Distinct intracellular localization of Lck and Fyn protein tyrosine kinases in human T lymphocytes.

Two src family kinases, lck and fyn, participate in the activation of T lymphocytes. Both of these protein tyrosine kinases are thought to function via their interaction with cell surface receptors. Thus, lck is associated with CD4, CD8, and Thy-1, whereas fyn is associated with the T cell antigen receptor and Thy-1. In this study, the intracellular localization of these two protein tyrosine kinases in T cells was analyzed by immunofluorescence and confocal microscopy. Lck was present at the plasma membrane, consistent with its proposed role in transmembrane signalling, and was also associated with pericentrosomal vesicles which co-localized with the cation-independent mannose 6-phosphate receptor. Surprisingly, fyn was not detected at the plasma membrane in either Jurkat T cells or T lymphoblasts but was closely associated with the centrosome and to microtubule bundles radiating from the centrosome. In mitotic cells, fyn co-localized with the mitotic spindle and poles. The essentially non-overlapping intracellular distributions of lck and fyn suggest that these kinases may be accessible to distinct regulatory proteins and substrates and, therefore, may regulate different aspects of T cell activation. Anti-phosphotyrosine antibody staining at the plasma membrane increases dramatically after CD3 cross-linking of Jurkat T cells. The localization of lck to the plasma membrane suggests that it may participate in mediating this increase in tyrosine phosphorylation, rather than fyn. Furthermore, the distribution of fyn in mitotic cells raises the possibility that it functions at the M phase of the cell cycle.

High level expression of tissue inhibitor of metalloproteinases in Chinese hamster ovary cells using glutamine synthetase gene amplification.

We have used a glutamine synthetase (GS) gene as an amplifiable marker in Chinese hamster ovary (CHO) cells. GS was combined with an efficient transcription unit to produce tissue inhibitor of metalloproteinases (TIMP). Initial transfectant cell-lines selected using a GS gene secreted up to 9 micrograms TIMP/10(6) cells/24h. After one round of GS gene amplification expression levels of 110 micrograms TIMP/10(6) cells/24h were achieved. These GS gene amplified CHO cells, when adapted to grow in suspension, accumulated 180mg/l in shake flask culture. This system therefore provides a rapid method of achieving high level gene expression in mammalian cells.

High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker.

We report a method for introducing a glutamine synthetase (GS) selectable marker into myeloma cells in which transfectants are selected by growth in a glutamine-free medium. Vector amplification can subsequently be selected using the specific inhibitor of GS, methionine sulphoximine (MSX). Using this system, DNA sequences encoding a chimeric B72.3 IgG4 antibody were expressed from hCMV-MIE promoters in NSO myeloma cells. A cell line was isolated after a single round of selection for vector amplification which contains approximately 4 copies of the vector, secretes 10-15 pg/cell/day cB72.3 antibody during exponential growth and can accumulate 560 mg/l antibody in a fed-batch air-lift fermentation system. Productivity is stable in the absence of MSX selection.

The use of engineered E1A genes to transactivate the hCMV-MIE promoter in permanent CHO cell lines.

Vectors expressing adenovirus 5 E1A or a domain 2 mutant E1A were introduced into CHO-K1 cells in order to transactivate the hCMV-MIE promoter in transient and stable transfections. Expression from the hCMV promoter was efficiently activated by both wild-type and mutant E1A in contrast to other viral promoters such as the SV40 early promoter which are repressed by E1A. E1A genes expressed from a strong promoter were inhibitory to the growth of CHO cells. Nevertheless, by the use of a weaker promoter, it was possible to isolate stably transfected cell lines containing a level of E1A compatible with both continued cell growth and significant transactivation of the hCMV promoter. By this means we have generated cell lines secreting tissue inhibitor of metalloproteinases (TIMP) at levels approaching those previously attained using gene amplification. CHO cell lines constitutively expressing wild-type and mutant E1A genes have been derived which can serve as new host cell lines for transient expression and efficient stable expression without gene amplification.

Drosophila larval fat body surfaces : Changes in transplant compatibility during development.

