Mucosal Immunology

Staphylococcus aureus persistently colonises the nasal tissue (NT) of a significant proportion of the population. The long-lasting impact that asymptomatic S. aureus exposure has on immune memory at colonised barrier sites is incompletely understood, potentially impacting vaccine responsiveness in a pre-exposed population. Tissue resident memory (TRM) cells are long-lived T-cells which remain poised at barrier sites for localised reactivation following antigen exposure. This study demonstrates an increase in NT CD4+ and {gamma}{delta}+ TRM cells in response to S. aureus colonisation, which undergo expansion and IL-17 production upon secondary S. aureus exposure. Interestingly, these cells were also capable of non-specific reactivation, with IL-17+ TRM cells in S. aureus colonised mice enhancing protection against K. pneumoniae infection. Ex-vivo data suggest that non-specific CD4+ TRM cell re-activation is pro-inflammatory cytokine dependent, but antigen independent. Overall, these findings demonstrate that S. aureus nasal colonisation shapes long-lasting TRM cell responses in the NT, which have the potential for non-specific bystander reactivation during subsequent heterologous infection.

Biological sex is a major determinant of antiviral immunity, yet how it shapes lung-resident antiviral immunity remains poorly understood. We report that prior mild SARS-CoV-2 BA.1 infection protects mice from lethal heterologous Delta challenge through distinct sex-biased immune mechanisms. Although heterologous protection is clinically equivalent between sexes, T- and B-cell depletions before BA.1 exposure abrogate protection only in males. Depletion with anti-CD4, but not anti-CD8, abolishes protection in both sexes, but co-depletion with anti-CD4/anti-CD8 partially restores protection in females only. Further, females exhibit enhanced IFN-gamma-associated myeloid inflammation upon secondary Delta infection. Using lymphoid-deficient mice that display female-biased resistance to Delta infection, IFN-gamma treatment provides partial protection to naive males against Delta. Female resistance is lost after previous BA.1 exposure, highlighting the importance of lymphoid-mediated inflammatory regulation. Together, equivalent antiviral protection against heterologous respiratory viral infection is governed by sexually dimorphic mechanisms, with significant implications for designing mucosal vaccines and immunotherapies.

Multiple sclerosis (MS) is a chronic neuroinflammatory disease characterized by demyelination, neurodegeneration, and progressive neurological disability. Galanin, a neuropeptide with immunomodulatory properties, signals through G protein-coupled receptors, among which galanin receptor 2 (GALR2) has been implicated with neuroprotective and anti-inflammatory functions. A rare homozygous single nucleotide variant in GALR2 (rs61745847; p.W249L) has been identified in a patient diagnosed with relapsing-remitting MS, however, the biological relevance of this variant in neuroinflammation remains unknown. Here, we investigated the impact of the orthologous GALR2 W248L mutation using a knock-in mouse model and experimental autoimmune encephalomyelitis (EAE). GALR2 W248L knock-in (KI) mice exhibited a more severe clinical course of EAE, accompanied by enhanced inflammatory infiltration, exacerbated demyelination, and increased microglial activation in the spinal cord compared with wild-type (WT) mice. Despite comparable lymphoid and myeloid cell frequencies in the central nervous system, alterations in microglial density and morphology suggested an important contribution of the innate immune system to disease exacerbation in the KI mice. Ex vivo analyses revealed that bone marrow-derived macrophages from KI animals exhibited a pronounced shift toward a pro-inflammatory phenotype, characterized by enhanced M1 polarization, impaired M2-associated responses, and increased NLRP3 inflammasome activation. In parallel, live-cell imaging of primary hippocampal neurons demonstrated reduced galanin binding in mutant cells, consistent with impaired GALR2 functional availability at the plasma membrane. Together, these findings identify GALR2 as a modulator of the neuroinflammatory response and indicate that disruption of galanin-GALR2 signaling promotes sustained innate immune activation, highlighting the relevance of this pathway for MS pathogenesis and its potential as a therapeutic target in neuroinflammatory disorders.

