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*
Interdisciplinary Graduate Program in Immunology and
Department of Pathology, University of Iowa College of Medicine, Iowa City, IA 52242; and
Department of Microbiology, Dartmouth Medical School, Lebanon, NH 03756
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The GC reaction is critically dependent upon cognate interactions
between Ag-specific B and T cells, and represents one of two
developmental pathways to which activated B cells commit during
T-dependent responses, the other being the differentiation to
Ab-secreting plasma cells. Despite the accumulating data regarding
molecular requirements for GC formation, such as the critical role for
CD40-CD40L interactions (9) and the developmental
requirements for TNF-
, TNFR1, LT-, LT-ß, and LT-ßR
(10, 11, 12, 13, 14), much remains unclear regarding the processes
associated with GCs. Little is known regarding the mechanisms that
regulate commitment of B cells to the GC, somatic hypermutation, and
differentiation to the memory cell or plasma cell stage. Although
current investigation is beginning to reveal candidate molecules that
participate in murine GC regulation and terminal differentiation, such
as OX40-OX40L and the transcription factor Blimp-1
(15, 16, 17, 18), a more detailed understanding of the various
stages of GC maturation is needed. An initial step toward this goal is
the phenotypic and functional characterization of GC B cell
subsets.
During primary B lymphopoiesis, committed precursors progress through successive stages of maturation and selection, a process accompanied by the acquisition of unique cell surface phenotypes determinable by multiparameter flow cytometry (19, 20, 21, 22). Indeed, the phenotypic characterization of distinct B cell precursor populations has greatly facilitated subsequent investigations of genes required for or regulating B lineage commitment and differentiation. Exploiting changes in cell surface phenotype following peripheral B cell activation, Pascual et al. (23) similarly utilized flow cytometry to identify phenotypes potentially corresponding to sequential stages of peripheral B cell differentiation in the human. Subsequent isolation and molecular and functional analysis have suggested developmental relationships between these subsets and permitted the construction of a linear model of Ag-driven B cell differentiation (4). In addition, recent in vitro studies have partially recapitulated Ag-induced developmental stages associated with the GC (24).
In the mouse, several groups have employed flow cytometry and/or immunohistology to describe phenotypic characteristics distinguishing GC B cells from other B cell subsets (25, 26, 27, 28). Initial studies examining GC B cell phenotypes elicited during primary or secondary humoral response utilized heterologous erythrocytes (SRBC) as Ag (25). More recently, the phenotype of GC B cells evoked during the genetically restricted response of C57BL/6 mice to the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) has been partially characterized (27, 28). However, no report explicitly investigating the ontogeny and distribution of selected activation Ags and surface Ig (sIg) isotypes within the GC B cell compartment has been published. Such a study would have the potential to identify discrete GC B cell subsets that possibly correspond to distinct stages of GC B cell ontogeny. The SRBC system, previously exploited by Nieuwenhuis and colleagues (8) to determine the oligoclonality of the GC reaction in rats and Kraal et al. (25) to partially examine murine GC B cell phenotypes, was selected for study due to its potential to induce robust polyclonal GC responses in an adjuvant-independent manner.
This work presents multiparameter flow-cytometric data resolving GC B cells (defined as B220+PNAhigh splenic mononuclear cells from immunized mice) into discrete subsets based on the differential expression of several cell surface markers, including BLA-1, sIgM, sIgD, sIgG1, CD23, and CD38. Examination of sIg heavy and light expression by GC B cells revealed that isotype switching preceded or was concomitant with GC formation, established IgM as the predominant isotype expressed by GC B cells, and revealed an apparent steady state between switched and nonswitched GC B cells. B220+PNAhigh B cells defined by sIgD, CD38, and CD23 expression provided evidence for protracted recruitment into GCs throughout the first 2–3 wk of the primary response, revealing populations potentially corresponding to early stages following commitment to, or differentiation within, the GC reaction. Expression of the activation Ag BLA-1 was dynamic during the GC response, and GC B cell subsets defined by BLA-1 expression or absence correlated with the inductive, established, and dissociative phases of the GC response. Further phenotypic heterogeneity within the GC B cell compartment was revealed by four-color flow cytometry, wherein B220+PNAhigh cells were simultaneously examined for the expression of combinations of the aforementioned surface determinants. Implications of these results on the further study and characterization of GC B cells are discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight- to 16-wk-old female BALB/c mice were obtained from Charles River Laboratories (Wilmington, MA) and housed in a specific pathogen-free facility. CD40L-/- mice were bred in our colony from breeding pairs kindly supplied by Dr. Jacques Peschon (Immunex, Seattle, WA). TNFR1-/- mice were provided by Dr. Charles Lutz (University of Iowa, Iowa City, IA). Following immunization, animals were housed in a clean housing facility until sacrificed on specific days postimmunization.
