The role of IL-21, produced mainly by Th17 cells and T follicular helper cells, has been intensively investigated in B cell differentiation and Ab class switch. However, how IL-21 regulates memory IgA+ B cell development and memory IgA responses in the intestines is still not completely understood. In this study, we found the total IgA+ B cells as well as CD38+CD138−IgA+ memory B cells were significantly increased in intestinal lamina propria (LP) of TCRβxδ−/− mice after transfer of microbiota Ag-specific Th17 cells but not Th1 cells. Although IL-21R−/− mice or IL-17R−/− mice showed decreased Ag-specific memory IgA production in the intestines upon infection with Citrobacter rodentium, the percentage of IgA+CD38+CD138- memory B cells in Peyer’s patches and LP was decreased only in IL-21R−/− mice, but not in IL-17R−/− mice, after reinfection with C. rodentium compared with wild-type mice. Blockade IL-21 in vivo suppressed intestinal C. rodentium–specific IgA production as well as IgA+CD38+CD138− memory B cells in Peyer’s patches and LP. Furthermore, IL-21 significantly induced B cell IgA production in vitro, with the increased expression of genes related with class-switching and memory B cell development, including Aicda, Ski, Bmi1, and Klf2. Consistently, Aicda and Ski expression was decreased in B cells of IL-21R−/− mice after C. rodentium reinfection. In conclusion, our study demonstrated that IL-21 promotes intestinal memory IgA B cell development, possibly through upregulating differentiation-related and class switching–related genes, indicating a potential role of IL-21 in memory IgA+ B cell responses in the intestines.
Secreted Abs are essential for providing immune protection in the intestine. The enrichment and maintenance of Ab production after Ag encounter have been considered as the result of the activation of limited memory B cells that are generated and sustained after the primary responses to the Ag (1, 2). B cells normally undergo somatic hypermutation and differentiation in the germinal center (GC) and emerge out as memory B cells or plasma cells. The selection into memory B cell fate is regulated by several mechanisms, of which one prominent mechanism is extrinsic signals from T cells, including cytokines and cell contact–dependent signals (3). Several transcription regulators, which are essential for T cell development and differentiation, have been showed to be critical for the survival of Ig class-specific memory B cells, such as T-bet and retinoic acid in IgG2a+ memory B cells, and receptor-related orphan receptor-α (RORα) in IgA+ memory B cells, probably by controlling cell-surface BCR transcription (4). Furthermore, IL-2 and IL-10 cooperated with CD40L, playing an important role in driving the transition of GC B cells to memory B cells (5).
IgA, one of the most enriched isotypes of Abs in the intestine, has been crucial in regulation of host–microbiota interaction to maintain the intestinal homeostasis (6). It has been reported that the generation and maintenance of intestinal IgA response are different from that of systemic IgG response (7). The microbiota Ag-specific IgA+ B cells can retain in the intestine for >16 wk, which suggests the unique relationship between IgA–producing cells and intestinal environments. There are abundant long-lived microbiota Ag-specific Th17 cells in intestinal mucosa, which provide support for the development of IgA+ B cells (8–10). It has been shown that Th17 cells facilitate B cell proliferation and promote the GC formation as well as Ab isotype switching to IgG1, IgG2a, IgG2b, and IgG3 (11). IL-17, which is predominately produced by Th17 cells, is suggested to promote the Ab production indirectly through inducing the production of B cell activators in other immune cells (12). IL-21, a signature cytokine of Th17 cells and T follicular helper cells, binds to IL-21R, which is widely expressed on T cells, B cells, and DCs, and is upregulated after activation by anti-CD40 mAb in memory B cells (13, 14). IL-21 has been shown to regulate B cell proliferation, class switch recombination, and plasma cell differentiation as well as IgA production (10, 15–17). IgA+ memory B cell response can be boosted rapidly upon Ag rechallenge, which is crucial in the host defense. It has been shown that IL-21 deficiency led to reduction of GC B cells (18). However, the roles of Th17 cells as well as IL-17 and IL-21 in regulating intestinal memory IgA+ B cell responses are still unclear.
