Prediction of the binding location between the nuclear inhibitor of DNA binding and differentiation 2 (ID2) and HSPA5.

Glucose-regulated protein 78 (GRP78), also termed HSPA5, was widely studied in cancer. It was recently approved that GRP78 has nuclear localization potential that sheds light on its role in cancer development. The inhibitor of DNA binding and differentiation 2 (ID2) is the nuclear component that associates with GRP78. The interaction between these two proteins is not understood clearly. In the current study, the binding pattern of GRP78/ID2 is predicted using computational methods. Protein-protein docking is used along with molecular dynamics simulation. The substrate binding domain ß of GRP78 can stably interact with the loop region (C42-S60) of ID2 as predicted in this study. This paves the way for a possible destabilizer for this association and cancer eradication.

The CDK1/TFCP2L1/ID2 cascade offers a novel combination therapy strategy in a preclinical model of bladder cancer.

Aberrant activation of embryogenesis-related molecular programs in urothelial bladder cancer (BC) is associated with stemness features related to oncogenic dedifferentiation and tumor metastasis. Recently, we reported that overexpression of transcription factor CP2-like protein-1 (TFCP2L1) and its phosphorylation at Thr177 by cyclin-dependent kinase-1 (CDK1) play key roles in regulating bladder carcinogenesis. However, the clinical relevance and therapeutic potential of this novel CDK1-TFCP2L1 molecular network remain elusive. Here, we demonstrated that inhibitor of DNA binding-2 (ID2) functions as a crucial mediator by acting as a direct repressive target of TFCP2L1 to modulate the stemness features and survival of BC cells. Low ID2 and high CDK1 expression were significantly associated with unfavorable clinical characteristics. TFCP2L1 downregulated ID2 by directly binding to its promoter region. Consistent with these findings, ectopic expression of ID2 or treatment with apigenin, a chemical activator of ID2, triggered apoptosis and impaired the proliferation, suppressed the stemness features, and reduced the invasive capacity of BC cells. Combination treatment with the specific CDK1 inhibitor RO-3306 and apigenin significantly suppressed tumor growth in an orthotopic BC xenograft animal model. This study demonstrates the biological role and clinical utility of ID2 as a direct target of the CDK1-TFCP2L1 pathway for modulating the stemness features of BC cells.

GDF-11 promotes human trophoblast cell invasion by increasing ID2-mediated MMP2 expression.

BACKGROUND: Growth differentiation factor-11 (GDF-11), also known as bone morphogenetic protein-11, belongs to the transforming growth factor-beta superfamily. GDF-11 was first identified as an important regulator during embryonic development. Increasing evidence has demonstrated that GDF-11 regulates the development of various organs and its aberrant expressions are associated with the risk of cardiovascular diseases and cancers. Extravillous trophoblast (EVT) cells invasion is a critical event for placenta development and needs to be finely regulated. However, to date, the biological function of GDF-11 in the human EVT cells remains unknown. METHODS: HTR-8/SVneo, a human EVT cell line, and primary cultures of human EVT cells were used to examine the effect of GDF-11 on matrix metalloproteinase 2 (MMP2) expression. Matrigel-coated transwell invasion assay was used to examine cell invasiveness. A series of in vitro experiments were applied to explore the underlying mechanisms that mediate the effect of GDF-11 on MMP2 expression and cell invasion. RESULTS: Treatment with GDF-11 stimulates MMP2 expression, in the HTR-8/SVneo and primary human EVT cells. Using a pharmacological inhibitor and siRNA-mediated knockdown approaches, our results demonstrated that the stimulatory effect of GDF-11 on MMP2 expression was mediated by the ALK4/5-SMAD2/3 signaling pathways. In addition, the expression of inhibitor of DNA-binding protein 2 (ID2) was upregulated by GDF-11 and that was required for the GDF-11-stimulated MMP2 expression and EVT cell invasion. CONCLUSIONS: These findings discover a new biological function and underlying molecular mechanisms of GDF-11 in the regulation of human EVT cell invasion. Video Abstract.

