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Interleukin-21 (IL-21) plays a key role in the late stage of B cell development, where it has been shown to induce growth and differentiation of mature B cells into Ig-secreting plasma cells. Since IL-21 receptors (IL-21R) have also been reported on bone marrow (BM) B cell progenitors, we investigated whether IL-21R influenced earlier stages of B cell development. IL-21R is functional as early as the proB cell stage and the strength of receptor-mediated signalling increases as cells mature. The addition of IL-21 to B cell progenitors in cell culture resulted in the accelerated appearance of mature B cell markers and was associated with the induction of Aid, Blimp1, and germline transcripts (GLTs). We also found that stimulation of both IL-21R and CD40 was sufficient to induce the maturation of early B cell progenitors into IgM- and IgG-secreting cells. Consistent with a role for IL-21 in promoting B cell differentiation, the number of B220+CD43+IgM− proB cells was increased and the number of mature IgMhiIgDhi cells was decreased in BM of IL-21R-deficient mice (ILR-21−/−). We also report here that IL-21 is expressed by BM CD4+ T cells. These results provide the first evidence that IL-21R is functional in B cell progenitors and indicate that IL-21 regulates B cell development.
The generation of mature, Ag-responsive B cells from pluripotent stem cells is directed by signals provided by the supportive microenvironment in the bone marrow (BM). Some of these signals are mediated by the action of soluble cytokines. At each developmental stage, B cells express a distinct profile of cytokine receptors on their cell surface to sense these signals. In mouse, one critical phase of B cell development in the BM is regulated by the cytokine IL-7. It has been established that IL-7 promotes proliferation, survival, and development of proB cells toward the preB cell stage (1–4). Furthermore, our lab has previously shown that once the preBCR is expressed at the cell surface, integrated signals from both the IL-7R and the preBCR allow cells to proliferate in reduced concentrations of IL-7 (5).
IL-7 is a member of the common γ-chain-dependent cytokine family, which includes IL-2, IL-4, IL-9, IL-15, and IL-21 (6). Like IL-7, IL-21 has also been shown to play a key role in B cell development. However, in contrast to IL-7, current data show that IL-21 exerts its effect at later stages when B cells differentiate into plasma cells (6–8). The first evidence for the importance of IL-21 in B cell differentiation was provided by studies carried out on IL-21R-deficient (IL-21R−/−) mice (9). IL-21R−/− mice exhibited a severe defect in IgG1 production, while the secretion of IgE was augmented, both at steady-state and following immunization with T cell-dependent Ag. Using human EBV-infected B cell lines, we have demonstrated that IL-21-mediated signalling through the JAK/STAT pathway was required for the differentiation of B cells into late plasmablasts/early plasma cells (10). In mature B cells, binding of IL-21 to the IL-21R induces activation of STAT1, STAT3, and to a lesser extent STAT5 (10, 11).
Multiple studies have demonstrated that the effects of IL-21 stimulation of B cells depend on the cell signalling context (12–14). For instance, in the presence of signalling through both the BCR and CD40, IL-21 promotes growth and differentiation of murine splenic B cells into Ig-secreting cells (12, 13). Conversely, growth arrest and apoptosis occur following addition of IL-21 to B cells stimulated with lipopolysaccharides (LPS), CpG, or anti-IgM in the absence of T cell help (12, 14). These and other results have led to a consensus that the IL-21-IL-21R system is context-dependent and plays an important role in maintaining B cell homeostasis. One consequence of a breakdown in this system could be impaired elimination of autoreactive B cells and the subsequent development of Ab-mediated autoimmune diseases like systemic lupus erythematosus (SLE). In this regard, lupus prone BXSB.B6-Yaa+/J mice showed elevated levels of serum IL-21 (13). In addition, a recent study has shown that BXSB-Yaa+/J mice deficient for IL-21R failed to develop the disease (15).
To date, the majority of IL-21 studies have focused on its role in the final stage of B cell differentiation. However, IL-21R has also been reported to be expressed on early B cells progenitors in the BM (12), although it is not known whether it is functional and involved in the regulation of B cell development in the BM. One study reported that IL-21R−/− mice have normal numbers and phenotypes of B cells in the BM (9), although several specific populations of B cell progenitors, of interest to us, were not examined. While this study shows that IL-21 does not have an essential, non-redundant role in B cell development, more recent data show that IL-21 can contribute to early hematopoiesis. It has been found that murine hematopoietic progenitor c-kit+sca+lin−/low (KSL) cells express low levels of IL-21R (16). When grown in vitro with a cocktail of c-kit ligand, Flt-3L and IL-7, proliferation of KSL cells is enhanced in the presence of IL-21 (16). In addition, overexpression of IL-21 in vivo increased the number of KSL cells in the spleen (16). Another study showed that IL-21 did not have any mitogenic effect on total murine BM cells. However, apoptosis of BM CD11b− lymphoid cells that expressed IL-21R was delayed when the cells were cultured in the presence of IL-21 (17). Based on this information and the fact that very few B220− cells express IL-21R (12), the authors hypothesized that IL-21 acts mainly on lymphoid B220+ B cell subsets in the BM. Finally, it has been reported that IL-21 transgenic mice have increased number of immature B cells in the spleen (13). One explanation for this phenotype could be increased maturation of BM B cell precursors. Collectively, these studies indicate that further investigation is required to determine the exact role of IL-21 in development of B cell progenitors in the BM.
