Group 3 innate lymphoid cells (ILC3s) exhibit dynamic plasticity, with their differentiation and function orchestrated by epigenetic mechanisms, including histone modifications and DNA methylation. We identify the histone demethylases UTX and JMJD3 as pivotal regulators of ILC3 specialization. Their deficiency disrupts subset balance: NKp46+ ILC3s are depleted with impaired IL-22 production, whereas CCR6+ ILC3s expand and exhibit enhanced IL-17A-mediated antifungal immunity. Single-cell profiling reveals that UTX/JMJD3 ablation epigenetically restricts double-negative (DN)-to-NKp46+ differentiation while potentiating CCR6+ polarization, rewiring ILC3 lineage trajectories through chromatin remodeling. Cleavage under targets and tagmentation (CUT&Tag) analysis demonstrates that UTX directly occupies enhancer regions upstream of Tcf7, where it catalyzes H3K27me3 demethylation to maintain an open chromatin state. Retroviral Tcf7 reconstitution rescues the NKp46+ ILC3 deficit and normalizes cytokine production, positioning TCF7 as the key effector downstream of UTX. These findings establish UTX/JMJD3 as central epigenetic gatekeepers of mucosal immunity, offering therapeutic avenues for inflammatory disorders driven by ILC3 dysregulation.

The development and functional maintenance of CD8+ T cells are metabolically regulated processes in which mitochondria serve as the central hub. Here, we identify glucose-regulated protein 75 (GRP75) as a critical mitochondrial regulator controlling these processes. Using T cell-specific Hspa9 (encodes GRP75) knockout mice, we demonstrate that GRP75 deficiency disrupts CD8+ T cell fate, leading to defective T cell homeostasis and impaired memory differentiation. Mechanistically, impaired mitochondrial function in GRP75-deficient CD8+ T cells leads to perturbation of IL-7R signaling and aberrant expression of effector-associated molecules. Further studies reveal that GRP75 deficiency leads to upregulation of interferon regulatory factor 4 (IRF4), a critical transcription factor for effector versus memory fate, which in turn suppresses memory CD8+ T cell differentiation. Our findings establish GRP75 as a pivotal mitochondrial checkpoint that coordinates metabolic state and functional fate in CD8+ T cells.

In humans, the loss of function in a single copy of the CTLA4 gene leads to an immune disorder characterized by a spectrum of immunodeficient and autoimmune manifestations. To investigate the pathogenicity of CTLA-4 haploinsufficiency, we utilized a heterozygous mouse model carrying a patient-derived Y139C mutation. We found that young heterozygous mice exhibited lymphoproliferative features and an activated T cell phenotype, without displaying autoimmunity. Co-culture assays revealed impaired trans-endocytosis. Despite this dysfunction, they remained resistant to induced inflammation. With age, however, they eventually developed spontaneous autoimmune manifestations that were more pronounced than those in age-matched littermates. Mechanistically, systemic activation induced CTLA-4-independent compensatory pathways, allowing Tregs to retain suppressive activity. Our study confirmed the pathogenicity of the Y139C heterozygous mutation, providing supporting evidence for CTLA-4 haploinsufficiency. Moreover, these results revealed that compensatory mechanisms arise in response to compromised trans-endocytosis. Further exploration may offer insights into disease progression and support the development of therapeutic strategies.

BackgroundTo report a refractory Takayasu arteritis (TAK) patient who developed a severe inflammatory flare after tocilizumab (TCZ) therapy and explore the underlying mechanism.Case presentationA 21-year-old female with active TAK received TCZ after insufficient response to prednisone. TCZ was temporarily withheld then re-added at 400 mg due to disease relapse. After TCZ reintroduction, the patient developed worsening vascular pain, new abdominal pain, dizziness, fever, and markedly elevated inflammatory markers. Imaging showed worsened arteritis and new superior mesenteric arteritis. Methylprednisolone pulse and infliximab failed to control the flare, but tofacitinib achieved complete symptom resolution and normalized inflammatory markers within one month.In vitro findingsPeripheral blood samples of the patient were collected and peripheral blood mononuclear cells (PBMCs) Fcγ receptor (FcγR) expression and downstream signaling were assessed upon TCZ stimulation. In vitro studies showed that increasing TCZ concentrations were associated with enhanced FcγRIIA, FcγRIIB, and FcγRI expression, along with increased spleen tyrosine kinase (SYK) phosphorylation in the patient’s PBMCs.ConclusionTCZ can paradoxically trigger an inflammatory flare in refractory TAK, with exploratory evidence suggesting possible involvement of excessive FcγR activation. Non-antibody-based Janus kinase (JAK) inhibitors like tofacitinib may be effective rescue therapy in such cases.

