Journal of Experimental Medicine

ELF4 is an ETS family transcription factor involved in immune regulation, and germline loss-of-function mutations in ELF4 have been known as deficiency in ELF4, X-linked (DEX). To date, ELF4-related disease has been exclusively associated with germline mutations. Here, we report a pediatric patient with recurrent mucocutaneous inflammation and periodic fever caused by a somatic truncating mutation in ELF4. By directly comparing ELF4-mutant and wild-type immune cells within the same individual using full-length single-cell RNA sequencing, we identified mutation-associated transcriptional alterations across multiple immune cell types. Pathway analyses revealed cell type-specific immune alterations, characterized by reduced antiviral and interferon-related signaling in NK cells and enhanced inflammatory pathways related to Th17 differentiation and inflammatory bowel disease in CD16 monocytes. This study expands the disease spectrum of ELF4 deficiency by identifying somatic truncation of ELF4 as a genetic mechanism underlying autoinflammatory diseases and biased immune programs.

Natural killer (NK) cells undergo stepwise differentiation from multipotent progenitors within secondary lymphoid tissues. Despite the central importance of the tissue microenvironment in their development, little is known about cell-cell interactions that regulate human NK cell trafficking and maturation. Here, we identify the chemokine receptor CXCR4 and its lig- and CXCL12 as regulators of stromal-NK cell interactions required for NK cell maturation. We demonstrate that CXCR4 is expressed throughout human NK cell development in peripheral blood and tonsil, and CXCL12 is enriched in stromal niches containing developing NK cells. Pharmacologic blockade or genetic disruption of CXCR4 resulted in diminished adhesion to integrin ligands and high-resolution imaging demonstrated crosstalk between CXCR4 and integrins, providing a mechanistic basis for chemokine-dependent modulation of adhesion. Further, CXCR4 blockade resulted in altered contact-dependent motility on stromal cells and integrin ligands, with decreased stable stromal engagement and increased cell speed. Consistent with a requirement for these interactions, treatment with the CXCR4 antagonist plerixafor (AMD3100) impaired NK cell generation from CD34+ precursors. Analysis of NK cells from WHIM syndrome patients with CXCR4 gain-of-function mutations treated with plerixafor revealed similar defects in migration and adhesion, supporting the in-vivo relevance CXCR4-dependent regulation of NK cell adhesion and motility.

RIG-I is a cytosolic immune receptor that provides the first line of defense by detecting viral RNA and triggering antiviral responses. Its physiological role in humans remains unclear, as no patients with complete RIG-I deficiency have yet been reported. We identified a critically ill COVID-19 patient with severe RIG-I deficiency caused by heterozygous RIG-I G731R, a novel dominant loss-of-function variant. The G731R mutation in helicase motif VI disrupts the arginine finger, impairing the ATPase activity of RIG-I, but not its RNA-binding ability. However, viral RNA binding fails to expose the signaling domains, thereby impairing the IFN-{beta} response of G731R. Instead, G731R competes with wild-type RIG-I, exerting a dominant negative effect. The loss-of-function is caused by bulky-charged substitutions at G731, as alanine or leucine substitution results in an unexpected gain-of-function phenotype. These findings highlight the importance of uncompromised RIG-I function for human antiviral immunity and the pleiotropic effects of single mutations.

T follicular helper (Tfh) cells are a specialized subset of CD4 T cells that localize to germinal centers (GC), where they provide critical help to B cells through the delivery of IL-21 and other cytokines. Here, we demonstrate that the tight control of the chromatin remodeler Special AT-rich sequence-binding protein 1 (Satb1) is key for this process, as overexpression of Satb1 drives lymphoproliferation and expansion of the T cell and B cell compartments in secondary lymphoid organs. Specifically, Satb1 overexpression induces a pronounced shift towards Tfh cell differentiation and increased GC formation accompanied by an increase in non-classed switched GC B cells and auto-antibody secretion. These findings highlight the importance of the precise regulation of Satb1 in fine-tuning CD4 T cells and B cells responses and suggest a potential role for dysregulation of Satb1 in the pathogenesis of autoimmune disease such as systemic lupus erythematodes (SLE).

