B lymphocyte-induced maturation protein 1 controls Th9 cell development, IL-9 production and allergic inflammation

Abstract

Background:

The transcriptional repressor Blimp-1 has a key role in terminal differentiation in various T cell subtypes. However, whether Blimp-1 regulates Th9 differentiation and its role in allergic inflammation are unknown.

Objective:

We aimed to investigate the role of Blimp-1 in Th9 differentiation and in the pathogenesis of allergic airway inflammation.

Methods:

In vitro Th9 differentiation, flow cytometry, ELISA and real-time PCR were used to investigate the effects of Blimp-1 on Th9 polarization. T cell-specific Blimp-1-deficient mice (CKO), a model of allergic airway inflammation, and T cell adoptive transfer to Rag-1^−/− mice were used to address the role of Blimp-1 in the pathogenesis of allergic inflammation.

Results:

We found that Blimp-1 regulates Th9 differentiation, as deleting Blimp-1 increased IL-9 production in CD4⁺ T cells in vitro. In addition, we showed that in CKO mice, deletion of Blimp-1 in T cells worsened airway disease, and this worsening was inhibited by the neutralization of IL-9. In asthmatic patients, CD4⁺ T cells in response to TGF-β plus IL-4 increased IL-9 expression and down-regulated Blimp-1 expression compared to those of healthy controls. Blimp-1 overexpression in human Th9 cells inhibited IL-9 expression.

Conclusion:

Blimp-1 is a pivotal negative regulator of Th9 differentiation and controls allergic inflammation.

Capsule summary

These findings reveal the role of Blimp-1 in Th9 differentiation. Blimp-1 controls Th9 cells and allergic inflammation and may have important clinical applications in therapy for allergic diseases.

Introduction

Adaptive immune responses are orchestrated by different T helper (Th) cell subsets, including Th1, Th2, Th17 and regulatory T cells (Tregs), after encountering antigens in different cytokine microenvironments. IL-9-producing Th9 cells are generated by transforming growth factor-β (TGF-β) plus IL-4 during cell stimulation^1–3. Th9 cells have been shown to play a role in allergic airway inflammation^{4, 5}, autoimmune diseases^{6, 7} and cancer^{8, 9}. Although the transcription factors PU.1¹⁰, interferon regulatory factor (IRF-4)¹¹, signal transducer and activator of transcription (STAT) 6¹² and basic leucine zipper ATF-like transcription factor (BATF)¹³ are all involved in Th9 cell differentiation, the transcriptional networks that control Th9 differentiation are not yet fully understood.

B lymphocyte-induced maturation protein 1 (Blimp-1) is a transcription factor that plays crucial roles in regulating B and T lymphocyte function^14–16. Blimp-1 regulates differentiation of cytotoxic T lymphocytes (CTLs)^{17, 18}, Th1 cells¹⁹, Th17 cells^{20, 21}, interleukin (IL)-10-expressing Tregs²², T follicular helper (Tfh) cells²³ and conventional T cells²⁴. In addition to transcription factors, the differentiation of T helper subsets involves various cytokines and costimulatory signals²⁵. TGF-β mediates the differentiation of Th17 cells and Tregs, depending on the presence or absence of IL-6, respectively²⁶. The expression of Blimp-1 in T cells can be induced by several cytokines, including IL-2, IL-12, and IL-4. Once expressed, Blimp-1 can control the expression of multiple transcription factors, including T-bet, IRF-4 and B cell lymphoma 6 (BCL-6), which are required for the functions of Th1, Treg and Tfh cells, respectively^{19, 22, 23}. Since Th9 cell differentiation requires IL-4 signaling, we investigated whether Blimp-1 plays a role in Th9 differentiation and the mechanisms that control the Th9 cell response in inflammatory conditions.

We found that Blimp-1 negatively regulates the differentiation of Th9 cells in mice and humans. Blimp-1 deletion in T cells increased the differentiation of naïve CD4⁺ T cells into Th9 cells and the severity of disease in a murine model of allergic airway inflammation. In conclusion, we propose that Blimp-1 is a suppressive transcription factor that plays a critical role in protection against allergic airway inflammation by modulating Th9 cells.

Methods

Mice.

C57BL/6 Prdm1^flox/flox and C57BL/6 CD4^Cre mice were obtained from The Jackson Laboratory. Rag-1^−/− mice, C57BL/6 Prdm1^flox/floxCD4^Cre+ (Blimp-1 deficient mice-CKO) and Prdm1^+/+CD4^Cre (WT) mice were bred in the animal facility at the University of São Paulo, Brazil, and maintained in a pathogen-free environment. All procedures were performed in the accordance with the International Guidelines for the Use of Animals and approved by the local Ethics Committee at the University of São Paulo, Brazil (123/2017).

