Antitumor effect of CAR-T cells targeting transmembrane tumor necrosis factor alpha combined with PD-1 mAb on breast cancers.

BACKGROUND: Our previous study showed that transmembrane tumor necrosis factor alpha (tmTNF-α) is overexpressed in primary breast cancers including triple-negative breast cancers (TNBCs). Chimeric antigen receptor engineered-T (CAR-T) cells have been successfully used mainly in B-cell malignancies. METHODS: We generated CAR-T cells targeting tmTNF-α but not secreted tumor necrosis factor alpha and assessed the antitumor effect of the CAR-T cells on tmTNF-α-expressing breast cancer cells in vitro and in vivo. RESULTS: Our tmTNF-α CAR-T cells showed potent cytotoxicity against tmTNF-α-expressing breast cancer cells but not tmTNF-α-negative tumor cells with increased secretion of interferon gamma (IFN-γ) and interleukin (IL)-2 in vitro. In tmTNF-α-overexpressing TNBC-bearing mice, the tmTNF-α CAR-T therapy induced evident tumor regression, prolonged survival and increased serum concentrations of IFN-γ and IL-2. However, we found thattmTNF-α induced programmed death-ligand 1 (PD-L1) expression through the p38 pathway via TNF receptor (TNFR) and through the NF-κB and AKT pathways via outside-to-inside (reverse) signaling, which might limit the efficacy of the CAR-T cell therapy. Blockage of the PD-L1/programmed death-1 (PD-1) pathway by PD-1 monoclonal antibody significantly enhanced the antitumor effect of the tmTNF-α CAR-T cell therapy in vitro and in vivo, and the combination was effective for antiprimary tumors and had a tendency to increase the antimetastasis effect of the CAR-T cell therapy. CONCLUSION: Our findings suggest a potent antitumor efficacy of the tmTNF-α CAR-T cells that can be enhanced by anti-PD-L1/PD-1 because high PD-L1 expression in TNBC was induced by the tmTNF-α signaling, indicating a promising individual therapy for tmTNF-α-positive breast cancers including TNBC.

Suppression of Transmembrane Tumor Necrosis Factor Alpha Processing by a Specific Antibody Protects Against Colitis-Associated Cancer.

Soluble tumor necrosis factor-α (sTNF-α) plays an important role in colitis-associated cancer (CAC); however, little is known about transmembrane TNF-α (tmTNF-α). Here, we observed an increase in sTNF-α mainly in colitis tissues from an azoxymethane/dextran sodium sulfate (DSS)-induced CAC mouse model whereas tmTNF-α levels were chiefly increased on epithelial cells at the tumor stage. The ratio of intracolonic tmTNF-α/sTNF-α was negatively correlated with the levels of pro-inflammatory mediators (IL-1ß, IL-6, and NO) and M1 macrophages but positively correlated with the infiltration of myeloid-derived suppressor cells, regulatory T cells, and the level of the anti-inflammatory cytokine IL-10, suggesting an anti-inflammatory effect of tmTNF-α. This effect of tmTNF-α was confirmed again by the induction of resistance to LPS in colonic epithelial cell lines NCM460 and HCoEpiC through the addition of exogenous tmTNF-α or transfection of the tmTNF-α leading sequence that lacks the extracellular segment but retains the intracellular domain of tmTNF-α. A tmTNF-α antibody was used to block tmTNF-α shedding after the first or second round of inflammation induction by DSS drinking to shift the time window of tmTNF-α expression ahead to the inflammation stage. Antibody treatment significantly alleviated inflammation and suppressed subsequent adenoma formation, accompanied by increased apoptosis. An antitumor effect was also observed when the antibody was administered at the malignant phase of CAC. Our results reveal tmTNF-α as a novel molecular marker for malignant transformation in CAC and provide a new insight into blocking the pathological process by targeting tmTNF-α processing.

Transmembrane tumor necrosis factor alpha attenuates pressure-overload cardiac hypertrophy via tumor necrosis factor receptor 2.

