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* Immune Regulation Group, Centenary Institute for Cancer Medicine and Cell Biology, and
University of Sydney, New South Wales, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, for switching to IgA1 and IgA2
(17, 18, 19). By contrast, the specific signal required for
switching to IgG2 remains elusive. Although IFN-
or combinations of
Staphylococcus aereus Cowan strain I, anti-CD40 mAb, and
IL-10 have been found to induce expression of transcripts of the 2
heavy chain region gene (20, 21, 22), CD40-stimulated human B
cells failed to secrete IgG2 following culture with any known
recombinant cytokine (21, 23). These B cells, however, did
secrete IgG2 when cultured with crude supernatant from activated T cell
clones (23, 24), suggesting that T cells are the source of
an as yet unidentified factor.
In most studies Ig isotype switching by human B cells is assessed by
measuring the amount of Ig present in 7- to 12-day culture supernatants
of B cells stimulated under various conditions. However, Ig secretion
is the net result of multiple facets of B cell activation, including
efficiency of activation, proliferation rate, isotype switching,
differentiation to an Ig-secreting cell, and the rate of cell death in
culture. As a result of this complexity, the precise contribution made
by individual cytokines to switching can be unclear, as they may exert
differential effects on some, or all, of these different parameters of
B cell activation (25). This problem has been recently
highlighted further by studies in mice that show that switching to all
Ig isotypes by naive B cells increases in frequency with consecutive
cell division (26, 27, 28, 29). Specifically, naive murine B cells
stimulated with CD40L and IL-4 switched to IgG1 with much greater
frequency after three divisions, while switching to IgE or IgG2a in the
presence of IL-4 or IFN-, respectively, increased markedly after
approximately five or six divisions (26, 27, 29).
Similarly, isotype switching to IgG3, and to IgG2b and IgA in the
presence of TGF-, by LPS- or CD40L-stimulated murine B cells was
also division-linked (28). Thus, treatments that promote B
cell proliferation alone may increase the number of switched cells
without being true switching factors. As human B cell switching
cytokines, such as IL-4, IL-10, and IL-13, can also promote
proliferation (11, 12, 13, 14, 25, 30, 31, 32), the question of
whether they are true switch factors by this new procedure needs to be
resolved. Here, we have examined the extent to which a division-linked
differentiation program applies to isotype switching by human B cells
and how individual cytokines simultaneously affect both isotype
switching and proliferation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Biotinylated mouse anti-human IgG1, IgG2, IgG3, IgG4, IgA, and IgE mAb; PE-conjugated anti-human IgM, IgD, IgG, and CD27 mAb; and isotype control mAb were purchased from BD PharMingen (San Diego, CA). PE-conjugated mouse anti-human CD19 mAb and mouse IgG1 isotype control were purchased from BD Biosciences (San Jose, CA). Streptavidin conjugated to Tricolor (SA-TC) and FITC (SA-FITC) were purchased from Caltag Laboratories (Burlingame, CA). The source of recombinant human CD40L was membranes prepared from the Sf21 insect cell line infected with baculovirus vector containing the CD40L cDNA (provided by Dr. M. Kehry, Boehringer Ingleheim, Ridgefield, CT) (33). Human recombinant IL-4 and IL-10 were provided by Dr. R. de Waal Malefyt (DNAX, Palo Alto, CA). IL-13 was purchased from PeproTech (Rocky Hill, NJ).
Cells
Normal human spleens were obtained from trauma victims
undergoing splenectomies (Royal Prince Alfred Hospital, Sydney,
Australia) or from organ donors (Australian Red Cross Blood Service,
Sydney, Australia). Mononuclear cells were prepared by slicing splenic
tissue into small pieces and disrupting the capsule by forcing the
tissue through a filter mesh. RBC were lysed, and the remaining cells
were washed twice and cryopreserved in liquid nitrogen until required.
