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International Immunology Advance Access originally published online on August 14, 2007
International Immunology 2007 19(10):1175-1182; doi:10.1093/intimm/dxm085
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© The Author 2007. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved.
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Forced expression of Id2 in fetal thymic T cell progenitors allows some of their progeny to adopt NK cell fate

Shinji Fujimoto1, Tomokatsu Ikawa1,3, Tatsuo Kina1 and Yoshifumi Yokota2

1 Department of Immunology, Institute for Frontier Medical Sciences, Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
2 Department of Biochemistry, School of Medicine, Fukui University, Fukui 910-1193, Japan
3 Present address: Laboratory for Lymphocyte Development, Riken Research Center for Allergy and Immunology, Yokohama 230-0045, Japan

Correspondence to: S. Fujimoto; E-mail: fujimoto{at}frontier.kyoto-u.ac.jp


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 References
 
The E proteins are indispensable for early T cell development. On the other hand, we previously demonstrated that their inhibitor Id2 is essential for NK lineage commitment from bipotent progenitors generating both T and NK cells (p-T/NK). To shed more light on the role of E proteins and Id2 in the development of early intrathymic progenitors, we performed a clonal analysis: individual fetal thymic CD4–CD8–CD44+CD25–CD122– (DN1CD122–) cells were retrovirally transduced with an Id2-internal ribosomal entry site (IRES)-green fluorescent protein (GFP) (Id2-GFP) gene or a control IRES-GFP (GFP) gene, and cultured in a modified fetal thymus organ culture able to support T and NK cell development. After the culture, both T and NK cells, T cells and no NK cells, NK cells and no T cells, or completely no cells were generated from single cells in each lobe. Hence, the seeded cells were regarded as p-T/NK, unipotent progenitors generating T cells (p-T), unipotent NK progenitors, or cells without progenitor activity, respectively. With Id2-GFP transduction, p-T disappeared and more p-T/NK emerged than with GFP transduction. This increase corresponded to the number of p-T that was counted when the vector-transduced-DN1CD122– cells of the same number were examined. Additionally, a fraction of GFP– NK cells obtained after Id2-GFP transduction underwent TCRß D–J rearrangement. Our data strongly suggest that forced expression of Id2 allows some progeny of p-T to adopt an NK cell fate, and that p-T retain a program for NK lineage development that can be implemented by inhibiting the function of E proteins.

Keywords: cell fate change, clonal assay, E proteins, Id2, NK lineage commitment, retrovirus vector, T lineage commitment


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
The presence of bipotent progenitors generating both T and NK cells (p-T/NK) has been demonstrated in the fetal thymus (FT) (1–4), and p-T/NK yield unipotent progenitors generating T cells (p-T) or NK cells (p-NK) (5–7). Gene-targeting studies have demonstrated that E protein transcription factors, consisting of E2A gene products (E12 and E47), HEB and E2-2 play a key role in early T cell development (8–11). The function of E proteins is negatively regulated by the Id factors (Id1, Id2, Id3 and Id4) (12, 13). Intriguingly, adult Id2–/– mice display a marked reduction in the NK cell population and a normal T cell population (14), and our clonal analysis of Id2-deficient fetal thymocytes revealed that the Id2 is required for NK lineage commitment of p-T/NK (15).

A recent study strongly suggested that E proteins and Notch signaling act cooperatively to promote T lineage specification because E47 activates the expression of an ensemble of genes associated with Notch signaling in vitro (16). Notch signaling is also critical during T cell development (17–22). Hence, it is likely that heightening the activity of E proteins leads p-T/NK to T lineage commitment, and lowering this activity to NK lineage commitment, in vivo. The OP9 bone marrow stromal cell line ectopically expressing the Notch ligand Delta-like 1 (DL1) (OP9-DL1) has acquired the capacity to induce the differentiation of fetal liver (FL) progenitor cells into CD4+CD8+ double-positive cells in vitro (23). However, E2A–/– FL progenitors are severely impaired in their ability to differentiate into CD4–CD8– double-negative (DN) CD44–CD25+ (DN3) cells when they are cultured on OP9-DL1 cells, and at the same time, they give rise to a substantial number of NK cells (16). Also, T cell development is hindered and NK cell development is enhanced, when human fetal thymocytes ectopically expressing Id3 are cultured in fetal thymus organ culture (FTOC) (24), and in HEBbm/bm knock-in mice, which express exclusively dominant-negative HEB molecules, T cell development is completely inhibited, but in this case, NK cells are also decreased about 10-fold (25).

