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TRAF1 is a pro-survival adaptor molecule in TNFR superfamily (TNFRSF) signaling. TRAF1 is overexpressed in many B cell cancers including refractory chronic lymphocytic leukemia (CLL). Little has been done to assess the role of TRAF1 in human cancer. Here we show that the protein kinase C related kinase Protein Kinase N1 (PKN1) is required to protect TRAF1 from cIAP-mediated degradation during constitutive CD40 signaling in lymphoma. We show that the active phospho-Thr774 form of PKN1 is constitutively expressed in CLL but minimally detected in unstimulated healthy donor B cells. Through a screen of 700 kinase inhibitors, we identified two inhibitors, OTSSP167, and XL-228, that inhibited PKN1 in the nanomolar range and induced dose-dependent loss of TRAF1 in RAJI cells. OTSSP167 or XL-228 treatment of primary patient CLL samples led to a reduction in TRAF1, pNF-κB p65, pS6, pERK, Mcl-1 and Bcl-2 proteins, and induction of activated caspase-3. OTSSP167 synergized with venetoclax in inducing CLL death, correlating with loss of TRAF1, Mcl-1, and Bcl-2. Although correlative, these findings suggest the PKN1-TRAF1 signaling axis as a potential new target for CLL. These findings also suggest the use of the orally available inhibitor OTSSP167 in combination treatment with venetoclax for TRAF1 overexpressing CLL.

Summary Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) is a critical regulatory molecule in modulation of T cell immune responses. Here we report the mouse GITR (mGITR) and mGITR ligand (mGITRL) complex structure and find that the binding interface of mGITR and mGITRL is distinct from the typical tumor necrosis factor superfamily (TNFSF)/TNF receptor superfamily (TNFRSF) members. mGITR binds to its ligand with a single domain, whereas the binding interface on mGITRL is located on the side, which is distal from conserved binding sites of TNFSF molecules. Mutational analysis reveals that the binding interface of GITR/GITRL in humans is conserved with that in the mouse. Substitution of key interacting D93-I94-V95 (DIV) in mGITR with the corresponding K93-F94-S95 (KFS) in human GITR enables cross-recognition with human GITRL and cross-activation of receptor signaling. The findings of this study substantially expand our understanding of the interaction of TNFSF/TNFRSF superfamily molecules and can benefit the future design of biologics by targeting GITR/GITRL.

The TNFR superfamily of receptors, the major focus of the recent TNFR Superfamily Conference held in June 2019, employ the TNFR-associated factor (TRAF) family of adaptor proteins in key aspects of their signaling pathways. Although many early studies investigated TRAF functions via exogenous overexpression in nonhematopoietic cell lines, it has subsequently become clear that whereas TRAFs share some overlap in function, each also plays unique biologic roles, that can be highly context dependent. This brief review summarizes the current state of knowledge of functions of each of the TRAF molecules that mediate important functions in T lymphocytes: TRAFs 1, 2, 3, 5, and 6. Due to our current appreciation of the contextual nature of TRAF function, our focus is upon findings made specifically in T lymphocytes. Key T cell functions for each TRAF are detailed, as well as future knowledge gaps of interest and importance.

Lymph nodes (LNs) are crucial for the orchestration of immune responses. LN reactions depend on interactions between incoming and local immune cells, and stromal cells. To mediate these cellular interactions an organized vascular network within the LN exists. In general, the LN vasculature can be divided into two components: blood vessels, which include the specialized high endothelial venules that recruit lymphocytes from the bloodstream, and lymphatic vessels. Signaling via TNF receptor (R) superfamily (SF) members has been implicated as crucial for the development and function of LNs and the LN vasculature. In recent years the role of cell-specific signaling of TNFRSF members in different endothelial cell (EC) subsets and their roles in development and maintenance of lymphoid organs has been elucidated. Here, we discuss recent insights into EC-specific TNFRSF member signaling and highlight its importance in different EC subsets in LN organogenesis and function during health, and in lymphocyte activation and tertiary lymphoid structure formation during inflammation.

