IL-21 produced by follicular Th (Tfh) cells is an important regulator of Tfh cell development and B cell responses, including germinal center (GC) formation. However, whether defective GC formation and Ab responses are a consequence of impaired Tfh cells development or a B cell-intrinsic defect in IL-21–deficient mice requires clarification. To address this question, we generated chimeric mice lacking IL-21R exclusively on B cells. In this study, we demonstrate that GC reaction and B cell responses induced by immunization with virus-like particles were strongly reduced in both global and B cell-specific IL-21R–deficient mice. Interestingly, the presence of TLR7 ligand within virus-like particles largely restored defective GC reaction and Ab responses in global as well as in B cell-specific IL-21R–deficient mice. Hence, IL-21 acts directly on B cells and cooperates with TLR signaling for optimal B cell responses.

Interleukin-21 is a member of the common γ-chain (γc)-signaling cytokines. The IL-21R comprises a unique IL-21–binding chain and the γc-signaling subunit (1). IL-21R is strongly expressed on B cells and can also be found on the surface of NK and activated T cells including NKT cells. Besides its involvement in CD4 T cell differentiation, an important role for IL-21 in the regulation of humoral immune responses has been established. It induces B cell Ig isotype switching toward IgG1 postimmunization with T cell-dependent (TD) Ags (2) and plasma cell differentiation due to its capacity to induce Blimp-1 and Bcl-6 expression (3, 4). In contrast to its fundamental role in driving B cell responses against model protein Ags, lack of IL-21 or its receptor had a limited influence on antiviral Ab responses (57).

Germinal centers (GCs) are specialized sites within B cell follicles of secondary lymphoid organs where B cells rapidly proliferate, undergo class-switch recombination, somatic hypermutation, and selection for expression of high-affinity BCRs (8, 9). During encounter of cognate Ag within GCs, follicular B cells receive help provided by CXCR5+ follicular Th (Tfh) cells (10) by interaction of CD40 and ICOS ligand on B cells with CD40L and ICOS respectively expressed on Tfh cells (1113). Recently, an IL-21–driven autocrine loop in Tfh cell differentiation has been described. Consequently, it has been proposed that IL-21 controls GC formation indirectly by promoting Tfh cell generation rather then directly acting on B cells (14, 15).

Virus-like particles (VLPs) are strong immunogens that can induce potent Ab responses. VLPs are T cell-independent Ags that are able to induce IgM responses in the absence of T help because of multimeric interactions with cognate BCRs that induce a strong activation signal in B cells (16). Recombinant expression of the envelope protein of the bacteriophage Qβ results in self-assembly of Qβ-VLPs loaded with bacterial RNA. This RNA can be experimentally removed, allowing investigation of B cell responses against the same Ag in the presence or absence of TLR7/8 ligands. In this study, we demonstrate that IL-21 acts directly on B cells and is crucially required for VLP-specific IgG responses. Presence of TLR7/8 ligands largely overcomes this dependency, demonstrating that B cells integrate innate and Th cell-derived signals for optimal IgG responses.

C57BL/6 mice were purchased from Harlan Netherlands (Horst, The Netherlands). IL-21R−/− mice were generated and backcrossed to C57BL/6 background as described previously (17). Experiments were conducted in accordance with protocols approved by the Swiss Federal Veterinary Office.

C57BL/6 and IL-21R−/− mice were immunized s.c. with 50 μg Qβ-VLP either containing RNA or devoid of RNA, whereas chimeric mice were immunized with 200 μg. Capsids of the RNA phage Qβ were cloned into pQβ10 vector and purified as described elsewhere (18). VLPs devoid of RNA were generated by disassembling the VLP particles with DTT and acidic treatment. The resulting dimmers were purified by size exclusion chromatography and reassembled with poly-glutamic acid.

ELISA plates (Nunc Immuno MaxiSorp, Nunc, Roskilde, Denmark) were coated with Qβ-VLP, and ELISAs were performed according to standard protocols using HRP-conjugated secondary Abs: goat anti-mouse IgG (Fcγ specific) and goat anti-mouse IgM (Jackson ImmunoResearch Laboratories, West Grove, PA); and rat anti-mouse IgG1 and rat anti-mouse IgG2a (BD Pharmingen, San Diego, CA).

