BAFF mediates survival of peripheral immature B lymphocytes

Abstract

B cell maturation is a very selective process that requires finely tuned differentiation and survival signals. B cell activation factor from the TNF family (BAFF) is a TNF family member that binds to B cells and potentiates B cell receptor (BCR)-mediated proliferation. A role for BAFF in B cell survival was suggested by the observation of reduced peripheral B cell numbers in mice treated with reagents blocking BAFF, and high Bcl-2 levels detected in B cells from BAFF transgenic (Tg) mice. We tested in vitro the survival effect of BAFF on lymphocytes derived from primary and secondary lymphoid organs. BAFF induced survival of a subset of splenic immature B cells, referred to as transitional type 2 (T2) B cells. BAFF treatment allowed T2 B cells to survive and differentiate into mature B cells in response to signals through the BCR. The T2 and the marginal zone (MZ) B cell compartments were particularly enlarged in BAFF Tg mice. Immature transitional B cells are targets for negative selection, a feature thought to promote self-tolerance. These findings support a model in which excessive BAFF-mediated survival of peripheral immature B cells contributes to the emergence and maturation of autoreactive B cells, skewed towards the MZ compartment. This work provides new clues on mechanisms regulating B cell maturation and tolerance.

Figure 1

Survival of BAFF-treated splenocytes in vitro. C57BL/6-derived splenocytes were cultured for 72 h with or without BAFF and analyzed by flow cytometry on FSC/SSC plots. Gates R1 (low FSC, dead cells) and R2 (high FSC, live cells) are indicated in each plot. (A) R1 and R2 populations in fresh and cultured splenocytes 72 h with or without BAFF. (B) The effect of rabbit anti-BAFF, control rabbit serum, or 2 μg/ml inactivated (boiled) BAFF on the size of the R2 population as indicated. The slight inhibition observed with control rabbit serum is due to the higher serum concentration in the culture and was similar to that of four other control rabbit sera tested in this assay (data not shown). These plots are representative of at least five experiments.

Figure 1

Survival of BAFF-treated splenocytes in vitro. C57BL/6-derived splenocytes were cultured for 72 h with or without BAFF and analyzed by flow cytometry on FSC/SSC plots. Gates R1 (low FSC, dead cells) and R2 (high FSC, live cells) are indicated in each plot. (A) R1 and R2 populations in fresh and cultured splenocytes 72 h with or without BAFF. (B) The effect of rabbit anti-BAFF, control rabbit serum, or 2 μg/ml inactivated (boiled) BAFF on the size of the R2 population as indicated. The slight inhibition observed with control rabbit serum is due to the higher serum concentration in the culture and was similar to that of four other control rabbit sera tested in this assay (data not shown). These plots are representative of at least five experiments.

Figure 2

BAFF specifically promotes the survival of splenic B cells. (A) Splenocytes from C57BL/6 mice were stained with antibodies to B220 and CD3. B220⁺ B cells and CD3⁺ T cells were purified by cell sorting and incubated for 72 h with BAFF (bottom) or without (top). The R1 (dead cells) and R2 (live cells) gates for sorted B cells (left) and sorted T cells (right) are drawn. (B) Lymphocytes were prepared from spleen, PLNs (inguinal and brachial LNs), MLNs, and PBLs, and incubated with BAFF (black bars) or without (white bars) for 72 h. Staining with annexin V/PI was analyzed by flow cytometry and percentages of annexin V/PI double-negative R2-gated cells (live cells) are shown for each lymphocyte preparation. The plots in A are a representation of three separate experiments and the graph in B represents the mean and standard deviation of six separate lymphocyte preparations. P values were obtained using analysis of variance (ANOVA).

