A transgenic immunoglobulin mu gene prevents rearrangement of endogenous genes

Transgenic mice containing a microinjected rearranged immunoglobulin (Ig) mu heavy chain gene were examined for the effects on DNA rearrangement of the endogenous Ig genes. Abelson murine leukemia virus (A-MuLV) cell lines were isolated from pre-B cells of transgenic mice and of normal littermates. Microinjected mu gene RNA and a mu heavy chain protein were synthesized in every transgenic A-MuLV cell line. Only 10% of normal mouse A-MuLV transformants synthesized mu protein. A germ-line JH allele was observed in 40% of the transgenic lines, demonstrating that the block to endogenous Ig DNA rearrangement occurred at the first step of heavy chain DNA joining. All alleles were rearranged in normal mouse A-MuLV lines. Germline JH alleles were also detected in 10% of the transgenic hybridomas derived from proliferating B cells. Our results support a model of active prevention of rearrangement by the product of successfully rearranged mu genes.     

Immunoglobulin of T lymphoma cells. Biosynthesis, surface representation, and partial characterization.

Four cloned continuously cultured mouse T lymphoma cell lines, WEHI-22.1, WEHI-7.1, S49.1, and EL-4.1, were examined for immunoglobulin biosynthesis and the presence of immunoglobulin on the cell surface. Incorporation of [-3H]leucine into cellular proteins followed by serological analysis showed that immunoglobulin constituted between 0.1 and 1.1 percent of protein synthesized by the different cell lines during a 6-hr period. Under the same conditions cultured cells of nonlymphoid origin, the mastocytoma P-815 X-2.1, did not synthesize any detectable immunoglobulin. Lactoperoxidase-catalyzed radioiodination was used to label proteins on the surface of viable lymphoma and mastocytoma cells. Although the lymphoma lines lacked immunoglobulin as assessed by fluorescent antibody staining, immunoglobulin was detected in surface proteins of all four lymphoma lines. Estimates of the number of immunoglobulin molecules on the cell surface were 1.1 times 10-4/cell for S49.1 and EL-4.1, 1.7 times 10-4 for WEHI-7.1, and 4.3 times 10-4 for WEHI-22.1. Electrophoretic mobilities in sodium dodecyl sulfate polyacrylamide gel indicated that intact cell surface immunoglobulin was slightly larger than IgG, and on disulfide bond reduction to dissociate into two components, one with the mobility of serum immunoglobulin light chain, the other with a mobility similar to that of mu heavy chain. The heavy chain from the T lymphoma cells possessed an apparent molecular weight of about 65,000 compared with 70,000 for mu chain, although both chains shared antigenic determinants characteristic of mu chains. These findings are interpreted as support for other reports that T lymphocytes carry immunoglobulin on their surface and as direct evidence that thymus-derived lymphoid cells synthesize an immunoglobulin resembling the 7S subunit of IgM.     

Synthesis, surface deposition, and secretion of immunoglobulins by Abelson virus-transformed lymphosarcoma cell lines.

Three Abelson virus-transformed lymphoma cell lines were established in tissue culture and the immunoglobulin biosynthesis by these cell lines was studied. Two of the cell lines (ABLS-1 and ABLS-5) were found to synthesize monomeric IgM molecules which were deposited in the cell membrane, probably to serve as an antigen receptor. The third cell line (ABLS-8) was found to synthesize membrane-associated IgM as well as cellular IgG molecules. In addition, these cell lines were found to synthesize a protein of 35,000 molecular weight which is also membrane-associated and which has the capability to bind the immunoglobulin (MAID). It is speculated that this protein might play a role in adapting the receptor immunoglobulin molecule to the hydrophobic environment of the cell membrane. The kinetics of amino acid incorporation into immunoglobulins by these cell lines show that they produce immunoglobulins at a rate which is two orders of magnitude smaller than plasmacytoma cells (MOPC 104E). These results suggest that Abelson virus transforms thymus-independent lymphocytes in various stages of maturation and these lymphocytes might be of B cell origin. The T lymphoma (P1798) used as a control cell line was found occasionally to produce minute amounts of immunoglobulin.     