Tumor necrosis factor-alpha (TNF-α) plays an important pathogenic role in cardiac hypertrophy and heart failure (HF); however, anti-TNF is paradoxically negative in clinical trials and even worsens HF, indicating a possible protective role of TNF-α in HF. TNF-α exists in transmembrane (tmTNF-α) and soluble (sTNF-α) forms. Herein, we found that TNF receptor 1 (TNFR1) knockout (KO) or knockdown (KD) by short hairpin RNA or small interfering RNA (siRNA) significantly alleviated cardiac hypertrophy, heart dysfunction, fibrosis, and inflammation with increased tmTNF-α expression, whereas TNFR2 KO or KD exacerbated the pathological phenomena with increased sTNF-α secretion in transverse aortic constriction (TAC)- and isoproterenol (ISO)-induced cardiac hypertrophy in vivo and in vitro, respectively, indicating the beneficial effects of TNFR2 associated with tmTNF-α. Suppressing TNF-α converting enzyme by TNF-α Protease Inhibitor-1 (TAPI-1) to increase endogenous tmTNF-α expression significantly alleviated TAC-induced cardiac hypertrophy. Importantly, direct addition of exogenous tmTNF-α into cardiomyocytes in vitro significantly reduced ISO-induced cardiac hypertrophy and transcription of the pro-inflammatory cytokines and induced proliferation. The beneficial effects of tmTNF-α were completely blocked by TNFR2 KD in H9C2 cells and TNFR2 KO in primary myocardial cells. Furthermore, we demonstrated that tmTNF-α displayed antihypertrophic and anti-inflammatory effects by activating the AKT pathway and inhibiting the nuclear factor (NF)-κB pathway via TNFR2. Our data suggest that tmTNF-α exerts cardioprotective effects via TNFR2. Specific targeting of tmTNF-α processing, rather than anti-TNF therapy, may be more useful for the treatment of hypertrophy and HF.

Inhibition of transmembrane TNF-α shedding by a specific antibody protects against septic shock.

Transmembrane TNF-α (tmTNF-α) and secretory TNF-α (sTNF-α) display opposite effects in septic shock. Reducing tmTNF-α shedding can offset the detrimental effects of sTNF-α and increase the beneficial effect of tmTNF-α. We previously developed a monoclonal antibody that is specific for tmTNF-α and does not cross-react with sTNF-α. In this study, we show that this antibody can specifically suppress tmTNF-α shedding by competing with a TNF-α converting enzyme that cleaves the tmTNF-α ectodomain to release sTNF-α. This tmTNF-α antibody significantly inhibited LPS-induced secretion of interleukin (IL)-1ß, IL-6, interferon-ß, and nitric oxide by monocytes/macrophages, and protected mice from septic shock induced by lipopolysaccharide (LPS) or cecal ligation and puncture, while reducing the bacterial load. The mechanism associated with the protective effect of this tmTNF-α antibody involved promotion of LPS-induced toll-like receptor 4 (TLR4) internalization and degradation by recruiting Triad3A to TLR4. Moreover, the tmTNF-α antibody inhibited LPS-induced activation of nuclear factor-κB and interferon regulatory factor 3 pathways by upregulating expression of A20 and monocyte chemotactic protein-induced protein 1. Similarly, treatment of macrophages with exogenous tmTNF-α suppressed LPS/TLR4 signaling and release of proinflammatory cytokines, indicating that increased levels of tmTNF-α promoted by the antibody contributed to its inhibitory effect. Thus, use of this tmTNF-α antibody for specific suppression of tmTNF-α shedding may be a promising strategy to treat septic shock.

Lactate inhibits ATP6V0d2 expression in tumor-associated macrophages to promote HIF-2α-mediated tumor progression.

Macrophages perform key functions in tissue homeostasis that are influenced by the local tissue environment. Within the tumor microenvironment, tumor-associated macrophages can be altered to acquire properties that enhance tumor growth. Here, we found that lactate, a metabolite found in high concentration within the anaerobic tumor environment, activated mTORC1 that subsequently suppressed TFEB-mediated expression of the macrophage-specific vacuolar ATPase subunit ATP6V0d2. Atp6v0d2-/- mice were more susceptible to tumor growth, with enhanced HIF-2α-mediated VEGF production in macrophages that display a more protumoral phenotype. We found that ATP6V0d2 targeted HIF-2α but not HIF-1α for lysosome-mediated degradation. Blockade of HIF-2α transcriptional activity reversed the susceptibility of Atp6v0d2-/- mice to tumor development. Furthermore, in a cohort of patients with lung adenocarcinoma, expression of ATP6V0d2 and HIF-2α was positively and negatively correlated with survival, respectively, suggesting a critical role of the macrophage lactate/ATP6V0d2/HIF-2α axis in maintaining tumor growth in human patients. Together, our results highlight the ability of tumor cells to modify the function of tumor-infiltrating macrophages to optimize the microenvironment for tumor growth.

