InVivoMAb mouse IgG1 isotype control, unknown specificity

CD4 tissue-resident memory T cells (TRM) are crucial adaptive immune components involved in preventing influenza A virus (IAV) infection. Despite their importance, their physiological role in the upper respiratory tract, the first site of contact with IAV, remains unclear. Here, we find that, after IAV infection, antigen-specific CD4 TRM persist in the nasal tissue (NT) compartment after infection and provide protection upon heterosubtypic challenge. Single-cell RNA-sequencing analysis reveals that NT CD4 TRM are heterogeneous and transcriptionally distinct as compared with their lung counterparts. Mechanistically, we demonstrate that the CXCR6-CXCL16 axis promotes CD4 TRM residency in the NT. Furthermore, we show that the NT of mice and humans contains a high frequency of Th17 CD4 TRM that aid in local viral clearance and in reducing tissue damage. Collectively, our results support a robust physiological role for NT CD4 TRM in local protection during heterosubtypic IAV infection.

High-grade serous ovarian cancer (HGSOC) is a lethal malignancy characterized by therapy resistance. Focal adhesion kinase (FAK) is highly expressed in HGSOC, yet its impact on tumor-immune communication remains incompletely defined. Using three syngeneic ovarian cancer models, we show that FAK inhibition (FAKi) increased macrophage CXCL13 expression and promoted peritoneal B cell infiltration. Combining FAKi with low-dose pegylated doxorubicin and anti-T cell immunoreceptor with Ig and ITIM domains (TIGIT) checkpoint blockade suppressed orthotopic ovarian tumor growth, extended survival, and induced tertiary lymphoid structures. Macrophage lineage factor GATA6 inactivation reduced CXCL13 expression, enhanced FAK-knockout tumor growth, and limited ascites B cell accumulation. Mechanistically, FAKi-treated or FAK-deficient tumor cells release exosomes enriched in omega-3 fatty acids that stimulated macrophage CXCL13 production. Exposure of macrophages to tumor-derived omega-3 lipids or eicosapentaenoic acid induced anti-tumor reprogramming and CXCL13 expression. Together, these findings reveal a tumor lipid-macrophage signaling axis activated by FAKi that supports B cell recruitment and anti-TIGIT immunotherapy.

Recurrence is a major factor limiting the long-term survival of patients with intrahepatic cholangiocarcinoma (ICC). The molecular characteristics and potential therapeutic targets in ICC remain largely undefined.

Modification of antibodies to chemically couple labels or immobilization reagents is essential for developing biosensors. Typically, conjugation occurs through chemical methods that leverage reactive amines and thiols on native antibodies; however, this nonspecific approach can interfere with antibody function. Microbial transglutaminase (mTG) is an enzyme that has been used for site-specific conjugation of chemical modifiers to the Fc region of native antibodies, but thus far mTG-mediated conjugation has been limited to production of antibody-drug conjugates with human IgGs. Here, we assessed the scope and versatility of mTG to target IgGs, with the goal of site-specific conjugation to facilitate oriented immobilization. A fluorescently labeled peptide was conjugated to several IgG host species and subclasses commonly used to produce monoclonal (e.g., mouse IgG1 and rat IgG1) and polyclonal (e.g., rabbit IgG and goat IgG) antibodies. SDS-PAGE confirmed site-specific conjugation of the peptide to each of these IgG subclasses. In addition, NH2-PEG4-biotin was chemo-enzymatically installed on the Fc region of each tested IgG, as confirmed by Western blot analysis. Site-specific biotinylated antibody was immobilized on a streptavidin-coated substrate to evaluate antigen binding activity in a functional assay. The site-specific conjugation of biotin enabled the formation of an oriented capture antibody layer to enhance antigen binding when compared to the performance of a functional assay constructed by immobilizing a randomly biotinylated antibody prepared by conventional chemical conjugation. These results highlight the broad scope of mTG to site-specifically conjugate native antibodies to improve analytical performance of biosensing platforms.

Interleukin-17A (IL-17A) aggravates poststroke neurological damage and enhances neutrophil extracellular traps (NETs) formation, yet the underlying mechanism remains unclear. This study aimed to elucidate how IL-17A regulates NETs generation after ischemic stroke.

