BCMA-BBZ-OX40 CAR-T Therapy Using an Instant Manufacturing Platform in Multiple Myeloma.

BACKGROUND: Chimeric antigen receptor (CAR)-T cell has revolutionary efficacy against relapsed/refractory multiple myeloma (R/R MM). However, current CAR-T cell therapy has several limitations including long vein-to-vein time and limited viability. METHODS: A 4-1BB-costimulated B-cell maturation antigen (BCMA) CAR-T integrating an independently-expressed OX40 (BCMA-BBZ-OX40) was designed and generated by a traditional manufacturing process (TraditionCART) or instant manufacturing platform (named InstanCART). The tumor-killing efficiency, differentiation, exhaustion, and expansion level were investigated in vitro and in tumor-bearing mice. An investigator-initiated clinical trial was performed in patients with R/R MM to evaluate the outcomes of both TraditionCART and InstanCART. The primary objective was safety within 1 month after CAR-T cell infusion. The secondary objective was the best overall response rate. RESULTS: Preclinical studies revealed that integrated OX40 conferred BCMA CAR-T cells with superior cytotoxicity and reduced exhaustion levels. InstanCART process further enhanced the proliferation and T-cell stemness of BCMA-BBZ-OX40 CAR-T cells. BCMA-BBZ-OX40 CAR-T cells were successfully administered in 22 patients with R/R MM, including 15 patients with TraditionCART and 7 patients with InstanCART. Up to 50% (11/22) patients had a high-risk cytogenetic profile and 36% (8/22) had extramedullary disease. CAR-T therapy caused grade 1-2 cytokine release syndrome in 19/22 (80%) patients, grade 1 neurotoxicity in 2/22 (9%) patients and led to ≥grade 3 adverse events including neutropenia (20/22, 91%), thrombocytopenia (15/22, 68%), anemia (12/22, 55%), creatinine increased (1/22, 5%), hepatic enzymes increased (5/22, 23%), and sepsis (1/22, 5%). The best overall response rate was 100%, and 64% (14/22) of the patients had a complete response or better. The median manufacturing time was shorter for InstanCART therapy (3 days) than for TraditionCART therapy (10 days). Expansion and duration were dramatically higher for InstanCART cells than for TraditionCART cells. CONCLUSIONS: BCMA-BBZ-OX40 CAR-T cells were well tolerated and exhibited potent responses in patients with R/R MM. InstanCART shortened the manufacturing period compared to TraditionCART, and improved the cellular kinetics. Our results demonstrated the potency and feasibility of OX40-modified BCMA CAR-T cells using InstanCART technology for R/R MM therapy. TRIAL REGISTRATION NUMBER: This trial was registered at www. CLINICALTRIALS: gov as #NCT04537442.

Pharmacologic HIF stabilization activates costimulatory receptor expression to increase antitumor efficacy of adoptive T cell therapy.

Adoptive cell transfer (ACT) is a therapeutic strategy to augment antitumor immunity. Here, we report that ex vivo treatment of mouse CD8+ T cells with dimethyloxalylglycine (DMOG), a stabilizer of hypoxia-inducible factors (HIFs), induced HIF binding to the genes encoding the costimulatory receptors CD81, GITR, OX40, and 4-1BB, leading to increased expression. DMOG treatment increased T cell killing of melanoma cells, which was further augmented by agonist antibodies targeting each costimulatory receptor. In tumor-bearing mice, ACT using T cells treated ex vivo with DMOG and agonist antibodies resulted in decreased tumor growth compared to ACT using control T cells and increased intratumoral markers of CD8+ T cells (CD7, CD8A, and CD8B1), natural killer cells (NCR1 and KLRK1), and cytolytic activity (perforin-1 and tumor necrosis factor-α). Costimulatory receptor gene expression was also induced when CD8+ T cells were treated with three highly selective HIF stabilizers that are currently in clinical use.

The role of OX40 ligand/OX40 axis signalling in atopic dermatitis.

