Search

The T-cell hybridoma 51H7D specifically binds the hapten azobenzenearsonate (ABA). An antiserum (anti-PCIF) specific for antigenic determinants shared by antigen-binding molecules of T cells (TABM) was used to precipitate polyribosomes containing mRNA for TABM from this T-cell hybridoma. The resultant mRNA was translated in vitro. The translated product (TrP51H7D) bound specifically ABA and was bound by both anti-PCIF and anti-T-cell suppressor factor. The parent lymphoma BW5147 yielded a similar translated product with the same antiserum used to isolate specific mRNA containing polysomes. This product (TrPBW) was bound by the antiserum used but did not bind ABA. The specific translated protein from both cells had a pI of approximately equal to 5.0, and apparent molecular weight of 71,000 (reduced) or 145,000 (nonreduced). Both protein products, when treated with guanidine to break down all noncovalent bonds, revealed an elemental peptide of Mr 23,500. The cDNA made from the isolated mRNA had 600-900 bases. mRNA of this size is expected for a protein of Mr 25,000. Our data indicate that a TABM specific for ABA is composed of peptides of Mr 23,500.

We have examined the arrangement of immunoglobulin heavy chain constant (CH) and joining (JH) region genes in murine T cell hybrid lines and in T lymphomas. CH genes derived from both parental cell types were present in all hybrids for which polymorphism in sequences flanking CH genes permitted us to distinguish parental CH genes. All T lymphomas and T cell hybrids retained the C alpha gene in germ-line configuration and all but one cell line had germ-line C mu genes. Novel DNA fragments reactive with JH probes were observed in six of nine T cell hybrids, as well as in two T lymphomas, WEHI7.1 and YAC-1, but not in the fusion parent, BW5147. No RNA homologous to C gamma 2b, C alpha, or lambda genes was detected in any of the T cell lines. T cell lines contained poly(A)+ RNA homologous to a C mu cDNA probe. More importantly, in several cell lines the C mu RNAs were associated with membrane-bound polyribosomes. These results suggest that both JH rearrangements and C mu RNA production occur in at least some mature, antigen-specific T cells. They may therefore reflect events in normal T cell development and function related to those involved in the generation of the T receptor for antigen.

The gene for the receptor for ecotropic murine leukemia virus (Rev) has been assigned to mouse chromosome 5. This determination was made possible by an analysis of somatic cell hybrids between mouse and Chinese hamster cells. The parents of these hybrids were A/HeJ or Mus poschiavinus peritoneal exudate cells or BALB/c primary embryo fibroblasts and E36, a Chinese hamster lung fibroblast deficient in hypoxanthine guanine phosphoribosyltransferase. Segregation of mouse chromosomes in these hybrids was analyzed by chromosome banding and isozyme expression. Cells were tested for their ability to absorb and replicate vesicular stomatitis virus (murine leukemia virus [MuLV]) pseudotype particles and ecotropic MuLV as measured by the XC test. The presence of chromosome 5 was essential for receptor expression as determined by three statistical procedures. Segregation of the receptor for ecotropic murine leukemia virus was also followed in two series of subclones. In both, receptor expression was syntenic with phosphoglucomutase-1, an isozyme which has been mapped to mouse chromosome 5.

Mice of the low leukemia (BALB/cJ x A/J)F1 hybrid (CAF1) strain express B-and N-tropic infectious murine leukemia virus (MuLV) after the age of 6 mo. Initation of a protracted immunological disorder, the graft-versus-host reaction (GVHR), at 7 wk of age, accelerates the induction of both these mouse-tropic endogenous viruses, and preferentially enhances the replication of B-tropic MuLV. The earlier appearance of B-tropic MuLV in a greater proportion of mice and in higher titer is thought to be casually related to the eventual development of lymphoreticular tumors in the GVHR mice, since previous studies have shown that these same tumors can be reproduced by inoculating syngeneic recipients with serially passaged GVHR extracts containing B-tropic MuLV.

Murine lymphoid cells were infected in vitro with WN 1802 B, a naturally occurring murine leukemia virus isolated from the spleen of an 18-month-old BALB/c mouse. Normal spleen and bone marrow cells were more susceptible to infection than were cells prepared from thymus and lymph node. Spleen cells from athymic nu/nu mice also could be readily infected with virus. Permissive cells did not ingest iron readily infected with virus. Permissive cells did not ingest iron filings and did not adhere to plastic. Exogenous replication of murine leukemia virus was enhanced in spleen and lymph node cells treated with lipopolysaccharide, a bone marrow-derived lymphocyte mitogen. Conversely, cells treated with the thymus-derived lymphocyte cell mitogens, phytohemagglutinin and concanavalin A, were less capable of supporting murine leukemia virus replication. These studies suggest that the natural host for WN 1802 B is the bone marrow-derived lymphocyte.

A Lyt-2+, trinitrophenyl-specific, lymphotoxin-secreting, cytotoxic T-cell line, PCl 55, mediates the digestion of target cell DNA into discretely sized fragments. This phenomenon manifests itself within 30 min after effector cell encounter as measured by the release of 3H counts from target cells prelabeled with [3H]deoxythymidine and occurs even at very low effector to target cell ratios (0.25:1). A Lyt-1+, ovalbumin-specific, lymphotoxin-secreting T-helper cell clone, 5.9.24, is also able to mediate fragmentation of target cell DNA over a time course essentially indistinguishable from the cytotoxic T lymphocyte-mediated hit. Cell-free lymphotoxin-containing supernatants also cause release of DNA from targets, although they require a longer time course, on the order of 24 hr. In contrast, lysis of cells by antibody plus complement or Triton X-100 does not result in DNA release even after extended periods of incubation (24 hr). All three treatments that result in the release of DNA from cells cause fragmentation of that DNA into discretely sized pieces that are multiples of 200 base pairs. The results thus suggest that cytotoxic T cells, lymphotoxin-secreting helper clones with cytolytic activity, and lymphotoxin all effect target cell destruction by means of a similar mechanism and that observed differences in time course and the absence of target cell specificity in killing mediated by lymphotoxin may simply reflect differences in the mode of toxin delivery.

We have isolated a monoclonal antibody against Escherichia coli single-stranded DNA binding protein (SSB) that recognizes the functional domain specified by the ssb-113 temperature-sensitive mutation, a domain which is distinct from the DNA-binding site. Although the ssb-113 and ssb-1 mutations result in many similar phenotypic defects, they differ significantly in others, indicating that they affect different functional domains of the protein. Whereas the SSB-1 mutant protein is clearly defective in tetramer formation and is also unable to bind single-stranded DNA at nonpermissive temperatures, no similar in vitro defects have yet been found in the SSB-113 mutant protein. In fact, the only reported in vitro effect of the ssb-113 mutation on the protein is a slight increase in its helix destabilizing ability. Competition radioimmunoassays using a monoclonal antibody demonstrated that SSB-113 mutant protein, containing a single amino acid substitution at position 176 (the penultimate residue), did not compete with SSB while SSB-1 protein (with a single change at position 55) did compete with SSB. This analysis was refined by studies with a proteolysis fragment and with peptides derived from both SSB and SSB-113. The results indicate that the antibody recognizes a determinant near the COOH-terminal end of the protein and that the SSB-113 mutation lies within or very close to this determinant.