The point mutations accumulate in a stepwise manner as the descendants of each B cell proliferate in the germinal center to form B-cell clones (Fig. 10.14). An altered receptor can affect the ability of a B cell to bind antigen and thus will affect the fate of the B cell in the germinal center. Most mutations have a negative impact on the ability of the B-cell receptor to bind the original antigen, by preventing the correct folding of the immunoglobulin molecule or by blocking the complementarity-determining regions from binding antigen. Detrimental mutations may alter conserved framework regions (see Fig. 4.7) and disrupt basic immunoglobulin structure. Cells that harbor such detrimental mutations are eliminated by apoptosis in a process of negative selection, either because they can no longer make a functional B-cell receptor or because they cannot take up antigen as well as sibling B cells (Fig. 10.15). Germinal centers are filled with apoptotic B cells that are quickly engulfed by macrophages, giving rise to the characteristic tingible body macrophages. These contain dark-staining nuclear debris in their cytoplasm. Negative selection is implied by the relative scarcity of amino acid replacements in the framework regions, reflecting the loss of cells that had mutated any one of the many residues that are critical for immunoglobulin V-region folding. This process prevents rapidly dividing B cells from expanding to numbers that would overwhelm the lymphoid tissues. Less frequently, mutations may improve the affinity of a B-cell receptor for antigen, and these mutations will be selectively expanded (see Fig. 10.15) because the cells expressing receptors with such mutations will have an increased survival rate compared with low-affinity cells. Positive selection is evident in the accumulation of numerous amino acid replacements in the complementarity-determining regions, which determine antibody specificity
Fig. 10.14 Somatic hypermutation introduces mutations into the rearranged immunoglobulin variable (V) regions that improve antigen binding. The process of somatic hypermutation can be tracked by sequencing immunoglobulin V regions from hybridomas (clones of antibody-producing cells; see Appendix I, Section A-7) established at different time points after the experimental immunization of mice. The result of one experiment is depicted here. Each V region sequenced is represented by a horizontal line. The complementarity-determining regions CDR1, CDR2, and CDR3 are shown by pink shading. Mutations that change the amino acid sequence are represented by red bars. Within a few days of immunization, the V regions within a particular clone of responding B cells begin to acquire mutations, and over the course of the next week more mutations accumulate (top panels). B cells whose V regions have accumulated deleterious mutations and can no longer bind antigen die. B cells whose V regions have acquired mutations that improve the affinity of the B-cell receptor for antigen are able to compete more effectively for antigen, and receive signals that drive their proliferation and expansion. The antibodies they produce also have this improved affinity. This process of mutation and selection can continue in the lymph node germinal center through multiple cycles in response to secondary and tertiary immune responses elicited by further immunization with the same antigen (center and bottom panels). In this way, the antigen-binding efficiency of the antibody response is improved over time.
and affinity (see Fig. 10.14), a process we discuss in the next section. The result of selection for enhanced binding to antigen is that the nucleotide changes that alter amino acid sequences, and thus protein structure, tend to be clustered in the CDRs of the immunoglobulin V-region genes, whereas silent, or neutral, mutations that preserve amino acid sequence and do not alter protein structure are scattered throughout the V region.