By
From the * Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research
Center, Denver, Colorado 80206; Tumor Immunology Program, German Cancer Research Center,
Im Neuenheimer Feld 280, 6900 Heidelberg, Germany; § Walter and Eliza Hall Institute of Medical
Research, Melbourne, Victoria 3050 Australia;
Department of Medicine, Microbiology and
Immunology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis,
Missouri 63198; and ¶ Department of Immunology, University of Colorado Health Sciences Center,
Denver, Colorado 80220
The mechanisms that establish immune tolerance in immature and mature B cells appear to be
distinct. Membrane-bound autoantigen is thought to induce developmental arrest and receptor
editing in immature B cells, whereas mature B cells have shortened lifespans when exposed to
the same stimulus. In this study, we used Eµ-bcl-2-22 transgenic (Tg) mice to test the prediction that enforced expression of the Bcl-2 apoptotic inhibitor in B cells would rescue mature, but not immature, B cells from tolerance induction. To monitor tolerance to the natural membrane autoantigen H-2Kb, we bred 3-83µ (anti-Kk,b) Ig Tg mice to H-2b mice or to mice expressing transgene-driven Kb in the periphery. In 3-83µ
/bcl-2 Tg mice, deletion of autoreactive B cells induced by peripheral Kb antigen expression in the liver (MT-Kb Tg) or epithelia
(KerIV-Kb Tg), was partly or completely inhibited, respectively. Furthermore, Bcl-2 protected
peritoneal B-2 B cells from deletion mediated by acute antigen exposure, but this protection
could be overcome by higher antigen dose. In contrast to its ability to block peripheral self-tolerance, Bcl-2 overexpression failed to inhibit central tolerance induced by bone marrow antigen expression, but instead, enhanced the receptor editing process. These studies indicate that
apoptosis plays distinct roles in central and peripheral B cell tolerance.
Apoptosis is an essential element in the development and
homeostasis of many tissues. In the immune system, a
number of important processes are regulated through the
control of cell death and survival (1). Among these processes is immunological tolerance in which encounter with
ligands that signal through antigen receptors affect the cell's
subsequent survival (5, 6). The lifespans of lymphocytes in
vivo vary widely, from 1-2 d to many weeks (7). Self-antigen
can shorten the lifespan of reactive B cells in a number of
ways: by promoting cell death through developmental arrest
(8, 9), by increasing cell turnover (10, 11), by putting cells
at a competitive disadvantage with nonautoreactive cells for
unknown resources (12), by making the cells sensitive to Fas
ligand-mediated killing by T cells (13), or apparently through
direct induction of apoptosis (11, 14).
One regulator of lymphocyte survival is the Bcl-2
protein (1), which is highly expressed in long-lived lymphocytes and is poorly expressed in cells destined to turn
over rapidly (19). Bcl-2, and the closely related Bcl-xL
(27), are key protein regulators of apoptosis. Overexpression or inappropriate expression of these proteins can play
a role in lymphoma development and can allow the continued survival of cells that would otherwise be lost through
apoptosis (28). However, some forms of apoptosis, including Fas-mediated killing of certain lymphoid cells (37), are not inhibitable by Bcl-2. An important physiological
role for Bcl-2 is evident from the phenotype of bcl-2-deficient mice, which manifest a catastrophic apoptotic loss of
mature lymphocytes (38, 39).
The ability of Bcl-2 to block B cell tolerance has been
studied in several systems in which ligands that bind to the
B cell receptor (BCR)1 can stimulate cell death in vivo or
in vitro (9, 15, 33, 40). In some cases, these data have
been contradictory, suggesting that the ability of Bcl-2 overexpression to block tolerance-mediated apoptosis may be
contingent upon the precise quality of the BCR-mediated signal and perhaps other signals that may differ in certain
experimental systems. In the well-characterized surface Ig+ B
lymphoma WEHI-231, cross-linking of the BCR with antiimmunoglobulin antibody leads to cell death (for review
see reference 18). In some studies (43, 44), but not others
(40), transfection of bcl-2 expression constructs protected the
cells from BCR-mediated cell death. When transgenic (Tg)
mice with enforced B cell overexpression of Bcl-2 were
analyzed for B cell tolerance, bone marrow tolerance was
perturbed in one study (9), but not in another (46), whereas peripheral tolerance was inhibited (15, 46). Similarly, germinal center B cells have been shown to be sensitive to killing induced by acute ligation of sIg, and this
death could be inhibited by enforced Bcl-2 expression in
one study (48), but not in another (47). These studies and
others focusing on T cell tolerance (3, 4) have suggested
that the ability of Bcl-2 to protect autoreactive lymphocytes
from cell death may be contingent upon many factors, including the developmental stage of the lymphocyte as well
as the nature of the antigen stimulus.
