Nature Immunology

Mouse CD8 T cell differentiation has been studied extensively in models of infections and cancer, yet no unified framework spans the full spectrum of immunological contexts. We present the CD8 immgenT framework, integrating >200,000 single-cell transcriptomes and 128-plex surface proteomes from 734 samples spanning multiple perturbations, tissues, and timepoints. Unbiased analysis identifies 21 states encompassing naive, effector, circulating memory, tissue-resident memory, progenitor-exhausted, and terminally-exhausted compartments, among others. These states re-emerge with striking molecular convergence across acute/chronic infections, cancer, autoimmunity, aging, and homeostasis, showing that near-identical transcriptional programs support protective or dysfunctional outcomes depending on developmental history and microenvironment. Classic archetypes map to discrete clusters but exhibit unappreciated heterogeneity and overlap, cautioning against rigid nomenclature. We provide validated combinatorial markers, flow cytometry gating strategies, and immgenT reference-based integration for reproducible annotation of new datasets. This universal coordinate system harmonizes fragmented CD8 T cell literature and clarifies relationships across diverse immune challenges.

Cell signalling networks govern fundamental cellular processes yet remain incompletely defined. Moreover, what is known is biased toward a limited subset of well-characterised components. Phosphoprotein-based interrogation methods, including mass spectrometry and targeted phosphosite panels, have limited utility in physiological settings dependent on cell-cell interactions because the signalling fluxes can be difficult to detect despite producing robust functional responses. Here we developed a perturbation-based experimental framework that infers signalling pathway architecture using quantitative functional outputs rather than direct measurements of effector state, e.g., phosphorylation levels. Using antigen-specific, NF-{kappa}B-GFP reporter-expressing transformed T-cells co-cultured with cellular targets, we performed an arrayed CRISPR-Cas9 screen targeting a curated signalome of kinases, phosphatases, adaptor and scaffolding proteins, totalling 706 genes. Quantitative effect-size profiling recovered canonical T-cell receptor regulators and revealed unequal, family-specific patterns of control over NF-{kappa}B activation. Comparing T-cell stimulation with low- and high-affinity antigen uncovered signal-strength-dependent buffering of proximal signalling nodes, exemplified by reduced sensitivity to perturbation of LCK under high-intensity stimulation. Targeted perturbation in primary human CD8 T-cells validated our findings and identified TRRAP and CTDSPL2 as negative regulators of T-cell effector output, whose disruption enhanced cytotoxicity, degranulation, and cytokine production in both polyclonal and TCR-engineered T cells. Together, these results establish a scalable strategy for mapping signalling pathway architecture in the setting of physiological T-cell activation.

Tumor-infiltrating lymphocytes (TIL) often fail to restrain tumor growth due to progressive differentiation to an exhausted state. In healthy tissues, tissue-resident memory T cells (TRM) maintain protection for years, and patient tumors that contain TIL with TRM features are associated with better prognosis. Proteomic and transcriptomic profiling of T cell populations identified proteostasis as a significant factor distinguishing TRM and progenitor-exhausted TIL from terminally-exhausted TIL, including loss of E3 ubiquitin ligases NEURL3, RNF149, and WSB1, with accumulation of unfolded proteins in spite of functional proteasome activity. Enforced expression of these ligases by TIL preserved stem-like TCF1+ populations and improved anti-tumor function, whereas their knockout impaired TIL and altered T cell differentiation in acute infection. Sustained ligase expression rescued accumulation of unfolded proteins in TIL and improved immunotherapy outcome in preclinical models, highlighting the critical role of proteostasis in TIL function and identifying new avenues for advancing cancer immunotherapy.

Cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction is traditionally framed within a dichotomy of health and disease, yet its systemic immune consequences across the spectrum of CFTR activity remain incompletely defined. Using multimodal immune profiling, we constructed a single-cell atlas of circulating immune cells in people with cystic fibrosis (pwCF), healthy F508del carriers and non-carriers. In pwCF, circulating immunity was markedly altered following treatment with elexacaftor-tezacaftor-ivacaftor, with broad reductions in pro-inflammatory cytokines and immune changes linked to improved clinical outcomes. Strikingly, healthy F508del carriers exhibited a CF-like immune signature characterised by low-grade systemic inflammation, including elevated IL-6, reduced mucosal-associated invariant T cells, and inflammatory monocyte features overlapping with pwCF. Together, these findings show that CFTR dysfunction spans a spectrum of systemic immune dysregulation, challenging a strict dichotomy between health and disease.

