Antibody-Secreting Plasma Cells Naive and Memory B Cells Into IL-21 Induces Differentiation of Human

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IL-21 Induces Differentiation of Human Naive and Memory B Cells into Antibody-Secreting Plasma Cells

This information is current as Rachel Ettinger, Gary P. Sims, Anna-Marie Fairhurst, Rachel of September 23, 2021. 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

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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*

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 Downloaded from 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. http://www.jimmunol.org/ nterleukin-21 is a type I cytokine that signals through a re- plasma cells (10). Taken together, these data indicate that IL-21 is ceptor composing of the IL-21R and the common cytokine a critical, although complex, regulator of B cell function in the receptor ␥-chain (␥ )3 (1–5). In humans, IL-21 has been I C mouse, although less is known about its activities in humans. shown to be produced by activated peripheral T cells, as well as Regulation of the differentiation of human naive B cells into ϩ spontaneously by CXCR5 follicular helper T cells (2, 6). The memory and plasma cells has not been as extensively characterized IL-21R is closely related to the IL-2R ␤-chain and is expressed by as that of their murine counterparts. However, a variety of studies T, B, and NK cells (1, 2). Unexpectedly, IL-21R-deficient mice have suggested that cytokines, such as IL-2, IL-6, and IL-10 are exhibited a severe defect in IgG1 production following Ag priming involved in human plasma cell differentiation (11–21). Moreover, by guest on September 23, 2021 (7), demonstrating its importance in murine B cell differentiation. in vitro and in vivo analyses have identified cellular interactions, In this regard, IL-21 exerts a number of activities on B cell func- including those mediated by CD40 and its ligand, CD154, as play- tion, including apoptosis, growth arrest, or costimulation depend- ing pivotal roles in the generation of both memory B cells and ing on the nature of the activation signals provided to B cells (8, plasma cells (22–24). Nonetheless, responsiveness of B cells and 9). For example, IL-21 enhances anti-CD40 stimulation but inhib- particularly naive B cells is minimal in the presence of purified its anti-IgM and IL-4 responses in both mouse and human (2, 7, costimulators (21, 25–28). The specific signals that drive the dif- 10). These effects appear to relate to the capacity of IL-21 to in- ferentiation of human naive B cells into memory cells and plasma duce apoptosis of resting and anti-IgM-stimulated murine B cells, cells, therefore, have not been completely defined, and an inte- whereas it paradoxically promotes differentiation of anti-CD40- grated view of these essential steps in human B cell biology has stimulated murine splenic B cells into postswitch memory and not been definitively established. Because IL-21 plays an important role in murine B cell differentiation into memory cells and plasma cells, we investigated *Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin the role of IL-21 in human B cell differentiation. In this study, Diseases and †Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892 we report that IL-21 costimulation not only is capable of in- ϩ Received for publication March 28, 2005. Accepted for publication September ducing plasma cell differentiation from CD27 memory B cells, 29, 2005. but also has the capacity to induce class switch recombination The costs of publication of this article were defrayed in part by the payment of page (CSR) and stimulate poorly responsive naive cord blood B cells charges. This article must therefore be hereby marked advertisement in accordance into IgG-secreting plasma cells. Importantly, IL-21 costimula- with 18 U.S.C. Section 1734 solely to indicate this fact. tion up-regulated expression of both activation-induced cyti- 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 dine deaminase (AID) and B lymphocyte-induced maturation Diseases. protein-1 (BLIMP-1), but did not induce somatic hypermutation 2 Address correspondence and reprint requests to Dr. Rachel Ettinger, National In- (SHM). Finally, the action of IL-21 was largely prevented by stitute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of another type I cytokine, IL-4. These results demonstrate that Health, Building 10, Room 6D-47B, Bethesda, MD 20892. E-mail address: [email protected] IL-21 is one of the major T cell influences that initiates CSR 3 ␥ ␥ and differentiation of human B cells into Ab-secreting plasma Abbreviations used in this paper: C, common cytokine receptor -chain; CSR, class switch recombination; AID, activation-induced cytidine deaminase; BLIMP-1, B cells. Moreover, the interplay of IL-21 and IL-4 may exert an lymphocyte-induced maturation protein-1; SHM, somatic hypermutation; PB, peripheral blood; SAC, Staphylococcus aureus Cowan I; PI, propidium iodide; HU, hy- essential function in determining the outcome of human hu- ␤ ␤ doxyurea; 2M, 2 microglobulin; int, intermediate. moral immune responses.

