A phase II trial of murine monoclonal antibody 17-1A and interferon-γ: Clinical and immunological data

Summary A group of 15 patients with metastatic colorectal adenocarcinoma received a combination of interferon (0.1 mg/m², days 1–15) and the murine monoclonal antibody 17-1A (400 mg, days 5, 7, 9 and 12). The treatment was tolerated with minimal toxicity. Of the 14 evaluable patients, 13 developed human antibody to murine 17-1A, with 11 patients demonstrating antibody to the variable region of 17-1A (anti-idiotype). Antibody to the variable region was inhibited by 17-1A but not by mouse immunoglobulin. Sera from patients with substantial anti-idiotype reactivity were capable of inhibiting the binding of murine 17-1A to antigen expressing LS174-T cells thus indicating the presence of antibody directed against the 17-1A combining site (mirror-image anti-idiotype). The median survival of the whole group was 56 weeks and there was no correlation between clinical response/survival and the development of anti-idiotype antibody.Supported by the Veterans Administration Medical Center and by Public Health Services grant CA 45 232 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services     

Sensitive, high-resolution chromatin and chromosome mapping in situ: presence and orientation of two closely integrated copies of EBV in a lymphoma line

Here we describe development and application of highly sensitive fluorescence methodology for localization of single-copy sequences in interphase nuclei and metaphase chromosomes by nonisotopic in situ hybridization. Application of this methodology to the investigation of Epstein-Barr virus integration in the Namalwa lymphoma cell line has revealed two EBV genomes closely integrated at the known site on chromosome 1. Detecting sequences as small as 5 kb, we further demonstrate resolution within interphase nuclei of two fragments of the viral genome spaced only 130 kb apart. Results indicate that the viral genomes are in opposite orientations and separated by roughly 340 kb of cellular DNA. This work demonstrates the feasibility and resolving power of interphase chromatin mapping to assess the proximity of closely spaced DNA sequences. Implications for virology, gene mapping, and investigation of nuclear organization are discussed.     

Phosphotyrosine-modified proteins are concentrated at the membranes of epithelial and endothelial cells during tissue development in chick embryos

We have used high affinity polyclonal antibodies specific for phosphotyrosine (PTyr) residues to examine the localization in various chick embryonic tissues in situ of PTyr-modified proteins by immunocytochemical methods. During the period from 9 to 21 d of development, most tissues exhibit elevated levels of PTyr-modified proteins as determined by immunoblotting experiments of tissue extracts with the anti-PTyr antibodies (Maher, P. A., and E. B. Pasquale. 1988. J. Cell Biol. 106:1747-1755). By immunofluorescence labeling of semithin frozen sections, the highest concentrations of PTyr immunolabeling in all of the embryonic tissues examined were localized to the membranes of the epithelial and endothelial cells with other cells showing no detectable labeling. These results were confirmed by immunoelectron microscopic labeling, which showed particularly high concentrations of PTyr-modified proteins close to the membranes at the apical junctions. The corresponding adult tissues showed no labeling. It is proposed that these results reflect the molecular basis for the functional plasticity of epithelial and endothelial cell junctions during embryonic development.     

Comparative study of the tyrosine phosphorylation of proteins in Swiss 3T3 fibroblasts stimulated by a variety of mitogenic agents

We have carried out a comparative study of the protein tyrosine phosphorylation induced by a wide range of mitogenic stimuli on a single cell type, Swiss 3T3 mouse fibroblasts. For this purpose we have used high-affinity antibodies directed to phosphotyrosine residues on proteins (Wang: Mol. Cell. Biol. 5:3640-3643, 1985) in immunoblotting and immunofluorescence microscopy experiments. Immunoblotting experiments showed that all of the mitogens tested, including epidermal growth factor, platelet-derived growth factor, basic fibroblast growth factor, insulin, fetal calf serum, trypsin, and 12-O-tetradecanoylphorbol-13-acetate, increased the phosphorylation on tyrosine of a number of proteins. Most of the increase in tyrosine phosphorylation induced by each factor involved a small set of proteins with apparent molecular weights (Mr) above 50,000. Following stimulation with epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor, increased phosphotyrosine modification of proteins with molecular weights corresponding to those of the respective receptors was observed. A protein band of apparent Mr 160,000 contained substantially increased levels of phosphotyrosine following insulin treatment, but tyrosine phosphorylation of the insulin receptor was apparently below the level of detectability. The phosphotyrosine content of proteins with apparent Mr of 220,000, 120,000, and 70,000 was increased by all the agents tested. Phosphorylation on tyrosine of most of the proteins increased within a few minutes of the mitogenic stimulation, reached a peak, and returned more slowly to basal levels. Immunofluorescence labeling with the antibodies specific for phosphotyrosine showed a substantial increase in the amount of phosphotyrosine containing proteins only in the presence of platelet-derived growth factor and fetal calf serum. This finding suggests that most of the proteins phosphorylated on tyrosine in Swiss 3T3 fibroblasts are not concentrated in specific subcellular structures, but rather are diffusely distributed throughout the cell and are therefore not detectable by immunofluorescence microscopy.     

