ICOS is a new member of the CD28 family of costimulatory molecules that is expressed on activated T cells. Its ligand B7RP-1 is constitutively expressed on B cells. Although the blockade of ICOS/B7RP-1 interaction inhibits T cell-dependent Ab production and germinal center formation, the mechanism remains unclear. We examined the contribution of ICOS/B7RP-1 to the generation of CXCR5+ follicular B helper T (TFH) cells in vivo, which preferentially migrate to the B cell zone where they provide cognate help to B cells. In the spleen, anti-B7RP-1 mAb-treated or ICOS-deficient mice showed substantially impaired development of CXCR5+ TFH cells and peanut agglutinin+ germinal center B cells in response to primary or secondary immunization with SRBC. Expression of CXCR5 on CD4+ T cells was associated with ICOS expression. Adoptive transfer experiments showed that the development of CXCR5+ TFH cells was enhanced by interaction with B cells, which was abrogated by anti-B7RP-1 mAb treatment. The development of CXCR5+ TFH cells in the lymph nodes was also inhibited by the anti-B7RP-1 mAb treatment. These results indicated that the ICOS/B7RP-1 interaction plays an essential role in the development of CXCR5+ TFH cells in vivo.

T-dependent humoral immune responses are characterized by the development of germinal center (GC)3 in B cell follicles of the secondary lymphoid organs. Isotype switching and affinity maturation of the Abs produced by B cells, and the development of memory B cells or plasma cells occur within the GC. These B cell maturational processes require cognate help provided by CD4+ T cells (1). It is well known that both CD28/B7 (CD80 and CD86) and CD40/CD40L (CD154) interactions are required for optimal activation of CD4+ T cells and B cells to support GC formation (2, 3). Recently, ICOS, a homologue of CD28, was cloned (4, 5, 6) and shown to be also involved in the GC development (7, 8). Expression of ICOS is restricted to activated T cells (4, 9). Its ligand B7RP-1 (also known as B7h, B7-H2, GL50, and LICOS) (9, 10, 11, 12, 13) was identified as the third member of the B7 family, which is constitutively expressed on B cells, macrophages, and dendritic cells. In certain circumstances, ICOS signaling regulates either Th1 or Th2 cell differentiation (7, 8). Moreover, ICOS/B7RP-1 interaction may be involved in T/B cell interaction because ICOS is expressed on GC T cells and B7RP-1 is expressed on resting B cells (4). Transgenic mice expressing a soluble B7RP-1-Ig fusion protein, which could engage ICOS signaling, were characterized by lymphoid hyperplasia in the spleen, lymph nodes (LN), and Peyer’s patches, and high levels of serum IgG (9). Moreover, it has been shown that ICOS- or B7RP-1-deficient mice developed fewer and smaller GC in response to immunization (14, 15, 16, 17, 18, 19). These mice had consistently lower levels of serum IgG and showed a defect in IgG1 Ab production in response to T-dependent Ags, whereas the responses against T-independent Ags were normal. These results have indicated that the ICOS/B7RP-1 interaction regulates the GC development and Ab production, but the exact mechanism remains unclear.

We speculated that the ICOS/B7RP-1 interaction might be required to up-regulate the expression of a chemokine receptor CXCR5 on CD4+ T cells. CXCR5 confers responsiveness to B lymphocyte chemokine (CXCL13), which is produced by follicular stroma cells in the spleen, LN, and Peyer’s patches (20, 21). CXCR5 is constitutively expressed by circulating B cells and is required for their migration into B cell follicles in the secondary lymphoid organs (22, 23). A subset of CD4+ T cells also express CXCR5, which mediates their migration to the B cell follicles where they provide cognate help to B cells (24, 25, 26, 27). Thus, CXCR5+CD4+ T cells are referred to as follicular B helper T (TFH) cells (25, 28). Previous studies have implicated OX40/OX40 ligand (OX40L) interaction, a pair of the TNFR/TNF family members (29), in the expression of CXCR5 on CD4+ T cells (30, 31). It has been also reported that OX40L-transgenic mice, expressing a large amount of OX40L on dendritic cells, developed an increased number of CD4+ T cells in the B cell follicles of secondary lymphoid organs in response to immunization (32). Therefore, in this study, we compared the contributions of ICOS/B7RP-1 and OX40/OX40L to the development of CXCR5+ TFH cells and GC B cells. Our present results indicated that the ICOS/B7RP-1 interaction plays an essential role of CXCR5+ TFH cells in the spleen and LN, but the GC formation in LN is not always dependent on CXCR5+ TFH cells. In contrast, a substantial contribution of OX40/OX40L interaction to the development of CXCR5+ TFH cells and GC B cells was observed only in LN of certain strains of mice, depending on differential expression of OX40 on CXCR5+ TFH cells.

Female BALB/c, C57BL/6, C57BL/10, B10.D2, CBA/N, C3H/He, DBA/1, A/J, and C.B-17/scid (SCID) mice were purchased from Charles River Japan and Japan SLC. OX40L-deficient mice on C57BL/6 or BALB/c background were obtained from Drs. N. Ishii and K. Sugamura (Tohoku University School of Medicine, Sendai, Japan) (33, 34). CD40-deficient mice on BALB/c background were gift from Dr. H. Kikutani (Osaka University, Osaka, Japan) (35). CD28-deficient mice on C57BL/6 background were purchased from The Jackson Laboratory. ICOS-deficient mice on C57BL/6 background have been described previously (36). These mice were bred and maintained in the Oriental Yeast Company. All mice were 6–8 wk old at the start of experiments and kept under specific pathogen-free conditions during the experiments.

