Enforced Expression of GATA-3 During T Cell Development Inhibits Maturation of CD8 Single-Positive Cells and Induces Thymic Lymphoma in Transgenic Mice

Mice

The GATA-3^+/nlslacZ mice in which one GATA-3 allele was replaced by a lacZ reporter have been described previously (22). For the generation of the CD2-GATA3 construct, the translation initiation site was mutated (ATG to GTG) in a murine GATA-3 cDNA clone and three hemagglutinin (HA) epitope tags were added along with a new ATG and Kozak’s consensus sequence. Subsequently, the ∼2-kb mGATA-3 was cloned into a human CD2 mini-gene Bluescript SK vector, with ∼5 kb of CD2 5′ promoter sequence and ∼5.5 kb of 3′ CD2 flanking sequences (38). The latter contained the 3′ untranslated sequence and poly(A) addition site of the CD2 gene, as well as the LCR, which was shown to confer T cell-specific, copy-dependent, integration site-independent expression in transgenic mice (37). A 13.2-kb linear fragment was injected into pronuclei of FVB × FVB fertilized oocytes at a concentration of ∼2 ng/μl. Founder mice were identified by genomic Southern blotting and crossed onto an FVB background. To determine the genotype of the subsequent generations, tail DNA was analyzed by Southern blotting of either EcoRI/XbaI double digests hybridized to a 2-kb HindIII CD2 LCR probe (39) or EcoRI digests hybridized to a 800-kb partial GATA-3 cDNA probe (21).

Western blotting analyses

Total nuclear protein extracts were prepared according to Andrews and Faller (40). Protein concentration in the nuclear extracts was determined using the bicinchoninic acid protein assay (Pierce, Rockford, IL). For Western blotting analysis, 50 μg of total nuclear protein was loaded per lane and separated on 10% SDS-PAGE gels under reducing conditions and transferred to polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA). Blots were blocked with 2% BSA in PBS (pH 7.0)/0.05% Tween 20 and incubated with first- and second-step reagents in 2% nonfat dry milk in PBS (pH 7.0)/0.05% Tween 20. The mouse anti-GATA-3 mAb Hg-3-31 and the polyclonal rabbit-anti-HA Ab Y11 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Second-step reagents were HRP-conjugated goat anti-mouse Ig and swine anti-rabbit Ig from Dako (Glostrup, Denmark). Peroxidase activity was visualized by ECL using standard procedures.

Flow cytometric analyses

The preparation of single-cell suspensions, determination of β-galactosidase activity using fluorescein-di-β-d-galactopyranoside (FDG), mAb incubations, and three- or four-color cytometry have been described previously (41). The following mAb were purchased from BD PharMingen (San Diego, CA): FITC-conjugated anti-CD3ε and anti-TCRαβ, PE-conjugated anti-CD4 (L3T4), anti-CD24/HSA, anti-CD25 (clone 3C7), anti-CD62L and anti-CD69, CyChrome-conjugated anti-CD4, anti-CD8 and anti-CD44, biotinylated anti-CD4 and anti-CD8, APC-labeled anti-CD3ε, and anti-CD4. Secondary Abs used were PE-, TriColor-, or APC-conjugated streptavidin (Caltag, Burlingame, CA). FDG and To-Pro3 were purchased from Molecular Probes Europe (Leiden, The Netherlands). FITC-labeled annexin V was obtained from Nexins Research (Hoeven, The Netherlands).

For intracellular detection of GATA-3 protein, cells were fixed and permeabilized using paraformaldehyde and saponin as described previously (42) and subsequently incubated with the Hg-3-31 anti-GATA-3 mAb (Santa Cruz Biotechnology) and FITC-labeled anti-mouse IgG1 (BD PharMingen) as a second step.

Simultaneous two-color staining of membrane CD4 and CD8, combined with a TUNEL technique to quantify apoptosis, was performed using fluorescein in situ cell death detection (Roche Molecular Biochemicals, Mannheim, Germany) as described elsewhere (43).