Cord blood was obtained from King George V Memorial Hospital for
Mothers and Babies (Sydney, Australia). Mononuclear cells were isolated
by centrifugation over Ficoll-Paque (Amersham Pharmacia Biotech,
Uppsala, Sweden). Total human B cells were isolated using CD19
Dynabeads (Dynal Biotech, Oslo, Norway) (34). The
resulting cell population was >98% CD19+. Naive
CD27- B cells (34, 35, 36) were
purified by labeling total splenic B cells with anti-CD27 mAb MACS
beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and passing through
a column held in a magnetic field (Miltenyi Biotec). Naive (unbound,
CD27-) B cells were collected in the
flow-through and contained >98% IgD+ B cells
and <1% CD27+ IgG/A/E+ B
cells (34). Naive splenic B cells were also isolated by
sorting using a FACStar Plus (BD Biosciences) by labeling with
PE-conjugated anti-CD27 mAb and biotinylated anti-IgG, IgA and
IgE, followed by SA-FITC. Gates were set to collect
CD27- B cells. On reanalysis, the sorted B cells
were >99% CD27- (see Fig. 3
, a and
b).
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Purified naive human B cells were labeled with CFSE (Molecular Probes, Eugene, OR) as previously described (37, 38). Briefly, B cells were resuspended at 1 x 107/ml in PBS containing 0.1% BSA. CFSE, dissolved in DMSO, was added at a final concentration of 5 µM. The cells were vortexed for 10 s and then incubated at 37°C for 10 min. Labeled cells were washed with cold PBS containing 0.1% BSA and resuspended in medium. CFSE-labeled naive B cells (2 x 105/500 µl) were cultured in 48-well plates (BD Biosciences, Franklin Lakes, NJ) with CD40L (final dilution, 1/250) in the absence or the presence of IL-4 (400 U/ml), IL-10 (100 U/ml), or IL-13 (10 ng/ml), alone or in combination. The specificity of the response induced by CD40L expressed by insect cells was confirmed in control experiments in which insect cells expressing a control protein failed to induce any proliferation of naive human B cells alone or in combination with exogenous cytokines (data not shown). B cells were cultured in RPMI 1640 containing L-glutamine (Life Technologies, Grand Island, NY), 10% FCS (CSL, Parkville, Australia), 10 mM HEPES (pH 7.4; Sigma-Aldrich, St. Louis, MO), 0.1 mM nonessential amino-acid solution (Sigma-Aldrich), 1 mM sodium pyruvate (Life Technologies), 60 µg/ml penicillin, 100 µg/ml streptomycin, and 40 µg/ml apo-transferrin (Sigma-Aldrich). All cultures were conducted at 37°C in a humidified atmosphere containing 5% CO2.
Immunofluorescent staining
In vitro-activated naive B cells were harvested from culture wells, and nonspecific binding sites were blocked by preincubation with normal mouse IgG. The cells were then incubated with PE-conjugated isotype control, anti-IgM, -IgD, or -IgG mAb on ice for 20 min. To determine the expression of IgG subclasses, cells were incubated on ice for 20 min with biotinylated anti-IgG1, -IgG2, -IgG3, or -IgG4 mAb; bound Ab was revealed by the addition of SA-TC. The cells were analyzed on a FACScan flow cytometer using CellQuest software (BD Biosciences, San Jose, CA). Surface staining was measured on a logarithmic scale. Cells present in different divisions were characterized by "division slicing." Gates were drawn around each of the peaks present in histograms of CFSE-labeled B cells, representing cells in different divisions. The proportion of cells within each gate or the expression of different surface Ig isotypes by these cells was determined by backgating and analyzing the differently divided populations, defined by CFSE dilution, using the analysis tools of CellQuest (BD Biosciences).