T cell isolation and in vitro T helper differentiation.

Naïve CD4⁺ (CD25⁻CD44^low) T cells were sorted from spleen and lymph node cell suspensions using a FACSAria III (BD Biosciences). CD4⁺ T cells were stimulated with anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) for 4 days in RPMI-1640 medium supplemented with 5% FBS (Gibco), 100 U/ml penicillin/100 μg/ml streptomycin, 1 mM sodium pyruvate, nonessential amino acids, L-glutamine and 50 μM 2-mercaptoethanol. For Th1 differentiation (IL-12, 5 ng/ml; anti-IL-4, 10 μg/ml and IL-2, 25 U/ml were used and Th2 conditions (IL-4, 10 ng/ml; anti-IFN-γ, 10 μg/ml and IL-2, 25 U/ml. Th9 conditions (IL-4, 10 ng/ml; TGF-β, 3 ng/ml and anti-IFN-γμg10 μg/ml) in the presence or absence of OX86 (OX40 agonist) (30 μg/ml) for 4 days. All recombinant cytokines were obtained from RD, and neutralizing antibodies were from Bio×Cell.

Flow cytometry assay.

For intracellular cytokine staining, cells were restimulated with phorbol 12-myristate 13-acetate (PMA) (50 ng/ml), ionomycin (500 ng/ml) (Sigma-Aldrich) and brefeldin A (Biolegend) for 4 h. Then, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% saponin. The antibodies used are listed in the Supplementary material and methods. Data acquisition and analysis were performed using FACSCanto II and FlowJo software, respectively.

Quantitative real-time PCR (qPCR).

Total RNA isolation and qPCR for mRNA expression analysis were conducted as detailed in the Supplementary material and methods. The primers are listed in Supplementary Table E1. The analyses were performed using the cycle threshold (Ct) method, which allows for quantitative expression analysis using the formula 2^−ΔΔCt.

Lymphocyte proliferation and death cell assays.

Carboxyfluorescein succinimidyl ester (CFSE)-labeled purified CD4⁺CD44^loCD25⁻ cells were stimulated with 2 μg/ml anti-CD3 and 1 μg/ml anti-CD28 in Th9 differentiation conditions for 1, 3 and 5 days. The proliferation index was evaluated via CFSE dilution (Sigma), and apoptosis was assessed by staining with annexin V (BD Bioscience).

Induction of allergic airway disease.

Mice were subcutaneously sensitized and boosted using 4 μg chicken OVA/1.6 mg aluminum hydroxide in 0.2 ml PBS on days 0 and 7. Airway inflammation was induced by two intranasal challenges with 10 mg OVA on days 14 and 21. For IL-9 and IL-4 neutralization, mice were injected intranasally with anti-IL-9 Ab (10 μg/dose) (R&D Systems), anti-IL-4 Ab (10 μg/dose) (Bio×cell) or immunoglobulin (Ig)G control Ab on days 14 and 21 before the challenge. For the adoptive transfer experiment, CD4⁺ T cells from WT and CKO mice sensitized as described above were intravenously transferred into Rag-1^−/− mice (10 × 10⁶ cells/mouse). Twenty-four hours later, the recipient mice were intranasally challenged with two doses of OVA (10 mg) at 7-day intervals. Experiments were performed 24 h after the last intranasal challenge with OVA.

The trachea was cannulated, and the lungs underwent lavage with 1 ml of cold PBS to assay IL-4 and IL-5 by a sandwich ELISA kit (R&D Systems). Cell numbers in the bronchoalveolar lavage (BAL) were counted using a hemocytometer. Cells were isolated from the lungs as described in the Supplementary material. Lung tissue was embedded in paraffin and stained with hematoxylin and eosin (H&E) for the analysis of inflammatory infiltrate.

Patients.

Human peripheral blood mononuclear cells (PBMCs) were obtained from allergic asthmatic patients who were seen at the Allergy Clinic at the Clinical Hospital of Ribeirão Preto Medical School, University of São Paulo and from healthy donors. All subjects signed an informed consent form releasing the use of their specimens in the study, which was approved by the Ethics Committee of Ribeirão Preto Medical School Hospital (2261/2011).

Lentivirus production.

HEK 293T Peak cells²⁷ were transfected with 0.5 μg of pLX_TRC317 plasmid (GeneWiz; empty or coding for PRDM1 cDNA), 0.375 μg of psPAX2 (Addgene; cat. 12259) and 0.125 μg of pMD2.G (Addgene; cat. 12259) as described in the Supplementary methods.

Statistical analysis.

The statistical analysis was performed using an unpaired t-test or ANOVA followed by Bonferroni’s multiple comparison tests. The significance of these parameters was calculated using a log-rank test (5.0 GraphPad Software). All values were considered significantly different at P < 0.05.

