Validating human lymphoid organoids and explants as 3Rs methods to reduce and replace animal use in immunology research

Pablo’s award will build on the foundations of his NC3Rs infrastructure grant to demonstrate the broader applicability of human lymphoid organoid and lymphoid tissue precision cut explant models developed in his lab. He will further characterise the cellular and biochemical profiles of the models using single-cell RNA sequencing, high-dimensional flow cytometry and high content microscopy to generate an open database of multimodal datasets for other researchers to use. Pablo will also partner with four new end-user labs at the Universities of Dundee, Oxford and Muenster to test the transferability and portability of the organoid and explant models – through these collaborations the models will be applied to study key immunological processes, including B-cell germinal centre reactions, antibacterial immune response and T-cell activation and generation, replacing the need for mouse models. 

Our overall goal is to establish the use of lymphoid organoids and lymphoid tissue precision cut explants (hereafter explants) as essential toolkits for the reduction and replacement of animal models in immunology research. While lymphoid organoids enable the reorganisation of cell suspensions into follicle-like structures, lymphoid explants preserve tissue architecture and cell niches ex vivo. Both 3Rs models have shown promise in the discovery of novel immune mechanisms regulating B cell activation and germinal centre (GC) reactions. However, their broader adoption is rather limited and a body of evidence supporting their application in various immunological and non-immunological settings is lacking. Here, we have drafted a workplan separated in three different work packages and aims put together to 1) benchmark and validate these methods; 2) to test their transferability and portability, and promote their adoption, and 3) to disseminate the knowledge and secure an immediate and longer term 3Rs impact. In WP1/aim 1, we will validate, and benchmark lymphoid organoids and explants derived from the same organ and donor, undergoing the same stimulation regime throughout the same timescale. We will use antigens modelling known viral and bacterial infections affecting the upper respiratory tract and palatine tonsils as stimulation. We will use established multimodal outcome measures of adaptive immunity, such as frequencies of GC-B cells or activated follicular helper T cells (TFHs) and GC-TFHs. By tracking the kinetics of cell subset changes we will generate a comprehensive portfolio of single-cell RNA-sequencing (scRNA-seq) data, high-dimensional flow cytometry data, and high-content microscopy data. In WP2/aim 2 we will partner with four end-users from University of Dundee, University of Muenster and the University of Oxford to test the transferability of the methods. We will harness the multimodal database generated in WP1 to select new primary and secondary outcome measures (e.g., survival and expansion of PD-1High TFHs), adapt our high-dimensional flow cytometry panels and test independent hypotheses with either or both 3Rs models. Therefore, WP2 will deliver independent research articles providing varied, robust, high-dimensional and multimodal evidence building up confidence on the methods and motivating their wider adoption in immunology research. Finally, our WP3/aim 3 compiles our dissemination efforts. With the resources of this award, we will officially launch the lymphoid and organoid explant hub, first locally at the University of Oxford, with the continued delivery of online seminars and in-person workshops. As part of our wider dissemination goals, we will generate a resource article and a publicly available evidence base that will help others chose the best 3Rs model to test their unique hypotheses. To succeed, we will facilitate access to raw and processed data, metadata and standardised protocols to help others navigate experimental design and identify new primary outcome measures best fitted to test their independent hypothesis. By building up confidence in these 3Rs models, we therefore aim to encourage their adoption for the testing of a variety of questions in immunology and beyond, benefiting researchers globally.