Platelets must balance hemostatic function with pathological thrombosis, particularly under metabolic stress conditions. Mitogen-activated protein kinases (MAPKs) are central to platelet responses, but how these platelet signals differentially regulate hemostasis remains poorly understood. To investigate the role of Traf2/Nck-interacting kinase (TNIK), we generated megakaryocyte/platelet-specific TNIK-knockout mice (Tnikf/f PF4-Cre+) and evaluated platelet function, hemostasis, and thrombosis under normal and hyperlipidemic conditions using chimeric Tnikf/f PF4-Cre+ Apoe-/-mice fed high-fat diets. TNIK-deficient mice exhibited prolonged bleeding times, delayed arterial thrombosis and platelet activation under normal conditions, primarily due to impaired dense granule secretion. Mechanistically, TNIK interacted with JNK-interacting protein 1 (JIP1) to promote MLK3/MKK4/JNK pathway activation during hemostatic responses. Surprisingly, under hyperlipidemic conditions, TNIK deficiency accelerated thrombosis and enhanced platelet responses to oxidized low-density lipoprotein (ox-LDL). In this context, TNIK specifically bound to protein kinase C epsilon (PKCε) and suppressed the NOX2/ROS/ERK5 pathway, thereby inhibiting excessive platelet activation. We conclude that TNIK functions as a molecular switch in platelets, promoting normal hemostasis while simultaneously preventing hyperlipidemia-associated thrombosis through distinct signaling pathways. This dual regulatory mechanism provides insight into how platelets balance hemostatic function with pathological thrombosis risk and identifies TNIK as a potential therapeutic target in metabolic thrombotic disorders.

T cell differentiation and selection in the thymus are pivotal for establishing antigen specificity and shaping the functional repertoire of peripheral T cells. Here, we present an in vitro protocol to investigate the roles of specific target genes during early thymic development in mice utilizing retroviral vectors. We describe steps for retroviral packaging, the isolation of primary murine thymic cells, retroviral transduction, and the subsequent in vitro differentiation of thymocytes. For complete details on the use and execution of this protocol, please refer to Fan Zhao et al.1.

Dysregulation of thymic T cell development compromises immune homeostasis and can lead to leukemic transformation, but the molecular mechanisms linking developmental signals, proliferative cues, and leukemogenesis remain incompletely understood. Here, we integrate deubiquitinase library screening and publicly available single-cell RNA sequencing to analyze mouse and human thymocytes. We find the deubiquitinase USP10 to be expressed in thymocytes, and also elevated in peripheral blood from patients with T-cell acute lymphoblastic leukemia (T-ALL) compared to healthy controls; by contrast, T cell-specific USP10 deficiency blocks mouse thymocyte proliferation and differentiation. Mechanistically, USP10 interacts with SOX4, de-ubiquitinating and protecting SOX4 from degradation to promote thymocyte proliferation, with SOX4 overexpression restoring thymocyte differentiation in USP10-deficient mice. Lastly, MYC induces Usp10 expression, and pharmacologic inhibition of USP10 delays MYC-driven leukemogenesis in a mouse leukemia model. Our results thus identify USP10 as coordinator of developmental signals and oncogenic processes in thymocytes, and implicate USP10 as a potential target for T-ALL therapy.

Importins could inhibit the condensation of RNA-binding proteins, while it remains unknown whether exportins elicit a similar function. Here, we identified that exportin CRM1 binds to the nuclear protein NPAT, which initiates and maintains the formation of the histone locus body (HLB), a membraneless nuclear body regulating histone transcription. CRM1 drives the nuclear export of NPAT by targeting a nuclear export signal (NES) within the LisH domain. The LisH domain contributes to NPAT condensation by mediating its self-association. Mechanistically, CRM1 competitively occupies the self-association sites in the NES motif, thereby suppressing NPAT condensation. In contrast, the two recurrent CRM1 E571K and E571G mutants could not regulate NPAT condensation and HLB remodeling due to their impaired binding to the NES of NPAT. Based on the "competitive occupation" model, we designed a LisH domain-derived short peptide that competes with homotypic intermolecular interactions of NPAT to perturb HLB formation. Our findings reveal that exportin regulates nuclear protein condensation via a competitive occupation strategy.