In this study, we show that IL-21 message is constitutively expressed in murine BM CD4+ T cells. IL-21R is expressed and is functional on all subsets of B cell progenitors, including proB, preB and immature/mature B cells. In vitro culture of B cell progenitors with IL-21 is sufficient to induce expression of Aid and Blimp1, and germline transcripts (GLTs) in these cells, and to accelerate their development into mature B cells. A role for IL-21 in promoting B cell maturation is further supported by data that show increase in numbers of proB cells and decrease in number of mature B cells in BM of IL-21R−/− mice. Finally, stimulation of the IL-21R and CD40 on B cell progenitors results in the formation of Ig secreting cells.
Female C57Bl/6 mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA) at 6–8 weeks of age. IL-21R−/− mice were provided by Dr. Warren J. Leonard. Mice were bred at the Ontario Cancer Institute (OCI) and sacrificed according to a protocol approved by the animal care committee of the OCI.
Single cells suspension from BM was prepared by crushing and gently grinding both femurs and tibiae using a mortar and pestle. Cells were pelleted and resuspended in red blood cell lysis buffer (150 mM NH4Cl, 100 mM NaHCO3, 1 mM EDTA pH 8.0) for 1 minute on ice. Recovered cells were filtered through a 40 μm cell strainer and cultured in freshly reconstituted OptiMEM supplemented with 10% non-heat inactivated FCS, 50 μM β-mercaptoethanol, 2.4 g/L NaHCO3, 100 μg/mL penicillin-streptomycin, and grown at 37°C in a 5% CO2 atmosphere. Supernatant from the stably transfected J558 line was used as a source of IL-7 (supplied by Dr. Ana Cumano, Institut Pasteur, Paris). 30 ng/mL IL-21 and/or 2 μg/mL anti-CD40 were added to the culture where indicated.
BM cells were isolated by gently grinding both femurs and tibiae using a mortar and pestle. The cells were then flushed with MACS buffer (PBS –Ca2+, −Mg2+, 1 mM EDTA, 0.25% BSA). B cell progenitors were isolated directly using anti-B220 Ab coupled to beads (clone RA3-6B2, Miltenyi). A VarioMACS magnet with an LS adaptor was used to positively select the cells. Cells were washed three times with MACS buffer and eluted in OptiMEM (Life Technologies). Typically, 8–14 × 106 B220+ cells are recovered from BM (two femurs and two tibiae) of a mouse.
B220+ cells isolated by MACS were cultured with IL-7 for 4 days (day 4IL-7) as described above. On day 4, cells were labeled with appropriate Abs and sorted using a FACS Aria (BD Bioscience) to a greater than 98% purity. In order to stain proB (CD2−κ/λ −), preB (CD2+κ/λ−), and immature/mature (CD2+κ/λ −) BM B cells, rat anti-mouse CD2-PE (RM2-5; eBioscience), rat anti-mouse κ-FITC (Southern Biotech) and rat anti-mouse λ-FITC (southern Biotech) were used.
For experiments presented in Fig.1, freshly isolated BM or BM B220− cells were stained with rat anti-mouse-B220-APC (RA3-6B2), -CD3-FITC (145-2C11), -CD4-biotin (GK1.5), -CD8-PE (53–6.7), -CD44-APC (IM7), -CD69-PE, and -NK1.1-APC (PK136) (BD Bioscience). Cells were then sorted into CD4 T cells (CD3+CD4+NK1.1−), CD8 T cells (CD3+CD8+NK1.1−), NK cells (NK1.1+) and B cells (B220+) (Fig.1B). To sort naive CD4 T cells (CD3+CD4+CD44loCD69−) and memory CD4 T cells (CD3+CD4+CD44hiCD69−) (Fig. 1C), BM from 10 mice was first enriched for CD4+ cells by negative selection using EasySep (StemCell Technologies) according to the manufacturer instructions.
