Extensive polymorphism in the extracellular domain of the mouse B cell differentiation antigen Lyb-2/CD72.

Lyb-2/CD72 is a 45-kDa mouse B cell surface protein that binds CD5 and has been shown to play a role in B cell proliferation and differentiation. Using the polymerase chain reaction we have isolated and sequenced cDNA clones encoding the serologically defined mouse Lyb-2a, Lyb-2b, and Lyb-2c alleles. We confirmed that our full length cDNA clones encode the Lyb-2a, -2b, and -2c alleles, respectively, by transfecting the isolated Lyb-2/CD72 cDNA clones into L cells and demonstrating that the transfectants bind only the appropriate allele specific anti-Lyb-2/CD72 antibodies. Sequence comparisons demonstrate that the Lyb-2/CD72 allels are highly conserved in their cytoplasmic and transmembrane domains but exhibit a high degree of polymorphism in their extracellular domains. This polymorphism in the extracellular region involves amino acid substitutions at a minimum of 20 residues and is concentrated primarily in the membrane distal region. cDNA sequence comparisons also demonstrate two distinct seven amino acid insertion/deletions among these allelic variants. A form of Lyb-2b cDNA lacking the sequence encoding the transmembrane region was isolated from a C57B1/6 mouse and a CH12.LX subline. The Lyb-2/CD72 PCR products from mRNA of mice expressing Lyb-2a and Lyb-2c contain a DNA fragment that corresponds in size to the transmembraneless form, suggesting that these mouse strains also express this mRNA.     

Ly-1 (CD5), a membrane glycoprotein of mouse T lymphocytes and a subset of B cells, is a natural ligand of the B cell surface protein Lyb-2 (CD72).

CD5 is a 67-kDa glycoprotein expressed on the cell surface membrane of all T lymphocytes and on a small proportion of B lymphocytes. The physiologic role of this Ag is still unknown. Structural and functional studies of CD5 suggest that it might act as a receptor for a positive signal. CD5-specific mAb augment CD3- or mitogen-induced T cell proliferation, IL-2 secretion, and IL-2R expression and induce a rise in intracellular [Ca2+]. In this report, we describe the purification of mouse CD5 protein (mCD5) and its use as a probe to search for the ligand of CD5. We demonstrate that mCD5 specifically interacts with the mouse B cell differentiation Ag CD72/Lyb-2. Three serologically defined allelic forms of mouse CD72/Lyb-2 can all interact with mCD5. We further show that mCD5 can interact with human CD72/Lyb-2, and similarly, that human CD5 can interact with mouse CD72/Lyb-2. These studies may have major implications for the mechanisms of T-B cell communication.     

Differentiation of murine B cell precursors in agar culture. II. Response of precursor-enriched populations to growth stimuli and demonstration that the clonable pre-B cell assay is limiting for the B cell precursor

We established culture conditions under which fetal liver-derived B cell progenitors divide and differentiate in semisolid agar, forming colonies containing antibody-secreting cells. An important feature of this assay system is that, under certain conditions, it is limiting for only one component. This was revealed by determining the slope of the least-squares log-log regression line generated when the initial seeding density was varied. When support for the growth of the clonable pre-B cells was provided by fetal liver-derived adherent accessory cells, the slope of the regression line was 1, consistent with the interpretation that only one component, the pre-B cell, was limiting under these conditions. This interpretation was tested in the present report by positive selection for cells expressing B220, Lyb-2, or AA4.1, surface markers known to be present on cell lines of the B lineage. In all cases, an increased incidence of clonable pre-B cells was observed. Furthermore, regression analysis of titration experiments undertaken with these enriched populations revealed that the assay was still limiting for a single component. A second set of growth conditions have been defined in which the clonable pre-B assay appears to be limiting for more than one component. These conditions are obtained when the adherent accessory cells are replaced by one of three distinct hemopoietic growth factors, including CSF-1 (macrophage growth factor), GM-CSF (neutrophil/macrophage growth factor), or Multi-HGF (multi-lineage hemopoietic growth factor, also called BPA or IL 3). The most straightforward interpretation of these data is that a second cell type, distinct from the B cell precursor and dependent on the growth factors, was limiting under these conditions. In the present report, this hypothesis was confirmed because growth factor responsiveness was completely absent in B220 and Lyb-2-enriched populations. Factor responsiveness was found in unseparated fetal liver and in AA4-enriched populations. However, the AA4-enriched populations, in contrast to the B220- or Lyb-2-enriched populations, also contained a large number of factor-responsive neutrophil/macrophage colonies, raising the possibility that the effect of factors on AA4+ clonable pre-B cells was accessory cell-mediated.     