Results

The frequency of Th9 cells increases in the periphery of Blimp-1-deficient mice

To investigate whether Blimp-1 could regulate Th9 cell function and/or differentiation, we generated T cell-specific Blimp-1-deficient mice (Prdm1^flox/floxCD4^cre - CKO) and evaluated the frequency of IL-9–producing cells and IL-9 production. First, we analyzed the total frequency of lymphocytes in the periphery by flow cytometry. We found that the frequencies of CD4⁺ and CD8⁺ T cells in CKO mice were similar to those of WT mice (Fig. 1A). Moreover, we found a substantially increased frequency of effector/memory (CD44⁺CD62L⁻) T cells (34.13 ± 1.45 versus 14.26 ± 1.42) as well as a reduction of naïve (CD62L⁺CD44⁻) T cells (51.60 ± 1.82 versus 73.23 ± 2.06) in CKO mice compared to WT mice (Fig. 1B). In addition, the T cell proliferation levels from splenocytes of both CKO and WT mice cultured with anti-CD3 plus anti-CD28 were similar (Fig. 1C). Upon splenocyte stimulation, the frequencies of CD4⁺IL-9⁺ cells were threefold higher in CKO mice (1.54 ± 0.18) than in WT mice (0.55 ± 0.04) (Fig. 1D). Moreover, IL-9 secreted in the culture supernatant was significantly higher in CKO cells (1123.40 ± 505.00) than in WT mouse cells (196.61 ± 120.80) (Fig. 1G). Moreover, the percentage of CD4⁺IL-4⁺ cells was comparable in cells from WT and CKO mice (Fig. 1E). As expected from previously published work²², we also found that the frequency of Tregs (CD4⁺Foxp3⁺) was higher in the spleens of CKO mice (18.65 ± 4.00) compared to WT mice (11.35 ± 4.00) (Fig. 1F). Together, these data suggest that Blimp-1 deletion in T cells favors the Th9 phenotype in vivo.

Fig. 1. Loss of Blimp-1 in T cells results in the accumulation of Th9.

Splenocytes from Blimp-1-deficient mice (CKO) and WT mice stained for (A) CD4 and CD8 gated on CD3 and (B) CD44 and CD62L gated on CD4⁺CD3⁺ and analyzed by flow cytometry. Numbers in the quadrants indicate the percentages of naïve (CD4⁺CD62L⁺) and effector/memory (CD4⁺CD44⁺) T cells. (C) The proliferation of CFSE-labeled splenocytes from CKO and WT mice activated with anti-CD3 and anti-CD28 for 3 days, presented as CFSE dilution. Splenocytes from CKO and WT were stimulated ex vivo with PMA + ionomycin for 4 h. Percentages of (D) IL-9-, (E) IL-4- and (F) Foxp3- expressing-CD4⁺ T cells were evaluated by flow cytometry and based on the control isotype (see Fig. E1A). (G) Splenocytes from CKO and WT were stimulated ex vivo with anti-CD3 and anti-CD28 for 72 h and analyzed for IL-9 in the supernatant by ELISA. Data (mean ± SD) are representative of two experiments. * P < 0.05.

Blimp-1 is a negative regulator of Th9 differentiation and IL-9 production

To further elucidate whether Blimp-1 is involved in Th9 cell differentiation, we cultured naïve CD4⁺ T cells from CKO and WT mice under Th9 conditions for 4 days. The frequency of IL-9-producing CD4⁺ T cells (Fig. 2A) generated from CKO mice was higher than that in WT mice (Fig. 2A) and was correlated with enhanced IL-9 mRNA expression (Fig. 2B) and with the levels of IL-9 secreted into the culture supernatants (Fig. 2C). The addition of an OX40 agonist (OX86 antibody), which has been previously shown to potentiate Th9 differentiation²⁸, led to the increased expression of IL-9, mainly in the absence of Blimp-1 (Figure 2B–C). However, Blimp-1 deletion in CD4⁺ T cells did not alter the proliferation (Fig. 2D) and survival (Fig. 2E) of Th9 cells during activation with anti-CD3 and anti-CD28. Corroborating these data, sort-purified CD44^highCD25⁻CD4⁺ cells from pooled splenocytes of CKO mice exhibited 72% more IL-9⁺ cells compared to controls (Fig. 2F). These data suggest that Blimp-1 represses the Th9 differentiation program and could be required to control the differentiation of Th9 cells in tissues.

Fig. 2. Blimp-1 deficiency enhances Th9 cell differentiation.

