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The Journal of Immunology, 2003, 170: 1299-1303.
Copyright © 2003 by The American Association of Immunologists

Entry into the Thymic Microenvironment Triggers Notch Activation in the Earliest Migrant T Cell Progenitors 1

Benjamin C. Harman, Eric J. Jenkinson and Graham Anderson2

Department of Anatomy, Medical Research Council Center for Immune Regulation, Division of Immunity and Infection, Medical School, University of Birmingham, Edgbaston, Birmingham, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interactions between T cell precursors and thymic stromal cells are essential during thymocyte development. However, the role of the thymus in initial commitment of lymphoid progenitors to the T lineage remains controversial, with data providing evidence for both extra- and intrathymic commitment mechanisms. In this context, it is clear that Notch1 is an important mediator during initiation of T cell development. Here we have analyzed the mechanisms regulating Notch activation in lymphoid precursors at extrathymic sites and in the thymus, including stages representing the first wave of embryonic thymus colonization on embryonic day 12 of gestation. We show that Notch activation in migrant lymphoid precursors requires entry into the thymic microenvironment where they are exposed to Notch ligands expressed by immature thymic epithelial cells. Moreover, continued Notch signaling in such precursors requires sustained interactions with Notch ligands. Collectively, these findings suggest a role for Notch in an intrathymic mechanism of T cell lineage commitment involving sustained interactions with Notch ligand bearing thymic epithelium.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Although the thymic microenvironment provides optimal conditions for thymocyte development, controversy exists over its role in inducing migrant precursors to undergo initial commitment to the T cell lineage (1, 2, 3). Thus, several studies have reported evidence suggesting prethymic commitment to the T cell lineage (2, 4). In particular, multilineage progenitor (MLP)3 assays, which allow clonal analysis of precursor potential (4), have demonstrated the existence of cells in fetal hemopoietic tissues with myeloid/T cell potential (MT progenitors) or myeloid/B cell potential (MB progenitors), but failed to identify precursors that only give rise to T and B cells (i.e., T/B or common lymphoid progenitors (CLP)) (4). In contrast, other reports show a requirement for interaction with the thymus during T cell commitment (5), and clonal assays have demonstrated the existence of CLPs in adult mouse bone marrow (6). In addition, recent evidence that signaling through the Notch1 receptor is a key factor in T lineage choice (7, 8) raises the important question of when Notch signaling occurs in relation to T cell commitment events.

Overexpression of a constitutively active form of Notch1 (IC-Notch) promotes extrathymic CD4+8+ cell development at the expense of B cell development (7). Conversely, Notch1-deficient bone marrow precursors fail to undergo the earliest steps of intrathymic T cell development (8). Instead, Notch1-/- precursors adopt a B cell fate in the thymus (9), favoring the idea that cells entering the thymus have the potential for either T or B cell development and that T lineage choice is normally dependent on intrathymic events. Disruption of hairy enhancer of Split (HES)-1, a known downstream target gene of Notch signaling (10), also leads to the disruption of T cell development, providing further evidence for the importance of Notch-triggered events in early T cell development (11). However, overexpression of Deltex1, another gene transcribed as a consequence of Notch signaling (12), has the converse effect of inhibiting T cell development while promoting B cell development (13). Thus, Notch activation may have differing effects depending upon variables such as signaling strength and duration, which may, in turn, be influenced by interaction with members of the different Notch ligand families. In this context, defining the stages when Notch signaling is active and the factors that initiate, sustain, and regulate this signaling is crucial to elucidating the role of Notch in fate determination and differentiation in lymphoid cells.

Activation of Notch is known to depend upon binding to ligands belonging to the Delta or Jagged/Serrate families. Although there is conflicting evidence on the expression of these ligands on thymocytes themselves, there is clear evidence for expression of Notch ligand genes, including Jagged1, Jagged2, and Delta like1 by thymic epithelial cells (14). In contrast, little is known regarding the location and extent to which these ligands are expressed in prethymic environments (either bone marrow or fetal liver) and their accessibility to developing lymphoid progenitors. Data on the roles of different Notch ligands in T cell development are also conflicting. Evidence that Delta, rather than Jagged, is important in T lineage commitment has come from studies demonstrating that bone marrow stromal cells transfected with Delta, but not those transfected with Jagged, constructs can induce the expression of some markers characteristic of T cell commitment when associated with human cord blood progenitors in vitro (15). In contrast, inhibition of T cell development by transgenic expression of lunatic fringe, a glycosyl transferase thought to modify Notch receptors in a way that inhibits interaction with Jagged and facilitates interaction with Delta, suggests that Jagged ligands are functionally more important for T cell development (16).