Cutaneous leishmaniasis, caused by protozoan parasites of the Leishmania genus, remains a significant health concern in endemic regions such as the Middle-East, Asia, Latin America, and North Africa. The disease affects millions of people worldwide, with over one million new infections reported annually. Despite its health impact, there is currently no approved vaccine largely due to limited understanding of immunological mechanisms underlying protective immunity and disease pathogenesis. We previously reported that long pentraxin 3 (PTX3), a pattern recognition molecule involved in inflammation, tissue repair, and wound healing, is a negative regulator of immunity in primary Leishmania major infection. Specifically, we showed that PTX3 exacerbates disease by suppressing protective Th17 responses. Here, we extend these findings by showing that PTX3 also influences secondary (memory) immunity to L. major. PTX3-deficient (PTX3-/-) mice which had resolved a primary infection exhibited enhanced resistance to secondary challenge compared to their wild-type (WT) controls. This enhanced resistance correlated with higher frequencies of effector memory CD4+ T cells in the spleens and draining lymph nodes. Upon re-infection, healed PTX3-/- mice produced significantly more IL-17A, while levels of IFN-γ, TNF-α, and IL-10 were similar. In vivo BrdU incorporation assays further revealed increased proliferation of IL-17+ CD4+ T cells in PTX3-/- mice. Importantly, neutralization of IL-17A during secondary challenge abolished the enhanced resistance observed in PTX3-/- mice, confirming a central role of IL-17 in PTX3-regulated secondary immunity. Collectively, our findings identify PTX3 as a key regulator of secondary immunity in cutaneous leishmaniasis and underscores the importance of IL-17 in this process.

Atopic diseases associated with allergens, as well as allergic diseases, frequently arise early in life; however, the age-dependent mechanisms governing immune responses to allergens remain poorly understood1. Here we find that in early life, exposure to common allergens triggers a distinct bifurcated immune response, simultaneously triggering type 17 inflammation in the skin and initiating canonical T helper 2 sensitization in the lymph nodes. This early-life γδ type 17-mediated dermatitis primes the exaggerated allergic lung inflammation upon secondary allergen exposure. Mechanistically, we find dendritic cell (DC)-mediated type 17 activation directly in the skin without requiring migration to lymph nodes; we term this state 'peripheral immune inducer' (pii) DC. CD301b+ conventional type 2 DCs acquire allergen, adopt the pii-DC state, produce IL-23 and activate local γδ type 17 cells independently of lymph-node engagement. The pii-DC state is enabled by the immature hypothalamic-pituitary-adrenal axis and physiologically low systemic glucocorticoids characteristic of early life2,3; DC-specific deletion of the glucocorticoid receptor recapitulates the pii-DC phenotype. These findings define a developmental checkpoint, set by neuroendocrine maturation, that enables in situ DC activation and immune induction, thereby shaping age-dependent responses to allergens.

Prostate cancer (PCa) is a malignancy with high heterogeneity arising from tumor microenvironment and histological subtypes. Identifying conserved progression drivers within such heterogeneity is essential for improving clinical outcomes. Using imaging mass cytometry, this study analyzes 38 proteins across paracancerous tissue and four histological subtypes: low-grade prostate acinar adenocarcinoma (LgPAC), high-grade PAC (HgPAC), intraductal carcinoma (IDC), and ductal adenocarcinoma (DAC). Results reveal that eIF1A is overexpressed in high-risk subtypes including HgPAC, IDC, and DAC and correlates with poor prognosis. In luminal cells, EIF1A knockdown and the translation inhibitor homoharringtonine (HHT) both suppress HIF-1α translation and tumor growth, while promoting infiltration of anticancer immune cells including PD-1- T cells and CD163- macrophages. Clinically, neoadjuvant HHT combined with androgen deprivation therapy reduces hypoxia and enhances immune cell infiltration, as shown by single-cell RNA sequencing. Collectively, this work defines conserved molecular features across PCa subtypes, providing promising insights for clinical management. This study was registered at Clinicaltrials.gov (NCT06834321).

