Germinal centers (GCs) provide a microenvironment that promotes and regulates the interactions of B cells with follicular Th (TFH) cells. In this study, we show that there are significantly higher frequencies of CXCR5+ICOS+ TFH cells in autoimmune BXD2 mice, and these cells express both IL-21R and IL-17RA. Although IL-17 and IL-21 are both important for the formation of spontaneous GCs and development of pathogenic autoantibodies, IL-21, but not IL-17, is required for the proper development of TFH cells in BXD2 mice. The total numbers of TFH cells and their ability to induce B cell responses in vitro were not affected by a deficiency of IL-17RA in BXD2-Il17ra−/− mice, the majority of CXCR5+ TFH cells from BXD2-Il17ra−/− mice were, however, not localized in the GC light zone (LZ). Interruption of IL-17 signaling, either acutely by AdIL-17R:Fc or chronically by Il17ra−/−, disrupted TFH–B interactions and abrogated the generation of autoantibody-forming B cells in BXD2 mice. IL-17 upregulated the expression of regulator of G-protein signaling 16 (RGS16) to promote the ability of TFH to form conjugates with B cells, which was abolished in TFH cells from BXD2-Rgs16−/− mice. The results suggests that IL-17 is an extrinsic stop signal that it acts on postdifferentiated IL-17RA+ TFH to enable its interaction with responder B cells in the LZ niche. These data suggest a novel concept that TFH differentiation and its stabilization in the LZ are two separate checkpoints and that IL-21 and IL-17 act at each checkpoint to enable pathogenic GC development.

Germinal centers (GCs) are essential in promoting T-dependent (TD) immune responses (1, 2). They provide a microenvironment that promotes and regulates the interactions of B cells with follicular Th (TFH) cells, which provide the cognate help required for the generation of high-affinity, Ab-producing plasma cells and memory B cells (3, 4). Spontaneous GC formation has been demonstrated in various mouse models of autoimmune disease (5), and dysregulated GC formation or function may contribute to autoimmune disease in humans (6, 7). Dysregulation of GCs is often caused by aberrant accumulation or function of TFH cells (8, 9), and an increase in the numbers of TFH cells has been correlated with the severity of disease in several autoimmune conditions, including systemic lupus erythematosus (1012). In mouse models of lupus, the elimination or defective function of GC TFH cells reduces or even abolishes the disease (5, 13).

High levels of IL-21 are found in the circulation of patients with systemic lupus erythematosus (14), and disruption of IL-21 signaling can inhibit GC formation and autoantibody production as well as ameliorate autoimmune disease in mice (15). Although IL-21 is important but not necessarily essential for TFH development (1618), IL-21 is considered a signature cytokine of TFH cells (15, 19, 20). IL-21 acts in an autocrine manner to stimulate the proliferation and survival of TFH cells and hence has a direct effect on their accumulation and the associated formation of GCs (21, 22). IL-21 directly induces high expression of Bcl6, the master transcription factor for TFH differentiation, which is important for the sustained expression of CXCR5 and also promotes the expression of other TFH costimulatory molecules supporting cognate help to B cells (20, 23, 24). The CXCL13–CXCR5 interaction mediates the migration of TFH cells to the GC light zone (LZ), where the affinity maturation of B cells takes place (25, 26).

Until recently, the potential role of IL-17A in GC formation and function received less attention, as TFH cells has been shown to not to produce IL-17A (19). High levels of IL-17 and Th17 cells are commonly found in patients with autoimmune disease (27) and have now been shown to drive GC and ectopic lymphoid follicle development in mouse models of autoimmune disease (28, 29). Using the BXD2 model of systemic autoimmune disease, we established that Th17 cells can be identified in the spontaneous GCs (28, 30) and that IL-17 promotes the formation of GCs in these mice (28). Analysis of the effects of IL-17 on B cells in the GCs indicate that it modulates the expression of regulator of G-protein signaling (RGS) proteins, which promote desensitization of B cell chemotaxis to chemokines CXCL12 and CXCL13 and stabilization of interactions of the GC B cells with nearby Th cells (31).

IL-17A signals through the IL-17RA–IL-17RC heterodimeric receptor complex (32). The original cloning of IL-17RA revealed that T cells as well as B cells can express high levels of Il17ra and can be responsive to IL-17 stimulation (33). Although it has been demonstrated that IL-17 is not required for the development of TFH cells (22) but may be involved in Ab production (29, 34), the possibility that IL-17 can act cooperatively with IL-21 to affect the function of TFH cells in regulating GC responses has not been explored. In this study, we provide evidence that, compared with normal B6 mice, there are significantly higher frequencies of TFH cells in autoimmune BXD2 mice, and these cells express high levels of both IL-21R and IL-17RA. Although TFH was dramatically deficient in BXD2-Il21−/− mice, the percentage and the direct B cell support function of TFH were not affected by IL-17RA deficiency. We have established that the IL-17RA signal is important for securing the localization of TFH in the GC LZ in BXD2-Il17ra−/− mice. Such anatomic stabilization is needed for TFH to induce its function and hence is a unique TFH checkpoint in autoreactive GCs.

C57BL/6 (B6) and BXD2 recombinant inbred mice were obtained from The Jackson Laboratory, B6-Il17ra−/− mice were obtained from Amgen (Thousand Oaks, CA), B6-Rgs16−/− mice were a gift from Dr. Kirk M. Druey, and B6-Il21−/− mice were obtained from the Mutant Mice Regional Resource Center (Davis, CA). These mice were backcrossed with BXD2 mice for seven generations by a marker-assisted speed congenic approach. All mice were housed in the University of Alabama at Birmingham Mouse Facility under specific pathogen-free conditions in a room equipped with an air-filtering system. The cages, bedding, water, and food were sterilized. All mouse procedures were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee.

AdIL-17R:Fc and AdLacZ (2 × 109 pfu/mouse), generous gifts from Dr. Jay Kolls (University of Pittsburgh, Pittsburgh, PA) (35), were administered i.v. Spleen tissue and cells were analyzed 10 d later.

Effector T cells (CD62L−/lo CD4 T) from BXD2 and BXD2-Rgs16−/− mice were sorted by FACSAria II (BD Biosciences) and i.v. injected into BXD2-Il17ra−/− mice (3 × 106 cells in 200 μl PBS per mouse). Recipients were sacrificed on days 6 and 21. Spleen tissue and spleen cells were collected for the indicated analysis.

B6 and B6-Il17ra−/− mice were immunized i.p. with 50 μg 4-hydroxy-3-nitrophenylacetyl hapten conjugated to chicken γ globulin (NP-CGG; BioSearch Technologies, Novato, CA) adsorbed to 1.3 mg aluminum hydroxide (Sigma-Aldrich) in a total volume of 100 μl NP-CGG aluminum hydroxide/PBS. Mice were sacrificed on days 6, 9, and 12. Spleen tissue and spleen cells were collected for the indicated analysis.

Single-cell suspensions of whole-spleen cells or migrated cells collected from the lower chamber in a cell migration assay were labeled by surface staining with the following Abs: Pacific Blue–, Alexa 488– (RM4-5 and GK1.5; BioLegend), or PE–anti-CD4 (RM4-5; BioLegend); Pacific Blue–anti-CD19 (6D5; BioLegend); Alexa 647–anti-GL-7 (GL7; eBioscience); allophycocyanin–anti-B220 (RA3-6B2; BioLegend); PE-Cy7–anti-CXCR5 (2G8; BD Biosciences); and FITC-, PE- (398.4A and 7E.17G9; eBioscience), or PE-Cy5–anti-ICOS (15F9; BioLegend). Abs against programmed cell death-1 (PD-1; RMP1-30; BioLegend), CD28 (37.51; eBioscience), CD40L (MR1; eBioscience), CD62L (MEL-14; BD Biosciences), CD44 (IM7; BioLegend), IL-21R (eBio4A9; eBioscience), IL-17RA (PAAJ-17R; eBioscience), and Fas (15A7; eBioscience) were all conjugated with PE. Peanut agglutinin (PNA) was conjugated with biotin (Vector Laboratories, Burlingame, CA) and detected by Alexa 450–conjugated streptavidin (Invitrogen).

For nuclear transcription factor staining, cells were labeled with surface markers and then fixed and permeabilized with the Foxp3 Staining Buffer Set (eBioscience), according to the manufacturer's instruction. Permeabilized cells were stained with PE–anti-Bcl6 (K112-91; BD Biosciences) and PE-anti–retinoic acid–related orphan receptor γt (RORγt) (Q31-378; BD Biosciences).