Figure 2

BAFF specifically promotes the survival of splenic B cells. (A) Splenocytes from C57BL/6 mice were stained with antibodies to B220 and CD3. B220⁺ B cells and CD3⁺ T cells were purified by cell sorting and incubated for 72 h with BAFF (bottom) or without (top). The R1 (dead cells) and R2 (live cells) gates for sorted B cells (left) and sorted T cells (right) are drawn. (B) Lymphocytes were prepared from spleen, PLNs (inguinal and brachial LNs), MLNs, and PBLs, and incubated with BAFF (black bars) or without (white bars) for 72 h. Staining with annexin V/PI was analyzed by flow cytometry and percentages of annexin V/PI double-negative R2-gated cells (live cells) are shown for each lymphocyte preparation. The plots in A are a representation of three separate experiments and the graph in B represents the mean and standard deviation of six separate lymphocyte preparations. P values were obtained using analysis of variance (ANOVA).

Figure 3

BAFF induces survival of T2 B cells. (A) C57BL/6-derived splenocytes were incubated for 72 h with BAFF, stained with antibodies to B220, IgM, IgD, HSA, and L-selectin, and analyzed by flow cytometry. Freshly prepared C57BL/6 splenocytes were stained in parallel. For HSA and L-selectin expression, B220⁺ gated B cells are shown. Histograms for fresh splenocytes (black line) and R2-gated BAFF-treated splenocytes (gray line) are overlayed for comparison. (B) Three-color flow cytometric analysis of fresh and R2-gated BAFF-stimulated splenocytes from A. Cells were stained with antibodies to IgM, CD21, and CD23 and were separated into CD23⁻ and CD23⁺ cells. CD23⁻ cells include CD21^loIgM^hi T1 cells and CD21^hiIgM^hi MZ B cells. CD23⁺ B cells include CD21^intIgM^dull mature B cells and CD21^hiIgM^hi T2 B cells. T1, T2, mature, and MZ B cell populations are indicated. (C) Four-color flow cytometric analysis of fresh and R2-gated BAFF-stimulated splenocytes from A. Cells were stained with antibodies to IgM, CD21, CD23, and HSA. Freshly prepared splenocytes were gated on the T1, T2, and mature B cell populations as described in B and analyzed for the expression of HSA. The mean fluorescence intensity (MFI) for HSA on gated T1, T2, and mature B cells (black bars) is compared with that of R2-gated BAFF-stimulated splenocytes (white bar). Experiments in A, B, and C are representative of at least 10 animals and cultures analyzed. (D) Freshly prepared splenocytes were stained with antibodies to CD21 and HSA. CD21^loHSA^hi T1 and CD21^hiHSA^hi T2 B cells were identified (Before sort, top), sorted (After sort, middle), and cultured for 72 h with BAFF (bottom). Cells were then analyzed by flow cytometry for the presence of surviving cells in the R2 gate on FSC/SSC plots as indicated (bottom). R1 and R2 populations are shown. Percentages of cells in R2 are indicated. This figure is representative of six different cell sorting experiments analyzed.

Figure 3

BAFF induces survival of T2 B cells. (A) C57BL/6-derived splenocytes were incubated for 72 h with BAFF, stained with antibodies to B220, IgM, IgD, HSA, and L-selectin, and analyzed by flow cytometry. Freshly prepared C57BL/6 splenocytes were stained in parallel. For HSA and L-selectin expression, B220⁺ gated B cells are shown. Histograms for fresh splenocytes (black line) and R2-gated BAFF-treated splenocytes (gray line) are overlayed for comparison. (B) Three-color flow cytometric analysis of fresh and R2-gated BAFF-stimulated splenocytes from A. Cells were stained with antibodies to IgM, CD21, and CD23 and were separated into CD23⁻ and CD23⁺ cells. CD23⁻ cells include CD21^loIgM^hi T1 cells and CD21^hiIgM^hi MZ B cells. CD23⁺ B cells include CD21^intIgM^dull mature B cells and CD21^hiIgM^hi T2 B cells. T1, T2, mature, and MZ B cell populations are indicated. (C) Four-color flow cytometric analysis of fresh and R2-gated BAFF-stimulated splenocytes from A. Cells were stained with antibodies to IgM, CD21, CD23, and HSA. Freshly prepared splenocytes were gated on the T1, T2, and mature B cell populations as described in B and analyzed for the expression of HSA. The mean fluorescence intensity (MFI) for HSA on gated T1, T2, and mature B cells (black bars) is compared with that of R2-gated BAFF-stimulated splenocytes (white bar). Experiments in A, B, and C are representative of at least 10 animals and cultures analyzed. (D) Freshly prepared splenocytes were stained with antibodies to CD21 and HSA. CD21^loHSA^hi T1 and CD21^hiHSA^hi T2 B cells were identified (Before sort, top), sorted (After sort, middle), and cultured for 72 h with BAFF (bottom). Cells were then analyzed by flow cytometry for the presence of surviving cells in the R2 gate on FSC/SSC plots as indicated (bottom). R1 and R2 populations are shown. Percentages of cells in R2 are indicated. This figure is representative of six different cell sorting experiments analyzed.

