Cellular & Molecular Immunology

Human genetic studies have identified defects in multiple mechanisms that predispose the risk of developing inflammatory bowel diseases (IBD), which include alterations in adaptive and innate immune responses, epithelial integrity and regulation of the intestinal mucus layer. Despite the importance of intestinal barrier integrity in the pathogenesis of IBD, essentially all current therapies modulate the immune responses. In this study, we determined the high resolution cryo-EM structure of human NXPE1, a IBD associated protein. Based on the structural homology, we identified NXPE1 as an O-acetyltransferase. Since NXPE1 is a pseudo gene in mouse, we generated knockout mouse model that lacked two of the mouse NXPE1 homologs, Nxpe2 and Nxpe4. The O-acetylation of sialic acid on red blood cells was abolished in the double knockout mice, confirming the sialic acid O-acetyltransferase function of NXPE1 family members. These findings underscore the potential of NXPE1 as a novel therapeutic target of the intestinal barrier functions for the treatment of IBD.

Aberrant activation of type I interferon (IFN-I) is closely related to the development of autoimmune diseases. The metabolic regulation of cytokine signaling is essential for immune homeostasis. In this study, we characterized Urolithin A(UA), a natural gut-derived metabolite, as an inhibitor of Janus kinase (JAK) signaling. UA was found to broadly dampen JAK phosphorylation and the downstream signaling induced by cytokines such as type I interferons (IFN-I), type II interferons (IFN-II), and interleukin-6 (IL-6). UA can directly bind to JAK1 JH1 domain and treatment with UA attenuated autoimmune pathogenesis in Trex1-KO mice, IMQ-induced SLE and psoriasis models. Our findings unveil that UA is an anti-inflammatory metabolite that promotes immune homeostasis and could be used to treat inflammatory and autoimmune diseases.

Streptococcal pyrogenic exotoxin C (SpeC) is a prototypical superantigen produced by group A Streptococcus. It potently activates a broad subset of T lymphocytes via a bridging interaction involving TCR{beta}-SpeC-MHC-II. Our recent work demonstrated that SpeC induced profound release of IL-8 from human pharyngeal epithelial cells and this effect was reversible through a specific point mutation in SpeC. This study systematically investigated cellular signaling pathways using integrated transcriptomic profiling and Western blot analysis, with a focus on membrane-associated receptors and downstream intracellular signaling effectors. Our results demonstrate that this biological process is critically associated with the activation of Erk1/2, p38 MAPK and NF-{kappa}B signaling cascade. This study identifies a novel mechanism through which a bacterial superantigen target epithelial cells-the body primary physical barrier and first line of innate immune defense.

The germinal center (GC) reaction requires tight regulation of B cell and T follicular helper (Tfh) cell interactions to ensure B cell expansion and antibody affinity maturation, while preventing oncogenesis. However, regulatory mechanisms fine-tuning B-T cell interactions within the GC to prevent aberrant activation and proliferation remain incompletely understood. Here, we identify Siglec-G, the mouse ortholog of human Siglec-10, as an immune checkpoint that restrains the GC by dampening B-T cell interactions. Selective and temporal ablation of Siglec-G on B cells after immunization triggers GC hyperplasia and enhanced plasma cell and antibody output. While Siglec-G is dispensable in B cell receptor (BCR)-mediated processes, it acts as an intrinsic inhibitory receptor of B-T cell interactions in the GC, ultimately limiting Myc and mTORC activation within positively selected GC B cells. Trans interactions of Siglec-G and its glycan ligands on Tfh likely contribute in fine-tuning the strength of bidirectional signaling following contact between GC B cells and Tfh cells. This interaction is further reinforced by glycan remodeling that occurs in the GC, resulting in concurrent decreased in glycan ligands on GC B cells and increased in glycan ligands on Tfh. This augmented binding of Siglec-G/10 on Tfh is mainly due to the upregulation of 2-6 linked sialic acid ligands. Moreover, APEX2-based proximity labeling revealed several candidate Siglec-G/10 binding partners on T cells, including BTLA, CD6, and Slamf6, which are known negative regulators of Tfh cell activation. Taken together, our findings identified that Siglec-G acts as a GC checkpoint receptor, restricting B cell proliferation by tuning T cell help following B-T cell interactions.

