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The function of Wtap in N6-adenosine methylation of mRNAs controls T cell receptor signaling and survival of T cells

Abstract

T cell antigen-receptor (TCR) signaling controls the development, activation and survival of T cells by involving several layers and numerous mechanisms of gene regulation. N6-methyladenosine (m6A) is the most prevalent messenger RNA modification affecting splicing, translation and stability of transcripts. In the present study, we describe the Wtap protein as essential for m6A methyltransferase complex function and reveal its crucial role in TCR signaling in mouse T cells. Wtap and m6A methyltransferase functions were required for the differentiation of thymocytes, control of activation-induced death of peripheral T cells and prevention of colitis by enabling gut RORγt+ regulatory T cell function. Transcriptome and epitranscriptomic analyses reveal that m6A modification destabilizes Orai1 and Ripk1 mRNAs. Lack of post-transcriptional repression of the encoded proteins correlated with increased store-operated calcium entry activity and diminished survival of T cells with conditional genetic inactivation of Wtap. These findings uncover how m6A modification impacts on TCR signal transduction and determines activation and survival of T cells.

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Fig. 1: Wtap promotes the generation of gut RORγt+ Treg cells.
Fig. 2: Wtap controls thymocyte development.
Fig. 3: Overlapping phenotypes of mice lacking Wtap or Virma.
Fig. 4: Wtap has a role in the expansion of T cells in response to TCR stimulation.
Fig. 5: The m6A methyltransferase complex is essential for the survival of activated T cells.
Fig. 6: Identification of m6A-modified transcripts.
Fig. 7: The m6A methylation correlates with Orai1 and Ripk1 downregulation.
Fig. 8: The m6A methyltransferase complex regulates TCR signaling.

Data availability

RNA-seq, m6A-CLIP and YTHDF2-iCLIP datasets have been deposited in the Gene Expression Omnibus under the accession no. GSE188854. All data are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank C. Keplinger (Helmholtz Zentrum München) and L. Esser (Ludwig-Maximilians−Universität) for excellent technical support. We thank the BMC Core Facility (Ludwig-Maximilians-Universität) for flow cytometry, H. Blum and S. Krebs (Ludwig-Maximilians-Universität) for mRNA- and CLIP-seq and N. Angstman for initial processing of sequencing data. The present study was supported by the German Research Foundation grant nos. HE3359/8-1 (#444891219), HE3359/7-1 (#432656284) and SFB 1054 (project A03, #210592381), SPP 1935 (#313381103) and TRR338 (project C02, #452881907) to V.H.

Author information

Authors and Affiliations

Authors

Contributions

T.I.-K. and V.H. designed the research and experiments with input from S.K., S.C., S.F., S.M., A.K. and J.K. T.I.-K., C.L., M.B., K.B. and G.A. performed the research. T.I.-K, C.L., M.B., K.B. and G.A. analyzed the data and S.C. analyzed the RNA-seq data. T.I.-K., G.C. and A.M. analyzed the CLIP data. R.M. performed experiments for histology. T.I.-K. and V.H. wrote the manuscript with critical input from S.F. and S.M.

Corresponding author

Correspondence to Vigo Heissmeyer.

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The authors declare no competing interests.

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Nature Immunology thanks Ziv Shulman, Martin Turner and Monika Wolkers 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 Wtap depletion in T cells induces inflammation of the gut.

