A Mouse Carrying Genetic Defect in the Choice Between T and B Lymphocytes

Yayoi Tokoro, Takehiko Sugawara, Hiroyuki Yaginuma, Hiromitsu Nakauchi, Cox Terhorst, Baoping Wang and Yousuke Takahama
J Immunol November 1, 1998, 161 (9) 4591-4598;

Abstract

Transgenic mice with human CD3ε gene have been shown to exhibit early arrest of T cell development in the thymus. The present study shows that, instead of T cells, B cells are generated in the thymus of a line, tgε26, of the human CD3ε transgenic mice. The accumulation of mature B cells in the thymus was found only in tgε26 mice, not in other human CD3ε transgenic mouse lines or other T cell-deficient mice, including CD3-ε knockout mice and TCR-β/TCR-δ double knockout mice. Hanging drop-mediated transfer into 2-deoxyguanosine-treated thymus lobes showed that lymphoid progenitor cells rather than thymus stromal cells were responsible for abnormal B cell development in tgε26 thymus, and that tgε26 fetal liver cells were destined to become B cells in normal thymus even in the presence of normal progenitor cells undergoing T cell development. These results indicate that lymphoid progenitor cells in tgε26 mice are genetically defective in thymic choice between T cells and B cells, generating B cells even in normal thymus environment. Interestingly, tgε26 thymocytes expressed GATA-3 and TCF-1, but not LEF-1 and PEBP-2α, among T cell-specific transcription factors that are involved in early T cell development, indicating that GATA-3 and TCF-1 expressed during thymocyte development do not necessarily determine the cell fate into T cell lineage. Thus, tgε26 mice provide a novel mouse model in that lineage choice between T and B lymphocytes is genetically defective.

Tand B lymphocytes are both originated from multipotent hemopoietic stem cells (1, 2, 3). It is generally believed that hemopoietic stem cells first differentiate into either lymphoid progenitor cells or myeloid progenitor cells, and that lymphoid progenitor cells further differentiate into T cells and B cells as well as NK cells and dendritic cells (4, 5, 6). Identification of lymphoid progenitor cells in primary hemopoietic organs such as fetal liver and adult bone marrow has still been an issue of controversy (7, 8, 9), although most immature lymphoid cells in the thymus are shown to exhibit a differentiation potential that corresponds to lymphoid progenitor cells, being capable of becoming T cells, B cells, NK cells, and dendritic cells (10, 11, 12, 13). It is also controversial whether lineage commitment of the progenitor cells into T cells takes place before or after migrating into the thymus (8, 14). Even less is understood how lymphoid progenitor cells are committed to either T cells or B cells.

We have found recently that B220, a CD45R determinant that is generally appreciated as a B cell-specific marker, is expressed by fetal liver progenitor cells that can generate T cells upon migration into the thymus, suggesting a possibility that some B cell-specific molecules are expressed by immature T-lymphopoietic progenitor cells perhaps before their commitment to T cell lineage (9). During further analysis of B cell-specific molecules expressed by immature T-lineage cells from various genetic backgrounds, we have found that B220 is highly expressed by most thymocytes in a human CD3ε transgenic mouse line, tgε26. Transgenic mice with high copy numbers of human CD3ε gene, including tgε26, have been shown to exhibit early arrest of T cell development as well as of NK cell development (15, 16), and it has been suggested that the block in T cell development is caused by the overexpression of human CD3ε proteins (15, 16). The present study describes that mature B cells expressing IgM are generated only in the thymus of tgε26 mice, not in other human CD3ε transgenic mouse lines or in other T cell-deficient mice. Our results show that lymphoid progenitor cells in tgε26 mice are responsible for abnormal B cell development in the thymus, and that tgε26 fetal liver cells are destined to become B cells even in normal thymus environment. Interestingly, tgε26 thymocytes express GATA-3 and TCF-1, T cell-specific transcription factors that are involved in early T cell development, indicating that the expression by thymocytes of GATA-3 and TCF-1 does not necessarily determine their destination into T cells. Thus, the present study describes a mutant transgenic mouse in that lymphoid progenitor cells exhibit the defect in the choice between T and B cells.

