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Regulatory T cells (T regs ) make major contributions to immune homeostasis. Because T reg dysfunction can lead to both allo- and autoimmunity, there is interest in correcting these disorders through T reg adoptive transfer. Two of the central challenges in clinically deploying T reg cellular therapies are ensuring phenotypic stability and maximizing potency. Here, we describe an approach to address both issues through the creation of OX40 ligand (OX40L)-specific chimeric antigen receptor (CAR)-T regs under the control of a synthetic forkhead box P3 ( FOXP3 ) promoter. The creation of these CAR-T regs enabled selective T reg stimulation by engagement of OX40L, a key activation antigen in alloimmunity, including both graft-versus-host disease and solid organ transplant rejection, and autoimmunity, including rheumatoid arthritis, systemic sclerosis, and systemic lupus erythematosus. We demonstrated that OX40L-CAR-T regs were robustly activated in the presence of OX40L-expressing cells, leading to up-regulation of T reg suppressive proteins without induction of proinflammatory cytokine production. Compared with control T regs , OX40L-CAR-T regs more potently suppressed alloreactive T cell proliferation in vitro and were directly inhibitory toward activated monocyte-derived dendritic cells (DCs). We identified trogocytosis as one of the central mechanisms by which these CAR-T regs effectively decrease extracellular display of OX40L, resulting in decreased DC stimulatory capacity. OX40L-CAR-T regs demonstrated an enhanced ability to control xenogeneic graft-versus-host disease compared with control T regs without abolishing the graft-versus-leukemia effect. These results suggest that OX40L-CAR-T regs may have wide applicability as a potent cellular therapy to control both allo- and autoimmune diseases.

Objective/main Outcome: To study the expression of OX40 on T follicular helper (Tfh) cells and the ligand OX40L on antigen-presenting cells (APCs) in peripheral blood of patients with type 1 diabetes mellitus (T1DM) and the role of OX40 signaling in promoting Tfh cells to assist B-cell differentiation., Design: Cross-sectional study., Setting: Endocrinology department of a university hospital., Participants: Twenty-five patients with T1DM and 35 with newly diagnosed type 2 diabetes mellitus (T2DM) from January 2021 to December 2021 (39 males, 21 females; mean age: 31.0 ± 4.5, range: 19-46 years)., Interventions: None., Methods: The peripheral blood proportion of CD4+CD25-CD127+CXCR5+PD1+ Tfh cells in patients with T1DM or T2DM and the OX40L expression in CD14+ monocytes and CD19+ B cells were analyzed by flow cytometry. The OX40 signal effect on Tfh-cell function was analyzed by coincubating B cells with Tfh cells under different conditions. Flow cytometry detected the ratio of CD19-CD138+ plasmacytes., Results: The Tfh cells ratio and intracellular IL-21 expression in peripheral blood was significantly higher in patients with T1DM than with T2DM, and the OX40 expression in peripheral Tfh cells and OX40L expression in APC were significantly higher in T1DM. After adding OX40L protein, the CD19-CD138+-plasmacytes percentage was significantly increased and higher in T1DM. Blocking of anti-OX40L monoclonal antibodies significantly reduced the plasmacytes ratio., Conclusion: The peripheral Tfh cells proportion increased and the OX40 expression in peripheral Tfh cells was upregulated in patients with T1DM vs patients with T2DM. OX40/OX40L signaling enhanced the Tfh-cell function to assist B-cell differentiation, which may contribute to the pathogenesis of T1DM., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society.)

Interleukin-33 (IL-33) is an alarmin released by epithelial cells in response to tissue damage. It activates resident immune sentinel cells, which then produce signals commonly associated with type 2 immune responses, particularly affecting infiltrating antigen-specific T cells. Given that mast cells (MCs) are a primary target of IL-33 and can shape T helper (Th) cell responses, we investigated the effect of IL-33 priming on the ability of MCs to influence Th cell cytokine production. To examine the Th cell/MC interaction, we developed human primary MC/memory CD4 + T-cell coculture systems involving both cognate and non-cognate interactions. Our results demonstrated that IL-33-primed MCs, whether as bystander cells cocultured with activated effector T cells or functioning as antigen-presenting cells, promoted IL-9 and increased IL-13 production in Th cells via an OX40L-dependent mechanism. This indicates that MCs sense IL-33-associated danger, prompting them to direct Th cells to produce the key type 2 effector cytokines IL-9 and IL-13., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Battut, Leveque, Valitutti, Cenac, Dietrich and Espinosa.)

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., Competing Interests: Conflicts of interest E.G.-Y. is an employee of Mount Sinai and has received research grants from LEO Pharma, Pfizer, Amgen, GSK, Incyte, Sanofi, Bristol Myers Squibb, Aslan, Regeneron, AnaptysBio, Concert, Janssen, Q32Bio, AbbVie, Eli Lilly, Arcutis and Inmagene Bio, and consulted for AbbVie, Almirall, Amgen, AnaptysBio, Apogee Therapeutics, Apollo Therapeutics Limited, Artax Biopharma Inc., AstraZeneca, Bristol Myers Squibb, Boehringer Ingelheim, Cara Therapeutics, Centrexion Therapeutics Corporation, Connect Biopharm, Eli Lilly, Enveda Biosciences, Escient Pharmaceuticals, Inc., Fairmount Funds Management LLC, FL2022-001, Inc., Galderma, Gate Bio, Google Ventures (GV), GSK Immunology, Inc., Incyte, Inmagene, Janssen Biotech, Japan Tobacco, Jasper Therapeutics, Kyowa Kirin, Kymera Therapeutics, Inc., LEO Pharma, Merck, Nektar Therapeutics, Novartis Pharmaceuticals Corporation, NUMAB Therapeutics AG, OrbiMed Advisors LLC, Otsuka, Pfizer, Pharmaxis Ltd, Pioneering Medicine VII, Inc., Proteologix US Inc, RAPT, RayThera, Inc, Regeneron Pharmaceuticals, Ribon Therapeutics, Inc., Sanofi, SATO, Schrödinger, Inc., Sun Pharma Advanced Research Company (SPARC), Teva Branded Pharmaceutical Products R&D, Inc. and UCB. M.C. has received consulting fees or speaking honoraria from Sanofi, Amgen, UCB, MedImmune, Millennium, Perseid, Celgene, Novo Nordisk, Merck, AbbVie, Tanabe, Zai Lab, Merrimack, Pfizer, AnaptysBio, Celsius Therapeutics, HifiBio, Kiniksa, Shattuck Labs, Prometheus Biosciences, Invectys, Virtici, Capella Bioscience and RAPT Therapeutics, and research funds from Kyowa Kirin, Bristol Myers Squibb, Janssen, and Eli Lilly. He has licensed patents on OX40 and OX40L. B.G. has received consulting fees or speaking honoraria from Sanofi, Regeneron, Pfizer, AbbVie, LEO Pharma, Eli Lilly, Novartis and Incyte, and research funds from Sanofi, Regeneron, LEO Pharma, Eli Lilly, Novartis, Incyte and Amgen. N.R., D.L., M.P., C.v.K., W.V. and H.X. are employees of Sanofi and may hold stock and/or stock options in the company. S.W. has received institutional research grants from LEO Pharma, Pfizer inc. and Sanofi, and has performed consulting work and lectures for AbbVie, Almirall, Boehringer, Eli Lilly, Galderma, GSK, Kymab, LEO Pharma, Pfizer, Sanofi and Regeneron., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Association of Dermatologists.)

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., (© 2024. The Author(s).)

