Waldemar Lernhardt

Clonal growth and maturation to immunoglobulin secretion in vitro of every growth-inducible B lymphocyte

The frequency of normal murine B lymphocytes initiating growth in diluted suspension cultures in the presence of a B cell mitogen, such as lipopolysaccharide, can be increased approximately 10(4) fold by the addition of 2 X 10(6) normal thymus cells per ml. This increase in the frequency of growing cells by thymus cells can also be observed with X63-AG8 myeloma tumor cells secreting IgG1. Thus thymus cells may not contribute growth-stimulating factors, but may supply growth-supporting factors. Culture medium and plastic dishes can be conditioned by preincubation with thymus cells for a day after which the thymus cells may be omitted from further culture for maximal B cell growth. Irradiation of thymus cell abolishes their growth-enhancing properties. Thymus cells can be syngeneic and allogeneic with the growing B cells. The frequency of growing LPS-reactive, normal B cells in spleen of 6-8 week old C3H/Tif mice was determined by limiting dilution analysis to be one of three splenic B cells. With this limiting dilution analysis, it was also shown that the cloning efficiency of XB3-AG8 myeloma tumor cells in suspension culture in the presence of thymus cells is practically 100%. Analysis of the growth kinetics of single clones of LPS-reactive, normal B cells shown that these B cells divide every 18 hr. Within the first 126 hr of growth, every B cell in the clone divides, and every dividing B cell in this clone secretes sufficient immonoglobulin to form a hemolytic plaque. The conditions of in vitro suspension cultures of murine B lymphocytes are therefore perfect to the extent that every B cell capable of growth will grow as a single clone.

Cloning of murine transformed cell lines in suspension culture with efficiencies near 100

Addition of 3 × 106 thymus cells from either syngeneic, allogeneic or xenogeneic animals increases the cloning efficiencies of murine thymomas (EL-4, WC-2), B-lymphomas (McPC 1748, 38C-13), Abelson-virus transformed cell lines (F and K), mastocytomas (P815), myelomas (AbPC22, X63-AG8, 5563, MOPC 104 E, RFC 5, W 3469) and hybrids of myelomas and normal B-lymphocytes (Sp-1), all adapted to tissue culture, to near 100%. Thymus cells also increase the efficiencies of growth initiation in primary in vitro cultures of myeloma tumor cells (S117) transplanted in vivo, and of cells fused between the azaguanine-resistant X63-AG8 myeloma cell line and normal, LPS-stimulated B-lymphocyte blasts.

Roles of surface-bound immunoglobulin molecules in regulating the replication and maturation to immunoglobulin secretion of b lymphocytes.

Immunoglobulin (Ig) molecules as recognition structures on the surface of lymphocytes have been postulated by the clonal seleetion theory (Jerne 1955, Burnet 1957), and were first experimentally demonstrated by Moller (1961). Clonal selection demands a lymphocyte to synthesize and express on its surface a single antigen-binding specificity, which is getietically determined. This postulate has been supported by the findings that surface-bound Ig molecules show allelic exclusion (Pernis et al. 1965, Sell 1970) and that subsets ofcells among the lymphocytes cover different antigenic specificities. It was expected that binding of antigen at the cell surface would initiate reactions leading to replication and maturation of lymphocytes, resulting in cell-mediated or humoral effector functions. A clone of antigen-specific lymphocytes responding to antigenic stimulation was expected, and later proven, to retain its original specificity and thereby expand its particular recognition capacity within the whole immune system (Dutton & Eady 1964, Makela & Cross 1970). This simple concept of antigen recognition was no longer tenable when it was found that three different types ofcells T-lymphocytes, B-lymphocytes and macrophages had to cooperate in many humoral Immune responses (Claman ct al. 1966, Miller & Mitchell 1967, Mosier 1967). B lymphocytes have the function of producing and secreting the Ig-molecules. But, mitogens were discovered which could activate, by circumventing of the antigen-binding step to surface Ig molecules, a large part of all B cells, irrespective of their antigen-

Fatty acid requirement of B lymphocytes activated in vitro

The lipid and fatty acid requirement of B lymphocytes activated in vitro was examined by replacing soybean lipids with various combinations of lecithins, fatty acids and cholesterol. It is reported here that linoleic acid is the sole fatty acid required to support the proliferation of B lymphocytes and maturation to immunoglobulin-secreting cells.

Activation and Cell Cycle Control of Murine B Lymphocytes

SUMMARY The cell cycle of activated murine B lymphocytes (B cells) is controlled by the occupancy of surface membrane-bound immunoglobulin (Ig) and by two types of growth factors, called α and β factors. These growth factors are produced in an endocrine fashion by the interaction of helper T lymphocytes (T cells) with antigen-presenting macrophages (A cells). Antigen is taken up, processed and presented on the surface of A cells in the context of class II major histocompatibility complex (MHC) glycoproteins. Helper T cells recognize this association of antigen and class II MHC molecules. A cells produce α factors and T cells produce β factors. The molecular nature of these factors and of the corresponding receptors on B cells has yet to be elucidated, although it can be shown that the complement component C3d replaces α factor action. Resting, G0 phase B cells are refractory to the action of α and β factors. They have to be excited, i.e. rendered susceptible to the action of these factors. This can be achieved by the interaction with helper T cells that recognize antigen, bound by surface membrane Ig, in the context of class II MHC glycoproteins on the surface of resting G0 B cells. Excitation can also occur in a polyclonal fashion by cross-linking of suface Ig with immobilized, Ig-specific antibodies, or by the interaction with polyclonal activators of B cells, such as lipopolysaccharides. Entry into the cell cycle is asynchronous. Activated, cycling B cells can be synchronized by size separation, using velocity sedimentation. Synchronized B cells will retain their synchrony for several divisions, when they are stimulated by immobilized Ig-specific antibodies, α and β factors. They divide every 20 h at 37°C. Omission of either of the three stimuli arrests B cells, though at different points in the cell cycle. Three restriction points are found: the first occurs immediately after mitosis and is controlled by the binding of immobilized Ig-specific antibodies to surface membrane-bound Ig. The second is observed in the G1 phase, around 6–8 h after mitosis and 2–4h before entry into S phase. It is controlled by α factors. The third is found 2–4 h before mitosis, in G2 phase, and is controlled by β factors.