TRAF2 Sets a threshold for extrinsic apoptosis by tagging caspase-8 with a ubiquitin shutoff timer.

Apoptotic caspase activation mechanisms are well defined, yet inactivation modes remain unclear. The death receptors (DRs), DR4, DR5, and Fas, transduce cell-extrinsic apoptotic signals by recruiting caspase-8 into a death-inducing signaling complex (DISC). At the DISC, Cullin3-dependent polyubiquitination on the small catalytic subunit of caspase-8 augments stimulation. Here we report that tumor necrosis factor receptor-associated factor 2 (TRAF2) interacts with caspase-8 at the DISC, downstream of Cullin3. TRAF2 directly mediates RING-dependent, K48-linked polyubiquitination on the large catalytic domain of caspase-8. This modification destines activated caspase-8 molecules to rapid proteasomal degradation upon autoprocessing and cytoplasmic translocation. TRAF2 depletion lowers the signal threshold for DR-mediated apoptosis, altering cell life versus death decisions in vitro and in vivo. Thus, TRAF2 sets a critical barrier for cell-extrinsic apoptosis commitment by tagging activated caspase-8 with a K48-ubiquitin shutoff timer. These results may have important implications for caspase regulation mechanisms.

Inflammasome-dependent and -independent IL-18 production mediates immunity to the ISCOMATRIX adjuvant.

Adjuvants are an essential component of modern vaccines and used for their ability to elicit immunity to coadministered Ags. Many adjuvants in clinical development are particulates, but how they drive innate and adaptive immune responses remains poorly understood. Studies have shown that a number of vaccine adjuvants activate inflammasome pathways in isolated APCs. However, the contribution of inflammasome activation to vaccine-mediated immunity in vivo remains controversial. In this study, we evaluated immune cell responses to the ISCOMATRIX adjuvant (IMX) in mice. Like other particulate vaccine adjuvants, IMX potently activated the NALP-3-ASC-Caspase-1 inflammasome in APCs, leading to IL-1ß and IL-18 production. The IL-18R pathway, but not IL-1R, was required for early innate and subsequent cellular immune responses to a model IMX vaccine. APCs directly exposed to IMX underwent an endosome-mediated cell-death response, which we propose initiates inflammatory events locally at the injection site. Importantly, both inflammasome-related and -unrelated pathways contributed to IL-18 dependence in vivo following IMX administration. TNF-α provided a physiological priming signal for inflammasome-dependent IL-18 production by APCs, which correlated with reduced vaccine-mediated immune cell responses in TNF-α- or TNFR-deficient mice. Taken together, our findings highlight an important disconnect between the mechanisms of vaccine adjuvant action in vitro versus in vivo.

Host genetic background impacts modulation of the TLR4 pathway by RON in tissue-associated macrophages.

Toll-like receptors (TLRs) enable metazoans to mount effective innate immune responses to microbial and viral pathogens, as well as to endogenous host-derived ligands. It is understood that genetic background of the host can influence TLR responsiveness, altering susceptibility to pathogen infection, autoimmunity and cancer. Macrophage stimulatory protein (MSP), which activates the receptor tyrosine kinase recepteur d'origine nantais (RON), promotes key macrophage functions such as motility and phagocytic activity. MSP also acts via RON to modulate signaling by TLR4, which recognizes a range of pathogen or endogenous host-derived molecules. Here, we show that RON exerts divergent control over TLR4 activity in macrophages from different mouse genetic backgrounds. RON potently modulated the TLR4 response in macrophages from M2-prone FVB mice, as compared with M1-skewed C57Bl6 mice. Moreover, global expression analysis revealed that RON suppresses the TLR4-dependent type-I interferon gene signature only in FVB macrophages. This leads to attenuated production of the potent inflammatory mediator, tumor necrosis factor-α. Eliminating RON kinase activity markedly decreased carcinogen-mediated tumorigenesis in M2/Th2-biased FVB mice. We propose that host genetic background influences RON function, thereby contributing to the variability in TLR4 responsiveness in rodents and, potentially, in humans. These findings provide novel insight into the complex interplay between genetic context and immune function.

Distinct involvement of the Gab1 and Grb2 adaptor proteins in signal transduction by the related receptor tyrosine kinases RON and MET.

