Deficiency in TNFRSF13B (TACI) expands T-follicular helper and germinal center B cells via increased ICOS-ligand expression but impairs plasma cell survival Xijun Oua, Shengli Xua,b, and Kong-Peng Lama,b,c,d,1 a
Immunology Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research, Singapore; Departments of bPhysiology, Microbiology, and dPediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117599
c
Edited* by Tasuku Honjo, Graduate School of Medicine, Kyoto University, Kyoto, Japan, and approved August 3, 2012 (received for review January 9, 2012)
costimulation
| T-cell–dependent humoral immunity | TNF receptor
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ransmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI/TNFRSF13B), B-cell activating factor (BAFF) receptor (BAFF-R/TNFRSF13C), and B-cell maturation antigen (BCMA/TNFRSF17) are closely related members of the TNF receptor superfamily and bind B-cell survival cytokines BAFF and APRIL (1). Although BAFF-R and BCMA have been shown to mediate the survival of follicular B cells (2) and plasma cells (3), respectively, a similar role for TACI in mediating the survival of B lymphocytes at any particular stage of B-cell differentiation has not been demonstrated definitively. However, mutations in TACI are thought to contribute to ∼10% of common variable immunodeficiency (CVID) in humans (4, 5). This syndrome is characterized by antibody deficiency in late childhood and early adulthood. Paradoxically, some patients with TACI-mutated CVID also develop autoimmune diseases (6). How TACI deficiency leads to antibody deficiencies on one hand and autoimmunity on the other hand is not well understood. Mice lacking TACI have been generated (7, 8), and initial characterizations of these mice revealed modest phenotypes. Taci−/− mice had increased numbers of B cells but exhibited decreased antibody responses to T-cell–independent type II antigens. However, they did not appear to manifest any defective antibody responses to T-cell–dependent or protein antigens, as www.pnas.org/cgi/doi/10.1073/pnas.1200386109
determined by ELISA measurements of serum antibody titers (7, 8). The host response to T-cell–dependent antigens is known to involve the generation of germinal centers (GCs), transient structures found in secondary lymphoid tissues in which T-cell– B-cell interactions occur (9). Here antigen-activated B cells expand and undergo antibody class-switching and affinity maturation, and also differentiate into memory B cells and plasma cells (9). GC B-cell differentiation is aided by a subset of CD4+ T cells known as T-follicular helper (Tfh) cells, which are characterized by their surface expression of the chemokine receptor CXCR5 and costimulation molecules ICOS and PD-1, as well as the production of IL-21 (10). Interestingly, the generation of Tfh cells is also dependent on ICOS ligand (ICOSL, or B7H2) found on B cells (11), suggesting that GC B cells and Tfh cells mutually costimulate their respective differentiation. Whether TACI has a role in the GC reaction is not known; however, TACI expression is up-regulated on activated B cells and plasma cells (12, 13). In the present study, we reexamined the role of TACI in T-cell– dependent humoral immune response. We found that loss of TACI leads to expansion of GC B and Tfh cells owing to increased B7H2 expression on Taci−/− B cells. Interestingly, despite the increased presence of antigen-specific B cells in Taci−/− mice, these mice exhibit impaired antibody response to T-cell–dependent antigens as a result of severe reductions in plasma cell numbers. We further demonstrated that TACI can deliver survival signals in plasma cells. Taken together, our data indicate that TACI has a dual role in B-cell terminal differentiation in limiting the expansion of GC B cells and mediating the survival of plasma cells. Results TACI Deficiency Leads to Expansion of Tfh and GC B cells. TACI mutations have been shown to affect Ig class-switching to IgA (7, 8, 14–16), an antibody isotype that is enriched in mucosal tissues. Whether TACI deficiency can affect immune cell populations at these sites is not known, however. To examine this question, we analyzed the Peyer’s patches (PP) of WT and Taci−/− mice. PPs are sites of chronic immune responses and exhibit sustained GC reactions owing to continuous interactions between gut pathogens and host immune cells (17, 18). In the GC, one can stain for antigen-activated GC B cells that are CD19+CD38−Fas+GL-7+ and Tfh cells that are CD4+TCRβ+PD-1high(hi)CXCR5+ (10, 19– 21). Our flow cytometry analyses indicate that Taci−/− mice have a significantly increased fraction (more than twofold) of Tfh cells
Author contributions: S.X. and K.-P.L. designed research; X.O. performed research; X.O., S.X., and K.-P.L. analyzed data; and X.O. and K.-P.L. wrote the paper. The authors declare no conflict of interest. *This Direct Submission article had a prearranged editor. 1
To whom correspondence should be addressed. E-mail:
[email protected]. sg.