Transmembrane TNF-alpha promotes chemoresistance in breast cancer cells.

Chemoresistance remains a major obstacle to successful treatment of breast cancer. Although soluble tumor necrosis factor-α (sTNF-α) has been implicated in mediating drug-resistance in human cancers, whether transmembrane tumor necrosis factor-α (tmTNF-α) plays a role in chemoresistance remains unclear. Here we found that over 50% of studied patients expressed tmTNF-α at high levels in breast cancer tissues and tmTNF-α expression positively correlated with resistance to anthracycline chemotherapy. Alteration of tmTNF-α expression changed the sensitivity of primary human breast cancer cells and breast cancer cell lines to doxorubicin (DOX). Overexpression of N-terminal fragment (NTF) of tmTNF-α, a mutant form with intact intracellular domain of tmTNF-α to transmit reverse signals, induced DOX-resistance. Mechanistically, the tmTNF-α/NTF-ERK-GST-π axis and tmTNF-α/NTF-NF-κB-mediated anti-apoptotic functions were required for tmTNF-α-induced DOX-resistance. In a xenograft mouse model, the combination of tmTNF-α suppression with chemotherapy significantly enhanced the efficacy of DOX. Our data indicate that tmTNF-α mediates DOX-resistance through reverse signaling and targeting tmTNF-α may be beneficial for the treatment of DOX-resistant breast cancer.

Cardioprotective Role of Myeloid-Derived Suppressor Cells in Heart Failure.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that expand in cancer, inflammation, and infection and negatively regulate inflammation and the immune response. Heart failure (HF) is a complex clinical syndrome wherein inflammation induction and incomplete resolution can potentially contribute to HF development and progression. However, the role of MDSCs in HF remains unclear. METHODS: The percentage of MDSCs in patients with HF and in mice with pressure overload-induced HF using isoproterenol infusion or transverse aortic constriction (TAC) was detected by flow cytometry. The effects of MDSCs on isoproterenol- or TAC-induced HF were observed on depleting MDSCs with 5-fluorouracil (50 mg/kg) or gemcitabine (120 mg/kg), transferring purified MDSCs, or enhancing endogenous MDSCs with rapamycin (2 mg·kg-1·d-1). Hypertrophic markers and inflammatory factors were detected by ELISA, real-time polymerase chain reaction, or Western blot. Cardiac functions were determined by echocardiography and hemodynamic analysis. RESULTS: The percentage of human leukocyte antigen-D-related (HLA-DR)-CD33+CD11b+ MDSCs in the blood of patients with HF was significantly increased and positively correlated with disease severity and increased plasma levels of cytokines, including interleukin-6, interleukin-10, and transforming growth factor-ß. Furthermore, MDSCs derived from patients with HF inhibited T-cell proliferation and interferon-γ secretion. Similar results were observed in TAC- and isoproterenol-induced HF in mice. Pharmaceutical depletion of MDSCs significantly exacerbated isoproterenol- and TAC-induced pathological cardiac remodeling and inflammation, whereas adoptive transfer of MDSCs prominently rescued isoproterenol- and TAC-induced HF. Consistently, administration of rapamycin significantly increased endogenous MDSCs by suppressing their differentiation and improved isoproterenol- and TAC-induced HF, but MDSC depletion mostly blocked beneficial rapamycin-mediated effects. Mechanistically, MDSC-secreted molecules suppressed isoproterenol-induced hypertrophy and proinflammatory gene expression in cardiomyocytes in a coculture system. Neutralization of interleukin-10 blunted both monocytic MDSC- and granulocytic MDSC-mediated anti-inflammatory and antihypertrophic effects, but treatment with a nitric oxide inhibitor only partially blocked the antihypertrophic effect of monocytic MDSCs. CONCLUSIONS: Our findings revealed a cardioprotective role of MDSCs in HF by their antihypertrophic effects on cardiomyocytes and anti-inflammatory effects through interleukin-10 and nitric oxide. Pharmacological targeting of MDSCs by rapamycin constitutes a promising therapeutic strategy for HF.

STAT1 mediates transmembrane TNF-alpha-induced formation of death-inducing signaling complex and apoptotic signaling via TNFR1.

