Activation/suppression of the immune response in vitro by antigen and dextran sulfate I. Analysis by limiting dilution methods

Abstract

Spleen cells of normal mice which were stimulated with either T-dependent or T-independent antigens and dextran sulfate (DxS) in microcultures at limiting dilution show at low cell densities an increased frequency of responding units (B cells) with both types of antigens. At higher cell densities, DxS induces a suppression of the immune response as assessed by a declining frequency of expressed B cells. When T cells were decreased in number, the stimulating effect of DxS on T-independent responses remained unaltered while the suppressive effect (dependent on cell density) was eliminated. It is suggested that DxS lowers the threshold of activation for regulatory T cells, i.e. of cells which under standard in vitro conditions remain unactivated, and which in the presence of DxS become activated. We conclude that the regulatory cells are rare and that they are not present in most low cell density cultures. The net effect is a potent activation in dilute cultures and lack of response in dense cultures.

Regulation by CD25+ lymphocytes of autoantigen-specific T-cell responses in Goodpasture's (anti-GBM) disease

“…This nonlinear relationship is thought to be due to cell types with opposing effects interacting in the culture systems. The decrease in responding wells is thought to be due to a population of regulatory cells, inhibiting the effectors [17,19,21]. The model that best fits the experimental data [21,22] interprets the nonlinear, multiple-hit kinetics as being due to two cell populations possibly competing for growth factors, one responding, the other regulating at low cell numbers and responding at higher cell numbers.…”

“…When the logarithm of the proportion of nonresponding wells is plotted against the responder cell number, a straight-line relationship passing through the origin results. However, in cases where mixed populations of responders are utilized, analysis of LDA curves may reveal a nonlinear relationship between the fraction of nonresponding wells and cell number, typically with a zigzag pattern [16][17][18][19][20]. This nonlinear relationship is thought to be due to cell types with opposing effects interacting in the culture systems.…”

“…A straight line through the origin indicates single-hit kinetics, suggesting a solitary population of responding cells. However, in cases where mixed populations of responders are present, LDA curves reveal a nonlinear relationship typically with a zigzag pattern [16][17][18][19][20]. In the patients with acute disease (N ϭ 4), the response to CS-GBM or purified ␣3(IV)NC1 demonstrated predominantly straight lines (Fig.…”

Proteome analysis of activated murine B-lymphocytes

“…From our earlier work [10,11] and from the literature [44,45] we know that at the end of the 5-day mitogenic stimulation of spleen cells only a negligible contaminating cell population is of non-B-cell origin. From our earlier work [10,11] and from the literature [44,45] we know that at the end of the 5-day mitogenic stimulation of spleen cells only a negligible contaminating cell population is of non-B-cell origin.…”

“…(IMDM) supplemented with 20% fetal bovine serum (FBS), L-alanyl-L-glutamine (2 mM Glutamax-I), 1% minimal essential medium (MEM) nonessential amino acids, 100 mg/mL kanamycin (all from Gibco, BRL, Grand Island, NY, USA) and 2-mercaptoethanol (5´10 ±5 M) and stimulated with lipopolysaccharide (LPS, from Escherichia coli, 50 mg/mL; Difco, Detroit, MI, USA) and 5 mg/mL dextran sulfate for five days [10,11]. During the last 18 h of culture, radiolabeling was performed, if required, using [ 35 S]methionine (Cat.…”