Route-dependent effects of GM-CSF on systemic and mucosal myeloid activation in rhesus macaques. Subcutaneous GM-CSF administration induced rapid systemic expansion of myeloid cells, accompanied by chemokine release (MCP-1, CXCL13 and IL-1RA) and enrichment of Fc receptor-bearing myeloid populations (CD64 and CD32) in circulation. These cells subsequently migrated to rectal and vaginal mucosa along chemokine gradients, resulting in sustained mucosal infiltration without overt systemic inflammation. In contrast, topical mucosal GM-CSF delivery elicited no detectable systemic expansion, chemokine induction or myeloid recruitment. Figure made in ©BioRender-biorender.com.

LAG3-MHC-II binding recruits LAG3 to the immune synapse, suppressing T-cell activation through induced formation of condensates between the LAG3 intracellular domain and that of the CD3ε subunit. The BiTS molecule designed by Du et al. mimics the effect of MHC-II binding by tethering LAG3 to the TCR.

Naive CD4 T cells are highly plastic cells that can differentiate into various T helper (Th) cell subsets upon activation. It is well accepted that the vital expression of specific transcription factors and effector cytokines that characterize the different Th cell fates can be stabilized by epigenetic mechanisms including DNA methylation. However, a global view on DNA methylation profiles in Th cell subsets is currently lacking. In this study, we identified the DNA methylomes of human naive T cells, Th1, nonclassic Th1, and Th17 cells by performing a whole-genome bisulfite sequencing analysis. Differentially methylated regions (DMRs) obtained by pairwise comparison of the Th cell methylomes indicate a close relationship between ncTh1 and Th17 cells on a genome-wide level. However, similar methylation patterns at key gene loci such as TBX21, IFNG, SLAMF7, and SLAMF8 may explain the functional proximity of ncTh1 to Th1 cells. Using luciferase reporter assays, we demonstrated that DNA methylation can modulate the transcriptional activity of promoter-located DMRs belonging to genes such as GSPT1, SRSF7, SLAMF7, SLAMF8, TIGIT, and PDCD1. Upon stimulation, SLAMF7 gene expression was upregulated exclusively in IFN-γ producing cells, with SLAMF7 cells appearing among both Th1 and ncTh1 cells. Taken together, the DNA methylomes of proinflammatory human Th cells provide useful data for better functional characterization of the lineages and identification of key genes for therapeutic intervention.

Silicosis is a progressive occupational lung disease marked by persistent silica-induced inflammation and irreversible pulmonary fibrosis. The NLRP3 inflammasome, an innate immune sensor, has been implicated as a key driver of silica-triggered inflammation and fibrosis in preclinical models. However, the specific role of NLRP3 in immune cells, particularly within myeloid cells (monocytes, macrophages and neutrophils), remains poorly defined. In this study, we investigated the in vivo contribution of myeloid-derived NLRP3 to silica-induced lung pathology using a conditional NLRP3 knockout mouse model (LysM Nlrp3). These mice exhibited efficient deletion of NLRP3 in both resident and infiltrating lung myeloid cells. Following intranasal delivery of 2 mg of silica, NLRP3 expression was upregulated in myeloid cells by day 3. Despite upregulation of NLRP3 in myeloid cells by day 3, early inflammasome activation in the tissue and BAL, including caspase-1 cleavage and IL-1β and IL-18 secretion, remained intact. During the chronic phase (days 14 and 28), myeloid NLRP3 deletion did not mitigate hallmark features of silicosis, including alveolitis, structural lung damage, airway remodeling or peribronchial alpha-smooth muscle actin expression. Furthermore, the formation and size of silicotic nodules were unaffected. These findings indicate that NLRP3 expression in myeloid cells is not essential for the development of silica-induced pulmonary inflammation, tissue damage or fibrosis. This work highlights the need to explore alternative cellular sources and mechanisms of NLRP3-driven pathology in silicosis.