Anti-mouse B7RP-1 (HK5.3) and anti-mouse OX40L (RM134L) mAbs were generated in our laboratory as previously described (37, 38). Control rat IgG was purchased from Sigma-Aldrich. FITC-conjugated anti-CD4 (RM4-5), biotin- or allophycocyanin-conjugated anti-CD45R/B220 (RA3-6B2), and PE-conjugated anti-ICOS (15F9) mAbs were purchased from eBioscience. Biotin-conjugated anti-OX40 (OX86) mAb, biotin- or PE-conjugated anti-CXCR5 (2G8) mAb, rat IgG isotype controls, hamster IgG control, and PE- or allophycocyanin-labeled streptavidin were purchased from BD Pharmingen. Biotin- or FITC-conjugated peanut agglutinin (PNA) was purchased from Vector Laboratories. SRBC were purchased from Nippon Bio-supply Center.

Groups of five mice were i.p. immunized with 2 × 108 SRBC in 0.2 ml of PBS to induce GC response in the spleen or immunized with 5 × 107 SRBC/50 μl in the footpads to induce GC response in the popliteal LN. In some groups, mice were i.p. administrated with 300 μg of anti-B7RP-1 mAb, anti-OX40L mAb, or control rat IgG at the time of immunization (day 0) and on days 2 and 4. Spleen cells or popliteal LN cells were collected at day 6 or 7, and the induction of PNA+B220+ GC B cells and CXCR5+ TFH cells was analyzed by flow cytometry. To measure the secondary response, mice were i.p. injected with 2 × 108 SRBC/0.2 ml on day 30 after the first immunization. Some mice were treated with 300 μg of anti-B7RP-1 mAb, anti-OX40L mAb, or control rat IgG on days 30 and 32. Three days after the second immunization, spleen cells were analyzed by flow cytometry.

Spleen or LN cells (0.5–1 × 106) were first preincubated with unlabeled anti-CD16/32 mAb (BD Pharmingen) to avoid nonspecific binding of mAbs to FcγR. The GC B cells were determined by staining with FITC-labeled PNA and allophycocyanin-labeled anti-B220 mAb. The TFH cells were determined by staining with FITC-labeled anti-CD4 and biotin-labeled anti-CXCR5 mAbs, followed by PE-labeled streptavidin. The expression of ICOS, OX40, and CXCR5 on CD4+ T cells was determined by staining with FITC-labeled anti-CD4 mAb and PE- or biotin-labeled mAbs for respective molecules, followed by allophycocyanin-labeled streptavidin. After washing with PBS, the stained cells (live-gated on the basis of forward and side scatter profiles and propidium iodide exclusion) were analyzed on a FACSCalibur (BD Biosciences), and data were processed using the CellQuest program (BD Biosciences).

The spleens from anti-B7RP-1- or rat IgG-treated BALB/c mice and ICOS-deficient or wild-type C57BL/6 mice on day 7 after SRBC immunization were embedded in Tissue-Tek OCT compound (Sakura Finetechnical), and were frozen in liquid nitrogen bath. PNA staining was conducted as previously described (39). Briefly, 3-μm cryostat sections were air dried and fixed with 8% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min at 4°C. Nonspecific binding sites were blocked by incubation for 30 min at room temperature in PBS containing 2% BSA fraction V (Sigma-Aldrich). After removing the solution, the sections were incubated with 5 μg/ml biotin-conjugated PNA for 1 h at 37°C. After washing with PBS, the slides were treated with 0.3% hydrogen peroxide in methanol for 20 min at room temperature to block endogenous peroxidase activity. The sections were incubated with avidin-biotin-peroxidase complex followed by further amplification with boyinyl tyramide (Catalyzed Signal Amplification system; DAKO) according to the manufacturer’s instructions. Subsequently, the peroxidase color reaction was performed by a 10-min application of freshly prepared 0.05% 3,3′-diaminobenzidine-0.01% hydrogen peroxide solution (WAKO). The sections were counterstained with hematoxylin. For immunohistochemistry, 3-μm cryostat sections were fixed with acetone for 10 min at 4°C. The sections were incubated with 2% BSA in PBS for 30 min at room temperature before incubation with Abs to reduce nonspecific binding of Abs. After removing the solution, the sections were incubated with 20 μg/ml biotin-conjugated anti-CD45R/B220 mAb for 1 h at 37°C. After washing with PBS, the sections were labeled with Alexa-Fluor 594-conjugated streptavidin (Molecular Probes) at 1:100 for 1 h at 37°C, and finally incubated with 20 μg/ml FITC-conjugated anti-CD4 mAb for 1 h at 37°C.

BALB/c mice were i.p. immunized with SRBC on days 0 and 15 and administrated with 300 μg of anti-B7RP-1 mAb, anti-OX40L mAb, or control rat IgG on days −1, 0, and 2. Serum anti-SRBC Abs were measured on day 22 by isotype-specific ELISA. Soluble SRBC Ags were prepared as described (40) and protein concentration was determined using the Bio-Rad Protein Assay reagent (Bio-Rad). Soluble SRBC Ags (5 μg/ml in carbonate buffer, pH 9.6) were coated onto 96-well Immulon 2HB plates (Thermo Labsystems). After blocking with 1% BSA in PBS, SRBC-specific IgM and IgG isotypes were determined by incubating serially diluted serum samples for 2 h at 37°C. After washing with 0.05% Tween 20 in PBS, wells were incubated with biotin-conjugated isotype-specific mAbs, including anti-mouse IgG1 (Serotec) or anti-mouse IgG2a, IgG2b, or IgG3 (BD Pharmingen), washed, and then developed with Vectastain ABC kit (Vector Laboratories) and o-phenylendiamine (WAKO). After terminating the reaction with 2N H2SO4, OD at 490/595 nm was measured on a microplate reader (Bio-Rad).

