The JI Acurri Cytometers
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rahman, Z. S. M.
Right arrow Articles by Manser, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rahman, Z. S. M.
Right arrow Articles by Manser, T.
The Journal of Immunology, 2004, 173: 6179-6188.
Copyright © 2004 by The American Association of Immunologists

B Cells Expressing Bcl-2 and a Signaling-Impaired BAFF-Specific Receptor Fail to Mature and Are Deficient in the Formation of Lymphoid Follicles and Germinal Centers1

Ziaur S. M. Rahman and Tim Manser2

Department of Microbiology and Immunology and The Kimmel Cancer Institute, Thomas Jefferson Medical College, Philadelphia, PA 19017


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The TNF family cytokine B cell-activating factor belonging to the TNF family (BAFF) (BLyS) plays a fundamental role in regulating peripheral B cell survival and homeostasis. A BAFF-specific receptor (BAFF-R; BR3) appears to mediate these functions via activation of the NF-{kappa}B2 pathway. Signaling by the BAFF-R is also required to sustain the germinal center (GC) reaction. Engagement of this receptor results in the induction of Bcl-2, suggesting that this antiapoptotic factor acts downstream of the BAFF-R and NF-{kappa}B2 pathway to promote peripheral B cell survival during primary and Ag-driven development. To test this idea, we created lines of mice coexpressing a Bcl-2 transgene and a signaling-deficient form of the BAFF-R derived from the B lymphopenic A/WySnJ strain. Surprisingly, although dramatically elevated numbers of B cells accumulate in the periphery of these mice, these B cells exhibit extremely perturbed primary development, formation of lymphoid microenvironments, and GC and IgG responses. Moreover, mice expressing the bcl-2 transgene alone display a loss of marginal zone B cells, an expansion of follicular B cells that appear immature, and alterations of the GC reaction. These results suggest that the BAFF-R and Bcl-2 regulate key and nonoverlapping aspects of peripheral B cell survival and development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
After completing development in the adult bone marrow (BM), 3 immature B cells migrate to peripheral sites, where they undergo additional steps in differentiation and selection before becoming mature cells that can participate in foreign Ag-driven immune responses (1, 2). Although many factors important for the Ag-independent and -dependent stages of peripheral B cell development have been identified, the regulation of these pathways remains incompletely understood. B cell-specific survival and maturation factors appear essential for these processes. Recent studies have identified B cell-activating factor belonging to the TNF family (BAFF; also called TALL-1, THANK, BLyS, and zTNF4) as such a factor. This TNF family cytokine is expressed in membrane-bound and secreted forms (3, 4) and was shown to play a pivotal role in peripheral B cell survival during primary development and homeostasis (5, 6). Three receptors, BCMA (7, 8), TACI (9, 10), and BAFF-R (BR3) (11, 12), expressed by B cells can potentially bind this cytokine. However, BAFF-R is expressed on all peripheral B cells and appears to be specific for BAFF (11, 12). BAFF-deficient (BAFF–/–) mice exhibit a loss of transitional type 2 (T2), follicular, and marginal zone (MZ) B cells, whereas BM B lymphopoiesis, transitional type 1 (T1) B cells, B1 cells, and other hemopoietic cell lineages remain unaffected (5). Mice treated with TACI-Ig display a similar phenotype, indicating a role for BAFF at the T1 to T2 step in peripheral developmental progression (6).

In comparison, BAFF transgenic mice were shown to have increased numbers of B cells in T1 and T2 transitional, follicular, and MZ B cell compartments (13). Elevated levels of serum BAFF were reported in autoimmune diseases (14, 15). In vitro B cell proliferation induced by anti-µ Ab was shown to be augmented by the addition of BAFF (3, 4), and such treatment increases the BAFF-binding capacity of B cells (16). BAFF stimulation also results in prolonged survival of mature resting B cells in vitro (13, 17). The addition of BAFF to cultures of B cells augments class switching to IgG (18). Together, these data suggest that BAFF regulates B cell survival and behavior beyond the transitional stage.

BAFF engagement of the BAFF-R on B cells results in signaling via the NEMO (NF-{kappa}B Essential MOdulator)-independent NF-{kappa}B pathway (19). This leads to the activation of NF-{kappa}B2 and the induction of the antiapoptotic factors Bcl-2 and Bcl-xL (13, 17). Mice deficient in NF-{kappa}B2 display pleiotropic defects in the B cell compartment, including B lymphopenia, poorly developed follicular and MZ regions of secondary lymphoid organs, and failure to mount germinal center (GC) responses (20, 21). Thus, reduced and enhanced activation of the NF-{kappa}B2 pathway and Bcl-2 expression may account for many of the abnormalities displayed by BAFF-deficient and BAFF transgenic mice, respectively.

A/WySnJ mice, a subline of the A strain, are B lymphopenic, displaying a phenotype comparable to that characteristic of BAFF–/– mice (22, 23, 24). The bcmd-1 (B cell maturation defect) locus (25) was shown to contribute to this B lymphopenia in A/WySnJ mice (22, 23). Recent studies have demonstrated that a mutated form of the BAFF-R is encoded by the bcmd-1 locus (9, 11, 12, 26). The last eight amino acids of the cytoplasmic tail of the bcmd-1 form of BAFF-R, a region containing a putative TNF receptor-associated factor binding site, have been replaced by 21 aa derived from a retrotransposon insertion (26). This form of the receptor appears to be expressed normally, but is deficient in activation of the NF-{kappa}B2 pathway (27).

Bcl-2 transgenic expression in the B lineage results in B cell hyperplasia without an induction of cell cycle progression in vivo, and enhanced survival of peripheral B cells in vitro (28, 29, 30). In addition, immunization of Bcl-2 transgenic mice elicits robust and prolonged immune responses (28, 30) and an extended period of memory (31). Similar results were obtained from the analysis of Bcl-xL transgenic mice (32). These results suggest that enforced expression of Bcl-2 or Bcl-xL can over-ride the homeostatic mechanisms that control peripheral B cell numbers. Recently, Amanna et al. (26) have shown that transduction of developing A/WySnJ B cells with a Bcl-xL-expressing retroviral vector could reverse the B lymphopenia and inefficient peripheral B cell maturation characteristic of these mice. Moreover, Sasaki et al. (33) have reported that the introduction of a Bcl-2 transgene into BAFF-R-deficient mice restored mature B cell numbers, but the phenotypic analysis of these cells was limited. In contrast, Tardivel et al. (34) found that although a Bcl-2 transgene could promote B cell survival in a line of TACI-Ig transgenic mice, B cell developmental defects persisted. Such data support the idea that Bcl-2 family members act downstream of the BAFF-R in promoting peripheral B cell survival, but whether such a pathway is involved in the regulation of B cell maturation is unclear.

