bcl-2 transgene expression inhibits apoptosis in the germinal center and reveals differences in the selection of memory B cells and bone marrow antibody-forming cells

Abstract

Immunization with T cell-dependent antigens generates long-lived memory B cells and antibody-forming cells (AFCs). Both populations originate in germinal centers and, predominantly, produce antibodies with high affinity for antigen. The means by which germinal center B cells are recruited into these populations remains unclear. We have examined affinity maturation of antigen-specific B cells in mice expressing the cell death inhibitor bcl-2 as a transgene. Such mice had reduced apoptosis in germinal centers and an excessive number of memory B cells with a low frequency of V gene somatic mutation, including those mutations encoding amino acid exchanges known to enhance affinity. Despite the frequency of AFCs being increased in bcl-2-transgenic mice, the fraction secreting high-affinity antibody in the bone marrow at day 42 remained unchanged compared with controls. The inability of BCL-2 to alter selection of bone marrow AFCs is consistent with these cells being selected within the germinal center on the basis of their affinity being above some threshold rather than their survival being due to a selective competition for an antigen-based signal. Continuous competition for antigen does, however, explain formation of the memory compartment.

Figure 1

Levels of CD38 on antigen-specific IgG1 B cells at day 42 of the primary response. (A) Spleens from mice immunized 42 d previously with 100 μg i.p. of NP–KLH were stained with the indicated antibodies and analyzed by flow cytometry. Viable cells having the phenotype IgM⁻IgD⁻ were electronically gated on (rectangle) and examined for expression of IgG1 and the ability to bind the immunizing hapten NP coupled to a fluorescent protein. Such double-positive cells were gated on (rectangle), and the level of CD38 was determined. This result is depicted as the solid histogram in this figure. Negative control staining was less than the fluorescence level marked by the dashed vertical line. The percentages of NP-binding IgG1⁺ cells expressing high levels of CD38 are indicated. Cells used in this experiment were pooled from three mice, and the data shown are representative of three experiments. (B) Germinal center cells in bcl-2–transgenic mice downregulate CD38. Mesenteric LN B cells were gated on by expression of CD45R. These cells were then examined for CD38 levels and binding of the lectin peanut agglutinin; such B cells are boxed.

Figure 1

Levels of CD38 on antigen-specific IgG1 B cells at day 42 of the primary response. (A) Spleens from mice immunized 42 d previously with 100 μg i.p. of NP–KLH were stained with the indicated antibodies and analyzed by flow cytometry. Viable cells having the phenotype IgM⁻IgD⁻ were electronically gated on (rectangle) and examined for expression of IgG1 and the ability to bind the immunizing hapten NP coupled to a fluorescent protein. Such double-positive cells were gated on (rectangle), and the level of CD38 was determined. This result is depicted as the solid histogram in this figure. Negative control staining was less than the fluorescence level marked by the dashed vertical line. The percentages of NP-binding IgG1⁺ cells expressing high levels of CD38 are indicated. Cells used in this experiment were pooled from three mice, and the data shown are representative of three experiments. (B) Germinal center cells in bcl-2–transgenic mice downregulate CD38. Mesenteric LN B cells were gated on by expression of CD45R. These cells were then examined for CD38 levels and binding of the lectin peanut agglutinin; such B cells are boxed.

Figure 2

Reduced frequency of V_H gene somatic mutations in NP-specific memory B cells from bcl-2–transgenic mice. The frequency of mutations in V_H186.2 genes from single, antigen-specific CD38⁺IgG1⁺ B cells was determined by nucleotide sequencing. Single cells were sorted from a pooled spleen preparation from each mouse strain using the criteria depicted in Fig. 1. All recovered sequences showed unique CDR3 junctions, indicating clonality. (B) Cumulative distribution of somatic mutations in control and bcl-2 NP-specific B cells. In both components of the figure, the number of sequences containing mutations giving rise to a tryptophan→leucine exchange at amino acid 33 (numbered according to reference 24) is depicted by the hatched segment of each column. Details of the sequences are summarized in Table . These sequence data are available from EMBL/GenBank/DDBJ under accession no. AF210258-AF210307.

Figure 2

Reduced frequency of V_H gene somatic mutations in NP-specific memory B cells from bcl-2–transgenic mice. The frequency of mutations in V_H186.2 genes from single, antigen-specific CD38⁺IgG1⁺ B cells was determined by nucleotide sequencing. Single cells were sorted from a pooled spleen preparation from each mouse strain using the criteria depicted in Fig. 1. All recovered sequences showed unique CDR3 junctions, indicating clonality. (B) Cumulative distribution of somatic mutations in control and bcl-2 NP-specific B cells. In both components of the figure, the number of sequences containing mutations giving rise to a tryptophan→leucine exchange at amino acid 33 (numbered according to reference 24) is depicted by the hatched segment of each column. Details of the sequences are summarized in Table . These sequence data are available from EMBL/GenBank/DDBJ under accession no. AF210258-AF210307.

Figure 3

The frequency of apoptotic cells in germinal centers is reduced in bcl-2–transgenic mice. 2 wk after immunization, LNs were sectioned and stained for the presence of apoptotic cells using the TUNEL technique. An example of one such experiment using LNs from immunized and unimmunized control and bcl-2–transgenic mice is shown. Arrows indicate the location of germinal centers. No germinal centers were observed in the LNs of unimmunized mice. Magnification is 100, and one representative section is shown. Quantification of this data is presented in Table .

Figure 4

Selection in the germinal center during the development of memory and AFCs in a primary immune response. A schematic representation of the forces acting to influence the outcome of the germinal center reaction during the primary immune response. Indicted are the fact that (a) high-affinity cells selectively become AFCs; (b) low-affinity cells apoptose, an event that can be blocked by BCL-2; and (c) intermediate affinity cells remain in the germinal center. As the response progresses, the affinity threshold for each differentiation pathway will increase as the cells compete with antibody in serum. It is predicted that this reaches a level where memory B cell production will be the favored outcome. This change may well involve FcγRII.