LAT-deficient Mice Research Articles

Mutations in linker for activation of T cells (LAT) lead to a novel form of severe combined immunodeficiency

Signaling through the T-cell receptor (TCR) is critical for T-cell development and function. Linker for activation of Tcells (LAT) is a transmembrane adaptor signaling molecule that is part of the TCR complex and essential for T-cell development, as demonstrated by LAT-deficient mice, which show a complete lack of peripheral Tcells. We describe a pedigree affected by a severe combined immunodeficiency phenotype with absent Tcells and normal B-cell and natural killer cell numbers. Anovel homozygous frameshift mutation in the gene encoding for LAT was identified in this kindred. Genetic, molecular, and functional analyses were used to identify and characterize the LAT defect. Clinical and immunologic analysis of patients was also performed and reported. Homozygosity mapping was used to identify potential defective genes. Sanger sequencing of the LAT gene showed a mutation that resulted in a premature stop codon and protein truncation leading to complete loss of function and loss of expression of LAT in the affected family members. We also demonstrate loss of LAT expression and lack of TCR signaling restoration in LAT-deficient cell lines reconstituted with a synthetic LAT gene bearing this severe combined immunodeficiency mutation. For the first time, the results of this study show that inherited LAT deficiency should be considered in patients withcombined immunodeficiency with T-cell abnormalities.

Quantitative Phosphoproteome Analysis Unveils LAT as a Modulator of CD3ζ and ZAP-70 Tyrosine Phosphorylation

Signaling through the T cell receptor (TCR) initiates adaptive immunity and its perturbation may results in autoimmunity. The plasma membrane scaffolding protein LAT acts as a central organizer of the TCR signaling machinery to activate many functional pathways. LAT-deficient mice develop an autoimmune syndrome but the mechanism of this pathology is unknown. In this work we have compared global dynamics of TCR signaling by MS-based quantitative phosphoproteomics in LAT-sufficient and LAT-defective Jurkat T cells. Surprisingly, we found that many TCR-induced phosphorylation events persist in the absence of LAT, despite ERK and PLCγ1 phosphorylation being repressed. Most importantly, the absence of LAT resulted in augmented and persistent tyrosine phosphorylation of CD3ζ and ZAP70. This indicates that LAT signaling hub is also implicated in negative feedback signals to modulate upstream phosphorylation events. Phosphorylation kinetics data resulting from this investigation is documented in a database (phosphoTCR) accessible online. The MS data have been deposited to the ProteomeXchange with identifier PXD000341.

The Importance of LAT in the Activation, Homeostasis, and Regulatory Function of T Cells

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.

Peripheral Thy1+ lymphocytes rearranging TCR‐γδ genes in LAT‐deficient mice

Linker for activation of T cells (LAT) is an adaptor molecule indispensable for development of alphabeta and gammadelta T lymphocytes. Surprisingly, using a new model of LAT-deficient mice we found that despite arrested thymic development, a discrete population of cells with active Lat promoter, expressing Thy1 molecules, accumulated in peripheral lymphoid organs of homozygous (Lat(Inv/Inv)) mutant mice. By measuring frequencies of TCR gene rearrangements in conjunction with a panel of cell surface Ag, we dissected two subsets of these Thy1(+) cells. Thy1(dull) cells expressed markers of NK lymphocytes and contained low frequency of TCR-gamma gene rearrangements without detectable TCR-delta rearrangements. Thy1(high) cells resembled immature CD44(+)CD25(+) thymocytes and contained high frequency of non-productive TCR-gamma and TCR-delta rearrangements, indicating that cells displaying molecular signatures of commitment toward gammadelta T-cell lineage can develop and populate lymphoid tissues of LAT-deficient mice. Phenotypically similar Thy1(high) cells were also found in lymph nodes of lymphocyte-deficient (Rag2(-/-)) mice but not in T lymphocyte proficient, heterozygous Lat(+/Inv) mice suggesting that Thy1(high) cells of LAT-deficient mice identified in this study accumulate in peripheral lymphoid organs as a result of congenital lymphopenia.