Induction of apoptosis and activation of JNK and p38 MAPK pathways in deoxynivalenol-treated cell lines

Deoxynivalenol (DON) is a mycotoxin produced by what are thought to be the most prevalent toxin-producing fungi of the Fusarium genus. Here, we present the results of apoptosis induction, phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs), and expression of the c-Jun protein after DON treatment, in a pre-B lymphocyte REH cell line. In addition, human pre-T lymphocyte Jurkat, hamster kidney-derived BHK21 and mouse hepatoma MH-22a cells were used in comparative experiments in vitro. We found that the DON effect was cell origin-dependent and dose-dependent, with a significant slow-down of cell proliferation and increase of apoptotic cells in blood cell lines. BHK21 and MH-22a cells were less sensitive to the DON effect. In blood-derived REH and Jurkat cells, DON-induced apoptotic changes were preceded by an increase in JNK and p38 MAPKs phosphorylation, as well as in c-Jun expression. However, the activation of JNK phosphorylation and c-Jun expression were transient, but did not coincide with each other. An inhibitor of JNK1/2, SP600125, had a negligible negative effect on REH cell viability after DON treatment, demonstrating that JNK does not contribute to DON-induced apoptosis. In contrast, studies on the role of p38 MAPK revealed that p38 signalling is required for DON-induced apoptosis in REH cells.     

Isolation, characterization, and expression of cDNA clones encoding the mouse Fc receptor for IgE (Fc epsilon RII)1

We have isolated cDNA clones encoding a mouse low affinity receptor for IgE (Fc epsilon RII) from a cDNA library of BALB/c splenic B cells activated with LPS and IL-4. The 2.2-kb cDNA clone encodes a 331 amino acid membrane glycoprotein that is homologous to human Fc epsilon RII (CD23) and a family of carbohydrate-binding proteins. COS7 cells transfected with the cDNA clones expressed a 45,000 m.w. protein that bound IgE and the anti-Fc epsilon RII mAb, B3B4. Fc epsilon RII mRNA was up-regulated in mouse B cells by culture with IL-4, but not in B cells cultured with IgE. Fc epsilon RII mRNA was detected in IgM+/IgD+ B cell lines, but not in pre-B cell lines or in B cell lines which have undergone differentiation to secrete Ig. The monocyte line P388D1, mast cell lines MC/9 and PT18, and peritoneal macrophages stimulated with IL-4 lacked detectable Fc epsilon RII mRNA, as did Thy-1.2+, CD4+, and CD8+ normal T cells and Thy-1.2+ T cells from Nippostrongylus brasiliensis-infected mice.     