Cells were washed with ice-cold PBS containing 3% (v/v) FCS and then incubated for 30 min on ice with predetermined concentration of FACS Abs in a total volume of 100 μL. The following Abs (clone) were used: IgM-biotin (33.60), B220-FITC, B220-APC (RA3-6B2; eBioscience), CD19-APC (MB19-1), CD2-PE (RM2-5; eBiosciene), CD43-PE (S7-5; BD Bioscience), IgD-FITC (clone SBA.1; Southern), IL-21R-biotin (eBio4A9; eBioscience), kappa-FITC (Southern Biotech), lambda-FITC (Southern Biotech), IgM (clone 33.60; made in-house), and rat IgG-biotin (R35-95; Pharmingen). For indirect staining, cells were washed twice after binding of the primary Ab and incubated with streptavidin-PerCP (BD Bioscience) for 15 min on ice. Samples were kept at 4°C in the dark and analyzed using a FASCcalibur (BD Bioscience). 10,000 cellular events were analyzed for each sample.
BM cells from 10 mice were incubated in red blood cell lysis buffer (150 mM NH4Cl, 100 mM NaHCO3, 1 mM EDTA pH 8.0) for 1 minute on ice and washed in PBS/FCS. To isolate the CD4+ population, BM cells were enriched for the CD4+ T cell population by negative selection using EasySep (StemCell Technologies) according to the manufacturer instructions and then labeled with an anti-CD4-PE Ab for sorting on a FACS Aria (BD Bioscience). Sorted BM CD4+ T cells were culture for 3 days with anti-CD3 (10 μg/mL), anti-CD28 (2 μg/mL) ± human IL-6 (100 ng/mL). On day 3, the supernatants were collected and the presence of IL-21 was detected by cytometric bead array (CBA) according to the manufacturer protocol (BD Bioscience), with the following modification: 75 μL of supernatant were added to 25 μL of beads.
Total RNA was isolated from total BM of C57Bl/6, Rag2−/− and TCRβ−/− mice or from BM B cell progenitors from C57Bl/6 mice using the Trizol reagent (GIBCO BRL) or RNeasy kit (Quiagen) according to the manufacturer’s instructions. First-strand cDNA was prepared from 0.5 to 3.0 μg of total RNA in 20 μL reaction volume using the Superscript II (Gibco Life Technology). After reverse transcription, Il21, Blimp1 and Aid were amplified by real-time PCR according to manufacturer instruction (Applied Biosystems). Amplification of actin was used for sample normalization. PCR primers used: Il21 5′-CGCCTCCTGATTAGACTTCG-3′ (sense) and 5′-TGGGTGTCCTTTTCTCATACG-3′ (anti-sense), Blimp1 5′-TAGACTTCACCGATGAGGGG-3′ (sense) and 5′-GTATGCTGCCAACAACAGCA-3′ (anti-sense), Aid 5′-GCGGACATTTTTGAAATGGTA-3′ (sense) and 5′-TTGGCCTAAGACTTTGAGGG-3′ (anti-sense), and β-Actin 5′-GCCAACCGTGAAAAGATGACCCAG-3′ (sense) and 5′-ACGACCAGAGGCATACAGGGACAG-3′ (anti-sense).
Semi-quantititative RT-PCR for Il21r and actin was performed on three serial dilutions of cDNA isolated from sorted into proB (CD2−LC−), preB (CD2+LC−), and immature/mature (CD2+LC+) B cell populations. PCR products were amplified using the following conditions: for Il21r: Ta = 67°C, 34 cycles, and for β-Actin: Ta = 58°C, 22 cycles. Amplification of β-Actin was used as a cDNA loading control. The β-Actin specific primers were 5′-TCCCTGGAGAAGAGCTACGA-3′ (sense) and 5′-ATCTGCTGGAAGGTGGACAG-3′ (anti-sense). Primers for Il21r were 5′-ATGCCCCGGGGCCCAGTGGCTG-3′ (sense) and 5′-CACAGCATAGGGGTCTCTGAGGTTC-3′ (anti-sense).
BM from C57Bl/6 was cultured for 4 days in IL-7 and then sorted into proB (B220+CD2−κλ −), preB (B220+CD2+κλ −), and immature/mature (B220+CD2+κλ+) B cells. Cells were then cultured without supplements (ctr), with IL-21 (30ng/mL), with anti-CD40 (2ug/ml), or anti-CD40/IL21 for 24 hours prior to RNA extraction. After cDNA synthesis samples were analysed for class-switch recombination. Primers for GLTγ2b were previously described (18). Amplification of GLTγ2b was done using the following conditions: for: Ta = 62°C, 40 cycles.
Sorted BM B cell progenitors or B220+ BM cells day 4IL-7 were stimulated with 50 ng/mL IL-21 or IL-7, for 15 min and then lysed in 1% NP40, 150 mM NaCl, 20 mM Tris-HCl (pH 7.4), 10 mM NaF, 1 mM sodium orthovanadate, 5 mM sodium pyrophosphate, 1 mM PMSF, 5 μg/mL aprotinin and leupeptin (Roche) on ice for 30 min. Equal amount of cell lysates were separated onto a 4–12% gradient NuPAGE gel, and then transferred to a PVDF membrane. Membranes were blocked with 5% milk in PBS/0.05% Tween/5% BSA (TBST) for 1 hour at room temperature and then probed overnight at 4°C for pSTAT3, pSTAT1, pSTAT5 (Cell signaling), or actin (NeoMarkers). After several washes in TBST, membranes were subsequently probed with a horseradish peroxidase-coupled goat anti-rabbit IgG Ab or peroxidase-coupled goat anti-mouse IgG diluted 1:10,000 in TBST containing 5% milk for 45 min. Detection was performed using the ECL substrate (Amersham Pharmacia Biotech) as described by the manufacturer.
