Science Immunology

Mucosa-associated invariant T (MAIT) cells are unconventional T cells with innate-like rapid antimicrobial effector functions and serve as resident sentinels at mucosal and non-mucosal barriers. However, their role in immune defense against Staphylococcus aureus and the impact of bacterial immune evasion mechanisms are incompletely understood. Here, we have investigated MAIT cell responses to S. aureus and the impact of its broadly expressed leukocidin toxin HlgAB on MAIT cell responses in different human tissue sites. MAIT cells respond to S. aureus with a complex polyfunctional profile spanning pro-inflammatory IL-17, TNF, and IFN{gamma}, anti-inflammatory IL-10, plus granzymes A, B, and K, perforin, and granulysin. The quality of responses was influenced by microbial dose and time of exposure and was dependent on both MR1-presented antigen and cytokine co-activation. CD56 MAIT cells displayed stronger effector responses and higher HlgAB sensitivity compared to CD56- cells. MAIT cells were partially resistant to HlgAB-toxicity compared to monocytes; blood-derived MAIT cells remained susceptible, whereas tonsillar MAIT cells showed minimal sensitivity. Notably, activation reduced the MAIT cell susceptibility to HlgAB, and such activation also afforded indirect protection to monocytes in co-cultures. The reduced susceptibility of tonsillar MAIT cells correlated with lower CCR2 and CXCR1 expression, a pattern shared with barrier tissues such as the lung and intestines. In conclusion, these findings indicate that MAIT cells exhibit tissue- and context-dependent responses to S. aureus and sensitivity to HlgAB-mediated immune evasion. ImportanceMAIT cells are an evolutionarily conserved unconventional T cell subset that responds to riboflavin pathway-derived antigens from a range of microbes. Here, we found that the human MAIT cell response to the pathogen S. aureus is robust with a polyfunctional complexity influenced by bacterial concentration and response kinetics. The ubiquitously expressed S. aureus immune-evasive toxin HlgAB attacks MAIT cells via CCR2. However, the sensitivity of MAIT cells to HlgAB varies depending on tissue localization, where in particular tissue-resident MAIT cells in tonsils are resistant. Antigen-specific activation of MAIT cells reduces HlgAB sensitivity, with protection also afforded to monocytes in the vicinity. These findings uncover the complex and dynamic interaction between an evolutionarily conserved arm of immunity, and immune evasion mechanisms of the important pathogen S. aureus.

Langerhans cells express the nonpolymorphic antigen-presenting molecule CD1a, positioning them as contributors to host immunity against Mycobacterium leprae in human leprosy. CD1a was originally shown to present non-canonical lipopeptide antigens such as dideoxymycobactin and chemically diverse hydrophobic ligands. Here, we generated CD4 T cell lines from leprosy lesions that recognized M. leprae in a CD1a-restricted manner. Unexpectedly, antigen recognition was protease-sensitive, prompting biochemical purification that identified two microbial protein antigens: LppX, a 25-kDa lipoglycoprotein, and Ag85A, a 30-kDa secreted protein with no known lipid modification. Recombinant proteins activated the corresponding T cell lines in a CD1a-dependent manner. Epitope mapping identified 12-mer peptides that fully reconstituted antigenicity, were conserved between M. leprae and M. tuberculosis, and elicited robust, dose-dependent IFN-{gamma} production and T cell proliferation, establishing that DNA-encoded, ribosomally translated peptides serve as CD1a-restricted cognate antigens. Biochemical analyses showed peptide binding to CD1a, supported by isoelectric focusing and surface plasmon resonance (KD [~]75 M for Ag85A). CD1a-peptide tetramers specifically stained cognate T cells, soluble CD1a was sufficient to present peptide antigen, and transfer of the LppX-specific TCR into naive T cells restored antigen responsiveness. Using CD1a-peptide tetramers, we identified antigen-specific T cells enriched in patients undergoing reversal reactions compared with patients with lepromatous leprosy and healthy donors. The CD1a-restricted T cell lines secreted IFN-{gamma} and IL-26, cytokines with established antimicrobial activity. Together, these findings demonstrate that CD1a can present canonical microbial peptides as part of a cell-mediated immune response in leprosy, extending the known spectrum of CD1a ligands. Because CD1a is nonpolymorphic and presents antigens to antimicrobial T cells, CD1a-peptide complexes may provide a broadly applicable platform for studying, detecting, and potentially targeting mycobacterial immunity.

