The Asian lineage of Zika virus (ZIKV) has recently caused epidemics and severe disease. Unraveling the mechanisms causing increased viral transmissibility and disease severity requires experimental ...systems. We report an infectious cDNA clone of ZIKV that was generated using a clinical isolate of the Asian lineage. The cDNA clone-derived RNA is infectious in cells, generating recombinant ZIKV. The recombinant virus is virulent in established ZIKV mouse models, leading to neurological signs relevant to human disease. Additionally, recombinant ZIKV is infectious for Aedes aegypti and thus provides a means to examine virus transmission. The infectious cDNA clone was further used to generate a luciferase ZIKV that exhibited sensitivity to a panflavivirus inhibitor, highlighting its potential utility for antiviral screening. This ZIKV reverse genetic system, together with mouse and mosquito infection models, may help identify viral determinants of human virulence and mosquito transmission as well as inform vaccine and therapeutic strategies.
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•An infectious cDNA clone of Zika virus and a luciferase reporter virus were developed•Recombinant Zika virus is virulent in A129 and AG129 mice•Recombinant Zika virus is highly infectious for Aedes aegypti mosquitos•The luciferase Zika virus can be used for antiviral drug discovery
Zika virus (ZIKV) is causing devastating epidemics with severe disease. Shan et al. generated an infectious cDNA clone of ZIKV as well as a luciferase reporter virus. Recombinant ZIKV is virulent in mice and infectious in Aedes aegypti and thus may help identify viral determinants of virulence and transmission.
Caspases are a family of cysteine proteases that act as molecular scissors to cleave substrates and regulate biological processes such as programmed cell death and inflammation. Extensive efforts ...have been made to identify caspase substrates and to determine factors that dictate substrate specificity. Thousands of putative substrates have been identified for caspases that regulate an immunologically silent type of cell death known as apoptosis, but less is known about substrates of the inflammatory caspases that regulate an immunostimulatory type of cell death called pyroptosis. Furthermore, much of our understanding of caspase substrate specificities is derived from work done with peptide substrates, which do not often translate to native protein substrates. Our knowledge of inflammatory caspase biology and substrates has recently expanded and here, we discuss the recent advances in our understanding of caspase substrate specificities, with a focus on inflammatory caspases. We highlight new substrates that have been discovered and discuss the factors that engender specificity. Recent evidence suggests that inflammatory caspases likely utilize two binding interfaces to recognize and process substrates, the active site and a conserved exosite.
Severe injuries to peripheral nerves are challenging to repair. Standard-of-care treatment for nerve gaps >2 to 3 centimeters is autografting; however, autografting can result in neuroma formation, ...loss of sensory function at the donor site, and increased operative time. To address the need for a synthetic nerve conduit to treat large nerve gaps, we investigated a biodegradable poly(caprolactone) (PCL) conduit with embedded double-walled polymeric microspheres encapsulating glial cell line-derived neurotrophic factor (GDNF) capable of providing a sustained release of GDNF for >50 days in a 5-centimeter nerve defect in a rhesus macaque model. The GDNF-eluting conduit (PCL/GDNF) was compared to a median nerve autograft and a PCL conduit containing empty microspheres (PCL/Empty). Functional testing demonstrated similar functional recovery between the PCL/GDNF-treated group (75.64 ± 10.28%) and the autograft-treated group (77.49 ± 19.28%); both groups were statistically improved compared to PCL/Empty-treated group (44.95 ± 26.94%). Nerve conduction velocity 1 year after surgery was increased in the PCL/GDNF-treated macaques (31.41 ± 15.34 meters/second) compared to autograft (25.45 ± 3.96 meters/second) and PCL/Empty (12.60 ± 3.89 meters/second) treatment. Histological analyses included assessment of Schwann cell presence, myelination of axons, nerve fiber density, and
-ratio. PCL/GDNF group exhibited a statistically greater average area occupied by individual Schwann cells at the distal nerve (11.60 ± 33.01 μm
) compared to autograft (4.62 ± 3.99 μm
) and PCL/Empty (4.52 ± 5.16 μm
) treatment groups. This study demonstrates the efficacious bridging of a long peripheral nerve gap in a nonhuman primate model using an acellular, biodegradable nerve conduit.