Here we have examined the range and availability of Notch ligands in both prethymic and thymic environments in relation to the potential role of Notch in intrathymic vs extrathymic mechanisms of T cell commitment. We show that lineage-negative (Lin-) IL-7R{alpha}+ lymphoid precursors present in fetal liver (FL) express Notch1, but show no evidence of Notch activation. Consistent with this, during initial colonization of the thymic rudiment by such cells on day 12 of gestation (E12), newly arrived cells located in the perithymic mesenchyme show little evidence of Notch activation, whereas precursors that have moved into the epithelial microenvironment were found to transcribe a number of indicators of active Notch signaling. This correlates with expression of the Notch ligands Jagged1, Jagged2, Delta-like1, and Delta-like4 by E12 thymic epithelium. Thus, our findings suggest that entry into the thymic epithelial microenvironment is required for transcriptional activation of several genes known to be downstream of Notch1, favoring the idea that the function of Notch in T lineage commitment occurs intrathymically as a result of exposure to Notch ligand-bearing thymic epithelium.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Mice were bred and maintained under specific pathogen-free conditions at the Biomedical Services Unit, Birmingham University. BALB/c and p56lck bcl-2 transgenic (bcl-2tg) mice (gift from Dr. S. J. Korsmeyer, Dana-Farber Cancer Institute, Boston, MA) were used as stated. The day of vaginal plug detection was designated day 0.

Abs and immunoconjugates

The following were used for flow cytometric analysis: anti-CD45 FITC (clone 30-F11; BD PharMingen, San Diego, CA), anti-IL-7R{alpha}-biotin (clone A7R34; eBioscience), and streptavidin-PE (Southern Biotechnology Associates, Birmingham, AL). For immunocytochemistry the following were used: rat anti-CD45 (clone M1/9; American Type Culture Collection, Manassas, VA), mouse anti-pan-cytokeratin (clones C-11, PCK-26, CY-90, Ks-1A3, M20, and A53-B/A2; Sigma-Aldrich, St. Louis, MO), sheep anti-rat Ig-biotin (Amersham Pharmacia Biotech, Arlington Heights, IL), extravidin-tetramethyl rhodamine isothiocyanate (TRITC; Sigma-Aldrich), and goat anti-mouse Ig-7-amino-4-methylcoumarin-3-acetic acid (AMCA; Chemicon International, Temecula, CA).

The following were coated onto anti-rat Ig magnetic beads (Dynal, Wirral, U.K.): anti-CD4 (clone L3T4; BD PharMingen), anti-CD8 (clone 53-6.7; BD PharMingen), anti-TER119 (BD PharMingen), anti-CD45 (clone M1/9; American Type Culture Collection), anti-CD25 (clone 7D4; BD PharMingen), and anti-IL-7R{alpha}-biotin (clone A7R34, eBioscience).

Immunohistochemistry

E12 BALB/c thymus lobes were prepared and analyzed immunohistochemically as described previously (17).

Isolation of progenitors from E12 thymus and perithymic mesenchyme

Lymphoid progenitors were defined on the basis of a Lin-IL7R{alpha}+ phenotype that is known to include CLPs in adult bone marrow (6). E12 thymic lobes and their surrounding mesenchyme were isolated from BALB/c embryos by microdissection. These were incubated at 37°C for 20 min in 2.5 mg/ml of collagenase/dispase, placed under a dissecting microscope, and aspirated gently using a glass pipette to separate perithymic mesenchyme from the inner epithelial rudiment. Perithymic mesenchyme and inner epithelial rudiments were transferred into fresh solutions of collagenase/dispase, and enzymatic treatment was continued until cell suspensions were produced. Progenitors in the mesenchymal fraction were isolated using anti-IL-7R{alpha}-coated Dynabeads. Progenitors from thymic epithelial rudiments were isolated using anti-CD45-coated Dynabeads. Sorted cells were snap-frozen and processed for RT-PCR analysis.