Immunoglobulin E (IgE) drives allergic disease, yet what restrains the persistence of IgE production remains poorly understood. Mouse studies suggest that BCR-induced apoptosis limits the survival of IgE-producing plasma cells (PCs). Whether this mechanism applies to human IgE PCs is unclear. Using a human IgE class-switching system, we show that BCR crosslinking preferentially kills IgE PCs compared to IgG1+ PCs. However, this selective sensitivity is not explained by surface BCR levels or proximal BCR signaling as suggested in mice. Instead, elevated PTEN expression in IgE PCs constrains PI3K/Akt pro-survival signaling and lowers the apoptotic threshold by upregulating BIM, while JNK signaling sustains PTEN expression and amplifies their apoptotic sensitivity. CRISPR/Cas9 targeting of PTEN or BIM, or JNK inhibition protects IgE PCs from BCR-mediated killing. Therapeutic anti-IgE antibodies, including omalizumab and extracellular membrane-proximal domain (EMPD)-targeting antibodies, exploit this sensitivity to selectively eliminate IgE PCs and suppress IgE production, providing a mechanistic rationale for depleting IgE PCs in allergic disease. SummaryRamadani et al. identify a JNK/PTEN/BIM signaling axis that intrinsically limits human IgE plasma cell survival and drives their preferential sensitivity to BCR-induced apoptosis. This mechanism is distinct from that established in mice and has direct implications for anti-IgE therapeutic strategies.

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.

Polymorphonuclear neutrophils (PMNs) serve as frontline defenders against injury and infection, eliminating pathogens and initiating mucosal tissue repair. However, excessive PMN transepithelial migration (TEpM) contributes to chronic mucosal inflammatory disorders, including inflammatory bowel disease. PMN pro-inflammatory and pro-repair functions are regulated by incompletely defined signaling cascades involving kinases and phosphatases. Here, we determined how the protein tyrosine phosphatase CD45/PTPRC regulates PMN trafficking and effector functions in the gut. Pharmacologic inhibition of CD45 significantly reduced PMN colonic TEpM in vitro and in vivo and decreased intestinal PMN trafficking was observed in transgenic mice with PMN-specific deletion of CD45 (MRP8-Cre;Cd45fl/fl). Beyond limiting TEpM, CD45 depletion impaired key antimicrobial functions, including degranulation and phagocytosis, indicating broader effects on PMN effector activity. Importantly, recovery from dextran sodium sulfate (DSS)-induced colitis and biopsy-induced colonic wounding was delayed in MRP8-Cre;Cd45fl/fl mice, linking altered PMN function to defective mucosal healing. Mechanistically, CD45 depletion reduced surface expression of the {beta}2 integrin CD11b/CD18 and inactivated the Src family kinase member Lyn. Together, data highlight a novel CD45-CD11b-Lyn signaling axis that regulates PMN trafficking and effector functions in the intestine and identify CD45 as a promising target for modulating PMN function to promote mucosal tissue repair.

Secondary lymphoid tissue, including tonsil, supports human NK cell development, but the spatial organization and tissue niches that drive this differentiation remain undefined. Here, we used single cell analysis of cyclic immunofluorescence to generate a comprehensive atlas of human NK cell development in tissue. By integrating regional localization, chemokine signaling, cytokine availability, and cell phenotype, we show that NK cell differentiation follows a reproducible spatial trajectory defined by stage-specific cell-cell interactions. Notably, CD34+ NK cell progenitors are found in the interfollicular domain in proximity to high endothelial venules and preferentially interact with lymphatic endothelial cells, suggesting their route of progenitor entry into tissue. Mature NK cells are primarily found in the T-cell rich parafollicular domain, where they interact with other NK cells and T cell subsets. Local inflammation increases NK cell frequency in tissue through both proliferation of NK progenitors and recruitment of circulating mature NK cells. Finally, we identify a subset of tonsil stromal cells that support differentiation of NK cells in vitro and proliferation of NK precursors in situ. Together, these findings demonstrate that spatial localization defines human NK cell development and provide an in situ definition of niches that support human NK cell differentiation in tonsil.