Figure 3

BAFF induces survival of T2 B cells. (A) C57BL/6-derived splenocytes were incubated for 72 h with BAFF, stained with antibodies to B220, IgM, IgD, HSA, and L-selectin, and analyzed by flow cytometry. Freshly prepared C57BL/6 splenocytes were stained in parallel. For HSA and L-selectin expression, B220⁺ gated B cells are shown. Histograms for fresh splenocytes (black line) and R2-gated BAFF-treated splenocytes (gray line) are overlayed for comparison. (B) Three-color flow cytometric analysis of fresh and R2-gated BAFF-stimulated splenocytes from A. Cells were stained with antibodies to IgM, CD21, and CD23 and were separated into CD23⁻ and CD23⁺ cells. CD23⁻ cells include CD21^loIgM^hi T1 cells and CD21^hiIgM^hi MZ B cells. CD23⁺ B cells include CD21^intIgM^dull mature B cells and CD21^hiIgM^hi T2 B cells. T1, T2, mature, and MZ B cell populations are indicated. (C) Four-color flow cytometric analysis of fresh and R2-gated BAFF-stimulated splenocytes from A. Cells were stained with antibodies to IgM, CD21, CD23, and HSA. Freshly prepared splenocytes were gated on the T1, T2, and mature B cell populations as described in B and analyzed for the expression of HSA. The mean fluorescence intensity (MFI) for HSA on gated T1, T2, and mature B cells (black bars) is compared with that of R2-gated BAFF-stimulated splenocytes (white bar). Experiments in A, B, and C are representative of at least 10 animals and cultures analyzed. (D) Freshly prepared splenocytes were stained with antibodies to CD21 and HSA. CD21^loHSA^hi T1 and CD21^hiHSA^hi T2 B cells were identified (Before sort, top), sorted (After sort, middle), and cultured for 72 h with BAFF (bottom). Cells were then analyzed by flow cytometry for the presence of surviving cells in the R2 gate on FSC/SSC plots as indicated (bottom). R1 and R2 populations are shown. Percentages of cells in R2 are indicated. This figure is representative of six different cell sorting experiments analyzed.

