An IGHG1 variant exhibits polarized prevalence and confers enhanced IgG1 antibody responses against life-threatening organisms.

Evolutionary pressures sculpt population genetics, whereas immune adaptation fortifies humans against life-threatening organisms. How the evolution of selective genetic variation in adaptive immune receptors orchestrates the adaptation of human populations to contextual perturbations remains elusive. Here, we show that the G396R coding variant within the human immunoglobulin G1 (IgG1) heavy chain presents a concentrated prevalence in Southeast Asian populations. We uncovered a 190-kb genomic linkage disequilibrium block peaked in close proximity to this variant, suggestive of potential Darwinian selection. This variant confers heightened immune resilience against various pathogens and viper toxins in mice. Mechanistic studies involving severe acute respiratory syndrome coronavirus 2 infection and vaccinated individuals reveal that this variant enhances pathogen-specific IgG1+ memory B cell activation and antibody production. This G396R variant may have arisen on a Neanderthal haplotype background. These findings underscore the importance of an IGHG1 variant in reinforcing IgG1 antibody responses against life-threatening organisms, unraveling the intricate interplay between human evolution and immune adaptation.

Intraepithelial mast cells drive gasdermin C-mediated type 2 immunity.

A specialized population of mast cells residing within epithelial layers, currently known as intraepithelial mast cells (IEMCs), was originally observed over a century ago, yet their physiological functions have remained enigmatic. In this study, we unveil an unexpected and crucial role of IEMCs in driving gasdermin C-mediated type 2 immunity. During helminth infection, αEß7 integrin-positive IEMCs engaged in extensive intercellular crosstalk with neighboring intestinal epithelial cells (IECs). Through the action of IEMC-derived proteases, gasdermin C proteins intrinsic to the epithelial cells underwent cleavage, leading to the release of a critical type 2 cytokine, interleukin-33 (IL-33). Notably, mast cell deficiency abolished the gasdermin C-mediated immune cascade initiated by epithelium. These findings shed light on the functions of IEMCs, uncover a previously unrecognized phase of type 2 immunity involving mast cell-epithelial cell crosstalk, and advance our understanding of the cellular mechanisms underlying gasdermin C activation.

Progressive polyadenylation and m6A modification of Ighg1 mRNA maintain IgG1 antibody homeostasis in antibody-secreting cells.

Antigen-specific antibodies are generated by antibody-secreting cells (ASCs). How RNA post-transcriptional modification affects antibody homeostasis remains unclear. Here, we found that mRNA polyadenylations and N6-methyladenosine (m6A) modifications maintain IgG1 antibody production in ASCs. IgG heavy-chain transcripts (Ighg) possessed a long 3' UTR with m6A sites, targeted by the m6A reader YTHDF1. B cell-specific deficiency of YTHDF1 impaired IgG production upon antigen immunization through reducing Ighg1 mRNA abundance in IgG1+ ASCs. Disrupting either the m6A modification of a nuclear-localized splicing intermediate Ighg1 or the nuclear localization of YTHDF1 reduced Ighg1 transcript stability. Single-cell RNA sequencing identified an ASC subset with excessive YTHDF1 expression in systemic lupus erythematosus patients, which was decreased upon therapy with immunosuppressive drugs. In a lupus mouse model, inhibiting YTHDF1-m6A interactions alleviated symptoms. Thus, we highlight a mechanism in ASCs to sustain the homeostasis of IgG antibody transcripts by integrating Ighg1 mRNA polyadenylation and m6A modification.

The Mevalonate Pathway Is a Druggable Target for Vaccine Adjuvant Discovery.

Motivated by the clinical observation that interruption of the mevalonate pathway stimulates immune responses, we hypothesized that this pathway may function as a druggable target for vaccine adjuvant discovery. We found that lipophilic statin drugs and rationally designed bisphosphonates that target three distinct enzymes in the mevalonate pathway have potent adjuvant activities in mice and cynomolgus monkeys. These inhibitors function independently of conventional "danger sensing." Instead, they inhibit the geranylgeranylation of small GTPases, including Rab5 in antigen-presenting cells, resulting in arrested endosomal maturation, prolonged antigen retention, enhanced antigen presentation, and T cell activation. Additionally, inhibiting the mevalonate pathway enhances antigen-specific anti-tumor immunity, inducing both Th1 and cytolytic T cell responses. As demonstrated in multiple mouse cancer models, the mevalonate pathway inhibitors are robust for cancer vaccinations and synergize with anti-PD-1 antibodies. Our research thus defines the mevalonate pathway as a druggable target for vaccine adjuvants and cancer immunotherapies.

