IL-21 Induces Differentiation of Human
Naive and Memory B Cells into
Antibody-Secreting Plasma Cells
This information is current as
of June 17, 2017.
Rachel Ettinger, Gary P. Sims, Anna-Marie Fairhurst, Rachel
Robbins, Yong Sing da Silva, Rosanne Spolski, Warren J.
Leonard and Peter E. Lipsky
J Immunol 2005; 175:7867-7879; ;
doi: 10.4049/jimmunol.175.12.7867
http://www.jimmunol.org/content/175/12/7867
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References
The Journal of Immunology
IL-21 Induces Differentiation of Human Naive and Memory B
Cells into Antibody-Secreting Plasma Cells1
Rachel Ettinger,2* Gary P. Sims,* Anna-Marie Fairhurst,* Rachel Robbins,*
Yong Sing da Silva,* Rosanne Spolski,† Warren J. Leonard,† and Peter E. Lipsky*
I
nterleukin-21 is a type I cytokine that signals through a receptor composing of the IL-21R and the common cytokine
receptor ␥-chain (␥C)3 (1–5). In humans, IL-21 has been
shown to be produced by activated peripheral T cells, as well as
spontaneously by CXCR5⫹ follicular helper T cells (2, 6). The
IL-21R is closely related to the IL-2R ␤-chain and is expressed by
T, B, and NK cells (1, 2). Unexpectedly, IL-21R-deficient mice
exhibited a severe defect in IgG1 production following Ag priming
(7), demonstrating its importance in murine B cell differentiation.
In this regard, IL-21 exerts a number of activities on B cell function, including apoptosis, growth arrest, or costimulation depending on the nature of the activation signals provided to B cells (8,
9). For example, IL-21 enhances anti-CD40 stimulation but inhibits anti-IgM and IL-4 responses in both mouse and human (2, 7,
10). These effects appear to relate to the capacity of IL-21 to induce apoptosis of resting and anti-IgM-stimulated murine B cells,
whereas it paradoxically promotes differentiation of anti-CD40stimulated murine splenic B cells into postswitch memory and
*Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin
Diseases and †Laboratory of Molecular Immunology, National Heart, Lung, and
Blood Institute, National Institutes of Health, Bethesda, MD 20892
Received for publication March 28, 2005. Accepted for publication September
29, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by the Intramural Research Program of the National
Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin
Diseases.
2
Address correspondence and reprint requests to Dr. Rachel Ettinger, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of
Health, Building 10, Room 6D-47B, Bethesda, MD 20892. E-mail address:
[email protected]
3
Abbreviations used in this paper: ␥C, common cytokine receptor ␥-chain; CSR, class
switch recombination; AID, activation-induced cytidine deaminase; BLIMP-1, B
lymphocyte-induced maturation protein-1; SHM, somatic hypermutation; PB, peripheral blood; SAC, Staphylococcus aureus Cowan I; PI, propidium iodide; HU, hydoxyurea; ␤2M, ␤2 microglobulin; int, intermediate.
Copyright © 2005 by The American Association of Immunologists, Inc.
plasma cells (10). Taken together, these data indicate that IL-21 is
a critical, although complex, regulator of B cell function in the
mouse, although less is known about its activities in humans.
Regulation of the differentiation of human naive B cells into
memory and plasma cells has not been as extensively characterized
as that of their murine counterparts. However, a variety of studies
have suggested that cytokines, such as IL-2, IL-6, and IL-10 are
involved in human plasma cell differentiation (11–21). Moreover,
in vitro and in vivo analyses have identified cellular interactions,
including those mediated by CD40 and its ligand, CD154, as playing pivotal roles in the generation of both memory B cells and
plasma cells (22–24). Nonetheless, responsiveness of B cells and
particularly naive B cells is minimal in the presence of purified
costimulators (21, 25–28). The specific signals that drive the differentiation of human naive B cells into memory cells and plasma
cells, therefore, have not been completely defined, and an integrated view of these essential steps in human B cell biology has
not been definitively established.
Because IL-21 plays an important role in murine B cell differentiation into memory cells and plasma cells, we investigated
the role of IL-21 in human B cell differentiation. In this study,
we report that IL-21 costimulation not only is capable of inducing plasma cell differentiation from CD27⫹ memory B cells,
but also has the capacity to induce class switch recombination
(CSR) and stimulate poorly responsive naive cord blood B cells
into IgG-secreting plasma cells. Importantly, IL-21 costimulation up-regulated expression of both activation-induced cytidine deaminase (AID) and B lymphocyte-induced maturation
protein-1 (BLIMP-1), but did not induce somatic hypermutation
(SHM). Finally, the action of IL-21 was largely prevented by
another type I cytokine, IL-4. These results demonstrate that
IL-21 is one of the major T cell influences that initiates CSR
and differentiation of human B cells into Ab-secreting plasma
cells. Moreover, the interplay of IL-21 and IL-4 may exert an
essential function in determining the outcome of human humoral immune responses.
0022-1767/05/$02.00
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IL-21 is a type I cytokine that influences the function of T cells, NK cells, and B cells. In this study, we report that IL-21 plays
a major role in stimulating the differentiation of human B cells. When human B cells were stimulated through the BCR, IL-21
induced minimal proliferation, IgD down-modulation, and small numbers of plasma cells. In contrast, after CD40 engagement,
IL-21 induced extensive proliferation, class switch recombination (CSR), and plasma cell differentiation. Upon cross-linking both
BCR and CD40, IL-21 induced the largest numbers of plasma cells. IL-21 drove both postswitch memory cells as well as poorly
responsive naive cord blood B cells to differentiate into plasma cells. The effect of IL-21 was more potent than the combination
of IL-2 and IL-10, especially when responsiveness of cord blood B cells was examined. IL-21 costimulation potently induced the
expression of both B lymphocyte-induced maturation protein-1 (BLIMP-1) and activation-induced cytidine deaminase as well as
the production of large amounts of IgG from B cells. Despite the induction of activation-induced cytidine deaminase and CSR,
IL-21 did not induce somatic hypermutation. Finally, IL-2 enhanced the effects of IL-21, whereas IL-4 inhibited IL-21-induced
plasma cell differentiation. Taken together, our data show that IL-21 plays a central role in CSR and plasma cell differentiation
during T cell-dependent B cell responses. The Journal of Immunology, 2005, 176: 7867–7879.
7868
IL-21 DRIVES HUMAN PLASMA CELL DIFFERENTIATION
Materials and Methods
absorbance was measured, and OD was quantified at 410 nM by a Powerwave X 96-well plate reader (Bio-Tek Instruments). IgE concentrations
were measured with the Human IgE ELISA kit (Bethyl Laboratories). Specific IgG isotypes were differentiated using the Human IgG Subclass Profile ELISA KIT (Zymed Laboratories).
Isolation of human B cells
All human studies have been approved by the Warren G. Magnuson Clinical Center Institutional Review Board, and informed consent was obtained
according to the declaration of Helsinki. Human peripheral B cells were
isolated from buffy coats of anonymous healthy donors drawn at the National Institutes of Health Division of Transfusion Medicine. Umbilical
cord blood samples were obtained from Advanced Bioscience Resources.
For most studies, human cord blood and peripheral B cells were isolated by
negative selection using the Rosette-sep technique following the manufacturer’s instructions (StemCell Technologies). Preparations were typically
⬎96% CD19⫹ from peripheral blood (PB). However, negatively selected
cord blood B cell isolations yielded less pure cell populations. Thus, we
positively selected B cells with anti-CD19 magnetic beads (Miltenyi Biotec) in some of the cord blood experiments, and these were typically ⬎95%
pure. For further purity of peripheral B cells, CD20⫹CD27⫺ naive B cells
or CD20⫹CD27⫹ memory B cells were isolated on a MoFlo cell sorter
(DakoCytomation). Briefly, negatively selected B cells were incubated at
107 cells/ml in staining buffer with mAbs specific for CD20 and CD27 and
in some experiments anti-IgD as well. Cells were incubated for 30 min at
4°C, washed, and sorted into CD20⫹CD27⫹ or CD20⫹CD27⫺ subsets.
Preparations were typically ⬎98% pure.
Purified B cells were cultured at 1 ⫻ 106 cells/ml in either 1 ml in 24-well
culture plates or 100 ␮l in 96-well round-bottom culture plates. The cells
were incubated with a combination of human IL-2 (100 U/ml; Roche),
human IL-4 (100 ng/ml; R&D Systems), human IL-21 (100 ng/ml; R&D
Systems and BioSource International), human IL-10 (25 ng/ml; R&D Systems), and either 1 ␮g/ml anti-human CD40 (R&D Systems), 5 ␮g/ml
anti-IgM (Jackson ImmunoResearch Laboratories), or 0.01% final dilution
of heat-killed, formalin-fixed Staphylococcus aureus Cowan I (SAC) (Calbiochem). When multiple cytokines and stimuli were used, all cytokines
and stimuli were added at the initiation of culture. In some experiments,
purified B cells were labeled with CFSE. In brief, purified B cells were
washed extensively in PBS to remove all FCS, and CFSE/PBS (Molecular
Probes) was added at a final concentration of 2.5 ␮M to 2–5 ⫻ 107 cells/ml
for 8 min. Labeling was quenched by addition of FCS, and cells were
washed four times in medium containing 10% FCS before culture. To
obtain an accurate comparison for the controls, cord blood B cells were
stimulated in the presence of IL-4, which facilitated cell survival but induced neither IgD down-modulation or plasma cell differentiation.