The Essential Role of LAT in Thymocyte Development during Transition from the Double-Positive to Single-Positive Stage

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 Plays Important Role in Thymocyte Survival and Mature T Cell Activation (87.35)

Abstract LAT (Linker for Activation of T-cells) is a membrane-associated adaptor protein that plays a critical role in linking T-cell receptor engagement to activation of its downstream signaling events. Upon tyrosine phosphorylation, LAT binds Grb2, Gads, and PLC-gamma1. Previous studies show that LAT is essential in thymocyte development as LAT-deficient mice completely lack mature T cells. In addition, LAT-deficient Jurkat cells have a severe defect in TCR-mediated signaling. In order to investigate the role of LAT in T cell activation, survival, and homeostasis, mice in which the LAT gene can be inducibly deleted from thymocytes and mature T cells by injection of tamoxifen were generated. Our data showed that tamoxifen treatment of these mice led to a dramatic loss of thymocytes, especially DP thymocytes. Deletion of LAT in mature T cells affected TCR-mediated signaling, IL-2 production, and proliferation. These data suggested that LAT plays an important role in thymocyte survival and TCR-mediated signaling in mature T cells.

Negative regulation of mast cell signaling and function by the adaptor LAB/NTAL.

Engagement of the Fcɛ receptor I (FcɛRI) on mast cells and basophils initiates signaling pathways leading to degranulation. Early activation events include tyrosine phosphorylation of two transmembrane adaptor proteins, linker for activation of T cells (LAT) and non–T cell activation linker (NTAL; also called LAB; a product of Wbscr5 gene). Previous studies showed that the secretory response was partially inhibited in bone marrow–derived mast cells (BMMCs) from LAT-deficient mice. To clarify the role of NTAL in mast cell degranulation, we compared FcɛRI-mediated signaling events in BMMCs from NTAL-deficient and wild-type mice. Although NTAL is structurally similar to LAT, antigen-mediated degranulation responses were unexpectedly increased in NTAL-deficient mast cells. The earliest event affected was enhanced tyrosine phosphorylation of LAT in antigen-activated cells. This was accompanied by enhanced tyrosine phosphorylation and enzymatic activity of phospholipase C γ1 and phospholipase C γ2, resulting in elevated levels of inositol 1,4,5-trisphosphate and free intracellular Ca2+. NTAL-deficient BMMCs also exhibited an enhanced activity of phosphatidylinositol 3-OH kinase and Src homology 2 domain–containing protein tyrosine phosphatase-2. Although both LAT and NTAL are considered to be localized in membrane rafts, immunogold electron microscopy on isolated membrane sheets demonstrated their independent clustering. The combined data show that NTAL is functionally and topographically different from LAT.

Positive and negative regulation of FcepsilonRI-mediated signaling by the adaptor protein LAB/NTAL.

Linker for activation of B cells (LAB, also called NTAL; a product of wbscr5 gene) is a newly identified transmembrane adaptor protein that is expressed in B cells, NK cells, and mast cells. Upon BCR activation, LAB is phosphorylated and interacts with Grb2. LAB is capable of rescuing thymocyte development in LAT-deficient mice. To study the in vivo function of LAB, LAB-deficient mice were generated. Although disruption of the Lab gene did not affect lymphocyte development, it caused mast cells to be hyperresponsive to stimulation via the FcɛRI, evidenced by enhanced Erk activation, calcium mobilization, degranulation, and cytokine production. These data suggested that LAB negatively regulates mast cell function. However, mast cells that lacked both linker for activation of T cells (LAT) and LAB proteins had a more severe block in FcɛRI-mediated signaling than LAT−/− mast cells, demonstrating that LAB also shares a redundant function with LAT to play a positive role in FcɛRI-mediated signaling.

The four distal tyrosines are required for LAT-dependent signaling in FcepsilonRI-mediated mast cell activation.

The linker for activation of T cells (LAT) is an adaptor protein critical for FcɛRI-mediated mast cell activation. LAT is a substrate of the tyrosine kinases activated after TCR and FcɛRI engagement. After phosphorylation of the cytosolic domain of LAT, multiple signaling molecules such as phospholipase C–γ1, Grb2, and Gads associate with phosphorylated LAT via their SH2 domains. The essential role of the four distal tyrosines in TCR-mediated signaling and T cell development has been demonstrated by experiments using LAT-deficient cell lines and genetically modified mice. To investigate the role of these four tyrosines of LAT in FcɛRI-mediated mast cell activation, bone marrow–derived mast cells from LAT-deficient mice were infected with retroviral vectors designed to express wild-type or mutant LAT. Examination of bone marrow–derived mast cells expressing various tyrosine to phenylalanine mutants in LAT demonstrates a differential requirement for these different binding sites. In these studies, assays of biochemical pathways, degranulation, and cytokine and chemokine release were performed. Finally, the role of these tyrosines was also evaluated in vivo using genetically modified animals. Deletion of all four distal tyrosines, and in particular, loss of the primary phospholipase C–γ-binding tyrosine had a significant effect on antigen-induced histamine release.