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.

Inflammasomes are multiprotein complexes that form and activate after exposure to pathogenic microbes and host danger signals that trigger an inflammatory response. Although NLRP1 and NLRP3 inflammasomes share structural similarities and can be activated by similar stimuli, no evidence of heterotypic inflammasome assemblies has been reported. Here, we identify a unique interaction between NLRP1 and NLRP3 in human cells, forming a hybrid inflammasome, to drive inflammation. NLRP1 is essential for this hydrid inflammasome activation and NLRP3-mediated Caspase-1 activation and release of IL-1{beta} and IL-18. The presence of the heterocomplex inflammasome was confirmed in blood samples from patients after kidney transplantation and is associated with inflammatory responses driven by NLRP3 and MEFV mutations that cause inflammasomopathies. Our findings reveal an unexpected level of intricacy in inflammasome composition, pinpointing hybrid targets that may pave the way for innovative pharmacological treatments for inflammatory disorders. Significance StatementPrevious findings showed interactions between NLRP3 and NLRC4 or NLRP3 and NLRP11 showing that would be possible the interaction of the inflammasomes as supercomplexes and not working alone. Now, we show a new inflammasome heterocomplex inflammasome between NLRP1 and NLRP3 which is associated to the inflammatory profile in autoimmune diseases patietns and transplanted patients. These findings could open a new research topic for the design of dual inflammasomes inhibitors.

Type I interferon (IFN-I) and interferon-{gamma} (IFN{gamma}) are central regulators of antiviral immunity, yet how they cooperatively govern CD8 T cell fate during chronic infection remains unresolved. Here, we uncover a previously unrecognized, spatially encoded interferon circuit that actively constrains progenitor exhausted CD8 T cells (Tpex) during chronic LCMV infection. Persistent IFN-I signaling indirectly restricts Tpex expansion by enforcing their sequestration within PDL1-rich B cell niches of lymphoid tissue and by suppressing T cell-derived IFN{gamma}. Blockade of IFN-I signaling enables Tpex migration into T cell zones of splenic follicles driving IFN{gamma} production, which in turn sustains PDL1 expression on myeloid cells to re-impose local inhibitory pressure. Combined IFN-I and IFN{gamma} blockade disrupts this feedback, promoting coordinated niche redistribution of Tpex and checkpoint remodeling that drives robust Tpex expansion. Single-cell transcriptomics reveal that this layered IFN-I-IFN{gamma} interplay establishes a regulatory balance that constrains Tpex proliferation while preserving effector-like transcriptional programs in their progeny effector CD8 T cells, ultimately preventing premature terminal differentiation. Thus, interferons orchestrate the coordinated T cell-myeloid regulatory circuit that integrates tissue organization, cytokine feedback, and checkpoint control to regulate CD8 T cell exhaustion during chronic infection.

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.

Caspases-mediated processing of cytokines coordinates cell-autonomous defenses and induction of systemic inflammation 1. While caspase-1 processes IL-1{beta} and IL-18 2-5, human caspase-4 processes IL-18 mainly in monocytes 6. Caspase-14 is an exception, specializing in epidermal differentiation7,8, yet no cytokine target has been firmly established for caspase-14. Here, we report that recognition and IL-1{beta} maturation of IL-1{beta} by caspase-14 in epithelial cells determined anti-bacterial humoral immunity against Yersina pseudotuberculosis (Y. pseudotuberculosis) infection. Upon TAK1 inhibition by YopJ, activated caspase-8 cleaved caspase-14 at Asp 146, generating an active 16-kDa fragment, whose exposed pocket directly interacted with and cleaves pro-IL-1{beta} at Cys132. Moreover, conditional knock-out of caspase-14 in epithelial cells or knock-in of a caspase-inactive caspase-14C136A mutant impaired Y. pseudotuberculosis induced IL-1{beta} production and eliminated the total anti-Y. pseudotuberculosis IgG production, leading to uncontrolled Y. pseudotuberculosis infection. Thus, our findings establish caspase-14 as a processor of IL-1{beta} in epithelial cells to propel anti-bacterial humoral immunity, providing insights into the inflammation and vaccine development.

