T follicular helper cells mediate expansion of regulatory B cells via IL-21 in Lupus-prone MRL/lpr mice

Abstract

T follicular helper (Tfh) cells can mediate humoral immune responses and augment autoimmunity, whereas the role of Tfh cells on regulatory B (B10) cells in autoimmunity diseases is not clear. Here, we investigated the percentages of Tfh cells and B10 cells in lupus-prone MRL/Mp-lpr/lpr (MRL/lpr) mice and examined the effects and mechanism of Tfh cell-derived interleukin-21 (IL-21) on IL-10 production during the differentiation of B10 cells. Both Tfh cells and B10 cells were expanded in spleens of MRL/lpr mice. In addition, a positive correlation between the proportions of Tfh cells and B10 cells was observed. Tfh cell-derived IL-21 from MRL/lpr mice could promote IL-10 production during the differentiation of B10 cells. Importantly, neutralization of IL-21 inhibited IL-10 production and expansion of B10 cells both in vitro and in vivo. IL-21 induced IL-10 production via activation of phosphorylated signal transduction and activator of transcription 3 (p-STAT3). Inhibition of p-STAT3 effectively blocked IL-10 production during the differentiation of B10 cells. Moreover, IL-21-induced IL-10 exerted a regulatory function by inhibiting the proliferation of T cells. These data suggest that Tfh cells not only mediate humoral immune responses and augment autoimmunity but also play a broader role in immune regulatory actions via the induction of IL-10 production.

Figure 1. Expansion of Tfh cells in MRL/lpr mice.

(A) Splenomegaly in MRL/lpr mice. (B) Splenocytes were isolated from MRL/lpr and B6 mice. After staining, cells were first gated for CD4⁺ T cells, and the CXCR5⁺PD-1⁺ cells were analyzed with a CD4⁺ gate by flow cytometry. (C) The percentages of CXCR5⁺PD-1⁺ cells among CD4⁺ T cells (n = 6 for each group). (D) IL-21 mRNA expression in fresh isolated splenocytes was determined by real-time RT-PCR (n = 6 for each group). (E) Bcl-6 mRNA expression in fresh isolated splenocytes was determined by real-time RT-PCR (n = 6 for each group). (F) Sorted CD4⁺CXCR5⁺PD-1⁺ Tfh cells from MRL/lpr and B6 mice were cultured in the presence of anti-CD3 and anti-CD28 for 2 days, IL-21 mRNA expression was determined by real-time RT-PCR. Results shown are representative of at least three independent experiments. (G) Sorted CD4⁺CXCR5⁺PD-1⁺ Tfh cells from MRL/lpr and B6 mice were cultured in the presence of anti-CD3 and anti-CD28 for 2 days, Bcl-6 mRNA expression was determined by real-time RT-PCR. Results shown are representative of at least three independent experiments. (H) IL-21 expression in spleens was confirmed by immunohistochemical staining. Further magnification of the black-bordered box shows the predominance of IL-21⁺ lymphocytes. The scale bar represents 50 μm.

Figure 2. Tfh cells are associated with autoantibody production in MRL/lpr mice.

(A) H&E staining of spleens from MRL/lpr and B6 mice (left). PNA⁺ GC cells were determined by immunohistochemical staining (right). The scale bar represents 50 μm. (B) A positive correlation between the percentages of CD4⁺CXCR5⁺PD-1⁺ T cells and the number of PNA⁺ GC cells in spleens of MRL/lpr mice (n = 6) was observed. (C) A positive correlation between the percentages of CD4⁺CXCR5⁺PD-1⁺ T cells and renal scores of MRL/lpr mice (n = 6) was observed. (D) A positive correlation between serum levels of IL-21 and ANA in MRL/lpr mice (n = 6) was found. (E) A positive correlation between serum levels of IL-21 and ds-DNA in MRL/lpr mice (n = 6) was found. (F) Sorted CD4⁺CXCR5⁺PD-1⁺ Tfh cells from MRL/lpr and B6 mice were cultured in the presence of anti-CD3 and anti-CD28 for 2 days, detection of IL-21 in the supernatants by ELISA. Results shown are representative of at least three independent experiments. (G) The concentrations of IgM and IgG₁ in sorted naive B cells from B6 mice following a 3-day induction with LPS, anti-CD40, anti-IgM, and 20% of the supernatants from cultured Tfh cells from MRL/lpr (mTfh) and B6 mice (bTfh) or vehicle (culture media with 2 µg/ml plate-bound anti-CD3 and 2 µg/ml soluble anti-CD28) with or without neutralization of IL-21. Results shown are representative of at least three independent experiments.

Figure 3. Expansion of B10 cells is positively related to increased Tfh cells in MRL/lpr mice.

