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

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 V_γ6⁺ Tγδ17 cell conversion into type 1 effectors, with loss of IL-17A production and partial RORγt downregulation. Multiome analysis revealed a trajectory along V_γ6⁺ 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 V_γ6⁺ Tγδ17 cell plasticity by stabilizing type 3 identity and restricting type 1 effector conversion.

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 R26^ZSG and Il17a^Cre R26^ZSG 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 R26^ZSG mice from 2 experiments, n=10. (c) γδ T cells (CD3⁺γδTCR⁺TCRβ⁻) from tissues of Il17a^Cre R26^TOM 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 Il17a^Cre R26^ZSG 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 Il17a^Cre R26^ZSG 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 4h 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 Il17a^Cre R26^TOM 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 R26^ZSG mice. Summary data include IFNγ expression after 4h 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 Il17a^Cre R26^TOM 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 Il17a^CreR26^ZSG mice after 4 h PMA/Ionomycin stimulation. Summary graph pooled from two independent experiments. (f) Same as in e but from S. typhimurium infected Il17a^CreR26^ZSG 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 Il17a^CreR26^ZSG deleter background at steady state (TF^+/+, TF^WT; TF^fl/+, TF^HET; TF^fl/fl, TF^KO): (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^+/+Il17a^CreR26^ZSG (Bach2^WT) and Bach2^fl/flIl17a^CreR26^ZSG (Bach2^KO) 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 24h culture with cytokines from coLP ZS⁺ Vγ6⁺ Tγδ17 cells sorted from Il17a^CreR26^ZSG 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.

Fig. 1.. Tγδ17 cell identity is stable at steady state and IFNγ production is restrained in intestinal T-bet⁺ Tγδ17 cells.

(a) Flow cytometric analysis of RORγt versus ZsGreen (ZS) expression in total γδ T cells (CD3⁺γδTCR⁺TCRβ⁻) from Rorc-Cre R26^ZSG mice. Flow cytometric analysis of cytokine production by (b) total γδ T cells (CD3⁺γδTCR⁺TCRβ⁻) and (c) ZS⁺ CD3⁺γδTCR⁺TCRβ⁻ cells from lymphoid (iLN, mLN) and nonlymphoid tissues (siLP, coLP, Lung, FRT) of Rorc-Cre R26^ZSG mice after 4 h of PMA/Ionomycin stimulation. Representative of three or more independent experiments with n=8, except lung n=5. (d) Flow cytometric analysis of RORγt and T-bet expression in siLP and coLP γδ T cells (CD3⁺γδTCR⁺TCRβ⁻) from Rorc-Cre R26^ZSG mice. (e) As in (a), except analysis of Vγ6 and ZsGreen expression among RORγt⁺T-bet⁺ γδ T cells. (f) Cytokine production after 4 h PMA/Ionomycin stimulation of siLP and coLP RORγt⁺T-bet⁺ ZS⁺ γδ T cells (top) and coLP RORγt⁻T-bet⁺ ZS⁻ γδ T cells (bottom). Three or more independent experiments performed. d, e, and f summary plots are from two independent experiments; (d) left n=7 siLP, 10 coLP, (d) right n=6, (e) left n=4, (e) right n=6 mice. All results represent mean ± s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant (two-tailed unpaired Student’s t-test). Numbers in flow plots represent percentages of cells in the gate.

Fig. 2.. Vγ6⁺ Tγδ17 cells are functionally plastic after intestinal S. typhimurium infection.