Materials and Methods

Mice

Human CD3ε transgenic mouse strains, tgε26, tgε600, and tgε2978, were described previously (15). C57BL/6 mice were purchased from SLC (Hamamatsu, Japan). TCR-β/TCR-δ double knockout mice (17) and RAG-1 knockout mice (18) were obtained from The Jackson Laboratory (Bar Harbor, ME). CD3-ε knockout mice will be described elsewhere (Wang et al., manuscript in preparation). These CD3-ε knockout mice are also deficient in the expression of CD3-γ and CD3-δ genes, similar to the recently described CD3-εΔ5/Δ5 mice (19). B6-Ly-5.1 mice were bred in a pathogen-free animal facility of Laboratory Animal Research Center at University of Tsukuba (Tsukuba, Japan).

Thymus organ cultures

Neonatal thymus organ culture was described previously (20). Briefly, thymus lobes obtained from newborn tgε26 mice on the day of birth were cultured on sponge-supported filter membranes at an interface between 5% CO2-humidified air and the RPMI 1640-based culture medium containing 10% FBS (Life Technologies, Gaithersburg, MD), 50 μM 2-ME, 2 mM l-glutamine, 1× nonessential amino acids, 10 mM HEPES, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies).

Hanging drop-mediated transfer and organ culture of either fetal thymocytes or fetal liver cells in 2-deoxyguanosine (dGuo)3-treated fetal thymus lobes were conducted as described (21, 22). Briefly, day 14 fetal thymus lobes from indicated mice were cultured for 5 to 7 days in the presence of 1.35 mM dGuo (Yamasa, Chiba, Japan) on sponge-supported filter membranes at an interface between 5% CO2-humidified air and RPMI 1640-based culture medium described above. The dGuo-treated thymus lobes were washed in fresh culture medium for three times over 2 h. Day 14 fetal thymocytes or fetal liver cells (105/well) were cultured with dGuo-treated thymus lobes (1 lobe/well) in hanging drops (20–25 μl/well) in Terasaki plate for 24 h at 37°C. Lobes were rinsed with culture medium, transferred to freshly prepared sponge-supported filter membranes, and organ cultured for indicated period.

Immunofluorescence staining and flow cytometry

Single cell suspensions were washed in PBS, pH 7.2, containing 0.2% BSA and 0.1% NaN3. Cells were first incubated with 2.4G2 anti-FcγR mAb (23) to block binding of Ig to FcγR, and stained with FITC-labeled Ab and biotinylated Ab for 30 min at 4°C. Cells were then stained with phycoerythrin (PE)-streptavidin for 10 min at 4°C. Following Abs were obtained from PharMingen (San Diego, CA): FITC anti-IgM (R6-60.2), FITC anti-B220 (RA3-6B2), FITC anti-Thy-1.2 (30-H12), FITC anti-CD4 (Rm4-5), FITC anti-BP-1 (6C3), FITC normal IgG, biotinylated anti-B220, biotinylated anti-CD8 (53-6.7), and biotinylated normal IgG. Anti-Ly-5.1 Ab (clone A20 (24)) was purified and biotinylated in our laboratory. Multicolor flow-cytometry analysis was performed using FACSort (Becton Dickinson, San Jose, CA). Data were obtained using either LYSYS II or Cellquest software on viable cells, as determined by forward light scatter intensity and propidium iodide exclusion. Cell sorting of thymocytes was conducted using FACSVantage (Becton Dickinson).

Immunohistochemistry

Adult thymus lobes from tgε26 mice or normal C57BL/6 mice were fixed in 4% paraformaldehyde and sliced for 7-μm sectioning. Fixed sections were incubated with biotinylated anti-B220 or anti-Thy-1.2 Ab, followed by streptavidin-peroxidase. Slides were developed in diaminobenzidine and counterstained in methyl green.

RT-PCR analysis of mRNA expression levels

Total cellular RNA was prepared by Isogen solution (Nippon Gene, Tokyo, Japan), followed by isopropanol precipitation. Poly(A) RNA was reverse transcribed to cDNA by oligo(dT) primers and M-MLV reverse transcriptase (Life Technologies). Equal amount of cDNA was PCR amplified for 40 cycles by using Taq polymerase (Takara, Tokyo, Japan) in the presence of indicated primers. Sequences for GATA-3 (25), TCF-1 (26), LEF-1 (26), Sox-4 (26), TCR-Cβ (9), CD3-ε (9), VpreB (9), and β2-microglobulin (9) primers were previously described. PCR primers for PEBP-2αA, 5′-AGTATGAGAGTAGGTGTCCCGCC-3′ and 5′-AAATGCTTGGGAACTGCCTGGGG-3′ (27), and for PEBP-2αB, 5′-CGCCACAAGTTGCCACCTACCAT-3′ and 5′-TGAAGGCGCCTGGGTAGTGCATG-3′ (28), were generously provided by Dr. Masanobu Satake, Tohoku University (Sendai, Japan). Amplified products were electrophoresed on 1.8% agarose gel and were visualized with ethidium bromide.

