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RAPID COMMUNICATION
Evolving Abundance and Clonal Pattern of Human Germinal Center B Cells
During Childhood
By Yadira Valles-Ayoub, Herman L. Govan 111, and Jonathan Braun
Childhood is a critical period for the development of the
memory B-lymphocyte repertoire necessary in protective
humoral immunity. This study addressed the natural history of memory B cells based on the previous identification
of germinal center and mantle zone cells as the probable
precursor and mature memory cell populations, respectively. Using flow cytometric quantitation of these B-cell
subpopulations in human tonsil, w e found that germinal
center cells were abundant (70% of tonsil B cells) during
early childhood (2 t o 3 years), but decline by early adolescence (8 t o 14 years) t o a low level (33%. P = .0003).To
study the clonal evolution of these B-cell subpopulations,
germinal center and mantle zone B cells were isolated
using a preparative magnetic immunobead method, and
analyzed using a novel polymerase chain reaction-based
quantitative assay t o measure the abundance of B-cell
clones bearing certain rearranged VH subfamilies. Two VH
subfamilies were informative: VH1 N clones were uniquely
deficient in germinal center B cells at the early age period,
but became abundant in later childhood; and VH3L clones
were absent among germinal center cells regardless of
age. In contrast, B-cell clones bearing each VH subfamily
were abundant in the mantle zone subpopulation throughout childhood. These findings suggest that the abundance
and clonal pattern of germinal center B cells evolves during
childhood, presumably due t o changing antigenic or ontogenic processes. Moreover, the distinct clonal pattern of
germinal center versus mantle zone B cells suggests that a
major phase of clonal selection occurs after germinal
center emigration.
0 1990 by The American Society of Hematology.
I
patterns.20,2’If local germinal center B cells are indeed the
precursors of regional memory cells, and if predominant
immunoselection initiates rather than follows memory cell
differentiation, then germinal center and mantle zone B cells
should display a common clonal pattern. To test this prediction, we have isolated these subpopulations a quantitatively
assayed V, utilization a t the genomic level using a novel
polymerase chain reaction (PCR)-based strategy. The present
study describes an unexpected difference in clonal pattern
between germinal center and mantle zone B cells, and its
evolution from early to later periods of childhood.
N HUMANS, large numbers of B lymphocytes are
produced daily beginning in the eighth week of gestation,
and the newborn appears to have a full complement of B cells
expressing all immunoglobulin (Ig) isotypes.’-’ In contrast,
the adult pattern of antibody responsiveness is not acquired
until several years after birth, generally attributed to the
expansion and diversification of memory B cells through
cumulative influence of environmental antigens and the
developing T-lymphocyte compartment.’-’ Intrinsic ontogenic factors may also play a role in this physiologic immune
deficit, but are difficult to assess due to the lack of definitive
markers for memory B cells.
This gap has been partially filled by recognition that the
germinal center is the site of a critical stage in the development of memory B cells.6-8 The germinal center is also
considered the major source of precursors for the mantle
zone, a site populated by long-lived B cells apparently
representing the quiescent memory cell pool.y*”However, the
presumed precursor-descendent relationship between germinal center and mantle zone cells is complicated by uncertainty as to whether B cells undergo one or multiple periods
of residence in the germinal center, and whether the same or
different antigenic and regional factors determine trafficking
to and inhabitation of germinal center versus mantle zone
sites.6-8.11.12
Accordingly, much effort has been devoted to the isolation
and characterization of B-cell subpopulations residing in
these microanatomic locations based on buoyant density,
peanut agglutinin (PNA) binding sites, and expression of a
various differentiation-specific membrane proteins.’’-” In
this report, we have used these phenotypic criteria to determine the abundance of germinal center and mantle zone B
cells, and as the basis of a rapid, large-scale fractionation
method using magnetic immunobeads.
