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A Novel Role for HEB Downstream or Parallel to the Pre-TCR Signaling Pathway During ß Thymopoiesis¹

Department of Immunology, Duke University Medical Center, Durham, NC 27710

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
TCR gene rearrangement and expression are central to the development of clonal T lymphocytes. The pre-TCR complex provides the first signal instructing differentiation and proliferation events during the transition from CD4^-CD8^-TCR^- double negative (DN) stage to CD4⁺CD8⁺ double positive (DP) stage. How the pre-TCR signal leads to downstream gene expression is not known. HeLa E-box binding protein (HEB), a basic helix-loop-helix transcription factor, is abundantly detected in thymocytes and is thought to regulate E-box sites present in many T cell-specific gene enhancers, including TCR-, TCR-ß, and CD4. Targeted disruption of HEB results in a 5- to 10-fold reduction in thymic cellularity that can be accounted for by a developmental block at the DN to DP stage transition. Specifically, a dramatic increase in the CD4^low/-CD8⁺CD5^lowHSA⁺TCR^low/- immature single positive population and a concomitant decrease in the subsequent DP population are observed. Adoptive transfer test shows that this defect is cell-autonomous and restricted to the ß T cell lineage. Introduction of an ß TCR transgene into the HEB^ko/ko background is not sufficient to rescue the developmental delay. In vivo CD3 cross-linking analysis of thymocytes indicates that TCR signaling pathway in the HEB^ko/ko mice appears intact. These findings suggest an essential function of HEB in early T cell development, downstream or parallel to the pre-TCR signaling pathway.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The development of T cells from hematopoietic precursors entails a dynamic set of differentiation steps that takes place, in large part, in the thymus and that allows for the generation of a diverse repertoire of Ag-specific T cells. These steps have been subdivided on the basis of specific and ordered surface molecule expression, as well as TCR gene rearrangements and expression. Canonical subdivisions break down thymocytes into at least four main CD4/CD8/TCR-expressing cell subpopulations: CD4^-CD8^-TCR^- double negative (DN),³ CD4⁺CD8⁺TCR^low/int double positive (DP), CD4⁺TCR^high single positive (SP), and CD8⁺TCR^high SP. Maturation proceeds from DN to DP stages, allowing for productive rearrangement of the TCR genes and subsequent positive/negative selection from the DP to either CD4⁺ or CD8⁺ SP stages (1).

In the case of the earliest developmental stage, DN cells have been subdivided more extensively on the basis of CD44 (hyaluronic acid receptor) and CD25 (IL-2R) expression (2). In brief, the earliest multipotential lymphoid precursor cell progresses from the CD44⁺CD25^- stage to the CD44⁺CD25⁺ pro-T cell stage and commits to the T cell lineage. TCR ß, , rearrangement is initiated as the committed T cell progresses to the CD44^-CD25⁺ pre-T cell stage. For the ß T cell lineage, signals mediated by a pre-TCR complex select cells that have productively rearranged TCR ß-chains (ß-selection) and allow for their subsequent cell expansion (3). During the CD25⁺ stage, cells that alternatively commit to the T cell lineage do not utilize a pre-TCR complex and do not progress through the remaining CD4/CD8 developmental steps of the ß lineage (4). The ß lineage continues through a CD4^low/-CD8⁺ immature SP (ISP) population before reaching the CD4⁺CD8⁺ DP stage (5, 6).

Clues into intracellular signaling events during the CD25⁺ DN to CD25^- DP transition have come mostly from transgenic and gene knockout experiments of the nonreceptor protein tyrosine kinase, p56^lck (7, 8). p56^lck lies downstream of pre-TCR/CD3-mediated signaling and has been suggested to be more important to cell proliferation/expansion rather than to differentiation events (9, 10, 11). Accordingly, other signaling molecules and pathways (Ras/MAPK, G-protein) have been implicated to account for the latter processes (12, 13).

Notwithstanding these important hallmarks of early T cell development, our understanding of these complex differentiation steps at the level of transcriptional regulation remains even more limited than the above proximal membrane-associated phenomena. Transcriptional regulation downstream of the pre-TCR/p56^lck pathway must be needed to transduce the environmental inputs into dramatic and distinct changes in gene expression of a developing T cell. While several transcription factors, e.g., EBF-1 (14), Pax-5 (15), and E2A (16, 17), have been shown to be critical for B cell development, fewer transcription factors have been shown to be indispensable for thymopoiesis, that is, whose absence has been shown to completely block development at a precise stage of thymopoiesis (18). Of note, dominant-negative and knockout mutations of zinc-finger proteins Ikaros (19) and Gata-3 (20), respectively, have been shown to be essential at the earliest differentiation events, while a knockout mutation of the high mobility group (HMG) protein TCF-1 and double knockout of LEF-1 and TCF-1 show a specific impairment and more complete arrest at the DN to DP transition, respectively (21, 22).

More recently, it has been suggested that bHLH (basic helix-loop-helix) E-proteins might play a critical role in thymocyte on-togeny. Specifically, the E2A gene products, E12 and E47, are thought to be necessary for thymocyte lineage commitment in mice (23) and humans (24). Further implicating the role of E-protein in thymopoiesis, it has been recognized that E2A knockout mice have an increased incidence of T cell tumors (23, 25). Yet, like many transcription factor knockout phenotypes, E2A^ko/ko thymocyte development is not completely blocked and is associated with hypocellularity but normal distribution of CD4/CD8 subpopulations (23, 25).

Another bHLH E-protein, HeLa E-box binding protein (HEB), is also highly expressed in the thymus (26, 27). HEB (also known as REB (28) and ME-1/Alf-1 (27)) is one of the three mammalian class A bHLH E-proteins (29). The other members include E2A (30) and E2-2 (31). These ubiquitously expressed E-proteins are thought to heterodimerize with other bHLHs via their HLH motif and bind DNA via the adjoining "basic" domain, which then leads to transactivation of downstream target genes containing the consensus CANNTG E-box site (32). Besides high thymic expression of HEB mRNA, an HEB/E2A heterodimer has been shown to bind to a tandem E-box site within the CD4 5' proximal enhancer, which is critical for CD4 expression (33). Most recently, the HEB^ko/ko mouse was generated and analyzed with respect to the overall E-protein contribution to B cell development, specifically the pro-B cell stage. A defect in thymocyte development was noted but not extensively investigated (34).

