Normal B cell homeostasis requires B cell activation factor production by radiation-resistant cells

Abstract

The cellular source of B cell activation factor (BAFF) required for peripheral B cell survival/maturation is unknown. To determine the nature of BAFF-producing cells we established and analyzed reciprocal bone marrow (BM) chimeras with wild-type (WT) and BAFF-deficient mice. The results revealed that BAFF production by radiation-resistant stromal cells is completely sufficient to provide a necessary signal for B cell survival/maturation, as BAFF-/- BM cells transferred into lethally irradiated WT mice gave rise to normal numbers of follicular (FO) and marginal zone (MZ) B cell subpopulations. On the other hand, transfer of WT BM into BAFF-/- lethally irradiated mice resulted only in minimal reconstitution of mature FO B cells and no restoration of MZ B cells. Thus, in the absence of BAFF+/+ stromal cells, BAFF production by BM-derived cells, presumably by macrophages, dendritic cells, and/or neutrophils, was not at all sufficient to support normal B cell homeostasis. Interestingly, immunization of both types of chimeras stimulated high levels of antigen-specific antibody secretion, indicating that either stromal cell- or hematopoietic cell-derived BAFF is sufficient for B cell antibody responses.

Figure 1.

Hematopoietic cells in BM chimeras are of donor origin. Lethally irradiated mice were reconstituted with 6.5 × 10⁶ BM cells. 8–12 wk after reconstitution, single cell suspensions were prepared by collagenase digestion of spleens from B6.SJL BM→BAFF KO C57BL/6 (A), BAFF KO C57BL/6 BM→B6.SJL (B), and B6.SJL BM→C57BL/6 (C) chimeric mice. After gating, B220⁺ (left), CD11c⁺ (middle), or CD11b⁺ CD11c⁻ (right) cells were examined for CD45.1 and CD45.2 expression. Graphs are representative of six mice per group examined.

Figure 2.

BAFF^+/+ stromal cells are necessary and sufficient for complete reconstitution of the B cell compartment. (A) B cell reconstitution of secondary lymphoid organs was evaluated by FACS^® analysis of spleens and mesenteric lymph nodes from different chimeric mice 8–12 weeks after BM transplantation. (B) Spleen cells from these mice were also evaluated for the expression of IgM and IgD (B), as well as CD21 and CD23 (C). CD21 and CD23 expression on B cell after gating on B220⁺ cells is also shown (D). Numbers in the quadrants or next to the boxes indicate percentages of the indicated population. MZ, FO, and NF denote the gates used to determine correspondingly MZ, FO, and newly formed B cell populations. Dot plots are representative of four mice per group. The experiment was repeated five times with similar results.

Figure 2.

BAFF^+/+ stromal cells are necessary and sufficient for complete reconstitution of the B cell compartment. (A) B cell reconstitution of secondary lymphoid organs was evaluated by FACS^® analysis of spleens and mesenteric lymph nodes from different chimeric mice 8–12 weeks after BM transplantation. (B) Spleen cells from these mice were also evaluated for the expression of IgM and IgD (B), as well as CD21 and CD23 (C). CD21 and CD23 expression on B cell after gating on B220⁺ cells is also shown (D). Numbers in the quadrants or next to the boxes indicate percentages of the indicated population. MZ, FO, and NF denote the gates used to determine correspondingly MZ, FO, and newly formed B cell populations. Dot plots are representative of four mice per group. The experiment was repeated five times with similar results.

Figure 3.

Organization of the splenic B cell follicle in spleen requires BAFF^+/+ stroma. Frozen sections of spleens from different chimeras were stained with anti-CD19 (green) and anti-CD4 plus anti-CD8 (red; top), anti-IgM (green), anti-IgD (red), and anti-CD11c plus CD11b (blue; middle), or anti–MOMA-1 (green) and anti-B220 (red). Images of one representative section out of five spleens per group are shown. ×200.

Figure 4.

Both BM-derived BAFF^+/+ and stroma BAFF^+/+ cells can provide BAFF signal during antigen-specific antibody response. (A) 10 d after the immunization with 100 μg NP₂₁-CGG in alum, spleen cells from various chimeras were analyzed for the presence of GC B cells. Numbers in the squares represent percent of GC (GL7⁺ IgD⁻) B cells among B220⁺ cells in spleens of the chimeric mice. Graphs representative of six mice per each group are shown. No significant number of GL7⁺ IgD⁻ B220⁺ cells could be detected in the absence of immunization. (B) Spleen cells from the mice immunized as in A were assayed for the frequency of NP₂- and NP₁₇-specific ASCs per 9 × 10⁵ splenocytes using ELISPOT assay (right). NP₂- and NP₁₇-specific titers of IgG1 antibodies in sera of different immunized chimeric mice were determined by ELISA and expressed as a reciprocal of standard sera dilution giving the same OD450 as the sample (left). 6–10 mice per group were used in the experiment. The values for all chimeric mice were statistically significantly higher than the values from BAFF^−/− (KO) mice (P < 0.05).

Figure 5.

Production of systemic levels of secreted BAFF in sera requires BAFF^+/+ stromal cells. Sera from different chimeric mice 8–12 wk after their reconstitution were assayed for BAFF levels by ELISA. Sera were collected from 6–10 mice per group. *, statistically significant (P < 0.05) difference relative to the WT→WT mice. Limit of detection of this ELISA was 5 ng/ml.