GATA-3 expression is strongly induced during positive selection of developing T cells

We have previously quantified the GATA-3 expression profile during T cell development in vivo by placing a lacZ reporter gene, containing a nuclear localization signal, under direct GATA-3 transcriptional control. In these GATA-3^+/nlslacZ mice, GATA-3-directed lacZ expression was analyzed by flow cytometry using FDG as a β-galactosidase substrate, and differential expression of GATA-3 in DP (∼16% lacZ⁺ cells), CD4 SP (∼84%), and CD8 SP (∼33%) cells was found (22). Since it has been shown that DP T cells differentiate into mature SP T cells via a series of phenotypically distinct subpopulations, reflecting the multistage process of positive selection and CD4/CD8 lineage commitment (2, 11, 12), we investigated GATA-3 gene expression in these subpopulations in more detail.

Upon MHC-TCRαβ interaction in DP cells, the surface expression of the CD69 marker is up-regulated (32, 33, 34, 35), followed by a down-regulation of the CD4/CD8 coreceptor surface expression (2). Therefore, we analyzed lacZ activity in conjunction with surface expression of CD4, CD8, and CD69 (Fig. 1⇓). The majority of DP cells (∼85%) did not express CD69 on the cell surface and in this CD69⁻ DP population lacZ was expressed in ∼19% of cells. By contrast, within the CD69⁺ DP subpopulation, lacZ was expressed in ∼54% of the cells. After subsequent down-regulation of coreceptor expression and transition into the CD4^lowCD8^low subpopulation, ∼80% of the CD69⁺ cells expressed lacZ (Fig. 1⇓). The CD4^lowCD8^low cells have been shown to subsequently enhance CD4 expression (2), thereby developing into the CD4⁺CD8^low subset, which still contains precursors for both CD4 and CD8 SP T cells (2, 44, 45, 46). LacZ expression was present in ∼86% of these CD4⁺CD8^lowCD69⁺ cells (Fig. 1⇓). In addition, we found that in the DP, CD4^lowCD8^low, and CD4⁺CD8^low subpopulations, lacZ expression correlated with the expression levels of TCRαβ or CD3 on the cell surface (shown for TCRαβ in Fig. 1⇓A). These results indicated that the induction of GATA-3 transcription coincides with CD3 and TCRαβ up-regulation and CD69 expression in DP cells.

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FIGURE 1.

Analysis of lacZ expression in thymocytes from GATA-3^+/nlslacZ mice by four-color flow cytometry. A, Thymus cell suspensions were loaded with the β-galactosidase substrate FDG and subsequently stained with anti-CD4 and anti-CD8 in combination with anti-CD69, anti-TCRαβ, or anti-HSA Abs. Cells were analyzed for the expression of CD4 and CD8, the indicated subpopulations A–E were gated and analyzed for lacZ expression and the fourth surface marker (CD69, TCRαβ, or HSA). The numbers indicate the percentage of lacZ-expressing cells in the subpopulations analyzed. All samples are lymphocyte gated by FSC and side scatter. B, Overview of the proportions of lacZ-expressing cells in the indicated thymocyte subpopulations in GATA-3^+/nlslacZ mice. The numbers are mean values ± SD (n = 3). The background percentages of β-galactosidase-positive cells, as determined in wild-type control mice, was <1% in all subpopulations.

GATA-3 gene expression is down-regulated after commitment to the CD8 lineage

Bipotential CD4⁺CD8^lowCD69⁺ T cells differentiate into either CD4 or CD8 SP cells by shutting down expression of the reciprocal coreceptor gene (2, 44, 45, 46). Final maturation of SP thymocytes is accompanied by down-regulation of CD69 and HSA on the cell surface and induction of high-level expression of CD62L and CD44 (36, 47, 48).

As shown in Fig. 1⇑, lacZ expression was present in ∼87% of the cells committed to the CD4 lineage (CD4⁺CD8⁻CD69⁺TCRαβ^high cells) and in ∼78% of the more mature CD4⁺ cells with a CD69⁻HSA^low surface profile. By contrast, during the maturation of CD8 lineage cells, ∼40% of the CD69⁺HSA⁺ and only ∼16% of the mature CD69⁻HSA^low CD8⁺TCRαβ^high cells expressed lacZ. Likewise, lacZ expression was found to be significantly down-regulated in mature CD44⁺ and CD62L⁺CD8⁺ cells (data not shown). The intensities of the fluorescence signals show that the lacZ expression levels per cell increased slightly during the maturation process of CD4 SP cells, whereas CD8 SP cells displayed lower and more heterogeneous lacZ expression levels (Fig. 1⇑A).

