The Journal of Immunology, 2001, 166: 3226-3230.
Copyright © 2001 by The American Association of Immunologists
The T Cell-Dependent B Cell Immune Response and Germinal Center Reaction Are Intact in A-myb-Deficient Mice1
Kalpit A. Vora2,*,
Vicky M. Lentz*,
William Monsell*,
Sambasiva P. Rao*,
Richard Mettus
,
Antonio Toscani,
E. Premkumar Reddy and
Tim Manser3,*
*
Kimmel Cancer Center and Department of Microbiology and Immunology, Jefferson Medical College, Philadelphia, PA 19107; and
Fels Institute for Cancer Research and Department of Biochemistry, Temple University Medical School, Philadelphia, PA 19140
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Abstract
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Expression of the protooncogene A-myb is restricted to the
developing CNS, adult testes, breasts in late pregnancy, and germinal
centers of secondary B cell follicles. The functional relevance of
A-myb expression at three of these sites has been demonstrated
previously via the generation and analysis of A-myb-deficient mice,
which display behavioral abnormalities, male sterility, and perturbed
breast development during pregnancy. In contrast, here we show that the
germinal center response driven by T cell-dependent Ag immunization and
the associated processes of Ab V gene somatic hypermutation, affinity
maturation, and heavy chain class switching are overtly normal in
A-myb-deficient mice. Nonetheless, these mice display mild splenic
white pulp hypoplasia and blunted primary serum Ab responses,
suggesting that although A-myb is not directly involved in the
regulation of the memory B cell response, it may play a role in
enhancing peripheral B cell survival or proliferative
capacity.
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Introduction
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A-myb
belongs to the myb family of transcription factors involved in the
regulation of cell proliferation and differentiation (1).
All members of this family (A-, B-, c-, and v-myb) share a high degree
of homology within their DNA binding domains (2). A-myb,
B-myb, and c-myb exhibit similar DNA binding specificities, and all
three proteins were found to transactivate reporter constructs
containing consensus Myb binding sequences (3). Of the
three members of this family, A-myb and c-myb show a restricted pattern
of expression, whereas B-myb appears to be expressed ubiquitously. The
highest levels of A-myb are seen in developing CNS, adult testis,
breast ductal epithelium during pregnancy, and in germinal center
(GC)4 B lymphocytes
(4, 5, 6). Male mice homozygous for a targeted germline
mutation ablating A-myb expression are infertile. Female
A-myb-/- mice have defective development of
breast tissue after pregnancy (7). These results confirm
the critical role of A-myb in spermatogenesis and mammary gland
development predicted from its restricted pattern of expression.
A-myb expression in human GCs has been sublocalized to the dark zone
resident centroblast population (8). In this population,
its expression has been further linked to the S and
G2/M phases of the cell cycle (8, 9). Moreover, A-myb up-regulation has not been seen in in vitro
stimulated B cells, and A-myb expression is rapidly down-regulated in
GC-derived B cells that have further differentiated to plasma or memory
B cell phenotypes (8). A-myb expression was also found to
be characteristic of certain subsets of mature B cell neoplasias
(Burkitt’s lymphoma, sIg+ B cell-acute
lymphocytic leukemia, subsets of chronic lymphocytic leukemia),
supporting their GC origin (8, 10). Calabretta and
colleagues found that ectopic expression of A-myb driven from a
transgene led to follicular hyperplasia in peripheral lymphoid organs
because of enhanced proliferation and accumulation of B cells bearing a
GC phenotype (11). Based on these observations, it has
been proposed that A-myb plays a critical role in the regulation of the
GC reaction, including promoting high-rate B cell proliferation and Ab
V gene somatic hypermutation (8). We have investigated
these issues by using a previously described line of
A-myb-deficient mice.
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Materials and Methods
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Mice
The line of A-myb-deficient mice used in these studies has been
described and was maintained by brother-sister mating of
A-myb+/- mice that had been generated on a mixed
C57BL6/129xJ background, and offspring were genotyped as described
earlier (7). Age-matched mice (8–12 wk old) were used in
all experiments.
