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A γδ T cell–IL-3 axis controls allergic responses through sensory neurons

Sokol, Caroline L.
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Data availability

scRNA-seq data generated in this project are deposited in the Gene Expression Omnibus database (under accession number GSE223220 ). scRNA-seq from human skin 29 , 30 ( https://vitiligo.dolphinnext.com/index.html and https://zenodo.org/record/4569496 (ref. 61 )) and bulk RNA-seq from mouse and human DRG 32 ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305999/ ) were obtained from publically available sources. In the mouse scRNA-seq experiment, raw sequencing data was aligned to the mouse reference genome (mm10, v.2020-A from 10X Genomics). In the mouse scTCR-seq experiment, raw sequencing data was aligned to the mouse immune repertoire genome (GRCm38, v.7.0.0 from 10X Genomics). Source data are provided with this paper.

Code availability

Source code for data analysis is available on GitHub ( https://github.com/villani-lab/gdt_allergic_response ). There are no access restrictions.

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Acknowledgements

This work was supported by grant no. T32HL116275 and a National Eczema Association Catalyst Research grant (to C.H.F.), grant nos. National Institutes of Health (NIH) K99/R00 HL151750, R01 HL158534, R01 AG082185 and the Cure Alzheimer’s Fund (to C. S. McAlpine), grant nos. R35 HL135752 (to F.K.S.), NIH R35 NS105076-01 and R01 AT011447 (to C.J.W.), grant nos. DP2CA247831 (to A.-C.V.), R01AI15116, AAAAI Foundation and D.Y.M. Leung/JACI Editors Faculty Development Award, Food Allergy Science Initiative, Massachusetts General Hospital Howard Goodman Scholarship, and the Broad Institute Next Generation Scholar (to C.L.S.) and Massachusetts General Hospital Transformative Scholar Award (to A.-C.V. and C.L.S.). C.L.S. receives extra sponsored research support from GlaxoSmithKline (GSK). We thank J. Kagan for his advice and mentorship. We thank J. Boyce for providing Cpa3 cre mice. We thank D. Mathis (Harvard Medical School) for providing Tcrd GDL mice. We thank S. Bromley (Massachusetts General Hospital) for providing Il4ra −/− mice. We thank D. Masopust for providing pet shop mice. We thank D. Mucida and B. Reis for providing Trgv4 −/− mice. E. Pondeville, MRC-University of Glasgow Centre for Virus Research, UK, kindly supplied mosquito eggs. Through the Harvard Catalyst programme, we consulted with a biostatistician to review our statistical approaches and methods. We thank the Massachusetts General Hospital Cancer Center Translational Cartography Core for supporting the imaging and analysis of confocal microscopic and RNA FISH images. Schematics in Fig. 1h and Extended Data Figs. 1a , 8a and 10f were created using BioRender.com .

Author information

Author notes

  1. These authors contributed equally: Isabela J. Kernin, Peri Matatia

Authors and Affiliations

  1. Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

    Cameron H. Flayer, Isabela J. Kernin, Peri R. Matatia, Cai Han, Parth R. Naik, Dean R. Buttaci, Pamela A. Aderhold, Xueping Zhu, Alice J. Tirard, John T. McGuire, Neal P. Smith, Alexandra-Chloe Villani & Caroline L. Sokol

  2. Department of Immunology, Harvard Medical School, Boston, MA, USA

    Peri R. Matatia & Ryan B. Camire

  3. FM Kirby Center, Boston Children’s Hospital and Department of Neurobiology, Harvard Medical School, Boston, MA, USA

    Xiangsunze Zeng, David A. Yarmolinsky & Clifford J. Woolf

  4. Virus Host Interaction Team, Skin Research Centre, University of York, York, UK

    Clive S. McKimmie

  5. Cardiovascular Research Institute and the Department of Medicine, Cardiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Cameron S. McAlpine & Filip K. Swirski

  6. Friedman Brain Institute and the Nash Family Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Cameron S. McAlpine

Authors

  1. Cameron H. Flayer

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  2. Isabela J. Kernin

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  3. Peri R. Matatia

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  4. Xiangsunze Zeng

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  5. David A. Yarmolinsky

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  6. Cai Han

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  7. Parth R. Naik

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  8. Dean R. Buttaci

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  9. Pamela A. Aderhold

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  10. Ryan B. Camire

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  11. Xueping Zhu

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  12. Alice J. Tirard

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  13. John T. McGuire

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  14. Neal P. Smith

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  15. Clive S. McKimmie

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  16. Cameron S. McAlpine

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  17. Filip K. Swirski

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  18. Clifford J. Woolf

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  19. Alexandra-Chloe Villani