Resting B cells encountering tissue-specific membrane-bound antigen in the periphery are eliminated, suggesting
that in this case, peripheral tolerance is mediated by an
apoptotic process (14). In contrast, it has been shown in
several systems that immature B cells encountering membrane-bound or nuclear self-antigens in the bone marrow
are blocked in their development (8, 9) and either undergo
receptor editing (49, 50) or are eliminated (50). Our recent finding that in vitro tolerance induction of immature
B cells stimulates intense receptor editing with little immediate cell death (53, 54) suggests that antigen-induced programmed cell death makes only a minor contribution to
central tolerance. Assuming that Bcl-2 is a key cell death
regulator in B cells, this model predicts that enforced Bcl-2
expression can block clonal elimination of mature, but not
immature, self-reactive B cells. To test this, peripheral and
central tolerance in 3-83µ Mice.
MT-Kb mice, in which Kb antigen expression is directed to hepatocytyes by the sheep metallothionein promoter
(55), and KerIV-Kb mice, in which Kb is expressed in epithelial
cells under the control of the keratin IV promoter (56), were bred
several generations onto the B10.D2 background (H-2d) before
breeding with other Tg or H-2 congenic mice. 3-83µ Mouse Typing.
PCR typing for 3-83µ Antibodies, Immunofluorescence, Cell Lines, and Intraperitoneal Injection Assays.
Preparation of lymphoid cells, antibody reagents,
fluorochrome conjugation, flow cytometry analysis, cell lines, and
intraperitoneal injection assay methods were as previously described (58). In brief, the A/J splenocyte × Sp2/0 hybridoma
4549 (which expresses Kk) and Sp2/0 (H-2d) parent hybridoma
were washed and resuspended at either 5 × 107 or 5 × 108 cells/ml
in PBS and injected intraperitoneally into 3-83µ Cell Culture.
Spleen or lymph node cells were cultured in
RPMI-1640 medium supplemented with 10% FCS and 50 µM
2-mercaptoethanol ± 50 µg/ml LPS (Sigma Chemical Co., St.
Louis, MO). The cell concentration was adjusted depending on
the number of 3-83 B cells in the organ according to the following guidelines: 106 cells/ml for H-2d 3-83 Tg, H-2d bcl-2/3-83
double-Tg, and bcl-2/3-83µ 5-bromo-2 The 3-83µ
To study
peripheral B cell tolerance to natural chronic autoantigen
exposure, we generated 3-83µ
In bcl-2/3-83µ The Eµ-bcl-2-22 transgene drives expression of
functionally active Bcl-2 in immature bone marrow B cells
that normally lack Bcl-2 expression (46). To test the effect
of this transgene on central B cell tolerance, we bred H-2b
mice to 3-83µ Table 1.
Effect of Bcl-2 Overexpression on Tolerance Induction and Receptor Editing in 3-83 Immunoglobulin Transgenic Mice
In some CDTg/bcl-2 mice (13/21), a population
of IgM
Bcl-2 overexpression increased the numbers of
nonautoreactive, Id
The elevated frequency of nonautoreactive B cells in the CDTg/bcl-2 mice could theoretically have been the result of expansion or prolonged
survival of B cells in the peripheral lymphoid organs. This
simple explanation would predict that the relative frequencies of B cells bearing endogenous Ig-
This study provides insight into the mechanisms by
which Bcl-2 can perturb B cell tolerance. First, we have
found that natural self-proteins expressed on epithelial cells
can mediate B cell tolerance by accelerating cell death
through a Bcl-2-inhibitable pathway. We have also found
that foreign antigens, administered acutely into the peritoneal cavity, can cause rapid B cell deletion that is rescued in
an antigen dose-dependent manner by enforced Bcl-2 expression. Finally, we have made the novel observation that
enforced Bcl-2 expression in immature B cells promotes receptor editing.