The immune system consists of diverse cell types that act in coordination, yet it remains unclear at what biological scale coherent immune organization emerges. Here we analyze single-cell transcriptomics from a large longitudinal cohort of healthy adults and demonstrate that transcription is coordinated across immune cells. Although baseline expression differs by cell type, transcriptional deviations spanning thousands of genes align across immune cells within donors. This alignment resolves into axes of transcriptional variation that organize both inter-donor differences and longitudinal change. Leading axes capture the largest fraction of variance and reflect dynamic shifts shared across donors, whereas higher-order axes encode more stable donor-specific structure. We refer to this shared multi-dimensional variance structure as the immune transcriptional landscape (ITL). An individuals position within the ITL reflects their transcriptional configuration and is recapitulated in matched serum proteomics, linking immune cell transcription to the circulating proteome. Together, these findings demonstrate that coherent immune organization is encoded in coordinated gene expression across immune cells, revealing a multi-dimensional, temporally stratified transcriptional landscape shared across individuals and over time.

Single-cell transcriptomic and proteomic technologies enable molecular profiling of immune cells at scale but provide limited access to cellular phenotypes shaped by spatial organisation, organelle architecture and cytoskeletal remodelling. Here we present TGlow, a scalable high-content imaging platform optimized for systematic single-cell phenotyping of primary human lymphocytes. TGlow integrates cyclic immunofluorescence, deep z-stack confocal imaging, and open-source data processing pipelines, including both classical and self-supervised vision transformer-based feature extraction, to jointly quantify cellular morphology, organelle organization, and immune activation states. Applied across over 400,000 primary human T cells spanning CD4+ activation time courses, drug perturbations, CRISPR knockouts and CD8+ T-cell exhaustion, TGlow resolves distinct and reproducible phenotypic states. We uncover dose-dependent and mechanism-specific drug phenotypes, such as defective endoplasmic reticulum polarisation under mycophenolic acid and tofacitinib. We show that mitochondrial clustering reveals activation- and cell-cycle-linked remodelling programs, CRISPR perturbations map gene-specific phenotypes that reposition cells along activation trajectories, and we identify a previously unrecognised collapse of cytoskeletal architecture in exhausted CD8+ T cells. TGlow provides a scalable framework for high-dimensional phenotyping of lymphocyte states advancing functional genomics, perturbation screening and population-level immune profiling by resolving the morphological and functional heterogeneity of lymphocytes and enabling systematic linkage of genetic and pharmacological perturbations to cellular function.

T cell activation results in profound proteome remodeling that programs T cells into distinct cellular states. The mechanistic target of rapamycin (mTOR) biases T cell differentiation toward a cytotoxic fate at the expense of memory-precursor formation, making mTOR inhibition an attractive strategy to boost T cell memory during vaccination. Here, we used matched time-resolved mRNA sequencing and quantitative mass spectrometry to define how the human T cell proteome is remodeled during the first 24 hours of activation. We found that human T cells rapidly remodel their proteome in distinct, temporally ordered modules that drive translation and proliferation while promoting a cytotoxic T cell state. Notably, mTOR inhibition during the first 24 hours of T cell activation perturbed these protein modules. Strikingly, transient mTOR inhibition limited to the first 16 hours of T cell priming was sufficient to imprint a memory-like T cell state, while preserving the capacity to produce inflammatory cytokines and mediate target cell killing. Together, these findings indicate that mTOR activity dictates stable functional trajectories during early T cell activation, revealing a therapeutic window to refine vaccination responses.

The oral mucosa is a prototypical human barrier reliant on neutrophils for homeostasis, as both neutrophil deficiency and excessive activation are linked to immunopathology. Yet, whether neutrophils acquire tissue-specific states in health or disease remains unclear. We incorporated single-cell RNA sequencing, spectral flow cytometry, and spatial proteomics across tooth-associated oral mucosa (gingiva) and interconnected compartments of blood and oral cavity to define neutrophil tissue specification in healthy individuals and patients with periodontitis, a neutrophil-dominated inflammatory disease. In health, mucosal neutrophils adopt discrete immunoregulatory states despite constant microbial exposure and mechanical injury. Periodontitis disrupts these programs through infiltration of blood-like neutrophil subsets, increased transcriptional noise, and heightened effector activation. Strikingly, oral inflammation systemically imprints on circulating neutrophils, marked by the expansion of a Rho-GTPase regulatory program that is shared across diverse human inflammatory conditions. Together, these findings establish a framework for understanding how localized tissue inflammation affects both neutrophil plasticity at barrier surfaces and conditioning of systemic neutrophil states with broad implications for inflammatory disease pathogenesis.