PCR analysis for TCR-β gene rearrangement

Genomic DNA (0.5 μg) was PCR amplified by EX Taq polymerase (Takara Biomedicals, Shiga, Japan) in the presence of Jβ2 primer and either one of Vβ8.2 or Dβ2 primer, as previously described (22, 29). Amplified DNA products were electrophoresed on 5% polyacrylamide gel, denatured in 0.4 M NaOH, and electrotransferred to Gene Screen Plus membranes (DuPont, Boston, MA). Membranes were hybridized with biotinylated Jβ2 probe (22). Hybridization was visualized by the PolarPlex chemoluminescence detection reagents (Millipore, Tokyo, Japan).

Results

B cells are generated in Tgε26 human CD3 transgenic thymus

To better understand molecular basis for early arrest of thymocyte development in tgε26 human CD3 transgenic mice (15), we first examined the expression of various surface molecules by tgε26 thymocytes. Consequently, we found that majority of tgε26 thymocytes expressed high levels of B220-CD45R determinant (Fig. 1⇓A). These B220+ thymocytes in tgε26 mice were IgM+IgD+CD19+ and Thy-1−CD3−CD4−CD8−, the phenotypes of mature B cells (Fig. 1⇓A). Although tgε26 thymus was disorganized, lacking cortex and medulla architectures (30), B220+ thymocytes were broadly localized within tgε26 thymus lobes (Fig. 1⇓B), ruling out a possibility that B cells in tgε26 thymocyte preparations might be derived from contaminated blood or proximal lymph nodes.

FIGURE 1.
FIGURE 1.

B cell accumulation in tgε26 thymus. A, Surface molecules expressed by tgε26+/+ thymocytes. Adult thymocytes from indicated mice were two-color stained with FITC-labeled Ab (x-axis) and biotinylated Ab (y-axis) with indicated specificity. Biotin staining was visualized with PE-streptavidin. Numbers within each box indicate the frequency of cells within that box. B, Immunohistochemical analysis of tgε26+/+ thymus. Fixed sections from adult thymus of tgε26 mice or normal C57BL/6 mice were incubated with biotinylated Ab specific for either B220 or Thy-1.2, followed by streptavidin-peroxidase. Slides were developed in diaminobenzidine and counterstained in methyl green. Shown are representative results from four (A) and three (B) independent experiments.

It has been shown that small numbers of B cells exist even in normal thymus (31; also shown in Fig. 1⇑A). To test whether B cells in tgε26 thymus reflect normal thymic B cells because of the lack of T cell development, we next measured the absolute numbers of B cells in tgε26 thymus and normal B6 thymus, as well as thymuses from TCR-β/TCR-δ double knockout mice, CD3-ε knockout mice, and RAG-1 knockout mice in which T cell development is arrested at early CD4−CD8− stage. As summarized in Table I⇓, IgM+B220+ B cells in tgε26 thymus were 1.2 × 106/head, ∼6 times more than the numbers of thymic B cells in normal B6 mice and ∼60 times more than B cell numbers in TCR-β/TCR-δ double knockout thymuses and CD3-ε knockout thymuses (Table I⇓). Thus, B cells are unusually accumulated in tgε26 thymus, neither simply reflecting the lack of T cell development nor reflecting the overrepresentation of normal thymic B cells.

View this table:
Table I.

Numbers of thymocytes from tgε26, tgε600, tgε2978, RAG-1 knockout, TCR-β/TCR-δ double knockout, and CD3ε knockout micea

To examine the origin of thymic B cells in tgε26 mice, we analyzed the ontogeny of B cells in tgε26 thymus. As shown in Figure 2⇓A, IgM+B220+ cells in tgε26 thymus were generated after birth by 1 wk old, and the frequency and numbers of B cells in tgε26 thymus reached to adult levels by 2 wk after birth. Transient increase of BP-1+B220+ cells, resembling pre-B cells, in tgε26 thymus at 1 wk old and their subsequent decrease suggested that temporal development of B cells occurs in tgε26 thymus. To directly examine whether B cells are generated from progenitor cells in tgε26 thymus, we performed organ culture of newborn tgε26 thymus lobes in which B220+ B-lineage cells were still not generated. As shown in Figure 2⇓B, IgM+B220+ cells were generated in the thymus lobes after 7 to 12 days of tgε26 thymus organ cultures. BP-1+B220+ cells were found more at day 7 than at day 12 in tgε26 thymus organ cultures (data not shown). Thus, B cells in tgε26 thymus are generated from immature progenitor cells within the thymus environment in situ, even without the supply of migrating B cells.