Persistence of memory T lymphocytes, in the apparent absence of antigen, is a hallmark of immune memory and key to adaptive immunity to recurrent infections. The signaling pathways ensuring survival and quiescence of the memory T cells are largely enigmatic. Here we show, by inhibition in vivo, that persistence of surface CD69+KLF2-tissue-resident memory T cells of murine bone marrow and spleen is blocked by antibodies to the integrins VLA-4 and LFA-1, connecting the memory T cells to VCAM1 and ICAM1 of stromal cells. Persistence requires the PI3K/AKT signaling pathway, since it is blocked by Wortmannin, and it involves PI3K-dependent survival genes. Surface CD69-KLF2+ memory T cells of the bone marrow are also dependent on integrin-mediated contact to stromal cells. Their persistence critically depends on the NF-kB pathway, their PI3K signaling pathway is not relevant. Blocking Jak1 and 3 of the interleukin-7 and -15 signaling pathways does affect memory T cells of the spleen, but not those of the bone marrow. Thus, tissue-resident KLF2+ and KLF2-memory T cells, and memory T cells of spleen and bone marrow, use different signaling pathways, adapting them to their respective tissues and reflecting an unexpected heterogeneity in the molecular mechanisms of persistence.

Visceral leishmaniasis (VL), a life-threatening parasitic disease caused by Leishmania donovani (LD), progresses primarily due to profound immunosuppression. However, the molecular and cellular mechanisms underlying this immune dysfunction remain poorly defined. Here we show that early production of IL-35 by dendritic cells (DCs) is critical for immunosuppression and disease pathogenesis during LD infection. LD stimulated IL-35 expression in DCs through the TIM-3 receptor and the downstream transcription factor STAT3. IL-35 produced by DCs subsequently suppressed DC maturation and T cell proliferation, propagated immunosuppression by inducing IL-35 expression in T cells, and impaired type-1 anti-leishmanial immunity, thereby promoting disease progression. Genetic or pharmacologic inhibition of STAT3 markedly reduced IL-35 production by DCs, restored protective type-1 T cell responses, and promoted parasite clearance in vivo. Notably, treatment with WP1066, an FDA-designated orphan STAT3 inhibitor, significantly lowered parasite burden and disease severity in infected mice. Together, these findings uncover a previously unrecognized TIM-3-STAT3-IL-35 axis that drives immunosuppression and pathogenesis in VL and highlight STAT3 inhibition as a promising therapeutic strategy to restore host immunity and control infection.

Multisystem inflammatory syndrome in children (MIS-C) is a pediatric hyperinflammatory disease manifesting 4-6 weeks after SARS-CoV-2 infection. While the immunological hallmarks of MIS-C have been defined, few details regarding the underlying disease pathology have been resolved. To address this, we used a multiomics approach to profile the plasma and peripheral immune cells of 13 acute MIS-C patients, 18 recovered MIS-C follow-ups resampled over multiple time points (1-18 months), and 15 healthy pediatric controls. Despite rapid clinical disease resolution, circulating pro-inflammatory (IL-8, IL-6, IL-1, IL-1{beta}, TNF-{beta}) and TH2-type cytokines (IL-4, IL-5, IL-13) remained elevated up to three months post-MIS-C onset, revealing a subclinical inflammatory state that endures in recovered children. Surprisingly, the majority of patient-expanded TCRs recognizing SARS-CoV-2 epitopes were cross-reactive (75%, 12/16 SARS-CoV-2 TCRs) for autoantigens related to prostaglandin biology and insulin metabolism, suggesting a breakdown of self-tolerance via SARS-CoV-2 molecular mimicry. Indeed, autoantibody screening confirmed that 13 gene targets with self-antigen peptides also exhibited elevated autoantibodies in MIS-C patients. Further, autoreactive TCR expansions lasted over time and correlated with cytokines involved in allergic inflammation. Together, our findings point to a mechanism of sustained autoimmunity wherein promiscuous TCRs recognize both viral and self-antigens that are activated during primary SARS-CoV-2 infection in children who develop MIS-C. Upon onset, these circulating cross-reactive T cells drive clinically apparent sterile autoinflammation that persists subclinically into convalescence.