Anti-VLP titers in serum were measured by endpoint titer by calculating the OD (450 nm) cutoff as 0.1.

Radiation BM chimeras in which all B cells were deficient in the expression of IL-21R were generated by i.v. injection of BM mixture containing 20% BM cells isolated from IL-21R−/− mice and 80% BM cells isolated from JH−/− mice (1–5 × 107 total injected cells) into JH−/− mice that had been lethally irradiated (950 rad γ) 1 d previously. Control chimeras with wild-type (WT) B cells were made using a combination of C57BL/6 and JH−/− donor BM.

Detection of VLP-specific B cells expressing surface Ig was performed as described (16) by incubation with Alexa 647-labeled VLPs. Isotype-switched B cells were detected with a mixture of FITC-conjugated Abs (rat anti-IgD; goat anti-IgM serum, Jackson ImmunoResearch Laboratories; rat anti-CD4; anti-CD8; anti-CD11b; anti–Gr-1) and PE-Cy7–conjugated rat anti-B220. Biotin peanut agglutinin (PNA) was purchased from Vector Laboratories (Burlingame, CA). Tfh cells were detected with allophycocyanin-labeled rat anti-CD4, biotin rat anti-CXCR5, and FITC-conjugated anti–programmed death-1. Biotinilated Abs were detected with PerCP-Cy5.5–labeled streptavidin. In all cases, FcRs were blocked with rat anti-mouse CD16/32. Abs were purchased from BD Biosciences (San Jose, CA) unless otherwise specified. Cells were acquired with a Canto cytometer (BD Biosciences).

Spleens were immersed in OCT and snap-frozen in liquid nitrogen. Tissue sections of 7-μm thickness were cut in a cryostat and fixed with acetone. For detection of VLP-specific B cells, Alexa 488-labeled VLPs were added. PNA-binding B cells were detected with biotinylated PNA. B cell follicles were identified with Alexa 647 rat anti-mouse B220 (BD Pharmingen). T cell follicles were identified with biotinylated rat anti-mouse CD4 (BD Pharmingen). Biotinilated Abs were detected with Alexa 546-labeled Streptavidin. Images were acquired with Axioplan 2 microscope (Zeiss, Oberkochen, Germany), and pictures were analyzed with Open laboratory software (Improvision, Waltham, MA).

A key role for IL-21 in Ab responses following immunization with TD Ags has been well established (2). However, antiviral Ab responses were marginally affected in IL-21R–deficient mice (57). In this study, we sought to investigate the role of IL-21 in Ab responses to VLPs, which are highly organized particulate Ags of viral structure that induce T cell-independent IgM and TD IgG responses (16). To this end, we measured the Ab responses generated in WT and IL-21R−/− mice immunized s.c. with VLPs devoid of bacterial RNA (RNA-free VLP). Fig. 1A, upper panel, shows poor IgG responses in IL-21R–deficient mice following immunization with RNA-free VLPs. In agreement with previous reports (2, 17), we also observed that the IgG1 subclass was most strongly affected by the absence of IL-21R signaling. Thus, similar to Ab responses against soluble proteins, IL-21 is crucial for IgG, and in particular IgG1, responses to particulate Ags such as VLPs in the absence of TLR ligands.

FIGURE 1.

Requirement of IL-21 or TLR stimuli for optimal induction of VLP-specific IgG and GC responses. Mice were immunized s.c. with 50 μg either RNA-free VLP or VLP loaded with RNA. A, Twenty days later, specific IgG, IgG1, and IgG2c levels were measured in serum by ELISA. Geometric mean ELISA titers + SEM are indicated. Statistical significance was assessed by two-sided unpaired Student t test. VLP-specific GC formation was assessed 10 d postimmunization by flow cytometry (B) and immunofluorescence histology (C). B, PNA binding by VLP+ isotype-switched B cells was determined in spleen. Isotype-switched B cells (R1) were identified as (IgD, IgM, CD4, CD8, CD11b, CD11c, Gr-1) B220+ B cells as shown in the top panel. C, Immunofluorescence detection of VLP-specific PNA-binding B cells (red) within B cell follicles stained with B220 (blue) in spleen sections of WT and IL-21R−/− mice (original magnification ×50). The data shown are representative of two independent experiments. *p < 0.05; **p < 0.01.

FIGURE 1.