Abstract: Objective This study aimed to systematically screen for potential candidate biomarkers and identify therapeutic targets associated with gouty arthritis (GA) through integrated analyses of single-cell and bulk RNA sequencing (RNA-seq) data. Methods The single-cell dataset GSE211783 and the bulk RNA-seq dataset GSE160170 were analyzed using a series of bioinformatic approaches, including cell clustering, differential expression analysis, immune cell infiltration assessment, protein-protein interaction network construction, gene set enrichment analysis, as well as drug sensitivity evaluation. To establish an animal model of GA, monosodium urate crystals were injected intra-articularly into experimental mice. Joint swelling was evaluated, and morphological changes in joint tissues were analyzed through hematoxylin-eosin staining. The presence of TREM1-positive cells was detected by immunohistochemistry and the level of TREM1 protein expression in joint tissues were assessed by Western blotting. Results We identified 102 differentially expressed genes (DEGs) and 14 signaling pathways associated with GA. The PPI network revealed 25 hub genes, of which 17 (including TREM1, TNF, PTGS2, and NLRP3) were highly expressed and 8 (including FCGR3B and CXCR6) showed low expression in the GA samples. These genes correlated significantly with the infiltration levels of macrophages. Among the hub genes, TREM1 was selected for further validation because it correlated significantly with all 14 differential pathways. In animal experiments, GA mice developed marked joint swelling and inflammatory tissue injury, along with a significant increase in TREM1-positive cells and TREM1 protein expression. Conclusion Integrative analysis of single-cell and bulk RNA-seq data identified 102 GA-related DEGs and 14 key pathways, from which 25 hub genes were screened. TREM1 is significantly upregulated in GA and may be linked to macrophage function, providing new insights into biomarker and therapeutic target discovery for GA.

PRV-101 is a multivalent formalin-inactivated Coxsackievirus B (CVB) vaccine developed to prevent CVB infections, which are associated with increased risk of islet autoimmunity. While PRV-101 induces robust neutralizing antibody responses, its T-cell immunogenicity is unknown. We analyzed peripheral blood mononuclear cells from 25 healthy adults receiving three high or low PRV-101 doses or placebo in a Phase I randomized, placebo-controlled trial. CVB-reactive CD8 T-cell responses were assessed using HLA Class I multimers, and CD4 and T follicular helper (Tfh) responses were measured by activation-induced marker assays following stimulation with a CVB peptide library. PRV-101 elicited minimal CVB-reactive CD8 T-cell responses but robust CD4 and Tfh responses, peaking at week 12 and persisting through week 32. Responses were observed in both seronegative and seropositive individuals, consistent with effective immune priming and boosting. Tfh frequencies correlated with neutralizing antibody titers. Female participants exhibited higher peak Tfh responses than males. We conclude that PRV-101 elicits a CVB-protective immune profile, dominated by Tfh responses supporting durable humoral immunity and devoid of potentially diabetogenic cytotoxic T-cell responses. This profile invites further investigations in vaccine trials for type 1 diabetes prevention.

Despite the availability of effective vaccines, pneumococcal disease remains a major global health concern. Pneumococcal vaccines are multivalent vaccines that have progressively increased in valency, a change associated with lower antibody titers to individual polysaccharide antigens. Whether increasing vaccine valency influences B cell responses through antigenic competition remains incompletely understood. Here, we studied pneumococcal polysaccharide-specific B cell responses in peripheral blood and lymph nodes of healthy adults following vaccination with the 13-valent pneumococcal conjugate vaccine. Antigen-specific memory B cells in peripheral blood expanded 2 weeks post-vaccination, whereas germinal center formation was delayed and peaked after 4 weeks. Notably, germinal center B cell responses were dominated by a limited number of specificities, in contrast to the more evenly distributed expansion observed in peripheral blood. Together, these data highlight the importance of extrafollicular responses in adult anti-pneumococcal polysaccharide immunity and provide evidence for antigenic competition during lymph node germinal center formation, which may have important implications in the context of multivalent vaccines.

Underprocessed oligomannose glycans on protein nanoparticle immunogens engage the innate immune system through mannose-binding lectin and complement, enhancing immunogen trafficking and B cell responses. However, the extent to which oligomannose glycans directly improve protective immunity has remained unclear. Here we generate a series of CSP-bearing I53-50 nanoparticle malaria vaccine candidates with defined numbers and types of engineered N-linked glycans and systematically evaluate their immunogenicity and protective efficacy. Oligomannose display enhanced early plasmablast and germinal center B cell responses, leading to increased CSP-specific memory B cells, long-lived plasma cells, and durable serum antibody titers. Furthermore, nanoparticles bearing oligomannose glycans conferred the strongest protection against sporozoite challenge. By comparing immunogens with defined glycoforms, we attribute improved immune responses and protection specifically to oligomannose rather than complex or truncated glycans. These results will help guide the development of general strategies for glycan engineering aimed at enhancing the protective efficacy of nanoparticle vaccines.

