Type 2 helper T cells convert into Interleukin-13-expressing follicular helper T cells after antigen repriming

Abstract

Anaphylaxis caused by allergen sensitization and vaccination is a serious health concern. The severity of anaphylaxis is associated with the presence of immunoglobulin E (IgE) antibodies that bind to allergens with a high affinity. Here, we report the development of a unique Interleukin-13 (IL-13)-producing follicular helper T (T_FH) cell subset, designated T_FH2 cells, which is tightly associated with the production of high-affinity IgE antibodies. T_FH2 cells had a transcriptionally hybrid phenotype between T_H2 and T_FH cells, which express GATA-binding protein 3 (Gata3) and B-cell/CLL lymphoma 6 (BCL-6), respectively. Adaptive transfer experiments demonstrated that type 2 helper T (T_H2) cells were capable of differentiating into T_FH2 cells upon secondary antigen stimulation. The type 2 T_FH (T_FH2) conversion process was entirely attenuated in T cell-specific deficient mice of Bcl6 (Bcl6^f/f cd4-cre; Bcl6^ΔT). Moreover, the transfer of IL-13 defective T_H2 cells partially inhibited IgE responses and significantly decreased the high-affinity IgE, even though T_FH development was intact. Therefore, IL-13 from T_FH2 cells controlled the selective enrichment of IgE⁺ B-cells with a high affinity for allergens. A previously undefined unique T_FH subset, T_FH2, likely contributes to IgE responses. This suggests that the accumulation of antigen-specific T_H2 cells in human patients with allergies is a high-risk factor for IgE-dependent allergic diseases.

Highlights

• T_FH2 cells, a unique T_FH subset, are a transcriptional hybrid phenotype of T_H2 and T_FH cells.

• T_FH2 cells differentiate from T_H2 cells in response to allergens.

• T_FH2 cells contribute to the production of pathogenic IgE antibodies by expressing high IL-13 levels.

• The plasticity of memory T_H2 cells contributes to the production of pathogenic IgE antibodies in patients with allergies.

Introduction

Anaphylaxis is a life-threatening allergic reaction caused by the degranulation of mast cells through immunoglobulin E (IgE)–Fc epsilon receptor (FcεR) crosslinking following allergen binding [1]. Food-mediated anaphylaxis has become a serious health concern, with an increasing number of deaths in children and adults. Insect sting anaphylaxis, including venom allergy, is a common potentially life-threatening condition. Studies of patients with food allergies and murine models indicate that IgE antibodies that bind with allergens with high affinity induce mast cell degranulation associated with anaphylaxis [1, 2]. IgE is the least abundant serum antibody, with the shortest serum half-life among all antibody isotypes, 2–3 days versus approximately 23 days for immunoglobulin G (IgG) [3, 4]. Soluble IgE binds to the high-affinity FcεRI expressed on tissue mast cells and circulating basophils [5,6,7,8,9], at which point its half-life becomes considerably longer, perhaps months [10, 11]. Mast cells and basophils are activated after the crosslinking of allergen-specific FcεRI-bound IgE, causing degranulation that releases various inflammatory chemical mediators, such as cytokines and chemokines, which are closely associated with anaphylaxis and food allergies.

Interleukin-4 (IL-4) is a key cytokine that controls the production of IgG1 and IgE antibodies in mice and IgG4 and IgE in humans [12,13,14]. IL-4 is secreted from two distinct T_H subsets, T_H2 and T_FH cells. T_H2 cells highly express GATA-binding protein 3 (GATA3), a master transcriptional regulator that controls the expression of type 2 cytokines IL-4, IL-5, and IL-13 [15,16,17], whereas the development of T_FH cells is strictly controlled by a basic zipper type transcriptional repressor, B-cell/CLL lymphoma 6 (BCL-6) [18]. T_FH cells, rather than T_H2 cells, are widely accepted to constitute a significant source of IL-4 responsible for the IgE responses in the germinal center (GC) [19,20,21,22]. Our team and other researchers have found that a specific enhancer controls IL-4 expression in T_FH cells, that is, consensus noncoding sequence-2 (CNS-2) [19, 20]. T_FH cells also secrete IL-21, a negative regulator of IgE production, by inhibiting IL-4-induced germline Cε transcription in B-cells [23, 24]. A previous study has described a rare population of T_FH cells in Dock8-deficient and normal mice, which the authors designated as T_FH13 cells, which expressed IL-13 and GATA3 [25]. IL-13 secreted from T_FH13 cells was reported to contribute to the generation of high-affinity IgE via sequential switching of high-affinity IgG1⁺ cells. However, conventional GC-T_FH cells do not express IL-13 or GATA-3, indicating that this unique T_FH subset uses a differentiation pathway distinct from that of conventional T_FH cells. Therefore, generation of IL-13-expressing T_FH cells remains unclear.