The role of complement receptor positive and complement receptor negative B cells in the primary and secondary immune response to thymus independent type 2 and thymus dependent antigens

Both complement receptor positive (CR+) and complement receptor negative (CR-) B cells have been shown to be involved in the primary immune response to PC-Hy (phosphocholine conjugated hemocyanin), a thymus dependent (TD) antigen which preferentially induces antibody secretion in Lyb-5+ B cells during a primary adoptive transfer assay. CR+ and CR- B cells also responded in a primary adoptive transfer assay to TNP-Ficoll, a thymus independent type 2 (TI-2) antigen which activates only Lyb-5+ B cells. When the secondary immune response to PC-Hy and TNP-Ficoll were analyzed, it was found that most of the immune memory to both antigens was present in the CR- B cell subset. The CR- B cell subset also dominated the secondary immune response to PC-Hy in immune defective (CBA/N X DBA/2N)F1 male mice. These data indicate that CR- B cells dominate the memory response in both the Lyb-5+ and Lyb-5- B cell subsets of normal and xid immune defective mice and suggest that Lyb-5+ and Lyb-5- B cells can be subdivided into CR+ and CR- subsets.     

Molecular structure of human lymphocyte receptor for immunoglobulin E

We have isolated and sequenced a cDNA clone encoding the human lymphocyte receptor for IgE (Fc epsilon R). The deduced protein sequence reveals that Fc epsilon R consists of 321 amino acids, without any signal sequence, and is oriented with its N-terminus on the cytoplasmic side and its C-terminus on the outside of the cell. This molecule shows striking sequence homology with chicken asialoglycoprotein receptor (hepatic lectin), suggesting a possible role for Fc epsilon R in endocytosis. Fc epsilon R mRNA is expressed in B cells, B cell lines, and macrophage cell lines. It is not expressed in T cells or T cell lines, with the exception of an HTLV-transformed T cell line. mRNAs expressed in a macrophage line and in the latter T cell line differ in size from mRNA expressed in B cells. Human BSF-1 (or IL-4) induces the expression of Fc epsilon R mRNA in B cells, but not in T cells.     

Expression of the Grb2-related protein of the lymphoid system in B cell subsets enhances B cell antigen receptor signaling through mitogen-activated protein kinase pathways

Adapter proteins play a critical role in regulating signals triggered by Ag receptor cross-linking. These small molecules link receptor proximal events with downstream signaling pathways. In this study, we explore the expression and function of the Grb2-related protein of the lymphoid system (GrpL)/Grb2-related adaptor downstream of Shc adapter protein in human B cells. GrpL is expressed in naive B cells and is down-regulated following B cell Ag receptor ligation. By contrast, germinal center and memory B cells express little or no GrpL. Using human B cell lines, we detected constitutive interactions between GrpL and B cell linker protein, Src homology (SH)2 domain-containing leukocyte protein of 76 kDa, hemopoietic progenitor kinase 1, and c-Cbl. The N-terminal SH3 domain of GrpL binds c-Cbl while the C-terminal SH3 domain binds B cell linker protein and SH2 domain-containing leukocyte protein of 76 kDa. Exogenous expression of GrpL in a GrpL-negative B cell line leads to enhanced Ag receptor-induced extracellular signal-related kinase and p38 mitogen-activated protein kinase phosphorylation. Thus, GrpL expression in human B cell subsets appears to regulate Ag receptor-mediated signaling events.     

A new lymphocyte-specific gene which encodes a putative Ca2+-binding protein is not expressed in transformed T lymphocyte lines

The LSP1 gene is a new lymphocyte-specific gene which is expressed in normal mouse B and T lymphocytes and in transformed B cells but not (or in much smaller amounts) in nine T lymphoma lines tested. No LSP1 mRNA is found in myeloid cells or in liver, kidney, or heart tissue. Inspection of the predicted LSP1 protein sequence reveals the presence of two putative Ca2+-binding domains in the LSP1 protein. Southern blotting analysis of genomic DNA from mouse liver suggests that the LSP1 gene is present as one copy per haploid genome. Similar analysis of genomic DNA extracted from three transformed B cell lines and five transformed T cell lines shows that the absence of LSP1 mRNA in T cell lines is not due to deletion or gross rearrangements of the LSP1 locus. With the use of the mouse LSP1 cDNA as a probe we can detect a cross-hybridizing RNA species in four normal human functional T cell lines but not in three transformed human T cell lines. This suggests that at least part of the DNA sequence and the expression pattern of the LSP1 gene is conserved between mouse and man. These conserved features, together with the particular expression pattern and the protein sequence homologies, suggest that the LSP1 protein is involved in a Ca2+-dependent aspect of normal T cell growth.     

