Tumor Necrosis Factor Receptor 2 Expression Research Articles

GRN Activates TNFR2 to Promote Macrophage M2 Polarization Aggravating Mycobacterium Tuberculosis Infection.

The polarization of macrophages plays a critical role in the immune response to infectious diseases, with M2 polarization shown to be particularly important in various pathological processes. However, the specific mechanisms of M2 macrophage polarization in Mycobacterium tuberculosis (Mtb) infection remain unclear. In particular, the roles of Granulin (GRN) and tumor necrosis factor receptor 2 (TNFR2) in the M2 polarization process have not been thoroughly studied. To investigate the effect of macrophage M2 polarization on Mtb infection and the mechanism of GRN and TNFR2 in M2 polarization. Forty patients with pulmonary tuberculosis (PTB) and 40 healthy volunteers were enrolled in this study, and peripheral blood samples were taken to detect the levels of TNFR2 and GRN mRNA by Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR); monocytes were isolated and then assessed by Flow Cytometry (FC) for M1 and M2 macrophage levels. To further validate the function of TNFR2 in macrophage polarization, we used interleukin 4 (IL-4) to induce mouse monocyte macrophages RAW264.7 to M2 polarized state. The expression of TNFR2 was detected by Western Blot and RT-qPCR. Next, we constructed a GRN knockdown plasmid and transfected it into IL-4-induced mouse monocyte macrophage RAW264.7, and detected the expression of TNFR2, M1 macrophage-associated factors tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), and interleukin 6 (IL-6), and the M2 macrophage-associated factors CD206, IL-10, and Arginase 1 (Arg1); Immunofluorescence staining was used to monitor the expression of CD86+ and CD206+, and FC was used to analyze the macrophage phenotype. Subsequently, immunoprecipitation was used to detect the binding role of GRN and TNFR2. Finally, the effects of GRN and TNFR2 in macrophage polarization were further explored by knocking down GRN and simultaneously overexpressing TNFR2 and observing the macrophage polarization status. The results of the study showed elevated expression of TNFR2 and GRN and predominance of M2 type in macrophages in PTB patients compared to healthy volunteers (p < 0.05). Moreover, TNFR2 was highly expressed in M2 macrophages (p < 0.05). Additionally, GRN knockdown was followed by elevated expression of M1 polarization markers TNF-α, iNOS and IL-6 (p < 0.05), decreased levels of M2 polarization-associated factors CD206, IL-10 and Arg1 (p < 0.05), and macrophage polarization towards M1. Subsequently, we found that GRN binds to TNFR2 and that GRN upregulates TNFR2 expression (p < 0.05). In addition, knockdown of GRN elevated M1 polarization marker expression, decreased M2 polarization marker expression, and increased M1 macrophages and decreased M2 macrophages, whereas concurrent overexpression of TNFR2 decreased M1 polarization marker expression, elevated M2 polarization marker expression, and decreased M1 macrophages and increased M2 macrophages. TNFR2 and GRN are highly expressed in PTB patients and GRN promotes macrophage M2 polarization by upregulating TNFR2 expression.

Microfibril-Associated Protein-3-Like Regulates TGFβ-Induced EMT Process via TNFR2/p38 MAPK Pathway in Endothelial Cells

Abdominal aortic inflammation (AAI) is a major arterial vasculitis characterized by chronic inflammation and fibrosis. Endothelial cells transform into mesenchymal cells (EMT) is one of the significant mechanisms of vasculitis fibrosis. Despite its importance, the molecular mechanism of EMT in AAI remains poorly understood. In this study, we induced AAI in mice through intraperitoneal injection of tumor necrosis factor-alpha (TNFα). To analyze protein expression, we performed Western blotting. Additionally, we extracted RNA using the nanomagnetic bead method to investigate the expression of functionally related genes. We conducted cell migration and invasion assays using scratch and Transwell techniques. Western blot analysis revealed the upregulation of microfibril-associated protein-3-like (MFAP3L) and tumor necrosis factor (TNF) receptor 2 (TNFR2), along with p38 signaling pathway activation. Notably, MFAP3L expression played a crucial role in the transforming growth factor-beta (TGFβ)-induced EMT process in endothelial cells. Furthermore, we identified that MFAP3L-mediated EMT relied on both TNFR2 expression and the activity of the TNFR2/p38 signaling pathway.