Cigarette smoke is a leading cause of lung cancer, promoting disease progression through remodeling of the immune microenvironment. This study explores the impact of cigarette smoke exposure on the mA reader YTHDC2, its role in inducing macrophage senescence, and the consequent formation of a tumor-supportive inflammatory niche in lung cancer. Single-cell RNA sequencing of lung cancer tissues revealed an enrichment of senescent macrophages with decreased YTHDC2 expression in smokers compared to non-smokers. In vitro experiments showed that cigarette smoke extract (CSE) suppressed YTHDC2 expression in macrophages, resulting in enhanced cellular senescence, increased secretion of pro-inflammatory cytokines and M2-like polarization. Overexpression of YTHDC2 attenuated macrophage senescence by regulating RPS8, thereby limiting the formation of a tumor-promoting microenvironment. In vivo studies using a cigarette smoke-exposed lung cancer model confirmed the role of YTHDC2 in smoke-induced immune microenvironment modulation and tumor progression. These findings identify YTHDC2 as a critical regulator of smoke-induced macrophage senescence and the tumor-promoting microenvironment, providing a potential therapeutic target for lung cancer in smokers.

Tumor-associated macrophages (TAMs) play critical roles in the progression of triple-negative breast cancer (TNBC), yet the mechanisms underlying their differentiation remain unclear. Heterogeneity of macrophages in TNBC tissues was comprehensively dissected using single-cell transcriptome analysis. The crucial role of Apolipoprotein C1 (APOC1) in macrophages was investigated through loss or gain-of-function experiments. Single-cell analysis revealed that myeloid cells were the second most metabolically active cell type in TNBC after epithelial cells, with a subset of lipid-associated macrophages (LA-TAMs) potentially linked to TNBC progression. Further analysis showed significant upregulation of APOC1 in myeloid cells and LA-TAMs, with pseudo-temporal expression profiling indicating that APOC1 tended to be expressed in the mid-late stages of macrophage development. KEGG analysis highlighted significant enrichment of APOC1 in glycolysis-related pathways. Cell experiments in vitro demonstrated that macrophages overexpressing APOC1 exhibited enhanced glycolytic activity, a skew toward an immunosuppressive M2 phenotype, and increased secretion of anti-inflammatory cytokines. APOC1-deficient macrophages effectively slowed the progression of TNBC by suppressing the proliferation, migration, and invasion of TNBC cells. These findings suggested that APOC1 promoted macrophage polarization toward the pro-tumor M2 phenotype by activating the glycolytic pathway, thereby facilitating the malignant progression of TNBC. This study provides new insights into the role of macrophages in TNBC and establishes a theoretical basis for developing immunotherapeutic strategies targeting APOC1.

This study assessed how the interaction between human monocyte dectin-1 and β-glucan induces CD83 expression using THP-1 cells as a model. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) were used to assess the dynamics of membrane-bound CD83 (mCD83) and soluble CD83 (sCD83) expression. Insoluble β-glucan induced CD83 expression more effectively than that of soluble β-glucan. Additionally, our findings indicate that the activation of nuclear factor-kappa B (NFκB) and nuclear factor of activated T cells (NFAT) plays a crucial role in the dectin-1 signaling pathway. sCD83 production is driven by metalloproteinases following mCD83 expression and inhibits mCD83 expression. This study offers novel insights into the immunoregulatory role of CD83 and its regulatory mechanisms, highlighting potential strategies for treating fungal infections and autoimmune diseases.

Activated PI3K delta syndrome 1 (APDS1) is caused by a heterozygous germline gain-of-function (GOF) variant in PIK3CD, which encodes the p110δ catalytic subunit of phosphoinositide 3-kinase (PI3K). APDS1 patients display a broad range of clinical manifestations and perturbations in cellular phenotype. One of the most striking features is the dysregulation of the T-cell compartment, characterized by an increase in memory T cells, including Tfh cells, and a concomitant decrease in naïve T cells. We have previously shown that many of these changes in T-cell populations were T-cell extrinsic and were also induced in WT T cells that developed in the presence of PI3K GOF cells. Here we dissected the drivers of dysregulated T-cell activation using a mouse model of APDS1. This revealed that PI3K GOF macrophages and DCs made little contribution to the aberrant T-cell activation. Instead, PI3K GOF T cells were able to drive the loss of WT naïve CD4 T cells, while dysregulated PI3K GOF B cells mediated an increase in Tfh cells. Surprisingly, despite previous reports of increased PI3K signalling driving dysregulated inflammatory Tregs, we saw no evidence for Pik3cd GOF Tregs acquiring an inflammatory phenotype and driving T-cell activation. These studies provide new insights into the role of PI3K in immune cells and how increased PI3K can drive T- and B-cell dysregulation and contribute to the phenotype of APDS1 patients.

