore monensin

Summary The carboxylic ionophore monensin has a biphasic effect on antibody-induced Thy-1 cap formation. At higher concentrations, 5×10^–6–5×10^–5 m monensin causes a significant inhibition of receptor capping similar to that previously found with the Ca²⁺ selective ionophore A23187. At lower concentrations, 5×10^–8–5×10^–7 m capping is stimulated. It is concluded that capping at lower ionophore concentrations is a secific response to the ability of monensin to induce a rise in intracellular Na⁺, which indirectly elevates intracellular Ca²⁺ activity. This in turn activates the contractile machinery required for the aggregation of surface receptors into capped structures. At higher concentrations monensin acts as a nonspecific detergent, which causes detrimental structural alterations in some of the membrane components involved in the capping process.     

Clonospecific structural heterogeneity in the Thy-1 molecule from mouse T lymphocytes

The Thy-1 molecule immunoprecipitated from detergent-solubilized, ¹²⁵I-labeled cell-surface proteins was shown to be processed in two distinct ways by mouse T lymphocytes: one leading to the expression by thymocytes, concanavalin A-activated spleen blasts, and six of nine T-cell clones of a molecule of 25–28 kd, and another, observed in three other T-cell clones, leading to the expression at their surface of a so far undescribed low M _r (23 kd) form of Thy-1. The results of two-dimensional gel electrophoresis and neuraminidase, endoglycosidase H, and endoglycosidase F treatment revealed that the observed heterogeneity of Thy-1 molecules from peripheral cloned T cells was due to major differences in the maturation and sialylation of their N-linked complex-type oligosaccharide residues. It was also found that a given T-cell clone could express T200, LFA.1, and transferrin receptor molecules with a low or high M _r. Furthermore, and in contrast to previously reported results, this study revealed that the differences in cell-surface glycoprotein profiles could not be correlated with the Lyt-2,3/T4 phenotypes, the specificity for allo-H-2, allo-I-A, allo-I-E, or GAT + I-A^k determinants, nor with the cytolytic or helper/amplifier potential of the various T-cell clones examined. The possible implications of these findings are discussed.     

Coordinate change in phenotype in a mouse cell line selected forCD8 expression

A CD4⁺, CD8⁺ derivative of the CD4⁺, CD8^– cell line SAKRTLS 12.1 was isolated by fluorescence activated cell sorting for CD8⁺ cells. This derivative showed a co-ordinate change in a number of independent characters: The parental cell line was CD4⁺, CD8^–, CD3⁺, CD5^hi, HSA⁺, DEX^R, CD44^hi, while the derivative was CD4⁺, CD8⁺, CD3^–, CD5¹⁰, HSA⁺, DEX^S, CD44¹⁰. The derivative expressed the Thy-1.1, Ly-2.1, and Ly-3.1 surface antigens, consistent with origin from the SAKRTLS 12.1 parental cell line, and showed a drug resistance profile identical to that of the parent. It was not possible to isolate revertants with a phenotype identical to that of the parental cell line. Activation of the structural gene coding for CD8 chain was correlated with demethylation at several sites. We interpret these results to mean that this CD8⁺ derivative of SAKRTLS 12.1 arose as a result of an alteration of a gene that coordinately regulates multiple genes whose expression changes during thymocyte differentiation. Gene methylation may contribute, directly or indirectly, to some or all of the changes in gene expression observed. Address correspondence and offprint requests to: R. Hyman.     

