Loss of TET function in T regulatory cells yields Tfh-like ex-Treg cells with the features of heterochromatin dysfunction 2266533

Abstract Introduction FOXP3 is the lineage-defining factor of T regulatory (Treg) cells, which can lose FOXP3 and convert into pathogenic ex-Tregs under strong inflammation. Ten-eleven translocation (TET) proteins maintain FOXP3 and CD4 expression by demethylating Foxp3 enhancers and the Cd4 locus. Methods CD4+ FOXP3+ Tregs from WT or CD4-Cre Tet2/3fl/fl mice were co-transferred with naïve CD4+ T cells into Rag1⁻/⁻ mice. This method allows us to isolate “pure” Tet2/3 double-knockout ex-Treg cells. Results WT Tregs maintained FOXP3 and CD4 expression, whereas Tet2/3 double-knockout (DKO) Tregs lost both, generating FOXP3⁻ CD4⁻ ex-Treg cells. Bulk and single-cell RNA-seq revealed that Tet2/3 DKO ex-Tregs expressed T follicular helper (Tfh)-related genes, exhibiting a Tfh-like phenotype. When transferred into immunocompetent WT mice, Tet2/3 DKO ex-Tregs expanded and induced severe inflammation, and there was also a striking expansion of plasma cells and CD8+ T cells. Base-resolution “6-base” sequencing revealed hypermethylation at Prdm1 and Bcl6 loci in Tet2/3 DKO ex-Tregs. Prdm1 mRNA was decreased as expected from the correlation of increased methylation with decreased gene expression; whereas Bcl6 expression increased, associated with decreased CTCF binding to the first intron of the Bcl6 gene. The Cd4 locus exhibited hypermethylation extending over several Kb. Although TET deficiency is expected to promote DNA methylation, heterochromatic regions in Tet2/3 DKO ex-Tregs underwent hypomethylation, accompanied by de-repression of transposable elements (TEs). Furthermore, ChIP-seq showed FOXP3 binding near TEs, suggesting that FOXP3 may regulate TEs expression. Conclusion Tet2/3 loss converts Tregs into Tfh-like ex-Tregs that trigger inflammation. Heterochromatic demethylation and TEs activation may underlie the strong pro-inflammatory phenotype of Tet2/3 DKO ex-Tregs, which FOXP3 may suppress. Funding Source the Tullie and Rickey Families SPARK Awards for Innovations in Immunology at La Jolla Institute Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)