In this study, we demonstrated that transfer of gut microbiota Ag-specific Th17 cells from CBir1 flagellin–specific TCR transgenic (CBir1 Tg) mice, which are specific for an immunodominant gut microbiota Ag CBir1 flagellin, significantly induced the CD19+CD38+CD138−IgA+ memory B cells in TCRβxδ−/− mice. In addition, upon Citrobacter rodentium infection, C. rodentium–specific IgA+ memory B cells as well as Ag-specific memory IgA production were impaired in IL-21R−/− mice. Our study thus indicates a critical role of IL-21 produced by Th17 cells in promoting the intestinal memory IgA responses.
Materials and Methods
Wild-type (WT) C57BL/6, TCRβxδ−/−, and IL-21R−/− mice were obtained from The Jackson Laboratory. IL-17R−/− mice were kindly provided by Amgen. CBir1 Tg mice were maintained and bred in the Animal Facilities at The University of Texas Medical Branch. All experiments were reviewed and approved by the Institutional Animal Care and Use Committees of The University of Texas Medical Branch. All mice are bred onto the C57BL/6 background. Eight to twelve-week-old mice were used for all experiments. All the mice used in this study were gender matched and maintained under specific pathogen-free conditions.
The following Abs were used for flow cytometry: FITC–anti-CD19 (6D5), PE/Cy7–anti–IL-17A (TC11-18H10.1), FITC–anti-B220 (RA3-6B2), Brilliant Violet 421–anti-CD4 (RM4-5), PE/Cy7–anti-CD38 (90), allophycocyanin–anti-CD138 (281-2), Percp/Cy5.5–anti–GL-7 (29F.1A12), and Percp/Cy5.5–IL-17 (TC11-18H10.1) were purchased from BioLegend. Allophycocyanin–anti–IL-21 (mhalx21) and Brilliant Violet 421–anti-CD95 (Jo2) were from BD Biosciences; PE-IgA (polyclonal) was from Southern Biotechnology. Foxp3 perm/fix kit for intracellular permeabilization and Live/Dead Fixable Dead Cell stain kit for gating live cells were from Thermo Fisher Scientific. Phorbolmyristate acetate was purchased from Sigma-Aldrich, and ionomycin was purchased from Invitrogen. Golgistop was purchased from BD Biosciences. Mouse rIL-6, IL-17, and IL-21 and human recombinant TGF-β1 were purchased from BioLegend. Anti–IFN-γ (XMG1.2), anti–IL-4 (11B11), and anti–IL-21R (4A9) were from BioXCell. Ab against IgD was purchased from Southern Biotechnology. Anti-μ was purchased from Jackson ImmunoResearch Laboratories. Antibiotin microbeads from Miltenyi Biotec were used to isolate naive IgD+ B cells. Anti-mouse phosphorylated Erk1/2, anti-mouse total Erk1/2, anti-mouse phosphorylated stat3, and anti-mouse total phosphorylated stat3 Abs were purchased from Cell Signaling Technology. Anti-CD4 Ab was purchased from Abcam.
Polarization of Th17 cells and Th1 cells
CD4+ T cells were isolated from spleens of CBir1 Tg mice using anti-mouse CD4 magnetic beads (GK1.5; BD Biosciences) as previously described (19). To polarize Th17 cells, CBir1-Tg CD4+ T cells were cultured for 5 d with 15 ng/ml TGF-β, 20 ng/ml IL-6, 10 μg/ml anti–IFN-γ, 10 μg/ml anti–IL-4, and 1 μg/ml CBir1 peptide with irradiated splenic APC (20). To polarize Th1 cells, CBir1-Tg CD4+ T cells were cultured with 10 ng/ml IL-12, 1 μg/ml CBir1 peptide, and irradiated APCs.