Immunotherapy is reportedly effective in a subset of colorectal cancers (CRCs) with high microsatellite instability (MSI-H). Exploring the expression patterns and clinical values of immunologic molecules is critical in defining the specific responsive candidates. Here, we performed comprehensive molecular profiling of the B7 and TNFR family genes across 6 CRC datasets with over 1,000 patients’ details using cBioPortal TCGA data. About 20% of patients had B7 and TNFR family gene alterations. The frequency of B7 gene mutations (2.2%–5%) were similar to copy number alterations (0.53%–5.46%). TNFR amplifications were relatively more common (5.45–11.32%) than that of B7 (0.09–2.73%). B7 and TNFR gene mRNAs were upregulated in 26% of cases (102/379) and 16% of cases (61/379), respectively. The mRNA levels of B7 and TNFR genes were inversely correlated with promoter methylation status. Clinically, both B7-H3 and TNFSF7 mRNA overexpression were associated with unfavorable clinical outcomes, and the B7-H3 expression was increased gradually in cases with gene amplifications. Moreover, patients with MSI-H had significantly higher PD-L1 or PD-1 expression. Most importantly, in MSI-H group, patients with PD-L1 or PD-1 upregulation had poorer survivals than those with PD-L1/PD-1 downregulation. This is the first study drawing the immune landscapes of the co-stimulator B7 and TNFR families in CRC and showing that MSI-H patients with PD-1/PD-L1 upregulation are associated with poor clinical outcomes, providing potential markers to stratify patients responsive to immune checkpoint therapy.

Tumor necrosis factor receptor (TNFR) associated factor 1 (TRAF1) is a signaling adaptor first identified as part of the TNFR2 signaling complex. TRAF1 plays a key role in pro-survival signaling downstream of TNFR superfamily members such as TNFR2, LMP1, 4-1BB, and CD40. Recent studies have uncovered another role for TRAF1, independent of its role in TNFR superfamily signaling, in negatively regulating Toll-like receptor and Nod-like receptor signaling, through sequestering the linear ubiquitin assembly complex, LUBAC. TRAF1 has diverse roles in human disease. TRAF1 is overexpressed in many B cell related cancers and single nucleotide polymorphisms (SNPs) in TRAF1 have been linked to non-Hodgkin's lymphoma. Genome wide association studies have identified an association between SNPs in the 5′ untranslated region of the TRAF1 gene with increased incidence and severity of rheumatoid arthritis and other rheumatic diseases. The loss of TRAF1 from chronically stimulated CD8 T cells results in desensitization of the 4-1BB signaling pathway, thereby contributing to T cell exhaustion during chronic infection. These apparently opposing roles of TRAF1 as both a positive and negative regulator of immune signaling have led to some confusion in the literature. Here we review the role of TRAF1 as a positive and negative regulator in different signaling pathways. Then we discuss the role of TRAF1 in human disease, attempting to reconcile seemingly contradictory roles based on current knowledge of TRAF1 signaling and biology. We also discuss avenues for future research to further clarify the impact of TRAF1 in human disease.

Manipulation of co-stimulatory or co-inhibitory checkpoint proteins allows for the reversal of tumor-induced T-cell anergy observed in cancer. The field has gained credence given success with CTLA-4 and PD-1 inhibitors. These molecules include immunoglobulin family members and the B7 subfamily as well as the TNF receptor family members. PD-L1 inhibitors and LAG-3 inhibitors have progressed through clinical trials. Other B7 family members have shown promise in preclinical models. TNFR superfamily members have shown variable success in preclinical and clinical studies. As clinical investigation in tumor immunology gains momentum, the next stage becomes learning how to combine checkpoint inhibitors and agonists with each other as well as with traditional chemotherapeutic agents.

