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The transcription factor c-Maf is essential for the commitment of IL-17-producing γδ T cells

A Publisher Correction to this article was published on 28 February 2019

This article has been updated

Abstract

γδ T cells that produce the cytokine IL-17 (Tγδ17 cells) are innate-like mediators of immunity that undergo effector programming in the thymus. While regulators of Tγδ17 specialization restricted to various Vγ subsets are known, a commitment factor essential to all Tγδ17 cells has remained undefined. In this study, we identified the transcription factor c-Maf as a universal regulator of Tγδ17 cell differentiation and maintenance. Maf deficiency caused an absolute lineage block at the immature CD24+CD45RBlo γδ thymocyte stage, which revealed a critical checkpoint in the acquisition of effector functions. Here, c-Maf enforced Tγδ17 cell identity by promoting chromatin accessibility and expression of key type 17 program genes, notably Rorc and Blk, while antagonizing the transcription factor TCF1, which promotes interferon-γ-producing γδ T cells (Tγδ1 cells). Furthermore, γδ T cell antigen receptor (γδTCR) signal strength tuned c-Maf expression, which indicates that c-Maf is a core node that connects γδTCR signals to Tγδ17 cell transcriptional programming.

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Fig. 1: Selective expression of c-Maf in Tγδ17 cells.
Fig. 2: Selective loss of peripheral Tγδ17 cells in the absence of c-Maf.
Fig. 3: c-Maf is required for Tγδ17 differentiation.
Fig. 4: c-Maf maintains Tγδ17 cell features.
Fig. 5: c-Maf controls the regulatory status of the Rorc locus in γδ thymocytes.
Fig. 6: c-Maf is essential for Tγδ17 programming.
Fig. 7: TCR signals modulate c-Maf expression during γδ diversification.

Data availability

Sequence data that support the findings of this study have been deposited in the Gene Expression Omnibus with the primary accession code GSE120427. Other sequence data referenced in this study are listed in the Online Methods.

Change history

  • 28 February 2019

    In the version of this article initially published, the top right plot in Figure 4a was aligned incorrectly. The error has been corrected in the HTML and PDF versions of the article. The original and corrected figures are provided in the accompanying Publisher Correction.

References

  1. 1.

    Shibata, K., Yamada, H., Hara, H., Kishihara, K. & Yoshikai, Y. Resident Vδ1+ γδ T cells control early infiltration of neutrophils after Escherichia coli infection via IL-17 production. J. Immunol. 178, 4466–4472 (2007).

    CAS  Article  Google Scholar 

  2. 2.

    Conti, H. R. et al. Oral-resident natural Th17 cells and γδ T cells control opportunistic Candida albicans infections. J. Exp. Med. 211, 2075–2084 (2014).

    CAS  Article  Google Scholar 

  3. 3.

    Papotto, P. H., Reinhardt, A., Prinz, I. & Silva-Santos, B. Innately versatile: γδ 17 T cells in inflammatory and autoimmune diseases. J. Autoimmun. 87, 26–37 (2017).

    Article  Google Scholar 

  4. 4.

    Ribot, J. C. et al. CD27 is a thymic determinant of the balance between interferon-γand interleukin 17-producing γδ T cell subsets. Nat. Immunol. 10, 427–436 (2009).

    CAS  Article  Google Scholar 

  5. 5.

    Havran, W. L. & Allison, J. P. Origin of Thy-1+ dendritic epidermal cells of adult mice from fetal thymic precursors. Nature 344, 68–70 (1990).

    CAS  Article  Google Scholar 

  6. 6.

    Itohara, S. et al. Homing of a γδ thymocyte subset with homogeneous T-cell receptors to mucosal epithelia. Nature 343, 754–757 (1990).

    CAS  Article  Google Scholar 

  7. 7.

    Xiong, N. & Raulet, D. H. Development and selection of γδ T cells. Immunol. Rev. 215, 15–31 (2007).

    CAS  Article  Google Scholar 

  8. 8.

    Haas, J. D. et al. Development of interleukin-17-producing γδ T cells is restricted to a functional embryonic wave. Immunity 37, 48–59 (2012).