Macroautophagy/autophagy is the main lysosomal pathway for the degradation and recycling of cytoplasmic cargo, with emerging roles in endothelial cell (EC) biology. While autophagy has been extensively studied in blood ECs, its function in lymphatic ECs (LECs) remains unexplored. Given the central role of the lymphatic system in antitumor immunity and metastatic spread, we investigated how LEC autophagy affects metastatic lung colonization. In line with previous reports showing that autophagy regulates the availability of the egress signal sphingosine 1 phosphate (S1P) in secondary lymphoid organs (SLOs), lungs of non-tumor-bearing mice with LEC-specific genetic deletion of Atg5 (ATG5LEC-KO mice) exhibited reduced lymphocyte infiltration. Remarkably, in tumor-bearing mice, either genetic loss of LEC-autophagy or pharmacological blockade of S1P lyase by 4-deoxypyridoxine (DOP) suppressed lung metastasis. Pulmonary immune profiling revealed that while DOP enhanced effector T cell activity despite lower numbers, LEC-autophagy-deficient mice markedly increased the B and CD8 T Cell abundance coupled with profound reduction of VEGFR3 expression in the lung lymphatic vasculature. Together, these findings uncover an autophagy-dependent remodeling of the lymphatic routes and immune niches that fosters metastatic seeding and growth in the lung.

Interferon regulatory factor 1 (IRF1), a transcription factor encoded within the 5q31 locus harboring systemic lupus erythematosus (SLE) associated variants, promotes inflammatory responses by T and myeloid cells. Although IFN{gamma}-activated B cells also express IRF1, its role in B cell biology and SLE is unclear. Here, we use a mouse SLE model, single-cell multiomics, and human B cells to show that IRF1 intrinsically regulates Irf4 gene chromatin accessibility and expression in B cells to control the balance between the antibody secreting cell (ASC) lineage commitment factor, IRF4, and the B cell identity factor, IRF8. We demonstrate that IRF1, through its integration of IFN{gamma} and TLR7 induced transcriptional programs, tips B cells toward a terminal effector inflammatory AC fate at the expense of preserving more stem-like, resting and regulatory B cells that do not elicit autoantibody-associated pathology in SLE. Thus, IRF1 serves as a central node controlling B cell-driven autoimmune disease.

Here we describe a damaging heterozygous variant in CDC45 in an individual with common variable immunodeficiency (CVID) and recurrent viral infections. This individual has variably decreased number of circulating NK cells, disruption in the ratio of CD56bright to CD56dim cells, and consistently decreased NK cell function. Interestingly, the inherited CDC45 variant is also present in a sibling with less severe clinical manifestations; we determined that allelic bias of the damaging allele accounts for this differential expressivity. As previously reported for other helicase variants that cause inborn errors of immunity (IEI), we found cell cycle defects in immune cells from the proband that lead to reduced survival of NK cells. Together, these findings link another member of the core replicative helicase complex to inborn errors of immunity and highlight the sensitivity of NK cells to these variants. They also define another case of allelic bias contributing to variable expressivity of an IEI gene.