Enzyme immunoabsorbant (EIA) plates (Costar; no. 3590) were coated with 5 μg/mL goat anti-mouse IgM (Jackson ImmunoResearch Laboratories), IgG1, IgG2a, IgG2b, IgG3 or IgA (Sigma) overnight at 4°C. Plates were washed with distilled water several times and blocked for 40 min at room temperature with 3%FCS/PBS. After washing, 50 μL of culture supernatant was added and plates were incubated at room temperature for 40 min. A standard curve was established using purified mouse IgM (Pharmingen), IgG1 κ (Sigma), IgG2a κ, IgG2b κ, IgG3 κ and IgA λ isotype standards (Pharmingen). Plates were washed several times with distilled water. Plate-bound Abs were detected after a 40 min incubation with anti-mouse IgM, anti-mouse IgG, or anti-mouse IgA conjugated to peroxydase (Sigma). After washing, BD OptEIA substrate (BD biosciences) was added to the plates and absorbance was read according to the manufacturer instructions.
Statistical significance was assessed by 2-tailed Student t test and the level of significance was established at p < 0.05.
To determine whether B cell progenitors could encounter IL-21 during development in the BM, we performed RT-PCR on BM cells isolated from WT mice kept in pathogen-free conditions. Our results show that Il21 mRNA was expressed in total BM of WT mice (Fig. 1A). To further define the source of Il21 message, we analysed BM of RAG−/− mice, which lack mature B and T cells but contain normal numbers of stromal cells and other hematopoietic cells, as well as BM of TCRβ−/− mice, which lack mature αβ T cells. We failed to detect the expression of Il21 mRNA in either RAG−/− and TCRβ−/− BM (Fig. 1A). These results suggest that Il21 message is expressed by BM lymphocyte subsets, most likely T cells. To establish whether BM T cells produce Il21 we sorted different populations of BM cells including CD4+ and CD8+ T cells. We found expression of Il21 mRNA only in the CD4+ T cell population (fig. 1B). To further characterize which subpopulation of T cells is producing Il21, we performed real-time PCR on sorted naive and memory BM T cells (Fig. 1C). We found that Il21 mRNA was produced mainly by memory T cells.
It has been reported that IL-21 protein is secreted by splenic CD4+ T cells activated with anti-CD3, -CD28, and IL-6 (18). Therefore to determine whether IL-21 protein was secreted by BM CD4+ T cells, we cultured BM CD4+ T cells with anti-CD3, anti-CD28 with or without IL-6. Data collected by CBA assay show that CD3- and CD28-mediated signals were sufficient to induce IL-21 secretion by BM CD4+ T cells (Fig. 1D). The greater number of cells observed in wells containing IL-6 likely explains the higher level of IL-21 detected in these wells.
The developmental stages of B lymphopoiesis can be characterized by different profiles of cell surface proteins. We have previously shown that CD2 and μ heavy chain, or CD2 and κ + λ light chains (LC), constitute reliable developmental markers to follow the progression of progenitors from proB to preB to immature and mature B cell stages in in vitro culture (19). In order to analyse the expression of IL-21R on different BM B cell subsets, B220+ BM cells were isolated and grown in IL-7 for 4 days (day 4IL-7). We used a combination of antibodies that recognize CD2, κ and λ light chains (anti-LC) to select different B cell populations, including CD2−LC− (proB), CD2+LC− (preB), and CD2−LC+ (immature/mature B cells). Semi-quantitative RT-PCR results show that Il21r mRNA was present at low levels in proB cells, and at higher levels in preB and immature/mature B cells (Fig. 2A). As expected, Il21r mRNA was not expressed in S17 stromal cells but was expressed in total spleen cells (Fig. 2A). Next, FACS analysis was performed on day 4IL-7 B220+ BM cells isolated from WT and IL-21R−/− mice to determine the expression of IL-21R on the cell surface of B cell progenitors. We found that IL-21R expression progressively increased with the developmental stage of B cell progenitors. IL-21R was below detection on CD2−LC− proB cells, but it was expressed at low levels on CD2+LC− preB cells and at high levels on CD2+LC+ immature/mature B cells (Fig. 2B – upper panel). This pattern of expression closely matched the expression pattern on freshly isolated BM B cells stained with the same markers (Fig. 2B – lower panel)