Materials and Methods absorbance was measured, and OD was quantified at 410 nM by a Pow- Isolation of human B cells erwave X 96-well plate reader (Bio-Tek Instruments). IgE concentrations were measured with the Human IgE ELISA kit (Bethyl Laboratories). Spe- All human studies have been approved by the Warren G. Magnuson Clin- cific IgG isotypes were differentiated using the Human IgG Subclass Pro- ical Center Institutional Review Board, and informed consent was obtained file ELISA KIT (Zymed Laboratories). according to the declaration of Helsinki. Human peripheral B cells were isolated from buffy coats of anonymous healthy donors drawn at the Na- B cell proliferation and cell cycling tional Institutes of Health Division of Transfusion Medicine. Umbilical To assess proliferative responses of cultured cells, 105 purified B cells were cord blood samples were obtained from Advanced Bioscience Resources. cultured as described above in 96-well round-bottom plates. After 3–5 days For most studies, human cord blood and peripheral B cells were isolated by of culture, [3H]thymidine (37 Kbq/well) was added to the cultures for an negative selection using the Rosette-sep technique following the manufac- additional 16 h. Thymidine uptake was measured using a liquid scintilla- turer’s instructions (StemCell Technologies). Preparations were typically tion counter. To assess whether IL-21-induced plasma cells were in cell Ͼ ϩ 96% CD19 from peripheral blood (PB). However, negatively selected cycle, two strategies were used. First, propidium iodide (PI) incorporation cord blood B cell isolations yielded less pure cell populations. Thus, we was used to determine the cell cycle position of the cells. Stimulated and positively selected B cells with anti-CD19 magnetic beads (Miltenyi Bio- control cultures were stained after various days with either anti-IgD-FITC, Ͼ tec) in some of the cord blood experiments, and these were typically 95% anti-CD19 PerCP-Cy5.5, and anti-CD38-APC to visualize plasma cells pure. For further purity of peripheral B cells, CD20ϩCD27Ϫ naive B cells ϩ ϩ based on IgD and CD38 expression or anti-CD27-FITC and anti-CD38- or CD20 CD27 memory B cells were isolated on a MoFlo cell sorter APC, followed by 1% paraformaldehyde solution for 15 min, followed by (DakoCytomation). Briefly, negatively selected B cells were incubated at ␮ 7 the addition of 100 g/ml PI solution in PBS with 0.1% Triton X-100 for 10 cells/ml in staining buffer with mAbs specific for CD20 and CD27 and at least 1 h before analyses. Second, hydroxyurea (HU) (Sigma Chemicals) in some experiments anti-IgD as well. Cells were incubated for 30 min at Ϫ2 ϩ ϩ ϩ Ϫ was added to some cultures at a final concentration of 10 M at either day 4°C, washed, and sorted into CD20 CD27 or CD20 CD27 subsets. 0 (that were subsequently either pulsed with [3H]thymidine after 3 days or Ͼ Downloaded from Preparations were typically 98% pure. stained for flow cytometric analysis or analyzed for IgG production after 8 Cell culture, activation, and CFSE labeling 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 Purified B cells were cultured at 1 ϫ 106 cells/ml in either 1 ml in 24-well concentration of HU has been shown to completely inhibit the proliferation culture plates or 100 ␮l in 96-well round-bottom culture plates. The cells of activated cycling human B cells (17). 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 Real-time quantitative PCR

Systems and BioSource International), human IL-10 (25 ng/ml; R&D Sys- http://www.jimmunol.org/ Negatively or positively purified B cells were stimulated as described tems), and either 1 ␮g/ml anti-human CD40 (R&D Systems), 5 ␮g/ml above. After 3 days in culture, cells were harvested and resuspended in anti-IgM (Jackson ImmunoResearch Laboratories), or 0.01% final dilution TRIzol (Invitrogen Life Technologies) and stored at Ϫ70°C. RNA was of heat-killed, formalin-fixed Staphylococcus aureus Cowan I (SAC) (Cal- isolated using the RNeasy mini kit (Qiagen). Purity was measured using biochem). When multiple cytokines and stimuli were used, all cytokines spectrophotometry. Reverse transcription reactions were prepared using the and stimuli were added at the initiation of culture. In some experiments, SuperScript One-Step PCR System with Platinum Taq Polymerase and purified B cells were labeled with CFSE. In brief, purified B cells were ROX reference dye (Invitrogen Life Technologies). Fifty nanograms of washed extensively in PBS to remove all FCS, and CFSE/PBS (Molecular isolated RNA was added per reaction with 1.2 mM MgSO . TaqMan As- Probes) was added at a final concentration of 2.5 ␮Mto2–5ϫ 107 cells/ml 4 says-on Demand Gene expression primer/probe sets (Applied Biosystems) for 8 min. Labeling was quenched by addition of FCS, and cells were were used for BLIMP-1 (Hs00153357_m1), Bcl6 (Hs00153368_m1), washed four times in medium containing 10% FCS before culture. To AICDA (Hs00221068_m1), PAX-5 (Hs00277134_m1), and ␤ micro- by guest on September 23, 2021 obtain an accurate comparison for the controls, cord blood B cells were 2 globulin (␤ M) (Hs99999907_m1). Final concentrations were 1.8 ␮M for stimulated in the presence of IL-4, which facilitated cell survival but in- 2 primers and 0.5 ␮M for probes. RT-PCR was performed using the ABI duced neither IgD down-modulation or plasma cell differentiation. Prism 7700 Sequence Detection System (Applied Biosystems), and cycle Flow cytometry conditions and relative quantification were completed as described by manufacturer’s instructions (Applied Biosystems). Expression of each tran- Four-color flow cytometry was performed using a FACSCalibur (BD Bio- scription factor was calculated using the comparative computerized tomog- sciences). Briefly, supernatants were collected, and then all cells were har- raphy method with efficiency calculations and with all mRNA levels vested 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-CD38- allophycocyanin (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 anti- CD31-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 anti- CD44-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 FIGURE 1. IL-21 induces plasma cell differentiation after stimulation with anti-CD40 and anti-IgM. Purified PB B cells were stimulated with no Secreted Ig in the culture supernatant was quantitated by ELISA. Briefly, polyclonal B cell activator or with the combination of anti-CD40 and anti- 96-well flat-bottom Nunc-Brand Immuno plates (Nalge Nunc Interna- IgM in the presence or absence of IL-21 as indicated. After 6 days of ␮ tional) were coated with 5 g/ml of either affinity-purified goat anti-human culture, B cells were identified based on CD19 expression and assessed for IgM or goat anti-human IgG-Fc (Bethyl Laboratories) overnight at 4°C. Ϫ high Wells were then washed and blocked with a 0.2% BSA/PBS, and then the expression of IgD and CD38. IgD CD38 plasma cells are present in the lower right quadrant of anti-CD40, anti-IgM, and IL-21-stimulated cul- culture supernatants were titered onto the treated plates and incubated over- ϩ night at 4°C. Bound Ig was detected with 0.5 ␮g/ml alkaline phosphatase- tures. The numbers in the quadrants indicate the percentage of CD19 cells conjugated goat anti-human IgM or IgG (Bethyl Laboratories) and devel- in each region. Cell surface phenotype of B cells before culture is also oped with p-nitrophenyl phosphate tablets (Sigma-Aldrich). Specific shown (day 0). The Journal of Immunology 7869