(A) Following isolation, splenocytes were stained and sorted first for CD19⁺ B cells. Then CD5⁺CD1d^high B cells were analyzed in a CD19⁺ gate by flow cytometry (left). The percentages of CD5⁺CD1d^high cells among CD19⁺ B cells are shown (right, n = 6 for each group). (B) A positive correlation between the proportions of CD4⁺CXCR5⁺PD-1⁺ T cells and CD19⁺CD5⁺CD1d^high B cells in spleens of MRL/lpr mice (n = 6) was found. (C) Splenocytes were isolated from MRL/lpr and B6 mice and stimulated with LPS plus PIB for 5 hours. CD19⁺IL-10⁺ cells among CD19⁺ B cells were detected by intracellular cytokine staining and flow cytometry analysis (n = 6 for each group). (D) Sorted CD19⁺CD5⁺CD1d^high B cells from MRL/lpr and B6 mice were stimulated with LPS for 48 hours and PIB for the last 5 hours, IL-10 mRNA expression was detected by real-time RT-PCR. Results shown are representative of at least three independent experiments. (E) IL-10 protein expression in spleens was confirmed by immunohistochemical staining. Further magnification of the black-bordered box shows the predominance of IL-10⁺ lymphocytes. The scale bar represents 50 μm. (F) A positive correlation between the numbers of IL-10⁺ and IL-21⁺ cells in spleens of MRL/lpr mice (n = 6) was observed. (G) A positive correlation between the serum levels of IL-21 and IL-10 in MRL/lpr mice (n = 6) was found. (H) The percentage of CD19⁺IL-10⁺ cells among B cells in spleens of MRL/lpr mice with neutralization of IL-21 or PBS vehicle control once per week for 4 weeks (n = 6) was analyzed by flow cytometry. (I) The concentrations of IL-10 in supernatants of cultured B6 mouse-derived B cells that were induced for 3 days by LPS plus 20% of supernatants of Tfh cell cultures from MRL/lpr (mTfh) and B6 mice (bTfh) or vehicle (culture media with 2 µg/ml plate-bound anti-CD3 and 2 µg/ml soluble anti-CD28) with or without neutralization of IL-21 and stimulated with PI for the last 5 hours were determined. Results shown are representative of at least three independent experiments.

Figure 4. IL-21 induces IL-10 production during the differentiation of B10 cells via activation of p-STAT3.

(A) Naïve B cells sorted from B6 mice were cultured in the presence of LPS with or without 10 ng/ml IL-21 for the indicated times and stimulated with PIB for the final 5 hours. STAT3 mRNA expression was detected by real-time RT-PCR. (B) The expression of p-STAT3 and STAT3 in sorted CD19⁺CD5⁺CD1d^high B cells from MRL/lpr mice and B6 mice was analyzed by Western blot. (C) Naïve B cells sorted from B6 mice were cultured with or without 10 ng/ml IL-21 for the indicated times. Then, p-STAT3 proteins were analyzed by Western blot. (D) Naïve B cells sorted from B6 mice were cultured with or without 10 ng/ml IL-21 or AG490 for the indicated times, and the levels of p-STAT3 and STAT3 proteins were determined by Western blot. (E) Naïve B cells sorted from B6 mice were cultured in the presence of LPS with or without 10 ng/ml IL-21 or AG490 for the indicated times and stimulated with PI for the final 5 hours. IL-10 in supernatants was detected by ELISA. (F) Naïve B cells sorted from B6 mice were cultured in the presence of LPS with or without the indicated concentrations of IL-21 or AG490 for 48 hours and stimulated with PI for the final 5 hours. IL-10 in supernatants was detected by ELISA. (G) Sorted CD19⁺CD5⁺CD1d^high B cells from MRL/lpr mice were cultured in the presence of LPS and 10 ng/ml IL-21 with or without AG490 for 48 hours, and IL-10 in supernatants was detected by ELISA. (H) Naïve B cells sorted from B6 mice were cultured with or without 10 ng/ml IL-21 or SPI for 15 minutes, p-STAT3 proteins were then analyzed by Western blot. (I) Naïve B cells sorted from B6 mice were cultured in the presence of LPS with or without 10 ng/ml IL-21 or SPI for the indicated times and stimulated with PI for the final 5 hours, IL-10 in supernatants was then detected by ELISA. (J) CFSE-labeled CD4⁺CD25^- T cells from MRL/lpr mice were cultured for 3 days with anti-CD3 and anti-CD28 antibodies in combination with 20% of the supernatants from IL-21-induced B10 cell cultures (from MRL/lpr mice) with or without neutralization of IL-10. The proliferation of these cells was determined by flow cytometry. All the above results shown are representative of at least three independent experiments.