    CAS  Article  Google Scholar 

  9. 9.

    Jensen, K. D. et al. Thymic selection determines γδ T cell effector fate: antigen-naive cells make interleukin-17 and antigen-experienced cells make interferon γ. Immunity 29, 90–100 (2008).

    CAS  Article  Google Scholar 

  10. 10.

    Turchinovich, G. & Hayday, A. C. Skint-1 identifies a common molecular mechanism for the development of interferon-γ-secreting versus interleukin-17-secreting γδ T cells. Immunity 35, 59–68 (2011).

    CAS  Article  Google Scholar 

  11. 11.

    Sumaria, N., Grandjean, C. L., Silva-Santos, B. & Pennington, D. J. Strong TCR γδ signaling prohibits thymic development of IL-17A-secreting γδ T cells. Cell Rep. 19, 2469–2476 (2017).

    CAS  Article  Google Scholar 

  12. 12.

    Malhotra, N. et al. A network of high-mobility group box transcription factors programs innate interleukin-17 production. Immunity 38, 681–693 (2013).

    CAS  Article  Google Scholar 

  13. 13.

    Shibata, K. et al. Notch-Hes1 pathway is required for the development of IL-17-producing γδ T cells. Blood 118, 586–593 (2011).

    CAS  Article  Google Scholar 

  14. 14.

    Do, J. S. et al. Cutting edge: spontaneous development of IL-17-producing γδ T cells in the thymus occurs via a TGF-β1-dependent mechanism. J. Immunol. 184, 1675–1679 (2010).

    CAS  Article  Google Scholar 

  15. 15.

    In, T. S. H. et al. HEB is required for the specification of fetal IL-17-producing γδ T cells. Nat. Commun. 8, 2004 (2017).

    Article  Google Scholar 

  16. 16.

    Lu, Y., Cao, X., Zhang, X. & Kovalovsky, D. PLZF controls the development of fetal-derived IL-17+ Vγ6+ γδ T cells. J. Immunol. 195, 4273–4281 (2015).

    CAS  Article  Google Scholar 

  17. 17.

    Bauquet, A. T. et al. The costimulatory molecule ICOS regulates the expression of c-Maf and IL-21 in the development of follicular T helper cells and TH-17 cells. Nat. Immunol. 10, 167–175 (2009).

    CAS  Article  Google Scholar 

  18. 18.

    Ho, I. C., Hodge, M. R., Rooney, J. W. & Glimcher, L. H. The proto-oncogene c-Maf is responsible for tissue-specific expression of interleukin-4. Cell 85, 973–983 (1996).

    CAS  Article  Google Scholar 

  19. 19.

    Rutz, S. et al. Transcription factor c-Maf mediates the TGF-β-dependent suppression of IL-22 production in TH17 cells. Nat. Immunol. 12, 1238–1245 (2011).

    CAS  Article  Google Scholar 

  20. 20.

    Ciofani, M. et al. A validated regulatory network for Th17 cell specification. Cell 151, 289–303 (2012).

    CAS  Article  Google Scholar 

  21. 21.

    Yu, J. S. et al. Differentiation of IL-17-producing invariant natural killer T cells requires expression of the transcription factor c-Maf. Front. Immunol. 8, 1399 (2017).

    Article  Google Scholar 

  22. 22.

    Wheaton, J. D., Yeh, C. H. & Ciofani, M. Cutting edge: c-Maf is required for regulatory T cells to adopt RORγt+ and follicular phenotypes. J. Immunol. 199, 3931–3936 (2017).

    CAS  Article  Google Scholar 

  23. 23.

    Xu, M. et al. c-MAF-dependent regulatory T cells mediate immunological tolerance to a gut pathobiont. Nature 554, 373–377 (2018).

    CAS  Article  Google Scholar 

  24. 24.

    Narayan, K. et al. Intrathymic programming of effector fates in three molecularly distinct γδ T cell subtypes. Nat. Immunol. 13, 511–518 (2012).

    CAS  Article  Google Scholar 

  25. 25.

    Barbee, S. D. et al. Skint-1 is a highly specific, unique selecting component for epidermal T cells. Proc. Natl Acad. Sci. USA 108, 3330–3335 (2011).