KSHV RTA Induces Degradation of the Host Transcription Repressor ID2 To Promote the Viral Lytic Cycle.

The immediate early viral protein replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for activating the lytic cycle of KSHV. RTA induces the KSHV lytic cycle by several mechanisms, acting as a viral transcription factor that directly induces viral and host genes and acting as a viral E3 ubiquitin ligase by degrading host proteins that block viral lytic replication. Recently, we have characterized the global gene expression changes in primary effusion lymphoma (PEL) upon lytic reactivation of KSHV, which also led to the identification of rapidly downregulated genes such as ID2, an inhibitor of basic helix-loop-helix transcription factors. Here, we demonstrate that ID2 overexpression in PEL ablates KSHV lytic reactivation, indicating that ID2 inhibits the KSHV lytic cycle. Furthermore, we show that while ID2 is highly expressed during latency, its protein level is rapidly reduced by 4 h postinduction during lytic reactivation. Our results indicate that RTA binds to ID2 and induces its degradation during the KSHV lytic cycle by N-terminal ubiquitination through the ubiquitin-proteasome pathway. Importantly, we found that not only KSHV RTA but also its Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68) homologs interact with ID2, and they can induce the degradation of all four members of the ID protein family, suggesting an evolutionarily conserved interplay between gammaherpesvirus RTAs and ID proteins. Taken together, we propose that ID2 acts as a repressor of the KSHV lytic cycle, which is counteracted by its RTA-mediated degradation. We also predict that ID proteins may act as restriction factors of the lytic phase of the other gammaherpesviruses as well. IMPORTANCE In addition to its transcription regulatory role, RTA is also known to have an E3 ubiquitin ligase activity, which RTA utilizes for inducing protein degradation. However, it is still largely unknown what host factors are downregulated during KSHV lytic reactivation by RTA-mediated protein degradation and what the biological significance of the degradation of these host factors is. In this study, we discovered that RTA employs N-terminal ubiquitination to induce degradation of ID2, a potent transcription repressor of host genes, via the ubiquitin-proteasome pathway to promote KSHV lytic reactivation in PEL cells. Furthermore, we found that not only KSHV RTA but also RTA of EBV and MHV68 gammaherpesviruses can induce the degradation of all four human ID proteins, indicating that the interplay between gammaherpesvirus RTAs and ID proteins is evolutionarily conserved.

Temporal Expression of Transcription Factor ID2 Improves Natural Killer Cell Differentiation from Human Pluripotent Stem Cells.

Natural killer (NK) cells are one type of innate lymphoid cells, and NK cell-based immunotherapy serves as a potentially curative therapy for cancers. However, the lack of reliable resources for a large amount of NK cells required for clinical infusion has limited the broader application of NK cells in targeted immunotherapy. Substantial effort has thus been made to generate NK-like cells from human pluripotent stem cells (hPSCs), but detailed molecular mechanisms regulating NK cell differentiation remain elusive, preventing us from developing robust strategies for NK cell production. Here, we genetically engineered hPSCs with inducible overexpression of transcription factors NFIL3, ID2, or SPI1 via CRISPR/Cas9-mediated gene knock-in and investigated their temporal roles during NK cell differentiation. Our results demonstrated ID2 overexpression significantly promoted NK cell generation compared with NFIL3 and SPI1 overexpression under a chemically defined, feeder-free culture condition. The resulting ID2 hPSC-derived NK cells exhibited various mature NK-specific markers and displayed effective tumor-killing activities, comparable to NK cells derived from wildtype hPSCs. Our study provides a new platform for efficient NK cell production, serving as a realistic off-the-shelf cell source for targeted cancer immunotherapy.

Inhibitor of DNA binding 2 knockdown inhibits the growth and liver metastasis of colorectal cancer.