Abs for flow cytometry
The following reagents were used during the flow-cytometric
studies: RA3-6B2, a rat IgG anti-mouse B220; 1D3, a rat IgG
anti-mouse CD19; clone 90, a rat IgG anti-mouse CD38; Jo2, a
hamster IgG anti-mouse CD95 (PharMingen, San Diego, CA); 53-10.1, a
rat IgG anti-mouse BLA-1; M1/69, a rat IgG anti-mouse
CD24 (heat-stable Ag (HSA)); 1C10, rat IgG anti-mouse
CD40; CY34.1.2, mouse IgG anti-mouse CD22; 7E9, rat IgG
anti-mouse CD21/CD35; PS/2, rat IgG anti-mouse
VLA-4; F441, rat IgG anti-mouse LFA-1; YN1, rat IgG anti-mouse
ICAM-1; b-7-6, rat IgG anti-mouse IgM; 11-26, rat IgG
anti-mouse IgD; B3B4, a rat IgG anti-mouse CD23; GL-1, rat IgG
anti-mouse CD86; GL-7, a rat IgM directed against an undefined
Ag; S7, a rat IgG anti-mouse CD43; M5114, a rat IgG anti-mouse
MHC II; 281-2, a rat anti-mouse Syndecan 1 (PharMingen);
polyclonal goat anti-mouse 
light chain; polyclonal goat
anti-mouse IgG1; PNA, specific for terminal galactosyl (ß-1,3)
N-acetylgalactoseamine residues. Biotin-conjugated
polyclonal goat anti-mouse IgG1, PE-conjugated polyclonal goat
anti-mouse light chain, and PE-streptavidin were purchased from
Southern Biotechnology Associates (Birmingham, AL). Chromatographically
purified rat and goat IgG (Jackson ImmunoResearch, West Grove, PA) were
used as isotype controls. FITC-PNA and FITC-streptavidin were purchased
from Vector Laboratories (Burlingame, CA). Texas Red-streptavidin was
purchased from Leinco Technologies (Ballwin, MO) and The Jackson
Laboratory (Bar Harbor, ME). mAbs were semipurified from HB101
serum-free supernatants by 50% ammonium sulfate precipitation and
conjugated to biotin, PE, Texas Red, or Cyanine 5.18 using standard
procedures. Cyanine 5.18-streptavidin was prepared in our laboratory
using standard procedures.
Immunizations
Mice were injected once i.p. with 0.2 ml of 10% v/v SRBC (Colorado Serum Company, Denver, CO) in balanced salt solution (BSS; equivalent to 1–5 x 108 SRBC) or 25 µg of TNP-Ficoll (Solid Phase Sciences, San Rafael, CA). Splenocytes were examined at 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 days postimmunization.
Cell preparation
On the selected days postimmunization, animals were anesthetized with Metofane (methoxyfluorane) and animals sacrificed by cervical dislocation. Spleens were removed and cell suspensions prepared by pressing the excised spleens between the frosted ends of two glass slides, followed by suspension in BSS. Cells were washed by centrifugation at 1500 rpm for 7 min at 4°C and resuspended in staining buffer (SB; BSS supplemented with 5% bovine calf serum and 0.1% sodium azide). Mononuclear cells were isolated by density centrifugation over FicoLite-LM (Atlanta Biologicals, Norcross, GA) for 20 min at 2500 rpm at room temperature. Cells at the interface were collected and resuspended in BSS and again washed by centrifugation at 1500 rpm for 7 min at 4°C. Viable cells were enumerated by hemocytometer and resuspended at a final concentration of 50 x 106 cells/ml in SB.
Flow-cytometric analysis
A total of 5 x 105 splenocytes in SB
was added to the appropriate mixture of biotinylated, FITC-, PE-,
cyanine 5.18-, or Texas Red-conjugated Abs, and fluorochrome-conjugated
PNA in the presence of 25 µg 2.4G2 (anti-Fc
R) and 10 µl
normal rat serum to minimize Fc receptor-dependent background staining.
Cells were incubated on ice for 20 min, then washed twice with SB.
Biotinylated reagents were visualized by the addition of appropriately
conjugated streptavidin. After 20 min on ice, cells were again washed
twice with SB, suspended in fixative (1% formaldehyde in 1.25x PBS),
and run on a FACSVantage flow cytometer (Becton Dickinson, Mountain
View, CA) featuring a primary argon ion laser and a rhodamine 6G CR559
dye head laser (Coherent, Palo Alto, CA) pumped by a second argon ion
laser. A minimum of 30,000 events was collected per sample. Low angle
and orthogonal light scatter were used to exclude dead cells and
debris, and electronic compensation was utilized to correct for
spectral overlap between FITC/PE and Texas Red/cyanine 5.18. Data were
stored on a VAX station 3200 computer equipped with DESK FACS analysis
software (kindly provided by Wayne Moore, Stanford University,
Stanford, CA). Two-color contours are represented as 2% probability
plots. Two-color contours from four-color flow-cytometric studies are
represented as 5% probability plots. In those experiments in which
goat anti- light chain or goat anti-IgG1 Abs were utilized,
it was necessary to first incubate cells with these reagents in the
absence of rat or mouse Abs and blocking agents. Cells were washed
after treatment with the goat Abs, followed by addition of blocking
agents, and stained as described above. To facilitate analysis and
comparison of populations defined by the expression or absence of
particular surface determinants, data for each time point presented in
Figs. 3
A, 4A, and 5A were derived from
the same mouse.
