Immunity

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.

The transcription coregulator OCA-B promotes CD4+ T cell memory recall responses and autoimmunity. OCA-B T cell deletion prevents spontaneous type-1 diabetes (T1D) onset in non-obese diabetic (NOD) mice and blunts T1D in a subset of more aggressive models. However, the role of OCA-B in diabetes induced by treatment with immune checkpoint inhibitors (ICIs), and the role of OCA-B in the control of tumors with and without ICI treatment, has not been studied. Here we show that islet and pancreatic lymph node T cells from T1D individuals express measurable POU2AF1 mRNA. Deletion of OCA-B in T cells fully insulates 8-week-old non-obese diabetic (NOD) mice against ICI-induced diabetes and partially protects 12-week-old mice. Salivary and lacrimal gland infiltration and inflammation were also reduced. Protection was associated with a block in the differentiation of progenitor exhausted CD8+ T cells (TPEX) into terminally exhausted CD8+ T cells (TEX). We show that OCA-B T cell loss preserves anti-tumor immune responses following PD-1 blockade in different tumors and mouse strains. These findings point to a potential therapeutic window in which pharmaceuticals targeting OCA-B could be used to block the emergence of both spontaneous and ICI-induced autoimmunity while sparing anti-tumor immunity. We develop first-in-class small molecule inhibitors of Oct1/OCA-B transcription complexes and show that administration into NOD mice also blocks diabetes emergence following PD-1 blockade. These results identify OCA-B as a promising therapeutic target for the prevention of autoimmunity and immune-related adverse events (irAEs).

Epstein-Barr virus (EBV) infection is nearly ubiquitous in humans and has been associated with multiple sclerosis (MS) and other immune-mediated diseases, yet mechanisms by which EBV-infected B-cells influence distal tissues remain incompletely understood. Extracellular vesicles (EVs) mediate intercellular communication during viral infection, but their integrated viral and host cargo has not been comprehensively defined in EBV-transformed B-cells generated from naturally infected individuals. Here, we performed an unbiased multiomic characterization of small EVs (sEVs) released from spontaneous lymphoblastoid cell lines (SLCLs) derived from normal donors and patients with stable or active MS, transformed ex vivo by endogenous wild-type EBV. Using surface profiling, quantitative proteomics, whole-genome sequencing of EV-associated DNA, total stranded RNA sequencing, droplet digital PCR, and super-resolution microscopy, we mapped the protein, DNA, and RNA cargo of these vesicles and determined their host and viral origins. SLCL sEVs contained canonical tetraspanins, B-cell markers, and were free of detectable virions. Proteomics identified over 6,000 shared proteins and revealed enrichment of nucleic acid-binding and chromatin-associated proteins. Whole-genome sequencing demonstrated abundant EV-associated DNA comprising two distinct compartments: high-molecular weight, DNase-sensitive DNA associated with the vesicle corona and DNase-resistant, nucleosome-sized ([~]130-150 bp) DNA protected within vesicles. In both compartments, DNA was overwhelmingly host-derived and broadly distributed across the genome, whereas EBV genomic DNA was minimal. RNA sequencing identified diverse EBV transcripts, with striking enrichment of the viral long noncoding RNA EBER1 across all lines. Super-resolution imaging and ddPCR confirmed EBER1 incorporation within individual vesicles. Notably, EBER1 has been detected in MS brain tissue in prior studies, and our findings provide a plausible vesicle-mediated mechanism for dissemination of this viral lncRNA from EBV-infected B-cells to distal sites. These findings establish a foundational multiomic profile of sEVs from wild-type EBV-transformed B-cells and reveal export of host DNA and EBER1, with broad implications for viral immunobiology and intercellular signaling in MS and beyond. SIGNIFICANCE STATEMENTEpstein-Barr virus (EBV) infects over 90% of adults worldwide and is strongly linked to multiple sclerosis (MS). How EBV-infected B-cells may communicate with distant tissues, including the central nervous system (CNS), remains unclear. We provide the first integrated multiomic profile of extracellular vesicles released from B-cells transformed by endogenous, wild-type EBV. These vesicles are enriched in nucleosome-associated host DNA and the viral long noncoding RNA EBER1 but contain minimal viral DNA. Because EBER1 has been detected in MS brain tissue, our findings suggest a vesicle-mediated mechanism by which EBV-infected B-cells could deliver viral RNA to the CNS independently of infectious virus. These results establish a framework for understanding how EBV latency reshapes intercellular communication in immune-mediated disease.