BACKGROUND: Liver metastasis of colorectal cancer (CRC) remains high mortality and the mechanism is still unknown. Here we investigated the effects of inhibitor of DNA binding 2 (Id2) on growth and liver metastasis of CRC. METHODS: qPCR and western blotting were used to demonstrate mRNA and protein expressions in Id2-knockdown HCT116 cells. Cell growth was observed by cell proliferation assay, colony formation assay and flow cytometry. Cell migration and invasion were observed with wound healing assay and transwell migration and invasion assay. The effects of Id2 knockdown on tumor growth and liver metastasis in vivo were evaluated respectively with subcutaneous tumor model and colorectal liver metastasis model by injecting HCT116 cells into the mesentery triangle of cecum in mice. RESULTS: Id2 overexpression was found in CRC cell lines. Id2 knockdown resulted in a reduction in the proliferation, colony formation, migration and invasion of HCT116 cells. The suppression of cell proliferation was accompanied by the cell cycle arrest in the G0/G1 phase with down-regulation of Cyclin D1, Cyclin E, p-Cdk2/3, Cdk6, p-p27 and up-regulation of p21 and p27. Id2 knockdown reversed epithelial-mesenchymal transition (EMT) through increasing E-Cadherin and inhibiting N-Cadherin, Vimentin, ß-catenin, Snail and Slug. Id2 was also found to inhibit CRC metastasis via MMP2, MMP9 and TIMP-1. Furthermore, Id2 knockdown suppressed CRC liver metastasis in vivo. CONCLUSION: Id2 promotes CRC growth through activation of the PI3K/AKT signaling pathway, and triggers EMT to enhance CRC migration and invasion.

ASCL1 phosphorylation and ID2 upregulation are roadblocks to glioblastoma stem cell differentiation.

The growth of glioblastoma (GBM), one of the deadliest adult cancers, is fuelled by a subpopulation of stem/progenitor cells, which are thought to be the source of resistance and relapse after treatment. Re-engagement of a latent capacity of these cells to re-enter a trajectory resulting in cell differentiation is a potential new therapeutic approach for this devastating disease. ASCL1, a proneural transcription factor, plays a key role in normal brain development and is also expressed in a subset of GBM cells, but fails to engage a full differentiation programme in this context. Here, we investigated the barriers to ASCL1-driven differentiation in GBM stem cells. We see that ASCL1 is highly phosphorylated in GBM stem cells where its expression is compatible with cell proliferation. However, overexpression of a form of ASCL1 that cannot be phosphorylated on Serine-Proline sites drives GBM cells down a neuronal lineage and out of cell cycle more efficiently than its wild-type counterpart, an effect further enhanced by deletion of the inhibitor of differentiation ID2, indicating mechanisms to reverse the block to GBM cell differentiation.

LncRNA IPW inhibits growth of ductal carcinoma in situ by downregulating ID2 through miR-29c.

BACKGROUND: Ductal carcinoma in situ (DCIS) of breast is the noninvasive lesion that has propensity to progress to the malignant form. At present, it is still unknown which lesions can potentially progress to invasive forms. In this study, we aimed to identify key lncRNAs involved in DCIS growth. METHODS: We employ disease-related lncProfiler array to identify IPW in specimens of DCIS and matching control samples and validate the observations in three DCIS-non-tumorigenic cell lines. Further, we examine the mechanism of IPW action and the downstream signaling in in vitro and in vivo assays. Importantly, we screened a library containing 390 natural compounds to identify candidate compound selectively inhibiting IPW low DCIS cells. RESULTS: We identified lncRNA IPW as a novel tumor suppressor critical for inhibiting DCIS growth. Ectopic expression of IPW in DCIS cells strongly inhibited cell proliferation, colony formation and cell cycle progression while silencing IPW in primary breast cells promoted their growth. Additionally, orthotropic implantation of cells with ectopic expression of IPW exhibited decreased tumor growth in vivo. Mechanistically, IPW epigenetically enhanced miR-29c expression by promoting H3K4me3 enrichment in its promoter region. Furthermore, we identified that miR-29c negatively regulated a stemness promoting gene, ID2, and diminished self-renewal ability of DCIS cells. Importantly, we screened a library containing 390 natural compounds and identified toyocamycin as a compound that selectively inhibited the growth of DCIS with low expression of IPW, while it did not affect DCIS with high IPW expression. Toyocamycin also suppressed genes associated with self-renewal ability and inhibited DCIS growth in vivo. CONCLUSION: Our findings revealed a critical role of the IPW-miR-29c-ID2 axis in DCIS formation and suggested potential clinical use of toyocamycin for the treatment of DCIS.

