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Runt-related transcription factor (RUNX) family members play critical roles in the development of multiple organs. Mammalian RUNX family members, consisting of RUNX1, RUNX2, and RUNX3, have distinct tissue-specific expression and function. In this study, we examined the spatiotemporal expression patterns of RUNX family members in developing kidneys and analyzed the role of RUNX1 during kidney development. In the developing mouse kidney, RUNX1 protein was strongly expressed in the ureteric bud (UB) tip and weakly expressed in the distal segment of the renal vesicle (RV), comma-shaped body (CSB), and S-shaped body (SSB). In contrast, RUNX2 protein was restricted to the stroma, and RUNX3 protein was only expressed in immune cells. We also analyzed the expression of RUNX family members in the cynomolgus monkey kidney. We found that expression patterns of RUNX2 and RUNX3 were conserved between rodents and primates, whereas RUNX1 was only expressed in the UB tip, not in the RV, CSB, or SSB of cynomolgus monkeys, suggesting a species differences. We further evaluated the roles of RUNX1 using two different conditional knockout mice: Runx1 f/f :HoxB7-Cre and Runx1 f/f :R26-CreER T2 and found no abnormalities in the kidney. Our findings showed that RUNX1, which is mainly expressed in the UB tip, is not essential for kidney development., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Motoko Yanagita reports financial support was provided by Japan Agency for Medical Research and Development. Motoko Yanagita reports a relationship with Mitsubishi Tanabe Pharma Corporation, Boehringer Ingelheim that includes: funding grants. None has patent pending to none. none If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Leukemia stem cells (LSCs) are widely believed to reside in well-characterized bone marrow (BM) niches; however, the capacity of the BM niches to accommodate LSCs is insufficient, and a significant proportion of LSCs are instead maintained in regions outside the BM. The molecular basis for this niche-independent behavior of LSCs remains elusive. Here, we show that integrin-α9 overexpression (ITGA9 OE) plays a pivotal role in the extramedullary maintenance of LSCs by molecularly mimicking the niche-interacting status, through the binding with its soluble ligand, osteopontin (OPN). Retroviral insertional mutagenesis conducted on leukemia-prone Runx-deficient mice identified Itga9 OE as a novel leukemogenic event. Itga9 OE activates Akt and p38MAPK signaling pathways. The elevated Myc expression subsequently enhances ribosomal biogenesis to overcome the cell integrity defect caused by the preexisting Runx alteration. The Itga9-Myc axis, originally discovered in mice, was further confirmed in multiple human acute myeloid leukemia (AML) subtypes, other than RUNX leukemias. In addition, ITGA9 was shown to be a functional LSC marker of the best prognostic value among 14 known LSC markers tested. Notably, the binding of ITGA9 with soluble OPN, a known negative regulator against HSC activation, induced LSC dormancy, while the disruption of ITGA9-soluble OPN interaction caused rapid cell propagation. These findings suggest that the ITGA9 OE increases both actively proliferating leukemia cells and dormant LSCs in a well-balanced manner, thereby maintaining LSCs. The ITGA9 OE would serve as a novel therapeutic target in AML., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Since its discovery over three decades ago, signal transducer and activator of transcription 1 (STAT1) has been extensively studied as a central mediator for interferons (IFNs) signaling and antiviral defense. Here, using genetic and biochemical assays, we unveil Thr 748 as a conserved IFN-independent phosphorylation switch in Stat1, which restricts IFN signaling and promotes innate inflammatory responses following the recognition of the bacterial-derived toxin lipopolysaccharide (LPS). Genetically engineered mice expressing phospho-deficient threonine748-to-alanine (T748A) mutant Stat1 are resistant to LPS-induced lethality. Of note, T748A mice exhibited undisturbed IFN signaling, as well as total expression of Stat1. Further, the T748A point mutation of Stat1 recapitulates the safeguard effect of the genetic ablation of Stat1 following LPS-induced lethality, indicating that the Thr 748 phosphorylation contributes inflammatory functionalities of Stat1. Mechanistically, LPS-induced Toll-like receptor 4 endocytosis activates a cell-intrinsic IκB kinase-mediated Thr 748 phosphorylation of Stat1, which promotes macrophage inflammatory response while restricting the IFN and anti-inflammatory responses. Depletion of macrophages restores the sensitivity of the T748A mice to LPS-induced lethality. Together, our study indicates a phosphorylation-dependent modular functionality of Stat1 in innate immune responses: IFN phospho-tyrosine dependent and inflammatory phospho-threonine dependent. Better understanding of the Thr 748 phosphorylation of Stat1 may uncover advanced pharmacologically targetable molecules and offer better treatment modalities for sepsis, a disease that claims millions of lives annually., Competing Interests: Competing interests statement:H. Metwally, T.O., and T.K. are inventors on the patent application (PCT/JP2023/022262) covering parts of the methodology in the presented work. Other authors declare no financial or commercial conflict of interest.