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Confocal microscopic analysis was performed as described previously (29). Briefly, spleens from unimmunized or SRBC-immunized animals were harvested and embedded in 4% agarose in HBSS and sectioned in a vibrating microtome (Microcut H12; Energy Beam Sciences, Agawam, MA), and 500-µm sections were incubated overnight at 4°C with FITC-conjugated anti-IgD (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) and cyanine 5.18-conjugated PNA (both used at a concentration of 10–20 µg/ml in PBS/1% BSA/0.02% sodium azide). Mouse IgG was included to minimize Fc-dependent background staining. After washing, the samples were mounted in slides with PBS/azide/10% glycerol and analyzed on the confocal microscope (Bio-Rad 1024; Bio-Rad, Hercules, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using multiparameter flow cytometry, we identified and enumerated
GC B cells 4–26 days following primary immunization of BALB/c mice
with SRBC. B220+PNAhigh
cells, previously defined by flow cytometry and immunofluorescence as
GC B cells (25, 26), were apparent by day 4
postimmunization, reached a peak frequency by days 6–8, and then
decreased to one-half this maximum frequency between days 12 and 14
postimmunization (Fig. 1).
B220+PNAhigh cells could
still be identified into the third and fourth week postimmunization,
but by day 42, only a residual
B220+PNAhigh population was
detected (Fig. 1, and data not shown). This kinetic profile is
consistent with previous immunohistologic, stathmokinetic, and
flow-cytometric analyses of GC evolution (28, 30, 31, 32).
These results also demonstrate the robust nature of the SRBC response
and the potential to analyze GC B cells in detail.
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, and data not shown). Likewise,
B220+PNAhigh cells were
undetectable following SRBC immunization of mice lacking CD40L or TNFR1
(data not shown).
To further substantiate the identity of the
B220+PNAhigh cells, we more
thoroughly characterized the phenotype of this population. The
B220+PNAhigh cells were
determined to consist of mature B cells because they uniformly
expressed the B cell lineage markers CD19, CD21/35, CD22, and CD40,
excluding contamination by either non-B cells or immature B cells (Fig. 2 and Table I). In addition, and consistent with
earlier findings (25, 28), the
B220+PNAhigh population was
not enriched for Ab-forming cells (AFCs), as demonstrated by the
virtual absence of AFC-associated markers such as CD43 and Syndecan-1
(CD138) (Table I). Furthermore, as shown in Fig. 2, the majority of
B220+PNAhigh cells
expressed heightened levels of CD95 (Fas) and the Ag recognized by the
mAb GL-7, as has been previously reported for GC B cells (5, 36). Although GL-7 expression roughly overlapped with high
avidity for PNA, GL-7 expression was heterogeneous within the
B220+PNAhigh population,
and a population of
B220+PNAlow cells
expressing GL-7 was detected (Fig. 2). The significance of GL-7
heterogeneity within the GC compartment, also apparent in
immunohistochemical analyses of GCs (37), is unclear at
this time. Collectively, these data demonstrate that
B220+PNAhigh cells are
mature B cells present only when GC formation occurs and which possess
the phenotypic attributes of GC B cells, but not AFCs.
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Having established a reproducible system for induction of GC
responses, we further analyzed the phenotype of murine GC B cells by
investigating the expression and distribution of selected cell surface
markers within the GC B cell compartment at consecutive time points
following primary immunization. Cohorts of mice were challenged with
SRBC, and 4–18 days following immunization, splenic mononuclear cells
were subjected to multiparameter flow-cytometric analysis in which mAbs
specific for one or two of the surface markers listed in Table I were
used in conjunction with anti-B220 mAb and fluorochrome-conjugated
PNA to delineate GC B cells. As shown in Fig. 2 and summarized in Table I, GC B cells prominently expressed CD19, CD21/35, CD22, CD40, and
CD95. In addition, GC B cells also were found to express high levels of
HSA (CD24) and class II MHC molecules, intermediate levels of CD11a and
CD54, and low levels of VLA-4 (Table I). However, these proteins were
uniformly expressed by GC B cells on all days examined (data not
shown), and discrete GC B cell subpopulations defined by the presence
or absence of these surface markers were therefore not evident. In
contrast, GC B cell subsets defined by the expression of sIg isotypes
and activation markers were readily detected.