Flow cytometry
Four-color flow cytometry was performed using a FACSCalibur (BD Biosciences). Briefly, supernatants were collected, and then all cells were harvested from 96-well cultures at the end of the incubation period and stained
for 30 min on ice with a combination of mAbs (BD Biosciences). The
combination of anti-IgD-FITC, anti-CD19-PerCP-Cy5.5, and anti-CD38allophycocyanin (clone HB7) was routinely used with either PE-conjugated Abs to CD27, CD40, CD95, HLA-DR, CD9, CD63, CD49D,
CD62L, CXCR4 (all obtained from BD Biosciences), or IL-6R-biotin
(Bender MedSystems) and B cell maturation Ag (BCMA)-biotin (R&D
Systems) followed by streptavidin-PE. In addition, the combination of antiCD31-FITC, anti-IgD-PE, anti-CD19-PerCP-Cy5.5, and anti-CD38-APC,
or anti-IgD-FITC, anti-CD38-PE, anti-CD19-PerCP-Cy5.5, and antiCD44-APC were also used (all obtained from BD Biosciences). Viable
cells were identified by gating on lymphocytes, and cells were analyzed
immediately. All samples were collected for 1 min, and as a result the
density of the dot plots reveals relative cell numbers. In some experiments,
to obtain total B cell numbers more accurately, AccuCount Particles
(Spherotech) were added before analyzing samples by flow cytometry, and
total cell numbers were determined as per the manufacturer’s instructions.
In some experiments, the combination of CFSE, anti-IgD-PE, anti-CD19
PerCP-Cy5.5, and anti-CD38-APC was used.
Determination of Ig levels
Secreted Ig in the culture supernatant was quantitated by ELISA. Briefly,
96-well flat-bottom Nunc-Brand Immuno plates (Nalge Nunc International) were coated with 5 ␮g/ml of either affinity-purified goat anti-human
IgM or goat anti-human IgG-Fc (Bethyl Laboratories) overnight at 4°C.
Wells were then washed and blocked with a 0.2% BSA/PBS, and then the
culture supernatants were titered onto the treated plates and incubated overnight at 4°C. Bound Ig was detected with 0.5 ␮g/ml alkaline phosphataseconjugated goat anti-human IgM or IgG (Bethyl Laboratories) and developed with p-nitrophenyl phosphate tablets (Sigma-Aldrich). Specific
To assess proliferative responses of cultured cells, 105 purified B cells were
cultured as described above in 96-well round-bottom plates. After 3–5 days
of culture, [3H]thymidine (37 Kbq/well) was added to the cultures for an
additional 16 h. Thymidine uptake was measured using a liquid scintillation counter. To assess whether IL-21-induced plasma cells were in cell
cycle, two strategies were used. First, propidium iodide (PI) incorporation
was used to determine the cell cycle position of the cells. Stimulated and
control cultures were stained after various days with either anti-IgD-FITC,
anti-CD19 PerCP-Cy5.5, and anti-CD38-APC to visualize plasma cells
based on IgD and CD38 expression or anti-CD27-FITC and anti-CD38APC, followed by 1% paraformaldehyde solution for 15 min, followed by
the addition of 100 ␮g/ml PI solution in PBS with 0.1% Triton X-100 for
at least 1 h before analyses. Second, hydroxyurea (HU) (Sigma Chemicals)
was added to some cultures at a final concentration of 10⫺2 M at either day
0 (that were subsequently either pulsed with [3H]thymidine after 3 days or
stained for flow cytometric analysis or analyzed for IgG production after 8
days) or at 8 days of culture (that were subsequently either stained for flow
cytometry or analyzed for IgG production at 11 days of culture). This
concentration of HU has been shown to completely inhibit the proliferation
of activated cycling human B cells (17).
Real-time quantitative PCR
Negatively or positively purified B cells were stimulated as described
above. After 3 days in culture, cells were harvested and resuspended in
TRIzol (Invitrogen Life Technologies) and stored at ⫺70°C. RNA was
isolated using the RNeasy mini kit (Qiagen). Purity was measured using
spectrophotometry. Reverse transcription reactions were prepared using the
SuperScript One-Step PCR System with Platinum Taq Polymerase and
ROX reference dye (Invitrogen Life Technologies). Fifty nanograms of
isolated RNA was added per reaction with 1.2 mM MgSO4. TaqMan Assays-on Demand Gene expression primer/probe sets (Applied Biosystems)
were used for BLIMP-1 (Hs00153357_m1), Bcl6 (Hs00153368_m1),
AICDA (Hs00221068_m1), PAX-5 (Hs00277134_m1), and ␤2 microglobulin (␤2M) (Hs99999907_m1). Final concentrations were 1.8 ␮M for
primers and 0.5 ␮M for probes. RT-PCR was performed using the ABI
Prism 7700 Sequence Detection System (Applied Biosystems), and cycle
conditions and relative quantification were completed as described by manufacturer’s instructions (Applied Biosystems). Expression of each transcription factor was calculated using the comparative computerized tomography method with efficiency calculations and with all mRNA levels
FIGURE 1. IL-21 induces plasma cell differentiation after stimulation
with anti-CD40 and anti-IgM. Purified PB B cells were stimulated with no
polyclonal B cell activator or with the combination of anti-CD40 and antiIgM in the presence or absence of IL-21 as indicated. After 6 days of
culture, B cells were identified based on CD19 expression and assessed for
expression of IgD and CD38. IgD⫺CD38high plasma cells are present in the
lower right quadrant of anti-CD40, anti-IgM, and IL-21-stimulated cultures. The numbers in the quadrants indicate the percentage of CD19⫹ cells
in each region. Cell surface phenotype of B cells before culture is also
shown (day 0).
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Cell culture, activation, and CFSE labeling
B cell proliferation and cell cycling
The Journal of Immunology
7869
normalized to ␤2M. All reported values were then further normalized to
control conditions, of cultures from PB B cells with no cytokine and no
stimuli or cultures from cord blood B cells with IL-2 (Biological Research
Branch, Division of Cancer Treatment and Diagnosis, National Cancer
Institute, Frederick, Maryland) and IL-4 (R&D Systems) without any stimuli, as a value of 1.
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FIGURE 2. IL-21 induces plasma cell differentiation.
A, Purified PB B cells stained with anti-IgD and anti-IgM
contain few (1.3%) IgD⫺CD38high plasma cells. B–D, Purified PB B cells were stimulated with anti-IgM, antiCD40, both stimuli, or neither in the presence or absence
of IL-2 and/or IL-21 as indicated. B, After 3 days of culture, proliferation was determined by incubating the cells
for 16 h with [3H]thymidine. C, After 6 days of culture,
the cells were stained and B cells identified based on
CD19 and CD38 expression as shown in Fig. 1, and analyzed for surface expression of IgD and CD38. Results
from a representative experiment of six similar experiments are shown. D, Purified B cells were first labeled
with CFSE before being cultured. After 7 days of culture,
CD19⫹ B cells were analyzed for CD38 expression and
CFSE dilution. Data are representative of results from
three similar experiments.
naive B cells in multiple individuals were considered putative polymorphic
variants and were excluded from the analysis. Data obtained from plasma
cells derived from anti-CD40 and anti-CD40 and anti-IgM-stimulated cultures were similar, and their data were pooled for the analysis.
Results
Somatic hypermutation
IL-21 induces plasma cell differentiation
B cells enriched from four cord blood samples were stimulated with IL-2
and IL-21 in the presence of anti-CD40 or anti-CD40 and anti-IgM for
7–12 days. Individual live, CD19⫹IgD⫺CD38high plasma cells were sorted,
and the rearranged Ig H chain V regions (VH) from genomic DNA of single
cells was amplified and directly sequenced as described previously (29).
The VH gene sequences from these IL-21-induced plasma cells, and unstimulated cord blood B cells collected immediately after purification, were
compared with germline genes and their polymorphic variants from our
own and public databases used to determine the closest germline gene and
the number of VH gene segment mismatches. Several undocumented but
recurring VH gene mismatches that we have found in genes isolated from
The capacity of IL-21 to costimulate responses of purified human
B cells was initially explored. Freshly obtained B cells or those
retrieved from cultures were stained with anti-CD19, anti-IgD, and
anti-CD38. CD19⫹ cells were gated and assessed for expression of
IgD and CD38 as shown in Fig. 1. Fresh CD19⫹ B cells or those
cultured without stimulation or with the combination of anti-CD40
and anti-IgM in the absence of cytokines contained few
IgD⫺CD38high plasma cells. In contrast, costimulation of purified
PB B cells with anti-CD40 and anti-IgM in the presence of IL-21
7870
IL-21 induces plasma cell differentiation from naive cord blood
B cells
CD27 expression denotes that human B cells have somatically mutated Ig genes and, therefore, are considered to be memory B cells
FIGURE 3. Large numbers of plasma cells are induced following coculture with IL-21. A, Purified PB B cells were cultured as described in Fig.