Whilst the majority of individuals infected with M. tuberculosis control the infection and remain asymptomatic, only 5-10% progress to active tuberculosis (TB)1. Previous or current infection with M. tuberculosis is detected using antigen-specific interferon (IFN)-{gamma} release assays (IGRA), which cannot identify those who will remain healthy or progress to active TB1. However, 18F-Fluorodeoxyglucose positron emission-computed tomography (PET-CT) can detect increased immune cell metabolic activity in lung parenchyma and intrathoracic lymph nodes associated with infection2. The local early immune factors dictating protection or disease progression have not been defined. To address this, we interrogated the airway immune response at single cell resolution in bronchoalveolar lavage (BAL) from extensively clinically characterised recent household contacts of TB patients, who either controlled the infection or progressed to TB disease. Using unbiased analysis of bulk and scRNA-seq of BAL samples, we define type I IFN-dependent and -independent neutrophil signatures in active TB patients and contacts that progressed to TB. We additionally report an inverse relationship between airway neutrophils and T cells, with T cells showing signatures of exhaustion, cytotoxicity and cell death in progressors and TB patients with a neutrophil dominated airway profile. Conversely, T cell signatures of protection in contacts who remained healthy were dominated by genes related to regulation, quiescence and a stem-like profile. We show that both the inflammatory neutrophil signature of TB progression and the stem-like T cell signature of non-progressors from human airways were recapitulated in scRNA-seq data from non-human primate (NHP) granulomas, associated with disease or immune protection, respectively. Our findings from early human airway responses in TB contacts reveal genes, pathways and cell states that may dictate infection outcome and inform strategies for host-directed therapy and vaccine studies.

Gene expression in regulatory T cells (Tregs) is context-dependent and maintains peripheral immune homeostasis. FOXP3 is lineage defining but not sufficient for Treg function or persistence. To define the cell-intrinsic roles of the FOXP3 paralogs FOXP1 and FOXP4, we generated and studied mice with Treg-specific deletion of Foxp1 and/or Foxp4. FOXP1 and FOXP4 are required to maintain the peripheral Treg pool through enhancing Il2ra transcription, thereby promoting sustained high-level expression of IL-2R and thus of the high-affinity IL-2R{beta}{gamma} complex. Integrating RNA-seq and ATAC-seq with previously published ChIA-PET and publicly available data, we propose a model of Il2ra transcriptional regulation in which in which FOXP1 and FOXP4 anchor chromatin looping of the Il2ra locus in mature Tregs, augment super-enhancer activity, and drive sustained CD25 expression. Our results reveal a unique role of FOXP1, and to a lesser extent FOXP4, in controlling Treg homeostasis. One Sentence SummaryFOXP1 and FOXP4 regulate chromatin architecture at the Il2ra locus, promoting sustained CD25 expression and maintaining the peripheral Treg pool.

Antibody responses to T-dependent antigens are suboptimal in young children, yet the evolution of T follicular helper cell (Tfh) function across the human lifespan remains poorly defined. Using human tonsils, a physiologically relevant and abundant source of Tfh, we investigated age-associated differences in their repertoire and functional programs. Pediatric tonsils were enriched for cytokine-expressing Tfh subsets with increased clonal diversity and phenotypic plasticity. However, in response to influenza antigens, they exhibited reduced Th1 polarization, diminished IL-21 production, and limited B cell help. Across ages, high neutralizing flu antibody responses were associated with robust Tfh1 activation, which was ICOS dependent in adults but not in children. Interestingly, Tfh depletion strategies revealed enhanced Tfh differentiation from distinct precursors in pediatric donors, yet antibody responses during early life were less reliant on Tfh help. Together, these findings define developmentally programmed differences in Tfh differentiation and function with implications for pediatric vaccine design.