Tumor necrosis factor-alpha (TNF-α) exists in two forms: secretory TNF-α (sTNF-α) and transmembrane TNF-α (tmTNF-α). Although both forms of TNF-α induce tumor cell apoptosis, tmTNF-α is able to kill tumor cells that are resistant to sTNF-α-mediated cytotoxicity, indicating their differences in signal transduction. Here, we demonstrate that internalization of TNFR1 is crucial for sTNF-α- but not for tmTNF-α-induced apoptosis. sTNF-α induces binding of tumor necrosis factor receptor type 1-associated death domain protein (TRADD) to the death domain (DD) of TNFR1 and subsequent activation of nuclear factor kappa B (NF-κB), and the formation of death-inducing signaling complexes (DISCs) in the cytoplasm after internalization. In contrast, tmTNF-α induces DISC formation on the membrane in a DD-independent manner. It leads to the binding of signal transducer and activator of transcription 1 (STAT1) to a region spanning amino acids 319-337 of TNFR1 and induces phosphorylation of serine at 727 of STAT1. The phosphorylation of STAT1 promotes its binding to TRADD, and thus recruits Fas-associated protein with DD (FADD) and caspase 8 to form DISC complexes. This STAT1-dependent signaling results in apoptosis but not NF-κB activation. STAT1-deficiency in U3A cells counteracts tmTNF-α-induced DISC formation and apoptosis. Conversely, reconstitution of STAT1 expression restores tmTNF-α-induced apoptotic signaling in the cell line. Consistently, tmTNF-α suppresses the growth of STAT1-containing HT1080 tumors, but not of STAT1-deficient U3A tumors in vivo. Our data reveal an unappreciated molecular mechanism of tmTNF-α-induced apoptosis and may provide a new clue for cancer therapy.

Transmembrane tumor necrosis factor-α promotes the recruitment of MDSCs to tumor tissue by upregulating CXCR4 expression via TNFR2.

Myeloid-derived suppressor cells (MDSCs) accumulated in tumor sites promote immune evasion. We found that TNFR deficiency-induced rejection of transplanted tumor was accompanied with markedly decreased accumulation of MDSCs. However, the mechanism(s) behind this phenomenon is not completely understood. Here, we demonstrated that TNFR deficiency did not affect the amount of MDSCs in bone marrow (BM), but decreased accumulation of Gr-1+CD11b+ MDSCs in the spleen and tumor tissues. The chemotaxis of Tnfr-/- MDSCs was prominently decreased in response to both tumor cell culture supernatants and tumor tissue homogenates from Tnfr-/- and wild-type mice, indicating an effect of TNFR signaling on chemokine receptor expression in MDSCs. We used real-time PCR to detect gene expression for several chemokine receptors in MDSCs from BM and found that CXCR4 was the most affected molecule at the transcriptional level in Tnfr-/- MDSCs. Neutralizing CXCR4 in wild-type MDSCs by a specific antibody blocked their chemotactic migration. Interestingly, it was tmTNF-α, but not sTNF-α, that induced CXCR4 expression in MDSCs. This effect of tmTNF-α was totally blocked in TNFR2-/- but not in TNFR1-/- MDSCs, and partially inhibited by PDTC or SB203580, an inhibitor of NF-κB or p38 MAPK pathway, respectively. Adoptive transfer of wild-type MDSCs restored MDSCs accumulation in tumors of Tnfr-/- mice, but this could be partially blocked by treatment with a CXCR4 inhibitor AMD3100. Our data suggest that tmTNF-α upregulates CXCR4 expression that promotes chemotaxis of MDSCs to tumor, and give a new insight into a novel mechanism by which tmTNF-α facilitates tumor immune evasion.

The effect of HMGB1 on the clinicopathological and prognostic features of non-small cell lung cancer.

Several studies have assessed the diagnostic and prognostic values of high mobility group protein box 1 (HMGB1) expression in non-small cell lung cancer (NSCLC), but these results remain controversial. The purpose of this study was to perform a meta-analysis of the gene microarray analyses of datasets from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) to evaluate the association of HMGB1 expression with the clinicopathological and prognostic features of patients with NSCLC. Furthermore, we investigated the underlying molecular mechanisms by bioinformatics analysis. Twenty relevant articles involving 2651 patients were included in this meta-analysis; the HMGB1 expression in NSCLC tissues was significantly higher than that in the healthy non-cancer control tissues. We also found an indication by microarray analysis and meta-analysis that HMGB1 expression was associated with the cancer TNM Staging System. In terms of prognostic features, a survival analysis from KM-Plotter tool revealed that the high HMGB1 expression group exhibited poorer survival in lung adenocarcinoma (ADC) and overall NSCLC patients. The survival and disease-free analyses from TCGA datasets also showed that HMGB1 mainly affected the development of patients with ADC. Therefore, we focused on how HMGB1 affected the prognosis and development of ADC using bioinformatics analyses and detected that the mitogen-activated protein kinases (MAPK), apoptosis and cell cycle signaling pathways were the key pathways that varied during HMGB1 up-regulation in ADC. Moreover, various genes such as PLCG2, the phosphatidylinositol-4, 5-bisphosphate 3-kinase superfamily (PI3Ks), protein kinase C (PKC) and DGKZ were selected as hub genes in the gene regulatory network. Our results indicated that HMGB1 is a potential biomarker to predict progression and survival of NSCLC, especially of ADC types.