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  20. Caroline L. Sokol

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Contributions

Experiments were designed, performed and analysed by C.H.F. (all), I.J.K. (Fig. 2a,b,g–j and Supplementary Figs. 1a,b , 2a–d and 4 ), P.R.M. (Extended Data Figs. 2d–g and 6b,c and Supplementary Fig. 2f ), X. Zeng (Fig. 3l,m ), D.A.Y. (Fig. 3l,m ), C.H. (Fig. 4e ), P.R.N. (Figs. 1b and 4h and Extended Data Figs. 1b , 3f and 10b ), D.R.B. (Extended Data Figs. 1b and 4g,h ), P.A.A. (Fig. 1a and Extended Data Fig. 7d ), R.B.C. (Fig. 1a and Extended Data Fig. 7d ), X. Zhu (Extended Data Fig. 4f ), A.J.T. (Fig. 2a,b,g–j and Supplementary Figs. 1a,b , 2a–d and 4 ), J.T.M. (Fig. 2a,b,g–j and Supplementary Figs. 1a,b , 2a–d and 4 ), N.P.S. (Fig. 2a,b,g–j and Supplementary Figs. 1a,b , 2a–d and 4 ) and C.L.S. (all). C. S. McKimmie generated the mosquito saliva and offered advice. C. S. McAlpine and F.K.S. generated and provided the Il3 and Il3ra mouse models and offered advice. C.J.W. designed and guided in vivo calcium imaging experiments. A.-C.V. designed and guided the single-cell transcriptomics. C.H.F. and C.L.S. wrote the paper. C.L.S. provided resources, reagents and funding. C.L.S. supervised the study.

Corresponding author

Correspondence to Caroline L. Sokol .

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Competing interests

C.L.S. is a paid consultant for Bayer and Merck and receives sponsored research support from GSK. P.A.A. is a current employee of Werewolf Therapeutics. C. S. McAlpine is a paid consultant of Granite Bio. C.J.W. is a founder of Nocion Therapeutics, QurAlis and BlackBox Bio, and is on the scientific advisory board of Lundbeck Pharma, Axonis and Tafalgie Therapeutics. A.-C.V. has a financial interest in 10X Genomics. The company designs and manufactures gene sequencing technology for use in research, and such technology is being used in this research. A.-C.V.’s interests were reviewed by The Massachusetts General Hospital and Mass General Brigham in accordance with their institutional policies.

Peer review

Peer review information

Nature thanks Brian Kim and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Mice lacking γδ T cells have no broad defect in itch, pain, or neuronal innervation in the skin.

a , Schematic of standard behaviour protocol. b , Cumulative spontaneous nonspecific scratch at any skin site (n = 6 Cpa3 +/+ ; n = 8 Cpa3 cre/+ ; n = 10 WT; n = 8 Rag2 −/− ; n = 12 Tcrd −/− ; n = 10 Tcra −/− ; n = 9 muMT −/− ). c - e , Cumulative cheek scratch bouts following intradermal (i.d.) injection as indicated ( c , n = 11 Cpa3 +/+ ; n = 10 Cpa3 cre/+ ; d , histamine: n = 10 WT; n = 12 Tcrd −/− ; d , chloroquine: n = 10 WT; n = 14 Tcrd −/− ; e , n = 9 WT; n = 12 Tcrd −/− ). f , Confocal microscopy z-stack of Tuj1 in naive WT and Tcrd −/− mice. The scale bar is 50 μm (n = 6 WT; n = 7 Tcrd −/− ). Symbols represent individual mice ( b - e ) or images ( f ). Violin plots show the median and quartiles. Data represent at least two independent experiments and were combined. Statistical tests: one-way ANOVA with Tukey’s multiple comparisons test ( b , WT versus indicated mouse strains), two-sided unpaired t -test ( b , Cpa3 ; c ; d , histamine; e , f ), or two-sided Mann Whitney U -test ( d , chloroquine). ns=not significant. Schematic in a created using BioRender ( https://biorender.com ).