The protective correlates of Mycobacterium tuberculosis (Mtb) infection-elicited host immune responses are incompletely understood. Here, we report pro-pathogenic crosstalk involving Ly6G+ granulocytes (Ly6G+Gra), IL-17, and COX2. We show that in the lungs of Mtb-infected wild-type mice, either BCG-vaccinated or not, most intracellular bacilli are Ly6G+Gra-resident 4 weeks post-infection onwards. In the genetically susceptible ifng-/- mice, excessive Ly6G+Gra infiltration correlates with severe bacteremia. Neutralizing IL-17 (anti-IL17mAb) and COX2 inhibition by celecoxib reverse Ly6G+Gra infiltration, associated pathology, and death in ifng-/- mice. Surprisingly, Ly6G+Gra also serves as the major source of IL-17 in the lungs of Mtb-infected WT or ifng-/- mice. The IL-17-COX2-Ly6G+Gra interplay also operates in WT mice. Inhibiting RORγt, the key transcription factor for IL-17 production or COX2, reduces the bacterial burden in Ly6G+Gra, leading to reduced bacterial burden and pathology in the lungs of WT mice. In the Mtb-infected WT mice, COX2 inhibition abrogates IL-17 levels in the lung homogenates and significantly enhances BCG's protective efficacy, mainly by targeting the Ly6G+Gra-resident Mtb pool, a phenotype also observed when IL-17 is blocked by RORγt inhibitor. Furthermore, in pulmonary TB patients, high neutrophil count and IL-17 correlated with adverse treatment outcomes. Together, our results suggest that IL-17 and PGE2 are the negative correlates of protection, and we propose targeting the pro-pathogenic IL-17-COX2-Ly6G+Gra axis for TB prevention and therapy.

Tumor-draining lymph nodes are a pivotal site for antitumor T-cell priming. However, their mechanistic roles in cancer immune surveillance and immunotherapy response remain poorly defined. Intratumor (IT) virotherapy generates antitumor T-cell immunity through multifaceted engagement of innate antiviral inflammation. In this study, we identify type-I interferon (IFNI) signaling in glioma-draining cervical lymph nodes as a mediator of IT polio virotherapy. Transient IFNI signaling after IT administration was rescued by cervical perilymphatic infusion (CPLI) virotherapy, targeting cervical lymph nodes directly. Dual-site (IT plus CPLI) virotherapy induced profound inflammatory reprogramming of cervical lymph nodes, enhanced viral RNA replication and IFNI signaling in dendritic cells and high endothelial venules, augmented antiglioma efficacy in mice, and was associated with T-cell activation in patients with recurrent glioblastoma. A phase II clinical trial of IT plus CPLI polio virotherapy is ongoing (NCT06177964). This study implicates the lymphatic system as a virotherapy target and demonstrates that CPLI virotherapy has the potential to complement brain tumor immunotherapy. See related Spotlight by Kaufman, p. 182.

Background: The switch to endothelial-to-mesenchymal transition (EndMT) in endothelial cells (ECs) induced by disturbed flow (d-flow) has been identified as the critical driver of the pathogenesis of inflammatory vascular disorders. We aimed to investigate the role of EndMT in abdominal aortic aneurysms (AAA) and the underlying mechanism. Methods: Immunoblotting, immunofluorescence and transmission electron microscope were used to assess d-flow-induced EndMT in human and mouse AAA models (Ang II/PPE). An Ibidi pump system was used to produce d-flow on human aortic endothelial cells (HAECs), and the expression of galectin-7 was enhanced and weakened using an adeno-associated virus. Furthermore, single-cell RNA sequencing was performed to explore the underlying mechanism of galectin-7-mediated EndMT. Results: EndMT induced by d-flow, which suppressed galectin-7 expression, was positively correlated with AAA. Enhanced galectin-7 expression inhibited d-flow-induced EndMT and AAA progression, whereas reduced galectin-7 expression resulted in the opposite effect. Mechanistically, we found a EndMT-related cluster in HAECs by single-cell RNA sequencing, and the SRGN gene in this cluster was considered the core gene. Galectin-7 bound competitively to the transcription factor CREB, resulting in the inhibition of SRGN transcription, which in turn prevented TGFβ/smad pathway activation, thereby restoring EndMT progression. Conclusions: EndMT transformation in ECs exposed to d-flow was the critical driver of AAA development. Furthermore, endothelium-enriched galectin-7 suppressed the EndMT process induced by d-flow and prevent AAA progression by transcriptionally inhibiting SRGN via competitive binding with CREB to restrict TGFβ/smad pathway.

Women diagnosed with metastatic triple negative breast cancer (mTNBC) have limited treatment options, are more prone to develop resistance and are associated with high mortality. A cold tumor immune microenvironment (TIME) characterized by low T cells and high tumor associated macrophages (TAMs) in mTNBC is associated with the failure of standard-of-care chemotherapy and immune checkpoint blockade (ICB) treatment. We demonstrate that the combination of immunomodulatory low-dose Cyclophosphamide (CTX) coupled with anti-CSF-1R antibody targeted therapy (SNDX-ms6352) and anti-PD-1 (ICB), was highly effective against aggressive metastatic Trp53 null TNBC transplantable syngeneic models that present with high macrophage infiltration. Mechanistically, CSF-1R inhibition along with CTX disrupted the M-CSF/CSF-1R axis which upregulated IL-17, IL-5 and type II interferon resulting in elevated B- and T cell infiltration. Addition of an anti-PD-1 maintenance dose helped overcome de novo PD-L1 intra-tumoral heterogeneity (ITH) associated recurrence in lung and liver mTNBC.

