A regulatory circuit controlled by extranuclear and nuclear retinoic acid receptor α determines T cell activation and function.

Ligation of retinoic acid receptor alpha (RARα) by RA promotes varied transcriptional programs associated with immune activation and tolerance, but genetic deletion approaches suggest the impact of RARα on TCR signaling. Here, we examined whether RARα would exert roles beyond transcriptional regulation. Specific deletion of the nuclear isoform of RARα revealed an RARα isoform in the cytoplasm of T cells. Extranuclear RARα was rapidly phosphorylated upon TCR stimulation and recruited to the TCR signalosome. RA interfered with extranuclear RARα signaling, causing suboptimal TCR activation while enhancing FOXP3+ regulatory T cell conversion. TCR activation induced the expression of CRABP2, which translocates RA to the nucleus. Deletion of Crabp2 led to increased RA in the cytoplasm and interfered with signalosome-RARα, resulting in impaired anti-pathogen immunity and suppressed autoimmune disease. Our findings underscore the significance of subcellular RA/RARα signaling in T cells and identify extranuclear RARα as a component of the TCR signalosome and a determinant of immune responses.

Identification of a novel enhancer essential for Satb1 expression in TH2 cells and activated ILC2s.

The genome organizer, special AT-rich binding protein-1 (SATB1), functions to globally regulate gene networks during primary T cell development and plays a pivotal role in lineage specification in CD4+ helper-, CD8+ cytotoxic-, and FOXP3+ regulatory-T cell subsets. However, it remains unclear how Satb1 gene expression is controlled, particularly in effector T cell function. Here, by using a novel reporter mouse strain expressing SATB1-Venus and genome editing, we have identified a cis-regulatory enhancer, essential for maintaining Satb1 expression specifically in TH2 cells. This enhancer is occupied by STAT6 and interacts with Satb1 promoters through chromatin looping in TH2 cells. Reduction of Satb1 expression, by the lack of this enhancer, resulted in elevated IL-5 expression in TH2 cells. In addition, we found that Satb1 is induced in activated group 2 innate lymphoid cells (ILC2s) through this enhancer. Collectively, these results provide novel insights into how Satb1 expression is regulated in TH2 cells and ILC2s during type 2 immune responses.

HVEM structures and mutants reveal distinct functions of binding to LIGHT and BTLA/CD160.

HVEM is a TNF (tumor necrosis factor) receptor contributing to a broad range of immune functions involving diverse cell types. It interacts with a TNF ligand, LIGHT, and immunoglobulin (Ig) superfamily members BTLA and CD160. Assessing the functional impact of HVEM binding to specific ligands in different settings has been complicated by the multiple interactions of HVEM and HVEM binding partners. To dissect the molecular basis for multiple functions, we determined crystal structures that reveal the distinct HVEM surfaces that engage LIGHT or BTLA/CD160, including the human HVEM-LIGHT-CD160 ternary complex, with HVEM interacting simultaneously with both binding partners. Based on these structures, we generated mouse HVEM mutants that selectively recognized either the TNF or Ig ligands in vitro. Knockin mice expressing these muteins maintain expression of all the proteins in the HVEM network, yet they demonstrate selective functions for LIGHT in the clearance of bacteria in the intestine and for the Ig ligands in the amelioration of liver inflammation.

Requirement for intron structures in activating the Cd8a locus.

During differentiation of CD4+CD8+ double-positive (DP) thymocytes into the CD4-CD8+ single-positive (CD8SP) thymocytes committed to the cytotoxic T cell lineage, Cd8a transcription is temporally terminated after positive selection and is subsequently reinitiated, a process known as coreceptor reversal. Despite the identification of a transcriptional enhancer in the Cd8a gene that directs reporter transgene expression specifically in CD8SP thymocytes, the molecular mechanisms controlling reactivation of the Cd8a gene are not fully understood. Here, we show that, after positive selection, hCD2 reporter expression from the Cd8a locus, which was generated by insertion of hCD2 cDNA into the first exon of the Cd8a gene, requires the incorporation of intron sequences into the hCD2 transcript. The presence of polyadenylation signals after hCD2 cDNA inhibited hCD2 expression in mature CD8+ T cells, whereas hCD2 expression in DP thymocytes recapitulated the Cd8a expression. Incorporation of the endogenous short intron structure and heterologous intron structure of the Cd4 locus restored hCD2 expression in mature CD8+ T cells in a variegated manner. Interestingly, stage-specific DNA demethylation was impaired in Cd8a reporter alleles that failed to express hCD2 in CD8+ T cells, and intron sequences lacking RNA splicing signals still restored hCD2 expression. These observations indicate that "intron-mediated enhancement" is involved in a stage-specific reactivation of the Cd8a locus harboring hCD2 cDNA. However, the Cd8a gene was transcribed in mature CD8+ T cells, albeit at a lower level, from a mutant Cd8a locus lacking intron structures, suggesting that protein-coding sequences in transcripts affect sensitivity to intron-mediated enhancement.