To determine whether IL-21R was functional, we carried out signalling experiments on different subsets of B cell progenitors. Day 4IL-7 B220+ cells were sorted as described above. CD2−LC− proB cells, CD2+LC− preB cells, and CD2+LC+ immature/mature B cells were stimulated with IL-21 (50 ng/mL for 15 min). Western blot analysis shows that the tyrosine phosphorylation of STAT3, STAT1, and STAT5 was induced after stimulation in all B cell populations (Fig. 3A). Moreover, the intensity of the signal correlated with the expression levels of IL-21R in the different B cell populations. It was the weakest in proB cells and the strongest in immature/mature B cells (Fig. 2B). To rule out the possibility that contaminating preB cells could account for the phosphorylation of STAT3 observed in the proB cell fraction, we spiked an IL-21 unresponsive B progenitor cell line with different numbers of sorted preB cells. We then measured the phosphorylation of STAT3 following IL-21 stimulation. Fig. 3B shows that contamination with preB cells greater than 10% was necessary to achieve the level of STAT3 phosphorylation observed in IL-21-stimulated proB cells. Since the purity of the sorted proB cell fraction was 99.98%, this result indicated that the source of the phosphorylated STAT3 was an IL-21 stimulated proB cell and not a contaminating preB cell. To further test this finding, we isolated B220+ BM cells from RAG−/− mice, which have a B cell developmental block at the proB cell stage. We found that IL-21 treatment of day4IL-7 RAG−/− B220+ BM cells activated phosphorylation of STAT3 (Fig. 3C). To exclude the possibility that a receptor other than the IL-21R was involved in the activation of STAT3, we stimulated proB, preB and immature/mature B cells from WT and IL-21R−/− mice with IL-21. Our results show that phosporylation of STAT3 occurred only in WT cells (Fig. 3D). Finally, we showed that phosphorylation of STAT3 could be induced by IL-7, but not IL-21 in IL-21R−/− cells indicating that STAT3 signalling is functional in IL-21R−/− cells (Fig. 3E). Together, these results clearly indicate that the IL-21R expressed on the surface of proB, preB and immature/mature B cells is functional, and that its stimulation can result in the phosphorylation of downstream proteins.
To examine whether IL-21-mediated signals affect development of B cell progenitors, day 4IL-7 BM B220+ cells isolated from WT and IL-21R−/− mice were sorted into proB, preB and immature/mature B cell populations as previously described, and cultured with or without IL-21 for 48 hours. Absolute numbers of viable cells for each population were determined using trypan blue exclusion. No significant difference in total cell numbers was observed between the control and IL-21-containing cultures (data not shown). However, FACS analysis showed that IL-21 treatment affected the percentages of maturing cells that arose from WT proB and WT preB cultures. In the cultures initiated from CD2−LC− proB cells, there were more preB cells and fewer proB cells in the presence of IL-21 after 48 hours (Fig. 4A and 4B). Similarly, we observed a trend towards increased percentages of immature/mature B cells and fewer preB cells in cultures initiated from CD2+LC− preB cells in the presence of IL-21 (Fig. 4C; top panel and and4D;4D; top panel). Further analysis showed that the percentage of immature/mature B cells expressing high levels of LC tended to be higher in the IL-21-containing wells than in the control wells (Fig. 4C; top panel; CD2+LChi population and 4D; lower panel). We hypothesized that this population (CD2+LChi) likely represents cells that express both IgM and IgD on the surface. In agreement with this, FACS analysis showed that the percentage of IgM+IgD+ mature B cells was higher in cultures grown with IL-21 than the percentage observed in the control cultures (Fig. 4C; lower panel). In support of the finding that the increase in percentages of maturing B cells is IL-21 mediated, we did not observe an increase in B cell development with IL-21 treatment of cultures initiated with IL-21R−/− cells (Fig. 4A). Together, these results show that IL-21 accelerates the transition of proB toward the preB cell stage and of preB cells toward the mature B cell stage.
To determine whether IL-21 affects B cell development in a similar way in vivo, we performed FACS analysis on BM B cell progenitors freshly isolated from WT and IL-21R−/− mice. In agreement with the in vitro analysis, we found that IL-21R−/− mice have increased proportion of B220+CD2−IgM− proB cells (p=0.0376) and smaller proportion of B220+CD2+IgM+ immature/mature B cells (p=0.0131) than WT mice (Fig. 5A-top panel). We confirmed this result using a different set of B cell development markers and found that both the percentage and absolute number of B220loCD43hiIgM− proB were increased (p<0.0001 and p=0.0021 respectively) and the percentage and absolute number of B220hiCD43−IgM+ mature B cells were decreased (p=0.0232 and p=0.0756 respectively) in IL-21R−/− (Fig. 5A and 5B-middle panel). The decrease in IgM+ cells was due to a decrease in the IgM+IgDhi mature B cell population, more specifically in the IgMhiIgDhi cell population (percentage: p=0.0355; absolute number: p=0.0053) (Fig. 5A and 5B-bottom panel). These results confirm that IL-21 has an impact on B cell development in the BM and support the hypothesis that IL-21 accelerates the maturation of B cell progenitors.