Spleen cells were collected on day 7 after immunization of BALB/c mice with SRBC. ICOS+CXCR5, ICOS+CXCR5+, and ICOSCXCR5 CD4+ T cells were isolated by FACS sorting and 2 × 105 cells per well were cultured in RPMI 1640 medium containing 10% FCS, 10 mM HEPES, 2 mM l-glutamine, 0.1 mg/ml penicillin and streptomycin, and 50 μM 2-ME on 5 μg/ml immobilized anti-CD3 mAb (2C11). To determine the production of cytokines, cell-free supernatants were collected at 48 h and assayed for IL-2, IL-4, IL-5, and IL-10 by ELISA using OptEIA kits (BD Pharmingen) and IFN-γ using Mouse IFN-γ ELISA Ready-SET-Go! kit (eBioscience) according to the manufacturer’s instructions.

CD62L+CD4+ naive T cells were purified from the spleen of BALB/c mice by passage through nylon wool columns (WAKO) and by using autoMACS columns with CD4+ T cell isolation kit and anti-CD62L-coupled microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. Small resting B cells were also purified from the spleen of BALB/c mice as previously described (38). Briefly, spleen cells were treated with a mixture of hybridoma supernatants (anti-Thy-1.2, anti-CD4, and anti-CD8) and low-tox rabbit complement (Cedarlane Laboratories). After Percoll (Amersham Biosciences) gradient centrifugation, small B cells were collected from the 60/70% interface. The purified CD4+ T cells (>95% CD4+CD62L+; 2 × 106 cells) with or without the purified B cells (>95% B220+; 1 × 106 cells) were i.v. injected into SCID mice (day −1). After 24 h, mice were i.p. immunized with 2 × 108 SRBC and then i.p. administrated with 300 μg of anti-B7RP-1 mAb or control rat IgG on days 0, 2, and 4. Seven days after the immunization, spleen cells were analyzed by flow cytometry.

The results are expressed as the mean ± SD of five mice in each group. Significant differences between two experimental groups were analyzed by the unpaired Student’s t test. Values of p < 0.01 were considered significant.

Because a previous report demonstrated an impaired GC formation in ICOS-deficient mice in response to immunization of SRBC (17), we followed the same protocol and used SRBC as an Ag in this study, which can induce robust polyclonal GC responses in an adjuvant-independent manner. The splenic GC formation was determined by flow cytometric analysis of PNA+B220+ cells, which have been defined as GC B cells (41). In our preliminary experiments, the PNA+B220+ GC B cells appeared in the spleen at a peak frequency between days 6 and 8 and disappeared on day 30 after i.p. immunization of 2 × 108 SRBC (data not shown).

To explore the contribution of ICOS/B7RP-1 and OX40/OX40L interactions to the development of GC B cells and CXCR5+CD4+ TFH cells in vivo, we administered a neutralizing anti-B7RP-1 mAb, a neutralizing anti-OX40L mAb, or control rat IgG on days 0, 2, and 4 after immunization of BALB/c mice with SRBC. Spleen cells were collected on day 7, and the development of PNA+B220+ GC B cells and CXCR5+ TFH cells was determined by two-color flow cytometry. As represented in Fig. 1,A and compiled in Fig. 1,B, the control IgG-treated mice developed substantial percentages of PNA+ GC B cells within total B220+ B cells (10.4 ± 1.3%) upon SRBC immunization. The anti-OX40L mAb treatment exhibited no significant effect on the development of PNA+B220+ GC B cell population (8.7 ± 2.3%). In contrast, the anti-B7RP-1 mAb treatment significantly reduced the PNA+B220+ GC B cell population (2.9 ± 0.3%). This inhibitory effect of anti-B7RP-1 mAb on GC formation was also confirmed by histological examination, in which the spleen sections from the anti-B7RP-1 mAb-treated mice had smaller GC as compared with those from the control IgG-treated mice (Fig. 2 A).

FIGURE 1.

Effect of anti-B7RP-1 and anti-OX40L mAbs on primary GC B cell and TFH cell induction in the spleen. BALB/c mice were i.p. immunized with SRBC and administrated with anti-B7RP-1 mAb, anti-OX40L mAb, or control rat IgG on days 0, 2, and 4. Spleen cells were collected on day 7. A, The GC B cell induction was determined by staining with FITC-labeled PNA and allophycocyanin-labeled anti-B220 mAb. The dot plots are representative of five mice in each group. The numbers in the upper right quadrant are the mean percentage ± SD of PNA+B220+ B cell population within total spleen cells. B, The data are compiled and expressed as the mean percentages ± SD of PNA+ cells within B220+ B cells from five in each group. C, The TFH cell induction was determined by staining with FITC-labeled anti-CD4 mAb and biotinylated anti-CXCR5 mAb, followed by PE-labeled streptavidin. The dot plots are representative of five mice in each group. The numbers in the upper right quadrant are the mean percentage ± SD of CXCR5+CD4+ T cell population within total spleen cells. D, The data are compiled and expressed as the mean percentages ± SD of CXCR5+ cells within CD4+ T cells from five mice in each group. Similar results were obtained in three independent experiments. ∗∗, p < 0.001. E, Kinetics of TFH cell induction. On the indicated days postimmunization, spleen cells were collected and stained with anti-CXCR5 and anti-CD4 mAbs. The percentages of CXCR5+ cells within CD4+ T cells are expressed as mean ± SD of five mice in each group.

FIGURE 1.