When the T cells leave the thymus, the proportions of GATA-3⁺ cells decrease to ∼20% of the CD4⁺ and to <1% of the CD8⁺ T cell populations in the spleen and lymph nodes (22). For the CD4⁺ lineage cells in the spleen, we did not observe a clear correlation between GATA-3 and the expression of the HSA, CD69, CD44, CD62L, or CD25 surface markers, which are instrumental to specify subpopulations of naive, activated, or memory T cells (49, 50).

As summarized in Fig. 1⇑B, the proportions of GATA-3-expressing cells were low in CD3⁻TCRαβ⁻CD69⁻ DP cells (∼19%) and increased at the onset of positive selection events characterized by up-regulation of CD3 and TCRαβ surface expression and induction of CD69. The proportions increased to ∼86% at the stage of the last uncommitted subset of CD4⁺CD8^lowCD69⁺cells and remained high for the most mature thymic CD69⁻CD4⁺ subpopulation. By contrast, commitment to the CD8 lineage was associated with down-regulation of GATA-3 expression, resulting in <20% GATA-3⁺ cells within the mature population of CD69⁻CD8⁺ cells.

Transgenic expression of GATA-3 driven by the human CD2 LCR

To modify GATA-3 expression in vivo, transgenic mice were generated in which the murine GATA-3 gene, 5′ tagged with three HA epitopes, was expressed under the control of the human CD2 LCR (38). Two independent CD2-GATA3-transgenic lines, TgA and TgB, were established that appeared to contain comparable numbers of transgene copies (data not shown). No differences were found between the two lines in any of the performed analyses. The offspring did not manifest developmental defects or any increased susceptibilities to infectious disease or malignancies for over 9 mo of age, with the exception of the observed thymic lymphomas discussed below.

Expression of the CD2-GATA3 transgene was analyzed in various lymphoid tissues by comparing transgenic and nontransgenic littermates. Western blotting experiments were performed on nuclear protein extracts from 2- to 3-mo-old mice using a mouse mAb specific for GATA-3 and a polyclonal Ab specific for the HA tag. The endogenous GATA-3 gene encodes a ∼47-kDa protein that was detected in nuclear extracts from thymus and spleen of wild-type as well as CD2-GATA3-transgenic mice (Fig. 2⇓A). The anti-GATA-3 Ab also detected a slightly larger ∼51-kDa band in the thymus samples from mice of both transgenic lines. Comparison of the intensities of the ∼47- and ∼51-kDa GATA-3-specific bands in these nuclear protein extracts from thymus samples showed that the expression level of the 3XHA-GATA-3 transgene-encoded protein was similar to that of the endogenous GATA-3 protein. In contrast, in the spleen samples, the ∼51-kDa GATA-3-specific band was very weak or absent (Fig. 2⇓A). The ∼51-kDa band in the thymus extracts of transgenic mice was also recognized by Ab against the HA tag, but the expression levels of transgene-encoded GATA-3 protein in the spleen and lymph node extracts were very low and often almost undetectable by Western blotting analyses (Fig. 2⇓B).

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FIGURE 2.

Expression of GATA-3 in lymphoid organs of wild-type and CD2-GATA3-transgenic mice. A, Western blotting analyses of GATA-3 protein expression in total nuclear protein extracts from thymus and spleen from wild-type (wt) and CD2-GATA3-transgenic mouse lines (tgA and tgB) as detected by anti-GATA-3 Abs (∼47-kDa band, endogenous (endo.) GATA-3; ∼51-kDa band, transgenic (trans.) GATA-3). B, Western blotting analyses of GATA-3 protein expression in nuclear extracts from the indicated tissues from wild-type and CD2-GATA3 tgA mice. GATA-3 is detected by anti-GATA3 Abs (upper half) or anti-HA Abs (lower half). In the anti-HA blot, thymus and spleen cell extracts from both nontransgenic and CD2-GATA3 transgenic mice displayed two weak background bands, just above the ∼51-kDa HA-GATA-3 band. C, Cell suspensions were stained for surface CD3, CD4, and CD8 expression, and subsequently for intracellular GATA-3 protein. The indicated T cell subpopulations were gated and analyzed for GATA-3 expression. The results are displayed as histograms of CD2-GATA3-transgenic mice (bold lines) along with those of nontransgenic control mice (thin lines). CD4⁻CD8⁻ populations were gated on CD3⁻ cells. CD4^lowCD8^low cells and CD4⁺CD8^low were gated on CD3⁺ cells. The numbers indicate the mean fluorescence intensities in nontransgenic (normal type) and CD2-GATA3-transgenic mice (bold type). Data shown are representative of six mice examined within each group.