RT-PCR analysis
Total RNA was prepared from splenocytes. After reverse
transcription, cDNAs were amplified by using a c-myb-specific primer
combination, run on agarose gels, blotted to nylon membranes, and
hybridized with a 32P-labeled internal
c-myb-specific DNA probe, all as described previously (7).
The hybridized membranes were analyzed on a Fuji (Tokyo, Japan)
phosphoimager to quantitate levels of cDNA.
Immunizations and serology
Mice were immunized with 100 µg of alum-precipitated
(4-hydroxy-3-nitrophenyl)acetyl chicken 
globulin (NP-CGG) i.p. for
primary responses and boosted with the same amount of Ag in PBS
i.p. Mice were bled via the retro-orbital sinus,
and the levels of anti-nucleoprotein (NP) Abs of various isotypes
were enumerated by ELISA as described previously (12). In
most of the assays, monoclonal anti-NP Abs of different H chain
isotypes but similar affinities for NP were used as standards, allowing
the results to be presented as microgram per milliliter equivalents of
these mAbs. In the IgM assay, relative levels of Ag-binding Abs in
different sera were determined by using serial dilutions. The points at
which the resulting OD curves were 50% maximal were then used to
calculate the relative dilution factor, giving an equivalent OD for
each serum sample. The data illustrated for different isotypes were
obtained by using sera pooled from at least three different mice of
each genotype. Isotype levels were determined from supernatants of in
vitro-stimulated B cells by ELISA, and relative affinities of serum Abs
were evaluated by using altered ligand density ELISA as described
earlier (13).
Immunohistochemistry, GC microdissection, V gene PCR amplification,
and sequencing
Processing of spleens for immunohistochemistry, sectioning, and
staining for NP+, 
+ B
cells and peanut agglutinin (PNA)+ GC has been
described in detail previously (14). Fifty to 100 cells
from Ag-specific GC were microdissected from sections, and their
genomic DNA was isolated as reported earlier (15). Two
rounds of PCR amplification were conducted with primers specific for
the V1 gene, and the conditions of
amplification were described by Jacobs et al. (16). The
amplified products were cloned into the pBluescript vector (Stratagene,
La Jolla,\E
CA), and inserts were sequenced as described (15).
Mitogen stimulation of small resting B cells in vitro
Small resting B cells were isolated from spleens via T cell
depletion, followed by purification of high-density cells on Percoll
gradients. The isolated B cells were then stimulated with various
concentrations of LPS, goat anti-mouse IgM
F(ab')2, or anti-CD40 mAb (FGK45). In some
experiments, recombinant IL-4 (PeproTech, Princeton, NJ) was included
in cultures at 50 ng/ml. Cell proliferation was assayed by pulsing
after 48 h with [3H]thymidine, harvesting
onto glass-fiber filters, and scintillation counting. All of these
procedures were performed as described before (12).
Flow cytometry
Lymphocyte suspensions were prepared as described previously
(17). Marrow cells were obtained from the two hind limbs
of each of two donor animals of each genotype. Cell surface staining
was then performed as described (17, 18). Splenocytes were
stained with anti-CD45R (B220) and anti-CD24 (heat-stable Ag),
and, in some experiments, with anti-IgD. Bone marrow cells were
stained with anti-IgM, anti-CD45R, and anti-IgD, and, in
some experiments, with anti-CD24. Stained cells were analyzed with
a Coulter Epics Elite (Coulter Pharmaceutical, Palo Alto, CA) with live
lymphocyte forward and side scatter gates. The proportions of immature
and mature splenic B cells were derived by determining the proportion
of CD24high and CD24low
(and in some experiments IgDhigh) cells,
respectively, among all CD45R+ splenocytes. The
proportions of cells in different stages of development in the bone
marrow were determined in one experiment as follows: mature B cells
were CD45R+, IgM+, and
IgD+; immature B cells were
CD45R+, IgM+, and
IgD-; and pro- and pre-B cells were
CD45R+, IgM-, and
IgD-. In a second experiment, mature B cells
were defined as CD45R+ and
IgD+; immature and pre-B cells as
CD45R+, CD24+ and
IgD-; and pro-B cells as
CD45R+, IgD-, and
CD24-.
