Judy Lieberman

Individual mammalian mRNAs and proteins are typically believed to originate from single genomic loci, with isoform diversity arising through cis -splicing of pre-mRNA. Whether mRNA from distant genes can undergo trans -splicing to generate functionally relevant chimeric transcripts has remained unclear. Here we develop a pipeline combining long-read direct RNA sequencing with non-targeted and targeted validation to identify chimeric transcripts in macrophages. Chromatin conformation capture studies reveal that inflammation induces interchromosomal DNA interactions, positioning parent genes proximally to facilitate the formation of chimeric mRNA. Notably, we identify a protein-coding chimeric mRNA representing a fusion between the pore-forming protein gasdermin D (GSDMD) ^1,2 and a C-terminal domain translated out of frame from Tmem106a ( Gsdmd - Tmem106a ) in mice. We show that inflammasome priming upregulates Gsdmd - Tmem106a , with the protein localizing to the plasma membrane. After activation of the inflammasome, GSDMD–TMEM106A directly interacts with canonical GSDMD N termini to accelerate and enhance pore formation and IL-1β release. Finally, we show that GSDMD–TMEM106A balances host defence and immunopathology in vivo: its loss protects against lethal sepsis but compromises antibacterial defence, whereas overexpression enhances host protection while increasing sepsis lethality. We establish that protein-coding chimeric mRNAs formed by regulated transcript fusion events are operative during inflammation and immunity.

Pyroptosis is defined as gasdermin-mediated lytic programmed cell death. Gasdermin E (GSDME), a substrate of the apoptotic caspase-3, can convert apoptosis into pyroptosis, with critical roles in antitumor immunity and chemotherapy-induced tissue damage. Despite its importance, the structural mechanism of GSDME pore formation and its regulation by posttranslational modifications remain largely unknown. Here, we present the cryo-electron microscopy (cryo-EM) structure at 3.16 Å resolution of the human GSDME N-terminal (NT) pore using proteins expressed from mammalian cells. The structure reveals a GSDME-NT pore assembled mainly as a 28-subunit homo-oligomer, and a dramatic conformational rearrangement from the autoinhibited state, with refolding of the two β-hairpins in each monomer to form a membrane-spanning β-barrel with an acidic conduit. Unexpectedly, we identify endogenous S-palmitoylation of C45, C168, and C180, required for membrane binding and pore formation. In addition, extra cryo-EM densities are visible adjacent to the C45 side chain, potentially corresponding to the flexibly linked palmitate chain. Structure-guided mutagenesis demonstrates that these palmitoylation sites synergistically control pore formation. The structure served as a molecular blueprint for analyzing cancer-associated mutations, known to disrupt GSDME function. These mutations cluster at functional hotspots in the oligomerization interfaces, membrane-contact regions, and the β-barrel, where they disrupt pore integrity. Collectively, our findings establish palmitoylation as an obligatory licensing step for membrane binding and pore formation, provide structural visualization of a palmitoylated gasdermin, and reveal how cancer-associated mutations impair pyroptotic function. This structure and these insights will be useful for developing strategies that target GSDME to treat cancer and inflammatory disease.

A major limitation on the development of a malaria vaccine is the lack of validated T cell epitope targets. Plasmodium falciparum is the most prevalent malaria parasite affecting humans in Africa, whereas Plasmodium vivax is more widespread and is the main species that causes malaria in the Americas and Asia¹. P. vivax exclusively infects peripheral-blood reticulocytes, which retain RNA and the capacity for host protein synthesis². We previously reported that reticulocytes infected with P. vivax express human leukocyte antigen class I (HLA-I), which enables recognition and killing of the parasite by CD8⁺ T cells³. Here we use immunopeptidomics to identify Plasmodium-antigen-derived peptides presented by HLA-I on infected reticulocytes. We identified 453 unique peptides, mapping to 166 proteins. Seventy-five antigens were housekeeping proteins that are constitutively expressed at multiple stages of the parasite’s life cycle and are highly conserved between Plasmodium species. Identical peptides were presented in different individuals by the same or distinct HLA-A, HLA-B and HLA-C alleles, as well as by the non-classical HLA-E allele. The antigenicity of the newly identified epitopes was validated in samples from both P. vivax-infected and P. falciparum-infected individuals. Furthermore, T cell responses to several of these antigens were observed in the blood and liver of non-human primates after infection with Plasmodium or immunization with attenuated parasites. Two antigens also induced protective CD8⁺ T cell-mediated immunity in rodents. Thus, these antigens have the potential for use in a cross-stage and cross-species malaria vaccine.