Maternal immunoglobulin G (IgG) transferred across the placenta is crucial for newborn immunity. IgG transfer efficiency modulated by Fc characteristics including subclass and glycosylation gives rise to diverse transfer profiles across the population, yet the molecular mechanisms driving this variation are incompletely understood. To disentangle multimodal molecular relationships driving population heterogeneity in maternal-fetal antibody transfer, we characterized placental and serological antibody features in matched human tissues from two geographically distinct cohorts. Unsupervised clustering of maternal clinical covariates in both cohorts revealed a distinct patient profile with reduced plasma C-reactive protein and pregravid BMI and enhanced transfer efficiency of IgG subclasses. Quantification of IgG Fc glycan structures in matched maternal and cord plasma by liquid chromatography-mass spectrometry revealed subclass-specific IgG glycosylation patterns which impacted placental transfer efficiency and correlated with these key clinical features. We profiled expression and colocalization of key Fc {gamma} Receptors (Fc{gamma}Rs) by multiplex immunohistochemistry, revealing cell type-specific expression patterns. Variable Fc{gamma}R expression across gestation was consistent in both cohorts, implicating Fc{gamma}Rs as key drivers of temporal antibody transfer dynamics. While Fc{gamma}Rs were not strongly variable across the clinical profiles, partial correlation analysis of matched samples controlling for gestational age and demographic covariates revealed correlations between Fc{gamma}R expression frequencies and glycan- and subclass-specific transfer efficiency. These data systematically define multi-factorial regulatory networks of antibody transfer by placental Fc receptors and maternal IgG Fc characteristics, which are further modulated by clinical covariates. This study provides a basis for the rational design of prenatal vaccination strategies, administration schedules, and potential lifestyle interventions to improve maternal-fetal immunity.

Neoantigens are cancer-specific antigens arising from genomic alterations. Single Amino Acid Variants (SAAVs) represent a primary class of these neoantigens. To evaluate the therapeutic potential of Neurofibromin 1 (NF1)-derived SAAVs - given that NF1 is frequently mutated in malignant brain tumors - we prioritized the 40 NF1 SAAVs determined to be HLA-A*02:01 binders using computational prediction coupled with experimental validation. To validate these predicted neoepitopes, we employed a two-tiered experimental approach in HLA-A*02:01 homozygous U87-MG cells. We first synthesized minigene constructs encoding the predicted neoepitopes, introduced them via lentiviral transfection and confirmed their expression by mass spectrometry (MS). Subsequently, we performed endogenous validation using pan-HLA immunoprecipitation mass spectrometry (IP-MS), confirming 4 (10 neoepitopes) of the 40 candidate SAAVs. We observed a discrepancy between in silico predictions and the observed sequences. Our endogenous peptidomics further revealed conserved peptide motifs and demonstrated that peptide selection for HLA presentation is transient. While our study substantiates the therapeutic feasibility of T-cell immunotherapies targeting NF1 mutations, these results underscore a limitation in current computational prediction. Our study highlights the necessity of experimental validation to refine neoantigen prioritization strategies.

Influenza neuraminidase (NA) is a promising target for universal flu vaccines, yet eliciting potent B-cell responses against its conserved epitopes remains challenging. Here, we developed a membrane-anchored, folding-domain-free NA (mNA) that elicited superior head-specific germinal center B cell and antibody responses compared to soluble tetrameric NA. In non-human primates, mNA immunization induced cross-reactive memory B cell (MBC) responses, expanding clones with the conserved "DR" motif in HCDR3, a hallmark of human broadly reactive NA antibodies. These MBCs conferred cross-inhibitory activity against diverse NA variants and in vivo cross-protection. Cryo-EM analysis revealed that the 554-C2 clone targets the conserved enzymatic pocket via the "DR" motif, while the 554-C1 clone recognizes previously uncharacterized epitopes at the interface between two adjacent N2 monomers, effectively reducing plaque formation by contemporary H3N2 strains. Our findings highlight the immunological advantages of membrane-anchoring, providing a robust strategy for designing next-generation vaccines against influenza and other pathogens.