Alveologenesis during early postnatal life requires tight coordination between epithelial differentiation and immune regulation, yet how immune cells contribute to this process remains unclear. Regulatory T cells (Tregs) are established mediators of immune homeostasis and tissue repair in adult lung injury, but their role in lung development is unknown. Here, we identify a transient wave of highly proliferative, activated Tregs that accumulates in the neonatal lung during an early postnatal window. Using inducible Treg ablation, we show that loss of this neonatal Treg population disrupts alveologenesis, resulting in enlarged airspaces, and persistent structural abnormalities later in life. Treg depletion also induces interferon-associated inflammatory programmes, promotes neutrophil accumulation, and is accompanied by a sustained imbalance in alveolar epithelial populations. Notably, neutrophil depletion partially rescues both epithelial composition and alveolar structure, identifying neutrophils as key downstream effectors of Treg-mediated regulation. Together, these findings show that neonatal Tregs are required for normal alveologenesis by restraining neutrophil-driven inflammation and preserving epithelial balance. Our study reveals a previously unappreciated role for immune regulation in lung organogenesis.

Innate-like lymphocyte subsets are generated predominantly during early-life windows, yet the mechanisms that restrict their development in adulthood remain unclear. Here we identify Cbfb2 gene dosage as a quantitative regulator of stage-specific lymphoid potential. We show that reduction of CBF{beta}2 levels unlocks fetal-like competence in adult hematopoietic progenitors, enabling robust generation of IL-17-producing {gamma}{delta} T (T{gamma}{delta}17) cells. Although Cbfb2 haploinsufficiency minimally alters steady-state transcription, chromatin profiling of H3K4me3 revealed promoter-level changes in adult lymphoid-primed multipotent progenitors consistent with altered developmental priming. In adult bone marrow chimeras, Cbfb+/2m progenitors efficiently generated functional V{gamma}2+ T{gamma}{delta}17 cells in lymph nodes and skin, and restoring Cbfb2 expression suppressed this capacity, establishing a dosage-dependent mechanism. Using an optimized in utero transplantation system, we further demonstrate that fetal niches amplify this latent competence and selectively favor IL-17-committed {gamma}{delta} T cell differentiation over conventional {beta} T cell output. Notch1 haploinsufficiency enhanced T{gamma}{delta}17 generation and phenocopied the effect of CBF{beta}2 dosage reduction, linking quantitative NOTCH1 signaling to innate-like lymphocyte developmental programming. Together, these findings reveal that fetal versus adult lymphopoiesis is governed by quantitative tuning of RUNX:CBF{beta} activity and uncover unexpected plasticity in adult hematopoiesis controlled by transcription factor dosage.

B cell development relies on stringent checkpoints that ensure immune competence and eliminate autoreactive clones. Transitional B cells (B220CD93), which emerge from the bone marrow, migrate to the spleen and differentiate into follicular (FO) or marginal zone (MZ) B cells, a process governed by B cell receptor (BCR) signaling strength, metabolic fitness, and survival cues. Here, we identify Folliculin Interacting Protein 1 (Fnip1) as a key regulator of this developmental transition. Using conditional Fnip1-deficient mice (Fnip1fl/flCD21Cre), loss of Fnip1 results in a developmental arrest at the transitional B220CD93mid stage, severely limiting differentiation into FO and MZ B cells and leading to accumulation of a distinct enlarged CD19high, RAG negative B cells. Fnip1 modulates BCR signaling thresholds and metabolic programming by regulating the AMPK/FLCN/TFEB and CD19/PI3K/Akt/mTORC1 pathways through restricting TFEB access to the nucleus. Using the MD4/mHEL/sHEL tolerance model, we show that Fnip1 is dispensable for negative selection but is essential for maintaining peripheral tolerance. Together, our findings define Fnip1 as a metabolic gatekeeper that integrates nutrient-sensing pathways with BCR signaling to orchestrate transitional B cell fate decisions, promote peripheral tolerance, and maintain immune homeostasis.