Fecal pellet preparation
Fecal pellets were collected every week from individual mice, weighed, and homogenized in cold PBS containing 0.04 mg/ml soybean trypsin inhibitor, 20 mM EDTA, and 2 mM PMSF and centrifuged at 15000 rpm for 15 min to remove bacteria and insoluble debris as described previously (21). The supernatants were kept in −20°C for further ELISA processing.
Ninety six–well plates were coated with 1 μg/ml anti-IgA (Kirkegaard and Perry Laboratories [KPL]) or 1 μg/ml C. rodentium bacterial lysate overnight at 4°. The plates were washed with PBS/Tween and blocked with 1% BSA/PBS at room temperature for 2 h, following with incubation of fecal pellets supernatant for 2 h. After washing, adherent Abs were detected by biotinylated anti-IgA (KPL) and HRP-labeled streptavidin. Plates were developed using a two-component TMB substrate (KPL) and read at 450 nm. Results were quantified by normalizing to standard concentrations of IgA (Southern Biotechnology).
Intestinal lamina propria cell isolation
Intestines were removed from individual mice, and the Peyer’s patches (PP) were excised as previously described (19). Intestines were sliced and incubated with 0.5 mM EDTA-PBS at 37° for 40 min to remove epithelial cells. Intestines were then digested by collagenase IV for 40 min. After going through 100-μm filter, the cell lysis was resuspended in 40% Percoll, overloaded onto 70% Percoll, and centrifuged at 2000 rpm for 20 min at room temperature. The interface containing lymphocytes was collected. Lymphocytes were directly stained for T cell and B cell analysis.
Flow cytometry staining
For CD4+ T cell staining, cells were activated with phorbolmyristate acetate (50 ng/ml) and ionomycin (750 ng/ml) for 2 h, followed by adding 0.7μl/ml Golgistop for another 3 h. Cells were then stained with live/dead and Ab against CD4. Subsequently, cells were permeabilized and fixed by Foxp3 perm/fix kit. Finally, cells were stained with intracellular Abs against IL-17 and IL-21.
For B cell staining, cells were stained with live/dead as well as surface markers of CD19, B220, CD38, CD38, CD95, and GL7. After permeabilization, cells were stained with Ab against IgA.
C. rodentium infection
The infection method was descripted previously (22). Briefly, C. rodentium strain DBS100 (American Type Culture Collection) was grown overnight in lysogeny broth at 37°C and subcultured (1:100 dilution) in fresh LB for another 5–6 h. Bacteria were harvested and resuspended in sterile PBS at 1 × 107 CFU/ml. Adult mice (6–8 wk old) were given 200 μl of bacterial suspension (2 × 106 CFU) by gavage. Fecal pellets were collected from individual mice and weighed; one part was homogenized in PBS and serial diluted onto MacConkey agar (BD Biosciences) for CFU counting, the other part was processed for fecal pellets supernatant. Mice were reinfected with the same amount of C. rodentium at 5 wk after initial infection, and the fecal pellets were collected and processed for bacteria quantification and supernatant preparation. During the experiment period, all the mice were monitored and weighed.
Quantitative real-time/reverse transcription PCR
RNA was extracted with TRIzol (Invitrogen) and followed by cDNA synthesis with Revertaid reverse transcriptase (Fermentas). Quantitative PCR was performed using SYBR Gene Expression Assays. Primers were synthesized from Integrated DNA Technologies, and data were normalized to Gapdh mRNA expression. Primer sequences are as follows: activation-induced cytidine deaminase (Aicda): forward, 5′-AGAAAGTCACGCTGGAGACC-3′, reverse, 5′-CTCCTCTTCACCACGTAGCA-3′; Bmi1: forward, 5′-ATGAGTCACCAGAGGGATGG-3′, reverse, 5′-AAGAGGTGGAGGGAACACCT-3′; Klf2: forward, 5′-GCCTGTGGGTTCGCTATAAA-3′, reverse, 5′-TTTCCCACTTGGGATACAGG-3′; Ski: forward, 5′-AAAAGCCCTCCGCTCTAGTC-3′, reverse, 5′-GACGTCAGGGCTTAGCAGTC-3′; Il17r: forward, 5′-AGTGTTTCCTCTACCCAGCAC-3′, reverse, 5′-GAAAACCGCCACCGCTTAC-3′; Il21r: forward, 5′-GGCTGCCTTACTCCTGCTG-3′, reverse, 5′-TCATCTTGCCAGGTGAGACTG-3′; and Gapdh: forward, 5′-CCATGGAGAAGGCTGGGG-3′, reverse, 5′-CAAAGTTGTCATGGATGACC-3. Aliquots of PCR products were visualized by electrophoresis on 1.5% agarose gels.
