Science Immunology

Whether human tumor-specific CD4 T cells can drive antitumor immunity in cancers lacking tumor-cell MHC class II expression has not been established. In MHC class II-negative melanoma and breast cancer, CD4 T cells with a transcriptional signature of tumor specificity correlated with myeloid and CD8 T cell activation. Using a patient-derived NY-ESO-specific, HLA-DP04-restricted TCR, we tested whether human CD4 T cells could control tumors through indirect antigen presentation. In vitro, CD4 T cells inhibited HLA-DP-negative melanoma growth through recognition of tumor-derived antigen on antigen presenting cells (APC). In humanized mice, CD4 T cell transfer induced regression of HLA-DP-negative tumors, and control required HLA-matched human APCs. CD4 T cell transfer induced endogenous CD8 T cell proliferation and myeloid activation that mirrored immune programs correlated with the presence of these cells in patients. These findings establish that human tumor-specific CD4 T cells coordinate antitumor immunity through APC without direct tumor recognition, supporting their therapeutic relevance in MHC class II-negative cancers.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1s), collectively known as group 1 innate lymphoid cells (ILCs), are critical mediators of anti-tumor immunity, yet their role in brain metastasis remains poorly defined. Using single-cell RNA sequencing and functional depletion models, we show that group 1 ILCs fail to provide adequate tumor control in breast cancer brain metastasis (BCBM). In contrast to the liver, where diverse, functionally competent group 1 ILC populations facilitate excellent metastatic control, group 1 ILCs in BCBM show limited diversity and profound tissue-specific dysfunction such that their depletion had no effect on tumor burden despite modest parenchymal infiltration. We attribute this group 1 ILC dysfunction to severely restricted IL-15 availability, with IL-15R protein detected at 500-fold lower concentration in the brain than in the liver. Local IL-15 complex administration restored group 1 ILC accumulation, cytotoxicity, and tissue residency, significantly reducing brain metastatic burden independent of T cells. Intracranial delivery of IL-15-secreting induced pluripotent stem-cell-derived (iPSC)-NK cells, already in clinical testing for non-small cell lung cancer, significantly reduced tumor burden. Collectively, these findings reveal how tissue-specific differences in cytokine availability shape group 1 ILC population diversity and disease response, establishing localized IL-15 signaling as a compelling strategy to unlock innate immunity against intracranial malignancies.

Oral premalignant lesions (OPLs) are common; however, histopathological grading incompletely identifies lesions destined for cancer. To define tissue ecosystems that precede malignant transformation, we integrated single-cell-resolution Xenium spatial transcriptomics of 20 HPV-negative biospecimens from 16 patients, with independent single-cell RNA sequencing and immunofluorescence data from 38 patients with OPLs after quality control. Progressive OPLs exhibit a coordinated epithelial-immune program comprising MX1/NOTCH3-high KRT14+/KRT15+ basal epithelial states, S100A9-high inflammatory macrophages, TIGIT-high, exhaustion-associated T cells, and altered dendritic cell states. Spatial analyses showed that T cells and dendritic cells were displaced from the basal epithelial interface, and that epithelial neighborhoods were depleted of dendritic cells but enriched for TIGIT-high T cells, revealing immune reorganization not captured by cellular abundance alone. Independent single-cell and protein-level analyses reproduced basal epithelial interferon/stress programs, S100A9-associated myeloid inflammation, TIGIT-high, exhaustion-associated T-cell programs, and dendritic cell redistribution. Ligand-receptor inference suggested convergent myeloid and lymphoid signals linked to epithelial stress, checkpoint regulation, and extracellular matrix remodeling. Functionally, recombinant S100A9 accelerated wound closure in oral epithelial and cancer cells, whereas S100A9 and S100A8/S100A9 enhanced oral cancer cell proliferation. These responses were attenuated by pharmacologic inhibition of TLR4 or RAGE. Together, these data define a spatially organized, myeloid-skewed, checkpoint-enriched ecosystem present before invasion and suggest S100A9-TLR4/RAGE signaling and epithelial-immune geometry as candidate mechanisms and biomarkers for oral cancer interception.

