Generation of regulatory T cells by reprogramming

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.

Our goal in this project is to develop efficient methods for making a class of immune-system cells known as regulatory T cells (Tregs), that have the potential to be useful in a wide variety of clinical situations. Tregs control the harmful immune responses seen in patients with autoimmune diseases including childhood (Type I) diabetes, rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease; they also suppress rejection of transplanted organs in patients given heart, liver or kidney transplants. We proposed to ask whether three proteins known as TET proteins, recently discovered in our laboratory, had a role in Treg development and function. We also proposed to develop efficient ways to make Tregs from different types of stem cells. With regard to the second objective, we have now made the interesting discovery that mice lacking two of the three Tet proteins develop an enlarged spleen and other features which suggest that they possess diminished numbers of functional Tregs. This will allow us to understand Treg development and function at a molecular level. With regard to the first objective, we are examining the entire set of transcripts present in different forms of Tregs and their precursors through next-generation sequencing of RNA. We anticipate that introduction of combinations of these factors into precursor cells of various types will lead to the successful generation of Tregs in cell culture. Although our research will be done mostly in animals (mice), we believe that it will be possible to translate it quickly into humans, and that if successful, it will address a pressing clinical need. For patients receiving transplanted organs, we hope to take their own normal T cells and turn them into Tregs. For patients suffering from autoimmune diseases, it would be useful to make Tregs artificially from their bone marrow stem cells, whereas for cancer patients who have been given chemotherapy followed by a bone marrow transplant from a different person, it would be useful to make Tregs from the same bone marrow cells that the patient receives, in the hope that these Tregs can suppress graft-versus-host disease. We believe that all these approaches are feasible in principle, given recent discoveries from our own and other labs.

In addition to the four main letters (bases) in the DNA alphabet – A, C, G and T – there is also a ‘fifth’ base known as 5-methylcytosine (5mC) that has a disproportionately crucial role, despite the fact that it is expressed at quite low levels in cells. 5mC is important in the development of the embryo, in the expression of genes, in brain function and in cancer. It was originally thought that 5mC was a very stable and unchanging mark, but our recent discovery that a class of proteins known as TET proteins converts 5mC to derivatives known as oxidized methylcytosines – 5hmC, 5fC and 5caC, thus proving that 5mC can indeed be altered dynamically. TET proteins and oxidized methylcytosines have important roles in embryonic development, normal cellular functions, and the properties of nerve cells in the brain. They also have a major role in the reprogramming of mouse fibroblasts into induced pluripotent stem (iPS) cells, which have great potential to treat many human diseases. Mutations in TET proteins lead to developmental abnormalities, infertility, genetic diseases and cancer. In our CIRM project, we had proposed to discover the role of TET proteins in a class of cells of the immune system known as T cells, and especially in a subclass of these known as regulatory T cells (Tregs). T cells are important because they are involved in fighting bacterial and viral infections as well as cancers; among their harmful features, however, is that they also reject transplanted organs and may cause “autoimmune” responses to the body’s own constituents and unnecessarily overactive responses to harmless components of the environment such as those that provoke food allergies and asthma. Tregs oppose the actions of normal T cells, and so are useful in preventing autoimmune disease and suppressing allergies and asthma, but are harmful since they also prevent the rejection of cancer cells by the immune system. TET proteins have been associated with human cancers, and we have now mimicked this process in mice. By generating mice that lack two of the three TET proteins, we found that these mice develop a striking and very rapidly progressing cancer that involves the uncontrolled growth of certain immune system cells. The mice may also possess diminished numbers or functions of Tregs. Further study of these mice should allow us to understand the role of TET proteins in Treg function as well as in the development of T cell leukemias and lymphomas. In addition, we have looked at the distribution of 5hmC during the development of normal T cells and Tregs in an organ known as the thymus. We find that the levels of 5hmC in genes correlate strongly with the degree to which those genes are expressed. We also find that during T cell and Treg development, 5hmC levels change rapidly at certain genes before the genes are most highly expressed. These are important new findings that may relate to the role of 5hmC in normal development as well as in cancer cells. Although our research has been performed mostly in mice, we believe that it will be possible to translate it quickly into humans, and that if successful, it will address some pressing clinical needs. For patients receiving transplanted organs, and for cancer patients who have been given chemotherapy followed by a bone marrow transplant from a different person, we may be able to build on our understanding of the role of TET proteins in Treg development to generate Tregs more efficiently. Our analysis suggests that these Tregs would also be more stable and more effective in suppressing rejection of the organs or the bone marrow transplant. In contrast for patients suffering from autoimmune diseases, or whose cancers have arisen due to problems with TET protein function, we might be able to increase Treg function and/or suppress cancer cell growth by increasing the activity of TET proteins in the Tregs and cancer cells. We believe that all these approaches are feasible in principle, given recent discoveries from our own and other labs.