We previously showed that despite their B lymphopenia, A/WySnJ mice have IgMlow,IgDhigh follicular B cells, and a GC response can be induced in these mice (35). However, this response is not sustained, and this defect is B cell autonomous, indicating that normal BAFF-R signaling in follicular B cells is required for progression of the GC response (35). A/WySnJ mice also have diminished T-dependent (TD) serum Ab responses, and their development of humoral memory is impaired (36). These results and the data discussed above suggested the possibility that both the inefficient primary and Ag-driven development characteristics of A/WySnJ B cells might be due to reduced Bcl-2 family member expression secondary to defective signaling through the bcmd-1 BAFF-R. To address this question, we examined whether constitutive expression of Bcl-2 in the B lineage could rescue efficient primary B cell development and the GC response in mice homozygous for the bcmd-1 locus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice, immunizations, and serology

A/J and A/WySnJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The M23 bcl-2 transgene (28) was crossed to the A strain for at least 12 generations to generate A.bcl-2 mice. These mice were used to generate A/W.bcl-2 mice by crossing and then backcrossing them with A/WySnJ mice. Animals were maintained in a pathogen-free barrier facility. A.bcl-2, A/W.bcl-2, and control mice were immunized i.p. with either SRBC (Lampire Biological Laboratories, Pipersville, PA) as previously described (35) or with 50 µg of (4-hydroxy-3-nitrophenyl) acetyl chicken {gamma} globulin (NP-CGG) in alum for primary responses and with 10 µg of NP-CGG in PBS for secondary responses, as previously described (37). Mice were bled via the retro-orbital sinus, and sera were assayed for anti-NP IgM and IgG Ab titers via ELISA as previously described (37). All mice used in these studies were 8–12 wk old.

Abs and other reagents

Abs and other reagents used for flow cytometry and immunohistology included FITC-GL7 and PE- and FITC-anti-B220 (clone RA3-6B2); biotin-anti-CD35 (clone 8C12); rat IgG Ab to mouse follicular dendritic cells (FDC-M1); streptavidin-CyChrome; biotin-anti-CD3; FITC-anti-CD21/35; PE-anti-CD1d; PE- and biotin-anti-CD23; PE-anti-CD19; FITC-anti-CD24 (heat-stable Ag (HSA)); PE-anti-CD22 and CD62L L-selectin (clone Mel-14); allophycocyanin-anti-B220 (clone RA3-6B2; BD Pharmingen, San Diego, CA); FITC-anti-mouse IgM (clone II/41); biotin-(Fab')2 mouse anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA); HRP-peanut agglutinin (PNA; Sigma-Aldrich, St. Louis, MO); HRP-anti-CD4 (clone GK1.5; made in-house); anti-Ki67 (clone TEC-3) and streptavidin-alkaline phosphatase (DakoCytomation, Glostrup, Denmark); biotin-anti-B220 (clone RA3-6B2); biotin-anti-IgD (clone 11–26; Southern Biotechnology Associates, Birmingham, AL) and streptavidin-PE (Molecular Probes, Eugene, OR); FITC-PNA (Vector Laboratories, Burlingame, CA); rat IgG Ab to mouse follicular dendritic cells (FDC-M2; ImmunoKontact, Abingdon, U.K.); and PE-Cy 5.5-anti-C1qRp (clone AA4.1; a gift from Dr. R. Hardy, Fox Chase Cancer Center, Philadelphia, PA).

Immunohistology

Spleen and lymph node (LN) cryostat sections (5–6 µm) were prepared as previously described (37). Immunohistology was performed using either visible dye or immunofluorescent staining, the stained sections were analyzed using light or fluorescence microscopy (Leitz Diaplan, Wetzlar, Germany), and images were captured as previously described (35).

TUNEL assay

A TUNEL assay was performed on spleen sections as previously described (35) using an ApopTag in situ apoptosis detection kit (Intergen, Purchase, NY). Alkaline phosphatase and HRP-labeled Abs were visualized as described above. GCs were then categorized into small, medium, and large sizes as previously described (35). The level of apoptosis was evaluated by enumerating the number of TUNEL-positive nuclei in small and medium GCs.

Flow cytometry

Flow cytometric analysis was performed on cell suspensions prepared from spleen, LNs, and PBL by staining with multiple combinations of the Abs listed above. Biotinylated Abs were detected with streptavidin-CyChrome. Stained cells were mainly analyzed using an EPICS XL/MCL analyzer (Beckman Coulter, Fullerton, CA). Cells stained with allophycocyanin- and PE-Cy 5.5-conjugated Abs were analyzed using a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA). These data were analyzed using FlowJo software (Treestar, San Carlos, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Bcl-2 promotes B cell accumulation in bcmd-1 mice, but formation of white pulp areas of the spleen and follicles in the LNs is severely impaired

We generated mice expressing the wild-type or bcmd-1 form of the BAFF-R and containing a bcl-2 transgene that is predominantly expressed in B-lineage cells (28). These mice are referred to as A.bcl-2 and A/W.bcl-2, respectively. Quantitation of B cell numbers in the spleen, LN, and peripheral blood of these mice as well as in age-matched A/J and A/WySnJ control mice was conducted by flow cytometry (Table I). In the spleen, both A.bcl-2 and A/W.bcl-2 mice had 3–4 times the total number of B220+ cells as A/J controls. The LNs of A/W.bcl-2 mice contained B220+ cells in numbers similar to those in A/J controls and did not display the mild hyperplasia characteristic of A.bcl-2 mice. Most strikingly, A/W.bcl-2 PBL contained ~35- and 70-fold more B220+ cells than A/J and A/WySnJ controls, respectively, a level of hyperplasia 2–3 times more severe than that observed in A.bcl-2 mice. Analysis of B cell numbers at the pro- and pre-B cell stages in the BM did not indicate significant expansion of any of these compartments in A.bcl-2 or A/W.bcl-2 mice (data not shown), indicating that the effects of enforced Bcl-2 expression and deficient BAFF-R signaling were confined to the immature B cell stage and beyond.


View this table:
[in this window]
[in a new window]
  		Table I. The percentage and total number of lymphocytes and B220– cells in the peripheral organs of A.bcl-2 and A/W.bcl-2 transgenic mice

 
Spleen sections from all four lines of mice were stained with anti-B220 and anti-CD4 Abs to evaluate lymphoid microarchitecture. As we previously reported (35), A/WySnJ mice have B cell follicles, but they are substantially reduced in size and frequency; however, T cell areas appeared relatively normal (Fig. 1, top panels). B cell areas in A.bcl-2 spleens were larger than those in A/J spleens, and increased numbers of B cells were observed in the red pulp, consistent with the data discussed above and with previous studies showing that the bcl-2 transgene induces mild B lymphoid hyperplasia (28, 30). Unexpectedly, in A/W.bcl-2 mice, focal B cell regions of the white pulp were small and infrequent, T cell zones were poorly formed, and the major fraction of B cells was seen scattered in the red pulp.



View larger version (92K):
[in this window]
[in a new window]
  		FIGURE 1. Perturbed secondary lymphoid organ microarchitecture in A/W.bcl-2 mice. Spleen (top and middle panels) and LN (bottom panels) sections from A.bcl-2 and A/J control and A/W.bcl-2 and A/WySnJ control mice were stained with anti-B220 (blue) and anti-CD4 (red). The data are representative of two or three mice from each group. Original magnification of images, x100.

 
Histological analysis of the LNs gave analogous results (Fig. 1, bottom panels). Well-formed follicles in cortical areas were observed in A/J LNs, and these regions were increased in size in A.bcl-2 mice. In A/WySnJ LNs, cortical follicles were observed, but they were smaller and much less frequent than in A/J LNs. In A/W.bcl-2 mice, the B cell areas of LNs were abnormal. Well-formed follicular areas of the cortex were not seen, and most B220+ cells were diffusely organized in the medullary region. In contrast to the situation in A/W.bcl-2 spleen, however, T cell areas of the paracortex appeared normal.