Atopic dermatitis (AD) is a heterogeneous inflammatory condition involving multiple immune pathways mediated by pathogenic T cells. OX40 ligand (OX40L) and OX40 are costimulatory immune checkpoint molecules that regulate effector and memory T-cell activity and promote sustained immune responses in multiple immunological pathways, including T helper (Th)2, Th1, Th17 and Th22. As such, OX40L/OX40 signalling between antigen-presenting cells (APCs) and activated T cells postantigen recognition promotes pathogenic T-cell proliferation and survival. Under inflammatory conditions, OX40L is upregulated on APCs, enhancing the magnitude of antigen-specific T-cell responses and secretion of proinflammatory cytokines. In AD, OX40L/OX40 signalling contributes to the amplification and chronic persistence of T-cell-mediated inflammation. Recent therapeutic success in clinical trials has highlighted the importance of the OX40L/OX40 axis as a promising target for the treatment of AD. Here, we discuss the many factors that are involved in the expression of OX40L and OX40, including the cytokine milieu, antigen presentation, the inflammatory environment in AD, and the therapeutic direction influenced by this costimulatory pathway.

Atopic dermatitis (AD) (also known as atopic eczema) is a common skin disease caused by inflammation, and affects 2­3 of every 10 people worldwide. AD affects people of all ages and can cause a range of symptoms, including dry thickened skin, itchiness, rashes and pain. Despite the recent addition of new targeted treatment options, there is still a need for new treatments for people with moderate-to-severe AD. New drugs are being studied that target two important signalling molecules in the immune system, called OX40 ligand (OX40L) and OX40. OX40L and OX40 bind together to continue the cycle of immune system activation, leading to increasing symptoms of AD. Blocking the OX40L and OX40 interaction may ease or stop symptoms of AD. This review outlines what is currently known about the causes of AD, including the role played by the immune system and specifically the role of OX40L and OX40. We also highlight the development of new treatments that target the OX40L and OX40 interaction to treat AD, and suggest what the future may hold for managing AD.

Therapeutic potential of co-signaling receptor modulation in hepatitis B.

Reversing CD8+ T cell dysfunction is crucial in treating chronic hepatitis B virus (HBV) infection, yet specific molecular targets remain unclear. Our study analyzed co-signaling receptors during hepatocellular priming and traced the trajectory and fate of dysfunctional HBV-specific CD8+ T cells. Early on, these cells upregulate PD-1, CTLA-4, LAG-3, OX40, 4-1BB, and ICOS. While blocking co-inhibitory receptors had minimal effect, activating 4-1BB and OX40 converted them into antiviral effectors. Prolonged stimulation led to a self-renewing, long-lived, heterogeneous population with a unique transcriptional profile. This includes dysfunctional progenitor/stem-like (TSL) cells and two distinct dysfunctional tissue-resident memory (TRM) populations. While 4-1BB expression is ubiquitously maintained, OX40 expression is limited to TSL. In chronic settings, only 4-1BB stimulation conferred antiviral activity. In HBeAg+ chronic patients, 4-1BB activation showed the highest potential to rejuvenate dysfunctional CD8+ T cells. Targeting all dysfunctional T cells, rather than only stem-like precursors, holds promise for treating chronic HBV infection.

ABIN1 is a negative regulator of effector functions in cytotoxic T cells.

T cells are pivotal in the adaptive immune defense, necessitating a delicate balance between robust response against infections and self-tolerance. Their activation involves intricate cross-talk among signaling pathways triggered by the T-cell antigen receptors (TCR) and co-stimulatory or inhibitory receptors. The molecular regulation of these complex signaling networks is still incompletely understood. Here, we identify the adaptor protein ABIN1 as a component of the signaling complexes of GITR and OX40 co-stimulation receptors. T cells lacking ABIN1 are hyper-responsive ex vivo, exhibit enhanced responses to cognate infections, and superior ability to induce experimental autoimmune diabetes in mice. ABIN1 negatively regulates p38 kinase activation and late NF-κB target genes. P38 is at least partially responsible for the upregulation of the key effector proteins IFNG and GZMB in ABIN1-deficient T cells after TCR stimulation. Our findings reveal the intricate role of ABIN1 in T-cell regulation.