Figure 3

BAFF induces survival of T2 B cells. (A) C57BL/6-derived splenocytes were incubated for 72 h with BAFF, stained with antibodies to B220, IgM, IgD, HSA, and L-selectin, and analyzed by flow cytometry. Freshly prepared C57BL/6 splenocytes were stained in parallel. For HSA and L-selectin expression, B220⁺ gated B cells are shown. Histograms for fresh splenocytes (black line) and R2-gated BAFF-treated splenocytes (gray line) are overlayed for comparison. (B) Three-color flow cytometric analysis of fresh and R2-gated BAFF-stimulated splenocytes from A. Cells were stained with antibodies to IgM, CD21, and CD23 and were separated into CD23⁻ and CD23⁺ cells. CD23⁻ cells include CD21^loIgM^hi T1 cells and CD21^hiIgM^hi MZ B cells. CD23⁺ B cells include CD21^intIgM^dull mature B cells and CD21^hiIgM^hi T2 B cells. T1, T2, mature, and MZ B cell populations are indicated. (C) Four-color flow cytometric analysis of fresh and R2-gated BAFF-stimulated splenocytes from A. Cells were stained with antibodies to IgM, CD21, CD23, and HSA. Freshly prepared splenocytes were gated on the T1, T2, and mature B cell populations as described in B and analyzed for the expression of HSA. The mean fluorescence intensity (MFI) for HSA on gated T1, T2, and mature B cells (black bars) is compared with that of R2-gated BAFF-stimulated splenocytes (white bar). Experiments in A, B, and C are representative of at least 10 animals and cultures analyzed. (D) Freshly prepared splenocytes were stained with antibodies to CD21 and HSA. CD21^loHSA^hi T1 and CD21^hiHSA^hi T2 B cells were identified (Before sort, top), sorted (After sort, middle), and cultured for 72 h with BAFF (bottom). Cells were then analyzed by flow cytometry for the presence of surviving cells in the R2 gate on FSC/SSC plots as indicated (bottom). R1 and R2 populations are shown. Percentages of cells in R2 are indicated. This figure is representative of six different cell sorting experiments analyzed.

Figure 4

BAFF and anti-μ antibodies promote the maturation of T2 B cells in vitro. (A) CD21^intHSA^lo/int mature and (B) CD21^hiHSA^hi T2 B cells from C57BL/6-derived splenocytes were sorted and incubated with or without BAFF at time 0 as indicated. After 50 h, anti-μ antibodies were added to the cultures, which were carried out for an additional 22 h. Cells were stained with antibodies to HSA, CD21, and IgD. Anti-IgM antibody could not be used for staining due to interferences with anti-μ antibodies used for stimulation. Mature B cells were defined as HSA^int/loCD21^intIgD⁺; T2 cells as HSA^hiCD21^hiIgD⁺; T1 cells as HSA^hiCD21^loIgD⁻; and MZ B cells as HSA^loCD21^hiIgD⁻. The top panel for each B cell subset (A and B) shows FSC/SSC plots indicating the R1 and R2 regions; the bottom panel shows analysis of HSA versus CD21 on cells from the R2 surviving population subgated on the IgD⁺ subpopulation. Boxes for T2 and mature B cells are drawn. This experiment is representative of three separate cell sorting experiments.

Figure 5

Expansion of the T2 and MZ B cell compartment in BAFF Tg mice. (A) Splenocytes freshly isolated from control littermates (Control) and BAFF Tg mice were stained with antibodies to IgM, CD21, and CD23, and were separated into CD23⁻ and CD23⁺ cells. T1, T2, mature, and MZ B cell regions were drawn as shown in Fig. 3 B. The top three histograms show all cells. Note the difference of phenotype of the T1 B cells in BAFF Tg mice (shown with arrows), which consistently show higher CD21 levels and slightly lower IgM levels compared with control T1 splenocytes. These plots are representative of 14 control littermates and 11 BAFF Tg mice analyzed. (B) Frozen tissue sections of spleens from a control littermate (left) and a BAFF Tg mouse (right) were stained with biotin-labeled anti-mouse IgM and revealed using horseradish peroxidase–labeled streptavidin (brown staining). Periarteriolar lymphoid sheath (PALS) or T cell zone (T) and B cell follicle (B) areas are indicated. The width of the MZ, showing a stronger IgM expression, is indicated with a black bar. These pictures are representative of at least six animals analyzed in each group. (C) Splenocytes freshly isolated from control littermates and BAFF Tg mice were stained with antibodies to IgM, CD1, CD21, and CD23, and were separated into CD23⁻ and CD23⁺ cells. T1, T2, mature, and MZ B cell regions were gated as shown in A and expression of CD1 for each B cells subset was overlayed on histogram plots. (D) A corresponding graph for each histogram plot in C shows the mean fluorescence intensity (MFI) of CD1 expression on mature, T1, T2, and MZ B cells. These results are representative of three animals analyzed per group. (E) Lymphocytes isolated as in the legend to Fig. 2 B from PLNs, MLNs, and PBLs of control littermates and BAFF Tg mice were stained and analyzed by flow cytometry for the presence of T1, T2, mature, and MZ B cells as described above in A. Boxes indicating each B cell subset have been drawn. These plots are representative of at least six animals analyzed per group.