Multiple sclerosis is a complex neurological disorder, involving both the adaptive and innate immune systems as well as the CNS. The interaction between these systems is complex, and as such, there is the potential for MS therapies to have conflicting effects in different tissues. It is therefore critical that in addition to tissue-specific studies, system-wide effects of potential therapeutic pathways are explored. The circulating protein Gas6 is a promising therapy to promote remyelination in people with multiple sclerosis. Gas6 is a ligand for the TAM family of receptor protein tyrosine kinases that are widely expressed in the immune system and in the CNS, highlighting the potential for multi-system effects as a result of Gas6 treatment. In this study, we demonstrate that global genetic deletion of either Gas6 or the Gas6 receptor Tyro3 results in reduced disease severity following induction of experimental immune encephalomyelitis in mice. The reduction in severity was accompanied by increased expression of both IL-4 and IL-17A in Tyro3 KO mice lymph node tissue and decreased expression of both cytokines in spinal cord tissues. IL-4 is a cytokine known to be protective in inflammatory demyelination in mice. Conversely, the cytokine IL-17A is known to be pathological. The overall shift to reduced disease severity highlights the multi-faceted role of TAM receptor signaling in inflammatory demyelination.

Tumor rejection is primarily mediated by cytotoxic T cells, making them critical targets for therapeutic cancer vaccines. Vaccine adjuvants can modulate innate immunity, influencing adaptive immune responses. For particulate adjuvants, such as polymeric nanoparticles, physicochemical properties-including size, charge, composition and antigen location within the formulation-can shape these responses. Free-soluble antigens typically fail to induce sufficient dendritic cell maturation and cross-presentation needed for robust CD8 T-cell activation. However, this can be enhanced by delivering antigen with nanoparticles of appropriate size. While adjuvants like oil-in-water emulsions do not require antigen association for vaccine efficacy, the importance of antigen location in the adjuvanticity of polymeric nanoparticles is less clear. We demonstrate that colocalization of antigen and polymeric nanoparticles through antigen adsorption enhances proliferation and activation of antigen-specific CD8 T cells following intramuscular vaccination. While type 1 conventional dendritic cells (cDC1) can prime CD8 T cells in other settings, their requirement with polymeric nanoparticles has not been fully addressed. We show that nanoparticle-induced CD8 T-cell responses rely on cDC1s. The therapeutic efficacy of a polymeric nanoparticle vaccine was significantly enhanced when antigen was adsorbed on nanoparticles, leading to reduced tumor growth and prolonged survival in mice challenged with immunologically hot (MC38) and cold (B16F10) tumors expressing ovalbumin. Furthermore, vaccination with nanoparticle-adsorbed antigen synergized with anti-PD-1 checkpoint blockade, enhancing protection, especially against B16F10-ovalbumin tumors. This work highlights the role of antigen association with polymeric nanoparticles in eliciting CD8 T-cell responses for the development of effective therapeutic cancer vaccines.

T follicular helper (T) cells are a helper T-cell subset that is defined by their localisation to B-cell areas of secondary lymphoid tissues, enabling them to provide their B-cell helper function. Precursors of T cells migrate to the B-cell follicles by upregulating CXCR5 and downregulating CCR7, a process that can be blocked by S1PR1 overexpression. T cells and their precursors also express the early activation antigen CD69, which is a negative regulator of S1PR1. In this study, we tested the hypothesis that CD69 expression by T cells is important for their differentiation and localisation after immunization. Genetic deletion of CD69 on T cells and a proportion of their precursors did not alter their formation, nor their ability to support high-affinity B-cell responses. This demonstrates that although CD69 is expressed highly on T cells, it is not necessary for their formation or their B-cell helper functions in lymph nodes (LNs).