Mast cells (MCs) are tissue-resident sentinels of the innate immune system that play pivotal roles in host defense and inflammation. Perivascular MCs exert a particularly strong influence on the onset and dynamics of inflammation through the rapid, directional release of proinflammatory mediators into the circulation. Yet, the mechanisms governing their attachment to the vessel wall - a prerequisite for intravascular degranulation - remain poorly defined. Using a conditional knockout of integrin {beta}1 (Itgb1) in MCs, we investigated how perivascular positioning, degranulation, and vasoactive function contribute to inflammatory responses. In vivo imaging revealed that Itgb1 is essential for positioning MCs within the perivascular niche, particularly around arterioles. The absence of Itgb1 markedly reduced directional MC degranulation into blood vessels during skin inflammation. In vitro, Itgb1-deficient MCs displayed impaired degranulation kinetics together with altered SHIP1/PI3K-AKT signaling and calcium influx upon P2X7 ligation by ATP. During contact hypersensitivity, mice lacking Itgb1 in MCs exhibited strongly diminished ear swelling and reduced recruitment of multiple leukocyte subsets. Mechanistically, disordered MC positioning and attenuated degranulation impaired endothelial activation, resulting in decreased leukocyte adhesion and extravasation. These findings uncover a dual role for Itgb1 in regulating MC responsiveness and pro-inflammatory vasoactive function, establishing Itgb1-mediated perivascular MC positioning as a key prerequisite for effective leukocyte recruitment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/723399v1_ufig1.gif" ALT="Figure 1"> View larger version (80K): org.highwire.dtl.DTLVardef@ada618org.highwire.dtl.DTLVardef@73a85forg.highwire.dtl.DTLVardef@1330cccorg.highwire.dtl.DTLVardef@8d3b3c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Juvenile dermatomyositis (JDM) is characterized by a type I interferon (IFN-I) signature associated with disease activity. We previously identified a link between SARS-CoV-2 infection and the onset or relapse of JDM. Here, we show that newly diagnosed JDM patients display an overexpression of IFIH1 (encoding MDA5 protein) at baseline, coupled with an altered response to dsRNA stimulation at proteomic and transcriptomic levels, indicating abnormal activation of this antiviral sensing pathway. Single-cell transcriptomic and chromatin accessibility profiling of peripheral blood mononuclear cells (PBMCs) further revealed myeloid-specific enrichment of interferon-stimulated genes (ISGs) and preferential disruption of this pathway at disease onset, supporting a dysregulated IFN-I state in this cell type. We identified SARS-CoV-2 RNA in muscle biopsies of two Covid-19 pandemic-onset JDM patients, strongly implicating viral infection as a potential trigger of the dysregulated MDA5 immune response. To extend these observations beyond SARS-CoV-2, we screened two independent retrospective cohorts for antibodies against 27 common childhood infections. In our discovery cohort JDM patients showed significantly increased exposure to 4 RNA viruses in line with our immunological findings. Increased exposure to RSV B was confirmed in an independent replication cohort supporting a robust association with JDM pathophysiology. Together, these findings integrate systemic, single-cell, and tissue-level analyses implicating RNA viral infection and biased antiviral sensing in shaping IFN-I responses at JDM onset, providing mechanistic insight into environmentally triggered pathogenesis. One sentence summaryType I interferon dysregulation at juvenile dermatomyositis onset implicates altered dsRNA sensing and RNA viral exposure as potential disease triggers.

Dendritic cells (DCs) are central to activating cytotoxic CD8 T cells, yet DC-based vaccines have achieved limited success against established tumors. To address this gap, we analyzed the transcriptomic and functional changes CD8 T cells undergo following interactions with DC subsets in lymphoid organs and tumor sites. This approach allowed us to map their trajectory from naive to fully cytotoxic effector cells. We found that classical DCs in lymphoid organs provide essential antigen presentation but fail to elicit cytotoxicity. Instead, antigenexperienced CD8 T cells require additional inflammatory signals, primarily through TNF, delivered at tumor sites by infiltrating myeloid DCs. Effective cytotoxic responses therefore depend on the synchronization of these distinct, temporally separated signals. Notably, tumor antigen-pulsed DC vaccines rapidly lose TNF expression after infiltrating tumors, limiting their efficacy. These findings establish a sequential model of T cell activation and suggest strategies to enhance the potency of DC-based immunotherapies.

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.

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.

