Richard A. Goldsby

Hybrid cell lines with T cell characteristics

THE hybridisation of established cell lines with differentiated cells provides a useful strategy for the production of cell lines having differentiated properties and unlimited growth potential. Clones of these hybrids could be particularly useful in the dissection of cellular interactions of the immune system, a network involving numerous specific cell types which display characteristic ensembles of surface and internal markers. We describe, here, the production and partial characterisation of hybrid cell lines with surface antigens characteristic of T lymphocytes.

Production of specific antibody without specific immunization

Under appropriate conditions, the fraction of B lymphocytes stimulated in a population of mouse spleen cells may be large enough to constitute a representative sample of the total repertoire of antibody specificities which reside in the entire set of B lymphocytes (1). However the transitory nature of the polyclonal response (depending on culture condition, it declines and eventually ceases after 4–8 days) has prevented its exploitation as a strategy for the production of useful amounts of antibodies to particular antigens. The demonstration (2) that continuous cultures secreting antibody could be obtained by the hybridization of mouse myeloma and mouse spleen cells suggested a means of preserving the polyclonal response for an indefinite period of time. It seemed reasonable to assume that the hybridization of spleen cells in which a polyclonal response had been elicited by LPS with an appropriate myeloma cell line would result in continuous cultures displaying polyclonal antibody production. Using this approach, it was possible to isolate from the fusion of unimmunized (Balb/c x SJL)F1 spleen cells with NS1, a nonsecreting, HAT sensitive variant of MOPC 21, hybrid populations in which the polyclonal response as evidenced by the elaboration of antibodies to human hemoglobin A, KLH, DNP and human RBC’s was preserved (see Figure 1). Furthermore monospecific production of anti-DNP antibody was successfully factored out of the polyspecific production of antibodies by a hybrid population through the use of cloning. The expansion and subsequent injection of an active anti-DNP producing clone into a (Balb/c x SJL)F1 mouse resulted in the formation of an antibody producing tumor.

Chapter 13 The Isolation and Replica Plating of Cell Clones

Publisher Summary This chapter describes a system that provides for the mechanical performance of such necessary operations in clone isolation and replica plating as seeding, feeding, pipetting, and the precise transfer of measured aliquots. The basic culture vessel employed in clone isolation and replica plating contains a matrix of 96 optically clear, flat-bottom wells. Generally, it is desirable to seed the matrix at an average density of 1 cell/well by adding 0.1 ml of a cell suspension which contains 10 cells/ml. At such low cell densities some wells receive no cells, many receive one cell, and a few receive more than one cell. While this seeding operation can be conducted with Pasteur pipettes outfitted with dropping bulbs, a considerable saving in time and effort, as well as a more uniform pattern of addition, is obtained when one uses the syringe replicator. Mechanical seeding is accomplished by immersing the syringe replicators matrix of transfer elements into a cell suspension of the desired concentration and drawing an appropriate volume of the seeding suspension into the syringes by rotation of the handle which drives the rack-and-pinion assembly.

Selective Expression of Loci in the I-J Region on T Cell Hybrids

Gene products of the major histocompatibility complex (MHC) in the mouse are responsible for a variety of effector and cooperator functions of immunocompetent lymphoid cells. Specifically, loci mapping to the I-region have been linked to suppression (1), MLR stimulator cells (2) and GVH reactions (3). Ia antigens, which are thought to be the products of the Ir genes, have been found on B lymphocytes, macrophages, sperm and a variety of functionally different subsets of T cells (4). The selective expression of I-region molecules on T cell subsets has led many to postulate that these molecules are important in T-B collaboration (5).

Characterization of a Human T-Cell Population Identified and Isolated by a Monoclonal Antibody

The surface properties of human lymphocytes are of great interest because they identify populations of lymphocytes which conduct different functions of the immune system. Thus B lymphocytes carry an array of immunoglobulin (Ig) molecules on their surface (Froland and Natvig, 1972; Fu et al., 1974). Human peripheral T cells and thymocytes bear receptors for sheep erythrocytes and are thus capable of forming rosettes when mixed with sheep red blood cells (Brain et al., 1970; Coombs et al., 1970; Jondal et al., 1972). With the application of appropriate rosetting techniques, complement receptors can be demonstrated on the surface of human B cells and null cells (Perlman et al., 1975). Additionally, immune-response-associated (Ia) alloantigens are expressed in human B cells (Jones et al., 1975; Winchester et al., 1975). Antisera to these determinants have been shown to block stimulation in mixed lymphocyte cultures (Winchester et al., 1975) and to inhibit antibody-dependent cellular cytotoxicity (Chess et al., 1976). These considerations make it apparent that highly specific, high-titer antibody preparations to particular human lymphocyte-surface antigens would assist greatly in extending our ability to detect changes in lymphocyte subpopulations.

Identification, isolation and characterization of a human T cell population by a monoclonal antibody

The hybridization of spleen cells from mice immunized with mononuclear leukocytes with the HAT-sensitive nonsecreting myeloma, NS1, resulted in the production of hybrid cell lines secreting monoclonal antibodies to lymphocyte surface antigens. One of these, anti-Ta, was shown by fluorescence-activated cell sorter analysis to be specific for a subpopulation of peripheral human T cells. Anti-Ta did not react with peripheral human B cells. Immunoprecipitation followed by two-dimensional gel analysis demonstrated that the T cell subpopulation-specific antigen recognized by this monoclonal antibody is part of, or firmly associated with, a protein of the plasma membrane.

Preservation of polyclonal antibody production by hybridization

The fusion of unimmunized (Balb/c × SJL)F1, mouse spleen cells in which a polyclonal response had been induced by bacterial lipopolysaccharide with a myeloma cell line resulted in hybrid cell populations. The hybrid populations obtained elaborated antibody activity to human hemoglobin A, Keyhole Limpet hemocyanin, the dinitrophenyl (DNP) hapten, and human erythrocytes, Thus, hybridization allowed preservation of the normally transitory polyclonal response induced in mouse B cells by lipopolysaccharide. Furthermore, monospecific production of anti-DNP antibody was successfully factored out of the polyspecific production of antibodies by cloning. The expansion and subsequent injection of one of these clones into a (Balb/c × SJL)F1 mouse resulted in the formation of an antibody-producing tumor that was successfully passed to other (Balb/c × SJL)F1 recipients. Collection of the serum from tumor-bearing mice provided useful quantities of an anti-DNP antiserum without resort to any program of immunization whatsoever.