The immune system uses a variety of DNA sensors, including endo-lysosomal Toll-like receptors 9 (TLR9) and cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthase (cGAS). These sensors activate immune responses by inducing the production of a variety of cytokines, including type I interferons (IFN). Activation of cGAS requires DNA-cGAS interaction. Accumulation of cGAMP activates the stimulator of interferon genes (STING), ultimately leading to pathogen clearance by type I IFN production. To prevent the sensing of endogenous nuclear DNA, cGAS is usually localized in the cytoplasm. In this work, we studied the interaction and activation of cGAS by DNA containing non-CpG methyl adducts N3-methyl-C (3mC) and 7-methyl-G (7mG). We report that while DNA with 3mC and 7mG interacts with cGAS, it fails to stimulate its activity in vitro. To gain mechanistic insight, we used synthetic oligonucleotides containing 3mC and 7mG for cGAS activation. We observed that the presence of these adducts was inhibitory to cGAS-catalyzed cGAMP production and type I IFN response in human monocyte cell line THP1. Thus, our study reveals that the specific DNA base methylation adducts 3mC and 7mG contribute to the regulation of cGAS activation and provide a potential strategy for delivering DNA without activating the cGAS pathway.

Advax is a delta inulin polysaccharide adjuvant shown in animal models to enhance and accelerate influenza vaccine protection. A clinical trial was conducted in 109 healthy adult participants aged 18-70 years randomized to receive a single intramuscular seasonal trivalent influenza vaccine (TIV) alone or formulated with 5 or 10 mg Advax adjuvant to explore the effect of the adjuvant on the humoral immune response. The addition of Advax 10 mg to TIV accelerated the rise in serum influenza-specific antibodies, with this group exhibiting significantly higher increases in hemagglutinin inhibition (HAI) against 3 of the 3 vaccine serotypes at 7 days post-vaccination (7 dpv), 2 at 14 dpv and 1 at 21 dpv. By 7 dpv, the Advax 10-mg group achieved HAI seroprotection rates of 96.9% against H1N1, 100% against H3N2 and 46.9% against influenza B versus rates of 86.1%, 100% and 22.2%, respectively, for the TIV alone group. The Advax-adjuvanted groups demonstrated an increased frequency of non-silent CDR3 mutations in the B cell receptor heavy chain of peripheral blood IgG and IgM plasmablasts at 7 dpv, consistent with the adjuvant enhancing B cell affinity maturation in IgM and IgG plasmablasts independently of class switch recombination. The ability of Advax adjuvant to accelerate humoral responses against influenza could be advantageous during influenza outbreaks when time to protection is of the essence. Further studies are needed into the mechanisms whereby delta inulin accelerates vaccine immunity.

CD4 tissue-resident memory T (T) cells are essential for immune protection in the lungs, providing rapid responses against respiratory pathogens. Unlike circulating memory T cells, CD4 T cells persist in the tissue parenchyma and possibly inducible lymphoid tissues, where they facilitate pathogen clearance through cytokine production and interactions with local immune cells. While CD8 T cells are well studied, the role of CD4 T cells in immunity remains less defined and is the focus of this review. Distinct subsets, based on the effector T1, T2, T17 and T follicular helper (T)-like tissue-resident helper (T) cells, contribute to antiviral, antibacterial, antifungal and vaccine-induced immunity. CD4 T cells play a key role in infections, enhancing immune responses and supporting antibody production. However, they are also implicated in chronic inflammation, allergies and fibrosis. Given their importance, vaccines aiming to elicit lung-resident CD4 T cells, particularly via mucosal delivery, have shown promise in inducing long-term protective immunity. Intranasal vaccination strategies, such as live-attenuated influenza virus and tuberculosis vaccines, have successfully generated CD4 T cells, highlighting their potential for respiratory pathogen control. In this review, we focus on CD4 T cells, their differentiation, maintenance and role, especially in the lungs.

Vγ9/Vδ2 T cells represent the largest γδ T-cell population in human blood and possess a unique responsiveness towards microbial organisms by sensing the metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) in the context of the butyrophilin family members BTN2A1 and BTN3A1. Curiously, the bacterium Staphylococcus aureus does not produce HMB-PP but appears to be capable of inducing activation, cytokine expression and proliferation of Vγ9/Vδ2 T cells regardless, through a largely unknown mechanism. We here provide a comprehensive review of the existing literature around Vγ9/Vδ2 T-cell responses to S. aureus and discuss potential pathways, ligands and biological functions.

The role of CD8 T cells, as cytotoxic cells, being critical against intracellular pathogens is well known. Through the killing of infected (target) cells, CD8 T cells impair intracellular pathogens' replication. However, extracellular pathogens are not directly targeted by CD8 T cells, since these pathogens do not express MHC-I-peptides, responsible for the activation of the cytotoxic activity of CD8 T cells. In this sense, how CD8 T cells affect the course of extracellular infections is discussed in this review, underscoring the important regulatory functions of CD8 T cells, killing phagocytes and other cells that are able to cross-present extracellular antigens. In addition, the role of CD8 T cells in the modulation of immune responses through the secretion of cytokines, such as gamma interferon (IFNγ), is also discussed in the context of extracellular infections.