Loss of MZ B cells in A.bcl-2 and A/W.bcl-2 mice

Conflicting results have been published regarding whether enforced Bcl-2 expression perturbs the development of MZ B cells (34, 38). Because B cells in the MZ are IgMhigh,IgDlow, spleen sections obtained from all four types of mice were stained with anti-IgM (green) and anti-IgD (red; Fig. 2A, upper panels), or anti-B220 (red) and metalophillic macrophage-1 (MOMA-1; green), specific for metalophillic cells (Fig. 2A, lower panels) that normally line the follicular border of the MZ. These data indicated a substantial reduction of MZ B cells in both A/W.bcl-2 and A.bcl2 mice, as evidenced by the lack of IgMhigh cells adjacent to IgDhigh follicular B cells (upper panels) and B220+ B cells outside the characteristic ring of MOMA-1 cell staining (lower panels). In addition, the ring of MOMA-1 staining was reduced and somewhat disordered in the large white pulp areas of A.bcl-2 spleens. In the small white pulp areas of A/W.bcl-2 spleens, MOMA-1 weakly stained only a central cluster of cells, indicating a complete lack of the MZ region. In contrast, IgMhigh cells adjacent to IgDhigh B cell areas and B220+ B cells outside of well-formed MOMA-1+ boundaries were clearly observed in A/WySnJ spleens.



View larger version (68K):
[in this window]
[in a new window]
  		FIGURE 2. A.bcl-2 and A/W.bcl-2 mice are deficient in MZ B cells. A, Two spleen sections from mice of each genotype were stained with anti-IgM (green) and anti-IgD (red; upper panels) and with B220 (red) and MOMA-1 (green; lower panels). Original magnification of images, x250. B, Splenocytes were stained with multiple combinations of mAbs specific for the indicated cell surface Ags and analyzed by flow cytometry. A gate is set around the MZ B cell population in each panel. The data represent two pooled spleens of each genotype. Due to the lower levels of expression of CD21, gates in the top and middle panels were set differently for data from A/WySnJ and bcl-2 transgenic mice.

 
In addition to being IgMhigh, MZ B cells are CD21high, CD23low, and CD1dhigh (39). Flow cytometric analysis showed only an ~2-fold reduction in the proportion of A/WySnJ B cells resident in this phenotypic compartment of the spleen (but their CD21 levels were lower than A/J B cells; see below). In contrast, A.bcl-2 mice revealed a substantially reduced frequency of these cells (Fig. 2B), and an almost complete loss of B cells of this cell surface phenotype was observed in A/W.bcl-2 (Fig. 2B) mice.

Many B cells in A/W.bcl-2 mice appear to be immature members of the follicular lineage

The data above demonstrate that the B cell hyperplasia of A/W.bcl-2 mice is not the result of expansion of the mature MZ compartment. In addition, flow cytometric analysis of cell surface markers characteristic of the B1 lineage (e.g., CD5 and CD43) on B cells in spleen, LN, and blood did not indicate elevated numbers of this B cell subset in A/W.bcl-2 mice (data not shown). As such, peripheral B cells were examined for the expression of a variety of cell surface molecules characteristic of the follicular lineage. Staining with the AA4.1 mAb, specific for the C1qRp Ag that is indicative of the immature and transitional stages of follicular development (40), revealed that the ratios of B cells expressing and lacking this marker were similar in all strains (Fig. 3A). However, further subdivision of the AA4.1+ population using CD23 and surface IgM (sIgM) levels showed that, as expected, A/WySnJ mice had expanded T1 subpopulations in spleen and PBL. A/W.bcl-2 mice contained increased T1, and decreased proportions of transitional type 3 (T3) cells, whereas A.bcl-2 mice had somewhat expanded T2 cell numbers in these two compartments. In the LN, the majority of all AA4.1+ cells appeared to be at the T3 stage in all four strains.



View larger version (54K):
[in this window]
[in a new window]
  		FIGURE 3. C1qRp-expressing and -nonexpressing peripheral B cell compartments in mice. Cells from the indicated organ and mouse sources were stained with anti-B220, anti-IgM, anti-CD23, and the anti-C1qRp AA4.1 mAb and subjected to flow cytometric analysis. A, Each left column shows the profiles of B220 and AA4.1 staining of cells in the lymphocyte gate, and the gates set to define AA4.1+ (immature) and AA4.1– (more mature) subpopulations are indicated. Each right column shows the distribution of anti-IgM and anti-CD23 staining among lymphocytes in the AA4.1+ gate. In these panels, gates corresponding to the three previously identified stages of transitional development are indicated. B, The sIgM and CD23 expression profiles of B220+ lymphocytes in the AA4.1– gate are shown. In these panels, cross-hairs have been set on the center of the distribution obtained from A/J mice to serve as a point of references for evaluating levels of expression of these two molecules on B cell in the other types of mice. The data presented were obtained from the pooled cells of three mice of each genotype and are representative of those obtained in three separate experiments.

 
Subdivision of the AA4.1– B cell subpopulation in spleen using sIgM and CD23 levels supported our previous conclusion (35) that A/WySnJ mice contain a significant number of relatively mature, follicular B cells (Fig. 3B). In contrast, most AA4.1– B cells in the spleen and PBL of A.bcl-2 mice displayed elevated levels of sIgM. This was also found to be true in A/W.bcl-2 mice, and most of these cells also expressed reduced levels of CD23. Levels of sIgD were also somewhat decreased on most splenic B cells in A.bcl-2 and A/W.bcl-2 mice (data not shown). In LN, CD23 levels on A/W.bcl-2 B cells were nearer to that expected of follicular B cells, but a major subpopulation of these cells expressed elevated levels of sIgM (Fig. 3B).

These data indicated that although enforced expression of Bcl-2 in mice with the bcmd-1 form of the BAFF-R promotes progression beyond the transitional stages of follicular development, the cells that have traversed these stages remain immature. To further address this idea, the expression of a variety of additional cell surface markers diagnostic of mature, follicular B cells were evaluated. Although many of these were found to be present at levels expected of follicular B cells, exceptions were CD21, expressed at somewhat reduced levels on most A/WySnJ and A.bcl-2 B cells and at substantially reduced levels on most A/W.bcl-2 B cells in all compartments; CD62L, expressed at reduced levels on all B cells in all compartments of A/WySnJ, A.bcl-2, and A/W.bcl-2 mice, with the possible exception of A/WySnJ LN B cells (Fig. 4); and CD24 (HSA) found at elevated levels on the majority of B cells in the PBL of A.bcl-2 and A/W.bcl-2 mice (data not shown). Finally, levels of expression of B220 on B cells in the spleen and blood of both A/W.bcl-2 and A.bcl-2 mice were somewhat lower than those on B cells in control mice (see positions of the B220/AA4.1 gates in Fig. 3A).



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 4. A/W.bcl-2 B cells express reduced levels of CD21 and CD62L. Cells from the indicated sources were stained with mAbs against B220, CD21, and CD62L (L-selectin) and analyzed by flow cytometry. Levels of expression of these markers on A/J (red lines), A/WySnJ (blue lines), A.bcl-2 (green lines), and A/W.bcl-2 (black lines) B220+ cells in the lymphocyte gate are shown. These data are representative of at least two experiments of pooled samples from two mice from each line.

 
Bcl-2 does not rescue, but rather exacerbates, the attenuated GC response resulting from expression of the bcmd-1 form of the BAFF-R

We next examined whether correction of B lymphopenia by the bcl-2 transgene would influence the attenuated Ag-driven GC response characteristic of mice expressing the bcmd-1 form of the BAFF-R (35). A.bcl-2 and A/J control mice and A/W.bcl-2 and A/WySnJ control mice were immunized with SRBC, and spleen sections obtained at multiple time points were stained with PNA and GL7 to identify GCs.

