Stromal cells are important bone marrow (BM) niche components that regulate immune cell homeostasis through the production of cytokines such as interleukin 15 (IL-15). Although stromal-derived IL-15 is known to support lymphocyte survival, it remains unclear which stromal cell subsets are capable of IL-15 transpresentation, and how they influence specific lymphocyte populations. By using conditional IL-15 receptor alpha (IL-15Rα) deletion models, we demonstrate that IL-15Rα expression by BM stromal cells is essential for the maintenance of multiple IL-15-dependent lymphocyte populations. Deletion of IL-15Rα in Lepr+ or IL-7+ stromal cells selectively reduced central memory CD8+ T cells in the BM, whereas deletion of IL-15Rα in Osx+ stromal cells resulted in a marked loss of natural killer T (NKT) cells and tissue-resident memory CD8+ T cells. Surprisingly, endothelial-specific IL-15Rα deletion did not affect lymphocyte maintenance in the BM, but specifically impaired natural killer (NK) maturation and survival in the periphery, uncovering a role of endothelial IL-15 in mature NK cell maintenance. Together, our findings establish that transpresentation of IL-15 by distinct BM stromal cell subsets creates functionally specialized BM niches to support specific lymphocyte populations.

With the death of Charles Weissmann, molecular biology has lost of one of its most productive, outstanding, and critical representatives. A molecular biologist who became famous through his ground-breaking discoveries, as well as his scientific presentations, which always included “little stories,” used to illustrate the main points of his talks in a persuasive and amusing fashion. He will be remembered for unraveling the replication of RNA bacteriophages; for developing the first example of targeted mutagenesis, using viral RNA; for cloning the first interferon gene; for his decisive contributions to the origin and pathogenic effects of prions; and for the superb quality of his experimental work and mentorship.

Vascularized skins were 3D printed using single donor human fibroblasts, pericytes, keratinocytes, and endothelial cells (ECs), the latter either unmodified (WT-ECs) or deleted of MHC molecules (KO-ECs). Adult MISTRG6 immunodeficient mice neonatally inoculated with adult human hematopoietic stem cells (HSCs) received printed skin allogeneic to the HSCs and were boosted 3 weeks after grafting with human PBMCs autologous to the HSCs. HSC inoculation alone produced low levels of circulating human myeloid and lymphoid cells without affecting grafts; PBMC boosting dramatically increased circulating human CD4+T cells and boosted CD8+ T cells only in mice with WT-EC grafts. These grafts became infiltrated by human macrophages, dendritic cells, CD4+ and CD8+ T cells and showed evidence of rejection. Shared T cell clones were present in skin and spleen. KO-EC grafts had minimal infiltration of graft or spleen without rejection, despite MHC molecule expression on other graft cell types.

Generating mice that express human antibodies is a lengthy and labor-intensive process. Knocking in human gene segments into mice has potential, but how large a gene segment can be knocked into a zygote? Nair et al. insert a 155-kb human VH locus directly into mouse zygotes by CRISPR-mediated homology-directed repair, generating functional humanized antibody mice in weeks rather than years.

We compared duodenal biopsies showing active celiac disease (CeD) to normal controls using single-cell RNA sequencing, cyclic immunofluorescence, RNAScope, and proximity ligation assays. There is increased infiltration of villous but not crypt epithelium T cells bearing either αβ or γδ T cell receptors (TCRs) in CeD. Both T cell subsets are activated cytotoxic T lymphocytes (CTLs) and are the predominant mucosal source of IFNγ. In response to this IFNγ, villous but not crypt enterocytes show an IFNγ signature, including nuclear phospho-STAT1 protein, class II HLA molecules and IFNγ-inducible chemokines known to recruit CTLs (e.g., CCL3, CCL4, CXCL10, and CXCL11) and receptors for these chemokines are expressed on the infiltrating CTLs. Villous enterocytes also display increased HLA-E and HLA-B mRNAs and proteins. Bioinformatic analyses (NICHES) and proximity ligation assays show frequent binding of both αβ and γδ TCRs with enterocyte HLA-E or HLA-B , but not HLA-DR . In contrast, NKG2C, proposed as an alternative trigger of CTL activation, is infrequently expressed and shows few interactions with HLA-E. Our data suggest that activated intraepithelial CTLs produce IFNγ which recruits additional CTLs and increases antigen-dependent killing of villous epithelium using either conventional or HLA-E antigen presentation.

