Oxidative stress induced necroptosis activation is involved in the pathogenesis of hyperoxic acute lung injury

Necroptosis has been found to be involved in the pathogenesis of some lung diseases, but its role in hyperoxic acute lung injury (HALI) is still unclear. This study aimed to investigate contribution of necroptosis to the pathogenesis of HALI induced by hyperbaric hyperoxia exposure in a rat model. Rats were divided into control group, HALI group, Nec-1 (necroptosis inhibitor) group and edaravone group. Rats were exposed to pure oxygen at 250?kPa for 6?h to induce HALI. At 30?min before hyperoxia exposure, rats were intraperitoneally injected with Nec-1 or edaravone, and sacrificed at 24?h after hyperoxia exposure. Lung injury was evaluated by histology, lung water to dry ratio (W/D) and bronchoalveolar lavage fluid (BALF) biochemistry; the serum and plasma oxidative stress, expression of RIP1, RIP3 and MLKL, and interaction between RIP1 and RIP3 were determined. Results showed hyperoxia exposure significantly caused damage to lung and increased necroptotic cells and the expression of RIP1, RIP3 and MLKL. Edaravone pre-treatment not only inhibited the oxidative stress in HALI, but also reduced necroptotic cells, decreased the expression of RIP1, RIP3 and MLKL and improved lung pathology. Nec-1 pretreatment inhibited necroptosis and improved lung pathology, but had little influence on oxidative stress. This study suggests hyperoxia exposure induces oxidative stress may activate necroptosis, involving in the pathology of HALI, and strategies targeting necroptosis may become promising treatments for HALI.     

Isotype-dependent inhibition of tumor growth in vivo by monoclonal antibodies to death receptor 4

To explore an approach for death receptor targeting in cancer, we developed murine mAbs to human death receptor 4 (DR4). The mAb 4H6 (IgG1) competed with Apo2L/TNF-related apoptosis-inducing ligand (DR4's ligand) for binding to DR4, whereas mAb 4G7 (IgG2a) did not. In vitro, both mAbs showed minimal intrinsic apoptosis-inducing activity, but each triggered potent apoptosis upon cross-linking. In a colon tumor nude mouse model in vivo, mAb 4H6 treatment without addition of exogenous linkers induced apoptosis in tumor cells and caused complete tumor regression, whereas mAb 4G7 partially inhibited tumor growth. An IgG2a isotype switch variant of mAb 4H6 was much less effective in vivo than the parent IgG1-4H6, despite similar binding affinities to DR4. The same conclusion was obtained by comparing other IgG1 and IgG2 mAbs to DR4 for their anti-tumor activities in vivo. Thus, the isotype of anti-DR4 mAb may be more important than DR4 binding affinity for tumor elimination in vivo. Anti-DR4 mAbs of the IgG1 isotype may provide a useful tool for investigating the therapeutic potential of death receptor targeting in cancer.     

Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death

Mixed lineage kinase domain-like protein (MLKL) was identified to function downstream of receptor interacting protein 3 (RIP3) in tumor necrosis factor-α (TNF)-induced necrosis (also called necroptosis). However, how MLKL functions to mediate necroptosis is unknown. By reconstitution of MLKL function in MLKL-knockout cells, we showed that the N-terminus of MLKL is required for its function in necroptosis. The oligomerization of MLKL in TNF-treated cells is essential for necroptosis, as artificially forcing MLKL together by using the hormone-binding domain (HBD*) triggers necroptosis. Notably, forcing together the N-terminal domain (ND) but not the C-terminal kinase domain of MLKL causes necroptosis. Further deletion analysis showed that the four-α-helix bundle of MLKL (1-130 amino acids) is sufficient to trigger necroptosis. Both the HBD*-mediated and TNF-induced complexes of MLKL(ND) or MLKL are tetramers, and translocation of these complexes to lipid rafts of the plasma membrane precedes cell death. The homo-oligomerization is required for MLKL translocation and the signal sequence for plasma membrane location is located in the junction of the first and second α-helices of MLKL. The plasma membrane translocation of MLKL or MLKL(ND) leads to sodium influx, and depletion of sodium from the cell culture medium inhibits necroptosis. All of the above phenomena were not seen in apoptosis. Thus, the MLKL oligomerization leads to translocation of MLKL to lipid rafts of plasma membrane, and the plasma membrane MLKL complex acts either by itself or via other proteins to increase the sodium influx, which increases osmotic pressure, eventually leading to membrane rupture.     

