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. 2017 Oct 19;153(1):105–117. doi: 10.1111/imm.12815

Transcriptional repressor Blimp1 regulates follicular regulatory T‐cell homeostasis and function

Guang Yang 1, Xiaosu Yang 1, Junmei Zhang 2, Guancheng Li 3, Dandan Zheng 1, Anjiao Peng 1, Jue Hu 4, Liqun Xu 1, Baifeng Yang 1, Huan Yang 1, Wenbin Zhou 1, Erdem Tuzun 5, Jing Li 1,✉
PMCID: PMC5721240  PMID: 28833081

Summary

The B‐lymphocyte‐induced maturation protein 1 (Blimp1) regulates T‐cell homeostasis and function. Loss of Blimp1 could double the proportion of follicular regulatory T (Tfr) cells. However, the effects that Blimp1 may have on the function of Tfr cells remain unknown. Here we document the function for Blimp1 in Tfr cells in vitro and in vivo. Data presented in this study demonstrate that Tfr cells indirectly inhibit the activation and differentiation of B cells by negatively regulating follicular helper T cells, so lowering the secretion of antibody. Lack of Blimp1 makes the immune suppression function of Tfr cells impaired in vitro. In the in vivo study, adoptive transfer of Tfr cells could reduce immune responses in germinal centres and relieve the muscle weakness symptoms of mice with experimental autoimmune myasthenia gravis. Blimp1 deficiency resulted in reduced suppressive ability of Tfr cells. This study identifies that Tfr cells are potent suppressors of immunity and are controlled by Blimp1.

Keywords: autoinflammatory disease, neuroimmunology, neuroinflammation, regulatory T cells, T follicular helper cell

Introduction

Myasthenia gravis is a T‐cell‐dependent, antibody‐mediated, complement‐involved, organ‐specific autoimmune disease, characterized by muscle fatigability, which develops as a result of impaired neuromuscular transmission.1, 2, 3, 4, 5 Abnormal development of high‐affinity autoantibody‐producing B cells in the germinal centres is a central feature of myasthenia gravis.6 Experimental autoimmune myasthenia gravis (EAMG) has been recognized as the animal model for myasthenia gravis. It can be induced by a synthetic peptide corresponding to regions 97–116 of the rat AChR α‐subunit (R97‐116 peptide).1, 7, 8, 9, 10

Germinal centres (GCs) are temporary structures in peripheral lymphatic tissue and play a crucial role in affinity maturation by somatic hypermutation and isotype switching.11, 12, 13 Follicular helper T cells (Tfh cells) are essential for helping cognate B cells form and maintain the GC reaction.14, 15, 16, 17, 18 Aberrant changes in the number or function of Tfh cells have been correlated with the pathogenesis and severity of autoimmune disease.16, 19, 20, 21, 22 It was reported that adoptive transfer of Tfh cells led to GC formation.16 How to limit the numbers of Tfh cells within GCs has become the focus of preventing the emergence of autoantibody.16, 23

Follicular regulatory T (Tfr) cells, which share phenotypic characteristics of Tfh and Foxp3+ regulatory T (Treg) cells, are required to suppress T‐cell proliferation and Tfh numbers, and control GC B‐cell selection.24 Tfr cells are crucial for the development of self‐tolerance and have become the major focus in many studies interpreting the pathogenesis of autoimmune disease. Tfr cells derive from Treg cell precursors, and concurrently express B‐lymphocyte‐induced maturation protein 1 (Blimp1) and B‐cell lymphoma 6 (Bcl‐6).24, 25, 26, 27 Bcl‐6 is essential for Tfr cell formation, and Blimp1 regulates the size of the Tfr cell population. Blimp1 is required for the differentiation of B cells and T cells, and it has been identified as an essential regulator of T‐cell homeostasis and activation.28, 29, 30 It has been shown that loss of Blimp1 could double the proportion of Tfr cells without any alteration in the proportion of Tfh cells.24 If deficiency of Blimp1 results in greater suppressive ability of Tfr cells, then Blimp1 will become an important target for the treatment of myasthenia gravis.