Macrophage-driven inflammation underlies many inflammatory diseases, yet the mechanisms that restrain macrophage activation within inflamed tissues remain poorly understood. Using mouse models of foreign body granuloma formation and macrophage activation syndrome, we show that neutrophils accumulate at sites of inflammation and metabolically restrain macrophage-driven pathology. Neutrophil depletion or genetic impairment exacerbated tissue pathology, whereas induced neutrophilia attenuated inflammatory damage. Tissue-infiltrating neutrophils acquired an alternatively polarized state characterized by high arginase 1 activity that was regulated by an IL4Ra-STAT3-NOX2 signaling axis. Functionally, polarized neutrophils depleted extracellular arginine in an arginase 1-dependent manner, thereby limiting macrophage effector programs. Human neutrophils exhibited conserved regulation of arginase activity, and alternatively polarized neutrophils were detected in patients with sarcoidosis and macrophage activation syndrome. Together, these findings identify an arginine-dependent metabolic mechanism through which alternatively polarized neutrophils restrain macrophage-driven inflammation and reveal a previously unrecognized immune-regulatory role for these cells in chronic inflammatory disease.

How endocrine states modulate local immune responses is a fundamental question in immunology. Sexual dimorphism is a striking example: females generally mount stronger immune responses than males, yet how sex hormones intersect with local signals is poorly understood. Here, we identify an endocrine-neuro-immune circuit in which sex hormones determine whether immune cells respond to nociceptor signals. In female skin, nociceptor-derived CGRP promotes dendritic cell (DC) migration and T cell priming indirectly through inflammatory monocytes. CGRP induces monocyte production of Activin A, which upregulates CXCR4 on DCs and promotes migration to draining lymph nodes. In males, androgen receptor signaling suppresses monocyte Activin A and silences this circuit. Disrupting the relay abolishes enhanced female immunity, whereas deleting androgen receptor in male monocytes restores neuronal control. Thus, sex hormones regulate immunity by determining how immune cells interpret neuronal signals, revealing how endocrine state can gate local neuroimmune communication to generate sex-specific immune responses.

Tertiary lymphoid structures (TLS) predict response to checkpoint inhibition, and conventional dendritic cells (cDC) are required to build and maintain them. What renders a niche permissive for their assembly is unknown: chemokines recruit the cellular constituents but do not specify how they organize. Here we show that the organizing signal is a matrix fragment. Proteolysis of the proteoglycan versican (VCAN) releases the matrikine versikine, constitutively in germinal center light zones and inducibly in tumor stroma. Versikine activates CD11b+ cDC and expands cDC1 to assemble TLS-archetypal triads, apposed DC: CD4+ T: CD8+ T units. Versikine-conditioned DC engage progenitor-exhausted CD8+ T cells (Tpex) and Th1-polarized follicular helper CD4+ T cells. Excess unproteolyzed VCAN inverts this toward DC-Treg crosstalk and Tpex loss. Versikine supplied as mRNA or recombinant protein sensitizes refractory tumors to PD-L1 blockade. VCAN proteolysis prospectively predicted outcomes after PD-1 blockade in metastatic colorectal cancer. Matrikines organize chemokine-recruited cells into functional immune niches.

Bacterial biofilms drive persistent infection and chronic inflammation, yet host sensing of biofilm-derived ligands remains poorly understood. Here we identify curli amyloid, an integral component of Enterobacteriaceae biofilms, as a novel activator of the non-canonical inflammasome in human and mouse dendritic cells (DCs). Curli both primes and activates the inflammasome, directly binding caspase-11 to engage NLRP3 and drive robust IL-1{beta} maturation without pyroptosis, establishing curli as a previously unrecognized trigger of DC hyperactivation. Mechanistically, this non-lytic signaling is marked by limited gasdermin D oligomerization without mitochondrial localization and NINJ1-dependent membrane rupture. Functionally, curli-hyperactivated DCs show enhanced migration to the draining lymph nodes in vivo, suggesting consequent augmented adaptive immunity. Finally, curli-containing biofilms also elicit IL-1{beta} secretion without cell death, identifying bacterial amyloid as a central driver of non-lytic inflammasome activation during immune recognition of biofilms.

