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Restriction of innate Tγδ17 cell plasticity by an AP-1 regulatory axis

Nature Immunology volume 26, pages 1299–1314 (2025)Cite this article

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Abstract

Interleukin-17 (IL-17)-producing γδ T (Tγδ17) cells are innate-like mediators of intestinal barrier immunity. Although IL-17-producing helper T cell and group 3 innate lymphoid cell plasticity have been extensively studied, the mechanisms governing Tγδ17 cell effector flexibility remain undefined. Here, we combined type 3 fate mapping with single-cell ATAC-sequencing/RNA-sequencing multiome profiling to define the cellular features and regulatory networks underlying Tγδ17 cell plasticity. During homeostasis, Tγδ17 cell effector identity was stable across tissues, including for intestinal T-bet+ Tγδ17 cells that restrained interferon-γ production. However, Salmonella enterica subsp. enterica serovar Typhimurium infection induced intestinal Vγ6+ Tγδ17 cell conversion into type 1 effectors, with loss of IL-17A production and partial RORγt downregulation. Multiome analysis revealed a trajectory along Vγ6+ Tγδ17 cell effector conversion, with TIM-3 marking ex-Tγδ17 cells with enhanced type 1 functionality. Last, we characterized and validated a critical AP-1 regulatory axis centered around JUNB and FOSL2 that controls Vγ6+ Tγδ17 cell plasticity by stabilizing type 3 identity and restricting type 1 effector conversion.

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Fig. 1: Tγδ17 cell identity is stable at steady state and IFNγ production is restrained in intestinal T-bet+ Tγδ17 cells.
Fig. 2: Vγ6+ Tγδ17 cells are functionally plastic after intestinal S. Typhimurium infection.
Fig. 3: Single-cell multiome characterization of Vγ6+ Tγδ17 plasticity.
Fig. 4: TIM-3 marks ex-Tγδ17 cells with type 1 functionality.
Fig. 5: bZIP TFs are dynamically regulated during Vγ6+ Tγδ17 cell effector plasticity.
Fig. 6: JUNB and FOSL2 stabilize Vγ6+ Tγδ17 cell identity.
Fig. 7: JUNB and FOSL2 limit type 1 plasticity in Vγ6+ Tγδ17 cells during S. Typhimurium infection.
Fig. 8: JUNB directly regulates type 1 plasticity.

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Data availability

All sequencing data that support the findings of this study have been deposited in Gene Expression Omnibus under primary accession code GSE290581. All other data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

We thank E. Park for assistance with gut preps. We also thank the Molecular Genomics Core for processing and running the 10x Genomics Multiome samples. We acknowledge the expert assistance of L. Martinek with flow cytometry sorting and S. Langdon at the Duke DNA Sequencing Facility. We also acknowledge the use of the Duke Compute Cluster. Last, we thank Y. Yoshikai (Kyushu University) for sharing the antibody to Vγ6. This work was funded by NIH R01 GM115474 and P01 AI102853 grants to M.C. M.E.P. was supported by F31 AI152457 and a Duke Training Grant in Digestive Diseases and Nutrition (5T32DK007568). N.U.M. was supported by F31 AI181082.

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Author notes

  1. These authors contributed equally: Morgan E. Parker, Naren U. Mehta.

Authors and Affiliations

  1. Department of Integrative Immunobiology, Duke University School of Medicine, Durham, NC, USA

    Morgan E. Parker, Naren U. Mehta, Tzu-Chieh Liao, William H. Tomaszewski, Stephanie A. Snyder, Julia Busch & Maria Ciofani

  2. Center for Advanced Genomic Technologies, Duke University, Durham, NC, USA

    Morgan E. Parker, Naren U. Mehta, Tzu-Chieh Liao, Stephanie A. Snyder, Julia Busch & Maria Ciofani

  3. Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, USA

    Maria Ciofani

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Contributions

M.E.P. and N.U.M. designed, performed and analyzed the experiments. T.-C.L. performed computational analysis for single-cell multiome experiments. W.H.T. and M.E.P. performed initial computational analyses for single-cell multiome experiments. T.-C.L. and J.B. performed the CUT&RUN analysis. J.B. and M.E.P performed the C. rodentium infection experiments. S.A.S. maintained the mouse colony and performed infections. M.E.P., N.U.M. and M.C. wrote the manuscript. M.C. conceived the study and designed, supervised and analyzed the experiments.