However, little is known about the effect of loss of Blimp1 on the function of Tfr cells. In this study, we demonstrated that Blimp1‐deficient Treg cells differentiated into more Tfr cells than did wild‐type Treg cells in vitro. Blimp1‐deficient Tfr cells in the spleen had a decreased ability to suppress both the activation of Tfh cells and antibody production in vitro. In addition, we found that adoptive transfer of Blimp1‐deficient Tfr cells could ameliorate clinical myasthenic symptoms in mice with ongoing EAMG, although this was inferior to wild‐type Tfr cells.

Materials and methods

Animal and antigen

Female C57BL/6 mice (6–8 weeks of age) were purchased from Laboratory Animal Co. Ltd. of Slack King in Hunan Province (license no. SCXK 2011‐0003). Animals were housed at the animal facility of the Division of Animal Care at Central South University. All animal experiments were approved by the Ethics Committee of the institute and performed in accordance with the Principles of Laboratory Animal Care of Xiangya Hospital. Animals were killed after deep anaesthesia obtained by carbon dioxide; low‐grade anaesthesia with 4% chloral hydrate (Sigma‐Aldrich, St Louis, MO) administered intraperitoneally was used for immunization and treatments.

The antigen used to induce EAMG was a synthetic peptide corresponding to region α 97–116 of the rat AChR α‐subunit (R97–116). The peptides R97–116 (DGDF AIVK FTKV LLDY TGHI) were synthesized by GL Biochem Ltd., Shanghai, China (Lot number: P140924‐MJ148487). Peptides were purified by reverse‐phase high‐performance liquid chromatography, and their synthesis was confirmed by mass spectroscopy.

Synthesis of siRNA‐Blimp1

Cholesterol‐conjugated Blimp1 small interfering RNA (sense: 5′‐CCCGAAUCAAUGAAGAAAUTT‐3′, antisense: 5′‐AUUUCUUCAUUGAUUCGGGTT‐3′), its negative control and Cy3‐siRNA were from RiboBio (Guangzhou, China).

Induction and clinical assessment of EAMG

Mice were randomly divided into two groups. Mice in the EAMG group were immunized by subcutaneous injection in the hind footpads and the shoulders with 50 μg AChR R97–116 peptide in 200 μl CFA (Sigma) supplemented with 1 mg of Mycobacterium tuberculosis strain H37RA (Difco Laboratories, Detroit, MI) on day 0 and boosted on days 30 and 60 with the same peptide in CFA. The control group was immunized with CFA emulsion, containing phosphate‐buffered saline (PBS) instead of the peptide (Figure S1).

Clinical scoring was based on the presence of tremor, hunched posture, muscle strength, fatigability, and was assessed after paw exercise (repetitive paw grips on the cage grid for 30 times). Disease severity was expressed as follows: grade 0, normal muscle strength and activities; grade 1, normal at rest, mildly decreased activity (characteristically shown by hunchback posture, and weak grip or backward movement), more evident at the end of exercise; grade 2, clinical signs present at rest (tremor, hunchback posture and weak grip or backward movement); grade 3, severe clinical signs present at rest, moribund with or without closure or secretions of the eyes; grade 4, dead. Mice with intermediate signs were assigned scores of 0·5, 1·5, 2·5 or 3·5.

Cell culture in vitro

To explore the effect of Blimp1 on the homeostasis and function of Tfr cells, spleen cells were harvested from the wild‐type C57BL/6 mice aged 6–8 weeks. CD4+ CD25+ Treg cells were purified with immunomagnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) from spleen cells; CD4+ CXCR5+ ICOS+ GITR+ Tfr and CD4+ CXCR5+ ICOS+ GITR‐Tfh cell populations were pre‐purified with CD4+ immunomagnetic beads from spleen cells, and then were sorted to a purity of 99% on a FACSAria II (BD Biosciences, San Diego, CA). B cells were then purified with immunomagnetic beads from the spleen cells of mice with EAMG (grade ≥ 1.5),and the purity of B cells was up to 98.5% (Figure S2). 2 × 105 Treg cells from the spleen of wild‐type mice were plated with 2 μg/ml soluble anti‐CD3 (BD Biosciences) and 100 U/ml interleukin‐2 (eBioscience, San Diego, CA), and then were transiently transfected by siRNA‐Blimp1 (400 nm final) and siRNA‐negative control (NC) (400 nm final). After 6 hr, fresh complete RPMI‐1640 was added supplemented with 20% fetal bovine serum, 2 μg/ml soluble anti‐CD3 and 100U/ml interleukin‐2. After 24 and 48 hr, cells were harvested for the detection of transcription and expression of Blimp1. After 5 days, cells were collected to identify the frequency of Tfr cells by flow cytometry. 1 × 105 Tfr cells from the spleens of wild‐type mice were plated with 2 μg/ml soluble anti‐CD3 (BD Bioscience), and were then transiently transfected by siRNA‐Blimp1 (400 nm final) and siRNA‐NC (400 nm final). After 6 hr, fresh complete RPMI‐1640 supplemented with 20% fetal bovine serum and 2 μg/ml soluble anti‐CD3. Twenty‐four hours after transient transfection of Tfr cells, B cells from EAMG mice were plated with Tfh cells and/or transfected Tfr cells pooled from wild‐type C57BL/6 mice and 2 μg/ml soluble anti‐CD3 and 5 μg/ml anti‐IgM (BD Bioscience). After 4 days, supernatants were collected for antibody analysis by ELISA, and cells were harvested to identify the frequency of B‐cell subsets and the surface activation molecules on B cells by flow cytometry.