Germinal-center development of memory B cells driven by IL-9 from follicular helper T cells.

Germinal centers (GCs) support high-affinity, long-lived humoral immunity. How memory B cells develop in GCs is not clear. Through the use of a cell-cycle-reporting system, we identified GC-derived memory precursor cells (GC-MP cells) that had quit cycling and reached G0 phase while in the GC, exhibited memory-associated phenotypes with signs of affinity maturation and localized toward the GC border. After being transferred into adoptive hosts, GC-MP cells reconstituted a secondary response like genuine memory B cells. GC-MP cells expressed the interleukin 9 (IL-9) receptor and responded to IL-9. Acute treatment with IL-9 or antibody to IL-9 accelerated or retarded the positioning of GC-MP cells toward the GC edge and exit from the GC, and enhanced or inhibited the development of memory B cells, which required B cell-intrinsic responsiveness to IL-9. Follicular helper T cells (TFH cells) produced IL-9, and deletion of IL-9 from T cells or, more specifically, from GC TFH cells led to impaired memory formation of B cells. Therefore, the GC development of memory B cells is promoted by TFH cell-derived IL-9.

A Structural Change in Butyrophilin upon Phosphoantigen Binding Underlies Phosphoantigen-Mediated Vγ9Vδ2 T Cell Activation.

Human Vγ9Vδ2 T cells respond to microbial infections and malignancy by sensing diphosphate-containing metabolites called phosphoantigens, which bind to the intracellular domain of butyrophilin 3A1, triggering extracellular interactions with the Vγ9Vδ2 T cell receptor (TCR). Here, we examined the molecular basis of this "inside-out" triggering mechanism. Crystal structures of intracellular butyrophilin 3A proteins alone or in complex with the potent microbial phosphoantigen HMBPP or a synthetic analog revealed key features of phosphoantigens and butyrophilins required for γδ T cell activation. Analyses with chemical probes and molecular dynamic simulations demonstrated that dimerized intracellular proteins cooperate in sensing HMBPP to enhance the efficiency of γδ T cell activation. HMBPP binding to butyrophilin doubled the binding force between a γδ T cell and a target cell during "outside" signaling, as measured by single-cell force microscopy. Our findings provide insight into the "inside-out" triggering of Vγ9Vδ2 T cell activation by phosphoantigen-bound butyrophilin, facilitating immunotherapeutic drug design.

Affinity-coupled CCL22 promotes positive selection in germinal centres.

Antibody affinity maturation depends on positive selection in germinal centres (GCs) of rare B cell clones that acquire higher-affinity B cell receptors via somatic hypermutation, present more antigen to follicular helper T (TFH) cells and, consequently, receive more contact-dependent T cell help1. As these GC B cells and TFH cells do not maintain long-lasting contacts in the chaotic GC environment2-4, it is unclear how sufficient T cell help is cumulatively focused onto those rare clones. Here we show that, upon stimulation of CD40, GC B cells upregulate the chemokine CCL22 and to a lesser extent CCL17. By engaging the chemokine receptor CCR4 on TFH cells, CCL22 and CCL17 can attract multiple helper cells from a distance, thus increasing the chance of productive help. During a GC response, B cells that acquire higher antigen-binding affinities express higher levels of CCL22, which in turn 'highlight' these high-affinity GC B cells. Acute increase or blockade of TFH cells helps to rapidly increase or decrease CCL22 expression by GC B cells, respectively. Therefore, a chemokine-based intercellular reaction circuit links the amount of T cell help that individual B cells have received recently to their subsequent ability to attract more help. When CCL22 and CCL17 are ablated in B cells, GCs form but B cells are not affinity-matured efficiently. When competing with wild-type B cells in the same reaction, B cells lacking CCL22 and CCL17 receive less T cell help to maintain GC participation or develop into bone-marrow plasma cells. By uncovering a chemokine-mediated mechanism that highlights affinity-improved B cells for preferential help from TFH cells, our study reveals a principle of spatiotemporal orchestration of GC positive selection.

Correction: Farnesyl pyrophosphate is a new danger signal inducing acute cell death.

[This corrects the article DOI: 10.1371/journal.pbio.3001134.].