Inflammasomes are multiprotein signaling complexes that activate the innate immune system. Canonical inflammasomes recruit and activate caspase-1, which then cleaves and activates IL-1β and IL-18, as ...well as gasdermin D (GSDMD) to induce pyroptosis. In contrast, non-canonical inflammasomes, caspases-4/-5 (CASP4/5) in humans and caspase-11 (CASP11) in mice, are known to cleave GSDMD, but their role in direct processing of other substrates besides GSDMD has remained unknown. Here, we show that CASP4/5 but not CASP11 can directly cleave and activate IL-18. However, CASP4/5/11 can all cleave IL-1β to generate a 27-kDa fragment that deactivates IL-1β signaling. Mechanistically, we demonstrate that the sequence identity of the tetrapeptide sequence adjacent to the caspase cleavage site regulates IL-18 and IL-1β recruitment and activation. Altogether, we have identified new substrates of the non-canonical inflammasomes and reveal key mechanistic details regulating inflammation that may aid in developing new therapeutics for immune-related disorders.
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•Human CASP4/5 directly process IL-18 at D36 to generate the activated cytokine•CASP4/5 cleave IL-1β at D27 into a p27 species that inactivates downstream IL-1R signaling•The tetrapeptide sequence of IL-18 and IL-1β regulates their processing by caspases•CASP11 can process IL-1β into the deactivating p27 species but does not process IL-18
Exconde et al. report that the non-canonical inflammasomes directly process IL-18 and IL-1β. The tetrapeptide sequence regulates IL-18 and IL-1β processing to generate an active or inactive cytokine respectively. These results suggest that the non-canonical inflammasomes directly modulate inflammation and may have a broader substrate repertoire than previously known.
Cancer immunotherapy is a novel pillar of cancer treatment that harnesses the immune system to fight tumors and generally results in robust antitumor immunity. Although immunotherapy has achieved ...remarkable clinical success for some patients, many patients do not respond, underscoring the need to develop new strategies to promote antitumor immunity. Pyroptosis is an immunostimulatory type of regulated cell death that activates the innate immune system. A hallmark of pyroptosis is the release of intracellular contents such as cytokines, alarmins, and chemokines that can stimulate adaptive immune activation. Recent studies suggest that pyroptosis promotes antitumor immunity. Here, we review the mechanisms by which pyroptosis can be induced and highlight new strategies to induce pyroptosis in cancer cells for antitumor defense. We discuss how pyroptosis modulates the tumor microenvironment to stimulate adaptive immunity and promote antitumor immunity. We also suggest research areas to focus on for continued development of pyroptosis as an anticancer treatment. Pyroptosis-based anticancer therapies offer a promising new avenue for treating immunologically 'cold' tumors.
Chimeric antigen receptor (CAR)-T cells represent a major breakthrough in cancer therapy, wherein a patient's own T cells are engineered to recognize a tumor antigen, resulting in activation of a ...local cytotoxic immune response. However, CAR-T cell therapies are currently limited to the treatment of B cell cancers and their effectiveness is hindered by resistance from antigen-negative tumor cells, immunosuppression in the tumor microenvironment, eventual exhaustion of T cell immunologic functions and frequent severe toxicities. To overcome these problems, we have developed a novel class of CAR-T cells engineered to express an enzyme that activates a systemically administered small-molecule prodrug in situ at a tumor site. We show that these synthetic enzyme-armed killer (SEAKER) cells exhibit enhanced anticancer activity with small-molecule prodrugs, both in vitro and in vivo in mouse tumor models. This modular platform enables combined targeting of cellular and small-molecule therapies to treat cancers and potentially a variety of other diseases.