A promising strategy in cancer immunotherapy is to restore or enhance the cytotoxicity of NK cells, among others, by activating the mechanism of antibody-dependent cellular cytotoxicity (ADCC). Monoclonal antibodies targeting tumor antigens, such as rituximab (targeting CD20), induce NK cell-mediated ADCC and have been used to treat B cell malignancies, such as non-Hodgkin lymphoma, but not always successfully. The aim of this study was to analyze the gene expression profile of the NK cells involved in the cytolytic response stimulated by rituximab. NK cells were co-cultured with rituximab-opsonized Raji cells. Sorting into responder and non-responder groups was based on the presence of CD107a, which is a degranulation marker. RNA-seq results showed that the KIT and TNFSF4 genes were strongly down-regulated in the degranulating population of NK cells (responders); this was further confirmed by qRT-PCR. Both genes encode surface proteins with cellular signaling abilities, namely c-KIT and the OX40 ligand. Consistent with our findings, c-KIT was previously reported to correlate inversely with cytokine production by activated NK cells. The significance of these findings for cancer immunotherapy seems essential, as the pharmacological inhibition of c-KIT and OX40L, or gene ablation, could be further tested for the enhancement of the anti-tumor activity of NK cells in response to rituximab.

Cervical cancer (CC) poses a significant health burden, particularly in low- and middle-income countries. NK cells play a crucial role against CC; however, they can become exhausted and lose their cytotoxic capacity. This work explores the expression of costimulatory receptors (ICOS, 4-1BB, OX-40) in exhausted NK cells from CC patients. Peripheral blood and tumor biopsies were collected, and flow cytometry was used to evaluate the expression of costimulatory receptors in exhausted NK cells. There is an increase of peripheral exhausted NK cells (PD-1 + TIGIT + ) in CC patients; this subpopulation has a selectively increased expression of the costimulatory receptors ICOS and 4-1BB. An exhausted population is also highly increased in tumor-infiltrating NK cells, and it shows a dramatically increased expression of the costimulatory receptors ICOS (>15×) and 4-1BB (>10×) compared to peripheral NK cells. The exhausted cells, both in the periphery and in the tumor infiltrating lymphocytes (TILs), are also more likely than non-exhausted NK cell populations (PD-1 - TIGIT - ) to express these costimulatory receptors; increases ranging from 2.0× ICOS, 2.4× 4-1BB, and 2.6× OX-40 in CD56 dim PBMCs to 1.5× ICOS, 5× 4-1BB, and 10× OX-40 in TILs were found. Our study demonstrates for the first time the increased expression of the costimulatory receptors ICOS, 4-1BB, and OX-40 in peripheral CD56 dim , CD56 bright , and tumor-infiltrating NK cells in CC. Targeting these receptors for stimulation could reverse exhaustion and be a promising immunotherapy strategy.

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., (© 2024 Wiley‐VCH GmbH.)

Background: Advancements in immunotherapeutic approaches only had a modest impact on the therapy of lung neuroendocrine neoplasms (LNENs). Our multicenter study aimed to investigate the expression patterns of novel immunotherapy targets in intermediate- and high-grade LNENs., Methods: The expressions of V-domain Ig suppressor of T cell activation (VISTA), OX40L, Glucocorticoid-induced TNF receptor (GITR), and T cell immunoglobulin and mucin domain 3 (TIM3) proteins were measured by immunohistochemistry in surgically resected tumor samples of 26 atypical carcinoid (AC), 49 large cell neuroendocrine lung cancer (LCNEC), and 66 small cell lung cancer (SCLC) patients. Tumor and immune cells were separately scored., Results: Tumor cell TIM3 expression was the highest in ACs (p < 0.001), whereas elevated tumor cell GITR levels were characteristic for both ACs and SCLCs (p < 0.001 and p = 0.011, respectively). OX40L expression of tumor cells was considerably lower in ACs (vs. SCLCs; p < 0.001). Tumor cell VISTA expression was consistently low in LNENs, with no significant differences across histological subtypes. ACs were the least immunogenic tumors concerning immune cell abundance (p < 0.001). Immune cell VISTA and GITR expressions were also significantly lower in these intermediate-grade malignancies than in SCLCs or in LCNECs. Immune cell TIM3 and GITR expressions were associated with borderline prognostic significance in our multivariate model (p = 0.057 and p = 0.071, respectively)., Conclusions: LNEN subtypes have characteristic and widely divergent VISTA, OX40L, GITR, and TIM3 protein expressions. By shedding light on the different expression patterns of these immunotherapy targets, the current multicenter study provides support for the future implementation of novel immunotherapeutic approaches., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

The parasitic helminth Schistosoma mansoni is a potent inducer of type 2 immune responses by stimulating dendritic cells (DCs) to prime T helper 2 (Th2) responses. We previously found that S. mansoni soluble egg antigens (SEA) promote the synthesis of Prostaglandin E 2 (PGE2) by DCs through ERK-dependent signaling via Dectin-1 and Dectin-2 that subsequently induces OX40L expression, licensing them for Th2 priming, yet the ligands present in SEA involved in driving this response and whether specific targeting of PGE2 synthesis by DCs could affect Th2 polarization are unknown. We here show that the ability of SEA to bind Dectin-2 and drive ERK phosphorylation, PGE2 synthesis, OX40L expression, and Th2 polarization is impaired upon cleavage of high-mannose glycans by Endoglycosidase H treatment. This identifies high-mannose glycans present on glycoproteins in SEA as important drivers of this signaling axis. Moreover, we find that OX40L expression and Th2 induction are abrogated when microsomal prostaglandin E synthase-1 (mPGES) is selectively inhibited, but not when a general COX-1/2 inhibitor is used. This shows that the de novo synthesis of PGE2 is vital for the Th2 priming function of SEA-stimulated DCs as well as points to the potential existence of other COX-dependent lipid mediators that antagonize PGE2-driven Th2 polarization. Lastly, specific PGE2 inhibition following immunization with S. mansoni eggs dampened the egg-specific Th cell response. In summary, our findings provide new insights in the molecular mechanisms underpinning Th2 induction by S. mansoni and identify druggable targets for potential control of helminth driven-Th2 responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Almeida, van Roey, Patente, Otto, Veldhuizen, Ghorasaini, van Diepen, Schramm, Liu, Idborg, Korotkova, Jakobsson, Giera, Hokke and Everts.)

We investigated expressions of PD-L1, LAG-3, TIM-3, and OX40L as immune checkpoint proteins, and MSI (repetitive short-DNA-sequences due to defective DNA-repair system) status were analyzed with immunohistochemistry from tissue blocks. Of 83 patients, PD-L1 expression was observed in 18.1% (n = 15) of the patients. None of the patients exhibited LAG-3 expression. TIM-3 expression was 4.9% (n = 4), OX40L was 22.9% (n = 19), and 8.4% (n = 7) of the patients had MSI tumor. A low-to-intermediate positive correlation was observed between PD-L1 and TIM-3 expressions (rho: 0.333, p  < 0.01). Although PD-L1 expression was higher in grade 3 NET/NEC, MSI status was prominent in grade 1/2 NET.

