Soluble antigen abrogates the appearance of anti-protein IgG1-forming cell precursors during primary immunization.

Abstract

The anti-human serum albumin (HSA) B-cell repertoire of C57BL/6 mice was examined by culturing splenocytes at limiting dilution following polyclonal stimulation with Escherichia coli lipopolysaccharide and a lymphokine mixture. The frequency of anti-HSA precursors was determined before and after immunization with alum-precipitated HSA and 109 killed Bordetella pertussis organisms, by submitting clonal supernatants to an ELISA. Anti-HSA IgGl-forming precursors were rare in unimmunized spleens, representing 1 in 500,000 splenocytes or only -100 cells per spleen. Between day 5 and day 7 after immunization, this figpre increased to -20,000 cells per spleen. Over the following 3 weeks, there was a progressive increase in the mean optical density generated in the clonal ELISA, presumably due to affmity maturation of the B-cell population. When freshly deaggregated HSA was injected before or even up to 4 days after challenge immunization, the appearance of anti-HSA IgGl-forming cell precursors was largely prevented. The effect was most marked with 5 mg or 1 mg of soluble HSA, but impressive partial effects could be seen with as little as 10 ,ug of HSA if administered before challenge immunization. Most of the few clones seen after the higher doses of the toleragen appeared to make antibody of low affinity. The capacity to influence the B-cell pool by soluble antigen administered just 1-2 days before the sudden appearance of IgG1 precursors argues against the totality of the effect being due to T-cell-mediated suppression and in favor of a direct effect on B cells.Previous work from this laboratory (1-3) has shown that the preimmune B-cell repertoire of murine spleen contains surprisingly few cells with sufficient affinity for protein antigens (1, 2) or syngeneic intracellular structures (3) to bind in an ELISA when IgG1 antibody is the clonal product. Five days after challenge immunization with protein antigens and adjuvants, there is a sudden burst of appearance of IgG1 antibody-forming cell precursors (AFCP), most of which had already undergone an isotype switch in vivo (2) and were surface IgM-negative IgG1-positive cells. The relationship between these newly appearing B cells and the anti-protein IgM AFCP noted in unimmunized mice is not clear, but the former are unlikely, on kinetic grounds, to be the direct, unmutated progeny of the latter. Linton et al. (4) (Sigma). Some groups of mice were serially bled from the orbital venous plexus, and serum samples were prepared for antibody assay.Cell Preparation and Culture. Five thousand 3T3 BALB/c fibroblasts (Commonwealth Serum Laboratories, Melbourne, Australia) were aliquoted into flat bottomed 96-well microtiter trays in 100 A.l. Culture medium (CM) was RPMI 1640 containing 100 AsM 2-mercaptoethanol and 5% (vol/vol) fetal calf serum (Flow Laboratories). Spleen cell suspensions were prepared as described (6, 7) including 0.83% NH4CI for erythrocyte lysis followed by damaged and dead cell removal. Splenocytes were dispensed in 100 Al of CM containing Escheri...

Autoantibodies associated with lupus induced by diverse drugs target a similar epitope in the (H2A-H2B)-DNA complex.

“…Alternatively, it has been proposed that low molecular weight forms of self-macromolecules may be normally involved in maintenance of nonneonatal immune tolerance to self (49). This view is consistent with the demonstration by Nossal and Karvelas (50) (26) or of 50-60 bp after shorter digestion times in the presence of 3 M urea (52). If a similar process occurs in vivo, the resultant subnucleosome particles may suppress chromatin immunogenicity.…”

IgG antibody response to polyethylene glycol-modified adenosine deaminase in patients with adenosine deaminase deficiency.

“…An empirical approach (21) (details to be described elsewhere) was successful in inducing apparent tolerance to PEG-ADA in patient 8 mechanism that involved T cell suppression (24). On the other hand, the response in patient 8 resembles the experimental abrogation of primary immunization by high doses of soluble antigen, which may have resulted from direct effects on B cells (25). Although the use ofsteroids in a patient with SCID should be carried out with caution, the tolerizing regimen used in the present patients was much less intensive than that employed in hemophilia patients with high levels of inhibitory antibodies to Factor VIII:C, which included cyclophosphamide and plasmapheresis or protein A adsorption to remove circulating antibody (26).…”

Th2 Activities Induced During Virgin T Cell Priming in the Absence of IL-4, IL-13, and B Cells

“…Alum-precipitated (4-hydroxy-3-nitrophenyl)acetyl (NP) 3 conjugated to chicken ␥-globulin (CGG) was prepared as previously described (17,21). Adult mice (8 -12 wk) were injected into both rear footpads with 20 g alum-precipitated NP-CGG plus 5 ϫ 10 8 heat-killed Bordetella pertussis (Evans Medical, Liverpool, U.K.).…”