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Correspondence to Maria Ciofani.

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Nature Immunology thanks Venuprasad Poojary, Bruno Silva-Santos and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: L. A. Dempsey, in collaboration with the Nature Immunology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Tγδ17 cells are stable at steady state and Vγ6+ Tγδ17 cells are plastic after S. typhimurium in mLN and coLP.

(a–i, k–n) Flow cytometric analysis across multiple tissues of fate-mapping mice. (a) RORγt, IL-17A, and ZsGreen expression in total γδ T cells from Rorc-Cre R26ZSG and Il17aCre R26ZSG mice. Representative of 3 experiments, n = 11. (b) Vγ6, Vγ4, and ZsGreen expression in total (left, top right) and ZsGreen+ (bottom right) γδ T cells from Rorc-Cre R26ZSG mice from 2 experiments, n = 10. (c) γδ T cells (CD3+γδTCR+TCRβ−) from tissues of Il17aCre R26TOM IFNγ-YFP mice at steady state; positive IFNγ-YFP control from coLP 4 days post-S. typhimurium (STm). Two experiments: n = 3 (FRT), 5 (siLP, coLP, lung, mLN, iLN). (d) RORγt and T-bet expression in γδ T cells from Il17aCre R26ZSG mice. 2 experiments, n = 6. (e) IL-17A and IFNγ in ZsGreen+ γδ T cells from coLP of naïve (n = 11) and STm (n = 12) infected Il17aCre R26ZSG mice; three experiments. (f) IL-17A−IFNγ+ ZsGreen− γδ T cells from coLP. n = 9 naïve, 10 STm; two experiments. (g) Frequency of IL-17A+IFNγ−, IL-17A+IFNγ+, IL-17A−IFNγ+ Vγ4+ ZsGreen+ γδ T cells from coLP. n = 9 naïve, 10 STm; two experiments. (h) Cytokine production by ZsGreen+ γδ T cells from mLN. Three experiments, n > 10. (i) Vγ4 or Vγ6 versus IFNγ-YFP in TOM+ γδ T cells from coLP of naïve and STm infected mice from 3 experiments, n = 10. (j) Splenic CFUs post-aroA− STm infection. n = 4/timepoint; one experiment. (k) IFNγ+ Vγ6+ ZsGreen+ γδ T cells after actA− L. monocytogenes (LmOva actA-) infection. n = 2-4/timepoint; one experiment. (l) IL-17A and IFNγ in CD4+ ZsGreen+ and Vγ6+ γδ T cells 15 days post-C. rodentium. Two experiments, summary from one, naïve (n = 4), Cr (n = 6). (m) Normalized RORγt in Vγ6+ ZsGreen+ γδ T cells from naïve (n = 11) and STm (n = 15) infected mice; three experiments. (n) IL-22 in CD4+ ZsGreen+ and Vγ6+ γδ T cells post-aroA− STm infection. n = 4/timepoint; one experiment. Cytokine expression in (a, e–h, k, l, n) measured after 4 h PMA/ionomycin stimulation. All results represent mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant (two-tailed unpaired Student’s t-test). Numbers in flow plots represent percentages of cells in the gate.

Extended Data Fig. 2 Single cell multiome characterization of γδ T cells and Vγ6+ Tγδ17 cell trajectories.

(a) Bar plot of number of cells in each cluster from each condition for total γδ T cells. (b, c) Violin plots of type 3 and 1 genes for γδ T cells clusters. (d) Volcano plot for cells from naïve condition for C0 vs C6 Vγ6+ Tγδ17 cells, with blue dots indicating significance based on p-val adj < 0.05 and log2FC > 0.25; FC, fold change. (e) zFP506 (ZsGreen) Feature plot for γδ T cell clusters. (f) Plot of per cell unspliced versus spliced Rorc transcript, RNA velocity for Rorc, and Rorc expression feature plot all for Vγ6+ Tγδ17 clusters C0, C6, C9 and C7. (g) Monocle 3 trajectory of Vγ6+ Tγδ17 clusters C0, C6, C9 and C7. (h) CD8+ T cell gene signatures (GSE9650) projected onto UMAP space with Seurat’s AddModuleScore function. Nebulosa Density plot displaying enrichment of gene signature. (Left) Genes upregulated in naive vs effector CD8+ T cells. (Right) Genes downregulated in naive vs effector CD8+ T cells. (i) Euclidian distances between γδ T cell clusters based on WNN UMAP. Seurat MAST test (GLM framework) was used for differential expression in d.

