Proinflammatory T helper type 17 cells are effective B-cell helpers

Abstract

T helper type 17 (TH17) cells are highly proinflammatory effector T cells that are characterized by the production of high amounts of IL-17A, IL-17F, IL-21, and IL-22. Furthermore, TH17 cells have been associated with a number of autoimmune diseases. However, it is not clear whether TH17 cells can also serve as effective helper cells. Here we show that TH17 cells can function as B-cell helpers in that they not only induce a strong proliferative response of B cells in vitro but also trigger antibody production with class switch recombination in vivo. Transfer of TH17 cells into WT or T-cell receptor alpha-deficient mice, which lack endogenous T cells, induces a pronounced antibody response with preferential isotype class switching to IgG1, IgG2a, IgG2b, and IgG3, as well as the formation of germinal centers. Conversely, blockade of IL-17 signaling results in a significant reduction in both number and size of germinal centers. Whereas IL-21 is known to help B cells, IL-17 on its own drives B cells to undergo preferential isotype class switching to IgG2a and IgG3 subtypes. These observations provide insights into the unappreciated role of TH17 cells and their signature cytokines in mediating B-cell differentiation and class switch recombination.

Fig. 1.

TH17 cells induce B-cell proliferation and isotype class switching in vitro. (A) TH0 or TH17 cells from 2D2 MOG_35-55-specific TCR transgenic mice were cocultured with purified, CFSE-labeled CD19⁺ B cells in the presence of MOG_35-55 (2 μg/mL). Overall survival of cultured cells was determined 3 d later by flow cytometry on the basis of their location in the forward and side scatter (Upper). Numbers in the lower right corners represent percentage of cells located within the live gate. Proliferation of B cells (CD19⁺CD4⁻7AAD⁻) was assessed determining CFSE dilution on day 3 (Lower). Numbers represent percentage of divided B cells. (B) Mitomycin C–treated TH0 or TH17 cells were cocultured with isolated B cells at various ratios in the presence of MOG_35-55 (2 μg/mL). All conditions were plated in triplicate. On day 10 supernatant was analyzed for antibody production and Ig class switching. Graphs show mean concentration (in nanograms per milliliter) ± SEM. *P < 0.05; **P < 0.01.

Fig. 2.

TH17 cells transferred into WT mice help B cells to class switch in vivo. TH0 or TH17 cells from 2D2 MOG_35-55-specific TCR transgenic mice were transferred i.v. into WT B6 mice. At the same time mice were immunized with 50 μg rMOG emulsified in incomplete Freund's adjuvant (IFA) s.c. in both flanks. Serum was obtained on day 7 after transfer/immunization, and MOG-specific antibody response and isotype switch was determined. Serum was applied in serial dilutions starting at 1:10. Each group represents three to five mice, and graphs show mean OD (at 450 nm) ± SEM. *P < 0.05, TH17 recipients vs. TH0.

Fig. 3.

TH17 cells transferred into WT mice induce GC formation. TH0 or TH17 cells from 2D2 MOG_35-55-specific TCR transgenic mice were transferred i.v. into WT or IL-17RA–deficient B6 mice. At the same time mice were immunized with 50 μg rMOG emulsified in IFA s.c. in both flanks. On day 7, formation of GCs was analyzed by staining of lymph node sections for GL-7-FITC and B220-PE. Transferred T cells were detected with a Vα3.2 antibody. (A) Lymph node sections were analyzed with a ×10 objective (Upper) and ×20 objective (Lower). (B) The number of GCs was calculated by evaluation of two sections of two to four draining lymph nodes or the spleen of each mouse. Size of GCs was analyzed by determining total pixels of the GL-7–positive area per two sections of draining lymph nodes or spleen using ImageJ software. Graphs represent mean ± SEM. Each group consisted of three to five mice. *P < 0.05; **P < 0.01.

Fig. 4.

TH17 cells are sufficient to induce class switching in TCRα KO mice. TH0 or TH17 cells from 2D2 MOG_35-55-specific TCR transgenic mice were transferred i.v. into TCRα KO B6 mice. At the same time mice were immunized with 50 μg rMOG emulsified in IFA s.c. in the flanks. Serum was obtained on day 7 after transfer/immunization, and MOG-specific antibody response and isotype switch was determined using a MOG-specific Ig ELISA. Serum was applied in serial dilutions starting at 1:10. Each group represents three to five mice, and graphs show mean OD (at 450 nm) ± SEM. *P < 0.05; **P < 0.01, TH17 condition vs. TH0.

Fig. 5.

IL-17 induces class switching in vitro. B cells were purified and activated in the presence of increasing doses of the indicated cytokines. Antibody production and class switch recombination were analyzed in response to stimulation with anti-CD40 (5 μg/mL)/IgM (5 μg/mL) plus IL-17 (0.1/1/10 ng/mL) or IL-21 (10/25/50 ng/mL) or no cytokine as a control. Columns for each cytokine represent increasing concentrations from left to right. Graphs show mean concentration (in nanograms per milliliter) ± SEM, representing 10 cultures per condition. *P < 0.05; **P < 0.01, IL-17 or IL-21 vs. no cytokine.