SIGNALS REGULATING T CELL DEVELOPMENT
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Paul Love, MD, PhD, Head, Section on Cellular and Developmental Biology Sandra Hayes, PhD, Senior Fellow |
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Our research is directed at understanding the cellular and genetic events that regulate T lymphocyte development. Current studies focus on the role of T lymphocyte signal-transducing molecules in immature T lymphocyte (thymocyte) selection, a process essential for formation of the mature T cell repertoire. To analyze the function of specific signal-transducing proteins in T lymphocyte development, we use transgenic and gene targeting methods to create overexpression, dominant negative, and loss-of-function mutants in mice. In addition, we are employing molecular genetic techniques to identify and characterize the function of novel genes that are expressed in T lymphocytes. T cell antigen receptor signaling in thymocyte development Frolova, El-Khoury, Love; in collaboration with Shores A major focus of our research has been the investigation of the role of T cell antigen reciptor (TCR) signal transduction in thymocyte development. Signal transduction sequences (termed Immunoreceptor Tyrosine-based Activation Motifs; ITAMs) are contained within four distinct subunits of the multimeric TCR complex (zeta, CD3-gamma, -delta, -epsilon). Di-tyrosine residues within ITAMs are phosphorylated upon TCR engagement and function to recruit signaling molecules, such as protein tyrosine kinases, to the TCR complex, thereby initiating the T cell activation cascade. Though conserved, ITAM sequences are nonidentical, raising the possibility that the diverse developmental and functional responses controlled by the TCR may be regulated, in part, by distinct ITAMs. To determine if TCR signal-transducing subunits perform distinct or analogous functions in development, we generated zeta-deficient and CD3-epsilon-deficient mice by gene targeting, genetically reconstituted the mice with transgenes encoding wild-type or signaling-deficient (ITAM-mutant) forms of zeta and CD3-epsilon, and then characterized the developmental and functional consequences of the alterations on TCR signaling. The results of our studies demonstrated that TCR-ITAMs are functionally equivalent but act in concert to amplify TCR signals. We found TCR signal amplification to be critical for thymocyte selection, the process by which potentially useful immature T cells are instructed to survive and differentiate further (positive selection). We also found that potentially auto-reactive cells that may cause auto-immune disease are deleted in the thymus (negative selection). Thus, the multisubunit structure of the TCR may have evolved to enable complex organisms to develop a broad, self-restricted yet auto-tolerant T cell repertoire.
Love PE, Chan AC. Regulation of thymocyte development: only the meek survive. Curr Opin Im- munol 2003;15:199-203. McFarland HI, Hansal SA, Morris DI, McVicar DW, Love PE, Rosenberg AS. Signaling through MHC in transgenic mice generates a population of memory phenotype cytolytic cells that lack TCR. Blood 2003;101:4520-4528. Mechanism of CD5-mediated TCR signal inhibition Park, Love The cell surface protein CD5 negatively regulates TCR signaling and thus participates in thymocyte selection. Examination of CD5 expression during T cell development revealed that surface levels of CD5 are regulated by TCR signal intensity as well as by the affinity of the TCR for self-peptide ligands in the thymus that mediate selection. To determine if the ability to regulate CD5 expression is important for thymocyte selection, we generated transgenic mice that constitutively express high levels of CD5 throughout development. Overexpression of CD5 significantly impaired positive selection of some thymocytes (those that would normally express low levels of CD5) but not of others (those that would normally express high levels of CD5). These findings support a role for CD5 in modulating TCR signal transduction and thereby influencing the outcome of thymocyte selection. The ability of individual thymocytes to regulate CD5 expression represents a mechanism for "fine tuning" the TCR signaling response during development. Our results indicate that this potential for signal modulation may be particularly useful for generating the maximum possible diversity in the mature T cell repertoire. Given that the activation-induced binding of regulatory molecule(s) to sequences within the CD5 cytoplasmic domain is a probable mechanism for CD5 function, we generated transgenic mice that express a tail-less form of CD5 (mCD5) and then used both the intact and mCD5 transgenes to reconstitute CD5 surface expression in