In the version of this article initially published, the institution name for affiliation 3 (Maryland Anderson Cancer Center) was incorrect. The correct institution is MD Anderson Cancer Center. The error has been corrected in the HTML and PDF versions of the article.

ObjectivesThis study aimed to explore the relationship between dermatomyositis (DM) and thyroid cancer and the role of transcriptional intermediary factor 1γ (TIF-1γ) in the pathogenesis of cancer-associated DM.MethodsFive cases of DM with thyroid cancer, including one with metastasis, were summarized and analyzed. Published cases of DM with thyroid cancer were reviewed. Subsequently, a bidirectional two-sample Mendelian randomization (MR) analysis was performed to investigate the relationship between thyroid cancer and myositis using inverse-variance weighting (IVW), MR-Egger, and weighted-median (WM) analyses. Finally, immunohistochemistry (IHC) and immunofluorescence (IF) analysis were performed to detect TIF-1γ in tumor tissues of a DM patient and a thyroid cancer patient without DM.ResultsThis article reports the first case of a patient with DM and metastatic papillary thyroid carcinoma (PTC), in whom recurrence of the rash prompted the detection of metastasis, with the rash resolving after surgical resection of the metastatic foci. MR analysis showed a potential causal relationship between thyroid cancer and myositis (OR = 1.398; 95% CI = 1.045–1.869; p = 0.029). A higher expression level of TIF-1γ was observed in tumor tissues of DM patients compared with those of thyroid cancer patients without DM by immunofluorescence (IF) analysis (p < 0.0001).ConclusionsDM symptoms might represent a paraneoplastic phenomenon in patients with thyroid cancer, with the underlying pathological mechanisms requiring further exploration and TIF-1γ overexpression potentially serving as a trigger.

T cell receptors(TCRs)serve key roles in the adaptive immune system by enabling recognition and response to pathogens and irregular cells.Various methods have been developed for TCR construction from single-cell RNA sequencing(scRNA-seq)datasets,each with its unique characteristics.Yet,a comprehensive evaluation of their relative performance under different conditions remains elusive.In this study,we conducted a benchmark analysis utilizing experimental single-cell immune profiling datasets.Additionally,we introduced a novel simulator,YASIM-scTCR(Yet Another SIMulator for single-cell TCR),capable of generating scTCR-seq reads containing diverse TCR-derived sequences with different sequencing depths and read lengths.Our results consistently showed that TRUST4 and MiXCR outperformed others across multiple datasets,while DeRR demonstrated considerable accu-racy.We also discovered that the sequencing depth inherently imposes a critical constraint on successful TCR construction from scRNA-seq data.In summary,we present a benchmark study to aid researchers in choosing the appropriate method for reconstructing TCRs from scRNA-seq data.

Systemic lupus erythematosus (SLE) remains refractory to conventional immunosuppression in a subset of patients. In treatment-refractory SLE, we show that peripheral CD19⁺ B cells and bone marrow CD19⁻BCMA⁺ long-lived plasma cells are dominant autoantibody sources, motivating dual CD19 and BCMA targeting. Here we report results from a cohort of patients (14 female, one male) in an ongoing phase 1 dose-escalation trial of co-infused autologous anti-CD19 and anti-BCMA chimeric antigen receptor (CAR) T cells after fludarabine/cyclophosphamide lymphodepletion. Primary endpoints were dose-limiting toxicities (DLTs) within 28 days and adverse events within 12 weeks; key secondary endpoints comprised attainment of Lupus Low Disease Activity State (LLDAS) and DORIS remission within 12 weeks and in vivo CAR-T persistence within 24 weeks. Exploratory endpoints were the duration of post-infusion B cell depletion and time to recovery, the kinetics of immune reconstitution and longitudinal changes in autoantibody titers and serum immunoglobulin concentrations after CAR-T therapy. Over a median 712-day follow-up (range, 613-1,134), no DLTs occurred. Grade 1 cytokine release syndrome developed in 86.7% of patients, with no neurotoxicity or treatment-related deaths. The most common grade 3 or higher adverse events were neutropenia (100%), thrombocytopenia (40%) and anemia (13.3%), all of which were reversible with supportive care. By week 12, 12 of 15 patients (80%) fulfilled both the LLDAS and DORIS remission criteria. Multiomic analyses confirmed elimination of autoreactive CD19⁺BCMA⁺ clones, reconstitution of naive IgM/IgD B cells and durable downregulation of interferon-stimulated and BAFF-dependent signatures, indicating improved immune homeostasis. Longitudinal monitoring of three patients for 1 year demonstrated sustained eradication of pathogenic clones, suggesting potential cure. Dual anti-CD19/anti-BCMA CAR-T cell therapy demonstrates good safety and promising clinical efficacy in treatment-refractory SLE. This study supports the further development of this treatment approach for patients with rSLE. ClinicalTrials.gov identifier: NCT05030779 .

