Thymocyte Sensitivity and Supramolecular Activation Cluster Formation Are Developmentally Regulated: A Partial Role for Sialylation 1

TCR reactivity is tuned during thymic development. Immature thymocytes respond to low-affinity self-ligands resulting in positive selection. Following differentiation, T cells no longer respond to low-affinity ligands, but respond well to high-affinity (foreign) ligands. We show in this study that this response includes integrin activation, supramolecular activation cluster formation, Ca2+ flux, and CD69 expression. Because glycosylation patterns are known to change during T cell development, we tested whether alterations in sialylation influence CD8 T cell sensitivity to low affinity TCR ligands. Using neuraminidase treatment or genetic deficiency in the ST3Gal-I sialyltransferase, we show that desialylation of mature CD8 T cells enhances their sensitivity to low-affinity ligands, although these treatments do not completely recapitulate the dynamic range of immature T cells. These studies identify sialylation as one of the factors that regulate CD8 T cell tuning during development.

[1]  M. Purbhoo,et al.  Sensory Adaptation in Naive Peripheral CD4 T Cells , 2001, The Journal of experimental medicine.

[2]  J. Chauvin,et al.  TCR signal initiation machinery is pre‐assembled and activated in a subset of membrane rafts , 2002, The EMBO journal.

[3]  J. Dennis,et al.  Negative regulation of T-cell activation and autoimmunity by Mgat5 N-glycosylation , 2001, Nature.

[4]  A. Rudensky,et al.  Dynamic Tuning of T Cell Reactivity by Self-Peptide–Major Histocompatibility Complex Ligands , 2001, The Journal of experimental medicine.

[5]  Andrea Iaboni,et al.  The immunological synapse and CD28-CD80 interactions , 2001, Nature Immunology.

[6]  B. Alarcón,et al.  Internalization and intracellular fate of TCR-CD3 complexes. , 2000, Critical reviews in immunology.

[7]  Colin R. F. Monks,et al.  Three-dimensional segregation of supramolecular activation clusters in T cells , 1998, Nature.

[8]  L. Borysiewicz,et al.  TCR signaling thresholds regulating T cell development and activation are dependent upon SHP-1. , 1999, Journal of immunology.

[9]  M. A. Alonso,et al.  The role of lipid rafts in signalling and membrane trafficking in T lymphocytes. , 2001, Journal of cell science.

[10]  K. Hogquist,et al.  Assays of thymic selection. Fetal thymus organ culture and in vitro thymocyte dulling assay. , 2001, Methods in molecular biology.

[11]  T. McKeithan,et al.  Kinetic proofreading in T-cell receptor signal transduction. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[12]  Mark M. Davis,et al.  Imaging synapse formation during thymocyte selection: inability of CD3zeta to form a stable central accumulation during negative selection. , 2002, Immunity.

[13]  D. Kioussis,et al.  T cell activation results in physical modification of the mouse CD8 beta chain. , 1994, Journal of immunology.

[14]  C. Thomas,et al.  Differential binding of fluorescein-labeled lectins to mouse thymocytes: subsets revealed by flow microfluorometry. , 1980, Journal of immunology.

[15]  J. Altman,et al.  CD8 binding to MHC class I molecules is influenced by T cell maturation and glycosylation. , 2001, Immunity.

[16]  B. Burgering,et al.  Binding of protein kinase B to the plakin family member periplakin , 2002, Journal of Cell Science.

[17]  E. Reinherz,et al.  Developmentally Regulated Glycosylation of the CD8αβ Coreceptor Stalk Modulates Ligand Binding , 2001, Cell.

[18]  M. Eichelberger,et al.  Viral neuraminidase treatment of dendritic cells enhances antigen-specific CD8(+) T cell proliferation, but does not account for the CD4(+) T cell independence of the CD8(+) T cell response during influenza virus infection. , 2001, Virology.

[19]  J. Marth,et al.  The ST3Gal-I sialyltransferase controls CD8+ T lymphocyte homeostasis by modulating O-glycan biosynthesis. , 2000, Immunity.

[20]  M. Linker-Israeli,et al.  Separation of mouse thymocytes into two subpopulations by the use of peanut agglutinin. , 1976, Cellular immunology.

[21]  T. Pozzan,et al.  Lipid rafts and T cell receptor signaling: a critical re‐evaluation , 2002, European journal of immunology.

[22]  B. Evavold,et al.  TCR Reserve: A Novel Principle of CD4 T Cell Activation by Weak Ligands 1 , 2003, The Journal of Immunology.

[23]  R. Dwek,et al.  Glycosylation and the immune system. , 2001, Science.

[24]  Kristin A. Hogquist,et al.  T cell receptor antagonist peptides induce positive selection , 1994, Cell.

[25]  Michael Loran Dustin,et al.  The immunological synapse , 2002, Arthritis research.

[26]  P. Ohashi,et al.  Degree of ERK activation influences both positive and negative thymocyte selection , 2000, European journal of immunology.

[27]  G. Anderson,et al.  Thymic epithelial cells provide unique signals for positive selection of CD4+CD8+ thymocytes in vitro , 1994, The Journal of experimental medicine.

[28]  S. Bromley,et al.  A supramolecular basis for CD45 tyrosine phosphatase regulation in sustained T cell activation. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[29]  S. Bromley,et al.  The immunological synapse: a molecular machine controlling T cell activation. , 1999, Science.

