Attenuation of apoptosis underlies B lymphocyte stimulator enhancement of humoral immune response

Abstract

B lymphocyte stimulator (BLyS) is a newly identified monocyte-specific TNF family cytokine. It has been implicated in the development of autoimmunity, and functions as a potent costimulator with antiimmunoglobulin M in B cell proliferation in vitro. Here we demonstrate that BLyS prominently enhances the humoral responses to both T cell-independent and T cell-dependent antigens, primarily by attenuation of apoptosis as evidenced by the prolonged survival of antigen-activated B cells in vivo and in vitro. BLyS acts on primary splenic B cells autonomously, and directly cooperates with CD40 ligand (CD40L) in B cell activation in vitro by protecting replicating B cells from apoptosis. Moreover, although BLyS alone cannot activate the cell cycle, it is sufficient to prolong the survival of naive resting B cells in vitro. Attenuation of apoptosis by BLyS correlates with changes in the ratios between Bcl-2 family proteins in favor of cell survival, predominantly by reducing the proapoptotic Bak and increasing its prosurvival partners, Bcl-2 and Bcl-xL. In either resting or CD40L-activated B cells, the NF-kappaB transcription factors RelB and p50 are specifically activated, suggesting that they may mediate BLyS signals for B cell survival. Together, these results provide direct evidence for BLyS enhancement of both T cell-independent and T cell-dependent humoral immune responses, and imply a role for BLyS in the conservation of the B cell repertoire. The ability of BLyS to increase B cell survival indiscriminately, at either a resting or activated state, and to cooperate with CD40L, further suggests that attenuation of apoptosis underlies BLyS enhancement of polyclonal autoimmunity as well as the physiologic humoral immune response.

Figure 1

BLyS enhances antibody response to Pneumovax. Primary Pneumovax 23–specific IgM (A), IgG (B), or IgA (C) responses in mice after BLyS administration. Pneumovax-specific Ig titers were determined by ELISA on sera collected from buffer control mice on day 0 (▵), from mice immunized with Pneumovax (○), or from mice immunized with Pneumovax and injected subcutaneously daily with BLyS (20 μg/mouse) on day 7 (•).

Figure 2

BLyS enhances antibody response to DNP-BSA and NP-CGG. (A) Primary DNP-BSA–specific IgM and IgG responses in mice after BLyS administration. Serum DNP–specific Ig titers were determined on day 14 by ELISA from PBS control mice (▵), DNP-BSA–immunized (○), BLyS alone (▴) or DNP-BSA–immunized and injected intraperitoneally daily with 20 μg/mouse BLyS (•). (B) Primary NP-CGG IgM and IgG responses in mice after BLyS administration. NP-specific Ig titers were determined by ELISA from sera collected from uninjected mice on day 0 (○), or from NP-CGG–immunized mice on day 8, which were coinjected with PBS (○) or 10 μg/mouse BLyS (•). (C) Primary NP-CGG–specific IgM- and IgG-secreting plasma cells after PBS (P) or BLyS (B) administration were determined by ELISPOT assay on day 8. Frequency of plasma cells (left) and total splenic plasma cells (right). These experiments have been performed three times.

Figure 4

BLyS enhances the activation of B cells by CD40L in vitro. (A) Resting mouse splenic B cells were cocultured with CD40L cells, in the presence (•) or absence (○) of BLyS (50 ng/ml). The accumulation of live cells was determined on the days indicated, by trypan blue staining and counting in triplicate. This experiment has been performed three times. (B) The accumulation of viable cell number on day 3 of coculture with CD40L cells in the presence of indicated concentrations of BLyS or without BLyS (N). (C) FACS^® analysis of DNA content on day 3 of coculture with CD40L cells in the presence of indicated concentrations of BLyS or without BLyS (N), and expressed as the percentage of cells in the S and G2/M phases of the cell cycle. (D) Analysis of BrdU incorporation in a 2-h period, at 12-h intervals of coculturing with CD40L cells in the presence (•) or absence (○) of BLyS (5 ng/ml).