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Mutations in TNFRSF13B, better known as transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), contribute to common variable immunodeficiency and autoimmunity in humans. How TACI regulates these two opposing conditions is unclear, however. TACI binds the cytokines BAFF and APRIL, and previous studies using gene KO mice indicated that loss of TACI affected only T-cell–independent antibody responses. Here we demonstrate that Taci −/− mice have expanded populations of T follicular helper (Tfh) and germinal center (GC) B cells in their spleens when immunized with T-cell–dependent antigen. The increased numbers of Tfh and GC B cells in Taci −/− mice are largely a result of up-regulation of inducible costimulator (ICOS) ligand on TACI-deficient B cells, given that ablation of one copy of the Icosl allele restores normal levels of Tfh and GC B cells in Taci −/− mice. Interestingly, despite the presence of increased Tfh and antigenspecific B cells, immunized Taci −/− mice demonstrate defective antigen-specific antibody responses resulting from significantly reduced numbers of antibody-secreting cells (ASCs). This effect is attributed to the failure to down-regulate the proapoptotic molecule BIM in Taci −/− plasma cells. Ablation of BIM could rescue ASC formation in Taci −/− mice, suggesting that TACI is more important for the survival of plasma cells than for the differentiation of these cells. Thus, our data reveal dual roles for TACI in B-cell terminal differentiation. On one hand, TACI modulates ICOS ligand expression and thereby limits the size of Tfh and GC B-cell compartments and prevents autoimmunity. On the other hand, it regulates the survival of ASCs and plays an important role in humoral immunity.
Fig. 1. TACI deficiency leads to expansion of Tfh and GC B cells. (A and B) Flow cytometry analyses of Tfh and GC B-cell populations in PPs of unchallenged WT and Taci−/− mice (A) and spleens of NP38-CGG–immunized WT and Taci−/− mice (B). Numbers depict PD-1hiCXCR5+ Tfh and CD38−Fas+ or GL-7+Fas+ GC B cells among CD4+TCRβ+ and CD19+ cells, respectively. (C) Graphical representations of the fraction and total number of Tfh cells (CD4+TCRβ+PD-1hiCXCR5+) and GC B cells (CD19+CD38−Fas+) in the spleens of NP38-CCG–immunized WT and Taci−/− mice. Each data point represents one mouse. ***P < 0.001.
compared with WT controls (Fig. 1A), along with a significant increase in the fraction of GC B cells. We next examined whether this phenomenon also occurred in an acute immune response to T-cell–dependent antigen. Thus, we challenged WT and Taci−/− mice with 4-hydroxy-3-nitrophenylacetyl hapten conjugated to chicken gamma globulin (NP-CGG). At 10 d postimmunization, we isolated splenocytes from these mice and examined their Tfh and GC B-cell compositions. Consistent with the PP data, we found an approximate threefold increase in Tfh and a twofold increase in GC B-cell fractions in the spleens of Taci−/− mice compared with WT controls (Fig. 1B). Enumeration of total Tfh and GC B cells also confirmed significantly increased numbers of these cells in mutant mice (Fig. 1C). Thus, TACI deficiency leads to expansion of Tfh and GC B cells in both acute and chronic immune responses.