BCL11B is a transcription factor with six C 2 H 2 -type zinc-finger domains. Studies in mice have shown that Bcl11b plays essential roles in T cell development. Several germline heterozygous BCL11B variants have been identified in human patients with inborn errors of immunity (IEI) patients. Among these, two de novo mis-sense variants cause asparagine (N) to lysine (K) replacement in distinct zinc-finger domains, BCL11B N441K and BCL11B N807K . To elucidate the pathogenesis of the BCL11B N807K variant, we generated a mouse model of BCL11B N807K by inserting the corresponding mutation, Bcl11b N797K , into the mouse genome. In Bcl11b +/N797K mice, the proportion of immature CD4 - CD8 + single-positive thymocytes was increased, and the development of invariant natural killer cells was severely inhibited in a T-cell-intrinsic manner. Under competitive conditions, γδT cell development was outcompeted by control cells. Bcl11b N797K/N797K mice died within one day of birth. Recipient mice reconstituted with Bcl11b N797K/N797K fetal liver cells nearly lacked CD4 + CD8 + double-positive thymocytes, which was consistent with the lack of their emergence in culture from Bcl11b N797K/N797K fetal liver progenitors. Interestingly, Bcl11b N797K/N797K progenitors gave rise to aberrant c-Kit + and CD44 + cells both in vivo and in vitro . The increase in the proportion of immature CD8 single-positive thymocytes in the Bcl11b N797K mutants is caused, in part, by the inefficient activation of the Cd4 gene due to the attenuated function of the two Cd4 enhancers via distinct mechanisms. Therefore, we conclude that immunodeficient patient-derived Bcl11b N797K mutant mice elucidated a novel role for Bcl11b in driving the appropriate transition of CD4 - CD8 - into CD4 + CD8 + thymocytes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Matsumoto, Okuyama, Sidwell, Yamashita, Endo, Satoh-Takayama, Ohno, Morio, Rothenberg and Taniuchi.)

T follicular helper (Tfh) cells are essential for the development of germinal center B cells and high-affinity antibody-producing B cells in humans and mice. Here, we identify the guanine nucleotide exchange factor (GEF) Rin-like (Rinl) as a negative regulator of Tfh generation. Loss of Rinl leads to an increase of Tfh in aging, upon in vivo immunization and acute LCMV Armstrong infection in mice, and in human CD4+ T cell in vitro cultures. Mechanistically, adoptive transfer experiments using WT and Rinl-KO naïve CD4+ T cells unraveled T cell-intrinsic GEF-dependent functions of Rinl. Further, Rinl regulates CD28 internalization and signaling, thereby shaping CD4+ T cell activation and differentiation. Thus, our results identify the GEF Rinl as a negative regulator of global Tfh differentiation in an immunological context and species-independent manner, and furthermore, connect Rinl with CD28 internalization and signaling pathways in CD4+ T cells, demonstrating for the first time the importance of endocytic processes for Tfh differentiation., (© 2023 Sandner et al.)

After activation, some invariant natural killer T (iNKT) cells are differentiated into Klrg1 + long-lived effector NKT1 cells. However, the regulation from the effector phase to the memory phase has not been elucidated. Zeb2 is a zinc finger E homeobox-binding transcription factor and is expressed in a variety of immune cells, but its function in iNKT cell differentiation remains also unknown. Here, we show that Zeb2 is dispensable for development of iNKT cells in the thymus and their maintenance in steady state peripheral tissues. After ligand stimulation, Zeb2 plays essential roles in the differentiation to and maintenance of Klrg1 + Cx3cr1 + GzmA + iNKT cell population derived from the NKT1 subset. Our results including single-cell-RNA-seq analysis indicate that Zeb2 regulates Klrg1 + long-lived iNKT cell differentiation by preventing apoptosis. Collectively, this study reveals the crucial transcriptional regulation by Zeb2 in establishment of the memory iNKT phase through driving differentiation of Klrg1 + Cx3cr1 + GzmA + iNKT population., (© 2023. Springer Nature Limited.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., (© 2023. Springer Nature Limited.)