The TNFα/TNFR2 axis mediates natural killer cell proliferation by promoting aerobic glycolysis

Natural killer (NK) cells are predominant innate lymphocytes that initiate the early immune response during infection. NK cells undergo a metabolic switch to fuel augmented proliferation and activation following infection. Tumor necrosis factor-alpha (TNFα) is a well-known inflammatory cytokine that enhances NK cell function; however, the mechanism underlying NK cell proliferation in response to TNFα is not well established. Here, we demonstrated that upon infection/inflammation, NK cells upregulate the expression of TNF receptor 2 (TNFR2), which is associated with increased proliferation, metabolic activity, and effector function. Notably, IL-18 can induce TNFR2 expression in NK cells, augmenting their sensitivity toward TNFα. Mechanistically, TNFα-TNFR2 signaling upregulates the expression of CD25 (IL-2Rα) and nutrient transporters in NK cells, leading to a metabolic switch toward aerobic glycolysis. Transcriptomic analysis revealed significantly reduced expression levels of genes involved in cellular metabolism and proliferation in NK cells from TNFR2 KO mice. Accordingly, our data affirmed that genetic ablation of TNFR2 curtails CD25 upregulation and TNFα-induced glycolysis, leading to impaired NK cell proliferation and antiviral function during MCMV infection in vivo. Collectively, our results delineate the crucial role of the TNFα-TNFR2 axis in NK cell proliferation, glycolysis, and effector function.

The expression of tumor necrosis factor receptor 2 is correlated with the prognosis of cancer: a systematic review and meta-analysis.

Tumor necrosis factor receptor 2 (TNFR2) is a subtype of the tumor necrosis factor receptors and is known to promote cancer progression by enhancing cancer cell proliferation and inducing immune suppression. More recently, there are reports that TNFR2 expression is related to the prognosis of patients with cancer, including lung, breast, esophageal, colorectal cancer, and lymphoma. In this study, the correlation between the expression of TNFR2 and the prognosis and clinicopathological factors of cancer was systematically evaluated. This study aimed at elucidating the relationship between TNFR2 and prognosis in patients with cancer. PubMed, Embase, and Cochrane Library were searched and a meta-analysis was performed to assess the prognostic and clinicopathological values of TNFR2 expression in patients with cancer. Nine studies with 2,229 patients were included. High expression of TNFR2 was significantly correlated with poor overall survival (OS) [hazard ratio (HR), 1.76; 95% confidence interval (CI): 1.37-2.27; P<0.001] and disease-free survival (DFS) (HR, 2.75; 95% CI: 1.92-3.92; P<0.001). High expression of TNFR2 was also significantly associated with higher tumor grade [odds ratio (OR), 1.58; 95% CI: 1.26-1.98; P<0.001], higher tumor stage (OR, 2.41; 95% CI: 1.62-3.60; P<0.001) and higher clinical stage (OR, 1.80; 95% CI: 1.44-2.23; P<0.001). High expression of TNFR2 was related to poor prognosis and could be a prognostic factor in patients with cancer.

TFNR2 in Ischemia-Reperfusion Injury, Rejection, and Tolerance in Transplantation.

Tumor necrosis factor receptor 2 (TNFR2) has been shown to play a crucial role in CD4+ T regulatory cells (CD4+Tregs) expansion and suppressive function. Increasing evidence has also demonstrated its role in a variety of immune regulatory cell subtypes such as CD8+ T regulatory cells (CD8+ Tregs), B regulatory cells (Bregs), and myeloid-derived suppressor cells (MDSCs). In solid organ transplantation, regulatory immune cells have been associated with decreased ischemia-reperfusion injury (IRI), improved graft survival, and improved overall outcomes. However, despite TNFR2 being studied in the context of autoimmune diseases, cancer, and hematopoietic stem cell transplantation, there remains paucity of data in the context of solid organ transplantation and islet cell transplantation. Interestingly, TNFR2 signaling has found a clinical application in islet transplantation which could guide its wider use. This article reviews the current literature on TNFR2 expression in immune modulatory cells as well as IRI, cell, and solid organ transplantation. Our results highlighted the positive impact of TNFR2 signaling especially in kidney and islet transplantation. However, further investigation of TNFR2 in all types of solid organ transplantation are required as well as dedicated studies on its therapeutic use during induction therapy or treatment of rejection.

Abstract 1810: Anti-human TNFR2 antagonist induces T cell proliferation and restores T cell balance to promote tumor killing in TNFR2 HuGEMM™ engrafted with MC38/OVA tumor cells

