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The mechanisms that regulate the T(H)9 subset of helper T cells and diseases mediated by T(H)9 cells remain poorly defined. Here we found that the costimulatory receptor OX40 was a powerful inducer of T(H)9 cells in vitro and T(H)9 cell-dependent airway inflammation in vivo. In polarizing conditions based on transforming growth factor-β (TGF-β), ligation of OX40 inhibited the production of induced regulatory T cells and the T(H)17 subset of helper T cells and diverted CD4(+)Foxp3(-) T cells to a T(H)9 phenotype. Mechanistically, OX40 activated the ubiquitin ligase TRAF6, which triggered induction of the kinase NIK in CD4(+) T cells and the noncanonical transcription factor NF-κB pathway; this subsequently led to the generation of T(H)9 cells. Thus, our study identifies a previously unknown mechanism for the induction of T(H)9 cells and may have important clinical implications in allergic inflammation.

OX40, a member of the tumor necrosis factor (TNF) receptor superfamily, is expressed on the surface of activated T cells. Upon interaction with its cognate ligand, OX40L, OX40 transmits costimulatory signals to antigen-primed T cells, promoting their activation, differentiation, and survival — processes essential for the establishment of adaptive immunity. Although the OX40-OX40L interaction has been extensively studied in the context of disease treatment, developing a substitute for the naturally expressed membrane-bound OX40L, particularly a multimerized OX40L trimers, that effectively regulates OX40-driven T cell responses remains a significant challenge. In this study, we successfully engineered soluble OX40L-fusion proteins capable of robustly activating OX40 on T cells. This was achieved by incorporating functional multimerization domains into the TNF homology domain of OX40L. These OX40L proteins bound to OX40, subsequently activated NF-κB signaling, and induced cytokine production by T cells in vitro . In vivo , mice treated with one of the OX40L-fusion proteins — comprising a single-chain OX40L trimer linked to the C-terminus of the human IgG1 Fc domain, forming a dimer of trimers — exhibited significantly enhanced clonal expansion of antigen-specific CD4 + T cells during the primary phase of the immune response. A comparable antibody-fusion single-chain TNF protein incorporating 4-1BBL, CD70 (CD27L), or GITRL in place of OX40L elicited similar in vivo T cell responses. Thus, we propose that optimizing the multimerization of OX40L proteins through innovative design strategies may facilitate the development of more effective agonists for targeted immunotherapies.

mRNA vaccines have demonstrated efficacy during the COVID-19 pandemic and are now being investigated for multiple diseases. However, concerns linger about the durability of immune responses, and the high incidence of breakthrough infections among vaccinated individuals highlights the need for improved mRNA vaccines. In this study, we investigated the effects of reinforcing costimulation via 4-1BB, a member of the TNF receptor superfamily, on immune responses elicited by mRNA vaccines. We first immunized mice with mRNA vaccines, followed by treatment with 4-1BB costimulatory antibodies to reinforce the 4-1BB pathway at different time points after vaccination. Consistent with prior studies, reinforcing 4-1BB costimulation on the day of vaccination did not result in a substantial improvement in vaccine responses. However, reinforcing 4-1BB costimulation on day 4 after vaccination, when 4-1BB expression levels were highest, resulted in a profound improvement in CD8+ T cell responses associated with enhanced protection against pathogen challenges. A similar clinical benefit was observed in a therapeutic cancer vaccine model. We also report time-dependent effects with OX40, another costimulatory molecule of the TNF receptor superfamily. These findings demonstrate that delayed reinforcement of costimulation may exert an immunologic benefit, providing insights for the development of more effective mRNA vaccines for infectious diseases and cancer.

Tumour-associated antigens (TAAs) comprise a large set of non-mutated cellular antigens recognized by T cells in human and murine cancers. Their potential as targets for immunotherapy has been explored for more than two decades 1 , yet the origins of TAA-specific T cells remain unclear. While tumour cells may be an important source of TAAs for T cell priming 2 , several recent studies suggest that infection with some viruses, including Epstein–Barr virus and influenza virus can elicit T cell responses against abnormally expressed cellular antigens that function as TAAs 3 , 4 . However, the cellular and molecular basis of such responses remains undefined. Here we show that expression of the Epstein–Barr virus signalling protein LMP1 in B cells provokes T cell responses to multiple TAAs. LMP1 signalling leads to overexpression of many cellular antigens previously shown to be TAAs, their presentation on major histocompatibility complex classes I (MHC-I) and II (MHC-II) (mainly through the endogenous pathway) and the upregulation of costimulatory ligands CD70 and OX40L, thereby inducing potent cytotoxic CD4 + and CD8 + T cell responses. These findings delineate a mechanism of infection-induced anti-tumour immunity. Furthermore, by ectopically expressing LMP1 in tumour B cells from patients with cancer and thereby enabling them to prime T cells, we develop a general approach for rapid production of autologous cytotoxic CD4 + T cells against a wide range of endogenous tumour antigens, such as TAAs and neoantigens, for treating B cell malignancies. This work stresses the need to revisit classical concepts concerning viral and tumour immunity, which will be critical to fully understand the impact of common infections on human health and to improve the rational design of immune approaches to treatment of cancers. Expression of the Epstein–Barr virus protein LMP1 in B cells increases expression of—and promotes T cell responses to—tumour-associated antigens, delineating a mechanism of infection-induced anti-tumour immunity, which could inform immune-based approaches to cancer treatment.

