Science Immunology

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

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.

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

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.

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

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

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

The interaction between the Major Histocompatibility Complex Class I (MHC-I) 3 domain and CD8 has classically been viewed as a positive coreceptor interaction that stabilizes TCR signaling during antigen recognition. However, its physiological function in mature peripheral CD8+ T cells in vivo remains incompletely understood. Here, we identify the MHC-I 3 domain-CD8 interaction as a previously unrecognized inhibitory pathway that tonically restrains peripheral CD8+ T-cell activation and maintains T-cell tolerance in vivo. Antibody-mediated disruption of the MHC-I 3 domain-CD8 interaction induced spontaneous activation of peripheral CD8+ T cells without impairing their survival, lowered the threshold for antigen-induced activation, and enhanced responsiveness to cognate peptide stimulation. In peptide-induced OT-I T-cell anergy models, blockade of either H-2Db or H-2Kb 3 domain interactions with CD8 prevented the induction of anergy and restored responsiveness of previously anergic T cells. Notably, blockade of the H-2Kb 3 domain enhanced OT-I responses despite simultaneously disrupting the classical positive coreceptor interaction within the TCR-peptide-MHC complex, indicating that tonic inhibitory signaling mediated by the MHC-I 3 domain predominates under these conditions. Together, these findings redefine the classical MHC-I-CD8 interaction as a bidirectional pathway that not only supports antigen recognition but also imposes tonic inhibitory control over peripheral CD8+ T cells. These results identify the MHC-I 3 domain-CD8 axis as a potential target for reversing T-cell tolerance and enhancing antitumor or antiviral immunity.

Acute respiratory distress syndrome (ARDS) is a devastating complication of respiratory infections; however, the biological mechanisms that initiate its onset are poorly defined. Here we show that TNFRSF13B polymorphisms increase the risk of ARDS following SARS-CoV-2 infection up to 7.4-fold compared to the WT genotype. The increased risk was not due to immune-deficiency or impaired virus neutralization. On the contrary, TNFRSF13B mutant subjects mounted better antibody neutralization compared to subjects with WT TNFRSF13B. However, IgG from subjects expressing TNFRSF13B variants had less sialic acid, terminal galactose, and fucose than IgG from subjects with a WT genotype. Moreover, IgG from TNFRSF13B mutant subjects exhibited increased recruitment of complement factors. Thus, besides well-known actions governing plasma cell differentiation, TNFRSF13B impacts both affinity maturation and effector functions of IgG in ways that independently govern complement activation controlling inflammatory responses known to trigger ARDS.

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.

Patients with a dominant mutation in the Rho GTPase RAC2, RAC2E62K, which hyperactivates the protein, suffer from a combined immunodeficiency characterized by recurrent bacterial and fungal infections and severe T cell lymphopenia. Patient neutrophils have elevated F-actin and superoxide production yet fail to control growth of S. aureus, and the mechanism underlying this killing defect is unknown. Here we report that hyperactive Rac2 primes neutrophils for primary granule degranulation, potentially depleting myeloperoxidase (MPO) needed for intraphagosomal microbial killing. Using a Rac2+/E62K mouse model, we show that mature bone marrow neutrophils have decreased side scatter, elevated surface CD63, and reduced intracellular MPO. Interestingly, bone marrow architecture and neutrophil development in the mice are normal. Rac2+/E62K neutrophils are hyperactivated, with increased CD11b expression, cell spreading, and bioparticle phagocytosis. In the spleen, Rac2+/E62K mice display extramedullary granulopoiesis and an accumulation of degranulating neutrophils. Splenic T cells, but not B cells, show elevated surface phosphatidylserine, an "eat me" signal that sensitizes them to phagocytic clearance and provides a candidate mechanism for the selective T cell lymphopenia. Together these findings suggest that hyperactive Rac2 compromises antimicrobial neutrophil function and drives selective T cell clearance in the spleen.