AIOLOS, encoded by IKZF3 , is a member of the IKZF family of proteins that plays an important role in regulating late B-cell differentiation. Human individuals heterozygous for the AIOLOS p.N160S variant displayed impaired humoral immune responses as well as impaired B and T cell development. We have previously reported that a mouse strain harboring an Ikzf3 N159S allele that corresponds to human IKZF3 N160S recapitulated immune-deficient phenotypes, such as impaired B cell development and loss of CD23 expression. In this study, we investigated the effect of the Ikzf3 N159S variant and found that B1a cell development was impaired in Ikzf3 N159S/N159S mice. In addition, CD62L expression was severely decreased in both B and T lymphocytes by the Ikzf3 N159S mutation, in a dose-dependent manner. Mixed bone marrow chimera experiments have revealed that most immunodeficient phenotypes, including low CD62L expression, occur in intrinsic cells. Interestingly, while Ikzf3 N159S/N159S lymphocytes were still present in the spleen, they were completely outcompeted by control cells in the lymph nodes, suggesting that the capacity for homing or retention in the lymph nodes was lost due to the Ikzf3 N159S mutation. The homing assay confirmed severely decreased homing abilities to lymph nodes of Ikzf3 N159S/N159S B and T lymphocytes but selective enrichment of CD62L expressing Ikzf3 N159S/N159S lymphocytes in lymph nodes. This finding suggests that impaired CD62L expression is the major reason for the impaired homing capacity caused by the Ikzf3 N159S mutation. Interestingly, an excess amount of Ikaros, but not Aiolos, restored CD62L expression in Ikzf3 N159S/N159S B cells. Together with the loss of CD62L expression due to Ikaros deficiency, the Aiolos N159S mutant protein likely interferes with Ikaros function through heterodimerization, at least in activating the Sell gene encoding CD62L expression. Thus, our results revealed that Aiolos N159S causes some immunodeficient phenotypes via the pathogenesis referred to as the heterodimeric interference as observed for Aiolos G158R variant., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chang, Yamashita, Padhi, Zhang and Taniuchi.)

While group-2 innate lymphoid cells (ILC2s) are highly proliferative in allergic inflammation, the removal of overactivated ILC2s in allergic diseases has not been investigated. We previously showed that chronic airway allergy induces "exhausted-like" dysfunctional ILC2s expressing T cell immunoreceptor with Ig and ITIM domains (TIGIT). However, the physiological relevance of these cells in chronic allergy remains elusive. To precisely identify and monitor TIGIT+ ILC2s, we generated TIGIT lineage tracer mice. Chronic allergy stably induced TIGIT+ ILC2s, which were highly activated, apoptotic, and were quickly removed from sites of chronic allergy. Transcripts from coding genes were globally suppressed in the cells, possibly due to reduced chromatin accessibility. Cell death in TIGIT+ ILC2s was enhanced by interactions with CD155 expressed on macrophages, whereas genetic ablation of Tigit or blockade by anti-TIGIT antagonistic antibodies promoted ILC2 survival, thereby deteriorating chronic allergic inflammation. Our work demonstrates that TIGIT shifts the fate of ILC2s toward activation-induced cell death, which could present a new therapeutic target for chronic allergies., (© 2023 Yamada et al.)

Posttranslational modification, such as phosphorylation, is an important biological event that modulates and diversifies protein function. Bcl11b protein is a zinc-finger transcription factor that plays a crucial role in early T cell development and the segregation of T cell subsets. Bcl11b possesses at least 25 serine/threonine (S/T) residues that can be phosphorylated upon TCR stimulation. To understand the physiological relevance of the phosphorylation on Bcl11b protein, we replaced S/T residues with alanine (A) by targeting murine Bcl11b gene in embryonic stem cells. By combinational targeting of exons 2 and 4 in the Bcl11b gene, we generated a mouse strain, Bcl11b-phosphorylation site mutation mice, in which 23 S/T residues were replaced with A residues. Such extensive manipulation left only five putative phosphorylated residues, two of which were specific for mutant protein, and resulted in reduced amounts of Bcl11b protein. However, primary T cell development in the thymus, as well as the maintenance of peripheral T cells, remained intact even after loss of major physiological phosphorylation. In addition, in vitro differentiation of CD4+ naive T cells into effector Th cell subsets-Th1, Th2, Th17, and regulatory T-was comparable between wild-type and Bcl11b-phosphorylation site mutation mice. These findings indicate that the physiological phosphorylation on major 23 S/T residues in Bcl11b is dispensable for Bcl11b functions in early T cell development and effector Th cell differentiation., (Copyright © 2023 by The American Association of Immunologists, Inc.)