For cytokine-producing T cell analysis, cells were stimulated for 5 h with PMA (50 ng/ml; Sigma-Aldrich) and ionomycin (750 ng/ml; Sigma-Aldrich) in the presence of GolgiPlug (BD Biosciences). Cells were stained for surface markers and then fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences) before intracellular staining with allophycocyanin-anti–IL-17 (eBio17B7; eBioscience) and recombinant murine (rm)IL-21R/h-Fc chimera (R&D Systems), followed by R-PE–conjugated goat anti-human IgG (Jackson ImmunoResearch Laboratories) (36). FACS data were acquired with an LSRII FACS analyzer (BD Biosciences) and analyzed with FlowJo software (Tree Star Ashland, OR).

Except for T–B conjugates analysis, all flow cytometry analysis was carried out using a combination use of forward light scatter and side scatter height, area, and width parameters to exclude aggregated cells.

Spleens from mice were collected, embedded in Frozen Tissue Media (Fisher Scientific), and snap-frozen in 2-methylbutane. Frozen sections (8-μm thick) were fixed and processed as previously described (28). Unless specified, all reagents and Abs were purchased from Invitrogen: biotin–PNA (Vector Laboratories) followed by streptavidin (SA)–Alexa 350; Alexa 555–anti-IgM; Alexa 488–anti-CD35/21 (clone 8C12; BD Biosciences); Dylight 649–goat-anti-mouse IgG (Poly4053; BioLegend); Alexa 647–anti-CD4 (GK1.5; BioLegend); and rat anti-mouse-CXCR5 (2G8; BD Biosciences), followed by goat anti-rat IgG–biotin (Southern Biotechnology Associates) and SA–Alexa 488; rat anti–IL-17 (TC11-18H10.1; BioLegend) and Alexa 555–goat–anti-rat IgG (A21434); goat anti-mouse IL-21 (AF594; R&D Systems), and anti–IL-17R (AF448; R&D Systems) followed by Alexa 647–donkey–anti-goat IgG. Application of chicken anti-mouse RGS16 (NB300-350; Novus Biologicals) and goat anti-chicken IgY–HRP (ab6877; Abcam) was followed by the tyramide signal amplification method (TSA Kit; T20931, Invitrogen, Eugene, OR) and SA–Alexa 488. Sections were mounted in the Slow Fade Gold Antifade reagent (Invitrogen), and images were captured with a Leica DMIRBE inverted Nomarski/epifluorescence microscope outfitted with Leica TCS NT laser confocal optics (Leica Microsystems). Confocal imaging intensity analysis was carried out using ImageJ software, version 1.4, developed by the National Institutes of Health (http://rsb.info.nih.gov/nih-image/), with background intensity subtracted from each image.

Cells were labeled with fluorochrome Abs against CD3, CD4, B220, CD19, CXCR5, ICOS, and IL-17RA as needed. For T–B conjugates analysis, the doublets based on forward light scatter and side scatter height, area, and width were included before gating on CD3+CD4+ cells. The phenotypes of CD4 T cells forming conjugates with B cells were then identified by FACS sorting of CD4+B220+ or CD4+CD19+ doublets. The collected conjugates were dissociated with 2 mM EDTA/PBS, as described (37). Cells were then washed and resuspended for flow cytometry acquisition of the cell population distribution.

RNA isolation, cDNA synthesis, and real-time PCR reactions were carried out as we described previously (28). The following primers were used: Rgs16, 5′-GGTACTTGCTACTCGCTTTTCC-3′ (forward) and 5′-CAGCCGCGTCTTGAACTCT-3′ (reverse); and Gapdh, 5′-AGGTCGGTGTGAACGGATTTG-3′ (forward) and 5′-TGTAGACCATGTAGTTGAGGTCA-3′ (reverse).

Spleen CD4+ T cells from BXD2 mice were purified using a MACS column (Miltenyi Biotec) and cultured with either medium alone or with rmIL-17 (30 ng/ml; R&D Systems) for 1 or 24 h. Collected cells were subjected to protein extraction and Western immunoblot analysis using the method we described previously (31). Chicken anti-mouse RGS16 (NB300-350; Novus Biologicals) was used at 1:2000 followed by goat anti-chicken IgY–HRP (ab6877; Abcam) at 1:1000. Rabbit anti-mouse GAPDH was used at 1:5000 followed by anti-rabbit HRP-conjugated Ab (Cell Signaling Technology) at a 1:3000 dilution. HRP Abs were detected using chemiluminescence reagent (Pierce).

Serum levels of autoantibodies and total IgG levels in cultured supernatant were determined by ELISA, as we described previously (38). BiP was purchased from Assay Designs and all other autoantigens from Sigma-Aldrich. Urinary albumin was analyzed using the competitive Albuwell M ELISA kit (Exocell), as described previously (38).

The ELISPOT procedure was carried out using the method we previously described (30). Briefly, polyvinylidene difluoride–backed 96-well plates (Millipore) were coated overnight at 4°C with 5 mg/ml indicated autoantigen. BiP was purchased from StressMarq Biosciences (Victoria, BC, Canada), NP7-BSA was purchased from BioSearch Technologies, and all other autoantigens were purchased from Sigma-Aldrich. Total spleen cells in 100 μl complete medium, containing 5 × 105 cells/well for detecting IgM and 2 × 106/well for detecting IgG, were cultured overnight in a 37°C incubator. Plated cells were then incubated at room temperature with HRP-labeled goat anti-mouse IgG/M (Southern Biotechnology Associates) in PBS with 1.5% BSA for 3 to 4 h. Spots were developed with 3-amino-9-ethylcarbazole. Plates were read by an automatic ELISPOT reader (CTL Shaker Heights, OH) and analyzed with Immunospot 3.1 software (CTL).

Single-cell suspensions of spleen CD4 T cells were purified using a MACS column (Miltenyi Biotec) and stimulated with medium alone or with rmIL-17 (30 ng/ml; R&D Systems) for 3 h. The cell migration assay was carried out as described (39). The stimulated cells (2 × 106) were loaded into the upper well insert (5-μm pore size) of a Transwell system (Costar), and medium alone or medium with CXCL13 (100 ng/ml; R&D Systems) was added to the bottom chamber. After incubation for 2 h at 37°C in an incubator, the cells that had migrated down to the lower chamber were collected and counted, and the percentage of CXCR5+ICOS+ CD4 T cells (TFH) was determined by flow cytometry in some experiments. The chemotaxis index of total CD4 T cells or TFH cells was calculated by dividing the number of cells that migrated in the presence of CXCL13 by the number of cells that migrated in the absence of CXCL13 (39).

Anti-CD19 MACS column–purified (Miltenyi Biotec) B cells from BXD2 were cocultured in a 3:1 ratio with FACS-sorted TFH cells and other CD4 T subsets based on CXCR5 and ICOS expression. T cells were stimulated with anti-CD3 (1 μg/ml) and anti-CD28 (2 μg/ml). T cells and B cells were cocultured for 2 to 3 d. One hour before collection, 5-ethynyl-2'-deoxyuridine (EDU; Invitrogen) was added to the cell culture. EDU intensity together with anti-B220 and anti-Thy1.2 staining were determined by flow cytometry analysis, according to the manufacturer’s instructions. For analysis of Ig secretion, T cells and B cells were cocultured for 3 d, supernatant was collected, and total IgG were detected by ELISA.

Kidney tissues collected from the mice were fixed in 10% buffered formalin and then processed to make a paraffin-embedded block. Tissue sections (5 μm) were stained with H&E and examined by light microscopy.

All results are shown as the mean ± SD. A two-tailed t test was used when two groups were compared for statistical differences. An ANOVA test was used when more than two groups were compared for statistical differences. The p values <0.05 were considered significant.