Epitope mapping is central to rational antibody drug design, affinity optimization and the anticipation of therapeutic resistance mechanisms. Here, we demonstrate the use of Sensor Integrated Proteome on Chip (SPOC) technology for single amino acid resolution epitope mapping. By generating high throughput (HTP) binding kinetics data, we identify important residues within the target epitope whose mutations alter drug-target interactions. The SPOC platform integrates simultaneous HTP cell-free production of folded proteins in nanowells from immobilized plasmid DNAs or linear expression cassettes and capture onto biosensor chips for subsequent label-free binding kinetic analysis using surface plasmon resonance (SPR). The model system comprised the extracellular domain (ECD) of CD20, a membrane-spanning 4-domain family protein, screened against its FDA-approved therapeutic monoclonal antibodies (thAbs) - rituximab and ocrelizumab. Using our proprietary POC protein nanofactory system, a partial deep mutationally scanned (DMS) CD20 ECD mutant library of 79 variants was produced on SPOC biosensor chips via rational single amino acid substitutions of the epitope and surrounding residues with alanine, aspartic acid, lysine, and serine, collectively representing four broad classes of amino acid side chain chemistries: nonpolar, acidic, basic, and polar neutral. The SPOC protein biosensor chip was then screened with both thAbs using SPOC SPR to generate kinetic affinity data, evaluate mutations that led to affinity loss or gain, and ultimately identify critical epitope residues that interface with the antibodies. Most mutations within the rituximab and ocrelizumab epitopes - EPANPSEK and YNCEPANPSEKNSPST, respectively - resulted in complete loss of binding or >25% increase in apparent KD. Notably, N171, P172, and S173 mutations, irrespective of side chain substitution, resulted in complete loss of rituximab binding while at least three diverse side chain substitutions at E168, P169, N171, P172, S173, E174, K175, and T180, led to complete loss of binding for ocrelizumab. These outcomes identify the listed residues as the most critical contact points for their respective antibodies. Interestingly, we also found that functional side-chain substitutions at some residues flanking the epitope increased affinity. This indicates that these non-epitope residues contribute to antibody contact, and that polarity at these sites is a tractable lever for affinity modulation by targeting the corresponding contact residues on the antibody CDRs. The proposed SPOC approach of screening drug candidates against on-chip library of mutationally-scanned therapeutic targets is relevant in the early phase of drug development to resolve epitopes at the residue-level to support more informed down-selection of candidates. It facilitates cost-effective improvement of thAbs, enhancing therapeutic efficacy across a wide array of therapeutic targets, including rare variants that might otherwise lead to therapeutic resistance.

Th9/Tc9 cells polarized with TGF-{beta} and IL-4 exhibit superior antitumor efficacy, prompting extensive efforts to optimize their differentiation protocols and augment therapeutic potency. In the current study, we identified IL-36{gamma} as a potent cytokine that synergizes with TGF-{beta} to robustly drive Th9 differentiation both in the presence and absence of IL-4. IL-36{gamma}-programmed Th9 cell subsets exhibited phenotypic and transcriptional profiles identical to classic Th9 cells. Mechanistically, IL-36{gamma} drove Th9 cell differentiation through amplifying key signaling pathways such as STAT6, STAT5, and NF-{kappa}B that are essential for classic Th9 programming. Notably, we uncovered a novel regulatory axis wherein IL-36{gamma} upregulates the transactivation factor I{kappa}B{zeta}, which directly governs Th9 lineage specification. In in adoptive cell therapy (ACT) models, IL-36{gamma}-polarized Th9 cell subsets demonstrated enhanced antitumor efficacy, attributable to their sustained persistence, reduced exhaustion markers and stem-like/memory properties. Collectively, this study elucidates a previously unrecognized I{kappa}B{zeta}-dependent mechanism underpinning Th9 differentiation and highlights the translational potential of IL-36{gamma}-engineered Th9 cells as a valuable ACT strategy for refractory tumors.