counterparts regardless of mouse immunization status (Fig. 2 A and B). On the other hand, B7H2 expression level was similar on WT and Taci−/− dendritic cells (CD11c+) (Fig. 2B). Moreover, the expression level of its receptor ICOS was also comparable in WT and Taci−/− T and Tfh cells (Fig. 2C). Taken together, these data suggest that the expansion of Tfh and GC B cells in Taci−/− mice possibly could be related to the increased expression of
B7H2 Expression Is Up-Regulated on Taci−/− B Cells. The de-
velopment of GC B and Tfh cells depends on cognate interactions involving various receptor–ligand pairs on the two cell types, including Fas–FasL, CD80/86–CD28, CD40–CD40L, B7H2–ICOS, PDL1/2–PD1, and others (10). Given that TACI is expressed mainly on B cells, we reasoned that TACI deficiency might affect the expression of some of these molecules and in turn lead to expansion of Tfh and GC B cells. Thus, we performed flow cytometry to examine the expression levels of the key molecules involved in B-cell–T-cell interactions. Our data indicate that the expression levels of major histocompatibility class II, CD40, Fas (CD95), CD80, CD86, PDL1, and PDL2 on follicular B cells or GC B cells were comparable in immunized WT and Taci−/− mice (Fig. S1A). Similarly, there were no differences in the expression levels of FasL, PD-1, CD28, or CD40L between WT and Taci−/− CD4+ T or Tfh cells (Fig. S1B). Interestingly, the expression of B7H2, previously shown to be critical for GC formation (22), was consistently up-regulated on Taci−/− B cells and GC B cells compared with their WT 15402 | www.pnas.org/cgi/doi/10.1073/pnas.1200386109
Fig. 2. Up-regulation of B7H2 expression on Taci−/− B cells. (A and B) Histogram depicting B7H2 expression level on GC B cells (CD19+CD38−Fas+) and non-GC B cells (CD19+CD38+Fas−) from spleens of day 10 NP38-CCG–immunized WT and Taci−/− mice (A) and CD19+ B and CD11c+ dendritic cells from naïve WT and Taci−/− mice (B). (C) Histogram depicting the level of ICOS expression on Tfh (CD4+TCRβ+PD-1hiCXCR5+) and CD4+TCRβ+ cells from WT and Taci−/− mice at day 10 postimmunization. Data shown are representative of more than three independent experiments.
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were then analyzed by flow cytometry at 10 d postimmunization. We found that reconstituted CD45.1 WT and CD45.2 Taci−/− B cells had comparable levels of B7H2 expression (Fig. S4B), which is also similar to that seen on B cells in CD45.1 WT and CD45.2 WT reconstituted mice. This suggests that TACI indirectly controls B7H2 expression on B cells and that as long as TACI is present in the system, even on neighboring WT B cells, there is no increase in B7H2 expression on Taci−/− B cells. These data are consistent with the hypothesized model of TACI sequestering away excess BAFF. Concomitant with the lack of B7H2 up-regulation in the reconstituted mice, we found no increase in the Tfh cell population in the chimeras (Fig. S4C). Interestingly, however, we observed a slight increase of CD45.2 Taci−/− GC B cells compared with CD45.1 WT GC B cells (Fig. S4 D and E), suggesting that TACI also may deliver some inhibitory signals to constrain GC B-cell development, consistent with previous reports (1).