Extended Data Fig. 3 Effector-converted Vγ6+ Tγδ17 cells have distinct transcriptional profiles compared to steady state.

(a) IFNγ-YFP expression in fate-mapped TIM-3− or TIM-3+ Vγ6+ TOM+ γδ T cells from naïve and S. typhimurium-infected Il17aCre R26TOM IFNγ-YFP mice. Summary plot pooled from two experiments, n = 8 mice. (b, c) Flow cytometric analysis of colonic Vγ6+ γδ T cells at 4 days post S. typhimurium infection, following daily intraperitoneal injections of either an isotype control or anti-TIM-3 antibody: (b) Fate-mapped Vγ6+ ZS+ γδ T cells from Rorc-Cre R26ZSG mice. Summary data include IFNγ expression after 4 h PMA/ionomycin stimulation and the absolute number of unstimulated Vγ6+ ZS+ γδ T cells (one experiment; n = 5 per condition). (c) Fate-mapped Vγ6+ TOM+ γδ T cells from Il17aCre R26TOM IFNγ-YFP mice. Data show IFNγ-YFP expression, Ki-67 expression, and the total number of Vγ6+ TOM+ γδ T cells (one experiment; n = 5 per condition). (d) Summary data for PD-1 gMFI in PD-1+TIM-3− or PD-1+TIM-3+ Vγ6+ ZS+ γδ T cells from three experiments, n = 13 mice. (e) Volcano plot for differentially expressed genes between C7 vs C9 Vγ6+ Tγδ17 cells with blue dots having p-val adj < 0.05 and log2FC > 0.25. (f) Volcano plot of differentially expressed transcription factors in type 1 converting Vγ6+ γδ T cell clusters (C7 + C9) compared to type 3 steady state clusters (C0 + C6) with red dots having p-val adj < 0.05 and log2FC > 0.25; FC, fold change. (g) Dot plot for Tγδ17 cell clusters for select transcriptional regulators downregulated (left) or upregulated (right) in Vγ6+ Tγδ17 cells with Vγ4+ Tγδ17 cells for comparison. All results represent mean ± s.e.m. Two-tailed paired t-test for a, d, two-tailed unpaired t-test for b, c, and Seurat MAST test (GLM framework) was used for differential expression in e, f. ***P < 0.001; ****P < 0.0001; ns, not significant (two-tailed paired Student’s t-test). Numbers in flow plots represent percentages of cells in the gate.

Extended Data Fig. 4 BACH2 and AP-1 TFs regulate Vγ6+ Tγδ17 plasticity in vitro.

(a) Schematic of Vγ6+ Tγδ17 cluster 0, 6, 9, and 7 for number of regions differentially accessible (DA) (p < 0.05) with regions increasing (UP, red) and decreasing (DOWN, blue) in accessibility. (b) Pseudobulk scATAC-seq CoveragePlots for Rorc and Ifng loci for Vγ6+ Tγδ17 cluster 0, 6, 9, and 7. Rectangle highlights regions with significant differential accessibility (p < 0.05) shown for decreasing (orange) or increasing (blue) in accessibility. Asterisks indicate JunB CUT&RUN binding in Vγ6+ Tγδ17 cells. (c) Motif activity dot plot of Vγ6+ Tγδ17 clusters using chromVAR with colored boxes highlighting specific TF families. (d) TFs in Vγ6+ Tγδ17 cell overexpression screen. X’s in RNA DEG column means the TF of interest is a DEG at some point along trajectory. X in Regulon column means the TF is significant in regulon analysis. X in Motif Activity column means TF has differential motif activity (chromVAR) during conversion. X in Literature column means TF is implicated in type 3 lymphocyte regulation. Blue TFs predicted to stabilize type 3 program and green TFs predicted to promote type 1 conversion. (e) Flow cytometric analysis of cytokine production from day 9 Tγδ17 mLN culture. Gated on transduced Vγ6+ (Vγ4−) Thy1.1+ ZS+ γδ T cells from steady state Il17aCreR26ZSG mice after 4 h PMA/Ionomycin stimulation. Summary graph pooled from two independent experiments. (f) Same as in e but from S. typhimurium infected Il17aCreR26ZSG mice. Summary graph from one independent experiment. For e, all conditions have n = 3 except SMAD3, LEF1, HIF1a, and ETV6, which have n = 2; for f, all conditions have n = 2. Statistical analyses included the LR framework test with Signac for differential accessibility calling in a, b, and chromVAR z-score based deviation test in c, and ordinary one-way ANOVA tests for e, f. Results represent mean ± s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001; DEG, differentially expressed gene; ns, not significant.

