Our research is directed at elucidating the cellular and molecular processes that regulate mammalian T lymphocyte development. Within this broad context, our studies focus on three main areas. The first involves characterization of the role of T cell antigen receptor (TCR) signals, particularly the role of individual TCR signal-transducing subunits and signal-transducing motifs in T cell development. Our studies employ several genetically altered mouse strains generated by gene targeting and transgenic technology. Second, we have extended our studies to include analysis of signal-transducing molecules that function downstream of the TCR (LAT and TLAP) or that inhibit TCR signaling (CD5). The aim of these studies is to understand how the 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. Third, we have begun to characterize the function of chemokine receptors that are expressed on developing T cells. These cell surface proteins mediate chemotaxis in response to specific ligands that are expressed in discrete regions of the thymus. Chemokine receptors are candidates for regulating the homing of progenitor cells to the thymus and the intrathymic migration of thymocytes.
T cell antigen receptor signaling in thymocyte development
El-Khoury, Love; in collaboration with Shores
Much of our research has concentrated on the role of 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 and CD3-gamma, -delta, and -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 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 for TCR signaling. The results 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) and by which potentially auto-reactive cells that may 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.
Pitcher LA, Mathis MA, Subramanian S, Young JA, Wakeland EK, Love PE, vanOers NS. Selective expression of the 21-kilodalton tyrosine-phosphorylated form of TCR zeta promotes the emergence of T cells with autoreactive potential. J Immunol 2005;174:6071-6079.
Mechanism of CD5-mediated TCR signal inhibition
Lesourne, Love
The cell surface protein CD5 negatively regulates TCR signaling and thus participates in thymocyte selection (Bhandoola et al., Eur J Immunol 2002;32:1811). Examination of CD5 expression during T cell development revealed that surface levels of CD5 are regulated by TCR signal intensity and by the affinity of the TCR for thymic self-peptide ligands 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 others (those that would normally express high levels of CD5). Our 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 the potential for signal modulation may be particularly useful for generating the maximum possible diversity in the mature T cell repertoire. Given that the probable mechanism of CD5 function is via the activation-induced binding of regulatory molecule(s) to sequences within the CD5 cytoplasmic domain, we generated transgenic mice that express a tail-less form of CD5 (mCD5). We 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. The laboratory is currently attempting to identify molecules that interact with CD5 in an effort to determine how CD5 regulates signal transduction by the TCR.
Role of LAT in T cell development
Song, 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 T cell antigen receptor (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; such LAT activity includes activation of the calcium and MAP kinase (MAPK) downstream signaling pathways. The 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 the pathways. We generated knockin mutant mice that express LAT proteins containing single or multiple tyrosinegphenylalanine mutations of the four critical tyrosine residues. Knockin mice that express the wild-type version of the protein exhibited normal T cell development, thereby validating the strategy of targeting specific LAT tyrosines. Inactivation of all four distal LAT tyrosines yielded a null phenotype (identical to the LAT knockout), demonstrating the critical role of the residues for T cell development. Surprisingly, the knockin 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 the mice revealed a severe defect in TCR-induced/phospholipase C-gamma–mediated calcium flux. However, MAPK signaling was intact in the 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 the coordinating function is essential for normal T cell homeostasis. Current studies are determining the role of calcium signaling in thymocyte selection by using the LAT tyr136 knockin mice and analyzing other LAT tyr knockin mutants generated in our laboratory
Sommers CL, Lee J, Steiner KL, Gurson JM, Depersis CL, El-Khoury D, Fuller CL, Shores EW, Love PE, Samelson LE. Mutation of the phospholipase C-gamma1-binding site of LAT affects both positive and negative thymocyte selection. J Exp Med 2005;201:1125-1134.
Sommers CL, Samelson LE, Love PE. LAT: a T lymphocyte adaptor protein that couples the antigen receptor to downstream signaling pathways. BioEssays 2004;26:61-67.
Structure and signaling potential of the gamma/delta TCR complex
Hayes, Li, Love
Most vertebrate species contain two 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. Our findings revealed 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/betaTCR as assessed by several criteria. The data suggest that the structural difference between alpha/beta and gamma/delta TCRs may influence the signaling potential of the TCR complex and may have important functional consequences for T cell activation. Indeed, in a study performed in the past year, we showed that, in developing thymocytes, TCR signal intensity plays a critical role in regulating alpha/beta versus gamma/delta lineage choice. Current studies involve further analysis of the effect of TCR subunit structure on signaling responses.
Hayes SM, Li L, Love PE. TCR signal strength influences alphabeta/gammadelta lineage fate. Immunity 2005;22:583-593.
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 the cells are exposed to different growth factors and signals. Chemokines are a group of small, structurally related molecules that regulate the 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 the 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 functional redundancy. We are currently testing the hypothesis on redundancy by generating mice deficient in both CCR9 and CXCR4, a second chemokine receptor highly expressed on developing thymocytes.
Uehara S, Farber JM, Love PE. Migration of T cell progenitors in the thymus. Curr Med Chem Anti-Inflamm Anti-Allergy Agents 2005 (in press).
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 Westphal). Although the 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, it was recently found that the Wnt signaling pathway contributes to T cell maturation, suggesting that Wnt proteins, as well as their receptors and inhibitors, likely have important functions during thymocyte development. We have initiated RT-PCR–based screening for expression of these genes in fetal and adult lymphoid tissues. An attractive feature of the study is that knockout mice and embryonic stem (ES) cells are already available for many of the genes. Thus, if the expression pattern of specific genes suggests a role in lymphopoiesis, our results can be rapidly extended by analyzing the lymphoid phenotype of knockout mice, or in the case of embryonic lethality, generating chimeric mice using knockout ES cells to study lymphopoiesis in particular.
Woodside KJ, Shen H, Muntzel C, Daller JA, Sommers CL, Love PE. Expression of Dlx and Lhx family homeobox genes in fetal thymus and thymocytes. Gene Exp Patterns 2004;4:315-320.
Collaborators
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, Center for Biologics Evaluation and Research, 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
For further information, contact pel@helix.nih.gov.