In this report, we have more fully characterized the thymocyte phenotype in HEB-deficient mice, placing it among other transcription factors that are recognized to be important for normal thymopoiesis. Disruption of HEB leads to an 5- to 10-fold reduction in total thymocyte numbers and a dramatic accumulation of ISP cells between the DN and DP stages. Likewise, the percentage of S and G₂/M phase ISP thymocytes in HEB-deficient mice is dramatically decreased. We demonstrate the function of HEB to be cell-autonomous and its specificity to the ß T cell compartment. More importantly, the block in ß T cell development by HEB^ko/ko cannot be rescued by forced expression of TCR genes to allow progression to the DP stage, suggesting that HEB plays a role either downstream or parallel to a TCR (or pre-TCR) signal, which normally leads to expansion of the DP compartment.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

The HEB^ko strain was generated in our laboratory as previously described (34). Both male and female homozygote mice are sterile, requiring that homozygous mutants be generated from heterozygote crossings. Mice are of a mixed 129/sv and C57BL/6 background. Animals analyzed ranged in age from E18.5 fetuses to 8-wk-old neonates. The RAG2^ko/ko strain (129/SvEv) was provided by Dr. Michael Krangel (Duke University), who purchased the strain from Taconic (Germantown, NY). The AND ß transgenic strain was provided by Dr. Carolyn Doyle (Duke University), who originally received permission to use this strain from Dr. Stephen Hedrick (University of California, San Diego, CA). A founder male (AND x H-2^b) was bred with HEB^ko/+ females. Subsequent HEB^ko/+ AND^tg male progeny were crossed with HEB^ko/+ females to generate HEB^ko/ko AND^tg+ mice. The B6/SJL/lyy5.1 strain was also obtained from C. Doyle, who originally purchased them from The Jackson Laboratory (Bar Harbor, ME). Two- to three-month-old animals were used as hosts in adoptive transfer experiments. All animals were maintained in specific pathogen-free facilities at Duke University (Durham, NC) before sacrifice. Mouse handling and experimental procedures were conducted in accordance with institutional guidelines for animal care and use.

Immunoblot analysis

Nuclear extracts were made from 20 x 10⁶ total thymocytes from age-matched mice. Thymocytes were washed 1x with PBS, resuspended in 10 µl of hypotonic buffer (50 mM HEPES (pH 7.9), 20 mM KCl, 2 mM EDTA, 5% glycerol, 0.1% Triton X-100, 1 mM DTT) and placed on ice for 10 min. A total of 100 µl of nuclear extract buffer was added (50 mM HEPES (pH 7.9), 0.55 M KCl, 20% glycerol, 10% sucrose, 5 mM MgCl₂, 0.05% Triton X-100, 10 mM DTT, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin), placed on ice for 10 min, and centrifuged at 13 K for 15 min at 4°C. Supernatants were kept at -70°C until further use. Protein concentrations were determined by Bio-Rad (Hercules, CA) protein assay.

A 10% SDS-PAGE minigel was loaded with 20 µg of nuclear extract, blotted to nitrocellulose with a Trans-Blot SD Electrophoretic Transfer Cell (Bio-Rad), and blocked with 10% nonfat dried milk in 1x TBS/0.5% Tween 20. Primary Abs included anti-HEB rabbit polyclonal antiserum purchased from Santa Cruz Biotechnology (San Diego, CA) and anti-E2A (32F) rabbit polyclonal antiserum, which was a gift from Tom Kadesch (Howard Hughes Medical Insitute, University of Pennsylvania, Philadelphia, PA) and were used at a 1:2000 dilution in blocking buffer. An alternative anti-HEB serum was provided by Dr. Dan Littman (HHMI, New York University, New York, NY). The secondary donkey anti-rabbit conjugated to HRP (Jackson ImmunoResearch Laboratory, West Grove, PA) was used at a 1:5000 dilution, followed by ECL treatment as suggested by the manufacturer (Amersham, Buckinghamshire, U.K.). Data was quantitated by National Institutes of Health (Bethesda, MD) Image software.

Histology

Neonatal thymi were harvested, embedded in OCT compound (Miles, Elkhart, IN), and frozen in a dry ice/ethanol bath. Samples were kept at -70°C until sectioning at a 4–5 micron thickness on a Jung CM1800 Cryostat (Leica, Deerfield, IL) onto 0.5% gelatin-coated slides and then acetone fixed. Sections were stained with hematoxylin and eosin and analyzed with a dissection scope at 10x and 25x magnification. Images were analyzed and stored using National Institutes of Health software.

Indirect immunofluorescent staining and confocal microscopy

Thymi were treated as above. Tissue sections were cut to a 10-micron thickness onto gelatin-coated slides and acetone fixed. In a humidified chamber, samples were rehydrated with 1x PBS, blocked with a 10% nonfat dried milk/1x TBS/0.1% Tween 20 solution supplemented with 5% bovine calf serum (HyClone, Logan, UT) and 5% goat serum (Sigma, St. Louis, MO), incubated with rabbit anti-HEB primary Ab (Santa Cruz Biotechnology) at a 1:50 dilution in blocking buffer, washed 3 times in 1x PBS, incubated with biotinylated goat anti-rabbit secondary Ab (Sigma) at a 1/80 dilution, washed as before, and incubated with Texas Red-conjugated streptavidin (Jackson ImmunoResearch) and PE-conjugated anti-mouse Thy1 (Sigma) at dilutions of 1/100 and 1/50, respectively. Slides were washed 3 times in 1x PBS, mounted with Vectashield mounting media (Vector Laboratories, Burlingame, CA), and stored in darkness before analysis with a Zeiss (Oberkochen, Germany) confocal microscope. Then, 40x and zoomed single scan images were overlaid and colors assigned with Adobe Photoshop software.

Flow cytometry

Primary isolated thymocytes were immediately placed in cold wash buffer (1x PBS/5% bovine calf serum) and kept on ice for the entire staining procedure. Staining was done with 1–2 x 10⁶ cells in 100 µl wash buffer with 1 µl of mAb for 30 min in darkness. The following mAbs were purchased from PharMingen (San Diego, CA): FITC-conjugated anti- T cell receptor, GL3; FITC- and CyChrome-conjugated anti-CD8, 53-6.7; CyChrome-conjugated anti-CD4, RM4-5; FITC-conjugated anti-CD69, H1.2F3; FITC-conjugated anti-CD45.2, 104; FITC-conjugated anti-CD25, 7D4; PE-conjugated anti-CD44, IM7; PE-conjugated anti-CD5, 53-7.3; CyChrome-conjugated anti-ß TCR, H57-597; biotinylated anti-HSA, M1/69. The following mAbs were purchased from Sigma: PE-conjugated anti-CD3, 29B; PE-conjugated anti-CD4, H129.19; PE-conjugated anti-TCR /ß, H57-597. 7-Aminoactinomycin D (7AAD; Molecular Probes, Leiden, The Netherlands) was used at 1 µg/ml in wash buffer to exclude dead cells. Cells were analyzed with a FACScan (Becton Dickinson, Mountain View, CA); data was stored and displayed with CellQuest software (Becton Dickinson). A total of 10,000–50,000 events was routinely collected.