Blockade of OX40/OX40L signaling using anti-OX40L alleviates murine lupus nephritis.

Genetic variants of the OX40 ligand (OX40L) locus are associated with the risk of systemic lupus erythematosus (SLE), it is unclear how the OX40L blockade delays the lupus phenotype. Therefore, we examined the effects of an anti-OX40L antibody in MRL/Lpr mice. Next, we investigated the effect of anti-OX40L on immunosuppression in keyhole limpet hemocyanin-immunized C57BL/6J mice. In vitro treatment of anti-OX40L in CD4+ T and B220+ B cells was used to explore the role of OX40L in the pathogenesis of SLE. Anti-OX40L alleviated murine lupus nephritis, accompanied by decreased production of anti-dsDNA and proteinuria, as well as lower frequencies of splenic T helper (Th) 1 and T-follicular helper cells (Tfh). In keyhole limpet hemocyanin-immunized mice, decreased levels of immunoglobulins and plasmablasts were observed in the anti-OX40L group. Anti-OX40L reduced the number and area of germinal centers. Compared with the control IgG group, anti-OX40L downregulated CD4+ T-cell differentiation into Th1 and Tfh cells and upregulated CD4+ T-cell differentiation into regulatory T cells in vitro. Furthermore, anti-OX40L inhibited toll-like receptor 7-mediated differentiation of antibody-secreting cells and antibody production through the regulation of the SPIB-BLIMP1-XBP1 axis in B cells. These results suggest that OX40L is a promising therapeutic target for SLE.

Facile generation of biepitopic antibodies with intrinsic agonism for activating tumor necrosis factor receptors.

Agonist antibodies are being pursued for therapeutic applications ranging from neurodegenerative diseases to cancer. For the tumor necrosis factor (TNF) receptor superfamily, higher-order clustering of three or more receptors is key to their activation, which can be achieved using antibodies that recognize two unique epitopes. However, the generation of biepitopic (i.e., biparatopic) antibodies typically requires animal immunization and is laborious and unpredictable. Here, we report a simple method for identifying biepitopic antibodies that potently activate TNF receptors without the need for additional animal immunization. Our approach uses existing, receptor-specific IgGs, which lack intrinsic agonist activity, to block their corresponding epitopes, then selects single-chain antibodies that bind accessible epitopes. The selected antibodies are fused to the light chains of IgGs to generate human tetravalent antibodies. We highlight the broad utility of this approach by converting several clinical-stage antibodies against OX40 and CD137 (4-1BB) into biepitopic antibodies with potent agonist activity.

High sensitivity of host Helios+/Neuropilin-1+ Treg to pretransplant conditioning hampers development of OX40bright/integrin-ß7+ regulatory cells in acute gastrointestinal GvHD.

This study sought to compare the behavior of Treg subsets displaying different coexpression patterns of Neuropilin-1 (Nrp1) and Helios, under the influence of gut stress unrelated to hematopoietic stem cell transplantation, pretransplantation conditioning, and posttransplant gastrointestinal acute graft versus host disease (GI-aGvHD). Host CD4+/CD25hi/Foxp3+ Treg cells, identified by flow cytometry, were isolated from various tissues of mice affected by these stressors. Expression of CD25, CTLA-4, CD39, OX40, integrin-ß7, LAG3, TGFß/LAP, granzyme-A, -B, and interleukin-10 was compared in four Treg subsets displaying Helios or Nrp1 only, both or none. Fluorescence-activated cell sorter-sorted Treg subsets, displaying markers affected in a conditioning- and GI-aGVHD-restricted manner, were further investigated by transcriptome profiling and T-cell suppression assays. We found that conditioning by irradiation greatly diminished the relative frequency of Helios+/Nrp1+ Treg, shifting the balance toward Helios-/Nrp1- Treg in the host. Upregulation of integrin-ß7 and OX40 occurred in GI-aGvHD-dependent manner in Helios+/Nrp1+ cells but not in Helios-/Nrp1- Treg. Sorted Treg subsets, confirmed to overexpress Nrp1, Helios, OX40, or integrin-ß7, displayed superior immunosuppressive activity and enrichment in activation-related messenger RNA transcripts. Our data suggest that conditioning-induced shrinkage of the Nrp1+/Helios+ Treg subset may contribute to the development of GI-GvHD by impairing gut homing and decreasing the efficiency of Treg-mediated immunosuppression.