Consistent with the notion that tolerance mechanisms differ in immature and mature B cells, Bcl-2 overexpression
was able to abrogate peripheral B cell tolerance, while only
subtly altering central B cell tolerance. The ability of Bcl-2
to confer protection from deletion induced by peripherally
expressed membrane self-antigen suggests that mature, autoreactive B cells are tolerized by apoptosis induction. These
results are partly in agreement with the studies of Honjo's
group (15, 46), who studied the effects of intraperitoneal injections of antiimmunoglobulins and self-erythrocytes, rather than foreign antigens, on peritoneal B cells that were largely of the B-1 lineage. The signals regulating apoptosis and immune tolerance in B-1 and B-2 subsets are likely to differ
(5, 6, 18, 60). In our 3-83 Tg mice, the Id+ B cells, including those in the peritoneal cavity, are almost entirely B-2 B
cells. Thus, our data are clear evidence for deletion of peritoneal B-2 B cells upon antigen encounter in vivo, which is inhibited by Bcl-2 overexpression in a dose-dependent
manner. No such dose dependence was found in the study
by Nisitani et al. (46), but analogous results have been obtained in bcl-2 transfected WEHI-231 cells in which anti-IgM-mediated death could be blocked at low but not high
antibody dose (43). These differences may reflect a difference in the density of tolerogenic antigen expression, in
BCR affinity for antigen, or in the relative tolerance susceptibility of B-1 and B-2 B cells.
Similar to the results with acute antigen administration,
peripheral B cells tolerized by chronic exposure to natural
autoantigen were variably rescued by enforced Bcl-2 overexpression. Furthermore, the extent of rescue afforded by the
Bcl-2 transgene appeared to be inversely correlated with the
degree of deletion in its absence: in MT-Kb mice, which
normally show complete deletion of Id+ B cells from lymph
nodes, enforced Bcl-2 only slightly inhibited deletion, whereas
in the KerIV-Kb mice, which may express lower amounts
of Kb in a relatively small subset of epithelial cells and in
which autoreactive B cells are eliminated at a slower pace
than in MT-Kb mice, B cell elimination was fully abrogated by Bcl-2. Because in this study the autoantigen and
antibody receptor were kept constant, and only the nature
and anatomical location of the antigenic stimulus were altered, the variable outcomes of Bcl-2 overexpression appear to be real effects, dependent on antigen density, concentration, in vivo location, or the developmental state of
the B cell at time of antigen encounter. These results seem
at odds with the paradigm developed from genetic studies
in Caenorhabditis elegans that places the action of the nematode Bcl-2 homologue ced-9 at a distal point in the death
signaling cascade (61). If this were also true in B cells, one
might predict that Bcl-2 should protect from apoptosis
over a wide range of antigen doses. It is therefore possible
that the function of Bcl-2 is involved with the downstream
integration of certain BCR signals. As Bcl-2 can titrate the
death activity of heterodimerization partners, such as Bax
(62), it is possible that the activity or quantity of these
molecules can be affected by antigen signaling. It is interesting in this regard that Bcl-2 is subject to inactivation by
inducible serine phosphorylation (65), a process that could
conceivably be influenced by BCR signaling. It will be important to determine if higher doses of Bcl-2 can provide
protection at higher antigen dose, or if cell death regulators
distinct from Bcl-2 become limiting. It is interesting to note
in this regard that in one study combined transgene expression of Bcl-2 and Bcl-xL had an additive, but still incomplete, effect in protection from anti-IgD-mediated B cell
elimination (66). An alternative interpretation of our data is
that strong tolerogenic signals through the BCR activate both Bcl-2-inhibitable and Bcl-2-resistant death pathways,
whereas weaker tolerance signals activate only Bcl-2-inhibitable death pathways.
The observed weak or negligible effect of Bcl-2 overexpression on the rescue from the bone marrow of autoreactive B cells is in agreement with the study of Hartley et al.
(9), who showed that Bcl-2 could delay turnover and permit limited peripheralization of immature autoreactive B cells,
but could not promote their continued development to
maturity. Young et al. have shown that Bcl-2 expression
permits ectoptic accumulation of pre-B cells in the spleen
(67). In RAG-sufficient mice, these peripheral, immature
B cells are presumably capable of undergoing receptor editing even in response to antigens expressed exclusively in the
periphery. This suggestion is supported by the upregulation of RAG-2 messenger RNA levels in spleens of CDTg/bcl-2/RAG-1 How might Bcl-2 promote receptor editing? The ability
of Bcl-2 to permit elevated steady-state numbers of immature and mature cells cannot alone explain the increased
proportion of One implication of our study is that altered or defective
apoptosis in B cells encountering self-antigen in the periphery could provide a pool of potentially functional autoreactive B cells. In this study we have observed that the cells
rescued from elimination by enforced Bcl-2 expression could
sometimes also acquire functional reactivity. Further studies
are needed to fully establish their functional capacity, but
these results further suggest that although multiple independent levels of regulation are available to limit autoreactivity at each step in differentiation, tolerance to certain autoantigens may be definitively abrogated by a Bcl-2-
inhibitable pathway.
