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Mycosis fungoides (MF) and Sézary syndrome (SS), the most common types of cutaneous T-cell lymphoma (CTCL), are characterized by proliferation of mature CD4+ T-helper cells. Patients with advanced-stage MF and SS have poor prognosis, with 5-year survival rates of 52%. Although a variety of systemic therapies are currently available, there are no curative options for such patients except for stem cell transplantation, and thus the treatment of advanced MF and SS still remains challenging. Therefore, elucidation of the pathophysiology of MF/SS and development of medical treatments are desired. In this study, we focused on a molecule called OX40. We examined OX40 and OX40L expression and function using clinical samples of MF and SS and CTCL cell lines. OX40 and OX40L were co-expressed on tumor cells of MF and SS. OX40 and OX40L expression was increased and correlated with disease severity markers in MF/SS patients. Anti-OX40 antibody and anti-OX40L antibody suppressed the proliferation of CTCL cell lines both in vitro and in vivo. These results suggest that OX40–OX40L interactions could contribute to the proliferation of MF/SS tumor cells and that the disruption of OX40–OX40L interactions could become a new therapeutic strategy for the treatment of MF/SS.

Oral squamous cell carcinoma (OSCC) is one of the most prevalent malignancies worldwide. More recently, the administration of immune checkpoint inhibitors has opened up more possibilities for cancer treatment. We utilized a weighted gene co-expression network and the single sample gene set enrichment analysis (ssGSEA) algorithm in the TCGA database and identified a module highly correlated with regulatory T cell (Treg) abundance in OSCC. Subsequently, we verified the results by tissue microarrays and utilized immunohistochemical staining (IHC) to test the relationship between the expression level and clinicopathological staging. CCK-8, transwell, and wound healing assays were utilized to detect the functions of OSCC cells. LCK, IL10RA, and TNFRSF1B were selected as biomarkers related to regulatory T cell infiltration. IHC staining showed significantly increased expression of or in OSCC patients, and the expression levels were associated with tumor stage, lymph node metastasis, pathological stage, clinical status and the overall survival. In vitro experiments showed that or knockdown efficiently impaired the proliferative, migrative, and invasive capacity in OSCC cell lines. We performed a series of bioinformatics analyses in OSCC and identified three oncogenic indicators: LCK, IL10RA, TNFRSF1B. These findings uncovered the potential prognostic values of hub genes, thus laying foundations for in-depth research in OSCC.

The tumor necrosis factor (TNF) receptor superfamily is a structurally and functionally related group of cell surface receptors that play crucial roles in various cellular processes, including apoptosis, cell survival, and immune regulation. This review paper synthesizes key findings from recent studies, highlighting the importance of clustering in TNF receptor superfamily signaling. We discuss the underlying molecular mechanisms of signaling, the functional consequences of receptor clustering, and potential therapeutic implications of targeting surface structures of receptor complexes.

[This corrects the article DOI: 10.3389/fimmu.2023.1194610.].

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptors TRAILR1/death receptor 4 (DR4) and TRAILR2/DR5 trigger cell death in many cancer cells but rarely exert cytotoxic activity on non-transformed cells. Against this background, a variety of recombinant TRAIL variants and anti-TRAIL death receptor antibodies have been developed and tested in preclinical and clinical studies. Despite promising results from mice tumor models, TRAIL death receptor targeting has failed so far in clinical studies to show satisfying anti-tumor efficacy. These disappointing results can largely be explained by two issues: First, tumor cells can acquire TRAIL resistance by several mechanisms defining a need for combination therapies with appropriate sensitizing drugs. Second, there is now growing preclinical evidence that soluble TRAIL variants but also bivalent anti-TRAIL death receptor antibodies typically require oligomerization or plasma membrane anchoring to achieve maximum activity. This review discusses the need for oligomerization and plasma membrane attachment for the activity of TRAIL death receptor agonists in view of what is known about the molecular mechanisms of how TRAIL death receptors trigger intracellular cell death signaling. In particular, it will be highlighted which consequences this has for the development of next generation TRAIL death receptor agonists and their potential clinical application.

