The immune system is capable of establishing an enormous repertoire of antibodies before its first contact with antigen. Most antibodies that express germ-line sequences are of relatively low affinity. Once antigen enters the system, it stimulates a somatic mutational mechanism that generates antibodies of higher affinity and selects for the expression of those antibodies to produce a more effective immune response. The details of the mechanism and regulation of somatic hypermutation remain to be elucidated.
The fully assembled gamma G globulin molecule consists of two heavy and two light polypeptide chains linked by disulfide bonds. The synthesis and assembly of this multichained molecule can be effectively studied in transplantable mouse plasma cell tumors which synthesize large amounts of homogeneous gamma globulin (1). In some of these tumors blocks at different stages in the assembly process (2, 3) make it possible to identify precursors of the assembled gamma globulin molecule.In previous studies Merwin plasma cell t u m o r -l l (MPC-11) 1 myeloma cells freshly dissociated from tumors were found to secrete fully assembled molecules (HzL2), half molecules (HI.), light chain dimers (L2), and free light chains (L) (4). The production of relatively large amounts of partially assembled molecules could have been due to significant numbers of nonviable cells and to the suboptimal metabolic conditions in the incubation mixture. Since some of the subunits appeared to be end products while others were precursors of the fully assembled molecule (4), it was important to determine whether all the precursors could be produced by an individual cell.These possibilities have now been explored in MPC-11 myeloma cells adapted to growth in continuous culture. Such cultures provided a uniform population of viable cells which could be studied during logarithmic growth to ensure
Cultured mouse myeloma cells have been cloned in soft agar using a modification of the method established by Pluznik and Sachs ('65, '66) and by Bradley and Metcalf ('66). A linear relationship existed between the number of cells plated and the number of colonies produced. Conditions for obtaining optimum cloning efficiency and colony size were determined for the MPC-11 cell line. Feeder cells of mouse, human and rabbit origin and conditioned growth medium obtained from mouse cultures all had a n enhancing effect on colony formation. Immunoglobulin production by cloncd cells was detected by overlaying the clones with anti-immunoglobulin antiserum. The antiserum had no adverse effect on cloning efficiency or colony size. A reconstruction experiment was performed to show that the plate assay could reliably detect rare variants of immunoglobulin producing cells. The plate assay was validated by studying immunoglobulin production following recovery of clones from dishes and their growth to mass suspension culture. Immunoglobulin formation i n these cultures was assessed by a Ouchterlony immunodiffusion of the supernatant medium. and bv incubating the cells with radioactive amino acids and analyzing the intracellular acrylamide gels.
In most instances, fusion of differentiated cell types with fibroblasts has resulted in the extinction of differentiation‐specific traits of the nonfibroblast parental cell. To explore the genetic basis of this phenomenon, we have used a series of somatic cell hybrids between myeloma cells and fibroblasts. Previous findings show that in these hybrids expression of the immunoglobulin (Ig) genes was extinguished at the transcriptional level. Our present results show that NF‐kappa B transcription factor, known to be critical for kappa‐chain enhancer activity, is present although in a lower amount, in the nucleus and in the cytosolic fraction of most of these hybrids (probably attached to the previously postulated I‐kappa B inhibitor). In contrast, the expression of the NF‐A2/OTF‐2 transcription factor encoded by the oct‐2 gene, which binds to the octameric motif located in the Ig promoters and heavy chain gene enhancer, is extinguished at the transcriptional level. Our data thus suggest that extinction of Ig genes expression occurs via an indirect mechanism in which a fibroblast factor suppresses transcription factor(s) which are critical for Ig transcription.
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