(anti-H-2k,b) Ig Tg mice were
compared to that occurring in 3-83µ
/Eµ-bcl-2-22 double-Tg mice, in which Bcl-2 is constitutively expressed in B-lineage cells.
Tg mice,
which express IgM and IgD forms of the 3-83 antibody, have been
described (14). 3-83µ
mice were backcrossed a minimum of 10 times onto the B10.D2 background. C57BL/6(H-2b), C3H-H-2o2/SfSn (C3H.OH: KdDk), and B10.D2/nSnJ (H-2d) mice were
purchased from Jackson Laboratory (Bar Harbor, ME). Mice
overexpressing the human bcl-2 gene in B-lineage cells were Tg lines
Eµ-bcl-2-22 (34) and bcl-2-Ig NL (36). These mice were backcrossed a minimum of three generations to B10.D2 and subsequently bred with 3-83µ
Tg mice. The phenotypes of mice bearing either of the bcl-2 transgenes were indistinguishable in our
study. Unless otherwise stated, the experiments described made use
of the Eµ-bcl-2-22 line. The 3-83µ
/RAG-1 knockout mice (57)
were bred with Eµ-bcl-2-22 Tg mice. F2 crosses were set up to
generate bcl-2/3-83µ
/RAG-1
/
mice with all transgenes hemizygous except where noted. All mice were bred and maintained
under specific pathogen-free conditions in the animal care facility
at the National Jewish Medical and Research Center (NJMRC,
Denver, CO).
, Kb, and bcl-2 transgenes was as described previously (14). RAG-1-deficient mice
were identified by their lack of T cells in flow cytometric analysis
of peripheral blood.
/H-2d or
3-83µ
/H-2d/bcl-2 Tg mice. Approximately 16 h later, the peritoneal cells were harvested with 10 ml HBSS, washed twice, and
stained for the presence of Id-reactive antibodies.
/KerIV-Kb triple-Tg mice; and 3-5 × 106 cells/ml for H-2b 3-83 Tg, H-2b bcl-2/3-83µ
, 3-83/
MT-Kb, 3-83/KerIV-Kb double-Tg, and bcl-2/3-83µ
/MT-Kb
triple-Tg mice. In some experiments, lymph node and spleen cells were T cell depleted with anti-Thy1 antibodies and rabbit complement and cultured at 5 × 105 cells/ml. Culture supernatants
were harvested on days 3 and 7 after initiation of culture and Ig
concentrations determined by ELISA, as described (14).
-deoxyuridine Incorporation Assay.
5-bromo-2
-deoxyuridine (BrdU; Sigma Chemical Co.) was provided in filtered,
deionized drinking water at a concentration of 1 mg/ml for 7 d.
Staining for the incorporation of BrdU was described previously (59).
Enforced Bcl-2 Expression Blocks B Cell Deletion Induced by
Acute Antigen Administration at Low Dose, but Not at High
Dose.
Ig transgene encodes a BCR that is
reactive to a number of MHC class I alloforms including
Kk, Dk, and Kb, but fails to bind to H-2d; thus, 3-83µ
/H-2d
mice contain a virtually monoclonal B cell population
bearing the 3-83 BCR and are called nondeleting (ND)Tg
mice (14). To probe the ability of enforced Bcl-2 expression to block tolerance to acute intraperitoneal antigen challenge, we injected NDTg and NDTg/bcl-2 mice with antigen-bearing cells, a protocol that stimulates rapid apoptosis
of fully mature antigen-specific peritoneal B cells (15, 16).
Injection of hybridoma cells bearing the high-affinity Kk antigen consistently led to massive loss (~80%) of the 3-83µ
peritoneal B cells over a 16-h period, whereas injection of
control H-2d cells did not (Fig. 1). At low antigen dose (5 × 106 cells), NDTg/bcl-2 B cells resisted deletion induced by
the Kk antigen (Fig. 1); however, this protection from death
afforded by Bcl-2 overexpression was overcome with a 10-fold increased antigen dose (Fig. 1).
Fig. 1.
Enforced Bcl-2 expression inhibits B cell deletion induced by
acute challenge with membrane antigen in an antigen dose-dependent manner. NDTg and NDTg/bcl-2 mice were injected intraperitoneally with either PBS control, Kk-expressing tumor cells, or Kd tumor cells, and
the peritoneal cells were analyzed 16 h later for the loss of 3-83 B cells using 54.1 anticlonotype and anti-IgM antibodies. The data from three experiments are presented as mean ± SEM. In some data points, error bars
are not apparent because of their small range.
