2023 Volume 5 Issue 1 Pages 1-12
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 (TFH) cell subset, designated TFH2 cells, which is tightly associated with the production of high-affinity IgE antibodies. TFH2 cells had a transcriptionally hybrid phenotype between TH2 and TFH 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 (TH2) cells were capable of differentiating into TFH2 cells upon secondary antigen stimulation. The type 2 TFH (TFH2) conversion process was entirely attenuated in T cell-specific deficient mice of Bcl6 (Bcl6f/f cd4-cre; Bcl6ΔT). Moreover, the transfer of IL-13 defective TH2 cells partially inhibited IgE responses and significantly decreased the high-affinity IgE, even though TFH development was intact. Therefore, IL-13 from TFH2 cells controlled the selective enrichment of IgE+ B-cells with a high affinity for allergens. A previously undefined unique TFH subset, TFH2, likely contributes to IgE responses. This suggests that the accumulation of antigen-specific TH2 cells in human patients with allergies is a high-risk factor for IgE-dependent allergic diseases.
• TFH2 cells, a unique TFH subset, are a transcriptional hybrid phenotype of TH2 and TFH cells.
• TFH2 cells differentiate from TH2 cells in response to allergens.
• TFH2 cells contribute to the production of pathogenic IgE antibodies by expressing high IL-13 levels.
• The plasticity of memory TH2 cells contributes to the production of pathogenic IgE antibodies in patients with allergies.
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 . 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 TH subsets, TH2 and TFH cells. TH2 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 TFH cells is strictly controlled by a basic zipper type transcriptional repressor, B-cell/CLL lymphoma 6 (BCL-6) . TFH cells, rather than TH2 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 TFH cells, that is, consensus noncoding sequence-2 (CNS-2) [19, 20]. TFH 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 TFH cells in Dock8-deficient and normal mice, which the authors designated as TFH13 cells, which expressed IL-13 and GATA3 . IL-13 secreted from TFH13 cells was reported to contribute to the generation of high-affinity IgE via sequential switching of high-affinity IgG1+ cells. However, conventional GC-TFH cells do not express IL-13 or GATA-3, indicating that this unique TFH subset uses a differentiation pathway distinct from that of conventional TFH cells. Therefore, generation of IL-13-expressing TFH cells remains unclear.
Previous studies have reported that TFH cells differentiate from TH2 cells in response to schistosome eggs and that these TFH cells express IL-5 and IL-13 . Similarly, human circulating TFH2 (hcTFH2) cells and murine TFH cells induced by house dust mite allergens express IL-13 and GATA3 [25, 27,28,29]. Some studies have also indicated that TH2 and TFH cells could be interchangeable during helminth type 2 responses [26, 30]. Therefore, these observations indicate the development of IL-13-expressing TFH cells; however, the developmental cues for TFH13 and other IL-13-expressing TFH cells remain elusive.
In the present study, TH2-derived TFH cells were discovered to have characteristics of both TFH and TH2 cells and express the TFH markers C-X-C chemokine receptor type 5 (CXCR5) and BCL-6, and the TH2 markers GATA3 and IL-13. Adaptive transfer experiments indicated that TH2 cells acquired several TFH characteristics after secondary antigen priming and migrated into B-cell follicles. Characteristic conversion of TH2 cells into TFH2 cells is associated with severe anaphylaxis, which largely depends on high-affinity anaphylactic IgE. Here, the developmental cue for TFH2 and the functional relationship between TH2 and TFH2 in IgE responses and allergic symptoms are discussed.
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, Bcl6floxCD4cre, 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 . 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.
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 . 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 . 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 220.127.116.11), 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.
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 TH2 differentiation. After six days, TH2 cells were sorted using a cell sorter, Moflo XDP (Beckman Coulter, Brea, CA, USA), and transferred into mice.
TH2 cells were derived from OT-II mice (OTII-TH2 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.
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 .
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).
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).
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.
RNA-seq data were deposited in the NCBI Gene Expression Omnibus (GEO) database under the accession number GEO: GSE137970.
All R scripts used to analyze the data reported in this publication are available from the corresponding authors upon request.
Recently, Gowthaman et al. identified a unique IL-13 secreting TFH subset, TFH13 cells, that contributes to high-affinity IgE antibody production . However, development of IL-13-expressing TFH cells remains unclear. We hypothesized that IL-13-expressing TFH cells develop from TH2 cells in which IL-13 is abundantly expressed. To test whether there is phenotypic conversion of TH2 into TFH 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+ TH2 cells was investigated after in vivo transfer into normal C57BL/6J (B6) mice. The transferred hCD2+ TH2 cells began to display a TFH 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 TH2-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 TH2 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.