GC B cell subsets delineated by IgM or IgG1 expression
Because GCs are associated with isotype switching (reviewed in
Ref. 38 ; see also Refs. 30, 32, 39), we
evaluated the distribution and kinetics of sIgM, sIgG1, and sIgG2a
expression following primary antigenic challenge. In Fig. 3A,
sIgM+ GC B cells were detected at day 4 and
represented approximately two-thirds of the GC B cell compartment, with
one-third being sIgM-. Correspondingly,
approximately one-fourth to one-third of the GC B cell population was
sIgG1+.
Surprisingly, despite the expansion and subsequent diminution of the GC
B cell compartment over time, the percentage of total GC B cells
expressing or lacking either sIgM or sIgG1 remained nearly constant
throughout the primary GC reaction, even 3–4 wk postimmunization (Fig. 3A). In contrast, and characteristic of the BALB/c response,
sIgG2a+ GC B cells were infrequent upon SRBC
immunization, and not always detected (data not shown). Using
four-color flow cytometry to simultaneously examine expression of sIgM
and sIgG1, a mutually exclusive pattern of sIgM and sIgG staining was
observed on days 4, 8, 12, and 18 (Fig. 3B). This
demonstrates the sIgG1+ cells to be bona fide
class-switched GC B cells, and not cells with Fc receptor-bound immune
complexes. In addition, the relative frequencies of
sIgM+ and sIgG1+ cells did
not change by treating cells with a low pH buffer before staining (data
not shown). Although IgG1 is the major target of isotype switching upon
SRBC challenge in BALB/c mice, the results in Fig. 3B
indicate switching to other isotypes. Because virtually all of the GC B
cells at all time points were light chain positive (data not shown)
(26, 40), the sIgM, sIgG1 negative cells are likely to
represent infrequent sIgG2+,
sIgA+, and sIgE+ cells.
The contemporaneous emergence of sIgM+ and sIgG1+ GC B cells, as evidenced by our flow-cytometric analyses, suggests that the initiation of isotype switching occurs very early after, concomitant with, or even before the induction of the GC reaction following SRBC immunization. Intriguingly, there was no progressive conversion, as measured by frequency, from sIgM+ to class-switched GC B cells during the primary response. Furthermore, the data presented in this work corroborate earlier immunohistochemical and flow-cytometric studies indicating that class switching precedes somatic hypermutation within the murine GC, a process that is not discernible by current methodologies until 6–8 days following immunization (30, 32). This is in apparent contrast to the human GC reaction, as Liu and colleagues have recently provided evidence that a subset of human GC B cells undergoes class switching, and that this occurs subsequent to the initiation of somatic hypermutation (41, 42).
BLA-1 expression is dynamic and correlates with distinct phases of the GC reaction
The uncharacterized 53-kDa cell surface determinant BLA-1 was
previously reported to be expressed by GC B cells (27, 43). Interestingly, BLA-1+ cells were
capable of adoptively transferring memory responses 6 days, but not 6
wk following immunization (43), and hapten-specific,
sIgG1+ B cells were found to express BLA-1 at day
6, but not at day 24 postimmunization (27). Together,
these data indicate that BLA-1 expression is modulated during the
evolution of the GC reaction. We therefore studied the kinetics and
distribution of BLA-1 expression on GC B cells. In Fig. 4A, the vast majority of GC B
cells were BLA-1+ at 4 days postimmunization,
consistent with previous phenotypic analyses. By day 8, however, BLA-1
expression became heterogeneous; approximately two-thirds of GC B cells
were BLA-1+, but one-third was now
BLA-1-. The frequency of
BLA-1+ GC B cells continued to decrease until by
day 12 approximately one-half of the GC B cells were
BLA-1+ and the other half
BLA-1-, a pattern also evident during the third
week postimmunization (Fig. 4B). By 24–26 days following
challenge, little or no surface BLA-1 was present on GC B cells (data
not shown). Therefore, the pattern of BLA-1 expression closely
correlated with the distinct phases of GC evolution, with uniform
expression typical of the inductive phase, heterogeneous expression
characteristic of the established phase, and relative absence
representative of the dissociative phase.
