Highlights d Cryo-EM structures of 7 intermediates in promoter opening pathway from RPc to RPo d Intermediates populated by using an inhibitor and a promoter with unstable RPo d RNAP and DNA conformational changes in mobile regions mark the steps in the pathway d Transient interactions identified in intermediates are not found in RPc or RPo

Activation of the Mcm2-7 replicative DNA helicase is the committed step in eukaryotic DNA replication initiation. Although Mcm2-7 activation requires binding of the helicase-activating proteins Cdc45 and GINS (forming the CMG complex), an additional protein, Mcm10, drives initial origin DNA unwinding by an unknown mechanism. We show that Mcm10 binds a conserved motif located between the oligonucleotide/oligosaccharide fold (OB-fold) and A subdomain of Mcm2. Although buried in the interface between these domains in Mcm2-7 structures, mutations predicted to separate the domains and expose this motif restore growth to conditional-lethal MCM10 mutant cells. We found that, in addition to stimulating initial DNA unwinding, Mcm10 stabilizes Cdc45 and GINS association with Mcm2-7 and stimulates replication elongation in vivo and in vitro. Furthermore, we identified a lethal allele of MCM10 that stimulates initial DNA unwinding but is defective in replication elongation and CMG binding. Our findings expand the roles of Mcm10 during DNA replication and suggest a new model for Mcm10 function as an activator of the CMG complex throughout DNA replication.

Summary RNA polymerase binds tightly to DNA to recognize promoters with high specificity but then releases these contacts during the initial stage of transcription. We report a site-specific crosslinking approach to map the DNA path in bacterial transcription intermediates at amino acid and nucleotide resolution. After validating the approach by showing that the DNA path in open complexes (RPO) is the same as in high resolution X-ray structures, we define the path following substrate addition in ‘scrunched’ complexes (RPITC). The DNA bulges that form within the transcription bubble in RPITC are positioned differently on the two strands. Our data suggest that the non-template strand bulge is extruded into solvent in complexes containing a 5-mer RNA, whereas the template strand bulge remains within the template strand tunnel, exerting stress on the β flap-β′ clamp-σ3.2 interaction. We propose that this stress contributes to σ3.2 displacement from the RNA exit channel, facilitating promoter escape.

Eukaryotic replication origin licensing, activation and timing are influenced by chromatin but a mechanistic understanding is lacking. Using reconstituted nucleosomal DNA replication assays, we assessed the impact of nucleosomes on replication initiation. To generate distinct nucleosomal landscapes, different chromatin-remodeling enzymes (CREs) were used to remodel nucleosomes on origin-DNA templates. Nucleosomal organization influenced two steps of replication initiation: origin licensing and helicase activation. Origin licensing assays showed that local nucleosome positioning enhanced origin specificity and modulated helicase loading by influencing ORC DNA binding. Interestingly, SWI/SNF- and RSC-remodeled nucleosomes were permissive for origin licensing but showed reduced helicase activation. Specific CREs rescued replication of these templates if added prior to helicase activation, indicating a permissive chromatin state must be established during origin licensing to allow efficient origin activation. Our studies show nucleosomes directly modulate origin licensing and activation through distinct mechanisms and provide insights into the regulation of replication initiation by chromatin.DOI: http://dx.doi.org/10.7554/eLife.22512.001

The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via G-protein coupled receptors S1P1–5 and LPA1–3 respectively, and are implicated in allergy. Eosinophils accumulate at innervating cholinergic nerves in asthma and adhere to nerve cells via intercellular adhesion molecule-1 (ICAM-1). IMR-32 neuroblastoma cells were used as an in vitro cholinergic nerve cell model. The Gi coupled receptors S1P1, S1P3, LPA1, LPA2 and LPA3 were expressed on IMR-32 cells. Both S1P and LPA induced ERK phosphorylation and ERK- and Gi-dependent up-regulation of ICAM-1 expression, with differing time courses. LPA also induced ERK- and Gi-dependent up-regulation of the eosinophil chemoattractant, CCL-26. The eosinophil granule protein eosinophil peroxidase (EPO) induced ERK-dependent up-regulation of transcription of S1P1, LPA1, LPA2 and LPA3, providing the situation whereby eosinophil granule proteins may enhance S1P- and/or LPA- induced eosinophil accumulation at nerve cells in allergic conditions.

BackgroundAntigen-specific CD8+ T cell activity is critical for mounting an effective immune response in a wide range of indications, including immune-oncology and infectious diseases.MethodsTo elicit antigen-specific CD8+ T cell activity, we used microfluidics cell squeezing (Cell Squeeze®) to deliver antigens directly to the cytosol of antigen presenting cells (APCs). Direct cytosolic delivery bypasses the need for cross-presentation and efficiently loads antigen into the major histocompatibility complex class I (MHC-I) pathway. The Cell Squeeze® platform is generally agnostic to cell type and material. Therefore, not only does microfluidic squeezing enable cell subsets within human peripheral blood mononuclear cells (PBMCs) to function as unconventional APCs, but it also enables us to efficiently investigate a wide range of antigens including whole protein, peptides, and mRNA. This ‘plug and play’ nature of the platform allows for broad application in multiple disease areas.ResultsIn human cells, we demonstrated that microfluidic squeezing of PBMCs enables effective delivery to the major cell subsets including T cells, B cells, NK cells and monocytes. Delivery of CMV and HPV16 synthetic long peptides (SLPs) resulted in robust in vitro responses of both CD8+ T cell clones and patient-derived memory populations. To broaden the impact of our PBMC-based cell therapy approach, we investigated several other antigens relevant to other disease areas. Additional materials we delivered via squeezing and demonstrated antigen presentation include neoantigens, M1 Influenza mRNA, and pp65 SLP. Cell Squeeze® platform is simple to use and amenable to scale up. We demonstrated that delivery and viability for research scale process (~2 × 106 cells) is equivalent to delivery and viability of PBMCs processed at manufacturing scale (~1 × 109 cells).ConclusionsMicrofluidic cell squeezing of human PBMCs with antigenic material can be tailored to produce APCs that drive robust CD8+ T cell response against targets across multiple disease areas and has been scaled up for clinical use. SQZ-PBMC-HPV are currently under clinical evaluation for treatment of HPV16+ tumors.