[View Larger Version of this Image (16K GIF file)]
mice bearing MT-Kb or
KerIV-Kb transgenes, which target cell surface expression of
the Kb protein to hepatocytes or epithelia, respectively (55,
56). Relative to antigen-free mice, antigen-bearing 3-83µ
mice had profoundly reduced B cell numbers in the lymph
nodes (Fig. 2 A, top; Fig. 2 B, compare H-2d to MT-Kb and
KerIV-Kb), and substantial, but on average, incomplete deletion of the B cells in the spleen (Fig. 2 C, top; Fig. 2 D
and reference 14). Control mice bearing antigen on all tissues (Fig. 2, H-2b) exhibited the phenotype of central B cell
tolerance in which antigen-reactive cells were absent from
the spleen. In the mice that demonstrated peripheral B cell
deletion (3-83µ
/MT-Kb or 3-83µ
/KerIV-Kb), the remaining splenic cells manifested rapid turnover as assessed by their BrdU uptake over a 1-wk labeling period (Fig. 3).
It is important to note that the MT-Kb/3-83µ
mice
showed a more profound tolerance than KerIV-Kb/3-
83µ
mice, and had more complete elimination of Id+ B
cells in the lymph nodes (Fig. 2) and of Id+ antibodies in
the serum (Fig. 4 C).
Fig. 2.
Enforced Bcl-2 expression blocks peripheral clonal
deletion in 3-83µ mice. (A
and C) 3-83µ
Tg mice (top) or
3-83µ
/ bcl-2 Tg mice (bottom)
bearing the indicated self-antigens were analyzed for Id+ B
cells in lymph node (A) and
spleen (C). (B and D) The compiled FACS® data as percentage
of IgM+/Id+ cells in the lymphoid analysis gate is shown.
Data are presented as mean ± SEM. The numbers below each
bar represent the number of
mice analyzed per group.
[View Larger Version of this Image (46K GIF file)]
Fig. 3.
Effect of bcl-2 transgene on B cell turnover in peripheral antigen-expressing 3-83µ Tg mice. Mice were fed BrdU-containing water
for 7 d followed by staining of isolated lymph node and spleen cells with
anti-BrdU and anti-B220 antibodies. Data are presented as percentage
(mean ± SEM) of B220+ cells that were BrdU positive. Numbers of mice
analyzed are indicated below bars. Mice lacking antigen were H-2d ND
controls.
[View Larger Version of this Image (31K GIF file)]
Fig. 4.
In vitro and in vivo analysis of antibody secretion. Lymph
node and spleen cells from mice of the indicated genotypes were cultured
in the presence or absence of 50 µg/ml LPS. After 3 d of culture, supernatants were analyzed by ELISA for Id+ (A) and IgM (B) antibodies. Antibody amounts were quantitated using 3-83 IgM transfectoma supernatant control. Similarly, Id+ (C) and IgM (D) serum antibodies were
quantitated by ELISA. Data are presented as mean ± SEM with number
of mice (n) analyzed shown. The table below the graph shows Tg genotypes (3-83, bcl-2, MT-Kb, and/or KerIV-Kb) and H-2 haplotypes (d, no
antigen; b, antigen). In C and D the RAG-1 genotype of analyzed mice
(+/+ or /
) is indicated.
[View Larger Version of this Image (36K GIF file)]
/MT-Kb and bcl-2/3-83µ
/KerIV-Kb
triple-Tg mice, enforced Bcl-2 expression partially or completely blocked peripheral B cell deletion to liver and skin
antigens, respectively, and, on average, increased numbers
of Id+ cells in both the lymph nodes and spleen (Fig. 2, A
and C, bottom; Fig. 2, B and D). Interestingly, in the bcl-2/
3-83µ
/MT-Kb mice in which Bcl-2 protection was partial, the high rate of turnover of the autoreactive B cells was
unchanged, whereas in the bcl-2/3-83µ
/KerIV-Kb triple-Tg mice in which B cell deletion was effectively blocked, B cell turnover was also normalized, and only ~20% of the
B cells had incorporated BrdU over a 1-wk period (Fig. 3).
Furthermore, lymph node cells from bcl-2/3-83µ
/KerIV-Kb
triple-Tg, but not those from bcl-2/3-83µ
/MT-Kb mice, acquired the ability to respond to LPS stimulation (Fig. 4 A).