After T lineage commitment, Notch signals are necessary for T cell specification and it has been suggested that without Notch signaling p-T adopt the NK cell lineage because NK cells but not T cells are produced when thymic DN CD44+CD25– (DN1) and CD44+CD25+ (DN2) cells are cultured on normal OP9 cells (26). However, the effect of inhibiting E protein function in p-T has not been elucidated yet.

In this study, in order to determine whether thymic NK cell development is enhanced or blocked by hampering the function of E proteins, we transduced FT DN1CD122– cells with the Id2 gene. The transduced cells included all three types of progenitors, p-T/NK, p-T and p-NK (27) and were cultured in a modified FTOC able to support both T and NK cell development. The results showed that constitutive expression of Id2 totally blocked T cell development and also reduced the number of NK cells. Furthermore, to examine the effect of forced expression of Id2 on individual thymic progenitors directly, we transduced single DN1CD122– cells. After Id2 transduction, progenitors that produced only T cells could not be observed and the number of progenitors that produced both T and NK cells increased compared with that in the control transduction. Notably, the increase in the number of p-T/NK was equal to the number of p-T observed with control transduction. Additionally, we detected TCRß gene rearrangement in NK cells generated with Id2-green fluorescent protein (GFP) transduction. Taken together, these findings imply that it is highly likely that inhibiting the function of E proteins in p-T allows some of their progeny to adopt an NK cell fate.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Mice
All animal procedures described in this study were performed in accordance with the guideline for animal experiments of the Institute for Frontier Medical Sciences, Kyoto University. C57BL/6 (B6) mice were purchased from SLC (Shizuoka, Japan). B6Ly5.1 mice were maintained in our animal facility.

Retroviral vectors, transduction of fetal thymic progenitors and FTOC
The retroviral bicistronic vector pMX-IRES-GFP was kindly donated by Dr T. Kitamura (University of Tokyo, Tokyo, Japan). A 1.0-kb cDNA fragment containing the entire Id2 coding region was ligated into the EcoRI site upstream of the internal ribosomal entry site (IRES) and the construct was termed pMX-Id2-IRES-GFP (Fig. 1A). Helper-free recombinant retrovirus was produced after transfection of the construct into the packaging cell line PLAT-E (28). The lineage marker– DN1CD122– cells were sorted from day 14 or 15 B6 FT as described (27). To transduce 300 DN1CD122– cells or a single DN1CD122– cell with virus supernatant, we used a deoxyguanosine (dGuo)-treated FT lobe (dGuo lobe) instead of stromal cells (Fig. 1B). Single dGuo lobes obtained from B6Ly5.1 mice were placed into the wells of a 96-well V-bottom plate, into which medium containing polybrene (10 µg ml–1), stem cell factor (SCF) (10 ng ml–1) and IL-7 (10 ng ml–1) was added and finally progenitors were seeded. To boost the transduction efficiency, the plates were centrifuged at 1800 r.p.m. for 1 h at 32°C (29), and then placed into a plastic bag (Ohmi Oder Air Service, Hikone, Japan) that was filled with a gas mixture containing high concentration of oxygen (70% O2, 25% N2 and 5% CO2) and incubated at 37°C [high oxygen submersion (HOS) culture] (30) for 16 h. After transduction, the medium was changed to a fresh one containing SCF, IL-7 and IL-15 (5 ng ml–1) to support both T and NK cell development (27). CD3–NK1.1+ cells which were harvested from the same IL-15-supplemented cultures under HOS conditions were shown to have cytolytic activity against the target cell line Yac-1 (27). In order not to disturb the interaction between the transduced cells and the thymic environments, we uninterruptedly cultured the cells using the same dGuo lobe under HOS conditions for 11 or 14 days in the case of transducing 300 or single DN1CD122– cells, respectively.