In mature B cells, stimulation of the IL-21R has been shown to induce the expression of B lymphocyte induced maturation protein 1 (BLIMP1) (13, 20). BLIMP1 is a DNA binding zinc finger protein that can associate with certain methyl transferases and is important in the regulation of plasma cell differentiation (21). IL-21 also induces the expression of activation-induced cytidine deaminase (AID), an enzyme essential for class switch recombination (CSR) and somatic hypermutation, when costimulated with anti-CD40 or anti-CD40 and anti-IgM (22–24). Although no evidence of somatic hypermutation has been reported, CSR to IgG and IgA has been observed in human cord blood and mature B cells in response to IL-21 and anti-CD40 (22, 24, 25). Since BLIMP1 and AID are typically expressed by the peripheral B cells, it was of interest to determine whether IL-21 can activate a similar pattern of genes in B cell progenitors from the BM. For this purpose, day 4IL-7 B220+ BM cells were stimulated with IL-21 for 24 hours prior to being sorted into proB, preB and immature/mature B cells. Real-time PCR results showed that IL-21 increased expression of Blimp1 in preB cells (Fig. 6A). In contrast, we did not observe any changes in Blimp1 expression in proB or immature/mature B cells. In addition, IL-21 clearly induced Aid expression in preB cells and immature/mature cells, but no significant effect was observed in the cultures initiated with proB cells (Fig. 6B).
In mature B cells, IL-21-induced expression of Aid has been associated with the initiation of class switch recombination (CSR) (25). CSR begins with the initiation of transcription of the germline transcripts (GLTs) from the promoter of a specific isotype. Therefore we measured this early hallmark of CSR by RT-PCR. We searched for GLTs in unstimulated B cells, B cells stimulated with anti-CD40, IL-21, or anti-CD40 and IL-21. For this purpose, day 4IL-7 B220+ BM cells were sorted into proB, preB, and immature/mature B cells and then stimulated for 24 hours. Interestingly, IL-21 treatment of sorted preB and sorted immature/mature B cells resulted in increased levels of GLTγ2b compared to the untreated cells (Fig 7A). Given that B cell progenitors continuously mature when cultured in vitro, we wanted to determine whether IL-21 exposure resulted in increased transcription of GLTs in the preB cells themselves or in immature/mature B cells that develop from preB cells. We treated bulk BM B220+ day 3IL-7 cultures with IL-21 for 24 hours and then sorted the proB, preB and immature/mature B population. Although there was a trend for increased GLT2γb in IL21 treated cells, we did not observe a significant difference between stimulated versus unstimulated cells in progenitor populations that were treated with IL-21 prior to sorting (data not shown). Under these conditions we also examined later hallmarks of CSR and did not observe IL-21-induced differences in postswitch transcripts (PSTs) and circular transcripts (CTs) (data not shown). Collectively, our data show that IL-21 induces aid in preB cells but suggest that IL-21-initiated CSR occurs at later stages of B cell development.
It is known that IL-21 induces plasma cell differentiation and Ig secretion of human cord blood and CD19+ peripheral blood and splenic B cells when used in combination with anti-CD40 (22, 24). IL-21-mediated induction of Blimp1, Aid, and GLTs described above is consistent with the hypothesis that IL-21 can drive the differentiation of BM B cell progenitors into Ig-secreting cells. To test this, we plated day 4IL-7 B220+ BM cells with IL-7, and combination of IL-21 and anti-CD40. In this experiment, IL-7 was included in the culture to allow for continued expansion of proB subset which kept maturing and replenishing preB and immature/mature subsets. This extended the survival of the overall cell culture, thereby giving it sufficient time to respond to IL-21 and anti-CD40. ELISAs were performed on supernatants collected on day 7 of culture. Fig. 7B (left panel) shows that both IL-21 and anti-CD40 were required to induce secretion by B220+ cells. In these cultures, we detected mostly IgM and IgG3, but also IgG1, IgG2b, and some IgG2a immunoglobulins. However, we did not detect IgA (data not shown).
To determine whether early B cell progenitors can differentiate into Ig-producing cells, we sorted day 4IL-7 B220+ BM cells into proB cells. ProB cells were then cultured with IL-7 and anti-CD40 with or without IL-21. ELISAs were performed on supernatants harvested on day 9 of culture. We show that IL-21 and CD40 signals induced differentiation of proB cells into cells that secreted mostly IgG3, but also IgM, IgG1, IgG2a, and IgG2b in the presence of IL-21 and anti-CD40. IgA was below detection level of our assay (Fig. 7B - right panel).