The gastrointestinal tract (GI) is the largest environmental mucosal interface and is exposed to diverse commensal and pathogenic microbes. B cells are a prevalent immune component of the GI tract and its associated secondary lymphoid organs, yet we know little about the diversity and stability of distinct transcriptional programs that modulate B cell responses against different classes of pathogens or environmental perturbations. A subset of B cells defined by expression of the transcription factor T-bet, has been canonically associated with antiviral immunity through IgG production. However, the role of T-bet expressing B cells in mucosal tissues, where IgA responses predominate, is poorly understood. Here, we identify a population of intestinal T-bet+ B cells that, in the absence of overt perturbation, constitutes a minor fraction of intestinal associated B cells and undergoes continuous turnover. In contrast, during enteric viral infection with murine norovirus (MNV), T-bet B cells undergo a marked expansion, with T-bet expression stably maintained in the majority of virus-specific B cells, including IgG2c and IgA switched B cells. Moreover, virus-reactive IgG2c and IgA B cells arise independently rather than through sequential switching, and B cell intrinsic T-bet expression is required for effective germinal center responses but dispensable for IgA class switching. Moreover, T-bet expressing B cells are required for the generation of all MNV-specific circulating IgG and mucosal IgA, and for protection upon re-encounter with the virus. Together, these findings establish T-bet expressing B cells as a specialized B cell subset essential for mucosal immunity and protection against norovirus infection.

Innate and adaptive immune responses play critical roles in the pathogenesis of inflammatory bowel disease (IBD), yet the molecular pathways integrating these responses remain elusive. Here, we identify DNAM-1 immunoreceptor as a central driver of colitis through distinct, cell type-specific mechanisms. Transcriptomic analyses of human and murine group 3 innate lymphoid cells (ILC3s) revealed DNAM-1 as a conserved IL-23-responsive surface molecule associated with inflammatory cytokine production. In an innate immune-driven anti-CD40 monoclonal antibody (mAb)-induced colitis model, DNAM-1 expressed on ILC3s promoted intestinal inflammation by enhancing IL-22 and GM-CSF production via the integration of the Akt-mTORC1-HIF-1 signaling pathway. Genetic ablation or antibody-mediated blockade of DNAM-1 attenuated inflammatory cytokine production and disease severity. Paradoxically, in T cell-dependent colitis, DNAM-1 expression on dendritic cells, but not on ILC3s or CD4 T cells, exacerbated disease by promoting dendritic cell activation and pathogenic Th1 and Th17 differentiation. Notably, therapeutic blockade of DNAM-1 ameliorated disease in both colitis models and exerted complementary effects when combined with anti-TNF therapy, accompanied by modulation of immune activation programs distinct from those regulated by TNF inhibition. Collectively, these findings establish DNAM-1 as a pivotal regulator of intestinal inflammation bridging innate and adaptive immunity and identify DNAM-1 blockade as a next-generation therapeutic strategy for IBD. Highlight{blacktriangleright} DNAM-1 is an IL-23-responsive receptor conserved in human and mouse ILC3s. {blacktriangleright}DNAM-1 on ILC3s drives innate colitis via Akt-mTORC1-HIF-1 signaling. {blacktriangleright}DNAM-1 on DCs promotes T cell-dependent colitis by inducing Th1/Th17 cells. {blacktriangleright}DNAM-1 blockade targets immune pathways distinct from TNF inhibition. {blacktriangleright}Combined DNAM-1 and TNF blockade shows additive therapeutic efficacy in colitis.

