MECHANISMS OF CLONAL ABORTION TOLEROGENESIS .3. ANTIGEN ABROGATES FUNCTIONAL MATURATION OF SURFACE IMMUNOGLOBULIN-NEGATIVE ADULT BONE-MARROW LYMPHOCYTES

The fluorescence-activated cell sorter (FACS) was used to fractionate adult CBA mouse bone marrow cells on the basis of their size and amount of surface immunoglobulin (sIg) expressed. A population of viable, small sIg- cells could generate up to 28% sIg+ cells within 24 h in tissue culture. Capacity of various cell fractions to respond to T[thymus-derived cell]-independent stimulation by antibody production was studied. Fractionated cells were placed into microculture at limiting dilution in the presence of thymus filler cells, stimulated with Escheria coli lipopolysaccharide (LPS) and after 3-5 days, assayed for presence of antibody-forming cell clones to the hapten (4-hydroxy-3-iodo-5-nitrophenyl) acetyl (NIP). Through appropriate statistical means, the number of anti-NIP precursors originally present was calculated. While the sIg+ fraction contained approximately 70 cells in 106 capable of immediate development into anti-NIP clones, the sIg- fraction contained very few reactive cells. The cloning capacity of this fraction increased markedly following 24 h of maturation in tissue culture. When bone marrow cells were exposed to NIP-coupled human .gamma.-globulin (NIP-HGG) acting as a B [bone marrow-derived] cell tolerogen, some tolerance was induced in vitro reflecting the relative immaturity of these cells. When the FACS-sorted sIg- cells were similarly treated, there was virtually complete abrogation of their functional maturation into clonable anti-NIP precursors. The effect of LPS on cells converting from the sIg- to sIg+ status was investigated. When LPS and NIP-HGG were concomitantly present, about 1/3 of the cells escaped tolerance induction. LPS thus rescued some cells form clonal abortion, but did not totally abolish tolerance induction. The significance of these results for the clonal abortion theory of B lymphocyte tolerance was briefly discussed. Cells converting from the sIg- to sIg+ status in the normal course of differentiation represent a prime target for tolerance induction.