TOB1 Blocks Intestinal Mucosal Inflammation Through Inducing ID2-Mediated Suppression of Th1/Th17 Cell Immune Responses in IBD.

BACKGROUND & AIMS: TOB1 is an anti-proliferative protein of Tob/BTG family and typically involved in the tumorigenesis and T cell activation. Although TOB1 is associated with T helper 17 cell-related autoimmunity, its role in modulating T cell-mediated immune responses in IBD remains poorly understood. Here, we explored its expression and the underlying mechanisms involved in the pathogenesis of inflammatory bowel disease (IBD). METHODS: TOB1 and ID2 expression in IBD patients was examined by quantitative real time polymerase chain reaction and immunohistochemistry. IBD CD4+ T cells were transfected with lentivirus expressing TOB1, ID2, TOB1 short hairpin RNA and ID2 short hairpin RNA, respectively, and Tob1-/-CD4+ T cells were transfected with lentivirus expressing Id2. Experimental colitis was established in Tob1-/- mice by trinitrobenzene sulfonic acid enema and in Rag1-/- mice reconstituted with Tob1-/-CD45RBhighCD4+ T cells to further explore the role of Tob1 in intestinal mucosal inflammation. Splenic CD4+ T cells of Tob1-/- mice were sorted to determine transcriptome differences by RNA sequencing. RESULTS: TOB1 expression was decreased in inflamed mucosa and peripheral blood CD4+ T cells of IBD patients compared with healthy subjects. Overexpression of TOB1 downregulated IBD CD4+ T cells to differentiate into Th1/Th17 cells compared with control subjects. Severe colitis was observed in Tob1-/- mice through trinitrobenzene sulfonic acid enema or in Rag1-/- mice reconstituted with Tob1-/-CD45RBhighCD4+ T cells, compared with control animals. RNA sequencing analysis revealed ID2 as functional target of TOB1 to inhibit IBD CD4+ T cell differentiation into Th1/Th17 cells. Mechanistically, TOB1 was associated with Smad4/5 to induce ID2 expression and restrain Th1/Th17 cell differentiation. CONCLUSIONS: TOB1 restrains intestinal mucosal inflammation through suppressing Th1/Th17 cell-mediated immune responses via the Smad4/5-ID2 pathway. It may serve as a novel therapeutic target for treatment of human IBD.

USP1 Promotes GC Metastasis via Stabilizing ID2.

Gastric cancer (GC) is one of the most common malignant tumors all over the world. And recurrence and metastasis are still the main causes of low survival rate for advanced GC. USP1 has been shown overexpressed in multiple cancers, which indicate its important biomarker in tumorigenesis and development. Our study is aimed at defining the exact role of USP1 on GC metastasis and the underlying mechanism. USP1 was firstly found overexpressed in GC tissues and relatively high-expression levels conferred poor survival rates. Then, real-time cellular analysis (RTCA) showed that USP1 knockdown inhibited GC metastasis both in vitro and in vivo. Mechanically, we demonstrated that USP1 promoted GC metastasis via upregulating ID2 expression and further confirmed that USP1 stabilized ID2 expression through deubiquitinating ID2 in GC. In conclusion, our study showed that USP1 promoted GC metastasis via stabilizing ID2 expression, which provides a potential biomarker and therapy target for GC.

Discovery of novel ID2 antagonists from pharmacophore-based virtual screening as potential therapeutics for glioma.

