mice were purchased from Jackson Laboratories (Bar Harbor, ME) and were crossed to at the University or college of Massachusetts Medical School. that this predominant populace of Eomes+ innate CD4SP thymocytes is largely absent in mice lacking CD1d-specific iNKT cells, with no effect on innate CD8SP thymocytes. In contrast, both subsets of innate Eomes+ T cells require the presence of a novel PLZF-expressing, SAP-dependent thymocyte populace Itga2 that is essential for the conversion of conventional CD4+ and CD8+ T cells into innate T cells with a memory phenotype. Introduction T cell development in the thymus produces a wide range of T cell subsets with varying functions in immune responses. In addition to standard na?ve CD4+ and CD8+ T cells, which require prolonged activation and differentiation to acquire protective effector functions, several subsets of T cells with innate effector functions are now known to develop in the thymus (1, 2). This latter group includes several distinct categories of T cells, CD1d-specific invariant natural killer (iNKT) cells, MR1-specific mucosal-associated invariant T (MAIT) cells, H2-M3-specific CD8+ T cells, and Foxp3+ regulatory T cells, among others (1, 2). While a thorough understanding of the signals giving LJ570 rise to each of these T cell lineages has not yet been achieved, recent studies indicate a role for the strength of TCR signaling, extrinsic signals provided by cytokines, as well as components of intrinsic developmental programming in this process (3-8). One important clue to dissecting the signals regulating T cell lineage development has come from LJ570 studies of genetically-altered mice. In the absence of LJ570 the Tec kinase, Itk, as well as in mice lacking the transcription factors Krppel-like factor 2 (Klf2), Inhibitor of DNA-binding 3 (Id3), and CREB-binding protein (CBP), standard CD8+ T cells developing in the thymus are converted into innate/memory-like T cells expressing high levels of the effector-promoting transcription factor, Eomesodermin (Eomes) (6, 8-10). While the involvement of Itk indicates a role for TCR signaling in this process, further evidence supporting this conclusion comes from studies demonstrating an identical phenotype in mice expressing a mutant form of the adapter protein SLP-76, SLP-76(Y145F), which lacks the ability to recruit Itk in response to TCR activation (7). LJ570 Together, these data indicate that intact TCR signaling pathways are critical for the normal development of conventional CD8+ T cells. Interestingly, impaired TCR signaling is not the only requirement for the development of innate/memory CD8+ thymocytes expressing high levels of Eomes. Studies by Hogquist and colleagues first exhibited a requirement for exogenous IL-4 to induce Eomes expression in CD8+ T cells (3, 6-8). Thus, in the absence of the IL-4R (CD124), Eomes is usually no longer expressed in mice (11, 12), it is not currently known whether NKT cells are required to induce Eomes expression in CD8+ T cells. Multiple cell types ( iNKT cells, MAIT cells, etc.) are capable of IL-4 production. Therefore, even though NKT cells are the most likely candidate for the excess IL-4 acting on CD8+ thymocytes, it remains possible that other cell types are contributing to this process. Additionally, the effect of thymicIL-4 around the development of conventional CD4+ T cells has not been addressed. While numerous studies have documented that mice have an increased frequency of activated CD4+ T cells, it is not known whether these cells are expressing Eomes similarly to mice, we demonstrate that this phenotype is not induced by T cells. Materials and Methods Mice Wild-type (WT) C57Bl/6 mice were purchased from either Taconic Farms, Inc. (Hudson, NY), Jackson Laboratories (Bar Harbor, ME), or Charles River Laboratories International, Inc. (Wilmington, MA). mice were previously explained (15-17) and housed at the.