Self-programmed dynamics of T cell receptor condensation.

A common event upon receptor-ligand engagement is the formation of receptor clusters on the cell surface, in which signaling molecules are specifically recruited or excluded to form signaling hubs to regulate cellular events. These clusters are often transient and can be disassembled to terminate signaling. Despite the general relevance of dynamic receptor clustering in cell signaling, the regulatory mechanism underlying the dynamics is still poorly understood. As a major antigen receptor in the immune system, T cell receptors (TCR) form spatiotemporally dynamic clusters to mediate robust yet temporal signaling to induce adaptive immune responses. Here we identify a phase separation mechanism controlling dynamic TCR clustering and signaling. The TCR signaling component CD3ε chain can condensate with Lck kinase through phase separation to form TCR signalosomes for active antigen signaling. Lck-mediated CD3ε phosphorylation, however, switched its binding preference to Csk, a functional suppressor of Lck, to cause the dissolvement of TCR signalosomes. Modulating TCR/Lck condensation by targeting CD3ε interactions with Lck or Csk directly affects T cell activation and function, highlighting the importance of the phase separation mechanism. The self-programmed condensation and dissolvement is thus a built-in mechanism of TCR signaling and might be relevant to other receptors.

Fn-Dps, a novel virulence factor of Fusobacterium nucleatum, disrupts erythrocytes and promotes metastasis in colorectal cancer.

Fusobacterium nucleatum (Fn) is a critical colorectal cancer (CRC)-associated bacterium. DNA hunger/stationary phase protective proteins (Dps) are bacterial ferritins that protect DNA from oxidative stress. However, little is known about the regulatory roles of Fn-Dps towards host cellular functions. Here, we identified Fn-Dps from the culture supernatant of Fn by mass spectrometry, and prepared the recombinant of Fn-Dps protein. We show a novel virulence protein of Fn, Fn-Dps, which lyses and disrupts erythrocytes by the competition for iron acquisition. Also, Fn-Dps facilitates intracellular survival of Fn in macrophages by upregulating the expression of the chemokine CCL2/CCL7. In addition, Fn-Dps can elicit a strong humoral immune response, and mucosal immunization with Fn-Dps conferred protection against Fn in the intestinal tract. Moreover, a high level of anti-Fn-Dps antibody was prevalent in populations, and elevated anti-Fn-Dps antibody levels were observed in CRC patients. Furthermore, Fn-Dps promotes the migration of CRC cells via the CCL2/CCL7-induced epithelial-mesenchymal transition (EMT) and promotes CRC metastasis in vivo.

Mechanical force determines chimeric antigen receptor microclustering and signaling.

Chimeric antigen receptor (CAR) T cells are activated to trigger the lytic machinery after antigen engagement, and this has been successfully applied clinically as therapy. The mechanism by which antigen binding leads to the initiation of CAR signaling remains poorly understood. Here, we used a set of short double-stranded DNA (dsDNA) tethers with mechanical forces ranging from ∼12 to ∼51 pN to manipulate the mechanical force of antigen tether and decouple the microclustering and signaling events. Our results revealed that antigen-binding-induced CAR microclustering and signaling are mechanical force dependent. Additionally, the mechanical force delivered to the antigen tether by the CAR for microclustering is generated by autonomous cell contractility. Mechanistically, the mechanical-force-induced strong adhesion and CAR diffusion confinement led to CAR microclustering. Moreover, cytotoxicity may have a lower mechanical force threshold than cytokine generation. Collectively, these results support a model of mechanical-force-induced CAR microclustering for signaling.

NAT10-mediated RNA ac4C acetylation contributes to the myocardial infarction-induced cardiac fibrosis.

