BAFF receptor signaling aids the differentiation of immature B cells into transitional B cells following tonic BCR signaling

Abstract

BAFF is an important prosurvival cytokine for mature B cells. However, previous studies have shown that BAFFR is already expressed at the immature B cell stage, and that the prosurvival protein Bcl-2 does not completely complement the B cell defects resulting from the absence of BAFFR or BAFF. Thus, we hypothesized that BAFF also functions to aid the differentiation of nonautoreactive immature B cells into transitional B cells and to promote their positive selection. We found that BAFFR is expressed at higher levels on nonautoreactive than on autoreactive immature B cells and that its expression correlates with that of surface IgM and with tonic BCR signaling. Our data indicate that BAFFR signaling enhances the generation of transitional CD23(-) B cells in vitro by increasing cell survival. In vivo, however, BAFFR signaling is dispensable for the generation of CD23(-) transitional B cells in the bone marrow, but it is important for the development of transitional CD23(-) T1 B cells in the spleen. Additionally, we show that BAFF is essential for the differentiation of CD23(-) into CD23(+) transitional B cells both in vitro and in vivo through a mechanism distinct from that mediating cell survival, but requiring tonic BCR signaling. In summary, our data indicate that BAFFR and tonic BCR signals cooperate to enable nonautoreactive immature B cells to differentiate into transitional B cells and to be positively selected into the naive B cell repertoire.

Figures

Figure 1. BAFF-R is differentially expressed by non-autoreactive and autoreactive immature B cells

(A) Bone marrow cells from non-autoreactive 3-83Igi, H-2d (non-aut) and autoreactive 3-83Igi, Rag1−/−, H-2b (aut) mice were analyzed by flow cytometry for expression of B220, IgD and BAFF-R. Dot plots show expression of B220 and IgD on live lymphoid cells. Histograms display BAFF-R levels on gated B220+IgD− cells (representing mostly immature B cells) from 3-83Igi, H-2d (non-aut, intact line) and 3-83Igi, Rag1−/−, H-2b (aut, dashed line) mice, and on B220− non-B cells (filled histogram). Data are representative of three mice per group analyzed in three independent experiments. (B) Bone marrow B220+IgD−CD23−CD43− immature B cells from two groups each of non-autoreactive 3-83Igi, H-2d (black bar) and autoreactive 3-83Igi, Rag1−/−, H-2b (gray bar) mice were sorted via Moflo to >95% purity and subjected to whole genome microarray analyses. The relative tnfrsf13c mRNA levels (arithmetic mean of normalized processed signals from two groups of mice) in the immature B cell samples are shown. Differences between the signals had P values <10−12.

Figure 2. Expression of BAFF-R correlates with that of surface IgM and tonic BCR signaling

(A) Flow cytometric analysis of IgM (top) and BAFF-R (bottom) expression on B220+IgD− bone marrow immature B cells that carry two types of non-autoreactive BCRs (B1-8/3-83Igi, H-2d mice, dual-non-aut, black intact line) or autoreactive and non-autoreactive BCRs (B1-8/3-83Igi, H-2b mice, dual-aut, black dashed line). Expression on B220− non-B cells (non-B, filled histograms) is shown as a negative control. Expression of BAFF-R on spleen CD19+ B cells from B1-8/3-83Igi, H-2d mice (dual-non-aut, SP, gray line) is shown as reference (spleen B cells from BALB/c mice had identical levels; data not shown). Data are representative of three mice per group collected in three independent experiments. (B) Flow cytometric analysis of IgM (top) and BAFF-R (bottom) expression on B220+IgD− bone marrow immature B cells that express normal levels (3-83Igi, H-2d mice, Normal, intact line) or low levels (3-83Igi-low mice, Low, dashed line) of non-autoreactive BCRs. Expression on B220− non-B cells (non-B, filled histograms) is shown as a negative control. Data are representative of four mice per group collected in three independent experiments. (C) Representative flow cytometric analysis of BAFF-R expression on immature B cells generated in culture with IL-7 and then transduced or not. Shown are BAFF-R levels on BCR-normal non-transduced cells from 3-83Igi, H-2d mice (Normal, filled histogram), and on BCR-low GFP control transduced (Low+GFP, dotted line) and N-RasD12 transduced (Low+RasD12, black line) cells from 3-83Igi-low mice. Immature B cells were gated as B220+IgD− (Normal) or B220+IgD−GFP+ (Low+GFP and Low+RasD12). (D) Geometric mean fluorescence intensity (MFI, ± SD, n = 4, from 4 independent experiments) of BAFF-R expression on immature B cells analyzed as described in (C). P-values of <0.05 (*) and <0.005 (**) are indicated. N.S. = not significant.