Single-cell studies have defined immune states in inflammatory bowel disease (IBD), but how adaptive receptor histories organize circulating immunity remains unclear. We generated a single-cell transcriptomic atlas of peripheral blood from 249 participants with Crohn's disease, ulcerative colitis, or non-IBD control status, including 182 with productive TCR and BCR recovery. Expanded TCR clonotypes marked inflammatory-memory and cytotoxic states, while distinct but similar paired TCRs shared inflammatory programs across participants. BCR lineage maturation linked IgA-associated mucosal and plasma B cell programs to somatic mutation and class switching, distinguishing maturation-associated biology from clonal expansion. Helper, regulatory, and cytotoxic T-cell programs covaried with B-cell states, and inferred interactions nominated reciprocal antigen-presentation and helper pathways. Repertoire-based machine learning distinguished diagnosis, inflammation, and contemporaneous six-month treatment-response status. Together, this atlas connects receptor architecture to coordinated systemic immune remodeling, establishes a foundation for repertoire-informed patient stratification, and prioritizes candidate mechanisms of IBD pathogenesis.

Intestinal V{gamma}4 {gamma}{delta} intraepithelial lymphocytes (IELs) persistently bind the constitutively expressed epithelial ligand BTNL3/8 through germline-encoded T cell receptor (TCR) determinants. While they resemble innate-like T cells such as NKT cells, unlike these populations, V{gamma}4 IELs encounter ligand only after thymic development. Moreover, unlike conventional {beta} T cells, V{gamma}4 IELs sustain persistent physiological ligand engagement without becoming exhausted. Here, using biophysical, functional, and multimodal single-cell approaches, we show how V{gamma}4 IELs address this challenge. While germline-encoded TCR regions broadly mediate BTNL3 recognition, productive activation requires additional non-germline TCR features that license responsiveness to BTNL3/8 and enable local selection in the gut. Rather than driving exhaustion, BTNL3/8 reactivity directly promotes expression of an NK-like program marked by adaptor molecules that license innate-like signaling in healthy tissue. These findings reveal how combined innate and adaptive features of the {gamma}{delta}TCR enable durable tissue specialization under conditions of persistent physiological ligand engagement.

Vaccination generates heterogenous B cell responses that collectively provide protective immunity, yet how this diversity is established remains poorly understood. Here, we identify the draining lymphatic network as a spatial organizer of humoral immunity. Vaccine dispersal across interconnected draining lymph nodes (dLNs) establishes gradients of antigen and inflammation that create distinct B cell differentiation and selection environments. dLNs proximal to the vaccination site preferentially support antibody-secreting cells and clonally diverse germinal center (GC) responses. In contrast, distal dLNs impose more stringent selection, favoring clonally restricted GCs enriched for high-affinity B cells. Spatially distinct inflammatory environments further determine antibody isotype, linking LN position to antibody effector class. These spatial biases persist upon recall and can be reprogrammed through vaccine design, including nanoparticle properties and LN-directed targeting. Thus, lymphatic networks diversify B cell fate, repertoire and affinity selection, and antibody isotype, revealing a programmable axis for tuning humoral immunity through vaccine design.

Human leukocyte antigen B*27 (HLA-B*27) is a major risk factor for autoimmune diseases such as axial spondyloarthritis (axSpA), acute anterior uveitis (AAU), and psoriatic arthritis (PsA). HLA-B*27 presents bacterial and self-antigens to disease-associated CD8+ T cells, however, how this leads to a pathogenic IL-17-mediated disease remains unclear. We performed mutational experiments in an established disease-associated TCR and demonstrated that the pathogenic motif encompasses a wider range of TCR sequences than previously appreciated. We next leveraged computational pairing of alpha/beta TCR sequencing (TCR-seq) to demonstrate that the broadened pathogenic motif distinguishes axSpA and AAU patients from healthy controls. Paired single-cell RNA sequencing and single-cell TCR sequencing datasets demonstrated that the pathogenic cells have a distinct transcriptional signature. We find a coupling of pathogenic TCRs to this transcriptional signature in public datasets from axSpA and PsA joint fluid and AAU ocular fluid. Finally, we detect the pathogenic CD8+ T cells in the gut and demonstrate that they express a Type 17 program. Our findings suggest that HLA-B*27+ axSpA, AAU, and PsA are initiated by HLA-B*27-restricted pathogenic CD8+ T cells that undergo Type 17 differentiation in response to intestinal antigens.