Kupffer-cell-expressed transmembrane TNF-α is a major contributor to lipopolysaccharide and D-galactosamine-induced liver injury.

Tumor necrosis factor (TNF)-α exists in two bioactive forms, a 26-kDa transmembrane form (tmTNF-α) and a 17-kDa soluble form (sTNF-α). sTNF-α has been recognized as a key regulator of hepatitis; however, serum sTNF-α disappears in mice during the development of severe liver injury, and high levels of serum sTNF-α do not necessarily result in liver damage. Interestingly, in a mouse model of acute hepatitis, we have found that tmTNF-α expression on Kupffer cells (KCs) significantly increases when mice develop severe liver injury caused by lipopolysaccharide (LPS)/D-galactosamine (D-gal), and the level of tmTNF-α expression is positively related to the activity of serum transaminases. Therefore, we hypothesized that KC-expressed tmTNF-α constitutes a pathomechanism in hepatitis and have explored the role of tmTNF-α in this disease model. Here, we have compared the impact of KCs(tmTNFlow) and KCs(tmTNFhigh) on acute hepatitis in vivo and ex vivo and have further demonstrated that KCs(tmTNFhigh), rather than KCs(tmTNFlow), not only exhibit an imbalance in secretion of pro- and anti-inflammatory cytokines, favoring inflammatory response and exacerbating liver injury, but also induce hepatocellular apoptosis via tmTNF-α and the expression of another pro-apoptotic factor, Fas ligand. Our data suggest that KC(tmTNFhigh) is a major contributor to liver injury in LPS/D-gal-induced hepatitis.

Transmembrane tumor necrosis factor-alpha sensitizes adipocytes to insulin.

Transmembrane TNF-α (tmTNF-α) acts both as a ligand, delivering 'forward signaling' via TNFR, and as a receptor, transducing 'reverse signaling'. The contradiction of available data regarding the effect of tmTNF-α on insulin resistance may be due to imbalance in both signals. Here, we demonstrated that high glucose-induced impairment of insulin-stimulated glucose uptake by 3T3-L1 adipocytes was concomitant with decreased tmTNF-α expression and increased soluble TNF-α (sTNF-α) secretion. However, when TACE was inhibited, preventing the conversion of tmTNF-α to sTNF-α, this insulin resistance was partially reversed, indicating a salutary role of tmTNF-α. Treatment of 3T3-L1 adipocytes with exogenous tmTNF-α promoted insulin-induced phosphorylation of IRS-1 and Akt, facilitated GLUT4 expression and membrane translocation, and increased glucose uptake while addition of sTNF-α resulted in the opposite effect. Furthermore, tmTNF-α downregulated the production of IL-6 and MCP-1 via NF-κB inactivation, as silencing of A20, an inhibitor for NF-κB, by siRNA, abolished this effect of tmTNF-α. However, tmTNF-α upregulated adiponectin expression through the PPAR-γ pathway, as inhibition of PPAR-γ by GW9662 abrogated both tmTNF-α-induced adiponectin transcription and glucose uptake. Our data suggest that tmTNF-α functions as an insulin sensitizer via forward signaling.

Transmembrane TNF-α promotes suppressive activities of myeloid-derived suppressor cells via TNFR2.