Figure 1
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  		Fig. 1. Retrovirus-mediated gene transfer into fetal thymic progenitors. (A) Both Id2 and GFP are expressed from this bicistronic retrovirus vector. (B) Fetal thymocytes were retrovirally transduced using a dGuo lobe. After the transduction, the same dGuo lobe was continuously employed in culture to nurture T and NK cell precursors. We also transduced single fetal thymocytes. In this case, the culture period was prolonged to 14 days.

 
Flow cytometric analysis and cell sorting
All mAbs and procedures for flow cytometric analysis and sorting with a FACS Vantage (BD Biosciences) have been described previously (20). The sorting efficiency was >99%, as determined by post-sort analysis.

PCR
After culturing 300 Id2-GFP-transduced or GFP-transduced DN1CD122– cells for 11 days, GFP–CD3+NK1.1–, GFP–CD3–NK1.1+ and GFP+CD3–NK1.1+ cells were sorted. Preparation of genomic DNA and PCR analysis of TCRß gene D–J rearrangement and TCR{gamma} gene V–J rearrangement were done as described previously (27). Other primers were Id2 sense, 5'-TCTGAGCTTATGTCGAATGATAGC-3'; Id2 antisense, 5'-CGTGTTCTCCTGGTGAAATGGCTG-3'; Rag2 sense, 5'-AGTGAATTGCACAGTCTTGCCAG-3'; Rag2 antisense, 5'-GGGTTTATTGAGCTCCGTTGAATAG-3'. The Rag2 gene was amplified to evaluate the amount of the genomic DNA. To amplify the endogenous Id2 gene and retrovirally transduced Id2 cDNA, thermocycling conditions were as follows: 5 min at 94°C, followed by 30 cycles of 1 min at 94°C, 1 min at 62°C and 2 min at 72°C. For detection of the Rag2 gene, the annealing temperature was 62°C and 35 cycles were performed. PCR products were electrophoresed on a 1.2% agarose gel and stained with ethidium bromide.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
Id2 gene transduction into fetal thymocytes with use of a dGuo lobe and its effect on T and NK cell development
We first examined whether inhibiting the functions of E proteins in thymic early progenitors results in generation of more NK cells. To this end, we retrovirally transferred the Id2 gene into 300 DN1CD122– fetal thymocytes. The DN1CD122– subset is the least mature subset in the thymus and includes all three types of progenitors, p-T/NK, p-T and p-NK (27). Use of the retrovirus vector pMX-IRES-GFP (GFP) (31) and our construct pMX-Id2-IRES-GFP (Id2-GFP) (32) enabled us to identify the cells expressing the transduced gene by virtue of bicistronically expressed GFP (Fig. 1A). Since murine leukemia virus-based vectors require cell proliferation for transduction (33), we added IL-7 and SCF to the medium and used a dGuo lobe, which is usually employed for FTOC, in place of stromal cells. Following the transduction, we immediately started an HOS culture (30), a type of modified FTOC, by changing the medium and utilizing the same dGuo lobe (Fig. 1B). To support both T cell development and NK cell development, we added IL-15 to the HOS culture.

After culturing for 11 days, the total cell numbers were 4.6 ± 0.4 x 103 (n = 4) and 5.8 ± 2.8 x 103 (n = 4) for Id2-GFP and control GFP transduction, respectively. No GFP+ CD3+NK1.1– cells were generated from Id2-GFP-transduced CD122– cells, although GFP– CD3+NK1.1– cells were generated (Fig. 2A). On the other hand, CD3–NK1.1+ cells emerged irrespective of GFP expression. However, the number of GFP+ CD3–NK1.1+ cells was lower than the number that emerged from vector-transduced DN1CD122– cells (Fig. 2A). In addition, flow cytometric analysis showed the absence of cells strongly expressing Id2-GFP (Fig. 2B). We previously obtained 2.4 x 104 cells from 100 DN1 cells without transduction, after culturing for 10 days under the same conditions except for using IL-2 (25 units ml–1) instead of IL-15 (15). Comparison of the numbers of CD3+NK1.1– cells (1010 ± 670) and CD3–NK1.1+ cells (860 ± 650) obtained in this study with those obtained in the previous ones (4300 and 1000, respectively) shows that our transduction method has some negative effect on proliferation, especially from T cell progenitors. Nevertheless, it appears that our novel transduction method using a dGuo lobe causes little, if any, skew in the progenitor activity because both CD3+NK1.1– cells and CD3–NK1.1+ cells differentiated irrespective of GFP expression with the control transduction.