TH2-derived TFH cells control IgE production and anaphylaxis. (a) TH2 cells were generated in CD45.2+ OTII-IL-4/hCD2 mice and purified hCD2+ TH2 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 TFH (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 TH2-transferred mice immunized with OVA. (d) OVA-specific serum IgG1and IgE titers in mice immunized with OVA in alum (no transfer; non n=2, TH2 transfer n=3). (e) Reduction in body temperature in non-transfer (Non n=3) and TH2 transferred (TH2 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+TH2 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.
TH2-converted TFH cells were designated as TFH2 cells and whether TFH2-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 TH2 cells were able to further differentiate into TFH cells controlled by secondary antigen sensitization. TFH2 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 TFH cells that develop from naïve CD4 T cells . Thus, the functional distinction between TFH2 and conventional TFH cells was determined. To compare the IgE response mediated by conventional TFH and TFH2 cells, naïve CD4 T cells or ex vivo TH2 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 TFH and GC-B cells (Fig. 2a). More than 60% of the transferred TH2 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 TH2 cells showed robust expression of the type 2 cytokines IL-4 and IL-13, and the TFH cytokine IL-21. RT-PCR showed the exclusive expression of Bcl6 and Blimp1 with and without OVA sensitization in TH2 cells (Fig. 2b).
TH2-derived TFH cells are critical for high-affinity IgE production. (a) TH2 cells were generated from CD45.2+ OTII-IL-4/hCD2 mice. The purified IL-4+ TH2 cells (TH2) 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 TFH (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 TFH cells and GC-B cells (n=3). (b) Expression of TH2 and TFH signature genes in the TH2 cells before (TH2) and after (TH2 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 TH2 (TH2 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 TH2 transfer system, B-cells in Cd28−/− host mice were unprimed. Therefore, IL-13 secreted by TFH2 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 TFH2 cells have a different role from conventional TFH cells in the initial priming of B-cells, the binding affinity of IgE and IgG1 antibodies generated by conventional TFH and TFH2 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 TFH2 cells, but not in the presence of conventional TFH 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 TFH2 cells (Fig.. 2c). These results indicate that TFH2 cells induced high-affinity IgE antibodies, even in the absence of high-affinity IgG1 antibodies.
Moreover, the mice that developed TFH2 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 TFH cells (Fig. 2e). These results suggest that TFH2 cells are a potentially distinctive TFH population capable of promoting high-affinity pathogenic IgE, leading to severe anaphylaxis, and that their plasticity is controlled by antigen sensitization.
Conventional TFH cells express a set of genes, including Bcl6, Tcf7, Tox2, and Il21, which are not expressed by TH2 cells . RNA-seq was performed on the donor-derived TFH population (CD28+ CXCR5+ PD-1+) sorted seven days after OVA + alum immunization and on ex vivo TH2 and in vivo CXCR5+ TFH2 cells. Pairwise comparisons indicated that TFH2 cells expressed specific TH2 signature genes, including Il13, Epas1, and Gata3 (Fig. 3a). In addition, a pairwise comparison between conventional TFH and TFH2 cells indicated that TFH2 cells also expressed several TFH signature genes, including Bcl6, Btla, Cd40lg, Cxcr5, and Tox2 (Fig. 3a). GO and Venn diagram analyses indicated that TFH2 and TFH cells had several pathways related to antibody and B-cell responses (Fig. 3b, Supplementary Fig.1).
RNA-Seq analysis of TH2, TFH2, and TFH cells. (a–d) RNA-seq, gene ontology (GO) enrichment, and principal component analyses of sorted ex vivo TH2 (n=3), TFH2 (n=1), and TFH (n=2) cells. Naïve CD4 T cells (CD4+, CD62Lhi, CD44lo, CD25−) were sorted from B6 mice (n=1). The ex vivo TH2 cells were sorted from Il4 Bac reporter mice as the IL-4 (hCD2) expressing CD4 T cells under TH2 in vitro skewing. TFH2 and TFH cells were prepared from TH2- 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, TH2, TFH, and TFH2 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 TH2, TFH, and TFH13 cells. (b) GO enrichment analysis to extract common biological processes among the gene clusters. (c) QuantSeq 3′ mRNA-Seq analysis of TFH2 (n=2) and TFHhi, TFHmid, and TFHlow(n=3 each) cells. CXCR5lo, CXCR5mid, and CXCR5hi populations were sorted based on the expression levels of CXCR5. Heatmaps of the transcripts per million (TPM) values were compared among CXCR5lo, CXCR5mid, and CXCR5hi populations for previously characterized signature genes for TH2, TFH, and TFH13. (d) Principal component analysis of TFH2 (n=2) TFHhi, TFHmid, and TFHlow bulk cell populations.