    CAS  Article  Google Scholar 

  26. 26.

    Schlenner, S. M. et al. Fate mapping reveals separate origins of T cells and myeloid lineages in the thymus. Immunity 32, 426–436 (2010).

    CAS  Article  Google Scholar 

  27. 27.

    Wende, H. et al. The transcription factor c-Maf controls touch receptor development and function. Science 335, 1373–1376 (2012).

    CAS  Article  Google Scholar 

  28. 28.

    Kashem, S. W. et al. Nociceptive sensory fibers drive interleukin-23 production from CD301b+ dermal dendritic cells and drive protective cutaneous immunity. Immunity 43, 515–526 (2015).

    CAS  Article  Google Scholar 

  29. 29.

    Laird, R. M., Laky, K. & Hayes, S. M. Unexpected role for the B cell-specific Src family kinase B lymphoid kinase in the development of IL-17-producing γδ T cells. J. Immunol. 185, 6518–6527 (2010).

    CAS  Article  Google Scholar 

  30. 30.

    Eberl, G. & Littman, D. R. Thymic origin of intestinal alphabeta T cells revealed by fate mapping of RORγt+ cells. Science 305, 248–251 (2004).

    CAS  Article  Google Scholar 

  31. 31.

    Visel, A. et al. ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457, 854–858 (2009).

    CAS  Article  Google Scholar 

  32. 32.

    Powolny-Budnicka, I. et al. RelA and RelB transcription factors in distinct thymocyte populations control lymphotoxin-dependent interleukin-17 production in γδ T cells. Immunity 34, 364–374 (2011).

    CAS  Article  Google Scholar 

  33. 33.

    Shibata, K. et al. IFN-γproducing and IL-17-producing γδ T cells differentiate at distinct developmental stages in murine fetal thymus. J. Immunol. 192, 2210–2218 (2014).

    CAS  Article  Google Scholar 

  34. 34.

    Muro, R. et al. γδ TCR recruits the Syk/PI3K axis to drive proinflammatory differentiation program. J. Clin. Invest. 128, 415–426 (2018).

    Article  Google Scholar 

  35. 35.

    Azzam, H. S. et al. CD5 expression is developmentally regulated by T cell receptor (TCR) signals and TCR avidity. J. Exp. Med. 188, 2301–2311 (1998).

    CAS  Article  Google Scholar 

  36. 36.

    Ito, K. et al. Different γδ T-cell receptors are expressed on thymocytes at different stages of development. Proc. Natl Acad. Sci. USA 86, 631–635 (1989).

    CAS  Article  Google Scholar 

  37. 37.

    Azuara, V., Lembezat, M. P. & Pereira, P. The homogeneity of the TCRδ repertoire expressed by the Thy-1dull γδ T cell population is due to cellular selection. Eur. J. Immunol. 28, 3456–3467 (1998).

    CAS  Article  Google Scholar 

  38. 38.

    Ciofani, M., Knowles, G. C., Wiest, D. L., von Boehmer, H. & Zuniga-Pflucker, J. C. Stage-specific and differential notch dependency at the αβ and γδ T lineage bifurcation. Immunity 25, 105–116 (2006).

    CAS  Article  Google Scholar 

  39. 39.

    Mombaerts, P., Anderson, S. J., Perlmutter, R. M., Mak, T. W. & Tonegawa, S. An activated lck transgene promotes thymocyte development in RAG-1 mutant mice. Immunity 1, 261–267 (1994).

    CAS  Article  Google Scholar 

  40. 40.

    Gabrysova, L. et al. c-Maf controls immune responses by regulating disease-specific gene networks and repressing IL-2 in CD4+ T cells. Nat. Immunol. 19, 497–507 (2018).

    CAS  Article  Google Scholar 

  41. 41.

    Vahedi, G. et al. STATs shape the active enhancer landscape of T cell populations. Cell 151, 981–993 (2012).

    CAS  Article  Google Scholar 

  42. 42.

    Barros-Martins, J. et al. Effector γδ T cell differentiation relies on master but not auxiliary Th cell transcription factors. J. Immunol. 196, 3642–3652 (2016).