The B7 family and tumor necrosis factor receptor (TNFR) superfamily play a vital role in the T-cell co-stimulatory and co-inhibitory pathways, regulating T-cell activation, tolerance, and exhaustion; therapeutic modulation of these pathways is translated into effective new cancer treatments. Better understanding of the immune molecular landscapes of the B7 and TNFR families would guide head and neck immuno-oncology clinical research. We performed comprehensive molecular profiling of 10 B7 and 6 TNFR family members in head and neck cancer. Over 20% of patients had B7 and TNFR gene alterations. B7 gene amplifications were relatively more common (3–11%) than TNFR gene amplifications (0–5%). Analysis of 496 sequenced samples revealed that all genes were upregulated: B7 and TNFR mRNA were upregulated in 158 cases (> 30%) and 83 cases (∼15%), respectively. B7-H1 (PD-L1) mRNA upregulation was the most common (∼10%). Promoter methylation analysis indicated an epigenetic basis for B7 and TNFR gene regulation (especially B7-H1, which was relatively strongly correlated with promoter methylation). B7-H1 expression was significantly associated with worse overall survival, and its expression was increased in cases with gene amplifications. Human papillomavirus (HPV) status correlated significantly with B7-H1 alterations at genetic level. Almost half (47.1%) of HPV-negative patients had deep or shallow B7-H1 deletion; >90% of HPV-positive patients had diploid, copy number gain, or amplification of B7-H1. This is the first study elucidating the immune molecular landscapes of the B7 and TNFR families in head and neck cancer, providing a potential novel rationale for clinical investigations.

Receptor clustering is important for signaling among the therapeutically relevant TNFR superfamily of receptors. In nature, this clustering is driven by trimeric ligands often presented in large numbers as cell surface proteins. Molecules capable of driving similar levels of clustering could make good agonists and hold therapeutic value. However, recapitulating such extensive clustering using typical biotherapeutic formats, such as antibodies, is difficult. Consequently, generating effective agonists of TNFR superfamily receptors is challenging. Toward addressing this challenge we have used lipid- and polyion complex-based micelles as platforms for presenting receptor-binding biologics in a multivalent format that facilitates receptor clustering and imparts strong agonist activity. We show that receptor-binding scFvs or small antibody mimetics that have no agonist activity on their own can be transformed into potent agonists through multivalent presentation on a micelle surface and that the activity of already active multivalent agonists can be enhanced. Using this strategy, we generated potent agonists against two different TNFR superfamily receptors and mouse tumor model studies demonstrate that these micellar agonists have therapeutic efficacy in vivo. Due to its ease of implementation and applicability independent of agonist molecular format, we anticipate that this strategy could be useful for developing agonists to a variety of receptors that rely on clustering to signal.

Glucocorticoid-induced TNFR-related protein (GITR) is a member of the TNFR superfamily, expressed in several cells and tissues including T lymphocytes, NK cells and antigen-presenting cells (APC). GITR activation, upon interaction with its ligand (GITRL), functions as a co-activating signal. GITRL is mainly expressed on APC and GITR/GITRL interaction is important for the development of immune response. This review summarizes recent results about the GITR/GITRL system, focusing on the interplay between APC, effector and regulatory T cells.