The hemocytes oftu-Sz ts melanotic tumor larvae ofDrosophila melanogaster encapsulate heterospecific and surface-modified homospecific tissue implants, but do not encapsulate unmodified homospecific implants (R. Rizki and Rizki 1980). In the present study we usedtu-Sz ts hosts to assay changes in larval fat body surfaces during development. Donor fat bodies from various ages of larvae were accepted (remained unencapsulated) intu-Sz ts hosts whereas fat bodies from donors with everted spiracles and all subsequent stages of development that were tested were rejected (encapsulated). Since the demarcation between acceptance and rejection by thetu-Sz ts blood cells did not coincide with the gross morphological changes that appear in the fat body during metamorphosis (dissolution of the basement membrane and dispersal of the freed fat body cells at pupation), we compared acceptable and nonacceptable fat body surfaces by three other methods. Fat body surface ultrastructure was examined, fat bodies were treated with fluorescein isothiocyanate-conjugated lectins, and fat body surfaces were reacted with a monoclonal antibody specific for basement membrane. These approaches did not uncover fat body surface changes associated with eversion of the anterior spiracles, suggesting that recognition of tissue surface heterogeneities by the insect hemocytes exceeds the resolving power of the other three methods. However, the monoclonal antibody fails to bind to the basement membrane ofD. virilis larvae, whose fat body is always rejected intu-Sz ts hosts. This supports our suggestion that the molecular architecture of the basement membrane may be important in eliciting the encapsulation response.

Amplification using CHO cell expression vectors.

The ability to select for integration of plasmid DNA into the host chromosome allows the generation of stably transfected cell lines. With transfection of a selectable marker linked to a nonselectable target gene (or by cotransfection of the two unlinked genes), high-level expression of the desired gene is obtained by selecting for amplification of the selectable marker. This unit presents two systems for gene amplification and expression. The first describes the dihydrofolate reductase (DHFR) selection system while the second is based on selection of the glutamine synthetase (GS) gene. The DHFR system is probably more widely used, and results in very high levels of amplification and expression; however, the DHFR amplification process is lengthy and may require several months to isolate and characterize a stable, amplified line. In contrast, the GS system typically requires only a single round of selection for amplification to achieve maximal expression levels.

Chimeric receptors providing both primary and costimulatory signaling in T cells from a single gene product.

Single chain Fv chimeric receptors, or T-bodies, are described with intracellular sequences comprising the costimulatory signaling domain of CD28 in series with the zeta-chain from the TCR complex. Using an engineered human single chain Fv derived from P67, an mAb with specificity for human CD33, and a spacer comprising an Ab hinge region with either Fcgamma or part of the CD28 extracellular region, fusion molecules were constructed to test the ability of single chain designs to mediate both primary signaling and costimulation from one extracellular binding event. Constructs with the CD28 signaling domain proximal and the zeta-chain distal to the membrane were found to express more efficiently in Jurkat than constructs with the opposite orientation and were capable of mediating up to 20 times more IL-2 production on stimulation with solid phase Ag when compared with transfectants expressing chimeric receptors with zeta-chain intracellular signaling domains only. IL-2 production was specific to Ag challenge and was completely inhibited by incubation with free Ab of the same specificity as the extracellular binding site of the construct, but not by an isotype-matched control Ab. The CD28 intracellular domain of these fusion proteins was shown to be capable of binding the p85 subunit of phosphatidylinositol 3'-kinase. These constructs represent the first of a new generation of single gene multidomain chimeric receptors capable of mediating both primary and costimulatory signaling specifically from a single extracellular recognition event.

Selecting and designing cell lines for improved physiological characteristics.

We have developed several approaches to create cell lines with improved characteristics in cell culture. In some cases it has been possible to isolate natural variants with useful properties. Cholesterol independent variants of the mouse NSO myeloma cell line were isolated by cloning in a selective medium. A glutamine independent variant of a hyridoma was isolated by continuous (chemostat) culture under glutamine limited conditions in the presence of glutamate. Choline independent cells were isolated from a choline limited chemostat. In an alternative approach to modifying cell behaviour, we have used recombinant DNA techniques to introduce the glutamine synthetase (GS) gene to a hybridoma. This resulted in glutamine independence and increased productivity.

Recombinant human antibodies: linkage of an Fab fragment from a combinatorial library to an Fc fragment for expression in mammalian cell culture.

The combinatorial phage library approach to immunoglobulin repertoire cloning recently made it possible to isolate gene fragments encoding human immunoglobulin G1 Fabs binding with high affinity to specific antigens. Here we describe the construction of genes encoding whole human anti-tetanus toxoid antibodies based on one of these gene fragments and the efficient expression of these constructs by co-transfection of separate heavy and light chain vectors into a Chinese hamster ovary cell line constitutively expressing a viral transactivator protein. This system will be generally useful for the rapid analysis of recombinant antibodies derived from combinatorial libraries.