OX40/OX40 ligand and its role in precision immune oncology.

Immune checkpoint inhibitors have changed the treatment landscape for various malignancies; however, their benefit is limited to a subset of patients. The immune machinery includes both mediators of suppression/immune evasion, such as PD-1, PD-L1, CTLA-4, and LAG-3, all of which can be inhibited by specific antibodies, and immune-stimulatory molecules, such as T-cell co-stimulatory receptors that belong to the tumor necrosis factor receptor superfamily (TNFRSF), including OX40 receptor (CD134; TNFRSF4), 4-1BB (CD137; TNFRSF9), and glucocorticoid-induced TNFR-related (GITR) protein (CD357; TNFRSF18). In particular, OX40 and its binding ligand OX40L (CD134L; TNFSF4; CD252) are critical for immunoregulation. When OX40 on activated T cells binds OX40L on antigen-presenting cells, T-cell activation and immune stimulation are initiated via enhanced T-cell survival, proliferation and cytotoxicity, memory T-cell formation, and abrogation of regulatory T cell (Treg) immunosuppressive functions. OX40 agonists are in clinical trials both as monotherapy and in combination with other immunotherapy agents, in particular specific checkpoint inhibitors, for cancer treatment. To date, however, only a minority of patients respond. Transcriptomic profiling reveals that OX40 and OX40L expression vary between and within tumor types, and that only ~ 17% of cancer patients have high OX40 and low OX40L, one of the expression patterns that might be theoretically amenable to OX40 agonist enhancement. Taken together, the data suggest that the OX40/OX40L machinery is a critical part of the immune stimulatory system and that understanding endogenous expression patterns of these molecules and co-existing checkpoints merits further investigation in the context of a precision immunotherapy strategy for cancer therapy.

OX40 in the Pathogenesis of Atopic Dermatitis-A New Therapeutic Target.

Atopic dermatitis (AD) is a chronic, heterogeneous, inflammatory disease characterized by skin lesions, pruritus, and pain. Patients with moderate-to-severe AD experience chronic symptoms, intensified by unpredictable flares, and often have comorbidities and secondary complications, which can result in significant clinical burden that impacts the patient's overall quality of life. The complex interplay of immune dysregulation and skin barrier disruption drives AD pathogenesis, of which T-cell-dependent inflammation plays a critical role in patients with AD. Despite new targeted therapies, many patients with moderate-to-severe AD fail to achieve or sustain their individual treatment goals and/or may not be suitable for or tolerate these therapies. There remains a need for a novel, efficacious, well-tolerated therapeutic option that can deliver durable benefits across a heterogeneous AD patient population. Expression of OX40 [tumor necrosis factor receptor superfamily, member 4 (TNFRSF4)], a prominent T-cell co-stimulatory molecule, and its ligand [OX40L; tumor necrosis factor superfamily, member 4 (TNFSF4)] is increased in AD. As the OX40 pathway is critical for expansion, differentiation, and survival of effector and memory T cells, its targeting might be a promising therapeutic approach to provide sustained inhibition of pathogenic T cells and associated inflammation and broad disease control. Antibodies against OX40 [rocatinlimab (AMG 451/KHK4083) and telazorlimab (GBR 830)] or OX40L [amlitelimab (KY1005)] have shown promising results in early-phase clinical studies of moderate-to-severe AD, highlighting the importance of OX40 signaling as a new therapeutic target in AD.

Noninvasive Imaging OX40+ Activated T Cells Provides Early Warning of Rheumatoid Arthritis.