B7H2 on Taci−/− B cells that enhances Tfh cell development, which in turn further supports GC B-cell differentiation (10, 23). B7H2 Up-Regulation Is Largely Responsible for the Expansion of Tfh and GC B Cells in Taci−/− Mice. To test the hypothesis that enhanced
expression of B7H2 on Taci−/− B cells drives the expansion of the Tfh cell population in Taci−/− mice, we first generated Taci−/− B7h2−/− and Taci−/−cd28−/− mice and immunized them and various controls with NP38-CGG. At 10 d postimmunization, we analyzed the fractions of Tfh and GC B cells in the spleens of these mice. Taci−/− mice again displayed increased populations of Tfh and GC B cells compared with WT controls. Taci−/−B7h2−/− and Taci−/−cd28−/− mice could not generate any substantial fraction of Tfh and GC B cells (Fig. S2). These data suggest that the increased numbers of Tfh and GC B cells in Taci−/− mice arise from cognate interactions between B cells and T cells. To further examine the contribution of B7H2 up-regulation in enhancing Tfh and GC Bcell development in Taci−/− mice, we introduced B7h2 heterozygosity into these mice to determine whether these populations are sensitive to changes in B7H2 abundance. B7h2 heterozygosity led to reduced B7H2 expression on Taci−/− B cells (Fig. 3A) to a level comparable to that seen on Taci+/+B7h2+/− cells. Interestingly, ablation of one allele of B7h2 was sufficient to modulate the increased formation of Tfh cells (Fig. 3B) and GC B cells (Fig. S3) in Taci−/− mice. These data suggest that the increased fraction of Tfh cells in Taci−/− mice is most likely mediated by increased expression of B7H2 on Taci−/− B cells, which provides extra stimulation for Tfh cell development. It is possible that TACI binding of BAFF or APRIL directly signals the down-regulation of B7H2 expression on B cells. To test this, we stimulated WT and Taci −/− B cells in vitro with APRIL or BAFF. APRIL had no effect on B7H2 expression, but BAFF stimulated equivalent up-regulation of B7H2 on WT and Taci−/− B cells (Fig. S4A). This finding suggests that excess BAFF could stimulate via BAFF-R for the up-regulation of B7H2 expression. This data led us to hypothesize that TACI binding of BAFF could perhaps lead to less BAFF for BAFF-R induction of B7H2 up-regulation. To test this hypothesis, we generated mixed bone marrow (BM) chimeras using CD45.1 WT and CD45.2 WT cells or CD45.1 WT and CD45.2 Taci−/− cells. Reconstituted mice were left for 6 wk before NP38-CGG immunization. Splenic GC B-cell numbers and B7H2 expression Ou et al.
TACI Deficiency Impairs T-Cell–Dependent Antibody Immune Response.
Tfh and GC B cells play critical roles in GC reactions during humoral immune responses by regulating antibody class-switching and memory B-cell and plasma cell differentiation (10). Our foregoing data indicate that loss of TACI leads to expansion of Tfh and GC B cells via increased B7H2 expression, which in principle should lead to enhanced T-cell–dependent immune responses. However, it was reported previously that TACI deficiency impairs T-cell–independent responses but does not affect T-cell–dependent responses (7, 8). To resolve this discrepancy, we reanalyzed the antibody responses of NP38-CGG–immunized Taci−/− mice. The high-affinity and total anti-NP antibodies elicited in C57BL/6 mice were detected using anti-NP2 and anti-NP17 ELISA, respectively. Surprisingly, in contrast to previous data indicating no impairment (7, 8) and our prediction of enhanced response, Taci−/− mice had much lower anti-NP antibody titers compared with WT mice. The ELISA data indicated that antiNP17 and anti-NP2 IgM and IgG1 antibodies (Fig. 4) and IgG2b and IgG3 antibodies (Fig. S5) in the sera of Taci−/− mice were significantly reduced across all time points examined compared with WT controls, suggesting that TACI is required for optimal T-cell–dependent humoral immune response. The reduced anti-NP antibody response seen in Taci−/− mice represents an enigma, given that we found expansion of Tfh and GC B cells in these mice during both acute and chronic immune responses (Fig. 1). To better understand this conundrum, we
Fig. 4. Taci−/− mice exhibit a defective T-cell–dependent antibody response. Groups of eight WT and Taci−/− mice were challenged with NP38-CCG, and their sera-specific antibody titers at day 7, 14, 28, and 42 were measured by ELISA using NP2 and NP17-BSA as coating antigens to detect NP-specific IgM and IgG1 antibodies, respectively. Sera were diluted 1,000-fold for IgM and 20,000-fold for IgG1 detection. *P < 0.05; **P < 0.01; ***P < 0.001.