Figure 2
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  		Fig. 2. Effects of forced Id2 expression in FT DN1CD122– cells. Three hundred FT DN1CD122– cells were transduced with control GFP or with Id2-GFP and cultured under the conditions supportive of both T and NK cell development for 11 days. (A) Cells generated were gated according to the GFP expression, and each population was analyzed for surface expression of CD3{epsilon} and NK1.1 and counted. The numbers of cells obtained from the GFP transduction versus the Id2-GFP transduction were as follows: GFP– CD3+NK1.1– cells (620 ± 380 versus 740 ± 170) and CD3–NK1.1+ cells (490 ± 290 versus 430 ± 170), and GFP+ CD3+NK1.1– cells (390 ± 290 versus 2.5 ± 5) and CD3–NK1.1+ cells (370 ± 360 versus 150 ± 30) (mean cell number ± SD of four experiments). (B) Representative FACS profiles. Numbers in the profiles represent percentages of cells in each quadrant.

 
Consistent with the results from the HEBbm/bm knock-in mouse (25), functional blockade of E proteins by Id2 over-expression generated no T cells and allowed a small number of NK cells to develop, but never enhanced development into NK cells. Moreover, excessive expression of Id2 rather prevents p-T/NK and p-NK from further development to NK cells since NK cells strongly expressing Id2-GFP were not obtained.

Transduction into single DN1CD122– cells
To evaluate the outcome of forced Id2 expression in FT p-T/NK, p-T and p-NK, we tried a clonal progenitor analysis of GFP-transduced and Id2-GFP-transduced FT DN1CD122– cells. First, 1 x 103 DN1CD122– cells were transduced with the GFP recombinant virus overnight, then GFP+ cells was isolated by cell sorting, and the progenitor activity of individual cells toward T and/or NK cells were examined by seeding single GFP+ cells into each well of a culture plate. However, the seeded cells hardly multiplied (data not shown), probably due to the loss of survival or growth signals from the thymic environments during the sorting step. To overcome this problem, we transduced single DN1CD122– cells individually using one dGuo lobe for each DN1CD122– cell and cultured them continuously with the same lobes.

Because the progenitor frequencies of p-T/NK, p-T or p-NK determined after vector transduction were comparable to those without transduction, respectively [Table 1 and (15)], it is unlikely that our transduction method itself alters the progenitor activity to a large extent. However, there were dGuo lobes containing only GFP– cells. This might have been due to unsuccessful transduction of the GFP gene. Alternatively, the transduction was successful, but the expression of the transduced gene was halted afterward. Since the Id2 gene cloned in the vector is of cDNA origin (32), it can be distinguished by PCR from the endogenous one that retains introns. We prepared genomic DNA from cells that proliferated in each dGuo lobe and investigated whether the transduced Id2 gene was integrated into the genome. From all the samples, two bands were amplified, a long one from the endogenous Id2 gene and a short one, which was undetectable in the control lanes, from the transduced Id2 gene. However, the proportion of transduced cells seemed to vary from sample to sample (Fig. 3A). Notably, even in samples containing exclusively GFP– cells, the existence of the transduced gene was detected (Fig. 3A, lanes 1, 6 and 8). Hence, the single DN1CD122– cell seeded into a dGuo lobe divided during the overnight transduction period and some cells were transduced, namely Id2-GFP genes were integrated into their genomic DNA, while the others were not transduced. Consequently, it is expected that non-transduced cells would obey the inherent program for differentiation, and that transduced cells would be under the influence of the forced expression of Id2. Two groups of cells started to differentiate and multiply in the same dGuo lobe. It was therefore conceivable that all or a part of the transduced cells stopped transcription of the Id2-GFP gene at some time point during the subsequent culture.