Purification of fetal liver precursors and CD4-8- thymocyte subsets

To isolate lymphoid precursors for RT-PCR analysis, isolated E12 FLs were disaggregated and depleted of lineage-positive (Lin+) cells using a cocktail of biotinylated anti-CD3{epsilon}, anti-B220, anti-Ly6C/Ly6G, anti-CD11b, and anti-TER119 Abs (BD PharMingen), streptavidin-coated microbeads, and MiniMACS separation columns (Miltenyi Biotech). Lin- cells were then rosetted on anti-IL-7R{alpha}-coated Dynabeads, to produce Lin-IL-7R{alpha}+ precursors. These preparations were snap-frozen for RT-PCR analysis.

Double-negative subset I (DNI) CD44+25- cells were sorted from thymocyte suspensions obtained from E14 day BALB/c fetal thymus lobes by depletion of CD25+ cells using anti-CD25-conjugated magnetic beads (Dynal).

DNII and DNIII subsets were purified from E15 BALB/c thymocyte suspensions by isolation of CD25+ cells using MiniMACS separation. To isolate CD44+25+ DNII cells, purified CD25+ cells was mixed with anti-CD44-conjugated magnetic beads (Dynal). Rosetted cells, representing CD25+44+ DNII cells, were then snap-frozen directly for RT-PCR analysis. To obtain CD25+44- DNIII cells, purified CD25+ cells were depleted of CD44+ cells using anti-CD44-coated Dynabeads.

CD25-44- DNIV thymocytes were sorted from E17 day BALB/c thymocyte suspensions using multiple rounds of magnetic beads coated with Abs to CD4, CD8, CD44, and CD25.

Purification of fetal thymic and fetal liver stromal cells

Fetal thymic stroma was prepared from E15 thymic lobes organ cultured in 1.35 mM 2-deoxyguanosine (dGuo) as previously described (18). MHC class II+ epithelial cells were sorted from dGuo-treated thymic lobes (19), snap-frozen, and used for RT-PCR analysis.

FL stroma was obtained from mechanically disaggregated E14 FL depleted of TER119+ and CD45+ cells using magnetic beads (Dynal). Stromal cell content was demonstrated by adherence in tissue culture. The resulting cells were then snap-frozen for RT-PCR analysis.

RT-PCR

RT-PCR analysis was performed as previously reported (20). Primer sequences used for RT-PCR were published previously (14), except in the case of HES-1 (forward, 5'-GCCAGTGTCAACACGACACCGG-3'; reverse, 5'-TCACCTCGTTCATGCACTCG-3'), Delta-like4 (forward, 5'-CAGAGACTTCGCCAGGAAAC-3'; reverse, 5'-ATCCATTCTTGCACGGAGAG-3'), and Pre-T{alpha} (forward, 5'-CATGCTTCTCCACGAGTG-3'; reverse, 5'-CTATGTCCAAATTCTGTGGGT G-3').

PCR products were analyzed by ethidium bromide agarose gel electrophoresis and were identified by fragment size. Band quantification by densitometric analysis was performed using GeneTools (Syngene, Cambridge, U.K.). Values of target gene expression are expressed per unit of {beta}-actin.