Unique N-terminal sequences in two Runx1 isoforms are dispensable for Runx1 function.

BACKGROUND: The Runt-related transcription factors (Runx) are a family of evolutionarily conserved transcriptional regulators that play multiple roles in the developmental control of various cell types. Among the three mammalian Runx proteins, Runx1 is essential for definitive hematopoiesis and its dysfunction leads to human leukemogenesis. There are two promoters, distal (P1) and proximal (P2), in the Runx1 gene, which produce two Runx1 isoforms with distinct N-terminal amino acid sequences, P1-Runx1 and P2-Runx1. However, it remains unclear whether P2-Runx specific N-terminal sequence have any specific function for Runx1 protein. RESULTS: To address the function of the P2-Runx1 isoform, we established novel mutant mouse models in which the translational initiation AUG (+1) codon for P2-Runx1 isoform was modulated. We found that a truncated P2-Runx1 isoform is translated from a downstream non-canonical AUG codon. Importantly, the truncated P2-Runx1 isoform is sufficient to support primary hematopoiesis, even in the absence of the P1-Runx1 isoform. Furthermore, the truncated P2-Runx1 isoform was able to restore defect in basophil development caused by loss of the P1-Runx1 isoform. The truncated P2-Runx1 isoform was more stable than the canonical P2-Runx1 isoform. CONCLUSIONS: Our results demonstrate that the N-terminal sequences specific for P2-Runx1 are dispensable for Runx1 function, and likely serve as a de-stabilization module to regulate Runx1 production.

Priming of lineage-specifying genes by Bcl11b is required for lineage choice in post-selection thymocytes.

T-lineage committed precursor thymocytes are screened by a fate-determination process mediated via T cell receptor (TCR) signals for differentiation into distinct lineages. However, it remains unclear whether any antecedent event is required to couple TCR signals with the transcriptional program governing lineage decisions. Here we show that Bcl11b, known as a T-lineage commitment factor, is essential for proper expression of ThPOK and Runx3, central regulators for the CD4-helper/CD8-cytotoxic lineage choice. Loss of Bcl11b results in random expression of these factors and, thereby, lineage scrambling that is disconnected from TCR restriction by MHC. Initial Thpok repression by Bcl11b prior to the pre-selection stage is independent of a known silencer for Thpok, and requires the last zinc-finger motif in Bcl11b protein, which by contrast is dispensable for T-lineage commitment. Collectively, our findings shed new light on the function of Bcl11b in priming lineage-specifying genes to integrate TCR signals into subsequent transcriptional regulatory mechanisms.CD4 and CD8 T cells develop in the thymus with their transcription programs controlled by ThPOK and Runx3, respectively. Here the authors show that a pre-commitment event modulated by the transcription factor, Bcl11b, is required for the proper expression of ThPOK and Runx3 and correct CD4/CD8 lineage commitment.

Essential Roles of SATB1 in Specifying T Lymphocyte Subsets.

T cell receptor (TCR) signaling by MHC class I and II induces thymocytes to acquire cytotoxic and helper fates via the induction of Runx3 and ThPOK transcription factors, respectively. The mechanisms by which TCR signaling is translated into transcriptional programs for each cell fate remain elusive. Here, we show that, in post-selection thymocytes, a genome organizer, SATB1, activates genes for lineage-specifying factors, including ThPOK, Runx3, CD4, CD8, and Treg factor Foxp3, via regulating enhancers in these genes in a locus-specific manner. Indeed, SATB1-deficient thymocytes are partially re-directed into inappropriate T lineages after both MHC class I- and II-mediated selection, and they fail to generate NKT and Treg subsets. Despite its essential role in activating enhancers for the gene encoding ThPOK in TCR-signaled thymocytes, SATB1 becomes dispensable for maintaining ThPOK in CD4+ T cells. Collectively, our findings demonstrate that SATB1 shapes the primary T cell pool by directing lineage-specific transcriptional programs in the thymus.

Requirement of Stat3 Signaling in the Postnatal Development of Thymic Medullary Epithelial Cells.

Thymic medullary regions are formed in neonatal mice as islet-like structures, which increase in size over time and eventually fuse a few weeks after birth into a continuous structure. The development of medullary thymic epithelial cells (TEC) is dependent on NF-κB associated signaling though other signaling pathways may contribute. Here, we demonstrate that Stat3-mediated signals determine medullary TEC cellularity, architectural organization and hence the size of the medulla. Deleting Stat3 expression selectively in thymic epithelia precludes the postnatal enlargement of the medulla retaining a neonatal architecture of small separate medullary islets. In contrast, loss of Stat3 expression in cortical TEC neither affects the cellularity or organization of the epithelia. Activation of Stat3 is mainly positioned downstream of EGF-R as its ablation in TEC phenocopies the loss of Stat3 expression in these cells. These results indicate that Stat3 meditated signal via EGF-R is required for the postnatal development of thymic medullary regions.