Ly-6.2: A new lymphocyte specificity of peripheral T-cells

A new cell-membrane alloantigen determining locus, Ly-6, has recently been described, and the single specificity Ly-6.2 has been defined by the serum (BALB/c× A)F₁ anti-CXBD. Using both fluorescence and cytotoxicity, we found this specificity predominantly on peripheral (extrathymic) T cells, as tissues react thus: thymus, 0–5 percent; spleen, 25 percent; lymph nodes, 69 percent; bone marrow, 15 percent. These reactions agree with the proportion of (Thy⁺, Ig^–) cells present in these tissues. Cortisone-resistant thymus cells were positive. Absorption studies with thymus cells demonstrated the sparse or absent representation of Ly-6.2 on intrathymic T cells. Examination of spleen and lymph node cells from T cell-depleted C57BL/6 mice (after in vitro treatment with anti-Thy-1 serum or examination of tissues of C57BL/6-nu/nu mice) also showed a depletion of Ly-6.2⁺ cells. Conversely, removal of Ig⁺ B cells, which caused a relative increase in the number of T cells in the residual population, also increased the number of Ly-6.2⁺ cells. Additive effects of anti-Thy-1.2 and anti-Ly-6.2 could not be demonstrated, which suggests that the same population was Thy-1.2⁺, Ly-6.2⁺. However, additive effects could be shown with an anti-Ia serum and anti-Ly-6.2. The Ly-6.2 specificity is not found on red cells, liver, brain, or antibody-forming cells, but has been identified on a T-cell (but not B-cell) tumor and on kidney. Ly-6.2 can therefore be considered to be a marker for peripheral T cells, and it differs from the Thy-1 and the Ly-1,2,3, and 5 specificities in its relative absence from the thymus.     

Biochemical and genetic analysis of the Oka blood group antigen

The monoclonal antibody TRA-1-85 recognizes a cell surface antigen which is expressed by all human cell types tested, including red blood cells (RBCs), but not by mouse cells. All the human RBCs tested were TRA-1-85 positive except those with the rare phenotype Ok(a^–). Oka is a blood group antigen of very high frequency and only three unrelated Ok(a^–) people are known. The red cells of all three propositi were negative with the TRA-1-85 antibody. To confirm the relationship between the TRA-1-85 antibody and anti-Ok^a, the immune antibody found in the serum of Ok(a^–) individuals, Western blot analysis was used: the TRA-1-85 antibody and anti-Ok^a gave identical but complex patterns of re-activity in Western blot analysis of human cell lysates or membranes. This suggests that the anti-Oka and TRA-1-85 antibodies recognize the same cell-surface determinant and implies that Ok^a is not restricted in its expression to the surface of RBCs but is expressed on white blood cells (WBCs) of Ok(a⁺) individuals and all human cell lines tested to date. WBCs from one of the Ok(a^–) propositi were tested and found to be negative with the TRA-1-85 antibody. Finally, the species specificity of the TRA-1-85 antibody has been exploited by the use of somatic cell hybrids and DNA transfection techniques to examine the genetic control of the Ok^a antigen defined by the TRA-1-85 antibody. We report that the determinant is controlled by a single gene OK present on human chromosome 19.     

Time course study of H-2 and other antigen expression by hybrids of a myeloma cell line with inflammatory macrophages

The hybrids (the CANS lines) between inflammatory macrophages from C57BL/6N (B6) mice (H-2^b) and BALB/c mouse (H-2^d)-derived myeloma cell line NS1 in the early period after cell fusion showed no macrophage functions. However, most of the hybrids expressed these functions after prolonged cultivation accompanied with chromosome loss. In contrast, the hybrids initially displaying myeloma functions ( light chain production) lost this function when they exhibited macrophage functions. We studied the expression of cell-surface antigens in these hybrids and found that hybrids in the early period after cell fusion codominantly expressed both parental cell H-2 antigens (H-2K^b, H-2K^d, and H-2D^d) but not the H-2D^b antigen. On the other hand, aged hybrids strongly expressed the H-2 d antigen but lacked the H-2K^b antigen. Alternatively, these aged hybrids with macrophage functions expressed antigen(s) as detected with antiaged CANS-196 cell sera and asialo GM1 antigen, both of which were thought to be found exclusively on macrophages. Thus, the expression of cell-surface antigens in these hybrids was greatly altered after cell fusion.     

Phenotype and distribution of T lymphocytes in Peyer's patches of athymic mice

Summary The distribution and phenotype of T (Thy-1.2^–) cells was examined in Peyer's patches of 8 and 16 week old athymic mice by peroxidase and two-color-fluorescence immunohistochemistry. Despite the generally recognized T cell deficiency of nude mice, some T cells consistently occurred in Peyer's patch domes in all mice. However, many Thy-1.2⁺ lymphocytes lacked cell surface markers for either helper T cells (L3T4) or cytotoxic/suppressor T cells (Lyt-2), indicating that these cells may be an immature subset of T cells. These cells may represent a population of resident T cell precursors delayed in maturation or T cells newly immigrated to Peyer's patches.     