MLKL in cancer: more than a necroptosis regulator

Mixed lineage kinase domain-like protein (MLKL) emerged as executioner of necroptosis, a RIPK3-dependent form of regulated necrosis. Cell death evasion is one of the hallmarks of cancer. Besides apoptosis, some cancers suppress necroptosis-associated mechanisms by for example epigenetic silencing of RIPK3 expression. Conversely, necroptosis-elicited inflammation by cancer cells can fuel tumor growth. Recently, necroptosis-independent functions of MLKL were unraveled in receptor internalization, ligand-receptor degradation, endosomal trafficking, extracellular vesicle formation, autophagy, nuclear functions, axon repair, neutrophil extracellular trap (NET) formation, and inflammasome regulation. Little is known about the precise role of MLKL in cancer and whether some of these functions are involved in cancer development and metastasis. Here, we discuss current knowledge and controversies on MLKL, its structure, necroptosis-independent functions, expression, mutations, and its potential role as a pro- or anti-cancerous factor. Analysis of MLKL expression patterns reveals that MLKL is upregulated by type I/II interferon, conditions of inflammation, and tissue injury. Overall, MLKL may affect cancer development and metastasis through necroptosis-dependent and -independent functions.Subject terms: Cancer genomics, Prognostic markers     

Necroptosis contributes to chronic inflammation and fibrosis in aging liver

Inflammaging, characterized by an increase in low‐grade chronic inflammation with age, is a hallmark of aging and is strongly associated with various age‐related diseases, including chronic liver disease (CLD) and hepatocellular carcinoma (HCC). Because necroptosis is a cell death pathway that induces inflammation through the release of DAMPs, we tested the hypothesis that age‐associated increase in necroptosis contributes to chronic inflammation in aging liver. Phosphorylation of MLKL and MLKL oligomers, markers of necroptosis, as well as phosphorylation of RIPK3 and RIPK1 were significantly upregulated in the livers of old mice relative to young mice and this increase occurred in the later half of life (i.e., after 18 months of age). Markers of M1 macrophages, expression of pro‐inflammatory cytokines (TNFα, IL6 and IL1β), and markers of fibrosis were all significantly upregulated in the liver with age and the change in necroptosis paralleled the changes in inflammation and fibrosis. Hepatocytes and liver macrophages isolated from old mice showed elevated levels of necroptosis markers as well as increased expression of pro‐inflammatory cytokines relative to young mice. Short‐term treatment with the necroptosis inhibitor, necrostatin‐1s (Nec‐1s), reduced necroptosis, markers of M1 macrophages, fibrosis, and cell senescence as well as reducing the expression of pro‐inflammatory cytokines in the livers of old mice. Thus, our data show for the first time that liver aging is associated with increased necroptosis and necroptosis contributes to chronic inflammation in the liver, which in turn appears to contribute to liver fibrosis and possibly CLD.     

Enhancement of CD147 on M1 macrophages induces differentiation of Th17 cells in the lung interstitial fibrosis

Lung interstitial fibrosis is a chronic lung disease, and few effective therapies are available to halt or reverse the progression of the disease. In murine and human lung fibrosis, the expression of CD147 is increased. However, the role of CD147 in lung fibrosis has not been identified, and it remains to be determined whether lung fibrosis would be improved by decreasing the expression of CD147. A murine bleomycin-induced lung interstitial fibrosis model was used in the experiments, and HAb18 mAbs and CsA were administered during the induction of lung fibrosis. In our study, we found that the HAb18 mAbs markedly reduced the collagen score and down-regulated M1 macrophages and Th17 cells. In vitro, flow cytometry analysis showed that M1 macrophages induced higher Th17 differentiation than M2 macrophages. After treatment with HAb18 mAbs or after reducing the expression of CD147 by lentivirus interference in M1 macrophages, the level of Th17 cells were significantly inhibited. In conclusion, HAb18 mAbs or CsA treatment ameliorates lung interstitial fibrosis. CD147 promoted M1 macrophage and induced the differentiation of Th17 cells in lung interstitial fibrosis, perhaps by regulating some cytokines such as IL-6, IL-1β, IL-12 and IL-23. These results indicated that CD147 may play an important role in the development of lung interstitial fibrosis.     

