Abstract
Allergic diseases are common and affect a large proportion of the population. Interleukin-13 (IL-13)-expressing follicular helper T (TFH13) cells are a newly identified population of TFH cells that have been associated with high-affinity IgE responses. However, the origins, developmental signals, transcriptional programming and precise functions of TFH13 cells are unknown. Here, we examined the developmental signals for TFH13 cells and found a direct and progressive differentiation pathway marked by the production of IL-21. These two pathways differed in kinetics and extrinsic requirements. However, both pathways converged, forming transcriptionally similar TFH13 cells that express the transcription factor JunB as a critical stabilizing factor. Using an intersectional genetics-based TFH13-diphtheria toxin receptor model to perturb these cells, we found that TFH13 cells were essential to drive broad germinal center responses and allergen-specific IgG and IgE. Moreover, we found that IL-21 is a broad positive regulator of allergen germinal center B cells and synergizes with IL-13 produced by TFH13 cells to amplify allergic responses. Thus, TFH13 cells orchestrate multiple features of allergic inflammation.
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Data availability
The sequencing data generated in this study have been deposited in the Gene Expression Omnibus database under GSE279058. All data are available from the corresponding author on reasonable request.
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Acknowledgements
We would like to thank E. Wagner (Medical University of Vienna), U. Höpken (Max-Delbrück Center for Molecular Medicine) and G. Kelsoe (Duke University) for kindly providing mice and reagents and the MicRoN (Microscopy Resources on the North Quad) Core, the Brigham and Women’s Hospital Center for Cellular Profiling and the Harvard Medical School Biopolymer facility for their support and assistance in this work. We would also like to thank the Beth Israel Deaconess Medical Center Flow Core staff for help with flow cytometry. This work was supported by grants from the National Institutes of Health (R01AI153124 to P.T.S., R01AI158413 to P.T.S., P01AI056299 to P.T.S. and V.K.K., T32AI007306 to C.S.N., R01AI065617 to T.C. and R01GM115474 to M.C.). Portions of figures were created using Biorender.com.
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P.C., C.S.N., M.A.P., C.B.C., M.G., J.-M.L., S.R., H.Z. and S.S. performed experiments and data analyses. C.S.N., M.A.P. and J.-M.L. performed bioinformatics analyses. V.K.K. and M.C. provided resources/models and key technical support. T.C. provided key technical support. P.C. and P.T.S. designed the study and wrote the manuscript. P.T.S. supervised the study and secured funding. All authors edited the manuscript.
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Extended data
Extended Data Fig. 1 Tfh13 cells express IL13 at the transcript and protein level.
Related to Fig. 1. a) HDM was administered to IL-13 fate mapping (Il13creRosa26LoxSTOPLoxYFP) mice and draining lymph nodes harvested on day 14. Tcon (CD19−CD4+CXCR5−), Tfh (gated as CD19−CD4+CXCR5+PD1+YFP−) or Tfh13 (gated as CD19−CD4+CXCR5+PD1+YFP+) cells were sorted and processed for low input bulk RNAseq. Rpm= Reads per million transcripts. b) IL-13 fate mapping (Il13creRosa26LoxSTOPLoxTdtomato) mice received HDM and draining lymph nodes harvested on day 14 (n = 3). Tfh (gated as CD19−CD4+CXCR5+PD1+Tdtomato−) and Tfh13 (gated as CD19−CD4+CXCR5+PD1+Tdtomato+) were sorted and cultured with total B cells and HDM in vitro for 4 days. After culture, supernatant was assessed for cytokine protein levels. (left) Schematic of experiment. (middle) Frequency of Tfh13 in indicated Tfh populations. (right) Quantification of IL-13 levels in supernatants. c) HDM was administered to IL-13 fate mapping mice (Il13creRosa26LoxSTOPLoxTdtomato) and dLNs harvested on day 15 (n = 4) as in Fig. 1a-c. (left) gating for IL-13 protein positivity. (right) quantification of percent positive in indicated populations. d) HDM was administered to WT mice (n = 3) which were intracellular stained for IL-13 as well as BCl6. (left) Identification of total Tfh cells. (right) Tfh13 cells identified by protein expression. e) Bcl6 MFI in indicated populations from d. MFI= mean fluorescence intensity. f) HDM was administered to IL-13 fate mapping (Il13creRosa26LoxSTOPLoxTdtomato) mice. Overlay of total CD4 T cells or Tfh13 cells on the same plot is shown. g-i) Il13GFP direct reporter mice received administration of HDM and draining lymph nodes harvested on day 14 (n = 3). Tfh13 (gated as CD19−CD4+CXCR5+PD1+GFP+) were sorted and cultured with total B cells and HDM in vitro for 4 days. Culture supernatant was assessed for cytokine protein levels. (g) Schematic of experiment. (h) Gating strategy and quantification of GFP+ cells after culture. (i) Quantification of IL-13 protein levels in culture supernatant. e. Unpaired two-tailed student’s t-test.