Culture of bcl-2tg thymocytes in the absence of the thymic microenvironment

Thymus lobes were isolated from E15 bcl-2tg embryos and teased apart using fine cataract knives. Cells were then either snap-frozen immediately for RT-PCR analysis or placed in culture. For culture in isolation, liberated cells were placed in 96-well, round-bottom plates (Sterilin, Teddington, U.K.) at 2.5 x 105 cells/well for 1 day. CD45+ thymocytes were recovered from these cultures using anti-CD45-coated Dynabeads to prevent stromal contamination and then analyzed by RT-PCR.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Correlating Notch1 signaling in precursors with notch ligand expression in intrathymic microenvironments

We first analyzed both lymphoid precursors and stromal cells from fetal liver and fetal thymus for the expression of Notch1, evidence of Notch signaling, and the availability of Notch ligands. Due to the lack of suitable Abs to Notch-related molecules, as in other studies (10, 21), we used RT-PCR to analyze expression (Fig. 1). As Notch1 signaling in thymocytes has been reported to induce transcriptional activation of a number of genes, including Deltex1 and HES-1 (10, 12), we used transcription of these genes as indicators of Notch activation. Thus, Lin-IL-7R{alpha}+ precursors from 12-day FL, containing lymphoid progenitors (22), were compared with intrathymic CD4-8- (double-negative (DN)) subsets for Notch expression and evidence of Notch activation.



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  		FIGURE 1. Differential activation of Notch signaling in T cell precursors from extrathymic and intrathymic microenvironments. Semiquantitative RT-PCR for Notch1, Deltex1, and HES-1 was performed on cDNAs obtained from purified Lin-IL-7R{alpha}+ FL precursors and thymocytes constituting the four CD4-8- (DNI–DNIV) subpopulations. In all cases, cDNA loading volumes were normalized by {beta}-actin expression, and PCR product sizes are shown in base pairs. Each experiment was performed three times with similar results.

 
For cells within the thymus, expression of the Notch1 receptor together with evidence of Notch-mediated signaling, as indicated by HES-1 and Deltex1 mRNA, was found in the earliest DN subset (DNI), as well as all subsequent DN subpopulations (Fig. 1). Moreover, maintenance of Notch signaling in CD4-8- cells was found to be dependent upon ongoing contact with thymic stromal cells, since removal of bcl-2tg (23) CD4-8- thymocytes from their surrounding thymic microenvironment resulted in a dramatic down-regulation of both Deltex1 and HES-1 expression in short term suspension cultures (Fig. 2). In marked contrast to the readily detectable signals for HES-1 and Deltex1 in intrathymic DN cells, Lin-IL-7R{alpha}+ FL precursors expressed only a slight signal for HES-1 and Deltex1, although they did express comparable levels of Notch1 (Fig. 1) and Presenilin-1 (data not shown), a molecule required for Notch signaling through release of the intracellular portion of the Notch1 receptor (Notch1-IC) (24). These data suggest that Notch signaling is active in intrathymic DN cells, but not in Lin-IL-7R{alpha}+ FL precursors, and that this is not simply due to the absence of Notch1 receptor expression or the inability of these cells to cleave the intracellular portion of the Notch1 molecule via Presenilin-1.



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  		FIGURE 2. Sustained contact with the thymic microenvironment is required to maintain Notch signaling in CD4-8- thymocytes. Isolated thymocytes from E15 p56lck bcl-2 transgenic embryos were analyzed for Deltex1 and Hes-1 expression either fresh or following overnight culture in wells as single-cell suspensions. In all cases, cDNA loading volumes were normalized by {beta}-actin expression, and PCR product sizes are shown in base pairs. Each experiment was performed three times with similar results.

 
To determine whether differential activation of Notch signaling in FL precursors and thymocytes correlated with the availability of Notch ligands in surrounding microenvironments, we next analyzed the expression of Jagged1 and -2, and Delta-like1, -3, and -4 by RT-PCR. In agreement with our previous observations on CD4+8+ thymocytes (14), neither FL precursors nor DNI-DNIV thymocyte subsets were found to express detectable mRNA for any of the Notch ligands analyzed (data not shown). This implies that Notch signaling in lymphoid precursors requires interaction with other cell types bearing Notch ligands and is consistent with an inductive signaling model of Notch activation in T cell commitment rather than a lateral inhibition model involving interaction between thymocytes (reviewed in Ref.25). In support of this, mRNA for all Notch ligands tested, with the exception of Delta-like3 (Fig. 3), was readily detected in functionally mature MHC class II+ thymic epithelial cells isolated from dGuo-treated thymus lobes. In contrast, FL stroma showed an absence of Jagged1 together with greatly reduced levels of Jagged2 and Delta-like1 and -4 (Fig. 3) relative to thymic epithelium. Thus, the absence of Notch signaling in Lin-IL-7R{alpha}+ FL precursors and the evidence for Notch activation in DNI-DNIV thymocyte subsets correlate with the relative abundance of Notch ligands in the extrathymic and intrathymic stromal microenvironments.