It has been reported that TNFR2 is involved in regulatory T cell induction and myeloid-derived suppressor cell (MDSC) accumulation, two kinds of immunosuppressive cells contributing to tumor immune evasion. Because transmembrane TNF-α (tmTNF-α) is the primary ligand for TNFR2, we hypothesized that tmTNF-α is mainly responsible for the activation of MDSCs. Indeed, we found that tmTNF-α, rather than secretory TNF-α (sTNF-α), activated MDSCs with enhanced suppressive activities, including upregulating arginase-1 and inducible NO synthase transcription, promoting secretion of NO, reactive oxygen species, IL-10, and TGF-ß, and enhancing inhibition of lymphocyte proliferation. This effect of tmTNF-α was mediated by TNFR2, as TNFR2 deficiency significantly impaired tmTNF-α-induced release of IL-10 and NO and inhibition of T cell proliferation by MDSC supernatant. Furthermore, tmTNF-α caused p38 phosphorylation and NF-κB activation, whereas inhibition of NF-κB or p38 with an inhibitor pyrrolidine dithiocarbamate or SB203580 abrogated tmTNF-α-mediated increased suppression of lymphocyte proliferation by MDSCs. Consistently, our in vivo study showed that ectopic expression of uncleavable tmTNF-α mutant by 4T1 cells significantly promoted tumor progression and angiogenesis, accompanied with more accumulation of MDSCs and regulatory T cells in the tumor site, increased production of NO, IL-10, and TGF-ß, as well as poor lymphocyte infiltration. In contrast, enforced expression of sTNF-α mutant by 4T1 cells that only released sTNF-α without expression of surface tmTNF-α markedly reduced MDSC accumulation and induced more lymphocyte infiltration instead, showing obvious tumor regression. Our data suggest that tmTNF-α acts as a potent activator of MDSCs via TNFR2 and reveals another novel immunosuppressive effect of this membrane molecule that promotes tumor immune escape.

Targeting transmembrane TNF-α suppresses breast cancer growth.

TNF antagonists may offer therapeutic potential in solid tumors, but patients who have high serum levels of TNF-α fail to respond to infliximab, suggesting consumption of the circulating antibody and loss of transmembrane TNF-α (tmTNF-α) on tumors by ectodomain shedding. Addressing this possibility, we developed a monoclonal antibody (mAb) that binds both full-length tmTNF-α and its N-terminal truncated fragment on the membrane after tmTNF-α processing but does not cross-react with soluble TNF-α. We documented high levels of tmTNF-α expression in primary breast cancers, lower levels in atypical hyperplasia or hyperplasia, but undetectable levels in normal breast tissue, consistent with the notion that tmTNF-α is a potential therapeutic target. Evaluations in vitro and in vivo further supported this assertion. tmTNF-α mAb triggered antibody-dependent cell-mediated cytotoxicity against tmTNF-α-expressing cells but not to tmTNF-α-negative cells. In tumor-bearing mice, tmTNF-α mAb delayed tumor growth, eliciting complete tumor regressions in some mice. Moreover, tmTNF-α mAb inhibited metastasis and expression of CD44v6, a prometastatic molecule. However, the antibody did not activate tmTNF-α-mediated reverse signaling, which facilitates tumor survival and resistance to apoptosis, but instead inhibited NF-κB activation and Bcl-2 expression by decreasing tmTNF-α-positive cells. Overall, our results established that tmTNF-α mAb exerts effective antitumor activities and offers a promising candidate to treat tmTNF-α-positive tumors, particularly in patients that are nonresponders to TNF antagonists.

The involvement of ß-actin in the signaling of transmembrane TNF-α-mediated cytotoxicity.

Actin cytoskeleton has been shown to play a regulating role in several signaling pathways, and disruption of actin filament has been reported to increase sTNF-α-induced cell death. However, whether actin is involved in tmTNF-α-mediated cytotoxicity remains unclear. Here, we demonstrated that pretreatment of HL-60 with CytD or LatA to depolymerize actin significantly suppressed tmTNF-α-mediated apoptosis. Interestingly, tmTNF-α increased the actin immunoprecipitated by anti-TNFR2 but not anti-TNFR1 antibody, and disruption of the actin filament totally blocked this effect. In addition, TNFR1 knockdown by siRNA did not affect tmTNF-α-mediated cytotoxicity and the inhibitory effect of CytD, suggesting that the involvement of actin in the tmTNF-α-induced apoptosis is linked to the TNFR2 pathway. Our results revealed further that tmTNF-α signaled the inhibition of IκB degradation and NF-κB activity by recruiting RIP1 to and uncoupling TRAF2 from the TNFR2 complex. Nevertheless, CytD totally reversed the tmTNF-α signaling and activated NF-κB by recruiting TRAF2 to and dissociating RIP1 from the TNFR2 complex. Furthermore, tmTNF-α led to activation of caspase-8 by dissociation of cFLIP from TNFR2 and inhibition of the cFLIP expression. Activated caspase-8 cleft RIP1 to suppress NF-κB activity and also mediated tmTNF-α-induced apoptosis. However, CytD blocked the tmTNF-α-induced uncoupling of cFLIP from TNFR2 and prevented caspase-8 activation and the resulting cleavage of RIP1, converting the signaling for tmTNF-α-mediated apoptosis into one for activating NF-κB to survive. These results suggest that the actin cytoskeleton functions in transmitting signals via TNFR2 to mediate tmTNF-α-induced apoptosis.