Cloning of a complementary DNA encoding a new mouse B lymphocyte differentiation antigen, homologous to the human B1 (CD20) antigen, and localization of the gene to chromosome 19

The human B1 (CD20) molecule is a differentiation Ag found only on the surface of B lymphocytes. This structurally unique phosphoprotein plays a role in the regulation of human B cell proliferation and differentiation. In order to determine whether this structure is also expressed by murine B cells, cDNA clones that encode the mouse equivalent of the B1 molecule were isolated. The longest murine cDNA clone isolated, pmB1-1, contained a 1.4-kb insert with an 873 base pair open reading frame that encodes a protein of 32 kDa. The predicted mouse B1 protein contains three hydrophobic domains that may span the membrane four times and shares a 73% amino acid sequence homology with the human B1 protein. The pmB1-1 cDNA probe was used to examine mB1 mRNA expression. Northern blot analysis indicated that pmB1-1 hybridized with two mRNA species of 2.3 and 3.0 kb that were expressed only in murine spleen lymphocytes, in B lineage cell lines representing mature B cells, and were weakly expressed in one of two plasmacytoma cell lines. pmB1-1 failed to hybridize with RNA isolated from murine T cell lines, thymus, and nonlymphoid tissues. Southern blot analysis indicated that mB1 was encoded by a single copy gene. In situ hybridization localized the mB1 gene to chromosome 19 band B, a region that also contains the genes that encode the Ly-1, Ly-10, and Ly-12 Ag. These results suggest that only B cells express this heretofore undescribed murine cell-surface protein that is structurally homologous with the membrane-embedded human B1 Ag.     

Affinity purification, peptide analysis, and cDNA sequence of the mouse interferon gamma receptor

The receptor for mouse interferon gamma (IFN-gamma) was purified from detergent-solubilized plasma membranes of EL-4, a thymoma cell line which expresses a high number of receptors on its cell surface. The purification was carried out by immunoaffinity chromatography using an anti-receptor monoclonal antibody. The purified receptor was subjected to NH2-terminal sequence analysis as well as sequencing of endopeptidase-generated peptides. One of the peptides was found to be identical to a portion of the published amino acid sequence of the human IFN-gamma receptor deduced from cDNA. This information was utilized to construct a mixed-sequence oligodeoxynucleotide probe which permitted the isolation of a full-length cDNA clone coding for the mouse IFN-gamma receptor. The mouse IFN-gamma receptor cDNA is comprised of 105 base pairs of the 5'-untranslated region, an open reading frame coding for a 477-amino acid serine-rich protein having calculated Mr 52,276, and a 3'-untranslated region of 539 base pairs. The receptor is first synthesized as a pre-protein from which a 25-amino acid signal peptide is cleaved. The receptor contains a hydrophobic transmembrane portion near the center of the molecule. Northern blot analysis of various cell lines showed that each contained a single 2.0-kilobase mRNA. A direct correlation between the amount of IFN-gamma receptor mRNA and the level of receptor expressed on the cell surface was observed. The mouse and human IFN-gamma receptors are structurally similar, showing 51% over-all homology in amino acid sequence. Mouse IFN-gamma receptor cDNA when inserted in a mammalian shuttle vector and transfected into COS-7 monkey cells was able to direct the expression of specific binding activity for mouse IFN-gamma.     