A novel lymphocyte function-associated antigen (LFA-1): cellular distribution, quantitative expression, and structure

The derivation of a new human pre-B cell line from the leukocytes of a girl in the leukemic phase of lymphoblastic lymphoma is reported. Cells of this line, termed SMS-SB, synthesized mu but not light chains. These mu-chains were found only in the cytoplasm and were not secreted. SMS-SB cells bore HLA-DR determinants. They did not express the common ALL antigen or terminal deoxynucleotidyl transferase and, therefore, differed from previously described human pre-B leukemia cell lines. SMS-SB cells resembled certain Abelson leukemia virus-transformed mouse bone marrow cells. Except for the lack of formed mouse bone marrow cells. Except for the lack of mu secretion, the phenotype of SMS-SB cells was the same as the major subpopulation of marrow pre-B cells.     

Altered actin and immunoglobulin C mu expression in nitrogen mustard-resistant human Burkitt lymphoma cells

Raji-HN2 is a B cell lymphoma (Burkitt lymphoma) line that was made resistant to nitrogen mustard. The drug-resistant phenotype was accompanied by changes in gene expression. The expression of four unrelated genes was examined by Northern blot analysis. Raji-HN2 cells were found to contain about twice the number of actin mRNA found in Raji cells. Both cell lines were found to contain equivalent amounts of beta 2-microglobulin, c-myc oncogene, and immunoglobulin C mu mRNAs. The C mu mRNA was, however, larger in size in Raji-HN2 cells. Alterations in actin and C mu mRNAs in Raji-HN2 cells were not due to gene amplification or rearrangement because Southern blot analysis revealed no changes in the genomic organization of these genes. The increased actin mRNA content was correlated with an increased actin content of Raji-HN2 cells. The F-actin (stained with 7-nitrobenz-2-oxa-1,3-diazolylphallacidin) content of single cells was quantitated in a meridian interactive laser cytometer. Raji-HN2 cells contained about twice the amount of F-actin present in the parental Raji cells. Similar results were obtained when large populations, 10(6) cells each, were examined in a flow cytometer.     

A temperature-sensitive mutant of Abelson murine leukemia virus confers inducibility of IgM expression to transformed lymphoid cells.

Lymphoid cell lines were isolated that were inducible for the expression of surface immunoglobulin by shift from 35.5 to 39.5 degrees C after infection of mouse bone marrow cells with a mutagen-treated Abelson murine leukemia virus. Virus produced by one of the cell lines (ts49) transmitted the temperature-sensitive phenotype to new lymphoid transformants as well as to NIH/3T3 cells. In addition, the tyrosine autophosphorylating activity of the p120gag-abl protein synthesized in ts49-transformed cells was found to be temperature-sensitive. Shift experiments using ts49-transformed lymphoid cells showed that at 39.5 degrees C they synthesize increased amounts of mu and kappa chain RNA and protein, and that they can be further induced to secrete IgM when treated with lipopolysaccharide.     

Two types of mu chain complexes are expressed during differentiation from pre-B to mature B cells.

Immunoglobulin mu chains synthesized in murine pre-B cells are known to be associated with surrogate light chains designated as omega (omega), iota (iota) and B34. In addition to these molecules, we identified the complexes of polypeptides (50, 40, 27 and 15.5 kd) associated with surface or intracellular mu chains of pre-B cell lines. Most of these polypeptides were continuously synthesized and associated with mu chains in virgin B cells lines, although some of them scarcely bound to the mu kappa dimer or mu 2 kappa 2 tetramer concomitantly present in the same clone or population. However, in mature B cells they were no longer detectable except B34. Cross-linking of micron chains on the surface of pre-B cells resulted in an increase in intracellular free Ca2+, indicating that the micron chain complex on the surface of pre-B cell lines acted as a signal transduction molecule. However, the receptor cross-linkage of pre-B cell lines did not induce the increased inositol phospholipid metabolism usually observed in virgin and mature B cell lines. These results suggest that, during the differentiation from pre-B to mature B cells, the cells express two types of mu chain complexes which exhibit different structures as a whole and possess different signal transducing capacities.     