Ig ELISAs
Ninety-six-well microtiter plates (Dynex, Chantilly, VA) were precoated with goat anti-human IgM or IgG polyclonal antisera (Southern Biotechnology Associates, Birmingham, AL), and nonspecific binding sites were blocked with 2% FCS prepared in PBS. Culture supernatants and Ig standards were added to the wells and incubated for 2 h at 37°C before addition of biotin-conjugated anti-human IgM or IgG antisera (Southern Biotechnology Associates). Bound Ab was detected by addition of SA-conjugated HRP (Jackson ImmunoResearch, West Grove, PA) and was visualized with HRP substrate ABTS (Sigma-Aldrich; 1 mg/ml) prepared in citrate buffer (pH 4.5) containing 0.03% H2O2.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To develop an assay for precisely quantifying the proliferative
effects of cytokines that induce Ig isotype switching in human B cells,
CFSE-labeled B cells were cultured with CD40L in the absence or the
presence of cytokines, and after 6 days their division profiles were
determined. In the presence of CD40L alone, the greatest proportion of
harvested cells (
30%) was found in the undivided fraction; the
remaining cells were spread throughout the divisions, with
the percentage of cells decreasing with each subsequent division (Fig. 1, a and g). When
cultures of CD40L-stimulated B cells were supplemented with IL-4,
IL-10, or IL-13, the proportion of harvested cells that remained
undivided was significantly reduced (10%), while that of cells in
later divisions was increased (Fig. 1, b–d and
h–j). Under these conditions, the greatest percentage of
cells was found in the second to fourth divisions, suggesting that
addition of these cytokines induced a greater proportion of activated B
cells to undergo additional rounds of cell division. The effects of
IL-4 and IL-10 on the distribution of CD40L-activated B cells across
division were comparable. In contrast, although IL-13 enhanced the
proliferation of CD40L-activated B cells, it was less effective than
IL-4 and IL-10 in inducing the same proportion of B cells to enter the
later divisions (Fig. 1, b–d and h–j). The
addition of IL-10 to B cells cultured with CD40L and IL-4 induced
greater proliferation than either signal alone and was accompanied by a
further decrease in the percentage of undivided cells, with the
greatest percentage of cells being found in the fourth or fifth
divisions (Fig. 1, e and k). In contrast,
proliferation in response to the combination of IL-10 and IL-13
did not greatly differ from that in response to IL-10 alone (Fig. 1, f and l).
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Human naive B cells were cultured with CD40L in the absence or the
presence of IL-4, IL-10, or IL-13, alone or in combination. After 6
days the cells were harvested, and the expression of surface IgG and
the amounts of secreted IgM and IgG in culture supernatants were
determined. Very few IgG+ cells (<0.8%) were
detected in cultures of naive B cells stimulated with CD40L alone. In
contrast, variable percentages of IgG-expressing cells were evident in
cultures of B cells stimulated with CD40L in combination with the
different cytokines. When the effects of individual cytokines were
assayed in isolation, IL-4 induced the greatest proportion of B cells
to undergo isotype switching. However, significant proportions of
IgG+ B cells were also generated from
CD40L-activated B cells in the presence of IL-10 and IL-13 (Table I). Although the frequency of
IgG-expressing cells appearing in cultures containing IL-10 was quite
low, it was consistently higher than that observed in cultures of B
cells stimulated with CD40L alone. The combination of IL-4 and IL-10
caused a 2- to 7-fold increase in the proportion of
IgG+ B cells compared with cultures of
CD40-activated B cells containing either cytokine on their own (Table I). In a parallel manner to the absence of an additive or synergistic
effect on proliferation, the combination of IL-10 and IL-13 did not
appreciably increase the frequency of IgG+ B
cells compared with cultures containing IL-10 or IL-13 alone
(Table I).
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). Addition of IL-4 or IL-10 resulted
in the appearance of low, but detectable, amounts of IgG, and the
combination of IL-4 and IL-10 usually increased the amount produced
(Table II). Consistent with the data for expression of IgG, IL-13
appeared to be less efficient than IL-4 in inducing IgG secretion
(Table II). Although IgG could be detected, the levels observed here
were several orders of magnitude less than those reported by other
investigators (31, 39, 40, 41) (see Discussion).