The generation of a diverse and self-tolerant B cell repertoire is essential for adaptive immunity and is achieved through V(D)J recombination. In mice, Ig{kappa} is the dominant light chain, whereas Ig{lambda} rearrangement typically occurs in response to nonproductive or autoreactive Ig{kappa} recombination, a process termed receptor editing. Recombination at the RS element deletes the Ig{kappa} constant exon, silencing the locus and enabling Ig{lambda} expression. However, the epigenetic regulatory framework that orchestrates and governs receptor editing remains poorly defined. Here, we identify a CTCF-binding insulator element (CBE) within the 3' Ig{kappa} super-enhancer (3'-SE{kappa}) that regulates receptor editing and directs the {kappa}-to-{lambda} switch required for Ig{lambda} B-cell development. Mechanistically, loss of this CBE activates an insulated enhancer within the 3'-SE{kappa}, causing aberrant V{kappa} rearrangements and altered chromatin interactions through disrupted loop extrusion dynamics. Notably, loss of this CBE in mice leads to increased autoantibody production by ten weeks of age, demonstrating that CBE-mediated chromatin architecture shapes B cell fate by constraining autoreactive potential. Collectively, our findings define a novel CTCF-dependent cis-regulatory insulation checkpoint that connects chromatin loop extrusion to antigen-driven receptor editing, thereby enforcing B-cell tolerance.

Many genetic variants associated with increased type 1 diabetes (T1D) risk are located within the SKAP2 gene; however, the mechanisms by which these variants confer disease risk remain unclear. SKAP2 encodes an adapter protein that functions within the integrin signaling pathway and is found at the highest levels in myeloid leukocytes. We recently identified a de novo gain-of-function SKAP2 mutation in an individual with T1D, leading to hyperactive integrin signaling in myeloid cells. To dissect the mechanisms by which this mutation may lead to T1D, we generated a knock-in mouse line containing the orthologous p.G153R substitution in mouse SKAP2 on the diabetes-prone nonobese diabetic (NOD) genetic background. Both female and male SKAP2G153R/G153R mice developed accelerated T1D. The SKAP2G153R/G153R mice also exhibited a unique spectrum of autoantibodies, leading to immune-complex nephritis. Accelerated infiltration of pancreatic islets by myeloid cells, B lymphocytes, and activated T cells was observed in SKAP2G153R/G153R mice. Single-cell RNA sequencing demonstrated a type 1 IFN{gamma}-driven inflammatory program within the pancreatic islets of SKAP2G153R/G153R mice. Dendritic cells from SKAP2G153R/G153R mice demonstrated increased antigen-presenting capacity, characterized by enhanced adhesion to T cells during immune synapse formation. Macrophages and neutrophils from SKAP2G153R/G153R mice also showed increased integrin signaling responses, with neutrophils expressing high levels of activated {beta}2 integrins on the cell surface. When backcrossed onto the C57BL/6J genetic background, the SKAP2G153R/G153R mice developed spontaneous autoantibody formation and exhibited accelerated autoimmunity, including nephritis, in the pristane-induced model of autoimmune disease. These findings demonstrate that dysregulation of leukocyte integrin signaling, through alterations in SKAP2, may increase the genetic risk for autoimmunity and T1D.