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 T H 2 cells. This enhancer is occupied by STAT6 and interacts with Satb1 promoters through chromatin looping in T H 2 cells. Reduction of Satb1 expression, by the lack of this enhancer, resulted in elevated IL-5 expression in T H 2 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 T H 2 cells and ILC2s during type 2 immune responses., (© 2023 Nomura et al.)

RUNX proteins, such as RUNX2, regulate the proliferation and differentiation of chondrocytes and osteoblasts. Haploinsufficiency of RUNX2 causes cleidocranial dysplasia, but a detailed analysis of Runx2 +/- mice has not been reported. Furthermore, CBFB is required for the stability and DNA binding of RUNX family proteins. CBFB has two isoforms, and CBFB2 plays a major role in skeletal development. The calvaria, femurs, vertebrae and ribs in Cbfb2 -/- mice were analyzed after birth, and compared with those in Runx2 +/- mice. Calvarial development was impaired in Runx2 +/- mice but mildly delayed in Cbfb2 -/- mice. In femurs, the cortical bone but not trabecular bone was reduced in Cbfb2 -/- mice, whereas both the trabecular and cortical bone were reduced in Runx2 +/- mice. The trabecular bone in vertebrae increased in Cbfb2 -/- mice but not in Runx2 +/- mice. Rib development was impaired in Cbfb2 -/- mice but not in Runx2 +/- mice. These differences were likely caused by differences in the indispensability of CBFB and RUNX2, the balance of bone formation and resorption, or the number and maturation stage of osteoblasts. Thus, different amounts of CBFB and RUNX2 were required among the bone tissues for proper bone development and maintenance.

The Runt-related transcription factor (RUNX) family of proteins are crucial for many developmental and immuno-physiological processes. Their importance in cellular and tissue development has been repeatedly demonstrated as they are often found mutated and implicated in tumorigenesis. Most importantly, RUNX have now emerged as critical regulators of lymphocyte function against pathogenic infections and tumorigenic cells, the latter has now revolutionized our current understandings as to how RUNX proteins contribute to control tumor pathogenicity. These multifunctional roles of RUNX in mammalian immune responses and tissue homeostasis have led us to appreciate their value in controlling anti-tumor immune responses. Here, we summarize and discuss the role of RUNX in regulating the development and function of lymphocytes responding to foreign and tumorigenic threats and highlight their key roles in anti-tumor immunity.

Among the BTB-ZF transcription factor family, three amino acids in the BTB domain make Thpok unique in repressing cytotoxic lineage-related genes via recruitment of the NuRD chormatin-remodeling complex (see the related Research Article by Gao et al. ).