CD5-/- mice. The experiments revealed a critical function for the cytoplasmic domain in CD5 signaling. In an effort to determine how CD5 regulates signal transduction by the TCR, the laboratory is currently attempting to identify molecules that interact with CD5. Azzam HS, DeJarnette JB, Huang K, Emmons R, Park CS, Sommers CL, El-Khoury D, Shores EW, Love PE. Fine tuning of TCR signaling by CD5. J Immunol 2001;166:5464-5472. Bhandoola A, Bosselut R, Yu Q, Cowan ML, Feigenbaum L, Love PE, Singer A. CD5-mediated inhibition of TCR signaling during intrathymic selection and development does not require the CD5 extracellular domain. Eur J Immunol 2002;32:1811-1817. Role of LAT in T cell development Park, Love; in collaboration with Samelson, Shores, Sommers Linker for Activation of T cells (LAT) is an integral membrane protein that functions as a critical adaptor linking the TCR to multiple downstream signaling pathways required for T cell activation. The distal four tyrosines in LAT (tyr136, tyr175, tyr195, tyr235) are necessary and sufficient for LAT activity in T cells, which includes activation of the calcium and MAP kinase (MAPK) downstream signaling pathways. These signaling pathways are also activated by a large number of other receptors and are required for the development and function of many cell types. Thus, their inactivation in all cells would likely result in embryonic lethality. However, by mutating specific LAT tyrosines, we have been able to uncouple the TCR from downstream signaling pathways in T cells without affecting the ability of other receptors or cells to use these pathways. We generated "knock-in" mutant mice that express LAT proteins containing single or multiple tyrosine-phenylalanine mutations of the four critical tyrosine residues. Knock-in mice that express the wild-type version of the protein exhibited normal T cell development, thereby validating the targeting strategy. Inactivation of all four distal LAT tyrosines yielded a null phenotype (identical to the LAT knock-out), demonstrating the critical role of the residues for T cell development. Surprisingly, knock-in mutation of the first tyr residue (tyr136) resulted in a fatal lymphoproliferative disorder characterized by expansion and multitissue infiltration of CD4+ T cells. Consistent with previous data demonstrating that tyr136 preferentially binds to phospholipase C-gamma, examination of the signaling response of T cells from these mice revealed a severe defect in TCR-induced/phospholipase C-gamma-mediated calcium flux. However, MAPK signaling was intact in these cells, indicating that the TCR was selectively uncoupled from the calcium but not from the MAPK pathway. The results reveal a critical role for LAT in coordinating downstream signals initiated by TCR engagement and demonstrate that this function is essential for normal T cell homeostasis. Current studies are using the LAT tyr136 knock-in mice to determine the role of calcium signaling in thymocyte selection and are analyzing other LAT tyr knock-in mutants generated in our laboratory. Sommers CL, Menon RK, Grinberg A, Zhang W, Samelson LE, Love PE. Knock-in mutation of the distal four tyrosines of linker for activation of T cells blocks murine T cell development. J Exp Med 2001;194:135-142. Sommers CL, Park CS, Lee J, Feng C, Fuller CL, Grinberg A, Hildebrand JA, Lacana E, Menon RK, Shores EW, Samelson LE, Love PE. A LAT mutation that inhibits T cell development yet induces lymphoproliferation. Science 2002;296:2040-2043. Structure and signaling potential of the gamma/delta TCR complex Hayes, Love; in collaboration with Fowlkes, Laky Most vertebrate species contain two separate lineages of T cells that are distinguished by the antigen-binding clonotype-specific chains contained within their TCRs: alpha-/beta-T cells and gamma-/delta-T cells. Although the more abundant alpha/beta TCR has been extensively characterized, much less is known about the structure or function of the gamma/ delta TCR, which is expressed on the smaller subset of gamma-delta T cells. We found that the subunit composition of the gamma/delta TCR differs from that of the alpha/beta TCR in that a component of the alpha/beta TCR, the CD3delta chain, is not present in gamma/delta TCRs, revealing a major difference in the subunit structure of the alpha/beta and gamma/delta TCRs. Interestingly, signal transduction by the gamma/delta TCR was found to be superior to that of the alpha/beta TCR as assessed by several criteria. Our data suggest that the structural difference between alpha/beta and gamma/delta TCRs may influence the signaling potential of the TCR complex and could have important functional consequences on T cell activation. Current studies involve further analysis of the effect of TCR subunit