BAFF selectively enhances the survival of plasmablasts generated from human memory B cells

Abstract

The generation of Ig-secreting cells (ISCs) from memory B cells requires interactions between antigen-specific (Ag-specific) B cells, T cells, and dendritic cells. This process must be strictly regulated to ensure sufficient humoral immunity while avoiding production of pathogenic autoantibodies. BAFF, a member of the TNF family, is a key regulator of B cell homeostasis. BAFF exerts its effect by binding to three receptors - transmembrane activator of and CAML interactor (TACI), B cell maturation antigen (BCMA), and BAFF receptor (BAFF-R). To elucidate the contribution of BAFF to the differentiation of B cells into ISCs, we tracked the fate of human memory B cells stimulated with BAFF or CD40L. BAFF and CD40L significantly increased the overall number of surviving B cells. This was achieved via distinct mechanisms. CD40L induced proliferation of nondifferentiated blasts, while BAFF prevented apoptosis of ISCs without enhancing proliferation. The altered responsiveness of activated memory B cells to CD40L and BAFF correlated with changes in surface phenotype such that expression of CD40 and BAFF-R were reduced on ISCs while BCMA was induced. These results suggest BAFF may enhance humoral immunity in vivo by promoting survival of ISCs via a BCMA-dependent mechanism. These findings have wide-ranging implications for the treatment of human immunodeficiencies as well as autoimmune diseases.

Figure 1

Expression of BAFF-Rs on human B cells. Human splenic B cells were incubated with (a) soluble BAFF or mAb specific for (b) BAFF-R, (c) TACI, or (d) BCMA. These results represent experiments performed using B cells from three to five different donor spleens. (e and f) 293T cells were transiently transfected with cDNA encoding human (e) BCMA or (f) TACI. Expression of the transfected protein was assessed using the anti-BCMA and anti-TACI mAb’s described in c and d. For each plot, the thick and thin lines represent the fluorescence of cells incubated with the specific or control reagent, respectively.

Figure 2

BAFF enhances survival but does not affect proliferation of human B cells. (a) Total splenic B cells were cultured in medium alone (squares) or with BAFF (circles; 2.5 μg/ml) and the number of viable cells determined after 2 and 4 days. (b) Total, naive, or memory B cells were cultured with media alone (black bars), BAFF (white bars), or CD40L (gray bars), and the number of surviving cells was quantitated after 4 days. Each point represents the mean ± SD of duplicate samples and is representative of three different experiments. Error bars are shown for all graphs; however, they are not always visible. For the experiment shown, BAFF increased the survival of total, naive, and memory B cells 1.95-, 1.4-, and 2.1-fold, respectively, while CD40L increased survival 9.2-, 9.7-, and 6.3-fold. (c) CFSE-labeled memory B cells were cultured for 5 days with media alone (squares), CD40L (diamonds), or BAFF (circles). The percentage of cells in each division was determined by division slicing. These results are representative of three different experiments.

Figure 3

BAFF promotes the generation of CD38⁺ B cells from activated memory B cells. (a) The scheme of the two-step culture system used. CFSE-labeled memory B cells were initially cultured with CD40L and IL-2/IL-10 for 4 days, harvested, washed, and then recultured with media (b–d) or IL-2/IL-10 (e–g) alone (black bars) or in the presence of CD40L (white bars) or BAFF (gray bars). After an additional 4 days, the total number of cells (b and e) and percentage of CD38⁺ B cells (c and f) in each culture were determined. The number of CD38^– and CD38⁺ B cells (d and g) was calculated by multiplying total cell number by the frequency of CD38^– and CD38⁺ cells, respectively. The values represent the mean ± SEM of four (b–d) or five (e–g) experiments. The horizontal lines in b and e indicate the mean number of B cells present at the beginning of the secondary culture.*P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

Differential effect of CD40L and BAFF on the generation and maintenance of populations of activated memory B cells. CFSE-labeled memory B cells were cultured with CD40L and IL-2/IL-10 for 4 days, washed, and then recultured for an additional 4 days with (a) IL-2/IL-10, (b) CD40L and IL-2/IL-10, or (c) BAFF and IL-2/IL-10. After the secondary culture, cell populations defined by CFSE dilution and CD38 expression were determined. Population (popn.) 1: undivided early divisions/CD38^–; population 2: late divisions/CD38^–; population 3: late divisions/CD38⁺. The values represent the mean percentage of cells (± SEM of seven independent experiments) comprising populations 1, 2, and 3. (d) The absolute number of cells present in populations 1 (black bars), 2 (white bars), and 3 (gray bars) in secondary cultures containing IL-2/IL-10, CD40L and IL-2/IL-10, or BAFF and IL-2/IL-10 was determined by multiplying the total cell number by the frequency of cells, using the gates illustrated in a–c. Each value represents the mean ± SEM of five independent experiments.