A defective GC response can be manifested by changes in GC size, frequency, and kinetics (35, 41, 42). Therefore, GCs were counted and categorized as small, medium, and large at multiple time points as previously described (35). Although the total number of GCs on days 6 and 9 of the response was comparable in A/J, A.bcl-2, and A/WySnJ mice, there were very few GCs in A/W.bcl-2 spleens at any time point (Fig. 5, Total). In A/W.bcl-2 spleens, there were no large GCs, very few medium GCs, and few small GCs, whereas in A/J and A.bcl-2 spleens, there was a comparable number of small, medium, and large GCs at all time points of the response (Fig. 5). Consistent with our previous data (35), in A/WySnJ spleens there were very few large GCs at any time point, and the number of small and medium GCs was comparable to those in A/J and A.bcl-2 mice at the early stages of the response (days 6 and 9), but total GC numbers decreased rapidly beginning at intermediate stages of the response (day 14).



View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 5. Reduced GC responses in A/W.bcl-2 mice. A/J ({cjs2108}), A.bcl-2 ({blacksquare}), A/WySnJ ({cjs2113}), and A/W.bcl-2 ({square}) mice were immunized with SRBC. Spleen sections from these mice were stained with PNA, and PNA+ small, medium, and large GCs were counted per x100 field. The data were obtained from two sections (four fields) per mouse at each time point; the two sections were at least 40–50 sections apart and included sections from at least three mice from each group.

 
Enforced Bcl-2 expression alone perturbs the GC response

During this histological analysis, we noted that despite being quantitatively and kinetically similar to the A/J response, the GC response in A.bcl-2 mice was qualitatively altered. Representative images of A/J and A.bcl-2 GCs on day 9 are shown in Fig. 6. Uniform and brightly stained GL7+,IgD– cells were observed in all A/J GCs (Fig. 6, A and C, left panels). In contrast, punctate and less intense staining by GL7 of IgD– B cells was observed in most A.bcl-2 GCs (Fig. 6, A and C, right panels). Similar results were noted for PNA staining (Fig. 6B). In addition, staining for the presence of mature FDC reticula using mAbs specific for the FDC-M1, FDC-M2, and Fc{gamma}RIIB (data not shown) markers demonstrated that such networks failed to develop in A.bcl-2 GCs (Fig. 6A, right panels). However, the primary follicular reticulum, GC T cells numbers and locale (Fig. 6C), and Bcl-6 expression by GC B cells (data not shown) appeared normal. These characteristics were also noted in the few GCs that developed after immunization of A/W.bcl-2 mice (data not shown).



View larger version (111K):
[in this window]
[in a new window]
  		FIGURE 6. Altered splenic GC response in A.bcl-2 mice. Spleen sections from A.bcl-2, A/WySnJ, and A/J control mice on day 9 of the SRBC response were stained A) with GL7 (green) and FDC-M1 and FDC-M2 (both in red); B) with PNA (green) and anti-IgD (red); and C) with anti-CD35 (CR1) in red and GL7 (green; upper panels) and with anti-CD3 (red) and GL7 (green; lower panels). The data are representative of three or four mice from each group. Original magnification of images, x250.

 
We recently reported altered Ki67 (a nuclear proliferation Ag) expression in A/WySnJ GCs despite the fact that A/WySnJ B cells harbor no generic defect in their ability to express high levels of Ki67 expression in vitro (35). To determine whether such a defect might account for the altered GC responses of A.bcl-2 and A/W.bcl-2 mice, the expression of Ki67 was monitored at multiple stages of the GC response in these mice. GL7+ cells in A.bcl-2 GCs were uniformly Ki67+ throughout the response (data not shown). Analysis of Ki67 expression in A/W.bcl-2 GCs was difficult due to their substantially reduced size, frequency, and GL7 staining intensity, but such GCs did appear to usually contain Ki67+ cells. In contrast, reduced or undetectable levels of Ki67 expression were observed in A/WySnJ GCs (data not shown), consistent with our previous data (35).

Decreased apoptosis in GCs of A/W.bcl-2 transgenic mice

To investigate whether the failure of Bcl-2 to rescue the A/WySnJ GC response might be due to inability to block GC B cell apoptosis in the context of expression of the defective BAFF-R, we performed TUNEL assays on spleen sections obtained from all four groups of mice on day 9 post-SRBC immunization. Only small GCs from A/W.bcl-2 mice and small and medium GCs from A/J, A.bcl-2, and A/WySnJ mice were considered in the analysis, because there were no large GCs in A/W.bcl-2 or A/WySnJ mice. The average number of apoptotic nuclei (Fig. 7, horizontal bars) in GCs of A/J and A/WySnJ control mice was comparable in small and medium GCs (Fig. 7), as previously reported (35). The number of apoptotic nuclei was decreased to similar levels in A.bcl-2 and A/W.bcl-2 GCs compared with these control levels.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 7. Levels of apoptosis in GCs. Eight randomly chosen small GCs in stained sections from A/W.bcl-2 and small and medium GCs from A.bcl-2 and control mice were analyzed. Apoptotic activity was assessed on day 9 of the SRBC response by enumerating TUNEL-positive nuclei. Horizontal bars represent mean values.

 
Altered TD serum Ab responses in A/W.bcl-2 mice

The severely attenuated and altered GC responses characteristic of A/W.bcl-2 and A.bcl-2 mice, respectively, suggested that the generation of class-switched serum Ab might be compromised in these mice. To test this idea, mice were immunized with the TD Ag NP-CGG, and amounts of IgM and IgG anti-NP serum Abs were assayed at various times thereafter. Table II shows that IgM anti-NP titers elicited in A/W.bcl-2 mice were elevated compared with those in A/J controls, but the serum IgG responses were similar. Because A/W.bcl-2 mice contain dramatically elevated numbers of B cells, this suggests that, on a per cell basis, the A/W.bcl-2 B cell compartment can mount a robust IgM, but not an IgG, response. Consistent with previously published data (30, 31, 34), A.bcl-2 mice produced substantially elevated levels of both serum IgM and IgG anti-NP Ab, indicating that the GC defects described above do not overtly affect class switching and Ab-forming cell formation.


View this table:
[in this window]
[in a new window]
  		Table II. Serum Ig titers in A.bcl-2 and A/W.bcl-2 transgenic mice immunized with NP-CGGa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Enormous numbers of B cells accumulate in the blood and affiliated tissues of A/W.bcl-2 mice, demonstrating that enforced Bcl-2 expression completely over-rides the deficiency in BAFF-R signaling that results in reduced peripheral B cell life span in A/WySnJ mice. Similarly, Amanna et al. (26) showed that retroviral expression of Bcl-xL in developing A/WySnJ B cells restores normal splenic B cell numbers. However, the B cells that populate the periphery of A/W.bcl-2 mice are phenotypically immature and are incompatible with the formation of splenic white pulp, LN follicles, and GCs as well as the generation of a robust TD IgG serum Ab response. Thus, contrary to our initial expectation that enforced expression of Bcl-2 would allow BAFF-R signaling-deficient B cells to more efficiently progress to the mature follicular phenotype, such expression exacerbates and expands the defects in peripheral homeostasis and development characteristic of these cells.