Localisation régionale des gènes humains IDHS,MDHS, PGK, α-GAL,G6PD par l'hybridation cellulaire interspécifique

Summary 22 independent man-hamster (HGPRT^–) hybrids using male human cells with balanced reciprocal translocation t(X;2)(p22;q32) were analysed for human genes localized on chromosome 2 (IDH_S, MDH_S), on chromosome X (PGK, GAL, G6PD) and for the different chromosomes in relation with the balanced reciprocal translocation (chr.2, chr.2q^–, chr.Xp⁺).The following results were obtained:The chromosomes 2 and 2q^– are absent in the 22 hybrids.In 9 hybrids, the absence of MDH_S in spite of the presence of the chromosome Xp⁺ indicates that the gene for MDH_S is not localized on this chromosome (or that the gene for MDH_S is not on the segment 2q32-2qter translocated on X).In 14 hybrids, the three markers of X (PGK, GAL, G6PD) and IDH_S are expressed in the presence of the chromosome Xp⁺. This result indicates that the genes for these markers are on Xp⁺ or that the genes PGK, GAL, G6PD are on X without the Xp22-Xter segment, translocated on the chr.2, and that the gene for IDH_S is on the 2q32-2qter segment translocated on X.In 8 hybrids, in the absence of the intact chromosome Xp⁺, the higher percentage of the presence of G6PD (7 hybrids) and the lower percentage of the presence of IDH_S (3 hybrids) are explained by the fact that these hybrids selected in HAT medium had to retain a segment of Xp⁺ bearing the human gene HGPRT. G6PD appeared very close to HGPRT and IDH_S very distant from HGPRT.The study of the different correlations between the presence and the absence of these four markers on Xp⁺ in the different hybrids indicates the following order on the chromosome Xp⁺ from p to q: IDH_s — PGK — GAL — G6PD.

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Groupe INSERM: Directeur J. Frézal

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Groupe CNRS, ER, 149: Directeur J. de Grouchy     

Kinetic studies on the stimulation of Na+−H+ exchange activity in renal brush border membranes isolated from thyroid hormone-treated rats

Summary Na⁺–H⁺ exchange activity in renal brush border membrane vesicles isolated from hyperthyroid rats was increased. When examined as a function of [Na⁺], treatment altered the initial rate of Na⁺ uptake by increasingV _m (hyperthyroid, 18.9±1.1 nmol Na⁺ · mg^–1 · 2 sec^–1; normal, 8.9±0.3 nmol Na⁺ · mg^–1 · 2 sec^–1), and not the apparent affinityK _Na ⁺ (hyperthyroid, 7.3±1.7mm; normal, 6.5±0.9mm). When examined as a function of [H⁺] and at a subsaturating [Na⁺] (1mm), hyperthyroidism resulted in the proportional increase in Na⁺ uptake at every intravesicular pH measured. A positive cooperative effect on Na⁺ uptake was found with increased intravesicular acidity in vesicles from both normal and hyperthyroid rats. When the data were analyzed by the Hill equation, it was found that hyperthyroidism did not change then (hyperthyroid, 1.2±0.06; normal, 1.2±0.07) or the [H⁺]_0.5 (hyperthyroid, 0.39±0.08 m; normal, 0.44±0.07 m) but increased the apparentV _m (hyperthyroid, 1.68±0.14 nmol Na⁺ · mg^–1 · 2 sec^–1; normal 0.96±0.10 nmol Na⁺ · mg^–1 · 2 sec^–1). The uptake of Na⁺ in exchange for H⁺ in membrane vesicles from normal and hyperthyroid animals was not influenced by membrane potential. H⁺ translocation or debinding was rate limiting for Na⁺–H⁺ exchange since Na⁺–Na⁺ exchange activity was greater than Na⁺–H⁺ exchange activity. Hyperthyroidism caused a proportional increase and hypothyroidism caused a proportional decrease in Na⁺–Na⁺ and Na⁺–H⁺ exchange. We conclude that hyperthyroidism leads to either an increase in the number of functional exchangers in the membrane or exactly proportional increases in the rate-limiting steps for Na⁺–Na⁺ and Na⁺–H⁺ exchange activity.