a, b, d Deletion efficiency of floxed Wtap alleles in thymocytes and splenic T cells showing an immunoblot (a) with Wtap-specific antibodies using Gapdh as a loading control or flow cytometry (b, d) with Wtap-specific antibodies on the indicated subpopulations, CD4+ T cells from lamina propria of the colon (d). (n = 3 in a, n = 6 in b, d three independent experiments). c, qPCR analysis of the indicated cytokine gene expression within mRNA prepared from colon tissue. Results are to Ywhaz expression and presented relative to wild-type. (n = 3, representative of three independent experiments with technical triplicates). e, f, IFNγ and IL-17a production in CD4+ T cells from the colon. Percentages of IFNγ+IL-17a+ cells are shown in e. (WT, n = 4; KO, n = 9, three independent experiments). g, h Representative images of mice, spleen and lymph nodes from 5-week-old Wtapfl/-; Foxp3-Cre and Wtapfl/fl; Foxp3-Cre mice. Body weight is shown in the bottom (g). Scale bar, 10 mm. (h) (n = 2 biological replicates) i, H&E stained colon tissue from 4week-old Wtapfl/-; Foxp3-Cre and Wtapfl/fl; Foxp3-Cre mice. Scale bar, 10 µm, magnification x10. (n = 3 biological replicates) j-m, Stainings to detect CD4+ T and Treg cells in spleen (j, k, l) and colon (m) from 5-week-old Foxp3-Cre and Wtapfl/fl; Foxp3-Cre mice. Numbers adjacent to outlined areas indicate the percentage of cells in each gate. (WT, n = 5-7; KO, n = 4, three independent experiments). n, Stainings to detect RORγt and Helios for CD4+YFP+Foxp3+ or YFPFoxp3+ T cells in mLNs from female Wtapfl/fl; Foxp3-Crehet mice. (n = 3, two independent experiments). o, IFNγ and IL-17a production in Treg cells in lamina propria of the colon. Percentages of IFNγ+ or IL17a+ cells are shown in the right panel. (WT, n = 4; KO, n = 3 biological replicates). All data are presented as mean values ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student’s t-test.

Source data

Extended Data Fig. 2 Analyzing Wtap effects on thymic Treg cells and intracellular TCRβ expression.

a, Staining to detect CD4+CD25+Foxp3+ Treg cells in thymus from 6-8-week-old Wtapfl/fl and Wtapfl/fl; Cd4Cre mice. Numbers adjacent to outlined areas indicate the percentage of cells in each gate. (n = 6, three independent experiments). b, Intracellular staining of TCRβ on gated CD4SP CD24- or CD8SP CD24- thymocytes. (n = 3, two independent experiments). c, d, Flow cytometry analysis (c) and cellularity (d) of thymocytes from bone marrow chimeras, assessed for expression of CD4 and CD8 (right) after gating on CD45.1 or CD45.2 (left). Numbers adjacent to outlined areas indicate percentage of cells in each gate. (n = 3 individual mice in one experiment). e, f, Cellularity of thymocytes (Left), CD4SP or CD8 SP thymocytes (Right) from Wtapfl/fl and Wtapfl/fl; Cd4-Cre mice expressing a transgene encoding the MHC class I-restricted OT-I TCR (e) or the MHC class II-restricted OT-II TCR (f). (WT, n = 3; KO, n = 5, two independent experiments). All data are presented as mean values ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student’s t-test.

Source data

Extended Data Fig. 3 Virma expression is essential for m6A modification.

Deletion efficiency of floxed Wtap alleles in splenic T cells showing flow cytometry with Wtap-specific antibodies on subsets gating on CD44 and CD62L in splenic CD4+ T cells. (n = 6, three independent experiments) b, Surface staining of TCRβ on subsets gating on CD44 and CD62L in splenic CD4+ T and CD8+ T cells. (n = 6, three independent experiments). c, Percentage of the different congenic marked hematopoietic CD45+ cells (c) and flow cytometry analysis of splenocytes (d) from bone marrow chimeras harbouring WT (CD45.1+) and Wtapfl/fl; Cd4-Cre (CD45.2+) hematopoietic cells, assessed for expression of CD4 and CD8 after identifying CD45.1 or CD45.2 congenic cells. Numbers adjacent to outlined areas indicate the percentages of cells in each gate. (n = 3 individual mice in one experiment). e, Surface staining of CD44 and CD62L on CD4+ and CD8+ T cells after gating on CD45.1 or CD45.2 congenic splenocytes of the bone marrow chimeras. Numbers adjacent to outlined areas indicate percentage of cells in each gate. (n = 3 individual mice in one experiment). f, Immunoblot analysis of Virma protein in extracts from MEF cells showing Gapdh as a loading control. (n = 3, two independent experiments). g, m6A abundance as determined by LC/MS/MS analysis in the oligo-dT-purified mRNAs of Virma-depleted and Cre expressing MEF cells. (n = 3 biological replicates), (mean ± s.e.m, unpaired two-tailed t-test). h, Stainings to identify CD4+CD25+Foxp3+ Treg cells among thymocytes of Virmafl/fl or Virmafl/fl; Cd4-Cre mice. Numbers adjacent to outlined areas indicate the percentages of cells in each gate. (WT, n = 5; KO, n = 4, two independent experiments).