PURPOSE: The goal of this study was to develop an imaging probe-IRDye-680RD-OX40 mAb-that can be used for noninvasive imaging and optical imaging of rheumatoid arthritis (RA). OX40/OX40 ligand (OX40L) interactions have been shown to exert potent costimulatory effects on T cell activation. Detectable change in T cell activation profiles was observed in early RA. METHODS: OX40 expression pattern was analyzed by flow cytometry. N-hydroxysuccinimide (NHS) esters are used to label proteins selectively on free amino groups of OX40 monoclonal antibody (mAb). Characterization of IRDye-680RD-OX40 mAb was measured and a fluorescence spectrum gathered. Cell binding assay was also performed between activated and naïve murine T cells. Longitudinal near-infrared fluorescence (NIRF) imaging of the probe was performed on day 8, day 9, day 10, and day 11 of adjuvant-induced arthritis (AIA) mouse model. Paw thickness and body weight were compared between the OX40 mAb and IgG injection groups. RESULTS: NIRF imaging with IRDye-680RD-OX40 mAb revealed strong OX40-positive responses with high specificity. Flow analysis showed that OX40 was specifically expressed on the surface of T cells in RP and spleen of AIA model. The AIA group was significantly differentiated from the control group at all time points with imaging monitoring. The region of interest (ROI) was in line with ex vivo imaging and biodistribution study. This study highlights the potential utility of the OX40 NIRF imaging as a new strategy for RA prediction and T cell monitoring. CONCLUSION: The results provide evidence that IRDye-680RD-OX40 mAb detects organized T cells activation in early RA. The optical probe was capable of detection of RA pathogenesis. It identified transcriptional responses to RA that mediate its immune functions. Thus, it may be an ideal probe for RA imaging.

Synovial Macrophages Expression of OX40L Is Required for Follicular Helper T Cells Differentiation in the Joint Microenvironment.

Signaling via the OX40/OX40L axis plays a key role in CD4+ T cell development, and OX40L expression is primarily restricted to antigen-presenting cells (APCs). This study was designed to assess the role of APC-mediated OX40L expression in the context of the development of rheumatoid arthritis (RA)-associated CD4+ T cell subsets. For these analyses, clinical samples were harvested from patients with osteoarthritis and RA, with additional analyses performed using OX40-/- mice and mice harboring monocyte/macrophage-specific deletions of OX40L. Together, these analyses revealed tissue-specific roles for OX40/OX40L signaling in RA. Specifically, higher levels of synovial macrophage OX40L expression were associated with the enhanced development of T follicular helper cells in the joint microenvironment, thereby contributing to the pathogenesis of RA. This Tfh differentiation was found to be OX40/OX40L-dependent in this synovial setting. Overall, these results indicate that the expression of OX40L by synovia macrophages is necessary to support Tfh differentiation in the joint tissues, thus offering new insight regarding the etiological basis for RA progression.

Structural Basis of a Novel Agonistic Anti-OX40 Antibody.

Agonistic antibodies targeting co-stimulating receptor OX40 on T cells are considered as important as (or complementary to) the immune checkpoint blockers in cancer treatment. However, none of these agonistic antibodies have reached the late stage of clinical development partially due to the lack of intrinsic potency with the correlation between binding epitope and activity of the antibody not well understood. Here, we identified a novel anti-OX40 agonistic antibody DF004, which stimulated the proliferation of human CD4+ T cells in vitro and inhibited tumor growth in a mouse model. Our crystallography structural studies showed that DF004 binds to the CRD2 region of OX40 while RG7888, an OX40 agonist antibody developed by Roche, binds to CRD3 of OX40 to the diametrically opposite position of DF004. This suggests that the agonistic activities of the antibodies are not necessarily epitope dependent. As their agonistic activities critically depend on clustering or cross-linking, our structural modeling indicates that the agonistic activity requires the optimal positioning of three Fc receptor/antibody/OX40 complexes on the cell membrane to facilitate the formation of one intracellular hexameric TRAF complex for downstream signal transduction, which is relatively inefficient. This may explain the lack of sufficient potency of these OX40 antibodies in a therapeutic setting and sheds light on the development of cross-linking-independent agonistic antibodies.