(A) Naïve CD4⁺ T cells from WT and CKO mice were differentiated into Th9 cells with TGF-β and IL-4 plus anti-IFN-γ (Th9) without or with the agonist OX40 (OX86) (Th9 + OX86). On day 4, the differentiated cells were stimulated with PMA + ionomycin for 4 h. The frequency of CD4⁺IL-9⁺ cells was determined by flow cytometry based on the control isotype (see Fig. E1B). (B) Expression of IL-9 mRNA in CD4⁺T cells from WT mice and CKO mice cultured for 4 days under neutral (Th0) or Th9-polarizing conditions, as described above. The results are presented relative to those of the control gene GAPDH. (C) Levels of IL-9 in culture supernatants from Th0- and Th9-polarizing conditions were measured by ELISA. (D) Proliferation and (E) survival of WT and CKO CD4⁺ T cells activated with anti-CD3 and anti-CD28 in Th9-polarizing conditions for 1, 3 and 5 days, as assessed by CFSE dilution and annexin V staining. Numbers above bracketed lines indicate percent cells that proliferated (top) or annexin V-positive cells (bottom). (F) Flow cytometry analysis of intracellular IL-9 in (CD4⁺CD44^high) effector/memory cells cultured with anti-CD3 and anti-CD28 for 18 h and stimulated with PMA and ionomycin for 4 h was determined based on the control isotype (see Fig. E1C). Data (mean ± SD) are representative of three (A-C) or two (D-F) experiments. * P < 0.05.

Blimp-1 expression is not induced in Th9 cells

We then investigated whether Blimp-1 expression is modulated during Th9 differentiation. Sorted naïve (CD25⁻CD44^low) CD4⁺ T cells from WT mice were stimulated under neutral, Th1, Th2, and Th9 conditions, and Blimp-1 mRNA expression was determined by qPCR. As expected, Th9 cells showed higher IL-9 mRNA expression than did Th1 and Th2 cells (Fig. 3A). As previously shown^19,29, the stimulation of naïve CD4⁺ T cells with anti-CD3 plus anti-CD28 induced low Blimp-1 expression, while cells cultured under Th1 and Th2 conditions expressed higher levels of Blimp-1 than did those in neutral conditions. In contrast, cells stimulated under Th9 conditions exhibited low Blimp-1 expression (Fig. 3B). We next investigated the factors involved in the regulation of Blimp-1 expression in Th9 cells. Wild-type naïve CD4⁺ T cells cultured with IL-12 or IL-4 showed high Blimp-1 mRNA expression (Fig. 3C). The addition of OX86 and TGF-β alone did not induce Blimp-1 expression, even in the presence of IL-4 (Fig. 3C). As expected, the expression of IL-9 mRNA and the production of IL-9 were very low in naïve CD4⁺ T cells cultured with IL-4, TGF-β and OX86 alone (Fig. 3D and E). However, the combination of IL-4 and TGF-β or OX86 and TGF-β induced significant IL-9 mRNA expression and IL-9 production but did not induce Blimp-1 expression. Moreover, the combination of IL-4 and TGF-β and OX86 potentiated IL-9 mRNA expression and IL-9 secretion into culture supernatants (Fig. 3D and E). To determine whether Blimp-1 expression was induced or inhibited in Th9 cells, we cultured CD4⁺ T cells with IL-2 and IL-12 for 3 days to induce Blimp-1 expression (Fig. 3F) and added IL-4+TGF-β+OX86 (Th9 condition) either at the same time (Th1 + Th9, IL-12+IL-2+IL-4+TGF-β+OX86) or after 3 days under Th1 conditions (Th1–3d/Th9). Under Th9 conditions, Blimp-1 expression was significantly reduced after 3 days of Th1 differentiation (Th1–3d/Th9) (72% inhibition, but was not induced when Th1 and Th9 conditions were imposed together (Th1 + Th9 condition) (Fig. 3F). Consistently, CD4⁺ T cells cultured under Th1 + Th9 conditions exhibited IL-9 production as high as that of Th9 cells, but those cultured under Th1–3d/Th9 conditions did not (Fig. 6G). Together, these results indicate that Blimp-1 not is induced during Th9 differentiation and that the inhibition of Blimp-1 by cytokines related to Th9 differentiation is essential for high IL-9 production.

Fig. 3. Blimp-1 expression is not induced during Th9-differentiation.

