Most Foxp3+ regulatory T (Treg) cells develop in the thymus as a functionally mature T cell subpopulation specialized for immune suppression. Their cell fate appears to be determined before Foxp3 expression; yet molecular events that prime Foxp3− Treg precursor cells are largely obscure. We found that Treg cell–specific super-enhancers (Treg-SEs), which were associated with Foxp3 and other Treg cell signature genes, began to be activated in Treg precursor cells. T cell–specific deficiency of the genome organizer Satb1 impaired Treg-SE activation and the subsequent expression of Treg signature genes, causing severe autoimmunity due to Treg cell deficiency. These results suggest that Satb1-dependent Treg-SE activation is crucial for Treg cell lineage specification in the thymus and that its perturbation is causative of autoimmune and other immunological diseases.

T-follicular helper (Tfh) cells differentiate through a multistep process, culminating in germinal center (GC) localized GC-Tfh cells that provide support to GC-B cells. T-follicular regulatory (Tfr) cells have critical roles in the control of Tfh cells and GC formation. Although Tfh-cell differentiation is inhibited by IL-2, regulatory T (Treg) cell differentiation and survival depend on it. Here, we describe a CD25− subpopulation within both murine and human PD1+CXCR5+Foxp3+ Tfr cells. It is preferentially located in the GC and can be clearly differentiated from CD25+ non–GC-Tfr, Tfh, and effector Treg (eTreg) cells by the expression of a wide range of molecules. In comparison to CD25+ Tfr and eTreg cells, CD25− Tfr cells partially down-regulate IL-2–dependent canonical Treg features, but retain suppressive function, while simultaneously up-regulating genes associated with Tfh and GC-Tfh cells. We suggest that, similar to Tfh cells, Tfr cells follow a differentiation pathway generating a mature GC-localized subpopulation, CD25− Tfr cells.

Foxp3-expressing CD25+CD4+ regulatory T cells (Tregs) are abundant in tumor tissues. Here, hypothesizing that tumor Tregs would clonally expand after they are activated by tumor-associated antigens to suppress antitumor immune responses, we performed single-cell analysis on tumor Tregs to characterize them by T cell receptor clonotype and gene-expression profiles. We found that multiclonal Tregs present in tumor tissues predominantly expressed the chemokine receptor CCR8. In mice and humans, CCR8+ Tregs constituted 30 to 80% of tumor Tregs in various cancers and less than 10% of Tregs in other tissues, whereas most tumor-infiltrating conventional T cells (Tconvs) were CCR8–. CCR8+ tumor Tregs were highly differentiated and functionally stable. Administration of cell-depleting anti-CCR8 monoclonal antibodies (mAbs) indeed selectively eliminated multiclonal tumor Tregs, leading to cure of established tumors in mice. The treatment resulted in the expansion of CD8+ effector Tconvs, including tumor antigen-specific ones, that were more activated and less exhausted than those induced by PD-1 immune checkpoint blockade. Anti-CCR8 mAb treatment also evoked strong secondary immune responses against the same tumor cell line inoculated several months after tumor eradication, indicating that elimination of tumor-reactive multiclonal Tregs was sufficient to induce memory-type tumor-specific effector Tconvs. Despite induction of such potent tumor immunity, anti-CCR8 mAb treatment elicited minimal autoimmunity in mice, contrasting with systemic Treg depletion, which eradicated tumors but induced severe autoimmune disease. Thus, specific removal of clonally expanding Tregs in tumor tissues for a limited period by cell-depleting anti-CCR8 mAb treatment can generate potent tumor immunity with long-lasting memory and without deleterious autoimmunity.

Reelin is a secreted glycoprotein whose function is regulated by proteolysis. One of the specific cleavage sites of Reelin, called C-t, is located approximately between the sixth and seventh Reelin repeat but its exact site was unknown. We here show that a metalloprotease present in the culture supernatant of cerebellar granular neurons (CGN) cleaves Reelin between Ala2688 and Asp2689. A Reelin mutant in which Asp2689 is replaced by Lys (Reelin-DK) is resistant to C-t cleavage by culture supernatant of CGN. From biochemical characteristics and the cleavage site preference, meprin α and meprin β were suggested candidate proteases and both were confirmed to cleave Reelin at the C-t site. Meprin α cleaved Reelin-DK but meprin β did not. Actinonin, a meprin α and meprin β inhibitor, did not inhibit the Reelin-cleaving activity of CGN and the amount of Reelin fragments in brains of meprin β knock-out mice was not significantly different from that of the wild-type, indicating that meprin β does not play a major role in Reelin cleavage under basal conditions. We propose that meprin α and meprin β join the modulators of Reelin signalling as they cleave Reelin at a specific site and are upregulated under specific pathological conditions.