Genes and Signals Regulating Mammalian Hematopoiesis

Paul Love, MD, PhD, Head, Section on Cellular and Developmental Biology
LiQi Li, MD, PhD, Research Fellow
Julia Pinkhasov, PhD, Postdoctoral Fellow
Renaud Lesourne, PhD, Visiting Fellow
Ki-Duk Song, PhD, Visiting Fellow
Dalal El-Khoury, BS, Technician
Jan Lee, BS, Technician

We aim to elucidate the cellular and molecular events that regulate mammalian hematopoiesis by (1) characterizing the role of T cell antigen receptor (TCR) signals, particularly individual TCR signal–transducing subunits and signal-transducing motifs in T cell development—we employ several genetically altered mouse strains generated by gene targeting and transgenic technology; (2) identifying and analyzing signal-transducing molecules that function downstream of the TCR or inhibit TCR signaling—we aim to understand how these molecules participate in TCR-mediated signaling and to determine the roles played by the molecules and associated signaling pathways in regulating T cell maturation and T cell activation; (3) examining the function of chemokine receptor signaling in developing T cells—chemokine receptors are cell surface proteins that mediate chemotaxis in response to specific ligands that are expressed in discrete regions of the thymus and are candidates for regulating the homing of progenitor cells to the thymus and the intrathymic migration of thymocytes; (4) studying hematopoietic stem cells (HSCs) by characterizing the genes important for the generation, maintenance, and self-renewal of HSCs—we have uncovered a critical function for one protein (Ldb1) in controlling the self-renewal/differentiation cell fate decision in HSCs.

T cell antigen receptor (TCR) signaling in thymocyte development

Song, Lee, El-Khoury, Love; in collaboration with Hayes

Much of our research focuses on investigating 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, CD3-gamma, CD3-delta, and CD3-epsilon). Di-tyrosine residues within ITAMs are phosphorylated upon TCR engagement, and their function is 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 partly regulated by distinct ITAMs. We previously generated zeta-deficient and CD3-epsilon–deficient mice by gene targeting. We genetically reconstituted the mice with transgenes encoding wild-type or signaling-deficient (ITAM-mutant) forms of zeta and CD3-epsilon and characterized the developmental and functional consequences of these alterations for TCR signaling. We found that TCR-ITAMs are functionally equivalent but act in concert to amplify TCR signals. TCR signal amplification was 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). Unexpectedly, we found that multiple TCR-ITAMs were not required for mature T cell effector functions. One possible explanation is that ITAM-mediated signal amplification is not required for mature T cell activation. Another possible explanation is that T cells in ITAM-mutant mice exhibit normal functional responsiveness because of compensatory mechanisms imposed during selection. To resolve this issue, we recently generated a TCR-zeta chain conditional knockin mouse in which T cell development and selection can occur without attenuation of TCR signaling (i.e., in the presence of wild-type 3 ITAM zeta chain), but in which mature, post-selection T cells may be induced to express TCRs containing signaling-defective (0 ITAM) zeta chains in lieu of wild-type zeta chain. Thus, mature T cell signaling should not be influenced by potential compensatory mechanisms that operate during T cell maturation such that T cells in these mice should be faithful indicators of the role of multiple TCR ITAMs in mediating specific, mature T cell responses. Preliminary experiments with the mice have confirmed that the knockin zeta locus functions as predicted. We are currently using this model system to evaluate the role of ITAM multiplicity in mature T cells.

A separate line of investigation centers on the importance of TCR signaling for gamma/delta T cell development. Most vertebrate species, including humans and mice, 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 signal transduction by the gamma/delta TCR to be superior to that by 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 on T cell activation. Indeed, in a study performed in the past year, we showed that TCR signal intensity plays a critical role in regulating alpha/beta versus gamma/delta lineage choice in developing thymocytes. Current studies involve further analysis of the effect of TCR subunit structure on signaling responses.

Erman B, Alag AS, Dahle O, van Laethem F, Sarafova SD, Guinter TI, Sharrow SO, Grinberg A, Love PE, Singer A. Coreceptor signal strength regulates positive selection but does not determine CD4/CD8 lineage choice in a physiologic in vivo model. J Immunol 2006;177:6613-6625.
Hayes SM, Love PE. A retrospective on the requirements for γδ T-cell development. Immunol Rev 2007;215:8-14.
Hayes SM, Love PE. Stoichiometry of the murine γδ T cell receptor. J Exp Med 2006;203:47-52.
Hayes SM, Love PE. Strength of signal: a fundamental mechanism for cell fate specification. Immunol Rev 2006;209:170-175.