To further investigate differential expression of GATA-3 in the individual stages of T cell development, intracellular flow cytometry experiments were performed using the mouse monoclonal antiserum specific for GATA-3. Although this technique is limited by a background signal of the GATA-3 Ab, it allows a comparison of GATA-3 expression levels in nontransgenic and CD2-GATA3-transgenic mice in separate T cell subpopulations. In the wild-type animals, the GATA-3 levels were low in DP cells, increased during positive selection in CD4^lowCD8^low cells, and were high in CD4 SP cells (Fig. 2⇑C), consistent with our findings in the GATA-3^+/nlslacZ mice (Fig. 1⇑). Expression of the CD2-GATA3 transgene was determined by comparison of the mean fluorescence intensities of intracellular GATA-3 staining in histogram overlays of transgenic and nontransgenic mice, revealing substantial GATA-3 overexpression in most thymocyte subpopulations (Fig. 2⇑C). GATA-3 protein levels were uniformly higher in DP, CD4^lowCD8^low, CD4⁺CD8^low, and CD4 SP thymic subpopulations from CD2-GATA3-transgenic mice as compared with wild-type mice. By contrast, for the CD8 SP cells in the thymus and the CD4⁺ or CD8⁺ T cells in the spleen, GATA-3 levels in the CD2-GATA3-transgenic mice were close to those observed in wild-type littermates. Therefore, these findings confirm the very low expression levels of the transgene-encoded GATA-3 protein in peripheral T cells that were observed in the Western blotting experiments. Since CD2 surface expression in the individual T cell subpopulations in thymus and spleen was comparable (data not shown), the observed modulated GATA-3 protein expression profile in the transgenic mice does not appear to reflect the activity of the CD2 LCR.

Collectively, these data show that the presence of the CD2-GATA3 transgene resulted in a modification of the expression pattern of GATA-3 during T cell development, without extreme overexpression of GATA-3 protein in any of the thymic subpopulations. Especially in the DP population, which normally show little GATA-3 expression, the presence of the CD2-GATA3 transgene strongly increased the GATA-3 protein levels.

CD2-GATA3-transgenic mice have decreased CD8⁺ T cell numbers in the periphery

To analyze the effect of the CD2-GATA3 transgene on T cell development, we examined the T cell populations in thymus, spleen, and mesenteric lymph nodes from 2- to 3-mo-old CD2-GATA3-transgenic mice and nontransgenic littermates by flow cytometry (Fig. 3⇓). In the CD2-GATA3 mice, the sizes of the main thymocyte subpopulations, the DN, DP, and SP cells, were within the normal ranges, indicating that the enforced GATA-3 expression did not dramatically impede thymocyte development (Fig. 3⇓). Moreover, thymus cellularity was not significantly different between transgenic mice (99 ± 34 × 10⁶, n = 21) and nontransgenic littermates (103 ± 37 × 10⁶, n = 14). No significant differences were detected between CD2-GATA3-transgenic mice and normal littermates within the DN subpopulations as defined by differential CD44 and CD25 expression (data not shown). In contrast, the CD2-GATA3-transgenic mice had fewer CD8⁺ T cells (∼50% of control) in spleen and lymph nodes (shown for spleen in Fig. 3⇓). The residual transgenic CD8⁺ T cells present exhibited a more heterogeneous CD8 expression and higher CD3 expression on the cell surface. The numbers of CD4⁺ T cells in the periphery were comparable between the two groups of mice.

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FIGURE 3.

