The Journal of Immunology Molecular Dissection of Prethymic Progenitor Entry into the T Lymphocyte Developmental Pathway1 C. Chace Tydell,2 Elizabeth-Sharon David-Fung,2,3 Jonathan E. Moore, Lee Rowen,4 Tom Taghon,5 and Ellen V. Rothenberg6 Notch signaling activates T lineage differentiation from hemopoietic progenitors, but relatively few regulators that initiate this program have been identified, e.g., GATA3 and T cell factor-1 (TCF-1) (gene name Tcf7). To identify additional regulators of T cell specification, a cDNA library from mouse Pro-T cells was screened for genes that are specifically up-regulated in intrathymic T cell precursors as compared with myeloid progenitors. Over 90 genes of interest were iden- tified, and 35 of 44 tested were confirmed to be more highly expressed in T lineage precursors relative to precursors of B and/or myeloid lineage. To a remarkable extent, however, expression of these T lineage-enriched genes, including zinc finger transcription factor, helicase, and signaling adaptor genes, was also shared by stem cells (Lin؊Sca-1؉Kit؉CD27؊) and multipotent progenitors (Lin؊Sca-1؉Kit؉CD27؉), although down-regulated in other lineages. Thus, a major fraction of these early T lineage genes are a regulatory legacy from stem cells. The few genes sharply up-regulated between multipotent progenitors and Pro-T cell stages included those encoding transcription factors Bcl11b, TCF-1 (Tcf7), and HEBalt, Notch target Deltex1, Deltex3L, Fkbp5, Eva1, and Tmem131. Like GATA3 and Deltex1, Bcl11b, Fkbp5, and Eva1 were dependent on Notch/Delta signaling for induction in fetal liver precursors, but only Bcl11b and HEBalt were up-regulated between the first two stages of intrathymic T cell development (double negative 1 and double negative 2) corresponding to T lineage specification. Bcl11b was uniquely T lineage restricted and induced by Notch/Delta signaling specifically upon entry into the T lineage differentiation pathway. The Journal of Immunology, 2007, 179: 421–438. he circulating population of mature T lymphocytes is con- cell type, loss of the transcription factor causes a selective block of stantly regenerated as hemopoietic progenitors leave the the developmental pathway while gain of function of the transcrip- T bone marrow (BM)7 and home to the thymus where de- tion factor can accelerate differentiation into the lineage. Equiva- velopment and maturation occur (1–3). Cell-intrinsic regulatory lent “master regulators” of T cell development have not yet been factors that are up-regulated in a lineage-specific way play domi- found. More than eight known transcription factors are essential nant roles in lineage choice of hemopoietic precursors. In RBC for T cell development (reviewed in Refs. 4 and 5), but none of development, GATA1 acts as a central mediator of erythroid gene these exhibits the ability to instruct or accelerate entry into the T expression, and it is known that B cells are instructed specifically cell program. by early B cell transcription factor and Pax5. These transcription Two transitions in early T cell development are of particular factors can be considered “master regulators” because, for each interest for lineage choice mechanisms: the onset of T lineage gene expression (“specification”), and the final exclusion of any fate except a T cell fate (“commitment”). Both transitions occur among Division of Biology, California Institute of Technology, Pasadena, CA 91125 intrathymic, early T lineage cell populations (Pro-T cells), which Received for publication December 18, 2006. Accepted for publication April are still negative for the mature T cell markers CD4 and CD8 and 16, 2007. do not yet express TCRs. These double-negative (DN) cells within The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance the mouse thymus are divided into four stages on the basis of their with 18 U.S.C. Section 1734 solely to indicate this fact. expression of the surface markers Kit, CD25, and CD44 reviewed 1 This work was supported by grants (to E.V.R.) from the National Science Founda- in Refs. 6–8. The first thymocyte population, DN1, maintains tion (MCB-9983129) and National Institutes of Health U.S. Public Health Service much lineage plasticity and, under special conditions, is capable of (R01 CA90233 and R01 CA98925), by National Institutes of Health U.S. Public Health Service awards K08 AI054699 (to C.C.T.) and F32 AI068366 (to J.E.M.); and producing macrophages, NK cells, or dendritic cells, with a minute from the DNA Sequencer Royalty Fund at the California Institute of Technology. subset apparently capable of generating B cells as well (9–12). It 2 C.C.T. and E.-S.D.