Human and Mouse Artificial Lymph Nodes: Novel technology to Reduce and Replace the use of animal models in clinical and developmental immunology
Lead Research Organisation:
University of York
Department Name: Biology
Abstract
The treatment of autoimmune and inflammatory disease has a major impact on the NHS and society and is estimated to cost the UK over 20 billion pounds per year. New treatments are required to treat these debilitating disorders. Currently, the development of novel therapeutics involves using rodent models of the disease and testing in non-human primates before initiating clinical trials in humans. This process utilises large numbers of animals, is expensive and is not necessarily very informative in how a treatment might work in humans. In this proposal in collaborate with a pharmaceutical manufactoring and testing organisation, ProBioGen Ag, we will develop and utilise a new technology we have termed an artificial human lymph nodes to replicate the human immune system in a test tube. This system will allow replacement of animals in the drug development process and reduction in animal numbers required to do basic research into how the immune system develops and functions.
Technical Summary
Lymph nodes have an essential role in the initiation of protective immune responses. In this proposal we will utilise human and mouse adipose derived stem cells (ADSCs) and peripheral blood lymphocytes and dendritic cells to generate artificial lymph node (ALN) structures in vitro. This represents a novel approach to the analysis of human immune function and mouse lymph node development, organisation and function. This technology will have key applications in toxicology and mechanistic studies of new therapeutics and as a tool to determine the role of specific signalling pathways and physical forces in LNs, thus replacing and reducing animals in research.
The development and application of human monoclonal antibody (mAb) to treat disease requires determination of toxicology and efficacy and understanding the mechanism of action. The current approach utilises large numbers of mice and non-human primate models and surrogate antibodies that are not the best predictor of human immune system. Through the development of ALNs we will generate a technology to address key questions on mAb safety and efficacy in vitro replacing and reducing mouse and non-human primates in therapeutic development. We have developed a methodology to differentiate ADSCs into T and B cell lymphoid stroma. ProBioGen has developed bioreactor technology that can maintain the survival and function of human lymphocytes over 4 weeks in culture. In this research program we will bring the two technologies together to construct a human ALN that contain all the key cell types and organisation found in vivo in highly structured 3D culture system. With the joint expertise in stromal cell culture, human immunology, 3D culture systems, bioreactors and measurements of immune responses in vitro we believe we can not only develop the technology to assay and visualise human immune responses in vitro but can commercialise the culture system, directly reducing the use of animals in drug development
The development and application of human monoclonal antibody (mAb) to treat disease requires determination of toxicology and efficacy and understanding the mechanism of action. The current approach utilises large numbers of mice and non-human primate models and surrogate antibodies that are not the best predictor of human immune system. Through the development of ALNs we will generate a technology to address key questions on mAb safety and efficacy in vitro replacing and reducing mouse and non-human primates in therapeutic development. We have developed a methodology to differentiate ADSCs into T and B cell lymphoid stroma. ProBioGen has developed bioreactor technology that can maintain the survival and function of human lymphocytes over 4 weeks in culture. In this research program we will bring the two technologies together to construct a human ALN that contain all the key cell types and organisation found in vivo in highly structured 3D culture system. With the joint expertise in stromal cell culture, human immunology, 3D culture systems, bioreactors and measurements of immune responses in vitro we believe we can not only develop the technology to assay and visualise human immune responses in vitro but can commercialise the culture system, directly reducing the use of animals in drug development
Planned Impact
The development of human and mouse artificial lymph nodes will permit specific reductions in animal usage in developmental studies and the replacement of animal models (rodent and non-human primates) in the testing and development of new therapeutics targeting immune system function.
