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Correlation analysis between frequencies of

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1406 H. Leyendeckers et al. Eur. J. Immunol. 1999. 29: 14061417

Correlation analysis between frequencies of

circulating antigen-specific IgG-bearing memory
B cells and serum titers of antigen-specific IgG
Heike Leyendeckers1, Marcus Odendahl2, Andrea Lhndorf3, Johannes Irsch1,
Michael Spangfort5, Stefan Miltenyi1, Nicolas Hunzelmann4, Mario Assenmacher1,
Andreas Radbruch2 and Jrgen Schmitz1
1
Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
2
Deutsches Rheuma Forschungszentrum, Berlin, Germany
3
Institute for Genetics, University of Cologne, Cologne, Germany
4
Department of Dermatology, University of Cologne, Cologne, Germany
5
ALK, Horsholm, Denmark

Recent studies in mice have indicated that the long-lasting specific antibody responses seen
after vaccination are probably due to the existence of long-lived plasma cells. Therefore,
because the maintenance of humoral immunity does not necessarily reflect continuous
restimulation of long-lived memory B cells, the question arises as to what degree antibody
immunity, as determined by measuring serum immunoglobulin titers against a particular
antigen, and memory B cell immunity, as determined by counting circulating memory B cells
with specificity for that same antigen, correlate. Here, using a new assay combining two-
step immunomagnetic enrichment with multiparameter flow cytometry to detect, enumerate
and characterize antigen-specific memory B cells, we show for tetanus toxin C-fragment in
blood of normal tetanus toxoid vaccinized donors, and for wasp venom phospholipase A1B
in blood of wasp venom-allergic donors undergoing an immune therapy with wasp venom,
that there is no statistically significant linear correlation between the frequencies of circulat-
ing antigen-specific IgG-bearing memory B cells and the serum titers of antigen-specific
IgG. This lack of a statistically significant linear correlation is in accordance with the idea that
B memory cells and plasma cells represent independently controlled forms of immunological
memory.
Received 21/9/98
Revised 11/1/99
Key words: Antigen-specific B cell / Magnetic cell sorting / Memory cell / Plasma cell / Tetanus Accepted 21/1/99
toxin

1 Introduction upon reexposure to pathogen, the presence of memory

Both, long-lasting specific Ab responses as well as long-
lived memory B cells may contribute to long-term pro-
tective immunity, but the relative importance of their con-
tribution is not well understood [1]. The presence of pre-
existing neutralizing and/or opsonizing Ab is the only
means of specifically preventing an infection and is the
most important protective mechanism against many viral
and most microbial infections [14]. Memory B cells can-
not prevent infection per se but since they rapidly prolif-
erate and differentiate into Ab-producing plasma cells
[I 18814]
Abbreviations: rTT.C: Recombinant tetanus toxin C-
fragment PLA1B: Phospholipase A1B Dig: Digoxigenin
B cells could be critical in controlling the extent of infec-
tion and preventing disease [1].
The half-live of secreted Ab is about 3 weeks [58].
Hence, continuous presence of Ab-producing plasma
cells is required to sustain elevated Ab levels. The major-
ity of Ab is produced by plasma cells in the BM [9, 10].
Conventional models postulate that plasma cells are
short-lived and continously generated de novo from
long-lived memory B cells by stimulation with persisting
Ag [1, 2, 1114]. Recent studies by Manz et al. [15, 16]
and Slifka et al. [17], however, demonstrate that a sub-
stantial fraction of plasma cells in the murine BM is long-
lived and can secrete Ab for extended periods of time
( G 1 year) in the absence of memory B cells. If long-lived
plasma cells are the cellular source for long-lasting Ab

0014-2980/99/0404-1406$17.50 + .50/0 WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999

Eur. J. Immunol. 1999. 29: 14061417 Enumeration of antigen-specific memory B lymphocytes 1407

titers, humoral immunity does not necessarily reflect
memory B cell immunity, but could represent an inde-
pendently controlled form of immunological memory.
The latter would be critical for the rational development
of vaccines and would have important implications for
the strategies on booster vaccination. The generation
and maintenance of elevated Ab titers in the serum is
usually the only correlate of immunity measured after
vaccination. Minimal titers are often defined as marking a
protective level and are used as unique criterion for
recommendations for booster vaccinations. This has not
always proved useful. When Ab is passively adminis-
tered, susceptibility to infection returns as Ab titers
decline. Protective immunity can persist, however, after
immunization with vaccines when Ab is not detectable
anymore [18]. Whether this is due to the persistence of
long-lived memory B cells is not clear.
In this study, we have investigated whether the amount
of Ag-specific IgG correlates with the frequency of Ag-
specific memory IgG+ B cells in the circulation. We have
found that frequencies of recombinant tetanus toxin C-
fragment (rTT.C)-specific memory IgG+ B cells and IgG
titers against rTT.C do not statistically significantly corre-
late in normal healthy blood donors and that frequencies
of phospholipase A1B (PLA1B)-specific memory IgG+ B
cells and levels of PLA1B-specific IgG do not statistically
significantly correlate in wasp venom-allergic donors
undergoing an immune therapy with wasp venom.