Another distinction between B-cell subpopulations is the
divergent pattern of antigenic and immunoregulatory selection they encounter, which in turn should create characteristic patterns of antibody repertoire and V gene clonal
Blood, Vol 76, No 1 (July 1). 1990: pp 17-23
MATERIALS AND METHODS
Reagents. Purified monoclonal mouse antibodies for human
IgD, CD3 (Leu-4), CD20 (Leu-16), and CD38 (Leu-17) were
purchased from Becton-Dickinson (Mountain View, CA); PNA was
purchased from E-Y Laboratories (San Mateo, CA). Leu-I6 was
obtained as a phycoerythrin conjugate. The other reagents were
obtained as fluorescein conjugates. Monoclonal mouse antibodies to
CD44 (HK23) and globotriosyl ceramide (5B5) were tissue culture
From the Department of Pathology and Jonsson Comprehensive
Cancer Center, UCLA School of Medicine; and Molecular Biology
Institute. UCLA, Los Angeles, CA.
Submitted February 9, 1990; accepted April 4, 1990.
Supported by US Public Health Service CAI2800 and a UCLA
AIDS Clinical Research Grant. H.L.G. I l l is a predoctoral fellow of
the Ford Foundation. National Research Council. Y.V.-A. is a
postdoctoral fellow of the Fogarty Program in AIDS Research at
UCLA. J.B. is a Scholar of the Leukemia Society of America, and a
recipient of an RJR-Nabisco Research Scholars Award.
Address reprint requests to Jonathan Braun. MD, PhD, Department of Pathology CHS 13-186, UCLA School of Medicine. Los
Angeles. CA 90024-1 732.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1990 by The American Society of Hematology.
0006-4971/90/7601-0033$3.00/0
17
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18
supernatantsI6 (generously provided by Dr M.H. Nahm, Washington University School of Medicine), and detected with fluoresceinFab’2-goat anti-mouse IgG (Tago, South San Francisco, CA). For
cell separations, monoclonal mouse antibodies for human IgD,
TA4-1,” and CD38, THB-7” were produced as mouse ascites and
tissue culture supernatant, and used without further purification.
Sheep anti-mouse IgG magnetic immunobeads (Dynabeads, Dynal,
Inc) were purchased from Robbins Scientific (Mountain View, CA).
Cell separation. Tonsil tissue was obtained as fresh tonsillectomy specimens from patients ranging in age from 2 to 14 years, and
processed in culture medium (RPMI-1640, 20% fetal calf serum
[Hyclone, Logan, UT], 1 mmol/L sodium pyruvate, 50 pmol/L
2-mercaptoethanol, and antibiotics). Enriched B cells were prepared
by Ficoll-Hypaque mononuclear cell isolation and sheep erythrocyte
T-cell depletion.24In some cases, mantle zone and germinal center
B-cell subpopulations were isolated. B cells, 2 x lo7,were incubated
with anti-CD38 or anti-IgD for 30 minutes on ice, then washed,
resuspended at 6 x 106/mL with anti-mouse IgG magnetic immunobeads (5 mg/mL), and incubated in a 15”
plastic conical tube on
a rocker for 45 minutes at 4OC. Immunobead-bound cells were
separated from unbound cells (CD38-depleted and IgD-depleted
cells, respectively) by twice decanting the solution in the presence of
a strong magnet on the wall of the tube. The different cell fractions
were washed and used without further efforts to remove free or
cell-bound immunobeads.
Now cytometry. Flow cytometry was performed on a FACS 440
(Becton-Dickinson) with 3.5 decade logarithmic amplification; list
mode data analysis was performed using a Digital MiniVAX system
(Boston, MA). For quantitation studies, fluorescence channel gates
were selected by setting a level of -3% positive cells with negative
control reagents (for single-step staining, an isotype-matched, fluorochrome-conjugated irrelevant monoclonal antibody; for two-step
staining [5B5 and HK231 fluorescein anti-IgG alone).
