Memory B lymphocytes migrate to bone marrow in humans

Abstract

IgM-bearing B lymphocytes with mature phenotype (CD10- CD24(lo) IgD+) are acquired after birth in the bone marrow of humans. These B cells are defined here as relatively large, nondividing lymphocytes, variable proportions of which express cell surface molecules indicative of relatively recent activation. Analysis of V(H)5(2) (heavy chain variable region) gene transcripts indicated point mutations throughout the Ig variable region from the mature IgM+ B population but not from the immature B cells in the bone marrow. The mutations were concentrated in the complementarity determining regions, and amino acid substitutions were favored over silent mutations, findings indicative of antigen selection within germinal centers in peripheral lymphoid tissues. The V(H) sequence analysis also revealed the existence of clonal relatives in individual bone marrow samples. These antigen-experienced lymphocytes did not secrete Ig spontaneously but could be induced to do so in vitro. The data suggest that a subpopulation of memory B lymphocytes generated during antigen responses recirculates to the bone marrow in humans.

Figure 1

The CD10⁻ IgM⁺ cells in the bone marrow are relatively large, nondividing cells that accumulate with age. (A) Representative dot plot of bone marrow lymphocytes stained with anti-IgM and anti-CD10; 5000 events are displayed. (B) Analysis of the frequency of CD10⁻ IgM⁺ cells in bone marrow as a function of age, expressed as proportion of total IgM⁺ bone marrow lymphocytes. Each dot represents a separate tissue sample. (C) Relative cell size of CD10⁻ IgM⁺ lymphocytes (solid line) and CD10⁺ IgM⁺ lymphocytes (dotted line) as estimated by forward light scatter; 5000 events are displayed. (D) Flow cytometric analysis of DNA content of CD10⁻ IgM⁺ lymphocytes in adult bone marrow; 10,000 events are displayed.

Figure 2

Phenotypic characterization of the CD10⁻ IgM⁺ bone marrow population. Bone marrow cells were stained with a panel of mAbs to activation and B-lineage markers. Lymphocytes, gated on the basis of forward and side light scatter and CD10⁻ IgM⁺ immunofluorescence characteristics, were analyzed; 5000 events are displayed. Dashed, open histograms represent background fluorescence, and solid histograms represent staining, with the activation and B-lineage markers indicated. The bone marrow donor was 46 years old.

Figure 3

V_H5₂-containing variable region [V(D)J_H] genes in the mature subpopulation of B cells within the bone marrow are frequently mutated. Diagrammatic representation of cloned sequences from CD10⁻ IgM⁺ (A) and CD10⁺ IgM⁺ (B) bone marrow B cells. For each clone, the line represents the deduced germ line V5-51 amino acid sequence. Solid boxes represent replacement amino acid mutations, and open boxes represent silent amino acid mutations. The J region gene of each V region clone is indicated; subscripts A, B, and D designate alleles.

Figure 4

Related clones of CD10⁻ IgM⁺ bone marrow cells in individual donors. Sequence comparison of the V5-51, J_H4_B, and J_H5_B germ line sequences with clones isolated from sorted CD10⁻ IgM⁺ bone marrow cells. Each set of related clones was isolated from an individual bone marrow donor.