Immunity

Viral pneumonia is perpetuated by inflammatory circuits between activated T cells and monocyte-derived alveolar macrophages (MoAM). T cells and macrophages express ORAI1 and STIM1, which form calcium release-activated calcium (CRAC) channels that allow extracellular calcium entry in response to endoplasmic reticulum calcium store depletion. In a randomized, placebo-controlled, multicenter phase 2 trial (CARDEA), Auxora, a CRAC channel inhibitor, reduced all-cause 30-day mortality by 56% in patients with severe SARS-CoV-2 pneumonia. Here, we report a multi-omics analysis of serially collected alveolar samples from unvaccinated patients with severe SARS-CoV-2 pneumonia treated with Auxora versus placebo. We found reductions in plasma levels of the monocyte- and T cell-chemokines, CCL8 and PDGF-AA. Using peripheral blood mononuclear cells (PBMC) from healthy volunteers, we show that Auxora directly targets T cells to inhibit the transcription of CCL8 and PDGFA in monocyte-derived macrophages, supporting a mechanism for its effects and a potential intermediate biomarker of efficacy.

Immune responses to infection and vaccination exhibit diversity between individuals that can be shaped by differences in their immune cell landscapes and the signalling, transcriptional, and genetic mechanisms that coordinate immune cell function. Specific antibody deficiency (SAD) and common variable immunodeficiency (CVID) are common forms of predominantly antibody deficiencies that result in poor responses to vaccination. While molecular and cellular causes of the immune dysfunction and poor vaccination responses for individuals with CVID have been reported, immune cell or molecular defects have not yet been identified in SAD. Here, we have used single-cell multi-omics to define the cellular landscapes, transcriptional states, adaptive immune repertoires and protein expression of patients with SAD and CVID before and after polysaccharide vaccination. We discovered that while SAD and CVID exhibit overlapping immune defects, including accumulation of exhausted NK memory cells and dysregulated expression of genes that mediate lipopolysaccharide sensing and clearance by monocytes, individuals with SAD have a unique expansion of cytotoxic CD4+ T cells that correlates with reduced regulatory T cells. In response to vaccination, we observed rapid changes in gene expression associated with lipopolysaccharide responses by monocytes and NF-kB pathway activation in B cells, and an apparent expansion of a CD95+ class-switched memory B cell population that does not occur in patients with lower antigen-specific responses. Together, our findings reveal cellular and molecular factors that underpin variability in vaccine responses and define SAD in a broader spectrum of immune dysfunction.

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease driven by uncensored B and T cell autoreactivity. Understanding this pathogenic process has been hampered by lack of studies of secondary lymphoid organs in human SLE. Using minimally invasive lymph node fine needle aspirates (LN-FNAs), we profiled tissue-resident immune cells from 59 SLE patients and 34 healthy controls through high-dimensional 43-color flow cytometry, antigen-specific tetramer probing, and sc-RNA sequencing with paired VH/VL repertoire analysis. Our findings reveal hyperactive lymph node immunity in SLE characterized by spontaneous germinal center (GC) activation, plasma cell accumulation enriched in mature CD19- and CD138+ antibody-secreting cells, and increased frequencies of both GC-TFH and PD-1+CXCR5- T extra-follicular helper cells. SLE lymph nodes harbored large oligoclonal B cell families with altered isotype usage, dominated by IgG1 and IgG4. Critically, self-reactive 9G4+ and Ro60+ B cells showed defective tolerance checkpoint control, accumulating in activated naive, GC, and plasma cell compartments with distinctive PD-1+Tox+ expression absent in viral-specific responses. Single-cell repertoire analysis revealed VH4-34 clones in SLE BGC and BPC, that in contrast to HD, had not experienced clonal redemption. Instead, SLE VH4-34 clones displayed low somatic hypermutation and preserved the AVY hydrophobic patch associated with autoreactivity. Monoclonal antibody testing confirmed that unmutated AVY+ VH4-34 clones retained polyreactivity against naive B cells, apoptotic cells, and multiple self-antigens. Together, these results define "clonal damnation" as a key mechanism in SLE whereby autoreactive VH4-34 clones of pathogenic potential escape tolerance checkpoints, expand in germinal centers, and differentiate into tissue plasma cells while preserving germline-encoded self-reactivity. Combined, our study defines critical mechanisms of tolerance breakdown in lupus pathogenesis.