(a–i) Flow cytometry of cells isolated from coLP. (a) IFNγ vs. ZsGreen expression in γδ T cells from naïve and S. typhimurium (STm) infected Rorc-Cre R26^ZSG mice (three experiments; n= 13 naïve, 15 STm). (b) Cytokine production in ZS⁻ and ZS⁺ γδ T cells from the coLP of naïve (n=9) or STm (n=10) infected Il17a^Cre R26^ZSG mice, further gated on Vγ4⁺ or Vγ6⁺ subsets; two experiments. (c) IL-17A and IFNγ production in ZsGreen⁺ Vγ6⁺ Tγδ17 cells from coLP of Il17a^Cre R26^ZSG mice at indicated days post-STm infection (one experiment; n= 4/timepoint). (d) As in (c), with attenuated aroA⁻ S. typhimurium (aroA⁻ STm), evaluated at the indicated time points (one experiment; n = 4/timepoint). (e) RORγt expression in ILCs (CD3⁻CD127⁺CCR6⁻) and γδ T cells for ZS⁻ and ZS⁺ populations in naïve and STm-infected Rorc-Cre R26^ZSG mice (two experiments; n= 6 naïve, 10 STm). (f) IL-17A and IFNγ production in ZS+ CD4+ T cells (CD3+CD4+) from Il17a^Cre R26^ZSG mice at day 6 post-infection with aroA⁻ STm or low dose STm (one experiment; n= 4 naïve, 5 aroA⁻ STm [aroA⁻], 7 STm). (g) As in (f), representative histograms and summary data of RORγt expression in Vγ6⁺ γδ T and CD4⁺ T cells from ZS− and ZS+ populations at day 6 post-infection (n= 4 naïve, 5 aroA⁻, 7 STm). (h) RORγt-E2-Crimson expression in CD127⁺ or CD127⁻ subsets of Vγ4⁻ T-bet⁻ZsGreen⁺ γδ T cells in RORγt-E2-Crimson reporter mice (one experiment; n= 4 naïve, 5 STm). (i) IFNγ and RORγt expression in Vγ6⁺ ZS⁺ and ZS⁻ γδ T cells from naïve and STm-infected Rorc-Cre R26^ZSG mice (one experiment; n= 5, representative of ≥2 experiments. a-c, d, f, i cytokine expression following 4 h PMA/ionomycin stimulation. Statistical analyses: Two-tailed unpaired Student’s t-tests for a, b, h; ordinary one-way ANOVA test for e, f-i. Results represent mean ± s.e.m. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Numbers in flow plots represent percentages of cells within gates.

Fig. 3.. Single cell multiome characterization of Vγ6⁺ Tγδ17 plasticity.

(a) Dimension reduction plot of γδ T cells reclustered with WNN reduction. (b) Dimension reduction plot of γδ T cells from each condition. (c) Feature plot showing Rorc and Tbx21 expression in γδ T cells clusters. (d) Feature plot showing Cd163l1 (Scart1), 5830411N06Rik (Scart2), and Trdv4 expression in γδ T cells clusters. (e) Violin plots of Rorc, Tbx21, Il17a, and Ifng expression for Vγ6⁺ Tγδ17 cell clusters. (f) Dot plot for select genes in Vγ6⁺ Tγδ17 cell clusters. (g) TSCAN pseudotime trajectory on a feature plot of Vγ6⁺ Tγδ17 clusters C0, C6, C9 and C7. (h) RNA Velocity (bottom) of Vγ6⁺ Tγδ17 clusters C0, C6, C9 and C7. (i) Groups of genes changing along TSCAN pseudotime as in (g). (j) Volcano plot of post-infection Vγ6⁺ Tγδ17 cell clusters (C7+C9) compared to steady state clusters (C0+C6) with red dots having p-val adj < 0.05 and log2FC > 0.25. One-way ANOVA Tukey test for e Rorc plot and Seurat MAST test (GLM framework) was used for differential expression in j. ***P < 0.001; ns, not significant.

Fig. 4.. TIM-3 marks ex-Tγδ17 cells with type 1 functionality.

(a) Volcano plot for differentially expressed genes for C7 versus C9 Vγ6⁺ Tγδ17 cells. Red dots denote significant differences with p-val adj < 0.05 and log₂FC > 0.25; FC, fold change. (b) Violin plots showing expression of select genes in Vγ6⁺ Tγδ17 cell clusters. (c) Flow cytometric analysis of PD-1 and TIM-3 expression on ZS⁺ Tγδ17 cells from naive and S. typhimurium (STm)-infected Il17a^Cre R26^ZSG mice. (Top) Vγ6⁺ Tγδ17 cells and (bottom) are Vγ4⁺ Tγδ17 cells gated as Vγ6⁻ Tγδ17 cells. Summary data from one experiment, representative of more than three experiments. (d) Flow cytometry plots showing cytokine production after ex vivo stimulation gated on TIM-3⁻ versus TIM-3⁺ Vγ6⁺ Tγδ17 cells from S. typhimurium (STm)-infected Il17a^Cre R26^ZSG mice. Summary plots from two independent experiments, n=10 mice. (e) Nebulosa density plot for Havcr2 and Mki67 for Vγ6⁺ Tγδ17 cell clusters. (f) Ki-67 frequency in ZS⁺ Tγδ17 cells from naive and S. typhimurium (STm)-infected Il17a^Cre R26^ZSG or Rorc-Cre R26^ZSG mice. Representative histograms gated on Vγ6⁺ or Vγ4⁺ Tγδ17 cells. Summary data compiled from two independent experiments, n=6 naive mice and n=9 STm mice. (g) Histogram gated on TIM-3⁻ versus TIM-3⁺ Vγ6⁺ Tγδ17 cells or Vγ6⁺ Tγδ17 cells from a naive mouse as a control. Summary data of percent Ki-67⁺ among TIM-3⁻ versus TIM-3⁺ Vγ6⁺ Tγδ17 cells from S. typhimurium (STm)-infected Il17a^Cre R26^ZSG or Rorc-Cre R26^ZSG mice, compiled from two independent experiments, n=9 mice. Statistical analyses included Seurat MAST test (GLM framework) for differential expression in a, Two-tailed unpaired Student’s t-test for c, f, and Two-tailed paired Student’s t-test for d, g. Results represent mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Numbers in flow plots represent percentages of cells in the gate.