Induction of anti-CD8 resistant cytotoxic T lymphocytes by anti-CD8 antibodies. Functional evidence for T cell signaling induced by multi-valent cross-linking of CD8 on precursor cells

The effector function of most MHC class I allospecific CTL is inhibited by anti-CD8 mAb. In the present study, we report the surprising observation that multi-valent cross-linking of CD8 molecules on precursor cells by specific antibody actively induces the generation of CD8+ class I allospecific CTL whose lytic function is resistant to anti-CD8 antibody inhibition, and actively induces down-modulation of cell surface CD8 expression on these cells. In marked contrast, bi-valent cross-linking of CD8 inhibits the generation of CD8+ CTL from precursor cells and fails to induce down-modulation of cell surface CD8 expression. These results demonstrate that CD8 can transduce net positive signals, but only when the molecule is extensively cross-linked.     

Nonequivalent effects of PKC activation by PMA on murine CD4 and CD8 cell-surface expression

The membrane glycoproteins CD4 (L3T4) and CD8 (Lyt2) are expressed on distinct populations of mature murine T lymphocytes, and are thought to be receptors for monomorphic determinants expressed on MHC class II and class I molecules, respectively. Although they differ in their ligand specificity, it has been presumed that CD4 and CD8 perform equivalent functions in the T cells that bear them. Since activation of protein kinase C (PKC) is known to cause rapid down-regulation of various receptors, including the T cell receptor complex (TcR complex), we treated cells with phorbol 12-myristate 13-acetate (PMA), a PKC activator, to determine whether cell-surface expression of CD4 and CD8 would be similarly affected by this intracellular mediator. Brief or relatively prolonged treatment with PMA induced mature murine T cells to reduce their surface expression of the TcR complex and of CD4, but not of CD8. Similarly, PMA rapidly induced transfected L cells to down-regulate surface CD4 expression, but had no effect on surface CD8 expression. Most significantly, PMA treatment induced CD4+CD8+ immature thymocytes to rapidly reduce their surface CD4 expression, but, again, it had no immediate effect on the surface expression of CD8. These results indicate that CD4 and TcR complex cell-surface expression are both sensitive to PKC activation by brief treatment with PMA, whereas CD8 expression is not, and suggest that CD4 and CD8 surface expression levels are regulated by distinct intracellular mechanisms.     

Inhibition of T-cell receptor beta-chain gene rearrangement by overexpression of the non-receptor protein tyrosine kinase p56lck.

The variable region genes of the T cell receptor (TCR) alpha and beta chains are assembled by somatic recombination of separate germline elements. During thymocyte development, gene rearrangements display both an ordered progression, with beta chain formation preceding alpha chain, and allelic exclusion, with each cell containing a single functional beta chain rearrangement. Although considerable evidence supports the view that the individual loci are regulated independently, signaling molecules that may participate in controlling TCR gene recombination remain unidentified. Here we report that the lymphocyte-specific protein tyrosine kinase p56lck, when overexpressed in developing thymocytes, provokes a reduction in V beta--D beta rearrangement while permitting normal juxtaposition of other TCR gene segments. Our data support a model in which p56lck activity impinges upon a signaling process that ordinarily permits allelic exclusion at the beta-chain locus.     

Determination for inflorescence development is a stable state,separable from determination for flower development in Pisum sativum L. buds

Terminal meristems of Pisum sativum (garden pea) transit from vegetative to inflorescence development, and begin producing floral axillary meristems. Determination for inflorescence development was assessed by culturing excised buds and meristems. The first node of floral initiation (NFI) for bud expiants developing in culture and for adventitious shoots forming on cultured meristems was compared with the NFI of intact control buds. When terminal buds having eight leaf primordia were excised from plants of different ages (i.e., number of unfolded leaves) and cultured on 6-benzylaminopurine and kinetin-supplemented medium, the NFI was a function of the age of the source plant. By age 3, all terminal buds were determined for inflorescence development. Determination occurred at least eight nodes before the first axillary flower was initiated. Thus, the axillary meristems contributing to the inflorescence had not formed at the time the bud was explanted. Similar results were obtained for cultured axillary buds. In addition, meristems excised without leaf primordia from axillary buds three nodes above the cotyledons of age-3 plants gave rise to adventitious buds with an NFI of 8.3 ±0.3 nodes. In contrast seed-derived plants had an NFI of 16.5 ±0.2. Thus cells within the meristem were determined for inflorescence development. These findings indicate that determination for inflorescence development in P. sativum is a stable developmental state, separable from determination for flower development, and occurring prior to initiation of the inflorescence at the level of meristems.