In vitro study of the comparative behaviour of a human acute lymphoblastic leukemia cell line and a reference normal cell line towards the effects of a mitogenic lectin

An in vitro study of the behaviour of a human acute lymphoblastoid leukemia cell line (REH) towards the action of a mitogenic lectin of Robinia pseudoacacia was carried out. The results were compared with those a reference cell line (LHN13) established from normal human lymphocytes. In both cell lines, the lectin induces agglutination (measured by counting the number of aggregates as well as the number of cells in each aggregate) and decrease of growth (measured by counting the number of cells and the incorporation of tritiated thymidine into TCA-precipitable material per 10(6) cells). The agglutination and the decrease of growth are produced at the doses of 0.5 and 1 microgram/ml of culture medium and after 4 h of exposure of cells to the lectin, respectively. These effects increase progressively with higher doses of lectin and continues throughout the culture. However, the REH line is less sensitive than the LHN13 line to the effects of lectin. Both agglutination and growth decrease of REH as well as LHN13 cell lines by the lectin are reversible; this is confirmed by the fact that the monospecific anti-Robinia lectin serum suppresses these effects.     

A role for Lyb-2 in B cell activation mediated by a B cell stimulatory factor

The Lyb-2 system of the mouse is involved in regulation of a proliferative step in the differentiation of B cells responding to T-dependent antigen. The present study concerns the role of Lyb-2 in an early phase of B cell activation with respect to B cell receptor functions for activation factors. It is shown that interaction of monoclonal anti (alpha)-Lyb-2 antibody with Lyb-2 on the B cell surface induces B cell proliferation by synergistic action with B cell growth factor II-containing factor or interleukin 1. In contrast, alpha-Lyb-2 antibody could not synergize with the Con A-induced culture supernatant of T cell hybridoma FS6-14.13 (FS6) containing B cell stimulatory factor-1 (BSF-1; formerly called BCGF I), and the effect of combining the two was only additive on B cell proliferation. Absorption studies showed that BSF-1 in FS6 could be absorbed by unstimulated B cells, about 95% of which were at Go phase of the cell cycle, but not by thymocytes, and more importantly that alpha-Lyb-2 antibody blocked the absorption in an Lyb-2-specific manner, possibly by competing with BSF-1. It is thus likely that alpha-Lyb-2 antibody may interact with a BSF-1 receptor on B cells or a molecule closely associated with it. Interestingly, alpha-Lyb-2 antibody mimicked the action of BSF-1 in a costimulator assay with affinity-purified goat alpha-mouse IgM antibody, but could not replace all the activities ascribed to BSF-1. Possible mechanisms involved are discussed.     

Analysis of glucocorticoid unresponsive cell variants using a mouse glucocorticoid receptor complementary DNA clone

We have used a glucocorticoid receptor cDNA isolated from a mouse lymphoma cell line to characterize receptor mRNA and genomic sequences present in wild type and mutant rat hepatoma (HTC) and mouse thymoma (S49 and WEHI7) cells. Wild type rat and mouse cell lines contain two receptor mRNAs, 5 and 7 kilobase pairs (kb) in length, which differ in the length of their 3'-untranslated regions. Levels of receptor mRNA present in mutant HTC, WEHI7, and S49 cells of the r- (receptorless) phenotype are decreased compared to wild type cells. This decreased level of receptor mRNA parallels the decreased level of total immunoreactive receptor protein found in these cells. S49 nt- (nuclear transfer minus) cells contain receptor mRNA levels which parallel their hormone binding and immunoreactive receptor levels. Cells of the r- and nt- phenotype contain no detectable deletions or rearrangements of the receptor gene. We conclude that r- cells have lesions which affect the expression of receptor mRNA. Surprisingly, HTC cells of the r- phenotype differ from WEHI7 and S49 r- cells in that HTC r- cells contain a lower level of receptor DNA than does their parental wild type cell line. Although these cells may contain multiple lesions, it appears that loss of receptor genomic sequences is responsible, in part, for the phenotype of the HTC r- cells. The S49 nti (nuclear transfer increase) cells produce glucocorticoid receptors of molecular weights 40,000 and 94,000. These cells produce, in addition to the wild type 5- and 7-kb receptor mRNAs, two other receptor messages 5.5 and 3.5 kb in length. RNA blot analysis using various portions of our receptor cDNA indicates that these are 5' truncated messages and suggests that the 40-kDa nti receptor is truncated at its NH2-terminal end. These data also indicate that the hormone and DNA-binding regions of the receptor are located in the COOH-terminal half of the protein.     