Self-reactive B cells are generated during normal development and can acquire increased pathogenicity through activation-induced cytidine deaminase (AID)-mediated diversification following activation. Clonal deletion is thought to eliminate these cells, yet how deletion is distributed across developmental and activation stages to prevent autoimmune disease remains unclear. Here, we show that clonal deletion is enforced through temporally distinct mitochondrial apoptosis (MOMP) checkpoints that differentially regulate autoreactive B cell fate and disease progression. Using conditional Bcl-2 expression to inhibit MOMP either before or after B cell activation, we find that early inhibition permits the survival and maturation of autoreactive B cells after peripheral egress, expanding the pool of cells available for activation. These cells subsequently undergo AID-dependent diversification, producing class-switched IgG autoantibodies with expanded antigen breadth that target a wider range of self-antigens and drive lethal, female-biased autoimmune disease characterized by complement activation and kidney pathology. In contrast, inhibition of MOMP only after activation allows the accumulation of germinal center, switched memory, and plasma cells and promotes autoantibody production, but results in more restricted IgG autoreactivity, limited complement activation and limited tissue damage, and normal survival. Notably, early MOMP inhibition does not expand immature bone marrow B cells, indicating that a major clonal deletion checkpoint operates in the periphery rather than during initial B cell generation. Together, these findings support a Distributed Clonal Deletion Model in which early checkpoints restrict the entry of autoreactive B cells into diversification pathways, while later checkpoints limit the persistence of diversified autoreactive clones, thereby constraining autoimmune disease progression. One Sentence SummaryDistributed clonal deletion prevents autoimmune disease progression by restricting the breadth of autoreactive clones entering immune responses, with early MOMP checkpoints limiting diversification and later checkpoints constraining persistence.

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.

Zika virus (ZIKV) encephalitis induces cytokine-mediated cognitive deficits which persist long-term. Here, we determined if NOD2-mediated conversion of monocytes from Ly6CHi inflammatory to Ly6CLo anti-inflammatory phenotypes during ZIKV encephalitis preserves neural correlates of learning and memory within the hippocampus. Short-term administration of the NOD2 agonist, muramyl dipeptide (MDP), during peak ZIKV infection prevents synapse elimination and loss of adult hippocampal neurogenesis, without impacting CNS virologic control. Transcriptomic analyses of forebrain immune cells in MDP-treated mice revealed functional modulation of infiltrated monocytes and T cells, reducing their expression of pro-inflammatory cytokines, with limited effects on microglia, compared to controls. Notably, NOD2 activation in peripheral immune cells alone balances innate immune signals, preserving synapses, and increasing macrophage phagocytic capacities that do not target synapses. Our findings identify infiltrating Ly6CHi monocytes as key drivers of long-term cognitive dysfunction following ZIKV encephalitis and as potential therapeutic targets for limiting synapse loss. HighlightsO_LINOD2 activation via MDP phenotypically shifts monocyte subsets from inflammatory to anti-inflammatory during the acute phase of ZIKV encephalitis. C_LIO_LIShort-term MDP administration increases phagocytic machinery and reduces inflammatory cytokine/chemokine production within macrophage populations. C_LIO_LISynapse elimination is attenuated within the hippocampus of ZIKV-infected animals treated with MDP. C_LIO_LIMDP derived effects are mediated through peripherally derived immune cells. C_LI

Antibiotic exposure is a significant risk factor for Crohns disease, yet the tissue-specific consequences of antibiotic-driven dysbiosis remain poorly defined. Adherent-invasive Escherichia coli (AIEC), a pathobiont enriched in Crohns disease, expands following antibiotic treatment, but whether discrete mucosal niches support this expansion is unknown. Peyers patches are specialized lymphoid structures that coordinate mucosal immunity and are frequently associated with early disease lesions, suggesting they may represent a vulnerable site for pathobiont colonization. Here, we show that vancomycin disrupts the Peyers patch-associated microbiome, creating a permissive niche that is selectively exploited by AIEC and associated with focal inflammation. Antibiotic treatment markedly increased AIEC burden within Peyers patches. AIEC localized within the lymphoid follicle was accompanied by focal tissue pathology and a distinct cytokine signature. In contrast, expansion of resident E. coli in the absence of AIEC did not elicit comparable inflammation, indicating that the pathogenic traits of AIEC are required to trigger disease-relevant responses in this niche. Supporting this, genetic disruption of flagellin, long polar fimbriae, or antimicrobial peptide resistance in AIEC attenuated Peyers patch colonization or inflammation, revealing separable mechanisms governing niche access and immunopathology. Together, these findings identify Peyers patches as a previously unrecognized reservoir for antibiotic-driven AIEC expansion and define a localized host-microbe interaction that links dysbiosis to focal intestinal inflammation. These results provide a mechanistic framework for understanding how antibiotic exposure may precipitate site-specific pathology in Crohns disease. Further, these findings highlight that mucosal lymphoid tissues should be considered when evaluating microbiome-targeted therapeutic interventions in Crohns disease.