2 Results
2.1 Immunomagnetic enrichment of rTT.C- and
PLA1B-specific CD19+ B cells
For the isolation of Ag-specific B cells, CD19+ B cells
were first isolated from whole blood or from PBMC by
magnetic labeling with CD19 mAb-conjugated releas-
able microbeads and enrichment of labeled cells by
magnetic cell sorting (MACS). The average purity of
CD19+ B cells, as determined by flow cytometry (Fig. 1),

Figure 1. Two-step immunomagnetic enrichment of Ag-specific B cells. (AC) rTT.C-specific B cells were enriched from PBMC
of a normal healthy blood donor. Histogram A shows CD19-PE staining of PBMC, dot plot B shows CD19-FITC vs. rTT.C-PE
staining of CD19+ B cells positively selected from PBMC, and dot plot C shows CD19-FITC vs. rTT.C-PE staining of rTT.C-
specific B cells enriched from pre-selected CD19+ B cells. (DF) PLA1B-specific B cells were enriched from blood of a wasp
venom-allergic donor. Histogram D shows CD19-FITC staining of CD19+ B cells isolated from whole blood, dot plot E shows
PLA1B-PE vs. CD9-Cychrome and PI staining of isolated CD19+ B cells, and dot plot F shows PLA1B-PE vs. CD9-Cychrome and
PI staining of PLA1B-specific B cells enriched from pre-selected CD19+ B cells. In (AD) cell debris, erythrocytes, thrombocytes
and dead cells were excluded by gating according to light-scatter signals and PI staining. In (E) and (F) cell debris, erythrocytes
and thrombocytes were excluded by forward vs. side scatter gating. CD9-Cychrome staining in (E) and (F) was performed to dis-
criminate between PLA1B-binding CD9+ basophilic granulocytes and PLA1B-specific CD9 B cells.
1408 H. Leyendeckers et al.
was 97.85 1.24 % in 47 experiments in which B cells
were isolated from PBMC and 75.38 13.83 % in 11
experiments in which B cells were isolated from whole
blood. The average recovery was 79.36 16.31 % in a
series of 38 experiments in which B cell recoveries were
determined upon isolation from PBMC. Due to difficulties
in accurately enumerating B cells in samples of whole
blood, recoveries for the isolation of B cells from whole
blood were not determined. The CD19+ B cells obtained
that way were incubated with a microbead-releasing
agent to remove the microbeads from the CD19 Ab on the
cell surface. This allowed the enrichment of Ag-specific
cells from the pre-enriched CD19+ B cells by another
round of magnetic labeling and separation with MACS.
Two strategies for magnetic labeling and fluorescence
staining were used to enrich and detect rTT.C-specific
B cells: (a) indirect labeling with digoxigenin (Dig)-
conjugated rTT.C as primary reagent and anti-Dig Ab-
conjugated microbeads plus anti-Dig Ab-conjugated PE
as secondary reagents, or (b) direct labeling with rTT.C-
conjugated microbeads and rTT.C-conjugated PE.
For comparison of both labeling strategies, rTT.C-
specific B cells were enriched from 2.5 107 pooled
CD19+ B cells of six normal healthy donors using both
methods (Fig. 2). Purity (6.41 % vs. 6.39 %) and yield
(1600 vs. 1720 cells) of rTT.C-specific B cells were about
the same, irrespective of whether the direct or indirect
labeling approach was used. Direct staining with rTT.C-
conjugated PE, however, gave a 15.4-fold increase in
mean PE fluorescence of rTT.C-specific B cells, com-
pared to indirect staining with Dig-conjugated rTT.C and
Figure 2. Direct vs. indirect immunomagnetic enrichment of
rTT.C-specific B cells. (A) Anti-Dig-PE vs. CD19-FITC stain-
ing of rTT.C-specific B cells enriched from pre-selected
CD19+ B cells of a normal healthy blood donor using rTT.C-
Dig and anti-Dig-microbeads. (B) rTT.C-PE vs. CD19-FITC
staining of rTT.C-specific B cells enriched from pre-selected
CD19+ B cells of the same donor using rTT.C-microbeads.
Live nucleated cells were gated according to light-scatter
signals and PI immunofluorescence.
Eur. J. Immunol. 1999. 29: 14061417
anti-Dig Ab-conjugated PE. For the enrichment and
staining of PLA1B-specific B cells, PLA1B-conjugated
microbeads and PLA1B-PE (Fig. 1), but no Dig-
conjugates of PLA1B were used.
To analyze whether the procedure for enrichment of Ag-
specific B cells provides reproducible results, rTT.C-
specific B cells were enriched from pooled B cells of six
normal healthy donors in a series of six parallel separa-
tions using the direct labeling method. The purity of
rTT.C-specific B cells ranged from 9.2 % to 11.2 % (aver-
age purity: 10.16 0.74 %) and the number of rTT.C-
specific B cells isolated from approximately 2 107 pre-
enriched B cells ranged from 2550 to 3020 (average
yield: 2743 159 cells).