Mice are key preclinical animal models in vaccine and immunological research, yet their predictive value for human immunity remains contested. Here, we evaluated the translatability of murine models across inactivated and subunit vaccination (influenza, hepatitis B), acute infection (S. aureus, E. coli), and injury (burns and trauma), integrating transcriptomic profiles with sequence evolution, cis-regulatory architecture, and functional annotation. Functional modules were more conserved between species than individual orthologous genes. Translational accuracy depended on stimulus intensity, as infections and injuries engaged conserved signatures, while single-dose vaccination diverged. We then built multilayer models to predict human expression rank and direction of change, and to classify shared leading-edge genes. Adding evolutionary and regulatory layers improved these predictions. We provide a step-by-step R Markdown notebook to apply the models to user data. The study code and datasets are available at https://github.com/wapsyed/mousetohuman_multilayer

Mitochondrial DNA (mtDNA) instability is an emerging feature of cancer, yet its biological effects on the tumor microenvironment (TME) and tumor progression remain incompletely defined. Using melanocyte-specific conditional silencing of mitochondrial transcription factor A (TFAM) in an autochthonous BrafV600E/Pten-/- melanoma model, we demonstrate that tumor cell-intrinsic mtDNA instability accelerates melanoma growth and metastatic dissemination in a cGAS-dependent manner. Transcriptomic profiling of early and late tumor stages revealed that mtDNA instability drives temporal remodeling of the tumor microenvironment (TME), characterized by an early type I interferon (IFN)-rich inflammatory state that licenses immunoregulatory programs as tumors progress. Tumor-associated macrophages emerged as the principal immune cell type reshaped by tumor cell-intrinsic mtDNA stress signaling, with early enrichment of inflammatory states and a shift toward efferocytosis-associated, tissue-adaptive states. The early mtDNA instability-associated macrophage signatures mapped to human melanoma states associated with favorable survival and therapy response, whereas the late signatures mapped to states associated with poor prognosis and therapy resistance across independent cohorts. Anti-PD-1 treatment delayed progression of mtDNA-instable tumors, consistent with a therapeutic sensitivity window associated with the early IFN-rich immune state. These findings establish tumor cell-intrinsic mtDNA instability as a driver of temporal immune-stromal remodeling that promotes melanoma progression and shapes therapeutic response.

The generation of appropriate responses to the intestinal microbiota is an essential aspect of immune homeostasis. Studies of microbes that elicit stereotyped, antigen-specific CD4 T cell responses support the view that immune recognition of the microbiota is specific, relatively limited, and largely skewed toward Tregs, but recent profiling efforts have suggested that recognition of the microbiota may be broader and less regulatory. Such studies have mostly tracked responses to single antigens or used mice that acquire their microbiota after weaning, which has made it difficult to generalize the rules that govern microbiota-mediated T cell responses. Here we comprehensively profile, for the first time, the T cell response to the microbiota at homeostasis, and develop a panel of tools to track multiple, endogenous microbiota-reactive CD4 T cell populations in vivo. Our profiling reveals a broad microbiota-reactive T cell response that is Treg-biased but that also contains non-Treg responses to specific microbes. We find that certain microbiota-reactive T cells require priming during early-life to efficiently establish their Treg identity, while others do not. Our findings illustrate an unappreciated antigen-specific complexity in programming of microbiota-reactive T cells and underscore the importance of studying responses to the microbiota under homeostatic conditions.