(A-B) Expression of IL-9 and Blimp-1 mRNA in naïve CD4⁺T cells from WT mice and stimulated with anti-CD3 and anti-CD28 under neutral (medium alone), Th1 (IL-12 and IL-2 plus anti-IL-4), Th2 (IL-4 and IL-2 plus anti-IFN-γ), Th9 (TGF-β and IL-4 plus anti-IFN-γ) and Th9 plus OX86 (TGF-β, IL-4, OX86 and anti-IFN-γ). The expression levels of the indicated genes were analyzed by qPCR. (C-E) Naïve CD4⁺ T cells from WT mice were cultured in different conditions: stimulation of TCR alone or in the presence of IL-4, TGF-β and OX86 or in combination as shown in Fig. 3E. On day 4, the RNA and culture supernatant were collected, the expression levels of (C) Blimp-1 and (D) IL-9 mRNA were analyzed by qPCR, and (E) the IL-9 levels were measured by ELISA. (F-G) CD4⁺ T cells were cultured with IL-12+IL-2 (Th1 conditions) for 3 days. In some wells, IL-4+TGF-β+OX86 (Th9 condition) was added along with IL-2+IL-12 (Th1+Th9) on the same day or after 3 days (Th1-3d/Th9) under Th1 conditions. Th1 or Th9 differentiated cells were used as positive or negative controls. On day 6, the RNA and culture supernatants were collected, the expression of (F) Blimp-1 mRNA was analyzed by qPCR, and (G) the IL-9 levels were measured by ELISA. Data (mean ± SD) are representative of three experiments. * P < 0.05; ^& P < 0.05 and ^# P < 0.05.

Fig. 6. Blimp-1 deletion in CD4⁺ T cells favors the proallergic Th9 phenotype.

WT and CKO mice were treated with IgG control, neutralizing anti-IL-9 or anti-IL-4 Abs and challenged with OVA, as outlined in the Methods. (A) Representative lung sections of WT (top panels) and CKO (bottom panels) mice were stained with H&E for analysis of inflammatory infiltrates. Original magnification × 200. The absolute number of (B) total leukocytes and (C) eosinophils (Siglec-F⁺GR-1⁻) gated on CD11b⁺MHCII⁻ and isolated from lung was determined by FACS. Expression of (D) IL-4 and (E) IL-5 mRNA of the lung tissues of WT and CKO mice was performed using qPCR. The concentrations of (F) IL-4 and (G) IL-5 of the BAL fluid from WT and CKO mice were determined by ELISA. Data are representative of the four mice per group. *P < 0.05.

Absence of Blimp-1 in T cells potentiates allergic airway inflammation

To determine whether the increase in Th9 cells caused by Blimp-1 deficiency affects the development of Th9-dependent inflammatory diseases, we investigated whether deletion of Blimp-1 in T cells aggravates allergic airway inflammation. We subjected WT and CKO mice to the standard protocol to develop ovalbumin (OVA)-induced allergic airway inflammation and then evaluated cell influx to the lungs. Compared with WT mice, the CKO mice exhibited an intense influx of eosinophils (Siglec-F⁺GR-1⁻) into airways (Fig. 4A). FACS analysis showed that the frequency and total number of leukocytes, more specifically eosinophils (Siglec-F⁺GR-1⁻), in lung tissues were significantly higher in CKO mice (2.47 ± 0.74) than in WT mice (0.85 ± 0.49) (Fig. 4B–D). Histological analysis confirmed that CKO mice had more intense lung parenchymal inflammation, characterized by diffuse cell infiltrates, than WT mice (Fig. 4E–F). Consistently, the number of CD4⁺IL-9⁺ T cells infiltrating the lungs was higher in CKO mice (10.62 ± 4.26) than in WT mice (3.25 ± 0.79) (Fig. 4G). In addition, together with the enhanced number of eosinophils, the number of lung CD4⁺ T cells producing IL-5 and IL-4 was higher in allergic CKO mice (22.61 ± 10.68; 19.00 ± 6.95, respectively) than in allergic WT mice (6.08 ± 2.09; 3.61 ± 1.73, respectively) (Fig. 4H and I). Immunization alone or challenge with OVA did not induce inflammatory infiltrate in the lungs of the WT and CKO mice (Fig. E2A–C). However, only challenge with OVA induces an increase in IL-9-producing CD4⁺ cells in the lungs of the CKO mice compared with the WT mice (Fig. E2D).

Fig. 4. Blimp-1 deficiency in T cells promotes the development of allergic inflammation.

(A) Differential counts of cytospin preparations from BAL cells of WT and CKO mice, 24 h after the last challenge with intranasal OVA, as outlined in the Methods. (B-D) Frequency and absolute numbers of eosinophils (Siglec-F⁺GR-1⁻) gated on CD11b⁺MHCII⁻ in the lungs of WT and CKO mice were determined by flow cytometry. (E-F) Representative lung sections of (E) WT and (F) CKO were stained with hematoxylin and eosin for analysis of inflammatory infiltrates. Original magnification × 200. Cells isolated from lungs were stimulated with PMA and ionomycin for 4 h for intracellular staining by FACS. The frequency and absolute number of CD4⁺ cells expressing (G) IL-9, (H) IL-5 and (I) IL-4, gated on CD3, were determined based on the control isotype (see Fig. E1D). Data (mean ± SD) are representative of three experiments with five experiments per group. * P < 0.05.