PRAME is a prominent member of the cancer testis antigen family of proteins, which triggers autologous T cell-mediated immune responses. Integrative genomic analysis in diffuse large B cell lymphoma ...(DLBCL) uncovered recurrent and highly focal deletions of 22q11.22, including the PRAME gene, which were associated with poor outcome. PRAME-deleted tumors showed cytotoxic T cell immune escape and were associated with cold tumor microenvironments. In addition, PRAME downmodulation was strongly associated with somatic EZH2 Y641 mutations in DLBCL. In turn, PRC2-regulated genes were repressed in isogenic PRAME-KO lymphoma cell lines, and PRAME was found to directly interact with EZH2 as a negative regulator. EZH2 inhibition with EPZ-6438 abrogated these extrinsic and intrinsic effects, leading to PRAME expression and microenvironment restoration in vivo. Our data highlight multiple functions of PRAME during lymphomagenesis and provide a preclinical rationale for synergistic therapies combining epigenetic reprogramming with PRAME-targeted therapies.
•“Orexi” CAR T cells in combination with an orthogonally targeted monoclonal antibody are more effective than traditional CAR T cells.•OrexiCAR T cells reversed immunosuppression of tumor ...microenvironment myeloid cells.
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Chimeric antigen receptor (CAR) T-cell therapy has shown success in the treatment of hematopoietic malignancies; however, relapse remains a significant issue. To overcome this, we engineered “Orexi” CAR T cells to locally secrete a high-affinity CD47 blocker, CV1, at the tumor and treated tumors in combination with an orthogonally targeted monoclonal antibody. Traditional CAR T cells plus the antibody had an additive effect in xenograft models, and this effect was potentiated by CAR T-cell local CV1 secretion. Furthermore, OrexiCAR-secreted CV1 reversed the immunosuppression of myelomonocytoid cells both in vitro and within the tumor microenvironment. Local secretion of the CD47 inhibitor bypasses the CD47 sink found on all cells in the body and may prevent systemic toxicities. This combination of CAR T-cell therapy, local CD47 blockade, and orthogonal antibody may be a combinatorial strategy to overcome the limitations of each monotherapy.
An immune-suppressive tumor microenvironment is a major barrier to the efficacy of antibody-based therapies. Dacek et al present a novel strategy to overcome this by enhancing the antibody-dependent killing of tumors through additional infusion of chimeric antigen receptor (CAR) T cells engineered to locally secrete a small anti-CD47 blocking agent that disrupts antiphagocytic signaling induced by tumor cell CD47 binding to macrophage signal-regulatory protein α. This increases macrophage-mediated antibody-dependent cellular phagocytosis or antibody-dependent cellular cytotoxicity and enables increased tumor cell lysis in a mouse model. Such strategies can now be tested in clinical trials.
Cellular therapies are engineered using foreign and synthetic protein sequences, such as chimeric antigen receptors (CARs). The frequently observed humoral responses to CAR T cells result in rapid ...clearance, especially after re-infusions. There is an unmet need to protect engineered cells from host-versus-graft rejection, particularly for the advancement of allogeneic cell therapies. Here, utilizing the immunoglobulin G (IgG) protease “IdeS,” we programmed CAR T cells to defeat humoral immune attacks. IdeS cleavage of host IgG averted Fc-dependent phagocytosis and lysis, and the residual F(ab′)2 fragments remained on the surface, providing cells with an inert shield from additional IgG deposition. “Shield” CAR T cells efficiently cleaved cytotoxic IgG, including anti-CAR antibodies, detected in patient samples and provided effective anti-tumor activity in the presence of anti-cell IgG in vivo. This technology may be useful for repeated human infusions of engineered cells, more complex engineered cells, and expanding widespread use of “off-the-shelf” allogeneic cellular therapies.
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Cellular therapies are engineered using foreign and synthetic protein sequences. Utilizing the IgG protease “IdeS,” we programmed CAR T cells to defeat humoral immune attacks. This technology may be useful for repeated human infusions of engineered cells, more complex engineered cells, and expanding widespread use of “off-the-shelf” allogeneic cellular therapies.
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