Allograft inflammatory factor-1 (AIF1) is a scaffold protein expressed predominantly in myeloid antigen-presenting cells (APCs) and associated with inflammatory disease. Although genetic studies have linked AIF1 loci to immune traits, its causal role in physiological immune responses remains poorly defined. We examined AIF1 deficiency using a conditional knockout model with deletion of AIF1 in hematopoietic cells (AIF1-cKO) during development and challenged mice with Listeria monocytogenes. AIF1 loss impaired bacterial clearance and diminished inflammatory responses, indicating reduced immune readiness during infection. AIF1-cKO mice exhibited impaired expansion of antigen-specific CD4+ and CD8+ T cells, accompanied by regulatory-associated phenotypic changes. These defects were associated with reduced cDC1 frequencies and transcriptional and phenotypic remodeling of splenic macrophages toward a less inflammatory state. Single-cell RNA sequencing revealed transcriptional alterations across multiple myeloid and lymphoid compartments despite AIF1 expression being largely restricted to myeloid APC, indicating broader immune remodeling. Increased Tgfbr1 expression was a recurrent feature across several immune populations. Consistent with this finding, AIF1-deficient cells displayed enhanced TGF{beta} responsiveness, while Tgfbr1 silencing partially restored inflammatory responses and T cell priming ex vivo. These findings establish AIF1 as a regulator of immune competence that promotes effective innate and adaptive immune responses.

Constitutive androstane receptor (CAR; encoded by Nr1i3) is a nuclear xenobiotic receptor that mediates hepatic drug and bile acid metabolism. We previously identified CAR as also operating in CD4+ T helper (TH) cells, where CAR-dependent gene expression mitigates bile acid toxicity and promotes a Foxp3-IL-10+type 1 regulatory (Tr1)-like phenotype in the small intestine. Here, we show that CAR acts early and specifically during the priming of type 1 immune responses to stabilize Tr1 lineage commitment. CAR-dependent Tr1 cells formed during type 1 (Th1-associated) but not type 3 (Th17-associated) intestinal inflammation. In vitro, IL-27 upregulated CAR expression during naive TH cell activation, which contributed to Il10 induction. Single-cell analysis revealed that naive TH cells primed with IL-27 adopt a multipotent "Th1/Tr1 precursor" (THR1p) transcriptional state, which subsequently diverges into Th1 or Tr1 developmental trajectories. CAR transcriptional activity peaked in THR1p cells, upregulating Tr1 genes, including Il10, and repressing Th1 genes. Moreover, glucocorticoid receptor activation--which increases CAR expression in hepatocytes--synergized with IL-27 to augment both CAR expression and CAR-dependent Tr1 differentiation. Together, these results suggest that CAR acts in a lineage-biased manner to enforce Tr1-mediated immune tolerance during type 1 intestinal inflammation, and this pathway is amplified by glucocorticoids.

Background and Aims: Fibroblast-neutrophil interactions are implicated in ulcerative colitis (UC) treatment resistance, but whether fibroblasts modify neutrophil inflammatory activity remains unclear. We investigated responses to fibroblast exposure, stromal depletion, and 5{beta}1-directed treatment. Methods: We integrated human and mouse colonic single-cell RNA sequencing (scRNA-seq), spatial protein profiling (CODEX), fibroblast-neutrophil coculture, and dextran sulfate sodium (DSS) mouse colitis with fibroblast activation protein (FAP) ablation or 5{beta}1-directed treatment. Readouts included cellular representation and protein fluorescence via flow cytometry, neutrophil extracellular trap (NET)-associated elastase activity, RNA programs and epithelial responses using scRNA-seq. Results: Inflamed UC tissue contained higher fractions of inflammatory fibroblasts and oncostatin M (OSM)- and CXCR4-associated neutrophils. Inflammatory fibroblasts had higher FAP-5{beta}1 module scores, and proximity to FAP-high fibroblasts was associated with higher neutrophil OSM and CXCR4 fluorescence. UC fibroblasts increased neutrophil OSM, CXCR4 and myeloperoxidase (MPO) fluorescence and NET-associated elastase activity relative to control fibroblasts, while 5{beta}1-directed treatment attenuated these responses. Both mouse FAP ablation and 5{beta}1 blockade in DSS colitis reduced histologic inflammation and overall neutrophil frequency. Ablation broadly reduced recovered neutrophil representation, whereas blockade increased OSM-positive and PADI4-positive percentages within neutrophils despite lower MPO fluorescence within these subsets. Both interventions were associated with lower epithelial chemokine scores but distinct absorptive and mucus/secretory responses. Conclusions: Fibroblast exposure modifies neutrophil inflammatory phenotype and effector-associated activity, identifying candidate therapeutic pathways in UC. Evaluating these responses alongside neutrophil representation and tissue inflammation could inform pharmacodynamic assessment of stromal-directed therapies.