The effects of these cytokines on IgM production by CD40L-stimulated
naive B cells were quite variable; they either increased or decreased
secretion in separate experiments (Table II).
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Ig isotype switching in murine naive B cells has been found to be
linked to cell division (26, 27, 28, 29). When isotype switching
of CD40L-stimulated naive B cells was analyzed with respect to cell
division, very few if any switched cells were present in any division
(Fig. 2, a and g).
However, by comparing IgG expression by human naive B cells activated
with CD40L and cytokines to CFSE intensity, it was apparent that the
IgG+ B cells accumulated in populations of B
cells that had undergone the greatest number of divisions (Fig. 2, a–f). Thus, in the presence of IL-4 or IL-13, B cells
expressing IgG were detected after approximately the third or fourth
division (Fig. 2, b and d, and h and
j). The percentage of IgG+ cells
increased with each successive division, such that 10–20% of CD40L-
and IL-4- or IL-13-stimulated B cells in the fifth to seventh divisions
were IgG+ (Fig. 2, h and
j). Consistent with the finding that IL-13 induced fewer
cells to switch to IgG than IL-4 at the bulk population level (Table I
), the rate of Ig isotype switching per division was less in the
presence of IL-13 than IL-4 (compare Fig. 2, h and
j). This cannot be attributed to IL-13 inducing fewer cells
to reach the later divisions, because there was a reduced percentage of
IgG+ cells per division in cultures containing
IL-13 than IL-4 (Fig. 2, h and j). A low level of
switching to IgG could also be demonstrated in the presence of IL-10
after the cells had undergone five or more divisions (Fig. 2i), which is in agreement with the data shown in Table I.
The increased percentage of IgG+ cells evident at
the population level in the presence of IL-4 and IL-10 (Table I) was
occasionally observed on a per division basis; in other words, a
greater proportion of IgG+ B cells were detected
per division in the presence of both IL-4 and IL-10 than in cultures
containing either cytokine alone (Fig. 2k). Under these
conditions, 20–40% of cells in divisions 5–7 were
IgG+ (Fig. 2k). In contrast, the
combination of IL-10 and IL-13 did not appreciably increase the
proportion of IgG+ B cells in each division
compared with IL-13 alone (Fig. 2l).
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The total human B cell population contains both naive B cells and
memory B cells, which can be distinguished by the differential
expression of CD27 (34, 35, 36). The memory
(CD27+) population also contains nonswitched and
isotype-switched cells (34) (Fig. 3a). These populations can be
resolved phenotypically, with naive B cells being
IgM+D++G/A/E-
CD27-, nonswitched memory B cells being
IgM++D+/-G/A/E-
CD27+, and isotype switched memory B cells being
IgM-D-G/A/E+
CD27+ (Fig. 3a and data not shown)
(34). Although phenotypic analysis of MACS-purified B cell
populations suggested an absence of residual
CD27+IgG/A/E+ memory cells,
a contribution to Ig isotype switching from a very small population of
contaminating cells could not be completely excluded. To check this,
naive B cells were purified by sorting
CD19+CD27-IgG/A/E-
B cells. The resulting naive B cells (>99.5% pure; Fig. 3b) were cultured with CD40L, IL-4, and IL-10, and after 6
days their pattern of isotype switching to IgG was determined compared
with that of B cells isolated from the same donor by MACS beads and
cultured under similar conditions. For both MACS- and sort-purified
naive B cell cultures, IgG+ cells appeared after
approximately the third or fourth division, with the percentage of
IgG+ cells increasing with each successive
division (Fig. 3c), thereby making a contribution from
residual memory B cells very unlikely.
To further exclude any possible effect of residual memory B cells on Ig
isotype switching, experiments were performed using cord blood and
infant peripheral blood as a source of naive B cells, both of
which are devoid of CD27-expressing B cells (34, 42, 43).
When these naive B cells were cultured with CD40L only, <0.5%
IgG+ B cells were detected (data not shown).