2, and the mean percentage (⫾SEM) of plasma cells from seven independent experiments is shown. B, The absolute plasma cell number following
7 days in culture from one representative experiment is also shown. There
was approximately a total of 500 plasma cells per well in the initial population of cells.
(31). These include both IgD⫹CD27⫹ B cells as well as
IgD⫺CD27⫹ postswitched B cells. To address whether IL-21 had
the capability to drive plasma cell differentiation from both naive
and memory B cells, cord blood B cells were used as a natural
source of naive CD27⫺IgD⫹ B cells, and CD27⫹ (IgD⫺ and
IgD⫹) memory B cells were isolated from PB by cell sorting. As
shown in Fig. 4, A and B, IL-21 costimulated considerable proliferation of both naive cord blood and memory B cells. Although
cord blood B cells costimulated with IL-21 proliferated less well
than adult CD27⫹ memory B cells (compare Fig. 4, B to A), the
capacity of IL-21 to down-modulate IgD and induce differentiation
of plasma cells was dramatic in both populations (Fig. 4, C and D).
IL-21 with anti-CD40 or anti-IgM and anti-CD40 (but not with
anti-IgM alone) had the capacity to induce the generation of
plasma cells from nearly all the CD27⫹IgD⫺ and CD27⫹IgD⫹
memory B cells (Fig. 4D). More notable was the finding that
nearly all surviving IL-21-stimulated cord blood B cells activated
with either anti-CD40 or anti-IgM and anti-CD40 had down-modulated IgD and most differentiated into plasma cells. It is important
to note that neither anti-CD40 nor anti-IgM and anti-CD40 stimulation of cord blood B cells alone led to the generation of any
plasma cells in the absence of IL-21 (Fig. 4C). However, IL-21
induced the down-modulation of IgD on stimulated cord blood B
cells within 3 days of culture (data not shown), and by day 6 many
plasma cells were observed (Fig. 4C). In contrast, IL-21-induced
differentiation of plasma cells from adult PB B cells could be observed within 4 days of culture (data not shown).
IL-21 induces Ig secretion from both naive and memory B cells
Analysis of culture supernatants confirmed that IL-21 induced the
secretion of Ig. Unlike IL-2, or cultures with no added cytokines,
IL-21 costimulated considerable production of both IgG and IgM
from total adult peripheral B cells, as well as naive cord blood B
cells activated with anti-CD40 or anti-IgM and anti-CD40 (Fig. 5,
A and B). The amount of Ig produced generally correlated with the
frequency of plasma cells in the cultures. IgM and IgG could be
assayed in the culture supernatants as early as day 4 of culture
(data not shown). It is notable that IL-21 induced large amounts of
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resulted in marked down-modulation of IgD and substantial differentiation of plasma cells that were phenotypically identified as
CD19low/⫹IgD⫺CD38high cells (Fig. 1).
The next experiments examined the impact of IL-21 in detail
and compared it with another type I cytokine that has been reported to support Ig production from human B cells, IL-2 (30). As
shown in Fig. 2A, only a small fraction (1.3%) of freshly isolated
peripheral B cells are IgD⫺CD38high plasma cells; the majority are
IgD⫹CD38low/int naive B cells, and a smaller percentage are
IgD⫺CD38⫺/low postswitched memory B cells. In the presence of
anti-CD40 (which can stimulate both naive and memory B cells),
IL-21 induced maximal proliferation of B cells, whereas IL-2 had
little effect (Fig. 2B). In contrast, in the presence of anti-IgM
(which stimulates IgD⫹/IgM⫹ B cells only), IL-21 induced only
minimal proliferation. Notably, IL-2 enhanced the proliferation of
B cells stimulated with anti-IgM and IL-21, whereas it had little
effect on B cells stimulated with anti-CD40 and IL-21. When B
cells were triggered through both CD40 and IgM, IL-21 induced a
proliferative response that was comparable to that noted with antiCD40 and IL-21. As with the anti-IgM and IL-21 costimulation,
IL-2 also increased the magnitude of the response when cells were
stimulated with the combination of IL-21, anti-IgM, and
anti-CD40.
Flow cytometric evaluation was conducted to assess the impact
of IL-21 on human B cells in greater detail. When negatively selected peripheral B cells were cultured with cytokines alone, modest changes in B cell phenotype were noted as determined by IgD
and CD38 expression (Fig. 2C, a– d). It was notable, however, that
IL-21 in the absence of any other stimulus increased the percentage of IgD⫺CD38high plasma cells modestly, and this effect was
augmented by IL-2 (Fig. 2C, c and d). The ability of IL-21 to
induce moderate plasma cell differentiation was also observed with
anti-IgM-stimulated B cells, and again there was an increase in the
presence of IL-2 (Fig. 2C, g and h). In addition, IL-21 induced a
striking loss in the numbers of B cells in anti-IgM-stimulated cultures (Fig. 2C, compare g to e), which was largely reversed by the
addition of IL-2 (Fig. 2C, compare h to g). Moreover, IL-21 or the
combination of IL-2 and IL-21 induced marked down-regulation
of surface IgD by anti-IgM-stimulated B cells (Fig. 2C, g and h).
IL-21 also induced IgD down-modulation by antiCD40-stimulated B cells, as well as a dramatic increase in cellular
expansion and plasma cell differentiation that was observed as
early as day 4 of culture (Fig. 2C, compare k and l to i and j and
s). T cell-dependent B cell responses involve engagement of both
surface Ig as well as CD40 by CD154 expressed by activated T
cells. It was therefore of interest to examine the impact of IL-21 on
B cells stimulated through both the BCR and CD40. We found that
the combination of anti-IgM and anti-CD40 in the presence of
IL-21 resulted in down-modulation of IgD by nearly all B cells
(Fig. 2C, compare o to m). Moreover, the largest percentage of
plasma cells was generated when both surface IgM and CD40 were
engaged, and the cells were costimulated with IL-21 in the presence or absence of IL-2 (Fig. 2C, o and p). Importantly, evaluation
of CFSE dilution revealed that IL-21 induced the differentiation of
plasma cells from a dividing B cell precursor that had diluted
CFSE with or without costimulation (Fig. 2D). Numeric calculation in repetitive experiments demonstrated the significant increase
in both the percentage (Fig. 3A) and absolute cell number (Fig. 3B)
of plasma cells in cultures costimulated with IL-21.
IL-21 DRIVES HUMAN PLASMA CELL DIFFERENTIATION
The Journal of Immunology
7871
IgG from both naive CD27⫺ adult and cord blood B cells (Fig. 5,
B and C), consistent with its capability to function as a switch
recombination factor for human B cells. All IgG isotypes were
produced from adult total PB B cells stimulated with anti-CD40
and anti-IgM in the presence of IL-21, whereas cord blood B cells
produced largely IgG3. Moreover, CD27⫺ naive adult B cells produced both IgG1 and IgG3 (Fig. 5C). A similar pattern was also
noted when the various B cell populations were stimulated with the
combination of IL-2, IL-21, anti-CD40, and anti-IgM (Fig. 5C),
indicating that IL-2 exerted no isotype specificity as shown previously (30). In contrast, IL-21 influenced naive B cells at different
stages of maturation to switch to specific Ig isotypes, IgG3 for cord
blood B cells and IgG1 and IgG3 for adult naive B cells. In addition, stimulation with IL-21 and anti-CD40 or IL-21, anti-CD40
and anti-IgM of PB B cells had no effect on IgE production (data
not shown), but did costimulate IgA production without or with
IL-2 (1–2 ␮g/ml vs 4 – 6 ␮g/ml IgA, respectively).
IL-21 induces the differentiation of both cycling and terminally
differentiated cells
We next addressed the state of the IL-21-induced plasma cells both
by cell surface phenotype and cell cycle analysis. In addition to
being CD19low/⫹IgD⫺CD38high, the majority of IL-21, anti-CD40,
and anti-IgM-induced plasma cells (red) were also found to express other typical plasma cells markers such as IL-6R and B cell
maturation Ag (BCMA) (Fig. 6). Furthermore, when compared
with IgD⫺CD38low cells (blue), these plasma cells were CD40⫹,
CD95low, HLA-DRlow, CD9high, CD63high, CXCR4low/⫹,
CD44high, CD49dlow, CD31high, and CD62Lhigh (Fig. 6). The majority of the IL-21-driven plasma cells were also CD19low,
CD20low, CD22low, CD21low, and CD27high (data not shown).