The ability of lymph node and spleen cells to secrete 3-83 antibodies in vitro was consistent with the levels of 3-83
antibodies measured in the sera of these mice, as Id+ antibody levels were negligible in the bcl-2/3-83µ
/MT-Kb
mice, but were significant in the bcl-2/3-83µ
/KerIV-Kb
triple-Tg mice (Fig. 4 C). The only differences noted between bcl-2/3-83µ
/KerIV-Kb triple-Tg mice and bcl-2/
3-83µ
ND controls were a consistent twofold reduction
in surface IgM expression in the bcl-2/3-83µ
/KerIV-Kb
mice, suggesting antigen encounter that did not result in
deletion (Fig. 2, A and C), and a lower percentage of cells
expressing CD21 (data not shown). In both bcl-2/3-83µ
/
KerIV-Kb and bcl-2/3-83µ
/MT-Kb mice, the B220+ B cells
coexpressed the maturation marker CD23 (data not shown). Collectively, these data demonstrated that Bcl-2 overexpression could inhibit peripheral B cell tolerance to both
acute and chronic antigen exposure, but the degree of protection varied depending upon the dose or anatomical location of the antigenic stimulus.
and bcl-2/3-83µ
mice, generating central
deleting (CD)Tg and CDTg/bcl-2 mice, respectively. Like
CDTg mice, CDTg/bcl-2 mice lacked Id+ B cells in the
peripheral lymphoid organs (Fig. 2, H-2b; Table 1) and Id+
antibodies in the sera (Fig. 4 C). In addition, no Id+ antibodies were found in the supernatants of LPS-stimulated
spleen cells from CDTg or CDTg/bcl-2 mice, whereas NDTg
and NDTg/bcl-2 controls had significant levels of Id+ antibodies in both the sera and LPS culture supernatants (Fig. 4 A;
data not shown). Since previous central tolerance studies investigating Bcl-2 overexpression used high-affinity antigens (KA ~109 M
1), we tested whether or not Bcl-2 overexpression had perhaps a subtle, antigen affinity-dependent
effect on tolerance induction by generating CDTg/bcl-2 mice
expressing the Dk class I molecule to which 3-83 has very
low affinity (KA ~104 M
1; reference 58). Again the CDTg/
bcl-2 mice had no detectable increase of Id+ B cells in the
peripheral lymphoid organs (Fig. 5) nor IgM idiotype in
the serum (data not shown).
Lymph Node
Spleen
Genotype
54.1+
IgDa,
+
B220+, IgM
54.1+
IgDa,
+
B220+, IgM
Cell content (× 105)
NDTg +/+
82 ± 16 (12)
0.6 ± 0.2 (11)
1.6 ± 0.4 (10)
280 ± 38 (11)
1.8 ± 0.7 (11)
9.9 ± 2.5 (11)
NDTg/bcl-2 +/+
160 ± 40 (9)
1.6 ± 0.4 (9)
6.3 ± 1.2 (9)
350 ± 70 (9)
4.4 ± 1.3 (9)
17 ± 7 (9)
NDTg
/
30 ± 2 (2)
0 (1)
0.7 ± 0.7 (2)
230 ± 40 (3)
0.7 ± 0.3 (2)
15 ± 7 (3)
NDTg/bcl-2
/
580 (1)
0 (1)
2.1 (1)
870 (1)
0 (1)
5.3 (1)
CDTg +/+
0.3 ± 0.1 (19)
3.3 ± 0.6 (19)
2.3 ± 0.4 (17)
5.8 ± 1.2 (19)
36 ± 5 (20)
31 ± 8 (19)
CDTg/bcl-2 +/+
0.6 ± 0.2 (19)
14 ± 3 (18)
9.9 ± 2.5 (16)
6.1 ± 1.7 (17)
66 ± 19 (19)
57 ± 17 (17)
CDTg
/
0.1 ± 0.1 (5)
0.1 ± 01 (3)
3.8 ± 1.4 (5)
1.9 ± 1.2 (5)
0 ± 0 (4)
45 ± 15 (5)
CDTg/bcl-2
/
0.8 ± 0.4 (6)
0.1 ± 0.1 (4)
11 ± 6 (6)
5.3 ± 3.3 (6)
0.2 ± 0.2 (5)
160 ± 28 (6)
Absolute number of idiotype-positive (54.1+), "edited" (IgDa, +), and immature (B220+, IgM
) B cells in 3-83 immunoglobulin transgenic mice
in the presence (CDTg) or absence (NDTg) of antigen in the bone marrow. In the genotype column +/+ refers to normal levels of RAG-1 whereas
/
refers to RAG-1 homozygous knock-out mice. All numbers shown are averages ± SEM divided by 105. The numbers in parenthesis refer to
sample size.