Ulcerative colitis (UC) is characterized by epithelial barrier dysfunction and dysregulated mucosal immune responses; however, the mechanisms driving disease onset remain poorly defined. Autoantibodies against the epithelial-restricted integrin v{beta}6 are a highly specific biomarker of UC that can precede clinical diagnosis by up to 10 years. Because v{beta}6 activates TGF{beta} at epithelial surfaces, we hypothesized that UC-associated v{beta}6 autoantibodies inhibit mucosal TGF{beta} activation and disrupt epithelial homeostasis. We showed that v{beta}6 autoantibodies were enriched in UC and that IgG from autoantibody-positive individuals inhibited v{beta}6-dependent activation of TGF{beta}. v{beta}6 blockade dampened TGF{beta} signaling and altered differentiation-associated gene programs in human intestinal epithelial cells. In mice, deletion of v caused expansion of inflammation-associated goblet cells in the colon and changes in intestinal immune cells. Using a novel mouse model, we showed that v{beta}6-specific autoantibody disrupted epithelial-immune crosstalk and increased susceptibility to DSS colitis. Together, these findings establish anti-v{beta}6 autoantibodies as active inhibitors of epithelial TGF{beta} signaling, constituting a de facto anti-cytokine response, rather than passive biomarkers. By linking preclinical seropositivity to impaired epithelial signaling and heightened susceptibility to colitis, this work identifies epithelial v{beta}6-dependent TGF{beta} activation as a pathway that may be leveraged to modify disease risk or limit disease severity. One Sentence SummaryUC-associated autoantibodies impair epithelial TGF{beta} activation, alter mucosal homeostasis, and predispose to colitis.

Blocking the programmed cell death 1 (PD-1) pathway using monoclonal antibodies reinvigorates exhausted T cells (Tex), enhancing control of chronic viral infections and cancer. Considerable effort has focused on evaluating different PD-1 blockade agents in preclinical and clinical cancer settings, but relatively little information exists on how to optimize the pharmacodynamic effects of PD-1 pathway blockade on reinvigorating Tex. To address this question, we performed longitudinal tracking of Tex reinvigoration during chronic infection with lymphocytic choriomeningitis virus (LCMV) following different regimens of PD-1 blockade. We compared single-cycle (2 weeks of treatment), long-term continuous PD-1 pathway blockade (i.e. 3 months), or blockade followed by a drug holiday and then re-blockade (intermittent treatment). These studies revealed little benefit of continuous versus single-cycle PD-1 blockade, with both resulting in a single peak of Tex reinvigoration and similar effects on viral replication. In contrast, intermittent blockade resulted in a new cycle of secondary Tex reinvigoration upon redosing after a washout and this secondary Tex reinvigoration improved disease control. Mechanistically, long-term blockade eroded the ability of Tex progenitor cells (Tpex) to give rise to downstream, more functional Tex intermediate (Tex-Int) progeny, whereas the drug holiday restored this Tpex proliferative and differentiation capacity. Tpex from long-term treated mice showed evidence of adaptive resistance and additional layers of negative regulation, including sustained expression of the inhibitory receptor CD22. Indeed, co-blockade of PD-1 and CD22 using combination antibodies or bispecific antibody approaches improved disease control and reinvigoration of Tex. These data have implications for clinical immune pharmacodynamics of PD-1 blockade and provide insights into the biology of Tex reinvigoration. One Sentence SummaryModifying the immunopharmacology of PD-1 blockade reveals a benefit of a drug holiday and identifies mechanisms of Tex progenitor deficiency provoked by prolonged loss of PD-1 signals including the inhibitory receptor CD22.

Tissue-specific peripheral tolerance mechanisms are essential to prevent autoimmunity. The cornea is immune privileged, and anterior chamber-associated immune deviation (ACAID) governs its inner surface. However, the mechanisms that apply to corneal epithelial (outer surface) antigens remain unknown. Using an inducible, cornea-restricted neoantigen mouse model, we found that the cornea relies on inducible regulatory T cells (Tregs) rather than ignorance or ACAID for its epithelial antigens. Although the cornea is both avascular and alymphatic, its epithelial antigens are still efficiently presented by ocular surface-derived antigen-presenting cells to T cells in draining lymph nodes under homeostatic conditions, leading to conventional antigen-specific Treg expansion without ocular pathology. This tolerance was not absolute: systemic immunization redirected antigen-specific responses toward pathogenic effector T cells that disrupted epithelial barrier function. These findings identify Treg induction as a dominant mechanism of corneal epithelial immune homeostasis and demonstrate that inflammatory priming can render a tolerated corneal antigen into an autoimmune target, providing mechanistic insight into dry eye pathogenesis. SummaryThis study shows that immune tolerance to corneal epithelial neoantigens relies not on immune privilege but on peripherally induced regulatory T cells in the draining lymph nodes that can be subverted by innate activation, shedding light on ocular surface disease pathophysiology.