Figure 3. BAFF augments the differentiation of immature B cells into transitional B cells in vitro

(A) Schematic of the system to study the differentiation of immature B cells in vitro. Briefly, bone marrow cells are cultured with IL-7 for 3–4 days and then transferred in cultures with or without BAFF for two days. (B) Representative flow cytometric analysis of CD21, CD23 (dot plots) and IgD (histograms) expression on 3-83Igi, H-2d non-autoreactive immature B cells that express normal levels of BCR, at the beginning (day 0) and end (day 2) of culture with or without BAFF. Cells from 3-83Igi-mb1−/− mice were used as a negative control as the B cell population of these mice contains only pro-B cells and is deficient of immature B cells. Numbers represent frequencies of live B220+ B cells in indicated gates. Frequencies in histograms are shown only for 3-83Igi, H-2d cells. (C) Mean frequency (± SD, n = 11 mice from 11 independent experiments) of CD21+CD23− (T1-like), CD21+CD23+ (T2-like), and IgD+ (T1-like and T2-like) B cells at days 0 and 2 of culture with or without BAFF as described in (B). (D) Absolute cell numbers (± SD, n = 5 mice from 2 independent experiments) of CD21−CD23− (immature), CD21+CD23− (T1-like), and CD21+CD23+ (T2-like) B cells as described in (B). P-values of <0.05 (*), <0.005 (**), and <0.001 (***) are indicated. N.S. = not significant.

Figure 4. BAFF is required for the differentiation of CD23− into CD23+ transitional B cells via a mechanism distinct from that mediating cell survival

(A) Representative flow cytometric analysis of CD21 and CD23 expression on Thy1.1 control and Bcl-2 transduced non-autoreactive BCR-normal immature B cells from 3-83Igi, H-2d mice, at the beginning (day 0) and end (day 2) of culture with or without BAFF. Numbers represent frequencies of live B220+Thy1.1+ B cells in indicated gates. (B) Mean frequency (± SD, n = 2 pooled mice in each of 4 independent experiments) of CD21+CD23− (T1-like) and CD21+CD23+ (T2-like) transitional B cells in the transduced Thy1.1+ population of Thy1.1 control (black bars) and Bcl-2 (white bars) 3-83Igi, H-2d B cells at day 2 of culture with or without BAFF, as described in (A). P-values of <0.05 (*) are indicated. N.S. = not significant.

Figure 5. Increased levels of active N-Ras, but not of BAFF-R expression, rescue the differentiation of BCR-low immature B cells