-F. contributed equally to this study. is not yet clear whether DN1 cells are distinct from prethymic 3 Current address: Theoretical Division, Los Alamos National Laboratory, Los precursors in gene expression pattern. However, as cells enter the Alamos, NM 87544. next stage, DN2, they express sharply increased levels of Pro-T 4 Current address: Institute for Systems Biology, Seattle, WA 98103. cell genes such as those encoding pT␣, CD3␧, CD25, Rag1, and 5 Current address: Department of Clinical Chemistry, Microbiology and Immunology, IL-7R␣ (CD127) (10, 13–16), and some rearrangement begins at University Hospital Ghent, Ghent University, Ghent, Belgium. the DJ␤ and VJ␥ TCR loci (17, 18). In hemopoietic precursors 6 Address correspondence and reprint requests to Dr. Ellen V. Rothenberg, Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125. E-mail (derived from fetal liver) that are differentiating in vitro in re- address: [email protected] sponse to Notch/Delta signaling, the first-appearing DN2 pheno- 7 Abbreviations used in this paper: BM, bone marrow; DN, double negative; Dtx3L, type cells display the same dramatic increase in expression of these Deltex3-like; pT␣, pre-TCR␣; qRT-PCR, quantitative real-time PCR; TCF-1, T cell genes (19). DN2 cells have undergone “specification” but are not factor-1. yet committed to the T lymphocyte pathway; a high proportion of Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 DN2 cells are still able to differentiate into NK cells, macrophages, www.jimmunol.org 422 GENE DISCOVERY FOR T CELL SPECIFICATION IN VIVO AND IN VITRO or dendritic cells (10, 20–23). At the DN3 stage, thymocytes stop Thymus and BM samples were taken from animals 5–7 wk old. The dividing, further increase expression of the Pro-T differentiation animals used were bred and maintained under sterile conditions at Caltech. genes as well as Notch target genes (24), and undergo extensive cDNA library TCR rearrangements. Only at this stage do they become committed to a T cell fate in vivo. Cells only progress beyond the DN3 stage The C.B-17-scid thymocyte, random-primed, cDNA library was con- through successful TCR gene rearrangement and TCR-dependent structed in the pSPORT1 vector (Invitrogen Life Technologies), and was arrayed and spotted at high density onto Hybond-Nϩ nylon filters selection, at which time they graduate from Pro-T cell status, to (Amersham Biosciences) using the Q-BOT robot (Genetix) as described give rise to up to five types of T cells: ␣␤ CD4, ␣␤ CD8, ␥␦, NKT, previously (31). or regulatory T cells. Of all these developmental transitions, surprisingly little is Cell populations for library generation and library screening known about the stages encompassing “T lineage specification,” The two types of cells used as sources of RNA for the subtraction protocol that is, the DN1 to DN2 transition. The regulatory participants in were a bulk population of Pro-T cells and a population of progenitor/ these early stages have not been sufficiently characterized to ex- premyeloid cells. To obtain large numbers of Pro-T cells in the DN1-DN3 plain the outcome, although Notch/Delta signaling plays a role (19, stages, we took advantage of the Rag2 knockout mouse, in which thymo- cyte development arrests at DN3. In the wild-type mouse thymus, DN3 25). Traditional methods to identify all the transcription factors cells account for only 1% of thymocytes, but even without sorting, Rag2 that play key roles in early stages of T lineage specification have knockout thymocytes consist of 90% DN3 Pro-T cells, with the remaining found limited success, in part because transcription factors are typ- cells being DN1, DN2, NK, or thymic stromal cells. Because these cells were not pure Pro-T cells, we refer to them in the text as “Pro-T plus.” A ically present in low copy numbers. T cell precursors at the earliest Ϫ ϩ stages are represented in vivo at any one time by tiny numbers of progenitor/premyeloid population was obtained from Lin Kit BM cells matured in culture toward a myeloid fate. Specifically, BM cells that were cells, providing very limiting material for standard microarray KitϩGr1ϪCD11bϪTer119ϪCD19Ϫ from Rag2ko mice were cultured for analysis. In addition, small fold changes in transcription factor 42 h at 37°C in 5% CO2 in IMDM with 10% heat-inactivated FCS sup- abundance or changes in transcription factor ratios may generate plemented with IL-3 (100 ␮l of WEHI-3B cell supernatant/ml medium), stem ␮ dramatic shifts in cell state. Identification of truly novel regulatory cell factor (Kit ligand) (100 l of BHK-MKL cell supernatant/ml medium), and 10 ng/ml rIL-6 (PeproTech). At the time of harvest, the morphological factors and novel isoforms of known factors has also been limited appearance of the cells ranged from undifferentiated, blast-like cells to mature by the comprehensiveness of the microarrays used (26–29) and by granulocytes. the microarray’s nucleotide probe design. To circumvent these problems, we used a subtractive hybrid- Cell populations for quantitative RNA expression analysis ization technique (30) to probe a mouse Pro-T cell cDNA mac- In addition to the subtraction populations, sorted cell populations were roarray library.