Type 2 high asthma is driven by coordinated GATA3 dependent programs in CD4+ T cells and group 2 innate lymphoid cells (ILC2). Although biologics targeting IL4, IL5, or IL13 benefit subsets of patients, many remain symptomatic, suggesting that upstream regulatory mechanisms may sustain type 2 inflammation. We investigated whether HuR (ELAVL1), an RNA-binding protein that stabilizes GATA3 and Th2 cytokines mRNA, regulates type 2 inflammatory programs in allergic asthma. Using a house dust mite (HDM) model in vivo, HuR inhibition with the small molecule KH3 reduced lung inflammation, suppressed Th2 cytokine expression, accelerated Gata3 mRNA decay in lung CD4+ T cells, and attenuated airway hyperresponsiveness toward control levels. In ex vivo activated human lung CD4+ T cells, KH3 accelerated GATA3 mRNA decay with minimal effects on RORC or TBX21 and selectively reduced Th2 cytokine secretion, while IL10 and IL2 were unchanged. Similarly, ILC2s isolated from peripheral blood mononuclear cells (PBMCs) of type 2 high asthmatic donors showed reduced GATA3 mRNA stability and diminished Th2 cytokine production following KH3 treatment. Single-cell transcriptomic analysis of bronchoalveolar lavage fluid after allergen challenge demonstrated co-enrichment of ELAVL1 and GATA3 within Th2 clusters in human airways. Together, these findings identify HuR as a post-transcriptional regulator of GATA3 driven type 2 inflammation in allergic asthma.

Chronic allergic diseases are driven by T helper type 2 (Th2) cells in barrier tissues. Despite their profound effects on tissue physiology, Th2 cells represent a rare cell population within tissues, suggesting mechanisms restraining Th2 cell expansion at barrier sites that remain ill defined. Using a murine model of allergic asthma, we demonstrate that effector Th2 cells promote cDC2 activation within the lungs, including expression of the CCR4 ligands that attract Foxp3+ regulatory T cells (Tregs). Selective deletion of Ccr4 in Tregs during the effector Th2 cell response led to increased lung Th2 cells, activated cDC2s, and allergic inflammation. Mechanistically, CCR4 promoted Treg trafficking efficiency and was required to specifically control tissue cDC2 co-stimulatory molecule expression. Lastly, in the airways of humans with allergy, the expression of the CCR4 ligands in activated cDCs correlated with Treg enrichment. In sum, we define a cDC2-Treg feedback circuit within a barrier tissue that restrains effector Th2 cell expansion, revealing a novel role for tissue cDC2s in controlling Th2 cell biology.

Acquired resistance to immune checkpoint blockade (ICB) is increasingly recognized as an active adaptive process. However, prior studies have typically focused on individual tumor models, limiting the ability to distinguish conserved mechanisms from model-specific observations. Here, we integrated four independent transcriptomic datasets of acquired ICB resistance, spanning human non-small cell lung cancer (NSCLC) biopsies, murine CT26 colorectal tumors, organoid-derived murine NSCLC tumors, and EMT6 breast cancer cells. Differential expression analysis was performed within each dataset, followed by an intersection-based consensus approach to identify reproducible resistance-associated programs. Contrary to the conventional cold tumor paradigm, acquired-resistance tumors consistently maintained interferon-{gamma} response and innate immune signaling while simultaneously activating cell-cycle programs and constitutive KRAS signaling signatures across all four models. We term this an apparent inflammatory-proliferative paradox: the persistence of IFN-{gamma}-driven inflammatory signatures, canonically associated with productive antitumor immunity, in tumors that have escaped immune control. Notably, this program was retained in immune-depleted organoid and cell-line models, supporting a tumor-cell-associated component maintained independently of the immediate immune microenvironment. Transcription factor activity inference identified a conserved regulatory backbone linking interferon-associated regulators (STAT2, IRF2) with proliferation drivers (E2F4, TFDP1) and suppression of lineage-specifying factors (HNF4A, EGR1). Integrated network analysis resolved these signals into three reinforcing modules, namely hyper-proliferative outgrowth, active inflammatory adaptation, and lineage identity loss. This architecture provides a systems-level framework for prioritizing combination strategies that simultaneously address interconnected resistance axes.

