Transitional B cell subsets in human bone marrow

Abstract

B cells originate from precursors in the bone marrow, and the first cells which migrate to the peripheral blood have been classified as 'transitional B cells'. Transitional B cells have been characterized in human blood with stage 1 (T1) and stage 2 (T2) subsets being proposed. In the present study, 27 normal human bone marrow samples were analysed for transitional B cell markers by eight-colour flow cytometry. T1 transitional B cells (CD45(+)CD19(+)CD10(+)IgM(+)IgD(lo)) and T2 transitional B cells (CD45(+)CD19(+)CD10(+)IgM(+)IgD(+)) were identified in normal bone marrow samples at a mean frequency of 3·2 and 3·1% of total B lineage cells, respectively. A majority of the bone marrow transitional B cells were CD24(hi)CD38(hi) , the phenotype of blood transitional B cells. Consistent with recent peripheral blood data, T2 B cells had a significantly higher CD21 expression compared with T1 B cells (72·4 versus 40·9%) in the bone marrow. These data raise the possibility that transitional B cells are capable of differentiating from T1 to T2 B cells within the bone marrow. Furthermore, transitional cells at either stages 1 or 2 might be capable of migrating out of the bone marrow.

Keywords: CD21; bone marrow; flow cytometry; human; transitional B cells.

Fig. 1

Identification of transitional B cell subsets in normal human bone marrow (BM). The dot-plots presented display the flow cytometric approach used to identify various B cell subsets in a representative sample. Each B cell subset was assigned a particular colour. (a) Phenotypic criteria for the characterization of B lineage subsets (b) CD19⁺ cells with low side-scatter (SSC) were gated as B cells. (b) All BM cells except those with high SSC. In (c–f), only B cells (gated in b) are shown. (c) Pre-B I cells (P1, red) were identified as CD45^loCD10^hi, and pre-B II cells (P2, yellow) were identified as CD45^intCD10⁺. (d) CD10⁺ B cells were gated as shown. The CD19^– T and natural killer (NK) cells (not shown) were used as a negative control to appropriately draw the CD10⁺ gate. (e) Identification of B cell subsets. The most immature B cells [CD19⁺CD10⁺ immunoglobulin (Ig)D^–IgM^–] were used as a negative control to define the IgD⁺ and IgM⁺ cell populations. Gates were placed to identify the IgD^–, IgD^lo and IgD⁺ cell populations; B cells with IgD intensities higher than pre-B cells (CD45^lo) and lower than mature B cells (CD10^–) were classified as low IgD intensity. (f) This is a duplicate of (e); however, only the CD10⁺IgM⁺ cells are shown. (g) The gating algorithms employed to derive B cell subsets are shown. Pre-B I cells (P1, red) were identified as CD45^loCD10^hi and pre-B II cells (P2, yellow) were identified as CD45^intCD10⁺. All other subsets were CD45^bright. Immature (P3, purple) B cells were CD10⁺ and IgD^–; T1 (dark blue) B cells were IgD^lo and CD10⁺; T2 (light green) B cells were IgD⁺ and CD10⁺. Mature CD10^– B cells were divided into IgD⁺ cells (maroon; naive and natural effector B cells), and IgD^– cells (light blue; IgM only, and switched memory B cells).

Fig. 2

Enumeration of B cell subsets in normal bone marrow (BM). The mean [± standard error of the mean (s.e.m.)] frequencies of various B cell subsets in the normal BM are presented in this chart (n = 27). The means (± s.e.m.) of each subset are as follows: pre-B I (4·7 ± 0·5%), pre-B II (28·7 ± 3·3%), immature (4 ± 0·6%), T1 (3·2 ± 0·3%), T2 (3·1 ± 0·4%), IgD⁺ mature B cells (naive and natural effector) (45·2 ± 3·6%) and IgD^– mature B cells (IgM only, and switched memory) (10·2 ± 1·1%).

Fig. 3

Bright CD24 and CD38 co-expression on bone marrow (BM) transitional B cell subsets. (a) A representative normal BM sample is presented here, and only transitional B cell subsets are displayed [T1 (blue; CD10⁺IgM⁺IgD^lo) and T2 (green; CD10⁺IgM⁺IgD⁺)]. (b) This chart displays the proportion of CD24^hiCD38^hi cells in the T1 and T2 B cell populations. The mean (± s.e.m.) frequency of T1 and T2 B cells that were CD24^hiCD38^hi in the normal BM was 85·2% (± 2·7) and 69% (± 4·1), respectively (n = 19). The lines join the two values in the same sample. CD24 and CD38 expression is significantly lower in the T2 B cells (P < 0·001).

Fig. 4

CD21 expression on bone marrow (BM) transitional B cell subsets. (a) A representative normal BM sample is presented here, and only transitional B cell subsets are displayed [T1 (blue; CD10⁺IgM⁺IgD^lo) and T2 (green; CD10⁺IgM⁺IgD⁺)]. (b) This chart displays the proportion of CD21⁺ cells in the T1 and T2 B cell populations. The means [± standard error of the mean (s.e.m.)] of CD21⁺ T1 and CD21⁺ T2 B cells in the normal BM were 40·9% (± 4·2) and 72·4% (± 3·4), respectively (n = 25). A higher percentage of T2 B cells express CD21 compared with T1 B cells (P < 0·0001).

Fig. 5

CD5 expression on bone marrow (BM) transitional B cell subsets. (a) A representative normal BM sample is presented here, and only transitional B cell subsets are displayed [T1 (blue; CD10⁺IgM⁺IgD^lo) and T2 (green; CD10⁺IgM⁺IgD⁺)]. (b) This chart illustrates CD5 expression on T1 and T2 B cells. The mean [± standard error of the mean (s.e.m.)] of CD5⁺ T1 and CD5⁺ T2 B cells in the normal BM was 43·1% (± 2·8) and 58·2% (± 3·6), respectively (n = 20). CD5 expression is significantly higher on T2 cells than T1 cells (P < 0·001).