FIGURE 2. IL-21 induces plasma cell differentiation. A, Puriﬁed PB B cells stained with anti-IgD and anti-IgM contain few (1.3%) IgDϪCD38high plasma cells. B–D,Pu-

rified PB B cells were stimulated with anti-IgM, anti- Downloaded from CD40, 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 http://www.jimmunol.org/ 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, CD19ϩ B cells were analyzed for CD38 expression and CFSE dilution. Data are representative of results from three similar experiments. by guest on September 23, 2021

␤ normalized to 2M. All reported values were then further normalized to naive B cells in multiple individuals were considered putative polymorphic control conditions, of cultures from PB B cells with no cytokine and no variants and were excluded from the analysis. Data obtained from plasma stimuli or cultures from cord blood B cells with IL-2 (Biological Research cells derived from anti-CD40 and anti-CD40 and anti-IgM-stimulated cul- Branch, Division of Cancer Treatment and Diagnosis, National Cancer tures were similar, and their data were pooled for the analysis. Institute, Frederick, Maryland) and IL-4 (R&D Systems) without any stimuli, as a value of 1. Results Somatic hypermutation IL-21 induces plasma cell differentiation B cells enriched from four cord blood samples were stimulated with IL-2 The capacity of IL-21 to costimulate responses of purified human and IL-21 in the presence of anti-CD40 or anti-CD40 and anti-IgM for ϩ Ϫ B cells was initially explored. Freshly obtained B cells or those 7–12 days. Individual live, CD19 IgD CD38high plasma cells were sorted, retrieved from cultures were stained with anti-CD19, anti-IgD, and and the rearranged Ig H chain V regions (VH) from genomic DNA of single ϩ cells was amplified and directly sequenced as described previously (29). anti-CD38. CD19 cells were gated and assessed for expression of ϩ The VH gene sequences from these IL-21-induced plasma cells, and un- IgD and CD38 as shown in Fig. 1. Fresh CD19 B cells or those stimulated cord blood B cells collected immediately after purification, were cultured without stimulation or with the combination of anti-CD40 compared with germline genes and their polymorphic variants from our own and public databases used to determine the closest germline gene and and anti-IgM in the absence of cytokines contained few IgDϪCD38high plasma cells. In contrast, costimulation of purified the number of VH gene segment mismatches. Several undocumented but recurring VH gene mismatches that we have found in genes isolated from PB B cells with anti-CD40 and anti-IgM in the presence of IL-21 7870 IL-21 DRIVES HUMAN PLASMA CELL DIFFERENTIATION resulted in marked down-modulation of IgD and substantial differentiation of plasma cells that were phenotypically identified as CD19low/ϩIgDϪCD38high 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 IgDϪCD38high plasma cells; the majority are IgDϩCD38low/int naive B cells, and a smaller percentage are IgDϪCD38Ϫ/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 IgDϩ/IgMϩ 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 FIGURE 3. Large numbers of plasma cells are induced following co- proliferative response that was comparable to that noted with anti- culture with IL-21. A, Purified PB B cells were cultured as described in Fig. Downloaded from CD40 and IL-21. As with the anti-IgM and IL-21 costimulation, 2, and the mean percentage (ϮSEM) of plasma cells from seven indepen- IL-2 also increased the magnitude of the response when cells were dent experiments is shown. B, The absolute plasma cell number following stimulated with the combination of IL-21, anti-IgM, and 7 days in culture from one representative experiment is also shown. There anti-CD40. was approximately a total of 500 plasma cells per well in the initial pop- Flow cytometric evaluation was conducted to assess the impact ulation of cells.