Adoptive transfer

The Tfr cells were pre‐purified with CD4+ immunomagnetic beads from spleen cells, and then were sorted to a purity of 99% on a FACSAria II (BD Biosciences). Then, 2 × 105 Tfr cells from the spleens of wild‐type mice were plated with soluble anti‐CD3 (as described above), and then were transiently transfected by siRNA‐Blimp1 and siRNA‐NC. 24 hr after transient transfection, Tfr cells were used for adoptive transfer.

On day 70 (after the first immunization) the EAMG mice (Grade ≥ 1.5) were divided randomly into three groups: EAMG‐PBS, EAMG‐siBlimp1‐Tfr and EAMG‐siNC‐Tfr groups. The mice in the EAMG‐siBlimp1‐Tfr group were treated with siRNA‐Blimp1‐Tfr cells (2 × 105/200 μl) by tail injection intravenously every 5 days for a total of three injections (days 0, 5, 10). The mice were treated with siRNA‐NC‐Tfr cells (2 × 105/200 μl) in the EAMG‐siNC‐Tfr group and PBS (200 μl) in the EAMG‐PBS group using the same method, and clinical assessment and myasthenic scoring of the mice in each group were recorded every day. At the same time the serum specimens were collected to detect the secretion level of anti‐R97‐116 IgG by ELISA. At day 15 after the first treatment on day 0, the mice were killed and spleen cells were collected. Frequencies of B‐cell subsets and T‐cell subsets were detected by flow cytometry. The expression of key protein in the mitogen‐activated protein kinase (MAPK) pathway and AKT pathway in mouse B cells was detected by Western blot.

Flow cytometry

Cells were resuspended in staining buffer (PBS containing 1% fetal calf serum and 2 mm EDTA) and were stained with the following directly labelled antibodies: FITC‐conjugated anti‐mouse CD4, allophyocyanin (APC) ‐conjugated anti‐mouse CXCR5, phycoerythrin (PE) ‐Cy7‐conjugated anti‐mouse ICOS, PE‐conjugated anti‐mouse Foxp3, PE‐conjugated anti‐mouse GITR, FITC‐conjugated anti‐mouse CD45R, APC‐conjugated anti‐mouse CD27, PE‐conjugated anti‐mouse CD138, PE‐conjugated anti‐mouse CD44, PE‐conjugated anti‐mouse CD40, PE‐conjugated anti‐mouse CD80, PE‐conjugated anti‐mouse CD86, APC‐conjugated anti‐mouse CD25, APC‐conjugated anti‐mouse CD3e, PE‐conjugated anti‐mouse interferon‐γ, PE‐conjugated anti‐mouse interleukin‐4, PE‐conjugated anti‐mouse interleukin‐17A, PE‐conjugated anti‐mouse CD 19, PE‐Cy7‐conjugated anti‐mouse CD5, PE‐conjugated anti‐mouse CD25, PE‐conjugated anti‐mouse CD69, PE‐conjugated anti‐mouse CD71. For nuclear transcription factor staining, a Foxp3 Fix/Perm kit (eBioscience) was used after surface staining. For cellular staining, Fixation/Permeabilization kit (BD Bioscience) was used after surface staining. All flow cytometry data were analysed with an LSR II (BD Bioscience) with standard filter sets and were further analysed with flowjo software (TreeStar, Ashland, OR).