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Fig. 3. Ablation of one copy of B7h2 allele modulates surface B7H2 expression and Tfh expansion in Taci−/− mice. (A) Histogram depicting B7H2 expression level on splenic B cells of WT and Taci−/− mice bearing two copies or one copy of the B7h2 allele. (B) Flow cytometry analyses of Tfh cell population in the spleens of 10 d NP38-CCG–immunized WT, Taci−/−, B7h2+/−, and Taci−/−B7h2+/− mice. Values shown are representative of two independent experiments.
examined the population of antigen-specific B cells in the GC of WT and Taci−/− mice by staining for them with anti-B220 and anti-IgG1 antibodies and NIP, an analog of the immunizing antigen, and using a mixture of antibodies (Dump) to gate away irrelevant cells. We identified a small population of NIP+IgG1+ B cells (∼2.2% of Dump−B220+ cells) in the spleens of WT mice (Fig. 5A). A similar fraction (∼2.5%) of NIP+IgG1+ B cells was detected in the spleens of immunized Taci−/− mice, suggesting that TACI deficiency did not impair the generation of antigenspecific B cells. In fact, enumeration of these antigen-specific IgG1 B cells revealed their presence in much greater abundance (an approximate threefold increase) in Taci−/− mice, owing to their increased splenocyte numbers. TACI Is Required for Plasma Cell Survival. Because Taci−/− mice have greater numbers of Tfh and GC B cells and generate greater numbers of antigen-specific B cells but yet have significantly reduced specific serum antibody titers, we hypothesize that TACI deficiency probably affects the generation or survival of plasma cells. To investigate this possibility, we performed a direct ELISPOT assay for the presence of NP-specific plasma cells in the spleen and BM of immunized Taci−/− mice. At 14 d and 28 d postimmunization with NP38-CGG, we detected a substantial number of NP-specific IgG1 antibody-secreting cells (ASCs) in the spleen and BM of WT mice (Fig. 5B). Interestingly, Taci−/− mice had greatly reduced numbers of anti-NP IgG1-secreting cells in the spleen at day 14 postimmunization and a further decrease by day 28. The reduction in IgG1 ASCs was even more pronounced in the BM of Taci−/− mice; these cells were hardly detectable at day 14 or 28 postimmunization. Thus, TACI deficiency impairs ASCs. The reduction in ASCs in the spleen and BM of Taci−/− mice could be due to inefficient generation of plasma cells from activated antigen-specific GC B cells or impaired survival of plasma cells. Because TACI binds BAFF and APRIL, which are B-cell survival factors (1), the latter possibility is more plausible. In
support of this idea, we found that TACI is expressed on WT plasma cells (Fig. 6A). Furthermore, we demonstrated in vitro that treatment with APRIL or BAFF could increase the survival of WT plasma cells by 1.5- to 2-fold (Fig. 6B and Fig. S6A). However, APRIL treatment did not lead to any increase in the survival of Taci−/− plasma cells (Fig. 6B), whereas BAFF treatment enhanced survival only slightly (Fig. S6A). APRIL and BAFF signaling can down-regulate BIM (24), a proapoptotic molecule whose down-regulation enhances cell survival (25). We next examined the expression level of BIM in APRIL- and BAFF-treated WT and Taci−/− plasma cells. Bim mRNA level was significantly reduced in WT, but not in Taci−/− plasma cells stimulated with APRIL (Fig. 6C). BAFF treatment significantly down-regulated Bim expression in WT plasma cells, but to a much lesser extent in Taci−/− plasma cells (Fig. S6B). Taken together, these data suggest that APRIL and BAFF deliver survival signals to plasma cells predominantly via TACI. To further investigate the role of TACI in plasma cell survival in vivo, we introduced BIM deficiency into Taci−/− mice. Taci−/− Bim−/− mice had comparable Tfh cells and more GC B cells compared with Taci−/−Bim+/+ mice (Fig. S6 C, D, and E). A greater number of ASCs were found in the spleen and BM of Taci−/− Bim−/− mice compared with WT and Taci−/− mice at day 14 postimmunization (Fig 6 D and E). These data suggest that TACI is required for the survival of plasma cells and not for their differentiation. If the latter were the case, then BIM deficiency would not restore the ASC compartment in Taci−/− mice. Discussion We report that loss of TACI leads to expansion of Tfh and GC B cells in mouse PPs and immunized spleens (Fig. 1). This phenomenon is attributed to increased B7H2 expression on Taci−/− B cells (Fig. 2), which enhances cognate interactions between B cells and T cells and further increases costimulation of Tfh cells. The expanded Tfh cells may provide further positive feedback to
Fig. 5. Presence of antigen-specific B cells and absence of plasma cells in immunized Taci−/− mice. (A) Increased numbers of antigen-specific B cells in Taci−/− mice. WT and Taci−/− mice on day 10 postimmunization with NP38-CGG were examined by flow cytometry for the presence of antigen-binding (NIP+) IgG1 B cells in their spleens, and the fraction and total number of these cells were quantified and graphed. (B) Significant reductions in the numbers of plasma cells in the spleen (SP) and BM of Taci−/− mice. The presence of antigen-specific IgG1 plasma cells in the spleen and BM of WT and Taci−/− mice on days 14 and 28 of antigenic challenge were examined via ELISPOT to detect NP2- and NP17-specific ASCs. The frequency and number of ASCs in the spleen and BM were graphed as well. Each dot represents one animal tested. **P < 0.01; ***P < 0.001.