Long-term in vivo production of therapeutic proteins and development of vaccines that elicit protective levels of broadly neutralizing antibodies (bNAbs) against major pathogens face challenges. In this study, we report on an alternative gene editing approach using small numbers of hematopoietic stem and progenitor cells (HSPCs) to direct long-term, high-level expression of antibodies or cargo proteins. In mice, edited B lymphocytes derived from transplanted HSPCs were activated by cognate antigen, underwent clonal expansion, and developed into specific antibody-synthesizing or cargo protein-synthesizing plasma cells. These cells produced long-lasting, therapeutic levels of serum antibody against HIV-1, malaria, or an anti-influenza virus bNAb that mediated universal protection from heterologous lethal challenge. Our data provide a paradigm for cell therapy approaches to prevent or treat disease using self-amplifying B cell protein factories.

Placental insufficiency affects fetomaternal health throughout life. Although the interaction between the maternal uterine immune milieu and fetus-derived cells plays a crucial role in placental formation, several aspects remain unclear. Therefore, we conducted this study to investigate the effects of interleukin (IL)-18, a distinctive cytokine with both proinflammatory and anti-inflammatory properties, on the uterine immune milieu and placental development. Our results identified pregnant uterine smooth muscle cells as an important source of IL-18, which supports homeostatic type 1 immune responses. IL-18 facilitates appropriate placental development through uterine vascular remodeling and placental angiogenesis. Smooth muscle cell-specific Il18 knockout dam mice exhibited excessive cytotoxicity of uterine natural killer (NK) cells, impaired fetoplacental growth, and elevated maternal blood pressure, reflecting preeclampsia-like phenotypes. Their offspring demonstrated a tendency toward excessive weight gain and delayed neurodevelopment. Overall, this study emphasizes the essential role of IL-18 in placental formation and its wider implications for fetomaternal health.

Macrophage-enteric neuron dialog is crucial for intestinal homeostasis, but its specific role and underlying mechanism in inflammatory bowel disease (IBD) pathogenesis remain elusive. Here, we demonstrate that colonic macrophage-mediated extracellular matrix (ECM) remodeling orchestrates enteric neuronal maturation and dictates colitis progression. The depletion of Pcif1, recognized as a unique methyltransferase for m6Am mRNA methylation, in macrophages attenuates colitis and protects enteric neurons from inflammation-driven degeneration. Specifically, Pcif1-deficient macrophages increase ECM accumulation, promoting their own extravasation into enteric neuronal plexuses and the maturation of enteric neurons, thereby curbing inflammation-driven neuronal injury and colitis progression. Mechanistically, Pcif1-mediated m6Am modification inhibits the translation of Znf219 mRNA and represses the Znf219-Egr1 axis, a master transcriptional module that governs ECM remodeling. Finally, pharmacological blockade of PCIF1 promotes macrophage-mediated ECM deposition and suppresses colitis and neuronal loss. Our findings reveal a novel mechanism whereby ECM remodeling in macrophages drives enteric neuronal maturation and illuminate the m6Am machinery as a promising therapeutic target for preventing neuronal loss in IBD patients.

Loss of function mutation in the human DPP9 gene causes Hatipoglu syndrome leading to severe inflammasomopathy. A key feature of the disease is pancytopenia and patients require bone marrow transplantation, but the mechanism of cell loss is unclear since Dpp9 mutant mice have normal hematopoiesis, suggesting that a distinct mechanism of disease occurs in humans. Here, we present a model of human DPP9 deficiency leveraging reverse genetics in the MISTRG6 humanized mice. We found that CRISPR editing of human CD34+ hematopoietic stem and progenitor cells (HSPCs) led to very efficient and persistent gene deletion in vivo. Human DPP9 deletion recapitulated cytopenia in peripheral blood and in the bone marrow, and cell loss was cell intrinsic. However, DPP9 deletion led to little transcriptional changes suggesting post-transcriptional regulation in human HSPCs. Mechanistically, DPP9 deficiency led to the activation of the CARD8 inflammasome resulting in HSPC pyroptosis, whereas NLRP1 was dispensable for cell death. Thus, our results reveal a unique human mechanism of disease and offer therapeutic insight for this inflammasomopathy.