DNA content analysis

A total of 10 x 10⁶ thymocytes were resuspended in RPMI 1640 with glutamine (Life Technologies, Gaithersburg, MD), supplemented with 5% FBS (HyClone) and 100 U/ml penicillin/100 µg/ml streptomycin (Life Technologies). Hoechst 33342 (Molecular Probes) was added to a final concentration of 6 µg/ml. Resuspended live cells were incubated for 30–60 min at 37°C, washed once with 1x PBS/5% BCS, and stained on ice with CyChrome-conjugated anti-CD4, PE-conjugated anti-TCR ß, and FITC-conjugated anti-CD8, as previously described. For AND^tg mice, the anti-TCR was substituted with biotinylated anti-HSA conjugated to PE-streptavidin (Jackson ImmunoResearch). A total of 50,000–100,000 events was collected with a FACStar^Plus (Becton Dickinson) with 488 argon I-90 and UV lasers.

ISP four-color analysis

CyChrome-conjugated anti-CD4, PE-conjugated anti-TCR ß, and FITC-conjugated anti-CD8 mAbs and biotinylated anti-HSA mAb with Texas-Red-streptavidin (Jackson ImmunoResearch) were used to stain thymocytes as previously described. A total of 50,000–100,000 events was collected using a FACStar^Plus, using argon I-90 and dye lasers (Coherent).

Irradiation and stem cell reconstitution

The B6/SJL/lyy5.1 mice (congenic for the Ly-5 allotype marker) or HEB^ko/ko mice were treated as previously described (35). In brief, host mice at 8–12 wk of age were irradiated with 1100 rads 1 day before stem cell transfusion and maintained in sterile bedding and with antibiotics thereafter. Donor cells were prepared from frozen stocks of fetal liver cells (E14.5 or E18.5). A total of 1–5 x 10⁵ total cells was delivered to the host in 0.2 ml 1x PBS through tail vein injection. For each donor, two to four recipients were used. Mice were sacrificed 6–8 wk after irradiation for FACS analysis, using FITC anti-CD45.2 (PharMingen) to detect host- vs donor-derived cells.

PCR genotyping

Mice were genotyped by a competitive PCR strategy as previously described (35). For HEB, the primer sequences are as follows: YZ-29, 5'-TCGCAGCGCATCGCCTTCTA-3' (neomycin sense, mutant primer); YZ-119, 5'-GACATCAAGGTCTCATCTAGG-3' (HEB sense, common primer); YZ-122, 5'-TCTCACTTGCTGTTCTAGACT-3' (HEB antisense, wild-type primer). Expected size of mutant and wild-type HEB PCR products are 2.8 and 2.1 kilobases, respectively.

Analysis of V(D)J rearrangements by PCR

DNA was isolated from total thymocytes (10⁶) or cell-sorted ISP thymocytes (2–10 x 10⁴) (see above for description). Cells were lysed in 10 mM Tris-HCl (pH 8.0)/1 mM EDTA (pH 8.0)/0.2 µg/ml proteinase K/0.2% Triton X-100 for 30 min at 55°C and then 10 min at 94°C. The following primers were used for PCR (36, 37):Vß8, 5'-GCATGGGCTGAGGCTGATCCATTA-3'; Vß5, 5'-CCCAGCAGATTCTCAGTCCAACAG-3'; Jß2, 5'-TGAGAGCTGTCTCCTACTATCGATT-3'. PCR reactions used 1 µl of the DNA lysate (ISP cells) or dilutions of the DNA lysate (total thymocytes) for template and contained 2.5 mM MgCl₂, 0.33 mM dNTPs, 1 µM of each primer (Vß and Jß2), 1.6 mM (NH4)₂SO₄, 67 mM Tris-HCl (pH 8.8), 0.01% Tween 20, and 1 U Taq DNA polymerase (Life Technologies). Amplification conditions (45 s at 94°C, 45 s at 57°C, and 1 min at 72°C) were repeated 30 times. Products were run on a 1.3% agarose gel, blotted with the TurboBlotter system (Schleicher & Schuell, Keene, NH) onto nitrocellulose, and probed with a Jß2-specific probe (1.4 kb HincII-SacII DNA fragment covering the entire Jß2 region). PCR for the E2A gene was completed with primers as described previously (35). In brief, equivalent amounts of DNA (as above) were used for a PCR of 30 amplification cycles (30 s at 93°C, 30 s at 57°C, and 3 min at 65°C). Products were run on a 0.8% agarose gel, blotted as above, and probed with an E2A-specific DNA probe. PhosphorImager and ImageQuant software (Molecular Dynamics) was used to analyze the blots.

In vivo anti-CD3 treatment

The 145-2C11 hybridoma (38) was purchased from American Type Culture Collection (Manassas, VA) and grown for production of the anti-CD3 mAb as described previously (39). The Ab supernatant was protein G column-purified, dialyzed in 1x PBS, and concentrated with a Centriprep-100 Concentrator (Amicon, Beverly, MA). The control hamster IgG (Armenian and Syrian) was purchased from Southern Biotechnology Associates (Birmingham, AL) then dialyzed in 1x PBS to remove sodium azide before injection. Then, 6- to 8-wk-old wild-type and HEB^ko/ko mice were injected with either 300 µg of 145-2C11 or control hamster IgG i.p., sacrificed 7 days later, and analyzed by flow cytometry. The control RAG2^ko/ko mice in this experiment were 6 mo old. In a second separate experiment, mice were injected with 50 µg of Ab and analyzed 2 days after injection.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
HEB^ko/ko strain expresses no HEB protein

To demonstrate whether the knockout allele abrogated expression of full-length HEB, we undertook Western blot analysis of total thymus nuclear extracts. Previously, the thymus from mouse, rat, and human, as well as various T cell lines had been shown to contain some of the highest levels of HEB mRNA (26, 27). Wild-type (HEB^+/+) and heterozygous (HEB^ko/+) mice had progressively lower levels of HEB protein, i.e., the heterozygote contained 44% of wild type, where wild type was arbitrarily set at 100%. Homozygous HEB knockout (HEB^ko/ko) mice displayed the complete absence of HEB protein (Fig. 1A, lane 3). This result was duplicated with another independently produced HEB antiserum (kindly provided by D. Littman (New York University, New York, NY) data not shown). The E2A level in HEB^ko/ko was increased 2-fold from wild type, and the HEB^ko/+ had a 10% increase in E2A levels from wild type (Fig. 1B). Coomassie-blue staining of an identical gel revealed equal loading of nuclear extract for each genotype (data not shown).