Enzyme‐linked immunosorbent assay

To detect the production of anti‐R97‐116 IgG, ELISA was performed with an anti‐mouse IgG (Immunology Consultants Laboratories, Portland, OR), as indicated by the instructions. Optical density (OD) was measured at 450 nm using an automated microplate ELISA reader. Results are expressed as OD at 450 nm.

Western blot analysis

Purified B cells from spleen of the three EAMG groups were prepared and analysed by SDS–PAGE. The following primary antibodies were used: rabbit anti‐phospho‐JNK (Thr183/Tyr185) antibody, rabbit anti‐phospho‐p44/42 MAPK (ERK1/2) (Thr202/Tyr204) antibody, rabbit anti‐phospho‐P38 (Thr180/Tyr182) antibody, rabbit anti‐phospho‐Akt (Ser473) antibody and rabbit anti‐Akt antibody (Cell Signal Technology, Beverley, MAUSA). The quantification of the bands was done by densitometry analysis.

Histological staining

Tissue samples from the spleen were immersed in 4% paraformaldehyde and embedded in paraffin wax. Sections were processed and stained with haematoxylin & eosin (H&E) to assess the number and size of GCs. Each sample was sliced into 2‐mm sections, and then was visualized using fluorescence microscopy (Nikon, Tokyo, Japan).

For GL7 staining, 4‐μm sections were prepared from paraffin‐embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0·3% H2O2 in 60% methanol for 30 min. After 30‐min blockade with 10% goat serum, anti‐rat GL7 monoclonal antibody (1 : 100 dilution in PBS; eBioscience) was incubated overnight at 4°, followed by a second incubation with rabbit anti‐rat IgG‐horseradish peroxidase for another 2 hr. Finally, DAB was used as a substrate‐chromogen that produces a brown colour in GL7‐positive cells. Sections were counterstained for 1 min with haematoxylin.

Statistical analysis

The data were presented as the mean ± standard deviation. All statistical analyses were carried out with the Statistical Package for Social Science, version 13.0 (SPSS Inc., Chicago, IL). Multiple comparisons were analysed using one‐way analysis of variance. P‐values of < 0·05 were considered statistically significant.

Result

Blimp1 regulates Tfr cell homeostasis

It has been confirmed that Tfr cells derive from Foxp3+ Treg cell precursors. We isolated Treg cells from mouse spleen cell suspension, and the purity of Treg cells was up to 91.3% (Fig 1a). To determine whether Blimp1 plays a role in Tfr homeostasis, Treg cells were transfected with siRNA‐Blimp1 to establish Blimp1‐deficient Treg cells. We defined Tfr cells as CD4+ CXCR5+ ICOS+ Foxp3+, a gating strategy that separated Tfr cells from CD4+ CXCR5+ ICOS+ Foxp3− Tfh cells. Five days after transfection, Blimp1‐deficient Treg cells developed more Tfr cells than the negative control group (Fig. 1). This is consistent with a recent report showing that loss of Blimp1 causes the proportion of Tfr cells to double.

Figure 1.

Figure 1

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Blimp1 in Foxp3+ regulatory T (Treg) cells limits generation of follicular regulatory T (Tfr) cells. (a) Expression of surface markers CD4+ CD25+ in spleen cells of C57BL/6 after magnetic bead isolation. The purity of CD4+ CD25+ Treg cells was 91 ± 2%. (b) Microscopy observation of small interfering RNA (siRNA) ‐transfected cells. The transfection efficiency of siRNA in Treg cells was over 90%. (c, d) Transcription and expression verification of Blimp1 was significantly decreased in siRNA‐Blimp1‐infected cells. (e) Quantification of Tfr cells (CD4+ CXCR5+ ICOS+ Foxp3+) in cultured Treg cells on day 5 after siRNA‐Blimp1 transfection. Blimp1‐deficient Treg cells developed more Tfr cells than negative control group Blimp1 controls. [Colour figure can be viewed at wileyonlinelibrary.com]

Blimp1 regulates Tfr cell activation

We constructed Blimp1‐deficient Tfr cells by transfection of siRNA‐Blimp1 to assess the function of Blimp1 on Tfr cells. Tfr cells have higher expression of the GITR on the surface than do Tfh cells, which allowed us to separate Tfr cells and Tfh cells in a similar manner to that of intracellular staining for Foxp3. To assess the ability of Blimp1‐deficient Tfr cells to suppress activation and antibody production by B cells in vitro, we cultured B cells from the spleen of EAMG mice with Tfh cells for 4 days in the presence or absence of Blimp1‐deficient Tfr cells (Tfh cells and Tfr cells from wild‐type mice), anti‐IgM and anti‐CD3.