In multiple sclerosis (MS), autoreactive B cells play a central role in driving CD4 T cell-mediated inflammatory damage to myelin (1). Here we investigated how disrupting Brutons tyrosine kinase (BTK) signaling exclusively in B cells shapes the course of experimental autoimmune encephalomyelitis (EAE), a model for MS, through alterations in B cell development and activity. B cell-specific BTK deletion significantly ameliorated both human MOG (hMOG) induced EAE (p = 0.0087) as well as spontaneous disease in 2D2+IgHMOG mice (p = 0.0004). Additionally, MOG-specific cells were found to be more sensitive to loss of BTK than tolerant clones (p = 0.0002) and production of anti-MOG immunoglobulins was also found to be diminished (p < 0.004) while overall IgG was unchanged (p = 0.44). B cells isolated from conditional knockout mice did not upregulate expression of co-stimulatory receptors or MHC II to the same extent as controls when cultured alongside MOG-specific CD4 T cells (p < 0.005) and were inferior at driving T cell proliferation (p < 0.0001) in vitro. Lastly, while BTK deletion diminished the proliferative and survival response of B cells following mitogen stimulation, B cell trafficking to the leptomeninges and organization into ectopic lymphoid tissues (ELTs) in 2D2+IgHMOG mice continued unabated. We identified that BTK signaling regulates several features adopted by autoreactive B cells that contribute to EAE pathogenesis. This study provides mechanistic insights into the therapeutic benefits of BTK inhibitors observed in clinical trials exploring BTK as a therapeutic target in the context of MS. Significance statementAutoreactive B cells contribute to the neuroinflammation that drives multiple sclerosis (MS) and related diseases, yet the molecular mechanisms enabling their pathogenicity remain incompletely understood. This study demonstrates that B cell-specific deletion of Brutons tyrosine kinase (BTK) markedly reduces disease severity in two complementary versions of experimental autoimmune encephalomyelitis (EAE), a widely used animal model for MS. Loss of BTK impairs autoreactive B cell survival, antibody production, antigen presentation to encephalitogenic T cells, and T cell activation, while leaving meningeal ectopic lymphoid tissue formation intact. These findings provide direct mechanistic evidence that BTK signaling in B cells promotes neuroinflammatory damage and supports the therapeutic targeting of BTK to limit B cell-driven pathology in MS.

SEZ6L2 autoantibodies have been identified in patients with subacute cerebellar ataxia, but the underlying immune mechanisms and pathogenic pathways remain poorly understood. We previously established a C57BL/6 mouse model of SEZ6L2 autoimmunity that recapitulates key features of the disease. Here, we evaluated whether genetic background influences the magnitude and organization of SEZ6L2-directed immune responses. Pilot screening of autoimmune-prone strains identified SJL mice as exhibiting accelerated and enhanced antibody responses following SEZ6L2 immunization. In a large-cohort study, SEZ6L2-immunized SJL mice developed robust and sustained antibody responses, along with antigen-specific CD4 and CD8 T-cell activation. Expanded immune profiling revealed increased CNS infiltration of multiple lymphocyte populations, including CD4 T cells, CD8 T cells, B cells, and dendritic cells, as well as the presence of SEZ6L2-specific B cells within the brain. In addition, SJL mice exhibited strain-specific immunodominant T-cell epitopes distinct from those observed in C57BL/6 mice. Functionally, SEZ6L2-immunized SJL mice developed motor deficits consistent with cerebellar dysfunction. Integration of behavioral outcomes demonstrated a consistent overall impairment, and multivariate analysis revealed that coordinated humoral and cellular immune responses were associated with behavioral deficits. Together, these findings demonstrate that SEZ6L2-directed immune responses produce coordinated adaptive immune activation linked to neurological dysfunction and establish the SJL strain as an enhanced model for studying SEZ6L2 autoimmunity. This model also provides a platform for investigating disease mechanisms and therapeutic strategies.

Mucosa-associated invariant T (MAIT) cells have been paradoxically implicated in both tissue repair and fibrosis. However, when and how they modulate fibrogenesis in the injured liver remain unclear. Here, using the carbon tetrachloride-induced model of liver injury in MR1- and MAIT cell-sufficient and -deficient mice, we identify MAIT cells as an early driver of fibrogenesis. The presence of MAIT cells exacerbated hepatocellular injury, myofibroblast activation, and matrix deposition early in the course of fibrosis development, but not at later stages. This was accompanied by rapid polarization of hepatic MAIT cells toward a MAIT17 phenotype and enrichment of pro-fibrotic transcriptional programs. Concurrently, MAIT cells acquired an exhaustion-associated phenotype while still retaining their effector functions. Mechanistically, we demonstrate that MAIT cells limit hepatic regulatory T (Treg) cell accumulation, accompanied by reduced Ki-67 and CXCR3 levels in the latter population, suggesting their impaired proliferation and tissue recruitment. Furthermore, Treg cell inactivation reversed MAIT cell-dependent differences in the severity of fibrosis, establishing Treg cells as a key downstream mediator. Together, these findings identify MAIT cells as early orchestrators of fibrogenesis and reveal a novel MAIT-Treg axis that can be considered a potential therapeutic target in the early stages of fibrotic diseases.