Source data

Extended Data Fig. 4 The m6A methyltransferase complex controls T cell survival.

a, Flow cytometry of CD4+ and CD8+ T cells from Cd4-CreERt2 and Wtapfl/fl; Cd4-CreERt2 mice stained with anti-WTAP antibody seven days after the last gavage. (n = 8, three independent experiments). b, c, Flow cytometry of cells from lymphoid organs of tamoxifen-gavaged Rag1−/− (b) or CD45.1 (c) mice 9 days after transfer of naive CD4+ T cells from Cd4-CreERt2 or Wtapfl/fl; Cd4-CreERt2 mice, assessed for expression of Wtap after gating on CD4 and TCRβ in b or CD45.1 and CD45.2 in c. (WT, n = 5; KO, n = 6, three independent experiments for spleen, WT, n = 3; KO, n = 4 two independent experiments for pLN and mLN, WT, n = 4; KO, n = 3, two independent experiments (f)). d, Histogram of Ki67 expression in CD4+ T cells from Cd4-CreERt2 and Wtapfl/fl; Cd4-CreERt2 mice that received tamoxifen gavage and anti-CD3 antibody injection. (WT, n = 5; KO, n = 4, three independent experiments). e, Cellularity of pLNs from Cd4-CreERt2 and Wtapfl/fl; Cd4-CreERt2 mice that received tamoxifen gavage and anti-CD3 antibody injection. (WT, n = 5; KO, n = 4, three independent experiments). (mean ± s.e.m, unpaired two-tailed t-test).

Source data

Extended Data Fig. 5 Analyzing TCR induced apoptosis and activation markers after Virma or Mettl3 deletion in CD4+ T cells.

a, Flow cytometry analysis of CTV-labelled CD4+ T cells from Cd4-CreERt2 and Wtapfl/fl; Cd4-CreERt2 mice, stained with Annexin V on day 4. (n = 3 biological replicates). b, Flow cytometry of CD4+ T cells from Cd4-CreERt2 and Virmafl/fl; Cd4-CreERt2 mice, stained with anti-VIRMA antibody three days after the last gavage. (n = 6, thee independent experiments). c, Flow cytometry of CD4+ T cells from Cd4-CreERt2 and Virmafl/fl; Cd4-CreERt2 mice, stained with AnnexinV and LIVE/DEAD Fixable dye on day 4. Percentage of AnnexinV+ population is shown in the right. (n = 6, thee independent experiments). d. Flow cytometry of naive CD4+ T cells from Cd4-CreERt2 and Wtapfl/fl; Cd4-CreERt2 mice, stained with AnnexinV and LIVE/DEAD Fixable dye on day 4. Percentage of AnnexinV+ population is shown in the right. (n = 10, thee independent experiments). e, f, Flow cytometry of naive CD4+ T cells from Cd4-CreERt2 and Wtapfl/fl; Cd4-CreERt2 mice that received tamoxifen gavage (e) or CD4+ T cells transfected with sgRNA for Cd90.2 or Mettl3 (f), assessed for expression of phospho-Stat5 after the stimulation with IL7 (10 ng/ml). (n = 6, two independent experiments). g, Surface staining of activation marker CD5 and CD25 in CD4+ T cells. (n = 4 biological replicates). h-k, In vitro co-culture of naive OTII-CD4+ T cells (CD45.2) from Cd4-CreERt2 and Wtapfl/fl; Cd4-CreERt2 mice with mutant (R9) (h) or WT (i-k) OVA323-339 peptide-loaded splenocytes/lymphocytes (CD45.1). Surface staining of CD69 after gating on CD45.2+CD4+ population (h), percentage of CD45.1 and CD45.2 (i), CTV profiles (j) and AnnexinV staining (k) are shown. (n = 6, two independent experiments). All data are presented as mean values ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student’s t-test.

