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Analysis of bacterial gene function commonly relies on gene disruption or replacement followed by phenotypic characterization of the resulting mutant strains. Deletion or replacement of targeted regions is commonly achieved via two homologous recombination (HR) events between the bacterial genome and a nonreplicating plasmid carrying DNA fragments flanking the region to be deleted. The counterselection of clones that have integrated the entire plasmid in their genome via a single HR event is crucial in this procedure. Various genetic tools and well-established protocols are available for this type of mutagenesis in model bacteria; however, these methods are not always efficiently applicable in less established systems. Here we describe the construction and application of versatile plasmid vectors pREDSIX and pTETSIX for marker replacement and markerless mutagenesis, respectively. Apart from an array of restriction sites optimized for cloning of GC-rich DNA fragments, the vector backbone contains a constitutively expressed gene for mCherry, enabling the rapid identification of clones originating from single or double HR events by fluorescence-assisted cell sorting (FACS). In parallel, we constructed a series of plasmids from which gene cassettes providing resistance against gentamicin, kanamycin, hygromycin B, streptomycin and spectinomycin, or tetracycline were excised for use with pREDSIX-based marker replacement mutagenesis. In proof-of-concept mutagenesis experiments, we demonstrated the potential for the use of the developed tools for gene deletion mutagenesis in the nitrogen-fixing soybean symbiont Bradyrhizobium diazoefficiens (formerly Bradyrhizobium japonicum) and three additional members of the alphaproteobacteria. IMPORTANCEMutation and phenotypic analysis are essential to the study of gene function. Efficient mutagenesis protocols and tools are available for many bacterial species, including various model organisms; however, genetic analysis of less-well-characterized organisms is often impaired by the lack of efficient methods. Here we describe a set of novel genetic tools for facilitated mutagenesis of the nitrogen-fixing soybean symbiont Bradyrhizobium diazoefficiens and related alphaproteobacteria. We demonstrated their usefulness by generating several mutant strains lacking defined genes. Isolation of both antibiotic resistance gene-containing and markerless deletion mutants is greatly facilitated because undesired clones which contain the entire mutagenic plasmid integrated in the genome can be identified on the basis of their fluorescent phenotype derived from the mCherry gene carried by the vector backbone. The possibility to generate markerless mutants assists with the isolation of strains carrying multiple deletions, which can be crucial while studying functionally redundant genes. Many functional studies of biological phenomena rely on the isolation of mutants and the availability of the respective genetic tools. Mutants are preferably constructed in a targeted manner, which traditi...

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Background Immune checkpoint inhibitors provide a new hope for cancer patients not responding to chemotherapy by removing the tumor protection against immune cell attacks. Nevertheless, only a subset of patients responds well to these therapies, and recent studies demonstrated that the mechanisms of action are more complex than expected, often involving multiple cell types of the immune system. Importantly, for a better understanding and assessment of immune cell eliciting cell tumor death, there is a need to have fully human, complex 3D in vitro models. We developed a 3D tumor-immune cell coculture model and show here acquired insights on the modulatory capacity of immune checkpoint inhibitors. Aim In the present study we have developed a 3D tumor - immune cell coculture model and evaluated its application to study the effect of immune checkpoint inhibitors on tumor viability, cytokine secretion, and immune cell infiltration. Material & Methods GFP-labelled A549 tumor cells were aggregated together with human dermal fibroblast to produce 3D tumor microtissues in Akura 96-well and 384-well format. Immune cells were pre-treated using different activation protocols to induce priming and pT-cell exhaustion. 3D tumor-immune cell cocultures were treated for 10 days with Nivolumab, its corresponding isotype and non-treated controls. Tumor viability was measured by GFP-fluorescence over time. Additionally, we have evaluated the effect of nivolumab by measuring the microtissue size by automatic stage fluorescence microscopy. Cytokine levels of IL-2, TNFα, IFNγ and GS-CSM were measured with a MAGPIXTM Luminex system and were monitored over time. The validation of immune cell infiltration and characterization of tumor and immune cell specific markers was based on histological analysis. Results Based on tumor fluorescence we have shown a superior effect of Nivolumab versus its isotype control antibody on tumor viability at the end of the treatment period. The cytokine analysis of supernatants has shown higher levels of IFNγ and GS-CSM in the wells treated with Nivolumab compared to the isotype control. CoCultures with PBMCs have shown stronger activation upon treatment compared to the T-cells alone. The histological analysis has demonstrated a higher number of T-cell infiltration and activation, as well as a stronger induction of apoptosis in the tumor microtissues treated with Nivolumab. Conclusion In summary, our novel in vitro immuno-oncology model system allows high throughput screening of immune check point inhibitors alone and in combination with other anti-cancer drugs for making it efficient for a broad range of tumor diseases. Citation Format: Nicole Buschmann, Silvan Strebel, Francesca Chiovaro, Patrick Guye, Irina Agarkova. Development of an in vitro 3D model system for testing PD-1/ PDL-1 immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 57.