Personalized T cell therapy empowered by chimeric antigen receptor (CAR) that recognizes specific tumor antigen has cured numerous blood cancer patients since its initial approval in 2017. However, its access to a broader population has been limited by the unavailability of an off-the-shelf product derived from an allogeneic donor that can evade immune rejection, which is mediated by polymorphic class I and class II human leukocyte antigens (HLAs). Since class II HLAs are only expressed in specialized antigen-presenting cells but not T cells, it might suffice to evade T cells by deleting the common class I HLA light chain Beta-2 Microglobulin (B2M) (1). However, B2M-deficient cells can trigger a "missing-self" response to activate natural killer (NK) cells (2), a second function that was evolved to compensate loss of T cell response. Inserting a less polymorphic class I HLA gene encoding a known NK inhibitory ligand, namely HLA-E or HLA-G (3), into the B2M locus so that the endogenous B2M expression is disrupted could theoretically allow evasion of both T and NK cells. Despite being a seemingly better candidate in that HLA-G is uniquely expressed in immune-privileged sites such as the placenta with a believed function in protecting the fetus from immune rejection by the pregnant mother, whereas ubiquitously-expressing HLA-E is known to bind both inhibitory and activating NK receptors (4, 5), only HLA-E engineering has been attempted yet without convincing success in vivo (6, 7). Here, we generate an off-the-shelf CAR-T product with B2M replaced by a gene fusion encoding an HLA-G single-chain trimer under minimally impacted B2M epigenetic landscape, and observe its immune evasion property and a tumor-inhibitory function that is equivalent to its autologous control using a humanized mouse model for the first time with T and NK cells reconstituted from a donor with a distant HLA haplotype. HLA-G engineering may thus reprogram T cells into an immune-privileged state that can be utilized for all cell-based therapies.

T cells are central players in killing virally infected and malignant cells. To achieve this, T cells depend on dynamic, tightly regulated alterations of the proteome. To decipher the rules instructing translation in T cells, we performed transcriptome and proteome analysis of polysome fractions from human effector CD8+ T cells. Transcriptome analysis informed on ribosomal occupancy of RNAs and uncovered the rapid and RNA-specific redistribution upon T cell activation. With machine learning, we identified RNA binding motifs that predict the RNA (re)distribution among the polysome fractions. Using matched proteome analysis, we identified polysome-associated RBPs and their swift shuttling across polysome fractions upon T cell activation. Integrating sequence feature analysis with polysome RBP localization uncovered PTBP1 as a positive translation regulator, through its interactions with cis-regulatory elements in 3'UTRs of target mRNAs. In conclusion, the multi-level analysis presented here identifies rules of selective translation control which shape the T cell proteome.

Although the endolysosome system is central to intracellular recycling, signal transduction, and intercellular communication via exocytosis, its role in immunoregulation remains incompletely defined. We recently identified that CD4+ T cell-specific depletion of BLOC1S1, a component of multiprotein complexes regulating endolysosomal biology, predisposes toward type 2 (Th2) immunity. We therefore hypothesized that the study of BLOC1S1-deficient CD4+ T cells would expand our understanding of endolysosomal dynamics in Th2 function. Here, we demonstrate that CD4+ T cell BLOC1S1 deficiency resulted in aberrant lysosomal distribution, accumulation of endosomal vesicles, and increased exocytosis, which collectively correlated with enhanced Th2 immune responses. The phenotype was associated with upregulation of key components of the exocytosis machinery, including RAB11 and VAMP7. Functional inhibition of these vesicle trafficking proteins following siRNA knockdown of RAB11 and VAMP7 significantly attenuated Th2 cytokine secretion in BLOC1S1-deficient CD4+ T cells, highlighting their essential role in exosome-mediated cytokine export. Furthermore, exosomes derived from BLOC1S1-deficient CD4+ T cells promoted Th2 polarization in recipient cells, indicating a mechanism of intracellular amplification. Together, these findings identify BLOC1S1 as a critical regulator of lysosomal dynamics and exocytic vesicle fusion, thereby linking intracellular trafficking mechanisms to Th2 immune regulation.