Cellular protein was extracted using radio-immunoprecipitation assay buffer, and the protein concentration was determined with a BCA Protein Assay kit (Thermo Fisher Scientific). Six micrograms of total protein per sample was loaded and separated by TGX gels (Bio-Rad) and then transferred to polyvinylidene difluoride membranes. After blocking, membranes were incubated with primary Abs (1:1000), followed by incubation with a secondary Ab (1:2000). Protein bands were developed using ECL assay.
For comparisons between samples, levels of significance were determined by Student t test or one-way ANOVA in Prism 8.0 (GraphPad). Mean ± SD is represented. A *p < 0.05 was considered as significant.
Gut microbiota-specific Th17 cells promote intestinal memory IgA+ B cell development and IgA production
To investigate how gut microbiota Ag-specific Th17 cells regulate intestinal memory IgA responses to microbiota, we generated Th17 cells from CBir1 Tg mice, which are specific for an immunodominant gut microbiota Ag CBir1 flagellin (23), in vitro by culture under Th17 polarization conditions with TGF-β and IL-6 and transferred them into TCRβxδ−/− mice, which lack T cells. To address the specific role of Th17, but not other effector T cells, in memory IgA responses, we transferred the in vitro–generated Th1 cells from CBir1 Tg mice into TCRβxδ−/− mice. We also gave another group of mice PBS to serve as controls. The fecal pellets were collected for measuring IgA on day 21. Transfer of Th17 cells significantly increased fecal IgA production in TCRβxδ−/− mice compared with transfer of Th1 cells and the controls with PBS (Fig. 1A). To investigate whether transferred T cells localize to PP, the recipient mice were sacrificed 21 d post–T cell transfer, and PPs were stained with anti-CD4 Ab. As shown in Supplemental Fig. 1A, CD4+ T cells presented in PPs from the mice received either Th1 or Th17 cells. Spleen, PP, and lamina propria (LP) cells from these mice were harvested for analysis of B cell phenotypes by flow cytometry (FACS). The percentage of CD95+GL7+ B cells in PP, which represent the GC B cells, was significantly higher in mice that received Th17 cells than that in control mice with PBS (Fig. 1B) and also higher than that in mice that received Th1 cells, although not reaching statistical significance (Fig. 1B). There were no significant differences of CD95+GL7+ B cells in PP between mice transferred with Th1 cells and control mice with PBS (Fig. 1B). The IgA+ B cells were significantly increased in spleen, PP, and LP in the recipient mice with Th17 cells but not with Th1 cells (Fig. 1C) and significantly higher in PP of the mice that received Th17 cells compared with the mice with Th1 cells (Fig. 1C). Importantly, total CD138−CD38+IgA+ memory B cells were significantly expanded in spleen, PP, and LP in the recipient mice with Th17 cells compared with control mice, whereas transfer of Th1 cells did not increase memory IgA+ B cells (Fig. 1D). In addition, CD138−CD38+IgA+ memory B cells in PP and LP were significantly increased in mice that received Th17 cells compared with those in mice that received Th1 cells (Fig. 1D). Similar results were observed on CD19−CD38−CD138+IgA+ plasma cells in spleen, PP, and LP (Supplemental Fig. 1B). Taken together, these data indicated that Th17 cells promoted IgA memory B cell development in the intestine. The levels of IL-17 and IL-21, which are signature cytokines of Th17 cells, in those Th17 cells before and after transfer were shown in Supplemental Fig. 1C and 1D.
