HIV‐1 infection of CD4 T cells impairs antigen‐specific B cell function

Failures to produce neutralizing antibodies upon HIV‐1 infection result in part from B‐cell dysfunction due to unspecific B‐cell activation. How HIV‐1 affects antigen‐specific B‐cell functions remains elusive. Using an adoptive transfer mouse model and ex vivo HIV infection of human tonsil tissue, we found that expression of the HIV‐1 pathogenesis factor NEF in CD4 T cells undermines their helper function and impairs cognate B‐cell functions including mounting of efficient specific IgG responses. NEF interfered with T cell help via a specific protein interaction motif that prevents polarized cytokine secretion at the T‐cell–B‐cell immune synapse. This interference reduced B‐cell activation and proliferation and thus disrupted germinal center formation and affinity maturation. These results identify NEF as a key component for HIV‐mediated dysfunction of antigen‐specific B cells. Therapeutic targeting of the identified molecular surface in NEF will facilitate host control of HIV infection.     

Mechanisms of resistance of normal cells to TRAIL induced apoptosis vary between different cell types

Resistance of normal cells to tumour necrosis factor related apoptosis inducing ligand (TRAIL) induced apoptosis is believed to be mediated by expression of two decoy receptors. Here we show that the expression and localisation of TRAIL receptors (TRAIL-Rs) vary between different cells and that resistance to TRAIL is mediated by different mechanisms. The decoy receptor, TRAIL-R3, appeared important in protection of endothelial cells, whereas lack of surface death receptor expression and as yet unknown intracellular inhibitor(s) of apoptosis downstream of caspase-3 may play a major role in protection of melanocytes and fibroblasts from TRAIL induced apoptosis, respectively. Differential subcellular location of decoy receptors may be an important determinant of their effectiveness in different types of normal cells.     

Histone chaperone CAF‐1 promotes HIV‐1 latency by leading the formation of phase‐separated suppressive nuclear bodies

HIV‐1 latency is a major obstacle to achieving a functional cure for AIDS. Reactivation of HIV‐1‐infected cells followed by their elimination via immune surveillance is one proposed strategy for eradicating the viral reservoir. However, current latency‐reversing agents (LRAs) show high toxicity and low efficiency, and new targets are needed to develop more promising LRAs. Here, we found that the histone chaperone CAF‐1 (chromatin assembly factor 1) is enriched on the HIV‐1 long terminal repeat (LTR) and forms nuclear bodies with liquid–liquid phase separation (LLPS) properties. CAF‐1 recruits epigenetic modifiers and histone chaperones to the nuclear bodies to establish and maintain HIV‐1 latency in different latency models and primary CD4⁺ T cells. Three disordered regions of the CHAF1A subunit are important for phase‐separated CAF‐1 nuclear body formation and play a key role in maintaining HIV‐1 latency. Disruption of phase‐separated CAF‐1 bodies could be a potential strategy to reactivate latent HIV‐1.     

Doxorubicin hydrochloride enhanced antitumour effect of CEA‐regulated oncolytic virotherapy in live cancer cells and a mouse model

Oncolytic adenovirus (OA) has attracted increasing attention due to their specific proliferation in tumour cells and resulting in lysis of tumour cells. To further improve the antitumour effect of OA, in this study, we combined CD55‐TRAIL‐IETD‐MnSOD (CD55‐TMn), a CEA‐controlled OA constructed previously, and chemotherapy to investigate their synergistic effect and possible mechanisms. MTT assay was performed to detect antitumour effects. Hoechst 33 342 and flow cytometric analysis were used to examine cell apoptosis. Western blotting was performed to examine cell pyroptosis and apoptosis mechanism. Animal experiment was used to detect antitumour effect of doxorubicin hydrochloride (Dox) combined with CD55‐TMn in vivo. We firstly found that Dox promotes gene expression mediated by CEA‐regulated OA and virus progeny replication by activating phosphorylation of Smad3, and Dox can enhance antitumour effect of CEA‐regulated CD55‐TMn by promoting cell apotopsis and cell pyroptosis. Thus, our results provide an experimental and theoretical basis on tumour therapy by combination treatment of the oncolytic virotherapy and chemotherapy and it is expected to become a novel strategy for liver cancer therapy.     