Chronic stimulation of CD8⁺ T cells within the tumor microenvironment (TME) induces a hypofunctional state characterized by diminished cytotoxicity and functionally impaired anti-tumor function, known as exhaustion. Exhaustion is associated with epigenetic changes that remain relatively stable despite interventions like immune checkpoint inhibition (ICI). Although epigenetic changes are potentially reversible, reports of therapeutic strategies to effectively restore function in exhausted CD8⁺ T cells remain limited. Here, we report DNA methyltransferase 1 (DNMT1) inhibition (DNMT1i) in counteracting CD8 + T cell dysfunction during the anti-tumor response. We show that DNMT1i synergizes with ICI to rescue the tumor cell killing activity of chronically stimulated CD8⁺ T cells in a melanoma model. DNMT1i mitigates transcriptional features of exhaustion while inducing a divergent effector program. DNMT1i attenuates the global increase in chromatin accessibility associated with exhaustion and enables epigenetic remodeling of the exhausted chromatin landscape upon restimulation. Finally, DNMT1i enhances the effector function of melanoma patient-derived tumor infiltrating lymphocytes after prolonged ex vivo expansion. These studies establish DNMT1 targeting as a promising strategy to counteract CD8⁺ T cell exhaustion and potentiate ICI efficacy.

Over one million patients receive cancer immunotherapy annually, yet the mechanisms underlying life-threatening immune-mediated toxicities remain poorly understood. Checkpoint inhibitor pneumonitis (CIP) is the leading cause of immunotherapy-related mortality, with a case fatality rate approaching 10%, and no genetic risk factors have been described to date. We identified Dipeptidyl-peptidase 9 (DPP9) as the first genetic susceptibility gene for CIP in a clinico-genomics cohort of 4,397 patients treated with immune checkpoint inhibitors. Mechanistically, DPP9 suppresses CARD8 inflammasome activation and IL-18 secretion in human monocytes, a pathway which is engaged prior to CIP onset, with IL-18 selectively elevated in the plasma of patients who subsequently develop CIP. Myeloid-restricted ablation of Dpp8 and Dpp9 in mice recapitulated the pulmonary histopathological and immunological hallmarks of CIP, including granuloma formation, accumulation of IFNγ-producing T cells and monocyte-derived macrophages. Each of these phenotypes were driven by excessive IL-18 secretion. Together, these findings establish DPP9 as a genetic determinant of CIP and nominate IL-18 blockade as a mechanistically rational therapeutic strategy.

BACKGROUND:Stearoyl-CoA desaturase 1 (SCD1) is a key enzyme in fatty acid (FA) metabolism that catalyzes the addition of a cis double bond to palmitic and stearic saturated FAs, producing palmitoleic and oleic monounsaturated FAs, respectively. Interleukin (IL)9-secreting CD4+ T-helper lymphocytes (Th9) exert antitumoral activity in preclinical cancer models. In the present study, we evaluated the role of SCD1 in Th9 differentiation and their antitumoral properties. RESULTS:A specific monounsaturated FA profile is found in Th9 lymphocytes compared with Th1, Th2, Th17 and regulatory T cell (Treg) lymphocyte subsets and is correlated to the induction of SCD1 expression in mouse and human Th9 differentiated in vitro with TGFβ1 and IL4. The expression of SCD1 is also detected in tumor-draining lymph nodes and tumor-infiltrating Th9 lymphocytes of mice. The canonical Smad3 and non-canonical Pi3K members of the TGFβ signaling drive SCD1 expression in combination with IL4 during Th9 polarization. The invalidation of SCD1 gene expression or inhibition of its activity blocks Th9 differentiation by disrupting Smad2/3 activation. Furthermore, the lipidomic analysis between Th9 and Th9 invalidated for SCD1 gene or activity reveals a change in the FA profile, specifically a decrease in palmitoleic and oleic acids. Nevertheless, only oleic acid restores Th9 differentiation in CD4+ T lymphocytes invalidated for SCD1 gene or activity under TGFβ1 and IL4 polarizing conditions. Finally, invalidation of the SCD1 gene or its activity leads to the loss of Th9 antitumoral functions and promotes tumor growth through the production of TGFβ1. CONCLUSION:We demonstrate that SCD1 contributes to Th9 differentiation and their antitumoral activity via the regulation of Smad2/3 signaling.