Stimulation of costimulatory receptors serves as an alternative immunotherapeutic strategy other than checkpoint inhibition. However, systemic administration of the agonistic antibodies is associated with severe toxicities, which is one of the major obstacles for their clinical application. This study aimed to develop a mesenchymal stem cell (MSC)-based system for tumor-targeted delivery of TNF superfamily ligands and assess their potential in enhancing antitumor immunity. Here we established an MSC-based system for tumor-targeted delivery of TNF superfamily ligands, including TNFSF4, 9 and 18. The TNFSF receptors (TNFRSFs) were evaluated in mouse models and patient samples for lung and colorectal cancers. TNFRSFs were all expressed at various levels on tumor-infiltrated lymphocytes, with TNFRSF18 being the most prevalent receptor. Human umbilical cord-derived MSCs expressing these costimulatory ligands (MSC-TNFSFs) effectively activated lymphocytes in vitro and elicited antitumor immunity in mice. TNFSF4 showed the least antitumor efficacy in both LLC1 and CT26 tumor models. MSC-TNFSF9 showed the most potent tumor-inhibiting effect in the LLC1 tumor model, while MSCs expressing TNFSF18 in combination with CXCL9 most significantly repressed CT26 tumor growth. Overall, TNFSF9 and TNFSF18 exhibited stronger lymphocyte-stimulating and antitumor activities than TNFSF4. Our study provides evidence that antitumor effects of agonism of different costimulatory receptors may vary in different tumor types and presents a promising approach for targeted delivery of TNF superfamily costimulatory ligands to avoid the systemic toxicities and side effects associated with immune agonist antibodies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Asthma, characterized by persistent inflammation and increased sensitivity of the airway, is the most common chronic condition among children. Novel, safe, and reliable treatment strategies are the focus of current research on pediatric asthma. Amygdalin, mainly present in bitter almonds, has anti-inflammatory and immunoregulatory potential, but its effect on asthma remains uninvestigated. Here, the impact of amygdalin on the thymic stromal lymphopoietin (TSLP)-dendritic cell (DC)-OX40L axis was investigated. A BALB/c mouse model for allergic asthma was established using the ovalbumin-sensitization method. Amygdalin treatment was administered between days 21 and 27 of the protocol. Cell numbers and hematoxylin and eosin (H&E) staining in bronchoalveolar lavage fluid (BALF) were used to observe the impact of amygdalin on airway inflammation. TSLP, IL-4, IL-5, IL-13, and IFN-γ concentrations were determined via Enzyme-linked immunosorbent assay (ELISA). TSLP, GATA-3, and T-bet proteins were measured using western blotting. Cell-surface receptor expression on DCs (MHC II, CD80, and CD86) was assessed via flow cytometry. OX40L mRNA and protein levels were detected using western blotting and qRT-PCR, respectively. Amygdalin treatment attenuated airway inflammation decreased BALF TSLP levels, inhibited DC maturation, restrained TSLP-induced DC surface marker expression (MHCII, CD80, and CD86), and further decreased OX40L levels in activated DCs. This occurred together with decreased Th2 cytokine levels (IL-4, IL-5, and IL-13) and GATA3 expression, whereas Th1 cytokine (IFN-γ) levels and T-bet expression increased. Amygdalin thus regulates the Th1/Th2 balance through the TSLP-DC-OX40L axis to participate in inflammation development in the airways, providing a basis for potential allergic asthma treatments.

OX40 (CD134, TNFRSF4) is a member of the tumor necrosis factor receptor superfamily that can be activated by its cognate ligand OX40L (CD252, TNFSF4) and functions as a pair of T cell costimulatory molecules. The interaction between OX40 and OX40L (OX40/OX40L) plays a critical role in regulating antitumor immunity, including promoting effector T cells expansion and survival, blocking natural regulatory T cells (T reg ) activity, and antagonizing inducible T reg generation. However, current OX40 agonists including anti-OX40 monoclonal antibodies (aOX40) have serious side effects after systemic administration, which limits their clinical success and application. Herein, we propose a strategy to reprogram tumor cells into OX40L-expressing "artificial" antigen-presenting cells (APCs) by OX40L plasmid-loaded nanoparticles for boosting antitumor immunity in situ. A novel gene transfection carrier was prepared by a modular hierarchical assembly method, which could efficiently transfect various tumor cells and express OX40L proteins on their surface. These surface-decorated OX40L proteins were proved to stimulate T cell proliferation in vitro while stimulating strong antitumor immune responses in vivo. Importantly, this in situ reprogramming strategy did not induce any toxicity as observed in aOX40 treatment, thus providing a novel method for immune checkpoint stimulator application.

NDV as an attractive candidate for oncolytic immunotherapy selectively lyses tumor cells but shows limited anti-tumor immunity. Immune co-stimulator OX40 ligand (OX40L) boosts anti-tumor immunity response by delivering a potent costimulatory signal to CD4 + and CD8 + T cells. To improve the anti-tumor immunity of NDV, the recombinant NDV expressing the murine OX40L (rNDV-mOX40L) was engineered. The viral growth kinetics was examined in CT26 cell lines. The ability of rNDV-mOX40L to express mOX40L was detected in the infected tumor cells and tumor tissues. The anti-tumor activity of rNDV-mOX40L was studied in the CT26 animal model. Tumor-specific CD4 + , CD8 + and OX40 + T cells were examined by immunohistochemistry staining. The virus growth curve showed that the insertion of the mOX40L gene did not affect the growth kinetics of NDV. rNDV-mOX40L expresses mOX40L and effectively inhibits the growth of CT26 colorectal cancer in vivo. The tumor inhibition rate of the rNDV-mOX40L-treated group was increased by 15.8% compared to that of  NDV-treated group in the CT26 model. Furthermore, immunohistochemistry staining of tumor tissues removed from the CT26 model revealed that intense infiltration of tumor-specific CD4 + , CD8 + T cells, especially OX40 + T cells were found in the rNDV-mOX40L-treated group. FACS showed that rNDV-mOX40L significantly enhanced the number of CD4 + and CD8 +  T cells in spleen. Moreover, compared to the NDV-treated group, the level of mouse IFN-γ protein in the tumor site increased significantly in the rNDV-mOX40L-treated group. Taken together, rNDV-mOX40L exhibited superior anti-tumor immunity by stimulating tumor-specific T cells and may be a promising agent for cancer immunotherapy., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

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 (OX40 high ) on AML blasts survived significantly shorter than OX40 low 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., (© 2022. The Author(s).)

Background: Kidney renal papillary cell carcinoma (KIRP) is a dangerous cancer, which accounts for 15-20% of all kidney malignancies. Ferroptosis is a rare kind of cell death that overcomes medication resistance. Ferroptosis-related long non-coding RNAs (LNCRNAs) in KIRP, remain unknown., Method: We wanted to express how ferroptosis-related LNCRNAs interact with immune cell infiltration in KIRP. Gene set enrichment analysis in the GO and KEGG databases were used to explore gene expression enrichment. The prognostic model was constructed using Lasso regression. In addition, we also analyzed the modifications in the tumor microenvironment (TME) and immunological association., Result: The expression of LNCRNA was closely connected to the ferroptosis, according to co-expression analyses. CASC19, AC090197.1, AC099850.3, AL033397.2, LINC00462, and B3GALT1-AS1 were found to be significantly increased in the high-risk group, indicating that all of these markers implicates the malignancy processes for KIRP patients and may be cancer-promoting variables. LNCTAM34A and AC024022.1 were shown to be significantly elevated in the low-risk group; these might represent as the KIRP tumor suppressor genes. According to the TCGA, CCR, and inflammation-promoting genes were considered to be significantly different between the low-risk and high-risk groups. The expression of CD160, TNFSF4, CD80, BTLA, and TNFRSF9 was different in the two risk groups., Conclusion: LNCRNAs associated with ferroptosis were linked to the occurrence and progression of KIRP. Ferroptosis-related LNCRNAs and immune cell infiltration in the TME may be potential biomarkers in KIRP that should be further investigated., (© 2022. The Author(s).)