Extended Data Fig. 5 JunB plays a more prominent role than Fosl2 in Vγ6+ Tγδ17 cell plasticity.

(a, b) Flow cytometric analysis was performed on coLP ZsGreen+ Vγ6+ Tγδ17 cells from mice with compound Junb and Fosl2 conditional deletions on the Il17aCreR26ZSG deleter background at steady state (TF+/+, TFWT; TFfl/+, TFHET; TFfl/fl, TFKO): (a) Representative flow cytometric analysis of the frequency of IL-17A and IFNγ producing cells following 4 h PMA/ionomycin stimulation. (n = 8 WT, 5 JunB KO Fosl2 KO; three independent experiments) (b) Representative flow cytometric analysis and summary plots of the frequency of IL-17A and IFNγ producing colonic ZS+ Vγ6+ Tγδ17 cells at steady state following 20 h stimulation with IL-23 and IL-1β. (n = 6 Fosl2 KO, 11 JunB KO, 4 Fosl2 KO JunB HET, 4 Fosl2 HET JunB KO, 5 JunB KO Fosl2 KO; four independent experiments). (c) Flow cytometric analysis was performed on ZS+ Vγ6+ from mLN of naïve Bach2+/+Il17aCreR26ZSG (Bach2WT) and Bach2fl/flIl17aCreR26ZSG (Bach2KO) mice on day 9 of Tγδ17 mLN culture. Summary plots of the frequency of IL-17A and IFNγ producing cells following 4 h PMA/ionomycin stimulation (n = 5 mice/genotype; three independent experiments). (d) Gating strategy for fate-mapped Vγ6+ Tγδ17 cells (CD3ε+γδTCR+TCRβ−ZS+Vγ6+). (e) JunB gMFI measured by flow cytometry after 24 h culture with cytokines from coLP ZS+ Vγ6+ Tγδ17 cells sorted from Il17aCreR26ZSG mice. (f) Representative CUT&RUN tracks for JunB and Fosl2 in Vγ6+ Tγδ17 cells and pseudobulk ATAC track for C0 Vγ6+ Tγδ17 cells. Orange bar represents significant peak called over IgG control. Break in gene intron represented by // or \\. Visualized in IGV. (g) Barplot showing JunB-bound or not bound DEGs between C7 vs C0 Vγ6+ Tγδ17 cells (p-val adj < 0.05 and log2FC > |0.58|). (a–c) Gating was performed on fate-mapped Vγ6+ Tγδ17 cells (CD3ε+γδTCR+TCRβ−ZS+Vγ4−). Statistical analyses include Two-tailed unpaired Student’s t-tests for (a, c) and an Ordinary one-way ANOVA test for (b). Results represent mean ± s.e.m. *P < 0.05; ****P < 0.0001; ns, not significant. Numbers in flow plots represent percentages of cells in the gate. DEG, differentially expressed gene.

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Parker, M.E., Mehta, N.U., Liao, TC. et al. Restriction of innate Tγδ17 cell plasticity by an AP-1 regulatory axis. Nat Immunol 26, 1299–1314 (2025). https://doi.org/10.1038/s41590-025-02206-7

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