Pulmonary vaccination has been proposed as a strategy to improve protection against tuberculosis, due to the generation of immune cells that more efficiently survey and eliminate infected cells within the lung. However, uncontrolled replication and excessive inflammation associated with mucosal delivery of live-attenuated vaccines highlight the need for alternative strategies that balance efficacy and safety. Here, we describe a prime-and-pull vaccination approach in which systemic immunity is established by subcutaneous BCG vaccination, followed by the induction of local lung immunity through mucosal delivery of lipid nanoparticle-formulated multi-antigen mRNA. Proof-of-concept studies using a model antigen demonstrated the induction of polyfunctional antigen-specific T cells in the lung with minimal inflammatory cell infiltration, compared with mucosal BCG vaccination. Eight Mycobacterium tuberculosis- and BCG-derived proteins were subsequently selected to generate four multi-antigen mRNA constructs. In vivo vaccination and challenge experiments demonstrated that this prime-and-pull strategy is well tolerated and confers protective immunity against tuberculosis in a murine model. Collectively, these data support a modular mRNA-based prime-and-pull vaccination strategy as a translational approach that bridges the improved immunogenicity and efficacy of mucosal BCG vaccination with the safety profile of parenteral BCG administration. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/739464v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@d5f991org.highwire.dtl.DTLVardef@1f3c4f9org.highwire.dtl.DTLVardef@108cae6org.highwire.dtl.DTLVardef@40d60f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Intraepithelial lymphocytes expressing the {gamma}{delta} T cell receptor ({gamma}{delta} IEL) continuously survey the intestinal epithelium to promote mucosal host defense. Although {gamma}{delta} IELs migrate in and out of the lateral intercellular space (LIS) between adjacent enterocytes, the molecular mechanisms governing their migratory behavior are incompletely understood. Based on the known role of CD47, or integrin associated protein (IAP), in mediating neutrophil transepithelial migration, we investigated whether CD47 expression reflects a conserved mechanism regulating {gamma}{delta} IEL surveillance behavior. Here, we report that conditional CD47 deletion on intestinal epithelial cells or {gamma}{delta} T cells had no effect on IEL composition. Using intravital imaging, we identified complementary roles for CD47 on {gamma}{delta} IELs and epithelial cells, with epithelial CD47 restricting {gamma}{delta} IEL motility and {gamma}{delta} T-cell-derived CD47 promoting cell migration. Further investigation revealed that both CD47 and CD18 contribute to {gamma}{delta} IEL surveillance behavior, although CD47 regulates {gamma}{delta} IEL migration in a CD18-independent manner.