Previous studies have reported that T_FH cells differentiate from T_H2 cells in response to schistosome eggs and that these T_FH cells express IL-5 and IL-13 [26]. Similarly, human circulating T_FH2 (hcT_FH2) cells and murine T_FH cells induced by house dust mite allergens express IL-13 and GATA3 [25, 27,28,29]. Some studies have also indicated that T_H2 and T_FH cells could be interchangeable during helminth type 2 responses [26, 30]. Therefore, these observations indicate the development of IL-13-expressing T_FH cells; however, the developmental cues for T_FH13 and other IL-13-expressing T_FH cells remain elusive.

In the present study, T_H2-derived T_FH cells were discovered to have characteristics of both T_FH and T_H2 cells and express the T_FH markers C-X-C chemokine receptor type 5 (CXCR5) and BCL-6, and the T_H2 markers GATA3 and IL-13. Adaptive transfer experiments indicated that T_H2 cells acquired several T_FH characteristics after secondary antigen priming and migrated into B-cell follicles. Characteristic conversion of T_H2 cells into T_FH2 cells is associated with severe anaphylaxis, which largely depends on high-affinity anaphylactic IgE. Here, the developmental cue for T_FH2 and the functional relationship between T_H2 and T_FH2 in IgE responses and allergic symptoms are discussed.

Materials and Methods

Mice

The mouse strains used in these experiments included C57BL/6J, ovalbumin (OVA)-specific T cell receptor (TCR) transgenic (OT-II) mice, IL-4-human CD2 reporter Bac transgenic (OTII-IL-4/hCD2 Tg) mice, Bcl6^floxCD4cre, a diphtheria toxin (DT)-based conditional deletion mice for Basophile (Bas-TRECK) and mast cells (Mas-TRECK) as described in previous reports [18, 19, 31, 32], and Il4^−/−, IL-13-tomato mice, which were kindly provided by G. Köhler and A. Mackenzie (MRC Laboratory of Molecular Biology), respectively [5, 33]. Cd28^−/− are described elsewhere [34]. All mice were maintained on a C57BL/6J background in our specific-pathogen-free facility, following institutional guidelines and protocols approved by the Animal Studies Committee at the Tokyo University of Science. All animals were maintained on 12-hr light cycles and housed at 70°F and 50% humidity. Experiments were performed with mice 6–12 weeks of age, with sex-matched littermates, whenever possible.

RNA-sequencing analysis (RNA-seq) and quantitative PCR (qPCR)

Total RNA was isolated using a kit (Zymo, Freiburg, Germany) and cDNA libraries were prepared using the NEBNext Ultra RNA Library Prep Kit (NEB, Ipswich, MA) for RNA-seq analysis or the QuantSeq 3′mRNA-Seq Library Prep Kit (Lexogen, Vienna, Austria) for QuantSeq 3′mRNA-Seq analysis [35]. Sequencing was performed using the HiSeq 1500 system (Illumina, San Diego, CA). Sequenced reads were mapped to the whole mouse genome using the annotation file of mouse mm9 acquired from the UCSC Table Browser [36]. Normalization of the RNA-seq data was performed by calculating the transcripts per million (TPM). Heatmaps were drawn for the Z-scores of the signature genes for different cell types. The intersecting genes were extracted from the Venn diagram, and common biological processes among the gene clusters were extracted by gene ontology (GO) enrichment analysis using the R package clusterProfiler and over-representation test [37, 38]. Principal component analysis (PCA) was performed based on TPM normalized gene expression.

DESeq2 (version 3.9) was used for the Venn diagram to extract differentially expressed genes. The data were filtered (p-adjusted <0.1, and log2 fold change >4) and subsequently, depending on the expression level compared to naïve T cells, the up- and downregulated genes were listed. Venn diagrams were drawn according to these listed genes using the R package gplots (version 3.0.1.14), to derive the gene expression intersection between the samples.

qRT-PCR was performed on a 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). Primers used were as follows: Il4: 5′-cttatcgatgaatccaggcatcg-3′ (forward) and 5′-catcggcattttgaacgaggtca-3′ (reverse), Il13: 5′-ggcccccactacggtctcca-3′ (forward) and 5′-gaaggggccgtggcgaaaca-3′ (reverse), Il21: 5′-gctccacaagatgtaaaggggc-3′ (forward) and 5′-ccacgaggtcaatgatgaatgtc-3′ (reverse), Bcl6: 5′-cctgtgaaatctgtggcactcg-3′ (forward) and 5′-cgcagttggcttttgtgacg-3′ (reverse), Gata3: 5′-agaaccggccccttatcaa-3′ (forward) and 5′-agttcgcgcaggatgtcc-3′ (reverse), Prdm1: 5′-acatagtgaacgaccacccctg-3′ (forward) and 5′-cttaccacgccaataacctctttg-3′ (reverse), and β-actin: 5′-actattggcaacgagcggttc-3′ (forward) and 5′-ggatgccacaggattccatac-3′ (reverse). The mRNA value of each gene was calculated based on normalization with β-actin mRNA levels.