of IL-21 on human B cells in greater detail. When negatively se- http://www.jimmunol.org/ lected peripheral B cells were cultured with cytokines alone, mod- ϩ ϩ est changes in B cell phenotype were noted as determined by IgD (31). These include both IgD CD27 B cells as well as Ϫ ϩ and CD38 expression (Fig. 2C, a–d). It was notable, however, that IgD CD27 postswitched B cells. To address whether IL-21 had IL-21 in the absence of any other stimulus increased the percent- the capability to drive plasma cell differentiation from both naive age of IgDϪCD38high plasma cells modestly, and this effect was and memory B cells, cord blood B cells were used as a natural Ϫ ϩ ϩ Ϫ augmented by IL-2 (Fig. 2C, c and d). The ability of IL-21 to source of naive CD27 IgD B cells, and CD27 (IgD and ϩ induce moderate plasma cell differentiation was also observed with IgD ) memory B cells were isolated from PB by cell sorting. As anti-IgM-stimulated B cells, and again there was an increase in the shown in Fig. 4, A and B, IL-21 costimulated considerable prolif- presence of IL-2 (Fig. 2C, g and h). In addition, IL-21 induced a eration of both naive cord blood and memory B cells. Although by guest on September 23, 2021 striking loss in the numbers of B cells in anti-IgM-stimulated cul- cord blood B cells costimulated with IL-21 proliferated less well ϩ tures (Fig. 2C, compare g to e), which was largely reversed by the than adult CD27 memory B cells (compare Fig. 4, B to A), the addition of IL-2 (Fig. 2C, compare h to g). Moreover, IL-21 or the capacity of IL-21 to down-modulate IgD and induce differentiation combination of IL-2 and IL-21 induced marked down-regulation of plasma cells was dramatic in both populations (Fig. 4, C and D). of surface IgD by anti-IgM-stimulated B cells (Fig. 2C, g and h). IL-21 with anti-CD40 or anti-IgM and anti-CD40 (but not with IL-21 also induced IgD down-modulation by anti- anti-IgM alone) had the capacity to induce the generation of ϩ Ϫ ϩ ϩ CD40-stimulated B cells, as well as a dramatic increase in cellular plasma cells from nearly all the CD27 IgD and CD27 IgD expansion and plasma cell differentiation that was observed as memory B cells (Fig. 4D). More notable was the finding that early as day 4 of culture (Fig. 2C, compare k and l to i and j and nearly all surviving IL-21-stimulated cord blood B cells activated s). T cell-dependent B cell responses involve engagement of both with either anti-CD40 or anti-IgM and anti-CD40 had down-mod- surface Ig as well as CD40 by CD154 expressed by activated T ulated IgD and most differentiated into plasma cells. It is important cells. It was therefore of interest to examine the impact of IL-21 on to note that neither anti-CD40 nor anti-IgM and anti-CD40 stim- B cells stimulated through both the BCR and CD40. We found that ulation of cord blood B cells alone led to the generation of any the combination of anti-IgM and anti-CD40 in the presence of plasma cells in the absence of IL-21 (Fig. 4C). However, IL-21 IL-21 resulted in down-modulation of IgD by nearly all B cells induced the down-modulation of IgD on stimulated cord blood B (Fig. 2C, compare o to m). Moreover, the largest percentage of cells within 3 days of culture (data not shown), and by day 6 many plasma cells was generated when both surface IgM and CD40 were plasma cells were observed (Fig. 4C). In contrast, IL-21-induced engaged, and the cells were costimulated with IL-21 in the pres- differentiation of plasma cells from adult PB B cells could be ob- ence or absence of IL-2 (Fig. 2C, o and p). Importantly, evaluation served within 4 days of culture (data not shown). of CFSE dilution revealed that IL-21 induced the differentiation of plasma cells from a dividing B cell precursor that had diluted IL-21 induces Ig secretion from both naive and memory B cells CFSE with or without costimulation (Fig. 2D). Numeric calcula- Analysis of culture supernatants confirmed that IL-21 induced the tion in repetitive experiments demonstrated the significant increase secretion of Ig. Unlike IL-2, or cultures with no added cytokines, in both the percentage (Fig. 3A) and absolute cell number (Fig. 3B) IL-21 costimulated considerable production of both IgG and IgM of plasma cells in cultures costimulated with IL-21. 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, IL-21 induces plasma cell differentiation from naive cord blood A and B). The amount of Ig produced generally correlated with the B cells frequency of plasma cells in the cultures. IgM and IgG could be CD27 expression denotes that human B cells have somatically mu- assayed in the culture supernatants as early as day 4 of culture tated Ig genes and, therefore, are considered to be memory B cells (data not shown). It is notable that IL-21 induced large amounts of The Journal of Immunology 7871

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 CD20ϩCD27ϩ 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 CD27ϩ memory B cells (B). The purity of the cord blood and CD20ϩCD27ϩ memory B cells before culture is shown in (C and D). Neither cord blood B cells nor CD20ϩCD27ϩ memory cells contained identifiable plasma cells before culture. After 6 days of culture, the Downloaded from 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 http://www.jimmunol.org/ for CD27ϩ-sorted PB B cells.

Ϫ IgG from both naive CD27 adult and cord blood B cells (Fig. 5, To evaluate whether IL-21-induced plasma cells were termi- by guest on September 23, 2021 B and C), consistent with its capability to function as a switch nally differentiated nondividing cells, cell cycle analysis was also recombination factor for human B cells. All IgG isotypes were conducted. To assess cell cycle position, B cells were stimulated produced from adult total PB B cells stimulated with anti-CD40 with IL-21, anti-IgM, and anti-CD40 and after 7 days of culture the and anti-IgM in the presence of IL-21, whereas cord blood B cells resultant plasma cells were identified either as IgDϪCD38high (Fig. Ϫ produced largely IgG3. Moreover, CD27 naive adult B cells pro- 7B) or CD27highCD38high cells that were differentiated from duced both IgG1 and IgG3 (Fig. 5C). A similar pattern was also CD27lowCD38low nonplasma cells (Fig. 7C). Both populations noted when the various B cell populations were stimulated with the were analyzed for cell cycle progression by PI staining (Fig. 7D). combination of IL-2, IL-21, anti-CD40, and anti-IgM (Fig. 5C), As shown in Fig. 7D, approximately one-third of IL-21-induced indicating that IL-2 exerted no isotype specificity as shown previ- plasma cells were cycling, whereas the majority were not cycling ously (30). In contrast, IL-21 influenced naive B cells at different as evidenced by the finding that they were not in the S, G ,orM stages of maturation to switch to specific Ig isotypes, IgG3 for cord 2 phase of the cell cycle. It is notable that approximately one-fourth blood B cells and IgG1 and IgG3 for adult naive B cells. In ad- of the other B cells in the culture were in cell cycle after 7 days of dition, stimulation with IL-21 and anti-CD40 or IL-21, anti-CD40 culture, although 46% of these nonplasma cells were found to be and anti-IgM of PB B cells had no effect on IgE production (data in S/G /M on day 3, before plasma cell differentiation (data not not shown), but did costimulate IgA production without or with 2 shown). IL-2 (1–2 ␮g/ml vs 4–6 ␮g/ml IgA, respectively). Finally, to address whether IL-21-induced plasma cells had dif- IL-21 induces the differentiation of both cycling and terminally ferentiated to nondividing Ig-secreting cells, Ig production was differentiated cells examined in the presence of the inhibitor of proliferation, HU. HU We next addressed the state of the IL-21-induced plasma cells both is an inhibitor of proliferation that prevents Ig secretion by cycling by cell surface phenotype and cell cycle analysis. In addition to but not noncycling Ig-secreting cells (17, 32). The addition of HU being CD19low/ϩIgDϪCD38high, the majority of IL-21, anti-CD40, at the initiation of culture resulted in a complete block of IL-21- and anti-IgM-induced plasma cells (red) were also found to ex- induced proliferation and plasma cell differentiation (Fig. 8, A and press other typical plasma cells markers such as IL-6R and B cell B). However, when HU was added to the cultures at day 8 after maturation Ag (BCMA) (Fig. 6). Furthermore, when compared differentiation of plasma cells, persistence of these cells was noted with IgDϪCD38low cells (blue), these plasma cells were CD40ϩ, over the next 3 days of culture (Fig. 8B). Importantly, the amount CD95low, HLA-DRlow, CD9high, CD63high, CXCR4low/ϩ, of Ig doubled during the last 3 days of culture, which was not CD44high, CD49dlow, CD31high, and CD62Lhigh (Fig. 6). The ma- blocked by HU, indicating that the ongoing production of Ig by jority of the IL-21-driven plasma cells were also CD19low, plasma cells was not dependent on ongoing proliferation. These CD20low, CD22low, CD21low, and CD27high (data not shown). data demonstrate that the production of Ig by IL-21-induced 7872 IL-21 DRIVES HUMAN PLASMA CELL DIFFERENTIATION

