Michael Cooke, Ph.D.
Director of Immunology
Immunology at GNF
Our Immunology Department aims to identify critical regulatory molecules that govern immune function. In collaboration with lead discovery scientists at GNF and Novartis, we develop these molecules into novel therapeutics that aim treat a variety of immune disorders, including asthma, allergies, arthritis, and dermatitis. We also have an active program that aims to find ways to prolong the survival of organ and bone marrow transplants and improve the outcome of transplantation.
With programs in innate immunity, adaptive immunity, and hematopoietic stem cell biology, our department brings to bear many of the advanced technologies and resources available at GNF, including whole genome-scale RNA expression profiling, high-resolution proteomics, large-scale arrayed cDNA and siRNA libraries, and forward and reverse genetics approaches in the mouse.
Goals/Aims of the research
A more complete understanding of the genes governing immune cell function is a first step towards developing new drugs to treat autoimmune diseases such as Type 1 diabetes and rheumatoid arthritis and new immunosuppressive drugs that will help transplanted organs survive. Our laboratory is looking for these genes by investigating the mechanisms that control lymphocyte development, lymphocyte activation, and hematopoiesis.
The Use of Large-Scale RNA Expression Profiling to Identify Candidate ImmunoRegulatory Genes
GNF has compiled a comprehensive RNA expression atlas of all predicted mouse and human genes across more than 100 tissues. In addition, our scientists have studied the in vitro and in vivo activation of several types of highly purified immune cells (e.g., T cells, B cells, mast cells, and hematopoietic stem sells). By mining these data for genes that are enriched in the immune system, we have identified a variety of potential immunoregulatory genes, and we are now looking for the functions of many of these candidate genes. Some of the methods we employ include using viral vectors to alter their expression via cDNA overexpression and applying siRNA knockdown coupled with in vitro or in vivo assays of immune function.
Hematopoietic Stem Cells
The goal of our hematopoietic stem cell (HSC) project is to find methods to prevent HSC differentiation during in vitro culture and understand the molecular signals that determine whether a hematopoietic stem cell will choose to differentiate or self renew. To this end, we have performed RNA expression profiling of highly purified mouse and human HSCs and their progeny cells to identify HSC-enriched genes. We are now testing the role of these candidate genes using retroviral and lentiviral vectors to modulate gene expression.
One interesting aspect of hematopoietic stem cells is that their number and activity varies greatly between inbred strains of mice. In collaboration with Dr Gerald de Haan at the University of Groningen in The Netherlands, we have combined both classical genetics (QTL mapping) and RNA expression profiling of purified HSCs from 22 different recombinant inbred strains of mice and have identified several genes and loci that control HSC function.
A second area of interest is exploring the mechanisms that control stem cell differentiation and self-renewal. Insights into how to influence stem cell fate decisions will have a major impact in the field of bone marrow transplantation and may provide important in vitro model systems for the identification of genes and compounds that can be used to regulate the process of stem cell differentiation.
Phenotypic and Functional Screening of Mutant Mice
GNF scientists have used random mutagenesis to create a panel of mutant mice for phenotypic screening. We screen mice with altered immune function using, for instance, flow cytometry to analyze lymphoid subsets in the blood, hematological analysis to determine the absolute number of red cells, white cells and platelets, and antibody levels following immunization.
We have established ~50 mouse lines with a wide range of immune and hematological disorders, including anemia, thrombocytosis, thrombocytopenia, T-cell or B-cell lymphopenia, and altered antibody responses. Once we have established these lines, we identify the genetic mutations responsible for the observed phenotypes using a GNF-specific genotyping panel. Using this approach, we have cloned causative genes for more than 20 mutant lines.
Recently we identified of a novel viable allele of the transcription factor c-Myb, which alters the development of several hematopoietic lineages and gives rise to 10-fold increases in the number of hematopoietic stem cells. Current follow-up work focuses on identifying how c-Myb regulates HSC self-renewal and differentiation.
We have also identified a critical role for the IP3 Kinase ITPKb, which converts IP3 to IP4, in lymphocyte development and activation. Mice lacking ITPKb have a 50-fold reduction in their number of peripheral T cells, resulting from a block in T cell positive selection in the thymus.
In collaboration with the protein sciences group at GNF we have used x-ray crystallography to determine the structure of this inositol kinase. These studies reveal that despite having no sequence identity to protein kinases, ITPKb has a clearly recognizable kinase fold with an inserted IP binding domain, which suggests a potential rationale for the activation of ITPKb by Ca2+/CaM. Our current work focuses on determining how Itpkb regulates lymphocyte development and activation and characterizing additional mutants identified using ENU mutagenesis.
- Sandberg ML, Sutton SE, Pletcher MT, Wiltshire T, Tarantino LM, Hogenesch JB, Cooke MP. c-Myb and p300 regulate hematopoietic stem cell proliferation and differentiation. Dev Cell 2005;8(2):153-66.
- Bystrykh L, Weersing E, Dontje B, Sutton S, Pletcher MT, Wiltshire T, Su AI, Vellenga E, Wang J, Manly KF, et al. Uncovering regulatory pathways that affect hematopoietic stem cell function using 'genetical genomics'. Nat Genet 2005;37(3):225-32.
- Wen BG, Pletcher MT, Warashina M, Choe SH, Ziaee N, Wiltshire T, Sauer K, Cooke MP. Inositol (1,4,5) trisphosphate 3 kinase B controls positive selection of T cells and modulates Erk activity. Proc Natl Acad Sci U S A 2004;101(15):5604-9.
- Aza-Blanc P, Cooper CL, Wagner K, Batalov S, Deveraux QL, Cooke MP. Identification of modulators of TRAIL-induced apoptosis via RNAi-based phenotypic screening. Mol Cell 2003;12(3):627-37.