Glioma, especially the most aggressive type glioblastoma multiforme, is a malignant cancer of the central nervous system with a poor prognosis. Traditional treatments are mainly surgery combined with radiotherapy and chemotherapy, which is still far from satisfactory. Therefore, it is of great clinical significance to find new therapeutic agents. Serving as an inhibitor of differentiation, protein ID2 (inhibitor of DNA binding 2) plays an important role in neurogenesis, neovascularization and malignant development of gliomas. It has been shown that ID2 affects the malignant progression of gliomas through different mechanisms. In this study, a pharmacophore-based virtual screening was carried out and 16 hit compounds were purchased for pharmacological evaluations on their ID2 inhibitory activities. Based on the cytotoxicity of these small-molecule compounds, two compounds were shown to effectively inhibit the viability of glioma cells in the micromolar range. Among them, AK-778-XXMU was chosen for further study due to its better solubility in water. A SPR (Surface Plasma Resonance) assay proved the high affinity between AK-778-XXMU and ID2 protein with the KD value as 129 nM. The plausible binding mode of ID2 was studied by molecular docking and it was found to match AGX51 very well in the same binding site. Subsequently, the cancer-suppressing potency of the compound was characterized both in vitro and in vivo. The data demonstrated that compound AK-778-XXMU is a potent ID2 antagonist which has the potential to be developed as a therapeutic agent against glioma.

Ubiquitin Specific Protease 1 Expression and Function in T Cell Immunity.

T cells are essential mediators of immune responses against infectious diseases and provide long-lived protection from reinfection. The differentiation of naive to effector T cells and the subsequent differentiation and persistence of memory T cell populations in response to infection is a highly regulated process. E protein transcription factors and their inhibitors, Id proteins, are important regulators of both CD4+ and CD8+ T cell responses; however, their regulation at the protein level has not been explored. Recently, the deubiquitinase USP1 was shown to stabilize Id2 and modulate cellular differentiation in osteosarcomas. In this study, we investigated a role for Usp1 in posttranslational control of Id2 and Id3 in murine T cells. We show that Usp1 was upregulated in T cells following activation in vitro or following infection in vivo, and the extent of Usp1 expression correlated with the degree of T cell expansion. Usp1 directly interacted with Id2 and Id3 following T cell activation. However, Usp1 deficiency did not impact Id protein abundance in effector T cells or alter effector T cell expansion or differentiation following a primary infection. Usp1 deficiency resulted in a gradual loss of memory CD8+ T cells over time and reduced Id2 protein levels and proliferation of effector CD8+ T cell following reinfection. Together, these results identify Usp1 as a player in modulating recall responses at the protein level and highlight differences in regulation of T cell responses between primary and subsequent infection encounters. Finally, our observations reveal differential regulation of Id2/3 proteins between immune versus nonimmune cell types.

Harmine targets inhibitor of DNA binding-2 and activator protein-1 to promote preosteoclast PDGF-BB production.

Osteoporosis is one of the most common metabolic bone diseases affecting millions of people. We previously found that harmine prevents bone loss in ovariectomized mice via increasing preosteoclast platelet-derived growth factor-BB (PDGF-BB) production and type H vessel formation. However, the molecular mechanisms by which harmine promotes preosteoclast PDGF-BB generation are still unclear. In this study, we revealed that inhibitor of DNA binding-2 (Id2) and activator protein-1 (AP-1) were important factors implicated in harmine-enhanced preosteoclast PDGF-BB production. Exposure of RANKL-induced Primary bone marrow macrophages (BMMs), isolated from tibiae and femora of mice, to harmine increased the protein levels of Id2 and AP-1. Knockdown of Id2 by Id2-siRNA reduced the number of preosteoclasts as well as secretion of PDGF-BB in RANKL-stimulated BMMs administrated with harmine. Inhibition of c-Fos or c-Jun (components of AP-1) both reversed the stimulatory effect of harmine on preosteoclast PDGF-BB production. Dual-luciferase reporter assay analyses determined that PDGF-BB was the direct target of AP-1 which was up-regulated by harmine treatment. In conclusion, our data demonstrated a novel mechanism involving in the production of PDGF-BB increased by harmine, which may provide potential therapeutic targets for bone loss diseases.