RORγt is a lineage-specifying transcription factor that is expressed by immune cells that are enriched in the gastrointestinal tract and promote immunity, inflammation and tissue homeostasis 1-15 . However, fundamental questions remain with regard to the cellular heterogeneity among these cell types, the mechanisms that control protective versus inflammatory properties and their functional redundancy. Here we define all RORγt + immune cells in the intestine at single-cell resolution and identify a subset of group 3 innate lymphoid cells (ILC3s) that expresses ZBTB46, a transcription factor specifying conventional dendritic cells 16-20 . ZBTB46 is robustly expressed by CCR6 + lymphoid-tissue-inducer-like ILC3s that are developmentally and phenotypically distinct from conventional dendritic cells, and its expression is imprinted by RORγt, fine-tuned by microbiota-derived signals and increased by pro-inflammatory cytokines. ZBTB46 restrains the inflammatory properties of ILC3s, including the OX40L-dependent expansion of T helper 17 cells and the exacerbated intestinal inflammation that occurs after enteric infection. Finally, ZBTB46 + ILC3s are a major source of IL-22, and selective depletion of this population renders mice susceptible to enteric infection and associated intestinal inflammation. These results show that ZBTB46 is a transcription factor that is shared between conventional dendritic cells and ILC3s, and identify a cell-intrinsic function for ZBTB46 in restraining the pro-inflammatory properties of ILC3s and a non-redundant role for ZBTB46 + ILC3s in orchestrating intestinal health., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Proprotein convertase subtilisin/kexin type 9 (PCSK9) participates in the development of various cancers, including hepatocellular carcinoma (HCC). Here, we attempted to reveal the underlying mechanism of PCSK9 in HCC., Methods: Tumor tissues and adjacent tissues were separated from HCC patients to detect PCSK9 expression. Then, PCSK9 was overexpressed or silenced in HCC cells (MHCC97H or Huh7), and then the cell supernatant was incubated with THP-1 macrophages. OX40L neutralizing antibody (nAb) was used to inhibit OX40L activity. The expression of macrophage markers was examined by immunohistochemical staining and flow cytometry. Finally, tumor-bearing mouse model was constructed by inoculation of LV-PCSK9 infected MHCC97H cells to verify the role of PCSK in HCC., Results: PCSK9 expression was decreased in tumor tissues of HCC patient specimens. HCC patients displayed M2 macrophage infiltration in tumor tissues. Moreover, PCSK9-silenced Huh7 cell supernatant promoted cell migration, and enhanced the proportion of CD206-positive cells and the expression of M2 macrophage markers IL-10 and ARG-1 in THP-1 macrophages. PCSK9-overexpressing MHCC97H cell supernatant inhibited THP-1 macrophage migration and M2-like tumor-associated macrophage (TAM) polarization, which was abolished by OX40L nAb treatment. PCSK9 overexpression enhanced the expression of OX40L in MHCC97H cells. In tumor-bearing mouse models, PCSK9 overexpression inhibited tumor growth and M2 polarization of TAMs in HCC by promoting OX40L expression. Conclusion: This work demonstrated that PCSK9 suppressed M2-like TAM polarization by regulating the secretion of OX40L from hepatocellular carcinoma cells. This study suggests that PCSK9 may be a potential target for HCC treatment.

High frequent metastasis is the major cause of breast cancer (BC) mortality among women. However, the molecular mechanisms underlying BC metastasis remain largely unknown. Here, we identified six hub BC metastasis driver genes (BEND5, HSD11B1, NEDD9, SAA2, SH2D2A and TNFSF4) through bioinformatics analysis, among which BEND5 is the most significant gene. Low BEND5 expression predicted advanced stage and shorter overall survival in BC patients. Functional experiments showed that BEND5 could suppress BC growth and metastasis in vitro and in vivo . Mechanistically, BEND5 inhibits Notch signaling via directly interacting with transcription factor RBPJ/CSL. BEN domain of BEND5 interacts with the N-terminal domain (NTD) domain of RBPJ, thus preventing mastermind like transcriptional coactivator (MAML) from forming a transcription activation complex with RBPJ. Our study provides a novel insight into regulatory mechanisms underlying Notch signaling and suggests that BEND5 may become a promising target for BC therapy., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Natural killer (NK) cell-based immunotherapy is a promising treatment approach for multiple myeloma (MM), but obtaining a sufficient number of activated NK cells remains challenging. Here, we report an improved method to generate ex vivo expanded NK (eNK) cells from MM patients based on genetic engineering of K562 cells to express OX40 ligand and membrane-bound (mb) IL-18 and IL-21., Methods: K562-OX40L-mbIL-18/-21 cells were generated by transducing K562-OX40L cells with a lentiviral vector encoding mbIL-18 and mbIL-21, and these were used as feeder cells to expand NK cells from peripheral blood mononuclear cells of healthy donors (HDs) and MM patients in the presence of IL-2/IL-15. Purity, expansion rate, receptor expression, and functions of eNK cells were determined over four weeks of culture., Results: NK cell expansion was enhanced by short exposure of soluble IL-18 and IL-21 with K562-OX40L cells. Co-culture of NK cells with K562-OX40L-mbIL-18/-21 cells resulted in remarkable expansion of NK cells from HDs (9,860-fold) and MM patients (4,929-fold) over the 28-day culture period. Moreover, eNK cells showed increased expression of major activation markers and enhanced cytotoxicity towards target K562, U266, and RPMI8226 cells., Conclusions: Our data suggest that genetically engineered K562 cells expressing OX40L, mbIL-18, and mbIL-21 improve the expansion of NK cells, increase activation signals, and enhance their cytolytic activity towards MM cells., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2022 Xiaoling Zhou et al.)

Asthma is an inflammatory disease. Th2 differentiation plays an important role in the pathogenesis of asthma. We explored the role and action mechanism of membrane-associated RING-CH 1 (March1) in the Th2 differentiation regulated by dendritic cells (DCs). Our data showed that the expression of March1 was higher in asthmatic children-derived DCs, asthmatic mice-derived DCs and house dust mites (HDMs)-treated DCs than that in control DCs. Increasing of March1 promoted the production of pro-inflammatory cytokines from HDMs-treated DCs, and enhanced the promotion of HDMs-treated DCs to CD4 + T cell proliferation and Th2 differentiation, whereas decreasing of March1 resulted in opposite effects. Furthermore, our data indicated that March1 positively regulated the expression of OX40 ligand (OX40L) and facilitated DCs-induced Th2 differentiation through OX40L. In asthmatic mice, March1-overexpressed DCs significantly aggravated the injury in lung tissues and promoted Th2 differentiation. Overall, our data proved that highly expressed March1 in DCs facilitated asthma development through inducing Th2 differentiation by facilitating OX40L expression. Our data might provide a new idea for the treatment of asthma., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Immune checkpoint blockade, an immunotherapy, has been applied in multiple systemic malignancies and has improved overall survival to a relatively great extent; whether it can be applied in breast cancer remains unknown. We endeavored to explore possible factors that may influence immunotherapy outcomes in breast cancer using several public databases. The possible treatment target TNF superfamily member 4 (TNFSF4) was selected from many candidates based on its abnormal expression profile, survival-associated status, and ability to predict immune system reactions. For the first time, we identified the oncogenic features of TNFSF4 in breast carcinoma. TNFSF4 was revealed to be closely related to treatment that induced antitumor immunity and to interact with multiple immune effector molecules and T cell signatures, which was independent of endocrine status and has not been reported previously. Moreover, the potential immunotherapeutic approach of TNFSF4 blockade showed underlying effects on stem cell expansion, which more strongly and specifically demonstrated the potential effects of applying TNFSF4 blockade-based immunotherapies in breast carcinomas. We identified potential targets that may contribute to breast cancer therapies through clinical analysis and real-world review and provided one potential but crucial tool for treating breast carcinoma that showed effects across subtypes and long-term effectiveness., (© 2021. The Author(s).)