Source Data

Extended Data Fig. 2 GD3 cells are Vγ5 - epidermal γδ T cells.

a , Flow cytometry of naive WT pinnae or dorsal root ganglia (DRG). b - d , f , g , Flow cytometric quantification of naive WT pinnae or DRG ( b ), naive WT dermis or epidermis ( c ), naive WT pinnae, cheek, or foot ( d ), or WT pinnae ( g ) ( b , n = 6 per group; c , n = 3 per group; d , n = 12 per group; f , g , n = 11 PBS; n = 10 papain). e , Pinnae thickness was measured following chronic papain exposure (n = 11 PBS; n = 10 papain). Symbols represent individual mice. Bar plots are mean ± SEM. Data represent at least two independent experiments and were combined, except in c . Statistical tests: two-sided unpaired t -test ( b - f ; g , dermal γδ T cells) or two-sided Mann Whitney U -test ( g , DETCs and GD3 cells). * p < 0.05, *** p < 0.001, **** p < 0.0001, ns=not significant.

Source Data

Extended Data Fig. 3 Dermal γδ T cells and DETCs are not required for allergic itch.

a , b , e , Flow cytometry of naive pinnae ( a , gating strategy corresponding to Fig. 1d ; e , gating strategy corresponding to Fig. 1f ). c , Flow cytometric quantification of naive WT pinnae (n = 9 iTcrd cont ; n = 10 iTcrd DTA ). d , f , g , Cumulative cheek scratch bouts following i.d. injection as indicated ( d , n = 9 iTcrd cont ; n = 10 iTcrd DTA ; f , n = 10 histamine and chloroquine; n = 15 HDM; g , n = 9 αCD3/28 −; n = 7 αCD3/28 +). Symbols represent individual mice. Bar plots are mean ± SEM. Data represent at least two independent experiments and were combined. Statistical tests: two-sided unpaired t -test ( c , g ) or two-sided Mann Whitney U -test ( d , f ). * p < 0.05, *** p < 0.001, ns=not significant.

Source Data

Extended Data Fig. 4 GD3 cells are regulated by Trgv4 , age, microbial factors, and dry skin.

a - c , e - h , Flow cytometric quantification from pinnae ( a , n = 12 WT; n = 11 Trgv4 -/- ; b , n = 11 8–12 weeks; n = 9 8–12 months; c , n = 12 per group; e , n = 16 per group; f , n = 4 WT; n = 7 per shop; g , n = 7 sham; n = 9 AEW; h , n = 11 sham; n = 15 AEW). d , Cumulative cheek scratch bouts following i.d. injection of papain (n = 12 per group). Symbols represent individual mice. Bar plots are the mean ± SEM. Violin plots show the median and quartiles. Data represent at least two independent experiments and were combined, except in f . Statistical tests: two-sided unpaired t -test ( a ; b , DETCs; f , dermal γδ T cells and DETCs; g ; h , DETCs) or two-sided Mann-Whitney U -test ( b , dermal γδ T cells and GD3 cells; c - e ; f , GD3 cells; h , dermal γδ T cells and GD3 cells). * p < 0.05, ** p < 0.01, *** p < 0.001, ns=not significant.

Source Data

Extended Data Fig. 5 Vγ4 + GD3 cells are the major source of IL-3 in naive skin.

a , Heatmap of multiplex assay of cell-free supernatant from FACS sorted γδ T cells stimulated as indicated (n = 3 per group). * symbols represent a cytokine or chemokine that significantly increased following stimulation. b , c , e , IL-17A or IL-3 ELISA of cell-free supernatant from FACS sorted γδ T cells or immune cells stimulated as indicated ( b , n = 4 per group, except stimulated GD3 cells (n = 6); c , n = 3 per group; e , n = 2 per group). d , Flow cytometry of FACS isolated γδ T cells. CTV = cell trace violet (viability: n = 3 per group; CTV: n = 2 per group). Symbols represent individual wells ( b ) or experimental replicates ( c - e ). Bar plots are mean ± SEM. Data represent at least two independent experiments combined, except in a , a single pilot experiment, and b , a representative experiment with n = 4–6 wells. Statistical tests: two-way ANOVA with Tukey’s multiple comparisons test ( a - c ) or two-way ANOVA ( d , viability). * p < 0.05, ** p < 0.01, **** p < 0.0001, ns=not significant.