We found that plasma cells, memory B cells and the expression of CD80 and CD86 were significantly increased when B cells were cultured with Tfh cells (Fig. 2). Plasma cells and the expression of the CD80 and CD86 were obviously decreased in the presence of Tfr cells. The inhibition of the Blimp1‐deficient Tfr cells were less than negative control Tfr cells.

Figure 2.

Figure 2

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Blimp1‐deficient follicular regulatory T (Tfr) cells are faulty in the suppression of activation of antibody production by B cells in vitro. Flow cytometry analysis of B‐cell subtypes and expression of surface activation markers of B cells (CD40, CD80, CD86) in cultures of B cells obtained from the spleen of experimental autoimmune myasthenia gravis (EAMG) mice (Grade ≥ 1·5), incubated with wild‐type follicular helper T (Tfh) cells in the presence or absence of Tfr cells/Blimp1‐deficient Tfr cells. (a) Flow cytometry analysis of CD45R+ CD27+ CD138+ plasma cells. In the B‐cell group it was 11·82 ± 1·27%, in the co‐culture group of Tfh + B cells it was 22·72 ± 1·94%, in the co‐culture group of Tfr + B cells it was 12·06 ± 0·94%, in the co‐culture group of Tfr + Tfh + B cells it was 10·92 ± 0·99%, and in the co‐culture group of siBlimp1‐Tfr + Tfh + B cells it was 13·3 ± 1·06%. (b) Flow cytometry analysis of CD45R+ CD27+ CD44+ memory B cells. In the B‐cell group it was 12·54 ± 1·61%, in the co‐culture group of Tfh + B cells it was 33·26 ± 1·68%, in the co‐culture group of Tfr + B cells it was 11·92 ± 1·29%, in the co‐culture group of Tfr + Tfh + B cells it was 31·58 ± 0·90%, and in the co‐culture group of siBlimp1‐Tfr + Tfh + B cells it was 33·0 ± 1·02%. (c) There were no significant differences in the expression of CD40 in all groups. (d) Flow cytometry analysis of CD80 showed that in the B‐cell group it was 40·64 ± 1·56%, in the co‐culture group of Tfh + B cells it was 44·94 ± 1·36%, in the co‐culture group of Tfr + B cells it was 39·74 ± 0·69%, in the co‐culture group of Tfr + Tfh + B cells it was 41·32 ± 1·18%, in the co‐culture group of siBlimp1‐Tfr + Tfh + B cells was 40·88 ± 1·33%. As for CD86 (e), in the B cells group it was 84·64 ± 1·61%, in the co‐culture group of Tfh + B cells it was 94·56 ± 1·58%, in the co‐culture group of Tfr + B cells it was 84·42 ± 1·03%, in the co‐culture group of Tfr + Tfh + B cells it was 83·08 ± 1·82%, in the co‐culture group of siBlimp1‐Tfr + Tfh + B cells it was 86·14 ± 1·63% (*P < 0·05). [Colour figure can be viewed at wileyonlinelibrary.com]

B cells produced large amounts of IgG when cultured with Tfh cells (Fig. 3). When we added the Tfr cells to the wells along with the Tfh cells, the production of IgG dramatically decreased. Blimp1‐deficient Tfr cells suppress IgG production less than negative control Tfr cells did at ratios of Tfr cells to Tfh cells of 1 : 1. This is consistent with the change of plasma cells.

Figure 3.

