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Is There a Typical Germinal Center? A
Large-Scale Immunohistological Study on the
Cellular Composition of Germinal Centers d
uring the Hapten-Carrier−Driven Primary
Immune Response in Mice
Nicole Wittenbrink, Anke Klein, Armin A. Weiser, Johannes
Schuchhardt and Michal Or-Guil
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Copyright © 2011 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2011; 187:6185-6196; Prepublished online 18
November 2011;
doi: 10.4049/jimmunol.1101440
http://www.jimmunol.org/content/187/12/6185
The Journal of Immunology
Is There a Typical Germinal Center? A Large-Scale
Immunohistological Study on the Cellular Composition of
Germinal Centers during the Hapten-Carrier–Driven
Primary Immune Response in Mice
Nicole Wittenbrink,*,† Anke Klein,*,†,1 Armin A. Weiser,*,† Johannes Schuchhardt,‡ and
Michal Or-Guil*,†
A
ntibody affinity maturation is a hallmark of the adaptive
immune response that requires formation of germinal
centers (GCs) in secondary lymphoid organs such as
spleen and lymph nodes. GCs are crucial sites because they finetune the B cell response with regard to amplitude and specificity,
and thereby lead to the generation of (long-lived) high-affinity
plasma cells and memory B cells (1, 2). Affinity maturation is accomplished through a microevolutionary process during which GC
B cells are subject to random somatic hypermutation (SHM) of
their BCR genes and subsequent affinity-based selection (1).
GCs are complex, multicell-type, transient structures that form
in response to antigenic stimulation. The primary GC response in
*Systems Immunology Laboratory, Department of Biology, Humboldt University
Berlin, D-10115 Berlin, Germany; †Research Center ImmunoSciences, Charité University Medicine Berlin, D-10115 Berlin, Germany; and ‡MicroDiscovery GmbH,
D-10405 Berlin, Germany
1
Current address: Rudolf Boehm Institute of Pharmacology and Toxicology, University Leipzig, Leipzig, Germany.
Received for publication May 17, 2011. Accepted for publication September 29,
2011.
This work was supported by the Volkswagen Foundation and the Bundesministerium
für Bildung und Forschung (Germany) (Grant 0315005B).
Address correspondence to Dr. Michal Or-Guil, Systems Immunology Laboratory,
Research Center ImmunoSciences, Charité University Medicine Berlin, Hessische
Strasse 3-4, D-10115 Berlin, Germany. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: CSA, chicken serum albumin; CV, coefficient of
variation; DRFZ, Deutsches Rheuma-Forschungszentrum Berlin; DZ, dark zone;
FDC, follicular dendritic cell; GC, germinal center; LZ, light zone; phOx, 2-phenyl-5-oxazolone; PNA, peanut agglutinin; ROI, region of interest; SHM, somatic
hypermutation; TBM, tingible body macrophage.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101440
the spleen has been previously shown to exhibit clearly defined kinetics with inductive, established, and dissociative phases (3–5).
Although B cells make up the majority of their cell population,
GCs also encompass T cells, macrophages, and stromal follicular
dendritic cells (FDCs) (1). Other cell types, such as dendritic cells
(6) and accessory CD4+ CD32 cells (7), might be present at times.
The fine processes of FDCs form a network that histologically
divides the GC into two compartments referred to as the dark zone
(DZ) and the light zone (LZ). As distinguished from the DZ, the
LZ shows a compact FDC network wherein FDCs serve as Ag
deposits that build upon trapping of immune complexes through
complement and FcRs (8–10). Because competition for Ag binding has been proposed to drive B cell selection (11), the concentration of Ag on the surface of FDCs in the LZ is commonly taken
as evidence for spatial confinement of selection to this zone. The
capability of FDCs to promote B cell survival through switching
off the apoptotic machinery in adhering cells (12) has further led
to the concept of selection as a result of BCR cross-linking by
FDC-trapped Ag (13, 14). Alternatively, other models presume
a role of follicular Th cells in selection (2, 15, 16). The general
idea is that BCR-bound Ag is first internalized and then presented
as peptide-MHC for follicular T cell recognition. The possible
outcomes of such an interaction with follicular Th cells, a subset
believed to accumulate in the LZ of GCs, are survival and differentiation of GC B cells.
SHM of BCRs generates both B cell variants with advantageous
and deleterious mutations. Therefore, deleterious mutations need to
be eliminated from the GC B cell population to achieve enrichment of high-affinity B cells. As a corollary of this condition, GCs
appear as sites of extensive proliferation on the one hand and
massive programmed cell death on the other hand (17). Classically,
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Germinal centers (GCs) are complex, multicell-type, transient structures that form in secondary lymphatic tissues in response to
T cell-dependent stimulation. This process is crucial to the adaptive immune response because it is the source of affinity maturation
and long-lived B cell memory. Our previous studies showed that the growth of murine splenic GCs is nonsynchronized, involving
broad-volume distributions of individual GCs at any time. This raises the question whether such a thing as a typical GC exists. To
address this matter, we acquired large-scale confocal data on GCs throughout the course of the 2-phenyl-5-oxazolone chicken serum
albumin-driven primary immune response in BALB/c mice. Semiautomated image analysis of 3457 GC sections revealed that,
although there is no typical GC in terms of size, GCs have a typical cellular composition in that the cell ratios of resident T cells,
macrophages, proliferating cells, and apoptotic nuclei are maintained during the established phase of the response. Moreover, our
data provide evidence that the dark zone (DZ) and light zone (LZ) compartments of GCs are about the same size and led us to
estimate that the minimal cell loss rate in GCs is 3% per hour. Furthermore, we found that the population of GC macrophages is
larger and more heterogeneous than previously thought, and that despite enrichment of T cells in the LZ, the DZ of murine splenic
GCs is not poor in T cells. DZ and LZ differ in the T cell-to-macrophage ratio rather than in the density of T cells. The Journal of
Immunology, 2011, 187: 6185–6196.
6186
Materials and Methods
Mice and immunization
Six- to 8-wk-old BALB/c mice were immunized with a single i.p. injection
of 100 mg phOx coupled to CSA at a ratio of 10:1 and precipitated onto
alum as described previously (27). BALB/c mice were purchased from the
Bundesinstitut für Risikobewertung, Berlin, Germany and were housed
under specific pathogen-free conditions at the animal facility of the
Deutsches Rheuma-Forschungszentrum Berlin (DRFZ), Berlin, Germany.
All animal experiments were performed in accordance with institutional,
state, and federal guidelines.