Requirement of IL-21 or TLR stimuli for optimal induction of VLP-specific IgG and GC responses. Mice were immunized s.c. with 50 μg either RNA-free VLP or VLP loaded with RNA. A, Twenty days later, specific IgG, IgG1, and IgG2c levels were measured in serum by ELISA. Geometric mean ELISA titers + SEM are indicated. Statistical significance was assessed by two-sided unpaired Student t test. VLP-specific GC formation was assessed 10 d postimmunization by flow cytometry (B) and immunofluorescence histology (C). B, PNA binding by VLP+ isotype-switched B cells was determined in spleen. Isotype-switched B cells (R1) were identified as (IgD, IgM, CD4, CD8, CD11b, CD11c, Gr-1) B220+ B cells as shown in the top panel. C, Immunofluorescence detection of VLP-specific PNA-binding B cells (red) within B cell follicles stained with B220 (blue) in spleen sections of WT and IL-21R−/− mice (original magnification ×50). The data shown are representative of two independent experiments. *p < 0.05; **p < 0.01.

Close modal

As mentioned above, untreated VLPs are loaded with Escherichia coli-derived ssRNA during its self-assembly process. We and others have shown that TLR ligands can strongly enhance Ab responses (19, 20). Furthermore, it has been shown that TLR ligands promote Ab responses by acting directly on B cells rather than dendritic cells or other cell types (1922).

Immunization of WT mice with RNA-loaded VLPs induced predominantly IgG2c (19, 22), which was indistinguishable in IL-21R−/− (Fig. 1A, lower panel). These data demonstrate that the presence of RNA in VLPs can largely overcome the deficiency of Ab responses observed in IL-21R−/− mice.

Next, we investigated the GC reaction to VLPs in IL-21R−/− mice. To this end, we immunized WT and IL-21R−/− mice s.c. either with RNA-loaded VLPs or with VLPs devoid of TLR stimuli and assessed GC formation 10 d later by flow cytometry. Fig. 1B shows that the frequency of PNA+ GC VLP-specific B cells was similar comparing WT and IL21R−/− mice (although PNA binding was reduced on VLP+ IL-21R−/− B cells). In contrast, immunization of IL-21R−/− mice with RNA-free VLPs resulted in reduced frequencies of specific B cells, and PNA binding was as low as on IgM+IgD+ naive B cells (Fig. 1B). These data obtained by flow cytometry were further confirmed by immunohistochemistry (Fig. 1C). Thus, absence of IL-21 abolished GC formation unless RNA was incorporated into VLPs.

In the next set of experiments, we investigated how IL-21R signaling controls GC formation and IgG production. It has been recently suggested that IL-21 promotes Ab responses by driving Tfh cell development and thereby GC formation (14, 15). To dissect the role of IL-21 on Tfh and B cells, we generated chimeras exhibiting IL-21R expression in all hematopoietic cells except B cells. To this end, B cell-deficient (JH−/−) mice were irradiated and reconstituted with a mixture of BM cells isolated from JH−/− and IL-21R−/− mice or from JH−/− and WT mice. Reconstituted chimeras were immunized either with RNA-free VLPs or RNA-loaded VLPs. Fig. 2A, upper panel, shows that following immunization with RNA-free VLP, specific Ab responses, including all IgG subclasses, were more than 10-fold reduced in chimeric mice harboring IL-21R–deficient B cells, and IgG1 was completely abolished (Fig. 2A, upper panel). In contrast, a potent IgG2c response was generated in IL-21R–deficient B cell chimeras immunized with RNA-loaded VLPs (Fig. 2A, lower panel). Furthermore, VLP+ IL-21R–deficient B cells expressed similar levels of PNA compared with WT B cells in the control chimeras immunized with RNA-loaded VLPs (Fig. 2B, lower panel). Conversely, PNA levels were similar to naive B cells in a great proportion of VLP+ IL-21R–deficient B cells immunized with RNA-free VLPs (Fig. 2B, upper panel). Note that quantification of total B cell numbers as well as B cell populations in the spleen of mixed BM chimeras revealed a similar degree of B cell reconstitution in both IL-21R−/− and WT B cell chimeras (Supplemental Fig. 1). Immunofluorescence histology confirmed that chimeric mice harboring WT B cells efficiently formed GCs (Fig. 2C), which were specific for VLPs (Supplemental Fig. 2). In addition, chimeric mice harboring IL-21R–deficient B cells failed to form VLP-specific GCs unless RNA was introduced into VLPs (Fig. 2C). This observation was largely corroborated by enumerating VLP-specific PNA+ GCs by histology (Fig. 2D). Similarly to day 10 shown in Fig. 2, also at day 6 postimmunization with RNA-free VLPs, B cell follicles of IL-21R−/− B cell chimeras were completely devoid of GC, which was largely restored by the addition of RNA (Fig. 3). This suggests that in the absence of IL-21R signaling by B cells, GC formation could not be initiated.