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B, sIgM and BLA-1 expression by
B220+PNAhigh-gated cells
defined four subsets at day 8 postimmunization. The predominant GC B
cell subset at this time expressed both sIgM and BLA-1 and
represented approximately one-half of the total GC B cell population
and three-quarters of sIgM+ GC B cells. A
prominent sIgM- BLA-1+
population was also evident at day 8, accounting for nearly one-third
of total GC B cells and greater than one-half of
sIgM- GC B cells. Two minor subpopulations,
namely sIgM+ BLA-1- and
sIgM- BLA-1-, each
accounted for less than one-fifth of total GC B cells at day 8. The
frequency of BLA-1+ GC B cells decreased between
days 8 and 12 postimmunization, and this reduction was observed in both
the sIgM+ and sIgM- GC B
cell populations. The relative frequencies of
sIgM+ and sIgM- GC B cells
expressing BLA-1 observed at day 12 were maintained as late as day 18
(Fig. 4B). These data indicate that BLA-1 expression is not
restricted to sIgM+ GC B cells and that BLA-1
expression decreases as a function of time among both
sIgM+ and sIgM- GC B cell
subsets. Subsets of B220+PNAhigh cells revealed by the expression of sIgD, CD23, and CD38
It is largely accepted that most GC B cells are derived from mature naive B cells expressing sIgM and sIgD (5). The vast majority of GC B cells, however, have been reported to be sIgD-, including those bearing sIgM (8, 25, 26). sIgD+ GC B cells from human tonsils have been previously described (44, 45) and more recently isolated and partially characterized (46, 47, 48, 49). GC B cells expressing both sIgM and sIgD most likely represent a transitional stage between follicular mantle and GC B cells and may correspond to putative GC founder cells (48). We sought to identify such GC founder cells by examining within the GC B cell compartment the expression and distribution of molecules associated with naive B cells, namely sIgD, CD23, and CD38. CD23 is expressed late in B cell ontogeny, but is absent on isotype-switched B cells (50, 51, 52). Expression of CD23 on GC B cells, as tested by immunohistochemical means, has been difficult to determine due to robust expression of this molecule on follicular dendritic cells (53, 54). CD38 is expressed by naive B cells, down-regulated by GC B cells, and subsequently up-regulated by memory B cells (55, 56, 57, 58).
As seen in Fig. 5A, subsets of
B220+PNAhigh cells both
expressing and lacking CD38, CD23, and sIgD were observed
following SRBC immunization. At day 4,
B220+PNAhigh cells
uniformly expressed CD38 (albeit at lower levels than on
B220+PNAlow cells), more
than one-half were CD23+, and nearly one-fifth
were sIgD+. By day 8, a
B220+PNAhigh population
expressing high levels of CD38 could be identified, and the emergence
of this prominent subset coincided with an increase in frequency of
sIgD+ cells, suggesting that the
CD38high cells also expressed sIgD. By days 12
and 18 of the primary response, the CD38high
subset was less numerous, representing approximately one-third of the
B220+PNAhigh population.
These data are thus consistent with previous reports demonstrating
diminished CD38 expression on GC B cells following immunization
(57, 58). Although the sIgD+ subset
had also decreased in relative frequency at these later time points,
approximately one-half or more of the
B220+PNAhigh cells remained
CD23+. Because
B220+PNAhigh cells
expressing CD23 were less numerous than those bearing sIgM, but more
numerous than cells displaying sIgD or CD38 (see Figs. 3A
and 5A), we speculated that the loss of sIgD and CD38
precedes that of CD23 within the sIgM+ GC B cell
population. We further speculated that simultaneous evaluation of CD23
and sIgD or CD38 would identify discrete or overlapping populations
possibly representing early intermediates in GC B cell ontogeny. This
was confirmed in Fig. 5B, in which
sIgD+B220+PNAhigh
cells were found to express high levels of CD23. A
CD23+sIgD- population was
also observed, in accordance with the data in Fig. 5A, and
suggesting that the loss of sIgD precedes that of CD23. The
sIgD+B220+PNAhigh
cells were also found to be CD38high and
sIgM+ (data not shown). In Fig. 5C,
simultaneous examination of CD23 and CD38 expression revealed a
CD38high population that similarly expressed high
levels of CD23 at most time points. Although CD38 expression within the
B220+PNAhigh compartment
was not bimodal at day 4, a subset of CD23high
cells expressing elevated levels of CD38 was detected. Fig. 5C also demonstrates a small CD23+CD38low
population, again indicating transition of GC B cells to a
CD38low phenotype precedes loss of CD23.
Together, these data indicate that the acquisition of PNA receptors can
occur before the loss of sIgD, CD23, and CD38, and the resulting
sIgM+sIgD+CD23+CD38highB220+PNAhigh
cells may represent the earliest determinable stage of commitment to
the GC reaction. This early stage is most likely followed by sequential
loss of IgD and CD38, leaving a
sIgM+sIgD-CD23+CD38low
intermediate stage GC B cell. It is also of interest that both the
sIgM+sIgD+CD23+CD38high
and
sIgM+sIgD-CD23+CD38low
GC subsets are present at all time points examined, suggesting that
founder cells are continually recruited into the GC reaction or
maintained with a founder phenotype once the GC has been
established.
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As the
IgD+B220+PNAhigh
cells were an unexpected finding, especially at the later time points,
we further explored the location of these cells relative to GCs.
Specifically, we used two-color confocal microscopy of fresh splenic
sections to determine whether these cells were within or outside of
established GCs. This technology was chosen because it leaves sensitive
epitopes intact and generally allows for greater sensitivity compared
with other techniques. Spleens were harvested from animals immunized 8
days previously with SRBC (corresponding to the peak of the primary GC
reaction and the time at which the
IgD+B220+PNAhigh
cells were most numerous in the previous flow-cytometric experiments).