Induced Developmental Arrest of Early Hematopoietic Progenitors Leads to the Generation of Leukocyte Stem Cells.

Self-renewal potential and multipotency are hallmarks of a stem cell. It is generally accepted that acquisition of such stemness requires rejuvenation of somatic cells through reprogramming of their genetic and epigenetic status.We show here that a simple block of cell differentiation is sufficient to induce and maintain stem cells. By overexpression of the transcriptional inhibitor ID3 in murine hematopoietic progenitor cells and cultivation under B cell induction conditions, the cells undergo developmental arrest and enter a self-renewal cycle. These cells can be maintained in vitro almost indefinitely, and the long-term cultured cells exhibit robust multi-lineage reconstitution when transferred into irradiated mice. These cells can be cloned and re-expanded with 50% plating efficiency, indicating that virtually all cells are self-renewing. Equivalent progenitors were produced from human cord blood stem cells, and these will ultimately be useful as a source of cells for immune cell therapy.

An essential developmental checkpoint for production of the T cell lineage.

In early T cell development, progenitors retaining the potential to generate myeloid and natural killer lineages are eventually determined to a specific T cell lineage. The molecular mechanisms that drive this determination step remain unclarified. We show that, when murine hematopoietic progenitors were cultured on immobilized Notch ligand DLL4 protein in the presence of a cocktail of cytokines including interleukin-7, progenitors developing toward T cells were arrested and the arrested cells entered a self-renewal cycle, maintaining non-T lineage potentials. Reduced concentrations of interleukin-7 promoted T cell lineage determination. A similar arrest and self-renewal of progenitors were observed in thymocytes of mice deficient in the transcription factor Bcl11b. Our study thus identifies the earliest checkpoint during T cell development and shows that it is Bcl11b-dependent.

A novel gene essential for the development of single positive thymocytes.

A critical step during intrathymic T-cell development is the transition of CD4(+) CD8(+) double-positive (DP) cells to the major histocompatibility complex class I (MHC-I)-restricted CD4(-) CD8(+) and MHC-II-restricted CD4(+) CD8(-) single-positive (SP) cell stage. Here, we identify a novel gene that is essential for this process. Through the T-cell phenotype-based screening of N-ethyl-N-nitrosourea (ENU)-induced mutant mice, we established a mouse line in which numbers of CD4 and CD8 SP thymocytes as well as peripheral CD4 and CD8 T cells were dramatically reduced. Using linkage analysis and DNA sequencing, we identified a missense point mutation in a gene, E430004N04Rik (also known as themis), that does not belong to any known gene family. This orphan gene is expressed specifically in DP and SP thymocytes and peripheral T cells, whereas in mutant thymocytes the levels of protein encoded by this gene were drastically reduced. We generated E430004N04Rik-deficient mice, and their phenotype was virtually identical to that of the ENU mutant mice, thereby confirming that this gene is essential for the development of SP thymocytes.

Adult T-cell progenitors retain myeloid potential.

During haematopoiesis, pluripotent haematopoietic stem cells are sequentially restricted to give rise to a variety of lineage-committed progenitors. The classical model of haematopoiesis postulates that, in the first step of differentiation, the stem cell generates common myelo-erythroid progenitors and common lymphoid progenitors (CLPs). However, our previous studies in fetal mice showed that myeloid potential persists even as the lineage branches segregate towards T and B cells. We therefore proposed the 'myeloid-based' model of haematopoiesis, in which the stem cell initially generates common myelo-erythroid progenitors and common myelo-lymphoid progenitors. T-cell and B-cell progenitors subsequently arise from common myelo-lymphoid progenitors through myeloid-T and myeloid-B stages, respectively. However, it has been unclear whether this myeloid-based model is also valid for adult haematopoiesis. Here we provide clonal evidence that the early cell populations in the adult thymus contain progenitors that have lost the potential to generate B cells but retain substantial macrophage potential as well as T-cell, natural killer (NK)-cell and dendritic-cell potential. We also show that such T-cell progenitors can give rise to macrophages in the thymic environment in vivo. Our findings argue against the classical dichotomy model in which T cells are derived from CLPs; instead, they support the validity of the myeloid-based model for both adult and fetal haematopoiesis.

Quantification of progenitors capable of generating T cells in human cord blood.