The murine homolog (Mph) of human herpesvirus entry protein B (HveB) mediates entry of pseudorabies virus but not herpes simplex virus types 1 and 2

A mouse member of the immunoglobulin superfamily, originally designated the murine poliovirus receptor homolog (Mph), was found to be a receptor for the porcine alphaherpesvirus pseudorabies virus (PRV). This mouse protein, designated here murine herpesvirus entry protein B (mHveB), is most similar to one of three related human alphaherpesvirus receptors, the one designated HveB and also known as poliovirus receptor-related protein 2. Hamster cells resistant to PRV entry became susceptible upon expression of a cDNA encoding mHveB. Anti-mHveB antibody and a soluble protein composed of the mHveB ectodomain inhibited mHveB-dependent PRV entry. Expression of mHveB mRNA was detected in a variety of mouse cell lines, but anti-mHveB antibody inhibited PRV infection in only a subset of these cell lines, indicating that mHveB is the principal mediator of PRV entry into some mouse cell types but not others. Coexpression of mHveB with PRV gD, but not herpes simplex virus type 1 (HSV-1) gD, inhibited entry activity, suggesting that PRV gD may interact directly with mHveB as a ligand that can cause interference. By analogy with HSV-1, envelope-associated PRV gD probably also interacts directly with mHveB during viral entry.     

Contribution of Lyb 5+ and Lyb 5- B cells to the primary and secondary phosphocholine-specific antibody response

Due to a mutation on their X-chromosome, CBA/N mice lack the Lyb-5+ subset of B cells. The loss of this B cell subset results in a profound alteration in the immune response of these mice to the hapten phosphocholine (PC). Thus, when these mice are immunized with high doses of PC-KLH (200 micrograms) in CFA, they: 1) fail to produce IgM anti-PC antibodies; 2) produce little or no anti-PC antibody bearing the normally predominant T15-idiotype; and 3) produce IgG anti-PC antibodies only late in the primary response. In order to more fully delineate this defect in responsiveness to PC, the splenic focus assay was used to analyze Lyb-5- B cell precursors from both normal and immune defective mice. Lyb-5- cells were obtained from normal (CBA/N x DBA/2)F1 (CD) female spleens by treatment with anti-Lyb-5 serum and complement. These normal Lyb-5- cells and Lyb-5- cells from immune defective CD male mice were stimulated in vitro with either PC-Hy or TNP-Hy in the presence of Hy-primed T helper cells. The results demonstrate that primary Lyb-5- PC-specific B cells fail to respond in the splenic focus assay, while secondary Lyb-5- PC-specific precursors respond normally, and that both primary and secondary Lyb-5- TNP-specific precursors respond in the splenic focus assay. These data suggest that Lyb-5- PC-specific precursors must differentiate into memory cells before they can be activated to secrete antibody, and they also indicate that the Lyb-5- B cell subset may be composed of two subsets with different activation requirements.     

人白细胞介素2受体(IL-2R)cDNA在哺乳类细胞中表达的初步研究

按DNA-磷酸钙共沉淀法将人白细胞介素2受体(IL-2R)cDNA转染小鼠成纤维细胞L929,经RNA点渍杂交分析、荧光标记IL-2染色和抗Tac(人IL-2受体α链)特异性玫瑰花环试验,均证明转入的IL-2R cDNA在L929细胞中表达,其产物具有结合IL-2和抗Tac抗体的能力。本文还报道了T细胞白血病Jukat细胞和Molt-4等细胞系异常表达IL-2R的结果,并对此作了分析和讨论。