NK cells can form clonal populations demonstrating features of adaptive immunity, including long-term memory and at least partial antigenic specificity. Given the limited individual diversity of activating receptors, the nature of NK cell antigenic specificity remains elusive. To explore this riddle, we combined scRNA-Seq of ex vivo FACS-sorted NK cell subsets expressing specific KIR receptors, single-cell cloning and bulk RNA-Seq of in vitro cultured KIR2DS4 NK cell clones, transcriptomic profiling of antigen-stimulated NK cells, and in silico modeling of glycosylated KIR2DS4-peptide-HLA complexes. scRNA-Seq resolved 12-15 clusters per KIR subset with highly heterogeneous KIR, KLRC and NCR expression patterns, consistent with clonal lineages. Notably, those clusters demonstrated over 30 differentially expressed glycosyltransferase genes, potentially involved in post-translational modification of NK cell receptors. Single-cell-derived KIR2DS4 cultures exhibited clone-specific cytotoxic, chemokine and KIR receptor genes, and transcriptional differences in > 40 glycosyltransferases. In peptide culturing autologous assays, SARS-CoV-2 (KTFPPTEPK) and EBV (CRAKFKHLL) peptides elicited NK cell proliferation and distinct transcriptional programs linking cytotoxicity genes, KIR2DS4 and glycosyltransferases. Structural modeling revealed that N-linked glycosyl residues in specific regions of KIR2DS4 may alter its contacts and interaction with MHCI and the presented peptide. We conclude that KIR human NK cells comprise clonally imprinted populations with distinct glycosyltransferase expression profiles, and site-specific KIR2DS4 glycosylation may modulate interaction with peptide-MHCI complexes, suggesting a post-translational layer of clonal NK cell diversification as a clue to their antigenic specificity.

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.

Neural regulation of innate immunity is increasingly recognized, yet how the nervous system controls immune resolution after pathogen clearance remains poorly understood. Using Caenorhabditis elegans as a genetically tractable model, we identify the AIA interneurons as critical regulators of both infection-phase homeostasis and post-infection recovery. Acute silencing or genetic ablation of AIA neurons during Salmonella enterica infection results in reduced host survival with heightened activation of conserved immune and stress pathways, including PMK-1/p38 MAPK, insulin/IGF-1 signaling, and the XBP-1-mediated unfolded protein response (UPR). AIA-deficient animals exhibit excessive immune and stress response gene expression and increased intestinal tissue damage, demonstrating that immune hyperactivation is detrimental. Strikingly, selective silencing of AIA neurons during the recovery phase after pathogen clearance significantly impairs survival, revealing that neural activity is required not only for defense but also for resolution. This recovery defect is rescued by knockdown of xbp-1, but not pmk-1 or daf-16, indicating that unresolved ER stress is the principal driver of post-infection mortality. Consistently, AIA silencing during recovery sustains UPR activation and exacerbates epithelial barrier damage. Together, our findings establish AIA interneurons as central coordinators of immune homeostasis that limit pathological stress responses during infection and actively promote infection resolution. These findings provide mechanistic insight into how the nervous system not only restrains excessive immune and stress responses during infection but also actively resolves stress signaling and preserves tissue integrity during post-infection recovery.

Despite the clinical success of cancer immunotherapies, the cellular interactions driving tumor regression remain incompletely understood. Here, we investigated the dynamic remodeling of the tumor immune microenvironment during regression of transplanted PyMT mammary tumors following STING agonist treatment. Using scRNA-seq of sorted CD8+ T cells and myeloid cells, combined with imaging approaches, we identified major changes in both lymphoid and myeloid compartments during tumor regression. Regressing tumors showed a transient accumulation of Ly6Chi monocyte populations associated with a decline in macrophage subsets, while effector and memory CD8+ T-cell populations increased at the expense of exhausted T cells. Interaction analyses predicted enhanced chemotactic and adhesion interactions between CXCL9+ Ly6Chi monocytes and effector CD8+ T cells. Consistently, dynamic imaging revealed increased CD8+ T-cell motility and infiltration into tumor cores following treatment. In particular, CXCR6+ effector CD8+ T cells transiently accumulated within tumor islets during regression before relocalizing to stromal regions. Together, these findings reveal a coordinated spatiotemporal remodeling of myeloid and CD8+ T-cell populations during immunotherapy-induced tumor regression and highlight cooperative interactions that may promote durable anti-tumor immunity.