Phenotype of CD2-GATA3-transgenic mice. T cell development in the presence of the CD2-GATA3 transgene results in reduced numbers of peripheral CD8⁺ cells. Flow cytometric analyses of the thymus and spleen of 2-mo-old wild-type and CD2-GATA3 mice. Single-cell suspensions were stained with anti-CD3, anti-CD4, and anti-CD8 Abs. Results are displayed as dot plots of lymphocyte gate cells; percentages of total cells within the indicated quadrants are given. Data shown are representative of >20 mice examined within each group.

Taken together, these results indicated that enforced expression of GATA-3 did not result in detectable adverse effects on CD4⁺ T cell development in 2- to 3-mo-old CD2-GATA3-transgenic mice. In contrast, mature CD8 SP cells manifested decreased survival, either within the thymus or shortly after leaving the thymus.

CD2-GATA3-transgenic mice develop thymic lymphomas

When the CD2-GATA3-transgenic mice were followed up to 9 mo of age, ∼50% (26 of 51) developed thymic lymphomas (Fig. 4⇓A). Typically, these lymphomas were noticed as mice displayed respiratory distress at the age of 6–8 mo, but in three cases such animals were observed at ∼3 mo of age. Tumor frequencies in the two independent transgenic lines were similar, whereas tumors were not seen in nontransgenic littermates. Several animals with a thymic lymphoma exhibited enlargement of spleen or lymph nodes. Lymphoma cells were found to be present in the spleen, liver, lymph nodes, and kidney, indicating that the thymic lymphomas metastasized to the periphery. This was confirmed by the presence of identical clonal TCRβ rearrangement patterns in Southern blotting analyses using probes specific for J_β1 or J_β2 gene segments (Fig. 4⇓B). In a fraction of the tumors analyzed, we observed J_β2 restriction fragment patterns that would be consistent with biclonality (see Fig. 4⇓B, TL5). Often particular restriction fragments were lost in metastases, suggesting ongoing TCRβ rearrangement or deletion (see Fig. 4⇓B, compare thymus and lymph node of TL2).

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FIGURE 4.

Characteristics of thymic lymphomas in CD2-GATA3-transgenic mice. A, Survival of wild-type mice (gray line, n = 42) and CD2-GATA3-transgenic mice (black line, n = 51), followed for 38 wk in a Kaplan-Meier curve as fraction of the total numbers of mice. B, Southern blotting analysis of TCRβ rearrangements in various lymphoblastoid tumor samples. EcoRI digests were hybridized to a TCR J_β2 probe. T, Thymus; S, spleen; L, lymph node. ∗, Position of the germline 2.4-kb EcoRI fragment; on the left the positions of λ X BsteII restriction fragments are indicated in kb. C, Western blotting analyses of GATA-3 protein expression in total nuclear extracts from the indicated tumor tissues as detected by anti-GATA-3 Abs. trans., ∼51-kDa transgenic GATA-3; endo., ∼47-kDa endogenous GATA-3; T, thymus; S, spleen; M, mesenteric lymph node; L, axillary lymph node.

When tumor cell samples from thymus, spleen, or lymph node were analyzed for the expression of GATA-3 in Western blotting experiments, high levels of transgenic HA-tagged GATA-3 were observed, often accompanied by high endogenous GATA-3 expression (Fig. 4⇑C). The ratio between transgenic and endogenous GATA-3 varied, not only between individual tumors but also between different metastases of a single tumor (see Fig. 4⇑C, compare mesenteric lymph node and spleen of TL 9).

Flow cytometric analyses demonstrated that the thymic lymphomas consisted of CD4⁺ lymphoblasts with variable levels of CD8 coexpression. Fig. 5⇓A illustrates four examples of thymic lymphomas (CD4⁺CD8^+/low cells), with different metastases in lymph nodes and spleen, showing the variability of surface CD4 and CD8 expression on the malignant cells. Immunohistochemical examination of thymic tumor tissue sections confirmed that the tumors mainly consisted of CD4⁺CD8⁺ lymphoblasts. Most of the tumors contained areas that had lost expression of CD8 and sometimes also CD4. A network of MHC class II-negative fibroblasts supported these lymphoblasts, whereas characteristic structures of epithelial cells expressing cortical or medullar cell markers were absent (data not shown).

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FIGURE 5.