Source Data

Extended Data Fig. 6 Expression and production of IL-3 by GD3 cells.

a , QPCR of Il3 (normalized to Gapdh ) from FACS sorted γδ T cells from naïve WT pinnae (n = 3 per group, except GD3 cells (n = 4)). b , Flow cytometric quantification of naive Il3 GFPfl/fl pinnae (n = 8 per group). c , d , Flow cytometric quantification of MACS enriched GD3 cells from Skint1 -/- FVB/N pinnae ( c ) or FACS sorted γδ T cells from Il3 GFPfl/fl pinnae ( d ) left unstimulated (unstim) or stimulated as indicated ( c , n = 3 per group; d , n = 2 per group). Symbols represent individual experimental replicates of pooled mice ( a , c , d ) or mice ( b ). Bar plots are mean ± SEM. Data represent at least two independent experiments and were combined. n = 3-4 experimental replicates of pooled mice ( a ), n = 8 mice ( b ), or n = 2-3 experimental replicates ( c , d ). Statistical tests: one-way ANOVA with Tukey’s multiple comparisons test ( a - c ). * p < 0.05, ** p < 0.01, ns=not significant.

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Extended Data Fig. 7 IL-3 targets PEP1 sensory neurons, which encode mixed itch and pain and are required for allergic itch.

a - c , Cumulative cheek scratch bouts following i.d. injection of recombinant cytokine, then allergen or IL-31 ( a , n = 8 HDM sham; n = 7 HDM IL-3; n = 12 Alternaria sham; n = 11 Alternaria IL-3; n = 8 fire ant; b , n = 11 sham; n = 9 IL-3; c , n = 12 papain sham; n = 8 papain IL-17A; n = 15 papain sham; n = 12 papain CCL3). d , Cumulative cheek scratch bouts following i.d. injection of papain (n = 19 WT; n = 20 Il4ra -/- ). e , Representative traces of ratiometric calcium imaging of DRG neurons sequentially stimulated as indicated. f , Venn diagram indicating the overlapping responsiveness of individual DRG neurons. g , Percent of DRG neurons that responded to the indicated stimuli (n = 14 per group). h , Percent of IL-3 responsive DRG neurons that also responded to papain, capsaicin, or AITC (n = 14 per group). i , Flow cytometric quantification of naive pinnae (n = 10 WT; n = 8 Trpv1 DTR ). j , Cumulative cheek wipe or scratch bouts following i.d. injection of CNO (n = 10 Tac1 cre/+ ; n = 8 Tac1 cre/+ Gq DREADD ). k , Cumulative cheek wipe or scratch bouts following i.d. injection of CNO, then papain or IL-31 (n = 9 Gi DREADD papain; n = 11 Tac1 cre/+ Gi DREADD papain; n = 7 Gi DREADD IL-31; n = 8 Tac1 cre/+ Gi DREADD IL-31). Symbols represent mice ( a - d , i , k ) or individual wells ( g - h ). Violin plots show the median and quartiles. Bar plots and time courses are mean ± SEM. Data represent at least two independent experiments and were combined. Statistical tests: two-sided unpaired t -test ( a , HDM and fire ant; b , c ; i , dermal γδ T cells and DETCs; k , IL-31), two-sided Mann Whitney U -test ( a , Alternaria; d ; i , GD3 cells; k , papain), one-way ANOVA with Tukey’s multiple comparisons test ( h ), or two-way ANOVA with Tukey’s multiple comparisons test ( j ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns=not significant.

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Extended Data Fig. 8 The absence of the GD3-IL-3-sensory neuron axis inhibits papain-induced Th2-skewing dendritic cell migration.

a , Schematic of the allergic immune response protocol. b , c , e , Flow cytometry of the draining lymph node (dLN) 24 h after immunization with ova ± papain (gating strategy corresponding to Fig. 4a, b, i, k ). d , f , g , Flow cytometric quantification of the draining lymph node (dLN) 24 h after immunization with ova ± papain ( d , n = 10 per group; f , n = 12 iTcrd cre/+ Il3 +/+ ; n = 11 iTcrd cre/+ Il3 fl/fl ; n = 10 Scn10a cre/+ Il3ra +/+ ; n = 11 Scn10a cre/+ Il3ra fl/fl ; g , n = 12 iso; n = 11 keta). Symbols represent individual mice. Symbols connected by lines indicate paired samples derived from the same mouse. Data represent at least two independent experiments and were combined. Statistical tests: two-way repeated measures ANOVA ( d , f , g ). * p < 0.05, ** p < 0.01, **** p < 0.0001, ns=not significant. Schematic in a created using BioRender ( https://biorender.com ).