p53 deficiency and Myc dysregulation are frequently associated with cancer. However, the molecular mechanisms linking these two major oncogenic events are poorly understood. Using an osteosarcoma model caused by p53 loss, we have recently shown that Runx3 aberrantly upregulates Myc via mR1, a Runx consensus site in the Myc promoter. Here, we focus on thymic lymphoma, a major tumour type caused by germline p53 deletion in mice, and examine whether the oncogenic Runx-Myc axis plays a notable role in the development of p53-deficient lymphoma. Mice lacking p53 specifically in thymocytes (LP mice) mostly succumbed to thymic lymphoma. Runx1 and Myc were upregulated in LP mouse lymphoma compared with the normal thymus. Depletion of Runx1 or Myc prolonged the lifespan of LP mice and suppressed lymphoma development. In lymphoma cells isolated from LP mice, knockdown of Runx1 led to Myc suppression, weakening their tumour forming ability in immunocompromised mice. The mR1 locus was enriched by both Runx1 and H3K27ac, an active chromatin marker. LP mice with mutated mR1 had a longer lifespan and a lower incidence of lymphoma. Treatment with AI-10-104, a Runx inhibitor, improved the survival of LP mice. These results suggest that Myc upregulation by Runx1 is a key event in p53-deficient thymic lymphoma development and provide a clinical rationale for targeting the Runx family in p53-deficient malignancies., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background & Aims: RUNX transcription factors play pivotal roles in embryonic development and neoplasia. We previously identified the single missense mutation R122C in RUNX3 from human gastric cancer. However, how RUNX3 R122C mutation disrupts stem cell homeostasis and promotes gastric carcinogenesis remained unclear., Methods: To understand the oncogenic nature of this mutation in vivo, we generated the RUNX3 R122C knock-in mice. Stomach tissues were harvested, followed by histologic and immunofluorescence staining, organoid culture, flow cytometry to isolate gastric corpus isthmus and nonisthmus epithelial cells, and RNA extraction for transcriptomic analysis., Results: The corpus tissue of RUNX3 R122C/R122C homozygous mice showed a precancerous phenotype such as spasmolytic polypeptide-expressing metaplasia. We observed mucous neck cell hyperplasia; massive reduction of pit, parietal, and chief cell populations; as well as a dramatic increase in the number of rapidly proliferating isthmus stem/progenitor cells in the corpus of RUNX3 R122C/R122C mice. Transcriptomic analyses of the isolated epithelial cells showed that the cell-cycle-related MYC target gene signature was enriched in the corpus epithelial cells of RUNX3 R122C/R122C mice compared with the wild-type corpus. Mechanistically, RUNX3 R122C mutant protein disrupted the regulation of the restriction point where cells decide to enter either a proliferative or quiescent state, thereby driving stem cell expansion and limiting the ability of cells to terminally differentiate., Conclusions: RUNX3 R122C missense mutation is associated with the continuous cycling of isthmus stem/progenitor cells, maturation arrest, and development of a precancerous state. This work highlights the importance of RUNX3 in the prevention of metaplasia and gastric cancer., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Osteosarcoma (OS) in human patients is characterized by genetic alteration of TP53. Osteoprogenitor-specific p53-deleted mice (OS mice) have been widely used to study the process of osteosarcomagenesis. However, the molecular mechanisms responsible for the development of OS upon p53 inactivation remain largely unknown. In this study, we detected prominent RUNX3/Runx3 expression in human and mouse p53-deficient OS. Myc was aberrantly upregulated by Runx3 via mR1, a consensus Runx site in the Myc promoter, in a manner dependent on p53 deficiency. Reduction of the Myc level by disruption of mR1 or Runx3 knockdown decreased the tumorigenicity of p53-deficient OS cells and effectively suppressed OS development in OS mice. Furthermore, Runx inhibitors exerted therapeutic effects on OS mice. Together, these results show that p53 deficiency promotes osteosarcomagenesis in human and mouse by allowing Runx3 to induce oncogenic Myc expression., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

AIOLOS/IKZF3 is a member of the IKAROS family of transcription factors. IKAROS/IKZF1 mutations have been previously associated with different forms of primary immunodeficiency. Here we describe a novel combined immunodeficiency due to an IKZF3 mutation in a family presenting with T and B cell involvement, Pneumocystis jirovecii pneumonia, and/or chronic lymphocytic leukemia. Patients carrying the AIOLOS p.N160S heterozygous variant displayed impaired humoral responses, abnormal B cell development (high percentage of CD21low B cells and negative CD23 expression), and abrogated CD40 responses. Naive T cells were increased, T cell differentiation was abnormal, and CD40L expression was dysregulated. In vitro studies demonstrated that the mutant protein failed DNA binding and pericentromeric targeting. The mutant was fully penetrant and had a dominant-negative effect over WT AIOLOS but not WT IKAROS. The human immunophenotype was recapitulated in a murine model carrying the corresponding human mutation. As demonstrated here, AIOLOS plays a key role in T and B cell development in humans, and the particular gene variant described is strongly associated with immunodeficiency and likely malignancy., Competing Interests: Disclosures:   C.M. Dutmer reported personal fees from Horizon Therapeutics and personal fees from Enzyvant Therapeutics outside the submitted work. No other disclosures were reported., (© 2021 Kuehn et al.)