Although early-life immunity was once considered immature, the human fetal immune system is dynamic and compartmentalized by the second trimester. By 21 weeks of gestation, T lymphocytes become a major immune population in the fetal small intestine (SI), yet their functional roles within this tissue remain largely undefined. To explore their unique contributions to intestinal development, we established an ex vivo co-culture system in which mucosal T cells isolated from fetal, neonatal, or adult SI donors were cultured with tissue-derived 3D SI organoids derived from various ages. Homeostatic early-life (fetal and neonatal) SI T cells uniquely promoted organoid generation, a metric of stem cell renewal, by upregulating cell cycle-associated gene programs. These early-life T cells also directed intestinal stem cell differentiation toward the secretory lineage in both growth and differentiation phase, highlighting that T cells poise stem cells to adopt secretory fates. T cells from infants with necrotizing enterocolitis (NEC), an inflammatory intestinal disease affecting predominantly preterm infants, failed to activate these same programs, suggesting a pathologic role for T cells in NEC. T cells from the adult SI similarly failed to support organoid growth or differentiation, revealing developmentally specialized, nonimmune functions for early-life T cells in the intestine. Similarly, T cells derived from cord blood did not enhance organoid generation, indicating that this function is not necessarily a generalized feature of early-life T cells but rather is restricted to mucosal T cells. Organoids derived from adults or NEC, however, could re-enter regenerative states when co-cultured with fetal T cells, indicating that fetal T cells can restore stem cell self-renewal across developmentally and disease-imposed states. We further identified that T cell-derived soluble factors alone were insufficient to modulate intestinal stem cell fate, implying the need for physical interactions. Concordant with this finding, we report that T cells heavily localize to the stem cell niche during prenatal development, where they express factors involved in Notch, Wnt, and growth factor signaling to support fetal stem cell function. Collectively, these findings reveal a coordinated developmental program in which fetal SI T cells balance stem cell self-renewal and differentiation, identifying a developmental immune-epithelial axis that can be harnessed to restore intestinal regeneration.

The gut-joint axis describes how impaired intestinal epithelial function and increased gut permeability allow luminal factors to enter circulation. This can drive inflammation in Rheumatoid Arthritis, a chronic condition affecting the joint with systemic features. What mechanisms contribute to disease persistence are, as yet, incompletely understood. In health, extensively Oglycosylated intestinal mucins are central to epithelial protection and immune homeostasis; however, whether mucin glycosylation is altered during arthritis has not been addressed. Here, we investigated whether arthritisassociated inflammation alters mucin Oglycosylation, potentially compromising intestinal barrier function. Using a collageninduced arthritis mouse model, we combined epithelial transcriptomics, mass spectrometry-based glycomics, and imaging approaches to profile intestinal glycosylation. We identified distinct glycan remodeling in the colon, characterized by reduced fucosylation, while the ileum remained largely unaffected. In vitro studies using 3D human epithelial cultures further demonstrated that inflammatory cues, particularly from TNFactivated stromal cells, are sufficient to reduce epithelial fucosylation. Together, these findings identify a stromal-inflammatory mechanism that disrupts mucin glycosylation during arthritis. Loss of colonic fucosylation emerges as a novel element of inflammatory arthritis, providing an additional mechanistic link between intestinal inflammation and fibroblast-dependent modulation of the tissue microenvironment.

Primarily recognized as the site for T cell development, the thymus supports a complex interplay between thymic stromal cells and developing thymocytes, which is essential for T cell maturation and the establishment of central tolerance. Emerging evidence indicates that thymic B cells contribute to tolerance induction by functioning as antigen-presenting cells. However, their developmental pathways and functional roles remain poorly understood. Using tissue mass cytometry, we localized B cells in the thymus and their orientation towards other cells in the medulla. We characterized the heterogeneity of thymic B cells using single-cell RNA sequencing of cells isolated from human thymi, identifying naive, germinal center-like, plasma cell, and multiple memory B cell populations. We identified a distinct pre-B cell subset with local thymic B cell development potential, that can develop due to the inhibition of Notch signaling by Deltex1, a Notch antagonist. Using BCR repertoire analysis, we explored clonal diversity, somatic hypermutation patterns and class switch recombination of thymic B cells. Spectral flow cytometry further validated the surface phenotype of thymic B cell populations and confirmed the presence of distinct CD21-CD27- memory compartments with heterogeneous surface immunoglobulin isotype usage. In doing so we provide novel insights into the unique biology of human thymic B cells, their development, and their differentiation in the human thymus. We conclude that the human thymus supports local B cell development and differentiation into medullary memory-like populations that undergo class switching with limited somatic hypermutation, suggesting secondary lymphoid-like B cell programs adapted to central tolerance induction. One Sentence SummaryThe human thymus is not only a primary lymphoid organ for T cell development, but also a secondary site for the development and maturation of unique populations of B cells.