Blocking TNF-α by combination of TNF-α- and TNFR-binding cyclic peptide ameliorates the severity of TNBS-induced colitis in rats.

Tumor necrosis factor alpha (TNF-α) has been implicated in the pathogenesis of Crohn's disease. TNF antagonists are effectively used to treat these patients, although the efficiency of different antagonists varies. In the present study we combined TNF-α binding cyclic peptide (TBCP) and TNFR1 binding cyclic peptide (TRBCP) to treat TNBS-induced colitis in rats for one week. The symptoms of colitis including bloody diarrhea, rectal prolapse, and a profound and sustained weight loss were significantly ameliorated and the colon inflammatory damage, both macroscopic and histological scores, MPO activity, and NO production were markedly decreased in rats by neutralization of TNF-α and blocking TNFR1, as compared with those in rats treated with irrelevant peptide or normal saline (P<0.05). The transcripts of IL-1ß and IL-8, and the protein expression of TNF-α in rats treated with both TBCP and TRBCP were also down-regulated (P<0.05), while these proinflammatory cytokines remained unchanged in rats treated with irrelevant peptide or normal saline. These findings suggest that the combination of TNF-α- and TNFR1-binding peptide effectively improves the symptoms of TNBS-induced colitis and alleviates colonic pathological damages in rats. This combination may be a potent candidate for clinical treatment of the inflammatory bowel disease.

Myeloid-derived suppressor cells prevent type 1 diabetes in murine models.

Effective immunotherapy for type 1 diabetes (T1D) relies on active induction of peripheral tolerance. Myeloid-derived suppressor cells (MDSCs) play a critical role in suppressing immune responses in various pathologic settings via multiple mechanisms, including expansion of regulatory T cells (Tregs). In this study, we investigated whether MDSCs could act as APCs to induce expansion of Ag-specific Tregs, suppress T cell proliferation, and prevent autoimmune T1D development. We found that MDSC-mediated expansion of Tregs and T cell suppression required MHC-dependent Ag presentation. A murine T1D model was established in INS-HA/RAG(-/-) mice in which animals received CD4-HA-TCR transgenic T cells via adoptive transfer. We found a significant reduction in the incidence of diabetes in recipients receiving MDSC plus HA, but not OVA peptide, leading to 75% diabetes-free mice among the treated animals. To test further whether MDSCs could prevent diabetes onset in NOD mice, nondiabetic NOD/SCID mice were injected with inflammatory T cells from diabetic NOD mice. MDSCs significantly prevented diabetes onset, and 60% of MDSC-treated mice remained diabetes free. The pancreata of treated mice showed significantly lower levels of lymphocyte infiltration in islet and less insulitis compared with that of the control groups. The protective effects of MDSCs might be mediated by inducing anergy in autoreactive T cells and the development of CD4(+)CD25(+)Foxp3(+) Tregs. Thist study demonstrates a remarkable capacity of transferred MDSCs to downregulate Ag-specific autoimmune responses and prevent diabetes onset, suggesting that MDSCs possess great potential as a novel cell-based tolerogenic therapy in the control of T1D and other autoimmune diseases.

Immune stimulatory receptor CD40 is required for T-cell suppression and T regulatory cell activation mediated by myeloid-derived suppressor cells in cancer.

Immune tolerance to tumors is often associated with accumulation of myeloid-derived suppressor cells (MDSC) and an increase in the number of T-regulatory cells (Treg). In tumor-bearing mice, MDSCs can themselves facilitate the generation of tumor-specific Tregs. In this study, we demonstrate that expression of the immune stimulatory receptor CD40 on MDSCs is required to induce T-cell tolerance and Treg accumulation. In an immune reconstitution model, adoptive transfer of Gr-1+CD115+ monocytic MDSCs derived from CD40-deficient mice failed to recapitulate the ability of wild-type MDSCs to induce tolerance and Treg development in vivo. Agonistic anti-CD40 antibodies phenocopied the effect of CD40 deficiency and also improved the therapeutic efficacy of IL-12 and 4-1BB immunotherapy in the treatment of advanced tumors. Our findings suggest that CD40 is essential not only for MDSC-mediated immune suppression but also for tumor-specific Treg expansion. Blockade of CD40-CD40L interaction between MDSC and Treg may provide a new strategy to ablate tumoral immune suppression and thereby heighten responses to immunotherapy.