IL-21R−/− mice and IL-17R−/− mice demonstrate impaired intestinal memory Ag-specific IgA production and reduced capacity of bacteria clearance upon enteric infection
As Th17 cells produce their signature cytokines IL-17 and IL-21, we asked whether IL-17 or IL-21 is involved in the regulation of intestinal memory IgA+ B cell development. We first infected WT mice with a low dose of C. rodentium (2 × 106 CFU/mouse) by oral gavage to avoid induction of intestinal inflammation, which could confound data interpretation, and reinfected with the same dose of C. rodentium on day 35. Mice were sacrificed 7 d post-reinfection. We found that CD4+ T cells produced both IL-17 and IL-21 in PP and LP (Fig. 2A). We then infected WT, IL-17R−/−, and IL-21R−/− mice with a low dose of C. rodentium (2 × 106 CFU/mouse) by oral gavage. Fecal pellets were collected weekly for analysis of intestinal IgA secretion. We found that C. rodentium–specific IgA levels were dramatically increased in feces of WT mice from day 21 post–initial infection, whereas intestinal C. rodentium–specific IgA secretion in IL-17R−/− mice and IL-21R−/− mice was significantly lower than that in WT mice (Fig. 2B, 2C). Once the specific Ab secretion dropped back to the relatively low levels in feces at 5 wk after initial infection (Fig. 2B, 2C), these mice were reinfected with same amount of C. rodentium for analysis of the memory B cell responses. Seven days after reinfection, the intestinal C. rodentium–specific IgA production was induced rapidly in WT mice but not in IL-17R−/− mice and IL-21R−/− mice (Fig. 2B, 2C), indicating the importance of IL-17 and IL-21 in the intestine memory IgA responses. Additionally, we found that IL-17R−/− and IL-21R−/− mice showed higher levels of fecal C. rodentium upon reinfection compared with WT mice (Fig. 2D, 2E).
IL-21R−/− mice but not IL-17R−/− mice show the impaired intestinal memory IgA+ B cells
To investigate the role of IL-17 in memory B cell development, we analyzed B cell phenotypes of WT and IL-17R−/− mice that were infected with C. rodentium (2 × 106 CFU/mouse) by oral gavage and reinfected mice 5 wk post–initial infection. One week after reinfection, WT and IL-17R−/− mice developed similar levels of GL7+CD95+ GC B cells in PP (Fig. 3A), with comparable levels of the total IgA+ B cells (Fig. 3B), B220−CD38−CD138+IgA+ plasma cells (Supplemental Fig. 2A), and B220+CD138−CD38+IgA+ memory B cells (Fig. 3C) in spleen, PP, and LP. Moreover, serum level of C. rodentium–specific IgA was similar between WT and IL-17R−/− mice (Supplemental Fig. 2B). These data indicated that decreased Ag-specific memory IgA secretion in IL-17R−/− mice is not due to the impaired memory IgA+ B cell development, which could be explained by our previous study showing the lower polymeric IgR (pIgR) expression, which facilitates the secretion of the IgA into lumen, in intestinal epithelial cells in IL-17R−/− mice (24).
Next, we investigated whether IL-21 is responsible for Th17 induction of memory IgA+ B cell responses. WT and IL-21R−/− mice were infected with C. rodentium (2 × 106 CFU/mouse) by oral gavage, and we reinfected mice 5 wk post–initial infection. One week after reinfection, the levels of CD95+GL7+ GC B cells were significantly lower in PP of IL-21R−/− mice than those of WT mice (Fig. 4A). The levels of IgA+ B cells and B220−CD38−CD138+IgA+ plasma cells were also decreased in spleen, PP, and LP of IL-21R−/− mice compared with WT mice (Fig. 4B, Supplemental Fig. 2D). Interestingly, IL-21R−/− mice showed reduced levels of B220+CD138−CD38+IgA+ memory B cells compared with WT mice (Fig. 4C). Taken together, these data demonstrated a crucial role of IL-21 in development of intestinal memory IgA+ B cells. Additionally, there was no difference of serum IL-21 levels between WT and IL-17R−/− mice (Supplemental Fig. 2C), indicating that reduced fecal IgA in IL-17R−/− was not attributed to decreased IL-21 production.