Fig. 5.
Enforced Bcl-2 expression fails to block central deletion induced by the ultralow affinity 3-83 ligand Dk. Lymph node and spleen
cells from mice of the indicated genotypes were double stained for the
presence of B cells bearing the 3-83 Tg BCR with 54.1 anti-Id and anti-IgM antibodies. The substantial population of IgM+ B cells present in the
CDTg mice were clonotype and are presumably the result of receptor
editing. The large percentage of B cells in the ND control in this experiment reflects the RAG deficiency of this particular mouse.
[View Larger Version of this Image (38K GIF file)]
, B220+ B Cells in the Peripheral Lymph
Organs.
, B220+ B cells was detected in the spleen and, to a
lesser extent, in the lymph nodes. To further characterize
this population in a context in which receptor editing
could not occur, we generated CDTg/bcl-2/RAG-1-deficient mice (57). Again, no strongly Id+ cells were detected
in the peripheral lymphoid organs (Fig. 6 A; Table 1), but a
significant population of IgM
, B220+, Idlo cells was detected in the spleen of the CDTg/bcl-2/RAG-1
/
mice
(Fig. 6, A and B, note arrows), and a similar population was
detected to a lesser extent in CDTg/RAG-1
/
mice
without Bcl-2 overexpression. These IgM
, B220+ cells
were CD23
,
lo, IgDlo, and CR1/2
(data not shown)
which is indicative of immature B cells as has been previously reported in Bcl-2 overexpressing Ig Tg mice (9). Also
indicative of immature B cells, these cells expressed RAG-2 messenger RNA transcripts (data not shown). These cells
failed to secrete antibodies in LPS cultures and in vivo, as
no antibodies were detected in the serum (Fig. 4 C). In
CDTg/RAG-1
/
mice, no serum IgM was detected because RAG deficiency prevented development of Id
B cells
(Fig. 4 D). These splenic immature B cells were short lived,
as ~50% of the cells had incorporated BrdU+ after 1 wk
compared to only ~20% of B220+ cells from non-Tg or
NDTg mice (data not shown). Thus, in contrast to its effect on peripheral tolerance, the bcl-2 transgene appeared to
have only a subtle effect on central B cell tolerance, allowing a subset of autoreactive nonfunctional, immature B cells
to survive in the spleen for a short time.
Fig. 6.
Appearance of immature B cells in spleen of CD (H-2b) bcl-2/RAG-1-deficient mice. Spleen cells from mice of the indicated genotypes were stained for presence of 3-83 B cells with anticlonotype (54.1) and anti-IgM antibodies (A), and anti-IgM and anti-B220 antibodies (B).
The immature B cells have low levels of Id and lack detectable sIgM (arrows). The NDTg control used in this particular experiment was 3-83µ
homozygous, which consistently show reduced B cell populations. Data
shown represent one of five similar experiments.
[View Larger Version of this Image (35K GIF file)]
B
Cells.
B cells that developed in CD H-2b/
3-83µ
Tg mice by two- to fivefold (Fig. 7 B; Table 1). In contrast to the results with the mice bearing antigen targeted to peripheral tissues in which autoreactive B cells
with enforced Bcl-2 expression were spared, the B cells appearing in the spleen and lymph nodes of CDTg/bcl-2 mice
were nonautoreactive and had undergone receptor editing
(Figs. 2 and 5). One clear indication of receptor editing was
the appearance of B cells bearing both the Tg heavy chain,
as detected with anti-IgDa, and endogenously encoded
light chains, detected with
chain-specific antibody (Fig. 7
A, and reference 49). The increase in the percentages of
"edited" cells was the result of an increase in the total number of these cells in the lymphoid organs (Table 1). Thus,
Bcl-2 overexpression apparently enhanced receptor editing
or allowed survival of cells that had undergone receptor editing, or both.
Fig. 7.
Increased percentage of
IgDa, + cells in CDTg mice with
enforced Bcl-2 expression. 3-83µ
Tg mice were bred with bcl-2 Tg
mice in the presence (CDTg) or absence (NDTg) of bone marrow antigen expression. To determine the
extent of receptor editing, lymph node cells were double stained for
Tg heavy chain (anti-IgDa) and endogenous light chain (anti-
). (A)
Each row illustrates FACS® analysis
from independent experiments. (B)
Summary data showing percentage
of IgDa-positive, Id
cells in both
CDTg and CDTg mice with Bcl-2
overexpression. Data are presented
as mean ± SEM.
