Paul Love, MD, PhD, Head, Section on Cellular and Developmental Biology
Sandra Hayes, PhD, Senior Fellow
Renaud Lesourne, PhD, Visiting Fellow
LiQi Li, PhD, Visiting Fellow
Ki-Duk Song, PhD, Visiting Fellow
Shoji Uehara, MD, PhD, Visiting Fellow
Dalal El-Khoury, BS, Technician
Jan Lee, BS, Technician

We focus on elucidating the cellular and molecular processes that regulate mammalian T lymphocyte development and we currently pursue work in three areas. The first involves characterization of the role of T cell antigen receptor (TCR) signals in T cell development. Our experiments employ a number of genetically altered mouse strains generated by gene targeting and transgenic technology. We have extended these studies to the analysis of signal-transducing molecules that function downstream of the TCR or that inhibit TCR signaling. The aim is to understand how these molecules participate in TCR-mediated signaling and to determine what roles they and the signaling pathways they regulate play in T cell maturation and T cell activation. Second, we have begun to characterize the function of chemokine receptors expressed on developing T cells. These cell-surface proteins mediate chemotaxis in response to specific ligands expressed in discrete regions of the thymus. Chemokine receptors are candidates for regulating homing of progenitor cells to the thymus as well as for regulating intrathymic migration of thymocytes. Finally, in collaboration with Heiner Westphal's laboratory, we have initiated studies examining the role of the nuclear protein Ldb1 in hematopoiesis.

T cell antigen receptor (TCR) signaling in thymocyte development

Lee, El-Khoury, Love; in collaboration with Samelson, Shores, Sommers

For many years, a major theme of our research has been the role of TCR signal transduction in thymocyte development. Signal transduction sequences (termed Immunoreceptor Tyrosine-based Activation Motifs or ITAMs) are contained within four distinct subunits of the multimeric TCR complex (zeta, CD3-gamma, CD3-delta, and CD3-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 non-identical, 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 reconstituting the mice with transgenes encoding wild-type or signaling-deficient (ITAM-mutant) forms of zeta and CD3-epsilon. We then characterized the developmental and functional consequences of these 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 that TCR signal amplification is 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 might cause auto-immune disease are deleted in the thymus (negative selection). Thus, the multi-subunit structure of the TCR may have evolved to enable complex organisms to develop a broad, self-restricted yet auto-tolerant T cell repertoire.

Linker for Activation of T cells (LAT) is an integral membrane protein that functions as a critical adaptor linking the TCR to several downstream signaling pathways required for T cell activation. The distal four tyrosines in LAT (tyr136, tyr175, tyr195, and tyr235) are necessary and sufficient for LAT activity in T cells, including the 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 were 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 knockin mutant mice that expressed LAT proteins containing single or multiple tyrosine-phenylalanine mutations of the four critical tyrosine residues. Knockin mice expressing 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 knockout), demonstrating the critical role of these residues in T cell development. Surprisingly, knockin mutation of the first tyosine 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, the signaling response of T cells from the knockin 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 calcium but not from the MAPK pathway. Our results reveal a critical role for LAT in coordinating downstream signals initiated by TCR engagement and demonstrate that such function is essential for normal T cell homeostasis. More recent work has shown that LAT-mediated signaling is also important for thymocyte selection.

Structure and signaling potential of the gamma/delta TCR complex

Hayes, Li, Love

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. These results revealed a major difference in the subunit structure of the alpha/beta and gamma/delta TCRs. Interestingly, we found that signal transduction by the gamma/delta TCR was superior to that of the alpha/betaTCR as assessed by several criteria. The data suggest that the structural difference between alpha/beta and gamma/delta TCRs influences the signaling potential of the TCR complex, which, in turn, may have important functional consequences for T cell activation. Indeed, we found in a recent study that TCR signal intensity plays a critical role in regulating alpha/beta versus gamma/delta lineage choice in developing thymocytes. Current efforts involve further analysis of the effect of TCR subunit structure on signaling responses.

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 thymocytes 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-deficient (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. Our results indicate that CCR9 participates in regulating 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 the redundancy hypothesis by generating mice deficient in several chemokine receptors (e.g., CCR9 and CCR7).

Role of Ldb1 in T cell development

Li, Love; in collaboration with Westphal

Lim domain binding protein-1 (Ldb1) is a ubiquitously expressed nuclear protein that contains a LIM-zinc-finger-protein interaction motif and a dimerization domain (see report by Westphal). In hematopoietic cells, Ldb1 functions by interacting with and/or recruiting specific partners (including LMO2, SCL, and GATA-1) to form multimolecular transcription complexes. Within the hematopoietic lineage, expression of Ldb1 is highest in lineage-negative, Sca1+c-kit+ (LSK) multipotent progenitors (which include hematopoietic stem cells), although such expression gradually decreases as cells commit to and mature within specific lineages. Ldb1-/- mice die between days 9 and 10 of gestation, excluding the possibility of directly studying the impact of loss of Ldb1 on fetal or adult hematopoiesis. We investigated the role of Ldb1 in hematopoiesis by following the fate of Ldb1-null embryonic stem (ES) cells in mouse blastocyst chimeras. Significantly, Ldb1-null ES cells were capable of generating LSKs in the fetal liver and could give rise to both myeloid and lymphoid lineage cells; however, the number of Ldb1-/- LSKs gradually diminished at later stages of development. Following adoptive transfer of fetal liver cells, Ldb1-null LSKs were rapidly lost over a period of three months, indicating failure of self-renewal or survival. More recent data indicate that the loss of Ldb1-null LSKs is likely attributable to accelerated hematopoietic stem cell differentiation. These data provide the first evidence that LIM domain binding proteins function in hematopoiesis and identify Ldb1 as a critical factor in regulating the maintenance/self-renewal of hematopoietic stem cell populations.

COLLABORATORS

Eugene Butcher, MD, Stanford University School of Medicine, Stanford, CA
Joshua Farber, MD, Laboratory of Clinical Investigation, NIAID, Bethesda, MD
B.J. Fowlkes, PhD, Laboratory of Cellular and Molecular Immunology, NIAID, Bethesda, MD
Lawrence Samelson, MD, Laboratory of Cellular and Molecular Biology, NCI, Bethesda, MD
Elizabeth W. Shores, PhD, Laboratory of Immunology, CBER, FDA, Bethesda, MD
Alfred Singer, MD, Experimental Immunology Branch, NCI, Bethesda, MD
Connie L. Sommers, PhD, Laboratory of Cellular and Molecular Biology, NCI, Bethesda, MD
Yousuke Takahama, PhD, University of Tokushima, Tokushima, Japan
Nicolai Van Oers, PhD, University of Texas Southwestern Medical Center at Dallas, Dallas, TX
Heiner Westphal, MD, Laboratory of Mammalian Genes and Development, NICHD, Bethesda, MD

For further information, contact lovep@mail.nih.gov.