Fig. 5.. bZIP TFs are dynamically regulated during Vγ6⁺ Tγδ17 cell effector plasticity.

(a) Regulon activity using SCENIC analysis on Vγ6⁺ Tγδ17 cell clusters C0, C6, C9, and C7. Colored genes draw attention to specific gene families. Red = AP-1 family. (b) Motif activity analysis on differentially accessible regions between Vγ6⁺ Tγδ17 cell clusters C7+C9 (post-infection) compared to C0+C6 (steady state). Vertical dotted line represents fold-change cut off at 1.25 for average difference in z-score in terms of fold-change between groups. Horizontal line represents p-val adjusted cut off at 5×10⁻⁵. Colored circles represent TF families. (c) Representative top motifs displayed as MotifPlots per TF family circled in (b). (d) Motif activity (red) and mRNA expression (black) for select TFs across Vγ6⁺ Tγδ17 pseudotime. (e) Flow cytometric analysis of cytokine production in day 9 of Tγδ17 mLN culture. Gated on transduced Vγ6⁺ Thy1.1⁺ ZS⁺ γδ T cells after 4 h PMA/Ionomycin stimulation. Top row is from naive mLN cultures from Il17a^CreR26^ZSG mice (n=3), and bottom row is from mLN cultures from S. typhimurium (STm)-infected Il17a^CreR26^ZSG mice (n=4 for EV, Bach2, Fos and n=3 for DN Fosl2, Fosl2). Summary graph is pooled from two independent experiments for both top and bottom. Statistical analyses include chromVAR z-score based deviation test for differential motif activity in b and an Ordinary one-way ANOVA test for e. Results represent mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Numbers in flow plots represent percentages of cells in the gate.

Fig. 6.. JunB and Fosl2 stabilize Vγ6⁺ Tγδ17 cell identity.

(a-i) Flow cytometric analysis was performed on colonic ZsGreen⁺ Vγ6⁺ Tγδ17 cells from mice with Junb, Fosl2, or Bach2 conditional deletions on the Il17a^CreR26^ZSG deleter background at steady state (TF^+/+, TF^WT; TF^fl/+, TF^HET; TF^fl/fl, TF^KO). (a) Histogram of T-bet expression, and (a,c,e) summary plots of normalized T-bet and RORγt expression. (b,d,f) Representative flow cytometric analysis and summary plots of the frequency of IL-17A and IFNγ producing cells following 4 h PMA/ionomycin stimulation. (a-b) Junb: (a) n = 9 WT, 11 HET, 6 KO; three experiments. (b) n = 7 WT, 7 HET, 5 KO; three experiments. (c-d) Fosl2: (c) n = 5 WT, 4 HET, 3 KO; two experiments. (d) n = 6 WT, 4 HET, 6 KO; two experiments. (e-f) Bach2: (e) n = 6 WT, 5 HET, 5 KO; two experiments. (f) n = 3 WT, 4 HET, 5 KO; two experiments. (g-i) Representative flow cytometric analysis and summary plots of the frequency of IL-17A and IFNγ producing cells at steady state following 20h stimulation with IL-23 and IL-1β for the following genotypes: (g) Junb (n = 8 WT, 6 KO; two experiments), (h) Fosl2 (n = 8 WT, 3 KO; two experiments), and (i) Bach2 (n = 9 WT, 9 KO; two experiments). Fate-mapped Vγ6⁺ Tγδ17 cells (CD3ε⁺γδTCR⁺TCRβ⁻ZS⁺) were gated as Vγ6⁺ (a-f) or Vγ4⁻ (g-i). Statistical analyses include Ordinary one-way ANOVA tests for (a-f) and Two-tailed unpaired Student’s t-tests for (g-i). Results represent mean ± s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant. Numbers in flow plots represent percentages of cells in the gate.

Fig. 7.. JunB and Fosl2 limit type 1 plasticity in Vγ6⁺ Tγδ17 cells during S. typhimurium infection.