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enlarge the GC B-cell compartment. Thus, TACI regulates the size of the Tfh and GC B-cell compartments by modulating B7H2 expression. Our data indicate that TACI negatively regulates B7H2 expression on B cells. However, it is not clear whether TACI actively signals B7H2 down-modulation or sequesters BAFF away from BAFF-R, which signals B7H2 up-regulation. We favor the latter, because we and others have shown that exogenous BAFF can upregulate B7H2 expression on B cells (26, 27) (Fig. S4A), and because elevated BAFF levels have been found in the sera of Taci−/− mice (28) and TACI-mutated patients (29). Thus, excess BAFF up-regulates B7H2 expression in Taci−/− mice and humans. In the BM chimera experiments, we found no increase in B7H2 expression on Taci−/− B cells compared with WT B cells (Fig. S4B). This is because both WT and Taci−/− B cells are present in the same organism, and there likely is no excess BAFF in the body. BAFF could be captured by neighboring TACI+ B cells in the microenvironment. It is also possible that “soluble TACI” (1) derived from TACI+ B cells can neutralize BAFF, although its existence remains questionable. Future experiments with B cells lacking BAFF-R, TACI, or both or expressing truncated TACI without its signaling domain would help resolve this issue. Intuitively, it would be expected that with increased Tfh and GC B cells, Taci−/− mice would mount an enhanced antigen-specific antibody response to a T-cell–dependent antigen, but this was not the case. We found that Taci−/− mice had reduced antibody titers when challenged with NP38-CGG (Fig. 4 and Fig. S4). We further demonstrated that Taci−/− mice had increased numbers of antigen-specific B cells but significantly reduced numbers of plasma cells (Fig. 5). Previous studies have shown that TACI is highly expressed in plasma cells, whereas BAFF-R expression is decreased upon B-cell differentiation to plasma cells (13, 30). These findings suggest that TACI could have a role in plasma cells. BAFF is known to down-regulate the proapoptotic molecule BIM in B cells (24). Consistent with this, we found that both APRIL and BAFF could significantly down-regulate BIM expression in Ou et al.