Abstract Introduction: Tumor necrosis factor receptor 2 (TNFR2) is a member of the TNF-receptor superfamily, which is abundantly expressed in regulatory T cells (Treg) and effector T cells (Teff) in the tumor microenvironment (TME) upon T cell activation. TNFR2 is also highly expressed on the surface of many human tumors. For hot tumors, the TME is infiltrated by immunosuppressive Treg cells that suppress Teff cells to allow tumor growth. However, antagonistic anti-TNFR2 inhibitors are believed to break this imbalance by promoting T cell activation and eliminating Tregs to improve cytotoxic T cell function. Taken together, TNFR2 is becoming a promising potential immunotherapy target in the TNF-receptor superfamily along with GITR, OX40, 4-1BB, etc. whether by stimulating T cells to restore Teff/Treg balance in the TME or by directly inducing tumor cell killing though NF-kappaB signaling. We have developed a humanized TNFR2 mouse model (TNFR2 HuGEMM) to evaluate antagonistic anti-TNFR2 inhibitors to induce tumor killing. Methods: We established TNFR2 HuGEMM in both C57BL/6 background and BALB/c background by inserting chimeric h/m TNFR2 CDS in the mouse Tnfrsf1b locus via CRISPR/Cas9-based gene engineering, in which the mouse TNFR2 extracellular domain was replaced by human counterparts, leading to expression of chimeric TNFR2 receptors with human extracellular domain and mouse trans-membrane and intracellular domains. Results: We profiled and characterized the surface expression of human/mouse chimeric TNFR2 on FoxP3+ Treg cells as well as on granulocytes (CD11b+Gr1hi/F4/80- and CD11b+Gr1-/F4/80+) and monocytes (CD11blowGr1-/F4/80low) by FACS analysis of peripheral blood and spleen in the heterozygous and homozygous TNFR2 HuGEMM mice. The expression of chimeric m/hTNFR2 on Tregs in HuGEMM mice can proportionally recapitulate the expression of mouse TNFR2 in wild-type mice as well as human TNFR2 expression in human PBMC. We then inoculated MC38-OVA syngeneic tumors into the female homozygous HuGEMM mice and treated the mice with anti-human TNFR2 antagonists (Ab1 and Ab2). Anti-hTNFR2 Ab1 led to ~80% tumor growth inhibition (TGI) and 14% of the mice (1/7) were eventually cured. By contrast, anti-hTNFR2 Ab2 only led to ~50% TGI and 28% of the mice (2/7) were cured, but 3 out of 7 mice were found dead upon treatment with Ab2. At the efficacy endpoint, FACS analysis of tumor infiltrating lymphocytes indicated that both anti-hTNFR2 antibodies promoted T cell proliferation and elevated Teff/Treg ratio in tumors. Conclusions: Here, we report the in vivo tumor killing by anti-hTNFR2 antibodies in a humanized TNFR2 mouse model engrafted with MC38-OVA. The TNFR2 HuGEMM is a useful preclinical animal model to evaluate the in vivo efficacy and safety of human TNFR2 agonists or antagonists potentially in combination with other immune modulators. Citation Format: Daniel X. He, Lei Zheng, Ruilin Sun, Annie X. An, Henry Q.x. Li, Davy Xuesong Ouyang. Anti-human TNFR2 antagonist induces T cell proliferation and restores T cell balance to promote tumor killing in TNFR2 HuGEMM™ engrafted with MC38/OVA tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1810.

Regulatory T cells express Tumor Necrosis Factor Receptor 2 with the highest intensity among CD4+ T cells in the draining lymph nodes of breast cancer

Tumor Necrosis Factor Receptor 2 (TNFR2) is one of the receptors of TNF-α, which is expressed on various cell types. TNFR2 signaling has a balancing role between regulatory and effector functions of T cells. Herein, we investigated the expression of TNFR2 on regulatory T cells (Tregs) and non-Tregs in breast tumor-draining lymph nodes. Mononuclear cells were isolated from 16 axillary lymph nodes, and the expressions of TNFR2, Foxp3 and CD25 were assessed in CD4+ T cells by flow cytometry. Our results showed that the majority of TNFR2+CD4+ T cells were Foxp3−CD25−. However, the percentage of TNFR2+ cells was significantly higher in Foxp3+CD25+CD4+ Tregs compared to Foxp3−CD25−CD4+, Foxp3+CD25−CD4+, and Foxp3−CD25+CD4+ T cell subsets. Among these subsets, Foxp3+CD25+TNFR2+CD4+ T cells were found to have the highest intensity of TNFR2 expression. The intensity of Foxp3 expression in Foxp3+CD25+TNFR2+CD4+ Treg cells was significantly higher than in their TNFR2− counterpart. Collectively, we showed that most of TNFR2+CD4+ T lymphocytes were Foxp3−CD25−, while the majority of Foxp3+CD25+CD4+ Tregs were TNFR2+, and they expressed TNFR2 with the highest intensity. This report highlights the importance of TNFR2 expression on Tregs and paves the way for further investigation of the effects of TNF-α on the suppressive activity of Tregs in the tumor microenvironment.

TNFR2 expression is a hallmark of human memory B cells with suppressive function.

Tumor Necrosis Factor Receptor 2 (TNFR2) expression is increasingly being linked to tolerogenic immune reactions and cells with suppressor function including a subset of T-regulatory cells. B-regulatory cells play an important role in control of T-cell responses and inflammation. Recently, we described TNFR2 as a marker for IL-10-producing B cells, a hallmark of this cell subset. Here, we demonstrate that proliferation of T cells is reduced in the presence of TNFR2 positive human memory B cells generated with TLR9 ligand, while TNFR2- and TNFR2+CD27- B cells display costimulatory activity. Our data further reveal that IL-10 secretion is characteristic of IgM+ naïve and memory B cells but suppressive activity is not restricted to IL-10: (i) the inhibitory effect of TNFR2+ switched memory B cells was comparable to that exerted by TNFR2+ IgM+ memory B cells although IL-10 secretion levels in the cocultures were lower; (ii) supernatants from TNFR2+ memory B cells failed to suppress T-cell proliferation. Based on our findings, we propose that formation of Breg is a specific characteristic of human memory B cells undergoing terminal differentiation. Our data further corroborate that TNFR2 represents a viable marker for identification of memory B cells with regulatory function.