Recoveries of Ag-specific B cells upon isolation by
MACS are difficult to determine directly due to their
extremely low frequency in blood (see below). To esti-
mate a minimum recovery, we derived benefit from the
fact that in allergic patients with allergy against PLA1B, in
addition to PLA1B-specific B cells, basophilic granulo-
cytes can be labeled with PLA1B. This is shown in Fig. 3:
Figure 3. Staining and magnetic enrichment of PLA1B-
binding basophilic granulocytes. Cells were stained with
anti-human IgE-FITC vs. PLA1B-PE. Dot plot A shows PBMC
of a normal healthy blood donor, dot plot B shows PBMC of
a wasp venom-allergic donor, dot plot C shows PBMC of a
wasp venom-allergic donor after depletion of PLA1B-binding
basophils (negative cell fraction), and dot plot D shows
PBMC of a wasp venom allergic donor after enrichment of
PLA1B-binding basophils (positive cell fraction). Live nucle-
ated cells were gated according to light-scatter signals and
PI staining.
Eur. J. Immunol. 1999. 29: 14061417
surface IgE+ basophils from an allergic donor are specifi-
cally stained with PLA1B-PE, whereas surface IgE+ baso-
phils from a normal donor are not stained. The receptor
for PLA1B on basophils from wasp venom-allergic
patients is Fc 4 RI-bound IgE with specificity for PLA1B
[19]. Basophils from a wasp venom-allergic donor could
be enriched to 96.62 % purity with a recovery of 98.57 %
using PLA1B-conjugated microbeads (Fig. 3). The mean
PE fluorescence intensity of the PLA1B-PE-stained baso-
phils was with a value of 346 in this experiment 1.2- to
4.3-fold lower as compared to the mean PE fluorescence
intensity values of PLA1B-specific B cells, which were in
the range 4221471 (n = 10). This indicates that the level
of PLA1B-specific Ig is lower on basophils than on
PLA1B-specific B cells, and it is reasonable to assume
that the recovery of PLA1B-specific B cells by magnetic
enrichment using the PLA1B-conjugated microbeads is
of the same magnitude or even higher than the recovery
of basophils.
To proof the specificity of the enrichment and detection
of Ag-specific B cells, enriched rTT.C- and PLA1B-
specific B cells from a normal and an allergic donor,
respectively, were stimulated at a density of 0.3 Ag-
specific IgG+ B cells per well in 96-well microplates with
50 000 irradiated EL-4 B5 thymoma cells and 10 % cul-
ture SN of PHA/PMA-co-activated T cells and mono-
Figure 4. Immunophenotyping of rTT.C-specific B cells. rTT.C-specific B cells were enriched from pooled PBMC of six normal
healthy blood donors. Dot plots show staining with rTT.C-PE and counterstaining with FITC-conjugated antibodies against sev-
eral lineage and differentiation markers. PI immunofluorescence and light scatter signals were used for gating of live cells.
Enumeration of antigen-specific memory B lymphocytes 1409
cytes [20]. After 10 days of stimulation, culture SN were
analyzed for secreted Ag-specific IgG by ELISA. The
proportion of wells positive for rTT.C-specific IgG in the
cultures of enriched rTT.C-specific B cells (23.95 %) and
the proportion of wells positive for PLA1B-specific IgG
in the cultures of enriched PLA1B-specific B cells
(21.30 %), was very close to the value of 26 % which is
expected according to Poisson-distributed frequencies if
100 % of the Ag-specific IgG+ B cells proliferate and dif-
ferentiate into IgG-secreting cells.
2.2 Phenotype of rTT.C- and PLA1B-specific
CD19+ B cells
According to forward and side scatter properties, most
of the enriched rTT.C- and PLA1B-specific CD19+ B cells
in the circulation are small, resting lymphocytes rather
than large, activated blasts (not shown). To classify them
with respect to their B cell differentiation stage and Ig
isotype expression, rTT.C- and PLA1B-specific B cells
were analyzed for the expression of several B cell differ-
entiation Ag and/or Ig classes (Figs. 4, 5).
Most rTT.C-specific CD19+ B cells express sIgG but not
sIgD. Only few rTT.C-specific CD19+ B cells express sIgD
but not sIgG. In two experiments performed with pooled
1410 H. Leyendeckers et al. Eur. J. Immunol. 1999. 29: 14061417

Figure 5. Ig isotype expression of PLA1B-specific B cells. PLA1B-specific B cells were enriched from a normal (AC) and two
wasp venom-allergic donors (DF) and (GI). Dot plots (A), (D), and (G) show PLA1B-PE vs. CD9-Cychrome and PI staining of the
enriched cells and gating of PLA1B-specific B cells. Dot plots (B), (E) and (H) show PLA1B-PE vs. anti-human IgE-FITC staining
and dot plots (C), (F), and (I) show PLA1B-PE vs. anti-human IgG-FITC staining of gated PLA1B-specific B cells.