Effect of anti-B7RP-1 and anti-OX40L mAbs on primary GC B cell and TFH cell induction in the spleen. BALB/c mice were i.p. immunized with SRBC and administrated with anti-B7RP-1 mAb, anti-OX40L mAb, or control rat IgG on days 0, 2, and 4. Spleen cells were collected on day 7. A, The GC B cell induction was determined by staining with FITC-labeled PNA and allophycocyanin-labeled anti-B220 mAb. The dot plots are representative of five mice in each group. The numbers in the upper right quadrant are the mean percentage ± SD of PNA+B220+ B cell population within total spleen cells. B, The data are compiled and expressed as the mean percentages ± SD of PNA+ cells within B220+ B cells from five in each group. C, The TFH cell induction was determined by staining with FITC-labeled anti-CD4 mAb and biotinylated anti-CXCR5 mAb, followed by PE-labeled streptavidin. The dot plots are representative of five mice in each group. The numbers in the upper right quadrant are the mean percentage ± SD of CXCR5+CD4+ T cell population within total spleen cells. D, The data are compiled and expressed as the mean percentages ± SD of CXCR5+ cells within CD4+ T cells from five mice in each group. Similar results were obtained in three independent experiments. ∗∗, p < 0.001. E, Kinetics of TFH cell induction. On the indicated days postimmunization, spleen cells were collected and stained with anti-CXCR5 and anti-CD4 mAbs. The percentages of CXCR5+ cells within CD4+ T cells are expressed as mean ± SD of five mice in each group.

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FIGURE 2.

Defective primary GC formation and reduced infiltration of CD4+ T cells in the splenic B cell follicles in anti-B7RP-1-treated mice and ICOS-deficient mice. A, Spleen from anti-B7RP-1- or rat IgG-treated BALB/c mice and ICOS-deficient or wild-type C57BL/6 mice on day 7 after SRBC immunization were sectioned and GC formation was analyzed by PNA staining (brown). B, Immunohistochemical analysis of a follicle (arrowhead) displayed in A. Sections were stained with Abs to CD4 (green) and CD45R/B220 (red). Original magnification: A, ×10; B, ×20. The results are representative of five animals in each group analyzed.

FIGURE 2.

Defective primary GC formation and reduced infiltration of CD4+ T cells in the splenic B cell follicles in anti-B7RP-1-treated mice and ICOS-deficient mice. A, Spleen from anti-B7RP-1- or rat IgG-treated BALB/c mice and ICOS-deficient or wild-type C57BL/6 mice on day 7 after SRBC immunization were sectioned and GC formation was analyzed by PNA staining (brown). B, Immunohistochemical analysis of a follicle (arrowhead) displayed in A. Sections were stained with Abs to CD4 (green) and CD45R/B220 (red). Original magnification: A, ×10; B, ×20. The results are representative of five animals in each group analyzed.

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Serum levels of SRBC-specific IgG1, IgG2a, and IgG2b Abs were significantly reduced in the anti-B7RP-1 mAb-treated mice as compared with the control IgG-treated mice, while the levels of IgM Abs were not significantly reduced (Fig. 3). This indicated that the ICOS/B7RP-1 interaction played an important role in inducing the production of Ag-specific IgG Abs, as described by previous reports (14, 15, 16, 18, 19).

FIGURE 3.

Anti-B7RP-1 mAb treatment inhibits SRBC-specific IgG Ab production. BALB/c mice were i.p. immunized with SRBC on days 0 and 15 and administrated with anti-B7RP-1 mAb, anti-OX40L mAb, or control rat IgG on days −1, 0, and 2. Serum levels of anti-SRBC Abs were determined using isotype-specific ELISA on day 22 and expressed as absorbance values at 490/595 nm. Data are represented as the mean ± SD of five mice in each group. Similar results were obtained in three independent experiments. ∗, p < 0.01; ∗∗, p < 0.001.

FIGURE 3.

Anti-B7RP-1 mAb treatment inhibits SRBC-specific IgG Ab production. BALB/c mice were i.p. immunized with SRBC on days 0 and 15 and administrated with anti-B7RP-1 mAb, anti-OX40L mAb, or control rat IgG on days −1, 0, and 2. Serum levels of anti-SRBC Abs were determined using isotype-specific ELISA on day 22 and expressed as absorbance values at 490/595 nm. Data are represented as the mean ± SD of five mice in each group. Similar results were obtained in three independent experiments. ∗, p < 0.01; ∗∗, p < 0.001.

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Concurrently, as represented in Fig. 1,C and compiled in Fig. 1,D, substantial numbers of CXCR5+ TFH cells were induced in the control IgG-treated mice (5.6 ± 0.9% within CD4+ cells) upon SRBC immunization. Again, the anti-OX40L mAb treatment did not significantly affect the development of CXCR5+ TFH cells (6.0 ± 0.3%). In contrast, the anti-B7RP-1 mAb treatment significantly inhibited the development of CXCR5+ TFH cell population (1.7 ± 0.1%). This inhibitory effect of anti-B7RP-1 mAb was also observed when the development of CXCR5+ TFH cells was monitored 2–10 days after SRBC immunization (Fig. 1,E). Immunohistological analysis showed that, whereas many CD4+ T cells were found in the splenic B cell follicles of the control IgG-treated mice, only a small number of CD4+ T cells infiltrated in the splenic B cell follicles of the anti-B7RP-1-treated mice (Fig. 2 B). Similar results were obtained when C57BL/6 mice were i.p. immunized with SRBC and treated with anti-B7RP-1 or anti-OX40L mAb (data not shown). These results indicated that the ICOS/B7RP-1 interaction was essential for the development of CXCR5+ TFH cells and PNA+ GC B cells in the spleen in response to immunization of SRBC. Unexpectedly, no significant contribution of the OX40/OX40L interaction to either GC formation or CXCR5+ TFH cell development was observed in this experimental system.

To further confirm the critical contribution of ICOS/B7RP-1, but not OX40/OX40L, interaction, we immunized ICOS- or OX40L-deficient mice with SRBC and examined the development of CXCR5+ TFH cells and PNA+B220+ GC B cells in the spleen 7 days after. We also included CD28- or CD40-deficient mice, which have been reported to have a defect in GC formation (42, 43), for comparison. As expected, CD40- or CD28-decifient mice did not developed PNA+B220+ GC B cells in response to SRBC immunization (Fig. 4, A and C). Correspondingly, CXCR5+ TFH cells were not developed in either CD40- or CD28-deficient mice (Fig. 4, B and D). OX40L-deficient mice on either BALB/c or C57BL/6 background did not exhibit a significant defect in the development of either PNA+B220+ GC B cells or CXCR5+ TFH cells (Fig. 4, AD). In contrast, the development of both GC B cells (Fig. 4,C) and CXCR5+ TFH cells (Fig. 4,D) were significantly impaired in ICOS-deficient mice. Immunohistological analysis showed that the development of GC and the infiltration of CD4+ T cells in the splenic B cell follicles were notably reduced in ICOS-deficient mice in response to SRBC immunization (Fig. 2). These results indicated that ICOS as well as CD40 and CD28, but not OX40, were essential for the generation of CXCR5+ TFH cells and GC B cells in the spleen in response to SRBC immunization.