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  		Table 1. The number of dGuo lobes in which T or/and NK cells were generated from single FT DN1CD122– cells transduced with GFP or Id2-GFP

 

Figure 3
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  		Fig. 3. Effects of forced Id2 expression in FT DN1CD122– single cells. Cells proliferated inside a dGuo lobe from a single Id2-GFP-transduced DN1CD122– cell were collected. (A) Genomic DNA was prepared and PCR analysis was done to detect the retrovirally transduced Id2 gene of cDNA origin. Results from 10 of 45 samples are shown. Control samples (lanes C1 and C2) were derived from single GFP-transduced cells. Above each lane, the type of cells that proliferated is indicated. In cases in which all the cells were GFP–, (–) is added. In the other cases, both GFP– and GFP+ cells were observed. (B) FACS profiles of representative samples are shown. GFP– and GFP+ cells were gated and surface expression of CD3{epsilon} and NK1.1 was examined.

 
Id2-GFP transduction did not seem to affect the overall progenitor frequency because every progenitor gave rise to untransduced cells in addition to transduced cells before the initiation of the HOS culture. Nevertheless, the frequency of p-T/NK, p-T or p-NK was likely to have been changed. In fact, the total number of progenitors for Id2-GFP transduction and that for GFP transduction were nearly the same for two series of experiments (Table 1). The average cell numbers obtained from single GFP-transduced cells and single Id2-GFP-transduced cells were 4.3 ± 4.0 x 103 and 7.1 ± 6.1 x 103, respectively.

Effects of forced Id2 expression in DN1CD122– single cells
As shown in Table 1, lobes containing exclusively T cells could not be observed in Id2-GFP-transduced groups, although 8 of 100 lobes contained only T cells in control GFP-transduced groups. On the other hand, more lobes which had been seeded with Id2-transduced-single cells yielded both T and NK cells (9 versus 7 for Experiment I and 19 versus 13 for Experiment II in Table 1). Intriguingly, these numbers of p-T after Id2-GFP transduction (9 and 19) were equal to those of p-T/NK plus p-T (7 + 2 for Experiment I and 13 + 6 for Experiment II) observed after GFP transduction. This strongly suggests that ectopic expression of Id2 made some cells of p-T origin give rise to NK cells.

We observed that GFP+ cells and GFP– cells co-existed inside a lobe and both T cells and NK cells were generated after Id2-GFP transduction. In this case, the GFP+ subset contained no T cells, and as shown in Fig. 3B, its FACS profile closely resembled the FACS profile in Fig. 2B. Exceptionally, a small number of GFP+ T cells were detected in one lobe; however, the small number of cells recovered from the lobe prevented further analysis (data not shown). The number of lobes in which exclusively NK cells developed was 9 for GFP transduction and 12 for Id2-GFP transduction. Thus, forced expression of Id2 in some NK lineage cells may give them progenitor activity afresh.

Status of the TCR gene rearrangement in NK cells derived from Id2-GFP-transduced DN1CD122– cells
As revealed above, some GFP– cells that proliferated in a lobe from a single cell after Id2-GFP transduction appeared to have transiently expressed Id2. Also, we detected the transduced Id2 gene in the GFP– NK cells generated after transducing 300 DN1CD122– cells with Id2-GFP but never with the empty vector (Fig. 4A). Thus, it is considered that a fraction of the GFP– NK cells were affected by transient expression of Id2. These GFP– NK cells may be related with T lineage cells because our data obtained from single-cell transduction suggest that some progeny of p-T adopt an NK cell fate as a result of ectopic expression of Id2. Actually, we detected D–J rearrangements of the TCRß gene, which are almost T cell specific, in the GFP– NK cells generated with Id2-GFP transduction (Fig. 4B). On the other hand, only one band corresponding to the germ line configuration was amplified from genomic DNA of the GFP+ NK cells obtained after Id2-GFP transduction, and also from that of both GFP+ and GFP– NK cells after empty vector transduction (Fig. 4B). Since thymocytes initiate the process of TCRß rearrangements during the DN2 to DN3 transition, though a small percentage of DN2 cells were shown to have rearranged constructs (34), it is highly likely that the recombination detected in the GFP– NK cells occurred during the culture under the influence of transient expression of Id2. We also examined the TCR{gamma} locus, which is rearranged not only in {gamma}{delta} T cells but also extensively in {alpha}ß T cells (35), and found recombined constructs exclusively in genomic DNA of the GFP– NK cells that proliferated after Id2-GFP transduction (Fig. 4B). These data indicate that a fraction of the GFP– NK cells generated through Id2-GFP transduction were in close proximity to T lineage cells, and supports the notion that forced transient expression of Id2 allows some progeny of p-T to change their fate into NK lineage cells.


