Macroautophagy (hereafter referred to as autophagy) is a well-conserved intracellular degradation process. Recent studies examining cells lacking the autophagy genes Atg5 and Atg7 have demonstrated that autophagy plays essential roles in cell survival during starvation, in innate cell clearance of microbial pathogens, and in neural cell maintenance. However, the role of autophagy in T lymphocyte development and survival is not known. Here, we demonstrate that autophagosomes form in primary mouse T lymphocytes. By generating Atg5−/− chimeric mice, we found that Atg5-deficient T lymphocytes underwent full maturation. However, the numbers of total thymocytes and peripheral T and B lymphocytes were reduced in Atg5 chimeras. In the periphery, Atg5−/− CD8+ T lymphocytes displayed dramatically increased cell death. Furthermore, Atg5−/− CD4+ and CD8+ T cells failed to undergo efficient proliferation after TCR stimulation. These results demonstrate a critical role for Atg5 in multiple aspects of lymphocyte development and function and suggest that autophagy may be essential for both T lymphocyte survival and proliferation.

Macroautophagy (hereafter referred to as autophagy) is a well-conserved intracellular degradation process. Recent studies examining cells lacking the autophagy genes Atg5 and Atg7 have demonstrated that autophagy plays essential roles in cell survival during starvation, in innate cell clearance of microbial pathogens, and in neural cell maintenance. However, the role of autophagy in T lymphocyte development and survival is not known. Here, we demonstrate that autophagosomes form in primary mouse T lymphocytes. By generating Atg5 −/− chimeric mice, we found that Atg5-defi cient T lymphocytes underwent full maturation. However, the numbers of total thymocytes and peripheral T and B lymphocytes were reduced in Atg5 chimeras. In the periphery, Atg5 −/− CD8 + T lymphocytes displayed dramatically increased cell death. Furthermore, Atg5 −/− CD4 + and CD8 + T cells failed to undergo effi cient proliferation after TCR stimulation. These results demonstrate a critical role for Atg5 in multiple aspects of lymphocyte development and function and suggest that autophagy may be essential for both T lymphocyte survival and proliferation.

The interaction between the linker for activation of T cells (LAT) with phospholipase C (PLC-γ1) is important for T cell receptor (TCR)-mediated Ca2+ signaling and MAPK activation. Knock-in mice harboring a mutation at the PLC-γ1 binding site (Y136) of LAT develop a severe lymphoproliferative syndrome. These mice have defective thymic development and selection and lack natural regulatory T cells, implicating a breakdown of both central and peripheral tolerance. To bypass this developmental defect, we developed a conditional knock-in line in which only LATY136F is expressed in mature T cells after deletion of the wildtype LAT allele. Analysis of LATY136F T cells indicated that the interaction between LAT and PLC-γ1 plays an important role in TCR-mediated signaling, proliferation, and IL-2 production. Furthermore, the deletion of LAT induced development of the lymphoproliferative syndrome in these mice. Although Foxp3+ natural Treg cells were present in these mice after deletion, they were unable to suppress the proliferation of conventional T cells. Our data indicated that the binding of LAT to PLC-γ1 is essential for the suppressive function of CD4+CD25+ regulatory T cells.

The linker for activation of T cells (LAT) is an adaptor protein that couples TCR engagement to downstream signaling cascades. LAT is important in early thymocyte development as LAT-deficient mice have a complete block at the double-negative (DN) 3 stage. To study the role of LAT beyond the DN3 stage, we generated mice in which the lat gene could be deleted by the Cre recombinase. Analysis of these mice showed that deletion of LAT after the DN3 stage allowed thymocytes to develop past the DN3 to DN4 checkpoint and to generate double-positive thymocytes. However, LAT-deficient DP thymocytes were severely defective in responding to stimulation via the TCR and failed to differentiate into single-positive thymocytes efficiently. Consequently, few LAT-deficient mature T cells could be found in the periphery. These T cells had undergone extensive homeostatic proliferation and expressed low levels of the TCR on their surface. Collectively, our data indicate that in addition to its role in pre-TCR signaling, LAT also plays an essential role in thymocyte development during transition from the double-positive to single-positive stage.