To evaluate whether IL-21-induced plasma cells were terminally differentiated nondividing cells, cell cycle analysis was also
conducted. To assess cell cycle position, B cells were stimulated
with IL-21, anti-IgM, and anti-CD40 and after 7 days of culture the
resultant plasma cells were identified either as IgD⫺CD38high (Fig.
7B) or CD27highCD38high cells that were differentiated from
CD27lowCD38low nonplasma cells (Fig. 7C). Both populations
were analyzed for cell cycle progression by PI staining (Fig. 7D).
As shown in Fig. 7D, approximately one-third of IL-21-induced
plasma cells were cycling, whereas the majority were not cycling
as evidenced by the finding that they were not in the S, G2, or M
phase of the cell cycle. It is notable that approximately one-fourth
of the other B cells in the culture were in cell cycle after 7 days of
culture, although 46% of these nonplasma cells were found to be
in S/G2/M on day 3, before plasma cell differentiation (data not
shown).
Finally, to address whether IL-21-induced plasma cells had differentiated to nondividing Ig-secreting cells, Ig production was
examined in the presence of the inhibitor of proliferation, HU. HU
is an inhibitor of proliferation that prevents Ig secretion by cycling
but not noncycling Ig-secreting cells (17, 32). The addition of HU
at the initiation of culture resulted in a complete block of IL-21induced proliferation and plasma cell differentiation (Fig. 8, A and
B). However, when HU was added to the cultures at day 8 after
differentiation of plasma cells, persistence of these cells was noted
over the next 3 days of culture (Fig. 8B). Importantly, the amount
of Ig doubled during the last 3 days of culture, which was not
blocked by HU, indicating that the ongoing production of Ig by
plasma cells was not dependent on ongoing proliferation. These
data demonstrate that the production of Ig by IL-21-induced
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FIGURE 4. IL-21 induces CSR and plasma cell differentiation from both naive and memory B cells. B cells
were positively selected from cord blood, or negatively
selected from PB, and the latter were further purified into
CD20⫹CD27⫹ memory B cells by cell sorting. All cells
were cultured with anti-IgM, anti-CD40, both stimuli, or
neither in the presence or absence of IL-21. B cell populations were stimulated as indicated, and proliferative responses were measured by [3H]thymidine incorporation
after 3 days in culture of either cord blood B cells (A) or
CD27⫹ memory B cells (B). The purity of the cord blood
and CD20⫹CD27⫹ memory B cells before culture is
shown in (C and D). Neither cord blood B cells nor
CD20⫹CD27⫹ memory cells contained identifiable
plasma cells before culture. After 6 days of culture, the
cells were stained and B cells identified based on CD19
and CD38 expression as shown in Fig. 1, and analyzed for
IgD and CD38 expression. No difference was observed in
proliferation or change in phenotype of cord blood B cells
isolated by negative vs positive selection. Data are representative of results from one of six experiments for purified cord blood B cells and one of five similar experiments
for CD27⫹-sorted PB B cells.
7872
IL-21 DRIVES HUMAN PLASMA CELL DIFFERENTIATION
plasma cells largely reflects secretory activity of terminally differentiated, nondividing cells (Fig. 8C).
IL-21 induces expression of BLIMP-1, AID, and Bcl-6 mRNA,
but not SHM
A variety of transcription factors are known to regulate specific
stages of B cell maturation. It was, therefore, of interest to determine whether IL-21 costimulation would up-regulate expression of
BLIMP-1, which is essential for plasma cell differentiation (33),
AID, which is involved in CSR (34), Bcl-6, which is involved in
germinal center reactions (35), or PAX-5, which is required for the
generation of B cells (36). IL-21 induced expression of both
BLIMP-1 (Fig. 9A, a) and AID (Fig. 9A, b) mRNA. BLIMP-1 was
induced by IL-21-costimulated B cells activated with anti-CD40 or
anti-IgM and anti-CD40, and less so with anti-IgM only (Fig. 9A,
a). However, in the presence of IL-2, IL-21 also induced BLIMP-1
expression without additional costimulation. AID mRNA was induced by IL-21 when B cells were activated with anti-IgM, antiCD40, or both anti-IgM and anti-CD40, and was enhanced by the
presence of IL-2. Bcl-6 mRNA was induced to a lesser degree by
IL-21 (Fig. 9A, c), whereas IL-21 had little effect on PAX-5
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FIGURE 5. IL-21 induces robust secretion of IgG and
IgM. Purified B cells were cultured with either anti-IgM,
anti-CD40, both stimuli, or neither in the presence or absence of IL-2, IL-21, or both IL-2 and IL-21. Cell supernatants were removed after 10 –11 days of culture (A and
B) or after 6 days (or 8 –12 days; data not shown) for cord
blood and 12 days for total PB or CD27⫺ and CD27⫹ B
cells (C). Production of total IgG or IgM from purified
total peripheral B cells (A) or cord blood B cells (B). Secretion of specific Ig isotypes from naive cord blood B
cells (CB) (C) as well as from PB naive (CD27⫺), memory (CD27⫹), and total (PB) B cells were quantitated. A
and B, IgM levels were not measured in any cultures containing anti-IgM. Data are mean concentration (⫾SD)
from one of seven and six representative experiments, for
A and B, respectively, and one representative experiment
of nine cord blood and three with CD27⫺ and CD27⫹
adult peripheral B cells (C).
mRNA (Fig. 9A, d). To determine whether IL-21 had the ability to
induce these transcription factors in naive B cells, experiments
using cord blood B cells were undertaken. To obtain sufficient
mRNA for analysis, cord blood B cells were cultured with viability
promoting cytokines, IL-2 and IL-4, which in preliminary experiments did not induce the mRNAs analyzed. IL-21 (in the presence
of IL-2) induced a significant increase in BLIMP-1 mRNA in naive anti-CD40 or anti-CD40 and anti-IgM-stimulated cord blood B
cells, whereas the combination of IL-2 and IL-4 exhibited no such
activity (Fig. 9B). IL-21 also costimulated AID mRNA expression,
but no more effectively than the combination of IL-2 and IL-4 (Fig.
9B). In contrast, IL-21 had little effect on Bcl-6 or PAX-5 mRNA
levels.
It was notable that IL-21 costimulation increased AID mRNA
levels, differentiation of IgD⫹ and IgD⫺ B cells, CSR, generation
of plasma cells, and Ig production, but it could not induce SHM.
Compared with unstimulated cord blood B cells, no significant
increase in the percentage of sequences with VH gene mismatches
was detected in isolated IgD⫺CD38high plasma cells induced from
cord blood B cells stimulated with IL-2 and IL-21 in the presence
of anti-CD40 or anti-CD40 and anti-IgM (Fig. 9C). The small
The Journal of Immunology
7873
IL-21 induces greater plasma cell differentiation than does the
combination of IL-2 and IL-10
The next experiments compared the impact of IL-21 with that of
the combination of IL-2 and IL-10 that has been shown to support
plasma cell differentiation under some circumstances (13, 20). As
shown in Fig. 11, stimulation of purified PB B cells with antiCD40 and the combination of IL-2 and IL-10 induced proliferation
(Fig. 11A), as well as the differentiation of plasma cells (Fig. 11B)
and IgG secretion (Fig. 11C), although the responses were not as
robust as those induced by anti-CD40 and IL-21. In contrast, stimulation of naive cord blood B cells with anti-CD40 and the combination of IL-2 and IL-10 failed to stimulate any of these responses, although IL-21 induced proliferation, plasma cell
generation, and IgG secretion from anti-CD40-stimulated cord
blood B cells (Fig. 11, A–C). Notably, however, the combination
of IL-2 and IL-10 induced up-regulation of both BLIMP-1 and
AID mRNA, although AID mRNA was induced substantially more
effectively by IL-21 (Fig. 11D).
FIGURE 6. Cell surface phenotype of IL-21-induced plasma cells. Purified PB B cells were isolated and cultured with IL-21, anti-IgM, and
anti-CD40. After 7 or 8 days of culture, CD19⫹ cells were subdivided into
IgD⫺CD38low cells (blue histogram) and IgD⫺CD38high plasma cells (red
histogram) and further analyzed for expression of the markers indicated.
The data are representative of results from one of two experiments.
percentage of sequences with mismatches most likely reflected unidentified VH gene polymorphisms or Taq-induced PCR misincorporation events, rather than bona fide somatic mutations.
IL-4 suppresses plasma cell generation promoted by IL-21
The effects of IL-21 can be modulated by other cytokines. As
shown in Fig. 10A, IL-4 partially inhibited the down-modulation of
surface IgD induced by IL-21 in cells stimulated with the polyclonal B cell activator, SAC (Fig. 10A, compare e to f). Moreover,
IL-4 also inhibited IgD down-modulation induced by IL-21 and
anti-IgM or the combination of anti-IgM and anti-CD40 (Fig. 10A,
compare h and n to i and o, respectively), but not by anti-CD40
alone (Fig. 10A, compare l to k). Importantly, IL-21-induced generation of plasma cells was also inhibited by IL-4 in cultures costimulated by SAC (Fig. 10A, compare e to f), or in cultures containing the combination of IL-21, anti-IgM, and anti-CD40 (Fig.