Fibroblasts are key organizers of tissue architecture and immune cell homeostasis, yet how they shape adaptive immune function within non-lymphoid tissues remains incompletely understood. CD8+ tissue-resident memory T cells (TRM) provide localized protection against pathogens and contribute to tumor control, but the microenvironmental signals that maintain their persistence and survival are poorly defined. Here, we identify fibroblast-derived TGF-{beta}3 as a conserved stromal niche factor that specifically sustains CD8+ TRM in both steady-state and disease settings. Across human single-cell cross-tissue atlases, CD8+ TRM preferentially correlated with fibroblast abundance in healthy barrier tissues and multiple tumor types, and TGFB3 emerged as a key fibroblast-enriched candidate mediator. In human and murine co-culture systems, fibroblast-derived TGF-{beta}3 promoted CD8 TRM-like differentiation in vitro. Using a novel genetic in vivo model, inducible fibroblast-specific deletion of Tgfb3 reduced CD8 TRM across barrier tissues at steady state and impaired antigen-specific CD8 TRM formation following viral infection. In tumor models, genetic loss or antibody mediated neutralization of TGF-{beta}3 impaired CD8 T cell residency and cytotoxicity, induced dysfunction via proteotoxic stress and apoptotic programs, and accelerated tumor growth. These findings provide mechanistic insight into the limited efficacy of pan-TGF-{beta} blockade in cancer therapy. Collectively, we describe a novel fibroblast-CD8 T cell axis mediated by TGF-{beta}3 that sustains residency and restrains proteotoxic stress in barrier tissues and tumors.

Age-associated B cells (ABCs) expand in systemic lupus erythematosus (SLE) and contribute to pathogenic humoral immunity, but the mechanisms that restrain their differentiation remain unclear. Here, we identify the transcription factor IKZF2 (Helios) as a regulator that limits ABC differentiation. Transcriptomic and functional analyses showed that suppression of oxidative phosphorylation (OXPHOS) in B cells promoted ABC differentiation and was accompanied by reduced IKZF2 expression. Pharmacologic modulation of mitochondrial metabolism further demonstrated that OXPHOS inhibition promoted, whereas OXPHOS activation restrained, ABC differentiation. Integrative analyses revealed reduced IKZF2 expression in selected B cell subsets from patients with SLE. Functional suppression of IKZF2 enhanced ABC differentiation and attenuated the inhibitory effects of OXPHOS activation, indicating that IKZF2 mediates metabolic control of B cell fate. Mechanistically, IKZF2 restrained early ABC-associated gene programs, including ITGAX and TBX21. Circulating miR-1285-3p in small extracellular vesicles, elevated in SLE, suppressed OXPHOS and recapitulated these effects. Together, these findings identify an OXPHOS-IKZF2 axis that restrains pathogenic B cell differentiation and links extracellular microRNA-mediated metabolic stress to ABC formation in SLE. One-sentence summarySmall EV-associated miR-1285-3p in SLE promotes ABC differentiation by suppressing OXPHOS and relieving IKZF2-mediated restraint.

High-risk neuroblastoma (HRNB) is a leading cause of pediatric cancer death. Current therapies center on intensive multimodal treatment including anti-GD2 therapy, with growing interest in harnessing T cell-mediated immunity. How T cells and their receptors (T-cell receptors, TCRs) are spatially organized and function within tumors remains poorly defined. To assess whether intratumoral location influences clonotype-specific T cell states, we profiled TCR repertoires across blood and tumor samples from 37 patients with HRNB using longitudinal bulk TCR sequencing. In a nested subset of 5 patients with paired pre- and post-therapy tumors, we integrated spatial transcriptomics with in situ TCR profiling. Across all tumors, T and B cells preferentially co-localized in immune-rich regions and showed reduced proximity to neuroblast cells. Despite this compartmentalized architecture, {gamma}{delta}T cells were more evenly distributed across tumor sections and showed greater proximity to neuroblast-rich regions than other T cell subsets. Within TCR clonotypes, spatial location was associated with distinct transcriptional states, with immune-rich regions supporting more progenitor-like programs. These findings identify spatial context as a key determinant of phenotype clonotype-specific T cell phenotype and highlight {gamma}{delta}T cells cells as a spatially distinct population with potential roles in neuroblastoma tumor-immune interactions.