Surface profile of lymphoblastoid tumor cells in CD2-GATA3-transgenic mice. A, Flow cytometric analyses of four different CD4⁺CD8^+/low thymic lymphoma primary tumor samples as well as metastases present in lymph node and spleen. B, Identification of an atypical GATA-3^highCD3⁺CD4⁺CD8^low lymphoblastoid cell population in the thymus of a CD2-GATA3-transgenic mouse, indicative for a thymic lymphoma (TL7). Nontransgenic and tumor-free CD2-GATA3-transgenic mice are shown as controls. Results are displayed as dot plots for CD4 and CD8. The given percentages of the gated CD4⁺CD8^low populations are of all thymocytes. C, Analysis of FSC, CD3, and intracellular GATA-3 expression in the gated CD4⁺CD8^low thymocyte subpopulation shown in B. The results are displayed as histograms of the CD2-GATA3-transgenic TL7 mouse (bold lines) along with those of a nontransgenic (dashed lines) and a tumor-free CD2-GATA3-transgenic mouse (thin lines). Cell suspensions were stained for CD3, CD4, and CD8 and subsequently for intracellular GATA-3. All samples are lymphocyte/lymphoblast gated by FSC and side scatter.

Among CD2-GATA3-transgenic mice that did not exhibit outward signs of illness nor manifested a macroscopically visible thymic tumor at ∼3 mo of age, we found evidence for early stages of tumor development in 6 of 32 cases (∼19%). In flow cytometric analyses of thymus cell suspensions, the CD4⁺CD8^+/low subsets contained atypical fractions of CD3⁺ lymphoblastoid cells with high forward scatter (FSC) characteristics suggestive of tumor growth. In the example shown in Fig. 5⇑, B and C, the lymphoblastoid cells had a CD3⁺CD4⁺CD8^low phenotype and expressed high levels of GATA-3 protein, as determined by intracellular flow cytometry. In these lymphoblastoid cells, CD69 expression was variable (data not shown).

These findings indicate that dysregulation of GATA-3 expression results in the formation of lymphoblastoid tumors at a specific stage of thymic development, i.e., the CD4⁺CD8^+/low thymocyte subpopulation.

GATA-3 enhances TCRαβ up-regulation during positive selection

As we observed a correlation between GATA-3 expression and TCRαβ or CD3 surface levels in GATA-3^+/nlslacZ mice, we investigated these parameters in the thymocyte subpopulations of the CD2-GATA3 mice (Fig. 6⇓A). The expression of CD69 in the DP, CD4^lowCD8^low, and CD4⁺CD8^low subpopulations was similar in CD2-GATA3 and wild-type mice. In contrast, the proportions of TCRαβ^high or CD3^high cells were significantly increased in CD2-GATA3 mice, particularly in the CD69⁺CD4^lowCD8^low subpopulation (shown for TCRαβ expression in Fig. 6⇓A). In the more mature fractions of CD4^lowCD8⁺ and SP cells, the expression levels of CD3 and TCRαβ were similar in transgenic animals and wild-type littermates.

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FIGURE 6.

The CD2-GATA3 transgene enhances TCRαβ up-regulation during positive selection. A, Cell suspensions were stained for CD4, CD8, and CD69 expression along with either CD3 or TCRαβ. The indicated T cell subpopulations (see also Fig. 1⇑A) were gated and analyzed for TCRαβ expression. The results are displayed as histograms of CD2-GATA3-transgenic mice (bold lines) along with those of nontransgenic control mice (thin lines). The percentages shown are the fractions of the CD69⁺CD4^lowD8^low cells that are in the indicated TCRαβ^high gate in wild-type mice (below marker) and CD2-GATA3-transgenic mice (above marker, bold type). B, The effect of the CD2-GATA3 transgene on the cell sizes of the DP thymocyte subpopulation. Cell suspensions were stained for CD4 and CD8. DP cells were gated and analyzed for FSC; the results are displayed as histogram overlays of a CD2-GATA3-transgenic (bold line) and nontransgenic control (thin line) mouse.