Antigen-specific CD8+ T cell priming is critical for mounting an effective immune response against altered self or foreign antigens that are found in most tumors and virally infected cells. T cell priming events require antigen presenting cells (APCs) to process and load antigenic peptides onto MHC molecules. One recently established method for providing APCs with foreign antigen is through delivery of mRNA. An advantage of using antigen-encoding mRNA, compared to protein or peptides, is the inherent protein amplification of mRNA translation; a single mRNA can be translated multiple times. In addition, when antigen-encoding mRNA is delivered directly to the cell, the antigens will have the appropriate post-translational modifications, potentially increasing the immunogenicity. Whereas typical mRNA vaccine platforms require modified nucleotides and lipid nanoparticle encapsulation, Cell Squeeze® technology allows for delivery of uncomplexed and unmodified mRNA directly into the cytosol. Here, we use ex vivo microfluidic Cell Squeeze® technology to deliver uncomplexed and unmodified mRNA encoding for disease related antigens into human peripheral blood mononuclear cells (PBMCs) to generate PBMC APCs. We demonstrate that squeezing PBMCs with mRNA results in effective delivery and translation of mRNA in major cell subsets including T cells, B cells, NK cells, and monocytes. Delivery of antigen-encoding mRNA to human PBMCs enables them to function as APCs, capable of presenting antigenic peptides on MHC molecules for activation of antigen-specific T cells. Specifically, we demonstrate squeezing antigen-encoding mRNA (e.g. HPV16 E7) into human PBMCs results in PBMC APCs that can elicit robust in vitro activation of antigen-specific CD8+ T cells. This approach to APC engineering demonstrates Cell Squeeze® technology's potential to leverage the modularity, cost-effectiveness, and streamlined manufacturing of multiple mRNAs to create cellular vaccines for treating various tumor and infectious disease targets. Citation Format: Michael F. Maloney, Emrah Ilker Ozay, Christian Yee, Amy Merino, Paul R. Dunbar, Mubeen Mosaheb, Kelly Volk, Carolyne Smith, Katherine J. Seidl, Howard Bernstein, Scott M. Loughhead. Cell Squeeze® delivery of antigen-encoding mRNA enables human PBMCs to drive antigen-specific CD8+ T cell responses for diverse clinical applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1523.

Antigen-specific CD8+ T cells are critical for mounting an effective immune response against tumors. Generation of antigen-specific T cells require interactions with multiple signals produced by antigen presenting cells (APCs). These signals are comprised of three components: (signal 1) the peptide-MHC complex binding to the T cell receptor, (signal 2) costimulatory molecules on the surface of APCs, and (signal 3) inflammatory cytokines binding to cognate receptors on T cells. To engineer all major cell subsets of human peripheral blood mononuclear cells (PBMCs) to become enhanced APCs (eAPCs), we used Cell Squeeze® technology to deliver multiple mRNAs encoding for non-self-antigens (signal 1), CD86 (signal 2), and/or membrane-bound cytokines (signal 3). The signal 3 molecules, membrane-bound IL-12 (mbIL-12) and membrane-bound IL-2 (mbIL-2), are chimeric proteins designed to increase the localized concentration of the cytokines at the immune synapse and limit off-target effects. Flow cytometry confirmed translation of delivered signal 2/3 mRNAs by all major subsets within PBMCs: T cells, B cells, NK cells, and monocytes. The potency of these SQZ™ eAPCs was assessed in vitro by culturing the eAPCs with antigen-specific T cells for multiple days before measuring the functionality of antigen-specific T cells via intracellular cytokine staining or ELISA. Using this approach, we demonstrate that Cell Squeeze® co-delivery of antigen mRNA and signal 2/3 mRNAs significantly enhances CD8+ T cell responses to a variety of antigens, including CMV pp65, Influenza M1, HPV16 E6, and HPV16 E7. Furthermore, we demonstrate that SQZ™ eAPCs drive significant expansion of antigen-specific CD8+ T cells in a humanized mouse model. Thus, we demonstrate that Cell Squeeze® can deliver multiple mRNAs encoding for signals 1, 2, and 3 to human PBMCs and has the potential to generate enhanced APCs that drive strong CD8+ T cell responses against multiple antigens. The versatility of this approach has the potential to enable rapid exchange of mRNA to encode for other antigens or T cell activation signals. Citation Format: Michael F. Maloney, Emrah Ilker Ozay, Katarina Blagovic, Carolyne Smith, Andrea A. Silva, Amber Martin, Sanjana Manja, Madhav Upadhyay, Lindsay J. Moore, Ryan Stagg, Henry Mack, Christine Trumpfheller, Pablo Umana, Armon Sharei, Howard Bernstein, Scott M. Loughhead. Co-delivery of antigen-encoding mRNA and signal 2/3 mRNAs to PBMCs by Cell Squeeze® technology generates SQZ™ eAPCs that prime CD8+T cells in a humanized mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2853.