10A, compare n to o) but not in cultures of IL-21 and anti-CD40
(Fig. 10A, compare k to i) reproducibly. This was reflected in the
quantity of secreted Ig (Fig. 10B), as well as the levels of AID or
BLIMP-1 mRNA (Fig. 10C). Thus, IL-4 inhibited IL-21-induced
up-regulation of Ig production as well as BLIMP-1 and AID mRNAs in cultures stimulated by SAC or anti-CD40 with anti-IgM,
but not reproducibly in those stimulated by anti-CD40. Notably,
IL-2 did not reproducibly affect Ig production or BLIMP-1 or AID
mRNA levels in these cultures. Finally, the addition of IL-2 did not
rescue IgG production in IL-4-suppressed cultures (Fig. 10B), but
did allow for the differentiation of IgM-producing plasma cells
(data not shown).
During T cell-dependent responses in a germinal center, naive B
cells receive a combination of signals from encounter with Ag and
T cells that result in clonal expansion, CSR, SHM, and the differentiation of memory and plasma cells. Studies in the mouse have
defined a number of these specific processes and factors. Recently,
IL-21 has been shown to be an essential factor in the generation of
plasma cells that produce IgG (10). Less is known about the control of these processes in humans. Although a number of cytokines
and T cell influences are known to regulate B cell responses, the
specific factors that stimulate the differentiation of naive human B
cells into plasma cells secreting IgG have not been well defined.
Indeed, both adult and especially cord blood naive human B cells
have been shown to have a poor propensity to differentiate into
plasma cells in vitro, especially in the presence of purified costimulators (11, 13, 21, 25, 27). However, a modest capacity to
differentiate into Ig-producing plasma cells has been reported
when naive B cells are stimulated through CD40, in the presence
of L cells, and supported by IL-10 (12) or activated by the combination of SAC, IL-2, IL-10, and cross-linked anti-CD40 (18).
Others have found no plasma cell generation when naive CD27⫺
B cells are stimulated with CD40L transfectants in combination
with IL-2, IL-4, IL-6, IL-10, and IL-12 (21).
We now show that IL-21 is a powerful costimulator of human
CSR and plasma cell differentiation, but not SHM. Importantly,
IL-21 can promote both CSR and plasma cell differentiation from
naive as well as memory B cells. Notably, the combination of
anti-IgM and anti-CD40, which most closely mimics B cell activation via Ag and T cell interaction, was the most effective signal
in promoting the maximal differentiation of plasma cells driven by
IL-21. Although anti-CD40 and IL-21 induced considerable CSR
and plasma cell differentiation, the “T cell only” signal was somewhat less effective than the combination. Isolated BCR cross-linking, which mimics the “Ag-only” signal, primed B cells for IL21-mediated death (Fig. 1C, g, and our unpublished observations).
These results suggest that IL-21 is pivotal in cell fate decisions by
activated B cells and may function to eliminate B cells that have
been activated by Ags or autoantigens in the absence of T cell
signals.
IL-21 has been reported to be a switch factor for IgG1 and IgG3
(26), although in this study we found that the nature of the B cell
population contributed to the specific Ig produced. Thus, IL-21stimulated cord blood B cells predominantly switched to IgG3,
whereas naive adult B cells switched to both IgG1 and IgG3. The
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Discussion
7874
IL-21 DRIVES HUMAN PLASMA CELL DIFFERENTIATION
molecular basis for the differences in these outcomes is currently
unknown, but clearly the nature of the B cell population contributes to the specificity of switch recombination. Notably, memory B
cells were induced by IL-21 to produce all IgG isotypes as well as
IgA. This is somewhat different from previous results with CD19⫹
splenic B cells in which IgA was not induced by IL-21 (26). The
explanation for this difference is currently unknown. This may
reflect differences in peripheral and splenic B cells.
The combination of IL-2 and IL-10 has been reported to induce
human plasma cell differentiation from memory, but not naive B
cells (13, 20, 25). In contrast to IL-21, a variety of other cytokines,
including IL-2, IL-4, IL-6, and IL-10 (data not shown), had minimal ability to support the generation of plasma cells from comparably stimulated B cells. Importantly, although the combination
of IL-2, IL-10, and anti-CD40 did induce some plasma cell differentiation from PB B cells, this stimulus could not induce plasma
cell differentiation from naive cord blood B cells and has been
reported previously (25). These data indicate that IL-21 was a far
more potent inducer of plasma cell differentiation than the combination of IL-2 and IL-10 and may be uniquely able to foster
differentiation of plasma cells from naive B cells.
BLIMP-1 is a transcriptional repressor that is necessary/sufficient for plasma cell differentiation in the mouse, and in human B
cell lines (33, 37, 38). Consistent with this, IL-21-driven plasma
cell differentiation from both naive cord blood B cells as well as
CD27⫹ memory B cells was preceded by the induction of
BLIMP-1 up-regulation. We have also found that IL-21 induced
BLIMP-1 expression in murine splenic B cells (10). The capacity
of IL-21 to up-regulate BLIMP-1 may explain its ability to drive
plasma cell differentiation. In addition, IL-21 costimulation induced AID expression, which is normally down-modulated by
BLIMP-1 (38). Although AID is required and believed to be the B
cell-specific factor that is sufficient for both CSR and SHM (34),
IL-21 costimulation induced CSR, but not SHM. Similar findings
have been reported with CD27⫺ B cells stimulated with the combination of SAC, IL-2, IL-10, and cross-linked anti-CD40 (18). Of
note, we found that IL-4 and CD40 ligation induced AID in cord
blood B cells, but neither CSR nor plasma cell differentiation. Previously, AID has been shown to be induced by IL-4, anti-CD40, or
the combination of both IL-4 and anti-CD40 in adult B cells (26,
39, 40). It is of particular interest that AID was induced comparably by IL-4 and IL-21, although only the latter induced CSR. In
this regard, engagement of CD40 induced both AID and SHM in
human Ramos B cell lines (41, 42). These results suggest that
up-regulation of AID may have different effects on B cells at different stages of differentiation. Moreover, our data indicate that the
induction of AID mRNA is not sufficient to induce CSR or SHM
in all circumstances. The property of IL-21 that permits it to induce
both AID and CSR is currently unknown, but of great interest. The
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FIGURE 7. The majority of IL-21-induced plasma
cells are not in cell cycle. Purified PB B cells were isolated and cultured with IL-21, anti-IgM, and anti-CD40.
After 7 days of culture, cells were stained with the combination of either anti-IgD, anti-CD27, anti-CD19, and antiCD38, or anti-CD27, anti-CD38, and PI. For the latter,
cells were subdivided into either CD27⫺ and CD27⫹,
CD38low nonplasma cells (non-PC) or CD27high, CD38high
plasma cells (PC) and cell cycle was determined. Cell surface phenotype of cells before culture is shown. Data are
representative of results from one of three experiments.
The Journal of Immunology
7875
finding that IL-21 can induce AID and CSR but not SHM is reminiscent of the recent delineation of AID function, indicating that
different portions of the molecule govern CSR and SHM. Presumably IL-21 can induce CSR, the activity of AID that is governed by
the C-terminal portion of the molecule and involves the capacity to
bind DNA-protein kinase catalytic subunit (43), whereas it is not
able to induce SHM that is regulated by the N-terminal of the
protein by unknown molecules (44). The biological basis of this
functional discrimination is an important area for future investigation. Finally, the combination of IL-2, IL-10, and anti-CD40 was
able to induce BLIMP-1 mRNA, but far less AID mRNA from PB
B cells, suggesting that this cytokine pair may be unable to induce
CSR, and may explain its inability to induce the differentiation of
postswitched plasma cells from naive cord blood B cells. Moreover, the data suggest that the capacity to induce BLIMP-1 may be
necessary for plasma cell differentiation, but may not be the sole
determinant of the ability of IL-21 to induce the differentiation of
large numbers of plasma cells from both naive and memory B
cells.
It was possible that IL-21 may have functioned as a plasma cell
survival factor, rather than as an initiator of plasma cell differen-
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FIGURE 8. Ig production from IL-21-induced plasma
cells is resistant to HU. Purified PB B cells were isolated
and (A) cultured in the presence of IL-21 with or without
anti-CD40 and anti-IgM and with or without HU (1 ⫻
10⫺2 M) as indicated. After 3 days of culture, proliferation
was determined by incubating the cells for 16 h with
[3H]thymidine. B, Purified PB B cells were isolated and
cultured with or without IL-21 in the presence of antiCD40 and anti-IgM, and HU was added (or not) on the
days indicated. The cultures were analyzed for IgD and
CD38 expression by CD19⫹ B cells after 8 or 11 days in
culture as indicated. C, Supernatants from IL-21, antiIgM, and anti-CD40-stimulated cultures in B were analyzed for IgG content. HU was added where indicated.