Antibodies
The following Abs and other reagents were used to visualize splenic architecture and to detect GC cell populations: unconjugated rat IgG2a to mouse Ki67 (clone Tec-3; Dako, Glostrup, Denmark); biotin-labeled Ab to mouse FDC
(clone FDC-M2; ImmunoK, Abingdon, U.K.); Alexa 488-labeled anti-mouse
CD3 (clone KT3; AbD Serotec, Düsseldorf, Germany); Alexa 488-labeled
anti-mouse CD68 (clone FA-11; AbD Serotec); Alexa 647-labeled anti-rat
IgG (Invitrogen, Karlsruhe, Germany); biotin-labeled anti-mouse B220 (clone
RA3.6B2, in-house conjugate, DRFZ); Cy5-labeled anti-mouse CD4 (clone
GK1.5, in-house conjugate, DRFZ); streptavidin-Alexa 546 and streptavidinAlexa 647 (Invitrogen); and rhodamine-labeled peanut agglutinin (PNA;
Vector Laboratories, Burlingame, CA).
Immunohistology
For cross-sectional evaluation of GC cell population kinetics, cohorts of two
to four immunized mice were killed on days 4, 6, 8, 10, 12, 14, 16, 18, and 21
postimmunization. Spleens were removed, bisected, and snap frozen, and
longitudinal cryostat spleen sections of 10 mm thickness were prepared as
described previously (25). Cryostat sections of NZB and NZB/W spleens
were kindly provided by Prof. Rudolf Manz (Institute for Systemic Inflammation Research, University of Lübeck, Lübeck, Germany). Spleen
sections were treated before staining by fixation in ice-cold 1% PFA for 30
min and permeabilization in ice-cold 1% sodium citrate containing 1%
Triton X-100 (Promega, Mannheim, Germany) for 2 min. After blocking
in PBS containing 3% BSA for 30 min, sections were triple stained with
anti–Ki-67, anti–FDC-M2, and either anti-CD3 (staining 1) or anti-CD68
(staining 2). Bound Ki-67 and biotinylated FDC-M2 Abs were detected
using Alexa 647 anti-rat IgG and streptavidin-Alexa 546, respectively. In
some experiments, sections were subjected to TUNEL assays (DeadEnd
Colorimetric TUNEL System; Promega). In these cases, biotinylated nucleotides were detected using streptavidin-Alexa 647, and sections were colabeled with PNA and anti-CD68 (staining 3). An overview of stainings 1,
2, and 3 is given in Fig. 1.
Quantitative immunohistology
Slides were viewed under a Leica DM Ire2 confocal laser-scanning microscope, and digital images of GCs were acquired using a 340 objective
and Leica LCS software (Leica, Wetzlar, Germany). In stainings 1 and 2,
GCs were identified as dense clusters of Ki-67+ proliferating cells in the
right anatomical context (white pulp, proximity to the periarterial lymphatic sheath, immediate vicinity to a FDC network). GC LZ and DZ were
distinguished by FDC polarity (28–30). The outer boundary of a GC was
defined by the stretch of the Ki-67+ cell cluster; its inner DZ-LZ border
was delineated based on the absence (DZ) and presence of a dense reticular
network of FDC processes (LZ; Fig. 1). In case of staining 3, GCs were
identified by PNA reactivity, and outer GC boundaries were defined by the
stretch of the PNA staining (Fig. 1). Outer GC and, where relevant, LZ
boundaries were assigned manually to each GC, and the areas included
were saved as regions of interest (ROIs); GC DZ ROIs were obtained by
subtraction of GC LZ ROIs from GC ROIs. ROI areas were measured
using ImageJ image analysis software (31). The numbers of Ki-67+ cells
and TUNEL+ nuclei within ROIs were determined automatically, applying
an adapted version of the Nucleus Counter plugin for the ImageJ image
analysis software; CD68+ macrophages and CD3+ T cells were counted
manually using an adapted version of the Cell Counter plugin. For each
mouse and each staining, two independent spleen sections (S1 and S2,
distance $ 400 mm) were analyzed.
GCImagePresenter
To make our data accessible to the public, we set up an online database
including all recorded confocal images and their associated data sheets. For
reasons of portability and fast access, we decided to use plain HTML pages
to represent this information. The scripting language Perl (version 5.10.0)
was used to generate HTML pages that compile images and tables from
∼30,000 source files. Access to the database is organized in a hierarchical fashion by grouping data according to the experimental setup, which
makes each of the roughly 10,000 Web pages accessible with a few mouse
clicks. The system was developed in collaboration with MicroDiscovery
GmbH and can be accessed at: http://sysimmtools.eu.
Statistics
Quantitative data were tested for normality using the D’Agostino–Pearson
omnibus test for normality. Association between variables in the data sets
was assessed by Spearman correlation (r) and linear regression statistics.
Significant differences among and within groups of mice or days were
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proliferation is thought to occur in the DZ of GCs (1), a notion
strongly supported by a recent multiphoton microscopy study by
Victora et al. (18). Cell death by apoptosis is described to be most
extensive among LZ B cells in rodents; however, in chronically
inflamed tonsillar GCs, apoptosis was reported to be most evident
at the interface between DZ and LZ, a region also referred to as
basal LZ (17). Apoptotic GC B cells are strong candidates as
sources of autoantigens, and their impaired clearance by macrophages has been linked to the pathogenesis of autoimmune diseases (19). GC-resident macrophages constitute a unique subset
known as tingible body macrophages (TBM) (20), which, besides
fulfilling an important scavenger function, is believed to play
a role in regulating the magnitude of the GC reaction (21). Although TBM express MHC class II molecules, they are believed
neither to function as APCs in GCs nor to be required for GC
formation (21).
Because of their crucial role in establishing an effective humoral
immune response, GC formation and function continue to be important subjects of research for immunologists. Assessing the role
of a designated protein in affinity maturation, for instance, often
includes comparison of GC growth in transgenic mice or mice deficient in said protein and wild-type animals; a few past examples of
such proteins include CD19 (22), BAFF (23), and Bcl-XL (24). The
results reported in this article, together with our previous findings
(25), however, alert us to caution when such a comparison is made
by cross-sectional profiling of GCs. At any time point, the crosssectional profile of an ensemble of GCs shows a broad size distribution of individual GCs (25). Although this is certainly due in large
part to a sectioning bias (i.e., the section plane does not necessarily
pass through the center of GCs), we have previously demonstrated
that such a profile also reflects a real-size distribution of GCs in
three dimensions (25). Simply put, the variety of cross-sectional
GCs in a tissue section is not the mere result of cutting these GCs
differently but also of these GCs being different. Accordingly, the
sampling of GCs for scoring is a main source of bias in the data.
Both the number of scored GCs and the criteria according to which
they are selected strongly influence the results and, in the worst case,
might even distort statistical inferences. Therefore, cross-sectional
scoring should ideally include all GCs, that is, the whole ensemble
and not a subjectively chosen subset thereof.
Because there are no such things as typical size (25) and typical clonal diversity (26) of a GC at a given time point, the question arises whether typicality exists at all for GCs. To address
this matter, this study acquired large-scale confocal data on cellular composition of GCs during the primary immune response to
2-phenyl-5-oxazolone (phOx)-chicken serum albumin (CSA) in
BALB/c mice. We evaluated the recorded images of GCs and their
DZ and LZ compartments quantitatively according to cross-sectional
size and counts of proliferating cells, T cells, macrophages, and
apoptotic nuclei. In particular, we show that during the established
phase of the phOx-CSA–driven GC response, GCs maintain
a typical cellular composition. This composition is independent of
GC size and the time elapsed since immunization.