It is well established that IL-21 strongly influences the differentiation of murine and human B cells. To date, this has been demonstrated at the end stages of B cell development where IL-21 induces the transition from fully mature B cells to Ig-secreting plasma cells. In this report we significantly extend the role of IL-21 by showing, for the first time, that IL-21 accelerates the maturation of murine B cell progenitors. Such cells are induced to express Blimp1 and Aid, genes normally expressed in peripheral mature B cells. In addition, we show that IL-21 induces the first step of CSR in B cell progenitors by inducing GLTs and, together with anti-CD40, enables cells to differentiate into Ig-secreting cells.
Careful analysis of expression of IL-21R on different BM B cell progenitors revealed a gradual increase of cell surface IL-21R from proB cells to immature/mature B cells, consistent with the previous observations reported by Jin et al (12). We extend these findings by showing that IL-21R is functional on these populations of B cell progenitors. The intensity of IL-21-induced STAT phosphorylation signals correlated with the level of IL-21R expression. The signals were the weakest in proB cells and the strongest in the immature/mature B cells. Even though we and others (12) failed to detect IL-21R protein at the surface of proB cells by FACS, the results from both RT-PCR and functional signalling experiments clearly show that IL-21R is also expressed on these cells. Given the unexpected nature of this finding, it was important to ensure that the observation was not based on contaminating preB cells. To examine this question we used an IL-21 unresponsive proB cell line to which we added different numbers of FACS sorted IL-21 responsive preB cells. We found that a contamination level of at least 10% preB was required to attain the level of STAT3 phosphorylation observed in sorted proB cells stimulated with IL-21. This threshold is much higher than the 0.5–1 % contamination levels we routinely achieve when we enrich for proB cells. Further, we found that IL-21 stimulation of BM B cells isolated from RAG−/− mice, where B cell development is blocked at the proB cell stage, also induces phosphorylation of STAT3. Finally, IL-21 stimulation failed to induce phoshorylation of STAT3 in IL-21R-deficient proB cells confirming that the phosphorylation of STAT3 in IL-21-stimulated proB cells occurs through the IL-21R.
We also found that Il21 message is constitutively expressed in murine BM, providing evidence that B cell progenitors may well encounter IL-21 during development. Activated Th17, T follicular helper (Tfh), and NKT cells are thought to be main sources of IL-21 in the periphery (26–29). Consistent with a T cell origin of the Il21 transcripts detected in the BM, we detected Il21 message specifically in CD4+ T cells. Furthermore, we showed that BM CD4+ T cells require fewer stimuli to secrete IL-21 protein than splenic CD4+ T cells. In contrast to splenic naïve and memory CD4+ T cells, which require stimulation through CD3, CD28 and IL-6R (18), IL-6R stimulation was not required to induce secretion of IL-21 protein in BM CD4+ T cells.
One of the main findings of our study is that IL-21 promotes the development of B cells progenitors. This is supported by in vivo data showing that IL-21R−/− mice have more proB cells which is likely a consequence of slower maturation. These mice also have fewer mature B cells in their BM than WT mice, although based on current models (30–32) we cannot distinguish whether this difference in IgMhiIgDhi cells comes from the newly arising B cells or from the recirculating B cells.
In further support of our in vivo observation, in vitro stimulation of sorted proB and preB progenitors by IL-21 accelerates their development. The increased percentage of B cells at a more advanced developmental stage is unlikely to be a consequence of enhanced proliferation or survival in response to IL-21. This is supported by the finding that the total number of cells recovered after 48 hours of culture is similar between IL-21-treated and control wells. Moreover, after 48 hours treatment with IL-21, annexin-V and 7-AAD staining showed variable cell survival with no observable trend in cultures initiated with proB or preB cells (data not shown). It should be noted that while the experiments reported here show an effect of IL-21 on purified B cell progenitors in tissue culture, it may be the case that other factors are involved in vivo. For example, it has been shown that IL-21 synergizes with Flt3L and IL-15 to increase proliferation and promote differentiation of NK cells from BM progenitors (33).
Several lines of evidence show that IL-21 can induce Blimp1 and, when used in combination with anti-CD40 or anti-IgM, IL-21 can induce Aid in different populations of mature B cells (13, 22–24). We show that IL-21 increases expression of Blimp1 in developing B cells, and in particular, in preB cells. It is possible that the increase in Blimp1 expression contributes to the acceleration of maturation of preB cell progenitors observed in presence of IL-21 by inducing genes normally found in mature B cells and repressing genes associated with early B cell progenitors. We also show that IL-21 alone induces Aid as early as the preB cell stage. This result contrasts with data obtained on other type of B cells where anti-CD40 or anti-CD40 and anti-IgM are required. While this is not the first report of early B lineage cells expressing Aid and Blimp1 (34–36), it is the first study to suggest a method of induction in early B cell progenitors.
