Statement of Benefit to California:
Year 1A prominent subset of white blood cells, named CD4 helper T cells, are critical in modulating the immune response against viral and bacterial pathogens. During HIV infection, the CD4 compartment is selectively reduced, suppressing the activity and response of cytolytic CD8 T cells, needed to abolish cells infected with the virus. Pharmaceutical therapies have been developed but they are not consistently effective and multidrug resistant viral strains are increasingly prevalent. Similarly, in vitro manipulated human dendritic cells are now being explored to tolerize against autoimmune disease or to stimulate antitumor responses. However, the number of dendritic cells that can be isolated form patients using current technologies is small. Consequently, different approaches need to be developed to enhance T cell reconstitution. In principle, multipotent hematopoietic progenitors could be derived from hESCs without long-term in vitro culture. A drawback is that the number of human hematopoietic progenitors derived from human ES cell cultures is limited using current culture conditions. Consequently, a subset of studies involving in vitro manipulated human cells would be difficult to perform. The transduction of human progenitor cells can be achieved using a tissue culture system in which human cord blood progenitors are differentiated in the presence of stromal cells that express the Notch ligand DL-1 towards the T cell lineage. However, the efficiency by which human progenitor cells differentiate into the T lineage cells is low. In the original application we proposed to develop a novel strategy that would permit the generation of large numbers of human T cell progenitors (up to 109) from human hematopoietic stem cells. To accomplish this objective we would target a critical regulator of early hematopoieisis, named E2A. Indeed during the two years period funded by CIRM we have demonstrated that murine hematopoietic progenitors that overexpress an inhibitor of E2A, named Id2, can be grown indefinitely in culture without losing their ability to generate many different types of white blood cells in the laboratory. This strategy is unconventional since it would permit the growth and isolation of large numbers of T cell progenitors, which has not been achieved so far by conventional culture conditions. We will continue these studies and use the same strategy to establish a long-term culture of human hematopoietic progenitor cells. If successful the approach would enable clinicians to reconstitute the hematopoietic compartments of patients carrying invading pathogens, including HIV infected patients, with large numbers of T cells that either express either a wild-type TCR repertoire or TCRs with specificities directed against invading pathogens. I expect this to succeed since we have already achieved this objective using murine progenitors as demonstrated during the past two years using funds obtained form the CIRM.