Moreover, enforced Bcl-2 expression does not appear to rectify attenuated progression through the transitional stages of follicular B cell development due to deficient signaling of the bcmd-1 form of the BAFF-R. The PBL, spleen, and even LNs of A/W.bcl-2 mice contain increased proportions of T1 and reduced proportions of T3 cells relative to A/J and A.bcl-2 mice. Nonetheless, AA4.1– B cells accumulate in large numbers in all these compartments of A/W.bcl-2 mice, indicating that the inhibition of apoptotic pathways conferred by enforced Bcl-2 expression allows transitional B cells that would normally die due to insufficient trophic factor stimulation to progress beyond the transitional stage. Thompson et al. (43) suggested that lymphocyte trophic factor signaling pathways are directly linked to those controlling cellular energetics, and in the absence of appropriate activity of the former, apoptotic pathways are triggered via the mitochondrial pathway of programmed cell death. If, however, this death pathway is blocked by enforced expression of Bcl-2 or Bcl-xL, trophic factor signaling-deprived lymphocytes survive, but in a crippled state due to insufficient acquisition and metabolism of extracellular energy sources such as glucose. Given these considerations, future studies to determine whether the phenotypic and functional defects characteristic of A/W.bcl-2 B cells that have progressed beyond the transitional stage are an indirect consequence of perturbation of cellular metabolic pathways seem warranted.

Like A/W.bcl-2 and A.bcl-2 mice, mice deficient in NF-{kappa}B2 also display disturbed primary development of peripheral B cells and B cell microenvironments, including the MZ (20, 21). Because A/WySnJ mice do not reveal major qualitative defects in this regard, the bcmd-1 form of the BAFF-R may allow levels of NF-{kappa}B2 activation sufficient to sustain reduced, but relatively normal, development of mature B cells and their peripheral microenvironments. In contrast, enforced Bcl-2 expression may qualitatively alter signaling via the NF-{kappa}B2 pathway, such that certain aspects of the peripheral B cell maturation program are modified, including formation of the MZ compartment and complete activation during the GC response.

Under normal circumstances, the NF-{kappa}B pathways regulate Bcl-2 expression, but when such expression is enforced, feedback effects might perturb NF-{kappa}B signaling networks, resulting in pleiotropic effects on B cell development. Given these scenarios, it will be important to evaluate whether the synergistic effects of enforced Bcl-2 and bcmd-1 BAFF-R expression on primary B cell development might be accounted for by the combination of qualitative and quantitative perturbations of the NF-{kappa}B2 pathway. In addition, because B cells play a nucleating role in generation of both B and T cell regions of the splenic white pulp (44, 45), future studies to determine whether the unusual B cells that accumulate in A/W.bcl-2 mice are deficient in production of the cytokines and chemokines required for formation of lymphoid microarchitecture are suggested. It is tempting to speculate that the massive buildup of B cells in the blood and its affiliated tissues of A/W.bcl-2 mice is due in part to an indirect effect of perturbed formation and population of the follicular and MZ microenvironments.

Despite the severely inhibited progression from the T1 to the T2 stage in A/WySnJ mice, follicular and MZ B cells develop, splenic and LN follicles form, and B cells appear to enter the recirculating pool. Nonetheless, these cells cannot produce a normal GC response, apparently due to a cell autonomous proliferative defect (35), and express reduced levels of CD21 and CD62L. This indicates that in the absence of normal BAFF-R signaling, most peripheral B cells are incapable of differentiating to full maturity, perhaps simply because they do not live long enough to do so. This idea is supported by our finding that all peripheral B cell compartments in the spleen, LN, and blood are substantially reduced in size in A/WySnJ mice as well as by previous BrdU labeling studies showing that the half-life of B cells in both immature and mature splenic compartments is decreased by the bcmd-1 mutation (23, 24, 26). Regarding the defective GC response, Gorelik et al. (46) have recently reported that BAFF dynamically regulates CD21 levels on peripheral B cells. In addition, Fischer et al. (47) have found that CD21-deficient B cells can enter GCs, but do not survive in this microenvironment. Therefore, whether reduced CD21 expression secondary to defective BAFF-R signaling contributes to the inability of A/WySnJ B cells to sustain the GC response is worthy of future investigation.

Probably, the abnormal secondary lymphoid architecture of A/W.bcl-2 mice is a major factor explaining their substantially reduced GC and inefficient TD serum IgG responses. Indeed, the few GCs that formed in these mice exhibited near-normal levels of Ki67 and Bcl-6 expression and reduced levels of apoptosis, characteristic of Bcl-2 transgenic mice. However, in A.bcl-2 mice, which have well-developed white pulp areas and large numbers of recirculating B cells, the splenic GC response is also abnormal, as evidenced by the lack of development of the secondary FDC reticulum and reduced levels of GC B cell expression of the ligands for PNA and the GL7 mAb. Although the defects in the GC response of A/WySnJ mice also include inhibited development of the secondary GC stroma, this is associated with reduced or undetectable Ki67 expression by GC B cells, but normal levels of staining with GL7 and PNA. In contrast, GCs in A.bcl-2 mice express normal levels of Ki67, indicating that the mechanisms responsible for alteration of the GC response in A/WySnJ and A.bcl-2 mice are distinct.

Enforced Bcl-2 expression inhibits GC B cell apoptosis, but also slows cell cycle progression (48, 49, 50). Indeed, GC B cells normally express very low levels of Bcl-2 (51, 52), and this might contribute both to the ability to undergo rapid progression through the cell cycle as well as the susceptibility to selection processes that act via apoptotic pathways. The combined effects of enforced Bcl-2 expression may allow a quantitatively normal GC response to develop in A.bcl-2 mice in a situation where the rate of proliferation of GC B cells is slowed, perhaps accounting for their reduced expression of the ligands for GL7 and PNA. Regarding the inhibition of development of the secondary FDC reticulum, given previous data suggesting an important role of MZ B cells in Ag and immune complex transport into B cell follicles and GCs during immune responses (53, 54), the near absence of this B cell subset in bcl-2 transgenic mice may reduce such transport, contributing to this defect. Because bcl-2 transgenic mice have been used by us (55, 56) and others (57, 58) to investigate the mechanisms of positive and negative selection that shape the composition of the memory B cell compartment, determining whether MZ B cell, GC B cell, or FDC developmental defects in such mice predominantly influence these selection processes is an important area for future analysis.

In contrast to the expression of the bcmd-1 form of BAFF-R, enforced Bcl-2 expression not only blocks MZ B cell development, but also results in accumulation of follicular B cells that appear less mature than conventional follicular cells. Like A/WySnJ B cells, bcl-2 transgenic B cells express reduced levels of CD21 and CD62L, but they also predominantly have an IgMint,IgDint phenotype in the spleen, unlike A/WySnJ follicular B cells. Because the majority of splenic and LN bcl-2 transgenic B cells do not express high levels of HSA, they seem to have progressed well beyond the T1 stage of development. This is particularly true in the LN, where their sIgM and sIgD levels are characteristic of mature follicular cells. Although a current subject of controversy, the T2 stage may include a period of cell cycle progression (40, 59, 60), and it has been suggested that this could reflect the positive selection of certain B cell clonotypes into the stable, peripheral pool of B cells via BCR-mediated signaling events (59, 60). In this regard, the idea that positive selection due to BCR engagement is important for the development of MZ B cells has received experimental support (61), and such a process may play a role in the maturation of follicular B cells as well (2). Thus, by disrupting cell cycle progression at the intermediate to late stages of transitional development, enforced Bcl-2 expression may preclude the BCR-mediated positive selection of B cells into the MZ compartment and inhibit certain BCR-induced events necessary for completion of follicular development.