Importantly, our study shows that IL-21 alone was sufficient to initiate early steps of CSR in BM B cell progenitors. This is in contrast to what has been reported for human cord blood B cells, in which both IL-21 and anti-CD40 are required for GLTs to occur (24, 25). However, similar effects on GLTs have been observed in bulk human CD19+ spleen cells even though Aid expression was absent (24). While expression and function of Aid is generally associated with the germinal centre reaction in the secondary lymphoid organs, a recent study has shown that aid message is expressed in early proB/preB cell progenitors in vivo, and is responsible for CSR observed in these cells (34). Moreover, other studies have shown that CSR can occur in some of the Abelson-transformed preB cell lines (37–39).
We believe it is highly unlikely that IL-21R, Aid, Blimp1, GLTγ2b messages come from the contaminating BM plasma and BM memory B cells. First, BM B cells used in our study are selected using an anti-B220 Ab, a molecule absent from BM plasma cells (40). Second, we used anti-κ and anti-λ Abs instead of anti-μ to negatively select proB and preB cells, and thereby avoid possible contamination of these populations with memory cells expressing IgM or any other heavy chain isotype.
Our results show that proB cells differentiate into plasma cells secreting IgM and IgG1, IgG2a, IgG2b and IgG3 Igs when costimulated with IL-21 and anti-CD40. Seagal et al. have proposed that isotype-switched B cell precursors are deleted under normal physiological conditions by a mechanism involving Fas/FasL interaction, presumably to prevent autoimmunity (41). However, presence of IgA and IgG in μMT mice (42, 43) suggests that at least some isotype-switched cells can bypass this mechanism. Two hypotheses have been proposed to explain the presence of CSR in BM B cell progenitors. One hypothesis is these cells could be the product of an alternative B cell development pathway (43). Alternatively, signals through BCR and TLRs can induce aid expression, which is involved in CSR observed in some B cell progenitors. Class-switched B cell progenitors could responsible for enhanced innate immunity by generating IgG- or IgA- producing cells (34).
Having demonstrated that developing B cell progenitors express IL21R and show accelerated maturation in response to IL-21, it is of critical importance to determine what role the IL-21/IL-21R pathway plays in the life of a B cell. One possibility is that T cell-derived IL-21 contributes to the “normal” development of B cells in the BM. CD8+ and CD4+ mature T cells constitute approximately 3–8% of total BM nucleated cells (44). Several studies reported that, at steady state, most CD4+ T cells in murine BM have an activated phenotype (45, 46). Maintenance of the activation state of BM T cells does not require Ag stimulation, but occurs in response to factors produced by the local microenvironment, such as IL-7, IL-15, and 4-1BBL (44, 46–49). There is evidence for interaction between BM T cells and BM B cells through CD40/CD40L that would be required for maintaining bone homeostasis (50). In addition, it has been reported that hematopoiesis is severely impaired in T cell-deficient nude mice, and that restoration of the BM CD4+ T cells rescued normal development of the myeloid compartment (46). Our discovery that IL-21 in the murine BM is produced by T cells reinforces the potential importance of a T cell-dependent B cell developmental pathway option.
An alternative, but not mutually exclusive, interpretation is that IL-21 influences B cell development in the context of an inflammatory response. It is known that leukocyte production is affected during infection and inflammation. Experimental inflammation caused by the injection of adjuvants leads to a remarkable decline in BM B cell development and a corresponding onset of extramedulary development in the spleen (51). This phenomenon is mediated by a rapid reduction of CXCL12 which is thought to be involved in the sequestration of developing B cell progenitors in the BM (51). Under these conditions, B cell progenitors might be found in areas with active CD4+ T cells in the spleen. Interaction of B cell progenitors with T cells through CD40-CD40 ligand interaction in the presence of IL-21 could allow B cell progenitors that have been mobilized to the periphery to continue their development outside the BM and participate in the immune response. It has been noted that such extramedulary development might bypass checkpoints which normally eliminate autoreactive cells in the BM (52). Indeed, there is a growing body of evidence linking IL-21 with the development of some autoimmune diseases with strong humoral component. For example, BXSB.B6-Yaa+/J murine model of SLE shows elevated levels of serum IL-21 (13). In contrast, BXSB-Yaa+ mice deficient for IL-21R do not develop SLE (15).
We do not yet know if our observations have uncovered a “normal” process that may contribute to the immune response or even a “dangerous” anomaly that may contribute to autoimmunity by allowing immature cells to bypass regulatory mechanisms that normally eliminate autoreactive cells. However our results clearly suggest that IL-21 regulates maturation of B cell progenitors and, in combination with anti-CD40, can lead to the formation of Ig-secreting cells.
We thank Rakesh Nayyar and Francis Tong for their help with cell sorting.
1This work was supported by grant #9862 from the Canadian Institutes of Health and Research (CIHR) and by the Terry Fox Foundation. NS acknowledges the support of a CIHR Doctoral Award.
The authors have no financial conflict of interest.