LAT (linker for activation of T cells) is a transmembrane adaptor protein that plays an essential role in TCR-mediated signaling and thymocyte development. Because LAT-deficient mice have an early block in thymocyte development, we utilized an inducible system to delete LAT in primary T cells to study LAT function in T cell activation, homeostasis, and survival. Deletion of LAT caused primary T cells to become unresponsive to stimulation from the TCR and impaired T cell homeostatic proliferation and long term survival. Furthermore, deletion of LAT led to reduced expression of Foxp3, CTLA-4, and CD25 in T reg cells and impaired their function. Consequently, mice with LAT deleted developed a lymphoproliferative syndrome similar to that in LATY136F mice, although less severe. Our data implicate that LAT has positive and negative roles in the regulation of mature T cells.Upon engagement of the TCR (T cell receptor), 3 LAT (linker for activation of T cells) is phosphorylated at multiple tyrosine residues by ZAP-70 kinase (1) and functions as a protein scaffold to assemble a large membrane-tethered signalosome (1-3). LAT binding of Gads leads to the membrane recruitment of SLP-76, which in turn interacts with Itk, as well as Vav1 and PLC-␥1. Through its interaction with LAT and SLP-76, PLC-␥1 is also brought to the plasma membrane. Upon activation by ZAP-70 and Itk, PLC-␥1 hydrolyzes phosphatidylinositol 4,5-bisphosphate into the secondary messengers inositol trisphosphate and diacylglycerol (4, 5). Inositol trisphosphate induces Ca 2ϩ mobilization, whereas diacylglycerol binds to RasGRP1 to activate the Ras-MAPK pathway (6, 7). LAT also contributes to Ras-MAPK activation through recruitment of the Grb2-Sos complex to the plasma membrane to activate Ras (1, 8). Initiation of these signaling cascades eventually leads to activation of transcription factors that regulate the genes critical for T cell proliferation and effector functions.The essential role of LAT in T cell activation was initially demonstrated in LAT-deficient Jurkat T cells. Although the proximal signals upstream of LAT phosphorylation remain intact in these cells, TCR-mediated calcium mobilization, Ras-MAPK activation, and NFAT activation are all abolished (9, 10). LAT function during T cell development has also been well characterized. Thymocyte development in LAT Ϫ/Ϫ mice is completely blocked at the CD25 ϩ CD44 Ϫ DN3 stage, indicating an absolute requirement for LAT in pre-TCR signaling (11). Our recent studies using LAT conditional knock-out mice show that LAT is also required during thymocyte development at the DP to SP transition, as positive selection in LAT-deficient DP thymocytes is markedly compromised in these mice. Consequently, the maturation of SP thymocytes is severely blocked (12). In addition, a point mutation at LAT tyrosine 136 (Y136F), which specifically abolishes the LAT-PLC-␥1 interaction, renders both positive and negative thymic selection processes defective in the LATY136F knock-in mice (13), highlighting the importance of LAT-m...

Linker for activation of T cells (LAT) is a transmembrane adaptor protein that is essential to bridge T cell receptor (TCR) engagement to downstream signaling events. The indispensable role of LAT in thymocyte development and T cell activation has been well characterized; however, the function of LAT in cytotoxic-T-lymphocyte (CTL) cytotoxicity remains unknown. We show here that LAT-deficient CTLs failed to upregulate FasL and produce gamma interferon after engagement with target cells and had impaired granule-mediated killing. We further dissected the effect of the LAT deletion on each step of granule exocytosis. LAT deficiency led to altered synapse formation, subsequently causing unstable T cell-antigen-presenting cell (APC) conjugates. Microtubule organizing center polarization and granule reorientation were also impaired by LAT deficiency, leading to reduced granule delivery. Despite these defects, granule release was still observed in LAT-deficient CTLs due to residual calcium flux and phospholipase C (PLC) activity. Our data demonstrated that LAT-mediated signaling intricately regulates CTL cytotoxicity at multiple steps.