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  		FIGURE 3. The paucity of Notch ligand expression by fetal liver stromal cells compared with thymic epithelial cells. cDNAs obtained from freshly prepared populations of MHC class II+ thymic epithelial cells and fetal liver stromal cells were analyzed by RT-PCR for the expression of the Notch ligands Jagged-1, Jagged-2, Delta-like1, Delta-like3, and Delta-like4. Band intensity was analyzed by densitometry and was calculated per unit {beta}-actin. These results are representative of three separate experiments.

 
Contact with thymic epithelium initiates Notch signaling in migrant precursors during the initial wave of thymus colonization

Data in the previous section argue strongly that, at least in embryonic development, activation and maintenance of Notch signaling in developing T cells, and thus Notch function in T cell commitment and differentiation, are dependent upon entry into the thymus and interaction with thymic epithelial cells. To obtain further evidence for this possibility we examined Notch activation during the process of thymus colonization in embryogenesis. This process involves the migration of lymphoid precursors from the fetal liver to the thymus, where they traverse the peripheral mesenchyme-derived layers of the nonvascularized thymic anlage to enter the central area comprising thymic epithelium (26). On E12, CD45+ cells are present in both the perithymic mesenchyme and the intraepithelial compartment (Fig. 4a), providing the opportunity to isolate and examine lymphoid progenitors just before and just after they have made contact with the thymic epithelium.



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  		FIGURE 4. Microanatomical and phenotypic analysis of initial thymus colonization. a, Thymus sections from E12 embryos were stained for CD45 (red) and pan-cytokeratin (blue) to detect migrant precursors colonizing the thymic epithelial primordium. The dashed line marks the perimeter of the thymic epithelial rudiment, and CD45+ precursor cells can be seen within the perithymic mesenchyme (arrowhead) as well as within the epithelial anlagen (arrow). Original magnification, x400. b, Flow cytometric analysis of CD45 and IL-7R{alpha} expression in cell suspensions prepared from E12 fetal liver and perithymic mesenchyme. Lower panels, IL-7R{alpha} expression on CD45+ populations gated in upper panels. Note the relative enrichment of IL-7R{alpha} cells in perithymic mesenchyme.

 
As shown in Fig. 4b, CD45+ cells isolated from the perithymic mesenchyme are enriched for IL-7R{alpha}+ lymphoid progenitors relative to the total CD45+ population in fetal liver, consistent with the selective recruitment of these cells to the thymus (27). By RT-PCR analysis, such cells, purified from both the perithymic and intraepithelial compartments, expressed Notch1 (Fig. 5a). However, while progenitors recovered from the perithymic mesenchyme were found to express Deltex1, they did not express HES-1 or Pre-T{alpha} (Fig. 5a). In contrast, the expression of all three of these genes, which are transcribed in response to Notch signaling (10, 12, 28), was detected in lymphoid progenitors recovered from the epithelium (Fig. 5a). Since thymic epithelial cells on E12 are of a mainly immature phenotype compared with those analyzed in the previous section (17), it was important to confirm Notch ligand expression at this stage. As shown in Fig. 5b, we were able to detect Jagged1 and -2, Delta-like1, and Delta-like4, but not Delta-like3, in E12 thymic epithelium. Thus, from the onset of thymus colonization, thymic epithelium expresses Notch ligands and appears capable of inducing Notch activation in migrant precursors.