To determine whether Blimp-1 deficiency in CD4⁺ T cells alone is sufficient to aggravate OVA-induced airway inflammation in a mouse model and whether Blimp-1 has an intrinsic role in controlling Th9-mediated inflammation in vivo, we sorted CD4⁺ T cells from CKO and WT mice and adoptively transferred them to Rag-1^−/− mice. Then, we evaluated the severity of allergic airway inflammation in the recipient mice after challenge with OVA. The histological analysis showed increased cell infiltration in the lungs from CKO mice compared to WT mice (Fig. 5A–B). We also detected higher numbers of eosinophils and CD4⁺IL-9⁺ T cells infiltrating into the lungs of Rag1^−/− mice that received CD4⁺ cells from CKO mice than from WT mice (Fig. 5C–D). The enhancement of CD4⁺IL-9⁺ T cells was accompanied by an increase in CD4⁺IL-5⁺ T cells (Fig. 5E) but not CD4⁺IL-4⁺ T cells (Fig. 5D). Therefore, Blimp-1 deletion in T cells is sufficient to increase allergic lung inflammation, which is mainly mediated by IL-9, although cytokines of the Th2 profile, such as IL-5, are also involved in the pathogenesis of allergic airway inflammation. Thus, the primary determinant of disease severity in Blimp-1-deficient mice is T cell intrinsic.

Fig. 5. T cell-intrinsic Blimp-1 deficiency in airway inflammation promotes Th9 cell accumulation.

CD4⁺ T cells from WT and CKO mice were adoptively transferred into Rag1-deficient recipients. Recipients were challenged intranasally with OVA, as described in the Methods, before analysis of pulmonary inflammation. (A-B) Representative lung sections of Rag-1^−/− recipient mice that received (A) WT and (B) CKO CD4⁺ cells were stained with hematoxylin and eosin for analysis of inflammatory infiltrates. Original magnification × 200. (C) Absolute numbers of eosinophils (Siglec-F⁺GR-1⁻), gated on CD11b⁺MHCII⁻, in the lungs of WT and CKO mice were determined by flow cytometry. (D-F) Cells isolated from the lungs were stimulated with PMA and ionomycin for 4 h for intracellular staining by FACS. The absolute numbers of (D) IL-9-, (E) IL-4- and (F) IL-5-expressing CD4⁺ cells gated on CD3 were determined. Data (mean ± SD) are representative of the four mice per group. * P < 0.05.

Blimp-1 regulates Th9 cell pathogenicity in allergic inflammation

We next investigated whether the severity of allergic inflammation in Blimp-1-deficient mice was mediated by IL-9. For this analysis, CKO and WT mice underwent induction of OVA-induced allergic airway inflammation and were treated with anti-IL-9 or anti-IL-4 Ab (10 μg/mouse) intranasally on days 1 and 2 of the challenge. Twenty-four hours after the last challenge, compared with mice treated with control IgG, the CKO mice treated with the anti-IL-9 Ab showed attenuated lung inflammation with decreased peribronchial and perivascular accumulation of leukocytes. In contrast, treatment with anti-IL-4 did not reduce airway inflammation in either experimental group (Fig. 6A). Flow cytometry analysis showed that treatment with the anti-IL-9 Ab reduced the numbers of leukocytes and eosinophils in the lungs (Fig. 6B and C). Moreover, IL-9 blockade was associated with a significant reduction in IL-4 and IL-5 expression in the pulmonary tissue (Fig. 6D and E) and in the BAL fluid (Fig. 6G and G) in CKO mice compared with those of control CKO mice. Taken together, a lack of Blimp-1 in T cells leads to the IL-9-mediated exacerbation of airway inflammation, indicating that Blimp-1 plays a nonredundant role in controlling Th9 responses in vivo.

Blimp-1 controls IL-9 production in human Th9 cells

Finally, we investigated whether Blimp-1 also acts as a repressor of Th9 differentiation in humans. We isolated CD4⁺ cells from the PBMCs of healthy donors and asthmatic patients and stimulated them with anti-CD3 plus anti-CD28 and Th9-polarizing cytokines. Similar to the results obtained with murine cells, TGF-β plus IL-4 induced IL-9 mRNA expression in CD4⁺ T cells from healthy donors and asthmatic patients (Fig. 7A). However, IL-9 mRNA expression was significantly higher in CD4⁺ T cells from asthmatic patients than in those from healthy donors (Fig. 7A). Consistent with these findings, Blimp-1 mRNA expression was not induced in human CD4⁺ T cells in response to TGF-β plus IL-4 (Th9) compared with that in neutral conditions (Th0) (Fig. 7B). These results clearly indicate that in both humans and mice, Th9 cells express low levels of Blimp-1. To directly test whether Blimp-1 plays a negative role in human Th9 cell differentiation, we isolated CD4⁺ T cells from healthy donors and asthmatic patients, stimulated them under Th9-polarizing conditions and transduced them with control (control-LV) or Blimp-1-expressing (Blimp-1-LV) lentivirus, as shown Fig. 7C. Th9 cells differentiated in vitro and overexpressing Blimp-1 showed lower IL-9 mRNA expression and IL-9 production than did control-LV-transfected Th9 cells (Fig. 7D–E). Thus, similar to the observations in murine cells, Blimp-1 repressed IL-9 expression in human CD4⁺ T cells.