FIGURE 2.
FIGURE 2.

B cell generation in tgε26 thymus. A, Ontogeny of tgε26 thymocytes. Thymocytes from tgε26+/+ mice at indicated age were two-color stained with FITC-labeled Ab (x-axis) and biotinylated Ab (y-axis) with indicated specificity. Biotin staining was visualized with PE-streptavidin. Numbers within each box of contour diagrams indicate the frequency of cells within that box. Numbers in parentheses indicate the numbers of thymocytes from one mouse at indicated age. B, Neonatal thymus organ culture of tgε26 thymocytes. Thymus lobes from newborn tgε26+/+ mice were cultured in organ for indicated days. Cells recovered from the cultures were two-color stained with FITC-labeled Ab (x-axis) and biotinylated Ab (y-axis) with indicated specificity. Shown are representative results from two (A) and three (B) independent experiments.

Tgε26 progenitor cells become B cells even in normal thymus environment

To examine whether unusual B cell generation in the thymus is caused by abnormal progenitor cells or abnormal environment in tgε26 thymus, we next set up hanging drop-mediated transfer of tgε26 progenitor thymocytes into normal fetal thymus lobes that had been treated with dGuo. We found that tgε26 fetal thymocytes generated B220+ cells in normal B6 thymus lobes as well as in tgε26 thymus lobes (Fig. 3⇓). In contrast, normal B6 fetal thymocytes generated Thy-1+CD4/CD8+ cells in tgε26 thymus lobes as well as in normal B6 thymus lobes (Fig. 3⇓). Thus, lymphoid progenitor cells rather than thymus environment are responsible for B cell generation in tgε26 thymus. It is interesting to note that many B220+ cells in tgε26-derived thymocytes expressed low levels of Thy-1, as exemplified in tgε26 → B6 cells (Fig. 3⇓), although B6 environment in tgε26 → B6 condition did not especially enrich Thy-1low cells or always retard growth and development (data not shown). Thy-1low expression by B220+ tgε26 thymocytes appeared to be more pronounced in immature B220+ cells than in mature IgM+B220+ B cells (Fig. 2⇑, A and B), consistent with previous findings that immature B-precursor cells express Thy-1 even during normal B cell development (32).

FIGURE 3.
FIGURE 3.

B cell generation in normal thymus stromal cells from tgε26 progenitor cells. Fetal thymocytes (105) from either tgε26+/+ (ε26) mice or normal B6 mice were transferred in hanging drops to dGuo-treated fetal thymus lobes from indicated mice, and organ cultured for 12 days. Cells recovered from thymus cultures were stained with FITC-labeled anti-Thy-1.2 or anti-CD4 Ab. Cells were also stained with biotinylated anti-B220 or anti-CD8 Ab, followed by PE-streptavidin. Numbers indicate the frequency of cells within the defined area. Shown are representative results from three independent experiments.

To analyze whether tgε26 progenitor cells generate B cells even in the presence of normal T cell development in the thymus, fetal liver progenitor cells from tgε26 mice were mixed at graded ratios with fetal liver cells from normal B6-Ly-5.1 mice, and transferred into dGuo-treated B6-Ly-5.1 thymus lobes (Fig. 4⇓). Tgε26 cells (Ly-5.1−2+) could be distinguished from B6-Ly-5.1 cells (Ly-5.1+2−) by allele-specific detection of CD45 (Ly5) molecules. As shown in Figure 4⇓, Ly-5.1− tgε26-derived cells became B220+ even in the presence of normal Ly-5.1+ thymocytes. It is interesting to note that the ratio of input cell numbers between tgε26 and B6-Ly-5.1 fetal liver cells correlated with the ratio between Ly-5.1− tgε26-derived B cells and Ly-5.1+ normal thymocytes generated in the thymus, suggesting that tgε26 progenitor cells are capable of generating B cells at an efficiency comparable with normal progenitor cells generating T cells in the thymus. These results indicate that tgε26 progenitor cells efficiently generate B cells even in the thymus in which normal T cell development occurs, and that immature lymphoid progenitor cells in tgε26 fetal liver are abnormal in generating B cells upon migration in the thymus environment.