Signaling through tumor necrosis receptor 2 induces stem cell marker in CD133+ regenerating tubular epithelial cells in acute cell-mediated rejection of human renal allografts.

Tumor necrosis factor receptor 2 (TNFR2) is strongly upregulated on renal tubular epithelial cells by acute cell-mediated rejection (ACR. In human kidney organ culture, TNFR2 signaling both upregulates TNFR2 expression and promotes cell cycle entry of tubular epithelial cells. We find significantly more cells express CD133 mRNA and protein, a putative stem cell marker, in allograft biopsy samples with ACR compared to acute tubular injury without rejection or pretransplant "normal kidney" biopsy samples. Of CD133+ cells, ~85% are within injured tubules and ~15% are interstitial. Both populations express stem cell marker TRA-1-60 and TNFR2, but only tubular CD133+ cells express proximal tubular markers megalin and aquaporin-1. TNFR2+ CD133+ cells in tubules express proliferation marker phospho-histone H3S10 (pH3S10 ). Tubular epithelial cells in normal kidney organ cultures respond to TNFR2 signaling by expressing CD133 mRNA and protein, stem cell marker TRA-1-60, and pH3S10 within 3hours of treatment. This rapid response time suggests that CD133+ cells in regenerating tubules of kidneys undergoing ACR represent proliferating tubular epithelial cells with TNFR2-induced stem cell markers rather than expansion of resident stem cells. Infiltrating host mononuclear cells are a likely source of TNF as these changes are absent in acute tubular injury .

Optimizing TNFR2 antagonism for immunotherapy with tumor microenvironment specificity.

Most approved cancer immunotherapies lack T-regulatory (Treg) or tumor specificity. TNF receptor 2 (TNFR2) antibody antagonism is emerging as an attractive immunotherapy due to its tumor microenvironment (TME) specificity. Here we show that the human TNFR2 receptor is overexpressed on both human tumor cells and on human tumor-residing Tregs, but negligibly expressed on beneficial T effectors (Teffs). Further, we found widespread, if variable, TNFR2 expression on 788 human tumor cell lines from diverse cancer tissues. These findings provided strong rationale for developing a targeted immunotherapy using a TNFR2 antibody antagonist. We designed a novel, human-directed TNFR2 antibody antagonist and tested it for function using three cell-based TME assays. The antagonist showed TME specificity by killing of TNFR2-expressing tumor cells and Tregs, but sparing Teffs, which proliferated. However, the antagonist shuffled between five isoforms, only one of which showed the desirable function. We designed and tested several new chimeric human versions of the antagonist, finding that the IgG2 isotype functioned better than the IgG1 isotype. To further improve function, we introduced targeted mutations to its amino acid sequence to stabilize the natural variability of the IgG2 isotype's hinge. Altogether, our findings suggest that optimal TNFR2 antagonists are of the human IgG2 isotype, have hinge stabilization, and have wide separation of antibody arms to bind to newly synthesized TNFR2 on rapidly growing tumor cells. Antagonistic antibodies with these characteristics, when bound to TNFR2, can form a nonsignaling cell surface dimer that functions with high TME specificity.

Synergistic Effects of Nanomedicine Targeting TNFR2 and DNA Demethylation Inhibitor-An Opportunity for Cancer Treatment.

Tumor necrosis factor receptor 2 (TNFR2) is expressed on some tumor cells, such as myeloma, Hodgkin lymphoma, colon cancer and ovarian cancer, as well as immunosuppressive cells. There is increasingly evidence that TNFR2 expression in cancer microenvironment has significant implications in cancer progression, metastasis and immune evasion. Although nanomedicine has been extensively studied as a carrier of cancer immunotherapeutic agents, no study to date has investigated TNFR2-targeting nanomedicine in cancer treatment. From an epigenetic perspective, previous studies indicate that DNA demethylation might be responsible for high expressions of TNFR2 in cancer models. This perspective review discusses a novel therapeutic strategy based on nanomedicine that has the capacity to target TNFR2 along with inhibition of DNA demethylation. This approach may maximize the anti-cancer potential of nanomedicine-based immunotherapy and, consequently, markedly improve the outcomes of the management of patients with malignancy.