Figure 3

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Titres of anti‐R97‐116 IgG in supernatants of cultures of B cells obtained from the spleens of experimental autoimmune myasthenia gravis (EAMG) mice (Grade ≥ 1·5), incubated with wild‐type follicular helper T (Tfh) cells in the presence or absence of follicular regulatory T (Tfr) cells/Blimp1‐deficient Tfr cells. In the B cells group it was 3·17 ± 0·08 (OD450 nm), in the co‐culture group of Tfh + B cells it was 3·88 ± 0·06, in the co‐culture group of Tfr + B cells it was 3·17 ± 0·08, in the co‐culture group of Tfr + Tfh + B cells it was 3·15 ± 0·07, and in the co‐culture group of siBlimp1‐Tfr + Tfh + B cells it was 3·24 ± 0·05 (*P < 0·05). [Colour figure can be viewed at wileyonlinelibrary.com]

Reduced suppression of Blimp1‐deficient Tfr cells in vivo

The results described above suggest that Blimp1 controls the activation of Tfr cells in vitro. To demonstrate that the deficiency of Blimp1 could attenuate the suppression of Tfr in vivo, Blimp1‐deficient Tfr cells and negative control Tfr cells were transferred into EAMG mice (Fig. 4a). By 15 days after first transfer, we observed less amelioration of the clinical severity in the EAMG mice that received Blimp1‐deficient Tfr cells than in the group treated with negative control Tfr cells (Fig. 4b). As expected, compared with the EAMG mice treated with negative control Tfr cells, the level of anti‐R97‐116 IgG was significantly higher in the EAMG mice that received Blimp1‐deficient Tfr cells (Fig. 4c).

Figure 4.

Figure 4

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Blimp1‐deficient follicular regulatory (Tfr) cells less potently regulate antibody in vivo. (a) Tfr cells were isolated from the spleens of wild‐type mice, and transfected by siRNA‐Blimp1 or siRNA‐NC, and then transferred into experimental autoimmune myasthenia gravis (EAMG) recipient mice (Grades ≥ 1·5) at days 0, 5 and 10. (b) Blimp1‐deficient Tfr cells less potently ameliorated clinical myasthenic symptoms in mice with ongoing EAMG than siRNA‐NC‐Tfr cells. At the end of the experiment, the mean clinical score (MCS) of the siRNA‐NC‐Tfr group was 0·560 ± 0·035, of the siRNA‐Blimp1‐Tfr group was 0·933 ± 0·076, and of the PBS group was 1·867 ± 0·021. (c) Titres of anti‐R97‐116 IgG in recipients of PBS or Tfr cells as in (a) (*P < 0·05). [Colour figure can be viewed at wileyonlinelibrary.com]

Blimp1 regulates Tfr cell activation in vivo

We next investigate the suppressive ability of Blimp1‐deficient Tfr cells in vivo. Negative control Tfr cells inhibited the generation of plasma cells (Fig. 5a), memory B cells (Fig. 5b), B1 cells (Fig. 5c), Th1 cells (Fig. 6a), Th17 cells (Fig. 6c) and Tfh cells (Fig. 7b), in the spleen to a greater extent than did Blimp1‐deficient Tfr cells in EAMG mice. Meanwhile, the proportions of Treg cells (Fig. 6d), Tfr cells (Fig. 7a) and Th2 cells (Fig. 6b) were significantly higher in the EAMG mice that received negative control Tfr cells than in the group transferred with Blimp1‐deficient Tfr cells. Together these data showed that Blimp1 deficiency resulted in reduced suppressive ability of Tfr cells.

Figure 5.

Figure 5

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The effect of adoptive transfer of follicular egulatory T (Tfr) cells/Blimp1‐deficient Tfr cells on B cells in experimental autoimmune myasthenia gravis (EAMG) mice. A representative FACS analysis of CD45R+ CD27+ CD138+ plasma cells, CD45R+ CD27+ CD44+ memory B cells and CD5+ CD19+ B1 cells among spleen mononuclear cells (MNCs) are shown in (a–c, gated on CD45R+). The percentages of plasma cells, memory B cells and B1 cells among spleen MNCs in the EAMG‐PBS group, EAMG‐siNC‐Tfr group and EAMG‐siBlimp1‐Tfr group are shown on the right of (a–c). The results are expressed as mean ± SD (n = 5 in each group) (*P < 0·05). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6.

Figure 6

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Distribution of T helper (Th) cells in vivo. Mononuclear cells (MNCs) were isolated from the spleen of experimental autoimmune myasthenia gravis (EAMG) mice in respective treatment groups. (a–d) FACS analysis of interferon‐γ (IFN‐γ), interleukin‐4 (IL‐4), IL‐17 and CD25+ Foxp3+ expression in CD4+ T cells harvested from the EAMG‐PBS group, EAMG‐siNC‐Tfr group and EAMG‐siBlimp1‐Tfr group. Data are expressed as mean ± SD (n = 5 in each group) (*P < 0·05). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 7.