Cardiac fibrosis is featured cardiac fibroblast activation and extracellular matrix accumulation. Ac4C acetylation is an important epigenetic regulation of RNAs that has been recently discovered, and it is solely carried out by NAT10, the exclusive enzyme used for the modification. However, the potential regulatory mechanisms of ac4C acetylation in myocardial fibrosis following myocardial infarction remain poorly understood. In our study, we activated fibroblasts in vitro using TGF-ß1 (20 ng/mL), followed by establishing a myocardial infarction mouse model to evaluate the impact of NAT10 on collagen synthesis and cardiac fibroblast proliferation. We utilized a NAT10 inhibitor, Remodelin, to attenuate the acetylation capacity of NAT10. In the cardiac fibrosis tissues of chronic myocardial infarction mice and cultured cardiac fibroblasts (CFs) in response to TGF-ß1 treatment, there was an elevation in the levels of NAT10 expression. This increase facilitated proliferation, the accumulation of collagens, as well as fibroblast-to-myofibroblast transition. Through the administration of Remodelin, we effectively reduced cardiac fibrosis in myocardial infarction mice by inhibiting NAT10's ability to acetylate mRNA. Inhibition of NAT10 resulted in changes in collagen-related gene expression and ac4C acetylation levels. Mechanistically, we found that NAT10 upregulates the acetylation modification of BCL-XL mRNA and enhances the stability of BCL-XL mRNA, thereby upregulating its protein expression, inhibiting the activation of Caspase3 and blocking the apoptosis of CFs. Therefore, the crucial involvement of NAT10-mediated ac4C acetylation is significant in the cardiac fibrosis progression, affording promising molecular targets for the treatment of fibrosis and relevant cardiac diseases.

Atomic Zn-Doping Induced Sabatier Optimum in NiZn0.03 Catalyst for CO2 Electroreduction at Industrial-Level Current Densities.

The Sabatier principle defines the essential criteria for an ideal catalyst in heterogeneous catalysis, while reaching the Sabatier optimum is still challenging in catalyst design. Herein, an elegant strategy is described to reach the Sabatier optimum of Ni electrocatalyst in CO2 reduction reaction (CO2 RR) by atomically Zn doping. The incorporation of 3% Zn single atom into Ni lattice leads to the moderate degrade of d-band center via Ni-Zn electronic coupling, which balances the bonding strengths of *COOH and *CO, resulting in a relative low energy barrier for CO2 activation while not being substantially poisoned by CO. Consequently, NiZn0.03 /C exhibits unique catalytic activity (jCO >100 mA cm-2 at -0.6 V), wide potential range for selective CO production (FECO >90% from -0.65 to -1.15 V), and outstanding long-term stability (FECO >90% during 85 h electrolysis at -0.85 V). The results provide valuable insights for the rational fabrication of superior non-noble bimetallic electrocatalysts in CO2 electroreduction.

Well-Dispersed CsPbBr3@TiO2 Heterostructure Nanocrystals from Asymmetric to Symmetric.

Metal halide perovskites (MHPs) have undergone rapid development in the fields of solar cells, light diodes, lasing, photodetectors, etc. However, the MHPs still face significant challenges, such as poor stability and heterocompositing with other functional materials at the single nanoparticle level. Herein, the successful synthesis of well-dispersed CsPbBr3@TiO2 heterostructure nanocrystals (NCs) is reported, in which each heterostructure NC has only one CsPbBr3 with a precise anatase TiO2 coating ranging from asymmetric to symmetric. Due to the protection of anatase TiO2, CsPbBr3 shows dramatically improved chemical stability and photostability. More significantly, the synthesized CsPbBr3@TiO2 heterostructure NCs form a type II heterojunction, which strongly promoted efficient photogenerated carrier separation between anatase TiO2 and CsPbBr3, hence leading to improved optoelectronic activity. This study provides a robust avenue for synthesizing stable and highly efficient MHPs@metal oxide heterostructure NCs, paving the way for the practical application of all inorganic perovskites.

Proinflammatory phenotype of B10 and B10pro cells elicited by TNF-α in rheumatoid arthritis.

OBJECTIVES: B10 and B10pro cells suppress immune responses via secreting interleukin (IL)-10. However, their regulators and underlying mechanisms, especially in human autoimmune diseases, are elusive. This study aimed to address these questions in rheumatoid arthritis (RA), one of the most common highly disabling autoimmune diseases. METHODS: The frequencies and functions of B10 and B10pro cells in healthy individuals and patients with RA were first analysed. The effects of proinflammatory cytokines, particularly tumour necrosis factor (TNF)-α on the quantity, stability and pathogenic phenotype of these cells, were then assessed in patients with RA before and after anti-TNF therapy. The underlying mechanisms were further investigated by scRNA-seq database reanalysis, transcriptome sequencing, TNF-α-/- and B cell-specific SHIP-1-/- mouse disease model studies. RESULTS: TNF-α was a key determinant for B10 cells. TNF-α elicited the proinflammatory feature of B10 and B10pro cells by downregulating IL-10, and upregulating interferon-γ and IL-17A. In patients with RA, B10 and B10pro cells were impaired with exacerbated proinflammatory phenotype, while anti-TNF therapy potently restored their frequencies and immunosuppressive functions, consistent with the increased B10 cells in TNF-α-/- mice. Mechanistically, TNF-α diminished B10 and B10pro cells by inhibiting their glycolysis and proliferation. TNF-α also regulated the phosphatidylinositol phosphate signalling of B10 and B10pro cells and dampened the expression of SHIP-1, a dominant phosphatidylinositol phosphatase regulator of these cells. CONCLUSIONS: TNF-α provoked the proinflammatory phenotype of B10 and B10pro cells by disturbing SHIP-1 in RA, contributing to the disease development. Reinstating the immunosuppressive property of B10 and B10pro cells might represent novel therapeutic approaches for RA.