Pokeweed mitogen (PWM)-induced Ig secretion. CD38- and
IgD-depleted B-cell fractions and autologous T cells were each
adjusted to 1 x lo6cells/mL in culture medium, and 50 pL of B- and
T-cell suspensions were added to U-bottom tissue culture microtiter
plates with 50 pL PWM (GIBCO, Grand Island, NY, 1:lOO in
culture medium). After a 4-day culture at 37OC/5% CO,, the culture
supernatants were collected and quantitated by isotype-specific
enzyme-linked immunosorbent assay (ELISA) for the level of Ig
secretion.’’
Quantitation of genomic V, subfamily utilization. Quantitative
PCR analysis of V, subfamily utilization was essentially as described by Govan et
Genomic DNA was prepared from -2 x
IO’ cells by standard methodsz7 Oligonucleotides were designed
based on members of tabulated V, gene families,’* and
were prepared by Dr Thomas Sutherland, UCLA Molecular Biology
Institute Oligonucleotide Synthesis Laboratory; VHlN:5’-dTCTGGGGCTGAGGTGAAGAAG (coding strand sequence, codons
7 to 13, FRl, VH1); VH3L: 5’-dCTGGTGGAGTCTGGGGGAGGC (coding strand sequence, FR1, codons 6 to 12, VH3);
VH46; 5’-dCCAGGACTGGTGAAGCC (coding strand sequence,
FRl, codons 9 to 14, VH4 and 6); VH5N: 5’-dCCTCCACAGTGAGTCTCC (coding strand sequence, 5’ flank, -68 to -50
upstream of translational start site, VH5); JHKOR: 5’-dAGTGCCAACCGCCTCACAGG (noncoding strand sequence, codons 108
to 2 nt 3’of codon 113A, consensus human JH).29
Amplification reactions were based on standard conditionsg0using
100 ng predenatured (94OC. 2 minutes) genomic DNA templates,
0.5 U Thermus aquaticus DNA polymerase (Taq) (Perkin-Elmer/
Cetus, Norwalk, CT), 100 pmol oligonucleotide (each), 200 pmol
dNTP, and 0.1 pCi 32P-[a]-dCTP(Amersham, Arlington Heights,
IL) in a final volume of 0.1 mL. Reaction mixtures were subjected to
40 successive cycles consisting of heat denaturation (92OC, 1.5
VALLES-AYOUB, GOVAN, AND BRAUN
minutes), annealing (5 l0C, 2 minutes), and primer extension (72OC,
1.5 minutes), using an Ericomp Twin-Block instrument (San Diego,
CA). PCR products were fractionated using l%/l% Nu-Sieve/LE
agarose (FMC) in 1X TAE (0.04 mol/L TRIS-acetate, 0.002
mol/L EDTA) buffer. Gels were briefly stained with ethidium
bromide to visualize bands; the apparent sizes of the amplified
products were: VHIN, 440 base pairs (bp); VH3L, 434 bp; VH46,
434 bp; VHSN, 583 bp. Agarose gels were vacuum-dried onto a
sandwich of neutral nylon filter (Hybond, Amersham) and 3-M
paper, and autoradiographed with XAR film (Kodak, Rochester,
NY) for 2 hours at room temperature. The autoradiographs were
used as templates to excise bands, and Cerenkov counts were
measured using a Beckman LS-7000 scintillation counter. Duplicate
samples were typically processed, and the values were reported as
arithmetic means; the standard errors were less than 10%.
All phases of the study were approved by the human subject
protection committees of Kaiser-Permanente and UCLA Medical
Center, the UCLA Chancellor’s Animal Research Committee, and
were in accordance with current guidelines of the Committee on
Care and Use of Laboratory Animals of the Institute of Laboratory
Animal Resources. National Research Council Maintenance.