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Western blot analysis of HEB (A) and E2A (B) proteins from total thymocyte nuclear extracts of wild-type (lane 1), HEBko/+ (lane 2), HEBko/ko (lane 3). The black dash indicates the HEB and E2A bands, respectively.

View larger version (82K): [in this window] [in a new window]   FIGURE 2. Indirect immunofluorescent staining and confocal microscopy for Thy1 (green) and HEB (red) from wild-type (A and B) and HEBko/ko (C and D) 10-micron thymus sections, and hematoxylin and eosin-stained wild-type (E and F) and HEBko/ko (G and H) 4-micron thymus sections from 2- to 3-wk-old mice. A 40x (area = 1 mm2) confocal image on the left (A and C) and a zoomed image on the right (B and D) are shown, where the white box indicates the location of the zoomed image in the original section. 10x (E and G) and 25x (F and H) images are shown, where arrows indicate the corticomedullar junction.

When the total number of thymocytes was compared between age-matched HEB^+/+ or HEB^ko/+ and HEB^ko/ko animals, the latter displayed a 5- to 10-fold reduction in total thymocyte numbers (Fig. 3A). Although the magnitude of the reduction varied from litter to litter, the hypocellular phenotype was always seen in the HEB^ko/ko with respect to control littermates (see Fig. 4, A and B for representative examples of total numbers for each genotype). Due to reduced postnatal survival of unknown cause (34) and a less penetrant exencephaly phenotype, it has been difficult to accumulate a larger data set consisting of older animals (4–6 wk) or of animals of the same age. Accordingly, data for mice ranging in age from 18–22 days are presented.

View larger version (32K): [in this window] [in a new window]   FIGURE 3. Preweaning age total thymocyte cellularity (A), fetal E18.5 total, DN and thymocyte cellularity (B), and /CD3 FACS plot of fetal E18.5 thymocytes (C) from wild-type or HEBko/+ and HEBko/ko mice. A and B, Four separate experiments were combined in each graph, where each circle represents an individual animal, and numbers (n) of each genotype are indicated. A, Litters varied in age from 18 to 22 days. For each group, the total cell number for each individual animal was presented as a percentage of the average cell number of wild-type and HEBko/+ thymocytes, where the average was arbitrarily set at 100%. The average percentage (± SDs) for the six wild-type/HEBko/+ and five HEBko/ko mice are 100 ± 36% and 16 ± 12%, respectively. B, The wild-type/HEBko/+ average cell number of the combined data sets was fixed at 100%, and cell numbers for each individual animal were converted to a percentage of this average. The average percentages (± SDs) for wild-type/HEBko/+ are: (total) 100 ± 49%, (DN) 15 ± 9%, and () 3 ± 1.6%, and for HEBko/ko are: (total) 30 ± 18%, (DN) 23 ± 14%, and () 3 ± 1.8%. C, Representative FACS plot where 50,000 events were gated for lymphocytes and to exclude dead 7AAD+ cells (data not shown), and the remaining 7AAD- cells were plotted with respect to FITC-conjugated anti- and PE-conjugated anti-CD3. The relative percentage and total number of CD3++ cells (the boxed cell population) are shown, as well as total thymocyte counts (lower right).

View larger version (30K): [in this window] [in a new window]   FIGURE 4. A, Three-color flow cytometry analysis of E18.5 fetal thymocytes from wild-type and HEBko/ko for CyChrome-conjugated anti-CD4, FITC-conjugated anti-CD8, and PE-conjugated anti-TCRß. Representative CD4/CD8 FACS plots are presented with total cell number of thymocytes (top of plot). Size scatters and TCR levels for the boxed region (R3) are presented as a histogram (right). A total of 10,000 events was collected. B, Three-color CD4, CD8, and TCR FACS analysis as in A of thymocytes from 3-wk-old wild-type and HEBko/ko littermates. Total cell number of thymocytes (top of plot) and relative percentage of each quadrant are given. TCR levels for each quadrant are presented as a histogram (right) and are in correspondence to the CD4/CD8 plot. A total of 10,000 events was collected. C, ISP vs CD8+ SP cell discrimination from the CD4low/-CD8+ population by four-color flow cytometry with CyChrome-conjugated anti-CD4, FITC-conjugated anti-CD8, TexasRed-conjugated anti-HSA, and PE-conjugated anti-TCR of 2-wk-old neonatal wild-type and HEBko/ko thymocytes. From a CD4/CD8 FACS plot, a gate was drawn for the CD4low/-CD8+ cells (left) and was analyzed with respect to HSA/TCR levels (right). Relative percentage and total number of HSA+TCR- and HSAlow/-TCR+ populations (boxes) are shown. A total of 100,000 events was collected. The data is representative of four separate experiments. Total thymocyte counts are indicated (left top).

Normal thymocyte numbers

The total number of thymocytes was similar in both HEB^ko/ko and control littermates of 18.5E fetal thymi, when analyzed for CD3⁺⁺7AAD^- cells by three-color flow cytometry (Fig. 3, B and C). In fact, an 2-fold increase in the thymocyte relative percentage with respect to the total 7AAD^- gate was seen in the HEB^ko/ko as compared with control littermates (Fig. 3C). This finding suggests that thymic development of the lineage remains unaffected by targeted disruption of HEB.

Decreased DP thymocyte subpopulation

The hypocellularity and morphology of HEB^ko/ko thymi pointed to an early HEB-dependent block in thymopoiesis. Three-color flow cytometry of HEB^ko/ko fetal and 3-wk-old thymocytes revealed a distinct CD4/CD8/TCR pattern when compared with wild-type littermates (Fig. 4, A and B). HEB^ko/ko mice displayed a severe reduction in their CD4⁺CD8⁺TCR^int DP cells by both relative percentage and total numbers, and a concomitant increase in the relative percentage of the heterogeneous CD4^-CD8^-TCR^- DN and CD4^low/-CD8⁺ populations.