The immune system mediates tissue responses under both physiological and pathological conditions. In addition to leukocyte subsets, non-hematopoietic tissue cell types actively contribute to shaping tissue responses, including the inflammatory, fibrogenic, and regenerative components. TWEAK and its receptor Fn14, members of the TNF/TNFR superfamily, have emerged as a prominent molecular axis regulating tissue responses (1). Generally leukocyte-derived, TWEAK signals through Fn14, which is highly induced in injured and disease tissues on the surface of parenchymal, vascular, stromal, and progenitor cells, thereby orchestrating a host of tissue-shaping processes, including inflammation, angiogenesis, cell proliferation, and death, and regulation of progenitor cells. Of the downstream signaling pathways, particular attention has been given to the non-canonical NFκB pathway, given that TWEAK induces acute activation of canonical NFκB but prolonged non-canonical NFκB activation. Thus, non-canonical NFκB signaling may be a key mechanism underlying TWEAK/Fn14-induced tissue responses. The non-canonical NFκB pathway is known to play a role in immunity and disease pathologies and is typically activated by only a subset of TNFR superfamily members, including Fn14, TNFR2, BAFFR, CD40, LTβR, and RANK. Thus, there is also broad interest in the role of this subset of TNFR superfamily members and their downstream signaling potentials in the regulation of processes underlying tissue remodeling in health and disease. This Research Topic is addressed in a compilation of 19 expert reviews and 1 original research article. The TWEAK/Fn14 pathway as an injury-inducible mediator of pleiotropic responses is introduced in a review of the work implicating sustained Fn14 signaling in disease pathogenesis and encompassing the current TWEAK/Fn14-targeting approaches for treatment of human disease (2). Broad relevance to neurological diseases is supported by a basic TWEAK/Fn14 role in regulating the structure and function of the neurovascular unit, thereby regulating blood–brain barrier (BBB) permeability (3). Furthermore, BBB damage appears to be an important component of neuropsychiatric systemic lupus erythematosus, and there is emerging evidence for a role for TWEAK/Fn14 in compromising the BBB in lupus (4). Also relevant to lupus is the pathogenic role of TWEAK/Fn14 in the renal manifestation of lupus nephritis. Indeed, evidence supporting TWEAK/Fn14-mediated pathological mechanisms in contexts of acute kidney injury and chronic kidney diseases is substantial and clinical targeting of TWEAK is ongoing in lupus nephritis (5). Also addressed in this Research Topic is the role of TWEAK/Fn14 in the pathological remodeling underlying other inflammatory diseases, namely cardiovascular diseases and obesity-associated Type-2 diabetes (6, 7), as well as in myocardial remodeling leading to heart failure (8), and a common theme also addressed in these articles is the potential use of soluble TWEAK as a biomarker for cardiovascular diseases. The expression of soluble TWEAK in biological fluids of patients with autoimmune/chronic inflammatory diseases and its potential as a biomarker of these diseases is also more broadly discussed (9). Given that TWEAK has emerged as a major cytokine regulating skeletal muscle biology, two articles are dedicated to its role in muscle wasting and mitochondrial dysfunction, and its complex regulation of myogenesis where distinct roles for canonical versus non-canonical NFκB signaling have been delineated (10, 11). Besides Fn14 upregulation in contexts of injury/disease, it is also highly expressed on tumor cells relative to normal tissue making it an attractive therapeutic target. Purcell et al. (12) review the growth inhibitory activity of an Fn14-specific antibody on an array of human tumor cells, differentially dependent on canonical and/or non-canonical NFκB, an approach that is currently being pursued as a novel cancer treatment. In summary, there is substantial evidence implicating TWEAK/Fn14 in the regulation of physiological and pathophysiological tissue responses, though there is still an incomplete understanding of the role of TWEAK/Fn14-induced canonical versus non-canonical NFκB in these various contexts. Since soluble TWEAK and membrane TWEAK differ in their capacity to induce canonical NFκB, distinct biological responses may reflect spatial and temporal differences in sources of TWEAK (13). New studies continue to inform the understanding of TWEAK-induced signaling, including ubiquitination events that are key to orchestrating canonical and non-canonical NFκB activation (14). The role of other TNF/TNFR superfamily members in shaping tissue responses in health and disease is also reviewed, concentrating on those that can activate non-canonical NFκB. In a review on TNFα signaling through TNFR2, Faustman and Davis (15) discuss the concept of leveraging TNFR2 agonism to reshape the T cell compartment in autoimmune disease, and to promote tissue regenerative processes. On the other hand, Gardam and Brink (16) review the importance of BAFF/BAFFR-mediated non-canonical NFκB signaling in peripheral B cell survival and maturation. Likewise, CD40L–CD40-mediated activation of non-canonical NFκB appears to be critical for B cell survival and possibly contributes to development of B cell malignancies (17). Both BAFFR and CD40-mediated non-canonical NFκB activation in B cells is restrained by the adaptor protein TRAF3, and the relationship between TRAF3 degradation and non-canonical NF-kB2 activation is delineated in an original research article (18). Beyond T and B cell activation, positioning cues are critical for proper immune system development and function. In this regard, LTβR plays a critical role in lymph node development and remodeling through its delivery of differentiation signals for reticular networks and vasculature (19). Tissue remodeling in the context of chronic liver diseases also features a prominent role for both LTβ and TWEAK in the crosstalk between liver progenitor cells and hepatic stellate cells, which can evolve into pathological fibrosis and hepatocellular carcinoma (20). Finally, Walsh and Choi (21) review the RANKL–RANK–OPG system, a preeminent player in the bone homeostasis, pathologies including mammary gland tumorigenesis, and in the interplay between bone and the immune system. This collection of expert reviews provides a current perspective of the role of this particular subset of TNF/TNFR family members that can activate non-canonical NFκB signaling in shaping tissue responses in contexts of development, homeostasis, and remodeling.