We established an IL-2 and IL-4 (IL2/4) - dependent adult T-cell leukemia/lymphoma (ATLL) cell line (YG-PLL) by adding poly-L-lysine (PLL) to the culture medium. YG-PLL originates from lymphoma cells and contains a defective HTLV-I proviral genome. Although YG-PLL cannot survive without IL-2/4, the follicular dendritic cell (FDC)-like cell line HK expressing OX40-ligand gene (OX40L + HK) inhibited their death in the presence of soluble neutral polymers. After the prevention of cell death, YG-PLL proliferated on OX40L + HK without IL2/4 in the presence of two kinds of positively or negatively charged polymers. In particular, dermatan sulfate and poly-L-histidine supported growth for more than 4 months. Therefore, the original lymphoma cells proliferated transiently in the presence of IL2/4, and their growth arrest was inhibited by the addition of PLL. Furthermore, YG-PLL lost IL2/4 dependency by the following 3-step procedure: preculture with IL2/4 and neutral polymers, 3-day culture with neutral polymer on OX40L + HK to inhibit cell death, and co-culture with OX40L + HK in the presence of the positively and negatively charged polymers. The extracellular environment made by soluble polymers plays a role in the growth of ATLL in vitro.

Alloreactive memory cells play a critical role after a second transplant and are difficult to suppress. This study investigated the effect of an immunotherapeutic strategy that combines anti-OX40L, rapamycin (Rapa), and a low dose of IL-2 in a memory cell-based adoptive model. In this model, the median survival time (MST) of the grafts of the combined treatment group was significantly extended compared to that of the control group and other treatment groups. A similar effect was observed regarding a reduction in memory T cells (Tm) and inflammatory cytokines production. Also, the percentages of Foxp3 + regulatory T cells (Tregs) increased in our model. In addition, mounting evidence has shown CD8 + CD122 + T cells are also Tregs. We found that the group of CD8 + CD122 + PD1 + T cells was markedly increased in the combined treatment group, especially in the graft. We further demonstrated that CD8 + CD122 + PD1 + T cells could suppress activated T cells. Our data suggest that anti-OX40L combined with Rapa and a low dose of IL-2 can suppress Tm, modulate CD4 and CD8 Tregs, and induce long-term heart allograft survival in sensitized mice., (Copyright © 2021. Published by Elsevier B.V.)

OX40 plays an essential role in maintaining late T-cell proliferation and survival by suppressing apoptosis and by inducing T-cell memory formation. Here, we report the results of the phase 1 study of KHK4083, a fully human antimonoclonal antibody specific for OX40. In this study, we aimed to assess the safety and tolerability of a single intravenous or subcutaneous administration of KHK4083 compared with placebo in healthy Japanese and Caucasian subjects and determined the pharmacokinetics (PK) and immunogenicity. Also, we assessed the preliminary efficacy and pharmacodynamics of multiple intravenous doses in Japanese patients with moderate to severe ulcerative colitis (UC). Drug-related treatment emergent adverse events occurred in 21 healthy subjects (58.3%) and 5 patients with UC (62.5%) after administration of KHK4083. There were no serious adverse events. The PK profile of a single intravenous dose of 10 mg/kg KHK4083 was similar in healthy Japanese and Caucasian subjects. Of 8 UC patients, a clinical response was observed in 3 patients (37.5%) and clinical remission in 2 patients (25.0%) in week 6. Our study demonstrated the safety and tolerability of single and multiple administrations of KHK4083 in healthy Japanese and Caucasian subjects and Japanese patients with moderate to severe UC. It also indicated favorable pharmacological properties of the drug., (© 2021 The Authors. Clinical Pharmacology in Drug Development published by Wiley Periodicals LLC on behalf of American College of Clinical Pharmacology.)

The epithelium-associated cytokine thymic stromal lymphopoietin (TSLP) can induce OX40L and CCL17 expression by myeloid dendritic cells (mDCs), which contributes to aberrant Th2-type immune responses. Herein, we report that such TSLP-induced Th2-type immune response can be effectively controlled by Dectin-1, a C-type lectin receptor expressed by mDCs. Dectin-1 stimulation induced STAT3 activation and decreased the transcriptional activity of p50-RelB, both of which resulted in reduced OX40L expression on TSLP-activated mDCs. Dectin-1 stimulation also suppressed TSLP-induced STAT6 activation, resulting in decreased expression of the Th2 chemoattractant CCL17. We further demonstrated that Dectin-1 activation was capable of suppressing ragweed allergen (Amb a 1)-specific Th2-type T cell response in allergy patients ex vivo and house dust mite allergen (Der p 1)-specific IgE response in non-human primates in vivo . Collectively, this study provides a molecular explanation of Dectin-1-mediated suppression of Th2-type inflammatory responses and suggests Dectin-1 as a target for controlling Th2-type inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gu, Upchurch, Horton, Wiest, Zurawski, Millard, Kane, Joo, Miller and Oh.)

T Follicular helper (Tfh) cells, a unique subset of CD4 + T cells, play an essential role in B cell development and the formation of germinal centers (GCs). Tfh differentiation depends on various factors including cytokines, transcription factors and multiple costimulatory molecules. Given that OX40 signaling is critical for costimulating T cell activation and function, its roles in regulating Tfh cells have attracted widespread attention. Recent data have shown that OX40/OX40L signaling can not only promote Tfh cell differentiation and maintain cell survival, but also enhance the helper function of Tfh for B cells. Moreover, upregulated OX40 signaling is related to abnormal Tfh activity that causes autoimmune diseases. This review describes the roles of OX40/OX40L in Tfh biology, including the mechanisms by which OX40 signaling regulates Tfh cell differentiation and functions, and their close relationship with autoimmune diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fu, Xie, Sun and Wang.)

Mucosal associated invariant T (MAIT) cells play a critical role in Helicobacter pylori ( H. pylori )-induced gastritis by promoting mucosal inflammation and aggravating mucosal injuries (1, 2). However, the underlying mechanism and key molecules involved are still uncertain. Here we identified OX40, a co-stimulatory molecule mainly expressed on T cells, as a critical regulator to promote proliferation and IL-9 production by MAIT cells and facilitate mucosal inflammation in H. pylori -positive gastritis patients. Serum examination revealed an increased level of IL-9 in gastritis patients. Meanwhile, OX40 expression was increased in mucosal MAIT cells, and its ligand OX40L was also up-regulated in mucosal dendritic cells (DCs) of gastritis patients, compared with healthy controls. Further results demonstrated that activation of the OX40/OX40L pathway promoted IL-9 production by MAIT cells, and MAIT cells displayed a highly-activated phenotype after the cross-linking of OX40 and OX40L. Moreover, the level of IL-9 produced by MAIT cells was positively correlated with inflammatory indexes in the gastric mucosa, suggesting the potential role of IL-9-producing MAIT cells in mucosal inflammation. Taken together, we elucidated that OX40/OX40L axis promoted mucosal MAIT cell proliferation and IL-9 production in H. pylori -induced gastritis, which may provide potential targeting strategies for gastritis treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ming, Zhang, Liang, Li, He, Chen, Zhang, Niu, Deng, Geng, Zhang, Gong and Wu.)

