IN-SITU STUDIES OF THE PRIMARY IMMUNE-RESPONSE TO (4-HYDROXY-3-NITROPHENYL)ACETYL .4. AFFINITY-DEPENDENT, ANTIGEN-DRIVEN B-CELL APOPTOSIS IN GERMINAL-CENTERS AS A MECHANISM FOR MAINTAINING SELF-TOLERANCE

Germinal centers (GCs) are the sites of antigen-driven V(D)J gene hypermutation and selection necessary for the generation of high affinity memory B lymphocytes. Despite the antigen dependence of this reaction, injection of soluble antigen during an established primary immune response induces massive apoptotic death in GC B cells, but not in clonally related populations of nonfollicular B lymphoblasts and plasmacytes. Cell death in GCs occurs predominantly among light zone centrocytes, is antigen specific, and peaks within 4-8 h after injection. Antigen-induced programmed death does not involve cellular interactions mediated by CD40 ligand (CD40L) or Fas; disruption of GCs by antibody specific for CD40L was not driven by apoptosis and C57BL/6.lpr mice, though unable to express the Fas death trigger, remained fully susceptible to soluble antigen. Single injections of antigen did not significantly decrease GC numbers or average size, but repeated injections during an 18-h period resulted in fewer and substantially smaller GCs. As cell loss appeared most extensive in the light zone, decreased GC cellularity after prolonged exposure to soluble antigen implies that the Ig(-) centroblasts of the dark zone may require replenishment from light zone cells that have survived antigenic selection. GC cell death is avidity-dependent; oligovalent antigen induced relatively little apoptosis and GC B cells that survived long exposures to multivalent antigen expressed atypical VDJ rearrangements unlikely to encode high affinity antibody. Antigen-induced apoptotic death in GCs may represent a mechanism for the peripheral deletion of autoreactive B cell mutants much as the combinatorial repertoire of immature B lymphocytes is censored in the bone marrow.