Satoshi Kojo

MHC-I- and MHC-II-selected CD4⁺CD8⁺ precursor thymocytes differentiate into cytotoxic CD8⁺ and helper CD4⁺ lineage T cells, during which suppression of Cd4 and Thpok genes by Runx-dependent-silencers in those genes is crucial to segregate the two lineages. However, how TCR signals are linked to cytotoxic-lineage-specific Cd4-Thpok silencing remains unclear. Here we show that the terminal Y residue within the evolutionarily conserved C-terminal WRPY motif in Runx1, which is essential for interacting with TLE co-repressor proteins, was phosphorylated more in CD4⁻CD8⁺ thymocytes than in CD4⁺CD8⁻ thymocytes, inducing an interaction with TLE co-repressors for cytotoxic-lineage specific Cd4-Thpok silencing. Non-receptor tyrosine kinases Lck and Zap70 interacted with Runx in the cytoplasm more in MHC-I-signaled CD4⁻CD8⁺ thymocytes than in CD4⁺CD8⁻ thymocytes. Collectively, these findings reveal that differential phosphorylation states at the terminal tyrosine residue in Runx connect MHC restriction with the helper versus cytotoxic T cell lineage choice.

Genetic studies in mice have shown that the zinc finger transcription factor BCL11B has an essential role in regulating early T cell development and neurogenesis. A de novo heterozygous missense BCL11B variant, BCL11B^N441K, was isolated from a patient with T cell deficiency and neurological disorders. Here, we show that mice harboring the corresponding Bcl11b^N440K mutation show the emergence of natural killer (NK)/group 1 innate lymphoid cell (ILC1)-like NKp46⁺ cells in the thymus and reduction in TBR1⁺ neurons in the neocortex, which are observed with loss of Bcl11a but not Bcl11b. Thus, the mutant BCL11B-N440K protein interferes with BCL11A function upon heterodimerization. Mechanistically, the Bcl11b^N440K mutation dampens the interaction of BCL11B with T cell factor 1 (TCF1) in thymocytes, resulting in weakened antagonism against TCF1 activity that supports the differentiation of NK/ILC1-like cells. Collectively, our results shed new light on the function of BCL11A in suppressing non-T lymphoid developmental potential and uncover the pathogenic mechanism by which BCL11B-N440K interferes with partner BCL11 family proteins.

Intestinal intraepithelial lymphocytes (IELs) reside in the gut epithelial layer, where they help in maintaining intestinal homeostasis. Peripheral CD4⁺ T cells can develop into CD4⁺CD8αα⁺ IELs upon arrival at the gut epithelium via the lamina propria (LP). Although this specific differentiation of T cells is well established, the mechanisms preventing it from occurring in the LP remain unclear. Here, we show that chemokine receptor 9 (CCR9) expression is low in epithelial CD4⁺CD8αα⁺ IELs, but CCR9 deficiency results in CD4⁺CD8αα⁺ over-differentiation in both the epithelium and the LP. Single-cell RNA sequencing shows an enriched precursor cell cluster for CD4⁺CD8αα⁺ IELs in Ccr9^−/− mice. CD4⁺ T cells isolated from the epithelium of Ccr9^−/− mice also display increased expression of Cbfβ2, and the genomic occupancy modification of Cbfβ2 expression reveals its important function in CD4⁺CD8αα⁺ differentiation. These results implicate a link between CCR9 downregulation and Cbfb2 splicing upregulation to enhance CD4⁺CD8αα⁺ IEL differentiation.

CCL5 is a unique chemokine with distinct stage and cell-type specificities for regulating inflammation, but how these specificities are achieved and how CCL5 modulates immune responses is not well understood. Here we identify two stage-specific enhancers: the proximal enhancer mediates the constitutive CCL5 expression during the steady state, while the distal enhancer located 1.35 Mb from the promoter induces CCL5 expression in activated cells. Both enhancers are antagonized by RUNX/CBFβ complexes, and SATB1 further mediates the long-distance interaction of the distal enhancer with the promoter. Deletion of the proximal enhancer decreases CCL5 expression and augments the cytotoxic activity of tissue-resident T and NK cells, which coincides with reduced melanoma metastasis in mouse models. By contrast, increased CCL5 expression resulting from RUNX3 mutation is associated with more tumor metastasis in the lung. Collectively, our results suggest that RUNX3-mediated CCL5 repression is critical for modulating anti-tumor immunity.