B cells from six donors each, 72.18 4.58 % of the
rTT.C-specific cells were IgG+ and 15.77 2.13 % were
IgD+. The mean rTT.C-PE fluorescence intensity of cells
expressing IgD was clearly lower than that of cells
expressing IgG, indicating that the latter may have a
higher affinity for rTT.C or a higher number of surface Ab
molecules per cell. The co-stimulatory molecules CD80
and CD86 are not expressed or are expressed at only
very low levels on rTT.C-specific B cells. The same is true
for: (a) CD10, which is restricted in its B cell lineage
expression to early B cell precursors and germinal center
B cells [21, 22]; (b) CD5, a pan T cell marker that is
weakly expressed on a subset of mature B cells [2325];
(c) CD38, an Ag that is found not on naive or memory B
cells but on germinal center B cells and plasma cells [21,
22]; and (d) CD77, which is restricted in its expression on
B cells to a subset of germinal center B cells [21, 22]. The
lymphocyte homing receptor CD44, an Ag which is
found on naive B cells, memory B cells and plasma cells
but not on germinal center B cells [21, 22, 26], is
expressed on all rTT.C-specific B cells. Most rTT.C-
specific B cells do not express CD23 (85.31 1.41 %,
n = 2), a marker which is expressed on naive B cells but
not on germinal center B cells, memory B cells and
plasma cells [21]. Very few rTT.C-specific B cells are
CD62L positive (3.03 2.18 %, n = 2).
Between 60 % and 100 % of the PLA1B-specific B cells
were positive for IgG (average of 10 samples: 86 13 %),
irrespective of whether the cells were obtained from nor-
mal or allergic donors. The predominant IgG subclass
is IgG1. Between 40 % and 71 % of the PLA1B-specific
B cells were IgG1+ (average of three samples from aller-
gic donors: 54 16 %). No IgE- (n = 13) and IgG4-
expressing (n = 3) PLA1B-specific B cells could be
detected, either in normal or in allergic donors.
The absence of IgE-expressing PLA1B-specific B cells in
the blood cell samples of patients with allergy against
PLA1B could be confirmed analyzing the SN of the limit-
ing dilution cultures described above for secreted
PLA1B-specific IgE. In the cultures of PLA1B-specific B
cells from an allergic donor, secreted PLA1B-specific IgE
was detectable in 21.30 % of the wells in which PLA1B-
specific IgG was detected, but in none of the wells in
which no PLA1B-specific IgG was detectable. For com-
parison, in the cultures of rTT.C-specific B cells from a
normal donor, secreted rTT.C-specific IgE was detect-
able in 23.95 % of the rTT.C-specific IgG-positive wells
but in none of the rTT.C-specific IgG-negative wells. This
shows that in no case were IgE-expressing cells present
before the culture period. Upon culturing, however, a
high proportion of the IgG-expressing B cells had
switched its Ig class to IgE, most likely because the 10 %
Eur. J. Immunol. 1999. 29: 14061417
culture SN of PHA/PMA-co-activated T cells and mono-
cytes used for culturing contained 27 pg/ml (0.27 U/ml)
of IL-4.
2.3 Frequencies of rTT.C- and PLA1B-specific
IgG+ B lymphocytes and serum titers of
rTT.C- and PLA1B-specific IgG
B cells with specificity for rTT.C were enriched from
blood samples of 24 normal healthy donors using the
indirect labeling method and PLA1B-specific B cells were
enriched from blood samples of 8 normal donors and 9
wasp venom-allergic donors undergoing an immune
therapy with wasp venom using the direct labeling
method. In Table 1 the number of Ag-specific IgG+ B
cells that has actually been detected in the magnetically
enriched cell fraction by flow cytometry is given for each
blood sample. For all samples, in which a minimum of
ten Ag-specific IgG+ B cells was actually detected, the
number of Ag-specific IgG+ B cells is also given as calcu-
lated on the basis of the flow cytometrically determined
total cell numbers and the frequencies of Ag-specific
IgG+ B cells. The calculated number of Ag-specific IgG+
B cells is always higher than the number of Ag-specific
IgG+ B cells that has been actually detected by flow
cytometry, as a result of the fact that only approximately
2550 % (between 125 ? l and 250 ? l) of the magnetically
enriched cell fraction (500 ? l) were collected and ana-
lyzed using the flow cytometer. Frequencies of Ag-
specific IgG+ B cells among CD19+ B cells were calcu-
lated on the basis of the calculated number of enriched
Figure 6. Correlation analysis between the frequencies of Ag-specific IgG+ B cells and the serum titers of Ag-specific IgG. ( 1 )
Data for rTT.C in normal donors; ( , ) data for PLA1B in normal donors; ( | ) data for PLA1B in allergic donors.
Enumeration of antigen-specific memory B lymphocytes 1411
Ag-specific IgG+ B cells and the total number of pre-
enriched CD19+ B cells in the sample. The frequencies of
IgG+ rTT.C-specific B cells among CD19+ B cells ranged
from 6.5 106 to 7.81 105 (mean 2.95 105, n = 13)
and the frequencies of IgG+ PLA1B-specific B cells
among CD19+ B cells ranged from 2.0 106 to
4.448 104 (mean 9.76 105, n = 10). The frequencies
of IgG+ PLA1B-specific B cells appeared to be higher in
allergic donors (mean 1.328 104, n = 7) as compared
to normal donors (mean 1.53 105, n = 3).