Splenic sections were then stained with FITC-conjugated anti-IgD
(green) and cyanine 5.18-conjugated PNA (blue) to delineate both
IgD+ cells and GCs. In spleens from unimmunized
control mice, IgD+ cells were limited to the
follicles, and PNA staining exhibited a background reticular pattern
consistent with quiescent spleen (data not shown). In a day 8 immunized
spleen, however, PNA+ GCs were readily apparent.
As illustrated in Fig. 6,
IgD+PNA+ double staining
cells were also evident (appearing as turquoise cells), and
surprisingly, could be found in several follicular sites. In addition
to a small number residing within the GC, clusters of
IgD+PNA+ cells were
observed in the mantle both at the GC mantle interface and in areas
more distant from the GC. This was a consistent finding in three
separate immunized mice (data not shown). Thus, while
IgD+PNA+ cells were indeed
part of the GC, a significant fraction of this population was found
outside of the GC. Although the meaning of this result is presently
unclear, the finding of non-GC
IgD+PNA+ cells may be
consistent with IgD+ cells being continually
recruited into the GC reaction (founder cells) or
IgD+ cells exiting the GC as post-GC or memory B
cells.
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We subsequently extended our phenotypic analysis of GC B cells to
include the relatively uncharacterized late phase of the GC response,
primarily to determine whether the GC B cell subsets that were
established early following immunization persisted throughout the
response. As shown in Fig. 1, B220+PNAhigh cells were
still consistently detected 16–26 days postimmunization.
Interestingly, and despite the reduction in the overall frequency of GC
B cells, the subsets defined by sIgM, CD23, and CD38 expression were
still present and in the relative frequencies observed upon their
establishment during the inductive phase (data not shown). sIgD
expression was less evident at these times with between 10 and 15% of
total B220+PNAhigh cells
expressing sIgD (data not shown). In addition, fewer GC B cells
expressed BLA-1 at these later time points, although 20–30% of GC B
cells were BLA-1+ as late as days 22–24 (data
not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The multiparameter flow-cytometric data presented in this study provide
a comprehensive phenotypic analysis of the murine GC B cell compartment
at consecutive intervals following primary immunization of BALB/c mice
with SRBC. We defined GC B cells as
B220+PNAhigh splenic
mononuclear cells, and the validity of this combination of surface
determinants was verified in several ways. In addition to demonstrating
this population’s Ag, T cell, and CD40 dependence,
B220+PNAhigh cells were
found to be comprised exclusively of mature B lineage cells that
expressed markers previously ascribed to GC B cells, but were devoid of
markers associated with AFCs (Figs. 1 and 2, Table I, and data not
shown). We subsequently resolved the GC B cell population into discrete
subsets based on the expression and distribution of various Ig isotypes
(sIgM, sIgD, and sIgG1), the activation marker BLA-1, and
differentiation Ags (CD23, CD38).
sIgM was found to be expressed by more than one-half of GC B cells
isolated 4–26 days following SRBC immunization (Fig. 3A,
data not shown). sIgG1+ cells were also evident
at day 4 postimmunization, and represented between one-quarter to
one-third of GC B cells at that and all subsequent time points (Fig. 3A). The sIgG1+ cells accounted for
the majority of sIgM- B cells, and few bona fide
sIg- cells were observed at any time
postimmunization (Fig. 3, data not shown). Strikingly, although the
frequency of the total GC B cell population waxed and waned, the
relative frequencies of sIgM+ and
sIgG1+ GC B cells remained nearly constant,
possibly reflecting the establishment of a dynamic steady state between
class-switched and nonswitched GC B cells (Fig. 3).
The kinetics and distribution of Ig isotypes observed during the primary anti-SRBC response are consistent with earlier studies demonstrating the rapidity of isotype switching in vivo (27, 30, 32, 59), but raise questions regarding the extent, kinetics, and duration of the isotype switch reaction with respect to GC formation and evolution. In immunohistochemical analyses of C57BL/6 mice responding to a thymus-dependent form of the hapten NP, isotype-switched cells were first detected within extrafollicular PALS foci (from which both AFCs and GC founder cells originate), and the majority of GCs observed (first seen in this report at day 8) were comprised almost entirely of isotype-switched cells, indicating a rapid and near total conversion from sIgM to sIgG in both PALS foci and GCs (30). Although subsequent studies by Kelsoe and colleagues reported GC formation as early as day 4 postimmunization (63), the heavy chain isotype expressed within these early GCs was not examined. Using multiparameter flow cytometry, other studies of the primary anti-NP response revealed that Ag-specific, isotype-switched GC and non-GC B cells displayed similar kinetics with respect to appearance and exponential expansion, but the relative frequencies of nonswitched vs switched NP-binding GC B cells were not evaluated (27, 32, 59).