Influence of reverse signaling via membrane TNF-alpha on cytotoxicity of NK92 cells.

Membrane tumor necrosis factor-alpha (mTNF-alpha) serves as a receptor transducing signals into mTNF-alpha-bearing cells. Among human peripheral blood mononuclear cells, natural killer (NK) cells have been reported to be the only cell type constitutively expressing mTNF-alpha, which is involved in the cytotoxicity of resting NK cells. Using an IL-2-dependent human NK cell line, NK92, which constitutively expresses mTNF-alpha, we examined the effect of reverse signaling via mTNF-alpha on cellular activities. When the cells were prestimulated with soluble TNFR1 (sTNFR1) which activated mTNF-alpha-mediated reverse signaling, the cytotoxicity of NK92 cells was significantly increased. Further investigation demonstrated that prestimulation with sTNFR1 augmented exocytosis and mRNA transcription of two cytotoxic molecules, perforin and granzyme B, which could serve as underlying molecular mechanisms by which mTNF-alpha-mediated reverse signaling promoted cytotoxicity of NK cells toward K562 cells. On the other hand, pretreatment of NK92 with sTNFR1 boosted the expression of FasL and TNF-alpha, including both the secretory and membrane forms. These molecules also contribute to the NK-mediated cytotoxicity, although K562 cells are Fas-negative and sTNF-alpha-resistant. Interestingly, the mTNF-alpha reverse signaling was found to act synergistically with IL-2 on NK-mediated cytotoxicity. This synergy markedly promoted the production of secretory as well as membrane cytotoxic molecules which may be responsible for the enhanced NK92-mediated cytotoxicity. Our observations suggest that, via reverse signaling, constitutively expressed mTNF-alpha may sensitize NK cells to activating stimuli, such as IL-2, resulting in increased NK-mediated cytotoxicity through promoting the production of multiple cytotoxic effector molecules.

Expression of TNF-alpha leader sequence renders MCF-7 tumor cells resistant to the cytotoxicity of soluble TNF-alpha.

Transmembrane TNF-alpha (tmTNF-alpha) contains a leader sequence (LS) that can be phosphorylated and cleaved at its cytoplasmic portion, inducing IL-12 production. We observed that the breast cancer cell line MDA-MB-231 expressing transmembrane TNF-alpha (tmTNF-alpha) at high level was resistant to soluble TNF-alpha (sTNF-alpha)-induced cytotoxicity, accompanied by constitutive NF-kappaB activation. In contrast, MCF-7 cells expressing tmTNF-alpha at very low level were sensitive to sTNF-alpha-induced cell death and had no detectable NF-kappaB activation. Consistently, siRNA-mediated tmTNF-alpha knockdown blocked NF-kappaB activation and rendered MDA-MB-231 sensitive. To test our hypothesis that TNF-LS may play an important role in determining the sensitivity of tumor cells to sTNF-alpha, we stably transfected MCF-7 cells with TNF-LS. We found that transfection of TNF-LS or wild-type TNF-alpha containing LS constitutively activated NF-kappaB and conferred the cytotoxic resistance of MCF-7 cells, while transfection of a mutant tmTNF-alpha lacking the cytoplasmic segment of LS neither activated NF-kappaB nor affected the sensitivity. However, NF-kappaB inhibitor PDTC suppressed NF-kappaB activation and reconstituted sensitivity of TNF-LS/MCF-7 cells. To check whether TNF-LS is required to be cleaved or internalized for NF-kappaB activation to occur, we used signal peptide peptidase inhibitor (Z-LL)(2)-ketone and receptor internalization inhibitor MDC to treat cells. Interestingly, both inhibitors increased TNF-LS expression on the cell surface and enhanced NF-kappaB activation. These results indicate that membrane-anchored TNF-LS contributes to constitutive activation of NF-kappaB and resistance to sTNF-alpha-induced cell death. Therefore, TNF-LS appears to be responsible for tmTNF-alpha-induced resistance in the breast cancer cells.