Fig. 6 shows the data on frequencies of Ag-specific IgG+
B cells among CD19+ B cells plotted against the titer of
Ag-specific IgG in the serum, as determined by ELISA
(Table 1). No statistically significant linear correlation is
seen, either for the frequency of rTT.C-specific IgG+ B
cells and the serum titer of rTT.C-specific IgG (r = 0.059,
p = 0.848, n = 13) in normal donors, or for the frequency
of IgG+ PLA1B-specific B cells and the titer of PLA1B-
specific IgG (r = 0.098, p = 0.834, n = 7) in wasp
venom-allergic donors. Since only three samples were
analyzed, no meaningful correlation data could be gener-
ated for the frequency of IgG+ PLA1B-specific B cells and
the serum titer of PLA1B-specific IgG in normal donors,
even though the three points nearly lie in a line (Fig. 6). A
very significant linear correlation is found between the
serum titer of PLA1B-specific IgE and the mean fluores-
cent intensity of staining of basophilic granulocytes with
PLA1B-PE (r = 0.939, p = X 0.0001, n = 9, data not
shown). The latter directly confirms the previous notion
[19] that staining of basophils with fluorochrome-
conjugated allergens occurs via Fc 4 RI-bound allergen-
specific IgE.
1412 H. Leyendeckers et al. Eur. J. Immunol. 1999. 29: 14061417

Table 1. Frequencies of Ag-specific IgG+ B cells and serum concentrations of Ag-specific IgG in blood of normal
and allergic donors
Eur. J. Immunol. 1999. 29: 14061417
3 Discussion
Efficient generation of memory B cells is believed to be
crucial in designing optimal vaccines [3]. To some extent
this is based on the notion that the prolonged Ab
responses seen after human vaccination are a key
aspect of protective immunity and generally reflect the
level of memory B cell immunity. Here we reinvestigated
this issue and studied whether the amount of specific Ab
correlates with the frequency of specific memory B cells
in the circulation.
As Ag, we chose rTT.C and PLA1B. Because rTT.C is a
soluble Ag to which human subjects are almost invariably
immunized with inert vaccines during childhood, blood of
normal adult blood donors could be used for a correlation
analysis between rTT.C-specific Ab and the frequency of
rTT.C-specific memory B cells. PLA1B, a prominent aller-
gen from wasp venom to which a major proportion of the
population is immunized by wasp stings, was chosen
because blood of wasp venom-allergic donors could be
analyzed in the course of a specific immune therapy with
purified wasp venom exactly 1 month after the patients
had received their last maintainance dose.
A new assay combining immunomagnetic enrichment
with multiparameter flow cytometric analysis was devel-
oped to quantitate and characterize Ag-specific B cells
ex vivo. The approach proved to be highly efficient, very
reliable and extremely specific. With the double-positive
magnetic cell sorting procedure, we achieved a consider-
able sample volume reduction as well as an up to 20 000-
fold enrichment of Ag-specific B cells. The recovery of
Ag-specific cells at this enrichment was higher than
98 %, as determined in the model system in which baso-
philic granulocytes from wasp venom-allergic donors
were enriched. However, the most important aspect
when evaluating the usefulness of a given cell sorting
technology is the proof of specificity. In the present
experiments, we have documented the specificity of the
assay in manifold ways: (a) the flow cytometric quantita-
tion of enriched rTT.C- and PLA1B-specific B cells was in
accordance with the enumeration upon in vitro prolifera-
tion and differentiation into Ab-secreting cells by limiting
dilution analysis; (b) the magnetic labeling and enrich-
ment of rTT.C-specific B cells with the Dig-conjugates of
rTT.C could be inhibited by pre-incubation with non-
conjugated rTT.C (data not shown); and (c) labeling of
basophilic granulocytes with PE- or microbead-
conjugates of PLA1B correlated well with the level of
PLA1B-specific IgE in the serum, i. e. it was clearly depen-
dent on arming of basophils with PLA1B-specific IgE.
The immunophenotype of enriched rTT.C-specific B cells
from normal adult volunteers was analyzed to determine
Enumeration of antigen-specific memory B lymphocytes 1413
which proportion of these cells are indeed memory B
lymphocytes. In a previous attempt to isolate tetanus
toxin (TT)-specific B cells following rosette formation
with large Ag-coated immunomagnetic beads, Oshiba et
al. [27] have shown evidence implying that TT-specific B
cells from blood of normal donors are predominantly
sIgM+ with low percentages of cells expressing other iso-
types, IgG, IgA, or IgE on the surface. The specificity of
the rosetting procedure, however, has been doubtful for
two reasons: about 10 % of the rosetting cells were iden-
tified as CD3+ T cells; and the mean percentage of TT-
specific B cells among B cells was, with a value of
0.34 %, much higher as compared to reports on the
determination of TT-specific Ab-producing cell precur-
sors by limiting dilution methods. In the study presented
here, we found that the vast majority of the rTT.C-
specific CD19+ B cells display indeed all the criteria of
isotype-switched memory B lymphocytes [21, 22, 26,
28]: these cells express CD19, CD44 and high levels of
sIgG, but neither sIgD nor CD38; they express neither
the germinal center markers CD10 and CD77, nor the fol-
licular mantle B cell markers CD5 and CD23; if at all, they
express very low levels of the accessory molecules
CD80 and CD86; they have the capacity to secrete Ag-
specific Ab upon further stimulation; and, according to
forward and side scatter properties, they are small rest-
ing lymphocytes rather than large activated blasts.