The tumor immune microenvironment (TIME) is often dysfunctional and complex, contributing to tumor metastasis and drug resistance. This study investigates the use of mRNA-based cancer agents as promising tools to combat and reverse refractory TIME conditions. We optimized and engineered an mRNA cancer agent encoding double tandemly repeated sequences of the T cell costimulator Oxford 40 ligand (diOX40L). The diOX40L mRNAs were encapsulated into lipid nanoparticles (LNPs) for effective delivery. The research explored its safety and antitumor effects through a series of in vivo and in vivo experiments. Our results demonstrate that diOX40L mRNAs efficiently express increased levels of OX40L proteins. The optimized diOX40L mRNA cancer agent generated potent immune costimulatory signals within the TIME, leading to decreased tumor growth and improved survival compared to the original sequence agent. OX40L expression in subcutaneous tumors promoted CD4 and CD8 T cell activation, resulting in heightened IFN-γ and IL-2 secretion and robust immune responses. Combination therapy involving PD-1 antibodies and diOX40L substantially enhanced antitumor efficacy, with increased infiltration of activated CD4 and CD8 T cells. In conclusion, our findings highlight the therapeutic potential of the optimized diOX40L mRNA cancer agent in cancer treatment and its potential as an innovative alternative to protein-based therapies. The study underscores the significance of mRNA-based agents in modulating the immune microenvironment and enhancing antitumor responses.

Regulatory T cells (Tregs) play a pivotal role in tumor immune evasion, and strategies to overcome their immunosuppressive activity are urgently needed. This study investigates the immunomodulatory effects of microwave ablation (MWA) on TNFRSF4+ Tregs, focusing on the OX40L/TNFRSF4 signaling axis as a potential therapeutic target. TNFRSF4+ Tregs were isolated from C57BL/6 mice and subjected to MWA-mimetic thermal stress. functional assays and LLC xenograft models were employed, with OX40 agonist intervention. Molecular mechanisms were analyzed via RT-qPCR, Western blot, and immunohistochemistry. The balance of tumor-infiltrating immune cells was quantified by multi-color flow cytometry. MWA induced three key effects: (1) Phenotypic shift: decreased CTLA-4+ (P<0.0001) Treg subsets, but increased OX40L+ (P<0.01) in LLC cells; (2) Functional impairment: reduced Treg-mediated support for LLC proliferation, migration, and invasion; (3) Enhanced CD8+ T cell cytotoxicity. , MWA reshaped the tumor microenvironment by significantly increasing the intratumoral CD8+/Treg ratio (P<0.001), indicating a shift toward an anti-tumor inflammatory state. Mechanistically, MWA suppressed NF-κB/IκBα/TRAF6 signaling, and these effects were amplified by an OX40 agonist, suggesting the pathway is potentially OX40L-dependent. This study demonstrates that MWA disrupts Treg immunosuppression, likely by activating OX40L/TNFRSF4 signaling, and favorably alters the balance of effector to suppressor cells, providing a novel rationale for combining thermal ablation with OX40-targeted immunotherapies in cancer treatment.

Immunity in the vaginal mucosa (VM) is of critical importance for the protection from infections and cancers. Dendritic cells (DCs) are the major antigen-presenting cells that can induce and control T cell responses. Interestingly, VM Langerhans cells (vLCs) and VM CD1c CD14 DCs (vDCs) polarize CD4 T cells toward Th2-type. However, the mechanisms underlying Th2 polarization by vDCs remain unknown. OX40L expression was determined in the human VM tissue sections, followed by the measurement of OX40L expression on vLCs, CD1c CD14 vDCs, and VM macrophages (vMØs) by flow cytometry. The roles of OX40L on vDC subsets in the induction of different types of CD4 T cell responses were assessed. Both vLCs and CD1c CD14 vDCs express surface OX40L. Neutralizing OX40L with anti-OX40L antibody significantly decreased the frequency of Th2-type CD4 T cells with a reduction of CD4 T cell proliferation, while increasing the frequency of IL - 10-producing CD4 T cell responses. Anti-OX40L did not affect vLC- or CD1c CD14 vDC-induced Th1-type T cell responses. OX40L also contributed to the induction of IL - 21 CD4 T cell responses by vLCs and CD1c CD14 vDCs. In contrast to vLCs and CD1c CD14 vDCs, vMØs expressed a minimal level of surface OX40L. Likewise, anti-OX40L did not significantly affect vMØ-induced CD4 T cell responses. OX40L contributes to vLC- and CD1c CD14 vDC-induced Th2 polarization. It also significantly affects the frequency of vLC- and CD1c CD14 vDC-induced IL - 10 and IL - 21 CD4 T cells. This study provides new insights into the immunological landscape of the human VM tissues, with implications for the development of targeted immunomodulatory strategies at this mucosal site.