Low-dose UV-radiation sensitizes keratinocytes to TRAIL-induced apoptosis

The impact of low-dose ultraviolet light (UV-light) on apoptotic susceptibility of keratinocytes (KCs) induced by TRAIL is unclear. Skin expresses a functional form of TRAIL, and while sun exposure influences TRAIL death receptors, a role for decoy receptors has not been evaluated. Unraveling mechanisms involving apoptotic sensitivity of KCs is important because skin is the first target of UV-light, and a site for commonly occurring cancers. Since apoptosis is a homeostatic process eliminating UV-light induced DNA damaged cells, elucidating molecular events regulating apoptosis enhances understanding of cutaneous photocarcinogenesis. Here we demonstrate low-dose UV-light enhances susceptibility of KCs to TRAIL-induced apoptosis. Low-dose UV-light selectively reduces decoy receptors, without influencing death receptor levels. UV-induced enhanced apoptotic susceptibility was reduced by over-expression of decoy receptor TRAIL-R4, but not TRAIL-R3; or treatment with thiol compound pyrrolidine dithiocarbamate (PDTC), which also enhanced TRAIL-R4 levels. Besides influencing decoy receptors, low-dose UV-light plus TRAIL also synergistically promoted cytochrome c and Smac release from mitochondria. Inhibitors directed against caspases 2, 3, 8, and 9 reduced the synergistic apoptotic response following low-dose UV-light plus TRAIL exposure; as did forced over-expression of Bcl-x and dominant negative (DN) constructs of FADD and caspase 9. Thus, relative levels of decoy receptors significantly influence susceptibility of KCs to TRAIL-induced apoptosis with concomitant low-dose UV-light exposure; in addition to the apoptotic pathway mediated by mitochondrial permeabilization.     

Aneuploid senescent cells activate NF‐κB to promote their immune clearance by NK cells

The immune system plays a major role in the protection against cancer. Identifying and characterizing the pathways mediating this immune surveillance are thus critical for understanding how cancer cells are recognized and eliminated. Aneuploidy is a hallmark of cancer, and we previously found that untransformed cells that had undergone senescence due to highly abnormal karyotypes are eliminated by natural killer (NK) cells in vitro. However, the mechanisms underlying this process remained elusive. Here, using an in vitro NK cell killing system, we show that non‐cell‐autonomous mechanisms in aneuploid cells predominantly mediate their clearance by NK cells. Our data indicate that in untransformed aneuploid cells, NF‐κB signaling upregulation is central to elicit this immune response. Inactivating NF‐κB abolishes NK cell‐mediated clearance of untransformed aneuploid cells. In cancer cell lines, NF‐κB upregulation also correlates with the degree of aneuploidy. However, such upregulation in cancer cells is not sufficient to trigger NK cell‐mediated clearance, suggesting that additional mechanisms might be at play during cancer evolution to counteract NF‐κB‐mediated immunogenicity.     

Endogenous Bak inhibitors Mcl-1 and Bcl-xL: differential impact on TRAIL resistance in Bax-deficient carcinoma

Tumor necrosis factor (α)–related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that preferentially kills tumor cells with limited cytotoxicity to nonmalignant cells. However, signaling from death receptors requires amplification via the mitochondrial apoptosis pathway (type II) in the majority of tumor cells. Thus, TRAIL-induced cell death entirely depends on the proapoptotic Bcl-2 family member Bax, which is often lost as a result of epigenetic inactivation or mutations. Consequently, Bax deficiency confers resistance against TRAIL-induced apoptosis. Despite expression of Bak, Bax-deficient cells are resistant to TRAIL-induced apoptosis. In this study, we show that the Bax dependency of TRAIL-induced apoptosis is determined by Mcl-1 but not Bcl-x_L. Both are antiapoptotic Bcl-2 family proteins that keep Bak in check. Nevertheless, knockdown of Mcl-1 but not Bcl-x_L overcame resistance to TRAIL, CD95/FasL and tumor necrosis factor (α) death receptor ligation in Bax-deficient cells, and enabled TRAIL to activate Bak, indicating that Mcl-1 rather than Bcl-x_L is a major target for sensitization of Bax-deficient tumors for death receptor–induced apoptosis via the Bak pathway.     