Furthermore, a comparison of CXCR5lo, CXCR5mid, and CXCR5hi populations among conventional TFH and TFH2 cells demonstrated that CXCR5lo cells retained a TH2 phenotype, whereas CXCR5hi cells did not, as several TH2 signature genes were downregulated. In contrast, only a subtle downregulation of Gata3 and Il13 was observed in the CXCR5hi population (Fig. 3c). PCA indicated that TFH2 cells were distant from TH2 cells and had a gene profile similar to that of conventional TFH cells (Fig. 3d). These data demonstrated that CXCR5hi TFH2 cells are a unique IL-13-expressing TFH subset that shares the transcriptional profile of TH2 cells.
The lineage commitment of TFH cells requires the transcription factor BCL-6, which has multifaceted effects on TFH-related gene expression. Thus, CD4 T cells lacking BCL-6 fail to differentiate into TFH cells and generate GC-B cells. The requirement for TFH conversion in Bcl6f/f cd4-cre (Bcl6ΔT) mice was evaluated. The ex vivo development of IL-4/hCD2+ OTII TH2 cells from Bcl6ΔT mice was comparable to that of IL-4+ TH2 cells from Bcl6-sufficient mice (Fig. 4a). The IL-4+ TH2 cells from Bcl6ΔT OTII mice (Bcl6ΔT OTII TH2) contained no CXCR5+ TFH2 cells, even after OVA immunization (Fig. 4a). Moreover, Bcl6ΔT TH2 mice had no GC-B cells and markedly reduced IgE antibody responses and anaphylaxis (Fig. 4a, 4b, and 4e).
Roles of BCL-6 and IL-4 in TFH2 development and function. (a) TH2 cytokine profiles of Bcl6-deficient and -sufficient TH2 cells. CD45.2+OTII TH2 (WT n=5) or Bcl6ΔTCD45.2+OTII TH2 cells (Bcl6ΔTn=5) were transferred into CD45.1 CD28−/−mice. Shown is the frequency of TFH (CXCR5+PD1+) and GC-B (FAS+GL7+) cells six days after immunization. Bar graphs indicate the percentage of TFH 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 TH2 (WT n=3) or Il4−/− CD45.2+OTII TH2 cells (Il4−/− n=5) were transferred into CD45.1 Cd28−/−mice. The flow cytometry plots show the percentage of TFH (CXCR5+PD1+) after immunization with NP-OVA in alum. Bar graphs show the TFH 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, Bcl6ΔT, Il4−/− n=4). (f) The ACA responses in sera was measured as described in Fig. 1e (WT n=5, Bcl6ΔT, Il4−/− n=4). All bar graph data are mean ± SD.
IL-4 derived from TFH cells is the primary driver of GC formation and the IgE response in vivo [41, 42]. Indeed, mice receiving OTII Il4−/− TH2 cells exhibited a marked reduction in GC formation, even though TFH2 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 Bcl6ΔT TH2 and Il4−/− TH2 cells showed no signs of vascular permeability after OVA challenge (Fig. 4f). Collectively, these results suggest that IL-4 derived from TFH2 cells is critical for GC formation and IgE antibody response.
Classically, in vivo transfer of TH2 cells promoted type 2 antibody responses, IgG1 and IgE. Previous studies suggested that IL-13 secreted by TFH13 cells contributes to the generation of high-affinity IgE antibodies . Thus, we hypothesized that IL-13 from TH2-derived TFH2 cells could explain the IgE antibody response induced by TH2 transfer. To investigate the role of IL-13 from TFH2 cells in IgG1 and IgE responses, naïve T cells and TH2 cells were obtained from OTII-IL-4/hCD2 mice crossed with IL-13 tomato knock-in (Il13tomato) mice and transferred into Cd28−/− mice. Tomato expression was found only in the CXCR5+ TFH population of the mice receiving TH2 cells, but not in recipients of naïve T cells (Fig. 5a), indicating that TFH2 cells are a significant source of IL-13 contributing to type 2 antibody responses.