Background: Patients receiving anti-TNF treatment for chronic inflammatory disease display impaired antibody responses, but it remains unclear how immune memory formation is affected. We evaluated antibody responses and memory B cells (Bmem) after COVID-19 booster vaccination in inflammatory bowel disease (IBD) patients receiving anti-TNF treatment. Methodology: Blood was sampled at baseline, 1, and 6 months after WH1/BA.5 bivalent or XBB.1.5 monovalent vaccination from 27 IBD patients receiving intravenous anti-TNF and 44 controls. Neutralizing antibodies were measured using an infectious virus assay. SARS-CoV-2 spike receptor binding domain (RBD)-specific serum IgG was quantified by ELISA, and RBD-specific Bmem were immunophenotyped by flow cytometry using recombinant proteins from ancestral, Omicron BA.1, BA.5, XBB.1.5, and JN.1 variants. Results: Serum IgG to vaccine RBD and neutralizing antibodies in patients increased pre to 1 month post-vaccination, but were lower than controls. Ancestral-, BA.5- and XBB.1.5-specific Bmem increased after vaccination but were significantly lower in patients than controls. Within RBD-specific Bmem, frequencies of recently activated CD21lo cells were increased after vaccination, and were higher in patients than controls. Fewer antigen-specific Bmem in patients expressed IgG4, and more expressed IgG3 or IgD following vaccination. Following vaccination, more RBD-specific Bmem recognized multiple viral variants. However, patients had fewer Bmem that could bind to subvariants than controls. Conclusion: Antibody and Bmem responses to COVID-19 booster vaccination in anti-TNF-treated IBD patients displayed reduced capacity, durability and cross-reactivity, suggesting impaired immune memory for protection against breakthrough infection. This supports the recommendation for annual booster vaccination to prevent severe disease and viral spread.

Intestinal organoids provide physiologically relevant models of the epithelial barrier, but lack the immune compartment that critically shapes host responses to infection. Here, we established a human colon organoid-derived monolayer co-culture system with macrophage-like THP-1 cells positioned directly beneath the epithelial layer. The model enabled controlled apical infection with Listeria monocytogenes and Salmonella Typhimurium while preserving epithelial barrier integrity. PMA-differentiated THP-1 cells reduced intracellular L. monocytogenes burden, whereas additional activation with IFN-{gamma} and LPS resulted in a pronounced reduction of both L. monocytogenes and S. Typhimurium, accompanied by decreased infection-associated cytotoxicity. Bulk RNA sequencing revealed a distinct co-culture transcriptional signature characterized by coordinated changes in inflammatory, antimicrobial, and epithelial lineage-associated programs. These included reduced HLA-D/MHC class II-associated gene expression, altered S100A8/S100A9 expression, and changes in epithelial lineage markers indicating a shift in epithelial cellular composition and differentiation state. Together, these findings establish a versatile human organoid-macrophage platform for dissecting epithelial-immune interactions and macrophage-associated control of invasive bacterial infection.

The transcriptional and cell repertoire changes of lymph node (LN) T cells following influenza vaccination have been under-investigated across different ethnicities. Draining (dLN) and non-draining (ndLN) axillary LN cells from 17 participants with genotypically varied African or Asian ancestry (ISRCTN13657999) were collected by fine needle aspiration (FNA) before, 5 days and 42 days after adjuvanted influenza vaccination and analysed by single-cell RNA-sequencing (scRNA-seq). The early dLN transcriptional profile and cellular dynamics were distinct from the ndLN; BACH2 expressing T cells were highly induced, exhibiting stem-like and unique quiescence signatures. Proliferating T follicular helper cells (Tfh) were enriched in dLN and Th1-like Tfh predominated with highly induced transcriptional changes. The BACH2+ T cell state and Th1-like Tfh cells, transcriptionally distinct from germinal centre (GC) T cells, were co-localised with LAMP3+ dendritic cells in the T cell zone of publicly available LN datasets, a region that was also distinct from GC Tfh niches. Together, these findings indicate that the induction of a robust, proliferative, and transcriptionally diverse Tfh response, alongside a discrete subset of BACH2+ T cells, represents a marked feature of the early T cell zone immune response to adjuvanted influenza vaccine in the dLN.