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 CD27Ϫ and CD27ϩ B Downloaded from cells (C). Production of total IgG or IgM from purified total peripheral B cells (A) or cord blood B cells (B). Se- cretion of specific Ig isotypes from naive cord blood B cells (CB) (C) as well as from PB naive (CD27Ϫ), memory (CD27ϩ), and total (PB) B cells were quantitated. A and B, IgM levels were not measured in any cultures con- Ϯ taining anti-IgM. Data are mean concentration ( SD) http://www.jimmunol.org/ from one of seven and six representative experiments, for A and B, respectively, and one representative experiment of nine cord blood and three with CD27Ϫ and CD27ϩ adult peripheral B cells (C). by guest on September 23, 2021

plasma cells largely reflects secretory activity of terminally differ- mRNA (Fig. 9A, d). To determine whether IL-21 had the ability to entiated, nondividing cells (Fig. 8C). induce these transcription factors in naive B cells, experiments using cord blood B cells were undertaken. To obtain sufficient IL-21 induces expression of BLIMP-1, AID, and Bcl-6 mRNA, mRNA for analysis, cord blood B cells were cultured with viability but not SHM promoting cytokines, IL-2 and IL-4, which in preliminary exper- A variety of transcription factors are known to regulate specific iments did not induce the mRNAs analyzed. IL-21 (in the presence stages of B cell maturation. It was, therefore, of interest to deter- of IL-2) induced a significant increase in BLIMP-1 mRNA in na- mine whether IL-21 costimulation would up-regulate expression of ive anti-CD40 or anti-CD40 and anti-IgM-stimulated cord blood B BLIMP-1, which is essential for plasma cell differentiation (33), cells, whereas the combination of IL-2 and IL-4 exhibited no such AID, which is involved in CSR (34), Bcl-6, which is involved in activity (Fig. 9B). IL-21 also costimulated AID mRNA expression, germinal center reactions (35), or PAX-5, which is required for the but no more effectively than the combination of IL-2 and IL-4 (Fig. generation of B cells (36). IL-21 induced expression of both 9B). In contrast, IL-21 had little effect on Bcl-6 or PAX-5 mRNA BLIMP-1 (Fig. 9A, a) and AID (Fig. 9A, b) mRNA. BLIMP-1 was levels. induced by IL-21-costimulated B cells activated with anti-CD40 or It was notable that IL-21 costimulation increased AID mRNA anti-IgM and anti-CD40, and less so with anti-IgM only (Fig. 9A, levels, differentiation of IgDϩ and IgDϪ B cells, CSR, generation a). However, in the presence of IL-2, IL-21 also induced BLIMP-1 of plasma cells, and Ig production, but it could not induce SHM. expression without additional costimulation. AID mRNA was in- Compared with unstimulated cord blood B cells, no significant duced by IL-21 when B cells were activated with anti-IgM, anti- increase in the percentage of sequences with VH gene mismatches CD40, or both anti-IgM and anti-CD40, and was enhanced by the was detected in isolated IgDϪCD38high plasma cells induced from presence of IL-2. Bcl-6 mRNA was induced to a lesser degree by cord blood B cells stimulated with IL-2 and IL-21 in the presence IL-21 (Fig. 9A, c), whereas IL-21 had little effect on PAX-5 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 anti- CD40 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). Downloaded from