IS THERE A TYPICAL GERMINAL CENTER?
The Journal of Immunology
6187
determined by Kruskal–Wallis ANOVA with Dunn’s posttest for n . 2 and
the two-tailed Mann–Whitney U test for n = 2. One-way ANOVA with
Tukey’s post test was used to test for differences among groups of means.
DZ and LZ data were compared using the two-tailed Mann–Whitney U
test. Significance levels were set at *p # 0.05, **p # 0.01, ***p # 0.001,
and ****p # 0.0001. Slope estimates and R2 values of linear regression
curves (y = my,x 3 x) fitted to each individual day’s GC composition
~ of GC composition paraparameters and theoretical slope estimates (m)
meters that were not recorded from the same staining are summarized
in Supplemental Table I. All statistical analyses were performed with
GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA).
Results
Histological GC image database and data evaluation
We investigated the dynamics of GC zoning after phOx-CSA immunization in BALB/c mice by immunohistology of spleens on
days 4, 6, 8, 10, 12, 14, 16, 18, and 21 postimmunization. In the
absence of immunization, the background level of environmental
Ag-induced GCs was minimal, and the sizes of B cell zones decreased significantly compared with immunized mice (Supplemental Fig. 2D, 2E), which indicates that the examined GCs were
induced by immunization. PhOx-CSA–induced GCs first became
detectable on day 4, with the majority (54%) exhibiting only an
LZ but no DZ (Fig. 2A). Thereafter, the proportion of GCs having
both DZ and LZ increased, reaching a plateau on day 8 (79%) that
was maintained until day 21. Thus, starting at day 8, the established phase of the response extends to at least day 21. As we have
reported previously for whole GCs (25), DZs and LZs also showed
broad area distributions that underwent dynamic changes over time
(Fig. 2B). Notably, although the variability in the relative sizes
of DZ and LZ among GCs turned out to be considerable at all
sampled time points (coefficient of variation [CV] . 67%), the
cumulative frequency curves of relative size are statistically indistinguishable between days 6 and 21 (p $ 0.05, Kruskal–Wallis
ANOVA; Fig. 2B). Likewise, LZ and DZ share similar average
growth kinetics with peaks on days 8 (10,983 6 2718 mm2) and 10
(11,290 6 2325 mm2), respectively (Fig. 2C).
Throughout the response, the numbers of Ki-67+ proliferating
cells in DZs and LZs were positively correlated to the size of the
respective compartment (Spearman r . 0.95; p , 0.0001; Fig.
2D). Interestingly, the slope estimates of linear regression curves
fitted to each day have a CV ,5% (Table III, Supplemental Table I), indicating that the association between size and number of
Table I. Overview of the histological image database
Staining,
Time (d)
1,
1,
1,
1,
1,
1,
1,
1,
1,
2,
2,
2,
2,
2,
2,
2,
2,
2,
3,
3,
3,
3,
3,
3,
3,
3,
3,
4
6
8
10
12
14
16
18
21
4
6
8
10
12
14
16
18
21
4
6
8
10
12
14
16
18
21
M1, S1
M1, S2
M2, S1
M2, S2
10
11
18
27
22
27
34
17
16
30
19
15
10
19
16
25
26
44
12
17
15
11
25
23
18
23
26
22
18
18
30
29
19
21
27
15
13
11
24
28
26
21
24
20
12
17
36
31
22
18
29
20
17
13
8
16
13
41
8
32
29
14
47
M3, S1
M3, S2
4
16
35
31
37
11
35
45
41
52
50
21
19
6
14
31
32
41
21
46
23
16
7
11
34
31
7
38
20
28
5
23
51
45
55
12
38
23
27
5
17
48
37
20
50
20
21
22
23
28
M4, S1
M4, S2
15
27
11
37
26
30
25
39
15
31
12
36
28
25
28
46
16
30
8
42
38
57
18
34
29
36
S
42
138
192
128
127
108
199
110
86
44
114
204
153
178
147
193
178
78
37
80
214
127
170
39
172
113
86
Indicated are the numbers of GCs evaluated per staining and time point. Two to four mice (M1–M4) were analyzed per
time point, and two independent spleen sections (S1 and S2, distance . 400 mm) were evaluated per individual. Staining 1:
Ki-67, FDC-M2, CD3; staining 2: Ki-67, FDC-M2, CD68; and staining 3: PNA, TUNEL, CD68.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
This study is based on a database of 3457 histological images of
murine splenic GCs recorded during the primary response of
BALB/c mice to phOx-CSA. The database includes three datasets of
GC image series derived from different triple-immunofluorescence
stainings with: 1) Ki-67, FDC-M2, and CD3 (S = 1130); 2) Ki-67,
FDC-M2, and CD68 (S = 1289); and 3) PNA, TUNEL, and CD68
(S = 1038). Summaries of composition and evaluation of the
database are given in Table I and Fig. 1; the recorded GC composition parameters are summarized in Table II. An online version
of the database, GCImagePresenter, including all images and
their associated data evaluation sheets is available at: http://
sysimmtools.eu.
Regarding individual mice, all recorded parameters showed
broad, nonnormal size and count distributions; however, distributions were found to be robust for mice analyzed at the same
time point, with differences usually being statistically insignificant
(Supplemental Fig. 1A). For the purposes of graphical presentation
and statistical analysis, data were therefore aggregated by day
postimmunization. The raw data for all recorded parameters are
displayed in Supplemental Fig. 1B. Slope estimates and R2 values
of linear regression curves fitted to our data are summarized in
Supplemental Table I.
Dynamics of GC zoning
6188
IS THERE A TYPICAL GERMINAL CENTER?
Ki-67+ proliferating cells is time invariant. Accordingly, the average densities of Ki-67+ cells in the LZ and the DZ remained
constant over time (Fig. 2F). However, the density of Ki-67+ cells
was significantly higher in the DZ than in the LZ at all sampled
time points (p # 0.05; Mann–Whitney U test; Fig. 2E).
Dynamics of accumulation and proliferation of GC T cells
The dynamics of T cell accumulation within splenic GCs were
assessed in BALB/c mice after immunization with phOx-CSA
(Table I). As exemplified in Fig. 1, GC T cells were identified
and enumerated as CD3+ cells within manually assigned ROI
boundaries including total GC area, DZ area, and LZ area. At all
sampled time points, there were marked differences in the numbers of CD3+ T cells among GCs and their DZ and LZ com-
Dynamics of accumulation of GC macrophages
Accumulation of macrophages within de novo–induced GCs was
assessed in BALB/c mice 4–21 d after primary immunization with
phOx-CSA (Table I). Distributions of GC macrophages, identified as CD68+ cells within ROI boundaries (Fig. 1), were broad
in terms of the numbers of CD68+ cells among GCs, LZs, and
DZs, at all sampled time points (CV . 62%; Fig. 4B). These distributions were each found to be statistically indistinguishable between days 8 and 12 and days 16 and 21 (p . 0.05 for GC, DZ,
and LZ; Kruskal–Wallis ANOVA). Macrophages were already detected in considerable numbers in nascent GCs on day 4 (Fig.