Expression of the immune checkpoint modulator OX40 indicates poor survival in acute myeloid leukemia.

Despite therapeutic advances, mortality of Acute Myeloid Leukemia (AML) is still high. Currently, the determination of prognosis which guides treatment decisions mainly relies on genetic markers. Besides molecular mechanisms, the ability of malignant cells to evade immune surveillance influences the disease outcome and, among others, the expression of checkpoints modulators contributes to this. In AML, functional expression of the checkpoint molecule OX40 was reported, but the prognostic relevance of OX40 and its ligand OX40L axis has so far not been investigated. Here we described expression and prognostic relevance of the checkpoint modulators OX40 and OX40L, analyzed on primary AML cells obtained from 92 therapy naïve patients. Substantial expression of OX40 and OX40L on AML blasts was detected in 29% and 32% of the investigated subjects, respectively, without correlation between the expression of the receptor and its ligand. Whereas OX40L expression was not associated with different survival, patients with high expression levels of the receptor (OX40high) on AML blasts survived significantly shorter than OX40low patients (p = 0.009, HR 0.46, 95% CI 0.24-0.86), which identifies OX40 as novel prognostic marker and a potential therapeutic target in AML patients.

Identification and validation of TNFRSF4 as a high-profile biomarker for prognosis and immunomodulation in endometrial carcinoma.

BACKGROUND: The interaction between tumor microenvironment (TME) and tumors offers various targets in mounting anti-tumor immunotherapies. However, the prognostic biomarkers in endometrial carcinoma (EC) are still limited. Here, we aimed to analyze the TME features and identify novel prognostic biomarkers for EC. METHODS: ESTIMATE, CIBERSORT, protein-protein interaction (PPI) network, univariate and multivariate Cox regression, and functional enrichment analysis were performed to identify immune- and survival-related hub genes as well as possible molecular mechanisms. The limma package and deconvolution algorithm were adopted to estimate the abundance of tumor-infiltrating immune cells (TICs) and their relationship with the target gene. In the validation section, tissue microarrays (TMAs) of EC and multiplex immunohistochemistry (m-IHC) were evaluated to validate the expression of TNFRSF4, and its correlation with immune markers, including CD4, CD8, and FOXP3. Besides, the receiver operating characteristic (ROC) curve was plotted to determine the diagnostic performance of TNFRSF4, CD4, CD8, and FOXP3 in EC. RESULTS: Two genes, TNFRSF4 and S1PR4, were screened out from 386 intersection differential expression genes (DEGs) shared by ImmuneScore and StromalScore in EC. Highlighted by TNFRSF4, we found that it was not only positively correlated with the TICs (mainly CD4+ T cells, CD8+ T cells, and Tregs) but significantly related to the prognosis in patients of EC, both verified by data from The Cancer Genome Altas (TCGA)-EC database and clinical samples. At the same time, the expression trend of TNFRSF4 was further confirmed by an integrated meta-analysis based on six microarrays from the Gene Expression Omnibus database (GEO). CONCLUSIONS: Collectively, TNFRSF4, a previously unrecognized key player in EC, could serve as a potential biomarker for prognosis prediction and immunomodulation of EC.

Clinical Significance of OX40 and OX40 Ligand in the Peripheral Blood of Patients with Myasthenia Gravis.

