Mitogenic Activation of Human Lymphocytes: a Protein A Plaque Assay Evaluation of Polyclonal B‐Cell Activators

Abstract

Using the protein A plaque assay, the capacity of various polyclonal B cell activators to induce differentiation in human B lymphocytes was investigated. Dextran sulphate and native dextran were both virtually devoid of mitogenic properties. Lipopolysaccharide, however, was found to be a potent mitogen in human cells that, although giving rise to low DNA synthetic response, induced high numbers of immunoglobulin-synthesizing cells. Mean plaque-forming cell (PFC) numbers in healthy blood donors assayed on the optimal day (days 5-7) were 23,493 IgM/10(6) cells, 11,288 IgG/10(6) cells, and 2643 IgA/10(6) cells. Values obtained in spleen cells, peaking at days 4-6, were slightly higher. Purified protein derivative (PPD) was equally or even more effective than lipopolysaccharide (LPS) in generating PFC of different subclasses in peripheral blood with mean of 29,241 IgM/10(6), 21,269 IgG/10(6), and 3681 IgA/10(6). PPD furthermore induced a marked DNA synthetic response in human lymphocytes. These data suggest that LPS and PPD may both be used as functional markers in human cells when analysing patients with a suspected immunodeficiency state. It is suggested that cultures should be assayed using the protein A plaque assay, thereby being able not only to investigate the individual immunoglobulin classes but also to avoid the possible hazards involved in measuring antigen-specific responses in patients whose prior immunization to the antigen tested can never be totally excluded.

Characterization of Human Lysozyme (Muramidase)-Releasing Cells

“…Thus, unless various purifica tion methods are used it must be born in mind that cultures normally thought to contain peripheral blood lymphocytes also contain other cell types. The tissue culture system employed in these experiments was optimal for Ig secretion [3,16,17,31]. Thus, it is probable that the use of a culture procedure deve loped for the maintenance of macrophages and mo nocytes [35] would have resulted in higher PFC values using antimuramidase Ig as a developing agent.…”

“…A similar kinetics for Ig secretion was recorded in control cultures containing purified protein deriva tive of tuberculin. The peak for Ig secretion using this and other mitogens in this culture system has been re ported to occur after approximately I week of culture [3,16,17,31]. AB serum and fetal calf serum were used as supplements and to some cultures the polyan ions dextran sulfate or sodium polyanethole sulfonate were added.…”

“…The effect of various lymphocyte-stimulatory agents such as phytohemagglutinin, pokeweed mito gen, purified protein derivative and Epstein-Barr vi rus, which have all been shown to activate human PBC to Ig secretion [3,16,17,31] was also tested. Since Epstein-Barr virus and C3 receptors have been shown to be associated [19,20], and since macro phages also carry C3 receptors [2], we found it rele vant to study this agent.…”

“…Since dextran sulfate has been re ported to stimulate phagocytic activity in bone mar row cell cultures [15] this and another polyanion, so dium polyanethole sulfonate, were added to some cul tures. Furthermore, since dextran sulfate, under cer tain circumstances, has been shown to induce the for mation of false plaques in the presence of AB serum, but not in the presence of fetal calf serum [17], these polyanions were only added to cultures supplemented with fetal calf serum.…”

“…The tissue culture system employed was the one found optimal for immunoglobulin secretion detected as PFC in the protein A plaque assay [3,16,17,31]. AB serum of fe tal calf serum in a final concentration of 10% was used as supplement.…”