(a-i, k-m) Flow cytometry of coLP from Il17a^CreR26^ZSG mice with conditional deletion of Junb, Fosl2, Bach2, or Rorct at steady state or after S. typhimurium infection. TF genotypes: TF^+/+, TF^WT; TF^fl/+, TF^HET; TF^fl/fl, TF^KO. (a-f, i, k-m) Mice were infected with S. typhimurium and Vγ6⁺ ZsGreen⁺ Tγδ17 cells were analyzed at day 4 or as indicated; two experiments/condition. (a, b, e) RORγt histograms, normalized RORγt and T-bet expression. (c, d, f) IL-17A and IFNγ production. (a, c) Junb: n=4 WT, 6 HET, 5 KO. (b, d) Fosl2: (b) n=6 WT, 7 HET, 4 KO; (d) 7 WT, 10 HET, 5 KO. (e, f) Bach2: (e) n=6 WT, 10 HET, 8 KO; (f) 6 WT, 8 HET, 8 KO. (g, h) Steady state Rorct WT versus HET mice; three experiments. (g) T-bet histograms, normalized T-bet and RORγt expression (n=10 WT, 7 HET). (h) IL-17A and IFNγ production (n=11 WT, 7 HET). (i) ZsGreen⁺ Vγ6⁺ Tγδ17 cell numbers at day 3 (n=8 WT, 7 Fosl2 KO, 6 Junb KO) and day 4 (n=9 WT, 9 Fosl2 KO, 7 Junb KO); two experiments/timepoint. (j-m) Fosl2 WT and KO mice; two experiments each. (j) Splenic CFUs at day 4 (n=11 WT, 9 KO). (k) Proportion and number of IFNγ⁺ ZsGreen⁺ Vγ6⁺ Tγδ17 and αβ T cells: day 3 (n=8 WT, 7 KO); day 4 (n=11 WT, 9 KO). (l) Frequency of CD4⁺ αβ T cells (n = 9 WT, 8 KO). (m) Frequency of IFNγ⁺ ZsGreen⁺ cells among Vγ6⁺ Tγδ17 (n=11 WT, 9 KO), αβ (n = 9 WT, 8 KO), and CD4⁺ (n=9 WT, 8 KO) T cells. Fate-mapped Vγ6⁺ Tγδ17 cells gated as CD3ε⁺γδTCR⁺TCRβ⁻ZS⁺ (Vγ6⁺ in a-f, i; Vγ4⁻ in g, h, k-m), αβ T cells as CD3ε⁺γδTCR⁻TCRβ⁺ZS⁺, and CD4⁺ T cells as CD3ε⁺γδTCR⁻TCRβ⁺CD4⁺ZS⁺. Cytokine expression in c, d, f, h, k, m measured following 4h PMA/ionomycin stimulation. Statistical analysis used one-way ANOVA (a-f, i), two-tailed unpaired Student’s t-test (g, h, j, l, m). Data represent mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Numbers in flow plots represent percentages of cells within gates.

Fig. 8.. JunB directly regulates type 1 plasticity.

(a) JunB and Fosl2 gMFI measured by flow cytometry after 42h culture with cytokines for ZS⁺ Vγ6⁺ Tγδ17 cells sorted from day 5 Tγδ17 mLN expansion culture from Il17a^CreR26^ZSG mice. Two independent experiments performed, n=4 (except IL-18+IL-2 and IL-18+IL-1β, n=2). Summary data pooled from two independent experiments for JunB and one experiment for Fosl2. (b) mRNA expression for select genes across Vγ6⁺ Tγδ17 pseudotime. (c-g) CUT&RUN for JunB and Fosl2 in Vγ6⁺ Tγδ17 cells sorted from day 5 Tγδ17 mLN expansion culture from Il17a^CreR26^ZSG mice. (c) Venn diagram for overlap of Fosl2 peaks with JunB peaks. (d) Median distance from peak summits for TF pairs. Th17 ChIP-seq: n=2 (JunB/BATF), 4 (JunB/Fosl2, JunB/RORγt, JunB/Maf); Vγ6⁺ Tγδ17 CUT&RUN: n=3 (JunB/JunB), 6 (JunB/Fosl2). (e) Network of select JunB-bound DEGs between C7 vs C0 Vγ6⁺ Tγδ17 cells visualized with Cytoscape (p-val adj < 0.05 and log2FC > |0.58|), grouped by function, color of nodes represents log₂FC. Dotted lines indicate a log₂FC<|0.58| for Il7r and Runx1. (f, g) Representative tracks for JunB CUT&RUN in Vγ6⁺ Tγδ17 cells and pseudobulk ATAC track for C0 Vγ6⁺ Tγδ17 cells visualized with IGV. Orange bar represents significant peak called over IgG control in at least 2 of 3 biological replicates. Conserved noncoding sequences (CNSs) labeled for distance in kilobases from transcription start site. Ordinary one-way ANOVA test for (a, d). Results represent mean ± s.e.m.**P < 0.01; ****P < 0.0001; DEG, differentially expressed gene; ns, not significant.