Neutrophil extracellular traps (NETs) are associated with cancer progression; however, the functional role and clinical importance of NET-DNA in therapeutic resistance remain unclear. Here, we show that chemotherapy and radiotherapy provoke NET-DNA formation in primary tumor and metastatic organs in breast cancer patients and mouse models, and the level of NET-DNA correlates with treatment resistance. Mechanistically, the cathepsin C in tumor debris generated by anticancer therapy is phagocytosed by macrophages and drives CXCL1/2 and complement factor B production via activating the TLR4/NF-κB signaling pathway, subsequently promoting NETosis and impairing therapeutic efficacy. Importantly, we demonstrate that NET-DNA sensor CCDC25 is indispensable in NET-mediated treatment resistance by inducing cancer cell epithelial-mesenchymal transition via pyruvate kinase isoform M2-mediated STAT3 phosphorylation. Clinically, tumoral CCDC25 abundance is closely associated with poor prognosis in patients who underwent chemotherapy. Overall, our data reveal the mechanism of NET formation and elucidate the interaction of NET-CCDC25 in therapy resistance, highlighting CCDC25 as an appealing target for anticancer interventions.

Head and neck squamous cell carcinoma (HNSCC) demonstrates suboptimal responses to current immune checkpoint inhibitors (ICIs), with objective response rates (ORRs) of merely 15-20%. The molecular mechanisms underlying these low ORRs remain incompletely defined. Here, two functionally distinct CD8⁺ T cell subsets are identified within the tumor microenvironment: precursor exhausted T (Texprog) cells and terminally exhausted T (Texterm) cells. Notably, although anti-PD-1 therapy reduced Texprog cell frequencies, it failed to reverse Texterm cells. Elevated Texterm cell infiltration correlated with advanced tumor-node-metastasis (TNM) staging and poor prognosis. Furthermore, non-responders exhibited significantly higher baseline Texterm proportions than responders before immunotherapy. Multivariate analysis established stromal Texterm cell density as both an independent prognostic factor and predictor of ICIs resistance. Mechanistically, Texterm cell infiltration strongly correlated with PD-L1 on tumor-derived extracellular vesicles (PD-L1+EVs). Most importantly, it is demonstrated that PD-L1+EVs drive Texterm cell differentiation by upregulating the basic leucine zipper transcription factor, ATF-like (BATF) in CD8⁺ T cells. Knocking out PD-L1 on EVs reduced Texterm cell infiltration and BATF expression. These findings elucidate an EV-mediated immune evasion axis and reveal actionable targets to overcome immunotherapy resistance.

The olfactory epithelium (OE) undergoes life-long renewal and regeneration. This process is supported by the globose basal cells (GBC) during the homeostatic state, as well as horizontal basal cells (HBC) during severe damage. Inflamm-aging refers to the low-grade, chronic and progressive state of heightened pro-inflammation associated with aging. However, the impact of inflamm-aging on OE homeostasis, regeneration, and the inflammatory microenvironment is not fully understood. In this study using mouse models, we elucidate the role of interleukin-17a (IL-17a) in OE regeneration and olfactory function. Our findings implicate that inflamm-aging in aged OE promotes the recruitment and activation of immune cells, accompanied by crosstalk between HBC and T cells. Elevated expression of IL-17a in aged OE triggers inflammatory signals and impairs olfactory function. Administration of IL-17a inhibitor Y-320 or neutralizing antibody promotes sensory neuronal regeneration and reverses age-related respiratory metaplasia in OE. Co-culturing mouse OE organoids with Th17 cells impairs neuronal generation and enhances the transformation towards respiratory cells, while neutralizing antibody against IL-17a alleviates neuronal loss and respiratory transformation. Additionally, conditional knockout of IL-17a in T cells facilitates OE regeneration by promoting HBC recruitment and differentiation into GBC. Collectively, our study identifies a function of IL-17a in OE regeneration and age-related deficits in olfactory function, providing evidence for further investigation of IL-17a as a possible therapeutic target against presbyosmia.

