Assessing B cell diversification by antigen receptor and precursor cell analysis

Abstract

A major element in the understanding of B cell specificity diversification is the extent of diversity present in mature and in developing B cell populations. Two general methods are currently used for assessing the specificity repertoire: (a) the enumeration of cells whose receptors can bind a specific antigen, and (b) the enumeration of cells which can respond to antigenic stimulation by antibody-forming cell clone production. Our laboratory has utilized the latter method to establish the frequency of B cells responsive to a wide variety of antigenic determinants. The findings indicate that: (a) the primary murine B cell specificity repertoire probably includes more than 10(7) clonotypes; (b) some clonotypes are represented by numerous B cells (40,000 TEPC 15 precursors per BALB/c mouse) while most are represented by fewer than to B cells per mouse; (c) the acquisition of the repertoire is apparently antigen-independent since germfree mice have repertoires similar to conventional mice and secondary B cells are easily distinguished from primary B cells; (d) the neonatal repertoire appears to contain only 10(4) clonotypes at birth, each represented by perhaps 200-400 cells; (e) the diversification process from neonatal to adult repertorie appears highly ordered and reproducible. Antigen binding cell studies have now used in conjunction with the splenic focus assay in an attempt to correlate these two techniques. The results indicate that the efficiency of the splenic focus assay used for precursor cell anlysis it 4-5% for both primary and secondary B cells and is similar to the percent of donor B cells lodged in recipient spleens. For certain antigens (DNP-BSA) the number of antigen-binding cells can represent 4% of the total B cells, and this number directly correlates with the concentration of antigen used; stimulation, on the other hand, appears to have an affinity threshold achieved by only 0,02% of the DNP-specific B cells. In contrast, PC-BSA antigen-binding cell and splenic focus precursor cell frequencies are identical. These findings are interpreted to indicate that antigen-binding cell analyses confirm the validity of the calculations used to estimate precursor frequencies in the splenic focus technique. However, for some antigens binding to cell receptors, one detects a large number of cells belonging either to a nonstimulatable B cell subclass or whose receptor affinity is too low to permit stimulation.