FIGURE 4.
FIGURE 4.

B cell generation in normal thymus environment from tgε26 progenitor cells. Fetal liver cells (105) from indicated mice were transferred to dGuo-treated day 14 fetal thymus lobes in hanging drops, and organ cultured for 8 days. Where indicated, the mixture (105 cells in total) of tgε26+/+ fetal liver cells and B6-Ly-5.1 fetal liver cells were transferred to dGuo-treated thymus lobe. Cells from thymus cultures were two-color stained with FITC-labeled anti-B220 and biotinylated anti-Ly-5.1 Ab, followed by PE-streptavidin. Adult thymocytes from tgε26+/+ and B6-Ly-5.1 mice were also stained with indicated Abs. Numbers indicate the frequency of cells within the defined area. Shown are representative results from three independent experiments.

Other human CD3ε transgenic mice do not exhibit excessive B cell development in the thymus

To gain insights into the mechanism by which tgε26 progenitor cells develop into B cells in the thymus, we examined whether two other strains of human CD3ε transgenic mice may exhibit the similar accumulation of thymic B cells. Tgε600 mice carry the same human CD3ε transgene as in tgε26 mice, whereas tgε2978 mice carry the transgene that encodes transmembrane and cytoplasmic regions of human CD3ε proteins lacking extracellular regions (15). Nonetheless, both tgε600 and tgε2978 mice exhibit severe defect in early T cell development (15). As shown in Table I⇑, however, both tgε600 and tgε2978 human CD3ε transgenic mice showed no increases in B cells in the thymus; in contrast, tgε26 thymocytes showed clear B cell accumulation. It should be noted that tgε2978+/+ mice, which carry higher copy numbers (80–100 copies) of the transgene than those in tgε26+/+ mice (40–60 copies), did not exhibit any accumulation of B cells in the thymus, suggesting that B cell generation in tgε26 mice is not a direct consequence of high copy numbers of human CD3ε transgene. Thus, thymic accumulation of B cells in tgε26 mice is a phenotype specific for tgε26 transgenic mice, not a phenotype commonly observed in other human CD3ε transgenic mice, including tgε600 and tgε2978 strains.

We next examined whether B cells are predominantly generated even in the thymus of F1 hybrid mice crossed between tgε26 homozygous transgenic mice and normal C57BL/6 (B6) mice. Unlike tgε26 homozygotes, (tgε26 × B6) F1 heterozygous mice exhibited T cell development in the thymus, although they had the thymus of 10∼20% cellularity of normal B6 mice (Table I⇑). Despite the generation of many T cells, however, tgε26+/− heterozygous thymocytes contained increased numbers of B cells as compared with normal B6 thymocytes (Table I⇑). Thus, the combination of complete T cell deficiency and increased B cell development in the thymus was found only in tgε26 transgenic homozygotes, not in tgε26 heterozygotes, whereas tgε26 heterozygotes showed a compromised T cell development and increased B cell generation in the thymus.