BXD2 mice spontaneously develop a lupus-like syndrome with high titers of autoantibodies and lupus nephritis (30, 40). The disease manifests with age and correlates with the formation of GCs in the spleen (31, 41). At 10–12 wk of age, the mice have well-defined GCs with a prominent LZ and dark zone (28, 41). Analysis of the CD4+ T cells in the spleens of BXD2 mice of this age indicated that the frequency and cell count of CXCR5+ICOS+ CD4+ T cells was significantly higher than in B6 mice (Fig. 1A, 1B, Supplemental Fig. 1A, 1B). This subpopulation of cells expressed higher levels of PD-1, CD28, CD40L, CD44, IL-21R (Fig.1C, Supplemental Fig. 1C), and Bcl6 (Fig. 1D) but lower levels of CD62L than CXCR5ICOS and CXCR5+ICOSCD4+ T cells. Approximately 60–70% of the CXCR5+ICOS+ CD4+ T cells expressed Bcl6, and the highest frequency of cells expressing IL-21 was found in this subpopulation of CD4+ T cells (Fig. 1E, Supplemental Fig. 1D, 1E). A subpopulation of the CXCR5+ICOS+ CD4+ T cell population expressed RORγt (Fig. 1D) and IL-17 (Fig. 1E). The CXCR5ICOS+ subpopulation of CD4 T cells predominately expressed IL-17 and contained a population of RORγt+ cells (Fig. 1D, 1E, Supplemental Fig. 1E). However, very few cells in either subpopulation expressed both cytokines (Fig. 1E). The percentage of IL-17+ or IL-21+ cells from either subpopulation was significantly higher in BXD2 mouse spleen compared with B6 (Supplemental Fig. 1D, 1E). The current results show that IL-17 and IL-21 are produced from two distinct populations of CD4 T cells and that a subset of TFH cells in the GCs of BXD2 mice may express IL-17 rather than IL-21.

FIGURE 1.

Increased CXCR5+ICOS+ TFH cells producing IL-21 or IL-17 in spleen GCs in naive BXD2 mice. (AE) Flow cytometry analysis of CD4 T subsets based on the expression of CXCR5 and ICOS in the spleens of female 10–12-wk-old mice. Cells were first gated for CD4 T cells. (A) Analysis of CXCR5 and ICOS. (B) Frequency of the indicated CD4 T subsets. Analysis of the indicated cell-surface markers (C) or Bcl6 and RORγt (D) on CD4 T subsets in BXD2 mice. Cells were gated based on the differential expression of CXCR5 and ICOS. (E) Analysis of intracellular IL-21 and IL-17 produced by the indicated CD4 T subsets from BXD2 mice. (FJ) Confocal imaging analysis of frozen spleen sections from 3-mo-old BXD2 mice. Sections were stained with fluorochrome-conjugated reagents or Abs. Follicular dendritic cells (FDC) marked with yellow arrows in (F). The GC LZ vicinity is marked by a white dashed line (G). A higher-magnification view of the boxed area is shown (HJ, bottom panels). White arrows indicate representative CXCR5+ CD4 T cells (G), IL-21+ CD4 T cells (H), IL-17+ CD4 T cells (I), or IL-17RA+ CD4 T cells (J) in the GC LZ. Data are representative of the analysis. Numbers in dot plots indicate the percentage of cells in each quadrant (A, E). Bar graph results from (B) are shown as mean ± SD; n ≥ 5/group. **p < 0.01, ***p < 0.005 between B6 and BXD2 (B). Ctrl, Control; DZ, dark zone; FO B, follicular B cells.

FIGURE 1.

Increased CXCR5+ICOS+ TFH cells producing IL-21 or IL-17 in spleen GCs in naive BXD2 mice. (AE) Flow cytometry analysis of CD4 T subsets based on the expression of CXCR5 and ICOS in the spleens of female 10–12-wk-old mice. Cells were first gated for CD4 T cells. (A) Analysis of CXCR5 and ICOS. (B) Frequency of the indicated CD4 T subsets. Analysis of the indicated cell-surface markers (C) or Bcl6 and RORγt (D) on CD4 T subsets in BXD2 mice. Cells were gated based on the differential expression of CXCR5 and ICOS. (E) Analysis of intracellular IL-21 and IL-17 produced by the indicated CD4 T subsets from BXD2 mice. (FJ) Confocal imaging analysis of frozen spleen sections from 3-mo-old BXD2 mice. Sections were stained with fluorochrome-conjugated reagents or Abs. Follicular dendritic cells (FDC) marked with yellow arrows in (F). The GC LZ vicinity is marked by a white dashed line (G). A higher-magnification view of the boxed area is shown (HJ, bottom panels). White arrows indicate representative CXCR5+ CD4 T cells (G), IL-21+ CD4 T cells (H), IL-17+ CD4 T cells (I), or IL-17RA+ CD4 T cells (J) in the GC LZ. Data are representative of the analysis. Numbers in dot plots indicate the percentage of cells in each quadrant (A, E). Bar graph results from (B) are shown as mean ± SD; n ≥ 5/group. **p < 0.01, ***p < 0.005 between B6 and BXD2 (B). Ctrl, Control; DZ, dark zone; FO B, follicular B cells.

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On analysis of the CXCR5+ CD4+ cells in the spleens of BXD2 mice, we found that a very high percentage (∼80–90%) of these cells expressed IL-17RA (Fig. 1C, Supplemental Fig. 1C), which indicates that the TFH cells in the spleen of BXD2 mice can potentially respond to IL-17. By confocal microscopic analysis of spleen sections, we found that the LZ contained an apparent population of CD4+ T cells in close proximity to CD35+ follicular dendritic cells, with a distinct CD4 T cell zone located adjacent to the dark zone of the GC (Fig. 1F). The majority of CD4 T cells localized in the LZ end of the GC were CXCR5+ (arrows indicated, yellow color, Fig. 1G). CD4 T cells localized to the LZ of the GC also expressed IL-21 (Fig. 1H), IL-17 (Fig. 1I), and IL-17RA (Fig. 1J). These results demonstrate that there was a distinct population of CXCR5+CD4+ T cells in the LZ that express IL-17RA and either IL-21 or IL-17 in the BXD2 mice. Such IL-17–producing T cells may represent a unique population of IL-17–producing TFH, as identified by their surface phenotype and location.

Although it is shown that blockade of IL-21 or IL-17 signaling can affect GC formation and ameliorate autoimmune disease, it is not known whether the effects of the two cytokines on these aspects of the immune response are identical in BXD2 mice. To answer this question, we generated BXD2-Il17ra−/− and BXD2-Il21−/− mice. Compared with wild-type BXD2 (BXD2-WT) mice, there were lower levels of anti-histone and anti-DNA autoantibodies of the IgM, IgG2b, and IgG2c isotype in both BXD2-Il17ra−/− and BXD2-Il21−/− mice (Fig. 2A). Kidney disease, as determined by the levels of urinary albumin (Fig. 2B), the extent of inflammatory infiltrates (Fig. 2C), and IgG deposition in kidney glomeruli (Fig. 2D, 2E) was alleviated in both BXD2-Il17ra−/− and BXD2-Il21−/− mice. Splenomegaly, which is a key autoimmune feature of BXD2-WT mice, was also reduced in both BXD2-Il17ra−/− and BXD2-Il21−/− mice (Supplemental Fig. 2A, 2B). The frequency of GC B cells (Fas+PNA+CD19+ cells) was significantly lower in BXD2-Il17ra−/− and BXD2-Il21−/− mice than in BXD2-WT mice (Fig. 2F). Defective formation of GCs in BXD2-Il17ra−/− and BXD2-Il21−/− mice was apparent by confocal imaging analysis (Fig. 2G) and quantitation of the fractional area of PNA+ B cells in each IgM+ spleen follicle by ImageJ analysis (National Institutes of Health) (Fig. 2H). These data indicated that both IL-17RA and IL-21 are required for optimal GC responses and autoimmune disease development in BXD2 mice.

FIGURE 2.

GC development and autoimmune disease severity were alleviated in BXD2-Il17ra−/− and BXD2-Il21−/− mice. (A) ELISA of IgM, IgG2b, and IgG2c autoantibodies specific for histone and DNA in the sera of 4- to 5-mo-old mice. (B) ELISA of urinary albumin in 4- to 5-mo-old mice. (C) H&E staining in paraffin-embedded kidney sections from 5-mo-old mice. Original magnification ×20. (D) Immunofluorescence analysis for IgG (red) deposition in glomeruli in frozen kidney sections from 5-mo-old mice. Original magnification ×20. Each image was equally magnified digitally to reveal the detail structure. (E) ImageJ (National Institutes of Health) quantitation of the intensity of IgG deposition per glomerulus in frozen kidney sections from 5-mo-old mice. (F) Flow cytometry analysis of the percentage of PNA+Fas+ GC B cells in the spleens of 2- to 3-mo-old mice. Cells were gated on CD19+ B cells. Numbers above the boxed region represent the frequency of PNA+Fas+ within CD19+ cells (mean ± SD). (G and H) Confocal imaging analysis of PNA+ GC B cells in frozen spleen sections from 2- to 3-mo-old mice. (G) Sections were stained with anti-IgM (red), anti-CD4 (green), and PNA (blue). Original magnification ×10. (H) ImageJ quantitation of the area of PNA+ staining versus the area of each IgM+ spleen follicle. All data are representative or an average of n ≥ 5 female mice/group. *p < 0.05, **p < 0.01, ***p < 0.005 compared with BXD2-WT mice.