However, in the presence of IL-4, 5–10% of cells harvested expressed
IgG (Fig. 4, a and
b). Similarly, IL-10 induced IgG expression by 1–2% of
cells (not shown). When analyzed on a per division basis, cord blood
and infant B cells stimulated with CD40L and IL-4 switched to IgG
following the third cell division. The proportion of positive cells
continued to increase with each subsequent division, reaching a maximum
of 15–20% IgG+ B cells in divisions 5 and
greater (Fig. 4, c and d). Moreover,
IgG+ B cells were detected in the later divisions
of cultures of cord blood and infant B cells stimulated with CD40L and
IL-10 (Fig. 4, c and d). Although the proportion
of IgG+ B cells generated in the presence of
IL-10 was quite low, it always exceeded the proportion of these cells
in cultures of CD40L alone (Fig. 4, c and d). The
rate of division-based switching exhibited by cord blood and infant
naive B cells therefore was comparable to that of adult splenic naive B
cells (see Fig. 2). Taken with the data from cultures of sorted
adult naive splenic B cells (Fig. 3c), it would appear that
the contribution of any contaminating pre- or nonswitched memory B
cells to isotype switching observed here was minimal, and the switching
results obtained reflected bona fide molecular events occurring in
naive B cells.
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Isotype switching by activated human naive B cells to the four IgG
subclasses was next investigated. In the presence of CD40L, IL-4, and
IL-10, naive B cells were induced to switch to IgG1, IgG2, and IgG3
(Fig. 5). The majority of
isotype-switched B cells (70%) were IgG1+,
with the remainder expressing IgG2 or IgG3. In contrast, there was
negligible switching to IgG4 (Fig. 5, a–e), with the
percentage of IgG4+ cells being approximately
equal to that of cells cultured in the presence of CD40L alone (data
not shown). When analyzed on a per division basis, the pattern of
switching to the IgG subclasses was similar to that for switching to
total IgG, with cells expressing IgG1, IgG2, or IgG3 appearing after
the third or fourth division, and the percentage of positive cells
increasing with each subsequent division (Fig. 5f). Thus,
the cells comprising the total IgG+ population
were predominantly IgG1+, with IgG2 and IgG3
being equally expressed (Fig. 5f). Importantly, the sum of
the percentage of cells expressing IgG1, IgG2, and IgG3 per division
equaled the percentage of total IgG+ cells per
division, demonstrating the reliability and specificity of the reagents
used. CD40L and IL-4 also induced switching to IgG1, IgG2, and IgG3,
but to a lesser extent than the combination of CD40L, IL-4, and IL-10
(data not shown). No switching to IgG1 was induced by CD40L alone, in
agreement with the finding that these culture conditions did not induce
switching to total IgG (data not shown, and Table I and Fig. 2).
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When naive B cells undergo isotype switching, the expression of
IgM and IgD is down-regulated (25). The expression of IgM
and IgD on CFSE-labeled human naive B cells cultured with CD40L, IL-4,
and IL-10 was therefore examined. In an equivalent manner to purified
naive B cells, undivided B cells as well as B cells that had undergone
one or two divisions continued to express a high level of both IgM and
IgD (Fig. 6, a and
b). After about three divisions, however, a population of
cells expressing reduced levels of IgM and IgD was detected. This
population continued to increase with each successive division (Fig. 6, a and b). To determine the phenotype of cells
expressing reduced levels of IgM, the cells were double stained with
mAbs specific for IgM and IgG. It was found that all
IgG+ cells were indeed
IgMlow (Fig. 6c), consistent with
isotype-switched B cells being unable to express multiple Ig isotypes.
By quantitating the proportions of IgMlow and
IgG+ B cells in each division it was found that
down-regulation of IgM occurred over the same divisions as isotype
switching to IgG (Fig. 6d). Thus, loss of IgM and IgD from
activated human B cells proved to be division-linked, as was observed
for isotype switching.