Figure 5

CD40L and BAFF have distinct effects on proliferation and survival of CD38^– and CD38⁺ B cells. CFSE-labeled memory B cells were cultured as described in Figure 3a. The percentage of (a) CD38^– and (b) CD38⁺ B cells present in different divisions after primary culture (day 4; thick line, squares) or following secondary culture with IL-2/IL-10 (diamonds), CD40L and IL-2/IL-10 (circles), or BAFF and IL-2/IL-10 (triangles) was determined by division slicing. Each value represents the mean ± SEM of five different experiments. (c) Ramos B cells were cultured overnight in the absence of serum. Expression of active caspase-3 by total cells (thin line), live cells (dotted line), and dead cells (bold line) was then determined by intracellular staining and flow cytometry using gates established according to forward- and side-scatter characteristics. (d) The percentage of populations 2 and 3 B cells expressing active caspase-3 following secondary culture with IL-2/IL-10 (black bars), CD40L and IL-2/IL-10 (white bars), or BAFF and IL-2/IL-10 (gray bars) was determined as described for c by gating on both live and dead cells. Each value represents the mean ± SEM of three different experiments. **P < 0.01.

Figure 6

BAFF increases the generation of ISC from activated memory B cells. (a and b) Memory B cells were preactivated with CD40L and IL-2/IL-10 for 4 days and then recultured with (a) media (black bars), or (b) IL-2/IL-10 alone (black bars) or in the presence of CD40L (white bars) or BAFF (gray bars). Each value represents the mean Ig secretion ± SEM of five (a) or seven (b) experiments using cells from different donors. *P < 0.05; **P < 0.01. (c) Secondary B cell cultures were performed in the absence (white bars) or presence (black bars) of soluble TACI-Ig (20 μg/ml). The values represent the mean IgA ± SD of duplicate samples. (d) Memory B cells were preactivated with CD40L/IL-2/IL-10 for 4 days and then recultured with IL-2/IL-10 alone or in the presence of BAFF. The total number of cells secreting IgM (black bars), IgG (white bars), and IgA (gray bars) was determined by ELISPOT. Expt, experiment. (e) IgM⁺ and (f) IgG/A/E⁺ memory B cells were isolated by cell sorting, and the amount of IgA secreted during secondary culture with IL-2/IL-10 (black bars), CD40L/IL-2/IL-10 (white bars), or BAFF/IL-2/IL-10 (gray bars) was determined. The scales of the y axes of these graphs are different to enable meaningful comparison. (g) Cells corresponding to populations 2 and 3 were isolated by sorting, recultured with IL-2/IL-10 (black bars), CD40L/IL-2/IL-10 (white bars), or BAFF/IL-2/IL-10 (gray bars), and the amount of IgA secreted was then determined.

Figure 7

Altered expression of BAFF-Rs and CD40L on activated human B cells. CFSE-labeled memory B cells were cultured as in Figure 3a. Cells were harvested and incubated with anti-CD38 mAb in combination with (a) soluble BAFF or mAb specific for (b) BAFF-R, (c) TACI, (d) BCMA, or (e) CD40. Expression of these receptors on B cells in populations 1 (left panel), 2 (middle panel), and 3 (right panel) was determined. For each plot, the thick and thin lines represent the fluorescence of cells incubated with the specific or isotype control mAb or protein, respectively. These results are representative of three independent experiments.

Figure 8

APRIL and BAFF are equally efficient at increasing survival of human ISCs. Memory B cells were cultured with CD40L and IL-2/IL-10 for 4 days, washed, and then recultured for an additional 4 days with IL-2/IL-10 alone (black bars) or in the presence of CD40L (white bars), BAFF (dark gray bars), or APRIL (light gray bars; 500 ng/ml). The (a) number of CD38⁺ B cells and (b) secretion of IgA was then determined. Each value represents the mean ± SD of duplicate samples.