Activation of Regulatory T Cells with IL-2 and TNF-α to Promote Homing to the Gut and to Improve Acute GvHD Prevention

INTRODUCTION Adoptive immunotherapy with donor conventional T cells (Tcons) and CD4+CD25+FOXP3+regulatory T cells (Tregs) is used to enhance immune reconstitution in clinical trials of haploidentical transplantation. Donor Tregs are infused to prevent adverse alloreactions mediated by Tcons against normal tissues, which cause GvHD, in the absence of any post transplant immune suppression. This strategy proved to be effective in preventing leukemia relapse and chronic GvHD, allowing for ~70% event-free survival in patients with acute leukemia (Pierini et al. submitted). However, acute GvHD occurs in ~30% of the patients. We are investigating if ex-vivo stimulation of Tregs with a combination of low-dose IL-2 and TNF-α before adoptive transfer could improve acute GvHD prevention. Tregs express higher levels of the TNF receptor 2 (TNFR2) compared with Tcons and TNFR2 expression defines a Treg subpopulation with maximal suppressive capacity. Recent studies showed that activation of TNFR2 signaling in mouse Tregs reduced GvHD lethality in mouse models. METHODS Human CD4+or CD4+CD25+T cells were incubated for 60 hours with IL-2 (10 UI/ml) with or without TNF-α (20 ng/ml). Treg proliferation in the presence of allogeneic irradiated peripheral blood mononuclear cells (PBMC), and Treg-mediated suppression of Tcon proliferation were evaluated by CFSE assay. Colon biopsies from patients with suspected GvHD were stained with anti-CD3 and anti-FOXP3 antibodies. RESULTS We developed a translatable in vitro system for improving human Treg function by adding a short term stimulation with low-dose IL-2 and TNF-α (IL-2/TNF) in the selection process. CD4+T cells were primed with IL-2/TNF before CD25 positive selection. When total CD4+T cells were stimulated with IL-2/TNF, CD25 expression was selectively upregulated on CD4+FOXP3+Tregs, but not on CD4+FOXP3- T cells. IL-2/TNF-primed Tregs maintained TNFR2 expression and expressed higher levels of FOXP3 and CD25 compared with Tregs stimulated with IL-2 alone (p&lt;.05). They also had enhanced proliferation in response to allogeneic PBMC (p&lt;.05) and suppressed Tcon proliferation more efficiently (p&lt;.01). Whole transcript expression analyses showed IL-2/TNF priming increased the expression of genes associated with Treg activation, proliferation and function compared to IL-2 alone, such us OX-40, 4-1BB, CD69, CCR8, CD39 genes and components of the NF-κB signaling pathway. We analyzed GvHD clinical signs of the last 20 patients who were diagnosed with grade II-IV acute GvHD after Treg/Tcon-based haploidentical transplantation at our institution. Nineteen presented with early-onset gut GvHD and 40% of those patients did not have any skin involvement. We hypothesized that preferential targeting to the gut was the consequence of defective homing of Tregs to the gut. Compared with Tcons, Tregs express higher levels of skin homing receptors (such as CCR4 and CLA-4), but lower levels of the gut homing receptor α4β7-integrin. We analyzed the infiltration of Tregs and Tcons in 31 colon biopsies from patients with suspected GvHD. The number of Tregs was higher in patients who were not diagnosed with acute GvHD compared with those who were (158.3/mm2 vs 86.3/mm2, p&lt;.05). A similar trend was observed for the Treg:Tcon ratio (0.27 vs 0.15, p&lt;.05). These results suggest that homing of Tregs to the gut plays an important role in the effectiveness of gut GvHD prevention. We also found that IL-2/TNF priming selectively upregulated α4β7-integrin expression on Tregs compared with freshly isolated cells (p&lt;.01). Experiments in humanized mouse models are underway to ascertain if IL-2/TNF priming improves Treg homing to the gut and site-specific GvHD prevention. CONCLUSION The present study shows that activation of TNFR2 signaling in human Tregs enhances their proliferation and suppressive function. It also suggests that Treg suppressive function exerted in periphery could be critical for tissue-specific GvHD control. Moreover, IL-2/TNF-priming could promote homing of Tregs to the gut and their capability to inhibit gut GvHD. Thus, IL-2/TNF-priming may be critical to optimize the ongoing clinical trial of adoptive immunotherapy with Tregs for GvHD prevention. Future studies will ascertain if IL-2/TNF-priming of Tregs can also allow for the infusion of higher doses of Tcons to further enhance immune reconstitution and leukemia killing without increasing the risk of GvHD. Disclosures No relevant conflicts of interest to declare.