Salt-inducible kinases inhibitor HG-9-91-01 targets RIPK3 kinase activity to alleviate necroptosis-mediated inflammatory injury

Receptor-interacting protein kinase 3 (RIPK3) functions as a central regulator of necroptosis, mediating signaling transduction to activate pseudokinase mixed lineage kinase domain-like protein (MLKL) phosphorylation. Increasing evidences show that RIPK3 contributes to the pathologies of inflammatory diseases including multiple sclerosis, infection and colitis. Here, we identified a novel small molecular compound Salt-inducible Kinases (SIKs) inhibitor HG-9-91-01 inhibiting necroptosis by targeting RIPK3 kinase activity. We found that SIKs inhibitor HG-9-91-01 could block TNF- or Toll-like receptors (TLRs)-mediated necroptosis independent of SIKs. We revealed that HG-9-91-01 dramatically decreased cellular activation of RIPK3 and MLKL. Meanwhile, HG-9-91-01 inhibited the association of RIPK3 with MLKL and oligomerization of downstream MLKL. Interestingly, we found that HG-9-91-01 also trigger RIPK3-RIPK1-caspase 1-caspase 8-dependent apoptosis, which activated cleavage of GSDME leading to its dependent pyroptosis. Mechanistic studies revealed that SIKs inhibitor HG-9-91-01 directly inhibited RIPK3 kinase activity to block necroptosis and interacted with RIPK3 and recruited RIPK1 to activate caspases leading to cleave GSDME. Importantly, mice pretreated with HG-9-91-01 showed resistance to TNF-induced systemic inflammatory response syndrome. Consistently, HG-9-91-01 treatment protected mice against Staphylococcus aureus-mediated lung damage through targeting RIPK3 kinase activity. Overall, our results revealed that SIKs inhibitor HG-9-91-01 is a novel inhibitor of RIPK3 kinase and a potential therapeutic target for the treatment of necroptosis-mediated inflammatory diseases.Subject terms: Necroptosis, Target validation     

Integrin α-3 ß-1’s central role in breast cancer,melanoma and glioblastoma cell aggregation revealed by antibodies with blocking activity

Breast cancer, melanoma and glioblastoma cells undergo cell-mediated aggregation and aggregate coalescence in a transparent 3D Matrigel environment. Cells from normal tissue and non-tumorigenic cell lines do not exhibit these behaviors. Here, 266 monoclonal antibodies (mAbs) demonstrated to interact with a wide variety of membrane, secreted and matrix proteins, have been screened for their capacity to block these tumorigenic cell-specific behaviors in a 3D environment. Remarkably, only six of the 266 tested mAbs exhibited blocking activity, four targeting integrin ß-1, one targeting integrin α-3 and one targeting CD44. Colocalization of integrins ß-1 and α-3 in fixed cells and in live aggregates suggests that the integrin α-3 ß-1 dimer plays a central role in cancer cell aggregation in the 3D environment provided by Matrigel. Our results suggest that blocking by anti-integrin and anti-CD44 mAbs involves interference in cell-cell interactions.     

The Lck inhibitor,AMG-47a,blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

Necroptosis is a form of caspase-independent programmed cell death that arises from disruption of cell membranes by the mixed lineage kinase domain-like (MLKL) pseudokinase after its activation by the upstream kinases, receptor interacting protein kinase (RIPK)-1 and RIPK3, within a complex known as the necrosome. Dysregulated necroptosis has been implicated in numerous inflammatory pathologies. As such, new small molecule necroptosis inhibitors are of great interest, particularly ones that operate downstream of MLKL activation, where the pathway is less well defined. To better understand the mechanisms involved in necroptosis downstream of MLKL activation, and potentially uncover new targets for inhibition, we screened known kinase inhibitors against an activated mouse MLKL mutant, leading us to identify the lymphocyte-specific protein tyrosine kinase (Lck) inhibitor AMG-47a as an inhibitor of necroptosis. We show that AMG-47a interacts with both RIPK1 and RIPK3, that its ability to protect from cell death is dependent on the strength of the necroptotic stimulus, and that it blocks necroptosis most effectively in human cells. Moreover, in human cell lines, we demonstrate that AMG-47a can protect against cell death caused by forced dimerisation of MLKL truncation mutants in the absence of any upstream signalling, validating that it targets a process downstream of MLKL activation. Surprisingly, however, we also found that the cell death driven by activated MLKL in this model was completely dependent on the presence of RIPK1, and to a lesser extent RIPK3, although it was not affected by known inhibitors of these kinases. Together, these results suggest an additional role for RIPK1, or the necrosome, in mediating human necroptosis after MLKL is phosphorylated by RIPK3 and provide further insight into reported differences in the progression of necroptosis between mouse and human cells.Subject terms: Kinases, Necroptosis     