Fig. 7. Blimp-1 inhibits differentiation of human Th9 cells.

(A) CD4⁺ T cells isolated from PBMCs of healthy donors and allergic asthmatic patients were differentiated under Th0- and Th9-polarizing conditions. After 4 days of culture, RNA was isolated, and (A) IL-9 and (B) Blimp-1 mRNA levels were determined using qPCR. Data (mean ± SD) are representative of six allergic asthmatic patients and the healthy donor. (C) Immunoblot analysis of Blimp-1 and β-actin in Th9 cells transduced with control lentivirus (Ctrl-LV) or Blimp-1-expressing lentivirus (Blimp-1-LV); the cells were from healthy donors. (D) IL-9 mRNA expression and (E) IL-9 levels in the culture supernatant of Th9 cells transduced with Ctrl-LV or Blimp-1-LV; the cells were from healthy donors (C1–4) or asthmatic patients (P1–4), and IL-9 mRNA expression and levels were determined by qPCR and ELISA, respectively. Data are representative of four asthmatic patients and four healthy donors.

Discussion

IL-9-producing Th cells, or Th9 cells, are a recently described CD4⁺ T cell subset that plays important roles in airway inflammation. The transcription factors STAT6, STAT5, BATF, PU.1 and IRF-4 have been implicated as positive regulators of Th9 differentiation³⁰. However, little is known about the negative regulators of Th9 programming. Our studies described here support the idea that the transcription factor Blimp-1 functions as a negative regulator of Th9 differentiation in vivo and that its expression in T cells is required to suppress the pathogenesis of asthma.

Our results showed that Blimp-1 deficiency in T cells promotes increased Th9 differentiation and accumulation in the periphery. The increase in Th9 differentiation from Blimp-1-deficient CD4⁺ T cells is unlikely to be secondary to Blimp-1’s previously described role in preventing T cell proliferation and survival^{14, 15}, as the proliferation and survival of Blimp-1–deficient and sufficient Th9 cells were comparable. The deletion of Blimp-1 in T cells in vivo favored the differentiation/accumulation of Th9 cells but not Th2 cells. Moreover, the forced expression of Blimp-1 in human Th9 cells was sufficient to repress IL-9 expression, further supporting the idea that Blimp-1 is a repressor of the Th9 differentiation program.

Blimp-1 expression is consistently higher in antigen-experienced Th1¹⁹ and Th2 cells³¹ than in those in neutral conditions. However, in Th9 cells, Blimp-1 mRNA expression remained low as cells differentiated. The impaired expression of Blimp-1 in Th9 cells is probably due to the presence of TGF-β, as TGF-β was previously shown to be a potent repressor of Blimp-1 expression in Th17 cells²⁰, and we further observed here that in the presence of TGF-β, Blimp-1 expression was consistently low, even in the presence of an OX40 agonist and IL-4. Furthermore, the previous induction of Blimp-1 in CD4⁺ cells, even under Th9-polarizing conditions, inhibits its expression and IL-9 production. Thus, the ideal conditions for high IL-9 expression do not favor Blimp-1 expression.

The mechanisms underlying the repression of IL-9 production/Th9 differentiation by Blimp-1 require further elucidation. Our observation that Blimp-1–deficient CD4⁺ T cells had increased IL-9 mRNA expression suggested that Blimp-1 could regulate IL-9 production at the transcriptional level. This idea is further supported by our observation that the forced expression of Blimp-1 in human CD4⁺ T cells resulted in the repression of IL-9 mRNA levels.

Our results also support the idea that the repression of IL-9 expression by Blimp-1 in T cells is required to prevent severe airway inflammation. Although Th9 cells have been reported to be important in allergic inflammation, autoimmune diseases and tumor immunity³⁰, the detection of Th9 cells is difficult, as the generation of these cells is transient.

The allergic airway inflammation model is typical and characterized by lung eosinophilia that are dependent on the excess activation of T cells, mucus production, and airway hyperreactivity³². However, we demonstrated that a lack of Blimp-1 in T cells promotes severe airway inflammation, including intense lung parenchymal inflammation, enhanced eosinophil recruitment and Th2 and Th9 cell responses. The increase in the number of Th2 and Th9 cells in the lungs can be explained by the need for cooperation of both helper T subtypes for the development of airway inflammation. The division of labor between these helper T subsets in the airway inflammation is still unclear. Our observations suggested the importance of Th9 cells in lung tissues for the induction of Th2 responses and severity of airway inflammatory disease.