Expression of GATA-3 and TCF-1, but not PEBP-2α and LEF-1, in Tgε26 thymus

We finally examined whether tgε26 thymus, which generated B cells instead of T cells, may express modulated expression of transcription factors that are involved in early development of T cells. To do so, RT-PCR analysis was performed for the expression of T cell-specific transcription factors such as GATA-3, TCF-1, PEBP-2αA, PEBP-2αB, LEF-1, and Sox-4 (Fig. 5⇓A). We have found that tgε26 thymocytes expressed GATA-3, TCF-1, and Sox-4, but failed to express PEBP-2αA, PEBP-2αB, and LEF-1 (Fig. 5⇓A). The expression of Sox-4 in B cell-generating tgε26 thymocytes is consistent with previous findings that Sox-4 is expressed in immature B cells as well as in T-lineage cells (33, 34). On the other hand, it has been shown that the expression of GATA-3 and TCF-1 transcription factors is restricted in T cell lineage (35, 36, 37, 38). It is therefore interesting to point out that GATA-3 and TCF-1 transcripts were detected even in IgM+B220+ mature B cells as well as in IgM−B220− cells within tgε26 thymus (Fig. 5⇓B). The detection of GATA-3 and TCF-1 transcripts in IgM+B220+ B cells in tgε26 thymus is not due to contaminated T-lineage precursor cells, since these transcripts were not detected in 10-fold- or 100-fold-diluted cDNA from IgM−B220− purified cells that contained equivalent amount of cDNA from IgM−B220− cells contaminated in IgM+B220+ preparations (Fig. 5⇓C). Moreover, in contrast to B cells in tgε26 thymus, GATA-3 and TCF-1 transcripts were not detected in IgM+B220+ B cells in the spleen of tgε26 mice (Fig. 5⇓D), supporting the possibility that the expression of GATA-3 and TCF-1 by B cells in tgε26 thymus reflects abnormal switch toward B cells of thymus-migrated progenitor cells that are otherwise directed into T-lineage development. Collectively, these results indicate that 1) GATA-3 and TCF-1 expressed by thymocytes are not sufficient for their final decision to enter T cell lineage, and that 2) the defect in tgε26 thymocytes is associated with the failure for thymocytes in expressing PEBP-2αA, PEBP-2αB, and LEF-1.

FIGURE 5.
FIGURE 5.

RT-PCR analysis of mRNA expression by tgε26 thymocytes. Oligo(dT)-primed cDNA prepared from indicated cell populations was employed for RT-PCR analysis using indicated primers. Water alone (−) or cDNA from adult B6 thymocytes (+) was also PCR amplified for negative and positive controls. Molecular weight marker (M) was 100-bp DNA ladder (Life Technologies). A, cDNA prepared from tgε26+/+ thymocytes was PCR amplified. For the positive control of VpreB detection, cDNA from day 14 fetal liver cells from B6 mice was used. Signals with the expected size derived from correctly spliced RNA are indicated with arrows. B and C, Thymocytes from tgε26+/+ mice were two-color stained for IgM and B220. IgM+B220+ cells and IgM−B220− cells were sorted by FACS-Vantage flow cytometry. Purity of sorted IgM+B220+ cells and IgM−B220− cells was 95.9 and 96.6% in B, and 99.6 and 98.1% in C, respectively. Equal numbers of IgM+B220+ cells and IgM−B220− cells were used for cDNA preparation, giving rise to largely comparable β2-microglobulin signals between the two groups. C shows that, unlike IgM+B220+ purified cells, 10-fold- or 100-fold-diluted cDNA from IgM−B220− purified cells failed to give rise to GATA-3 signals, ruling out the possibility that the GATA-3 signal from IgM+B220+ purified cells could be derived from <10% of contaminated IgM−B220− cells. D, Spleen cells from tgε26+/+ mice were purified for IgM+B220+ (99.2% purity). Shown are representative results from six (A), three (B), two (C), and two (D) independent experiments.

It is also interesting to note that tgε26 thymocytes expressed TCR-Cβ transcripts, although they did not express endogenous CD3-ε, another T cell-specific molecule, and instead they expressed VpreB, a B cell-specific molecule (Fig. 5⇑A). To examine whether TCR-β gene locus is rearranged in tgε26 thymocytes, DNA prepared from tgε26 thymocytes was PCR amplified with either Dβ- and Jβ-specific primers or Vβ8.2- and Jβ-specific primers. Unlike normal thymocytes, tgε26 thymocytes did not contain any detectable rearrangement of TCR-β gene locus, including D-J rearrangement (Fig. 6⇓). These results suggest that TCR-Cβ transcripts expressed by tgε26 thymocytes represent germline Cβ transcripts of unrearranged TCR-β gene.

FIGURE 6.
FIGURE 6.