To further confirm the roles of IL-17 and IL-21 for development of intestinal IgA memory B cells, we analyzed the B cell phenotypes in IL-17R−/− and IL-21R−/− mice under steady condition. Interestingly, IL-21R−/− mice showed fewer B220+CD138−CD38+IgA+ memory B cells in PP and LP (Supplemental Fig. 3B). Furthermore, PP CD95+GL7+ GC B cells as well as B220−CD38−CD138+IgA+ plasma cells in PP and LP were also decreased in IL-21R−/− mice (Supplemental Fig. 3A, 3C). However, there were no differences of PP CD95+GL7+ GC B cells, B220+CD138−CD38+IgA+ memory B cells, and B220−CD38−CD138+IgA+ plasma cells between WT and IL-17R−/− mice (Supplemental Fig. 3A–C).
Blockade of IL-21 suppresses intestinal memory IgA+ B cell development
To further investigate the role of IL-21 in regulating memory IgA+ B cell development, we infected WT mice orally with C. rodentium (2 × 106 CFU/mouse) on day 0 and reinfected on day 35. A group of mice was given anti–IL-21R Ab or control anti-IgG Ab i.p. every other day from the day of reinfection. The mice were sacrificed 1 wk post-reinfection. Administration of anti–IL-21R Ab suppressed fecal C. rodentium–specific IgA (Fig. 5A) and inhibited bacterial clearance (Fig. 5B). Treatment of anti–IL-21R Ab decreased GL7+CD95+ GC B cells in PP (Fig. 5C) and suppressed total IgA+ B cells and B220+CD138−CD38+IgA+ memory B cells in spleen, PP, and LP (Fig. 5D, 5E). In addition, B220−CD38−CD138+IgA+ plasma cells were also decreased after treatment of anti–IL-21R Ab (Supplemental Fig. 4).
IL-21 promotes the memory B cell development–related gene expression
To investigate the mechanisms by which IL-21 promotes IgA+ memory B cell development, we treated splenic naive IgD+ B cells in vitro with IL-21 and/or IL-17. We first confirmed the B cell expression of IL-17R and IL-21R (Fig. 6A). IL-17 and IL-21 activated their downstream Erk1/2 (Fig. 6B) and Stat3 (Fig. 6C) in B cells, respectively, indicating that IL-17 and IL-21 at the tested dose of 10 ng/ml stimulates their signal pathways. Interestingly, IL-21, but not IL-17, promoted IgA production (Fig. 6D), which is consistent with our previous report (10). IL-21 treatment also increased IgA+ B cells in in vitro cultures (Fig. 6E). We then analyzed the genes, which have been associated with development of memory B cells, Krüppel-like factor 2 (Klf-2), Ski, and Bmi1 as well as class switch–related gene, Aicda. IL-21 treatment promoted the expression of those genes in B cells (Fig. 6F). Consistently, the expression of Ski and Aicda in B cells in PP and spleen of IL-21R−/− mice infected with C. rodentium was significantly decreased compared with C. rodentium–infected WT mice (Fig. 6G, 6H).
The secretory IgA production in the intestine is pivotal to keep microbiota symbiosis and maintain intestine hemostasis. In this report, we demonstrated that IL-21, a signature cytokine of Th17 cells, promotes intestinal memory IgA+ B cell development and memory IgA production. Our results thus revealed a potential aspect of Th17 cells on the development of intestinal memory IgA+ B cells, in addition to the impacts on total IgA production in the intestines.