FIGURE 2.

IL-21R expression on B cells is required for optimal IgG responses against VLPs. JH−/− mice were irradiated and reconstituted with BM cells from JH−/− mice mixed with BM cells from either C57BL/6 (WT B cells) or IL-21R−/− (IL-21R−/− B cells) mice. Ten weeks after reconstitution, chimeric mice were immunized s.c. either with 200 μg RNA-free VLPs or RNA-loaded VLPs. A, Ten days later, specific IgG, IgG1, and IgG2c titers were determined in serum by ELISA. Geometric mean ELISA titers + SEM are indicated. Statistical significance was assessed by two-sided unpaired Student t test. VLP-specific GC formation was assessed 10 d postimmunization by flow cytometry (B) and immunofluorescence histology (C). B, Analysis of PNA binding on VLP+ isotype-switched B cells was determined in spleen. C, Immunofluorescence detection of VLP+ PNA-binding B cells (red) within B cell follicles stained with B220 (blue) in spleen sections of WT and IL21R−/− B cells chimeras (original magnification ×50). D, Enumeration of VLP-specific GCs by histological analysis. The data shown are representative of two independent experiments. *p < 0.05; **p < 0.01. n.d., not detectable.

FIGURE 2.

IL-21R expression on B cells is required for optimal IgG responses against VLPs. JH−/− mice were irradiated and reconstituted with BM cells from JH−/− mice mixed with BM cells from either C57BL/6 (WT B cells) or IL-21R−/− (IL-21R−/− B cells) mice. Ten weeks after reconstitution, chimeric mice were immunized s.c. either with 200 μg RNA-free VLPs or RNA-loaded VLPs. A, Ten days later, specific IgG, IgG1, and IgG2c titers were determined in serum by ELISA. Geometric mean ELISA titers + SEM are indicated. Statistical significance was assessed by two-sided unpaired Student t test. VLP-specific GC formation was assessed 10 d postimmunization by flow cytometry (B) and immunofluorescence histology (C). B, Analysis of PNA binding on VLP+ isotype-switched B cells was determined in spleen. C, Immunofluorescence detection of VLP+ PNA-binding B cells (red) within B cell follicles stained with B220 (blue) in spleen sections of WT and IL21R−/− B cells chimeras (original magnification ×50). D, Enumeration of VLP-specific GCs by histological analysis. The data shown are representative of two independent experiments. *p < 0.05; **p < 0.01. n.d., not detectable.

Close modal
FIGURE 3.

Lack of IL-21 signaling in B cells prevents initial GC formation. Chimeric mice reconstituted with either WT or IL-21R−/− B cells were immunized s.c. with either RNA-free VLP (upper panel) or with RNA-loaded VLP (lower panel), and 6 d later, GC formation was assessed by immunofluorescence histology. Detection of VLP-specific PNA-binding GC B cells (red) within B cell follicles stained with B220 (blue) in spleen sections is shown (original magnification ×50).

FIGURE 3.

Lack of IL-21 signaling in B cells prevents initial GC formation. Chimeric mice reconstituted with either WT or IL-21R−/− B cells were immunized s.c. with either RNA-free VLP (upper panel) or with RNA-loaded VLP (lower panel), and 6 d later, GC formation was assessed by immunofluorescence histology. Detection of VLP-specific PNA-binding GC B cells (red) within B cell follicles stained with B220 (blue) in spleen sections is shown (original magnification ×50).

Close modal

In contrast to B cell responses, generation of Tfh cells was not affected by the absence of IL-21R on B cells (Fig. 4). Taken together, these data clearly show that IL-21 controls Ab responses by acting directly on B cells and not by promoting Tfh cell generation.

FIGURE 4.