D-Jβ and V-DJβ rearrangement in tgε26 thymocytes. Genomic DNA from adult thymocytes of indicated mice was subject to TCR-β gene rearrangement analysis using indicated PCR primers. PCR amplification was conducted for 25 cycles (Dβ2-Jβ2) and 30 cycles (Vβ8.2-Jβ2). PCR products containing Jβ2 segments were visualized with biotinylated probe hybridization. Equal amount (0.5 μg/reaction) of genomic DNA was used for PCR amplification to minimize possible deviation among multiple PCR reactions. The sensitivity of every PCR detection was normalized by pretitrating PCR cycle numbers, so that rearranged signals can be visualized when 0.1 to 1% of rearranged DNA from adult thymocytes was mixed in liver DNA (22). For the analysis of D-Jβ rearrangement, unrearranged DNA gives 1.8-kb signals corresponding to germline D-Jβ2, whereas the rearrangement gives six discrete signals corresponding to rearranged D-J genes using six Jβ2 segments (22). For the analysis of Vβ8.2-DJβ rearrangements, unrearranged DNA gives no signals, whereas the rearrangement gives discrete signals corresponding to rearranged genes (22). Biotinylated HinfI fragments of pX174 DNA (726–24 bp; Life Technologies) were used as a m.w. marker. Shown are representative results from two independent experiments.

Discussion

The present study shows that, instead of T cells, B cells are generated in the thymus of tgε26 human CD3ε transgenic mice. Selective B cell generation in the thymus was found only in tgε26 mice, not in other T cell-deficient mice, including two other human CD3ε transgenic mouse lines. The transfer of fetal liver progenitor cells into thymus lobes showed that tgε26 lymphoid progenitor cells were destined to become B cells even in normal thymus environment, indicating that lymphoid progenitor cells in tgε26 mice are genetically defective in thymic choice between T cells and B cells. Interestingly, tgε26 thymocytes expressed GATA-3 and TCF-1, indicating that GATA-3 and TCF-1 expressed during thymocyte development do not necessarily determine the cell fate into T cells. Thus, tgε26 mice provide a novel mouse model in that lineage choice between T and B lymphocytes is genetically defective.

The present results show that abnormal lymphoid progenitor cells in tgε26 mice are responsible for unusual accumulation of B cells in the thymus. The accumulation of B cells in tgε26 thymus is not merely due to the overrepresentation of normal thymic B cells by the lack of T cells that otherwise dominate thymocytes, since 1) such an accumulation of thymic B cells was not found in other T cell-deficient mice, including CD3-ε knockout mice and TCR-β/TCR-δ double knockout mice (Table I⇑); 2) the numbers of B cells in small tgε26 thymus were approximately sixfold higher than those of B cells in normal large thymus (Table I⇑); and 3) B cells in tgε26 thymus mostly belong to CD5−/low conventional B cells (Tokoro and Takahama, unpublished results), unlike normal thymic B cells that are reported to be mostly CD5+ B-1 cells (39). Instead, immature lymphoid progenitor cells of tgε26 mice either from fetal liver or fetal thymus efficiently and exclusively gave rise to B cells in the thymus (Figs. 3⇑ and 4⇑). Moreover, in vitro fetal thymus organ culture experiments (Fig. 4⇑) and in vivo bone marrow transfer experiments (B.W., unpublished results) indicated that such a selective generation of B cells by tgε26 progenitor cells was found even in normal thymus environment in which normal progenitor cells undergo normal T cell development. Furthermore, the early immigrants in tgε26 thymus were still B220−BP1−Thy1−, capable of generating temporal progression of pre-B and B cell differentiation in the thymus (Fig. 2⇑), suggesting that the abnormal B cell development in tgε26 thymus is due to thymic migration of abnormal lymphoid progenitor cells rather than abnormal thymic migration of further differentiated B-lineage cells. Together, we think that tgε26 lymphoid progenitor cells are destined to become B cells, being defective in the choice for T cells upon migration into the thymus.

Although tgε26 progenitor cells are destined to become B cells in the thymus, tgε26 thymocytes expressed several T cell-specific transcripts, including GATA-3, TCF-1, and unrearranged TCR-β. The expression of these T cell-specific molecules by B cell-oriented tgε26 thymocytes suggests that these molecules may be expressed during early thymocyte development before the final commitment to become T cells. Together with our previous findings that several B cell-specific molecules are expressed during early T cell development in normal fetal liver (9), it is possible that lymphoid progenitor cells that can become either T or B cells may first express several T cell- and B cell-specific molecules together, before reaching to T/B-branching point at which cells would terminate the expression of molecules specific for alternative lineage.

More interestingly, the results showing that GATA-3 and TCF-1 are expressed by tgε26 thymocytes indicate that the expression of GATA-3 and TCF-1 by lymphoid progenitor cells is not sufficient for final decision for T cell development even after the migration into the thymus. The expression of GATA-3 and TCF-1 even by IgM+ B cells in tgε26 thymus further indicates that the expression of these T cell-specific transcription factors does not prohibit the process of B cell development. On the other hand, it has been shown that GATA-3 and TCF-1 are both essential for normal T cell development (26, 36, 38). Thus, it is conceivable that GATA-3 and TCF-1 are required for supporting earliest stages of T cell differentiation, but are sufficient for neither supporting T cell development nor inducing the commitment to T cell lineage.