The role of IL-9 in allergic asthma is currently recognized. IL-9 expression is increased in the lungs of asthmatic patients^{33, 34}, and transgenic expression of IL-9 results in allergic inflammation³⁵. Our results showed that blockade of IL-9 inhibited the development of the inflammatory profile of Th2 cells and consequently eosinophilia. However, blocking IL-4 did not affect the response of the Th9 cells and pulmonary inflammation. Interestingly, IL-9 induced during allergic inflammation is the initial trigger for the development of an efficient Th2 response. Previous experiments have suggested that IL-9 promotes type 2 cytokine production by innate lymphoid cells in the lungs³⁶, and IL-13-deficiency inhibits the allergic inflammation initiated by an IL-9 transgene³⁷.

In addition, allergic airway inflammation is characterized by eosinophilia that are dependent on IL-9 production. IL-9 blockade in allergic CKO mice reduces eosinophilia but also impairs IL-5 production. Thus, it remains to be determined if IL-9 has a direct effect on eosinophils. Previous reports showed that IL-9 increased the expression of IL-5R on eosinophils and inhibited eosinophil apoptosis, enhancing eosinophil development and promoting eosinophil maturation in synergism with IL-5^{38, 39}. The expression of IL-9 in transgenic mice under the control of a lung-specific promoter resulted in severe airway inflammation with eosinophils and lymphocytes as well as mast cell hyperplasia⁴⁰. Thus, it is difficult to develop an ordered model of Th9 and Th2 cell functions, and each subset likely contributes to the development of allergic inflammation. Our observations further confirm the importance of Th9 cells in the lung tissue for the induction of Th2 responses and the severity of airway inflammatory disease.

Atopic diseases, including atopic dermatitis and asthma, are most commonly associated with Th2 cytokine responses³⁰. However, the adoptive transfer of Th9 cells leads to an increase in eosinophil recruitment after OVA challenge and favors allergic airway disease⁴¹. Recent reports have identified Th9 cells as major contributors to atopic diseases in humans⁴². The adoptive transfer model in Rag1-KO mice, which received Blimp-1-deficient CD4⁺ cells, aggravated the pathogenesis of allergic airway inflammation, including increases in eosinophils and Th9 cells, suggesting that IL-9 produced by Th9 cells is essential for the development of disease independent of IL-4, although Th2 cells is also in the development of airway inflammation.

In addition, our data showed that Th9 cells from allergic asthma patients that were differentiated in vitro induce higher IL-9 expression than do those from healthy donors. In addition, similar to the observations in murine cells, Blimp-1 expression was very low in in vitro-differentiated human Th9 cells. Additionally, the overexpression of Blimp-1 in CD4⁺ T cells from healthy donors and asthmatic patients inhibited IL-9 production during Th9 differentiation, suggesting that Blimp-1 could play a role in regulating asthma-associated pathogenesis in humans, similar to the observations in our murine experimental model. Overall, the results we describe here uncover a new role for Blimp-1 in repressing Th9 differentiation and thus controlling allergic airway inflammation. Collectively, these findings may have important implications for the development of new therapeutic approaches to control allergic airway inflammation.

Key Messages.

1. Blimp-1 regulates Th9 differentiation and IL-9 production.

2. Blimp-1-deficient mice show severe IL-9-mediated allergic airway inflammation.

3. Blimp-1 overexpression in human Th9 cells represses IL-9 expression.

Abbreviations:

Ab

Antibody

BAL

Bronchoalveolar lavage

BATF

Basic leucine zipper ATF-like transcription factor

BCL-6

B cell lymphoma 6 protein

Blimp-1

B lymphocyte-induced maturation protein 1

CFSE

Carboxyfluorescein succinimidyl ester

CKO

T cell-specific Blimp-1-deficient mice

CTLs

Cytotoxic T lymphocytes

FOXP3

Forkhead box protein 3

H&E

Hematoxylin and eosin

Ig

Immunoglobulin

IL

Interleukin

IRF-4

Interferon regulatory factor

LV

Lentivirus

OVA

Ovalbumin

OX86

OX40 agonist

PBMC

Peripheral blood mononuclear cell

PBS

Phosphate-buffered saline

PMA

Phorbol 12-myristate 13-acetate

qPCR

Quantitative real-time PCR

Rag-1

Recombination-activating gene 1

STAT

Signal transducer and activator of transcription

T-bet

T-box-containing protein

TGF-β

Transforming growth factor beta

Th

T helper

Thf

T follicular helper

Treg

Regulatory T cell