The transcriptional repressor ID2 supports natural killer cell maturation by controlling TCF1 amplitude.

Gaining a mechanistic understanding of the expansion and maturation program of natural killer (NK) cells will provide opportunities for harnessing their inflammation-inducing and oncolytic capacity for therapeutic purposes. Here, we demonstrated that ID2, a transcriptional regulatory protein constitutively expressed in NK cells, supports NK cell effector maturation by controlling the amplitude and temporal dynamics of the transcription factor TCF1. TCF1 promotes immature NK cell expansion and restrains differentiation. The increased TCF1 expression in ID2-deficient NK cells arrests their maturation and alters cell surface receptor expression. Moreover, TCF1 limits NK cell functions, such as cytokine-induced IFN-γ production and the ability to clear metastatic melanoma in ID2-deficient NK cells. Our data demonstrate that ID2 sets a threshold for TCF1 during NK cell development, thus controlling the balance of immature and terminally differentiated cells that support future NK cell responses.

Gut microbial metabolites facilitate anticancer therapy efficacy by modulating cytotoxic CD8+ T cell immunity.

Recent studies in both mice and humans have suggested that gut microbiota could modulate tumor responsiveness to chemo- or immunotherapies. However, the underlying mechanism is not clear yet. Here, we found that gut microbial metabolites, especially butyrate, could promote the efficacy of oxaliplatin by modulating CD8+ T cell function in the tumor microenvironment. Butyrate treatment directly boosted the antitumor cytotoxic CD8+ T cell responses both in vitro and in vivo in an ID2-dependent manner by promoting the IL-12 signaling pathway. In humans, the oxaliplatin responder cancer patients exhibited a higher amount of serum butyrate than did non-responders, which could also increase ID2 expression and function of human CD8+ T cells. Together, our findings suggest that the gut microbial metabolite butyrate could promote antitumor therapeutic efficacy through the ID2-dependent regulation of CD8+ T cell immunity, indicating that gut microbial metabolites could be effective as a part of cancer therapy.

RORα is a critical checkpoint for T cell and ILC2 commitment in the embryonic thymus.

Type 2 innate lymphoid cells (ILC2) contribute to immune homeostasis, protective immunity and tissue repair. Here we demonstrate that functional ILC2 cells can arise in the embryonic thymus from shared T cell precursors, preceding the emergence of CD4+CD8+ (double-positive) T cells. Thymic ILC2 cells migrated to mucosal tissues, with colonization of the intestinal lamina propria. Expression of the transcription factor RORα repressed T cell development while promoting ILC2 development in the thymus. From RNA-seq, assay for transposase-accessible chromatin sequencing (ATAC-seq) and chromatin immunoprecipitation followed by sequencing (ChIP-seq) data, we propose a revised transcriptional circuit to explain the co-development of T cells and ILC2 cells from common progenitors in the thymus. When Notch signaling is present, BCL11B dampens Nfil3 and Id2 expression, permitting E protein-directed T cell commitment. However, concomitant expression of RORα overrides the repression of Nfil3 and Id2 repression, allowing ID2 to repress E proteins and promote ILC2 differentiation. Thus, we demonstrate that RORα expression represents a critical checkpoint at the bifurcation of the T cell and ILC2 lineages in the embryonic thymus.

Effect of cAMP Signaling Regulation in Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells.