FIGURE 2.

GC development and autoimmune disease severity were alleviated in BXD2-Il17ra−/− and BXD2-Il21−/− mice. (A) ELISA of IgM, IgG2b, and IgG2c autoantibodies specific for histone and DNA in the sera of 4- to 5-mo-old mice. (B) ELISA of urinary albumin in 4- to 5-mo-old mice. (C) H&E staining in paraffin-embedded kidney sections from 5-mo-old mice. Original magnification ×20. (D) Immunofluorescence analysis for IgG (red) deposition in glomeruli in frozen kidney sections from 5-mo-old mice. Original magnification ×20. Each image was equally magnified digitally to reveal the detail structure. (E) ImageJ (National Institutes of Health) quantitation of the intensity of IgG deposition per glomerulus in frozen kidney sections from 5-mo-old mice. (F) Flow cytometry analysis of the percentage of PNA+Fas+ GC B cells in the spleens of 2- to 3-mo-old mice. Cells were gated on CD19+ B cells. Numbers above the boxed region represent the frequency of PNA+Fas+ within CD19+ cells (mean ± SD). (G and H) Confocal imaging analysis of PNA+ GC B cells in frozen spleen sections from 2- to 3-mo-old mice. (G) Sections were stained with anti-IgM (red), anti-CD4 (green), and PNA (blue). Original magnification ×10. (H) ImageJ quantitation of the area of PNA+ staining versus the area of each IgM+ spleen follicle. All data are representative or an average of n ≥ 5 female mice/group. *p < 0.05, **p < 0.01, ***p < 0.005 compared with BXD2-WT mice.

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IL-21 is important for the development of TFH (21, 22), especially in chronic autoimmune response (15), whereas IL-17 is not required for the development of TFH cells in normal mice (22). The total CD4+ T cell counts in the spleens of BXD2-Il21−/− and BXD2-Il17ra−/− mice were lower than in BXD2-WT mice (Supplemental Fig. 2C). There were significantly fewer CXCR5+ICOS+ TFH cells in the spleens of BXD2-Il21−/− mice than in BXD2-WT mice (Fig. 3A, 3B, Supplemental Fig. 2D, 2E). The deficiency in IL-21, however, also resulted in a reduction in the frequency of IL-17+CXCR5+ICOS+ CD4+ T cells (Fig. 3C), which is consistent with the report that IL-21 can act to promote the formation of Th17 cells (42, 43).

FIGURE 3.

Increased frequency of TFH cells in the spleens of BXD2-Il17ra−/− mice. Single-cell suspensions were prepared from the spleens of female 10–12-wk-old mice. (A) Flow cytometry analysis of CXCR5 and ICOS on CD4 T cells. (B) Bar graphs showing the frequency (left panel) and the total cell count (right panel) of CXCR5+ICOS+ TFH cells per spleen. (C) Top panel: flow cytometry analysis of intracellular IL-21 and IL-17 produced by CXCR5+ICOS+ TFH cells. Cells were gated on CD4+ T cells first. Bottom panel: quantitation of the percentage of CXCR5+ICOS+ TFH that expressed either IL-17 or IL-21. (D) Flow cytometry analysis of the indicated cell-surface markers on CXCR5+ICOS+ CD4 T cells. (E and F) Functional assay of T cells in vitro. Sorted TFH cells and other CD4 T subsets based on CXCR5 and ICOS expression from BXD2-WT or Il17ra−/− mice, either unstimulated or stimulated with anti-CD3 plus anti-CD28, were cocultured with B cells from BXD2 mice. Flow cytometry analysis of the proliferative responses indicated by EDU+ staining was carried out on day 3 for B cells (E, top panel) and day 2 for T cells (E, bottom panel). (F) ELISA analysis of IgG secretion in the coculture supernatant on day 3 after subtraction of the background levels without anti-CD3 and anti-CD28 stimulation. Numbers in quadrants or above gate indicate the percentage of cells (A, C) or the mean percentage ± SD (E, F). Data are representative of the analysis of n = 5 mice/group (A, C, top panel, D), two independent experiments with n = 3 mice/group (E, F), or are the mean ± SD of n = 5 mice/group (B, C, bottom panel). *p < 0.05, **p < 0.01, ***p < 0.005 compared with BXD2-WT mice (B, C), the unstimulated group (E), or the CXCR5+ICOS+ CD4 T subset (F). Ctrl, Control.

FIGURE 3.

Increased frequency of TFH cells in the spleens of BXD2-Il17ra−/− mice. Single-cell suspensions were prepared from the spleens of female 10–12-wk-old mice. (A) Flow cytometry analysis of CXCR5 and ICOS on CD4 T cells. (B) Bar graphs showing the frequency (left panel) and the total cell count (right panel) of CXCR5+ICOS+ TFH cells per spleen. (C) Top panel: flow cytometry analysis of intracellular IL-21 and IL-17 produced by CXCR5+ICOS+ TFH cells. Cells were gated on CD4+ T cells first. Bottom panel: quantitation of the percentage of CXCR5+ICOS+ TFH that expressed either IL-17 or IL-21. (D) Flow cytometry analysis of the indicated cell-surface markers on CXCR5+ICOS+ CD4 T cells. (E and F) Functional assay of T cells in vitro. Sorted TFH cells and other CD4 T subsets based on CXCR5 and ICOS expression from BXD2-WT or Il17ra−/− mice, either unstimulated or stimulated with anti-CD3 plus anti-CD28, were cocultured with B cells from BXD2 mice. Flow cytometry analysis of the proliferative responses indicated by EDU+ staining was carried out on day 3 for B cells (E, top panel) and day 2 for T cells (E, bottom panel). (F) ELISA analysis of IgG secretion in the coculture supernatant on day 3 after subtraction of the background levels without anti-CD3 and anti-CD28 stimulation. Numbers in quadrants or above gate indicate the percentage of cells (A, C) or the mean percentage ± SD (E, F). Data are representative of the analysis of n = 5 mice/group (A, C, top panel, D), two independent experiments with n = 3 mice/group (E, F), or are the mean ± SD of n = 5 mice/group (B, C, bottom panel). *p < 0.05, **p < 0.01, ***p < 0.005 compared with BXD2-WT mice (B, C), the unstimulated group (E), or the CXCR5+ICOS+ CD4 T subset (F). Ctrl, Control.

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Surprisingly, the frequency of CXCR5+ICOS+ TFH cells was significantly higher in the spleens of BXD2-Il17ra−/− mice than in BXD2-WT mice, which resulted in an equivalent cell count for this subset in both strains of mice (Fig. 3A, 3B, Supplemental Fig. 2D, 2E). The frequency of IL-21+ cells within the CXCR5+ICOS+ CD4 T subset from BXD2-Il17ra−/− mice was equivalent to that in the BXD2-WT mice, whereas the frequency of IL-17–expressing cells was significantly higher (Fig. 3C). The expression of CD28, CD40L, CD62L, CD44, and IL-21R was similar in CXCR5+ICOS+ TFH cells from BXD2-WT and BXD2-Il17ra−/− mice, although the expression intensity of PD-1 was slightly, but not significantly, lower (Fig. 3D, Supplemental Fig. 2F).