Discussion 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 differ-

entiation of memory and plasma cells. Studies in the mouse have http://www.jimmunol.org/ defined a number of these specific processes and factors. Recently, IL-21 has been shown to be an essential factor in the generation of FIGURE 6. Cell surface phenotype of IL-21-induced plasma cells. Pu- plasma cells that produce IgG (10). Less is known about the con- rified PB B cells were isolated and cultured with IL-21, anti-IgM, and trol of these processes in humans. Although a number of cytokines anti-CD40. After 7 or 8 days of culture, CD19ϩ cells were subdivided into and T cell influences are known to regulate B cell responses, the Ϫ Ϫ IgD CD38low cells (blue histogram) and IgD CD38high plasma cells (red specific factors that stimulate the differentiation of naive human B histogram) and further analyzed for expression of the markers indicated. cells into plasma cells secreting IgG have not been well defined. The data are representative of results from one of two experiments. Indeed, both adult and especially cord blood naive human B cells have been shown to have a poor propensity to differentiate into by guest on September 23, 2021 plasma cells in vitro, especially in the presence of purified costimulators (11, 13, 21, 25, 27). However, a modest capacity to percentage of sequences with mismatches most likely reflected un- differentiate into Ig-producing plasma cells has been reported identified VH gene polymorphisms or Taq-induced PCR misincor- when naive B cells are stimulated through CD40, in the presence poration events, rather than bona fide somatic mutations. 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). IL-4 suppresses plasma cell generation promoted by IL-21 Others have found no plasma cell generation when naive CD27Ϫ The effects of IL-21 can be modulated by other cytokines. As B cells are stimulated with CD40L transfectants in combination shown in Fig. 10A, IL-4 partially inhibited the down-modulation of with IL-2, IL-4, IL-6, IL-10, and IL-12 (21). surface IgD induced by IL-21 in cells stimulated with the poly- We now show that IL-21 is a powerful costimulator of human clonal B cell activator, SAC (Fig. 10A, compare e to f). Moreover, CSR and plasma cell differentiation, but not SHM. Importantly, IL-4 also inhibited IgD down-modulation induced by IL-21 and IL-21 can promote both CSR and plasma cell differentiation from anti-IgM or the combination of anti-IgM and anti-CD40 (Fig. 10A, naive as well as memory B cells. Notably, the combination of compare h and n to i and o, respectively), but not by anti-CD40 anti-IgM and anti-CD40, which most closely mimics B cell acti- alone (Fig. 10A, compare l to k). Importantly, IL-21-induced gen- vation via Ag and T cell interaction, was the most effective signal eration of plasma cells was also inhibited by IL-4 in cultures co- in promoting the maximal differentiation of plasma cells driven by stimulated by SAC (Fig. 10A, compare e to f), or in cultures con- IL-21. Although anti-CD40 and IL-21 induced considerable CSR taining the combination of IL-21, anti-IgM, and anti-CD40 (Fig. and plasma cell differentiation, the “T cell only” signal was some- 10A, compare n to o) but not in cultures of IL-21 and anti-CD40 what less effective than the combination. Isolated BCR cross-link- (Fig. 10A, compare k to i) reproducibly. This was reflected in the ing, which mimics the “Ag-only” signal, primed B cells for IL- quantity of secreted Ig (Fig. 10B), as well as the levels of AID or 21-mediated death (Fig. 1C, g, and our unpublished observations). BLIMP-1 mRNA (Fig. 10C). Thus, IL-4 inhibited IL-21-induced These results suggest that IL-21 is pivotal in cell fate decisions by up-regulation of Ig production as well as BLIMP-1 and AID mR- activated B cells and may function to eliminate B cells that have NAs in cultures stimulated by SAC or anti-CD40 with anti-IgM, been activated by Ags or autoantigens in the absence of T cell but not reproducibly in those stimulated by anti-CD40. Notably, signals. IL-2 did not reproducibly affect Ig production or BLIMP-1 or AID IL-21 has been reported to be a switch factor for IgG1 and IgG3 mRNA levels in these cultures. Finally, the addition of IL-2 did not (26), although in this study we found that the nature of the B cell rescue IgG production in IL-4-suppressed cultures (Fig. 10B), but population contributed to the specific Ig produced. Thus, IL-21- did allow for the differentiation of IgM-producing plasma cells stimulated cord blood B cells predominantly switched to IgG3, (data not shown). whereas naive adult B cells switched to both IgG1 and IgG3. The 7874 IL-21 DRIVES HUMAN PLASMA CELL DIFFERENTIATION

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 anti-

CD38, or anti-CD27, anti-CD38, and PI. For the latter, Downloaded from cells were subdivided into either CD27Ϫ and CD27ϩ, 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. http://www.jimmunol.org/ by guest on September 23, 2021

molecular basis for the differences in these outcomes is currently CD27ϩ memory B cells was preceded by the induction of unknown, but clearly the nature of the B cell population contrib- BLIMP-1 up-regulation. We have also found that IL-21 induced utes to the specificity of switch recombination. Notably, memory B BLIMP-1 expression in murine splenic B cells (10). The capacity cells were induced by IL-21 to produce all IgG isotypes as well as of IL-21 to up-regulate BLIMP-1 may explain its ability to drive ϩ IgA. This is somewhat different from previous results with CD19 plasma cell differentiation. In addition, IL-21 costimulation in- splenic B cells in which IgA was not induced by IL-21 (26). The duced AID expression, which is normally down-modulated by explanation for this difference is currently unknown. This may BLIMP-1 (38). Although AID is required and believed to be the B reflect differences in peripheral and splenic B cells. cell-specific factor that is sufficient for both CSR and SHM (34), The combination of IL-2 and IL-10 has been reported to induce IL-21 costimulation induced CSR, but not SHM. Similar findings human plasma cell differentiation from memory, but not naive B have been reported with CD27Ϫ B cells stimulated with the com- cells (13, 20, 25). In contrast to IL-21, a variety of other cytokines, bination of SAC, IL-2, IL-10, and cross-linked anti-CD40 (18). Of including IL-2, IL-4, IL-6, and IL-10 (data not shown), had min- note, we found that IL-4 and CD40 ligation induced AID in cord imal ability to support the generation of plasma cells from com- blood B cells, but neither CSR nor plasma cell differentiation. Pre- parably stimulated B cells. Importantly, although the combination viously, AID has been shown to be induced by IL-4, anti-CD40, or of IL-2, IL-10, and anti-CD40 did induce some plasma cell differentiation from PB B cells, this stimulus could not induce plasma the combination of both IL-4 and anti-CD40 in adult B cells (26, cell differentiation from naive cord blood B cells and has been 39, 40). It is of particular interest that AID was induced compa- reported previously (25). These data indicate that IL-21 was a far rably by IL-4 and IL-21, although only the latter induced CSR. In more potent inducer of plasma cell differentiation than the com- this regard, engagement of CD40 induced both AID and SHM in bination of IL-2 and IL-10 and may be uniquely able to foster human Ramos B cell lines (41, 42). These results suggest that differentiation of plasma cells from naive B cells. up-regulation of AID may have different effects on B cells at dif- BLIMP-1 is a transcriptional repressor that is necessary/suffi- ferent stages of differentiation. Moreover, our data indicate that the cient for plasma cell differentiation in the mouse, and in human B induction of AID mRNA is not sufficient to induce CSR or SHM cell lines (33, 37, 38). Consistent with this, IL-21-driven plasma in all circumstances. The property of IL-21 that permits it to induce cell differentiation from both naive cord blood B cells as well as both AID and CSR is currently unknown, but of great interest. The The Journal of Immunology 7875