Group 2 innate lymphoid cells (ILC2s) regulate type 2 immune responses partly by recruiting eosinophils, but the mechanisms underlying this process remain incompletely understood. Although both ILC2s and type 2 T helper (Th2) cells are capable of expressing IL-5, a cytokine critical for recruiting eosinophils, ILC2s are more potent than Th2 cells in this process. Here, we show that the transcription factor Bhlhe40 promotes GM-CSF production in ILC2s, and Bhlhe40 expression in ILC2s is required for efficient eosinophil recruitment during both papain- and helminth-induced type 2 immune responses. However, Bhlhe40 deficiency in ILC2s had no effect on the production of classical type 2 cytokines, including IL-4, IL-5 and IL-13, despite Bhlhe40 being required for type 2 cytokine production by Th2 cells. Furthermore, in contrast to ILC2s, Th2 cells produced little GM-CSF and administration of GM-CSF rescued eosinophil recruitment in ILC2-specific Bhlhe40-deficient mice. In addition to promoting GM-CSF expression, Bhlhe40 repressed IL-10 production in ILC2s as it did in Th2 cells, particularly during chronic inflammation. Single-cell transcriptomic analyses further supported this regulatory network, and the ChIP-Seq data revealed direct binding of Bhlhe40 to the enhancer and promoter regions within the Il10 and Csf2 loci. Collectively, our findings show that Bhlhe40 exerts distinct gene regulatory functions in ILC2s and Th2 cells, and Bhlhe40 modulates the inflammatory and anti-inflammatory functions of ILC2s by promoting GM-CSF while suppressing IL-10 production.

Substantial inter-individual variation in susceptibility to viral infection persists despite widespread vaccination, and its immunological basis remains poorly understood. We investigated how innate and adaptive immune responses contribute to susceptibility to SARS-CoV-2 infection during the early COVID-19 pandemic. We compared two groups of vaccinated individuals who either remained uninfected or became infected during the first Omicron wave. Blood samples were collected at baseline and 24 weeks later. Peripheral blood mononuclear cells (PBMCs) and polymorphonuclear neutrophils (PMNs) were isolated and stimulated with the TLR7/8 agonist R848 to assess innate responses. PBMCs were stimulated with SARS-CoV-2 peptide pools and highly purified inactivated viruses (ancestral and Omicron BA.1) to evaluate adaptive immunity. Prior to infection, individuals in the infected group exhibited reduced CD4 and CD8 T cells proliferative responses, alongside with increased TNF production across all stimulation conditions, despite largely comparable immune phenotypes, indicating a pre-existing functional immune deficit. Following infection, T-cell proliferation and IFN-gamma production were partially restored in response to viral antigens, although responses to Omicron BA.1 remained suboptimal. This functional deficit was accompanied by heightened inflammatory activity, including increased TNF and IFN-gamma production, elevated anti-nucleocapsid IgG3 levels, higher frequencies of B cells and myeloid cells, reduced circulating interferon-inducible T-cell Alpha Chemoattractant (I-TAC) concentrations, and a modest impairment in PMN IL-8 responses. Notably, these alterations were detectable prior to infection and persisted thereafter, indicating that they represent determinants rather than consequences of viral infection. Importantly, beyond differences in the magnitude of immune responses, protection was associated with the degree of functional coordination within the humoral compartment, as reflected by the relationship between Spike-binding antibodies and neutralizing activity. Together, these results demonstrate that susceptibility to Omicron infection is linked to a pre-existing and persistent functional immune imbalance affecting both innate and adaptive arms of immunity.

Interactions between intraepithelial lymphocytes (IELs) and the intestinal epithelium are central to mucosal homeostasis and disease. However, mechanistic in vitro studies describing their crosstalk in humans are limited by scarceness of primary material and insufficient knowledge about co-culture requirements. Here, we establish an autologous human duodenal IEL-organoid co-culture system encompassing expandable and bankable IEL and organoid protocols, with co-culture conditions that allow viability of both cell types. This system enables successive interrogation of lympho-epithelial interactions starting from minimal biopsy material. Under baseline conditions, CD45CD8CD103TCR{beta} IELs retain tissue-residency and effector features and induce an epithelial interferon response and chemokine production, without overt epithelial apoptosis. IL-15 and IL-21, essential cytokines involved in IEL-activation in intestinal enteropathies like celiac disease, increases granzyme B expression and interferon-{gamma} secretion but do not trigger epithelial cell death. However, enforcing IEL-epithelial contact using an anti-CD3-anti-Ep-CAM bispecific antibody induces epithelial apoptosis accompanied by increased tumor necrosis factor (TNF) and FAS-ligand (FASLG) secretion. These findings validate the platforms ability to resolve non-destructive and cytotoxic lympho-epithelial interaction and provide a tractable system for studying intestinal inflammation and immune-mediated epithelial cell death.