CD8+ T cell differentiation has been associated with changes in the expression of long noncoding RNAs (lncRNAs). Yet, which and how lncRNAs regulate CD8+ T cell responses following infection in vivo remains incompletely understood. We performed deep RNA-seq to map the lncRNA expression landscape of CD8+ T cell subsets during infection and generated lncRNA knockout mouse models to evaluate the in vivo relevance of six lncRNAs. We identified Rroid2 to regulate effector CD8+ T cell function and effector-to-memory differentiation. Rroid2-deficient mice displayed increased CD44dim Foxp3+ regulatory T cells while the development of other immune cells, such as natural killer cells, was not affected. In CD8+ T cells, Rroid2 deficiency resulted in a fine-tuned downregulation of transcription factors Id2 and T-bet and impaired KLRG1+ and KLRG1- effector CD8+ T cell proliferation and cytotoxicity as well as effector-to-memory CD8+ T cell differentiation. The human orthologue of Rroid2, LINC01814, is also upstream of the transcriptional regulator ID2 and is highly expressed in human memory CD8+ T cells. Taken together, Rroid2 represents a key regulatory layer that controls CD8+ T cell differentiation.

ABSTRACT:Immune-driven fibrotic skin diseases, including scleroderma/systemic sclerosis (SSc) and chronic graft-versus-host disease (GVHD), cause skin stiffening that has a major impact on patient quality of life and associated patient mortality. Therapies to improve sclerotic skin resulting from these diseases are largely ineffective. We previously showed that epiregulin (EREG), a dendritic cell type 3-derived epidermal growth factor receptor (EGFR) ligand, is elevated in the skin and lungs of patients with SSc and required for the maintenance of skin fibrosis. Here, we developed a fully human anti-EREG neutralizing antibody that has both high affinity and specificity. We found this therapeutic antibody to be functional and safe in vivo using human EREG knockin mice. To understand the antifibrotic mechanism of targeting EREG, we aligned skin single-cell transcriptomic profiles of SSc, morphea (localized scleroderma), and sclerotic GVHD (SclGVHD) with disease biomarkers. EREG expression in the skin was elevated in all 3 fibrotic diseases and is a driver of tenascin C (TNC) production by myofibroblasts. TNC is a proinflammatory extracellular glycoprotein that functions as an endogenous Toll-like receptor 4 (TLR4) ligand, which induces expression of TLR4 target genes CCL2 and interleukin-6. Examination of skin explants from patients with active SclGVHD treated with anti-EREG therapeutic antibody by spatial transcriptomics demonstrated upregulation of matrix degradation by increased MMP and decreased TIMP1 expression. Protein measurements showed reduced secretion of EREG targets TNC, CCL2, and TIMP1 in all patients and type I collagen and FN1 in three-fourths of patients. Thus, sclerotic skin treated with the anti-EREG therapeutic antibody reduced inflammatory and fibrosis biomarkers associated with EGFR and TLR4 signaling.