The major effector cells of antitumor immunity are killer lymphocytes that recognize and eliminate tumor cells. The fact that tumor cells look a lot like normal cells poses a challenge to antitumor immune control. A danger signal from the tumor or from antigen-presenting cells that have taken up dying tumor cells is needed to distinguish tumor cells from normal cells to fully activate killer cell effector functionality and memory and thereby control the tumor. How a tumor cell dies strongly affects whether the immune system sees it as dangerous. Activation of innate immunity in the tumor, including interferon signaling and necrotic cell death (e.g., necroptosis and pyroptosis), sounds a potent immune alarm. Pyroptosis plays an important role in tumor immunity by generating an inflamed tumor microenvironment. However, it is a double-edged sword that can both promote tumorigenesis and increase the effectiveness and cytotoxicity of cancer therapy. In this article, we review what is known about the role of tumor cell pyroptosis, which is arguably the most inflammatory type of cell death, in antitumor immunity and discuss whether it could be safely harnessed to broaden the range of tumors that respond to immunotherapy.

A recent paper in Nature shows that SARS-CoV-2 mRNA vaccination within 100 days of beginning immune checkpoint inhibitor immunotherapy improves survival of cancer patients by systemically inducing Type I interferons, suggesting that an off-the-shelf cancer vaccine that potently stimulates innate immunity may not need to be customized to contain tumor-associated antigens to be effective.

The successes of cancer immunotherapy have inspired research aiming to increase the number of immune-responsive cancers. The first effective immunotherapeutic strategies—immune checkpoint blockade (ICB) and CAR T cells—were designed to overcome limitations in CD8 ⁺ T cell recognition and killing of tumor cells. However, most solid tumors still do not respond to these measures and new treatment approaches are needed. Tumors evolve many strategies to avoid immune control. One way to identify immunotherapy strategies is to study what distinguishes immunotherapy-responsive and -unresponsive tumors. Another way is to identify the differences in tumors that emerge after carcinogen exposure in immunocompetent versus immunodeficient hosts. Still another way is to identify changes in gene expression in emerging tumors that enable them to escape immunosurveillance (known as tumor immunoediting). Evolving tumors suppress antigen processing and presentation to avoid triggering tumor-specific T cells but also repress key innate immune genes that transmit danger signals to immune cells. In this perspective, we discuss the roles of innate immunity in anti-tumor responses and consider how innate immunity could be harnessed to make tumors more immune-responsive.

Toll-like receptor 4 (TLR4), a pattern-recognition receptor located on the plasma membrane, senses extracellular danger signals to initiate inflammatory immune responses. It is initially synthesized in the endoplasmic reticulum (ER), undergoes N-linked glycosylation, and is subsequently transported to the Golgi before ultimately reaching the plasma membrane. However, the mechanisms underlying the processing and maturation of TLR4 in the ER remain elusive. Through whole genome-wide CRISPR screening, CCDC134 was identified as a critical and essential factor for TLR4-dependent inflammatory response. Localization of CCDC134 in the ER lumen rather than its exosome-mediated secretion is required for its role in TLR4 signaling. Loss of CCDC134 results in the retention of TLR4 in the ER for subsequent ER-associated degradation, and thus blockade of TLR4 maturation and plasma membrane trafficking. Defects in TLR4 processing and maturation in the ER in CCDC134-depleted cells are caused by aberrant hyperglycosylation and destabilization of glycoprotein 96 (gp96), a key chaperone of TLR4. These results suggest that CCDC134 controls gp96 glycosylation to facilitate TLR4 maturation in the ER.

Malaria is one of the most prevalent parasitic diseases in the world. In 2023, 263 million malaria cases were estimated worldwide. Two species of Plasmodium, P. falciparum and P. vivax, cause most human malaria. Despite the licensing of two partially protective vaccines for P. falciparum, there is no vaccine capable of providing long-term control or elimination. A major limitation for vaccine development is the lack of validated T cell epitopes for either species that could be targeted by vaccines. P. vivax is the most widespread human malaria parasite and is the major species causing malaria in the Americas and Asia while P. falciparum is more prevalent in Africa1. P. vivax exclusively infects reticulocytes in peripheral blood, which, unlike the mature erythrocytes infected by P. falciparum, still retain RNA and therefore retain host protein translation capabilities. We previously reported that P. vivax-infected reticulocytes express the major human leukocyte antigen class I (HLA-I), which allows parasite sensing by CD8+ T cells and consequent killing of parasite-infected host cells and intracellular parasites. Here we report by immunopeptidomic analysis the first unbiased identification of Plasmodium spp. antigens presented via HLA-I on infected reticulocytes. We identified 453 unique peptides that mapped to 166 different proteins. Most of these antigens were housekeeping proteins that are constitutively expressed at multiple stages of the parasite life cycle. Common peptides were presented in different individuals by the same or distinct HLA-ABC alleles as well as by non-classical HLA-E. Many peptide sequences were highly conserved in P. falciparum and P. vivax. The immunogenicity of the newly identified epitopes was validated in both P. vivax- and P. falciparum-infected patient samples. Furthermore, several of these antigens were immunogenic in the blood and liver of non-human primates following Plasmodium infection and attenuated parasite immunization. Two antigens were also the target of protective CD8+ T cell-mediated immunity in rodents. Thus, these antigens have potential for use in a cross-stage and cross-species malaria vaccine.