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  		FIGURE 5. Contact with thymic epithelium induces Notch signaling during initial thymus colonization and correlates with Notch ligand expression by thymic epithelium. a, cDNA obtained from IL-7R{alpha}+ perithymic mesenchyme-resident precursors and CD45+ thymic epithelial-resident precursors were analyzed by fixed point RT-PCR for expression of Notch1 and downstream Notch signaling molecules: lane 1, {beta}-actin; lane 2, Notch1; lane 3, HES-1; lane 4, Deltex1; lane 5, Pre-T{alpha}. b, cDNA obtained from E12 thymic epithelial anlagen were analyzed for the expression of the five presently known Notch ligands using fixed point RT-PCR: lane 1, {beta}-actin; lane 2, Jagged1; lane 3, Jagged2; lane 4, Delta-like1; lane 5, Delta-like3; lane 6, Delta-like4. In all cases, samples were run for 35 cycles. PCR product sizes are shown in base pairs. These experiments were performed three times with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Intrathymic generation of T cells requires interactions between developing thymocytes and a complex heterogeneity of stromal cells, including cortical and medullary epithelium, and mesenchymal fibroblasts (1). Whether the precursors that seed the thymus are committed to the T cell lineage before thymus colonization is unclear, and evidence exists to support both extra- and intrathymic models of T cell commitment (2, 4, 5). Recent evidence that signaling through Notch1 plays a pivotal role in the T/B lineage choice, with Notch signaling driving commitment of precursors to the T cell lineage (7, 8, 16), also needs to be considered in this context. Given the uncertainty surrounding spatial and temporal aspects of T cell commitment, here we have investigated Notch signaling in T cell progenitor populations and its relationship to the availability of Notch ligands in the anatomical sites associated with the production and maturation of precursors with T cell potential Thus we have examined the Notch signaling status of T cells and their progenitors before, during and after thymus colonization.

Our findings show that lymphoid progenitors in fetal prethymic hemopoietic tissues do not display evidence of active or ongoing Notch signaling and that this is not initiated until these cells enter the thymus and make contact with thymic epithelium. This lack of Notch signaling until progenitors enter the thymus is not due to the absence of Notch itself or of presenilin that is necessary for Notch cleavage and activation, but correlates well with the relative paucity of Notch ligands in the fetal liver stroma compared with thymic epithelium.

Our observations support evidence from studies of animals with induced Notch deficiencies suggesting that the role of Notch in T vs B lineage choice occurs after precursors have entered the thymus (9). However, they are difficult to reconcile with evidence for prethymic commitment to the T cell lineage (2, 4) and a role for Notch at this stage. One possible explanation for this discrepancy is that T cell commitment at the prethymic stage is driven by factors other than Notch, but is only revealed after Notch signaling in the thymus. In addition, our evidence indicates that Notch signaling is ongoing throughout the CD4-8- stage, and we have shown that such signaling, at least within unseparated E15 CD4-8- thymocytes, is dependent upon interaction with thymic epithelial cells. As the currently available inducible Notch knockout animals result in deletion either before or toward the end of the DN stage (8, 29, 30), the physiological relevance of this activity remains to be determined. Likewise, the roles of individual Notch ligands expressed by thymic epithelium in regulating intrathymic Notch activity for T/B lineage choice and for other lineage choices (e.g., {alpha}{beta}/{gamma}{delta} vs NK) within the T pathway are important issues still to be resolved. We have recently developed techniques for efficient adenoviral transfection of isolated thymic epithelial cells that will allow manipulation of the expression of individual Notch ligands. Used in conjunction with reaggregate organ culture techniques, this will provide a direct approach to address these issues.


   Acknowledgments
 
We are grateful to Sonia Parnell for skilled technical support.


   Footnotes
 
1 This work was supported by a Medical Research Council (U.K.) Program Grant (to E.J.J. and G.A.). Back

2 Address correspondence and reprint requests to Dr. Graham Anderson, Department of Anatomy, Medical School, University of Birmingham, Edgbaston, Birmingham, U.K. B15 2TT. E-mail address: g.anderson{at}bham.ac.uk Back

3 Abbreviations used in this paper: MLP, multilineage progenitor assay; bcl-2tg, p56lck bcl-2 transgenic; CLP, common lymphoid progenitor; dGuo, 2-deoxyguanosine; DN, double-negative; E12, embryonic day 12; FL, fetal liver; Lin, lineage marker; HES-1, hairy enhancer of Split-1. Back

Received for publication September 3, 2002. Accepted for publication November 20, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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