    CAS  Article  Google Scholar 

  43. 43.

    Munoz-Ruiz, M., Sumaria, N., Pennington, D. J. & Silva-Santos, B. Thymic determinants of γδ T cell differentiation. Trends Immunol. 38, 336–344 (2017).

    CAS  Article  Google Scholar 

  44. 44.

    Tanaka, S. et al. Sox5 and c-Maf cooperatively induce Th17 cell differentiation via RORγt induction as downstream targets of Stat3. J. Exp. Med. 211, 1857–1874 (2014).

    CAS  Article  Google Scholar 

  45. 45.

    Rajaram, N. & Kerppola, T. K. Synergistic transcription activation by Maf and Sox and their subnuclear localization are disrupted by a mutation in Maf that causes cataract. Mol. Cell. Biol. 24, 5694–5709 (2004).

    CAS  Article  Google Scholar 

  46. 46.

    Yu, Q., Sharma, A., Ghosh, A. & Sen, J. M. T cell factor-1 negatively regulates expression of IL-17 family of cytokines and protects mice from experimental autoimmune encephalomyelitis. J. Immunol. 186, 3946–3952 (2011).

    CAS  Article  Google Scholar 

  47. 47.

    Lee, S. Y. et al. Noncanonical mode of ERK action controls alternative αβ and γδ T cell lineage fates. Immunity 41, 934–946 (2014).

    CAS  Article  Google Scholar 

  48. 48.

    Auderset, F. et al. Notch signaling regulates follicular helper T cell differentiation. J. Immunol. 191, 2344–2350 (2013).

    CAS  Article  Google Scholar 

  49. 49.

    Zhang, Y. E. Non-Smad pathways in TGF-β signaling. Cell Res. 19, 128–139 (2009).

    CAS  Article  Google Scholar 

  50. 50.

    Rossi, F. M., Kringstein, A. M., Spicher, A., Guicherit, O. M. & Blau, H. M. Transcriptional control: rheostat converted to on/off switch. Mol. Cell 6, 723–728 (2000).

    CAS  Article  Google Scholar 

  51. 51.

    Carr, T. M., Wheaton, J. D., Houtz, G. M. & Ciofani, M. JunB promotes Th17 cell identity and restrains alternative CD4+ T-cell programs during inflammation. Nat. Commun. 8, 301 (2017).

    Article  Google Scholar 

  52. 52.

    Ciofani, M. et al. Obligatory role for cooperative signaling by pre-TCR and Notch during thymocyte differentiation. J. Immunol. 172, 5230–5239 (2004).

    CAS  Article  Google Scholar 

  53. 53.

    Morita, S., Kojima, T. & Kitamura, T. Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther. 7, 1063–1066 (2000).

    CAS  Article  Google Scholar 

  54. 54.

    Ramsdell, F., Zúñiga‐Pflücker, J. C. & Takahama, Y. Curr. Protoc. Immunol. In vitro systems for the study of T cell development: fetal thymus organ culture and OP9-DL1 cell coculture. 71, 3.18.1–3.18.18 (2006)..

  55. 55.

    Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    CAS  Article  Google Scholar 

  56. 56.

    Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).

    CAS  Article  Google Scholar 

  57. 57.

    Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

    CAS  Article  Google Scholar 

  58. 58.

    Corces, M. R. et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat. Methods 14, 959–962 (2017).

    CAS  Article  Google Scholar 

  59. 59.

    Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    CAS  Article  Google Scholar 

  60. 60.

    Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    CAS  Article  Google Scholar 

  61. 61.

    Ramirez, F. et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44, W160–W165 (2016).

    CAS  Article  Google Scholar 

  62. 62.

    Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

  63. 63.

    Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome. Biol. 9, R137 (2008).

    Article  Google Scholar 

  64. 64.

    Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

    CAS  Article  Google Scholar 

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Acknowledgements

We thank C. Birchmeier (Max Delbrück Center for Molecular Medicine, Germany) for providing Maf conditional mice; J.C. Zúñiga-Pflücker (University of Toronto) for providing OP9-DL1 cells; H. Kawamoto (Kyoto University, Japan) for providing TCF1 antibody; J. Heitman (Duke University) for providing C. albicans strain SC5314; and R. DePooter for critical reading of the manuscript. We acknowledge the expert assistance of N. Martin and L. Martinek with flow cytometry. This work was funded by a Whitehead Scholar Award (to M.C.). J.D.W. and M.C. were supported by NIH grant R01 GM115474. M.C. was supported by a Career Development Award from the Crohn’s and Colitis Foundation of America.

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M.K.Z., M.E.P., and M.C. designed, performed, and analyzed experiments. J.D.W. performed computational analyses of RNA-seq, ChIP-seq, and ATAC-seq data. H.R.S., J.R.E., and E.P. performed experiments. M.C. conceived the study and wrote the manuscript.

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Correspondence to Maria Ciofani.

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Integrated supplementary information

Supplementary Figure 1 c-Maf expression in fetal γδ T cell subsets.

(a) Flow cytometric analysis of intracellular staining of c-Maf is shown for fetal γδ thymocytes on the indicated day of gestation. Previous subsetting of γδ effector groups based on cytokine production and CD25 and CD27 expression shown and as described in text. (b) Flow cytometry showing developmental time series of RORγt and c-Maf expression relative to CD27 gated on γδ thymocytes. (a) and (b) are representative of four experiments. (c) Flow cytometric analysis of the indicated sort-purified subsets (i, ii, and iii) analyzed after 4 days of culture with OP9-DL1 stromal cells. Representative of two experiments. (d) c-Maf expression in E18 FT Vγ T cell subsets. Representative of four experiments. All flow cytometric plots are gated for CD4- CD8- CD3ε+ γδTCR+ thymocytes.

Supplementary Figure 2 Characterization of T cell subsets in Maffl/flIl7rCre mice at steady state.

(a) Flow cytometry plots gated for CD4-CD8-NK1.1-CD3ε- (top) and total live cells from Maffl/flIl7rCre (KO) versus Il7rCre (WT) adult mice (bottom). (b) Cellularity of total γδ T cells (CD3ε+ TCRγδ+) and of γδ T cell subsets from spleen, SILP, and iLN of adult WT and KO or thymus of E18 WT and KO fetus, as indicated. Cytokine producing cells were analyzed after 4h of stimulation in vitro. (c) c-Maf expression shown for a representative Maffl/flIl7rCre mouse with incomplete deletion of Maf from splenocytes gated for CD3ε+ γδTCR+ cells. (d) Plots are gated as indicated on RORγt+ subsets of αβ T cell and innate lymphoid cells (ILC) in the SILP from WT and KO mice, Lin-, Lineage (CD3ε, CD8α, CD19, Gr-1, CD11b, CD11c). (e) Flow cytometry for Vγ distribution in peripheral tissues in WT and KO mice gated for CD3ε+ γδTCR+ cells. All summary data are representative of 3 independent experiments. (a, b, e) n=8 biological replicates except T-bet+ γδ T cell data n=5. Mean ± SEM. ns, not significant; * p<0.05, *** p<0.001, **** p<0.0001. Statistical significance determined by unpaired two-tailed Student’s t-test (cellularity comparisons) or two-way ANOVA with Fisher’s LSD posttest (Vγ distribution).

Supplementary Figure 3 Role of c-Maf during γδ T cell maturation and specialization.

(a) Developmental analysis of RTOCs 10 days post reconstitution with equivalent mixtures of C57Bl/6 CD45.1+ DN2 cells and either Maf+/+Il7rCre (WT) or Maffl/flIl7rCre (KO) DN2 cells (CD45.2+ cells identified as CD45.1-). Plots gated for CD4-CD8-CD3ε+γδTCR+ cells and subsequently for CD45.1+ or CD45.1- expression. Thymic stroma obtained from Rag1-/- FT. Representative of 2 independent experiments. (b) Proportion of Vγ1+, Vγ2+, and Vγ3+ cells in WT and KO E17 FT. Flow cytometry plots gated for CD3ε+ γδTCR+ cells. (b-c) Summary graphs representative of 2 experiments with n=4 per group (c) Proportion of mature CD24lo γδ T cells in WT versus KO E17 FT for total γδTCR+ cells or the indicated Vγ subset. (d) Developmental analysis of RTOCs 8 days post reconstitution with purified Thy1.1+ (transduced) E16 FT γδTCR+CD45RBint cells overexpressing empty vector, c-Maf, or RORγt. Plots gated for CD4-CD8-CD3ε+ γδTCR+Thy1.1+ cells. Representative of 3 independent experiments. For summary data mean ± SEM. * p<0.05, *** p<0.001 unpaired two-tailed Student’s t-test.