Multi-omics study reveals different pathogenesis of the generation of skin lesions in SLE and IDLE patients.

Lupus erythematosus (LE) is a heterogeneous, antibody-mediated autoimmune disease. Isolate discoid LE (IDLE) and systematic LE (SLE) are traditionally regarded as the two ends of the spectrum, ranging from skin-limited damage to life-threatening multi-organ involvement. Both belong to LE, but IDLE and SLE differ in appearance of skin lesions, autoantibody panels, pathological changes, treatments, and immunopathogenesis. Is discoid lupus truly a form of LE or is it a completely separate entity? This question has not been fully elucidated. We compared the clinical data of IDLE and SLE from our center, applied multi-omics technology, such as immune repertoire sequencing, high-resolution HLA alleles sequencing and multi-spectrum pathological system to explore cellular and molecular phenotypes in skin and peripheral blood from LE patients. Based on the data from 136 LE patients from 8 hospitals in China, we observed higher damage scores and fewer LE specific autoantibodies in IDLE than SLE patients, more uCDR3 sharing between PBMCs and skin lesion from SLE than IDLE patients, elevated diversity of V-J recombination in IDLE skin lesion and SLE PBMCs, increased SHM frequency and class switch ratio in IDLE skin lesion, decreased SHM frequency but increased class switch ratio in SLE PBMCs, HLA-DRB1*03:01:01:01, HLA-B*58:01:01:01, HLA-C*03:02:02:01, and HLA-DQB1*02:01:01:01 positively associated with SLE patients, and expanded Tfh-like cells with ectopic germinal center structures in IDLE skin lesions. These findings suggest a significant difference in the immunopathogenesis of skin lesions between SLE and IDLE patients. SLE is a B cell-predominate systemic immune disorder, while IDLE appears limited to the skin. Our findings provide novel insights into the pathogenesis of IDLE and other types of LE, which may direct more accurate diagnosis and novel therapeutic strategies.

Farnesyl pyrophosphate is a new danger signal inducing acute cell death.

Cell death is a vital event in life. Infections and injuries cause lytic cell death, which gives rise to danger signals that can further induce cell death, inflammation, and tissue damage. The mevalonate (MVA) pathway is an essential, highly conserved and dynamic metabolic pathway. Here, we discover that farnesyl pyrophosphate (FPP), a metabolic intermediate of the MVA pathway, functions as a newly identified danger signal to trigger acute cell death leading to neuron loss in stroke. Harboring both a hydrophobic 15-carbon isoprenyl chain and a heavily charged pyrophosphate head, FPP leads to acute cell death independent of its downstream metabolic pathways. Mechanistically, extracellular calcium influx and the cation channel transient receptor potential melastatin 2 (TRPM2) exhibit essential roles in FPP-induced cell death. FPP activates TRPM2 opening for ion influx. Furthermore, in terms of a mouse model constructing by middle cerebral artery occlusion (MCAO), FPP accumulates in the brain, which indicates the function of the FPP and TRPM2 danger signal axis in ischemic injury. Overall, our data have revealed a novel function of the MVA pathway intermediate metabolite FPP as a danger signal via transient receptor potential cation channels.

Development of Redox-Active Lyotropic Lipid Cubic Phases for Biosensing Platforms.