Promyelocytic leukemia protein targets MK2 to promote cytotoxicity

Promyelocytic leukemia protein (PML) is a tumor suppressor possessing multiple modes of action, including induction of apoptosis. We unexpectedly find that PML promotes necroptosis in addition to apoptosis, with Pml ^−/− macrophages being more resistant to TNF‐mediated necroptosis than wild‐type counterparts and PML‐deficient mice displaying resistance to TNF‐induced systemic inflammatory response syndrome. Reduced necroptosis in PML‐deficient cells is associated with attenuated receptor‐interacting protein kinase 1 (RIPK1) activation, as revealed by reduced RIPK1[S166] phosphorylation, and attenuated RIPK1‐RIPK3‐MLKL necrosome complex formation. We show that PML deficiency leads to enhanced TNF‐induced MAPK‐activated kinase 2 (MK2) activation and elevated RIPK1[S321] phosphorylation, which suppresses necrosome formation. MK2 inhibitor treatment or MK2 knockout abrogates resistance to cell death induction in PML‐null cells and mice. PML binds MK2 and p38 MAPK, thereby inhibiting p38‐MK2 interaction and MK2 activation. Moreover, PML participates in autocrine production of TNF induced by cellular inhibitors of apoptosis 1 (cIAP1)/cIAP2 degradation, since PML‐knockout attenuates autocrine TNF. Thus, by targeting MK2 activation and autocrine TNF, PML promotes necroptosis and apoptosis, representing a novel tumor‐suppressive activity for PML.     

Escape of HIV-1-Infected Dendritic Cells from TRAIL-Mediated NK Cell Cytotoxicity during NK-DC Cross-Talk—A Pivotal Role of HMGB1

Early stages of Human Immunodeficiency Virus-1 (HIV-1) infection are associated with local recruitment and activation of important effectors of innate immunity, i.e. natural killer (NK) cells and dendritic cells (DCs). Immature DCs (iDCs) capture HIV-1 through specific receptors and can disseminate the infection to lymphoid tissues following their migration, which is associated to a maturation process. This process is dependent on NK cells, whose role is to keep in check the quality and the quantity of DCs undergoing maturation. If DC maturation is inappropriate, NK cells will kill them (“editing process”) at sites of tissue inflammation, thus optimizing the adaptive immunity. In the context of a viral infection, NK-dependent killing of infected-DCs is a crucial event required for early elimination of infected target cells. Here, we report that NK-mediated editing of iDCs is impaired if DCs are infected with HIV-1. We first addressed the question of the mechanisms involved in iDC editing, and we show that cognate NK-iDC interaction triggers apoptosis via the TNF-related apoptosis-inducing ligand (TRAIL)-Death Receptor 4 (DR4) pathway and not via the perforin pathway. Nevertheless, once infected with HIV-1, DC_HIV become resistant to NK-induced TRAIL-mediated apoptosis. This resistance occurs despite normal amounts of TRAIL released by NK cells and comparable DR4 expression on DC_HIV. The escape of DC_HIV from NK killing is due to the upregulation of two anti-apoptotic molecules, the cellular-Flice like inhibitory protein (c-FLIP) and the cellular inhibitor of apoptosis 2 (c-IAP2), induced by NK-DC_HIV cognate interaction. High-mobility group box 1 (HMGB1), an alarmin and a key mediator of NK-DC cross-talk, was found to play a pivotal role in NK-dependent upregulation of c-FLIP and c-IAP2 in DC_HIV. Finally, we demonstrate that restoration of DC_HIV susceptibility to NK-induced TRAIL killing can be obtained either by silencing c-FLIP and c-IAP2 by specific siRNA, or by inhibiting HMGB1 with blocking antibodies or glycyrrhizin, arguing for a key role of HMGB1 in TRAIL resistance and DC_HIV survival. These findings provide evidence for a new strategy developed by HIV to escape immune attack, they challenge the question of the involvement of HMGB1 in the establishment of viral reservoirs in DCs, and they identify potential therapeutic targets to eliminate infected DCs.     