Macrophage (M-), granulocyte (G-), and granulocyte-macrophage (GM-) colony-stimulating factors (CSFs) regulate myeloid cell function, yet their relative roles during inflammation remain poorly defined. To uncover how CSFs shape spatial immune niches in Crohns disease, we performed Xenium single-cell spatial transcriptomics on ileal tissues, revealing cell-type-specific expression and source-target interactions for each CSF. GM-CSF, unlike M-CSF or G-CSF, was locally enriched in ulcerated regions where lymphocytes adjacent to macrophage aggregates signaled through STAT5 phosphorylation. To study functional consequences, we developed a csf2rb-/- zebrafish model of intestinal injury. Using this model, we found that loss of GM-CSF signaling exacerbated epithelial damage and inflammation, whereas recombinant human GM-CSF limited injury by restraining ILC1 expansion, sustaining ILC3 maintenance, and promoting IL-22 production. Cross-species single-cell analysis revealed conserved ILC gene modules and GM-CSF-dependent transcriptional networks linking lymphoid and myeloid populations. These findings establish GM-CSF as a critical spatial regulator of myeloid-lymphoid crosstalk and intestinal immune homeostasis in Crohns disease.

ABSTRACTSHeterozygous gain-of-function (GOF) mutations in signal transducer and activator of transcription 1 (STAT1) cause an inborn error of immunity characterized by immune dysregulation, recurrent infections and various autoimmune manifestations. However, the precise pathogenic mechanism by which STAT1 GOF contributes to autoimmunity remains elusive. In our cohort, STAT1-GOF patients exhibit biased circulating follicular helper T (cTfh) populations with CXCR3+ Tfh1-like features. Using a Stat1 GOF mouse model that spontaneously developed autoimmunity, we found that overactivated STAT1 promotes Tfh differentiation and disrupted T cell-dependent humoral responses with skewed immunoglobulin class switching towards IgG2. Furthermore, STAT1 GOF directly targets to Tfh and Th1 cell signature genes and thereby drives the development of Tfh1 cells with excessive IFN-{gamma} production, which implicated in autoantibody production and the development of autoimmunity. Notably, IFN-{gamma} neutralization significantly alleviated autoimmune cellular responses and autoantibody levels in mutant mice, highlighting IFN-{gamma} blockade as a promising targeted therapy for the STAT1-GOF patients with autoimmunity. Our findings suggest that proper regulation of STAT1 activity within a reasonable magnitude is crucial for ensuring optimal host-protective humoral immunity. One-sentence summaryOveractivated STAT1 promotes Tfh1 differentiation to drive autoimmunity.

Osteopontin (OPN) is a secreted phosphoprotein implicated in colorectal cancer liver metastasis (CRCLM), yet the distinct spatial contributions of host-and tumor-derived OPN in driving this disease remain unclear. Using a 2 x 2 genetic knockout mouse model targeting OPN in host and tumor compartments, combined with spatial transcriptomics, we investigated compartment-specific OPN functions in CRCLM. Tumor-derived OPN promotes tumor proliferation through MEK/ERK signaling. Host OPN licenses monocyte-to-macrophage differentiation, while tumor OPN polarizes macrophages towards an M2-like state. Both host and tumor OPN suppress T cells in the tumor microenvironment, whereas loss of host OPN reveals an interferon-driven, anti-tumor niche. Translational studies using OPN-blockade immunotherapy in syngeneic and patient-derived xenograft mouse models reduced tumor burden and enhanced T cell infiltration. Together, these findings redefine the OPN-myeloid paradigm in CRC and nominate OPN as a potential therapeutic target.

Innate immune detection of Gram-negative bacteria depends on sensing of cytosolic lipopolysaccharide (cLPS) by the non-canonical inflammasome, mediated in humans by NLRP11 and caspase-4 (CASP4). Activation of this pathway in human macrophages triggers gasdermin-D activation and pyroptotic cell death. Although CASP4 directly binds LPS in vitro, additional host factors are required for efficient activation in vivo. Here, we show that NLRP11, a primate-specific pattern recognition receptor, facilitates CASP4 recognition of cLPS and promotes non-canonical inflammasome activation. NLRP11 functions upstream CASP4, forming an ASC-independent complex that requires a conserved CASP4 p20 residue, binds cLPS, and enhances CASP4-dependent LPS recognition. Mutational analyses demonstrate that in human macrophages, in addition to LPS binding and CASP4 catalytic activity, CASP4 interaction with NLRP11 is essential for efficient pyroptosis. Together, these findings establish NLRP11 as a primate-specific determinant that enhances CASP4-mediated cLPS detection and non-canonical inflammasome activation, revealing a mechanism for human-specific regulation of innate immunity.