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Extended Data Fig. 9 The absence of the GD3-IL-3-sensory neuron axis impairs papain-induced Th2 differentiation.

a , Flow cytometry of the draining lymph node (dLN) 5 days after immunization with ova ± papain (gating strategy corresponding to Fig. 4c, j ). b - e , Flow cytometric quantification of the draining lymph node (dLN) 5 days after immunization with ova ± papain (n = 10 WT; n = 12 Tcrd -/- ; n = 11 WT; n = 11 Il3 -/- ; n = 10 Il3ra -/- ; n = 13 iTcrd cre/+ Il3 +/+ ; n = 14 iTcrd cre/+ Il3 fl/fl ; n = 11 Scn10a cre/+ Il3ra +/+ ; n = 8 Scn10a cre/+ Il3ra fl/fl ). f , Pinnae thickness after papain challenge following the induction of delayed-type hypersensitivity (n = 10 WT; n = 6 Tcrd -/- ). Symbols represent individual mice. Symbols connected by lines indicate paired samples. Bar plots and line graphs are mean ± SEM. Data represent at least two independent experiments and were combined. Statistical tests: two-way ANOVA with Tukey’s multiple comparisons test ( b - d ; e , WT v Tcrd -/- ; g ), two-way repeated measures ANOVA ( i ), one-way ANOVA with Tukey’s multiple comparisons test ( b - d ; e , WT v Il3 -/- v Il3ra -/- ), two-sided unpaired t -test ( b , c ; d , iTcrd cre/+ and Scn10a cre/+ ), or two-sided Mann Whitney U -test ( e ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns=not significant.

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Extended Data Fig. 10 Inhibition of the JAK2-STAT5 pathway reduces the responsiveness of sensory neurons to allergens.

a , Representative fluorescence microscopic images of pSTAT5 in DRG neurons. The scale bar is 20 μm. b , Cumulative cheek scratch bouts following i.d. injection of papain or histamine (papain: n = 7 DMSO; n = 6 upad; histamine: n = 8 DMSO; n = 9 fed). c , e , Flow cytometric quantification of the draining lymph node (dLN) 24 h after immunization as indicated ( c , n = 7 per group; e , n = 5 per group). d , Flow cytometric quantification of the draining lymph node (dLN) 5 days after immunization with ova ± papain (n = 8 DMSO ova; n = 12 DMSO ova+papain; n = 7 fedratinib ova; n = 12 fedratinib ova+papain). f , Graphical abstract: a γδ T cell-IL-3 axis controls allergic responses through sensory neurons. Vγ4 + GD3 cells are the major source of IL-3 in naïve skin and control the allergen responsiveness of Il3ra -expressing sensory neurons. Upon allergen exposure, IL-3-primed sensory neurons respond with robust itch, Th2-skewing DC migration to the dLN, and Th2 differentiation in the dLN. Mechanistically, IL-3 activates JAK2 and STAT5, leading to the transcription of the substance P gene Tac1 . While JAK2 is necessary for allergic itch and the initiation of the allergic immune response, STAT5 is only required for the initiation of the allergic immune response. Together, the GD3-IL-3 axis controls the initial sensory neuronal response to allergens, dictating whether an organism is resistant or sensitive to allergens. Symbols represent individual mice ( b , d ), while symbols connected by lines indicate paired samples ( c , e ). Violin plots show the median and quartiles. Bar plots are mean ± SEM. Data are representative of at least two experiments and combined. Statistical tests: two-way repeated measures ANOVA ( c , e ), two-way ANOVA with Tukey’s multiple comparisons test ( d ), or two-sided unpaired t -test ( b ). * p < 0.05, ** p < 0.01, **** p < 0.0001, ns=not significant. Schematic in f created using BioRender ( https://biorender.com ).

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Flayer, C.H., Kernin, I.J., Matatia, P.R. et al. A γδ T cell–IL-3 axis controls allergic responses through sensory neurons. Nature (2024). https://doi.org/10.1038/s41586-024-07869-0

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