In the primary anti-SRBC response of BALB/c mice described in this
study, sIgG1+ and sIgM+ GC
B cells emerged at the same time as GC B cells first become
discernible, and the relative frequencies of these two populations did
not change appreciably over the ensuing 2–3 wk of the response.
Surprisingly, no gradual (or sudden) diminution in the
sIgM+ population coincident with an equivalent
increase in the sIgG1+ population was observed
(Fig. 3). These findings could be interpreted to mean that the isotype
switch reaction not only begins to occur before or concomitant with the
induction of GCs, but that class switching is restricted to this early
phase of B cell activation. Thus, the relative frequencies of the
switched and nonswitched populations established during the early stage
of B cell activation would remain essentially unchanged after this
window of opportunity for class switching had passed. Alternatively,
the present data could imply that isotype switching involving µ
and/or other heavy chain genes may occur continuously throughout the GC
response, with regulatory mechanisms maintaining a nearly constant
frequency of both switched and sIgM+ B cells.
Although it is unclear which mechanism accounts for the almost fixed frequency of switched and nonswitched cells in response to SRBC challenge, evidence for both exists in other systems. Specifically, studies have provided support for isotype switching at extrafollicular sites early in the response as well as in the GC later after Ag challenge. As discussed above, immunohistochemical studies by Kelsoe and colleagues have provided evidence for switching in both PALS foci and GC (30). Toellner et al. (39) and Peakman and Maizels (64), combining RT-PCR or in situ hybridization with immunohistochemistry, respectively, examined the localization of switch transcript production in situ and reported the predominant site of isotype switching during anti-NP responses to be in the extrafollicular T cell regions of immunized spleen. Peakman and Maizels (64) documented that peak transcript production preceded GC formation, although switch transcripts were detected within GCs subsequent to their appearance, suggesting GCs also have the potential to serve as sites of isotype switching. Consistent with this, Zinkernagel and colleagues (65), performing immunohistochemical studies following primary infection with vesicular stomatitis virus, reported both switched and nonswitched GC B cell populations at day 12 following infection. This is of interest because PALS foci are not observed in response to vesicular stomatitis virus immunization. Finally, evidence for isotype switching within the GC also comes from human studies in which switch transcripts and DNA excision loops, intermediates in deletional isotype switch recombinations, were detected in sort-purified GC B cell subsets (41, 42). It is thus possible that the present results could be explained by either switching before GC formation, within the GC, or both. Experiments examining sIgM+ and sIgG1+ GC B cells at various times after SRBC immunization for intermediates of the isotype switching reaction are in progress to directly address this issue.
The persistence of sIgM+ cells throughout all stages of the primary response was particularly striking, and could also be explained by a protracted recruitment of mature sIgM+ cells into the GC reaction, although some of the sIgM+ cells may well represent the precursors of sIgM+ memory cells, direct evidence for which has been reported in the human (66, 67, 68, 69). However, most sIgM+ GC B cells also expressed CD23 (data not shown), a determinant expressed late in B cell ontogeny, but absent from isotype-switched or sIgM+ memory B cells (50, 51, 67). Moreover, and as discussed below, a significant proportion of sIgM+ GC B cells simultaneously expressed sIgD, CD23, and levels of CD38 comparable with follicular B cells. Collectively, these phenotypic data are consistent with many of the sIgM+ cells corresponding to GC founder cells or other early stages of GC B cell differentiation.
Recently, human GC founder cells have been identified and partially
characterized (46, 47, 48, 49). This population exhibited a
phenotype reminiscent of both follicular and GC B cells, including the
expression of sIgD, sIgM, CD23, and CD38. In our studies, mAbs directed
against murine sIgD, CD23, and CD38 revealed subpopulations of
B220+PNAhigh cells
expressing these respective markers (Fig. 5A). This
phenotype is virtually identical to that described for human GC founder
cells, allowing for the possibility that murine
sIgD+CD23+CD38highB220+PNAhigh
cells likewise represent the first stage of GC commitment.
Surprisingly, B220+PNAhigh
cells expressing sIgD, CD38, or CD23 were detected after day 10, when
the peak of the GC response (as measured by the frequency of
B220+PNAhigh cells) had
diminished (Fig. 5A). Although the significance of this
observation is currently unclear, it could suggest that recruitment
from the follicular B cell pool may occur for an extended period of
time following SRBC challenge.
Further phenotypic analysis revealed that CD23, CD38, and sIgD
demarcated discrete but overlapping populations potentially
corresponding to early stages of GC B cell ontogeny (Fig. 5, B and C, and data not shown). From the kinetic
analyses and the four-color flow-cytometric data, it would appear that
during the process of commitment to the GC reaction,
sIgM+sIgD+CD23+CD38high
follicular B cells acquired receptors for PNA before the loss of sIgD.