Toxin-Induced Necroptosis Is a Major Mechanism of Staphylococcus aureus Lung Damage

Staphylococcus aureus USA300 strains cause a highly inflammatory necrotizing pneumonia. The virulence of this strain has been attributed to its expression of multiple toxins that have diverse targets including ADAM10, NLRP3 and CD11b. We demonstrate that induction of necroptosis through RIP1/RIP3/MLKL signaling is a major consequence of S. aureus toxin production. Cytotoxicity could be prevented by inhibiting either RIP1 or MLKL signaling and S. aureus mutants lacking agr, hla or Hla pore formation, lukAB or psms were deficient in inducing cell death in human and murine immune cells. Toxin-associated pore formation was essential, as cell death was blocked by exogenous K+ or dextran. MLKL inhibition also blocked caspase-1 and IL-1β production, suggesting a link to the inflammasome. Rip3 ^-/- mice exhibited significantly improved staphylococcal clearance and retained an alveolar macrophage population with CD200R and CD206 markers in the setting of acute infection, suggesting increased susceptibility of these leukocytes to necroptosis. The importance of this anti-inflammatory signaling was indicated by the correlation between improved outcome and significantly decreased expression of KC, IL-6, TNF, IL-1α and IL-1β in infected mice. These findings indicate that toxin-induced necroptosis is a major cause of lung pathology in S. aureus pneumonia and suggest the possibility of targeting components of this signaling pathway as a therapeutic strategy.     

Suppression of RIP3-dependent Necroptosis by Human Cytomegalovirus

Necroptosis is an alternate programmed cell death pathway that is unleashed by caspase-8 compromise and mediated by receptor-interacting protein kinase 3 (RIP3). Murine cytomegalovirus (CMV) and herpes simplex virus (HSV) encode caspase-8 inhibitors that prevent apoptosis together with competitors of RIP homotypic interaction motif (RHIM)-dependent signal transduction to interrupt the necroptosis. Here, we show that pro-necrotic murine CMV M45 mutant virus drives virus-induced necroptosis during nonproductive infection of RIP3-expressing human fibroblasts, whereas WT virus does not. Thus, M45-encoded RHIM competitor, viral inhibitor of RIP activation, sustains viability of human cells like it is known to function in infected mouse cells. Importantly, human CMV is shown to block necroptosis induced by either TNF or M45 mutant murine CMV in RIP3-expressing human cells. Human CMV blocks TNF-induced necroptosis after RIP3 activation and phosphorylation of the mixed lineage kinase domain-like (MLKL) pseudokinase. An early, IE1-regulated viral gene product acts on a necroptosis step that follows MLKL phosphorylation prior to membrane leakage. This suppression strategy is distinct from RHIM signaling competition by murine CMV or HSV and interrupts an execution process that has not yet been fully elaborated.     

Emodin-Induced Necroptosis in Prostate Cancer Cells via the Mitochondrial Fission HSP90/MLKL/PGAM Pathway

Currently, prostate cancer is one of the major malignant tumors in males. Recurrence and metastasis are the main obstacles that prevent the effective treatment of prostate cancer. In the present study, we aimed to evaluate emodin (EG) against human prostate cancer PC3 and DU145 cells. Our study showed that EG significantly decreased the cell viability of PC3 and DU145 cells and strikingly induced non-apoptotic cell death via necroptosis that was visualized through colony formation assay, Hoechst 33258 staining, and TEM analysis. Furthermore, RNA-sequencing and KEGG functional enrichment analysis revealed that the necroptosis-related pathway was activated upon EG treatment in PC3 cells. mRNA and protein expression of necroptosis markers were analyzed by qPCR and immunoblotting, which implied that EG-induced cell necroptosis via enhancing the expression of MLKL and HSP90AA1 activating PGAM pathway which is considered as a key mediator of mitochondrial fission and leading to ROS generation in PC3 and DU145 cells. Thus, our findings suggested that EG is a new small molecule agonist that induced necroptosis in prostate cancer cells via the mitochondrial fission HSP90/MLKL/PGAM pathway.     