Supplementary Figure 4 Lack of peripheral Tγδ17 cells following Rorc-Cre deletion of c-Maf.

(a) Flow cytometric analysis of CD3ε+ γδTCR+ γδ T cells in indicated peripheral tissues of adult Maf +/+Rorc-Cre (WT) and Maffl/flRorc-Cre (KO) mice. Cytokine production is shown for in vitro stimulated splenocytes. Summary statistics are shown for the percentage of RORγt+, IL-17A+, and IFNγ+ splenic γδ T cells; data compiled from two independent experiments, n=5. (b) Histograms of c-Maf expression gated for RORγt+ γδ thymocytes (CD4-CD8-CD3ε+γδTCR+) or DN thymocytes (negative control for c-Maf staining) at E18 and neonatal day 1 and 2. (c) Summary data for cell numbers for total γδ T cells, RORγt+ γδ T cells, and IL-17A+ γδ T cells for thymi from day 2 Maf +/+Rorc-Cre (WT) and Maffl/flRorc-Cre (KO) neonates, n=6.(d) Intracellular expression of Blk for DN2 thymocytes transduced with empty or Blk retrovirus and cultured with OP9-DL1 stroma for 5 days. Plots are gated for Thy1.1 transduced cells; data representative of two independent experiments. Mean ± SEM. ns, not significant; * p<0.05, **** p<0.0001 unpaired two-tailed Student’s t-test.

Supplementary Figure 5 Conserved TF consensus sites at Rorc CNS+10.

Schematic of Rorc locus showing relative positions and sequences for conserved TF consensus sites present in CNS+10. CNS name label provides distance of genomic region relative to Rorc(t) transcription start site. MARE, Maf recognition element; TCF, TCF consensus site; RORE, ROR response element. The conservation track defines regions of over 50% conservation with human RORC.

Supplementary Figure 6 c-Maf is an activator of type 17 programming in γδ T cells.