FIGURE 4.

CD40, CD28, and ICOS, but not OX40, are essential for induction of primary GC B cells and ThF cells. OX40L- or CD40-deficient BALB/c mice and wild-type BALB/c mice (A and B) and OX40L-, ICOS-, or CD28-deficient C57BL/6 mice and wild-type C57BL/6 mice (C and D) were i.p. immunized with SRBC. Spleen cells were collected on day 7. The induction of GC B cells was determined by staining with PNA and anti-B220 mAb, and expressed as the mean percentages ± SD of PNA+ cells within B220+ cells from five mice in each group (A and C). The induction of TFH cells was determined by staining with anti-CXCR5 and anti-CD4 mAbs, and expressed as the mean percentages ± SD of CXCR5+ cells within CD4+ cells from five mice in each group (B and D). Similar results were obtained in three independent experiments. ∗, p < 0.01; ∗∗, p < 0.001.

FIGURE 4.

CD40, CD28, and ICOS, but not OX40, are essential for induction of primary GC B cells and ThF cells. OX40L- or CD40-deficient BALB/c mice and wild-type BALB/c mice (A and B) and OX40L-, ICOS-, or CD28-deficient C57BL/6 mice and wild-type C57BL/6 mice (C and D) were i.p. immunized with SRBC. Spleen cells were collected on day 7. The induction of GC B cells was determined by staining with PNA and anti-B220 mAb, and expressed as the mean percentages ± SD of PNA+ cells within B220+ cells from five mice in each group (A and C). The induction of TFH cells was determined by staining with anti-CXCR5 and anti-CD4 mAbs, and expressed as the mean percentages ± SD of CXCR5+ cells within CD4+ cells from five mice in each group (B and D). Similar results were obtained in three independent experiments. ∗, p < 0.01; ∗∗, p < 0.001.

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We further investigated the roles of ICOS/B7RP-1 and OX40/OX40L interactions in the secondary response to SRBC immunization. BALB/c mice were i.p. immunized again with SRBC on day 30 after the first immunization, when almost no primary GC B cells remained in the spleen (data not shown), and treated with anti-B7RP-1 mAb, anti-OX40L mAb, or control IgG on days 30 and 32. Three days after the secondary immunization, the development of PNA+B220+ GC B cells and CXCR5+ TFH cells in the spleen was analyzed by flow cytometry. As shown in Fig. 5, the control IgG-treated mice quickly developed both PNA+ GC B cells and CXCR5+ TFH cells. The anti-B7RP-1 mAb treatment significantly inhibited the induction of both PNA+ GC B cells and CXCR5+ TFH cells, while anti-OX40L mAb treatment showed no significant effect. These results indicated that the ICOS/B7RP-1, but not OX40/OX40L, interaction was also essential for the development of GC B cells and TFH cells in the spleen in response to secondary immunization with SRBC.

FIGURE 5.

Involvement of ICOS, but not OX40, in induction of GC B cells and TFH cells after secondary immunization. BALB/c mice were i.p. immunized with SRBC on days 0 and 30 and administrated with anti-B7RP-1 mAb, anti-OX40L mAb, or control rat IgG on days 30 and 32. Spleen cells were collected on day 33. A, The induction of GC B cells was determined by staining with PNA and anti-B220 mAb, and expressed as the mean percentages ± SD of PNA+ cells within B220+ cells from five mice in each group. B, The induction of TFH cells was determined by staining with anti-CXCR5 and anti-CD4 mAbs, and expressed as the mean percentages ± SD of CXCR5+ cells within CD4+ cells from five mice each group. Similar results were obtained in three independent experiments. ∗, p < 0.01; ∗∗, p < 0.001.

FIGURE 5.

Involvement of ICOS, but not OX40, in induction of GC B cells and TFH cells after secondary immunization. BALB/c mice were i.p. immunized with SRBC on days 0 and 30 and administrated with anti-B7RP-1 mAb, anti-OX40L mAb, or control rat IgG on days 30 and 32. Spleen cells were collected on day 33. A, The induction of GC B cells was determined by staining with PNA and anti-B220 mAb, and expressed as the mean percentages ± SD of PNA+ cells within B220+ cells from five mice in each group. B, The induction of TFH cells was determined by staining with anti-CXCR5 and anti-CD4 mAbs, and expressed as the mean percentages ± SD of CXCR5+ cells within CD4+ cells from five mice each group. Similar results were obtained in three independent experiments. ∗, p < 0.01; ∗∗, p < 0.001.

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To explore the mechanism by which the development of CXCR5+ TFH cells depended on the ICOS/B7RP-1 interaction rather than the OX40/OX40L interaction, we examined correlation in the expression of CXCR5, ICOS, and OX40 on splenic CD4+ T cells 7 days after primary SRBC immunization. As shown in Fig. 6,A, the expression of CXCR5 was preferentially detected on a substantial part of ICOS+ T cells, but not on OX40+ T cells, in the spleen of SRBC-immunized BALB/c mice. A similar pattern was observed in the spleen of SRBC-immunized C57BL/6 mice (Fig. 6 B). These results suggested that the CXCR5+ TFH cells preferentially developed from the ICOS+OX40 subset of activated CD4+ T cells in the spleen.

