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Tet2 and Tet3 in B cells are required to repress CD86 and prevent autoimmunity

A Publisher Correction to this article was published on 21 September 2020

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Abstract

A contribution of epigenetic modifications to B cell tolerance has been proposed but not directly tested. Here we report that deficiency of ten–eleven translocation (Tet) DNA demethylase family members Tet2 and Tet3 in B cells led to hyperactivation of B and T cells, autoantibody production and lupus-like disease in mice. Mechanistically, in the absence of Tet2 and Tet3, downregulation of CD86, which normally occurs following chronic exposure of self-reactive B cells to self-antigen, did not take place. The importance of dysregulated CD86 expression in Tet2- and Tet3-deficient B cells was further demonstrated by the restriction, albeit not complete, on aberrant T and B cell activation following anti-CD86 blockade. Tet2- and Tet3-deficient B cells had decreased accumulation of histone deacetylase 1 (HDAC1) and HDAC2 at the Cd86 locus. Thus, our findings suggest that Tet2- and Tet3-mediated chromatin modification participates in repression of CD86 on chronically stimulated self-reactive B cells, which contributes, at least in part, to preventing autoimmunity.

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Fig. 1: Spontaneous immune cell activation in bDKO mice.
Fig. 2: Lupus-like autoimmune phenotype in bDKO mice.
Fig. 3: Requirement for T–B interaction in initiation and maintenance of autoimmune inflammation.
Fig. 4: Contribution of dysregulated CD86 expression to induction of autoimmune inflammation.
Fig. 5: A diverse BCR repertoire is required to induce autoimmune inflammation.
Fig. 6: Derepressed CD86 on Tet2- and Tet3-deficient B cells in a peripheral tolerance model.
Fig. 7: Integrative analysis for DNA methylation, HDAC binding and acetylated histones regulated by Tet2 and Tet3.
Fig. 8: Molecular mechanism for CD86 repression by Tet2 and Tet3.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. The RNA-seq, WGBS-seq and ChIP–seq data reported in this paper are available under Gene Expression Omnibus accession numbers GSE137914, GSE137621 and GSE137666, respectively. Source data for Figs. 16 and 8 are provided with the paper.

Change history

  • 21 September 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank A.C. Chan (Genentech) for providing the anti-CD20 monoclonal antibody, K. Rajewsky (Max Delbruck Center for Molecular Medicine) for the Cd19-Cre and μMT mice, C.C. Goodnow (Garvan Institute of Medical Research) for MD4 and ML5 mice, A. O’Garra (Francis Crick Institute) for TCR7 TCR-transgenic mice, T. Shimaoka (Tokyo University of Science) for anti-CD4 monoclonal antibody, H. Fukuyama for assistance with the RNA-seq experiment, A. Baba for plasmid DNA construction, C. Kawai for assistance with experiments, K. Tanaka for assistance with the computational analysis, and N. Yakushiji-Kaminatsui and H. Sugishita for assistance with the ChIP–seq experiment and data processing. We appreciate the technical assistance from the Research Support Center at the Kyushu University Graduate School of Medical Sciences. This research was partially supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under grant no. JP19am0101103 and JP19am0101105. This work is supported by research grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and AMED (Grant-in-Aid for Scientific Research (S) for T.K., AMED under grant no. 19gm6110004h0003 and Grant-in-Aid for Scientific Research (B) for Y.B. and Grant-in-Aid for Scientific Research (C) for S.T.).

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Authors and Affiliations

Authors

Contributions

S.T. conceived the project, designed the study, performed experiments, analyzed data, prepared and organized the figures and wrote the manuscript. W.I. designed and performed experiments and analyzed data. T. Inoue contributed to BCR repertoire analysis, and A.I. and K. Fujii assisted with experiments. C.O., Y.S. and S.S. performed pathology experiments. M.K. supported experiments. I.M. performed SNP analysis to verify genetic background. J.S. contributed to the ChIP analysis. M.N. generated the targeting constructs for the mice with loxP-flanked Tet genes. H.K. provided the mice with loxP-flanked Tet genes and wrote the manuscript (Fig. 7). P.A.K. provided the mice with loxP-flanked H2-Ab1. I.R. and Q.-Z.L. performed the autoantibody array. K. Fujiki and R.N. contributed to ChIP–seq experiments. K.S. organized the ChIP–seq experiments. H.A. and F.M. analyzed the WGBS-seq data. T. Ito organized the WGBS-seq experiments. E.K. performed almost all of the secondary bioinformatic analysis related to RNA-seq, WGBS-seq and ChIP–seq experiments. Y.B. designed the study, supported experiments and provided important inputs. T.K. designed the study, supported experiments, provided important inputs and wrote the manuscript.

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Correspondence to Yoshihiro Baba or Tomohiro Kurosaki.

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Peer review information L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Deletion of Tet genes in B cells.

Schematic diagrams of floxed and deletion alleles of Tet2 and Tet3 genes with primer sets for detection of genomic deletion (Upper panel). Genomic PCR for detection of floxed and deletion alleles with genomic DNA purified from T (TCR-b+) and B (CD19+) cells isolated from Tet2 f/f, Tet3 f/f, CD19 Cre-/- (Cre-, n=4) and Tet2 f/f, Tet3 f/f, CD19 Cre+/- (Cre+, n=4) mice (Lower left panels). mRNA expression of Tet genes in T and B cells isolated from bDKO mice measured by quantitative PCR (Lower right panels). β-actin was used for internal control. Data are mean ± SD.

Extended Data Fig. 2 Spleen and lymph nodes of control and bDKO mice.

Spleen and lymph nodes of 6-week-old control (CD19 Cre+/-) and bDKO mice (Tet2 f/f, Tet3 f/f, CD19 Cre+/-). Data are representative of more than five independent experiments.

Extended Data Fig. 3 Gene expression analysis of control and DKO B cell subsets.

MA plots and enrichment GO terms for differential gene expression (analyzed by DEseq2, FDR<0.1) of B220+ AA4.1+, B220+ AA4.1- CD23+ CD21Int and B220+ AA4.1- CD23- CD21lo B cells isolated, by FACS, from spleen of 4-week-old age matched control and bDKO mice (n=3 each). The fast preranked gene set enrichment analysis (GSEA) was performed by using an algorithm for cumulative GSEA-statistic calculation. The significantly affected pathways were represented as red bars.

Supplementary information

Supplementary Information

Supplementary Figs. 1–6.

Reporting Summary

Supplementary Tables 1–19

Supplementary Table 20

Basic statistics for methylome data.

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Supplementary Data 1

Statistical source data for Supplementary Fig. 1.

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Supplementary Data 4

Statistical source data for Supplementary Fig. 6.

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Tanaka, S., Ise, W., Inoue, T. et al. Tet2 and Tet3 in B cells are required to repress CD86 and prevent autoimmunity. Nat Immunol 21, 950–961 (2020). https://doi.org/10.1038/s41590-020-0700-y

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