Most of the enriched PLA1B-specific B cells displayed
light scatter properties of small lymphocytes and
expressed sIgG, indicating that they also represent
isotype-switched memory B lymphocytes. We could not
detect any IgG4- or IgE-expressing PLA1B-specific B
cells. The reason for this could be that the frequency of
IgG4- and IgE-expressing B cells is too low to detect
these cells, analyzing a maximum of 406 PLA1B-specific
B cells per sample (donor N32). However, it might well be
that IgE memory B lymphocytes do not exist in the circu-
lation, because from blood samples of patients with
Hyper-IgE syndrome we could enrich and detect IgE+
basophils, but not a single IgE+ memory B cell, using
anti-human IgE-microbeads and PE-conjugated anti-
human IgE (manuscript in preparation).
The frequencies of IgG+ rTT.C-specific B cells among
CD19+ B cells from normal donors ranged from
6.5 10 6 to 7.81 105 (n = 13) and the frequencies of
PLA1B-specific B cells among CD19+ B cells from aller-
gic donors ranged from 2.65 105 to 4.45 104 (n = 7).
These results are consistent with previous data assaying
Ag-specific B cells by limiting dilution analysis [29].
We did not find a statistically significant linear correlation
between the frequency of IgG+ Ag-specific B cells
among CD19+ B cells and the serum titer of Ag-specific
1414 H. Leyendeckers et al.
IgG, neither for rTT.C in normal donors nor for PLA1B in
allergic donors. This lacking statistically significant linear
correlation is in accordance with the recent hypothesis
based on studies in mice, that long-term persistence of
circulating Ab is not provided by continuous differentia-
tion of long-lived memory B cells into short-lived Ab-
secreting plasma cells, but simply by long-lived plasma
cells [1517]. Furthermore, it supports rather than con-
tradicts the idea that humoral and memory B cell immu-
nity could represent independently controlled forms of
immunological memory. However, some objections to
this way of interpreting the results may be raised: (a) if
memory B cells were capable of accumulating in other
parts of the body, blood may not be representative for
the frequency of memory B cells, (b) if in addition to
memory B cells themselves other cells, e. g. memory
CD4+ T helper cells or follicular dendritic cells, control the
generation of Ab-secreting plasma cells from memory B
cells, no statistically significant linear correlation may be
found anymore, and (c) if antibody secretion rates con-
siderably differ in different individuals, the same titer of
circulating Ab may not necessarily reflect the same fre-
quency of plasma cells.
If memory B cells are indeed not required to maintain
protective elevated levels of Ab, a legitimate question is
whether and how memory B cells contribute to protec-
tive immunity. Follow-up studies of hepatitis B (HB) vac-
cinees have shown that protection can persist after anti-
HB Ab responses have declined to undetectable levels
and that rapid recall anti-HB Ab responses can be
induced in subjects who no longer have detectable anti-
HB Ab [18, 3032]. The accelerated Ab responses are
most likely due to the persistence of both memory Th
and B cells, but the mechanism of protection in the
absence of neutralizing Ab might be much more complex
with both rapid recall Ab responses and cytotoxic T cell
responses playing a part. In general, memory B cells dis-
play several intrinsic differences as compared to naive B
cells, which may play a role in confering protection: less
stringent activation requirements [33]; more efficient dif-
ferentiation into plasma cells [34]; capacity to present Ag
directly to T cells [28], and capability of colonizing unique
Ag-draining sites like mucosal epithelium [28].
Studies on the relative importance of memory B cells in
comparison to long-lived plasma cells and memory T
cells for protective immunity are needed for the develop-
ment of vaccines and guidelines for booster vaccination.
The method described here for detection and enumera-
tion of memory B cells might be very helpful to this end.
Eur. J. Immunol. 1999. 29: 14061417
4 Materials and methods
4.1 Patients and blood specimen
Samples of blood (2040 ml) were obtained with informed
consent from patients with severe allergy against wasp
venom. Except for one (patient A3), all allergic patients were
analyzed during the course of a specific immune therapy
with purified wasp venom (ALK, Copenhagen, Denmark)
immediately before the patients received their monthly
maintenance dose. The blood samples were drawn into
Vacutainer tubes (Becton Dickinson, San Jose, CA) contain-
ing EDTA as anticoagulant, and processed within 6 h after
collection. Buffy coats (about 60 ml) from normal healthy
volunteers were obtained from the Institute for Transfusion
medicine (Hospital Merheim, Cologne, Germany) and pro-
cessed within 6 h. Samples of whole blood and buffy coats
were spun down at 400 g for 35 min. Serum was collected
and frozen at 20 C. Mononuclear cells (PBMC) were
obtained by density gradient centrifugation over Ficoll
Paque (Pharmacia, Freiburg, Germany).