Figure 7

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Adoptive transfer of follicular regulatory T (Tfr) cells/Blimp1‐deficient Tfr cells decreased follicular helper T (Tfh) cells and increased Tfr cells in experimental autoimmune myasthenia gravis (EAMG) mice. Mononuclear cells (MNCs) were isolated from the spleens of EAMG mice in the respective treatment groups. Expression of Tfh cells (CD4+ CXCR5+ ICOS+ Foxp3–) and Tfr cells (CD4+ CXCR5+ ICOS+ Foxp3+) (a, b) was assessed by FACS as indicated. Data are expressed as mean ± SD of n = 5 mice/group representative of three independent experiments (*P < 0·05). [Colour figure can be viewed at wileyonlinelibrary.com]

Diminished suppression of GC formation by transfer of Blimp1‐deficient Tfr cells into EAMG mice

Transfer of negative control Tfr cells significantly reduced the size of the spleen and the size of GCs in the spleen and lymph node compared with transfer of Blimp1‐deficient Tfr cells (Fig. 8a–c). GL7 is the marker of B cells in GCs. In our study, we detected the expression of GL7 in the spleen and lymph nodes harvested from the three groups. We demonstrated that activated B cells in GCs of mice treated with Blimp1‐deficient Tfr cells were significantly more than in the negative control Tfr‐cell‐treated group (Fig. 9). Finally, we explored the possible mechanisms of diminished suppression of GC formation by transfer of Blimp1‐deficient Tfr. By 15 days after first transfer, B cells were purified with magnetic beads from spleens of the different treatment groups, and B‐cell lysates were analysed by Western blot analysis for several signalling axes. Our results demonstrated that p‐AKT activation was significantly dampened in splenic B cells from the Blimp1‐deficient Tfr‐treated mice compared with that in the negative control Tfr group (Fig. 8d). We failed to find a difference in the signal pathways of p‐MAPK between the two groups (Fig. 8e).

Figure 8.

Figure 8

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Decrease in germinal centre (GC) formation and expression of p‐AKT of B cells in experimental autoimmune myasthenia gravis (EAMG) mice that received adoptive transfer of follicular regulatory T (Tfr) cells/Blimp1‐deficient Tfr cells. The number and size of GCs were analysed by haematoxylin & eosin staining using paraffin wax lymph node sections (a) and spleen sections (b). The gross image of the spleen from a representative mouse is shown in (c). CD45R+ B cells were purified with immunomagnetic beads from spleen cells of EAMG mice in the respective treatment groups, p‐AKT (d) and proteins in the signal pathway of phosphorylated mitogen‐activated protein kinase (e) expression detected by Western blotting. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 9.

Figure 9

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Effects of adoptive transfer of follicular regulatory T (Tfr) cells/Blimp1‐deficient Tfr cells on the B cells in germinal centres (GCs) of experimental autoimmune myasthenia gravis (EAMG) mice. Localization of GL7+ cell masses was analysed by immunohistochemistry using paraffin‐embedded lymph nodes (a) and spleen sections (b). Magnification × 100. [Colour figure can be viewed at wileyonlinelibrary.com]

Discussion

An imbalance between Tfr cells and Tfh cells has been observed in autoimmune diseases.16, 19, 20, 21, 22, 31 Decreased numbers of Tfr cells have been confirmed in EAMG and in patients with myasthenia gravis.18, 32, 33 However, little is known about how the Tfr cells function. Tfr cells express high amounts of the prototypic Tfh genes Cxcr5, Cxcl13, Icos, Bcl6 and Pdcd1.24 However, Tfr cells more closely resembled Treg cells than Tfh cells.18, 24, 26, 27, 31, 33 Blimp1 is required to control the numbers of Tfr cells in the GC.24 In our study and published data, deficiency of Blimp1 could increase the proportion of Tfr cells.24 If lack of Blimp1 results in greater suppressive ability of Tfr cells, Blimp1 will become an important target for the treatment of myasthenia gravis.