Figure 4
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  		Fig. 4. Effect of forced Id2 expression on TCRß and TCR{gamma} gene rearrangements in NK cells. (A) GFP– T and NK cells developed from FT DN1CD122– cells which had been transduced with Id2-GFP or control vector were sorted, and the presence of the cDNA-type Id2 gene was examined by PCR. After culturing Id2-GFP-transduced cells, a fraction of GFP– NK cells and a much smaller fraction of GFP– T cells retained the retrovirus-mediated Id2 gene. (B) GFP+ and GFP– NK cells developed from FT DN1CD122– cells transduced with Id2-GFP or GFP were sorted, and the rearrangement status of the TCRß and TCR{gamma} genes was examined by PCR. Rearranged TCRß DJ and TCR{gamma} VJ bands were amplified only from GFP– NK cells generated from Id2-GFP-transduced cells in addition to the germline band. On the other hand, no rearranged bands were detected for the GFP+ NK cells and for the GFP– and GFP+ cells obtained after GFP transduction. Control amplification was done for the Rag2 gene.

 
We also detected a very faint band corresponding to the transduced Id2 gene in genomic DNA of the GFP– T cells developed from Id2-GFP-transduced DN1CD122– cells (Fig. 4A). Thus, transient expression of Id2 in some T cell progenitors may be permissive for their further development to T cells.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
Previous data have indicated that E protein activity is essential at the point of T lineage commitment from p-T/NK (16, 24, 25). However, whether E proteins are required after the commitment has not been completely clarified. In this study, we have shown that ectopic expression of Id2 in murine FT DN1CD122– p-T allows some of their progeny to adopt an NK cell fate, implying that the function of E proteins is needed for p-T to further differentiate. Our findings also suggest that p-T still maintain a developmental program for the generation of NK cells.

It has been reported that E2A–/– Lin– FL cells generate not T cells but NK cells when cultured on OP9-DL1 cells (16). This indicates that in the absence of E2A gene products, T cell progenitors cannot further develop as T cells, and adopt an NK cell fate as a default pathway. Heemskerk et al. (24) have reported that over-expression of Id3 in human CD1a–CD34+ fetal thymocytes completely blocks T cell development and enhances NK cell development in an FTOC. However, the effects of E2A deficiency or Id3 over-expression on p-T/NK and those on p-T were not examined separately. In order to assess the effects of inhibiting E proteins on individual progenitors, we have transduced FT DN1CD122– single cells with the Id2 gene and clonally explored progenitor activity toward T and/or NK cells. Our retrovirus-mediated gene transfer into single cells and subsequent culture with a modified FTOC always gave rise to two kinds of cells: cells with the transduced Id2-GFP gene and cells without the transduced gene. It is therefore expected that the former developed under the influence of constitutive or transient expression of Id2, and that the latter developed according to the built-in developmental schedule of the seeded DN1CD122– progenitor. Actually, p-T was not observed and p-T/NK increased with Id2-GFP transduction. Remarkably, the increment in the number of p-T/NK was the same as the number of p-T observed with control transduction (Table 1). Thus, it is highly likely that forced expression of Id2 changes the fate of some T lineage cells into the NK lineage. This notion was further supported by the detection of rearranged constructs of the TCRß and TCR{gamma} genes in GFP– NK cells after Id2-GFP transduction (Fig. 4B). Usually, TCR gene rearrangements occur only in T lineage cells.