Glucocorticoid-induced TNFR-related gene (GITR; TNFRSF18), a receptor belonging to the TNFR superfamily (TNFRSF), is activated by GITRL. GITR is expressed at low levels on resting responder T lymphocytes and is up-regulated in T regulatory cells (Treg cells) and in activated T cells. GITRL is expressed in endothelial and antigen-presenting cells. The cytoplasmic region of GITR has a striking homology with other TNFRSF members (4-1BB, CD27, OX40) and binds TRAF molecules and Siva. Over recent years, the role of GITR in the development and in the pathophysiology of the immune system has been actively explored by several groups. GITR triggering induces both pro- and anti-apoptotic effects, abrogates the suppressive activity of Treg cells and co-stimulates responder T cells, with the latter activities over-stimulating the immune system. In vivo, GITR activation causes development of autoimmune diseases and restores immune responses in a persistent retroviral infection model and in a tumor model. Intriguingly, GITR knockout mice demonstrate lower mortality in an ischemia model. The GITR-GITRL system appears crucial in regulating immunity and warrants further study.

Targeted disruption of death receptor (DR)6 results in enhanced CD4(+) T cell expansion and T helper cell type 2 differentiation after stimulation. Similar to T cells, DR6 is expressed on resting B cells but is down-regulated upon activation. We examined DR6(-/-) B cell responses both in vitro and in vivo. In vitro, DR6(-/-) B cells undergo increased proliferation in response to anti-immunoglobulin M, anti-CD40, and lipopolysaccharide. This hyperproliferative response was due, at least in part, to both increased cell division and reduced cell apoptosis when compared with wild-type B cells. Consistent with these observations, increased nuclear levels and activity of nuclear factor kappaB transcription factor, c-Rel, and elevated Bcl-x(l) expression were observed in DR6(-/-) B cells upon stimulation. In addition, DR6(-/-) B cells exhibited higher surface levels of CD86 upon activation and were more effective as antigen-presenting cells in an allogeneic T cell proliferation response. DR6(-/-) mice exhibited enhanced germinal center formation and increased titers of immunoglobulins to T-dependent as well as T-independent type I and II antigens. This is the first demonstration of a regulatory role of DR6 in the activation and function of B cells.

CD30 is up-regulated in several human diseases and viral infections but its role in immune regulation is poorly understood. Here, we report the expression of a functional soluble CD30 homologue, viral CD30 (vCD30), encoded by ectromelia (mousepox) virus, a poxvirus that causes a severe disease related to human smallpox. We show that vCD30 is a 12-kD secreted protein that not only binds CD30L with high affinity and prevents its interaction with CD30, but it also induces reverse signaling in cells expressing CD30L. vCD30 blocked the generation of interferon γ–producing cells in vitro and was a potent inhibitor of T helper cell (Th)1- but not Th2-mediated inflammation in vivo. The finding of a CD30 homologue encoded by ectromelia virus suggests a role for CD30 in antiviral defense. Characterization of the immunological properties of vCD30 has uncovered a role of CD30–CD30L interactions in the generation of inflammatory responses.