Preparation of T_H2 cells

CD4 T cells were isolated from the spleens of OT-II mice using a magnetic cell separation system, Mojosort Mouse CD4 T Cell Isolation Kit (BioLegend, San Diego, CA, USA). OTII CD4 T cells (1 × 10⁶/mL) and irradiated B6 splenic cells were cultured with the OVA peptide (323–339; 1 µM) and IL-2 (30 U/mL) for 48 hr. After two days, the cells were cultured in the presence of recombinant mouse IL-4 (rmIL-4) (10 ng/mL; PeproTech, Rocky Hill, NJ, USA) and anti-IFN-γ (10 ng/mL; R4-6A2, BD, San Jose, CA, USA) for T_H2 differentiation. After six days, T_H2 cells were sorted using a cell sorter, Moflo XDP (Beckman Coulter, Brea, CA, USA), and transferred into mice.

Active cutaneous anaphylaxis (ACA)

T_H2 cells were derived from OT-II mice (OTII-T_H2 cells) and transferred into mice, which were then immunized with OVA (100 µg, grade V; Sigma, St. Louis, MO) or 11 of 4-hydroxy-3-nitrophenyl acetyl (NP)-conjugated OVA (NP11-OVA) (100 µg) plus alum adjuvant (FUJIFILM Wako Pure Chemical Co, Tokyo, Japan) through intraperitoneal (i.p.) injection. One month later, the right ears of the mice were sensitized with OVA (100 µg) in PBS through subcutaneous (s.c.) injection. Simultaneously, the mice were injected with 0.5% Evans blue dye (FUJIFILM Wako Pure Chemical Co.). The rectal temperature was measured every 5 min. After 20 min, the ears were corrected and extracted by immersion in formamide solution, and the dye concentrations were measured using an iMark (BIO-RAD, Hercules, CA, USA) at OD 600 nm. Serum histamine concentrations were measured using a Histamine ELISA (RE59221, IBL International, Hamburg, Germany) 20 min after sensitization.

Deletion of mast cells and basophils

To eliminate mast cells, Mas-TRECK mice were injected intraperitoneally with 250 ng diphtheria toxin (DT) (Sigma, St Louis, MO, USA) for five consecutive days. To eliminate basophils, Bas-TRECK mice were injected intraperitoneally with 500 ng DT. Complete deletion of mast cells and basophils was sustained for three weeks and three days, respectively [32].

Flow cytometry and Immunofluorescence staining

After blocking of Fc receptors with anti-mouse CD16/32 mAb (2.4G2, BD, San Jose, CA, USA), the cells were stained with the following mAbs: hCD2 (RPA-2.10) and PD-1 (RMP1-30) were purchased from BioLegend (San Diego, CA, USA); and CD4 (RM4-5), B220 (RA30-B2), Fas (15A7), GL-7, and IL-13Rα1 (13MOKa) were purchased from eBioscience (San Diego, CA, USA). CXCR5 (2G8) and IL-4Rα (M1) were purchased from BD Biosciences. Flow cytometry was performed on a FACSCalibur (BD) and data were analyzed using FlowJo (Tree Star, Ashland, OR, USA).

The spleens were fixed with 4% PFA overnight at 4°C and then penetrated with a 10% sucrose solution. The fixed spleens were frozen in OCT compound (4583, Sakura Finetek, Japan). Sections (8-µm thick) were prepared using a cryostat (Leica, Wetzlar, Germany) and fixed onto glass slides. After permeabilization (0.1% Triton X-100) and blocking (3% BSA–PBS), sections were stained with B220 (RA3-6B2)-PE, CD4 (RM4-5)-FITC, and GL-7 (GL-7)-APC mAbs. Images were captured using a fluorescence microscope BZ-X710 (Keyence, Osaka, Japan).

Antibody measurement and affinity assay

OVA- or NP-specific IgG1 and IgE concentrations were measured by ELISA. Plates were coated with OVA (100 µg/mL) and NP29-BSA (1 µg/mL for IgG1 and 10 mg/mL for IgE) in 0.1 M carbonate–bicarbonate buffer. For the measurement of anti-NP high-affinity antibodies, NP1-BSA (1 µg/mL for IgG1 and 10 µg/mL for IgE) were coated. After blocking with 5% BSA, the serum samples were collected. HRP-conjugated rabbit anti-mouse IgG antibody (anti-IgG1-HRP) (A90-105p; BETHEL Montgomery, TX, USA), biotinylated rat anti-mouse IgE antibody (clone R35-118, BD), StAv-HRP (Invitrogen, Waltham, MA, USA), and OptEIA (BD) were used as secondary antibodies and HRP detection reagents. OVA-specific IgE was measured using the Mouse IgE ELISA OVA (DS Pharma Biomedical, Osaka, Japan).

Statistical analysis

All data are presented as mean ± SD, and statistical analysis was performed using Student’s t-test. Differences were considered significant at a p-value of <0.05.

Data availability

RNA-seq data were deposited in the NCBI Gene Expression Omnibus (GEO) database under the accession number GEO: GSE137970.

Code availability

All R scripts used to analyze the data reported in this publication are available from the corresponding authors upon request.