To test the B cell helper function of CXCR5+ICOS+ CD4+ T cells from BXD2-Il17ra−/− mice, we cocultured anti-CD3 plus anti-CD28–stimulated CXCR5+ICOS+ TFH cells and other CD4 T subsets based on CXCR5 and ICOS expression from either BXD2-WT or BXD2-Il17ra−/− mice with B cells from BXD2-WT mice. The ability of CXCR5+ICOS+ CD4 T cells from BXD2-Il17ra−/− to promote B cell activation and IgG secretion was equivalent to those from BXD2-WT mice (Fig. 3E, top panel, 3F). The ability of CXCR5+ICOS+ CD4 T cells from both groups to promote IgG secretion was greater compared with other subsets of CD4 T cells, supporting the role of CXCR5+ICOS+ CD4 T cells as the TFH subset (Fig. 3F). The proliferative response of CXCR5+ICOS+CD4+ T cells from BXD2-Il17ra−/− mice to anti-CD3 plus anti-CD28 stimulation was even higher than those from BXD2-WT mice (Fig. 3E, bottom panel). Thus, despite the dramatically reduced development of spontaneous GCs in the BXD2-Il17ra−/− mice, the numbers of TFH cells from these mice were not reduced, and these cells exhibited equivalent ability, compared with those from WT mice, to support the activation and IgG secretion of B cells in vitro.

Confocal imaging analysis confirmed that, in the spleen follicles of BXD2-WT mice, the majority of CXCR5+CD4+ T cells were localized in the LZ of the PNA+ GC area (Fig. 4A, left panels). Although intact white pulp follicles were found in the spleens of BXD2-Il21−/− mice, PNA+ GC structures could not be identified, and almost no CXCR5+ TFH cells were present (Fig. 4A, right panels). By contrast, marked mislocalization of the CXCR5+CD4+ T cells was apparent in the spleens of BXD2-Il17ra−/− mice. In these mice, many CXCR5+CD4+ T cells were located outside the PNA+ GC area and were not in immediate contact with PNA+ GC B cells (Fig. 4A, middle panels).

FIGURE 4.

Abnormal localization of TFH cells and defective TFH–B cell conjugates in the spleens of BXD2-Il17ra−/− mice. (A and B) Immunofluorescence microscopy of frozen sections from spleens of naive 3-mo-old mice (A) or from spleens of 2.5-mo-old mice 10 d postadministration of either AdLacZ or AdIL-17R:Fc (B). Sections were stained with anti-CXCR5 (red), anti-CD4 (green), and PNA (blue). The GC border is marked by a white dashed line (B, top panel), and the white rectangle regions from the top panels were further magnified to show the distribution of CXCR5+ CD4 T cells (yellow, arrows). The GC LZ vicinity is marked by a white dashed line (all bottom panels). Original magnification ×20. (CE) Analysis of conjugates formed between CXCR5+ICOS+CD4 T and B cells in the spleens of 3-mo-old mice. (C) FACS sorting of CD4–B220 cell conjugates. Top panel: cells gated on CD3+CD4+ T cells prior to acquisition of CD4+B220+ doublets. Middle panel: conjugated cells were dissociated with EDTA/PBS as described (37) to yield CD4 and B200 single cells. Bottom panel: analysis of expression of ICOS and CXCR5 within the dissociated CD4 T cells; numbers in quadrants indicate frequency. (D) Frequency of CD4+B220+ doublets in the CD3+CD4+ T cells in each strain. (E) Frequency of CXCR5+ICOS+ or CXCR5ICOS+ cells in dissociated CD4 T cells. Data are representative of the analysis of n = 5 mice/group (A), two independent experiments with n = 3 mice/group (B, C), or indicate mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005 compared with BXD2-WT mice (D, E).

FIGURE 4.

Abnormal localization of TFH cells and defective TFH–B cell conjugates in the spleens of BXD2-Il17ra−/− mice. (A and B) Immunofluorescence microscopy of frozen sections from spleens of naive 3-mo-old mice (A) or from spleens of 2.5-mo-old mice 10 d postadministration of either AdLacZ or AdIL-17R:Fc (B). Sections were stained with anti-CXCR5 (red), anti-CD4 (green), and PNA (blue). The GC border is marked by a white dashed line (B, top panel), and the white rectangle regions from the top panels were further magnified to show the distribution of CXCR5+ CD4 T cells (yellow, arrows). The GC LZ vicinity is marked by a white dashed line (all bottom panels). Original magnification ×20. (CE) Analysis of conjugates formed between CXCR5+ICOS+CD4 T and B cells in the spleens of 3-mo-old mice. (C) FACS sorting of CD4–B220 cell conjugates. Top panel: cells gated on CD3+CD4+ T cells prior to acquisition of CD4+B220+ doublets. Middle panel: conjugated cells were dissociated with EDTA/PBS as described (37) to yield CD4 and B200 single cells. Bottom panel: analysis of expression of ICOS and CXCR5 within the dissociated CD4 T cells; numbers in quadrants indicate frequency. (D) Frequency of CD4+B220+ doublets in the CD3+CD4+ T cells in each strain. (E) Frequency of CXCR5+ICOS+ or CXCR5ICOS+ cells in dissociated CD4 T cells. Data are representative of the analysis of n = 5 mice/group (A), two independent experiments with n = 3 mice/group (B, C), or indicate mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005 compared with BXD2-WT mice (D, E).

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To determine whether IL-17RA signaling is required for localization of TFH in GC LZ, we neutralized endogenous IL-17 in WT BXD2 mice by administration of AdIL-17R:Fc. Following such treatment, there was a dramatic reduction in the localization of CXCR5+CD4+ T cells to the GC LZ, and the majority of these cells were not in close contact with PNA+ GC B cells (Fig. 4B, right panel). By contrast, the frequency and location of CXCR5+ CD4 T cells in spleens of control AdLacZ-treated BXD2 mice were similar to those observed in untreated BXD2 mice, with the majority of these cells found in the LZ of the GCs (Fig. 4B, left panel). Consistent with our previous results (25), the size, weight, and cellularity of the spleen and the numbers of autoantibody-producing B cells were significantly reduced in mice treated with AdIL-17R:Fc compared with mice treated with AdLacZ (data not shown). These results suggest that neutralization of IL-17 also disrupted the interaction of TFH and GC B cells and perturbed the generation of autoantibody-producing B cells.

We next determined whether mislocation of TFH cells was associated with loss of CD4+ T–B cell conjugate formation in the spleens of BXD2-Il17ra−/− mice. A significantly higher percentage of B cell–CD4 T cell doublets was isolated from the spleens of BXD2-WT mice than of B6 mice (Fig. 4C, top panel, 4D) and the frequency of CXCR5+ICOS+ TFH and CXCR5ICOS+ CD4 T cells in the disassociated conjugates was higher in BXD2-WT mice than in B6 or BXD2-Il21−/− mice (Fig. 4C, bottom panel, 4E). Interestingly, although there was an increased frequency of CXCR5+ICOS+ TFH and CXCR5ICOS+ CD4 T cells in the spleens of BXD2-Il17ra−/− mice, the frequency of these CD4 T cells forming conjugates with B cells was lower in BXD2-Il17ra−/− than in BXD2 mice (Fig. 4C, bottom panel, 4E). These results indicate that TFH cells in BXD2-Il17ra−/− mice were unable to locate to the GC LZ and that their ability to interact with B cells was compromised.

It has been reported that upregulation of RGS16 is associated with a desensitization of both B cells and CD4+ T cells to CXCL12 and CXCL13 migratory cues (39, 44), thereby prolonging retention of both cells in the GC for efficient interaction. IL-17 can induce upregulation of RGS16 proteins in B cells, resulting in migration arrest (31). We investigated the association between IL-17 signaling and RGS16 expression in CD4+ T cells. IL-17 stimulation of CD4+ T cells isolated from the spleens of BXD2 mice resulted in a significant increase in RGS16 at both the mRNA (Fig. 5A) and protein levels (Fig. 5B). Analysis of IL-17 modulation of the TFH chemotactic response to CXCL13 showed that pretreatment with IL-17 inhibited CXCL13-induced migration of TFH cells from BXD2-WT mice in response to CXCL13 in the transwell assay (Fig. 5C). A similar result was found in CXCR5+ICOS CD4 T cells that also expressed both CXCR5 and IL-17RA (data not shown), suggesting that the migration desensitizing effect of IL-17 is not limited to CXCR5+ICOS+ TFH cells. In contrast, pretreatment with IL-17 did not inhibit migration of TFH from BXD2-Rgs16−/− mice (Fig. 5C). The possibility that TFH cells from BXD2-Rgs16−/− mice expressed lower levels of IL-17RA compared with TFH cells from BXD2-WT mice was ruled out by FACS analysis, which showed an equivalent intensity of IL-17RA (Supplemental Fig. 3A). These results suggest that RGS16 is involved in the migration arrest of TFH cells stimulated by IL-17.

FIGURE 5.