4.2 Antigens
rTT.C was purchased from Boehringer Mannheim (Mann-
heim, Germany). PLA1B from wasp venom was kindly pro-
vided by ALK (Horsholm, Denmark). The Ag were conju-
gated to PE (Cyanotech Corporation, Kaila-Kona, HI), Dig
(Boehringer Mannheim) and colloidal super-paramagnetic
microbeads (approximately 50 nm in diameter, Miltenyi Bio-
tec GmbH, Bergisch-Gladbach, Germany) as previously
described [19, 3537].
4.3 Cell sorting
rTT.C- and PLA1B-specific B cells were enriched using a
two-step high-gradient magnetic cell sorting procedure.
Briefly, either non-diluted pelleted cells from 2040 ml blood
or between 1.3 108 and 7.9 108 PBMC at a concentra-
tion of about 5 108 cells/ml of buffer (PBS, 5 mM EDTA,
0.5 % BSA), were incubated with CD19 multisort micro-
beads (Miltenyi Biotec) for 15 min at 4 C at a final dilution of
microbeads with buffer of 1 : 35 and 1 : 5, respectively. After
washing (300 g, 10 min), the cells were applied onto a LS+
column placed in a MidiMACS magnet (Miltenyi Biotec). The
column was rinsed with buffer and CD19+ B cells were
eluted from the column after removing it from the magnetic
field. To release the magnetic particles from the enriched
CD19+ B cells, the cells were washed and incubated in 1 ml
buffer with 20 ? l release reagent (Miltenyi Biotec) for 10 min
at 4 C. To remove any remaining magnetically labeled cells,
the sample was applied onto an MS+ column placed in a
MiniMACS magnet (Miltenyi Biotec), the column was rinsed
with buffer and the non-magnetic, released cell fraction was
collected.
Eur. J. Immunol. 1999. 29: 14061417
For magnetic labeling and enrichment of rTT.C-specific
B cells, the released CD19+ B cells were washed and
resuspended in 80 ? l buffer. Either 20 ? l rTT.C-Dig or
rTT.C-microbeads were added and the cells were incu-
bated for 15 min at 4 C. If rTT.C-Dig was used, the cells
were washed again and incubated with anti-Dig Ab-
microbeads (Miltenyi Biotec) in a volume of 100 ? l at a
final dilution of microbeads with buffer of 1 : 5 for 15 min
at 4 C. After washing, the cells were applied onto a MS+
column placed in a MiniMACS magnet. The negative
cells were washed off the column with buffer and the
retained cells were eluted from the column outside of the
magnetic field with 1 ml of buffer. To achieve a higher
enrichment rate, the eluted cells from the first column
were applied to another MS+ column and the magnetic
separation was repeated.
For the magnetic labeling and enrichment of PLA1B-specific
B cells, the released CD19+ B cells were washed and incu-
bated with PLA1B-microbeads in a volume of 100 ? l at a
dilution of microbeads with buffer of 1 : 5 for 15 min at 4 C.
Cells were separated as described above.
For the magnetic labeling and enrichment of PLA1B-binding
basophilic granulocytes from blood of allergic patients,
PBMC were incubated with PLA1B-microbeads at a cell con-
centration of 108 cells/ml of buffer and a final dilution of
microbeads with buffer of 1 : 5 for 15 min at 4 C. The mag-
netic separation was performed as described above.
4.4 Flow cytometric analysis
A FACScan or FACScalibur (Becton Dickinson) was used for
flow cytometry. Data of 5 103 105 cells per sample were
collected and analyzed using CellQuest software (Becton
Dickinson).
To detect, enumerate and further characterize rTT.C- and
PLA1B-specific B cells by two- or three-color immunofluores-
cence the following reagents were used: rTT.C-Dig, rTT.C-
PE, PLA1B-BE, anti-Dig-PE (Miltenyi Biotec), Streptavidin-
Cychrome (Pharmingen, San Diego, CA), CD9-biotin (clone
M-L13, Pharmingen), anti-human IgD-FITC (clone TA 4.1,
Becton Dickinson), anti-human IgG-FITC (clone JDC-10,
Southern Biotechnology Associates, Birmingham, AL), CD5-
FITC (clone CLB-T1/1, CLB, Amsterdam, The Netherlands),
CD10-FITC (clone W8E7, Becton Dickinson), CD19-FITC
(clone SJ25-C1, Cymbus, Hampshire, GB), CD23-FITC
(clone 9P25, Immunotech, Marseilles, France), CD38-FITC
(clone T16, Immunotech), CD44-FITC (clone IM7, kindly pro-
vided by J. Moll, Karlsruhe, Germany), CD62L-FITC (clone
145.bl, kindly provided by A. Thiel, Berlin, Germany), CD77
(clone 38.13, Immunotech), anti-rat IgM-FITC (clone MARM-
4, Serotech, Oxford, GB), CD80-FITC (clone MAB104, Immu-
notech) and CD86-FITC (clone IT2.2, Pharmingen), anti-
human IgG1-FITC (clone JDC-1, Southern Biotechnology
Enumeration of antigen-specific memory B lymphocytes 1415
Associates), anti-human IgG4-FITC (clone AC3-BB2, South-
ern Biotechnology Associates) and anti-human IgE-FITC
(clone G7-18, Pharmingen).
