Human immunoglobulin (Ig)M+IgD+ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells

Abstract

Immunoglobulin (Ig)M+IgD+ B cells are generally assumed to represent antigen-inexperienced, naive B cells expressing variable (V) region genes without somatic mutations. We report here that human IgM+IgD+ peripheral blood (PB) B cells expressing the CD27 cell surface antigen carry mutated V genes, in contrast to CD27-negative IgM+IgD+ B cells. IgM+IgD+CD27(+) B cells resemble class-switched and IgM-only memory cells in terms of cell phenotype, and comprise approximately 15% of PB B lymphocytes in healthy adults. Moreover, a very small population (<1% of PB B cells) of highly mutated IgD-only B cells was detected, which likely represent the PB counterpart of IgD-only tonsillar germinal center and plasma cells. Overall, the B cell pool in the PB of adults consists of approximately 40% mutated memory B cells and 60% unmutated, naive IgD+CD27(-) B cells (including CD5(+) B cells). In the somatically mutated B cells, VH region genes carry a two- to threefold higher load of somatic mutation than rearranged Vkappa genes. This might be due to an intrinsically lower mutation rate in kappa light chain genes compared with heavy chain genes and/or result from kappa light chain gene rearrangements in GC B cells. A common feature of the somatically mutated B cell subsets is the expression of the CD27 cell surface antigen which therefore may represent a general marker for memory B cells in humans.

Figure 1

Fluorescence analysis of IgD⁺ PB B cell subsets with regard to the expression of CD23, CD5, and IgM. CD19⁺ PB B cells enriched by magnetic cell separation (99% purity) were stained with FITC- or PE-conjugated anti-CD27, anti-IgD–CyChrome, and anti-CD23–FITC, anti-CD5–FITC, or anti-IgM–PE. (Top) Windows were set around the IgD⁺CD27⁺ and the IgD⁺CD27⁻ populations and analyzed for CD23, CD5, and IgM staining within the respective fractions (histograms).

Figure 2

Fluorescence analysis of B cells derived from the PB of a healthy adult. Anti-IgD/ anti-CD27 two-color staining of CD19⁺ PB B cells enriched by magnetic cell separation (>98% purity). Indicated are the gates set for sorting of single IgD⁺CD27⁺ and IgD⁺CD27⁻ cells.

Figure 3

Fluorescence analysis of PB B cells with regard to the expression of CD27. Anti-IgD/anti-IgM double-fluorescence analysis of CD19⁺ PB B cells enriched by magnetic cell separation (99% purity). (Top) A window is set around the IgD-only population analyzed for CD27 staining compared with all B cells (histograms). The sample depicted here showed an exceptionally high percentage of IgD-only cells, which is usually in the range of 0.5% of all PB B lymphocytes. In total, 10⁵ events were collected on a FACScan^®.

Figure 4

The PB B cell pool and its presumptive generation. The unmutated B cell compartment (CD27⁻) is distinguished into CD5-positive IgM⁺IgD⁺ cells, whose derivation is debated, and CD5-negative IgM⁺IgD⁺, bone marrow–derived, naive B cells. Whereas the CD5⁺ B cells usually do not participate in T cell–dependent immune responses, naive B cells upon antigen encounter are activated and establish GC. In the course of the GC reaction, somatically mutated class-switched, IgM-only, and IgM⁺IgD⁺ cells, all of which express the CD27 membrane antigen, are generated. After selection, they leave the GC as memory B cells. In some GC, IgD-only B cells are generated, which carry an exceptionally high load of somatic mutation. Indicated are the percentages of the respective populations among all PB B lymphocytes.