(a) Tγδ17-enriched CD25-CD27- γδ thymocytes populations sort purified from E17-18 Maf +/+Il7rCre (WT) and Maffl/flIl7rCre (KO) FT for RNA-seq. Plots are gated CD3ε+ γδTCR+. (b) RNA expression values for type 17 signature genes from WT and KO RNA-seq of CD25-CD27- γδ thymocytes. RPKM, reads per kilobase million. ♦, significant differential expression at FDR < 0.05. (c) Gene set enrichment analysis (GSEA; Broad Institute) for KO relative to WT CD25-CD27- γδ thymocytes demonstrating significant enrichment of top-ranking Th17 cell network targets among genes downregulated in the absence of c-Maf (p < 1 x 10−4). ES, enrichment score. (d) Ingenuity canonical pathway analysis for genes differentially expressed in Maffl/flIl7rCre relative to Maf +/+Il7rCre (WT) E17-18 CD25- CD27- Tγδ17 cells (a-d, n=2 biological replicates). (e) Sox13 functions upstream of c-Maf. shRNA-mediated knockdown of Sox13 following retroviral transduction of fetal liver hematopoietic progenitor cells (HPC) and γδ T cell differentiation with OP9-DL1. Left: qPCR of Sox13 cDNA prepared from sort-purified Thy1.1+ CD4- CD8- cells on day 10 of culture and normalized to actb. Sox13 western blot demonstrating 50% reduction in protein relative to luciferase control shRNA (Luc). CD4+CD8+ DP thymocytes represent a Sox13 non-expressing control. Knockdown evaluation performed for two experiments. Ratio of ImageJ pixel intensity is provided for Sox13 relative to Actin. Middle: Flow cytometry of day 10 cultures gated for γδ T cells (CD4- CD8- CD3ε+ γδTCR+). DN in histogram gated CD4- CD8- CD3ε- γδTCR-. Data are representative of three independent experiments. Right: qPCR evaluation of Maf transcripts NM_001025577 (RefSeq) and ENSMUST00000069009.6 (Ensembl) for samples shown left. A no reverse transcription (RT) control was included for intronless NM_0010255. Mean shown for two experiments. (f) Results of known motif analysis among regions of differential ATAC accessibility (FDR < 0.05) in Maffl/flIl7rCre vs. Maf +/+Il7rCre CD24+ CD45RBlo immature γδ thymocytes using Homer. –log10(p-value) plotted for the top ten enriched motifs (n=2 biological replicates for f-h) (g) Proportion of regions with differential accessibility (DA; FDR < 0.05) and non-DA that overlap the indicated motifs. (h) Proportion of total ATAC peaks overlapping the indicated motifs which display differential accessibility (FDR < 0.05; left) and violin plot of log2 fold-changes among differential peaks (FDR < 0.05) in each motif-based category, with median indicated by horizontal line. P-value, Kruskal Wallis test followed by pairwise Wilcoxon rank-sum tests. (i) Differential mRNA expression in Rorc(t)+/+ (WT) and Rorc(t)GFP/GFP (KO) E18 CD25-CD27- γδ thymocytes displayed as a volcano plot of log2 fold change vs. the –log10(p-value) for each gene. Genes considered significant (FDR < 0.05) in orange and select type 17-associated genes in blue. p-value capped at 10−75 (n=4 biological replicates) (j) IL-17A production displayed for stimulated Rorc(t)+/+ and Rorc(t)GFP/GFP E18 γδ thymocytes isolated from single FT. Plots gated CD4- CD8- CD3ε+ γδTCR+; data representative of two independent experiments with a total 6 embryos in each group. (k) Luciferase reporter assay of enhancer or silencer activity for select CNS in Maf-dependent loci in CD45RBlo γδ T cells sort purified from day 11 OP9-DL1 cultures of C57BL/6 or Maf +/+Il7rCre (WT n=5 except Tcf7 CNS+16 n=3) and Maffl/flIl7rCre (KO n=2) fetal liver HPCs. Mean ± SEM of at least two experiments. nd, not done, ** p<0.01, **** p<0.0001 unpaired two-tailed Student’s t-test.

Supplementary Figure 7 c-Maf expression is inversely related to γδTCR strength during γδ effector acquisition.

(a) Flow cytometric analysis of fetal thymic organ cultures (FTOC) analyzed 7 days post reconstitution with Rag1-/- E15 fetal thymocytes transduced to express the indicated γδTCR chains gated for CD4- CD8- γδTCR+ cells. RORγt versus c-Maf plots are additionally gated for CD45RBlo expression. Representative of 2 independent experiments. (b) Flow cytometric analysis for purified γδTCR-transduced culture-derived Rag1-/- DN3 cells after 7 days of OP9-DL1 culture gated for CD4- CD8- γδTCR+ transduced cells. RORγt versus c-Maf plots are additionally gated for CD45RBlo expression. Data is representative of three independent experiments.

Supplementary Figure 8 Gating strategy for flow cytometric analyses.

(a) Flow cytometry gating scheme for all peripheral analyses of γδ T cells at steady state (wild-type and conditional Maf mutant mice). The example plots are shown for adult thymus. (b) Flow cytometry gating scheme for DN and DP populations from fetal thymus that is the basis for the c-Maf expression analysis in Fig. 1. (c) Gating strategy for CD4- CD8- CD3ε+ γδTCR+ γδ T cells from fetal and neonatal thymus. (d) Gating strategy to identify γδ T cells that have differentiated from DN progenitors transduced with Thy1.1-expressing retrovirus post OP9-DL1 cell culture.

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Zuberbuehler, M.K., Parker, M.E., Wheaton, J.D. et al. The transcription factor c-Maf is essential for the commitment of IL-17-producing γδ T cells. Nat Immunol 20, 73–85 (2019). https://doi.org/10.1038/s41590-018-0274-0

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