RGS16 is involved in IL-17–induced location of CXCR5+ CD4 T cells. (AC) MACS-purified CD4 T cells from spleens were cultured with/without IL-17 (30 ng/ml) in vitro. Quantitative RT-PCR analysis of Rgs16 (A) and Western blot and ImageJ quantitation (B) of RGS16 at the indicated times. (C) Chemotactic response of CXCR5+ICOS+ CD4 T cells. Culture medium or IL-17–preincubated CD4 T cells in response to CXCL13 or medium were analyzed. Migrated cells were counted, and the percentage of CXCR5+ICOS+ TFH cells was determined by flow cytometry. The chemotaxis index was calculated as previously described (39). (D) Immunofluorescence microscopy of frozen spleen sections of naive mice. For both strains, the same spleen follicle is presented for each staining. Arrows indicate representative RGS16+ CD4 T cells (yellow, top panel) or CXCR5+CD4 T cells (yellow, bottom panel) in the GC LZ. Original magnification ×20. (E) Immunofluorescence microscopy of frozen spleen sections showing the localization of CXCR5+CD4 T cells in naive mice. Original magnification ×20. GC LZ vicinity is marked by a white dashed line (D, E). (F and G) B cell–T cell conjugate analysis of the indicated strains. (F) Frequency of CD4+CD19+ doublets in spleen cells. (G) Frequency of CXCR5ICOS+ or CXCR5+ICOS+ CD4 T cells dissociated from the CD4+CD19+ doublets. Data are representative of the analysis of three to four mice (2.5–3 mo old) per group (D, E) or indicate mean ± SD of two to three mice per group for three repeated experiments. *p < 0.05, **p < 0.01, ***p < 0.005 between the groups (A–C, F, G). mIL-17, murine IL-17.

FIGURE 5.

RGS16 is involved in IL-17–induced location of CXCR5+ CD4 T cells. (AC) MACS-purified CD4 T cells from spleens were cultured with/without IL-17 (30 ng/ml) in vitro. Quantitative RT-PCR analysis of Rgs16 (A) and Western blot and ImageJ quantitation (B) of RGS16 at the indicated times. (C) Chemotactic response of CXCR5+ICOS+ CD4 T cells. Culture medium or IL-17–preincubated CD4 T cells in response to CXCL13 or medium were analyzed. Migrated cells were counted, and the percentage of CXCR5+ICOS+ TFH cells was determined by flow cytometry. The chemotaxis index was calculated as previously described (39). (D) Immunofluorescence microscopy of frozen spleen sections of naive mice. For both strains, the same spleen follicle is presented for each staining. Arrows indicate representative RGS16+ CD4 T cells (yellow, top panel) or CXCR5+CD4 T cells (yellow, bottom panel) in the GC LZ. Original magnification ×20. (E) Immunofluorescence microscopy of frozen spleen sections showing the localization of CXCR5+CD4 T cells in naive mice. Original magnification ×20. GC LZ vicinity is marked by a white dashed line (D, E). (F and G) B cell–T cell conjugate analysis of the indicated strains. (F) Frequency of CD4+CD19+ doublets in spleen cells. (G) Frequency of CXCR5ICOS+ or CXCR5+ICOS+ CD4 T cells dissociated from the CD4+CD19+ doublets. Data are representative of the analysis of three to four mice (2.5–3 mo old) per group (D, E) or indicate mean ± SD of two to three mice per group for three repeated experiments. *p < 0.05, **p < 0.01, ***p < 0.005 between the groups (A–C, F, G). mIL-17, murine IL-17.

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To further determine the function of IL-17–induced upregulation of RGS16 in the localization of TFH in vivo, we performed confocal imaging analysis. These studies revealed that RGS16 was highly expressed by cells in spleen follicles, including CD4 T cells, in the GC LZ of BXD2-WT mice but not in BXD2-Il17ra−/− mice (Fig. 5D, top panels). Moreover, localization of RGS16+CD4 T cells in GC LZ mirrored that of CXCR5+ CD4 T cells in the GC LZ in the spleens of BXD2 mice (Fig. 5D, bottom left panel). By contrast, in BXD2-Il17ra−/− mice, few RGS16+ cells were detected in spleen follicles, and this was associated with fewer CXCR5+CD4 T cells localized in the GC LZ (Fig. 5D, bottom right panel). We also observed reduced numbers of CXCR5+ CD4 T cells in the spleen GC LZ of BXD2-Rgs16−/− (Fig. 5E, right panel) compared with BXD2-WT mice (Fig. 5E, left panel). FACS analysis revealed a lower frequency of TFH cells in the spleens of BXD2-Rgs16−/− mice than in the spleens of BXD2-WT mice (Supplemental Fig. 3B), and confocal imaging demonstrated that the size of the GCs in the spleens of BXD2-Rgs16−/− mice was also smaller than the size of the GCs formed in BXD2 mice (Fig. 5E). Analysis of T–B cell conjugates further showed that there was reduced in vivo formation of CD4 T–B cell conjugates (Fig. 5F), with a lower frequency of CXCR5+ICOS+ CD4 T and CXCR5ICOS+ CD4 T cells in the conjugates isolated from the spleens of BXD2-Rgs16−/− compared with those isolated from BXD2 mice (Fig. 5G, Supplemental Fig. 3C). Collectively, these results suggest that TFH cells stimulated by IL-17 increased expression of a desensitizing regulator of chemotaxis, RGS16, resulting in migration arrest in the splenic GC LZ of BXD2 mice.

We previously showed that IL-17 acts through the canonical NF-ĸB pathway to upregulate RGS16 in B cells, thereby modulating B cell migration responses (31). To rule out the possibility that the IL-17–induced stabilization of TFH cells in the GCs is secondary to its effects on B cells, effector CD4 T cells (CD62L−/lo) from BXD2-WT and BXD2-Rgs16−/− mice were adoptively transferred into BXD2-Il17ra−/− mice. This strategy enabled us to discriminate between IL-17RA+RGS16+ and IL-17RA+RGS16 TFH cells in an IL-17hi but IL-17RA–deficient microenvironment. Six days following transfer, it was apparent that donor effector CD4+ T cells from BXD2-WT mice caused an increased percentage of Fas+GL7+ GC B cells and splenomegaly in BXD2-Il17ra−/− recipients. By contrast, donor cells from BXD2-Rgs16−/− mice did not lead to significant changes in these phenotypes in the BXD2-Il17ra−/− recipients (Fig. 6A, Supplemental Fig. 4A). ELISPOT analysis of autoantibody-producing cells on day 21 also indicated that BXD2-WT, but not BXD2-Rgs16−/−, donor cells promoted autoantibody production associated with dramatically elevated numbers of autoantibody-forming B cells in BXD2-Il17ra−/− recipients (Fig. 6B, Supplemental Fig. 4B).

FIGURE 6.

RGS16+IL-17RA+, but not RGS16IL-17RA+, CD4 T cells resumed GC formation in BXD2-Il17ra−/− mice. Effector CD62L−/lo CD4 T cells from 2.5-mo-old BXD2 or BXD2-Rgs16−/− mice were FACS-sorted and transferred into age/sex-matched BXD2-Il17ra−/− mice by i.v. injection (3 × 106 cells in 200 μl PBS/mouse). (A) Flow cytometry analysis of GL-7+Fas+ GC B cells from the spleens of recipients on day 6. (B) ELISPOT assay of the autoantibody-producing B cells from the spleens of recipients on day 21. (C) The ratio of CD4+–B220+ doublets versus CD4+–B220 singlets (left panel) and the ratio of CXCR5+ICOS+CD4+–B220+ doublets versus CXCR5+ICOS+CD4+–B220 singlets (right panel) determined by flow cytometry sorting and analysis on day 6 in recipient spleens. Donor IL-17RA+ CD4 T cells and CXCR5+ICOS+ TFH cells were separated from IL-17RA recipient cells by IL-17RA staining. Ratios were calculated using the absolute cell count. (D) Quantitative RT-PCR analysis of Rgs16 in conjugated and nonconjugated FACS-sorted CD4 T cells from BXD2 donor or BXD2-Il17ra−/− recipients on day 6. Data are representative (A) or indicate mean ± SD of two independent experiments with n = 3 mice/group. *p < 0.05, **p < 0.01 among the groups (B–D). Ctrl, Control.

FIGURE 6.