RESULTS
Enriched tonsil B cells were labeled with antibodies
detecting surface antigens known to distinguish between
germinal center a n d mantle zone B cells, a n d analyzed by
flow cytometry (Fig 1). Each of these antigens (Leu-16, IgD,
Leu-17, and PNA) divided t h e tonsil B cells into bright and
dim populations. As expected, in each specimen there was a
correlation between the abundance of cells with phenotypes
characteristic of the germinal center (bright for Leu- 16,
Leu-17, a n d PNA; dim for IgD) and mantle zone (dim for
Leu-16, Leu-17, and PNA; bright for IgD). However, after
examination of a series of patients, we were surprised t o find
that the proportion of germinal center to mantle zone cells
differed between individual patients, but was consistent
according to age (Table 1). In young children (2 to 4 years
old), the ratio of germinal center t o mantle zone cells was 2:1,
whereas in older children (8 to 1 4 years old), t h e ratio was
reversed. In contrast, there was no age effect on the fraction
of B a n d T cells in the total mononuclear cell population
(Table 1) or the total number of tonsil lymphocytes recovered (data not shown).
T o directly analyze germinal center and mantle zone B
cells, we isolated these two populations by magnetic immunobead fractionation. W e expected t h a t germinal center B cells
would be enriched in the IgD-depleted a n d CD38-bound
fraction. Conversely, mantle zone B cells would be enriched
in t h e CD38-depleted and IgD-bound fraction. In fact, the
relative yield of cells in each fraction corresponded to the
ratios predicted by flow cytometry, a n d were nearly quantitative (greater than 90% recovery) (data not shown). T h e
purity of the fractionated populations was assessed by flow
cytometry (Table 2, top) using several subpopulation-specific
markers for germinal center (CD20high,5B5, and Leu-17) or
mantle zone ( C D 4 4 or IgD) cells. Expression of these
antigens was heterogenous: while all t h e markers demonstrated enrichment of t h e appropriate subpopulation, estimates of purity were generally greater than 90%, but ranged
from 75% t o 95%.
T h e identity of the isolated subpopulations was further
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19
DEVELOPMENTALLY REGULATED B CELLS
Leu-17
Leu-16
PNA
2 year old
d
W
m
8 year old
60
Il#
1BB
LtI
6s
IPI
tea
M B
68
IC0
lam
t4B
6@
lea
I)@
118
FLUORESCENCE CHANNEL NUMBER (LOG SCALE)
Fig 1. Phenotype of tonsil B cells during childhood. T-cell-depleted mononuclear cells from tonsil specimens of a 2- or E-year-old
patient were labeled with antibodies to the indicated antigens or with the PNA lectin. and analyzed by single-color flow cytometry.
characterized by testing for PWM-induced Ig secretion
(Table 2, bottom). CD38-depleted (mantle zone) cells responded with high level secretion for all tested Ig isotypes. In
contrast, IgD-depleted (germinal center) cells were completely unresponsive. These findings are in accordance with
previous studies indicating the distinctive unresponsiveness
of germinal center (but not mantle zone) B cells to PWMinduced Ig secretion,’’ and suggest a high level of purity.
The V, clonal patterns of the highly purified germinal
center (CD38-bound) and mantle zone (IgD-bound) B cells
were analyzed using a quantitative, V, subfamily-specific
PCR assay to characterize the relative abundance of index
Table 1. Abundance of Follicular B-Cell Subpopulations in
Young and Old Children
Age lyl
Patient
% GC
% MZ
%T
Younger age group
1
2
3
4
5
Average’
21.5
20.2
2
78.5
42.7
18.1
2
57.3
18.6
20.3
3
81.4
45.4
19.0
3
51.6
21.0
21.4
3
79.0
2.6 0.45 69.6 A 6.3 29.8 f 5.8 19.8 0.6
*
*
Older age group
6
7
8
9
10
Average’
8
9
14
9
9
9.8 k 1.1
69.5
19.6
30.5
20
42.2
57.8
78.3
18.5
21.7
17.7
35.3
64.7
62.8
20.3
37.2
33.4 * 3.5 66.6 f 3.5 19.2 + 0.5
Paired, two-tailed r-test (ranked)
P = ,0018 P = ,0004 P = ,0003
P = ,0141
Human tonsillar lymphocytes were analyzed by flow cytometry for the
fraction of T cells (CD3’ lymphocytes), and the fraction of B cells either
bright (germinal center) or dim (mantle zone) for Leu-16.
