Kathryn Haskins: T cell lines to study autoimmunity, Transgenic Mice to study diabetes onset, and Translation to Human Patients
by Adam Burrack, PhD
Relevant literature:
Diabetes 37(10): 1444-1448, PNAS 86(20): 8000-8004, Nature Immunology 11(3):225-231, Diabetes 60(9): 2325-2330, and Journal of Autoimmunity 50:38-41
In previous posts in our ‘immunology of type 1 diabetes’ series I have described T cells of the immune system as key players in the autoimmune response that destroys insulin-producing beta cells in the pancreas. I’ve also described the Barbara Davis Center for Childhood Diabetes in Denver, Colorado, as one of the critical loci of both clinical and basic science research on the pathogenesis of the disease. In this edition I will describe a series of critical discoveries by Katie Haskins, PhD, describing a set of tools that have greatly aided the study of autoreactive T cell responses in general and the pathogenesis of type 1 diabetes in particular. One of these tools are autoreactive T cells isolated from the spleen of diabetic NOD mice. These cells facilitate the study of the exact same autoreactive T cells in cell culture, over time, in different laboratories around the world. An additional tool derived from these T cells are TCR transgenic mice, which are a type of genetically modified mouse used for biomedical science research. These mice are engineered to produce only one T cell in their thymus and provide an animal model in which to study the behavior of specific T cells in different health and disease settings.
In a seminal paper in the journal Diabetes in 1988, Dr Haskins described the derivation of a T cell line that was associated with development of diabetes in the mouse model of type 1 diabetes. This T cell line provided researchers, for the first time, with a critical tool to investigate the cell biology and regulation of autoimmunity in an animal model. This T cell line was named the BDC2.5 T cell, for the institution where it was discovered and the round of limiting dilution assay which isolated the T cell after isolation of cells from the spleen. The BDC2.5 T cell has been studied by dozens of researchers around the world investigating the biology of T cells, the sensitivity of the T cell receptor to the presence of its specific target, and the biology of type 1 diabetes. Since the initial characterization of the cell line, the BDC2.5 has been a true work-horse in the fields of T cell biology and autoimmune diabetes.
In a subsequent manuscript in the journal of the National Academy of the United States in 1989, Dr Haskins described several additional T cell lines generated from the spleens of diabetic NOD mice. All of these T cell lines derived from the spleen and lymph nodes of NOD mice exhibited the hall-mark behavior of autoreactive T cells: the cells strongly responded to (underwent several rounds of division and produced effector molecules) the presence of pancreatic islets, including the ability to transfer disease. When these cells are injected into a mouse lacking its own immune system for genetic reasons, these cells will specifically attack insulin-producing beta cells, precipitating the development of type 1 diabetes. This follow-up paper validated the methods of the initial approach and described several additional T cell lines which have been useful cell biology reagents for multiple laboratories.
Despite the straight-forward process used by Dr Haskins to initially locate and characterize these autoreactive CD4+ T cells, determining what they specifically respond to proved to be a daunting challenge of >20 years. After several false starts and much debate over the years as to whether the BDC2.5 T cell receptor was relevant to disease, was a regulatory T cell or a pathogenic T cell, Dr Haskins recruited a chemist with expertise in separation of proteins by chromatography. Dr Haskins and her post-doctoral researcher Thomas Delong used this technique to determine what peptides the BDC2.5 T cell line was responding to, and determined the target of this response to be a peptide derived from chromagranin A molecule. The chromagranin A molecule is present within the secretory granules that insulin is packaged within before the entire granule is expelled from the beta cell into the circulation. This discovery answered a >20 year mystery and demonstrated that while insulin was a critical target of the autoreactive T cell response (see our post about the career of George Eisenbarth), it was not the only peptide target which could precipitate a CD4 T cell response which promotes autoreactivity and the development of type 1 diabetes in the mouse model. A subsequent paper with clinical collaborations at the BDC and other institutions has demonstrated that chromagranin A is a relevant target in human subjects with type 1 diabetes, validating the NOD mouse and the BDC2.5 T cell as clinically relevant research reagents.
As mentioned above, the BDC2.5 T cell line was not the only T cell that Dr Haskins and colleagues isolated from diabetic NOD mice in the late 1980’s. In her 1989 paper Dr Haskins described several T cell lines, all of which “responded” to the presence of pancreatic islets by producing effector molecules called cytokines. Cytokines can be thought of as broadly similar to hormones (the primary role of hormones is to instruct other cell types how to behave during growth and development), but more specific for cells of the immune system. In this role, cytokines are critical to both initiating and concluding immune system responses against pathogens. The cytokines produced by BDC T cells, following recognition of the peptides against which they specifically respond, promote strong immune responses against insulin-producing cells. These immune responses are exquisitely specific: beta cells which produce insulin are destroyed but alpha cells which produce glucagon, physically located immediately adjacent to beta cells, are left intact and functional.
Using similar techniques to their 2010 paper in Nature Immunology, two post-doctoral researchers from Dr Haskins’ group determined the peptide against which another of the BDC T cell panel responds in a 2011 publication in the journal Diabetes. For this manuscript, Rocky Baker worked closely with Thomas Delong to determine the specific target of the BDC5.2.9 CD4 T cell. Rocky and Thomas determined that the BDC5.2.9 T cell specifically responds against a protein derived from a different insulin secretory granule protein, islet amyloid polypeptide (IAPP), with insulin-derived peptide a close second when they quantified the cytokine response. A current direction of the Haskins laboratory is determining the peptide specificity of the other CD4 T cells in the BDC panel. This work is critical because determining the targets of the autoreactive response provides therapeutic windows for researchers to attempt to re-establish ‘tolerance’ of the immune to targets against which there should never have been an immune response. It is likely that no single peptide target will provide a ‘magic bullet’ to reverse disease state (with the possible exception of insulin), rather that tolerance will have to re-established to a broad range of insulin granule-related proteins to re-educate the immune about what is ‘self’. Knowledge of the peptides targeted by autoreactive T cells allows researchers to design therapies with the goal re-establishing immune tolerance to those specific peptides while not perturbing the broader immune response to bacteria, viruses, parasites, or malignancies. Dr Haskins discoveries in this field make this type of strategy feasible.