WT but not Taci−/− plasma cells, and that the impairment of ASCs in Taci−/− mice could be rectified by ablation of BIM (Fig. 6). This suggests that TACI regulates the survival, rather than the differentiation, of plasma cells. This finding is significant, given that BCMA, a related tumor necrosis factor receptor superfamily (TNFRSF) member, was once thought to regulate the survival of long-lived plasma cells (3). However, immunization of Bcma−/− mice with NP-CGG seemed to elicit normal sera antibody titers (3, 31). In contrast, our current data indicate that Taci−/− mice exhibited reductions in both antibody titers and plasma cell numbers when challenged with NP38-CGG. Given that APRIL could not improve the survival of Taci−/− plasma cells in vitro in which BCMA is present (Fig. 6B), TACI might play a more important role in regulating plasma cell survival. While this manuscript was in preparation, Tsuji et al. (32) reported that TACI plays an important role in plasma cells and signals Blimp-1 expression. However, that study was not clear as to whether TACI signals the survival or differentiation of plasma cells, because Blimp1 is a critical transcriptional factor involved in plasma cell development. Our study complements and extends that work by showing that APRIL engagement of TACI down-regulates BIM expression and increased plasma cell survival and by generating Taci−/−Bim−/− mice to demonstrate that TACI is indeed important for plasma cell survival. TACI mutations in human give rise to CVID and autoimmunity (4–6). Our findings suggest how these two seemingly opposing conditions could arise in patients. TACI regulates plasma cell survival and is thus important for humoral immune response. On the other hand, the loss of TACI leads to increased B7H2 expression and expands Tfh and GC B cells. Increased B7H2 expression (33, 34) and expansion of Tfh and GC B cells (35, 36) have been reported to underlie various autoimmune syndromes. Materials and Methods Mice. Taci−/− mice were obtained from Vishva Dixit (Genentech); C57BL/6 CD45.1, Cd28−/−, and Bim−/− mice were obtained from Jackson Laboratory;
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Fig. 6. TACI is critical for the down-regulation of BIM and plasma cell survival. (A) Plasma cells express TACI. Purified WT and Taci−/− B cells were treated with 20 μg/mL of LPS for 3 d, and TACI expression on plasma cells (B220lowCD138hi) was analyzed by flow cytometry. (B) APRIL stimulation of TACI enhanced plasma cell survival. WT and Taci−/− plasma cells generated via LPS treatment were purified using anti-CD138 microbeads, and their survival was monitored after overnight culture with or without treatment with APRIL, 400 ng/mL. Cell viability was analyzed by annexin-V staining. Numbers indicate the percentage of annexin-V–negative live cells. (C) Quantitative real-time PCR analyses of Bim mRNA expression in WT and Taci−/− plasma cells stimulated as in B. Results are presented relative to the expression of Gapdh mRNA. *P < 0.05. NS, not significant. (D and E) ELISPOT analyses of NP17- and NP2-specific ASCs (D) and quantification of the antigen-specific numbers (E) in the spleen and BM of WT, Taci−/−, Bim−/−, and Taci−/−Bim−/− mice on day 14 postimmunization. Data are representative of two or three independent experiments.
and B7h2−/− mice were generated in the laboratory as described previously (22). Mice were bred to a C57BL/6 background and maintained under specific pathogen-free conditions. The mouse experiments were approved by the A*STAR Biological Resource Centre (BRC) Institutional Animal Care and Use Committee. For analyses of T-cell–dependent antibody responses, mice were immunized i.p. with 100 μg of alum-precipitated NP 38 -CGG (Biosearch Technologies). Flow Cytometry. Single-cell suspensions from spleens, BM, and PPs were prepared and stained with various combinations of fluorochrome-conjugated antibodies to CD4, CD19, CD38, CD45.1, CD45.2, and B220 (BioLegend) and CD40, TCR-β, ICOS, PD-1, CXCR5, Fas, GL7, and B7H2 (BD Biosciences). Antigen-specific B cells were detected as described previously (21) with antiB220 and anti-IgG1 antibodies and NIP and using a mixture of antibodies as Dump. Samples were acquired on an LSRII cytometer (BD Biosciences) and analyzed with FlowJo software (TreeStar). BM Chimeras. BM reconstitution was performed as described previously with minor modifications (37). In brief, 3 × 106 mixed BM cells of CD45.1 WT and CD45.2 WT (1:1) or CD45.1 WT and CD45.2 Taci−/− (1:1) origins were injected i.v. into lethally irradiated C57BL/6 CD45.1 mice (1,000 rads). Recipient mice were left for 6 wk before NP38-CGG immunization. 