Previous studies have attempted to rescue B cell development in mice with deficiencies in BCR expression or signaling via enforced Bcl-2 expression. A bcl-2 transgene was shown to promote peripheral accumulation of surface BCR negative B lymphocytes in scid, Rag1–/–, and µMT mice (62, 63), but the levels of CD21 and CD23 on these cells were very low. When a functional IgM H chain transgene was introduced into Rag-2–/– mice expressing this bcl-2 transgene, B cells populated splenic follicles, but their expressions of B220, CD21, CD22, and CD23 were reduced compared with those of mature B cells (64). Bcl-2 expression expands the T2-like B cell compartment in Btk-deficient xid mice, but the cells that accumulate continue to resemble and behave like T2 transitional cells (65, 66). Finally, enforced Bcl-2 expression increases the numbers of B cells in the BM and spleen of mice deficient in the B cell-specific transcriptional coactivator BOB.1/OBF.1, but does not reverse the deficiencies in MZ B cells and pre-GC B cells characteristic of these mice (38). Our data suggest that interpretation of the role of BCR signaling in driving B cell maturation using this approach could be confounded by the fact that in and of itself, enforced Bcl-2 expression perturbs this process.


   Acknowledgments
 
We thank Scot Fenn for technical assistance, and all members of the Manser laboratory for their indirect contributions. We also thank Dr. Randy Hardy (Fox Chase Cancer Center, Philadelphia, PA) for providing the AA4.1 mAb.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant AI46806 (to T.M.). Back

2 Address correspondence and reprint requests to Dr. Tim Manser, Department of Microbiology and Immunology and The Kimmel Cancer Center, Jefferson Medical College, Bluemle Life Sciences Building 708, 233 South 10th Street, Philadelphia, PA 19017-5541. E-mail address: manser{at}mail.jci.tju.edu Back

3 Abbreviations used in this paper: BM, bone marrow; BAFF, B cell-activating factor belonging to the TNF family; Bcmd, B cell maturation defect; FDC, follicular dendritic cell; GC, germinal center; HSA, heat-stable Ag; LN, lymph node; MOMA-1, metalophillic macrophage-1; MZ, marginal zone; PNA, peanut agglutinin; sIgM, surface IgM; T1, transitional type 1; T2, transitional type 2; T3, transitional type 3; NP-CGG (4-hydroxy-3-nitrophenyl) acetyl chicken {gamma} globulin; TD, T dependent. Back