Differential roles of TNF\u03b1-TNFR1 and TNF\u03b1-TNFR2 in the differentiation and function of CD4+Foxp3+ induced Treg cells in vitro and in vivo periphery in autoimmune diseases

Tumor Necrosis Factor (TNF) α is a multifunctional cytokine with pro-inflammatory and anti-inflammatory characteristics. Increasing evidence suggests that thymus-derived, natural regulatory T cells (nTreg) express a remarkably high level of TNF Receptor 2 (TNFR2) and TNFα modulates the number or function of nTreg via TNFR2 in autoimmune diseases. Nonetheless, Treg cells consist of at least nTreg and iTreg that are induced in the periphery or in vitro and two subsets may have different biological characteristics. However, the role of TNF-TNFR signaling in development and function of these iTreg cells is less clear. In this study, we systemically studied the effect of TNFα and its receptor signals on iTreg differentiation, proliferation, and function in vitro and in vivo. We further investigated the expression and requirement of TNFR1 or TNFR2 expression on iTreg by utilizing TNFR1−/− and TNFR2−/− mice. We found that exogenous TNFα facilitated iTreg differentiation and function in vitro. TNFR2 deficiency hampered iTreg differentiation, proliferation, and function, while TNFR1 deficiency decreased the differentiation of inflammatory T cells such as Th1 and Th17 cells but maintained the regulatory capabilities of iTreg both in vitro and in vivo. Using colitis model, we also revealed TNFR2 but not TNFR1 deficiency compromised the iTreg functionality. Interestingly, inflammation affects TNFR expression on nTreg but not iTreg subset. Our results demonstrate that exogenous TNFα may enhance the differentiation and function of iTreg via TNFR2 signaling. The expression of TNFR2 on Treg might be downregulated in some autoimmune diseases, accompanied by an increased level of TNFR1. Thus, TNFR2 agonists or TNFR1-specific antagonists hold a potential promise for clinical application in treating patients with autoimmune diseases.

TNFR2 ligation in human T regulatory cells enhances IL2-induced cell proliferation through the non-canonical NF-\u03baB pathway

Human T regulatory cells (T regs) express high levels of TNF receptor 2 (TNFR2). Ligation of TNFR2 with TNF, which can recognise both TNFR1 and TNFR2, or with a TNFR2-selective binding molecule, DARPin 18 (D18) activates canonical NF-κB signalling, assessed by IκBα degradation, and the magnitude of the response correlates with the level of TNFR2 expression. RNA-seq analysis of TNF- or D18-treated human T regs revealed that TNFR2 ligation induces transcription of NFKB2 and RELB, encoding proteins that form the non-canonical NF-κB transcription factor. In combination with IL2, D18 treatment is specific for T regs in (1) stabilising NF-κB-inducing kinase protein, the activator of non-canonical NF-κB signalling, (2) inducing translocation of RelB from cytosol to nucleus, (3) increasing cell cycle entry, and (4) increasing cell numbers. However, the regulatory function of the expanded T regs is unaltered. Inhibition of RelB nuclear translocation blocks the proliferative response. We conclude that ligation of TNFR2 by D18 enhances IL2-induced T regs proliferation and expansion in cell number through the non-canonical NF-κB pathway.

Clinical significance of tumor necrosis factor receptor 2 in middle and lower thoracic esophageal squamous cell carcinoma.

High expression and role of tumor necrosis factor receptor 2 (TNFR2) in cancer progression and prognosis has been reported in several types of tumors. However, its role in esophageal carcinoma (EC) remains unknown. In the present study, TNFR2 expression in middle and lower thoracic esophageal squamous cell carcinoma (ESCC) was detected using immunohistochemistry (IHC). Chi-square test revealed that TNFR2 was positively correlated with invasion depth, advanced clinical stage and low differentiation degree. Furthermore, survival analysis revealed that TNFR2 was positively correlated with poor overall survival (OS). Moreover, univariate COX regression analysis revealed that clinical stage, lymph node involvement, and invasion depth can affect the OS of ESCC patients, while multivariate COX regression analysis revealed that lymph node involvement and invasion depth can affect the OS of ESCC patients. In middle thoracic ESCC patients, TNFR2 was positively correlated with invasion depth, advanced clinical stage and poor OS. Furthermore, univariate and multivariate COX regression analysis both revealed that clinical stage, lymph node involvement, and invasion depth can affect OS. In lower thoracic ESCC patients, TNFR2 was positively correlated with low differentiation degree. Furthermore, the positive correlation of TNFR2 with poor OS did not reach statistical significance. In addition, univariate COX regression analysis revealed that only lymph node involvement could affect OS. All the results suggest that TNFR2 can play an important role in the progression and poor prognosis of ESCC patients. Moreover, the role of TNFR2 in the prognosis of middle thoracic ESCC patients was earlier and stronger than in lower thoracic ESCC patients.

Increased Regulatory T Cells in Peripheral Blood of Acute Myeloid Leukemia Patients Rely on Tumor Necrosis Factor (TNF)-α-TNF Receptor-2 Pathway.