FIGURE 6.

Associated expression of CXCR5 with ICOS, but not OX40, on splenic CD4+ cells. Spleen cells were isolated from BALB/c mice (A) or C57BL/6 mice (B) on day 7 after primary SRBC immunization, and stained with anti-CXCR5, anti-ICOS, anti-OX40, and anti-CD4 mAbs. The dot plots were derived from CD4+-gated cells and are representative of three mice with similar results.

FIGURE 6.

Associated expression of CXCR5 with ICOS, but not OX40, on splenic CD4+ cells. Spleen cells were isolated from BALB/c mice (A) or C57BL/6 mice (B) on day 7 after primary SRBC immunization, and stained with anti-CXCR5, anti-ICOS, anti-OX40, and anti-CD4 mAbs. The dot plots were derived from CD4+-gated cells and are representative of three mice with similar results.

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We also examined cytokine production by isolated ICOS+CXCR5+, ICOS+CXCR5, and ICOSCXCR5 CD4+ T cells after stimulation with immobilized anti-CD3 mAb. As shown in Fig. 7, the ICOS+CXCR5+ cells produced a high level of IFN-γ and low levels of IL-2 and IL-10 but did not produce detectable levels of IL-4 or IL-5. This agrees with the results obtained with human blood and tonsil CXCR5+ T cells (25, 44). In contrast, ICOS+CXCR5 cells produced high levels of IL-2, IL-4, IL-5, IL-10, and IFN-γ (Fig. 7).

FIGURE 7.

Cytokine production by CD4+ T cells expressing ICOS and CXCR5. ICOS+CXCR5, ICOS+CXCR5+, and ICOSCXCR5 CD4+ T cells were isolated from the spleen on day 7 after immunization of BALB/c mice with SRBC and stimulated with immobilized anti-CD3 mAb for 48 h. Concentrations of the indicated cytokines in the culture supernatants were determined by ELISA. Data are expressed as the mean ± SD of triplicate samples. Similar results were obtained in two independent experiments.

FIGURE 7.

Cytokine production by CD4+ T cells expressing ICOS and CXCR5. ICOS+CXCR5, ICOS+CXCR5+, and ICOSCXCR5 CD4+ T cells were isolated from the spleen on day 7 after immunization of BALB/c mice with SRBC and stimulated with immobilized anti-CD3 mAb for 48 h. Concentrations of the indicated cytokines in the culture supernatants were determined by ELISA. Data are expressed as the mean ± SD of triplicate samples. Similar results were obtained in two independent experiments.

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Splenic B cells constitutively express B7RP-1 (9). Thus, it is likely that the CXCR5+ TFH cell development is regulated by ICOS/B7RP-1 through T/B cell cognate interaction. To address this possibility, purified naive CD4+ T cells with or without purified naive B cells from BALB/c mice were adoptively transferred into SCID mice (day −1), which were then i.p. immunized with SRBC on day 0 and treated with anti-B7RP-1 mAb or control IgG on days 0, 2, and 4. Seven days after the immunization, spleen cells were analyzed for the development of CXCR5+ TFH cells by flow cytometry. As shown in Fig. 8, CXCR5+ TFH cells were only marginally induced by SRBC immunization when SCID mice were reconstituted with CD4+ T cells alone. The cotransfer of B cells markedly enhanced the development of CXCR5+ TFH cells, which was abrogated by the anti-B7RP-1 mAb treatment. These results suggested that the ICOS/B7RP-1 interaction was involved in the development of TFH cells through cognate T/B cell interaction.

FIGURE 8.

ICOS/B7RP-1 interaction is required for splenic ThF cell induction through T/B cell interaction. Purified CD4+ T cells with or without purified B cells from naive BALB/c mice were i.v. injected into SCID mice on day −1. These mice were i.p. immunized with SRBC on day 0 and administrated with anti-B7RP-1 mAb or control rat IgG on days 0, 2, and 4. Spleen cells were collected on day 7. The induction of TFH cells was determined by staining with anti-CXCR5 and anti-CD4 mAbs, and expressed as the mean percentages ± SD of CXCR5+ cells within CD4+ cells from five mice in each group. Similar results were obtained in two independent experiments. ∗, p < 0.01; ∗∗, p < 0.001.

FIGURE 8.

ICOS/B7RP-1 interaction is required for splenic ThF cell induction through T/B cell interaction. Purified CD4+ T cells with or without purified B cells from naive BALB/c mice were i.v. injected into SCID mice on day −1. These mice were i.p. immunized with SRBC on day 0 and administrated with anti-B7RP-1 mAb or control rat IgG on days 0, 2, and 4. Spleen cells were collected on day 7. The induction of TFH cells was determined by staining with anti-CXCR5 and anti-CD4 mAbs, and expressed as the mean percentages ± SD of CXCR5+ cells within CD4+ cells from five mice in each group. Similar results were obtained in two independent experiments. ∗, p < 0.01; ∗∗, p < 0.001.

Close modal

We next examined the contribution of ICOS/B7RP-1 and OX40/OX40L interactions to the development of PNA+B220+ GC B cells and CXCR5+ TFH cells in LN. BALB/c and C57BL/6 mice were immunized with SRBC in the footpads and treated with control IgG or anti-B7RP-1 and/or anti-OX40L mAbs on days 0, 2, and 4. Six days after the immunization, the induction of PNA+B220+ cells and CXCR5+CD4+ cells in the popliteal LN was analyzed by flow cytometry. In BALB/c mice, the treatment with anti-B7RP-1 mAb, but not anti-OX40L mAb, significantly inhibited the development of CXCR5+ TFH cells in the LN (Fig. 9,B) as in the spleen (Fig. 1,D). However, the development of PNA+ GC B cells in LN was not significantly inhibited by anti-B7RP-1 and/or anti-OX40L mAbs (Fig. 9,A) unlike in the spleen (Fig. 1,B). In contrast, in the LN of C57BL/6 mice, the development of both PNA+ GC B cells and CXCR5+ TFH cells was substantially inhibited by either anti-B7RP-1 or anti-OX40L mAb alone (Fig. 9, C and D). A further inhibition was observed by mixture of both mAbs. These results suggested that while the ICOS/B7RP-1 interaction played the major role in the development of TFH cells in LN, the OX40/OX40L interaction also partially contributed to the development of both GC B cells and TFH cells in LN of some strains of mice. It was also noted that the development of GC B cells was not always associated with the development of TFH cells, as represented by the LN of anti-B7RP-1 mAb-treated BALB/c mice.