A prominent ISP-like subpopulation found in HEB^ko/ko

An ISP population, transitional thymocytes between the DN and DP stages, had been differentiated from more mature SP thymocytes based on its TCR^low, CD5^low, HSA⁺ surface staining (5, 6). A transitional population of thymocytes with ISP-like features was accumulated in HEB^ko/ko mice. As shown in Fig. 4, A and B, the HEB^ko/ko ISP-like cells were CD4^low/-, TCR^low/-, and larger in size, indicating that they are at a developmental stage earlier than the DP stage. The HEB^ko/ko ISP-like cells also express low levels of CD5, an Ag that is highly expressed in true DP cells (34). These cells were first detected in fetal thymus before the appearance of the true DP cells (Fig. 4A). At the same fetal stage, the cellularity for HEB^ko/ko is significantly lower than the wild type, further indicating that the ISP-like cells in HEB^ko/ko mice are not equivalent to the true DP cells. Concurrent with the accumulation of ISP-like cells, the appearance of DP and SP cells in HEB^ko/ko mice is significantly delayed (compare Fig. 4A and 4B). The numbers of DP and SP cells in HEB^ko/ko mice are also lower than the age-matched wild-type mice (data not shown).

In older animals, the ISP cells cannot be easily separated from the SP cells in two-color CD4/CD8 FACS analysis. To resolve more definitively the CD4^low/-CD8⁺ ISP and SP thymocytes, cells were analyzed by four-color flow cytometry for HSA, TCR, CD4, and CD8, as well as by three-color flow and UV for their TCR, CD4, CD8, and DNA content. The mixed CD4^low/- CD8⁺ population in both HEB^+/+ and HEB^ko/ko was resolved by their HSA and TCR levels into SP and ISP subpopulations (Fig. 4C). Confirming that the "transitional" population was of a more immature stage of thymocyte development (as suggested above), the analysis showed an 2- to 3-fold increase in the total number of CD4^low/-CD8⁺HSA^high TCR^low ISP cells and a 5- to 6-fold decrease in the CD4^-CD8⁺HSA^lowTCR^high SP cells of HEB^ko/ko vs HEB^+/+ and/or HEB^ko/+.

The ISP population has been observed to be mostly cycling cells, which is in sharp contrast to other thymocyte subpopulations (6). In one typical experiment of three independent and reproducible analyses (Table I), HEB^+/+ mice had 13% CD8⁺ SP vs 52% ISP cells in S and G₂/M phases, as assessed by Hoechst 33342-staining of live cells (Fig. 5, bottom left). Surprisingly, HEB^ko/ko mice had 13% SP vs 15% ISP cells, respectively (Fig. 5, bottom right). Consistent with DNA content analysis, forward/side scatter plots show that the majority of cells is large in wild type and is small in HEB^ko/ko (data not shown). Both HEB^+/+ and HEB^ko/ko had comparable percentages of DN (21.5 ± 2.0% vs 16.3 ± 4.7%), DP (10.1 ± 3.4% vs 10.5 ± 3.1%), and CD4⁺ SP (3.7 ± 0.6% vs 5.5 ± 1.0%) cells in cycle, respectively (data not shown). Such a reduced proliferation capacity specific to the ISP stage indicates an important regulatory role for HEB in driving expansion toward the DP stage.

View larger version (36K): [in this window] [in a new window]   FIGURE 5. DNA content analysis of ISP and CD8+ SP populations. Simultaneous three-color flow cytometry (as in part A but without HSA) and Hoechst 33342-staining: (top) CD4/CD8 FACS plot used to create a R1 gate of the CD4low/-CD8+ population with relative percentage of total cell number shown; (middle) TCR levels of the R1 gate where R2 = TCRlow/- cells and R3 = TCR int/high cells, with relative percentages shown; (bottom) R2 and R3 gates analyzed for DNA content, with percentage of cells in S and G2/M shown for each subpopulation. A total of 100,000 events was collected, and the data are representative of three separate experiments.

The relative percentage of CD4^-CD8^-TCR^- DN cells with respect to the HEB^+/+/HEB^ko/+ combined average total cell number (i.e., the absolute DN number for each animal divided by the average total thymocyte number of HEB^+/+/HEB^ko/+), was increased 2-fold in E18.5 fetal HEB^ko/ko mice (Fig. 3B). Similar analysis of 3-wk-old animals demonstrated an attenuation of this phenotype, where the absolute number of DN cells was not significantly different between the neonatal HEB^ko/ko and HEB^+/+/HEB^ko/+ genotypes (1.7 ± 0.5 x 10⁶ vs 1.6 ± 0.9 x 10⁶, respectively). However, an increase in the relative percentage of the DN population with respect to other CD4/CD8 subpopulations persisted in the neonatal animals (Fig. 4B).

To further subdivide the DN population on the basis of CD44 and CD25 expression, three-color flow cytometry was done on 2-day-old neonatal thymocytes from HEB^+/+, HEB^ko/+, and HEB^ko/ko, where one fluorescent channel was used as an exclusion channel for cells with high levels of CD4, CD8, and/or TCR (Fig. 6, left panels). An increase in the percentage of the CD44^-CD25⁺ DN cells (HEB^+/+ or HEB^ko/+ vs HEB^ko/ko, 53 ± 4% vs 69 ± 4%, respectively) was observed (Fig. 6, right panels), which is similar to the RAG-2 knockout mice (Fig. 6, lower right). A slight increase in the percentage of CD44⁺CD25⁺ cells was also observed in HEB^ko/ko mice, but this phenotype disappears in 2- to 4-wk-old animals (data not shown).

View larger version (66K): [in this window] [in a new window]   FIGURE 6. CD44/CD25 expression pattern of DN cells. Three-color flow cytometry for PE-conjugated anti-CD44, FITC-conjugated anti-CD25, and CyChrome-conjugated anti-CD4, anti-CD8, and anti-TCRß (exclusion channel) on neonatal 2-day thymocytes from wild-type/HEBko/+ (n = 7) and HEBko/ko (n = 3) mice. Inclusion gate is shown on the right with relative percentage of total events. Low to intermediate CyChrome-staining cells are plotted for CD44 and CD25. Representative FACS plots of 50,000 total events are shown with relative percentage of each quadrant. A 7-day-old RAG-2ko/ko is shown as a control.