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Results and Discussion
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Mild hypoplasia of the splenic white pulp in naive
A-myb-/- mice
Histological studies revealed that naive
A-myb-/- mice have mild splenic hypoplasia
(Fig. 1
). This hypoplasia appeared to be
confined to the B cell follicular and marginal zone regions and
resulted in smaller average spleen sizes and expanded red pulp areas.
The hypoplasia seemed to involve all areas of the white pulp. The
frequency of "spontaneous" GCs in naive spleens of
A-myb-/- mice were also severalfold lower than
observed in A-myb+/+ animals.
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FIGURE 1. Mild splenic white pulp hypoplasia in A-myb-/- mice.
Spleens from naive A-myb+/+ (A) and
A-myb-/- (B) littermates were processed
for histology and sections stained with anti-IgM (blue) to
elaborated B cell areas and PNA (red) to elaborate GC. Original
magnification of images was x10.
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Flow cytometric analysis revealed that splenic B cell percentages in
A-myb-/- mice were 
2-fold lower than in
A-myb+/- and A-myb+/+
littermates (Table I), in agreement with
the immunohistochemical results. Five A-myb+/+
and A-myb+/- animals had an average splenic B
cell percentage of 30, whereas three
A-myb-/- animals had an average splenic B cell
percentage of 16. Despite the reduced numbers of splenic B cells,
the proportions of splenic immature and mature B cells were comparable
in all groups of animals. Analysis of bone marrow cells in two separate
experiments showed no reproducible abnormalities in the numbers of
pro-B, pre-B, immature, and mature B cell subpopulations (Table II). Therefore, the splenic white pulp
hypoplasia does not appear to result from defects in primary B cell
differentiation or selective loss of B cells at a particular stage of
development. Collectively, these data suggest that A-myb is involved in
regulation of peripheral B cell survival or homing to follicular areas.
This result was somewhat unexpected given that previous studies have
suggested that high levels of A-myb expression are only observed in
GCs. Nonetheless, it is consistent with the studies of Calabretta and
colleagues (11) showing that overexpression of A-myb leads
to follicular hyperplasia.
Serum Ab response to a T-dependent Ag in A-myb-/-
mice
Cohorts of A-myb-/- and
A-myb+/- mice were immunized with the T
cell-dependent Ag (4-hydroxy-3-nitrophenyl)acetyl chicken globulin
(NP-CGG) in alum and were bled at various times thereafter. At day 7,
the levels of -bearing anti-NP Abs (characteristic of the
anti-NP response) did not significantly differ in the two groups of
animals, but subsequently, A-myb-/- mice
displayed lower primary serum Ab levels (Fig. 2). After boosting the serum anti-NP
levels were comparable between the two groups of animals. Analysis of
serum Ab isotypes did not reveal any major differences in the isotypes
being produced in primary and secon-dary responses (Fig. 3). In addition, the early primary IgM
response appeared to be comparable in both groups of animals. However,
primary serum levels of IgG3 and IgG1 (the major isotypes in this
response), as well as IgG2a, appeared uniformly lower in
A-myb-/- as compared with
A-myb+/- mice.
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FIGURE 3. H chain isotype levels in the primary anti-NP response of
A-myb-/- mice. Cohorts of at least three aged-matched
A-myb+/- and A-myb-/- were immunized with
NP-CGG, bled at the days indicated during the primary response, sera
pooled, and levels of the various anti-NP H chain isotypes
indicated measured by ELISA as described in Materials and
Methods. Note that the scales on the y-axes
differ for the different isotypes.