Local antibody response in Peyer's patches to the orally administered dietary protein antigen

To understand local antibody production to dietary protein antigens in the gut, the reactivity of the monoclonal antibodies (mAbs) from Peyer's patches of BALB/c mice raised against orally administered hen egg lysozyme (HEL) was studied. These mAbs were of IgG1 (7 clones), IgA (5 clones) and IgM (13 clones) isotypes. Some of the HEL-binding mAbs preferentially reacted with reduced, carboxy-methylated HEL, rather than with native HEL. MAbs of the IgA and IgM isotypes had cross-reactivity with other unrelated environmental antigens such as E. coli, single-strand DNA, and soluble components of mouse food. In contrast, the IgG1 mAbs did not cross-react with these antigens. The average of the Kd values for HEL of these mAbs was in the order of 10(-6) M, which is moderately higher than those of mAbs from the preimmune repertoire. These results suggest that, under normal physiological conditions, orally administered dietary proteins predominantly induce the local production of polyreactive IgA/IgM antibodies cross-reacting with environmental luminal antigens.     

Heat shock protein A12A activates migration of hepatocellular carcinoma cells in a monocarboxylate transporter 4–dependent manner

Metastasis is responsible for most of the hepatocellular carcinoma (HCC)–associated death. However, its underlying mechanism has yet to be fully elucidated. Glycolysis-derived lactate has been shown to be a powerful regulator of cancer metastasis. Heat shock protein A12A (HSPA12A) encodes a novel member of HSP70 family. We have recently demonstrated that heat shock protein A12A (HSPA12A) inhibited renal cancer cell migration by suppressing lactate output and glycolytic activity, which were mediated by unstabilizing CD147 and promoting its degradation. By striking contrast, here we demonstrated that HSPA12A promoted migration of human HCC cells. Extracellular acidification, lactate export, and glycolytic activity in HCC cells were also promoted following HSPA12A overexpression. Further analysis revealed that HSPA12A interacted with MCT4 and increased its membrane localization, thereby promoting export of lactate generated from glycolysis; this led, ultimately, to HCC cell migration. Our results revealed the opposite effect of HSPA12A on migration of renal cancer cells and that of HCC cells. Of note, in contrast to the inhibitory effect on CD147 expression in renal cancer cells, we found that HSPA12A increased CD147 expression in HCC cells, indicating that the expression of CD147 might exist heterogeneity in different cancer cell types. Taken together, we identified HSPA12A as an activator of HCC migration, a role opposite to that of renal cancer cells. Inhibiting HSPA12A might be a potential therapeutic intervention for HCC metastasis.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-021-01251-z.     

TKI治疗晚期非小细胞肺癌患者免疫功能的变化及意义

目的:通过观察晚期非小细胞肺癌患者TKI治疗前后外周血IgG、IgM、IgA、C3、C4、C-反应蛋白及CD3+、CD4+、CD8+、CD4+/CD8+细胞的表达变化,探讨TKI治疗对晚期非小细胞肺癌患者免疫功能的影响及意义。方法:检测TKI组30例非小细胞肺癌患者TKI治疗前、治疗一个月后外周血IgG、IgM、IgA、C3、C4、C-反应蛋白及CD3+、CD4+、CD8+、CD4+/CD8+细胞表达水平,分析表达变化及与疗效的关系。30例非小细胞肺癌患者作为对照组。结果:治疗前,TKI组与对照组IgG、IgM、IgA、C3、C4、C-反应蛋白水平基本正常,但CD4+细胞数量减低、CD4+/CD8+比值较低、CD8+细胞数量增高,两组相比IgG、IgM、IgA、C3、C4、C-反应蛋白、CD3+、CD4+、CD8+、CD4+/CD8+差异均无统计学意义(P0.05);TKI治疗一个月后,TKI组与对照组IgG、IgM、IgA、C3、C4、C-反应蛋白水平无明显变化,而CD4+细胞数量增多、CD4+/CD8+较前增高,CD8+细胞数量较前减低,两组相比CD3+、IgG、IgM、IgA、C3、C4、C-反应蛋白差异无统计学意义(P0.05),而CD4+、CD4+/CD8+、CD8+差异有统计学意义(P0.01)。结论:TKI治疗后,晚期非小细胞肺癌患者细胞免疫功能得到改善,体现在CD4+、CD8+细胞数量的变化上,且TKI治疗的疗效可通过比较外周血CD4+、CD4+/CD8+、CD8+细胞表达变化体现。     