[30]  M. Ginsberg,et al.  Integrins in the immune system. , 1999, Advances in immunology.

[31]  Z. Grossman,et al.  Autoreactivity, dynamic tuning and selectivity. , 2001, Current opinion in immunology.

[32]  Jianzhu Chen,et al.  Soluble peptide–MHC monomers cause activation of CD8+ T cells through transfer of the peptide to T cell MHC molecules , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[33]  C. Smith,et al.  Characterization of the activation-associated isoform of CD43 on murine T lymphocytes. , 1994, Journal of immunology.

[34]  B. Freiberg,et al.  Formation of supramolecular activation clusters on fresh ex vivo CD8+ T cells after engagement of the T cell antigen receptor and CD8 by antigen-presenting cells , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[35]  P. W. Janes,et al.  The role of lipid rafts in T cell antigen receptor (TCR) signalling. , 2000, Seminars in immunology.

[36]  M. Bevan,et al.  Clone-specific T cell receptor antagonists of major histocompatibility complex class I-restricted cytotoxic T cells , 1993, The Journal of experimental medicine.

[37]  R. Germain,et al.  Information transfer at the immunological synapse , 2000, Current Biology.

[38]  D. Zaller,et al.  Staging and resetting T cell activation in SMACs , 2002, Nature Immunology.

[39]  Philippe Bousso,et al.  Dynamics of Thymocyte-Stromal Cell Interactions Visualized by Two-Photon Microscopy , 2002, Science.

[40]  G. Crabtree.  Generic Signals and Specific Outcomes Signaling through Ca2+, Calcineurin, and NF-AT , 1999, Cell.

[41]  M. Bachmann,et al.  Cd2 Sets Quantitative Thresholds in T Cell Activation , 1999, The Journal of experimental medicine.

[42]  Timothy K Starr,et al.  Positive and negative selection of T cells. , 2003, Annual review of immunology.

[43]  Peter Ebert,et al.  Immature CD4+CD8+ Thymocytes Do Not Polarize Lipid Rafts in Response to TCR-Mediated Signals1 , 2000, The Journal of Immunology.

[44]  A. Lanzavecchia,et al.  T lymphocyte costimulation mediated by reorganization of membrane microdomains. , 1999, Science.

[45]  S. Jameson,et al.  Positive Selection Is Limited by Available Peptide-Dependent MHC Conformations1 , 2000, The Journal of Immunology.

[46]  Kai Simons,et al.  Lipid Domain Structure of the Plasma Membrane Revealed by Patching of Membrane Components , 1998, The Journal of cell biology.

[47]  H. Ploegh,et al.  Class I negative CD8 T cells reveal the confounding role of peptide-transfer onto CD8 T cells stimulated with soluble H2-Kb molecules , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[48]  E. Kabat,et al.  Immunochemical studies on the specificity of the peanut (Arachis hypogaea) agglutinin. , 1976, Carbohydrate research.

[49]  J. Sprent,et al.  Antigen‐presenting Cells for CD8+ T Cells , 1990, Immunological reviews.

[50]  S. Jameson,et al.  Preselection Thymocytes Are More Sensitive to T Cell Receptor Stimulation Than Mature T Cells , 1998, The Journal of experimental medicine.

[51]  G. Hart,et al.  Cell surface sialic acid influences tumor cell recognition in the mixed lymphocyte reaction. , 1987, Journal of immunology.

[52]  Mark E. Rogers,et al.  Sialic Acid Capping of CD8β Core 1-O-Glycans Controls Thymocyte-Major Histocompatibility Complex Class I Interaction* , 2003, The Journal of Biological Chemistry.

[53]  B. Lucas,et al.  Developmental regulation of TCR efficiency , 2000, European journal of immunology.

[54]  C. June,et al.  Human CD8+ T cells do not require the polarization of lipid rafts for activation and proliferation , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[55]  Michael Loran Dustin,et al.  T Cell Receptor Signaling Precedes Immunological Synapse Formation , 2002, Science.

[56]  I. Weissman,et al.  T cell receptor-mediated negative selection of autoreactive T lymphocyte precursors occurs after commitment to the CD4 or CD8 lineages , 1990, The Journal of experimental medicine.

[57]  R. Zaru,et al.  Cutting Edge: TCR Engagement and Triggering in the Absence of Large-Scale Molecular Segregation at the T Cell-APC Contact Site1 , 2002, The Journal of Immunology.

[58]  R. Testi,et al.  The CD69 receptor: a multipurpose cell-surface trigger for hematopoietic cells. , 1994, Immunology today.

[59]  R. Germain,et al.  Divergent changes in the sensitivity of maturing T cells to structurally related ligands underlies formation of a useful T cell repertoire. , 1999, Immunity.

[60]  Y. Shimizu,et al.  Regulation of integrin function by T cell activation , 1999, Immunologic research.

[61]  S. Jameson,et al.  The impact of duration versus extent of TCR occupancy on T cell activation: a revision of the kinetic proofreading model. , 2001, Immunity.

[62]  R. Germain,et al.  Opening a Window on Thymic Positive Selection: Developmental Changes in the Influence of Cosignaling by Integrins and CD28 on Selection Events Induced by TCR Engagement , 2000, The Journal of Immunology.

[63]  Michael Loran Dustin,et al.  Immature CD4(+)CD8(+) thymocytes form a multifocal immunological synapse with sustained tyrosine phosphorylation. , 2002, Immunity.