Blimp1, an essential regulator of T‐cell homeostasis and activation, controls T‐cell proliferation and apoptosis.28, 29, 30 Loss of Blimp1 led to expansion of CD4+ T cells, which were due to diminished activation‐induced cell death.28 However, the function of Blimp1 in Treg cells remains controversial.28, 30 Kallies et al. reported that Blimp1‐deficient Treg cells are present and functional in in vitro suppression assay and an in vivo model.28 Meanwhile, Martins et al. found Blimp1‐deficient Treg cells were defective in blocking the development of colitis in vivo.30 In our study, we found that Blimp1‐deficient Tfr cells are faulty in the suppression of activation and antibody production by B cells in vitro and protecting against EAMG in vivo, which was consistent with the study of Martins et al.30

It has been reported that Tfr cells from the blood are potent at inhibiting Tfh cell‐mediated production of antibody, even when relatively few Tfr cells were transferred.26, 31 Our data demonstrate that in EAMG mice, adoptive transfer of Tfr cells could regulate antibody production and GC formation by preferential expansion of negative regulatory cells (Tfr cells, Treg cells and Th2 cells) relative to inflammatory cells (Tfh cells, Th1 cells and Th17 cells) in vivo. Blimp1‐deficient Tfr cells attenuated antibody production in vivo to a lesser extent than did negative control Tfr cells. Furthermore, we explored how modulation of Tfr cells was used therapeutically to protect immunity and inhibit autoimmune disease. Mitogen‐activated protein kinases are essential in orchestrating a variety of cellular processes such as changes in cell proliferation, differentiation and survival.34 They are present and active in most cell types and respond to a range of stimuli including growth factors, cytokines and oxidative stress.35, 36, 37 We failed to find abnormal activation of the MAPKs signalling pathway in B cells of EAMG mice. The phosphoinositide 3‐kinase‐AKT pathway is an important signalling pathway that regulates the growth, proliferation and survival of lymphocytes. Abnormalities in this pathway are sufficient to alter lymphocyte homeostasis.38, 39 It has been reported that mesenchymal stem cell transplantation suppressed abnormal activation of AKT/GSK 3β signalling pathway in T cells from mice in a mouse model of systemic lupus erythematosus.40 Several recent studies showed that abnormal activation of the AKT signalling pathway was associated with the development, activation and proliferation of B cells.41, 42 In line with these reports, our results showed that transfer of Tfr cells could inactivate AKT by down‐regulating the phosphorylation of AKT in the B cells from the spleen of EAMG mice to a greater extent than Blimp1‐deficient Tfr cells in vivo. Taken together, Tfr cells are potent suppressors of immunity and are controlled by Blimp1.

Our work has identified that Blimp1 is essential for the suppression of Tfr cells in vitro and in vivo. We have also demonstrated that Tfr cells could inhibit antibody production by down‐regulating the phosphorylation of AKT in the B cells of the spleens of EAMG mice, to a greater extent than Blimp1‐deficient Tfr cells. Our data provide a basis for further work to elucidate the function of Tfr cells.

Author contributions

GY, JZ, XY and JL conceived and designed the experiments; GY, DZ, AP and BY performed the experiments; GY, LX and JH analysed the data; GL, HY, WZ and TE contributed reagents/materials/analysis tools; and GY wrote the paper. All the authors reviewed the manuscript.

Disclosures

The authors declare no competing financial interests.

Supporting information

Figure S1. Evaluation of experimental autoimmune myasthenia gravis. (a) The incidence of EAMG mouse. (b) The severity of EAMG mouse. (c) Posture of EAMG mouse with grade 2 weakness. (d) Posture of the mouse in CFA group. (e) Titers of anti‐R97‐116 lgG in EAMG group and CFA group.

Click here for additional data file. (442.3KB, jpg)

Figure S2. Purity of isolated B cells from mouse spleen cell suspension.

Click here for additional data file. (112.7KB, jpg)

 

Click here for additional data file. (309.5KB, jpg)

 

Click here for additional data file. (105.2KB, jpg)

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (No. 81271325, No 81471225).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. Evaluation of experimental autoimmune myasthenia gravis. (a) The incidence of EAMG mouse. (b) The severity of EAMG mouse. (c) Posture of EAMG mouse with grade 2 weakness. (d) Posture of the mouse in CFA group. (e) Titers of anti‐R97‐116 lgG in EAMG group and CFA group.

Click here for additional data file. (442.3KB, jpg)

Figure S2. Purity of isolated B cells from mouse spleen cell suspension.

Click here for additional data file. (112.7KB, jpg)

 

Click here for additional data file. (309.5KB, jpg)

 

Click here for additional data file. (105.2KB, jpg)

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