The differentiation of effector CD8+ T cells from their naive precursors requires global reprogramming of gene expression and broad changes in the chromatin state of the cell. We have recently shown that AP-1 family transcription factor (TF) BATF is absolutely required for effector CD8 T cell differentiation. Our data suggest that regulation by BATF is an initiating event in the reprogramming of naive CD8 T cells into effector cells. However, the exact mechanism by which BATF regulates the switch to effector differentiation is not known. During the development of other tissues, a subset of TFs known as pioneer factors initiate differentiation. Pioneer TF are able to bind to regions of chromatin tha are inaccessible to other TFs and transcriptionally silent because of tight compaction of the DNA around nucleosomes. Pioneer factors are required to 1) initiate development; 2) open the chromatin, and 3) allow the subsequent binding of other TFs that activate gene expression. In this proposal, we will test the hypothesis that BATF regulates effector CD8+ T cell differentiation as a pioneer factor, acting early in effector differentiation, to increase chromatin accessibility and allow other TFs to bind to their target regions.
In Aim 1 we will ask if BATF is required only to transiently initiate effector differentiation or also to permanently maintain the effector and memory CD8+ T cell state. We will test whether continued BATF expression is also required to maintain effector and memory differentiation, using a series of inducible and conditional knockout studies in vivo.
In Aim 2 we will test the hypothesis that BATF is required to alter chromatin accessibility in developing effector CD8 T cells. We will assay chromatin accessibility and active chromatin modifications in WT and Batf-/- CD8 T cells during effector differentiation, using a novel assay of chromatin accessibility, ATAC-Seq. Furthermore will test whether enforced expression of BATF or other TFs that regulate effector differentiation (Tbet, Eomes, Blimp1, Id2, Runx3) can restore chromatin accessibility in Batf-/- CD8 T cells.
In Aim 3, we will test whether BATF binding overlaps with other effector TF binding and whether BATF enhances their binding. We will assay TF occupancy by ChIP-seq for a set of key effector TFs (Tbet, Eomes, Blimp1, Id2, Runx 3) in WT and Batf-/- effector CD8+ T cells and test whether these TFs bind at regions that overlap with BATF. If BATF is a pioneer we would expect that the binding of these TFs would be reduced at regions of shared binding in Batf-/- effector CD8 T cells. Finally we will test if normal effector differentiation can be achieved in Batf-/- CD8+ T cels by enforced expression of Tbet, Eomes, Blimp1, Id2 or Runx3. These experiments will establish the mechanism by which BATF operates and create the definitive map of transcriptional regulation of effector CD8 T cells. Such a map will be essential to understand how to optimally generate effector T cells in response to vaccines and engineer T cells for adoptive immunotherapy.
During the immune response to infection, a set of immune cells called effector CD8 T cells develop in a process that involves immense changes to their function and gene expression. This project will determine whether a key transcription factor called BATF acts to initiate this developmental process by serving as a 'pioneer' transcription factor. Understanding how this crucial part of the immune response is governed will help identify how to make vaccines and immunotherapies more effective.
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