Chikungunya virus (CHIKV) infection causes acute febrile illness that frequently progresses to chronic arthralgia. Although monocytes are CHIKV targets, whether viral exposure drives their differentiation into macrophages to sustain persistent inflammation remains incompletely understood. To address this, we integrated transcriptomic profiling of pediatric CHIKV patients during acute and convalescent phases, in vitro differentiation of primary human monocytes into macrophages under CHIKV infection, and single-cell RNA-sequencing (scRNA-seq) of joint-associated macrophages from an immunocompetent mouse model of chronic CHIKV arthritis at 28 days post-infection. In acute patients, we observed a marked upregulation of monocyte/macrophage markers alongside robust, IFN-dependent JAK-STAT signaling. In vitro, CHIKV-infected monocytes differentiated into persistently infected macrophages (MDM-CHIKV) that closely aligned with an M1-polarized transcriptional profile. These cells exhibited STAT1/NF-{kappa}B-dependent inflammation, ISG induction, and enhanced antigen presentation capacity. Remarkably, MDM-CHIKV uniquely produced IFN-{pi}/IL-27, revealing an alternative non-canonical axis where JAK-STAT activation persists despite the absence of classical type I IFNs. Functionally, MDM-CHIKV displayed an enhanced respiratory burst upon immune complex uptake, pointing to Fc{gamma}R-mediated pathology, as well as complement activation and T-cell stimulatory capacity. However, upon TLR4 challenge, these cells retained partial regulatory responses via IL-10 and TGF-{beta}; this hybrid immunophenotype offers a potential mechanistic explanation for relapsing arthralgia. Additionally, scRNA-seq of murine joints confirmed that persistent CHIKV replication was restricted to a distinct macrophage subset that resembles MDM-CHIKV, establishing these cells as the primary in vivo viral reservoir. Mirroring our human findings, joint-associated murine macrophage subsets exhibited elevated IFN-{pi}/IL-27 expression alongside robust NF-{kappa}B/STAT1-dependent inflammatory signatures (Tnf, Il1{beta}, Il6, Ccl2, Ccl5, Cxcl9, Cxcl10, Cxcl1-3, and/or Ptgs2), underscoring their critical role in sustaining chronic arthritis. Collectively, our findings demonstrate that CHIKV actively drives monocyte-to-macrophage differentiation into a persistently infected, M1-skewed hybrid phenotype that serves as a viral reservoir and fuels pro-arthritic inflammation. Consequently, macrophage reprogramming, the non-canonical IFN-{pi}/IL-27-STAT1 axis, Fc{gamma}R-mediated phagocytosis, and complement activation emerge as central pathogenic nodes and promising therapeutic targets for mitigating chronic post-CHIKV arthropathy.

Chronic or persistent T cell activation is widely thought to culminate in T cell exhaustion, yet how autoreactive CD8+ T cells remain functionally intact despite sustained self-antigen exposure is poorly understood. In chronic infection and cancer this durability is attributed to stem-like, TCF1+ PD-1+ progenitor-exhausted (Tpex) cells that reside in lymphoid tissue and continuously replenish functional effectors; whether such a reservoir operates within a chronically inflamed peripheral organ during sterile autoimmunity is unknown. Here we show that mice lacking the lysosomal nuclease DNase2a together with the type I interferon receptor Ifnar1 (Dnase2a-/- Ifnar1-/-) develop spontaneous, progressive hepatic inflammation. This hepatitis was dependent on the endosomal DNA sensor TLR9, identifying self-DNA as the initiating ligand. Inflammation was marked by expansion of inflammatory macrophages and accumulation of CD8+ T cells co-expressing PD-1 and TOX that retained, rather than lost, effector function. Paired single-cell TCR and transcriptomic analysis revealed progenitor-exhausted T cells within the liver itself that shared clonotypes with clonally expanded PD-1+ TOX+ T cells, defining a locally operating progenitor-to-effector pipeline. Spatial transcriptomics positioned these intrahepatic T cell clusters adjacent to IL-27-expressing inflammatory macrophages, and disruption of IL-27 or its receptor in bone marrow-derived cells reduced PD-1+ CD8+ T cells and their effector functions. These findings define a novel mechanism for sustaining autoreactive T cells in the liver via TLR9-driven macrophage-induced IL-27 circuit that sustains a functional, self-renewing autoreactive PD-1+ CD8+ T cell program in situ.