RGS16+IL-17RA+, but not RGS16IL-17RA+, CD4 T cells resumed GC formation in BXD2-Il17ra−/− mice. Effector CD62L−/lo CD4 T cells from 2.5-mo-old BXD2 or BXD2-Rgs16−/− mice were FACS-sorted and transferred into age/sex-matched BXD2-Il17ra−/− mice by i.v. injection (3 × 106 cells in 200 μl PBS/mouse). (A) Flow cytometry analysis of GL-7+Fas+ GC B cells from the spleens of recipients on day 6. (B) ELISPOT assay of the autoantibody-producing B cells from the spleens of recipients on day 21. (C) The ratio of CD4+–B220+ doublets versus CD4+–B220 singlets (left panel) and the ratio of CXCR5+ICOS+CD4+–B220+ doublets versus CXCR5+ICOS+CD4+–B220 singlets (right panel) determined by flow cytometry sorting and analysis on day 6 in recipient spleens. Donor IL-17RA+ CD4 T cells and CXCR5+ICOS+ TFH cells were separated from IL-17RA recipient cells by IL-17RA staining. Ratios were calculated using the absolute cell count. (D) Quantitative RT-PCR analysis of Rgs16 in conjugated and nonconjugated FACS-sorted CD4 T cells from BXD2 donor or BXD2-Il17ra−/− recipients on day 6. Data are representative (A) or indicate mean ± SD of two independent experiments with n = 3 mice/group. *p < 0.05, **p < 0.01 among the groups (B–D). Ctrl, Control.

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Analysis of conjugate formation further revealed that donor IL-17RA+ CD4+ T cells and CXCR5+ICOS+ TFH cells from BXD2-WT mice had a significantly enhanced ability to form conjugates with recipient B cells compared with the same population of CD4+ T cells from either BXD2-Rgs16−/− donors or from endogenous recipient BXD2-Il17ra−/− cells (Fig. 6C, Supplemental Fig. 4C). Real-time RT-PCR showed that, on day 6 posttransfer of BXD2-WT effector CD4+ T cells, there was higher expression of Rgs16 in conjugated donor IL-17RA+ CD4+ T cells than in singlet donor cells and in recipient IL-17RACD4+ T cells (Fig. 6D). These results further indicate that intact RGS16 in the IL-17RA+CD4+ T effector cells is needed to promote the formation of spontaneous GCs, even in an environment in which all other cells are IL-17RA deficient.

It is unknown whether IL-17RA–IL-17 signaling affects TFH in a similar pattern in the TD response in nonautoimmune mice. To address this, we analyzed the effect of IL-17 on CD4 T cells and TFH cells in normal B6 mice. CXCR5+ICOS+ TFH cells from B6 expressed the highest level of IL-17RA followed by the CXCR5+ICOS CD4 T subset, whereas the other CD4 T subsets expressed low levels of IL-17RA (Fig. 7A). IL-17 stimulation of CD4+ T cells isolated from the spleens of B6 mice resulted in a significant increase in Rgs16 at the 4-h time point (Fig. 7B) and also inhibited migration of CD4 T cells in response to CXCL13 in the transwell assay (Fig. 7C). To further investigate TFH response in B6-Il17ra−/− mice during a TD response, B6-Il17ra−/− mice and WT B6 mice were immunized with NP (21)-CGG and sacrificed on days 6, 9, and 12. The results indicate that the frequency of TFH peaked on day 9 and began to decrease by day 12 after NP-CGG immunization. The TFH frequency was synchronized with that of anti-NP IgG Ab level in sera, whereas the GC B population peaked on day 12. On day 9, the frequency of CXCR5+ICOS+ TFH was approximately two-fold higher in B6-Il17ra−/− mice, compared with WT B6 (Fig. 7D, left panel). Although IL-21 production was comparable, production of IL-17 by CXCR5+ICOS+ TFH cells was significantly increased in B6-Il17ra−/− mice, compared with WT B6 (Fig. 7D, middle panel). The frequency of GC B cells was, however, higher in WT compared with B6-Il17ra−/− mice (Fig. 7D, right panel), which is consistent with the lower number of high-affinity anti-NP IgG Ab-producing B cells detected by ELISPOT in B6-Il17ra−/− mice (Fig. 7E). Confocal image analysis showed that, on day 9, CXCR5+ CD4 T cells could be localized in the LZ of the GCs in the spleen of WT B6 mice (Fig. 7F, left panel, yellow color). By contrast, although many CXCR5+ CD4 T cells were identified in the spleen of B6-Il17ra−/− mice, these CXCR5+ CD4 T cells were not localized to the LZ of the smaller GCs found in these mice (Fig. 7F, right panel, yellow color). These results indicate that, even in normal mice, IL-17RA–IL-17 signaling can regulate TFH cell localization in GC LZ potentially via upregulation of Rgs16 in TFH cells to promote GC development and high-affinity Ab production.

FIGURE 7.

The increased percentage of TFH cells in B6-Il17ra−/− mice also exhibited a lower ability to promote a TD immunization response. (A) FACS analysis of IL-17RA in the indicated subsets. Cells were first gated for CD4 T cells. (B) Quantitative RT-PCR analysis of Rgs16 in purified CD4 T cells with or without IL-17 (30 ng/ml) stimulation. (C) CXCL13-mediated chemotactic response of CD4 T cells with and without IL-17 pretreatment. (DF) The 2.5-mo-old B6 and B6-Il17ra−/− mice were immunized with NP (21)-CGG by i.p. Mice with vehicle injection were used as control. (D) Flow cytometry analysis of TFH (left panel), expression of IL-21 and IL-17 in TFH (gated on CXCR5+ICOS+ CD4 T cells, middle panel), and GC B cells (gated on CD19+ cells, right panel) on day 9 after NP-CGG immunization in the indicated mice. Numbers in quadrants indicate mean frequency ± SD. (E) ELISPOT quantitation of the IgG anti-NP7 Ab-producing B cells at the indicated days (D) post–NP-CGG immunization. (F) Confocal imaging on spleen tissue for GC B cells and TFH on day 9. GC LZ is indicated by a white dashed line. Original magnification ×20. Representative data (A, D, F) or mean ± SD (B, C, E) are shown. All experiments were carried out using three mice per group for two experiments (2.5 mo old). *p < 0.05, **p < 0.01.

FIGURE 7.

The increased percentage of TFH cells in B6-Il17ra−/− mice also exhibited a lower ability to promote a TD immunization response. (A) FACS analysis of IL-17RA in the indicated subsets. Cells were first gated for CD4 T cells. (B) Quantitative RT-PCR analysis of Rgs16 in purified CD4 T cells with or without IL-17 (30 ng/ml) stimulation. (C) CXCL13-mediated chemotactic response of CD4 T cells with and without IL-17 pretreatment. (DF) The 2.5-mo-old B6 and B6-Il17ra−/− mice were immunized with NP (21)-CGG by i.p. Mice with vehicle injection were used as control. (D) Flow cytometry analysis of TFH (left panel), expression of IL-21 and IL-17 in TFH (gated on CXCR5+ICOS+ CD4 T cells, middle panel), and GC B cells (gated on CD19+ cells, right panel) on day 9 after NP-CGG immunization in the indicated mice. Numbers in quadrants indicate mean frequency ± SD. (E) ELISPOT quantitation of the IgG anti-NP7 Ab-producing B cells at the indicated days (D) post–NP-CGG immunization. (F) Confocal imaging on spleen tissue for GC B cells and TFH on day 9. GC LZ is indicated by a white dashed line. Original magnification ×20. Representative data (A, D, F) or mean ± SD (B, C, E) are shown. All experiments were carried out using three mice per group for two experiments (2.5 mo old). *p < 0.05, **p < 0.01.

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Analysis of the CD4+ T subpopulations in the spleens of BXD2 mice provided several unique insights into the regulation of TFH cell function. First, we found that the majority of cells that expressed the high levels of CXCR5 typical of TFH cells also expressed high levels of IL-17RA and thus were potentially capable of responding to IL-17. Second, our data suggest that in BXD2 mice, a subset of CD4+ T cells with phenotypic characteristics of TFH cells was capable of producing IL-17 rather than IL-21. CD4+ T cells with the characteristics of Th17 cells (CXCR5ICOS+) were also present. Third, the loss of IL-17RA affected the stabilization of TFH in GC LZ. These results together suggest that IL-17 plays an essential role in stabilizing TFH location and close contact with B cells in GCs. IL-17 thus complements the effects of IL-21, and both cytokines are needed for pathogenic GC development in BXD2 mice, as loss of either cytokine dramatically abrogated this response.