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The mild follicular hypoplasia characteristic of A-myb mice may account
for their blunted primary serum Ab responses. However, the anamnestic
serum Ab response appears overtly normal in
A-myb-/- mice, indicating that the memory B
cell differentiation pathway is not perturbed by lack of A-myb.
The GC reaction is qualitatively intact in A-myb-/-
mice
To characterize the GC reaction in
A-myb-/- mice we used a
immunohistochemical/microdissection/PCR approach. The frequency of
total GCs and GCs containing -expressing B cells at days 9 and 12
after NP-CGG in alum immunization were evaluated in the spleens of four
A-myb+/- and four
A-myb-/- mice (two at each time point). A
spleen section was randomly chosen from each mouse, and the number of
GCs in each of three randomly chosen 10x fields that stained with
either PNA-HRP or PNA-HRP and anti--biotin (bio) were counted.
This analysis revealed 5.7 ± 0.7 and 5.3 ± 0.5
PNA+ GCs per field in
A-myb+/- and A-myb-/-
spleen sections, respectively; and 4.5 ± 0.7 and 4.1 ± 1.0
PNA+, + GCs per field in
A-myb+/- and A-myb-/-
spleen sections, respectively. Thus, neither the number of total or
+ GCs were significantly different in
A-myb-/- and A-myb+/-
mice (total GC numbers were similar in both types of mice at the day 9
and 12 time points, but day 9 GCs were uniformly smaller). Although the
GCs observed in the A-myb-/- mice appeared
slightly smaller on average than in A-myb+/-
littermates at both time points, these minor differences were variable
from mouse to mouse (data not shown).
Adjacent spleen sections obtained from A-myb+/+
and A-myb-/- animals at day 12 after
immunization with NP-CGG in alum were stained with NP-CGG-bio/PNA-HRP
and anti--bio/PNA-HRP to identify NP+,
+ GCs. Individual GCs of this type were
microdissected from the sections and genomic DNA PCR-amplified with
V1 gene-specific primers, and the PCR products were cloned and
sequenced. In five NP+,
+ GCs from three
A-myb+/+ mice, an average V1 mutation
frequency of 0.54% was observed (Table III). Analysis of seven
NP+, + GC from two
A-myb-/- mice yielded a mutation frequency of
0.52%. The mutation frequencies observed in these two samples were not
significantly different as evaluated by a Student’s t test
(90% confidence level). In addition, we did not detect any obvious
differences in the locations or chemical nature of mutations in V1
genes recovered from A-myb+/+ and
A-myb-/- GCs.
To evaluate whether the selection of high-affinity Ab variants
generated by V gene hypermutation resulting in serum Ab affinity
maturation was altered in A-myb-/- mice, we
assayed the relative affinities of serum Abs produced during the
anti-NP response by altered ligand-density ELISA. Affinity-matured
serum anti-NP Abs were detected at later time points during primary
(data not shown) and early secondary (Table IV) immune responses in both
A-myb-/- and A-myb+/-
mice. Thus, two processes fundamental to the development of B cell
memory, V gene somatic hypermutation and Ag affinity-based positive
selection, seem normal in A-myb-/- mice. These
data are concordant with those discussed above in indicating that the
GC reaction and memory B cell pathway can operate efficiently in the
absence of A-myb.
In vitro proliferative and isotype switching responses of
A-myb-/- B cells
The mild B cell hypoplasia and lower primary serum IgG Ab levels
observed in A-myb-/- mice prompted us to
evaluate the proliferative and H chain class switching potential of
A-myb-/- B cells in vitro. Small resting B
cells were purified from A-myb+/+,
A-myb+/-, and A-myb-/-
mice and stimulated with LPS, anti-CD40, or anti-IgM in vitro.
B-cells from A-myb-/- mice proliferated as well
(data not shown) or better (Fig. 4) than
A-myb+/- and A-myb+/+ B
cells when stimulated with high concentrations of LPS, but no
differences were observed at low LPS concentrations and in response to
any concentration of anti-CD40 and anti-IgM (Fig. 4).