HIV escapes sterilizing immunity through a variety of mechanisms, including the downregulation of MHC-I expression by HIV Nef and Vpu to counteract CD8+ T cell responses. While reduced MHC-I expression would be expected to support targeting by NK cells, a subpopulation of infected CD4+ T cells consistently resists multiple rounds of NK cell natural and antibody-dependent cytotoxicity. Studies further reveal that the HIV accessory protein Vpr induces expression of TNFRSF10B (TRAIL-R2) in CD4+ T cells, with survivors of NK cell targeting exhibiting relatively higher MHC-I and weaker expression of TRAIL-R2. In fact, reverse TRAIL signaling in NK cells leads to the release of perforin and granzymes, a pathway limited when TRAIL-R2 expression is diminished. Thus, independent of canonical death receptor signaling, TRAIL-R2 serves as an activating ligand that augments NK cell killing. These observations demonstrate that through Vpr, HIV can regulate the TRAIL/TRAIL-R2 axis to control NK cell functionality.

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.

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.

Viral infection polarizes monocyte-derived dendritic cells (moDC) to initiate type 1 immunity. The availability of overlapping (homologous) MHC class I and II epitopes, an occurrence frequently and primarily associated with intracellular infection, significantly enhances this process; however, the underlying mechanism(s) are unclear. We demonstrate that moDC loaded with homologous MHC epitopes acquire a cDC1-like phenotype in a process governed by mTORC1. mTORC1 pathway inhibition leads to NF-{kappa}B-mediated expression of IL-12 and other type I immune polarizing genes. The observed cDC1-like gene signature was also significantly enhanced in clinical moDC vaccine products made through methodologies that enforced class I and II antigenic homology. Collectively, these findings reveal a novel and previously unrecognized mechanism of immune governance that might also be exploited in cancer immunotherapy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/716309v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1399bedorg.highwire.dtl.DTLVardef@12c177forg.highwire.dtl.DTLVardef@1bac19corg.highwire.dtl.DTLVardef@1fcff31_HPS_FORMAT_FIGEXP M_FIG C_FIG

The mouse epidermis harbors two key resident immune populations--dendritic epidermal T cells (DETCs), a subset of invariant {gamma}{delta} T cells, and Langerhans cells (LCs), specialized tissue-resident macrophages--both of which play critical roles in immune surveillance, barrier integrity, and tissue homeostasis. While the fetal origin of both cell types has been defined, the cellular and molecular mechanisms that govern their postnatal fates following colonization of the epidermis around birth remain incompletely understood. Here, we present a combination of immunophenotyping- and transcriptome-resolved single-cell map of DETC and LC development in the mouse epidermis from late embryogenesis through adulthood. We delineate differentiation trajectories for both cell types, marked by distinct changes in morphology, proliferation, and transcriptional programming. Using mice deficient in {gamma}{delta} T cells, which lack canonical DETCs, we demonstrate that LCs develop independently of canonical DETCs likely due to the presence of {beta}DETCs. Moreover, analysis of germ-free mice and wildlings reveals that the postnatal development of both DETCs and LCs is independent of microbial colonization. Together, our findings define the core principles underlying the establishment of the mouse epidermal immune niche. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/716534v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@401a75org.highwire.dtl.DTLVardef@890bf8org.highwire.dtl.DTLVardef@170cb28org.highwire.dtl.DTLVardef@29df05_HPS_FORMAT_FIGEXP M_FIG C_FIG