4A, 4B), and their average numbers more than doubled until day
10, reaching 18 6 7 CD68+ cells at the peak; thereafter, their
Table II. Summary of recorded GC composition parameters
Parameter
Symbol
Da
da
Staining
Area
Ki-67+ cells
CD3+ T cells
CD3+Ki-67+ T cells
CD68+ macrophages
TUNEL+ nuclei
a
Ki
T
TKi
M
A
GC, LZ
GC, LZ
DZ, LZ
DZ, LZ
DZ, LZ
GC
DZ = GC 2 LZ
DZ = GC 2 LZ
GC = DZ + LZ
GC = DZ + LZ
GC = DZ + LZ
1, 2, 3b
1, 2
1
1
2
3
a
Listed are the directly measured (D) and derived (d) parameters (obtained by either subtraction or addition). Staining 1:
Ki-67, FDC-M2, CD3; staining 2: Ki-67, FDC-M2, CD68; and staining 3: PNA, TUNEL, CD68.
b
In the case of staining 3, only the total GC area was recorded.
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FIGURE 1. Overview of histological image data sets and their quantitative evaluation. This study is based on a database of 3457 histological
images of murine splenic GCs recorded during the primary response of
BALB/c mice to phOx-CSA. The database includes three GC image series
derived from different triple-immunofluorescence stainings: Ki-67, FDCM2, and CD3 (staining 1, number of images [S] = 1130); Ki-67, FDC-M2,
and CD68 (staining 2, S = 1,289); PNA, TUNEL, and CD68 (staining 3,
S = 1038). For each image, ROI boundaries outlining the GC and its LZ
(if present) were drawn and saved; DZ ROIs were obtained by subtraction.
ROI areas were measured, and cell numbers within ROIs were either
counted manually (CD3+, CD3+Ki-67+, CD3+Ki-672, CD68+) or automatically using the Nucleus Counter plugin of the ImageJ image analysis
software (Ki-67+, TUNEL+). Symbols in parentheses are the same as in
Table II. Scale bar, 100 mm.
partments (CV . 71%; Fig. 3B). Such broad distributions of CD3+
T cell numbers were subject to change over time; however, between days 10 and 21, the cumulative frequency curves of the
numbers of CD3+ GC T cells were statistically indistinguishable
(p . 0.05 for GC, DZ, and LZ; Kruskal–Wallis ANOVA). CD3+
T cells, although low in number, were already detectable in nascent GCs (Fig. 3A, day 4), and their average numbers increased
progressively, reaching 27 6 1 T cells per GC on day 8 (average
calculated across mice). The numbers of CD3+ T cells in DZ and
LZ followed this kinetics, with peak numbers of 13 6 2 and 16 6
2 CD3+ T cells (Fig. 3C). Correlation analysis further revealed
a positive association between the number of CD3+ T cells and
GC or compartment size (Spearman rGC . 0.84, rDZ . 0.71, and
rLZ . 0.77; p , 0.0001; Fig. 3D). Likewise, there was a positive
correlation between the numbers of CD3+ T cells and Ki-67+
proliferating cells (Spearman rGC . 0.82, rDZ . 0.64, and rLZ .
0.79; p , 0.0001). The slope estimates of linear regressions between T cell numbers and GC compartment size were very similar
for all time points (CV , 19%; Fig. 3D, Table III, Supplemental Table I), but the average densities of CD3+ T cells in the DZ
and the LZ showed a slight increase over time (Fig. 3F). However, when the groups of means were compared statistically, the
changes were insignificant (p . 0.05, one-way ANOVA). Most
importantly, from day 10 onward (1151 6 222 CD3+ cells/mm2
DZ and 1438 6 75 CD3+ cells/mm2 LZ), the density of CD3+
T cells was significantly higher in the LZ than in the DZ (p #
0.05, Mann–Whitney U tests; Fig. 3E).
We also investigated GC T cell proliferation by counting Ki-67+
cells among CD3+ T cells. The number of proliferating GC T cells
per GC was generally low, ranging from 0 to 15, and 79% of all
GCs showed #3 proliferating GC T cells (Supplemental Fig.
3A). At the peak on day 8, GCs contained 3.2 6 0.9 proliferating
T cells, on average, but the highest density of proliferating T cells
was already observed on day 6 (248 6 50 CD3+Ki-67+ cells/mm2;
Supplemental Fig. 3A). Also, the frequency of proliferating per
total GC T cells peaked on day 6 (17 6 2%; Supplemental Fig.
3A).
The Journal of Immunology
6189
numbers gradually declined to 14 6 2 CD68+ cells on day 21 (Fig.
4C). The numbers of macrophages in DZs and LZs followed the
same kinetics, with peak numbers of 12 6 4 and 7 6 2 CD68+
cells on day 10 (Fig. 4C). Correlation analysis revealed a significant positive relationship between CD68+ cell numbers and GC or
compartment size (Spearman rGC . 0.81, rDZ . 0.83, and rLZ .
0.76; p , 0.0001; Fig. 4D). Furthermore, CD68+ cell numbers
were positively correlated to Ki-67+ proliferating cell numbers
(Spearman rGC . 0.74, rDZ . 0.79, and rLZ . 0.77; p , 0.0001).
The slope estimates of linear regressions between CD68+ cell
numbers and GC compartment size showed only small variation in
the course of the immune response (CV , 17%; Fig. 4D, Table
III, Supplemental Table I), and the average densities of macrophages in DZs and LZs proved to be constant (e.g., 1124 6 170
cells/mm2 DZ and 840 6 20 cells/mm2 LZ on day 10; Fig. 4F). As
of day 8, the density of CD68+ macrophages in the DZ was significantly increased compared with that of the LZ (p # 0.05,
Mann–Whitney U tests; Fig. 4E).
Whereas B cell zones of naive mice were sparse in CD3+ T cells,
they already contained numerous small CD68+ macrophages (Supplemental Fig. 2A, 2B). Indeed, we found that the density of
CD68+ cells in B cell zones was even slightly higher in naive than
in immunized mice (1443 6 213 versus 1130 6 60; p , 0.0001,
Mann–Whitney U test; Supplemental Fig. 2D, 2F). Thus, mac-
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FIGURE 2. Dynamics of GC zoning. Mice were immunized with phOx-CSA and sacrificed 4–21 d later. Spleen sections were prepared, stained, and
evaluated for sizes of GCs as described in Materials and Methods. Two sections at least 400 mm apart were scored for each spleen, and two to four mice
were evaluated per time point (Table I). A, Proportions of GCs showing only a DZ, only an LZ, or both DZ and LZ at different points in time after
immunization. GCs were identified as Ki-67+ cell clusters and by anatomical location in triple-immunofluorescence stainings of proliferating cells (Ki-67,
blue), T cells (CD3, green), and FDC networks (FDC-M2, red). GC LZ and DZ were distinguished by the presence of FDCs in the LZ. Photomicrographs
are representative of day 18 GCs. Scale bar, 100 mm. B, Size distributions and size ratio of LZ and DZ plotted as cumulative frequency curves. Dotted
vertical lines indicate medians. C, Average size kinetics of GCs (filled area), DZ (filled squares), and LZ (open squares), expressed as mean and SD of two
to four mice. D, The numbers of Ki-67+ proliferating cells in DZ and LZ are positively correlated to the size of DZ and LZ, respectively (Spearman r .