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Ig isotype switching by murine B cells has been found to be
independent of time in culture (26, 27, 28, 29). That is, although
the proportion of cells in each division differs at different times,
the percentage of switched cells in the corresponding divisions remains
constant, suggesting that the proportion of B cells undergoing isotype
switching during each cell division is determined by the number of
times the cells divide (i.e., the division history of the cells) rather
than by the time of in vitro culture or exposure to a stimulus. As
switching to IgG by human B cells was found to be division-linked, it
was necessary to determine whether isotype switching was also
independent of time in culture. To achieve this, CFSE-labeled human
naive B cells were cultured with CD40L, IL-4, and IL-10 and then
harvested on sequential days, after 5 and 6 days of stimulation. At
these different times (i.e., 5 and 6 days), the greatest percentage of
cells was found in the third and fifth divisions, respectively, thereby
demonstrating that the cells had undergone several division cycles
during this 24-h period (Fig. 7a). Although the distribution
of cells across division differed for these two times, the percentage
of IgG+ cells in each division remained the same
(Fig. 7b). Thus, like mouse B cells (26),
isotype switching by human B cells is independent of time of culture.
This also indicates that this process is not affected by time-dependent
events, such as production of endogenous factors by activated B cells
that may accumulate over time or the asynchronous rate of proliferation
exhibited by activated naive B cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human CD40L was shown to induce human B cells to
proliferate in the absence of additional cytokines. The proliferative
response, however, was augmented by addition of IL-4, IL-10, or IL-13
(Fig. 1). The greater degree of proliferation of cytokine-stimulated B
cells, revealed by dilution of CFSE staining, not only confirmed the
results from earlier studies using
[3H]thymidine incorporation to monitor cell
proliferation (13, 30, 31, 39, 40), but indicated that the
increase reflects the ability of IL-4, IL-10, and IL-13 to promote a
greater proportion of cells to 1) enter division and 2) undergo further
rounds of division (Fig. 1). Furthermore, although the proportions of
CD40L-stimulated B cells in each division in cultures containing IL-4
or IL-10 were similar, IL-13 induced fewer activated B cells to undergo
a similar number of divisions. This is consistent with previous reports
showing that [3H]thymidine incorporation by
CD40L-stimulated B cells cultured with IL-4 or IL-10 was comparable
(30, 31, 40), whereas incorporation by IL-13-stimulated B
cells was less than that by IL-4- or IL-10-stimulated B cells
(13, 31, 45). Thus, even though IL-4 and IL-13 share many
biological functions (46), IL-13 is a less efficient
promoter of B cell growth than IL-4.
In addition to promoting proliferation, IL-4, IL-10, and IL-13 induced
CD40L-activated naive B cells to switch to IgG in a division-linked,
time-independent manner, as well as to secrete low amounts of IgG
(Figs. 2 and 7 and Tables I and II). The appearance of
IgG+ cells coincided with the appearance of
IgMlow/- B cells, thereby showing that
down-regulation of IgM (and IgD) was also division-linked (Fig. 6).
There appeared to be a hierarchy to cytokine-mediated isotype switching
to IgG, with the rate being greatest in the presence of IL-4, less with
IL-13 (in agreement with data from previous studies (13, 31, 32, 47)), and very low in the presence of IL-10 (Fig. 2). The reason
for the difference in potency between IL-13 and IL-4 is unclear, but
does not appear to reflect differences in the expression of receptors
for these cytokines, because naive B cells have been reported to
express abundant levels of the IL-13R 
1-chain (45, 48).
The proportion of IgG+ cells at the bulk
population level, and for some donors on a per division basis as well,
was greater in the presence of IL-4 and IL-10 than with either cytokine
alone (Table I and Fig. 2), similar to the effect of the combination on
B cell proliferation (Fig. 1). This accorded with earlier data of
Punnonen et al. (49), who reported that IL-10 synergized
with IL-4 to induce IgG secretion by PBMC activated by anti-CD40
mAbs. However, because their B cell population contained memory as well
as naive B cells, it was unclear whether the increase in secreted IgG
resulted from 1) a synergistic effect of IL-4 and IL-10 on isotype
switching, 2) induction of Ig secretion by the memory B cells present
in the PBMC, or 3) both. By contrast, the use of highly purified naive
B cells here allowed us to conclude that IL-4 and IL-10 can function
synergistically to induce Ig isotype switching in these cells.
