T to Myeloid Lineage Reprogramming by the Synergistic Action of C/EBP and PU.1.

During hematopoiesis, transcription factors can not only direct differentiation of committed progenitors but also drive lineage commitment decisions from uncommitted multipotent progenitors. To further study their instructive role, we asked whether critical steps in the hematopoietic differentiation pathway could be reversed, and more specifically, whether myeloid transcription factors, C/EBP alpha and C/EBP beta, could re-direct committed T lymphoid progenitors into a myeloid fate. Previously in our laboratory, it was shown that C/EBP alpha efficiently reprograms CD19+ B cells to Mac1+ Gr1+ functional macrophages (Xie et al. (2004) Cell 117:663). To address lineage conversion of T to myeloid cells, we purified T cell subsets by MACS enrichment or FACS sorting and infected these cells with retroviruses expressing C/EBP alpha or C/EBP beta. Cells were cultured on myeloid supporting stroma with myeloid cytokines and analyzed for expression of antigens and marker genes by flow cytometry and RT-PCR. To rule out the presence of contaminating myeloid progenitors in our cultures, we started with a marked population of T lymphoid progenitors derived from Lck-Cre Rosa-EYFP reporter mice in which T cells are irreversibly marked with EYFP upon T cell commitment. T cell origin was further verified using PCR to detect TCRbeta DJ rearrangements. Using double negative DN3 and DN4 T cells, we show that a large proportion of EYFP+ DN cells infected with C/EBP alpha or C/EBP beta are induced to express surface antigens Mac1 and Gr1, express myeloid cytokine receptors and Fc receptors, and exhibit a myeloid morphology, compared to control infected cells. The reprogrammed cells are also functional as they can bind and phagocytose sheep red blood cells. In addition, the pan-T cell marker Thy1 and other T lineage genes including Lck, Rag2, and preT alpha are downregulated in reprogrammed cells. Furthermore, using a conditional knockout approach developed in Daniel Tenen’s laboratory, we show that endogenous expression of PU.1 is required for activation of Mac1 during this process. In conclusion, C/EBP alpha acts alone to extinguish the T lymphoid program while, in concert with PU.1, it activates the myeloid program leading to reprogramming of the T cell phenotype.