Enzyme-based electrochemical biosensors play an important role in point-of-care diagnostics for personalized medicine. For such devices, lipid cubic phases (LCP) represent an attractive method to immobilize enzymes onto conductive surfaces with no need for chemical linking. However, research has been held back by the lack of effective strategies to stably co-immobilize enzymes with a redox shuttle that enhances the electrical connection between the enzyme redox center and the electrode. In this study, we show that a monoolein (MO) LCP system doped with an amphiphilic redox mediator (ferrocenylmethyl)dodecyldimethylammonium bromide (Fc12) can be used for enzyme immobilization to generate an effective biosensing platform. Small-angle X-ray scattering (SAXS) showed that MO LCP can incorporate Fc12 while maintaining the Pn3m symmetry morphology. Cyclic voltammograms of Fc12/MO showed quasi-reversible behavior, which implied that Fc12 was able to freely diffuse in the lipid membrane of LCP with a diffusion coefficient of 1.9 ± 0.2 × 10-8 cm2 s-1 at room temperature. Glucose oxidase (GOx) was then chosen as a model enzyme and incorporated into 0.2%Fc12/MO to evaluate the activity of the platform. GOx hosted in 0.2%Fc12/MO followed Michaelis-Menten kinetics toward glucose with a KM and Imax of 8.9 ± 0.5 mM and 1.4 ± 0.2 µA, respectively, and a linearity range of 2-17 mM glucose. Our results therefore demonstrate that GOx immobilized onto 0.2% Fc12/MO is a suitable platform for the electrochemical detection of glucose.

Risk factors for peripherally inserted central catheter-related venous thrombosis in adult patients with cancer.

PURPOSE: The purpose of this study was to understand and analyze the risk factors of peripherally inserted central catheter (PICC)-related venous thrombosis in adult patients with cancer. METHODS: This observational cohort study included adult patients with cancer who underwent color Doppler ultrasound at the Xiangya Hospital of Central South University, Hunan Provincial Maternal and Child Healthcare Hospital, and Xiangya Changde Hospital, Hunan Province, from January 1, 2017 to December 31, 2021. Univariate and multivariate logistic regression analyses were performed to determine the risk factors of PICC-related venous thrombosis. RESULTS: After risk adjustment, multivariate logistic regression analysis revealed statistically significant associations between PICC-related venous thrombosis and age > 65 years old (OR: 1.791, CI: 1.343-2.389), male sex (OR: 1.398, CI: 1.057-1.849), white blood cell count > 9.5 × 109 /L (OR: 1.422, CI: 1.041-1.942), APTT < 25 s (OR: 2.006, CI: 1.431-2.811), gastrointestinal tumor (OR: 2.191, CI: 1.406-3.414), infection (OR:7.619, CI: 5.783-10.037), the use of cisplatin (OR: 2.374, CI: 1.714-3.214), vincristine (OR: 2.329, CI: 1.447-3.749), the use of polyurethane (OR: 2.449, CI: 1.863-3.219) and open-ended catheters (OR:1.660, CI: 1.131-2.439), keeping time of the catheter (days) (OR: 1.003, CI: 1.001-1.005) were associated with PICC-related venous thrombosis. CONCLUSION: We identified that the presence of age > 65 years old, male sex, white blood cell count > 9.5 × 109 /L, APTT < 25 s, gastrointestinal tumor, infection, the use of cisplatin and vincristine, the use of polyurethane, open-ended catheters and keeping time of the catheter (days), were associated with PICC-related venous thrombosis.

M2e-specific antibodies protect against influenza PR8 virus in an isotype and route dependent manner.

The ectodomain of influenza matrix protein 2 (M2e) is a promising target for the development of universal prophylactic and therapeutic agents against influenza viruses of different subtypes. We constructed three M2e-specific monoclonal antibody variants, M2A1-1 (IgG1), M2A1-2a (IgG2a), M2A1-2b (IgG2b), which have the same Fab region targeting the M2e epitope but different isotypes, and compared their protective efficacy in influenza PR8-infected mice. We found that anti-M2e antibodies provided protection against influenza virus in a subtype-dependent manner, with the IgG2a variant providing significantly better protection with lower virus titers and milder lung injury than IgG1 and IgG2b isotypes. Additionally, we observed that the protective efficacy was dependent on the administration routes, with intranasal administration of antibody providing better protection than intraperitoneal administration. The timing of administration was also critical in determining the protective efficacy; while all the antibody isotypes provided protection when administered before influenza challenge, only IgG2a provided minimal protection when the antibodies were administered after virus challenge. These results provide valuable information for optimizing the therapeutics usage of M2e-based antibodies and furthering the development of M2e-based universal influenza vaccines.