Background: A previous study on thymomas in myasthenia gravis (MG) patients indicated that OX40 expression may be upregulated in thymic tissues adjacent to germinal centers (GCs) and thymomas, and OX40 may interact with OX40L in GCs to enhance anti-acetylcholine receptor antibody production. However, little is known about the clinical significance of the expression of OX40 and OX40L in the peripheral blood of patients with MG. We aimed to characterize the expression of membrane-bound and soluble OX40 and OX40L in the peripheral blood of patients with MG and to identify their clinical significance. Methods: For membrane molecules, we collected peripheral blood (PB) from 39 MG patients at baseline, 22 patients in relapse, and 42 patients in remission, as well as from 36 healthy participants as controls. For soluble molecules, plasma from 37 MG patients at baseline, 34 patients in relapse, and 30 patients in remission, as well as plasma from 36 healthy controls (HC), was retrospectively collected from the sample bank of the First Hospital of Soochow University. The expression of membrane-bound OX40 and OX40L (mOX40 and mOX40L) by immune cells was measured using flow cytometry. Plasma levels of soluble OX40 and OX40L (sOX40 and sOX40L) were measured by ELISA. Results: (1) The expression of OX40 on CD4+ T cells and that of OX40L on B cells and monocytes were significantly increased, and the levels of sOX40 were significantly decreased in MG patients at baseline compared with HC, while the expression of sOX40L was not significantly different between the two groups. (2) Dynamic observation of the molecules showed significantly higher expression of OX40 on CD4+ T cells and higher levels of sOX40 in MG patients in relapse than in MG patients at baseline and MG patients in remission. Furthermore, the expression levels of sOX40 were significantly elevated in MG patients in remission compared with MG patients at baseline, and the expression of sOX40L was significantly lower in MG patients in remission than in MG patients at baseline and MG patients in relapse. (3) Plasma levels of sOX40 and sOX40L were significantly decreased in 13 patients with relapsed MG after immunosuppressive treatment compared with those before treatment. (4) Correlation analysis showed that the expression of OX40 on CD4+ T cells in patients with relapsed MG was positively correlated with the concentration of acetylcholine receptor antibodies (AchR-Ab), whereas the expression of OX40L on CD19+ B cells and CD14+ monocytes was negatively correlated with disease duration. (5) Binary regression analysis showed that patients with high CD4+ OX40 expression and high sOX40L levels had an increased risk of relapse. Conclusions: OX40 and OX40L are abnormally expressed in the peripheral blood of patients with MG and may be closely associated with disease status and treatment. The OX40/OX40L pathway may be involved in the immunopathological process of MG and may play a role mainly in the later stage of MG.

Biomimetic nanoparticles deliver mRNAs encoding costimulatory receptors and enhance T cell mediated cancer immunotherapy.

Antibodies targeting costimulatory receptors of T cells have been developed for the activation of T cell immunity in cancer immunotherapy. However, costimulatory molecule expression is often lacking in tumor-infiltrating immune cells, which can impede antibody-mediated immunotherapy. Here, we hypothesize that delivery of costimulatory receptor mRNA to tumor-infiltrating T cells will enhance the antitumor effects of antibodies. We first design a library of biomimetic nanoparticles and find that phospholipid nanoparticles (PL1) effectively deliver costimulatory receptor mRNA (CD137 or OX40) to T cells. Then, we demonstrate that the combination of PL1-OX40 mRNA and anti-OX40 antibody exhibits significantly improved antitumor activity compared to anti-OX40 antibody alone in multiple tumor models. This treatment regimen results in a 60% complete response rate in the A20 tumor model, with these mice being resistant to rechallenge by A20 tumor cells. Additionally, the combination of PL1-OX40 mRNA and anti-OX40 antibody significantly boosts the antitumor immune response to anti-PD-1 + anti-CTLA-4 antibodies in the B16F10 tumor model. This study supports the concept of delivering mRNA encoding costimulatory receptors in combination with the corresponding agonistic antibody as a strategy to enhance cancer immunotherapy.

Tnfrsf4-expressing regulatory T cells promote immune escape of chronic myeloid leukemia stem cells.