Appearance of the LAT protein at an early stage of B-cell development and its possible role.

The linker protein LAT is expressed mainly in T and natural killer (NK) cells. LAT-deficient mice have an arrest of intrathymic T-cell development at the CD4+ CD8+ stage and lack mature T cells in the periphery. However, no gross abnormality in development and function of the B and NK cells has been described. Here we report that LAT is expressed in mouse progenitor B (pro-B) and precursor B (pre-B) cells, but not in immature or mature B cells. LAT in pre-B cells becomes tyrosine phosphorylated upon cross-linking of the pre-B-cell receptor (pre-BCR) by anti- micro antibody. Incubation of 1xN/2b (mouse pre-B-cell line) cells or bone marrow cells from microMT/ microMT mice, which lack B cells after the small pre-B-cell stage, with anti-Ig beta antibody resulted in the downregulation of LAT expression. Transgenic mice which expressed LAT protein in B-lineage cells showed an increased proportion of pro- and large pre-B cells in the bone marrow and a remarkable reduction in the numbers of mature B cells in peripheral lymphoid tissues. Collectively, the present results indicate that LAT is expressed in the cells at the early stages of B-lineage development, but is absent in immature and mature B cells. LAT may play a crucial role in the negative regulation of B-cell development at the transition from pre-B to mature B-cell stages, and signal(s) via the pre-BCR may extinguish LAT expression, thus allowing pre-B-cell differentiation towards the mature B-cell stage.

Minimal requirement of tyrosine residues of linker for activation of T cells in TCR signaling and thymocyte development.

Linker for activation of T cells (LAT) is a membrane-associated adaptor protein that is phosphorylated on multiple tyrosines upon TCR cross-linking. Previous studies show that LAT is essential for TCR-mediated signaling and thymocyte development. In this study, we expressed a series of LAT Tyr to Phe mutants in LAT-deficient J.CaM2.5 cells and examined their tyrosine phosphorylation; association with Grb2, Gads, and phospholipase C (PLC)-gamma1; and function in T cell activation. Our results showed that the five membrane-distal tyrosines were phosphorylated upon T cell activation. Grb2, Gads, and PLC-gamma1 associated with LAT preferentially via different sets of tyrosine residues; however, they failed to interact with LAT mutants containing only one tyrosine. We also determined the minimal requirement of LAT tyrosine residues in T cell activation and thymocyte development. Our results showed that a minimum of three tyrosines is required for LAT to function in T cell activation and thymocyte development. LAT mutants that were capable of binding Grb2 and PLC-gamma1 could reconstitute T cell activation in LAT-deficient cells and thymocyte development in LAT-deficient mice.

The transmembrane adapter LAT plays a central role in immune receptor signalling.

The transmembrane adapter LAT (linker for activation of T cells) plays a central role in signalling by ITAM bearing receptors expressed on T cells, natural killer cells, mast cells and platelets. Receptor engagement leads to the phosphorylation of tyrosine residues present in the intracellular domain of LAT and formation of a multiprotein complex with other adapter molecules and enzymes including Grb2, Gads/SLP-76 and PLCgamma isoforms. These signalling events predominantly take place in glycolipid-enriched membrane domains. The constitutive presence of LAT in GEMs enables its function as the main scaffolding protein for the organization of GEM-localized signalling. The study of LAT-deficient mice and LAT-deficient cell lines further emphasizes the importance of LAT for these signalling cascades but also defines the existence of LAT-independent events downstream of the Syk-family kinase-ITAM complex.

The Adapter Protein LAT Enhances Fcγ Receptor-mediated Signal Transduction in Myeloid Cells