Data are representative of results from one of three experiments with similar results.
tiation. This seems unlikely for a number of reasons. First, IL-21
alone had little capacity to maintain the few plasma cells that were
found in the initial population of peripheral CD19⫹ cells. Secondly, in cultures costimulated with IL-21, plasma cells appeared
after 3– 6 days, whereas in the absence of IL-21 few if any cells
with the phenotype of plasma cells were noted. This was most
notable in cultures of cord blood B cells. Therefore, it is unlikely
that IL-21 functions as a growth factor for plasmablasts induced by
anti-IgM or/and anti-CD40 signaling, but rather plays an essential
role in the decision to differentiate into nondividing, high-rate Igsecreting cells. The capacity of IL-21 costimulation, but not antiCD40 or anti-IgM alone, to induce BLIMP-1 mRNA is consistent
with this conclusion.
The effects of other cytokines that bind ␥C-containing cytokine
receptors on the actions of IL-21 were noteworthy. IL-2 has previously been shown to drive plasma cell differentiation when B
cells are activated with SAC (30), as well as costimulate plasma
cell differentiation in the presence of anti-CD40 and IL-10 (13, 25)
or IL-6 (14, 17). IL-4, in contrast, has been shown to both promote
and inhibit B cell responsiveness to various stimuli (13, 23, 45–
7876
IL-21 DRIVES HUMAN PLASMA CELL DIFFERENTIATION
48). Most notable is the ability of IL-4 to inhibit SAC and IL-2driven plasma cell differentiation (45, 47). We found that IL-2
enhanced the effect of IL-21 on plasma cell differentiation induced
by the combination of anti-IgM and anti-CD40, whereas IL-4 inhibited it. These results suggest an antagonistic effect of IL-4 on
the actions of IL-21 in B cells stimulated by anti-IgM or the combination of anti-IgM and anti-CD40. It is notable that antagonism
between IL-4 and IL-21 was not consistently observed when cells
were stimulated with anti-CD40 alone, indicating that the nature of
the stimulus influences the ability of B cells to respond to the
combination of IL-4 and IL-21. Moreover, the origin of the B cells
may play a role because IL-4 has been shown to enhance Ig production by IL-21 and anti-CD40-activated CD27⫹ human splenic
memory B cells (26). These differences may reflect variability in
the cytokine responsiveness of B cells from various compartments
because IL-4 did not consistently inhibit or enhance anti-CD40 and
IL-21-induced plasma cell differentiation by peripheral B cells.
These findings make it unlikely that receptor competition can explain these results, owing to the fact that both IL-21 and IL-4 use
the ␥C for signaling (49). In addition, IL-2, which also signals
FIGURE 10. IL-4 inhibits IL-21-induced plasma cell
differentiation. Purified peripheral B cells were isolated
and cultured with the stimuli and cytokines indicated. Before culture, the B cells contained 78% naive B cells, 20%
IgD- B cells, and 0.4% plasma cells. A, Cell surface expression of IgD and CD38 by CD19⫹ B cells after 6 –7
days of culture is shown. B, IgG was quantified from culture supernatants after 7–11 days of incubation, and data
are shown as a mean concentration (⫾SEM). C, mRNA
was isolated and BLIMP-1 and AID expression was determined after 3 days of culture. All data in C were normalized to ␤2M mRNA and shown as fold change compared with cultures incubated with only IL-2 and IL-4.
Data are shown as a mean fold change (⫾SEM). Data are
representative of results from cultures of two similar experiments with SAC, three with anti-IgM, five with antiCD40, seven with anti-IgM and anti-CD40 (A), two with
SAC, and eight with anti-IgM and anti-CD40 (B). B, For
anti-CD40 stimulation, three of six experiments are shown
in which four resulted in IL-4-induced suppression of IgG
production and two did not. The data in C are representative of four similar experiments.
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FIGURE 9. IL-21 induces both BLIMP and AID expression, but not SHM. Purified PB B cells (A) or cord blood B
cells (B and C) were cultured with the stimuli and cytokines
indicated. After 3 days of culture, cells were harvested, verified for cell surface phenotype, RNA isolated, and quantitative
RT-PCR was conducted to identify BLIMP-1, AID, Bcl-6,
and PAX-5 mRNA. All data were normalized to ␤2M mRNA,
and data shown represent fold change compared with samples
that were incubated with no stimuli (nil) (A), or to unstimulated, primary cord blood B cells (B). Data are representative
of results from six similar experiments for PB B cells, and two
for cord blood B cells. Mean fold change (⫾SEM) of triplicate
mRNA samples from one representative experiment is shown.
C, Negatively selected B cells from four cord blood samples
were cultured for 7–12 days with IL-2, IL-21, and anti-CD40
with or without anti-IgM to induce plasma cell differentiation.
After stimulation, IgD⫺CD38high plasma cells were isolated
by cell sorting, and the Ig VH genes were amplified from
genomic DNA by single-cell PCR, sequenced, and compared
with the closest germline VH gene or polymorphic variant to
determine the number of mismatches. The data indicate the
percentage of all sequences from each population with 0, 1, 2,
or 3 or more mismatches. The distribution of sequences with
the indicated number of mismatches from 189 sequences
(98 from anti-CD40- and 91 from anti-CD40 and anti-IgMstimulated cultures) from IL-21-induced plasma cells was not
significantly different from 141 sequences from unstimulated
cord blood B cells examined immediately ex vivo (␹2 test, p ⫽
0.2745). Combined results from four experiments are shown.
The Journal of Immunology
7877
through a receptor-containing ␥C, reversed the IL-4 mediated-inhibition of IgM-producing plasma cell generation (data not
shown), making the idea of receptor competition more remote.
Furthermore, IL-2 synergized with IL-21 in fostering growth of
anti-IgM and anti-CD40-stimulated B cells.
Taken together, these results suggest that the stimulus-dependent inhibition of IL-21 responses by IL-4 relates to the downstream signals generated by engagement of the IL-4R. In this regard, IL-4 is known to costimulate IgE synthesis by human B cells
(48), and also induce the expression of CD23 on mature B cells
(50). However, IL-4 also has been reported to repress human B cell
responses (45, 47), and inhibit BLIMP-1 induction as well as
plasma cell differentiation in mice (51). It is notable that in our
studies, with the appropriate costimulation, IL-4 repressed
BLIMP-1 expression, and inhibited plasma cell differentiation,
whereas IL-21 induced both BLIMP-1 and plasma cell differenti-
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FIGURE 11. IL-21 induces greater proliferation, plasma cell differentiation, and IgG production from anti-CD40-stimulated PB and
cord blood B cells than does IL-2 and IL-10.
A–C, Purified naive cord blood B cells (CB) or
PB B cells (PB) (A–D) were cultured with the
stimuli and cytokines indicated for 3 days (A),
7 days (B), 9 days for CB B cells (C), and 10
days for PB B cells, and 3 days (D). At the end
of the incubation period [3H]thymidine incorporation (A), absolute plasma cell number (B),
and IgG secretion (C) were determined. In D,
relative fold change of mRNA was determined
for BLIMP-1 and AID. All samples were first
normalized to ␤2M mRNA and then to mRNA
of samples that were not stimulated and did not
contain cytokine (nil, nil). Data are shown as
mean fold change (⫾SEM). Data are representative of results from one of two similar
experiments.
ation. In the appropriate signaling context, as discussed above, the
actions of the two cytokines appear to be mutually inhibitory of
one another. In this regard, IL-21 has been reported to inhibit proliferation of both murine and human B cells stimulated with antiIgM and IL-4 (2, 7, 10) and to inhibit IL-4-induced IgE transcription (52). In this study, IL-4 repressed IL-21-driven plasma cell
differentiation following either SAC or anti-IgM and anti-CD40
stimulation. It is of considerable interest that IL-4R ligation induces primarily STAT6, which is essential for IL-4 signaling, including IL-4-induced up-regulation of AID in both mice and humans (39, 40), whereas IL-21R engagement activates primarily
STAT1 and -3 and more weakly STAT5 (1, 53, 54). Although the
level of antagonism is unknown, the data suggest that IL-21 and
IL-4 inhibit one another’s actions after receptor engagement. Biologically, T cell-dependent B cell responses may be regulated by
T cells that either produce IL-21, IL-4, or other cytokines at
7878
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
Acknowledgments
We gratefully acknowledge the editorial assistance of Iris Pratt, the technical help of Randy T. Fisher, and help with acquiring cord blood samples
by Nancy Longo. We also thank James Simone and Derek Hewgill of the
National Institute of Arthritis and Musculoskeletal and Skin Diseases flow
cytometry facility for B cell sorting.
24.
25.
26.
Disclosures
The authors have no financial conflict of interest.
27.
References
28.