We have also found that tgε26 thymocytes expressed TCR-Cβ transcripts. Since the sensitive PCR assay for D-Jβ rearrangement failed to detect any TCR-β rearrangement in tgε26 thymocytes, our results suggest that tgε26 thymocytes express unrearranged sterile TCR-Cβ transcripts. These results suggest that the expression of germline TCR-Cβ transcripts may occur before branching point between T and B cells, and does not necessarily reflect the commitment to T cells. In addition, the failure to detect other T cell-specific transcription factors such as LEF-1, PEBPα2A, and PEBP2αB in tgε26 thymocytes suggests that 1) the failure in expressing these transcription factors may be involved in early arrest of T cell development in tgε26 mice, and 2) the expression of these transcription factors may not be required for the expression of germline TCR-Cβ transcripts.

How are lymphoid progenitor cells in tgε26 mice destined to become B cells even in thymus environment? Our results show that the aberrant switch from T cells to B cells in the thymus occurs only in tgε26 mice, not in other two lines of human CD3ε transgenic mice generated in the same laboratory, including a transgenic line carrying higher copy numbers of the transgene (Table I⇑). It has been shown previously that the copy numbers of the human CD3ε transgene correlate well with the expression levels of the CD3ε transgenic proteins (40), suggesting that B cell generation in tgε26 thymus is due to neither high copy numbers of the transgene nor high expression of the transgenic products. Rather, our results show that the aberrant B cell development in the thymus is limited only in the tgε26 line among CD3ε transgenic mouse strains. Thus, we think that the defect of lymphoid progenitor cells in the choice between T and B cells may be a consequence of transgene insertion into a gene locus that is crucial for the commitment to either T cells or B cells, although it is still possible that the phenotype is caused by a unique pattern of expression of transgenic CD3ε protein in early progenitor cells in tgε26 mice. Our results also show that heterozygous tgε26+/− mice still show the accumulation of B cells in the thymus, compatible with the possibility that abnormal B cell development in tgε26 thymus is a genetically dominant phenotype. It is thus possible that the enhancer and promoter in the human CD3ε transgene may drive the expression of a nearby gene in thymus-migrated lymphoid progenitor cells, which in turn results in the aberrant conversion of the developmental direction from T cell lineage to B cell lineage. Alternatively, it is also possible that a gene at the site of transgenic insertion may be disrupted, resulting in the switch from T cell development to B cell development in the thymus. To better understand the molecular mechanism causing this defect in the choice between T and B cells, we are currently attempting to identify the transgene-inserted gene locus in tgε26 mice.

In conclusion, the present study describes a mouse strain in that lymphoid progenitor cells are destined to become B cells in the thymus. These mice serve a unique model for the genetic defect in the choice between T and B lymphocytes. Understanding molecular basis causing the defect in tgε26 mice will provide a useful clue to reveal the molecular mechanism underlying the lineage commitment between T cells and B cells.

Acknowledgments

We thank Drs. Masanobu Satake and Masayuki Yamamoto for polymerase chain reaction primers; Dr. Hiroyuki Ichijo for helping immunohistochemical analysis; and Drs. Willem van Ewijk, Masanobu Satake, and Alfred Singer for helpful discussion.

Footnotes

  • ↵1 The present study was supported by University of Tsukuba Research Projects, The Naito Foundation, Uehara Memorial Foundation, Kanahara Ichiro Memorial Foundation, PRESTO Research Project Unit Process and Combined Circuit, and Ministry of Education, Science, Sports, and Culture of Japan.

  • ↵2 Address correspondence and reprint requests to Dr. Y. Takahama, PRESTO Research Project and Institute of Basic Medical Sciences, TARA Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, 305-8577, Japan. E-mail address: takahama{at}tara.tsukuba.ac.jp

  • ↵3 Abbreviations used in this paper: dGuo, 2-deoxyguanosine; PE, phycoerythrin.

  • Received March 23, 1998.
  • Accepted June 25, 1998.

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The Journal of Immunology
Vol. 161, Issue 9
1 Nov 1998
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