Identification and characterization of proteins important for TCR “fine tuning” and TCR signaling

Lesourne, Pinkhasov, Love

We have extended our analysis of TCR signaling subunits to other molecules that participate in or influence the TCR signaling response. The cell-surface protein CD5 negatively regulates TCR signaling and functions in thymocyte selection. 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 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. Reasoning that other molecules besides CD5 participate in TCR tuning, we initiated microarray-based screening for genes differentially expressed in developing T cells under conditions of high- or low-affinity TCR interactions and have identified several candidate genes for further study.

Using a subtractive cDNA library–screening approach, we recently identified a novel T cell–specific protein, T Lymphocyte Adaptor Protein (TLAP). We have cloned this protein, which we renamed AGAPE (Another Grb-Associated Protein Enigma), and generated AGAPE knockdown cell lines, AGAPE knockout mice, and AGAPE transgenic mice. Analysis of the effects of modulating AGAPE expression revealed a function for AGAPE in the TCR signaling pathway. In addition, we discovered an important role for in T cell development.

Role of the chemokine receptor CCR9 in T cell development

Uehara,¹ Love; in collaboration with Farber, Takahama, Butcher, Bhandoola

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 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. These 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 such a hypothesis by generating mice deficient in several chemokine receptors (e.g., CXCR4 and CCR7).

Liu C, Saito F, Liu Z, Lei Y, Uehara S, Love P, Lipp M, Kondo S, Manley N, Takahama Y. Coordination between CCR7- and CCR9-mediated chemokine signals in prevascular fetal thymus colonization. Blood 2006;108:2531-2539.
Schwarz BA, Sambandam A, Maillard I, Harman BC, Love PE, Bhandoola A. Selective thymus settling regulated by cytokine and chemokine receptors. J Immunol 2007;178:2008-2017.
Staton TL, Habtezion A, Winslow MM, Sato T, Love PE, Butcher EC. CD8⁺ recent thymic emigrants home to and efficiently repopulate the small intestine epithelium. Nat Immunol 2006;7:482-488.
Uehara S, Hayes SM, Li L, El-Khoury D, Canelles M, Fowlkes BJ, Love PE. Premature expression of chemokine receptor CCR9 impairs T cell development. J Immunol 2006;176:75-84.

Role of Ldb1 in T cell development

Li, Lee, 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 the LIM-only protein LMO2 and the transcription factors SCL and GATA-1) to form multimolecular transcription complexes. Within the hematopoietic lineage, expression of Ldb1 is highest in lineage-negative, Sca1⁺c-kit⁺ multipotent progenitors, which include HSCs, but gradually decreases as cells commit to and mature within specific lineages. Ldb1^−/− mice die between day 9 and day 10 of gestation, preventing us from 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 and by conditional, stage-specific deletion of Ldb1. Significantly, Ldb1-null ES cells were capable of generating HSCs, which could give rise to both myeloid and lymphoid lineage cells; however, the number of Ldb1^−/− HSCs 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 a failure of self-renewal or survival. More recent data indicate that the loss of Ldb1-null HSCs results from accelerated HSC differentiation. Although expressed in embryonic stem cells (ESC), Ldb1 is not required for ESC maintenance, indicating a selective requirement in adult stem cell populations. Our results provide the first example of a gene that directly regulates HSCs’ cell-fate decision whether to differentiate or self-renew.

¹Shoji Uehara, MD, PhD, former Visiting Fellow

Collaborators

Avinash Bhandoola, PhD, University of Pennsylvania, Philadelphia, PA
Eugene Butcher, MD, Stanford University, Stanford, CA
Joshua Farber, MD, Laboratory of Clinical Investigation, NIAID, Bethesda, MD
Sandra M. Hayes, MD, SUNY Upstate Medical University, Syracuse, NY
Alfred Singer, MD, Experimental Immunology Branch, NCI, Bethesda, MD
Yousuke Takahama, PhD, University of Tokushima, Tokushima, Japan
Heiner Westphal, MD, Program in Genomics of Differentiation, NICHD, Bethesda, MD

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