Results

Antigen sensitization causes the conversion of T_H2 cells into T_FH cells

Recently, Gowthaman et al. identified a unique IL-13 secreting TFH subset, T_FH13 cells, that contributes to high-affinity IgE antibody production [25]. However, development of IL-13-expressing T_FH cells remains unclear. We hypothesized that IL-13-expressing T_FH cells develop from T_H2 cells in which IL-13 is abundantly expressed. To test whether there is phenotypic conversion of T_H2 into T_FH cells, a combined system of OT-II and IL-4/hCD2 (OTII-IL-4/hCD2) mice was used and the phenotype of committed OVA-specific IL-4⁺ T_H2 cells was investigated after in vivo transfer into normal C57BL/6J (B6) mice. The transferred hCD2⁺ T_H2 cells began to display a T_FH phenotype, expressing high levels of CXCR5 and IL-4 after immunization with OVA + alum (Fig. 1a). A significant increase in GL-7⁺ GC-B cells in T_H2-transferred mice was observed after OVA+ alum immunization (Fig. 1b). The transferred T cells migrated from the T cell zone and T–B border area of the spleen into B-cell follicles among the GL-7⁺ GC-B cells (Fig. 1c). The T_H2 transferred mice displayed a high titer of anti-OVA IgE (Fig. 1d) and a severe anaphylactic reaction with a striking increase in serum histamine levels and vascular permeability after OVA challenge (Fig. 1e); however, this was not observed in non-tranferred control mice.

Fig. 1.

T_H2-derived TFH cells control IgE production and anaphylaxis. (a) T_H2 cells were generated in CD45.2⁺ OTII-IL-4/hCD2 mice and purified hCD2+ T_H2 cells were transferred into CD45.1⁺ B6 mice. Recipient mice were immunized with OVA (100 µg) in alum, and CXCR5 expression was assessed in high and low IL-4 (hCD2) populations among CD45.2⁺ CD4 T cells obtained from the spleen (right panel). The flow cytometry profiles are representative data from three independent experiments. (b) The percentage of T_FH (CXCR5⁺ PD1⁺) cells among CD45.2 and GC-B (FAS⁺ GL-7⁺) cells among B-cells (n=3) on day 6 after immunization with or without OVA in alum. (c) Immunohistochemical staining of spleen sections. (Left image: green, B220; red, CD45.2. blue GL-7). The two right bar graphs indicate cell numbers (Mean ± SD) in a 0.3 mm² section (no stim n=4, OVA n=5) in the indicated region of the spleen section. The figure shows a representative spleen from T_H2-transferred mice immunized with OVA. (d) OVA-specific serum IgG1and IgE titers in mice immunized with OVA in alum (no transfer; non n=2, T_H2 transfer n=3). (e) Reduction in body temperature in non-transfer (Non n=3) and T_H2 transferred (T_H2 n=5) mice after OVA challenge. ACA responses were assessed by extravasated Evans blue in skin tissue 30 min after the challenge (left bar graph, n=3), and histamine concentrations in sera were measured (right bar graph, n=3). (f) hCD2⁺T_H2 cells were transferred into Mas-TRECK CD28^−/− or Bas-TRECK CD28^−/− mice. Body temperature change in C57BL/6J (control), Mas-TRECK (n=5), or Bas-TRECK mice (n=5) (upper panel). ACA responses (lower left) and histamine concentrations in sera (lower right) were measured as described in (e). All bar graph data are presented as the mean ± SD. “N.D.” indicates not detected.

T_H2-converted T_FH cells were designated as T_FH2 cells and whether T_FH2-mediated anaphylaxis depended on mast cells and/or basophils was determined. To answer this question, Mas-TRECK or Bas-TRECK mice were used in which DT deleted either mast cells or basophils in vivo. The anaphylactic response, including increased vascular permeability and histamine release, was completely attenuated in mast cell-deleted mice (Mas-TRECK in Cd28^−/−) but not in basophil-deleted mice (Bas-TRECK in Cd28^−/−) (Fig.. 1f). These results indicate that effector T_H2 cells were able to further differentiate into T_FH cells controlled by secondary antigen sensitization. T_FH2 cells are essential for mast cell-dependent anaphylaxis, which depends largely on IgE levels.

The IgE antibody response largely depends on the GC response and T_FH cells that develop from naïve CD4 T cells [39]. Thus, the functional distinction between T_FH2 and conventional T_FH cells was determined. To compare the IgE response mediated by conventional T_FH and T_FH2 cells, naïve CD4 T cells or ex vivo T_H2 cells were transferred from OTII-IL-4/hCD2 mice into Cd28^−/− mice as the host mouse lacked T cell response. Both groups displayed a similar pattern in the development of T_FH and GC-B cells (Fig. 2a). More than 60% of the transferred T_H2 cells co-expressed PD-1 and CXCR5 after immunization with NP-OVA. PD-1⁺ CXCR5⁺ IL-4/hCD2⁺ CD4 T cells appeared 5 d after immunization. The transferred T_H2 cells showed robust expression of the type 2 cytokines IL-4 and IL-13, and the T_FH cytokine IL-21. RT-PCR showed the exclusive expression of Bcl6 and Blimp1 with and without OVA sensitization in T_H2 cells (Fig. 2b).