0.95; p , 0.0001). Lines represent linear fits of compiled data for each day (see Supplemental Table I for individual slopes m and R2 values); mean slopes
(days 8–21) are indicated by m. E, Density of Ki-67+ proliferating cells in DZ (squares) and LZ (circles). Each symbol represents a single DZ or LZ, and the
data are compilations of all calculated values for a given day; bars show mean and SD. Statistically significant differences between DZ and LZ are indicated: *p # 0.05, **p # 0.01, ****p # 0.0001, Mann–Whitney U test. F, Average kinetics of the densities of Ki-67+ proliferating cells in GCs (filled
area), DZ (filled squares), and LZ (open squares). Data are expressed as mean and SD of two to four mice.
6190
IS THERE A TYPICAL GERMINAL CENTER?
Table III. Mean slope estimates for GC composition parameters
GC
Estimate
mKi;a
mT ;a
mM;a
mA;a
mT ;Ki
mM;Ki
~ M;T
m
~ A;M
m
m
~ A;T
~ A;Ki
m
DZ
LZ
Mean 6 SD
CV (%)
Mean 6 SD
CV (%)
Mean 6 SD
CV (%)
6
6
6
6
6
6
6
6
6
6
4.0
15.2
13.0
11.6
12.3
14.9
10.8
16.1
18.1
10.7
0.01293 6 0.00057
0.00127 6 0.00022
0.00112 6 0.00012
4.4
17.6
10.9
0.01085 6 0.00054
0.00169 6 0.00028
0.00085 6 0.00014
5.0
16.3
16.8
0.09644 6 0.01419
0.08904 6 0.00991
0.89747 6 0.14098
14.7
11.1
15.7
0.15131 6 0.02055
0.07235 6 0.01559
0.50851 6 0.04627
13.6
21.6
9.1
0.01182
0.00151
0.00101
0.00231
0.12446
0.08245
0.67083
2.32895
1.55889
0.19578
0.00047
0.00023
0.00013
0.00027
0.01527
0.01231
0.07219
0.37440
0.28183
0.02094
Mean slope estimates (m) of linear regression curves (y = my,x 3 x) fitted to each individual day’s GC composition parameters. Further listed are the
~ of GC composition parameters that were not recorded from the same staining. Indicated means were calculated
theoretical mean slope estimates (m)
taking into account data from days 8 to 21 postimmunization. Symbols are the same as in Table II.
Dynamics of GC B cell death and uptake of apoptotic cells
GCs are sites of significant cell death, at which apoptosis is thought
to reflect negative selection of lower affinity and autoreactive
B cells. To monitor the dynamics of cell death within GCs, we
applied the TUNEL assay to spleen tissue derived from BALB/c
mice 4–21 d after phOx-CSA immunization (Fig. 1, Table I). In
accordance with previous studies, TUNEL+ signals in GCs were
confined exclusively to macrophages (33), with the number of
apoptotic nuclei engulfed by a macrophage defining its size (Fig.
5A, 5D). Next to the highly loaded large macrophages commonly
referred to as TBM, we observed considerable numbers of small
macrophages at all time points (Fig. 5A). Although large and small
macrophages were detected consistently in both DZs and LZs, the
TBM-like macrophages preferentially resided at the interface
between DZ and LZ (Fig. 4A).
Automated quantification of cell death (Fig. 1) showed that the
distributions of the numbers of TUNEL+ nuclei among GCs were
broad at all sampled time points (CV . 55%) and changed with
time (Fig. 5B). Between days 8 and 10 and days 12 and 21,
however, these changes were found to be statistically insignificant
(p . 0.05, Kruskal–Wallis ANOVA). Correlation analysis further
demonstrated a positive relationship between TUNEL+ nuclei and
GC size (Spearman rGC . 0.84; p , 0.0001; Fig. 5E), with very
similar slope estimates for linear regression curves from days 8 to
21 (CV , 12%; Table III, Supplemental Table I). Despite the
established correlation, we occasionally observed GCs of the same
size in a tissue section showing strikingly arbitrary numbers of
TUNEL+ nuclei. Visually, these GCs can additionally be disting–
uished by the size of their macrophages (Fig. 5D). The levels of
cell death in B cell zones of naive mice and in nascent GCs of
immunized mice were low and indistinguishable from other
regions of the spleen (Fig. 5A, Supplemental Fig. 2C). A steep
increase in the average numbers of TUNEL+ nuclei by a factor of
3 was first observed between days 6 and 8 (Fig. 5C), coextensive
with the believed onset of SHM. Over the same period, the density
of TUNEL+ nuclei also doubled significantly (p # 0.001, Mann–
Whitney U test; Fig. 5F, 5G). Whereas the average number of
TUNEL+ nuclei per GCs showed a peak on day 10 (64 6 12
TUNEL+ nuclei; Fig. 5D), the average density of TUNEL+ nuclei
in GCs remained constant between days 8 and 21 (e.g., 2357 6
167 TUNEL+ nuclei/mm2 on day 10; Fig. 5G).
Discussion
The typical GC
Although cross-sectional profiling does not justify specifying a
typical GC in terms of absolute size and cell numbers, it allows
delineation of the typical cellular composition of GCs. In this study,
the recorded numbers of T cells, macrophages, and apoptotic nuclei
per GC all feature large dispersion from the mean of the ensemble
(CV . 55%) and by no means follow a Gaussian distribution. We
therefore believe that definite quantitative statements, for instance,
that the typical GC on day 10 has a size of 0.019 mm2 and shows
∼22 T cells, on average, are questionable and not particularly
meaningful. Just as an example, on day 10, ,20% of phOx-CSA–
induced GCs have a size in the range of 0.019 mm2 6 25%.
However, common to all recorded cell types is the strong correlation of their cell numbers with GC size. These correlations are
remarkably stable during weeks 2 and 3 postimmunization, as
indicated by small SDs (4–22%) of the mean slope estimates of
linear regression curves fitted to each day’s GC composition parameters (Table III). This means that the quantitative relations
between the cellular players of GCs are maintained. Thus, during
the established phase of the phOx-CSA–induced GC response
(days 8–21), the cellular composition of a GC is always roughly
the same (summarized in Table IV). Moreover, because we have
previously shown that the cross-sectional area distributions of spleen
sections reflect broad real-size distributions of GCs, it is valid to
argue that the typical cellular composition is independent of
GC size.
Because there are no systemic quantitative data on the cellular
composition of GCs, this study was designed as a baseline study.