Leukemia stem cells (LSCs) promote the disease and seem resistant to therapy and immune control. Why LSCs are selectively resistant against elimination by CD8+ cytotoxic T cells (CTLs) is still unknown. In this study, we demonstrate that LSCs in chronic myeloid leukemia (CML) can be recognized and killed by CD8+ CTLs in vitro. However, Tregs, which preferentially localized close to CD8+ CTLs in CML BM, protected LSCs from MHC class I-dependent CD8+ CTL-mediated elimination in vivo. BM Tregs in CML were characterized by the selective expression of tumor necrosis factor receptor 4 (Tnfrsf4). Stimulation of Tnfrsf4 signaling did not deplete Tregs but reduced the capacity of Tregs to protect LSCs from CD8+ CTL-mediated killing. In the BM of newly diagnosed CML patients, TNFRSF4 mRNA levels were significantly increased and correlated with the expression of the Treg-restricted transcription factor FOXP3. Overall, these results identify Tregs as key regulators of immune escape of LSCs and TNFRSF4 as a potential target to reduce the function of Tregs and boost antileukemic immunity in CML.

SCFAs promote intestinal double-negative T cells to regulate the inflammatory response mediated by NLRP3 inflammasome.

Short-chain fatty acids (SCFAs) are a product of intestinal bacteria metabolism. Our previous study has found that intestinal bacteria in patients with Alzheimer's disease (AD) can promote the activation of NLRP3 inflammasome and mediate neuroinflammation. In this study, we mainly explored the regulation of intestinal microenvironmental immunity by intestinal bacterial metabolite SCFAs and the mechanism of NLRP3 activation. First, wild-type (WT) and APP/PS1 mice were intervened with SCFAs. As a result, the proportion of double-negative T cells (CD3+CD4-CD8-, DNTs) in the intestine was increased, SCFAs could promote the expression of intestinal NLRP3 and inflammatory factors (IL-18, IL-6 and TNF-α). Moreover, SCAFs could also promote the level of inflammatory factors in the cerebrospinal fluid (CSF) of mice and aggravate the cognitive impairment in AD mice. CD3+ T cells isolated from the spleen were pre-treated with SCFAs, followed by detection of the proportion of DNTs. Consequently, SCFAs could promote the formation of DNTs, activate OX40 signal and simultaneously up-regulate the protein expression of Bcl-2, Bcl-xl and Survivin. Knockdown of OX40 could inhibit SCFAs-induced differentiation of DNTs. The co-culture of DNTs and intestinal macrophages showed that DNTs could activate Fas/FasL-TNF-α signal and induce the activation of NLRP3 inflammasome. In AD mouse models, treatment with Fas and TNFR1 inhibitors could significantly inhibit SCFAs-induced NLRP3 activation and inflammatory factors, while attenuate the inflammatory response in the brain tissue of mice and improve the cognitive ability of mice, however, without significant effect on the level of DNTs. The present study showed that SCFAs can promote the formation of DNTs through OX40. DNTs could induce the activation of NLRP3 inflammasome and the release of inflammatory factors in macrophages through Fas/FasL-TNF-α signals, thereby increasing the level of inflammatory factors in the central nervous system. When Fas and TNFR1 were inhibited by suppressing the functions of DNTs and macrophages, the activation of NLRP3 was inhibited. DNTs are affected by SCFAs, which is a new mechanism of neuroinflammation in AD.

Tumor eradicated by combination of imiquimod and OX40 agonist for in situ vaccination.

Various cancer vaccines have been developed to generate and amplify antigen-specific T cell responses against malignancy. Among them, in situ vaccination is one of the most practical types as it can trigger immune responses without previous antigen identification. Here we reported a novel in situ vaccine by intratumoral injection of imiquimod and OX40 agonist. In mice bearing hepatic carcinoma, both the injected tumor and the noninjected tumor in the distant lesion of the same mice were suppressed after vaccination. Further studies found that this in situ vaccine triggered systemic tumor-specific responses, with one-fold increase of effector memory T cells properties and stronger toxicity of lymphocytes in spleen. Besides, we found that imiquimod upregulated the expression of OX40 on CD4+ T cells and thus enhanced the effectiveness of OX40 agonist. Five immune-positive-related pathways were activated after vaccination. This in situ vaccine caused little harm to normal organs and provided long-term protection against the same syngeneic tumor rechallenge. Due to its effectiveness, feasibility and safety, this strategy could potentially be applied to various types of late-stage solid tumors and worthy of further clinical research.