Fig. 2.

T_H2-derived T_FH cells are critical for high-affinity IgE production. (a) T_H2 cells were generated from CD45.2⁺ OTII-IL-4/hCD2 mice. The purified IL-4⁺ T_H2 cells (T_H2) or naïve CD4 T cells (Naïve T) from OTII-IL-4/hCD2 mice were transferred into CD28^−/− mice. The mice were immunized with OVA in alum and donor cells were analyzed one week after transfer. The flow cytometry plots show the percentage of T_FH (CXCR5⁺ PD1⁺) cells among CD45.2 cells and GC-B (FAS⁺ GL-7⁺) cells among B-cells six days after immunization. Bar graphs show the percentage of T_FH cells and GC-B cells (n=3). (b) Expression of T_H2 and T_FH signature genes in the T_H2 cells before (T_H2) and after (T_H2 OVA) transfer and in vivo priming (n=3) was assessed by qPCR. Data were normalized by β-actin copy number. (c) Serum titers of OVA- or NP-specific (NP29 and NP1), IgG1 and IgE levels and the NP1/NP29 ratios, were assessed in the mice receiving naïve T (Naïve n=5) or T_H2 (T_H2 n=25) as described in (a). (d) Mice were challenged with NP-OVA intradermally on day 14. Body temperature was measured immediately after the challenge. Graphs show the reduction in body temperature. (e) ACA responses (left panel) and histamine concentrations in sera (right panel) were measured as described in Fig. 1e (n=3). All bar graph data are mean ± SD.

In this T_H2 transfer system, B-cells in Cd28^−/− host mice were unprimed. Therefore, IL-13 secreted by T_FH2 cells was speculated to have the potential to promote high-affinity IgE production, even in the primary response following initial B-cell activation. To test whether T_FH2 cells have a different role from conventional T_FH cells in the initial priming of B-cells, the binding affinity of IgE and IgG1 antibodies generated by conventional T_FH and T_FH2 cells to the haptens NP1 and NP29 were compared (Fig. 2c). High levels of IgE antibodies capable of binding to NP1-BSA were found in the presence of T_FH2 cells, but not in the presence of conventional T_FH cells derived from naïve OTII cells (Fig. 2c). Remarkably, NP1-binding IgE antibodies emerged in the primary B-cell response to NP. In contrast, no increase in NP1 binding IgG1 antibodies was noted at this early time point in mice with emerging T_FH2 cells (Fig.. 2c). These results indicate that T_FH2 cells induced high-affinity IgE antibodies, even in the absence of high-affinity IgG1 antibodies.

Moreover, the mice that developed T_FH2 cells had a more severe anaphylactic reaction, including severely decreased body temperature (Fig. 2d) and increased histamine concentration and vascular permeability, than the mice that developed conventional T_FH cells (Fig. 2e). These results suggest that T_FH2 cells are a potentially distinctive T_FH population capable of promoting high-affinity pathogenic IgE, leading to severe anaphylaxis, and that their plasticity is controlled by antigen sensitization.

T_FH2 cells have transcriptionally hybrid phenotype between T_H2 and T_FH cells

Conventional T_FH cells express a set of genes, including Bcl6, Tcf7, Tox2, and Il21, which are not expressed by T_H2 cells [40]. RNA-seq was performed on the donor-derived T_FH population (CD28⁺ CXCR5⁺ PD-1⁺) sorted seven days after OVA + alum immunization and on ex vivo T_H2 and in vivo CXCR5⁺ T_FH2 cells. Pairwise comparisons indicated that T_FH2 cells expressed specific T_H2 signature genes, including Il13, Epas1, and Gata3 (Fig. 3a). In addition, a pairwise comparison between conventional T_FH and T_FH2 cells indicated that T_FH2 cells also expressed several T_FH signature genes, including Bcl6, Btla, Cd40lg, Cxcr5, and Tox2 (Fig. 3a). GO and Venn diagram analyses indicated that T_FH2 and T_FH cells had several pathways related to antibody and B-cell responses (Fig. 3b, Supplementary Fig.1).

Fig. 3.