Targeting Tregs is a potential strategy to improve cancer therapies. However, which Tregs accumulate in response to tumoral processes, and how tumors affect their phenotype, is poorly understood. Here we show that tumor Tregs are equivalent to effector tissue Tregs in steady state organs. We used a mouse model of intestinal neoplasia to demonstrate that one early event in carcinogenesis is sufficient to induce local accumulation of Tregs resembling human tumor Tregs. Treg accumulation was driven by TCR-dependent oligoclonal expansion of tissue Tregs with an effector Treg phenotype. Treg expansion was independent of CCR8, IL33R and CD137, which were previously linked to tumor Treg. In contrast, GATA3 was required for effector tissue Tregs and for their expansion in response to neoplasia. Our findings identify GATA3-dependent clonal expansion of effector tissue Tregs as a key event in promoting tumor growth. HighlightsO_LIAn early tumorigenic event alone drives accumulation of effector tissue Tregs C_LIO_LITregs in tumors are phenotypically akin to effector tissue Tregs C_LIO_LIThe accumulation of Tregs is driven by TCR-dependent oligoclonal expansion C_LIO_LIGATA3 controls tumor-promoting effector tissue Tregs C_LI

Tissue regeneration requires tight control of immune cell behavior, yet the mechanisms that restrain immune-driven regenerative responses remain poorly defined. Here, we identify the rhomboid intramembrane serine protease Rhbdl2 as a critical regulator of regeneration in zebrafish. We generated rhbdl2 mutants by CRISPR-Cas9 and found that it does not affect normal development, but triggers enhanced regenerative growth following injury, accompanied by increased macrophage accumulation at the wound site, which is accompanied by increased early apoptosis and proliferation. Proteomic analyses reveal increased Rac2 protein levels in rhbdl2 mutants, indicating dysregulated immune signaling. Functionally, Rac2 morpholino oligonucleotides-mediated knockdown in rhbdl2 mutant larvae suppresses the elevated macrophage recruitment and enhanced tissue regenerative phenotype. Together, these findings uncover Rhbdl2 as an immune checkpoint that constrains macrophage-driven enhanced regeneration, with vast implications for inflammatory disease, fibrosis, and tumor-immune interactions.

Regulatory T cells (Treg cells) maintain peripheral immune tolerance but display considerable plasticity in peripheral tissues. The molecular mechanisms governing their function and plasticity, particularly under inflammatory conditions, remain poorly defined. Here, we identify the NF-{kappa}B-associated transcriptional cofactor I{kappa}B{zeta} as a critical regulator of Treg cell plasticity and function. Enforced expression of I{kappa}B{zeta} in Treg cells triggered the excessive expansion of functionally impaired Treg cells, resulting in lymphadenopathy, splenomegaly, and systemic type 2 inflammation, most prominently in the lung. Mechanistically, I{kappa}B{zeta} modified BATF expression and function, thereby driving the cell-intrinsic production of Th2-associated cytokines by Treg cells. Conversely, Treg-specific deletion of I{kappa}B{zeta} constrained IL-33-mediated expansion of tissue Treg cells and surprisingly attenuated type 2 inflammation. Thus, I{kappa}B{zeta} functions as a molecular switch that reprograms regulatory T cells into Th2-like Treg cells, thereby perturbing peripheral immune tolerance. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/707402v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1ef0b17org.highwire.dtl.DTLVardef@c12eaaorg.highwire.dtl.DTLVardef@decc86org.highwire.dtl.DTLVardef@1458767_HPS_FORMAT_FIGEXP M_FIG C_FIG