Yael Kaufmann

Lymphoid Cell Surface Interaction Structures Detected Using Cytolysis‐Inhibiting Monoclonal Antibodies

We screened monoclonal antibodies obtained by xenogeneic immunization for their capacity to inhibit T cell-mediated cytolysis. These antibodies fell into two classes according to the cell structures they recognized, of 30-35 K and 94-180 K apparent molecular weight, respectively. The main features of these structures and of their interaction with the corresponding antibodies were reviewed. The inhibition of cytolysis by these antibodies was shown to occur mainly at the effector cell level, at the recognition stage of cytolysis, and to depend on the nature of target cells, effector cells, and link between these cells. T cell functions other than cytolysis were also inhibited by some of these antibodies. We considered various possible mechanisms to account for the inhibition of cytolysis by these mAb. We favor an hypothesis based on inhibition by these mAb of lymphoid cell surface interaction structures. This hypothesis was discussed within the general framework of cell interaction structures in immunological and non-immunological experimental systems.

Protection of E. coli ribosomes against colicin E3-induced inactivation by bound aminoacyl-tRNA.

Colicin E3 inactivates bacterial ribosomes by introducing a single endonucleolytic break in the 16 S ribosomal RNA [ 1, 21. The cleavage is observed only when colicin acts on 70 S ribosomes. Isolated 30 S subunits or purified 16 S RNA are not substrates for the reaction [3,4]. It has recently been reported that streptomycin, tetracycline and gentamycin, antibiotics known to interact specifically with the 30 S ribosomal subunit, counteract the effect of colicin both in vivo and in vitro [S]. In the experiments described in this communication we tested the ability of molecules that normally bind to ribosomes during polypeptide synthesis to provide protection against colicin E3-induced inactivation. It has been found that ribosomes complexed with PhetRNA* and poly U are resistant to the inactivation by colicin. The protection was observed irrespective of the mode of binding; nonenzymatically and enzymatitally bound aminoacyl-tRNA’s were similarly effective.

Blocking by cyclosporine of antigen-induced maturation and lymphokine secretion by cytotoxic T lymphocyte hybridomas.

We have investigated the effect of cyclosporine (Cys) on the maturation of cytotoxic T lymphocytes (CTL), and on the induction of interleukin-2 (IL-2) secretion using two bifunctional CTL hybridomas. Both hybridomas can be stimulated with allogeneic cells to secrete IL-2 and to specifically kill target cells. Cys, at 10–50 ng/ml, eliminates the induction of both functions (secretion and lysis). Although maturation of both specific and lectin-mediated killing by the hybrid cells exhibits high sensitivity to Cys, the actual killing of target cells by previously activated cells is less affected. Our results suggest that pharmacological levels of Cys directly interfere with the antigen-responsiveness of helper-independent cytotoxic T cells, as represented by these hybridomas, and prevent their maturation.

In vivo inactivation of some ribosomal functions in a potassium depleted mutant ofE. coli

Isolated E. coli ribosomes require K’ ions (NH:, Rb+ and Cs’ are also effective) to maintain a number of specific functions, e.g. peptidyl transferase activity of 50 S subunits [ 11, aminoacyl-tRNA binding to 30 S subunits [2] and the ability of both subunits to bind certain antibiotics [3-61. Removal of the specific monovalent cation inactivates these functions, but they can be restored by heating the inactive ribo somes in the presence of K+, NH: or Rb* [ 1, 21. It was suggested that these interconversions may reflect a physiological behaviour of ribosomes [ 11. To test this hypothesis we have studied some rib@ somal activities in a mutant of E. coli B that fails to accumulate potassium [7]. When mutant cells are depleted of potassium, cell division and protein synthesis stop, but RNA synthesis and its degradation continue [7,8], We have found that the maintenance of peptidyl transferase activity in vivo depends on the intracellular concentration of potassium ions. Inactivation of this function as well as of the related ability to bind chloramphenicol and erythromycin took place when the bacterial growth medium was depleted of potassium. Moreover, these properties could be restored to the mutant ribosomes by activating them in vitro. These results may also explain the inhibition of protein synthesis in potassium depleted cells.