Pediatric tonsillar hypertrophy is a leading cause of sleep disordered breathing in children, yet the immune mechanisms sustaining tonsillar enlargement remain unclear. Using integrated single cell and spatial profiling of pediatric tonsils in a discovery cohort (n = 45) and independent validation cohort (n = 32), we identify a selective expansion and follicular retention of FCRL4+ memory B cells within the mantle zone of hypertrophic tonsils. These cells exhibit a quiescent, tissue-adapted phenotype characterized by NFATC1 repression and reciprocal RUNX1/RUNX2 regulation. Their accumulation is orchestrated by follicular type 1 regulatory T (Tr1) cells, which engage CTLA-4/CD86 checkpoint interactions to suppress plasma cell differentiation and reinforce a local immune tolerance program. Together, these interactions establish a persistent follicular niche enriched in regulatory FCRL4+ memory B cells. Our findings delineate a mechanistic Tr1/FCRL4+ B cell axis underlying follicular hypertrophy and highlight potential immunomodulatory targets for nonsurgical management of pediatric sleep-disordered breathing.

The bronchial epithelium in asthma is vulnerable to damage and has impaired barrier function, but the mechanisms by which it contributes to airway inflammation and remodelling remain unclear. Here, we dissect these epithelial and immunological disease mechanisms by establishing a comprehensive single cell atlas of the bronchial wall from 21 patients with childhood-onset asthma and 25 matched healthy controls. We identify a novel asthma-associated non-canonical epithelial differentiation trajectory in which a repair-associated KLF4+ basal cell subset differentiates into KRT13+ hillock-like cells through a proliferative KRT14+ intermediate. In vitro cultured matched primary bronchial epithelial cells show that this trajectory is retained in absence of exogenous factors. We find that IL-13 induces hillock-like cell differentiation into CEACAM5hi goblet cells, driving goblet cell metaplasia. Repair-associated basal cells and transitioning CEACAM5hi hillock-like cells strongly contribute to airway inflammation and remodelling. In turn, dendritic cells and mast cells promote a state of highly active epithelial differentiation, which shows increased multiciliated cell fate decisions, in concordance with an increase in multiciliated cell death observed in asthma. Proportions of the epithelial cells of the non-canonical differentiation trajectory are associated with clinical outcomes such as disease severity, FeNO, and small airway function.

Systemic iron sequestration occurs frequently in cancer due to inflammation-driven expression of the iron-regulatory hormone hepcidin. The impact of systemic iron availability on tumoral immunity is unclear. Here, we show that elevated serum hepcidin is associated with reduced survival and decreased intratumoral CD8+ T cells in patients with pancreas cancer. While hepcidin is not induced in murine tumor models, administration of a hepcidin mimetic phenocopies the T-cell-depleted tumor microenvironment seen in patients. Mechanistically, chronic antigen-driven mitochondrial dysfunction disrupts iron metabolism and selectively depletes high avidity CD8+ T cells during iron restriction. These findings establish a direct link between hepcidin-mediated iron sequestration and tumoral immunity and nominate systemic iron dysregulation as a therapeutic target to enhance anti-tumoral CD8+ T cell responses.

Lactation is a hallmark of mammalian evolution, imposing extraordinary physiological demands on mothers to nourish their offspring. Yet how mothers maintain their own health while meeting these demands remains poorly understood. Here, we discover that lactation establishes a preemptive innate defense program in the maternal lung through enhanced epithelial production of surfactant proteins and mucin, vascular remodeling that supports granulocyte recruitment and accumulation of activated intravascular neutrophils. These tissue-wide adaptations safeguard lactating dams against diverse respiratory pathogens. We further identify prolactin and CGRP as neuroendocrine signals contributing to pulmonary remodeling and uncover parallel lactation-associated granulocyte adaptations in marsupial and non-human primate models. Together, our findings expand the framework of reproductive immunology, revealing that reproduction couples systemic fetal tolerance with tissue-specific immune adaptations that safeguard maternal health during lactation.