Replacement: Animal models are currently used to develop and test new therapeutics targeting autoimmune and inflammatory disease pathologies. They also are used to determine immune activation by novel drugs and cosmetics. There is an urgent requirement for new tests as animal models in cosmetic safety testing are being phased out in 2013. We estimate that this system might lead to a 10-20% decrease in the use of animals in immune-therapeutic development and toxicology screening. We are particularly hopeful that the technology will eventually significantly reduce the use of non-human primates for the testing of human monoclonal antibody therapies as the hALN can replicate the complexity of the in vivo immune system. This new research and development tool will have a big impact on the wider research community and industry. We will work closely with GSK (Fund 2 BBSRC CASE studentships in MC's laboratory) and MedImmune (STROMA ITN partner) to incorporate the assay into their development pipeline. ProBioGen will take lead in the commercialisation of the technology as they have both the facilities and experience to develop the technology for industrial application.
Refinement: Understanding mechanisms of lymphoid tissue development has involved the development of increasingly complex tissue specific knock-out mice. The process of generating sufficient embryos to do meaningful experiments requires large numbers of breeding cages resulting from the complex genetics and very inefficient generation of embryos. Through use of mouse ADSCs one mouse is sufficient to generate sufficient ADSCs for large numbers of experiments. By obtaining fat pads from flox/flox mice we can inducibly delete alleles using adenovirus Cre permitting analysis of signalling pathways in lymphoid tissue formation based on 1-3 mice without having to cross to tissue specific cre. By collaborating with colleagues we will not have to import or breed new mouse lines in York, fat can be isolated and transported to York for ADSC isolation significantly reducing the numbers of mice required to study lymphoid tissue development. Additionally it is possible to use lentivirus shRNAs to knock-down genes in ADSCs widening the potential number of genes that can be targeted in developmental studies. This approach will lead to a ~50% reduction in the number animals used in experiments in MC laboratory group as we will not need to import new mice lines and cross to stromal cell specific cre recombinase mice. This will lead to a reduction of >1000 mice per year. We believe this methodology is highly applicable to studying the role of mesenchymal stromal cells in development, organisation of a number of different tissues and will thus have a significant impact on the greater research community.
Replacement: Animal models are currently used to develop and test new therapeutics targeting autoimmune and inflammatory disease pathologies. They also are used to determine immune activation by novel drugs and cosmetics. There is an urgent requirement for new tests as animal models in cosmetic safety testing are being phased out in 2013. We estimate that this system might lead to a 10-20% decrease in the use of animals in immune-therapeutic development and toxicology screening. We are particularly hopeful that the technology will eventually significantly reduce the use of non-human primates for the testing of human monoclonal antibody therapies as the hALN can replicate the complexity of the in vivo immune system. This new research and development tool will have a big impact on the wider research community and industry. We will work closely with GSK (Fund 2 BBSRC CASE studentships in MC's laboratory) and MedImmune (STROMA ITN partner) to incorporate the assay into their development pipeline. ProBioGen will take lead in the commercialisation of the technology as they have both the facilities and experience to develop the technology for industrial application.
Refinement: Understanding mechanisms of lymphoid tissue development has involved the development of increasingly complex tissue specific knock-out mice. The process of generating sufficient embryos to do meaningful experiments requires large numbers of breeding cages resulting from the complex genetics and very inefficient generation of embryos. Through use of mouse ADSCs one mouse is sufficient to generate sufficient ADSCs for large numbers of experiments. By obtaining fat pads from flox/flox mice we can inducibly delete alleles using adenovirus Cre permitting analysis of signalling pathways in lymphoid tissue formation based on 1-3 mice without having to cross to tissue specific cre. By collaborating with colleagues we will not have to import or breed new mouse lines in York, fat can be isolated and transported to York for ADSC isolation significantly reducing the numbers of mice required to study lymphoid tissue development. Additionally it is possible to use lentivirus shRNAs to knock-down genes in ADSCs widening the potential number of genes that can be targeted in developmental studies. This approach will lead to a ~50% reduction in the number animals used in experiments in MC laboratory group as we will not need to import new mice lines and cross to stromal cell specific cre recombinase mice. This will lead to a reduction of >1000 mice per year. We believe this methodology is highly applicable to studying the role of mesenchymal stromal cells in development, organisation of a number of different tissues and will thus have a significant impact on the greater research community.