Behcets disease (BD) is a systemic inflammatory disease. It is considered as an autoinflammatory disease triggered by innate immunity rather than adaptive immunity. Human leukocyte antigen-B51 (HLA-B51) is the strongest genetic factor associated with BD. This study investigated how HLA class 1 molecules interact with innate immune cells and induce cytokine secretion. For this purpose, 293T cells transfected with a plasmid encoding HLA-B51 were cultured with natural killer (NK) cells obtained from healthy human donors. Within 24 h, the concentrations of interleukin-4 (IL-4), IL-8, and interferon-{gamma} (IFN-{gamma}) in the medium increased, indicating that NK cells secreted cytokines without undergoing cellular expansion for cytolysis. NK cells stimulated by nonself HLA-B51 produced IFN-{gamma} levels comparable to those produced by NK cells stimulated by self HLA-B51. NK cells carrying HLA-B51 were accurately recognized by overexpressing HLA-B51 on 293T cells. Moreover, ample intracellular IFN-{gamma} levels were detected in NK cells after stimulation with phorbol 12-myristate-13-acetate (PMA) plus ionomycin. KLRK1 (CD314)-positive cells mainly primarily accounted for IFN-{gamma}-producing cells, whereas KLRK1-negative cells did not. In contrast, both NCR1 (CD335)-positive and -negative cells contributed to IFN-{gamma} production. We next investigated whether HLA-B51 on the surface of 293T cells stimulates KLRK1 as a ligand causing IFN-{gamma} secretion. In masking experiments using anti-KLRK1 antibodies, NK cells with high levels of cell surface KLRK1 decreased the production of IFN-{gamma}. Conversely, human NK cell line KHYG1 cells also produced IFN-{gamma} in culture with 293T cells, but did not increase IFN-{gamma} through HLA-B51 stimulation. The mRNA expression of the signal adaptor protein HCST (DAP10) in KHYG1 cells was lower than that in NK cells, whereas the relative expression of IL-2RA in KHYG1 cells was higher than that in NK cells. These findings suggest that HLA-B51 can interact with KLRK1 on the NK cells inducing IFN-{gamma} secretion, whereas IL-2 signals outweigh HLA-51 stimulation in KHYG1 cells.

Based on the success in pre-clinical models, methods that reduce sialylation in tumors have progressed to clinical trials, as this improves anti-tumor cellular responses. Immune responses against cancer can also be mediated by soluble, non-cellular mechanisms, such as the complement system. Dysregulation of the complement cascade and hypersialylation are hallmarks found across tumor types. Sialic acids are known to interact with complement proteins. However, the downstream pathways involved in the regulation of the complement cascade when reducing sialylation in tumors remain unclear. Here, using human melanoma cell lines and patient samples, we show that metabolic or enzymatic targeting of sialylation directly increases the activation of the complement, enhancing C3 opsonization of tumor cells and the formation of the membrane attack complex. This is mediated by the classical pathway of the complement system, in line with increased binding of immunoglobulins to tumor cells when sialylation is impaired. Our work positions the complement cascade as a relevant anti-tumor response playing a role when sialylation is targeted for cancer treatment. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/723302v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@8ef68forg.highwire.dtl.DTLVardef@1dd30d4org.highwire.dtl.DTLVardef@b0c9ecorg.highwire.dtl.DTLVardef@98cc49_HPS_FORMAT_FIGEXP M_FIG C_FIG

Orthohantaviruses cause severe human diseases including hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), with case fatality rates up to 40%. No FDA-approved therapeutics are currently available, highlighting urgent need for drug development following recent outbreak events. We systematically examined host protease dependencies in hantavirus replication, focusing on Signal Peptidase (SP) and Signal Peptide Peptidase (SPP) essential for viral glycoprotein maturation. Through comprehensive database mining and molecular docking analysis, we identified six potential protease inhibitors, with Compound E achieving the highest binding confidence score (-0.28) against SPP. Our analysis reveals that targeting host ER proteases represents a viable antiviral strategy, providing a systematic framework for protease-targeted antihantavirus drug development and identifying specific lead compounds for experimental validation.

Immune checkpoint inhibitors (ICI) have revolutionized cancer therapy, yet response rates remain suboptimal across many solid tumors, and resistance mechanisms, particularly those involving glycans, are not fully understood. Recent studies have identified sialic acid-containing glycans and their interactions with Siglec receptors on tumor-associated macrophages as an important contributor to immune suppression within the tumor microenvironment (TME). Targeting this sialic acid-Siglec axis by glycan engineering with sialidases and other glycosidases has shown therapeutic potential in preclinical models. However, safe and effective delivery of sialidases to tumors remains a challenge. Here, we present a novel approach using adeno-associated virus (AAV)-mediated therapy to deliver sialidases (AAVSia) and other glycosidases, including fucosidase, directly to the TME. Intratumoral administration of AAVSia in mouse models resulted in significant tumor growth reduction, enhanced survival, and robust systemic antitumor immunity through improved cross-presentation and dendritic cell activation. Furthermore, combining local sialidase expression with fucosidase treatment and classical PD-1 blockade allowed a synergistic effect, amplifying antitumor response. Our findings highlight the therapeutic promise of glycoengineering the TME using local delivery systems and support the development of combination strategies to overcome glycan-mediated resistance in cancer immunotherapy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/720097v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@dc9d72org.highwire.dtl.DTLVardef@1e4e455org.highwire.dtl.DTLVardef@4a8f93org.highwire.dtl.DTLVardef@11813a3_HPS_FORMAT_FIGEXP M_FIG C_FIG