Temporal down-regulation of the CD8 co-receptor after receiving positive-selection signals has been proposed to serve as an important determinant to segregate helper versus cytotoxic lineages by generating differences in the duration of TCR signaling between MHC-I and MHC-II selected thymocytes. By contrast, little is known about whether CD8 also modulates TCR signaling engaged by the non-classical MHC-I–like molecule, CD1d, during development of invariant natural killer T (iNKT) cells. Here, we show that constitutive transgenic CD8 expression resulted in enhanced differentiation of innate memory-like CD8⁺ thymocytes in both a cell-intrinsic and cell-extrinsic manner, the latter being accomplished by an increase in the IL-4–producing iNKT2 subset. Skewed iNKT2 differentiation requires cysteine residues in the intracellular domain of CD8α that are essential for transmitting cellular signaling. Collectively, these findings shed a new light on the relevance of CD8 down-regulation in shaping the balance of iNKT-cell subsets by modulating TCR signaling.

The original version of this Article contained an error in the author affiliations. Affiliation 5 incorrectly read ‘Laboratory for Prediction of Cell Systems Dynamics, RIKEN Center for Biosystems Dynamics Research (BDR), Suite, Hyogo 565-0874, Japan.’ This has now been corrected in both the PDF and HTML versions of the Article.

Group 2 innate lymphoid cells (ILC2s) have tissue-resident competence and contribute to the pathogenesis of allergic diseases. However, the mechanisms regulating prolonged ILC2-mediated T_H2 cytokine production under chronic inflammatory conditions are unclear. Here we show that, at homeostasis, Runx deficiency induces excessive ILC2 activation due to overly active GATA-3 functions. By contrast, during allergic inflammation, the absence of Runx impairs the ability of ILC2s to proliferate and produce effector T_H2 cytokines and chemokines. Instead, functional deletion of Runx induces the expression of exhaustion markers, such as IL-10 and TIGIT, on ILC2s. Finally, these ‘exhausted-like’ ILC2s are unable to induce type 2 immune responses to repeated allergen exposures. Thus, Runx confers competence for sustained ILC2 activity at the mucosa, and contributes to allergic pathogenesis.

An intronic silencer, S4, in the Cd4 gene has been shown to be responsible for the helper-lineage-specific expression of CD4; S4 requires Runx complex binding to exert its silencer function against the enhancer-mediated Cd4 activation by modulating the epigenetic state of the Cd4 gene. Here we identify a late-acting maturation enhancer. Bcl11b plays essential roles for activation of both the early-acting proximal enhancer and maturation enhancer of Cd4. Notably, Runx complexes suppress these enhancers by distinct mechanisms. Whereas repression of the proximal enhancer depends on the S4 silencer, the maturation enhancer is repressed by Runx in the absence of S4. Moreover, ThPOK, known to antagonize S4-mediated Cd4 repression, assists Runx complexes to restrain maturation enhancer activation. Distinct modes of S4 silencer action upon distinct enhancers thus unravel a pathway that restricts CD4 expression to helper-lineage cells by silencer-independent and Runx-dependent repression of maturation enhancer activity in cytotoxic-lineage cells.

Multipotent hematopoietic progenitors must acquire thymus-homing capacity to initiate T lymphocyte development. Despite its importance, the transcriptional program underlying this process remains elusive. Cbfβ forms transcription factor complexes with Runx proteins, and here we show that Cbfβ2, encoded by an RNA splice variant of the Cbfb gene, is essential for extrathymic differentiation of T cell progenitors. Furthermore, Cbfβ2 endows extrathymic progenitors with thymus-homing capacity by inducing expression of the principal thymus-homing receptor, Ccr9. This occurs via direct binding of Cbfβ2 to cell type–specific enhancers, as is observed in Rorγt induction during differentiation of lymphoid tissue inducer cells by activation of an intronic enhancer. As in mice, an alternative splicing event in zebrafish generates a Cbfβ2-specific mRNA, important for ccr9 expression. Thus, despite phylogenetically and ontogenetically variable sites of origin of T cell progenitors, their robust thymus-homing capacity is ensured by an evolutionarily conserved mechanism emerging from functional diversification of Runx transcription factor complexes by acquisition of a novel splice variant.