Although the signal transduction mechanisms of the receptor tyrosine kinase MET are well defined, less is known about its close relative RON. MET initiates intracellular signaling by autophosphorylation on specific cytoplasmic tyrosines that form docking sites for the adaptor proteins Grb2 and Gab1. Grb2 binds directly and is essential for all of the biological activities of MET. Gab1 docks either directly or indirectly via Grb2 and controls only a subset of MET functions. Because MET and RON possess similar adaptor binding sites, it was anticipated that their adaptor interactions would be conserved. Here we show that in contrast to MET, RON relies primarily on Gab1 for signal transmission. Surprisingly, disruption of the Grb2 docking site of RON or Grb2 depletion augments activity, whereas enhancement of Grb2 binding attenuates Gab1 recruitment and signaling. Hence, RON and MET differ in their adaptor interactions; furthermore, Grb2 performs a novel antagonistic role in the context of RON signaling.

ISCOMATRIX vaccines mediate CD8+ T-cell cross-priming by a MyD88-dependent signaling pathway.

Generating a cytotoxic CD8(+) T-cell response that can eradicate malignant cells is the primary objective of cancer vaccine strategies. In this study we have characterized the innate and adaptive immune response to the ISCOMATRIX adjuvant, and the ability of vaccine antigens formulated with this adjuvant to promote antitumor immunity. ISCOMATRIX adjuvant led to a rapid innate immune cell response at the injection site, followed by the activation of natural killer and dendritic cells (DC) in regional draining lymph nodes. Strikingly, major histocompatibility complex (MHC) class I cross-presentation by CD8α(+) and CD8α(-) DCs was enhanced by up to 100-fold when antigen was formulated with ISCOMATRIX adjuvant. These coordinated features enabled efficient CD8(+) T-cell cross-priming, which exhibited prophylactic and therapeutic tumoricidal activity. The therapeutic efficacy of an ISCOMATRIX vaccine was further improved when co-administered with an anti-CD40 agonist antibody, suggesting that ISCOMATRIX-based vaccines may combine favorably with other immune modifiers in clinical development to treat cancer. Finally, we identified a requirement for the myeloid differentiation primary response gene 88 (MyD88) adapter protein for both innate and adaptive immune responses to ISCOMATRIX vaccines in vivo. Taken together, our findings support the utility of the ISCOMATRIX adjuvant for use in the development of novel vaccines, particularly those requiring strong CD8(+) T-cell immune responses, such as therapeutic cancer vaccines.

Local Delivery of OncoVEXmGM-CSF Generates Systemic Antitumor Immune Responses Enhanced by Cytotoxic T-Lymphocyte-Associated Protein Blockade.

Purpose: Talimogene laherparepvec, a new oncolytic immunotherapy, has been recently approved for the treatment of melanoma. Using a murine version of the virus, we characterized local and systemic antitumor immune responses driving efficacy in murine syngeneic models.Experimental Design: The activity of talimogene laherparepvec was characterized against melanoma cell lines using an in vitro viability assay. Efficacy of OncoVEXmGM-CSF (talimogene laherparepvec with the mouse granulocyte-macrophage colony-stimulating factor transgene) alone or in combination with checkpoint blockade was characterized in A20 and CT-26 contralateral murine tumor models. CD8+ depletion, adoptive T-cell transfers, and Enzyme-Linked ImmunoSpot assays were used to study the mechanism of action (MOA) of systemic immune responses.Results: Treatment with OncoVEXmGM-CSF cured all injected A20 tumors and half of contralateral tumors. Viral presence was limited to injected tumors and was not responsible for systemic efficacy. A significant increase in T cells (CD3+/CD8+) was observed in injected and contralateral tumors at 168 hours. Ex vivo analyses showed these cytotoxic T lymphocytes were tumor-specific. Increased neutrophils, monocytes, and chemokines were observed in injected tumors only. Importantly, depletion of CD8+ T cells abolished all systemic efficacy and significantly decreased local efficacy. In addition, immune cell transfer from OncoVEXmGM-CSF-cured mice significantly protected from tumor challenge. Finally, combination of OncoVEXmGM-CSF and checkpoint blockade resulted in increased tumor-specific CD8+ anti-AH1 T cells and systemic efficacy.Conclusions: The data support a dual MOA for OncoVEXmGM-CSF that involves direct oncolysis of injected tumors and activation of a CD8+-dependent systemic response that clears injected and contralateral tumors when combined with checkpoint inhibition. Clin Cancer Res; 23(20); 6190-202. ©2017 AACR.

Proapoptotic activation of death receptor 5 on tumor endothelial cells disrupts the vasculature and reduces tumor growth.