FIGURE 9.

Effect of anti-B7RP-1 and anti-OX40L mAbs on induction of GC B cells and TFH cells in LN. BALB/c (A and B) or C57BL/6 (C and D) mice were immunized with SRBC in the footpads and administrated with anti-B7RP-1 mAb, anti-OX40L mAb, or control rat IgG on days 0, 2, and 4. Popliteal LN cells were collected on day 6. The induction of GC B cells was determined by staining with PNA and anti-B220 mAb, and expressed as the mean percentages ± SD of PNA+ cells within B220+ cells from five mice in each group (A and C). The induction of TFH cells was determined by staining with anti-CXCR5 and anti-CD4 mAbs, and expressed as the mean percentages ± SD of CXCR5+ cells within CD4+ cells from five mice in each group (B and D). Similar results were obtained in three independent experiments. ∗, p < 0.01; ∗∗, p < 0.001.

FIGURE 9.

Effect of anti-B7RP-1 and anti-OX40L mAbs on induction of GC B cells and TFH cells in LN. BALB/c (A and B) or C57BL/6 (C and D) mice were immunized with SRBC in the footpads and administrated with anti-B7RP-1 mAb, anti-OX40L mAb, or control rat IgG on days 0, 2, and 4. Popliteal LN cells were collected on day 6. The induction of GC B cells was determined by staining with PNA and anti-B220 mAb, and expressed as the mean percentages ± SD of PNA+ cells within B220+ cells from five mice in each group (A and C). The induction of TFH cells was determined by staining with anti-CXCR5 and anti-CD4 mAbs, and expressed as the mean percentages ± SD of CXCR5+ cells within CD4+ cells from five mice in each group (B and D). Similar results were obtained in three independent experiments. ∗, p < 0.01; ∗∗, p < 0.001.

Close modal

To explore the mechanism for the differential contribution of OX40/OX40L interaction to the TFH cell development in LN between BALB/c and C57BL/6 mice, we examined the expression of CXCR5, ICOS, and OX40 on CD4+ T cells in the LN of BALB/c and C57BL/6 mice at 6 days after the immunization. As shown in Fig. 10,A, CXCR5 was preferentially expressed on CD4+ T cells expressing ICOS but not OX40 in the LN of BALB/c mice, as observed in the spleen (Fig. 6,A). In contrast, as shown in Fig. 10,B, CXCR5-expressing CD4+ T cells in the LN of C57BL/6 mice expressed both ICOS and OX40, while those in the spleen did not express OX40 (Fig. 6,B). We then examined the expression of OX40 on the CXCR5+ TFH cells in the LN and spleen of various strains of mice after SRBC immunization. As shown in Fig. 10,C, CXCR5+ TFH cells in the LN of C57BL/6 and C3H/He mice distinctively expressed OX40. In contrast, no significant expression of OX40 was observed on splenic CXCR5+CD4+ T cells in all strains tested (Fig. 10 D). These results indicated that the expression of OX40 on CXCR5+ TFH cells in LN was variable among mouse strains. This might be responsible for the differential contribution of OX40/OX40L interaction to the development of TFH cells and GC B cells in LN of certain strains of mice.

FIGURE 10.

Expression of OX40 on TFH cells in LN is variable among mouse strains. The indicated eight strains of mice were immunized with 5 × 107 SRBC in the footpads (AC) or i.p. immunized with 2 × 108 SRBC (D). Popliteal LN cells (AC) or spleen cells (D) were isolated 6 days after immunization and stained with anti-CXCR5, anti-ICOS, anti-OX40, and anti-CD4 mAbs. The dot plots represent CD4+-gated LN cells from BALB/c (A) or C57BL/6 (B) mice. Mean fluorescence intensity (MFI) of OX40 expression on CXCR5+CD4+ T cells in LN (C) or spleen (D) is expressed as the mean ± SD of five mice in each group. Similar results were obtained in two independent experiments.

FIGURE 10.

Expression of OX40 on TFH cells in LN is variable among mouse strains. The indicated eight strains of mice were immunized with 5 × 107 SRBC in the footpads (AC) or i.p. immunized with 2 × 108 SRBC (D). Popliteal LN cells (AC) or spleen cells (D) were isolated 6 days after immunization and stained with anti-CXCR5, anti-ICOS, anti-OX40, and anti-CD4 mAbs. The dot plots represent CD4+-gated LN cells from BALB/c (A) or C57BL/6 (B) mice. Mean fluorescence intensity (MFI) of OX40 expression on CXCR5+CD4+ T cells in LN (C) or spleen (D) is expressed as the mean ± SD of five mice in each group. Similar results were obtained in two independent experiments.

Close modal

ICOS and its ligand B7RP-1 have been implicated in GC formation and Ab production in response to T-dependent Ags (14, 15, 16, 17, 18, 19). In this study, we found that blockade of ICOS/B7RP-1 interaction by neutralizing anti-B7RP-1 mAb or ICOS deficiency abolished the development of CXCR5+ TFH cells as well as PNA+B220+ GC B cells in the spleen in response to primary or secondary immunization with SRBC. These results suggest that the critical role played by ICOS/B7RP-1 is to induce the CXCR5+ TFH cells that control GC formation and Ab production.

