Mechanisms of clonal abortion tolerogenesis. I. Response of immature hapten- specific B lymphocytes

Abstract

B lymphocytes with receptors specific for the hapten fluorescein (FLU) were prepared from the spleens of mice of various ages. For most experiments, a one-step fractionation procedure based on the adherence of FLU-specific cells to FLU-gelatin was used. For some experiments, a subset of higher FLU-binding capacity was prepared from the FLU-gelatin binding population through the use of the fluorescence-activated cell sorter (FACS). FLU-specific B cells were placed into microculture with either FLU(3.6)-human gamma globulin (FLU(3.6)HGG) or FLU(12)HGG usually for 24 h at 37 degrees C. The tolerogen was then removed and 0.1 μg/ml of a T-independent antigen, FLU-polymerized flagellin, was substituted. 3 days later, cells were harvested from the microcultures and assayed for FLU-specific plaque-forming cells to determine any reduction in clonable hapten-specific B cells which the tolerogenesis treatment might have induced. The results showed that with FLU(3.6)HGG, hapten-specific newborn B cells could be tolerized at 1,000-fold lower tolerogen concentrations than adult splenic B cells of equal antigen-binding capacity. The high-avidity subset was even more susceptible to tolerance induction. Tolerance could be induced within 8 but not within 2 h, and at lower tolerogen concentrations, longer periods of tolerogenesis were required for a given effect. Using a 24-h tolerogenesis phase, 50 percent reduction in clone frequency among newborn FLU-gelatin fractionated cells was achieved at 0.08 μg/ml of FLU(3.6)HGG. Tolerance induction in immature B cells was inhibited by the concomitant presence of a polyclonal B-cell activator, Escherichia coli lipopolysaccharide (LPS) but tolerance once induced, was stable to challenge with LPS. Tolerogenesis was hapten specific. The proportion of tolerizable cells in spleens decreased with increasing age, reaching 50 percent at around 9 days. FLUI(12)HGG proved a more powerful tolerogen than FLU(3.6)HGG. It had an effect on adult cells, 50 percent reduction in clone frequency being noted at around 1 μg/ml. However, and in contrast to results claimed for other T- independent systems, there still was a major difference between immature and mature B cells, the immature cells displaying much greater sensitivity to tolerogenesis.

CpG DNA stimulates autoreactive immature B cells in the bone marrow

“…Results from several early experiments have shown that immature B cells that are isolated from neonates or adult spleens or grown in BM cultures, respond to stimulation with LPS by proliferation and antibody production [22][23][24][25][26]. These findings suggest that immature B cells are responsive to mitogenic stimuli.…”

“…The in vitro and the ex vivo data obtained from our experiments suggest that these nontolerant immature B cells can be driven to secrete autoantibodies in response to stimulation with CpG DNA and with LPS (data not shown). Although LPS has been shown to activate immature B cells to secrete IgM antibodies [22][23][24][25][26], it is now clear that a significant proportion of these cells are autoreactive. Our results, therefore, suggest that developing B cells in the BM are responsive to TLR stimuli and that such activation is physiologically relevant.…”

Lipopolysaccharide prevents apoptosis and induces responsiveness to antigen receptor cross‐linking in immature B cells

“…2,5 Since LPS has been shown to interfere with B-cell tolerance induction, [11][12][13] we examined its ability to overcome the unresponsiveness of immature B cells to BCR cross-linking in vitro. Immature B cells (purified from the spleens of 2-3-dayold mice) and mature B cells (from 8-10-week-old mice) were cultured for 48 hr with no stimulus, 50 m g/ml of goat anti-m F(ab') 2 fragments, 50 m g/ml of LPS, or anti-m plus LPS.…”

“…For example, unresponsiveness induced in adult animals by polysaccharides or deaggregated proteins can be overcome by administration of LPS within several days of exposure to the tolerogen. 11,12 In addition, LPS can prevent clonal deletion of immature B cells in response to antigen 13 and growth inhibition of immature B-cell lines in response to antiimmunoglobulin stimulation. 14 Finally, in transgenic models of Bcell clonal anergy, LPS stimulation has been shown to partially overcome the anergic state.…”

The Rearranged VH Domain of a Physiologically Selected Anti-Single-Stranded DNA Antibody as a Precursor for Formation of IgM and IgG Antibodies to Diverse Antigens

“…Processes that occur during B cell development shape that balance and give rise to a very nonrandom repertoire of new B cells even before exposure to foreign Ag. The best defined of the early processes are those that test for effective association of a -chain with pseudo L chain (1,2) and those that abrogate potentially damaging autoreactivity by cell deletion, anergy, or receptor editing (3)(4)(5)(6)(7)(8).…”