4.5 Flow cytometric cell counting
Due to the constant flow rates ( ? l/s) of a FACScan and
FACScalibur in each flow mode (fast, medium, slow), abso-
lute cell numbers in a cell sample can be determined on the
basis of the sample volume ( ? l), the flow rate ( ? l/s) and the
cell rate (n/s) as follows: no. of cells in sample (n) = sample
volume ( ? l) [average cell rate (n/s)/flow rate ( ? l/s)]. The
flow rate of the flow cytometers in each mode are deter-
mined using cell samples with known cell concentrations
according to the following formula: flow rate ( ? l/s) = [aver-
age cell rate (n/s)/cell concentration (n/ ? l)]. To obtain accu-
rate cell counts using the flow cytometric measurement it is
important that the cell samples are well suspended.
4.6 ELISA
Concentration of Ag-specific IgG and IgE in sera or cell cul-
ture SN were measured by ELISA as described elsewhere
[38]. For quantification of rTT.C- and PLA1B-specific IgG,
ELISA plates (Greiner, Solingen, Germany) were coated with
rTT.C and PLA1B, respectively, blocked, and then incubated
with serial dilutions of serum or culture SN. Bound IgG was
detected with biotinylated goat F(ab')2 anti-human IgG
(Southern Biotechnology Associates) and plates were devel-
oped with streptavidin-conjugated alkaline phosphatase
(Boehringer Mannheim) and substrate (4-nitrophenyl-
phosphate, Merck, Darmstadt, Germany). Concentrations of
rTT.C-specific IgG were determined using Tetagam N (Behr-
ing, Marburg, Germany) as standard. Concentrations of
PLA1B-specific IgG were calculated relative to the serum of
the allergic patient A4.
To quantify rTT.C- and PLA1B-specific IgE, ELISA plates
were coated with anti-human IgE (clone G7-18, Pharmin-
gen), blocked, then serially diluted serum or cell culture
supernatant was added and bound rTT.C- and PLA1B-
specific IgE was detected with biotinylated rTT.C and
PLA1B, respectively. Concentrations of PLA1B-specific IgE
were calculated relative to the serum of the allergic pa-
tient A4.
4.7 Limiting dilution culture assay
Limiting dilution assays were performed as previously de-
scribed [20]. Briefly, enriched Ag-specific B cells were cul-
tured at limiting dilution in flat-bottom 96-well plates (Corn-
ing Costar, Acton, MA) in the presence of 5 104 irradiated
(5000 rad) EL4 B5 cells (kindly provided by R. H. Zubler,
Geneva, Switzerland) in a final volume of 200 ? l RPMI 1640
(Life Technologies, Paisley, GB) supplemented with
1416 H. Leyendeckers et al.
10 % FCS (Life Technologies), 1 ? M L-alanyl-glutamine (Life
Technologies), 10 mM Hepes (Life Technologies), 100 U/ml
penicillin/streptomycin (Life Technologies), 0.05 mM 2-ME
(Life Technologies) and 10 % SN from a mixed T cell and
monocytes culture stimulated with PHA (5 ? g/ml, Sigma, St.
Louis, MO) and PMA (10 ng/ml, Sigma). The SN was pre-
pared as follows: T cells and monocytes were isolated from
PBMC of normal healthy donors using a T cell (Miltenyi Bio-
tec) and monocyte isolation kit (Miltenyi Biotec), respec-
tively, and were separately cultured for 48 h in medium at a
cell concentration of 106 T cells per ml medium and 3 105
monocytes/ml medium. Then, 107 T cells were added to
3 106 monocytes in a final volume of 10 ml medium and
the cell mixture was cultured for 36 h in the presence of PHA
and PMA. The culture SN was stored at 70 C. As deter-
mined by an ELISA assay for human IL-4 (CLB) the IL-4 con-
centration in the SN was 0.27 U/ml.
Acknowledgements: We are grateful to Drs. R. Zubler,
A. Thiel, U. Klein, and J. Moll for generous gifts of Ab, Ag
and cell lines. This study was supported by the Bundesmi-
nisterium fr Bildung, Wissenschaft, Forschung und Tech-
nologie.
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Correspondence: Jrgen Schmitz, Miltenyi Biotec GmbH,
Friedrich-Ebert-Str. 68, D-51429 Bergisch Gladbach, Ger-
many
Fax: +49-2204-8306489
e-mail: JuergenS MiltenyiBiotec.de