ABSTRACT
In contrast to the well-characterized T cell receptor (TCR) signaling pathways that induce genes that drive T cell development or polarization of naïve CD4 T cells into the diverse T(H)1, T(H)2, T(H)17 and T(reg) lineages, it is unclear what signals maintain specific gene expression in mature resting T cells. Resting T cells residing in peripheral lymphoid organs exhibit low-level constitutive signaling. Whereas tonic signals in B cells are known to be critical for survival, the roles of tonic signals in peripheral T cells are unknown. Here we demonstrate that constitutive signals in Jurkat T cell lines are transduced via the adapter molecule LAT and the Ras exchange factor RasGRP1 to maintain expression of TCRα mRNA and surface expression of the TCR/CD3 complex. Independent approaches of reducing basal activity through the LAT-diacylglycerol-RasGRP pathway led to reduced constitutive Ras-MEK-ERK signals and decreased TCRα mRNA and surface TCR expression in Jurkat cells. However, loss of TCR expression takes several days in these cell line experiments. In agreement with these in vitro approaches, inducible deletion of Lat in vivo results in reduced TCRα mRNA- and surface TCR-expression in a delayed temporal manner as well. Lastly, we demonstrate that loss of basal LAT-RasGRP signals appears to lead to silencing or repression of TCRα transcription. We postulate that basal LAT-diacylglycerol-RasGRP signals fulfill a regulatory function in peripheral T lymphocytes by maintaining proper gene expression programs.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , DNA-Binding Proteins/metabolism , Diglycerides/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Membrane Proteins/metabolism , Receptors, Antigen, T-Cell/metabolism , Signal Transduction/physiology , T-Lymphocytes/metabolism , Adaptor Proteins, Signal Transducing/genetics , Blotting, Western , Cell Line , DNA-Binding Proteins/genetics , Guanine Nucleotide Exchange Factors/genetics , Humans , Jurkat Cells/metabolism , Membrane Proteins/genetics , Polymerase Chain Reaction , Receptors, Antigen, T-Cell/genetics , Signal Transduction/geneticsABSTRACT
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.
Subject(s)
Adaptor Proteins, Signal Transducing/immunology , Homeostasis/immunology , Lymphocyte Activation/immunology , Membrane Proteins/immunology , Phosphoproteins/immunology , T-Lymphocytes/immunology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Autoimmune Lymphoproliferative Syndrome/genetics , Autoimmune Lymphoproliferative Syndrome/immunology , Autoimmune Lymphoproliferative Syndrome/metabolism , Blotting, Western , Cell Proliferation/drug effects , Cell Survival/drug effects , Cell Survival/immunology , Female , Flow Cytometry , Forkhead Transcription Factors/immunology , Forkhead Transcription Factors/metabolism , Homeostasis/drug effects , Lymphocyte Activation/drug effects , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Knockout , Phosphoproteins/genetics , Phosphoproteins/metabolism , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , Signal Transduction/immunology , Spleen/cytology , Spleen/immunology , Spleen/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Tamoxifen/pharmacology , Thymus Gland/cytology , Thymus Gland/immunology , Thymus Gland/metabolismABSTRACT
BACKGROUND: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) can involve MHC-restricted presentation of a drug or its metabolites for T-cell activation. HLA-B(*)1502 tightly associated with carbamazepine (CBZ) induced these conditions in a Han Chinese population. OBJECTIVE: We sought to identify HLA-B(*)1502-bound peptides that might be involved in CBZ-induced SJS/TEN. METHODS: Soluble HLA-B(*)1502 was used to identify bound peptides in the presence and absence of CBZ by using liquid chromatography-tandem mass spectrometry. Peptide-binding assays were performed to detect the specific interaction between the HLA molecule and the identified peptides. Mass spectra were compared to detect CBZ-modified peptides. RESULTS: We identified more than 145 peptides bound to HLA-B(*)1502. In 13 of 15 peptides examined, we functionally confirmed their specificity with binding assays. Preferable uses of these peptides at the anchoring residues P2 and P9 were similar to those observed in other HLA-B alleles in the Han Chinese population. However, the preferable use of serine residues at the nonanchoring position (P) 5, P6, P7, and P8 appeared to be unique for the B(*)1502 peptides. No specific CBZ-modified peptides were detected when we compared the mass spectra of peptides detected in the presence or absence of the drug. CONCLUSION: Noncovalent interaction between a drug and an HLA complex might contribute to cytotoxic T cell-mediated cell death in patients with SJS/TEN. CLINICAL IMPLICATIONS: An understanding of pharmacologic interaction of drugs with an HLA complex might lead to safer drugs that avoid SJS/TEN.