1. Ozaki, K., K. Kikly, D. Michalovich, P. R. Young, and W. J. Leonard. 2000.
Cloning of a type I cytokine receptor most related to the IL-2 receptor ␤ chain.
Proc. Natl. Acad. Sci. USA 97: 11439 –11444.
2. Parrish-Novak, J., S. R. Dillon, A. Nelson, A. Hammond, C. Sprecher,
J. A. Gross, J. Johnston, K. Madden, W. Xu, J. West, et al. 2000. Interleukin 21
29.
and its receptor are involved in NK cell expansion and regulation of lymphocyte
function. Nature 408: 57– 63.
Asao, H., C. Okuyama, S. Kumaki, N. Ishii, S. Tsuchiya, D. Foster, and
K. Sugamura. 2001. Cutting edge: the common ␥-chain is an indispensable subunit of the IL-21 receptor complex. J. Immunol. 167: 1–5.
Collins, M., M. J. Whitters, and D. A. Young. 2003. IL-21 and IL-21 receptor: a
new cytokine pathway modulates innate and adaptive immunity. Immunol. Res.
28: 131–140.
Leonard, W. J. 2001. Cytokines and immunodeficiency diseases. Nat. Rev. Immunol. 1: 200 –208.
Chtanova, T., S. G. Tangye, R. Newton, N. Frank, M. R. Hodge, M. S. Rolph, and
C. R. Mackay. 2004. T follicular helper cells express a distinctive transcriptional
profile, reflecting their role as non-Th1/Th2 effector cells that provide help for B
cells. J. Immunol. 173: 68 –78.
Ozaki, K., R. Spolski, C. G. Feng, C. F. Qi, J. Cheng, A. Sher, H. C. Morse, 3rd,
C. Liu, P. L. Schwartzberg, and W. J. Leonard. 2002. A critical role for IL-21 in
regulating immunoglobulin production. Science 298: 1630 –1634.
Jin, H., R. Carrio, A. Yu, and T. R. Malek. 2004. Distinct activation signals
determine whether IL-21 induces B cell costimulation, growth arrest, or Bimdependent apoptosis. J. Immunol. 173: 657– 665.
Mehta, D. S., A. L. Wurster, M. J. Whitters, D. A. Young, M. Collins, and
M. J. Grusby. 2003. IL-21 induces the apoptosis of resting and activated primary
B cells. J. Immunol. 170: 4111– 4118.
Ozaki, K., R. Spolski, R. Ettinger, H. P. Kim, G. Wang, C. F. Qi, P. Hwu,
D. J. Shaffer, S. Akilesh, D. C. Roopenian, et al. 2004. Regulation of B cell
differentiation and plasma cell generation by IL-21, a novel inducer of Blimp-1
and Bcl-6. J. Immunol. 173: 5361–5371.
Kindler, V., and R. H. Zubler. 1997. Memory, but not naive, peripheral blood B
lymphocytes differentiate into Ig-secreting cells after CD40 ligation and costimulation with IL-4 and the differentiation factors IL-2, IL-10, and IL-3. J. Immunol.
159: 2085–2090.
Briere, F., C. Servet-Delprat, J. M. Bridon, J. M. Saint-Remy, and J. Banchereau.
1994. Human interleukin 10 induces naive surface immunoglobulin D⫹sIgD⫹ B
cells to secrete IgG1 and IgG3. J. Exp. Med. 179: 757–762.
Arpin, C., J. Banchereau, and Y.-J. Liu. 1997. Memory B cells are biased towards
terminal differentiation: a strategy that may prevent repertoire freezing. J. Exp.
Med. 186: 931–940.
Jego, G., R. Bataille, and C. Pellat-Deceunynck. 2001. Interleukin-6 is a growth
factor for nonmalignant human plasmablasts. Blood 97: 1817–1822.
Burdin, N., C. Van Kooten, L. Galibert, J. Abrams, J. Wijdenes, J. Banchereau,
and F. Rousset. 1995. Endogenous IL-6 and IL-10 contribute to the differentiation
of CD40-activated human B lymphocytes. J. Immunol. 154: 2533–2544.
Rousset, F., E. Garcia, T. Defrance, C. Peronne, N. Vezzio, D.-H. Hsu,
R. Kastelein, K. W. Moore, and J. Banchereau. 1992. Interleukin 10 is a potent
growth and differentiation factor for activated human B lymphocytes. Proc. Natl.
Acad. Sci. USA 89: 1890 –1893.
Vernino, L., L. M. McAnally, J. Ramberg, and P. E. Lipsky. 1992. Generation of
nondividing high rate Ig-secreting plasma cells in cultures of human B cells
stimulated with anti-CD3-activated T cells. J. Immunol. 148: 404 – 410.
Nagumo, H., K. Agematsu, N. Kobayashi, K. Shinozaki, S. Hokibara, H. Nagase,
M. Takamoto, K. Yasui, K. Sugane, and A. Komiyama. 2002. The different
process of class switching and somatic hypermutation: a novel analysis by
CD27⫺ naive B cells. Blood 99: 567–575.
Fujieda, S., A. Saxon, and K. Zhang. 1996. Direct evidence that ␥1 and ␥3
switching in human B cells is interleukin-10 dependent. Mol. Immunol. 33:
1335–1343.
Arpin, C., J. Dechanet, C. Van Kooten, P. Merville, G. Grouard, F. Briere,
J. Banchereau, and Y.-J. Liu. 1995. Generation of memory B cells and plasma
cells in vitro. Science 268: 720 –722.
Tarte, K., J. De Vos, T. Thykjaer, F. Zhan, G. Fiol, V. Costes, T. Rème,
E. Legouffe, J.-F. Rossi, J. Shaughnessy, Jr., et al. 2002. Generation of polyclonal
plasmablasts from peripheral blood B cells: a normal counterpart of malignant
plasmablasts. Blood 100: 1113–1122.
Ramesh, N., R. Fuleihan, and R. Geha. 1994. Molecular pathology of X-linked
immunoglobulin deficiency with normal or elevated IgM (HIGMX-1). Immunol.
Rev. 138: 87–104.
Splawski, J. B., S. M. Fu, and P. E. Lipsky. 1993. Immunoregulatory role of
CD40 in human B cell differentiation. J. Immunol. 150: 1276 –1285.
Foy, T. M., A. Aruffo, J. Bajorath, J. E. Buhlmann, and R. J. Noelle. 1996.
Immune regulation by CD40 and its ligand GP39. Annu. Rev. Immunol. 14:
591– 617.
Splawski, J. B., K. Yamamoto, and P. E. Lipsky. 1998. Deficient interleukin-10
production by neonatal T cells does not explain their ineffectiveness at promoting
neonatal B cell differentiation. Eur. J. Immunol. 28: 4248 – 4256.
Pene, J., J. F. Gauchat, S. Lecart, E. Drouet, P. Guglielmi, V. Boulay, A. Delwail,
D. Foster, J. C. Lecron, and H. Yssel. 2004. Cutting edge: IL-21 is a switch factor
for the production of IgG1 and IgG3 by human B cells. J. Immunol. 172:
5154 –5157.
Fecteau, J. F., and S. Neron. 2003. CD40 stimulation of human peripheral B
lymphocytes: distinct response from naive and memory cells. J. Immunol. 171:
4621– 4629.
Miyashita, T., M. J. McIlraith, A. C. Grammer, Y. Miura, J. F. Attrep,
Y. Shimaoka, and P. E. Lipsky. 1997. Bidirectional regulation of human B cell
responses by CD40-CD40 ligand interactions. J. Immunol. 158: 4620 – 4633.
Sims, G. P., R. Ettinger, Y. Shirota, C. H. Yarboro, G. Illei, and P. E. Lipsky.
2005. Identification and characterization of circulating human transitional B cells.
Blood 105: 4390 – 4398.
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different times during an immune response, or different subsets of
T cells that express different cytokine profiles. In support of this,
IL-21 has been described to be produced by a restricted subset of
follicular Th cells that express CXCR5 in humans (6).
Our final question was whether IL-21 had the ability to induce
terminally differentiated nondividing plasma cells or merely dividing Ig-secreting plasmablasts. Ig-secreting cells have been shown
to be quite heterogeneous, and different stages of plasma cell development have been described that can be identified phenotypically (55, 56). The majority of plasma cells in the germinal center
are short lived, produce Ab rapidly and briefly, and then die in
secondary lymphoid tissue. In contrast, a subpopulation matures
into terminally differentiated nondividing plasma cells that exit
lymphoid tissue and migrate to the bone marrow where they reside
as long-lived plasma cells that produce Ab for long periods of time
(55). These stages of plasma cells can be identified both functionally and phenotypically (56). It is notable that by phenotypic analysis, the IL-21-induced plasma cells could not clearly be identified
as typical Ig-secreting plasma cells found in any one tissue compartment. Thus, they have some characteristic features of all
plasma cells, including decreased expression of CD19, CD20,
CD21, CD22, and CD23, as well as increased expression of CD27,
CD38, and CD63. However, they have some features of tonsillar
plasma cells, such as bright expression CD9 and CD31 and decreased expression of CD49d, as well as some features of blood
plasma cells, including bright expression of CD44 and CD62L. In
addition, subpopulations of IL-21-induced plasma cells express
features of bone marrow plasma cells, including diminished expression of HLA-DR and increased expression of CXCR4. The
explanation for this mixed plasma cell phenotype is uncertain. It is
not clear whether other features of the local microenvironment
might contribute to the phenotype of resident plasma cells. Indeed,
cytokines, such as B cell activation factor of the TNF family
(BAFF) (57) or potentially IL-21 itself could affect the phenotype
of plasma cells. Importantly, however, the plasma cells generated
in response to IL-21 were postswitched nondividing, Ig-secreting
cells as evidenced by their ability to continue to secrete Ig even
when proliferation was inhibited by HU. These data, therefore,
indicate that IL-21 costimulation drives naive and memory B cells
into terminally differentiated nondividing Ig-secreting plasma
cells.