Due the dynamic interplay between the thymic stromal environment and thymocytes throughout their development (40), we pursued stem cell adoptive transfer experiments to address whether the above phenotypes were cell autonomous and/or dependent on stromal elements. Equal numbers of fetal liver-derived "stem cells" from HEB^+/+ and HEB^ko/ko mice were tail vein-injected into lethally irradiated recipients, which are allelic for CD45 (CD45.1) with respect to the donor (CD45.2). After 6–8 wk, reconstituted mice were sacrificed, and their bone marrow, spleen, and thymus were analyzed by three-color flow cytometry. In most cases, >95% of lymphocytes were CD45.2⁺ cells of donor origin (Fig. 7A, left panels). For thymocytes, mice reconstituted with wild-type stem cells gave a normal CD4/CD8 pattern, whereas those mice receiving HEB^ko/ko stem cells recapitulated the previously observed abnormal distribution of thymocyte subpopulations (Fig. 7A, right panels). The small population of CD45.2^- cells (of host origin) that were found in mice receiving HEB^ko/ko stem cells gave a normal CD4/CD8 pattern (data not shown).

View larger version (64K): [in this window] [in a new window]   FIGURE 7. Adoptive transfer of HEBko/ko stem cells into a wild-type host and wild-type stem cells into a HEBko/ko host. Three-color flow cytometry for FITC-conjugated anti-CD45.2 (donor allelic marker), PE-conjugated anti-CD4, and CyChrome-conjugated anti-CD8 was completed on thymocytes 8 wk postadoptive transfer. Representative FACS plot for CD45 and CD8 is shown (left), where the CD45.2+ (A) or CD45.2- (B) cell gate is given with its relative percentage with respect to total thymocytes. FACS plot for CD4/CD8 (right) of the gated CD45.2+ (top and middle) or CD45.2- (bottom) cells is shown with the relative percentage for each quadrant; the absolute number of donor-derived cells is shown above the plot. *, The mouse used in this experiment was doubly deficient in HEB and Id3, i.e., HEBko/ko Id3ko/ko genotype. We have found that Id3-deficiency (but not Id1-deficiency) significantly and specifically increases the numbers of surviving HEBko/ko neonates. The HEBko/ko T cell phenotype is not perturbed by Id3-deficiency (data not shown).

TCR transgene cannot bypass HEB^ko/ko arrest

TCR gene rearrangement and expression are obligatory events in the transition from the DN to DP stage. Given the involvement of E2A proteins in Ig gene transcription (17, 41, 42), we hypothesized that HEB may play a parallel role in TCR gene transcription. By providing the HEB^ko/ko mice with a rearranged TCR transgene, we may have been able to rescue the block in thymopoiesis. Rescue of blocked DN thymocytes to the DP stage (as well as to the SP stage) by introduction of a TCR ß transgene has been demonstrated for several knockout mutations, such as RAG-2 and IL-7-R, using the chicken OVA TCR and H-Y TCR, respectively (43, 44). More recently, the AND ß transgene has been shown to rescue thymocyte development in H-2^b RAG-2^ko/ko strain (45, 46). The AND ß TCR transgene is specific for a PCC peptide on MHC II I-E^k and selects the majority of cells to the CD4 lineage in both H-2^b and H-2^k backgrounds with endogenous peptides (47, 48). To test whether the AND transgene could rescue the HEB^ko/ko DN thymocytes to the DP stage in an analogous manner, we crossed HEB^ko/+ mice with HEB^ko/+ AND^tg animals (both of mixed B6/129sv background).

E18.5 fetal thymocytes from the six possible genotypes were analyzed by three-color flow cytometry. The CD4/CD8 FACS profile of HEB^ko/ko thymocytes was relatively unchanged by the introduction of the ß AND transgene (Fig. 8A). Like HEB^ko/ko, few SP and DP cells could be found in the transgenic HEB^ko/ko mice. The overall HEB^ko/ko AND^tg thymus remained hypocellular with respect to wild-type AND^tg, but showed a slight increase with respect to nontransgenic HEB^ko/ko. This increase in HEB^ko/ko AND^tg cellularity may be due to the higher DN cell numbers that were observed in all AND^tg animals (wild type or HEB^ko/ko). The transgene-positive thymocytes expressed high TCR (presumably AND) and CD3 levels (Fig. 8A, right, and data not shown, respectively), indicating that expression of the transgene was not HEB-dependent and occurred at the DN stage.

View larger version (49K): [in this window] [in a new window]   FIGURE 8. ß AND transgene rescue. A, Fetal E18.5 thymocytes were analyzed by three-color flow cytometry for CyChrome-conjugated anti-CD4, FITC-conjugated anti-CD8, and conjugated-PE anti-TCR. Representative CD4/CD8 FACS plots and TCR histograms of total thymocytes for HEBko/+, HEBko/+ ANDtg, HEBko/ko, and HEBko/ko ANDtg mice. Number of mice = 8, 8, 8, and 3, respectively. Total thymocyte numbers and relative percentage of each quadrant are shown. B, Adoptive transfer of HEBko/ko ANDtg fetal stem cells to wild-type donors. Donor cell genotypes are shown. Three-color flow cytometry and analysis was completed as for Fig. 7. Total number of CD45.2+ thymocytes and relative percentages for each quadrant are shown. Number of animals transferred with a specific genotype of stem cell = two wild-type/ANDtg, two HEBko/ko, and threeHEBko/ko ANDtg.

The negative outcome of the TCR transgenic experiment does not completely rule out the possibility that HEB is still required for TCR expression and rearrangement. To further evaluate this possible role, the relative levels of endogenous rearrangement at the ß locus were analyzed. A PCR assay was used with a 5' Vß-specific primer (Vß8 or Vß5) and a primer that is 3' to the Jß2 cluster (36, 37). The source of DNA template was genomic DNA isolated either from cell-sorted CD4^low/-CD8⁺HSA⁺TCR^- ISP cells or from total thymocytes of neonatal wild-type and HEB^ko/ko mice. This assay revealed no obvious difference in the relative levels of rearrangement between wild-type and mutant cell populations (ISP and total thymocytes) for either of the Vßs analyzed (Fig. 9). This result, together with the TCR transgenic result, suggests a role for HEB downstream or parallel to the pre-TCR signaling pathway, dissociated from TCR rearrangement and expression.