Nevertheless, the question whether the presented findings are specific to the hapten-carrier–induced response or equally valid for
other model systems is certainly relevant. The literature provides
instances of differences in the immune response as to strain of
mouse and nature of Ag; however, we believe that the parameters
reported in this study look unlikely to vary significantly for two
reasons. First, the laboratory of T. J. Waldschmidt previously
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rophages are present in B cell follicles before antigenic stimulation, that is, when GCs are absent.
To examine whether the stable macrophage densities are tied
to our model system and immunization protocol, we additionally
analyzed spleen sections from systemic lupus erythematosus-prone
mouse strains (NZB, NZB/W) that spontaneously develop GCs in
the absence of either purposeful immunization or infection (32).
Importantly, the mean macrophage densities in B cell zones of
young NZB (1150 6 149 cells/mm2) and NZB/W mice (1387 6
130 cells/mm2) turned out to be very similar to those of immunized (1130 6 60 cells/mm2) or naive BALB/c mice (1443 6 213
cells/mm2; Supplemental Fig. 2G). However, disease progression
in NZB/W mice involved a decrease in macrophage density of
more than one third (845 6 59 cells/mm2; Supplemental Fig. 2G).
The Journal of Immunology
6191
demonstrated that the GC response shows signs of a high degree
of regulation that are strain and Ag independent (3, 4, 34). In particular, they found a steady ratio of nonswitched to switched
GC B cells that is maintained throughout the response. This holds
equally true for immunization with SRBCs, hapten-carrier conjugates, PE, and mouse-adapted influenza A virus (3). Second, we
could show that the remarkably stable macrophage density in
B cell zones is strain independent and not tied to our model system
(Supplemental Fig. 2G). Importantly, the mean macrophage densities in B cell zones of systemic lupus erythematosus-prone NZB
and NZB/W mice, which spontaneously develop GCs in the ab-
sence of either purposeful immunization or infection (32), are very
similar to those of immunized or naive BALB/c mice (Supplemental Fig. 2G). However, disease progression in NZB/W mice
appears to involve a severe decrease in the density of macrophages
(Supplemental Fig. 2G), underscoring the importance of maintenance of cell ratios during the response.
Identification of GCs
All Ab markers, or combinations thereof, used in this study were
chosen by two main criteria: 1) unambiguity of their staining
pattern, and 2) suitability for semiautomated cell counting. Al-
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FIGURE 3. Dynamics of CD3+ GC T cells. Mice were immunized with phOx-CSA and sacrificed 4–21 d later. Spleen sections were prepared, stained,
and scored as described in the legend for Fig. 1 and in Materials and Methods. A, Photomicrographs representative of GCs recorded on days 4, 6, 10, 14,
and 21 after immunization. GCs were identified as Ki-67+ cell clusters and by anatomical location in triple-immunofluorescence stainings of proliferating
cells (Ki-67, blue), T cells (CD3, green), and FDC networks (FDC-M2, red). LZ and DZ were distinguished by the presence of FDCs in the LZ. Scale bar,
100 mm. B, Time-dependent changes in numbers of CD3+ T cells in GCs, DZ, and LZ plotted as cumulative frequency curves. C, Average kinetics of CD3+
T cells present in GCs (filled area), DZ (filled squares), and LZ (open squares) expressed as mean and SD of two to four mice. D, The numbers of CD3+
T cells in GCs, DZ, and LZ are positively correlated to the size of the respective compartment (Spearman r . 0.75; p , 0.0001). Lines represent linear fits
of compiled data for each day (see Supplemental Table I for individual slopes m and R2 values); mean slopes (days 8–21) are indicated by m. E, Density of
CD3+ T cells in DZ (squares) and LZ (circles). Each symbol represents a single DZ or LZ, and the data are compilations of all calculated values for a given
day; bars show mean and SD. Statistically significant differences between DZ and LZ are indicated: **p # 0.01, ***p # 0.001, ****p # 0.0001, Mann–
Whitney U test. F, Average kinetics of the densities of CD3+ T cells in GCs (filled area), DZ (filled squares), and LZ (open squares). Data are expressed as
mean and SD of two to four mice.
6192
IS THERE A TYPICAL GERMINAL CENTER?
though PNA is the most commonly used marker for detection of GC
by immunohistology, we opted for Ki-67 staining in two of the
three staining series. PNA staining is sticky and the separation of
PNA-high GC B cells from surrounding PNA-low resting B cells
is distinctly “blurred” (a circumstance well reflected by the GC
images of staining series 3 in our database). These characteristics
make for difficulties in assigning “tight/accurate” outer GC boundaries to GCs visualized by PNA. In terms of measuring GC size,
Ki-67 outdoes PNA for two reasons: 1) clear separation of GC
B cells (Ki-67+) from surrounding resting B cells (Ki-672), and 2)
applicability for automated cell counting. Finally, because it is
a nuclear stain, Ki-67 facilitates counting of other surface-stained
cells (in our case, CD3+ T cells and CD68+ macrophages in staining series 1 and 2).
The Ki-67 Ag is a well-known proliferation marker that stains
the growth fraction of cell populations, that is, cells that are in any
phase of the cell cycle other than G0 (35). However, although not
all B cells in a GC are actively proliferating, almost all GC B cells
stain positive for Ki-67. Although this statement may appear a
contradiction, it is experimentally validated: in 2003, Rahman
et al. (23) showed that all splenic GCs of SRBC-immunized A/J
and C57BL/6 mice stain extensively for Ki-67. In agreement with
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FIGURE 4. Dynamics of CD68+ GC macrophages. Mice were immunized with phOx-CSA and sacrificed 4–21 d later. Spleen sections were prepared,
stained, and scored as described in the legend for Fig. 1 and in Materials and Methods. A, Photomicrographs representative of GCs recorded on days 4, 6,
10, 14, and 21 postimmunization. GCs were identified as Ki-67+ cell clusters and by anatomical location in triple-immunofluorescence stainings of
proliferating cells (Ki-67, blue), macrophages (CD68, green), and FDC networks (FDC-M2, red). LZ and DZ were distinguished by the presence of FDCs
in the LZ. Scale bar, 100 mm. B, Time-dependent changes in numbers of CD68+ macrophages in GCs, DZ, and LZ plotted as cumulative frequency curves.
C, Average kinetics of CD68+ macrophages present in GCs (filled area), DZ (filled squares), and LZ (open squares), expressed as mean and SD of two to
four mice. D, The numbers of CD68+ macrophages in GCs, DZ, and LZ are positively correlated to the size of the respective compartment (Spearman r .
0.78; p , 0.0001). Lines represent linear fits of compiled data for each day (see Supplemental Table I for individual slopes m and R2 values); mean slopes
(days 8–21) are indicated by m. E, Density of CD68+ macrophages in DZ (squares) and LZ (circles). Each symbol represents a single DZ or LZ, and the
data are compilations of all calculated values for a given day; bars show mean and SD. Statistically significant differences between DZ and LZ are indicated: **p # 0.01, ***p # 0.001, ****p # 0.0001, Mann–Whitney U test. F, Average kinetics of the densities of CD68+ macrophages in GCs (filled
area), DZ (filled squares), and LZ (open squares). Data are expressed as mean and SD of two to four mice.