Abbreviations: GC. germinal center; MZ,mantle zone.
*Arithmetic mean f SE.
B-cell clones. The rationale for this type of analysis has been
presented previously.26 Briefly, oligonucleotide probes have
been selected to detect subsets (“subfamilies”) of several V,
gene families. When 5’-3’ V, subfamily and 3’-5’J, oligonucleotides are used to amplify genomic DNA, product occurs
only with fully rearranged V, subfamily segments (ie,
juxtaposed to a J, segment by VDJ rearrangement). In
addition, the concentration of genomic D N A is reduced to a
range where the amount of amplified product (judged by
incorporated radioactivity) is proportional to the amount of
genomic DNA included in the reaction mixture. In the case
of tonsil B-cell DNA, titration studies showed a linear
relationship between genomic D N A and amplified product
when genomic DNA was between 20 and 200 ng/O.l
reaction (-200 to 2,000 cell equivalents). Finally, because
human B cells usually bear only one fully rearranged allele,3’
the abundance of VDJ segments for a particular V, subfamily closely approximates the abundance of B cells in the
population expressing that V, subfamily. For these reasons,
this PCR assay allows a quantitative comparison of the
relative number of V, subfamily B cells in different sample
populations.
The results of a typical analysis of a young and old child
are shown in Fig 2. B cells bearing rearranged V H l N or
VH3L were abundant in mantle zone B cells of both young
and old children. In contrast, while germinal center B cells
bearing rearranged VH 1N were abundant in older children,
they were rare in younger children. Moreover, germinal
center cells bearing rearranged VH3L were rare in both age
groups. These findings were consistent for three additional
children in each age group (Fig 3). The data from these
groups were compiled to obtain the following quantitative
description of these differences. For germinal center VH 1N,
the average signal was 113 f 21 (cpm, mean f SE) and
4,301 529 for the young and old groups, respectively; the
ratio (young:old) was 2.6% + 0.2%. For VH3L, the average
signal was 142 f 18 and 4,575 f 56 for germinal center and
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20
VALLESAYOUB, GOVAN, AND BRAUN
Table 2. Characterization of Fractionated B Cells
Surface Antigen Phenotype
PWM-Induced Ig Secretion (nglmL)
CD2OW
585
CD38
CD44
IgD
CD3
IgM
1
6
IeA
74.2
5.2
91.2
55.1
6.5
81.4
72.3
ND
93.0
35.5
74.0
17.1
30.7
88.4
ND
6.7
ND
7.7
150
0.5
350
0.5
200
Unfractionated
CD38-depleted
IgD-depleted
0.5
Enriched 8 cells were labeled with antiCD38 or anti-lgD, and separated with anti-mouse lg-magnetic immunobeads to obtain bound and unbound
(depleted)fractions. (Left) Surface antigen phenotype. The depleted fractions were analyzed by flow cytometry for the percent cells expressing germinal
center (CD20h'*, 585, CD38) or mantle zone (CD44, IgD) membrane antigens. The bound fractions were not analyzed due to artifacts caused by residual
bound antibody and immunobeads. (Right) PWM-induced Ig secretion. Fractionated 8 cells were cultured with autologous T cells and PWM for 4 days,
and culture supernatants were analyzedfor secreted Ig by isotype-specific ELSA. The levels of secreted Ig in cultures without PWM were less than 0.5
ng/mL (germinal center) and 0.5 ng/mL (mantlezone).
mantle zone B cells, respectively; the ratio (germinal center:
mantle zone) was 3.1% * 0.4%. In contrast, B cells bearing
two other rearranged V, subfamilies (VH46 and VH5N)
were abundant in both B-cell subpopulations, regardless of
age (Fig 3).