RNA-Seq analysis of T_H2, T_FH2, and T_FH cells. (a–d) RNA-seq, gene ontology (GO) enrichment, and principal component analyses of sorted ex vivo T_H2 (n=3), T_FH2 (n=1), and T_FH (n=2) cells. Naïve CD4 T cells (CD4⁺, CD62L^hi, CD44^lo, CD25⁻) were sorted from B6 mice (n=1). The ex vivo T_H2 cells were sorted from Il4 Bac reporter mice as the IL-4 (hCD2) expressing CD4 T cells under T_H2 in vitro skewing. T_FH2 and T_FH cells were prepared from T_H2- and naïve T cell-transferred CD45.1 B6 mice, respectively, on day 6 after the initial immunization. (a) Heatmap of the transcripts per million (TPM) values for Naïve, T_H2, T_FH, and T_FH2 bulk cell populations. The hierarchical clustering of genes is shown as dendrograms. Heatmaps were drawn for the Z-scores of the signature genes for previously characterized signature genes for T_H2, T_FH, and T_FH13 cells. (b) GO enrichment analysis to extract common biological processes among the gene clusters. (c) QuantSeq 3′ mRNA-Seq analysis of T_FH2 (n=2) and T_FH^hi, T_FH^mid, and T_FH^low(n=3 each) cells. CXCR5^lo, CXCR5^mid, and CXCR5^hi populations were sorted based on the expression levels of CXCR5. Heatmaps of the transcripts per million (TPM) values were compared among CXCR5^lo, CXCR5^mid, and CXCR5^hi populations for previously characterized signature genes for T_H2, T_FH, and T_FH13. (d) Principal component analysis of T_FH2 (n=2) T_FH^hi, T_FH^mid, and T_FH^low bulk cell populations.

Furthermore, a comparison of CXCR5^lo, CXCR5^mid, and CXCR5^hi populations among conventional T_FH and T_FH2 cells demonstrated that CXCR5^lo cells retained a T_H2 phenotype, whereas CXCR5^hi cells did not, as several T_H2 signature genes were downregulated. In contrast, only a subtle downregulation of Gata3 and Il13 was observed in the CXCR5^hi population (Fig. 3c). PCA indicated that T_FH2 cells were distant from T_H2 cells and had a gene profile similar to that of conventional T_FH cells (Fig. 3d). These data demonstrated that CXCR5^hi T_FH2 cells are a unique IL-13-expressing T_FH subset that shares the transcriptional profile of T_H2 cells.

Role of IL-4 and IL-13 in GC formation and IgE responses

The lineage commitment of T_FH cells requires the transcription factor BCL-6, which has multifaceted effects on T_FH-related gene expression. Thus, CD4 T cells lacking BCL-6 fail to differentiate into T_FH cells and generate GC-B cells. The requirement for T_FH conversion in Bcl6f/f cd4-cre (Bcl6^ΔT) mice was evaluated. The ex vivo development of IL-4/hCD2⁺ OTII T_H2 cells from Bcl6^ΔT mice was comparable to that of IL-4⁺ T_H2 cells from Bcl6-sufficient mice (Fig. 4a). The IL-4⁺ T_H2 cells from Bcl6^ΔT OTII mice (Bcl6^ΔT OTII T_H2) contained no CXCR5⁺ T_FH2 cells, even after OVA immunization (Fig. 4a). Moreover, Bcl6^ΔT T_H2 mice had no GC-B cells and markedly reduced IgE antibody responses and anaphylaxis (Fig. 4a, 4b, and 4e).

Fig. 4.

Roles of BCL-6 and IL-4 in T_FH2 development and function. (a) T_H2 cytokine profiles of Bcl6-deficient and -sufficient T_H2 cells. CD45.2⁺OTII T_H2 (WT n=5) or Bcl6^ΔTCD45.2⁺OTII T_H2 cells (Bcl6^ΔTn=5) were transferred into CD45.1 CD28^−/−mice. Shown is the frequency of T_FH (CXCR5⁺PD1⁺) and GC-B (FAS⁺GL7⁺) cells six days after immunization. Bar graphs indicate the percentage of T_FH cells and GC-B cells. (b) The two left panels indicate OVA-specific IgG1 and IgE titers after immunization with NP-OVA in alum. The two right panels indicate NP-specific IgE concentrations and the NP1/NP29 ratios. (c) CD45.2⁺OTII T_H2 (WT n=3) or Il4^−/− CD45.2⁺OTII T_H2 cells (Il4^−/− n=5) were transferred into CD45.1 Cd28^−/−mice. The flow cytometry plots show the percentage of T_FH (CXCR5⁺PD1⁺) after immunization with NP-OVA in alum. Bar graphs show the T_FH and GC-B cell percentages. (d) The top two panels indicate OVA-specific IgG1 and IgE titers after immunization with NP-OVA in alum. The bottom two bar graphs indicate NP-specific IgE concentrations and the NP1/NP29 ratios on day 6. (e) Body temperature was measured after NP-OVA challenge (WT n=5, Bcl6^ΔT, Il4^−/− n=4). (f) The ACA responses in sera was measured as described in Fig. 1e (WT n=5, Bcl6^ΔT, Il4^−/− n=4). All bar graph data are mean ± SD.

IL-4 derived from T_FH cells is the primary driver of GC formation and the IgE response in vivo [41, 42]. Indeed, mice receiving OTII Il4^−/− T_H2 cells exhibited a marked reduction in GC formation, even though T_FH2 development was not completely inhibited (Fig. 4c). No IgE antibody response and a substantial reduction in anaphylaxis (Fig. 4d and 4e) were observed. The mice injected with Bcl6^ΔT T_H2 and Il4^−/− T_H2 cells showed no signs of vascular permeability after OVA challenge (Fig. 4f). Collectively, these results suggest that IL-4 derived from T_FH2 cells is critical for GC formation and IgE antibody response.