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 ϫ 10Ϫ2 M) as indicated. After 3 days of culture, proliferation was determined by incubating the cells for 16 h with Downloaded from [3H]thymidine. B, Puriﬁed PB B cells were isolated and cultured with or without IL-21 in the presence of anti- CD40 and anti-IgM, and HU was added (or not) on the days indicated. The cultures were analyzed for IgD and CD38 expression by CD19ϩ B cells after 8 or 11 days in culture as indicated. C, Supernatants from IL-21, anti- IgM, and anti-CD40-stimulated cultures in B were ana- http://www.jimmunol.org/ lyzed for IgG content. HU was added where indicated. Data are representative of results from one of three experiments with similar results. by guest on September 23, 2021

ﬁnding that IL-21 can induce AID and CSR but not SHM is rem- tiation. This seems unlikely for a number of reasons. First, IL-21 iniscent of the recent delineation of AID function, indicating that alone had little capacity to maintain the few plasma cells that were different portions of the molecule govern CSR and SHM. Presum- found in the initial population of peripheral CD19ϩ cells. Sec- ably IL-21 can induce CSR, the activity of AID that is governed by ondly, in cultures costimulated with IL-21, plasma cells appeared the C-terminal portion of the molecule and involves the capacity to after 3–6 days, whereas in the absence of IL-21 few if any cells bind DNA-protein kinase catalytic subunit (43), whereas it is not with the phenotype of plasma cells were noted. This was most able to induce SHM that is regulated by the N-terminal of the notable in cultures of cord blood B cells. Therefore, it is unlikely protein by unknown molecules (44). The biological basis of this that IL-21 functions as a growth factor for plasmablasts induced by functional discrimination is an important area for future investi- anti-IgM or/and anti-CD40 signaling, but rather plays an essential gation. Finally, the combination of IL-2, IL-10, and anti-CD40 was role in the decision to differentiate into nondividing, high-rate Ig- 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 secreting cells. The capacity of IL-21 costimulation, but not anti- CSR, and may explain its inability to induce the differentiation of CD40 or anti-IgM alone, to induce BLIMP-1 mRNA is consistent postswitched plasma cells from naive cord blood B cells. More- with this conclusion. ␥ over, the data suggest that the capacity to induce BLIMP-1 may be The effects of other cytokines that bind C-containing cytokine necessary for plasma cell differentiation, but may not be the sole receptors on the actions of IL-21 were noteworthy. IL-2 has pre- determinant of the ability of IL-21 to induce the differentiation of viously been shown to drive plasma cell differentiation when B large numbers of plasma cells from both naive and memory B cells are activated with SAC (30), as well as costimulate plasma cells. cell differentiation in the presence of anti-CD40 and IL-10 (13, 25) It was possible that IL-21 may have functioned as a plasma cell or IL-6 (14, 17). IL-4, in contrast, has been shown to both promote survival factor, rather than as an initiator of plasma cell differen- and inhibit B cell responsiveness to various stimuli (13, 23, 45– 7876 IL-21 DRIVES HUMAN PLASMA CELL DIFFERENTIATION

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, veri- fied 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 (ϮSEM) 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, IgDϪCD38high 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 Downloaded from 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-IgM- stimulated cultures) from IL-21-induced plasma cells was not significantly different from 141 sequences from unstimulated http://www.jimmunol.org/ cord blood B cells examined immediately ex vivo (␹2 test, p ϭ 0.2745). Combined results from four experiments are shown.

48). Most notable is the ability of IL-4 to inhibit SAC and IL-2- combination of IL-4 and IL-21. Moreover, the origin of the B cells driven plasma cell differentiation (45, 47). We found that IL-2 may play a role because IL-4 has been shown to enhance Ig pro- enhanced the effect of IL-21 on plasma cell differentiation induced duction by IL-21 and anti-CD40-activated CD27ϩ human splenic by the combination of anti-IgM and anti-CD40, whereas IL-4 in- memory B cells (26). These differences may reflect variability in hibited it. These results suggest an antagonistic effect of IL-4 on the cytokine responsiveness of B cells from various compartments by guest on September 23, 2021 the actions of IL-21 in B cells stimulated by anti-IgM or the com- because IL-4 did not consistently inhibit or enhance anti-CD40 and bination of anti-IgM and anti-CD40. It is notable that antagonism IL-21-induced plasma cell differentiation by peripheral B cells. between IL-4 and IL-21 was not consistently observed when cells These findings make it unlikely that receptor competition can ex- were stimulated with anti-CD40 alone, indicating that the nature of plain these results, owing to the fact that both IL-21 and IL-4 use ␥ the stimulus influences the ability of B cells to respond to the 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. Be- fore 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 CD19ϩ 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 (ϮSEM). C, mRNA was isolated and BLIMP-1 and AID expression was determined after 3 days of culture. All data in C were nor- ␤ malized 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 (ϮSEM). Data are representative of results from cultures of two similar experiments with SAC, three with anti-IgM, five with anti- CD40, 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. The Journal of Immunology 7877