CD4 + T cells share common developmental pathways with CD8 + T cells, and upon maturation, CD4 + T conventional T (Tconv) cells lack phenotypic markers that distinguish these cells from FoxP3 + T regulatory cells. We developed a tamoxifen-inducible ThPOK CreERT2.hCD2 line with Frt sites inserted on either side of the CreERT2-hCD2 cassette, and a Foxp3 Ametrine-FlpO strain, expressing Ametrine and FlpO in Foxp3 + cells. Breeding these mice resulted in a CD4conv iCreERT2-hCD2 line that allows for the specific manipulation of a gene in CD4 + Tconv cells. As FlpO removes the CreERT2-hCD2 cassette, CD4 + Treg cells are spared from Cre activity, which we refer to as allele conditioning. Comparison with an E8I iCreERT2.GFP mouse that enables inducible targeting of CD8 + T cells, and deletion of two inhibitory receptors, PD-1 and LAG-3, in a melanoma model, support the fidelity of these lines. These engineered mouse strains present a resource for the temporal manipulation of genes in CD4 + T cells and CD4 + Tconv cells., Competing Interests: Declaration of interests D.A.A.V. is a cofounder and stockholder in Novasenta, Tizona, Trishula, and Potenza; a stockholder in Oncorus, Werewolf, and Apeximmune; has patents licensed and royalties in Astellas and BMS; is a scientific advisory board member of Tizona, Werewolf, F-Star, Bicara, and Apeximmune; a consultant to Astellas, BMS, Almirall, Incyte, G1 Therapeutics; and has received research funding from BMS, Astellas, and Novasenta. A.H.S. has patents/pending royalties on the PD-1 pathway from Roche and Novartis. A.H.S. is on the advisory boards of Surface Oncology, Elstar, SQZ Biotechnologies, Elpiscience, Selecta, Bicara, and Monopteros; consults for Novartis; and has received research funding from Novartis, Roche, Quark, Merck, and AbbVie that is unrelated to this project. E.J.W. has consulting agreements with and/or is on the scientific advisory boards of Merck, Elstar, Janssen, Related Sciences, Synthekine, and Surface Oncology; is a founder of Surface Oncology and Arsenal Biosciences; and has a patent licensing agreement on the PD-1 pathway with Roche/Genentech., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In the present study, we report a human-inherited, impaired, adaptive immunity disorder, which predominantly manifested as a B cell differentiation defect, caused by a heterozygous IKZF3 missense variant, resulting in a glycine-to-arginine replacement within the DNA-binding domain of the encoded AIOLOS protein. Using mice that bear the corresponding variant and recapitulate the B and T cell phenotypes, we show that the mutant AIOLOS homodimers and AIOLOS-IKAROS heterodimers did not bind the canonical AIOLOS-IKAROS DNA sequence. In addition, homodimers and heterodimers containing one mutant AIOLOS bound to genomic regions lacking both canonical motifs. However, the removal of the dimerization capacity from mutant AIOLOS restored B cell development. Hence, the adaptive immunity defect is caused by the AIOLOS variant hijacking IKAROS function. Heterodimeric interference is a new mechanism of autosomal dominance that causes inborn errors of immunity by impairing protein function via the mutation of its heterodimeric partner.

The immune system uses two distinct defence strategies against infections: microbe-directed pathogen destruction characterized by type 1 immunity 1 , and host-directed pathogen containment exemplified by type 2 immunity in induction of tissue repair 2 . Similar to infectious diseases, cancer progresses with self-propagating cancer cells inflicting host-tissue damage. The immunological mechanisms of cancer cell destruction are well defined 3-5 , but whether immune-mediated cancer cell containment can be induced remains poorly understood. Here we show that depletion of transforming growth factor-β receptor 2 (TGFBR2) in CD4 + T cells, but not CD8 + T cells, halts cancer progression as a result of tissue healing and remodelling of the blood vasculature, causing cancer cell hypoxia and death in distant avascular regions. Notably, the host-directed protective response is dependent on the T helper 2 cytokine interleukin-4 (IL-4), but not the T helper 1 cytokine interferon-γ (IFN-γ). Thus, type 2 immunity can be mobilized as an effective tissue-level defence mechanism against cancer.