structure on signaling responses and aim to determine if TCR subunit composition influences T cell development and T cell lineage commitment. Hayes SM, Laky K, El-Khoury D, Kappes DJ, Fowlkes BJ, Love PE. Activation-induced modification in the CD3 complex of the gammadelta T cell receptor. J Exp Med 2002;196:1355-1361. Hayes, SM, Love, PE. Distinct structure and signaling potential of the gamma delta TCR complex. Immunity 2002;16:827-838. Hayes SM, Shores EW, Love PE. An architectural perspective on signaling by the pre-, alphabeta and gammadelta T cell receptors. Immunol Rev 2003;191:28-37. Role of the chemokine receptor CCR9 in T cell development Uehara, Love; in collaboration with Farber T cell development continues into adulthood and requires the periodic migration of T-progenitor cells from the bone marrow to the thymus. The ordered progression of thymocytes through distinct stages of development is also associated with migration into and between different thymus microenvironments where they are exposed to different growth factors and signals. Chemokines are a group of small, structurally related molecules that regulate trafficking of leukocytes through interactions with a subset of seven-transmembrane, G protein-coupled receptors. The chemokine CCL25 is highly expressed in the thymus and small intestine, the two known sites of T lymphopoesis. CCR9, the receptor for CCL25, is expressed on the majority of thymocytes, raising the possibility that CCR9 and its ligand may play an important role in thymocyte development. To investigate the role of CCR9 during lymphocyte development, we generated CCR9-decient (CCR9-/-) and CCR9-transgenic mice. Surprisingly, both T cell and B cell development appeared normal in CCR9-/- mice. However, bone marrow transplantation experiments demonstrated that lymphocyte progenitors from CCR9-/- mice had a markedly reduced capacity to repopulate the thymus when forced to compete with progenitor cells from CCR9+/+ mice. In other experiments, overexpression of CCR9 in transgenic mice inhibited early thymocyte development and blocked the normal migration of immature thymocytes within the thymus. The results indicate that CCR9 participates in regulating both the migration of progenitor cells to the thymus and the migration of developing thymocytes within the thymus. However, CCR9 is not essential for normal T cell development, suggesting possible functional redundancy. We are currently testing this hypothesis by generating mice decient in both CCR9 and CXCR4, a second chemokine receptor highly expressed on developing thymocytes. Wright TM, Iwasaki A, Weng Y, DeMartino JA, Elkins KL, Farber JM. The CXC chemokine murine monokine induced by IFN-gamma (CXC chemokine ligand 9) is made by APCs, targets lymphocytes including activated B cells, and supports antibody responses to a bacterial pathogen in vivo. J Immunol 2002;169:1433-1443. Uehara S, Grinberg A, Farber JM, Love PE. A role for CCR9 in T lymphocyte development and migration. J Immunol 2002;168:2811-2819. Uehara S, Song K, Farber JM, Love PE. Characterization of CCR9 expression and CCL25/thymus- expressed chemokine responsiveness during T cell development: CD3(high)CD69+ thymocytes and gammadeltaTCR+ thymocytes preferentially respond to CCL25. J Immunol 2002;168:134-142. Exploring the function of developmental transcription factors in T cell development Li, Love Lhx genes encode a conserved family of proteins that function as transcription factors during embryonic development (see report by Heiner Westphal on page 244). Although these genes have been shown to play critical roles in the development of various organ systems, their possible role in lymphopoiesis has not been systematically examined. In addition, the Wnt signaling pathway has recently been found to contribute to T cell maturation, suggesting that Wnt proteins, as well as their receptors and inhibitors, are likely to have important functions during thymocyte development. We have initiated an RT-PCR-based screening for expression of these genes in fetal and adult lymphoid tissues. An attractive feature of the study is that knock-out mice and embryonic stem (ES) cells are already available for many of these genes. Thus, if the expression pattern of specific genes suggests a role in lymphopoiesis, we can rapidly extend our results by analyzing the lymphoid phenotype of knock-out mice or, in the case of embryonic lethality, generating chimeric mice using knock-out ES cells to study lymphopoiesis specifically. COLLABORATORS Joshua Farber, MD, Laboratory of Clinical Investigation, NIAID, Bethesda MD Alfred Singer, MD, Experimental Immunology Branch, NCI, Bethesda MD For further information, contact pel@helix.nih.gov |
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