Regulatory T (Treg) cells maintain immune tolerance via contact-dependent inhibition and soluble mediators. While canonical suppressive mechanisms are well characterized, their hierarchical spatiotemporal organization remains unclear. We previously showed Foxp3 represses ER Ca2+ channel RyR2, reducing cytoplasmic Ca2+ to inactivate m-Calpain (Calpain-2) and stabilize high-affinity LFA-1-mediated Treg-dendritic cell (DC) adhesion--an early step blocking DC antigen presentation. Using m-Calpain as a molecular switch, we generated mechanically deficient Tregs (TregFD) with constitutively active m-Calpain that abrogated high-force Treg-DC adhesion. TregFD retained a near-wild-type transcriptome (differential expression limited to adhesion pathways), yet failed to suppress autoimmunity or DC-driven T cell proliferation. Stable adhesion was required for localized TGF-{beta}/IL-10 delivery to DCs. Single-cell RNA sequencing of rescued Scurfy mouse lymphoid tissues revealed organ-specific division of labor: non-mechanical modules support homeostasis/tissue repair, while mechanical modules dominate epithelial barrier/antigen presentation suppression. Combined TregFD and RyR2-deficient Tconv transfer restored wild-type Treg activity, demonstrating module synergism. These findings establish a mechanical force-centered, two-tiered hierarchical model of Treg suppression, providing a framework for targeting Treg mechanics in autoimmunity and inflammation.

Mucosal IgA is critical for controlling the microbiota and preventing pathogenic infection of the epithelium. The innate signals that regulate the generation of IgA remain poorly defined. Here, we identified TRIF and IL-1R1 as immune checkpoints for intestinal IgA production. In the absence of infection, Trif-/- and Il1r1-/- mice exhibited markedly elevated stool IgA and IgA-bound commensals. We show that IL-1R1 restricts IgA class-switching in a B cell-intrinsic manner, while TRIF acts extrinsically of B cells and shapes the Peyers patch microenvironment. Loss of either Trif or Il1r1 enhanced retinoic acid metabolism and allowed premature Ccnd3 upregulation in naive B cells, favoring both IgA class-switching and differentiation into germinal center B cells. During oral vaccination, the absence or blockade of the TRIF/IL-1R1 pathway increased antigen-specific IgA production without affecting seric antigen-specific IgG levels. These findings unveil novel and local signaling targets to promote robust antigen-specific mucosal immunity.

Tumor-specific exhausted CD8 T cells (Tex) adopt diverse phenotypes across human cancers, but the drivers of this heterogeneity remain poorly understood. Using flow cytometry and single-cell RNA and T cell receptor (TCR) sequencing of 106,667 tumor-infiltrating CD8 T cells from head and neck squamous cell carcinoma (HNSCC) tumors, we identified and validated three Tex subsets, each with distinct clonotypes: (1) Tex-Conv, expressing conventional exhaustion genes; (2) Tex-CCR6, distinguished by CCR6 and Tc17-like genes; and (3) Tex-KLR, marked by killer cell lectin-like receptors (KLRs) and particularly high immune checkpoint expression. Through multiplexed immunofluorescence, we found that Tex-KLR cells preferentially localized within tumor nests in direct contact with malignant cells. Due to the elevated checkpoint expression, unique clonotypes, and intra-tumoral localization of Tex-KLR cells, we hypothesized that high-avidity TCR signaling induces this state. We therefore developed an in vitro co-culture system to model TCR avidity in primary human CD8 T cells and identified high-avidity, NFAT-dependent TCR signaling as a key driver of the Tex-KLR signature. Strikingly, in HNSCC and breast cancer patients, we found that Tex-KLR cells are associated with response to neoadjuvant anti-PD-1 therapy. Together, our findings demonstrate that high-avidity, NFAT-dependent TCR signaling shapes Tex phenotypes and promotes the Tex-KLR signature. These data support further investigation of the Tex-KLR subtype and its role, dynamics, and targetable translational applications to cancer immunotherapy.