Received for publication March 18, 2004. Accepted for publication September 14, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rajewsky, K.. 1996. Clonal selection and learning in the antibody system. Nature 381:751.[Medline]
  2. Pillai, S.. 1999. The chosen few? Positive selection and the generation of naive B lymphocytes. Immunity 10:493.[Medline]
  3. Schneider, P., F. MacKay, V. Steiner, K. Hofmann, J. L. Bodmer, N. Holler, C. Ambrose, P. Lawton, S. Bixler, H. Acha-Orbea, et al 1999. BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth. J. Exp. Med. 189:1747.[Abstract/Free Full Text]
  4. Moore, P. A., O. Belvedere, A. Orr, K. Pieri, D. W. LaFleur, P. Feng, D. Soppet, M. Charters, R. Gentz, D. Parmelee, et al 1999. BLyS: member of the tumor necrosis factor family and B lymphocyte stimulator. Science 285:260.[Abstract/Free Full Text]
  5. Schiemann, B., J. L. Gommerman, K. Vora, T. G. Cachero, S. Shulga-Morskaya, M. Dobles, E. Frew, M. L. Scott. 2001. An essential role for BAFF in the normal development of B cells through a BCMA-independent pathway. Science 293:2111.[Abstract/Free Full Text]
  6. Gross, J. A., S. R. Dillon, S. Mudri, J. Johnston, A. Littau, R. Roque, M. Rixon, O. Schou, K. P. Foley, H. Haugen, et al 2001. TACI-Ig neutralizes molecules critical for B cell development and autoimmune disease: impaired B cell maturation in mice lacking BLyS. Immunity 15:289.[Medline]
  7. Gross, J. A., J. Johnston, S. Mudri, R. Enselman, S. R. Dillon, K. Madden, W. Xu, J. Parrish-Novak, D. Foster, C. Lofton-Day, et al 2000. TACI and BCMA are receptors for a TNF homologue implicated in B-cell autoimmune disease. Nature 404:995.[Medline]
  8. Shu, H. B., H. Johnson. 2000. B cell maturation protein is a receptor for the tumor necrosis factor family member TALL-1. Proc. Natl. Acad. Sci. USA 97:9156.[Abstract/Free Full Text]
  9. Yan, M., S. A. Marsters, I. S. Grewal, H. Wang, A. Ashkenazi, V. M. Dixit. 2000. Identification of a receptor for BLyS demonstrates a crucial role in humoral immunity. Nat. Immunol. 1:37.[Medline]
  10. Xia, X. Z., J. Treanor, G. Senaldi, S. D. Khare, T. Boone, M. Kelley, L. E. Theill, A. Colombero, I. Solovyev, F. Lee, et al 2000. TACI is a TRAF-interacting receptor for TALL-1, a tumor necrosis factor family member involved in B cell regulation. J. Exp. Med. 192:137.[Abstract/Free Full Text]
  11. Thompson, J. S., S. A. Bixler, F. Qian, K. Vora, M. L. Scott, T. G. Cachero, C. Hession, P. Schneider, I. D. Sizing, C. Mullen, et al 2001. BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF. Science 293:2108.[Abstract/Free Full Text]
  12. Yan, M., J. R. Brady, B. Chan, W. P. Lee, B. Hsu, S. Harless, M. Cancro, I. S. Grewal, V. M. Dixit. 2001. Identification of a novel receptor for B lymphocyte stimulator that is mutated in a mouse strain with severe B cell deficiency. Curr. Biol. 11:1547.[Medline]
  13. Batten, M., J. Groom, T. G. Cachero, F. Qian, P. Schneider, J. Tschopp, J. L. Browning, F. Mackay. 2000. BAFF mediates survival of peripheral immature B lymphocytes. J. Exp. Med. 192:1453.[Abstract/Free Full Text]
  14. Zhang, J., V. Roschke, K. P. Baker, Z. Wang, G. S. Alarcon, B. J. Fessler, H. Bastian, R. P. Kimberly, T. Zhou. 2001. Cutting edge: a role for B lymphocyte stimulator in systemic lupus erythematosus. J. Immunol. 166:6.[Abstract/Free Full Text]
  15. Groom, J., S. L. Kalled, A. H. Cutler, C. Olson, S. A. Woodcock, P. Schneider, J. Tschopp, T. G. Cachero, M. Batten, J. Wheway, et al 2002. Association of BAFF/BLyS overexpression and altered B cell differentiation with Sjogren’s syndrome. J. Clin. Invest. 109:59.[Medline]
  16. Smith, S. H., M. P. Cancro. 2003. Cutting edge: B cell receptor signals regulate BLyS receptor levels in mature B cells and their immediate progenitors. J. Immunol. 170:5820.[Abstract/Free Full Text]
  17. Do, R. K., E. Hatada, H. Lee, M. R. Tourigny, D. Hilbert, S. Chen-Kiang. 2000. Attenuation of apoptosis underlies B lymphocyte stimulator enhancement of humoral immune response. J. Exp. Med. 192:953.[Abstract/Free Full Text]
  18. Litinskiy, M. B., B. Nardelli, D. M. Hilbert, B. He, A. Schaffer, P. Casali, A. Cerutti. 2002. DCs induce CD40-independent immunoglobulin class switching through BLyS and APRIL. Nat. Immunol. 3:822.[Medline]
  19. Claudio, E., K. Brown, S. Park, H. Wang, U. Siebenlist. 2002. BAFF-induced NEMO-independent processing of NF-{kappa}B2 in maturing B cells. Nat. Immunol. 3:958.[Medline]
  20. Caamano, J. H., C. A. Rizzo, S. K. Durham, D. S. Barton, C. Raventos-Suarez, C. M. Snapper, R. Bravo. 1998. Nuclear factor (NF)-{kappa}B2 (p100/p52) is required for normal splenic microarchitecture and B cell-mediated immune responses. J. Exp. Med. 187:185.[Abstract/Free Full Text]
  21. Franzoso, G., L. Carlson, L. Poljak, E. W. Shores, S. Epstein, A. Leonardi, A. Grinberg, T. Tran, T. Scharton-Kersten, M. Anver, et al 1998. Mice deficient in nuclear factor (NF)-{kappa}B/p52 present with defects in humoral responses, germinal center reactions, and splenic microarchitecture. J. Exp. Med. 187:147.[Abstract/Free Full Text]
  22. Miller, D. J., C. E. Hayes. 1991. Phenotypic and genetic characterization of a unique B lymphocyte deficiency in strain A/WySnJ mice. Eur. J. Immunol. 21:1123.[Medline]
  23. Lentz, V. M., M. P. Cancro, F. E. Nashold, C. E. Hayes. 1996. Bcmd governs recruitment of new B cells into the stable peripheral B cell pool in the A/WySnJ mouse. J. Immunol. 157:598.[Abstract]
  24. Lentz, V. M., C. E. Hayes, M. P. Cancro. 1998. Bcmd decreases the life span of B-2 but not B-1 cells in A/WySnJ mice. J. Immunol. 160:3743.[Abstract/Free Full Text]
  25. Hoag, K. A., K. Clise-Dwyer, Y. H. Lim, F. E. Nashold, J. Gestwicki, M. P. Cancro, C. E. Hayes. 2000. A quantitative-trait locus controlling peripheral B-cell deficiency maps to mouse chromosome 15. Immunogenetics 51:924.[Medline]
  26. Amanna, I. J., J. P. Dingwall, C. E. Hayes. 2003. Enforced bcl-xL gene expression restored splenic B lymphocyte development in BAFF-R mutant mice. J. Immunol. 170:4593.[Abstract/Free Full Text]
  27. Kayagaki, N., M. Yan, D. Seshasayee, H. Wang, W. Lee, D. M. French, I. S. Grewal, A. G. Cochran, N. C. Gordon, J. Yin, et al 2002. BAFF/BLyS receptor 3 binds the B cell survival factor BAFF ligand through a discrete surface loop and promotes processing of NF-{kappa}B2. Immunity 17:515.[Medline]
  28. McDonnell, T. J., N. Deane, F. M. Platt, G. Nunez, U. Jaeger, J. P. McKearn, S. J. Korsmeyer. 1989. Bcl-2-immunoglobulin transgenic mice demonstrate extended B cell survival and follicular lymphoproliferation. Cell 57:79.[Medline]
  29. McDonnell, T. J., G. Nunez, F. M. Platt, D. Hockenberry, L. London, J. P. McKearn, S. J. Korsmeyer. 1990. Deregulated Bcl-2-immunoglobulin transgene expands a resting but responsive immunoglobulin M and D-expressing B-cell population. Mol. Cell. Biol. 10:1901.[Abstract/Free Full Text]
  30. Strasser, A., S. Whittingham, D. L. Vaux, M. L. Bath, J. M. Adams, S. Cory, A. W. Harris. 1991. Enforced BCL2 expression in B-lymphoid cells prolongs antibody responses and elicits autoimmune disease. Proc. Natl. Acad. Sci. USA 88:8661.[Abstract/Free Full Text]
  31. Nunez, G., D. Hockenbery, T. J. McDonnell, C. M. Sorensen, S. J. Korsmeyer. 1991. Bcl-2 maintains B cell memory. Nature 353:71.[Medline]
  32. Grillot, D. A., R. Merino, J. C. Pena, W. C. Fanslow, F. D. Finkelman, C. B. Thompson, G. Nunez. 1996. Bcl-x exhibits regulated expression during B cell development and activation and modulates lymphocyte survival in transgenic mice. J. Exp. Med. 183:381.[Abstract/Free Full Text]
  33. Sasaki, Y., S. Casola, J. L. Kutok, K. Rajewsky, M. Schmidt-Supprian. 2004. TNF family member B cell-activating factor (BAFF) receptor-dependent and -independent roles for BAFF in B cell physiology. J. Immunol. 173:2245.[Abstract/Free Full Text]
  34. Tardivel, A., A. Tinel, S. Lens, Q. G. Steiner, E. Sauberli, A. Wilson, F. Mackay, A. G. Rolink, F. Beermann, J. Tschopp, et al 2004. The anti-apoptotic factor Bcl-2 can functionally substitute for the B cell survival but not for the marginal zone B cell differentiation activity of BAFF. Eur. J. Immunol. 34:509.[Medline]
  35. Rahman, Z. S., S. P. Rao, S. L. Kalled, T. Manser. 2003. Normal induction but attenuated progression of germinal center responses in BAFF and BAFF-R signaling-deficient mice. J. Exp. Med. 198:1157.[Abstract/Free Full Text]