1. Mackay F, Schneider P (2008) TACI, an enigmatic BAFF/APRIL receptor, with new unappreciated biochemical and biological properties. Cytokine Growth Factor Rev 19: 263–276. 2. Shulga-Morskaya S, et al. (2004) B cell-activating factor belonging to the TNF family acts through separate receptors to support B cell survival and T cell-independent antibody formation. J Immunol 173:2331–2341. 3. O’Connor BP, et al. (2004) BCMA is essential for the survival of long-lived bone marrow plasma cells. J Exp Med 199:91–98. 4. Salzer U, et al. (2005) Mutations in TNFRSF13B encoding TACI are associated with common variable immunodeficiency in humans. Nat Genet 37:820–828. 5. Castigli E, et al. (2005) TACI is mutant in common variable immunodeficiency and IgA deficiency. Nat Genet 37:829–834. 6. Cunningham-Rundles C, Bodian C (1999) Common variable immunodeficiency: Clinical and immunological features of 248 patients. Clin Immunol 92:34–48. 7. von Bülow GU, van Deursen JM, Bram RJ (2001) Regulation of the T-independent humoral response by TACI. Immunity 14:573–582. 8. Yan M, et al. (2001) Activation and accumulation of B cells in TACI-deficient mice. Nat Immunol 2:638–643. 9. Klein U, Dalla-Favera R (2008) Germinal centres: Role in B-cell physiology and malignancy. Nat Rev Immunol 8:22–33. 10. Crotty S (2011) Follicular helper CD4 T cells (TFH). Annu Rev Immunol 29:621–663. 11. Nurieva RI, et al. (2008) Generation of T follicular helper cells is mediated by interleukin-21 but independent of T helper 1, 2, or 17 cell lineages. Immunity 29: 138–149. 12. Ng LG, et al. (2004) B cell-activating factor belonging to the TNF family (BAFF)-R is the principal BAFF receptor facilitating BAFF costimulation of circulating T and B cells. J Immunol 173:807–817. 13. Darce JR, Arendt BK, Wu X, Jelinek DF (2007) Regulated expression of BAFF-binding receptors during human B cell differentiation. J Immunol 179:7276–7286. 14. Castigli E, et al. (2005) TACI and BAFF-R mediate isotype switching in B cells. J Exp Med 201:35–39. 15. Lee JJ, et al. (2009) The murine equivalent of the A181E TACI mutation associated with common variable immunodeficiency severely impairs B-cell function. Blood 114: 2254–2262. 16. Lee JJ, et al. (2010) The C104R mutant impairs the function of transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) through haploinsufficiency. J Allergy Clin Immunol 126:1234–1241. 17. Butcher EC, et al. (1982) Surface phenotype of Peyer’s patch germinal center cells: Implications for the role of germinal centers in B cell differentiation. J Immunol 129: 2698–2707. 18. Weinstein PD, Cebra JJ (1991) The preference for switching to IgA expression by Peyer’s patch germinal center B cells is likely due to the intrinsic influence of their microenvironment. J Immunol 147:4126–4135. 19. Dengler HS, et al. (2008) Distinct functions for the transcription factor Foxo1 at various stages of B cell differentiation. Nat Immunol 9:1388–1398.
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ELISA and ELISPOT. NP-specific antibodies and ASCs were detected via ELISA and ELISPOT, respectively, as described previously (21). Cell Isolation and Culture. Total B cells were isolated from the spleens using anti-CD43 antibody-conjugated microbeads (Miltenyi Biotec) and cultured with 20 μg/mL of LPS for 3 d. Plasma cells were purified with anti-CD138 microbeads and cultured with or without 400 ng/mL of APRIL or BAFF (R&D Systems) overnight for the subsequent survival and RT-PCR analysis. Quantitative Real-Time PCR. Total RNA was isolated using isopropanol precipitation, and the cDNA was prepared with the RevertAid H Minus FirstStrand cDNA Synthesis Kit (Fermentas). SYBR Green Master Mix (Applied Biosystems) was used for real-time PCR. The primer sequences were as follows: Gapdh forward, 5′-TGTGTCCGTCGTGGATCTGA-3′, Gapdh reverse, 5′TTGCTGTTGAAGTCGCAGGAG-3′; Bim forward, 5′-CGACAGTCTCAGGAGGAACC-3′, Bim reverse, 5′-CAATGCCTTCTCCATACCAGA-3′. Statistical Analysis Two-tailed unpaired t tests were performed using GraphPadPrism software. ACKNOWLEDGMENTS. We thank members of the K.-P.L. laboratory for insightful discussion and the A*STAR Biomedical Research Council for grant support.
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