An optimal GC response requires cognate interactions between Ag-specific T cells and B cells (1). T cells are trapped and activated by APCs in the T cell zone. When B cells migrate into lymphoid organs, they first enter the T cell zone. Most of the B cells move quickly through the T cell zone into the B cell zone (primary follicle), but those B cells that have bound Ag are trapped. Thus, at the border between the T cell zone and the B cell zone, Ag-specific T cells and B cells interact to initiate the GC response (45). It is most likely that the ICOS/B7RP-1 interaction is involved in this process. In the present study, we have dissected the role of ICOS/B7RP-1 interaction in the GC response, especially focusing on the development of CXCR5+ TFH cells, which migrate to the B cell zone where they provide cognate help to B cells (28). Our adoptive transfer experiments showed that the development of CXCR5+CD4+ T cells was enhanced by B cells in an ICOS/B7RP-1-dependent manner (Fig. 8). Therefore, the defect in GC formation and Ab production in ICOS- or B7RP-1-deficient mice might be primarily due to the impaired development of TFH cells. However, it should be noted that it is not yet clear whether ICOS signaling directly induces the CXCR5 expression on CD4+ T cells. So far we tested that an apparent induction of CXCR5+CD4+ T cells was not observed when naive CD4+ T cells were stimulated with anti-CD3 and anti-CD28 mAbs in the presence of B7RP-1-transfected P815 cells in vitro (data not shown). We also could not find the CXCR5-expressing CD4+ T cells even when whole spleen cells were stimulated with anti-CD3 mAb or Con A in vitro (data not shown). Therefore, some other signals provided by the splenic microenvironment might be required for the expression of CXCR5 on CD4+ T cells in addition to the ICOS signal. Further studies are needed to address this possibility.

Although recent studies have shown the impaired GC formation and Ab production in ICOS- or B7RP-1-deficient mice in response to T-dependent Ags (14, 15, 16, 17, 18, 19), it remained unclear whether the ICOS/B7RP-1 interaction was involved in the secondary GC response mediated by memory T cells and memory B cells. In the present study, we demonstrated that the blockade of ICOS/B7RP-1 interaction with anti-B7RP-1 mAb at the secondary immunization also abrogated the development of both TFH cells and GC B cells (Fig. 5). This clearly indicates that the ICOS/B7RP-1 interaction also plays a critical role in the secondary Ab response mediated memory T cells and memory B cells.

It was notable that the anti-B7RP-1 mAb treatment abrogated the development of TFH cells while sparing the development of GC B cells in the LN of BALB/c mice (Fig. 9, A and B). This suggests that CXCR5+ TFH cells are not always needed for the development of PNA+ GC B cells in LN. Consistent with this notion, an impaired development of GC in the spleen, but not LN, was observed in CXCR5-deficient mice (22). The mechanisms for the B7RP-1- and TFH cell-independent GC formation in the LN of BALB/c mice remain to be determined.

Previous studies have suggested that OX40 plays a critical role in the regulation of T cell migration into B cell follicles. In particular, OX40 signaling up-regulated CXCR5 mRNA in CD4+ T cells (30). It has been suggested that the impaired GC formation in CD28-deficient mice may be due to compromised OX40 expression on CD4+ T cells (31). Moreover, OX40L-transgenic mice demonstrated an accumulation of OX40+CD4+ T cells in the B cell follicles of secondary lymphoid organs (32). Furthermore, blockade of OX40/OX40L interaction by OX40-Ig in chronic intestinal inflammation has shown a marked reduction of CXCR5+CD4+ T cells in the lamina propria (46). In contrast, a recent study has indicated that CXCR5 expression on Ag-specific T cells and their migration into the B cell zone were comparable between wild-type and OX40-deficient mice when inoculated with OVA or Heligmosomoides polygyrus (47). In our present study, OX40 expression was not found on splenic CXCR5+CD4+ T cells from eight mouse strains when i.p. inoculated with SRBC (Figs. 6 and 10,D). Consistently, the development of CXCR5+CD4+ T cells and GC B cells in the spleen was not significantly affected in anti-OX40L mAb-treated or OX40L-deficient BALB/c and C57BL/6 mice (Figs. 1 and 4). In contrast, CXCR5+CD4+ T cells in the LN of C57BL/6 and C3H/He mice uniquely expressed OX40, while those from the other strains including BALB/c did not (Fig. 10, AC). Consistent with the OX40 expression, development of both CXCR5+CD4+ T cells and GC B cells was partially inhibited by anti-OX40L mAb treatment in the LN of C57BL/6, but not BALB/c, mice (Fig. 9). These results indicate that the expression of OX40 on TFH cells and the contribution of OX40/OX40L interaction to the development of TFH cells and GC B cells are variable among secondary lymphoid organs and among mouse strains. The mechanism for this differential expression of OX40 is presently unknown.

Initial functional studies have suggested that ICOS is important for regulating Th2 immune responses, but recent studies have demonstrated that ICOS is also involved in the regulation of Th1 immune responses (7, 8). In addition, we and others have demonstrated that OX40/OX40L interaction plays critical roles in both Th1 and Th2 disease models (29). In the present study, we found subpopulations of activated CD4+ T cells (CXCR5+ICOS+OX40 cells, CXCR5ICOS+OX40 cells, CXCR5ICOSOX40+ cells, and CXCR5ICOS+OX40+ cells) in the spleen and LN (Figs. 6 and 10). It is possible that the three CXCR5 populations may represent functionally distinct subsets producing Th1 or Th2 cytokines in vivo. Further studies are now under way to address this possibility.

We thank Drs. N. Ishii, K. Sugamura, and H. Kikutani for mice and Dr. S. Ishikawa for helpful discussion.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

3

Abbreviations used in this paper: GC, germinal center; LN, lymph node; PNA, peanut agglutinin; TFH, follicular B helper T cell; OX40L, OX40 ligand.

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