Developmental programs establish tissue identity and organization, but how their reactivation contributes to adult tissue responses remains poorly understood. Here, we identify a functional contribution of apolipoprotein A-I (APOA1), which marks a late fetal colonic epithelial state that reappears after injury. Spatial and temporal profiling localized Apoa1 transcription predominantly to epithelial cells at the tips of developing proximal colonic folds. Apoa1 loss delayed fold maturation and altered the abundance and composition of developing enteroendocrine populations, linking this apolipoprotein to mucosal architecture and epithelial differentiation. APOA1 protein closely interacted with WNT5A during development and injury, while Apoa1 deficiency increased Wnt5a expression and altered its epithelial protein distribution. Following colonic injury, single-cell and spatial profiling identified genotype-associated transcriptional changes involving stress responses, barrier and immune functions, and metabolism, with a shared component across males and females. These changes accompanied reduced enteroendocrine representation and increased representation of inflammation-associated macrophages. Finally, regional and compartmental expression analyses, together with hindgut explant perturbation, identified HOXB7 transcription factor as a regulator of developmental Apoa1 expression. Together, these findings establish functional relevance for a component of a reactivated late fetal epithelial program, connecting APOA1 to colonic maturation and the epithelial and inflammatory organization of injured tissue.

Herpes zoster (HZ), caused by varicella zoster virus (VZV) reactivation, is common in older adults with declining cell-mediated immune protection. A modestly efficacious live attenuated (ZVL) and a highly efficacious recombinant adjuvanted (RZV) zoster vaccine protect against HZ. To uncover the mechanism(s) associated with the superior efficacy of RZV, we developed a controlled human infection model consisting of intradermal administration of attenuated vOka VZV to older adults previously vaccinated with RZV or ZVL. Infiltration of VZV-specific CD4+ and CD8+ T cell clonotypes at the infection site was quicker and higher in RZV than ZVL recipients [≥]5 years post-immunization due to the persistence of RZV-induced gE-reactive CD4+ and CD8+ T cell clonotypes in blood. High CD4+ cytotoxic T lymphocyte frequencies in blood on Day 1 post-inoculation predicted control of VZV replication. Persistence of circulating RZV-induced CD4+ and CD8+ CTL and rapid migration to sites of VZV replication are potential mechanisms underlying durable protection against HZ.

Inflammatory bowel disease (IBD) often involves disrupted intestinal epithelial barrier, but therapies specifically targeting this barrier are limited. We found that downregulated AUF1 (HNRNPD) contributes to defective barrier integrity in ulcerative colitis (UC). Compared to controls, its expression level was decreased and inversely correlated with clinical severity. Knocking down AUF1 in human and mouse colonic organoids led to impaired barrier function, with reduced Occludin and upregulated Claudin-2, mimicking characteristic IBD-associated mucosal alterations. Distinct RNA-binding activity of AUF1 protein isoforms contributed to these changes: p37 stabilized Occludin mRNA and blocked microRNA-122/Ago2-mediated repression, whereas p40 promoted Claudin-2 mRNA degradation via ubiquitin-proteasome pathway. Restoring AUF1 expression in organoids enhanced epithelial properties and, when transplanted into mice with established colitis, accelerated mucosal healing and epithelial regeneration in recipient mice and decreased fibrosis. Our study unravelled a post-transcriptional mechanism important for intestinal homeostasis and demonstrated a concept of using engineered organoids for treating IBD.