TRAF6 is divergent from other members of the TRAF family. Therefore, TRAF6 was expected to play physiological roles distinct from those of other TRAFs. In this chapter, we focused on the physiological functions specific to TRAF6 but not other TRAFs in immune system, formation of skin appendices, and nervous system development by describing abnormal phenotypes observed in TRAF6-deficient mice. The role of TRAF6 in osteoclastogenesis and the molecular mechanisms of TRAF6-mediated signal transduction are described in other chapters.

We have recently identified a gene named GITR (glucocorticoid-induced TNF receptor related gene). GITR is expressed in different cells and tissues such as T lymphocytes from thymus and spleen and lymph nodes, and also in the lung. GITR ligand (GITRL) is expressed in several cells including macrophages, B cells, dendritic cells, and endothelial cells. In the present study, by comparing the responses in wild-type (WT) mice (GITR+/+) and GITR-deficient mice (GITR-/-), we investigated the role played by GITR-GITRL interaction in the development of chronic lung injury caused by bleomycin instillation. When compared with bleomycin-treated GITR+/+ mice, bleomycin-treated GITR-/- mice exhibited a reduced degree of i) lung infiltration with polymorphonuclear neutrophils (MPO activity); ii) edema formation; iii) histological evidence of lung injury; iv) TNF-alpha and interleukin (IL)-1beta production; v) nitrotyrosine formation; and vi) NF-kappaB activation. The cotreatment of GITR+/+ mice with Fc-GITR fusion protein (6.25 microg/mouse) also significantly attenuated all of the above indicators of lung damage and inflammation. Our results clearly demonstrate that GITR-GITRL interaction plays an important role in the chronic lung injury induced by bleomycin in the mice.

2583 Background: Death receptor 5 (DR5) is a member of the TNFR superfamily that initiates the extrinsic apoptotic pathway. CS-1008 is a humanised, monoclonal IgG1 agonistic antibody (Ab) to human DR5 created by CDR grafting of the murine Ab TRA-8. The aim of this study was to investigate the impact of CS-1008 dose on biodistribution, quantitative tumor uptake and anti-tumor response in pts with mCRC. Methods: Pts with mCRC were treated with weekly IV CS-1008 in 5 non-sequential cohorts (Co). Different loading doses were used on days 1 and 8 (0.2 to 6 mg/kg), followed by a weekly dose of 2 mg/kg. D1 and D36 doses were trace-labeled with 111In, followed by whole-body planar and regional SPECT imaging over 10 days. Primary endpoints: initial biodistribution, pharmacokinetic (PK) and tumor uptake following single infusion; changes in biodistribution, PK and tumor uptake following sequential doses. Secondary endpoints: tumor response; changes in tumor metabolism by FDG-PET; serum apoptosis biomarkers and tumor response markers. Results: 19 pts (median age 64 yrs; M:F 11:8; 2-6 prior chemotherapy regimens) were enrolled as follows: Co 1, n=2; Co 2, n=4; Co 3, n=5; Co 4, n=3; and Co 5, n=5. Tumor uptake was variable: 7 pts had no uptake, 11 pts had uptake in all sites of disease but liver, 1 pt showed liver uptake. Tumor uptake and PK were not affected by dose or repeated drug administration. No anti-CS-1008 Ab responses were detected. DR5 expression in archived samples did not correlate with uptake or response. 111In‐CS‐1008 biodistribution showed gradual blood pool clearance and no abnormal uptake in normal tissue. Mean % change in SUVmax from baseline in lesions with uptake was higher than in those with no uptake. There were 8 SD, 1 PR and 10 PD. Duration of PR was 3.7 months (mo). Mean duration of SD was 4 mo. Disease control rate (SD + PR) in pts with uptake was 58% vs 28% of pts with no uptake. Lesions with no uptake were more likely to progress, with a PD risk of 3.4 times higher than those lesions with uptake. Conclusions: Tumor DR5 expression, assessed by 111In-CS-1008 imaging, revealed real-time heterogeneous DR5 expression and appeared to be a promising predictive imaging biomarker of clinical benefit in pts with mCRC receiving CS-1008. Clinical trial information: NCT01220999.

International audience; No abstract available