View larger version (77K): [in this window] [in a new window]   FIGURE 9. PCR analysis of the relative levels of TCRß V(D)J rearrangement in ISP and total thymocytes. Top panel, PCR products were run on a 1.3% agarose gel, blotted, and hybridized with a Jß2 probe. The 5' primer is either Vß8- (left) or Vß5-specific (right), and the 3' primer lies downstream of the Jß2 cluster. Expected products are listed on the right and size markers on the left. +/+ indicates wild-type (lanes 1, 3, 5, and 7), and ko/ko indicates HEBko/ko.mice (lanes 2, 4, 6, and 8). ISP cells are thymocytes sorted and collected with a CD4low/-CD8+HSA+TCRlow/- phenotype (see Fig. 4B). Bottom panel, Blot hybridization of the PCR product for the E2A gene, used to compare the relative amount of genomic DNA in the above TCRß V(D)J PCR.

In vivo anti-CD3 treatment depletes the HEB^ko/ko ISP and DP thymocytes

To test whether TCR/CD3-mediated signaling was intact in HEB^ko/ko mice, we made i.p. injections of wild-type and HEB^ko/ko mice with either the 145-2C11 anti-CD3 mAb or a hamster IgG control Ab. It has been shown that CD3 cross-linking will lead to depletion of DP cells in wild-type animals and differentiation of the DN cells to the DP stage in RAG2^-/- mice (49). Animals were analyzed by flow cytometry 7 or 2 days postinjection with 300 µg and 50 µg of Ab, respectively. The HEB^ko/ko mice displayed a severe reduction in thymic cellularity (Fig. 10, A and B), similar to or worse than wild-type controls, respectively, and as noted by others (50, 51). The RAG-2^-/- mice displayed an efficient rescue and expansion from the DN to the DP stage. Depletion of the ISP thymocytes in the HEB^ko/ko mice suggest that this cell population can be affected in a similar fashion as DP thymocytes, and that the signaling pathways, which are believed to lead to the loss of these immature populations, are not compromised.

View larger version (39K): [in this window] [in a new window]   FIGURE 10. Depletion of HEBko/ko ISP and DP thymocytes by in vivo anti-CD3 i.p. injection. A, Wild-type (left), HEBko/ko (center), and RAG2ko/ko (right) mice were treated with 300 µg of hamster IgG (top) or of 145-2C11 (middle) Ab, and their thymi were analyzed 1 wk later. B, Wild-type and HEBko/ko mice were treated with 50 µg of 145-2C11 and analyzed 2 days later. Representative CD4/CD8 FACS dot plots for each genotype/condition are shown with the total number of recovered thymocytes (when determined) and relative percentage per quadrant.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

A role downstream of TCR signaling

Surface expression of TCRß has been shown to be necessary and sufficient (assuming its pairing with the p-T -chain to form the pre-TCR) to drive T cell differentiation from the DN to DP stage, and with TCR (in the context of ß TCR) from the DP to SP stage (52). For instance, a ß transgene can rescue arrested RAG-2 knockout DN thymocytes to the DP (and SP) stage(s) (43). In contrast, thymocyte development of p56^lck knockout mice was not restored by a TCR ß or ß transgene (10), placing it genetically downstream of TCR. This latter observation mirrors our failure of the AND ß transgene to circumvent the inefficient transition out of the ISP stage in HEB^ko/ko mice (Fig. 8). In accord with the cytofluorometric data, DNA content analysis of the CD4^low/-CD8⁺HSA⁺ ISP population of 4-wk-old HEB^ko/ko AND⁺ neonates revealed the same decrease in the percentage of S and G₂/M phase cells when compared with the same population in controls (data not shown). The trivial argument that failure to rescue is due to peculiarity of the AND transgene seems less likely due to the ability of AND to rescue the RAG-2 knockout phenotype (45, 46).

It remains unclear which pre-TCR-mediated downstream events (or pre-TCR-independent events) require HEB for efficient progression to the DP stage, e.g., proliferation and CD4/CD8 coreceptor expression. At least four nonmutually exclusive possibilities may exist. First, HEB may be downstream of an unknown pathway that is independent of TCR (pre-TCR) signals at this transition. Second, proliferation of the ß-selected ISP cells may be compromised due to HEB-dependent expression of a limiting factor that plays a direct role in the signaling pathway. Alternatively, proliferation signals may remain intact while subsequent, more distal stage-specific gene expression is delayed. Initial semiquantitative RT-PCR analysis of a few known T cell-specific genes (e.g., RAG-2, SLP-76, p56^lck, and CD3///) in both HEB^ko/ko and wild-type ISP populations indicates no dramatic differences in their expression (data not shown). The observation of an increased number of ISP cells in G₁ phase of cell cycle and a reduction in the S and G₂/M pool in the HEB knockout mice when compared with wild-type ISP, lends support to an impairment in proliferation. Yet it cannot be ruled out that the noncycling ISP cells represent a postproliferation population with more limited cycling potential. Finally, cells that have failed to enter the DP stage may undergo apoptosis. Our flow cytometry analyses using either Hoechst 33342 or 7AAD did not detect any obvious increase in the numbers of apoptotic cells from HEB^ko/ko thymus (data not shown); therefore, apoptosis may not be sufficient to explain the deficit of total thymocytes. As noted by Surh and Sprent (53), efficient clearance of apoptotic thymocytes by resident macrophages may explain our inability to detect increased apoptosis in HEB^ko/ko. TUNEL assay of thymus sections does indicate increased apoptosis in HEB^ko/ko mice relative to wild-type controls (data not shown). Whether this is a direct or an indirect effect of HEB-deficiency on any particular apoptotic pathway is unclear. Attempts to rescue the phenotype by expression of a T cell-specific Bcl-2 transgene are underway.

In other systems, it has been possible to bypass the requisite expression of rearranged TCR chain(s) at the DN to DP transition by injection of anti-CD3 Ab. This is due to the presence of low levels of CD3 complexes ( or ) that remain unassociated with TCR chains (54) but that retain interactions with the intracellular signaling molecules, e.g., src- and syk-family PTKs (55). For TCRß^-/-, RAG2^-/-, and CD3^-/- (56, 49, 57), expansion and "differentiation" (loss of CD25 and gain of coreceptor expression) are seen upon anti-CD3 treatment, but for RAG1^-/-CD3^-/- and RAG1^-/-lck^-/- mice, "differentiation" was noted with and without accompanying proliferation, respectively (9). In the case of HEB^ko/ko, the ISP thymocytes were depleted, rather than expanded and differentiated. This suggests that the HEB^ko/ko ISP share characteristics, i.e., apoptosis sensitivity, more closely with DP than DN thymocytes. Yet the ISP cells do not express CD4 and CD5 and are greatly reduced in total cell number, unlike canonical DP thymocytes. In this regard, the HEB^ko/ko ISP population display limited cycling potential or may represent a postcycling population. The data leaves open the possibility that the ISP cells were rescued to the DP stage before death, although the data at early time points, when RAG-2^ko/ko mice are thought to have incomplete DN to DP rescue (49), make this less likely. The in vivo results could be attributed directly to the anti-CD3 effect on thymocytes or a completely unrelated and indirectly induced cytokine-mediated death pathway (50). In any event, the anti-CD3 effect suggests the competence of these signaling pathways.