Protective immunity against many infectious diseases develops following primary infection, called infection induced immunity (III), and provides a blueprint for vaccination. However, many vaccination strategies have failed. In the parasitic Leishmania major model of self-healing cutaneous disease, control of secondary challenge infection relies on pre-existing T helper (Th)1-dependent activation of skin-infiltrating monocytes for elimination of intracellular parasites. To better understand immune-evasion of pre-existing Th1 immunity by pathogens, we investigated the pathogen-niche established following non-healing challenge infection with the L. amazonensis parasite in a setting of pre-existing III. Following secondary challenge, pre-existing Th1 III initially controlled infection but ultimately failed. Loss of protection was not overtly STAT6- or IL-10-mediated. Rather, monocyte-lineage tracing revealed inflammatory monocyte-derived PD-L1+PD-L2+ macrophages provide an intracellular pathogen-niche and facilitate evasion of pre-existing Th1 immunity. Anti-PD-1 immune checkpoint blockade enhanced uninfected, but not infected, monocyte-derived cell activation and depletion of monocyte-derived precursors improved parasite control. These observations suggest that evasion of pre-existing Th1 immunity in this setting is not due to a failure of the Th1 response, but rather due to infected-cell intrinsic defects in activation.

Tuberculosis (TB) disease consistently causes the highest number of annual deaths from infectious disease and is responsible for over 100 million disability-adjusted-life-years annually. Despite the dramatic impact on human health, TB treatments are intensive and moderately effective, while Bacille Calmette-Guerin (BCG), the primary TB vaccine strategy and the oldest vaccine in circulation, needs updating. One gap in knowledge is that the vaccine primed lung immune environment has not been systematically profiled following TB challenge, making it challenging to interpret how various vaccine strategies provide protection. Here we profiled the male and female mouse lung immune environment using Mass Cytometry by Time of Flight (CyTOF) and bacterial burden (colony forming units, CFU), to systematically identify immune cells contributing to Mycobacterium tuberculosis (Mtb) infection or vaccine-induced protection. We investigated the mouse immune system after various infection outcomes including primary Mtb infection, Mtb reinfection, Mtb challenge after BCG vaccination and Mtb challenge after vaccination with a live attenuated Mtb vaccine. Across all conditions we identified Thy1.2 expression on NK cells as a new candidate for a sex-specific correlate of protection. Our results further replicate and support known immune responses for vaccine-induced protection, such as T-cell activation and infiltration. These findings highlight the pan-immune response to vaccine-induced protection, showcase the importance of sex in rodent models of TB, and identify novel host immune candidates to protect the lung against TB disease.

Cutaneous leishmaniasis is characterized by chronic inflammatory skin lesions in which CD8{square} T cells exhibit paradoxical functions. While IFN-{gamma}-producing CD8{square} T cells contribute to the development of protective immunity in the draining lymph node, CD8{square} T cells recruited to the infected skin lose their ability to produce IFN-{gamma} and instead acquire cytotoxic functions that exacerbate tissue damage. We previously demonstrated that the hypoxic microenvironment of leishmanial lesions promotes CD8{square} T cell cytotoxicity through induction of Blimp-1. Whether hypoxia also suppresses protective CD8{square} T cell functions, however, is unknown. Here, we show that hypoxia simultaneously suppresses production of the protective cytokines IFN-{gamma} and TNF- while enhancing expression of granzyme B and perforin in activated CD8{square} T cells. In vitro, HIF-1, but not HIF-2, was required for hypoxia-induced expression of granzyme B, perforin, and Blimp-1, whereas suppression of IFN-{gamma} and TNF- occurred independently of HIF signaling, indicating that distinct oxygen-related pathways regulate pathogenic and protective CD8{square} T cell functions. Hypoxia also increased expression of multiple inhibitory receptors on CD8{square} T cells, although lesional CD8{square} T cells lacked expression of the terminal exhaustion-associated transcription factor TOX, suggesting that hypoxia promotes an inhibitory phenotype distinct from terminal exhaustion. Finally, adoptive transfer studies demonstrated that in vivo both HIF-1 and HIF-2 expression in CD8{square} T cells contributed to immunopathology during cutaneous leishmaniasis. Together, these findings identify hypoxia as a key regulator that functionally reprograms CD8{square} T cells by promoting pathogenic cytotoxicity while suppressing protective cytokine production within lesions.