DISCUSSION
In this study of chronic tonsillitis specimens, germinal
center and mantle zone B-cell subpopulations were found to
contain distinct clonal patterns that consistently recurred in
different age-matched individuals. In particular, VH 1N
clones were rare among the germinal center cells of young
but not old children, and VH3L clones were rare regardless
A Ethidium Bromide
- SI77
434
B 35S[a]dCrP autoradiography
- SXl
NXY)
c Gel
z
Age (years)
R cell ubpopul;ttion
V I { probe
Quantitation
x
SIZ
I
2
x
2
GC
V113L
x
GC
I
2
x
31%
VIIIN
Fig 2. Effect of age and B-cell subpopulation on t h e abundance
of B cells bearing fully rearranged VHSL and V H l N subfamily
genes. (A) Direct UV-fluorescence photograph of gel stained w i t h
ethidium bromide. (6) Autoradiography of same gel. (C) Quantitation of Cerenkov counts of excised gel bands (434bp).
of age. In contrast, all index clones were abundant among the
mantle zone cells throughout childhood.
This study introduces a preparative magnetic immunobead method for separating germinal center and mantle zone
subpopulations, based on the reciprocal expression by these
subpopulations of CD38 (Leu-17) and IgD.I7 In addition to
the criteria of CD38 and IgD expression used in the
separation procedure, the purity of fractionated cells was
confirmed by three criteria: flow cytometry for a panel of
subpopulation-specific markers; responsiveness or anergy to
PWM-induced Ig secretion in mantle zone and germinal
center cells, respectively; and restriction of the unique deficit
of VH3L clones to the germinal center cell fraction. These
findings verify the identity of the fractionated populations
with a level of purity comparable with other preparative
method^.'^*'**'^ In our hands, the flexibility, speed, and high
yield of the immunobead fractionation is an improvement
over panning or antibody-rosette procedures. Greater purity
can be achieved by fluorescence-activatedcell ~ o r t i n g , ' ~but
.'~
the drawbacks include long separation times, low yield, and
dependence on sophisticated instrumentation.
A PCR-based assay was introduced to measure the abundance of B cells bearing rearranged VDJ segments of
selected V, subfamilies. This approach differs from the
typical assessment of V, utilization patterns based on levels
of V, specific Ig transcripts. While the latter approach is a
fair representation of the expressed repertoire, it does not
necessarily give an accurate estimate of the clonal abundance
of V,-expressing cells because it systematically overestimates the abundance of secretory (high-transcript level) B
cells and underestimates that of quiescent (low-transcript
level) cells. To overcome this issue, several strategies have
been developed, most commonly generation of hybridoma or
Epstein-Barr virus (EBV)-transformed B-cell librarie~.'.'~
The present assay, which was developed in parallel with other
PCR-based studies of V gene
has several advantages: (1) it detects only genomic VDJ segments, which are a
direct measure of clonal abundance; (2) it allows direct
analysis of small numbers (500 to 5,000 cell equivalents) of
fresh B lymphocytes; and (3) it obviates the technical
requirements for primary cell tissue culture and the low
efficiency and potential clonal skewing of hybridoma/EBV
transformation.