Id2-GFP transduction into 300 DN1CD122– cells and culturing of the transduced cells in a modified FTOC resulted in the generation of no GFP+ T cells and fewer GFP+ NK cells than generated with vector transduction (Fig. 2A). This is consistent with the finding that no T cells are present and NK cells are decreased about 10-fold in HEBbm/bm knock-in mice (25). Moreover, we found no GFPhi cells after Id2-GFP transduction (Fig. 2B). These results indicate that continuous expression of Id2 does not enhance NK cell development from p-T/NK. Rather, forced expression of Id2 at high levels may restrain p-T/NK and p-NK from developing further. Id3-GFP over-expression in human CD1a–CD34+ fetal thymocytes hindered the development of GFP+ T cells (24). However, Spits et al. (36) have reported that Id3-GFP transduction into human pre-T cells which have already commenced TCR gene rearrangement can yield GFP+ {gamma}{delta} T cells. It is therefore suggested that E proteins are dispensable for some {gamma}{delta} T lineage cells after the initiation of their TCR gene rearrangement. We also observed GFP+ CD3+NK1.1– cells, which might have been expressing {gamma}{delta} TCR, from one out of 99 Id2-GFP-transduced cells. These GFP+ T cells generated with our Id2-GFP transduction may have been derived from CD122– cells with rearranged TCR{gamma} and {delta} genes because we have detected V–J rearrangements of the TCR{gamma} gene from FT DN1 thymocytes and a single-cell assay has revealed the existence of progenitors capable of generating exclusively {gamma}{delta} T cells in the DN1 subset at very low frequency (our unpublished results).

When DN2 thymocytes are cultured on OP9 stromal cells, they do not generate T cells but do generate NK cells due to the lack of Notch signals, and the NK cells do not rearrange their TCRß genes (26). In contrast, we demonstrated that a fraction of NK cells, which were probably generated from p-T as a result of transient expression of transduced Id2, underwent ß rearrangements (Fig. 4B). Because the periods and levels of Id2 expression seemed to differ considerably among the transduced cells in our experiments, it is probable that the extent of the inhibitory function of Id2 against E proteins varied resulting in heterogeneous outputs for the further development of the cells. For this reason, we consider that our Id2 transduction induced, in some cases, the emergence of NK cells with rearranged TCRß genes from p-T, and in other cases NK cells without rearranged TCRß genes. Recently, Porritt et al. (37) demonstrated that adult DN1 cells can be further classified into five subsets according to their different levels of c-kit and CD24 expression, and that canonical p-T and p-NK are included in two subsets and cells in the remaining subsets do not exhibit substantial growth potential. Interestingly, rearranged TCRß D–J constructs were detected from the latter subsets. Thus, we cannot rule out the possibility that our GFP– NK cells with rearranged TCRß genes were derived from non-canonical p-T. However, this may not be the case because we could not amplify rearranged TCRß D–J constructs from 14 dpc FT DN1 cells by PCR (our unpublished data).

Cell recoveries from GFP-transduced and Id2-GFP-transduced DN1CD122– single cells after culture for 14 days were 4.3 ± 4.0 x 103 and 7.4 ± 6.1 x 103, respectively. Comparison of those numbers with those from 300 cells after culture for 11 days (5.8 ± 2.8 x 103 and 4.6 ± 0.4 x 103 for GFP and Id2-GFP transduction, respectively) reveals that there was remarkably vigorous proliferation from Id2-GFP-transduced DN1CD122– single cells. However, Fig. 3 shows that non-transduced cells tended to expand better than transduced cells. Since it has been reported that Id2 suppresses cell differentiation and facilitates cell cycling in various tissues (38), Id2-GFP-expressing cells, many of which would disappear later due to the lack of functional E proteins, might transiently increase in number during the early culture period and contribute to the survival of non-transduced cells through cell-to-cell interaction. Finally, more non-transduced cells were assumed to survive after Id2-GFP transduction than after control transduction. On the other hand, in the case of transducing 300 cells, the cell-to-cell interaction facilitating survival of the progenitors among them may have been sufficient from the beginning. Thus, for Id2-GFP transduction, only the negative effect of constitutive Id2 expression is thought to have contributed to the final smaller yield of the cells.