Dendritic cells (DCs) orchestrate anti-tumor immune responses, yet the full extent of their phenotypic diversity, and spatial dynamics within the tumor microenvironment (TME) remains incompletely understood. Here, we constructed an integrated atlas of tumor-infiltrating DCs by harmonizing single-cell transcriptomic data from 12 murine tumor studies and 28 published human cancer datasets, together with newly generated single-cell-resolved multiplexed tissue imaging across immunotherapy conditions in a murine model. We noted conserved transcriptional states across species, including canonical conventional type 1 DCs (cDC1s), diverse type 2 DC (cDC2) subpopulations, and two activation states characterized by CCR7 expression (CCR7+ DCs) or interferon-stimulated gene expression (ISG DCs). Spatial transcriptomics analyses from human TMEs revealed that CCR7+ DCs and ISG DCs reside in distinct T cell-enriched regions that are embedded within distinct signaling environments. High-dimensional multiplexed proteomic imaging demonstrated that these DC-T cell niches undergo divergent remodeling across multiple immunotherapy conditions. Notably, this spatial reorganization occurred despite minimal detectable changes in DC transcriptional states. This study delineates conserved DC activation states and their spatial organization within tumors and captures the therapy-dependent remodeling, providing a framework for studying therapy-associated remodeling of DC immune programs in cancer.

Systemic lupus erythematosus (SLE) is characterised by aberrant neutrophil activation and pathogenic B-cell responses that drive organ damage, particularly in lupus nephritis (LN). Here, we identify neutrophil-derived mitochondrial DNA (mtDNA) as a metabolic driver of B-cell dysregulation in LN. Using single-cell transcriptomics of human blood and kidney tissue together with functional co-culture assays, we show that neutrophils from patients with active LN release extracellular traps enriched in mtDNA that induce mitochondrial oxidative stress in B cells. NET-derived mtDNA suppresses IL-10-producing regulatory B cells while promoting pro-inflammatory and plasmablast differentiation through nucleic acid-dependent signalling. Mechanistically, oxidative stress drives maladaptive NADPH utilisation, diverting NADPH from cholesterol biosynthesis toward antioxidant defence, resulting in altered lipid trafficking, mitochondrial dysfunction, and impaired regulatory B-cell differentiation. Consistent with this model, kidney B cells from patients with LN exhibit transcriptional signatures of disrupted redox and lipid metabolism, and NADPH deficiency is associated with reduced regulatory B-cell differentiation in humans. These findings identify a neutrophil-driven metabolic checkpoint that governs B-cell fate in lupus nephritis.

The intestinal epithelium plays a pivotal role in balancing immune tolerance and inflammation, yet how it communicates tissue state to the adaptive immune system remains unclear. Here, we show that intestinal epithelial cells (IECs) encode tissue identity and injury into the major histocompatibility complex class II (MHC II) ligandome. We employed integrated single cell transcriptomics, quantitative proteomics, and high-depth in vivo immunopeptidomics to map the MHC class II self-peptidome of the mouse small intestine across epithelial and immune compartments. Mature enterocytes and intestinal stem cells (ISCs) emerged as the dominant epithelial antigen-presenting cells (APCs), displaying a compartmentalized repertoire of endogenous self-immunopeptides reflecting epithelial differentiation and function. Disruption of epithelial MHC II expression led to loss of antigenic compartmentalization, immune infiltration, extracellular matrix remodeling, and emergence of inflammation-associated immune ligands, demonstrating that epithelial MHC II is required to maintain homeostasis. Functionally, a subset of ISC-derived self-immunopeptides preferentially promotes regulatory CD4{square} T cell responses, linking epithelial antigen presentation and peripheral tolerance. During gut inflammation, the epithelial MHC II landscape shifted toward damage-associated antigens. Together, these findings establish epithelial MHC II presentation as a context-dependent tissue-immune communication system that promotes tolerance in homeostasis and alerts to tissue injury during inflammation.

Bacille Calmette-Guerin (BCG) is the only licensed vaccine against tuberculosis (TB) but provides inconsistent protection against disease. Studies in non-human primates show that intravenous BCG confers superior protection compared to intradermal vaccination, yet how different vaccination routes reprogram lung immunity in mice - the primary model for mechanistic studies - remains incompletely defined at single cell or spatial resolution. Moreover, while lung macrophages are key effectors during Mycobacterium tuberculosis infection, most studies have focused on alveolar macrophages, leaving interstitial macrophage responses to vaccination largely unexplored. Here, we define the cellular and spatial immune landscape shaped by three BCG vaccination routes in mice using flow cytometry, single-cell RNA sequencing, and spatial transcriptomics. Comparing intratracheal (IT), intravenous (IV), and subcutaneous (SC) vaccination, we demonstrate that mucosal IT delivery uniquely reprograms interstitial macrophages, establishes spatially organized immune hubs with CD4 T cells, and provides superior protection against infectious challenge. These findings identify IM as key mediators of mucosal vaccine-induced protection and provide a framework for the rational optimization of vaccines against respiratory pathogens.