Topical immunotherapy with diphenylcyclopropenone (DPCP) is considered to be the most effective treatment of severe AA. However, the mechanism is unclear and an early predictor for the efficacy needs to be explored. The TSLP/OX40L/IL-13 pathway is an important pathway to initiate and maintain Th2 immune responses. Our previous work suggests this pathway may play a role in severe AA treated with DPCP. Thus, to further investigate the mechanism of TSLP/OX40L/IL-13 pathway in severe AA treated with DPCP and explore the predictor for the efficacy of DPCP therapy, we conducted a prospective study to compare expression levels of TSLP, OX40L, Th2 cytokines IL-4, IL-5 and IL13, and Th1 cytokine IFN-γ in severe AA patients before and after the treatment. Results showed that 21 AA patients were responsive (responders) to the DPCP therapy and 12 were not responsive (non-responders). Responders had lower levels of TSLP, OX40L and IL-13 than non-responders before the treatment. After the DPCP treatment, TSLP, IL-5 and IL-13 increased and IFN-γ decreased in responders while there were no changes of TSLP, IL-4, IL-13 and IFN-γ in non-responders. Our data suggest that the TSLP/OX40L/IL-13 pathway is down-regulated in some severe AA patients and DPCP might play a therapeutic role by up-regulating the pathway in these severe AA patients. The TSLP/OX40L/IL-13 pathway could be a predictor of response to the DPCP therapy for severe AA patients., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In a retrospective study, the expression of mRNA of membrane receptor OX40 and its ligand OX40L in liver tissues was analyzed in 34 patients with hepatocellular carcinoma in order to assess their clinical implications and prognostic value. Expression of mRNA was analyzed by reverse transcription PCR and TaqMan probes. Expression of OX40 mRNA was significantly higher in tumor specimens in paired comparison with the samples of adjacent non-tumor tissue or normal liver tissue of control patients. In contrast, expression of OX40L mRNA was lower in tumor tissue in paired comparison with the samples of adjacent non-tumor tissue or normal liver tissue. The clinical and pathological analysis showed that expression of OX40 mRNA significantly correlated with the degree of tumor differentiation; there was an insignificant decreasing trend in the length of recurrence-free period. It was hypothesized that microenvironment of hepatocellular carcinoma can induce immunosuppression due to dysregulation of the expression of OX40 and OX40L in tumor tissue, which promotes tumor growth.

Stress conditions induced by routine treatments might affect cancer-associated fibroblasts in lung adenocarcinoma. The present study tried to explore transcriptome changes in lung fibroblasts under chemotherapeutics, irradiation, and hypoxia, which were induced by chemotherapy, radiotherapy, and anti-angiogenesis therapy, respectively. We established three in vitro models to mimic the stress conditions for lung fibroblasts. Interestingly, one of the secretory molecules, tumor necrosis factor superfamily member 4 (TNFSF4, also known as OX40L), was significantly up-regulated in lung fibroblasts under stress environments. Lung adenocarcinoma patients received chemotherapy and radiotherapy had a higher expression level of TNFSF4 in serum and tumor tissues. There was a negative correlation between the increase of serum TNFSF4 levels and the shrink of the tumor after chemotherapy. TNFSF4 could promote cisplatin resistance and inhibit the apoptosis of lung adenocarcinoma cells. Furthermore, TNFSF4 could significantly increase the activity of NF-κB/BCL-XL pathway in lung adenocarcinoma cells, which could be counteracted by knocking down the expression of TNFRSF4 (receptor of TNFSF4). In conclusion, TNFSF4, secreted by cancer-associated fibroblasts under stress conditions, could facilitate chemoresistance of lung adenocarcinoma through inhibiting apoptosis of tumor cells., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Introduction: Osteoarthritis (OA) is a common musculoskeletal disease characterized by pain, stiffness, limited activity, occasional effusion, and local inflammation. MiR-146 is one of the noncoding RNA closely related to OA, but the role of miR-146 in OA remains controversial. The tumour necrosis factor receptor OX40 is activated by its cognate ligand OX40L (TNFSF4) and functions as a T-cell costimulatory molecule. The T-cell functions, including cytokine production, expansion, and survival, are enhanced by the OX40 costimulatory signals., Methods: We established an inflammatory model of condylar chondrocytes induced by IL-1β and TNF-α and detected the expression of miRNA by miRNA sequencing. Then, cell transfection was used to study the role of miR146a-5p in OA. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and database analysis were used to screen out potential target genes of miR-146a-5p. A dual luciferase activity assay tested whether ox40l is the target gene of miR-146a-5p., Results: MiR-146a-5p and OX40L was upregulated after induced by IL-1β and TNF-α, miR-146a-5p reduced the production of inflammatory factors but had no effect on chondrophenotypic factors, and ox40l was targeted by miR-146a-5p., Conclusion: OX40L and miR-146a-5p of condylar chondrocytes in the inflammatory environment (induced by IL-1β and TNF-α) were significantly increased, miR-146a-5p is a protective factor in the inflammatory response, which can reduce the production of inflammatory factors, and miR-146a-5p may regulate T-cell-mediated immunity through targeting of ox40l in OA., (© 2020 S. Karger AG, Basel.)

TNF receptor superfamily comprises many T-cell costimulatory receptors, including TNFRSF1, TNFRSF2, TNFRSF4 (OX40), TNFRSF9 (4-1BB), TNFRSF18 (GITR), and TNFRSF7 (CD27). Signaling through these costimulatory stimulatory receptors can promote conventional T-cell (Tconv) proliferation, and effector functions in an antigen-dependent manner. Thus, agonistic antibodies and ligands for OX40, 4-1BB, GITR, and CD27 have been tested for inducing T-cell-mediated antitumor responses in several cancers. However, recently emerging reports show critical role for TNFR signaling in regulatory T-cell (Treg) differentiation and expansion, which might suppress effector T-cell proliferation and functions. Here, we show preferential over expression of TNFR2, OX40, 4-1BB, and GITR in Treg cells over Tconv cells, and the ability of OX40L and GITRL to induce selective proliferation of Treg cells, but not Tconv cells, in an antigen-independent manner. We describe the standard protocols used for Affymetrix gene expression profiling, T-cell isolation, and Cell Trace Violet-based cell proliferation assay.

This study's aim was to demonstrate that the combination of patient immune profiling and testing in a humanized mouse model of ulcerative colitis (UC) might lead to patient stratification for treatment with oxelumab. First, immunological profiles of UC patients and non-UC donors were analyzed for CD4+ T cells expressing OX40 (CD134; also known as TNFRSF4) and CD14+ monocytes expressing OX40L (CD252; also known as TNFSF4) by flow cytometric analysis. A significant difference was observed between the groups for CD14+ OX40L+ (UC: n=11, 85.44±21.17, mean±s.d.; non-UC: n=5, 30.7±34.92; P=0.02), whereas no significant difference was detected for CD4+ OX40+. CD14+ OX40L+ monocytes were correlated significantly with T helper 1 and 2 cells. Second, NOD/Scid IL2Rγ null mice were reconstituted with peripheral blood mononuclear cells from UC donors exhibiting elevated levels of OX40L, and the efficacy of oxelumab was compared with that of adalimumab. The clinical, colon and histological scores and the serum concentrations of IL-6, IL-1β and glutamic acid were assessed. Treatment with oxelumab or adalimumab resulted in significantly reduced clinical, colon and histological scores, reduced serum concentrations of IL-6 and reduced frequencies of splenic human effector memory T cells and switched B cells. Comparison of the efficacy of adalimumab and oxelumab by orthogonal partial least squares discrimination analysis revealed that oxelumab was slightly superior to adalimumab; however, elevated serum concentrations of glutamic acid suggested ongoing inflammation. These results suggest that oxelumab addresses the pro-inflammatory arm of inflammation while promoting the remodeling arm and that patients exhibiting elevated levels of OX40L might benefit from treatment with oxelumab., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Dendritic cells (DCs) are the main mediators of Th2 immune responses in allergic asthma, and Fms-like tyrosine kinase 3 ligand (Flt3L) is an important growth factor for the development and homeostasis of DCs. This study identified the DC populations that primarily cause the initiation and development of allergic lung inflammation using Fms-like tyrosine kinase 3 (Flt3) knockout (KO) mice with allergen-induced allergic asthma. We observed type 2 allergic lung inflammation with goblet cell hyperplasia in Flt3 KO mice, despite a significant reduction in total DCs, particularly CD103 + DCs, which was barely detected. In addition, bone marrow-derived dendritic cells (BMDCs) from Flt3 KO mice directed Th2 immune responses in vitro, and the adoptive transfer of these BMDCs exacerbated allergic asthma with more marked Th2 responses than that of BMDCs from wild-type (WT) mice. Furthermore, we found that Flt3L regulated the in vitro expression of OX40 ligand (OX40L) in DCs, which is correlated with DC phenotype in in vivo models. In conclusion, we revealed that Flt3-independent CD11b + DCs direct Th2 responses with the elevated OX40L and are the primary cause of allergic asthma. Our findings suggest that Flt3 is required to control type 2 allergic inflammation.