When IgG subclasses were examined, IL-4 alone or in combination with
IL-10 induced CD40L-stimulated naive B cells to express IgG1, IgG2, and
IgG3, but little IgG4. These Ig isotypes appeared in a similar
division-linked pattern to that of total IgG (Fig. 5). The majority
(70%) of IgG+ cells expressed IgG1, with
fewer cells expressing IgG2 or IgG3, and very few if any expressing
IgG4 (Fig. 5). When naive IgD+ B cells were
cultured with an activated T cell clone rather than a defined cytokine,
the percentages of total secreted IgG of IgG1, IgG2, IgG3, or IgG4
isotypes were 70, 15, 10, and 5%, respectively (24).
Since IgG1 is the most abundant IgG subclass present in human serum
(60–65% of total IgG) (50), these in vitro data point
to a greater rate of switching to this isotype in vivo than to the
other IgG subclasses.
Our experiments with defined naive B cells yielded several unexpected
results that contrasted with the findings of previous investigations.
For example, IL-10 has been reported to induce isotype switching to
IgG1 and IgG3 (14, 15, 16), and IL-4 to induce isotype
switching to IgG4 (11, 12, 13, 51). Furthermore, none of the
known recombinant cytokines has been shown to induce the secretion of
IgG2 (24), although IFN- or combinations of S.
aereus Cowan strain I, anti-CD40 mAb, and IL-10 induced the
expression of germline transcripts of the 2 heavy chain constant
region gene (18, 20, 21). Thus, our observation that IL-4
induced expression in CD40L-stimulated B cells of IgG2 as well as IgG1
and IgG3 was surprising. One explanation for these discrepancies lies
in the use of different experimental procedures. First, our studies
employed recombinant trimeric CD40L, which is known to provide a
stronger stimulus to B cells than divalent anti-CD40 mAb, which was
used in previous studies (52). Consequently, this improved
signaling may have influenced the expression of different IgG subclass
isotypes. Second, splenic B cells were used in our studies, while
previous investigations largely used peripheral blood or tonsil B
cells. Intrinsic differences between B cells isolated from different
tissues cannot be excluded without detailed comparisons of the
responses of these different B cell populations. Third, and most
importantly, different B cell isolation and activation systems were
employed in some of the previous studies mentioned above, including
purification of naive B cells on the basis of IgD positivity or
addition to cultures of CD4+ T cell clones as a
source of T cell-derived factors (11). Such methods may
provide activation signals in addition to CD40L. Indeed, coactivation
of CD40-stimulated human B cells via surface Ig can induce very high
levels of secreted Ig (14, 17, 22, 41), and
membrane TNF- expressedby activated
CD4+ T cells can provide a costimulatory
signal for Ig production by human B cells (9, 53). This
may explain our finding that the amounts of IgG secreted by
CD40L-activated naive B cells were 10- to 100-fold less than
those previously reported from total B cell populations (Table II)
(31, 39, 40, 41). Lastly, the negligible isotype switching to
IgG4 observed may reflect a requirement for a different form of
activation, because the production of significant amounts of IgG4 in
vivo has only been observed following repeated antigenic exposure
(54). Thus, the induction of IgG4 may require more
signals, and over a longer period of time, than those provided by CD40L
in the current study.
An alternative or complementary explanation for the differences
observed between our data and those previously published may reflect
the complex regulation of Ig secretion by isotype-switched B cells. For
instance, the expression of a particular Ig isotype may not correlate
with Ig secretion. It has been found that anti-Ig-induced murine B
cell blasts stimulated with LPS and IL-4 could express IgG1, but failed
to secrete detectable levels of Ig (55). More recently, a
similar reciprocal relationship was reported for Ig secretion and
isotype switching by activated human B cells (56).