FcgammaR clustering in monocytes initiates a cascade of signaling events that culminate in biological responses such as phagocytosis, production of inflammatory cytokines, and generation of reactive oxygen species. We have identified and determined the function of the adapter protein linker of activation of T cell (LAT) in FcgammaR-mediated signaling and function. Clustering of FcgammaRs on the human monocytic cell line, THP-1, induces phosphorylation of a major 36-kDa protein which immunoreacts with anti-LAT antisera. Our data indicate that although both the 36-kDa and 38-kDa isoforms of LAT are expressed in THP-1 and U937 human monocytic cells, FcgammaR clustering induces phosphorylation of the 36-kDa isoform only. Co-immunoprecipitation experiments revealed a constitutive association of p36 LAT with both FcgammaRI and FcgammaRIIa immunoprecipitates, and an activation-induced association of LAT with PLCgamma1, Grb2, and the p85 subunit of phosphatidylinositol 3-kinase. Transient transfection experiments in COS-7 cells indicated that overexpression of a wild type but not a dominant-negative LAT, that is incapable of binding to p85, enhances phagocytosis by FcgammaRI. Furthermore, bone marrow-derived macrophages from LAT-deficient mice displayed reduced phagocytic efficiency in comparison to the macrophages from wild-type mice. Thus, we conclude that p36 LAT serves to enhance FcgammaR-induced signal transduction in myeloid cells.

LAT is Important for FcϵRI Signaling

The linker protein LAT functions as a signaling network hub where several proteins bind and become activated. The importance of LAT in T cell-receptor signaling is well documented; however, the role of LAT in other immune cells has not been so intensively examined. Saitoh et al . show that LAT has an important function in FcϵRI signaling in mast cells. Mast cells derived from LAT-deficient mice are able to partially signal through FcϵRI cross-linking and can activate Syk and Vav; however, activation of SLP-76, of both isoforms of PLC-γ, and of specific MAPK pathways was greatly reduced. In addition, Ca 2+ mobilization, exocytotic degranulation, and the expression of several cytokines were impaired in LAT-deficient mast cells. These results suggest that LAT is important for proper signaling by FcϵRI and for physiological responses to antigens. Saitoh, S., Arudchandran, R., Manetz, T.S., Zhang, W., Sommers, C.L., Love, P.E., Rivera, J., and Samelson, L.E. (2000) LAT is essential for FcϵRI-mediated mast cell activation. Immunity 12 : 525-535. [Online Journal]

LAT Is Essential for FcεRI-Mediated Mast Cell Activation

The linker molecule LAT is a substrate of the tyrosine kinases activated following TCR engagement of T cells. LAT is also expressed in platelets, NK, and mast cells. Although LAT-deficient mice contain normal numbers of mast cells, we found that LAT-deficient mice were resistant to IgE-mediated passive systemic anaphylaxis. LAT-deficient bone marrow–derived mast cells (BMMC) showed normal growth and development. Whereas tyrosine phosphorylation of FcεRI, Syk, and Vav was intact in LAT-deficient BMMCs following FcεRI engagement, tyrosine phosphorylation of SLP-76, PLC-γ1, and PLC-γ2 and calcium mobilization were dramatically reduced. LAT-deficient BMMCs also exhibited profound defects in activation of MAPK, degranulation, and cytokine production after FcεRI cross-linking. These results show that LAT plays a critical role in FcεRI-mediated signaling in mast cells.

The role of membrane-associated adaptors in T cell receptor signalling

Engagement of the T cell receptor leads to activation of several tyrosine kinases and phosphorylation of many intracellular proteins. This is followed by Ca2+Copyright 2000 Academic Pressmobilization and activation of multiple biochemical pathways, including the Ras/MAPK cascade, and several downstream serine/threonine kinases. Membrane-associated adaptor proteins play an important role in T cell activation by coupling TCR ligation at the membrane to distal signalling cascades. Several new membrane associated adaptors have been identified in recent years. LAT (linker for activation of T cells) is an adaptor molecule, which following its phosphorylation associates with Grb2, Gads, PLC-γ1, and other signalling molecules. The functional importance of this molecule has been demonstrated by the study of LAT-deficient cell lines and LAT-deficient mice. Two other recently identified adaptor proteins, TRIM (T cell receptor interacting molecule) and SIT (SHP2-interacting transmembrane adaptor protein), which constitutively associate with several surface molecules, bind to PI3K and SHP2, respectively, after T cell activation and might also function in the TCR signalling pathway.

Essential Role of LAT in T Cell Development

The linker molecule LAT is a substrate of the tyrosine kinases activated following TCR engagement. Phosphorylated LAT binds many critical signaling molecules. The central role of this molecule in TCR-mediated signaling has been demonstrated by experiments in a LAT-deficient cell line. To probe the role of LAT in T cell development, the LAT gene was disrupted by targeting. LAT-deficient mice appeared healthy. Flow cytometric analysis revealed normal B cell populations but the absence of any mature peripheral T cells. Intrathymic development was blocked within the CD4−CD8− stage. No gross abnormality of NK or platelet function was observed. LAT is thus critical to both T cell activation and development.