Major obstacles in immunotherapies include toxicities associated with systemic administration of therapeutic agents, as well as low tumor lymphocyte infiltration that hampers the efficacies. In this study, we report a mesenchymal stem cell (MSC)-based immunotherapeutic strategy in which MSCs specifically deliver T/natural killer (NK) cell-targeting chemokine CXCL9 and immunostimulatory factor OX40 ligand (OX40L)/tumor necrosis factor superfamily member 4 (TNFSF4) to tumor sites in syngeneic subcutaneous and azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced spontaneous colon cancer mouse models. This approach generated potent local antitumor immunity by increasing the ratios of tumor-infiltrating CD8 + T and NK cells and production of antitumor cytokines and cytolytic proteins in the tumor microenvironment. Moreover, it improved the efficacy of programmed death-1 (PD-1) blockade in a syngeneic mouse model and significantly suppressed the growth of major histocompatibility complex class I (MHC class I)-deficient tumors. Our MSC-based immunotherapeutic strategy simultaneously recruits and activates immune effector cells at the tumor site, thus overcoming the problems with toxicities of systemic therapeutic agents and low lymphocyte infiltration of solid tumors., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Immune checkpoint inhibitors improved the survival rate of patients with unresectable melanoma. However, some patients do not respond, and variable immune-related adverse events have been reported. Therefore, more effective and antigen-specific immune therapies are urgently needed. We previously reported the efficacy of an immune cell therapy with immortalized myeloid cells derived from induced pluripotent stem cells (iPS-ML). In this study, we generated OX40L-overexpressing iPS-ML (iPS-ML-Zsgreen-OX40L) and investigated their characteristics and in vivo efficacy against mouse melanoma. We found that iPS-ML-Zsgreen-OX40L suppressed the progression of B16-BL6 melanoma, and prolonged survival of mice with ovalbumin (OVA)-expressing B16 melanoma (MO4). The number of antigen-specific CD8 + T cells was higher in spleen cells treated with OVA peptide-pulsed iPS-ML-Zsgreen-OX40L than in those without OX40L. The OVA peptide-pulsed iPS-ML-Zsgreen-OX40L significantly increased the number of tumor-infiltrating T lymphocytes (TILs) in MO4 tumor. Flow cytometry showed decreased regulatory T cells but increased effector and effector memory T cells among the TILs. Although we plan to use allogeneic iPS-ML in the clinical applications, iPS-ML showed the tumorgenicity in the syngeneic mice model. Incorporating the suicide gene is necessary to ensure the safety in the future study. Collectively, these results indicate that iPS-ML-Zsgreen-OX40L therapy might be a new method for antigen-specific cancer immunotherapy., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Endogenous control mechanisms, including immune checkpoints and immunosuppressive cells, are exploited in the process of tumorigenesis to weaken the anti-tumor immune response. Cancer treatment by chemotherapy or immune checkpoint inhibition can lead to changes of checkpoint expression, which influences therapy success. Peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) were isolated from head and neck squamous cell carcinoma (HNSCC) patients ( n = 23) and compared to healthy donors ( n = 23). Immune checkpoint expression (programmed cell death ligand 1 (PD-1), tumor necrosis factor receptor (TNFR)-related (GITR), CD137, tumor necrosis factor receptor superfamily member 4 (TNFRSF4) (OX40), t-cell immunoglobulin and mucin-domain containing-3 (TIM3), B- and T-lymphocyte attenuator (BTLA), lymphocyte-activation gene 3 (LAG3)) was determined on immune cells by flow cytometry. PD-L1 expression was detected on tumor tissue by immunohistochemistry. Immune cells were treated with immuno- and chemotherapeutics to investigate treatment-specific change in immune checkpoint expression, in vitro. Specific changes of immune checkpoint expression were identified on PBL and TIL of HNSCC patients compared to healthy donors. Various chemotherapeutics acted differently on the expression of immune checkpoints. Changes of checkpoint expression were significantly less pronounced on regulatory T cells compared to other lymphocyte populations. Nivolumab treatment significantly reduced the receptor PD-1 on all analyzed T cell populations, in vitro. The specific immune checkpoint expression patterns in HNSCC patients and the investigated effects of immunomodulatory agents may improve the development and efficacy of targeted immunotherapy.

The OX40-OX40L pathway provides crucial co-stimulatory signals for CD4 T cell responses, however the precise cellular interactions critical for OX40L provision in vivo and when these occur, remains unclear. Here, we demonstrate that provision of OX40L by dendritic cells (DCs), but not T cells, B cells nor group 3 innate lymphoid cells (ILC3s), is critical specifically for the effector Th1 response to an acute systemic infection with Listeria monocytogenes (Lm). OX40L expression by DCs is regulated by cross-talk with NK cells, with IFNγ signalling to the DC to enhance OX40L in a mechanism conserved in both mouse and human DCs. Strikingly, DC expression of OX40L is redundant in a chronic intestinal Th1 response and expression by ILC3s is necessary. Collectively these data reveal tissue specific compartmentalisation of the cellular provision of OX40L and define a mechanism controlling DC expression of OX40L in vivo.

Atopic dermatitis (AD) is a chronic, or chronically relapsing, inflammatory skin disease associated with asthma and allergic rhinitis, and is dominated by Th2 cells. The co-stimulatory T-cell receptor OX40 and its ligand, OX40L, play a central role in the pathogenesis of AD, as their interactions are crucial for the generation of TH2 memory cells. Using enzyme-linked immunoassay (ELISA) and flow cytometry on blood samples from patients with AD and healthy volunteers, this study shows that the serum level of soluble (s) OX40 is decreased in patients with AD, and the expression of OX40 by activated skin-homing CD4+ T cells is increased. This study further shows, using immunofluorescence on skin biopsies, that OX40+ and OX40L+ cells are co-located within the dermis, indicating local activity of OX40/OX40L. Serum levels of sOX40 were associated with atopic diseases and, together, these results support that the OX40 system is important for chronic inflammation in AD skin.

Our study aimed to identify differentially methylated CpGs/regions and their enriched genomic pathways associated with underlying chronic musculoskeletal pain in older individuals. We recruited cognitively healthy older adults with ( n  = 20) and without ( n  = 9) self-reported musculoskeletal pain and collected DNA from peripheral blood that was analyzed using MethylationEPIC arrays. We identified 31,739 hypermethylated CpG and 10,811 hypomethylated CpG probes ( p s ≤ 0.05). All CpG probes were clustered into 5966 regions, among which 600 regions were differentially methylated at p ≤ 0.05 level, including 294 hypermethylated regions and 306 hypomethylated regions (differentially methylated regions). Ingenuity pathway enrichment analysis revealed that the pain-related differentially methylated regions were enriched across multiple pathways. The top 10 canonical pathways were linked to cellular signaling processes related to immune responses (i.e. antigen presentation, programed cell death 1 receptor/PD-1 ligand 1, interleukin-4, OX40 signaling, T cell exhaustion, and apoptosis) and gamma-aminobutyric acid receptor signaling. Further, Weighted Gene Correlation Network Analysis revealed a comethylation network module in the pain group that was not preserved in the control group, where the hub gene was the cyclic adenosine monophosphate-dependent transcription factor ATF-2. Our preliminary findings provide new epigenetic insights into the role of aberrant immune signaling in musculoskeletal pain in older adults while further supporting involvement of dysfunctional GABAergic signaling mechanisms in chronic pain. Our findings need to be urgently replicated in larger cohorts as they may serve as a basis for developing and targeting future interventions.