Epithelial regeneration and barrier integrity are impaired in inflammatory bowel diseases, including Crohns disease (CD), yet current therapies largely target immune inflammation without directly promoting mucosal repair. While regulatory T cells are classically immunomodulatory, their capacity to directly support human intestinal stem cells (ISCs) and barrier function remains unclear. In this study, we tested the hypothesis that FOXP3-expressing regulatory T cells--engineered CD4LVFOXP3 and thymic-derived Treg (tTreg)--directly support human ISC maintenance and restore epithelial barrier function independent of their immunomodulatory function. Using CD patient ISCs-derived enteroids that display disease-associated damage, we established co-culture with FOXP3-engineered Treg cell-CD4LVFOXP3 or thymic-derived Treg (tTreg). The presence of either CD4LVFOXP3 or tTreg cells enhanced enteroid growth, improved epithelial barrier function, and restored apical-basal polarity of ISCs, indicating reparative capacity. Conversely, activated conventional CD4+ T cells reduced barrier function and abrogated apical-basal polarity. Integrating secretome profiling with ligand add-back and receptor or ligand blockade, we identify the PDGF-AA-PDGFR axis as a key regulator of Treg-mediated intestinal epithelial barrier integrity, but dispensable for Treg suppressive capacity. Collectively, our data delineate a direct, human tissue-intrinsic role of FOXP3-driven Treg in the interaction with ISCs via PDGF-AA-PDGFR, enhancing epithelial barrier function and positioning CD4LVFOXP3 as a treatment approach coupling immunoregulation with epithelial repair. One Sentence SummaryRegulatory T cells improve the intestinal epithelial barrier function, primarily, through the PDGF-AA-PDGFR axis

Interleukin-1 (IL-1) is a central driver of autoinflammatory disease, yet IL-1 blockade often provides incomplete benefit in complex, neutrophil-driven conditions. Here we identify a licensing circuit in which common {gamma}-chain ({gamma}c) cytokines provide a redundant signal required for maximal IL-1-driven neutrophil inflammation. IL-1 and {gamma}c cytokines synergize to drive inflammatory cytokine production exceeding either stimulus alone, and these signals engage the MEK/ERK pathway, an effect substantially suppressed by pharmacological MEK inhibition. We validated this circuit in vivo in a mouse model of IL-1-driven neutrophil-dominant autoinflammation. Ablation of the shared {gamma}c receptor markedly prolonged survival and attenuated pathology, whereas deletion of individual {gamma}c cytokine pathways had no major effect--demonstrating in vivo necessity and functional redundancy. Analysis of public phospho-proteomic and transcriptomic datasets confirms MEK/ERK as a conserved neutrophil response to diverse inflammatory stimuli and coordinated IL-1, {gamma}c, and MEK/ERK activation in neutrophils from patients with systemic juvenile idiopathic arthritis (sJIA) and in lesional skin from hidradenitis suppurativa. Together, these findings define a signaling architecture in which redundant {gamma}c inputs enhance MEK/ERK-dependent inflammatory output, identify the {gamma}c receptor as an in vivo disease-modifying node, and position MEK/ERK as a mechanistically grounded therapeutic target. eTOC SummaryLorenzo et al. show that common {gamma}-chain ({gamma}c) cytokines provide redundant licensing signals that amplify IL-1-driven neutrophil inflammation through MEK/ERK convergence. Blocking any single {gamma}c cytokine fails to suppress disease, but ablating the shared {gamma}c receptor or inhibiting MEK/ERK markedly attenuates pathology, identifying these nodes as therapeutic targets in autoinflammatory disease.