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

Many new immunotherapeutic approaches aim on the stimulatory targeting of receptors of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) using antibodies with intrinsic or conditional agonism. There is an initial need to characterize corresponding TNFRSF receptor (TNFR)-targeting antibodies with respect to affinity, ligand binding, receptor activation and the epitope recognized. Here, we report a collection of simple and matched protocols enabling the detailed investigation of these aspects by help of Gaussia princeps luciferase (GpL) fusion proteins and analysis of interleukin-8 (IL8) production as an easily measurable readout of TNFR activation. In a first step, the antibodies and antibody variants of interest are transiently expressed in human embryonal kidney 293 cells, either in non-modified form or as fusion proteins with GpL as a reporter domain. The supernatants containing the antibody-GpL fusion proteins can then be used without further purification in cell-free and/or cellular binding studies to determine affinity. Similarly, binding studies with mutated TNFR variants enable the characterization of the antibody binding site within the TNFR ectodomain. Furthermore, in cellular binding studies with GpL fusion proteins of soluble TNFL molecules, the ability of the non-modified antibody variants to interfere with TNFL-TNFR interaction can be analyzed. Last but not least, we describe a protocol to determine the intrinsic and the Fc gamma receptor (FcγR)-dependent agonism of anti-TNFR antibodies which exploits i) the capability of TNFRs to trigger IL8 production in tumor cell lines lacking expression of FcγRs and ii) vector- and FcγR-transfected cells, which produce no or only very low amounts of human IL8. The presented protocols only require standard molecular biological equipment, eukaryotic cell culture and plate readers for the quantification of luminescent and colorimetric signals.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily and has been shown to induce apoptosis in cancer cells but not normal cells. TRAIL triggers apoptosis through binding to its receptors DR4 and KILLER/DR5. Chemo or radiotherapy induces apoptosis through activation of p53 in response to cellular damage, whereas TRAIL induces apoptosis independent of p53. Mutations or deletions of p53 occurred in more than half of human tumors confer resistance to chemo-radiotherapy. Treatment of TRAIL-resistant tumors with agents targeting death receptors, intrinsic Bcl-2 family members, inhibitor of apoptosis proteins or PI3K/Akt pathway restores the sensitivity to TRAIL-induced apoptosis. Combination of rhTRAIL or the agonist antibody for TRAIL receptor with conventional chemotherapeutic agents results in enhanced efficacy in preventing tumor progression and metastasis. Therefore, the rational design of TRAIL-based therapy combining with other modality that either synergizes to apoptosis induction or overcomes the resistance represents a challenging strategy to achieve the systemic tumor targeting and augment the antitumor activity of cancer therapeutics.

Fibroblast growth factor (FGF)-inducible 14 (Fn14) activates the classical and alternative NFκB (nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells) signaling pathway but also enhances tumor necrosis factor (TNF)-induced cell death. Fn14 expression is upregulated in non-hematopoietic cells during tissue injury and is also often highly expressed in solid cancers. In view of the latter, there were and are considerable preclinical efforts to target Fn14 for tumor therapy, either by exploiting Fn14 as a target for antibodies with cytotoxic activity (e.g. antibody-dependent cellular cytotoxicity (ADCC)-inducing IgG variants, antibody drug conjugates) or by blocking antibodies with the aim to interfere with protumoral Fn14 activities. Noteworthy, there are yet no attempts to target Fn14 with agonistic Fc effector function silenced antibodies to unleash the proinflammatory and cell death-enhancing activities of this receptor for tumor therapy. This is certainly not at least due to the fact that anti-Fn14 antibodies only act as effective agonists when they are presented bound to Fcγ receptors (FcγR). Thus, there are so far no antibodies that robustly and selectively engage Fn14 signaling without triggering unwanted FcγR-mediated activities. In this study, we investigated a panel of variants of the anti-Fn14 antibody 18D1 of different valencies and domain architectures with respect to their inherent FcγR-independent ability to trigger Fn14-associated signaling pathways. In contrast to conventional 18D1, the majority of 18D1 antibody variants with four or more Fn14 binding sites displayed a strong ability to trigger the alternative NFκB pathway and to enhance TNF-induced cell death and therefore resemble in their activity soluble (TNF)-like weak inducer of apoptosis (TWEAK), one form of the natural occurring ligand of Fn14. Noteworthy, activation of the classical NFκB pathway, which naturally is predominately triggered by membrane-bound TWEAK but not soluble TWEAK, was preferentially observed with a subset of constructs containing Fn14 binding sites at opposing sites of the IgG scaffold, e.g. IgG1-scFv fusion proteins. A superior ability of IgG1-scFv fusion proteins to trigger classical NFκB signaling was also observed with the anti-Fn14 antibody PDL192 suggesting that we identified generic structures for Fn14 antibody variants mimicking soluble and membrane-bound TWEAK.

TNF family members and their receptors contribute to increased gene expression for inflammatory processes and intracellular cascades leading to programmed cell death, both via activation of NF-κB. TNF receptor (TNFR)-associated factors (TRAFs) are cytoplasmic adaptor proteins binding to various receptors of the TNFR family. In an attempt to delineate the role of individual TRAFs, we compared NF-κB activation by CD40wtand CD40 mutants with different TRAF recruitment patterns. Recognized only recently, NF-κB signaling occurs at least via two different pathways. Each pathway results in nuclear translocation of two different Reldimers, the canonical p50/RelA and the noncanonical p52/RelB. Here, we show that via TRAF6, CD40 mediates only the activation of the canonical NF-κB pathway. Via TRAF2/5, CD40 activates both the canonical and the noncanonical NF-κB pathways. We observed that TRAF3 specifically blocked the NF-κB activation via TRAF2/5. This inhibitory effect of TRAF3 depends on the presence of an intact zinc finger domain. Paradoxically, suppression of TRAF2/5-mediated NF-κB activation by TRAF3 resulted in enhanced transcriptional activity of TRAF6-mediated canonical NF-κB emanating from CD40. We also observed that 12 TNFR family members (p75TNFR, LTβR, RANK, HVEM, CD40, CD30, CD27, 4- 1BB, GITR, BCMA, OX40, and TACI) are each capable of activating the alternative NF-κB pathway and conclude that TRAF3 serves as a negative regulator of this pathway for all tested receptors.