Interferon regulatory factor 1 (IRF1) has long been recognized as a tumor suppressor; however, recent studies have revealed context-specific and sometimes opposing roles in cancer progression. Here, we describe a T cell-specific mechanism underlying the antitumor activity of IRF1. Unlike germline Irf1-deficient mice, T cell-specific loss of IRF1 does not lead to a deficiency in cytotoxic CD8 T cells. Nevertheless, tumor burden remains elevated in these mice, associated with reduced CD8 T cell infiltration driven by impaired activation and proliferation in the absence of IRF1. Transcriptomic analysis of activated Irf1-deficient T cells identified NFATc1 as a key gene significantly downregulated upon IRF1 loss. Analysis of human melanoma datasets further corroborated this finding, highlighting a previously unappreciated role for IRF1 in regulating T cell activation and antitumor immunity.

Resident dermal macrophages (DMs) play essential roles in maintaining skin homeostasis and initiating inflammatory responses during tissue injury and against infectious agents. However, studies of their cellular mechanisms have been limited by their low abundance in steady-state skin and by technical challenges in isolating resident DMs. Here, we describe the generation and characterization of a novel DM cell line, termed SB89. F4/80+ skin-resident DMs were sorted and immortalized using J2 retroviral transduction. SB89 cells display a stable, homogeneous macrophage phenotype and distinct surface markers compared with Langerhans cells and alveolar macrophages. Functionally, SB89 cells efficiently phagocytose methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, zymosan particles, and apoptotic cells, and effectively kill MRSA. Importantly, SB89 cells respond to LPS, as evidenced by production of IL-6, TNF, and IL-10, and by MRSA-induced production of inflammatory cytokines, chemokines, and eicosanoids. RNA-seq and gene ontology analyses revealed that SB89 cells elicit stronger responses in innate immunity, cell signaling, and epigenetic regulation than immortalized bone marrow-derived macrophages. SB89 cells are genetically tractable, amenable to gene silencing via RNAi and gene introduction via plasmid transfection. Overall, SB89 cells provide a renewable, dermis-imprinted macrophage model that preserves key functional and transcriptional features of resident DMs while reducing reliance on primary cells and animal models. This cell line represents a powerful platform for mechanistic, genetic, and translational studies in skin immunobiology.

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.

Immune dysfunction is a major driver of morbidity and mortality in critical illness syndromes including sepsis. Specifically, CD8+ T cell dysfunction has been linked to organ failure and death. To characterize the immune substructure of circulating CD8+ T cells in critical illness at high dimension, we used single-cell RNA sequencing of peripheral blood CD8+ T cells from 38 critically ill patients and 9 healthy controls. We annotated seven CD8+ T cell clusters, which included a CD8+ effector subset, termed T effector state 2 (TEff-2), that was only present in critically ill patients and associated with more severe respiratory failure and higher mortality. TEff-2 showed effector activation and inflammatory stress conditioning yet had markedly reduced metabolic transcripts without canonical features of exhaustion. Trajectory analyses positioned TEff-2 as a terminal CD8+ T effector cell fate driven in part by DDIT4 and DUSP1, which negatively regulate mTOR and MAPK signaling, respectively. Interestingly, this transcriptional program was indistinguishable by classical protein cytometry methods. These results, including the mortality association, were validated in a larger (n=91) independent external cohort of critically ill patients with sepsis. In summary, TEff-2 represents a latent transcriptional program that delineates a clinically high-risk CD8+ T cell state in critical illness.

Macrophages maintain tissue homeostasis by phagocytosing spent cells, recycling nutrients, and mounting antimicrobial responses to eliminate pathogens. Yet, they can also act as a cellular niche and organize granulomas that enable intracellular bacteria, such as Salmonella enterica, to persist in infected tissues. Here, using a murine Salmonella Typhimurium (STm) infection model, we find that granuloma formation and bacterial persistence are dependent on SPIC, which controls development of VCAM1+ macrophages critical for erythrocyte, heme, and iron recycling. VCAM1+ macrophages markedly increase in infected spleens and have high levels of erythrophagocytosis, intracellular bacteria, and T-cell co-stimulatory ligands. Using SPIC-deficient mice generated from CRISPR gene editing, we show that SPIC is required for macrophage co-stimulatory ligand expression and formation of a VCAM1+ macrophage zone that produces CXCL9 retaining T cells at the granuloma periphery. SPIC deletion abolishes this granuloma cellular architecture and reduces bacterial persistence. We propose that SPIC-dependent erythrophagocytic macrophages drive granuloma formation and bacterial tissue persistence.