Putative targets of HEB

Although our experiments with the AND transgene demonstrate that HEB is not essential for expression of the previously rearranged transgenes for the TCR - and ß-chains, it is still possible that HEB plays a role in TCR expression and regulation at the endogenous and ß enhancers, whose loci undergo V(D)J rearrangements and presumably more complex transcriptional regulation. Yet, limited analysis of TCRß and TCR V(D)J recombination within the ISP population and total thymocytes of HEB^ko/ko and wild-type mice indicates similar levels of rearrangement (Fig. 9, and data not shown). The unique involvement of HEB in rearrangement seems unlikely due to the leakiness of the HEB-deficient phenotype. Nevertheless, it should be noted that E-box binding sites (CANNTG) can be located in both the TCR ß (ßE3 and ßE6) and TCR (T3 and T4) enhancers (58, 59, 60). Furthermore, the E2A-encoded E-protein, E47, has been shown to induce IgH sterile transcripts in a pre-T cell line (41) and nonlymphoid cell lines (42), suggesting a role for the bHLH E-proteins in Ig transcription in B cells. In T cells, HEB may play an analogous role. However, as an added complication in T cell development, a role for HEB in regulating TCR gene expression may not be specific, or at least, may be compensated for by other E-proteins, such as E2A gene products E12 and E47 (Fig. 1B), which are coexpressed with HEB in thymocytes. Such functional compensation among same class or unrelated transcription factors may account for the significant yet reduced thymopoiesis in HEB^ko/ko mice.

The multifaceted phenotype of HEB^ko/ko may point to other direct gene targets of HEB. For instance, HEB^ko/ko fetal thymocytes had been shown to express lower levels of CD5 at the DN to DP transition (34), as did neonatal animals in our current experiments (data not shown). Initial dissection of the CD5 promoter has been done (61), and E-box sites can be identified in the promoter. Likewise, the observed dysregulation of CD4 surface levels in HEB^ko/ko ISP and DP subpopulations correlates with the previous observation of the requirement for an HEB/E2A heterodimer at the CD4 minimal enhancer (33). In fact, our HEB^ko/ko phenotypic analysis may indicate that CD4 derepression (at the DN to DP stages) has a specific HEB requirement that distinguishes HEB from E2A function, while at later stages E2A-dependent dimers can act equivalently at the enhancer for constitutive expression in CD4⁺ SP T cells. Alternatively, this enhancer may become inactive at the more mature stages with expression determined from other cis-elements, e.g., 5' distal enhancer (62), and consequently another set of transcription factors. In any event, CD4⁺ SP cells are able to further mature in the HEB^ko/ko thymus (Fig. 4), populate the periphery, and express high CD4 levels indistinguishable from their wild-type counterparts with only minor reductions in splenic and lymph node T cell cellularity, e.g., a decrease in the ratio of CD4⁺:CD8⁺ T lymphocytes and an increase in the ratio of B-:T-lymphocytes (data not shown, and Ref. 34).

Importantly, TCR - (52), CD5- (63), and CD4-deficient (64) mice do not reveal any obvious phenotype in thymocyte development at the same early developmental stages as HEB^ko/ko. Therefore, altered gene expression in these loci is not sufficient to account for the dramatic accumulation of ISP-like cells and reduction of total thymic cellularity in HEB^ko/ko mice. These developmental defects may be attributed to HEB-dependent gene regulation of other unknown targets or multiple downstream targets.

Functional relationship between HEB and TCF

A similar noncycling CD4^low/-CD8⁺ ISP phenotype and concomitant normal T cell phenotype were reported for mice with targeted mutations in the TCF-1 gene (21, 65), which encodes for a HMG transcription factor with LEF-1-like DNA-binding activity (66), but with a T cell-specific expression pattern (67). More recently, the double knockout of TCF-1 and LEF-1 revealed a more dramatic ISP phenotype and a severe reduction in TCR gene transcription (22). In fact, E-boxes and HMG-binding sites are found in the T cell-specific enhancers of several genes, including TCR and ß genes (56), adenosine deanimase (68), and CD4 coreceptor (69). For the latter, the HMG-protein consensus site (CD4-2) lies upstream of the tandem E-box site (CD4-3) within the CD4 5' proximal enhancer (69). From these coincident phenotypes of HEB^ko/ko and TCF^ko/ko mice, it is suggestive that these two classes of transcription factors act synergistically in a combinatorial array at the CD4 enhancer and other gene regulatory sequences at the early stages of thymocyte development.

The HEB^ko/ko T lymphocyte developmental phenotype provides an in vivo nodal point of E-protein function in the processes of differentiation, proliferation, and apoptosis. Our attempts to bypass or rescue the TCR pathway were unsuccessful in affecting the HEB^ko/ko phenotype. Consequently, a distinct and direct role for HEB on known T lymphocyte gene expression and participation in the pre-TCR/TCR signaling pathway remains unclear. Importantly, the phenotypic results for HEB highlight the limited knowledge of and appreciation for the critical interplay between tissue-specific and more fundamental cellular pathways in the immune system.

	Acknowledgments

 
We thank Lihua Pan, Dr. Michael Krangel, Dr. Carolyn Doyle, and Dr. Cristina Hernandez-Munain for critical reading of the manuscript. We are grateful to Dr. Gabriel Bikah and the Duke Cancer Center Flow Cytometry Facility for technical support.

	Footnotes

 
¹ This work was supported by the Whitehead Scholarship and National Cancer Institute Grant R01CA72433-01 (to Y.Z.).

² Address correspondence and reprint requests to Dr. Yuan Zhuang, Department of Immunology, Box 3010, Duke University Medical Center, Durham, NC 27710. E-mail:

³ Abbreviations used in this paper: DN, double negative; DP, double positive; SP, single positive; ISP, immature SP; HEB, HeLa E-box binding protein; HMG, high mobility group; bHLH, basic helix-loop-helix; 7AAD, 7-aminoactinomycin D.

Received for publication April 15, 1999. Accepted for publication July 7, 1999.

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