Normal Tfh cell development in IL-21R−/− B cell chimeras. Chimeric mice reconstituted with either WT or IL21R−/− B cells were immunized s.c. with RNA-free VLPs, and 10 d later, development of Tfh cells was assessed by flow cytometry. Contour plots of Tfh cells identified by CXCR5 and programmed death-1 staining on CD4+B220 lymphocytes are shown. Mean percentages ± 1 SD (n = 4) is shown. Tfh cells staining from a naive mouse is shown as control. The data shown are representative of two independent experiments.

FIGURE 4.

Normal Tfh cell development in IL-21R−/− B cell chimeras. Chimeric mice reconstituted with either WT or IL21R−/− B cells were immunized s.c. with RNA-free VLPs, and 10 d later, development of Tfh cells was assessed by flow cytometry. Contour plots of Tfh cells identified by CXCR5 and programmed death-1 staining on CD4+B220 lymphocytes are shown. Mean percentages ± 1 SD (n = 4) is shown. Tfh cells staining from a naive mouse is shown as control. The data shown are representative of two independent experiments.

Close modal

Besides the follicular GC response, B cells can also migrate to the splenic red pulp and locally form extrafollicular foci of plasmablasts (PBs), which predominantly secrete IgM (23). To confirm that the mixed BM chimeras had an intrinsic defect in GC formation upon immunization with RNA-free VLPs and rule out the possibility that the initial B cell response was abrogated, we assessed the extrafollicular B cell response in these mice. To this end, we compared VLP-specific IgM titers in serum 5 d post RNA-free VLP immunization (prior GC formation). Fig. 5A shows that IL-21R−/− B cell chimeras elicited similar IgM titers as WT B cell chimeras and also produced comparable numbers of IgM secreting specific PBs (not shown). The extrafollicular response was further investigated by histology. Fig. 5B shows that similar numbers of VLP-specific B cells could be observed in the red pulp of both WT and IL-21R−/− B cell chimeras. Collectively, these data confirm that the IL-21R−/− B cell chimeras display a specific defect in GC reaction, whereas the extrafollicular response remains largely normal. This finding fits well with a previous report showing that extrafollicular PBs can interact with extrafollicular Ag-specific Th cells and locally expand in an IL-21–independent manner (24).

FIGURE 5.

Lack of IL-21 signaling on B cells does not affect extrafollicular B cell responses. A, Chimeric mice reconstituted with either WT or IL21R−/− B cells were immunized s.c. with RNA-free VLPs, and 5 d later, IgM titers were measured in serum by ELISA. B, Immunofluorescence detection of specific B cells (green) in red pulp (original magnification ×100). Red pulp was identified as B220 (blue, B cell follicle) and CD4 (red, T cell zone) negative area. The data shown are representative of two independent similar experiments.

FIGURE 5.

Lack of IL-21 signaling on B cells does not affect extrafollicular B cell responses. A, Chimeric mice reconstituted with either WT or IL21R−/− B cells were immunized s.c. with RNA-free VLPs, and 5 d later, IgM titers were measured in serum by ELISA. B, Immunofluorescence detection of specific B cells (green) in red pulp (original magnification ×100). Red pulp was identified as B220 (blue, B cell follicle) and CD4 (red, T cell zone) negative area. The data shown are representative of two independent similar experiments.

Close modal

In conclusion, our data demonstrate that B cell-intrinsic IL-21R signaling critically regulates IgG Ab responses as well as GC formation as has been observed in two very recent studies (25, 26). Importantly, our data reveal that TLR7/8 signaling is able to largely overcome this defect, explaining why IL-21 plays a minor role for the generation of antiviral B cell responses. Thus, innate signals are able to largely replace IL-21 generated by the adaptive Th cell response.

We thank Philippe Saudan for helpful discussions and Begonia Fudrini, Ala’a Ragab, Anna Flace, Franziska Wagen, and Alexander Titz for excellent technical assistance.

Disclosures M.F.B. and J.B. are employees of Cytos Biotechnology and may hold shares or share options in the company.

The online version of this article contains supplemental material.

Abbreviations used in this paper:

BM

bone marrow

GC

germinal center

n.d.

not detectable

PB

plasmablast

PNA

peanut agglutinin

TD

T cell-dependent

Tfh

follicular Th

VLP

virus-like particle

WT

wild-type.

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