OBJECTIVE: For transplantation of cord blood (CB) cells, it is important to select a CB sample that can reconstitute not only myelo-erythropoiesis but also lymphopoiesis in recipients. However, until now the reconstitution ability of CB samples has been assessed by colony forming unit-culture (CFU-C) assay or by simply counting CD34+ cells. The present study aims at establishing a method capable of assessing the potential of T lymphopoieses of CB samples. METHODS: CD34+ CD38- cells sorted from CB were cultured on a monolayer of murine stromal cell line TSt-4, transduced with the human Delta-like 1 gene. RESULTS: Immature T cells expressing CD5 and/or CD7 were generated in the culture. As these immature T cells can easily be discriminated from mature T cells that are included in the mononuclear cell population (MNCs), we can use the MNCs as starting material for quantification of progenitors capable of generating T cells (TGP). By applying a limiting dilution analysis, we succeeded in determining the frequency of TGP in MNCs. It was found that the ratios for the number of TGP vs. that of CFU-C differ among CB samples maximally by 3.5 times. CONCLUSION: The present assay system provides a novel tool for the evaluation of CB samples, especially for their T-cell-generating potential.

T cell lineage determination precedes the initiation of TCR beta gene rearrangement.

Loss of dendritic cell potential is one of the major events in intrathymic T cell development, during which the progenitors become determined to the T cell lineage. However, it remains unclear whether this event occurs in synchrony with another important event, TCRbeta chain gene rearrangement, which has been considered the definitive sign of irreversible T cell lineage commitment. To address this issue, we used transgenic mice in which GFP expression is controlled by the lck proximal promoter. We found that the double-negative (DN) 2 stage can be subdivided into GFP- and GFP+ populations, representing functionally different developmental stages in that the GFP-DN2, but not GFP+DN2, cells retain dendritic cell potential. The GFP+DN2 cells were found to undergo several rounds of proliferation before the initiation of TCRbeta rearrangement as evidenced by the diversity of D-Jbeta rearrangements seen in T cells derived from a single GFP+DN2 progenitor. These results indicated that the determination step of progenitors to the T cell lineage is a separable event from TCRbeta rearrangement.

Prethymic T-cell development defined by the expression of paired immunoglobulin-like receptors.

T cells are produced in the thymus from progenitors of extrathymic origin. As no specific markers are available, the developmental pathway of progenitors preceding thymic colonization remains unclear. Here we show that progenitors in murine fetal liver and blood, which are capable of giving rise to T cells, NK cells and dendritic cells, but not B cells, can be isolated by their surface expression of paired immunoglobulin-like receptors (PIR). PIR expression is maintained until the earliest intrathymic stage, then downregulated before the onset of CD25 expression. Unlike intrathymic progenitors, generation of prethymic PIR(+) progenitors does not require Hes1-mediated Notch signaling. These findings disclose a prethymic stage of T-cell development programmed for immigration of the thymus, which is genetically separable from intrathymic stages.

Activation of CEA-CAM-1-mediated cell adhesion via CD98: involvement of PKCdelta.

CD98 is a multifunctional protein involved in amino acid transport and regulation of integrin-mediated cell adhesion. Herein, we demonstrated that CD98 stimulation by anti-CD98 antibodies induced CEA-CAM-1-mediated cell adhesion in BaF3 cells expressing CEA-CAM-1, and suggest that this might be responsible for compact clumping of F9 embryonic carcinoma cells by CD98 stimulation. CEA-CAM-1 was co-immunoprecipitated by anti-CD98 antibody. CD98 stimulation induced the translocation of cytoplasmic protein kinase Cdelta (PKCdelta) to the cell adhesion sites, and rottlerin that inhibited the PKCdelta translocation abolished the cell aggregation without affecting integrin activation. The results suggested that CD98 stimulation could activate CEA-CAM-1-mediated cell adhesion independently of integrins.

Myeloproliferative stem cell disorders by deregulated Rap1 activation in SPA-1-deficient mice.

SPA-1 (signal-induced proliferation-associated gene-1) is a principal Rap1 GTPase-activating protein in hematopoietic progenitors. SPA-1-deficient mice developed a spectrum of myeloid disorders that resembled human chronic myelogenous leukemia (CML) in chronic phase, CML in blast crisis, and myelodysplastic syndrome as well as anemia. Preleukemic SPA-1-deficient mice revealed selective expansion of marrow pluripotential hematopoietic progenitors, which showed abnormal Rap1GTP accumulation. Overexpression of an active form of Rap1 promoted the proliferation of normal hematopoietic progenitors, while SPA-1 overexpression markedly suppressed it. Furthermore, restoring SPA-1 gene in a SPA-1-deficient leukemic blast cell line resulted in the dissolution of Rap1GTP accumulation and concomitant loss of the leukemogenicity in vivo. These results unveiled a role of Rap1 in myeloproliferative stem cell disorders and a tumor suppressor function of SPA-1.