Altered human aldo-keto reductase family 1 member C3 (AKR1C3) expression has been associated with poor prognosis in diverse cancers, ferroptosis resistance, and metabolic diseases. Despite its clinical significance, the endogenous biochemical roles of AKR1C3 remain incompletely defined. Using untargeted metabolomics, we identified a major transformation mediated by AKR1C3, in which a spermine oxidation product ''sperminal'' is reduced to ''sperminol.'' Sperminal causes DNA damage and activates the DNA double-strand break response, whereas sperminol induces autophagy in vitro. AKR1C3 also pulls down acyl-pyrones and pyrone-211 inhibits AKR1C3 activity. Through G protein-coupled receptor ligand screening, we determined that pyrone-211 is also a potent agonist of the semi-orphan receptor GPR84. Strikingly, mammalian fatty acid synthase produces acyl-pyrones in vitro, and this production is modulated by NADPH. Taken together, our studies support a regulatory role of AKR1C3 in an expanded polyamine pathway and a model linking fatty acid synthesis and NADPH levels to GPR84 signaling.

Bone marrow (BM) mesenchymal stromal cells (MSC) provide microenvironmental niches that support hematopoietic stem cells and regulate hematopoiesis. Whether functional heterogeneity among BM MSCs contributes to the development and survival of distinct immune cell lineages remains incompletely understood. Here, we use an Il15 knockin reporter and multiple conditional deletion mouse models to show distinct differences in IL-15 expression between BM MSC subtypes. Conditional deletion of Il15 in Osx+ stromal cells results in decreased natural killer (NK) cell precursors, memory CD8+ T cells and NKT cells but not mature NK cells. Lepr+ stromal cells support the survival of mature NK cells and memory CD8+ T cells in the BM of older mice, while endothelial cells support mature NK cells and memory CD8+ T cells in the blood but not in the BM. Thus, our data suggest that MSC subtypes differentially regulate the development and survival of IL-15-dependent immune cell lineages in the BM.

Pulmonary large cell neuroendocrine carcinoma (LCNEC) is a rare, aggressive lung tumor marked by significant molecular heterogeneity. In a study of 590 patients across two independent cohorts, we observe comparable overall survival across treatment regimens (chemotherapy, chemoimmunotherapy, immunotherapy) without unexpected adverse events. Genomic analysis identifies distinct non-small cell lung cancer-like (NSCLC-like, KEAP1, KRAS, STK11 mutations) and SCLC-like (RB1, TP53 mutations) LCNEC subtypes, with 80% aligning with SCLC transcriptional profiles. Serial sampling reveals stable mutational but shifting transcriptomic landscapes over time. Here we show, elevated FGL-1 (a LAG-3 ligand) and SPINK1 expression in NSCLC-like LCNECs, and higher levels of DLL3 in SCLC-like LCNECs. Immunofluorescence confirms FGL-1 expression in NSCLC-like LCNECs, and H&E slide analyses indicates fewer tumor-infiltrating lymphocytes in LCNECs versus other lung cancers. These findings highlight LCNEC's distinct immunogenomic profile, supporting future investigations into LAG-3, SPINK1, and DLL3-targeted therapies.

There is a constant worldwide need for blood products, traditionally obtained from donations. In vitro red blood cell (RBC) production could supplement this demand and offer benefits such as thorough screening for improved safety, the possibility of genetic manipulation to restore genetic deficiencies, and therapeutic loading. Induced pluripotent stem cells (iPSCs) are a promising cell source for transfusable RBCs and blood products due to their immortality and independence from donors. However, current iPSC differentiation protocols—including both monolayer and embryoid body-based systems—have failed to produce sufficient erythroid cells (1,012 per unit) for therapeutic application, primarily due to developmental immaturity, inefficient enucleation (<20%), and suboptimal, static culture conditions lacking physiologic relevance. Here, we developed a hematopoietic organoid production protocol through embryoid body formation starting from iPSCs using low growth factor culture conditions. Optimization of this—iPSC to RBC differentiation platform—and its step-by-step translation process to complete dynamic culture conditions allows scalability and bioreactor application. The optimized dynamic culture yields ∼5 × 103 RBC/iPSC, requiring an estimated ∼5 × 107 iPSCs to produce a minitransfusion unit, achieving a consistent ∼70% enucleation rate and bona fide function, demonstrated by both in vitro and in vivo assays. Our feeder-free, GMP-compatible system accomplishes an enucleated RBC production rate sufficient for large-scale application and serves as a bridge to large-scale bioreactor RBC production, facilitating clinical application.