TRAIL mediates apoptosis in cancerous but not normal primary cultured cells of the human reproductive tract

Cancer of the reproductive tract encompasses malignancies of the uterine corpus, cervix, ovary, Fallopian tube, among others and accounts for 15% of female cancer mortalities. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates apoptosis by binding to death receptors and offers a promising cancer treatment. The goal of this study was to investigate and characterize the effect of TRAIL in endometrial cancer cell lines and normal (non-cancerous) epithelial cells of endometrial origin. We also examined the effect of TRAIL in other primary cultured cancers and normal cells of the human female reproductive tract and evaluated if TRAIL mediated apoptosis correlated with death receptors and decoy receptors 1 and 2. Herein, we demonstrate that TRAIL at concentrations which kill cancerous cells, does not mediate apoptosis or alter cell viability in normal human endometrium, ovary, cervix or Fallopian tube. The partial inhibition by a caspase 9 inhibitor and the total inhibition by a caspase 8 inhibitor demonstrates the dependency on the extrinsic apoptotic pathway. The selective mortality does not correlate with the presence of death or decoy receptors. These results suggest that TRAIL may be an effective treatment for endometrial cancer and other female reproductive cancers, with minimal secondary effects on healthy tissue. This work was supported by a grant from the Wellcome Trust GR071469 (GIO) and the Chilean national science grants FONDECYT 1060495 (GIO) and 1020675 (MC). An erratum to this article is available at .     

RGD-avidin–biotin pretargeting to α<Subscript>v</Subscript>β<Subscript>3</Subscript> integrin enhances the proapoptotic activity of TNFα related apoptosis inducing ligand (TRAIL)

Recombinant TNF-related apoptosis-inducing ligand (TRAIL) is considered a powerful and selective inducer of tumor cell death. We hypothesize that TRAIL’s potential as anticancer agent can be enhanced further by promoting its accumulation in tumor tissue. For this purpose, we developed TRAIL complexes that bind to angiogenic endothelial cells. We employed an avidin–biotin pretargeting approach, in which biotinylated TRAIL interacted with RGD-equipped avidin. The assembled complexes killed tumor cells (Jurkat T cells) via apoptosis induction. Furthermore, we demonstrated that the association of the RGD-avidin-TRAIL complex onto endothelial cells enhanced the tumor cell killing activity. Endothelial cells were not killed by TRAIL nor its derived complexes. Our approach can facilitate the enrichment of TRAIL onto angiogenic blood vessels, which may enhance intratumoral accumulation. Furthermore, it offers a versatile technology for the complexation of targeting ligands with therapeutic recombinant proteins and by this a novel way to enhance their specificity and activity.     

Gossypol Induces Death Receptor-5 through Activation of the ROS-ERK-CHOP Pathway and Sensitizes Colon Cancer Cells to TRAIL

Development of resistance to TRAIL, an apoptosis-inducing cytokine, is one of the major problems in its development for cancer treatment. Thus, pharmacological agents that are safe and can sensitize the tumor cells to TRAIL are urgently needed. We investigated whether gossypol, a BH3 mimetic that is currently in the clinic, can potentiate TRAIL-induced apoptosis. Intracellular esterase activity, sub-G₁ cell cycle arrest, and caspase-8, -9, and -3 activity assays revealed that gossypol potentiated TRAIL-induced apoptosis in human colon cancer cells. Gossypol also down-regulated cell survival proteins (Bcl-x_L, Bcl-2, survivin, XIAP, and cFLIP) and dramatically up-regulated TRAIL death receptor (DR)-5 expression but had no effect on DR4 and decoy receptors. Gossypol-induced receptor induction was not cell type-specific, as DR5 induction was observed in other cell types. Deletion of DR5 by siRNA significantly reduced the apoptosis induced by TRAIL and gossypol. Gossypol induction of the death receptor required the induction of CHOP, and thus, gene silencing of CHOP abolished gossypol-induced DR5 expression and associated potentiation of apoptosis. ERK1/2 (but not p38 MAPK or JNK) activation was also required for gossypol-induced TRAIL receptor induction; gene silencing of ERK abolished both DR5 induction and potentiation of apoptosis by TRAIL. We also found that reactive oxygen species produced by gossypol treatment was critical for TRAIL receptor induction and apoptosis potentiation. Overall, our results show that gossypol enhances TRAIL-induced apoptosis through the down-regulation of cell survival proteins and the up-regulation of TRAIL death receptors through the ROS-ERK-CHOP-DR5 pathway.