Source data

Extended Data Fig. 6 Wtap depletion induces apoptosis in vitro.

a, Experimental scheme of in vitro deletion of Wtap induced by 4’-hydroxy-tamoxifen in iKO CD4+ T cells. b, Flow cytometry of CD4+ T cells prepared from in vitro 4’OH-tamoxifen induced Wtap deletion, stained with anti-WTAP antibody on day 4. c, Flow cytometry of iKO and control CD4+ T cells, stained with AnnexinV and LIVE/DEAD Fixable dye on day 4. Percentage of AnnexinV+ cell populations is shown in the right panel. (n = 5, two independent experiments). (mean ± s.e.m, unpaired two-tailed t-test). d, Measurement of m6A abundance on mRNAs in CD4+ T cells prepared from in vitro 4’OH-tamoxifen induced Wtap deletion. (n = 3 biological replicates). (mean ± s.e.m, unpaired two-tailed t-test).

Source data

Extended Data Fig. 7 Strategy and validation of the CLIP analyses.

a, Reproducibility of enriched peaks in m6A- or Ythdf2-iCLIP. Data were prepared from three biological replicate for m6A-CLIP and two replicate for Ythdf2-CLIP. b, Flow chart of the CLIP analysis. c, Venn diagram showing the overlap of m6A target mRNAs from Yao et al.21 and this study. d, Integrative Genomics Viewer (IGV) tracks of Tnf displaying m6A-CLIP and Ythdf2-CLIP reads distribution. The read coverage is shown for merged replicates.

Extended Data Fig. 8 Regulation of m6A modified mRNAs by Wtap deletion.

a, qPCR analysis of iKO and control CD4+ T cells. Results are presented relative to Ywhaz expression. (WT, n = 4; KO, n = 3, two independent experiments with technical triplicates). (mean ± s.e.m, unpaired two-tailed t-test). b, mRNA decay curves of Tnfrsf1b mRNA in iKO and control CD4+ T cells using Actinomycin D to terminate transcription of newly synthesized mRNAs. (n = 3, two to three independent experiments with technical triplicates). (mean ± s.e.m, two-way ANOVA).

Source data

Extended Data Fig. 9

a, Integrative Genomics Viewer (IGV) tracks displaying m6A-CLIP and Ythdf2-iCLIP read distribution. The read coverage is shown for merged replicates. b, Flow cytometry of CD4+ T cells transduced with eGFP, Orai1 or Ripk1 encoding retroviruses, stained with Thy1.1 two days after TCR re-stimulation. Grey histogram shows Thy1.1 negative cells. (n = 5-6, three independent experiments).

Extended Data Fig. 10 Exemplary gating strategy.

Cells were pre-gated on lymphocytes (FSC-A/SSC-A), single cells (FSC-H/FSC-W and SSC-H/SSC-W) and live cells (LIVE/DEAD staining -) prior to gating on cell populations of interest.

Supplementary information

Supplementary Information

Supplementary Table 3.

Reporting Summary

Peer Review File

Supplementary Table 1

List of 1,577 DEGs.

Supplementary Table 2

List of genes identified by annotating each GO term.

Source data

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Source Data Extended Data Fig. 1

Statistical source data, uncropped blots.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data, uncropped blots.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 6

Statistical source data, uncropped blots.

Source Data Extended Data Fig. 8

Statistical source data, uncropped blots.

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Ito-Kureha, T., Leoni, C., Borland, K. et al. The function of Wtap in N6-adenosine methylation of mRNAs controls T cell receptor signaling and survival of T cells. Nat Immunol 23, 1208–1221 (2022). https://doi.org/10.1038/s41590-022-01268-1

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