Classically, in vivo transfer of T_H2 cells promoted type 2 antibody responses, IgG1 and IgE. Previous studies suggested that IL-13 secreted by T_FH13 cells contributes to the generation of high-affinity IgE antibodies [25]. Thus, we hypothesized that IL-13 from T_H2-derived T_FH2 cells could explain the IgE antibody response induced by T_H2 transfer. To investigate the role of IL-13 from T_FH2 cells in IgG1 and IgE responses, naïve T cells and T_H2 cells were obtained from OTII-IL-4/hCD2 mice crossed with IL-13 tomato knock-in (Il13^tomato) mice and transferred into Cd28^−/− mice. Tomato expression was found only in the CXCR5⁺ T_FH population of the mice receiving T_H2 cells, but not in recipients of naïve T cells (Fig. 5a), indicating that T_FH2 cells are a significant source of IL-13 contributing to type 2 antibody responses.

Fig. 5.

Roles of IL-13 in T_FH2 development and function. (a) CD45.2⁺ OTII Il13^tomato naïve T cells (n=6) or CD45.2⁺ OTII Il13^tomato T_H2 cells (n=5) were transferred into CD45.1⁺CD28^−/−mice. The representative flow cytometry plots and bar graphs show IL-13-tomato expression in the CXCR5⁺ PD-1⁺ T_FH population after OVA immunization. (b) IL-4 and IL-13 expression in T_H2 cells prepared from the CD4 T cells of CD45.2⁺ OTII T_H2 (T_H2) or Il13^{tomato/tomato} CD45.2⁺ OTII T_H2 (IL-13KO T_H2). T_H2 (n=12) or IL-13KO T_H2 (n=12) cells were transferred into CD45.1⁺CD28^-/−mice. (c) The bar graphs represent NP-specific IgG1 and IgE titers and the NP1/NP29 ratios on day 6. (d) The ACA responses in sera was measured as described in Fig. 1e (T_H2 n=5, IL-13KO T_H2 n=4). Body temperature (left upper panel) and survival percentage (left lower panel) are shown. All bar graph data are mean ± SD.

Thereafter, we investigated whether IL-13 expression in the GC was required to produce IgE antibodies capable of binding to NP1. To this end, T_H2 cells lacking IL-13 expression were generated from OTII mice crossed with Il13-deficient (Il13^{tomato/tomato}) mice and transferred to Cd28^−/− mice (IL-13KO-T_FH2) (Fig. 5b). Seven days after NP-OVA sensitization, IL-13KO-T_H2 mice had comparable levels of total IgG1 and slightly lower levels of IgE than IL13-sufficient mice. Importantly, complete abrogation of high-affinity IgE antibodies capable of binding to NP1 was found in IL-13KO-T_FH2 mice (Fig. 5c). After intradermal challenge with NP-OVA, a marked reduction in anaphylaxis, decreased vascular permeability, and increased survival was observed in IL-13KO-T_FH2 mice (Fig. 5d). These results support our hypothesis that the conversion of T_H2 cells into T_FH2 cells is essential for antigen-specific IgE responses, including the generation of high-affinity IgE antibodies that determine anaphylaxis severity.

Discussion

High-affinity IgE antibodies are highly pathogenic and can cause life-threatening acute cutaneous anaphylaxis. In the present study, we addressed the mechanism by which allergen sensitization of T_H2 cells led to these high-affinity pathogenic IgE antibodies. Allergen sensitization was demonstrated to promote the plasticity of conventional T_H2 cells into T_FH2 cells, which promoted high-affinity pathogenic IgE through the expression of IL-13. T_FH2 cells have the hybrid transcriptional features of both T_FH and T_H2 cells, which express IL-4, IL-13, IL-21, CXCR5, Gata3, and BCL-6. Adaptive transfer of BCL-6- and IL-4-deficient T_H2 cells demonstrated that IL-4 secreted by T_FH cells is essential for GC formation and IgE response. In contrast, IL-4 and IL-13 secreted by T_FH2 have different roles in IgE responses, whereas neither cytokine affects T_FH2 development. IL-4 controls the class switching of IgE, whereas IL-13 plays a specific role in the generation of high-affinity IgE that is generated independently from the high-affinity IgG1 memory B cells. These results suggest that antigen exposure of T_H2 cells that are continually enriched in allergic patients is a critical risk factor for the induction of high-affinity IgE antibodies, determining the severity of IgE-dependent allergic responses.