  36. Miller, D. J., K. D. Hanson, J. A. Carman, C. E. Hayes. 1992. A single autosomal gene defect severely limits IgG but not IgM responses in B lymphocyte-deficient A/WySnJ mice. Eur. J. Immunol. 22:373.[Medline]
  37. Vora, K. A., J. V. Ravetch, T. Manser. 1997. Amplified follicular immune complex deposition in mice lacking the Fc receptor {gamma}-chain does not alter maturation of the B cell response. J. Immunol. 159:2116.[Abstract/Free Full Text]
  38. Brunner, C., D. Marinkovic, J. Klein, T. Samardzic, L. Nitschke, T. Wirth. 2003. B cell-specific transgenic expression of Bcl2 rescues early B lymphopoiesis but not B cell responses in BOB.1/OBF.1-deficient mice. J. Exp. Med. 197:1205.[Abstract/Free Full Text]
  39. Martin, F., J. F. Kearney. 1999. CD21high IgMhigh splenic B cells enriched in the marginal zone: distinct phenotypes and functions. Curr. Top. Microbiol. Immunol. 246:45.[Medline]
  40. Allman, D., R. C. Lindsley, W. DeMuth, K. Rudd, S. A. Shinton, R. R. Hardy. 2001. Resolution of three nonproliferative immature splenic B cell subsets reveals multiple selection points during peripheral B cell maturation. J. Immunol. 167:6834.[Abstract/Free Full Text]
  41. Alabyev, B., T. Manser. 2002. Bcl-2 rescues the germinal center response but does not alter the V gene somatic hypermutation spectrum in MSH2-deficient mice. J. Immunol. 169:3819.[Abstract/Free Full Text]
  42. Han, S., K. Hathcock, B. Zheng, T. B. Kepler, R. Hodes, G. Kelsoe. 1995. Cellular interaction in germinal centers. Roles of CD40 ligand and B7-2 in established germinal centers. J. Immunol. 155:556.[Abstract]
  43. Rathmell, J. C., M. G. Vander Heiden, M. H. Harris, K. A. Frauwirth, C. B. Thompson. 2000. In the absence of extrinsic signals, nutrient utilization by lymphocytes is insufficient to maintain either cell size or viability. Mol. Cell 6:683.[Medline]
  44. Fu, Y. X., D. D. Chaplin. 1999. Development and maturation of secondary lymphoid tissues. Annu. Rev. Immunol. 17:399.[Medline]
  45. Ngo, V. N., R. J. Cornall, J. G. Cyster. 2001. Splenic T zone development is B cell dependent. J. Exp. Med. 194:1649.[Abstract/Free Full Text]
  46. Gorelik, L., A. H. Cutler, G. Thill, S. D. Miklasz, D. E. Shea, C. Ambrose, S. A. Bixler, L. Su, M. L. Scott, S. L. Kalled. 2004. Cutting Edge: BAFF regulates CD21/35 and CD23 expression independent of its B cell survival function. J. Immunol. 172:762.[Abstract/Free Full Text]
  47. Fischer, M. B., S. Goerg, L. Shen, A. P. Prodeus, C. C. Goodnow, G. Kelsoe, M. C. Carroll. 1998. Dependence of germinal center B cells on expression of CD21/CD35 for survival. Science 280:582.[Abstract/Free Full Text]
  48. O’Reilly, L. A., A. W. Harris, D. M. Tarlinton, L. M. Corcoran, A. Strasser. 1997. Expression of a bcl-2 transgene reduces proliferation and slows turnover of developing B lymphocytes in vivo. J. Immunol. 159:2301.[Abstract/Free Full Text]
  49. O’Reilly, L. A., D. C. Huang, A. Strasser. 1996. The cell death inhibitor Bcl-2 and its homologues influence control of cell cycle entry. EMBO J. 15:6979.[Medline]
  50. Mazel, S., D. Burtrum, H. T. Petrie. 1996. Regulation of cell division cycle progression by bcl-2 expression: a potential mechanism for inhibition of programmed cell death. J. Exp. Med. 183:2219.[Abstract/Free Full Text]
  51. Klein, U., Y. Tu, G. A. Stolovitzky, J. L. Keller, J. Haddad, Jr, V. Miljkovic, G. Cattoretti, A. Califano, R. Dalla-Favera. 2003. Transcriptional analysis of the B cell germinal center reaction. Proc. Natl. Acad. Sci. USA 100:2639.[Abstract/Free Full Text]
  52. Martinez-Valdez, H., C. Guret, O. de Bouteiller, I. Fugier, J. Banchereau, Y. J. Liu. 1996. Human germinal center B cells express the apoptosis-inducing genes Fas, c-myc, P53, and Bax but not the survival gene bcl-2. J. Exp. Med. 183:971.[Abstract/Free Full Text]
  53. Gray, D., I. McConnell, D. S. Kumararatne, I. C. MacLennan, J. H. Humphrey, H. Bazin. 1984. Marginal zone B cells express CR1 and CR2 receptors. Eur. J. Immunol. 14:47.[Medline]
  54. Heinen, E., M. Braun, P. G. Coulie, J. Van Snick, M. Moeremans, N. Cormann, C. Kinet-Denoel, L. J. Simar. 1986. Transfer of immune complexes from lymphocytes to follicular dendritic cells. Eur. J. Immunol. 16:167.[Medline]
  55. Notidis, E., L. Heltemes, T. Manser. 2002. Dominant, hierarchical induction of peripheral tolerance during foreign antigen-driven B cell development. Immunity 17:317.[Medline]
  56. Hande, S., E. Notidis, T. Manser. 1998. Bcl-2 obstructs negative selection of autoreactive, hypermutated antibody V regions during memory B cell development. Immunity 8:189.[Medline]
  57. Smith, K. G., A. Light, L. A. O’Reilly, S. M. Ang, A. Strasser, D. Tarlinton. 2000. Bcl-2 transgene expression inhibits apoptosis in the germinal center and reveals differences in the selection of memory B cells and bone marrow antibody-forming cells. J. Exp. Med. 191:475.[Abstract/Free Full Text]
  58. Smith, K. G., U. Weiss, K. Rajewsky, G. J. Nossal, D. M. Tarlinton. 1994. Bcl-2 increases memory B cell recruitment but does not perturb selection in germinal centers. Immunity 1:803.[Medline]
  59. Loder, F., B. Mutschler, R. J. Ray, C. J. Paige, P. Sideras, R. Torres, M. C. Lamers, R. Carsetti. 1999. B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals. J. Exp. Med. 190:75.[Abstract/Free Full Text]
  60. Su, T. T., D. J. Rawlings. 2002. Transitional B lymphocyte subsets operate as distinct checkpoints in murine splenic B cell development. J. Immunol. 168:2101.[Abstract/Free Full Text]
  61. Martin, F., J. F. Kearney. 2000. Positive selection from newly formed to marginal zone B cells depends on the rate of clonal production, CD19, and btk. Immunity 12:39.[Medline]
  62. Strasser, A., A. W. Harris, L. M. Corcoran, S. Cory. 1994. Bcl-2 expression promotes B- but not T-lymphoid development in scid mice. Nature 368:457.[Medline]
  63. Tarlinton, D. M., L. M. Corcoran, A. Strasser. 1997. Continued differentiation during B lymphopoiesis requires signals in addition to cell survival. Int. Immunol. 9:1481.[Abstract/Free Full Text]
  64. Young, F., E. Mizoguchi, A. K. Bhan, F. W. Alt. 1997. Constitutive Bcl-2 expression during immunoglobulin heavy chain-promoted B cell differentiation expands novel precursor B cells. Immunity 6:23.[Medline]
  65. Woodland, R. T., M. R. Schmidt, S. J. Korsmeyer, K. A. Gravel. 1996. Regulation of B cell survival in xid mice by the proto-oncogene bcl-2. J. Immunol. 156:2143.[Abstract]
  66. Middendorp, S., R. W. Hendriks. 2004. Cellular maturation defects in Bruton’s tyrosine kinase-deficient immature B cells are amplified by premature B cell receptor expression and reduced by receptor editing. J. Immunol. 172:1371.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
G. Chattopadhyay, A. Q. Khan, G. Sen, J. Colino, W. duBois, A. Rubtsov, R. M. Torres, M. Potter, and C. M. Snapper
Transgenic Expression of Bcl-xL or Bcl-2 by Murine B Cells Enhances the In Vivo Antipolysaccharide, but Not Antiprotein, Response to Intact Streptococcus pneumoniae
J. Immunol., December 1, 2007; 179(11): 7523 - 7534.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
P. Neri, S. Kumar, M. T. Fulciniti, S. Vallet, S. Chhetri, S. Mukherjee, Y. Tai, D. Chauhan, P. Tassone, S. Venuta, et al.
Neutralizing B-Cell Activating Factor Antibody Improves Survival and Inhibits Osteoclastogenesis in a Severe Combined Immunodeficient Human Multiple Myeloma Model
Clin. Cancer Res., October 1, 2007; 13(19): 5903 - 5909.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Z. S. M. Rahman, B. Alabyev, and T. Manser
Fc{gamma}RIIB Regulates Autoreactive Primary Antibody-Forming Cell, but Not Germinal Center B Cell, Activity
J. Immunol., January 15, 2007; 178(2): 897 - 907.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. O. Jacob, L. Pricop, C. Putterman, M. N. Koss, Y. Liu, M. Kollaros, S. A. Bixler, C. M. Ambrose, M. L. Scott, and W. Stohl
Paucity of Clinical Disease despite Serological Autoimmunity and Kidney Pathology in Lupus-Prone New Zealand Mixed 2328 Mice Deficient in BAFF
J. Immunol., August 15, 2006; 177(4): 2671 - 2680.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. P. Miller, J. E. Stadanlick, and M. P. Cancro
Space, Selection, and Surveillance: Setting Boundaries with BLyS.
J. Immunol., June 1, 2006; 176(11): 6405 - 6410.
[Abstract] [Full Text] [PDF]