In conclusion, our studies demonstrate that IL-21 has pleiotropic
effects on human B cells and strongly suggest that IL-21 is the
major T cell-derived cytokine that drives plasma cell differentiation upon CD40 ligation. The ability of IL-21 to induce human
plasma cell differentiation has implications not only in the treatment of immunocompromised individuals but also in autoimmune
diseases where IL-21 blockade may have efficacy.
IL-21 DRIVES HUMAN PLASMA CELL DIFFERENTIATION
The Journal of Immunology
44. Shinkura, R., S. Ito, N. A. Begum, H. Nagaoka, M. Muramatsu, K. Kinoshita,
Y. Sakakibara, H. Hijikata, and T. Honjo. 2004. Separate domains of AID are
required for somatic hypermutation and class-switch recombination. Nat. Immunol. 7: 707–712.
45. Jelinek, D. F., and P. E. Lipsky. 1988. Inhibitory influence of IL-4 on human B
cell responsiveness. J. Immunol. 141: 164 –173.
46. Fujieda, S., K. Zhang, and A. Saxon. 1995. IL-4 plus CD40 monoclonal antibody
induces human B cells ␥ subclass-specific isotype switch: switching to ␥1, ␥3,
and ␥4, but not ␥2. J. Immunol. 155: 2318 –2328.
47. Splawski, J. B., D. F. Jelinek, and P. E. Lipsky. 1989. Immunomodulatory role of
IL-4 on the secretion of Ig by human B cells. J. Immunol. 142: 1569 –1575.
48. Jabara, H. H., S. M. Fu, R. S. Geha, and D. Vercelli. 1990. CD40 and IgE:
synergism between anti-CD40 monoclonal antibody and interleukin 4 in the induction of IgE synthesis by highly purified human B cells. J. Exp. Med. 172:
1861–1864.
49. Zhang, J. L., D. Foster, and W. Sebald. 2003. Human IL-21 and IL-4 bind to
partially overlapping epitopes of common ␥-chain. Biochem. Biophys. Res. Commun. 300: 291–296.
50. Nelms, K., A. D. Keegan, J. Zamorano, J. J. Ryan, and W. E. Paul. 1999. The
IL-4 receptor: signaling mechanisms and biologic functions. Annu. Rev. Immunol.
17: 701–738.
51. Knodel, M., A. W. Kuss, I. Berberich, and A. Schimpl. 2001. Blimp-1 overexpression abrogates IL-4- and CD40-mediated suppression of terminal B cell
differentiation but arrests isotype switching. Eur. J. Immunol. 31: 1972–1980.
52. Suto, A., H. Nakajima, K. Hirose, K. Suzuki, S. Kagami, Y. Seto, A. Hoshimoto,
Y. Saito, D. C. Foster, and I. Iwamoto. 2002. Interleukin 21 prevents antigeninduced IgE production by inhibiting germ line C⑀ transcription of IL-4-stimulated B cells. Blood 100: 4565– 4573.
53. Habib, T., S. Senadheera, K. Weinberg, and K. Kaushansky. 2002. The common
␥ chain (␥c) is a required signaling component of the IL-21 receptor and supports
IL-21-induced cell proliferation via JAK3. Biochemistry 41: 8725– 8731.
54. Habib, T., A. Nelson, and K. Kaushansky. 2003. IL-21: a novel IL-2-family
lymphokine that modulates B, T, and natural killer cell responses. J. Allergy Clin.
Immunol. 112: 1033–1045.
55. Shapiro-Shelef, M., and K. Calame. 2005. Regulation of plasma cell development. Nat. Rev. Immunol. 5: 230 –242.
56. Medina, F., C. Segundo, A. Campos-Caro, I. González-Garcı́a, and J. A. Brieva.
2002. The heterogeneity shown by human plasma cells from tonsil, blood, and
bone marrow reveals graded stages of increasing maturity, but local profiles of
adhesion molecule expression. Blood 99: 2154 –2161.
57. Gorelik, L., A. H. Cutler, G. Thill, S. D. Miklasz, D. E. Shea, C. Ambrose,
S. A. Bixler, L. Su, M. L. Scott, and S. L. Kalled. 2004. Cutting edge: BAFF
regulates CD21/35 and CD23 expression independent of its B cell survival function. J. Immunol. 172: 762–766.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
30. McIlraith, M. J., and P. E. Lipsky. 1996. Interleukin-2. In Cytokine Regulation of
Humoral Immunity: Basic and Clinical Aspects. C. M. Snapper, ed. John Wiley
and Sons, Hoboken, pp. 159 –193.
31. Klein, U., K. Rajewsky, and R. Kuppers. 1998. Human immunoglobulin
IgM⫹IgD⫹ peripheral blood B cells expressing the CD27 cell surface antigen
carry somatically mutated variable region genes: CD27 as a general marker for
somatically mutated (memory) B cells. J. Exp. Med. 188: 1679 –1689.
32. Jelinek, D. F., and P. E. Lipsky. 1983. The role of B cell proliferation in the
generation of immunoglobulin-secreting cells in man. J. Immunol. 130:
2597–2604.
33. Turner, C. A. J., D. H. Mack, and M. M. Davis. 1994. Blimp-1, a novel zinc
finger-containing protein that can drive the maturation of B lymphocytes into
immunoglobulin-secreting. Cell 77: 297–306.
34. Honjo, T., M. Muramatsu, and S. Fagarasan. 2004. AID: how does it aid antibody
diversity? Immunity 20: 659 – 668.
35. Ye, B. H., G. Cattoretti, Q. Shen, J. Zhang, N. Hawe, R. de Waard, C. Leung,
M. Nouri-Shirazi, A. Orazi, R. S. Chaganti, et al. 1997. The BCL-6 proto-oncogene controls germinal-centre formation and Th2-type inflammation. Nat. Genet.
16: 161–170.
36. Rolink, A. G., C. Schaniel, and F. Melchers. 2002. Stability and plasticity of
wild-type and Pax5-deficient precursor B cells. Immunol. Rev. 187: 87–95.
37. Shapiro-Shelef, M., K. Lin, L. J. McHeyzer-Williams, M. G. McHeyzer-Williams, and K. Calame. 2003. Blimp-1 is required for the formation of immunoglobulin secreting plasma cells and pre-plasma memory B cells. Immunity 19:
607– 620.
38. Shaffer, A. L., K. I. Lin, T. C. Kuo, X. Yu, E. M. Hurt, A. Rosenwald, J. M.
Giltnane, L. Yang, H. Zhao, K. Calame, and L. M. Staudt. 2002. Blimp-1 orchestrates plasma cell differentiation by extinguishing the mature B cell gene
expression program. Immunity 17: 51– 62.
39. Zhou, C., A. Saxon, and K. Zhang. 2003. Human activation-induced cytidine
deaminase is induced by IL-4 and negatively regulated by CD45: implication of
CD45 as a Janus kinase phosphatase in antibody diversification. J. Immunol. 170:
1887–1893.
40. Dedeoglu, F., B. Horwitz, J. Chaudhuri, F. W. Alt, and R. S. Geha. 2004. Induction of activation-induced cytidine deaminase gene expression by IL-4 and
CD40 ligation is dependent on STAT6 and NF␬B. Int. Immunol. 16: 395– 404.
41. Zan, H., X. Wu, A. Komori, W. K. Holloman, and P. Casali. 2003. AID-dependent generation of resected double-strand DNA breaks and recruitment of Rad52/
Rad51 in somatic hypermutation. Immunity 18: 727–738.
42. Faili, A., S. Aoufouchi, Q. Gueranger, C. Zober, A. Leon, B. Bertocci,
J. C. Weill, and C. A. Reynaud. 2002. AID-dependent somatic hypermutation
occurs as a DNA single-strand event in the BL2 cell line. Nat. Immunol. 3:
815– 821.
43. Wu, X., P. Geraldes, J. L. Platt, and M. Cascalho. 2005. The double-edged sword
of activation-induced cytidine deaminase. J. Immunol. 174: 934 –941.
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