Consequently, the lack of expression of IgG4 by human B cells activated
with CD40L and IL-4 does not mean that the cells are incapable of
secreting IgG4. Similarly, CD40L-activated B cell stimulated with IL-4
may express IgG1, IgG2, and IgG3, but may not secrete these isotypes.
In support of this idea is the observation that although IL-4 induced
the transcription of germline 
mRNA by CD40-stimulated human B
cells, two-thirds of these B cells failed to produce IgE
(57). Moreover, according to recent evidence, dendritic
cells (DC) and follicular DC can influence B cell activation by
modulating survival, proliferation, differentiation, isotype switching,
and Ig secretion (56, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67). Based on these
observations, a model can be proposed whereby initial interactions with
T cell-derived signals such as CD40L and IL-4 induce the expression of
switched isotypes on the surface of activated B cells, but further
signals delivered via follicular DC or DC are required for Ig
secretion. The validity of such a model is currently being
explored.
Ig secretion by activated human B cells can be modulated by other
cytokines and PGs. For example, IL-5 (68), IL-6
(69), IL-7 (70), IL-9 (71), and
TNF- (57) enhance Ig production, while IFN-, IFN-
(51), TGF- (17, 57), IL-12
(72), and PGE2 (51, 73)
inhibit it. The ability to describe Ig isotype switching with reference
to division history now makes it possible to delineate whether this
array of factors modulates Ig secretion by affecting cell division, the
division-based rate of isotype switching, the amount of Ig secreted by
isotype-switched B cells, or a combination of these effects. A similar
approach may be applied to studying the pathogenesis of human diseases
involving defective or disturbed Ig production. One example is common
variable immunodeficiency (CVID), which is characterized by impaired Ab
responses in vivo and in vitro due to an intrinsic B cell defect
(49). Compared with B cells from healthy controls, the
production of Ig by CVID B cells in response to stimulation via CD40 in
the presence of IL-4, IL-10, or IL-13 was greatly reduced
(49). However, this defect could be overcome by the
combined stimulus of CD40, IL-4, and IL-10 (49). The
mechanism by which these cytokines correct the functional defect could
be resolved by studying Ig isotype switching of naive B cells from CVID
patients in relation to cell division. Furthermore, the ability to
examine isotype switching to IgG using cell division as a reference may
facilitate our understanding of the complex events involved in
switching to other Ig isotypes (IgA and IgE), Ig production, and the
overall regulation of the humoral immune response in health and in
conditions such as allergy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stuart G. Tangye, Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag Number 6, Newtown, New South Wales 2042, Australia. E-mail address: s.tangye{at}centenary.usyd.edu.au
3 Current address: Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia.
4 Abbreviations used in this paper: CD40L, CD40 ligand; CVID, common variable immunodeficiency; DC, dendritic cell; SA-FITC, streptavidin conjugated to FITC; SA-TC, streptavidin conjugated to Tricolor.
Received for publication June 24, 2002. Accepted for publication August 20, 2002.
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) were
cultured with CD40L, IL-4, and IL-10; after 6 days the percentage of
sIgG+ cells in each division was determined by division
slicing. The expression of IgG per division by sorted B cells was
compared with that of MACS-purified naive B cells (
) prepared from
the same donor spleen and cultured under the same conditions.
) or IL-10 (
). a and b,
The expression of sIgG by dividing B cells was determined by flow
cytometry. These contour plots are from cultures of B cells stimulated
with CD40L and IL-4 and are representative of three (a)
or two (b) independent experiments. c and
d, The percentage of sIgG+ cells in each
division was determined by division slicing. Each point in
c represents the mean ± SEM of three experiments
using cells from different donors; the data in d are
representative of two separate experiments using cells from the same
donor.
), or IgG4 (