Fibroblasts play a dual role in shaping tissue homeostasis and immune responses during inflammatory perturbations. Manipulating fibroblast behavior has therefore emerged as a promising strategy for autoimmune diseases. Here, through integrated multimodal single-cell transcriptomic and proteomic profiling of synovial tissue combined with prospective clinical data from 54 patients with rheumatoid arthritis, we identify C-X-C motif chemokine 12 (CXCL12)hi Apolipoprotein C1 (APOC1)+ fibroblasts as a pathogenic cell population driving refractory synovitis. CXCL12hi APOC1+ fibroblasts construct local niche in spatial coordinates with plasmablasts via the CXCL12-CXCR4 axis. APOC1 orchestrates senescent inflammatory cancer-associated fibroblast(iCAF)-like properties of this cluster through activation of the STAT3-C/EBP pathway. Therapeutic elimination of senescent cells, either alone or in combination with TNF inhibition, significantly ameliorates experimental arthritis. Together, these findings uncover a mechanistic basis for treatment resistance in rheumatoid arthritis and highlight senescent iCAF-like fibroblasts as a promising therapeutic target.

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.

Systemic vaccination induces serum antibodies and circulating memory B cells but provides limited protection in the upper respiratory tract, where many respiratory pathogens initiate infection. How systemic memory B cells contribute to mucosal immunity remains unclear. Using multiparametric flow cytometry, single-cell RNA and V(D)J sequencing, and functional analyses of paired blood and nasal/oropharyngeal samples, we characterized human B cells across systemic and mucosal compartments. Swab-derived B cells transcriptionally overlap with circulating activated memory B cells while exhibiting distinct features of activation, tissue retention, and spontaneous IgA/IgG secretion. Approximately 6% of mucosal B-cell clones were shared with blood, indicating systemic-mucosal connectivity. Both infection and vaccination expanded two circulating antigen-specific activated memory B cells subsets, whereas antigen-specific B cells accumulated in the upper respiratory tract only following local inflammation. The finding that B-cell recruitment is reactive rather than preemptive may explain the limited efficacy of parenteral vaccines and provides a rationale for developing integrated systemic-mucosal vaccination strategies.

The contributions of antigen compartmentalization to recognition differences between CD4 and CD8 T cells have long been appreciated, but little is known of how subcellular localization of different antigens expressed by a single pathogen impacts T cell immunity. By tracking a clonal CD4 T cell response to its cognate epitope shuttled between different virulence proteins of the enteropathogenic bacterium, Citrobacter rodentium (Cr), we find a remarkable bias in the magnitude and quality of the response contingent on whether antigen remains bacterially associated or is introduced into intestinal epithelial cells colonized by the bacterium. Only proteins injected into the cytosol of colonocytes via the type 3 secretion system (T3SS) of Cr were found to recruit robust antigen-specific T cell responses to the infected mucosa and give rise to CD4 resident memory T (Trm) cells that populate the mucosal epithelium--and this required direct presentation of these antigens by infected epithelial cells. Single-cell transcriptomic analyses revealed that sustained, bidirectional epithelial-T cell communication was required both to elicit epithelial barrier-protective T cell help and to promote transcriptional networks that program a tissue-residency rather than central memory fate. These results establish a central role for antigen presentation by non-professional APCs in controlling memory fate decisions by CD4 T cells, with important implications for development of successful mucosal vaccines.

Immunological memory depends on the maintenance of stem cell-like memory CD8 T cells, which require sustained expression of the transcription factors TCF1. Here, I identify MLL1 as a key regulator of CD8 T cell memory. In activated T cells, MLL1 sustains Tox transcription through interaction with MENIN, thereby maintaining BTLA expression and restraining cytokine-driven AKT activation. Loss of MLL1 or disruption of the MLL1-MENIN interaction accelerates AKT-driven loss of TCF1, leading to impaired memory potential. MLL1-deficient T cells fail to reconstitute lymphopenic hosts and are unable to mediate graft-versus-host disease, while exhibiting increased expansion of virtual memory T cells. Unexpectedly, MLL1 regulates Tox, Btla and Tcf7 independently of its methyltransferase activity and MOF-mediated H4K16 acetylation. These findings define a pathway in which the MLL1-MENIN complex restrains cytokine signaling to preserve CD8 T cell memory and identify a noncanonical function of MLL1 in transcriptional maintenance.