PROJECT SUMMARY Regulatory T cells (Tregs) are lymphocytes that constitute ~5% of CD4+ T cells which suppress the activation of other immune cells. While there is a detailed transcriptional understanding of T cell development, including the development of Tregs, historically there has been little understanding of the translational regulation of T cell lineage commitment and function, including that of Tregs. We recently demonstrated that Treg development involves a novel, specialized translation mechanism carried out by a poorly investigated translation factor complex we termed DAP5/eIF3d, that is independent of canonical mTORC1 and eIF4E. DAP5/eIF3d is an alternate mechanism of cap-dependent mRNA translation which we discovered and recently showed is unique to Tregs among T cell types. This work lays a foundation for studies in this renewal application to understand how the novel DAP5/eIF3d mechanism controls Treg cell plasticity, function and stability, and integrates other molecular regulatory events in Treg development and function. We hypothesize that DAP5/eIF3d specialized mRNA translation is required for the differentiation of Tregs (tTregs, pTregs, iTregs) and involves specific m6A modification of Treg mRNAs. Studies are described in this application to now more fully characterize how DAP5/eIF3d specialized mRNA translation regulates Treg lineage commitment, the molecular mechanisms by which Treg mRNAs are selectively translated, including by an m6A- DAP5/eIF3d mechanism. There is currently little understanding of translational regulation of Tregs, and no understanding of the translational regulation of exTregs, which will also be investigated. Four Specific Aims have been developed to investigate the role of canonical eIF4E/mTORC1 versus specialized DAP5/eIF3d mediated mRNA translation in Treg translational control of lineage determination, stability and function. Aim 1. Characterize DAP5 dependent mRNA translation in development and stability of tTreg and pTreg cell subtypes in the mouse. Aim 2. Investigate the role of DAP5 expression level in generation of exTregs and Treg-exTreg phenotypic plasticity. Aim 3. Characterize mRNA translational reprogramming in Treg subtype lineage commitment and function. Aim 4. Determine the role of m6A with DAP5 in selective translation of Treg cell lineage determining mRNAs, suppression function and Treg cell stability.