The successful implementation of adipose-derived mesenchymal stem cells (ADSCs) in bone regeneration depends on efficient osteogenic differentiation. However, a literature survey and our own experience demonstrated that current differentiation methods are not effective enough. Since the differentiation of mesenchymal stem cells (MSCs) into osteoblasts and adipocytes can be regulated by cyclic adenosine monophosphate (cAMP) signaling, we investigated the effects of cAMP activator, forskolin, and inhibitor, SQ 22,536, on the early and late osteogenic differentiation of ADSCs cultured in spheroids or in a monolayer. Intracellular cAMP concentration, protein kinase A (PKA) activity, and inhibitor of DNA binding 2 (ID2) expression examination confirmed cAMP up- and downregulation. cAMP upregulation inhibited the cell cycle and protected ADSCs from osteogenic medium (OM)-induced apoptosis. Surprisingly, the upregulation of cAMP level at the early stages of osteogenic differentiation downregulated the expression of osteogenic markers RUNX2, Osterix, and IBSP, which was more significant in spheroids, and it is used for the more efficient commitment of ADSCs into preosteoblasts, according to the previously reported protocol. However, cAMP upregulation in a culture of ADSCs in spheroids resulted in significantly increased osteocalcin production and mineralization. Thus, undifferentiated and predifferentiated ADSCs respond differently to cAMP pathway stimulation in terms of osteogenesis, which might explain the ambiguous results from the literature.

Heterogenous Populations of Tissue-Resident CD8+ T Cells Are Generated in Response to Infection and Malignancy.

Tissue-resident memory CD8+ T cells (Trm) provide host protection through continuous surveillance of non-lymphoid tissues. Using single-cell RNA-sequencing (scRNA-seq) and genetic reporter mice, we identified discrete lineages of intestinal antigen-specific CD8+ T cells, including a Blimp1hiId3lo tissue-resident effector cell population most prominent in the early phase of acute viral and bacterial infections and a molecularly distinct Blimp1loId3hi tissue-resident memory population that subsequently accumulated at later infection time points. These Trm populations exhibited distinct cytokine production, secondary memory potential, and transcriptional programs including differential roles for transcriptional regulators Blimp1, T-bet, Id2, and Id3 in supporting and maintaining intestinal Trm. Extending our analysis to malignant tissue, we also identified discrete populations of effector-like and memory-like CD8+ T cell populations with tissue-resident gene-expression signatures that shared features of terminally exhausted and progenitor-exhausted T cells, respectively. Our findings provide insight into the development and functional heterogeneity of Trm cells, which has implications for enhancing vaccination and immunotherapy approaches.

Macrophage development and activation involve coordinated intron retention in key inflammatory regulators.

Monocytes and macrophages are essential components of the innate immune system. Herein, we report that intron retention (IR) plays an important role in the development and function of these cells. Using Illumina mRNA sequencing, Nanopore direct cDNA sequencing and proteomics analysis, we identify IR events that affect the expression of key genes/proteins involved in macrophage development and function. We demonstrate that decreased IR in nuclear-detained mRNA is coupled with increased expression of genes encoding regulators of macrophage transcription, phagocytosis and inflammatory signalling, including ID2, IRF7, ENG and LAT. We further show that this dynamic IR program persists during the polarisation of resting macrophages into activated macrophages. In the presence of proinflammatory stimuli, intron-retaining CXCL2 and NFKBIZ transcripts are rapidly spliced, enabling timely expression of these key inflammatory regulators by macrophages. Our study provides novel insights into the molecular factors controlling vital regulators of the innate immune response.

Differential Expression of the Transcription Factor GATA3 Specifies Lineage and Functions of Innate Lymphoid Cells.

Lymphoid tissue inducer (LTi) cells are regarded as a subset of innate lymphoid cells (ILCs). However, these cells are not derived from the ILC common progenitor, which generates other ILC subsets and is defined by the expression of the transcription factor PLZF. Here, we examined transcription factor(s) determining the fate of LTi progenitors versus non-LTi ILC progenitors. Conditional deletion of Gata3 resulted in the loss of PLZF+ non-LTi progenitors but not the LTi progenitors that expressed the transcription factor RORγt. Consistently, PLZF+ non-LTi progenitors expressed high amounts of GATA3, whereas GATA3 expression was low in RORγt+ LTi progenitors. The generation of both progenitors required the transcriptional regulator Id2, which defines the common helper-like innate lymphoid progenitor (ChILP), but not cytokine signaling. Nevertheless, low GATA3 expression was necessary for the generation of functionally mature LTi cells. Thus, differential expression of GATA3 determines the fates and functions of distinct ILC progenitors.