Interaction of Th17 cells and B cells has been well studied previously on promoting the Ab class-switching and B cell differentiation. However, it is still not completely clear whether and how Th17 cells influence the development and proliferation of different B cell populations in the intestines. Unlike Th1 and Th2 cells, which are known to provide help on B cells through their signature cytokines IFN-γ and IL-4 to induce class switch recombination to IgG2a or IgG1 and IgE, respectively (25, 26), Th17 cells have distinct roles in triggering B cell proliferation and promoting the formation of GC (11). IgA in the intestine is essential in the maintenance of intestinal homeostasis and mucosal host defense (27). Besides, IgA plays a role in promoting specific bacteria colonization in the gut, such as Bacteroides fragilis, beyond its role in pathogen clearance (28). In this study, we used gut microbiota Ag-specific T cells from CBir1 Tg mice, which are specific for an immunodominant gut microbiota Ag CBir1 flagellin, to investigate how Th17 cells regulate intestinal memory in IgA+ B cells. Although CBir1 Tg T cells do not respond to luminal CBir1 Ag when transferred into WT mice (19, 29), which is attributed to the presence of intestinal IgA blocking direct contact between T cells and gut microbiota/gut microbiota Ags, these T cells do respond robustly when transferred into IgA-deficient mice or immunocompromised mice whose intestinal IgA response was impaired completely or partially, such as RAG−/− mice and TCRβxδ−/− mice (19, 24, 30). This also indicates a crucial role of intestinal IgA response in regulation of intestinal T cell response to gut microbiota. We reported in this article that in the TCRβxδ−/− mice, transfer of gut microbiota Ag-specific Th17 cells increased fecal IgA production as well as the IgA+ B cells in the spleen, PP, and LP compared with control mice. Th17 cells promoted GC formation in PP. Interestingly, the CD19+CD138−CD38+IgA+ memory B cells were significantly induced by Th17 cells in spleen, PP, and LP. Similar effects were demonstrated on CD19−CD38−CD138+IgA+ plasma cells as well. CBir1 Ag is just one of the gut microbiota Ags. Although we showed in this study that CBir1-specific Th17 cells induce intestinal IgA responses once activated, our results do not exclude the possibility that Th17 cells reactive to other gut microbiota Ags could also induce IgA responses. Our results thus suggest that gut microbiota Ag-specific Th17 cells have potential roles to promote the development of intestinal IgA+ B cells, more specifically, the memory IgA+ B cells.
It has been shown that IL-17 promotes B cell class switch recombination to IgG2a and IgG3, whereas IL-21 promotes the switch recombination to IgG2b and IgG1 (11). In addition, IL-17 regulates the expression of pIgR on intestinal epithelial cells, which mediates the translocation of IgA into intestinal lumen, thus indirectly inducing the intestine IgA production (24, 31, 32). IL-21, in cooperation with either IL-4 and IL-10 or TGF-β and retinoid acid, regulates Ab isotype switching to subclasses of IgG and IgA in B cells (10, 16). In this study, we showed that IL-21 alone increased B cell IgA production in vitro, whereas the IL-17 alone showed limited effect. The combination of IL-21 and IL-17 showed similar effects on promoting IgA production as IL-21 alone. These results suggest that the induction of B cell IgA production by Th17 cells may mainly rely on IL-21. Moreover, IL-21R−/− mice demonstrated significantly lower GC B cells in PP compared with WT (p < 0.05). Interestingly, the B220+CD138−CD38+IgA+ memory B cells were lower in IL-21R−/− mice compared with WT mice. IL-21 was previously found to be involved in plasma cell differentiation from both naive and memory B cells and was believed to play a role in the maintenance of serologic memory (33, 34). Nevertheless, our data suggested that IL-21 plays a crucial role in development of intestinal memory IgA responses. Interestingly, it has been reported that through promoting IgA responses to atypical commensals intestine, IL-21 also contributes to altering the microbiota composition and mucosal immune response (35).