Figure 5

Expansion of the T2 and MZ B cell compartment in BAFF Tg mice. (A) Splenocytes freshly isolated from control littermates (Control) and BAFF Tg mice were stained with antibodies to IgM, CD21, and CD23, and were separated into CD23⁻ and CD23⁺ cells. T1, T2, mature, and MZ B cell regions were drawn as shown in Fig. 3 B. The top three histograms show all cells. Note the difference of phenotype of the T1 B cells in BAFF Tg mice (shown with arrows), which consistently show higher CD21 levels and slightly lower IgM levels compared with control T1 splenocytes. These plots are representative of 14 control littermates and 11 BAFF Tg mice analyzed. (B) Frozen tissue sections of spleens from a control littermate (left) and a BAFF Tg mouse (right) were stained with biotin-labeled anti-mouse IgM and revealed using horseradish peroxidase–labeled streptavidin (brown staining). Periarteriolar lymphoid sheath (PALS) or T cell zone (T) and B cell follicle (B) areas are indicated. The width of the MZ, showing a stronger IgM expression, is indicated with a black bar. These pictures are representative of at least six animals analyzed in each group. (C) Splenocytes freshly isolated from control littermates and BAFF Tg mice were stained with antibodies to IgM, CD1, CD21, and CD23, and were separated into CD23⁻ and CD23⁺ cells. T1, T2, mature, and MZ B cell regions were gated as shown in A and expression of CD1 for each B cells subset was overlayed on histogram plots. (D) A corresponding graph for each histogram plot in C shows the mean fluorescence intensity (MFI) of CD1 expression on mature, T1, T2, and MZ B cells. These results are representative of three animals analyzed per group. (E) Lymphocytes isolated as in the legend to Fig. 2 B from PLNs, MLNs, and PBLs of control littermates and BAFF Tg mice were stained and analyzed by flow cytometry for the presence of T1, T2, mature, and MZ B cells as described above in A. Boxes indicating each B cell subset have been drawn. These plots are representative of at least six animals analyzed per group.

Figure 5

Expansion of the T2 and MZ B cell compartment in BAFF Tg mice. (A) Splenocytes freshly isolated from control littermates (Control) and BAFF Tg mice were stained with antibodies to IgM, CD21, and CD23, and were separated into CD23⁻ and CD23⁺ cells. T1, T2, mature, and MZ B cell regions were drawn as shown in Fig. 3 B. The top three histograms show all cells. Note the difference of phenotype of the T1 B cells in BAFF Tg mice (shown with arrows), which consistently show higher CD21 levels and slightly lower IgM levels compared with control T1 splenocytes. These plots are representative of 14 control littermates and 11 BAFF Tg mice analyzed. (B) Frozen tissue sections of spleens from a control littermate (left) and a BAFF Tg mouse (right) were stained with biotin-labeled anti-mouse IgM and revealed using horseradish peroxidase–labeled streptavidin (brown staining). Periarteriolar lymphoid sheath (PALS) or T cell zone (T) and B cell follicle (B) areas are indicated. The width of the MZ, showing a stronger IgM expression, is indicated with a black bar. These pictures are representative of at least six animals analyzed in each group. (C) Splenocytes freshly isolated from control littermates and BAFF Tg mice were stained with antibodies to IgM, CD1, CD21, and CD23, and were separated into CD23⁻ and CD23⁺ cells. T1, T2, mature, and MZ B cell regions were gated as shown in A and expression of CD1 for each B cells subset was overlayed on histogram plots. (D) A corresponding graph for each histogram plot in C shows the mean fluorescence intensity (MFI) of CD1 expression on mature, T1, T2, and MZ B cells. These results are representative of three animals analyzed per group. (E) Lymphocytes isolated as in the legend to Fig. 2 B from PLNs, MLNs, and PBLs of control littermates and BAFF Tg mice were stained and analyzed by flow cytometry for the presence of T1, T2, mature, and MZ B cells as described above in A. Boxes indicating each B cell subset have been drawn. These plots are representative of at least six animals analyzed per group.

