Lupus is a devastating disease predominantly affecting young women. Lupus autoimmunity is prototypical of many different autoimmune diseases, which together affects 3-5% of the popula- tion. T cells, whose activation depends on both B cells and innate immune sensing, are pivotal in promoting disease. Despite that T cells are critical in driving virtually all aspects of systemic autoimmune disease, their identity, specificity, function and fate remain very poorly defined. Au- toreactive T cells provide help to autoreactive B cells and also cause tissue damage by target organ infiltration. While we have a deep understanding of the specificity, regulation and fate of autoreactive B cells, the same cannot be said for autoreactive T cells. In particular, we would like to elucidate how self-reactive T cells are regulated by self-tolerance mechanisms and how, upon activation, they contribute to various aspects of autoimmunity. With the current proposal we hope to make significant inroads into this important yet understud- ied area. We will first use a novel strategy to isolate new clones of autoreactive T cells that rec- ognize nuclear components (“ANA T cells”) from both normal and autoimmune prone genetic backgrounds (Aim 1A); this will provide insights into the repertoire of autoimmune B-helper T cell repertoire. We will then clone the TCRs of selected T cells into retroviral expression vectors to make “retrogenic” (Rg) mice. We will then determine (Aim 1B) how these autoreactive cells develop, are subject to a variety of tolerance mechanisms, and are potentially spontaneously activated. This will be studied according to both the origin of the T cell as well as the context in which the T cell is expressed (normal or autoimmune background). Finally in Aim 1(C), we will convert selected TCRs into full-fledged TCR transgenic (Tg) mice, which will be required for more robust and detailed mechanistic studies. In Aim 2 we will then use these Tg mice T cells to study mechanisms of activation, T-B interaction and Toll-like receptor (TLR) dependence. This will be done by a series of mixed bone marrow chimeras that will allow us to interrogate the fate and function of ANA T cells in a more natural milieu in which the T cells will develop and operate in the context of polyclonal autoimmunity driven by the other cells in the chimera that will vary in expression of cognate BCRs as well as TLR signaling capability. At the conclusion of these studies we will not only have a truly unique set of new tools in the form of clones, Rg mice and Tg mice on normal and autoimmune backgrounds, but we will have very significantly advanced the field in terms of understanding how ANA T cells are activated and regulated, how they promote disease, and how they interact with innate immune signals.