Our previous clonal analysis of fetal thymocytes revealed that p-T/NK, p-T and p-NK coexist in the DN1CD122– population, while the DN1CD122+ population contains p-NK but not p-T/NK or p-T (27). The latter population is missing from Id2-deficient embryos (15) and adult Id2–/– mice retain few NK cells (14). Moreover, all the progenitors in the Id2–/– DN1CD122– population were shown to generate exclusively T cells. Interestingly, the progenitor frequency of Id2–/– CD122– p-T corresponded to the sum of the frequencies of wild-type p-T, p-T/NK and p-NK (15), suggesting that the first point at which Id2 is required for NK cell development is NK lineage commitment from p-T/NK. Two additional important facts are that the cellularity of Id2–/– FT is lower than that of Id2+/+ FT and the number of Id2+/– fetal thymocytes is between those of Id2–/– and Id2+/+ fetal thymocytes (15). In conjunction with this, Id2 is expressed at low levels in the DN1CD122– population (15) and Id2 generally stimulates cell cycling of immature cells (38). Thus, Id2 may allow p-T/NK to self-renew. Based on the data obtained in this study and our previously reported data (15), we propose that self-renewal of p-T/NK or emergence of p-T or p-NK from p-T/NK is closely related to the expression level of Id2. The most immature p-T/NK in the thymus may be expressing Id2 at a low level. As long as this level remains unchanged, p-T/NK continues to self-renew. If the expression level of Id2 in p-T/NK is lowered, functional E proteins accumulate and p-T may be generated. However, these p-T are not irreversibly committed because they still possess a program for NK cell development. The lack of Notch signaling has also been suggested to start a program for NK lineage development in p-T because FT DN2 progenitors, largely p-T, generate NK cells instead of T cells when they are cultured on OP9 cells (26). Since E proteins are thought to activate the transcription of a group of genes related to Notch signal transduction (16), the fate change plan put into effect when the function of E proteins is inhibited may be overlapping with one that commences in the absence of DL1–Notch interactions. In contrast, when the expression level of Id2 rises in p-T/NK, several target genes of E proteins whose products are indispensable for T cell development are down-regulated, and NK lineage commitment may occur. However, expression of Id2 exceeding a certain level decreases functional E proteins to below the minimum amount necessary for early NK cell development, and consequently, no progeny of p-T/NK can survive. It has been shown that E2A+/– Lin– FL cells cultured on OP9-DL1 cells produce fewer T cells than E2A+/+ Lin– FL cells and also fewer NK cells than E2A–/– Lin– FL cells (21). Thus, it is plausible that the amount of E2A gene products is positively related to T cell development and inversely related to NK cell development. Since high levels of Id2 molecules cause lower functional activity of E proteins, the data concerning E2A+/– progenitors are well consistent with our model.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
This work was supported in part by the Ministry of Education, Science, Sports and culture of Japan (11470086) to SF.


   Acknowledgements
 
We thank Drs Y. Katsura, K. Ikuta, B. Malissen and W. T. V. Germeraad for critical reading and helpful comments on the manuscript.

Funding to pay the Open Access publication charges for this article was provided by the Ministry of Education, Science, Sports and culture of Japan.


   Abbreviations
 
B6, C57BL/6
dGuo, deoxyguanosine
DL1, Delta-like 1
dGuo lobe, dGuo-treated FT lobe
DN, double negative
FT, fetal thymus
FTOC, fetal thymus organ culture
FL, fetal liver
GFP, green fluorescent protein
HOS, high oxygen submersion
IRES, internal ribosomal entry site
p-NK, unipotent progenitors generating NK cells
p-T, unipotent progenitors generating T cells
p-T/NK, bipotent progenitors generating both T and NK cells
SCF, stem cell factor

   Notes
 
Transmitting editor: T. Saito

Received 27 November 2006, accepted 3 July 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

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