Figure 5

Expansion of the T2 and MZ B cell compartment in BAFF Tg mice. (A) Splenocytes freshly isolated from control littermates (Control) and BAFF Tg mice were stained with antibodies to IgM, CD21, and CD23, and were separated into CD23⁻ and CD23⁺ cells. T1, T2, mature, and MZ B cell regions were drawn as shown in Fig. 3 B. The top three histograms show all cells. Note the difference of phenotype of the T1 B cells in BAFF Tg mice (shown with arrows), which consistently show higher CD21 levels and slightly lower IgM levels compared with control T1 splenocytes. These plots are representative of 14 control littermates and 11 BAFF Tg mice analyzed. (B) Frozen tissue sections of spleens from a control littermate (left) and a BAFF Tg mouse (right) were stained with biotin-labeled anti-mouse IgM and revealed using horseradish peroxidase–labeled streptavidin (brown staining). Periarteriolar lymphoid sheath (PALS) or T cell zone (T) and B cell follicle (B) areas are indicated. The width of the MZ, showing a stronger IgM expression, is indicated with a black bar. These pictures are representative of at least six animals analyzed in each group. (C) Splenocytes freshly isolated from control littermates and BAFF Tg mice were stained with antibodies to IgM, CD1, CD21, and CD23, and were separated into CD23⁻ and CD23⁺ cells. T1, T2, mature, and MZ B cell regions were gated as shown in A and expression of CD1 for each B cells subset was overlayed on histogram plots. (D) A corresponding graph for each histogram plot in C shows the mean fluorescence intensity (MFI) of CD1 expression on mature, T1, T2, and MZ B cells. These results are representative of three animals analyzed per group. (E) Lymphocytes isolated as in the legend to Fig. 2 B from PLNs, MLNs, and PBLs of control littermates and BAFF Tg mice were stained and analyzed by flow cytometry for the presence of T1, T2, mature, and MZ B cells as described above in A. Boxes indicating each B cell subset have been drawn. These plots are representative of at least six animals analyzed per group.

Figure 6

Immature B cells from splenocytes isolated from BAFF Tg mice survive in vitro in the absence of exogenous stimuli. Splenocytes isolated from control littermates were cultured for 72 h with or without BAFF as indicated. Splenocytes from BAFF Tg mice were plated in parallel in normal nonsupplemented medium. The R1 and R2 populations were analyzed by flow cytometry on FSC/SSC plots. The R1 and R2 gates are drawn on the plots. As in the legend to Fig. 3 A, expression of B220, IgM, IgD, HSA and L-selectin was analyzed on control freshly prepared splenocytes and BAFF Tg mice–derived splenocytes cultured for 72 h in normal medium. Histograms for fresh control splenocytes (gray line indicated with an arrowhead) and R2-gated BAFF Tg mice–cultured splenocytes (black line) are overlayed for comparison.

Figure 7

Proposed mechanism for one origin of autoimmune B cells in the spleen of BAFF Tg mice. BAFF induces excessive survival of all B cells in the spleen with a preferential role on immature T2 B cells. Autoreactive T2 B cells fail to respond to censoring death signals and massively escape to differentiate into CD1^hi autoreactive MZ B cells. In contact with autoantigens (AutoAg), autoreactive MZ B cells are activated and become plasma cells or undergo affinity maturation in a germinal center to further differentiate into memory B cells, some of which will relocate into the MZ.