The Journal of Immunology
6193
this, Wang et al. (22) and Linterman et al. (36) report proportions
of Ki-67–expressing cells among GC B cells of 90–100% in
SRBC-immunized 129/Sv or C57BL/6 mice. Most importantly,
the immunohistological results published by Wang et al. (22) reveal that anti–Ki-67 and PNA stain and cover identical GC areas.
Altogether, this proves Ki-67 as an eligible marker for immunohistological identification of GCs.
FDC-M2 is a well-established and widely used FDC marker that
is also lowly expressed on white pulp reticular cells and some
uncharacterized perivascular cells (37). However, the latter does
not interfere with detection of FDC networks or other GC cell
populations. This and the fact that FDC-M2 expression is constant
and independent of whether FDCs are involved in GC reactions
(38) made it the marker of choice for our study.
GC zoning
Table IV. The typical GC
Ratio
T:Ki
M:Ki
A:Ki
M:T
T:A
M:A
DZ:LZ (volume to volume)b
GCa
DZ
LZ
12.5:100
8.3:100
19.6:100
6.7:10
6.4:10
4.3:10
1:1
9.6:100
8.9:100
15.1:100
7.2:100
9.0:10
5.1:10
a
Cellular composition of the typical GC and its DZ and LZ. Indicated are the
ratios of the different cellular players as derived from the mean slope estimates for
GC composition parameters. Symbols are the same as in Table II.
b
Estimated from 70 previously three-dimensional–reconstructed day 10 GCs (Supplemental Fig. 3B).
Recent live-imaging studies using a photoactivatable fluorescent
reporter emphasize once more that GCs are highly polarized
with respect to function, whereas the DZ specializes in cell division,
Ag-driven selection takes places in the LZ (18). Such segregation
suggests a DZ-LZ interdependence model for GC action wherein
the two zones are connected by B cell migration (39–41). In line
with this, we find evidence for a dependency between DZ and LZ
in terms of their growth kinetics. First, DZs and LZs grow and
decay in parallel during the established phase of the response (Fig.
2B, 2C). Moreover, the overall ratio of B cells in the DZ and the
LZ is fixed over time, as indicated by stable DZ/LZ area curves
over days (Fig. 2B). Interestingly, a great proportion of crosssectional GCs show a DZ/LZ size ratio of ∼1 (Fig. 2B), which
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FIGURE 5. Dynamics of cell death within GCs. Mice were immunized with phOx-CSA and sacrificed 4–21 d later. Spleen sections were prepared,
stained, and scored as described in the legend for Fig. 1 and in Materials and Methods. A, Photomicrographs representative of GCs recorded on days 4, 6,
10, 16, and 21 after immunization. GCs were identified by PNA reactivity and anatomical location in triple-immunofluorescence stainings of GC B cells
(PNA, blue), macrophages (CD68, green), and apoptotic nuclei (TUNEL assay, red). Scale bar, 100 mm. B, Time-dependent changes in numbers of
TUNEL+ nuclei in GCs plotted as cumulative frequency curves. C, Average kinetics of TUNEL+ nuclei present in GCs (filled area), expressed as mean and
SD of two to four mice. D, GCs of the same cross-sectional size harbor arbitrary numbers of apoptotic nuclei (TUNEL assay, red) and differ in the size of
GC macrophages (CD68, green). Scale bar, 100 mm. Images are representative of three GCs on day 10 postimmunization. E, The number of TUNEL+
nuclei in GCs is positively correlated to GC size (Spearman r . 0.84; p , 0.0001). Lines represent linear fits of compiled data for each day (see Supplemental Table I for individual slopes m and R2 values); mean slopes (days 8–21) are indicated by m. F, Density of TUNEL+ nuclei in GCs. Each symbol
represents a single GC, and the data are compilations of all calculated values for a given day; bars show mean and SD. G, Average kinetics of the densities
of TUNEL+ nuclei in GCs. Data are expressed as mean and SD of two to four mice.
6194
led us to conjecture that the volumes occupied by the DZ and the
LZ are about the same. Indeed, when we determined the DZ-to-LZ
volume ratios of 70 three-dimensional–reconstructed day 10 GCs
(25), the values ranged from 0.5 to 1.9 with a mean of 1.0 6 0.4
for GCs with a size $2.5 mm3 (Supplemental Fig. 3B). GC formation starts in the LZ (Fig. 2A) (22) and occurs over an extended
period (25). Therefore, small GCs tend to show DZ/LZ ratios ,1
(Supplemental Fig. 3B).
GC T cells
fraction of macrophages also has an impact on the available T cell
help, and that the T cell-to-macrophage ratio constitutes a selective
force.
GC macrophages
The presence of large phagocytic cells in GCs has been recognized
for 125 y (52). Early light and electron microscopic studies
revealed that these characteristic phagocytes contain many tingible bodies of lymphocyte origin in their cytoplasm, which led
to their naming as TBM (53, 54). The use of CD68 Abs to investigate GC macrophages in this study brought out results that conflict with previous research using Abs specific to the Mac-2 Ag
(21). Most importantly, CD68 reveals considerable numbers of
macrophages in both primary follicles and GCs that are missed by
Mac-2.
Regarding GCs, macrophage populations are larger and more
heterogeneous than previously thought. This finding manifests
itself in an increased macrophage-to-B cell ratio (8:100 versus
1:350) (21) and in the abundance of small macrophages at all time
points. Hence TBM as defined earlier make up only a subpopulation of GC macrophages. Furthermore, in contrast with current concepts (20, 21), macrophages are present in follicles before
immunologic stimulation and may, after all, play a role in the induction of de novo GC formation, for instance, by functioning as
APCs. Unlike the large TBM-like macrophages (20–30 mm in major axis length) stained by anti–Mac-2 Ab (21), the Mac-22
macrophages in primary follicles are typically of small size (10–
15 mm in major axis length) and have not yet taken up apoptotic
cells. Given the differential expression of Mac-2 during macrophage differentiation (55) and its variation with strength of inflammatory stimuli (56), it is tempting to speculate that follicular
macrophages upregulate Mac-2 in response to uptake of dying
cells.
The copresence of both Mac-2+ and Mac-22 macrophages in
GCs may be significant because shifts in phenotypes of macrophages during tumor growth have been shown to be associated
with immunoregulation (57, 58). In this context, Mac-2+ macrophages produce large amounts of PGE2, an arachidonic acid metabolite capable of suppressing B cell proliferation (59, 60). Interestingly, Mac-22 macrophages counteract this suppression (57,
58). As we have shown, the apoptotic load of GCs increases with
their size. At the same time, increasing numbers of apoptotic
cells lead to accumulation of large macrophages. That is, if uptake
of apoptotic cells in GCs induces a phenotypic shift from small
(nonsuppressive) Mac-22 to large (suppressive) Mac-2+ macrophages, this could provide a regulatory framework for GC B cell
homeostasis. Indeed, two lines of evidence support the presence of
a feedback relation between GC B cells and macrophages: first,
Mac-2+ TBM have been identified as a rich source of PGs (61);
second, the macrophage-to-B cell ratio is remarkably constant
throughout the GC response, emphasizing the importance of stable
macrophage–B cell interactions.