The management of bronchiectasis-asthma overlap (BAO) is an important clinical issue to be addressed. Little is known regarding the endotype of BAO. Here we recruit patients with a primary diagnosis of bronchiectasis and co-existing asthma. The levels of interleukin (IL)-17C are positively correlated with the levels of IL-17A or group 3 innate lymphoid cells (ILC3s) in peripheral blood samples from patients with BAO. An in vivo mouse model of Pseudomonas aeruginosa chronic lower respiratory tract infection followed by ovalbumin-induced asthma shows that IL-17C potentiates IL-17A expression via interacting with IL-17 receptor E in ILC3s. Additionally, ablation of Il17re in mice attenuates ILC3 responses and IL-17A-mediated asthma endotype switching towards neutrophilic asthma driven by P. aeruginosa chronic lower respiratory tract infection. Lastly, impaired epithelial barrier integrity by P. aeruginosa exposure is associated with IL-17C production in vitro. Collectively, our study implicates evidence for IL-17C governing IL-17A pathogenicity and promoting asthma endotype switching in bronchiectasis, implicating IL-17C as a potential therapeutic target for individuals with BAO.

Nucleoside-modified messenger RNA (mRNA) vaccines elicit protective antibodies through their ability to promote T follicular helper (Tfh) cell differentiation. The lipid nanoparticles (LNPs) of mRNA vaccines possess inherent adjuvant activity. However, the extent to which the nucleoside-modified mRNA is sensed and contributes to Tfh cell responses remains undefined. Herein, we deconvolute the signals induced by LNPs and mRNA that instruct dendritic cells (DCs) to promote Tfh cell differentiation. We demonstrate that the mRNA drives the production of type I interferons, which act on DCs to enhance their maturation and Tfh cell differentiation, and favors plasma cells and memory B cell responses. In parallel, LNPs, which allow for mRNA uptake by DCs within the draining lymph node, also modulate Tfh cell responses by shaping the localization of CD25+ DCs. Our work unravels distinct adjuvant features of mRNA and LNPs necessary for the induction of Tfh cells, with implications for rational vaccine design.

We recently showed that cell surface translocation of the endoplasmic reticulum-resident protein GRP78, when bound by activated α 2-macroglobulin (α2M*), induces pro-fibrotic responses in glomerular mesangial cells in response to high glucose and regulates activation of the pro-fibrotic cytokine transforming growth factor-β1 (TGF-β1), implicating a pathogenic role in glomerulosclerosis. Interstitial fibrosis, largely mediated by proximal tubular epithelial cells (PTEC) and renal fibroblasts, develops later in kidney disease and correlates with functional decline. Here we investigated whether interstitial fibrosis was mediated by cell surface GRP78 (csGRP78)/α2M*. High glucose and TGF-β1 increased csGRP78 and α2M* in PTEC and renal fibroblasts, and their inhibition prevented fibrotic protein production. Interestingly, for TGF-β1, this depended on inhibition of noncanonical signaling through YAP/TAZ, with Smad3 activation unaffected. In vivo, type 1 diabetic Akita mice overexpressing TGF-β1 were treated with either a neutralizing antibody for csGRP78 (C38) or α2M* (Fα2M) or an inhibitory peptide blocking csGRP78/α2M* interaction, and mice with unilateral ureteral obstruction were treated with Fα2M or inhibitory peptide. Consistently, inhibition by antibody or peptide attenuated fibrosis and pro-fibrotic signaling. These findings show an important role for csGRP78/α2M* in mediating tubulointerstitial fibrosis in both diabetic and nondiabetic kidney disease and support their inhibition as a potential antifibrotic therapeutic intervention.

The microenvironment of distant organs affects the colonization and growth of disseminated tumor cells. It remains unclear how tumor-associated neutrophils are influenced by the microenvironment of distant organs. Here, we demonstrate that mature low-density neutrophils in colorectal cancer patients abnormally accumulate neutral lipids and induce the reactivation of dormant tumor cells, a process regulated by hepatic stellate cells. Mechanistically, activated hepatic stellate cells increased DGAT1/2-dependent lipid droplet synthesis in low-density neutrophils through the secretion of IL33, thereby maintaining the survival and immunosuppressive function of these neutrophils. The uptake of lipids from lipid-laden low-density neutrophils drives dormant tumor cell reactivation through the potentiation of β-oxidation and the stimulation of protumorigenic eicosanoid synthesis. In mouse models, targeting IL33 blocked neutrophil lipid synthesis, decreased the colonization of colorectal cancer cells in the liver, and enhanced the efficacy of immunotherapy. Overall, our study revealed that lipid accumulation in mature low-density neutrophils regulates the growth of dormant tumor cells and antitumor immunity to facilitate colorectal cancer liver metastasis. Targeting IL33 could be a promising therapeutic approach for colorectal cancer liver metastases.