Background: Diabetes mellitus (DM) worsens pulmonary tuberculosis (TB) and drives systemic hyper-inflammation, but the underlying mechanisms remain unknown. Neutrophils have key roles in TB immunopathology and lung cavitation. Here, we determine the role of neutrophils in DMTB patients and in driving TB immunopathology. Methods: Sputum and plasma from 30 TB and 30 DMTB patients were analysed for proteases and cytokines using Luminex bead array. Whole blood transcriptomics identified transcriptional differences. Single-cell RNA sequencing characterised neutrophil subsets and dysregulated pathways. Neutrophil function of poorly-controlled DM patients (HbA1c>8%) and healthy controls (HC) were examined following Mycobacterium tuberculosis stimulation, including reactive oxygen species (ROS), neutrophil extracellular traps (NETs), and phagocytosis. Pathways were interrogated using chemical inhibitors, protein array and western blot. Results: Compared to non-diabetic TB patients, poorly-controlled DMTB patients showed up-regulated sputum MMP-8 and MMP-9, associated with increased collagen-destruction and lung cavity formation. Circulating neutrophil count and neutrophil-derived plasma MMP-8 were up-regulated, alongside transcriptional enrichment of extracellular matrix degradation and inflammatory pathways including TNF and RAGE. Single-cell profiling identified reduced cycling neutrophil subset and myelocytes in DMTB, with overall reduced antibacterial and cell-killing signatures. Ex vivo mycobacterial stimulation of DM neutrophils increased ROS and MMP-9 with impaired NETs and delayed phagocytosis. TNFR1, TNFR2, and RAGE were up-regulated. RAGE inhibition with rosiglitazone mitigated Mtb-induced ROS and MMP-8 release. Conclusion: DM worsens neutrophil-driven tissue destruction and inflammation in TB via dysregulated TNF and RAGE-signalling, priming neutrophils towards immunopathology. Targeting RAGE alongside tight glycaemic control may dampen neutrophil hyper-inflammatory responses to limit tissue destruction.

CD96 is a member of the immunoglobulin superfamily including TIGIT and CD226 that bind to a shared ligand, CD155. This family of co-receptors and its ligands are dysregulated in various autoimmune conditions and cancers, highlighting therapeutic potential. While TIGIT and CD226 are recognised co-inhibitory and co-stimulatory receptors respectively, the function of CD96 remains incompletely defined. Here, we assessed CD96-CD155 interaction and downstream trafficking to define a novel CD96 mechanism of action. Using primary human T cells and engineered cell models, we demonstrate that CD96 mediates uptake and internalisation of both soluble and cell-associated CD155. CRISPR-Cas9-mediated receptor knockout in primary human T cells revealed that CD155 uptake was uniquely dependent on CD96, but not TIGIT or CD226, identifying CD96 as the dominant mediator of CD155 internalisation in human T cells. This uptake process was dependent on active receptor cycling, which was partially facilitated by the CD96 cytoplasmic domain. Furthermore, CD96 variant 2 exhibited enhanced ligand binding and uptake efficiency compared with variant 1. Mechanistically, internalised CD155 trafficked to lysosomal compartments and associated with autophagy-related proteins, consistent with degradative processing. These findings reveal a previously unrecognised mechanism by which CD96 may regulate ligand availability through ligand internalisation and trafficking, with potential implications for T cell function and immune regulation. This process may promote changes in the immunoregulatory balance and could inform new targeted therapeutic development.

CD8 T cells mediate protective immune responses. However, persisting antigens such as chronic viruses or tumors redirect CD8 T cell differentiation to a sub-optimal, epigenetically defined state called exhaustion. Exhausted T cells (TEX) lose their ability to persist long-term and to initiate functional memory responses. Checkpoint inhibitor blockade temporarily restores effector functions, but immune reinvigoration is not long-lasting, due to the epigenetic stability of TEX. Therefore, epigenetic reprogramming of TEX leading to durable T cell responses is essential to improve disease control. Here, we demonstrate that a single microRNA (miR), miR-29a, epigenetically re-directs TEX differentiation and preserves TEX into a stem-like state, leading to long-term persisting progenitor TEX. MiR-29a rewires epigenetic maintenance programs, including downregulation of key exhaustion-associated regulators (Dnmt1, Dnmt3a, and Dnmt3b), alongside increased expression of progenitor- and stemness-associated genes such as Tcf7 and Il7r. These reprogrammed CD8 T cells are more sensitive to PD-L1 checkpoint blockade. Ectopic expression of miR-29a combined with aPD-L1 treatments enhances effector responses, while preserving T cell stemness. Together, our findings suggest that miR-29a can be leveraged to overcome current barriers to immune checkpoint blockade. HighlightsO_LIMiR-29a rewires key exhaustion-associated epigenetic maintenance programs, while enhancing stemness-associated transcriptional circuits. C_LIO_LIMiR-29a drives extensive remodeling of accessible chromatin in TEX. C_LIO_LIMiR-29a preserves newly generated progenitor TEX in a durable, epigenetically defined stem-like state with increased effector function. C_LIO_LIMiR-29a synergizes with aPD-L1; while miR-29a preserves progenitor TEX state, addition of aPD-L1 enhances the cytotoxic potential of these progenitor TEX cells. C_LI

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.

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.