Cell death in GCs and clearance of apoptotic cells
Where, when, and how fast do GC B cells die? The answers to these
questions are of importance because they may provide information
on the spatiotemporal regulation of GC selection. Marked apoptosis
beyond basal levels is first detected by the end of the first week
postimmunization (Fig. 5C) and thus coincides with the onset of
SHM and selection (62). Thereafter, the apoptotic (TUNEL+)-toproliferating (Ki-67+) B cell ratio remains fixed at ∼1:5 (Table
III), an observation consistent with either stable average susceptibility of GC B cells to apoptosis or steady selection stringency.
As to the where, the traces of cell death are spread across GCs.
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The GC reaction depends critically on T cell help because the
latter not only affects initiation and maintenance of GCs but also
differentiation of high-affinity GC B cells into memory B cells or
Ab-forming cells (16, 42). Within the population of follicularhoming T cells that is commonly distinguished by expression of
the CXCR5, different subsets of follicular T cells have been
identified (reviewed in Ref. 16); however, in this study, we did
not consider subsets but evaluated the entirety of T cells within
the confines of GCs. To visualize follicular/GC T cells, we chose
CD3 instead of CD4 as a marker because it has previously been
demonstrated that many CD4-immunoreactive cells in B cell follicles are not T cells but belong to a CD4+CD32 population of
accessory cells (7).
The observation that CD3+ T cells are absent or only sporadically present in B cell zones of naive mice suggests that follicular localization of T cells depends on immunization. This
runs counter to previous research that identifies substantial numbers of T cells in B cell follicles before immunization (43).
The discrepancy is probably due to the different markers used to
visualize follicular T cells (e.g., like CD4, Thy1 has been reported to be expressed by follicular non-T CD4+CD32 accessory
cells). Colonization of nascent GCs by T cells was found to be
followed by an early phase of T cell proliferation reaching its
peak by the end of the first week. This early proliferative response of T cells in GCs may not be related to cognate B–T interaction and provision of B cell help because T cell accumulation in follicles is not directed by B cells but by DCs (43), and
the early phase of GC formation takes place in the absence of
T cells (44, 45). It is more likely that proliferation of T cells
during GC formation is linked to regular T cell differentiation
pathways including clonal expansion and formation of CD4 memory (46–49).
Interactions between GC B and T cells are required for positive
selection of high-affinity GC B cells and maintenance of GCs (18,
45). Recent insights obtained by real-time imaging of GCs support
a model in which competition among GC B cells for cognate T cell
help is one aspect of selection (50). Because T cells are believed to
be most abundant in the LZ, these events have been attributed especially to this GC compartment. However, although enrichment of
T cells in the LZ is certainly real (and statistically significant),
another reality is that the DZ of murine GCs is anything but poor in
T cells. In stark contrast with chronically inflamed human tonsils,
where T cell numbers in the DZ are vanishingly small (51), the
T cell density in the DZ of murine GCs is more than two thirds
of that in the LZ (Fig. 3F). DZ and LZ differ in the T cell-tomacrophage ratio rather than in the density of T cells: in the LZ,
two T cells must share one macrophage, whereas in the DZ, every
T cell has its own (Table IV). The former might be of importance
because dying GC B cells have been reported to release cytoplasmic blebs that can be picked up by macrophages and T cells alike.
Based on the latter observation, it was suggested that apoptotic
B cell blebs influence the availability of T cell help to live GC
B cells, thereby driving selection (50). However, because both
T cells and macrophages pick up blebs, it seems possible that the
IS THERE A TYPICAL GERMINAL CENTER?
The Journal of Immunology
Acknowledgments
We especially thank the DRFZ, a Leibnitz Institute, in Berlin for providing
laboratory space, equipment, and generous support during the project. We
also thank Prof. Rudolf Manz and Dr. Katrin Moser for providing cryostat
sections of NZB and NZB/W spleens.
Disclosures
The authors have no financial conflicts of interest.
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Although we did not quantify the numbers of TUNEL+ nuclei in
different GC compartments in this study, we qualitatively observed two regions of elevated cell death. Both the interface between DZ and LZ (also referred to as the basal LZ) and the
interface between DZ and the periarterial lymphatic sheath are
often rich in large, TBM-like macrophages (staining series 2) that
colocalize with large clusters of TUNEL+ nuclei (staining series
3). However, the finding that TUNEL+ nuclei of dying GC B cells
are only present in association with macrophage scavengers but
not “freely” has two substantial implications for the interpretation
of the data: 1) the clearance time of apoptotic GC B cells is very
likely to be short, and 2) the spatial distribution of TUNEL+
signals in GCs need not necessarily reflect where GC B cells are
programmed to die. Provided that dying GC B cells are rapidly
engulfed and degraded by macrophages, they ought to leave the
site of death almost immediately, especially if cell intermixture is
quick, as has been shown for GCs (63). Thus, TUNEL detection
alone gives insufficient evidence as to where the initial selective
signals that eventually lead to apoptosis of GC B cells are (or, for
that matter, are not) delivered.
Indeed, where calculated, clearance times of cells undergoing
apoptosis are surprisingly short, ranging from 1 to 3 h for different
tissues (64). Given an incidence, i, of TUNEL+ nuclei among GC
B cells of 20% (1:5) and assuming a clearance time, d, of 3 h and
a correction factor, f = 0.5, to account for the formation of more
than one TUNEL+ nucleus from an apoptotic cell, the minimal cell
ipf
@ 3%=h.
loss rate, r (65), in GCs by apoptosis is given by r ¼
d
In our previous work we found that the growth of de novo–
induced GCs is nonsynchronized and further characterized by
broad GC volume distributions at anytime (25). Given this heterogeneity in size, and probably also age, the stable cellular
composition of GCs we report in this article is remarkable. GCs
can be viewed as open systems (30) that continuously deal with
factors such as high proliferative and apoptotic activities, internal
cell migration, and cell influx and efflux. The observed maintenance of quantitative relations between the cellular players during
the life span of GCs therefore implies the existence of a tightly
regulated network of cellular interactions and communication. It
will be particularly interesting to explore how this network achieves its robustness in the highly dynamic environment of a GC.
We believe that integrative mathematical models, combining
quantitative data on cellular composition, cell migration, clonal
diversity, and overall GC growth kinetics, will be essential for
understanding the complex regulation of GC cell dynamics and its
linkage with GC function. Our study makes a contribution toward
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modeling GC dynamics, such as assessment of aberrations from
the “perfect” GC reaction, we suggest that monitoring changes in
the ratios of GC cells to each other is valuable and more sensitive
than following average values.
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IS THERE A TYPICAL GERMINAL CENTER?