Immunoglobulin gene expression and DNA methylation in murine pre-B cell lines

DNA modification accompanying immunoglobulin gene expression was examined in various Abelson murine leukemia virus (A-MuLV)-transformed cell lines, which were able to differentiate from the mu- to mu+ stage or to undergo an isotype switch during in vitro culture. The C mu genes were relatively demethylated in the A-MuLV-transformed cell lines examined irrespective of whether or not the C mu genes were expressed. Normal IgM-bearing B cells, as well as a T cell line, also showed a similar DNA methylation pattern and the C mu genes were relatively demethylated. In one of the mu+ clones, however, the expressed C mu gene was heavily methylated. The DNA methylation pattern did not change and remained hypermethylated before and after gamma 2b expression in the two cell lines which underwent class switch to gamma 2b during in vitro culture, suggesting that expression of the gamma 2b gene was not accompanied by demethylation of the C gamma 2b gene. Taken together, these results indicate that DNA demethylation within and around the CH gene may not be necessary for its expression.     

Murine T lymphoma cells express a novel membrane-associated antigen with unique features

A novel membrane-associated antigen expressed on various murine T lymphoma cells has been detected by a rat monoclonal antibody. The antibody YE6/6 initially produced against Moloney leukemia virus-transformed T lymphoma line MBL-2, reacted with several other lymphoma lines including non-T lymphoma lines as well as thymocytes from leukemic AKR mice, but it did not show significant reactivities with resting or mitogen-activated normal lymphocytes by flow cytometric analysis. The antibody did not bind to some Abelson leukemia-transformed cells, which express Moloney virus antigens, suggesting that the antigen is unlikely to be encoded by Moloney virus genome. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the antigen molecules immunoprecipitated by the antibody revealed three major polypeptides. Two of the polypeptides, with approximate m.w. of 95,000 and 35,000, can be labeled by the cell surface iodination and, therefore, seem to be exposed on the cell surface. The third polypeptide, with approximate m.w. of 65,000, is not labeled by the surface iodination but it is readily detected by [35S]methionine labeling. The third polypeptide was labeled with [32P]orthophosphate indicating that it is a phosphoprotein. Western blot analysis showed that YE6/6 antibody primarily reacts with 35,000 m.w. polypeptide. Furthermore, the same 35,000 m.w. protein was also detected in concanavalin A-activated spleen cells at a low level by Western blot, but normal resting lymphocytes were negative. These results suggest that the antigen detected by YE6/6 antibody may be a cell proliferation-associated antigen and its expression is highly elevated on transformed lymphoma cells as compared to normal mitogen-activated lymphocytes.     

Differentiation antigens on normal and Abelson virus transformed lymphocytes.

Rabbit antisera to Abelson leukemia virus (A-MuLV)-induced murine lymphomas have been analyzed by absorption with a variety of murine lymphoma lines. Antibody binding to a panel of cell lines and normal lymphocytes was visualized by using hapten-sandwich indirect membrane immunofluorescence. Novel membrane antigens thereby detected are shared between lymphosarcomas, B lymphomas, normal B lymphocytes, and normal membrane immunoglobulin negative (sIg-) bone marrow cells, but are not found on T cells, thymic lymphomas, plasmacytoid lymphomas, or myelomas. The existence of such shared differentiation antigens suggests that sIg- A-MuLV-induced lymphosarcomas may be transformed B cell precursors. Since differences in the expression of these antigens on individual plasma-cytoid lymphoma lines were found, this category of lymphomas may include cells at a variety of differentiation states.     

Immunoglobulin synthesis by lymphoid cells transformed in vitro by Abelson murine leukemia virus.

The majority of cell lines derived by infection of murine bone marrow cells with Abelson murine leukemia virus (A-MuLV) synthesize a mu chain but no detectable light chain. Aside from this mu-only phenotype, lines that make only light chain, both chains or no immunoglobulin-related polypeptides have also been found. Two lines have been studied in detail: one that makes only mu chain and one that makes only kappa light chain. Synthesis of both polypeptides can be increased by modifying the culture conditions so as to decrease the growth rate of the cells. Although some kappa chain secretion was observed, neither secreted nor surface mu was detected. We suggest that the mu- only phenotype may be an early normal step in the pathway of B lymphocyte maturation.