Quantitative analysis of phosphoproteome in necroptosis reveals a role of TRIM28 phosphorylation in promoting necroptosis-induced cytokine production

Necroptosis is a form of regulated necrotic cell death that promotes inflammation. In cells undergoing necroptosis, activated RIPK1 kinase mediates the formation of RIPK1/RIPK3/MLKL complex to promote MLKL oligomerization and execution of necroptosis. RIPK1 kinase activity also promotes cell-autonomous activation of proinflammatory cytokine production in necroptosis. However, the signaling pathways downstream of RIPK1 kinase in necroptosis and how RIPK1 kinase activation controls inflammatory response induced by necroptosis are still largely unknown. Here, we quantitatively measured the temporal dynamics of over 7000 confident phosphorylation-sites during necroptosis using mass spectrometry. Our study defined a RIPK1-dependent phosphorylation pattern in late necroptosis that is associated with a proinflammatory component marked by p-S473 TRIM28. We show that the activation of p38 MAPK mediated by oligomerized MLKL promotes the phosphorylation of S473 TRIM28, which in turn mediates inflammation during late necroptosis. Taken together, our study illustrates a mechanism by which p38 MAPK may be activated by oligomerized MLKL to promote inflammation in necroptosis.Subject terms: Cell biology, Immunology     

HAb18G/CD147 functions in invasion and metastasis of hepatocellular carcinoma

CD147 molecule is reported to be correlated with the malignancy of some cancers; however, it remains unclear whether it is involved in the progression of hepatocellular carcinoma (HCC). Here, we investigated the function of HAb18G/CD147, a member of CD147 family, and its antibodies, HAb18 and LICARTIN, in HCC invasion and metastasis. We observed that HAb18G/CD147 gene silence in HCC cells significantly decreased the secretion of matrix metalloproteinase (MMP) and the invasive potential of HCC cells (P < 0.001). MMP silence in HCC cells also significantly suppressed the invasion of the cells when cocultured with fibroblasts; however, its inhibitory effect was significantly weaker than that of both HAb18G/CD147 silence in HCC cells and that of MMP silence in fibroblasts (P < 0.001). Blocking theHAb18G/CD147 molecule on HCC cells with HAb18 monoclonal antibody resulted in a similar suppressive effect on MMP secretion and cell invasion, but with no significant effects on the cell growth. (131)I-labeled HAb18 F(ab')(2) (LICARTIN), however, significantly inhibited the in vitro growth of HCC cells (P < 0.001). In an orthotopic model of HCC in nude mice, HAb18 and LICARTIN treatment effectively reduced the tumor growth and metastasis as well as the expression of three major factors in the HCC microenviroment (MMPs, vascular endothelial growth factor, and fibroblast surface protein) in the paracancer tissues. Overall, these results suggest that HAb18G/CD147 plays an important role in HCC invasion and metastasis mainly via modulating fibroblasts, as well as HCC cells themselves to disrupt the HCC microenviroment. LICARTIN can be used as a drug targeting to HAb18G/CD147 in antimetastasis and recurrence therapy of HCC.     

The Smac mimetic BV6 cooperates with STING to induce necroptosis in apoptosis-resistant pancreatic carcinoma cells

Pancreatic cancer (PC) still remains a major cause of cancer-related death worldwide and alternative treatments are urgently required. A common problem of PC is the development of resistance against apoptosis that limits therapeutic success. Here we demonstrate that the prototypical Smac mimetic BV6 cooperates with the stimulator of interferon (IFN) genes (STING) ligand 2′,3′-cyclic guanosine monophosphate–adenosine monophosphate (2′3′-cGAMP) to trigger necroptosis in apoptosis-deficient PC cells. Pharmacological inhibition of key components of necroptosis signaling, such as receptor-interacting protein 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL), significantly rescues PC cells from 2′3′-cGAMP/BV6/zVAD.fmk-mediated cell death, suggesting the induction of necroptosis. Consistently, 2′3′-cGAMP/BV6 co-treatment promotes phosphorylation of MLKL. Furthermore, we show that 2′3′-cGAMP stimulates the production of type I IFNs, which cooperate with BV6 to trigger necroptosis in apoptosis-deficient settings. STING silencing via siRNA or CRISPR/Cas9-mediated gene knockout protects PC cells from 2′3′-cGAMP/BV6/zVAD.fmk-mediated cell death. Interestingly, we demonstrate that nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNFα), and IFN-regulatory factor 1 (IRF1) signaling are involved in triggering 2′3′-cGAMP/BV6/zVAD.fmk-induced necroptosis. In conclusion, we show that activated STING and BV6 act together to exert antitumor effects on PC cells with important implications for the design of new PC treatment concepts.Subject terms: Cancer, Cancer     