[View Larger Version of this Image (36K GIF file)]
/
Ratio Suggests that Bcl-2 Overexpression Promotes Receptor Editing.
and Ig-
light chains
would be the same in both CDTg and CDTg/bcl-2 mice.
To test this notion, we measured the frequencies of Id
B
cells that expressed
or
light chains. Fig. 8 A shows that most of the increase in peripheral B cells of the CDTg/bcl-2
mice could be accounted for by an increase in
+ B cells,
suggesting that the bcl-2 transgene influences B cells undergoing light chain gene rearrangement. Interestingly, this elevated
expression was also observed in bcl-2 Tg mice lacking Ig transgenes (Fig. 8 B), which consistently had a
significantly higher percentage (~17%) of
+ B cells compared to non-Tg mice (~6%
+).
Fig. 8.
Bcl-2 overexpression increases /
ratio of B cells in both 3-83
CDTg mice and non-Ig Tg mice. (A) Lymph node cells from CDTg and
CDTg/bcl-2 mice were double stained with anti-IgDa and anti-
antibodies. Percentage of lymph node cells staining positive for IgDa and negative
for
were scored as
positive. (B) Increased percentage of
-expressing
B cells in (non-Ig Tg) bcl-2 Tg mice. The percentage of IgM+ cells that
were also
positive was determined. Data presented as mean ± SEM.
Numbers above bars reflect number of mice examined.
[View Larger Versions of these Images (17 + 34K GIF file)]
/
mice, which have a significant population of
these immature B220+ cells (data not shown), and by the
appearance of Id
, IgM+ B cells in spleens of 3-83/MT-Kb/bcl-2 mice (Fig. 1 C). These Id
, IgM+ cells express Tg
heavy chain (data not shown) and are presumed to be the
product of receptor editing. Collectively, these data show
that Bcl-2 overexpression can manifest varying tolerance phenotypes depending on the maturational stage at which
the B cell encounters autoantigen, promoting receptor editing in immature cells, and rescuing functional, mature
cells from apoptosis.
+ B cells because increased numbers of cells
undergoing editing, or surviving after editing, would not
be expected to alter the Ig light chain
/
ratio. A more
likely explanation is that the extended lifespan of autoreactive bone marrow B cells that is conferred by the bcl-2 transgene may provide an extended time window per cell for
light chain gene rearrangement. The current models of murine light chain rearrangement suggest that
rearrangements generally follow
rearrangement by ~24 h (68),
and that most cells that turn over in the bone marrow do so
before
rearrangements are complete (69). This so-called
crash-factor (70) suggests that mouse B cells normally have
too little time to take full advantage of potential
rearrangements that could rescue nonfunctional or autoreactive
B cells. We propose that the bcl-2 transgene expression allows autoreactive B cells more potential rearrangement attempts, promoting the development of more nonautoreactive B cells bearing "edited" BCRs. This is consistent with
data concerning transgene-driven Bcl-2 overexpression in
thymocytes in which positive selection is enhanced as a result of increased endogenous TCR rearrangements, presumably due to the increased lifespan of the double-positive thymocytes expressing the bcl-2 transgene (1). The finding that Eu-bcl-2-22 Tg mice have consistently high percentages of
+ B cells suggests that receptor editing is enhanced
even in the absence of antibody transgenes. Data from
Rolink et al. have indicated that the Eµ-bcl-2-22 transgene
prolongs in vitro survival of B cells undergoing light chain
gene rearrangement resulting in high
/
ratios in cultured B
cells (71).
Address correspondence to Dr. Nemazee, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206. Phone: 303-398-1623; FAX: 303-398-1225; E-mail: nemazeed{at}njc.org
Received for publication 23 July 1997 and in revised form 4 September 1997.
1 Abbreviations used in this paper: BCR, B cell receptor; BrdU, 5-bromo-2The authors are grateful to Shirley Sobus and Bill Townsend for flow cytometry assistance, Leigh Landskroner for illustrations, Russell Yawger and Alicia Kuhl for technical assistance, and members of our laboratory for critical review of the manuscript.
This work was supported by a Howard Hughes Medical Institute Predoctoral Fellowship (to J. Lang), by the Arthritis Foundation (to D. Nemazee), the National Institutes of Health (R01 GM 44809, R01 AI 33608, and K04 AI 01161; to D. Nemazee), and the National Health and Medical Research Council (Canberra, Australia; to A. Strasser). A. Strasser is a scholar of the Leukemia Society of America and a recipient of a Clinical Investigator Award from the Cancer Research Institute.
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