We noticed that in the CD2-GATA3-transgenic mice, the cells within the DP subpopulations had increased average FSC values closer to those of normal SP cells (Fig. 6⇑B). The increased size of CD2-GATA3-transgenic DP cells did not reflect an enhanced activation status of these cells, as we failed to detect activated cells with high Th2 cytokine production (26) in immunohistochemical analyses of the thymi of CD2-GATA3 mice. We also did not find evidence for a direct effect of transgenic GATA-3 on the cell cycle in DP cells, as flow cytometric analyses, using anti-CD4, anti-CD8, and To-Pro3, did not reveal differences in the cell cycle between CD2-GATA3-transgenic animals and their wild-type littermates (data not shown). The development of CD3/TCRαβ^low DP into CD3/TCRαβ^high SP cells is normally accompanied by an increase in the average cell size. Therefore, the findings of the small increase in DP cell size and the slightly accelerated up-regulation of surface TCRαβ and CD3 expression in CD2-GATA3-transgenic mice suggest that enforced GATA-3 expression may influence the kinetics of positive selection.

GATA-3 inhibits maturation of CD8 SP T cells

As the reduction of peripheral CD8⁺ T cell numbers in the CD2-GATA3 transgenic mice suggested increased cell death or hampered maturation of CD8 SP cells in the thymus, we analyzed the thymic CD8 SP compartment in more detail and specifically evaluated the final maturation steps of CD8 SP cells.

To analyze the extent of apoptosis in the SP subpopulations, we determined the fraction of cells that were annexin V-positive in CD2-GATA3-transgenic mice and their nontransgenic littermates. In addition, we performed TUNEL assays in conjunction with surface CD4/CD8 staining. Using these techniques, we found that the thymi of CD2-GATA3-transgenic mice contained higher numbers of apoptotic cells, not only in the CD8 SP but to some extent also in the CD4 SP subpopulations (Fig. 7⇓, A and B).

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FIGURE 7.

Enforced expression of GATA-3 induces apoptosis and inhibits the maturation of CD69^lowHSA^low CD8 SP cells in the thymus. Thymus cell suspensions were stained for CD4, CD8, and annexin V (A) or TUNEL (B). Thymocytes were analyzed for the expression for CD4 and CD8; the indicated SP subpopulations were gated and analyzed for FSC and annexin V or TUNEL. The numbers indicate the percentage of annexin V-positive (A) or TUNEL-positive (B) cells in the subpopulations analyzed. C, In four-color flow cytometry experiments, thymus cell suspensions were stained for CD4 and CD8 expression along with anti-HSA and anti-CD69 or with anti-CD62L and anti-CD44. The CD4 and CD8 SP T cells were gated and analyzed for the expression of the indicated markers. The results are displayed as histograms of CD2-GATA3-transgenic mice (bold lines) along with those of nontransgenic control mice (thin lines).

It has been reported that final maturation of SP T cells is accompanied by a down-regulation of CD69 and HSA expression (36, 48). The enforced GATA-3 expression appeared to inhibit the final maturation of CD8⁺ cells, as a selective deficiency of CD69^lowHSA^low cells was observed, when CD2-GATA3-transgenic and wild-type littermates were compared (Fig. 7⇑C). For CD4⁺ cells, the enforced GATA-3 expression only mildly affected the final thymic maturation steps. In addition, in the CD2-GATA3-transgenic mice an increase in the surface expression of CD44, a marker for activated or memory T cells, was observed both in the CD4 and the CD8 SP population (Fig. 7⇑C). This phenomenon was also seen in the mature CD4⁺ and CD8⁺ T cells in the spleen (see accompanying paper). Finally, the expression of L-selectin (CD62L), a marker which is expressed at high levels on naive T cells and which is essential for homing to peripheral lymphoid organs (51), was comparable in transgenic and nontransgenic animals. Therefore, the decrease in peripheral CD8⁺ T cell numbers in CD2-GATA3-transgenic mice cannot be explained by a reduced capacity of the mature CD8 SP T cells to leave the thymus.

In summary, we observed a substantial increase of apoptotic CD8 SP cells and a decrease of mature CD69^lowHSA^low cells in the thymic CD8 SP subpopulation, as well as reduced numbers of CD8⁺ T cells in the peripheral organs. These findings indicate that enforced GATA-3 expression resulted in a partial differentiation arrest of CD8⁺ cells associated with significant cell death in the thymus.