TKI治疗晚期tPd,细胞肺癌患者免疫功能的变化及意义

目的：通过观察晚期非小细胞肺癌患者TKI治疗前后外周血IgG、[gM、IgA、C3、c4、C-反应蛋白及CD3＋、CD4＋、CD8＋、CD4＋／CD8＋细胞的表达变化,探讨TKI治疗对晚期非小细胞肺癌患者免疫功能的影响及意义。方法：检测TKI组30例非小细胞肺癌患者TKI治疗前、治疗一个月后外周血IgG、IgM、IgA、C3、C4、C-反应蛋白及CD3＋、CD4＋,CD8＋、CD4＋／CD8＋细胞表达水平,分析表达变化及与疗效的关系。30例非小细胞肺癌患者作为对照组。结果：治疗前,TKI组与对照组IgG、IgM、IgA、C3、C4、c＋反应蛋白水平基本正常,但CD4＋细胞数量减低、CD4＋／CD8＋比值较低、CD8＋细胞数量增高,两组相比IgG、IgM、IgA、C3、C4、C-反应蛋白、CD3＋、CD4＋．CD8＋、CD4＋／CD8‘差异均无统计学意义（P〉0．05）;TKI治疗一个月后,TKI组与对照组IgG、IgM、IgA、C3、C4、C-反应蛋白水平无明显变化,而CD4＋细胞数量增多、CD4＋／CD8＋较前增高,CD8＋细胞数量较前减低,两组相比CD3＋、IgG、IgM、IgA、C3、C4、c-反应蛋白差异无统计学意义（P〉0．05）,而CD4＋．CD4＋／CD8＋、CD8＋差异有统计学意义（P〈O．01）。结论：TKI治疗后,晚期非小细胞肺癌患者细胞免疫功能得到改善,体现在CD4＋,CD8＋细胞数量的变化上,且TKI治疗的疗效可通过比较外周血CD4＋、CD4＋／CD8＋、cD8＋细胞表达变化体现。     

Analysis of Tn antigenicity with a panel of new IgM and IgG1 monoclonal antibodies raised against leukemic cells

CD175 or Tn antigen is a carbohydrate moiety of N-acetylgalactosamine (GalNAc)α1-O- linked to the residue of amino acid serine or threonine in a polypeptide chain. Despite the chemical simplicity of the Tn antigen, its antigenic structure is considered to be complex and the clear determinants of Tn antigenicity remain poorly understood. As a consequence, a broad variety of anti-Tn monoclonal antibodies (mAbs) have been generated. To further investigate the nature and complexity of the Tn antigen, we generated seven different anti-Tn mAbs of IgM and IgG classes raised against human Jurkat T cells, which are Tn-positive due to the low activity of T-synthase and mutation in specific chaperone Cosmc. The binding analysis of anti-Tn mAbs with the array of synthetic saccharides, glycopeptides and O-glycoproteins revealed unexpected differences in specificities of anti-Tn mAbs. IgM mAbs bound the terminal GalNAc residue of the Tn antigen irrespective of the peptide context or with low selectivity to the glycoproteins. In contrast, IgG mAbs recognized the Tn antigen in the context of a specific peptide motif. Particularly, JA3 mAb reacted to the GSPP or GSPAPP, and JA5 mAb recognized specifically the GSP motif (glycosylation sites are underlined). The major O-glycan carrier proteins CD43 and CD162 and isoforms of CD45 expressed on Jurkat cells were precipitated by anti-Tn mAbs with different affinities. In summary, our data suggest that Tn antigen-Ab binding capacity is determined by the peptide context of the Tn antigen, antigenic specificity of the Ab and class of the immunoglobulin. The newly generated anti-Tn IgG mAbs with the strong specificity to glycoprotein CD43 can be particularly interesting for the application in leukemia diagnostics and therapy.