Protein phosphatase 2A (PP2A) is one of the most abundant serine/threonine phosphatases and plays critical roles in regulating cell fate and function. We previously showed that PP2A regulates the differentiation of CD4+ T cells and the development of thymocytes. Nevertheless, its role in CD8+ T cells remains elusive. By ablating the catalytic subunit α (Cα) of PP2A in CD8+ T cells, we revealed the essential role of PP2A in promoting the effector functions of CD8+ T cells. Notably, PP2A Cα-deficient CD8+ T cells exhibit reduced proliferation and decreased cytokine production upon stimulation in vitro. In vivo, mice lacking PP2A Cα in T cells displayed defective immune responses against lymphocytic choriomeningitis virus infection, associated with reduced CD8+ T cell expansion and decreased cytokine production. Consistently, the ablation of the PP2A Cα subunit in CD8+ T cells results in attenuated antitumor activity in mice. There is a notable decrease in the infiltration of PP2A Cα-deficient CD8+ T cells within the tumor microenvironment, and the cells that do infiltrate exhibit diminished effector functions. Mechanistically, PP2A Cα deficiency impedes CD28-induced AKT Ser473 phosphorylation, thus impairing CD8+ T cell costimulation signal. Collectively, our findings underscore the critical role of phosphatase PP2A as a propeller for CD28-mediated costimulation signaling in CD8+ T cell effector function by fine-tuning T cell activation.

Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer, known for its early onset, strong metastatic tendencies, and poor prognosis. Conventional treatments, including chemotherapy and immunotherapy, often face challenges such as limited efficacy, adverse side effects, and high recurrence rates. To address these limitations, a family of tri-block chimeric proteins, cysteine-tagged silk-elastin-like proteins (cSELPs), is designed to form responsive in situ hydrogels for treating late-stage and metastatic TNBC. These cSELPs are de novo designed to integrate multiple functional protein motifs, including a photothermal agent binding motif, silk-inspired crosslinking motifs, and elastin-like thermo-responsive motifs. This unique sequence design enables the cSELP hydrogels to exhibit in situ gelation, photothermal responsive release of chemotherapeutic agent doxorubicin and immune checkpoint inhibitor anti-PD-L1, and antibacterial properties, leading to effective tumor microenvironment remodeling. By promoting immunogenic cell death and stimulating immune activation, this approach converts immunosuppressive "cold" tumors into immunologically active "hot" tumors. The cSELP hydrogel system demonstrates potent therapeutic efficacy against both primary and metastatic TNBC while maintaining excellent biocompatibility and long-term safety. This protein material platform offers an innovative strategy to reshape the tumor microenvironment and combine multimodal treatments into a single biocompatible system, highlighting its potential for clinical translation.