Conventional T_FH cells secrete IL-4, thus controlling IgE class switching by GC-B cells [19, 20, 22]. Our team and other researchers have demonstrated that T_FH13 and T_FH2 cells are unique IL-13 expressing T_FH subset and IL-13 promotes the generation of high-affinity IgE responses. Previous studies have reported that T_FH13 cells could be identified as a rare population of T_FH cells in Dock8-deficient mice and that other IL-13 expressing T_FH cells could be induced by specific antigens, such as schistosome egg antigens and house dust mite (HDM) allergens [25, 26]. Helminths and HDM are well known to induce T_H2-polarized responses [43]. HDM is a type 2 antigen that induces T_H2 polarization due to the increased penetration of allergens due to barrier dysfunction [44, 45]. Therefore, we hypothesized that T_FH cells differentiate from T_H2 cells, which are potent producers of IL-13. In this study, the transfer experiments using committed T_H2 cells support the notion that IL-13-expressing T_FH2 cells are derived from T_H2 cells. The T_H2-derived T_FH2 cells had the characteristics of both T_H2 and T_FH cells, expressing a set of T_FH gene signatures, including Bcl6, Btla, Cd40lg, Cxcr5, Tox2, and Il21, as well as T_H2 gene signatures, including Il13, Epas1, and Gata3. In contrast, the transferred naïve OTII T cells developed into conventional T_FH cells without T_H2 gene signatures. Therefore, we concluded that T_H2 cells are a potent precursor of T_FH2 cells and that antigen sensitization is a critical developmental cue for the plasticity of T_H2 cells.

Previous studies have demonstrated that conventional T_FH and T_H2 cells play distinct roles in allergic responses. Primary immune responses against soluble antigens mainly generate conventional T_FH cells, rather than T_H2 cells, in the blood, suggesting that the T_FH program is a major developmental pathway for antigen-specific helper T cells. T_H2 cells become dominant in many patients with allergies because of continuous exposure to allergens in conjunction with IL-33 and TSLP [22, 46, 47]. T_H2-derived T_FH2 cells were discovered to be a significant source of IL-13, which directly regulates the production of high-affinity IgE. Therefore, T_FH2 conversion from T_H2 cells is a key process in eliciting life-threatening anaphylaxis when patients with allergies are repeatedly exposed to allergen sensitization, leading to T_H2 cell accumulation. Moreover, T_FH2 cells derived from IL-13-defective T_H2 cells exhibited abrogation of high-affinity IgE, even though T_FH development and T_FH2-derived IL-4 were intact. In contrast, T_FH2-derived IL-4 failed to induce IgE responses or GC formation, which is consistent with results of studies on influenza virus infection [48]. Therefore, IL-4 and IL-13 from T_FH2 cells control IgE responses differently. Previous studies have reported that human circulating T_FH2 (hcT_FH2) cells have characteristics similar to those of the mouse T_FH2 cells reported here [27, 28]. In patients with allergies, the role of IL-13 is typically ascribed to circulating T_FH2 cells. Therefore, a likely scenario in patients with allergies is that the accumulated T_H2 cells are a major differentiation pathway for hcT_FH2 cells.

In general, helminth infection and intraperitoneal immunization with haptenated OVA in alum generates low-affinity IgE antibodies during the primary response. This process has been demonstrated using three independent IgE reporter mouse systems in which IgE switching occurs within GCs and IgE⁺ GC-B cells quickly differentiate into PCs [38, 41, 42]. The high-affinity IgE is mainly secreted by IgE⁺ PCs that originate from the sequential switching of high-affinity IgG1 memory cells generated after secondary immunization [39]. However, our CD28^−/− transfer system demonstrated that the generation of high-affinity anti-NP IgE antibodies, even after intraperitoneal immunization with NP-OVA, was a primary response for B-cells. These results indicate that T_H2-derived T_FH2 cells can promote high-affinity IgE responses without the generation of high-affinity IgG1⁺ memory cells. Therefore, secondary activation of fully committed antigen-specific T_H2 cells has the potential to generate high-affinity IgE B-cells, even if cognate B-cells are in a primary response.

In conclusion, we have identified that the differentiation cue for converting T_H2 cells into T_FH2 cells is antigen repriming, and that IL-13 expression by T_FH2 cells is critical for the generation of high-affinity IgE antibodies in primary responses to the hapten. Therefore, biological targeting of IL-13 function in patients with IgE-dependent allergic diseases could be a promising clinical strategy. Recently, Lebrikizumab, the anti-IL-13 antibody biologic targeting IL-13, has shown a pivotal inhibitory effect on moderate to severe forms of atopic dermatitis (AD) in adults and IgE responses [49, 50]. Moreover, mouse IL-13 kinoids vaccines capable of inducing the antibody against IL-13, by coupling with diphtheria “cross-reactive material 197,” inhibit IgE responses and asthmatic diseases [51]. Therefore, targeting IL-13 from T_FH2 cells represents a promising and important therapeutic strategy for IgE-dependent allergic disorders and AD inflammation.

Conflict of Interests

None.

Acknowledgments

We thank Ki Sewon and Y. Suzuki for technical support and animal maintenance. All authors were involved in drafting the manuscript or critically revising it for important intellectual content. All authors approved the final version to be published. Dr. Kubo had full access to all the data in the study and takes responsibility for the integrity and accuracy of the data analysis. This work was supported by AMED-CREST (19gm1310002) for HU and MK.

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