Acute myeloid leukemia (AML) harbors an immune suppression environment, featured by increased regulatory T cells (Tregs). The expression of tumor necrosis factor receptor-2 (TNFR2) on Tregs could be used to identify the maximally suppressive Treg population, and TNF-α furtherly promoted the expansion and function of Tregs via TNFR2 in mice. However, the role of TNF-α has not been determined in AML patients. In view of high levels of TNF-α and Tregs in AML patients, we hypothesized that the increased frequency of Tregs may rely on TNF-α–TNFR2 pathway. We investigated the levels of TNFR2+ Tregs and TNF-α secreted by T cells in peripheral blood (PB) of AML by flow cytometry and enzyme-linked immunosorbent assay, respectively. Our results showed the elevated plasma TNF-α in PB of newly diagnosed (ND) AML patients. The production of TNF-α by CD4+ T cells, especially by T helper (Th)17 cells was remarkably higher in ND AML patients than in complete remission (CR) patients and healthy controls. Then, we found that the circulating frequencies of CD4+CD25+ Tregs and CD4+CD25high Tregs in AML patients were elevated compared with those in healthy controls and CR patients. TNFR2 expression was much higher on Tregs in AML patients and was preferentially expressed on CD4+CD25high T cells. Furthermore, we confirmed that, in vitro, the additional TNF-α can increase the frequency of Tregs through TNFR2 in both AML patients and healthy controls. Summarily, in AML patients, the abnormally elevated level of TNF-α secreted by CD4+ T especially Th17 cells promoted the higher Tregs frequency via the TNF-α–TNFR2 pathway.

Expression of Tumor Necrosis Factor Receptor 2 Characterizes TLR9-Driven Formation of Interleukin-10-Producing B Cells.

B cell-derived interleukin-10 (IL-10) production has been described as a hallmark for regulatory function in B lymphocytes. However, there is an ongoing debate on the origin of IL-10-secreting B cells and lack of specific surface markers has turned into an important obstacle for studying human B regulatory cells. In this study, we propose that tumor necrosis factor receptor 2 (TNFR2) expression can be used for enrichment of IL-10-secreting B cells. Our data confirm that IL-10 production can be induced by TLR9 stimulation with CpG ODN and that IL-10 secretion accompanies differentiation of peripheral blood B cells into plasma blasts. We further show that CpG ODN stimulation induces TNFR2 expression, which correlates with IL-10 secretion and terminal differentiation. Indeed, flow cytometric sorting of TNFR2+ B cells revealed that TNFR2+ and TNFR2− fractions correspond to IL-10+ and IL-10− fractions, respectively. Furthermore, CpG-induced TNFR2+ B cells were predominantly found in the IgM+ CD27+ B cell subset and spontaneously released immunoglobulin. Finally, our data corroborate the functional impact of TNFR2 by demonstrating that stimulation with a TNFR2 agonist significantly augments IL-10 and IL-6 production in B cells. Altogether, our data highlight a new role for TNFR2 in IL-10-secreting human B lymphocytes along with the potential to exploit this finding for sorting and isolation of this currently ill-defined B cell subset.

Abstract 21146: Late Pregnancy is Associated With a Decrease in Global Circumferential Strain and Tumor Necrosis Factor Receptor 2

Background: The hearts of pregnant women and rodents undergo physiological hypertrophy with preserved function. Recently, we have demonstrated that the hearts of late pregnant (LP) rodents are more prone to ischemia reperfusion injury compared to non-pregnant (NP) rodents. Currently, measurements such as ejection fraction (EF) are not sensitive enough to determine subclinical myocardial dysfunction. However, speckle-tracking echocardiography (STE), a novel imaging tool, has been demonstrated to assess myocardial function with greater sensitivity. Tumor Necrosis Factor Receptor 2 (TNFR2), a membrane receptor that has been implicated in cardioprotection and has been demonstrated as marker for subclinical myocardial dysfunction in patients. Thus, we hypothesized that global circumferential strain (GCS) measured by STE can detect subclinical myocardial dysfunction in pregnancy that is associated with a decrease in TNFR2 in rats. Methods: LP (20 days pregnant) and NP female Sprague-Dawley rats were used for the study (n=14). Left ventricular EF (LVEF, %), fractional shortening (FS, %), heart rate (HR, bpm), and GCS (%) were obtained using echocardiography (Visualsonics, Vevo 2100) (n=7/group). GCS was obtained with STE and was measured in triplicates. Total ventricular weight (VW) was recorded and LV was isolated to measure LV hypertrophy (LV/VW) (n=6/group). TNFR2 expression was measured using qPCR. Results: LP rats showed slightly reduced but insignificant LVEF (LP: 67.65+/-1.41% vs. NP: 71.35+/-1.52%, P=0.10) and FS (39.07+/-0.90 % vs. 42.643+/-1.76 %, P=0.09) compared to NP rats. Additionally, no difference in HR between LP and NP rats were found (352.50+/-16.36 bpm vs. 333.75+/-4.78 bpm, respectively, p=0.314).However, hearts from LP rats had evidence of hypertrophy evident by an increase in LV/VW compared to NP (0.57+/-0.03 g vs. 0.48+/-0.02 g). LP rats had a significantly reduced GCS compared to NP rats (-27.50+/-1.40 % vs. -34.375+/-1.95 %, p&lt;0.01) and reduced TNFR2 expression compared to NP. Conclusions: Our results demonstrate that late pregnancy is associated with a decrease in GCS with no significant change in EF and a decrease in TNFR2, which may be a subclinical marker for myocardial dysfunction.