Thymic stromal lymphopoietin (TSLP) potently induces deregulation of Th2 responses, a hallmark feature of allergic inflammatory diseases such as asthma, atopic dermatitis, and allergic rhinitis. However, direct downstream in vivo mediators in the TSLP-induced atopic immune cascade have not been identified. In our current study, we have shown that OX40 ligand (OX40L) is a critical in vivo mediator of TSLP-mediated Th2 responses. Treating mice with OX40L-blocking antibodies substantially inhibited immune responses induced by TSLP in the lung and skin, including Th2 inflammatory cell infiltration, cytokine secretion, and IgE production. OX40L-blocking antibodies also inhibited antigen-driven Th2 inflammation in mouse and nonhuman primate models of asthma. This treatment resulted in both blockade of the OX40-OX40L receptor-ligand interaction and depletion of OX40L-positive cells. The use of a blocking, OX40L-specific mAb thus presents a promising strategy for the treatment of allergic diseases associated with pathologic Th2 immune responses.

Background Glioblastoma is the most malignant human brain tumor and has a dismal prognosis; however, some patients show long-term survival. The interaction between the costimulatory molecule OX40 and its ligand OX40L generates key signals for T-cell activation. The augmentation of this interaction enhances antitumor immunity. In this present study, we explored whether OX40 signaling is responsible for antitumor adaptive immunity against glioblastoma and also established therapeutic antiglioma vaccination therapy. Methods Tumor specimens were obtained from patients with primary glioblastoma (n = 110) and grade III glioma (n = 34). Quantitative polymerase chain reaction (PCR), flow cytometry, and immunohistochemistry were used to analyze OX40L expression in human glioblastoma specimens. Functional consequences of OX40 signaling were studied using glioblastoma cell lines, mouse models of glioma, and T cells isolated from human subjects and mice. Cytokine production assay with mouse regulatory T cells was conducted under hypoxic conditions (1.5% O 2 ). Results OX40L mRNA was expressed in glioblastoma specimens and higher levels were associated with prolonged progression-free survival of patients with glioblastoma, who had undergone gross total resection. In this regard, OX40L protein was expressed in A172 human glioblastoma cells and its expression was induced under hypoxia, which mimics the microenvironment of glioblastoma. Notably, human CD4 T cells were activated when cocultured in anti-CD3-coated plates with A172 cells expressing OX40L, as judged by the increased production of interferon-γ. To confirm the survival advantage of OX40L expression, we then used mouse glioma models. Mice bearing glioma cells forced to express OX40L did not die during the observed period after intracranial transplantation, whereas all mice bearing glioma cells lacking OX40L died. Such a survival benefit of OX40L was not detected in nude mice with an impaired immune system. Moreover, compared with systemic intraperitoneal injection, the subcutaneous injection of the OX40 agonist antibody together with glioma cell lysates elicited stronger antitumor immunity and prolonged the survival of mice bearing glioma or glioma-initiating cell-like cells. Finally, OX40 triggering activated regulatory T cells cultured under hypoxia led to the induction of the immunosuppressive cytokine IL10. Conclusion Glioblastoma directs immunostimulation or immunosuppression through OX40 signaling, depending on its microenvironment.

Genetically modified tumor cells harboring immunomodulators may be used as therapeutic vaccines to stimulate antitumor immunity. The therapeutic benefit of these tumor vaccines is extensively investigated and mechanisms by which they boost antitumor response may be further explored. Tumor cells are large secretors of extracellular vesicles (EVs). These EVs are able to vehiculate RNA and proteins to target cells, and engineered EVs also vehiculate recombinant proteins. In this study, we explore immunomodulatory properties of EVs derived from antitumor vaccines expressing the TNFSF ligands 4-1BBL and OX40L, modulating immune response mediated by immune cells and eliminating tumors. Our results suggest that the EVs secreted by genetically modified tumor cells harboring TNFSF ligands can induce T cell proliferation, inhibit the transcription factor FoxP3, associated with the maintenance of Treg phenotype, and enhance antitumor activity mediated by immune cells. The immunomodulatory extracellular vesicles have potential to be further engineered for developing new approaches for cancer therapy.