CD8+ suppressor T-cells play important roles in the maintenance of immune homeostasis. However, the phenotypic and functional characteristics of these cells remains poorly characterized relative to their CD4 regulatory T cell counterparts, even though some evidence suggests CD8 regulators can be more important than CD4 T regulatory cells. We have been investigating the cellular determinants limiting autoimmune attack in mice against otherwise healthy tissue by introducing and activating self-reactive T cells into a normally tolerant and regulated environment. A key finding is that inflammation in the tissue is quickly extinguished limiting cellular damage to vital structures. Early in the inflammatory process immune cells from the tolerant repertoire are recruited into the tissues harboring autoimmune CTL. Using genetic depletion strategies, we learned that CD8+ regulators are more critical to limiting autoimmune attack in this setting than are CD4+ regulators. Importantly, regulation can be restored in our model by adoptive transfer of CD8+ spleen cells from otherwise unmanipulated animals. This approach identified IL10 and perforin as critical factors determining the ability to limit the damage by autoimmune attack. However, we failed to find evidence of IL10 gene expression the CD8 ?regulatory? cell population in inflamed tissue using an IL10 reporter suggesting that IL10 may act earlier in the process. Furthermore, we found that fixing the T cell receptor specificity of the regulatory population did not ablate their ability to limit autoimmune attack. Accordingly, we have developed the overarching hypothesis that IL10 is essential for the generation of CD8+ suppressor cells and that the mechanism of suppression is through a perforin-dependent killing or inactivation of antigen specific effector T- cells mediated through a TcR independent recognition process. In this study, we seek to acquire needed fundamental information about the role IL10 in the generation and function of CD8 regulatory T cells. Our approach will be to (1) identify the cellular surface phenotypic markers associated with the regulatory cells, (2) identify important sources of IL10 in the generation and function of these CD8 regulatory T cells, and (3) learn where and when these regulatory cells are formed. Along the way we will examine alternative in vitro models to enable efficient definition of the molecular mechanisms giving rise to CD8 regulators and to their ability to limit the functions of activated CTL. Understanding the basis of this strong immune regulatory function could provide insights into how to augment or limit CTL attack for the benefit of cancer or autoimmune disease patients, respectively.