Recent advances in single-cell technology enable the simultaneous capture of T cell receptor (TCR) sequences and gene expression (GEX), providing an integrated view of T cell function. However, linking TCRαβ information and T cell phenotypes at the population level to elucidate their disease association remains an unaddressed gap. Here, by constructing a large-scale reference of paired single-cell RNA/TCR sequencing (scRNA/TCR-seq) comprising more than 2 million T cells from 70 studies, 1017 biological samples, 583 individuals, and 46 disease conditions, along with their single-cell transcriptome, full-length paired TCR, and human leukocyte antigen (HLA) genotypes, we revealed the intrinsic features of germline-encoded TCR-major histocompatibility complex (MHC) restriction in CD4+/CD8+ lineages. We also observed widely existing public TCRαβs across the population, associated with higher clonal expansion levels and shared HLA alleles. The most publicly shared TCRs are likely to target epitopes from common viruses, such as Epstein-Barr virus (EBV), cytomegalovirus (CMV), and influenza A virus (IAV). Furthermore, we introduced TCR-DeepInsight, a computational framework to identify HLA-shared and disease-associated TCRαβ clusters that exhibit similar TCR sequence and GEX profiles, extensible for researchers to incorporate their data with our reference and characterize potentially functional TCRs. In summary, our work presents a panoramic scTCRαβ reference and computational methods for TCR study.

Group 3 innate lymphoid cells (ILC3s) serve as critical guardians of mucosal immunity. However, the transcriptional networks governing their function remain incompletely characterized. Here, we demonstrate that interferon regulatory factor 4 (IRF4) is essential for maintaining intestinal ILC3 homeostasis and function. IRF4-deficient mice exhibit reduced NKp46+ ILC3s, expanded precursor-like NKp46-CCR6- ILC3s, and impaired interleukin-22 (IL-22)/IL-17A production, increasing susceptibility to infections. Furthermore, IRF4 loss disrupted major histocompatibility complex (MHC)-class II-associated transcriptional signatures in ILC3s, particularly in CCR6+ ILC3s, accompanied by downregulation of MHC class II protein expression. This perturbation consequently diminished ILC-mediated apoptosis of effector CD4+ T cells. Sequencing and trajectory analysis link IRF4 to NKp46+ ILC3 maintenance and Tbx21 regulation. ATAC-seq/CUT&Tag reveal direct IRF4 binding to Batf, Tbx21, Il22, Il17a, and MHC II loci. Overexpression of T-bet partially rescued the differentiation defects in intestinal ILC3s, whereas Batf overexpression partially restored functional impairments and significantly enhanced MHC class II expression in ILC3s.

T cell function is defined by both T cell receptors (TCR) and T cell gene expression (GEX). Although single-cell technology enables the simultaneous capture of TCR and GEX information, the lack of a reference atlas and computational tools hinders our ability to uncover the fundamental TCR usage rules and to efficiently characterize disease-associated TCRs. Here, through the collection of million-scale reference atlas of paired single-cell RNA/TCR sequencing (scRNA/TCR-seq) comprising 46 diverse disease conditions, we revealed the intrinsic features of TCR-MHC restriction in CD4+/CD8+ lineages, and observed the widely-existing public TCRαβ pairs across individuals, which is correlated to higher clonal expansion levels and similar gene expression profiles. Building upon the reference atlas, we introduced TCR-DeepInsight, a computational framework featuring both GEX and TCR, with a disease specificity scoring system that enables the characterization of disease-associated TCR clusters with similar TCR sequences and transcriptome profiles. Our study provides a valuable tool for researchers to integrate their datasets with the large-scale scRNA/TCR-seq reference atlas and identify disease-associated TCR sequences comprehensively and robustly. National Natural Science Foundation of China 31930038, 32100718, U21A20199, 32170551. Fundamental Research Funds for the Central Universities 226-2022-00134. Tencent AI Lab Rhino Bird Research Funding RBFR2022015. Computational and Systems Immunology (COMP)

Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) plays a crucial role in maintaining peripheral immune tolerance, but its mechanisms of action are highly complicated. Here, through the generation of a gene knock-in (KI) mouse carrying a CTLA-4 Y139C human patient-derived pathogenic mutation, we phenocopied the lethal autoimmune diseases in Ctla4 KO mice due to the impairment of Treg functions. Interestingly, although both KO and KI Treg cells lost the ability to endocytose B7 molecules, the KO and KI mice differed in terms of T-cell proliferation since the KI mutation retained its ability to transmit inhibitory signals. Therefore, this study not only dissected the two distinct immunoregulatory mechanisms of CTLA-4 but also provided genetic evidence highlighting the importance of ligand trans-endocytosis in the function of CTLA-4. Our findings enhance our understanding of CTLA-4 function and CTLA-4 insufficiency disease, providing valuable insights for advancing improved immunotherapy strategies targeting CTLA-4.