Interleukin 6 Present in Inflammatory Ascites from Advanced Epithelial Ovarian Cancer Patients Promotes Tumor Necrosis Factor Receptor 2-Expressing Regulatory T Cells.

Epithelial ovarian cancer (EOC) remains a highly lethal gynecological malignancy. Ascites, an accumulation of peritoneal fluid present in one-third of patients at presentation, is linked to poor prognosis. High levels of regulatory T cells (Tregs) in ascites are correlated with tumor progression and reduced survival. Malignant ascites harbors high levels of Tregs expressing the tumor necrosis factor receptor 2 (TNFR2), as well as pro-inflammatory factors such as interleukin 6 (IL-6) and tumor necrosis factor (TNF). IL-6 is also associated with poor prognosis. Herein, we study the effect of IL-6 and TNF present in ascites on the modulation of TNFR2 expression on T cells, and specifically Tregs. Ascites and respective peripheral blood sera were collected from 18 patients with advanced EOC and soluble biomarkers, including IL-6, sTNFR2, IL-10, TGF-β, and TNF, were quantified using multiplexed bead-based immunoassay. Peripheral blood mononuclear cells (PBMC) from healthy donors were incubated with cell-free ascites for 48 h (or media as a negative control). In some experiments, IL-6 or TNF within the ascites were neutralized by using monoclonal antibodies. The phenotype of TNFR2+ Tregs and TNFR2- Tregs were characterized post incubation in ascites. In some experiments, cell sorted Tregs were utilized instead of PBMC. High levels of immunosuppressive (sTNFR2, IL-10, and TGF-β) and pro-inflammatory cytokines (IL-6 and TNF) were present in malignant ascites. TNFR2 expression on all T cell subsets was higher in post culture in ascites and highest on CD4+CD25hiFoxP3+ Tregs, resulting in an increased TNFR2+ Treg/effector T cell ratio. Furthermore, TNFR2+ Tregs conditioned in ascites expressed higher levels of the functional immunosuppressive molecules programmed cell death ligand-1, CTLA-4, and GARP. Functionally, TNFR2+ Treg frequency was inversely correlated with interferon-gamma (IFN-γ) production by effector T cells, and was uniquely able to suppress TNFR2+ T effectors. Blockade of IL-6, but not TNF, within ascites decreased TNFR2+ Treg frequency. Results indicating malignant ascites promotes TNFR2 expression, and increased suppressive Treg activity using PBMC were confirmed using purified Treg subsets. IL-6 present in malignant ovarian cancer ascites promotes increased TNFR2 expression and frequency of highly suppressive Tregs.

Abstract 4321: TNF receptor 2 is essential for RIP1-dependent cell death in refractory leukemia

Abstract The identification of molecular determinants that regulate sensitivity to specific agents is essential for the development of new therapeutic approaches in cancer. We have earlier shown that a subset of refractory acute lymphoblastic leukaemia (ALL) samples respond to SMAC-mimetic (SM) induced IAP depletion by concurrently inducing RIP1-dependent apoptosis and necroptosis. Comparative gene expression profiling indicated a correlation of sensitivity to SM with the expression of TNF receptor 2 (TNFR2) in primary ALL. Using an independent cohort of primary chemotherapy-resistant ALL samples, we found that presence of high TNFR2 expression identified by qPCR predicted ex vivo-sensitivity to SM. High TNFR2 levels also correlated with higher expression of TNFR1. Deletion of either TNFR1 or TNFR2 using CRISPR/Cas9 in primary ALL conferred resistance to treatment with SM, indicating that TNFR1 and 2 are both functionally required for cell death. Concomitant with an important role for TNFR2 in the response to SM, the overexpression of TNFR2 leads to increased sensitivity to TNF through increased activation of the TNFR1/RIP1 death axis. On the mechanistic level, SM induced recruitment of RIP1 to TNFR1, which was abolished in cells deficient for TNFR2. Taken together, our data reveal a novel function of TNFR2 in cell death signalling, as TNFR2 predicts sensitivity to SMAC mimetics and plays a key role in modulating a switch from RIP1-controlled cell survival to cell death. Citation Format: Julia Aguadé-Gorgorio, Scott McComb, Cornelia Eckert, Maria Pamela Dobay, Gunnar Cario, Caterina Mezzatesta, Arend Von Stackelberg, Martin Stanulla, Schrappe Martin, Jean-Pierre Bourquin, Beat C. Bornhauser. TNF receptor 2 is essential for RIP1-dependent cell death in refractory leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4321. doi:10.1158/1538-7445.AM2017-4321