Figure 3

BLyS enhances activated B cell expansion and reduces apoptosis in vivo and in vitro. (A) Spleens were weighed (weights shown in grams) on day 8 of NP-CGG immunization, with coinjection of PBS (○) or 10 μg/mouse BLyS (•). (B) Viable splenic B cells in PBS and BLyS–coinjected mice were determined on day 8 of NP-CGG immunization (n = 3). (C) Annexin V binding of high density (H) and low density (L) splenic B cells isolated on day 11 after immunization with NP-CGG and coinjected with PBS (○) or 10 μg/mouse BLyS (•) (n = 3). (D) Annexin V binding of low density splenic B cells were isolated on day 8 of NP-CGG–immunized mice, coinjected with PBS or BLyS as indicated, and cultured in vitro for 24 h in media alone (○) or with 100 ng/ml BLyS, n = 3 (•). (E) Expression of CD40 and Fas on high (H) and low density (L) B cells without immunization (C), or on day 8 after immunization with NP-CGG coinjected with PBS or BLyS. These experiments have been performed three times.

Figure 5

BLyS enhances the expansion of CD40L-activated B cells through the attenuation of apoptosis. (A) The accumulation of live cells and the percentage of dead B cells were determined by trypan blue staining on days of culturing with CD40L cells at the indicated ratios in the presence (•) or absence (○) of BLyS (5 ng/ml). (B) The accumulation of live cells and the percentage of cells expressing annexin V binding activity were determined on B cells continuously cocultured with CD40L cells at a ratio of 10:1, or (C) cultured in media alone after 2 d of coculture with CD40L cells, in the presence (•) or absence (○) of BLyS (5 ng/ml). These experiments were repeated three times.

Figure 6

BLyS does not regulate CD40 or Fas expression in vitro. FACS^® analysis of CD40 and Fas expression on the indicated days of in vitro culturing in the presence of CD40L, alone or together with BLyS (5 ng/ml). These experiments were repeated three times.

Figure 7

BLyS prolongs the survival of resting B cells in vitro. (A) The number of live resting B cells and the percentage of cells expressing Annexin V binding activity were determined on the days indicated during culture in media in the presence (▴) or absence (▵) of BLyS (5 ng/ml). (B) FACS^® analysis of DNA content of day 0 and 3 B cells. (C) Immunostaining of intracellular IgM in B cells cocultured with CD40L cells or BLyS (5 or 50 ng/ml), or both, on the days indicated. These experiments have been performed three times.

Figure 8

Regulation of Bcl-2 family proteins by BLyS. (A) Expression of Bak and Bcl-xL in high density (H) and low density (L) splenic B cells isolated from mice administered with BLyS or PBS on day 11 of immunization with NP-CGG. (B) Expression of Bak, Bcl-xL, and Bcl-2 on high density splenic B cells, either freshly isolated (lane 1), after 24 h of in vitro culture with media alone (lanes 2 and 3), or after 48 h of coculture with CD40L cells (lanes 4 and 5), in the presence or absence of BLyS. ns, nonspecific bands used as controls for sample loading.

Figure 9

BLyS activates NF-κB in resting and CD40L-activated B cells. (A) EMSA of the NF-κB DNA binding activity present in whole cell extracts prepared from freshly isolated resting splenic mouse B cells (0 d), cells cultured in media alone for 1 d (lane 2), or on days of coculture with CD40L cells in the presence or absence of 5 ng/ml BLyS. The migration of specific NF-κB complexes is marked. (B) EMSA of extracts prepared from B cells cocultured with CD40L cells in the presence or absence of BLyS (5 ng/ml) for 3 d, with or without preincubation with indicated antibodies. (C) EMSA of NF-κB DNA binding activity present in whole cell extracts prepared from freshly isolated resting splenic B cells (lane 1), or cultured for hours in media in the presence or absence of 100 ng/ml of BLyS (lanes 2–9). RelB and p50 lanes represent preincubation of extracts with the antibodies.