The absence of VHlN and VH3L clones in the germinal
center B-cell subpopulation was a striking and unexpected
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21
DEVELOPMENTALLY REGULATED B CELLS
1
12000 7
VHlN
loo00
vH3L
GC
-.MZ
8000
E
8
6000
4000
2000
0
2
2
3
8
9
14
2
2
3
8
9
14
2
2
3
8
9
14
8
9
14
12000
AGE
2
2
3
AGE
Fig 3. V, subfamily quantitation in fractionated germinal center (CD38-bound) and mantle zone (IgD-bound) B cells from six different
patients of the indicated age in years.
finding. A simple interpretation stems from the fact that
individual germinal centers are composed of oligoclonal or
monoclonal B-cell populations specific for the ongoing response to local
Accordingly, germinal centers
from chronic tonsillitis specimens would be expected to be
composed of B cells specific for the pathogens responsible for
the inflammatory state. Following this reasoning, the VH46
and VHSN subfamilies presumably include V, genes suitable for the generation of relevant antibodies to these
pathogens, whereas the VH3L subfamily does not. Similarly,
the age-dependent occurrence of VH 1N germinal center
cells may reflect a shift in pathogens causing tonsillitis during
childhood, the VH 1 N subfamily presumably suitable only
for pathogens prevalent during late childhood.
There are certain limitations and discordances with this
explanation. (1) The microbiologic premise is uncertain:
while the decline in tonsillitis due to Hemophilus influenzae
coincides with the appearance VH 1N germinal center cells,
the major pathogens for chronic tonsillitis (Streptococcus
pneumoniae and adenovirus) do not show an age-specific
(2) It is surprising that subsets of the very small
VH4, VH6, and VH5 families are abundant in the presumed
antigen response, whereas subsets of the large and immunodominant VH1 and VH3
are almost completely unrepresented. However, we note that each subfamily
may not be functionally representative of its aggregate
family.39 (3) A recent examination of fractionated B-cell
subpopulations from mesenteric nodes of a 40-year-old with
ulcerative colitis demonstrated the same clonal patterns of
the older-child tonsil, including the absence of germinal
center VH3L clones (Valles et al, unpublished observations,
October 1989). This suggests that the germinal center clonal
pattern may be generated by factors independent of childhood and the tonsil antigenic environment.
Alternatively, the evolving abundance and V, clonal
pattern of germinal center cells during childhood may be a
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22
VALLES-AYOUB, GOVAN, AND BRAUN
manifestation of B-cell ontogeny. In humans, ontogenic
uncertain whether germinal center emigrants ever return to
shifts have been observed for V, ~ t i l i z a t i o n ~and
~ ~pat~ ~ ~ ’ ~the germinal center. A key prediction of this scenario is the
terns of antigen responsi~eness.~~~
Notably, the late appeardeficit of V H l N and VH3L clones in germinal center cell
ance of germinal center V H l N clones corresponds to the
precursors; efforts to test this prediction are a current
period of childhood in which serum IgAZ and humoral
laboratory focus.
responses to H injuenzae infection4 shift to adult levels. In
this line of thinking, the early deficit of V H l N and VH3L
ACKNOWLEDGMENT
clones would suggest that these clones are uncommon among
We are grateful to Lily King for her many insightful contributions
germinal center cell precursors (such as recent bone marrow
in development of the detection and quantitation strategies for
emigrants). Accordingly, their abundance in mantle zone
rearranged V, subfamily genes; Dr Gerald Higgins and members of
cells would indicate that strong positive selection for these
the Department of Pathology, Kaiser-Permanente Hospital, Panclones occurs after germinal center emigration. If so, the
orama City, for tonsil specimens; Dr Lynn Gordon for design of the
continued absence of these clones in the germinal center cell
VH3L oligonucleotide; Dr Andrew Saxon for performing ELISA
subpopulation would imply that most germinal center coloniquantitations of human Ig; and Dr Moon H. Nahm for gifts of HK23
zation involves immature precursors, rather than returning
and 5B5 antibodies. We thank Lance Hultin for assistance and
mantle zone cells. This issue is meaningful because it remains
advice in flow cytometry, and Carol Appleton for photographic work.
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1990 76: 17-23
Evolving abundance and clonal pattern of human germinal center B
cells during childhood
Y Valles-Ayoub, HL 3d Govan and J Braun
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