LYT-2 negative and T cell growth factor independent cytotoxic T lymphocyte hybridomas.

Homogeneous populations of cytotoxic T lymphocytes (CTL) are essential for studying the CTL receptor and killing mechanism. Such populations are obtained mainly through continuous CTL lines. However, the limiting amounts of cells thus produced, the months required to establish these lines and the need for repeated stimuli to maintain CTL growth and activity represent major drawbacks. To circumvent these problems we developed functional CTL-hybridomas which proliferate autonomously both in culture and in vivo (1–3). Previous attempts to generate T cell growth factor (TCGF)-independent CTL-hybridomas were unsuccessful (4,5), possibly due to CTL-induced nonspecific lysis of the fusion partner during hybridization. Our approach was based on the assumption that nonspecific lysis during hybridization may be prevented by a transient inactivation of CTL with trypsin prior to fusion and cytotoxicity regenerated in the hybrid cells after fusion, as is the case with trypsin-inactivated parental CTL (1). Using this approach, we have generated functional CTL-hybridomas which constitutively express their killing potential.

Antigen/Mitogen Induced Cytolytic Activity and IL-2 Secretion in Memory-Like CTL-Hybridomas

Memory-like monoclonal CTL hybridomas, derived from fusion of the AKR thymoma BW5147 with secondary CTL generated in vivo or in MLC cultures, have been used to study the mechanism whereby antigen/mitogen induces anamnestic CTL responses. Specifically, we have asked whether induction of cytolytic activity can be promoted by an antigenic/mitogenic signal without involvement of IL-2 receptors, IL-2, or other extrinsic factors. We have found that antigen/lectin alone can trigger the cytolytic potential of the hybridomas and induce IL-2 secretion. Pure IL-2 and conditioned medium were ineffective inducers of cytotoxicity. Moreover, IL-2 receptors were not detected on the hybrid cells before and after antigenic stimulation, demonstrating that expression of IL-2 receptors and induction of specific killing activity are not genetically linked. Non-activated and activated cells conjugated with target cells equally well, suggesting that induction of cytolytic activity involves a post target cell binding step. Close linkage between cytotoxicity and IL-2 secretion has been observed: induction of killing was consistently associated with IL-2 secretion and stimulation of both activities could be blocked by Cyclosporin A. IL-2 was secreted by the CTL hybrids as early as 3 h following stimulation. We propose that the immediate supply of IL-2 by such memory CTL enhances antigenic response of other, IL-2-dependent T cells.

Functional Relationships of Lymphocyte Membrane Structures Probed with Cytolysis and/or Proliferation-Inhibiting H35-27.9 and H35-89.9 Monoclonal Antibodies

The mechanism(s) of T lymphocyte “functions” such as cytolysis (1–4) or proliferation involve those cell surface structures that insure specific recognition and may involve other cell surface structures as well. Detection of these may be via the use of monoclonal antibodies (mAb) selected for their ability to inhibit lymphocyte functions. Indeed, anti-Lyt-2 mAb have been extensively studied as to their inhibitory effect on mouse T cell-mediated cytolysis, with repeated suggestions that Lyt-2 itself may be related to the T cell specific receptor (5–10). We have developed a range of xenogeneic rat anti-mouse mAb selected for their ability to inhibit T cell-mediated cycolysis (11,12). Three of them will be used in the present report: H35-17.2 mAb, which is most probably an anti- Lyt-2 mAb, as an experimental counterpoint to the two other mAb; H35-27.9 mAb, which differs from an anti-Lyt-2 mAb at least by the tissue distribution of the structures it recognizes; and H35-89.9 mAb, which immunoprecipitates from lymphoid cell surfaces two polypeptides of 180K and 94K molecular weight.