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 Downloaded from 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, http://www.jimmunol.org/ 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 (ϮSEM). Data are representative of results from one of two similar experiments. by guest on September 23, 2021

␥ through a receptor-containing C, reversed the IL-4 mediated-in- ation. In the appropriate signaling context, as discussed above, the hibition of IgM-producing plasma cell generation (data not actions of the two cytokines appear to be mutually inhibitory of shown), making the idea of receptor competition more remote. one another. In this regard, IL-21 has been reported to inhibit pro- Furthermore, IL-2 synergized with IL-21 in fostering growth of liferation of both murine and human B cells stimulated with anti- anti-IgM and anti-CD40-stimulated B cells. IgM and IL-4 (2, 7, 10) and to inhibit IL-4-induced IgE transcrip- Taken together, these results suggest that the stimulus-depen- tion (52). In this study, IL-4 repressed IL-21-driven plasma cell dent inhibition of IL-21 responses by IL-4 relates to the down- differentiation following either SAC or anti-IgM and anti-CD40 stream signals generated by engagement of the IL-4R. In this re- stimulation. It is of considerable interest that IL-4R ligation in- gard, IL-4 is known to costimulate IgE synthesis by human B cells duces primarily STAT6, which is essential for IL-4 signaling, in- (48), and also induce the expression of CD23 on mature B cells cluding IL-4-induced up-regulation of AID in both mice and hu- (50). However, IL-4 also has been reported to repress human B cell mans (39, 40), whereas IL-21R engagement activates primarily responses (45, 47), and inhibit BLIMP-1 induction as well as STAT1 and -3 and more weakly STAT5 (1, 53, 54). Although the plasma cell differentiation in mice (51). It is notable that in our level of antagonism is unknown, the data suggest that IL-21 and studies, with the appropriate costimulation, IL-4 repressed IL-4 inhibit one another’s actions after receptor engagement. Bi- BLIMP-1 expression, and inhibited plasma cell differentiation, ologically, T cell-dependent B cell responses may be regulated by whereas IL-21 induced both BLIMP-1 and plasma cell differenti- T cells that either produce IL-21, IL-4, or other cytokines at 7878 IL-21 DRIVES HUMAN PLASMA CELL DIFFERENTIATION different times during an immune response, or different subsets of and its receptor are involved in NK cell expansion and regulation of lymphocyte T cells that express different cytokine profiles. In support of this, function. Nature 408: 57–63. 3. Asao, H., C. Okuyama, S. Kumaki, N. Ishii, S. Tsuchiya, D. Foster, and IL-21 has been described to be produced by a restricted subset of K. Sugamura. 2001. Cutting edge: the common ␥-chain is an indispensable sub- follicular Th cells that express CXCR5 in humans (6). unit of the IL-21 receptor complex. J. Immunol. 167: 1–5. 4. Collins, M., M. J. Whitters, and D. A. Young. 2003. IL-21 and IL-21 receptor: a Our final question was whether IL-21 had the ability to induce new cytokine pathway modulates innate and adaptive immunity. Immunol. Res. terminally differentiated nondividing plasma cells or merely divid- 28: 131–140. ing Ig-secreting plasmablasts. Ig-secreting cells have been shown 5. Leonard, W. J. 2001. Cytokines and immunodeficiency diseases. Nat. Rev. Im- munol. 1: 200–208. to be quite heterogeneous, and different stages of plasma cell de- 6. Chtanova, T., S. G. Tangye, R. Newton, N. Frank, M. R. Hodge, M. S. Rolph, and velopment have been described that can be identified phenotypi- C. R. Mackay. 2004. T follicular helper cells express a distinctive transcriptional cally (55, 56). The majority of plasma cells in the germinal center profile, reflecting their role as non-Th1/Th2 effector cells that provide help for B cells. J. Immunol. 173: 68–78. are short lived, produce Ab rapidly and briefly, and then die in 7. Ozaki, K., R. Spolski, C. G. Feng, C. F. Qi, J. Cheng, A. Sher, H. C. Morse, 3rd, secondary lymphoid tissue. In contrast, a subpopulation matures C. Liu, P. L. Schwartzberg, and W. J. Leonard. 2002. A critical role for IL-21 in into terminally differentiated nondividing plasma cells that exit regulating immunoglobulin production. Science 298: 1630–1634. 8. Jin, H., R. Carrio, A. Yu, and T. R. Malek. 2004. Distinct activation signals lymphoid tissue and migrate to the bone marrow where they reside determine whether IL-21 induces B cell costimulation, growth arrest, or Bim- as long-lived plasma cells that produce Ab for long periods of time dependent apoptosis. J. Immunol. 173: 657–665. 9. Mehta, D. S., A. L. Wurster, M. J. Whitters, D. A. Young, M. Collins, and (55). These stages of plasma cells can be identified both function- M. J. Grusby. 2003. IL-21 induces the apoptosis of resting and activated primary ally and phenotypically (56). It is notable that by phenotypic anal- B cells. J. 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