Therefore, A-myb is not required to achieve normal rates of B cell
proliferation in vitro.
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FIGURE 4. In vitro proliferative responses of A-myb-/- B cells.
Purified small resting B cells were prepared and stimulated in vitro
with the indicated concentrations of anti-IgM, LPS, and mitogenic
anti-CD40 mAb, and proliferation was assessed by
[3H]thymidine incorporation after 48 h. Assays were
performed in triplicate and error bars are shown.
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To induce H chain class switching in vitro,
A-myb+/- and A-myb-/- B
cells were stimulated with high concentrations of LPS or LPS and IL-4.
After 5 days, supernatants were assayed for levels of IgG3 and IgG1
Abs. Fig. 5 shows that
A-myb-/- B cells produced 2-fold higher
levels of IgG3 in response to LPS, perhaps as a consequence of their
sometimes slightly enhanced proliferative response under these
conditions. Stimulation of both A-myb-/- and
A-myb+/- B cells with LPS and IL-4 resulted in
suppression of production of IgG3 and dramatic enhancement of
production of IgG1. These data indicate that regulation of class
switching is not altered by the A-myb deficiency.
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FIGURE 5. H chain class switching by A-myb-/- B cells in vitro.
Purified small resting B cells were prepared and stimulated in vitro
with LPS or LPS and IL-4 for 5 days and supernatants then collected and
assayed for levels of IgG3 and IgG1 Abs, all as described in
Materials and Methods.
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A-myb is dispensable for the GC reaction and maturation of the B
cell response
A-myb expression and functional importance are strongly correlated
in testes and in the breast in pregnancy (7). In addition,
A-myb-/- mice display behavioral abnormalities,
suggesting that A-myb expression in the CNS is functionally relevant
(K.A.V. and E.P.R., unpublished observations). In contrast, whereas
expression of A-myb in the immune system is highly restricted to GC B
cells, our data show that its absence does not overtly perturb the GC
reaction and related processes necessary for memory B cell development.
It will be important to determine whether this is due to redundancy of
function of other myb family members and A-myb during the GC reaction.
Preliminary analyses of c-myb mRNA levels in the splenocytes of
A-myb-/-, A-myb+/-, and
A-myb+/+ mice revealed no significant differences
(data not shown), but detailed studies of the expression of c-myb and
other myb family members in GC B cells at various stages of the immune
response will be required to appropriately address this issue.
Moreover, further studies will be required to determine whether GC
formation or pathways are altered by A-myb deficiency under conditions
of less robust antigenic stimulation and whether the memory B cells
produced in A-myb-/- mice are phenotypically
and functionally identical with those that develop in normal mice.
Nonetheless, previous hypotheses that ascribe a crucial role for A-myb
in the GC reaction need to be revised given the data presented
here.
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Acknowledgments
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We thank David Dicker (Kimmel Cancer Institute flow cytometry
facility) and Kate Dugan for technical assistance, and all members of
the Manser laboratory for their indirect contributions to this work.
This manuscript is dedicated to the memory of Dr. Antonio Toscani, who
constructed and initially characterized the A-myb null line of mice in
the laboratory of E.P.R.
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Footnotes
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1 This work was supported by a National Institutes of Health Grants to T.M. (AI23739) and E.P.R. (CA79085). During portions of this study K.A.V. was support by a training grant from the National Institutes of Health (CA09678). 
2 Current address: Biogen Corporation, Inflammation and Immunology Group, 12 Cambridge Center, Boston, MA 02142.
3 Address correspondence and reprint requests to Dr. Tim Manser, Jefferson Medical College, BLSB 708, 233 South 10th Street, Philadelphia, PA 19107.
4 Abbreviations used in this paper: GC, germinal center; PNA, peanut agglutinin; NP-CGG, (4-hydroxy-3-nitrophenyl)acetyl chicken globulin; bio, biotin.
Received for publication July 19, 2000.
Accepted for publication December 11, 2000.
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Abstract
Introduction