Based on the comparative frequencies of sIgM, sIgD, CD23, and
CD38-expressing GC B cells, it is likely that both sIgD and CD38 were
subsequently down-regulated by the sIgM+ GC B
cells. In contrast, CD23 expression was maintained until the
sIgM+ GC B cells either underwent isotype
switching or proceeded further along the GC developmental trajectory
toward nonswitched memory B cells, as few
sIgM+CD23- GC B cells were
detected until about day 8 (data not shown).
Although
sIgD+B220+PNAhigh
cells have previously been detected 12 days following immunization of
C57BL/6 mice with NP keyhole limpet hemocyanin (70) and
IgD+ cells can be found within presumptive GCs 9
days after immunization with pigeon cytochrome c
(71), IgD expression by murine GC B cells has not been
well described. To more thoroughly investigate the
IgD+B220+PNAhigh
population observed by flow cytometry, we employed two-color confocal
microscopy of splenic sections to examine cellular localization.
IgD+PNAhigh cells detected
8 days following immunization were found within the follicular mantle
of secondary follicles, along the border of the follicular mantle and
the subjacent GC, and within the GC (Fig. 6). The presence of non-GC
IgD+PNAhigh cells is of
particular interest, and can be interpreted several ways. One could
suggest these cells to be at an early stage of activation and
commitment to the GC, and hence founder cells, as discussed above.
Alternatively, these cells might be IgD+ GC B
cells emigrating from the GC, and hence represent a population of
memory cells, especially at later time points. It is also possible that
both processes are going on simultaneously.
That murine IgD+PNAhigh cells might represent post-GC memory cells is all the more intriguing with the recent revelation of a population of human memory B cells expressing not only sIgM, but also sIgD (68, 69). In the human studies, CD27 expression was found to distinguish human memory B cells (both sIgM+ and sIgM-) from naive B cells (68, 69), raising the possibility that this marker may prove useful in murine studies as well. In preliminary experiments, however, few if any murine GC B cells were found to express detectable levels of CD27 at days 4, 8, 12, and 18 postimmunization (Shinall and Waldschmidt, unpublished observations).
Unlike the situation described above for isotype distribution,
expression of the activation Ag BLA-1 within the GC B cell compartment
was found to be dynamic over time. BLA-1 expression was initially
uniform, but became heterogeneous by the end of the first week. By day
12 and thereafter, approximately one-half of the GC B cell population
expressed BLA-1, and by days 24–26, most GC B cells were devoid of
this marker (Fig. 4A, and data not shown). Intriguingly,
simultaneous evaluation of sIgM and BLA-1 expression at different times
following immunization revealed four GC B cell subsets with the
predominant GC B cell population, expressing both sIgM and BLA-1 (Fig. 4B). BLA-1 expression decreased among both
sIgM+ and sIgM- cells as a
function of time. These data confirm and extend earlier studies that
documented expression of BLA-1 in GCs at early but not late time points
following immunization (27, 43).
The identity, function, and regulation of expression of BLA-1 remain unclear, although polyclonal activators such as LPS can induce BLA-1 expression on cultured B cells and such BLA-1+ cells have the forward light scatter characteristics of B cell blasts (43). Given the distribution and kinetics of BLA-1 expression within the GC compartment and the restriction of BLA-1 to LPS blasts in vitro, it is conceivable that BLA-1 expression may coincide with proliferating GC B cells. Alternatively, expression of this Ag may be induced early during GC B cell ontogeny, and subsequently become down-regulated as a function of time, cell division, or availability of stimuli (e.g., Ag, T cell-derived costimulation), and thus may serve to distinguish recently recruited from resident GC B cells. We are currently investigating the relationship between BLA-1 expression and proliferative and differentiational status within the murine GC.
In summary, the present work identifies GC B cell subsets potentially corresponding to distinct stages of GC B cell ontogeny defined by the differential expression of isotypes (IgM, IgD, IgG1), the activation marker BLA-1, and differentiation Ags (CD38, CD23) as well as the kinetics of their induction and persistence following primary immunization of BALB/c mice with SRBC. Importantly, in comparative studies, the subsets identified in this study were conserved in other strains of mice immunized with SRBC and in BALB/c mice immunized with other thymus-dependent Ags, suggesting that these subsets are general rather than unique to the SRBC response (S. Shinall and T. Waldschmidt, manuscript in preparation). The generality of the findings may therefore allow for further work in the mouse describing subsets or stages of GC B cells in which distinct genetic programs are turned on, leading to affinity maturation and memory cell formation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas J. Waldschmidt, Department of Pathology, University of Iowa College of Medicine, Iowa City, IA 52242.
3 Abbreviations used in this paper: GC, germinal center; AFC, Ab-forming cell; BSS, balanced salt solution; HSA, heat-stable Ag; LT, lymphotoxin; NP, (4-hydroxy-3-nitrophenyl)acetyl; PALS, periarteriolar lymphatic sheaths; PNA, peanut agglutinin; SB, staining buffer; sIg, surface Ig.
Received for publication June 21, 1999. Accepted for publication March 14, 2000.
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