Extended Data Fig. 2 Additional assessment of Tfh13 subpopulations.
Related to Fig. 2. a) Il13CreRosa26LoxSTOPLoxTdtomatoIl21VFP mice received HDM administration and dLN was harvested on indicated days for analysis (n = 6 for 0 d, n = 7 for 5 d, 8 d, 11 d and n = 9 for 14 d). Plots are pregated on CD4+CD19−CXCR5+PD1+GITR− cells. b) Immunofluorescence microscopy images of dLN GCs from Il13CreRosa26LoxSTOPLoxTdtomatoIl21VFP mice that received HDM administration, similar to Fig. 2d, Scale bar = 150 μM c) Schematic of experiment to assess role of IL-13 in Tfh differentiation. Il13CreRosa26LoxSTOPLoxTdtomatoIl21VFP mice were given HDM and on day 7 dLNs were harvested and DN (IL13−IL21−) or 21SP (IL13−IL21+) Tfh were sorted and cultured with B cells, HDM and anti-IL13 or control IgG. Tfh13 development was assessed in n = 3 independent experiment. d) Gating of Tfh13 cells from experiment in c. Plots are pregated on CD4+MHCII− cells (left). Quantification of Tfh13 development (right). Data are represented as mean ± s.e.m. Unpaired two-tailed student’s t-test.
Extended Data Fig. 4 Additional characterization of Tfh13-DTR mice.
Related to Fig. 5. a) Control or Tfh13-DTR mice (n = 9) (as in Fig. 5b) were reanalyzed linking Tfh that are not Tfh13 (‘Tfh’) and Tfh13 cells from the same mouse (indicated by connecting line). b) Additional controls for Tfh13 deletion (n = 4). (left) Genotypes for specific groups. (right) Frequencies of Tfh13 cells or serological HDM specific IgG or IgE are shown. P value indicates unpaired Student’s t test. c) Schematic of samples from Fig. 5g used for BCR sequencing. d) Number of mutations in the Vh CDR3 region of IgG+ GC B cells from c. P value indicates Mann Whitney test.
Extended Data Fig. 5 IL-21 is a broad positive regulator of allergy induced GC B cells and synergizes with IL-13 signaling.
Related to Fig. 6. a) Schematic of in vitro assay to assess combinatorial effects of cytokines on transcriptional programs in allergy GC B cells, as in Fig. 6h. b) Groups of similar gene expression patterns across indicated groups. c) Expression of indicated genes from bulk RNAseq as in Fig. 6i. rpm=reads per million. d) Schematic of loci changes in Il21LoxDTRLox mice, which were made by inserting a LoxP-IRES-DTR-LoxP cassette immediately downstream of the stop codon of the Il21 gene. e) WT (Il21wt) or 21DTR (Il21LoxDTRLox) mice (n = 6) were sensitized with HDM and DT given starting on day 2 and HDM challenge starting on day 7. Organs were harvested on day 14. (left) Gating strategy and (right) quantification of total Tfh cells (gated as CD4+CD19−CXCR5+PD1+). f) Quantification of GC B cells (defined as CD19+GL7+CD38−) in dLN of WT or 21LDL mice. P value indicates unpaired Student’s t test.
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Chandrakar, P., Nelson, C.S., Podestà, M.A. et al. Progressively differentiated TFH13 cells are stabilized by JunB to mediate allergen germinal center responses. Nat Immunol 26, 473–483 (2025). https://doi.org/10.1038/s41590-025-02077-y
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DOI: https://doi.org/10.1038/s41590-025-02077-y
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