Upon re-exposure to same Ag, gut memory B cells migrate into multiple sites in the GALT, synchronize the response, and exhibit strong cross-protection against related pathogens (36, 37). Our previous studies showed that the deficiency of IL-17 or IL-21 decreased intestinal total IgA production. To further elucidate the roles of IL-17 and IL-21 in development of Ag-specific memory IgA+ B cell development and IgA production, we infected IL-17R−/− and IL-21R−/− mice as well as WT mice with a low dose of C. rodentium, and both IL-21R−/− and IL-17R−/− mice showed impaired IgA responses in the gut. Moreover, the IL-21R−/− mice did not acquire the specific IgA responses after reinfection, whereas the WT mice showed a rapid elevated IgA response against C. rodentium, and no changes were observed on the IgA levels in IL-17R−/− mice as time progressed. A previous study has indicated that the intestine may adapt its memory IgA response to the predominant commensal species dominant in the lumen (7). Upon rechallenge of C. rodentium, total IgA+ B cells and B220+CD138−CD38+IgA+ memory B cells in the spleen, PP, and LP were lower in IL-21R−/− mice when compared with WT mice. In contrast, IgA+ B cells in spleen, PP, and LP were similar between WT and IL-17R−/− mice, which suggested that IL-17 did not directly induce IgA+ B cell differentiation. The lower secretory IgA production in the intestinal lumen is mainly due to lower pIgR expression in IL-17R−/− mice, which limits the IgA translocation across intestinal epithelial cells into intestinal lumen (24, 32, 38). Previous studies also suggested that IL-21 plays an important role in driving B cell differentiation into memory cells and plasma cells in both murine and human (15, 39). Thus, our data provided new evidence of IL-21 in promoting the memory IgA responses in the intestine.
Upregulation of genes involved in activation, costimulation, and survival of memory B cells has been shown in increasing frequencies of Ag-specific precursor cells, which rapidly acquire T cell help upon Ag re-encounter (40, 41). The constitutive expression of Aicda in memory B cells indicates that they remain poised to undergo additional rounds of Ab isotype switching and somatic hypermutation upon Ag re-exposure. In our study, transfer of microbiota Ag-specific Th17 cells significantly increased Aicda expression in both PP and spleen B cells compared with those in control mice. The Aicda expression in B cells isolated from the PP and spleen of WT mice after re-exposure to C. rodentium was significantly higher compared with IL-21R−/− mice. Moreover, the IL-21 treatment promoted the Aicda expression in B cells in vitro. Polycomb group gene Bmi-1 and Krüppel-like factor 2 (Klf2) have been suggested to function in memory B cells, which is important for controlling the differentiation of immune stimulation after pathogens re-enter (40, 42). Our data demonstrated that IL-21 promoted the Bmi-1 and Klf2 expression in B cells, which provided further support for signifying the role of IL-21 in driving the development of memory B cells. Ski, a transcriptional factor, is highly expressed in memory B cells compared with naive B cells, suggesting it may play a role in memory B cell differentiation (40). In our study, IL-21 treatment increased Ski expression in B cells. Furthermore, reinfection with C. rodentium induced Ski expression in PP and spleen B cells, which is positively correlated with the memory IgA production in WT mice. In contrast, the Ski expression as well as C. rodentium–specific memory IgA response were defected in IL-21R−/− mice, suggesting that Ski may play a role in IL-21 induction of memory IgA+ B cell development.
This work was supported by National Institutes of Health Grants DK105585, DK112436, DK125011, AI150210 and DK124132, a Science and Technology Acquisition and Retention award from The University of Texas System (to Y.C.), and a McLaughlin postdoctoral fellowship from The University of Texas Medical Branch (to X.H.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
activation-induced cytidine deaminase
- CBir1 Tg
CBir1 flagellin–specific TCR transgenic
Kirkegaard and Perry Laboratories
The authors have no financial conflicts of interest.