Generation of regulatory T cells by reprogramming

Year 1

Our overall objective in this grant was to investigate the function of a class of T cells, known as regulatory T cells or Tregs, that damp down immune function. There is hope that when given to patients, Tregs would prevent rejection of haematopoietic stem cell and other stem cell transplants in humans, and could also be used to prevent autoimmune diseases such as Type 1 diabetes, rheumatoid arthritis and multiple sclerosis. It is known that a minor chemical modification – addition of a methyl group to DNA – has surprisingly large effects on gene expression during normal development as well as in stem cells and cancer cells. We recently discovered a small family of enzymes, known as TET proteins, which change the methyl group by successively adding oxygen to it. There is much evidence that the presence or absence of these additional modifications affect stem cell function and cancer cell growth. Our first objective in this grant, therefore, was to test the importance of TET proteins in Treg function. To this end, we developed mouse strains that lack a single Tet protein, and analysed their immune function. We find that mice that lack two of the proteins, Tet2 and Tet3, have impaired development not only of Tregs, but also of cells that produce a pro-inflammatory cytokine (hormone) known as interleukin 17. However the expression of another pro-inflammatory cytokine, interferon-gamma, is increased. These experiments were performed in the test-tube using T cells taken out of the mouse, and we will need to perform additional experiments to determine how Tregs lacking Tet2 or Tet3 will function during an actual immune response. During the coming year, we will perform these experiments, and also breed the Tet2- and Tet3-deficient mice together to generate and analyse mice that lack both Tet proteins. Our second objective was to develop methods for efficient Treg generation by “reprogramming” murine and human T cells, haematopoietic stem cells (HSC), embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC). We proposed to optimize methods for Treg generation by introducing (“transducing”) combinations of transcription factors into these different types of precursor cells, and monitoring the transduced cells for expression of the transcription factor FOXP3 (characteristic of Tregs). We have a small list of likely transcription factors that have been cloned into the appropriate vectors and are ready to introduce into the precursor cells. In addition, we are conducting next-generation sequencing experiments on RNA obtained from different types of Tregs, so as to identify additional candidate transcription factors that would induce reprogramming of the precursor cells to Tregs. The results should be available shortly, and we expect to initiate the reprogramming experiments next year. Our third objective was to test the function of the reprogrammed Tregs in mouse models of transplant rejection and autoimmune disease. These experiments ensure that the reprogramming creates Tregs whose function is stable in the mice, and that suppress immune responses effectively. We have performed many test experiments and are ready to perform these studies once efficient reprogramming is achieved.