Myeloid cells play critical roles as Fc effector cells in antibody-mediated immunity. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that promotes the recruitment and activation of multiple myeloid populations and has been used in combination with vaccines/treatments against infectious diseases, inflammatory conditions and cancers. To evaluate GM-CSF-mediated kinetics of immune cell expansion and immune outcomes, we compared subcutaneous (subQ) and topical hypoosmolar (intravaginal/intrarectal) administration in vivo using rhesus macaques (RM), as they provide easy access to longitudinal mucosal tissue sampling and are a critical model species for vaccines/therapeutics development. While topical GM-CSF did not result in a major change, neutrophils, eosinophils and monocytes were elevated within 1-3 days of subQ GM-CSF administration, with peak eosinophil and neutrophil enrichment in blood at days 7 and 8, respectively. Corresponding elevations of neutrophils, eosinophils, total CD64 and total CD32 were observed at days 7 and 14 in rectal biopsies, indicating general Fc effector cell accumulation in these animals. Histological evaluations of vaginal biopsies showed myeloid cell infiltration at day 14 of subQ GM-CSF treatment. Further, subQ GM-CSF administration resulted in myeloid cell activation and trafficking, as evidenced by elevated levels of cytokines (CXCL13, MCP-1, IL-1RA). Importantly, neither subQ nor topical GM-CSF administration induced overt systemic inflammation or adverse clinical impacts. Overall, our findings delineate the kinetics of systemic and mucosal myeloid cell expansion, activation and trafficking achieved by subQ GM-CSF administration in RM. These findings will inform the use of GM-CSF as an adjuvant in clinical applications where myeloid cell mobilization is advantageous.

Optic neuritis (ON), characterized by inflammation and demyelination of the optic nerve, is a leading cause of vision impairment. It frequently manifests as the initial symptom and a recurrent syndrome in patients with myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), neuromyelitis optica spectrum disorder (NMOSD) and multiple sclerosis (MS). While the diagnosis of ON is relatively straightforward, predicting relapses and managing the disease remain significant challenges. The myelination of retinal ganglion cell axons is essential for the efficient and accurate transmission of signals between optic neurons. Oligodendrocytes, specialized glial cells responsible for myelination, engage in metabolic interactions with neurons. Demyelination and inflammation of the optic nerve locally release a variety of metabolites and inflammatory factors within this unique anatomical region. Cerebrospinal fluid (CSF), in close proximity to ON lesions, is a critical source for identifying metabolic and inflammatory biomarkers that are essential for tracking disease activity and guiding therapeutic decisions. This comprehensive review examines the structure, myelination and demyelination of the optic nerve, as well as the immunopathological mechanisms underlying ON. It also explores changes in CSF constituents, including pleocytosis, antibodies, cytokines, proteins, metabolites and extracellular vesicles. Special emphasis is placed on elucidating the pathological contributions of metabolites in MOGAD, MS and NMOSD. By advancing our understanding of these mechanisms, this review elucidates the potential predictive roles of CSF constituents in acute attacks and relapses of optic neuritis.

Early career researchers (ECRs) are often faced with uncertainty about their professional futures, a challenge exacerbated by the increasing pressures within the academic research landscape. As ECRs navigate their next steps in science, mentorship is crucial, particularly as they face points of decision-making and possible career diversions from the traditional postdoctoral-to-professor pathway. In response to these challenges, the second iteration of the Australian and New Zealand Society of Immunology (ASI) Mentor-Mentee Program aimed to provide mentorship and training to ECRs about academic career pathways in research and education to bridge the professional communities, values and advice of these two often independent career choices. As a component of the program, three eminent Australian immunologists in research-intensive, teaching and research (TnR) and education-focused careers shared their professional journeys and experiences which led to their chosen career pathways in national workshops. Here, we share their insights, lessons learned and professional development tips to establish an academic career. By outlining the three primary scientific academic career pathways available post-PhD, we aim to inform and inspire the next generation of immunologists as they consider the diversity of possible academic careers ahead.