During mammalian T cell development, CD4 + CD8 + double-positive (DP) thymocytes must make a lineage choice to become either conventional CD4 + or CD8 + T cells, dependent on their specificity for MHC-II or MHC-I, respectively. Alternatively, DP thymocytes can decide to become innate-like T cells in response to nonclassical MHC-I molecules. A key feature is the downregulation of CD8, which causes transient T cell receptor (TCR) signaling in MHC-I-selected DP thymocytes. Hence, this kinetic signaling model postulates that short or long duration of TCR signals during positive selection can direct the development of cytotoxic or helper T cell lineages. In this opinion article, we discuss the effects of constitutive expression of transgenic CD8 and prolonged TCR signaling on T cell lineage choice in MHC-I selected mouse thymocytes., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

CD4/CD8 T-cell lineage differentiation is a key process in immune system development; however, a defined regulator(s) that converts the signal from T-cell receptor and co-receptor complexes into lineage differentiation remains unclear. Here, we show that Twist2 is a critical factor in CD4/CD8 thymocyte differentiation. Twist2 expression is differentially regulated by T-cell receptor signaling, leading to differentiation into the CD4 or CD8 lineage. Forced Twist2 expression perturbed CD4 + thymocyte differentiation while enhancing CD8 + thymocyte differentiation. Furthermore, Twist2 expression produced mature CD8 + thymocytes in B 2 m -/- mice, while its deficiency significantly impaired CD8 + cells in MHC class-II -/ - and TCR transgenic mice, favoring CD8 T-cell differentiation. During CD8 lineage differentiation, Twist2 interacted with Runx3 to bind to the silencer region of the ThPOK locus, thereby blocking ThPOK expression. These findings indicate that Twist2 is a part of the transcription factor network controlling CD8 lineage differentiation.

Mechanisms of resistance to cancer immunotherapy remain poorly understood. Lymphocyte activation gene-3 (LAG3) signaling is regulated by a disintegrin and metalloprotease domain-containing protein-10 (ADAM10)- and ADAM17-mediated cell surface shedding. Here, we show that mice expressing a metalloprotease-resistant, noncleavable LAG3 mutant (LAG3 NC ) are resistant to PD1 blockade and fail to mount an effective antitumor immune response. Expression of LAG3 NC intrinsically perturbs CD4 + T conventional cells (T convs ), limiting their capacity to provide CD8 + T cell help. Furthermore, the translational relevance for these observations is highlighted with an inverse correlation between high LAG3 and low ADAM10 expression on CD4 + T convs in the peripheral blood of patients with head and neck squamous cell carcinoma, which corresponded with poor prognosis. This correlation was also observed in a cohort of patients with skin cancers and was associated with increased disease progression after standard-of-care immunotherapy. These data suggest that subtle changes in LAG3 inhibitory receptor signaling can act as a resistance mechanism with a substantive effect on patient responsiveness to immunotherapy., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Core Binding Factors (CBFs) are a small group of heterodimeric transcription factor complexes composed of DNA binding proteins, RUNXs, and a non-DNA binding protein, CBFB. The LH surge increases the expression of Runx1 and Runx2 in ovulatory follicles, while Cbfb is constitutively expressed. To investigate the physiological significance of CBFs, we generated a conditional mutant mouse model in which granulosa cell expression of Runx2 and Cbfb was deleted by the Esr2Cre. Female Cbfb flox/flox ;Esr2 cre/+ ;Runx2 flox/flox mice were infertile; follicles developed to the preovulatory follicle stage but failed to ovulate. RNA-seq analysis of mutant mouse ovaries collected at 11 h post-hCG unveiled numerous CBFs-downstream genes that are associated with inflammation, matrix remodeling, wnt signaling, and steroid metabolism. Mutant mice also failed to develop corpora lutea, as evident by the lack of luteal marker gene expression, marked reduction of vascularization, and excessive apoptotic staining in unruptured poorly luteinized follicles, consistent with dramatic reduction of progesterone by 24 h after hCG administration. The present study provides in vivo evidence that CBFs act as essential transcriptional regulators of both ovulation and luteinization by regulating the expression of key genes that are involved in inflammation, matrix remodeling, cell differentiation, vascularization, and steroid metabolisms in mice.

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.

The Runt-related transcription factors (RUNX) transcription factors have been known for their critical roles in numerous developmental processes and diseases such as autoimmune disorders and cancer. Especially, RUNX proteins are best known for their roles in hematopoiesis, particularly during the development of T cells. As scientists discover more types of new immune cells, the functional diversity of RUNX proteins also has been increased over time. Furthermore, recent research has revealed complicated transcriptional networks involving RUNX proteins by the current technical advances. Databases established by next generation sequencing data analysis has identified ever increasing numbers of potential targets for RUNX proteins and other transcription factors. Here, we summarize diverse functions of RUNX proteins mainly on lymphoid lineage cells by incorporating recent discoveries.

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., (© 2020 Kojo et al.)