The proapoptotic death receptor DR5 has been studied extensively in cancer cells, but its action in the tumor microenvironment is not well defined. Here, we uncover a role for DR5 signaling in tumor endothelial cells (ECs). We detected DR5 expression in ECs within tumors but not normal tissues. Treatment of tumor-bearing mice with an oligomeric form of the DR5 ligand Apo2L/TRAIL induced apoptosis in tumor ECs, collapsing blood vessels and reducing tumor growth: Vascular disruption and antitumor activity required DR5 expression on tumor ECs but not malignant cells. These results establish a therapeutic paradigm for proapoptotic receptor agonists as selective tumor vascular disruption agents, providing an alternative, perhaps complementary, strategy to their use as activators of apoptosis in malignant cells.

An Fcγ receptor-dependent mechanism drives antibody-mediated target-receptor signaling in cancer cells.

Antibodies to cell-surface antigens trigger activatory Fcγ receptor (FcγR)-mediated retrograde signals in leukocytes to control immune effector functions. Here, we uncover an FcγR mechanism that drives antibody-dependent forward signaling in target cells. Agonistic antibodies to death receptor 5 (DR5) induce cancer-cell apoptosis and are in clinical trials; however, their mechanism of action in vivo is not fully defined. Interaction of the DR5-agonistic antibody drozitumab with leukocyte FcγRs promoted DR5-mediated tumor-cell apoptosis. Whereas the anti-CD20 antibody rituximab required activatory FcγRs for tumoricidal function, drozitumab was effective in the context of either activatory or inhibitory FcγRs. A CD40-agonistic antibody required similar FcγR interactions to stimulate nuclear factor-κB activity in B cells. Thus, FcγRs can drive antibody-mediated receptor signaling in target cells.

Cooperation of the proapoptotic receptor agonist rhApo2L/TRAIL with the CD20 antibody rituximab against non-Hodgkin lymphoma xenografts.

Recombinant human rhApo2L/TRAIL selectively stimulates apoptosis in various cancer cells through its receptors DR4 and DR5, and is currently in clinical trials. Preclinical studies have established antitumor activity of rhApo2L/TRAIL in models of epithelial cancers; however, efficacy in non-Hodgkin lymphoma (NHL) models is not well studied. Of 7 NHL cell lines tested in vitro, rhApo2L/TRAIL stimulated apoptosis in BJAB, Ramos RA1, and DoHH-2 cells. Rituximab, a CD20 antibody used to treat certain types of NHL, augmented rhApo2L/TRAIL-induced caspase activation in Ramos RA1 and DoHH2 but not BJAB or SC-1 cells, through modulation of intrinsic rather than extrinsic apoptosis signaling. In vivo, rhApo2L/TRAIL and rituximab cooperated to attenuate or reverse growth of tumor xenografts of all 4 of these cell lines. Depletion of natural killer (NK) cells or serum complement substantially reduced combined efficacy against Ramos RA1 tumors, suggesting involvement of antibody-dependent cell- and complement-mediated cytotoxicity. Both agents exhibited greater activity against disseminated than subcutaneous BJAB xenografts, and worked together to inhibit or abolish disseminated tumors and increase survival. Moreover, rhApo2L/TRAIL helped circumvent acquired rituximab resistance of a Ramos variant. These findings provide a strong rationale for clinical investigation of rhApo2L/TRAIL in combination with rituximab as a novel strategy for NHL therapy.

TWEAK attenuates the transition from innate to adaptive immunity.

Innate immunity is the first line of defense against infection, protecting the host during the development of adaptive immunity and critically affecting the nature of the adaptive response. We show that, in contrast to tumor necrosis factor alpha (TNF-alpha), the related protein TWEAK attenuates the transition from innate to adaptive mechanisms. TWEAK-/- mice had overabundant natural killer (NK) cells and displayed hypersensitivity to bacterial endotoxin, with their innate immune cells producing excess interferon (IFN)-gamma and interleukin (IL)-12. TWEAK inhibited stimulation of the transcriptional activator STAT-1 and induced p65 nuclear factor (NF)-kappaB association with histone deacetylase 1, repressing cytokine production. TWEAK-/- mice developed oversized spleens with expanded memory and T helper 1 (TH1) subtype cells upon aging and mounted stronger innate and adaptive TH1-based responses against tumor challenge. Thus, TWEAK suppresses production of IFN-gamma and IL-12, curtailing the innate response and its transition to adaptive TH1 immunity.