Despite continuous exposure to trillions of microbes, the intestinal immune system protects the mucosa by balancing barrier protection, tolerance, and immunity. As both sentinel and effector, the mucosal innate immune system plays a central role in coordinating these responses. By integrating signals from the intestinal microbiota, mononuclear phagocytes (MNPs) serve as a critical link in regulating effector functions of group 3 innate lymphoid cells (ILC3s). Our recent work identified the role for MNP production of the IBD-linked protein TNF-like ligand 1A (TL1A) in modulating microbial regulation of ILC3 barrier immunity. These findings highlight a broader role for ILC3s in local control of T cell immunity and their potential role in the pathogenesis and treatment of inflammatory disease.

Objectives: Analysis of tumor immune infiltration has been suggested to outperform tumor, node, metastasis staging in predicting clinical course of colorectal cancer (CRC). Infiltration by cells expressing OX40, a member of the tumor necrosis factor receptor family, or CD16, expressed by natural killer cells, monocytes, and dendritic cells, has been associated with favorable prognosis in patients with CRC. We hypothesized that assessment of CRC infiltration by both OX40+ and CD16+ cells might result in enhanced prognostic significance., Methods: Colorectal cancer infiltration by OX40 and CD16 expressing cells was investigated in 441 primary CRCs using tissue microarrays and specific antibodies, by immunohistochemistry. Patients' survival was evaluated by Kaplan-Meier and log-rank tests. Multivariate Cox regression analysis, hazard ratios, and 95% confidence intervals were also used to evaluate prognostic significance of OX40+ and CD16+ cell infiltration., Results: Colorectal cancer infiltration by OX40+ and CD16+ cells was subclassified into 4 groups with high or low infiltration levels in all possible combinations. High levels of infiltration by both OX40+ and CD16+ cells were associated with lower pT stage, absence of peritumoral lymphocytic (PTL) inflammation, and a positive prognostic impact. Patients bearing tumors with high infiltration by CD16+ and OX40+ cells were also characterized by significantly longer overall survival, as compared with the other groups. These results were confirmed by analyzing an independent validation cohort., Conclusions: Combined infiltration by OX40+ and CD16+ immune cells is an independent favorable prognostic marker in CRC. The prognostic value of CD16+ immune cell infiltration is significantly improved by the combined analysis with OX40+ cell infiltration.

Hepatitis C virus (HCV) infection is among the leading causes of hepatocellular carcinoma and liver cirrhosis globally, with a high economic burden. The disease progression is well established, but less is known about the spontaneous HCV infection clearance. This study tries to establish the relationship between codon biasness and expression of HCV clearance candidate genes in normal and HCV infected liver tissues. A total of 112 coding sequences comprising 151 679 codons were subjected to the computation of codon indices, namely relative synonymous codon usage, an effective number of codon (Nc), frequency of optimal codon, codon adaptation index, codon bias index, and base compositions. Codon indices report of GC3s, GC12, hydropathicity, and aromaticity implicates both mutational and translational selection in the candidate gene set. This was further correlated with the differentially expressed genes among the selected genes using BioGPS. A significant correlation is observed between the gene expression of normal liver and cancerous liver tissues with codon bias (Nc). Gene expression is also correlated with relative codon bias values, indicating that CCL5, APOA2, CD28, IFITM1, and TNFSF4 genes have higher expression. These results are quite encouraging in selecting the high responsive genes in HCV clearance. However, there could be additional genes which could also orchestrate the clearance role with the above mentioned first line of defensive genes., (© 2019 Wiley Periodicals, Inc.)

Foxp3 + T regulatory cells (Tregs) control autoimmune response by suppressing proliferation and effector functions of self-reactive Foxp3 - CD4 + /CD8 + T cells and thereby maintain the critical balance between self-tolerance and autoimmunity. Earlier, we had shown that OX40L-JAG1 cosignaling mediated through their cognate receptors OX40 and Notch3 preferentially expressed on murine Tregs can selectively induce their proliferation in the absence of TCR stimulation. However, the differential molecular mechanisms regulating TCR-independent versus TCR-dependent Treg proliferation and lineage stability of the expanded Tregs remained unknown. In this study, we show that OX40L-JAG1 treatment induced TCR-independent proliferation of Tregs in the thymus and periphery. The use of Src kinase inhibitor permitted us to demonstrate selective inhibition of TCR-dependent T cell proliferation with little to no effect on OX40L-JAG1-induced TCR-independent Treg expansion in vitro, which was critically dependent on noncanonical NF-κB signaling. OX40L-JAG1-expanded Tregs showed sustained lineage stability as indicated by stable demethylation marks in Treg signature genes such as Foxp3, Il2ra, Ctla4, Ikzf2, and Ikzf4. Furthermore, OX40L-JAG1 treatment significantly increased CTLA4 + and TIGIT + Tregs and alleviated experimental autoimmune thyroiditis in mice. Relevance of our findings to humans became apparent when human OX40L and JAG1 induced TCR-independent selective expansion of human Tregs in thymocyte cultures and increased human Tregs in the liver tissue of humanized NSG mice. Our findings suggest that OX40L-JAG1-induced TCR-independent Treg proliferation is a conserved mechanism that can be used to expand lineage-stable Tregs to treat autoimmune diseases., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Systemic lupus erythematosus (SLE) is characterized by systemic end-organ damage. We investigated the involvement of IRF5 , TLR-7 , MECP2 , STAT4 , and TNFSF4 genes and TNF- α , IFN- γ , IL-2, IL-12, IL-6, and IL-10 cytokines in SLE pathogenesis and in organ damage in Jordanian patients. Blood was collected from 51 patients and 50 controls. Expression levels of SLE genes in PBMCs and cytokine levels were determined using RT-PCR and ELISA, respectively. Expression levels of all genes and levels of TNF- α , IL-12, IL-6, and IL-10 were higher in SLE patients than those in controls ( p < 0.05), whereas IL-2 level was lower. High STAT4 ( α ), TNFSF4 , and IL-10 levels correlated with cardiovascular damage, and high MECP2 ( α ) and TNF- α correlated with renal damage. Pulmonary and musculoskeletal damages correlated with high levels of TNFSF4 . We concluded that STAT4 and TNFSF4 genes with TNF- α and IL-10 cytokines could be used as biomarkers to assess SLE activity and manage treatment., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2019 Amal H. Uzrail et al.)

Immunotherapy with checkpoint inhibitors revolutionized melanoma treatment in both the adjuvant and metastatic setting, yet not all metastatic patients respond, and metastatic disease still often recurs among immunotherapy-treated patients with locally advanced disease. TNFSF4 is a co-stimulatory checkpoint protein expressed by several types of immune and non-immune cells, and was shown in the past to enhance the anti-neoplastic activity of T cells. Here, we assessed its expression in melanoma and its association with outcome in locally advanced and metastatic disease. We used publicly available data from The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia (CCLE), and RNA sequencing data from anti-PD1-treated patients at Sheba medical center. TNFSF4 mRNA is expressed in melanoma cell lines and melanoma samples, including those with low lymphocytic infiltrates, and is not associated with the ulceration status of the primary tumor. Low expression of TNFSF4 mRNA is associated with worse prognosis in all melanoma patients and in the cohorts of stage III and stage IIIc-IV patients. Low expression of TNFSF4 mRNAs is also associated with worse prognosis in the subgroup of patients with low lymphocytic infiltrates, suggesting that tumoral TNFSF4 is associated with outcome. TNFSF4 expression was not correlated with the expression of other known checkpoint mRNAs. Last, metastatic patients with TNFSF4 mRNA expression within the lowest quartile have significantly worse outcome on anti-PD1 treatment, and a significantly lower response rate to these agents. Our current work points to TNFSF4 expression in melanoma as a potential determinant of prognosis, and warrants further translational and clinical research.