(A) Representative flow cytometric analysis of BAFF-R expression on BCR-low immature B cells from 3-83Igi-low mice generated in cultures with IL-7 and transduced with either GFP only control (filled histogram) or BAFF-R (black line). Immature B cells were gated as live B220+IgD−GFP+. (B) Representative flow cytometric analysis of CD21 and CD23 expression on GFP control or BAFF-R transduced 3-83Igi-low B cells before (day 0) and after (day 2) culture with BAFF. Numbers represent frequencies of live B220+GFP+ B cells in indicated gates. (C) Mean frequency (± SD, n = 2 pooled mice in each of 3 independent experiments) of CD21+CD23− (T1-like) and CD21+CD23+ (T2-like) transitional B cells in GFP control (white bars) or BAFF-R (black bars) transduced (GFP+) 3-83Igi-low B cell populations at days 0 and 2 of culture with BAFF, as described in (B). (D) Representative flow cytometric analysis of CD21 and CD23 expression on GFP control or N-RasD12 transduced 3-83Igi-low B cells before (day 0) and after (day 2) culture with or without BAFF. Numbers represent frequencies of live B220+GFP+ B cells in indicated gates. (E) Mean frequency (± SD, n = 2 pooled mice in each of 7 independent experiments) of CD21+CD23− (T1-like) and CD21+CD23+ (T2-like) transitional B cells in GFP control (white bars) or N-RasD12 (black bars) transduced (GFP+) 3-83Igi-low B cell populations at days 0 and 2 of culture with BAFF, as described in (D). P-values of <0.05 (*) and <0.005 (**) are indicated. N.S. = not significant.

Figure 6. BAFF is required for optimal differentiation of wild-type T1, as well as T2, splenic transitional B cells

(A) Schematic for the generation of BAFF-R-deficient and -sufficient mixed bone marrow chimeras. Bone marrow cells from Ly5.1 and Ly5.2 congenic mice were used for these experiments. (B) B cells from mixed bone marrow chimeras generated as described in (A) were analyzed by flow cytometry for expression of the congenic marker Ly5.2. The bar graphs represent the mean frequencies (± SD, n = 6 chimera mice) of Ly5.2+ cells (BAFF-R+/+, black bars; BAFF-R−/−, white bars) in B cell populations from bone marrow and spleen tissues analyzed ex-vivo. Similar results were obtained in an additional independent experiment with three mice per group (data not shown). The following surface markers were used to discriminate B cell subsets within the total B220+ cell population in bone marrow: pro-B: IgM−CD43+; pre-B: IgM−CD2+; immature: IgM+IgD−; transitional T1-like: IgMhighIgDlowCD23−; transitional T2-like: IgMhighIgDlowCD23+; and in spleen: immature + transitional T1: CD24highCD23−; transitional T2+T3: CD24highCD23+; follicular (Fo.): CD24lowCD23+. The gating strategy used in the analysis is depicted in Figure S5A. P-values of <0.05 (*), <0.005 (**), and <0.001 (***) are indicated. N.S. = not significant.

Figure 7. IL-7 neither promotes nor blocks the differentiation of immature B cells into transitional B cells in vitro

(A) Bone marrow cells from 3-83Igi, H-2d mice were cultured in the presence of IL-7 for 3 days to generate BCR-normal non-autoreactive immature B cells. Immature B cells were then washed with PBS and re-cultured with or without BAFF or IL-7, as indicated. Dot plots are representative flow cytometric analyses of CD21 and CD23 expression on B cells at the beginning (day 0) and end (day 2) of culture with the indicated cytokines. Numbers represent frequencies of live B220+ B cells in indicated gates. IL-7 was used approximately at 250 pg/ml (low IL-7) and 10 ng/ml (high IL-7). (B) Immature B cells from 3-83Igi, H-2d mice were generated in the presence of IL-7 as described in (A) and then re-cultured in the presence of IL-7 and BAFF, as indicated. Cells were analyzed as described in (A). (C) Mean frequency (± SD, n = 3 mice, one in each of 3 independent experiments) of CD21+CD23− (T1-like) and CD21+CD23+ (T2-like) 3-83Igi, H-2d transitional B cells at days 0 and 2 of culture with indicated cytokines, as described in (A) and (B). (D) Absolute cell numbers (± SD, n = 3 mice from 1 experiment) of CD21−CD23− (immature), CD21+CD23− (T1-like), and CD21+CD23+ (T2-like) B cells at days 0 and 2 of culture with indicated cytokines as described in (A) and (B). P-values of <0.05 (*), <0.005 (**), and <0.001 (***) are indicated. N.S. = not significant.