The parameters that should be used to define TFH cells are currently a matter of debate. Moreover, the relationship of TFH cells to other CD4+ T cell populations, including Th17 cells, is unclear, and it has been suggested that there is a considerable degree of plasticity (19, 45). A recent study shows that, in human tonsils, whereas IL-21 was the major cytokine produced by CXCR5hi TFH cells, IL-17 was produced mainly by CXCR5lo/− CD4 T cells (24). However, in autoimmune BXD2 mouse spleen, IL-17 and IL-21 could be produced by two separate subsets of CXCR5+ICOS+ TFH cells, and IL-17 could be further identified in CXCR5ICOS+ CD4 T cells. Interestingly, these IL-17–producing TFH cells are different from the conventional Th17 by their surface phenotype and location.

In agreement with previous studies (22), we found that IL-21, but not IL-17, was important for the proper development of TFH cells in this autoimmune mouse model. Although there were almost no GCs in BXD2-Il21−/− mice, the deficiency of IL-21 did not appear to perturb the structure of the follicles in the spleens in that there was a normal distribution of CD4+ T cells surrounded by IgM+ B cells. Development of Th17 cells was significantly reduced in BXD2-Il21−/− mice, which is also consistent with the previous finding that IL-21 exhibits a unique effect in promoting Th17 differentiation (42, 43). In contrast, the frequency and total numbers of TFH cells were not reduced by lack of IL-17RA expression in the BXD2-Il17ra−/− mice as well as in NP-CGG–immunized B6-Il17ra−/− mice. The proliferative response of TFH cells from BXD2-Il17ra−/− and their ability to induce B cell proliferation and IgG secretion in vitro were also intact. Surprisingly, the sera titers of IgG autoantibodies were similar in the BXD2-Il17ra−/− and BXD2-Il21−/− mice. Confocal imaging analysis confirmed our previous report (28) of a dissipation of B cells in the follicles of the BXD2-Il17ra−/− mice and further indicated that the majority of CXCR5+ TFH cells were not localized in the GC LZ. The present study suggests that IL-21 acts at an early checkpoint to cue the development of TFH, whereas IL-17 acts at a later checkpoint at the LZ to enable prolonged interaction of differentiated TFH to help GC B cell maturation.

Under optimal conditions for generating Ab-forming B cells, both GC B cells and TFH exhibit a tendency to migrate toward the same LZ compartment, thereby enhancing their opportunity for cognate interactions (46). Although recruitment to the GC LZ is enabled by CXCL13, special migration stop signaling events are required to induce conjugate formation. Prolonged interactions between CD4 T cells and APCs have been shown to enable the generation of autoreactive T cells, and such immobilization is associated with TCR signals and can affect the future activation fate of T cells (47). Similarly, stable interaction between CD4 T cells and cognate B cells mediated by SAP is another intrinsic mechanism to retain CD4 T cells in a nascent GC for sustained GC reaction (48). The present study, however, suggests that IL-17 is an extrinsic stop signal in that it acts on postdifferentiated IL-17RA+ TFH as a braking trigger to enable its interaction with responder B cells in the LZ niche. This unique property is especially important for a GC response, for which T cell help is proposed to be the limiting factor in the establishment of stable conjugates (49). This braking effect of IL-17 in BXD2 mice may preferentially occur in the LZ because IL-17+ CD4 cells are mainly located in this region. It has been reported that GC CD4+ T cells use the RGS13 and RGS16 signaling proteins to modulate their trafficking and that GC B cells use RGS1, RGS13, and RGS16 during CXCL12/13-induced formation of GCs (39, 44). In this study, we show that, in the BXD2 mouse, RGS16 is responsible for the IL-17–mediated migration arrest of TFH cells in the GC LZ, providing the necessary cell anchorage for cognate B–T cell interactions and GC development. We found that IL-17 stimulated the expression of RGS16 in CD4 T cells at both the transcriptional and protein levels and that IL-17 stimulation inhibited CXCL13-mediated chemotaxis of TFH cells isolated from RGS16+ BXD2 mice but not BXD2-Rgs16−/− mice. The lack of aggregating TFH cells in GC LZ in the spleen of BXD2-Il17ra−/− mice was recapitulated in BXD2-Rgs16−/− mice. Furthermore, TFH cells from BXD2-WT, but not BXD2-Rgs16−/−, mice that were adoptively transferred into BXD2-Il17ra−/− formed more conjugates with B cells and supported the resumption of GC formation in the recipients. These observations provide further evidence that IL-17–induced upregulation of RGS16 plays a dominant role in the proper localization of TFH cells in GC LZ. Under these experimental conditions, the B cells as well as all other host cells were IL-17RA deficient. Together with our previous findings concerning the effects of IL-17 on B cells (28, 31), the current results suggest that TFH–B conjugate formation in GCs can be regulated by modification of the chemotactic responses of either cell type at the postreceptor level.

Why is regulation of the location of TFH cells and their prolonged interactions with GC B cells important for development of highly pathogenic autoantibodies in BXD2 mice that can passively transfer autoimmunity (30)? TFH cells located within GCs drive positive selection of B cells that have acquired high-affinity receptors (3, 4). The cognate contact between TFH and GC B cells, mediated by a series of cell-surface costimulator interactions, such as CD28–CD86, ICOS–ICOS ligand, and CD40L–CD40, is required for class-switch recombination and somatic hypermutation (25). New evidence further suggests that the ability to induce somatic hypermutation plays an important role in the survival of self-reactive B cells in the GC (50). The present results indicate that perturbation of IL-17 signaling, either acutely by AdIL-17R:Fc or chronically by Il17ra−/−, disrupts TFH–B interactions and abrogates the generation of autoantibody-forming B cells in BXD2 mice. As a soluble mediator, IL-17 potentially defies TCR specificity and unselectively arrests all IL-17R+ TFH cells in the LZ niche, enhancing the opportunity of cognate interactions of TFH–B cells to drive the development of pathogenic polyreactive autoantibody-forming B cells in BXD2 mice (28, 30). Because the TFH phenotype and numbers are normal in BXD2-Il17ra−/− mice, the current study further suggests that TFH cell differentiation can be dissociated from GCs but that their anatomical location in the GC LZ is indispensable for their function in supporting autoreactive GC B cell maturation.

We thank Drs. Jay Kolls (University of Pittsburgh) and Paul Robbins (University of Pittsburgh) for providing AdIL-17 and AdLacZ. We also thank Amgen Inc. (Thousand Oaks, CA) and the Mutant Mice Regional Resource Center (Davis, CA) for providing IL-17RA and IL-21–deficient mice, respectively; Drs. Fiona Hunter and Paul Todd for review of the manuscript; Dr. Chander Raman for critical discussion; and Karen Beeching for excellent secretarial assistance.

This work was supported by Veterans Affairs Merit Review Grant 1I01BX000600-01; the National Institutes of Health/National Institute of Allergy and Infectious Diseases Grants 1AI 071110, ARRA 3RO1AI71110-02S1, 1RO1 AI083705, and U01 AI082966; American College of Rheumatology-Within-Our-Reach, the Rheumatology Research Foundation; the Alliance for Lupus Research; the Arthritis Foundation; the Lupus Research Institute; and in part by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. Flow cytometry and confocal imaging data acquisition were carried out at the University of Alabama at Birmingham Comprehensive Flow Cytometry Core (supported by National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases [NIAMS] Grant P30 AR048311 and National Institutes of Health/National Institute of Allergy and Infectious Diseases Grant P30 AI027767) and University of Alabama at Birmingham Analytic Imaging and Immunoreagents Core (NIAMS Grant P30 AR048311), respectively.

The online version of this article contains supplemental material.

Abbreviations used in this article:

B6

C57BL/6

BXD2-WT

wild-type BXD2

EDU

5-ethynyl-2'-deoxyuridine

GC

germinal center

LZ

light zone

NP-CGG

4-hydroxy-3-nitrophenylacetyl hapten conjugated to chicken γ globulin

PD-1

programmed cell death-1

PNA

peanut agglutinin

RGS

regulators of G-protein signaling

rm

recombinant murine

RORγt

retinoic acid–related orphan receptor γt

SA

streptavidin

TD

T-dependent

TFH

follicular Th.

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The authors have no financial conflicts of interest.