Roles of IL-13 in TFH2 development and function. (a) CD45.2+ OTII Il13tomato naïve T cells (n=6) or CD45.2+ OTII Il13tomato TH2 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+ TFH population after OVA immunization. (b) IL-4 and IL-13 expression in TH2 cells prepared from the CD4 T cells of CD45.2+ OTII TH2 (TH2) or Il13tomato/tomato CD45.2+ OTII TH2 (IL-13KO TH2). TH2 (n=12) or IL-13KO TH2 (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 (TH2 n=5, IL-13KO TH2 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, TH2 cells lacking IL-13 expression were generated from OTII mice crossed with Il13-deficient (Il13tomato/tomato) mice and transferred to Cd28−/− mice (IL-13KO-TFH2) (Fig. 5b). Seven days after NP-OVA sensitization, IL-13KO-TH2 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-TFH2 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-TFH2 mice (Fig. 5d). These results support our hypothesis that the conversion of TH2 cells into TFH2 cells is essential for antigen-specific IgE responses, including the generation of high-affinity IgE antibodies that determine anaphylaxis severity.
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 TH2 cells led to these high-affinity pathogenic IgE antibodies. Allergen sensitization was demonstrated to promote the plasticity of conventional TH2 cells into TFH2 cells, which promoted high-affinity pathogenic IgE through the expression of IL-13. TFH2 cells have the hybrid transcriptional features of both TFH and TH2 cells, which express IL-4, IL-13, IL-21, CXCR5, Gata3, and BCL-6. Adaptive transfer of BCL-6- and IL-4-deficient TH2 cells demonstrated that IL-4 secreted by TFH cells is essential for GC formation and IgE response. In contrast, IL-4 and IL-13 secreted by TFH2 have different roles in IgE responses, whereas neither cytokine affects TFH2 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 TH2 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 TFH cells secrete IL-4, thus controlling IgE class switching by GC-B cells [19, 20, 22]. Our team and other researchers have demonstrated that TFH13 and TFH2 cells are unique IL-13 expressing TFH subset and IL-13 promotes the generation of high-affinity IgE responses. Previous studies have reported that TFH13 cells could be identified as a rare population of TFH cells in Dock8-deficient mice and that other IL-13 expressing TFH 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 TH2-polarized responses . HDM is a type 2 antigen that induces TH2 polarization due to the increased penetration of allergens due to barrier dysfunction [44, 45]. Therefore, we hypothesized that TFH cells differentiate from TH2 cells, which are potent producers of IL-13. In this study, the transfer experiments using committed TH2 cells support the notion that IL-13-expressing TFH2 cells are derived from TH2 cells. The TH2-derived TFH2 cells had the characteristics of both TH2 and TFH cells, expressing a set of TFH gene signatures, including Bcl6, Btla, Cd40lg, Cxcr5, Tox2, and Il21, as well as TH2 gene signatures, including Il13, Epas1, and Gata3. In contrast, the transferred naïve OTII T cells developed into conventional TFH cells without TH2 gene signatures. Therefore, we concluded that TH2 cells are a potent precursor of TFH2 cells and that antigen sensitization is a critical developmental cue for the plasticity of TH2 cells.
Previous studies have demonstrated that conventional TFH and TH2 cells play distinct roles in allergic responses. Primary immune responses against soluble antigens mainly generate conventional TFH cells, rather than TH2 cells, in the blood, suggesting that the TFH program is a major developmental pathway for antigen-specific helper T cells. TH2 cells become dominant in many patients with allergies because of continuous exposure to allergens in conjunction with IL-33 and TSLP [22, 46, 47]. TH2-derived TFH2 cells were discovered to be a significant source of IL-13, which directly regulates the production of high-affinity IgE. Therefore, TFH2 conversion from TH2 cells is a key process in eliciting life-threatening anaphylaxis when patients with allergies are repeatedly exposed to allergen sensitization, leading to TH2 cell accumulation. Moreover, TFH2 cells derived from IL-13-defective TH2 cells exhibited abrogation of high-affinity IgE, even though TFH development and TFH2-derived IL-4 were intact. In contrast, TFH2-derived IL-4 failed to induce IgE responses or GC formation, which is consistent with results of studies on influenza virus infection . Therefore, IL-4 and IL-13 from TFH2 cells control IgE responses differently. Previous studies have reported that human circulating TFH2 (hcTFH2) cells have characteristics similar to those of the mouse TFH2 cells reported here [27, 28]. In patients with allergies, the role of IL-13 is typically ascribed to circulating TFH2 cells. Therefore, a likely scenario in patients with allergies is that the accumulated TH2 cells are a major differentiation pathway for hcTFH2 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 . 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 TH2-derived TFH2 cells can promote high-affinity IgE responses without the generation of high-affinity IgG1+ memory cells. Therefore, secondary activation of fully committed antigen-specific TH2 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 TH2 cells into TFH2 cells is antigen repriming, and that IL-13 expression by TFH2 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 . Therefore, targeting IL-13 from TFH2 cells represents a promising and important therapeutic strategy for IgE-dependent allergic disorders and AD inflammation.
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.